The molecular mechanisms underlying and determining egg developmental competence remain poorly understood in vertebrates. Nucleoplasmin (Npm2) is one of the few known maternal effect genes in mammals, but this maternal effect has never been demonstrated in nonmammalian species. A link between developmental competence and the abundance of npm2 maternal mRNA in the egg was previously established using a teleost fish model for egg quality. The importance of maternal npm2 mRNA for egg developmental competence remains unknown in any vertebrate species. In the present study, we aimed to characterize the contribution of npm2 maternal mRNA to early developmental success in zebrafish using a knockdown strategy. We report here the oocyte-specific expression of npm2 and maternal inheritance of npm2 mRNA in zebrafish eggs. The knockdown of the protein translated from this maternal mRNA results in developmental arrest before the onset of epiboly and subsequent embryonic death, a phenotype also observed in embryos lacking zygotic transcription. Npm2 knockdown also results in impaired transcription of the first-wave zygotic genes. Our results show that npm2 is also a maternal effect gene in a nonmammalian vertebrate species and that maternally inherited npm2 mRNA is crucial for egg developmental competence. We also show that de novo protein synthesis from npm2 maternal mRNA is critical for developmental success beyond the blastula stage and required for zygotic genome activation. Finally, our results suggest that npm2 maternal mRNA is an important molecular factor of egg quality in fish and possibly in all vertebrates.

Mammalian blastocysts comprise three distinct lineages, namely, trophoblast, hypoblast, and epiblast, which develop into fetal placenta, extraembryonic yolk sac, and embryo proper, respectively. Pluripotent embryonic stem cells, capable of forming all adult cell types, can only be derived from the epiblast. In mouse and rat, this process is promoted by the double inhibition (2i) of mitogen-activated protein kinase kinase (MAP2K), which antagonizes FGF signaling, and glycogen synthase kinase 3 (GSK3), which stimulates the WNT pathway. We investigated variations of the 2i treatment on lineage segregation and pluripotency-related gene expression in bovine blastocysts. In vitro-fertilized embryos were cultured either in the presence of inhibitors of GSK3 (3 μM CHIR) and MAP2K (0.4 vs. 10 μM PD0325901, designated 2i and 2i , respectively) or in 2i/2i with FGFR inhibitor (0.1 μM PD173074, designated 3i [2i and PD173074] and 3i [2i and PD173074]). Compared with 2i, both 2i and 3i potentiated the improvement in blastocyst morphology. Using an automated platform for multiplexed digital mRNA profiling, we simultaneously counted transcripts of 76 candidate genes in bovine blastocysts treated with multiple kinase inhibitors. We show that 2i medium specifically increased FGF4 and NANOG while reducing PDGFRalpha and SOX17 levels. The shift from a hypoblast to an epiblast gene expression signature was confirmed by quantitative PCR. A wide range of functionally related genes, including candidates involved in DNA methylation, were not significantly changed. This well-defined 2i effect was not observed after pharmacologically inhibiting FGF receptor or related MAP kinases (p38, JNK, and ERK5). In summary, our data suggest that increased MAP2K inhibition exerts its pluripotency-promoting effects through as yet unidentified signals.

The endometrium undergoes continuous repair and regeneration without scarring throughout the reproductive life of women. However, the mechanisms responsible for this complete restoration remain mostly unexplored. We hypothesized that the ischemic state and local hypoxia present after parturition may create a special microenvironment for endometrial healing, and that this ischemia might be caused by reduction in organ volume via postpartum uterine contraction. Here, we developed a mouse model using a combination of cesarean section and the administration of a beta 2 adrenergic receptor agonist (ritodrine hydrochloride) in postpartum mice that had been ovariectomized to exclude the effect of ovarian hormones. Our results revealed that transient hypoxia indeed occurred in postpartum uteri. Furthermore, we found that the number of M2 macrophages, which play a central role in wound healing, peaked on Postpartum Day 3 and gradually decreased thereafter in hypoxic injury sites. Almost concurrently, significant upregulation of vascular endothelial growth factor and transforming growth factor beta (TGFbeta) was observed. In particular, the antifibrotic factor TGFbeta3 was released during the endometrial healing process. These changes were significantly suppressed by inhibition of uterine contraction. Taken together, these results suggest that uterine contraction is essential, not only for hemostasis, but also for endometrial regeneration, leading to a process that involves the activation of macrophages, increased endometrial cell proliferation, and upregulation of nonfibrotic growth factors. This study paves the way to a novel approach for investigating the process of scarless wound healing.

