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M. A. Driancourt, R. Webb and R. C. Fry

Summary. The process by which a single follicle is selected to ovulate while others regress is unknown in ewes. If the dominant follicle secretes substances that directly inhibit the growth of other follicles, the superovulatory response to the administration of exogenous gonadotrophins may be blunted. Administration of 1250 iu pregnant mares' serum gonadotrophin (PMSG) before or after the emergence of the dominant follicle in the follicular phase, or 1000 iu PMSG in the presence or absence of a large healthy or atretic follicle during the luteal phase did not affect the induced ovulatory response. Comparisons between the ovary with or without the dominant follicle did not reveal any differences in ovulatory response to PMSG. The in-vitro features (i.e. mitotic index, oestradiol and testosterone production) of follicles ipsilateral or contralateral to the dominant follicle during the early and late follicular phases were also similar.

If the dominant follicle secretes substances detrimental to the other follicles, this could be mimicked in vitro. Co-culture of small follicles with the largest follicles in a closed system did not reduce their incorporation of 3H thymidine in granulosa cells, compared with small follicles cultured alone.

These data suggest that dominance is probably not operative in sheep. The administration of 500 iu of PMSG during the midfollicular phase increased ovulation rate in Merino ewes, indicating that dominance is essentially passive in ewes and can easily be overcome by raising gonadotrophin concentration.

Keywords: follicle; ovulation; gonadotrophin; paracrine regulation; sheep

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M. A. Driancourt, P. Guet, K. Reynaud, A. Chadli and M. G. Catelli

In cattle, it has been suggested that follicular fluid has direct modulatory effects on follicular growth and maturation. In the first part of this study, an in vitro test using aromatase activity of follicular wall fragments as an end point was validated for cattle follicles and was used to test whether follicular fluid (from dominant or non-dominant follicles) modulates aromatase activity. Fluid from dominant follicles at a concentration of 24 or 12% (obtained during the luteal and follicular phases, respectively) significantly inhibited aromatase activity. Inhibitory activity was low or absent in fluid from non-dominant follicles. FSH-stimulated aromatase activity was also reduced by fluid from dominant follicles, but not to a greater extent than in basal conditions. Finally, charcoal-treated fluid from dominant follicles retained its inhibitory activity. In contrast, ovarian venous serum draining a dominant follicle had no activity at the three concentrations tested (6, 12 and 24%). In the second part of the study, identification of the compounds involved in this modulatory activity was attempted using SDS-PAGE. Comparison of the fluorographs from de novo synthesized proteins stored in follicular fluid (inhibitory medium) with those secreted in incubation medium (inactive medium) demonstrated that one protein (90 kDa, pI 5.8) was significantly (P < 0.05) more abundant in fluid from dominant follicles (2.0 ± 0.09%) than in the culture medium (1.3 ± 0.1% of the total proteins). This protein had characteristics similar to those of heat shock protein 90 (hsp 90). Therefore, in the final part of the study, the presence of hsp 90 in ovarian cells and follicular fluid was investigated using immunohistochemistry and western blot analysis. After immunohistochemistry, a positive signal was detected mainly in the granulosa cells of larger follicles and to a smaller extent in thecal cells and oocytes. Western blot analysis also demonstrated the presence of hsp 90 in follicular wall fragments and fluid. When blotting was achieved on a sample of follicular fluid resolved by two-dimensional PAGE, the spot detected had a similar location to that at 90 kDa and pI 5.8. Addition of purified hsp to bovine follicles in vitro depressed aroaromatase altering the ltalue (and Kmossibly the poss value) oVmaxe enzyme. It is proposed that rop 90 is a functional regulator of follicular maturation through its action on aromatase.

