Third Annual Meeting

March 22-25, 1978

AMERICAN SOCIETY

OF

ANDROLOGY

Hyatt Regency

623 Union Street

Nashville, Tennessee

AMERI CAN SOC I ETY OF ANDROLOGY

-Th i rd Annual Meeti ng

March 22-25 , 1978

Hya tt Regency

Nas hvi l l e , Tennessee

SPONSOR

Vanderbil t Un i vers i ty School of Medi ci ne

i

Th i s p rogram has been s uppo rted i n pa rt th ro ugh a grant from Organon , I nc .

ii

PROGRAM COMMI TTEE

Mari e-Cl ai re O rgebi n- Cri s t , Ph . D . , Chai rman I an M . Burr, M . D .

Cl aude Desj a rdi ns , Ph . D . La rry Ewi ng , Ph . D .

Davi d W . Hami l ton , Ph . D . S tuart S . H owards , M . D .

Cl a i re H ucki ns , Ph . D . Anth ony R . Means , Ph . D.

Wi l l i am D . Ode l l , M . D . , Ph . D . Davi d Rabi n , M . D .

LOCAL ARRANGEMENTS COMM ITTEE

Ma ri e-Cl ai re Orgebi n- Cri s t , Ph . D . , Chai rma n Lonn i e S . Burne tt , M . D .

Benjami n J . Danza, Ph . D . Davi d L . Garbers , Ph . D . Loren H . Hoffman , Ph . D .

Gary E . O l s on , Ph . D . Davi d Rabi n , M . D .

Robert K . Rhamy , M . D . P i e rre Soupa rt , M . D . ,Ph . D .

AMERI CAN SOCI ETY OF ANDROLOGY

1 977- 1 978

OFFI CERS

President - Don W . Fawcett, M.D . Vice President - C . Al vi n Paul se n , M . D .

secretary - E . S . E . Hafez, Ph . D . Treasurer - Nancy J . Al exande r , Ph . D .

E XE CUTI VE COUNCI L

Rupert P . Amann , Ph . D . Andrzej Bartke, Ph . D .

Joseph N . Corri e re , J r . , M . D . Torrmy N . Evans , M . D .

La rry Ewi ng , Ph . D . Stuart S . Howards, M . D . E uge ni a Rosembe rg, M . D .

Ri cha rd J . Sheri ns , M . D . Anna Stei nbe rger, Ph . D .

Phi l i p Troen, M . D .

COMMITTEES

By-Laws - C . Al vi n Paul sen, M . D . Fiscal - Nancy J . Al exande r , Ph . D .

Liaison - J oseph N . Corri e re , J r . , M . D . Membership - Andrzej Bartke , Ph . D . Nomination - C . Al vi n Pa ul sen , M . D .

Postgraduate - Emi l Ste i nberge r , M . D . Program - M . - C . Orgebi n-Cri s t, Ph . D . Publication - Eugeni a Rosemberg , M . D .

i i i

iv

POSTGRADUATE COURSE

Emil Steinberger, M.D., Director

The Postgraduate Course on ''Recent Advances i n Androl ogy11 wi l l be hel d from 4 : 30 p . m . to 6 : 30 p . m. on March 22 i n Su i te 6 ( 6th fl oor) and from 4 : 30 p . m . to 6 : 30 p.m . on March 23 and 24 i n Sui te 4 ( 4th fl oor) . Thos e who have not reg i s tered i n advance for th i s cou rse may do so at the Reg i s trati on Des k .

As an organi zati on accredi ted for conti nui ng medi cal educati o n , the Uni vers i ty of Texas Hea l th Sc ien ce Cente r, Offi ce of Conti nui ng Educati on for the Medi cal School certi fi es that th i s conti nui ng medi cal education offe r­i ng meets the cri teri a fo r 6 credi t hours i n Category I of the Phys i c ian's Recogni ti on Award of the Ameri can Medi cal As s oci ati on and for 1 0 cogna tes by the Ame ri can Col l ege of Obs tetri ci ans and Gynecol ogi s ts , p rov i ded i t i s use d and comp l eted as desi gned .

AMERICAN SOCIETY OF ANDRI HYATI REGEi

March 2:

Registrat.

Tuesday, March 21, 6 to 8 p.i

Wednesday, March 22 Thursday

7:50 to 8:00 a.m.

WELCOME

8:00 to

REPRODUCTIVE 8:00 to 11:00 Researc

REPRODUCTIVE PHYSIOLOGY and MORPHOLOGY I Research Papers

I

11:30 to 12:30

" CHEMICAL PATHOLOGY OF THE 11:30

MALE REPRODUCTIVE TRACT - "PHOTOPERIOD RELATIONSHIP TO INFERTILITY" OF REPRODUCT

Guest Speaker Guest

Thaddeus Mann, Michael Men

C.B,E.,M.D., Sc.D.,Ph.D.,F.R.S.

_l_J r

2:00 to 4:30 p.m. 2 :00 to REPRODUCTIVE PHYSIOLOGY REPRODUCTIVE

and MORPHOLOGY II Reseaz Research Papers

4:30 to 6:30 4:30

POSTGRADUATE COURSE POSTGRADl "IMMUNOLOGIC PHENOMENON "ARTIFICIAL J

IN HUMAN INFERTILITY" CRYOPRESERVATH

Rudi Ansbacher, M.D., M.S. s. J. Bel

_.!;_!

6:30

7:00 to 8:00 BUSINE�

COCKTAIL RECEPTION

8.

BAI

...!...!

u

QUICK REFERENCE PROGRAM

IGY - 3rd Annual Meeting ' NASHVILLE �5. 1978

1 Begins:

. Levidson Foyer (2nd floor}

�arch 23 Friday, March 24

•00 a.m. 8:00 to 11:00 a.m.

lOCRINOLOGY I CLINICAL STUDIES IN ANDROLOGY I >apers Research Papers

11:30 to 12:30 12:30 "STUDIES OF SERTOLI CELL FUNCTION REGULATION IN THE RAT WITH HEREDITARY

: FUNCTION" SEMINIFEROUS TUBULAR FAILURE"

!aker Guest Speaker �r, Ph.D. c. Wayne Bardin, M.D.

:30 p.m. 2:00 to 4:30 p.m.

JO CR I NOLOGY II CLINICAL STUDIES IN ANDROLOGY II Papers Research Papers

6:30 4:30 to 6:30

E COURSE POSTGRADUATE COURSE EMINATION AND "PATHOPHYSIOLOGY OF BENIGN OF HUMAN SEMEN" PROSTATIC HYPERTROPHY"

ian, M.D. Avery A. Sandberg, M.D.

' 7:30

MEETING

I

ET

v

MEMBERSHIP I NFORMATION

Thi s i s an i nvi ta ti on to joi n the Ame ri can Soci ­e ty of Andro l ogy . The ASA was organi zed i n 1 975 an d i s composed of s ci e nti s ts and c l i ni ci ans i n­te reste d i n the mal e reproducti ve sys tem. Our members h i p now i ncl udes anatomi s ts , an ima l s ci ­enti s ts , bi ochemi s ts , endocri nol ogi s ts , gynecol ­ogi s ts , i nterni s ts , pathol ogi s ts , phys i o l ogi s ts , psychi a tri s ts , uro l ogi s ts , vete ri nari ans , and o the rs , i ncl udi ng many s tudents .

Thi s soci e ty was founde d to mee t the need of concentra ti ng a ttenti on and exchang i ng sci en­ti fi c i nfonna ti o n i n the a rea of mal e reproduc­ti on i n i ts broadest s ense . I nte rests of members range from fundamental mo rphol ogi cal , b i ochemi ­cal , e ndo cri ne , neuroe ndoc ri ne , and phys i ol ogi cal s tudies to cl i ni cal , d i agnos ti c , therapeuti c , s u rg ica l , and beh av ioral s tudies .

We i nvi te you to joi n the Soci ety and take part i n i ts annual mee ti ngs , the next of whi ch wi l l be i n Hous ton , Texas i n 1 979. Your contri buti on to the Soc i e ty ' s growth wi l l be mos t wel come .

App l i cat i on forms are avai l abl e at the Regi s tra­ti on Des k , or from

Dr. Andrzej Bartke

Membership Chairman, ASA

Worcester Foundation

for Experimental Biology

222 Maple Avenue

Shrewsbury, Massachusetts 01545

vi

GENERAL I NFORMATION

Headquarters for the 1 978 Meeti ng of the Ame ri can Soc i e ty of An drol ogy wi l l be at the Hyatt Regency , 623 Un i on Street , Nas hv i l l e , Tennessee , whe re a l l mee tings w i l l be hel d .

For tho se who have not reg i s tered i n advance , the Reg i s trati on Des k wi l l be l ocated i n the Dav i ds on Foye r ( 2nd fl oor) on Tuesday , March 2 1 , and wi l l be open from 6 : 00 p . m . to 8 : 00 p .m . The reafte r , the Regi s tration Des k wi l l b e l ocated i n the Wes t Foyer ( 2nd fl oor) and wi l l be open from 7 : 00 a . m . to 4 : 30 p . m . on Wednes day , March 22 , a nd from 7 : 30 a . m . to 4 : 30 p.m . on Th urs day and Fri day , March 23 a nd 24 . Parti c i pants who have reg i s tered i n advance may obtai n thei r badges at the Reg i s trati on Des k .

vii

COCKTAIL RECEPTION

A no- hos t Cocktai l Reception { open cas h bar) wi l l be hel d from 7 : 00 p . m . to 8 : 00 p.m. on Wednesday, March 2 2 , i n Regency I V ( 2nd fl oo r) . Al l regi s ­tra nts , gues ts , and s pouses are wel come .

ANNUAL BUS INESS MEETI NG

The Annual Bus i ness Meeti ng of the Ame ri can So­c iety of Androl ogy wi l l be he l d from 6 : 30 p.m. to 7 : 30 p .m . on Thurs day , March 2 3 , i n Regency I I I and I V ( 2nd fl oor ). Commi ttee reports , un­fi ni shed and new bus i nes s wi l l be p resented. Atte ndance i s l i mi ted to membe rs of the Soci e ty ; they are urged to attend .

ANNUAL BANQUET

The Annua l Banque t wi l l ta ke pl ace on Thu rs day , March 2 3 , at 8 : 00 p .m. i n Regency I and I I { 2n d fl oor ) . OUTBOUN D FRE I GHT wi l l entertai n wi th bl uegras s mus i c .

vii i

Wednesday , March 22 , 1 978

"CHEMI CAL PATHOLOGY OF THE MALE REPRODUCTIVE TRACT -

RELATIONSH I P TO I NFERTI L I TY"

Thaddeus Mann , C . B . E. , M . D. , Sc. D . , Ph . D . ,F . R. S. Emeri tus Professor of

Phys i ol ogy of Reproducti on Uni vers i ty of Cambri dge , Eng l and

V i s i ti ng Sci enti s t at the Nati onal I nsti tutes of Heal th

Thursday , March 23 , 1 978

11 PHOTOPE RIODIC REGULATION OF REPRODUCTI VE FUNCTI ON 1 1

Mi chael Mena ker , Ph . D . Professor o f Zoo l ogy

The Un ive rs i ty of Texas at Aus ti n Aus ti n , Texas

ix

x

Fri day , Ma rch 24 , 1978

" STUDI ES OF SERTOLI CELL FUNCTI ON I N THE RAT WI TH

HE RE DI TARY SEMI N I FE ROUS TUBULAR FAI LURE"

C . Wayne Bardi n , M . D . Pro fes sor o f Medi ci ne and

Ch i ef, Di v i s i on of E ndocri nol ogy The Pen nsyl van i a State Un i ve rs i ty

Hers hey , Pennsyl van i a

xi

AME RI CAN SOCI ElY OF ANDROLOGY - 3rd Annual Meeti ng

Tue s day , March 2 1 , 1 978

6:00 p . m. Regi stra tion Desk Ope ns Davi dson Foyer (2nd floor)

Wednesday , Ma rch 22 , 1 978

7:00 a . m .

7:50

Regi stra tion We st Foyer (2nd floor)

WELCOME Regency I V ( 2nd fl oor ) M . -c. Orgebin -Cri st, Ph . D. Progra m Chairman

John E. Chap man, M . D . Dean, School o f Medi cine Vanderbil t Uni versi ty

REPRODUCT I VE PHYSI OLOGY AND MORPHOLOGY I Rege ncy I V ( 2nd fl oor ) Modera tors: Davi d w. Hami l ton , Ph . D.

Davi d L. Garbers, Ph . D .

( * = Presenter) (,cl..- � &- r G-8: oo 80

C-FfrilJA!i;J

��? Observations on the i ni ti ati on of s perm mo ti 1 i ty T. T. Turner* and s. s. Howards

8:15 73 Bovi ne s pe rm cycl i c AMP phospho-d ieste ras e : Phys i cal properti es a nd deve l opmental changes D . T . S tephens*, Jun -lan Wang, and D. D. Hoskin s

xii

8: 30 55 Modi fi cati on of cal ci um b i ndi ng

1 0 _ I.A � spe rm cel l membranes

� L . Nel son* and M. Young

8: 45 60 In vi tro s ti mul ati on of rabbi t s pe rm moti l i ty by L-argi n i ne E. s. Radany* and R . w. A t herton

9 : 00 56 B i ochemi ca l changes i n the rat s pe rmatozoan pl asma membrane duri ng epi di dymal tra ns i t G . E. Ol son * and B . J. Dan zo

9: 15 35 Label l i ng of a s urface cycl i c-AMP recep to r s i te on rabb i t sperm uti l i z i ng a photo­affi ni ty ana l og G . L . Hahn*, K . w . Me t z , R . w. A therton, and B . E. Hal ey

9 : 30 79 Presence of sol ubl e B- type cyto-ch rome i n human semi nal p l asma N . P. Tri funac*, A. F . B rodi e , and G . s . Bernstein

9: 45 5 2 Acrosoma l ami nopepti dase and a

10: 00

10: 15

non-acros i n endoprotei nase from rabbi t s permatozoa D. K. McRori e*, M . M. Bradford, and R . A . McRorie

33 The proacros i n , acros i n and acrosi n i nh i bi tor sys tem of human s pennatozoa J. C. Goodpa sture *, K. L. Polako ski , and L . J . D. Zaneveld

2 Effect of cal ci um on the di s ­sol uti on o f rabbi t zona pe l ­l uc i da by s pe rm ac rosomal extract S . R . Akruk* and P . N. Sri va stava

1 0 : 30

1 0 : 45

11 : 00

11 : 30

1 2: 30

xiii

66 Characteri sti cs of ca+2_depe nde nt bi ndi ng of mouse sperm to the zona pel l u cida of the mouse egg P . M. Saling* and B . T. Storey

75 The acros ome reacti on i n boa r spermatozoa Dani el S z ol l osi * and R.H .F . H unter

B REAK

1 1 CHEMICAL PATHOLOGY OF THE MALE RE PRODUCTI VE TRACT - RELATI ON­SH I P TO I NFE RT I L I TY1 1

Thadde us Mann , C.B .E. ,M. D. ,sc.D. ,Ph .D. , F.R . S . Emeri tus Professor of Physiol ogy of Reproducti on Uni versi ty of Cambri dge, England Vi si ting Scien ti st at the National Insti tutes of Heal th

LUNCH

REPRODUCTI VE PHYSI OLOGY AND MORPHOLOGY I I Rege ncy I V (2nd fl oor)

Modera tor s: Marti n Dym , Ph .D. Nancy J. Alexander, Ph . D .

( * = Presen ter)

2 : 0 0 p .m. 32 Ce l l ul ar i nte rrel ati ons h i ps i n the semi ni fe rous cords of the pos tna tal rabbi t tes ti s B . Gondos*, C . J. Connel l , M. F. McKoon , and c. E. Lai

xi v

2 : 15 65 E l i mi nati on of cytop l asm from the spermati d i n the head re­g i on duri ng l ate s pe rmi ogene s i s L. D . Russell *

2: 30 24 I nfl uence of age on spermato-gon i al s tem ce l l n umber and funct ion in the rat B. H. Eri ckson * and Debra Conna t se r

2: 45 11 In vi tro characte ri zation of rat spermatogonia l chal one E . Bustos-Obregon * and A . Thu mann

3:00 39 Hormonal , h i sto l ogi c and b i o-chemi cal characteri s ti cs of spermatogenes i s ( S g ) i n vi ta­mi n A defi ci ent ( VAD) and i n p ost vi tami n A ( PVA) treated rats H . F. S. Huang* and w. c. Hembree

3 : 15 B REAK

3 : 30 26 Fa i l u re of exogenous androge n to prevent regress i on of the i n i ti al segments of the epi ­di dymis after effere nt duct l i gati on or cas trati on D. w. Fawce tt* and A . P. Hoffer

3 : 45 15 Lumi nal perfusi on of the rat's epi di dymi s T. G. Cooper* and G . M. H. Wai tes

4 : 00 48 Semi nal ves i cl e membrane trans-port in vi vo i n gui nea pi gs N . Le vine * and H. Kelly

xv

4: 15 40 Cannul ati on and i ntra vas

4: 30

7 : 0 0

defe re ns trea tment of the rat pros tate w. L. Hun t * and N. Ni chol son

POSTGRADUATE COURSE Sui te 6 ( 6th fl oor)

RECE NT DEVELOPME NTS IN ANDROLOGY Director : Emi l S teinberger, M.D.

11I MMUNOLOG IC PHENOMENON I N H UMAN I NFERTI L I TY "

Rudi Ansbacher, M. D. , M.S. Chi ef, Obste tri cs & Gynecology Le tterman A rmy Medi cal Center San Franci sco , Cali forni a

Nancy J . Alexander, Ph . D . Oregon Regional Pri ma te Re search Cen ter Beaverton , Oregon

COCKTAI L RE CE PTI ON Rege ncy I V ( 2nd fl oo r)

xvi

AME R I CAN SOCIETY OF ANDROLOGY - 3rd Annua l Meeti ng

Thurs day , March 2 3 , 1 978

7 : 30 a . m. Regi stra tion De sk Opens We st Foyer (2nd floor)

REPRODUCTI VE E NDOCRI NOLOGY I Regency I I I and I V

Modera tors: Euge nia Rosemberg , M. D. S tuart s. Howards, M. D.

(* = Pre senter)

8 : 0 0

8 : 15

36 Reprod ucti ve system of the di a-beti c mal e rat n r:;/�...f J M.::jry E. 11arri s * lUJr't'e�fer {;(,. - -Jf>./ CJC;v l St.

S it r�sbtoy1 f?i Ir o I S:-(.{s-29 Importance of sero ton in i n the

regul ati on of gonadotropi n i n ma n F. Frai oli *, v. Bonifacio , s. Pall otti , D. Paol ucci , and A . Isi dori

8 : 30 30 Effects of i ntravenous admi n i s -trati on o f bra i n phos phol i pi ds on gonadotro pi n rel ease i n man F. Fraioli * , D. Paol ucci , M. B. Me sina , s. Morrone , and A. Isi dori

8 : 45 83 Phys i ol ogi c concentra ti ons of tes tos terone and the i r effect on LH secreti on i n the mal e mongrel dog D. L. Vincen t *, T. Kepi c , and R . E . Fal vo

xvii

9 : 00 67 Response of ram tes ti s to c l omi phe ne and gonadotropi n rel eas i ng hormone B. D. Schanba cher*

9 : 15 10 Acute and chroni c al te rati ons

9 : 30

9 : 45

10 : 00

10 : 15

i n tes ti cul ar feedback : E ffects on LH respons i veness to LHRH i n the ram W. Bremner*, J. Findl ay , I . Cumming , and D. de Kre tser

6 I nh i bi ti on of reproducti ve pro­ces ses i n the immature and mature mal e rat wi th an LH RH agoni s t F . J . Bex* and A . Corbin

57 I nh i bi ti on of spermatoge nesi s i n the rat by treatment wi th [D-ALA6 , DES-GLY-NH2lDJLHRH e thyl ami de G. Pel leti er*, L. Cusan , P . A . Kell y , L. Desy , and F. Labrie

4 I nteracti on be tween LH and PRL i n the contro l of tes ti cul ar LH and PRL receptor l eve l s c. A uclai r*, P. A . Kell y , and F . Labrie

17 Tes ti cul ar gonadotropi n receptor l oss and i nhi bi ti on of spermato­ge nes i s fol l owi ng l ong-term treatment wi th an LHRH agon i st i n the rat L. Cusan *, c. Aucl ai r , P . A. Kelly , M. Garon , and F. Labrie

xviii

10 : 30

10 : 4 5

11 : 00

11: 30

12 : 30

13 Sel ecti ve i nh i bi ti on of FSH synthes i s by the Sertol i cel l factor ( SCF ) i n vi tro M . Chowdhury *, A . Steinberger, and E. S teinberger

61 Effects of pas s i ve immun i ty to FSH on mal e reproducti on i n the i mma ture and adul t rat H . E. Ma dhwa Ra j, M . Dym*, H. E. Cheme s, N . J. Ko ti te , s. N. Nayfeh , and F . s. French

BREAK

1 1 PHOTOPERIOD I C REGULAT I ON OF RE PRODUCT! VE FUN CTI ON1 1

Mi chael Menaker, Ph . D . P rofessor of Zoology The Uni versi ty of Texa s a t A u stin A u sti n , Texas

LUNCH

REPRO DUCTIVE ENDOCRINOLOGY I I Rege ncy I I I and I V ( 2nd fl oor)

Modera tors : An thony R . Mean s, Ph . D. Larry L. Ewing , Ph . D.

(* = Pre sen ter)

2 : 0 0 p.m. 7 7 In vi tro me tabo l i sm of tes tos­terone by cul tured Serto l i cel l s of 36- day ol d rats wi th and wi tho ut FSH s ti mu l ati on R. K. Tcholaki an* and A . S teinberger

2 : 15 72 Evi den ce that Sa- reducti on of

xi x

�4-3- ketostero i ds may be more i mpo rtant for the i r a ndrogen i c than LH-i nh i bi ti ng acti v i ty R. E. Steel e * , F. Dida to, an d B. G. S teine t z

2 : 30 69 Pe ri fus i on of Leydi g tumor cel l s : HCG effects on s teroi dogenes i s Deborah L. Segaloff*, Mari o A scoli , and Davi d Puett

2 : 45 38 Effect of p i tui tary grafts on testos terone- s timul ated growth i n rat pros tate J. M. Hol land*, K. A . Han sen , and C. Lee

3 : 00 45 Speci fi c bi ndi ng of prol acti n and di hydrotes tos terone ( DHT) by mal e accessory sex o rgans E. J . Keenan *, E. D. Kemp , E. E. Ramsey , L. B . Garri son , H. D. Pearse , and C. V. Hodges

3 : 15 BREAK

3: 30 54 Androgen-i nduced s ti mul ati on of three new protei ns i n mouse ki dney N. c. Mil l s*, T. M . Mil l s, and C. W. Bardin

3 : 4 5 58 Receptor bi ndi ng and enzyme i nduc i ng acti v i ty of R 1 881 (methyl tri e nol one ) i n P seudo­monas testosteroni Ake Pousette * and Kjell Carl strom

xx

4 : 0 0 28 B i ndi ng of e s tramus ti ne , a n i tro-gen mus tard deri vati ve of estra ­di ol - 1 78 , i n cytosol from rat ve ntral prostate . B jorn Forsgren, Ake Pousette*, Jan-Ake Gu stafsson , and Bertil Ho gberg

4 : 15 19 Speci fi c b i n d i n g of es trad i o l to

4: 30

6: 30

8 : 00

epi di dymal cytosol from mature rabbi ts Benja min J. Danzo* and Mari e-Clai re Orgebin-Cri st

POSTGRADUATE COURSE Sui te 4 ( 4th fl oor)

RE CENT DE VELOPME NTS I N ANDROLOGY Di re ctor: Emil Steinberger, M . D .

1 1ART IFI CIAL I NSEMINATI ON AND CRYOPRESERVATI ON OF HUMAN SEMEN11

s. J . Behrman , M. D . Chi ef, Obste tri cs & G ynecology Willi a m Beaumont Hospi tal Royal Oaks, Mi chi gan and Professor of Obste tri cs & G yne cology Wayne Sta te Uni versi t y Detroi t, Mi chi gan

B US I N ESS ME ET I NG Regen cy I I I a nd I V ( 2n d f l oor )

BANQUET Regency I and I I ( 2nd fl oor )

xxi

AME RI CAN SOCI ETY OF ANDROLOGY - 3rd Annual Meeti ng

Fri day , March 24 , 1 978

7 : 30 a . m . Regi st ra ti on Desk Open s West Fo ye r (2nd floor)

CL I N I CAL STUDI ES IN ANDROLOGY I Regency I I I and I V ( 2nd fl oor)

Mode ra tors: Emil Steinbe rger, M. D. Ri cha rd J. Sheri n s, M. D.

(* = Presente r)

8 : 00 44 Eva l uati on of ci rcu l ati ng pat-terns of tes tos terone i n mal e rats mai ntai ned on total paren­teral nutri t i on ( TPN ) -An ex­peri me ntal model R. J . Kea ti n g*, R. K. Tcholaki an, and E. Steinbe rger

8 : 1 5 63 In vi tro tes ti cul ar s teroi d me-tabo l i sm and peri phe ral hormone l evel s in i nferti l e men L. J . Rodri guez -Ri gau*, D. B . Wei ss, K. D. Smi th, and E. Steinbe rge r

8 : 30 4 2 I nves ti gation of the axi s hypo-thal amus -hypophys i s - gonads i n 20 di a lysed men E. Je ch t *, E. Kl upp, and R . Bei tl er

8: 45 12 Chromosomal abnormal i ti e s associ -ated wi th the absence of dyne i n a rms i n the s permatozoa of two s teri l e men A. Chapdelaine *, C. -L. Ri che r, K. D. Robe rts, and G . Bleau

xx i i

9:0 0 5 Ki neti cs of s teroi d metabo l i sm i n a phenotypi c man wi th embry­on ic tes ti cul ar reg ress i on G. W. Bate s*, c. D. Ed man, and P . c. Ma cDonal d

9:1 5 70 Functi on of a human tes ti c l e after transpl antati on to a n anarch i c i de nti cal twi n bro the r S. J . Si lber*

9:30 71 A new mi croscopi c tech ni que for vasoepi di dymostomy : A prel i m­i na ry report s. J. Si lber*

9:45 51 Safe and successful re ctal probe

1 0 :0 0

1 0 :1 5

1 0 :30

1 0 :4 5

e l ectrosti mul a ti on of e recti on i n the human mal e D . E. Martin *, H . warner, R. T. Cren shaw, and T. L. Crenshaw

1 Gynecomas ti a - A l ate conse­quence of mumps orchi ti s E. J . Ai man *, P . F . Brenner, and P . C. Ma cDonald

53 Use of bovi ne cervi ca l mucus i n the in vi tro sperm pene trati on me thod A. c. Men ge* and N. E. Medl e y

2 5 Facto rs underlyi ng i nferti l i ty i n the al coho l i c w . E. Farn sworth *, A . H . Cavanaugh, J . R . B rown, I. Al varez, and L. M. Lewandowski

23 Argi n i ne aspartate and ma l e hypo­fe rti l i ty - A doubl e bl i nd s tudy J . C. Emperai re* and A . JM. Audebert

1 1 : 00

1 1 : 30

1 2 : 30

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B REAK

1 1STUDI ES ON SERTOLI CELL FUNCTION IN THE RAT WITH HEREDITARY SEMI ­N IFEROUS TUBULAR FAI LURE 1 1

c. Wayne Bardin, M. D. *, Neal A . Musto, Ph . D. , and Glen G unsal u s, Ph .D.

*Profe ssor of Medi cine and Chief, Di vi si on of Endocrinol o gy The Pennsylvani a State Uni versi ty Hershey, Penn syl vani a

LUNCH

CL I N I CAL STUDI ES I N ANDROLOGY I I Rege ncy I I I and I V { 2n d fl oo r ) Moderator s: C. Alvin Paul sen , M . D.

Davi d Rabin, M. D.

