Reduction of germ cell number induced in vitro by the testis in fetal rat ovary may be caused by factors other than anti-Müllerian hormone

Fetal testes explanted at 16.5 days and cultured with female genital tracts from 13.5-day-old rat fetuses strongly inhibited the Müllerian ducts and reduced the number of ovarian germ cells. Such a reduction was not obtained during cultures with testes from 13.5 days, even though they clearly inhibited Müllerian ducts. When testes from 16.5 days were cultured at distance from the female tracts only the loss of germ cells was observed. These results suggest that testes from 16.5 days produce a diffusible factor distinct from AMH and which reduces the number of germ cells in cultured ovaries.     

Detection of anti-Müllerian activity in boar rete testis fluid

Boar rete testis fluid was tested for its capacity to induce Müllerian regression in 14.5-day-old rat Müllerian ducts. Weak activity was present in crude RTF, but after gel filtration 5-fold concentration, greater activity was detected in 1 our of 7 pools of the eluted fractions. The biologically active fraction (mol. wt 160 000-310 000) coincided with the elution of authentic labelled anti-Müllerian hormone, obtained from bovine fetal testes. These results indicate that a small amount of anti-Müllerian hormone is still synthesized in post-natal life.     

Limitation on the number of germ cells in the fetal ovary of the rat by older ovaries, in vitro

Ovaries from 9-day-old rats co-cultured for 4 days in vitro with ovaries from 13.5-day-old fetal rats prevent the proliferation of the oogonia in the fetal ovaries (germinostatic effect) and produce the anti-Müllerian substance. The hypothesis that the germinostatic and anti-Müllerian activities are not due to the same factor is discussed.     

Cell-specific knockout of steroidogenic factor 1 reveals its essential roles in gonadal function

Knockout (KO) mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a compound endocrine phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To inactivate a conditional SF-1 allele in the gonads, we targeted the expression of Cre recombinase with a knock-in allele of the anti-Müllerian hormone type 2 receptor locus. In testes, Cre was expressed in Leydig cells. The testes of adult gonad-specific SF-1 KO mice remained at the level of the bladder and were markedly hypoplastic, due at least partly to impaired spermatogenesis. Histological abnormalities of the testes were seen from early developmental stages and were associated with markedly decreased Leydig cell expression of two essential components of testosterone biosynthesis, Cyp11a and the steroidogenic acute regulatory protein. In females, the anti-Müllerian hormone type 2 receptor-Cre allele directed Cre expression to granulosa cells. Although wild-type and SF-1 KO ovaries were indistinguishable during embryogenesis and at birth, adult females were sterile and their ovaries lacked corpora lutea and contained hemorrhagic cysts resembling those in estrogen receptor alpha and aromatase KO mice. Collectively, these studies establish definitively that SF-1 expression in the gonads is essential for normal reproductive development and function.     

Influence of age and medium on formation of epithelial cords in the rat fetal ovary in vitro

Fetal ovaries of 14.5-day-old rats were cultured for periods of up to 19 days in control medium or in medium conditioned by the preliminary culture of testes from fetal or young rats. In all ovaries, after 12 days of culture in either medium, epithelial cords were noted having an aspect identical to that of seminiferous cords present in fetal testes explanted at 14.5 days and also cultured for 12 days, i.e. the epithelial cords appeared in ovaries when there was no 'male' or testicular influence. The appearance of histological preparations suggested that the disappearance of the germ cells might bring about a reorganization of the follicular cells in epithelial cords during the differentiation period of the first follicles. With ovaries cultured in conditioned medium, degeneration of the germ cells was more marked, follicles were rare and intra-ovarian cords were greater in number than in ovaries cultured in control medium. The ovaries thus transformed produced the anti-Müllerian hormone (AMH) although they lacked the "germinostatic activity" normally developed by testes of fetal or young rats. This germinostatic activity prevents the multiplication of oogonia when the testes and ovaries are co-cultured in vitro. The transformed ovaries therefore do not have all the functional capacities of fetal testes.     

Functional redundancy of TGF-beta family type I receptors and receptor-Smads in mediating anti-Mullerian hormone-induced Mullerian duct regression in the mouse

