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.

Time course of female-to-male sex reversal in 38,XX fetal and postnatal pigs.

In an attempt to understand the etiology of intersexuality in pigs, we thoroughly analyzed the gonads of 38,XX (SRY negative) female to male sex-reversed animals at different developmental stages: during fetal life [50 and 70 days postcoitum (dpc)], just after birth [35 days postpartum (dpp)] and during adulthood. For each animal studied, we performed parallel histological and ultrastructural analyses on one gonad and RT-PCR analysis on the other gonad in order to define the expression profiles of sexually regulated genes: SOX9, 3beta-HSD, P450 aromatase, AMH, FOXL2, and Wnt4. Light and electron microscopic examination showed that testicular cords differentiated in XX sex-reversed gonads but were hypoplastic. Although the testicular cords contained gonia at the fetal stages, the germ cells had all died through apoptosis within a few weeks after birth. Ultrastructurally normal Leydig cells also differentiated, but later, and enclosed whorl-like residual bodies. At the fetal stages, three of the six genes studied in the intersex gonads presented, as early as 50 dpc, a modified expression profile corresponding to an elevated expression of SOX9 and the beginning of AMH and P450 aromatase gene transcription. In addition to genes involved in the testicular pathway, the same gonads expressed FOXL2, an ovarian-specific factor. The ovaries of true hermaphrodites were ineffective in ensuring correct folliculogenesis and presented abnormal expression profiles of ovarian specific genes after birth. These results indicate that the genes involved in this pathology act very early during gonadogenesis and affect the ovary-differentiating pathway with variable expressivity from ovarian germ cell depletion through to trans-differentiation into testicular structures.

Contribution of domestic animals to the identification of new genes involved in sex determination.

Among farm animals, two species present an intersex condition at a relatively high frequency: pig and goat. Both are known to contain XX sex-reversed individuals which are genetically female but with a true hermaphrodite or male phenotype. It has been clearly demonstrated that the SRY gene is not involved in these phenotypes. Consequently, autosomal or X-linked mutations in the sex-determining pathway may explain these sex-reversed phenotypes. A mutation referred to as "polled" has been characterized in goats by the suppression of horn formation and abnormal sexual differentiation. The Polled Intersex Syndrome locus (PIS) was initially located in the distal region of goat chromosome 1. The homologous human region has been precisely identified as an HSA 3q23 DNA segment containing the Blepharophimosis Ptosis Epicanthus locus (BPES), a syndrome combining Premature Ovarian Failure (POF) and an excess of epidermis of the eyelids. In order to isolate genes involved in pig intersexuality, a similar genetic approach was attempted in pigs using genome scanning of resource families. Genetic analyses suggest that pig intersexuality is controlled multigenically. Parallel to this work, gonads of fetal intersex animals have been studied during development by light and electron microscopy. The development of testicular tissue and reduction of germ cell number by apoptosis, which simultaneously occurs as soon as 50 days post coïtum, also suggests that several separate genes could be involved in pig intersexuality.

Pre-harvest accumulation of deoxynivalenol in sweet corn ears inoculated with Fusarium graminearum.

Three types of commercial sweet corn hybrids [surgary (su1), shrunken or 'supersweet' (sh2) and surgary enhancer (se1)] were silk channel inoculated in 1996 and 1997 with a macroconidial suspension of Fusarium graminearum to determine how early the mycotoxin deoxynivalenol accumulates in kernels. Disease symptoms rapidly developed on all hybrids and were apparent 4 days after inoculation. Symptoms stabilized by 28 days after inoculation. Toxin levels were greater than 1 microgram/g in kernels as early as 2 weeks after silk emergence and rapidly increased to extremely high levels. Susceptibility in all hybrids decreased as the silk dried out. Deoxynivalenol concentrations were correlated to disease severity. There was some indication that the sh2 genotype was more susceptible than the su1 or se1 genotypes. These results suggest that improvement needs to be made in sweet corn with respect to resistance to gibberella ear rot.

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.

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.

Cloning and expression of the chick anti-Müllerian hormone gene.

Müllerian duct regression in male embryos is due to early production by fetal Sertoli cells of anti-Müllerian hormone, a homodimeric protein of the transforming growth factor- beta superfamily. In mammals, both female Müllerian ducts develop into the uterus and Fallopian tubes, whereas in birds, the right oviduct does not develop. To gain insight into sex differentiation in birds, we have cloned the cDNA for chick anti-Müllerian hormone using antibodies raised against the partially purified protein. Expression cloning was required because of the lack of cross-hybridization between mammalian and chick anti-Müllerian hormone DNA. The chick DNA and protein are significantly longer, due to insertions that abolish nucleotide homology, except in the cDNA coding for the C-terminal, bioactive part of the protein. Nevertheless, the general structure of the gene, sequenced from the transcription initiation to the polyadenylation site, and the main features of the protein are conserved between the chick and mammals. The chick anti-Müllerian hormone gene is expressed at high levels in Sertoli cells of the embryonic testes and in lower amounts in both ovaries, higher levels being reached on the left side after 10 days of incubation.

