Reproductive genes are known to evolve more rapidly than genes expressed in other organs. In this paper we present an overview and bring some new data on the evolutionary study of reproduction-related genes by integrating phylogeny with gene genomic localization. We focus on the gene evolutionary processes of gene birth, death, and divergence. We show that phylogenetic gene birth is confirmed by gene location in genomes, which definitively localized the “place of birth” of new genes (such as Obox and KHDC1/DPPA5/ECAT1/OOEP gene families). By finding their “place of death” in genomes, it also demonstrates that ZP genes TGM4 and OVGP1 have been lost in certain species during vertebrate evolution. Moreover, in the case of gene divergence, comparison of gene locations across different genomes establishes orthologous relationships that are weakly supported by the phylogenetic tree. Specifically, genomic localization demonstrates that the fish and bird mtnr1c (Mel1C) receptor is orthologous to mammalian GPR50, and that ungulate genomes contain new seminal vesicle-specific BSP genes that are not present in other species. Overall, the phylogenomic approach to gene evolution presented in this paper offers more insight into gene function, such as species-specific duplications for speciation, changes in gene expression due to gene divergence, and functional loss by gene death.

Dietary supply of nutrients, both periconception and during pregnancy, influence the growth and development of the fetus and offspring and their health into adult life. Despite the importance of research efforts surrounding the developmental origins of health and disease hypothesis, the biological mechanisms involved remain elusive. Mitochondria are of major importance in the oocyte and early embryo, particularly as a source of ATP generation, and perturbations in their function have been related to reduced embryo quality. The present study examined embryo development following periconception exposure of females to a high-protein diet (HPD) or a low-protein diet (LPD) relative to a medium-protein diet (MPD; control), and we hypothesized that perturbed mitochondrial metabolism in the mouse embryo may be responsible for the impaired embryo and fetal development reported by others. Although the rate of development to the blastocyst stage did not differ between diets, both the HPD and LPD reduced the number of inner cell mass cells in the blastocyst-stage embryo. Furthermore, mitochondrial membrane potential was reduced and mitochondrial calcium levels increased in the 2-cell embryo. Embryos from HPD females had elevated levels of reactive oxygen species and ADP concentrations, indicative of metabolic stress and, potentially, the uncoupling of oxidative phosphorylation, whereas embryos from LPD females had reduced mitochondrial clustering around the nucleus, suggestive of an overall quietening of metabolism. Thus, although periconception dietary supply of different levels of protein is permissive of development, mitochondrial metabolism is altered in the early embryo, and the nature of the perturbation differs between HPD and LPD exposure.

Precocious male puberty is a significant problem in Atlantic cod aquaculture. While photoperiod manipulation can inhibit testis growth, a detailed analysis of effects on spermatogenesis is missing. Starting July 1, 2004, prepubertal fish were exposed to different photoperiod regimens in indoor tanks for 17 mo. Testis histology, germ cell dynamics (proliferation and apoptosis), and plasma androgen levels were analyzed. In the natural light (NL) group, testis growth started in September 2004 and was completed in February 2005, when a 2-mo spawning period started. In the constant light (LL) group, none or very few spermatogenic cysts were recruited into spermatogenesis, and apoptotic germ cell loss was high. A change of photoperiod from NL to LL at winter solstice (December 21, 2004) resulted in premature (2 mo) completion of the reproductive cycle, while changing from LL to NL at winter solstice triggered faster than normal testis development. Plasma testosterone levels increased in the NL group from spermatogonial proliferation toward meiosis, while those of 11-ketotestosterone increased toward spermiogenesis and spermiation. Plasma androgen levels did not rise under LL conditions. Comparing fish with developing testes from all groups indicated that low androgen levels were associated with a high incidence of spermatogonial apoptosis; we also found that androgen receptor mRNA expression was most prominent in Sertoli cells in contact with growing spermatogonial clones. Our data show that an inhibitory photoperiod (LL) reduced or blocked differentiation of spermatogonia, increased apoptosis (particularly among proliferating spermatogonia), and was associated with reduced androgen levels, a situation possibly reflecting insufficient gonadotropic stimulation.

