Characterization of the Period of Sensitivity of Fetal Male Sexual Development to Vinclozolin

Cynthia J. Wolf*,{dagger}, Gerald A. LeBlanc{dagger}, Joseph S. Ostby* and L. Earl Gray, Jr.*,1

* Endocrinology Branch, MD 72, Reproductive Toxicology Division, Endocrinology Branch, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and {dagger} Department of Toxicology, North Carolina State University, Raleigh, North Carolina 27695

Received November 3, 1999; accepted January 10, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vinclozolin is a fungicide whose metabolites are androgen receptor (AR) antagonists. Previous work in our laboratory showed that perinatal administration of vinclozolin to rats results in malformations of the external genitalia, permanent nipples, reduced anogenital distance (AGD), and reduced seminal vesicle, ventral prostate, and epididymal weights. The objectives of this study were to determine the most sensitive period of fetal development to antiandrogenic effects of vinclozolin and to identify a dosing regime that would induce malformations in all of the male offspring. Pregnant rats were dosed with 400 mg vinclozolin/kg/day on either GD 12–13, GD 14–15, GD 16–17, GD 18–19, or GD 20–21, or with corn oil (2.5 ml/kg) from GD 12 through GD 21 (Experiment 1). All 2-day periods in which significant effects were produced were included in an extended dosing period, GD 14 through GD 19, in which pregnant rats were dosed with 200 or 400 mg vinclozolin/kg (Experiment 2). In Experiment 1, significant effects of vinclozolin were observed in rats dosed on gestation days (GD) 14–15, GD 16–17, and GD 18–19, while the most significant effects were observed in rats treated on GD 16–17. These effects include reduced AGD; presence of areolas, nipples, and malformations of the phallus; and reduced levator ani/bulbocavernosus weight. In contrast, ventral prostate weight was reduced only in the GD 18–19 group. The expanded dosing regime (Experiment 2) increased the percentage of male offspring with genital malformations (> 92%), and retained nipples (100%), further reduced the weight of the ventral prostate, and reduced the weight of the seminal vesicles. In addition, malformations were more severe and included vaginal pouch and ectopic/undescended testes. The latter was induced only in the 400 mg/kg group. These data indicate that the reproductive system of the fetal male rat is most sensitive to antiandrogenic effects of vinclozolin on GD 16 and 17, although effects are more severe and 100 % of male offspring are affected with administration of vinclozolin from GD 14 through GD 19.

Key Words: vinclozolin; antiandrogen; androgen receptor; male reproductive development; critical period; levator ani; hypospadias; anogenital distance.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endocrine disruptors, or EDCs (endocrine disrupting compounds), have been associated with adverse effects on the populations and reproductive processes of wildlife such as egg shell thinning in eagles and gulls (Feyk and Giesy, 1998Go; Grier, 1982Go; Hunt and Hunt, 1973Go; Lundholm, 1984Go), female-female pairing of gulls and terns (Hunt and Hunt, 1973Go; 1977Go), imposex in gastropods (Gibbs et al., 1991Go), intersex in crustaceans near sewage discharge (Moore and Stevenson, 1991Go), small phallus in Florida alligators (Guillette et al., 1994Go), and cryptorchidism in the Florida panther (Facemire et al., 1995Go). In humans, trends in reproductive system abnormalities such as declining sperm counts and cryptorchidism (Carlsen et al., 1992Go), testicular cancer (Weir et al., 1999Go), prostate cancer (Fleming et al., 1999Go), and hypospadias (Chambers et al., 1999) have been linked to EDCs. Alterations in the human reproductive system continue to appear. Two surveillance areas in the United States reported a rise in the incidence of hypospadias in newborn boys from 1968 to 1993 (Dolk, 1998Go) and the incidence of testicular germ cell cancer is reported to have risen from 1964 to 1996 in Canada (Weir et al., 1999Go) and most other countries as well (Skakkebaek et al., 1998Go). EDCs, which can act as hormone mimics or antagonists to disrupt the reproductive system, can be especially injurious during gestation because of the undifferentiated state of the reproductive tract and the lack of compensatory homeostatic mechanisms. Vinclozolin is an EDC that acts as an antiandrogen, impairing development of the male rat reproductive system when administered during sexual differentiation (Gray et al., 1994Go; 1999aGo; 1999bGo).

