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 urogenital sinus (UGS), which forms prostate in males, consists of urogenital sinus mesenchyme (UGM), which gives rise to prostatic stromal cells, and urogenital sinus epithelium (UGE), which forms prostatic epithelium. UGM is an instructive inducer, and can induce UGE or other tissues (e.g., bladder epithelium) to become prostatic epithelium. We have demonstrated that when stem/progenitor spermatogonia were recombined with UGM and grown as tissue recombinants in vivo, they differentiated into functional prostatic epithelium. The critical question now is to understand how spermatogonia differentiate into different cell types. This could involve dedifferentiation of stem/progenitor spermatogonia into embryonic stem cell-like cells and then into prostatic epithelium. Conversely, since spermatogonial cells are relatively undifferentiated (multipotent), a direct differentiation of spermatogonia into prostatic stem cells and/or prostatic epithelium may occur under the instructive influence of the UGM. UGS obtained from fetuses at day 16.5 of gestation were trypsinized and separated into UGM and UGE. Stem/progenitor spermatogonia were isolated from 5- to 6-day-old male pups and recombined with UGM on agar plates (1% agar in DMEM with fetal bovine serum). Twenty four hours later, the tissue recombinants were grafted under the renal capsule of adult syngeneic male mice and harvested at day 2, 4 and 8 of grafting. To track cell lineages, transgenic mice expressing green fluorescent protein ubiquitously were used. NKX3.1 is a transcription factor expressed early in prostatic development that drives prostatic epithelial morphogenesis; NKX3.1 was used as the primary indicator of prostatic development. Our results indicated that NKX3.1 was expressed within the first week of grafting and in some grafts as early as 4 days, although mRNA expression of NKX3.1 was not detected after 24 h of tissue recombination or 2 days after grafting. Some tissue recombinant grafts developed histologically identifiable prostatic epithelium by day 8. mRNA expression for pluripotency markers (SSEA-1, Oct3/4, Nanog and Sox2) was not detected in the grafts at any time points tested. mRNA expression of p63, a marker of basal prostatic cells, was detected by day 4 in tissue recombinants. Basal cells are prostatic stem cells and p63 is required for prostatic development. Thus, these results are consistent with the hypothesis that stem/progenitor spermatogonia are directly differentiating into prostatic epithelial cells expressing NKX3.1 and p63 without transiting through an embryonic stem cell-like stage and expressing pluripotency markers. Supported by The Billie A. Field Endowment, University of Illinois (PSC). (platform)

Soy-based infant formulas are widely used in the United States and some other countries. These formulas contain high levels of the estrogenic isoflavone genistein, leading to concern that neonatal genistein exposure could cause acute and/or long-term adverse effects on reproductive and other organs. However, previous work to assess genistein effects in rodent models has not typically replicated the route of delivery and/or serum genistein concentrations reported for soy formula-fed human infants. Our objective was to develop a mouse model that more closely mimics the oral genistein exposure and total serum genistein concentrations observed in soy formula-fed infants. Mouse pups were dosed orally with genistein in a soy formula-corn oil emulsion from Postnatal Day (PND) 1 to PND 5, then effects on reproductive and nonreproductive organs were assessed after dosing and during subsequent development. Neonatal treatment resulted in changes both at the completion of dosing (PND 5) and in adult animals. At PND 5, neonatal genistein treatment caused increased relative uterine weight and down-regulation of progesterone receptor in uterine epithelia. Estrogenic effects of genistein were also seen in the neonatal ovary and thymus, which had an increase in the incidence of multioocyte follicles (MOFs) and a decrease in thymic weight relative to body weight, respectively. The increased incidence of MOFs persisted into adulthood for neonatally treated genistein females, and estrous cycle abnormalities were seen at 6 mo of age despite normal fertility in these mice. The immediate and long-term effects in this neonatal animal model raise concerns that high serum concentrations of genistein are estrogenic and could potentially impact the development of human infants fed soy formula.

Spermatogonial stem cells are the only stem cells in the body that transmit genetic information to offspring. Although growth factors responsible for self-renewal of these cells are known, the factors and mechanisms that attract and physically maintain these cells within their microenvironment are poorly understood. Mice with targeted disruption of Ets variant gene 5 (Etv5) show total loss of stem/progenitor spermatogonia following the first wave of spermatogenesis, resulting in a Sertoli cell-only phenotype and aspermia. Microarray analysis of primary Sertoli cells from Etv5 knockout (Etv5(-/-)) versus wildtype (WT) mice revealed significant decreases in expression of several chemokines. Chemotaxis assays demonstrated that migration of stem/progenitor spermatogonia toward Etv5(-/-) Sertoli cells was significantly decreased compared to migration toward WT Sertoli cells. Interestingly, differentiating spermatogonia, spermatocytes, and round spermatids were not chemoattracted by WT Sertoli cells, whereas stem/progenitor spermatogonia showed a high and significant chemotactic index. Rescue assays using recombinant chemokines indicated that C-C-motif ligand 9 (CCL9) facilitates Sertoli cell chemoattraction of stem/progenitor spermatogonia, which express C-C-receptor type 1 (CCR1). In addition, there is protein-DNA interaction between ETV5 and Ccl9, suggesting that ETV5 might be a direct regulator of Ccl9 expression. Taken together, our data show for the first time that Sertoli cells are chemoattractive for stem/progenitor spermatogonia, and that production of specific chemokines is regulated by ETV5. Therefore, changes in chemokine production and consequent decreases in chemoattraction by Etv5(-/-) Sertoli cells helps to explain stem/progenitor spermatogonia loss in Etv5(-/-) mice. STEM CELLS 2010; 28: 1882-1892

