Does the type II glucocorticoid receptor mediate cortisol-induced suppression in pituitary responsiveness to gonadotropin-releasing hormone?

Stress-like elevations in plasma cortisol suppress LH pulse amplitude in ovariectomized ewes by inhibiting pituitary responsiveness to GnRH. Here we sought to identify the receptor mediating this effect. In a preliminary experiment GnRH and LH pulses were monitored in ovariectomized ewes treated with cortisol plus spironolactone, which antagonizes the type I mineralocorticoid receptor (MR), or with cortisol plus RU486, which antagonizes both the type II glucocorticoid receptor (GR) and the progesterone receptor (PR). Cortisol alone reduced LH pulse amplitude, but not pulsatile GnRH secretion, indicating that it reduced pituitary responsiveness to endogenous GnRH. RU486, but not spironolactone, reversed this suppression. We next tested whether RU486 reverses the inhibitory effect of cortisol on pituitary responsiveness to exogenous GnRH pulses of fixed amplitude, frequency, and duration. Hourly GnRH pulses were delivered to ovariectomized ewes in which endogenous GnRH pulses were blocked by estradiol during seasonal anestrus. Cortisol alone reduced the amplitude of LH pulses driven by the exogenous GnRH pulses. RU486, but not an antagonist of PR (Organon 31710), prevented this suppression. Thus, the efficacy of RU486 in blocking the suppressive effect of cortisol is attributed to antagonism of GR, not PR. Together, these observations imply that the type II GR mediates cortisol-induced suppression of pituitary responsiveness to GnRH.     

Psychosocial stress inhibits amplitude of gonadotropin-releasing hormone pulses independent of cortisol action on the type II glucocorticoid receptor

Our laboratory has developed a paradigm of psychosocial stress (sequential layering of isolation, blindfold, and predator cues) that robustly elevates cortisol secretion and decreases LH pulse amplitude in ovariectomized ewes. This decrease in LH pulse amplitude is due, at least in part, to a reduction in pituitary responsiveness to GnRH, caused by cortisol acting via the type II glucocorticoid receptor (GR). The first experiment of the current study aimed to determine whether this layered psychosocial stress also inhibits pulsatile GnRH release into pituitary portal blood. The stress paradigm significantly reduced GnRH pulse amplitude compared with nonstressed ovariectomized ewes. The second experiment tested if this stress-induced decrease in GnRH pulse amplitude is mediated by cortisol action on the type II GR. Ovariectomized ewes were allocated to three groups: nonstress control, stress, and stress plus the type II GR antagonist RU486. The layered psychosocial stress paradigm decreased GnRH and LH pulse amplitude compared with nonstress controls. Importantly, the stress also lowered GnRH pulse amplitude to a comparable extent in ewes in which cortisol action via the type II GR was antagonized. Therefore, we conclude that psychosocial stress reduces the amplitude of GnRH pulses independent of cortisol action on the type II GR. The present findings, combined with our recent observations, suggest that the mechanisms by which psychosocial stress inhibits reproductive neuroendocrine activity at the hypothalamic and pituitary levels are fundamentally different.     

Does cortisol acting via the type II glucocorticoid receptor mediate suppression of pulsatile luteinizing hormone secretion in response to psychosocial stress?

This study assessed the importance of cortisol in mediating inhibition of pulsatile LH secretion in sheep exposed to a psychosocial stress. First, we developed an acute psychosocial stress model that involves sequential layering of novel stressors over 3-4 h. This layered-stress paradigm robustly activated the hypothalamic-pituitary-adrenal axis and unambiguously inhibited pulsatile LH secretion. We next used this paradigm to test the hypothesis that cortisol, acting via the type II glucocorticoid receptor (GR), mediates stress-induced suppression of pulsatile LH secretion. Our approach was to determine whether an antagonist of the type II GR (RU486) reverses inhibition of LH pulsatility in response to the layered stress. We used two animal models to assess different aspects of LH pulse regulation. With the first model (ovariectomized ewe), LH pulse characteristics could vary as a function of both altered GnRH pulses and pituitary responsiveness to GnRH. In this case, antagonism of the type II GR did not prevent stress-induced inhibition of pulsatile LH secretion. With the second model (pituitary-clamped ovariectomized ewe), pulsatile GnRH input to the pituitary was fixed to enable assessment of stress effects specifically at the pituitary level. In this case, the layered stress inhibited pituitary responsiveness to GnRH and antagonism of the type II GR reversed the effect. Collectively, these findings indicate acute psychosocial stress inhibits pulsatile LH secretion, at least in part, by reducing pituitary responsiveness to GnRH. Cortisol, acting via the type II GR, is an obligatory mediator of this effect. However, under conditions in which GnRH input to the pituitary is not clamped, antagonism of the type II GR does not prevent stress-induced inhibition of LH pulsatility, implicating an additional pathway of suppression that is independent of cortisol acting via this receptor.     

