New approaches for high-yield purification of Müllerian inhibiting substance improve its bioactivity.

We have established a new method to purify Müllerian inhibiting substance (MIS) with higher purity and recovery over existing procedures. Recombinant human MIS was expressed in Chinese hamster ovary cells and secreted into chemically defined serum-free media. The secreted products were concentrated by either precipitation with ammonium sulfate or lectin-affinity chromatography, each of which was followed by anion-exchange chromatography. Further separation of the active carboxy-terminal domain of MIS was achieved after cleavage by plasmin followed by lectin-affinity chromatography. This method may be applicable to other members of the transforming growth factor beta family with which MIS shares sequence homology.

Effect of single and compound knockouts of estrogen receptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes.

The functions of estrogen receptors (ERs) in mouse ovary and genital tracts were investigated by generating null mutants for ERalpha (ERalphaKO), ERbeta (ERbetaKO) and both ERs (ERalphabetaKO). All ERalphaKO females are sterile, whereas ERbetaKO females are either infertile or exhibit variable degrees of subfertility. Mast cells present in adult ERalphaKO and ERalphabetaKO ovaries could participate in the generation of hemorrhagic cysts. Folliculogenesis proceeds normally up to the large antral stage in both ERalphaKO and ERbetaKO adults, whereas large antral follicles of ERalpha+/-ERbetaKO and ERalphabetaKO adults are markedly deficient in granulosa cells. Similarly, prematurely developed follicles found in prepubertal ERalphaKO ovaries appear normal, but their ERalphabetaKO counterparts display only few granulosa cell layers. Upon superovulation treatment, all prepubertal ERalphaKO females form numerous preovulatory follicles of which the vast majority do not ovulate. The same treatment fails to elicit the formation of preovulatory follicles in half of the ERbetaKO mice and in all ERalpha+/-/ERbetaKO mice. These and other results reveal a functional redundancy between ERalpha and ERbeta for ovarian folliculogenesis, and strongly suggest that (1) ERbeta plays an important role in mediating the stimulatory effects of estrogens on granulosa cell proliferation, (2) ERalpha is not required for follicle growth under wild type conditions, while it is indispensable for ovulation, and (3) ERalpha is also necessary for interstitial glandular cell development. Our data also indicate that ERbeta exerts some function in ERalphaKO uterus and vagina. ERalphabetaKO granulosa cells localized within degenerating follicles transform into cells displaying junctions that are unique to testicular Sertoli cells. From the distribution pattern of anti-Müllerian hormone (AMH) in ERalphabetaKO ovaries, it is unlikely that an elevated AMH level is the cause of Sertoli cell differentiation. Our results also show that cell proliferation in the prostate and urinary bladder of old ERbetaKO and ERalphabetaKO males is apparently normal.

The novel epididymal secretory protein ESP13.2 in Macaca fascicularis.

Newly synthesized mammalian spermatozoa undergo critical modifications as they pass along the epididymis. The modifications endow spermatozoa with fertilizing ability and occur largely as a consequence of epididymal gene expression. With this in mind, we here employed a cDNA cloning strategy designed to identify key epididymal gene products. We describe a novel cynomolgus monkey (Macaca fascicularis) epididymal transcript designated cy-ESP13.2, of 690 nucleotides. The putative human ortholog was cloned and is highly conserved. Both cDNA sequences predict small, secretory proteins with a disulfide-stabilized core. Anti-peptide polyclonal antibodies were raised to a predicted cy-ESP13.2 surface loop. Western blotting with these antibodies revealed high-level, epididymis-specific expression of cy-ESP13.2, consistent with the pattern of cy-ESP13.2 mRNA expression assessed by Northern blotting. cy-ESP13.2 protein was of 30 kDa and was readily detectable in epithelial cells lining the efferent ductules, initial segment, and cauda regions of the epididymis, but not on spermatozoa. Similarities to members of the four-disulfide-core family suggest clues to ESP13.2 function.

The presence of Müllerian inhibiting substance binding sites in human sperm.

