Testis Wt. (g)Testis Wt. (g) 3434 4949 3.73.7 6.46.4

Efficiency of Sperm Efficiency of Sperm Production (10Production (10 6 6 / g ) / g )

4.44.4 2323 2424 2525

Sperm Production Per Sperm Production Per Male (10 Male (10 66 ) )

125125 11001100 8686 160160

Sperm in Caudae Sperm in Caudae Epididymis (10 Epididymis (10 66 ) )

420420 57005700 440440 16001600

Germ Germ CellCell

MigrationMigration

Sertoli Cell MultiplicationSertoli Cell MultiplicationGerm Cell DevelopmentGerm Cell Development

Initiation and Initiation and Expansion ofExpansion of

spermatogenesisspermatogenesis

SpermatogenesisSpermatogenesisSperm ReleaseSperm Release

Sperm MaturationSperm Maturation

Birth Puberty AdulthoodBirth Puberty Adulthood

Diagramatic illustration of developmental windows of susceptibility of spermatogenesisDiagramatic illustration of developmental windows of susceptibility of spermatogenesisTo disruption. Each of the grey-shaded boxes shows the approximate timing of a particularTo disruption. Each of the grey-shaded boxes shows the approximate timing of a particularStage of development of either the germ cells and/or the Sertoli cells. Quantitativly andStage of development of either the germ cells and/or the Sertoli cells. Quantitativly andQualitatively normal spermatogenesis in adulthood (whit box) depends on the successful Qualitatively normal spermatogenesis in adulthood (whit box) depends on the successful Completion of each of the earlier stages of development. There are well-documented Completion of each of the earlier stages of development. There are well-documented Examples in humans and animals of the consequences of the failure of each one of theseExamples in humans and animals of the consequences of the failure of each one of theseSteps, all of which lead to complete or partial loss of fertility.Steps, all of which lead to complete or partial loss of fertility.

SertoliSertoliCell Cell

DifferentiationDifferentiation

MouseMouse HamsterHamster RatRat RabbitRabbit

DogDog

(beagle)(beagle)

MonkeyMonkey

(rhesus)(rhesus) ManMan

Duration of cycle of semeniferous epithilium, daysDuration of cycle of semeniferous epithilium, days 8.98.9 8.78.7 12.912.9 10.710.7 13.613.6 9.59.5 16.016.0

Lifespan, daysLifespan, days

ß-Spermatogoniaß-Spermatogonia 1.51.5 1.61.6 2.02.0 1.31.3 4.04.0 2.92.9 6.36.3

Leptotene SpermatocytesLeptotene Spermatocytes 2.02.0 0.80.8 1.71.7 2.22.2 3.83.8 2.12.1 3.83.8

Pachytene spematocytesPachytene spematocytes 8.08.0 8.18.1 11.911.9 10.710.7 12.412.4 9.59.5 12.612.6

Golgi spermatidsGolgi spermatids 1.71.7 2.32.3 2.92.9 2.12.1 6.96.9 1.81.8 7.97.9

Cap spermatidsCap spermatids 3.63.6 3.53.5 5.05.0 5.25.2 3.03.0 3.73.7 1.61.6

Testis wt, gmTestis wt, gm 0.20.2 3.03.0 3.73.7 6.46.4 12.012.0 4949 3434

Daily sperm productionDaily sperm production

Per gram testis, 10Per gram testis, 1066/g/g 2828 2424 2424 2525 2020 2323 4.44.4

Per male, 10Per male, 1066 55 7474 8686 160160 290290 11001100 125125

Sperm reserves in cauda (paired sides at sexual rest) 10Sperm reserves in cauda (paired sides at sexual rest) 1066 4949 10201020 440440 16001600 21002100 57005700 420420

Epididymal transit (at sexual rest), daysEpididymal transit (at sexual rest), days 14.814.8 8.18.1 12.712.7 11.311.3 10.510.5 5.55.5

Evaluation possible ofEvaluation possible of

Serum Hormones (LH, Testosterone)Serum Hormones (LH, Testosterone) YesYes YesYes YesYes YesYes YesYes YesYes YesYes

