Oocyte specific oolemmal SAS1B involved in sperm binding through intra-acrosomal SLLP1 during fertilization.

Molecular mechanisms by which fertilization competent acrosome-reacted sperm bind to the oolemma remain uncharacterized. To identify oolemmal binding partner(s) for sperm acrosomal ligands, affinity panning was performed with mouse oocyte lysates using sperm acrosomal protein, SLLP1 as a target. An oocyte specific membrane metalloproteinase, SAS1B (Sperm Acrosomal SLLP1 Binding), was identified as a SLLP1 binding partner. cDNA cloning revealed six SAS1B splice variants, each containing a zinc binding active site and a putative transmembrane domain, with signal peptides in three variants. SAS1B transcripts were ovary specific. SAS1B protein was first detected in early secondary follicles in day 3 ovaries. Immunofluorescence localized SAS1B to the microvillar oolemma of M2 oocytes. After fertilization, SAS1B decreased on the oolemma and became virtually undetectable in blastocysts. In transfected CHO-K1 cells SAS1B localized to the surface of unpermeabilized cells. Recombinant and native SLLP1 co-localized with SAS1B to the microvillar domain of ovulated M2 oocytes. Molecular interactions between mouse SLLP1 and SAS1B were demonstrated by surface plasmon resonance, far-western, yeast two-hybrid, recombinant- and native-co-IP analyses. SAS1B bound to SLLP1 with high affinity. SAS1B had protease activity, and SAS1B protein or antibody significantly inhibited fertilization. SAS1B knockout female mice showed a 34% reduction in fertility. The study identified SAS1B-SLLP1 as a pair of novel sperm-egg binding partners involving the oolemma and intra-acrosomal compartment during fertilization.

Non-invasive translational Cynomolgus model for studying folliculogenesis and ovulation using color Doppler ultrasonography.

BACKGROUND: In women, different events of folliculogenesis can be measured and evaluated using ultrasound (US) technology. The availability of a non-invasive translational non-human primate model to study these processes would represent a major contribution to further advance R&D efforts toward novel therapies in assisted reproduction. METHODS: In our study, follicular growth and ovulation was measured in six cyclic Cynomolgus monkeys using abdominal Doppler US. RESULTS: The mean follicular diameter on cycle day -6 (cycle day 0=day of ovulation) was 3.7mm that increased to 6.8mm on cycle day -1. After ovulation, the mean diameter decreased to 4.6mm, confirming ovulation. The mean percentage of follicular size reduction after ovulation was 31%. CONCLUSION: Ultrasonography in combination with color-flow Doppler imaging was shown to be a useful, non-invasive translational method to measure ovarian follicular growth and occurrence and timing of follicular rupture in Cynomolgus monkeys.

Pre-ovulatory LH/hCG surge decreases C-type natriuretic peptide secretion by ovarian granulosa cells to promote meiotic resumption of pre-ovulatory oocytes.

BACKGROUND: In mammalian follicles, oocytes are arrested at the diplotene stage of prophase I until meiotic resumption following the LH surge. Recently, C-type natriuretic peptide (CNP), encoded by natriuretic peptide precursor type C (NPPC) was found to suppress mouse oocyte maturation by promoting cyclic guanosine 5'-monophospate (cGMP) production in cumulus cells. However, regulation of NPPC/CNP expression during the pre-ovulatory period and their regulation by the LH surge have not been investigated. METHODS AND RESULTS: Based on genome-wide analysis of DNA microarray data sets using samples from periovulatory ovaries, we found increases in NPPC transcripts in granulosa cells during pre-ovulatory follicle growth in mice and a rapid decline induced by the pre-ovulatory LH/hCG stimulation. Treatment of pre-ovulatory animals with hCG decreased ovarian CNP content. In isolated ovarian cells, NPPC mRNA was predominantly expressed in mural granulosa cells exhibiting similar regulation following gonadotrophin treatment. In cultured mouse pre-ovulatory follicles, meiosis resumption in oocytes by hCG treatment was accompanied by decreases in NPPC transcript levels. In cultured mouse cumulus cell-oocyte complexes, CNP treatment inhibited the resumption of meiosis with increases in cGMP levels in both cumulus cells and oocytes. In human ovaries, CNP levels in ovarian follicular fluid were also decreased following treatment of patients with an ovulatory dose of hCG. CONCLUSIONS: Our findings demonstrate gonadotrophins regulation of NPPC/CNP expression in mouse and human ovaries and confirm the role of CNP as a potent paracrine oocyte maturation inhibitor.

