Interferon gamma (IFNG) is a proinflammatory cytokine secreted in the uterus during early pregnancy. It is abundantly produced by uterine natural killer cells in maternal endometrium but also by trophoblasts in some species. In normal pregnancies of mice, IFNG plays critical roles that include initiation of endometrial vasculature remodeling, angiogenesis at implantation sites, and maintenance of the decidual (maternal) component of the placenta. In livestock and in humans, deviations in these processes are thought to contribute to serious gestational complications, such as fetal loss or preeclampsia. Interferon gamma has broader roles in activation of innate and adaptive immune responses to viruses and tumors, in part through upregulating transcription of genes involved in cell cycle regulation, apoptosis, and antigen processing/presentation. Despite this, rodent and human trophoblast cells show dampened responses to IFNG that reflect the resistance of these cells to IFNG-mediated activation of major histocompatibility complex (MHC) class II transplantation antigen expression. Lack of MHC class II antigens on trophoblasts is thought to facilitate survival of the semiallogeneic conceptus in the presence of maternal lymphocytes. This review describes the dynamic roles of IFNG in successful pregnancy and briefly summarizes data on IFNG in gestational pathologies.

Neonatal gonocytes are the precursors of both spermatogonial stem cells and spermatogonia; thus, any persistant DNA damage in these cells may lead to heritable mutations. We investigated the response of male mouse neonatal germ cells to ionizing radiation. Both gonocytes and spermatogonia died in large numbers by apoptosis. However, we found that the gonocytes were significantly more sensitive than spermatogonia and somatic cells to radiation-induced double-strand breaks (DSBs), as assayed by the number of gamma-H2AFX foci. In contrast, gonocytes irradiated in G2 phase seemed to repair DSBs faster than spermatogonia. Moreover, when irradiated in S phase, gonocytes arrested their cell cycle at the G1/S phase transition, whereas spermatogonia were mostly blocked in G2/M phase. Despite these differences, both cell types expressed high levels of proteins involved in DSB signaling and repair. Within the first hours after irradiation, the expression of Atr, Mre11a, H2afx, Xrcc6, and Xrcc4 was downregulated in neonatal spermatogonia, whereas, in gonocytes, most gene expression was unaffected. Together, these results suggest that the response of neonatal testis to genotoxic stress is regulated by different mechanisms according to the cell type and the differentiation status.

Intrauterine or intraperitoneal administration of lipopolysaccharide (LPS) into normal mice at midgestation induces preterm delivery (PTD) within 24 h through a mechanism dependent on Toll-like receptor signaling and expression of inflammatory cytokines. The exact participants in the cellular network involved in PTD are not known. Although the activities of innate immune cells are thought to be important, the extent to which this process depends on T and B cells has yet to be examined. Mice deficient in T and B cells due to genetic deficiency in the recombination activating gene 1 (Rag1^−/−) were given LPS intraperitoneally on Day 15 of gestation and found to be susceptible to LPS-induced PTD. This was found to involve many of the inflammatory mediators reported as important in normal mice. Moreover, at a low dose (3 μg), pregnant Rag1^−/− mice were found to be more susceptible to PTD than a cohort of normal mice on the same genetic background. This increased susceptibility was partially reversed by transfer, on Day 10 of gestation, of whole lymphocytes or purified CD4 T cells. Transfer of purified CD4 T cells to Rag1^−/− mice resulted in a uterine draining node population of FOXP3 cells, suggesting that these cells may contribute to resistance to LPS-induced PTD. Overall, the data suggest that, although T and B lymphocytes are not critical positive regulators of LPS-induced PTD, CD4 T cells play a protective and regulatory role, and thus could be a target for preventive or therapeutic manipulation.

Diethylhexylphthalate (DEHP) has been classified as an antiandrogen. However, whether in utero and lactational exposures of DEHP affect Leydig cells has not been well established. In the present study, the effects of DEHP exposures on fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) were assessed. Pregnant dams of Long-Evans rats were treated with 0, 10, and 750 mg/kg body weight DEHP from Gestational Day 12.5 to Postnatal Day (PND) 21.5. Fetal Leydig cell clustering and FLC-specific gene expression were examined. Anogenital distances (AGDs) of male pups were assessed at PND 2. Serum testosterone levels of male pups and mRNA levels of ALC-specific genes were measured at PNDs 21 and 49. The AGDs of male pups were significantly shorter in the group treated with 750 mg/kg DEHP (mean ± SEM, 3.68 ± 0.16 mm) compared with control (4.62 ± 0.13 mm). The FLCs were aggregated after 10 and 750 mg/kg DEHP exposures. Several FLC-specific genes, including luteinizing hormone receptor (Lhcgr) and steroidogenic enzyme genes, were downregulated at both doses. Serum testosterone levels were significantly lower compared with control at PND 21 after treatment of 10 or 750 mg/kg DEHP, and continued to be lower even up to 49 days postpartum at the higher dose. The mRNA levels for Lhcgr and steroidogenic enzyme genes were significantly lower at both doses of DEHP at PND 21, whereas there were no significant differences for these genes at PND 49. In conclusion, in utero and continued lactational exposures to DEHP exert long-term disruption of steroidogenesis of ALCs.

