Female Offspring From Chronic Hyperandrogenemic Dams Exhibit Delayed Puberty and Impaired Ovarian Reserve

Female Offspring From Chronic Hyperandrogenemic Dams Exhibit Delayed Puberty and Impaired Ovarian... Abstract Female offspring of many species exposed to high doses of androgens in utero experience endocrine dysfunction during adulthood. The phenotype of offspring from females with prepregnancy hyperandrogenemia and impaired ovulation, however, has not been examined. We developed a mouse model of hyperandrogenemia by implanting a low-dose dihydrotestosterone (DHT) pellet 15 days before conception. Female offspring born to dams with hyperandrogenemia (DHT daughters) had delayed puberty (P < 0.05) with first estrus on postnatal day (PND) 41 compared with daughters from dams with physiological levels of DHT (non-DHT daughters, PND37.5). Serum follicle-stimulating hormone (FSH) levels in the DHT daughters were fourfold higher (P < 0.05) on PND21, and anti-Müllerian hormone levels were higher (P < 0.05) on PND26 than in non-DHT daughters (controls). DHT daughters showed an extended time in metestrus/diestrus and a shorter time in the proestrus/estrus phase compared with non-DHT daughters (P < 0.05). To examine ovarian response to gonadotropins, superovulation was induced and in vitro fertilization (IVF) was performed. Fewer numbers of oocytes were retrieved from the DHT daughters compared with non-DHT daughters (P < 0.05). At IVF, there was no difference in rates of fertilization or cleavage of oocytes from either group. There were fewer (P < 0.01) primordial follicles (6.5 ± 0.8 vs 14.5 ± 2.1 per ovary) in the ovaries of DHT daughters compared with non-DHT daughters. Daughters from hyperandrogenemic females exhibited elevated prepubertal FSH levels, diminished ovarian response to superovulation, impaired estrous cyclicity, delayed onset of puberty, and reduced ovarian reserve, suggesting that fetal androgen exposure had lasting effects on female reproductive function. Androgens including testosterone (T) and dihydrotestosterone (DHT) play vital roles in development of the male reproductive system and secondary sexual features (1, 2). Androgens also play important roles in female physiology by influencing reproductive organs, liver, kidney, bone, and muscle (3). Hyperandrogenism or androgen excess is a feature of polycystic ovary syndrome (PCOS), a common endocrine disorder that affects ∼15% of reproductive aged women (4). Women with PCOS exhibit androgen excess, oligo-anovulation, and/or multiple antral follicles. The etiology of PCOS is unknown. Experimentally induced animal models in monkeys, sheep, and rodents (5, 6) have shown that female fetuses exposed to fetal male androgen levels during the intrauterine period develop PCOS-like traits later in life. Naturally occurring PCOS-like traits in female monkeys were also associated with fetal androgen excess (7). Similarly, female fetuses of women with congenital adrenal hyperplasia or defects in the aromatase gene, which causes androgenization, also develop PCOS later in life even after aggressively lowering the androgen excess during pregnancy with targeted therapies (8, 9). These extensive animal studies as well as clinical evidence from human populations suggest that fetal exposure to excess maternal androgens may induce PCOS features in female offspring (10). Longitudinal follow-up clinical studies of offspring born to hyperandrogenic mothers, however, are limited due to the time and expense required. Rodent models are appropriate to examine this question because rodents have known genetic backgrounds, are easy to handle, have low cost of maintenance, and exhibit shorter generation times compare with other mammalian models. Rodent and other animal models published to date have limitations that do not reflect the comprehensive nature of female offspring born to women with PCOS for the following reasons: (1) these models do not accurately reflect the prepregnancy hyperandrogenism of women with PCOS; (2) these models do not account for exposure to androgen excess of the egg and zygote during folliculogenesis, oogenesis, fertilization, and peri-implantation; and (3) the extremely high doses of T or DHT during gestation used may not reflect androgen environment of PCOS. In this study, we used a PCOS-like model by administering a low dose of DHT to adult female mice 15 days prior to breeding with males of proven fertility. The serum levels of DHT were twofold higher in mice with this DHT implant (DHT mice), compared with that of mice without DHT (non-DHT mice)—levels that resemble the elevated ratio observed in women with PCOS (11, 12). We chose DHT instead of T because DHT is nonaromatizable and thus acts via the androgen receptor only, rather than estrogen receptor α. T is metabolized to estrogens by the placenta in women and by the ovaries in rodents during pregnancy. Thus, the effects of T on pregnancy may be partly mediated by estrogen (13). Unlike most current PCOS-like models that cause infertility and obesity, our DHT mice exhibited impaired reproductive and metabolic function without altering body weight and body mass (11, 12). These DHT-exposed females spontaneously conceive and give birth to pups, which reflects the fact that hyperandrogenic women become pregnant spontaneously. Using this model, we are able to investigate and assess effects of preconception androgen excess from PCOS-like dams on the puberty and reproductive function of their female offspring. Materials and Methods Generation of hyperandrogenemic females and their female offspring Mice were maintained in a mixed background (C57/B6, CD1, and 129Sv) in the Johns Hopkins Animal Facility, as previously described (11, 14). All procedures were performed with approval of the Johns Hopkins Animal Care and Use Committee. To generate hyperandrogenemic females, 2-month-old adult female mice had a 4-mm length (pellet) of crystalline 5α-DHT (A8380-1G; Sigma-Aldrich, St. Louis, MO) inserted subcutaneously, or, as control, a no-DHT pellet was inserted, as described previously (11, 12). The pellets were incubated in saline for 24 hours at 37°C for equilibration prior to insertion (15, 16). To maintain a constant level of androgen exposure to dams, DHT or no-DHT pellets were replaced every month for the duration of experiments. The females were mated with proven fertile males 15 days after DHT, or no-DHT, pellet insertion. Pups from unexposed pregnant mice without DHT and spontaneously pregnant mice with DHT were weaned on postnatal day (PND) 21. The experimental design is presented in Fig. 1A. Figure 1. View largeDownload slide (A) Schematic diagram of the study. Numbers above the timeline represent days before birth (−) or postnatal days. Tests are listed below the timeline at times performed. (B) Serum DHT levels (y-axis) of control (open bars) and DHT-implanted (black bars) female mice before and during pregnancy. Values are mean ± standard error of the mean. n = 5 to 8 per group. Figure 1. View largeDownload slide (A) Schematic diagram of the study. Numbers above the timeline represent days before birth (−) or postnatal days. Tests are listed below the timeline at times performed. (B) Serum DHT levels (y-axis) of control (open bars) and DHT-implanted (black bars) female mice before and during pregnancy. Values are mean ± standard error of the mean. n = 5 to 8 per group. Assessment of puberty and estrous cyclicity in female offspring born to the hyperandrogenemic dams Puberty is a critical period for female reproductive development. The first observable evidence of puberty in females is estrogen dependent: vaginal introitus opening and a cornified vaginal smear. To investigate the effects of maternal androgen excess on puberty in female offspring, we assessed the puberty of DHT daughters and non-DHT daughters by checking vaginal opening and vaginal cytology. Puberty was evaluated beginning from PND21 by visual inspection of vaginal opening and identifying the age of first estrus (E) (17). Briefly, vaginal cells were collected daily by gentle lavage after the vagina opened and until the first E emerged, as indicated by presence of cornified cells only. This day was recorded as the time of first E. It was reported that higher doses of T exposure during gestation disrupted estrous cyclicity of female offspring (18). To examine whether estrous cyclicity was disrupted in female offspring born to dams exposed to DHT before conception, we assessed the estrous cyclicity of both DHT-exposed daughters and non-DHT–exposed daughters. Estrous cyclicity was assessed using vaginal smear cytology (19, 20) from PND100 for 15 consecutive days. The stage of the estrous cycle was determined and classified as proestrus (P), E, and metestrus/diestrus (M/D) based on observed ratios of cornified epithelial, nucleated epithelial, and polymorphonuclear leukocytes, as described in Nelson et al. (21). The examiners were blinded to genotypes during data collection. Hormone assays To explore whether maternal androgen excess affected hormone secretion of their daughters, we measured the serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), T, estradiol (E2), and anti-Müllerian hormone (AMH) levels in serum collected on PND21, 26, and 70. Blood was collected between 9 and 10 am. These PNDs were chosen because hormone levels may change in pups upon weaning (PND21) and upon vaginal opening (PND26), which is estrogen dependent. We assessed vaginal cytology (P, E, or M/D) on PND70 to correlate with hormone levels. LH and FSH levels were measured on a Luminex 200IS platform using a MILLIPLEXMAP Mouse