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M. A. Driancourt, P. Philipon, A. Locatelli, E. Jacques and R. Webb

Summary. Despite differences in FSH concentrations ranging from 1·5 ng/ml (Romanov ewes) to 4 ng/ml (Ile-de-France ewes) between the follicular and luteal phases, follicular growth (numbers of follicles growing, growth rates, maximum size reached) was morphologically similar between the two stages of the cycle. Injection of 750 i.u. hCG at Day 6 or 16 of the cycle triggered ovulation of 4·1 ± 0·7 and 4·0 ± 1·3 follicles in Romanov and 2·2 ± 0·5 and 1·7 ± 0·5 follicles in Ile-de-France ewes, respectively, demonstrating that functional differentiation was similar between the two stages of the cycle. As gonadotrophin environment differs between these two stages of the cycle, this suggests that there is a wide flexibility in the amount of gonadotrophins required to trigger terminal follicular growth and that ovarian requirements for gonadotrophins might work through thresholds. When Romanov and Ile-de-France ewes were given similar amounts of exogenous gonadotrophins (1250 i.u. PMSG, 750 i.u. hCG) after hypophysectomy, ovulation rates were close to the usual values (Romanov, 5·5 ± 3·9; Ile-de-France, 1·4 ± 0·5), demonstrating that differences in gonadotrophin concentrations during the follicular phase do not play a major role in the high ovulation of the Romanov compared to the Ile-de-France ewes.

Keywords: FSH; ovulation rate; follicular growth; prolific ewes; stage of the cycle

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M. A. Driancourt, H. Quesnel, G. Meduri, A. Prunier and D. Hermier

This experiment was conducted to determine why follicles luteinize faster in the Meishan breed than in the Large White breed of pig. Follicles were recovered during the late follicular phase from ovaries of both breeds before and after administration of hCG given to mimic the LH surge. First, the patterns of cholesterol transporters (high and low density lipoproteins: HDL and LDL) were compared. Cholesterol transporters detected in follicular fluid consisted of HDL only. Similar amounts of Apolipoprotein A-I were found in all samples. There was no obvious breed effect on minor lipoproteins found in the HDL-rich fraction, and this pattern was altered similarly by hCG in the two breeds. The LDL-rich samples of serum from both breeds contained similar amounts of protein. Second, three steroidogenic enzymes, adrenodoxin, 17α-hydroxylase–lyase (P45017α) and 3β-hydroxysteroid-dehydrogenase (3β-HSD) were detected by immunohistochemistry and quantified by image analysis on sections of the two largest follicles. Before hCG treatment, theca interna cells demonstrated immunoreactivities for adrenodoxin (strong), P45017α and 3β-HSD (very strong), whereas granulosa cells displayed immunoreactivities for adrenodoxin only. After hCG treatment, the localization of the enzymes was unchanged but the staining intensity of adrenodoxin on granulosa cells and 3β-HSD on theca cells increased (P < 0.01 and P < 0.05, respectively). Breed effects were detected for the amounts of adrenoxin in theca cells (Meishan > Large White; P < 0.05) and of 17α-hydroxylase (Large White > Meishan, P < 0.01). Breed × treatment interactions were never detected. Finally, gelatinases, plasminogen activator, plasminogen activator inhibitor, tissue inhibitors of metalloproteases (TIMP-1 and TIMP-2) were visualized by direct or reverse zymography or western blotting. Whatever the stage relative to LH administration, follicular fluid from Large White gilts contained more TIMP-1, and TIMP-2 (P < 0.02 and P < 0.01, respectively). No breed effect was detected for the amounts of gelatinases and plasminogen activator inhibitor 1. However, for these parameters, a significant breed × time interaction was obvious, as the Meishan follicles had a greater response to hCG (P < 0.01). Since proteolysis plays a key role in the bioavailability of growth factors such as insulin-like growth factor 1, fibroblast growth factor and transforming growth factor β, which have the ability to alter gonadotrophin-induced progesterone production in pigs, the differences observed in its control in the present study may explain, at least in part, the different patterns of luteinization observed in Meishan and Large White follicles.

