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Male factors, including those of autoimmune origin, contribute to approximately 50% of infertility cases in humans. However, the mechanisms underlying autoimmune male infertility are poorly understood. Deficiency in autoimmune regulator (AIRE) impairs central immune tolerance because of diminished expression of self-antigens in the thymus. Humans with AIRE mutations and mice with engineered ablation of Aire develop multiorgan autoimmunity and infertility. To determine the immune targets contributing to infertility in male Aire-deficient ((−/−)) mice, Aire(−/−) or wild-type (WT) males were paired with WT females. Aire(−/−) males exhibited dramatically reduced mating frequency and fertility, hypogonadism, and reduced serum testosterone. Approximately 15% of mice exhibited lymphocytic infiltration into the testis, accompanied by atrophy, azoospermia, and reduced numbers of mitotically active germ cells; the remaining mice showed normal testicular morphology, sperm counts, and motility. However, spermatozoa from all Aire(−/−) mice were defective in their ability to fertilize WT oocytes in vitro. Lymphocytic infiltration into the epididymis, seminal vesicle, and prostate gland was evident. Aire(−/−) male mice generated autoreactive antibodies in an age-dependent manner against sperm, testis, epididymis, prostate gland, and seminal vesicle. Finally, expression of Aire was evident in the seminiferous epithelium in an age-dependent manner, as well as in the prostate gland. These findings suggest that Aire-dependent central tolerance plays a critical role in maintaining male fertility by stemming autoimmunity against multiple reproductive targets.

Epigenetic modifications regulate normal physiological, as well as pathological processes in various organs, including the uterus and placenta. Both organs undergo dramatic and rapid restructuring that depends upon precise orchestration of events. Epigenetic changes that alter transcription and translation of gene-sets regulate such responses. Histone modifications alter the chromatin structure, thereby affecting transcription factor access to gene promoter regions. Binding of histones to DNA is regulated by addition or removal of subunit methyl and other groups, which can inhibit or stimulate transcription. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb repressive complex 2 (PRC2) that catalyzes tri-methylation of histone H3 at Lys 27 (H3K27me3) and subsequently suppresses transcription of genes bound by such histones. Uterine EZH2 expression exerts a critical role in development and function of this organ with deletion of this gene resulting in uterine hyperplasia and expression of cancer-associated transcripts. Elucidating the roles of EZH2 in uterus and placenta is essential as EZH2 dysregulation is associated with several uterine and placental pathologies. Herein, we discuss EZH2 functions in uterus and placenta, emphasizing its physiological and pathological importance.

Development of uterine glands (adenogenesis) in mammals typically begins during the early post-natal period and involves budding of nascent glands from the luminal epithelium and extensive cell proliferation in these structures as they grow into the surrounding stroma, elongate and mature. Uterine glands are essential for pregnancy, as demonstrated by the infertility that results from inhibiting the development of these glands through gene mutation or epigenetic strategies. Several genes, including forkhead box A2, beta-catenin and members of the Wnt and Hox gene families, are implicated in uterine gland development. Progestins inhibit uterine epithelial proliferation, and this has been employed as a strategy to develop a model in which progestin treatment of ewes for 8 weeks from birth produces infertile adults lacking uterine glands. More recently, mouse models have been developed in which neonatal progestin treatment was used to permanently inhibit adenogenesis and adult fertility. These studies revealed a narrow and well-defined window in which progestin treatments induced permanent infertility by impairing neonatal gland development and establishing endometrial changes that result in implantation defects. These model systems are being utilized to better understand the molecular mechanisms underlying uterine adenogenesis and endometrial function. The ability of neonatal progestin treatment in sheep and mice to produce infertility suggests that an approach of this kind may provide a contraceptive strategy with application in other species. Recent studies have defined the temporal patterns of adenogenesis in uteri of neonatal and juvenile dogs and work is underway to determine whether neonatal progestin or other steroid hormone treatments might be a viable contraceptive approach in this species.