PURPOSE: The binding of Müllerian inhibiting substance (MIS) to human sperm was investigated using immunohistological techniques. MATERIALS AND METHODS: Sperm from 5 normal donors and 6 subfertile men were studied. Whole or thin-sectioned sperm were incubated without or with recombinant human MIS (0.5 microg./ml.). MIS binding was identified under light microscopy (LM) using rabbit anti-human MIS antibodies tagged with goat IgG-horseradish peroxidase and diaminobenzidine as substrate, or by scanning and transmission electron microscopy (SEM and TEM) using gold labeled goat IgG. Intracellular MIS binding in sperm sections was examined by TEM. Antibodies were omitted in the controls. RESULTS: Under LM, DAB staining was present on sperm incubated with or without MIS and absent on controls. Using SEM, gold particles were found primarily on the surfaces of the sperm head with less binding to the tail. With TEM, the clustering of gold particles around the head of sperm represents MIS binding, but very few or no gold particles could be found associated with the sperm tail. MIS binding was also found associated with intracellular structures, but only within the head of the sperm. Overall, less gold particle binding was present in subfertile compared with normal sperm. CONCLUSIONS: These results suggest that MIS is bound to the sperm surface and sperm from normally fertile men have increased MIS binding. The function of MIS in sperm is unknown, but the presence of MIS binding suggests a direct role(s) in sperm function.

The 26 kD protein recognized on rat cauda epididymal sperm by monoclonal antibody 4E9 has internal peptide sequence that is identical to the secreted form of epididymal protein E.

MAb 4E9, raised against a detergent extract of rat cauda epididymal sperm, recognizes a 26 kD glycoprotein that is found on the plasma membrane of the sperm tail in cauda, but not caput, sperm (Moore et al., 1994). It also recognizes an epididymis-secreted protein that has been shown to be protein E (Xu and Hamilton, 1996). It is felt that the secreted protein becomes associated with sperm, but there has been no biochemical evidence of molecular identity between the secreted and membrane proteins. In this report, the membrane form of the antigen has been purified by reverse phase HPLC. Cyanogen bromide cleavage of the purified protein yielded 3 peptides that were purified, also by reverse phase HPLC. One of the peptides yielded an unambiguous sequence of 34 amino acids that is identical to an internal peptide of the protein found in epididymal fluid. This is the first report showing sequence identity between an epididymis-secreted protein and a protein of the sperm plasma membrane.

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.

Müllerian inhibiting substance as a model for the transforming growth factor-beta family: development of new treatment strategies.

The study of ligand receptor interactions and receptor function often requires multifaceted experimental approaches. In the course of studying the function and mechanism of action of müllerian inhibiting substance (MIS), we have used a wide range of molecular and cellular techniques. These have led to the identification, cloning, and characterization of the MIS receptors and of other receptors for the transforming growth factor-beta (TGF beta) family. This article describes the use of polymerase chain reaction (PCR) cloning to isolate candidate receptor genes, transfection and flow cytometry to study ligand binding, nonhomologous recombination targeted gene disruption (knockout) to analyze receptor function, and yeast genetics to identify other proteins that interact with the receptor complex. Together these techniques have led to the development of therapeutics and therapeutic strategies that are ready for clinical application.

Identification of the rat epididymis-secreted 4E9 antigen as protein E: further biochemical characterization of the highly homologous epididymal secretory proteins D and E.

Epididymis-secreted proteins D and E have been purified to homogeneity and partially characterized, and it is shown that monoclonal antibody (MAb) 4E9 (raised against a detergent extract of rat caudal epididymal sperm [Moore et al., 1994: Mol Reprod Dev 37(2):181-194]) recognizes protein E, but not protein D. The molecular weight of protein D (approximately 30 kD) is approximately 2 kD lower than protein E (approximately 32 kD). The NH2-terminus of each protein is blocked; however, microsequencing of internal peptides confirms earlier reports of significant sequence identity between the two proteins. High performance liquid chromatography tryptic peptide mapping showed peak differences between the two proteins, but it was not possible to obtain amino acid sequence in the peaks that were different. The epitope for MAb 4E9 was localized in the blocked NH2-terminus-CNBr peptide derived from protein E. The epitope was destroyed by protease treatment of protein E. Removal of N-linked oligosaccharides did not destroy the epitope for MAb 4E9 and did not affect the molecular weight difference between the proteins.