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L. P. Cahill, M. A. Driancourt, W. A. Chamley and J. K. Findlay

Summary. Manipulation of circulating concentrations of hormones and ovarian follicle status was carried out on Day 11–12 of the oestrous cycle in sheep. All follicles visible on the ovary were ablated by cautery and ewes were treated with oestradiol or ovine follicular fluid (oFF) to suppress FSH or with PMSG to increase circulating gonadotrophic activity. One group underwent unilateral ovariectomy which greatly increased endogenous FSH and was the only treatment which significantly affected LH pulse frequency. The size distribution of antral follicles, the extent of atresia and the mitotic index of granulosa cells of follicles on Day 15 showed that (a) treatment with oFF inhibited the growth of follicles beyond 2 mm diameter by suppressing the mitotic index of the granulosa cells and (b) the concentration of FSH in peripheral plasma was related to the ability of small antral follicles to grow during the late luteal—early follicular phase of the cycle.

Subsequently, it was demonstrated that oFF inhibits, in a dose-dependent manner, folliculogenesis sustained by PMSG in ewes on Days 12–15. Inhibition of folliculogenesis was represented by a decrease in those follicles > 4 mm, an increase in the relative proportion of follicles < 2 mm, and minimal change in the average number of follicles visible on the ovarian surface, and a decrease in the mitotic index of granulosa cells of follicles < 2 mm. There was no change in the extent of atresia. This supports the concept of a mitotic inhibitor present in charcoal-treated oFF, which can act at the ovarian level to prevent follicles passing beyond 2 mm diameter, even in the presence of excess exogenous gonadotrophin. Such a factor could be involved in selection of the follicle(s) destined to ovulate.

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K. H. Al-Gubory, M-A. Driancourt, M. Antoine, J. Martal and N. Neimer

Porcine and ovine follicular tissues were used to investigate, in vitro, the effect of charcoal-treated aqueous extract from ovine corpora lutea of pregnancy on aromatase activity as determined by the conversion of [3H]testosterone to oestradiol by follicular walls and measurement of 3H2O release. Extract (500 μg protein) prepared from corpora lutea of day 112 of pregnancy but not extract (500 μg) prepared from ovine fetal cotyledonary tissue obtained at a similar time significantly decreased (P < 0.02) aromatase activity of pig follicles in the absence of FSH. These results demonstrate that a non-steroidal factor in the corpora lutea of late pregnancy directly inhibits aromatase activity. When the effects of different doses (300, 600 or 1200 μg) of luteal extract from corpora lutea of day 100 of pregnancy on aromatase activity of pig follicles were studied, the dose by treatment (presence or absence of FSH) interaction was not significant. Luteal extract dose at 300 μg did not affect aromatase activity but a significant decrease in activity occurred at 600 μg of luteal extract (600 versus 300 μg, P < 0.02). There was no further significant increase in the inhibitory effect with 1200 μg luteal extract. When the effects of 600 μg luteal extract from corpora lutea of days 15, 75 or 100 of pregnancy on aromatase activity of pig follicles were studied, a significant (P < 0.05) stage of pregnancy effect was detected, but the stage of pregnancy by treatment (presence or absence of FSH) interaction was not significant. No effect was noted with day 15 or day 75 luteal extract. In contrast, aromatase activity in the presence of day 100 luteal extract was significantly reduced compared with that of control (P < 0.01) and day 15 luteal extract (P < 0.05). A significant (P < 0.05) stage of pregnancy effect was also observed on aromatase activity of sheep follicles. Aromatase activity of sheep follicles was significantly reduced in the presence of day 100 luteal extract compared with that of control (P < 0.05) and day 15 luteal extract (P < 0.02). These data suggest that the stimulus triggering the synthesis of the aromatase inhibitor appears after mid-pregnancy. The aromatase-inhibiting activity was lost from luteal extract of corpora lutea of day 100 of pregnancy after treatment with proteolytic enzymes, demonstrating the proteic nature of the aromatase inhibitor. These experiments provide evidence for the existence in ovine corpora lutea of late pregnancy of a non-steroidal factor that reduces follicular aromatase activity. We propose the term aromatase-inhibiting factor or AIF to describe this activity.

