Enhanced purification of Mullerian inhibiting substance by lectin affinity chromatography

Mullerian inhibiting substance (MIS), a secreted testicular product responsible for regression of the Mullerian ducts in the male mammalian embryo, was purified 7000 fold, exploiting the glycoprotein nature of this important fetal regressor to achieve enhanced purification. The present procedure employs media incubation of newborn calf testis, passage through DEAE Bio-Gel A and CM Bio-Gel A and sequential lectin affinity chromatography on wheat germ lectin (WGL)-Sepharose 6MB and concanavalin A (Con A)-Sepharose 4B. Strongly bioactive MIS was released from both lectin columns in the bound glycoprotein fraction only after elution with lectin-specific sugar. Carbohydrate analysis of the highly purified glycoprotein fraction eluted from Con A indicated the presence of both N-acetyl glucosamine and mannose, as would be expected from its sequential lectin affinity, as well as of galactose, galactosamine and N-acetyl neuraminic acid. Electrophoresis of this fraction on polyacrylamide-SDS gels showed an identical band pattern after staining with either Coomassie blue or periodic acid-Schiff reagent, further indicating that MIS is a glycoprotein.     

Involution of the female Mullerian duct of the fetal rat in the organ-culture assay for the detection of Mullerian Inhibiting Substance

The study of Mullerian Inhibiting Substance (MIS) has been made possible because of the organ-culture bioassay devised by Picon ('69) for detecting MIS in vitro. We have studied the degeneration of the female Mullerian duct of the rat fetus, the target tissue of the assay, with electron microscopy. We have observed that the involution of the female Mullerian duct in the organ-culture assay follows a pattern of degeneration similar to the normal involution of the male Mullerian duct under the influence of MIS from the fetal testis (Price et al., '77). This involution involves alterations in the duct epithelium subsequent to a response of the mesenchyme surrounding the duct. The degeneration of a specific organ system under the direct influence of a specific factor, Mullerian Inhibiting Substance, represents an example of "programmed cell death."     

The role of nucleotide pyrophosphatase in Mullerian duct regression

Mullerian inhibiting substance (MIS), a glycoprotein from the fetal testis causing regression of the embryonic Mullerian duct, can be inhibited in vitro in the presence of Mn²⁺ by a wide range of nucleotides including GTP, NAD, ATP, AMP, and several nonhydrolyzable synthetic ATP analogs. Extracellular nucleotide pyrophosphatase (NPPase), an enzyme able to hydrolyze the wide variety of the nucleotides and analogs found to inhibit Mullerian duct regression, was studied by histochemical staining (H. Sierakowska and D. Shugar (1963) to determine if NPPase localized in or around the Mullerian duct during regression. Frozen sections of urogenital ridges from to rat fetuses (n = 77) were incubated with a-naphthyl thymidine-5′-phosphate (naphthyl TMP) and Fast Red TR. Nucleotide pyrophosphatase hydrolyzes naphthyl TMP, releasing naphthol, which then reacts with Fast Red to produce color at the enzyme site. Nucleotide hydrolysis was detected around regressing male (n = 16) Mullerian duct cells at days of gestation, but no hydrolysis was detected around female (n = 17) Mullerian duct cells at any stage. Controls (n = 24) incubated without substrate did not stain. Addition of exogenous ATP (n = 20) to the histochemical incubation medium inhibited nucleotide hydrolysis on male Mullerian ducts, suggesting that this staining is specific for pyrophosphatase activity. Results in vivo were confirmed in vitro by incubating day female rat urogenital ridges with MIS for 72 hr prior to histochemical staining. The addition of testosterone to MIS was obligatory to detect staining in vitro (n = 10). The localized NPPase activity around the regressing Mullerian duct suggests that NPPase may appear as a consequence of duct regression and may act to control the degree of membrane phosphorylation by degrading excess trinucleotides.     

Müllerian inhibiting substance activity in bovine fetal, newborn and prepubertal testes

Mullerian Inhibiting Substance (MIS) activity in bovine fetal, newborn, and prepubertal testes was investigated. MIS was present in all fetuses studied during the 1st 6 weeks after birth. Activity was present but diminished at 8 weeks. Loss of activity appeared to coincide with the time of weaning. Electron microscopic studies of the neonate testes revealed highly active Sertoli cells with a morphology suggestive of a protein secreting cell, and implicate the Sertoli cell as the source of MIS. It is concluded that the neonate calf testes may be an excellent source of material for the isolation and biochemical characterization of MIS.     

The critical period for mullerian duct regression in the dog embryo.