(* = Presenter)

2 : 00 50 Immunos uppress i ve acti v i ti es o f mal e reproducti ve tract com­ponents z. H. Marcus*, J . H. Herman , and E. V. Hess

2 : 1 5 3 7 I nh i bi ti on of semi nal fl ui d DNA polymerase by 7S i mmunogl obul i n G i so l ated from h uma n post­vasectomy semen P . J. Hi ggi n s*, s. s. Wi tkin , a n d A . Bendi ch

2 : 30 78 Tes ti cul ar anti bodi es i n func-ti onal azoospe rmi a M . H . E. Ton sy *, H . Abdelal , A. A . Ha fiez, and o. Kandil

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2 :45 82 Decreased spennatogenes i s after experi menta l vari cocel e creati on i n the dog and rat R . L. Urry *, B. Deter, K. Dougherty, and A . T. K . Cocke tt

3 :0 0 8 4 Leydi g cel l functi on i n ol i go-

3 :1 5

s pe nni c me n wi th vari cocel e D. B . Wei ss*, L. J. Rodri guez­Ri ga u , K. D. Smi th , and E. Steinberger

B REAK

3 :30 49 Contact s crota l the rmography i n ferti l e men a nd i n subfe rti l e men wi th and wi tho ut cl i n i ca l vari cocel e R . w. Lewi s* and R . M. Harri son

3 : 45 20 Res ul ts of va ri coce le trans-catheter s cl eros i ng trea tment M . P . de Ca stro* and s. S . Li ma

4 :00 1 6 The p resence of exfol i ated geni touri nary trac t ce l l s i n h uman seme n Margare t L. Cou ture * and Matthew Fre und

4 :1 5 1 8 Spenn freezi ng i n 0-5 a n d 0-25 ml s traws - A comparative study F. Czyglik and J. c. Emperai re *

4 :30

POSTGRADUATE COURSE Sui te 4 ( 4th fl oor )

RECENT DEVELOPMENTS IN ANDROLOGY Di rector: Emil Steinbe rge r, M. D.

xxv

"PATHOPHYS IOLOGY OF BENIGN PROS­TATI C HYPERTROPHY"

Ave ry A . Sandbe rg, M. D. Chi ef, Department of Medi ci ne C Roswel l Park Me mori al In sti tute Buffalo , New York

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GYNECOMASTIA - A LATE CONSEQUENCE OF MUMPS ORCHITIS. E .J. Aiman, P .F. Brenner, and P .C. MacDonald. The Cecil H. and Ida Green Center for Re­productive Biology Sciences and the Department of Ob­stetrics and Gynecology, University of Texas South­western Medical School, Dallas, Texas.

Gynecomastia appeared 30 years after the occurrence of testicular atrophy due to mumps orchitis in 2 men, ages 63 and 68 years. The plasma production rates of testosterone (PR-T, 837 µg/day and 1409 µg/day) and androstenedione (PR-A, 2158 µg/day and 1395 µg/day) were 20% and 60% the rates of adult men without gyne­comastia. These values for the PR-T are still only 30% the reported values of older men without gynecomastia (Vermeulen et. al. J Cl in Endocrinol Metab 34: 730,

1972) . � �

The production rates of estradiol (PR-E2, 36 µg/day and 37 µg/day) and of estrone (PR-El, 84 µg/day and 39 µg/day) are comparable to values observed in adult males without gynecomastia. Extraglandular conversion of prehormones accounted for all the estradiol in both subjects and all the estrone in one of the two men. In the other man, glandular secretion of estrone accounted for 19 µg/day of the total PR-El of 84 µg/day.

We propose that mumps orchitis impairs the capacity of Leydig cells to secrete testosterone but does not alter the capacity to form estrogen, most of which is normally formed in extraglandular sites from circulating prehor­mones. Superimposed on this impairment of Leydig cell function is a progressive decline in testosterone secre­tion and an increase in extraglandular aromatization with advancing age and, in time, the PR-T becomes in­sufficient to antagonize the effect of estrogen. This hypothesis is supported by the findings of normal es­trone and estradiol production rates, but markedly di­minished production rates of androstenedione and testos­terone in both men. The ratio PR-T/PR-E2 is 130 in adult men without gynecomastia, 90 in elderly men with­out gynecomastia, but 26 and 23 in these two men. Gynecomastia is the consequence.

EFFECT OF C ALCIUM ON THE DISSOLUTION OF RABBIT ZONA PELLUCI DA BY SPERM ACROSOMAL EXTRACT. S .R. Akruk and P .N. Srivastava. Reproduction Research Laboratories, Department of Biochemistry, University of Georgia, Athens, GA

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The acrosomal extract from rabbit spermatozoa was more effective in dissolving rabbit zon a pellucida in the absence of ca++ than in the presence of ca++. Fol­licle cell-f°ree ova were incubated with the MgCl2 ex­tract of spermatozoa (Srivastava, P.N., 1973, J. Reprod. Fert. 33:323) in 50 mM Tris-HCl buffer at various pH levels with or without ca++ , at 37°C. The acrosomal ex­tract had a high Azocoll, dyed collagen (prote olytic activity), but low BAEE, benzoyl L-arginine ethyl ester (esterolytic activity), at neutral pH. Pretreatment of the extract at pH 3 reduced the Azocoll but enhanced the BAEE activity. In the absence of ca++, BAEE activ­ity was low but Azocoll activity was high, while in the presence of ca++, BAEE activity increased and Azocoll activity was reduced. In the absence of ca++ zona dis­solution was faster, and required 1 h, than in the pre­sence of 25 mM ca++ which required 8 h, using the same amount of the extract (87 µg protein) . The incubation mixture had an enzyme activity of 20 mu BAEE and 80 U of Azocoll in the absence of ca++, while in the presence of ca++ it had 200 mu BAEE and 16 U of Azocoll. Zona dissolution was more effective at pH 8.6 than pH 8 or 7 .4. Control eggs incubated in 0.15 M NaCl and the buf­fer showed n o signs of morphological change in the zona or vitellus whether in the presence or absence of ca++ after 24 h of incubation . Neither the pH nor the enzyme activity of the incubation mixture changed after 24 h of incubation. Freshly prepared MgCl2 extract was more effective in dissolving the zona than stored freeze­dried extracts. These preliminary results seem to sug­gest that acrosomal proteinase(s) other than acrosin may be involved in the zona penetration of rabbit eggs. (Supported by NIH Grant #ROl HD1087-0l and a Fore Foundation Grant #680-0805A) .

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ANTIBODIES AND FERTILITY IN RHESUS MONKEYS. N . J . Alexander and D . L . Fulgham. Oregon Regional Primate Res earch Center , Beaverton , Oregon .

We conducted s tudies to determine how antisperm ant ibodies affect male fertility , i . e . , whether sperm from infert ile vasovasos tomized rhesus monkeys (Macaca mulatta) have a shorter life span or reduced mo tility over t ime as a result of antisperm ant ibodies present in the s eminal plasma and whether the mot ility of normal monkey sperm incubated in seminal plasma from vasectomized or vasovasostomized animals with circu­lating ant isperm ant ibod ies is reduced over time .

S era from vasectomized and vasovasos tomized monkeys were tested for antisperm activity by means of sperm inunobilizat ion and indirect inununof luorescence as says . Semen samp les were obtained from those rhesus monkeys with high levels of c ir culating antisperm antibodies . Sperm samp les were incubated at 37°C , and their mot ility was determined at selected intervals there­after .

The ant isperm antibodies were sperm-specific , and ant isperm activity was limited to the immunoglobulin components . Homologous epididymal or ej aculated sp erm but not liver powder adsorbed out this activity . Ant isperm antibody levels in serum or seminal p lasma did not correlate with total innnunoglobulin levels , nor did seminal p lasma innnunoglobulin levels correlate with s erum levels . Because the various semen s amples responded differently in the motility tes t s , we could make no gener alizations . Some seminal p lasma samples appeared to impede motility , but these were not necessarily the same samples that exhib ited high innnunoglobulin levels . Spermatozoa incubated in sera containing antisp erm activity did exhibit reduced penetration and migration through cervical mucus in vitro .

Supported by a contract from the Program for App lied Research on Fertility Regulation (PARFR-70N) and NIH Grant RR-00163 .

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INTERACTION BETWEEN LH AND PRL IN THE CONTROL OF TESTI -1 CULAR LH AND PRL RECEPTOR LEVELS. C. Auclair, P.A. Kel­.lY and F. Labrie. MRC Group in Molecular Endocrinology, Le Centre Hospitalier de l 1Universite Laval. Quebec.

A marked decrease of testicular LH receptors and steroidogenic response to gonadotropins has been obser­ved in the rat after systemic administration of LH or hCG. More recently, we found that treatment of adult male rats with a potent analog of LHRH ,[D-Leu6,Des-Gly­NH210JLHRH ethylamide, led, somewhat unexpectedly to a marked reduction of testicular LH and prolactin (PRL) receptor levels accompanied by decreased testicular weight and plasma testosterone levels. A single injec­tion of the LHRH agonist or hCG was found to induce a long-lasting loss of testis LH binding sites with re­ceptor levels returning to control values at 8 days. Testis and seminal vesicle weight as well as plasma te� tosterone levels were also significantly reduced. Since such dramatic changes of peptide receptor levels are likely to play a major role in testicular desensitiza­tion after LHRH agonist administration, we have studied the hormonal factors involved in the control of testi­cular LH and PRL receptor levels using hypophysectomi­zed rats. Groups of animals were injected twice a day for 5 days with hCG (2IU), oPRL (250µg), oFSH (lµg), 17s-estradiol (E2, 0.5µg), testosterone (T, 200µg) or dihydrotestosterone (DHT, 200µg) alone or in combina­tion. The animals were killed at least 18 hours after the last injection. hCG led to a 80% reduction of LH receptor levels while PRL alone caused a 2.7-fold in­crease of LH receptors and partially reversed the inhi­bitory effect of hCG. PRL receptor concentration was markedly reduced (45% of control) after hypophysectomy, this decrease being completely reversible by hCG. At the doses used, 17s-E2, T and DHT had no significant influence on the level of peptide hormone receptors. Although the mechanism is unclear, the present data in­dicate that testicular LH and PRL receptor levels are under stimulatory influence of PRL and LH, respectively, and suggest an important interaction between these two hormones in the control of testicular function.

5

KINETICS OF STEROID METABOLISM IN A PHENOTYPIC MAN WITH EMBRYONIC TESTICULAR REGRESSION, G.W .Bates, C.D.Edman, and P.C.MacDonald, Cecil H. and Ida Green Center for Reproductive Biology Sciences, De­partment of Obstetrics-Gynecology, Southwestern Medical School, Dallas, Tx.

We have investigated the kinetics of steroid metabolism in a 24 year old phenotypic male with embryonic testicular regression by measuring the production rate (PR) of andro-· stenedione (A), testosterone (T), estrone (El), and estradiol (E2); and we have computed the transfer constant of conversion [p] of A +El, T+E2, and El+E2. In this man the PR-T was 222 µg/day, of which 60 µg was derived from extraglandular conversion of plasma A and 162 µg was not. Following a five day challenge with human chorionic gonadotropin, there was no increase in plasma testosterone. The PR-El (18.2 µg/day) could be accounted for from the extraglandular aromatization of plasma A, and similarly, all E2 was derived from plasma El. At postmortem examin­ation following accidental death, the wolffian duct-derived structures (vas deferens, epididymis, and seminal vesicles) were normal in structure and location. There were no mUllerian remnants, and no ectopic gonadal tissue was identified.

The role of the embryonic testis in directing sexual differentiation is well understood. The male gonads secrete two substances: (l) mullerian duct regression factor that causes regression of the female internal genital anlage and (2) testosterone that stimulates development of the wolffian duct structures, and as a prohormone, is converted to dihydrotestosterone that causes development of the external genital structures. We conclude from this study (1) that testicular regression occurred following the elaboration and action of these substances; (2) that the kinetics of extra­glandular metabolism of steroids in this subject are similar to those found in normal men; and (3) that the minimal production of testosterone could not be accounted for by the extraglandular metabolism of androstenedione, and that small amounts of testosterone were secreted by Leydig cells that could not be visualized.

INHIBITION OF REPRODUCTIVE PROCESSES IN THE IMMATURE AND MATURE MALE RAT WITH AN LHRH AGONIST. F . J . Bex and A. Corbin. Wyeth Laboratories, Philadelphia, PA.

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The e ffect o f chronic administration of a highly po­tent luteinizing hormone releasing hormone (LHRH) ago­nist, D-ALA6-DES-GLY 10- PR09-ethylamide-LHRH (Wy-18,481) on sexual maturation o f immature ma le rats and on re­productive function in adult male rats was evaluated.

Prepubertal males received daily sc inj ections o f l.Omg Wy- 18,481 from day 25 through day 35 o f age. Ani­mals were autops ied on day 39 o f age.

Adult males received a dai ly sc dose of l.Omg Wy-18 ,48 l for either 7, 14, 21, 28 or 35 days. All ani­mals with in a given treatment group were autops ied the day fol lowing the last injection. Vehicle (corn oil) injected anima ls served as controls in both studies.

Testes weights o f immature rats treated for 1 1 days with Wy-18,481 were s igni ficantly less (p<0. 0 1 ) than controls. However, weights o f accessory sex organs and the p ituitary were unaffected. In adult males, the agonist dramatically reduced the weight o f the testes within 7 days and those o f the seminal vesicles and ventral prostate within 14 days. These differences were maintained for the duration o f treatment and were corre lated with a loss o f mating behavior. Histo logi­cal evaluation of testes taken from representative animals during each o f the intervals o f Wy-18,481 treatment revealed progres sive disorganization of the semini ferous tubules, loss of sperm in the tubular lumen, and loss o f staining characteristics o f sperma­togonia and o f interstitial ce lls.

Results demonstrate the paradoxical anti-reproduc­tive effect in male rats of an extremely potent LHRH agonist which putatively releases LH and induces ovula­tion, and are o f particular importance in view o f the current c linical use o f such agents for treatment of the infertile or hypogonadal state .

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REDUCED MOTILE LONGEVITY IN THAWED HUMAN SPERMATOZOA . J . B . Black, B . A . Keel , Biology Department , Augus ta Col­lege , M. F . Keel , XYTEX Corporation , Augusta , Georgia .

Artificial ins emination with donor semen (AID) is a succ es sful therapeutic alternative for those coup les who are identified as having male factor infertility . Cryopreserved donor semen af fords numerous advantages in quality control and convenience over fresh semen . However , a number of physicians report it to be less succes s ful in achieving pregnancy than is fresh . On the o ther hand , s ome users of fro zen semen report suc­cess rates comparable to that ob tained with fresh . Of the pregnancies reported using cryopreserved semen the maj ority are achieved by multiple inseminations . To investigate the possibility that the freezing pro cess reduces the survival time of the thawed spermato zoa one hundred ej aculates from donors serving on an AID fro zen donor panel were evaluated as fo llows: the fresh ej aculate was scaled for mo tility (percent mov­ing ) and kinetics (forward progres sion , s cored 0-4 with 0 indicating no meaningful progression) . A 0 . 25 ml aliquot was then diluted with 0 . 5 ml Tyrode solu­tion and incubated in a 100% Tyrode atmosphere at 3 7°C . Motility and kinetic s were monitored initially and at subsequent 3 hour intervals . The remainder of the ej aculate was fro zen and thawed 24 hours later accord­ing to accep table techniques for human sperm . A 0 . 25 ml aliquot of the thawed semen was diluted , incubated and evaluated as before . Results are given as percent motile at each time interval based on initial motility of the fresh sample and initial post thaw motility of the frozen sample . This normalization all.ows between group comparison taking into account the expected at­trition during cryopreservation . The comparison show­ed a s tatis tically significant (0 . 05 at 1 hour ; 0 . 01 at all other times ) reduction in sperm motility in the thawed samp les . The kinetic s score was slightly lower in the thawed group but the decrease was not s tatis­tically significant . These results may help explain reduced pregnancy rates using cryopreserved semen , and point to the need for multiple insemina tions p er cycle to achieve better pr egnancy ratP.s.

RESPONSE TO LHRH STIMULATION IN VASECTOMISED MEN. G . Bleau, K . D . Roberts and A. Chapdelaine . Department of Obs tetrics and Gynecology . Univ . of Montreal and Maisonneuve-Ro s emont Hospital , Montreal , CANADA.

8

Vasectomy can influence spermatogenesis and modify the endocrine funct ion in s everal animal specie s . In men, however , normal levels of plasma FSH, LH and andro� gens have be�n reported following vasectomy . In this s tudy, the pi tuitary-testicular response to LHRH stimu­lation has been as sessed .

One group cons isted o f f ive men who had a bilateral vas ectomy performed 6 months to 6 years prior to tes­ting . One subj ect had recanalisation which had been successfully corrected ; another had significant sperm inunobilizing antibody titers not associated with any clinical symptom . Other clinical complicat ions were not found . These f ive men and a group of ten control non-vasec tomised men received a subcutaneous inj ection of lO()µg LHRH. Venous blood was collected at interval s over a period of 8 hours. Plasma FSH , LH and 178-ol­andro gens were analysed by radioinununoas say .

For the control group , mean basal levels were: FSH: 4 . 8 7 ± 1 . 7 6 m!U/ml ; LH: 10 . 6 ± 5 . 23 mIU/ml ; 17S-ol-androgens: 423.0 ± 107 . 2 ng% . For the vas ecto­mised men , the corresponding mean values were: FSH: 7.38 ± 3.55 mIU/ml ; LH: 1 0.9 ± 6 . 07 mIU/ml ; 178-ol­androgens: 4 3 9 ± 44 . 0 ng% . These d ifferences are no t statistically significant . Following the injec tion o f LHRH ,. the response curves showing the increase in FSH , LH and androgens were identical for both vasectomised and non-vasectomised men .

I t is concluded that in vasectomised men , the pituitary responds normally to synthe tic LHRH and the testicular response to endogenous gonadotropins is normal .

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CHARACTERIZATION OF AVAILABLE (R) AND OCCUPIE D (RA) ANDROGEN BINDING COMPONENTS OF THE HYPER­PLASTIC HUMAN PROSTATE. Robert W. Boesel and Sydney A. Shain. Southwest Foundation for Research and Education, San Antonio, TX.

Saturation protocols, employing the synthetic androgen methyl­trienolone ( R 1881) as probe, were developed for quantitation of available and total (R plus RA) androgen binding components of the hyperplastic human prostate. Available cytoplasmic binding site concentration, 2 hr incubation at 2°, and apparent affinity for R 1881 were 1 a.7 ± 1.4 fmoles per mg cytosol protein and 9.6 ± a.8 x 1 a8 M-1 • Specificity studies demonstrated potent androgens and progestins to be effective inhibitors of R 1881 binding, whereas estradiol, cortisol, and weak androgens were ineffective. The synthetic progestin R5a2a bound to available cytoplasmic binding sites of similar steroid specificity and concentration, 14.3 ± 3.2 fmoles per mg cytosol protein, with affinity of 1.6 ± a.4 x 1 a8 M-1. Total (R plus RA) cytoplasmic binding site concentration (2a - 24 hr at 15°) and apparent affinity for R 1881 were 49 ± 1 fmoles per mg protein and 2.7 ± a.6 x 1 a7 M-1• Specificity studies demonstrated potent androgens to be effective inhibitors of R 1881 binding, whereas progestins, estradiol, cortisol, and weak androgens were either poor or ineffective inhibitors. R5a2a binding sites were not detectable subsequent to incubation at 15°. Tissue mince saturation analysis (4 hr at 37°), employing 5a-DHT as probe, demonstrated a single class of KCI extractable nuclear binding sites with an apparent affinity for 5a-DHT of 1.a x 1 as M-1 and a concentration of 15 fmoles per 1 aa µg DNA. The nuclear binding component sedimented at 4-5S on linear sucrose gradients containing a.6 M KCI. Greater than 9a percent of the bound steroid was identified as 5a-DHT by TLC. 19-Nortestosterone and R1881 were effective inhibitors of nuclear 5a-DHT binding, whereas progesterone, estradiol, and cortisol were ineffective. Nuclear androgen binding sites were also demonstrated following incubation of KCI extracts of crude nuclear pellets with R 1881 for 2a - 24 hr at 15°. Nuclear site concentration and apparent R1881 affinity determined by this method were 29 ± 8 fmoles per 1 aa µg DNA and 3.1 ± a.4 x 1 as M-1• Binding site speci­ficity was indistinguishable from that determined by in vitro mince incubation with 5a-DHT. Available KCI extractable nuclear binding sites (2 hr incubation at 2°) for R 1881 were not detected. Compara­tive studies demonstrated that the properties of the cytoplasmic and nuclear androgen binding components of human hyperplastic prostate were remarkably similar to those of the androgen receptors of rat and canine prostate.

ACUTE AND CHRONIC ALTERATIONS IN TESTICULAR FEEDBACK: EFFECTS ON LR RESPONS IVENESS TO LHRH IN THE RAM . W. Bremner , J. Findlay, I . Cumming and D . de Kretser . Prince Henry ' s Hospital , Melbourne , Aus tralia .

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During pro longed , cons tant adnimistration of LHRH t o rams , plasma LH levels increase in two phases (s imilar to the pattern demons trated in man and cons is tent with the existence of two pools of pituitary LH) ; after 2- 3 hrs , LH levels progres s ively decl ine in spite o f con­tinued LHRH s timulat ion (Bremner , et al , Bio l . Reprod. 15: 141 , 19 7 6 ) . The possible role of the feedback effects of the teste s in determining this pattern of LH response was inves t igated in the present s tudy . Syn­thetic LHRH was infused i . v. at O . Spg/min. for 4-6 hrs to 23 rams . Nine rams were intact controls , 4 were chronic ()6 mo s ) castrates and 4 were acute (2 hrs ) castrates . S ix other intact animals had been pretreated with tes tos terone (T) either as short term i . v . infus­ions (83 µg/min. for 4 hrs ) or as i . m . inj ections of T enanthate 25 0mg twice weekly for 2 weeks . Blood was sampled at 15 min. intervals during the LHRH infus ions ; LH and T levels were measured by RIA .

Acute changes in tes ticular feedback, induced e ither by castrat ion 2 hrs before LHRH administrat ion or by 4 hrs of T infusion into normal animal s did not alter the pattern of LR increase and decrease during LHRH infus­ion . In contrast , LH release from the iunnediately re­leasable pool was markedly increased in chronically cas t�ated animals and decreased in animals treated with T for 2 weeks ; s imilar but smaller changes were noted in LR relea s e from the second pool in these animals . Conclusions: 1 . In the ram , alterations in tes ticular hormone p roduction require longer than 2 to 4 hrs to affect p ituitary respons ivenes s to LHRH . 2 . Testicular feedback in this species exerts a greater effect on the iunnedia tely releasable pool of p ituitary LR than on the pool requiring longer LHRH st imulation for release . 3 . The p ituitary refrac toriness that develops after 2-3 hrs of LHRH s timulat ion in normal rams is not caused by the increas ing plasma T levels , but is an intrins ic p ituitary phenomenon which could be due to an adverse effect of LHRH on its own receptors .

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"IN VITRO" CHARACTERIZATION OF RAT SPERMATO­GONIAL CHALONE. E. Bustos-Obreg6n and A. Thu­mann. Department of Cell Biology and Genetics University of Chile, Santiago, Chile.

Testicular samples £rom young adult rats, irradiated to the scrotum (300 r ) were incu­bated in a chemically defined medium contai­ning 20 uc of tritiated thymidine (TdR-H3 ) , and testicular extract (T.E. ) (105.000 g su­pernatant of testicular homogenate ).

The inhibited fraction (I.F. ) of testicu­lar DNA synthesis was 0.42, when compared to samples incubated with saline added instead of T.E. Liophylized T.E. causes a similar effect (I.F.=0.40 ) whereas T.E. heated at 100°c for 10 min. shows no effect. Tissue specificity of the inhibiting factor (sperma­togonial chalone ) was demonstrated using li­ver and spleen extracts, which showed no e­ffect on testicular TdR-H3 uptake.

The fraction obtained with ethanol (50-75% v/v ) showing highest chalone activity con­tains glycoproteins of an approximate molecu­lar weight of 50.000 (as determined by poly­acrilamide gel electrophoresis ) .

considering cell cycle duration for type A spermatogonia and the short incubation ti­mes tested (3-5 hs. ) , it can be postulated that the testicular chalone is of the G1 ty­pe. However, existence of a G2-chalone can not be excluded in this system.

( Supported by Grant 62.139.1.77 PLAMIRH ) .

CHROMOSOMAL ABNORMALITIES AS SOCIATED WITH THE ABSENCE OF DYNEIN ARMS IN THE SPERMATOZOA OF TWO STERILE MEN . A . Chapde laine , Claude-Lise Richer , K. D . Rober ts and G . Bleau. Faculty of Med icine , Univ . of Montreal , Quebec , CANADA .

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Cong enital absence of dynein arms causes s terility (immotile spermato zoa) and is responsible for the symp­toms resulting from incompetent ciliary func tion. Normal peripheral karyotypes have been reported in 5 pat ients afflic ted by this genetic d efect .

We repor t on our study of two sterile men presen­ting the typ ical Kartagener triad . Semen parameters were normal with the exception of v�able but immot ile spermatozoa . No immobilizing antibodies could be detec­ted in their sera and sperm maturation was normal as measured by the rate of sperm decondensation and by the number of sulfhydryl groups per cell . Electron­microscopy of ej aculated spermato zoa or of testicular biopsies conf irmed the abs ence of the dynein arms .

Chromosome analyses were made on peripheral blood lymphocytes . In the f irst patient , 47 chromo somes were found in about 7% of the 157 analysable metaphases .

·The extra chromo some proved to be a chromo some 12 , com­plete and free (R-banding and BrdU incorporation) . Cultured skin f ibroblasts did not show the trisomy .

In the s econd patien t , a normal karyotype was founti in per ipheral blood lymphocytes . However , a study of the chromosomes during meiosis showed about 8% of the 62 analysable metaphases I to contain an additional monovalent . One cell contained a trivalent (probably chromosome 12). In all cases the extra chromosome belonged to group C (possibly chromo some 1 2) .

Thus , in two cases o f sterility caused by the pro­duct ion of immotile spermatozoa , a heterogeneous trisomv 12 mo saicism was demonstrated in one of the patients and is highly suspected in the other . I t remains to be eluc idated if , and how, this chromosomal abnormal ity is related to the synthesis or attachment of dynein proteih in cilia and flagella .

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�ELECTIVE INHIBITION OF FSH SYNTHESIS BY THE SERTOLI CELL FACTOR ( SCF) IN VITRO . M. Chowdhury , A . S teinberger and E . S teinberger . Department of Reproduc tive Medicine and Biology U . T . Med ical School , Hous ton , Texas .

Recent evidenc es sugges t that the tubular compartment of tes tis produces a substanc e called inhibin which regulates the secret ion of FSH . A subs tance ( SCF) with inh ibin like properties has been shown to be s ecreted by ser toli c ells in culture which s electively suppress­es FSH s ecretion in vitro (Steinberger , A . and Stein­berger , E . : Endocrinol , �. 9 18 , 1 9 7 6 ) . I t was not clear:, however , whether the effect of the S CF was on the mechanism of FSH release or it ' s synthes is . The pre­sent report provides data concerning effect of SCF on the de novo synthesis of gonadotrophins .

S ertoli cells isolated from 29-3 3 day old rats were cultured in Eagle ' s chemically defined med ium (MEM) . The spent culture med ia ( SCF) were collec ted after 2-5 days of incubation and tes ted for their effect on go­nadotrophin synthesis in vitro . For this purpo s e an­ter ior p ituitar ies fro;-adult (90 day) male rats were organ-cultured for 24-48 hr with either fresh MEM (con­trol) or SCF . 3H-leuc ine was added to the cultures dur­ing the las t 6 hr of incubation . Sub sequently total go­nadotrophins were immunoprecipitated with rat LR or FSH ant ibody . Data were presented as dpm/µg FSH or LR .

In f ive separate exper iments , incubat ion of pitui­taries with the SCF resulted in a s ignificant suppres­sion of 3H-leuc ine incorporation into FSH compared to control cultures , although the degree of inhib ition var ied from experiment to exper iment probably depending on the concentration of S CF . In mos t ins tances the SCF increased 3H-leuc ine incorporat ion into LR ; except in one experiment where a 5-day cul ture media inhib ited the incorporation into FSH as well as LR , possibly due to high concentrat ion of SCF .

Pres ent preliminary data ind icate that S CF can s elec­tively reduce the incorporation of 3H-leuc ine precur­sors into FSH without decreasing the incorporation into LR , suggesting that the main effect of the SCF on FSH secret ion may be to suppress it ' s de nova synthesis .

Supported by Grant # 5 P50 HD08338 NICHHD .

GLUCOCORTICOIDS INVOLVEMENT IN THE TESTES FUNCTION OF THE RAT E . L . COOK AND K . F . A . SOLIMAN School of Pharmacy , Florida A & M University , Tallahassee , FL .

1 4

Male Spragu e Dawley rats maintained on 14L : l0D light-dard cycle were employed in this experiment . Drugs used were cortisone , progesterone , dexamethasone , corticosterone and metropirone . The rats were killed after 10 days of treatment and the weight of the testes , adrenals and seminal vesicles were determined . Significant reduction (P . OS ) was observed in the testes and seminal vesicles weights after treatment with cortisone , corticosterone or dexamethasone . In the dexamethasone treated group , corticosterone and/or progesterone administration alleviated the dexametha ­sone inhibiting effect . Progesterone inj ection alone was sufficient in maintaining normal testicular function in cortisone or corticosterone-treated animals. Metropirone administration resulted in no significant change in the testicular function . Results of this experiment indicate that adrenal progesterone may be required for n ormal testicular function . < supported by NIH-MBS Grant No . RR-811 1 )

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LUI1I1IAL PERFUSIOIT OF THE RAT ' S EPIDIDYHIS . T. G. Co oper and G. r :. H. Uai tes . Depart!nent of Physiology & Bio­chemiGtr,y , University of Reading, Berkshire , England.

