a histochemical study of steroid hormone synthesis in the … · 2017-01-05 · b. the use or...
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Loyola University ChicagoLoyola eCommons
Master's Theses Theses and Dissertations
1966
A Histochemical Study of Steroid HormoneSynthesis in the Gonads of the Brown LeghornChick EmbryoDavid Glenn RabuckLoyola University Chicago
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This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.Copyright © 1966 David Glenn Rabuck
Recommended CitationRabuck, David Glenn, "A Histochemical Study of Steroid Hormone Synthesis in the Gonads of the Brown Leghorn Chick Embryo"(1966). Master's Theses. Paper 2060.http://ecommons.luc.edu/luc_theses/2060
A nIBl'oamaCAL mUDY OF STJmOII) JllRHDm
snmmsm II mE 00lWJ8 t6 H
BRa!m lmDUI CUICK IIMBR!O
A baa SWnttted to the Facult7 of the a .. luate ScbcxU
of ~ Un1'IU'8.'L~ 1ft ~ Ful.f:U.l.mf.mt of
the ~ tor tbe Degree of
Heater of Science
Dav.ld Glenn Raback was bom on December JO, 1940 in Geneva, nUno1,..
He was graduated f1"OO1 Evanston Tovn8b1p High School, Evaneton, Illinois,
in June 1958. He attendecl Nortb Central College, Napel"Ville, lllinoia, cd
WU 8IIrU'Cled the BKhelor of Art. degree in June, 1963.
In Sept.ewbel". 1963 be began his ~ atwUee in the Department 01
AnatCll:Q', Loyola Un1ftHit,. Stritch School of Medicine, Chicago, Illinois.
He 18 the recipient of a Lo701& univere1t7 Gradute Stwient Teaching Assist
antatdp arid ia CUft"entl7 also Nl"'ri.ng _ & ieeearcb Assistant in Anatorq.
He is an Auociate Member of the American Chemical Societ,r.
To Dr. LiDcoln V. DOIIII. ITer .. sOJ" o.t Anat.a.a.Y, and. Chairman. DepaJ"taent
ot Aut.cm.r. my spouol". I w1ah to exteJxl JII7 siDeel"e appI"eCht10l'l tor b1a
auggeat10n at the problem. h1a «UDlp1e aDd h1a pa t1ent help al1l1 guid.aAoe
th:rouchou.t th1a studT.
To Dr. J .... E. Woods. Reaeal"Oh Jutsac1ate 111 AnatcB7. I wiah to extend
lIf3' heaJ"ttelt thaw tor h1a encourag-.ent and generoa1t," ot h1a t1me. without
whoM C0UD881 this UDlertaldJli would. haw bMft cou1c1erabq more d1tt1oult.
I alao would Uke to Gpl'U8 Jq appnc1aUOI1 to Dr. Lesl1e A. EDaeJ"t,
A.s1atant ProtessOl" of Anatc:a;y. tOJ" h1a thoughttul. mUos... and 8UiiesUou
on h1atoobem1oal aJ¥l teolm1oal p:robl..aa; to Dr. wcia _lte. Seldol" TeohD1-
oian 11'1 Autc:a;y, tor bel" oona1clel"ed adv:1oe ocmcel"ft1Da t1uue preparaUona,
aDd to Mr. John MaUl"8J". Medical Sohool Photographer. tor b1a expert aclv1ce
oonoel'ld.ltg 00101" photc.1orograph;y.
last, but not least. to -q w1te. Loralee. tor bel" 1.Ul8Dd1Di help and
.0000al support. I w1ah to give l1l7 8incere thaw. The studT was npported 18 part b.Y USPHS •• BIli. ReaeaJ"Oh Grant. AH 0:3895
aDd AM 09926. ancl a USPBS. t NIH. GeIleRl Rea_rob Support Grant. Grants ad ....
m1n1atel'eCl by Dr. L.i.nooln V. Daa.
TABLE OF CONTERTS PAGE
INTRODUCTION ••••••••••••••••••••••••••••••••••••••••••••••••••••••• 1
REVIEW OF THE RELATED LITERATURE •••••••••••••••••••••••••••••••••••
A. EARLl OBSERVATIONS AND THEORIES............................ , B. EXPElUHEltl'A.L VERIFICA.TIOJ AND TESTIIG or THEORIES.......... 4
1. TRlRSPLARTATIOR........................................ 4 2. TISSUE cutraRE AND CASTRATION.......................... 5
,. ~TIOI or HORMONES............................. 6
4. BIOCHEMICAL rINDIIGS................................... 8 s. HISTOCHEMICAL OBSERVATIONS............................. 9
a. TECHRIQUES EHPLOIED................................ 11 ~TERIALS AID MBrHQDS.............................................. 1)
EXPERIHIIfAL RBSULrS............................................... 18 A. THE HISTOCHEMICAL METHOD fOR ~ -~Oll'STEROID
DEH!DROGERASE............................................. 19
B. THE USE or DEHIDROEP:wmROSTERONE (DBA) AS THE SUBSTRATE.. 19
1. THE PnuoD 01 :3 DAYS TO SEX DJli'P'ERDTIA.TIOI........... 19
2. THE PE2.IOD P'i.oH SEX DIFFERDTIATIOI TO DAY 1)...... ••• 21
). THI PERIOD or DAY 1) TO HATCHING...................... 21
C. THE USE OF PREGIEIOLOHE AS THE stmSTRA.TE.................. 22
DISCUSSION......................................................... 23 SUHHARI AND CONCLUSIONS............................................ 21
LITERATURE CITED................................................... 29 TAStES............................................................. )4
PLATES AND rIGORES................................................. J7
1"
1. 'ftB Immau'lf ~ ~ DiiJI06lnOB II ,65 .. ~ PBEP.AaAflOHS DAlS ) 10 6 ••••••••••••••••••••••••••••••••••••••• l4
2. 'lIB nlmmn f:I FC8HA.Wf DD'061T1ON IN D.'-~ PllPAiATlORS DA18 7 !O 9 ••••••••••••••••••••••••••••••••••••••• lS
3. TiS lIm.tlliIT! f6 romWWI ~ II'! L::.5_~ POBPAfiA!XONS DAlS 11 TO aD ••••••••••••••••••••••••••••••••••••• 36
2. ~AL ~B fit TlfRJiB DAt lHJBlD SWWH II FIOUltI ot8 momD IBAC!IOB ~ UfMW.. '!UBI, ~ Ali) LIKtIG or our •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 31
3. A ~L ~ GI T.t~ DAY m4BR.1t> mtOWI IN rlCO'BES OWE AND TWO 10 SHOW ~ or ~ ~ ••••••••••••••••••••••••••••• 3B
4. A I:IlGiIm IfAGlD'ICAUOB or UPPEi. cmIIYAL RmGE ~ IU fIG'I.mE ~ to <X*PllII ~ 0'1 PO.8HAZAIi ~ ••••••••••••••••••••• 3S
5. ~ ~B OF A QOlW) or , rm DAY Jlllm'fO stfOWDIl SCA~ PO.8HAZAIi GRA.N01.BS 'HIlOOIIOUT mE amm.u RIDGE All) auT ~UH. ••••••••••••••••••••••••••••••••••••••••••••••••••••••• 39
6. ~AL ~ C1I A SIVER DAY OOHAD auaam DlP'FI:1S! Bur ~ GRAlULAf.JOu. ••••••••••••••••••••••••••••••••••••••••• 4Q
7. ~~» OF A lIfE DAY GOIAD ~ REACfIOH Ifi WI'iAf HAt m: MBDULLARY ~sm.' OF OVARY... ........ ......... ...... 40
8. ~AL ~ or It. 14 DAY aVAil SlD'1m It. PATCHY auatJ'OH II J.l)()SE fQlflOft 0'1 MmU'lJ..AllY Q.JGIOlBIr. AIl) vma !lSI9£ ISAC'llON IN 1HE oct4PAOT JIOJrt.l'OR. •••••••••••••••••••••••••••••••••• 41
9. HIQma MAGKIFlCAUOI or A ~ or J.£.IC.lag Himf1Ll.AlY PtIlTimi SiQlH IN PIWIm UCIrt stfOWDIl HmUIJ'.NtY tACU&\ AJI) THR ItEACTION II THE ~(ZS ~ KIIlu.t.tARI LA.CUHAB All) CORDS ••••••••••••••• 41
lO. ~AL ~ at A 16 OAt TIfBllS SDmI't A filUCflON THfI)tr.»1OUT !HE (BlAH .AS v!ELL AS ~ nBACTm 'ATClIDSS.......... 42 U. HllJEft HAOHIFlCAf'lOH at MM& TISSUE SECtlON AS IN FlOOR! USN '.to
SHOW Ii. PATCH OF :rJrJ.SB I"01IIAZAB llSPOSftIOl ••••••••••••••••••••••• 42
l2. ~AL ~ OF ",Ii SACRAL PORTION C1f A 17 DAt BmR'lfi ~'OO R OVARY \*l1.U JmPOSI'lS :m PATCHSS D !!f& MlDuu:.A. AID IN ~ ADdACEHf ~ ••••••••••••••••••••••••••••••••••••• 43
13. Im:lG MAGH.tFlCA.!J.O,W or SICTlOtl or OVARY SlOlH III FIGURE 12 ~ CI~ OF GRMlJtAR PATams ••••••••••••••••••••••••••• 43
14. EXPBBl!mtl1'AL ~ or A 3'J DAY msm 5HOWDIl QfWIJJ..A'l1OU ~Ll IN 'J.Hi: cmf1BR (JI 1HIS 0BGA..l1...... ••••••••••.•• ...... .•••• 44
lS. ~ ~C'llOU OF A 20 DAY OVARY ~ BILA'flVJ.LI ~ <XIt'l'£I All) LOOSLY ~ ~ \-lIm A PATCHt QRAII.tLI ~~ ••••••••••••••••••••••••••••••••••••••••••••••••••••• 44
It i8 wll known that. in at aut ~ of the wrtobratea. ax b()l'r.acm~8
aN .l..abontod b)* U. gonad. d.urln,g t..b& e~ period.. how" ... the exact
time in d,eft1opMnt at whickl the_ ~Ma &1"e firat prod.u.e~ is not known
'With 0el"ta:i.nt3_ U1 .. ocheruical and bioobem1oal method. should :reveal their
pre_Me earl.:i.er in a.m.-nt t.llal bioulNII.)" t.ec.bni..... Howver I this i8
not. the caso with ngud to the eb1Qk ~J70 gmad.
In t.he cidck ~ !e.!!.Y2. tranaplantation ~ilMtni6 l'iaYlt indicated
tbfi initial syntlles1a ard 1"181 ... at IIIaX honnone8 07 the t"Onflda at &1&ht dqs
(Wer.1i1}ir. 1,65).. On t.he ~ han4, b1ocham.1cal t.ec~$ hAve not. disclosed
the pt"eG8nOe of e.vopmr in embryonic. chick gonad exil"lIOta Wltil the tenth
dq of incubation (aaliien atd 14 FouJ.goo, 19'7).