Does cortisol inhibit pulsatile luteinizing hormone secretion at the hypothalamic or pituitary level?

Elevations in glucocorticoids suppress pulsatile LH secretion in sheep, but the neuroendocrine sites and mechanisms of this disruption remain unclear. Here, we conducted two experiments in ovariectomized ewes to determine whether an acute increase in plasma cortisol inhibits pulsatile LH secretion by suppressing GnRH release into pituitary portal blood or by inhibiting pituitary responsiveness to GnRH. First, we sampled pituitary portal and peripheral blood after administration of cortisol to mimic the elevation stimulated by an immune/inflammatory stress. Within 1 h, cortisol inhibited LH pulse amplitude. LH pulse frequency, however, was unaffected. In contrast, cortisol did not suppress either parameter of GnRH secretion. Next, we assessed the effect of cortisol on pituitary responsiveness to exogenous GnRH pulses of fixed amplitude, duration, and frequency. Hourly pulses of GnRH were delivered to ewes in which endogenous GnRH secretion was blocked by estradiol. Cortisol, again, rapidly and robustly suppressed the amplitude of GnRH-induced LH pulses. We conclude that, in the ovariectomized ewe, cortisol suppresses pulsatile LH secretion by inhibiting pituitary responsiveness to GnRH rather than by suppressing hypothalamic GnRH release.     

Endotoxin inhibits pituitary responsiveness to gonadotropin-releasing hormone

Immune/inflammatory challenges powerfully suppress reproductive neuroendocrine activity. This inhibition is generally considered to be centrally mediated via mechanisms that regulate GnRH secretion. The present study provides two lines of evidence that bacterial endotoxin, a commonly used model of immune/inflammatory challenge, also acts to inhibit pituitary responsiveness to GNRH: In the first experiment, pulsatile secretion of GnRH into pituitary portal blood and LH into peripheral blood were monitored in ovariectomized ewes treated with a low dose of endotoxin. Although this treatment only marginally suppressed GnRH pulsatile secretion, it markedly disrupted LH pulsatility. In extreme cases, the low dose of endotoxin blocked LH pulses without inhibiting endogenous GnRH pulses, thereby uncoupling GnRH and LH pulsatile suppression. In the second experiment, we tested the hypothesis that endotoxin inhibits pituitary responsiveness to exogenous GnRH pulses. Hourly pulses of GnRH were delivered to ovariectomized ewes in which endogenous GnRH secretion was blocked. Endotoxin suppressed the amplitude of GnRH-induced LH pulses. Together, these observations support the conclusion that endotoxin inhibits pituitary responsiveness to GNRH:     

Pulsatile administration of gonadotrophin-releasing hormone stimulates, in oestrogen-treated anaesthetized ovariectomized ewes, a surge release of LH qualitatively and quantitatively different from that induced by oestradiol in conscious ovariectomized ewes

