Treatment with bovine follicular fluid suppresses follicular development in gonadotrophin-releasing hormone-treated anoestrous ewes

The role of FSH in gonadotrophin-releasing hormone (GnRH)-induced follicular development in anoestrous ewes was investigated using injections of bovine follicular fluid (bFF) to reduce plasma FSH levels. Groups of five animals were treated for 12 h with GnRH (250 ng at 2-h intervals) alone, GnRH plus bFF or saline alone, or for 36 h with GnRH alone, GnRH plus bFF or bFF alone. The administration of bFF (1.5 ml s.c. at 8-h intervals) significantly (P less than 0.05) reduced mean plasma FSH levels, but with the exception of animals treated with bFF alone, had no effect on LH levels. Treatment with bFF alone for 36 h resulted in a significant (P less than 0.05) increase in LH concentrations. There was considerable variation in the number of follicles greater than or equal to 2 mm in diameter in the treatment groups. The mean diameter, oestradiol secretion and number of 'oestrogenic' follicles were significantly (P less than 0.01) reduced in ewes treated with GnRH plus bFF or bFF alone for 36 h compared with those treated with GnRH alone. Testosterone secretion by the follicles was not affected by treatment. These results confirm previous findings that treatment with bFF decreases circulating FSH levels in anoestrous ewes and, moreover, that concurrent administration of bFF and GnRH inhibits the follicular maturation that is induced by treatment with GnRH alone, suggesting that FSH as well as LH is required for follicular maturation in the ewe.     

Effects of long-term treatment with LH on induction of cyclic ovarian activity in seasonally anoestrous ewes

The aim of the present study was to establish whether cyclic ovarian activity could be induced and then maintained in anoestrous Romney ewes by the long-term administration of regular intravenous pulses of LH (10 micrograms ovine LH i.v. once every 1 or 2 h for 29-91 days). The LH pulse regimen was designed to generate plasma profiles of LH that were comparable to those experienced during the luteal and follicular phases of the oestrous cycle. The results showed that the LH treatments were capable of inducing cyclic ovarian activity, as assessed from the concentrations of progesterone in plasma, but that the treatments were inadequate for sustaining cyclic activity beyond two consecutive progestational phases. After 35-56 days of treatment, the plasma concentrations of FSH declined significantly (P less than 0.05) relative to those in the untreated animals. These data suggest that FSH supplementation as well as LH might be required for the long-term maintenance of cyclic ovarian activity in seasonally anoestrous ewes.     

Changes in FSH and the pulsatile secretion of LH during treatment of ewes with bovine follicular fluid throughout the luteal phase of the oestrous cycle

Treatment of Damline ewes with twice-daily i.v. injections of bovine follicular fluid during the luteal phase for 10 or 2 days before prostaglandin-induced luteolysis resulted in a delay in the onset of oestrous behaviour and a marginal increase in ovulation rate. During the treatment cycle, blood samples were withdrawn at 15-min intervals for 25 h from 08.00 h on days 1, 6 and 10 (day 0 = oestrus). At all three stages of the luteal phase, plasma FSH concentrations were suppressed relative to controls 3 h after the 09.00 h injection of follicular fluid and remained low until 06.00 h on the following day. In the 10-day treatment group LH pulse amplitude was significantly greater than that of controls on days 6 and 10. Pulse frequency remained high throughout treatment and was significantly higher relative to controls on day 10 despite normal progesterone levels. The results suggest that the higher pulsatile LH secretion during the luteal phase is due to reduced negative feedback effects of oestradiol occurring as a result of the follicular fluid-induced reduction in FSH.     

Inadequate function of corpora lutea following the induction of ovulation with monensin and FSH in seasonally anoestrous ewes