A t echnique for perfusing the lumen o f the epididy­mis has been developed to study the movanent of mo l e cules to it from blood. Pento barbitone-anaestheti.sed rat s were tracheotomised and their epididymides expo sed through scrotal incisions. A PVC cannula (0. 5mm OD ) on a gauge ';fl needle was tied into the lumen of the proximal vas deferens. A loop of the tubule in the distal cauda epididymidis was withdrawn through a cap sular incision and severed. The epididymal contents were flushed out through the cut with 1 68mM sodium chloride. When emptied , a drawn-out PVC can..11ula was t ied into the epididymal tubule and the perfusion was started in the reverse direction. Perfusion rat es varied from about 4 to 25µ.l/min.

No protein (<O. 5µ.g/µ.1) bound sialic acid (<5pno1/µ.l) or chol estero l (<0. 1 µ.g/µl � was detectable in pooled effluates collected above 1 Wl/min and tritiated inulin inj ected into the bloodstream was excluded from the perfusate , indicating that no leakage from blood o ccurred.

Glucose was detected by the gluco se oxidase method. Reducing the perfusi on rate from 24 to 1 1 µ.1/min increased glucose concent ratio n from 0 . 1 2mM to o. 31 mM ( i . e . 2-4% of blood levels) . Radioactivity ent ered the perf'usate to reach 4% of blood level s durLllg systemic infusion of carbon-labelled gluco se. Below this perfus ion rate glucose co ncentration was reduced, and stopped-flow experiment s confirmed that it s accumulat­ion never reached levels expe cted if a consta..�t secretion were as sumed. These result s suggest that metabo lism of luminal gluco se o c curs. Luminal perfusion of gl ucose indicated that the sugar could move in or out of the lumen, depending on the perfu sion rate and concent ratio n rela tive to blood.

Luminal glucose , entering from the blood , may be an important substrat e for spermatozo a in .illJ!.

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THE P RESENCE OF EXFOLI ATED GENITOURI NARY TRACT CE LLS I N HUMAN SEMEN . Margaret L . Couture and Matthew Freund . Dep artment of Phys iology , Southern I l l inoi s Un iversity , Carbonda l e , I l l ino i s .

To date , cytologi cal examination of human semen in ­vo lves morpho l o gi cal categori z at i on and quantitation of immature germ ce l l s and white b l ood ce l l s . These dat a are then corre lated with the presence of in fert i l i ty and/ or infe ct i on p rob l ems . Th i s routine method of s emen analys i s does not consider the potential diagno s ­t i c s i gnificance of exfo l i ated genitourinary tract ce l l s in semen . The pres ent s tudy was tmdertaken to determine the pre senc e and types of exfo l i ated genito­urinary tract ce l l s in the semen o f .vas ectomi zed and non-vasectom i zed men . Specimens were examined for the pres ence of sperm and sperm counts were made on the s emen spe cimens from non -vas ectomi zed men . Each semen s amp l e was fixed in a spe cial pres ervat ive s o lut i on (Muco l exx) w i th in three hours of co l le ct i on . The semen

was then fi ltered (Nuc l epore , 8 mi cron) to remove mos t o f the spermatozoa and white b l ood ce l l s ; retaining mos t of the exfo l i ated geni tourinary t ract ce l l s on the fi l ter . The fi lter with attached ce l l s was then s t ained by the Papan i co l aou s taining method . These ce l l s to dat e inc lude : b l adder transition a l and decoy ce l l s , urethral squamous epithe l i al ce l l s and prostat e c e l l s . The exfo l i ated prostate and b l adder ce l l s are we l l pres erved and may be used for cyt o logical rating and ana lys i s . Thes e re sults sugge s t that rout ine cyt o­l o gi ca l examinat i on o f s emen may be modified to include examinat i on of exfo l i ated genitourinary t ract cel l s . Such ce l l s in the pas t h ave not b een recogni zed as ex­fo l i ated cel l s and have mos t l ik e ly been rated as im­mature germ ce l l s and/or white b l ood c e l l s . It is pro­posed that this technique may be us ed to improve diag­nos t i c accuracy in inferti l i ty and infect ion prob l ems . Mos t important is the p otenti a l us e of thi s technique as a type o f "PAP" smear for men to detect at an earl y s tage the pre s ence of genitourinary tract mali gnancy and b en i gn prostatic hyperp l as i a . C l inical trial s have been s t arted to test the practical app l i c ation of thi s new approach .

1 7

TEST I CULAR GONADOTROP IN RECEPTOR LOSS AND I NH I B IT I ON OF SPERMATOGENES IS FOLLOW I NG LONG-TERM TREATMENT WITH AN LHRH AGON I ST I N THE RAT . L . Cusan , C . Aucl a i r , P . A . Kel ly, M . Garon and F . Labri e . MRC Group i n Mol e­cul ar Endocri nol ogy , Le Centre Hos pi tal i er de 1 1 Uni ver­s i te Laval , Quebec Gl V 4G2 .

We have recently found that acute treatment of adu l t mal e rats wi th potent LHRH agon i s ts l ed to a mar­ked decrease of testi cu lar LH and pro l acti n receptor l evel s accompani ed by reduced pl a sma tes tosterone con ­centrat ion and te sti s , semi nal ves i c l e and ventral prosta te wei ghts (Auc l a i r et a l . , B i ochem . B i ophys . Res� Commun . 76 : 855 ) . I t then became of i nterest to study the effect of l ong-term treatment wi th an LHRH agon i s t o n te sti c u l ar gonadotropi n receptor l evel s and seconda­ry sex organ wei g hts . Adu l t mal e Sprague-Dawl ey rats were i njected s . c . wi th 1 00 ng of [ D-Al a6 , des -Gly-NH2 l OJ LHRH ethyl ami de twi ce a week for peri ods extend­i ng from 1 to 1 2 weeks . Such treatment l ed to a 70-80% reducti on of testi cu l ar LH/ hCG receptors duri ng the fi rs t week , wi th a maxi ma l i nh i b i tory effect bei ng observed at four weeks . Test icu l ar prol acti n receptor l evel s were a l so s i gn i fi can tly reduced after four wee ks of treatment wh i l e no effect was detected on FSH receptors . Treatment wi th the LHRH agon i s t a l so caused a progress i ve decrease in testi s wei ght down to 50% of control at the end of the 1 2-week peri od , para l l e l ed by a 30 to 40% l oss i n semi nal ves i c l e and ventra l prosta ­te we i g ht . P l asma LH and FSH l evel s were found to be i ncreas ed by treatmen t wi th the LHRH agon i st whi l e p l a sma tes tos terone concentrati on was reduced . Marked h i s to l og i ca l degenerati ve changes observed . i n the semi ­n i fe rous tubu l es accompan i ed testi s wei ght l oss . The present data c l early show that the testi s i s h i ghly sen s i ti ve to i ncreased ci rcu l ati ng gonadotropi n l eve l s and s uggest that therapeuti ca l approa ches u s i ng LHRH and i ts agoni sts i n the treatment of ma l e i nferti l i ty s hou l d take i nto account these somewhat unexpected i n­h ib i tory effects on tes ti cu lar functi on .

1 8

SPERM FREEZING IN 0-5 AND 0-25 ml . STRAWS = A comparative S tudy . F . CZYGLIK CECOS Bicetre , Paris and JC . EMPERAIRE , CECOS Aqui taine , Bordeaux , France .

The mos t widely used method for cryopre ­serva t ion of both human and bovine semen invo lves a fas t cool ing technique by expos ing the sperm mixed wi th a protective medium in 0-5 (human) and /or 0-25 ml (bovine) plas t i c straws to l iquid n i trogen vapors . The use of the smaller s traw has given bet ter resul ts wi th bovine semen cryo-preservat ion . A comparat ive s tudy performed by us on 32 ej aculates could not conf irm thi s ob servation in human sperm. Each ej acul ate mixed with Ackerman pro tective medium was d ivided into both types of s traws , which were fro zen in the same operation ; pos t-thaw mo t i l i ty was evaluated on a double b l ind pro­cedure . The re nei ther seems to be a s i gnif icant di ffe­rence berween the two types of s traws when comparing the spermato zoal recovery rate , nor the pregnancy rate . There fore , wi th an ident ical effic iency , i t i s po ssible to obtain twi ce as many insemina tion do ses from the s ame ej acul ate .

1 9

SPECIFIC BINDING OF ESTRADIOL TO EPIDIDYMAL CYTOSOL FROM MATURE RABBITS . Benjamin J . Danzo and Mari e­Clai re Orgebin-Cri s t . Departmen t o f Obstetrics and Gynecol ogy , Vanderbi l t Uni versi ty , Nashvi l l e , Tenn .

Cytosol prepared from the epi di dymi des of castrated adul t rabbi ts contains a speci fic es trogen binding com­ponent (s) . This component (s) sedimen ts as a 4- 5 S peak on l ow i oni c s trength sucrose gra di ents . In contras t , the estrogen receptor from the immature rabbi t epi didy­mi s sediments as an 8 S speci es under i dentical condi ­ti ons . Binding of [3HJ estradiol to the 4- 5 S compo­nent i s i nhibi ted by unl abeled estrogens , but not by unl abeled 5a-dihydrotes tos terone (5a-DHT) . The speci f­i c binding component i s not detectabl e on sucrose gra­di ents unless the cytosol- [3H] es tradiol mixture i s fi rst extracted with charcoal to remove uncomplexed and losely "bo und l i gand from the sampl e . The speci fi c es­trogen binding moi ety can also be demons trated when charcoal extracted sampl es are chroma tographed on Sephadex G- 200 . Using this sys tem, three peaks of radi oacti vi ty can be detected in the el ua tes . The fi rs t peak is excl uded from the gel and thus has an ap­parent mol ecular wei ght of 200 , 000 Dal tons or grea ter , the second peak has an apparent molecular wei ght of 80-100 , 0 00 Da l tons , the third peak represents unbo und radi oacti vi ty . The second peak represents 70- 80% of the macromol ecular bound hormone in charcoal extracted samples . Binding of [3HJ estradiol to both macromol ec­ular peaks is dimini shed by the inclusion of unlabel ed estradiol , but not by the incl usion of unlabeled 5a-DHT in the i ncuba tion mixt ure . The macromol ecular binding that remains after charcoal extraction dissoci a tes slowl y . Thi s was shown by the fa ct tha t the a ddi ti on of a 1 00 0- fol d excess of unl abel ed es tra diol to the in­cubation mixt ure after binding equilibri um had occurred di d not resul t in a decrease in ra dioa cti vi ty associ a ted wi th the peaks . Estradi ol binding components in rabbi t plasma are non-specifi c and do not exhibi t the charac­teri sti cs tha t we have described here for the binder in c::pi di dymal cytosol .

Supported by NIH Grants HD- 0 8295 & HD- 0 3820 and a gran t from the Vanderbi l t Research Council .

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RESULT S OF VAR I C O C ELE TRAN SCATHETER S CLERO S ­I NG TREATMENT . M . P . d e Castro and S . S . Lima . G ynec o l o g ic C l inic Univer s ity o f Sao Pau lo Med ical S c hoo l and Ho sp ital Albert Einst ein , S�o Pau l o - BRAS I L .

S elec t e d infert ile men with c l inically a nd / or v e Do graphic a l l y d et ec t ed var icoc e l e wer e subm it ed to s c l ero s i s of t he s p erma t ic v e in s by mean s o f transcatheter inj ec t io n o f 5 0 % hyp ertonic gluco s e so lut ion fo llowed by the inj ec t io n o f mono ethano lamine o l eat e , immed iately after t he s el ec t iv e s p ermatic v e nogr aphy , in the same interv entio n .

No compl ica t ions wer e documented dur ing and after t h i s tr eatment . The pa t ient s wer e kept in b ed r e s t for 4 to 6 hour s a nd t hen d i scharg ed .

S ix mo nths t o one year fo l low-up of 2 0 pat i en t s s ho wed :

a ) no var icoc el e ma intenanc e or recurr enc e in a l l of them ;

b ) s ig nif icant increa s e in t he mean s p erm count , mo til ity ind ex and p erc entage of oval c ell s in 14 subj ect s ; s ev e n pr egna nc i e s in t h i s group ;

c ) inc o n s i s t ent or no improvement in s perm qual it y in 6 pat ient s .

In v i ew o f t he r e sult s obtained and due to the inno c u it y of the method we b e l i ev e t he spermat ic v e in s c l ero s is i s a quit e r ecommendable pro c edur e in mal e inf er t il ity r e lated w it h var ico c el e .

S l id e s and 3 5mm mot ion picture will b e pr e s ented .

2 1

A S IMPL I F I ED TECHN IQUE FOR EVALUAT I NG SPERM MORPHOLOGY . K . A . Dougherty, L . B . V . Emi l son , R . L . Urry, and A . T . K . Cockett . D i vi s i on of Urol ogy , The Uni vers i ty of Rochester , School of Medi c i ne and Denti s try , Rochester , New York .

Determi nat i on of s perm morphol ogy and v iab i l i ty are usefu l techni q ues for assessment of s emen qual i ty . I n thi s report , eos i n-ni gros i n i s compared to hemotoxyl i n­eos i n for effecti venes s i n characteri zi ng s permatozoa .

The res u l ts i ndi cate eos i n-ni gros i n and hemotoxyl i n­eos i n g i ve comparabl e res u l ts i n di fferenti ati ng s permatozoa . I n add i t i on , eos i n-ni gros i n permi ts determi nati on of the percent of vi abl e cel l s in each morpho l og i ca l category . The eos i n-ni gros i n s tai n i ng techn i que i s a s i mpl e , rap i d method wh ich may gi ve add i ti ona l i nformation for the assessment of ma l e fert i l i ty potenti a l .

MALATE DEHYDROGENASE I N HUMAN SEMI NAL PLASMA AND SPERMATOZOA . R . E l i asson , L . E l i asson , and N . V i rj i . Department of phys i ol ogy , Ka rol inska i nsti tu tet ,

S- 1 040 1 Stockho l m , Sweden .

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Human semi nal pl asma from more than 1 00 men ( i nfer­ti l i ty , prostati ti s , heal thy vol unteers ) wa s anal yzed for ma l ate dehydrogena se ( MDH , 1 . 1 . 1 . 37 ) . The mean acti v i ty was appr . l , 300 mI U/ml ( range 400-21 00 ) . From spl i t ej acu l ates i t cou l d be concl uded that the ma i n part of the MDH acti vi ty ori g i nated from the prostate . There was a cl ose correl ati on between the MDH acti vi ty and the z i nc and magnes i um concentra­t i ons . The occurrence of i soenzymes was stud i ed by agar gel el ectrophoresi s at pH 8 . 6 . The MDH acti v i ty was l oca l i zed wi th the N-BT method . The semi nal p l asma s howed one band mi grati ng towa rds the cathode . Some semen s pec i mens a l so had an i soenzyme wh i ch mi grated towards the anode . Th i s i soenzyme ori g i nated from the s permatozoa , probab l y the mi tochondri a , s i nce the rel ati ve proporti on of the i soenzyme was i ncreas­ed when the spermatozoa had been ex posed to phys i cal stress l i ke centri fugati on , di l uti on wi th d i sti l l ed water , a nd u l trasoni fi cati on . The cathoda l i soenzyme was i nh i bi ted l ess than that wi th anodal propert i es by 5 to 25 umol es of oxal oaceti c . Onl y the anodi c i soenzyme was i nh i b i ted by the addi ti on of p-hydroxy­mercu ri benzoate . The former i soenzyme wa s thus s i mi -1 ar to the cytopl asmi c and the other to the mi tochon­dri a l MDH i soenzymes . Semen sampl es from d i fferent men had vari ous proporti on of the anodal i soenzyme i n the semi nal pl asma and i t i s therefore suggested that a quanti tati ve measu rement of th i s i soenzyme cou l d be usefu l i n the study of the i ntegri ty of the s permatozoa l mi tochondri a . Studi es a l ong th i s l i ne a re i n progress .

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1 ARGININE ASPARTATE AND MALE HYPOFERTILITY - A DOUBLE BLIND STUDY - J . C . EMPERAIRE and A. JM . AUDEBERT Ins t i tut Aqui tain de Recherches et d ' E tude de la Re­production Humaine , 40 , Cours de Verdun 3 3000 BORDEAUX France .

The e ffic iency of Arginine aspartate as a spermato� ne tic factor is still controvers ial . In vivo s tudies are contradic tory , and , as a whole , do no t confirm in vi tro observa ti ons nor animal experiment s . A s tudy was undertaken to assess the validity of the treatment in i diopathic male hypofert i l i ty . Various degrees of oligo- , as theno , necro and/or terratospermia were considered as idi opathic in fifty males = there were no anatomi cal abno rmal i t ies such as test i cular atrophy , infectious desease or varicocele ; pl asma gonado tro­p ins , testosterone and pro lact ine were wi thin normal limi ts ; when performed , tes t icular biopsy showed va­rious degrees of spermatogene tic arrest . The subj ects were divi ded at random into two equal group s : The f irst group was put on Arginine Aspar tate per-os , 5 mg three time s a day for 100 days ; the other group received pl acebo . The me an of at least 3 spermiograms at monthly interval before treatment was compared wi th ano ther spermiogram within 20 days after complet ion of therapy . S t atis tical analysis of the res ults showed no signifi cant difference between the Arginine and the pl acebo groups . There fo re , Arginine Asp artate does not appear as an efficient therapy of spermatogenic disorders .

I N FLUENCE O F AGE ON S PERMATOGON IAL' STEM CEL L NUMB ER AND FUNCTI ON I N THE RAT . B . H . Eri ckson and Debra Connatser . Comparati ve An i mal Res earch La boratory , Oa k Ri dge , TN .

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It has been s hown i n a number of mammal i an species that s perm producti on di mi ni s hes duri n g the l ast ha l f of l i fe . The rol e of the s tem cel l i n thi s reducti on has not been exami ned . Stem cel l s and di fferenti ati n g s permato goni a were enumerated i n who l e-mounts of the s emi ni fero us tubul es of Spra gue- Dawl ey rats when ei ther 6 or 24 months ol d . Stem cel l s were i denti fi e d acco rdi n g to the cri teri a o f Hucki ns , and 1 2 an ima l s were emp l oyed at each s tage . .

Number o f s tem s permatogon i a per 1 00 Sertol i cel l s i n tubul es o f yo ung rats exceeded that of ol d rats ( 92% of yo un g ) at both Sta ge I and Sta ges V I I and VI I I o f the s permato geni c cycl e , but at a s tati sti cal l y nons i gni fi cant l evel ( P > 0 . 20 ) . The n umber o f s per­matogoni al pai rs ( the fi rst res ul t' o f a stem cel l mi ­tos i s ) i n aged testes equal ed that o f youn g testes at Stage I but exceeded youn g testes by a factor of 1 . 32 at Sta ges V I I and V I I I ( P < 0 . 1 0 ) . A1 spermatogoni a , whi ch res ul t from the mi to ses of pai rs , were more nu­mero us i n the tes tes of the young a s were A2 and A4 spermato goni a ( P < 0 . 01 ) . Yo ung exceeded o l d in num­ber o f di fferenti ati ng s permato goni a by factors o f 1 . 3 (A1 ) , 1 . 5 (A2 ) , and 1 . 3 (A4 ) . Hence the s permato­goni al contri bution to decrements i n the s perm produc­ti on .o f the aged rat is apparentl y due to the di ffer­enti a ti n g types and not to the stem cel l .

Res earch s ponsored by US Department of Energy Con­tract No . EY- 76- C- 05-0242 wi th the Uni vers i ty of Ten ­nes see .

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FAC'IDRS UNDERLYING INFERTILITY IN THE AICOHOLIC . W.E . Fam5Worth, A . H . Cavanaugh, J .R . Brown, .!. ­Alvarez and · L �M� · rewanaow5ki . v .A . Hosp . and School of Medicine, State Univ . of New York at Buffalo , 3495 Bailey Ave . , Buffalo, New York 14215 .

In an effort to identify the factor (s) contribut­ing to loss in sexual a::mpetence in alcoholics , a pilot study was made of the interrelationships of hepatic disa:tse , plasma hormone levels and impotence in 35 male patients in an alcohol unit . Levels of total blood testosterone and estradiol were determinerl by radioi.rmrunoassay, apparent free testosterone and sex honnone-biming globulin capacity by the methcrl of Farnsworth and Brown . Liver disease was estimated fran serum enzyme levels , biopsy and clinical find­ings . Contrary to previous reports , impotence was not a direct concanitant of hepatic disease, elevated sex hormone-binding globulin capacity or hyperestrin­ism. The irost significant aberration found was a nearly 30% lower mean free testosterone concentration which appeared to be secondary to a mean total testos­terone concentraticn 20% below that of the subjects with nonnal sexual function . We conclude fran this that impotence results fran testicular secretion impaired by the action of alcohol or its metabolite , acetaldehyde . (Supported by VA funds) .

26

FAILURE OF EXOGENOUS ANDROGEN TO PREVENT REGRE S SION OF THE INITIAL SEGMENTS OF THE EPIDIDYMI S AFTER EFFERENT DUCT LIGATION OR CASTRATION. D . W . Fawcett and A . P . Hoffer , Department o f Anatomy , Harvard Medical School , Bos ton , Ma .

The inves tigation was undertaken to explore further the dependence of the initial segments of the epididy­mis upon sub s tances in the tes ticular fluid . The exper­imental groups consist ed of rats cas trated ; rat s with ligated ductuli efferentes ; and sham operated controls . The epididymides were examined by light and electron microscopy 3 and 6 weeks pos toperatively . The initial segments of the duct in both ligated and cas trated ani­mals exhib ited ( 1 ) s triking decrease in duc t diame ter and epithelial height , ( 2 ) a marked decrease in endo­plasmic reticulum in the apical cytoplasm, ( 3 ) evidence of diminished activity of the Golgi complex , but (4) persis tence o f fluid up take by pinocytosis .

Daily adminis tration o f 500 ug . testos terone propr i­onate beginning on the firs t pos toperative day main­tained the accessory gland weights at normal or supra­normal values , but was ineffective in preventing the cytological regress ion of the initial segments of the caput epididymidis .

In an effort to achieve levels o f androgen in the blood comparable to those normally found in the rete testis fluid , castrated and ligated rats were implanted subcutaneously with 30 cm . of silas tic tubing contain­ing testosterone , estimated to release 4 mg/day (Bernt­son et al . , 1974) . P lasma tes tos terone levels when the rats were killed were 8 to 10 times normal . Despite these high levels of circulating androgen , cytological dedifferentiation of the initial segments was no t pre­vented . Mor e distal segments of the duct were essenti­ally normal in dimensions and appearance .

I t is concluded that the initial segments o f the rat epididymis require high intraluminal concentrations o f androgen bound t o androgen binding pro tein . The pos s i­bility that they are dependent upon some o ther , as yet unidentified , cons tituent of tes ticular fluid is no t ruled out .

2 7

E LE C TR O N M I C R O S C O P E C Y TO C H E M I C A L S T U D Y O F R A B B I T S P E R M A TO G E N E S I S U S I N G A PAS - E Q U I VA L ... E N T S TA I N I N G T E C H N I Q U E . J . E . F l � ch o n. D e p ar t m e n t of P h y s i c logy , I N R A , J ou y - e n - J osas , F r a n ce .

A c l ass i fi c a t i o n of s pe r m a toge n i c stages m a i n l y b ased on m orp h o l o g y of t h e form i n g acrosome was estab l i s h e d for the f i rst t i m e at l i g h t m i cros cope l e ve l u s i n g per iod i c a c i d ­S c h i ff 1 s r e a g e n t (PAS ) te c h n i q u e i n d i ffe re n t m o m m a I i a n spe c i es ( Le b l o n d a n d C l e r m o n t , 1 9 52 . Amer .J . An at . 9 0 : 1 6 7-2 1 6 ) , a n d at e l e ctro n m i cros cope I e v e I i n r abbi t (P l oe n , 1 9 7 1 . Z . Ze l l fo rs c h . M i kros k . An a t . , 1 1 5 :553-564) . U s i n g phosph otu ngs t i c a n d (P TA)s t a i n i n g at l ow "P H on t h i n se c t i o ns i n g l yco l m e thacry l ate we com b i ned cyto c h e m i c a l a nd u l tra-' s tr u c t u r e I appro a c h e s to spermato g e n es i s in tabbi t . As PT A stai n i n g used i n d e f i n e d c o nd i t i o ns (F l e c ho n , 1 9 74 . J . M i cr9 s co p i e 1 9 :2 07-2 1 2 )showed t h e s a m e aff i n i ,., as PAS , w e cou I d fo l l ow t h e s u c cess i ve stages i n f i ne l o c a l i z a t i o n of.:. L

acrosom a l g l y copro te i ns. C o n te n ts of G o l g i apparatus a nd a crosom a I ves i c l e w e re sta i ned i n r o u nd spermat ids . T h e a croso m a I gra n u l e r a p i d l y appe ared l ess s ta i ned t h an c o nd e n • s i ng m ater i a l of t h e acroso m a l ca p . l n e l o ngat i ng s pe r m a t i d s stai n i n g w as f a r l ess i n tense i n t h e i n ne r .part m argi n a l t h i c k ­e n i n g d e r i ved from t h e a crosom a l gr a nu l e t h a n i n a n te r i or and e q u ator i a l s e gm e n ts . More o ver i n m aturat i o n s tages a th i n i n ne r l ay e r of a nter i o r seg m e n t appe ared u ns ta i ne d , w h e re a s t h e e q u ator i a l s e g m e n t l oo ked re l ati ve l y t h i c k . G l y coprote i ns d i s appe ar from the e q u ator i a l se g m e n t d u ri ng e p i d i d y m a I transport (F l e c h o n , 1 9 75 i n T h e B i o l ogy of S p e r m ­ato z o a , T h i ba u I t a n d H afez eds , K arge r , 36 - 45 ) . O u r obser­vati o n s a nd sperm a toge n i c s tages as know n from PAS s tai n i n !!J and u l t r astru ctu r a l s tu d i es m a y be e as i l y corre l ated . G l y co ­pro te i n l o c a l i z at i o n i n the d e ve l o •l:i i n g a c rosome a s se e n i n r ab b i t (a nd a l so i n r a m , m a n ) agreed w i t h t h e few resu I ts p u b -1 i s h e d o n o t h e r s pe c i es (S vs i e t a I . , 1 9 7 1 • Ame r . J . Anat . l 3 0 : 2 5 1 - 2 6 8 ; S andoz , 1 9 77, T h e s i s , U n i ve rs i ty of Par i s). S e gre g ­ati o n of a croso m a l prote i ns and g l y coprote i ns m ay b e r e l ated to d i str i b u ti o n of s u ch e n z y m e s as h y a l u ro n i d ase , a cros i n .

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BI ND I NG OF ESTRAMUST I N E , A N I TROGEN MUSTARD DERI VATIVE OF ESTRADIOL- l 7 s , I N CYTOSOL FROM RAI VENTRAL PROSTATE . Bj�rn Fors gren 1 , �ke Pousette 2 , Jan-�ke Gus tafsson2 , and Bertil Angber�1 • 1A8 LEO Research Laborato ri es , He l s i ngborg , and Department of Chemi s try , Karo l i nska I n sti tutet , S tockhol m , Swe den .

Es tramus t i n i s a n i trogen deri vati ve of es tra d i o l -l 7 S tha t i s use d i n the thera py o f pros tati c carci noma ( trade n ame Es tracytR ) . 3H-es tramusti n was found to b i nd to a macromo l ec u l e i n ra t ventra l prostate cytos o l l . The formed compl ex had a sedi menta ti on coeffi ci ent o f 3 . 5-4 S a n d a n i soel ectri c po i nt of pH 5 . Gel fi l tra­t ion data i ndi cated that the macromol ecul e was di ffere�t from those b i n di ng 5a- di hydro-testos te rone , and had a mol ecul a r we i ght of approxima tely 50 ,000 dal tons . The estramus ti n bi n di ng p rote i n was puri fi ed to homogeni ty . The puri fi cati on protocol i nc l uded ch romatography on DEAE- ce l l u l ose , Ul trogel ACA-44 , Octyl -Sepharose , hy­droxyl a pati te and DEAE-Sepharose perfo rmed at pH 8 . 5 . The protei n p repara ti on was homogeneous as j udged from polyacryl ami d ge l el ectrophores i s .

