Ovarian steroid hormone involvement in endogenous opioid modulation of LH secretion in mature ewes during the breeding and non-breeding seasons

The opioid antagonist WIN-44441-3 (WIN-3, Sterling-Winthrop) caused significant increases in LH secretion in ovariectomized ewes treated with progesterone but not in ovariectomized animals treated with oestradiol-17 beta. In the non-breeding season, plasma LH concentrations in ovariectomized ewes without steroid therapy, given oestradiol-17 beta or oestradiol-17 beta and progesterone together were not affected by treatment with WIN-3 on Day 6 after ovariectomy (there was a significant increase in LH as a result of WIN-3 treatment 13 days after ovariectomy in sheep given no steroid therapy). However, WIN-3 treatment of ovariectomized sheep given progesterone resulted in a significant increase in plasma LH. WIN-3 was ineffective when given to intact ewes treated with progesterone during the non-breeding season. With ovariectomized sheep during the breeding season there was again no response to WIN-3 at 6 days after ovariectomy in sheep given oestradiol-17 beta, but significant LH elevations in animals given no steroid, those given progesterone and those given progesterone + oestradiol-17 beta. The lack of an LH response to WIN-3 in ovariectomized sheep treated with oestradiol-17 beta did not result from a reduced pituitary response to GnRH since such animals responded normally to exogenous GnRH treatment. Overall, these results are consistent with the idea that, irrespective of the time of year, progesterone exerts negative feedback upon LH release at least in part through an opioidergic mechanism, whereas oestradiol-17 beta exerts negative feedback through steps unlikely to involve opioids. Progesterone can override the effect of oestradiol-17 beta during the breeding season only. Further, there appears to be a steroid-independent opioid involvement in LH suppression, operating at both times of year.     

Effect of LHRH immunoneutralization on follicular development, the LH surge and luteal function in the stumptailed macaque monkey (Macaca arctoides)

A dose of 100 microliter of a potent ovine LHRH gamma globulin inhibited ovulation in the cyclic rat when administered at 12:00 h on the day of pro-oestrus. A dose of 10 ml of the preparation was administered i.v. to female stumptailed macaques to achieve circulating antibody titres 3-4-fold higher than in the rat. In an ovariectomized macaque, this caused a marked fall in serum concentrations of LH to less than 10% of pretreatment values and also a significant, though less pronounced, fall in FSH. Six monkeys were treated with the LHRH gamma globulin during the mid-late follicular phase of the cycle. In 2 monkeys in which serum oestradiol concentrations were less than 100 pg/ml at the time of antibody administration, the rising oestradiol levels were abruptly suppressed and the normal mid-cycle LH surge failed to occur. Serum concentrations of LH and FSH declined to low levels for 8-10 days after which time normal follicular development occurred. In the remaining 4 monkeys in which follicular development was more advanced as indicated by serum oestradiol concentrations of greater than 100 pg/ml, the antibodies induced either a transient decline or had no effect on the rising serum concentration of oestradiol. An LH/FSH surge followed by a rise in serum progesterone occurred in these macaques. When the antibodies were administered to a further 6 macaques, which had also been treated with oestradiol benzoate during the early follicular phase to induce an LH surge, the neutralization of LHRH again failed to block the surge even when the dose of antibody was increased to 20 ml. The results show that LHRH antibodies were unable to block the LH surge in the macaque. They contrast with results obtained with LHRH immunoneutralization in the sheep, rat, hamster, mouse and bird and suggest that the ability of oestrogen to induce an LH surge by acting directly on the LHRH-primed anterior pituitary gland is more dominant in the primate.     

