Insensitivity to anti-müllerian hormone due to a mutation in the human anti-müllerian hormone receptor.

Anti-Müllerian hormone (AMH) and its receptor are involved in the regression of Müllerian ducts in male fetuses. We have now cloned and mapped the human AMH receptor gene and provide genetic proof that it is required for AMH signalling, by identifying a mutation in the AMH receptor in a patient with persistent Müllerian duct syndrome. The mutation destroys the invariant dinucleotide at the 5' end of the second intron, generating two abnormal mRNAs, one missing the second exon, required for ligand binding, and the other incorporating the first 12 bases of the second intron. The similar phenotypes observed in AMH-deficient and AMH receptor-deficient individuals indicate that the AMH signalling machinery is remarkably simple, consisting of one ligand and one type II receptor.

Lhx9 expression during gonadal morphogenesis as related to the state of cell differentiation.

Lhx9 (LIM/Homeobox gene 9) encodes a transcription factor implicated in various developmental processes, including gonadogenesis. Our observations in the rat show that Lhx9 expression present in undifferentiated gonads disappears as epithelial cells differentiate into Sertoli cells and begin to express AMH. In rat and in chick testes, Lhx9 expression present in interstitial cells decreases progressively to become undetectable after birth. In the female rat, Lhx9 is highly expressed in epithelial ovigerous cords of the fetal ovary. Its expression is down-regulated as epithelial cells differentiate into granulosa cells during the process of folliculogenesis occurring at birth. If this process is impaired by the lack of oocytes, ovigerous cord organization is maintained together with Lhx9 expression. In conclusion, Lhx9 expression can be inversely correlated with the commitment into a differentiation pathway of the different categories of mesothelium-derived cells of the gonad.

[Molecular biology of normal and pathologic anti-müllerian hormone]. / Biologie moléculaire de l'hormone anti-müllérienne normale et pathologique.

Anti-Müllerian hormone, responsible for Müllerian regression in male fetuses, is a glycoprotein dimer with two 72 kD subunits. The AMH gene is a small (2,800 bp) gene with 5 exons, localized on the tip of the short arm of chromosome 19, band p 133, and transcribed in a 2,000 kbp mRNA. Persistent Müllerian duct syndrome, a rare form of male pseudohermaphroditism characterized by the presence of uterus and Fallopian tubes in patients with normally virilized genitalia, may result from defective AMH gene or from target-organ insensitivity. Four mutations were identified in the AMH gene, 3 are point mutations (2 stop codons, the third altering the secondary structure of the molecule), the last is a 14 bp deletion, leading to alteration of the reading frame of the mRNA.

Processing of the precursor of protamine P2 in mouse. Identification of intermediates by their insolubility in the presence of sodium dodecyl sulfate.

Two basic proteins, protamines P1 and P2, are present in chromatin of mouse spermatozoa. Protamine P1, the less abundant protein in mouse, has a homolog in most mammals, and its synthesis follows a conventional route. In contrast, protamine P2 has been found only in certain other mammals, including humans, and it is synthesized as a precursor nearly twice as long as the mature protein. Processing of this precursor is not yet understood, although it necessarily takes place in elongating spermatids and is likely to play a role in the chromatin condensation occurring in these haploid cells. We have fractionated basic proteins from mouse testis chromatin and have identified six proteins on electrophoretic gels which, like protamines, are insoluble in SDS. All six were also soluble at the same trichloroacetic acid concentration as protamine P2 and were present in chromatin of elongating spermatids. Radioactive labelling patterns acquired by these SDS-insoluble proteins during translation in vitro of testis RNA indicate that the largest represents the precursor of protamine P2, and suggest that the others represent intermediates generated by proteolytic cleavage of the precursor. Results from pulse 3H labelling in vivo were also consistent with the conclusion that a precursor/product relationship exists between these proteins and protamine P2. Conclusions concerning the kinetics of processing have, in addition, been drawn from this data. Hypotheses concerning possible functional roles played by the precursor are presented.

The persistent müllerian duct syndrome: a rare cause of cryptorchidism.

The persistent Müllerian duct syndrome is characterized by the retention of Müllerian derivatives in patients otherwise normally virilized. Clinically, the persistence of uterus and tubes leads either to cryptorchidism or inguinal hernia, depending on whether or not the Müllerian derivatives can be mobilized during testicular descent. The condition is usually discovered at surgery, however preoperative sonography could allow the diagnosis to be made preoperatively. The molecular basis of the persistent Müllerian duct syndrome is heterogeneous, and is reflected by wide variations in the serum concentration of anti-Müllerian hormone. Some cases are apparently due to end-organ resistance, and are associated with normal serum levels of the hormone. Others, characterized by absent or low hormone concentrations, can be explained by mutations of the gene coding for anti-Müllerian hormone, which are distributed along the whole length of the coding region.

