Rat Granulosa Cell Differentiation: An in Vitro Model*

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Abstract
An in vitro model of granulosa cell differentiation has been established. When FSH is added in vitro to granulosa cells from preantral follicles of diethylstilbestrol (DES)-primed rats, the LH/hCG receptor content increases from 0.725 pg hCG bound (50 cpm)/μg granulosa cell DNA to over 75 pg hCG bound (5000 cpm)/μg DNA. The animal model and the cell culture conditions are described. Briefly, in comparison with ovaries from DES-primed, hypophysectomized rats, ovaries from primed intact rats provided more granulosa cells per ovary. These cells from intact rats had less LH/hCG receptor at the initiation of culture and showed a greater LH/hCG receptor increase in vitro in response to FSH. Priming rats with a 3-cm DES capsule for 3 days was found to be optimal and superior to priming with a 1-cm capsule for 5 days in minimizing atresia in vivo and in maximizing LH/hCG receptor increase in vitro. LH/hCG receptor induction by FSH occurs in granulosa cells cultured in several types of plastic or glass tubes. Greater LH/hCG receptor increases were observed with Dulbecco's Modified Eagle's Medium mixed 1:1 with Ham's nutrient F–12 medium than with McCoy's 5a modified medium. The addition of insulin, transferrin, fibronectin, or hydrocortisone to the medium inhibited the induction of LH/hCG receptor by FSH. The maximally effective concentration of ovine FSH (15 × NIH-FSH-Sl) was 20 ng/ml. The LH/hCG receptor content was found to be greatest 72–80 h after the initiation of culture. Autoradiography revealed heterogeneity in LH/hCG receptor content among these cells, with single cells as well as clustered cells having acquired receptor. Available FSH receptor on cultured cells declined rapidly with FSH in the medium, and FSH receptor was only 30% of starting values by 72 h, with or without FSH. The receptors for LH/hCG induced in vitro are functional, as demonstrated by the ability of hCG to increase progesterone accumulation and ornithine decarboxylase activity. The development of granulosa cells in this in vitro model appears to closely resemble that seen in situ.