1,721,017 research outputs found
Hyaluronic acid synthesis by mural granulosa cells and cumulus cells in vitro is selectively stimulated by a factor produced by oocytes and by transforming growth factor-beta
No full textIn ovarian antral follicles cumulus cells (approximately 1,000/follicle) closely surround the oocyte, and mural granulosa cells (approximately 50,000/follicle) are distributed at the periphery. Previous work (Salustri, A., Yanagishita, M., and Hascall, V. C. (1990) Dev. Biol. 138, 26-32) showed that oocytes produce a factor(s) which stimulates hyaluronic acid (HA) synthesis by cumulus cells during expansion of the cumulus cell-oocyte complex. We now show that mural granulosa cells also respond in vitro to the oocyte factor(s) with greatly increased HA synthesis. As with cumulus cells, a factor(s) present in fetal calf serum is required to retain newly synthesized HA in the extracellular matrix. Unlike cumulus cells, follicle-stimulating hormone (FSH) is not required for maximal stimulation, in part because mural granulosa cells synthesize prostaglandin E2 which can substitute for FSH in promoting cumulus cell-oocyte complex expansion. Of several growth factors studied, only transforming growth factor-beta 1 (TGF-beta 1) stimulated HA synthesis in both cell types. However, the stimulation of HA synthesis by TGF-beta 1 was additive with that for the oocyte factor(s), and neutralizing antibodies to TGF-beta did not inhibit the response to the oocyte factor(s). The results indicate that the oocyte factor(s) and TGF-beta 1 are not the same and that they operate through different receptors in stimulating HA synthesis. Epidermal growth factor was able to replace FSH in amplifying the response of cumulus cells to the oocyte factor(s) and in stimulating synthesis of dermatan sulfate proteoglycans
Synthesis and accumulation of hyaluronic acid and proteoglycans in the mouse cumulus cell-oocyte complex during follicle-stimulating hormone-induced mucification
No full textIn most mammalian ovaries, the cumulus cell-oocyte complex (COC) expands at the time of ovulation by depositing an extensive extracellular matrix between the cumulus cells. This phenomenon can be reproduced in vitro by culturing COCs with follicle-stimulating hormone (FSH) and serum. Biosynthesis of hyaluronic acid (HA) and proteoglycans by mouse COCs in vitro was studied using [3H]glucosamine and [35S]sulfate as metabolic precursors. Radiolabeled complex carbohydrates were analyzed by ion exchange chromatography, specific enzyme digestion followed by high performance liquid chromatography, and gel filtration. The specific activities of [3H]hexosamines in the labeled molecules were determined by measuring the incorporation of 3H and 35S into chondroitin 4-sulfate disaccharides. When COCs were stimulated with FSH, HA biosynthesis increased 20-30-fold between 3-12 h later when expansion occurs, reaching a maximum rate of approximately 780 pmol (as glucosamine)/COC/h compared with the unstimulated rate of approximately 26 pmol/COC/h. The final concentration of HA in the expanded COC was calculated to be approximately 250 micrograms/ml. The effects of dibutyryl cyclic AMP (Bt2cAMP) on COC expansion and HA synthesis were similar to those of FSH, suggesting that the effects of FSH are mediated by cAMP. However, FSH significantly decreased the specific activity of the incorporated hexosamines while Bt2cAMP did not. Serum is necessary for the accumulation of HA in the COC matrix. HA synthesis in FSH-stimulated COCs was as high or higher in the absence of serum, but most was recovered in the medium and not in the COC matrix. The molecular size of the HA was greater than 2 million dalton in either case, suggesting that the serum did not alter physical properties of HA. Stimulation of proteoglycan biosynthesis by either FSH or Bt2cAMP was less pronounced (three to four times control) than for HA and was sustained throughout an 18-h culture period. A reduction of 80% in the deposition of newly synthesized PGs in the COC matrix by 0.5 mM beta-xyloside treatment did not affect the expansion of the cumulus
Mouse oocytes regulate hyaluronic acid synthesis and mucification by FSH-stimulated cumulus cells
No full textMucification (or expansion) of the cumulus cells surrounding the oocyte is thought to depend on the direct action of gonadotropins in stimulating production and deposition of hyaluronic acid (HA) in the extracellular matrix. We now report that the oocyte is essential for this process. Either follicle-stimulating hormone (FSH) at 1 micrograms/ml or dibutyryl cAMP at 2 mM induces mucification of intact cumulus cell-oocyte complexes (COCs) in vitro, but fails to stimulate mucification of isolated cumulus cells. HA synthesis by FSH-stimulated cumulus cells is only approximately 3.5% of the value achieved by FSH-stimulated COCs. Isolated oocytes cultured with or without FSH do not synthesize detectable amounts of HA but induce isolated cumulus cells to increase HA synthesis approximately 13-fold in cocultures with FSH. Medium conditioned by isolated oocytes for 5 hr induces nearly the same level of HA synthesis by cumulus cells under the same culture conditions. FSH also stimulates cumulus cells to increase synthesis of dermatan sulfate proteoglycans (DS-PGs) approximately 3-fold, but this stimulation does not depend upon the presence of oocytes. The results indicate that oocytes produce a soluble factor(s) essential in combination with FSH to stimulate HA, but not DS-PG, synthesis by cumulus cells in vitro and that this factor(s) acts independently or downstream from the FSH-induced formation of cAMP
Coding sequence of a hyaluronan synthase homologue expressed during expansion of the mouse cumulus-oocyte complex
