Karina F. Rodriguez

Estrogen receptor-α mediates an intraovarian negative feedback loop on thecal cell steroidogenesis via modulation of Cyp17a1 (cytochrome P450, steroid 17α-hydroxylase/17,20 lyase) expression

Excess androgen synthesis by thecal cells is invariably detrimental to preovulatory follicles in the ovary and is considered a fundamental characteristic of polycystic ovary syndrome in women. Investigators have long postulated that granulosa cell‐derived estrogens modulate thecal cell steroidogenesis via a short negative‐feedback loop within the follicle. To test this hypothesis, we assessed the steroidogenic capacity of individual wild‐type (WT) and estrogen receptor‐ (ERα)‐null follicles when cultured in vitro under comparable conditions. Late‐stage ERα‐null follicles exhibited markedly increased expression of the thecal cell enzyme CYP17A1 and secreted much greater amounts of its end product, androstenedione. This phenotype was reproduced in WT follicles when exposed to an aromatase inhibitor or ER‐antagonist, and prevented when the former treatment was supplemented with an ERα‐specific agonist. ERα‐null follicles also exhibited increased testosterone synthesis due to ectopic expression of hydroxysteroid (17ß) dehydrogenase type 3 (HSD17B3), a testis‐specific androgenic enzyme. These data indicate that ER17α functions within thecal cells to negatively modulate the capacity for androgen synthesis by repressing Cyp17a1 expression, and the biological activity of androgens produced by inhibiting Hsd17b3 expression. Hence, these findings provide novel evidence of an intraovarian ERα function that may be critical to the latter stages of folliculogenesis and overall ovarian function.—Taniguchi, F., Couse, J. F., Rodriguez, K. F., Emmen, J. M. A., Poirier. D., Korach, K. S. Estrogen receptor‐17α mediates an intraovarian negative feedback loop on thecal cell steroidogenesis via modulation of CYP17A1 (cytochrome P450, steroid 17α‐hydroxylase/17,20 μlyase) expression FASEB J. 21, 586 −595 (2007)

Role of Estrogen Receptor Signaling Required for Endometriosis-Like Lesion Establishment in a Mouse Model

Endometriosis results from ectopic invasion of endometrial tissue within the peritoneal cavity. Aberrant levels of the estrogen receptor (ER), ERα and ERβ, and higher incidence of autoimmune disorders are observed in women with endometriosis. An immunocompetent mouse model of endometriosis was used in which minced uterine tissue from a donor was dispersed into the peritoneal cavity of a recipient. Wild-type (WT), ERα-knockout (αERKO), and βERKO mice were donors or recipients to investigate the roles of ERα, ERβ, and estradiol-mediated signaling on endometriosis-like disease. Mice were treated with vehicle or estradiol, and resulting location, number, and size of endometriosis-like lesions were assessed. In comparison with WT lesions in WT hosts, αERKO lesions in WT hosts were smaller and fewer in number. The effect of ER status and estradiol treatment on nuclear receptor status, proliferation, organization, and inflammation within lesions were examined. αERKO lesions in WT hosts did not form distal to the incision site, respond to estradiol, or proliferate but did have increased inflammation. WT lesions in αERKO hosts did respond to estradiol, proliferate, and show decreased inflammation with treatment, but surprisingly, progesterone receptor expression and localization remained unchanged. Only minor differences were observed between WT lesions in βERKO hosts and βERKO lesions in WT hosts, demonstrating the estradiol-mediated signaling responses are predominately through ERα. In sum, these results suggest ER in both endometriosis-like lesions and their environment influence lesion characteristics, and understanding these interactions may play a critical role in elucidating this enigmatic disease.

