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.

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.

Effect of estradiol-17 beta on peripheral plasma concentration of 15-keto-13,14-dihydro PGF2 alpha and luteolysis in cyclic cattle.

Friesian heifers (n = 10) were assigned randomly to receive an intravenous injection of estradiol-17 beta (E2; 3 mg) or saline:ethanol vehicle solution (6 ml; 1:1) on day 13 of the estrous cycle. Blood was collected from the jugular vein by venipuncture into heparinized vacutainer tubes at 30 minute intervals for 2 hours (h) preinjection, 10.5 h postinjection and then at 3 h intervals until estrus. Repeated hormone measurements of 15-keto-13,14-dihydro-PGF2 alpha (PGFM) and progesterone (P4) were evaluated by split-plot analysis of variance. Mean concentration of PGFM for the 12.5 h acute sampling phase was 164.1 +/- .14 pg/ml. A treatment by time interaction was detected (P less than .01). After treatment with E2, PGFM concentrations began to increase at approximately 3.5 h, reached a mean peak of 330.4 +/- 44.5 pg/ml (n = 5) at 5.5 +/- .3 h, and returned to basal concentration by 9.0 +/- .6 h. Vehicle treatment did not alter concentrations of PGFM. Injection of E2 on day 13 of the estrous cycle caused luteolysis (P4 concentration less than 1 ng/ml) to occur earlier following injection (96.9 +/- 10.6 h less than 153.6 +/- 17.7 h; P less than 0.05) than did the vehicle control treatment. During the chronic sampling phase of 3 h intervals, 39 of 606 samples (6.4%) were classified as PGFM spikes (323.0 +/- 50.0 pg/ml); 21 (53%) of the spikes occurred at a mean interval of 18.9 +/- 3.86 h before the time of completed luteolysis. Exogenous E2 induced an acute increase in PGFM that may be indicative of uterine PGF2 alpha production. Peaks of PGFM in plasma were temporally associated with luteolysis on a within cow basis.

[Ultrastructural changes in female germ cells during the meiotic prophase in the rat: a special study of membranes after cryofracture]. / Modifications ultrastructurales des cellules germinales femelles au cours de la prophase méiotique chez le rat: étude particulière des membranes après cryofracture.

Ultrastructural changes in the nuclear and cytoplasmic elements in the germ cells of female rats were followed before meiotic prophase (15.50 days post-coïtum and 17.25 days post-coïtum) and during it (17.75 days post-coïtum to birth). We observed: modifications in the nuclear envelope which was thick during the oogonial stage, becoming thinner when the chromosomes entered preleptotene stage. The thinning of the envelope was due to the disappearance of the chromatin material lining it; variations in the number and distribution of germ cell nuclear pores according to stage; the pores were first scattered in small clusters of 6 to 8 over the entire nuclear membrane. From the preleptotene to zygotene stage, these clusters enriched in pores to form large areas. Finally, in the pachytene and diplotene stages, clusters of more than 100 pores were seen; nucleolar fragmentation from the preleptotene stage, followed by the formation of a new active nucleole in the diplotene; polarization of the mitochondria in the oldest oogonia just before the beginning of meiotic prophase. This polarization disappeared after the onset of the meiotic processes, then appeared again near the developing Golgi apparatus at the end of the pachytene stage; the formation of large gap junctions and numerous bands of tight junctions between the somatic cells; these formations contrasted with small gap junctions, and the tight junctions became scarce just before the meiotic process began. These observations, as well as those concerning nuclear pore distribution were made using the cryofracture technique.

[Female germ cell development during the meiotic prophase in the rat: criteria for cytoplasmic and nuclear identification of the different stages on thin histological slides]. / Evolution des cellules germinales femelles au cours de la prophase méiotique chez le rat: critères de reconnaissances cytoplasmique et nucléaire des différents stades en histologie fine.