Interactions between inhibin, oestradiol and progesterone in the control of gonadotrophin secretion in the ewe

Experiments were carried out to test the hypothesis that inhibin and oestradiol act synergistically to inhibit the secretion of FSH, to test for effects of progesterone, and to compare the FSH and LH responses to ovarian feedback. In Exp. 1, with 11 ovariectomized and 12 intact Romanov ewes during the anoestrous season, doses of oestradiol (administered by means of subcutaneous implants) that restored normal LH pulse frequencies were insufficient to restore normal concentrations of FSH. In Exp. 2, with 48 ovariectomized Welsh Mountain ewes during the breeding season, a factorial design with 4 ewes per cell was used to assess the responses in LH and FSH to 3 doses of oestradiol (s.c. implants) and 4 doses of bovine follicular fluid ('inhibin', 0.2-1.6 ml s.c. every 8 h). This was done initially in the absence of progesterone and then after 7 days of treatment with progesterone (s.c. implants). Analysis of variance revealed a significant synergistic interaction between oestradiol and inhibin on the plasma concentrations of FSH. Progesterone had little effect. In contrast, there was a significant synergistic interaction between oestradiol and progesterone on the concentrations of LH. 'Inhibin' also inhibited LH secretion but this effect was independent of the two steroids. We conclude that there are basic differences in the way that ovarian feedback acts to control the secretion of LH and FSH in the ewe. FSH secretion appears to be primarily controlled by the synergistic action of oestradiol and inhibin on the anterior pituitary gland, while the secretion of LH is inhibited during the follicular phase by an effect of oestrogen at pituitary level and during the luteal phase by the synergistic action of oestradiol and progesterone at the hypothalamic level. Inhibin, or another non-steroidal factor in follicular fluid, may also play a minor role in the control of LH secretion.     

Initiation of follicular maturation during mid-pregnancy by the administration of LH in the rat

Pregnant rats were injected twice daily for 1-3 days (Days 13-16 of pregnancy) with various doses of ovine LH. Follicular maturation was determined by the ability of the follicles to ovulate in response to 10 i.u. hCG as well as by endogenous production of oestradiol-17 beta and inhibin. In control animals, no ovulation was induced by hCG given on Day 16 of pregnancy. An injection of hCG on Day 16 of pregnancy, however, induced ovulation in LH-treated animals (6.25-50.0 micrograms LH per injection, s.c. at 12-h intervals from Days 13 to 16). Concentrations of oestradiol-17 beta and inhibin activity in ovarian venous plasma increased after the administration of LH, indicating that development of ovulatory follicles had been induced. Abolishing the decline in plasma LH values therefore induced maturation of a new set of follicles or prevented the atresia of large antral follicles usually seen at this time of pregnancy. Plasma and pituitary concentrations of FSH decreased in LH-treated animals compared with those in control animals. Concentrations of progesterone, testosterone and oestradiol-17 beta in the peripheral plasma were not significantly different between the two groups. These results suggest that the increase in inhibin secretion from the ovary containing maturing follicles after LH treatment may suppress the secretion of FSH from the pituitary gland. These findings indicate that (1) the development of ovulatory follicles can be induced by the administration of exogenous LH during mid-pregnancy in the rat and (2) basal concentrations of FSH are enough to initiate follicular maturation even in the presence of active corpora lutea of pregnancy, when appropriate amounts of plasma LH are present.     

Effects of ageing and exogenous melatonin on pituitary responsiveness to GnRH in rats

The effect of age and melatonin on the activity of the neuroendocrine reproductive system was studied in young cyclic (3-5 months-old), and old acyclic (23-25 month-old) female rats. Pituitary responsiveness to a bolus of GnRH (50 ng per 100 g body weight) was assessed at both reproductive stages in control and melatonin-treated (150 micrograms melatonin per 100 g body weight each day for 1 month) groups. After this experiment, female rats were treated for another month to study the influence of ageing and melatonin on the reproductive axis. Plasma LH, FSH, prolactin, oestradiol and progesterone were measured. A positive LH response to GnRH was observed in both control groups (cyclic and acyclic). However, a response of greater magnitude was observed in old acyclic rats. Melatonin treatment reduced this increased response in acyclic rats and produced a pituitary responsiveness similar to that of young cyclic rats. FSH secretion was independent of GnRH administration in all groups, indicating desynchronization between LH and FSH secretion in response to GnRH in young animals and during senescence. No effect on prolactin was observed. Significantly higher LH (3009.11 +/- 1275.08 pg ml(-1); P < 0.05) and FSH concentrations (5879.28 +/- 1631.68 pg ml(-1); P < 0.01) were seen in acyclic control rats. After melatonin treatment, LH (811.11 +/- 89.71 pg ml(-1)) and FSH concentrations (2070 +/- 301.62 pg ml(-1)) decreased to amounts similar to those observed in young cyclic rats. However, plasma concentrations of oestradiol and progesterone were not reduced. In conclusion, the results of the present study indicate that, during ageing, the effect of melatonin is exerted primarily at the hypothalamo-pituitary axis rather than on the ovary. Melatonin restored the basal concentrations of pituitary hormones and pituitary responsiveness to similar values to those observed in young rats.     