Histoci~eal tGoh~8 llaw not NV8~ at4re1d honnc.me S1llthests
prior to t.be eigbth d.a7 of incab\t.Um 1n ()rJ.ek flmbl",J'OS. Th0 p ... 8ilmCe of
histoob&mi~ally demonst.l"able cholesterol haG btMn Nportod at .ight dill_ in
the eontde of both 8I!:lO)$ (Cbieffi !tAl., 19114),. &in at oigbt dqtJ in the
ovary al'Jit4n dqa Ulthtt tGet10 (Narb&.tts and sabatini. 196;3). L1 5_~t,!y", r.:
pa~at$Nid dO~gt'lnQe (..6 J_)j3-HS» e.cti vit7 has been reported in U.
~'nbl'7on1~ g<».aa of both seD& 1'l"Ol~ right da.?:s ol.!W8.rC:t by Chietti (1964), whil$
Narba1ts am. Jtolodxqr (l<}64) f1nt obnr'Md tJw p.t'I)I!leJ"lCO Qt~ this en~ at
~ight days in t;,be ovwy m1 at tw 4&.rB in t.he tonie.
Tn. abeence of ldst.oo~1e&J.l;r <iemo:riStrablA /j.,5 ... 3J3-HSD act! vity in the
~on_. ot tbe ohick ~ betore $1,bt dqs ot incubation rtlJJ:j ponibly ~
1
ascriOOd. to the fact that this ~thod is a t~5hQld reaction and the enz;tlso
r::!J.y ·be present before this time but uot avail.able in su1'fici~:::lt quantities
for reduction of the tetruolltWl salt. Al&o, other 1nve31#igators may llot
have cut their sections thick OU()l.tgh.. or l...'1cubated thola long enough ill ttB3
A rec0ntly developed it:munotl udr'08eent technique for the histological
visualization of androf,'8nic sun'oids (Weoos arld Dotm" 1966) illl probably,. like
many l.rnt"d.tflological procedures, n sene.itiw and speoific indication for this
one class of steroids a.nd ths ell1Jl.u.sion of others .. ~
Tl';.t;~' -~HSD teehnique, demonstrating u it does an essential step il'l r
th~ sy.l1thesi5 of !Steroid hCrfJQI"AS .. genel"&l1y can provide a broeder spect~ of
ird'or:;;ation concerninz steroid hOl'ZiOno synt~$is than the i1'r!TI1U!wfluoreecllOlnt
a bio-s)"nthBtic pathwe.y does not :1$¢essarily in.~ successful syntto:.sis nor
release of termed horm:ar.e (Lobel !!. !l., 1962). ii{oreo'Wll'. this teohnique
does not provide information as to the chUacte%' of th~ elaborated steroid
tion of the hiatochl.!)mica.1. aspects of this problem 'WOW-a. settlll to be indicated.
In the present study, chick embryo ~;onad sections _r~ ~;d.ned for the l'
presence of 6' -3p-HSD in order to dourrdno tl» period durini:: e'fhryonie
devslop::;cnt 1'li116:i steroid hOrJUOlW syntheais first OCC1U"f> loll these organs.
1b8 t1Nt ftOOI"docl ~ .. at.W1baUna .. d1tt ... nt1&Uon to h0r
mone. ~1ng frrc:D t.b8 ~c ~ .. that of Bad,n an4 Ancel (1903).
bee ~ ........ 1nkNUual ..ua of L~ 10 • ., .. toI7
Rate in t.I8 tuU.. of \be 30 - pl& ambr;o.
The tint ~ 1n ~ of tb1a ~.u .... ~ bT 1.1111.
(1916, 1917) ad Iell.er -' ~ (1916) 1f1th"saN to the ~
of ~_ b oat.u.. !be7 ~ that. the ~d.c ~ .. of
tw1na oooatd«tllq ... eo clolel,r ueooiatecl tbat ~e or .......... and bloocl W88Dla ooOVNd, ttl&e ,..t.ttins • miI::I.ns of t.bIJ1r blood. U the
t1ld.ne ... a ~ ptilI-, the m&l.ta ckmtlopIcl ~ 1IIbUe t.ba ~
cmoU". ~ of tile t.ale .. ~ Of' ......... to be an ~
The non;al de'talbpi8llt of tlw 0'IW1an corte:& -.t of the MuJ.l.u:lan dwJt.a WN
1nb1b1W ad ~ aid ~ of tho Woltt.lan dwJta ...
t.u1t.at.ed in the fa:il&l.e. In .. at ... to .. 1 .... n th18 C«d1t1011 the ..
1nve8ti.ptoN poatul.at.ed that. 1n &lOb cue. tho male boNoue lIM pI'OCIwIe4 1D
the ~ prior to that or t.be tflD'la1e boI'morI8 arxl t.We U8I'ted it. iDtl.l.\fImce
.arliel' in ~ t.tmt. \"he pba~ c:U4 not OCCNl' in tbe "nee
of ~ anutaaoIG. ot ~ ~.
A e:lmSl .. ~ OCOUJIG, but to ala ..... 8Jdient, in tt. pis (Hu.gbee,
1«;29). Mekcd. (19". 19'5) &leo noted tbe ~iD ~ in cattle,
but d,i..1 tif't. oCana it 1n ~p .or f:oats# &ppaI'Ontly a-"e to a e~"ly
at. (,)stabll~nt of vas~lW t\.~c$1s of 8t'~n1Q ::mlb~. in the
Ul.l1o (1917) stated that it vou.ld be ~oNW7 t.o ~w hie theory
~ntally btU'ct"O it could ~ aceop~.
S1ne~ t~ p1"$_nt in"feAiL:~at1otj. 1lU 'bien lir.dtod to too b1.rd, tbt rol
lawJ..,.~ liturature l"evi\R( j.s altIO sc linited.
Pztucf of t.he pN_:n@ of .~do tIlO.Z j;tOrli.\Ol~ ~s1s iIJ bes1c to
tho ellt.abllst.lll.lO~t 01' t.h~ r~lOnal tl~ of G«X d1tt$l"entiat1Qn. Sewral
~ae.lw. haN b«tn el~oyed by a 1~ (If i;:1WGt~"'atOMS in an $lfOl"'t to
e~li$b t!» prcoduutj.O!l of the~ h~"'tiI)f~s dul"J..'1S, the q::i:lryor.d.c period.
1~\i0ra.'1O of oldck «;;1n706 (1 .. .1t~ra, 1921; ~A.~, 1925; ~, 1927~
"'~w.. 1927; jj~y, 1941). \-;llliol" (ll)Zt) craf"tod u.odiftfi'ei:ft.1at''id" as
!Jr..-llMy, O,941}. :";'Jti~j QI~;:a.tdQaal l·fJl1.ird_tlo~~ oJ.' testes i..u1der the
innu.~ of ~~ o~ ga.t~ O';1to the cbcri~\tcdc tr~~ of boat
tor tJa theoI7 tIlat .. hoII.Iond ... ~ trsr .. d1tt~.
vb:Ue t,he IIIOI'k of ~. of Kemp ani ot Wills ..... a flOt, ~ th1a
theol7.
Woltt (1941) ~t.d. ~ tftD 6-U .,. ~ We the coelan
of 48-52 1lQUI'...,.,.. He obtai DId femtmM.tion o! l1o$t. tAotoll (dow~
of a oort.a:) tv ~ 01'11'188 atd 1r.h1biUon of ~ or the
~ duct8 o! ~ hoGto b:r ~ tootoa.
a.o.nend1jK.adJ-.s (1960) ~ ~ ob1ck gen. l'Nrt
3-6 _ doI1OI'8 bt.o the ~ of 4-8 ..,. boat.. S:1Me _ dAt~ ~ pt.beftd.
_.tare u. al.nmtb _ of lDGu.bat1c:m of an.v hoA. t:bfI NaIlta 1rJd1eate that
with tI8 ~ safte hOftiOD8 ~ took p.lale ., an wdetelrd.Md
U. bot' .. t.be t.wlttb .,. of~. \ld.a ~igAt.cr alAo made
ebor.ln-ell_otc ~J how ... onlf an :.I.nf:3cqJl.et.e ftduct.1on of Mull.
man duet. we ~ Tb1a can41t1on .. con.a!d.ele4 to be due to the
tact. tb& 1t _ iapoatd:blo to \NDaplant enou&b t.e1lUa onto ,be ciaorio
allantoio ....... to .... CICIDpleM Nd.w:t1on of tb1a 4uot, .,....
2. lh!!! laMIa IiIl ~1.
'l'tIe reaut.e of t.hrI M JiL!a rutUR of ~ eSde b7 a:lcle. cd or __ a and taJwrt, ~ ~ etrictl,r tndit.1ona1 ~ to'
0ftIUn and Jll8fIO'altnt.at1on 'b7 teat1tN1aP.~ bea ft8\llt. ....
simSl .. to tho_ ~ tltca outNtion uparimanta. ~ toe aot.1on
01 ... ~. on ~t. 0Jtg8n8 1. 1nteI't ..... 1d.th b7 ~ thea
...... 1on tbftA&gb ~n (Woltt .... Woltt. 1949, 1951) oro .. ~ the
twett organa atd CNlUv.at1ng them a !Ua the a-e1lllU .. the _. Talpt
~OF~;.');t 1..'1 tl» di~ut.ion of the $IX cr tf~<:ultiU"e bcxn:£t<')~ (V(oltt !!.~.,
19521 Lut~-oDto.rt_, 195£,).
o~:le6 !e. !.~ and UtArwat'd t..~ t,r.e l~iJ.!~ tfA" estro,slO-n$ by tkw lli'ln
lkli"1 ~t.hoo. Esi;.;ro,sen.e ~ fil-st d~tt~c~ in. a l~iiEI. in ud . .ch ~v~n ~
ovlF.1.cl int'lua~ in tranaple...'1t. and U$8U0 cult~ ~rlr"lfIntG. Altll.<xlgl~
t~lGN .. some di!t~nCGa. the 8ifdlar1ty is quit.. evidoant.
In ~J ~etrot~t'm$ "n _dn~ in ~_ly emb~li:, lite fentin
~ 'to:' teawa by bI'1~r.,!. about tn. di.r!~l¥mt.:UIt1on of a C(~l't.u. &14 in
extl"W".JJ ca.., by <w.~,ing the ~ot;Gel ch.ttNct~"111tio8 oj' th$ tttlttis
80 th4lt it 'tIifae ind.i.t.i."'l~ehaba trm. an~. In f~~B" the right
~erian dttct., Wd.t::h nm"'~ u:n&~rt:oe. inv~l;utiont ie ~ntn1t_ and both
l¥lebt L"ld ~tt duct 1.11\). h~"pe~ d~ndjng on t.be dO~$.ini~
(D4ntt'Jbe1toft; \!!illlVt Gel.lat!ner and Koi.t.b; \~}olf':t and Ointlll'lt~:t'. 19~5).
iiGn-* d.1ft~'ntiat1011 0% t.he ~$ .. not afr$\ltod or c.b&~. ~mt
(1923), ~~a (1m), T~r. (19;4) .. ~d 1..ol-rh14 (19;~5), st~t that el¥trugona
~ to be ....,apor~sihl$ tor t~ .inhibition G.t' ~h .tld diffmti«LiO't) of the
right G\S"'lJ. Weltt.1d ~'f (l951), otl t~ ~:#tt14r b4."lI1. N~ t.hat the
gr~1&l f:}pitl'~,llw;i 1IU t~tatl'Jedaild di.ff61"'1U'ttiat4d irlto an ovar1ari cor+~x
on tl1G :rigbt $1dli _~~ 'MY injef.l'.ted e$t.rot;:er.:.s itlto tikl$ ~d}l70 ootoN tliA11
tou.rtb dq. I4vis (1'46) ~ LMd. •• Jd tlcr~l (1948) t~ ct.lOK ~"ll)l"1O. (ff
4 ... 10 dars lJw\t'baUoti. 'Id tkt ~atl"i)gan$ &~ obtaL"'led .. InodWeat.1on o1~ the
~~ .:Jd [{lOO:l.i.tal. t~;'lbe~ in t,.~ t~.:J.u ~.:¢tlot); .. 1micAUng that trt;$
~~ t~ff.al!lil ~te .. 1. de·tWi~,1twd by ~~~:6ru.}. t:dn~ tk&8SI'J retlfl.llts ~
mr..il..-to tho. ~ in ne1"'rn.u deV".)l~nt, t~y ~ to ;uldic~ tna.t
the ~j'Onl.c 8.0:*18 ~ ar1 1:ntb.at\ilC" on tWX dif:t$r.nt,iation u a l"'9$'J.lt of
the .~$1s erld Nl.ease of. $iX r~lOoo".
