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Biology of Reproduction, Vol 25, 421-431, Copyright © 1981 by Society for the Study of Reproduction
1 Department of Obstetrics and Gynecology and Department of Physiology,
Duke University Medical Center,
Durham, North Carolina 27710 Insulin is a requisite for the FSH-mediated induction of LH/hCG receptors in porcine granulosa
cell monolayers maintained in serum-containing medium (May et al, 1980). In this report we
describe further 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. 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, however, 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 innoculum. 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. However, 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
(MSA), compounds possessing insulin-like structure and activity, respectively, were incapable of
replacing insulin as a requisite for FSH-mediated LH/hCG receptor induction or gonadotropinstimulated progesterone secretion. Our results indicate that insulin 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.
Note:
ACKNOWLEDGMENTS
The authors wish to acknowledge Dr. O. David
Sherwood of the University of Illinois for his material
support and guidance in the studies dealing with relaxin.
The authors also wish to acknowledge the invaluable technical assistance of Anne Brown, Sherry Bass,
and Rebecca Addison. We also wish to thank Donna
Sanders for her secretarial assistance.
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