ToxSci Advance Access originally published online on January 11, 2007
Toxicological Sciences 2007 96(2):335-345; doi:10.1093/toxsci/kfm002
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Published by Oxford University Press 2007.
Prenatal Testosterone Exposure Permanently Masculinizes Anogenital Distance, Nipple Development, and Reproductive Tract Morphology in Female Sprague-Dawley Rats
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* Department of Zoology, North Carolina State University, Raleigh, North Carolina 27695
Reproductive Toxicology Division, Endocrinology Branch, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
USEPA/NCSU Cooperative Training agreement, Raleigh, North Carolina 27695
Merck Research Laboratories, West Point, Pennsylvania 19486
1 To whom correspondence should be addressed at Reproductive Toxicology Division, Endocrinology Branch, MD 72, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-4017. E-mail: gray.earl{at}epa.gov.
Received October 25, 2006; accepted January 2, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In mammals, abnormal increases in fetal androgens disrupt normal development of the female phenotype. Due to the recent concern regarding environmental androgen-active chemicals, there is a need to identify sources of fetal androgen variation and sensitive developmental markers for androgenic activity in female rats. Anogenital distances (AGD), nipple retention, reproductive tract, and external genitalia are morphological parameters organized by prenatal androgens and are predictive of altered masculinized/defeminized phenotype in adult female mice and rats. The objectives of this study were to (1) characterize the natural prenatal androgen environment of rats including the magnitude of the intrauterine position (IUP) effect, (2) characterize the permanent effects of prenatal androgen exposure on female rats, and (3) determine the ability of AGD and areolas to predict these permanent androgenic alterations in female rats. Untreated male fetal rats had higher tissue testosterone (T) concentrations than females in the amniotic fluid, reproductive tract, gonad, and fetal body. The intrauterine position (IUP) of male and female fetuses did not affect T concentrations or AGD in male or female rats at gestational day (GD) 22. Female offspring exposed to 0, 1.5, and 2.5 mg/kg/day testosterone propionate (TP) on GDs 14–18 displayed increased AGD at postnatal day (PND) 2 and decreased nipples at PND 13 and as adults. TP-induced changes in neonatal AGD and infant areola number were reliable indicators of permanently altered adult phenotype in female rats. Further, females in the two high-dose groups displayed increased incidences of external genital malformations and the presence of prostatic tissue, not normally found in female rats.
Key Words: AGD; areola; masculinization; reproductive development; fetal androgen; intrauterine position.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is increasing concern about the potential for environmental endocrine-disrupting chemicals (EDCs) to alter mammalian sexual differentiation. Although initial research focused on environmental estrogens and antiandrogens, androgenic activity has now been described in water from kraft pulp and paper mills and concentrated animal feed operations in the United States and Europe (Orlando et al., 2004
; Parks et al., 2001). Reports have shown an androgenic growth promoter used in livestock, Trenbolone, has a half-life of greater than 260 days in animal waste (Hotchkiss and Nelson, 2006; Schiffer et al., 2001; Wilson et al., 2002). Further, in humans, the developmental effects of prenatal androgens due to medical conditions such as polycystic ovarian syndrome or congenital adrenal hyperplasia remain an active area of investigation (Blank et al., 2006; Otten et al., 2005). Due to these medical conditions as well as reports of environmental androgenic chemicals, natural sources of variation in masculinization and sensitive end points of androgenic activity need to be identified.
Prenatal exposure of female rodents to exogenous androgens results in physiological and behavioral masculinization (Greene et al., 1939; Huffman and Hendricks, 1981; Rhees et al., 1997; Slob et al., 1983; Wolf et al., 2002). In addition, natural variation in adult female reproductive phenotype, consistent with prenatal androgen exposure, has been reported in numerous studies (reviewed in Ryan and Vandenbergh, 2002). Examination of androgen concentrations of fetal rats shows that males have roughly twice the whole animal content of androgens as females (Baum et al., 1991; Houtsmuller et al., 1995; Weisz and Ward, 1980). This difference is maintained over the later part of gestation from gestational days (GDs) 16–21 and reaches its peak around the critical developmental window of approximately postnatal days (PNDs) 18–19 (Baum et al., 1991). While the testis is the primary source of testosterone (T) in the male, in the developing female the source of androgens is unclear, but appears to be primarily of maternal-placental origin (Baum et al., 1991; Houtsmuller et al., 1995; Weisz and Ward, 1980). There is no evidence that the developing female ovary could be the source of androgens (Slob et al., 1980; Warren et al., 1973). Therefore, extraovarian sources of androgens have been proposed, including the placenta (Baum et al., 1991; Vreeburg et al., 1983), fetal adrenals (Stahl et al., 1991), maternal sources (vom Saal, 1989), or male fetuses via the intrauterine position or IUP (reviewed in Ryan and Vandenbergh, 2002).