29

IMPORTANCE OF SEROTONIN IN THE REGULATION OF GONADOTRO­PIN IN MAN . F . Fraioli , V . Bonifacio , S . Pallo t t i , D . Paolucci , and A . Isidori . Clinica Medica IV , Univ . of Rome , Italy .

Gonado tropin (LH and FSH) levels were measured by radioimmunoassay in 6 volunteers after the administra­t ion of 100 µg of LH-RH i . v . , in basal conditions and after pretreatment of 0 . 5 m of methysergide and 1 6 mg of cyprohep tad ine . Gonado tropin levels were also meas­ured in unmedicated schizophrenic patients after the administration of the same dose of LH-RH . Cond it ions have been s tandardized for time administration (9 ·am) , age of the subj ec t s and reduction of the assay error . Schizophrenic patients were s trictly

. s elected on the

basis of a supposed low serotonin act ivity in the brain ; for this purpose we have detected in each of them plate� let MAO and skin hystaminenaeaction . Normal subj ec ts showed 8 . 2 ± 3 . 5 mIU/ml (2 IRP-HMG) for LH and 6 . 5 ± 2 . 8 mIU/ml for FSH in basal conditions . After 20 min the LH was 8 0 . 7 ± 21 . 5 mIU/ml and FSH 38 . 5 ± 12 . 6 . The pretreatment with 0 . 5 mg of methysergide gave 65 . 0 ± 18 . 5 mIU /ml at 2 0 min for LH , and 4 . 5 ± 2 . 5 for FSH in response to the same dose of LH-RH . Cyproheptadine yielded s imilar results , but in 3 out of the 7 subj ec ts an elevation in FSH s tatis tically significant from the baseline , was observed between 45 and 60 min from the LH-RH inj ection . In the schizophrenic patients the LH was 5 . 8 ± 3 . 2 mIU/ml in basal conditions and 75 . 0 ± 15 . 0 mIU/ml after 20 min from the LH-RH inj ec tion , while the FSH was respect ively 4 . 0 ± 2 . 5 and 5 . 2 ± 3 . 0 after eo min . It is to notice that only the patients s elect­ed according to the above crytheria showed . this absence in the FSH response , while randomized pat ients showed a variable response . From the above results the follow­ing remarks can be made : 1 ) the administration of anti­sero toninergic drugs result s in a diminished FSH respon­se to an effec tive dose of LH-RH and 2 ) in unmed icated schizophrenic patients selec ted on the basis of reduced sero tonin activity in the brain , s imilar results can be ob served . These results confirm the importance of brain sero tonin in gonado tropin release in man .

30

EFFECTS OF INTRAVENOUS ADMINISTRATION OF BRAIN PHOSPHO­LIPIDS ON GONADOTROPIN RELEASE IN MAN . F . Fraioli , D . Paolucci , M . B . Mesina , S . Morrone , and A . Isidor i . Clinica Medica IV , University of Rome , Italy .

Brain pho spholipids are ef fective in mod ifying the brain catecholamine pattern . The aim of our experi­ments was to ascer tain if any variat ion in gonado tropin release exis t s . Eight normal volunteers aged 20-30 yr were inj ectea i . v . with a phospholipid preparation in a dose o f 600 mg and blood collec tions were made every 1 0 min f o r 240 min . LH , FSH , and testosterone were evalu­at ed by radioimmunoassay and the result s calculated by a computer program . Intra-assay and interas say vari­at ions d id not exceed 8% and 12 % respectively , expres­sed as coefficient of variat ion . The s ame sub j ects were also inj ected with placebo with the same experi­mental d etails . No apparent significant changes were observed in LH and FSH secretion after the phospholip id inj ect ion while a signif icant fall in testosterone was observed in 6 out of the 8 subj ects s tudied (175 ± 32 ng%ml after 90 min , being the basal value 580 ± 112 ng%ml ) . No changes were observed in the placebo treat­ed subj ects . The gonado tropin pattern as s tressed above appear s to be normal from a quantitative point o f view , but by applying variance analysis t o the data obtained a marked reduced pulsatility in gonadotropin output has b een observed .

We conclude that phospholipids given intravenously at p�armacological doses act on the phasic release mechanism of the gonadotropin , probably through changes in catecholamine contents in the brain , and that the phas ic release of gonado tropin is neces sary for its biological activity .

31

FURTHER STUD I E S ON THE ANT I TEST I CULAR ACT I ON OF DL-6 - ( N- 2 -P I PECOLI NOMETHYL ) - 5-HYDROXY­INDANE ( PMH I ) I N THE RAT . N . Furuh ashi and V i c t o r S . F an g . Dep ar tment of Med i c i n e , University of Ch i c ago , Ch i c ago , I L .

We h ave p rev i ous l y reported t h at t h e ant i ­t es t i cu l ar ac t io n o f PMHI was n o t med i at e d b y a n e n do cr i n e mech a n i sm ( Endo c r i n o logy 99 : 35 8 , 1 9 76 ) . I n t h e p resent s t ud i es , we h ave further inve s t i gat e d the action o f PMHI i n rats and i n v i t ro . �We u s e d 1 2 5 I - l ab e l e d PMH I prep ared by ch l or­ami n e T method and puri f i ed o n Sephadex G- 10 co l umn . Four h af t er an iv o r s c i n j e ct ion o f 1 2 5 I -PMH I , t h e t i s sue radioact iv i t y i n creased in t e s t e s , ep i d i dymides , and s em i n al ves i c l es wh i l e d e c l i n e d i n l iver , k i dney s , lungs , pro­st a t e , and b l ood . Rat s received s c i n j e c t i o n o f equal amount o f r ad i o act i v i t y o f 1 2 5 I -PMH I or Na 1 2 5 I and s acr i f i ced at d i f f erent t ime i n t e rv a l s . The i r s em i n i f erous t ub u l e s were sep ar at e d f rom i n t e rs t i t i a l t i s sue . The up t ake of r a d i o ac t iv i t y ( cpm / µ g pro t e i n ) by s em i n i ­ferous t ubu l e s was s l igh t l y h i gher i n r a t s t re a t e d w i t h 1 2 5 I -P MH I t h an r a t s w i t h N a 1 2 5 I at 4 h and t h e d i f f erence i n up t ake b e tween the two groups b e c ame s ign i f i cant at 8 h . I n v i t ro i n cub a t i o n o f i s o l at ed t ubu l e s wi th � 12 5 I -PMH I f ai l ed t o p roduce s ign i f i cant up t ake o f t h e i s o t ope . Rat s r e c e i ved a s c i n j ect ion o f 1 2 5 I -P MH I and s acr i f i ced at 4 h . The i r t e s t es were homogenized i n TEM buf f e r a n d pro­ceeded w i th sub c e l lul ar f r act i o n at ion o n sucr­o s e gradient u l tr acent r i fugat ion . The radio­act iv i t y was only on t h e top l ay e r .

These resu l t s sugges t t h at 1 ) t e s t e s h ave s e l e c t ive up t ak e o f radioact ive PMHI , 2 ) s em i ­n i f er ous tubu l e s a r e r e spons ib l e f o r t h e s e l e c t ive up t ake , a n d 3 ) there i s no s p e c i f i c b i n d i ng sub s t an ce i n s em i n i f erous t ubules me idat i n g t he e f fe c t o f PMH I .

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CELLULAR I NTERRELAT IONSH I PS I N THE SEM I N I FEROUS CORDS OF TH E POSTNATAL RABB I T TEST IS . B . Gondos , C . J . Connel l , M . F . McKoon and C . E . Lai . Department of Pathol ogy and Reproducti ve Endocri nol ogy Center , Uni ve rs i ty o f Cal i forni a , San Franci sco , CA .

A s tudy was undertaken to determi ne the pattern of deve l opment of Sertol i cel l juncti ons duri ng the postnatal p repubertal peri od i n the rabbi t . Freeze­fracture , l a�thanum tracer and s tandard e l ectron mi croscopi c techni ques were uti l i zed i n observati ons on New Zeal a nd whi te rabbi ts from bi rth to 75 days of age . Spermatogenesi s begi ns at 7-8 weeks i n the rabbi t . We previous ly reported that , i n the fetal rabb i t testi s , occl us i ve tight j unct i ons are not p resent between Serto l i cel l s , whi ch s how only s cattered s hort rows of parti cl es on thei r membrane s urfaces ( Go ndos and Connel l , Arch . Andro l . 1 : 19 , 1978 ) . A s i mi l ar appearance was obs erved i n-the present i nves ti gati on duri ng the fi rst week after bi rth . However , at 7 days , compl ex anastomosi ng s trands of parti cl es resembl i ng ti ght j uncti ons were found between Sertol i cel l s . Lanthanum preparati ons i ndi cated that the tracer was not excl uded by these j uncti ons . Thi s was the case up to 60-65 days of age , a t whi ch t ime occl usi ve compl exes composed of ti ght and s eptate j uncti ons fi rst became evi dent . Thi s was s hortly after the formati on of s permatocytes . The functi on of the occl us i ve j uncti ons i s a ssumed to rel ate to the establ i s hment and mai ntenance of the b l ood-testi s barri er . The j uncti ons between Sertol i cel l s i n the pers permatogeni c testi s ev i dently do not have s uch a functi on . The preci se rol e of these j uncti ons rema i ns to be determi ned , but may rel ate to cel l ul ar movement and reorgani zati on wi th i n the devel opi ng s em in i ferous cords .

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THE PRO-ACROS I N , ACROS I N AND ACROS I N I N H I B ITOR SYSTEM OF

HUMAN SPERMATOZOA . J . C . Goodpasture , K . L . Pol a koski and L . J . D . Zaneve l d . Department of Phys i o l ogy & B i ophys ics , Un1 vers 1 ty of I l l i noi s at the Medi cal Center , Chi cago , I l . , and Department of Obstetri cs & Gynecol ogy , Wash­i ng ton Uni vers i ty School of Medi c i ne , St . Lou i s , Mo .

The optimal method of extracti on of acros i n ( an en­zyme i n v ol ved i n s permatoz oon penetration through the zona pe l l uci da of the ovum ) , proacros i n ( PA ) and acros i n i nh i b i tor { I ) was found to be a s fol l ows : add i t i on of benz ami d i ne t o semen , sucrose dens i ty centr i fugati on to rem ove s em i na l pl a sma , resuspens ion of the s perm pel l et i n g lycero l and benzami d i n e , homogen i zat i on , ac id ext­ract ion , and d i a lys i s to remove benzami d i ne and g lycerol . [he acros i n assay method used coul d _be .appl i ed .to an exJ tract from as few as l mi l l i on spermatozoa . A l arge

umber of poo l ed s emen samp l es ( 62 ) were anal yzed . I n each of these poo l ed samp l es approximately 90% o f the acro s i n was i n zymogen ( PA ) form and , after compl ete act i vat i on , a pprox imately 60% of the acros i n was bound to I . Ana lyses of i nd i v i dual semen s amp l es gave s imi l a� res u l ts al though the vari abi l i ty i n I content was i n­

reased and a bout 1 0% of the samp les possessed g reatly ecreas ed %PA . The %PA d i d not change when s permatozoa ere kept i n semi n a l p l asma at room temperature for as

l ong as 24 hrs wi thout benzami d i ne , but remova l of se­i na l p l asma i n the absence of benzami d i ne caused rapi d � !s pontaneou s acti vation of PA to acros i n . Freez ing of

semen caused the %PA to decrease over ti�e unt i l only 20 to 30% remai ned after 6 months at -20 C; the % of i n� � i bi ted acros i n i ncreased substant i al l y . These changes coul d be prevented by pre-addti on of benzami d i ne to se­�en . Extracted PA (much more so than acros3n ) was fou9 d to be unstab l e to freez i ng or s torage at 27 C , or at 4 d for l onger peri ods of time . The % of i n h i b i ted acros i n

j was al so affected by these condi t ions . Acti vati on of PA at pH 8 and room temperature resu l ted i n an i ncrease i n act i vi ty that took the form o f a s i gmoi d shaped . curve; compl ete acti vation requ i red 5 to 1 0 m inutes . Cal c i um i ons i nhi bi ted PA act i vation but enhanced acro-s i n act i v i ty , wh i l e z i nc i ons i nh i bi ted both PA acti va­t i on and acro s i n acti v i ty . Supported by N I H HD 09868 .

RELAT I ON O F SPERM SURFACE CHARGE TO SPERM FUN CTiON . K . G . Goul d , Yerkes Primate Cente r , Emory Uni vers i ty , Atl anta , GA .

34

We have begun uti l i z i ng s cann i ng el ectron mi croscop� & energy di s pers i ve X-ray anal ys i s ( SEM/ EDX ) to i nves ­ti gate changes i n spennatozoon membranes as soci ated wi th the nonna l ferti l i zati on p rocess & wi th i nferti l ­i ty . Pos i ti ve & negati ve cha rge i ron col l o i ds , s tabl e at a vari ety of p H val ues between 1 . 6 and 8 . 5 are bei ng used to measure net s urface charge of spermato­zoa s ub seq uent to tho rough wash i ng by pas sage th rough 7 . 5% Fi co l l and gl utaral dehyde fi xati on . Changes i n di s tri b uti on & magnit ude of surface charge are corre-1 ated wi th s tructural al terati on re veal ed by SEM and TEM , wi th s emi nal pl asma compos i ti on meas ured by PAGE separati on ( p H 8 . 3 : Tri sGlyci ne buffer , 7 . 5% gel s ) , & compared a fte r enzyme treatment .

Res ul ts i ncl ude ; 1 ) the di s tri buti on o f net surfa ce charge among i n di vi dual sperm i s not uni fo rm & us ual l y the popul ati on i s t ri moda l . 2 ) Some i nferti l e mal es possess spe nn wi th a surface charge much l ess than the ' no rmal 1 range . Such semen s ampl es s ti l l demonstrate a mul ti mo dal di stri buti on , imp lyi ng that the change i n charge i s a res ul t of al teration i n materi al coati ng the spe rmatozoa . 3 ) Prote i n patte rns obtai ned us i ng PAGE are vari abl e , howeve r th ere i s a gro up of aci di c protei ns mi grati ng cl ose to the tracki ng dye wh i ch i s apparently of epi di dymal origi n . In some i ndi vi dual s absence , o r marked a l terati on , of these bands i s asso­ci ated wi th abnormal i ty i n amo unt &/o r di stri buti on of charge . 4 ) En zymes so far tes ted i ncl ude a -Gl ucuron i ­dase types V & I I ; a & 8 amyl ase ; a Chymotryps i n & Hy­a l uroni dase . In b oth human & ch impan zee spenn the or­de r of . effect i n reduci ng s urface cha rge is B-Gl uc type I I > a -Gl uc type V > a amyl ase > Hyal uroni dase . a� amyl ase & a Chymotryps i n i ncrease the Zn & Cl detected in the regi on of the s perm head . aChymotryps i n (2 un i ts /ml ) , on expos ure of >2h rs at 37°C res ul ts i n di s rup­ti o n of mi dpi ece membranes & severance of heads from tai l s . The structural changes may acco unt fo r detection of Zn . Thes e res ul t s i ndi cate the appl i cabi l i ty of S EM) EDX to studi es of spermatozoan s truct ure & functi on .

35

LABEi.LING OF A SURFACE CYCLIC-AMP RECEPIOR SITE ON RABBIT SPERM UTILIZING A PHOIO-AFFINITY ANAiffi . G. L. Hahn , K. W . Metz , R . W. Atherton , and B . E . Halev* . University of Wyaning , Laramie , Wyoming .

Recent studies have implicated cyclic nucleotides as possible regulatory molecules in the control of mam­malian spenn maturation and development of motility . These studies have centered on the stinrulatory effects of increased cyclic adenosine 3 ' , 5 ' monophosphate levels . Tradit ionally it has been asst.m1ed that the levels of this nucleotide are under the control of two enzymes , Adenyl cyclase and Phosphodiesterase . These enzymes are presumably activated by a first messenger , usually a honnone , acting at the cell membrane . This system utilizing cAMP as a second messenger has been outlined by Hoskins and Casillas ( 1975 ) . But to date no first messenger has been identified nor has its existence been denonstrated . A report by Rosado , et al . ( 1977 ) has shown that human spenn may possess a surface receptor for cAMP , leading to speculation that c.AMP may occupy a role as the first messenger .

The purpose of this study was to analyze the c.AMP receptors in rabbit spenn. To do this we have utilized a photo-affinity analog of cyclic AMP (8-N3..-c.AMP(32p ) ) . This canpound, when activated by UV light , -has the ability to covalently link specifically to cAMP receptor proteins . Using this technique we have been able to radioactively label the cAMP receptor proteins on the surface of intact rabbit spenn. Labelled proteins were subsequently separated by SDS slab gel electrophoresis and analyzed by autoradiography and liquid scintilla­tion counting . The results substantiate the existence of a surface receptor for cAMP . The receptor protein has a '&WI of 48 , 000 , displays saturation kinetics and can be canpeted for by native 3 ' , 5 ' cyclic AMP . We con­clude that ej aculated rabbit spenn have a surface re­ceptor for c.AMP and propose that this molecule may act as a first messenger in the regulation of rnarrmalian 1:3penn development . Supported by NIH grant GM21998-03* and the Division of Basic Research , College of Arts and Sciences , University of Wyaning .

36

REPRODUCTIVE SYSTEM OF THE DIABETIC MALE RAT . Mary E .

Harri s , Worcester Fdn for Exptl Bio , Shrewsbury , MA

Although sexual disturbance s are known to occur i n diabet ic men , very little is known concerning the mechanism by which diabetes affect s male reproduction . Male CD rats ( 62 days old ) were rendered experimentally diabetic ( blood gluc ose >300mg/100ml ) by an iv inj ec­tion of strepto zotocin ( S ) ( 75mg/kg ) . Half of the group were also treated with insulin ( 0 . 5 IU/kg ) daily either beginning on the day of S or beginning 28 days after S . Rat s were s acrifi ced at 14 , 28 , 42 and 56 days post S . Throughout the 56 days , diabetic rats showed decreases in body, ventral prostate , s eminal ves icle , c aput epididymi s and testis weight s . Testos­terone ( T ) and dibydrotestosterone (DHT ) concentrations ( ng /ml ) in rete test i s fluid (RTF ) were also decreas e d during the period with signific ant decreases oc curring at 28 days ( 4 3 . 65+2 . 5 5 vs 2 5 . 12+8 . 41 , P< . 02 , for T and 2 . 21+0 . 51 vs 0 . 88+0 . 5 5 , P< . 02 , for DHT ) and at 56 day s ( 49 . 54+12 . 30 v s 2 0 . 83+9 . 35 , P< . 05 , for T and 2 . 38+0 . 32 vs 0 . 87+0 . 4 5 , P< . 001 , for DHT ) post S inj ection . Peripheral blood levels of T were also consi stently lower in the diabetic rats compared to controls through+ out the eXIJeriment . Inj ection of insulin for 28 days restored RTF T and DHT to levels not s ignificantly different from those in control rats . This restora­tive effect of insulin was also seen in plasma levels of T and testis weight , however body weight and weight of sex ac c e s sories remained significantly lower than controls . Histological examination of testes showed decreases in preleptotene and pachytene spermatocytes and step 7 spermatids i n all diabetic rat s as well as rats inj ected with insulin . T propionate ( 12mg /kg per day for 14 days ) or HCG ( l . 5 IU/ kg per day for 14 day s ) restored these cell count s to tho s e seen in controls . Results suggest that sexual disturbances in diabetic men may be the result of severe depre s s ion of several maj or para.meter s of testicular function and that treat-1 • • • rent with insulin restores or at least improves the �ndocrine function of the testis but does not reverse �he effect of diabetes on spermatogenesis . jl NIH grant HD09584 and ACS grant IN-100-B )

37

INHIBITION OF SEMINAL FLUI D DNA POLYMERASE BY 7S IMMUNOGLOBULIN G ISOLATED FROM HUMAN P OST-VASECTOMY SEMEN . P . J . Higgins, S . S . Witkin, and A . Bendich . M emori al Sloan -Kettering Cancer Center, New York, N . Y .

As part o f a study to assess the immunological con­sequences of vasectomy , immunoglobulin G (I gG) was isolated from semen of intact and vasectomized males . All of the I gG (1 . 5 mg) derived from ej aculates of in­tact men by precipitation with ammonium sulfate and ion -exchange chromatography on DEAE-cellulose sedimen­ted as a single 7S peak in sucrose velocity gradients . Only 36% o f the corresponding fraction (0 . 4 o f 1 . 1 mg) isolated from the same volume of seminal fluid from vasectomized males was 7S I gG . The remaining immuno­l�gically unidentifiable protein was considerably smaller and heterogeneous in size although it co -chro ­matographed with post-vasectomy seminal fluid IgG . Semen and serum I gG of intact males, as well as the smaller than 7S fraction from post-vasectomy ej acu­lates, did not inhibit the activity of a parti ally puri fied seminal fluid DNA polymerase from intact males at concentrations o f up to 15 µg/75 µl of reaction mix­ture . In contrast, 10 µg o f 7S I gG derived from the semen of vasectomized men produced a 50% reduction in the activity of the seminal fluid dT12 - 18 · POly rA­templated DNA polymerase derived from intact males . Additionally , the activity of the seminal fluid DNA polymerase in vasectomy-derived semen was reduced 3 -4 fold as contrasted to that in prevasectomy fluid from the same individual . These data indicate that anti­bodies reacting with the human seminal fluid DNA poly ­merase are recoverable in the semen of vasectomized males . Inhibiti on studi es revealed that the seminal fluid enzyme is antigenically related to a DNA poly ­merase that is presen� in sperm nuclei . The inhibitor� I gG in vasectomy semen may thus reflect an auto immune response to the sperm enzyme . Supported by Public Health Service grant CA08748 from the N . C . I . , the National Institute of Child Health and Human Development grant #1 ROl HD10826 -0l, and the N . I . H . Biomedical Research Support Grant # 5S0 7RR0 5 5 3 4 .

EFFECT OF PITUITARY GRAFTS ON TESTOSTERONE-STIMULATED GROWTH IN RAT PROSTATE. J .M. Holland , K . A . Hansen, and c. Lee , Depar tment of Urology , Northwes tern University Medical School , Chic ago, Illinois 60611

38

To determine if exces s endogenous prolactin secre ted by pitui t ary transplants could affect the growth of pros tates in rats given normal and low doses of tes to­sterone , pituit aries from two adult female rats were grafted under the kidney capsule of 30-day- old cas tra­ted males . The contro l cas trates underwent s urgic al exposure of the kidney . Pituitary recipients and control r ats were each further divided into two groups , one group receiving s . c . implants o f 0 . 5 cm s i l astic tubing containing tes tos terone , and ·the other receiving 2 . 0 cm o f such tubing . Tubed tes tos terone ( TT) 2 . 0 cm s . c . provides a continuous physiologic tes tos terone blood leve l , whereas 0 . 5 cm TT re leases sub-physiologic levels .

Three weeks l ater , all rats were s acri ficed by de ­capita tion. In rats receiving only 0 . 5 cm or 2 . 0 cm TT the lateral pros tates weighed 25 . 6 ± 1 1 . 5 mg and 5 1 . 9 ± 6 . 5 mg , respectively . In pi tui tary transplanted animals , a signi ficant increas e in serum prolactin was observed ( P<0 . 01 ) and was accompanied by a s igni ficant increase in the weight of the lateral pros tate (P<0 . 01), which was 46 . 7 ± 4 . 6 mg for those carrying 0 . 5 cm TT and 6 6 . 3 ± 8 . 6 mg wi th 2 . 0 cm TT . There was a corres ­ponding increas e in total DNA and RNA levels in the lateral pros tate o f those rats with pituitary trans ­plant s . Pi tui tary grafts did not increase the weight of ventral or dorsal pros tate beyond that s timulated by the TT . The effec t of these grafts on the growth o f the lateral pros t ate was more pronounced (P<0 . 01 ) in animals which received 0 . 5 cm TT than in those with 2 . 0 cm TT .

The data s eem to indicate tha t the excess prolac t in secreted by pi tuitary grafts created a testos terone­sparing e ffec t on the lateral pros tate of rats given low-dose testosterone . ( Supported by PHS Grant CA 10286 ) .

39

HORMONAL , HISTOLOGIC AND BIOCHEMICAL CHARACTERISTICS OF SPERMATOGENESIS (Sg ) IN VITAMIN A DEFICIENT (VAD ) AND IN POST VITAMIN A (PVA) TREATED RATS . H . F . S . Huang and W . C . Hembree , Depts . of Med /Obs-Gyn . , Columbia University t New York , N . Y . VAD rats have a.meio tic azoospermia whi ch i s revers ible PVA. We have studied both VA-independent and VA-depen­dent spermatogonial divisions , as well as the kinetics of PVA germ cell regeneration . Male Holt zman rats were given a VAD diet from 21 days of age and retinoic acid after the onset3of VA def iciency . DNA synthes is was assessed us ing H-th�idine and mitosis by radioauto­graphy . At 120 days H-DNA specific activity (SA) was 37 2±75 (SE) DPM/µg in VAD rats vs . 7 60±58 DPM/µg in normals . The number of c ells labelled/ tubule was 7 . 8± 1 . 9 and spermatogonial metaphases were observed . DNA synthesis doubled (808±158 DPM/µg) (p<0 . 01 ) within 24 hours PVA, returned to baseline (335±132 DPM/µg) by 4 days and ro se again thereaf ter . Radioautography con­f irmed this b iphasic germ cell response . Kinetics of Sg in PVA-VAD rats was histologically normal . A peak of 3H-labelled s perm appeared in the caput epididymidis 52-58 days PVA, or 10-14 days later than ob served in normal rat s . 80 , 100 and 150 days PVA , average e�ididy­mal sperm counts (N=8) were 3 . 7 , 12 7 and 180xl0 ; coun�s in 80 and 120 day old normal rats were 359 and 505xl0 , respectively . Liter size was normal in PVA rats , FSH values in VAD rats were higher (p<0 . 005 ) than age­matched normals (501±66 vs . 349±15 ng/nl ) and LH levels were l ower (p<0 . 005 ) (86±10 vs . 12 7±6 ng/ml ) . �es to­sterone in each of 3 serum pools (n=lO) from VAD rats was 0 . 86 , 1 . 1 7 and 1 . 15 ng/ml . (Normal pool= 2 . 53 ng /ml ) . Conclus ions : l)VAD causes revers ible germ cell deplet­ion in spite of high FSH and low normal LR-testos terone l evels . 2 ) A population of VA-independent spermatogonia accounts for the rever'sib ility . 3 )VA-dependent DNA syn­thesis in A1 or A0 spermatogonia rei�itiates kinetic­allyDJ:JS:miilll Sg PVA . 4 ) Total sperm production PVA is sub­normal , though normal liter size suggests normal sperm function . 5 ) The PVA-VAD rat provides a precise model for the in vivo s tudy of the biochemical kinetics o f each s tage of S g and , perhaps , of human oligospermia .

CANNULATION AND INTRA VAS DEFERENS TREATMENT OF THE RAT PROSTATE . W . L . Hunt and N . Nicholson . G . D . Searle & Co . , Chicago , IL .

40

The obvious exposure of the pros tate to secretions of the male reproductive glands was the basis for es tabl ishing an animal model in which the effects of compounds f lushed through the pros tatic urethra could be s tud ied . Cannulation of the vas deferens was performed on 46 male rats with Tygon tub ing , O . D . 0 . 89 1IDll , I . D . 0 . 50 1ID11 . The indwelling cannulat ion tube was sutured so that solutions could be f lushed anteriorly through the pros tatic urethra for an extended treatment per iod . From tr ials it was found that f lushings contained many mo tile sperm cells . Als o a retrograde flow occurred from the vas into the indwelling tub e .

In a p ilot s tudy , 2 groups of rats wer e flushed daily , 5 days /week , for 3 weeks . Controls received a 1 ml saline f lush ; treated animals re­ceived 1 ml sal ine containing Premarin® (50 µg /ml) . Compared to controls , es trogen treated rats showed a s light increase in pros tate and seminal ves icle weights ; no tewor thy was the highly s ignifi­cant decrease in the s ize of tes tes , unilaterally , on the cannulated side .

A 6 week treatment ( 1 ml f lush/day , 5 days/ week) o f 3 groups of cannulated males involved vehicle , 100 µg 1 7 S-estradiol /ml/day , and 1 mg 5a-d ihydro testos terone/ml/day . Marked reduct ion of a ll r eproduc tive organs resulted in the 1 7 S­es tradiol rats . The average weight of the pros­tates were lower in the 5a-dihydrotes tos terone rats than in control s .

This animal model provides new ins ights into the ef fects of both endogenous and exogenous intra vas agents on male reproductive organs .

