PARACRINE CONTROL OF STEROIDOGENESIS BY MIS

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Mullerian inhibiting substance (MIS) is a glycoprotein hormone in the TGFbeta family of growth and differentiation factors and is required for normal development of the male reproductive tract. MIS signals through a heteromeric complex of type I and type II single transmembrane serine/theronine kinase receptors and causes regression of the Mullerian duct, which if left unfettered would lead to development of female reproductive tract structures, the Fallopian tubes, uterus and upper vagina. Expression of MIS in the newly formed Sertoli cells of the developing testis begins early (approximately 7 weeks gestation) in humans and is a hallmark of the SRY-initiated events in the fetal gonad that lead to testis differentiation. MIS is also expressed, albeit at lower levels, at adult ovaries and testes, roles for which remain unclear. However, adult transgenic mice chronically over-expressing high levels of testosterone leading to incomplete virilization. The hypothesis that MIS physiologically regulates steroidogenesis will be tested. We have cloned the rat MIS type II receptor and found that it is expressed in Mullerian ducts, in Sertoli and granulosa cells of fetal and adult gonads, as well as in adult Leydig cells, which produce testosterone in the male testis. We have also shown that the receptor is expressed in the MIS-responsive R2C cells, a transformed rat Leydig cell line and in MA-10 cells, an MIS- responsive mouse Leydig cell tumor line. These cells can now be used to great advantage to investigate the molecular signals that might be regulating the level of sex steroids. We found that recombinant human MIS causes a dramatic decline in testosterone secretion by these cells into the media and a decrease in the steady-state levels of mRNA for three key enzymes in the testosterone biosynthetic pathway, P450scc, 3betaHSD and P450c17. Further investigation with Cyp17 promoter/luciferase reporter minigenes revealed that MIS caused a marked decrease in c17 mRNA at the transcriptional level. To test the hypothesis that MIS, which is reciprocally expressed with respect to testosterone after birth, may have a physiological role in suppressing steroidogenesis, we propose to (I) define the cis-actin element and trans-acting factors involved in transcriptional regulation by MIS of the Cyp17 gene. (II) Define the type II receptor in Leydig cells during development and determine whether administration of MIS can reduce testosterone production in the adult in vivo as a prelude to considering MIS a therapeutic where reducing testosterone might be beneficial such as isosexual precocity or in prostatic carcinoma.