'tb.e N.u.t"" of ~~rir.r.ente in lrIt..ich atl4rot~ns _" ~.1n.1.ate~ VtJ
len ele~t at¥! not oam!; ~l«i!}.ed. l.ee,w.te~'te.ld ardrostJ\1l.rona WiU"~
ob$1)r'V~ to .Lir~ tf~ l;foltfiAr!. dU.ct$ C:;' both ~., (Dant()hAl<ott; l'lj.llif.!:,r",
001 .... a.n4 I'oeh.; WQ1ff aat. OlDgll ..... , 193') .. w.u .. the Mul.lerian
4u.ctfJ 1n ~ ~, ~ ... ob~ to 08W'IWI the 1nei ..
p1ellt acrtex of 'tiM left teat1e to 41.ftaWlt1ate 1ato a Slau'. CCI."tex, tbWI
~. about tbe __ lqaerJt of a twtca.1. ovoteat1a.
AltbouP ~ NaUlta .. aot too Wll cIef'1M4; ......... 1aU.oat1caa
tbat t.be et.tect.a at ,*,Id~ _I'" 1'81. 1'·18 t.bI:tIae of the aallOC1atJ.-.
~ ...... JA ~lD .wi !I. I£CI, ~l¥ v1tb ~ to ~
(lcIelJra IDl Qall .... , 1934; ~, 193', Wol1't 8DIl OIDSU~, .193'1
WUU .. 11 11., .1931; 1.Md.a, l.~). 1'De tact that tbe ftfNl.t.a do DOt eari •• •
pQrI4 CCIIIJ~ .., taUcate that tM _ .. al efteota .... !B. v;l;ta .. !a vivo aft tJ.-. ft8U1te of _ ~1cD ot ........... ~ _ tile
___ ...." .. ~. i!ae puri.'t7 at t.be .'.tld ......... ~ IIltIV alIJo
b.twIt bela .. cr1t1Cal ~.
1..eftI7 (l9It8) extnct.e4 .. ~ tNIa cb1ak ..."..,. of 14-19 cJa.l'8
~ tlbiOb. we active CD tbI CCIIb of tbe oepGD, UI41cat1D6 tl1e pre_ ..
OIl ......... UIJ.11Ia w.ocm.sca1. ....... , Stoll aa4 ~ (1956) ~
the al~c 8D4 .... ot1c tl.u1d8 or eb1ck ....,. fat! .11"~.
'.l'.hcee of tbaIe ........ ~li_ ~ .... n.nt ~ at 81x
.. , tba tIl'IDUIlt s.aar .... _ f)!(D ..... to 13 cII.\Ya. 00"
• aa4 lAJ ~
(1m) uae4 ~ .. ~ to detect. -.t .Ii"" pbar.aoU.c .tar
o14a ~ trr.a ovarSM of 10, 13 rat 2l. _ 014 cb1ck.....,.. TbNIt
atero1U ... ~ to ~ 1D ~ td.tb ~ ...
9
Tbe tJ.rat 1'd.lIFtCCbeIdcal ~QD, ~ haI'IIale p!I."OduatioD b7
the ~c .... !avo1.W14 tM 1aIDt1t1cat1_ of l1p148, .. cbolestAl'ol, tbe
pl"1I8'7 ~ of all ............
lDltlal13 SCbe1b (l956a, b) 1mut.l __ tot&l. l1pl4 CQ'I1teDt 1a 6, 12,
14, 18 aD4 GO _ Gl4 ah1c1r. ...,.. ~ SUda B. L.1.p14a ... v18ual1.a. .f.n
tile ...... at aU __ ....... 8D4 ... ~ ~ to tbe ~ cc:a
~ of bOth ...... 1tWI ~ lAttII' (8cbe1b, J.m) tBdD84 U.
paCta of ~ t.rcIr4 WI8l ... of lDoubatica to batCJdns b7 .... at Su4ttD
Ift;aSDJas- De DOte4 t.bat l:lptu ftnt ~ 18 tbe .. .tun.,. ........ fJ.t tbe
teat1a GIl .. ...,., ... til t.be .cluJ 1.,. ~It; at tbe fNf!Il"/I, "at tbe U.
of ~OCSea.1. _ ~1a\."
~t. aa4 Sabat:"" (l963&) t....s tbe audaD BlMk ~ Ie u.ptU
8124 the 41s1tca1a ~ 'bM ~ _ tile .... of cld.dt ...,.. of
1-12 .. r:4 i._.lbat1cll. III tbe fI'ftI/I:"1 both ~ 'I111III'8 leaD em ta. 81(#1tb
.., of 1Bcubatlca, 1tb1lAt SA tile ~ ~ .. WN clI.a"Ye4 em",,~, but
~ .. ___ uatU the teatb _.
Jlar!batta .. SOatsaS (l,slb) l"8IIIIMI!4 aaaaI. t'I.'cIa Cb1ck ...,.. at a1x
8l1l&I ... __ fJIp .. cult ... tbam Ja n;&rg tcr tour..,... ~ .......... ..... D1J4 _ ._ of tJ.18 41s1tGD1D tedad..- rar cbolestAl'ol. b NINlta
4lUeTea ~ ...... tns CD Wb1ch at tw CNltUftt laU .... ~.
CbOleeterOl 8plC!ftc pnc1pit..attan we .. __ oa the s1:atJl _ 1a &IV of t.be
aa-l. oulture4 til Voltt ... ~'. (19;2) ~ ..u.., but. 1t .. .-
1A,. or tile C8IM cult.\n4 best-as OIl tbI ~ dIV'. c. the ~ ... ,
10
a lIPIC1t1c Pl"'8c1pitat1oD .. aeeIl 1D ~ or the .... CUl.ture4 18 steDaItr
Battea'. lfo. It6 entbet1c ..u., (BteDaar ad. BafteD 1957) beginoiag vlth
the .uth ..,., aa4 ill e» of tbe .... eultun4 befJSnrt111S vlt.h the aewmth
cIIV. The poa1t1ve :nact1c:m ".. ~ 41at1"1'bute4 ~ the tHt1a,
but ill the 0Nr¥ 1t .. l1Id.te4 to the .. 'Unary ~. TIIe.11mtattp.
ten ... DOt able to ......... tile leX til tile ..... Sa aU 1rIataI2ce8, but
the7 attr1bute4 the laeJl fit ~ ~ fit their l'UUltt to .. ttrad'Di
~ feacr1be4 b7 IaJo'batta ..... ...,....D1 (1963&), -1J", tbat tbe fI'I8r1
..... the pn...,. of ~ t'tIO ........ u. tba the tNti.. !Ilua it
...... tbat, depeDls ... CD tile ..u_ ute4 r_ culture, tbe pIN .... of
chole~ 08Il be ~ 1a tis ...... 4a¥ 014 ......,aa1e ctd.ck ......
wb1ch bMe .... CNl.tuN4 !a n:ta tar tour..,.. Cb1eff1!1!:\.., (~) alao
u..a the 8u4aa B teebD1 .. tt:w l1p!.... Be ~ ..... at ch10lt --OJOI of
It. to 7, 7i, 8, 10, la, 15, 17 .. 2l ... of ...... obtene4 potiUve Nee-
tiC121 1Jes;twnc with 'the e1&tltb _ ot 1BOUb&tlcn_
lIIr'ba1t ... 1ol0lllW (1gtM), - Cb1ett1 !1 !l., (1g6Jt.) _ .. 11_ .......
of cb:1ck ~ foil tbe pre ... of tJ.~4 ~8Ia ••
(b.'."'BBD). '.l'bU ~ ~ the CCIIlV8N1eG otd-~ IIter014a
toA""3.~ <-'11 !1 11-, 1951). S1Dce the active f'GI'Il of' au . ,.
Ifte'r014 1Knacae. 11 the is. -3-keto ten, .. i. d8r1ve4 ftiCII a precut"IIQl" or th1. fta'Ia, 1t 1 • .,.-.at that U -3p-lIID 1. _ e1ll8Dt1al, ~ in the b1o
~e ~ Of 8tero14 I.1ta'lIICD88. 5!leretWe, 1t 11 pl"UeIlt Sa au tt ......... artaro14 ..... ~a1. ta1rea place. ...be.tt. 8DI1 EAlO!!b7¥
:tapte4 that tbe ..,. WI __ 1D the 0V8I!1 at e1gbt tap ot 1Dcubat1cm
aa4 18 the teat1. bq1D1daa at tea ..,.. tbue ~ pnv10ua l"ejOI"ta
u
~ tile ,,-.... fit Up""" _ ~ (~" ... .........sid, )9Sl&, b). CIdefft .. J:J:.., (1.96'), ",., NJICIl't.1 1_ JI'8 .... sa l:M:t\t.l
C7IfJt:':t ... t.Mt18 ~1 .. at e1Sb't __ of i~.
DIe teab1d .. ,.. Aift01OSS.Jl¥ -n.-.USS .. "- _1 .. Me ot6'''~ aoUv.s.'Q' I wll.lfW4 ita 'tilda ~ .. 'tJwt tit ~ (l958) ..
IIOIU.t1e4 Iv 'I.eY11l. &, (~). .....u. .... mil III: ts .... , t.'-i~
~ 11a • .u.. OC'IIt4dalDl ..... ~ < ........ j ............ , ........
....,. ...... ), 1d00'U ........ cli ... leotitlt (:lAD), lNftIII" (:pi &'0), a
tcttI'lilOU .. _t. ( ...... ) .... 1IOl\Wt. <...-.). DIe 1VOl8S11: lCDJ • ..."..,
tJNlI .. ~ tv _ ~1J11" .... ~ to tbe aD, U. ,.,.
tae JIUII to tJIa tetzuoU._ Rlt 1Ib.1cI:1l, ~ U. ~ ..... 1u
f-. 8DI4 ~te.te4 ... .-ular tar ...... lIItloat1_ CIIllvl..w local.:I..u..
of .... ~. ~ ~ tile p .... f1I • poeiUw ~ ita
tJIa ~ of 1M *-1 ....... , !atent2..'UAl oe.Ua or ~ of tile -..u., .....,. cella of tbiflllflr:f, .... Jrl~ 18 t.te cel.la or .. U ... ~
lIIf'I7llll-.. (l9J9) IIOIU.t1e4 vat ..... '. zwtIa04 _ MItt1tu't1D8 pro.
WlaDe ~ :. ..... , ~ _ .u. at. • l.oIer pi! (7.1-7.4), ..
