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Antonietta Salustri - One of the best experts on this subject based on the ideXlab platform.
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cyclic amp elevating agents promote Cumulus Cell survival and hyaluronan matrix stability thereby prolonging the time of mouse oocyte fertilizability
Journal of Biological Chemistry, 2016Co-Authors: Monica Di Giacomo, Antonella Camaioni, Francesca Gioia Klinger, Rita Bonfiglio, Antonietta SalustriAbstract:Cumulus Cells sustain the development and fertilization of the mammalian oocyte. These Cells are retained around the oocyte by a hyaluronan-rich extraCellular matrix synthesized before ovulation, a process called Cumulus Cell-oocyte complex (COC) expansion. Hyaluronan release and dispersion of the Cumulus Cells progressively occur after ovulation, paralleling the decline of oocyte fertilization. We show here that, in mice, postovulatory changes of matrix are temporally correlated to Cumulus Cell death. Cumulus Cell apoptosis and matrix disassembly also occurred in ovulated COCs cultured in vitro. COCs expanded in vitro with FSH or EGF underwent the same changes, whereas those expanded with 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP) maintained integrity for a longer time. It is noteworthy that 8-Br-cAMP treatment was also effective on ovulated COCs cultured in vitro, prolonging the vitality of the Cumulus Cells and the stability of the matrix from a few hours to >2 days. Stimulation of endogenous adenylate cyclase with forskolin or inhibition of phosphodiesterase with rolipram produced similar effects. The treatment with selective cAMP analogues suggests that the effects of cAMP elevation are exerted through an EPAC-independent, PKA type II-dependent signaling pathway, probably acting at the post-transcriptional level. Finally, overnight culture of ovulated COCs with 8-Br-cAMP significantly counteracted the decrease of fertilization rate, doubling the number of fertilized oocytes compared with control conditions. In conclusion, these studies suggest that cAMP-elevating agents prevent Cumulus Cell senescence and allow them to continue to exert beneficial effects on oocyte and sperm, thereby extending in vitro the time frame of oocyte fertilizability.
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activation of Cumulus Cell smad2 3 and epidermal growth factor receptor pathways are involved in porcine oocyte Cumulus Cell expansion and steroidogenesis
Molecular Reproduction and Development, 2011Co-Authors: Eva Nagyova, Antonella Camaioni, Sona Scsukova, Alzbeta Mlynarcikova, Radek Prochazka, Lucie Nemcova, Antonietta SalustriAbstract:SUMMARY Several lines of evidence suggest that in mice the activation of SMAD2/3 signaling by oocyte secreted factors, together with epidermal growth factor receptor (EGFR) activation, is essential to induce Cumulus expansion. Here we show that inhibition of EGFR kinase in follicle stimulating hormone (FSH)-stimulated porcine oocyte– Cumulus Cell complex (OCCs) strongly decreases hyaluronan (HA) synthesis and its retention in the matrix, as well as progesterone synthesis. Although porcine Cumulus Cells undergo expansion independently of oocytes, we use biochemical and gene expression analyses to show that they do require activation of SMAD2/3 for optimal stimulation of HA synthesis and proteins involved in the organization of this polymer in the expanded matrix. Furthermore, FSH-induced progesterone synthesis by porcine Cumulus Cells was increased by blocking SMAD2/3 activation. In conclusion, these results support the hypothesis that an FSH–EGF autocrine loop is active in porcine OCCs, and provide the first evidence that the SMAD2/3 signaling pathway is induced by paracrine/autocrine factors in porcine Cumulus Cells and is involved in the control of both Cumulus expansion and steroidogenesis.
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Activation of Cumulus Cell SMAD2/3 and epidermal growth factor receptor pathways are involved in porcine oocyte-Cumulus Cell expansion and steroidogenesis.
