Obstetric use of the antimalarial drug mefloquine has historically been discouraged during the first trimester and immediately before conception owing to concerns of potential fetal harm. With the rise of resistance to the antimalarial drug sulfadoxine-pyrimethamine (SP), mefloquine is now being considered as a replacement for SP for universal antenatal administration to women from malaria-endemic regions. Recent recommendations have also suggested that mefloquine may be used cautiously among pregnant travelers who cannot otherwise avoid visiting these areas. Mefloquine has been demonstrated to cause blockade of gap junction protein alpha 1 (GJA1) gap junction intercellular communication (GJIC), and recent evidence suggests that GJA1 GJIC is critical to successful embryonic implantation and early placental development. During routine use, mefloquine accumulates in organ and peripheral tissue, crosses the blood-placental barrier, and may plausibly accumulate in developing decidua and trophoblast at concentrations sufficient to interfere with GJA1 GJIC and, thus, cause deleterious effects on fetal outcomes. This conclusion is supported by epidemiological evidence that demonstrates use of the drug during early development is associated with an increased risk of miscarriage and stillbirth. Confirmatory studies are pending, but the available experimental and epidemiological evidence support renewed adherence, where feasible, to existing mefloquine package insert guidance that women avoid the drug during the periconceptional period.

Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. In the present study, we cloned cDNAs of cAMP-dependent protein-kinase (PKA) subunits from porcine oocytes and analyzed the involvement of the PKA regulation mechanism in the meiotic incompetence of GOs at the molecular level. We found a cAMP-independent high PKA activity in GOs throughout the in vitro culture using a porcine PKA assay system we established, and inhibition of the activity by injection of the antisense RNA of the PKA catalytic subunit (PKA-C) induced meiotic resumption in GOs. Then we examined the possibility that the amount of the PKA regulatory subunit (PKA-R), which can bind and inhibit PKA-C, was insufficient to suppress PKA activity in GOs because of the overexpression of two PKA-Rs, PRKAR1A and PRKAR2A. We found that neither of them affected PKA activity and induced meiotic resumption in GO although PRKAR2A could inhibit PKA activity and induce meiosis in cAMP-treated full-grown oocytes (FGOs). Finally, we analyzed the subcellular localization of PKA subunits and found that all the subunits were localized in the cytoplasm during meiotic arrest and that PKA-C and PRKAR2A, but not PRKAR1A, entered into the nucleus just before meiotic resumption in FGOs, whereas all of them remained in the cytoplasm in GOs throughout the culture period. Our findings suggest that the continuous high PKA activity is a primary cause of the meiotic incompetence of porcine GOs and that this PKA activity is not simply caused by an insufficient expression level of PKA-R, but can be attributed to more complex spatial-temporal regulation mechanisms.

Glucose is an important monosaccharide required to generate energy in all cells. After entry into cells, glucose is phosphorylated to glucose-6-phosphate and then transformed into glycogen or metabolized to produce energy. Glucose phosphate isomerase (GPI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. Without GPI activity or fructose-6-phosphate, many steps of glucose metabolism would not occur. The requirement for GPI activity for normal functioning of primordial germ cells (PGCs) needs to be identified. In this study, we first examined the expression of chicken GPI during early embryonic development and germ cell development. GPI expression was strongly and ubiquitously detected in chicken early embryos and embryonic tissues at Embryonic Day 6.5 (E6.5). Continuous GPI expression was detected in PGCs and germ cells of both sexes during gonadal development. Specifically, GPI expression was stronger in male germ cells than in female germ cells during embryonic development and the majority of post-hatching development. Then, we used siRNA-1499 to knock down GPI expression in PGCs. siRNA-1499 caused an 85% knockdown in GPI, and PGC proliferation was also affected 48 h after transfection. We further examined the knockdown effects on 28 genes related to the glycolysis/gluconeogenesis pathway and the endogenous glucose level in chicken PGCs. Among genes related to glycolysis/gluconeogenesis, 20 genes showed approximately 3-fold lower expression, 4 showed approximately 10-fold lower, and 2 showed approximately 100-fold lower expression in knockdown PGCs. The endogenous glucose level was significantly reduced in knockdown PGCs. We conclude that the GPI gene is crucial for maintaining glycolysis and supplying energy to developing PGCs.

