Sertoli cells isolated from the adult mouse and human testis resume proliferation in culture. After 20 days of culture in Dulbecco modified Eagle medium/Ham F12 (DMEM/F12) medium containing 5%% fetal calf serum, about 36%% of the mouse Sertoli cells, identified by their immunohistochemical staining for the Sertoli cell marker vimentin, incorporated bromodeoxyuridine (BrdU). The renewed proliferation was associated with a 70%% decrease in expression of the cell cycle inhibitor CDKN1B (P27^kip1) and a 2-fold increase in the levels of the proliferation inducer ID2. In vivo, the balance between cell cycle inhibitors and inducers probably is such that the cells remain quiescent, whereas in culture the balance is disturbed such that Sertoli cells start to proliferate again. The renewed proliferative activity of Sertoli cells in culture was further confirmed by double staining for BrdU and the Sertoli cell marker clusterin (CLU), showing about 25%% of the CLU-positive Sertoli cells to be also positive for BrdU after 13 days of culture. Radiobiologically, Sertoli cells are also different from other quiescent somatic cells in the testis because they express several DNA repair proteins (XRCC1, PARP1, and others). Indeed, a comet assay on irradiated Sertoli cells revealed a 70%% reduction in tail length and tail moment at 20 h after irradiation. Hence, Sertoli cells repair DNA damage, whereas other quiescent somatic testicular cells do not. This repair may be accomplished by nonhomologous end joining via XRCC1 and PARP1. In conclusion, cell kinetic and radiobiological data indicate that Sertoli cells more resemble arrested proliferating cells than the classic postmitotic and terminally differentiated somatic cells that they have always been assumed to be.

During mammalian fertilization, the contact between sperm and egg triggers increases in intracellular Ca² concentration ([Ca² ]_i) in sperm. Voltage-gated Ca² channels (Ca_Vs) are believed to mediate the initial phase of [Ca² ]_i increases in sperm induced by egg coat (zona pellucida [ZP]) glycoproteins, while store depletion-activated Ca² entry is thought to mediate the sustained phase. Using patch-clamp recording and Ca² imaging, we show herein that Ca_V channel currents, while found in spermatogenic cells, are not detectable in epididymal sperm and are not essential for the ZP-induced [Ca² ]_i changes. Instead, CATSPER channels localized in the distal portion of sperm (the principal piece) are required for the ZP-induced [Ca² ]_i increases. Furthermore, the ZP-induced [Ca² ]_i increase starts from the sperm tail and propagates toward the head.

Expression of GJA1 (commonly known as connexin43 or Cx43), a major myometrial gap junction protein, is upregulated before the onset of delivery, suggesting an essential role for Cx43-mediated gap junctional intercellular communication (GJIC) in normal uterine contraction during parturition. To determine how a disease-linked Cx43 mutation affects myometrial function, we studied a mutant mouse model carrying an autosomal dominant mutation (Gja1^Jrt) in the gene encoding Cx43 that displays features of the human genetic disease oculodentodigital dysplasia. We found that Cx43 level, specifically the phosphorylated species of the protein, is significantly reduced in the myometrium of the mutant mice (Gja1^Jrt/ ), as revealed by Western blotting and immunostaining. Patch-clamp electrophysiological measurements demonstrated that coupling between myometrial smooth muscle cells is reduced to <15% of wild-type, indicating that the mutant protein acts dominantly on its wild-type counterpart. The phosphorylated species of Cx43 in the mutant myometrium failed to increase prior to parturition as well as in response to exogenous estrogen. Correspondingly, in vitro experiments with uterine strips revealed weaker contraction of the mutant myometrium and reduced responsiveness to oxytocin, providing an explanation for the prolonged gestation and presence of suffocated fetuses in the uteri that were observed in some of the mutant mice. We conclude that the Gja1^Jrt mutation has a dominant-negative effect on Cx43 function in the myometrium, severely reducing GJIC, leading to impaired parturition.

During embryo implantation, a complex dialog exists between the mother and the fetus. However, little is known about the molecules that participate in this process. Among various factors secreted at the maternal-fetal interface, the adipose tissue-derived leptin is now considered a placental growth factor. Adiponectin is another adipocyte-derived signaling molecule known to exert antiproliferative effects in various cell types. In this work, we studied adiponectin sensitivity and effects on JEG-3 and BeWo choriocarcinoma cell lines. First, we showed that JEG-3 and BeWo cells express the specific adiponectin receptors ADIPOR1 and ADIPOR2 and respond to human recombinant adiponectin by AMP-activated protein kinase (PRKA, also known as AMPK) activation. Second, we demonstrated that adiponectin induces a reduction in cell number and in [³H]-thymidine incorporation, demonstrating that adiponectin has antiproliferative effects on trophoblastic cells. Furthermore, these effects of adiponectin seem to be, at least in part, mediated by the mitogen-activated protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K) signaling pathways. We describe herein the direct effects of adiponectin in the control of trophoblastic cell proliferation.