Molecular reproduction and development, 2011Co-Authors: Eva Nagyova, Antonella Camaioni, Sona Scsukova, Alzbeta Mlynarcikova, Radek Prochazka, Lucie Nemcova, Antonietta SalustriAbstract:SUMMARY Several lines of evidence suggest that in mice the activation of SMAD2/3 signaling by oocyte secreted factors, together with epidermal growth factor receptor (EGFR) activation, is essential to induce Cumulus expansion. Here we show that inhibition of EGFR kinase in follicle stimulating hormone (FSH)-stimulated porcine oocyte– Cumulus Cell complex (OCCs) strongly decreases hyaluronan (HA) synthesis and its retention in the matrix, as well as progesterone synthesis. Although porcine Cumulus Cells undergo expansion independently of oocytes, we use biochemical and gene expression analyses to show that they do require activation of SMAD2/3 for optimal stimulation of HA synthesis and proteins involved in the organization of this polymer in the expanded matrix. Furthermore, FSH-induced progesterone synthesis by porcine Cumulus Cells was increased by blocking SMAD2/3 activation. In conclusion, these results support the hypothesis that an FSH–EGF autocrine loop is active in porcine OCCs, and provide the first evidence that the SMAD2/3 signaling pathway is induced by paracrine/autocrine factors in porcine Cumulus Cells and is involved in the control of both Cumulus expansion and steroidogenesis.
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two distinct populations of tumor necrosis factor stimulated gene 6 protein in the extraCellular matrix of expanded mouse Cumulus Cell oocyte complexes
Archives of Biochemistry and Biophysics, 2001Co-Authors: Durba Mukhopadhyay, V C Hascall, Antonietta Salustri, Anthony J Day, Csaba FulopAbstract:After the luteinizing hormone surge, the Cumulus Cell-oocyte complexes (COCs) in the preovulatory follicles produce a viscoelastic extraCellular matrix, a process that requires the synthesis of hyaluronan as well as the incorporation of some components of the inter-alpha-trypsin inhibitor (IalphaI) family. In this study we report, that a hyaluronan-binding protein, the translated product of tumor necrosis factor-stimulated gene-6 (TSG-6), is also specifically accumulated in this matrix. TSG-6 mRNA expression is quickly upregulated and peaks at approximately 1500 copies/Cell 4 h after the ovulatory stimuli as assessed by quantitative reverse transcription-polymerase chain reaction. Immunohistochemistry reveals the colocalization of the TSG-6 protein and hyaluronan around the Cumulus and granulosa Cells. The TSG-6 protein exists in two distinct populations in the COC matrix as demonstrated by Western-blot analysis. One population is a monomer that is anchored to the matrix by a noncovalent interaction. The second population is a covalent complex with either of the heavy chains of IalphaI and is bound to hyaluronan through a strong interaction that is resistant to denaturing conditions. The specific incorporation of the TSG-6 protein into the COC matrix suggests a structural role for this molecule.
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proteoglycans and proteins in the extraCellular matrix of mouse Cumulus Cell oocyte complexes
Archives of Biochemistry and Biophysics, 1996Co-Authors: Antonella Camaioni, Antonietta Salustri, Masaki Yanagishita, V C HascallAbstract:Abstract In the preovulatory follicle, the oocyte is surrounded by ∼1000 closely associated Cumulus Cells forming the compact form of the Cumulus Cell–oocyte complex (COC). In response to the gonadotropin surge, the COC in a follicle destined for ovulation undergoes expansion when the Cumulus Cells synthesize and organize an extensive extraCellular matrix enriched in hyaluronan. Successful expansion of the COC appears to be essential for ovulation and ultimately for fertilization. We studied this process in vitro by isolating compact COCs from preovulatory mouse follicles and incubating them under conditions which promote COC expansion by retention of newly synthesized hyaluronan (HA) in the extraCellular matrix around the Cells. [ 3 H]Leucine and [ 35 S]sulfate were used as precursors to label macromolecules synthesized by the Cells that may be necessary for organizing the HA in this matrix. After labeling, expanded COCs were washed to remove medium and any labeled molecules that were not associated with the matrix. Macromolecules selectively associated with the matrix were then solubilized by digesting the expanded COCs briefly with Streptomyces hyaluronidase, an enzyme that specifically cleaves HA. Cells were removed by centrifugation, and the digest supernate was analyzed by molecular sieve chromatography and SDS–PAGE. A dermatan sulfate proteoglycan of large hydrodynamic size (>1 million Da) and a ∼46-kDa protein were the predominant labeled species identified. The proteoglycan has properties similar to proteoglycans such as aggrecan and versican which interact specifically with HA. The ∼46-kDa protein has the same molecular size as the link protein which interacts with HA and HA-binding proteoglycans to form stable ternary complexes in a variety of extraCellular matrices. We propose that the dermatan sulfate proteoglycan and the ∼46-kDa protein synthesized by the Cumulus Cells form similar ternary complexes that are necessary for retaining HA in the COC matrix and hence are required for successful COC expansion.