The luteolytic effects of exogenous prostaglandin F2alpha (PGF) that did and did not simulate natural 13,14-dihydro-15-keto-PGF (PGFM) pulses were studied during mid-diestrus in 42 Holstein heifers. Plasma concentrations of PGF were assessed by assay of PGFM. In experiment 1, a single intrauterine injection of 4.0 mg of PGF into the uterine horn ipsilateral to the corpus luteum resulted in a precipitous progesterone decline, whereas sequential injections of 0.25 or 1.0 mg every 12 h resulted in a stepwise decrease (P < 0.05) following each injection. A progesterone increase occurred during the first 5 min before the luteolytic decrease but only for the 4.0-mg dose. From the results of experiment 2, a 2-h intrauterine infusion of a total of 0.5 mg of PGF was judged to best simulate a natural PGFM pulse. In experiment 3, simulation of sequential pulses at 12-h intervals resulted in a continuous precipitous decrease in progesterone to <1 ng/ml by the beginning of the fourth simulated pulse. In contrast, a single simulated pulse resulted in a 6-h progesterone decrease to a constant concentration for 3 days after treatment, followed by a return to control concentrations. The mean ± SEM interval between the pretreatment and posttreatment ovulations was shorter (P < 0.05) in the group with sequential simulated pulses (14 ± 1 day) than in the group with a single pulse (21 ± 1 day). Results indicated that excessive PGF doses may stimulate nonphysiologic progesterone responses and supported the hypothesis that sequential PGF pulses are required to stimulate natural luteolysis in cattle.

The cause of reduced fecundity in women with endometriosis is unknown. Expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by both ectopic and eutopic endometrium reportedly has a role in the pathogenesis of endometriosis. We hypothesize that anomalous endometriotic TIMP protein synthesis, secretion, and localization also cause reproductive pathologies resulting in reduced fecundity. An established rat model for endometriosis (Endo) compared with nonendometriotic controls (Shams) was used to investigate reduced fecundity in endometriosis. Comparing Endo and Sham rats, Endo rats had altered ovarian dynamics, including fewer ovarian follicles and corpora lutea with luteinized unruptured follicles. Furthermore, in vivo anomalies in postovulatory oocyte structure and preimplantation embryo development, including misaligned chromosomes, nuclear and cytoplasmic fragmentation, and delayed or arrested cleavage, as well as spontaneous abortions, were found only in Endo rats. A causative role for TIMP1 in these phenomena is supported by our findings that Endo rats have more TIMP1 in their peritoneal fluid as detected by ELISA and more TIMP1 immunolocalization in the theca of antral follicles as measured by computer-assisted morphometric analysis. These data suggest that in endometriosis the accumulation of TIMP1 disrupts the normal MMP/TIMP enzymatic milieu in the peritoneal cavity and negatively affects ovarian dynamics, oocyte quality, and preimplantation embryo development, thereby decreasing fecundity. Most intriguingly, daughters of Endo rats that had no experimental interventions exhibited these same reproductive abnormalities. We predict that developmental exposure to endometriosis leads to permanent epigenetic changes in subsequent generations.

11Beta-hydroxysteroid dehydrogenase (HSD11B) enzymes have important roles in regulating cortisol availability in target tissues. We previously demonstrated that HSD11B1 is expressed and active in bovine endometrium and that cortisol suppresses prostaglandin (PG) F2alpha and PGE2 production in cultured bovine endometrial stromal cells. The present study was conducted to examine whether locally synthesized PGF2alpha and/or PGE2 regulates the enzymatic bioactivity and/or the expression of HSD11B1 in bovine endometrium. The conversion rate of cortisone to cortisol in cultured endometrial stromal cells was significantly stimulated by PGF2alpha (1 and 10 μM). In a dose-dependent manner, PGF2alpha but not PGE2 increased the net conversion of cortisone to cortisol in stromal cells after 4 h of treatment. In addition, the bioactivity of HSD11B1 was significantly inhibited by indomethacin (10 μM). The inhibitory effect of indomethacin on HSD11B1 bioactivity was abolished by PGF2alpha (1 μM) but not by PGE2. Although PGF2alpha (1 μM) did not affect the expression of HSD11B1 mRNA in cultured stromal cells, it significantly stimulated the protein expression of HSD11B1. Cycloheximide, a general translational inhibitor, abolished the stimulatory effects of PGF2alpha on HSD11B1 protein expression in endometrial stromal cells, indicating that PGF2alpha increases HSD11B1 protein expression by stimulating a posttranscriptional process rather than a transcriptional mechanism. These results demonstrate that PGF2alpha but not PGE2 increases HSD11B1 bioactivity and protein expression by stimulating a posttranscriptional mechanism in stromal cells and suggest that cortisol has a physiologically relevant role in preventing excessive uterine PG production in nonpregnant bovine endometrium.