An in vitro system was used to analyze the effects of sex steroids on the development of primary (late perinucleolar stage) and early secondary, previtellogenic (early cortical alveolus stage) ovarian follicles of coho salmon cultured for up to 21 days. Late perinucleolar-stage follicles increased significantly in size after 7 days of treatment with low concentrations of 11-ketotestosterone (11-KT), a nonaromatizable androgen. An androgen receptor antagonist (flutamide) inhibited this growth-promoting effect, and the highest concentration resulted in atresia of follicles, implicating androgens as survival factors at this stage. Testosterone (T) was less effective than 11-KT in promoting growth, but blocking aromatization with exemestane resulted in a growth response similar to that of 11-KT. Estradiol-17beta (E2) had no effect on growth at this stage. After 21 days of culture, E2 was the most potent steroid in increasing the number of follicles containing cortical alveoli and the number of cortical alveoli within those follicles. At the early cortical alveolus stage, low doses of E2 promoted growth and strongly stimulated synthesis of cortical alveoli, actions that were inhibited by an estrogen receptor antagonist (tamoxifen). 11-KT displayed moderate growth-promoting effects, and 11-KT and T stimulated moderate to substantial increases in abundance of cortical alveoli. This study shows that the predominant role of androgens is the promotion of growth of late perinucleolar-stage follicles, while E2 stimulates both the growth and accumulation of cortical alveoli in early cortical alveolus-stage follicles.

The sperm connecting piece is a complex structure that, from a mechanical perspective, appears to play a role in stabilizing the proximal part of the sperm tail. We report the three-dimensional structure of the intact bovine sperm connecting piece, revealing an intricate, asymmetrical architecture with the segmented columns held together by filamentous linkages. The columns fuse, at the proximal end, with each other into structures that form the centriolar vault, and at the distal end, with the outer dense fibers (ODFs). The grouping of the fibers into these structures is consistent with bending only in the plane of the head. Structures reminiscent of the proximal centriole were observed in the vault, while the association of a novel bar structure with ODFs 3 and 8 organizes the distal centriolar vault. It has been proposed that the elastic compliance of the connecting piece provides the underlying mechanism behind initiation of the sperm beat cycle and bend propagation. According to the basal sliding theory of sperm movement, distortion of the connecting piece may store energy that initiates a new beat. The intersegment linkers could serve as mechanosensitive elements that regulate alternation of the sperm tail's bending direction in the beat cycle in addition to providing structural stabilization for the connecting piece segmented structures. On the other hand, our video recordings of the bull sperm movement show little bending of the head with respect to the tail, so it appears that there may be normally little strain within the connecting piece.

Electron cryotomography of the structure of the sperm connecting piece yields novel insights into the motility of spermatozoa.

In most vertebrates, fully grown oocytes are arrested in meiotic prophase I and only resume the cell cycle upon external stimuli, such as hormones. The proper arrest and resumption of the meiotic cycle is critical for reproduction. A Galpha_S signaling pathway essential for the arrest is conserved in organisms from Xenopus to mouse and human. A previous gene association study implicated that mutations of human ACSL6 may be related to premature ovarian failure. However, functional roles of ACSL6 in human infertility have yet to be reported. In the present study, we found that triacsin C, a potent and specific inhibitor for ACSL, triggers maturation in Xenopus and mouse oocytes in the absence of hormone, suggesting ACSL activity is required for the oocyte arrest. In Xenopus, acsl1b may fulfill a major role in the process, because inhibition of acsl1b by knocking down its RNA results in abnormal acceleration of oocyte maturation. Such abnormally matured eggs cannot support early embryonic development. Moreover, direct inhibition of protein palmitoylation, which lies downstream of ACSLs, also causes oocyte maturation. Furthermore, palmitoylation of Galpha_s, which is essential for its function, is inhibited when the ACSL activity is blocked by triacsin C in Xenopus. Thus, disruption of ACSL activity causes inhibition of the Galpha_s signaling pathway in the oocytes, which may result in premature ovarian failure in human.