Sixteen ewes in mid-seasonal anoestrus were stimulated to ovulate using sequential injections of FSH (total dose 10 mg) over a 4-day period. Half of the ewes received a dietary growth promotant (monensin) known to enhance the ovarian response to exogenous gonadotrophins. The ewes were ovariectomized on day 5 or 11 (day 0 = the initiation of FSH treatment). Serial blood samples were taken in half of the ewes to determine peripheral concentrations of LH and a single sample of ovarian venous blood was collected before ovariectomy. All luteal structures were dissected from the ovaries, counted and incubated in vitro to determine progesterone production. The luteal structures were then examined histologically for the abundance of luteal cells. The physical appearance of the ovary, along with plasma concentrations of LH and ovarian venous oestradiol indicated that the monensin-treated ewes ovulated before control ewes. The corpora lutea from control ewes produced significantly (P less than 0.05) more progesterone than did the corpora lutea from the monensin-treated group. Furthermore, only 7% of the remaining luteal structures in the monensin-treated group produced significant amounts of progesterone on day 11, whereas 61% of the luteal structures in the control group were actively secreting progesterone. The mean number of granulosa cells in the follicles was similar at ovulation in the two groups, but the mean numbers of large and small luteal cells were significantly (P less than 0.05) lower in luteal structures from the monensin-treated ewes than in those from the control ewes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Episodic ovarian inhibin secretion is not due to LH pulses in anoestrous ewes

In sheep, secretion of oestradiol by the ovary is stimulated by pulses of LH but the factors controlling ovarian inhibin secretion are not well understood. We have investigated the effect of a single injection of LH on the ovarian secretion of inhibin. Six anoestrous Finn-Merino ewes which had one ovary autotransplanted to a site in the neck had jugular and timed ovarian venous blood samples collected at 10-min intervals for a total of 5 h. The secretion rates of both inhibin (1-3 ng/min) and oestradiol (0.5-8 ng/min) were similar to those observed during the breeding season indicating significant follicular development in these animals. After injection of 2.5 micrograms NIH-LH-S25 intravenously the concentration of LH in plasma rose from a baseline of 1.8 +/- 0.1 (S.E.M.) microgram/l to a peak of 3.9 +/- 0.3 micrograms/l (P less than 0.01). This LH pulse stimulated a corresponding increase (P less than 0.01) in oestradiol secretion from a basal level of 0.9 +/- 0.2 ng/min to a peak of 4.6 +/- 0.6 ng/min that occurred within 30 min of injection. Although inhibin secretion was episodic in nature, increases were not related to either exogenous or endogenous LH pulses. We conclude that, in contrast to oestradiol, the secretion of inhibin by the ovary is not controlled acutely by changes in plasma levels of LH during anoestrus.     

Differential suppression of FSH and LH secretion by follicular fluid in the presence or absence of GnRH

The differential role of porcine follicular fluid (pFF) in regulating follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release in vivo in situations of different gonadotropin releasing hormone (GnRH) backgrounds was studied. In experiment 1, 2-week ovariectomized rats injected intravenously with 4, 16 or 64 mg of protein from pFF, showed a dose-dependent suppression of FSH over time, with a maximal suppression to 40% of control values by 10 h. LH levels were slightly, but significantly, elevated by the two lower doses, but not by the highest dose of pFF. In experiment 2, 64 mg pFF was superimposed (i.v. injection) in ovariectomized rats injected subcutaneously with a high dose of GnRH antagonist (500 micrograms) 24 h earlier. The pFF suppressed FSH 35% below the level achieved in the absence of GnRH stimulation, with no effect on LH. In experiment 3, the rise in FSH secretion in acutely ovariectomized rats was shown to be inhibited by 8 or 32 mg pFF administered intravenously 3.5 h after surgery. Injection of GnRH (250 or 1,000 ng) 4.5 h after pFF could not overcome the inhibitory action of pFF on FSH, although non-pFF-treated controls responded in a dose-dependent fashion to GnRH stimulation. The expected LH response to GnRH was not affected by pFF, except in the group receiving 1,000 ng GnRH and 8 mg pFF. In these rats, LH was enhanced in one trial, but suppressed in a replicate trial, illustrating the inconsistent effects of pFF on LH under conditions of high GnRH stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of ovine plasma FSH by bovine follicular fluid: neutralization by plasma from ewes immunized against an inhibin-enriched preparation from bovine follicular fluid

Adult Merino ewes were immunized against an inhibin-enriched preparation (bFFI) obtained by affinity chromatography of bovine follicular fluid (bFF). Plasma was obtained in early luteal phase from these ewes and from control ewes immunized against bovine serum albumin. Ten months after ovariectomy the plasma concentration of FSH, but not LH, in control ewes was decreased by four s.c. injections of 8 ml bFFI (17,500 units inhibin/injection). There was no decrease in plasma concentrations of FSH or LH in immunized ewes with the same dose of bFFI. In a second study with long-term ovariectomized ewes, four injections of 20 ml plasma from the immunized ewes significantly reduced the decrease in FSH concentration caused by four injections of steroid-free bFF (2500 units inhibin/injection) in comparison with similar ewes injected with plasma from control ewes. These results show that the plasma of ewes immunized against bFFI contains substances, presumably antibodies, which neutralize the FSH-suppressive effects of bFF and bFFI in ovariectomized ewes.     