The different stages of meiotic prophase in germ cells of the rat ovary were studied cytologically at definite times when there was a dominant nuclear stage. Each stage was identified in 1 mu sections. The usual references for the definition of meiotic nuclear stages according to chromosomal structures wee used and have been described in more detail. It has also been shown that cytological observations such as the organization and distribution of cytoplasmic organelles (mitochondria, Golgi apparatus) equally contributes to the identification of the stages of meiotic prophase.

Sister-chromatid exchanges and cell kinetics in human and rabbit lymphocytes exposed in vivo and in vitro to 2-bromo-alpha-ergocryptine.

The possible mutagenic and DNA-synthesis inhibitory effects of 2-bromo-alpha-ergocryptine, a new semi-synthetic ergot alkaloid, was studied in human and rabbit lymphocytes exposed to it in vivo and in vitro. The analysis of SCE was mainly used to evaluate potential mutagenicity, and the mitotic and DNA-synthesis inhibition was explored by examining the proportions of first-, second- and third-division metaphases in the corresponding lymphocyte cultures. The results obtained show that 2-bromo-alpha-ergocryptine does not induce SCE in the cell systems tested, or structural chromosome aberrations in human lymphocytes in vivo. On the other hand, a marked mitotic inhibitory effect and associated cell kinetic changes could be clearly attributed to the drug, probably related to its cytotoxicity.

Influence of dietary intake and weight variation on LH release after a gonadotrophin-releasing hormone (GnRH) injection during the post-partum period of the nursing cow.

The experiment was conducted to measure the effects of restricting dietary intake on LH release following a GnRH injection during the post-partum period in nursing cows. Eighteen multiparous Charolais cows were fed a low ration from 45 days pre-partum to 45 days post-partum, and received an intravenous injection of synthetic GnRH (55 micrograms) at 5, 15 and 30 days post-partum. Plasma LH concentration was measured during the 4 hrs following each injection. The cows were separated into 2 groups depending on whether their mean daily gain was negative (group 1) or positive (group 2). The peak plasma LH concentration and the total release of LH after a GnRH injection tended to increase during the post-partum period irrespective of the group. The maximum LH value and the total LH release following GnRH were higher in Group 1 than in Group 2 (p less than 0.05), at 5, 15 and 30 days post-partum. Furthermore, the maximal plasma LH concentrations recorded after GnRH were highly correlated with the mean daily weight gain post-partum (day 15, r = -0.50; day 30, r = -0.54).

Interaction between two luteal cell types from the corpus luteum of the sow in progesterone synthesis in vitro.

Populations of two types of luteal cell (large and small) were prepared from CL of sows at 60 days of gestation. When the two types were recombined and incubated for 2 h, the amount of progesterone released into the medium was almost twice the sum of that released by each cell type alone. When the cells were superfused in vitro, in an "in series" arrangement such that the superfusate from one cell type then passed through the chamber containing the second cell type, the small cells were responsible for increasing the production of progesterone by the large cells. When cell population were superfused with media containing inhibitors of progesterone synthesis, trilostane completely blocked progesterone release but aminoglutethimide only reduced progesterone output by half, even when the concentration of inhibitor was increased. Addition of pregnenolone to the superfusion medium increased progesterone production by both cell types approximately 3-fold, whereas addition of cholesterol only increased progesterone production by the large cells.

Hormonal and follicular relationships in ewes of high and low ovulation rates.

Total follicular populations and peripheral plasma concentrations of LH, FSH, prolactin, oestradiol-17 beta and progesterone during the preceding cycle were studied in two breeds of sheep (Romanov and Ile-de-France) which differed widely in their ovulation rates (3.2 and 1.5 respectively). No LH parameters could be correlated with the follicular details measured. The second peak of FSH occurring 20-30 h after the preovulatory surge of LH was significantly larger in the Romanov ewes and the area under this peak was correlated (P less than 0.01) with the number of antral follicles present in the ovary 17 days later. This suggests that formation of the antrum during the follicular growth phase is under the control of FSH. The discharge of prolactin preceding the LH peak, although not significantly different between breeds, was correlated with several of the follicular classes measured, including the number of preantral follicles. The peak value of oestradiol-17 beta measured before the LH peak was significantly higher (P less than 0.05) in the Romanov ewes and was correlated with the number of the largest follicles present. There was no significant difference between breeds in the concentration of oestradiol at the onset of oestrus. The progesterone concentration during the luteal phase was highly correlated with the number of preovulatory follicles.

