Cumulus Oocyte Complex Matrix Research Articles

Compromised Cumulus-Oocyte Complex Matrix Organization and Expansion in Women with PCOS

The cumulus-oocyte complex (COC) matrix plays a critical role in the ovulation and fertilization process and a major predictor of oocyte quality. Proteomics studies of follicular fluid showed differential expression of COC matrix proteins in women with polycystic ovary syndrome (PCOS), indicating altered COC matrix in these women. In the present study, we aimed to understand COC matrix gene induction in humans and its probable dysfunction in women with PCOS. Animal studies have shown that amphiregulin (AREG) and growth differentiation factor-9 (GDF-9) are important in the induction of COC matrix genes which are involved in cumulus expansion. The effects of AREG and GDF-9 on expression of tumor necrosis factor alpha induced protein 6 (TNFAIP6) and hyaluronan synthase 2 (HAS2) on human cumulus granulosa cells (CGCs) and murine COC expansion were evaluated. Further time-dependent effects of growth factor supplementation on these gene expressions in CGCs from PCOS and control women were compared. Follicular fluid from PCOS showed reduced COC matrix expansion capacity, using murine COCs. Expression of COC matrix genes TNFAIP6 and HAS2 were significantly reduced in CGCs of PCOS. Treatment of CGCs with AREG and GDF-9 together induced expression of both these genes in controls and could only restore HAS2 but not TNFAIP6 expression in PCOS. Our results suggest that the reduced potential of follicular fluid to support COC expansion, altered expression of structural constituents, and intrinsic defects in granulosa cells of women with PCOS may contribute to the aberrant COC organization and expansion in PCOS, thus affecting fertilization.

Differential activation of noncanonical SMAD2/SMAD3 signaling by bone morphogenetic proteins causes disproportionate induction of hyaluronan production in immortalized human granulosa cells

Successful fertilization depends upon proper cumulus–oocyte complex (COC) expansion. Synthesized by hyaluronan synthases (HASs), hyaluronan forms the backbone of the COC matrix and plays a critical role in COC expansion. This study investigated the effects and mechanisms of ovarian BMPs on HAS expression and hyaluronan production in human granulosa cells. Treatment with BMP4, BMP6, BMP7 or BMP15 induced differing levels of noncanonical SMAD2/3, but equal levels of canonical SMAD1/5/8, phosphorylation which were mirrored by differing levels of HAS2 up-regulation and hyaluronan production. The effects of BMP4 and BMP15 on HAS2 mRNA were partially reversed by knockdown of SMAD3, and blocked by knockdown of SMAD2+SMAD3 or SMAD4. BMP4-induced SMAD2/3 phosphorylation and HAS2 mRNA up-regulation were mediated by both BMP and activin/transforming growth factor-β type I receptors. Our results suggest differential activation of noncanonical SMAD2/SMAD3 signaling by BMPs causes disproportionate induction of HAS2 expression and hyaluronan production in immortalized human granulosa cells.

ADAMTS1 Cleavage of Versican Mediates Essential Structural Remodeling of the Ovarian Follicle and Cumulus-Oocyte Matrix During Ovulation in Mice1

Remodeling of ovarian follicle extracellular matrix is essential for ovulation and vascularization of the corpus luteum (CL). Formation of the cumulus matrix around oocytes also plays an important role in ovulation and subsequent fertilization of oocytes. ADAMTS1 is an extracellular metalloprotease induced in ovarian follicles by ovulatory hormones and is required for fertility. In this study, we identified ADAMTS1-mediated structural and morphological changes in remodeling of the follicle and cumulus oocyte complex (COC). In Adamts1(-/-) mice, the ovulation rate was 77% reduced and fertilization of ovulated oocytes was reduced a further 63%, resulting in a reduced number of litters and pups per litter. Morphological assessment of peri-ovulatory ovaries revealed abnormal morphogenesis with a lack of thecal/vascular invagination in the basal region of follicles. Cleavage of the ADAMTS1 substrate, versican, at these invaginating regions was abundant in Adamts1(+/-) but undetectable in Adamts1(-/-) ovaries, indicating that processing of versican by ADAMTS1 is involved in ovulating follicle remodeling. Versican and hyaluronan localization was abnormal during COC matrix expansion, and versican persisted beyond the expected time of fertilization in Adamts1(-/-) but was catabolized and cleared from control COC. The results demonstrate that ADAMTS1 is critical in both ovulation and fertilization processes in vivo. The protease activity of ADAMTS1 mediates neomorphogenesis of the ovulating follicle wall and COC matrix necessary for successful ovulation and fertilization, as well as subsequent catabolism of versican required for degradation of COC matrix after fertilization.