In species shown to have significant IUP effects, there is uncertainty as to how androgens may be transferred from males to females in these litter-bearing species. It is unclear whether androgens are transferred through uterine blood flow in the rostral direction (caudal model) or transferred through fetal membranes (contiguous model). The IUP has been the focus of discussions in toxicology because of the potential for the IUP to alter susceptibility of fetuses to endogenous hormones and EDCs (Clark et al., 1993a; Howdeshell et al., 1999). In this regard, it also has been suggested that failure to account for IUP in EDC toxicology studies could lead to false-negative results, especially when adverse alterations are produced in low dosage levels in fetuses from only one IUP (Howdeshell et al., 1999; Welshons et al., 1999). To date, few studies have accounted for the IUP in rat toxicological studies of EDCs.
External biomarkers of prenatal androgen exposure or disruption have been described in rodents. These biomarkers include the anogenital distance or AGD and the prepubertal nipple/areolae number. The AGD is defined as the distance between the genital papilla and the anus; male rodents have AGDs that are approximately twice the length as those of females (Gray et al., 1999; Vandenbergh and Huggett, 1995). In mice, the AGD in females varies with IUP (Hotchkiss and Vandenbergh, 2005; Vandenbergh and Huggett, 1995) and is predictive of a number of adult physiological and behavioral characteristics (Vandenbergh and Hotchkiss, 2001). Interestingly, in humans, the AGD is also sexually dimorphic (Salazar-Martinez et al., 2004) and is increased in female infants viralized by androgens resulting from congenital adrenal hyperplasia (Callegari et al., 1987). Areolae (areolas) are dark areas surrounding the nipple bud and are indicative of adult nipples. Female rats typically have 12 nipples whereas males have none. Both of these biomarkers vary with prenatal exposure to androgens and antiandrogens in females and males, respectively (Gray et al., 1999).
Our objectives were to (1) characterize the natural prenatal androgen environment of rats including the magnitude of the IUP effect, (2) further characterize the effects of prenatal androgenization on reproductive development, and (3) determine the utility of AGD and areola number for predicting alterations to the adult reproductive phenotype. Further, identification of sensitive end points of masculinization in females exposed prenatally to T will provide a standard against which adverse effects of putative environmental androgens can be assessed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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General Methods
Pregnant Sprague-Dawley (SD) rats (Charles River Breeding Laboratory, Raleigh, NC) were shipped on the day after mating and housed individually in clear plastic cages (20 x 25 x 47 cm) with laboratory grade pine shavings as bedding (Northeastern Products, Warrensburg, NY) in an AAALAC-approved facility. The day after mating was designated day 1 of gestation. Animals were provided Purina Rat Chow (5008 during pregnancy and lactation and 5001 as juveniles and adults) and filtered (5 µm) water, ad libitum, in a room with a 14:10 h (light/dark, lights off at 1100 h eastern standard time [EST]) photoperiod and temperature of 20–22°C with a relative humidity of 45–55%. All measurements described were taken by an experimenter blind to the treatment groups of the animals. These studies were conducted under a protocol that had been approved by the National Health and Environmental Effects Research Laboratory institutional animal care and use committee.
Study 1—Variation in T Concentrations and AGD in Untreated Fetal Rats
Anogenital Distance
At GD 22 (or the day before parturition), 18 pregnant females were anesthetized using CO2 and euthanized by decapitation. C-sections were performed, the fetuses removed, and IUPs recorded. Fetuses were then sexed (based on AGD length), their body weights recorded, and AGD measurements taken using a dissecting microscope with an ocular micrometer (x15). The AGD was defined as the distance between the base of the genital papilla and the rostral end of the anal opening.