Chlamydia trachomatis is a common sexually transmitted bacterial infection that results in health care costs in the United States that exceed $2 billion per year. Chlamydia infections cause damage to the oviducts, resulting in ectopic pregnancy and tubal factor infertility, but the reasons for defective oviduct function are poorly understood. We have investigated the role of oviduct contractions in egg transport and found that underlying electrical pacemaker activity is responsible for oviduct motility and egg transport. Specialized pacemaker cells, referred to as oviduct interstitial cells of Cajal (ICC-OVI), are responsible for pacemaker activity. The ICC-OVI, labeled with antibodies to KIT protein, form a dense network associated with the smooth muscle cells along the entire length of the oviduct. Selective removal of ICC-OVI with KIT-neutralizing antibody resulted in loss of electrical rhythmicity and loss of propulsive contractions of the oviduct. We tested whether infection might adversely affect the ICC-OVI. Mice infected with Chlamydia muridarum displayed dilation of oviducts, pyosalpinx, and loss of spontaneous contractile activity. Morphological inspection showed disruption of ICC-OVI networks, and electrophysiological recordings showed loss of intrinsic pacemaker activity without change in basal smooth muscle membrane potential. Chlamydia infection also was associated with upregulation of NOS2 (iNOS) and PTGS2 (COX II) in leukocytes. Loss of ICC-OVI and pacemaker activity causes oviduct pseudo-obstruction and loss of propulsive contractions for oocytes. This, accompanied by retention of oviduct secretions, may contribute to the development of tubal factor infertility.

Comparative analyses of differentially expressed genes between somatic cell nuclear transfer (SCNT) embryos and zygote-developing (ZD) embryos are important for understanding the molecular mechanism underlying the reprogramming processes. Herein, we used the suppression subtractive hybridization approach and from more than 2900 clones identified 96 differentially expressed genes between the SCNT and ZD embryos at the dome stage in zebrafish. We report the first database of differentially expressed genes in zebrafish SCNT embryos. Collectively, our findings demonstrate that zebrafish SCNT embryos undergo significant reprogramming processes during the dome stage. However, most differentially expressed genes are down-regulated in SCNT embryos, indicating failure of reprogramming. Based on Ensembl description and Gene Ontology Consortium annotation, the problems of reprogramming at the dome stage may occur during nuclear remodeling, translation initiation, and regulation of the cell cycle. The importance of regulation from recipient oocytes in cloning should not be underestimated in zebrafish.

Androgens have distinct physiological functions within the ovary. The biological action of androgens is primarily exerted through transcriptional regulation by the nuclear androgen receptor (AR), but the molecular cascades governed by AR remain largely unknown. At present, there is imminent concern that environmental man-made chemicals with antiandrogenic properties, among others, are capable of modulating hormonal responses, thereby interfering with normal physiological processes that are critical to fertility. In the present study, we aimed to further characterize a standardized and reproducible follicle culture system in terms of AR expression during in vitro folliculogenesis to be able to use it as a bioassay to study effects of antiandrogens on follicular and oocyte growth, steroid secretion profile, and oocyte meiotic maturation capacity. Immunohistochemical analysis revealed that cytoplasmic AR protein was translocated to the nucleus of granulosa and theca cells in response to endogenous androgen production in theca cells during preantral follicular development. During the antral phase in vitro, AR was differentially expressed in mural and cumulus cells, implying an oocyte-mediated regulation. Treatment of follicles with hydroxyflutamide or bicalutamide, two model antiandrogenic compounds, resulted in reduced follicular growth during the preantral phase, altered steroidogenic environment, and arrest in oocyte meiotic maturation in response to human chorionic gonadotropin. Androgen receptor expression in the culture model corresponded well to what is described in vivo, and this system revealed several ovarian functions targeted by AR antagonists that can be further investigated using more in-depth molecular techniques.

We describe a novel epididymis-specific cDNA named Glb1l4, which was isolated from rat epididymis by differential display of mRNAs. Glb1l4 cDNA contains 2607 nucleotides and encodes a 637-amino acid protein with 50% similarity to mouse beta-galactosidase. The gene is located on chromosome 8q13, spanning 21 exons. Northern blot analysis reveals that Glb1l4 is specifically expressed in the caput region of epididymis and upregulated by androgen. A specific polyclonal antiserum against the N-terminal peptide of GLB1L4 has been produced. Western blot analysis and immunohistochemistry assay reveal that GLB1L4 is specifically expressed in the principal cells of the caput epididymis. Interestingly, its expression peaks at Postnatal Day 45 in mRNA level and at Postnatal Day 60 in protein level while the epididymis column cells undergo differentiation. Moreover, within this very period this secretory protein is confined inside the cell with a change of subcellular distribution pattern, which implies its important roles in the cell differentiation process. Only after the epididymal epithelium differentiation is completed and the spermatozoa enter the epididymal lumen is the GLB1L4 secreted into the luminal fluid and bound on the sperm head. Our results suggest that GLB1L4 may play various roles in principal cell differentiation and sperm maturation.