Amniotes, regardless of genetic sex, develop two sets of genital ducts: the Wolffian and Müllerian ducts. For normal sexual development to occur, one duct must differentiate into its corresponding organs, and the other must regress. In mammals, the Wolffian duct differentiates into the male reproductive tract, mainly the vasa deferentia, epididymides, and seminal vesicles, whereas the Müllerian duct develops into the four components of the female reproductive tract, the oviducts, uterus, cervix, and upper third of the vagina. In males, the fetal Leydig cells produce testosterone, which stimulates the differentiation of the Wolffian duct, whereas the Sertoli cells of the fetal testes express anti-Müllerian hormone, which activates the regression of the Müllerian duct. Anti-Müllerian hormone is a member of the transforming growth factor-beta (TGF-beta) family of secreted signaling molecules and has been shown to signal through the BMP pathway. It binds to its type II receptor, anti-Müllerian hormone receptor 2 (AMHR2), in the Müllerian duct mesenchyme and through an unknown mechanism(s); the mesenchyme induces the regression of the Müllerian duct mesoepithelium. Using tissue-specific gene inactivation with an Amhr2-Cre allele, we have determined that two TGF-beta type I receptors (Acvr1 and Bmpr1a) and all three BMP receptor-Smads (Smad1, Smad5, and Smad8) function redundantly in transducing the anti-Müllerian hormone signal required for Müllerian duct regression. Loss of these genes in the Müllerian duct mesenchyme results in male infertility due to retention of Müllerian duct derivatives in an otherwise virilized male.     

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.     

Expression of anti-Müllerian hormone (AMH) in equine granulosa-cell tumors and in normal equine ovaries

Anti-Müllerian hormone (AMH), also known as Müllerian inhibiting substance (MIS), is expressed by granulosa cells in females of many mammalian species, and circulating AMH concentrations have been used to monitor granulosa-cell tumors (GCT) in women. The objective was to characterize expression of AMH in equine GCT, and in normal equine ovaries, based upon immunohistochemistry (IHC), using a polyclonal primary antibody directed against human AMH. Equine GCT (n=27) and normal equine ovaries (n=10) were examined by IHC. In addition, sera from four mares with GCT were characterized for AMH bioactivity, based upon suppression of Müllerian duct development in the fetal rat. Immunolabeling with alpha-AMH was localized to granulosa cells in equine GCT, as well as within antral follicles in normal ovaries. Expression of AMH first appeared in granulosa cells of small growing follicles and was most intense in small antral follicles; large antral or atretic follicles had reduced immunolabeling. Omission of the primary antibody or incubation of the primary antibody with the corresponding blocking peptide eliminated immunolabeling of granulosa cells in GCT and in normal antral follicles, confirming the specificity of the immunolabel. Sera from mares with GCT had increased AMH bioactivity compared to control sera. In conclusion, AMH was strongly expressed by granulosa cells in equine GCT and in normal antral follicles. Therefore, anti-Müllerian hormone may be a useful biomarker for detection of GCT in the horse.     

Sexually dimorphic expression of a teleost homologue of Müllerian inhibiting substance during gonadal sex differentiation in Japanese flounder, Paralichthys olivaceus

Müllerian inhibiting substance (MIS), also known as anti-Müllerian hormone, is a glycoprotein belonging to transforming growth factor beta superfamily. In mammals, MIS is responsible for regression of Müllerian ducts, anlagen of the female reproductive ducts, in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fishes, which do not have the Müllerian ducts, has yet to be clarified. To address the role of MIS on gonadal sex differentiation in fishes, we isolated a MIS cDNA from the Japanese flounder testis and examined the expression pattern of MIS mRNA in gonads of both sexes during sex differentiation period. In this study, we present the first demonstration of sexually dimorphic expression of MIS mRNA during sex differentiation in teleost fishes, similarly to amniote vertebrates which possess the Müllerian ducts.     

Absence of anti-Müllerian activity of inhibin in the chick embryo

Inhibin extracted from bovine follicular fluid and administered to chick embryos at a dosage increasing from 0.4 to 30 micrograms per embryo did not induce the regression of the Müllerian ducts of treated females. This result contrasts with that obtained with a testis graft which acts through its anti-Müllerian hormone. Although both hormones were of glycoproteic nature and secreted by the same cells, this study shows no functional analogy between them.     

Abnormal sex-duct development in female moles: the role of anti-Müllerian hormone and testosterone

We have performed a morphological, hormonal and molecular study of the development of the sex ducts in the mole Talpa occidentalis. Females develop bilateral ovotestes with a functional ovarian portion and disgenic testicular tissue. The Müllerian ducts develop normally in females and their regression is very fast in males, suggesting a powerful action of the anti-Müllerian hormone in the mole. RT-PCR demonstrated that the gene governing this hormone begins to be expressed in males coinciding with testis differentiation, and expression continues until shortly after birth. Immunohistochemical studies showed that expression occurs in the Sertoli cells of testes. No expression was detected in females. Wolffian duct development was normal in males and degenerate in prenatal females, but developmental recovery after birth gave rise to the formation of rudimentary epididymides. This event coincides in time with increasing serum testosterone levels and Leydig cell differentiation in the female gonad, thus suggesting that testosterone produced by the ovotestes is responsible for masculinisation of female moles. During postnatal development, serum testosterone concentrations decreased in males but increased in females, thus approaching the levels that adult males and females have during the non-breeding season.     

