PARACRINE CONTROL OF STEROIDOGENESIS BY MIS

苗勒氏抑制物质是生长和分化因子家族中的一种糖蛋白激素，对男性生殖道的正常发育是必需的。MI通过I型和II型单一跨膜丝氨酸/丝氨酸激酶受体的异构体复合体传递信号，导致苗勒管退化，如果任其发展，将导致女性生殖道结构、输卵管、子宫和上阴道的发育。在人类早期(约7周妊娠)发育中的睾丸中新形成的支持细胞表达的MIS是SRY启动的胎儿性腺中导致睾丸分化的事件的标志。MI在成人卵巢和睾丸中也有表达，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的睾酮会导致不完全的男性化。该假说，即管理信息系统的生理调节类固醇的生成将得到检验。我们已经克隆了大鼠MISII型受体，发现它表达于苗勒管、胎儿和成人性腺的支持细胞和颗粒细胞，以及在男性睾丸产生睾酮的成人间质细胞。我们还发现该受体在MIS型反应的R2C细胞和MA-10细胞中表达，R2C细胞是转化的大鼠间质细胞系，MA-10细胞是MIS型反应的小鼠间质细胞瘤系。这些细胞现在可以用来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导致这些细胞分泌的睾酮急剧下降，并导致睾酮生物合成途径中三个关键酶P450scc、3betaHSD和P450c17的mRNA稳态水平下降。通过对细胞色素P17启动子/荧光素酶报告基因的进一步研究发现，在转录水平上，MISI导致C17基因的转录水平显著降低。为了验证这样一种假设，即在出生后与睾酮相互表达的MI可能在抑制类固醇生成方面具有生理作用，我们建议：(I)定义参与CyP17基因转录调控的顺式肌动蛋白元件和反式作用因子。(Ii)确定间质细胞在发育过程中的II型受体，并确定在体内给药是否可以减少成人体内睾酮的产生，以此作为认为降低睾酮可能有益的治疗方法的前奏，例如同性早熟或前列腺癌。

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.