A 11.7-kb deletion triggers intersexuality and polledness in goats.

Mammalian sex determination is governed by the presence of the sex determining region Y gene (SRY) on the Y chromosome. Familial cases of SRY-negative XX sex reversal are rare in humans, often hampering the discovery of new sex-determining genes. The mouse model is also insufficient to correctly apprehend the sex-determination cascade, as the human pathway is much more sensitive to gene dosage. Other species might therefore be considered in this respect. In goats, the polled intersex syndrome (PIS) mutation associates polledness and intersexuality. The sex reversal affects exclusively the XX individuals in a recessive manner, whereas the absence of horns is dominant in both sexes. The syndrome is caused by an autosomal gene located at chromosome band 1q43 (ref. 9), shown to be homologous to human chromosome band 3q23 (ref. 10). Through a positional cloning approach, we demonstrate that the mutation underlying PIS is the deletion of a critical 11.7-kb DNA element containing mainly repetitive sequences. This deletion affects the transcription of at least two genes: PISRT1, encoding a 1.5-kb mRNA devoid of open reading frame (ORF), and FOXL2, recently shown to be responsible for blepharophimosis ptosis epicanthus inversus syndrome (BPES) in humans. These two genes are located 20 and 200 kb telomeric from the deletion, respectively.

Cloning, expression, and alternative splicing of the receptor for anti-Müllerian hormone.

Anti-Müllerian hormone, also called Müllerian-inhibiting substance or factor, is a glycoprotein dimer belonging to the transforming growth factor-beta superfamily and synthesized by immature Sertoli cells and postnatal granulosa cells. Anti-Müllerian hormone plays a key role in sex differentiation by inducing the regression of Müllerian ducts in the male fetus. It is also responsible for the stunting and masculinization of fetal ovaries in bovine freemartin fetuses and may be involved in the control of follicular maturation in the postnatal ovary. Using a degenerate probe for a consensus region of the transforming growth factor-beta receptor superfamily to screen a complementary DNA library from rabbit fetal ovaries, we cloned a complementary DNA coding for a transmembrane serine/threonine kinase, which is expressed around the fetal Müllerian duct, in fetal and adult granulosa cells, and in immature Sertoli cells. Two transcripts, generated by alternative splicing of an exon coding for an N-terminal 61-amino acid domain, are strongly expressed in anti-Müllerian hormone target organs and Sertoli cells. The longer, 569-amino acid, isoform binds anti-Müllerian hormone when transiently expressed in COS cells and is believed to encode its functional receptor.

Mullerian inhibiting substance requires its N-terminal domain for maintenance of biological activity, a novel finding within the transforming growth factor-beta superfamily.

Mullerian inhibiting substance (MIS)/anti-Mullerian hormone is a differentiation factor that causes regression of the Mullerian duct in the developing male fetus and an apparent sex reversal of the fetal ovary when inappropriately exposed to it. The purified product is a 140-kilodalton glycoprotein composed of two identical subunits. We show that a C-terminal fragment of MIS, which shares homology with transforming growth factor-beta, causes regression of the Mullerian duct and inhibits the biosynthesis of aromatase in the fetal ovary. However, both activities are enhanced dramatically by addition of the N-terminal portion of MIS. Under conditions where potentiation occurs, the N- and C-terminal domains of MIS reassociate. These results indicate that the N-terminus of MIS, unlike that of the other members of the transforming growth factor-beta family, plays a role in maintaining the biological activity of the C-terminus.

A monoclonal antibody against bovine anti-Müllerian hormone.

Anti-Müllerian hormone (AMH) was partially purified from incubation medium of calf fetal testes and injected into a BALB/c mouse, whose splenocytes were fused with Sp2/Ag8 myeloma cells. Hybridomas were screened for specific antibody production by double antibody precipitation of labeled AMH, which was obtained by incubating fetal calf testes in the presence of tritiated fucose and submitting the medium to the standard procedure of purification. In spite of the extremely low concentration of AMH in the preparation used for immunization, three hybridomas gave positive results in the screening assay. One was cloned and grown in mice. The monoclonal antibody purified from ascites fluid abolished anti-Müllerian activity of partially purified AMH, whether or not the immune complex was removed from solution by a second, antimouse immunoglobulin antibody. The monoclonal antibody also blocked anti-Müllerian activity of calf but not rat fetal testes. Our results indicate that the monoclonal antibody is species specific and is directed towards the antigenic determinant responsible for biological activity.

