Mü llerian inhibiting substance (MIS), MIS,1 a member of the TGF-␤ family of hormones, causes regression of the epithelial-mesenchymal unit of the Mü llerian duct in the embryonic male urogenital ridge. In females the Mü llerian duct autonomously differentiates into the uterus, Fallopian tubes, and upper vagina (1). The Mü llerian ducts of both male and female embryos are responsive to MIS only during a critical period in development after which they lose sensitivity (1, 2). However, MIS is produced at high levels by Sertoli cells of the testis even after the regression of the Mü llerian duct, decreases at adolescence, and is produced throughout adult life. In females, it is synthesized by postnatal granulosa cells of the ovary and can be detected in the female serum until menopause (3, 4). Cleavage of MIS by a kex-like enzyme is required to manifest MIS activity since a noncleavable mutant is devoid of biological function (5).The MIS type II receptor gene, a highly conserved single transmembrane serine threonine kinase that is homologous to members of the TGF-␤ family of type II receptors, encodes a 2.0-kilobase mRNA (6 -8). It is expressed at high levels in the Mü llerian duct, Sertoli cells, and granulosa cells of the embryonic and adult gonads and in the uterus (8). The developmental significance of the MIS type II receptor was demonstrated in MIS type II receptor null male mice, which develop normally but have a persistent Mü llerian duct that forms a uterus and oviducts; this phenotype resembles that of MIS ligand-deficient mice (9 -11). Female MIS type II receptor or MIS ligand-deficient mice are normal and fertile as young adults.The detection of MIS in the serum of males and females even after the regression and differentiation of the Mü llerian duct (3, 4) suggests that MIS might be a multifunctional hormone. MIS is a negative regulatory factor in fetal rat lung maturation, where it inhibits the production of pulmonary surfactant both in vitro and in vivo (12, 13). MIS also inhibits oocyte meiosis in vitro (14,15). The presence of MIS type II receptor in non-Mü llerian tissues such as the ovary, lung, and Leydig cells of the testis (16 -18) and the occurrence of Leydig cell hyperplasia and Leydig cell tumors in receptor null male mice (11) also suggest that the biological action of MIS is not limited to the Mü llerian duct. Furthermore, MIS has been shown to inhibit the growth of tumor cells in vitro and in vivo (19 -22). Masiakos et al. (17), using ovarian epithelial cells derived from the ascites of ovarian cancer patients, correlated MIS type II receptor expression with MIS-mediated growth inhibition. Furthermore, exogenous MIS has been shown to inhibit metastases of the ocular melanoma cell line OM431 (23) and to block the growth of the vulvar epidermoid carcinoma cell line A431 and the OM431 cell line in vivo (21,24).Expression of MIS-related proteins including TGF-␤, activin, inhibin, and BMP ligands and their receptors has been demonstrated in human breast cancer cell lines and in breast cancer ...

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A 25-kilodalton dimeric carboxy-terminal fragment of the recombinant human Mullerian inhibiting substance protein (rhMIS) was produced by proteolytic cleavage with plasmin and purified by size-exclusion chromatography. The identity of the isolated dimer as the carboxy-terminal fragment was confirmed by gel electrophoresis and Western analysis. As was true of every sample of the holo molecule, all preparations of the carboxy-terminal domain of rhMIS (n = 10), when added in the 0.5-5.0 micrograms/ml range, exhibited a dose-dependent partial to complete regression of the 14.5-day fetal rat Mullerian duct in an organ culture assay. The carboxy-terminal dimer also inhibited, in a dose-dependent manner, the growth of A431 cells in monolayer cultures. Daily addition of 5, 10, or 20 micrograms carboxy-terminus for 3 days resulted in 0%, 25%, and 100% inhibition of cell proliferation, respectively. Similar and higher doses of holo rhMIS had no or inconsistent antiproliferative activity (0-34% inhibition), even though the preparations caused Mullerian duct regression. All amino-terminal fragments prepared using this separation protocol were found to be inactive in these assays. These findings suggest that the bioactivity of rhMIS as a regressor of fetal Mullerian ducts and an inhibitor of A431 cell growth resides in its carboxy-terminal domain. These results indicate that the urogenital ridge tissue, but not A431 cells in culture, may be capable of cleaving intact MIS to a biologically active conformation.