Testis Wt. (g) Testis Wt. (g) 34 34 49 49 3.7 3.7 6.4 6.4 Efficiency of Efficiency of Sperm Production Sperm Production (10 (10 6 6 / g ) / g ) 4.4 4.4 23 23 24 24 25 25 Sperm Production Sperm Production Per Male (10 Per Male (10 6 ) ) 125 125 1100 1100 86 86 160 160 Sperm in Caudae Sperm in Caudae Epididymis (10 Epididymis (10 6 ) ) 420 420 5700 5700 440 440 1600 1600
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Testis Wt. (g) 34493.76.4 Efficiency of Sperm Production (10 6 / g ) 4.4232425 Sperm Production Per Male (10 6 ) 125110086160 Sperm in Caudae Epididymis.
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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
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
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
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
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.