Cumulus Cell-oocyte Complexes Research Articles

Cumulus Cells are an Essential Mediator of Ovulation Stimuli from Granulosa Cells to Oocyte

Abstract: In preovulatory follicles, oocytes are surrounded by numerous layers of cumulus cells in a known as the cumulus cell-oocyte complex (COC). After stimulation of ovulation by the LH surge, the morphology of COC is dramatically changed as a hyaluronan rich matrix accumulates within cumulus cell layer, and the oocyte resumes meiosis by progressing to the metaphase II. Although both changes induced by LH surge are essential for successful fertilization in vivo, the expression of LH receptors is not detected in the oocyte and is minimal (negligible) in cumulus cells compared with granulosa cells. However, cumulus cells express members of the EGF receptor family (ErbB family), prostaglandin receptors (EP2 and EP4) and cytokine family receptors that respond to specific ligands secreted by granulosa cells during the ovulation process. By these intermediary steps, the cumulus cells mediate LH signaling from granulosa cells to induce oocyte maturation. This minireview focuses on the role of cumulus cells i...

Overexpression of VEGF165b in mouse ovary results in reduced litter size

Angiogenesis in the ovary facilitates oxygen, nutrient and hormone delivery. VEGF, which plays a key role in regulation of ovarian follicle development, is differentially spliced in the terminal exon to form two families of proteins, VEGFxxx (angiogenic) and VEGFxxxb (anti‐angiogenic). To identify the function of VEGF165b in the ovary, nine female transgenic mice (TG) overexpressing VEGF165b under the control of the MMTV promoter and 10 wild type (WT) female littermates were mated with WT mice. RT‐PCR and immunohistochemistry indicated strong expression of VEGF165b in ovaries of TG females. TG mothers produced litters of half the size of WT dams (3.3±0.4 vs. 5.8±0.7 pups/litter), or TG males (6.1±0.7 pups/litter). There was no developmental defect identified in either the WT or TG pups from TG mothers. The number of embryos released into oviducts by TG females were reduced to 82±6% of control mice. 38.5% of TG females had embryos lacked the Cumulus Cell‐Oocyte Complex, while none from WT females. Overexpression of VEGF165b appears to reduce fertility, possibly by its anti‐angiogenic actions on follicular development.Supported by the Wellcome Trust and British Heart Foundation.

The Nuclear Receptor Cofactor Receptor-Interacting Protein 140 Is a Positive Regulator of Amphiregulin Expression and Cumulus Cell-Oocyte Complex Expansion in the Mouse Ovary

The nuclear receptor cofactor receptor-interacting protein 140 (RIP140) is essential for cumulus cell-oocyte complex (COC) expansion, follicular rupture, and oocyte release during ovulation. The expression of many genes necessary for COC expansion is impaired in the absence of RIP140, but the studies herein document that their expression can be restored and COC expansion rescued by treatment with the epidermal growth factor (EGF)-like factor amphiregulin (AREG) both in vitro and in vivo. We demonstrate by several approaches that RIP140 is required for the expression of the EGF-like factors in granulosa cells, but the dependence of genes involved in cumulus expansion, including Ptgs2 Has2, Tnfaip6, and Ptx3, is indirect because they are induced by AREG. Treatment of granulosa cells with forskolin to mimic the effects of LH increases AREG promoter activity in a RIP140-dependent manner that 1) requires an intact cAMP response element in the proximal promoter region of the Areg gene and 2) involves its actions as a coactivator for cAMP response element-binding protein/c-Jun transcription factors. Although human chorionic gonadotropin and AREG coadministration is sufficient to restore ovulation fully in RIP140 heterozygous mice in vivo, both follicular rupture and ovulation remain impaired in the RIP140 null mice. Thus, we conclude that although the level of RIP140 expression in the ovary is a crucial factor required for the transient expression of EGF-like factors necessary for cumulus expansion, it also plays a role in other signaling pathways that induce follicular rupture.

The Cumulus Cell-Secreted Factor Neuregulin 1 Maintains the Cumulus Cell Function in Ovulated COCs To Enhance Fertilization and Developmental Competence in Porcine Oocytes.

