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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 non-reproductive 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.

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

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. Disclosure of potential conflicts of interest is found at the end of this article.

Ets variant gene 5 (ETV5) 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 inositol 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. TNFalpha and IL-1beta 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.

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.

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 wild-type (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.

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.

The simplicity and specialization of the cell motility machinery of Ascaris sperm provides a powerful system in which to probe the basic molecular mechanism of amoeboid cell motility. Although Ascaris sperm locomotion closely resembles that seen in many other types of crawling cell, movement is generated by modulation of a cytoskeleton based on the major sperm protein (MSP) rather than the actin present in other cell types. The Ascaris motility machinery can be studied conveniently in a cell-free in vitro system based on the movement of plasma membrane vesicles by fibres constructed from bundles of MSP filaments. In addition to ATP, MSP and a plasma membrane protein, reconstitution of MSP motility in this cell-free extract requires cytosolic proteins to orchestrate the site-specific assembly and bundling of MSP filaments that generates locomotion. One of these proteins, MFP2, accelerates the rate of movement in this assay. Here, we describe crystal structures of two isoforms of MFP2 and show that both are constructed from two domains that have the same fold based on a novel, compact beta sheet arrangement. Patterns of conservation observed in a structure-based analysis of MFP2 sequences from different nematode species identified regions that may be putative functional interfaces involved both in interactions between MFP2 domains and also with other components of the sperm motility machinery. Analysis of the growth of fibres in vitro in the presence of added MFP2 indicated that MFP2 increases the rate of locomotion by enhancing the effective rate of MSP filament polymerization. This observation, together with the structural data, suggests that MFP2 may function in a manner analogous to formins in actin-based motility.

Although Ascaris sperm motility closely resembles that seen in many other types of crawling cells, the lamellipodial dynamics that drive movement result from modulation of a cytoskeleton based on the major sperm protein (MSP) rather than actin. The dynamics of the Ascaris sperm cytoskeleton can be studied in a cell-free in vitro system based on the movement of plasma membrane vesicles by fibers constructed from bundles of MSP filaments. In addition to ATP, MSP, and a plasma membrane protein, reconstitution of MSP motility in this cell-free extract requires cytosolic proteins that orchestrate the site-specific assembly and bundling of MSP filaments that generates locomotion. Here, we identify a fraction of cytosol that is comprised of a small number of proteins but contains all of the soluble components required to assemble fibers. We have purified two of these proteins, designated MSP fiber proteins (MFPs) 1 and 2 and demonstrated by immunolabeling that both are located in the MSP cytoskeleton in cells and in fibers. These proteins had reciprocal effects on fiber assembly in vitro: MFP1 decreased the rate of fiber growth, whereas MFP2 increased the growth rate.