Granulosa Cell Differentiation in vitro: Effect of Insulin on Growth and Functional Integrity1

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Abstract
Insulin is a requisite for the FSH-mediated induction of LH/hCG [luteinizing hormone/human chorionic gonadotropin] receptors in porcine granulosa cell monolayers maintained in serum-containing medium. The role of insulin on this process and on other parameters of cell monolayer performance, e.g., plating efficiency, cell growth, basal and gonadotropin-stimulated progesterone production and aromatase activity is further described. Insulin did not affect the plating efficiency (> 90%) of freshly harvested, viable granulosa cells. During the first 2 days of culture, monolayer cell content increased slightly under insulin-free conditions. Insulin did not alter this slow rate of cell division. After the first 2 days cell and protein content of monolayers deprived of insulin decreased significantly (60%, P < 0.001), relative to insulin-treated monolayers in which cell and protein content were either maintained or increased relative to the initial inoculum. Basal progesterone secretion declined with time in culture but was maintained or slightly increased under the influence of insulin. Both FSH- and hCG-stimulated progesterone production were significantly enhanced (P < 0.001) by insulin after the first 2 days of culture. FSH-stimulated progesterone secretion was dose-dependent with respect to insulin as was FSH-mediated LH/hCG receptor induction. The immature granulosa cells used in these studies assumed a fibroblastic morphology in the absence of added hormones. Insulin induced an epithelioid morphology in vitro, characteristic of more mature cells. LH and FSH alone were incapable of altering the fibroblastic morphology and did not affect the change brought about by insulin. Thus, gross morphological maturation of granulosa cells in vitro correlated with insulin-mediated biochemical differentiation, e.g., LH/hCG receptor induction and steroidogenic capacity. Although insulin enhanced several markers of cell differentiation, it did not ameliorate the loss of aromatase activity during culture, a characteristic of this model system.During the first 2 days of culture and in the presence of testosterone, granulosa cell monolayers secreted significant amounts of estrogen. This capability declined rapidly with time in culture. Neither insulin alone nor insulin combined with FSH acutely stimulated aromatase activity, nor did these treatments protect against the loss of aromatase activity. Highly purified porcine relaxin and commercially available multiplication-stimulating activity, compounds possessing insulin-like structure and activity, respectively, were incapable of replacing insulin as a requisite for FSH-mediated LH/hCG receptor induction or gonadotropin-stimulated progesterone secretion. Insulin apparently is critical for the maintenance of several functional properties of porcine granulosa cell monolayers, and thus should be considered for routine use in studies of the regulation of ovarian function in vitro.

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