The embryonic period during which Mullerian duct regression and Mullerian Inhibiting Substance (MIS) secretion occur was determined in canine embryos removed from timed pregnancies (32, 36, 37, 39, 42, and 46 days gestation). Sex chromosomes of each embryo were identified in metaphase spreads prepared from fibroblast cultures. Testicular differentiation, defined by seminiferous tubule formation and the presence of Sertoli cells and Leydig cells, and the degree of Mullerian duct regression were determined by careful morphologic analysis of histologic sections of canine embryonic gonads (n = 20) and Mullerian ducts (n = 20). MIS was detected immunohistochemically in embryonic testes using avidin-biotin complex enhancement of a specific rabbit polyclonal anti-MIS antibody. Testicular differentiation was observed at 36 days gestation. The earliest evidence of Mullerian duct regression in male embryos was observed at 36 days gestation, and regression was completed by 46 days gestation. Positive staining for MIS was present in testes from 36 to 46 days (n = 9). Staining was absent in the undifferentiated testis (n = 1) at 32 days gestation and in ovaries at all ages tested (n = 10). Thus, MIS is normally present throughout the critical period for Mullerian duct regression in the embryonic male dog.     

Proteolytic processing of mullerian inhibiting substance produces a transforming growth factor-beta-like fragment

Mullerian inhibiting substance (MIS) is a differentiation factor that causes the Mullerian duct to regress during the development of the male reproductive tract. The active form is a disulfide-linked dimer consisting of two identical 70-kDa subunits. Recently, the amino acid sequence for MIS was deduced from its gene sequence and revealed that the carboxyl-terminal region shares homology with transforming growth factor (TGF)-beta. Since TGF-beta is produced as a large latent precursor that requires proteolytic activation for activity, we sought to determine if MIS might undergo a similar processing event. Here we demonstrate that typically 5 to 20% of the protein in MIS preparations is cleaved at a site 109 amino acids from the carboxyl terminus. Concurrent cleavages from both chains of the MIS dimer produces a 25-kDa TGF-beta-like fragment and a high molecular mass complex derived from the amino terminus of the protein. Although the two fragments are noncovalently linked, they remain tightly associated after cleavage, and thus are structurally organized like TGF-beta within its precursor. The same cleavage products also can be generated by limited proteolysis with plasmin, which provides a simple method for converting the entire preparation into the cleaved form. The plasmin-digested MIS is fully active in the organ culture assay.     

The ontogeny of mullerian inhibiting substance in granulosa cells of the bovine ovarian follicle

Mullerian Inhibiting Substance (MIS) previously detected in the Sertoli cells of the calf testis has been localized in the granulosa cells of the ovarian Graafian follicle by using an immunoperoxidase technique and a monoclonal antibody (IG8) to MIS that almost completely blocks its biological activity. The immunoperoxidase technique (avidin-biotin complex method) demonstrated specific localization of MIS in the cytoplasm of the ovarian granulosa cells in the bovine Graafian follicles over a wide age span, i.e. one day, one week, three months, two-and-a-half years and five years. The presence of MIS in the ovary implies a function that is as yet unknown.     

Immunochromatography on insolubilized antibodies of very low affinity: application to immunoadsorbence of bovine alpha-fetoprotein.

Cross-reactive antibodies were utilized to prepare immunoadsorbents possessing a very low affinity to bovine alpha-fetoprotein (AFP). A goat anti-human AFP serum cross-reactive with bovine AFP was first depleted of antibodies reactive with bovine AFP in immunodiffusion. The remaining antibodies from this serum and gamma-globulin from a sheep antiserum against rabbit AFP, without prior absorption, were coupled to Sepharose. Chromatography of fetal calf serum on these adsorbents resulted in retardation of bovine AFP relative to other proteins. A major part of the AFP eluted from the columns with phosphate-buffered saline. The rest eluted as a sharp peak with a small quantity of 4 or 6 M urea. The elution of AFP with the initial column buffer has made it possible to prepare pure AFP that has not been subjected to the chaotropic elution buffers usually employed in affinity chromatography. Elimination of the washing step and the ease of elution has allowed purification of gram amounts of AFP. The fact that immunoadsorbents prepared from antibodies with no detectable reactivity in immunodiffusion still caused delayed elution in chromatography suggests that this procedure may be useful in search of proteins cross-reactive with a known protein.     

The preservation of Mullerian inhibiting substance during long-term freezing of testicular fragments

Mullerian Inhibiting Substance, a fetal testicular hormone found in most mammalian species, causes regression in the male of the Mullerian duct, the anlagen of the fallopian tube, uterus, and upper vagina. Limitations to study of this substance in the past have been posed by its short period of production and by its localized and specific action. We have been able to store testicular fragments that continue to demonstrate detectable Mullerian Inhibiting Substance activity for up to 5 months by using techniques of slow freezing which approximate 1 °C/min, cryoprotective additives, storage in liquid nitrogen, and rapid thawing. These fragments then can be pooled for biochemical and endocrinological studies. In addition, unknown fragments can be transported long distances for assay of Mullerian Inhibiting Substance.     

