GnRH-induced gonadotrophin secretion in ovariectomized Booroola ewes with hypothalamic-pituitary disconnection

To test whether the F gene-specific differences in the plasma concentrations of FSH and LH are due to differences in the pituitary responsiveness to exogenous GnRH, ovariectomized Booroola ewes with hypothalamic-pituitary disconnection (HPD-ovx) were treated with GnRH (250 ng i.v.) once every 2 h for up to 5 weeks. In Exp. 1, jugular venous blood was collected once weekly from 13 FF and 14 ++ HPD-ovx ewes for 6 weeks before GnRH treatment and every 2nd, 3rd or 6th day for 5 weeks during treatment. In Exp. 2, jugular venous blood was collected from another 8 FF and 7 ++ HPD-ovx ewes at 5- or 10-min intervals over 4 GnRH pulses (250 ng i.v. once every 2 h) on 3 separate occasions after the animals had been subjected to the GnRH pulse regimen for approximately 7 days beforehand. Also in Exp. 2, the animals were extensively sampled around a larger (10 micrograms) i.v. injection of GnRH and the pituitary FSH and LH contents assessed after the animals had been re-exposed to the once every 2 h GnRH (250 ng i.v.) pulse regimen for several days following the larger GnRH bolus. In Exp. 3 the distributions of mean plasma concentrations of FSH and LH in individual GnRH-treated HPD-ovx ewes were compared with those in ovariectomized and ovary-intact FF and ++ ewes. During the 6 weeks before GnRH treatment (Exp. 1), the plasma concentrations of FSH (approximately 1 ng/ml) and LH (less than or equal to 0.8 ng/ml) were not different between the genotypes. After GnRH treatment both the mean FSH and LH concentrations increased significantly (P less than 0.01) above basal values after 2 days with F gene-specific differences being noted for FSH but not LH (FSH; FF greater than ++; P less than 0.05). Thereafter, the mean FSH but not LH concentrations increased at a faster rate in FF than in ++ ewes with the overall mean FSH concentrations between the genotypes being significantly different (P less than 0.05). In Exp. 2 considerable between-animal variation in the pulsatile pattern of FSH but not LH concentrations was seen in ewes of both genotypes during GnRH treatment. The overall mean FSH concentrations were higher in FF than in ++ ewes (P less than 0.05) and the mean FSH response to each GnRH pulse was significantly higher in FF than in ++ ewes (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)     

Differences in the plasma concentrations of FSH and LH in ovariectomized Booroola FF and ++ ewes

During 12 sampling days before ovariectomy the mean plasma FSH but not LH concentrations in FF ewes were higher (P less than 0.01) than those in ++ ewes (16 ewes/genotype). After ovariectomy increases in the concentrations of FSH and LH were noted for ewes of both genotypes within 3-4 h and the rates of increase of FSH and LH were 0.18 ng ml-1 h-1 and 0.09 ng ml-1 h-1 respectively for the first 15 h. From Days 1 to 12 after ovariectomy, the overall mean +/- s.e.m. concentrations for FSH in the FF and ++ ewes were 8.1 +/- 0.6 and 7.1 +/- 0.4 ng/ml respectively and for LH they were 2.7 +/- 0.3 and 2.1 +/- 0.2 ng/ml: these differences were not statistically significant (P = 0.09 for both FSH and LH; Student's t test). However, when the frequencies of high FSH or LH values after ovariectomy were compared with respect to genotype over time, significant F gene-specific differences were noted (P less than 0.01 for both FSH and LH; median test). In Exp. 2 another 21 ewes/genotype were blood sampled every 2nd day from Days 2 to 60 after ovariectomy and the plasma concentrations of FSH and LH were more frequently higher in FF than in ++ ewes (P less than 0.01 for FSH and LH). The F gene-specific differences in LH concentration, observed at 21-36 days after ovariectomy were due to higher mean LH amplitudes (P less than 0.025) but not LH peak frequency in FF than in ++ ewes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association between surges of follicle-stimulating hormone and the emergence of follicular waves in heifers.