Effects of bovine follicular fluid on gonadotrophin secretion in intact and chronically ovariectomized ewes before and after desensitization of pituitary gonadotrophs to gonadotrophin-releasing hormone

Intact and chronically ovariectomized ewes were treated for 4 days with charcoal-treated bovine follicular fluid (FF) or charcoal-treated bovine serum during the late-anoestrous period, and the effects on basal and gonadotrophin-releasing hormone (GnRH)-induced secretion of LH and FSH observed. Subsequently, ewes received s.c. implants containing a sustained-release formulation of a potent GnRH agonist D-Ser(But)6-Azgly10-LHRH (ICI 118630) to desensitize pituitary gonadotrophs to hypothalamic stimulation, and the effects of bovine FF and bovine serum were re-assessed 2 weeks later. Chronic exposure (for 2-3 weeks) to ICI 118630 significantly reduced basal levels of LH and FSH in both intact and ovariectomized ewes and completely abolished both spontaneous LH pulses as well as exogenous GnRH-induced acute increases in plasma LH and FSH levels. Treatment with bovine FF significantly reduced plasma FSH levels, but not LH levels, in both intact and ovariectomized ewes before and after chronic exposure to ICI 118630. In intact ewes before exposure to ICI 118630, treatment with bovine FF actually enhanced pulsatile LH secretion and raised mean plasma LH levels by 240% (P less than 0.05). No such stimulatory effect of bovine FF on LH secretion was observed in intact ewes exposed to ICI 118630 or in ovariectomized ewes before or after exposure to ICI 118630, suggesting that the effect probably involved an alteration in ovarian steroid feedback affecting hypothalamic GnRH output. Treatment with bovine FF did not significantly affect the magnitude of GnRH-induced surges of LH or of FSH observed in either intact or ovariectomized ewes before exposure to ICI 118630.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of level of food intake of ewes on the secretion of LH and FSH and on the pituitary response to gonadotrophin-releasing hormone in ovariectomized ewes

The effect of level of food intake on LH and FSH profiles and pituitary sensitivity to gonadotrophin-releasing hormone (GnRH) was investigated in two groups of 12 ovariectomized ewes. Ewes with a high intake (group H) had a mean daily intake (+/- S.E.M.) of 1.99 +/- 0.075 kg dry matter (DM)/head per day while ewes with a moderate intake (group M) consumed a mean of 1.02 +/- 0.021 kg DM/head per day. Ovaries were surgically removed from six ewes of each group on day 11 of the luteal phase and from the remainder 30 h after an injection of 100 micrograms prostaglandin analogue given on day 11 to induce luteolysis. During both the luteal phase and the follicular phase, mean LH and FSH concentrations and LH pulse frequencies and amplitudes were unaffected by the level of intake but mean plasma prolactin concentrations were higher (P less than 0.05) in group H than in group M ewes in the follicular phase. Mean LH and FSH concentrations at day 2 after ovariectomy were unaffected by treatment while mean prolactin concentrations were higher (P less than 0.05) in group H than in group M ewes. At day 7 after ovariectomy, mean LH and FSH concentrations were lower (P less than 0.05) in group H than in group M ewes although mean LH pulse frequencies and pulse amplitudes were not significantly affected by the level of intake at either time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of an effect of morphine or naloxone on the oestrogen-induced LH surge in anoestrous ewes