Fetal T
Four experimental blocks of five time-pregnant females were ordered and shipped to our animal facility on GD 2. Animals were then allowed to acclimatize in the animal facility until the morning of GD 18. Pregnant females were euthanized by exposure to CO2 and subsequent decapitation between 0800 h and 1100 h EST on the morning of GD 18. We selected GD 18 for assessing fetal T concentrations because this is the period of peak T content (Baum et al., 1991). Maternal serum was collected and frozen at – 70°C for later hormone analysis. Fetuses were then removed from the uterus, and their IUP were recorded and placed on ice. Amniotic fluid was collected and fetuses were sexed by presence of testes or ovaries. The gonads, reproductive tracts, and the remaining fetal bodies were then collected. Fetal body weights minus all other tissues were determined. Reproductive tracts were defined as the internal and external (genital tubercle included) reproductive tissues minus the gonads. Placentas were collected only for one experimental block (hereafter referred to as "block") and weighed on the day of necropsy. Tissues were then frozen on dry ice and subsequently stored in a – 70°C freezer for future extraction and hormone analysis.
Fetal Extractions
Extraction of T from fetal carcasses.
Extractions and radioimmunoassay (RIA) analysis were adapted from the methods described by Parks et al. (2001).
Extraction of T from gonads and adrenals.
Each gonad or adrenal was placed in a 12 x 75–mm glass tube, homogenized with a plastic pestle in 100 µl of deionized water, then extracted (2x) with 0.5 ml of ethyl ether, vortexed for 20 s, and allowed to separate for 10 min. Samples were snap frozen in an acetone/dry ice bath until the aqueous portion was frozen. The supernatant (ethyl ether fraction) was then poured into a clean 12 x 75–mm glass tube and the ether was evaporated off all samples overnight in a fume hood.
Reproductive tract and placental extractions.
Each reproductive tract or placenta was homogenized with 500 µl of deionized water and then extracted twice with 1.0 ml of ethyl ether, vortexed for 20–30 s, and centrifuged at 2200 rpm (1000 x g) for 10 min (Beckman GS-6R). Samples were snap frozen in acetone/dry ice bath and processed as described for the gonads and adrenals.
Amniotic fluid extractions.
Two hundred microliters of amniotic fluid was transferred into a clean 12 x 75–mm glass tube, extracted (2x) with 500 µl of ethyl ether, vortexed for 20 s, allowed to separate for 10 min, snap frozen in acetone, and processed as described for the gonads above.
Hormone analysis.
T levels in the fetuses were determined by RIA using the Coat-a-Count method (Diagnostic Products Corporation, Los Angeles, CA). Samples were resuspended with zero standard (provided in the RIA kit for establishing a standard curve), vortexed, and then assayed for T. The cross reactivity of the assay was 3.3% for dihydrotestosterone and 0% for androsterone. The sensitivity of the assay was 4 ng/dl. Extraction efficiencies as measured by radioisotope recovery were estimated between 66.0 and 70.0%. Intraassay coefficient of variation was 2.6%. Maternal serum as well as reproductive tracts, gonads, adrenals, placentas, and placental tissues were assayed using the methods previously described (Kelce et al., 1997). Intraassay coefficient of variation was between 4.2 and 7.6%. Data are expressed as ng/ml for maternal serum and amniotic fluid, ng/g for carcasses and placentas, and ng/tissue for reproductive tracts and gonads.
Adrenal T content.
Adrenals were obtained from two separate litters at a later date using the same protocol as outlined above. Necropsy was done on the morning of GD 18, and adrenals were removed from fetuses and frozen for later hormone analysis by RIA.
Study 2—Fetal T Concentrations in TP-Exposed Rats
Hormone Analysis
Animals.