The effects of ablation of a dominant follicle and treatment with follicular fluid on circulating concentrations of follicle-stimulating hormone (FSH) were studied and the temporal relationships between surges of FSH and follicular waves were studied in heifers with two or three follicular waves/interovulatory interval. Cauterization of the dominant follicle on Day 3 or Day 5 (ovulation on Day 0) (six control and six treated heifers/day) resulted in a surge (P less than 0.05) in FSH beginning the day after cautery. The FSH surge prior to wave 2 (first post-treatment follicular wave) occurred 4 days (Day 3 cautery) and 2 days (Day 5 cautery) before the surge in control groups, corresponding to a 4-day and a 2-day advance in emergence of wave 2 compared with controls. It was concluded that the dominant follicle on Day 3 and Day 5 was associated with the suppression of circulating FSH concentrations. Heifers (n = 4/group) were untreated or treated intravenously with a proteinaceous fraction of bovine follicular fluid on Days 0-3, 3-6, or 6-11. Concentrations of FSH were suppressed (P less than 0.05) for the duration of treatment, regardless of the days of treatment. Cessation of treatment was followed within 1 day by the start of a surge in FSH. The FSH surge prior to wave 2 occurred 2 days earlier (treatment on Days 0-3), 1 day later (treatment on Days 3-6), and 6 days later (treatment on Days 6-11) than in controls, corresponding to an equivalent advance or delay, respectively, in the emergence of wave 2 compared with controls. The results suggest that the effects of exogenous follicular fluid on follicular development were mediated, in whole or in part, by altering plasma FSH concentrations. Control heifers combined for the two experiments were separated into those with 2-wave (n = 11) or 3-wave (n = 5) interovulatory intervals. Two-wave heifers had two FSH surges and 3-wave heifers had three apparent FSH surges during the interovulatory interval. Results of the cautery and follicular fluid experiments indicated that a surge in FSH necessarily preceded the emergence of a wave. The FSH surges in treated and control heifers began 2-4 days before the detectable (ultrasound) emergence of a follicular wave (follicles of 4 and 5 mm), peaked 1 or 2 days before emergence and began to decrease approximately when the follicles of a wave begin to diverge into a dominant follicle and subordinate follicles (follicles 6-7 mm).     

Plasma follicle stimulating hormone and luteinizing hormone patterns during the estrous cycle of ewes

Fifteen Suffolk ewes were used in three experiments to compare plasma follicle stimulating hormone (FSH) and luteinizing hormone (LH) patterns during the estrous cycle and to determine whether FSH levels undergo changes in pulse frequency. Luteinizing hormone changed inversely with progesterone levels whereas FSH and progesterone concentrations revealed no obvious relationship. Unlike LH, FSH levels did not pulsate during the follicular phase. Higher FSH levels were detected on days 1, 6 and 12 and lower levels on days 0, 4 and 16. Coincident preovulatory LH and FSH surges were observed and this was the only time FSH and LH levels appeared to be jointly controlled.     

Effects of oestradiol-17 beta, progesterone or bovine follicular fluid on the plasma concentrations of FSH and LH in ovariectomized Booroola ewes which were homozygous carriers or non-carriers of a fecundity gene

The plasma concentrations of FSH and LH were measured in ovariectomized Booroola FF and ++ ewes before and after treatment with subcutaneous implants of oestradiol-17 beta (0, 2 or 8 cm Silastic capsules; 5 ewes/genotype per dose) or progesterone (0, 1 or 3 Silastic envelopes; 5 ewes/genotype per dose) or subcutaneous injections of steroid-free bovine follicular fluid (bFF; 0, 0.5, 1.0, 2.5 or 5 ml; 4 ewes/genotype per dose). During the first 50 h after implantation of oestradiol or progesterone, or the first 24 h after bFF treatment, the FSH and LH concentrations in plasma were not different between the genotypes although there were significant effects of the steriods and bFF with respect to dose (P less than 0.05). At 6 days after steroid implantation, no gene-specific effects were noted for the plasma concentrations of FSH although significant effects of dose of oestradiol (P less than 0.01) but not progesterone were noted. Also at 6 days after steroid implantation, no gene-specific differences in the pulsatile patterns (i.e. peak frequency or amplitude) of plasma LH concentrations were noted although there were significant effects of steriod dose (P less than 0.05) on frequency and/or amplitude. It is concluded that the higher ovulation-rate in FF than ++ Booroola ewes is unlikely to be due to gene-specific differences in the sensitivity of the hypothalamic-pituitary axis to ovarian hormones.     