To characterize the molecular phenotype of spermatogonial stem cells (SSCs), we examined genes that are differentially expressed in the stem/progenitor spermatogonia compared to nonstem spermatogonia. We isolated type A spermatogonia (stem and nonstem type A) from 6-day-old mice using sedimentation velocity at unit gravity and further selected the stem/progenitor cell subpopulation by magnetic activated cell sorting with an antibody to GDNF-receptor-alpha-1 (GFRA1). It has been previously shown that GFRA1 is expressed in SSCs and is required for their stemness. The purity of the isolated cells was approximately 95% to 99% as indicated by immunocytochemistry using anti-GFRA1. Comparison of GFRA1-positive and GFRA1-negative spermatogonia by microarray analysis revealed 99 known genes and 12 uncharacterized transcripts that are overexpressed in the former cell population with a ?>2-fold change. Interestingly, the highest level of overexpression was observed for Csf1r, encoding the receptor for macrophage colony-stimulating factor (M-CSF, official symbol CSF1), which has a well-established role in the regulation of myeloid progenitor cells. Analysis of our microarray data with a bioinformatics software program (Ingenuity Systems) revealed the potential role of various signaling pathways in stem/progenitor spermatogonia and suggested a common pathway for GFRA1 and CSF1R that may lead to their proliferation. Further investigation to test this hypothesis has shown that CSF1 promotes cell proliferation in primary cultures of the isolated type A spermatogonia and in the spermatogonial-derived stem cell line C18–4. Semiquantitative RT-PCR and immunohistochemistry confirmed the previously mentioned microarray data. Collectively, this study provides novel molecular signatures for stem/progenitor spermatogonia and demonstrates a role for CSF1/CSF1R signaling in regulating their proliferation.

Prenatal testosterone excess leads to neuroendocrine and periovulatory disruptions in the offspring culminating in progressive loss of cyclicity. It is unknown whether the mediary of these disruptions is androgen or estrogen, because testosterone can be aromatized to estrogen. Taking a reproductive life span approach of studying control, prenatal testosterone, and dihydrotestosterone-treated offspring, this study tested the hypothesis that disruptions in estradiol-negative but not -positive feedback effects are programmed by androgenic actions of testosterone and that these disruptions in turn will have an impact on the periovulatory hormonal dynamics. The approach was to test estradiol-negative and -positive feedback responses of all three groups of ovary-intact females during prepubertal age and then compare the periovulatory dynamics of luteinizing hormone, follicle-stimulating hormone, estradiol, and progesterone during the first breeding season. The findings show that estradiol-negative but not estradiol-positive feedback disruptions in prenatal testosterone-treated females are programmed by androgenic actions of prenatal testosterone excess and that follicular phase estradiol and gonadotropins surge disruptions during reproductive life are consistent with estrogenic programming. Additional studies carried out testing estradiol-positive feedback response over time found progressive deterioration of estradiol-positive feedback in prenatal testosterone-treated sheep until the time of puberty. Together, these findings provide insight into the mechanisms by which prenatal testosterone disrupts the reproductive axis. The findings may be of translational relevance since daughters of mothers with hyperandrogenism are at risk of increased exposure to androgens.

Prenatal testosterone excess programs an array of adult reproductive disorders including luteinizing hormone excess, functional hyperandrogenism, neuroendocrine defects, polycystic ovarian morphology, and corpus luteum dysfunction, culminating in early reproductive failure. Polycystic ovarian morphology originates from enhanced follicular recruitment and follicular persistence. We tested to determine whether prenatal testosterone treatment, by its androgenic actions, enhances follicular recruitment, causes early depletion of follicular reserve, and disrupts the ovarian architecture. Pregnant sheep were given twice-weekly injections of testosterone or dihydrotestosterone (DHT), a nonaromatizable androgen, from Days 30 to 90 of gestation. Ovaries were obtained from Day-90 and Day-140 fetuses, and from 10-mo-old females during a synchronized follicular phase (n = 5–9 per treatment). Stereological techniques were used to quantify changes in ovarian follicle/germ cell populations. Results revealed no differences in numbers of oocytes and follicles between the three groups on Fetal Day 90. Greater numbers of early growing follicles were found in prenatal testosterone- and DHT-treated fetuses on Day 140. Increased numbers of growing follicles and reduced numbers of primordial follicles were found in 10-mo-old, prenatal testosterone-treated females, but not in those treated with DHT. Antral follicles of prenatal testosterone-treated females, but not those treated with DHT, manifested several abnormalities, which included the appearance of hemorrhagic and luteinized follicles and abnormal early antrum formation. Both treatment groups showed morphological differences in the rete ovarii. These findings suggest that increased follicular recruitment and morphologic changes in the rete ovarii of prenatal testosterone-treated females are facilitated by androgenic programming, but that postpubertal follicular growth, antral follicular disruptions, and follicular depletion largely occur through estrogenic programming.