In ruminants, prostaglandin F2 alpha (PGF_2alpha) is synthesized and released in a pulsatile pattern from the endometrial luminal epithelial (LE) cells during the process of luteolysis. Interferon tau (IFNT) is a Type 1 IFN secreted by the trophoblast cells of the developing conceptus. IFNT acts locally on endometrial LE cells to inhibit pulsatile releases of PGF_2alpha and thus establish an endocrine environment for recognition of pregnancy. Cell signaling pathways through which IFNT stimulates expression of multiple genes or proteins in endometrial LE are largely unknown. Results of the present investigation indicate that intrauterine administration of IFNT inhibits pulsatile release of PGF_2alpha, while coadministration IFNT and ERK 1/2 inhibitor U0126 restores luteolytic PGF_2alpha pulses in sheep. IFNT increases phosphorylation of ERK1/2 proteins and increases its interaction with PGT proteins in endometrial LE. Blockade of ERK1/2 pathways inhibits IFNT action, decreases pERK1/2 and PGT protein interactions, and re-establishes the spatial expression of the oxytocin receptor protein completely and the estrogen receptor protein partially without modulating the expression of interferon regulatory factor-2 (IRF-2) protein in endometrial LE. IFNT does not decrease expression of COX-2, PGDH, or PGT protein in endometrial LE. Our results provide important new insights into IFNT signaling and the molecular endocrine control of PGF_2alpha release at the time of establishment of pregnancy in ruminants. This novel IFNT-ERK1/2 signaling module needs to be explored in future studies to understand molecular and cellular mechanisms of IFNT action in endometrial LE in ruminants.

The Mos-MAPK signaling pathway involving the Mos-MEK1/2-ERK1/2-RSK1/2/3 or MSK1-EMI2 cascade is directly linked to metaphase-II arrest of vertebrate oocytes. In this study, we examined whether p38, a member of the MAPK subfamily, is regulated under the control of Mos and contributes to metaphase-II arrest in the mouse oocyte. Morpholino oligonucleotide-mediated depletion of Mos revealed a remarkable decrease in phosphorylation of p38. Simultaneous treatment of oocytes with two chemical inhibitors of p38 and MEK1/2 induced both release from metaphase II and degradation of cyclin B1, whereas the treatment with each of these two inhibitors had little effect. Moreover, phosphorylation of EMI2 was dramatically abolished by addition of the two inhibitors. Indeed, MNK1, a kinase downstream of p38, exhibited the ability to phosphorylate EMI2. These results suggest that in addition to the Mos-MEK1/2 pathway, the Mos-mediated p38 pathway may be implicated in metaphase-II arrest.

Oocyte in vitro maturation (IVM) is an important assisted reproductive technology and research tool. The adoption of IVM into routine clinical practice has been hindered by its significantly lower success rates compared to conventional in vitro fertilization. Cyclic AMP (cAMP) modulation and follicle-stimulating hormone (FSH), independently, have long been known to improve IVM oocyte developmental competence. This study comprehensively examined the effects of FSH and cAMP/cGMP modulation, alone and in combination, on IVM oocyte metabolism and developmental outcomes. Mouse cumulus-oocyte complexes (COCs) were subjected to a 1 h prematuration phase ± the cAMP modulator forskolin and cAMP/cGMP modulator 3-isobutyl-1-methylxanthine followed by IVM ± FSH. Prematuration with these cyclic nucleotide modulators or IVM with FSH significantly improved oocyte developmental competence and reduced spindle abnormalities compared to spontaneous IVM (no treatment); however, these two treatments in combination endowed even greater developmental competence (improved subsequent blastocyst rates and quality; P < 0.05), albeit blastocyst yield and quality remained significantly lower than that of oocytes matured in vivo. A significant additive effect of combined IVM treatments was evident as increased COC lactate production and oxygen consumption and enhanced oocyte oxidative metabolism, ATP production, ATP:ADP ratio, and glutathione levels (P < 0.05). Nevertheless, IVM increased reactive oxygen species production, particularly as a consequence of FSH addition, relative to in vivo matured oocytes. In conclusion, improvements in the embryo yield following IVM is associated with increased COC oxygen consumption and oocyte oxidative metabolism, but these remain metabolically and developmentally less competent relative to in vivo derived oocytes.

