RAT SERTOLI-CELL AROMATASE CYTOCHROME-P450 - REGULATION BY CELL-CULTURE CONDITIONS AND RELATIONSHIP TO THE STATE OF CELL-DIFFERENTIATION

Primary cultures of immature rat Sertoli cells in plastic dishes are h ighly responsive to follicle stimulating hormone (FSH) and its second messenger, cAMP, in metabolizing testosterone to estradiol, thus indic ating the presence of an active, hormone-regulated aromatase cytochrom e P450 (P450arom). However, in vivo studies indicated that P450arom is FSH-responsive only in very young animals, where the cells have not y et 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 coat ed with Matrigel, assume structural and functional characteristics mor e similar to that of in vivo differentiated Sertoli cells. When the ce lls were cultured on laminin or Matrigel, the FSH- and cAMP-induced es tradiol production was greatly reduced by 30 and 60%, respectively. Wh en Sertoli cells were cultured in bicameral chambers coated with Matri gel, no induction of testosterone aromatization by FSH or cAMP was obs erved. 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 ass umption by Sertoli cells of a phenotype closer that of the differentia ted cells in vivo (tall columnar and highly polarized) suppress the in duction of P450arom by FSH and cAMP. We then examined the mechanism(s) by which cell phenotype affects p450arom activity. Northern blot anal yses of Sertoli cell RNA revealed one major band of 1.9 Kb and two min or bands of 3.3 and 5.2 Kb. However, there were no changes at the leve l of the expression of P450arom messenger RNA under the different cult ure conditions. No differences were found in P450arom enzymatic activi ty measured by the (H2O)-H-3 release method in microsomes prepared fro m Sertoli cells cultured under the various conditions. Similarly, no d ifferences were observed in the amount of protein detected by immunobl ot analysis of Sertoli cell extracts using an antiserum raised against the human placental enzyme. Recombination experiments using microsome s from cells cultured on plastic or in the chambers and cytosol from c ontrol or FSH-treated cells cultured on plastic also proved inadequate in inducing P450arom activity. These data suggest that: a) P450arom a ctivity could be used as a specific marker for Sertoli cell differenti ation, and b) the differentiation process in Sertoli cells is associat ed with specific changes in the microenvironment or the regulation of P450arom, or both, that rendered the enzyme insensitive to FSH or cAMP induction.