Enumerate testis spermatidsEnumerate testis spermatids YesYes YesYes YesYes YesYes YesYes YesYes YesYes

Evaluate Testis HistologyEvaluate Testis Histology YesYes YesYes YesYes YesYes YesYes YesYes YesYes

Evaluate Epididymis HistologyEvaluate Epididymis Histology YesYes YesYes YesYes YesYes YesYes YesYes YesYes

Enumerate Epididymal Sperm ReservesEnumerate Epididymal Sperm Reserves YesYes YesYes YesYes YesYes YesYes YesYes YesYes

Evaluate Epididymal / Ejaculated Sperm MotilityEvaluate Epididymal / Ejaculated Sperm Motility YesYes YesYes YesYes YesYes YesYes YesYes YesYes

Evaluate Epididymal / Ejaculated Sperm MorphologyEvaluate Epididymal / Ejaculated Sperm Morphology YesYes YesYes YesYes YesYes YesYes YesYes YesYes

Evaluate Epididymal / Ejaculated Sperm ProteomeEvaluate Epididymal / Ejaculated Sperm Proteome YesYes YesYes YesYes YesYes YesYes YesYes YesYes

Fertility test feasibleFertility test feasible

Natural matingNatural mating YesYes YesYes YesYes YesYes NoNo NoNo NoNo

Artificial inseminationArtificial insemination YesYesbb YesYesbb YesYesbb YesYes NoNo NoNo NoNo

Models and Endpoints for Studying Alterations of Male Reproduction

IN UTERO INSEMINATIONIN UTERO INSEMINATION

Synchronize adult female rats with LHRH agonistSynchronize adult female rats with LHRH agonist

Cervically stimulate receptive females using vasectomized malesCervically stimulate receptive females using vasectomized males

Recover cauda sperm in AI medium and inject 5 x 10Recover cauda sperm in AI medium and inject 5 x 10 6 6 per uterine horn per uterine horn

Assess fertilty on gestation day 9 (implants / corpora lutea) Assess fertilty on gestation day 9 (implants / corpora lutea)

00 22 44 66 88 1010 1212 1414 161600

2020

4040

6060

8080

100100

% O

F E

GG

S F

ER

TIL

IZE

D%

OF

EG

GS

FE

RT

ILIZ

ED

SPERM CONCENTRATION ( X 10 )SPERM CONCENTRATION ( X 10 )66

Fertility = Max Fertility = Max ** ( 1- e ( 1- e – k – k ** s s ) )Max = 104.3 ± 5.2Max = 104.3 ± 5.2K = 0.261 ± 0.000229K = 0.261 ± 0.000229EC 50 = 2.66 x 10 EC 50 = 2.66 x 10 66