Estradiol Induction of Spermatogenesis Is Mediated via an Estrogen Receptor-α Mechanism Involving Neuroendocrine Activation of Follicle-Stimulating Hormone Secretion

Both testosterone and its nonaromatizable metabolite dihydrotestosterone (DHT) induce spermatogenesis in gonadotropin-deficient hpg mice. Surprisingly, because aromatization is not required, estradiol (E2) also induces spermatogenesis and increases circulating FSH in hpg mice, but the mechanism remains unclear. We studied E2-induced spermatogenesis in hpg mice on an estrogen receptor (ER)-alpha (hpg/alphaERKO) or ERbeta (hpg/betaERKO) knockout or wild-type ER (hpg/WT) background treated with subdermal E2 or DHT implants for 6 wk. In hpg/WT and hpg/betaERKO, but not hpg/alphaERKO mice, E2 increased testis and epididymal weight, whereas DHT-induced increases were unaffected by ERalpha or ERbeta inactivation. E2 but not DHT treatment increased serum FSH (but not LH) in hpg/WT and hpg/betaERKO but not hpg/alphaERKO hpg mice. DHT or E2 alone increased (premeiotic) spermatogonia and (meiotic) spermatocytes without significant change in Sertoli cell numbers. DHT alone increased postmeiotic spermatids, regardless of ER presence, compared with variable ERalpha-dependent E2 postmeiotic responses. An ERalpha-mediated effect was confirmed by treating hpg mice for 6 wk by subdermal selective ER-alpha (16alpha-LE(2)) or ERbeta (8beta-VE(2)) agonist implants. ERalpha (but not ERbeta) agonist increased testis and epididymal weight, Sertoli cell, spermatogonia, meiotic, and postmeiotic germ cell numbers. Only ERalpha agonist markedly increased serum FSH, whereas either agonist induced small rises in serum LH. Administration of ERalpha agonist or E2 in the presence of functional ERalpha induced prominent gene expression of specific Sertoli (Eppin, Rhox5) and Leydig cell (Cyp11a1, Hsd3b1) markers. We conclude that E2-induced spermatogenesis in hpg mice involves an ERalpha-dependent neuroendocrine mechanism increasing blood FSH and Sertoli cell function.

Uterine Gland Formation in Mice Is a Continuous Process, Requiring the Ovary after Puberty, But Not after Parturition

Uterine gland formation occurs postnatally in an ovary- and steroid-independent manner in many species, including humans. Uterine glands secrete substances that are essential for embryo survival. Disruption of gland development during the postnatal period prevents gland formation, resulting in infertility. Interestingly, stabilization of beta-catenin (CTNNB1) in the uterine stroma causes a delay in gland formation rather than a complete absence of uterine glands. Thus, to determine if a critical postnatal window for gland development exists in mice, we tested the effects of extending the endocrine environment of pregnancy on uterine gland formation by treating neonatal mice with estradiol, progesterone, or oil for 5 days. One uterine horn was removed before puberty, and the other was collected at maturity. Some mice were also ovariectomized before puberty. The hormone-treated mice exhibited a delay in uterine gland formation. Hormone-treatment increased the abundance of uterine CTNNB1 and estrogen receptor alpha (ESR1) before puberty, indicating possible mechanisms for delayed gland formation. Despite having fewer glands, progesterone-treated mice were fertile, suggesting that a threshold number of glands is required for pregnancy. Mice that were ovariectomized before puberty did not undergo further uterine growth or gland development. Finally, to establish the role of the ovary in postpartum uterine gland regeneration, mice were either ovariectomized or given a sham surgery after parturition, and uteri were evaluated 1 wk later. We found that the ovary is not required for uterine growth or gland development following parturition. Thus, uterine gland development occurs continuously in mice and requires the ovary after puberty, but not after parturition.