41

RELATIO"! S H IF BET .. !EZ'1>I S EM PTA L F P. O S 'l'A G LA M D PT S

A"·TD REF �ODUC T IVE Pmrc'l' I O"! I'1\T MAM . A . I s idori ' D . Con t e and F . Dond ero , I s t i tuto C l i n ica �e­d i ca V , Univers ity of Roma ( I t a ly ) .

The s i gn i fi cance of t h e pres ence o f the pros t a g l a n d in s ( Fr. s ) in the huma n e j a cu l a t e , a s far a s the funct ion of te s t i s and/or a d­nexa l g l an n s i s concern ed , ha s been ma t t er o f controver s i a l s pecu l a t i on . Th i s ma i n ly in view of t he many d i f feren t and ofte� un re l i a b l e method s employed in t he a s s e s smen t o f t he PGs i n t he s em in a l f luid . U s ing a s en s i t ive g a s­chroma t o graphic procedure , s l ight ly mod i f i ed , the Aut hors ha ve a s sayed the mo s t phys i o l o g i­ca l ly impnrtant PG s ( PGE a n d 1 9- 0H-PGE ) in the e j a cu l a t e of 1 0 norma l a du l t ma l e s and of 50 infert i le , dys permi c pat ien t s . The se l a t t er s e­l ected a ccord ing to s t r ict ly s t a n d a rd i z ed cry­t er i a . A fter having a s s es sed a range of nor­ma l i ty , t he Aut hors have compa red the s e va lues with the common e s t semina l paramet er s ( concen­t r a t ion , mo t i l ity and morphology of s perma to-

+ zoa ; fructose a n d TRK sub s t ances l eve l s ) an d w i t h t h e pla sma an drogens ( t e s t o s terone a n d DHT ) l evel s . Accord ing to the resu l t s obt a i­ned , i t s eems t ha t t he mot i l i ty ( and fa r l e s s the con centra t ion ) o f t he s perma t ozoa i s a f­fected by the va riat ion s of the s emin a l PG s . In a d d i c t ion , i t seems to exi s t a d irect rela­t i o-1 s h i p between ], evel s of s em in a l PG s and p l a sma androgen concen tra t i on .

42

I �VESTIGAT ION OF THE AX IS HYPOTHALAMUS-HYPOPHYSIS­GONADS IN 20 D IALYSED MEN . E . Jecht , E . Kl u�p , Departmen t of Derma to l ogy , On1 vers 1 ty of Er angen , FRG , and R . Hei tl er , IV . Department of Internal Med i c i ne , Nuremberg Genera l Hos p i ta l , Nuremberg , FRG .

Whi l e there i s conc l u s i ve evi dence for damage to the tes ti cu l ar ti ssue i n d i a lysed pati ents , l i ttle is known wi th regard to the funct i on of the hypo­phys i s i n thes e pati ents . Gonadotropi ns a nd testo­sterone were determi ned by RIA in 20 d i a lysed ma l e pati ents pri or to and fol l owi ng st imul ati on wi th GnR H . When compared wi th the pattern of non-d i a l ysed ma l e pati ents , the fol l owi ng di fferEnces were noted i n d i a lys ed pati ents : �asa l va l ues of LH were el eva ted whi l e the i ncrease of LH after s ti mul ati on wi th GnRH was l ess pronounced , and al most no decl i ne was seen wi thi n the observati on peri od ( 180 mi n . fol l owi ng s ti mu l ati on ) ; basa l val ues of FSH were sl i ghtly el evated , there was , however , no change of FSH after st imu l ati on wi th GnRH ; ba sal val ues of tes tos tero ne were l ow , and s ti mu l a ti on wi th GnRH l ead to a s l i ght i ncrease of testosterone .

These resu l ts i ndi cate a d i s turbance of the hypo­phys i s i n add i ti on to the gonadal l es i on i n d i alysed pati ents . There was no obvi ous correl ation of hormone va 1 ues aid sexua 1 h i s tory when i nd i vi dua 1 data were

·

compared .

43

HYDROGEN ION PRODUCTION BY MAMMALIAN CELLS US ING BOVINE SPERMATOZOA AS A MODEL . R . S . Jeyendran and E . F . Graham . University o f Minnesota , St . Paul , Minneso t a .

Hydrogen ion production b y mannnalian cells i s important not only to gain basic knowledge but also for cryo preservation . By us ing bovine spermatozoa we have determined the amount of hydrogen ions produced per 10 9 cells . Dur ing 3 7C incubation of s emen the percent o f sp erm ce lls pas s ing through a s ephadex f il ter and the pH was measured at frequent intervals . The number of cells that died due to natural aging was calculated by incub ating the extended semen in a pH 6 . 5 buffer and count ing the cells by the filteration procedure . Conversion o f percent cells to a 109 and pH to hydrogen ions shows that 109 cells will produce 1 6 . 7 3Xlo- 7 grams of hydrogen ions per liter over 60 minute s .

The number o f cells that were immobili zed and d ied due to natural aging and pH change (b rought about by the metabolic ac t iv ity of the sperm cell) was calculated at frequent intervals during incub at ion us ing the filteration procedure . In the same way the number of cells that could be revived after exposure to extenders with d ifferent pH values and sub sequent exposure to initial pH ( 6 . 5 ) was determined . The results revealed that the number of cells recovered by the fil teration procedure decreas ed as the pH was lowered from 6 . 50 to 6 . 00 . The number of cells that could be revived inc reased from zero at pH 6 . 50 (because ini tial pH was 6 . 50) to a maximum at pH 6 . 20 and then gradually decreased to nearly zero at pH 6 . 00 .

44

EVALUAT ION OF C I RCULATING PATTERNS OF TESTOSTERONE I N MALE RATS MAI NTAI NED O N TOTAL PARENTERAL NUTRIT ION ( TPN� AN EXPERIMENTAL MODEL . R . J . Keat ing , R . K . Tchol a k ian , and E . Ste i nberger , Dept . Reproducti ve Med i c i ne and B i ol ogy , Univ . of 1 exas f·ledi cal School at Houston ,Texas .

Two groups of mal e Long-Evans rat s ( 90d ) were fi t ted with i n-dwel l i ng j ugul ar cannul as and housed i n metabo-1 ic cages under control l ed cond i ti ons . One group served as stock-fed control s ; the other recei ved a l l nutri t i on intravenou s l y ( TPN } . The parentera l sol ut i on con s i sted of gl u cose ( 25%) , amino ac ids ( 3 . 5% } , v itami ns and el ectrol ytes . The sol ut i on was i nfused at the rate o f 4 0 c . c . per day .

Rat s on TPN were uti l ized as our· model an i mal syste� to i nvest i gate : 1 ) whether any changes occur i n test i ­cul ar funct i on between control and TPN rats wi th i n a 7 day per i od . Test i cul ar and accessory organ we i ghts as wel l as pl asma Testosterone (T) were me asured and compared to control ; 2 ) i n v i vo vari at i on s i n pl asma T patterns i n i n d i v i dual """"'iin imal s duri ng a ) a 30 min . per i od wi th sampl i ng every 2 minutes ; b ) a 24 hr . per ­i od wi th s ampl ing every l � or 3 hours ; c ) a seven day peri od wi t h da i l y sa�pl ing . Bl ood samp l e s ( 0 . 1 5-0 . 2 c c � were obta i ned from TPN and control an i mal s for T-RIA whi l e the an ima l s were unrestrai ned and unanthes ized duri ng experimentation .

There was no stati st i ca l l y s i gn i fi cant d i fference i n testes , semi nal ves i cl e , ventral prostate we i ghts and pl asma T concentrat i on s between TPN and control rats . T concentrat i on s i n sampl es obta i ned every 2 mi n . from TPN and control rat s showed rapi d osci l l at i on s remi n i s ­cent of patterns observed i n other species . I nd i v idua l var i at i on s i n pl asma concentrat i on s and patterns of T were observed during a 24 hr . and 7d sampl ing peri od . The data obta ined from these exper i ments demonstrate the poten t i a l of th i s model system as a u n i que approach to studyi ng in v i vo testi cu l ar functi ons under preci se­ly contro l l ecr--cond i t i on s .

( Suppo rted by N I H grant P50-HD-08338 ) .

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SPE C I F I C B INDING OF PROLACTI N AN D D I HYDROTE STOSTE RONE ( DHT) BY MALE ACCESSORY S E X ORGANS . E . J . Keenan , E . D . Kemp , E . E . Ramsey, L . B . Garr i s on , H . D . Pearse , and----C:-V . HOciges . Departmen t s of Surgery and Pharmacology an� Div i s i on of Uro l ogy, Oreg on Hea lth S c i ences Center , Port l and , Oregon .

Pro lactin augments androgen action in certain ma l e acces s ory sex organs and this interaction may be de ­pendent upon the binding of both pro l act in and DHT . The pres ent stud ies examined specific 1 25 I -pro l actin b inding t o membrane part i c l e s ob tained from the pros ­t ate g lands and seminal v e s i c les of mice , guinea pigs and rats ( 3 - 4 months o ld) and examin_ed the r e l at i onship be tween pro l�ct in and DHT b inding in the rat ven tral pro stat e . 1 5 I -pro lactin wh i ch was displaced by un­l abeled pro lactin was con si dered sp ecifical l y bound and e xpre s s ed as a percentage of the total pro lactin incu ­bated per 0 . 1 mg protein . Spe c ific b inding of 1 2 5 I ­pro l actin was not de tectable in mous e or guinea p i g pro s tate , the mus c l e o f the guinea p i g s emina l �Ss i c le or in rat s eminal ve s i cl e . However , sp ecifi c 1 I -pro ­l actin b inding was pres ent in mous e s eminal ve s i c l e ( 0 . 5± 0 . 0 5 % ) , the ep ithel i um o f the guinea p i g s emina l ves i c le ( 1 . 1± 0 . 1 % ) and i n ventral �rostate o f the young rat ( 3 . 6± 0 . 3% ) . Specific 1 2 I -pro lactin binding a c t iv i ty in the ventral p ros t ate of aged rats ( 1 0 mon ths old) was s i gnifi cant l y l ower ( 1 . 0± 0 . 1 % ) . Incu­bation o f ventral prostate cytos o l with 3H - DHT ( 1 0 nM) demonstrated that age did not a l ter specific 3H - DHT b inding ( 1 3± 2 fm/mg pro t . ) in non- cas trated rat s . How­ever 2 4 h our s after castrat i on , l arger increas es in specific 3H- DHT binding were observed in the ventra l pro s t at e s of young ( 6 0± 5 fm/mg prot . ) as compared to aged rats ( 2 7±4 fm/mg pro t . ) . Th ese studies have dem­on s trated that spec ifi c pro l actin binding s i tes are s e l e ct ive ly d i s tribute'd in mal e acces s ory sex organs and further sugg e s t that pro l actin b ind ing activity may be rel ated to the increas es in DHT b inding whi ch o ccur in the rat pros tate short ly after cas trat i on .

S upported by NIH grant RR 00334 , MRF grant 3 8 . 1 7 , and the P l easants Prostate C ancer Re s e arch Center .

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CADMIUM AND L EAD INJECTION I NTO PROSTATE . N . K . Khare , R . Der , G . Ros s , Jr . , and M . S . Fah i m . Departments o f Obs tetr i cs and Gynecol ogy and Surgery ( Urol ogy) , Uni ­versi ty of M i ssouri Medi cal Schoo l , Col umb i a , MO .

Rec entl y cadm i um concentrati ons have been found to be mar kedly i ncreased i n carci nomatous human prostate ( J . Endocr . , 7 1 : 1 33-1 41 , 1 976) . Forty sexual l y mature mal e rats were di vi ded equa l l y i nto 4 grou ps . Group 1 served as control s ; Groups 2 , 3 , and 4 were i njected i n prosta te wi th l ead aceta te ( 1 mg ) , cadm i um chl ori de ( 1 mg) , a nd a mi xture of l ead acetate ( 500y) and cadmi um chl ori de ( 500y) res pecti vel y . Rats were sacr i fi ced 60 days a fter treatment . Tes tes , epi d i dymi s , and pros ta te decreased i n wei ght s i gn i fi cantl y ( p < 0 . 001 ) i n Groups 3 a nd 4 . Group 2 had mi n i mal cha nges i n the tes tes , epi di dymi s , and sem i na l vesi cl es . Pros tate had chal k­l i ke whi te depos i ts and th ei r l i ver showed marked focal necros i s . Group 3 had a trophy o f test i s but s i gni fi ­cant hyper pl a s i a of i ntersti ti um of testi s . Sem ina l ves i cl e and epi di dymi s were a troph i ed . Prosta te was marked l y a troph i ed and had acute and chroni c i nfl amma ­tory c hanges . L i ver had m i n i ma l focal necros i s . Gro up 4 had changes in the tes ti s , epi di dymi s , s emi nal ves i ­cl e , and p rosta te s i mi l ar to Group 3 but l i ver s howed s i gnifi cant focal necros i s . Long term study of effect of cadmi um and l ead on pros ta te i s under i nves ti gati on to cl a ri fy whether bo th trace substances act as carc i n­ogeni c agents .

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AN ANALYS IS OF 50 HYPOFERTILE MALES WITH VARICOCELE . J . A . LaNasa, Jr . Depar tment of Urology , Louisiana State Univers ity Medical Center , New Orleans , LA

The evaluation of 1 7 5 infert ile couples in the past 2 years yielded 50 males with a varicocele . The analysis of these patients revealed 5/50 to have azoospermia , 6/50 to have b ilat eral varicoceles , 8 / 50 had a sperrnat ic vein ligat ion prior to their init ial visit to our clinic . The testes volume using a caliper and orchidometer was approximat ed in every pat ient and revealed ipsilat eral testicular atrophy in 40/50 and contralat eral t est icular atrophy in 2 7 / 50 . The " S tress Pat tern" on s eminal cyto logy as def ined by MacLeod was pres ent in only 1 5 /50 . Sperm densities less than 20 million/cc were present in 44/50 . The rat ionale for prophylatic spermat ic vein ligation before the onset of test icular atrophy and oligosper­mia will be str essed based on this data .

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SEMINAL VES ICLE MEMBRANE TRANSPORT IN VIVO IN GUINEA PIGS . N . Levine and H. Ke l ly. Department o f Physiology, New York Med ical Co l lege, Valha l la, N. Y.

We have previous ly demons trated that the guinea pig semina l ves icle ( SV) when mounted in vitro in an Us sing type chamber can actively transpor�c 1- from serosa t o mucosa (Levine et a l . , J. Phys iol . 24 6, 197, 1975 ) . This s tudy present s results o f investigations per formed in vivo on water and ion transport by SV. A standard bicarbonate buffer (pH=7 . 4 ) was inj ected into an SV that had been flushed o f its contents . F luid was recollected a fter a suitab le time and net water f lux (V) calculated from changes in 3H-Inulin concentration . Na+ and 1(1- net f luxes (S) were calculated from change s in concentration and volume . Measurements were per­formed in normal animals (NA) , animals cas trated for 5 - 6 days ( C ) and animals rep laced with either testos­terone (T) or d ihydrotestosterone (DT) . Results are presen ted in following tab le.

V §� � K (µL/min, cm) (nEq /min, cm)

NA�SEM(n) o . 3.s"to . 04 (1 7 ) 6otlO ( l l ) 5±1 ( 11) ctsEM(n) -o . 1oto . 03 (8 ) 9±10 (8) -o . 4±0 . 1 (8 ) TtsEM (n ) o . 3 7±0 . 07 ( 7 ) 7o±l0 (7 ) .5"tl (7 ) D'I±SEM(n) 0 . 3 7±0 . 10 ( 5 ) 6o±2 0 (5 ) 4 . o±l (5 ) Concentrat ions o f Na+ and IC'" a fter 2 1 /2 hours in recollected per fusate (RP) from NA were lower than in initia l perfusate ( IP) (in mEq/ L RPNa=l22±2 vs . IPNa= 138 ; RPK_=l . 2±0 . 1 vs . IPNa=4 . 6) . Reabsorba te had higher concentrations o f these ions than IP, RP or p lasm3 (Na=220mEq / L and K=2 1mE q /L) . Data indicate that SV is capable o f reabsorption as wel l as secretion that is androgen d ependent and lend credence to our hypothesis that a fter ej aculation SV secretes relatively large volume electrolyte so lution that is subsequently reab ­sorbed . Hypertonic salt reabsorption is probably coupled to secretion o f nonreabsorbab le organic mo le­cules that are concentrated in lumen to ma intain osmo ­lality . O smo lality cons iderations indicate there is about 2 00 mM o f unidentified organic mo lecule in SV p lasma. Supported in part by NIH Grant HD09010.

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CONTACT SCROTAL THERMOGRAPHY IN FERTILE MEN AND IN SUBFERTILE MEN WITH AND WITHOUT CLINICAL VARICOCELE . R . W . Lewis and R . M . Harrison , Department o f Uro logy , De l ta Regiona l Primate Research Center , Tulane Univ . , Covington, Louis iana .

A c linica l s tudy was conduc t ed to determine if therma l s ens itive p lates cou ld be us ed to detect intra� scrotal t empera ture differences due to vascular change s econdary to varicocele or o ther scrotal patho logy . A C lark Contact Thermograph , made to our speci fications , was used in the Tulane Medical Center Ferti lity C linic � Tulane Univers i ty S choo l of Medic ine , and Louis iana Charity Hospital in New Orleans . This equipment al lows the inves tigators t o obtain a permanent record o f scrotal t emperatures in erect or supine patients .

Each interchangab le p late produces five co lors within its spec i fic temperature range , 30-35oc , 34-o 0 3 5 C , or 35 -36 C . The narrow range p lates showed t emp erature differences ass ociated with the left pam­pini form p lexus and co l laterals to the right scrotum in patients with varicocele . Eleva ted t emperatures were detected in patients with epididyrnitis or tumors , over the scrotum o f the affected side . Lower t emper­atures were found in tors ion and hydroceles on the side o f the patho logy . Pos t varicocele ligation patients were eva luated in the post surgica l p eriod . No differences in scrotal temperatures were detec ted in norma l men .

This equipment provides an excellent means to detect sma l l temperature changes in the scrotum by a noninvas ive technique and great ly as sis ts in the diagnosis of a sub c l inical vari coc e le in o ligospermic ma les with semen analysis simi lar to pat ients with c linica lly detec tab le varicoc e l e .

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IMMUNOSUPPRES SIVE ACTIVITIES OF MALE REPRODUCTIVE TRACT COMPONENTS . Z . H . Marcus, J . H . Herman and E . V . Hes s . Department o f Medicine , Division o f Immunology , University of Cincinnati College of Medicine, Cincin­nati , Ohio .

In the normal fertile male and female, tolerance exists to sp ermatozoa (HuSp) and s eminal plasma (HuSeP l ) constituents . The basis underlying this tol­erogen ic s tate remains unknown . Lately s everal author s reported that male genital constituents (MGC) pos ses s immunosuppressive activities . Thi s local im­munosuppression can explain the tolerogenic s tate to MGC . It also explains the lack of success in the ef­forts of documentating the cell mediated immune (CMI ) response to HuSp using conventional in vitro tes t s .

In the current s tudy CMI response t o MGC has been evaluated in normal and pos t-vasectomized human sub­j ects ( sens itive to HuSp) in an attempt to identify factor s c apable of spec ifically or non-specifically modulating results of in vitro assay systems . S ensi­tization to spermatozoa! antigens was documented in 4 of 10 subj ects evaluated over 2 years pos t-vasectomy . Each subj ect was studied on 5 s eparate occasions at weekly intervals employing leukocyte migration inhibi­tory , lymphocytotoxicity and blastogenic assay systems . Spermatozoa and chromatographed s eminal plasma f rac­tions were shown capable , on a dos e dependent basis , of inhibitory spontaneous and mitogen-induced lymphocyte DNA synthesis thus blocking the specific antigenic response to MCG . However , neuraminidase and o< -methyl­D-mannos ide (AMM) p re-treatment of sperm abrogated suppres so r properties . Results indicate that both HuSp and HuS eP l from normal donors contain immunosuppress ive activities that can modulate the in vitro immune re­sponse . Inactivation of cell surface rec eptors by AMM or neuram inidase should b e considered when using thes e antigens i n immunologic assay s . It remains now to determine whether lack of or presence of this immuno­suppressi ve fac to r ( s) in MGC may be a cause in human infertility .

Supported by NIH Grant NOl-HD-3-2709 .

51

SAFE AND SUCCESSFUL RECTAL PROBE EL ECTROSTIMULAT ION --u-t ERECT ION IN THE HUMAN MALE . D . E . Marti n*t , H . Warnert , R . T . Crens h aw+ , and T . L . Crenshaw+ . *Georgi a State Un i ­vers i ty , Atl anta , GA ; tYerkes Primate Cente r , Emory Uni • vers i ty , Atl anta , GA ; +Crens haw Cl i n i c San Di ego , CA .

Our objecti ve was to de fine the e l ectri cal pa rameter$ req ui red to induce erecti ons i n cons ci ous hea l thy and sp i na l cord-i nj ure d mal es . Previ ous work on 26 speci es of primates had been success ful i n l ) i nduci ng e rection� and/or s emi nal emi s s i ons , 2) provi di ng knowl edge of a l l e l ectri c a l sti mul us parameters ( st imul us wavefo rm , fre­q uency , vol tage , c urrent and res i stance ) i nvol ved , and 3 ) ensuri ng safety to the ani ma l s i nvol ved .

Two p robes were constructed , each wi th p l ati num el ec+ trodes mo un ted on a 1 9mm di ameter p l asti c body . One p robe had 4 el ectrodes 90° apart , ea ch wi th 3 1 2mm2 s ur­face a re a ; the other had 2 e l ectrodes 1 20° apart , each wi th 350mm2 surface area . Current del i ve ry was thus ori • ented ci rcul arl y w i th respe ct to the rectal axi s , and the effects of di recti ona l i zat i on of el ectri cal current co ul d be studi ed . El ectri cal shock hazard was mi nimi zed th ro ugh the use of i so l at i on transforme rs wi th el ectro­stati c s h i e l di ng and spec i a l current l i mi ti ng devi ces .

Res ul ts i ncl ude the fo l l owi ng : i n 6 heal thy mal e s , 1 ) e recti on was mos t eas i ly i nduced with e l ectrodes or­i ente d 1 20° apart and di rected away from the spi na l cord , 2 ) a p robe stem narrowi ng to 6mm i n the regi on o f the ana l sphi ncte r was mandato ry t o prevent di s comfo rt from sph i ncter s pa sm , 3 ) l ower frequenci es ( 0 . 25Hz to 20Hz AC ) el i c i ted e recti ons at sti mul us th resho l ds wh i ch produced di s comfort at h i gher freq uencies , and 4 ) typi ca l va l ues fo r el i ci ti ng erecti on were 31 mamps at 6 vo l ts , with 1 96 ohms resi s tan ce . These s tudi es a re bei ng extended to sp i nal cord- i nj ured ma l es and othe r p ati ents who may be phys i ol ogi cal l y i mpotent . Res u l ts wi th 4 parap l eg i cs ( l es i ons bel ow T6 ) i ndi cated addi ­ti ona l l y th a t , 1 ) erecti on co ul d be i nvoked , but at a consi derab ly h i ghe r stimul us i ntens i ty , and 2 ) h i ghe r sti mul us frequenci es (20Hz to 60Hz AC) were j ust as effecti ve as l ower freq uencies . Th us , recta l probe el ectro s timul ati on shows promi se a s an effecti ve tool i n i nvest i gati on of phys i ol ogi cal impotency .

52

ACROSOMAL A MINOPEPTI DASE AND A NON-ACROSIN ENDOPROTEIN­ASE FROM RABBIT SPERMATO ZOA . D . K . McRorie, M . M . Bradford, and R . A . McRorie . Reproduction Rese arch Laboratories, Department of Biochemistry, University of Georgia, Athens, GA .

In examining the proteinases present in rabbit sperm acrosomes, we have determined that there are at least two different enzymes which will hydrolyze the substrate L-leucyl- S -naphthylamide (LBNA) . Disc gel electrophoresis of total acrosomal extracts followed by activity staining reveals two different bands of LBNA hydrolyzing activity . When LBNA is N-succinylated to y ield SLBNA and used in the same assay, one of the activity bands disappears, indicating that one of the enzymes is an endopeptidase and the other an exopeptidase . Sequential extraction of rabbit acrosomes shows that the exopeptidase, acrosomal aminopeptidase, activity is solubilized by ions prorroting acrosomal activation . Endopeptidase activity is extractable by subsequent detergent treatment indicatin g that the enzyme is membrane boun d . The functions of these two proteinases are as yet undetermined ; however, it appears that membrane bound enzymes function in zona pellucida penetration while enzymes solubilized during acrosomal activation are involved in membran e dissolution during the acrosome reaction and penetration of cellular investments of the ovum .

Supported by NIH Grant # HD10456 and Ford Foundation Gran t # 680-0SOSA .

53

USE OF BOVINE CERVICAL MUCUS IN THE IN VITRO SPERM PENETRATION METHOD . A. C . Menge and N . E . Medley. De­partment of Obs tetrics and Gynecology , University of Michigan , Ann Arbor , MI .

The in vitro cervical mucus p enetration (CMP ) technique�s a useful method in evaluating human sperm quali ty . Becaus e of the difficulty in ob taining suffi­cient quantity o f midcycle human cervical mucus (HCM) we have evaluated the use of estrous bovine cervical mucus (BCM) in the CMP method . CM was drawn into rec­tangular glass capillaries ( 0 . 2 mm ID x 50 nnn 1) that were s ealed at one end with plas ticene and then placed upright into small tubes containing. the semen samples . CMP was rated a s the farthest distance that sperm had penetrated after 30 min incubat ion at 37°c . The rat­ings were as fo llow s : p oor , < 9 nnn ; fair , 10-19 mm ; good , � 20 mm . Human sperm motility , forward progres­s ion , and survival appeared to be no different between BCM and good quali ty HCM. In a comparison of CMP by sperm samp les from 16 men of infertile coup les between BCM and HCM (wif e ' s) in which the latter had good pen­etration by sperm of a donor , the ratings changed in only two cases . Both went from poor for HCMP to fair for BCMP . CMP analysis of sperm from infertile men with sperm antibodies in their sera showed a higher incidence of poor and fair p enetrations with both HCM and BCM. The BCMP result s with donor sperm treated with the seminal p lasmas of 40 men were not signifi­cantly differen t than results with the HCMP (wife ' s) . sperm ant ibodies in the seminal plasmas caused poor to fair BCMP by donor sperm which correlated well wi th the original HCMP (wife ' s) data . Our results indicate that es trous BCM is a good sub s titute for midcycle HCM in the sperm-cervical micus penetration technique .

54

ANDROGEN-INDUCED STIMULATION OF THREE NEW PROTEINS IN MOUSE KIDNEY . N . C . Mills , T . M . Mills , and C . W . Bardin . Dept . of Medicine , Div . of Endocrinology , M . S . Hershey Med . C t r . , Penn . S tate Univ . , Hershey , PA .

Androgen administration to female mice for 25 days causes a rapid increas e in kidney wet weight . This weight incr ease is due to protein and RNA but not DNA synthes is . Although known androgen respons ive end points such as a-glucuronidase and alcohol dehydrogen­ase respond rapidly and increas e many-fold per animal by 25 days , they do no t represent enough of the total proteins to account for the large increase in kidney weight . We , therefore , sought to identify maj or pro­teins which increase with androgen treatment . Analysis of total kidney proteins on 1 0% polyacrylamide-SDS gels revealed a small protein band of 4 3 , 000 molecular weight in androgen-treated kidney which was absent in untreated animals . Because the function o f this pro­tein was not known , it was tentatively labeled Tl s ince testos terone treatment induces its appearance . Further analysis showed the protein band to be present as early as 2 days following androgen treatment . In an effort t o localize this protein , subcellular frac­tionat ion was performed which demonstrated the presence of this protein in the mitochondrial-lysosomal frac- . t ion . Another protein band of 60 , 000 daltons des ig­nated T2 , was also observed in this fraction . Analysis of the microsomal fract ion revealed another protein , T3 , at 54 , 0 00 molecular weight in the androgen-treated kidney . Testosterone treatment produced no new pro­teins in the soluble fraction that could be identified by SDS gel electrophores is . This is of particular note s ince a-glucuronidase which increased 35-fold and rep­resented approximately 0 . 3% of total kidney protein was not observed in these analyses . In summary : ( 1 ) at leas t 3 new kidney proteins have been identified which increased many-fold during androgen treatment ; (2 ) all of these proteins are undetectable in uns timulated ani­mals and increase to levels in treated animals that can be quantified on SDS gels . We conclude that the T prot eins will be important markers for the study of androgen-induced renal hypert rophy .

55

MODIFI CATION OF CALCIUM BINDING BY SPERM CELL MEMBRANES . L . Nel son and M. Young. Department of Physiology , Medical Col lege of Ohio , Toledo , OH .