5 x 10 5 x 10 66 sperm = 75% Fertility sperm = 75% Fertilityr r 22 = 0.96 = 0.96

Natural MatingFertility Outcome

# Ejaculated Sperm

Intra Uterine Insemination Fertility Outcome

Sertoli cellsABPLEYDIG

CELLS

BLOODVESSEL

SEMINIFEROUSTUBULE

ABP

ANDROGEN

ANTERIORPITUITARY

GnRH

HYPOTHALMUS

LH

FSH

Stimulates synthesis of ABP and E

Stimulatessynthesis of T

Negative feedbackof T and E on the hypothalmus

T and E

T E

ABP

cAMP ATP

FreeCholesterolTransport

Lipoprotein

De Novo Synthesis

Cholesterol Esters

Plasma Membrane

Pegnenolone 3ßHSD

Progesterone 17α -Hydroxylase

17α– Hydroxyprogesterone C17-20 Lyase

Androstendione 17KSR

Testosteronep450arom

EstradiolPlasma Membrane

P450c17SERMitochondrion

LH

EX-VIVO AND IN-VITRO ASSESSMENT OFTESTICULAR STEROIDOGENESIS

In Vivo ExposureIn Vivo Exposure

MediumMediumTestis ParanchymaTestis Paranchyma

± LH ± LH

5% CO5% CO22

34 34 00CC

X X NN

In Vivo ExposureIn Vivo Exposure

MediumMediumTestis ParanchymaTestis Paranchyma

ToxicantToxicant± LH ± LH

5% CO5% CO22

34 34 00CC

X X NN

INCUBATE 2 HOURSINCUBATE 2 HOURS

INCUBATE 2 HOURSINCUBATE 2 HOURS

ASSAY ASSAY STEROIDSSTEROIDS

ASSAY ASSAY STEROIDSSTEROIDS

CellularCellular

AttributesAttributes FETALFETAL PROGENITORPROGENITOR IMMATUREIMMATURE ADULTADULT

Age WhenAge When

TypicalTypical

GD 15-19GD 15-19 PND 21PND 21 PND 35PND 35 PND 90PND 90

MitoticMitotic

ActivityActivity

LowLow HighHigh OnceOnce LittleLittle

TestosteroneTestosterone

MetabolismMetabolism

LowLow HighHigh HighestHighest LowLow

TestosteroneTestosterone

SynthesisSynthesis

HighHigh LowLow SomeSome HighHigh

LH ReceptorLH Receptor

NumberNumber

HighHigh LowLow SomeSome HighHigh

AndrogenAndrogen

ReceptorReceptor

NumberNumber

LowLow HighHigh HighHigh LowLow

CAPUT

CORPUS

PROXIMALCAUDA

DISTALCAUDA

VASDEFERENS

PROXIMAL CAUDAPROXIMAL CAUDA

( First site of Fertilizing Ability )( First site of Fertilizing Ability )

DISTAL CAUDADISTAL CAUDA

CORPUSCORPUS

CAPUTCAPUT

TRANSIT TIMETRANSIT TIME

= 4 DAYS= 4 DAYS

INTACT ( EDS, CEMS, EPI )INTACT ( EDS, CEMS, EPI )

CASTRATED + T ( HFLUT )CASTRATED + T ( HFLUT )

EPIDIDYMISEPIDIDYMIS

TRANSPORTTRANSPORT

STORAGESTORAGE

MATURATIONMATURATION

MOTILITYMOTILITY

FERTILIZINGFERTILIZINGABILITYABILITY

NORMALNORMALDEVELOPMENTDEVELOPMENT

HALOACETIC ACIDS PRODUCEHALOACETIC ACIDS PRODUCE

DELAYED SPERMIATIONDELAYED SPERMIATION

SPERM FUSIONSPERM FUSION

ALTERATIONS IN SPERM MORPHOLOGYALTERATIONS IN SPERM MORPHOLOGY

FORMATION OF ATYPICAL RESIDUALFORMATION OF ATYPICAL RESIDUAL BODIES BODIES

IN UTERO INSEMINATION SPERM MOTION ANALYSIS

SPERM PROTEIN QUANTITATION

0, 8, 24, 72 mg/kg Bromochloroacetic Acid0, 8, 24, 72 mg/kg Bromochloroacetic AcidDaily for 14 daysDaily for 14 days

*

*

*

** *

00

10001000

20002000

30003000

40004000

50005000

60006000

70007000

80008000

90009000

1000010000 SP 22SP 22

Inte

gra

ted

Op

tica

l Den

sity

In

teg

rate

d O

pti

cal D

ensi

ty ControlControl

BCA 8.0 mg/kgBCA 8.0 mg/kg

BCA 24.0 mg/kgBCA 24.0 mg/kg

BCA 72.0 mg/kgBCA 72.0 mg/kg

0

1010

2020

3030

40405050

6060

7070

8080

9090 FertilityFertility

Imp

lan

ts /

CL

s (

%)

Im

pla

nts

/ C

Ls

(%

)

ControlControl

BCA 8.0 mg/kgBCA 8.0 mg/kg

BCA 24.0 mg/kgBCA 24.0 mg/kg

BCA 72.0 mg/kgBCA 72.0 mg/kg

Developmental Reproductive Study ofDevelopmental Reproductive Study ofDBA in Rats via Drinking WaterDBA in Rats via Drinking Water