Although substantial evidence exists that sperm ATP production via glycolysis is required for mammalian sperm function and male fertility, conflicting reports involving multiple species have appeared regarding the ability of individual glycolytic or mitochondrial substrates to support the physiological changes that occur during capacitation. Several mouse models with defects in the signaling pathways required for capacitation exhibit reductions in sperm ATP levels, suggesting regulatory interactions between sperm metabolism and signal transduction cascades. To better understand these interactions, we conducted quantitative studies of mouse sperm throughout a 2-h in vitro capacitation period and compared the effects of single substrates assayed under identical conditions. Multiple glycolytic and nonglycolytic substrates maintained sperm ATP levels and comparable percentages of motility, but only glucose and mannose supported hyperactivation. These monosaccharides and fructose supported the full pattern of tyrosine phosphorylation, whereas nonglycolytic substrates supported at least partial tyrosine phosphorylation. Inhibition of glycolysis impaired motility in the presence of glucose, fructose, or pyruvate but not in the presence of hydroxybutyrate. Addition of an uncoupler of oxidative phosphorylation reduced motility with pyruvate or hydroxybutyrate as substrates but unexpectedly stimulated hyperactivation with fructose. Investigating differences between glucose and fructose in more detail, we demonstrated that hyperactivation results from the active metabolism of glucose. Differences between glucose and fructose appeared to be downstream of changes in intracellular pH, which rose to comparable levels during incubation with either substrate. Sperm redox pathways were differentially affected, with higher levels of associated metabolites and reactive oxygen species generated during incubations with fructose than during incubations with glucose.

Follistatin 1 (Fst1) is a binding protein of activin and some other members of the transforming growth factor beta superfamily. It plays a key role in the regulation of gonadal function in vertebrates. An oocyte-specific promoter, derived from the zona pellucida 3 (zp3) gene, was used to create transgenic fst1 zebrafish (Danio rerio). Three independent oocyte-specific overexpression fst1 transgenic zebrafish lines were generated. Decreased levels of phosphorylated Smad3 were observed in ovarian tissues in fst1 transgenic fish compared with those from their control female siblings. Analyses on the numbers of mature eggs also indicated the attenuated oocyte maturation in the fst1 transgenic fish and in the females administered recombinant human Fst protein. Remarkably, when raised in the same tank with their control siblings, a significantly larger proportion of the fst1 transgenic population developed as males compared to the controls. Moreover, assessing the levels of active caspase 3 in gonadal tissues at 30 days postfertilization, we observed increased levels of apoptosis in the transitioning gonads of the transgenic fish compared to nontransgenic control siblings. Our results demonstrate that zebrafish Fst1 not only acts as an inhibitory binding protein of activin in the regulation of oocyte maturation in adult females but also plays a potential role in the masculinization of juveniles. Overall, the present study contributes to our understanding of the paracrine roles of fst1 as well as normal oocyte maturation and gonadal differentiation.

Fish vitellogenin synthesized and released from the liver of oviparous animals is taken up into oocytes by the vitellogenin receptor. This is an essential process in providing nutrient yolk to developing embryos to ensure successful reproduction. Here we disclose the full length vtgr cDNA sequence for largemouth bass (LMB) that reveals greater than 90% sequence homology with other fish vtgr sequences. We classify LMB Vtgr as a member of the low density lipoprotein receptor superfamily based on conserved domains and categorize as the short variant that is devoid of the O-glycan segment. Phylogenetic analysis places LMB Vtgr sequence into a well-supported monophyletic group of fish Vtgr. Real-time PCR showed that the greatest levels of LMB vtgr mRNA expression occurred in previtellogenic ovarian tissues. In addition, we reveal the effects of insulin, 17beta-estradiol (E₂), and 11-ketotestosterone (11-KT) in modulation of vtgr, esr, and ar mRNAs in previtellogenic oocytes. Insulin increased vtgr expression levels in follicles ex vivo while exposure to E₂ or 11-KT did not result in modulation of expression. However, both steroids were able to repress insulin-induced vtgr transcript levels. Coexposure with insulin and E₂ or of insulin and 11-KT increased ovarian esr2b and ar mRNA levels, respectively, which suggest a role for these nuclear receptors in insulin-mediated signaling pathways. These data provide the first evidence for the ordered stage-specific expression of LMB vtgr during the normal reproductive process and the hormonal influence of insulin and sex steroids on controlling vtgr transcript levels in ovarian tissues.