Processing of the precursor of protamine P2 in mouse. Peptide mapping and N-terminal sequence analysis of intermediates.

Protamine P2, the major basic chromosomal protein of mouse spermatozoa, is synthesized as a precursor almost twice as long as the mature protein, its extra length arising from an N-terminal extension of 44 amino acid residues. This precursor is integrated into chromatin of spermatids, and the extension is processed during chromatin condensation in the haploid cells. We have studied processing in the mouse and have identified two intermediates generated by proteolytic cleavage of the precursor. H.p.l.c. separated protamine P2 from four other spermatid proteins, including the precursor and three proteins known to possess physiological characteristics expected of processing intermediates. Peptide mapping indicated that all of these proteins were structurally similar. Two major proteins were further purified by PAGE, transferred to poly(vinylidene difluoride) membranes and submitted to automated N-terminal sequence analysis. Both sequences were found within the deduced sequence of the precursor extension. The N-terminus of the larger intermediate, PP2C, was Gly-12, whereas the N-terminus of the smaller, PP2D, was His-21. Both processing sites involved a peptide bond in which the carbonyl function was contributed by an acidic amino acid.

Molecular genetics of the persistent müllerian duct syndrome: a study of 19 families.

A rare form of familial male pseudohermaphroditism, the persistent Müllerian duct syndrome (PMDS) is characterized by persistence of uterus and Fallopian tubes in 46,XY phenotypic males and is ascribed to defects in the synthesis or action of anti-Müllerian hormone (AMH). Biologically, PMDS is heterogeneous: in some cases, bioactive AMH is normally expressed by testicular tissue while, in others, no AMH is produced, suggesting the possibility of an AMH gene mutation, several of which have already been described. Molecular analysis of the AMH gene has now been performed in 21 additional patients and their families. In 6 patients, with normal serum concentration of AMH, the AMH gene was normal or contained only polymorphisms and silent mutations, supporting the hypothesis that the condition is due to end-organ resistance. Nine novel mutations were discovered in the remaining subjects, with low or undetectable levels of serum AMH. These mutations, when present in homozygotes or compound heterozygotes, were associated with the PMDS phenotype, the same mutation never being observed in two different families. The three first exons of the AMH gene appear particularly mutation-prone, although they are less GC rich than the 2 last ones and code for the N-terminal part of the AMH protein, which is not in itself essential to bioactivity.

Early expression of AMH in chicken embryonic gonads precedes testicular SOX9 expression.

In mammals, anti-Müllerian hormone (AMH) is produced by Sertoli cells from the onset of testicular differentiation and by granulosa cells only after birth. SOX9, a transcription factor related to the testis-determining factor SRY, is expressed in mouse testis 1 day before AMH. To determine the relationship between AMH and SOX9 in birds, we cloned the AMH promoter in search of SOX9 response elements, and we compared the expression of AMH and SOX9 in the gonads of chick embryos using in situ hybridization. Potential SOX response elements were found in the AMH promoter; however, AMH is expressed in both sexes at stage 25, 1 day before the first SOX9 transcripts appear in the male gonads. SOX9 is never expressed in the female. These results do not support the hypothesis that SOX9 could trigger the expression of testicular AMH in the chick but does not exclude a later role in testis development.

Variants of the anti-Müllerian hormone gene in a compound heterozygote with the persistent Müllerian duct syndrome and his family.

The persistent Müllerian duct syndrome (PMDS) is a rare form of male pseudohermaphroditism, characterized by the persistence of Müllerian derivatives in otherwise normal males. Two mutations, present in the homozygous state, have been previously described in such patients. The present observation is the first example of compound heterozygosity in this condition. DNA was obtained from a 3-month-old patient with PMDS, in whom no serum anti-Müllerian hormone (AMH) could be detected by enzyme-linked immunosorbent assay. Sequencing of cloned polymerase chain reaction amplified fragments of the AMH gene revealed a 14-bp deletion in the second exon of the maternal allele; this deletion disrupted the open reading frame. It occurred at a site containing two 8-bp direct repeats flanking a 6-bp sequence and removed one whole repeat plus all of the intervening sequence. It may be the result of a slipped mispairing at the DNA replication fork. The paternal allele contains a stop mutation in the third exon. These two mutations, impairing both AMH alleles, are consistent with the occurrence of PMDS, and are shared with a phenotypically normal younger sister. In this family, various other mutations, devoid of physiological significance, suggest that the AMH gene is highly polymorphic.