Interleukin 10 (IL10) is associated with maternal immunotolerance. IL10 also down-regulates decidual cell tissue factor expression, the main molecule triggering coagulation activation: this antithrombotic effect may protect the umbilicoplacental vasculature from the 10th wk of gestation onward. IL10 down-regulation may thus dispose to early pregnancy loss (PL) due to maternal immunotolerance defect or late pregnancy failure due to placental vascular insufficiency. IL10 gene promoter polymorphisms associated with cytokine down-regulation may help to identify the actual and probable mechanisms of IL10 modulation in pregnancy outcomes. We investigated the following four IL10 promoter polymorphisms associated with IL10 down-regulation: two single-nucleotide polymorphisms rs1800871 and rs1800872 and two polymorphic CA repeat microsatellites IL10 X78437.2:g8134CA(14_29) and IL10 X78437.2:g.5325CA(11_15). Each microsatellite was analyzed as a biallelic polymorphism. Based on a review of the literature, we define a short allele and a long allele for each microsatellite. We compared their frequencies in early PL occurring before 10 wk of amenorrhea (n = 342) and in PL occurring later on (n = 123). The mutated alleles rs1800871T (odds ratio, 3.083; 95% confidence interval, 1.984–4.792) and rs1800872A (odds ratio, 3.013; 95% confidence interval, 1.924–4.719) were associated with early PL. The haplotype rs1800872A/rs1800871T/X78437.2:g.8134CA[14_25]/X78437.2:g.5325CA[11_13], which includes the two mutated alleles, was significantly associated with the risk of early PL in a dose-dependent manner. Positivity for one haplotype was significantly associated with a 5.6-fold increase in the risk of early pregnancy failure, and positivity for two haplotypes was associated with an 8-fold increase in risk. In women with PL, some polymorphisms of the IL10 gene promoter seem to be constitutional risk factors for early (embryonic) pregnancy failure.

Fetal adaptations to periods of substrate deprivation can result in the programming of glucose intolerance, insulin resistance, and metabolic dysfunction in later life. Placental insufficiency can be associated with either sparing or sacrifice of fetal liver growth, and these different responses may have different metabolic consequences. It is unclear what intrahepatic mechanisms determine the differential responses of the fetal liver to substrate restriction. We investigated the effects of placental restriction (PR) on liver growth and the hepatic expression of SLC2A1, IGF1, IGF2, IGF1R, IGF2R, PPARGC1A, PPARA, PRKAA1, PRKAA2, PCK2, and HSDL1 mRNA in fetal sheep at 140–145 days of gestation. A mean gestational arterial partial pressure of oxygen less than 17 mmHg was defined as hypoxic, and a relative liver of weight more than 2 SD below the mean liver weight of controls was defined as reduced liver growth. Fetuses therefore were defined as control-normoxic (C-N; n = 9), PR-normoxic (PR-N; n = 7), PR-hypoxic (PR-H; n = 8), or PR-hypoxic reduced liver growth (PR-H RLG; n = 4). Hepatic SLC2A1 mRNA expression was highest (P < 0.05) in the PR-H fetuses, in which liver growth was maintained. Expression of IGF1 mRNA was decreased (P < 0.05) only in the PR-H RLG group. Hepatic expression of HSDL1, PPARGC1A, and PCK2 mRNA also were increased (P < 0.05) in the PR-H RLG fetuses. The present study highlights that intrahepatic responses to fetal substrate restriction may exist that protect the liver from decreased growth and, potentially, from a decreased responsiveness to the actions of insulin in postnatal life.

A surge of gonadotropin-releasing hormone (GnRH) release from the brain triggers the luteinizing hormone (LH) surge that causes ovulation. The GnRH surge is initiated by a switch in estradiol action from negative to positive feedback. Estradiol signals critical for the surge are likely transmitted to GnRH neurons at least in part via estradiol-sensitive afferents. Using an ovariectomized estradiol-treated (OVX E) mouse model that exhibits daily LH surges, we examined changes in glutamate transmission to GnRH neurons during negative feedback and positive feedback. Spontaneous glutamatergic excitatory postsynaptic currents (EPSCs) mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/kainate receptors (AMPA/KA Rs) or N-methyl-d-aspartate receptors (NMDARs) were recorded in GnRH neurons from OVX E and OVX mice. There were no diurnal changes in the percentage of GnRH neurons from OVX mice exhibiting EPSCs. In cells from OVX E mice, the profile of AMPA/KA R-mediated and NMDAR-mediated EPSCs showed changes dependent on time of day. Comparison of AMPA/KA R-mediated EPSC frequency in OVX E and OVX cells showed that estradiol suppressed transmission during negative feedback but had no effect during positive feedback. Tetrodotoxin treatment to block action potential firing did not affect AMPA/KA R-mediated EPSC frequency in OVX cells during negative feedback or in OVX E cells during positive feedback, suggesting that estradiol-induced suppression of glutamate transmission may be primarily due to activity-independent changes. The diurnal removal of estradiol-induced suppression of AMPA/KA R-mediated glutamate transmission to GnRH neurons during positive feedback suggests that the primary role for estradiol-induced changes in glutamate transmission may be in mediating negative feedback.