.,.,."., ... CKaOIIltiI"att1C1D8 of DAD, I1tro-Br .... ~. stldj .. ,... ...
.. rat a'INDala" ..... el tile ___ till lIoUl..... TbeIe ~
~ va~. fSDtUap, lNt. 'tbe7 &1.10 .... ,... • pott1U_ ~
18 tba epi'tbeUal cella of _ CW'14:uct.
A anat c1e&1. of ll' .. ~., 1IlI:ft .. 1:JeeA .. _ • )'11...- of ~tcn
(wnw .... ar:Lt.t1tba, JS(i4j CIdefft 11 &1 1~1 Iar1Md.ts aD41:Ol.oclaV, l~$
12
Preol. 1IIl'f-. a)..". 19$; Ittlbin fit al.. 196;; Ba.1J 11.0 ot. &1... 1965-l966; Ool4raaD ~ ........ ' ........... ' ............ '
!1 !:!:..~ 1966). at fI.POC1aJ. in:t ... 'Oat 10 tbrI st~ of Baillie &D4 ~tba
(1~), 1n wh1cb both ~~ (DBA) aad. ~ll.xw Wft uae4
a.a subatrate. tc.: tbI h1~eal ~ of 6.' -3p-lIJ'J) aetlvttJ in tta
1rltera'Ut1a1 oelJ.a CIt l8Jt.U& of teta11l1.01.We teatee. WbeD pre.-olex» we
~ .... subatrate, .. poa,tl". b1atooJwD1cal NaCtlC11 .. ob8erwI4 tour
..,. ... l1er 1D .... l~ U. (U clap) tba vbeD DBA. ... uee4 ... the
subatrate (1; ..,>. Tbwt, It ~. tIta either pftSMDOlane 18 a ...
eptC1ftc subatrate tar 1:':.; -3p-BSD, C8' tbat tbaN ... ~ two ____
1DYol'Vd, tbe cr.e wb1dl utU1_ ~c.me beiDs pnteBt at a ~
~c ... , at. vb1cb U. the ___ 1Ib1cb utW •• DBA .. ~ 18
~.
• of the ~ at. tbe tt. ~ ......ut0l ~ _ actual tSIII tM
............. in tl8~. ~ ..... MGl'iftce4 trcra .. ~
tbrousb ba'tch1D8. 'lbe .... of tbe ... lJeat ...,.. WN "NI'1tSe4 b7" of
tbe etec11DS tecbD.1t.ue till ~ ... u.uta (1951). SaaI:1.ftcea,.. per-
torr ... vltb1n tour boun fit the &I,peC1fte4 maber 01 aa,a. All st+asSDS aDd.
lum4UDl of ti-..a __ ftJr ~ puI'JOINMI .. C8ft"1e4 out .. ~
.. poatdh1.e, ..,., in het, _ ef'tort ...... to 0III!Il7 ~ tbl. ~CI.\
w:tb1D tt_ adDU:&:" of ~ of ~.
00DaclII wre ~ ill 'tIrI:M...,.: (1) !be f1Nt of tIaut ... by
tftIea1Qs the erat1ft ....,., 11& _ uteIJde4 poelt1cm .. the f1'eeUD8 IItaaI of a
~. (2) GaDa4e wn NCOWIIN4 attacbe4 to tbe ....... 'boJl'.u. A
Net_suI .. fJiICt1CIl till the cIcII!'Ml ~ wJ.l, nth tile ~ V8Atnl to tbe
E:JC2IDI48 aa4 the ek1Il of tbe .....". IItJrt8CIt 1'eIIIWe4 to obta1n bettal' 11qp14
ccatact tt1f' ~ .. m.ID 1"."j"~ a...1111de ctorm. .. tbe ftwz1Ds
.taae till a~. 1'bNe t,i ... 'block8 wn ...... W'IWPI4 v1th multiple
l.ele1'8 of ne:.1dble plaatic ... aa4 atoI!'e4 111 aeale4 bottle8 at. - fIlIPc. ~
~ aect1adns. (3) 'l'Iae ........ wn u..ate4, -iDs cafttul to
~ "t"Mm frcm ed3aoertt t.1aue.. '!be ftnt &CD84 r""""" we p1 .... GIl a
at«Saleu 8teel wi8bSus ap&tUla 8Dl f'loode4 v1th a 4I'op of col4 ilOtcXt1c
eel'ue. 1'be aeCCD1 CIIB .. ~ and placed 1.a the _ drqp at HlS ...
'fbe ..... &1 ... tMD plMeI. 1D tt. ~I ......... IIOletUN we
''',. ~ pr:Lor to ~ tw ~UDs u. .... fill tb8 ~ OIl __
1d.tIl ~ ~. Be ...... ,.. ~ to tI» fI._tas 8'teat,
.1~ b:f J.:I.f'Uas ~ AuI ... ~lla Wltb t.be ___ at .. __ til 411:1
......,. tcMtl ... _ t.be :pc'.d.aU OIl .. pea, of ...... ~.ff ~ Ja owta.lD
ca.a u.a ..... pla'le4 \WCIl tile ... ...rea. ., tbe ofJ_1Q1 ataea vIdle
twl&Saa .", cepillal7 ....,. fl'OI tbe ~ III aU .... atw .....
• ...,... ... to 'Cbe ~ 8tia8It t..t. ....... mil. vlwa ~ ..
88CCIIIlt • ts.. •
... ta. SaoJ.at.a ........ too -.u to __ ~ CD • .,..
stet ~ .. ~ _ .... ~ u. WIUJ4 .u. ,. J"IpS4 ..... .., '._U'D8 ot the u. ___ ......... 1'-~ at __ p1ecea 1/11 t1_ .. CIQD8f~
but 4'~ ___ tit .. lMk of ~_ ot tile ........ of
,..,1 Sill. OelaUa .. ~ ~, ...... IDl tn •• to IIbapa
~ ~ tlae _Ill'.' .. tt. -...., .... uUl1aa4. bee ........
po •• e. tbI ~OII 011 bOth UQi~:"'" ... lIDUId,*, a'I&lJ/IlIbtl1t7.
~ 8UCb ~ ... '-' ~ tb1a .-,... A,. ~ _ .. latta
iD ... :I.e .... 11114 1:>7 .... me ..... ~ta h1ock..... _NIl,
.... tld.8 .... tI"aU.cJD .. ~I tbe ~ ... e1~ Cl'.llll,W'II:I8'l ..
cau.l4 ... be olmISaaI s.a .nat.~. .... 'tile pNbl_ ., .. to be GlIB flit
too .,.. a tJ.exllxUS. of U. blDc:Ik, .. ~_ oIIIJtlat1D .. ~
to a.5- fbl8 ~.. I!bat .... ~ ~ ""*' bloc ICII,
but *- bloo1ctt -.1te4 too li.,iJ¥ ...... ,.. ~.
A c."nlrrc1a1.MtIIUIII.a_ ... __ ~ ~.iOD" Mila
88CtSOldIlS (~'O.C.~.") .. touId to ___ taut 1'4MU1ta. ~.as.. ie
avatlM1e 111 tiI&W snIe'u 1..1 II aid Ill" 10 "._DB .... of .... _
ftC(I fl$ld ftJI: 1te '*_ Ia OUI" __ 1:Iotb O.c.!J!. I (tea' te'l ....... of tP to-15Ocl.) tDl 0.0.':. II (,.~ erE -1, to -](Pc.) __ .... ..
pI'O..a ".". ~ __ \ad td.tb.1a tbIt .......... t.~ ... .
fJ1e o.O.t. I ..uua we N"" .-~ ~ tbe o.C.T. a tar
~ ....... , (1) __ ~ .... of cP to -UOc .... Q1t,~ cut
ft8UJ.te _ttl "Irlal ~ (2) t.bMe bJ.oaU cti4 _.-l.t UI'fI8ISl¥--
Adl., fIDI (:S) t.be bS ...................... :I~ ~ 1v the
~ ..,.d\A8 til ... ~ 1'0 PIli'" tbe bl.ocIt.fJ, a 1lOl4 ....
~ a atI1p of~" .. 1'1 ........ the ..... CD tbe ~
...... _ • ...w_ .as_ JQIt.I'N4 iatO .. I8l4. A ~ ~
~ to tile .u of ... ~ .. tbaD ~ QIIto t.be top at ...
tbe .-1.4 ~ .., .. tbe ~ ...,... iA slt1ple ~ or ~ ...... rt .. '* 1IIbel8d ... ~ SA ,. ••• 184 bottle at -acPc. tfIr future
~.
ft'C •• 11 b10ab .... iDiti&11¥ <Nt CD a 14,-. fnlellr1D8~. a.-.-., _ ~ 0gt10a1. fI~l ........ ,.. tba .... pQI'UcIt or 0Qtt
~1GD. seotiaa8 tJt a to laO ..... W4}la If. ,.. <Nt ~ ., ....
poeaSble, in ~. lbHe ~ ... e1~ l.ifte4 onto til __ ...oIdl) ..
.ua. til! ~, 'tiJb.f.eh ... tb8l ftln-aPRd A'QI ..... ___ cal tbe BeCJt1aD
all ... to 4r'/I CI'l the al.Sde, or a .... al148 we ~ 1ato ~ w1th
the ~ ti-. ~ to t.ba kDU'e. • U ... .uoe ...... to .. ~ted
CD tile fIlJ.cIe. AU ~ .... aw...arJ.tN1 ~ at lea8t f'J.tteeb ~.
'l'be toU_DB teatatq,ue we ~ to 4I'lRId_ ..... r.A5 .. 3p-lIJ0
16
aoUv1tl_ 5l1des wJ.th ~nt fBCtiona'iJUN placed. in & ~ (,:r,OC.)
butt.. solution (pi 7.2 dO.1) tor 15 to 30 minute. in oNer to ~ elldo
~ ~te8 (wat~ 1958) and to d1arolw the material used .. a
aJppOrt tor t.be t.181Wt l!lb1l.e outt,1ng. Follow.ll:lg We, 88Ct:1ona WI"e .1ncu.ba
ted in the a&betnte med1\Sl tor periocla at one-balt to twnty..ei_ houra.
In t.he majorit;r of 0". tbe ~ time we one~. Prol.ooged incu
bation periacta 1IIIIID'e u.eed tor gonads fran 1OWlSGI' ~8 in oJIdW to ccmpen
sate tor the analJ .. C'flant.:1t,' 0: ~ present. ~~fCne (DHA
wu tho ~ ~ with ~ ~ eanSJlIiJd.. HQwwr, pNgnenolone
we ueed. ., an alt.erna.te ~ tor ~ of' the ~.. Tt.le 1ncul:tat1on
~um .. prepuw4 accOl"d1ng to the method ot la",- &.Il.., (1959), as
tollowe.
£I.l~ MRfllf. De~~ (DNA) 0.2 ~
or Pl'e~
N1cotll~ diwcleot1de 2.4 ~ (lAD)
Rlt..l'O-BT 1.0 ~
~ btltter (pH 7.2) 4.0 uJ.
1lI.~DQJ!