Marsupial anti-Mullerian hormone gene structure,regulatory elements,and expression

During male sexual development in reptiles, birds, and mammals, anti-Müllerian hormone (AMH) induces the regression of the Müllerian ducts that normally form the primordia of the female reproductive tract. Whereas Müllerian duct regression occurs during fetal development in eutherian mammals, in marsupial mammals this process occurs after birth. To investigate AMH in a marsupial, we isolated an orthologue from the tammar wallaby (Macropus eugenii) and characterized its expression in the testes and ovaries during development. The wallaby AMH gene is highly conserved with the eutherian orthologues that have been studied, particularly within the encoded C-terminal mature domain. The N-terminus of marsupial AMH is divergent and larger than that of eutherian species. It is located on chromosome 3/4, consistent with its autosomal localization in other species. The wallaby 5' regulatory region, like eutherian AMH genes, contains binding sites for SF1, SOX9, and GATA factors but also contains a putative SRY-binding site. AMH expression in the developing testis begins at the time of seminiferous cord formation at 2 days post partum, and Müllerian duct regression begins shortly afterward. In the developing testis, AMH is localized in the cytoplasm of the Sertoli cells but is lost by adulthood. In the developing ovary, there is no detectable AMH expression, but in adults it is produced by the granulosa cells of primary and secondary follicles. It is not detectable in atretic follicles. Collectively, these studies suggest that AMH expression has been conserved during mammalian evolution and is intimately linked to upstream sex determination mechanisms.     

Decrease in the secretion of anti-Müllerian hormone by the chick testis during development

At the end of embryonic life the chick embryonic testis possesses a low anti-Müllerian activity, as evidenced by the grafting method to female hosts. The percentage of grafted embryos presenting a Müllerian duct regression is not increased by administration of an anti-estrogenic drug (tamoxifen). This observation does not favour the hypothesis according to which the low percentage of regression could be due to a protection of Müllerian ducts by estrogens from the host ovary. It shows rather that the anti-Müllerian hormone secretion actually decreases during development.     

Somatic cell mapping of the genes for anti-müllerian hormone and osteonectin in cattle: identification of a new bovine syntenic group

DNA probes from the bovine anti-Müllerian hormone and osteonectin genes were hybridized onto Southern blots containing DNAs from cow-hamster and cow-mouse hybrid somatic cell lines segregating bovine chromosomes. Bovine anti-Müllerian hormone and osteonectin loci were fully concordant with each other in 96 hybrid somatic cell lines, but were not concordant with any other bovine syntenic group described to date. As such, these two genes represent another syntenic group in cattle, bringing to 27 the number of autosomal syntenic groups identified thus far.     

Characterization of bovine fetal Leydig cells by KIT expression

The origin of fetal Leydig cells (FLC) and whether they share a common lineage with adult Leydig cells (ALC) is still under debate, and a marker to reliably track and isolate fetal Leydig precursor cells remains to be identified. We analyzed KIT positive (KIT+) cells in gonads from bovine fetuses with crown-rump-length (CRL) 2.5–85 cm by immunohistochemistry, and found that KIT expression was gender-specific. In female gonads, expression was mainly associated with epithelial cell cords, which extended from the surface epithelium towards the KIT-negative inner stroma. In male gonads of fetuses, after CRL 2.9 cm, KIT expression was strikingly strong in interstitial cells (IC). Only a few KIT+ cells were detected in the epithelial cell cords and in the stromal layer under the surface epithelium after CRL 3.5 cm. In the male fetuses, KIT expression in IC was a continuous and characteristic feature until full term. At all developmental stages KIT+ areas alternated with anti-Müllerian hormone-positive areas. Platelet-derived growth factor receptor α production was initiated after the expression of KIT at CRL 4.5 cm. Detection of cytochrome P450 side chain cleavage enzyme and steroidogenic acute regulatory protein in KIT+ IC identified them as FLC. KIT+ cells, isolated from testes by magnetic-activated cell sorting, retained their steroidogenic capacity in vitro. Together, these findings show that KIT+ IC of fetal testis correspond to FLC, which can be successfully cultivated for advanced studies.     

Biochemical analysis of bovine testicular anti-Müllerian hormone

Direct biochemical analysis has been applied to bovine testicular anti-Müllerian hormone (AMH), purified from incubation medium of bovine fetal testes by immunochromatography on a monoclonal antibody. The hormone contains a high proportion of hydrophobic amino acids and 13.5% carbohydrate. The oligosaccharide composition suggests that both N- and O-glycosidically linked chains are present. The molecular extinction coefficient is 3.27 +/- 0.06. One RIA unit, defined as the amount of hormone released by 1 g fetal bovine testicular tissue incubated during 4 h, corresponds to 3.06 +/- 0.17 microgram protein.