Antonella Camaioni - One of the best experts on this subject based on the ideXlab platform.
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cyclic amp elevating agents promote Cumulus Cell survival and hyaluronan matrix stability thereby prolonging the time of mouse oocyte fertilizability
Journal of Biological Chemistry, 2016Co-Authors: Monica Di Giacomo, Antonella Camaioni, Francesca Gioia Klinger, Rita Bonfiglio, Antonietta SalustriAbstract:Cumulus Cells sustain the development and fertilization of the mammalian oocyte. These Cells are retained around the oocyte by a hyaluronan-rich extraCellular matrix synthesized before ovulation, a process called Cumulus Cell-oocyte complex (COC) expansion. Hyaluronan release and dispersion of the Cumulus Cells progressively occur after ovulation, paralleling the decline of oocyte fertilization. We show here that, in mice, postovulatory changes of matrix are temporally correlated to Cumulus Cell death. Cumulus Cell apoptosis and matrix disassembly also occurred in ovulated COCs cultured in vitro. COCs expanded in vitro with FSH or EGF underwent the same changes, whereas those expanded with 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP) maintained integrity for a longer time. It is noteworthy that 8-Br-cAMP treatment was also effective on ovulated COCs cultured in vitro, prolonging the vitality of the Cumulus Cells and the stability of the matrix from a few hours to >2 days. Stimulation of endogenous adenylate cyclase with forskolin or inhibition of phosphodiesterase with rolipram produced similar effects. The treatment with selective cAMP analogues suggests that the effects of cAMP elevation are exerted through an EPAC-independent, PKA type II-dependent signaling pathway, probably acting at the post-transcriptional level. Finally, overnight culture of ovulated COCs with 8-Br-cAMP significantly counteracted the decrease of fertilization rate, doubling the number of fertilized oocytes compared with control conditions. In conclusion, these studies suggest that cAMP-elevating agents prevent Cumulus Cell senescence and allow them to continue to exert beneficial effects on oocyte and sperm, thereby extending in vitro the time frame of oocyte fertilizability.
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activation of Cumulus Cell smad2 3 and epidermal growth factor receptor pathways are involved in porcine oocyte Cumulus Cell expansion and steroidogenesis
Molecular Reproduction and Development, 2011Co-Authors: Eva Nagyova, Antonella Camaioni, Sona Scsukova, Alzbeta Mlynarcikova, Radek Prochazka, Lucie Nemcova, Antonietta SalustriAbstract:SUMMARY Several lines of evidence suggest that in mice the activation of SMAD2/3 signaling by oocyte secreted factors, together with epidermal growth factor receptor (EGFR) activation, is essential to induce Cumulus expansion. Here we show that inhibition of EGFR kinase in follicle stimulating hormone (FSH)-stimulated porcine oocyte– Cumulus Cell complex (OCCs) strongly decreases hyaluronan (HA) synthesis and its retention in the matrix, as well as progesterone synthesis. Although porcine Cumulus Cells undergo expansion independently of oocytes, we use biochemical and gene expression analyses to show that they do require activation of SMAD2/3 for optimal stimulation of HA synthesis and proteins involved in the organization of this polymer in the expanded matrix. Furthermore, FSH-induced progesterone synthesis by porcine Cumulus Cells was increased by blocking SMAD2/3 activation. In conclusion, these results support the hypothesis that an FSH–EGF autocrine loop is active in porcine OCCs, and provide the first evidence that the SMAD2/3 signaling pathway is induced by paracrine/autocrine factors in porcine Cumulus Cells and is involved in the control of both Cumulus expansion and steroidogenesis.
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Activation of Cumulus Cell SMAD2/3 and epidermal growth factor receptor pathways are involved in porcine oocyte-Cumulus Cell expansion and steroidogenesis.