Preputial SeparationPreputial Separation

Fertility by AIFertility by AI

HistologyHistology

Organ WtsOrgan Wts

Serum HormonesSerum Hormones

Sperm ProteinsSperm Proteins

Begin assessing Begin assessing

Preputial Preputial

in Utero in Utero Birth Weaning Separation Puberty Adult Birth Weaning Separation Puberty Adult

gd 15 pnd 0 21 37 56-57 117-120gd 15 pnd 0 21 37 56-57 117-120

ResultsResults

Body weight independant delays in PPS and VO with Body weight independant delays in PPS and VO with continuous exposurecontinuous exposure

Reduced fertility and sperm proteins with continuous Reduced fertility and sperm proteins with continuous exposureexposure

0, 4, 40, 400 ppm

GD 15 – PND 56GD 15 – PND 56 GD 15 - PND 21GD 15 - PND 21

*A

ge (

Day

sA

ge (

Day

s ) )Preputial Preputial SeparationSeparation

4040

4444

4848

5252

0ppm0ppm

4ppm4ppm

40ppm40ppm

400ppm400ppm

Onset of Puberty Onset of Puberty

0

5000

10000

15000

20000

25000

0 ppm

4 ppm

40 ppm

400 ppm

SP 22 LevelsSP 22 Levels

I.O

.D.

I.O

.D.

GD 15 Thru AdultGD 15 Thru Adult GD 15 thru PND 21GD 15 thru PND 21

****

00

0.050.05

0.10.1

0.150.15

0.20.2

0.250.25

ControlControl 11 55 5050

** ** **

SP22 (ELISA)SP22 (ELISA)SP22 (ELISA)SP22 (ELISA)

Failed to fertilize both Failed to fertilize both test femalestest females

Failed to fertilize both Failed to fertilize both test femalestest females

TreatmentTreatment(mg DBA/kg)(mg DBA/kg)

TreatmentTreatment(mg DBA/kg)(mg DBA/kg)

(17/20)(17/20)

(10/18)(10/18)

(13/20)(13/20)

(9/16)(9/16)

Percent conception Percent conception (after A I)(after A I)

Percent conception Percent conception (after A I)(after A I)

0/100/10

0/90/9

1/101/10

2/8 2/8

0/100/10

0/90/9

1/101/10

2/8 2/8

FertilityFertilityFertilityFertility

8585

5555

6565

55 55

8585

5555

6565

55 55

00

11

55

5050

00

11

55

5050 **

**

Developmental Repro Study of DBA in the RabbitDevelopmental Repro Study of DBA in the Rabbit

A

R

A

RDHT

HRE TATA Luciferase

LuciferaseExpression

A

R

A

RH Flut

HRE TATA Luciferase

LuciferaseExpression

ANDROGEN

ANTIANDROGEN

xxx

Comparison of observed effects at low doses of vinclozolin 24.5 mg/kg, flutamide 0.77 mg/kg, and procymidone 14.1 mg/kg on weights of muscle LABC (A), seminal vesicles (B), prostate (C), and PBP C3 gene expression (D) with the effects of Mix 3, the mixture that combines the three

chemicals at these low doses

Comparison of observed effects at low doses of vinclozolin 24.5 mg/kg, flutamide 0.77 mg/kg, and procymidone 14.1 mg/kg on weights of muscle LABC (A), seminal vesicles (B), prostate (C), and PBP C3 gene expression (D) with the effects of Mix 3, the mixture that combines the three

chemicals at these low doses

FIG. 4. Fetal testicular testosterone concentration of fetal testes collected on GD 19 from control and DBP-exposed fetuses. Values are expressed relative to control values and represent the average 6 SEM from three to four separate rat fetuses from one to four dams per treatment group. *p 5 0.05.

FIG. 2. Western analyses of testicular protein collected onGD19 from control and DBP-exposed fetuses. (A) Representative immunoblots for SR-B1, P450scc, StAR, and CYP17. (B) Average relative protein expression levels 6 SEM from four separate rat fetuses from different dams per treatment group. *P50.05

FIG. 2. Western analyses of testicular protein collected onGD19 from control and DBP-exposed fetuses. (A) Representative immunoblots for SR-B1, P450scc, StAR, and CYP17. (B) Average relative protein expression levels 6 SEM from four separate rat fetuses from different dams per treatment group. *P50.05

Figure 4. Comparison of testicular testosterone levels at E19.5 and E21.5 (top) and testicular immunoexpression of P450scc at E17.5 in controls (N=5) and in rats exposed in utero to DBP (N=5). Note the marked reduction in intensity of immunoexpression of P450scc in DBP-exposed males. Testosterone values are the mean ± SEM for 4-7 animals per group.*p<0.05, in comparison with respective control value. Scale bar shows 100μm.