Hormone responses to exogenous GnRH pulses in post-partum dairy cows

Nine Friesian dairy cows were treated with 2.5 micrograms GnRH i.v. at 2-h intervals for 48 h commencing between Days 3 and 8 post partum. Hormone concentrations were measured in jugular venous plasma. An episodic pattern of LH release was induced in all animals and there was no significant change in amplitude during treatment. However, cows treated between Days 7 and 8 ('late') showed higher LH episode peaks than did those treated between Days 3 and 6 ('early'). Plasma FSH concentrations showed a less clear episodic pattern in response to GnRH injection. The mean height of FSH responses to GnRH tended to be higher in the 'early' group than in the 'late' group, as did mean FSH concentrations during the pretreatment sampling period. Although clear episodic changes were not observed, GnRH treatment induced a rapid sustained rise in plasma oestradiol-17 beta concentrations, indicating the responsiveness of ovarian follicles to gonadotrophin stimulation early in the post-partum period. There was no difference in oestradiol-17 beta concentrations between the 'early' and 'late' groups during the treatment period. Only one cow exhibited preovulatory-type LH, FSH and oestradiol-17 beta surges during the 96-h post-treatment sampling period. It is concluded that: (1) responsiveness to GnRH pulses increases significantly and FSH responsiveness tends to decrease with time post partum, (2) ovarian follicles are able to secrete oestradiol-17 beta in response to GnRH-induced LH and FSH release during the early post-partum period and there is no time-dependent change in responsiveness; and (3) the lack of preovulatory surges, except in one cow, may reflect a temporary defect in the positive-feedback mechanism by which high concentrations of oestradiol-17 beta induce preovulatory gonadotrophin release.     

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.     

Oestradiol administration raises luteal LH receptor levels in intact and hysterectomized pigs

Occupied and unoccupied LH receptors in corpora lutea, and LH and progesterone concentrations in circulating plasma, were measured in non-pregnant gilts that had been treated with oestradiol-17 beta benzoate to prolong luteal function. Oestradiol benzoate (5 mg, administered on Day 12 after oestrus) delayed luteal regression and the decline in LH receptor levels at luteolysis and raised unoccupied receptor levels from 11.8 +/- 1.14 fmol/mg protein on Days 10--15 after oestrus to 31.8 +/- 3.26 fmol/mg protein on Days 15--21. There was no simultaneous rise in occupied receptor levels and occupancy decreased from 29.8 +/- 3.01 to 11.5 +/- 1.26%. Basal plasma LH concentrations were unchanged by oestradiol, but mean corpus luteum weight and plasma progesterone concentrations were slightly reduced. Oestradiol benzoate on Day 12 caused a similar increase in unoccupied receptor levels in gilts hysterectomized on Days 6--9 after oestrus, from 17.0 +/- 5.83 to 34.5 +/- 6.00 fmol/mg protein, determined on Days 15--18. Plasma concentrations of LH and progesterone were unchanged by oestradiol. Unoccupied receptor levels in corpora lutea and plasma LH and progesterone were unaltered by hysterectomy in untreated gilts. Occupied receptor levels were not influenced by hysterectomy or oestradiol. It is concluded that oestradiol-17 beta raises luteal LH receptor levels by a mechanism independent of the uterus.     

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.     