Molecular reproduction and development, 2011Co-Authors: Eva Nagyova, Antonella Camaioni, Sona Scsukova, Alzbeta Mlynarcikova, Radek Prochazka, Lucie Nemcova, Antonietta SalustriAbstract:SUMMARY Several lines of evidence suggest that in mice the activation of SMAD2/3 signaling by oocyte secreted factors, together with epidermal growth factor receptor (EGFR) activation, is essential to induce Cumulus expansion. Here we show that inhibition of EGFR kinase in follicle stimulating hormone (FSH)-stimulated porcine oocyte– Cumulus Cell complex (OCCs) strongly decreases hyaluronan (HA) synthesis and its retention in the matrix, as well as progesterone synthesis. Although porcine Cumulus Cells undergo expansion independently of oocytes, we use biochemical and gene expression analyses to show that they do require activation of SMAD2/3 for optimal stimulation of HA synthesis and proteins involved in the organization of this polymer in the expanded matrix. Furthermore, FSH-induced progesterone synthesis by porcine Cumulus Cells was increased by blocking SMAD2/3 activation. In conclusion, these results support the hypothesis that an FSH–EGF autocrine loop is active in porcine OCCs, and provide the first evidence that the SMAD2/3 signaling pathway is induced by paracrine/autocrine factors in porcine Cumulus Cells and is involved in the control of both Cumulus expansion and steroidogenesis.
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proteoglycans and proteins in the extraCellular matrix of mouse Cumulus Cell oocyte complexes
Archives of Biochemistry and Biophysics, 1996Co-Authors: Antonella Camaioni, Antonietta Salustri, Masaki Yanagishita, V C HascallAbstract:Abstract In the preovulatory follicle, the oocyte is surrounded by ∼1000 closely associated Cumulus Cells forming the compact form of the Cumulus Cell–oocyte complex (COC). In response to the gonadotropin surge, the COC in a follicle destined for ovulation undergoes expansion when the Cumulus Cells synthesize and organize an extensive extraCellular matrix enriched in hyaluronan. Successful expansion of the COC appears to be essential for ovulation and ultimately for fertilization. We studied this process in vitro by isolating compact COCs from preovulatory mouse follicles and incubating them under conditions which promote COC expansion by retention of newly synthesized hyaluronan (HA) in the extraCellular matrix around the Cells. [ 3 H]Leucine and [ 35 S]sulfate were used as precursors to label macromolecules synthesized by the Cells that may be necessary for organizing the HA in this matrix. After labeling, expanded COCs were washed to remove medium and any labeled molecules that were not associated with the matrix. Macromolecules selectively associated with the matrix were then solubilized by digesting the expanded COCs briefly with Streptomyces hyaluronidase, an enzyme that specifically cleaves HA. Cells were removed by centrifugation, and the digest supernate was analyzed by molecular sieve chromatography and SDS–PAGE. A dermatan sulfate proteoglycan of large hydrodynamic size (>1 million Da) and a ∼46-kDa protein were the predominant labeled species identified. The proteoglycan has properties similar to proteoglycans such as aggrecan and versican which interact specifically with HA. The ∼46-kDa protein has the same molecular size as the link protein which interacts with HA and HA-binding proteoglycans to form stable ternary complexes in a variety of extraCellular matrices. We propose that the dermatan sulfate proteoglycan and the ∼46-kDa protein synthesized by the Cumulus Cells form similar ternary complexes that are necessary for retaining HA in the COC matrix and hence are required for successful COC expansion.
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effects of exogenous hyaluronic acid and serum on matrix organization and stability in the mouse Cumulus Cell oocyte complex
Journal of Biological Chemistry, 1993Co-Authors: Antonella Camaioni, V C Hascall, Masaki Yanagishita, A SalustriAbstract:Compact Cumulus Cell-oocyte complexes (COCs) isolated from preovulatory mouse follicles undergo expansion in vitro when high levels of hyaluronic acid (HA) are synthesized and organized into an extraCellular matrix. We studied the effects of fetal bovine serum (FBS) and of exogenous HA and HA-oligomers on the expansion process. Maximum retention of HA in the COC matrix, and hence complete COC expansion, occurs when 1% FBS is continuously present during the first 18 h of culture. Irrespective of the culture time, HA synthesized when serum is absent is primarily in the medium, whereas HA synthesized when serum is present is primarily in the Cell matrix. These findings support the hypothesis that the serum factor, identified as an inter-alpha-trypsin inhibitor by Chen et al. (Chen, L., Mao, S. J., and Larsen, W. J. (1992) J. Biol. Chem. 267, 12380-12386), is a structural component of the matrix. Addition of exogenous HA or of HA oligomers of decasaccharide size (GlcUA-GlcNAc)5 or larger effectively displaces endogenously synthesized HA from the matrix into the medium, thereby preventing COC expansion. Addition of exogenous chondroitin sulfate affects neither matrix organization nor COC expansion, thus indicating specificity of the binding of some structural component(s) to HA. Fully expanded COCs disassemble when cultured longer than 18 h, a process which occurs also in vivo and which correlates with loss of oocyte fertilizability both in vivo and in vitro. This process involves release of macromolecular HA from the matrix into the medium, with loss of 50% of the HA in the first 8 h of incubation after full expansion. The release is not facilitated when HA oligomers, long enough to prevent matrix formation, are added to the culture medium after the COCs are fully expanded. This suggests that cooperative binding to HA of either the serum factor, an endogenously synthesized factor(s), or both is required to stabilize the fully expanded COC matrix.
