Characterization of long-term monolayer cultures of human granulosa cells from follicles of different size and exposed in vivo to clomiphene citrate and hCG

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Abstract
To determine the effects of prolonged hCG [human chorionic gonadotropin] treatment in vitro upon granulosa cells from follicles of various sizes previously exposed to clomiphene citrate and hCG in vivo, progesterone and relaxin concentrations of spent media were correlated with light microscopic and ultrastrucutral characteristics. Intact, freshly dispersed cells were characterized by numerous lipid droplets, elliptical mitochondria with tubular or lamellar cristae, moderate rough-surfaced endoplasmic reticulum (RER), sparse smooth-surfaced endoplasmic reticulum (SER), and few Golgi. After 10-24 days in culture, 2 morphologically distinct cell types, granulosa-type and luteal-type, were noted at the light microscopic level. Ultrastructurally, lipid droplets decreased in number, mitochonidria became pleomorphic, RER became more prominent and dilated, and Golgi became more widely dispersed. Tubular SER became abundant and annular nexuses became more numerous after hCG treatment in vitro. Granulosa cells generated from all follicles responded to hCG treatment with significantly increased progesterone secretion after 4 days in culture. Relaxin was not detectable in any sample of medium. Human granulosa cells from 15- to 25-mm follicles evidently retain their differentiated function of progsterone secretion in long-term culture and recover responsiveness to hCG in vitro, as demonstrated by enhanced progesterone secretion and development of prominent SER and increased annular nexuses.