It has been known that cumulus cells support oocyte meiotic and cytoplasmic maturation. Our previous study showed that the EGF like factors (amphiregulin, epiregulin and betacellulin) are expressed in granulosa cells and cumulus cells and act on cumulus cells to induce cumulus cell-oocyte complex (COC) expansion and oocyte maturation in mice and in pigs. Additionally, when ovulated mouse COCs were cultured with amphiregulin, this factor activated/phosphorylated the EGF receptor (ErbB1) and maintained cumulus expansion and the expression of specific genes when expression of endogenous amphiregulin is decreased markedly. Moreover, we have shown recently that the maintenance of the COC matrix and the secretion of specific chemokines from cumulus cells enhance successful fertilization via supporting sperm motility and induction of sperm capacitation. These data suggested that additional ligand members for the ErbB family are required to act to maintain cumulus cell function after ovulation. However, there is little information about what mechanisms regulate cumulus cells in oviductal COCs. Therefore, this study was designed to determine the kinetic expression patterns of ErbB family and the ligand in cumulus cells in vivo and in vitro, and then activate the ErbB signaling pathway to stimulate oocyte maturation in COCs in culture. For this, intact follicles and cumulus cells were collected at early, antral and ovulatory stages of follicle, or from COCs in the oviduct at specific time intervals following eCG and hCG treatment of gilts. FSH/eCG increased levels of EGF receptor and ErbB3 protein in cumulus cells, whereas ErbB2 remained constant and ErbB4 was down regulated. Immuno-histochemistry localized ErbB3 to cumulus cells after eCG and hCG stimulation. Because we found high expression of neuregulin 1 (Nrg1) mRNA in cumulus cells from our rat COC micro-array database, and because RT-PCR analyses showed that Nrg1 expression in cumulus cells of porcine COCs was increased after hCG stimulation and reached maximum levels after ovulation, we sought to determine if NGR1 might regulate cumulus cell functions via ErbB3. When porcine COCs were cultured in vitro with FSH alone, the expression of Nrg1 in cumulus cells did not increase. Therefore, to determine if NRG1 might enhance the actions of FSH, NRG1 was added to FSH-containing medium at 24 hr of culture and COC expansion and oocyte maturation were analyzed after an additional 20-hr culture (total 44 hr culture period). The addition of NRG1 strongly induced the phosphorylation of ErbB3 but not ErbB1, which resulted in the activation of both ERK1/2 and PI 3-kinase-PKB pathways in cumulus cells. The COCs exhibited improved cumulus cell survival (no apoptosis) and sustained expansion. The developmental competence of fertilized oocytes to the blastocyst stage was also enhanced by NGR1. From these results, we show for the first time that NRG1 is a novel COC secreted EGF-like factor that acts via autocrine mechanisms to activate ErbB3 expressed on cumulus cells within COCs after ovulation, and thereby helps maintain cumulus cell function to improve oocyte developmental competence, maybe due to suppress oocyte aging. Supported in part by JSPS-18688016 (MS). (poster)

Interleukin-6: an autocrine regulator of the mouse cumulus cell-oocyte complex expansion process.

Ovulation has long been regarded as a process resembling an inflammatory response. Recent studies indicate that genes associated with innate immune responses were also expressed during the ovulation process. Because the innate immune genes are induced in cumulus cell oocyte complexes (COCs) later than the inflammation-associated genes, we hypothesize that COC expansion is dependent on specific sequential changes in cumulus cells. Because IL-6 is a potent mediator of immune responses, we sought to determine what factors regulate the induction of Il6 mRNA in COCs and what impact IL-6 alone would have on COC expansion. We found that the levels of Il6 mRNA increased dramatically during COC expansion, both in vivo and in vitro. Moreover, IL-6, together with its soluble receptor (IL-6SR), could bypass the need for either amphiregulin and/or prostaglandin E2 to induce the expansion of COCs. This ability of IL-6/IL-6SR to induce COC expansion was blocked by the inhibitors to p38MAPK, MAPK kinase 1/2, and Janus kinase. More importantly, when COCs were in vitro maturated in the presence of IL-6, they had a significantly higher embryo transfer rate than the ones without IL-6 and comparable with in vivo matured oocytes. IL-6/IL-6SR activated multiple signaling pathways (Janus kinase/signal transducer and activator of transcription, ERK1/2, p38MAPK, and AKT) and progressively induced genes known to impact COC expansion, genes related to inflammation and immune responses, and some transcription factors. Collectively, these data indicate that IL-6 alone can act as a potent autocrine regulator of ovarian cumulus cell function, COC expansion, and oocyte competence.