Production of anti-Müllerian hormone: another homology between Sertoli and granulosa cells.

Anti-Müllerian hormone (AMH) has been detected by RIA in the follicular fluid of mature bovine ovaries and in incubation medium of bovine granulosa cells. Purification of AMH from two independent batches of follicular fluid was achieved with a yield of 11% and 15% respectively. Both ovarian and control testicular AMH produced near-complete regression of fetal rat Müllerian ducts exposed to it in culture at a final concentration of 200-300 mU/ml and were recognized by the same monoclonal and polyclonal antibodies. These findings indicate that adult mammalian granulosa cells are capable of producing immunoreactive and bioactive AMH at a rate apparently similar to that already demonstrated for mature Sertoli cells and add yet another item to the homologies reported between male and female somatic gonadal cells.

A sensitive radioimmunoassay for bovine anti-Müllerian hormone, allowing its detection in male and freemartin fetal serum.

Two monoclonal antibodies have been used to set up a solid-phase RIA for bovine anti-Müllerian hormone (bAMH). One AMH unit is defined as the amount released by 1 g of bovine fetal testicular tissue during a 4 h incubation period. Calibration curves were prepared using aliquots of a standard 500 ml pool of incubation medium, containing 200 AMH mU/ml, diluted either in 50% pig testes incubation medium, 5% horse serum, 10% female calf fetal serum or pure female calf fetal serum. Linearization of the calibration curves was achieved through "logit-log" transformation, all four lines were parallel. Within and between-assay variability were less than 5%. The RIA is at least 600 times more sensitive than the bioassay for anti-Müllerian activity and can detect AMH in male and freemartin fetal serum.

Effect of anti-Mullerian hormone on Sertoli and Leydig cell functions in fetal and immature rats.

Anti-Mullerian hormone (AMH) is mainly involved in the regression of Mullerian ducts in male fetuses, but it may have other functions linked to gonadal development. The present study examines the effect of AMH on steroidogenesis by Sertoli and Leydig cells in fetal and immature rats during the period where AMH is physiologically produced in the testis. The basal aromatase activity of Sertoli cells in primary culture was strongly stimulated (77-91%) by cAMP. AMH (35 nM) reduced cAMP-stimulated aromatase activity by 49-69% as early as fetal day 16 and until postnatal day 20. This effect was dose dependent and was seen after 48 h in culture. AMH also blocked the Sertoli cell aromatase activity stimulated by FSH, but LH did not stimulate this activity, confirming that the aromatase activity effectively resulted from Sertoli cells and not from contaminating Leydig cells. RT-PCR analysis showed that AMH reduced aromatase activity by decreasing the amount of aromatase messenger RNA. AMH also inhibited the LH-stimulated testosterone production by dispersed fetal Leydig cells in culture in a dose-dependent manner. The inhibitory effect of AMH did not depend on the fetal stage studied (16 or 20 days postconception) and resulted from a drop in the steroidogenic activity of each Leydig cell without affecting the number of 3beta-hydroxysteroid dehydrogenase-positive cells. These data provide the first evidence that AMH, like other members of the transforming growth factor-beta family, has an autocrine/paracrine effect on testicular steroidogenic function during the fetal and prepubertal periods.

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.

A monoclonal antibody against human testicular anti-müllerian hormone.

Using immunochromatography on a polyclonal antibody, testicular anti-Müllerian hormone (AMH) was purified from homogenates of human fetal testicular tissue and used as an antigen for hybridoma production. Two IgM clones were obtained. Both recognized AMH on biopsies of human testicular tissue and one blocked its anti-Müllerian activity. This monoclonal antibody (MAb) exhibited a relatively high affinity for AMH, and was studied further. It is interspecific, recognizing AMH in other mammalian species. The study of this MAb in relation to an IgM MAb raised against bovine AMH (bAMH) indicates that both MAbs recognize different but related epitopes on bAMH.