Liz Simon1, Marie-Claude Hofmann2 and Paul S. Cooke3 1Department of Biomedical Sciences, College of Veterinary Medicine, Nursing and Allied Health, Tuskegee University, Tuskegee, AL 2Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 3Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL USA

Uterine gland development (adenogenesis) in mice begins on Postnatal Day (PND) 5 and is completed in adulthood. Adenogenesis depends on estrogen receptor 1, and progesterone (P4) inhibits mitogenic effects of estrogen on uterine epithelium. This progestin-induced effect has been used to inhibit uterine gland development; progestin treatment of ewes for 8 wk from birth has produced infertile adults lacking uterine glands. The goals of the present study were to determine if a window of susceptibility to P4-mediated inhibition of uterine gland development exists in mice and whether early P4 treatment abolishes adenogenesis and fertility. Mice were injected daily with P4 (40 μg/g) or vehicle during various postnatal windows. Adenogenesis, cell proliferation, and expression of key morphoregulatory transcripts and proteins were examined in uteri at PNDs 10 and 20. Additionally, adenogenesis was assessed in isolated uterine epithelium. Treatment during PNDs 3-9, 5-9, or 3-7 abolished adenogenesis at PND 10, whereas treatments during PNDs 3-5 and 7-9 did not. Critically, mice treated during PNDs 3-9 lacked glands in adulthood, indicating that adenogenesis did not resume after this treatment. However, glands were present by PND 20 and later following treatment during PNDs 5-9 or 3-7, whereas treatment during PNDs 10-16 produced partial inhibition of adenogenesis at PND 20 and later. Epithelial proliferation at PND 10 was low following P4 treatment (PNDs 3-9) but exceeded that in controls at PND 20, indicating a rebound of epithelial proliferation following treatment. Messenger RNA for Wnt, Fzd, and Hox genes was altered by neonatal P4 treatment. All groups cycled during adulthood. Mice treated with P4 during PNDs 3-9, but not during other developmental windows, showed minimal fertility in adulthood. In summary, brief P4 treatment (7 days) during a critical neonatal window (PNDs 3-9) transiently inhibited epithelial proliferation but totally and permanently blocked adenogenesis and adult fertility. This resulted in permanent loss of uterine glands and, essentially, total infertility during adulthood. The narrow window for inhibition of adenogenesis identified here may have implications for development of this methodology as a contraceptive strategy for animals.

The study objective was to determine whether stromal and/or epithelial estrogen receptor-alpha (ERalpha) is required for relaxin to promote proliferation of stromal and epithelial cells in the mouse cervix. Four types of tissue recombinants were prepared with cervical stroma (St) and epithelium (Ep) from wild-type (wt) and ERalpha knockout (ko) mice: wt-St+wt-Ep, wt-St+ko-Ep, ko-St+wt-Ep and ko-St+ko-Ep. Tissue recombinants were grafted under the renal capsule of syngeneic female mice. After 3 wk of transplant growth, hosts were ovariectomized and fitted with silicon implants containing 17beta-estradiol (treatment d 1). Animals were injected sc with relaxin or vehicle PBS at 6-h intervals from 0600 h on d 8 through 0600 h on d 10. To evaluate cell proliferation, 5-bromo-2'-deoxyuridine was injected sc 10 h before tissue recombinants were collected at 1000 h on d 10. Relaxin promoted marked proliferation of both epithelial and stromal cells in tissue recombinants containing wt St (P0.001) but far lower proliferation in recombinants prepared with ko St, regardless of whether Ep was derived from wt or ko mice. An additional experiment using mice expressing wt ERalpha, a mutant of ERalpha that selectively lacks classical signaling through estrogen response element binding, or no ERalpha demonstrated that ERalpha must bind to an estrogen response element to enable relaxin's proliferative effects. In conclusion, this study shows that ERalpha-expressing cells in St, using a classical signaling pathway, are necessary for relaxin to promote marked proliferation in both stromal and epithelial cells of the mouse cervix.