O.lIU
1then ina~O!1$ \\el"e cor:.f}leto, fNctio.'lIJ 1IfIN'e f':1aJd tor lS to )0 tfdn
utes 1n a 1~un.t oonoitst:1ne; ot 1"'" tonaalU~ S% glac1al acetic acid ard S~
of W eth31 alcohol, (F.A..A.), ~ :in tap water to nBiOV8 ros1daal. fia
tive .m $ta:I.ned far S-lO re:dnutes isl ~r'. AJ.:.un~. AttGr a
~ tap wat.er ~, t1._ .ctions WN de~ated in • gNded attPi •• of
al.oobola, cleared in qlem m:l ~ td.th llWleco
0VIIiI:'2e8 _ teatee __ a.1JIo '-18I!t.tItc1 18 pRaft'1Il erd 8taSIMa4 vl:tb u.r:te'
ieMtG'lf'UQ all EaGa _ p.8'pQ1 •• or _al h:.I.etolastce.l ~
11
m..to1~ «&eI1aau. _ ~...,. ... 08ft's.t out 'b7 .'EW ott ..
~ ~ ~ ~ at Ito, lOO .. 450, eD4lQlO X ~
t1caa. PIaOtoa1 ...... ..,. ........ § .. ~ .. Ifolaool .. t1la
am4 ~ t.t.lwe Ott ... t'.I..ft,b to ... ,~ft.ftb 011 ........
A t.otal of 49 prepaNticma of oldck embI70 gonad. were exavdned. 'i'he7
~ in ~ t:nD three to 20 ~ of 1ncu.bation. E1sl'rt of tbe. _1"8 teste
13 0"f8'P.L •• , atd Z1 of sona:ta pr.1or to au: diffe.rent1at.1on.
For t.be exaud nat,ion of cb:iok ~. JOWlPI" than ton 481'8, croea ,.0-t.100a of tbe entJ.re embz70 18n twnd to be t.be most oOllWnient. tOftl of pN
paNtion, in terrlJ8 of orientation of the gonada u _11 as their aaace1at1on
with other U ..... 1n the ~.
'l'l18 t1n •• tOUD4 to be .,mal tal ftat.1on 1n F .A.A. wre 1S miml.tes t
aact10na up to lS}l ... )0 mimlte. tor tboee Weier thin is micra.
__ ditteNnt1at.1on 1. -.id to begin en tl1e t1fth aI e1xth de.7 or incu
bation (Stdtt, 1916, llanberg Cl1 a.w.cId., 1938), the 88'fentb ..,. ("--,lanie
l886; P1zoloIt, 1914), or the e1ght.h .,. (1I'1rke't, 1920). In this ~ it ..
t1ret t«Uld to .. noticeable poeal.7 tOWIUd the end of tile ~ _ and
m:lol'OtlCopioa.l.l1' .. abGlt nine... The onton. tam:t moet uae:talin
d18t:1ngu1abing tbe au ot the ~ ....nuM were. (1) '.lbe pre_nee 1n
OYariea 01 a ~ 1dl1ch ...... nt or greatq J:I'I!Iduoed 1n teetea, al¥1 .. two
~ ll8dullI., tb8 inner ~ of 1IJb1ch bas m8n7 l.u>ge l8ounM, g1'V1ng it a
lao_ ~, the ou.ter ~ cona1st1ng or c~t Us.... After nine
daia t.be ecrtex 8bowa a .a0lldal7 prolUeraUon of CORle whicb haw 1nvag1na.
ted. fltcIn tJ» ~ ap1tb1U1a. (2) The tellte. pNaant a ~ more
~DeO\l.S picture. TheT oona1at entirely of a oonnecti'N t181W1 st.rcea in
to 1IJbich ~ (medIl118f7) ooI'd8 bava 1n~ .tJotcu the gernd..nal epithe-
18
19
lium pr.i.oIt to the ~ dIV. 'l.'he. 1,reclulllll'7 cOfda bN<:m» ..,. b,- deJ
eisbt, comrolutGCl b7 _ U, ard t'.1.nall7 branch, ~. am. tarm a,..U
oul..um. __ U. lJtb.,. (3) '1'be tNrtaoe of teste. 18 IBQOt,b &rd ~ by a
~ th1ckan04 twdca. ~ Wb1ch ~ 1ncNU1ngl.J ~
wlth advft.nc1ng ._ 1ibU.e tbt fNl'taoe of t.h8 0V8I7 18 'I.UWflJU, g1~ it a
roughened ~.
A. :a.. !i!&!&~ ~ t2£ LlS_~~
The resulta toll.ow1ng tho u_ of thie method aN moat _aningtul when
d.1v1ded into three period. ao~ to inoubation age J (1) tile period prior
to eex d.1tteNnt1at.1on, (2) the period. .tJrcm S8X illterent.1ation to dar 13 and
(.;) t.ho period ban .,. 1.3 to hatch1ng~ '.lbe time of eex d1tte ... nt1at,1on
1Dll4 .... to be a ~~. WtJ,al point ot division, sine" toll.ow1ng
th1. period the gonad • ..,. 4u.'terent ~ one another, .., well .. fran their
prre-d1tferen'tUt1on tOftll. Tbirteen d.Iq'8 of 1ncub&t1on 1s " aign1.r1cant
8eC01'd point. ot division DeC __ it divide. the incubation per1od., . fI'Cm the
time of eex d.1ft .. nt1ation to hatching, into t.wo equal. t.ime 1nteJ'Yals and
a1ao 18 t.!» -... at Wb1ch intentit1al t1&fJUe, otten aa1d to be involved in
stuooid hormone fql'!tbeG1s. 18 bel..itrNd to appear (SwUt, 1(16).
1. ll!1 hast S?! ltD.! 12m. ~ ~ Dir'.J.l!lJ!~ rue pt.t1'1od wu o~ b7 an .~ finding. A ~
ger.l8P&l1Rd pattern ot tOJlmllAn granule deposit.ion .. aoen tbrcNgnOu.t most
at the t~$ 1n Ol"O_eact1ons of tbfte dIq' ~., beoardng lese general-
izecl in d.1str.2.bltion on the tollotdng da¥a ot 1noabat.1on up through the
pericd ot JJEIIX .u,tterent1at.1on. 'l'1eales other than tm genital ridgea axh1b1
1ng t~ gnr:ulaa WfI8 the gut, notochord, flIIIUNl tube, spleen ~~,
atd tlw genoral._:nchJmal ti ..... (figs. 1 W 2). The pnital 1"1d.ge. I1howad
t~ d.epolitioA to ale ..... ext.ent than the otJ'lIN" tisouee mentioned. &rI1 to
the __ oxt.ent .. the ~ ._nctomal t1_u .. an:1 the cl.oaeq adJacent
doNal _ald.e17 (fig. 1). '!'be control al.1d.e. ohoNed an abfllUnce ot foru .• _,n depoa1t:lon in all cue. (fige. l en:l 4). The int.en81tl of J"GaotJ..on in tbe
eeotJ.ons ~dJ:»d ... rated acc~ to .. 0 to ++++ acal.8, (table 1). The
gonad. and adjacent roacUw t~. e:sb1b1ted an :t.m:.ene1tl' ot ++, 1I1b1le tbe
~ NactJ..ne duet ep1\hel1a ~ • +H- do~ of N&Cd.iYit.7. The.
tJ.eaue ~ WN made on whol.e ~ SlCtJ..ooa wb:.i.ch WH cut at a
thiclme .. or 30 P. ard ~ 1011/' ~l~"ly s1x ~ 1n the h:letocn.n;..
10al mod1um.
~l ~ £1w, t1» nacUon hid bec<lmIt lllON local1secl arr1 was fJefm w1th
an 1ntens1t1' of ++ in the gonada (ag. 5), ep1tbel1al. l.1niIl.8 of the stcmech,
.rd~al lJQer of the neural tube, lJoltt1an duct am. d1tf\uJe13 :in tho .eonapbro8, afIi with an :1ntenB1ty or +++ in the adrenals. Again, the control
BlCt.1on8 shotl.Jd • eotlPlete absence of e;rawle depotd.tion. Sect:.1ona or _ tour ~s bid an ~ ~&te between those ot dIo'e t..bNe &rd
fi:w.
At"t4lr dq tift, ttw weeonepbl*os, including the gona:Ia, was uaueJl.,.
ditJeeoted .t"l'ee ot ot..her t1aeuos in prepant.1on tor aadnation. It cantimled.
to allow. til» aD1 VGJ!'3 d1tfUae t~ d.epoedtion. Tb:ls react!ftDfUJ8 was
21
also ~ ~l'l itl tho roe~~ of control sl.1des. thus m:1.rdJ~ng
it. fd.grd..fioenoG 1n th15~. Adrenal tissue olton "'ca~ 88CI"&l aug
menta an::1 uwally ~ an 1ntenee ~t1on. Sections o! eonadl of a1x
~ n1M d.&)'$ at 1rl:u.bation ~ a ++ react1:wMaa wilen cut at. ten Jl
ard ~ in the 14atoe~al ~Wlt tor t.hnG~. ~Jo~._N
seen :1n tho dor8Il rteOtlntel'7 .. nG'l'lNl tu.be or ue_ncl~ at the. ages (ii{;s.
6 ani 7).
In ov~. ~ ~t1on abowad. e tefdenc7. wlth advancing aee. to
beoQiJ'iJ looal i zed in patchaa. pre8.UQ8bJ.N in tt. CORia, witbin the ~ of
tM 0"rIt!II7. Te8fAa cad:d,bU.ed a ~ d.epoa1t1cm of + to +v, Um1ted ~"iilly
to tlw ~ co.nla. It> aubatant1al1ncrea. .in tl» anount or acUvit.y in
OVS"1ea .'11 testes we aMIn dUJ"ing this age l"angu. ".tbi cont1"ol aUdea sbowd
no l"NCtion. T.l'Na, it ~ that e Nlat1wl.7 atab.l.e anount oZ &~ 1.
p$etmt 1n tbe 0VIII!:t be~n tbB pll"iod ot StU: d1tteNnt1at1on atd 13 ~
'l."be ovflr.l abow8 a ~ ina" __ in tbG eou..."1t or ... aot1v1t, ~t
tb1a poPiod (table 1). (f'lga. 8 &.'11 15). The reactivitl WM eeen ~ ex
al.tu:d.wl7:in the ~ ~ of tho oVlll!1. althol.lgb there also 'NUN
eome f~ depoa1te in tbe cort.ex. The depoe1t1on was ~11 heaV7 in
the Qlter, l~ ~ port1w or t.he ~~Jl817 c~ in the 14 dq ~
bu.t it ws al.8o .. n 1n ..u ~ or cells vJ.tbin the ~ corda of tile
inner lC'«le pcrticr. of the reedulll'. (rise. C w.l! S}.
It. tfJra"6.1.Ued rc..activ.neu .... ~ ~t the atror:ua at the teati.
with the ~at btena1t.I111 the eor4-. 'fb1e generalized reaction had an
1n'tcma1t)' jw:1gecl at........ An.~ 1nten8e ~t of t~ ~.
wu .. n in occu1on&l cella wit.hin the HX COld. of teetea. 'Ibi8 cwree
pcm48 to ~one in the tCllJM1e, 1dd.ch ooW.d parhapa be anticipated
Id.noe tho teat1a aDd tb8 OYal'1an llIKlul1. are ~ at.ru.ctuMa. Thi8
depoe1t. Me ReI1 at about +++ ~t71n a 16 de¥ tHUs (tis •• 10 and ll)~
17 ... ' 0'1_1 ard teati. (fig •• 12 ard 13). arid in a 20 .., 0YII7 and, teau. (t'1&a. 14 ad. lS). The 16 ., ta.tu ... Clit at 4. J1 .. :1.noubatGcl tor one
bctir IIb.i.l$ t,he 17 60Cl .ao daI gonMa .... cu.t at 10 mora ml incubated for
tbrIN hcQra.
