Bernard Vigier

Secretion of anti-Müllerian hormone by immature bovine Sertoli cells in primary culture, studied by a competition-type radioimmunoassay: lack of modulation by either FSH or testosterone

Secretion of anti-Müllerian hormone (AMH) by immature bovine Sertoli cells in primary culture was studied through a competition-type RIA employing a polyclonal antibody and 125I-labelled purified AMH. This RIA is approximately 10 times more sensitive than the solid-phase two-site monoclonal antibody-based RIA described previously. Biosynthesis and secretion of AMH by cultured Sertoli cells require approximately 48 h, are not influenced by FSH or testosterone and steadily decrease over a one-week period of culture. Cyclic AMP response to FSH stimulation is normal in cultured cells. Whether the factors responsible for the extinction of AMH production in vitro are in any way related to those operating during normal maturation, which lead to repression of AMH biosynthesis in adult Sertoli cells, is not known at the present time and deserves further study.

Persistence of Müllerian ducts in male rabbits passively immunized against bovine anti-Müllerian hormone during fetal life

A female rabbit was immunized against purified bovine AMH and mated. Booster injections were given at Day 8 of pregnancy to ensure a high titer of anti-AMH antibodies at the time the rabbit fetal testis begins to produce AMH. In three consecutive litters, the immunized female produced a total of 12 males, 9 of which had persistent Müllerian duct derivatives. No other significant abnormalities were detected in these animals, which were compared to the offspring of a control saline-injected female. In particular, testicular morphology was normal in most animals, and serum FSH levels did not differ from controls. This experimental model lends no support to the hypothesis that AMH controls extra-Müllerian events of male sex differentiation, nor that of the existence of a regulatory mechanism for synthesis of AMH by Sertoli cells, but it does not definitely exclude these possibilities, inasmuch as our tentative conclusions are based upon study of only one immunized female.

Anti-Müllerian hormone: a local or long-distance morphogenetic factor?

SummaryIn an attempt to determine whether anti-Müllerian hormone could exert long-distance effects, we studied the anti-Müllerian activity of gonads from bovine Freemartin fetuses. Anti-Müllerian activity was detected in 3 out of the 7 animals studied: one was 62 days old, and its gonald contained undifferentiated tissue only; the 2 others were 110 and 130 days old respectively, and their gonads contained seminiferous tubules. The gonads devoid of anti-Müllerian activity contained only rete tubules or fibrous tissue. Anti-Müllerian activity was absent in fetal male and Freemartin serum, except in 2 cases, in which low activity was present after 37-fold purification by lectin affinity chromatography.The presence of anti-Müllerian activity in Freemartin gonads with seminiferous tubules is an indication that gonadal virilization in these fetuses is functional as well as morphological. Further experiments are needed to determine whether regression of the Müllerian ducts in the Freemartin is due to anti-Müllerian hormone produced by the Freemartin gonads in situ.

18 NEITHER TESTOSTERONE NOR FSH ARE RESPONSIBLE FOR DECREASED PRODUCTION OP AMH BY BOVINE SERTOLI CELLS IN PRIMARY CULTURE

Production of anti-Müllerian hormone (AMH) by Sertoli cells is maximal during the fetal and neonatal period, and tapers off in the course of postnatal development. To determine the factors responsible for the repression of AMH secretion after birth, Sertoli cells, isolated from immature calves, were plated in an hormonally defined medium and cultured in the presence of 0.1 mM MIX. AMH was assayed in the culture medium using a competition-type RIA capable of detecting 1.5 ng. The proportion of neosynthesized AMH was calculated by determining the amount of immunoreactive AMH unaffected by cycloheximide treatment in conditions where 92 % of protein neosynthesis is inhibited. AMH production by Sertoli cells, expressed per 24 hrs and per 106 cells, fell from 90 ng on the day following plating to 1.5 at day 7. The proportion of AMH synthesized after day 1 was 35 % on day 2 and 88 % on day 3. FSH, 2.5 μg/ml, and testosterone, 2 μM, were added to the culture medium on days 2 and 3, immunoreactive AMH was measured in the culture medium at day 3. Mean daily production of AMH by 106 Sertoli cells was 28.6±2.6 ng in control cultures, 24.6±2.8 in FSH-treated ones and 26.6±3.4 after testosterone treatment. Differences were not significant by analysis of variance. In the same conditions, FSH increased cyclic AMP production approximately tenfold. Further studies are in progress to determine which factors are essential to the continued production of AMH by Sertoli cells in vitro.

Polyclonal versus monoclonal antibodies: application to the study of human anti-M|[uuml]|llerian hormone

Monoclonal antibodies, raised against partially purified AMH, have allowed the purification of the hormone to homogeneity, and the development of radioimmunological and immunocytochemical methods, which could however be applied only to bovine AMH (bAMH), because of the zoospecificity of the first generation of monoclonal antibodies. Wishing to use these methods in clinical studies, we have sought to obtain antibodies recognizing human AMH. Two methods have been used. A second generation of monoclonal antibodies was obtained by immunization with pure bAMH. Thirteen clones, screened through an immunodot procedure using bAMH as antigen, have been identified. Their zoospecificity has been tested by using them as first antibody in an immunocytochemical reaction performed on testicular sections of different species. Caprine and ovine AMH are recognized by all monoclonal antibodies reacting with bAMH, most of these are IgG clones with high affinity. Three IgM clones recognize pig AMH, and one binds to human AMH. In parallel, a polyclonal antibody has been raised in a rabbit immunized with pure bovine AMH. This antibody is not zoospecific, and has allowed an immunocytochemical study of AMH in human testicular tissue, through an avidin-biotin-immunoperoxidase method, performed upon cryostat sections of testicular biopsies or autopsy material. AMH has been identified in the Sertoli cells of 6 human fetuses, aged 16 to 22 weeks, and 4 boys aged 1 month to 5 years. This method can be used to study testicular AMH in intersex disorders.

A monoclonal antibody against anti-müllerian hormone

Anti-müllerian hormone (AMH) has been partially purified from incubation medium of calf fetal testes (Picard and Josso, 1981). It produces müllerian regression in organ culture and contains one major radioactive protein, when testes have been incubated in the presence of tritiated fucose. Identity between AMH and this labelled marker protein has been postulated, and, in consequence, the latter has been used to screen hybridomas for anti-AMH activity. Splenocytes of a BALB/c mouse, injected with partially purified AMH, produced 297 hybridomas after fusion with myeloma cells. Culture medium from 94 was tested, in a double antibody precipitation test, for its capacity to bind the labelled protein contained in partially purified medium. Three IgG hybridomas gave positive results and one was cloned and used to produce ascites in BALB/c mice. Ascites IgG, used as first antibody in a double antibody precipitation test, precipitated the labelled protein and removed anti-müllerian activity from medium containing bioactive AMH. The monoclonal antibody also blocked anti-müllerian activity of calf fetal testicular tissue, indicating that it is directed against the biologically active portion of the AMH molecule. These results prove the identity between AMH and its biochemical marker, and should greatly hasten the isolation and quantitative assay of the hormone.