The serine/threonine kinase 5′ adenosine monophosphate-activated protein kinase (AMPK), a heterotrimeric protein known as a metabolic switch, is involved in oocyte nuclear maturation in mice, cattle, and swine. The present study analyzed AMPK activation in cumulus cell expansion during in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COC). 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) is a well-known activator of AMPK. It inhibited oocyte meiotic resumption in COC. Moreover, cumulus cell expansion did not occur in the presence of AICAR, demonstrating its marked impact on cumulus cells. Activation of AMPK was supported by AICAR-mediated phosphorylation of alpha AMPK subunits. Furthermore, the presence of AICAR increased glucose uptake, a classical response to activation of this metabolic switch in response to depleted cellular energy levels. Neither nuclear maturation nor cumulus expansion was reversed by glucosamine, an alternative substrate in hyaluronic acid synthesis, through the hexosamine biosynthetic pathway, which ruled out possible depletion of substrates. Both increased gap junction communication and phosphodiesterase activity in COC are dependent on protein synthesis during the initial hours of IVM; however, both were inhibited in the presence of AICAR, which supports the finding that activation of AMPK by AICAR mediated inhibition of protein synthesis. Moreover, this protein synthesis inhibition was equivalent to that of the well-known protein synthesis inhibitor cycloheximide, as observed on cumulus expansion and protein concentration. Finally, the phosphorylation level of selected kinases was investigated. The pattern of raptor phosphorylation is supportive of activation of AMPK-mediated inhibition of protein synthesis. In conclusion, AICAR-mediated AMPK activation in porcine COC inhibited cumulus cell expansion and protein synthesis. These results bring new considerations to the importance of this kinase in ovarian physiology and to the development of new oocyte culture medium.

Neurotensin (NT) has multiple functions, ranging from acting as a neurotransmitter to regulating intestinal movement. However, its function in reproductive physiology is unknown. Here, we confirmed the expression and localization of NT receptors (NTR1) in mouse epididymal spermatozoa and investigated the effect of NT on sperm function. Sperm protein tyrosine phosphorylation, one of the indices of sperm capacitation, was facilitated dose-dependently by NT administration. In addition, the acrosome reaction was promoted in capacitated spermatozoa, and addition of a selective antagonist of NTR1 and NTR2 blocked the induction. Furthermore, intracellular calcium mobilization by NT addition was observed. This showed that NT was an accelerator of sperm function via its functional receptors. The presence of NT was confirmed by immunohistochemistry and its localization was observed in epithelia of the uterus and oviduct isthmus and ampulla, which correspond to the fertilization route of spermatozoa. The NT mRNA level in ovulated cumulus cell was remarkably increased by treatment with human chorionic gonadotropin (hCG). Using an in vitro maturation model, we analyzed the effects of FSH, epidermal growth factor (EGF), estradiol, and progesterone in NT production in cumulus cells. We found that FSH and EGF upregulated NT release and mRNA expression. Both FSH- and EGF-induced upregulation were inhibited by U0126, an MAPK kinase inhibitor, indicating that FSH and EGF regulate NT expression via a MAPK-dependent pathway. This evidence suggests that NT can act as a promoter of sperm capacitation and the acrosome reaction in the female reproductive tract.

Adrenomedullin (ADM) may regulate seminal vesicle fluid secretion, and this may affect sperm quality. In this study, we investigated the effect of ADM on chloride secretion in the mouse seminal vesicle. The presence of ADM in mouse seminal vesicle was confirmed using immunostaining, and the molecular species was determined using gel filtration chromatography coupled with enzyme-linked assay for ADM. The effects of ADM on chloride secretion were studied by short-circuit current technique in a whole-mount preparation of mouse seminal vesicle in an Ussing chamber. The effects of specific ADM and calcitonin gene-related peptide (CGRP) receptor antagonists were investigated. Whether the ADM effect depended on the cAMP- and/or calcium-activated chloride channel was also studied using specific chloride channel blockers. The results showed that ADM was present in seminal vesicle epithelial cells. The major molecular species was precursor in the mouse seminal vesicle. ADM increased short-circuit current through the calcium-activated chloride channel in mouse seminal vesicle, and CGRP receptor was involved. We conclude that ADM may regulate chloride and fluid secretion from the seminal vesicle, which may affect the composition of the seminal plasma bathing the sperm and, hence, fertility.