146. MOLECULAR FILTRATION PROPERTIES OF THE EXPANDED CUMULUS MATRIX: CONTROLLED SUPPLY OF METABOLITES AND EXTRACELLULAR SIGNALS TO CUMULUS CELLS AND THE OOCYTE

Cumulus matrix genes are positively correlated with oocyte competence [1]. Formation of the expanded cumulus matrix during oocyte maturation is well described; however its function remains elusive. We investigated whether cumulus matrix acts as a molecular filter, based on recognised filtration properties of analogous matrices. We found that cumulus matrix controls metabolite supply to the oocyte and retains prostaglandin E2 (PGE2), which is critical in oocyte maturation. The uptake of fluorescently labelled hydrophilic and hydrophobic metabolites showed that cumulus matrix formation significantly impeded diffusion to the oocyte. Expanded in vivo matured cumulus oocyte complexes (COCs, eCG+hCG16h) resisted uptake of glucose and cholesterol compared to unexpanded (eCG44h, P < 0.05), as assessed by confocal microscopy and spatial quantitation of fluorescence (P < 0.05). In vitro maturation (IVM) results in pronounced compositional deficiency of cumulus matrix proteins [2] and poor oocyte quality. Glucose and cholesterol were transported more readily into cumulus cells and the oocyte of IVM COCs (matured in αMEM/5% FCS/50 mIU/mL FSH, 16 h) compared to in vivo matured COCs (P < 0.05 and P = 0.08, respectively). Taking the inverse approach we found that PGE2 synthesised by cumulus cells is retained within the matrix compartment of in vivo matured COCs but IVM COCs did not retain PGE2 and secreted 4.3-fold more into the media. The relationship of retained to secreted PGE2 was significantly higher after in vivo maturation vs IVM COCs (P < 0.0001). This property of the COC matrix reveals a potential mechanism whereby the prostaglandin signal intensifies through a physicochemical mechanism rather than gene regulation. This is the first demonstration that cumulus matrix regulates diffusion toward and secretion from the COC, thus excluding glucose, known to negatively affect oocyte quality, and trapping factors, including PGE2, with critical roles in oocyte maturation and fertilisation. Thus, IVM may reduce oocyte quality due to poor trafficking of metabolites and signalling molecules. (1) McKenzie LJ, et al. Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF. Hum Reprod 2004; 19: 2869–2874.(2) Dunning KR, et al. Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes. Hum Reprod 2007; 22: 2842–2850.

155. CD44 SIGNALLING IN THE CUMULUS OOCYTE COMPLEX DURING OVULATION

Oocytes develop within ovarian follicles that nurture and regulate oocyte maturation. The LH surge induces a cascade of gene expression leading to formation of the hyaluronan (HA) rich cumulus matrix around the oocyte. This cumulus oocyte complex (COC) is composed of high concentrations of HA cross-linked by several HA-binding proteins. Null mutation of several COC matrix genes results in ovulation defects demonstrating the importance of the composition and structure of the COC; but the mechanisms by which the matrix promotes ovulation are unknown. We hypothesised that HA, via activation of its receptor CD44 on cumulus cells, regulates cytoskeletal function, cell adhesion and migration, and that acquired cumulus cell motility facilitates ovulation. We investigated cellular signaling and cellular phenotypes occurring in response to the formation of the HA-rich COC matrix. Expression of CD44 was upregulated 5 to 6-fold in cumulus cells following 6 or 12h hCG (LH analog) stimulation. Signal transducers of CD44 action; Tiam1, a guanine exchange factor, and Rac1, an actin cytoskeleton remodelling Rho-family GTPase, were present in cumulus cells but not regulated by hCG. Induction of migratory and invasive capacity of cumulus cells by hCG was demonstrated using transwell migration and ECM invasion assays. Cumulus cell migration increased 8-fold 10h after hCG compared with cumulus cells from untreated mice. These cumulus cells also showed the capacity to invade through a matrigel barrier. Inhibitors of the CD44-assembled cell migration complex demonstrated the importance of this pathway in the migratory and invasive phenotype of cumulus cells. These results demonstrate that CD44 is a key factor in the assembly of a macromolecular complex facillitating cell motility in cumulus cells at the time of ovulation, and suggest that cumulus cells in the expanded COC undergo epithelial-mesenchymal transition to become invasive motile cells which may play a key role mediating ovulation.

