Mullerian duct regression and antiproliferative bioactivities of mullerian inhibiting substance reside in its carboxy-terminal domain.

Abstract

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.

Mutations of the Anti-Müllerian Hormone Gene in Patients with Persistent Müllerian Duct Syndrome: Biosynthesis, Secretion, and Processing of the Abnormal Proteins and Analysis Using a Three-Dimensional Model

“…The homology of AMH to other members of the TGF-␤ family is restricted to the C-terminal fragment, the bioactive domain of the molecule (6,7). Like other members of this superfamily, the C-terminal domain of AMH presumably interacts with a transmembrane serine/threonine kinase receptor complex, containing type I and type II components.…”

Müllerian Inhibiting Substance Inhibits Testosterone Synthesis in Adult Rats

“…The recombinant human MIS is quantified in a urogenital ridge bioassay and an ELISA specific for human and nonhuman primate MIS (Donahoe et al, 1977;Hudson et al, 1990). Recombinant human MIS has been shown in 2 different MIS bioassays in the rat and mouse (di Clemente et al, 1992;MacLaughlin et al, 1992) and in other in vitro systems to be bioactive across species (Vigier et al, 1989;Racine et al, 1998;Lee et al, 1999).…”

Direct diffusion of anti‐Müllerian hormone from both the cranial and caudal regions of the testis during early gonadal development in mice

“…16,17 Then, proAMH is cleaved by subtilisin/kexin proprotein convertases, yielding an N-terminal prodomain (AMH N ) and a C-terminal mature domain (AMH C ), the latter of which is capable of binding anti-Müllerian hormone receptor Type 2 (AMHR2). [18][19][20] AMHR2, a serine/threonine kinase, is expressed in the coelomic epithelial cells and subjacent mesenchymal cells lateral to the MD. [21][22][23] In male mice, AMHR2 expression begins around the most cranial part of the MD at 13.0 dpc and reaches the most caudal part by 13.5 dpc.…”

“…AMH is synthesized as a proprotein precursor (proAMH) in Sertoli cells, and in mice AMH mRNA expression starts around 12.0 dpc 16,17 . Then, proAMH is cleaved by subtilisin/kexin proprotein convertases, yielding an N‐terminal prodomain (AMH N ) and a C‐terminal mature domain (AMH C ), the latter of which is capable of binding anti‐Müllerian hormone receptor Type 2 (AMHR2) 18–20 . AMHR2, a serine/threonine kinase, is expressed in the coelomic epithelial cells and subjacent mesenchymal cells lateral to the MD 21–23 .…”