Rat Sertoli Cell Aromatase Cytochrome P450: Regulation by Cell Culture Conditions and Relationship to the State of Cell Differentiation

• Published in: In vitro cellular & developmental biology. Animal 1993-12, Vol.29A (12), p.943-949
• Main Authors: Papadopoulos, Vassilios, Meng-Chu Jia, Culty, Martine, Hall, Peter F., Dym, Martin
• Format: Article
• Language: English
• Subjects: Androgens; Animals; Aromatase - metabolism; Bucladesine - pharmacology; Cell culture techniques; Cell Differentiation - drug effects; Cellular differentiation; Collagen; Cultured cells; DNA - analysis; Drug Combinations; Enzymes; Estradiol - metabolism; Estrogens; Follicle Stimulating Hormone - pharmacology; Growth, Differentiation, and Senescence; Laminin; Leydig cells; Male; Microsomes; Plastics; Proteoglycans; Rats; Rats, Sprague-Dawley; RNA, Messenger - analysis; Sertoli cells; Sertoli Cells - cytology; Sertoli Cells - drug effects; Sertoli Cells - enzymology; Testosterone; Testosterone - metabolism
• ISSN: 1071-2690; 1543-706X
• DOI: 10.1007/BF02634233

Primary cultures of immature rat Sertoli cells in plastic dishes are highly responsive to follicle stimulating hormone (FSH) and its second messenger, cAMP, in metabolizing testosterone to estradiol, thus indicating the presence of an active, hormone-regulated aromatase cytochrome P450 (P450arom). However, in vivo studies indicated that P450arom is FSH-responsive only in very young animals, where the cells have not yet differentiated, but they lose this ability later on in development. Sertoli cells grown on Matrigel (a reconstituted basement membrane), laminin (a basement membrane component), or in bicameral chambers coated with Matrigel, assume structural and functional characteristics more similar to that of in vivo differentiated Sertoli cells. When the cells were cultured on laminin or Matrigel, the FSH- and cAMP-induced estradiol production was greatly reduced by 30 and 60%, respectively. When Sertoli cells were cultured in bicameral chambers coated with Matrigel, no induction of testosterone aromatization by FSH or cAMP was observed. However, FSH-induced cAMP formation was greater when the cells were cultured on basement membrane or in the chambers than on plastic dishes. These results suggest that culture conditions favoring the assumption by Sertoli cells of a phenotype closer that of the differentiated cells in vivo (tall columnar and highly polarized) suppress the induction of P450arom by FSH and cAMP. We then examined the mechanism(s) by which cell phenotype affects p450arom activity. Northern blot analyses of Sertoli cell RNA revealed one major band of 1.9 Kb and two minor bands of 3.3 and 5.2 Kb. However, there were no changes at the level of the expression of P450arom messenger RNA under the different culture conditions. No differences were found in P450arom enzymatic activity measured by the3H 2Orelease method in microsomes prepared from Sertoli cells cultured under the various conditions. Similarly, no differences were observed in the amount of protein detected by immunoblot analysis of Sertoli cell extracts using an antiserum raised against the human placental enzyme. Recombination experiments using microsomes from cells cultured on plastic or in the chambers and cytosol from control or FSH-treated cells cultured on plastic also proved inadequate in inducing P450arom activity. These data suggest that: a) P450arom activity could be used as a specific marker for Sertoli cell differentiation, and b) the differentiation process in Sertoli cel