Vinclozolin, or 3-(3,5-dichlorophenyl)-5-methyl-5-vinyloxazolidine-2,4-dione, is a fungicide used on fruits, vegetables, turfgrass, and ornamental plants (U.S. EPA, 1998Go). Antiandrogenic effects of vinclozolin exposure have been found in our laboratory and by others in several mammalian species such as the rabbit, mouse, rat, and dog (U.S. EPA, 1993Go). Vinclozolin degrades in the environment or is metabolized in the body to several metabolites, two of which (M1 and M2) have been shown to competitively inhibit the androgen receptor (AR) in vitro (Kelce et al., 1994Go; Wong et al. 1995Go). In vivo, vinclozolin inhibits AR-dependent gene expression (Kelce et al., 1997Go) and produces a spectrum of anatomical defects. Administration of 50 and 100 mg/kg vinclozolin to rats on gestational day (GD) 14 through postnatal day (PND) 3 resulted in effects in the male offspring similar to those caused by flutamide, a well known AR antagonist. These effects include reduced anogenital distance (AGD); persistent nipples; cleft phallus; hypospadias; reduced weights of the ventral prostate, seminal vesicles and epididymis; and reduced sperm counts (Gray et al., 1994Go; 1999aGo). In addition, peripubertal administration of 100 mg/kg vinclozolin to male rats delays puberty and reduces weights of androgen-dependent tissues including the ventral prostate, seminal vesicles, and epididymis (Monosson et al., 1999Go). Doses as low as 3 to 12.5 mg/kg administered perinatally reduce AGD and ventral prostate weight and induce areolas in males (Gray et al., 1999aGo). These responses in male offspring to low doses of vinclozolin emphasize the sensitivity of the developing fetus and pup to vinclozolin.

The heightened susceptibility of the fetus to antiandrogenic insult can be explained by the onset of AR expression during sexual differentiation, and the fact that normal AR function is essential for male reproductive tract differentiation. AR appear first in the rat testes at GD 14 (Bentvelsen et al., 1995Go) or GD 15 (You et al., 1998Go) and in the mesenchyme of the reproductive tract nearest the testes at GD 14 in the rat (Bentvelsen, et al., 1995Go) and at GD 12 in the mouse (Cooke et al., 1991Go; Prins and Birch, 1995Go). AR first appear in the mesenchyme of the murine mammary anlage at about GD 12 (Wasner et al., 1983Go) and the rat mammary anlage is responsive to androgens at the time the fetal testes produce testosterone (Imagawa et al., 1994Go), which is GD 14 (Picon, 1976Go). The appearance of AR along the reproductive tract is also coincident with the production of testosterone (T) by the testes and the onset of sexual differentiation of the tract (Wilson et al., 1995Go). Sexual differentiation is dependent on the androgens T or dihydrotestosterone (DHT), and on AR function. The role of androgens in male sexual development is made evident from human genetic and animal studies. Human males with mutations of the AR display androgen insensitivity syndrome, in which males have testes but display female-like external genitalia and breast development with variable development of the internal male reproductive tract (Quigley et al., 1995Go). Males lacking 5{alpha}-reductase, which converts T to DHT, also display female-like genitalia but have normal Wolffian duct structures (Wilson et al., 1981). Similar results have been obtained in animal studies using antiandrogens that either inhibit the AR, such as flutamide or vinclozolin, or inhibit the 5{alpha}-reductase conversion of T to DHT, as with finasteride. The development of the Wolffian duct system, which includes the epididymides, vas deferens, and seminal vesicles, is much more affected by an AR inhibitor, flutamide, than by a 5{alpha}-reductase inhibitor, finasteride (Imperato-McGinley et al., 1992Go), which illustrates T dependency in the Wolffian duct system. Testicular descent (Husmann and McPhaul, 1991aGo; Imperato-McGinley et al., 1992Go; Spencer et al., 1991Go) and levator ani weight (Breedlove and Arnold, 1983Go; Tobin and Joubert, 1991Go; van der Schoot, 1992Go) also appear to be mediated by T. In contrast, perinatal finasteride exposure did not affect the epididymis or vas deferens, and the incidence of undescended testes was much lower, although finasteride treatment greatly reduced the prostate weight, prevented closure of the penile folds as evidenced by a cleft phallus, and allowed for development of nipples and a vaginal pouch (Clark et al., 1993Go; Imperato-McGinley et al., 1992Go), which reflects a dependency of the urogenital sinus, perineum and mammary anlage on DHT. Finasteride or flutamide treatment also induce hypospadias, microphallus, and preputial adhesion in rodents and monkeys (Prahalada et al., 1997Go; Silversides et al., 1995Go).