Different cells and cell lines produce factors that modulate Sertoli cell function.

Peritubular myoid cells derived from immature rat testes produce factors that modulate Sertoli cell function (P-Mod-S). The secretion of these factors is controlled in part by androgens. Cultured prostatic stromal cells strongly resemble peritubular myoid cells and produce mediators with similar activity. Here we investigated whether myoid cell lines can be used as a source of P-Mod-S-like factors. Rat kidney fibroblast (NRK) and mouse fibroblast (3T3) cell lines were used as non-myoid controls. Surprisingly, serum-free media conditioned by all cell lines studied modulated Sertoli cell function in a similar fashion as media conditioned by peritubular cells (PTCM) or stromal cells (STCM). Using Sertoli cell transferrin secretion as an endpoint for P-Mod-S-like activity, the nature of the active principles involved was further explored. The observed activity could not be explained by residual contamination with fetal calf serum. Moreover, the effects of the conditioned media could not be mimicked by classical growth factors (IGF-I, bFGF, EGF, TGF-beta, NGF, PDGF-BB) added singly or in combination with submaximally effective concentrations of PTCM. Finally, the possibility that conditioned media might indirectly enhance Sertoli cell function by promoting the production or deposition of extracellular matrix elements was made unlikely by the demonstration that the observed effects were not mimicked by Matrigel and were unaffected when Sertoli cells were seeded on Matrigel. Superdex 75 chromatography after analytical reversed-phase chromatography indicates that the factors from different origin have a similar size (45-50 kDa). It is concluded that mediators with P-Mod-S-like activity are produced by various cells and cell lines both with and without smooth muscle cell characteristics. Whether the active principles involved are really identical requires further investigation.

Müllerian inhibiting substance: new perspectives and future directions.

MIS, as a differentiate and antiproliferative agent, is precisely regulated, for example, at the transcriptional level by such transacting factors as SRY, and posttranslationally by testosterone. Processing of MIS most likely requires an as yet unknown in vivo protease which probably serves to control cleavage of MIS and hence its activation at specific sites wherein a localized program of cell death is initiated via a receptor mediated event. Progress has been made in understanding the molecular domains of MIS; current efforts are focused on characterizing the wild type MIS receptor as well as cloning and expressing the MIS receptor. We need now to understand how to target and efficiently activate MIS at its projected site of action. We must focus, after structural analysis of its receptor, on elucidating the MIS initiated intracellular signals which result in localized cell inhibition. Understanding of these mechanisms will permit design of antitumor agents and therapeutic strategies. Similarly, understanding regulation of MIS expression may lead to therapeutic induction of expression in those states where depressed expression is associated with tumorigenesis, sexual ambiguity, or infertility.

Isolation and characterization of a 54-kilodalton precursor of caltrin, the calcium transport inhibitor protein from seminal vesicles of the rat.

A basic 54-kDa protein (pI approximately 8.8) that cross-reacts with anti-caltrin antisera has been detected and isolated by gel filtration and cation exchange chromatography from seminal vesicle content of the rat. The soluble protein spontaneously precipitated in NaHCO3-buffered solution at pH 7.8, but it was kept soluble in imidazole buffer containing EDTA and dithiothreitol at pH 7.0. In addition to the main band of 54 kDa, two faint immunoreactive fractions with molecular weights around 45,000 and 14,000 were also revealed by Western blotting. The presence of the rat caltrin sequence within the primary structure of the 54-kDa molecule has been investigated by sequencing the peptides generated by trypsin digestion. The sequence of the first 46 amino acid residues of rat caltrin has been found in one of the fragments produced by enzymatic cleavage. However, the exact location of the caltrin sequence in the whole 54-kDa protein has not been determined. The purified 54-kDa protein did not inhibit Ca2+ uptake by epididymal spermatozoa. Results indicated that this molecule represents an inactive precursor of caltrin and is enzymatically processed in the lumen of the seminal vesicle to the small and active calcium transport inhibitor protein. The immunoreactive proteins with intermediate molecular weights (45,000 and 14,000) could represent partially degraded products of the precursor. The lack of inhibitory activity of the precursor may be related to the molecule's having a size and conformation that would make it unable to interact with caltrin receptors on the sperm surface.