Effects of oestradiol on LH, FSH and prolactin in ovariectomized ewes

This study was conducted to test the hypothesis that the rate (dose/time) at which oestradiol-17 beta (oestradiol) is presented to the hypothalamo-pituitary axis influences secretion of LH, FSH and prolactin. A computer-controlled infusion system was used to produce linearly increasing serum concentrations of oestradiol in ovariectomized ewes over a period of 60 h. Serum samples were collected from ewes every 2 h from 8 h before to 92 h after start of infusion, and assayed for oestradiol, LH, FSH and prolactin. Rates of oestradiol increase were categorized into high (0.61-1.78 pg/h), medium (0.13-0.60 pg/h) and low (0.01-0.12 pg/h). Ewes receiving high rates of oestradiol (N = 11) responded with a surge of LH 12.7 +/- 2.0 h after oestradiol began to increase, whereas ewes receiving medium (N = 15) and low (N = 11) rates of oestradiol responded with a surge of LH at 19.4 +/- 1.7 and 30.9 +/- 2.0 h, respectively. None of the surges of LH was accompanied by a surge of FSH. Serum concentrations of FSH decreased and prolactin increased in ewes receiving high and medium rates of oestradiol, when compared to saline-infused ewes (N = 8; P less than 0.05). We conclude that rate of increase in serum concentrations of oestradiol controls the time of the surge of LH and secretion of prolactin and FSH in ovariectomized ewes. We also suggest that the mechanism by which oestradiol induces a surge of LH may be different from the mechanism by which oestradiol induces a surge of FSH.     

Secretion of LH, FSH and oestradiol-17 beta during the follicular phase of the oestrous cycle in the ewe

Plasma concentrations of LH, FSH and oestradiol-17 beta were measured in blood samples taken at 15 min intervals for 48 h during the follicular phase of four Merino ewes. The amplitude of pulses of LH and the mean concentration of LH were higher at the beginning of the follicular phase, 36-24 h before the preovulatory surge of LH (amplitude 2.4 ng ml-1, mean concentration 3.9 ng ml-1), than at the end, 24-0 h before the preovulatory surge (amplitude 1.2 +/- 0.1 ng ml-1; mean concentration 1.4 +/- 0.1 ng ml-1). There was no change in the inter-pulse interval during this time (mean 74 +/- 5 min). Over the same period, oestradiol levels increased from 7-8 pg ml-1 to a peak of 10-15 pg ml-1. Mean FSH concentrations declined (36-24 h: 3.6 ng ml-1 vs 24-0 h: 1.8 +/- 0.3 ng ml-1) before rising at the time of the preovulatory surge of LH and again 24 h later. It was concluded that the biphasic response of LH to oestrogen that is seen in ovariectomized ewes may also operate during the follicular phase of the oestrous cycle in entire ewes.     

Plasma gonadotrophin and ovarian steroid concentrations in women with menstrual cycles with a short luteal phase

Daily plasma concentrations of FSH, LH, oestradiol-17 beta and progesterone were compared for 12 cycles with a short luteal phase and 19 cycles with a luteal phase of normal length (i.e. cycles in which the luteal phase lasted 12 or more days). FSH and LH concentrations were suppressed in short luteal-phase cycles in the early follicular phase and the length of the follicular phase was prolonged (median duration, 14.5 days, range 13-21 days: compared with 12 days, range 9-17, in control cycles; P less than 0.025). Preovulatory oestradiol-17 beta values and the mid-cycle concentrations of FSH and LH were similar in both groups. Plasma progesterone values in the luteal phase were similar in both groups over the 2nd to 5th days inclusive after the midcycle LH peak but declined in the short luteal phases thereafter. In short luteal-phase cycles, menstruation occurred in the presence of higher levels of oestradiol-17 beta and progesterone than in cycles of normal length and the rise of gonadotrophin in the late luteal phase of the cycle was delayed. These findings suggest that in cycles with a short luteal phase there is a lack of synchrony between the ovarian and menstrual events.     

Effects of removal in late pregnancy of the corpus luteum, graafian follicle or ovaries on plasma progesterone, oestradiol, LH, parturition and post-partum oestrus in the tammar wallaby, Macropus eugenii

Concentrations of oestradiol-17 beta, progesterone, and luteinizing hormone (LH) were measured in plasma collected at 6- to 12-h intervals from tammars around the time of parturition and post-partum oestrus. Parturition occurred on Day 26 or 27 after reactivation of lactation-delayed pregnancy and coincided with a precipitous decline in progesterone levels. A sharp rise in oestradiol, from basal concentrations of less than 10 pg/ml to a peak of 13 to 32 pg/ml, as well as oestrus, followed the drop in progesterone by 8.3 and 9.8 h, respectively. The LH surge was dependent on the oestradiol rise and followed it by 7 h. Ovulation followed mating by about 30 h and the LH surge by 24 h. Removal of the ovary with the large Graafian follicle prevented the oestradiol rise, oestrus and the LH surge, but not parturition. Peripartum changes in peripheral oestradiol do not appear to be involved in initiation of parturition but the oestradiol rise and associated change in the oestradiol:progesterone ratio are important signals for post-partum oestrus and the LH surge.     