PN~looa .. \l8t .. " ... b.-ttI tor 6 5_3jW_ wlth the hope tbat
the MUv.:lt1 of tb1. tlnfQt'llt cwl4 be traca4 to a ~ 1ncubat1on __ than
that ~ tIben 001 WM uertd, thus extending to ~ epec1ea tho ob88r
vat10M ~ by &aUl1.. a\11 Or.t.tt1t.he (1964) on the teatu of \be mouse
~. The Nault •• ~7('t Wft not .. ant.ie1p&ted. A raacUw ~
depc81t1O!:'l .. 8INn only 1,~ ce.l'tain .xpa'ri..trantel al1dee, whoN. en ~17
~ 1Ie.:tion (++++) WM ~ when DUA was ~ as tbJ ItIbstrate •
.An ~G 01 We 18 the Q8aII ot an adNnal .f.'Itom a 19 _ tema1lt ~
which reve.Qled ..... reaction With prif)gnenolO1W, wtdle with DHA tho .cUon
4'IPJ'l'Ituod ~ c~ 0J*Jl0 u a retNlt of t~., tleponta. The
conVole tor both the pI'8~ end twA ~nt. wn negat.1w.
'l'b1a ~1.Q.at1ta WI ~ tar the pUl'p08e of ~ b7
~eal _, ?be t1lll of 1D1t1a1. t:rtero14 hanaara ~ ill _
.... ~ tbe ch1*~.
b reaulta ~ with ~ ~_ tI.'Ine __ or 1DCUbatloD _
the PII'1Ol or .x ~1e.t1al (~ .... ~ 6-'1 &e.;rs) .... DOt _
___ tel • .6.'.~ ect1vJ.t,-, ~ lIIA .... ~, VM DOt 111111te4
'to ~ u. ... WSob .. Jmt.Ml to ~ tb18 ~ 1 ••• ,~, ~,
•• ".,.... .... 11,.. (la'¥J .. 11-, lm). _II c ... ~ wre &lao ......
~ tbIt aut" atUI'I4 tube, .. wU .. sa tbI ~, ~ ~
......" 1D bt..st7 1I1th _~ .. uatU ~ a. :tU'th ..., tbfr:f hal
~ tr. tbe ~, epl.eeD ~_, tbe ~ tube, UIOIIgt
tbe ~ laJc, eat the __ ~. 'l'bNe 48pQfJ1ta b.a4 &lao c1:l~
'tV W. t1me sa ~ ,,*, ~ ~ tba epl~lJal l:la1-. 1'b1a ~1at1_
sa tbeee tt_ Ie .. UIlUBU81 aids .... 'two pQII:lbla ~ JII\Q' be
011 ~~. 1tae tact that. tile "_ .. le8II 4.1.f'tel"eat.1ate4 1Jl
___ .. iQftft:l..t'e leae ~a1S_ 1a ~ ~ -v e.1.lt.IrI t'be t1MuM
to~ a L1' ... ~ ~DS~ sa"" ..... to
iIItlfr ...... GUbetftte. A eectOII4 ...-WUti' is t.bat the .6' -~ S. ..... .u~ 1'oLI.1d 1&\ mare ~a1SI84 loaat1cal 1n V8'I!7 ~~, ~ ~ of
tile leu ~lI3i_ ...... fill ~ ~ __ • 1'bU wu.l4 ~ a ~
'9
potential1ty of the tissue to 81Dtbee1- atel"oid hormones, ~ or which no
d~bt. 10_ this potential as thel bec~ spec1&Used. steroid hormone ~
theei. in the gODld.. alone probab17 take. place f,I"Id\lally and mq be indic ....
ted b7 the 1nereaae in granule deposition in the gonads aeen in oa.u- 1nwlti
gatim sho1"tly before the onset ot ee.x 41f'tennt1ation, t.hat i., at abotlt six
The Naulta wlth ~c gonad. at , •• bet .... n sex dif'teNntiat1on
am hatching aN atn1ghtt~ in nature. An apparentl,y st.ead117 increas
ing concentrat1on of ems,.. .. obeerwd in the gonad. with 1Idva.ncin& age.
Tbe tact that tbe 88X homIones __ to be IQnthosiACl moetl.J b1 the
meclul.la17 cemponent of the ovary or teet111 ~ to COtilim the reSlllts of
m :!.UD atw:U.ea in wh1eh ch1ek gcmala .... <NltllNCl in betero8lDWtl ccmb1n6-
Uon and gODlds and acC888Ol7 aez orpne vue aaaoe1atecl (Woltt ani Hatten,
1952, We~r, 1962). It .. tOWld that the principal bo~e lnvol'1Nd in
ditt.rent:1at1on of the reprodacti ft ~ in the chiok are the arr.trogena.
The Hu.ll.erian c1uct grew cd d1tterent1ated no1'mAll7 in the absence ot hor
memoe, 'tIlbUe the pre_nee of atxirogena cau.ae4 degeneration, as ~
OOCUl"S wben differentiation takes place in the male direction. Thaa, the
direction of d1tterent1at1on oan be 4eterm1ned b,. the pre_nee 01" .. nee Qf
male bomonee. The fact that the te.us i8 tem1n1zed by female honaonee ia
prob8bl..y not as 1mport.ant .. the Mullerian c1uct phenomenon, s1noe .. high
concentr&t1on of eet.rogena i8 not ~ enco.mt.ered in the male.
()uo reeulta, tJlen, contbm the .tind.1nga ot Clrl.ett1 !1 !l., (1964), am Narbaita am ~ (1964) 1riIho obaerftd the presence ot b,. S -lJl-HSl in the
25
~n1c gonad. :lJIued.iate17 after gonI1d d1tteNnt1ation m:.l thus ebortly prio .
to dUtaNnt1at:lon of the *ller1an ducta. li.CMt'YV, _ haw al80 .. ned
L1S_~ act1111t7 in the und1ttenmt:1at«1 8OD84. 'Wh:1ch would III'JPMI" to 1D11-
caw tbat tho ~lc mechald.J!me t(1l! steroid bol:'mcne avnthea18 are preeent
in the.. organa acme time in Id.vc!ce ot secretion of the sex boJ'morlea. This
i.e a paJ"t.:1oular17 important po1nt in t.bat it :18 .. ftI"1tication ot the timing
ot one event in .. ~ ot .... nt.. Betore the ~a can be Nle&Md
thq tmlat tint be 8)nthes1ad b7 the gonIIi. aM betore t.hey can b7 qnt.be1Siz-
4M1. the .~. 1nvolVlld. in their Qnthes18 must be preeont. All of the.
ewnta ahould precede in tble the actual MCl"etion of fOl'mlld. hol:'alOl8 .. deter
nd.ned. 07 b10uaq techniqa •••
The a1gn1:t1canc. ot thi. work, tben, 11 •• in the tact that, whU. up to
the PI"8-nt the &bow cons1f.leratione WJ."e tppIIl'ttnt, the actual secpence ot
e'Vent. had not been e.:x:pe1'1mentall7 d«IImonatra.t.ed, i.e., the reau.lta ot biochem
ical and hiat.ochomical tecbn1c;pes on tbu one hw.d and b1~ l:'eSl.1lt. on the
other, cUd not reveal .. propel" t.ime ~nc.. It now appear. tr.i&t the aJtrl;be
tio meoban1ana tor atercd4 bo~ formation .... p"".nt prior to differentia
tion ot the gonad ••
• ~ the ta.il.u.N of the substrate pregnenolone to yield the
apected NSI.llt., it 1. to be noted that the solwnt uaed in our irMtsti.gation
wu ditterent tram t.b&t ueed on tetal 1tlO\laG gOMdrs b7 BaUll. aut Gl"i.£tiths
(1964). Tbeme worl4ara u.eed. propylen6 glycol in both their pregnenolone and
~p1Aunroat.erone (mlA) ~l1t8, enJPlo;ying the modification ot ~
n 11-, (1959) of Wattenberg'a (1958) orlginal~. tJe 11eec1 ,,\.one, u
26
did Wattenberg in hi. irrveet1gat1on. L"1 the L acotone-pregnenolone med1wn a
u~ieNte aoount ot prec1p.1tat.. wu ioieen on tm bottonl of the sta1ni.r.z jw,
'Which .. rwt _an 1..., t.he 0&\ Jar. It i. conceivable ~ltat. this pncip1tate
oaUd be preg"Mnolc. .. s1nc$ the madium oontatmd .. high pa.rcentese ot WtliWr.
~gr.~J.or.a ani IlHA .",.. both _ter .1Ju,olub19; ~vv, pregnenolone 1.
~ less eolabl.. 1...'1. ""'let l'if!'41um UHd. tha."1 i8 DIll, poeldbly to t.b1J point
~ it 1s rllOt a.va1.l.abls in aat.t1c1ent conc.mtr&t1on tor t.ltillution as a
SlbAr&t.e. It might be availabl. in ~ C(;~nt.rat1on it a cl1tterent
aolverrt wre lllled t. .... l>7 pN"mit:ting ocapleUi eolu.bil1z&t1on.
1. '0I't.7-nin11 ~ £Orals of to. B1ngle ccnb, ~. lagbcm
tOl4 f'Ita;l t.hNe ~ of 1lxr~on to batcb.'lng Wft exae1ned by the +--"'" tdque of Wat~ (1958) tor the pNeenoe of .6S_~
~n.Iti8e, an ~ neotJeaa:ry tor t.ba qnt.tles18 of atertdd ~a.
'rttIo ~ate. WN ~, _~ arx1 preanenoJ.one.
a. Vil'tual.l¥ no entQJIlS .,.c1t1t: gMDllat10n was seen in the tiastM ~
with /progncmola. ~ duo to it. relative 1neol.v.b11i:ty in the ace
tone C'Aldiwu~. ~ ~ .. t.b& aolwnt uGGd. 1n pre'fious
1nveet1gationa (BaUU. aD! G1'1ttitbs, 1964).
3. Bellllts ~ in t.iw'ee "-' ~, w1tb tbe uae 01 d8~
~ as tI:l8 ~, ~ t~ ~ in tbe iUt am
t~1an ducts, u wll .. tlJe ~ .... J.a.ver of t.be ~ tuba, dof'
eel r»oonter:f, ctpJ.ee......" ~ur.l, SI..L'l"l"CW'd1ng ab1cni.:nal Zl.,.,mbJmal
t1aet1e, and the genital r1dges.
4. ~ da.J'8 ~ IR'l tive tlr.t ce:nenl1S8d reactiwneee bee .. l'.OOl"e
Natnoted. to the· epithelia of :tb& woltt1an d;!.1cts, ntNNl tube, gr,lt ani
to tho genital ~G.
5. ~~nt to d88 tift, t~ deposit. ~ lXJlI8 inttmee in loeal-
1-.1 patches or oel.l.a arou.nd laewlIt9 in the ~ c~nt of the
0'I8J:'Y and wakl7 ~ in the 0Y8riar1 cOl"tex a1d the ~
2£
the emb170 trom. da;y t.bl"ee to five. 1s clue to the relati wl,y undifferentia
ted nature or embr)'onic etNoturea duri.ng thia period of deftlOJ:.mt be
caaee ot which &1l t18su.ea roA7l (1) poases. the potential tor steroid
s~a1. bl Y:l.l"'tue or the p ..... nee or certain ens,.,. involved in the
biogenesis of stMo14 hormones including ~ s_~ or (2) reepond. to
the teat eu.bstrate by producing .. ~5_~.