Experiment 2 was conducted in two blocks of six to seven pregnant females per block (two to three females per treatment) as above. For each block, females were ordered time pregnant and shipped to our animal facility on GD 2. On GD 13, animals were weight ranked and assigned randomly to treatment in a manner that provided similar means in body weights for the different treatment groups. Pregnant rats were treated with laboratory-grade corn oil (CAS # 8001-30-7, Sigma Aldrich, St. Louis, MO lot # 107H1649) or T propionate (CAS # 57-85-2, Sigma Aldrich, St. Louis, MO, lot # 98H0566) sc from GDs 14–18 at 0, 1.5, or 2.5 mg/kg/day in 0.1 ml of corn oil adjusted daily for body weight. These doses were used because higher doses induced adverse effects including loss of litters, delay of delivery, and decreased pup weight and extensive mortality in F1 females after weaning due to reproductive tract malformations (Wolf et al., 2002). This dosing window was selected based on previous data showing that it is a critical period for differentiation of male and female reproductive phenotypes (Baum et al., 1991; Weisz and Ward, 1980). After dosing on the morning of GD 18, pregnant females were anesthetized by exposure to CO2 and subsequently decapitated in between 0800 h and 1100 h EST. A total of two to three fetuses per sex were necropsied per litter. Body mass, amniotic fluid, gonads, reproductive tracts, and carcasses were collected as described above.
Study 3—Postnatal Effects of Fetal TP Exposure in Female Rats
Animals
Twenty-four pregnant SD rats (eight females per treatment group) were shipped on the day after mating and housed as described above.
Maternal Dosing
Methods and doses were identical to those outlined for study 2.
Neonatal and Pubertal Data
On PND 2 (morning after delivery was defined as PND 1), pups were sexed, body weight recorded, and AGD measurements taken. At this time, pups were also tattooed on the paws with black India ink within litters to keep track of individuals.
At PND 13, animals were reweighed and examined for presence or absence of areolae (dark areas lacking hair found in the region of the developing nipple bud). On PND 23 female animals were weaned, housed with two to three littermates, and weighed, and the AGD was measured with calipers to the nearest 0.1 mm.
The onset of puberty was assessed in females by monitoring animals daily for vaginal opening (VO) (normally an indicator of puberty in rats) from PNDs 29–45. Animals were weighed daily until VO was achieved. Data were analyzed both on an individual and litter means basis. Treated females without a vaginal orifice were not included in data analysis for age at VO.
Estrous Cyclicity
Two females that displayed VO were selected randomly from each litter for assessment of estrous cyclicity by daily vaginal lavages (smears). Monitoring began on PND 50 and continued for 17 days. Smears were taken between 0830 h and 1030 h each morning, examined, unstained, by light microscopy (x20), and assessed for relative abundance of leukocytes, nucleated epithelial cells, and cornified epithelial cells. Assessment of the cyclicity was determined by evaluating the number of estrous smears displayed by each animal over the monitoring period.
Necropsy
At approximately 6 months of age, females were weighed, anesthetized with CO2, and decapitated. The ventral surface of each animal was shaved, the AGD was measured, and the number of permanent nipples was counted. In addition to AGD, the distance between the caudal opening of the vagina and the genital papilla (VOG) was measured with calipers. External malformations such as lack of VO, presence of cleft phallus, hypospadias, or vaginal thread were noted. The uterus (with and without fluid) and ovaries were removed from the animal, trimmed of fat, and weighed. Finally, females were examined for presence of male-like structures such as levator ani bulbocavernosus muscles, ventral prostate, bulbourethral glands, or seminal vesicles. If any male structures were found, they were weighed and collected for subsequent histological evaluation.
Correlations between developmental markers (neonatal AGD and infant areolae) and malformations in F1 females
Correlations were generated using individual values for AGDs, total areola number, malformations, and reproductive organ weights in adults. Malformations were noted as either present or absent for each animal. For analysis, both external and total malformations were examined for significance. External malformations included hypospadias, absence of VO, or cleft phallus. Total malformations were calculated using the external malformation score + presence or absence of the ventral prostate. In addition to correlation analysis on AGD data for individual animals, data were separated into categories for graphical purposes. AGDs were separated into six different categories spanning the entire range of values with category 1 being an neonatal AGD of 1.2 mm and smaller, category 2 = 1.2–1.4 mm, category 3 = 1.4–1.6 mm, category 4 = 1.6–1.8 mm, category 5 = 1.8–2.0 mm, and category 6 = 2.0 mm and larger. Adult nipple retention was not included when analyzing for infant areola correlations with adult malformations.