No full textMaturation of mammalian cumulus cell-oocyte complex in the preovulatory follicle involves the deposition of a hyaluronan-rich extracellular matrix by the cumulus cells. In this study, we report the complete coding sequence of a novel mouse hyaluronan synthase homologue expressed in cumulus cell-oocyte complexes induced to expand in vivo. The time frame of mRNA expression of this molecule correlates well with previous biochemical and immunohistochemical findings on the initiation of hyaluronan synthesis in maturing preovulatory follicles. Evolutionary comparison of the vertebrate hyaluronan synthase homologues indicates that there are at least two genes that code for these proteins
High-resolution separation of disaccharide and oligosaccharide alditols from chondroitin sulphate, dermatan sulphate and hyaluronan using CarboPac PA1 chromatography
No full textRecent literature indicates that specific glycosaminoglycan structures are involved in various biological processes, such as anticoagulation, growth factor activation and viral infection. The initial step in the structural analysis of glycosaminoglycans is a definitive compositional analysis of its characteristic disaccharide repeat structures. Current chromatographic or electrophoretic procedures may have limitations in analysing glycosaminoglycan samples that are in low abundance, contain novel structures that need to be further characterized, or are metabolically labelled from radioactive precursors as a result of biosynthetic experiments. This study presents a new methodology for analysing disaccharides and oligosaccharides derived from chondroitin sulphate, dermatan sulphate and hyaluronan that fulfils the above criteria. The procedure involves the separation of reduced forms of these glycoconjugates on a CarboPac PA1 column using alkaline eluants. This study adopted a strategy which uses specific enzymes to release these disaccharides from their glycosaminoglycan forms. A borohydride reduction reaction was modified to be compatible with the buffer conditions commonly used with these enzymes in order to quantitatively reduce the disaccharides to their alditol forms (thereby stabilizing them to alkaline pH). Chromatography conditions were established which separated all known disaccharide alditol structures from chondroitin sulphate, dermatan sulphate and hyaluronan with extremely high resolution in a single run. Integrated pulsed amperometry was compared to UV absorbance measurement at 232 nm as two sensitive methods for detecting these reduced disaccharides; most of them could be routinely detected in the range of 50-500 ng. Data are presented applying this method to quantify hyaluronan in a biological sample which contains approximately 5000 cells and only approximately 10 ng of hyaluronan. Additional data are presented to demonstrate that this procedure will also separate oligosaccharide alditols derived from hyaluronan
Localization and synthesis of hyaluronic acid in the cumulus cells and mural granulosa cells of the preovulatory follicle
No full textMural and cumulus granulosa cells synthesize hyaluronic acid (HA) and expand in vitro in response to follicle-stimulating hormone and a soluble factor(s) produced by fully grown oocytes. In the present study we examined HA synthesis and extracellular matrix organization by the two cell populations in vivo during the preovulatory period. After injection of human chorionic gonadotropin into pregnant mares' serum gonadotropin-primed animals, a progressive increase in HA synthesis was observed by the cumulus cell-oocyte complex (COC), and by the mural granulosa cells adjacent to the antrum (antral granulosa cells). The outermost layers of mural granulosa cells (peripheral granulosa cells) did not synthesize HA. Net HA synthesis was approximately 4 pg/cell for COCs isolated after full expansion induced either in vivo or in vitro, whereas the total HA content and cell number in the ovulated COC (approximately 11 ng HA and approximately 3000 cells per COC) were about threefold higher than for COCs expanded in vitro (approximately 4 ng HA and approximately 1000 cells per COC). The increased cell content of ovulated COCs appears to be primarily the result of inclusion of proximal mural granulosa cells which synthesize HA in response to the oocyte factor(s) and become incorporated in the expanded COC extracellular matrix mass. Media conditioned by oocytes enclosed in the cumulus cell mass (intact COCs) contained only 10-20% of the HA-stimulatory activity of media conditioned by an equal number of isolated oocytes when tested on mural granulosa cell cultures. Further, HA-stimulatory activity of media conditioned by isolated oocytes was dramatically reduced (approximately 70%) by preincubation for 5 hr with cumulus cells compared to preincubation in the absence of cells. The results suggest that differences in HA synthesis between subregions of membrana granulosa depend on a diffusion gradient of the oocyte factor(s)
Hyaluronan production by epidermal keratinocytes: Up-regulation during differentiation of rat keratinocytes in an artifical epidermis at the air-liquid interface
No full textTwo distinct populations of tumor necrosis factor-stimulated gene-6 protein in the extracellular matrix of expanded mouse cumulus cell-oocyte complexes
No full textAfter 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
Hyaluronan and proteoglycans in ovarian follicles
No full textProteoglycans are macromolecules formed by a protein backbone to which one or more glycosaminoglycan side chains are co-valently attached. They can be secreted by the cells, retained at the cell surface, or stored in intracellular vacuoles. Hyaluronan is an extremely long glycosaminoglycan which, at variance with other glycosaminoglycans, is released into the extracellular matrix as a free polysaccharide not co-valently linked to a core protein. Both proteoglycans and hyaluronan influence many aspects of cell behaviour by multiple interactions with other molecules. They are involved in matrix formation, cell-cell and cell-matrix adhesion, cell proliferation and migration, and show co-receptor activity for growth factors. Both proteoglycan and hyaluranon synthesis change significantly during ovarian follicle development and atresia. This review describes the structure of these molecules and their possible function in ovarian physiology
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