We hypothesize that the development of any of these structures can be most effectively altered by an AR inhibitor such as vinclozolin at the time of onset of differentiation of each structure and coincident appearance of AR in the mesenchyme. In amendment of Paracelsus' statement "The right dose differentiates a poison from a remedy" (Gallo and Doull, 1993Go), dose alone does not determine the poison. The timing, duration and dose of the antiandrogenic agent used will influence the severity and spectrum of effects. The objectives of this study were to establish the critical period(s) during fetal development within which male offspring are most sensitive to the effects of vinclozolin on the reproductive organs, and to characterize the spectrum and level of antiandrogenic effects obtained from these dosing regimes for use in future studies. We dosed several groups of rats in separate 2-day time frames during gestation with high concentrations of vinclozolin (400 mg/kg) in order to identify the critical time to dose. We then extended the dose period and lowered the concentration of the dose in order to find a dose regimen that would significantly affect all of the offspring.


    METHODS AND MATERIALS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
Two-Day Window Study (Experiment 1)
Two sets (n = 32 and n = 33) of 90-day-old female Long Evans hooded rats from Charles River Laboratories (Raleigh, NC) arrived at the animal facility on gestational day (GD) 3 (day of sperm-positive smear = GD 1) on 2 separate dates. They were housed one per cage in polycarbonate cages (20 cm x 25 cm x 47 cm) with laboratory grade pine shavings as bedding, in an atmosphere of 68–72°F, 40–50% relative humidity, and a reversed light schedule (14-h light:10-h dark; lights off at 11:00 A.M. EST). Rats were fed rat chow (Purina chow 5001) and water ad libitum. On GD 10, dams were weighed and weight ranked, and 30 dams from each set were randomly assigned to treatment groups that were equilibrated with respect to body weight (bw). Dams were dosed with 400 mg/kg bw vinclozolin (Crescent Chemical; Hauppauge, NY; lot #10560) by oral gavage on GDs 12 and 13 (GD 12–13; n = 11), 14 and 15 (GD 14–15; n = 10), 16 and 17 (GD 16–17; n = 10), 18 and 19 (GD 18–19; n = 10), or 20 and 21 (GD 20–21; n = 11), or with corn oil vehicle (Sigma; 2.5 ml/kg bw) on GD 12 through 21 (control; n = 11). On postnatal day (PND) 1 (day of delivery) pups were counted, weighed, and sexed. Anogenital distance (AGD) was measured in a blind fashion using a dissecting scope fitted with an ocular micrometer. On PND 13, male pups were checked for areolas, and a nipple or an areola was reported as an areola. Male offspring were weaned on PND 23 and littermates were housed 2–3 per cage. Females were euthanized at weaning age. On PND 77–102, males (n = 73; all 28 from set 1, 44 from set 2) were sacrificed using decapitation, shaved for viewing nipples, and necropsied. Malformations of the genitalia, including cleft prepuce, cleft phallus, exposed os penis, hypospadias, vaginal pouch, and undescended and ectopic testes were identified and recorded. Vaginal pouch was recorded if severe (characterized by a large, deep, unambiguous orifice at the base of the phallus). The right testis and epididymis, the ventral prostate (VP), seminal vesicles (fluid-filled, + coagulating gland; SV), and levator ani with bulbocavernosus muscles (LA/BC) were weighed. On PND 175–176, necropsy was performed as above on the remaining rats from set 2 (n = 35), except that the right testis and epididymis, which appeared unaffected at PND 77–102, were not weighed. In set 2, randomly selected male representatives of each litter were included in both age groups.

Extended Window Study (Experiment 2)
Methods were the same as for the above experiment except for the following: (1) the experiment was performed on one set of animals; (2) dams were dosed with 200 mg/kg bw vinclozolin (n = 10), 400 mg/kg bw vinclozolin (n = 10), or corn oil vehicle (control; n = 10) on GD 14–19; (3) male offspring were weaned on PND 22; (4) males were sacrificed on PNDs 107–128 (n = 105); (5) testis and epididymis were not weighed but were observed for abnormalities; and (6) serum was collected for radioimmunoassay of T levels by a method described previously (Kelce et al., 1991Go).