Human endometrium is a highly regenerative tissue undergoing more than 400 cycles of growth, differentiation, and shedding during a woman's reproductive years. Endometrial regeneration is likely mediated by adult stem/progenitor cells. This study investigated key stem cell properties of individual clonogenic epithelial and stromal cells obtained from human endometrium. Single-cell suspensions of endometrial epithelial or stromal cells were obtained from hysterectomy tissues from 15 women experiencing normal menstrual cycles, and were cultured at clonal density (10 cells/cm²) or limiting dilution. The adult stem cell properties—self-renewal, high proliferative potential, and differentiation of single epithelial and stromal cells—were assessed by harvesting individual colonies and undertaking serial clonal culture, serial passaging, and culture in differentiation-induction media, respectively. Lineage differentiation markers were examined by RT-PCR, immunocytochemistry, and flow cytometry. Rare single human endometrial EpCAM epithelial cells and EpCAM⁻ stromal cells demonstrated self-renewal by serially cloning >3 times and underwent >30 population doublings over 4 mo in culture. Clonally derived epithelial cells differentiated into cytokeratin gland-like structures in three dimensional culture. Single stromal cells were multipotent, as their progeny differentiated into smooth muscle cells, adipocytes, chondrocytes, and osteoblasts. Stromal clones expressed mesenchymal stem cell (MSC) markers ITGB1 (CD29), CD44, NT5E (CD73), THY1 (CD90), ENG (CD105), PDGFRB (CD140B), MCAM (CD146) but not endothelial or hemopoietic markers PECAM1 (CD31), CD34, PTPRC (CD45). Adult human endometrium contains rare epithelial progenitors and MSCs, likely responsible for its immense regenerative capacity, which may also have critical roles in the development of endometriosis and endometrial cancer. Human endometrium may provide a readily available source of MSCs for cell-based therapies.

In many species, the reproductive centers of the brain are profoundly affected by sociosexual stimuli. This is particularly evident in female ungulates such as sheep, in which exposure to males switches them from reproductively quiescent to fertile. In two experiments with female sheep, we tested whether the brain centers that control gonadotropin-releasing hormone (GnRH) neuronal activity respond differentially to “novel” vs. familiar males and whether the neuroendocrine response is associated with increased cell proliferation in the hippocampus, a site associated with memory formation. In experiment 1, groups of 10 female sheep that had previously been habituated to males for 3 mo were re-exposed to familiar males or were exposed to novel males. Only the novel males increased luteinizing hormone (LH) pulse frequency, indicating stimulation of GnRH neuronal activity. In experiment 2, groups of six female sheep were injected with bromodeoxyuridine (BrdU) and then maintained in isolation from males or exposed to novel males. Two days later, the hippocampus and hypothalamus were removed and processed for fluorescence immunohistochemistry. Again, exposure to males increased LH pulse frequency. Most important, male exposure also doubled the number of BrdU-positive cells in the dentate gyrus of the hippocampus. No BrdU-positive cells were detected in the hypothalamus. We conclude that the stimulus from novel males switches on the reproductive centers of the brain of female sheep and rapidly doubles the rate of cell proliferation in the hippocampus. The rapidity of this response contrasts with rodents, in which several days of exposure to male pheromones seem necessary for an effect on neurogenesis.

Prenatal androgens masculinize postnatal reproductive neuroendocrine function and behavior in sheep. Testosterone treatment of pregnant ewes during midgestation masculinizes sexual behavior and luteinizing hormone secretion in female lambs, presumably in part via aromatization and estrogen receptor (ESR) binding in the brain. We hypothesized that male and female sheep also differ in the number and distribution of ESR-containing neurons. If so, ESR expression should be sensitive to prenatal hormones delivered exogenously or in situ. ESR alpha (ESR1) was compared by immunocytochemistry in male and female lambs at the end of gestation, as well as in fetal females exposed prenatally to testosterone or dihydrotestosterone. ESR1-positive neurons were abundant in the posteromedial bed nucleus of the stria terminalis (BSTpm), medial preoptic area (MPOA), posterior medial amygdaloid nucleus (MeP), amygdalohippocampal area (AHi), ventromedial hypothalamic nuclei (VMH), and arcuate hypothalamic nuclei (ARC). In females, the ARC had the largest number of stained cells (mean ± SEM, 475.6 ± 57.4 cells/0.173 mm²), while staining intensity was greatest in the MPOA (mean ± SEM gray level, 31.3 ± 5.3). The mean ± SEM integrated gray level (IGL) was high in the ARC (0.63 ± 0.13) and in the MPOA (0.51 ± 0.08). The mean ± SEM IGL was low in the MeP (0.31 ± 0.10) and in the BSTpm (0.21 ± 0.06), while it was intermediate in the AHi (0.36 ± 0.10) and in the VMH (0.37 ± 0.07). ESR immunostaining was not significantly different in male and female fetal lambs, nor in females fetuses exposed prenatally to androgens (P > 0.05). However, ESR1 staining was significantly increased in the ARC, MPOA, and AHi of adult rams vs. adult ewes. These results suggest that brain ESR immunoreactivity in fetal lambs is unlikely to account for postnatal sex differences in reproductive function. Instead, sex differences in ESR emerge postnatally.