Neovascularization is necessary for formation of the corpus luteum (CL) and includes angiogenesis and vasculogenesis. Vasculogenesis is the formation of new blood vessels by bone marrow-derived endothelial progenitor cells. Here we investigated whether vasculogenesis occurs in neovascularization during CL formation. Mice transplanted with bone marrow from transgenic mice expressing green fluorescent protein (GFP) were injected with equine chorionic gonadotropin and human chorionic gonadotropin (hCG) to induce ovulation and subsequent CL formation. Immunohistochemistry was performed on the ovaries obtained before hCG injection and at 6, 12, and 24 h after hCG injection using antibodies for CD34 or CD31 (an endothelial cell marker), platelet-derived growth factor receptor beta (PDGFR-beta, a pericyte marker), F4/80 (a macrophage marker), and GFP (a bone marrow-derived cell marker). Cells immunostained for CD34, PDGFR-beta, F4/80, and GFP were present in the theca cell layer of the preovulatory follicle before hCG injection. Each of these cell types invaded the granulosa cell layer after hCG injection, and a number of them were observed in the CL 24 h after hCG injection. Fluorescence-based immunohistochemistry or double immunohistochemical staining revealed that a few CD34/CD31-positive cells and PDGFR-beta-positive cells were also positive for GFP in the preovulatory follicle and CL, and that many of the GFP-positive cells recruited to the CL during CL formation were F4/80-positive macrophages. In conclusion, bone marrow-derived vascular progenitor cells and macrophages contribute to neovascularization during CL formation.

The actions of different concentrations of insulin alone or in combination with follicle-stimulating hormone (FSH) were evaluated by in vitro follicular development and mRNA expression of cytochrome P450 aromatase (CYP19A1) and as receptors for insulin (INSR) and FSH (FSHR) from isolated, cultured goat preantral follicles. Goat preantral follicles were microdissected and cultured for 18 days in the absence or presence of insulin (5 and 10 ng/ml or 10 μg/ml) alone or in combination with FSH. After 18 days, the addition of the maximum concentration of insulin to the culture medium reduced follicular survival and antrum formation rates significantly compared to the other treatments. However, when FSH was added to the culture medium, no differences between these two parameters were observed. Preantral and antral follicles from the fresh control as well as from all cultured follicles still presented a normal ultrastructural pattern. In medium supplemented with FSH, only insulin at 10 ng/ml presented oocytes with higher rates of meiosis resumption compared to control, as well as oocytes in metaphase II. Treatment with insulin (10 ng/ml) plus FSH resulted in significantly increased levels of INSR and CYP19A1 mRNA compared to that with other treatments. In conclusion, 10 ng/ml insulin associated with FSH was more efficient in promoting resumption of oocyte meiosis, maintaining survival, stimulating follicular development, and increasing expression of the INSR and CYP19A1 genes in goat preantral follicles.

Low concentration of insulin with follicle-stimulating hormone is essential for promoting oocyte meiosis resumption in goat preantral follicles.