Calc ium b inding by washed , ej aculated bull sperm cells may be s ignificantly altered by a var iety of agents that af fect the propert ies of cellular membranes . Calc ium and other divalent cat ions elicit a signal from the fluorescent probe , chlortetracycline , which is relat ed to the degree o f b inding to membranes . The s ignal is measured spectrof luoropho tome trically at an excitat ion wavelength of 428 nm, emiss ion 517nm, slit wid th 15nm . Ul trasonic disrup t ion of the sperm cells for 30 s econds increases the fluorescent signal by 25-30% apparently as a cons equence of increased e�posur e of cell surfaces . Subs tances which alter calc ium binding or up take also affect the s trength of the s ignal . Tho se agents which compete with calcium for b inding sites reduce the calc ium-induced fluores­cence . The lanthanum ion reduced s ignal of about two­thirds may be at least part ially reversed by addition of more calc ium to the suspens ion . Both physost igmine and acetylcholine , on the o ther hand , enhance the s ignal by about 10% . While procaine ' s effect is rela t ively small , dibucaine causes nearly a 40% depression . Decamethonium, the cholinergic receptor depolar izer , decreases the calcium induced s ignal by 12 % , while at the same concentration , the cholinomimetic agent , nico tine , appears to be twice as effec t ive , causing about a 25% drop . Other agent s (a-bungarotoxin , hemicholinium) are being tes ted to confirm the correla­tion between calcium transport and cholinergic regula­t ion of sperm cell mo tility .

�6

BIOCHEMICAL CHANGES IN THE RAT SPERMATOZOAN PLASMA MEM­BRANE DURING EPIDIDYMAL TRANSIT . G . E . Olson and B . J. Danzo . Depts . of Ana tomy and Obs te tri cs & Gynecol ogy , Vanderbi l t Uni versi ty , Nashvi l l e , Tenn .

We have used the binding of tri ti a ted lectins and en­zyme catal y zed radi olabeling of external l y ori en ted membrane proteins to detennine mol ecular al terati ons i n the pl asma membrane of ra t spermatozoa whi ch occur during thei r passage thro ugh the epi di dymi s . [H3] -Con­canavalin A binds to cauda and caput epi di dymal sperm wi th typi cal satura tion kineti cs and both sa turate a t about 3 . 1 x 1 0 7 Con A mol ecules per sperma tozoon . 0 . 1 M �-me thyl -D-mannosi de inhibits [H3] -Con A binding to sperm by > 9 0% . Further studies were performed to i den­ti fy membrane gl ycoproteins whi ch bind Con A . Enzyma ti � i odination of sperma tozoan pl asma membrane pro teins wi th l actoperoxi dase and [Il 2 5] -NaI was followed by SDS gel electrophoresis of membrane fractions . Cauda sperm demons tra ted two ma cromolecular peaks of radioactivi ty , one of abo u t 45, 000 MW and the o ther of 37 , 000 MW , as well as a peak of radi oacti vi ty tha t mi gra ted near the tracki n g dye . Caput spermatozoa l abeled by the same procedure showed no l abel ed 37 K component . The gala c­tose oxidase- [H3] -sodi um borohydri de techni que was used to label gal actosyl an d N-ace tyl galactosaminyl resi dues on sperm membranes . SDS gels of galactose oxi dase- [H3] ­NaBH4 l abel e d sperm reveal ed the 3 7 K componen t on ca uda sperma tozoa , but not on caput sperma tozoa . Afini ty •chroma tography on Con A-sepharose or Ri cinus Communi s .120-agarose col umns of detergen t sol ubi l i zed membrane ,components from gal actose oxi dase label ed cauda epi ­di dymal sperma tozoa re vealed a peak of radioacti vi ty el uted with the speci fi c sa cchari de compe ti tor . SDS gels of the sacchari de el uted fra ction revealed the only l abele d peak was the 37 K componen t . We conclude that the 37 K component is a membrane associ ated glyco­pro tein , wi th binding si tes for Con-A and R . communis , whi ch appears on the sperm surface during epi didymal transi t .

Supported by BSRG #RR- 05424 to Vanderbi l t Uni versi ty .

57

INH I B IT I ON OF SPERMATOGENES IS I N THE RAT BY TREATMENT W ITH [ 0-ALA6 , DES -GLY-NH2l D J LHRH ETHYLAMIDE . G . Pel ­l eti e r , L . Cusan , P . A . Kel ly , L . D�sy and F . Labri e , MRC Group i n Mo l ecu lar Endocri nol ogy , CHUL , Quebec .

A marked decrease of LH receptor l evel s i n rat testi s has recentl y been observed after one week of treatment wi th the LHRH agon i st [ 0-Leu6 , Des-Gl y-NH2 l O J LHRH ethy l ami de ( Aucl a i r et a l . , B i ochem . B i ophys . Res . Commun . 76 : 855 ( 1 977 ) . Th i s treatment was a l so accom­pan i ed b:Y-a decrease in testi s wei ght and pl asma tes ­tos terone l evel s . Si nce th i s l os s i n testi cul ar we i ght was l i ke l y to refl ect some defect i n spermatogenesi s , i t seemed i mportant to i nvesti gate the ce l l u l ar changes occurri ng i n rat testi s fol l owi ng the admi n i s trati on of [ 0-Al a6 , des-Gl y-NH2l O J LHRH ethyl ami de , a LHRH agoni st of potency s i mi l ar to the above-menti oned pept i de ana­l og . We th us s tudi ed the effect of thi s agoni s t ( l OOng twi ce a week fo r up to e i ght weeks ) on testi cul ar mor­phol ogy and testi c u l a r receptor l evel s . After two weeks of treatment , s i gn i fi cant degenerati ve changes coul d be observed i n many semi ni ferous tubul es . More dramati c changes were observed after four weeks of treatmen t , a l most a l l tubul es exhi b i t i ng s i gns of ma r­ked regres s i on . At· ei ght weeks , a l l tubu l es showed advanced degenerat i ve c hanges and very few spermatozoa coul d be detected i n the epi di dymi s . At a l l ti me i n ­terva l s s tudi ed , no s i gn i fi cant morphol og i cal change coul d be detected i n the Leyd i g cel l s . I n a l l treated groups , a marked l oss of testi cul ar LH receptor l evel s and decreased ( up to 60% ) testes we i ght were observed . Al though the mechani sms by wh i ch admi ni strati on of the LHRH agon i st i nduces degenerati on of the semi n i ferous tubu l es l argel y rema ins to be el uci dated , the changes observed i n the rat and the rel ati ve l ack of therapeu­ti c s uccess obtai ned wi th LHRH i n i nferti l e men suggest that deta i l ed fundamental and c l i n i cal stud i es are re ­qui red i n order to obta i n optimal response to treatment wi th LHRH or i ts agon i s ts .

5 8

RECE PTOR B I NDI NG AND ENZYME INDUCI NG ACTIVITY OF R 1 8 8 1 (METHYLTRIENOLONE) IN PSEUDOMONAS TE STOSTERON I . Ake Pouset te and Kj e l l Car l s t ro m . Department of Chemis try , K aro l inska Inst itutet and Department of Obs t et ri cs & Gyneco logy , S abbat sberg Hospital , Stockh o l m , Sweden .

R 1 8 8 1 ( methylt rienol one) was shown to increas e s t e ­ro i d � 1 - dehydrogenase act iv ity when added t o cul tur e s of Ps eudomonas testo steron i at concentrat i ons of 1 0- l O - l o -B M . When incubated with a s o l ub l e e xtract o f P . testos teron i , [ 3H]R 1 8 8 1 b ound t o a macromo l e cu l e w i th high affin ity ( Kd 0 . 6 x l 0- 9 M) and l ow capa city (num­ber of binding s i t e s 1 20 x l 0- 1 5 mol es /mg of pro tein) . The [ 3H]R 1 88 1 macromo l ecule comp l e x was p art ial ly de stroyed fol l owing treatment w i th proteas e , was pre ­cip i tated by addi t i on of ammon ium s ulphate to 20% o f saturat ion , s e dimented at 6 . 3 S both in 0 . 01 and 0 . 4 M KCl so l ut i on s , and had an i s oe l e ct ri c point of 6 . 3 . The comp l e x was par t i a l l y bound to DNA- ce l l ulose . Analys i s by sucro s e gradient centri fuga tion indi cat e d that n e ith e r [ 3H]testost erone , [ 3H]R 5 0 2 0 , [ 3H]es tra­diol - l 7� n or [ 3H] cort i costerone is botmd w i th h igh affinity t o the [ 3H]R 1 881 - b inding macromo lecul e . I t may b e sugges ted that the partia l ly characterized R 1 88 1 -b inding macromo l ec ul e , whi ch at l east in certain respe cts resemb l es androgen receptors des cribed in mammal i an ce l l s , may be invo lved in the inductive effect of R 1 8 8 1 on the � 1 - dehydrogenas e activity in P . testos teroni .

59

ANDROGEN �:;YNTHESIS IN THE BABOON TESTIS . J . P . Preslock. Dept . Reprod . Med . & Biol . , U Texas Med . Sehl . Houston , Texas .

The testis of the baboon Pap io anubis converts pregne-­nolone to t estos terone primarily through the 6 4 pathwa� The following s tud ies were undertaken to det ermine the metabolism of s teroid intermed ia tes of the 64 and 6 5

pathways . Testicular fragments ( 5 0 mg) from mature ?apio anub is were incuba ted with 10 µCi of the following 3H-labeled substrates : pregnenolone , 17a-hydroxypregne­nolone , proges terone , 17a-hydroxyproges terone , dehydro­ep iandrosterone , androstened ione , or tes tosterone . In­cuba t ions were in duplicate for 3 hr in fort ified Krebs­Ringer b icarbonate buffer , pH 7 . 4 . Me tabolites were iso­lated by chromatography , with ident ity and % conver s ion det ermined by recrys tall izat ion . Pregnenolone was con­ver ted to testost erone ( 1 . 1%) primarily through the 6 4

pathway with ac cumulation of prog esterone (30 . 5%) , 1 7a ­hydroxyproges terone (19 . 3%) , 20a-d ihydroproges terone ( 24 . 2%) and andros tenedione ( 1 . 5%) . Proges terone was metabolized primarily to 1 7a-hydroxyproges terone (23 . 7%) and 20a-d ihydroproges terone ( 3 6 . 3%) with 29 . 5% of the s ub s trat e not metabolized . Tes tosterone was 1 . 1 % and androst ened ione was 1 . 9% . 1 7a-Hydroxyproges terone was no t wel l me tabol ized as 86 . 5% of the sub s trate remained with testos terone ( 2 . 0%) and androstened ione ( 5 . 7%) identif ied in the incubates . However , 17a-hydroxypregne­nolone was effic iently conver ted into testosterone ( 28 . 2 %) and andro s tenedione ( 6 2 . 1%) . Dehydroepiandros­t erone was also ef f ic iently converted into testos terone ( 27 . 9%) and androstened ione (59 . 8%) . Androstenedione was converted into testo sterone ( 8 6 . 9%) , while tes tos­terone was poor ly metabolized into androstenedione ( 4 . 9%) . A subs tant ial por t ion of the testosterone ( 8 7 . 1� was not metabol ized . These results suggest tha t although the b ab oon tes tis effic iently converts 6 5 intermediates into tes tos terone , the 64 pathway may be predominant apparently because pregnenolone is no t effic iently con­verted into 17a-hydroxypregneno lone . Suppor ted by NICHD Grant P50 HD08 3 38 .

IN VITRO STIMUIATION OF RABBIT SPERM MOf ILITY BY l.r­ARGININE , E . W. Radany , R . W . Atherton . Department of Zoology-Physiology , University of Wyoming , Laramie , Wyoming .

60

In vitro Lr-Arginine stimulation of rabbit spenn rro­tility was quantitated at 22°c by a spectrophotanetric technique based upon the optical anisotropic properties of spenn in a flow. Stimulation of sperm rrotility was shown to be a function of in vitro age of the semen , concent ration of Lr-Arginine, and seminal plasma glucose concentration . A dose response stimulation by Arginine ( 1 , 5 , 10 , 50 , 100 mM ) of spe:rm rrotili ty was correlated to both the in vitro age of the serren and to the initial spe:rm rrotility . Maximum stimulat ion

· of rabbit spenn

rmtility occurred at 100 mM Arginine and spe:rm samples showing high initial rrotility showed less stimulation by Lr-Arginine than did samples exhibiting poor initial rrotility . Stimulation of spe:rm rrotility by Lr-Arginine increased significantly with in vitro age of the serren . As an example , at 3 , 4 , and 5 hours of in vitro age , 100 nM Arginine stimulated spenn rrotility 225%, 340%, and 500%, respect ively , over control values . By twenty hours of in vitro age , semen no longer exhibited spe:rm rrotility but when treated with 100 nM Arginine spe:rm motility was restored to initial control values . In all cases Arginine stimulation of rrotility occurred within 1 to 3 minutes and maintained rrotility for a minimum of 30 minutes after treatment . No ultrastructural changes were apparent in 100 nM Arginine treated spe:rm. Arginine stimulation was also shown to be correlated to seminal plasma glucose concentration . Seminal plasna glucose concentration and spenn rrotility decreased as a function of in vitro age but . the ability of Arginine to stimulate spenn rrotility increased. 'Ihese studies sup­port a hypothesis that as available glucose decreases in semen , sperm have the ability to ut ilize Arginine as an energy source to maintain rrotility . This observation that L-Arginine significantly increases rabbit sperm rmt ility suggests that it may be of clinical importance in dealing with spe:rm of subno:rmal rrotility . Supported by the Division of Basic Research , College of Arts and Sciences , Univ. Wyo .

61

EFFECTS O F PASS IVE IMMUNI TY TO F S H O N MALE REP RODUCTION I N THE IMMATURE AND ADULT RAT . H . G . Madhwa Ra j * , M . Dym* * , H . E . Cheme s * * , N . J . Kot i te * , S . N . Nayfeh * , and F . S . French * . Departments of Obstetrics and Gyneco logy , Pediatri c s , and Biochemi stry , Univers i ty o f North Carol ina Schoo l of Medic ine * , Chapel H i l l , NC and Department of Anatomy , Harvard Med i c a l School * * , Boston , MA .

Anti s erum to rat FSH has been prepared in rabb its and made monospec i fic by sui table ab­sorption . I t b inds rat I l 2 5 FSH , but no t I l 2 5 LH , and i s bio logica l ly active a s de termined by the HCG augmentation a s s ay . Ef fec ts of se­lect ive neutra l i z ation of FSH in the immature and adul t rat were inve stigated using th is an­t i s erum . Administrat ion o f the antiserum to immature rats from 2 0 to 3 4 days of age de­creased the te s t i s wei ght by 5 0 % without any reduct ion in we ights o f epididymi s and sex ac­c e s sory organs or serum testos terone leve l s . The sma l l er te s ti s was due to a 2 0 % reduction in the s eminif e rous tubule diameter and a loss o f pachytene s permatocytes and spermatids . There was a l so a 5 0 % reduction in the leve l s o f and rogen binding protein (ABP ) in the epi­didymi s a fter FSH was neutra l i zed for 14 days . Fol lowing administra tion o f antiserum to FSH to adu l t rats ( 9 0 day old ) over a 2 -week per i­od , prel iminary results s ugge st that there i s a s l ight reduc tion in te s t i s we i ght , germ cell number s , and ABP leve l s in testis and epididy­mi s . The se observat ions indicate a relative ins ens itivity o f the testis to wi thdrawal o f F S H in the adult rat , when compared t o the im­mat ure rat . Long term treatment o f adult rats with FSH anti s erum is in progress to de termine whe ther furthe r reduc tions in the above mea s ur ed parame ters wi l l be found . ( Supported in part by N ICHD grant No . HD 1 0 0 0 4 ) .

6 2

OCCURRENCE AND B I OLOG ICAL ACTIV ITY OF A H I GH MOLECULAR WEIGHT ANTI FERT I L I TY AGENT IN HUMAN SEMI NAL PLASMA . J . M . Reddy, R . A . Stark and L . J . D . Zanevel d . Department of Phys i ol ogy and B i ophys i cs and Department of Anatomy , Un i vers i ty of I l l i no i s at the Med i ca l Center , Chi cago , I l l i no i s .

Semi nal p l asma conta i ns a h i gh mol ecu l ar wei ght anti ferti l i ty compound termed " Decapaci tati on Factor" ( OF) . The obj ecti ve of th i s s tudy was to veri fy the presence of OF in human semen and to determi ne s ome of i ts phys i o l og i ca l acti ons . Spermatoz oa and other part i cu l ate matter were removed fran human sem i na l p l asma by l ow s peed centri fugat i on fi rs t at 2 , 500 rpm and subsequent ly at 6 , 000 rpm . The resu l t i ng s uper­natant was then centri fuged at 104 ,000 g for 4 hours to obta i n a pel l et of h i gh mol ecul ar wei gh t materia l . The anti ferti l i ty acti v i ty of thi s prec i pi tate was eva l u ated i n a mouse in vitPo ferti l i zation sys tem uti l i z i ng capaci tated s permatozoa i ncubated wi th the prec i pi tate . A s i gn i fi ca nt decrease i n ferti l i ty was obta i ned wh en treated s permatozoa were added to i ntact ova as compared to untreated c ontrol s , s howi ng the presence of OF i n the prec i p i tate . OF a l so reduced ferti l i ty when the fo l l i c l e ce l l s were d i s persed from the ovum keepi ng the zona pel l uc ida i ntact . However ; s perm-ovum fus i on occurred norma l l y when a l l the l ayers surround i ng the ovum were removed . Thus human OF appears to act by preventi ng sperm penetrati on through the egg i nvestments , but has no effect on the fus i on of t he spermatozoon with the v i tel l i ne membrane . Was h i ng treated spermat ozoa redu ced the anti ferti l i ty effect of OF but d i d not el imi nate i t , i nd i cati ng that OF cl osely adheres to spermatozoa . Human OF i s heat l ab i l e and i ts acti vi ty i s dose dependent . Thi s s tudy a l so shows the usefu l ness of the mouse in vitPo ferti l i zati on system as an assay method to mon i tor the puri fi cat i on of OF from human semi nal p l asma . Supported by N I H HD 09868 .

63

IN V ITRO TESTI CULAR STEROID f 1ETABOL ISM AND PERI PHERAL HORMONE LEVELS IN INFERT I LE MEN . L . J . Rodriguez-Rigau , D . B . We i ss , K . D . Smi th and E . Ste i nberger . Dept . of Reproduct i ve Med i c i ne & B i ol ogy , U . T . Med i cal School at Houston , Texas .

In 24 con secut i vely s tud i ed i nferti l e mal es the pat­tern of i n v i tro pregneno l one metabo l i sm was correl ated with per i pheral l evel s of testosterone ( T ) and LH , and compared to s i mi l ar data in normal s . The i nfert i l e men were arb i trari l y d i v i ded i nto 2 groups : 1 6 pat i ents wi tll T l eve l s above 400 ng% ( group A) and 8 pati ents wi th T bel ow 400ng% ( group B ) . Metabol i sm of pregnenol one was d i fferent i n the 2 group s . I n grou p . B total s tero i do ­genes i s ( a l l i ntermedi ary metabol i tes ) was depres sed and testosterone synthes i s was d i mi n i shed when compared to norma l and to subjects i n group A . In the l atter tota l stero i dogenes i s �1a s i ncreased above norma l , i nter­med i ates were ma rkedly i ncreased , wh i l e testosterone and androstened i one were i n the l ow-normal range . Pl a sma LH l evel s were bel ow normal i n group B and above normal i n group A . H i stol ogi c eval uat i on of the b i ops i es showed i ncreased numbers of Leydi g eel l s i n group A . T:1ese dat1� suggest that the 2 groups may be exampl es of d i s s imi l ar pathophys i o l ogy . I n group B , the marked ly decreased stero i dogenes i s and androgen product i on , but no change i n pattern of stero i d metabol i sm wh i ch wa s associ ated with s l i ght , but s i gn i f i cant , decrease i n peri phe ral T and LH l evel s suggests poss i bl e hypotha l ami c-p i tu i tary eti o l ogy for the dysfunction . Pati ents i n group A , on the other hand , s howed i ncreased s tero i dogenes i s but no i ncrease i n androgen product i on . Th i s was associ ated with s i gn i fi cant i ncrease i n pl asma LH l evel s suggest­i ng testi cul ar , probably Leydi g cel l , eti o l ogy for the dys funct i on . In th i s group 7 of the 1 6 pat i ents showed a spec i fi c and i so l ated defi c i ency of 3 S-hydroxystero i d dehydrogenase . I n 5 of the rema i n i ng 9 subjects 1 7-20 l yase acti v i ty was decreased . These resul ts demons trate that i n s p i te of T and LH l evel s \'Ji t h i n normal range , Leyd i g cel l dysfunct ion and hypothal ami c-pitui tary d i s ­turbances may be present , and a di agnos i s can be made when other parameters are a l so i n ves ti gated . Supported in part by NIH Grant HD-08338 and Ford Foundation.

64

AN IMAL EXPERIMENTS ( RATS ) FOR REVERS IBLE VAS OCCLUS I ON WITH DI FFER­E NT METAL DEVI CES ; A L I GHT- AND EL ECTRONMI CROSCOPI CAL EVALUATION. E . Rovan , T. Bende , H . Au l i tzky, and J . Fri ck . Uni vers i tat Salzburg, Zool ogis ches I nstitut , Sal zburg , Aus tri a .

U p to now vasectomy i s the most s ucces sful method for ma l e fer­ti l i ty control . Lee ( 1 976 ) refe rs , that i n the l as t few years 20 mi l l i on men have undergone spontaneous vasectomy .

There a re l i ttle di fferences between the various operati ng techni ques , and the compl i cation rate is l ow .

Two p robl ems a re sti l l unsol ved , the revers i bi l i ty, a nd the i mmunol og i ca l reacti ons concerni ng spermaggl uti ni ns and s permim­mobi l i s i ns .

P robably the demand for refe rti l i sation by vasovasos tomy wi l l i nc rease correspondi ng to the enl arged number of vasectomi zed per­sons . I n the report of Lee ( 1 976 ) and Al exander ( 1 976) the rate of pre gnanc i es after anastomos i s of vasectomized oati ents by vaso­vasos tomY was about 25% ( Al exander) to 33% ( Lee ) .

At thi s poi nt i t i s not enti rely clear i f th i s l ow p regna ncy rate i s caused by morphol ogi cal or functi onal al terati ons of the tes te s , epi d i dymi s and vas defere ns . The i nterrupti on i n the con­ti nui ty of the vas , the bl ockage of sperm, and the depre s s i on of s permatogenes i s mus t a lso be consi dered as causal factors . I n the l as t few years several authors have tried to so lve these probl ems us i ng vari o us devi ces (S i l i cones , Ta ntal umcl i ps , Metaloop s , etc . ) appl i e d to the vas . However, a temporary occl us i on o f the vas deferens coul d not be avoi ded , a nd the pathol ogi cal reac ti on of the vas epi thel i um , the sperm prol i ferati ng epi thel i um , a nd the epi di dymi s cel l l ayer remained the s ame as in the case of va­sectomy .

Cons i deri ng these experi ences , we made two cl a i ms o n the de­vi ces i n our experi ments to guara ntee l ) the ful l conti nui ty of the vas deferens throughout the whol e period of the reve rs i bl e s teri l i sati o n , and 2 ) the total sperm i mmobi l i sation .

We assumed that co i l s made of copper, s i l ver, or pl a ti num wi res woul d ful fi l l these two requi rements . The authors have then tested these dev i ces by i ncubation i n a human ejacu l ate , a nd found a s i gn i fi cant decrease o f the spermatozoa moti l i ty rate .

Duri ng our experiments we have impl anted these metal coi l s i nto the vas deferens of ferti l e mal e rats . I n mos t cases , thi s was done i nto one o f the ducts , the other one was secti one d . One week after the ope ration each ma l e was brought together wi th two fema les for 27 or 28 days . Afterwards the mal e s we re reoperated, and the di fferent ki nds of t issue reacti ons were reporte d . Whi l e si l ver and pl ati num coi l s show no reacti ons , the copper coi l s i nduced marked granul omatous reacti ons .

Several reoperated anima l s we re sacri ficed, and the h i s to­l ogica l s i tuation of the vas epi thel i um , the sperm p rol i ferati ng epi the l i um , and the epi di dymi s epi thel i um was exami ned by l i gh t­a nd el ectron mi croscopi cal techniques .

We ca n now demonstrate that i n the region of the meta l coi l (medi a n vas ) the epi thel i um i s damaged , whi l e i n front of thi s regi on and beh i nd i t the cel l confi gurati on i s comparabl e with con­trol s .

Therefore , we can assume tha t the conti nui ty of the vas deferens exi s ted o ver the whole period of steri l i sation ; neverthe­l es s , no femal es became preg nant .

The vas a of the rema i ni ng anima l s were reanas tomose d . Four days a fter reanas tomos i s they were brought together wi th fema l es aga i n and are sti l l unde r observati on .

65

EL IMINATION OF CYTOPLASM FROM THE SPERMATID IN THE HEAD REGION DURING LATE SPERMIOGENESIS . L . D . Rus sell Department of Phys iology and School of Medic ine , Southern Illinois Univers ity, Carbondale , IL 62901

The concept that a maj or portion o f the cytoplasm of the sp ermatozoan is eliminated pr ior t o its release int o the tubular lumen is widely accepted . The excess cytoplasm ( residual cytoplasm) is s eparated from the neck and p roximal f lagellar regions at the t ime of sperm r elease . Once freed this residual cytoplasm, now termed the res idual body , is transpor ted to the base of the seminif erous tubule where it is degraded by the Ser toli cel l . Ob servat ions f rom several mammalian species ind icate tha t , in addition to the process des­cr ibed above, cytoplasm is also eliminated from the spermat id at it ' s head region . Pr ior to the formation of the pos terior ring (area of contact of the nuclear and the p lasma membranes ) the cytoplasm surrounding the tail and neck regions of the spermat id is continuous with the cytop lasm around the head ( per inuclear cyto­plasm) . Long tubular proj ec tions of the spermatid with bulbous endings , the tubulobulbar complexes (Russell and C lermon t , 1 9 7 6 ) , form by invaginating into the Sertoli cel l . The material within the bulbous and tubular port ions of this complex is in d irect continu­ity with the cytoplasm surrounding the head of the sperma t id . Devo id of organelles it has an appearance similar to the perinuclear cytoplasm with which it is continuous . Tubulobulbar complexes which form for several days p rior to sperm release are continually phagocytized by the Ser toli cell . Af ter the regression of t ubulobulbar c omplexes the per inuclear space around the head is cons i derably reduced allowing a tight f it of the s perma tid plasma membrane and acrosome around the nuc leus . At the same time the volume of residual cytop lasm around the flagellum is subs tantially re­duced and as a result the cytoplasmic components wi thin this s t ruc ture become crowded . Thes e features s trongly sugg e s t a flow of "watery" cytoplasm from the tail region to the head reg ion whereupon this por t ion of the cytoplasm is extruded via tubulobulbar complexes . Support ed by NIH Grant HD11823-0l .

CHARACTERI STICS OF ca+2-DEPENDENT BINDING OF MOUSE SPERM TO THE ZONA PELLUC IDA OF THE MOUSE EGG . P . M . Saling and B . T . S torey, Dept s . of Physiology and Obstetrics and Gynecology , University of Pennsylvania S choo l of Medic ine , Philadelphia , PA 1 9104 .

6 6

Binding o f mouse sperm t o the zonae pellucidae of cumulus-free mous e eggs occurs in NaCl/Tris buf fer if the sperm are preincubated in the presence of 2 mM ca+2 but no t in i t s �bsence . Similar preincubation in the presence of La+ does n� t inhibit binding , and La+3 can subst itute f or 2 mM ca+ at 5 µM (Saling et al . [ 1977 1 Biophys . J . 1 7 : 27 6a) . EGTA reverses the effect of ca+2 and inhibits the ability to b ind , and this inhibition can in turn be reversed by the subsequent re-addit ion of ca+2 to the preincubation medium. The reaction con­f erring on the sperm the ab ility to bind is thus rever­s ible . It i s also rap id , requiring no t more than pre­incubat ion f or 10 min in the presence of ca+2 for vir­tually immed iate b inding upon the introduction of eggs . Replacement of ca+2 by Mg+Z leads to decreased binding ab ility . Moreover , Mg+2 has an inhibitory effect on the Ca+2-dependent b inding reaction . Spermine substi­tutes for c a+2 at 2 mM and s ignificantly enhances b inding when sperm are preincubated in spermine plus ca+2 • Attemp ts to assess the effect of zn+2 on the b inding react ion were thwarted by the cytotoxicity of this cation toward mouse sperm : binding is observed , but the sperm membrane s appear damaged at a zn+2 con­centration o f 17 µM. The reversibility , rap idity and ionic selec t ivi ty of the ca+2-dependent binding reaction imply that this is a plasma membrane surface reaction which precedes the proces ses leading to the acrosome r eaction . Supported by USPHS HD-062 7 4 and GM-00205 , and NSF PCM-7 7-1596 5 .