Pituitary Magnetic Bead Panel (MPTMAG-49K; Millipore, Billerica, MA) (22). The intra-assay coefficients of variation for LH and FSH were 3.1% and 8.7%, respectively. The interassay coefficients of variation for LH and FSH were 7% to 8% and 9% to 11%, respectively. E2 levels were measured with two different estradiol enzyme-linked immunosorbent assay (ELISA) kits with two independent sets of samples, respectively (catalog no. ES180S-100; Calbiotech EI, Cajon, CA; catalog no. 582251; Cayman Chemical) following the manufacturer’s instructions (22). T levels were measured with the T rat/mouse ELISA kit (catalog no. B79174; IBL America, Minneapolis, MN) following the user’s manual (20). DHT levels were quantified by the DHT ELISA kit (catalog no. 1940; Alpha Diagnostic International, San Antonio, TX) (11, 12). AMH levels were determined by AMH (rat and mouse) ELISA kit (Ansh Labs; catalog no. AL-113; Webster, TX) (23). Body weight analysis Puberty in rodents is dependent on body weight (24). To evaluate the growth rate of female pups, the weights of female offspring were assessed weekly starting from PND 7 to 70. We began weighing the pups on PND7 to decrease frequency of feticide as a result of handling by humans during the first few days after delivery. Oocyte retrieval and ovarian histology To examine whether ovarian follicle development and ovarian responses to superovulation were impaired in adult female offspring born to hyperandrogenemic mothers, female offspring were superovulated at ∼146 days after birth. Briefly, 10 IU pregnant mare’s serum gonadotropin (catalog no. G4877-1000 IU; Sigma-Aldrich) was injected in the afternoon, followed by 10 IU human chorionic gonadotropin (catalog no. C1063-1VL; Sigma-Aldrich) 48 hours after pregnant mare’s serum gonadotropin (25). Female mice were euthanized between 14 and 16 hours after the human chorionic gonadotropin injection, and the oviducts were collected. The transparent ampulla region of each oviduct was incised to release the cumulus–oocyte complexes into the human tubal fluid (HTF; catalog no. MR-070-D; Embry Max HTF; Millipore). Cumulus–oocyte complexes were transferred and cultured in potassium-supplemented simplex optimized medium [KSOM (KSOM Embryo culture medium, catalog no. MR-020P-5F; Millipore)] in 5% CO2 at 37°C until further experimental use (26, 27). Ovaries were collected after oocyte retrieval, preserved in 10% formalin phosphate-buffered saline, and dissected, as previously described (11). Because preantral follicles are gonadotropin independent and will not be affected by superovulation, we counted the number of follicles at the primodial, primary, and secondary stages in the ovaries. We serially sectioned the ovaries at 5 µm thickness. Once the ovarian tissue was reached, we cut total 10 sections and discarded these 10 sections. After this, we collected every 10th section for a total of 5 sections per ovary. We counted (blinded to genotyping and treatment) all the primordial, primary, secondary, and antral follicles on each of the 5 sections. A follicle was defined as containing of the nucleus of an oocyte. We added all follicles in each category from 5 sections. We only reported the maximum number of corpora lutea (CL) among the 5 sections (for example, if sections 1, 2, 3, and 5 have 2 to 3 CL per section, but section 4 has 5 CL, we will chose 5 as the number of CL). We use this method to avoid repeated counting of follicles. In vitro fertilization and embryo culture To evaluate whether maternal hyperandrogenemia affected the ovarian function in daughters, we performed in vitro fertilization (IVF) experiments. Twelve-week-old proven fertile male mice were euthanized by CO2, followed by CO2 and cervical dislocation. Each epididymis was dissected and placed in the central well of an IVF dish with HTF medium. After making five to seven longitudinal cuts on the epididymis using a needle-affixed syringe, tissue samples were incubated for 20 minutes at 37°C in 5% CO2 atmosphere to allow for sperm dispersion. The sperm suspensions were incubated for 1 hour at 37°C in 5% CO2 air to allow for capacitation. For IVF, metaphase II oocytes were inseminated with 2 × 104 sperm in a droplet of 150 µL HTF medium for 4.5 hours. Fertilized oocytes were subsequently cultured in a drop of 20 μL KOSM medium at 37°C at 5% CO2 atmosphere and high humidity (25, 27). Development of the fertilized oocytes was monitored under an inverted microscope (Nikon; TMS no. 201089) for the formation of two-cell–, four-cell–, morula-, and blastocyst-stage embryos at various intervals for up to 5 days. Statistical analyses Statistical analyses were carried out by two-tailed, unpaired Student t tests with GraphPad Prism version 6.0 (GraphPad Software, Inc.). Data were analyzed as indicated in the figure legends and presented as the mean ± standard error of the mean; P < 0.05 was considered statistically significant. Results DHT levels were increased from prepregnancy through pregnancy We measured DHT level of dams at 14 days after DHT insertion before pairing and ∼7 days (gestational day 14 ± 1) before delivery. The DHT level was twofold higher in DHT dams compared with non-DHT dams before pregnancy. Both treated and untreated groups of pregnant dams showed dramatically increased DHT levels from prepregnancy levels. Serum DHT levels were significantly higher in DHT dams before (P < 0.005) and during gestation than in non-DHT–exposed dams (P < 0.05) (Fig. 1B). Female offspring from chronically hyperandrogenized dams had a lower body weight Pups delivered vaginally from both groups were weighed and recorded. Female offspring (DHT daughters) born to DHT dams had lower (P < 0.05) body weights compared with female offspring (non-DHT daughters) from the non-DHT dam (Fig. 2) during the first 10 weeks of life. Figure 2. View largeDownload slide Prepregnancy maternal DHT treatment resulted in reduced body weight in female offspring. Body weight (y-axis) was measured on postnatal days, as shown (x-axis). *Represents significant difference (P < 0.05); **P < 0.01; ***P < 0.001 at each time point. Values are mean ± standard error of the mean. n = 9 to 14 per group. control, circles; DHT-exposed offspring, squares. Figure 2. View largeDownload slide Prepregnancy maternal DHT treatment resulted in reduced body weight in female offspring. Body weight (y-axis) was measured on postnatal days, as shown (x-axis). *Represents significant difference (P < 0.05); **P < 0.01; ***P < 0.001 at each time point. Values are mean ± standard error of the mean. n = 9 to 14 per group. control, circles; DHT-exposed offspring, squares. Female offspring from chronically hyperandrogenized dams exhibited delayed puberty We found no difference between the two groups regarding age at vaginal opening (PND 26.50 ± 0.26 vs 26.54 ± 0.27; Fig. 3A). The age of first E, however, was later (P < 0.05) in DHT daughters compared with non-DHT daughters (PND 37.8 ± 1.9 vs 41.0 ± 1.0; Fig. 3B). Given that opening of the vaginal introitus is estrogen dependent, we measured serum E2 level on PND21 and PND26. There was no difference in serum E2 levels in DHT daughter and non-DHT daughters (Fig. 4A; data from assay of Calbiotech), although E2 was increased in DHT daughters during this time, but not significantly. Taken together, these data suggest that maternal androgen excess delayed maturation of the hypothalamic-pituitary-ovarian axis in exposed daughters. Figure 3. View largeDownload slide Prepregnant maternal DHT treatment delayed the onset of puberty in female offspring. (A) Vaginal opening noted by daily assessment (y-axis) in control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). (B) Day at first E as assessed by vaginal cytology (y-axis) in control (open bars) or DHT-exposed (black bars) offspring. Values are mean ± standard error of the mean. n = 4 to 13 per group. NS, not significant. Figure 3. View largeDownload slide Prepregnant maternal DHT treatment delayed the onset of puberty in female offspring. (A) Vaginal opening noted by daily assessment (y-axis) in control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). (B) Day at first E as assessed by vaginal cytology (y-axis) in control (open bars) or DHT-exposed (black bars) offspring. Values are mean ± standard error of the mean. n = 4 to 13 per group. NS, not significant. Figure 4. View largeDownload slide Hormone levels during prepubertal/peripuberty. Blood was collected at PNDs 21 and 26 in the morning at M/D stage. (A) Serum estradiol levels were measured by ELISA (Calbiotech), (B) serum LH, (C) serum FSH, and (D) serum AMH levels. Values are mean ± standard error of the mean. n = 5 to 11 per group. For (A)–(E): control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). NS, not significant. Figure 4. View largeDownload slide Hormone levels during prepubertal/peripuberty. Blood was collected at PNDs 21 and 26 in the morning at M/D stage. (A) Serum estradiol levels were measured by ELISA (Calbiotech), (B) serum LH, (C) serum FSH, and (D) serum AMH levels. Values are mean ± standard error of the mean. n = 5 to 11 per group. For (A)–(E): control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). NS, not significant. Female offspring from chronically hyperandrogenized dams showed altered prepubertal hormonal levels We measured the LH, FSH, T, and AMH levels in serum collected on PND 21 and 26. There was no significant difference in serum LH levels for both DHT daughters and non-DHT daughters on PND21 and PND26 (Fig. 4B). DHT daughters, however, exhibited much higher FSH levels than non-DHT daughters on PND21, but not on PND26 (Fig. 