In this study, it was hypothesized that progesterone (P4) acts as a survival factor primarily by actions of the classic nuclear progesterone receptor (PGR) signaling pathway in rat periovulatory granulosa cells. Granulosa cells were isolated from immature female rats primed with equine chorionic gonadotropin/human chorionic gonadotropin and treated in vitro with PGR antagonists. As little as 10 nM of two different PGR antagonists (Org 31710 and RU 486) increased apoptosis measured as caspase 3/7 activity, which was reversed by cotreatment with the progestin R5020. Concurrently, P4 synthesis was decreased. Inhibition of P4 synthesis by cyanoketone similarly induced apoptosis but required greater inhibition of P4 synthesis than that seen after treatment with PGR antagonists. Therefore, the induction of apoptosis by PGR antagonists cannot be explained by decreased P4 synthesis alone. Low concentrations of R5020 also completely reversed the effects of cyanoketone. Inhibition of P4 synthesis was more effective in inducing apoptosis than treatment with PGR antagonists. However, cotreatment with PGR antagonists protected cells from the additional effects of cyanoketone, indicating partial agonist effects of the antagonists and a dominating role for PGR in P4-mediated regulation of apoptosis. Progesterone receptor membrane component 1 (PGRMC1) was expressed in granulosa cells; however, an anti-PGRMC1 antibody did not induce apoptosis in periovulatory granulosa cells. Neither anti-PGRMC1 nor P4 or cyanoketone affected apoptosis of immature granulosa cells. In conclusion, we show that P4 regulates apoptosis in periovulatory granulosa cells by acting via the classic nuclear receptor.

Peroxiredoxin 2 (PRDX2) is a highly efficient redox protein that neutralizes hydrogen peroxide, resulting in protection of cells from oxidative damage and in regulation of peroxide-mediated signal transduction events. The oxidized form of PRDX2 is reverted back to the reduced form by the thioredoxin system. In the present study, we investigated the presence of PRDX2 in mouse and boar spermatozoa and in mouse spermatids using proteomic techniques and immunocytochemistry. Sperm and spermatid extracts displayed a 20-kDa PRDX2 band on Western blotting. PRDX2 occurred as a Triton-soluble form in spermatids and as a Triton-insoluble form in mature spermatozoa. Boar seminiferous tubule extracts were immunoprecipitated with PRDX2 antibody and separated by SDS-PAGE. Peptide mass fingerprinting by matrix-assisted laser desorption ionization-time of flight (TOF) and microsequencing by nanospray quadrupole-quadrupole TOF tandem mass spectrometry revealed the presence of PRDX2 ions in the immunoprecipitated band, along with sperm mitochondria-associated cysteine-rich protein, cellular nucleic acid-binding protein, and glutathione peroxidase 4. In mouse spermatocytes and spermatids, diffuse labeling of PRDX2 was observed in the cytoplasm and residual bodies. After spermiation, PRDX2 localization became confined to the mitochondrial sheath of the sperm tail midpiece. Boar spermatozoa displayed similar PRDX2 localization as in mouse spermatozoa. Boar spermatozoa with disrupted acrosomes expressed PRDX2 in the postacrosomal sheath region. Peroxidase enzyme activity of boar sperm extracts was evaluated by estimating the rate of NADPH oxidation in the presence or absence of a glutathione depletor (diethyl maleate) or a glutathione reductase inhibitor (carmustine). Diethyl maleate partially inhibited peroxidase activity, whereas carmustine showed an insignificant effect. These observations suggest that glutathione and glutathione reductase activity contribute only partially to the total peroxidase activity of the sperm extract. While the specific role of PRDX2 in the total peroxidase activity of sperm extract is still an open question, the present study for the first time (to our knowledge) shows the presence of PRDX2 in mammalian spermatozoa. Peroxidase activity in sperm extracts is not due to the glutathione system and therefore possibly involves PRDX2 and other peroxiredoxins.

In most mammals, the expression of SRY (sex-determining region on the Y chromosome) initiates the development of testes, and thus determines the sex of the individual. However, despite the pivotal role of SRY, its mechanism of action remains elusive. One important missing piece of the puzzle is the identification of genes regulated by SRY. In this study we used chromatin immunoprecipitation to identify direct SRY target genes. Anti-mouse SRY antibody precipitated a region 7.5 kb upstream of the transcriptional start site of cerebellin 4 precursor (Cbln4), which encodes a secreted protein. Cbln4 is expressed in Sertoli cells in the developing gonad, with a profile mimicking that of the testis-determining gene SRY-box containing gene 9 (Sox9). In transgenic XY mouse embryos with reduced Sox9 expression, Cbln4 expression also was reduced, whereas overexpression of Sox9 in XX mice caused an upregulation of Cbln4 expression. Finally, ectopic upregulation of SRY in vivo resulted in ectopic expression of Cbln4. Our findings suggest that both SRY and SOX9 contribute to the male-specific upregulation of Cbln4 in the developing testis, and they identified a direct in vivo target gene of SRY.