The nature of the gonadotrophin-releasing hormone (GnRH) stimulus of the pituitary necessary for the oestrogen-induced plasma LH surge was studied in ovariectomized ewes. The sheep were treated with oestradiol benzoate (50 micrograms i.m.) at 0 h, and the hypothalamic contribution to the LH surge was blocked by pentobarbitone anaesthesia over the time during which the surge was expected (11-31 h). Pituitary responsiveness to exogenous GnRH (100 ng) administered i.v. in a pulsatile mode (once per hour or once per 20 min) over the period 15-30 h was assessed from plasma concentrations of LH. Neither of the GnRH treatments induced patterns of LH secretion similar to those seen in conscious ovariectomized ewes given oestrogen only. Plasma LH secretion in response to hourly GnRH pulses was less (P less than 0.01) than that associated with oestrogen-induced plasma LH surges in conscious control ewes. With pulses of GnRH administered every 20 min the amount of LH released was greater (P less than 0.05) than that in oestrogen-treated conscious control ewes. In contrast to the single surge induced by oestradiol in conscious ewes, GnRH pulses given every 20 min elicited phasic patterns of LH secretion consisting of two or three distinct surges. The failure of GnRH treatment to elicit an LH surge similar to an oestrogen-induced surge could reflect inappropriate GnRH treatment regimens, and/or inadequate priming of the pituitary with GnRH after induction of anaesthesia but before GnRH treatment.     

Sex difference in the suppressive effect of cortisol on pulsatile secretion of luteinizing hormone in sheep

We tested the hypothesis that there are sex differences in the inhibitory effect of cortisol on pulsatile LH secretion and pituitary responsiveness to GnRH in gonadectomized sheep. In experiment 1, pulsatile LH secretion was examined in gonadectomized ewes and rams infused with either saline, a low (250 microg/kg.h) or a high (500 microg/kg.h) dose of cortisol for 30 h. In experiment 2, direct pituitary actions of cortisol were assessed by monitoring LH pulse amplitude in response to exogenous GnRH in hypothalamo-pituitary disconnected ewes and rams infused with the low dose of cortisol. In experiment 1, the mean (+/-sem) plasma LH concentration was (P<0.05) reduced significantly during cortisol infusion in both sexes, but the effect was greater in rams. In ewes, LH pulse amplitude and frequency were reduced (P<0.05) at the high, but not the low, cortisol dose, whereas total LH output (LH pulse amplitude multiplied by frequency) was reduced (P<0.05) at both doses. In rams, LH pulse frequency and amplitude and total LH output were (P<0.05) reduced significantly at both cortisol doses. In experiment 2, plasma LH concentration and pulse amplitude in response to exogenous GnRH were not affected by infusion of cortisol in either sex. We conclude that gonadectomized rams are more sensitive than gonadectomized ewes to the effects of cortisol to inhibit LH secretion and that sex differences exist in the specific actions of cortisol on LH pulses. The results of experiment 2 suggest that intact hypothalamic input to the pituitary is necessary for cortisol to inhibit pituitary responsiveness to GnRH.     

Cortisol reduces gonadotropin-releasing hormone pulse frequency in follicular phase ewes: influence of ovarian steroids

Stress-like elevations in plasma glucocorticoids suppress gonadotropin secretion and can disrupt ovarian cyclicity. In sheep, cortisol acts at the pituitary to reduce responsiveness to GnRH but does not affect GnRH pulse frequency in the absence of ovarian hormones. However, in ewes during the follicular phase of the estrous cycle, cortisol reduces LH pulse frequency. To test the hypothesis that cortisol reduces GnRH pulse frequency in the presence of ovarian steroids, the effect of cortisol on GnRH secretion was monitored directly in pituitary portal blood of follicular phase sheep in the presence and absence of a cortisol treatment that elevated plasma cortisol to a level observed during stress. An acute (6 h) cortisol increase in the midfollicular phase did not lower GnRH pulse frequency. However, a more prolonged (27 h) increase in cortisol beginning just before the decrease in progesterone reduced GnRH pulse frequency by 45% and delayed the preovulatory LH surge by 10 h. To determine whether the gonadal steroid milieu of the follicular phase enables cortisol to reduce GnRH pulse frequency, GnRH was monitored in ovariectomized ewes treated with estradiol and progesterone to create an artificial follicular phase. A sustained increment in plasma cortisol reduced GnRH pulse frequency by 70% in this artificial follicular phase, in contrast to the lack of an effect in untreated ovariectomized ewes as seen previously. Thus, a sustained stress-like level of cortisol suppresses GnRH pulse frequency in follicular phase ewes, and this appears to be dependent upon the presence of ovarian steroids.     