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Y. Cognie, F. Benoit, N. Poulin, H. Khatir and M. A. Driancourt

Booroola ewes have a major gene that affects ovulation rate. Gene expression has consequences on ovarian somatic cells but it is unknown whether it also affects germ cells in the adult ovary. Hence, the present study examined (1) whether oocyte growth was similar in Fec B Fec B and Fec + Fec + oocytes during preantral and antral follicular growth, (2) whether the patterns of proteins neosynthesized by oocytes of these two genotypes were identical, (3) whether the ability of the oocytes to resume meiosis was unaffected by genotype and (4) whether, after IVF, oocytes from both genotypes could develop to the blastocyst stage at similar rates. Histological examination of the respective sizes of the oocyte and of the follicle demonstrated that oocytes were larger in Fec B Fec B versus Fec + Fec + preantral follicles. Resolution of the proteins neosynthesized by Fec B Fec B and Fec + Fec + oocytes by one-dimensional PAGE and image analysis demonstrated that quantitative (but not qualitative) differences could be observed between genotypes for bands at 74, 59, 35 and 25 kDa. In addition, a genotype by oocyte size interaction was detected for two additional bands at 45 and 43 kDa. After 24 h of culture in vitro in TCM-199 plus 100 ng ml−1 FSH plus 10% sheep follicular fluid, oocytes from Fec B Fec + follicles gained the ability to resume meiosis at a smaller size and a higher proportion of them reached metaphase II irrespective of the size class studied compared with Fec + Fec + follicles. In addition, the developmental rate of eggs after IVF was also affected by follicle size and genotype, since Fec B Fec + oocytes originating from 1.0–3.5 mm follicles had a greater ability (P < 0.05) to develop to the blastocyst stage than Fec + Fec + oocytes. It is concluded that the Fec B gene, in addition to its effects on granulosa cell maturation, also affects oocyte development and function. Whether these alterations are related requires further investigation.

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M. A. Driancourt, R. C. Fry, I. J. Clarke and L. P. Cahill

Summary. Ewes were hypophysectomized on Day 0 and ovariectomized 1, 2, 4 or 8 days later. There was no effect of hypophysectomy on the overall population of follicles > 0·8 mm in diameter during the time studied. However, the growth of healthy follicles >2 mm in diameter was prevented by Day 2. Turnover of follicles was very active in the ovaries of hypophysectomized ewes as shown by peaks in the proportion of follicles in early atresia at Day 4 and in advanced atresia at Days 1 and 8. By Day 8, most of the measures of the population of follicles 0·8 to 2 mm in diameter were back to the values of Day 0 ewes. The mitotic index of the granulosa cells of the healthy follicles exhibited a similar pattern with a nadir at Day 2 followed by a return at Days 4 and 8 to values similar to Day 0 ewes.

Ink-marked preovulatory follicles underwent a steady decrease in their histological size after hypophysectomy and this was associated with time-related changes in the health status of these follicles. By Day 1, 4 out of 7 follicles were still healthy while at Days 2, 4 and 8, all follicles were in advanced, late and collapsing atresia respectively. There was no evidence of an ability of PMSG (1000 i.u.) to rescue large follicles in advanced atresia (48 h after hypophysectomy). Furthermore, at 24 h after hypophysectomy, only 2 out of 5 follicles were maintained. However, PMSG partly overcame the depressing effects of hypophysectomy on the population of follicles 0·8 to 2 mm in diameter.

It is concluded that (1) active follicular turnover can occur in the absence of gonadotrophins; (2) it takes about 8 days for a large follicle to disappear in the ovarian stroma; and (3) it is unlikely that rescue of atretic follicles massively contributes to the ovulatory response after PMSG.