Epididymal protease inhibitor (EPPIN) is found on the surface of spermatozoa and works as a central hub for a sperm surface protein complex (EPPIN protein complex [EPC]) that inhibits sperm motility on the binding of semenogelin I (SEMG1) during ejaculation. Here, we identify EPPIN's amino acids involved in the interactions within the EPC and demonstrate that EPPIN's sequence C102-P133 contains the major binding site for SEMG1. Within the same region, the sequence F117-P133 binds the EPC-associated protein lactotransferrin (LTF). We show that residues Cys102, Tyr107, and Phe117 in the EPPIN C-terminus are required for SEMG1 binding. Additionally, residues Tyr107 and Phe117 are critically involved in the interaction between EPPIN and LTF. Our findings demonstrate that EPPIN is a key player in the protein-protein interactions within the EPC. Target identification is an important step toward the development of a novel male contraceptive, and the functionality of EPPIN's residues Cys102, Tyr107, and Phe117 offers novel opportunities for contraceptive compounds that inhibit sperm motility by targeting this region of the molecule.

Bull fertility is an important target for genetic improvement, and early prediction using genetic markers is therefore a goal for livestock breeding. We performed genome-wide association studies to identify genes associated with fertility traits measured in young bulls. Data from 1118 Brahman bulls were collected for six traits: blood hormone levels of inhibin (IN) at 4 mo, luteinizing hormone (LH) following a gonadotropin-releasing hormone challenge at 4 mo, and insulin-like growth factor 1 (IGF1) at 6 mo, scrotal circumference (SC) at 12 mo, ability to produce sperm (Sperm) at 18 mo, and percentage of normal sperm (PNS) at 24 mo. All the bulls were genotyped with the BovineSNP50 chip. Sires and dams of the bull population (n = 304) were genotyped with the high-density chip (∼800 000 polymorphisms) to allow for imputation, thereby contributing detail on genome regions of interest. Polymorphism associations were discovered for all traits, except for Sperm. Chromosome 2 harbored polymorphisms associated with IN. For LH, associated polymorphisms were located in five different chromosomes. A region of chromosome 14 contained polymorphisms associated with IGF1 and SC. Regions of the X chromosome showed associations with SC and PNS. Associated polymorphisms yielded candidate genes in chromosomes 2, 14, and X. These findings will contribute to the development of genetic markers to help select cattle with improved fertility and will lead to better annotation of gene function in the context of reproductive biology.

Extravillous trophoblast (EVT) cells of the human placenta invade the uterine decidua and utero-placental arteries to establish an efficient exchange of key molecules between maternal and fetal blood. Trophoblast invasion is stringently regulated in situ both positively and negatively by a variety of factors at the fetal-maternal interface to maintain a healthy utero-placental homeostasis. One such factor, decorin, a transforming growth factor (TGF)-beta binding, leucine-rich proteoglycan produced by the decidua, negatively regulates EVT proliferation, migration, and invasiveness independent of TGF-beta. We reported that these decorin actions were mediated by its binding to multiple tyrosine kinase receptors, including vascular endothelial growth factor receptor (VEGFR)-2. The present study explores the mechanisms underlying decorin antagonism of VEGF (VEGF-A) stimulation of endovascular differentiation of EVT using our EVT cell line, HTR-8/SVneo. We observe that decorin inhibits VEGF-induced EVT cell migration and endothelial-like tube formation on matrigel. VEGF activates MAPKs (p38 MAPK, MEK3/6, and ERK1/2) in EVT cells, and the activation is blocked in both cases by decorin. Employing selective MAPK inhibitors, we show that both p38 and ERK pathways contribute independently to VEGF-induced EVT migration and capillary-like tube formation. VEGF upregulates the vascular endothelial (VE) markers VE-cadherin and beta-catenin in EVT and endothelial cells, and this upregulation is blocked by decorin and MAPK inhibitors. These results suggest that decorin inhibits VEGF-A stimulation of trophoblast migration and endovascular differentiation by interfering with p38 MAPK and ERK1/2 activation. Thus decorin-mediated dual impediment of endovascular differentiation of the EVT and angiogenesis may have implications for pathogenesis of preeclampsia, a hypoinvasive trophoblast disorder in pregnancy.