Direct effects of ovine follicular fluid on ovarian steroid secretion and expression of markers of cellular differentiation in sheep

The objective of this study was to assess the effect of ovine follicular fluid (FF) treatment (with or without FSH replacement) during the late follicular phase on plasma concentrations of gonadotrophins and the development of the ovulatory follicle. Ovarian steroid secretion and expression of mRNA encoding inhibin alpha and beta A, beta B subunits, P450 aromatase and P450 17 alpha-hydroxylase were used as endpoints. After induction of luteolysis by injection of 100 micrograms cloprostenol on days 10-12, Scottish Blackface ewes were allocated to one of three groups: (1) control (n = 7): no further treatment; (2) FF (n = 9): subcutaneous injections of 3 ml steroid-free ovine follicular fluid at 9 h intervals, 18 and 27 h after cloprostenol injection; (3) FF + FSH (n = 8): injections of follicular fluid as above plus subcutaneous injections of 0.36 iu ovine FSH at 6 h intervals, 18, 24, and 30 h after cloprostenol injection. Jugular venous blood samples were obtained via indwelling cannulae at 6 h intervals from 0 to 36 h after cloprostenol injection, and at 10 min intervals from 12 to 18 h (control phase) and from 30 to 36 h after cloprostenol injection (treatment phase). At laparotomy, 36 h after cloprostenol injection, ovarian venous blood was collected and ovaries were removed and processed for in situ hybridization. Plasma concentrations of FSH, luteinizing hormone (LH) and oestradiol were determined by radioimmunoassay. Follicular fluid treatment resulted in a decrease (P < 0.001) in FSH concentrations associated with an acute decrease in ovarian steroid secretion (P < 0.01) and a specific depression in P450 aromatase, (P < 0.001), inhibin-activin beta B subunit (P < 0.05) and thecal LH receptor (P < 0.001) expression. Follicular fluid treatment had no effect on inhibin-activin alpha and beta A, subunit or P450 17 alpha-hydroxylase expression. FSH co-treatment with follicular fluid restored circulating FSH concentrations to normal values and reversed some of the effects of follicular fluid (androstenedione, testosterone and progesterone secretion, and inhibin beta B and thecal LH receptor expression) but not oestradiol secretion or P450 aromatase expression. It was concluded that the actions of follicular fluid are mediated via both central effects on pituitary FSH secretion and by direct ovarian effects on granulosa cell aromatase activity. The results indicate that follicular fluid contains a factor that inhibits aromatase activity of granulosa cells directly and may play a role in the selection of the dominant follicle.     

Effect of castration on plasma concentrations of luteinizing hormone and follicle-stimulating hormone in adult Merino rams which were homozygous carriers or non-carriers of the Booroola fecundity gene

Before castration, the mean plasma concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) did not differ between FF and ++ Booroola rams. After castration, mean LH and FSH concentrations increased after 8 h, and for the next 14 days the rate of increase in FSH, but not LH, secretion was significantly faster in FF than in ++ rams (P less than 0.05). Mean FSH concentrations over this period were significantly higher in FF than in ++ rams (P less than 0.05). In both genotypes, the ranked FSH values did not significantly change their order over time, i.e. a significant within-ram effect was noted (P less than 0.05). Repeated-measures analysis of variance indicated a significant effect of genotype on mean FSH secretion (P less than 0.05) and a significant effect of sire in the FF (P less than 0.05), but not the ++ (P = 0.76), genotype. From Day 28 to Day 58 after castration, FSH and LH concentrations were variable and no overall increases in concentrations were observed. The mean concentrations of both hormones over this period were not related to genotype. There were no gene-specific differences in pulsatile LH secretion 14 weeks after castration. However, the mean LH, but not FSH, response to a bolus injection of 25 micrograms of gonadotrophin-releasing hormone (GnRH) was significantly higher in FF than in ++ rams (P less than 0.05) and this was not significantly affected by sire. These studies support the hypothesis that the F gene is expressed in adult rams, in terms of pituitary responsiveness to an injection of GnRH and to the removal of the testes, but it is not clear from this study whether the influence of sire is related to or independent of the apparent gene-specific differences.     

Reduced intrafollicular androstenedione and estradiol levels in early-treated prenatally androgenized female rhesus monkeys receiving follicle-stimulating hormone therapy for in vitro fertilization