Previous research with female sheep indicates that exposure to excess testosterone for 60 days (from Gestational Days 30–90 of the 147-day gestation) leads to virilized genitalia, severe neuroendocrine deficits, as well as masculinization and defeminization of sexual behavior (T60 females). In contrast, 30 days of testosterone exposure (Gestational Days 60–90) produce animals with female-typical genitalia, less severe neuroendocrine alterations, and variable gender patterns of sexual behavior (T30 females). Variation in adult sexual behavior of male ungulates is influenced by early social experience, but this has never been tested in females. Here we investigate the influence of rank in the dominance hierarchy on the expression of adult sexual behavior in females. Specifically, we hypothesized that juvenile rank would predict the amount of male- and female-typical mating behavior exhibited by adult female sheep. This hypothesis was tested in two treatment groups and their controls (group 1: T60 females; group 2: T30 females). Dominance hierarchies were determined by observing competition over resources. Both groups of prenatal testosterone-treated females were higher ranking than controls (T60: P = 0.05; T30: P < 0.01). During the breeding season, both T60 and T30 females exhibited more male-typical mating behavior than did controls; however, the T30 animals also exhibited female-typical behavior. For the T60 group, prenatal treatment, not juvenile rank, best predicted male-typical sex behavior (P = 0.007), while juvenile rank better predicted male mating behavior for the T30 group (P = 0.006). Rank did not predict female mating behavior in the hormone-treated or control ewes. We conclude that the effect of prenatal testosterone exposure on adult male-specific but not female-specific mating behavior is modulated by juvenile social experiences.

Maternal B-vitamin status and homocysteinemia can affect fertility and pregnancy establishment, although the direct effects on ovarian follicle and oocyte development are not known. We report on the effects of restricting the supply of vitamin B₁₂ and methionine from the diet of mature female sheep on ovarian folliculogenesis following follicle-stimulating hormone (FSH) stimulation. The study was split into three batches and involved 76 animals. Surprisingly, the number of growing, estrogen-active antral follicles following FSH treatment was enhanced (P = 0.005) following this dietary intervention. This increase occurred even in the presence of modest live-weight loss (batch 1 only) and depressed plasma insulin concentrations, suggesting a breakdown in the regulation of follicular responsiveness to FSH. This dietary intervention also increased plasma homocysteine concentrations. Physiological concentrations of homocysteine increased granulosa cell proliferation (P < 0.001), estradiol production (P = 0.05), and FSHR transcript expression (P = 0.017) during culture. Transcript levels for growth differentiation factor 9 and bone morphogenetic protein 15 in oocytes from treated ewes were increased (P < 0.05) in the first two batches. Furthermore, regression of BMP receptor 2 (BMPR2) transcript expression and diet on follicle number revealed a significant interaction (P = 0.01); BMPR2 transcript expression was associated with follicle number only in vitamin B₁₂/methionine-restricted animals. Because FSHR transcript expression also was positively (P = 0.007) related to follicle number, the effects of diet may have arisen through enhanced FSH and BMP signaling. Although this remains to be confirmed, the data support an intraovarian impact of vitamin B₁₂/methionine-deficient diets.

Several studies have shown that dopamine and other catecholamines are present in oviduct luminal fluid. We recently reported that dopamine type 2 receptors (DRD2) are present in a wide range of mammalian sperm, suggesting a role for dopaminergic signaling in events such as fertilization, capacitation, and sperm motility. In the present study, we used Western blot analysis to show that boar sperm express DRD2 and that their activation with dopamine (100 nM) has a positive effect on cell viability that can be correlated with AKT/PKB phosphorylation. Bromocriptine (100 nM) and dopamine (100 nM and 10 μM) increased tyrosine phosphorylation during the capacitation period. Immunofluorescence analysis indicated that DRD2 localization is dynamic and depends on the capacitation stage, colocalizing with tyrosine phosphorylated proteins in the acrosome and midpiece region of capacitated boar sperm. This association was confirmed by coimmunoprecipitation analysis. We also showed that bromocriptine (100 nM) and low-concentration dopamine (100 nM and 10 μM) increased total and progressive motility of sperm. However, high concentrations of dopamine (1 mM) decreased tyrosine phosphorylation and motility in in vitro sperm capacitation assays. This can be explained by the presence of the dopamine transporters (DAT, official symbol SLC6A3) in sperm, as demonstrated by Western blot analysis and immunocytochemistry. Taken together, our results support the idea that dopamine may have a fundamental role during sperm capacitation and motility in situ in the female upper reproductive tract.

