Mutations of the anti-mullerian hormone gene in patients with persistent mullerian duct syndrome: biosynthesis, secretion, and processing of the abnormal proteins and analysis using a three-dimensional model.

Anti-Müllerian hormone (AMH), a TGF-beta family member, determines whether an individual develops a uterus and Fallopian tubes. Mutations in the AMH gene lead to persistent Müllerian duct syndrome in males. The wild-type human AMH protein is synthesized as a disulfide-linked dimer of two identical 70-kDa polypeptides, which undergoes proteolytic processing to generate a 110-kDa N-terminal dimer and a bioactive 25-kDa TGF-beta-like C-terminal dimer. We have studied the biosynthesis and secretion of wild-type AMH and of seven persistent Müllerian duct syndrome proteins, containing mutations in either the N- or C-terminal domain. Mutant proteins lacking the C-terminal domain are secreted more rapidly than full-length AMH, whereas single amino acid changes in both domains can have profound effects on protein stability and folding. The addition of a cysteine in an N-terminal domain mutant, R194C, prevents proper folding, whereas the elimination of the cysteine involved in forming the interchain disulfide bond, in a C-terminal domain mutant, C525Y, leads to a truncation at the C terminus. A molecular model of the AMH C-terminal domain provides insights into how some mutations could affect biosynthesis and function.

[Inhibins, activins and anti-Müllerian hormone: structure, signalling pathways, roles and predictive value in reproductive medicine]. / Inhibines, activines et hormone anti-müllerienne : structure, signalisation, rôles et valeur prédictive en médecine de la reproduction.

Anti-Müllerian hormone (AMH), inhibins and activins are members of the transforming growth factor (TGFbeta) superfamily and are known to have a variety of actions concerning reproduction, hormonogenesis, development processes and differentiation. Inhibins and activins are dimeric glycoproteins that are defined by their actions on the pituitary gonadotroph cells. AMH, inhibins and activins have a vast array of actions usually exerted through paracrine and endocrine mechanisms. The recent availability of specific inhibin assays has demonstrated that inhibin B is the relevant circulating inhibin form in the human male. Inhibin B seems to be a useful marker of spermatogenesis, but serum and seminal inhibin B levels are not predictive parameters for the selection of azoospermic men as candidates for testicular sperm extraction (TESE). AMH in seminal plasma may be important for sperm production, and is a good marker for sertoli cell development. It might be the only one seminal marker of spermatogenesis in non-obstructive azoospermia. Nevertheless, many of these studies were carried out with small patient numbers, and consequently must be interpreted with caution. In women ongoing assisted reproductive therapy (ART), day 3 inhibin B and AMH levels predict the number of oocytes retrieved, but cannot predict likelihood of pregnancy. Further studies are needed to determine if AMH and inhibin predict ART outcomes better than classical parameters (age, FSH levels and follicular ultrasonography). AMH and inhibin are also specific markers of Sertoli- and granulosa-cell origin in gonadal tumors.

Identification of a rabbit epididymal protein gene.

A protein designated as BE-20 was purified from cauda epididymal fluid of the rabbit by preparative polyacrylamide gel electrophoresis and HPLC on a mono Q HR5/5 anion exchange column. The purified protein migrated with an estimated Mt of 20,000 when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequence of the N-terminus of the BE-20 protein was determined. The initial eight amino acid residues were His-Gly-Ala-Asp-Lys-Pro-Gly-Val. The corresponding 23 mer oligonucleotide (5'-CATGGCGCTGACAAGCCTGGGGT-3') was synthesized and used as sense primer with rabbit epididymal mRNA as template in the RT-PCR system. The purified BE-20 cDNA consisted of 499 bp with an open reading frame of 285 bp encoding a deduced polypeptide composed of 95 amino acids. The BE-20 cDNA had 78.5% identity in 479 bp overlap with human epididymis-specific HE4 cDNA. The amino acid sequences of the initial 30 amino acid residues of the N-terminus of the purified protein and the deduced polypeptides were as follows: N-His-Gly-Ala-Asp-Lys-Pro-Gly-Val-Cys-Pro-Gln-Leu-Ser-Ala-Asp-Leu-Asn-Cy s- Thr-Gln-Asp-Cys-Arg-Ala-Asp-Gln-Asp-Cys-Ala-Glu. The deduced polypeptide contained 16 cysteine residues and had partial sequence homology with proteins belonging to the four-disulfide core family of extracellular proteinase inhibitors. The BE-20 protein may play a role in sperm maturation and/or capacitation.