Five early-treated and four late-treated prenatally androgenized and five normal female rhesus monkeys were studied to determine whether prenatal testosterone propionate exposure beginning Gestational Days 40-44 (early-treated) or 100-115 (late-treated) affects follicular steroidogenesis during recombinant human FSH (rhFSH) treatment. All monkeys underwent rhFSH injections, without human chorionic gonadotropin administration, followed by oocyte retrieval. Serum FSH, LH, estradiol (E2), progesterone (P), 17alpha-hydroxyprogesterone (17 OHP), androstenedione (A4), testosterone, and dihydrotestosterone were measured basally during rhFSH therapy and at oocyte retrieval. Follicle fluid (FF) sex steroids, oocyte fertilization, and embryo development were analyzed. Circulating FSH, E2, 17 OHP, A4, and dihydrotestosterone levels increased similarly in all females. Serum LH levels decreased from basal levels in normal and late-treated prenatally androgenized females but were unchanged in early-treated prenatally androgenized females. Serum P levels at oocyte retrieval were comparable with those before FSH treatment in all females. All prenatally androgenized females showed reduced FF levels of A4 and E2 but not P or dihydrotestosterone. Intrafollicular T concentrations also were significantly lower in late-treated compared with early-treated prenatally androgenized females or normal females. In early-treated prenatally androgenized females, but not the other female groups, intrafollicular A4 and E2 levels were reduced in follicles containing oocytes that failed fertilization or produced zygotes with cleavage arrest before or at the five- to eight-cell embryo stage. Therefore, in monkeys receiving rhFSH therapy alone without human chorionic gonadotropin administration, early prenatal androgenization reduced FF concentrations of E2 and A4 in association with abnormal oocyte development, without having an effect on P, testosterone, or dihydrotestosterone concentrations.     

Oestradiol-17beta, progesterone, FSH and LH in prepubertal calves induced to superovulate

Fluorogestone acetate (vaginal sponge for 4 days) and PMSG (i.m. injection at the time of sponge insertion) treatment was administered to seven 3-month-old calves to induce superovulation. Samples of peripheral plasma were taken every 4 h during treatment (4 days) and then every 2 h for 7 days. FSH, LH, oestradiol and progesterone were measured by radioimmunoassays. In all calves oestradiol concentrations increased 24 h after PMSG injection and reached the highest levels (41-502 pg/ml) during the preovulatory surge of both gonadotropins. The surge of LH and FSH occurred from 12 to 22 h after cessation of treatment. The maximum levels of LH and FSH were 11-72 ng/ml and 23-40 ng/ml respectively and occurred within 4 h of each other. Between 40 and 68 h after the LH peak the concentrations of progesterone began to increase from basal values, reaching 24.0-101.7 ng/ml when the animals were killed. A quantitative relationship was found between plasma oestradiol concentration and the numbers of ovulating follicles. Progesterone levels seemed to be related to the numbers of corpora lutea and also to the numbers of unovulated follicles. Gonadotrophin output was not quantitatively related to ovarian activity or to steroid secretion.     

The physiological role of beta-endorphin in porcine ovarian follicles

Beta-endorphin-like immunoreactivity (beta-END-LI) was measured by radioimmunoassay in porcine ovarian follicular fluid (FF) from small, medium and large follicles throughout the oestrous cycle. The concentration of beta-END-LI in FF from small follicles collected on days 1-5 of the cycle was at least tenfold higher than in the fluid from any other follicles independently from their size and the period of the cycle. The level of beta-END-LI in small follicles on days 6-10 was drastically decreased. Subsequently, on days 11-16 its concentration was enhanced and reduced again in pre-ovulatory period of the cycle. Concentrations of beta-END-LI in FF from medium follicles were relatively equal throughout the cycle (days 6-21). No significant differences in beta-END-LI levels were found between small, medium and large follicles from days 17-21. However, beta-END-LI concentrations in medium follicles on days 11-13 and 14-16 were statistically lower than those in small follicles. Moreover, the effects of FSH, prolactin (PRL), progesterone (P4), testosterone (T) and 17 beta-oestradiol (E2) on beta-END-LI release by granulosa cells (GCs) from large follicles and, on the other hand, the effects of the opioid agonist FK 33-824 alone or in combination with FSH, PRL or naloxone (NAL) on follicular steroidogenesis were studied. FSH drastically increased beta-END-LI output in a dose-dependent fashion. This stimulatory effect of the gonadotrophin was inhibited by the highest dose of P4 (10(-5) M). The effect of PRL and the steroids added to the cultures on beta-END-LI release was negligible. FSH- or PRL-induced P4 secretion by GCs was essentially abolished by both FK 33-824 and NAL. However, androstenedione (A4) and testosterone output by the cells was greatly potentiated by FK 33-824. In the presence of NAL, FSH or PRL, A4 release stimulated by FK 33-824 was suppressed to the basal level. Secretion of E2 was completely free from the influence of FK 33-824 or NAL; only oestrone (E1) output was modulated by them in cultures where FSH or PRL was present. In conclusion, FSH appears to be the key regulator of beta-END-LI secretion by porcine granulosa cells. Moreover, steroidogenesis in pig granulosa cells is modulated by opioid peptides acting both alone and by way of interaction with FSH or PRL.     