The brain of teleosts is known for its strong aromatase expression, exhibiting unique features compared with other vertebrates. Among these features is the high sensitivity of aromatase B (the product of cyp19a1b) to estrogens. This effect involves the binding of estrogen receptors on an estrogen-responsive element (ERE) of the cyp19a1b promoter. Given the presence of potential androgen-responsive elements (AREs) on this promoter, in vivo and in vitro effects of androgens were studied. Using immunohistochemistry and quantitative PCR on zebrafish embryos, we found that cyp19a1b is upregulated by testosterone, an aromatizable androgen, and by 5alpha-dihydrotestosterone (DHT), a nonaromatizable androgen, suggesting a potential androgenic regulation of cyp19a1b through androgen receptors (ARs). To assess a putative direct regulation of the cyp19a1b gene by ARs, we transfected U251MG cells with zebrafish AR together with a luciferase reporter gene driven by 3000 bp of the proximal cyp19a1b promoter containing the ERE and potential AREs. Interestingly, although zebrafish AR activated luciferase reporter genes controlled by AREs, they failed to induce the cyp19a1b-luciferase construct. These data suggest that the androgenic regulation of cyp19a1b does not involve AR. We further showed that regulation of the cyp19a1b gene by testosterone is, in fact, due to aromatization, whereas the effect of DHT involves conversion into 5alpha-androstane-3beta,17beta-diol (betadiol), a metabolite of DHT with known estrogenic activity. The blockage of the androgen regulation of cyp19a1b expression using antiestrogens further confirmed the involvement of estrogen receptors in mediating these effects.

Mammalian sperm become fertile after completing capacitation, a process associated with cholesterol loss and changes in the biophysical properties of the sperm membranes that prepares the sperm to undergo the acrosome reaction. Different laboratories have hypothesized that cholesterol efflux can influence the extent and/or movement of lipid raft microdomains. In a previous study, our laboratory investigated the identity of sperm proteins putatively associated with rafts. After extraction with Triton X-100 and ultracentrifugation in sucrose gradients, proteins distributing to the light buoyant-density fractions were cored from polyacrylamide gels and microsequenced. In this study, a subset of these proteins (TEX101, basigin, hexokinase 1, facilitated glucose transporter 3, IZUMO, and SPAM1) and other molecules known to be enriched in membrane rafts (caveolin 2, flotillin 1, flotillin 2, and the ganglioside GM3) were selected to investigate their localization in the sperm and their behavior during capacitation and the acrosome reaction. These molecules localize to multiple sperm domains, including the acrosomal cap (IZUMO, caveolin 2, and flotillin 2), equatorial segment (GM3), cytoplasmic droplet (TEX101), midpiece (basigin, facilitated glucose transporter 3, and flotillin 2), and principal piece (facilitated glucose transporter 3). Some of these markers modified their immunofluorescence pattern after sperm incubation under capacitating conditions, and these changes correlated with the occurrence of the acrosome reaction. While GM3 and caveolin 2 were not detected after the acrosome reaction, flotillin 2 was found in the equatorial segment of acrosome-reacted sperm, and IZUMO distributed along the sperm head, reaching the post- and para-acrosomal areas. Taking into consideration the requirement of the acrosome reaction for sperm to become fusogenic, these results suggest that membrane raft dynamics may have a role in sperm-egg membrane interaction.

Nuclear receptor subfamily 6, group A, member 1 (NR6A1) is an orphan member of the nuclear receptor superfamily and is required for normal mouse embryonic development. In adult mice, NR6A1 is predominantly expressed in spermatogenic cells and growing oocytes of the gonads and has a role in female reproduction by modulating the transcription of the oocyte-specific genes bone morphogenetic protein 15 (Bmp15) and growth differentiation factor 9 (Gdf9). In our goal to further understand the functional role of NR6A1 during postnatal development, we generated a Nr6a1:beta-galactosidase (LacZ) knockin reporter (Nr6a1^LacZ/ ) mouse line in which the Nr6a1:LacZ fusion gene was expressed and then characterized Nr6a1 expression in these reporter mice by performing LacZ staining. Our RT-PCR analyses showed that Nr6a1 was expressed in a variety of somatic tissues (e.g., oviduct and lung) other than gonads of normal adult mice. In adult Nr6a1^LacZ/ mice, robust LacZ staining was observed in the gametes of gonads. Strong positive LacZ staining was also observed in the sperm of the epididymis, epithelial cells of the oviduct, and bronchioles within the lung. In adult Nr6a1^LacZ/ mice, positive LacZ staining was observed in other somatic tissues, including hippocampus, cerebral cortex, cerebellum, and thalamus of brain; pars intermedia and pars anterior of pituitary; parathyroid; and islet of pancreas. NR6A1 expression in sperm within the epididymis, epithelial cells in the oviduct, and bronchioles of the lung was further confirmed by immunohistochemical studies. Nr6a1 is expressed not only in the germ cells of mouse gonads but also in a variety of somatic tissues, including epididymis, oviduct, brain, and pituitary. The extra-germ cell expression of NR6A1 makes it a less attractive contraceptive.