Ontogeny of reproductive abnormalities induced by deregulation of anti-müllerian hormone expression in transgenic mice.

Anti-müllerian hormone, normally responsible for the regression of müllerian ducts in male fetuses, induces stunting, germ cell loss, and seminiferous tubule formation in ovaries of bovine freemartin fetuses and of transgenic mice, which express the human anti müllerian hormone gene under the control of the metallothionein promoter. Because the latter have been studied only after birth, we undertook a detailed chronological study of their reproductive organs. Müllerian ducts of transgenic female fetuses regressed at the same time as those of normal or transgenic males. Maximal reduction of germ cell number occurred between 16 days postcoitus and birth, when most transgenic oocytes were still in the leptotene stage of the meiotic prophase, whereas normal oocytes had already reached the pachytene phase. Interference with progression of the meiotic prophase and germ cell loss in the fetal ovary are probably responsible for subsequent ovarian regression and retardation of follicle growth. Seminiferous tubule formation was not detectable prior to birth and occurred only rarely in postnatal ovaries. Aromatase activity of fetal transgenic ovaries was decreased, as well as serum concentration of testosterone in adult transgenic males, suggesting that high levels of anti-müllerian hormone may impair Leydig cell steroidogenesis.

Development of the gubernaculum and processus vaginalis in freemartinism: further evidence in support of a specific fetal testis hormone governing male-specific gubernacular development.

BACKGROUND: Freemartinism occurs in some species of ruminants and affects most female bovine fetuses in heterosexual, multiple pregnancies owing to fusion of the chorionic blood circulations soon after implantation. Maldevelopment of the ovaries and Müllerian ducts have been described and recognized as resulting from exposure of their respective primordia to an excess of anti-Müllerian hormone. The present study aimed to analyse the prenatal growth and development of the gubernaculum in freemartins to find out its possible affliction through foetal testis hormones derived from their male co-twin. METHODS: Histological sections of young and drawings and photographs of further developed freemartins and control male and female bovine foetuses were analysed. The specimens had been collected earlier for analysis of the time course of male and female gonadal and genital development and its impairment associated with freemartinism. RESULTS: The gubernaculum of 35-40-day-old male and female fetuses was in the initial stage of development and of similar appearance in all specimens. Gubernacula of 60-70-day-old male fetuses differed from those of females of similar age in various respects: the male gubernaculum size was larger and extension of the processus vaginalis was deeper. Freemartins showed an intermediate development with some individuals resembling male and others resembling female agemates. During further development, gubernacula in males developed into muscular cremaster sacs, whereas those in females generally did not develop beyond the size and structural complexity of 70-day-old foetuses. Beyond day 70 of fetal life, gubernaculum development in freemartins definitely showed male characteristics with respect to size and growth of a processus vaginalis with a cremaster muscular wall. The male-like pattern of the outgrowth of the processus vaginalis changed during the second half of prenatal life. Rather than its further deepening as in males, this structure became inverted to become emerging as a papilla-like structure from the inguinal abdomen bottom. An explanation is proposed for this unprecedented inversion, taking into account: (1) the faster and higher reaching rightsided ascent of the kidneys and gonads, (2) the femalelike outgrowth of the cranial gonadal suspensory ligaments, and (3) the absence of scrotum development. The ovaries and mesonephric remnants in developing freemartins, during their ascent together with the kidneys while remaining attached to the bottom of the developing processus vaginalis sacs via the gubernaculum ligament, are proposed to act together to pull up the bottom of the processus vaginalis sacs. From this action, "inverted hernia sacs" result as the irreversible consequence. CONCLUSION: The data support the concept that foetal testes act, via as an yet unidentified third hormone, to establish malelike development of gubernacula into muscular cremaster sacs. Further work is required to reveal the identity of this hormone. Furthermore, the apparent similarity of the freemartins' inverted processus vaginalis sacs and the fetal rodents' gubernacular cones suggests that the ruminants' and rodents' processus vaginalis are essentially similar structures. Thus there is no longer an urgent need to distinguish between two different types of gubernaculum development and testis descent in rodents and ruminants, respectively, and involving or not fetal gubernacular cones. The present observations may thus contribute to the development of a unified hypothesis for sexually dimorphic development of the gubernaculum throughout the mammalian class.

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 quantitative and interspecific test for biological activity of anti-müllerian hormone: the fetal ovary aromatase assay.