Effect of changes in FSH induced by bovine follicular fluid and FSH infusion in the preovulatory phase on subsequent ovulation rate and corpus luteum function in the ewe

Treatment of Damline ewes with i.v. injections of various doses (2, 5 or 10 ml) of bovine follicular fluid for 72 h after prostaglandin-induced luteal regression resulted in a significant decrease in plasma concentrations of FSH after a 1.5-2 h delay but did not affect LH. The half life of this decrease in plasma FSH levels (156 min) after injection of follicular fluid was similar to that for clearance (159 min) of ovine FSH after infusion. A significant rebound increase in plasma FSH levels occurred by 13 h after all follicular fluid injections, and the magnitude of this rebound was inversely related to the dose of follicular fluid injected. A significant delay in the onset of oestrus occurred only with 5 and 10 ml bovine follicular fluid. There was no significant effect on ovulation rate or subsequent corpus luteum function as measured by plasma concentrations of progesterone. Infusion of ovine FSH (50 micrograms/h for 48 h) during the period of follicular fluid treatment prevented the delay in onset of oestrus and resulted in a substantial (2-10-fold) increase in ovulation rate. Corpus luteum function in terms of progesterone secretion was also enhanced. These results show that (1) intermittent suppression of FSH during the preovulatory period in the ewe does not affect subsequent ovulation rate or corpus luteum function and (2) the delay in the onset of oestrus induced by bovine follicular fluid can be prevented by exogenous FSH.     

Plasma concentrations of gonadotrophins, preovulatory follicular development and luteal function associated with bovine follicular fluid-induced delay of oestrus in heifers

In Exp. 1, injections of 10 ml bovine follicular fluid (bFF, i.v. or s.c.), given twice daily for 3 days after injection of a luteolytic dose of PGF-2 alpha, delayed the onset of oestrus in 3 of 6 heifers to 8 or 9 days after PGF-2 alpha, as compared with 2 or 3 days after PGF-2 alpha in control heifers. Mean plasma concentrations of FSH and LH during the injection period were not different from those in saline-injected heifers. In Exp. 2, i.v. injections of 20 ml bFF twice daily for 3 days uniformly delayed oestrus to 8 days after PGF-2 alpha (N = 4) and injections of 20 ml bFF i.v. every 6 h for 24h on the day of PGF-2 alpha injection delayed oestrus to 5.0 +/- 0.6 days after PGF-2 alpha as compared with 2.8 +/- 0.3 days for control heifers. In both treatment groups, plasma concentrations of FSH were suppressed during the injection period and increased transiently after treatment, but plasma concentrations of LH during the injection period were not different from those of control heifers. Plasma levels of oestradiol in heifers given bFF remained basal for 2 or 3 days after treatment, then increased several days before the delayed oestrus, in a manner similar to that in control heifers, and elicited normal preovulatory surges of LH and FSH. Plasma concentrations of progesterone and the length of the next oestrous cycle were normal, indicating formation of functional corpora lutea. Therefore, bFF treatments appear to delay oestrus by selectively suppressing plasma FSH, without affecting LH, and delaying the development of the preovulatory follicle. These results suggest that FSH may be critical to support the growth and development of the preovulatory follicle after luteolysis in cows.     

Gonadotrophins, fecundity genes and ovarian follicular function

The Booroola Merino is a sheep breed having a major gene(s) (F) influencing its ovulation-rate. Homozygous (FF), heterozygous (F+) and non-carriers (++) of the gene have ovulation-rates of greater than or equal to 5, 3 or 4 and 1 or 2 respectively with the durations of each oestrous cycle and oestrous behaviour being similar in all genotypes. Although the principal site(s) of gene expression are obscure, FF genotypes have mean plasma concentrations of FSH and LH which are higher than in the F+ ewes, which in turn are higher than in the ++ animals. Thus, the FF and F+ animals provide a unique system in which to examine ovarian function under continual exposure to elevated gonadotrophin concentrations. At the ovarian level, F gene-specific differences in follicular development and function were noted. In small follicles (0.1-1.0 mm dia.), the basal levels of cAMP and the in vitro synthesis of cAMP, progesterone, androstenedione and oestradiol-17 beta in response to LH and FSH were significantly influenced by genotype (FF greater than F+ greater than ++; P less than 0.05). In larger follicles (1-4.5 mm dia.) the granulosa cells from FF and F+ ewes were more responsive to FSH and/or LH than in ++ ewes with respect to cAMP synthesis and they also had higher levels of aromatase activity. In vivo, the ovarian secretion-rates of oestradiol from greater than or equal to 5 ("oestrogenic") follicles in FF ewes, 3-4 such follicles in F+ ewes, and 1-2 such follicles in ++ animals during the follicular phase were similar. In FF and F+ ewes, the preovulatory follicles ovulated at a smaller diameter (i.e. 3-5 mm) than in ++ ewes (greater than 5 mm diam.) and also produced smaller corpora lutea. Thus, after continual exposure to elevated levels of gonadotrophins, follicles may synthesize steroid and mature at smaller diameters compared to those exposed to normal levels of FSH and LH.     