John J. Eppig - One of the best experts on this subject based on the ideXlab platform.
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Cooperative Effects of 17β-Estradiol and Oocyte-Derived Paracrine Factors on the Transcriptome of Mouse Cumulus Cells
Endocrinology, 2013Co-Authors: Chihiro Emori, John J. Eppig, Karen Wigglesworth, Wataru Fujii, Kunihiko Naito, Koji SugiuraAbstract:Oocyte-derived paracrine factors (ODPFs) and estrogens are both essential for the development and function of ovarian follicles in mammals. Cooperation of these two factors was assessed in vitro using intact Cumulus-oocyte complexes, Cumulus Cells cultured after the removal of oocytes [oocytectomized (OOX) Cumulus Cells], and OOX Cumulus Cells cocultured with denuded oocytes, all in the presence or absence of 17β-estradiol (E2). Effects on the Cumulus Cell transcriptome were assessed by microarray analysis. There was no significant difference between the Cumulus Cell transcriptomes of either OOX Cumulus Cells cocultured with oocytes or intact Cumulus-oocyte complexes. Therefore, oocyte-mediated regulation of the Cumulus Cell transcriptome is mediated primarily by ODPFs and not by gap junctional communication between oocytes and Cumulus Cells. Gene ontology analysis revealed that both ODPFs and E2 strongly affected the biological processes associated with Cell proliferation in Cumulus Cells. E2 had limited effects on ODPF-regulated biological processes. However, in sharp contrast, ODPFs significantly affected biological processes regulated by E2 in Cumulus Cells. For example, only in the presence of ODPFs did E2 significantly promote the biological processes related to phosphorylation-mediated signal transduction in Cumulus Cells, such as the signaling pathways of epidermal growth factor, vascular endothelial growth factor, and platelet-derived growth factor. Therefore, ODPFs and E2 cooperate to regulate the Cumulus Cell transcriptome and, in general, oocytes modulate the effects of estrogens on Cumulus Cell function.
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targeted suppression of has2 mrna in mouse Cumulus Cell oocyte complexes by adenovirus mediated short hairpin rna expression
Molecular Reproduction and Development, 2009Co-Authors: Koji Sugiura, John J. EppigAbstract: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.
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Oocyte regulation of metabolic cooperativity between mouse Cumulus Cells and oocytes: BMP15 and GDF9 control cholesterol biosynthesis in Cumulus Cells.
Development, 2007Co-Authors: Koji Sugiura, Stephanie A Pangas, Martin M Matzuk, Karen Wigglesworth, Marilyn J. O'brien, Jason P. Affourtit, John J. EppigAbstract:Oocyte-derived bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are key regulators of follicular development. Here we show that these factors control Cumulus Cell metabolism, particularly glycolysis and cholesterol biosynthesis before the preovulatory surge of luteinizing hormone. Transcripts encoding enzymes for cholesterol biosynthesis were downregulated in both Bmp15 -/- and Bmp15 -/- Gdf9 +/- double mutant Cumulus Cells, and in wild-type Cumulus Cells after removal of oocytes from Cumulus-Cell-oocyte complexes. Similarly, cholesterol synthesized de novo was reduced in these Cumulus Cells. This indicates that oocytes regulate Cumulus Cell cholesterol biosynthesis by promoting the expression of relevant transcripts. Furthermore, in wild-type mice, Mvk, Pmvk, Fdps, Sqle, Cyp51, Sc4mol and Ebp, which encode enzymes required for cholesterol synthesis, were highly expressed in Cumulus Cells compared with oocytes; and oocytes, in the absence of the surrounding Cumulus Cells, synthesized barely detectable levels of cholesterol. Furthermore, coincident with reduced cholesterol synthesis in double mutant Cumulus Cells, lower levels were also detected in Cumulus-Cell-enclosed double mutant oocytes compared with wild-type oocytes. Levels of cholesterol synthesis in double mutant Cumulus Cells and oocytes were partially restored by co-culturing with wild-type oocytes. Together, these results indicate that mouse oocytes are deficient in synthesizing cholesterol and require Cumulus Cells to provide products of the cholesterol biosynthetic pathway. Therefore, oocyte-derived paracrine factors, particularly, BMP15 and GDF9, promote cholesterol biosynthesis in Cumulus Cells, probably as compensation for oocyte deficiencies in cholesterol production.