Statistical Analysis
All analyses were done using both litter means and individual animal values. A significant litter effect was detected for both the AGD study and the T analyses. This necessitated our consideration of the litter effects in the overall model as suggested by others (Zorrilla, 1997). Data and statistics are presented for litter means. As there was a significant difference in body weight between male and female fetuses on GD 22 and the correlation between body weight and AGD was significant, body weight was used as a covariate in the analysis of AGD in experiment 1. Block was considered in all analyses. Analysis and statistics were calculated by analysis of variance using PROC GLM Statistical Analysis Software (SAS version 6.08, Cary, NC) on the EPA RTP IBM mainframe. If overall analysis of variance was significant (p < 0.05), a LSMEANS (Fisher) post hoc test was then used to further investigate differences between groups. Correlation analyses were done using the data from individual animals and the PROC CORR option on SAS that included all females from all treatments for each dose of TP.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Study 1—Variation in T Concentrations and AGD in Untreated Fetal Rats
AGD and Body Weight on GD 22
The AGD (p < 0.0001) and body weight (p < 0.0001) of fetuses on GD 22 were found to be significantly different between males and females (Fig. 1). Analysis of the effect of the gender of the contiguous pups in the uterus on the AGD did not show a significant effect (Fig. 1; 0M = 1M = 2M). Analysis of the AGD data in terms of the caudal hypothesis also did not show a significant effect due to fetal positioning in utero (Fig. 1; – CM vs. + CM). Analysis of the fetal weights revealed an effect for only caudal positioning of females (p = 0.014) (Fig. 1). No correlations between sex ratio of the litter, sex ratio of the horn, dam blood T content, and AGD of the developing pups were observed (data not shown).
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Fetal T Concentration on GD 18
There was a significant effect of sex (male > female) on androgen content in all tissues examined except the placenta. Litter mean values for tissues examined (gonads, reproductive tract, carcass, amniotic fluid, and placenta) are presented in Table 1. T concentrations for fetal adrenals did not differ significantly between males (0.025 ng/adrenal) and females (0.022 ng/adrenal). One block of fetal carcasses was not included in the analysis due to abnormally low T content based on previous studies. However, inclusion of this block in the analysis did not alter the interpretation of the data. Overall, the analysis did not reveal any significant effect of IUP with either the contiguous or caudal models for either individual or litter mean values. Any trend observed that appears to be in accordance with IUP was not statistically significant, and the amount of variability explained by the contiguous IUP was less than 3% (r2 < 0.03) in all cases.
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In addition to a robust sex effect in most of the tissues, there was an effect of litter on the hormone levels seen in some tissues. Specifically, in the females, there was a significant litter effect on fetal weight (p = 0.0008), fetal T (p = 0.012), and placental T (p = 0.025). Carcass weights also did not differ due to the IUP of the particular individual in either females or males, and there was no significant difference due to sex (Table 1). Also, placental T levels did not correlate with fetal tissue T levels. Correlation analysis of the influence of sex ratio did not reveal consistent results with the influences of the number of neighboring males on either male or female androgen content.
Study 2—Fetal T Concentrations in TP-Exposed Rats
Confirmation of Elevated Fetal T on GD 18
Fetal T concentrations were significantly higher in males than in females (Table 2) (p < 0.05). Amniotic fluid T concentrations were significantly different between males and females in only the control group (Table 2). Analysis of the treatment effect in the females revealed that carcass T in the 1.5- and 2.5-mg/kg/day dose groups was significantly elevated as compared to untreated controls (Table 2). Female amniotic fluid T in the 2.5-mg/kg/day dose group was significantly greater (one-way ANOVA) than control values.
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Although not reaching significance (p = 0.11), TP treatment tended to decrease the number of live fetuses on GD 18 and increased the numbers of fetal resorptions observed. In the 1.5-mg/kg/day treatment group, one of five litters had at least one resorption, whereas in the 2.5-mg/kg/day group, all four litters had at least one resorption. In contrast, the control group had no resorptions.
Study 3—Postnatal Effects of Fetal TP Exposure in Female Rats
Maternal and Pregnancy Data
T treatment significantly reduced body weight gain in pregnant females for both doses of TP (Table 3). Implantation numbers, litter size, and pup mortality in treated litters were not significantly different from controls (Table 3). One dam in the 1.5-mg dose group did not deliver despite having a normal number of implantation scars (data not shown). In addition, another litter of pups in the 1.5-mg/kg/day group did not survive to weaning (data not shown). Female pup mortality in the control, 1.5-mg/kg/day, and 2.5-mg/kg/day groups was 25.4, 23.8, and 36.2%, respectively. Similar losses were recorded in the males (data not shown).