Airway Smooth Muscle Cells Synthesize Hyaluronan Cable Structures Independent of Inter-α-inhibitor Heavy Chain Attachment

The covalent association of inter-alpha-inhibitor-derived heavy chains (HCs) with hyaluronan was first described in synovial fluid from arthritic patients and later described as a structural and functional component of hyaluronan "cable" structures produced by many different cells and stimuli. HC transfer has been shown to be mediated by the protein product of TSG-6 (tumor necrosis factor-stimulated gene 6). Considering the accumulation of hyaluronan in airways following asthmatic attacks and the subsequent infiltration of leukocytes, we sought to characterize HC substitution of hyaluronan "cables" in primary mouse airway smooth muscle cells (MASM) and primary human airway smooth muscle cells (HASM). We found that cells derived from mice lacking TSG-6 had no defect in hyaluronan production or hyaluronan-mediated leukocyte adhesion when treated with the viral mimic poly(I,C). Functional hyaluronan cables were induced by cycloheximide in the confirmed absence of protein synthesis, with or without simultaneous treatment with poly(I,C). We characterized the species specificity of the antibody other investigators used to describe the HC-hyaluronan complex of hyaluronan cables and found minimal affinity to bovine-derived HCs in contrast to HCs from mouse and human sera. Thus, we cultured MASM and HASM cells in serum from these three sources and analyzed hyaluronan extracts for HCs and other hyaluronan-binding proteins, using parallel cumulus cell-oocyte complex (COC) extracts as positive controls. We conclude that, if hyaluronan cables derived from MASM and HASM cells are substituted with HCs, the amount of substitution is significantly below the limit of detection when compared with COC extracts of similar hyaluronan mass.

037. IMMUNE-LIKE MECHANISMS ASSOCIATED WITH OVULATION

Ovulation is the unique biological process by which a mature oocyte and surrounding somatic cells, the cumulus cell-oocyte complex (COC), are released from the surface of the ovary into the oviduct for transport and fertilization. Ovulation is similar to an inflammatory response: the follicles become hyperemic, produce prostaglandins and synthesize a hyaluronan-rich extracellular matrix. However, this view of ovulation may be too restrictive and need to be broadened to encompass the innate immune cell surveillance response system. This hypothesis is being proposed because ovarian granulosa cells and cumulus cells express and respond to innate immune cell related surveillance proteins (Toll-like receptors 2 and 4) and cytokines such as interleukin 6 (IL6) during ovulation. In addition, recent studies indicate that the ovulation process that is set in motion by the surge of luteinizing hormone (LH) is mediated, in large part, by the EGF-like factors (Amphiregulin, epiregulin and betacellulin) and their critical activation of RAS, most probably KRAS that is expressed at high levels in granulosa cells, and the mitogen activated protein kinases, MAP3/1 (ERK1/2). Mice in which granulosa cells are depleted of ERK1/2 fail to ovulate, oocyte meiosis does not resume, COC expansion is impaired and luteinization is blocked. Thus the global molecular reprogramming of granulosa cell gene expression patterns is completely derailed. Supported, in part by NIH-HD-16229, -16272 and -07495 (SCCPIR).

Targeted suppression of Has2 mRNA in mouse cumulus cell-oocyte complexes by adenovirus-mediated short-hairpin RNA expression.

RNA interference (RNAi) is an effective tool for studying gene function in oocytes, but no studies have targeted somatic cells of primary cultured cumulus cell-oocyte complexes (COCs). This is probably due to difficulty in introducing RNAi-inducing molecules, such as a short-hairpin RNA (shRNA) gene, into COCs by commonly used transfection reagents. We therefore tested whether a developmental process of intact COCs could be suppressed by adenovirus-mediated shRNA expression. Has2, encoding hyaluronan synthase 2, was selected as the target transcript, because the process of cumulus expansion depends upon expression of Has2 mRNA and this process is easily evaluated in vitro. Intact COCs were infected with replication-incompetent adenoviruses containing an expression sequence of shRNA targeting either Has2 (Has2 shRNA) or a control transcript not expressed in cumulus cells, and the effects on epidermal growth factor (EGF)-stimulated cumulus expansion were determined. Has2 shRNA expression suppressed Has2 mRNA levels in COCs by more than 70%, without affecting expression levels of Ptgs2, Ptx3, Tnfaip6 mRNAs, which are also required for cumulus expansion, or other transcripts not related to expansion. Interestingly, levels of Areg and Ereg mRNAs were decreased in COCs expressing Has2 shRNA when compared with those in controls, while Btc mRNA levels remained unaffected. Furthermore, the degree of cumulus expansion by Has2 shRNA-expressing COCs was significantly less than that of controls. Thus adenovirus-mediated introduction of shRNA produces specific gene silencing and a phenotype in intact COCs, providing proof of principle that this method will be a helpful tool for understanding mechanisms of COC development.

Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro

In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression of LHCGR and PGR in cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-induced LHCGR expression in cumulus cells in culture. The expression of LHCGR mRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation of PGR and LHCGR in cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observed in vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.

Targeted Suppression of Has2 mRNA in Mouse Cumulus Cell-oocyte Complexes (COCs) by Adenovirus-mediated Short-hairpin RNA (shRNA) Expression.

Targeted Suppression of Has2 mRNA in Mouse Cumulus Cell-oocyte Complexes (COCs) by Adenovirus-mediated Short-hairpin RNA (shRNA) Expression.

Immune-like mechanisms in ovulation

Ovulation is the unique biological process by which a mature oocyte (egg) and surrounding somatic cells, the cumulus cell-oocyte complex (COC), are released from the surface of the ovary into the oviduct for transport and fertilization. Ovulation is similar to an inflammatory response: the follicles become hyperemic, produce prostaglandins and synthesize a hyaluronan-rich extracellular matrix. However, this view of ovulation might be too restrictive and needs to be broadened to encompass the innate immune cell surveillance-response system. This hypothesis is being proposed because ovarian granulosa cells and cumulus cells express and respond to innate immune cell-related surveillance proteins (Toll-like receptors 2 and 4) and cytokines, such as interleukin 6 (IL-6), during ovulation.

Hormone-Induced Expression of Tumor Necrosis Factor α-Converting Enzyme/A Disintegrin and Metalloprotease-17 Impacts Porcine Cumulus Cell Oocyte Complex Expansion and Meiotic Maturation via Ligand Activation of the Epidermal Growth Factor Receptor

The epidermal growth factor (EGF)-like growth factors, amphiregulin (AREG) and epiregulin (EREG), are expressed in murine cumulus oocyte complexes (COCs) where they impact the function of cumulus cells and oocyte maturation during LH-mediated ovulation. Because TNFalpha-converting enzyme (TACE)/a disintegrin and metalloprotease-17 (ADAM17) is essential for ectodomain shedding of AREG and EREG from the surface of other cell types, the expression and function of TACE/ADAM17 was analyzed in a porcine COC culture system in which FSH- and LH-mediated expansion and oocyte meiotic maturation have been well characterized and shown to occur between 20 and 40 h. In this model, Areg, Ereg, and Tace/Adam17 mRNAs increased significantly with maximal levels observed between 5 and 20 h of culture with FSH plus LH. TACE/ADAM17 protein and protease activity were up-regulated markedly at 10 h and maintained to 40 h. Treatment of COCs with the TACE/ADAM17-selective inhibitor TNFalpha-processing inhibitor-2 (TAPI-2) significantly suppressed in a time-dependent manner downstream targets of EGF receptor activation such as ERK1/2 phosphorylation, Ptgs2, Has2, and Tnfaip6 mRNA expression, hormone-induced COC expansion, and meiotic maturation of the oocytes. Addition of EGF to COCs cultured in the presence of FSH/LH reversed the inhibitory effects of TAPI-2 on these ovulation-related processes. Gonadotropin-induced phosphorylation of ERK1/2 was also inhibited in rat granulosa cells treated with TAPI-2 or after transfection with Tace/Adam17 small interfering RNA. Induced expression of Tnfaip6 mRNA was also reduced by Tace/Adam17 small interfering RNA. Thus, TACE/ADAM17 is induced and the activity is involved in porcine COC expansion as well as oocyte meiotic maturation through the activation of EGF receptor in cumulus cells.