4C). In contrast, AMH levels were higher (P < 0.05) in DHT daughters compared with non-DHT daughters on PND26, but not on PND21 (Fig. 4D). We observed that DHT daughters had lower (P < 0.05) T compared with non-DHT daughters on PND26, whereas both groups had equivalent levels of T on PND21 (Fig. 4E). Adult female offspring from chronically hyperandrogenized dams showed elevated serum T and disturbed estrous cycling To investigate whether maternal hyperandrogenemia can cause increased T in their adult female offspring, we also measured serum T levels at M/D on PND70. We observed that DHT daughters had higher (P < 0.05) T levels compared with non-DHT daughters on PND70 (Fig. 5A). There were, however, no differences in E2, AMH, LH, and FSH on PND70 between DHT daughters and non-DHT daughters (Fig. 5B–5D). Figure 5. View largeDownload slide Hormone levels at PND70. (A) Serum T levels. (B) Serum estradiol levels measured by ELISA (Calbiotech, Inc.). (C) Serum AMH levels. (D) Serum levels of LH and FSH at different stages of the estrous cycle. Values are mean ± standard error of the mean. n = 5 to 11 per group. For (A)–(D): control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). NS, not significant; P/E, proestrus/estrus. Figure 5. View largeDownload slide Hormone levels at PND70. (A) Serum T levels. (B) Serum estradiol levels measured by ELISA (Calbiotech, Inc.). (C) Serum AMH levels. (D) Serum levels of LH and FSH at different stages of the estrous cycle. Values are mean ± standard error of the mean. n = 5 to 11 per group. For (A)–(D): control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). NS, not significant; P/E, proestrus/estrus. All non-DHT daughters had at least two cycles within 15 consecutive days, whereas only 70% of DHT daughters had two cycles. Forty percent of non-DHT daughters experienced three cycles, but none of DHT daughters experienced three cycles (Fig. 6A). Moreover, DHT daughters spent significantly longer time in M/D and less time in P and E phases, compared with non-DHT daughters (Fig. 6B). Collectively, these data suggest maternal hyperandrogenemia disturbed estrous cycling in their adult daughters. Figure 6. View largeDownload slide Chronic maternal androgen excess leads to disturbed cyclicity in adult daughters. (A) The total completed E cycles (y-axis) within 15 days as measured by cytology. x-axis = number of cycles. (B) The percentage of time spent at each estrous stage (y-axis) was analyzed. Values are mean ± standard error of the mean. n = 5 to 9 per group. Values are mean ± standard error of the mean. Control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). P/E, proestrus/estrus. Figure 6. View largeDownload slide Chronic maternal androgen excess leads to disturbed cyclicity in adult daughters. (A) The total completed E cycles (y-axis) within 15 days as measured by cytology. x-axis = number of cycles. (B) The percentage of time spent at each estrous stage (y-axis) was analyzed. Values are mean ± standard error of the mean. n = 5 to 9 per group. Values are mean ± standard error of the mean. Control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). P/E, proestrus/estrus. Female offspring from chronically hyperandrogenized dams exhibited diminished ovarian reserve Hyperandrogenemia has been reported to impair reproductive function of females (28), but the degree to which hyperandrogenemia affects their offspring is controversial. We observed fewer (P < 0.05) oocytes were retrieved from the ovaries of DHT daughters than those of non-DHT daughters following superovulation. Furthermore, among oocytes retrieved, there were fewer (P < 0.05) mature oocytes from the ovaries of DHT daughters than those of non-DHT daughters (Fig. 7A). There were, however, no differences in the percentage of fertilized oocytes or cleavage stage embryos between DHT daughters and non-DHT daughters (Fig. 7B). We found fewer (P < 0.005) primordial follicles and more (P < 0.05) primary follicles in the ovaries of DHT daughters than those of non-DHT daughters (Fig. 7C). We observed fewer antral follicles in both control and DHT-exposed daughters in females after superovulation, compared with females not superovulated (because more antral follicles develop into preovulatory follicles and release eggs after superovulation). We only counted oocytes, but not CL after superovulation, to avoid misleading readers because some newly ovulated follicles after superovulation do not have enough time to form CL. To eliminate the effect of superovulation on the follicle development, we counted the follicles in the ovaries without exposure to superovulation and found a similar pattern of primodial, primary, secondary, and antral follicles to that after superovulation (Fig. 7D). Moreover, the ovaries of DHT daughters contained fewer (P < 0.05) CL than non-DHT daughters (maximum number of CL: 4.00 ± 0.49 vs 6.20 ± 0.58 per section per ovary). Figure 7. View largeDownload slide IVF and ovarian follicle counts. IVF was performed with the DHT daughter and non-DHT daughter mice. (A) The total number of retrieved oocytes and mature eggs (x-axis) after superovulation, which were counted (y-axis) from two ovaries per mouse. (B) The percentages (y-axis) of fertilized oocytes (number of fertilized oocytes divided mature oocytes) and cleavage stage embryos (≥2 cells stage; number of embryos divided fertilized oocytes; x-axis) are shown for treated and control groups. (C) Total numbers of follicles (y-axis) of different stages in the ovarian sections (see Materials and Methods) after superovulation were quantified as primordial, primary, secondary, or antral (x-axis). CL were not determined for these sections. (D) Total numbers of follicles (y-axis) were quantified in the ovarian sections from mice that had not undergone superovulation. Values are mean ± standard error of the mean. n = 5 to 13 per group. NS, not significant. Figure 7. View largeDownload slide IVF and ovarian follicle counts. IVF was performed with the DHT daughter and non-DHT daughter mice. (A) The total number of retrieved oocytes and mature eggs (x-axis) after superovulation, which were counted (y-axis) from two ovaries per mouse. (B) The percentages (y-axis) of fertilized oocytes (number of fertilized oocytes divided mature oocytes) and cleavage stage embryos (≥2 cells stage; number of embryos divided fertilized oocytes; x-axis) are shown for treated and control groups. (C) Total numbers of follicles (y-axis) of different stages in the ovarian sections (see Materials and Methods) after superovulation were quantified as primordial, primary, secondary, or antral (x-axis). CL were not determined for these sections. (D) Total numbers of follicles (y-axis) were quantified in the ovarian sections from mice that had not undergone superovulation. Values are mean ± standard error of the mean. n = 5 to 13 per group. NS, not significant. Discussion Hyperandrogenism is a feature of several endocrine disorders that affect women of reproductive age. Previous animal studies of gestational androgen excess have shown that female offspring exhibit metabolic and reproductive dysfunction into adulthood (18, 28–30). However, these models do not account for the prepregnancy hyperandrogenism of mothers that are present in PCOS. That is, the effect of maternal hyperandrogenism in early development, from preimplantation to gastrulation, has not been examined (31). In this study, we investigated female offspring from chronically hyperandrogenic dams (11). The DHT levels in serum were maintained for 4 weeks after the DHT implant, and the implants were replaced monthly; thus, the dams received a constant level of exogenous DHT during the experiment. Our data showed circulating DHT levels were elevated during pregnancy in both groups and were ∼1.7-fold higher in DHT-exposed dams than in control dams (no DHT) during gestation (Fig. 1B). This observation parallels what is observed in humans during pregnancy, in which total T rises, whereas free T does not rise until the third trimester (32). Additionally, serum androgens, including T, androstenedione, and/or dehydroepiandrosterone, are higher in pregnant women with PCOS (33–36). DHT levels during human pregnancy have not been systematically investigated. The rise in androgen experienced by the pregnant mice may mimic the rise in androgen experienced during pregnancy in women with PCOS. For both species, the degree to which maternal serum androgens cross the placenta to affect the fetus is not known. We could not measure DHT levels in exposed fetuses. Thus, it is not clear that DHT treatment of mice completely mimicked the human fetal environment of a PCOS pregnancy. It is unlikely that higher insulin levels associated with androgens stimulated ovarian production in the fetus, as the fetuses who experience hyperinsulinemia are generally large for gestational age. We observed that DHT daughters gained weight slower than non-DHT daughters from postnatal 1 to 10 weeks (Fig. 2). From the growth curve, we speculate that DHT daughters had a lower birth body weight than non-DHT daughters. This suggests that preconceptional androgen excess might impair intrauterine growth and that this persists postnatally. This resembles the higher prevalence of small for gestational age infants born to PCOS mothers that suffer from hyperandrogenemia, compared with infants of reproductively normal control women (37, 38). Possibly, gestational hyperandrogenemia can cause intrauterine growth retardation and lower birth weight, due to higher gestational androgens that promote placental differentiation and limit blood flow (39). In contrast