Births of freemartins derived from embryos reconstructed with ear fibroblasts.

Although the combination of artificial insemination (AI) and embryo transfer (ET) is effective for preventing large offspring syndrome in clone cattle production, it may cause freemartinism. In this study, 51 reconstructed embryos were transferred to artificially inseminated recipients. Of those 9 twin pregnancies, three delivered male and female offsprings. The females had tufts of long coarse hair and short blind pouch at the vaginal end. At necropsy, hypoplastic testicles and epididymis, which connected to the uterus through the spermatic cord, were found and seminal vesicles were also noted. All females had mixed sex chromosome configuration (60, XX and 60, XY). These results suggest that the combined ET program can cause freemartinism, which reduces the efficiency of clone cattle production.

Attempts surgically to induce chorionic fusion between embryos.

Intercornual fistulas were made in the uteri of 19 ewes in an attempt to induce chorionic fusion between fetuses. Ewes were 28 to 40 days pregnant with at least one fetus per horn. Sections were removed from the dorsomedial wall of each uterine horn, and left and right horns were sutured together around the openings to produce the fistulas. In some cases a doughnut-shaped sheet of silastic was inserted between the horns, and in most cases the chorioallantoic membranes of the respective fetuses were sutured to one another prior to suturing the uterine walls. In the 16 animals in which intercornual fistulas were constructed in the area of the intercornual ligament, no chorionic anastomosis was induced. In one of the three animals in which fistulas were produced distal to that ligament, the fistula remained patent 25 days post-surgery, and membranes of two female fetuses were joined, through the opening, by a narrow strand of chorionic tissue. We concluded that this technique was not a promising one for induction of choriovascular anastomosis.

[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.

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.