Synergistic Effect of Insulin and Follicle-Stimulating Hormone on Biochemical and Morphological Differentiation of Porcine Granulosa Cells in Vitro1

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Abstract
Insulin and follicle-stimulating hormone (FSH) have been shown to facilitate granulosa cell differentiation in vitro. To gain insight into this process, we evaluated the effects of these hormones, alone and in combination, upon the biochemical parameters of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor induction and progesterone secretion concomitantly with morphometric analysis of granulosa cell ultrastructure and LH/hCG receptor distribution by quantitative autoradiography under light microscopy. Granulosa cells isolated from small antral follicles (controls) cultured in the absence of exogenous hormones exhibited few microvilli and gap junctions; the mitochondria, endoplasmic reticulum, and Golgi complex were all poorly developed. Progesterone secretion was negligible and the cells bound little [125I]iodo-hCG. Insulin treatment increased gap junction formation, and the extent of smooth and rough endoplasmic reticulum and Golgi complex development (all p less than 0.05) but did not affect mitochondrial ultrastructure or volume. Insulin treatment modestly but significantly increased [125I]iodo-hCG binding and progesterone secretion relative to controls (p less than 0.001). FSH treatment had a similar effect to insulin on cell ultrastructure and additionally enhanced development of the mitochondria and smooth endoplasmic reticulum as well as formation of the microvilli (p less than 0.05). FSH significantly increased [125I]iodo-hCG binding and progesterone secretion relative to insulin-treated samples (p less than 0.001). Combined treatment with insulin and FSH markedly increased gap junction and microvilli formation and enhanced the development of the smooth endoplasmic reticulum and the Golgi complex relative to treatment with either hormone alone (p less than 0.05). Additionally, the combined treatment produced larger mitochondria with tubular christae. Consistent with the morphological development, the combined treatment of insulin and FSH significantly increased progesterone secretion and [125I]iodo-hCG binding (p less than 0.001). Autoradiographic analysis showed that aggregated cells in general exhibited higher LH/hCG receptor density than nonaggregated cells, and a significantly higher overall receptor density compared to nonaggregated cells or to cells treated either with insulin or FSH alone. Our results indicate that insulin and FSH facilitate morphological differentiation of the granulosa cell in a synergistic manner, stimulating gap junctions and microvilli formation and enhancing development of the mitochondria, endoplasmic reticulum, and Golgi complex.(ABSTRACT TRUNCATED AT 400 WORDS)