Paul S. Cooke

Paracrine Regulation of Epithelial Progesterone Receptor by Estradiol in the Mouse Female Reproductive Tract

Abstract Regulation of progesterone receptor (PR) by estradiol-17β (E2) in mouse uterine and vaginal epithelia was studied. In ovariectomized mice, PR expression was low in both vaginal stroma and epithelium, but high in uterine epithelium. E2 induced PR in vaginal epithelium and stroma, but down-regulated PR in uterine epithelium. Analysis of estrogen receptor α (ERα) knockout (ERKO) mice showed that ERα is essential for E2-induced PR expression in both vaginal epithelium and stroma, and for E2-induced down-regulation, but not constitutive expression of PR in uterine epithelium. Regulation of PR by E2 was studied in vaginal and uterine tissue recombinants made with epithelium and stroma from wild-type and ERKO mice. In the vaginal tissue recombinants, PR was induced by E2 only in wild-type epithelium and/or stroma. Hence, in vagina, E2 induces PR directly via ERα within the tissue. Conversely, E2 down-regulated epithelial PR only in uterine tissue recombinants constructed with wild-type stroma. Therefore, down-regulation of uterine epithelial PR by E2 requires stromal, but not epithelial, ERα. In vitro, isolated uterine epithelial cells retained a high PR level with or without E2, which is consistent with an indirect regulation of uterine epithelial PR in vivo. Thus, E2 down-regulates PR in uterine epithelium through paracrine mechanisms mediated by stromal ERα.

Proliferation of Adult Sertoli Cells Following Conditional Knockout of the Gap Junctional Protein GJA1 (Connexin 43) in Mice

Abstract GJA1 (also known and referred to here as connexin 43 and abbreviated CX43) is the predominant testicular gap junction protein, and CX43 may regulate Sertoli cell maturation and spermatogenesis. We hypothesized that lack of CX43 would inhibit Sertoli cell differentiation and extend proliferation. To test this, a Sertoli cell-specific Cx43 knockout (SC-Cx43 KO) mouse was generated using Cre-lox technology. Immunohistochemistry indicated that CX43 was not expressed in the Sertoli cells of SC-Cx43 KO mice, but was normal in organs such as the heart. Testicular weight was reduced by 41% and 76% in SC-Cx43 KO mice at 20 and 60 days, respectively, vs. wild-type (wt) mice. Seminiferous tubules of SC-Cx43 KO mice contained only Sertoli cells and actively proliferating early spermatogonia. Sertoli cells normally cease proliferation at 2 wk of age in mice and become terminally differentiated. However, proliferating Sertoli cells were present in SC-Cx43 KO but not wt mice at 20 and 60 days of age. Thyroid hormone receptor alpha (THRA) is high in proliferating Sertoli cells, then declines sharply in adulthood. Thra mRNA expression was increased in 20-day SC-Cx43 KO vs. wt mice, and it showed a trend toward an increase in 60-day mice, indicating that loss of CX43 in Sertoli cells inhibited their maturation. In conclusion, we have generated mice lacking CX43 in Sertoli cells but not other tissues. Our data indicate that CX43 in Sertoli cells is essential for spermatogenesis but not spermatogonial maintenance/proliferation. SC-Cx43 KO mice showed continued Sertoli cell proliferation and delayed maturation in adulthood, indicating that CX43 plays key roles in Sertoli cell development.

Regulation of Progesterone Receptors and Decidualization in Uterine Stroma of the Estrogen Receptor-α Knockout Mouse

Abstract Regulation of progesterone receptor (PR) in uterine stroma (endometrial stroma plus myometrium) by estrogen was investigated in estrogen receptor-α (ERα) knockout (αERKO) mice. 17β-Estradiol (E2) increased PR levels in uterine stroma of ovariectomized αERKO mice, and ICI 182 780 (ICI) inhibited this E2-induced PR expression. Estrogen receptor-β (ERβ) was detected in both uterine epithelium and stroma of wild-type and αERKO mice by immunohistochemistry. In organ cultures of αERKO uterus, both E2 and diethylstilbestrol induced stromal PR, and ICI inhibited this induction. These findings suggest that estrogen induces stromal PR via ERβ in αERKO uterus. However, this process is not mediated exclusively by ERβ, because in ERβ knockout mice, which express ERα, PR was up-regulated by E2 in uterine stroma. In both wild-type and αERKO mice, progesterone and mechanical traumatization were essential and sufficient to induce decidual cells, even though E2 and ERα were also required for increase in uterine weight. Progesterone receptor was strongly expressed in decidual cells in αERKO mice, and ICI did not inhibit decidualization or PR expression. This study suggests that up-regulation of PR in endometrial stroma is mediated through at least three mechanisms: 1) classical estrogen signaling through ERα, 2) estrogen signaling through ERβ, and 3) as a result of mechanical stimulation plus progesterone, which induces stromal cells to differentiate into decidual cells. Each of these pathways can function independently of the others.