Spermatogenesis is a temperature-dependent process, and increases in scrotal temperature can disrupt its progression. We previously showed that heat stress causes DNA damage in germ cells, an increase in germ cell death (as seen on TUNEL staining), and subfertility. The present study evaluated the stress response in mouse testes following a single mild transient scrotal heat exposure (40°C or 42°C for 30 min). We investigated markers of three types of stress response, namely, hypoxia, oxidative stress, and apoptosis. Heat stress caused an increase in expression of hypoxia-inducible factor 1 alpha (Hif1a) mRNA expression and translocation of HIF1A protein to the germ cell nucleus, consistent with hypoxic stress. Increased expression of heme oxygenase 1 (Hmox1) and the antioxidant enzymes glutathione peroxidase 1 (GPX1) and glutathione S-transferase alpha (GSTA) was consistent with a robust oxidative stress response. Germ cell death was associated with an increase in expression of the effector caspase cleaved caspase 3 and a decrease in expression of the protein inhibitor of caspase-activated DNase (ICAD). Reduced expression of ICAD contributes to increased activity of caspase-activated DNase and is consistent with the increased rates of DNA fragmentation that have been detected previously using TUNEL staining. These studies confirmed that transient mild testicular hyperthermia results in temperature-dependent germ cell death and demonstrated that elevated temperature results in a complex stress response, including induction of genes associated with oxidative stress and hypoxia.

Hormonal contraceptives are unsuitable for many women; thus, the development of new, nonhormonal contraceptives is of great interest. In women, uterine epithelial expression of interleukin 11 (IL11) and its receptor (IL11RA) suggests IL11 is critical for blastocyst attachment during implantation. Il11ra-deficient mice are infertile due to a defective decidualization response to the blastocyst, leading to total pregnancy loss. We examined the effect of administering a PEGylated IL11 antagonist, PEGIL11A (where PEG is polyethylene glycol), on pregnancy outcomes in mice and IL11 signaling in human endometrial epithelial cells (HES). PEGIL11A was detected in sera up to 72 h after intraperitoneal (IP) injection versus up to 2 h for the non-PEGylated antagonist. Following IP injection, PEGIL11A localized to uterine decidual cells and reduced immunoreactive cyclin D3 (IL11 decidual target). To inhibit IL11 action during early decidualization, PEGIL11A or control were administered IP on Days 3–6 (beginning just prior to maximal decidual Il11 expression). On Day 6, mesometrial decidualization was disturbed in PEGIL11A-treated animals with regions of hemorrhage visible in the mesometrial decidua. On Day 10, severe decidual destruction was visible: implantation sites contained significant hemorrhage, and the uterine luminal epithelium had reformed, suggesting a return to estrous cycling. These results demonstrate that PEGIL11A blocked IL11 action in the decidua during early decidualization, which totally abolished pregnancy and which is equivalent to the Il11ra^−/− mouse. PEGIL11A significantly diminished STAT3 phosphorylation in HES cells in vitro (P ≤ 0.05). This study provides valuable information for PEGIL11A that could lead to the development of this protein as a nonhormonal contraceptive.

The appropriate timing of the onset of labor is critical to a successful pregnancy, with potentially devastating consequences resulting to both the mother and child with the onset of preterm labor. In this study, we tested the central hypothesis that progesterone maintains uterine quiescence through regulation of active uterine caspase 3. Using the mouse as our model system, we examined, by Western blot analysis, levels of active caspase 3 and its association with the degradation of uterine contractile proteins during pregnancy. Our data demonstrate that caspase 3-specific cleavage fragments of uterine myocyte contractile proteins are elevated in late gestation. Prior to the onset of labor, active caspase 3 levels and fragmentation of the uterine myocyte contractile proteins decline. We postulate that uterine caspase 3 acts as an anticontractile agent maintaining uterine quiescence through degradation of uterine contractile proteins during late pregnancy. We propose that decreased progesterone action during the final days of pregnancy controls the timing of the onset of uterine contractions by removing the anticontractile action of the apoptotic protein caspase 3 locally in the pregnant myometrium.