Statistics
Data were analyzed on a litter-means basis using one-way ANOVA (Experiment 2), or 2-way ANOVA when the experiment was performed in 2 blocks, representing 2 sets of dams and their offspring (Experiment 1), using general linear models procedure PROC GLM on SAS for Windows 95TM version 3.0.554 (Cary, NC). AGD and organ weights were analyzed with body weight as a covariate. When general differences (p < 0.05 in ANOVA) for treatment effects were found, specific differences between groups were analyzed with a 2-tailed t test, using least-square means. Data given in percentages (areola, nipple and malformation data) were analyzed after arcsin transformation of litter means, in order to correct for heterogeneity of variance typical of percentage data. Fisher's Exact test was also performed on categorical data (malformations, vaginal pouch, and ectopic/undescended testes) on an individual basis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
Experiment 1
Pre-weaning litter data.
Dystocia and late delivery occurred only in the treated groups, although the incidences were very low (Table 1Go). Live litter size and sex ratio were not affected by vinclozolin treatment. Pup weight was slightly reduced in only one of the treated groups, GD 20–21, although overall, pup weights did not significantly differ between control and vinclozolin-treated groups. Pup survival to PND 13 or to weaning was low in all groups but was not significantly reduced by vinclozolin treatment. Litters not viable at weaning (n = 3) occurred only in treated groups (Table 1Go) but these numbers appear too low to indicate a treatment effect.


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TABLE 1 Dam Fertility and Preweaning Pup Weight and Viability (Experiment 1) following 2-Day Prenatal Administration of Vinclozolin (400 mg/kg bw)
 
Anogenital distance.
AGD was significantly reduced on PND 1 in the GD 16–17 and GD 18–19 groups, although the greatest reduction was observed in the GD 16–17 group (Table 2Go, Fig. 1Go). The reductions in AGD were not due to a reduction in pup size since body weight was not reduced in these groups (Table 1Go) and AGD was found by statistical analysis not to be correlated with body weight (covariate F[2,16] = 3.90; p > 0.06; Fig. 1Go, Table 2Go).


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TABLE 2 External Abnormalities in Male Offspring Induced by 2-Day Prenatal Administration of Vinclozolin (400 mg/kg) (Experiment 1)
 


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FIG. 1. Summary of the effects observed in Experiment 1 given in percentages, arranged by dose group to illustrate the overall window in which effects were found. Bars represent litter means; *percent reduction of AGD in males relative to average female AGD.

 
Areolas.
Areolas in the offspring were most easily observed on PND 13. Areolas can appear occasionally in untreated male pups and were detected in the control group (23%). This percentage, which is on a litter-means basis, is deceptively high since only 2/12 control pups had an areola but represented 2/5 of control litters. However, the percentage of pups with areolas increased significantly in the treated groups exposed on GD 14–15 and on GD 16–17, and did not appear at all in the GD 12–13 or GD 20–21 groups (Table 2Go). The number of areolas per pup, out of a possible 12 areolas, also increased significantly in these groups from the control value (0.33), with the highest value of 8.84 areolas appearing in the GD 16–17 group (Table 2Go).

Nipples.
Since untreated male rat pups can present areolas early in life but do not retain nipples, it was important to monitor the presence of nipples in adulthood. Nipples were observed on adult male offspring in the GD 14–15, GD 16–17, and GD 18–19 groups only. The percentage of males with nipples was significant in the GD 16–17 and GD 18–19 groups, while the highest incidence appeared in the GD 16–17 group. The number of nipples per animal was also highest in the GD 16–17 group (Fig. 1Go, Table 2Go).

Malformations of genitalia.
Malformations of the external genitalia included cleft prepuce, incomplete preputial separation, cleft phallus with accompanying exposed os, and hypospadias. These malformations were observed only in the GD 14–15, GD 16–17, and GD 18–19 groups. One animal from the GD 16–17 group displayed ectopic testis (Table 2Go). The incidence of malformations was significant only in the GD 16–17 group, by both ANOVA (p < 0.005) and Fisher's Exact test (12/24 individuals, vs. 0/17 individuals in control group; p < 0.001; Fig. 1Go; Table 2Go).

Body weight.
Body weight was not significantly affected in any dose group (p > 0.85; Table 3Go).


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TABLE 3 Effects of 2-Day Prenatal Administration of 400 mg/kg Vinclozolin on Reproductive Organ Weights at Necropsy (Experiment 1)
 
Organ weights.
Organ weight data was analyzed in 2 sets representing 2 age ranges. The testis, epididymis, and seminal vesicle were not affected in any group. No agenesis of the VP or SV was found. The ventral prostate was slightly reduced only in the GD 18–19 group evaluated at age PND 77–102, and was not reduced in any group evaluated (no data for GD 18–19 group) at age PND 175–176. The weight of the levator ani/bulbocavernosus muscle was significantly reduced in the GD 14–15, GD 16–17, and GD 18–19 groups at PND 77–102, while the greatest reduction was obtained in the GD 16–17 group (Fig. 1Go, Table 3Go). The LA/BC weight was also reduced in the GD 16–17 group at PND 175–176. Reductions in VP and LA/BC weights were independent of body weight by covariate analysis on a litter-means basis. Reductions in VP and LA/BC weights in the earlier, large age range were independent of age as determined by covariate analysis on an individual basis (VP, covariate p > 0.919; LA/BC covariate p > 0.617).