Anti-Müllerian hormone (AMH), also known as Müllerian-inhibiting substance or factor, has previously been shown to sex-reverse the steroidogenic pattern of fetal mammalian ovaries through repression of aromatase biosynthesis. Study of the ontogeny of the response of cyclic AMP-stimulated aromatase activity of rat fetal ovaries to AMH has allowed us to develop a quantitative bioassay for the hormone. Linear responses as a function of the logarithm of AMH concentration were observed over ranges of 0.2-7.5 micrograms/ml for the bovine protein and 0.15-2 micrograms/ml for the human protein, with a maximal decrease in aromatase activity of 90% for both proteins. Under the same in vitro conditions, AMH treatment did not affect cyclic AMP-stimulated fetal rat testicular aromatase activity. Partially purified chick AMH also decreased rat ovarian aromatase activity, allowing us to use this test to study AMH ontogeny in chick gonads. Analysis of the species specificity of AMH repression of ovarian aromatase activity indicated that turtle and rat fetal ovaries responded to AMH of other vertebrate classes, whereas aromatase activity of chick embryo ovaries could be repressed only by the homospecific hormone.

[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 produces endocrine sex reversal of fetal ovaries.

We have previously reported that anti-Müllerian hormone (AMH), also known as Müllerian-inhibiting substance, the testicular glycoprotein involved in regression of the Müllerian ducts of the male fetus, induces the formation of seminiferous cord-like structures in fetal ovaries exposed to it in organ culture. We have now investigated the effect of bovine AMH, purified to homogeneity, on ovarian endocrine differentiation. Ovine fetal ovaries exposed to AMH release testosterone instead of estradiol, an endocrine sex reversal due to suppression of aromatase activity. AMH dramatically decreases the conversion rate of testosterone to estradiol and also decreases total aromatase activity, as measured by the tritiated water technique. AMH acts by decreasing aromatase biosynthesis rather than by blocking enzyme activity, as suggested by the relatively long period of AMH exposure required to produce an effect. In the rabbit fetal ovary, aromatase activity is AMH-responsive during the whole gestational period. The basal steroidogenic activity of rat fetal ovaries is extremely low but can be markedly increased by cAMP. AMH completely blocks the effect of cAMP. Taken together, our results suggest that AMH plays a pivotal role in both morphological and endocrine gonadal sex differentiation.

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.

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.

Anti-müllerian hormone and freemartinism: inhibition of germ cell development and induction of seminiferous cord-like structures in rat fetal ovaries exposed in vitro to purified bovine AMH.

In 13 and 14-day old fetal rat ovaries maintained 3 to 10 days in organ culture, purified bovine anti-Müllerian hormone (AMH) (1.5 to 3 micrograms/ml) induced a characteristic freemartin effect. Gonadal volume and germ cell number were significantly reduced, compared to control ovaries cultured in anhormonal medium, and epithelial cells with large clear cytoplasm linked by interdigitations differentiated in the gonadal blastema. These cells resembling rat fetal Sertoli cells became polarized and formed seminiferous cord-like structures delineated by a basal membrane containing laminin and fibronectin as is the case of testicular seminiferous cords at the first step of their differentiation. These data indicate that AMH is probably the testicular factor responsible for the morphological modifications of bovine freemartin gonads and suggest that this hormone could also be involved in normal morphological differentiation of the testis. In contrast, in fetal rat ovaries, AMH did not trigger the testosterone production which occurs in freemartin gonads at an early stage of the gestation.

Immunocytochemical study of anti-Müllerian hormone in sheep ovarian follicles during fetal and post-natal development.

Anti-Müllerian hormone (AMH) was detected in perinatal and postnatal sheep ovaries, using avidin-biotin immunohistochemistry with a monoclonal antibody specific for ruminant AMH. Immunoreactivity was limited to granulosa cells, and was influenced both by the degree of follicular development, and by the age of the animal. In the fetus, only the most advanced follicles exhibited a faint immunoreactivity at 120 days gestation, and no reaction was observed in younger animals. Immediately before and after birth, primordial follicles were still negative, but a faint reaction was elicited in young growing follicles, increasing with follicle size. Strong immunoreactivity was visible in antral follicles, especially in the innermost granulosa cell layers, close to the oocyte and lining the antral cavity.

Purified bovine AMH induces a characteristic freemartin effect in fetal rat prospective ovaries exposed to it in vitro.

To determine whether anti-Müllerian hormone (AMH) is responsible for the gonadal lesions observed in bovine genetic females united by placental anastomoses to male twins (freemartins), prospective ovaries of fetal rats were exposed to purified bovine AMH in vitro. In cultures initiated at 14 days p.c. and maintained 3 to 10 days, AMH consistently induced a characteristic 'freemartin effect', namely reduction of gonadal volume, germ cell depletion and differentiation, in the gonadal blastema, of epithelial cells with large clear cytoplasm linked by interdigitations, resembling rat fetal Sertoli cells. These cells tend to become polarized and form cords, delineated by a continuous basal membrane containing laminin and fibronectin. Such structures, resembling developing seminiferous cords, were not detected in control ovarian cultures. These data strongly suggest that AMH is the testicular factor responsible for triggering the morphological abnormalities of freemartin gonads.