Gonadotropin stimulation of ovarian fractalkine expression and fractalkine augmentation of progesterone biosynthesis by luteinizing granulosa cells.

Recent studies indicated that ovarian functions are regulated by diverse paracrine factors induced by the preovulatory increases in circulating LH. Based on DNA microarray analyses and real-time RT-PCR, we found a major increase in the transcript levels of a chemokine fractalkine after human chorionic gonadotropin (hCG) treatment during the preovulatory period in gonadotropin-primed immature mice and rats. Although CX3CR1, the seven-transmembrane receptor for fractalkine, was also found in murine ovaries, its transcripts displayed minimal changes. Using tandem RT-PCR and immunohistochemistry, fractalkine transcripts and proteins were localized in cumulus, mural granulosa, and theca cells as well as the oocytes, whereas CX3CR1 was found in the same cells except the oocyte. Real-time RT-PCR further indicated the hCG induction of fractalkine transcripts in different ovarian compartments, with the highest increases found in granulosa cells. In cultured granulosa cells, treatment with fractalkine augmented hCG stimulation of progesterone but not estradiol and cAMP biosynthesis with concomitant increases in transcript levels for key steroidogenic enzymes (steroidogenic acute regulatory protein, CYP11A, and 3beta-hydroxysteroid dehydrogenase). In cultured preovulatory follicles, treatment with fractalkine also augmented progesterone production stimulated by hCG. Furthermore, treatment with fractalkine augmented the phosphorylation of P38 MAPK in cultured granulosa cells. The present data demonstrated that increases in preovulatory LH/hCG induce the expression of fractalkine to augment the luteinization of preovulatory granulosa cells and suggest the fractalkine/CX3CR1 signaling system plays a potential paracrine/autocrine role in preovulatory follicles.

Cyclic guanosine 5'-monophosphate-dependent protein kinase II is induced by luteinizing hormone and progesterone receptor-dependent mechanisms in granulosa cells and cumulus oocyte complexes of ovulating follicles.

Cyclic GMP (cGMP)-dependent protein kinase II (Prkg2, cGK II) was identified as a potential target of the progesterone receptor (Nr3c3) in the mouse ovary based on microarray analyses. To document this further, the expression patterns of cGK II and other components of the cGMP signaling pathway were analyzed during follicular development and ovulation using the pregnant mare serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG)-primed immature mice. Levels of cGK II mRNA were low in ovaries of immature mice, increased 4-fold in response to pregnant mare serum gonadotropin and 5-fold more within 12 h after hCG, the time of ovulation. In situ hybridization localized cGK II mRNA to granulosa cells and cumulus oocyte complexes of periovulatory follicles. In progesterone receptor (PR) null mice, cGK II mRNA was reduced significantly at 12 h after hCG in contrast to heterozygous littermates. In primary granulosa cell cultures, cGK II mRNA was induced by phorbol 12-myristate 13-acetate enhanced by adenoviral expression of PR-A and blocked by RU486 and trilostane. PR-A in the absence of phorbol 12-myristate 13-acetate was insufficient to induce cGK II. Expression of cGK I (Prkg1) was restricted to the residual tissue and not regulated by hormones. Guanylate cyclase-A (Npr1; GC-A) mRNA expression increased 6-fold by 4 h after hCG treatment in contrast to pregnant mare serum gonadotropin alone and was localized to granulosa cells of preovulatory follicles. Collectively, these data show for the first time that cGK II (not cGK I) and GC-A are selectively induced in granulosa cells of preovulatory follicles by LH- and PR-dependent mechanisms, thereby providing a pathway for cGMP function during ovulation.

Intraovarian tumor necrosis factor-related weak inducer of apoptosis/fibroblast growth factor-inducible-14 ligand-receptor system limits ovarian preovulatory follicles from excessive luteinization.

