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Recent studies indicate that LH stimulates production of ovarian paracrine factors that induce meiosis of the oocyte. DNA microarray analyses of ovarian transcripts were performed in mice and major increases of a short isoform of leptin receptor, ObRa, were identified by the preovulatory LH/human chorionic gonadotrophin (HCG) surge. In oocytes, the level of ObRa transcripts was increased shortly after HCG stimulation, whereas the level of ObRb transcripts was not changed. Leptin was produced by cumulus, granulosa, theca and interstitial cells of ovaries and its transcript level was not regulated during gonadotrophin treatment. Treatment with leptin promoted germinal vesicle breakdown (GVBD) in oocytes within preovulatory follicles, and enhance first polar body extrusion in both cumulus-oocyte complexes and denuded oocytes. The leptin-promoted GVBD and first polar body extrusion were blocked by a mitogen-activated protein kinase extracellular signal regulated kinase kinases (MEK)1/2 inhibitor, U0126, but not its inactive analogue U0124. Furthermore, leptin promoted fertilization of oocytes and the in-vitro development of zygotes to preimplantation embryos. These findings suggest paracrine roles of leptin in the enhancement of nuclear maturation of oocytes through MEK1/2 signalling, and in the promotion of cytoplasmic maturation essential for successful oocyte development to the preimplantation embryos.

Survival and apoptosis of cells in preimplantation embryos are fundamental for successful pregnancy. Relevant to these processes, tumor necrosis factor (TNF) and transforming growth factor alpha (TGFA) are produced by mammalian oviducts and uteri. In early embryos, TNF induces apoptosis, whereas TGFA could act as a survival factor. Here we investigated the TNF regulation of apoptosis in early mouse embryos and its antagonism by TGFA. TNF receptor superfamily, member 1a mRNA was detectable throughout early embryonic stages, with an increase after the early blastocyst stage, whereas the expression of TNF receptor superfamily, member 1b transcripts were detected only at the expanded blastocyst stage. Although pregnant uteri produced TNF, physiologic levels were low during the preimplantation period. Treatment with TNF inhibited the development of two-cell stage embryos to blastocysts showing decreased proliferation and increased apoptosis both in vitro and in vivo. These detrimental effects of TNF on early embryo development and survival were blocked by a neutralizing anti-TNF antibody. In addition to the death receptor-mediated pathway, TNF-induced apoptosis was further mediated by disruption of mitochondrial functions, characterized by release of cytochrome c and activation of caspase 9. The proapoptotic effects of TNF in blastocysts were counteracted by cotreatment with TGFA. The antagonistic effect of TGFA on TNF-induced apoptosis was blocked by phosphatidylionsitol 3-kinase (PI3K) inhibitors. The present findings demonstrate the stage-selective susceptibility to the apoptosis-inducing effect of TNF in mouse preimplantation embryos and that the TGFA/PI3K signaling system has an important role in the control of TNF-induced apoptosis in blastocysts.

Transforming growth factor alpha (TGFA) is produced by epithelial cells in the oviducts and uteri and has the potential to act as an anti-apoptotic factor on preimplantation embryos expressing its receptor. Previously, we demonstrated that survivin (also known as BIRC5), an anti-apoptotic gene expressed in mouse preimplantation embryos, protects embryos from apoptosis. In this study, we investigated the role of survivin on TGFA-mediated inhibition of apoptosis in mouse blastocysts. Under the suboptimal conditions produced by single embryo culture, blastocysts showed an increase of apoptosis that correlated with a decrease of survivin expression. TGFA treatment significantly decreased apoptosis and increased the levels of survivin mRNA in a dose-dependent manner in blastocyst, and conversely, these activities were neutralized by an anti-TGFA antibody. Antibody treatment alone exerted little effect on either the occurrence of apoptosis or the levels of survivin mRNA. Upregulation of survivin expression by TGFA treatment was insignificant before the blastocyst stage. Using an antisense approach, we examined whether upregulation of survivin is responsible for the anti-apoptotic effect of TGFA in blastocysts. Apoptosis was inhibited by TGFA treatment in blastocysts, but the effect was abrogated by cotreatment with antisense oligonucleotides directed against survivin. These data suggest that survivin contributes to the anti-apoptotic activities of TGFA in blastocysts. We also found that the upregulation of survivin expression was mediated by activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Thus, TGFA inhibits apoptosis in mouse blastocysts through upregulation of survivin expression via the PI3K pathway.