The effect of restricted food intake and refeeding on the ovarian follicle population of the pre-puberal Wistar rat.

A 50 p. 100 restriction of food intake in female Wistar rats from day 21 to day 42 of life prevented ovulation and altered the size distribution and numbers of ovarian follicles. The rate of atresia of non-growing oocytes in primordial follicles was retarded resulting in more oocytes per ovary. The number of follicles initiated to grow was reduced. Semi-starved rats allowed free access to food from day 42 of life achieved the body weight of ad libitum-fed controls at 66 days of age. A delayed puberty occurred. The numbers of non-growing oocytes in primordial follicles per ovary declined but remained significantly greater than control values at 66 days of age. Refeeding increased the numbers of follicles undergoing growth but within the size distribution found in age-matched controls. Thus semi-starvation followed by refeeding rendered the ovary developmentally younger but only in terms of total oocyte numbers. The ovarian response to starvation and refeeding is discussed in relation to pituitary function and provides new information on the potential lability of the oocyte population.

A study of the population of primordial and small follicles in the sheep.

The population of primordial and small follicles in adult ewes of high (3.1) and low (1.6) ovulation rate was studied by histological methods. The small follicle population which has a skewed frequency distribution in relation to either follicle or oocyte size can be divided according to morphological features into dormant, transitory and growth phases. It is suggested that initiation of follicle growth involves firstly the passage of follicles from the dormant to the transitory category and (if it occurs) is independent of gonadotrophins; and secondly, the passage from the transitory category to the growth phase. The second step is dependent on gonadotrophins and the rate of such passage is higher in ewes with a high ovulation rate. The overall mean follicle size of small follicles is larger in ewes with a low ovulation rate, suggesting that either they have structurally different small follicles or their reserve of small follicles is less depleted than in ewes with a high ovulation rate.

[Influence of undernutrition in the lactating cow on ovulation induction by serum gonadotropins (PMSG) during the post-partum period]. / Influence d'une sous-alimentation de la vache allaitante sur l'induction de l'ovulation par l'hormone gonadotrope sérique (PMSG) au cours du post-partum.

Two experiments were carried out to assess the influence of undernutrition on postpartum ovulation in nursing Charolais cows after PMSG injection. In each experiment, the nursing cows were divided into 2 groups: one fed at a low nutritional level and the other at a normal nutritional level. In the first experiment, 19 animals were injected on post-partum days 15 and 30 with 600 IU of PMSG; in the second experiment, 34 received the same injection on post-partum day 54 after 9 days of priming with a Norgestomet implant. On post-partum day 15, only one cow in each group ovulated. At post-partum day 30, 1 out of 8 cows at the low nutritional plane ovulated vs 5 out of 9 at the normal nutritional plane (P less than 0.05). Likewise, on post-partum day 54, 5 out of 14 cows at the low nutritional plane ovulated vs 17 out of 17 at the normal nutritional level (P less than 0.05). Therefore, there is a time during the post-partum period when the nursing cow ovary does not respond to PMSG by ovulation. The length of this time is increased by undernutrition.

LH receptors in ovine corpora lutea in relation to various physiological states and effects PGF-2 alpha on LH-induced steroidogenesis in vitro.

The LH binding properties (determined using tritiated methylated LH) and the in-vitro steroidogenic activity of CL from ewes in the oestrous cycle or early pregnancy (Day 18) were compared. No significant alteration in the Kd values was observed. However, the number of sites was maximal at Day 10 of the cycle and in early pregnant animals which had not been pregnant for at least 3 months (dry ewes). Non-lactating or suckling ewes had half the numbers of binding sites. The increase of the number of receptor sites was accompanied by a steroidogenic response at lower LH concentration. During incubation or superfusion for 5 h, a refractoriness to LH stimulation appeared after 1 h with high LH concentrations and after 3 h with low concentrations. The opposite effect of the addition of indomethacin or PGF-2 alpha suggests the intervention of PGs in this phenomenon.