Use of bovine follicular fluid to increase ovulation rate or prevent ovulation in sheep

Romney ewes were injected intramuscularly once or twice daily for 3 days with 0, 0.1, 0.5, 1 or 5 ml of bovine follicular fluid (bFF) treated with dextran-coated charcoal, starting immediately after injection of cloprostenol to initiate luteolysis on Day 10 of the oestrous cycle. There was a dose-related suppression of plasma concentrations of FSH, but not LH, during the treatment period. On stopping the bFF treatment, plasma FSH concentrations 'rebounded' to levels up to 3-fold higher than pretreatment values. The mean time to the onset of oestrus was also increased in a dose-related manner by up to 11 days. The mean ovulation rates of ewes receiving 1.0 ml bFF twice daily (1.9 +/- 0.2 ovulations/ewe, mean +/- s.e.m. for N = 34) or 5.0 ml once daily (2.0 +/- 0.2 ovulations/ewe, N = 25) were significantly higher than that of control ewes (1.4 +/- 0.1 ovulations/ewe, N = 35). Comparison of the ovaries of ewes treated with bFF for 24 or 48 h with the ovaries of control ewes revealed no differences in the number or size distribution of antral follicles. However, the large follicles (greater than or equal to 5 mm diam.) of bFF-treated ewes had lower concentrations of oestradiol-17 beta in follicular fluid, contained fewer granulosa cells and the granulosa cells had a reduced capacity to aromatize testosterone to oestradiol-17 beta and produce cyclic AMP when challenged with FSH or LH. No significant effects of bFF treatment were observed in small (1-2.5 mm diam.) or medium (3-4.5 mm diam.) sized follicles. Ewes receiving 5 ml bFF once daily for 27 days, from the onset of luteolysis, were rendered infertile during this treatment period. Oestrus was not observed and ovulation did not occur. Median concentrations of plasma FSH fell to 20% of pretreatment values within 2 days. Thereafter they gradually rose over the next 8 days to reach 60% of pretreatment values where they remained for the rest of the 27-day treatment period. Median concentrations of plasma LH increased during the treatment period to levels up to 6-fold higher than pretreatment values. When bFF treatment was stopped, plasma concentrations of FSH and LH quickly returned to control levels, and oestrus was observed within 2 weeks. The ewes were mated at this first oestrus and each subsequently delivered a single lamb.     

Changes in plasma oestradiol-17beta, progesterone, testosterone, LH and fertility after treatment with I.C.I. 80, 996.

Fifty heifers were twice injected with I.C.I. 80, 996 and inseminated 72 h and 96 h after the second administration. Twenty eight of them (56%) became pregnant. Changes in plasma oestradiol-17 beta, progesterone and LH concentrations around the oestrus following the second injection were similar to those occurring in spontaneous oestrus. The pattern of testosterone secretion resembled that of oestradiol;-17 beta. The highest testosterone concentration (135 +/- 24 pg/ml) was measured on the third day after treatment with I.C.I. 80, 996.     

Induction of follicular development and ovulation in seasonally acyclic mares using gonadotrophin-releasing hormones and progesterone.