Uterine receptivity to embryo implantation depends on appropriate progesterone (P4) and estrogen stimulation. P4 rapidly stimulates production of the morphogen Indian hedgehog (IHH) in murine uterine epithelium as well as downstream molecules in the hedgehog pathway such as Patched homolog 1 (PTCH1) and nuclear receptor subfamily 2, group F, member 2 (NR2F2) in uterine stroma. Studies using IHH-null mice indicate that IHH is obligatory for the normal P4 response in the uterus. To determine whether IHH induction in uterine epithelium is mediated through P4 receptor (PR) in epithelium (E) and/or stroma (S), we produced tissue recombinants using uteri from neonatal PR knockout (ko) mice and wild-type (wt) mice containing PR in S and/or E or lacking PR altogether using a tissue recombinant methodology and assessed their response to P4. In tissue recombinants containing wt-S (wt-S + wt-E and wt-S + ko-E), P4 induced Ihh mRNA expression at 6 h that was 6-fold greater than in oil-treated controls (P < 0.05; n = 6) in both types of tissue recombinants despite the absence of epithelial PR in wt-S + ko-E grafts. Conversely, Ihh mRNA expression was unaffected by P4 in ko-S + ko-E and ko-S + wt-E grafts despite epithelial PR expression in the latter. Nr2f2 and Ptch1 mRNA expression was similar in that it was stimulated by P4 only in recombinants containing stromal PR. These results indicate that stromal PR is both necessary and sufficient for P4 stimulation of epithelial IHH as well as downstream events such as PTCH1 and NR2F2 increases in stroma.

Pluripotent stem cells have great clinical potential for tissue regeneration/repair in humans. The use of embryonic stem (ES) cells is ethically controversial, leading to searches for other sources of pluripotent stem cells. Testicular spermatogonial stem cells (SSCs) produce the spermatogenic lineage. Under in vitro conditions, SSCs have the ability to give rise to pluripotent ES-like cells. We hypothesized that stem/progenitor spermatogonia could directly transdifferentiate into different tissue types if they were recombined with inductive mesenchymes from fetal/neonatal organs using a tissue separation/recombination methodology and grown in vivo. Green fluorescent protein transgenic mice were used to track cell lineages. Our results indicate that stem/progenitor spermatogonia recombined with the appropriate mesenchyme can directly transdifferentiate in vivo into tissues of all germ layers, including prostatic, uterine, and skin epithelium. In addition, transdifferentiated tissue expressed molecular, histological, and functional markers of the appropriate epithelium. The ability of stem/progenitor spermatogonia to directly generate various epithelia emphasizes their clinical potential, and if adult human SSCs have similar properties, this may have applications in human regenerative medicine. STEM CELLS 2009; 27: 1666-1675

Uterine gland development in mice begins on day 5 postnatal, when rapidly proliferating luminal epithelium begins forming epithelial buds that invade the underlying stroma, and is complete by day 60 postnatal. Gland development is obligatorily dependent on estrogen receptor alpha, as mice lacking this receptor lack uterine glands. Progesterone (P) inhibits many processes stimulated by estrogens, and blocks mitogenic effects of estrogen on uterine epithelium. This ability of P to inhibit estrogen-induced epithelial proliferation can be exploited to inhibit uterine gland development. For example, progestin treatment of neonatal ewes for 8 weeks from birth has been shown to produce adult sheep that lack uterine glands and are infertile. The goals of this study were to determine if there is a window of susceptibility in mice to a P-mediated inhibition of uterine gland development, and to determine whether early P treatment abolishes uterine gland development in mice as it does in sheep. Mice (C75Bl/6) were injected daily with P (40 ug/g/day) dissolved in ethanol: corn oil (1:9) or vehicle alone from days 3-9, 3-5, 3-7, 5-7, 5-9, 7-9 and 10-16 postnatally (n=12-20/group). Uteri at days 10 and 20 were examined histologically and by Ki-67 immunostaining to assess gland development and cell proliferation, or the intact epithelium was separated from the surrounding stroma by enzymatic dissociation to examine the extent of gland development. Treatment from days 3-9, 5-9 or 3-7 totally abolished gland development at day 10. Critically, the lack of glands in these mice persisted at day 20, indicating that gland morphogenesis did not resume after P treatment during these critical periods. Treatment from days 3-5, 5-7, 7-9 or 10-16 produced partial inhibition of gland development relative to controls at both day 10 and 20. Epithelial proliferation at day 10 was minimal following P treatment from days 3-9 or 5-9, with a labeling index (LI) of under 5%. In contrast, epithelial proliferation was marked in control uteri, with an epithelial LI of 70-80%. Glandular epithelium in control mice was proliferating rapidly, while histological examination confirmed results obtained with enzymatic separation of intact epithelia that no glands developed following P treatment from days 3-7, 3-9 or 5-9. Stromal proliferation was significant (LI=25-30%) in controls at day 10, and P treatment (days 3-9 and 5-9) further stimulated stromal proliferation (LI=35-40%). In contrast to day 10, epithelial and stromal proliferation at day 20 were both much lower in controls. Furthermore, both stromal and epithelial proliferation were comparable to controls in all P-treated groups at day 20, indicating that inhibitory effects on epithelial proliferation at day 10 following neonatal P treatment did not persist until day 20. Results indicate that a brief P treatment (as little as 5 days) during critical neonatal periods totally blocks initiation of murine uterine gland development. Shorter treatments or treatments at ages outside of the critical window retard but do not abolish gland development. Importantly, even though inhibitory effects on epithelial proliferation are reversed following cessation of P treatment, gland development is not subsequently initiated in mice treated during the critical window of gland genesis. This results in permanent losses of uterine glands, despite the recovery of normal epithelial proliferation after P treatment.