Nuclear transfer has been regarded as the only reliable tool for studying nuclear reprogramming of mammalian somatic cells by oocytes. However, nuclear transfer is not well suited for biochemical analyses of the molecular mechanisms of reprogramming. A cell-free system from oocytes is an attractive alternative way to mimic reprogramming in vitro, since a large number of cells can be treated and analyzed. Nevertheless, a cell-free system using oocytes has not been developed in mammals. Here, cell extracts from porcine oocytes were prepared and their ability to induce nuclear reprogramming was evaluated. Extracts from metaphase II (MII) oocytes erased the machinery for regulating gene expression in reversibly permeabilized somatic cells. For example, the extracts caused histone deacetylation and the disappearance of TATA box-binding protein from the nuclei. However, MII-extract-treated cells did not show any obvious changes after cell culture. In contrast, extracts from germinal vesicle (GV) oocytes activated pluripotent marker genes, especially NANOG, and induced partial dedifferentiation after cell culture. The activation of pluripotent marker genes by GV extracts was associated with histone acetylation that was induced during extract treatment. These results indicate that GV- and MII-oocyte extracts have different roles on nuclear reprogramming. Furthermore, both oocyte extracts induced site-specific demethylation in the upstream region of NANOG. These results indicate that cell-free extracts derived from GV- and MII-oocytes could be useful for studying the mechanisms involved in nuclear reprogramming.

DDK syndrome is the polar-lethal embryonic death that occurs at the morula-blastocyst transition when female mice of the DDK strain are mated with males from many other inbred strains (so-called alien males). Embryonic death is caused by incompatibility between a DDK oocyte factor and an alien male gene, both of which map to the Om locus on mouse chromosome 11. We compared global transcription patterns of DDK × DDK embryos (high viability) and DDK × C57BL/6 embryos (low viability) at the morula stage, approximately 24 h before any morphological manifestations of DDK syndrome are observed. Of the transcripts that are differentially more abundant in the DDK × C57BL/6 embryos, many are the products of genes induced by the “unfolded protein response.” We confirmed by quantitative RT-PCR that a number of genes in this pathway are upregulated in the DDK × C57BL/6 embryos. Immunostaining of the endoplasmic reticulum (ER) marker BIP/GRP78 (immunoglobin-binding protein/glucose-regulated protein of 78 kDa), official symbol HSPA5, heat shock protein 5 revealed an accompanying abnormal HSPA5 accumulation and ER structure in the DDK × C57BL/6 embryos. Immunostaining for HERPUD1 (homocysteine-inducible, ER stress-inducible, ubiquitin-like domain member 1) and ATF4 (activating transcription factor 4) also revealed accumulation of these stress-response products. Our results indicate that the unfolded protein response is induced in embryos destined to die of DDK syndrome and that the embryonic death observed is associated with inability to resolve the associated ER stress.

The mechanisms whereby the high variation in numbers of morphologically healthy oocytes and follicles in ovaries (ovarian reserve) may have an impact onovarian function, oocyte quality, and fertility are poorly understood. The objective was to determine whether previously validated biomarkers for follicular differentiation and function, as well as oocyte quality differed between cattle with low versus a high antral follicle count (AFC). Ovaries were removed (n = 5 per group) near the beginning of the nonovulatory follicular wave, before follicles could be identified via ultrasonography as being dominant, from heifers with high versus a low AFC. The F1, F2, and F3 follicles were dissected and diameters determined. Follicular fluid and thecal, granulosal, and cumulus cells and the oocyte were isolated and subjected to biomarker analyses. Although the size and numerous biomarkers of differentiation, such as mRNAs for the gonadotropin receptors, were similar, intrafollicular concentrations of estradiol and the abundance of mRNAs for CYP19A1 in granulosal cells and ESR1, ESR2, and CTSB in cumulus cells were greater, whereas mRNAs for AMH in granulosal cells and TBC1D1 in thecal cells were lower for animals with low versus a high AFC during follicle waves. Hence, variation in the ovarian reserve may have an impact on follicular function and oocyte quality via alterations in intrafollicular estradiol production and expression of key genes involved in follicle-stimulating hormone action (AMH) and estradiol (CYP19A1) production by granulosal cells, function and survival of thecal cells (TBC1D1), responsiveness of cumulus cells to estradiol (ESR1, ESR2), and cumulus cell determinants of oocyte quality (CTSB).