Regulation of Neonatal Sertoli Cell Development by Thyroid Hormone Receptor α1

Abstract Neonatal hypothyroidism increases adult Sertoli cell populations by extending Sertoli cell proliferation. Conversely, hyperthyroidism induces premature cessation of Sertoli cell proliferation and stimulates maturational events like seminiferous tubule canalization. Thyroid hormone receptors α1 and β1, which are commonly referred to as TRα1 and TRβ1, respectively, are expressed in neonatal Sertoli cells. We determined the relative roles of TRα1 and TRβ1 in the thyroid hormone effect on testicular development and Sertoli cell proliferation using Thra knockout (TRαKO), Thrb knockout (TRβKO), and wild-type (WT) mice. Triiodothyronine (T3) treatment from birth until Postnatal Day 10 reduced Sertoli cell proliferation to minimal levels in WT and TRβKO mice versus that in their untreated controls, whereas T3 had a diminished effect on TRαKO Sertoli cell proliferation. Seminiferous tubule patency and luminal diameter were increased in T3-treated WT and TRβKO testes. In contrast, T3 had no effect on these parameters in TRαKO mice. In untreated adult TRαKO mice, Sertoli cell number, testis weight, and daily sperm production were increased or trended toward an increase, but the increase in magnitude was smaller than that seen in WT mice following neonatal hypothyroidism. Conversely, in TRβKO mice, Sertoli cell number, testis weight, and daily sperm production were similar to those in untreated WT mice. In addition, Sertoli cell number and testis weight in adult WT and TRβKO mice showed comparable increases following hypothyroidism. Our results show that TRαKO mice have testicular effects similar to those seen in WT mice following neonatal hypothyroidism and that TRβKO mice, but not TRαKO mice, have normal Sertoli cell responsiveness to T3. Thus, effects of exogenous manipulation of T3 on neonatal Sertoli cell development are predominately mediated through TRα1.

Uterine glands: development, function and experimental model systems

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.

Brief Exposure to Progesterone During a Critical Neonatal Window Prevents Uterine Gland Formation in Mice

ABSTRACT 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.

Estrogens in Male Physiology

Estrogens have historically been associated with female reproduction, but work over the last two decades established that estrogens and their main nuclear receptors (ESR1 and ESR2) and G protein-coupled estrogen receptor (GPER) also regulate male reproductive and nonreproductive organs. 17β-Estradiol (E2) is measureable in blood of men and males of other species, but in rete testis fluids, E2 reaches concentrations normally found only in females and in some species nanomolar concentrations of estrone sulfate are found in semen. Aromatase, which converts androgens to estrogens, is expressed in Leydig cells, seminiferous epithelium, and other male organs. Early studies showed E2 binding in numerous male tissues, and ESR1 and ESR2 each show unique distributions and actions in males. Exogenous estrogen treatment produced male reproductive pathologies in laboratory animals and men, especially during development, and studies with transgenic mice with compromised estrogen signaling demonstrated an E2 role in normal male physiology. Efferent ductules and epididymal functions are dependent on estrogen signaling through ESR1, whose loss impaired ion transport and water reabsorption, resulting in abnormal sperm. Loss of ESR1 or aromatase also produces effects on nonreproductive targets such as brain, adipose, skeletal muscle, bone, cardiovascular, and immune tissues. Expression of GPER is extensive in male tracts, suggesting a possible role for E2 signaling through this receptor in male reproduction. Recent evidence also indicates that membrane ESR1 has critical roles in male reproduction. Thus estrogens are important physiological regulators in males, and future studies may reveal additional roles for estrogen signaling in various target tissues.