269. CD44 signaling in mouse ovulatory cumulus - oocyte complexes

Oocytes grow and develop within ovarian follicles, providing a nurturing environment before their release (ovulation) into the oviduct for fertilisation. For ovulation to occur the ovarian follicle responds to LH from the pituitary, leading to a cascade of regulated gene expression and formation of the hyaluronan rich cumulus matrix around the oocyte. This COC (cumulus oocyte complex) matrix is composed of high concentrations of the ECM glycosaminoglycan hyaluronan (HA) cross-linked by several HA-binding proteins. Several of the COC matrix components are essential for ovulation, since null gene mutations in mice lead to ovulation defects. Mechanisms by which the COC matrix controls ovulation however, are unknown. We have investigated cellular signalling and cellular phenotypes that occur as part of the formation of the COC matrix. The transmembrane HA receptor CD44 was significantly upregulated in cumulus cells from 6 h after hCG (LH analogue) treatment (9.8 ± 1.5 fold) until ovulation at 12 h post hCG (11.8 ± 2.9-fold). In many cell types CD44 activates the intracellular Rho-family GTPase Rac1 and its activator, the guanine exchange factor Tiam1, pleiotropic regulators of cytoskeletal function, cell-cell adhesion and migration. We found both Rac1 and Tiam1 were strongly detected in cumulus cells, but not regulated by hCG. These observations show that at the time of ovulation a macro-molecular complex associated with cell motility is assembled through the extracellular interaction of the COC matrix and cell surface proteins. We investigated the migratory and invasive activity of COCs from hormonally stimulated mice. Migration of cumulus cells from hCG treated mice was significantly increased compared with untreated COCs. Furthermore the hCG-stimulated cumulus cells were able to invade a range of ECM substrates including collagen and laminin. These results suggest the cumulus cells in the expanded COC transition to a motile cell phenotype that may play a key role in promoting ovulation.

Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes

In vitro maturation (IVM) of mammalian oocytes has potential health benefits for patients undergoing assisted reproduction as an alternative to gonadotrophin treatment. This procedure is also useful for studying the process of oocyte and early embryo development. However, oocytes undergoing IVM have much lower competence than in vivo matured oocytes. Efforts to optimize IVM success have focused on replicating in vivo timing, hormonal milieu and cumulus cell responses associated with maturing oocytes. We have previously identified two extracellular matrix proteins, the protease Adamts1 and hyaluronan-binding proteoglycan Versican, produced by mural granulosa cells that selectively incorporate into the periovulatory cumulus-oocyte complex (COC). Murine COC were cultured in the presence of epidermal growth factor and/or FSH. mRNA and protein were measured by real time PCR and Western blot and compared to in vivo derived COC. COCs from mice that underwent IVM for 6 or 20 h in the presence of epidermal growth factor, FSH or in combination had a > 10-fold reduction in mRNA (P < 0.05) for Adamts1 and Vcan when compared with in vivo matured COCs. Hyaluronan synthase 2 expression was up-regulated up to 8-fold (P < 0.05) over the unstimulated control, demonstrating successful induction of cumulus gene expression by the IVM conditions. While in vivo matured COCs showed abundant levels of these proteins, COCs that underwent IVM had neither detectable Adamts1, nor intact or Adamts1-cleaved Vcan. Human cumulus and granulosa cells matured in vivo contained abundant mRNA for Adamts1 and Vcan, demonstrating the potential relevance to human IVM. These results indicate that extensively altered COC matrix composition is present during IVM and may contribute to the observed poorer competence of the derived oocytes.