Plasma LH, FSH and testosterone concentrations in adult rams which were homozygous carriers or non-carriers of the Booroola fecundity gene

No gene-specific differences were found with respect to LH or testosterone pulsatile secretion (over 12 h), or in 12 hourly mean FSH concentrations in adult Booroola FF and ++ rams. Also, no differences between genotypes in the LH response to an injection of testosterone propionate, the FSH response to an infusion of bovine follicular fluid, or the testosterone response to injections of PMSG were noted. However, during the phase of seasonal testicular development, mean testosterone pulse amplitude (over 12 h) and the FSH response to 25 micrograms GnRH were higher in FF than in ++ rams (P less than 0.05); there were also significant effects of sire (P less than 0.05 in FF genotype only) and litter size (P less than 0.05) on testosterone pulse amplitude and GnRH-stimulated FSH release, respectively. During the breeding season, mean LH, but not FSH, concentrations were higher in FF than in ++ rams, after an injection of 0.5 micrograms GnRH; LH release was not affected by sire or litter size (P greater than 0.05). Long-term studies revealed that the FF rams were born in significantly larger litters, they weighed significantly less than ++ rams (P less than 0.05), and that bodyweight was significantly correlated (P less than 0.05) with litter size. There were no differences in testis size, and testis size was not significantly correlated with bodyweight. There was a strong tendency (P = 0.056) for overall mean FSH concentrations, measured weekly for 9 months, to be highest more often in FF than in ++ rams.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of the secondary FSH surge with bovine follicular fluid is associated with delayed ovarian follicular development in heifers

Holstein heifers were given 5 injections (twice/day) of 10 ml charcoal-extracted bovine follicular fluid (bFF; N = 6) or 10 ml saline (N = 5) beginning 12 h after the onset of oestrus. Blood samples were collected for determination of plasma concentrations of FSH, LH, progesterone and oestradiol-17 beta. Treatment with bFF suppressed the secondary FSH surge (P less than 0.01). Cessation of bFF injections was followed by a rebound period during which FSH was elevated compared with controls (P less than 0.01). Daily ultrasonographic examinations revealed that follicular growth occurred in waves, with 4 of 5 control heifers exhibiting 3 waves and the other 2 waves. In contrast, 5 of 6 bFF-treated animals exhibited 2 waves and the other 3 waves. Appearance of follicles in the first wave was delayed in bFF-treated heifers (Day 3.3 +/- 0.3 compared with Day 1.4 +/- 0.2; P less than 0.0001) and appearance of the dominant follicle of the first wave was delayed (Day 4.5 +/- 0.3 compared with Day 1.8 +/- 0.2; P less than 0.0001). Follicles in the second wave appeared later in animals treated with bFF (Day 12.7 +/- 0.4 compared with Day 10.4 +/- 0.6; P less than 0.01), and the dominant follicle of this wave also appeared later (Day 13.0 +/- 0.5 compared with Day 10.6 +/- 0.5; P less than 0.01). Oestradiol-17 beta increased during the early luteal phase, but this increase occurred later in heifers treated with bFF (peak concentrations on Day 6.3 +/- 0.6 compared with Day 4.2 +/- 0.2; P less than 0.05). LH, progesterone and cycle length were not affected by bFF. Delayed follicular growth associated with suppression of FSH suggests that the secondary FSH surge is important in the initiation of follicular development early in the bovine oestrous cycle, and thus may play a role in the regulation of ovarian follicular dynamics.     