to the observation that overweight/obesity is increased in girls with PCOS (40), we observed that DHT daughters were lighter in body weight compared with non-DHT daughters. The disparity in growth patterns between humans and our model might be because pregnant women with PCOS often have coexisting overweight/obesity preceding and during pregnancy to affect postnatal weight gain in human infants. In our model, the dams do not have prepregnancy obesity (12). Serum T levels were elevated in adult DHT female offspring (Fig. 5A), implying that androgen excess alone may not be sufficient to lead to obesity in female offspring. Serum T levels were similar between DHT daughters and non-DHT daughters on PND21, whereas serum T levels were lower in DHT daughters than non-DHT daughters on PND26. This indicates that maternal androgen excess resulted in lower levels of serum T in daughters before puberty. Pups at PND26 are recently weaned, so the continuous exposure to higher DHT in these pups during suckling may temporarily suppress endogenous T production. We found that maternal androgen excess resulted in higher serum T in adult daughters (Fig. 5A), which matches other prenatal androgenized models (41, 42). Chronically DHT-exposed mice have reduced Cyp17a1 messenger RNA expression (a rate-limited enzyme in androgenesis) in ovary (11, 41). Thus, the temporary suppression of serum T at PND26 might be due to a delay in Cyp17a1 expression from maternal DHT. To what extent breastfeeding contributed to this model’s phenotype remains elusive and could be investigated by feeding these pups using foster moms, which did not have the DHT implant. These experiments are beyond the scope of the current study. Body weight may affect pubertal development in rodents (43). We observed that first E emerged remarkably later for DHT daughters compared with non-DHT daughters, although there was no difference in timing of vaginal opening (Fig. 3). Serum E2 levels can regulate the vaginal opening, so we also examined serum E2 levels, which were similar between DHT daughters and non-DHT daughters (Fig. 4A). This is consistent with the vaginal opening observation. LH levels at PND 21 and 26 were not significantly different among groups (Fig. 4B), implying that signals regulating early puberty were normal between groups. First E as determined by vaginal cytology not only requires mature vaginal cells but also fully mature hypothalamic, pituitary, ovarian signaling with positive and negative feedback capabilities. Indeed, the FSH levels at PND26 were different between DHT-treated and nontreated groups, implying that the hypothalamic-pituitary-ovarian signaling was not fully mature. Low body weight disrupts hypothalamic-pituitary-ovarian signaling and may also be a factor in the delayed first E in DHT daughters. Both delayed and advanced puberty have been reported in animal models of prenatal androgenized female offspring. Whether delayed or early puberty is observed seems to depend upon the timing of the fetal androgen exposure. In Rhesus monkeys, female offspring showed delayed puberty if they received T or DHT at between days 40 and 55 of gestation (early exposure) (30, 44). The same pattern also was observed in guinea pig and porcine female offspring that received T at 29 to 35 or 39 to 45 gestational days, respectably (45, 46). Prenatal injection of mice with DHT on embryonic days 16.5 to 18.5 showed advanced puberty without effects on T or estrous cyclicity in adult offspring. The advanced puberty was not regulated by androgen receptor in the central nervous system or in kisspeptin neurons (47). The advanced puberty was also observed in rats injected with T at late gestational days (48). In general, early prenatal androgen treatment resulted in delayed puberty (30, 44–46), but late prenatal androgen treatment resulted in advanced puberty (47, 48) regardless of body weight. We also examined serum LH and FSH levels on PND70 for both groups. Although there are studies indicating that the daughters of PCOS women have higher basal and leuprolide-stimulated LH (49, 50), there were no differences in our study between control and treated groups of offspring. Hyperandrogenemia causes subfertility or infertility in women. Whether maternal hyperandrogenemia would impair the fertility of their daughters remained elusive. Although there are some clues from human and animal model studies to suggest that maternal androgen excess may affect the reproductive function of female offspring (48, 51–53), data in women exposed to androgen excess in utero are not available. In this study, we carried out IVF experiments to evaluate whether maternal hyperandrogenemia would impair the reproductive function of their daughters. We used IVF to estimate the reproductive capacity because IVF can measure the quality and quantity of oocytes, avoiding factors that may influence fertility during pairing, such as mating behaviors. IVF experiments revealed that fewer total oocytes and mature oocytes were retrieved from the ovaries of DHT daughters than that of non-DHT daughters, whereas there were no differences in the number and the percentage rate of fertilized oocytes and cleaved embryos (Fig. 7). These data suggest that maternal androgen excess jeopardizes the ovarian response to FSH. Moreover, we also counted the follicles in ovaries and found that the number of primordial follicles was significantly lower in DHT daughters than that in non-DHT daughters. There were significantly greater primary follicles in DHT daughters compared with that in non-DHT daughters (Fig. 7C). The ovaries of DHT daughters show a relatively earlier depletion of their reserve of primordial follicles (4 to 5 months old). Reduced primordial follicles in adulthood may result from premature exhaustion of the follicle pool, a diminished ovarian reserve at birth, or an accelerated rate of primordial follicle recruitment, and increased atresia or destruction of follicles by high androgens. Furthermore, at PND26 (but not in the adult), AMH levels were increased in DHT daughters compared with non-DHT daughters. We speculate that the increased level of AMH may be caused by the increased amount of granulosa cells (growing follicles) at prepuberty associated with DHT and FSH level (54, 55) because AMH is secreted by granulosa cells of ovary and is a marker of follicular development. AMH regulates early follicle growth and initial and cyclic follicle recruitment (56). Interestingly, girls born to mothers with PCOS also exhibit AMH overproduction, which persists from 2 months old to prepubertal life (52, 57, 58). These girls and our DHT-exposed daughters show evidence of an altered follicular development during infancy and childhood. Women with PCOS also show a significant increase in the percentage of primary follicles and a reciprocal decrease in the proportion of primordial follicles compared with normal ovaries (59). Androgens have been shown to promote the differentiation of primordial to primary follicles in the primate and sheep (53, 60, 61), although early follicular depletion is more severe in animals prenatally androgenized by T rather than DHT. Alternatively, the higher T level in adult DHT daughters compared with that in non-DHT daughters in our study may have contributed to the ovarian dysfunction. T exposure was reported to rapidly increase primordial follicles to primary follicles in mouse (by >twofold) through phosphatidylinositol 3-kinase/protein kinase B pathway (62). These data also suggest that more primordial follicles become activated to develop to primary follicles, but more follicles of poor quality are depleted during maturation, and that maternal androgen excess undermined ovarian reserve that leads to the subfertility. In summary, we found that maternal hyperandrogenemia induced by a low dose of DHT caused slower growth, delayed puberty, and altered hormone secretion, which may result in impaired reproductive function by disrupting E cycling and accelerating loss primordial follicles. Abbreviations: AMH anti-Müllerian hormone CL corpora lutea DHT dihydrotestosterone E estrus E2 estradiol ELISA enzyme-linked immunosorbent assay FSH follicle-stimulating hormone HTF human tubal fluid IVF in vitro fertilization KSMO potassium-supplemented simplex optimized medium LH luteinizing hormone M/D metestrus/diestrus P proestrus PCOS polycystic ovary syndrome PND postnatal day T testosterone. Acknowledgments We thank Dr. David Abbott (Professor, University of Wisconsin Obstetrics and Gynecology, Madison, Wisconsin) for editorial comments. Financial Support: This work was supported by National Institutes of Health Grant R00-HD068130 (to S.W.) and the Baltimore Diabetes Research Center: Pilots and Feasibility Grant (to S.W.). Author Contributions: Z.W. and S.W. contributed to the experimental design, conducted the experiments, and wrote and edited the manuscript. M.S. and P.X. contributed to performing some of the experiments, analyzing the corresponding data, and writing part of the manuscript. S.A.D. and J.S. contributed to the experimental design and reviewed and edited the manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Davey RA, Grossmann M. Androgen receptor structure, function and biology: from bench to bedside. Clin Biochem Rev . 2016; 37( 1): 3– 15. Google Scholar PubMed  2. Nef S, Parada LF. Hormones in male sexual development. 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Google Scholar CrossRef Search ADS PubMed  Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Endocrinology Oxford University Press