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Neonatal and Infant Data
Body weights of treated males and females were significantly reduced on PND 2 (Table 3). Compared to control, male body weights were reduced 17.0% in the 1.5-mg/kg/day TP-treated group and 19.5% in the 2.5-mg/kg/day TP-treated group (Table 3). Females were similarly affected with a 15.2 and 20.0% reduction for the 1.5- and 2.5-mg/kg/day dose groups, respectively (Table 3).
Neonatal AGD was significantly increased by 14.8% in females of the 1.5-mg/kg/day TP-treated group and by 21.6% in the 2.5-mg/kg/day TP-treated group (Fig. 2). In males, AGD was not significantly altered in TP-exposed groups. Prenatal exposure to T decreased the mean number of areolae in F1 females in the 2.5-mg/kg/day TP group by 93% and by 25.8% in the 1.5-mg TP group (Fig. 3). Examination of the mean number of faint or small areolae present in each of the treatment groups yielded similar results. The 1.5- and 2.5-mg/kg/day TP groups had, on average, a 6.12-fold and 16.6-fold (respectively) increase in small or faint areolae.
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Prepubertal and Adult Data
AGDs were slightly, though not significantly, affected in T-treated females at weaning (Table 4). Body weights at this age were reduced (from approximately 51 g to 45 g) by treatment, but this was not significant. However, considering body weight as a covariate in the analysis revealed that treatment did have a significant effect on AGD at weaning (p = 0.0009).
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There was a significant delay in the onset of VO in the 2.5-mg/kg/day group by approximately 2 days (Table 5). Onset of VO appeared to be independent of any effect due to the weight of the animal as weights of the females at VO were similar across treatment groups (Table 5).
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Assessment of estrous cyclicity data did not reveal a significant decrease in the number of observed estrous cycles over a 17-day period for both treatment groups (Table 5). However, there was a trend in the reduction in the number of estrous cycles consistent with the observation of a significant decrease (p < 0.05: one-tailed t-test) in the number of animals judged to be cycling normally (judged by observing two or more cycles over the indicated time period) (Table 5). In the 1.5-mg/kg/day TP group, approximately 75% of the animals displayed two or more cycles and 66% of the animals in the 2.5-mg/kg/day group showed two or more cycles (Table 5).
Adult Necropsy Data
External quantitative measurements.
Female rats prenatally exposed to T had decreased numbers of normal nipples and increased incidence of faint or missing nipples. These effects were dose related with the 2.5-mg/kg/day TP treatment group being the most affected with a mean number of irregular nipples followed by the 1.5-mg/kg/day TP group and finally the control females (Table 4). AGDs were significantly increased in androgen-treated animals (1.5-mg/kg/day TP: 8.8%; 2.5-mg/kg/day TP: 11.5%) (Table 4). In addition, the distance from the top of the VO to genital papilla was significantly decreased in TP-treated animals (Table 4).
External malformations of the genitalia.
Dose-dependent malformations of the genital area were observed including cleft phallus, hypospadias, and absence of VO (Fig. 4). In the 1.5-mg/kg/day TP group, 30.6% of the animals had cleft phallus. All animals in this group had normal VOs and no hypospadias (Fig. 5). In the 2.5-mg/kg/day TP group, 95.8% had cleft phallus, 28.1% hypospadias, and 14.0% complete agenesis of the lower vagina and vaginal orifice (Fig. 5).
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Internal reproductive tissue weights and agenesis of female reproductive tissues.
There was no apparent difference in uterine weight (control: 0.57 ± 0.04 g; 1.5 mg: 0.58 ± 0.04 g; 2.5 mg/kg/day: 0.56 ± 0.06 g) or paired ovarian weights (control:0.12 ± 0.006 g; 1.5 mg/kg/day: 0.11 ± 0.002 g; 2.5 mg/kg/day: 0.11 ± 0.005 g) between treatment groups or control animals. Male-like structures such as bulbourethral glands or seminal vesicles were not apparent in TP-treated or control groups. However, ventral prostate tissue was present in both the 1.5- and 2.5-mg/kg/day TP groups (35.0 and 82.3%, respectively) (Fig. 5).
Correlation of early developmental parameters with permanent effects.