Neuroendocrine control of follicle-stimulating hormone (FSH) secretion: III. Is there a gonadotropin-releasing hormone-independent component of episodic FSH secretion in ovariectomized and luteal phase ewes?

Our previous studies in ovariectomized ewes have provided direct evidence that FSH secretion is comprised of basal and episodic modes. In those studies, each GnRH pulse coincided with an FSH pulse, but additional FSH pulses were noted. To determine whether non-GnRH-associated pulses of FSH represent a GnRH-independent component of FSH secretion, we determined whether episodic FSH secretion persists after blockade of GnRH action with a GnRH antagonist. Hypophyseal portal and jugular blood was collected from five ovariectomized and six luteal phase ewes at 5-min intervals for 6 h before and 6 h after a single iv injection of Nal-Glu (10 micro g/kg body weight). Hypophyseal portal LH and FSH and jugular patterns of FSH were compared with patterns of GnRH. Before Nal-Glu, in both models, there was a one-to-one concordance between GnRH and portal LH pulses, and each GnRH pulse was associated with a FSH pulse. However, additional non-GnRH-associated pulses of FSH were present. Nal-Glu administration eliminated LH but not FSH pulsatility. Nal-Glu inhibited interaction of GnRH I with GnRH type I receptor but not interaction of GnRH II with type II receptor. These studies provide the first direct evidence of the existence of an acute GnRH I-independent component of episodic FSH secretion.     

The oestrogen-induced surge of LH requires a 'signal' pattern of gonadotrophin-releasing hormone input to the pituitary gland in the ewe

Two experiments were conducted with ovariectomized and hypothalamo-pituitary disconnected (HPD) ewes to ascertain the pattern of inputs, to the pituitary gland, of gonadotrophin-releasing hormone (GnRH) necessary for the full expression of an oestrogen-induced LH surge. The standard GnRH replacement to these sheep was to give pulses of 250 ng (i.v.) every 2h; at the onset of experimentation, pulses were given hourly. In experiment 1, groups of sheep (n = 7) were given an i.m. injection of 50 micrograms oestradiol benzoate, and after 10 h the GnRH pulse frequency or pulse amplitude was doubled. Monitoring of plasma LH concentrations showed that a doubling of pulse frequency produced a marked increase in baseline values, whereas a doubling of amplitude had little effect on the LH response. In a second experiment, ovariectomized HPD sheep that had received hourly pulses of GnRH for 16 h after an i.m. injection of oil or 50 micrograms oestradiol benzoate were given either a 'bolus' (2.25 micrograms GnRH) or a 'volley' (500 ng GnRH pulses 10 min apart for 30 min, plus a 500 ng pulse 15 min later). Both groups then received GnRH pulses (250 ng) every 30 min for the next 13 h. Oestrogen enhanced the LH responses to the GnRH treatments, and the amount of LH released was similar in ovariectomized HPD ewes given oestrogen plus bolus or volley GnRH treatments and ovariectomized hypothalamo-pituitary intact ewes given oestrogen. These results suggest that the oestrogen-induced LH surge is initiated by a 'signal' pattern of GnRH secretion from the hypothalamus.     

Does melatonin alter pituitary responsiveness to gonadotropin-releasing hormone in the ewe?

The diurnal secretion of melatonin from the pineal gland transduces information about day length to the reproductive axis of many seasonal breeders including the ewe. In the sheep the target for melatonin is thought to be neural, such that the hormone acts through the GnRH pulse generator to produce seasonal alterations in the frequency of pulsatile LH secretion. These effects on the pulse generation mechanism take approximately 50 days to become evident. It is possible that melatonin also exerts direct effects at the level of the pituitary gland to alter responsiveness to GnRH. Such effects have been noted in other species. The site of action of melatonin to regulate pulsatile LH secretion was assessed in the ewe by determining whether the animal's endogenous melatonin acutely modifies pituitary responsiveness to sustained pulsatile administration of GnRH. Using an animal model in which endogenous GnRH was blocked, pituitary responsiveness to hourly pulses of exogenous GnRH was assessed under conditions of both high (dark period) and low (light period) melatonin. No evidence for acute effects of melatonin on pituitary response to GnRH was found. In another experiment, the amplitude and frequency of endogenously generated LH pulses in ovariectomized ewes was found not to change during the 24-hour light/dark cycle. These data lead to the conclusion that melatonin does not act at the pituitary gland to produce acute effects on LH secretion. Rather, our findings are consistent with the hypothesis that the action of melatonin, in this short-day breeder is long term, and is directed towards the neural elements of the hypothalamic pulse-generating mechanism.     