Female Offspring From Chronic Hyperandrogenemic Dams Exhibit Delayed Puberty and Impaired Ovarian Reserve

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Abstract

Abstract Female offspring of many species exposed to high doses of androgens in utero experience endocrine dysfunction during adulthood. The phenotype of offspring from females with prepregnancy hyperandrogenemia and impaired ovulation, however, has not been examined. We developed a mouse model of hyperandrogenemia by implanting a low-dose dihydrotestosterone (DHT) pellet 15 days before conception. Female offspring born to dams with hyperandrogenemia (DHT daughters) had delayed puberty (P < 0.05) with first estrus on postnatal day (PND) 41 compared with daughters from dams with physiological levels of DHT (non-DHT daughters, PND37.5). Serum follicle-stimulating hormone (FSH) levels in the DHT daughters were fourfold higher (P < 0.05) on PND21, and anti-Müllerian hormone levels were higher (P < 0.05) on PND26 than in non-DHT daughters (controls). DHT daughters showed an extended time in metestrus/diestrus and a shorter time in the proestrus/estrus phase compared with non-DHT daughters (P < 0.05). To examine ovarian response to gonadotropins, superovulation was induced and in vitro fertilization (IVF) was performed. Fewer numbers of oocytes were retrieved from the DHT daughters compared with non-DHT daughters (P < 0.05). At IVF, there was no difference in rates of fertilization or cleavage of oocytes from either group. There were fewer (P < 0.01) primordial follicles (6.5 ± 0.8 vs 14.5 ± 2.1 per ovary) in the ovaries of DHT daughters compared with non-DHT daughters. Daughters from hyperandrogenemic females exhibited elevated prepubertal FSH levels, diminished ovarian response to superovulation, impaired estrous cyclicity, delayed onset of puberty, and reduced ovarian reserve, suggesting that fetal androgen exposure had lasting effects on female reproductive function. Androgens including testosterone (T) and dihydrotestosterone (DHT) play vital roles in development of the male reproductive system and secondary sexual features (1, 2). Androgens also play important roles in female physiology by influencing reproductive organs, liver, kidney, bone, and muscle (3). Hyperandrogenism or androgen excess is a feature of polycystic ovary syndrome (PCOS), a common endocrine disorder that affects ∼15% of reproductive aged women (4). Women with PCOS exhibit androgen excess, oligo-anovulation, and/or multiple antral follicles. The etiology of PCOS is unknown. Experimentally induced animal models in monkeys, sheep, and rodents (5, 6) have shown that female fetuses exposed to fetal male androgen levels during the intrauterine period develop PCOS-like traits later in life. Naturally occurring PCOS-like traits in female monkeys were also associated with fetal androgen excess (7). Similarly, female fetuses of women with congenital adrenal hyperplasia or defects in the aromatase gene, which causes androgenization, also develop PCOS later in life even after aggressively lowering the androgen excess during pregnancy with targeted therapies (8, 9). These extensive animal studies as well as clinical evidence from human populations suggest that fetal exposure to excess maternal androgens may induce PCOS features in female offspring (10). Longitudinal follow-up clinical studies of offspring born to hyperandrogenic mothers, however, are limited due to the time and expense required. Rodent models are appropriate to examine this question because rodents have known genetic backgrounds, are easy to handle, have low cost of maintenance, and exhibit shorter generation times compare with other mammalian models. Rodent and other animal models published to date have limitations that do not reflect the comprehensive nature of female offspring born to women with PCOS for the following reasons: (1) these models do not accurately reflect the prepregnancy hyperandrogenism of women with PCOS; (2) these models do not account for exposure to androgen excess of the egg and zygote during folliculogenesis, oogenesis, fertilization, and peri-implantation; and (3) the extremely high doses of T or DHT during gestation used may not reflect androgen environment of PCOS. In this study, we used a PCOS-like model by administering a low dose of DHT to adult female mice 15 days prior to breeding with males of proven fertility. The serum levels of DHT were twofold higher in mice with this DHT implant (DHT mice), compared with that of mice without DHT (non-DHT mice)—levels that resemble the elevated ratio observed in women with PCOS (11, 12). We chose DHT instead of T because DHT is nonaromatizable and thus acts via the androgen receptor only, rather than estrogen receptor α. T is metabolized to estrogens by the placenta in women and by the ovaries in rodents during pregnancy. Thus, the effects of T on pregnancy may be partly mediated by estrogen (13). Unlike most current PCOS-like models that cause infertility and obesity, our DHT mice exhibited impaired reproductive and metabolic function without altering body weight and body mass (11, 12). These DHT-exposed females spontaneously conceive and give birth to pups, which reflects the fact that hyperandrogenic women become pregnant spontaneously. Using this model, we are able to investigate and assess effects of preconception androgen excess from PCOS-like dams on the puberty and reproductive function of their female offspring. Materials and Methods Generation of hyperandrogenemic females and their female offspring Mice were maintained in a mixed background (C57/B6, CD1, and 129Sv) in the Johns Hopkins Animal Facility, as previously described (11, 14). All procedures were performed with approval of the Johns Hopkins Animal Care and Use Committee. To generate hyperandrogenemic females, 2-month-old adult female mice had a 4-mm length (pellet) of crystalline 5α-DHT (A8380-1G; Sigma-Aldrich, St. Louis, MO) inserted subcutaneously, or, as control, a no-DHT pellet was inserted, as described previously (11, 12). The pellets were incubated in saline for 24 hours at 37°C for equilibration prior to insertion (15, 16). To maintain a constant level of androgen exposure to dams, DHT or no-DHT pellets were replaced every month for the duration of experiments. The females were mated with proven fertile males 15 days after DHT, or no-DHT, pellet insertion. Pups from unexposed pregnant mice without DHT and spontaneously pregnant mice with DHT were weaned on postnatal day (PND) 21. The experimental design is presented in Fig. 1A. Figure 1. View largeDownload slide (A) Schematic diagram of the study. Numbers above the timeline represent days before birth (−) or postnatal days. Tests are listed below the timeline at times performed. (B) Serum DHT levels (y-axis) of control (open bars) and DHT-implanted (black bars) female mice before and during pregnancy. Values are mean ± standard error of the mean. n = 5 to 8 per group. Figure 1. View largeDownload slide (A) Schematic diagram of the study. Numbers above the timeline represent days before birth (−) or postnatal days. Tests are listed below the timeline at times performed. (B) Serum DHT levels (y-axis) of control (open bars) and DHT-implanted (black bars) female mice before and during pregnancy. Values are mean ± standard error of the mean. n = 5 to 8 per group. Assessment of puberty and estrous cyclicity in female offspring born to the hyperandrogenemic dams Puberty is a critical period for female reproductive development. The first observable evidence of puberty in females is estrogen dependent: vaginal introitus opening and a cornified vaginal smear. To investigate the effects of maternal androgen excess on puberty in female offspring, we assessed the puberty of DHT daughters and non-DHT daughters by checking vaginal opening and vaginal cytology. Puberty was evaluated beginning from PND21 by visual inspection of vaginal opening and identifying the age of first estrus (E) (17). Briefly, vaginal cells were collected daily by gentle lavage after the vagina opened and until the first E emerged, as indicated by presence of cornified cells only. This day was recorded as the time of first E. It was reported that higher doses of T exposure during gestation disrupted estrous cyclicity of female offspring (18). To examine whether estrous cyclicity was disrupted in female offspring born to dams exposed to DHT before conception, we assessed the estrous cyclicity of both DHT-exposed daughters and non-DHT–exposed daughters. Estrous cyclicity was assessed using vaginal smear cytology (19, 20) from PND100 for 15 consecutive days. The stage of the estrous cycle was determined and classified as proestrus (P), E, and metestrus/diestrus (M/D) based on observed ratios of cornified epithelial, nucleated epithelial, and polymorphonuclear leukocytes, as described in Nelson et al. (21). The examiners were blinded to genotypes during data collection. Hormone assays To explore whether maternal androgen excess affected hormone secretion of their daughters, we measured the serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), T, estradiol (E2), and anti-Müllerian hormone (AMH) levels in serum collected on PND21, 26, and 70. Blood was collected between 9 and 10 am. These PNDs were chosen because hormone levels may change in pups upon weaning (PND21) and upon vaginal opening (PND26), which is estrogen dependent. We assessed vaginal cytology (P, E, or M/D) on PND70 to correlate with hormone levels. LH and FSH levels were measured on a Luminex 200IS platform using a MILLIPLEXMAP Mouse Pituitary Magnetic Bead Panel (MPTMAG-49K; Millipore, Billerica, MA) (22). The