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Oocytes are required for the preantral granulosa Cell to Cumulus Cell transition in mice.
Developmental biology, 2007Co-Authors: Francisco Javier Diaz, Karen Wigglesworth, John J. EppigAbstract:Preantral granulosa Cells (PAGCs) differentiate into Cumulus Cells following antrum formation. Cumulus Cells, but not PAGCs, are competent to undergo expansion. Experiments reported here tested the respective roles of both oocytes and FSH in the transition of preantral granulosa Cells to Cumulus Cells competent to undergo expansion. PAGC-oocyte complexes were cultured with or without a low dose of FSH (0.005 IU/ml) and isolated PAGCs were cultured with or without oocytes. At the end of culture, complexes or isolated PAGCs were tested for their ability to undergo Cumulus expansion and upregulate expansion transcripts in response to EGF or FSH (0.5 IU/ml). The ability to undergo expansion in response to EGF required the presence of oocytes but not FSH during the culture period. Likewise, complexes isolated from the ovaries of hypogonadal mice, which lack circulating gonadotropins, underwent expansion in response to EGF, but not FSH. In contrast, the ability to activate MAPK3/1 and MAPK14 and undergo expansion in response to FSH required prior exposure to low doses of FSH. However, these low levels (0.005 or 0.025 IU FSH/ml) suppressed expression of Slc38a3 and Amh, two transcripts highly expressed in Cumulus Cells, suggesting opposing effects of FSH on Cumulus Cell differentiation. In conclusion, the ability to undergo expansion in response to FSH requires prior exposure to FSH during development, while oocyte-derived factors alone are sufficient to promote the ability to undergo expansion in response to EGF. These results highlight the crucial role of oocytes in driving the differentiation of PAGCs into Cumulus Cells during the preantral to antral follicle transition.
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synergistic roles of bmp15 and gdf9 in the development and function of the oocyte Cumulus Cell complex in mice genetic evidence for an oocyte granulosa Cell regulatory loop
Developmental Biology, 2004Co-Authors: Marilyn J Obrien, Martin M Matzuk, Frank L Pendola, James N Denegre, John J. EppigAbstract:Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific growth factors that appear to play key roles in granulosa Cell development and fertility in most mammalian species. We have evaluated the role(s) of these paracrine factors in the development and function of both the Cumulus Cells and oocytes by assessing Cumulus expansion, oocyte maturation, fertilization, and preimplantation embryogenesis in Gdf9+/-Bmp15-/- [hereafter, double mutant (DM)] mice. We found that Cumulus expansion, as well as the expression of hyaluronon synthase 2 (Has2) mRNA was impaired in DM oocyte-Cumulus Cell complexes. This aberrant Cumulus expansion was not remedied by coculture with normal wild-type (WT) oocytes, indicating that the development and/or differentiation of Cumulus Cells in the DM, up to the stage of the preovulatory luteinizing hormone (LH) surge, is impaired. In addition, DM oocytes failed to enable FSH to induce Cumulus expansion in WT oocytectomized (OOX) Cumulus. Moreover, LH-induced oocyte meiotic resumption was significantly delayed in vivo, and this delayed resumption of meiosis was correlated with the reduced activation of mitogen-activated protein kinase (MAPK) in the Cumulus Cells, thus suggesting that GDF9 and BMP15 also regulate the function of Cumulus Cells after the preovulatory LH surge. Although spontaneous in vitro oocyte maturation occurred normally, oocyte fertilization and preimplantation embryogenesis were significantly altered in the DM, suggesting that the full complement of both GDF9 and BMP15 are essential for the development and function of oocytes. Because receptors for GDF9 and BMP15 have not yet been identified in mouse oocytes, the effects of the mutations in the Bmp15 and Gdf9 genes on oocyte development and functions must be produced indirectly by first affecting the granulosa Cells and then the oocyte. Therefore, this study provides further evidence for the existence and functioning of an oocyte-granulosa Cell regulatory loop.