To determine whether opioid mechanisms modulate the positive feedback effect of oestrogen on LH secretion, anoestrous ewes were given a single injection of 50 micrograms oestradiol benzoate (OB), followed by infusions of morphine or naloxone. All sheep were injected i.m. with 50 micrograms OB at 00.00 h. In experiment 1, sheep were given i.v. infusions of the following: group 1, 12 ml saline/h from 09.00 to 15.00 h (n = 12); group 2, 40 mg naloxone/h from 09.00 to 12.00 h (n = 5); group 3, 40 mg naloxone/h from 10.00 to 14.00 h (n = 5); group 4, 10 mg morphine/h from 09.00 to 15.00 h (n = 5); and group 5, 20 mg morphine/h from 09.00 to 15.00 h (n = 5). Jugular blood samples were taken at 30-min intervals to monitor LH surges, which commenced 13.0 +/- 0.6 h after injection of OB in control (OB plus saline) ewes. The infusions of naloxone or morphine did not affect the timing or magnitude of the oestrogen-induced LH surge. To examine the possibility that opioidergic regulation of the LH surge occurred earlier than the infusion regimens in experiment 1, sheep were infused from the time of the OB injection (00.00 h) until 15.00 h. In this experiment (experiment 2), sheep were given i.v. infusions of the following: group 1, 4.2 ml saline/h (n = 5); group 2, 20 mg naloxone/h (n = 5); and group 3, 20 mg morphine/h (n = 5). As in experiment 1, treatment with neither the opioid agonist or antagonist was able to alter the positive feedback response of OB.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the body condition of ewes on the secretion of LH and FSH and the pituitary response to gonadotrophin-releasing hormone

The effects of body fat content (body condition) of ewes on hypothalamic activity and gonadotrophin-releasing hormone (GnRH) secretion and on pituitary sensitivity to GnRH were investigated using Scottish Blackface ewes. Two groups of 12 ewes were fed so that they achieved either a high body condition score (2.98, S.E.M. = 0.046; approximately 27% of empty body weight as fat) or a low body condition score (1.94, S.E.M. = 0.031; approximately 19% of empty body weight as fat) by 4 weeks before the period of study. Thereafter, they were differentially fed so that the difference in mean condition score was maintained. Oestrus was synchronized, and on day 11 of the subsequent cycle half of the ewes of each group were ovariectomized. On day 12, the remaining ewes were injected (i.m.) with 100 micrograms prostaglandin F2 alpha analogue and ovariectomized 30 h later. Numbers of large ovarian follicles and corpora lutea present at ovariectomy were recorded. Blood samples were collected at 15-min intervals for 12 h on day 10 of the cycle (luteal phase) and at 10-min intervals from 24 to 30 h after prostaglandin injection (follicular phase). At days 2 and 7 after ovariectomy, samples were collected at 15-min intervals for 8 h and ewes were then injected with 10 micrograms GnRH and samples were collected for a further 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the secretion of LH pulses, FSH and prolactin during the preovulatory phase of the oestrous cycle of the ewe and the influence of treatment with bovine follicular fluid during the luteal phase

It has previously been shown that treatment of ewes with bovine follicular fluid (bFF) throughout the luteal phase of the oestrous cycle lowers plasma levels of FSH but increases the frequency and amplitude of the pulses of LH. Under these conditions, ovarian follicles grow to a maximum diameter of 2.7 mm and have a reduced capacity to release oestradiol. We have examined the nature of the gonadotrophin signals controlling follicular development in the normally cycling ewe and have investigated the effects of previous exposure to bFF on these signals and the follicular responses to them. Control ewes (n = 7) were injected i.v. with 9 ml bovine serum and treated ewes were injected with 9 ml bFF, twice daily from days 1 to 10 of the luteal phase (day 0 = oestrus). The ewes were injected with prostaglandin analogue on day 11 of the cycle to induce luteolysis and the gonadotrophin patterns were studied in blood sampled from these animals every 10 min for up to 72 h during the subsequent follicular phase. Following luteolysis (and the end of bFF treatment), LH pulse frequency increased rapidly in both groups and reached 1 pulse/h within 6 h. Thereafter, pulse frequency increased marginally and reached 1 pulse/50 min by the onset of the LH surge. This pattern was not affected by previous treatment with bFF. In the control ewes, the amplitude of the LH pulses did not change significantly following luteolysis or at any time during the follicular phase, while the levels of FSH declined slowly until the onset of the surge. In the treated ewes, on the other hand, there was an immediate increase in both LH pulse amplitude and the concentration of FSH immediately after the end of bFF treatment at luteolysis, and they remained above control levels for 24 and 16 h respectively. Plasma prolactin levels did not appear to change around the time of luteolysis but showed a marked and significant diurnal rhythm (nadir around noon and peak around midnight) in both groups. The concentrations of prolactin were significantly (P less than 0.001) lower and the preovulatory peak was delayed and reduced in the bFF-treated ewes relative to controls. The onset of oestrus was also significantly (P less than 0.01) delayed by bFF treatment, but the ovulation rates did not differ between the groups. Furthermore, comparisons within or between groups revealed no significant relationships between any of the variables of plasma LH secretion during the follicular phase and the subsequent ovulation rate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of FSH and LH on adenylate cyclase activity in rat granulosa cell membranes during follicular maturation