Experiment 2
Dam and pre-weaning pup data.
No dams presented dystocia or delivered late. Dam weight gain through the dosing period (GD 14–GD 19) was slightly reduced by vinclozolin treatment at 400 mg/kg (p < 0.05; Table 4Go). Litter size and pup weight on PND 1 and pup survival to PND 13 and to weaning were not significantly reduced by vinclozolin treatment (Table 4Go).


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TABLE 4 Dam Fertility and Preweaning Pup Viability following Vinclozolin Administration from GD 14 to GD 19 (Experiment 2)
 
Anogenital distance.
AGD on PND 1 was significantly reduced by vinclozolin treatment at 200 and 400 mg/kg/day and was not related to pup weight (covariate F(2,16) = 0.04; p > 0.80). The two vinclozolin-treated groups were not significantly different from each other (Table 5Go).


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TABLE 5 Malformations and Reduced Reproductive Organ Weights at PND 107–128 in Male Offspring Induced by Vinclozolin Administration from GD 14 to GD 19 (Experiment 2)
 
Areolas.
Presence of areolas was significantly increased in both treated groups. While only 7.2% of male offspring in the control group (n = 5/34 individuals or 4/7 litters) displayed areolas on PND 13, nearly all the males in the vinclozolin-treated groups displayed areolas. The mean number of areolas per pup was also significantly higher in the 200 mg/kg group and the 400 mg/kg group. The 2 treated groups were not significantly different from each other (Table 5Go).

Nipples.
No adult control male had nipples while 100% of the treated males had nipples. The number of nipples per animal, out of possible 12 nipples, was 9.5 in the 200 mg/kg group and 9.6 in the 400 mg/kg group, both significantly different from controls (Table 5Go).

Malformations of the genitalia.
While no malformations were observed in the control group, virtually all animals in the vinclozolin-treated groups displayed at least one malformation of the reproductive tract, and this treatment effect was significant (Table 5Go). Malformations included cleft prepuce, cleft phallus accompanied by exposed os and hypospadias, vaginal pouch, and ectopic and undescended testes. The degree to which each of these malformations manifested varied between individuals, and the incidence and severity of some malformations were higher in the 400 mg/kg than in the 200 mg/kg dose group. The incidence of severe vaginal pouch increased in a dose-dependent fashion and was significant in both the 200 mg/kg and 400 mg/kg groups by Fisher's Exact test (Fig. 2Go) and in the 400 mg/kg group by ANOVA (F[2,18] = 7.3; p < 0.005). Ectopic or undescended testes occurred only in the 400 mg/kg group and was significant by Fisher's Exact test (Fig. 2Go). The abnormal testicular position found in this study was always near the inguinal region as opposed to the kidney area, but was outside the scrotum in a suprainguinal position either immediately rostral to the inguinal ring in the abdomen (undescended) or outside the abdominal muscle wall in the abdominal or pubic area immediately under the skin (ectopic).



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FIG. 2. Percentage of dose-dependent malformations of the genitalia induced by vinclozolin, Experiment 2: dose-dependent effects of vinclozolin administered from GD 14 to GD 19 on genitalia of male offspring. The incidences of certain types of malformations, severe vaginal pouch and ectopic/undescended testes, increase at the 400 mg/kg dose. Bars represent litter means. Significant differences were analyzed on an individual basis by Fisher's Exact test; *p < 0.02, **p < 0.0001.

 
Body weight.
Body weight at necropsy was slightly reduced in the 400 mg/kg dose group (p < 0.05; Table 5Go).

Organ weights.
Vinclozolin treatment at 200 and 400 mg/kg significantly reduced the weights of the seminal vesicles, ventral prostate and LA/BC by p < 0.0001 and there was no significant difference between the 200 and 400 mg/kg groups (Table 5Go). Also, ventral prostate agenesis occurred in one animal in the 200 mg/kg group and in one animal in the 400 mg/kg group. Significant reductions were independent of body weight as found by covariate analysis.