Epididymosomes are small membranous vesicles secreted by epithelial cells within the luminal compartment of the epididymis. In bovine, many proteins are associated with epididymosomes, and some of them, such as the glycosylphosphatidylinositol (GPI)-anchored protein P25b, macrophage migration inhibitory factor (MIF), and aldose reductase (AKR1B1), are transferred to spermatozoa during the epididymal maturation process. P25b is associated with detergent-resistant membrane (DRM) domains of epididymal spermatozoa, whereas MIF and AKR1B1 are cytosolic proteins associated with detergent-soluble fractions. In this study, we tested the hypothesis that DRM domains are also present in the epididymosomes and that P25b DRM-associated proteins in these vesicles are transferred to the DRMs of spermatozoa. The presence of DRMs in epididymosomes was confirmed by their insolubility in cold Triton X-100 and their low buoyant density in sucrose gradient. Furthermore, DRMs isolated from epididymosomes are characterized by the exclusive presence of ganglioside GM1 and by high levels of cholesterol and sphingomyelin. Biochemical analysis indicated that P25b is linked to DRM in epididymosomes, whereas MIF and AKR1B1 are completely excluded from these membrane domains. Proteolytic treatment of epididymosomes and immunoblotting studies showed that P25b is affected by trypsin or pronase proteolysis. In contrast, MIF and AKR1B1 are not degraded by proteases, suggesting that they are localized within epididymosomes. Interaction studies between epididymosomes and epididymal spermatozoa demonstrated that P25b is transferred from the DRM of epididymosomes to the DRM of the caput epididymal spermatozoa as a GPI-anchored protein. Together, these data suggest that specific localization and compartmentalization of proteins in the epididymosomes coordinate the association of epididymal proteins with the different functional structures of spermatozoa.

The natural clonal loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) is diploid (2n = 50) and produces genetically identical unreduced eggs, which develop into diploid individuals without any genetic contribution from sperm. Artificially sex-reversed clones created by the administration of 17alpha-methyltestosterone produce clonal diploid sperm. In metaphase spreads from testicular cells of the sex-reversed clones, spermatocytes had twice the normal number of chromosomes (50 bivalents) compared with those of normal diploids (25 bivalents). Thus, the production of unreduced diploid spermatozoa is initiated by premeiotic endomitosis (or endoreduplication), chromosome doubling before meiosis, and is followed by two quasinormal divisions. Larger nuclei in the germ cells were observed in all stages of type B spermatogonia in the testes of the sex-reversed clones. In contrast, besides having larger type A spermatogonia, the sex-reversed clones also had the type A spermatogonia that were the same size as those of normal diploids. It follows that chromosome duplication causing unreduced spermatogenesis occurred in the type A spermatogonia. The presence of tetraploid type A and early type B spermatogonia, identified by labeling with antispermatogonia-specific antigen 1, was verified using DNA content flow cytometry. These results support the conclusion that chromosome doubling occurs at the type A spermatogonial stage in diploid spermatogenesis in the clonal fish.

Prostaglandin F2 alpha (PGF_2alpha) brings about regression of the bovine corpus luteum (CL). This luteolytic property of PGF_2alpha is used in beef and dairy cattle to synchronize estrus. A limitation of this protocol is insensitivity of the early CL to luteolytic actions of PGF_2alpha. The mechanisms underlying this differential luteal sensitivity are poorly understood. The developing CL has a maximum number of PGF_2alpha receptors; therefore, differences in signaling events may be responsible for luteal insensitivity. Hence, differential gene expression at two developmental stages of CL, Day 4 (D-4) and D-10 after estrus, might account for differences in signal transduction pathways associated with luteal sensitivity. This possibility was examined in these studies. Microarray analysis (n = 3 cows per stage) identified 167 genes that were differentially expressed (P < 0.05). These were categorized into genes involved in protein biosynthesis and modification (18.5%), transcription regulation and DNA biosynthesis (18.5%), miscellaneous (17.0%), cell signaling (12.0%), steroidogenesis and metabolism (10.2%), extracellular matrix and cytoskeletal proteins (9.5%), unknown functions (6.0%), protein degradation (5.3%), and antioxidant property (3.0%). Real-time PCR confirmed the differential expression of nine selected genes, including tyrosine 3-monooxygenase/tryptophan 5-monooxygense activation protein zeta polypeptide (YWHAZ) and regulator of G protein signaling 2 24-kDa (RGS2), observed in microarray. Furthermore, the in vivo effect of exogenous PGF_2alpha (n = 3 cows per stage) on selected genes that were found to be differentially expressed during this developmental transition was examined. PGF_2alpha increased the expression of a guanine nucleotide-binding protein (G protein) beta polypeptide 1 (GNB1) in D-4 CL and calcium/calmodulin-dependent kinase kinase 2 beta (CAMKK2) in D-10 CL. Therefore, GNB1, CAMKK2, YWHAZ, and RGS2 are candidate genes that may have a significant role in acquisition of luteal sensitivity to PGF_2alpha. Additional evidence supporting the significance of the microarray data was obtained from the observation that the amount of CAMKK2 paralleled the differential mRNA expression observed for this gene when examined by microarray analysis and by real-time RT-PCR. Furthermore, the two types of luteal steroidogenic cells known to be targets for PGF_2alpha actions were demonstrated to be a cellular source for CAMKK2.