Induced Expression of Pattern Recognition Receptors in Cumulus Oocyte Complexes: Novel Evidence for Innate Immune-Like Functions during Ovulation

Ovulation is the complex, inflammatory-like process by which the cumulus oocyte complex (COC) is released from a mature, preovulatory follicle through a rupture site at the ovarian surface and requires expression of genes that generate and stabilize the expanded extracellular COC matrix. Gene profiling analyses of COCs at selected time intervals during ovulation revealed that many genes associated with immune related surveillance functions were also induced in cumulus cells. Specifically, cell surface signaling molecules known as pattern recognition receptors that act as sensors of the external environment important for the innate immune system to detect self from nonself or altered self are induced and/or expressed in cumulus cells as well as granulosa cells. These include the complement factor q1, CD14, and the Toll-like receptors (TLRs) 4, 8, and 9 as well as mediators of TLR activation, myeloid differentiation primary response gene 88 and interferon regulatory factor 3. COCs exposed to bacterial lipopolysaccharide exhibit enhanced phosphorylation of p38MAPK, ERK1/2 and nuclear factor-kappaB and increased expression of Il6 and Tnfa target genes, documenting that the TLR pathway is functional. Cumulus cells and granulosa cells also express the scavenger receptors CD36 and scavenger receptor type B1 and exhibited phagocytic uptake of fluorescently tagged bacterial particles. Collectively, these results provide novel evidence that cumulus cells as well as granulosa cells express innate immune related genes that may play critical roles in surveillance and cell survival during the ovulation process.

Significance of Mammalian Cumulus-Oocyte Complex Matrix in Oocyte Meiotic Maturation: Review of the Synthetic Control and Possible Roles of Hyaluronan (HA) and HA-binding Protein

In most mammals, the growth and development of the oocyte and its surrounding somatic cell compartment in the follicle occur in a highly coordinated and mutually dependent manner. Oocytes acquire developmental competence sequentially during follicle growth, finally gaining the ability to undergo complete meiotic and cytoplasmic maturation at the final stage of the preovulatory follicle. Fully-grown immature oocytes are tightly surrounded by compact layers of specialized granulosa cells called cumulus cells that form the cumulus-oocyte complex (COC). After a preovulatory surge of gonadotrophin, the cumulus cells organize a special muco-elastic extracellular matrix (ECM) that requires synthesis and deposition of a large amount of hyaluronan (HA) and HA-binding matrix glycoproteins. Many studies have reported that the formation of the COC matrix mass plays important roles in a variety of reproductive phenomenons: oocyte meiotic maturation with changes of junctional communication and cytoskeletal modification in COC, ovulation, fertilization and early embryo development. Recently, we identified the expressions of HA synthases and the HA receptor, CD44, in the porcine COC matrix. The interaction of HA and CD44 appears to be closely related to gap-junctional communications and meiotic resumption during oocyte maturation. This review describes the recent findings on the regulation and the presumptive mechanism of COC matrix molecules, and physiological features in COC expansion.