Deeply acyclic (seasonally anovulatory) mares were treated with GnRH or a GnRH analogue to induce follicular development and ovulation. Courses of GnRH (3--4) were administered at approximately 10-day intervals to reproduce the gonadotrophin surges which precede ovulation in the normal cycle. Exogenous progesterone was administered in an attempt to reproduce the luteal phase pattern. Induced serum FSH concentrations were comparable to those causing follicular development in the normal cycle, but induced LH levels were lower and of shorter duration than those of the periovulatory surge. Three of 4 mares treated with GnRH appeared to ovulate, but did not establish CL. Nine of 10 mares given GnRH analogue also developed follicles during the final treatment course, as did mares treated with progesterone only, while only 1 of 5 untreated control mares showed any ovarian development. Failure to induce final follicular maturation and CL development by this treatment regimen may be due to an inadequate LH surge at the time of the expected ovulation associated with the low preovulatory oestradiol-17 beta surge, possibly caused by the preceding FSH stimulation being inadequate or inappropriate. Progesterone treatment increased baseline FSH concentrations in GnRH-treated mares, and also stimulated follicular development in mares not treated with GnRH, indicating a possible role for progesterone in folliculogenesis and, indirectly, ovulation.     

Effect of FSH infusion on follicle development in GnRH agonist-treated gilts

The aim was to investigate the effect of infusion of purified FSH alone on follicle development in hypogonadotrophic GnRH agonist-treated gilts. Large-White hybrid gilts (n = 12) were treated during the mid-luteal phase and again after 28 days (day 0) with a potent slow releasing GnRH agonist. On day 3, seven gilts were infused for 168 h with 1.5 S1 units oFSH h-1 (equivalent to 1.5 units of bioactivity of NIH-FSH-S1 standard) and blood samples were collected. Ovaries were then recovered and all follicles > or = 1 mm in diameter were dissected and incubated for 2 h in 1 ml Eagle's minimum essential medium. The ovaries were recovered from the remaining five GnRH agonist-treated gilts on day 10 and also from five cyclic gilts during the late follicular phase (controls). Plasma FSH concentrations in GnRH agonist-treated gilts were lower (P < 0.01) than in follicular phase controls, increased (P < 0.001) after 1 h of FSH infusion and reached a plateau similar (P > 0.1) to that of controls after 8 h. Basal LH concentrations were similar (P > 0.1) between GnRH agonist-treated and control gilts and remained unchanged (P > 0.1) throughout the infusion period. GnRH agonist treatment reduced (P < 0.01) basal oestradiol concentrations compared with control gilts. Infusion with FSH alone increased (P < 0.001) plasma oestradiol concentrations after 96 h compared with those before infusion; when the animals were killed oestradiol concentrations were higher (P < 0.01) in GnRH agonist-treated gilts infused with FSH than in controls. This was also apparent by vulval swelling and behavioural oestrus. There were more follicles > or 1 mm in diameter in the GnRH agonist-treated groups than in the controls (184, 153 and 86 per animal; P < 0.01). Infusion with FSH increased the maximum follicle diameter (GnRH agonist: < 4 mm; FSH infused: < 12 mm; controls: < 10 mm) and tended to increase (P < 0.07) the mean number of follicles > or = 6 mm diameter per animal (FSH infused: 53; controls: 21). Total oestradiol production in vitro by follicles > or = 1 mm was higher (P < 0.01) in GnRH agonist-treated gilts infused with FSH and in follicular phase controls than in animals treated with GnRH agonist alone. However, oestradiol and testosterone secretion in vitro per follicle > or = 6 mm in diameter was lower (P < 0.05) in FSH-infused animals than in controls. In summary, although infusion of FSH alone stimulated the growth of multiple follicles of preovulatory size in GnRH agonist-treated gilts, steroidogenic output by individual follicles was impaired.     

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.     

Effects of oestradiol and some of its esters on gonadotrophin release and ovarian follicular dynamics in CIDR-treated beef cattle