The establishment of pregnancy requires bidirectional communication between the developing conceptus and the uterine endometrium. The aim of this study was to establish an in vitro coculture system with bovine trophoblast cells and uterine epithelial cells (EECs) that mimics the in vivo attachment process. We previously reported that expression of interferon tau (IFNT), a major secretory product from the trophectoderm, decreases with changes in chromatin structure when the conceptus successfully attaches to the uterine epithelium. Thus, IFNT is a good marker to assess whether attachment has successfully occurred. In this study, bovine trophoblast CT-1 cells were cultured to generate spheroids, which were then placed on type I collagen-coated plates (monoculture) or bovine EECs (coculture) with or without uterine flushings collected from Day 15 cyclic or Days 15, 17, or 19 pregnant animals. In the coculture but not the monoculture, addition of uterine flushings from Day 15 or 17 pregnant animals resulted in decreased IFNT and CDX2 mRNA expression in CT-1 spheroids, accompanied with changes in histone modifications. In monocultured CT-1 spheroids, integrin subunit ITGA8 and ITGB3 mRNAs were minimally expressed but were induced in cocultured CT-1 spheroids with or without uterine flushings. Expression of CDH2, another marker for bovine conceptus attachment to the uterine epithelium, was also induced in the cocultured CT-1 spheroids. These results suggest that this in vitro coculture system could be used to isolate processes essential for conceptus attachment to uterine EECs.

Accumulating evidence strongly supports the premise that testosterone may be a key player in fetal programming on hypertension. Studies have shown that gestational protein restriction doubles the plasma testosterone levels in pregnant rats. In this study, we hypothesized that elevated testosterone levels in response to gestational protein restriction were caused by enhanced expression of steroidogenic enzymes or impaired expression of Hsd17b2, a known testosterone inactivator that converts testosterone to androstenedione in placenta. Pregnant Sprague-Dawley rats were fed normal (20% protein, control; n = 10) or a low-protein diet (6% protein, PR; n = 10) from Day 1 of pregnancy until killed at Days 14, 18, or 21. Junctional (JZ) and labyrinth (LZ) zones of placenta were collected for expression assay on steroidogenic genes (Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b2, and Srd5a1) by real-time PCR. The main findings include the following: 1) expressions of Cyp11a1, Hsd3b1, and Cyp17a1 in JZ were not affected by diet but were affected by day of pregnancy; 2) expression of Hsd17b2 in both female and male JZs was remarkably increased by PR at Days 18 and 21 of pregnancy; 3) expressions of Hsd17b2 were reduced by PR in both female and male LZ at Day 18 of pregnancy and in female LZ at Day 21 of pregnancy; and 4) expression of Srd5a1in LZ was not affected by day of pregnancy, gender, or diet. These results indicate that in response to gestational protein restriction, Hsd17b2 may be a key regulator of testosterone levels and associated activities in placental zones, apparently in a paradoxical manner.

Amniotes, mammals, reptiles, and birds form common extraembryonic membranes during development to perform essential functions, such as protection, nutrient transfer, gas exchange, and waste removal. Together with the maternal uterus, extraembryonic membranes of viviparous (live-bearing) amniotes develop as an endocrine placenta that synthesizes and responds to steroid hormones critical for development. The ability of these membranes to synthesize and respond to steroid hormone signaling has traditionally been considered an innovation of placental amniotes. However, our laboratory recently demonstrated that this ability extends to the chorioallantoic membrane (CAM) of an oviparous (egg-laying) amniote, the domestic chicken, and we hypothesized that steroidogenic extraembryonic membranes could be an evolutionarily conserved characteristic of all amniotes because of similarities in basic structure, function, and shared evolutionary ancestry. In this study, we examined steroid hormone synthesis and signaling in the CAM of another oviparous amniote, the American alligator (Alligator mississippiensis). We quantified mRNA expression of a steroidogenic factor involved in the regulation of steroidogenesis (NR5A1), the key steroidogenic enzymes involved in the synthesis of progestins (HSD3B1), androgens (CYP17A1), and estrogens (CYP19A1), and the receptors involved in the signaling of progestins (PR), androgens (AR), estrogens (ESR1 and ESR2), and glucocorticoids (GR). Furthermore, we performed protein immunolocalization for PR and ESR1. Collectively, our findings indicate that the alligator CAM has the capability to regulate, synthesize, and respond to steroid hormone signaling, thus, supporting our hypothesis that the extraembryonic membranes of Amniota share a unifying characteristic, that is, the ability to synthesize and respond to steroid hormones.

