Dien Tran

The Antimüllerian Hormone

Publisher Summary This chapter discusses the macromolecular nature of antimullerian hormone (AMH), gel filtration of AMH, ultracentrifugation in a density gradient, and ion-exchange chromatography. The antimullerian hormone is synthesized by the fetal Sertoli cells very early in fetal life, as soon as seminiferous tubules are recognizable under the light microscope and before the appearance of fetal Leydig cells. AMH production ceases in the perinatal period and is not resumed at puberty. The biochemical structure of AMH has not yet been elucidated, mainly because the bioassay used up to now is not quantitative and, therefore, not ideally suited to techniques of protein purification. The macromolecular nature of the hormone has been firmly established and its protein nature is highly probable. A molecular weight of 200,000 to 300,000 has been demonstrated by gel filtration, whereas by density gradient ultracentrifugation, lower values, from 120,000 to 200,000, are found. This discrepancy is compatible with the hypothesis that AMH is a glycoprotein.

Immunocytochemical detection of anti-müllerian hormone in Sertoli cells of various mammalian species including human.

An immunocytochemical method, based on the use of a polyclonal antibody raised against purified bovine anti-Müllerian hormone (AMH), was used to detect AMH in Sertoli cell cytoplasm of various mammalian species, including human. Immunopurification of antiserum by AMH-affinity chromatography, although not mandatory, leads to better results and increased sensitivity. In human testicular tissue, AMH is detectable up to 6 years of age. In rats, AMH production is initiated at 13 days post coitum, peaks between 15 and 17 days, and is no longer detectable 1 week after birth. The reaction is strongest in Sertoli cells of calves, sheep, goats, and pigs, species characterized by a high degree of development of the rough endoplasmic reticulum. It is fainter in human, rat, rabbit, and cat Sertoli cells, in which the rough endoplasmic reticulum is not as abundant. This correlation is not unexpected, in view of the localization of reaction product in this cytoplasmic organelle. Preliminary results indicate that there may be a relationship between the amount of immunoreactive AMH present in testicular biopsies of intersex patients and the degree of regression of the Müllerian duct on the ipsilateral side. This may help to elucidate whether persistence of Müllerian ducts results from lack of testicular production of AMH or from peripheral resistance of the Müllerian primordia to the hormone.

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-idiotypic antibodies to a monoclonal antibody raised against anti-müllerian hormone exhibit anti-müllerian biological activity.

Polyclonal anti-idiotypic antibodies were raised to three monoclonal antibodies to bovine anti-Müllerian hormone, and purified by affinity chromatography. All anti-idiotypes inhibited binding of labelled anti-Müllerian hormone to the monoclonal antibody against which they were directed; in addition, the anti-idiotypes directed against a non-zoospecific monoclonal antibody inhibited binding of labelled anti-Müllerian hormone to a monoclonal antibody raised against human testicular AMH, indicating that the idiotype against which these anti-idiotypes are directed recognizes a conserved epitope on the anti-Müllerian hormone molecule. These anti-idiotypes, but not those directed against other monoclonals, exhibit hormone-like activity in a bioassay for anti-Müllerian activity, and we suggest that they may act as antibodies against the anti-Müllerian hormone receptor.

141 STUDY OF TESTICULAR ANTI-MULLERIANHORMONE IN PERSISTENT MULLERIAN DOCT SYNDROME

Persistent Müllerian duct syndrome was diagnosed at surgery for inguinal hernia and/or bilateral cryptorchidism in 6 otherwise normal XY boys aged 2 mos to 7 yrs, including 3 brothers. In two cases, the inguinal hernia contained the uterus and tubes, tightly attached to both testes. In the other patients, the hernial sac contained initially only the homolateral testis and tube, but gentle traction exteriorized the uterus and contralateral testis, suggesting that, in this syndrome, testicular position depends on the location of the fallopian tube. Testicular biopsies were studied by immunohistochemistry using a polyclonal antibody specific for anti-Müllerlan hormone (AMH), and by AMH bioassay, using the rat fetal Müllerian duct as target organ. In normal boys, immunohistochemistry detects AMH up to 6 yrs (Tran & all., 1987). In 4 patients from 2 sibships, both techniques gave negative results, indicating that persistence of Müllerian derivates resulted from AMH deficiency. In two unrelated cases, however, immunoreactive AMH was present, and biologically active, suggesting either target organ insensitivity to AMH or fetal testicular AMH production starting after the end of the AMH-sensitive window of Müllerian development. We conclude that the persistent Müllerian duct syndrome is etiologically heterogeneous and may therefor follow different patterns of inheritance.

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.

Evidence for the glycoprotein nature of anti-m|[uuml]|llerian hormone

Tritiated fucose incorporated into proteins released by foetal calf testes into incubation medium proved to be a marker for anti-müllerian hormone (AMH) once non-specific glycoproteins had been eliminated by partial purification. When partially purified incubation medium from foetal calf testes was fractionated by various procedures, a single radioactive protein peak co-purified with anti-müllerian activity. Partially purified medium from bull testes - which are devoid of anti-müllerian activity - has a much lower fucose content than that derived from foetal testes. Antisera directed against “foetal” partially purified incubation medium, and capable of blocking anti-müllerian activity, precipitated the radioactive protein peak. The MW of labelled AMH was 215,000 when determined by gel filtration and 124,000 when determined by density gradient sedimentation. By SDS-PAGE the MW of labelled AMH was 123,000 and dissociation into a 72,000 subunit was demonstrated under reducing conditions.