intra-assay coefficients of variation for LH and FSH were 3.1% and 8.7%, respectively. The interassay coefficients of variation for LH and FSH were 7% to 8% and 9% to 11%, respectively. E2 levels were measured with two different estradiol enzyme-linked immunosorbent assay (ELISA) kits with two independent sets of samples, respectively (catalog no. ES180S-100; Calbiotech EI, Cajon, CA; catalog no. 582251; Cayman Chemical) following the manufacturer’s instructions (22). T levels were measured with the T rat/mouse ELISA kit (catalog no. B79174; IBL America, Minneapolis, MN) following the user’s manual (20). DHT levels were quantified by the DHT ELISA kit (catalog no. 1940; Alpha Diagnostic International, San Antonio, TX) (11, 12). AMH levels were determined by AMH (rat and mouse) ELISA kit (Ansh Labs; catalog no. AL-113; Webster, TX) (23). Body weight analysis Puberty in rodents is dependent on body weight (24). To evaluate the growth rate of female pups, the weights of female offspring were assessed weekly starting from PND 7 to 70. We began weighing the pups on PND7 to decrease frequency of feticide as a result of handling by humans during the first few days after delivery. Oocyte retrieval and ovarian histology To examine whether ovarian follicle development and ovarian responses to superovulation were impaired in adult female offspring born to hyperandrogenemic mothers, female offspring were superovulated at ∼146 days after birth. Briefly, 10 IU pregnant mare’s serum gonadotropin (catalog no. G4877-1000 IU; Sigma-Aldrich) was injected in the afternoon, followed by 10 IU human chorionic gonadotropin (catalog no. C1063-1VL; Sigma-Aldrich) 48 hours after pregnant mare’s serum gonadotropin (25). Female mice were euthanized between 14 and 16 hours after the human chorionic gonadotropin injection, and the oviducts were collected. The transparent ampulla region of each oviduct was incised to release the cumulus–oocyte complexes into the human tubal fluid (HTF; catalog no. MR-070-D; Embry Max HTF; Millipore). Cumulus–oocyte complexes were transferred and cultured in potassium-supplemented simplex optimized medium [KSOM (KSOM Embryo culture medium, catalog no. MR-020P-5F; Millipore)] in 5% CO2 at 37°C until further experimental use (26, 27). Ovaries were collected after oocyte retrieval, preserved in 10% formalin phosphate-buffered saline, and dissected, as previously described (11). Because preantral follicles are gonadotropin independent and will not be affected by superovulation, we counted the number of follicles at the primodial, primary, and secondary stages in the ovaries. We serially sectioned the ovaries at 5 µm thickness. Once the ovarian tissue was reached, we cut total 10 sections and discarded these 10 sections. After this, we collected every 10th section for a total of 5 sections per ovary. We counted (blinded to genotyping and treatment) all the primordial, primary, secondary, and antral follicles on each of the 5 sections. A follicle was defined as containing of the nucleus of an oocyte. We added all follicles in each category from 5 sections. We only reported the maximum number of corpora lutea (CL) among the 5 sections (for example, if sections 1, 2, 3, and 5 have 2 to 3 CL per section, but section 4 has 5 CL, we will chose 5 as the number of CL). We use this method to avoid repeated counting of follicles. In vitro fertilization and embryo culture To evaluate whether maternal hyperandrogenemia affected the ovarian function in daughters, we performed in vitro fertilization (IVF) experiments. Twelve-week-old proven fertile male mice were euthanized by CO2, followed by CO2 and cervical dislocation. Each epididymis was dissected and placed in the central well of an IVF dish with HTF medium. After making five to seven longitudinal cuts on the epididymis using a needle-affixed syringe, tissue samples were incubated for 20 minutes at 37°C in 5% CO2 atmosphere to allow for sperm dispersion. The sperm suspensions were incubated for 1 hour at 37°C in 5% CO2 air to allow for capacitation. For IVF, metaphase II oocytes were inseminated with 2 × 104 sperm in a droplet of 150 µL HTF medium for 4.5 hours. Fertilized oocytes were subsequently cultured in a drop of 20 μL KOSM medium at 37°C at 5% CO2 atmosphere and high humidity (25, 27). Development of the fertilized oocytes was monitored under an inverted microscope (Nikon; TMS no. 201089) for the formation of two-cell–, four-cell–, morula-, and blastocyst-stage embryos at various intervals for up to 5 days. Statistical analyses Statistical analyses were carried out by two-tailed, unpaired Student t tests with GraphPad Prism version 6.0 (GraphPad Software, Inc.). Data were analyzed as indicated in the figure legends and presented as the mean ± standard error of the mean; P < 0.05 was considered statistically significant. Results DHT levels were increased from prepregnancy through pregnancy We measured DHT level of dams at 14 days after DHT insertion before pairing and ∼7 days (gestational day 14 ± 1) before delivery. The DHT level was twofold higher in DHT dams compared with non-DHT dams before pregnancy. Both treated and untreated groups of pregnant dams showed dramatically increased DHT levels from prepregnancy levels. Serum DHT levels were significantly higher in DHT dams before (P < 0.005) and during gestation than in non-DHT–exposed dams (P < 0.05) (Fig. 1B). Female offspring from chronically hyperandrogenized dams had a lower body weight Pups delivered vaginally from both groups were weighed and recorded. Female offspring (DHT daughters) born to DHT dams had lower (P < 0.05) body weights compared with female offspring (non-DHT daughters) from the non-DHT dam (Fig. 2) during the first 10 weeks of life. Figure 2. View largeDownload slide Prepregnancy maternal DHT treatment resulted in reduced body weight in female offspring. Body weight (y-axis) was measured on postnatal days, as shown (x-axis). *Represents significant difference (P < 0.05); **P < 0.01; ***P < 0.001 at each time point. Values are mean ± standard error of the mean. n = 9 to 14 per group. control, circles; DHT-exposed offspring, squares. Figure 2. View largeDownload slide Prepregnancy maternal DHT treatment resulted in reduced body weight in female offspring. Body weight (y-axis) was measured on postnatal days, as shown (x-axis). *Represents significant difference (P < 0.05); **P < 0.01; ***P < 0.001 at each time point. Values are mean ± standard error of the mean. n = 9 to 14 per group. control, circles; DHT-exposed offspring, squares. Female offspring from chronically hyperandrogenized dams exhibited delayed puberty We found no difference between the two groups regarding age at vaginal opening (PND 26.50 ± 0.26 vs 26.54 ± 0.27; Fig. 3A). The age of first E, however, was later (P < 0.05) in DHT daughters compared with non-DHT daughters (PND 37.8 ± 1.9 vs 41.0 ± 1.0; Fig. 3B). Given that opening of the vaginal introitus is estrogen dependent, we measured serum E2 level on PND21 and PND26. There was no difference in serum E2 levels in DHT daughter and non-DHT daughters (Fig. 4A; data from assay of Calbiotech), although E2 was increased in DHT daughters during this time, but not significantly. Taken together, these data suggest that maternal androgen excess delayed maturation of the hypothalamic-pituitary-ovarian axis in exposed daughters. Figure 3. View largeDownload slide Prepregnant maternal DHT treatment delayed the onset of puberty in female offspring. (A) Vaginal opening noted by daily assessment (y-axis) in control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). (B) Day at first E as assessed by vaginal cytology (y-axis) in control (open bars) or DHT-exposed (black bars) offspring. Values are mean ± standard error of the mean. n = 4 to 13 per group. NS, not significant. Figure 3. View largeDownload slide Prepregnant maternal DHT treatment delayed the onset of puberty in female offspring. (A) Vaginal opening noted by daily assessment (y-axis) in control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). (B) Day at first E as assessed by vaginal cytology (y-axis) in control (open bars) or DHT-exposed (black bars) offspring. Values are mean ± standard error of the mean. n = 4 to 13 per group. NS, not significant. Figure 4. View largeDownload slide Hormone levels during prepubertal/peripuberty. Blood was collected at PNDs 21 and 26 in the morning at M/D stage. (A) Serum estradiol levels were measured by ELISA (Calbiotech), (B) serum LH, (C) serum FSH, and (D) serum AMH levels. Values are mean ± standard error of the mean. n = 5 to 11 per group. For (A)–(E): control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). NS, not significant. Figure 4. View largeDownload slide Hormone levels during prepubertal/peripuberty. Blood was collected at PNDs 21 and 26 in the morning at M/D stage. (A) Serum estradiol levels were measured by ELISA (Calbiotech), (B) serum LH, (C) serum FSH, and (D) serum AMH levels. Values are mean ± standard error of the mean. n = 5 to 11 per group. For (A)–(E): control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). NS, not significant. Female offspring from chronically hyperandrogenized dams showed altered prepubertal hormonal levels We measured the LH, FSH, T, and AMH levels in serum collected on PND 21 and 26. There was no significant difference in serum LH levels for both DHT daughters and non-DHT daughters on PND21 and PND26 (Fig. 4B). DHT daughters, however, exhibited much higher FSH levels than non-DHT daughters on PND21, but not on PND26 (Fig. 4C). In contrast, AMH levels were higher (P < 0.05) in DHT daughters compared with