Wnt genes are involved in critical developmental and growth processes. The present study comprehensively analyzed temporal and spatial alterations in Wnt and Fzd gene expression in the mouse uterus during peri-implantation of pregnancy. Expression of Wnt4, Wnt5a, Wnt7a, Wnt7b, Wnt11, Wnt16, Fzd2, Fzd4, and Fzd6 was detected in the uterus during implantation. Wnt4 mRNA was most abundant in the decidua, whereas Wnt5a mRNA was restricted to the mesometrial decidua during decidualization. Wnt7a, Wnt7b, and Wnt11 mRNAs were abundantly detected in the endometrial epithelia. The expression of Wnt7b was robust in the luminal epithelium (LE) at the implantation site on Gestational Day 5, whereas Wnt11 mRNA disappeared in the LE adjacent to the embryo in the antimesometrial implantation chamber but remained abundant in the LE. Wnt16 mRNA was localized to the stroma surrounding the LE on Day 4 and remained in the stroma adjacent to the LE but not in areas undergoing the decidual reaction. Fzd2 mRNA was detected in the decidua, Fzd4 mRNA was in the vessels and stroma surrounding the embryo, and Fzd6 mRNA was observed in the endometrial epithelia, stroma, and some blood vessels during implantation. Ovarian steroid hormone treatment was found to regulate Wnt genes and Fzd receptors in ovariectomized mice. Especially, single injections of progesterone stimulated Wnt11 mRNA, and estrogen stimulated Wnt4 and Wnt7b. The temporal and spatial alterations in Wnt genes likely play a critical role during implantation and decidualization in mice.

A Disintegrin And Metalloprotease (ADAM) family members expressed in male reproductive tissues are divided phylogenetically into three major groups. In the present study, we analyzed six ADAMs in one of the groups (ADAMs 4, 6, 24, 26, 29, and 30) of which function is largely unknown. Our results showed that most of the ADAMs undergo unique processing during sperm maturation and are located at the surface of sperm head. We found that the levels of ADAM4 and ADAM6 are dramatically reduced in Adam2 and Adam3 knockout sperm defective in various fertilization processes. We observed premature processing of ADAM4 in the Adam3-null mice. Furthermore, we obtained a result showing complex formation of ADAM6 with ADAM2 and ADAM3 in testis. Taken together, these results disclose involvement of ADAM4 and ADAM6 in a reproductive ADAM system that functions in fertilization.

Reproductive aging of the male is characterized by decreasing fertility; however, factors that protect against reproductive aging in the male are largely unknown. Previous work has demonstrated that both female presence and aging have a dramatic effect on fertility in the male; yet, the effect of female presence on fertility in the aging male mouse is unknown. The objective of this work was to determine the effect of long-term isolation or cohabitation with females on fertility in aged male mice. Male mice were housed with or without females until between 16 and 32 mo of age. Males were subjected to fertility tests at specific ages, after which serum and testes were isolated for radioimmunoassay and histological analysis. We show that male mice continuously housed with females remain fertile longer (∼20% of the reproductive lifespan) than male mice housed alone. Fertility became significantly reduced 6 mo sooner for males housed alone compared with males housed with females; however, the rate of decline was the same for males housed with or without females once fertility began to decrease. Testis weight decreased as the mice aged, and a nearly significant positive effect of female presence was observed. Additionally, histological analysis indicated that abnormal spermatogenesis occurred sooner in isolated males, suggesting that defects in spermatogenesis may play a role in the greater decrease in fertility in isolated males. These results have significant implications for the maintenance of male fertility in wildlife, livestock, and human populations.

Extracellular matrix (ECM) formation by cumulus cells is an important process that determines fertilization and embryo quality. Several collagen types are present in the ovarian follicular ECM and are related to proliferation, steroidogenesis, and luteinization. In vitro mouse follicles can optimally grow and provide developmentally competent oocytes with 10 IU/L recombinant follicle-stimulating hormone (rFSH). As a model for superovulation, experiments with 100 IU/L rFSH or 100 IU/L highly purified menotropin (HP-hMG) exposure during antral growth were undertaken. Col4a1, Col4a2, and Col6a2 expression levels were analyzed at three time points during antral growth and at a 4-h interval up to 16 h after ovulation induction using quantitative PCR. The presence and induction of the collagen mRNA and protein were confirmed in cumulus from in vivo- and in vitro-grown follicles, and TGFBs 1 and 2 were assayed as potential regulators. The study revealed that exposure to 100 IU/L FSH, as in both superovulation conditions, significantly influenced the follicle morphology and slowed down nuclear maturation and mucification (P < 0.05). This coincided with an increased expression of the three collagens in the cumulus-oocyte complex at the end of antral growth and in the first hours following the ovulatory dose of human chorionic gonadotropin (P < 0.05). The increased expression might reflect a differentiation but is most likely due to a precocious luteinization of the cumulus. Growth in HP-hMG resulted in higher Tgfb1 mRNA and protein levels, fewer COCs with an increased collagen expression and with a more synchronous nuclear maturation. This suggests that the presence of luteinizing hormone activity tempered the effect of the elevated FSH dose.

