At the beginning of an ovulatory cycle, the murine oocyte is surrounded by tightly associated cumulus cells (the compact cumulus cell-oocyte complex, or COC). The cells are induced to synthesize and organize an extensive extracellular matrix with hyaluronan (HA) as the major component. The COC rapidly expands and is ovulated 10-12 hours later. HA synthesis decreases and stops during the next few hours. We have studied COC expansion in vivo (by isolating COCs at different time after inducing an ovulatory cycle) and in vitro (by culturing compact COCs and inducing matrix formation). We have used RT-PCR to identify mRNAs for: 1) a novel HA synthase (HAS2), 2) tumor necrosis factor stimulated gene-6 protein (TSG-6), and 3) versican, molecules that appear necessary for HA synthesis (HAS2), and for matrix formation (TSG-6 and versican). Preliminary data indicate the mRNAs for HAS2 and TSG-6 are absent in compact COCs, appear 1-2 hours prior to the onset of HA synthesis, and increase dramatically during the time when HA synthesis is sustained at maximal rates. Both TSG-6 and versican contain classical HA- binding motifs, and TSG-6 reacts specifically with inter-a-trypsin inhibitor (lal), the component in serum required for organizing HA in the COC matix. We have also shown that the cells synthesize a large chondroitin/dermatan sulfate proteoglycan and approximately 45 kDa protein that are incorporated into the matrix during COC expansion. We hypothesize that the large CS/DS-PG is versican and approximately 45kDa protein is TSG-6, and that both, with lal, are essential for organizing HA in the matrix. We have also shown that maximal HA synthesis in vitro requires exposure of cumulus cells to both follicle stimulating hormone (FSH) and a soluble factor produced by the oocytes; the FSH is required only during the first 2 hours (before the onset of HA synthesis) while the oocyte factor is required continuously. Epidermal growth factor can substitute for FSH and transforming growth factor (beta 1) partially (approximately 70%) for the oocyte factor. We hypothesize that these factors control HA synthesis primarily, if not exclusively, by regulating transcription of HAS2.
Our specific aims propose to: 1) quantitate copy numbers of mRNAs for HAS2, TSG-6 and versican relative to GAPDH in COCs isolated at times during expansion in vitro and in vivo, and in COCs in which HA synthesis is modulated by the presence or absence of the factors; 2) determine if the large CS/DS-PG is versican and approximately 45kDa protein is TSG-6, and to study their incorporation into the matrix; 3) use immunolocalization and in situ hybridization to identify TSG-6, HAS2 and versican, and their mRNAs in sections of ovaries isolated at different times during COC expansion; and 4) determine properties of HAS2 in cumulus cells (number of synthetic sites, association with the cell surface). The results should provide novel insight into fundamental mechanisms involved in regulating HA synthesis and organizing HA matrices in many biological processes such as tissue morphogenesis, wound healing and arterial restinosis.
| Mukhopadhyay, D; Hascall, V C; Day, A J et al. (2001) Two distinct populations of tumor necrosis factor-stimulated gene-6 protein in the extracellular matrix of expanded mouse cumulus cell-oocyte complexes. Arch Biochem Biophys 394:173-81 |
| Calabro, A; Hascall, V C; Midura, R J (2000) Adaptation of FACE methodology for microanalysis of total hyaluronan and chondroitin sulfate composition from cartilage. Glycobiology 10:283-93 |
| Calabro, A; Benavides, M; Tammi, M et al. (2000) Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE). Glycobiology 10:273-81 |
| Salustri, A; Camaioni, A; Di Giacomo, M et al. (1999) Hyaluronan and proteoglycans in ovarian follicles. Hum Reprod Update 5:293-301 |