230. Altered matrix composition of cumulus oocyte complexes following in vitro maturation

The luteinizing hormone (LH) surge initiates cumulus expansion, through synthesis of hyaluronan and cross-linking proteins including versican, which stabilise the cumulus oocyte complex (COC) matrix. Versican is a substrate for the protease ADAMTS-1 and mRNA for each are localised to granulosa cells (GCs) and greatly induced following the LH surge. In humans, the use of in vitro maturation (IVM) of oocytes is an appealing option, reducing costs and risk of side effects associated with in vitro fertilisation. IVM oocytes are of poorer quality, likely resulting from altered gene expression and environmental conditions during oocyte maturation. Real-time PCR showed that IVM and immature COCs from Balb/c mice have 12 and 13 fold-reduced levels of ADAMTS-1 and versican expression respectively compared to in vivo matured COCs (PMSG+hCG 12h). Ovulated COCs (PMSG+hCG 15h) had similar low levels of ADAMTS-1 and versican. Samples isolated from F1 C57Bl/6xCBA mice showed similar reduced versican and ADAMTS-1 mRNA. Western blot analysis revealed that full length and cleaved versican, from ADAMTS-1/4 activity, was not detected in immature COCs, was present in in vivo matured COCs isolated from follicles, but strongest in ovulated COCs. IVM COCs had no detectable versican protein, supporting the mRNA data. Full-length versican was also present in GCs after PMSG+hCG 12h or 15h. ADAMTS-1 protein was most abundant in in vivo matured COCs with reduced levels seen in ovulated COCs, but was absent from IVM and immature COCs. These results indicate that ADAMTS-1 and versican are secreted products of granulosa cells that bind and incorporate into the COC matrix. The presence of versican and ADAMTS-1 is not essential for cumulus matrix expansion in vitro, but may contribute to oocyte maturation, ovulation of the COC and/or interaction with sperm during fertilisation.

Processing and Localization of ADAMTS-1 and Proteolytic Cleavage of Versican during Cumulus Matrix Expansion and Ovulation

ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs-1) is a member of the ADAMTS family of metalloproteases which, together with ADAMTS-4 and ADAMTS-5, has been shown to degrade members of the lectican family of proteoglycans. ADAMTS-1 mRNA is induced in granulosa cells of periovulatory follicles by the luteinizing hormone surge through a progesterone receptor-dependent mechanism. Female progesterone receptor knockout (PRKO) mice are infertile primarily due to ovulatory failure and lack the normal periovulatory induction of ADAMTS-1 mRNA. We therefore investigated the protein localization and function of ADAMTS-1 in ovulating ovaries. Antibodies against two specific peptide regions, the pro-domain and the metalloprotease domain of ADAMTS-1, were generated. Pro-ADAMTS-1 of 110 kDa was identified in mural granulosa cells and appears localized to cytoplasmic secretory vesicles. The mature (85-kDa pro-domain truncated) form accumulated in the extracellular matrix of the cumulus oocyte complex (COC) during the process of matrix expansion. Each form of ADAMTS-1 protein increased >10-fold after the ovulatory luteinizing hormone surge in wild-type but not PRKO mice. Versican is also localized selectively to the ovulating COC matrix and was found to be cleaved yielding a 70-kDa N-terminal fragment immunopositive for the neoepitope DPEAAE generated by ADAMTS-1 and ADAMTS-4 protease activity. This extracellular processing of versican was reduced in ADAMTS-1-deficient PRKO mouse ovaries. These observations suggest that one function of ADAMTS-1 in ovulation is to cleave versican in the expanded COC matrix and that the anovulatory phenotype of PRKO mice is at least partially due to loss of this function.

TSG-6 is concentrated in the extracellular matrix of mouse cumulus oocyte complexes through hyaluronan and inter-alpha-inhibitor binding.

During development of ovarian follicles in mammals, cumulus cells and the oocyte form a mucoelastic mass that detaches itself from peripheral granulosa cell layers upon an ovulatory surge. The integrity of this cumulus-oocyte complex (COC) relies on the cohesiveness of a hyaluronan (HA)-enriched extracellular matrix (ECM). We previously identified a serum glycoprotein, inter-alpha-inhibitor (IalphaI), that is critical in organizing and stabilizing this matrix. Following an ovulatory stimulus, IalphaI diffuses into the follicular fluid and becomes integrated in the ECM through its association with HA. TSG-6 (the secreted product of the tumor necrosis factor-stimulated gene 6), another HA binding protein, forms a complex with IalphaI in synovial fluid. The purpose of this study was to investigate whether TSG-6 is involved in the ECM organization of COCs. Immunolocalization of TSG-6 and IalphaI in mouse COCs at different ovulatory stages was analyzed by immunofluorescence and laser confocal microscopy. IalphaI, TSG-6, and HA colocolized in the cumulus ECM. Western blot analyses were consistent with the presence of both TSG-6 and TSG-6/IalphaI complexes in ovulated COCs. These results suggest that TSG-6 has a structural role in COC matrix formation possibly mediating cross-linking of separate HA molecules through its binding to IalphaI.