Serum T-levels.
Testosterone levels were not significantly altered by vinclozolin treatment (p > 0.22; Table 5Go).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
In utero exposure to the AR-antagonist vinclozolin results in permanent modifications of androgen-dependent organs and structures of the male reproductive tract. In this study we sought to define the critical prenatal period during which vinclozolin exposure alters sexual differentiation, to relate these effects to the onset of AR expression in the developing tissues as reported in the literature, and to draw conclusions regarding the sensitivity of each tissue to vinclozolin. We identified GD 16–17 as the 2-day period during gestation in which fetal males are most sensitive to insult from vinclozolin, and GD 14–19 as the entire sensitive period for the reproductive effects of vinclozolin in the male.

The endpoints most affected by 2-day dosing include AGD, presence of areolas and permanent nipples, weight of the levator ani/bulbocavernosus muscles, and malformations of the external genitalia. These results not only show the critical 2 day period for sensitivity of sexual development to vinclozolin, but show that AGD, nipple retention, and levator ani/bulbocavernosus weight are sensitive indicators of antiandrogenicity. We also found that a 200 mg vinclozolin/kg dam body weight, administered from GD 14 through GD 19, is sufficient to significantly affect the reproductive development of every male rat fetus. In addition, a dose of 400 mg/kg vinclozolin administered in this time period is required to significantly induce ectopic or undescended testes. The fact that serum T levels in the adult were unaltered by prenatal vinclozolin treatment at a time when androgen-dependent organs were reduced indicates that the decreased organ weights are not due to a decreased T level, but to an organizational effect during sexual differentiation.

The effects observed in this study can be related to the timing of appearance of AR in the male reproductive tract, and illustrate the dose-dependent response of the tissues in the genital region.

The sensitive periods found with 2-day AR inhibition by vinclozolin correlate with the onset of appearance of ARs in the mesenchyme of each tissue rather than the epithelium, as described above. Appearance of ARs in the mesenchyme directs the development of reproductive tissues (Cunha et al., 1992Go). Morphological development in the seminal vesicle, the prostate, and the Wolffian duct begin after AR appear in the mesenchyme and before AR appear in the epithelial portion of these tissues. Epithelial proliferation and cytodifferentiation do not occur until testosterone binds mesenchymal cells nearest to the epithelial layer (reviewed by Cunha et al., 1992). Furthermore, mammary glands of the fetal mouse contain ARs in the mesenchyme surrounding the epithelial buds and not in the epithelium at the time of its responsiveness to testosterone and outgrowth of the primordial bud (Wasner et al., 1983Go).

AGD has been shown to be a sensitive indicator of antiandrogenicity. Doses as low as 3 mg/kg vinclozolin administered perinatally reduced the AGD on PND 2 in male rats (Gray et al., 1999aGo). AGD is used commonly as an indicator of antiandrogenicity and has been found by many to be sensitive with various types of antiandrogens (Clark et al., 1993Go; Ema et al., 1998Go; Gray et al., 1999bGo; Mylchreest et al., 1998Go; Ostby et al., 1999Go; You et al., 1998Go). In the current study, AGD proved a sensitive endpoint by its ability to be significantly reduced with only a 2-day administration of vinclozolin, and by the significance of these reductions. Also, the temporal effect on AGD in our study correlates with the appearance of AR in the mesenchyme of the affected tissue. The urogenital tubercle, folds, and swellings, which give rise to the phallus and perineum, first express mesenchymal ARs in the rat on GD 14 (Bentvelsen et al., 1995Go). AGD, a measurement in the perineal region, was first reduced with administration of vinclozolin on GD 14–15, and was further reduced on GD 16–17. The perineum on GD 18–19 still appeared responsive, since AGD was slightly reduced on GD 18–19.

The appearance of nipples has also been reported in studies on antiandrogens (Gray et al., 1999bGo; Kelce et al., 1994Go; Mylchreest et al., 1998Go; You et al., 1998Go) as a definitive indicator of antiandrogenicity. Nipple retention was a sensitive endpoint to vinclozolin toxicity in this study, shown by its inducibility at several 2-day dosing periods and the great significance of the effect. Although many studies report only thoracic nipples, we observed nipples at every point along the "milk line," or all 12 nipples. Male adult rats do not normally display nipples, and the presence of nipples indicates an interference of androgen action in this tissue. In the male rodent fetus, the mesenchyme of the mammary anlage contain ARs and respond to the rise in circulating testosterone, resulting in the condensation of the mesenchyme and disintegration and rupture of the epithelial stalk leading to the external skin (Topper et al., 1980). This response is to DHT rather than to T since 5{alpha}-reductase inhibition results in the presence of nipples in the male rat (Imperato-McGinley et al., 1992Go). Most investigation of mammary gland and nipple development has been conducted in the mouse where the onset of appearance of AR in the mammary anlage is on GD 12 (Wasner et al., 1983Go), which translates to GD 141/2 in the rat (Butler and Juurlink, 1987Go). Also, condensation of the mesenchyme of the mammary gland occurs at the time of testosterone production, or GD 14 in the rat. This time point correlates with the effects on nipple development in our study. Nipples were present in male adults after exposure to vinclozolin at GD 14–15 and increased in number, percent males affected, and significance at GD 16–17, when testosterone concentrations rise systemically.