Mice that are ets variant gene 5 (ETV5) null (Etv5(-/-)) undergo the first wave of spermatogenesis but lose all spermatogonial stem cells (SSCs) during this time. The SSC loss in Etv5(-/)-mice begins during the neonatal period, suggesting a role for ETV5 in SSC self-renewal during this period. Herein, we show that Etv5 mRNA was present in perinatal mouse testis and that ETV5 was expressed in fetal Sertoli cells and by germ cells and Sertoli cells during the neonatal period. Transplantation of Etv5(-/)-germ cells failed to establish spermatogenesis in W/W-v mice testes, indicating that germ cell ETV5 has a key role in establishment or self-renewal of transplanted SSCs. The SSC self-renewal is stimulated by glial cell-derived neurotrophic factor (GDNF) acting through the RET/GDNF family receptor alpha 1 (GFRA1) receptor complex in SSCs. Immunohistochemistry, quantitative PCR, and laser capture microdissection revealed decreased RET mRNA and protein expression in spermatogonia of neonatal Etv5-/-mice by Postnatal Days 4-8, indicating that disrupted GDNF/RET/GFRA1 signaling may occur before initial spermatogonial stem/progenitor cell decrease. Etv5(-/)-spermatogonia had reduced proliferation in vivo and in vitro. Decreased cell proliferation may cause the observed decreases in the number of type A spermatogonia (Postnatal Day 17) and daily sperm production (Postnatal Day 30) in Etv5(-/)-mice, indicating quantitative impairments in the first wave of spermatogenesis. In conclusion, ETV5 is expressed beginning in fetal Sertoli cells and can potentially have effects on neonatal Sertoli cells and germ cells. In addition, ETV5 has critical effects on neonatal spermatogonial proliferation, which may involve impaired signaling through the RET receptor.

Ets variant gene 5 (ETVS) and glial cell-derived neurotrophic factor (GDNF) are produced in Sertoli cells and required for maintenance and self-renewal of spermatogonial stem cells (SSCs) in mice. Fibroblast growth factors (FGFs) have been reported to stimulate Etv5 mRNA expression, and FSH was shown to stimulate Gdnf mRNA in Sertoli cell cultures, but there is no other information on factors that regulate these key Sertoli cell proteins necessary for stem cell maintenance. In this study, we investigated regulation of ETV5 and GDNF using the TM4 murine Sertoli cell line. FGF2 stimulated a time- and dose-dependent increase in Etv5 mRNA expression, with a maximal 8.3-fold increase at 6 h following 25 ng/ml FGF2 treatment. This FGF2 dose also stimulated Gdnf mRNA at 48 h. FGF2 effects on Etv5 and Gdnf mRNA were partially mediated through mitogen-activated protein kinase (MAPK) and phosphatidyl mositol 3-kinase (PI3K)-signaling cascades. Specific inhibitors of MAPK (PD98059) and PI3K (wortmannin) pathways reduced Etv5 and Gdnf mRNA expression in FGF2-treated cells. Epidermal growth factor (EGF) stimulated Etv5 mRNA but not Gdnf mRNA. TNF alpha and IL-1 beta stimulated Gdnf mRNA, but had no effect on Etv5 mRNA. Other hormonal regulators of Sertoli cells such as testosterone, triiodothyronine and activin A did not affect Etv5 or Gdnf mRNA expression. Results with primary Sertoli cell cultures confirmed findings obtained with the TM4 cell line, validating the use of the TM4 model to examine regulation of Etv5 and Gdnf mRNA expression. In conclusion, we have identified common and unique pathways that regulate Etv5 and Gdnf mRNA in Sertoli cells, and FGFs are emerging as key regulators of the Sertoli cell proteins that control SSCs. (c) 2007 Elsevier Inc. All rights reserved.