We previously showed that fetal adrenal fetal zone growth was increased and the number of follicles in the fetal ovary reduced in baboons in which estradiol was suppressed by treatment with the aromatase inhibitor letrozole between mid and late gestation periods. Because adrenal/ovarian development was restored in animals treated with letrozole and estradiol, and both tissues express estrogen receptor, we proposed that estrogen regulates fetal adrenal/ovary development via a direct action. However, because prolactin can modulate fetal adrenal and adult pituitary/ovarian function, the current study determined whether estrogen action involved estradiol-regulated changes in fetal prolactin/luteinizing hormone (LH) expression. Fetal prolactin levels and the number of prolactin-positive fetal pituitary cells (per 0.37 mm²) were increased (P < 0.01) between mid (6 ± 1 ng/ml; 15.8 ± 2.4) and late (257 ± 28 ng/ml; 57.3 ± 5.1) gestation, reduced (P < 0.01) in late-gestation fetuses in which estradiol was suppressed (>95%) by letrozole (61 ± 11 ng/ml; 19.3 ± 2.0), and minimally but not significantly increased by letrozole and estradiol (99 ± 11 ng/ml; 32.7 ± 5.2). In contrast, the number of LH-positive fetal pituitary cells decreased (P < 0.01) between mid (48.8 ± 9.5) and late (17.4 ± 3.2) gestation, remained elevated (P < 0.01) in estrogen-suppressed animals (56.6 ± 5.1), and was partially but not significantly decreased by letrozole-estradiol (28.8 ± 5.2). We conclude that estrogen regulates fetal pituitary prolactin and LH expression and fetal prolactin levels. However, because prolactin and LH expressions in estrogen-suppressed fetuses were inversely related to previously demonstrated changes in adrenal/ovarian development, we propose that estrogen regulates the fetal ovary and adrenal gland directly and not via action on the fetal pituitary gland.

The availability of specific neutral and acidic amino acids in the uterine lumen of ewes increased significantly during the peri-implantation period, but mechanisms for their transport into the uterine lumen and uptake by conceptuses are not established in any species. In this study, effects of pregnancy, progesterone (P4), and interferon tau (IFNT) on expression of neutral and acidic amino acid transporters in uteri of cyclic and pregnant ewes and conceptuses were studied. SLC1A2, SLC1A3, SLC3A1, SLC6A14, SLC6A19, SLC7A6, SLC38A3, and SLC38A6 mRNAs were only weakly expressed in the ovine endometrium. However, SLC1A4, SLC1A5, SLC7A8, and SLC43A2 mRNAs were detectable in uterine luminal epithelia (LE), superficial glandular epithelia (sGE), and/or glandular epithelia (GE). SLC1A1 and SLC7A5 mRNAs were most abundant in LE/sGE and GE. SLC1A3 and SLC38A4 mRNAs were most abundant in uterine stroma. SLC38A6 mRNA was detected only in cells with a stromal distribution suggesting immune lineage. SLC1A5 mRNA was expressed primarily in LE/sGE and stromal cells, and it was more abundant in uteri of pregnant ewes (day × status interaction; P < 0.05). Furthermore, P4 induced and IFNT further stimulated SLC1A5 expression in LE/sGE. Endometrial SLC1A1, SLC7A5, and SLC43A2 mRNAs demonstrated both temporal and cellSLCspecific changes. Several mRNAs were detectable in trophectoderm (SLC6A19, SLC7A5, SLC7A6, and SLC43A2), while others were more abundant in endoderm (SLC1A4, SLC1A5, SLC6A19, SLC7A5, SLC7A6, SLC7A8, and SLC43A2) of conceptuses. These results document coordinate changes in expression of transporters that are likely responsible for increases in amounts of neutral and acidic amino acids in the uterine lumen to support conceptus growth, development, and survival.