Human müllerian inhibiting substance: enhanced purification imparts biochemical stability and restores antiproliferative effects.

Separation of copurifying protease activity from recombinant human Müllerian inhibiting substance (rhMIS) bound to a monoclonal antibody immunoaffinity column by a high-salt wash results in cleaner preparations of rhMIS resistant to cleavage upon storage. In addition, an inhibitor of rhMIS antiproliferative activity is removed. Proteolytic cleavages produced by either a copurifying protease or exogenous plasmin occur at residues 229 and 427 but do not abolish rhMIS biological activity. This report details the modified immunoaffinity column isolation protocol suitable for proteins such as rhMIS and describes the biochemical and antiproliferative properties of this protein.

Sex-specific fetal lung development and müllerian inhibiting substance.

Male neonates develop respiratory distress syndrome (RDS) with a greater incidence and mortality than do female neonates; the cause of this male disadvantage remains obscure. Male fetuses are exposed to higher levels of androgens and Müllerian inhibiting substance (MIS). Androgens have been shown to inhibit fetal lung maturation, and recent evidence in vitro indicates that MIS, a Sertoli cell-derived glycoprotein made early in ontogeny of the testis, may also inhibit lung development. To study whether this fetal regressor might inhibit maturation of the fetal lung in vivo, we injected human recombinant MIS (rMIS) into fetal rats, measured serum levels of rMIS using an enzyme-linked immunosorbent assay, and analyzed fetal lung tissue histologically and for protein, glycogen, DNA, and disaturated phosphatidylcholine content. Peak serum levels of recombinant MIS were measured at 6 h, with an apparent elimination half-life of 3 h, and without leakage into adjacent littermates injected with vehicle alone. Female fetal rat lung tissue exposed to recombinant MIS (10(-9) M, 10(-8) M) revealed depressed disaturated phosphatidylcholine content both 48 and 72 h after injection compared with female vehicle-injected littermates. Male lungs of the same gestational age appeared inhibited at a higher (10(-8) M) rMIS dose. These inhibitory effects observed in vivo confirm those previously seen in vitro and suggest that MIS, as well as androgens, may play a causative or important ancillary role in the sexual dimorphism that characterizes the neonatal respiratory distress syndrome.

Human recombinant mullerian inhibiting substance inhibition of rat oocyte meiosis is reversed by epidermal growth factor in vitro.

Mullerian inhibiting substance (MIS), a glycoprotein responsible for the regression of Mullerian duct in the male mammalian embryo, was recently localized not only in the fetal and newborn testis, but also in the older ovary throughout reproductive life. Bovine MIS purified from newborn testicular tissue inhibited spontaneous oocyte meiosis in vitro in the rat. These studies show that partially purified recombinant MIS produced from a human MIS genomic construct caused inhibition of oocyte meiosis, but when purified to homogeneity, the effect was lost. Addition of low concentrations of the detergent Nonidet P-40, used to maintain stability in the purified bovine preparations, however, restored the MIS inhibitory effect to the human preparation, which could, in turn, be blocked by a polyclonal antibody raised to human recombinant MIS; Nonidet P-40 itself caused no inhibition. Since we have shown that epidermal growth factor (EGF) and MIS are antagonistic in a number of other systems, we tested the effect of EGF on the ability of MIS to inhibit oocyte meiosis. EGF added to the medium at a dose that caused no effect on oocytes (25 ng/ml) blocked the MIS inhibitory action on spontaneous rat oocyte meiosis, while EGF had no effect on a known oocyte meiosis inhibitor, 3-isobutyl-1-methylxanthine. These data indicate that human recombinant MIS can inhibit oocyte meiosis in the ovary and that its regulatory effect can be modulated by EGF, which appears to be an antagonist of MIS.