67

RESPONSE OF RAM TESTIS TO CLOMIPHENE AND GONADOTROP I N RELEAS I NG HORMONE . B . D . Schanbacher . Reproducti on Uni t , U . S . Meat An imal Research Center , ARS-USDA , Cl ay Center , NE .

Cl omi phene c i t rate ( C l om i d ) and gonadotrop i n rel eas ­i ng hormone ( Gn RH ) stimul ate gonadotropi n secreti on and testi cu l ar funct i on i n normal men . S imi l ar i nformati on was l ac k i n g for the ram ; therefore , concentrati ons of serum testosterone (T ) were determi ned duri ng Apr i l i n mature cros sbred rams ( approx . 1 20 kg ) whi ch recei ved i ntramuscul ar i nj ecti on s of Cl omi d and Gn RH .

Dose- res pon se data were obtai ned from 25 rams after rece i vi ng dai l y i nj ecti ons of Cl omi d for one week . Groups of rams ( fi ve eac h ) were i njected wi th ei ther sa l i ne ( I ) , 0 . 1 mg/da ( I I ) , 1 mg/da ( I I I ) , 1 0 mg/da ( I V ) , or 1 00 mg/da ( V ) . Testosterone concentrat i ons i n serum samp l es col l ected at 1 , 4 , 8 , 1 2 and 2 4 h after the l as t i nject i on d i d not s how a temporal rel at i on s h i p ( i ncrease or decrease ) rel ati ve to the t i me o f C l omi d i nj ecti on . Reduced ( P<0 . 05 ) concent rati on s of T i n rams recei v ing 1 00 mg/da d i d s uggest , however , that T secreti on may be s uppres sed by h i gh dosages of Cl omi d . Means ( ±SEM) for serum T i n groups I - V respect i vely were : 3 . 2±0 . 3 , 2 . 8±0 . 2 , 3 . 3±0 . 5 , 2 . 4±0 . 3 and 1 . 8±0 . 4 ng/m l .

Serum T was h i gher ( P<O. l ) i n ei ght rams rece i vi ng twi ce da i l y 0 . 05 mg i nj ecti ons of Gn RH ( 4 . 5±0 . 5 ng/ml ) than i n seven rams i njected wi th sal i ne ( 2 . 8±0 . 8 ng/ml ) . To determi ne the effects o f chroni c GnRH admi ni stra­ti on , twi ce da i l y i nj ecti ons were conti nued i n these 1 5 rams . After 60 days of treatment , serum T remai ned h i gher i n GnRH treated rams ( 7 . 8±0 . 8 vs 3 . 9±0 . 6 ng/ml , P<0 . 01 ) . Not on ly was an i ncrease i n"""'"fhe stero i dogen i c capaci ty o f the GnRH stimul ated testi s apparent , but a 1 5% i ncrease ( P<0 . 01 ) i n scrotal c i rcumference (only 2% i n sal i ne control s ) and 1 3% greater sperm numbers i n semen samp l es o f treated rams suggested a para l l el i n ­crease i n t hei r spermatogen i c capac i ty . H i stol ogi cal assessment and cal cu l ati on of da i l y sperm producti on rates ( 1 8 . 9± 1 . 8 x 1 09 vs 1 5 . 0± 1 . 0 x 1 09 ; P<0 . 05 ) veri ­fi ed a greater spermatogen i c yi e l d i n rams chron i cal l y treated w ith Gn RH .

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S EMEN COLLECTION AND EVALUATION I N SOME SPECIES O F CAP­TIVE WILD FELIDAE . S . W . J . SEAGER and C . C . PLATZ . Ins titute of Comparative Medicine , Baylor Co llege of Medicine , Hous ton , Texas .

Obj ectives of the study were to investigate t echni­ques of s emen collection , preservation and artific ial inseminat ion in breeding of zoo animals with special emphas is placed on selec ted endangered species . The maj ority of ej aculates were collected by electroej acu­lation after the animals were anes thetized . After in­sertion of the appropriate size probe into the rectum electrical s timulation was applied . The rectal probes used in this proj ect consis ted of s t�inless s teel or aluminum electrodes of varying numbers , lengths and diameters . Probe d iameter varied from 1 to 2 . 5 cm . , depending upon s ize of animal . A small number of the animals were collected by manual s timulation using the artificial vagina . This lat ter method was used in the t iger , ocelot , and leopard . Once an ej aculate was ob­tained , it was firs t evaluated for the following param­eters : color, viscos ity , pH , volume , and presence of spermatozoa . The ej aculate was then evaluated for percent motility , speed of progress ion on a scale of z ero to five (five being opt imal) , sperm count per ml . , total sp erm count and morphology . The following species were collect ed : African lion (Panthera leo) , Puma (Felis concolor) , North Chinese leopard CPanthera

ardus j aponensis) , Canadian lynx (Felis � canaden­is) , Bobcat (Felis rufa) , Temminck ' s Golden cat (Felis emminckii ) , Ocelot (F;iis pardalis) , Geof froy ' s cat

(Felis geoffroyi ) , Clouded leopard (Neofelis nebulosa) , engal t iger (Panthera tigris tigris ) , S iberian tiger

(Panthera t igris altaica) , Leopard (Panthera pardus ) , aguar (Panthera onca) , African cheetah (Acinonyx

" ubatus ) , Leopard cat (Felis bengalensis) , and Margay (Felis wiedii) .

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PERIFUS ION OF LEYDI G TUMOR CELLS : HCG EFFECTS ON S TE RO I DOGENE S I S . Deborah L . Sega lo f f , Mar i o Asco l i , and D avid Pue tt . Department o f Biochemi s try , Vande rbi lt Un ive r s i ty , Nash­vi l le , TN .

The transplantable M5 4 8 0 murine Leydig tumor has retained re sponsivene s s to gonado ­t rop in s , �' human choriogonadotropin ( hCG) and lutropin , unde r in vitro condition s . The ce l l s , which c an be di sper sed wi thout enzymic tre a tment , contain app roximate ly 2 0 0 0 recep ­tor s e ach ; the interaction wi th hCG i s highly spe ci f i c and i s characte ri z ed by an apparent �i s soci ation constant o f S x 1 0 - l l M . A sig­n i f i c ant increase is observed in proge s te rone stimul ation fol lowing hCG binding .

A peri fusion system has been deve loped in whi ch the tumor ce l l s are interdi sper sed wi th polyacry l amide beads in a Bio-Ge l P - 2 column equi l i br ated and deve loped with Medium 1 9 9 containing 0 . 1 % bovine se rum al bumin at 3 7 ° C . Expo sure o f the ce l l s to hCG ( 2 ng/ml ) for 1 0 min le ads to a rapid incre ase in proge sterone product i on . Upon removal o f the hormone from the pe r fu s ing medium , proge sterone stimula­t ion , a s de termined by radio immunoa s s ay o f the perfusate , i s detectable within 1 0 min and a (maximum ) plateau occurs between 9 0 and 1 3 0 min ; thi s i s fol lowe d by a s low return to the b a s al leve l . A do se-response c urve can be obtained us ing various hCG concentrations and me a suring the re sulting s t imulation o f stero i dogene s i s .

Thi s sys tem provi de s a new approach for studying gonadotrop in actions under dynami c condi t ions using a unique hormonal ly-re spon­s i ve te sticu l a r tumor . ( Supported by AM1 5 8 3 8 and RR05 4 2 4 ; D . L . S . acknowl edge s Training Grant HD 0 7 0 4 3 and D . P . i s the recip ient of an RCDA , AMO O OS S . )

70

FUNCT I ON OF A HUMAN TESTICLE AFTER TRANS­PLANTAT ION TO AN ANORCH I C I DENTICAL TWIN BROT HE R . S . J . S i lber . Department of Urology , S t . Luke ' s Hos pi tal-West , S t . Louis , Mo .

One o f two 3 0 year old , gene tically i denti­c al twins had b een born w i th two norma l tes t i ­c l es and th e oth er with none . I n the anorch i c twin preoperative s erum F S H and LH leve l s were extreme ly h i gh , and serum tes tos terone was e xtreme ly low . In the twin with two tes t i c l e s however , preoper ative s erum FSH , L H and tes­tos terone leve l s were normal .

After success ful transplantation o f a tes ticle from th e twin with two tes ticles to the twin w i th no tes ticles us ing mi crovas cular techn ique , the donor twin showed no s i gni fi­c ant a l teration . The recipient twin developed a normal serum tes tos terone wi th in two hours o f surgery , b ut FSH and LH levels came down to a normal range more s lowly over th e ensuing four weeks . Therea fter , hormone leve l s rema ined pers is tently normal in both twins .

Pre- and pos toperative sperm counts were normal in the donor . Sperm count o f th e recipient went up ini ti a l ly ( sperm produced wh ile tes ticle was in donor ) , dec lined over the next s everal months , and then began to r ise a gain .

71

A NEW MICROSCOPIC TECHNI QUE FOR VASOEP I D I DY­MOSTOMY : A P RELIMINARY REPORT . S . J . S i lber . De partment o f Uro logy , St . Luke ' s Hospital­We s t , S t . Louis , Mo .

Ten patients wi th epid idymal ob s truction c au s i ng azospermia we re sub j ec ted to a new mi cros copic te chnique for vas oepididymo s tomy . Nine out o f ten now h ave s perm in the ej acu­late pos t-operative ly . One remains a zo­s pe rmi c . O f th e 9 0 % with s pe rm, 7 0 % have norma l sperm counts and norma l mot i l i ty and the o ther 2 0 % are too early po s t-op ( 3 months ) to know whe ther the i r spe rm count wi ll improve . It is also too early to know what the fert i l i ty rate w i l l b e .

The technique cons i s ts o f a direct mi cro­ana s tomo s i s o f the inner lumen of th e vas de ferens d i re c tly to the epididyma l tubule . The outer mus c le l ayer o f the vas de ferens is then s epara te ly s utured to the outer tunic of the epididvmis . Conventional vasoepididy­mo s tomy would s imp ly inc i s e the tuni c of the epid idymis , cut into the convoluted tubule in a s trai gh t line ( thus dividing it helter skel ter in many areas ) , and hope th at s perm wou ld leak out o f the uppe rmo s t c ut of the tub u l e (becaus e it ce rtai nly couldn ' t l eak ou t o f any o f the other tubu les ) and form a f i s tula .

The new mi croscopi c technique is more pre c i s e and ana tomic , and wi l l provide an i ntere s ti ng s e ries for s tudying the e f fects o f var ious leve l s of the epidi dymi s on fe rti l i ty o f s perm in the human .

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EVIDENCE THAT Sa-REDUCTION OF 6 4 - 3-KETOSTEROIDS MAY BE MORE IMPORTANT F OR THEIR ANDROGENIC THAN LR- INHIBITING ACTIVITY . R . E . STEELE , F . DIDATO, and B . G . STE INETZ . Depar tment o f Metabolic Diseases, C iba-Geigy Cor p . Ards ley , N . Y .

The synthetic anabolic steroid methandro s t enolone (M; 17�-hydroxy-17a-me thyl-andro sta- 1 : 4-dien-3-one ) cannot b e me tabolized to its androgenically more potent Sa­reduced form (17�-hydroxy- 17a-methyl-Sa-androst-l-ene-3-one ) . M d ecreased ventral pros tate (VP ) weights and plasma test o s terone ( T ) concentrations in intac t rat s and lowered LR concentrations in cas trated rat s ( S teele et al , S teroids 29 : 33 1 , 19 77 ) . I t w�s pos tulated that M

- -

reduced VP weights by indirect ly lowering plasma T via its e ffect o n LR . Because M does have weak androgenic activity, this hypothesis presupposes that the LR- inhib i ting activity o f M relative to it s androgenic act iv­i ty on the VP must be greater than that of the endoge­nous testicular androgens being secreted� predominant ly T . The pres ent s tudy was designed to compare the rela­t ive LR- inh ibiting to androgenic act ivity o f M and T . Male rat s were cas trated and 7 days later inj ected sc for 7 days with ei ther 4S O , 300 , or lSO µg T/100 g bwt or 13SO , 9 00 , or 4S O µg M/100 g bwt . Twenty- four hour s after the l a s t inj ection the rat s were anes thetiz ed, . bled by cardiac puncture , and killed . The VP were ex­c ised , weighed , and assayed for DNA, RNA, and pro tein content . The plasma was assayed for LR . For do ses of M and T having comparab le LR- inhib it ing activity , the androgenic ac tivity o f M was significantly less than that of T; For example :

Group LR VP ng/ml mg/lOOg

Castrate

DNA µg/lOOg

RNA IJg/lOOg

Protein mg/lOOg

Control s 32S 10 41 4S 1 . 08 M 900 µg llS 2 6 S 8 174 2 . 3 1 T 300 µ g 1 0 4 4 6 7 4 441 3 . 90

� These findings support the above mentioned hypothe sis , and further suggest that the dis parity between the rela ­t ive LR- inhib iting and androgenic ac tivities of M and T may result from the lack o f Sa reduc tion o f M.

7 3

BOVINE SPERM CYCLIC AMP PHOSPHODIESTERASE : PHYSICAL PROPERT IES AND DEVELOPMENTAL CHANGES . D . T . Stephens , Jun-lan Wang, and D . D . Hoskins . Reprod . Phys . Dep t . , Ore . R eg . Primate Res . Ctr . , Beaverton , Oregon .

The involvement of cyclic AMP in the ini tiation of bovine sperm mot ility has been sugges ted by the observ­a tions that cyclic AMP phosphodies terase (PDE) inhibit­ors induce mo tion in sperm from the caput epid idymidis , marked incr eases in cyclic AMP levels oc cur in caudal sperm when activa ted by dilut ion , and more of this nucleotide is found in caudal than in caput sp erm . This s tudy was undertaken to see if changes in the forms or proper t ies of PDE during ep ididymal d evelop­ment ar e related to the lat ter event . Properties of the caudal enzyme were first describ ed . The enzyme has been shown to be largely particula te ( 67%) following sonic irradiation and centrifugation of cell suspens­ions . The soluble fraction displays subs trate satura­t ion kinetics indicative of 4-5 multiple forms . The exist ence of these forms was confirmed by separation on Bio-Gel A-0 . 5m and flatbed isoelectric focus ing in the pH range 3-7 (pl values ; 4 . 0 , · 4 . 1 , 4 . 5 , and 5 . 0) . The particulate enzyme disp layed class ical M-M kinetics while each soluble iso focused form showed curvilinear reciprocal plots sugges tive of negative cooperativity . Caput sperm PDE shared many of these proper ties . I t is only s lightly less par ticula te ( 51%) and the soluble forms are also s eparable by isoelectric focusing into 4 fract ions with s imilar pI ' s and anomolous kinetic s . The latter prop erty precludes comparison of kinetic cons tants of caput and caudal enzymes . However , spec­i fic activities of each focused caput form were higher at physiological cyclic AMP levels . In addition , in­tact c ell PDE activity is twice as great in capljt as in caudal sperm ( 3 2 . 5 ± 4 . 2 vs 15 . 5 ± 4 . 2 units/10 sp erm , n=l2) . Similarly , who le caput sonicates con9ain more activity (56 . 4 ± 0 . 7 vs 41 . 2 ± 0 . 3 units/10 sperm) . Thes e resul ts sugges t that cyclic AMP levels increase 4 in developing ep id idymal sperm because of a decrease in PDE l evels rather than chang es in the nature of the enzyme . Supported by NIH grant HD05969

LACTATE , PYRUVATE AND MALATE AS OXIDATIVE SUBSTRATES FOR RABBIT SPERM MITOCHONDRIA . B . T . S torey, and

7 4

F . J . Kayne , Depts . o f Obstetr ics and Gynecology , Physi­o logy , and B iochemis try and Biophys ics , Univer sity of P ennsylvania School of Medicine , Philadelphia, PA 19104 .

Rabbi t sp ermatozoa have lactate dehydrogenase (LDH) in the matrix of the ir mitochondria , whi ch enables them to us e lactate directly as oxidizable substrate (S torey and Kayne ( 19 7 7 ] Bio l . Reprod . 16 , 549) , but also allows pyruvate to be an oxidant for intramitochondrial NADH , which should inhib it Oz uptake . The interact ions between lactate , pyruvate and malate as mito chondrial substrates h ave been examined in hypo tonically treated rabbit epididymal spermatozoa (HTRES) by measuring Oz up take rates polarographically and reduction levels of intramitochondrial NADH fluorimetrically . Pyruvate is a potent inhib itor of lactate oxidation ; the inhib ition is overcome by malate . Pyruvate alone is oxidized s lowly , but in the presence o f malate is oxidized rap­idly . Lactate alone is oxid ized rapidly , but malate is needed f or maximal Oz uptake . Intramitochondr ial NAD is highly reduced with lactate as subs trate , and the reduction level is further increased by added malate . Pyruvate reoxidizes intramitochondrial NAD reduced by lactate while it inhibit s o2 uptake ; malate partially restores the NAD reduction while it reversed the inhib­ition . Mala te plus pyruvate maintain a level of NAD reduction suff icient for rap id Oz uptake . Pyruvate is consumed anaerobically by rabbit sperm mitochondria by d isproport ionation to lactate , COz and acetyl CoA (Terner (195 9] Biochim. Biophy s . Acta 36 , 479) , a reac­t ion which is the sum of the reactions catalyzed by pyruvate dehydrogenase (PDH) and LDH . It can occur only in mit ochondria which have intramito chondr ial LDH with access to the same pool of NAD as does PDH . Suppor ted by USPHS HD-062 7 4 , K04-GM-702 4 4 , and NSF PCM-74-1 2358 .

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THE A CROSOME REACTION IN BOAR SPERMA TOZOA . Daniel Szol l osi and R . H . F . H unter, Sta tion centrale de Physiol ogi e anima l e , I . N . R . A . , 78350 Jouy-en-Josas , France , School of Agricul ture , Uni versi ty of Edinb urgh , Scotl and.

The pol yspermi c condi tion created in the domes ti c pi g allows a recons truction of vari ous de tails of the acro­some reacti on a s i t occurs in the na tural envi ronmen t for ferti li zati on , after ascen t of sperma tozoa along the oviductal i s thmus and entry into the ampulla . These events are :

1 . Sli ght swel ling of the acrosome , 2 . Loss of free acrosomal con tents (hyal uroni dase)

before reaching the zona whi l e the sperm mem­branes are s ti l l apparently intact ,

3. Ini ti a ti on of vesi culation a t the apex of the sperma tozoon of the s urfa ce of the zona pell uci da �

4 . Compl e ti on of vesicul a tion aro und the acrosome reaching the equa torial segment whil s t on the s urfa ce of the zona ,

5 . Denuda ti on of the sperma tozoon from i ts membrane vesi cl es as i t en ters the ma tri x of the zone , and

6. Secondary vesi c ul a tion a t the equa tori al segmen t (a crosome col lar) in the more compact portion of the zona .

These observa tions on sperm head modi fi ca tions es­tabli sh a para l l elism be tween the res ul ts under in vi vo condi ti ons devel oped for these e xperi ments and those reported pre vi o usly under in vi tro condi tions . Because of the close parallel , it can be concl uded tha t the sequence of membranous events i n the two si tuations is very s imilar or identi cal .

76

THE E FFECTS OF LOW ORAL DOSES OF 2 , 6-CIS-DI PHENYLHEX­AMETHYLCYCLOTETRASI LOXANE ON MALE RATS AND RABB ITS . C . D . Taras e k , K . J . Zi mmerman , T . A . A ire , and B . G . Crabo , Depar tment of Animal Sci ence , Uni ver s i ty � Mi nnesota , S t . Paul , MN 5 5108

The contracepti ve effect of dai ly oral doses of 2 , 6-c i s-di phenyl hexamethyl cycl otetras i l oxane ( 2 , 6-ci s ) i n corn oi l was i nvesti gated i n rats and rabbi ts . Fer­ti l i ty was decreased by 1 . 0 and 2 . 0 mg/ kg/day and ceased by 5 . 0 mg/ kg/day i n rats after 2 weeks on the drug . A drug-free per i od of 2 weeks after the l ower doses and one of 5 weeks after 5 . 0 mg/ kg/day restored ferti l i ty to pre-drug l evel s . I n rabbi ts , 1 . 0 mg/ kg/ day i nh i bi ted and 0 . 5 mg/ kg/day l owered ferti l i ty . Long term stud i es of l i b i do , p l asma tes tos terone , and s emi nal parameters reveal ed that l i b i do was probably not depressed by 0 . 1 - 1 . 0 mg/ kg/day al though the pl asma tes tos terone l evel s were l owered at 14 , 28 and 3 5 weeks by 1 . 0 mg/ kg/day of the drug ( P<0 . 025 ) . A dose of 0 . 5 mg/ kg/day l owered peri pheral tes tos terone at 14 a nd 28 weeks ( P< 0 . 10 ) but the l owest dose was wi thout effect . The hi gh dose resul ted i n decreas ed s emi nal vol ume and s perm concentrati on , i ncreased frequency of tai l abnormal i ti es and detached sperm heads and u sual l y ceased s perm moti l i ty . S i mi l ar . changes but to a l esser degree were seen after 0 . 5 mg/ kg/day . C i rcul ar moti l i ty was the only noti ceabl e change caused by the l owes t dose .

The only s i de effec t observed was depress ed appeti te and a concomi tant wei ght l oss . Th i s effect was mos t pronounced early i n the dosi ng per i od and l ater l evel ed off .

The reproducti ve organs of rats and rabbi ts showed d i fferent s us cepti bi l i ty for 2 , 6-ci s at the mi n imum effecti ve d ose ( 1 . 0 mg/ kg/day for 21 day s i n rabbi ts and 5 . 0 mg/ kg/day for 14 days i n rats ) . S perma togen­es i s i n the ra t tes tes a ppeared normal whereas deger­erati ve cha nges wi th compl ete absence of s permatids were s een i n the s emi ni ferous tubu l es of the rabbi t .

Supported by PARFR- P5 .

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I N V ITRO METABOL ISM OF TESTOSTERONE BY CULTURED SERTOL I CELLS OF 36-DAY OLD RATS lf lTH AND lH THOUT FSH STIMULA­TION . R . K . Tchol ak ian and A . Ste inberger , Dept . Repro ­duct i ve Med . and B i ol . , Uni v . of Texas Medi cal School at Hou ston , Texas .

Two-day ol d cul tures of Sertol i cel l s de ri ved from testes o f 36-day ol d Long Evans rats were used i n these experimen ts . The cul tures were i ncubated w ith 1 4C­tes to sterone ( 1 µCi/5x l o- 'M) in 2 ml of Eagl e ' s m in imum es sent i a l medi um at 34C for 6h . Several cul tures were pre - i n cu bated wi th 5 µg/ml FSH ( N I H-FSH-Sl l ) at 34C for 6h before incubat i on with 1 4C-T pl us 5 µg/ml FSH .

The fol l owing stero ids were i sol at�d and crysta l l i ­zed to constant spec i f i c acti v i ty fro1;i control and FSH ­treated cu l tu res : T as unconverted substrate ; andros­tened i one ; 5a-androstan-3a , 1 7S-d i ol . Di hydrotestosterone crysta l l i zed i n control cul tures on l y . The 1 4C act i v i ty tentat i ve l y i denti fied as estrone ( E 1 ) and estradi o l ( E 2 ) metabol i tes o f T through sequenti al chromatogra­phy systems d i d not crystal l ize to constant spec i f i c act i v i ty wh i l e the 3H-E 1 and 3 H - E 2 used as recovery tracers d i d . Th i s i nd i cated that no aromat i zation of the 1 4 C -T substrate occurred ei ther in control or FSH­treated cu l tures .

The product i on of E 2 from T ( 5xl 0- 7M) i n s i mi l a r cul tures with and wi th out FSH for 72h was al so i nvesti � gated by RIA . Analys i s of vari ance of these data showed that the concentrat ion of E2 over a 72h peri od : 1 ) d i d n ot change s i gn i fi cantl y i n the control or the FSH­treated cul ture s ; 2 ) was s i mi l ar i n both groups of cul • tures ; 3 ) and wa s no d i fferent from the RIA bl an ks .

The se data demon strate that : 1 ) cu l tured Sertol i cel l s from immature ( 36d ) rats conta i n 5a-reductase , 3a- and 1 7S-hydroxy stero i d dehydrogenase acti v i ti es ; 2 ) the total metabo l i sm i n these cul tures i s s l i ghtly suppres sed by FSH ; and 3 ) cul tured Serto l i cel l s l ack the capac i ty t o aromati ze T to E1 and E2 even i n the presence of FSH .

( Supported by N I H Grant P 50-HD-08338 ) .

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TESTICULAR ANT IBODIES IN FUNCTIONAL AZOOSPERMIA . M . H . E . Tansy , H . Abdelal , A . A . Haf iez and O . Kandil . C . S . Mot t Center for Human Gr owth and Development , Wayne S tate Univers ity , Detro it , Michigan and Al Azhar Universi ty , Cairo , Egyp t .

Patients with azoospermia , small testes , normal secondary sexual characteristics and without evidence of obs truct ion of the duct system were selected . Test icular b iop si es were perf ormed and sections were s tained with hematoxyl in and eos in and with p eroxidase labelled ant ihuman gamma g lobulins antiserum . To evaluate spec if ic ity of any reaction in sect ions s tained with the labelled ant iserum , contro l s ect ions were first incubated in unlabelled antiserum then with the enzyme l abelled antiserum in order to block the receptors of testicular immunoglobulins . Histochem­ical localizat ion of the peroxidase enzyme marked the s ites of testicular immunog lobulins by a dark brown color . Th is color was markedly reduced in the controls indicat ing that the react ion was spec if ic for testicu­lar immunoglobul ins . In eight cases there was remark­able internal thickening of the basement membrane of the s eminiferou s tubules whi ch were populated with Sertoli cell s only and devoid of any germinal cell s . Two cases showed spermatogenic arrest and the tubular basement membrane was of normal thickness . Thickening o f the basement membrane , assoc iated with an increase in immunoglobulin concentration , was at tr ibuted to an auto immune response directed to the basement membrane . Lysi s of the germinal epithelium could be secondary result t o d isturbanc e of the blood-testis barrier ; only Sertoli cells could survive such pathology . Auto­immunity should be considered in evaluation of male inf ertil ity . ( Suppor t ed by WHO postdoctoral fellowship and Ford Foundation grant)

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PRESENCE OF SOLUBLE B-TYPE CYTOCHROME I N HUMAN SEMINAL PLASMA . N . P . Tri funac , A . F . Brodi e , and G . S . Berns tei n . Departmen t of Obs tetri cs an d Gyneco l ogy and Department of Bi ochemi s try , Uni vers i ty of Southern Cal i forni a Schoo l o f Medi ci ne , Los Angel es Coun ty-Uni vers i ty of Southern Cal i forni a Medi cal Cen te r , Los Angel es , CA.

In 1 95 1 Th adeus Mann reported that cytoch romes a , b , an d c were p res en t i n human spermatozoa . He noted that the cytochromes we re con fi ned to the sperm and were not found i n the semina l p l asma. Contrary to that report , we have obse rved a sol ub l e cy toch rome b ( SCB ) i n h uman s emi nal p l asma. SCB has the typi cal spectrum of a reduced cytochrome b but can be di s tingui s hed from the b- type cytoch romes from other mammal i an ti ss ues . I t has not been des cri bed befo re . · The spectrum o f reduced SCB has peaks at 4290A0 and

5620A0 • The reducti on can be i nduced chemi cal ly wi th s odi um d i thi on i te or enzymati cal ly us i ng NADH o r NADPH as el ectron dono rs . FAD i s necess ary for the enzy­mati c acti vi ty s ugges ti ng th at SCB i s a fl avoprotei n and the en zymati c reducti on can be i nhi bi ted by o­phenan th rol ene , whi ch sugges ts the i nvol vemen t of n on-heme i ron . The reduced SCB can bi nd CO gi vi ng an abs orp ti on peak i n the range 41 4-425A0 •

SCB was p resen t i n semen from vas ectomi zed men and i n ferti l i ty pati ents wi th azoospermi a as wel l as i n the semi nal pl asma o f normal donors . Thi s i n di cates that the en zyme does n ot en ter the semen by l eakage from s permatozoa. Th i s i s an unusual fi ndi ng because cytochrome b i s general l y bound wi thi n the ce l l and i s not found i n body fl ui ds .