Glucocorticoid excess in utero inhibits fetal growth and programs adverse outcomes in adult offspring. Access of maternal glucocorticoid to the glucocorticoid receptor (NR3C1) in the placenta and fetus is regulated by metabolism via the 11beta-hydroxysteroid dehydrogenase (HSD11B) enzymes, as well as multidrug resistance P-glycoprotein (ABCB1)-mediated efflux of glucocorticoids from the syncytiotrophoblast. This study determined expression of genes encoding the two HSD11B isoforms (Hsd11b1 and Hsd11b2), the two ABCB1 isoforms (Abcb1a and Abcb1b), and Nr3c1 in the junctional and labyrinth zones of rat placentas at Days 16 and 22 of normal gestation (Day 23 is term). To assess possible regulation of the Hsd11b and Abcb1 isoforms by glucocorticoids and progesterone, their placental expression was also measured at Day 22 after partial progesterone withdrawal from Day 16 (maternal ovariectomy plus full estrogen and partial progesterone replacement) or after treatment with dexamethasone acetate (1 μg/ml of drinking water from Day 13). Expression of Hsd11b1 mRNA increased in the labyrinth zone (the site of maternal-fetal exchange) from Day 16 to Day 22, whereas that of Hsd11b2 fell dramatically. Consistent with these changes, corticosterone levels increased 10-fold in the labyrinth zone over this period. Expression of both Abcb1a and Abcb1b was markedly higher in the labyrinth zone compared with the junctional zone on both days, consistent with the proposed barrier role of ABCB1 in the placenta. Nr3c1 mRNA expression was similar in the two placental zones at Day 16 but increased 3-fold in the labyrinth zone by Day 22. Partial progesterone withdrawal increased Hsd11b1 mRNA and protein expression in the labyrinth zone but decreased Nr3c1 mRNA expression. These data show that the dynamic expression patterns of the placental HSD11Bs in late gestation are associated with dramatic shifts in placental corticosterone. Moreover, the late gestational rise in labyrinthine Hsd11b1 seems to be driven by the normal prepartum fall in progesterone level.

Recently, F0 embryonic stem (ES) cell mice have been produced by injection of ES cells into eight-cell embryos using either laser- or piezo-assisted injection systems. To simplify the injection procedure, we have optimized the conventional blastocyst injection method, free of laser- or piezo-assisted micromanipulation systems, to produce F0 ES cell pups. To increase the efficiency of producing mice from ES cell injection into eight-cell and blastocyst stage embryos, we have tested: 1) the effect of activating ES cell before injection, 2) the effect of in vitro culture in medium optimized for the survival of both ES cells and embryos, and 3) the effect of transferring the micromanipulated embryos into the oviduct versus into the uterus of CD1 foster mice. Two B6D2 hybrid ES cell lines were used for injection in a multifactorial analysis to evaluate the efficiency of producing live chimeric and F0 ES cell mice. Our results demonstrate that the activation of ES cells and the appropriate culture conditions are crucial parameters influencing the generation of F0 ES cell offspring. Transfer of blastocysts injected with ES cells into the oviduct of 0.5-day postcoitum pseudopregnant females increased the number of live animals with higher chimera proportion. Under these conditions, injections into eight-cell embryos produce a high number of F0 ES mice, and the conventional blastocyst injection method produces a lower number of F0 ES cell pups; however, the efficiency of production of chimeric mice with germline transmission was high. We have developed an economical and efficient technique for producing fully ES cell-derived F0 mice with full germline transmission that can be applied in many laboratories without the use of piezo or laser instruments.

Interferon tau gene (IFNT) is expressed only by mononuclear trophectoderm cells in ruminant ungulates. To our knowledge, its epigenetic regulation and interaction with trophectoderm lineage-specific caudal-related homeobox 2 transcription factor (CDX2) have not been characterized. Herein, we studied differences in chromatin structures and transcription of endogenous bovine IFNT in bovine trophoblast BT-1 and CT-1 cells and in nontrophoblast MDBK cells. Transcripts from endogenous IFNT and CDX2 genes were found in BT-1 and CT-1 cells but not in MDBK cells. Chromatin immunoprecipitation study revealed that CDX2 binding sites exist in proximal upstream regions of IFNT (IFN-tau-c1). Endogenous IFNT transcription in BT-1 cells was increased with CDX2 overexpression but was reduced with short interfering RNA specific for the CDX2 transcript. In chromatin immunoprecipitation studies, histone H3K18 acetylation of IFNT was higher in CT-1 cells than in MDBK cells, while histone H3K9 methylation was lower in CT-1 cells than in nontrophoblast cells. In MDBK cells (but not in CT-1 cells), histone deacetylases were bound to IFNT, which was reversed with trichostatin A treatment; treatment with trichostatin A and CDX2 then increased IFNT mRNA levels that resulted from abundant CDX2 mRNA expression. These data provide evidence that significant increase in endogenous IFNT transcription in MDBK cells (which do not normally express IFNT) can be induced through CDX2 overexpression and high H3K18 acetylation, but lowering of H3K9 methylation could also be required for the degree of IFNT transcription seen in trophoblast cells.