Increased gonadotropin-releasing hormone pulse frequency associated with estrogen-induced luteinizing hormone surges in ovariectomized ewes

Hypophyseal portal blood samples were taken from ovariectomized (OVX) ewes given 50 micrograms estradiol benzoate. This estrogen treatment elicited a biphasic alteration (decrease then increase) in LH secretion. During the negative feedback phase, pulsatile GnRH secretion continued; at this time the interpulse interval for the GnRH pulses (49.5 +/- 5.7 min, mean +/- SE, n = 6) was similar to that in 7 control OVX ewes (53.4 +/- 8.7 min). During the positive feedback phase the GnRH interpulse interval (26.8 +/- 9.8 min; n = 6) was significantly (P less than 0.05) less than in the controls. In 3/7 cases the GnRH pulse frequency in OVX controls was within the range observed for estrogen-treated sheep during the positive feedback phase. These data suggest that, in most cases, the LH surge that can be induced by estrogen in OVX ewes, is associated with an increased GnRH pulse frequency. In some animals the inherent GnRH pulse frequency may already be at a rate that is high enough to permit an LH surge by action of estrogen on the pituitary. In general, the mean concentrations of GnRH in portal blood during the LH surge were higher than those in untreated animals, suggesting an overall increase in GnRH output during the LH surge. Pulsatile GnRH secretion continues throughout the early negative feedback phase, suggesting that the predominant effect of estrogen at this time is at the pituitary level.     

Absence of an estradiol-induced surge of luteinizing hormone in pigs receiving unvarying pulsatile gonadotropin-releasing hormone stimulation

The goal of this study was to pharmacologically block central nervous system (CNS) input to gonadotropes in mature ovariectomized gilts to determine the direct actions of estradiol (E2) on pituitary LH release when given at a dose sufficient to elicit a gonadotropin surge. Feeding AIMAX [N-methyl-N'-(1-methyl-2-propenyl)1,2-hydrazinedicarbothioamide; 125 mg/day] for 7 days reduced serum LH concentrations from 1.25 +/- 0.13 (mean +/- SE) to less than 0.18 ng/ml, abolished LH pulses, but did not compromise LH release in response to exogenous GnRH. Serum FSH concentrations were reduced by 27%, whereas serum concentrations of PRL, GH, thyroid hormones and cortisol were not affected after 7 days of AIMAX treatment. Behavior was not altered, aside from a slightly reduced appetite. The LH surge that peaked 48-80 h after injecting E2 benzoate (E2B) into control gilts was blocked in five of eight gilts given AIMAX. Giving GnRH pulses (1 microgram every 45 min) to AIMAX-treated gilts restored mean serum LH concentrations as well as the frequency and amplitude of LH pulses to those of untreated ovariectomized gilts. E2B suppressed the LH response to these GnRH pulses by 88% at 12 h, whereas from 24-96 h after E2B treatment, the LH response to GnRH and mean serum concentrations of LH were again similar to those of controls not given estradiol. These data indicate that induction of the gonadotropin surge by E2 in the gilt requires CNS input. The action of E2 on the pituitary in the presence of unvarying GnRH pulsation may, however, be limited to an early transient inhibition of responsiveness to GnRH, with no subsequent direct stimulation during the period of the surge.     