Alterations in intrafollicular regulatory factors and apoptosis during selection of follicles in the first follicular wave of the bovine estrous cycle

Changes in follicular fluid (FF) concentrations of estradiol, inhibin forms, and insulin-like growth factor binding proteins (IGFBPs), percentage of apoptotic granulosa cells (%A), and follicular size for individual follicles in a growing cohort were determined throughout the first wave of follicular development during the bovine estrous cycle and related to FSH decline. Four groups of heifers (n = 31) were ovariectomized between Days 1.5 and 4.5 of the estrous cycle at 5 +/- 1, 33 +/- 2, 53 +/- 1, and 84 +/- 2 h after the periovulatory peak in FSH concentrations. Follicles > or = 2.5 mm were dissected, measured, and FF aspirated. The five largest follicles were ranked based on their diameter (F1 to F5). Diameters of F1 to F5 were positively correlated with interval from FSH peak (r > or = 0.6, P < 0.05). Five hours after the FSH peak, follicular diameter and FF concentrations of estradiol, inhibins, and IGFBPs were similar for F1 to F5. From 5 to 33 h, amounts of the six precursor inhibin forms (> or = 48 kDa) increased (P < 0.05) in F1 follicles. The IGFBPs in F1 follicles remained low at all time periods. At 33 h, amounts of IGFBP-4 and -5 were higher (P < 0.05) in F4 and F5 compared with F1 follicles. At 84 h, IGFBP-2, -4, and -5 were increased (P < 0.05) in F3, F4, and F5 compared with F1. At 5, 33, or 53 h, %A was not different between follicles in any size class. At 84 h %A was increased (P < 0.05) in follicles <6 mm in diameter. However, at that time, %A did not differ between the selected DF and the largest subordinate follicle. For individual heifers, the selected DF at 84 h was largest in size, highest in estradiol, and lowest in IGFBP-2 and -4. The F1 follicle had highest estradiol in 23 of 27 heifers irrespective of stage of the wave and lowest IGFBP-4 in 19 of 21 heifers from 33 h. We concluded that the earliest intrafollicular changes that differentiate a dominant-like follicle from the growing cohort are enhanced capacity to produce estradiol and maintenance of low levels of IGFBPs.     

Effect of progesterone on basal LH and episodic LH and FSH secretion in heifers

Heifers between Days 6 and 10 of the cycle were allocated at random to groups of 8 and treated with (i) a 4% progesterone-releasing intravaginal device (PRID) + oestrogen capsule for 12 days; (ii) 4% PRID for 12 days; (iii) 20% PRID for 12 days; (iv) 4% for 14 days; or (v) 20% PRID for 14 days. Blood was obtained daily during treatment and at 2- or 4-h intervals for 72 h after removal of PRIDs. Some animals were sampled every 20 min for 4.676 h on the 3rd day after PRID insertion, and 1 day before and 36 h after removal of the PRID insertion, and 1 day before and 36 h after removal of the PRID. During progesterone treatment there was: (i) no correlation between concentrations of progesterone and LH within days; (ii) a significant negative correlation between progesterone and days (P less than 0.01) and also between progesterone and LH over days (P less than 0.01); (iii) the overall correlation co-efficient between LH and days was positive (P less than 0.05). The amplitude of LH or FSH episodes was not affected as progesterone concentrations declined during PRID treatment, but the number of LH (but not FSH) episodes was increased (p less than 0.01). After PRID removal, the amplitude of both LH and FSH episodes increased (P less than 0.01). We suggest that progesterone is part of a negative feedback complex on LH secretion in cattle and that this effect is apparently mediated through frequency of episodic LH release.     

Differences in gonadotrophin concentrations and pituitary responsiveness to GnRH between Booroola ewes which were homozygous (FF), heterozygous (F+) and non-carriers (++) of a major gene influencing their ovulation rate