Mitochondria are dynamic organelles that undergo fusion, fission, and translocation. The dynamic property is essential for establishing energy-consuming biological processes including cellular differentiation. Early ultrastructural studies have shown that mitochondria of mammalian spermatogenic cells dramatically change their number, size, distribution, and internal structure. However, its regulatory mechanism is largely unknown. In course of searching for molecules involved in the mitochondrial morphogenesis in spermatogenesis, we identified mouse gametogenetin-binding protein 1 (GGNBP1), a DUF1055 domain-containing protein of unknown function, as a mitochondrial protein. When GGNBP1 was expressed in COS7 cells, it was localized in the intermembrane space and induced an extensive fragmentation of mitochondria in the manner dependent on the activity of the mitochondrial fission factor DNM1L. Deletion mutant analyses demonstrated that the N-terminal region is required for its mitochondrial targeting and that the C-terminal region including the DUF1055 domain is responsible for the mitochondrial fragmentation activity. Immunohistochemistry of mouse testis revealed that GGNBP1 is highly expressed in the late pachytene spermatocytes and early round spermatids. However, a subcellular fractionation study showed that it is localized to not only mitochondria but also other membranous compartments in vivo. These results suggest that GGNBP1 is involved in spermatogenesis by modifying mitochondrial dynamics and morphology.

Programmed germ cell death is critical for functional spermatogenesis. Increased germ cell apoptosis can be triggered by various regulatory stimuli, including testicular hyperthermia or deprivation of gonadotropins and intratesticular testosterone. We have previously shown the involvement of the mitogen-activated protein kinase (MAPK) 14 in apoptotic signaling of male germ cells across species after hormone deprivation. This study investigates the role of MAPK14 in germ cell apoptosis in rats triggered by testicular hyperthermia. The contributions of the MAPK1/3 and the MAPK8 to male germ cell death were also examined after this intervention. We show that 1) testicular hyperthermia results in induction of both MAPK1/3 and MAPK14 but not MAPK8; 2) inhibition of MAPK1/3 has no effect on the incidence of heat-induced germ cell apoptosis, suggesting that MAPK1/3 signaling may be dispensable for heat-induced male germ cell apoptosis; and 3) activation of MAPK14 and BCL2 phosphorylation are critical for heat-induced male germ cell apoptosis in rats. Thus, unlike the hormone deprivation model, heat stress through activation of the MAPK14 signaling promotes germ cell apoptosis by provoking BCL2 phosphorylation, leading to its inactivation and the subsequent activation of the mitochondria-dependent death pathway. These novel findings point to a critical role of MAPK14 in stage- and cell-specific activation of male germ cell apoptosis triggered by hormone deprivation or heat stress.

Untimely rupture of the fetal membranes (FMs) is a major precipitant of preterm birth. Although the mechanism of FM weakening leading to rupture is not completely understood, proinflammatory cytokines, including tumor necrosis factor (TNF) and interleukin 1 beta (IL1B), have been shown to weaken FMs concomitant with the induction of reactive oxygen species, collagen remodeling, and prostaglandin release. We hypothesized that alpha-lipoic acid, a dietary antioxidant, may block the effect of inflammatory mediators and thereby inhibit FM weakening. Full-thickness FM fragments were incubated with control media or TNF, with or without alpha-lipoic acid pretreatment. Fetal membrane rupture strength and the release of matrix metalloproteinase 9 (MMP9) and prostaglandin E₂ (PGE₂) from the full-thickness FM fragments were determined. The two constituent cell populations in amnion, the mechanically strongest FM component, were similarly examined. Amnion epithelial and mesenchymal cells were treated with TNF or IL1B, with or without alpha-lipoic acid pretreatment. MMP9 and PGE₂ were analyzed by ELISA, Western blot, and zymography. TNF decreased FM rupture strength 50% while increasing MMP9 and PGE₂ release. Lipoic acid inhibited these TNF-induced effects. Lipoic acid pretreatment also inhibited TNF- and IL1B-induced increases in MMP9 protein activity and release in amnion epithelial cells, as well as PGE₂ increases in both amnion epithelial and mesenchymal cells. In summary, lipoic acid pretreatment inhibited TNF-induced weakening of FM and cytokine-induced MMP9 and PGE₂ in both intact FM and amnion cells. We speculate that dietary supplementation with alpha-lipoic acid might prove clinically useful in prevention of preterm premature rupture of fetal membranes.