Effects of progesterone on pulsatile luteinizing hormone (LH) secretion and LH subunit messenger ribonucleic acid during lactation in the rat

In ovarian-intact lactating rats, removal of the suckling stimulus leads to restoration of pituitary LH beta mRNA levels and pulsatile LH secretion after 72 h, which correlates with a sharp decrease in plasma progesterone concentrations to basal levels. In contrast, in ovariectomized lactating rats, the increase in pituitary LH function is observed by 24 h after pup removal. To determine if progesterone secretion from the ovary participates in the delayed recovery of LH secretion, we treated lactating rats with the progesterone antagonist RU 486 and determined the effects on the time course of recovery of pulsatile LH secretion and LH subunit mRNA after pup removal and on pituitary responsiveness to GnRH. In ovarian-intact lactating rats treated with RU 486, pulsatile LH secretion was observed in about 40% of the rats within 24 h after pup removal (LH interpulse interval, 43.7 +/- 8.3 min) and in about 90% of the rats within 48 h after pup removal (LH interpulse interval, 46.1 +/- 3.6 min). The mean plasma LH level in the RU 486-treated rats was 10.1 +/- 2.2 ng/ml 24 h after removal of pups (control, less than 5 ng/ml) and had increased to 35.1 +/- 6.4 ng/ml 48 h after pup removal (control, 9.1 +/- 2.5 ng/ml). However, RU 486 treatment had no significant effect on LH mRNA subunit levels. To determine whether progesterone acts at the pituitary to block GnRH stimulation of LH secretion, we tested the effects of RU 486 on LH secretion in response to 2- and 5-ng pulses of GnRH. Pituitary responsiveness was tested 24 h after pup removal. We found that both doses of GnRH were effective in stimulating pulsatile LH secretion, and treatment with RU 486 had no significant effect on this response. We conclude from these studies that progesterone secretion from the ovary contributes to the inhibition of LH secretion that occurs after pup removal, since antagonizing progesterone's action resulted in an earlier restoration of pulsatile LH secretion. The increase in LH secretion occurred in the absence of any significant changes in responsiveness of the pituitary to GnRH stimulation or in LH subunit mRNA levels. Therefore, the primary site of action of progesterone would appear to be at the hypothalamus to suppress pulsatile GnRH secretion.     

Morphine decreases LH secretion in ovariectomized ewes only after steroid priming and not by direct pituitary action

To determine whether opiates directly modulate pituitary LH secretion in vivo, morphine was administered to hypothalamo-pituitary-disconnected (HPD) ewes which were receiving exogenous pulses of GnRH. To define the steroidal background which is permissive to a morphine-induced decrease in LH secretion, ovariectomized (OVX) ewes were treated as follows in groups of four: group 1, no implant; group 2, small 17 beta-estradiol (E2) (1 cm long x 0.33 diameter) and progesterone (P) implants; group 3, medium E2 (1 cm long x 0.46 diameter) and P implants, and group 4, medium E2 implants. Jugular blood samples were taken at 10-min intervals for 9 h, during which there was a 3-hour pretreatment period, a 3-hour treatment period when the sheep were given six intravenous injections of 10 mg morphine every 30 min, and a 3-hour run-off period. Morphine inhibited the mean plasma concentrations of LH and LH pulse frequency in group 3 only, and in 2/4 ewes in this group LH secretion was abolished and did not return to a pulsatile mode during the 3-hour run-off sampling period. In a second experiment designed to test the pituitary action of morphine, OVX-HPD ewes were primed with medium E2 and P implants and were given hourly pulses of 250 ng GnRH intravenously. Jugular blood samples were taken around each GnRH pulse over an 8-hour period. The first three pulses served as a control sampling period, after which the sheep were treated with morphine (six intravenous injections of 10 mg morphine every 30 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

Biphasic response in the secretion of gonadotrophin-releasing hormone in ovariectomized ewes injected with oestradiol