[Chronology of development of the genital tract of the calf fetus]. / Chronologie du développement de l'appareil génital du foetus de veau

The chronology and the modalities of the differentiation of the genital apparatus were studied in 187 calf fetuses whose insemination age was exactly known (between 32 and 110 days). Male. In the male, the first seminiferous cords form around days 41-42, the interstitial cells appear 2 or 3 days later. From days 60-70 on, the rete testis is made of tubules with an open lumen, which connect the seminiferous cords. The masculinisation of the external genitalia begins as early as day 47 by a rapid increase of the anogenital distance: on day 60, the penis opens under the umbilicus and the scrotum is well differentiated. The regression of the Müllerian ducts starts at the level of their anterior (tubal) part on day 50, when their diameter decreases. From day 58 on, the oviducts become discontinuous and they are almost completely absent by day 63; the uterine horns and the vagina have dissappeared by day 80. The masculinization of the internal genitalia occurs during two phases: 1) between days 56 and 58: the early buds of the seminal vesicles and of the prostate appear, as well as the first differences at the level of the urogenital connections and of the Cowper's glands; a supra-urethral diverticulum develops at the level of the posterior prostatic urethral flexure; the non sexual urethra remains short. 2) After day 70 take place: the differentiation of the epididymides the branching of the seminal vesicles and the stabilization of the Wolffian ducts (after a transitory diminution of their diameter between days 60 and 80). Female. In the female, sexual organogenesis proceeds later than in males. The Müllerian ducts (which show a transitory reduction of their diameter at their tubal level between days 50 and 60) develop steadily after day 60 at the level of their uterine horns and of the vagina; simultaneously the non sexual urethra lengthens rapidly. A suburethral diverticulum, which is absent in the male, develops at the level of the urogenital connections, between days 60-70. The regression of the Wolffian ducts takes place after day 70 when the mesonephros disappear; they first become discontinuous in their median part (at the uterine level) between days 77-80, but important remains of these ducts still persist on day 110, in the posterior part of the vagina. The first primordial ovarian follicles form only after day 100 approximately.

[Meiotic prophase in germinal cells of rat ovarian primordium cultivated in vitro in anhormonal medium]. / Prophase méiotique dans les cellules germinales de l'ébauche ovarienne de Rat cultivée in vitro en milieu anhormonal

Gonads of female Rat fetuses were cultured in vitro in an anhormonal medium with the whole or parts of the mesonephros, at an age of 12, 13 or 14 days, i.e. 2 to 4 days before the onset of meiosis. Under these conditions the meiotic prophase takes place and proceeds to the dictyate phase, obeying a somewhat delayed chronology in comparison with controls in vivo.

Anti-müllerian hormone in relation to the growth and differentiation of the gubernacular primordia in mice.

The present study analysed gubernaculum development in mice that had been induced, through transgenesis, to express human anti-Müllerian hormone (h-AMH) throughout prenatal life. Growth and differentiation of the gubernacular primordia were assessed through the analysis of serial, transverse or sagittal, histological sections of the lower abdomen. Transgenic males and females expressed biologically active amounts of h-AMH as measured by sensitive and specific ELISA and evidenced through the regression, in females, of Müllerian ducts after day 13 of prenatal life. Gubernacular primordia became distinguishable at the same age in control and transgenic male and female fetuses on day 12 after coitus. In both groups gubernacular cords (inguinal folds of the genital mesenteries) increased in length more in females than in males while gubernacular cones showed larger growth in males. h-AMH thus appeared not to affect the sexually dimorphic pattern of growth and development of these structures. Growth and differentiation of the gubernacular primordia was further examined in 18-day-old control and h-AMH transgenic fetuses that had been exposed to testosterone propionate injected into their mothers on days 12 and 14 of pregnancy. Testosterone treatment affected, to a minor extent, the growth of the female gubernacular cords: these were reduced in length (but had a larger surface area) compared with controls. The gubernacular cones were slightly increased in length but male-like differentiation of the tissues of the cones into a muscular and mesenchymal component was not noticed to any extent. The observations thus add experimental support to the contention that AMH, even in combination with testosterone, is not effective in establishing the male pattern of gubernacular primordia development.

[Persistence of Mullerian ducts in the male rabbit by passive immunization against anti-mullerian hormone during fetal life]. / Maintien des canaux de Müller chez le Lapin mâle par immunisation passive contre l'hormone anti-müllérienne pendant la vie foetale.

A female Rabbit immunized against bovine anti-Müllerian hormone (AMH) was mated and gave birth to 3 litters, containing a total of 13 males. Ten of these presented with Müllerian derivatives, of variable development. Testicular position and structure, Wolffian development and virilization of the urogenital sinus were apparently normal in males with persistent Müllerian ducts.