The proteasome is a multicatalytic cellular complex present in human sperm that plays a significant role during several steps of mammalian fertilization. Here, we present evidence that the proteasome is involved in human sperm capacitation. Aliquots of highly motile sperm were incubated with proteasome inhibitors MG132 or epoxomicin. The percentage of capacitated sperm, the chymotrypsin-like activity of the proteasome, cAMP content, and the pattern of protein phosphorylation were assayed by using the chlortetracycline hydrochloride assay, a fluorogenic substrate, the cAMP enzyme immunoassay kit, and Western blot analysis, respectively. Our results indicate that treatment of sperm with proteasome inhibitors blocks the capacitation process, does not alter cAMP concentration, and changes the pattern of protein phosphorylation. To elucidate how proteasome activity is regulated during capacitation, sperm were incubated with: 1) tyrosine kinase (TK) inhibitors (genistein or herbimycin A); 2) protein kinase (PK) A inhibitors or activators (H89 and Rp-cAMPS, and 8-Br-cAMP, respectively); or 3) PKC inhibitors (tamoxifen or staurosporin) at different capacitation times. The chymotrypsin-like activity and degree of phosphorylation of the proteasome were then assayed. The results indicate that sperm treatment with TK and PKA inhibitors significantly decreases the chymotrypsin-like activity of the proteasome during capacitation. Immunoprecipitation and Western blot results suggest that the proteasome is phosphorylated during capacitation in a TK- and PKA-dependent pathway. In conclusion, we suggest that the sperm proteasome participates in the capacitation process, and that its activity is modulated by PKs.

T regulatory (Treg) cells are implicated in maternal immune tolerance of the conceptus at implantation; however, the antigenic and regulatory signals controlling Treg cells in early pregnancy are undefined. To examine the role of male seminal fluid in tolerance induction, the effect of exposure to seminal fluid at mating on responsiveness to paternal alloantigens was examined using paternal tumor cell grafts and by delayed-type hypersensitivity (DTH) challenge on Day 3.5 postcoitum. Exposure to seminal fluid inhibited rejection of paternal tumor cells, independently of fertilization and embryo development, while seminal fluid from major histocompatability complex (MHC)-dissimilar males was less effective. Similarly, mating with intact males suppressed the DTH response to paternal alloantigens in an MHC-specific fashion. Excision of the seminal vesicle glands diminished the tolerance-inducing activity of seminal fluid. Mating with intact males caused an increase in CD4 CD25 cells expressing FOXP3 in the para-aortic lymph nodes draining the uterus, beyond the estrus-associated peak in cycling mice. The increase in CD4 CD25 cells was abrogated when males were vasectomized or seminal vesicles were excised. Collectively, these data provide evidence that exposure to seminal fluid at mating promotes a state of functional tolerance to paternal alloantigens that may facilitate maternal acceptance of the conceptus at implantation, and the effects of seminal fluid are likely to be mediated by expansion of the Treg cell pool. Both seminal plasma and sperm components of the seminal fluid are necessary to confer full tolerance and elicit the Treg cell response, potentially through provision of immune-deviating cytokines and antigens, respectively.

Linearized p-eGFP (plasmid-enhanced green fluorescent protein) or p-hFSH (plasmid human FSH) sequences with the corresponding restriction enzyme were lipofected into sperm genomic DNA. Sperm transfected with p-eGFP were used for artificial insemination in hens, and in 17 out of 19 of the resultant chicks, the exogenous DNA was detected in their lymphocytes as determined by PCR and expressed in tissues as determined by (a) PCR, (b) specific emission of green fluorescence by the eGFP, and (c) Southern blot analysis. A complete homology was found between the Aequorea Victoria eGFP DNA and a 313-bp PCR product of extracted DNA from chick blood cells. Following insemination with sperm lipofected with p-hFSH, transgenic offspring were obtained for two generations as determined by detection of the transgene for human FSH (PCR) and expression of the gene (RT-PCR and quantitative real-time PCR) and the presence of the protein in blood (radioimmunoassay). Data demonstrate that lipofection of plasmid DNA with restriction enzyme is a highly efficient method for the production of transfected sperm to produce transgenic offspring by direct artificial insemination.