Figure 1. Representative photomicrographs showing immunoexpression of Insl3 in Leydig cells of testes from fetal and adult rats. Strong immuno-staining was detected in testes from control E17.5 (a) and adult (c) animals, but this was reduced markedly in E17.5 rats exposed in utero to DBP (b) and was absent in adults in which Leydig cellshad been ablated by treatment with EDS (d). Insets show tissue sections incubated with pre-immune serum. Scale bar shows 100 μm.

Figure 1 Proposed model for the formation of dysgenetic areas and Sertoli-cell-only (SCO) tubules in male rats exposed in utero to 500 mg/kg di(n-butyl) phthalate (DBP). At embryonic day 21.5 large aggregations of Leydig cells (LCs) are evident with Sertoli cells and presumably other cell types ‘trapped’ within them. This is illustrated using double immunofluorescence for 3b-hydroxysteroid dehydrogenase (3b-HSD) (red) and antimullerian hormone (green) and ‘trapped’ Sertoli cells are indicated by the white arrows. Inset shows a similarly stained testicular section from a control animal. By post-natal day 4 the large clusters of intermingled cells in testes of DBP-exposed animals attempt to form seminiferous tubules, resulting in the formation of dysgenetic cords/areas. LCs become ‘trapped’ within these malformed tubules and by adulthood (3b-HSD immunostaining;brown) intratubular LCs (black arrows) but no germ cells (SCO) are seen within dysgenetic tubules. sc, seminiferous cords. Scale bar, 50 lm.

Laser capture microdissection of fetal rat testes

Compartment-selective effects of DBP exposure in utero on immunoexpression of inhibin-{alpha}, CRABP2, and PEBP at e19.5 in the fetal rat testis

Percentage of seminiferous tubules graded as:Percentage of seminiferous tubules graded as:

nn 00 11 22 33 44 5-75-7Degree of Degree of germinal epithelial germinal epithelial loss loss aa

In utero and adolescent exposuresIn utero and adolescent exposures

ControlControl 66 88.088.0 9.29.2 2.72.7 __ __ __ 3.6 ± 0.23.6 ± 0.2

In uteroIn utero 66 79.779.7 14.814.8 5.05.0 0.20.2 0.30.3 __ 6.7 ± 1.0 *6.7 ± 1.0 *

AdolescentAdolescent 66 81.081.0 15.715.7 4.24.2 __ 0.20.2 __ 6.2 ± 1.0 *6.2 ± 1.0 *

Postpubertal exposurePostpubertal exposure

ControlControl 66 86.286.2 9.89.8 2.22.2 __ __ __ 3.5 ± 0.33.5 ± 0.3

DBPDBP 66 81.281.2 16.516.5 2.22.2 0.20.2 __ __ 5.3 ± 0.7 *5.3 ± 0.7 *

a Values represent mean ± SEM.* P < 0.05 and ** p < 0.01 using t-test

Histopathological Changes in the Seminiferous Epithelium after in Utero, AdolescentOr Postpubertal Exposure to 400 mg DBP/kg/dy

FIG. 3. Sperm acrosomal and nuclear defects in rabbits exposed to DBP in utero, during adolescence, or after puberty. Differential interference contrast (A, B), and electron microscopy (C) revealed that these defects included dysplasia and vesiculation of acrosomes and incomplete condensation and vacuolation of sperm nuclei. That these defects originated during spermiogenesis, and not during epididymal transit, is evident from histological examination of testicular sections (D). Arrows: acrosomal vesiculation, Arrowheads: incomplete nuclear condensation. Original magnification (A) 930; (B, D) 1030; (C) 5000.