Changes in luminal fluid protein composition in the rat cauda epididymidis following partial sympathetic denervation.

Sympathetic denervation of the rat cauda epididymidis by surgical removal of the inferior mesenteric ganglion (IMG) results in an excessive accumulation of sperm in the cauda epididymidis as well as significant changes in cauda sperm motility and cauda epididymal gross histology. The objective of the present study was to determine if the cauda-specific changes in sperm storage, sperm motility, and epididymal histology following the loss of sympathetic innervation were accompanied by changes in the protein composition of epididymal fluid. One and 4 weeks after surgical IMG removal or sham operations, luminal fluid obtained from the caput and cauda epididymidis and cauda epididymal sperm-associated proteins were subjected to two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and silver-stained proteins were quantitated. One week after IMG removal, two cauda epididymal fluid (CEF) proteins (2 and 13) had increased 43% and 49%, respectively, whereas four CEF proteins (5, 8, 9, and 19) had decreased between 30% and 73% compared to controls. Four weeks after IMG removal, changes in CEF proteins observed 1 week following surgery were no longer present, but the staining intensities of three additional CEF proteins (11, 12, and 18) were reduced an average of 70% compared to control CEF proteins. By obstructing the cauda epididymidis, we confirmed that the changes in CEF protein composition observed following IMG removal were not the result of sperm accumulation but were due directly to the loss of innervation; the staining intensity of CEF protein 2 increased as a result of excessive sperm accumulation in the cauda epididymidis both in the presence and absence of innervation from the IMG. No significant changes in caput epididymal fluid proteins or cauda epididymal sperm-associated proteins were detected following IMG removal. These data show that the protein composition of rat CEF is significantly affected by the loss of sympathetic innervation and suggest that neuronal input may play an important role in the maintenance of epididymal function.

The thermosome of Thermoplasma acidophilum and its relationship to the eukaryotic chaperonin TRiC.

A high molecular-mass protein complex from the archaebacterium Thermoplasma acidophilum, referred to here as the 'thermosome', is built from two subunits (M(r) 58 and 60). The thermosome has been purified to homogeneity. The molecular mass of the native complex was determined to be 1061 +/- 30 Da by scanning transmission electron microscopy. It shows a weak ATPase activity and is able to bind denatured polypeptides. Averages obtained from electron micrographs of negatively stained molecules in the end-on and side-on orientations, respectively, were compared with those of the t-complex polypeptide 1 ring complex (TRiC), isolated from bovine testes. Both molecules consist of two stacked pseudo eightfold symmetric rings which build up a cylindrical particle with a large cavity in the center. Sequence alignments of peptides generated from both subunits of the thermosome and different subunits of TRiC reveal a high partial similarity to each other and to the archaebacterial chaperonin thermophilic factor 55 from Sulfolobus shibatae as well as to eukaryotic TCP1 proteins. These striking structural similarities confirm the proposition that all these molecules belong to a single protein family which is structurally and functionally related to the GroEL class of molecular chaperones.

Mullerian inhibiting substance: a gonadal hormone with multiple functions.

Mullerian inhibiting substance (MIS) is the gonadal hormone that causes regression of the Mullerian ducts, the anlagen of the female internal reproductive structures, during male embryogenesis. MIS is a member of the large transforming growth factor-beta (TGF beta) multigene family of glycoproteins that are involved in the regulation of growth and differentiation. The proteins in this gene family are all produced as dimeric precursors and undergo posttranslational processing for activation, requiring cleavage and dissociation to release bioactive C-terminal fragments. Similarly, the 140 kilodalton (kDa) disulfide-linked homodimer of MIS is proteolytically cleaved to generate its active C-terminal fragments. The sexually dimorphic expression of MIS in Sertoli cells of the testis and granulosa cells of the ovary is critical for normal differentiation of the internal reproductive tract structures. A number of extra-Mullerian functions such as control of germ cell maturation and gonadal morphogenesis, induction of the abdominal phase of testicular descent, suppression of lung maturation, and growth inhibition of transformed cells have also been proposed for this growth-inhibitory hormone and will be discussed. This article will summarize the current understanding of the biology and multiple functions of MIS including its activation, regulation, and mechanism of action and discuss areas of interest in ongoing research.

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

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.