Eva Nagyova - One of the best experts on this subject based on the ideXlab platform.
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activation of Cumulus Cell smad2 3 and epidermal growth factor receptor pathways are involved in porcine oocyte Cumulus Cell expansion and steroidogenesis
Molecular Reproduction and Development, 2011Co-Authors: Eva Nagyova, Antonella Camaioni, Sona Scsukova, Alzbeta Mlynarcikova, Radek Prochazka, Lucie Nemcova, Antonietta SalustriAbstract:SUMMARY Several lines of evidence suggest that in mice the activation of SMAD2/3 signaling by oocyte secreted factors, together with epidermal growth factor receptor (EGFR) activation, is essential to induce Cumulus expansion. Here we show that inhibition of EGFR kinase in follicle stimulating hormone (FSH)-stimulated porcine oocyte– Cumulus Cell complex (OCCs) strongly decreases hyaluronan (HA) synthesis and its retention in the matrix, as well as progesterone synthesis. Although porcine Cumulus Cells undergo expansion independently of oocytes, we use biochemical and gene expression analyses to show that they do require activation of SMAD2/3 for optimal stimulation of HA synthesis and proteins involved in the organization of this polymer in the expanded matrix. Furthermore, FSH-induced progesterone synthesis by porcine Cumulus Cells was increased by blocking SMAD2/3 activation. In conclusion, these results support the hypothesis that an FSH–EGF autocrine loop is active in porcine OCCs, and provide the first evidence that the SMAD2/3 signaling pathway is induced by paracrine/autocrine factors in porcine Cumulus Cells and is involved in the control of both Cumulus expansion and steroidogenesis.
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Activation of Cumulus Cell SMAD2/3 and epidermal growth factor receptor pathways are involved in porcine oocyte-Cumulus Cell expansion and steroidogenesis.
Molecular reproduction and development, 2011Co-Authors: Eva Nagyova, Antonella Camaioni, Sona Scsukova, Alzbeta Mlynarcikova, Radek Prochazka, Lucie Nemcova, Antonietta SalustriAbstract:SUMMARY Several lines of evidence suggest that in mice the activation of SMAD2/3 signaling by oocyte secreted factors, together with epidermal growth factor receptor (EGFR) activation, is essential to induce Cumulus expansion. Here we show that inhibition of EGFR kinase in follicle stimulating hormone (FSH)-stimulated porcine oocyte– Cumulus Cell complex (OCCs) strongly decreases hyaluronan (HA) synthesis and its retention in the matrix, as well as progesterone synthesis. Although porcine Cumulus Cells undergo expansion independently of oocytes, we use biochemical and gene expression analyses to show that they do require activation of SMAD2/3 for optimal stimulation of HA synthesis and proteins involved in the organization of this polymer in the expanded matrix. Furthermore, FSH-induced progesterone synthesis by porcine Cumulus Cells was increased by blocking SMAD2/3 activation. In conclusion, these results support the hypothesis that an FSH–EGF autocrine loop is active in porcine OCCs, and provide the first evidence that the SMAD2/3 signaling pathway is induced by paracrine/autocrine factors in porcine Cumulus Cells and is involved in the control of both Cumulus expansion and steroidogenesis.
Jo Anne S. Richards - One of the best experts on this subject based on the ideXlab platform.