Another significant finding is the sensitivity of the levator ani/bulbocavernosus muscle group to vinclozolin. The levator ani (LA) is sexually dimorphic at birth in rats and humans, which illustrates that the fetal period is the time of susceptibility to antiandrogenic insult. At GD 22 in the rat, the number of muscle units (MUs) is greater in the male than in the female (Tobin and Joubert, 1991Go) and the LA is larger in weight and cross sectional area in males (Jordan et al., 1997Go). The male rat LA muscle at PND 7 is highly immunoreactive to testosterone and contains a high number of AR (Jordan et al., 1997Go). This response is directly to testosterone and not DHT, as there is no 5{alpha}-reductase activity in this muscle. The onset of expression of AR in the fetal rat LA and reactivity of the fetal LA to androgens or antiandrogens has not been studied. We found the timing of effects in the LA/BC to be similar to that found in the nearby external genitalia. The LA/BC weight was significantly reduced at GD 14–15, and the reduction was even more significant at GD 16–17, while GD 18–19 dosing still resulted in a significantly reduced weight. These results provide indirect evidence that AR appear in the levator ani/bulbocavernosus muscles in the rat on GD 14 and increase in number and/or activity by GD 16–17.

In the current study, ventral prostate weight was less sensitive to vinclozolin than the above endpoints. The prostate is derived form the DHT-dependent urogenital sinus (UGS). ARs first appear in the UGS in the rat on GD 16 but greatly increase in expression by GD 18 (Bentvelsen et al., 1995Go; Hayward et al., 1996Go), when rapid prostate development begins. In our study, the effect occurred with GD 18–19 dosing, which correlates with the rise in AR and rapid proliferation of cells in the prostate rather than at the onset of appearance of AR, and it is then that AR may be more functional.

The external genitalia displayed abnormalities in response to vinclozolin exposure in this study, consistent with the effects observed in other studies, and the temporal response to vinclozolin correlated with the appearance of ARs in the mesenchyme of the tissue. The mesenchyme of the external genitalia express AR on GD 14 and expression increases thereafter (Bentvelsen et al., 1995Go). These AR directly affect development of the penis since AR continue to be present in the penis of the immature and adult rat and are responsive to T (Rajfer et al., 1980Go). The incidence of malformations of the external genitalia occurred to a significant degree with GD 16–17 dosing. This response also occurs on treatment with the 5{alpha}-reductase inhibitor, finasteride, and it is most sensitive at this same gestational time point (Clark et al., 1990). As observed in the ventral prostate, the effect on the external genitalia correlated with the rise in AR rather than in the onset of appearance of AR.

The timing of appearance of AR in the Wolffian duct system could not be directly correlated with effects, since a large portion of the Wolffian-derived structures was unaffected by 2-day vinclozolin dosing. Bentvelsen et al. (rat; 1995) and Cooke et al. (mouse; 1991) both observed a correlation between the onset of appearance of AR and the distance from the testes in the male reproductive tract: the more proximal to the testes, the earlier AR appeared in the mesenchyme. In the Wolffian duct system of the rat, AR appear first in the efferent ductules and epididymides around GD 14 (Bentvelsen et al., 1995Go) or GD 15 (You and Sar, 1998Go), in the vas on GD 19 or 20, and in the seminal vesicles on GD 21 (Bentvelsen et al., 1995Go). Although vinclozolin administration included these time points, no effects were observed in the testes, epididymides, or seminal vesicles. Gray et al. (1994) observed that the Wolffian duct appeared more resistant to insult by vinclozolin, and Imperato-McGinley et al. (1992) found that much higher doses of flutamide were required to affect the testes, epididymides, and vas deferens.

Most of the aforementioned effects did not become more significant as the dose and duration of dosing were increased. Some effects, ectopic/undescended testes and vaginal pouch, were observed only when both the dose and duration of dosing were increased.

