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Multiple pregnancies were obtained in cows treated with gonadotrophic hormones and the fetuses were removed 39 to 62 days after mating. This covers the period of sexual organogenesis in males from the initial differentiation of the testes to the final establishment of most male structures. Twenty-nine pregnancies resulting in heterosexual fetuses with fused chorions and vascular anastomoses provided forty-eight presumptive freemartins. The genital tract was studied histologically and gonadal size was determined by the paper weight method.

No difference was observed between control females and presumptive freemartins up to 48 days. Thereafter, growth of the presumptive ovaries practically stopped in freemartins and the diameter of the upper part of their Müllerian ducts decreased, as in males. Contrary to older views, the first freemartin effects occurred at the same stage (49 to 52 days) in all the presumptive freemartins.

Ovarian and Müllerian inhibition begins simultaneously and a significant correlation between the degree of inhibition of both structures was obtained. The penis, scrotum and prostate were absent in all freemartins until Day 60. In only one case was the ano-genital distance slightly increased. A delayed appearance of seminal vesicles was found in three out of seven freemartins at 61 and 62 days.

There was a notable absence of seminiferous tubules in the gonads of all these freemartins. It is suggested that if these tubules are present after birth it is because they have developed later.

The factors responsible for the freemartin effect still remain to be discovered. Ovarian and Müllerian inhibitions occur during the period of time when the external genitalia of males become masculinized. This suggests that a factor of male origin which inhibits both the ovaries and the Müllerian ducts and which is distinct from the masculinizing hormone could be involved. Other possibilities are also discussed.

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L. Lyet, B. Vigier, and P. van der Schoot

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.

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B. Vigier, D. Tran, L. Legeai, Jacqueline Bézard, and Nathalie Josso

Summary. The origin of AMH responsible for Müllerian duct regression in bovine freemartins has been reinvestigated, using a sensitive RIA for this hormone. Between 50 and 80 days, Müllerian duct regression occurs simultaneously in males and freemartins. Both twins exhibited high and positively correlated serum AMH concentrations, whereas gonadal in-vitro production of AMH and biological anti-Müllerian activity were detectable at a low level only in 2 out of 13 freemartins. In the gonads of approximately half the freemartins after 80 days, seminiferous tubules differentiated and the gonads produced AMH, but the output was very low compared to that of the male twin. These data suggest that regression of Müllerian duct in freemartins is essentially mediated by AMH produced by the testes of the male twin.

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J. Bézard, B. Vigier, D. Tran, P. Mauléon, and Nathalie Josso

Summary. 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.

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B. Vigier, Dien Tran, F. du Mesnil du Buisson, Y. Heyman, and Nathalie Josso

Summary. Monoclonal antibodies against bovine anti-Müllerian hormone (AMH) were used to study the hormone in cattle. Anti-Müllerian activity of testicular tissue, immunoreactive testicular AMH, serum AMH concentration and AMH production by incubated testicular tissue were detectable from 42 days, i.e. at the time of seminiferous tubule differentiation, and peaked between 50 and 80 days, when the Müllerian ducts regress in the male fetus. All the values stabilized at a lower level until 30 days after birth and then slowly decreased. At 18 months, only traces of AMH immunoreactivity were detectable in testicular tissue and serum concentration and AMH production by incubated testicular tissue were negligible; the main source of AMH in the adult animal was the rete testis fluid. Study of the disappearance rate of AMH from the serum of castrated calves gave a half-life of approximately 2 days for bovine AMH.