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egf like factors induce expansion of the Cumulus Cell oocyte complexes by activating calpain mediated Cell movement
Endocrinology, 2012Co-Authors: Ikko Kawashima, Jo Anne S. Richards, Zhilin Liu, Lisa K Mullany, Toshihiro Mihara, Masayuki ShimadaAbstract:Cumulus Cell-oocyte complex (COC) expansion is obligatory for LH-induced ovulation and is initiated by LH induction of the epidermal growth factor (EGF)-like factors that mediate the synthesis of the hyaluronan-rich matrix and hyaluronan-stabilizing factors. COC expansion also involves the movement of Cumulus Cells within the matrix by mechanisms that have not been characterized. We document herein that two proteases, calpain 2 and to a lesser extent calpain 1, are expressed in Cumulus Cells and that the proteolytic activity of these enzymes is rapidly and significantly increased in COC isolated from human chorionic gonadotropin-induced ovulatory follicles in vivo. Stimulation of calpain activity was associated with proteolytic degradation of paxillin and talin (two components of focal adhesion complexes), Cell detachment, and the formation of Cell surface bleb-like protrusions. Injection of a calpain inhibitor in vivo reduced 1) human chorionic gonadotropin-stimulated calpain enzyme activity, 2) Cell detachment, 3) membrane protrusion formation, and 4) COC expansion by mechanisms that did not alter Has2 expression. During EGF-like factor induction of COC expansion in culture, calpain activity was increased by ERK1/2 and intraCellular Ca2+ signaling pathways. Inhibition of calpain activity in cultured COC blocked Cumulus Cell detachment, protrusion formation, and the vigorous movement of Cumulus Cells. As a consequence, COC expansion was impaired. Collectively, these results show that two highly coordinated processes control COC expansion. One process involves the synthesis of the hyaluronan matrix, and the other mediates Cumulus Cell detachment and movement. The latter are controlled by calpain activation downstream of the EGF receptor activation of the Ca2+ pathway and ERK1/2 pathways.
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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
Endocrinology, 2010Co-Authors: Jaya Nautiyal, Meritxell Rosell, Jo Anne S. Richards, Francesco J Demayo, Jennifer H Steel, Roger White, Evanthia Nikolopoulou, Malcolm G. ParkerAbstract: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.
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interleukin 6 an autocrine regulator of the mouse Cumulus Cell oocyte complex expansion process
Endocrinology, 2009Co-Authors: Zhilin Liu, Masayuki Shimada, Daniel Gustavo De Matos, Hengyu Fan, Stephen S Palmer, Jo Anne S. RichardsAbstract: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.
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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?
Molecular Endocrinology, 2006Co-Authors: Inmaculada Hernandez-gonzalez, Ignacio Gonzalez-robayna, Scott A Ochsner, Chad M. Wayne, Lisa White, Masayuki Shimada, Jo Anne S. RichardsAbstract: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.
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disrupted function of tumor necrosis factor α stimulated gene 6 blocks Cumulus Cell oocyte complex expansion
Endocrinology, 2003Co-Authors: Scott A Ochsner, Anthony J Day, Marilyn S Rugg, Richard M Breyer, Richard H Gomer, Jo Anne S. RichardsAbstract:During ovulation, the oocyte and surrounding somatic Cumulus Cells contained within a specialized, mucoid matrix are released from the ovary. One matrix component, TNF--stimulated gene 6 (TSG-6), is a hyaluronan binding protein induced in Cumulus Cells of preovulatory follicles by the LH surge and is decreased in Cumulus Cells of COX-2 and prostaglandin E2 (PGE2) receptor subtype EP2 null mice that exhibit impaired ovulation and Cumulus expansion. To determine if TSG-6 was hormonally induced in Cumulus Cells in vitro and was functional during the formation of the expanded matrix, we established a Cumulus Cell-oocyte complex (COC) culture system. This system was used to analyze the effects of FSH, PGE2, EP2 receptor, and selected protein kinase inhibitors on TSG-6 production as well as specific antibodies to the TSG-6 link module on TSG-6 function. We document that TSG-6 message and protein are induced by cAMP/protein kinase A/MAPK signaling pathways and that blocking these cascades prevents expansion and the production of TSG-6. FSH but not PGE2 rescued expansion and production of TSG-6 in the EP2 null COCs, indicating that generation of a cAMP signal is essential. Furthermore, disruption of the functional interactions between TSG-6, inter- trypsin inhibitor, and hyaluronan with specific antibodies severely altered matrix formation and Cumulus expansion, as recorded by time-lapse imaging. Collectively, these results indicate that TSG-6 mRNA is induced in Cumulus Cells in culture by cAMP and that the secreted TSG-6 protein is a key structural component of the mouse COC matrix. (Endocrinology 144: 4376 – 4384, 2003)