Cryptorchidism, or undescended testis, is a common male genital anomaly of unclear etiology. Hormonal stimulation of the developing fetal gubernaculum by testicular androgens and insulin-like 3 (INSL3) is required for testicular descent. In studies of the orl fetal rat, one of several reported strains with inherited cryptorchidism, we studied hormone levels, gene expression in intact and hormone-stimulated gubernaculum, and imaging of the developing cremaster muscle facilitated by a tissue clearing protocol to further characterize development of the orl gubernaculum. Abnormal localization of the inverted gubernaculum was visible soon after birth. In the orl fetus, testicular testosterone, gubernacular androgen-responsive transcript levels, and muscle-specific gene expression were reduced. However, the in vitro transcriptional response of the orl gubernaculum to androgen was largely comparable to wild type (wt). In contrast, increases in serum INSL3, gubernacular INSL3-responsive transcript levels, expression of the INSL3 receptor, Rxfp2, and the response of the orl gubernaculum to INSL3 in vitro all suggest enhanced activation of INSL3/RXFP2 signaling in the orl rat. However, DNA sequence analysis did not identify functional variants in orl Insl3. Finally, combined analysis of the present and previous studies of the orl transcriptome confirmed altered expression of muscle and cellular motility genes, and whole mount imaging revealed aberrant muscle pattern formation in the orl fetal gubernaculum. The nature and prevalence of developmental muscle defects in the orl gubernaculum are consistent with the cryptorchid phenotype in this strain. These data suggest impaired androgen and enhanced INSL3 signaling in the orl fetus accompanied by defective cremaster muscle development.

The sex differentiation mechanisms in zebrafish (Danio rerio) remains elusive, partly because of the absence of sex chromosomes but also because the process appears to depend on the synchrony of multiple genes and possibly environmental factors. Zebrafish gonadal development is initiated through the development of immature oocytes. Depending on multiple signaling cues, in about half of the individuals, the juvenile ovaries degenerate or undergo apoptosis to initiate testes development while the other half maintains the oogenic pathway. We have previously shown that activation of NFκB and prostaglandin synthase 2 (ptgs2) results in female-biased sex ratios. Prostaglandin synthase and prostaglandins are involved in multiple physiological functions, including cell survival and apoptosis. In the present study, we show that inhibition of ptgs2 by meloxicam results in male-biased sex ratios. On further evaluation, we observed that exposure with the prostaglandin D₂ (PGD₂) analogue BW-245C induced SRY-box containing gene 9a (sox9a) and resulted in male-biased sex ratios. On the other hand, prostaglandin E₂ (PGE₂) treatment resulted in female-biased sex ratios and involved activation of NFκB and the β-catenin pathway as well as inhibition of sox9. Exposure to the β-catenin inhibitor PNU-74654 resulted in up-regulation of ptgds and male-biased sex ratios, further confirming the involvement of β-catenin in the female differentiation pathway. In this study, we show that PGD₂ and PGE₂ can program the gonads to either the testis or the ovary differentiation pathways, indicating that prostaglandins are involved in the regulation of zebrafish gonadal differentiation.

Liver receptor homolog-1 (LRH-1) is a conserved member of the NR5A subfamily in vertebrates and a potential regulator of estrogen synthesis in the ovarian granulosa cells. An Lrh-1 homologue was obtained from the orange-spotted grouper Epinephelus coioides that contains the conserved structural features of NR5A and is phylogenetically closely related to NR5A2. The expression of the orange-spotted grouper Lrh-1 is tissue-specific with relatively higher levels in the liver and ovary. The immunoreactive signals for Lrh-1 and Cyp19a1a were present in the ovarian follicular cells and germ cells. In the ovarian follicular cells, Lrh-1 was present both in the nucleus and cytoplasm, and colocalized with Cyp19a1a. The expression levels of both increased during vitellogenesis whereas only Cyp19a1a dramatically decreased toward maturation when Lrh-1 was localized almost exclusively to the cytoplasm of the follicular cells. The orange-spotted grouper Lrh-1 could up-regulate cyp19a1a transcription in vitro via the two conserved Ftz-f1 sites in cyp19a1a promoter. Chromatin immunoprecipitation analysis showed that the orange-spotted grouper Lrh-1 could bind cyp19a1a promoter in vivo with a higher abundance in the vitellogenic ovary, whereas the binding was dramatically decreased in the mature ovary. Taken together, the results of present study demonstrate that Lrh-1 plays an important role in up-regulating cyp19a1a gene in the ovarian follicular cells during vitellogenesis, and the sequestration of Lrh-1 to the cytoplasm may down-regulate cyp19a1a expression in the mature ovary. This mechanism for modifying transcriptional roles of the orange-spotted grouper Lrh-1 may shed new light on the regulation of Cyp19a1 expression in other vertebrates as well.