Insufficient Luteinizing Hormone-Induced Intracellular Signaling Disrupts Ovulation in Preovulatory Follicles Lacking Estrogen Receptor-β

Gonadotropin-stimulated estrogen receptor-beta (ERbeta)-null preovulatory follicles exhibit submaximal estradiol production, insufficient acquisition of LH receptor, and attenuated expression of essential ovulatory genes. These observations lead to low ovulatory rates compared with wild-type (WT) follicles. We hypothesize that insufficient LH receptor results in reduced cAMP production after an ovulatory stimulus. Individual preantral follicles were cultured with FSH for 4 d and then induced to ovulate with a single dose of human chorionic gonadotropin (hCG). cAMP levels 1 h after hCG were 50% lower in ERbeta-null than WT follicles. To determine whether the lack of LH receptor, and resulting lack of cAMP, could be bypassed by direct activation of adenylyl cyclase, WT and ERbeta-null follicles were induced to ovulate with forskolin. Ten micromolar forskolin doubled the ovulatory rate of ERbeta-null follicles compared with treatment with hCG ( approximately 50 vs. 25%, respectively). In WT follicles, 10 microm forskolin reduced the ovulation rate compared with hCG (14 vs. 83%, respectively), indicating that high doses of forskolin inhibited WT ovulation. A 10 microm concentration of forskolin induced cAMP levels in ERbeta-null follicles that were comparable to levels produced in WT follicles after hCG and either partially or completely rescued the attenuated expression of LH-responsive genes. These data indicate that direct activation of adenylyl cyclase, resulting in increased production of cAMP, partially rescues the ovulatory response of ERbeta-null follicles, suggesting that insufficient LH receptor and low cAMP levels contribute to their poor ovulatory rates. We also determined that ERbeta-null ovaries exhibit an alteration in the activation of ERK1/2. Our evaluation of the ERbeta-null ovarian phenotype indicates that ERbeta plays a role in facilitating folliculogenesis. We show that expression of ERbeta in preovulatory follicles is required for adequate cAMP production and propose that an optimal level of cAMP is required for hCG-stimulated ovulation.

Effects of in Utero Exposure to Arsenic during the Second Half of Gestation on Reproductive End Points and Metabolic Parameters in Female CD-1 Mice

Background Mice exposed to high levels of arsenic in utero have increased susceptibility to tumors such as hepatic and pulmonary carcinomas when they reach adulthood. However, the effects of in utero arsenic exposure on general physiological functions such as reproduction and metabolism remain unclear. Objectives We evaluated the effects of in utero exposure to inorganic arsenic at the U.S. Environmental Protection Agency (EPA) drinking water standard (10 ppb) and at tumor-inducing levels (42.5 ppm) on reproductive end points and metabolic parameters when the exposed females reached adulthood. Methods Pregnant CD-1 mice were exposed to sodium arsenite [none (control), 10 ppb, or 42.5 ppm] in drinking water from gestational day 10 to birth, the window of organ formation. At birth, exposed offspring were fostered to unexposed dams. We examined reproductive end points (age at vaginal opening, reproductive hormone levels, estrous cyclicity, and fertility) and metabolic parameters (body weight changes, hormone levels, body fat content, and glucose tolerance) in the exposed females when they reached adulthood. Results Arsenic-exposed females (10 ppb and 42.5 ppm) exhibited early onset of vaginal opening. Fertility was not affected when females were exposed to the 10-ppb dose. However, the number of litters per female was decreased in females exposed to 42.5 ppm of arsenic in utero. In both 10-ppb and 42.5-ppm groups, arsenic-exposed females had significantly greater body weight gain, body fat content, and glucose intolerance. Conclusion Our findings revealed unexpected effects of in utero exposure to arsenic: exposure to both a human-relevant low dose and a tumor-inducing level led to early onset of vaginal opening and to obesity in female CD-1 mice. Citation Rodriguez KF, Ungewitter EK, Crespo-Mejias Y, Liu C, Nicol B, Kissling GE, Yao HH. 2016. Effects of in utero exposure to arsenic during the second half of gestation on reproductive end points and metabolic parameters in female CD-1 mice. Environ Health Perspect 124:336–343; http://dx.doi.org/10.1289/ehp.1509703

The absence of ER-β results in altered gene expression in ovarian granulosa cells isolated from in vivo preovulatory follicles.