non-DHT daughters on PND26, but not on PND21 (Fig. 4D). We observed that DHT daughters had lower (P < 0.05) T compared with non-DHT daughters on PND26, whereas both groups had equivalent levels of T on PND21 (Fig. 4E). Adult female offspring from chronically hyperandrogenized dams showed elevated serum T and disturbed estrous cycling To investigate whether maternal hyperandrogenemia can cause increased T in their adult female offspring, we also measured serum T levels at M/D on PND70. We observed that DHT daughters had higher (P < 0.05) T levels compared with non-DHT daughters on PND70 (Fig. 5A). There were, however, no differences in E2, AMH, LH, and FSH on PND70 between DHT daughters and non-DHT daughters (Fig. 5B–5D). Figure 5. View largeDownload slide Hormone levels at PND70. (A) Serum T levels. (B) Serum estradiol levels measured by ELISA (Calbiotech, Inc.). (C) Serum AMH levels. (D) Serum levels of LH and FSH at different stages of the estrous cycle. Values are mean ± standard error of the mean. n = 5 to 11 per group. For (A)–(D): control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). NS, not significant; P/E, proestrus/estrus. Figure 5. View largeDownload slide Hormone levels at PND70. (A) Serum T levels. (B) Serum estradiol levels measured by ELISA (Calbiotech, Inc.). (C) Serum AMH levels. (D) Serum levels of LH and FSH at different stages of the estrous cycle. Values are mean ± standard error of the mean. n = 5 to 11 per group. For (A)–(D): control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). NS, not significant; P/E, proestrus/estrus. All non-DHT daughters had at least two cycles within 15 consecutive days, whereas only 70% of DHT daughters had two cycles. Forty percent of non-DHT daughters experienced three cycles, but none of DHT daughters experienced three cycles (Fig. 6A). Moreover, DHT daughters spent significantly longer time in M/D and less time in P and E phases, compared with non-DHT daughters (Fig. 6B). Collectively, these data suggest maternal hyperandrogenemia disturbed estrous cycling in their adult daughters. Figure 6. View largeDownload slide Chronic maternal androgen excess leads to disturbed cyclicity in adult daughters. (A) The total completed E cycles (y-axis) within 15 days as measured by cytology. x-axis = number of cycles. (B) The percentage of time spent at each estrous stage (y-axis) was analyzed. Values are mean ± standard error of the mean. n = 5 to 9 per group. Values are mean ± standard error of the mean. Control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). P/E, proestrus/estrus. Figure 6. View largeDownload slide Chronic maternal androgen excess leads to disturbed cyclicity in adult daughters. (A) The total completed E cycles (y-axis) within 15 days as measured by cytology. x-axis = number of cycles. (B) The percentage of time spent at each estrous stage (y-axis) was analyzed. Values are mean ± standard error of the mean. n = 5 to 9 per group. Values are mean ± standard error of the mean. Control pellet (non-DHT; open bars) or DHT-exposed offspring (black bars). P/E, proestrus/estrus. Female offspring from chronically hyperandrogenized dams exhibited diminished ovarian reserve Hyperandrogenemia has been reported to impair reproductive function of females (28), but the degree to which hyperandrogenemia affects their offspring is controversial. We observed fewer (P < 0.05) oocytes were retrieved from the ovaries of DHT daughters than those of non-DHT daughters following superovulation. Furthermore, among oocytes retrieved, there were fewer (P < 0.05) mature oocytes from the ovaries of DHT daughters than those of non-DHT daughters (Fig. 7A). There were, however, no differences in the percentage of fertilized oocytes or cleavage stage embryos between DHT daughters and non-DHT daughters (Fig. 7B). We found fewer (P < 0.005) primordial follicles and more (P < 0.05) primary follicles in the ovaries of DHT daughters than those of non-DHT daughters (Fig. 7C). We observed fewer antral follicles in both control and DHT-exposed daughters in females after superovulation, compared with females not superovulated (because more antral follicles develop into preovulatory follicles and release eggs after superovulation). We only counted oocytes, but not CL after superovulation, to avoid misleading readers because some newly ovulated follicles after superovulation do not have enough time to form CL. To eliminate the effect of superovulation on the follicle development, we counted the follicles in the ovaries without exposure to superovulation and found a similar pattern of primodial, primary, secondary, and antral follicles to that after superovulation (Fig. 7D). Moreover, the ovaries of DHT daughters contained fewer (P < 0.05) CL than non-DHT daughters (maximum number of CL: 4.00 ± 0.49 vs 6.20 ± 0.58 per section per ovary). Figure 7. View largeDownload slide IVF and ovarian follicle counts. IVF was performed with the DHT daughter and non-DHT daughter mice. (A) The total number of retrieved oocytes and mature eggs (x-axis) after superovulation, which were counted (y-axis) from two ovaries per mouse. (B) The percentages (y-axis) of fertilized oocytes (number of fertilized oocytes divided mature oocytes) and cleavage stage embryos (≥2 cells stage; number of embryos divided fertilized oocytes; x-axis) are shown for treated and control groups. (C) Total numbers of follicles (y-axis) of different stages in the ovarian sections (see Materials and Methods) after superovulation were quantified as primordial, primary, secondary, or antral (x-axis). CL were not determined for these sections. (D) Total numbers of follicles (y-axis) were quantified in the ovarian sections from mice that had not undergone superovulation. Values are mean ± standard error of the mean. n = 5 to 13 per group. NS, not significant. Figure 7. View largeDownload slide IVF and ovarian follicle counts. IVF was performed with the DHT daughter and non-DHT daughter mice. (A) The total number of retrieved oocytes and mature eggs (x-axis) after superovulation, which were counted (y-axis) from two ovaries per mouse. (B) The percentages (y-axis) of fertilized oocytes (number of fertilized oocytes divided mature oocytes) and cleavage stage embryos (≥2 cells stage; number of embryos divided fertilized oocytes; x-axis) are shown for treated and control groups. (C) Total numbers of follicles (y-axis) of different stages in the ovarian sections (see Materials and Methods) after superovulation were quantified as primordial, primary, secondary, or antral (x-axis). CL were not determined for these sections. (D) Total numbers of follicles (y-axis) were quantified in the ovarian sections from mice that had not undergone superovulation. Values are mean ± standard error of the mean. n = 5 to 13 per group. NS, not significant. Discussion Hyperandrogenism is a feature of several endocrine disorders that affect women of reproductive age. Previous animal studies of gestational androgen excess have shown that female offspring exhibit metabolic and reproductive dysfunction into adulthood (18, 28–30). However, these models do not account for the prepregnancy hyperandrogenism of mothers that are present in PCOS. That is, the effect of maternal hyperandrogenism in early development, from preimplantation to gastrulation, has not been examined (31). In this study, we investigated female offspring from chronically hyperandrogenic dams (11). The DHT levels in serum were maintained for 4 weeks after the DHT implant, and the implants were replaced monthly; thus, the dams received a constant level of exogenous DHT during the experiment. Our data showed circulating DHT levels were elevated during pregnancy in both groups and were ∼1.7-fold higher in DHT-exposed dams than in control dams (no DHT) during gestation (Fig. 1B). This observation parallels what is observed in humans during pregnancy, in which total T rises, whereas free T does not rise until the third trimester (32). Additionally, serum androgens, including T, androstenedione, and/or dehydroepiandrosterone, are higher in pregnant women with PCOS (33–36). DHT levels during human pregnancy have not been systematically investigated. The rise in androgen experienced by the pregnant mice may mimic the rise in androgen experienced during pregnancy in women with PCOS. For both species, the degree to which maternal serum androgens cross the placenta to affect the fetus is not known. We could not measure DHT levels in exposed fetuses. Thus, it is not clear that DHT treatment of mice completely mimicked the human fetal environment of a PCOS pregnancy. It is unlikely that higher insulin levels associated with androgens stimulated ovarian production in the fetus, as the fetuses who experience hyperinsulinemia are generally large for gestational age. We observed that DHT daughters gained weight slower than non-DHT daughters from postnatal 1 to 10 weeks (Fig. 2). From the growth curve, we speculate that DHT daughters had a lower birth body weight than non-DHT daughters. This suggests that preconceptional androgen excess might impair intrauterine growth and that this persists postnatally. This resembles the higher prevalence of small for gestational age infants born to PCOS mothers that suffer from hyperandrogenemia, compared with infants of reproductively normal control women (37, 38). Possibly, gestational hyperandrogenemia can cause intrauterine growth retardation and lower birth weight, due to higher gestational androgens that promote placental differentiation and limit blood flow (39). In contrast to the observation that overweight/obesity is increased in girls