Meiosis is essential for generation of healthy gametes in both sexes and involves recombination and segregation of homologous chromosomes to produce haploid gametes. The initiation of meiosis in both sexes relies upon retinoic acid (RA) (Griswold MD, Hogarth CA, Bowles J, Koopman P. Initiating Meiosis: The Case for Retinoic Acid. Biol Reprod 2012; 86(35):1–7). Previous studies have demonstrated that the stimulated by retinoic acid gene 8 (Stra8) was required for meiotic progression in both the mouse ovary and postnatal testis. To identify additional candidates that may play a role during meiosis, we used microarray databases to generate lists of transcripts with expression profiles similar to that of Stra8 in the embryonic ovary and postnatal testis. One such gene, establishment of cohesion 1 homolog 2 (Saccharomyces cerevisiae) (Esco2), has been described as a regulator of sister chromatid cohesion during mitosis. This study describes the first in-depth analysis of ESCO2 localization and regulation during meiosis in both males and females. ESCO2 colocalized with the gamma H2A histone family member X (H2AFX) in pachytene spermatocytes, indicating that ESCO2 is a component of the XY body. In pachytene cells of the embryonic ovary, ESCO2 colocalized with H2AFX, which is consistent with the presence of ESCO2 in areas of double-stranded breaks. In addition, the expression of Esco2 was found to be regulated by RA in the postnatal testis. These data indicate that ESCO2 may play a vital role in meiosis in both males and females.

Oxidative stress has been implicated in pathogenesis of many diseases, but few studies describe its influence on spermatogenesis. In this study, we analyzed the direct influence of hypoxanthine (Hx)-induced reactive oxygen species (ROS) on spermatogenesis in fish using the Japanese eel (Anguilla japonica) testicular organ culture system. Testicular fragments of eels were cultured in 0.1–100 μM Hx with or without 10 ng/ml 11-ketotestosterone (11-KT). Immunohistochemistry for 5-bromo-2-deoxyuridine showed that Hx treatment at a low dose (1 μM) already inhibits 11-KT-induced germ cell proliferation after culture. An in situ TUNEL assay and 8-hydroxy-2′-deoxyguanosine immunohistochemistry revealed an intense germ cell apoptosis and high oxidative DNA damage in testicular fragments cultured at the highest dose of Hx (100 μM) with 11-KT. A total superoxide dismutase (SOD) activity assay showed a decrease in SOD activity in testicular fragments cultured with 11-KT. These data suggest that ROS may directly inhibit spermatogenesis, and that decreased SOD activity renders proliferating spermatogonia susceptible to ROS, hence leading to apoptosis.

In eukaryotes, DNA synthesis is preceded by licensing of replication origins. We examined the subcellular localization of two licensing proteins, ORC2 and MCM7, in the mouse zygotes and two-cell embryos. In somatic cells ORC2 remains bound to DNA replication origins throughout the cell cycle, while MCM7 is one of the last proteins to bind to the licensing complex. We found that MCM7 but not ORC2 was bound to DNA in metaphase II oocytes and remained associated with the DNA until S-phase. Shortly after fertilization, ORC2 was detectable at the metaphase II spindle poles and then between the separating chromosomes. Neither protein was present in the sperm cell at fertilization. As the sperm head decondensed, MCM7 was bound to DNA, but no ORC2 was seen. By 4 h after fertilization, both pronuclei contained DNA bound ORC2 and MCM7. As expected, during S-phase of the first zygotic cell cycle, MCM7 was released from the DNA, but ORC2 remained bound. During zygotic mitosis, ORC2 again localized first to the spindle poles, then to the area between the separating chromosomes. ORC2 then formed a ring around the developing two-cell nuclei before entering the nucleus. Only soluble MCM7 was present in the G2 pronuclei, but by zygotic metaphase it was bound to DNA, again apparently before ORC2. In G1 of the two-cell stage, both nuclei had salt-resistant ORC2 and MCM7. These data suggest that licensing follows a unique pattern in the early zygote that differs from what has been described for other mammalian cells that have been studied.