Dynamic changes in fetal Leydig cell populations influence adult Leydig cell populations in mice

Testes contain two distinct Leydig cell populations during development: fetal and adult Leydig cells (FLCs and ALCs, respectively). ALCs are not derived from FLCs, and it is unknown whether these two populations share common progenitors. We discovered that hedgehog (Hh) signaling is responsible for transforming steroidogenic factor 1‐positive (SF1+) progenitors into FLCs. However, not all SF1+ progenitors become FLCs, and some remain undifferentiated through fetal development. We therefore hypothesized that if FLCs and ALCs share SF1+ progenitors, increased Hh pathway activation in SF1+ progenitor cells could change the dynamics and distribution of SF1+ progenitors, FLCs, and ALCs. Using a genetic model involving constitutive activation of Hh pathway in SF1+ cells, we observed reduced numbers of SF1+ progenitor cells and increased FLCs. Conversely, increased Hh activation led to decreased ALC populations prepubertally, while adult ALC numbers were comparable to control testes. Hence, reduction in SF1+ progenitors temporarily affects ALC numbers, suggesting that SF1+ progenitors in fetal testes are a potential source of both FLCs and ALCs. Besides transient ALC defects, adult animals with Hh activation in SF1+ progenitors had reduced testicular weight, oligospermia, and decreased sperm mobility. These defects highlight the importance of properly regulated Hh signaling in Leydig cell development and testicular functions.— Barsoum, I. B., Kaur, J. Ge, R. S., Cooke, P. S., Yao, H. H.‐C. Dynamic changes in fetal Leydig cell populations influence adult Leydig cell populations in mice. FASEB J. 27, 2657‐2666 (2013). www.fasebj.org

Vaginal and uterine stroma maintain their inductive properties following primary culture.

SummaryVaginal and uterine stromal (VS and UtS) cells have been cultured in a collagen gel matrix, and the ability of the cells to retain their identity and interact normally with epithelia after culture was examined. Stromal explant from 2-d-old mice were plated onto an extracellular matrix covered with collagen, and maintained in Ham’s F12∶DMEM (1∶1) containing 15% fetal bovine serum. The fibroblastic stromal cells invaded and eventually filled the overlying collagen during the 4-wk growth period, and the total DNA of the UtS and VS cultures increased 3.5- and 4-fold, respectively. To assess the ability of the cultured stroma to perform its normal functions after the in vitro period, recombinations of cultured stroma and fresh epithelia were preparaed and transplanted under the renal capsule of female hosts and grown for 4 wk. The epithelium in recombinants of cultured VS + vaginal epithelium (VE) and cultured UtS + uterine epithelium (UtE) was histologically normal and proliferated in response to estrogen. Cultured stroma also instructively induced heterologous epithelium; VS induced UtE to undergo vaginal differentiation, and UtS induced VE to undergo uterine differentiation. These results indicate that UtS and VS retain their identity and do not irreversibly dedifferentiate in culture. Stromal cells grown in a colagen gel matrix form a functional stroma; they interact normally with epithelium after culture and express normal permissive and instructive inductive functions when reassociated with epithelium and grown in vivo.

Therapeutic effects of progesterone and its metabolites in traumatic brain injury may involve non-classical signaling mechanisms

Traumatic brain injury (TBI) is an important and costly medical problem for which no clinically proven treatment currently exists. Studies in rodents and humans have shown beneficial effects of progesterone (P4) on both mortality and functional outcomes following TBI. Neuroprotective effects of P4 in TBI likely involve the classical nuclear progesterone receptors (Pgr) that are widely distributed in both glial cells and neurons of the brain. However, P4 may have critical effects not mediated through Pgr. In the brain, P4 is converted to a metabolite, allopregnanolone (ALLO), whose beneficial effects equal or exceed those of P4 in TBI. ALLO does not bind Pgr, suggesting it acts through non-classical pathways. ALLO has effects on GABAA and pregnane X receptors, as well as on the mitochondrial permeability transition pore. In addition, ALLO is metabolized to another compound, 5alpha-dihydroprogesterone, which binds Pgr, suggesting ALLO actions may involve signaling through Pgr as well as the aforementioned mechanisms of action. P4 and ALLO also signal through a number of membrane receptors (progesterone receptor membrane component 1, and membrane progesterone receptors (mPRs) alpha, beta, gamma, delta, and epsilon) in the brain that are distinct from Pgr, although the role of these receptors in the normal brain and in the therapeutic response to P4 and ALLO following TBI is unclear. In summary, P4 has the potential to become the first clinically effective treatment for TBI, and the effects of P4 and its metabolite ALLO in TBI may involve Pgr, mPRs, and other signaling pathways. Elucidating these mechanisms will more clearly reveal the potential of classical and non-classical pathways to mediate important effects of P4 and its metabolites, and potentially offer new therapeutic approaches to TBI.