In addition to gonadotropins, many ovarian paracrine factors are crucial for optimal follicle rupture, oocyte maturation, and luteinization. Based on DNA microarray analyses, we found that transcripts for the fibroblast growth factor-inducible-14 (Fn14) receptor are increased after LH/human chorionic gonadotropin (hCG) treatment of gonadotropin-primed immature mice or rats. Fn14 is the cognate receptor for TNF-related weak inducer of apoptosis (TWEAK), a TNF superfamily member. TWEAK transcripts also were detected in the ovary; however, their levels were not regulated by gonadotropins. In situ hybridization analyses indicated that the Fn14 receptor is expressed in the granulosa and cumulus cells of preovulatory follicles and, to a lesser extent, in theca cells. In contrast, in situ hybridization analyses revealed that TWEAK is primarily expressed in theca cells. In cultured granulosa cells pretreated with hCG to induce Fn14 receptor expression, treatment with TWEAK suppressed progesterone synthesis without accompanying changes in cAMP production. Furthermore, intrabursal injection of TWEAK suppressed ovarian progesterone content in gonadotropin-primed rats. In contrast, preovulatory follicles cultured in the presence of the Fn14 decoy, a recombinant protein containing the ligand-binding domain of Fn14, led to increases in progesterone production, presumably by antagonizing the actions of endogenous TWEAK. Likewise, ip injection of the Fn14 decoy enhanced serum progesterone levels with accompanying increases in transcript levels for several key steroidogenic enzymes. The present findings demonstrate a suppressive role of the TWEAK/Fn14 signaling system in the ovary. Following gonadotropin induction of ovulation, Fn14 is induced and could protect preovulatory follicles from excessive luteinization.

Basic helix-loop-helix transcription factor Tcfl5 interacts with the Calmegin gene promoter in mouse spermatogenesis.

In mouse spermatogenesis, differentiating germ line cells initiate expression of specific genes at subsequent developmental steps. The Calmegin (Clgn) gene is first expressed in meiotic prophase, in primary spermatocytes, and encodes a protein that acts as a chaperone. To identify testis-specific transcription factors that control expression of the Clgn gene in spermatogenesis, we performed a yeast one-hybrid screening with a Clgn promoter sequence as bait DNA. This screening resulted in the identification of mouse Tcfl5 as a candidate Clgn promoter-binding protein. Tcfl5 is a member of the basic helix-loop-helix (bHLH) family of transcription factors, and mouse Tcfl5 shows 83% amino acid sequence identity with human TCFL5. Gel-shift and yeast one-hybrid experiments showed that Tcfl5 interacts with a non-canonical CACGCG site that is present in the Clgn promoter. By using northern blot, RT-PCR and in situ hybridization, mouse Tcfl5 mRNA was detected only in testis, with the highest expression level in primary spermatocytes and round spermatids. The highest level of Tcfl5 protein was found in primary spermatocytes at the diplotene stage of meiotic prophase, where the protein colocalizes with transcriptionally active chromatin.

Hormone-regulated expression and localization of versican in the rodent ovary.

During ovulation, production of a specialized hyaluronan (HA)-rich matrix cross-linked by associated HA binding factors causes expansion of the cumulus oocyte complex. Versican is a member of the hyalectan family that binds HA, provides structure and elasticity to tissues, and impacts cell motility and adhesion. In these studies, we sought to determine whether versican is synthesized by ovulating follicles and localizes along with HA in the expanded cumulus oocyte complex matrix in rodent ovaries and whether its expression and/or localization is altered in anovulatory mutant mice. Analysis of mRNA and protein identified isoforms V0, V1, and V3 versican in mouse and rat ovaries throughout follicular development. In situ hybridization localized versican mRNA most specifically to the granulosa cells. Expression was not significantly altered by estradiol or FSH treatment but was increased up to 10-fold during the periovulatory period after human chorionic gonadotropin treatment. In cultured granulosa cells, forskolin and phorbol 12 myristate 13-acetate or FSH + testosterone increased expression of versican. Immunohistochemical analyses verified versican protein in ovulating follicles localized to the expanded cumulus matrix as well as adjacent to the basement membrane. After ovulation, versican was localized around newly formed corpora lutea and vasculature. Unexpectedly, immunohistochemical analyses also demonstrated versican protein on granulosa cells in early primary and small antral follicles. Versican expression and localization were not altered in progesterone receptor or cyclooxygenase-2 null mice, suggesting that transcription of the versican gene is not a target of these two ovulatory mediators. These observations suggest that versican (V0, V1, and V3) is a matrix component of the granulosa layer throughout folliculogenesis and is enriched in remodeling matrices during ovulation and neovascularization of the corpora lutea.