An intrafollicular increase in proteolytic activity drives ovulatory events. Surprisingly, the periovulatory expression profile of the membrane-type matrix metalloproteinases (MT-MMPs), unique proteases anchored to the cell surface, has not been extensively examined. Expression profiles of the MT-MMPs were investigated in ovarian tissue from well-characterized rat and macaque periovulatory models and naturally cycling women across the periovulatory period. Among the six known MT-MMPs, mRNA expression of Mmp14, Mmp16, and Mmp25 was increased after human chorionic gonadotropin (hCG) administration in rats. In human granulosa cells, mRNA expression of MMP14 and MMP16 increased following hCG treatment. In contrast, mRNA levels of MMP16 and MMP25 in human theca cells were unchanged before ovulation but declined by the postovulatory stage. In macaque granulosa cells, hCG increased mRNA for MMP16 but not MMP14. Immunoblotting showed that protein levels of MMP14 and MMP16 in rats increased, similar to their mRNA expression. In macaque granulosa cells, only the active form of the MMP14 protein increased after hCG, unlike its mRNA or the proprotein. By immunohistochemistry, both MMP14 and MMP16 localized to the different ovarian cell types in rats and humans. Treatment with hCG resulted in intense immunoreactivity of MMP14 and MMP16 proteins in the granulosa and theca cells. The present study shows that MMP14 and MMP16 are increased by hCG administration in the ovulating follicle, demonstrating that these MMPs are conserved among rats, macaques, and humans. These findings suggest that MT-MMPs could have an important role in promoting ovulation and remodeling of the ovulated follicle into the corpus luteum.

The primary aims of this study were to utilize a specialized culture system to further elucidate the functional significance of pericellular hypoxia within the granulosa cell (GC) compartment of growing follicles, and to clarify its effects on the production of vascular endothelial growth factor (VEGF)-A isoforms and steroid hormones. Multilaminar clusters formed rapidly in ovine GCs seeded at high density (HD), and Hypoxyprobe-1 protein adducts appeared markedly more abundant and HIF-1 activation significantly (P < 0.001) greater than in cells seeded at low density (LD). Four proangiogenic VEGF mRNA transcript variants were identified in cultured GCs. Most abundant were VEGF₁₂₀ and VEGF₁₆₄, but VEGF₁₈₂ and VEGF₁₈₈ were also detected. Total VEGF mRNA was shown to be up-regulated transiently in the HD cells (P < 0.001) and VEGF₁₆₄ mRNA appeared to contribute most to this. The hypoxia mimetic cobalt chloride also induced marked increases in HIF-1 activation (P < 0.01) and total VEGF mRNA (P < 0.01) production. HD cells increased levels of HIF-1alpha (P < 0.001) and VEGF receptor type 1 (P < 0.05), but not VEGF receptor type 2 mRNA, compared to LD cells or cells grown under chemically induced hypoxia. Both 17beta-estradiol (E2) and progesterone (P4) were markedly lower (P < 0.001) in the HD, cells but though cobalt chloride treatment accompanied significantly reduced P4 production (P < 0.05), E2 levels remained similar to those in untreated cells. These outcomes suggest that pericellular hypoxia may be an important mediator of VEGF production in the GCs of growing follicles, but that local regulation is complex and may involve multiple mechanisms such as mediation by steroid hormones and differential variant mRNA production.