Ectopic and undescended testes were observed in our study to a significant degree only with GD 14–19 administration of vinclozolin and only in the 400 mg/kg group. Thus, this T-dependent process (Imperato-McGinley et al., 1992Go) appears to be less sensitive to antiandrogenic insult than other events studied here. Testicular descent has been described in 2 stages, transabdominal and transscrotal, and both these events may be under androgenic control. However, factors such as intraabdominal pressure, suppression of the cranial suspensory ligament, neural control, and hormones other than androgens may play roles in testicular descent (Levy and Husmann, 1995Go). The gubernaculum is at least partially responsible. ARs are present in the gubernaculum (Husmann and McPhaul, 1991bGo) and flutamide administered on GDs 15 to 17 prevented gubernaculum cord regression in the rat (Cain et al., 1995Go). The abnormal testicular position found in this study was always near the inguinal region, i.e., the testes had accomplished transabdominal migration while transinguinal migration was interfered with. It appeared more specifically that the positioning of the gubernaculum was in the abdominal wall rather than in the scrotum. Anomalies such as these have been described in rodents after antiandrogenic treatment (Neuman et al., 1970; van der Schoot, 1992Go). Also, inguinal hernias were produced in wallabies treated with flutamide during sexual development (Lucas et al., 1997Go).

The fact that the presence of a vaginal orifice was observed only with extended dosing in Experiment 2 and that the severity of this malformation increased with dose illustrates both the dose-dependency of this tissue on androgen action and its relative insensitivity to antiandrogenic insult. This finding is consistent with those of other AR inhibitors. The AR inhibitor flutamide demasculinized the external genitalia more completely than did the 5{alpha}-reductase inhibitor finasteride (Imperato-McGinley et al., 1992Go). Vaginal orifices appeared in rats after 12-day prenatal dosing using 10 mg flutamide per dam (van der Schoot, 1992Go), which is comparable in potency to the dosing regime used in our Experiment 2. Although 25–100 mg/kg/day of procymidone, a fungicide and AR inhibitor, significantly affected AGD and nipple formation, vaginal pouch was significantly affected only at 200 mg/kg from GD 14–PND 3 (Ostby et al., 1999Go). Also, while AGD was decreased and nipples were induced, vaginal pouches were not observed in male offspring exposed perinatally to p,p'-DDE (You et al., 1998Go), nor in males treated with various antiandrogens that displayed hypospadias (Gray et al., 1999bGo).

Extending the dosing period to include gestational days 14 through 19 did not increase the significance of the effect in the more sensitive endpoints (i.e., AGD, nipples, LA/BC weight), even at half the dose. However, this dosing regimen increased the significance of other effects and induced still others, including the reduced weight of the Wolffian duct-derived seminal vesicle. This is not to say other Wolffian duct derivatives were not affected. The epididymis, which was not weighed, could have been affected with the extended dose regime in our study, since administration of vinclozolin at 200 mg/kg from GD 14 through PND 4 reduced the weight of the epididymis (Gray et al., 1999bGo). The greater overall effect in androgen-dependent target tissues with the extended dosing period may show that effects are dependent upon the cumulative dose, or area under the curve (AUC), in addition to the timing or concentration of the dose alone (e.g., 6 days of 200 mg, or 1200 mg > 2 days of 400 mg, or 800 mg). In support of this theory, raising the dose level to 400 mg/kg vinclozolin for 6 days further affected development of the male genitalia. The effects may also be dependent on clearance of vinclozolin and its metabolites, requiring a continuous dose of the reversible AR-inhibitor over the course of a developmental event to ensure an effect in that organ. Since dosing on GD 12–13, a stage immediately before sexual differentiation and expression of AR, did not affect any tissues, clearance of vinclozolin and its metabolites from the rat may take place within a day. In light of these two considerations, some antiandrogenic effects found in Experiment 2 may actually be more significant if the dosing period is extended, or even moved to a later time frame.

This study illustrates the sensitivity of the fetus to vinclozolin as evident in the significance and spectrum of the effects. These effects and the timing of the effects are consistent with other studies (Clark et al., 1990; Husmann and McPhaul, 1991aGo; Silversides et al., 1995Go). The tissues affected by vinclozolin are most sensitive at or soon after the time they are acquiring AR in the mesenchyme, and are further sensitive to the dose and duration of dosing during this sensitive period. By the time ARs are no longer located in the mesenchyme but rather primarily in the epithelium, malformations cannot be induced. Indeed, studies in which neonates are dosed with high levels of EDCs are unable to induce gross malformations (Prins, 1995). The information obtained with the current study is invaluable to further investigation into the mechanisms of vinclozolin-induced antiandrogenicity in the developing fetus.


    NOTES
 
The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

1 To whom correspondence should be addressed. Fax: (919) 541-4017. E-mail: gray.earl{at}epa.gov. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
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