In ovariectomized ewes, an injection of oestrogen initially inhibits the tonic secretion of LH, and then induces a large release of LH similar to the preovulatory surge in intact ewes. The pattern of hypothalamic secretion of gonadotrophin-releasing hormone (GnRH) into the pituitary portal blood during this biphasic response to oestrogen was investigated in conscious, unrestrained, ovariectomized adult Ile-de-France ewes during the breeding season. The ewes were ovariectomized and implanted with cannulae for portal blood collection on the same day. Seven days later, portal and peripheral blood samples were collected simultaneously every 5 min for 25 h. The ewes were injected with oestradiol-17 beta (25 micrograms i.v. and 25 micrograms i.m.) 6.25 h after the start of sampling. GnRH and LH were measured by radioimmunoassay in portal and jugular plasma samples respectively. A clear pulsatile pattern of LH secretion was observed before the oestradiol injection in all the ewes, followed by the typical biphasic decrease (negative feedback) and increase (positive feedback) in mean concentrations. The sampling period was divided, for analysis, into pretreatment, negative feedback and positive feedback phases. Before injection with oestradiol, the GnRH pulses were clearly defined in portal blood and were always synchronized with LH pulses in the peripheral circulation. The frequency was 5.9 +/- 0.6 pulses/6 h (mean +/- S.E.M.), and the amplitude was 31.6 +/- 7.6 pmol/l. During negative feedback, both the frequency (4.2 +/- 0.5 pulses/6 h, P less than 0.01) and amplitude (15.2 +/- 4.6 pmol/l, P less than 0.05) of the GnRH pulses decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term negative feedback effects of oestrogen and progesterone on the pituitary gland of the long-term ovariectomized ewe

The effects of long-term treatment with physiological doses of oestradiol or oestradiol plus progesterone on plasma gonadotrophin levels and pituitary content of LH and gonadotrophin-releasing hormone (GnRH) receptors were studied in ovariectomized-hypothalamo-pituitary disconnected ewes given 250 ng pulses of GnRH every 2 h (i.v.). A pilot experiment showed that 3 cm long Silastic implants (s.c.) reduced both LH pulse frequency and pulse amplitude in long-term (greater than 6 months) ovariectomized ewes. The main experiment was conducted over 3 weeks in ovariectomized-hypothalamo-pituitary disconnected ewes that had received pulsatile GnRH replacement for 1 week after pituitary surgery. Group 1 (n = 5) received GnRH pulses alone throughout the study. Group 2 (n = 6) received oestradiol in week 2 and oestradiol plus progesterone in week 3 and in group 3 (n = 6) the steroid treatments were reversed. Oestradiol reduced (P less than 0.05) the mean (+/- S.E.M.) amplitude of LH in pulses in group 2 (from 8.2 +/- 1.6 to 5.0 +/- 0.5 micrograms/l) and group 3 (from 11.6 +/- 1.2 to 9.3 +/- 1.0 micrograms 1): an additional effect of progesterone was seen in group 2 but not group 3. The amplitudes of the LH pulses did not change in the control ewes. Plasma concentrations of FSH were reduced by approximately 50% by the oestradiol treatments with no additional effects of progesterone. There was no effect of steroidal treatment on pituitary content of LH or pituitary levels of GnRH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Absence of regular pulsatile LH secretion during pre- and post-implantation periods in sheep

Two experiments were conducted to determine the patterns of LH secretion and to evaluate the LH responses to pulsatile administration of GnRH during early pregnancy in ewes. In experiment 1, pregnant ewes (n=16) were used to determine the concentration of LH in plasma of jugular blood samples collected every 15 min for 6h before (day 10 post-mating) and after (days 20 and 30 post-mating) implantation. In experiment 2, the pituitary LH responses to exogenous pulsatile administration of GnRH were examined on day 10 post-mating in 4 pregnant ewes. A small dose of GnRH (200 ng/ml saline) was injected (i.v.) every 3h and jugular blood samples were collected every 15 min for 12h beginning at the onset of GnRH administration and continuing through the 4th GnRH pulse. During the frequent-sample bleeding at any of the stages of pregnancy examined, LH concentrations oscillated in a pulsatile manner. However, pulsatile LH release occurred irregularly and infrequently. Overall mean LH concentrations, frequency and amplitude of LH pulses were not significantly different between any of the stages of pregnancy examined. Pulsatile administration of GnRH on day 10 post-mating induced regular pulses of LH. In conclusion, these data demonstrate that: (i) pulsatile LH secretion occurs irregularly during early pregnancy, and (ii) the absence of regular pulsatile LH release during early pregnancy is not attributed to a lack of pituitary responsiveness to GnRH.