Regulation of early embryonal development during fertilization and implantation is crucial for mammalian reproduction. Several studies have described cell death during preimplantation embryogenesis in a range of mammalian species, both in vivo and in vitro. Therefore, apoptosis may be involved in early embryonic arrest and the characteristic cytoplasmic fragments are the equivalents of apoptotic bodies, the end-product of apoptosis. Although apoptosis is expected to associate with fragmentation in early preimplantation embryos, the mechanism through which this fragmentation occurs has not been elucidated. Recently, second mitochondria-derived activator of caspase/Direct IAP Binding Protein with Low pI (Smac/DIABLO) was identified as a mitochondrial protein that is released into the cytosol during apoptosis. Once released, the Smac/DIABLO blocks the anti-apoptotic activity of inhibitor of apoptosis proteins (IAPs). We hypothesized that the Smac/DIABLO may be involved in the fragmentation of mouse preimplantation embryos. Therefore, we investigated the expression of Smac/DIABLO mRNA and protein and its localization in mouse oocytes and preimplantation embryos. Smac/DIABLO mRNA was detected by RT-PCR in the oocytes and the preimplantation embryos. Immunohistochemistry studies showed that the Smac/DIABLO protein localized in mitochondria and was released into the cytosol in both fragmented embryos and embryos in which apoptosis was induced by staurosporine. These observations indicate that the Smac/DIABLO is involved in the fragmentation and apoptosis of preimplantation embryos.

Although ghrelin acts as a modulator of feeding behavior and energy metabolism in the central nervous system, recent studies have implicated the peripheral actions of ghrelin in reproductive tissues. Here, we investigated the expression of ghrelin and its receptor (GHS-R) in mouse oocyte and preimplantation embryos, and we examined the role of ghrelin in the regulation of early embryo development. Both ghrelin and GHS-R mRNAs were detected in morula or more advanced embryo stages. As for the origin of ghrelin, both ghrelin mRNA and protein were identified in the uterine endometrium. The levels of ghrelin in uterine fluid as well as plasma were significantly increased in fasting mice compared with animals with free access to foods. Addition of ghrelin to culture media inhibited the development of two-cell embryos to the hatched blastocysts, and the inhibitory effects of ghrelin were abolished by an antagonist for the GHS-R. In addition, ghrelin significantly decreased the number of total cells, inner cell mass, and trophectoderm cells in blastocysts. These observations suggest that ghrelin could inhibit the development of preimplantation embryos during fasting. Thus, ghrelin may act as a peripheral factor to avoid the excess metabolic demands imposed by pregnancy during malnutritional states.

Apoptosis is an essential physiologic process used in almost all tissues to remove damaged or superfluous cells. However, the early embryos are unique because no cell death is found up to the blastocyst stage during normal development. Survivin, a member of the IAP family, is capable of binding to caspases to modulate their functions. Here, we investigated the expression of survivin, and its role in preventing apoptosis in mouse preimplantation embryos. Transcripts for survivin and a splice variant lacking exon 2 were detected from unfertilized oocytes up to hatched blastocyst stage. At the protein level, survivin was also detected at all stages of early embryos. The antisense approach was used to demonstrate the role of survivin on embryo development. Development of early embryos treated with antisense survivin oligonucleotides was arrested at the morula or early blastocyst stage with disruption of tubulin formation and abnormal nuclei, associated with apoptosis. The effect of the antisense was enhanced by cotreatment with an apoptosis-inducing reagent, staurosporine. In contrast, apoptosis induced by the antisense treatment was inhibited by caspase-3 and -9 inhibitors. These results indicate that survivin is an essential antiapoptotic gene expressed in preimplantation embryos and could protect the embryos from apoptosis by inhibiting an apoptotic pathway involving caspases., (Copyright 2003 Elsevier Science (USA))