Immunopurified anti-müllerian hormone does not inhibit spontaneous resumption of meiosis in vitro of rat oocytes.

The ability of immunopurified, biologically potent anti-Müllerian hormone (AMH) to inhibit the spontaneous resumption of meiosis of rat oocytes was tested in vitro. Two different batches of AMH in the range of 0.75-9.0 micrograms/protein did not suppress the spontaneous resumption of meiosis. Neither did AMH (6 micrograms/ml) induce meiotic resumption of follicle-enclosed oocytes in culture. It is concluded, therefore, that AMH has no oocyte meiosis-inhibiting activity.

Purification of a paracrine factor, P-Mod-S, produced by testicular peritubular cells that modulates Sertoli cell function.

A testicular paracrine factor, P-Mod-S, was purified from conditioned medium obtained from serum-free cultures of peritubular cells. Stimulation of testicular transferrin production by cultured Sertoli cells was utilized as a bio-assay for P-Mod-S. A bioactive protein with an apparent molecular weight of 50,000 under physiological conditions was isolated by high pressure size exclusion chromatography. P-Mod-S was found to have an affinity for heparin and bound to a heparin affinity column. Two forms of P-Mod-S were purified with reverse-phase chromatography. The less hydrophobic form was referred to as P-Mod-S (A) and is a 56,000 molecular weight protein. The more hydrophobic form was referred to as P-Mod-S (B) and is a 59,000 molecular weight protein. Purification of P-Mod-S (A) and P-Mod-S (B) from peritubular cell-radiolabeled secreted proteins revealed that both proteins contain radioactivity. This result demonstrates active synthesis and secretion of P-Mod-S by peritubular cells. Although the amino acid composition of the two proteins indicates distinct differences in the content of several amino acids, the relationship of P-Mod-S (A) and P-Mod-S (B) is unknown at present. A greater than 1000-fold increase in the specific activity of P-Mod-S was achieved with the purification procedure utilized. P-Mod-S can account for essentially all the bioactivity present in crude peritubular cell-secreted protein preparations. The effects of the two forms of P-Mod-S on both transferrin and androgen-binding protein production by Sertoli cells was examined. Purified forms of P-Mod-S were found to have a greater effect on Sertoli cell function than any individual regulatory agent previously known to influence the cell, including follicle-stimulating hormone. The significance of peritubular cell-Sertoli cell interactions mediated via P-Mod-S to spermatogenesis and testicular function is discussed, as well as insight provided into general mesenchymal-epithelial cell interactions.

Isolation, characterization and amino-acid sequence of gamma-seminoprotein, a glycoprotein from human seminal plasma.

gamma-Seminoprotein (gamma-SM), a glycoprotein from human seminal plasma, was isolated in highly purified form by ion-exchange chromatography on a Mono Q column. The main form, fraction M, was homogeneous by PAGE at pH 8.3 and by SDS-PAGE. The complete amino acid sequence of gamma-SM was determined with the aid of fragments generated by cleavages with cyanogen bromide, clostripain, chymotrypsin and Staphylococcus aureus V8 protease. The fragments were aligned with overlapping sequences. The single polypeptide chain of gamma-SM contains 237 amino acids with a calculated Mr of 26079. A single N-linked carbohydrate side chain is attached to Asn45. The complex structure of this oligosaccharide has been determined recently [van Halbeek H. et al. (1985) Biochem. Biophys. Res. Commun. 131, 507-514]. Sequence comparison with serine proteases shows a high degree of homology, especially with the kallikrein family. The residues in the vicinity of the active site of serine proteases are also highly conserved in gamma-SM, indicating the participation of His41, Asp96 and Ser189 in its active site. gamma-SM hydrolyzed M-casein with a pH optimum at 8.0, but failed to hydrolyze any of the synthetic substrates tested. This proteolytic activity could be inhibited with diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, Zn2+ or Hg2+ ions.