PTX3 Interacts with Inter-α-trypsin Inhibitor

Pentraxin 3 (PTX3) and heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI) are essential for hyaluronan (HA) organization within the extracellular matrix of the cumulus oophorus, which is critical for in vivo oocyte fertilization and female fertility. In this study, we examined the possibility that these molecules interact and cooperate in this function. We show that HCs and PTX3 colocalize in the cumulus matrix and coimmunoprecipitate from cumulus matrix extracts. Coimmunoprecipitation experiments and solid-phase binding assays performed with purified human IalphaI and recombinant PTX3 demonstrate that their interaction is direct and not mediated by other matrix components. PTX3 does not bind to IalphaI subcomponent bikunin and, accordingly, bikunin does not compete for the binding of PTX3 to IalphaI, indicating that PTX3 interacts with IalphaI subcomponent HC only. Recombinant PTX3-specific N-terminal region, but not the PTX3-pentraxin C-terminal domain, showed the same ability as full-length protein to bind to HCs and to enable HA organization and matrix formation by Ptx3(-/-) cumulus cell oocyte complexes cultured in vitro. Furthermore, a monoclonal antibody raised against PTX3 N terminus, which inhibits PTX3/IalphaI interaction, also prevents recombinant full-length PTX3 from restoring a normal phenotype to in vitro-cultured Ptx3(-/-) cumuli. These results indicate that PTX3 directly interacts with HCs of IalphaI and that such interaction is essential for organizing HA in the viscoelastic matrix of cumulus oophorus, highlighting a direct functional link between the two molecules.

Gene Expression Profiles of Cumulus Cell Oocyte Complexes during Ovulation Reveal Cumulus Cells Express Neuronal and Immune-Related Genes: Does this Expand Their Role in the Ovulation Process?

Ovulation is a complex process initiated by the preovulatory LH surge, characterized by cumulus oocyte complex (COC) expansion and completed by the release of a mature oocyte. Although many ovarian genes that impact ovulation have been identified, we hypothesized that genes selectively expressed in COCs would be overlooked by approaches using whole ovary or granulosa cell samples. RNA isolated from COCs collected from preovulatory follicles of equine chorionic gonadotropin (CG) primed mice and at selected times after human CG treatment was subjected to microarray analyses and results confirmed by RT-PCR analyses, Western blotting, and immunofluorescent studies. A remarkable number of genes were up-regulated in COCs including Areg, Ereg, and Btc. Several genes selectively expressed in cumulus cells compared with granulosa cells were related to neuronal (Mbp, Tnc, Nts) or immune (Alcam, Pdcd1, Cd34, Cd52, and Cxcr4) cell function. In addition to Sfrp2, other members of the Wnt/Fzd family (Sfrp4, Fdz1 and Fdz2) were expressed in COCs. Thus, there is a cumulus cell-specific, terminal differentiation process. Furthermore, immunofluorescent analyses documented that cumulus cells are highly mitotic for 4-8 h after human CG and then cease dividing in association with reduced levels of Ccnd2 mRNA. Other down-regulated genes included: Cyp19a1, Fshr, Inhb, and the oocyte factors Zp1-3 and Gja4. In summary, the vast number of matrix, neuronal, and especially immune cell-related genes identified by the gene- profiling data of COCs constitutes strong and novel evidence that cumulus cells possess a repertoire of immune functions that could be far greater than simply mediating an inflammatory-like response.

Epidermal growth factor receptor downregulation in cultured bovine cumulus cells: reconstitution of calcium signaling and stimulated membrane permeabilization

Cumulus cell-oocyte complexes (COCs), cultured in vitro, are competent for maturation and fertilization. Inclusion of epidermal growth factor (EGF) in the COC culture medium enhances in vitro maturation and subsequent embryonic development. It has been shown that isolated COCs exposed to EGF respond with a prolonged and pulsatile release of Ca2+ into the extra-cellular medium and that cumulus cells (CCs) of complexes exhibit both a slow rise in intracellular [Ca2+] ([Ca2+]i) and plasma membrane permeabilization in response to EGF. These unusual signaling responses were examined in isolated, cultured bovine CCs. Few individual CCs showed [Ca2+]i increases; the lack of response was found to be due to decrease of expression of endogenous EGF receptors after dissociation. CCs transfected with a human EGF receptor-GFP fusion protein showed robust, prolonged, EGF-stimulated [Ca2+]i elevations characteristic of CC responses in intact COCs. Many CCs that responded to EGF stimulation with a [Ca2+]i rise also released entrapped fura-2 dye at the peak of the [Ca2+]i response, suggesting that CC permeabilization and death follows activation of the EGF receptor. The [Ca2+]i elevation due to EGF stimulation and subsequent membrane permeabilization was shown to be mediated by the inositol triphosphate signaling pathway.