Estrogens play an important role in prostatic development, health, and disease. While estrogen signaling is essential for normal postnatal prostate development, little is known about its prenatal role in control animals. We tested the hypothesis that estrogen signaling is needed for normal male prostatic bud patterning. Budding patterns were examined by scanning electron microscopy of urogenital sinus epithelium from wild-type mice, mice lacking estrogen receptor (ER)alpha, ERbeta, or both, and wild-type mice exposed to the antiestrogen ICI 182,780. Budding phenotypes did not detectably differ among any of these groups, strongly suggesting that estrogen signaling is not needed to establish the prototypical prostatic budding pattern seen in control males. This finding contributes to our understanding of the effects of low-level estrogen exposure on early prostate development. In utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can greatly alter the pattern in which prostatic buds form and reduce their number. For several reasons, including a prior observation that inhibitory effects of TCDD on prostatic budding in rats depend heavily on the sex of adjacent fetuses, we tested the hypothesis that estrogen signaling is needed for TCDD to disrupt prostatic budding. However, budding did not detectably differ among wild-type mice, or mice lacking ERalpha, ERbeta, or both, that were exposed prenatally to TCDD (5 microg/kg on embryonic day 13.5). Nor did ICI 182,780 detectably affect the response to TCDD. These results strongly suggest that estrogen signaling is not needed for TCDD to inhibit prostatic epithelial budding.

Relaxin and estrogen are secreted by the ovary during the second half of pregnancy in rats and mice. Relaxin promotes marked growth of the lower reproductive tract in both species. Relaxin promotes accumulation of epithelial and stromal cells in the cervix and vagina by both stimulating cell proliferation and inhibiting apoptosis. Estrogen acting through estrogen receptor alpha (ERalpha) plays an essential permissive role in relaxin's actions. A fundamental step toward understanding the actions of relaxin and estrogen is to identify the tissue compartments that initiate their effects. Limited studies using either antibodies to human relaxin receptor (LGR7, RXFP1) or an IRES-LacZ reporter cassette in the LGR7 gene revealed relaxin receptors in subepithelial stroma cells and smooth muscle cells but not in epithelial cells in rodent vaginal and/or cervical tissues. ERalpha has been reported in both stromal and epithelial compartments in the rodent reproductive tract. This chapter describes ongoing studies that use relaxin bioactivity as a means of identifying the tissue compartment(s) that initiates the actions of relaxin and estrogen on the lower reproductive tract. Specifically, a tissue separation-recombination methodology in combination with LGR7 knockout mice was initially used to obtain functional evidence that stromal LGR7 is both necessary and sufficient to promote proliferation and inhibit apoptosis in both stromal and epithelial cells in mouse cervix and vagina. The tissue separation-recombination method is currently being used in conjunction with ERalpha knockout mice to determine if the obligatory permissive effect of estrogen on relaxin-induced cell proliferation occurs through stromal and/or epithelial ERalpha.

Estrogen regulates the amount of white adipose tissue (WAT) in females, but its role in males and whether WAT effects involve estrogen receptor-alpha (ERalpha) or ERbeta were unclear. We analyzed the role of ERalpha in WAT and brown adipose tissue by comparing these tissues in wild-type (WT) and ERalpha-knockout (alphaERKO) male and female mice. Brown adipose tissue weight was similar in alphaERKO and WT males at all ages. Progressive increases in WAT were seen in alphaERKO males with advancing age. Epididymal, perirenal, and inguinal WAT weighed 139-185% more in alphaERKO than in WT males by 270-360 days of age. Epididymal and perirenal adipocyte size was increased 20% in alphaERKO males. Adipocyte number was 82-168% greater in fat pads of alphaERKO vs. WT males. Compared with WT, 90-day-old alphaERKO females had increases in fat pad weights (54-103%), adipocyte size, and number. Both alphaERKO males and females had insulin resistance and impaired glucose tolerance, similar to humans lacking ERalpha or aromatase. Energy intake was equal in WT and alphaERKO males, indicating that obesity was not induced by hyperphagia. In contrast, energy expenditure was reduced by 11% in alphaERKO compared with WT males, indicating that altered energy expenditure may be important for the observed obesity. In summary, ERalpha absence causes adipocyte hyperplasia and hypertrophy, insulin resistance, and glucose intolerance in both sexes. These results are evidence that estrogen/ERalpha signaling is critical in female and male WAT; obesity in alphaERKO males involves a mechanism of reduced energy expenditure rather than increased energy intake.

