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.

Uterine gland development (adenogenesis) in mice begins on Postnatal Day (PND) 5 and is completed in adulthood. Adenogenesis depends on estrogen recep

Estrogen receptor 1 (ESR1) mediates major reproductive functions of 17β-estradiol (E2). Male Esr1 knockout (Esr1KO) mice are infertile due to efferent ductule and epididymal abnormalities. The majority of ESR1 is nuclear/cytoplasmic; however, a small fraction is palmitoylated at cysteine 451 in mice and localized to cell membranes, in which it mediates rapid E2 actions. This study used an Esr1 knock-in mouse containing an altered palmitoylation site (C451A) in ESR1 that prevented cell membrane localization, although nuclear ESR1 was expressed. These nuclear-only estrogen receptor 1 (NOER) mice were used to determine the roles of membrane ESR1 in males. Epididymal sperm motility was reduced 85% in 8-month-old NOER mice compared with wild-type controls. The NOER mice had decreased epididymal sperm viability and greater than 95% of sperm had abnormalities, including coiled midpieces and tails, absent heads, and folded tails; this was comparable to 4-month Esr1KO males. At 8 months, daily sperm production in NOER males was reduced 62% compared with controls. The NOER mice had histological changes in the rete testes, efferent ductules, and seminiferous tubules that were comparable with those previously observed in Esr1KO males. Serum T was increased in NOER males, but FSH, LH, and E2 were unchanged. Critically, NOER males were initially subfertile, becoming infertile with advancing age. These findings identify a previously unknown role for membrane ESR1 in the development of normal sperm and providing an adequate environment for spermatogenesis.

Neonatal uterus and vagina express estrogen receptor 1 (ESR1), and respond mitogenically to exogenous estrogens. However, neonatal ovariectomy does not inhibit pre-weaning uterine cell proliferation, indicating that this process is estrogen independent. Extensive literature suggests ESR1 can be activated by growth factors in a ligand-independent manner and drive uterine cell proliferation. Alternatively, neonatal uterine cell proliferation could be ESR1 independent despite its obligatory role in adult luminal epithelial proliferation. To determine ESR1's role in uterine and vaginal development, we analyzed cell proliferation, apoptosis and uterine gland development (adenogenesis) in wild-type (WT) and Esr1 knockout (Esr1KO) mice from Days 2-60 postnatally. Uterine and vaginal cell proliferation, apoptosis and uterine adenogenesis were comparable in WT and Esr1KO mice before weaning. By Day 29-60, glands had regressed and uterine cell proliferation was reduced in Esr1KO mice, in contrast to continued adenogenesis and proliferation in WT. Apoptosis in Esr1KO uterine epithelium was not increased compared to WT at any age, indicating differences in cell proliferation, rather than apoptosis, cause divergence of uterine size in these two groups at puberty. Similarly, vaginal epithelial proliferation was reduced and the epithelium became atrophic in Esr1KO mice by 29 days of age and later in Esr1KO mice. These results indicate that pre-weaning uterine and vaginal development is ESR1-independent, but becomes dependent on ESR1 by Day 29 onwards. It is not yet clear what mechanisms drive pre-weaning vaginal and uterine development, but ligand-independent activation of ESR1 is not involved.