The on ly water sol ubl e b- type cytochromes of mamma­l i an o ri gi n are bs and P450 . The l a tte r i s an acti ve componen t i n ce l l ul ar s teroi d hydro xyl a ti ons . By anal ogy , SCB may be i nvol ved i n the con vers i on of tes tos terone to DHT , but i ts actual func ti on i n semi n al pl as ma i s un known at the presen t ti me .

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OBSERVATIONS ON THE INITIATION OF SPERM MOTILITY T . T . Turner , and s . s . Howards . Department of Urology ,

university of Virginia Medical School , Charlottesville ,

Virginia The innnotile spermatozoa in the cauda epididymidis

(CE ) have full capacity for progessive motility ; how­ever , they do not actua lly become motile until ej acu­lation occurs . The present study was undertaken to determine the effects of different ions and naturally occurring compounds on the initiation of rat sperm motility .

Samp les of CE content (fluid + spermatozoa ) were obtained by micropuncture . The 1 )ll samples were diluted 1 : 10 with 12 different ionic solutions contain­ing a 300 m:>sm concentration of a specific inorganic compound . The pH of these solutions ranged from 4 . 2 to 10 . 2 . 1 : 10 dilutions of CE samples were also ma.de with 300 mOsm nonionic solutions of urea , sucrose , and ma.n ­n i tol . Additionally , l ,..ul C E samples were exposed to sodium chloride and calcium chloride crystals without any diluent effect . Other CE samples were diluted 1 : 10 with physiologic solutions of glycerylphosphoryl­choline (GPC ) and/or carnitine .

Dilution of immotile CE spermatozoa with any of the 12 ionic solutions allowed for the initiation of motil­ity . Motility only appeared to be limited at pH ex- . tremes . 300 mOsm sodium chloride was the best diluent for motility in this system . ca++ previously hypothesized to be required in the initiation of sperm motility was of no benefit to the initiation of rat sperm motility . Spermatozoa did not become motile at all when exposed to salt crystals alone and only transiently when diluted with nonionic solutions . Solutions of GPC , carnitine , and GPC+carnitine significantly inhibited the initiation of sperm motil­ity (p < . 01) . Addition of 5 mg/ml BSA to the diluent did not protect the spermatozoa from this inhibition . It is proposed that spermatozoa are rendered innnotile in the ma. le tract by high concentrations of GPC and/or carnitine . The dilution of these compounds at ej acu­lation p lus the concomitant addition of ions allows for the ini tiation of rat sperm motility .

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PROLACTIN LEVELS I N OL I GOSPERMIA . V . A . Tzi ngoun i s , and M . F . Aksu . Department of Endocri nol ogy , Medi cal Col l ege of Georg i a , Au gus ta , GA .

Serum and semi nal p l asma prol acti n ( PRL ) concen tra­ti ons were meas ured radio immunol og ical l y in 1 2 ol i go ­spermi c ( s perm count < 20 mi l l i on/ml ) and i n 1 5 normo­s permi c mal es who served as contro l s . Al l men s tudi ed had normal serum gonadotropi n s and testosterone val ues . I n o l i gospenn i c subjec ts the semen PRL l evel s ( mean ± S . D . ) ( 21 . 7 ± 4 . 1 ng/ml ) were s i gni fi cantl y hi gher than those of control s ( 1 2 . 5 ± 5 . 2 ng/ml ) at the l evel of P < 0 . 001 , wh i l e serum PRL l evel s d i d not show any s i gn i fi cant d i fference . A · negative l i near regres s i on was found between the sperm count and semen PRL concentrati ons ( r = -0 . 63 , P < 0 . 05 ) but no s i gn i ­fi cant correl ation wa s found between sperm count and serum PRL . Furthermore , no s i gn i fi cant correl ation was fo und between semi nal pl asma and serum PRL l evel s . Th i s data does not support the s ugges tion tha t PRL l evel s are hi gher i n semen havi ng adequate sperm count ( Sheft et al . Ferti l . Steri l . 26 : 905 , 1 97 5 ) but rather sugges ts that h i gh l evelS of pro l actin i n the semen may i nterfere wi th testi cul a r spermatogen i c acti v i ty .

DECREASED SPERMATOGENESIS AFTER EXPERIMENTAL VARICOCELE CREATION IN THE DOG AND RAT. R.L. Urry , B. Deter , K. Dougherty, and A.T.K. Cockett . Depart­ment of Zoology , Brigham Young University , Provo , Utah , and Division of Urology , Roche ster School of Medicin e , Rochester , New York.

82

For the past three years we have been using various animal models to evaluate the effect of an " experi­mental varicocele" on testicular function. The following studies were undertaken to investigate the effect of the varicocele on spermatogenesis and to determine the role of the adrenal gland in any changes that might be observed. Baseline semen samples were obtained from sixteen dogs , biweekly for two months. An experimental varicocele wa s then created in eight of the dogs by partially restricting the renal vein proximal to the j unction of the spermatic vein. Semen samples were then obtained twice weekly for twelve weeks. The animals with the experimental varicocele had a reduction in sperm count , motility score , and in the percent of active , viable and oval spermatozoa. Testicular biops ies were also abnormal in four of the eight dogs.

In the s econd experimen t 48 rats underwent a vasocystostomy. A 24-hour sperm count was obtained from all of the animals each week for two months. At the end of the baseline period 12 of the animals underwent a partial left renal ve in ligation to create an experimental varicocele , 12 underwent the vein constriction coupled with an adrenal ve in ligation , 12 underwent only an adrenal vein ligation and the remaining 1 2 served as sham operated controls. Sperm counts were then followed in each animal for 12 additional weeks. Creation of the varicocele significantly lowered the sperm count in both the varicocele and varicocele plus adrenal vein ligation groups.

The results sugge st that the creation of an ex­perimental varicocele has a detrimental effect on sperm production . It also appears that the adrenal gland does not play a role in the dimin ished function of the tP � r Ps aftP.r crea tion of the var�co_cele_. __

83

PHYSI OLOGIC CONCENTRATIONS OF TESTOSTERONE Al.1D THEIR EFFECT ON LH SECRETION IN THE MALE MONGREL DOG . D . L . V i nc ent , T . Kepi c , and R . E . Falvo . S chool of Medic ine , Southern Illinois Un ivers ity , Carbondale , I L .

In order t o invest igate the hypothalami c-pituit ary­t esti cular axi s of the male mongre l dog , the fol lowing s tudi e s were done . Dogs ( N=4 ) were c astrated and plasma l evel s of testo sterone ( T ) and LH were determined by RIA from samples c ollected every 4hr ( for 48hr ) and dai ly ( for t he next 8-12 day s ) post-castrati on ( PC ) . In all dogs within 2 4 hr PC , T levels fell to non-de­t ectable ( ND ) and remained ND for the remainde r of the s tudy . LH was ND for the first 15-24hr PC and began to r i s e t hereafter . By 48hr PC the mean LH level was 1 0 . 5ng/ml whi ch i s s i gni fi cantly greater than that s een i n i nt ac t dogs .

In a s e c ond study the effect of T-filled S i last i c implant s on T and LH levels was determined in the i n­t ac t and c astrat ed dog . Dogs were treated as follows : GROUP I ( N= 4 ) received 1 empty c ap sule ; GROUP I I ( N=4 ) received 1 T-fi lled c apsule ; and GROUP I I I ( N= 5 ) re­c ei ved 3-T fi lled cap s ules . Plasma was c ollect ed for : 5 days pre-implant at i on , 7 days post-implant at i on , 7 days P C , and 4 day s after capsule removal . During the 7 days following implantat i on , whereas T levels were not s igni fi c ant ly di fferent in all groups as c ompared t o pre-implantati on l evels , LH level s were unaffected in GROUP I and lower i n GROUPS I I &I I I . Following c as ­trati on , T levels fell i n all groups : GROUP I , 0 . 7 to ND , GROUP I I , 1 . 2 to 0 . 6 and GROUP I I I , 1 . 3 to 0 . 5ng / ml respect ively . In all groups PC , the respons e of LH was proport i onal to the amount of T releas e d . In GROUP I , LH value s rose rap i dly t o castrat i on levels and c ontinued to ri s e unti l the end of the study . In GROUP I I the LH ros e above pre- implant ati on levels but was below those s ee n in GROUP I . In GROUP I I I , castrat i on h ad n o e ffe ct on the LH leve l . Following c ap sule re­moval the T levels in GROUPS I I & I I I be came ND while the LH levels rose to castrated values . The s e experi­meL1t s sugges·r; that rep lacement of T within phys i ologi­c al limit s i n the short-term castrated male dog can mai nt ai n LH at physiol ogi c al level s .

84

LEYDI G CELL FUNCTI ON IN OL I GOSPERMI C MEN WITH VARICOCEL� D . B . Wei s s , L . J . Rodriguez-Rigau , K . D . Smi th , and � S teinberger . Dept . of Reproducti ve Med i c i ne and B i o l ogy, U . T . Medical School at Hous ton , Texas .

Al though vari cocel e has l ong been known to be. as soc­i ated wi th mal e i nferti l i ty , the pathogenes i s rema i n s uncerta i n . I n severe ly o l i gospermi c men the respon se t o vari cocel ectomy i s poo r . The a i m o f thi s study was to eval uate Leydi g cel l function i n 1 6 young men wi th sperm counts < 1 0 m i l l i on/ml and with vary i ng degrees of vari ­cocel e . Tes t i cu l ar ti ssue obtai ned by routi ne testicul ar b i opsy was i ncubated wi th 3 H- pregnenol one uti l i z ing a prev ious ly described techn i que ( Rodri guez-R i gau et al . , S tero i d s 29 : 771 , 1 97 7 ) . Testosteron e formed was quanti � tated by i so tope d i l ution fol l owing puri fi cati on and crystal l i za t i on to constant spec i fi c act iv i ty . As a nor� mal control , testi cul ar ti s sue obtained from a young normal vol unteer wa s s tud i ed . In 1 3 of the 1 6 cases ( 81 % ) , testosterone synth es i s was depressed . Al though i n a l l subj ects ci rcul ating testosterone and LH l evel s were wi th i n normal range , testosterone synthes i s was found to corre l ate wi th c i rcu l ating testosterone and gonadotropi n l �vel s . S perm moti l i ty was h i g hest in sub­jects wi th h i ghest testos terone synthes i s rates . Mo cor ... rel ation wi th age , s i ze of vari cocel e , degree of ol igo­s permia or h i s to l og i c a ppea rance of the sem i n i ferous epi thel i um cou l d be demon s trated . An increased Leydi g cel l number was observed i n testes wi th hi ghest . testos­terone format ion ; s i nce tes ticul ar vol ume was l owes t i n th is group , th i s fi nding cou l d represent rel ative rath�r than absol u te i ncrease i n Leydi g cel l densi ty . Fol l owi n� vari coce l ectomy , no improvement i n s perm counts or mot i -1 i ty wa s observed , and no pregnanc ies occurred . Thi s study demonstrates a di sturbance of androgen biosynthe­s i s i n the testes of men wi th sperm counts < 1 0 mi l l ior! ml and vari cocel e . The l ack of response to vari cocel ec­tomy cou l d be due to i rreversi bl e damage of the semi ni ­ferous epi thel i um , or to an unrel ated d i sorder of Leyd i � cel l funct i on . Thus , Leyd i g cel l function s hou l d be car�­ful l y eval uated in ol i gos permic pati ents , regardl ess ot the presence of a vari cocel e . P l a sma testos terone may no t be suffi c i ent for thi s purpose .

85

THE FERT I L I Z I NG CAPAC ITY OF EPIDIDYMAL SPERM FROM THE GUINEA P I G . B . R . Wi l liamson , J . Martan , and B . A . Shepherd. Department of Zoology , Southern I l l inoi s University , Carbondal e , I l l inois .

Ferti l ity s tudies of a variety of mammal ian sp erm including the rat ( Blandau and Rumery , 196 1 , 1964) , rab . bit (Bedford, 1966; Orgeb in-Crist , 1967) , and hamster (Horan and Bedford, 19 72) have shown that the capacity

to fert i l i ze ova is acquired in the epididymis . Fawcet� and Hol lenberg ( 1963) inferred that sperm in the proxi­mal portions of the guinea pig epididymis were immature , bas ed on the shape of the sperm heads in contrast to the shape o f mature sperm in roul eau . Young ( 1931) ass ayed the ferti l ity of epididymal sperm from the guinea pig after arbitrar i ly dividing the duct into proximal and distal portions . He reported an increas e in fert i l i zin� cap acity of sperm from the dis tal half of the duct fol­lowing artificial insemination . However , no attempt was made to insure that only immature sperm were pres ent in the proximal region . The obj ective of the present study was to ass ess the fert i li z ing capacity of morpho logi cal ­ly immature sperm from the epididymis as characteri zed by Fawcett and Hollenberg . Epididymides from 16 s exual­ly mature guinea pigs were removed and gross ly s eparateq into regions , s imi lar to those reported by Fawcett and Hol lenberg . Small pieces of the duct were removed pro­gres s ing proximal ly unt i l s ing le sperm in the abs ence o� roul eaux were obs erved . The remaining proximal portion of the duct was macerated in warm Tyrode ' s solut ion , and estrous femal es were artificially ins eminated intraut e­rine . Smears were made to confirm whether or not a l l sperm were s ingl e . Females receiving s ingle sperm only did not conceive , while 4 of 9 females receiving sperm containing up to 1 . 2% rouleaux became pregnant . Resultd obtained indi cate that morpho logically immature sperma­tozoa in the guinea pig have not acquired fertili zing capacity, and suggest that the rouleau condition may be us ed as a reliab l e indication of phys iological maturity J

86

INDIRECT RADIO I MMUNOASSAY FOR THE DETECTI ON O F ANT I ­BODIES T O SPERMATOZOA . L . G . Young . Department of Phys i ol ogy , Emory Un i ve rs i ty Sch oo l of Me di ci ne , Atl ant& , GA .

I n o rder to detect i n h uman sera anti bodi es to sper­matozoa , we devel oped an i ndi rect radi o immunoassay ( I RIA ) based on the method des cri bed by Rosenthal et al . ( Appl . Mi crob i al . , 1 973) . Human sperm ( anti gen-poSTt1 ve cel l s ) and bul l sperm ( negati ve contro l cel l s ) were affi xed by drying on to the bottom s urface of the wel l s o f mi cro­ti ter pl a tes . Two fo l d seri al d i l uti ons of sera were added and a l l owed �o react . The presence of anti spenn anti bodi es was dete nni ned by meas uri ng the speci fi c b i ndi ng of 1 2 5 1 - l abel l ed rabbi t anti human i mmunogl obul i n . An tigen c oncentrati on , 1 2 5 1 - immunogl obul i n concentra ti on and othe r fac tors wh i ch i nfl uence the tes t were i nvesti ­gated . As de termi ned by I R I A , the presen ce of anti bodi es to sperm i n the sera of 1 6 i nfe rti l e i ndi vi dual s was compared to res ul ts obtai ned wi th ' these sera i n other 1 abora tories by gel , tray and s l i de a�gl uti nati on , mi cro­scop ic and macroscop i c immob i l i zation and cyto toxi c i ty tests ( Boettche r et a l . , Cl i n . Exp . Immuno l . , 1 977 ) . N i ne of the s e ra were found to be nega ti ve for anti s perm anti bodi e s by I RI A and by at l east fi ve of the other s i x tes ts . Three o f the sera ha d pos i ti ve ti ters i n al l of the tes ts , and th ree were pos i ti ve by I RI A and by fi ve of the o ther s i x tes ts . I R I A i s an operati onal ly s i mpl e method wh i ch p rovi des an objecti ve and q uanti ta ti ve means for ass es s i ng i n human sera the presence of anti ­podi es to s pe rm .

Supported by the Worl d Heal th Organi zati on Smal l Re­search Contract No . 7645 and by an Emory Un i vers i ty Bi o­medi ca 1 Resea rch Support Grant .

xxvi

AUTHOR I NDEX

Abstract No .

Abdel a l , H . . . • . . • . . . . . • • . . . . . . . • . . . 78 Ai man , E. J . . . . . . . . . . . . . . . . . . . . . . . . l Ai re , T . A . . . . . • . . . . • . • .. . . . . . . . . . . . 7 6 Akru k , S . R . . . . . . . . . . . . . . . . . . . . . . . . 2 Aks u , M . F . . . . . . . . . . . . . . . . . . . . . . . . . 81 Al exander , N . J . . . . . . . . . . . . . . . . . . . . 3 Al varez , I . . . . . . . . . . . . . . . . . . . . . . . . . 25 As co l i , M . • . • . • . . • • . • . • • . . • . . . • . • • • 69 Atherton , R . W . . • . . . • . . . . . • • . • . . . 35 , 60 Aucl ai r , C • • • • • • • • • • • • • • • • • • • • • • • 4 , 1 7 Audebert , A . JM • • • • • • • • • • . • • • • • • • • • 23 Au 1 i tz ky , H . . . . . . . • . • . . . • . . . . . . . . . . 64 Bardi n , C . W . . . . . . . . . . • . . . . . . . . . • . . 54 Bates , G . W . . . . . . . . . . . . . . . . . . . . . . . . 5 Bende , T . . . . . • . . . . . . . . . • . . . . . . . . . . • 64 Bendi ch , A . . • . . . . . . . . . . . . . . . . . . . • . • 37 Bernstei n , G . S . . . . . . . • . . . . . . . . . . . • 79 Bex , F . J . . . . . . . . . . . . . . . . . . . . . . . . . . 6 B l ack , J . B . . . . . . . . . . . . . . . .. . . . . . . . . 7 B l eau , G . . . . . . . . . . . . . . . . . . . . . . . . . 8 , 1 2 Boesel , R . W . . . . . . . . . . . . . . . . . . . . . . . . 9 Bon i fa ci o , V . . . . . . . . . . . . . . . . . . . . . . . 29 Bradford , M . M . . • . . . . • • . . . . . • . . . . . • 52 Bremner , W . . . . . . . • . . • . . • . . . . . . . • . . . 1 0 B re nne r , P . F . . . . . . . . • . . . . . • . . . . • . • 1 Brodie , A . F . . . . . . . . . . . . . . . . . . . . . . . 79 Brown , J . R . . . . . • . . . . . • • • . . . . . • . . • • 25 Bustos-Obreg6n , E . . . . . . • . . • . • • • • • • . 1 1 Car l s trom , K • . . . . . . • • . • . . . • . . . . • • • . 58 Cavanaugh , A . H . • . . . . . . • . . . . . . . • . . . 25 Chapde l a i ne , A . . • . . • . . . • . . . • . . • • . 8 , 1 2 Chemes , H . E . . . . . • . . . . . . . . . . . . . . . . . 61 Chowdhury , M . . . . • . . • . . • . . . . . . . • • . . • 1 3 Cockett, A . T . K . . • . . • . . . • . . . . • • • • . 21 , 82 Connatser , D • . • . • • . . • . . • . . • . • • . . • . • 24 Conne 1 1 , C . J . . . . . . . . . . . . . . . . . . . . . . 32 Con te , D. . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1

xxvi i

Abstract No .

Coo k , E . L . 1 4 Coo per, T . G . . . . . . . . . . 1 5 Corbi n , A . . . . . . . . . . . . . 6 Couture , M . L . 1 6 Crabo , B . G . 76 Cre ns haw , R . T . 5 1 Crenshaw , T . L . 51 Cummi ng , I . 1 0 Cus a n , L . 1 7 , 5 7 Czygl i k , F . 1 8 Danzo , B . J . 1 9 , 56 de Cas tro , M. P . 20 de Kretser , D . . . ·. 1 0 Der , R . . . . . . . . 46 Desy , L . 5 7 Dete r , B . 82 Di dato , F . . . 72 Do ndero , F . 41 Dougherty , K. A . 21 , 82 Dym , M . 6 1 Edma n , C . D . 5 E l i assen , L . 22 E l i as se n , R . 22 Emi l son , L . B . V . 2 1 Empe ra i re , J . C . 18 , 2 3 Eri ckso n , B . H . 24 Fah i m , M . s . 46 Fal vo , R . E . 83 Fang , V . S . 31 Farnswor th , w . E . 25 Fawcett , D . W . 26 Fi ndl ay , J . 1 0 Fl e chon , J . E . 27 Forsgre n , B . 28 French , F . S . 6 1 Freund , M . 1 6 Frai ol i , F . 29 , 30 Fri ck , J . 64 Ful gham , D . L . . . . . . . . 3

xxvi i i

Abs tract No .

Fu ruhash i , N • . . • . . . . . . . . . . . • . . • . . . . 31 Garon , M. . . . . . . . . . . . . . . . . . . . . . . . . . . 1 7 Garri son , L . B . . . . . . . . . . . . . • . • . . . • . 45 Gondos , B . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Goodpas ture , J . C • . • . . . • . . . . . • . . • • . 33 Go u 1 d , K . G • • . . • . . • . . • • • • • . • . . • . . . • 3 4 G raham , E . F . . . • . . . . . . • . . . . . • . • . • . . 43 Gustafsso n , J . -A . • • • . . . . . . . . . . . . . . . 28 Hafi e z , A . A . . . . . . . . . . . . . . . . . . . . . . . 78 Hah n , G . L . . . . .. . . . . . . . . . . . . . . . . . . . . 35 Ha 1 ey , B . E . . . . . . . . . . . . . . . . . . . . . . . . 35 H ansen , K . A . . . . . . . . . . . . . . . . . . . . . . . 38 H a rri s , M . E . . . . . . . . . . . . . . . . . . . . . . . 36 Harri son , R . M . • • . . • . . . . . • . . • • . . • . • 49 Hei tl er , R . . . . . . . . . • . . . . . . . . . . • • . . • 42 Hembree , W . C . . • • . . . • . . . . . . • • . . . . . . 39 Hennan , J . H . . . . . . . . . . . • . . . . . . . . . • . 50 Hes s , E . V . . . . . . . . . . . . . . . . . . . . . . . . . 50 H i gg i ns , P . J . . . . . . . . . • . . • . • • . • . • . . 3 7 Hodges , C . V . . . . . . • . . . . . . . . . . . . • . • . 45 Hoffe r , A . P . . • . . . • . . . . . . . . . . • . . . . • 26 Hogbe rg , B . . . . . . . . . . . . . . . . . . . . . . . . . 28 Hol l and , J . M . . . . . . . . . . . . . . . . . . . . . • 38 Hos k i ns , D. D . . . . . . . . . . . . . . • . . • . . . . 73 Howards , S . S . . . . . . • . . . • . . . . . • • . • . . 80 H uang , H . F . S . . . . . . . . . . . . . . . . . . . . . . . 39 Hunt , W . L . . . . . . . . . . . . . . . . . . . . . . . . . 40 Hunte r , R . H . F . . • . . . . • . . . . . • . • • • . . • • 75 I s i do ri , A . . . . . . . . . . . . . . . . . . . 29 , 30 ,4 1 Jech t , E . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Jeye ndran , R . S . • . . . . . . . . . . • . • . . . . . 43 Kandi l , 0 . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Kayne , F . J . . . . . . . . . . . . . . . . . . . . . . . . 7 4 Kea ti ng , R . J . . • . . . . . . . . . . . • . • • . . . • 44 Kee l , B . A . . . . . . . . . . . . . . . . . . . . . . . . . 7 Kee l , M . F . . . . . . . . . . . . . . . . . . . . . . . . . 7 Keenan , E . J • . • . . . . . . . • • . . • . . • . • • • • 45 Ke l ly , H . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Kel l y , P . A • . • . . . . . • . • . • . . . . • . 4 , 1 7 , 5 7

Kemp , Kep i c , Kha re ,

E . D . T . N . K .

K l upp , E . . . . . . . . . .

J r .

L .

Ko t i te , N . J . Labri e , F . Lai , C . E . LaNasa , J . A . , Lee , C . Lev i ne , N . Lewandows k i Lewi s , R . W . Li ma , S . S . MacDona l d , P . Marcus , Z . H . Martan , J . Marti n , D . E . McKoon , M . F . McRori e , D . K . McRori e , R . A . Med 1 ey , N . E . Menge , A . C . Mes i na , M . B . Metz , K . W .

M .

Mi 1 1 s , N . C . Mi l l s , T . M . Mor rone , S . Nayf eh , S • N . Nel son , L . Ni chol s on , N . Ol s on , G . E . Orgebi n-Cri s t , Pal l otti , S . Pao l ucci , D . Pea rse , H . D . Pel l et i e r , G . P l a tz , C . C . Pol ako s k i K . Pou sette , A .

c .

M . - C .

L .

xxix

Abstract No .

45 83 46 42 6 1

4 , 1 7 , 5 7 3 2 4 7 38 48 25 49 20

1 , 5 50 85 5 1 32 5 2 52 5 3 5 3 30 35 54 54 30 61 55 40 56 1 9 29

29 , 30 45 57 68 33

28, 58

xxx

Abstract No .

Pre s l oc k , J . P . . . . . . . . . . . . . . . . . . . . . 59 P ue tt , D. . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Radany , E . W . . . . . . . . . . . . . . . . . . . . . . . 60 Raj , H . G • M. . . . . . . . . . . . . . . . . . . . . . . . . 6 1 Ramsey , E . E . . . . . . . . . . . . . . . . . . . . . . . 45 Reddy , J . M. . . . . . . . . . . . . . . . . . . . . . . . 62 Ri cher , C . -L . . . . . . . . . . . . . . . . . . . . . . . 1 2 Roberts , K . D . . . . . . . . . . . . . . . . . . • . 8 , 1 2 Rod ri guez-Ri gau , L . J . . . . . . . . • . . 63 , 84 Ros s , G . , J r . . . . • . . . . . . . . . . . . . . . . . . 46 Rovan , E . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Russel l , L . D . . . . . . . . . . . . . . . . . . . . . . 65 Sa 1 i ng , P . M. . . . . . . . . . . . . . . . . . . . • . . 66 Schanbache r , B . D . . . . . . . . . . . . . . . . . . 67 Seage r , S . W . J . . . . . . . . . . . . . . . . . . . . . . 68 Sega l o ff , D . L . . . . . . . . . . . . . . . . . . . . . 69 Sha i n , S . A. . . . . . . . . . . . . . . . . . . . . . . . 9 Shepherd , B. A . . . . . . . . . . . . . . . . . . . . . 85 S i l be r , S . J . • . . • . . . . . . . . . . . • . . . 70 , 71 Smi th , K . D . . . . . . . . . . . . . . . . . . . . . 6 3 , 83 So 1 i man , K . F . A . . . . . . . . . . . . . . . . . . . . . 1 4 Sri vas tava , P . N . . . . . • . . . . . . . . . . . . . 2 Sta rk , R . A . . . . . . . . . . . . . . . . . . . . . . . . 62 S tee 1 e , R . E . • . . • • . . • • . . . . • • . . • • • . . 7 2 Ste i nberger, A . . . . . . . . . . . . . . . . . . 1 3 , 77 S te i nberge r , E . . . . . . . . . • . . 1 3 ,44 , 63 , 84 Ste i ne tz , B . G . . . . . . . . . . . . . . . . . . . . • 7 2 Stephens , D . T . . • . . . . . • . . . . . • . . • . . . 73 S to rey , B . T . . . . . . . . . . . . . . . . . . . . 66 , 7 4 SzOl l Os i , D . . . . . . . . . . . . . . . . . . . . . . . . 75 Tarasek , C . D . . . . . . . . . . . . • . . . . . . . . . 76 Tcho l a k i a n , R . K . . . . . . . . . . . . • . . • 44 , 7 7 Thumann , A . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 Tansy , M . H . E . . . . . . . . . . . . . . . . . . . . . . . 78 Tri funac , N . P . . . • . . . . . . . . . . . . . • • . . 79 Turner , T. T . . . . . . . . . . . . . . . . . . . . . . . 80 Tzi n gou ni s , V . A . . • . . . . . . . . . . . . . . . . 81 U rry , R . L . . . . . . . . . . . . . . . . . . . . . . 21 , 82 Vi n cent , D . L . . . . . . . . . . . . . . . . . . . . . . 83

xx xi

Abs tra ct No .

Vi rj i , N . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Wa i tes , G . M . H . . . . . . . . . . . . . . . . . . . . . . 1 5 Wang , J . - L . . . . . . . . . . . . . . . . . . . . . . . . . 73 Warne r , H. . . . . . . . . . . . . . . . . . . . . . . . . . 5 1 Wei s s , D . B . . . . . . . . . . . . . . . . . . . . . 6 3 , 84 Wi l l i amson , B . R . . . . . . . . . . . . • . . . . . . 85 Wi tki n , S . S . . . . . . . . . . . . . . . . . . . . . . . 37 Young , L . G . . . . . . . . . . . . . . . . . . . . . . . . 86 Young , M . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Zanevel d , L . J . D . . . . . . . . . . . . . . . . . 33 ,62 Zi mmerman , K . J . . • . . . . . • . . • . • • . . • . . 76