7. It 1. tentat1~ concluded that the reault. of this inftstigatlon indi
cate a capaoit7 tor eteroid ho.rmcne Qnthtts18 in the gonad ot the chick
"1'70 beg1nning on about t.he s1xt.h dq of incubation. At thia U. the
generally distributed reactivenes., _n in tIIOR of the tissue. d.u1"lng
the 8ar11 embJ7on1c J'8r1od., beg1na to diMPP'ar and reactive.s. in the
,onada beg1n8 to increase. Af'ter t.M sixth day, zreactivene •• in the
goMd.. appaaJ"8d to show a steld7 1ncreue up to the time of batching.
IUllSe, A. s.., .... It. Clrit.titbe 15164 ~ ~lIar. ta tile toeUl. ..... IaJdIa oau. J. 1NlOCI'la., a= 6).66.
BailUe, A. L, L C. CeJ.., 11. K. ........ D. MeL a.t 19S6 II1.tJto. ~ utSU..u_ fill 31(-, 6JI... u"-, 12cx:-, ]6£(., ... , 11 cr.., ace. .... at.<~. J ........ , a, 1-12.
wnw, A. u., S. JL D. en • __ , L QrUt1* ... D. JfaL Iat"t 1065 3,.. ~ ~".'I' sa till IIII'-.l __ .. ~ t • ...... , Jl: ..,..".
ltIItaCd.t, J. lSI3 '" nrl~ ..... 111F.tla1 d.v. _ ~ ~ ;pPMOOe .. la poula.......... c. a .... 1IcIQ., JJ!: 3IQ-3IM.
1Oo1e, l., ... P. ADael l903 .. 14 ~ .. la al-ate ~1 ... UelJIt Au te8Ueule ~". c. B. SOO. BiOle, 22,: ~
..u." a. II. 19I4 ... MI • .-1 .. _ fit ab1_ ... ctuaIi. fIIIJII'Ie _ ."uIlS" sa .... ...uo .. ~ ~ ...., .... 1 ... 1IIat.. _., li' 501--.
CId.etft, 0., lL _1],", v. *"~ .. 1. NiIIftItQUa 196tt SuUa ~"'u.Scar fill col-.o1D • dIIllIIi t:. .P.i4l'oNl ..... ~ •• t _Ua f!P.... ~ .. JOllo. ltLo. We, .us, as Tl
DIIltobeWt, v. ~ .. 1t~ _81la.~ CIa 1' ....... __ 1~ ... 1'""" .. poul8t.. C. B. AoaL 8c1., a 1S63-lS8'J.
lela, L. v. 19f1l ....... ' •• WI ~ ... tba JIfttbl- fill _ ~ f.D _ ftiIfl. J. Ibp. ZGol., !j,& ~173 •
....... J. IL, .. J. IC. CIIIWe1d. 1938 OIl .. ~ ad ~Il IIIlt at V. cIIttSa1Uw ... .u. la .. 4r. I eUe foWl. ____ J ...... CIJat. ~.,!i:~
ftIIIIet, I. 1.9':2.0 ~ .. 1'...,.8' ..... slm1lH __ uee .. lea _.... U. ... Blcal., al 39)?l6.
Qa1U .. , 1,. .. aal M. ~. 1a I'o&laoo 19'1l DRect1cIl .. ~ et ookW~ ................ "- lee .. I .... ~ .. fOulet. C. B. IOC. Bled.., m,: J.088.l.afJ9.
Goldman, 4. s., w. c. Yakovac and. A. K. Bongiovanni 1960 Deve10pent of activity of ~ deh)rdrogenue in human fetal tiaaNee and in two .'"'18DCephaUc l'l81Ifbome. J. Clin. Endocrin. Metab., ii' 14-22.
Greenwood, A. W. 1925 Gonad gatts in embryonic chicu and their relation to _:mal differentiation. Brit. J. E.1Ip. &io1., it 165-187.
GroeneDClijk-Hu1jben, 14.. H. 1960 Fwlctional. characteristics of t.he testicular homone 1n the chick embr:JO. J.nat.. £tec., In.' 231-249.
Hamburger, V. cd H. L. HamUton 1951 A _ri •• of noma]. stages in the <le'Velop.aent of the chick embryo. d. Horph., !!a 49-92.
He:U.ton, H. L. 1952 .. Ullie .... Developnent ot the chick. Holt, B:1nehfart and w'1nston, Cbep. 13, 488-5Ol.
Hughea, W. 1929 The treeme.rUn condition in swine. Anat. Ree., It.\.. 213-245
Keller, L, ar¥1 J. T.xUer 1916 Uber d.aa Vel"halten del" &ibalUte be1 del" ZWill:tngstracbtigba1t dee tinde.. h1.en. TierU"Zt1. tlochenachr., l' 513-5.26.
Jtemfi, T. 19111 it.dier OWl" Ionakarakterer Hoe FoatN. 1t6benha'tn. 1-117.
lomte14, w., and .A.. v. tf&1..bandov 1956 Endocrine intl.llences u.pon the growth of the l"Ild1ment.arygonad of fowl. Anat. n.c., !J2,. 619-6Y1.
tcoaelka, A. 1'4., and T. F. Gallagher 19.34 Eftect ot we hol"ftl.Ol'Jll extracts theelin and theelol on t..bt chick: embryo. Proc. Soc. Exp. B1ol. and Heel., ll,a ll.43-ll44.
Lanlanie, M. l886 Sur 1& mod.e d 'evolution et 1a valeur de 1 'epitbeliUl"n germ1natif 4a.ns 1e test1cule ~re due poW.et. C. R. Soc. 8101., li' tn-s<J.
Leroy, M. 1948 Rflet aoorogene d, t extra1ts erobryonnairee de pUet au- la crete cbl Chapon. c. It. Acad. Sci., iiA' 540-522.
IBvy, n., u. w. Daane atx1 n. L. &\bin 1959 Viau.allzation ot steroid-~ldetqdrogenue acti'rit.l in ti88U.eS ot int.act and hypo~ct.adzed rats. Endocrinolog, 65 I 932-943.
lBwia, 1.. B. 1946 A 8'tu.ci1 ot aa.ne etfects ot sex hon>JOU6a upon the embryonic ftpI'Oductiw qat.em ot the vbite ptldn duck. Ph3s1ol. Zool., !2.: 282-,329.
Lew1s, 1.. a. aDd 1.. V. Don1n 1948 Sexual t.ra."l8to.rmat1on of the 088i)U8 l:W.la in the clUck embJ.7o tolJ..ow1ng admhdatration of ecrtl'Ogena. Phy81ol. Zool., a= 65-69.
31
Ull1e, F. R. 191.6 The tbeor7 ot the trMma:rUn. Science. I;l.' 6U-6lJ.
LUl1e, F. R. 1917 The f"reemart.1nl • ~ ot the action ot sex hormone. in the foetal l1te of cattle. J. Exp. Zool., ill 371-452.
l.Dbe1, :a. !itl!; W. Deane and. S. 1.. Ratlney 1962 Ins,m,o b1stocberu:1atz7 ot the • portion of the human placenta fra'.l rd.x ","lea or gestation to tem. Am. J. abet. a,necol., QI 295-299.
lutz-Oetertas, Y. 1954 Cont.r1bu.tion. 1 'etude de developDtmt at de 18 l'eBN88ion .s canaux de ~ Chell 1 'embr7on d 'cd_au. Bull. B1ol., •• 334-1.12.
M1nc:lw'&, T. 1921 A 8tud7 of te8t18 am 0'f8'I'7 grana on the hen'. eU and tbail" eft.eta on the em'bl7o. J. b:p. Zool., J.J.. 1-61.
Moore, C. R. 1944 Gonad. boI"monea and .. diftventiat:1on. Am. Iatu.r., 711 97-JJO.
Narbait., 1'-, ard 1.. Iol.ocin7 1964. ~5_~ deiQdro~ in cU.fteNnt1at1n& chick gonad.. Z. Zell.toncb.., 211 6l2-6l7.
liarbaitll, n., ant K. T. sabatini. 196.)& Histochemical demonstration ot claolesterol in d1tte ... nt1at1ng cb1ck Sona:la. z. Zelll"orsch., 22,t 1-5.
iiaJlbaits, L, am M. T. sabat1n1 1963b Histochemical demonetration of choleat.erol in ~c chick gonId.s d1tt.,.nt1at.ed a viHR. z. ZellfOl"SCh., 911 52-55.
ht.akoi, P. G. 1953& Cord1t1ons ot the 1nt,ra..,utenne cle'nl~ of i2! MI# Cale:1.Dg pbencl'S8n& or~.. DAN SS8It Proc. Acad. lei. U. .s.a., 221 693.
htllco1, P. G. 1953b ~c parabioais in goats and abIllep. Proc. Ib1rov A9'1. lnet., 1- 63.
Petllkoi, P. G. 19S5 ~c paNbioa1a and llIiltlt.tat.ion ot farm mhi .. ) s. Trer1a. ln8t. NDrph. An1mala Acad. Se::1 •• U.S.S.B.., ~. 44-
PNsl, J., J. Jtruek, J. Honk;y ard H. Henzl 1965 Obaervationa on ateJ'01d-3J3-o1~ activitl" in tbe OYar'¥ dur1ng early poatnatAl .ft1-opIIl8nt. in the..... J. Endocrin., a. 2!J'J.4!J4.
Bcmanott, A. 1.. 1960 The Avian BmbJ7o. ~ Co., New York, R. Y. Cbap. 10.
albin, B. L., J. A. Hadlton, F. J. Karlson and R. I. Tu.f'aro 1965 &tteat 01' the , ..... of the Nt at the time ot hem1cutration on the wight, errtir;1&ted Il8CNt0J7 actint, am. ~5_~ro1d ~naae actint:r or the remain1n8 OVV7. Endoel"1noloQ, XLI t:p:jJj16.
32
S8m.lel., 1.. T., Ii. 1.. Helmre1cb, M. a. I .... ~r aM H. Beich 1951 An ~ in endocrine ti ..... 1fh1ch ox1d.1 .. e ~ -~1"01d.8 to cr ,,, u,Ileatu.rat.ed .tone.. SCience, W,t 490-491
Scheib, D. 1958a &u' 1& genese at la repartition dee llpide. aane 10. gonale. ~e ctu PotJJ.et. I. Gonadee teme.Ue •• e. a. Soc. Mol. lSl7'J!-Tt1!I.
Scheib, D. 19S8b Sur la pne .. et la repartition d •• lipid •• <1$18 le gonade. ~. du Poulet. II. OoNdea Halee. C. a. Soc. Mol., 1 762-7(lJ.
Scheib, D. 1959 SQr 1& detection de. lipide. ti&Uft8 dena lea gonadea de IfembJ70h de Pwlet. Am. H18toebem., 1&' .33-50.
stenpr...Hatten, L 1957 Etude dee beeoine 1'Iltr1t1te de. gonad •• ~ na1res eI'ot •• culti ..... en m:U1eux Qnthet1qwte. Arch • .&Dat. K1er. Morph. -., ii' S2'J.-.6C1l.
stoll, a. f and B. Ha:raucl 1956 Ex1ateneo d 'hormone. ateJ"Ollq.wa c:la1e 1'0 ..... pm._ de 1 f~ de Poulet au own de 88 d1tterenciation I!IIJfJXI&elle, C. R. Aoed.. Sci., ~I l23S-J.2.37.