Adenylate cyclase activity was measured in membranes prepared of granulosa cells isolated from PMSG-treated immature rats and the effects of ovine, highly purified human and rat gonadotrophins were compared. Furthermore, a comparison of the effects of the human preparations (hLH, hFSH) on the adenylate cyclase activity in membranes prepared from granulosa cells isolated at different stages of follicular maturation, was performed. The adenylate cyclase in membranes of immature granulosa cells was stimulable with FSH but not with LH, while in pre-ovulatory granulosa cell membranes, both gonadotrophins were stimulatory with FSH generally being more effective than LH. Surprisingly, the dose-response curve for ovine LH (oLH) was biphasic with a plateau at a level of adenylate cyclase activity corresponding to the maximal stimulatory effect of hCG. With increasing oLH concentrations the response resumed and the maximal stimulation corresponded to that of oFSH. With highly purified rat gonadotrophins the FSH response was significantly higher than the response to LH at all concentrations tested. Using highly purified human gonadotrophins the maximal FSH response was 50% higher than the maximal LH response and by adding increasing concentrations of hFSH to a maximally stimulatory concentration of hLH it was possible to mimic the biphasic dose-response curve for oLH. When the membranes were prepared from granulosa cells isolated after the pro-oestrus LH/FSH surge there was clear increase in the sensitivity of the adenylate cyclase to stimulation with LH although the maximal response was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of the Hypothalamus and FSH and LH secretion in the ferret

The acute effects of hypothalamic electrical stimulation on the secretion of FSH and LH have been examined in anestrous and estrous ferrets. Basal levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were higher in the anestrous than in the estrous animals; the anestrous animals showed a more marked increase in gonadotropin levels following hypothalamic stimulation than did the estrous animals whether the stimulation current was constant or increased in the course of the experiment. It is concluded that the physiological difference in the control of gonadotropin secretion in the estrous (vs the anestrous) ferret is not reflected in an increased sensitivity of the hypothalamo-hypophysial axis to stimulation.     

The role of LH in the autofeedback inhibition of LH and FSH secretion in ovariectomized rats

This study examined the role of exogenous LH in the autofeedback regulation of LH and FSH release in ovariectomized rats. The rats were implanted with third ventricular cannulae three weeks after ovariectomy and fitted with silastic jugular cannulae one week later. Baseline hormone levels were established on the day of experimentation in conscious, unrestrained animals. Thereafter, experimental animals received intraventricularly (IVT) either a 9 ug or 20 ug dose of a purified preparation of human (h)LH that did not crossreact in our rLH RIA. In response to 20 ug, but not 9 ug of LH, plasma levels of both LH and FSH were significantly reduced during the following one hour period compared to values in controls receiving buffer IVT. Administration of ovine (o)LH (6 ug, IVT), a preparation which crossreacts in the rLH RIA, significantly elevated plasma levels of detectable LH during the experimental period indicating that LH reaches the blood stream from the third ventricle and, thus, may effect endogenous hormone release at either the pituitary or hypothalamic levels. However, in animals preinjected with 9 or 20 ug of hLH IVT one hour earlier the surge of both LH and FSH in response to LHRH (25 ng iv) was not different from that in the buffer-injected controls receiving LHRH which indicates that pituitary responsiveness was not suppressed by the effective dose of hLH. The results of this study suggest that the inhibitory shortloop feedback of LH on endogenous LH and FSH secretion in ovariectomized rats occurs at the level of the hypothalamus.