Expression and localization of secreted frizzled-related protein-4 in the rodent ovary: evidence for selective up-regulation in luteinized granulosa cells.

Secreted frizzled-related protein-4 (sFRP-4) belongs to a family of soluble proteins that have a Frizzled-like cysteine-rich domain and function as modulators of Wnt-Frizzled (Fz) signals. As several Wnts and Fz are expressed at defined stages of follicular development in rodent ovaries, these studies were undertaken to evaluate the hormone-regulated expression and localization of sFRP-4. In the mouse ovary, the expression of sFRP-4 mRNA was up-regulated in granulosa cells of large antral follicles after human chorionic gonadotropin administration and was also elevated in corpora lutea, as determined by RT-PCR and in situ hybridization analyses. In hypophysectomized rat ovaries, sFRP-4 expression was similarly induced by human chorionic gonadotropin and further up-regulated by PRL. PRL also stimulated the secretion of sFRP-4 protein from luteinized rat granulosa cells in culture. Therefore, regulation of sFRP-4 by LH and PRL may be important for modulating Fz-1, which is known to be expressed in periovulatory follicles, and Wnt-4/Fz-4, which are expressed in corpora lutea.

Intraovarian thrombin and activated protein C signaling system regulates steroidogenesis during the periovulatory period.

In addition to its role in blood coagulation, thrombin directly stimulates protease-activated receptors (PAR) or interacts with thrombomodulin (THBD) to activate membrane-bound protein C which stimulates PAR1 and PAR4 receptors to promote downstream pleiotropic effects. Our DNA microarray, RT-PCR, and immunostaining analyses demonstrated ovarian expression of THBD, activated protein C (APC) receptor [endothelial protein C receptor (EPCR)], as well as PAR1 and PAR4 receptors in mice. After treatment of gonadotropin-primed immature mice with an ovulatory dose of human chorionic gonadotropin (hCG) (a LH surrogate), major increases in the expression of THBD, EPCR, PAR1, and PAR4 were detected in granulosa and cumulus cells of preovulatory follicles. Immunoassay analyses demonstrated sustained increases in ovarian prothrombin and APC levels after hCG stimulation. We obtained luteinizing granulosa cells from mice treated sequentially with equine CG and hCG. Treatment of these cells with thrombin or agonists for PAR1 or PAR4 decreased basal and forskolin-induced cAMP biosynthesis and suppressed hCG-stimulated progesterone production. In cultured preovulatory follicles, treatment with hirudin (a thrombin antagonist) and SCH79797 (a PAR1 antagonist) augmented hCG-stimulated progesterone biosynthesis, suggesting a suppressive role of endogenous thrombin in steroidogenesis. Furthermore, intrabursal injection with hirudin or SCH79797 led to ipsilateral increases in ovarian progesterone content. Our findings demonstrated increased ovarian expression of key components of the thrombin-APC-PAR1/4 signaling system after LH/hCG stimulation, and this signaling pathway may allow optimal luteinization of preovulatory follicles. In addition to assessing the role of thrombin and associated genes in progesterone production by the periovulatory ovary, these findings provide a model with which to study molecular mechanisms underlying thrombin-APC-PAR1/4 signaling.

Regulated expression of ADAM8 (a disintegrin and metalloprotease domain 8) in the mouse ovary: evidence for a regulatory role of luteinizing hormone, progesterone receptor, and epidermal growth factor-like growth factors.