In a search for genes involved in regulation of uterine contractility, we cloned a novel calcium-activated chloride channel gene, named rat Clca4, from pregnant rat uterus. The gene shares approximately 83% and 70% nucleotide homology with mouse Clca6 and human CLCA4, respectively, and was expressed primarily in rat uterus. The transcripts were upregulated at Gestational Day 22 (prior to parturition), implying a functional involvement in parturition. Western blot analysis showed that rat CLCA4 protein was present in uterus, lung, and heart, but not in any other tissues examined. Confocal microscopy revealed that rat CLCA4 is localized in cell membrane and could not be removed by alkaline or PBS washing. Transient transfection of rat CLCA4-enhanced green fluorescent protein in Chinese hamster ovary cells resulted in production of characteristic Cl⁻ currents that could be activated by Ca² and ionomycin but inhibited by niflumic acid, a CLCA-channel blocker. The identification and characterization of rat Clca4 help decipher the contribution of Ca² -activated Cl⁻ conductance in myometrial contractility.

Meiotic maturation in oocytes is a prolonged process that is unique because of cell cycle arrests at prophase of meiosis I (MI) and at metaphase of meiosis II (MII). Fluctuations in cyclin-dependent kinase 1 (CDK1/CDC2A) activity govern meiotic progression, yet little is known about how these fluctuations are achieved. CDC14 is a highly conserved dual-specificity phosphatase that counteracts the function of proteins phosphorylated by CDK. Mammals contain two CDC14 homologs, CDC14A and CDC14B. We report that CDC14B localizes with the meiotic spindle in mouse oocytes, and (unlike somatic cells) it does not localize in the nucleolus. Oocytes that overexpress CDC14B are significantly delayed in resuming meiosis and fail to progress to MII, whereas oocytes depleted of CDC14B spontaneously resume meiosis under conditions that normally inhibit meiotic resumption. Depletion of FZR1 (CDH1), a regulatory subunit of the anaphase-promoting complex/cyclosome that targets cyclin B1 (CCNB1) for ubiquitin-mediated proteolysis, partially restores normal timing of meiotic resumption in oocytes with excess CDC14B. These studies also reveal that experimentally altering CDC14B levels generates eggs with abnormal spindles and with chromosome alignment perturbations. Our data indicate that CDC14B is a negative regulator of meiotic resumption and may regulate MI in mouse oocytes.

In mice, male germ cells enter mitotic arrest beginning at 13.5 days postcoitum (dpc), and remain suspended in the G₀/G₁ cell cycle stage until after birth. During this period, male germ cells undergo extensive epigenetic reprogramming, which is essential for their subsequent function as male gametes. A global reorganization and spatial clustering of constitutive heterochromatin has been implicated in epigenetic plasticity during cellular differentiation. Here, we have studied the dynamics of heterochromatin in fetal (12.5–19.5 dpc) and neonatal (4 days postpartum) male germ cells. We monitored constitutive heterochromatin-specific markers, and observed changes in the association of histone H3 trimethylation of lysine 9 (H3K9me3), binding of heterochromatin protein 1, and patterns of 4′,6-diamino-2-phenylindole staining in pericentric regions of chromosomes, along with a coincident loss of chromocenters in fetal prospermatogonia during mitotic arrest. We also observed a transient loss of H3K9me3 associated with major and minor satellite repeat sequences, plus inactivation of histone methyltransferases (Suv39h1 and Suv39h2), and transient activation of histone demethylase (Jmjd2b) in these same cells. These epigenetic changes were correlated with relocation of centromeric regions toward the nuclear periphery in prospermatogonia during mitotic arrest. Taken together, these results show that constitutive heterochromatin undergoes dramatic reorganization during prespermatogenesis. We suggest that these dynamic changes in heterochromatin contribute to normal epigenetic reprogramming of the paternal genome in fetal prospermatogonia suspended in the G₀/G₁ stage, and that this also represents an epigenomic state that is particularly amenable to reprogramming.