Leptin acts as a modulator of diverse reproductive functions, and recent studies have implicated involvement of leptin in the early embryo development in mammal. The aim of this study was to investigate the expression of leptin and its receptor (OB-R) in mouse oocyte and preimplantation embryo, and to examine whether leptin influenced the early embryo development. Leptin mRNA was detected in blastocyst and hatched blastocyst, and two splice variants of OB-R (OB-Ra and OB-Rb) mRNAs were detected in oocytes, 1-cell, 2-cell, morula, blastocyst, and hatched blastocyst. As for the origin of leptin, both leptin mRNA and protein were identified in the oviduct epithelium and endometrium of pregnant mouse. In the pregnant mouse, the levels of leptin in uterine fluid were higher than those in nonpregnant mouse. Addition of leptin to embryo culture media promotes the development from 2-cell stage embryos to the blastocysts, fully expanded blastocysts and hatched blastocysts. This effect was neutralized by an antibody against the extracellular domain of OB-R. Leptin significantly increased the total cell number of blastocysts, and the effect was preferentially observed in the trophectoderm. These findings raise the possibility of a paracrine/autocrine leptin signaling system regulating the development of mouse preimplantation embryo.

Abstract: Optimal maturation of oocytes and successful development of preimplantation embryos is essential for reproduction. We performed DNA microarray analyses of ovarian transcripts and identified glial cell line-derived neurotrophic factor (GDNF) secreted by cumulus, granulosa, and theca cells as an ovarian factor stimulated by the preovulatory LH/hCG surge. Treatment of cumulus–oocyte complexes with GDNF enhanced first polar body extrusion with increase in cyclin B1 synthesis and the GDNF actions are likely mediated by its receptor GDNF family receptor-alpha1 (GFRA1) and a co-receptor ret proto-oncogene (Ret), both expressed in oocytes. However, treatment with GDNF did not affect germinal vesicle breakdown and cytoplasmic maturation of oocytes. During the preimplantation stages, GDNF was expressed in pregnant oviducts and uteri, whereas GFRA1 and Ret were expressed in embryos throughout early development with an increase after the early blastocyst stage. In blastocysts, both GDNF and GFRA1 were exclusively localized in trophectoderm cells, whereas Ret was detected in both cell lineages. Treatment with GDNF promoted the development of two-cell-stage embryos into blastocysts showing increased cell proliferation and decreased apoptosis mainly in trophectoderm cells. Our findings suggest potential paracrine roles of GDNF in the promotion of completion of meiosis I and the development of early embryos. [Copyright &y& Elsevier]

Abstract: Hormonal factors secreted by embryos and reproductive tracts are important for successful development of preimplantation embryos. We found expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) transcripts at its highest levels in the blastocyst stages. The transcripts for their receptor, TrkB, were detectable throughout the early embryonic stages with an increase after the early blastocyst stage. Both BDNF and TrkB are expressed in trophectoderm cells, whereas ligand-binding studies indicated specific binding of BDNF to trophectoderm cells. Furthermore, BDNF and NT-4/5 were produced in pregnant oviducts and uteri. Treatment with BDNF promoted the development of two-cell-stage embryos into blastocysts showing increased proliferation and decreased apoptosis. The effects of BDNF were blocked by the TrkB ectodomain or a Trk receptor inhibitor, K252a. Studies using specific inhibitors demonstrated the roles of the PI3K, but not the ERK, pathway in mediating BDNF actions. Under high-density embryo cultures, treatment with the TrkB ectodomain or K252a alone also inhibited embryonic development and survival, suggesting potential autocrine actions of BDNF produced by the embryo. In vivo experiments further demonstrated that K252a treatment suppressed early embryo development by inhibiting blastocyst cell numbers, and increasing blastocyst apoptosis. Our findings suggested that BDNF signaling plays important paracrine roles during blastocyst development by promoting the development of preimplantation embryos. [Copyright &y& Elsevier]