with PCOS (40), we observed that DHT daughters were lighter in body weight compared with non-DHT daughters. The disparity in growth patterns between humans and our model might be because pregnant women with PCOS often have coexisting overweight/obesity preceding and during pregnancy to affect postnatal weight gain in human infants. In our model, the dams do not have prepregnancy obesity (12). Serum T levels were elevated in adult DHT female offspring (Fig. 5A), implying that androgen excess alone may not be sufficient to lead to obesity in female offspring. Serum T levels were similar between DHT daughters and non-DHT daughters on PND21, whereas serum T levels were lower in DHT daughters than non-DHT daughters on PND26. This indicates that maternal androgen excess resulted in lower levels of serum T in daughters before puberty. Pups at PND26 are recently weaned, so the continuous exposure to higher DHT in these pups during suckling may temporarily suppress endogenous T production. We found that maternal androgen excess resulted in higher serum T in adult daughters (Fig. 5A), which matches other prenatal androgenized models (41, 42). Chronically DHT-exposed mice have reduced Cyp17a1 messenger RNA expression (a rate-limited enzyme in androgenesis) in ovary (11, 41). Thus, the temporary suppression of serum T at PND26 might be due to a delay in Cyp17a1 expression from maternal DHT. To what extent breastfeeding contributed to this model’s phenotype remains elusive and could be investigated by feeding these pups using foster moms, which did not have the DHT implant. These experiments are beyond the scope of the current study. Body weight may affect pubertal development in rodents (43). We observed that first E emerged remarkably later for DHT daughters compared with non-DHT daughters, although there was no difference in timing of vaginal opening (Fig. 3). Serum E2 levels can regulate the vaginal opening, so we also examined serum E2 levels, which were similar between DHT daughters and non-DHT daughters (Fig. 4A). This is consistent with the vaginal opening observation. LH levels at PND 21 and 26 were not significantly different among groups (Fig. 4B), implying that signals regulating early puberty were normal between groups. First E as determined by vaginal cytology not only requires mature vaginal cells but also fully mature hypothalamic, pituitary, ovarian signaling with positive and negative feedback capabilities. Indeed, the FSH levels at PND26 were different between DHT-treated and nontreated groups, implying that the hypothalamic-pituitary-ovarian signaling was not fully mature. Low body weight disrupts hypothalamic-pituitary-ovarian signaling and may also be a factor in the delayed first E in DHT daughters. Both delayed and advanced puberty have been reported in animal models of prenatal androgenized female offspring. Whether delayed or early puberty is observed seems to depend upon the timing of the fetal androgen exposure. In Rhesus monkeys, female offspring showed delayed puberty if they received T or DHT at between days 40 and 55 of gestation (early exposure) (30, 44). The same pattern also was observed in guinea pig and porcine female offspring that received T at 29 to 35 or 39 to 45 gestational days, respectably (45, 46). Prenatal injection of mice with DHT on embryonic days 16.5 to 18.5 showed advanced puberty without effects on T or estrous cyclicity in adult offspring. The advanced puberty was not regulated by androgen receptor in the central nervous system or in kisspeptin neurons (47). The advanced puberty was also observed in rats injected with T at late gestational days (48). In general, early prenatal androgen treatment resulted in delayed puberty (30, 44–46), but late prenatal androgen treatment resulted in advanced puberty (47, 48) regardless of body weight. We also examined serum LH and FSH levels on PND70 for both groups. Although there are studies indicating that the daughters of PCOS women have higher basal and leuprolide-stimulated LH (49, 50), there were no differences in our study between control and treated groups of offspring. Hyperandrogenemia causes subfertility or infertility in women. Whether maternal hyperandrogenemia would impair the fertility of their daughters remained elusive. Although there are some clues from human and animal model studies to suggest that maternal androgen excess may affect the reproductive function of female offspring (48, 51–53), data in women exposed to androgen excess in utero are not available. In this study, we carried out IVF experiments to evaluate whether maternal hyperandrogenemia would impair the reproductive function of their daughters. We used IVF to estimate the reproductive capacity because IVF can measure the quality and quantity of oocytes, avoiding factors that may influence fertility during pairing, such as mating behaviors. IVF experiments revealed that fewer total oocytes and mature oocytes were retrieved from the ovaries of DHT daughters than that of non-DHT daughters, whereas there were no differences in the number and the percentage rate of fertilized oocytes and cleaved embryos (Fig. 7). These data suggest that maternal androgen excess jeopardizes the ovarian response to FSH. Moreover, we also counted the follicles in ovaries and found that the number of primordial follicles was significantly lower in DHT daughters than that in non-DHT daughters. There were significantly greater primary follicles in DHT daughters compared with that in non-DHT daughters (Fig. 7C). The ovaries of DHT daughters show a relatively earlier depletion of their reserve of primordial follicles (4 to 5 months old). Reduced primordial follicles in adulthood may result from premature exhaustion of the follicle pool, a diminished ovarian reserve at birth, or an accelerated rate of primordial follicle recruitment, and increased atresia or destruction of follicles by high androgens. Furthermore, at PND26 (but not in the adult), AMH levels were increased in DHT daughters compared with non-DHT daughters. We speculate that the increased level of AMH may be caused by the increased amount of granulosa cells (growing follicles) at prepuberty associated with DHT and FSH level (54, 55) because AMH is secreted by granulosa cells of ovary and is a marker of follicular development. AMH regulates early follicle growth and initial and cyclic follicle recruitment (56). Interestingly, girls born to mothers with PCOS also exhibit AMH overproduction, which persists from 2 months old to prepubertal life (52, 57, 58). These girls and our DHT-exposed daughters show evidence of an altered follicular development during infancy and childhood. Women with PCOS also show a significant increase in the percentage of primary follicles and a reciprocal decrease in the proportion of primordial follicles compared with normal ovaries (59). Androgens have been shown to promote the differentiation of primordial to primary follicles in the primate and sheep (53, 60, 61), although early follicular depletion is more severe in animals prenatally androgenized by T rather than DHT. Alternatively, the higher T level in adult DHT daughters compared with that in non-DHT daughters in our study may have contributed to the ovarian dysfunction. T exposure was reported to rapidly increase primordial follicles to primary follicles in mouse (by >twofold) through phosphatidylinositol 3-kinase/protein kinase B pathway (62). These data also suggest that more primordial follicles become activated to develop to primary follicles, but more follicles of poor quality are depleted during maturation, and that maternal androgen excess undermined ovarian reserve that leads to the subfertility. In summary, we found that maternal hyperandrogenemia induced by a low dose of DHT caused slower growth, delayed puberty, and altered hormone secretion, which may result in impaired reproductive function by disrupting E cycling and accelerating loss primordial follicles. Abbreviations: AMH anti-Müllerian hormone CL corpora lutea DHT dihydrotestosterone E estrus E2 estradiol ELISA enzyme-linked immunosorbent assay FSH follicle-stimulating hormone HTF human tubal fluid IVF in vitro fertilization KSMO potassium-supplemented simplex optimized medium LH luteinizing hormone M/D metestrus/diestrus P proestrus PCOS polycystic ovary syndrome PND postnatal day T testosterone. Acknowledgments We thank Dr. David Abbott (Professor, University of Wisconsin Obstetrics and Gynecology, Madison, Wisconsin) for editorial comments. Financial Support: This work was supported by National Institutes of Health Grant R00-HD068130 (to S.W.) and the Baltimore Diabetes Research Center: Pilots and Feasibility Grant (to S.W.). Author Contributions: Z.W. and S.W. contributed to the experimental design, conducted the experiments, and wrote and edited the manuscript. M.S. and P.X. contributed to performing some of the experiments, analyzing the corresponding data, and writing part of the manuscript. S.A.D. and J.S. contributed to the experimental design and reviewed and edited the manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Davey RA, Grossmann M. Androgen receptor structure, function and biology: from bench to bedside. Clin Biochem Rev . 2016; 37( 1): 3– 15. Google Scholar PubMed  2. Nef S, Parada LF. Hormones in male sexual development. 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Google Scholar CrossRef Search ADS PubMed  Copyright © 2018 Endocrine Society

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EndocrinologyOxford University Press

Published: Feb 1, 2018

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