Frequency and types of induced and spontaneous chromosome aberrations in relation to cell kinetics.

Induced and spontaneous structural chromosome aberrations (SCA) were studied in a child accidentally radiated with a high dose of 192Ir, and in three sibs with Fanconi's anemia, analyzing by separate first division metaphases (FDM) and second division metaphases (SDM). The results showed that the number of SCA, number of cells with aberrations, and SCA per cell were markedly higher in FDM in all patients. Furthermore, for some type of structural changes like dicentric chromosomes and chromatid interchanges, the differences were particularly striking. The importance of ascertaining FDM identified with proper techniques, for the study of the clastogenic effect of environmental agents and some aspects related to the differences in cytogenetic features found in diverse tissues in Fanconi's anemia are discussed.

Influences of season, cycle and breed on follicular growth rates in sheep.

Follicular growth was studied in 16 ewes of different breeds (Romanov, mean ovulation rate 3.0, and Ile-de-France, mean ovulation rate 1.6), stage of cycle (Day 0 or 7) and season (December and June). The follicular growth rates, determined by measuring the mitotic index before and 2 h after colchicine treatment, varied greatly between animals studied and did not vary significantly between breeds, time of cycle or season. From 3 layers of granulosa cells until antrum formation the mitotic index increased slowly but then the follicles grew rapidly reaching maximum growth rate at a follicular diameter of 0.85 mm; thereafter the mitotic index decreased almost to zero in preovulatory follicles. The mean time for a follicle to pass from 3 layers of granulosa cells (200 cells) to preovulatory size (3 x 10(6) cells) was estimated to be about 6 months. The total number of normal follicles with > 3 layers of granulosa cells in Ile-de-France ewes was similar in the anoestrous (3 ovaries studied) and breeding (3 ovaries) seasons, but there were more antral follicles in the latter. Highly significant correlations existed in each ewe between the number of follicles and the mean mitotic index per class, suggesting the existence of an intraovarian mechanism regulating folliculogenesis.

Short- and long-term effects of hypophysectomy and unilateral ovariectomy on ovarian follicular populations in sheep.

The effects of hypophysectomy and unilateral ovariectomy on the total number of follicles with greater than 3 layers of granulosa cells were determined at 4 and 70 days following treatment. The population of preantral follicles (less than 0.23 min diam.) was found to be under the control of gonadotrophins but such control was only evident on a long-term basis. At 70 days after unilateral ovariectomy there was a large increase in the number of preantral follicles but at 70 days after hypophysectomy there was a large decrease. The population of antral follicles (greater than 0.23 mm diam.) was under the immediate control of gonadotrophins. By 4 days after hypophysectomy all large antral follicles had become atretic and the number of antral follicles was further decreased at 70 days after treatment. At 70 days after unilateral ovariectomy there was an increase in the number of antral follicles. The follicular growth rates at 70 days following treatment were decreased in hypophysectomized ewes but increased in ewes after unilateral ovariectomy.

Total follicular populations in ewes of high and low ovulation rates.

The total ovarian follicular populations were studied in two breeds of ewes which differed greatly in their ovulation rates. Thus 8 Romanov (mean ovulation rate 3.1) and 12 Ile-de-France ewes (mean ovulation rate 1.4) were ovariectomized at oestrus during the breeding season. Each right ovary and 3 left ovaries were sectioned at 7 micron and examined microscopically. The number of small follicles, i.e. with 2 or less layers of granulosa cells, was estimated by a tested sampling procedure whilst all larger follicles were measured and arranged into classes. There were half as many small follicles but 1.5--2 times more large follicles in the ovaries of the Romanov ewes compared to those of the Ile-de-France ewes. The number of atretic follicles was approximately the same in both breeds and does not explain the difference observed in ovulation rate. It is concluded that the higher ovulation rate in the Romanov ewe is due to the greater number of large follicles available to be stimulated for ovulation.