Progesterone receptor membrane component 2 (Pgrmc2) mRNA was detected in the immature rat ovary. By 48 h after eCG, Pgrmc2 mRNA levels decreased by 40% and were maintained at 48 h post-hCG. Immunohistochemical studies detected PGRMC2 in oocytes and ovarian surface epithelial, interstitial, thecal, granulosa, and luteal cells. PGRMC2 was also present in spontaneously immortalized granulosa cells, localizing to the cytoplasm of interphase cells and apparently to the mitotic spindle of cells in metaphase. Interestingly, PGRMC2 levels appeared to decrease during the G₁ stage of the cell cycle. Moreover, overexpression of PGRMC2 suppressed entry into the cell cycle, possibly by binding the p58 form of cyclin dependent kinase 11b. Conversely, Pgrmc2 small interfering RNA (siRNA) treatment increased the percentage of cells in G₁ and M stage but did not increase the number of cells, which was likely due to an increase in apoptosis. Depleting PGRMC2 did not inhibit cellular ³H-progesterone binding, but attenuated the ability of progesterone to suppress mitosis and apoptosis. Taken together these studies suggest that PGRMC2 affects granulosa cell mitosis by acting at two specific stages of the cell cycle. First, PGRMC2 regulates the progression from the G₀ into the G₁ stage of the cell cycle. Second, PGRMC2 appears to localize to the mitotic spindle, where it likely promotes the final stages of mitosis. Finally, siRNA knockdown studies indicate that PGRMC2 is required for progesterone to slow the rate of granulosa cell mitosis and apoptosis. These findings support a role for PGRMC2 in ovarian follicle development.

Both DICER and DROSHA are RNase III enzymes involved in the biogenesis of small noncoding RNAs. DROSHA cleaves the stem-loop portion of the primary miRNAs and produces precursor miRNAs in the nucleus, whereas DICER processes double-stranded RNA precursors into mature miRNAs and endogenous small interference RNAs in the cytoplasm. Selective inactivation of Dicer in growing oocytes of primary follicles leads to female infertility due to oocyte spindle defects. However, it remains unknown if oocyte Dicer expression in the fetal ovary is required for proper follicular development in the postnatal ovary. Moreover, the role of Drosha in folliculogenesis has never been investigated. Here, we report that conditional knockout of Dicer in prophase I oocytes of the fetal ovary led to compromised folliculogenesis, premature ovarian failure, and female infertility in the adult ovary, whereas selective inactivation of Drosha in oocytes of either the fetal or the developing ovary had no effects on normal folliculogenesis and female fertility in adulthood. Our data indicate that oocyte DICER expression in the fetal ovary is required, and oocyte DROSHA is dispensable, for postnatal follicular development and female fertility in adulthood.

INTELECTIN (ITLN) is an adipokine involved in the regulation of insulin sensitivity and inflammatory and immunity responses. Serum ITLN levels are lower in obese, diabetic, and polycystic ovary syndrome (PCOS) women than in control subjects. ITLN has never been studied in ovarian cells. Here, we identified ITLN1 in human ovarian follicles and investigated the molecular mechanisms involved in the regulation of its expression in response to the insulin sensitizers metformin and rosiglitazone, in human granulosa-lutein cells (hGLCs) and in a human ovarian granulosa-like tumor cell line (KGN). We also studied the effects of human recombinant ITLN1 (hRom1) on steroid production and on the activation of various signaling pathways. Using RT-PCR, immunoblotting, and immunohistochemistry, we found that INTL1 is present in human follicular cells. Using ELISA, we showed that INTL levels are similar in plasma and follicular fluid (FF) in control patients, whereas they are higher in FF than in plasma in PCOS patients. In KGN cells and hGLCs, insulin (10⁻⁸ M), insulin-like growth factor-1 (IGF-1; 10⁻⁸ M), and metformin (10⁻² M or 10⁻³ M) increased INTL1 expression (mRNA and protein) after 12 and 24 h of stimulation. For metformin, this effect was mediated by adenosine monophosphate-activated kinase (PRKA). Furthermore, hRom1 increased nicotinamide phosphoribosyltransferase (NAMPT) expression in KGN and hGLCs. We also showed that hRom1 increased IGF-1-induced progesterone and estradiol secretion and this was associated with an increase in the STAR and CYP19A1 protein levels and an increase in IGF-1R signaling. Furthermore, all these data were abolished when NAMPT was knocked down in KGN cells, suggesting that INTL1 improves IGF-1-induced steroidogenesis through induction of NAMPT in hGLCs.

