Abstract
In 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.
Highlights
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 previously showed that a soluble factor(s) produced by isolated oocytes is essential in combination with follicle-stimulating hormone (FSH) for HA synthesis during expansion of cumulus cell-oocyte complexes basal
(+oocyte-conditioned media (OCM)) (1 oocyte/lrl cultured for 9 h) or TGF-01 (+TGF-fi) (0.3 rig/ml)
Summary
Materials-The following were obtained from the indicated sources: female Swiss CD-1 mice from Charles River; Eagle’s minimum essentialmedium (with Earle’ssalt), glutamine, HEPES, and FCS from Life Technologies/GIBCO; bovine serum albumin (BSA). 5.~1 aliquots of the cell suspension were added to 15-kl droplets of culture medium containing 5% FCS, isotopes, and other substances as described in the protocols. Oocyte-conditioned medium was obtained by culturing isolated oocytes (2 oocyte/pl unless specified in the text) in culture medium plus 5% FCS for 9 h (unless specified in the text) After this time period, the oocytes were removed and the conditioned medium was distributed in 10.~1 droplets under dimethyl polysiloxane. In the experiments in which the absence of serum was studied, FCS was replaced with 3 mg/ml BSA Other test substances such as FSH, PGEI, growth factors, and antibodies were added at the concentrations specified in the text. At least two different experiments were done for each protocol
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