Overall, both neonatal AGD and infant areola number were correlated with malformations across treatments. External malformations correlated with AGD (r = 0.55, p < 0.001) and areolae (r = –0.77, p < 0.001). Internal and external malformations also correlated with AGD (r = 0.63, p < 0.001) and areolae (r = –0.76, p < 0.001) (Fig. 6). Within–treatment group analysis suggested that AGD and areolae were still predictive of adult phenotype, but with less certainty than that seen within treatment groups (data not shown). In most cases, areolae were more predictive than AGD in terms of both number of significant end points and r values. Both neonatal AGD and infant areolae correlated significantly with alterations in the adult AGD (r = 0.59, p < 0.0001) and nipples (r = 0.44, p < 0.0001). Neonatal AGD and infant areolae correlated significantly with each other (r = –0.50, p < 0.0001).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results of the current study, using a relatively large number of litters (n = 20), demonstrate that tissue T concentrations in female fetal rats are not affected by the gender of contiguous fetuses in the uterus. There was no IUP effect on T levels or AGD in either male or female SD rat fetuses. Further, exposure of pregnant rats to TP elevated T levels in female but not male amniotic fluid and carcass compartments. Treated female rat fetuses displayed T levels approaching those of normal male fetal rat levels, and the increase in T was sufficient to masculinize the female AGD, nipple development, and reproductive tract morphology. Treated females displayed longer AGDs, reduced numbers of nipples, retained male prostatic tissue, delayed VO, and vaginal agenesis. The effects on female neonatal AGD and the numbers of nipples in infant life were permanent, and these early alterations were predictive of adult reproductive tract malformations.
Both caudal (androgen transfer through uterine blood flow) and contiguous (androgen transfer through fetal membranes) models of IUP were considered as a source of the variation in tissue T, but neither was found to be a significant factor in the female's AGD. In addition, measurable levels of T were detected in all of the tissues examined in both males and females on GD 18. Interestingly, in females, important areas for sexual development such as the reproductive tract and gonads had detectable levels of T on GD 18. The source of these androgens in rats does not appear to be the IUP as we did not find evidence of significant transfer of androgens between rat fetuses. However, the relatively large amount of T found in the placenta makes it a likely source for the androgens in the female rat fetuses. Lack of a finding of an IUP effect in rats in this study does not preclude interfetal communication in utero (Ryan and Vandenbergh, 2002; Vandenbergh and Hotchkiss, 2001) via hormones other than T or at different gestational periods or in other species. However, intrauterine positioning does not appear to be an important factor to control in studies of the developmental effects of environmental androgens and antiandrogens or chemicals that affect AGD in SD rats.
In utero administration of 2.5-mg/kg/day TP during the fetal period delayed the onset of puberty in females, indicative of neuroendocrine masculinization during critical perinatal development. Altered onset of puberty due to prenatal/neonatal androgens has been observed in mice (Ryan and Vandenbergh, 2002; Vandenbergh and Huggett, 1995), rats (Rhees et al., 1997), and gerbils (Clark et al., 1993a). Although all adult females displayed estrous cycle variations, rats exposed to the higher dose of androgen displayed a disruption of ovarian cyclicity as indicated by increased length of estrous cycles. TP administration may partially disrupt the neural circuitry regulating the generation of normal pulses from the hypothalamus in female rats, although the reported sensitive period for central nervous system sex differentiation in regards to hypothalamic and ovarian cyclicity appears to be early postnatal, not prenatal. Although others have suggested that ovarian cycles are not as sensitive to disruption by prenatal androgens as in neonatal life (Greene et al., 1939; Rhees et al., 1997; Wolf et al., 2002), these studies only examined the presence of corpora lutea in ovaries or responsiveness to gonadotropin-releasing hormone treatment, and not daily cyclicity, using vaginal smears.
Malformations observed in androgen-exposed adult female rats were consistent with increased masculinization of phenotype (Swanson and Werff ten Bosch, 1965; Wolf et al., 2002). Externally, AGDs were increased and there was an elevated incidence of malformations; internally, male sex accessory structures were observed in treated female offspring. The most sensitive end point for retention of male internal structures, the ventral prostate (Greene et al., 1939; Wolf et al., 2002), may be related to the development of this tissue from the urogenital sinus instead of the Wolffian tract from which seminal vesicle and other reproductive tissues differentiate.