[Purification of bovine antimullerian hormone using a monoclonal antibody]. / Purification de l'hormone antimüllérienne bovine à l'aide d'un anticorps monoclonal.

Bovine antimüllerian hormone (AMH) has been purified from incubation medium of bovine fetal testes, by immunoaffinity chromatography using a monoclonal antibody. Presence of AMH in the column eluate is demonstrated by its high antimüllerian activity. Biochemical analysis, by SDS polyacrylamide gel electrophoresis, indicates that the testicular protein eluted from the column co-electrophoreses with native tritiated AMH which, as previously shown, is a dimer of 124,000 +/- 15,000 MW.

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

In 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 gonad 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.

[The first manifestations of freemartinism in the calf fetus do not depend on XX/XY chromosomal chimerism]. / Les premières manifestations du "freemartinisme" chez le foetus de veau ne dépendent pas du chimérisme chromosomique XX/XY

In order to investigate the eventual role of XX/XY chimerism in freemartinism the vascular anastomoses between twin fetuses were surgically suppressed in litters with several fetuses, before the appearance of the first sexual anomalies. In three female fetuses isolated from their co-twins on days 37 and 45, the initial freemartin effect of gonadal and Müllerian inhibition was absent, in spite of an important XX/XY chimerism in the liver.

[Anti-mullerian hormone and natural and experimental freemartin effect]. / Hormone anti-mullerienne et effet freemartin naturel et expérimental.

To determine whether anti-Müllerian hormone (AMH) is responsible for the gonadal anomalies observed in bovine female "freemartins" united by placental anastomosis to a male twin, fetal rat and ovine prospective ovaries were exposed to purified bovine AMH in organ culture. We show that AMH reproduces the caracteristical "freemartin effect"; namely the initial inhibition of gonadal and germ cell development, and the differentiation of fetal Sertoli cells forming seminiferous cord-like structures and producing immuno and bioactive AMH. In addition, fetal ovine ovaries submitted to AMH release testosterone instead of estradiol due to an inhibiting effect upon the biosynthesis of their aromatase enzyme. Taken together, these results indicate that AMH is probably the testicular factor responsible for the abnormalities of freemartin gonads and suggest that this hormone could play a pivotal role in normal testis differentiation.

Anti-müllerian hormone in sheep follicles.

The purpose of this work was to study anti-Müllerian hormone (AMH) secretion during ovarian development in sheep before and after birth. We used avidinbiotin immunocytochemistry and a monoclonal antibody specific for ruminant AMH. Only granulosa cells have an immunoreactivity; this immunoreactivity was influenced by animal age and by the degree of follicular development. In the fetus, no immunoreactivity was detected in somatic cells of ovigerous cords at 70 days post-coitum (p.c.) or in primordial and growing follicles at 100 and 120 days p.c. A faint reaction was only seen occasionally in a few cells belonging to preantral follicles at 120 days p.c. AMH was never detected in primordial follicles in ovaries of 144 days p.c., at birth, at 8, 97, 145 days post-natal or in adult ovaries. A faint reaction, elicited in small growing follicles, increased with follicle size to become more intense in antral follicles. Immunoreactivity was strongly positive in granulosa cells, especially in those lining the antral cavity and close to the oocyte, whereas there was little or no reactivity in peripheral cells near the basal membrane. Follicles without AMH reactivity were found at all times and their number decreased with age.

Monoclonal antibodies raised against bovine anti-müllerian hormone: bovine, ovine, and caprine hormones share a set of identical epitopes.

Monoclonal antibodies (Mabs) have been raised against purified bovine anti-Müllerian hormone (bAMH) in an effort to obtain nonzoospecific reagents. Although the majority of the resulting hybridomas resembled those obtained previously insofar as they recognized only bovine, ovine and caprine AMH, four others, all immunoglobulin Ms, were directed against an epitope shared with AMH of other species, namely rabbit, pig and cat. Both the zoospecific and the conserved epitopes were located close to the site required for biological activity. It is suggested that the similarity between the immunogenic characteristics of bovine, ovine and caprine AMH is in some way related to the fact that AMH in these species is disseminated in the blood stream and may produce freemartinism.

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.