ADAM8 (a disintegrin and metalloprotease domain 8) is expressed in immune, neuronal, and bone progenitor cells and is thought to be involved in the tissue-remodeling process. Microarray analyses indicate that Adam8 is a potential target of the progesterone receptor (Pgr) in murine ovary. Further studies document that Adam8 mRNA and protein are expressed in granulosa cells and cumulus cells of periovulatory follicles whereas expression is significantly reduced in Pgr null mice that fail to ovulate. There is a reduced expression in granulosa cells from cultured, in vitro ovulated follicles exposed to inhibitors of progesterone or epidermal growth factor signaling while epiregulin induced its expression in the absence of hCG. In vitro studies with primary mouse granulosa cells document that Adam8 is induced in response to forskolin (Fo) and phorbol ester (PMA) or Fo and Amphiregulin treatment. To understand the transcriptional regulation of the Adam8, we amplified 1 kb of the mouse Adam8 promoter by PCR and subcloned it into a pGL3-luciferase reporter construct. The Adam8 promoter-luciferase constructs are induced by Fo and PMA treatment after transfection into rat granulosa cells, and cotransfection with a PGR-A expression vector further augment basal and Fo/PMA inducibility. Site-specific mutations within the -615/+50 promoter document that a GC-rich region, NF-1 (nuclear factor-1) site, and putative TATA box are critical for Adam8 promoter activation by Fo/PMA. Thus, ADAM8 is expressed in a stage-specific manner and is hormonally regulated in ovulating follicles by the coordinate actions of LH and PGR. To our knowledge, ADAM8 is the first member of the ADAM family shown to be hormonally regulated.

Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos.

Optimal development of fertilized eggs into preimplantation embryos is essential for reproduction. Although mammalian oocytes ovulated after luteinizing hormone (LH) stimulation can be fertilized and promoted into early embryos in vitro, little is known about ovarian factors important for the conditioning of eggs for early embryo development. Because LH interacts only with ovarian somatic cells, its potential regulation of oocyte functions is presumably mediated by local paracrine factors. We performed DNA microarray analyses of ovarian transcripts and identified brain-derived neurotrophic factor (BDNF) secreted by granulosa and cumulus cells as an ovarian factor stimulated by the preovulatory LH surge. Ovarian BDNF acts on TrkB receptors expressed exclusively in oocytes to enhance first polar body extrusion of oocytes and to promote the in vitro development of zygotes into preimplantation embryos. Furthermore, in vivo treatment with a Trk receptor inhibitor suppressed first polar body extrusion and the progression of zygotes into blastocysts. Thus, ovarian BDNF is important to nuclear and cytoplasmic maturation of the oocyte, which is essential for successful oocyte development into preimplantation embryos. Treatment with BDNF could condition the cultured oocytes for optimal progression into the totipotent blastocysts.

The mouse germ-cell-specific leucine-rich repeat protein NALP14: a member of the NACHT nucleoside triphosphatase family.

Microscopy of sectioned neonatal mouse ovaries established the predominance of primordial follicles in Day 3 samples and the predominance of primary follicles by Day 8. To identify genetic determinants of the primordial to primary follicle transition, the transcriptome of Day 1 or Day 3 mouse ovaries was contrasted by differential display with that of Day 8 ovaries. This manuscript examines one of the up-regulated genes, the novel Nalp14 gene, whose transcript displayed 18- and 127-fold increments from Day 1 to Days 3 and 8, respectively. First noted by in situ hybridization in oocytes encased by primary follicles, Nalp14 transcripts were continuously expressed through the preovulatory stage. The transcripts declined when meiotic maturation resumed, and they were markedly diminished by the 2-cell embryo stage. The corresponding 3281-base pair, full-length cDNA coded for a 993 residue/104.6-kDa germ cell-specific protein. A member of the multifunctional NACHT NTPase family, the NALP14 protein featured 14 iterations of the leucine-rich-repeat domain, a region implicated in protein-protein interaction. Protein BLAST analysis of the NALP14 sequence revealed 2 previously reported germ cell-specific homologs (i.e., MATER [Maternal Antigen That Embryos Require], RNH2 [RiboNuclease/Angiogenin Inhibitor 2], and NALP4c). The structural attributes, expression pattern, and cellular localization of MATER and RNH2 largely conformed to those reported for NALP14.