Determining the spatial and temporal expression of genes involved in the ovulatory pathway is critical for the understanding of the role of each estrogen receptor in the modulation of folliculogenesis and ovulation. Estrogen receptor (ER)-β is highly expressed in ovarian granulosa cells, and mice lacking ER-β are subfertile due to inefficient ovulation. Previous work has focused on isolated granulosa cells or cultured follicles and, although informative, provides confounding results due to the heterogeneous cell types present including granulosa and theca cells and oocytes and exposure to in vitro conditions. Herein we isolated preovulatory granulosa cells from wild-type (WT) and ERβ-null mice using laser capture microdissection to examine the genomic transcriptional response downstream of pregnant mare serum gonadotropin (mimicking FSH) and pregnant mare serum gonadotropin/human chorionic gonadotropin (mimicking LH) stimulation. This allows for a direct comparison of in vivo granulosa cells at the same stage of development from both WT and ERβ-null ovaries. ERβ-null granulosa cells showed altered expression of genes known to be regulated by FSH (Akap12 and Runx2) as well as not previously reported (Arnt2 and Pou5f1) in WT granulosa cells. Our analysis also identified 304 genes not previously associated with ERβ in granulosa cells. LH-responsive genes including Abcb1b and Fam110c show reduced expression in ERβ-null granulosa cells; however, novel genes including Rassf2 and Megf10 were also identified as being downstream of LH signaling in granulosa cells. Collectively, our data suggest that granulosa cells from ERβ-null ovaries may not be appropriately differentiated and are unable to respond properly to gonadotropin stimulation.

Mapping lineage progression of somatic progenitor cells in the mouse fetal testis.

Testis morphogenesis is a highly orchestrated process involving lineage determination of male germ cells and somatic cell types. Although the origin and differentiation of germ cells are known, the developmental course specific for each somatic cell lineage has not been clearly defined. Here, we construct a comprehensive map of somatic cell lineage progression in the mouse testis. Both supporting and interstitial cell lineages arise from WT1+ somatic progenitor pools in the gonadal primordium. A subpopulation of WT1+ progenitor cells acquire SOX9 expression and become Sertoli cells that form testis cords, whereas the remaining WT1+ cells contribute to progenitor cells in the testis interstitium. Interstitial progenitor cells diversify through the acquisition of HES1, an indication of Notch activation, at the onset of sex determination. HES1+ interstitial progenitors, through the action of Sertoli cell-derived Hedgehog signals, become positive for GLI1. The GLI1+ interstitial cells eventually develop into two cell lineages: steroid-producing fetal Leydig cells and non-steroidogenic cells. The fetal Leydig cell population is restricted by Notch2 signaling from the neighboring somatic cells. The non-steroidogenic progenitor cells retain their undifferentiated state during fetal stage and become adult Leydig cells in post-pubertal testis. These results provide the first lineage progression map that illustrates the sequential establishment of somatic cell populations during testis morphogenesis. Highlighted Article: Somatic progenitor cell populations in the mouse testis are defined by progressive lineage-specific acquirement of WT1, HES1, SOX9 and GLI1 beginning at the time of sex determination.

Elimination of the male reproductive tract in the female embryo is promoted by COUP-TFII in mice

The makings of the reproductive tract Every embryo, regardless of its sex, contains both male and female primitive reproductive tracts before sexual differentiation. To establish a sex-specific reproductive system, female embryos need to remove the components of male tracts. The general consensus contends that removal of the male tracts occurs by default, a passive outcome owing to a lack of testis-derived androgens. Working in mice, Zhao et al. discovered that this process instead was actively promoted by the transcription factor COUP-TFII (see the Perspective by Swain). Without the action of this factor, embryos retained male reproductive tracts, independently of androgen action. These findings unveil unexpected mechanisms underlying the sexually dimorphic establishment of reproductive tracts. Science, this issue p. 717; see also p. 648 A transcription factor drives the process by which female mouse embryos establish their sex-specific reproductive structures. The sexual differentiation paradigm contends that the female pattern of the reproductive system is established by default because the male reproductive tracts (Wolffian ducts) in the female degenerate owing to a lack of androgen. Here, we discovered that female mouse embryos lacking Coup-tfII (chicken ovalbumin upstream promoter transcription factor II) in the Wolffian duct mesenchyme became intersex—possessing both female and male reproductive tracts. Retention of Wolffian ducts was not caused by ectopic androgen production or action. Instead, enhanced phosphorylated extracellular signal-regulated kinase signaling in Wolffian duct epithelium was responsible for the retention of male structures in an androgen-independent manner. We thus suggest that elimination of Wolffian ducts in female embryos is actively promoted by COUP-TFII, which suppresses a mesenchyme-epithelium cross-talk responsible for Wolffian duct maintenance.