The mean plasma concentrations of FSH and LH were significantly higher in FF ewes than in ++ ewes with those F+ animals being consistently in between. These gene-specific differences were found during anoestrus, the luteal phase and during a cloprostenol-induced follicular phase, suggesting that the ovaries of ewes with the F-gene are more often exposed to elevated concentrations of FSH and LH than are the ovaries of ewes without the gene. The gene-specific differences in LH secretion arose because the mean LH amplitudes were 2-3 times greater in FF compared to ++ ewes with the LH amplitudes for F+ ewes being in between. The LH pulse frequencies were similar. In these studies the pulsatile nature of FSH secretion was not defined. The pituitary contents of LH during the luteal phase, were similar in all genotypes whereas for FSH they were significantly higher in the F-gene carriers compared to ++ ewes. The pituitary sensitivity to exogenous GnRH (0.1, 0.5 and 25 micrograms i.v.) was related to genotype. Overall the LH responses to GnRH were lower in FF ewes than in ++ ewes with the results for the F+ ewes being in between. The FSH responses to all GnRH doses in the FF genotype were minimal (i.e. less than 2-fold). In the other genotypes a greater than 2-fold response was noted only at the highest GnRH dose (i.e. 25 micrograms). Treatment of FF and F+ but not ++ ewes with GnRH eventually led to a reduced FSH output, suggesting that the pituitary responses to endogenous GnRH were being down-regulated in the F-gene carriers whereas this was not the case in the non-carriers. Collectively these data confirm that peripheral plasma and the pituitary together with the ovary are compartments in which F-gene differences can be observed. In conclusion, these findings raise the possibility that F-gene-specific differences may also extend to the hypothalamus and/or other regions of the brain.     

Suppression of follicle wave emergence in cyclic ewes by supraphysiologic concentrations of estradiol-17beta and induction with a physiologic dose of exogenous ovine follicle-stimulating hormone

Follicle waves are preceded by follicle-stimulating hormone (FSH) peaks in ewes. The purpose of the present study was to see whether estradiol implant treatment would block FSH peaks to create a model in which the effect of the timing and mode of FSH peaks could be studied by ovine FSH (oFSH) injection. In Experiment 1, 10 ewes received estradiol-17beta implants on Day 4 after ovulation (Day 0, day of ovulation); five ewes received large implants, and five ewes received small implants. Five control ewes received empty implants. In Experiment 2, 12 ewes received large implants on Day 4. On Day 9, six ewes received oFSH twice, 8 h apart (0.5 microg/kg; s.c.). Implants were left in place for 10 days in both experiments. In both studies, ovarian ultrasonography and blood sampling was done daily. In Experiment 1, estradiol concentrations were significantly higher in ewes with large implants (10.4 +/- 0.7 pg/ml) compared with controls (3.9 +/- 0.7 pg/ml) and ewes with small implants (5.4 +/- 0.7 pg/ml; P < 0.001). A significant reduction was found in mean FSH peak concentration (31%; P < 0.05) and FSH peak amplitude (45%; P < 0.05) in ewes with large implants compared with controls. Mean and basal FSH concentrations were unaffected by the large implants. The large implants halted follicle-wave emergence between Day 0 and 8 after implant insertion. The small follicle pool (2-3 mm in diameter) was unaffected by the large implants. When oFSH was injected into ewes with large implants, a follicle wave emerged 1.5 +/- 0.5 days after injection; however, in ewes given saline alone, a follicle wave emerged 4.8 +/- 0.8 days after injection (P < 0.01). We concluded that truncation of FSH peaks by estradiol implants prevented follicle-wave emergence, but injection of physiologic concentrations of oFSH reinitiated follicle-wave emergence.     

Administration of a gonadotropin-releasing hormone antagonist to mares at different times during the luteal phase of the estrous cycle

The GnRH antagonist cetrorelix was given during the early (Days 1-5), mid (Days 6-10 or 5-12) or for the entire (Days 1-16) luteal phase of mares to inhibit the secretion of FSH and LH (Day 0=ovulation). Frequent blood sampling from Day 6 to Day 14 was used to determine the precise time-course of the suppression (cetrorelix given Days 6-10). Cetrorelix treatment caused a decrease in FSH and LH concentrations by 8 and 16 h, respectively, and an obliteration of the response to exogenous GnRH given 24h after treatment onset. Treatment never suppressed gonadotropin concentrations to undetectable levels; e.g. frequent sampling showed that the nadirs reached in FSH and LH were 46.2±6% and 33.1±11%, respectively, of pre-treatment concentrations. Daily FSH concentrations were decreased in all treatment groups but daily LH concentrations were lower only when treatment commenced at the beginning of the luteal phase; progesterone concentrations depended on the time of cetrorelix administration, but the changes suggested a role for LH in corpus luteum function. The inter-ovulatory interval was longer than controls when cetrorelix was given in the mid- or for the entire luteal phase, but was unaffected by treatment in the early phase. Nevertheless, in all groups, FSH concentrations were higher (P<0.05 when compared to Day 0, subsequent ovulation) approximately 6-10 days before this next ovulation. This consistent relationship suggests a stringent requirement for a GnRH-induced elevation of FSH above a threshold at, but only at, this time; i.e. approximately 6-10 days before ovulation.