Bisphenol A (BPA) is an estrogenic chemical used to manufacture many commonly used plastic and epoxy resin-based products. BPA ubiquitously binds to estrogen receptors throughout the body, including estrogen receptor alpha (ESR1) in the ovary. Few studies have investigated the effects of BPA on ovarian antral follicles. Thus, we tested the hypothesis that BPA alters cell cycle regulators and induces atresia in antral follicles via the genomic estrogenic pathway, inhibiting follicle growth. To test this hypothesis, we isolated antral follicles from 32- to 35-day-old control and Esr1-overexpressing mice and cultured them with vehicle control (dimethylsulfoxide [DMSO]) or BPA (1–100 μg/ml). Additionally, antral follicles were isolated from 32- to 35-day-old FVB mice and cultured with DMSO, BPA (1–100 μg/ml), estradiol (10 nM), ICI 182,780 (ICI; 1 μM), BPA plus ICI, or BPA plus estradiol. Follicles were measured for growth every 24 h for 96–120 h and processed either for analysis of estrogen receptor, cell cycle, and/or atresia factor mRNA expression, or for histological evaluation of atresia. Results indicate that estradiol and ICI do not protect follicles from BPA-induced growth inhibition and that estradiol does not protect follicles from BPA-induced atresia. Furthermore, overexpressing Esr1 does not increase susceptibility of follicles to BPA-induced growth inhibition. Additionally, BPA up-regulates Cdk4, Ccne1, and Trp53 expression, whereas it down-regulates Ccnd2 expression. BPA also up-regulates Bax and Bcl2 expression while inducing atresia in antral follicles. These data indicate that BPA abnormally regulates cell cycle and atresia factors, and this may lead to atresia and inhibited follicle growth independently of the genomic estrogenic pathway.

Deep functional changes occurring within the endometrium during implantation are orchestrated by embryonic and maternal signals. Human chorionic gonadotropin (hCG), a major embryonic signal, plays a critical role in the initiation and maintenance of pregnancy. Interleukin (IL) 1, one of the earliest embryonic signals, appears to exert a direct impact on the receptive endometrium and to induce major molecular changes that are essential for embryo implantation. Herein we investigate whether hCG can modulate endometrial stromal cell (ESC) receptivity to IL1 during the implantation window and assess the impact on angiogenesis in vitro. Primary cultures of ESCs from normal fertile women during the implantation window were treated for 24 h with different concentrations of hCG (0–100 ng/ml) and stimulated for 24 h with IL1B (0–0.1 ng/ml). IL1 receptors (IL1Rs), IL1R antagonist (IL1RA), and monocyte chemotactic protein (MCP) 1 were analyzed by real-time PCR, ELISA, and Western blotting. The angiogenic activity in vitro was studied using human microvascular endothelial cell line, scratch wound assay, and cell proliferation via BrdU incorporation into DNA. Human CG induced a dose-dependent imbalance in ESC receptivity to IL1 by significantly upregulating the functional signaling IL1R1 and concomitantly downregulating the decoy inhibitory IL1R2 and IL1RA upon subsequent exposure to IL1B. Prior exposure to hCG amplified MCP1 secretion by ESCs in response to IL1B and triggered the release of angiogenic activity in vitro in which MCP1 appeared to play a significant role. Overexpression of IL1R2 using cell transfection inhibited IL1 and hCG/IL1B-mediated MCP1 secretion. These findings suggest that hCG coordinates embryonic signal interaction with the maternal endometrium, and point to a new possible pathway by which it may promote embryonic growth.