Proliferating cells express cyclins, cell cycle regulatory proteins that regulate the activity of cyclin-dependent kinases (CDKs). The actions of CDKs are regulated by specific inhibitors, the CDK inhibitors (CDKIs), which are comprised of the Cip/Kip and INK4 families. Expression of the Cip/Kip CDKI 1B (Cdkn1b, encoding protein CDKN1B, also called p27^kip1) in developing Leydig cells (LCs) has been reported, but the function of CDKN1B in LCs is unclear. The goal of the present study was to determine the effects of CDKN1B on LC proliferation and steroidogenesis by examining these parameters in Cdkn1b knockout (Cdkn1b^−/−) mice. LC proliferation was measured by bromodeoxyuridine incorporation. Testicular testosterone levels, mRNA levels, and enzyme activities of steroidogenic enzymes were compared in Cdkn1b^−/− and Cdkn1b ^/ mice. The labeling index of LCs in Cdkn1b^−/− mice was 1.5% ± 0.2%, almost 7-fold higher than 0.2% ± 0.08% (P < 0.001) in the Cdkn1b ^/ control mice. LC number per testis in Cdkn1b^−/− mice was 2-fold that seen in the Cdkn1b ^/ control mice. However, testicular testosterone levels, mRNA levels of steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), and 3beta-hydroxtsteroid dehydrogenase 6 (Hsd3b6), and their respective proteins, were significantly lower in Cdkn1b^−/− mice. We conclude that deficiency of CDKN1B increased LC proliferation, but decreased steroidogenesis. Thus, CDKN1B is an important regulator of LC development and function.

Protein tyrosine kinases have important roles in spermatozoa; however, little is known about the presence and regulation in these cells of their counterparts in signaling, namely, protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). The objectives of the present study were to identify PTPs and DSPs in boar, stallion, and dog spermatozoa; to characterize their subcellular distribution; and to investigate the roles of tyrosine phosphatases in maintenance of protein tyrosine phosphorylation level and in sperm motility. Using Western blotting with specific antibodies in boar and stallion sperm lysates, we unequivocally identified two PTPs (PTPRB and PTPN11) and two DSPs (DUSP3 and DUSP4). In dog sperm lysates, only PTPN11, DUSP3, and DUSP4 were detected. In all these species, we did not detect the specific signal with anti-PTPRC (CD45), CDKN3, DUSP1, DUSP2, DUSP6, DUSP9, PTPN1, PTPN3, PTPN6, PTPN7, PTPN13, PTPRA, PTPRG, PTPRJ, PTPRK, or PTPRZ antibodies. Positive matches were further investigated by indirect immunofluorescence and confocal microscopy. Results showed that PTPRB was associated with the plasma membrane in the head and tail of boar and stallion spermatozoa. In agreement with Western blotting results, PTPRB antibodies did not show immunoreactivity in dog sperm analyzed by immunofluorescence. In the three species, DUSP4 was mainly found in the tail of spermatozoa, with little or no immunoreactivity in the head. PTPN11 was mainly located in the postacrosomal region in the head, whereas DUSP3 immunoreactivity was extended within the acrosome. PTPN11 and DUSP3 showed immunoreactivity in the tail that was restricted to the midpiece. Finally, we incubated boar, stallion, and dog spermatozoa with pervanadate and sodium orthovanadate, two PTP inhibitors, and analyzed overall protein tyrosine phosphorylation and assessed sperm motility. Sodium orthovanadate and pervanadate showed concentration-dependent inhibition of sperm motility that was rapid and reversible. Pervanadate also increased tyrosine phosphorylation of different proteins in capacitated and noncapacitated spermatozoa. Results showed that the phosphatases PTPN11, DUSP4, and DUSP3 are present in boar, stallion, and dog spermatozoa. PTPRB is also present in boar and stallion spermatozoa but was not detected in dog. The subcellular distribution of the identified phosphatases is diverse, suggesting that they likely have specific roles in sperm. Finally, PTP activity has a positive role in the regulation of motility and is involved in protein tyrosine phosphorylation in mammalian sperm.

To study the rapid action of estrogen on the male reproductive system in teleost, a full-length cDNA homologous to the seven-transmembrane receptor GPER of humans and rodents was cloned from the testis of zebrafish. Biological characterization of this cloned zebrafish gper was performed based on its functional expression in cultured eukaryotic cells. Saturation analysis and Scatchard plotting of [³H]-estradiol binding to plasma membranes of gper-transfected COS-7 cells and cAMP response element transactivation assay demonstrated the biological function of the cloned gper as an estrogen receptor. In addition, treatment of gper-transfected COS-7 cells with 17beta-estradiol increased the phosphorylation of MAPK3/MAPK1. However, the inactivity of Gper in the FOS promoter transactivation study indicated some functional difference between the zebrafish and human receptors. We found gper to be highly expressed in the brain and testis by RT-PCR analysis. Results of in situ hybridization demonstrated the localization of gper in specific brain regions and in early germ cells of the testis, including the spermatogonia, spermatocytes, and somatic cells such as Sertoli cells in adult male zebrafish. Subsequent RT-PCR analysis in cells derived from laser capture microdissection microscopy further confirmed the high expression of gper in early germ cells of the testis. The present study demonstrates the existence of a functionally active Gper in zebrafish and suggests a putative role in mediating the rapid action of estrogen in male reproduction.