Immunocytochemical localization of Mullerian inhibiting substance in the rough endoplasmic reticulum and Golgi apparatus in Sertoli cells of the neonatal calf testis using a monoclonal antibody

Mullerian Inhibiting Substance (MIS) has been localized in the Sertoli cells of the neonatal calf testis using preembedding immunoperoxidase techniques and a monoclonal antibody which almost completely blocks the biological activity of MIS. Both the peroxidase-labeled antibody method using a peroxidase-conjugated F(ab')2 fragment of IgG as a second antibody and the unlabeled antibody peroxidase-antiperoxidase (PAP) method using Fab fragments of the PAP complex were employed. With both methods, MIS was demonstrated within the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus. In the Golgi, MIS was concentrated in the transmost cisternae especially at their peripheral expansions. This study indicates that MIS is synthesized in the RER and transported to the Golgi apparatus, presumably for glycosidation, before secretion from Golgi derived vacuoles.     

Lectin bindings and diethylstilbestrol effects on the recognition of mullerian inhibiting substance (MIS) on chick mullerian ducts by MIS-antiserum

A high intensity of lectin bindings was demonstrated on the epithelial cells and serosa cells of the regressing right Mullerian ducts (Mds) in the female chick embryos. The strong lectin bindings occurs on, or in the regressing Md cells along with marked surface MIS bindings at the age of day 13. However, at the age of days 5-7 1/2, bindings of lectins were weak. Neither Wheat-germ agglutinin (WGA) or Concanavalin A (Con-A) labelings before MIS-antiserum (MIS-Ab) incubation can block antibody recognitions to the antigens, including MIS and growth hormone at the age of day 13. Our previous studies indicated that after WGA labeling on the surfaces of Md epithelial cells prior to the incubation of MIS-Ab at day 10 did not prevent the recognition of MIS-Ab (Wang 1989). On the contrary, at day 7 1/2, the specific binding of MIS was eliminated after preincubations with lectins and prenatal diethylstilbestrol (DES) treatment at the age of day 5. It is suggested that DES provides a protection to the Mds from MIS-induced regression by preventing the MIS binding to its specific membrane receptors. An increase of extra- and intracellular glycoproteins or carbohydrates of regressing Md epithelial cells were suggested. Internalization of WGA but not MIS molecules was found in Md epithelial cells. The Golgi saccules were negative of lectin bindings.     

Lectin bindings and diethylstilbestrol effects on the recognition of mullerian inhibiting substance (MIS) on chick mullerian ducts by MIS-antiserum

Summary A high intensity of lectin bindings was demonstrated on the epithelial cells and serosa cells of the regressing right Mullerian ducts (Mds) in the female chick embryos. The strong lectin bindings occurs on, or in the regressing Md cells along with marked surface MIS bindings at the age of day 13. However, at the age of days 5–7 1/2, bindings of lectins were weak. Neither Wheat-germ agglutinin (WGA) or Concanavalin A (Con-A) labelings before MIS-antiserum (MIS-Ab) incubation can block antibody recognitions to the antigens, including MIS and growth hormone at the age of day 13. Our previous studies indicated that after WGA labeling on the surfaces of Md epithelial cells prior to the incubation of MIS-Ab at day 10 did not prevent the recognition of MIS-Ab (Wang 1989). On the contrary, at day 7 1/2, the specific binding of MIS was eliminated after preincubations with lectins and prenatal diethylstilbestrol (DES) treatment at the age of day 5. It is suggested that DES provides a protection to the Mds from MIS-induced regression by preventing the MIS binding to its specific membrane receptors. An increase of extra- and intracellular glycoproteins or carbohydrates of regressing Md epithelial cells were suggested. Internalization of WGA but not MIS molecules was found in Md epithelial cells. The Golgi saccules were negative of lectin bindings.     

Müllerian-inhibiting substance inhibits rat Leydig cell regeneration after ethylene dimethanesulphonate ablation

The postnatal development of Leydig cell precursors is postulated to be controlled by Sertoli cell secreted factors, which may have a determinative influence on Leydig cell number and function in sexually mature animals. One such hormone, Mullerian inhibiting substance (MIS), has been shown to inhibit DNA synthesis and steroidogenesis in primary Leydig cells and Leydig cell tumor lines. To further delineate the effects of MIS on Leydig cell proliferation and steroidogenesis, we employed the established ethylene dimethanesulphonate (EDS) model of Leydig cell regeneration. Following EDS ablation of differentiated Leydig cells in young adult rats, recombinant MIS or vehicle was delivered by intratesticular injection for 4 days (Days 11-14 after EDS). On Days 15 and 35 after EDS (1 and 21 days post-MIS injections), endocrine function was assessed and testes were collected for stereology, immunohistochemistry, and assessment of proliferation and steroidogenesis. Although serum testosterone and luteinizing hormone (LH) were no different, intratesticular testosterone was higher on Day 35 in MIS-treated animals. At both time points, intratesticular 5alpha-androstan-3alpha,17beta-diol concentrations were much higher than that of testosterone. MIS-treated animals had fewer mesenchymal precursors on Day 15 and fewer differentiated Leydig cells on Day 35 with decreased numbers of BrdU+ nuclei. Apoptotic interstitial cells were observed only in the MIS-treated testes, not in the vehicle-treated group on Day 15. These data suggest that MIS inhibits regeneration of Leydig cells in EDS-treated rats by enhancing apoptotic cell death as well as by decreasing proliferative capacity.     