The bovine trophoblast produces significant amounts of estrogens. In maternal and fetal blood, estrogens occur predominantly in sulfonated forms, which are unable to bind to estrogen receptors (ESRs). However, estrogens may act as local factors in ESR-positive trophoblast cells or in the adjacent caruncular epithelium, which in addition to ESR highly expresses steroid sulfatase. Estrogen sulfonation is catalyzed by the cytosolic enzyme SULT1E1. Previous studies clearly indicated the trophoblast as the primary site of estrogen sulfonation. However, investigations into the cellular localization of SULT1E1 yielded conflicting results. In situ hybridization studies detected SULT1E1 mRNA only in trophoblast giant cells (TGCs), whereas in immunohistochemical experiments the SULT1E1 protein was virtually restricted to uninucleated trophoblast cells (UTCs). The aim of this work was to resolve this conflict by analyzing SULT1E1 expression in isolated UTCs and TGCs. Highly enriched pools of UTCs and TGCs were obtained from four bovine placentas (Days 118–130 of gestation) using an optimized fluorescence-activated cell sorting procedure. UTC and TGC pools were analyzed by quantitative RT-PCR and Western blot experiments to measure the amounts of SULT1E1 transcript and protein, respectively. In contrast to previously published results, both SULT1E1 transcript and SULT1E1 protein were clearly present in the UTC and TGC pools. However, some evidence indicated a higher transcript concentration in TGCs and a higher amount of protein in UTCs. Thus, our results resolve the conflicting results on the localization of SULT1E1 from earlier studies and suggest that posttranscriptional mechanisms play an important role in the control of SULT1E1 expression during TGC differentiation.

In the present study, we identify and describe an obese phenotype in mice as a long-term consequence of a suboptimal in vitro culture that resulted from the addition of fetal calf serum (FCS) into the culture medium. Mice produced with FCS displayed a high mortality rate (approximately 55% versus 15% in control mice within 20 mo) and increased sensitivity to the development of obesity in adulthood when fed either a standard or a high-fat diet. These mice developed hyperplastic obesity that was characterized by a significant expansion of the fat pads (approximately 25% and 32% higher body weight in male and female mice over controls, respectively) with unchanged adipocyte size. We observed a sexual dimorphism in the development of obesity in the mice produced with FCS. Whereas the female mice displayed hypertension, hyperleptinemia, and fatty liver, the male mice only displayed glucose intolerance. The mRNA expression of metabolically relevant genes in the adipose tissue was also affected. The males produced with FCS expressed higher mRNA levels of the genes that activate fatty acid oxidation (peroxisome proliferator-activated receptor alpha [Ppara, PPARalpha] and acyl-CoA oxidase 1 [Acox1, ACOX1]) and thermogenesis (uncoupling protein 1 [Ucp1, UCP1]), which may counteract the metabolic phenotype. Conversely, the females produced with FCS generally expressed lower levels of these metabolic genes. In the females, the obese phenotype was associated with inhibition of the lipogenic pathway (peroxisome proliferator-activated receptor gamma [Pparg, PPARgamma] and fatty acid synthase [Fasn, FAS]), indicating a saturation of the storage capacity of the adipose tissue. Overall, our data indicate that the exposure to suboptimal in vitro culture conditions can lead to the sexually dimorphic development of obesity in adulthood.

Chromosome aneuploidies commonly arise in embryos produced by assisted reproductive technologies and represent a major cause of implantation failure and miscarriage. Currently, preimplantation genetic diagnosis (PGD) is performed by array-based methods to identify euploid embryos for transfer to the patient. We speculated that a combination of next-generation sequencing technologies and sophisticated bioinformatics would deliver a more comprehensive and accurate methodology to improve the overall efficacy of embryo testing. To meet this challenge, we developed a high-resolution copy number variation (CNV) sequencing pipeline suitable for single-cell analysis. In validation studies, we showed that CNV-Seq was highly sensitive and specific for detection of euploidy, aneuploidy, and segmental imbalances in 24 whole genome amplification samples from PGD embryos that were originally diagnosed by gold standard array comparative genomic hybridization. In addition, CNV-Seq was capable of detecting, mapping, and accurately quantifying terminal chromosome imbalances down to 1 Mb in size originating from abnormal segregation of translocation chromosomes. These validation studies indicate that CNV-Seq displays the hallmarks of an accurate and reliable embryo test with the potential to further improve the overall efficacy of PGD.

