Identification of Subpopulations of Rat Granulosa Cells: Sedimentation Properties and Hormonal Responsiveness*

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Abstract
Ovarian granulosa cells from small follicles have generally been considered to comprise a homogeneous cell population; however, stratified arrangements of hormone receptors have been found in antral and mural granulosa cells of Graafian follicles. Using cultured granulosa cells derived from immature, hypophysectomized, estrogen-treated rats, it has been shown that vasoactive intestinal peptide (VIP) as well as FSH stimulates steroid production by these cells. Dose-response analysis indicated that the actions of these hormones were additive, suggesting the presence of subpopulations of granulosa cells. Using a continuous (0-30%) Metrizamide density gradient, 3 populations of granulosa cells with different sedimentation properties were identified. After centrifugation for 15 min at 1500 .times. g, cells sedimented at Metrizamide concentrations of 13%, 18% and 20% (peaks A, B and C, respectively). The subpopulation with lowest density (peak A) comprised 5% of the total cells, whereas the remaining cells were distributed approximately equally in the other 2 peaks. The profiles of estrogen and progesterone production by these cells in response to FSH and VIP indicated that FSH preferentially stimulated steroid production in cells with the highest density (peak C), whereas VIP mainly induced steroidogenic responses in cells of intermediate density (peak B). Cells with the lowest density (peak A) were unresponsive to either hormone. Treatment with forskolin, a universal adenylate cyclase activator, induced steroid production in both subpopulatios B and C. Further studies demonstrated that LH[luteinizing hormone]/human CG [chorionic gonadotropin] receptors were induced by FSH and forskolin in cells from peak C, whereas VIP treatment did not induce LH/human CG receptors in cells from peak B. In unfractionated cultured cells, GnRH [gonadotropin releasing hormone] potently antagonized FSH- but not VIP-induced steroidogenesis. Upon density-gradient fractionation, the profile of GnRH receptor content correlated well with GnRH effects since FSH-responsive cells (peak C) contained the majority of GnRH receptors. Granulosa cells from immature follicles are heterogeneous and consist of 2 major subpopulations of cells with differential responsiveness to FSH and VIP.