A comparative study of the differentiation and involution of the Mullerian duct and Wolffian duct in the male and female fetal mouse

The present investigation has examined the ultrastructural differentiation of the genital ducts of both sexes of fetal mice. The emphasis of observations was placed on the phenomenon of morphogenetic cytolysis, particularly during the critical periods of Wolffian duct stabilization and Mullerian duct involution. Both developing and regressing genital ducts evidence extensive cytolysis. Autophagy appears to be the mechanism of morphogenetic changes in the developing male Wolffian duct. Autophagy, heterophagy, and degeneration in situ are all prominent cytolytic activities in female Wollfian duct involution. The developing female Mullerian duct undergoes extensive morphogenetic remodeling by the mechanisms of autophagy, heterophagy, and degeneration in situ. In the male Mullerian duct, autophagy, heterophagy, and degeneration in situ are also prominent. In addition, whole degenerated epithelial cells are extruded from the duct early in regression which may be realted to the transformation of periductal mesenchymal cells into an "epithelioid cell cuff" which does not form around the regressing Wolffian duct. The formation of this mesenchymal condensation surrounding the duct is also accompanied by the protrusion of Mullerian epithelial cell cytoplasm into the mesenchymal cells. These observations may evidence a complex epithelial-mesenchymal interaction occurring during male Mullerian duct involution.     

Reduced testicular steroidogenesis in tumor necrosis factor-alpha knockout mice

We previously demonstrated that the expression of Mullerian inhibiting substance (MIS) in Sertoli cells is downregulated by tumor necrosis factor alpha (TNF-alpha), which is secreted by meiotic germ cells, in mouse testes. Several studies have reported that MIS that is secreted by Sertoli cells inhibits steroidogenesis and, thus, the synthesis of testosterone in testicular Leydig cells. Here, we demonstrate that in TNF-alpha knockout testes, which show high levels of MIS, steroidogenesis is decreased compared to that in wild-type testes. The levels of testosterone and the mRNA levels of steroidogenesis-related genes were significantly lower after puberty in TNF-alpha knockout testes than in wild-type testes. Furthermore, the number of sperm was reduced in TNF-alpha knockout mice. Histological analysis revealed that spermatogenesis is also delayed in TNF-alpha knockout testes. In conclusion, TNF-alpha knockout mice show reduced testicular steroidogenesis, which is likely due to the high level of testicular MIS compared to that seen in wild-type mice.     

Forskolin and the meiosis inducing substance synergistically initiate meiosis in fetal male germ cells

We have shown that Meiosis Inducing Substance (MIS) and forskolin synergistically and dose dependently induce meiosis in germ cells of cultured fetal mouse testes. We used a bioassay which consists of fetal mouse testes and ovaries cultured for 6 days. In this study MIS media are spent culture media from 24 hour cultures of minced adult mouse testes. In the bioassay one gonad of each fetus is cultured either in MIS medium, in control medium with forskolin, or in MIS medium with forskolin. The other gonad serves as the control and is cultured in control medium. After culture the gonads are fixed, squashed, and DNA-stained. In these preparations germ cells and somatic cells can be distinguished, and the number of germ cells in the different stages of meiosis is counted as is the number of somatic cells in mitosis. MIS activity is defined to be present in a medium when meiosis is induced in male germ cells during culture. We found that MIS media as well as forskolin induced meiosis in fetal male germ cells in a dose-dependent manner. In addition, MIS media and forskolin acted synergistically by inducing meiosis. Female germ cells seem to be unaffected by the various culture media. These findings indicate that receptors for stimuli of meiotic initiation may exist in germ cells or neighbouring somatic cells. In addition to induction of meiosis, MIS media and forskolin also dose dependently increase the number of male germ cells compared to controls. This increase is correlated with induction of advanced stages of meiosis: Male germ cells seem to survive better if they are triggered to enter meiosis. Neither MIS media nor forskolin affected the growth of somatic cells. We therefore propose that MIS media has a growth factor activity with a specific effect on meiotic initiation. © 1993 Wiley-Liss, Inc.