TSG-6 Protein Binding to Glycosaminoglycans: FORMATION OF STABLE COMPLEXES WITH HYALURONAN AND BINDING TO CHONDROITIN SULFATES

TSG-6 protein, up-regulated in inflammatory lesions and in the ovary during ovulation, shows anti-inflammatory activity and plays an essential role in female fertility. Studies in murine models of acute inflammation and experimental arthritis demonstrated that TSG-6 has a strong anti-inflammatory and chondroprotective effect. TSG-6 protein is composed of the N-terminal link module that binds hyaluronan and a C-terminal CUB domain, present in a variety of proteins. Interactions between the isolated link module and hyaluronan have been studied extensively, but little is known about the binding of full-length TSG-6 protein to hyaluronan and other glycosaminoglycans. We show that TSG-6 protein and hyaluronan, in a temperature-dependent fashion, form a stable complex that is resistant to dissociating agents. The formation of such stable complexes may underlie the activities of TSG-6 protein in inflammation and fertility, e.g. the TSG-6-dependent cross-linking of hyaluronan in the cumulus cell-oocyte complex during ovulation. Because adhesion to hyaluronan is involved in cell trafficking in inflammatory processes, we also studied the effect of TSG-6 on cell adhesion. TSG-6 binding to immobilized hyaluronan did not interfere with subsequent adhesion of lymphoid cells. In addition to immobilized hyaluronan, full-length TSG-6 also binds free hyaluronan and all chondroitin sulfate isoforms under physiological conditions. These interactions may contribute to the localization of TSG-6 in cartilage and to its chondroprotective and anti-inflammatory effects in models of arthritis.

Regulated Expression of ADAMTS Family Members in Follicles and Cumulus Oocyte Complexes: Evidence for Specific and Redundant Patterns During Ovulation1

Protease cascades are essential for many biological events, including the LH-induced process of ovulation. ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin-like repeats-1) is expressed and hormonally regulated in the ovary by LH and the progesterone receptor. To determine whether other family members might be expressed and regulated in the rodent ovary, those closely related to ADAMTS1 (ADAMTS4 and ADAMTS5) were analyzed in the mouse ovary by reverse transcription-polymerase chain reaction as well as by Western blot, immunohistochemical, and immunocytochemical analyses using highly specific antibodies. Prior to ovulation, ADAMTS4 and ADAMTS5 were coexpressed in granulosa cells of most follicles, whereas ADAMTS5 was also present in granulosa cells of atretic follicles. Following ovulation, ADAMTS1 and ADAMTS4 (but not ADAMTS5) were expressed in multiple cell types, including those within the highly vascular ovulation cone that marks the site of follicle rupture, endothelial cells of newly forming corpora lutea, and cumulus cells within the ovulated cumulus cell-oocyte complex (COC). Versican, a substrate for ADAMTS1 and ADAMTS4, colocalized with these proteases and hylauronan on the cumulus cell surface. To further characterize induction of these proteases and associated molecules, COCs and granulosa cells were isolated from preovulatory follicles and treated with FSH. In expanded COCs and differentiated granulosa cells, FSH induced expression of ADAMTS4 and versican message and protein, whereas increased levels of ADAMTS1 protein was observed in the media of granulosa cells where it was stabilized by heparin in this in vitro system. These studies provide the first evidence that ADAMTS1, ADAMTS4, and ADAMTS5 are expressed in spatiotemporal patterns that suggest distinct as well as some overlapping functions that relate to the broad expression pattern of versican in granulosa cells of small follicles, expanded COCs, and endothelial cells of the mouse ovary.

Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells: energy metabolism

Intercellular communication between oocytes and granulosa cells is essential for normal follicular differentiation and oocyte development. Subtraction hybridization was used to identify genes more highly expressed in cumulus cells than in mural granulosa cells of mouse antral follicles. This screen identified six genes involved in glycolysis: Eno1, Pkm2, Tpi, Aldoa, Ldh1, and Pfkp. When oocytes were microsurgically removed from cumulus cell–oocyte complexes, the isolated cumulus cells exhibited decreased expression levels of genes encoding glycolytic enzymes, glycolysis and activity of the tricarboxylic acid (TCA) cycle. These decreases were prevented by culturing the cumulus cells with paracrine factors secreted by fully grown oocytes. Paracrine factors from fully grown oocytes exhibited greater ability than those from growing oocytes to promote expression of genes encoding glycolytic enzymes and glycolysis in the granulosa cells of preantral follicles. However, neither fully grown nor growing oocytes secreted paracrine factors affecting activity of the TCA cycle. These results indicate that oocytes regulate glycolysis and the TCA cycle in granulosa cells in a manner specific to the population of granulosa cells and to the stage of growth and development of the oocyte. Oocytes control glycolysis in granulosa cells by regulating expression levels of genes encoding glycolytic enzymes. Therefore, mouse oocytes control the intercellular metabolic cooperativity between cumulus cells and oocytes needed for energy production by granulosa cells and required for oocyte and follicular development.

Effects of adding luteinizing hormone to a medium containing follicle stimulating hormone on progesterone‐induced differentiation of cumulus cells during meiotic resumption of porcine oocytes

ABSTRACTIn the present study, we investigated the effects of adding luteinizing hormone (LH) to a medium containing follicle stimulating hormone (FSH) on the shift in expression of progesterone receptor (PR) isoforms (PR‐A and PR‐B) and the roles in function of cumulus cells of cumulus‐oocyte complexes (COC). The level of PR‐B mRNA in cumulus cells was up‐regulated by FSH during the first 16‐h cultivation but the level was significantly decreased at 20 h. The decrease of PR‐B mRNA was accelerated when COC were cultured with FSH and LH. Still, a high level of total PR mRNA was maintained in cumulus cells cultured with or without the addition of LH up to 20 h, suggesting that the expression of PR isoforms was shifted from PR‐B to PR‐A in cumulus cells. The reduction of PR‐B was also induced by addition of progesterone to FSH‐containing medium. The addition of LH or progesterone to FSH‐containing medium stimulated cumulus expansion of COC as compared with that of COC cultured with FSH. In the expanded COC, ADAMTS‐1 which is expressed in granulosa cells and cumulus cells in rodent follicles through LH‐induced progesterone‐ and PR‐dependent pathway, was more accumulated within the COC matrix. These results suggest that the addition of LH or progesterone to FSH‐containing medium is required for the differentiation of cumulus cells, such as cumulus expansion, mediated by the shift from PR‐B to PR‐A in them.

Expression of two progesterone receptor isoforms in cumulus cells and their roles during meiotic resumption of porcine oocytes.

The present study aimed to investigate progesterone receptor (PR) gene expression in cumulus cells and their roles during meiotic resumption of porcine oocytes. The amount of PR-A or PR-B mRNA was analyzed by RT-PCR using primer sets for the PR-B region or the PR-A/B common region. The level of PR-B mRNA in cumulus cells was up-regulated by FSH and LH during the first 8 h of cultivation but the level significantly decreased at 12 h. However, a high level of total PR mRNA was maintained up to a cultivation period of 20 h. The level of PR-B protein in cumulus cells reached its maximum at 4 to 12 h, whereas PR-A predominated in cumulus cells of cumulus-oocyte complexes (COCs) at 20 h. Accompanying the shift in expression of PR isoforms, progesterone production in cumulus cells was significantly increased, and both the proliferative activity of cumulus cells during a 10- to 20-h cultivation period and the level of connexin-43, a major component of the gap junction, in cumulus cells significantly decreased. When COCs were cultured with FSH and LH for 10 h and then further cultured with additional RU486, there was a significant suppression in the shift in PR isoforms and in progesterone production, a loss of proliferative activity, and a decrease in connexin-43 mRNA in cumulus cells. Moreover, treatment with RU486 after 10-h cultivation of COCs inhibited the meiotic resumption of oocytes and cumulus cell expansion. These results suggest that the induction of PR isoforms in cumulus cells and their binding to progesterone appear to impact on proliferation and differentiation in a time-dependent manner, and the shift from PR-B to PR-A may help mediate certain events.