Response to "Comment on 'Effects of in Utero Exposure to Arsenic during the Second Half of Gestation on Reproductive End Points and Metabolic Parameters in Female CD-1 Mice'".

In a letter in response to our paper, Williams and DeSesso questioned why a significant weight gain in arsenic (As)-exposed offspring was found in our study but not in others (Markowski et al. 2011; Markowski et al. 2012; Tokar et al. 2010; Waalkes et al. 2003; Waalkes et al. 2004; Waalkes et al. 2006). In addition, they point out that early onset of vaginal opening in response to gestational As exposure was not observed by Markowski et al. (2012) or Gandhi et al. (2012). As we mentioned in the Discussion section of our paper, some of the results under our exposure scheme do not recapitulate those observed in other studies. The main difference that could contribute to these discrepancies is that the offspring in our study were fostered to dams that were not exposed to As during gestation. Dams exposed gestationally to As are known to produce lower-quality milk, which can result in weight deficits in their pups (Kozul-Horvath et al. 2012). In contrast, the studies mentioned by Williams and DeSesso left offspring with their As-exposed mothers. It is therefore possible that the impacts of gestational As exposure on milk quality could offset the effects of As on offspring weight gain and vaginal opening. Regarding the lack of a dose response, our study was designed to examine the impact of two specific As doses: 10 ppb (the U.S. Environmental Protection Agency drinking water standard) and 42.5 ppm (tumor-inducing concentration). Dose–response experiments are usually performed to identify either the proper dose for further experiments or the mode of action of a particular chemical (linear, biphasic, or others). Neither of these two parameters were an end point of our study. We do not have an explanation for the different responses between the 10-ppb and 42.5-ppm treatment groups, and further studies are definitely required. Williams and DeSesso further suggest that the control pups in our study may have been unusually small, such that our results reflect a statistical anomaly. However, data on CD-1 female weights (Lang and White 1996) indicate that the weight of our control mice at 25 weeks (approximately 34.4 g) falls in the normal range of approximately 31–42 g. Williams and DeSesso also questioned whether an increase in body weight could contribute to the early onset of vaginal opening. This argument is indeed the focal point of our experiments, as we described in the Results and Discussion sections of the paper. Based on the analyses that examined the association between weight at weaning (postnatal day 21) and age at vaginal opening (Figure 2D of our paper), we observed that the 42.5-ppm treatment and control groups showed a positive association between weight at weaning and onset of vaginal opening. This association was not found in the 10-ppb treatment group. Although we did not have the weight records at the time of vaginal opening, we believe the population data in Figure 2D are sufficient for us to make a valid conclusion regarding the associations. The two studies mentioned by Williams and DeSesso (Markowski et al. 2012 using B6 mice; Gandhi et al. 2012 using rats) found no effect of in utero exposure to As on vaginal opening. Strain and species differences may contribute to these discrepancies. In summary, in our Discussion section we fully recognize the differences between our results and those of other studies. We agree with Williams and DeSesso that the discrepancies could result from experimental conditions such as diet, strain, and species. Like all animal studies, our study provides observations on a particular strain of mouse under specific experimental conditions. The differences among studies only strengthen the point that more studies are needed to understand the mechanisms of action of As and how these different experimental conditions influence the outcomes.