Sw.U't, C. H. 1916 Origin ot t.ba 8e»oC0l'de and det1n1tift ... ~a in the male ch1ck. Am. J. Anat., i2,1 .37.5-410.
Taber, I., cd L W. Bal.le7 19S4 The .ttecta of sax hol'monea on the dew1-opaent of the r1gbt gonai in tea&l.e fowl. ~Q'I &1 4ls-424.
wattenberg, 1.. 1958 M:S.Cl"Oacop1c bistochemical ~1on or atero1d.-3~ o1-4elQdropnue in ti ... _otiona. J. Hlstoeham. c,toehem., it 225-232.
Weniger, J-P. 1962 DUtus1on"'8 hoJ'Imone. gcnadicpe. cte 1'~ de Poulet dana le m1l1eu de eu.ltuN. .Arch. Anat. M1cJt. Norph., 211 32S-335.
Weniger, J-p. 1964 LtOY&i.N dt~ de Pculet cu.lt1w inYat.E2 eeCJl8t.e \U1G hoI'r1.icma .. atropn.. C. a. Soc. Mol., Yl* 17s-rre.
\ien1gel', J-P. 1965a Ixt.,..,uon dtune lt1batance oeatl'Oge ... de mUiewt sur-1e.81. out. ete cul.t1wes des gonatea d.roitee teme1les, atroph:l.qQes, dt~ de Pou.let. C. L Soc. Bio1., utt 464-466.
Weniger, J-P. 1965b Extraction dtuna eu.bstance oestrogene (teat d t Al.l.4tn et Dol., poatlt.1t) t.le mU.1eux 1m" l.eeqt181a lee gonale. d 'embJ7OlUl de Pollet temaUea de 7 jOUZ's t\arent CNlU ... es pendant 24th. c. R. .leal. 501., ailt 809-812.
it'1ll181', S. ii. 1m The 8plJc1fio1t;yot sex, ot OJ'ganization and ot d1tter-
33
8DUaticD fit ~ cbiek ...... IIbc:Ma __ antt1A6 ~. J. _. UIOl., ~l ~.
Willi_ .. B. !l., T • .,. Qall ...... ,. O. Iactb 193' lea ...uti .. ,_ ia t.be obiok ~ 1UUl~J.as ,.. ~ of ale aa4 ,.... bcl'anna. fttOC. at. Aca4. SC1., a~ 625-631-
Woltt, I. 1"'7 laot.' ....... 1·~..u.. ~ pIQlulw .".. 1& ~ ... ~ ........ a 1·....,.. Ie poulft. AI'ob. AMt • • cr. ~ _ • .11 ~: 69-90.
VQl.ft, II., 8DIl L BId'teD 19!J2 ... la ..., •• 1.- .. la .... ~ ... ClI ••• de ,.,1 ..... 1 • ....,.. .. ~ .. l'acUaD IIii""he-.- ...... c. a. 100. ll1Ol., a: 1793-1195.
Voltt, B., I. ~ _ K. 1faftea 1951lt SUI' la ..., ••• lca .. 1& ..... ill ~ ......... &till_ .. 1'~ .. poulet. ... l' ...... ~ I ... n .. a1ea. c. a. -. J:llQl., l'.tt: 1193-1.195.
Volft, a ..... B. V01ft J9It9 ~ .. 1& ~ .. ~ a 1', ft~ .. .,...." ......... <Ie l'eat.1Y1te __ .. p .... ~ .. 1& .,..sa tit le ~ __ 'tal. o. a. 1Oe. Jlo1..,
J!i: '29-'31. Volft, I., .. B. WQ1tt 19.f1 DIe etftIcte of ~ .. \d.&t4....,..
I. _. ZOO1., al ~. Wolft, B., .. B. Wo1ft ~ 1a ~_ de 1& 41f'fIftIltJ.lIU.OD. 8U&1-
ella _ 1& .,.... Au -..4 cul:" ... Ja!'J!l:!- BuU. aol.., IV 315-350.
tfQ1tt" I., .. B. VOltf' 195a AaUc:a .. l'~ ,..11 .... 1& 4S.t:teIw.o1attta ••• 11 •• 1& .,n.r.. tie -... oul:tl:,.. &a "'tI't- C. R. IOD. B101., lIt6c m-w.
Vo1ft, B., .. B. 1fG1tt 1"3 Cu.1tiV8'UGla" t.U.ft'lftauatlGD 06 tbe .,..sax ot t.be duet III.,.. CNlU ...... v&MI- Poult.. 8oi., 2' 3118-3'1.
WoocIa, J. a., .. L. V. AlIa 1966 A ~'oa.l1tlllat.1t1oat1_ at tM alllJlQiI'~ CIIIll8 ill tile _pia of tbe hl • .u.a t'Wl.. AMt • .... uta ,.,.....,.. a1IIot -. CoIp. ~, (Ill ...... ).
THE Dfmasrn OF PORVAZAN ~ITIOO Itf b. 5 -~"iSD PDEP~1'IOlI3
DAIS :3 to 6,
~ DQa of No. of ~U- Th1ctc.ea Period ~ ~ .eM 9sDtIlI 2!E1t1 If$, {I&D2 " .. 2
:3 1 ~ )0 6
1 .+CJ2 10 2 3/4
4 1 -HG2 .30 18 1/2
1 ....a2 10 31/2-
5 4 i-+tG2 20-40 2-4
1 Gonad not eaenGl 10 41/2
1 ..en 10 1
6 4 ~ J.a 1-3 (one 17)
1 ++ 40 28
1 ++ 10 1 3/4
2 0 I.,D 21/2-4
-, ... j . • •• ~ ••••• "'1'" ..... ..... ,---. a ... • I .....,. ..............
'1'Hr~Drmr:llm fI ~J UEJII06trIOtl Ui 6 5 -~ PRoEPAHATlOtfS
DAIS 7 to 9
35
:tncu.batJ.on DfIra 01: No. of UrJd1.tfeNnti- ~_ PeriOd
la?lIJatoJ:itl tif'sdf1lUl Eft ~ 2D£1I1 II"'. ClAm} UJeH'fl n
7 4 t ...... t e I 30 1
1 ++ 10 21/2
1 0 30 1
1 ++ 10 4
1 0 20 1
1 + 12 1
2 ++ ++ 12 1
1 ++ 10 3
9 .3 6&10 1
'I'A'B18 3
'l1iE llJm.'I3l'l1' aF ~, DEPOSITION IIj b. S -~1SD PBEPARATIolti
OAm 11 to 20
)6
Incubation Dare of No. of UrJ21ttenmt.1- tb1clmeas Pv1od. 6ta~Qn ~ ItIJI S9JIdI 0D[il.g l!1trU , Qq.ea.) UiSurll
11
12
14
15
16
17
18
19
20
1
1
1
1
1
1
1
1
1
1
2
1
1
2
+
+
++
6
10
6
10
»4
40
10
Z4
10
10
2
10
1.0
1
3
1
1
3
1
23/4
11/3
1
11/3
3
1
11/3
3 1 _________ '_"_-'1 _. ______ ._tlWl _____ M_. _._ .. _'_._n ......... __ · ____ . _, ___ _
1 Experimental oross-section of .. .3 <1a1' ~ cut at .30 miera and inoubated tor 6i hours 8how1ng +++ reaction of ~'-~ in genital ridge (G), Woltt1an duct (W), fdjacent meaenc~ tissue (14). coelomic epithelium (e). ani donal meaenter;y (D). x 450 •
.2 Experir:.ental cr'Ose-aection of .. .3 day embryo shown in tigw:"e 1 showing ++ reaction throughout neural tube, (Nt), notochord (Itt), and lining of gilt (0). X 450.
31
1
PIQtJRl£ 4
5 E.1Ijper1mental Cl'Q • ......-ction of & gonad (G) of a 5 dq -170 cut at 10 miera aM incubated at 4t bc:Iun _owing scatteNd tOlll8Ull granules tbJoo\lghout the genital r1d.ge am aut epithelium (E.) at ++ 1nten81ty. X 450.
6 ~ntal crou-.ction at a 7 d.a¥ gonad cut at 12 micn. and incubated 1 hour ahoW:I.ng d.1tttlee but d.et1n1te grcml&t1on Judged at ++ intensity. .l 450.
7 R1cptl"1mental cro .... ction of & 9 da.J gonad au",. Ilt 6 micra ani incubated 1 hour sho'tdng rcaa(;iol1 (R) in what mq be the msdull.ar;t ~nent of an OVlil'y. X 100.
40
•
PlAm;
8 Experimental cl"OIIJS-IJiIotion ot a 14 dq ova'Q' cut at 40 miora and incubated 2 3/4 hoar. showing a pateq reaction in 100 .. ponion of medullaI"f cc=ponent. (Hl), and a wry dense reaction in the c~t portion (Me). There 1a a emall amount of reaction in the corta: (0). X 100.
9 Higher nlAgldfication of • cron-section of 10088 rned.ull.a17 ponion shown ill f4,JUre 8 sho1d.ng ~ lacuna (1.), ani the reaction (R) in the ;1nt.eraticea betwen med,.ll.,. lacu.nae an:l corda. X 450.
41
rIGURE 8
rmuaa 9
10 ....... W .'.1"'u.. ., .16 .., '-'4a _, at. It ...... .. t __ W 1 ....... d., ....... t4a ........ , ........ ...u ...... 111 •••• U ............ (a). 1-.,0.
U ,. .. of ___ ttl I ............ (0) ia .. u. ... -'I.e .. .... la t.tcue lO. a. ta ...... lal. 1 •• l1t.aid.e fit tile dIpN1u ..., ....... 0lU t 0111 .... 1 lOGO.
PLA.'1E7
'PtauaE, 12
F,ICURB 13
14 SX;per:i..mental ~ction ot a 20 da.T teetis cut at 10 micra ard incubated :3 hours 8howing gramllation <G) moatl1' in center ot this organ. X 100.
15 Experimental crees-section ot a 20 da7 over; cut .t 10 m:l.cra C1d 1ncru.bated :3 hours ahow1ng relatively cOmpact cOl'te.x (C) and 100sly ~ medulla <H> with a patch7 t~ granule deposition (D). X 100. .
44
PIOUS 14
The thesis St.lbm1tted b;V David Glenn aa:b:.Lck baa been read ard approved.
by tour membe1"S of the tacuJ.t;v ot the Gradt.l.at.e School of 1.o1Ola university.
The final copie. have been e.xM1ned by tbe d1rector of the thesi. and
the signature which 8pp8al'1J below ".rUle. the tact that any neceHU7
ohange. have been incorporated. W that the thesis ls nov given tinal
a:pproval w.1th reteNnce to oontent, tom, and mechanical acCUl"8C7.
The theai. 18 theretore accepted in pu'ti&l tult1llment ot tl»
nquil"eml8nta tor the Depree of Mute.r 01 Science.
/~, . \
... _-"'--.. J.e'A<~' ,~.!? Dat~
/ ,I , .
, / ;>
__ ,,,~~';t/~,L.- .c·-Lt.·..L.-S •• d ::::