Gonadotropins have been widely used in human-assisted reproduction and animal science for the past four decades. However, the effects of gonadotropins on oocyte maturation at the molecular and biochemical levels are poorly understood. To determine the effects of gonadotropins (recombinant follicle stimulating hormone and urinary human menopausal gonadotropin) on oocyte maturation, we used the bovine oocyte in vitro maturation model. First, we studied the effects of increasing gonadotropin concentrations on nuclear maturation and mitochondrial function in oocytes. Gonadotropins at concentrations of 0.075 and 0.75 IU/ml improved nuclear maturation and increased inner mitochondrial membrane potential and ATP levels; however, there were no beneficial effects at concentrations of 7.5 and 75 IU/ml. Second, we studied the effects of increasing gonadotropin concentrations on the status of methylation in matured (MII) oocytes. Aberrant methylation and demethylation of H19, SNRPN, and PEG3 genes were observed in MII oocytes at all concentrations except 0.075 IU/ml. The expression of genes that function in spindle formation, cell cycle control, and methylation was also downregulated by high gonadotropin concentrations. In conclusion, we established the optimal gonadotropin concentration (i.e., 0.075 IU/ml) to be used for bovine oocyte in vitro maturation studies. These results may provide a guide for clinical stimulation protocols and help to reduce the risks associated with gonadotropin administration during in vitro fertilization treatment.

The Niemann-Pick-type C1 (Npc1) protein mobilizes LDL-derived cholesterol from lysosomes. Npc1 deficiency disease is a panethnic autosomal recessive disorder of intracellular cholesterol trafficking, leading to accumulation of cholesterol in endosomes/lysosomes. This report assesses the effects of a spontaneous inactivating mutation of the Npc1 gene on spermatogenesis and cholesterol homeostasis in mice. We quantified 1) free and esterified cholesterol levels by enzymatic analysis, 2) cholesterol enzymes and transporter protein expression by Western blotting, and 3) the number of Apostain-labeled apoptotic germ cells and apoptosis levels by ELISA in seminiferous tubule-enriched fractions. In wild-type (WT) mice, esterified cholesterol was elevated when Npc1 expression was low during puberty, while in adulthood, the levels were low (P < 0.05) when Npc1 expression was high (P < 0.01). In Npc1−/− mice, free and esterified cholesterol were significantly elevated. The abundance of cholesterol regulatory proteins, HMGR ACAT1, ACAT2, SR-BI, and ABCA1 was significantly higher in Npc1−/− than in WT mice. The level of apoptosis determined by ELISA and the number of Apostain-labeled cells/tubule were higher in Npc1−/− than in WT mice. Circulating testosterone levels in the Npc1−/− males were threefold lower than those observed in the WT. Deleting the Npc1 gene is accompanied by an increase in germ cell apoptosis and compensatory imbalances in the expression of cholesterol enzymatic and transporter factors and is associated with esterified cholesterol accumulation in seminiferous tubules.

To evaluate the association of variants related to spermatogenesis with susceptibility to Chinese idiopathic nonobstructive azoospermia (NOA), seventeen tag single-nucleotide polymorphisms (SNPs) in CREM, ACT, KIF17b, and SPAG8 were analyzed in 361 NOA patients and 368 controls by Sequenom iplex technology. The results showed that two CREM SNPs, rs4934540 and rs22954152, were significantly associated with NOA and played protective roles against the disease (P value with Bonferroni correction = 0.00017, odds ratio [OR] = 0.624 and P = 0.012, OR = 0.686, respectively). Haplotype analysis of CREM gene variants suggested that haplotype CGTG of the SNPs, rs4934540, rs2295415, rs11592356, and rs1148247, exhibited significant protective effect against the occurrence of NOA (P = 0.001, OR = 0.659). The haplotype TATG conferred a significantly increased risk of NOA (P = 0.011, OR = 1.317). Furthermore, making use of quantitative RT-PCR, we demonstrated that relative mRNA expression of CREM in NOA patients with maturation arrest was only one-third of that in the controls with normal spermatogenesis (P < 0.0001). Our findings indicated that the polymorphisms of CREM gene were associated with NOA in the Chinese population and low CREM expression might be involved in the pathogenesis of spermatogenesis maturation arrest.