PARACRINE CONTROL OF STEROIDOGENESIS BY MIS

缪勒管抑制物质（MIS）是tgfβ生长分化因子家族中的一种糖蛋白激素，是男性生殖道正常发育所必需的。MIS通过I型和II型单跨膜丝氨酸/theronine激酶受体的异质复合体发出信号，并引起苗勒管的退化，如果不加以约束，将导致女性生殖道结构、输卵管、子宫和上阴道的发育。在人类发育中的睾丸中新形成的支持细胞中，MIS的表达开始得很早（大约在妊娠7周），并且是sry引发的胎儿性腺事件导致睾丸分化的标志。成人卵巢和睾丸也表达MIS，尽管表达水平较低，但其作用尚不清楚。然而，成年转基因小鼠长期过度表达高水平睾酮导致不完全男性化。MIS生理调控类固醇生成的假说将被验证。我们克隆了大鼠MIS II型受体，发现它在苗勒管中表达，在胎儿和成年性腺的支持细胞和颗粒细胞中表达，以及在成年雄性睾丸中产生睾酮的间质细胞中表达。我们还发现该受体在MIS应答的R2C细胞（一种转化的大鼠间质细胞系）和MA-10细胞（一种MIS应答的小鼠间质细胞肿瘤系）中表达。这些细胞现在可以用于研究可能调节性类固醇水平的分子信号。我们发现重组人MIS导致这些细胞向培养基中分泌睾酮的量急剧下降，睾酮生物合成途径中三个关键酶P450scc、3betaHSD和P450c17 mRNA的稳态水平下降。对Cyp17启动子/荧光素酶报告基因的进一步研究表明，MIS在转录水平上导致c17 mRNA的显著降低。为了验证出生后与睾酮相互表达的MIS可能在抑制类固醇生成中具有生理作用的假设，我们提出(1)定义参与Cyp17基因MIS转录调控的顺式肌动蛋白元件和反式作用因子。（II）在发育过程中确定间质细胞中的II型受体，并确定MIS的使用是否可以减少成人体内睾酮的产生，作为考虑MIS治疗的前驱，其中减少睾酮可能是有益的，如同性性早熟或前列腺癌。

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.