It is interesting that the concentration of T was not significantly increased in male fetuses of TP-treated dams, yet female fetuses of TP-treated dams display elevated concentrations of T. The reason for this discrepancy is unknown, but the phenomenon has been reported previously (Wolf et al., 2002, 2004). Although doses approximating those used in this study are capable of significantly increasing T in dams, use of a higher dose is not feasible due to maternal toxicity (Wolf et al., 2002). It is probable that the placenta plays a critical role in both metabolizing T to other steroids (that we did not measure, herein) and in regulating T concentrations in fetuses. Other studies have shown that exogenous T delivered to the dam is not equivalent to the dose received by the fetus at least partially because the placenta is capable of modulating (blocking and/or metabolizing) the T reaching the fetus (Slob et al., 1983; Vreeburg et al., 1983). Therefore, the placenta may serve an important role in regulating the T exposure of males (and possibly females at high doses). This may explain why the finding that exogenous T treatment was not able to "rescue" male fetuses exposed to the androgen receptor antagonist, vincolozolin (Wolf et al., 2004). In essence, administration of T in males is not able to increase T and therefore offset the adverse effects of an androgen receptor antagonist during development.
In this study, both the increase in AGD and the reduction in the number of normally formed areolae (reflective of adult nipples) were permanent effects lasting into adulthood. It has been suggested that some of these changes are simply transient effects that disappear in adult animals (Clark et al., 1990, 1993b). However, the data presented here in the female and elsewhere in male rats (Bowman et al., 2003; Hotchkiss et al., 2004; McIntyre et al., 2001) provide evidence to the contrary. Both neonatal AGD and infant areola number are predictive across treatments of external and internal alterations in the adult animal (Faber and Hughes, 1992; Gray et al., 1999; Hotchkiss et al., 2004).
Current testing guidelines for EDCs include only measuring AGD in F2 generation in a multigenerational study if reproductive effects are seen in the F1. However, when in vitro or short-term in vivo screening data indicate that a chemical has androgenic potential, neonatal AGD and infant nipple numbers should be measured in F1 females, not only in the F2 since the F2 may not be followed past weaning. In addition, a number of malformations were seen in TP-exposed F1 female rat offspring that could be missed in a standard multigenerational study unless the prosectors were alerted to the potential lesions that result from prenatal androgen exposures. A list of recommended end points affected by prenatal androgens has been developed from our studies (Table 6), and these additional androgen-sensitive end points may be useful to detect low levels of androgenic activity in standard multigenerational toxicity studies. However, since T crosses the placenta so poorly, it is possible that reproductive end points would be affected in the parent generation (P0) at lower doses than those required here to induce malformations in the developing female rat. In fact, whereas 0.1-mg/kg/day TP was not effective in masculinizing the female rat in utero, this dose does produce androgenic responses when castrate-immature male rats are treated for 10 days sc with this dose level (Owens et al., 2006).
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Considering the information presented here, both AGD and areolae should be considered permanent alterations and as indicators of other EDC androgen-mediated effects in the adult animal. The determination of areola number, not included to date in any test guideline, can be used as supporting evidence for an androgen-mediated effect. Due to the sensitivity and ease with which both of these biomarkers can be measured and their highly predictive nature, these end points should be included in test guidelines designed to test for EDC activity.
In regard to standard multigenerational toxicology testing, observations that androgens occur in the environment (Owens et al., 2006; Parks et al., 2001; Schiffer et al., 2001) require both a clear understanding of the effects of exogenous androgens on reproductive physiology of females and adequate end points to assess their activity. This study shows that alterations in the AGD of the newborn female SD rat are permanent and correlate highly with other, more serious malformations. In light of recent data suggesting chemicals may impact human AGDs (Swan et al., 2005), increased attention should be given to the potential health effects of these populations shown to be exposed to endocrine-active chemicals that disrupt the androgen signaling pathway.
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Disclaimer: The research described in the article has been reviewed by the National Health and Environmental Effects Laboratory, US 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.
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ACKNOWLEDGMENTS |
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The authors would like to thank Dr C. J. Wolf for assistance provided throughout this project. Funding was provided by the USEPA/NCSU Cooperative Training agreement CT826512010 (for A.K.H.).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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