Estradiol-17β (E 2 ) acts through the estrogen receptor (ER) to regulate uterine growth and functional differentiation. To determine whether E 2 elicits epithelial mitogenesis through epithelial ER versus indirectly via ER-positive stromal cells, uteri from adult ER-deficient ER knockout (ko) mice and neonatal ER-positive wild-type (wt) BALB/c mice were used to produce the following tissue recombinants containing ER in epithelium (E) and/or stroma (S), or lacking ER altogether: wt-S + wt-E, wt-S + ko-E, ko-S + ko-E, and ko-S + wt-E. Tissue recombinants were grown for 4 weeks as subrenal capsule grafts in intact female nude mice, then the hosts were treated with either E 2 or oil a week after ovariectomy. Epithelial labeling index and ER expression were determined by [ 3 H]thymidine autoradiography and immunohistochemistry, respectively. In tissue recombinants containing wt-S (wt-S + wt-E, wt-S + ko-E), E 2 induced a similar large increase in epithelial labeling index compared with oil-treated controls in both types of tissue recombinants despite the absence of epithelial ER in wt-S + ko-E tissue recombinants. This proliferative effect was blocked by an ER antagonist, indicating it was mediated through ER. In contrast, in tissue recombinants prepared with ko-S (ko-S + ko-E and ko-S + wt-E), epithelial labeling index was low and not stimulated by E 2 despite epithelial ER expression in ko-S + wt-E grafts. In conclusion, these data demonstrate that epithelial ER is neither necessary nor sufficient for E 2 -induced uterine epithelial proliferation. Instead, E 2 induction of epithelial proliferation appears to be a paracrine event mediated by ER-positive stroma. These data in the uterus and similar studies in the prostate suggest that epithelial mitogenesis in both estrogen and androgen target organs are stromally mediated events.

We have investigated the capacity of vaginal and uterine epithelia (VE and UE) to reexpress normal morphology and hormone responsiveness following cell culture. VE and UE from adult ovariectomized mice were grown in a collagen gel matrix with serum-free medium for 7-10 days. Proliferation of these cells occurs in the absence of 17 beta-estradiol and is not stimulated by 17 beta-estradiol; the VE usually does not keratinize or stratify in vitro. Cultured VE and UE were recombined with homologous vaginal or uterine stroma (VS and US, respectively) and these recombinants were grown under the renal capsule of female hosts for 4 weeks. The epithelium of the VS + VE recombinants cycled, proliferated and stratified in response to estrogen and mucified normally in response to progesterone. The UE that was grown in vivo with cultured US also showed normal morphology and estrogen responsiveness. These changes in the UE and VE were not simply a result of return to the in vivo environment, as epithelia alone in collagen gels transplanted under the renal capsule did not survive. These results indicate that both VE and UE, which are not estrogen-dependent in vitro, reexpress their estrogen dependency and normal morphology when recombined with homologous stroma and grown in vivo. Thus, the changes these cells show when grown in culture are the result of altered conditions in vitro rather than an irreversible alteration in the cells themselves or the selection of specific subpopulations that are not mitogenically or morphologically responsive to estrogen under any condition.