The objective of this study was to determine whether beta human chorionic gonadotropin (hCG) (CGB) subunits and alpha hCG (CGA) subunits are expressed and the hCG dimer is produced in normal human cyclic endometrium. Endometrial specimens were collected for histological dating from women undergoing treatment in our division of human reproduction. RNA from normal secretory endometrium was extracted, and CGB and CGA gene expression was assessed by semiquantitative PCR. Adequate secretory endometrial specimens were homogenized using protease inhibitors. Proteins present in the supernatant were separated electrophoretically, and molecular hCG isoforms were detected by Western blot. The supernatant hCG concentrations were measured by ELISA. We characterized hCG and leukocytes in endometrial specimens by immunohistochemistry. Uterine flushing was performed to confirm endometrial hCG secretion into the uterine fluid. A full-length CGB mRNA encompassing the exon 1 promoter region and the structure exons 2 and 3 (including the C-terminal peptide) was expressed in normal secretory endometrial specimens (similar to CGA) during the early secretory phase of the menstrual cycle, up to an optimum at the midsecretory to late secretory phases. In homogenate supernatants obtained from normal secretory endometrium, hormone concentrations of dimeric hCG were approximately 5 mU per 10 mg of tissue, compared with considerably smaller concentrations of corresponding single free CGB subunit. Single chains of CGB, CGA, and dimeric molecular hCG isoforms were found in endometrial specimens by Western blot. Glandular endometrial hCG production is demonstrated immunohistochemically, with an increase toward the late secretory phase vs. the early secretory phase of the normal secretory menstrual cycle. However, glandular hCG release is diminished or absent in the dyssynchronous or missing endometrial secretory transformation. Endogenous endometrial hCG may be important for implantation and maintenance of pregnancy.

Growing evidence suggests that exposure to bisphenol A (BPA) has the ability to disrupt several different stages of oocyte development. To date, most attention has focused on the effects of BPA on the periovulatory oocyte, and considerable variation is evident in the results of these studies. In our own laboratory, variation in the results of BPA studies conducted at different times appeared to correlate with changes in mill dates of animal feed. This observation, coupled with reports by others that dietary estrogens in feed are a confounding variable in studies of endocrine-disrupting chemicals, prompted us to evaluate the effect of diet on the results of BPA studies of the periovulatory oocyte. Genetically identical females were placed on a high- or low-phytoestrogen diet prior to mating. Their female offspring were exposed to BPA, oocytes collected, and meiotic spindle and chromosome characteristics compared between control and BPA-treated females. We observed significant diet-related variation in both the frequency of abnormalities in oocytes from untreated females and in the response to BPA. Our results demonstrate that the impact of BPA on meiosis depends, at least in part, on diet. We suggest that variation in the conclusions of recent BPA studies reflects differences in the diets used, as well as other methodological differences. Because meiotic disturbances are a feature of all studies to date, however, we conclude that low levels of BPA adversely affect the meiotic process.

Oocyte-cumulus cell bidirectional communication is essential for normal development of the oocyte and cumulus cells (CCs) within the follicle. We showed recently that addition of recombinant growth differentiation factor 9 (GDF9), which signals through the SMAD2/3 pathway, during mouse oocyte in vitro maturation (IVM) increased fetal viability. This study thus aimed to observe the effects of disrupting oocyte-CC bidirectional communication during IVM on oocyte developmental competence and fetal outcomes. Cumulus-oocyte complexes (COCs) from equine chorionic gonadotropin-primed prepubertal (CBA/C57BL6) mice were cultured with or without 50 mIU/ml follicle-stimulating hormone (FSH) and 10 ng/ml epidermal growth factor (EGF) or 4 μM SMAD2/3 inhibitor SB-431542. Cumulus expansion and first polar body extrusion were then assessed, or COCs were fertilized and stained to evaluate sperm entry or cultured to the blastocyst stage. Embryo development and blastocyst quality were assessed, and Day 4.5 blastocysts were transferred to pseudopregnant recipients to analyze fetal outcomes. SMAD2/3 inhibition or FSH/EGF absence during IVM resulted in decreased cumulus expansion. First polar body extrusion and sperm entry were decreased in the absence of FSH/EGF, whereas only sperm entry was affected in SB-431542-matured COCs. Embryo development and blastocyst rates were unaffected; however, blastocyst quality was significantly altered, with reduced inner cell mass cell numbers in embryos derived from COCs matured in both treatments. When COCs were matured with SB-431542 in the absence of FSH/EGF, cumulus expansion was reduced, but fertilization, embryo development, and embryo quality were not. Inhibition of SMAD2/3 signaling in the presence of FSH/EGF significantly reduced fetal survival but had no effect on implantation or fetal and placental dimensions and morphology.