Three experiments were conducted to: (1) compare the effect of three oestradiol formulations on gonadotrophin release in ovariectomised cows; (2) compare the effects of either oestradiol-17beta (E-17beta) or oestradiol benzoate (EB), given at two doses, on the synchrony of ovarian follicular wave emergence in CIDR-treated beef cattle; and (3) determine the timing of ovulation of the dominant follicle of a synchronised follicular wave following administration of E-17beta or EB 24h after progesterone withdrawal. In Experiment 1, ovariectomised cows (n = 16) received a once-used CIDR on Day 0 (beginning of the experiment) and were allocated randomly to receive 5mg of E-17beta, EB or oestradiol valerate (EV) plus 100mg progesterone i.m. The CIDR inserts were removed on Day 7. There were effects of time, and a treatment-by-time interaction (P < 0.0001) for plasma concentrations of both oestradiol and FSH. Plasma oestradiol concentrations peaked 12h after treatment, with highest (P < 0.01) peak concentrations in cows given E-17beta; estradiol concentrations subsequently returned to baseline by 36 h in E-17beta-treated cows and by 96 h in EB- and EV-treated cows. Plasma FSH concentrations decreased by 12h after oestradiol treatment in all groups (P < 0.0001), reached a nadir at 24h, and increased by 60 h in all groups; plasma FSH reached higher (P < 0.02) concentrations in E-17beta-treated than in EB- or EV-treated cows. In Experiment 2, non-lactating Hereford cows (n = 29) received a new CIDR on Day 0 (beginning of the experiment), and were assigned randomly to receive 1 or 5mg of E-17beta or EB i.m. on Day 1. On Day 8, CIDR were removed and PGF was given. Transrectal ultrasonography was done once daily from 2 days before CIDR insertion to 2 days after CIDR removal, and then twice-daily to ovulation. Although there was no difference among groups in the interval from oestradiol treatment to follicular wave emergence (4.2 +/- 0.3 days; P = 0.5), 5mg of E-17beta resulted in the least variable interval to wave emergence (P < 0.005), compared with the other treatment groups which were not different (P = 0.1). For the interval from CIDR removal to ovulation, there were no differences among groups for either means (P = 0.5) or variances (P = 0.1). In Experiment 3, beef heifers (n = 32) received a once-used CIDR on Day 0 (beginning of the experiment) plus 100mg progesterone i.m. and were assigned randomly to receive 5mg E-17beta or 1mg EB i.m. On Day 7, CIDR were removed and all heifers received PGF. On Day 8 (24h after CIDR removal), each group was subdivided randomly to receive 1mg of either E-17beta or EB i.m. There was no effect of oestradiol formulation on interval from treatment to follicular wave emergence (4.1 +/- 0.2 days; P = 0.7) or on the median interval (76.6h; P = 0.7) or range (72-120 h; P = 0.08) from CIDR removal to ovulation. In summary, oestradiol treatments suppressed FSH in ovariectomised cows, with the duration of suppression dependent on the oestradiol formulation. Both E-17beta and EB effectively synchronised ovarian follicular wave emergence and ovulation in CIDR-treated cattle, and the interval from CIDR removal to ovulation did not differ in heifers given either E-17beta or EB 24h after CIDR removal.     

Effect of the peripubertal pattern of LH and FSH secretion on in-vitro oestradiol-17 beta secretion and follicular growth within juvenile rat ovaries

Juvenile rat ovaries were placed in perfusion culture and exposed to (1) no gonadotrophin, (2) tonic NIH-FSH (200 ng RP-1 equiv./ml) or (3) NIH-FSH + NIH-LH pulses (2/h, amplitude = 80 ng RP-1 equiv./ml). After 3 h of perifusion, the ovaries were prepared for histological analysis and the perifusate assayed for oestradiol-17 beta. Since the NIH-FSH preparation is contaminated with LH, a second experiment was conducted using recombinant bovine LH and FSH. Ovaries were perifused for 3 h with (1) no hormones, (2) recombinant FSH (200 ng/ml) or (3) recombinant FSH plus 25 ng recombinant LH/ml. NIH-FSH alone increased the number of mid-size antral follicles (P less than 0.05) and decreased the number of small antral follicles (P less than 0.05). Pulsatile LH in the presence of FSH increased the number of mid-size antral follicles without reducing the number of small antral follicles. Studies with recombinant FSH and LH demonstrated that both FSH and LH are necessary to stimulate follicles to grow, indicating that the growth-promoting property of the NIH-FSH is due to the presence of both FSH and LH. Regardless of whether NIH or recombinant gonadotrophins were used, follicular growth was induced without increasing oestradiol secretion. These results demonstrate that enhanced oestradiol secretion is not essential for the induction of follicular growth, while both LH and FSH are necessary to stimulate small antral follicles to grow into mid-size antral follicles.(ABSTRACT TRUNCATED AT 250 WORDS)