Clomiphene citrate (CC) therapy for disorders of anovulatory infertility has been linked to an increased frequency of tubal ectopic pregnancy. Although CC enhances apoptotic processes in the ovaries, villi, and decidual tissues, its effect on apoptosis in the fallopian tube is unknown. Here, we show that chronic treatment with CC induces tubal apoptosis, but not necrosis, through an intrinsic mitochondria-dependent signaling pathway in vivo. The apoptosis was specific to epithelial cells in the isthmus, and the damage was reversed with 17beta-estradiol (E2); however, pretreatment or concomitant treatment with E2 did not protect against tubal apoptosis induced by chronic treatment with CC. Chronic treatment activated estrogen receptors (ESRs), particularly cilia-localized ESR2A (formerly ERbeta2). In contrast to E2, acute treatment of superovulating rats with a high dose of CC or the ESR2-selective agonist 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN) significantly delayed the transport of oocyte-cumulus complexes through the fallopian tube. Our findings suggest that in response to chronic CC therapy, isthmus-specific apoptosis of epithelial cells and activation of cilia-ESR2A act in parallel to block gamete and embryo passage through the fallopian tube, eventually resulting in tubal ectopic pregnancy.

Low progesterone concentrations and diminished ovarian reserves (total number of healthy oocytes) during reproductive cycles are linked to infertility in single-ovulating species like cattle. However, the extent and mechanisms whereby the inherently high variation in ovarian reserves may negatively affect progesterone production are unknown. Cattle were chosen to address these questions because the size of their ovarian reserves can be predicted based on an antral follicle count (AFC) during follicular waves. The present study determined if progesterone concentrations, differentiation and function of the corpus luteum (CL), and endometrial thickness differed during reproductive cycles of age-matched healthy young adult cattle with low versus high AFC during follicular waves. The results showed that, despite enhanced LH secretion, progesterone concentrations were lower during estrous cycles for animals with low versus high AFC. Animals with low versus high AFC also had a decreased basal, LH-, and 25-hydroxycholesterol-induced capacity of luteal and granulosal cells to produce progesterone, reduced amounts of STAR and mRNAs for STAR and LH receptor in the CL, and no change in endometrial thickness during estrous cycles. Taken together, these results 1) supported the conclusion that high variation in ovarian reserves of young adults is associated with alterations in differentiation and function of the CL and 2) provided insight into the potential factors that may cause suboptimal luteal function (e.g., heightened LH secretion and desensitization of the LH receptor, diminished LH responsiveness, diminished STAR, inherent deficiency in capacity of granulosal cells to undergo luteinization) and infertility (e.g., low progesterone, poor endometrial growth) in individuals with diminished ovarian reserves.

Follicle-stimulating hormone (FSH) regulation of aromatase gene expression in vitro requires the transcriptional coactivator beta-catenin. To ascertain the physiological significance of beta-catenin in granulosa cells during folliculogenesis, mice homozygous for floxed alleles of beta-catenin were intercrossed with Amhr2cre mice. Conditional deletion of beta-catenin in 8-wk-old females occurred in derivatives of the Müllerian duct, granulosa cells and, surprisingly, in brain, pituitary, heart, liver, and tail. Female mice deficient for beta-catenin were infertile, despite reaching puberty and ovulating at the expected age, indications of apparently normal ovarian function. In contrast, their oviducts were grossly distended, with fewer but healthy oocytes. In addition, their uteri lacked implantation sites. Together, these two phenotypes could explain the complete loss of fertility. Nevertheless, although the ovary appeared normal, with serum estradiol concentrations in the normal range, there was marked animal-to-animal variation of mRNAs encoding beta-catenin and aromatase. Similarly, inhibin-alpha and luteinizing hormone receptor mRNAs varied considerably in whole ovaries, whereas pituitary Fshb mRNA was significantly reduced. Collectively, these features suggested cyclization recombination (CRE)-mediated recombination of beta-catenin may be unstable in proliferating granulosa cells, and therefore may mask the suspected steroidogenic requirement for beta-catenin. We tested this possibility by transducing primary cultures of granulosa cells from mice homozygous for floxed alleles of beta-catenin with a CRE-expressing adenovirus. Reduction of beta-catenin significantly compromised FSH stimulation of aromatase mRNA and subsequent production of estradiol. Collectively, these data suggest that FSH regulation of steroidogenesis requires beta-catenin, a role that remains hidden when tested through Amhr2cre-mediated recombination in vivo.

Single intraperitoneal injection of busulfan at 20 mg/kg body weight to mature male mice induced the deletion of the spermatogenic cells, followed by the restoration of the spermatogenesis by the surviving undifferentiated spermatogonia. The changes of the protein contents in testis during these processes were analyzed by two-dimensional gel electrophoresis in order to identify the proteins expressed at the specific stages of spermatogenesis. An acidic protein that disappeared and recovered in the same time course as spermatids after the busulfan treatment was identified as CABS1 by mass spectrometry. It was found that CABS1 was specifically expressed in the elongate spermatids at steps 13 to 16 in stages I to VIII of the seminiferous epithelium cycle of the mouse, and then it localized to the principal piece of flagellum of the mature sperm in the cauda epididymis. We have found for the first time that CABS1 is a calcium-binding protein that binds calcium during the maturation in the epididymis.