Abnormal regulation of placental apoptosis and proliferation has been implicated in placental disorders. Recently, several DNA-damaging agents were reported to induce excessive apoptosis and reduce cell proliferation in the placenta; however, the molecular pathways of these toxic effects on the placenta are unclear. The aim of the present study was to determine the involvement of TRP53, a tumor suppressor that mediates cellular responses to DNA damage, in the induction of apoptosis and cell cycle arrest in the developing placenta. For this purpose, we treated pregnant mice on Day 12 of gestation with 10 mg/kg of etoposide and 5-Gy gamma irradiation, potent inducers of DNA damage. We found an increase in the number of trophoblastic apoptoses 8 and 24 h after etoposide injection and 6 and 24 h after irradiation in the placental labyrinth zone. The number of mitoses and DNA syntheses in trophoblasts decreased after treatment. The accumulation and phosphorylation of TRP53 protein were detected 8 and 6 h after etoposide injection and irradiation, respectively. In Trp53-deficient placentas, the induction of etoposide-induced trophoblastic apoptosis is abrogated, while the reduction of proliferation occurred similarly as in wild-type placentas. CDC2A, a regulator of G2/M progression, was inactivated by phosphorylation after etoposide injection and irradiation, suggesting that the cell cycle was arrested at the G2/M border by treatment. Our study demonstrated that etoposide injection induced TRP53-dependent apoptosis and TRP53-independent cell cycle arrest in labyrinthine trophoblasts, providing insights into the molecular pathway of placental disorders.

The present study was conducted to explore the source of acetylcholine (ACH) in the corpus luteum (CL) and to test our hypothesis of an antiapoptotic role of ACH in the bovine CL and, further, to investigate whether nerve growth factor (NGF), insulin-like growth factor 1 (IGF1), and transforming growth factor beta1 (TGFB1) influence the expression of choline acetyltransferase (CHAT), the biosynthetic enzyme of ACH, in cultured bovine luteal cells. Protein expression and immunolocalization of CHAT were carried out at different stages throughout the luteal phase and in cultured luteal and endothelial cells. ACH was measured in luteal tissue at the different luteal stages and in luteal cells cultured for 8 and 24 h. Cell viability and TUNEL assays were performed on cultured midluteal cells treated with or without tumor necrosis factor alpha (TNF)/interferon gamma (IFNG) in the presence of ACH and its muscarinic (atropine) and nicotinic (mecamylamine) receptor antagonists. The CL was devoid of cholinergic nerve fibers. CHAT immunostaining was evident in luteal, endothelial, and stromal cells in luteal tissue sections and in cultured luteal and endothelial cells. CHAT protein was expressed throughout the cycle without any significant changes. ACH concentration in luteal tissue was not changed during the luteal stages but increased over time and with increased cell numbers in luteal cell cultures. ACH increased cell viability and prevented cell death induced by TNF/IFNG. Atropine significantly attenuated ACH action, whereas mecamylamine had no effect. TNF/IFNG treatment downregulated CHAT expression, whereas NGF, IGF1, and TGFB1 upregulated CHAT expression, in cultured luteal cells. The overall findings strongly suggest a nonneural source and antiapoptotic role of ACH in the bovine CL. Locally produced ACH appears to be regulated by NGF, IGF1, and TGFB1.

The involvement of egg integrins in mammalian sperm-egg interactions has been controversial, with data from integrin inhibitor studies contrasting with evidence from knockouts showing that specific integrin subunits are not essential for fertility. An alpha₄/alpha₉ (ITGA4/ITGA9) integrin subfamily member has been implicated in fertilization but not extensively examined, so we tested the following three hypotheses: 1) an ITGA4/ITGA9 integrin participates in sperm-egg interactions, 2) short-term acute knockdown by RNA interference of integrin subunits would result in a fertilization phenotype differing from that of chronic depletion via knockout, and 3) detection of a fertilization phenotype is sensitive to in vitro fertilization (IVF) assay conditions. We show that mouse and human eggs express the alpha₉ integrin subunit (ITGA9). RNA interference-mediated knockdown resulted in reduced levels of Itga9 mRNA and surface protein in mouse eggs. RNA interference attempts to knockdown ITGA9′s likely beta partner, beta₁ (ITGB1), resulted in reduced Itgb1 mRNA but no reduction in ITGB1 surface protein. Therefore, studies using a function-blocking anti-ITGB1 antibody tested the hypothesis that ITGB1 participates in gamete interactions. Analyses of sperm-egg interactions with Itga9-knockdown eggs and anti-ITGB1 antibody-treated eggs in IVF assays using specific sperm:egg ratios revealed the following: 1) a reduction, but not complete loss, of sperm-egg binding and fusion was observed and 2) the reduction of sperm-egg binding and fusion was not detected in inseminations with high sperm:egg ratios. These data demonstrate that ITGA9 and ITGB1 participate in sperm-egg interactions but clearly are not the only molecules involved. This also shows that careful design of IVF parameters allows detection of deficiencies in gamete interactions.