Equine oocyte competence after in vitro maturation (IVM) was investigated in terms of the diameter of the follicle of origin and the stage of the estrous cycle, with three criteria of maturation: nuclear stage after DNA Hoechst staining, meiotic spindle morphology after tubulin immunocytochemical staining, and cortical granule localization after lectin labeling. Seven successive in vivo ultrasound-guided follicular punctures were performed on 10 cyclic saddle mares, alternatively at the end of the follicular phase (after induction of ovulation with a gonadotropin injection) and in midluteal phase (with or without a gonadotropin injection). Expanded cumulus-oocyte complexes (COCs) were stained at collection, and compact COCs were stained after in vitro culture. They were observed under a confocal microscope. Successive punctures on one mare provided 0.9 preovulatory COCs and 8 immature COCs per 22 days. Among the preovulatory oocytes, 55% had completed nuclear and cytoplasmic maturation, 86% of which displayed a normal meiotic spindle. Of the 262 oocytes cultured in vitro, 37% completed nuclear maturation. The nuclear and cytoplasmic maturation rate significantly increased with follicle diameter. The IVM rate tended to be higher in follicular phase and tended to increase in luteal phase with the gonadotropin injection. The meiotic spindle morphology was not significantly different between the classes of follicular diameters. This study provided the opportunity to increase the number of characterized oocytes collected per cycle and per mare. This is the first report showing the progressive acquisition of meiotic competence in the equine oocyte during antral follicle growth and is the only description of the equine meiotic spindle.

We have previously reported that antiMullerian hormone (AMH), also known as Mfillerianinhibiting substance, the testicular glycoprotein involved in regression of the Mullerian ducts of the mnale 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. New Zealand rabbits were sacrificed between day 16 and 28 of gestation. Prior to day 20, fetal sex was determined by a sex chromatin test performed on a fragment of the amniotic membrane (12); in older fetuses, sex was easily identified under the dissecting microscope. Wistar rat fetuses were used at 15 days postcoitum; sex was recognized macroscopically. Fetal age was counted from the day of mating, which was considered day 0.Reagents. Bovine AMH was purified by immunochromatography on a monoclonal antibody-conjugated matrix (3) and quantified by radioimmunoassay (13). Other unlabeled reagents were purchased from Sigma; [7-3H]testosterone (24.5 Ci/mmol; 1 Ci = 37 GBq) and [1,3,213-3H] Leshin et al. (14) to a specific activity of 23.2 Ci/mmol.Organ Culture. Fetal gonads were explanted in organ culture (7); they were separated from the mesonephros except in 29-day-old ovine fetal gonads, half of which were cultured together with the mesonephros. No difference was seen between gonads explanted with or without mesonephros, and the results were pooled. Culture medium was CMRL 1066 containing bovine serum albumin at 0.5 mg/ml and, for rat fetal gonads, 0.1 mM 3-isobutyl-1-methylxanthine; 1 mM N6,02'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2-cAMP) was added to culture medium in some cases. One gonad was placed in control medium, and the other one was placed in medium containing purified bovine AMH (2.25-3 Abbreviations: AMH, anti-Mullerian hormone; Bt2cAMP, N6,02'-dibutyryladenosine 3',5'-cyclic monophosphate. 3684The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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.

Le comportement d'oestrus cyclique et l'activité ovarienne ont été étudiés chez les mêmes brebis de races Ile-de-F y ance et Préalpes pendant deux années consécutives. Il existe des différences raciales dans la durée de l'anoestrus saisonnier. Les brebis de race Préalpes ont un anoestrus de 114 jours, et celles de race Ile-de-France, de 179 jours. Des différences individuelles ont été mises en évidence. Ainsi, certaines brebis de race Préalpes n'ont pas d'anoestrus, d'autres au contraire ont un anoestrus très long. Toutes les brebis de race Ile-de-France ont une période d'anoéstrus mais la durée de celle-ci peut varier du simple au double. Une reprise de l'activité ovarienne, contrôlée par caelioscopie, est observée chez les deux races fin avril-début mai. Dans les 2 races, l'activité ovarienne reprend au mois de juin, en jours longs croissants, contrairement à ce que les études antérieures sur le photopériodisme suggéraient. Chez les brebis de race Ile-de-Prance, pendant la période d'ancestrus, les teneurs hypophysaires en FSH et LH sont 2 fois plus faibles que celles trouvées dans les hypophyses pendant la saison sexuelle.