Effects of an opioid antagonist on pulsatile luteinizing hormone secretion in the ewe vary with changes in steroid negative feedback

In ewes during the breeding season, estradiol (E) and progesterone (P) synergistically regulate pulsatile luteinizing hormone (LH) secretion. E primarily inhibits LH pulse amplitude and P inhibits LH pulse frequency. To determine if endogenous opioid peptides (EOP) mediate these negative feedback effects, we administered the long-acting opioid antagonist WIN 44,441-3 (WIN) to intact ewes during the luteal and follicular phases of the estrous cycle and to ovariectomized ewes treated with no steroids, E, P, or E plus P. Steroid levels were maintained at levels seen during the estrous cycle by Silastic implants placed shortly after surgery. WIN increased LH pulse frequency, but not amplitude, in luteal phase ewes. In contrast, during the follicular phase, LH pulse amplitude was increased by WIN and pulse frequency was unchanged. Neither LH pulse frequency nor pulse amplitude was affected by WIN in long-term ovariectomized ewes untreated with steroids. In contrast, WIN slightly increased LH pulse frequency in short-term ovariectomized ewes. WIN also increased LH pulse frequency in ovariectomized ewes treated with P or E plus P. WIN did not affect pulse frequency but did increase LH pulse amplitude in E-treated ewes. These results support the hypothesis that EOP participate in the negative feedback effects of E and P on pulsatile LH secretion during the breeding season and that the inhibitory effects of EOP may persist for some time after ovariectomy.     

Seasonal changes in pulsatile luteinizing hormone (LH) secretion in the ewe: relationship of frequency of LH pulses to day length and response to estradiol negative feedback

Seasonal changes in pulsatile luteinizing hormone (LH) secretion in ovariectomized ewes were examined over the course of 2 yr in relation to annual changes in environmental photoperiod, shifts in response to estradiol negative feedback control of LH secretion, and timing of the breeding season. Under natural environmental conditions, the frequency of LH pulses in individual ovariectomized ewes changed gradually and in close association with the annual cycle of day length. As days became shorter in late summer and autumn, LH pulse frequency increased; conversely, as day length increased in late winter and spring, frequency declined. Under artificial conditions in which ovariectomized ewes were exposed to different photoperiods, a similar inverse relationship was observed between day length and LH pulse frequency. The seasonal changes in frequency of LH pulses in ovariectomized ewes, although symmetric with the annual photoperiodic cycle, were not temporally coupled to the dramatic shifts in response to estradiol feedback inhibition of LH secretion at the transitions between breeding season and anestrus. The feedback shifts occurred abruptly and at times when LH pulse frequency in ovariectomized ewes was at, or near, the annual maximum or minimum. The tight coupling between LH pulse frequency and photoperiod leads to the conclusion that there is a photoperiodic drive to the LH pulse-generating system of the ewe. The temporal dissociation between changes in this photoperiodic drive and the seasonal shifts in response to estradiol negative feedback support the hypothesis that the neuroendocrine basis for these two phenomena is not one and the same.     

Progesterone does not affect the amount of mRNA for gonadotropins in the anterior pituitary gland of ovariectomized ewes

To evaluate the effect of progesterone on the synthesis and secretion of gonadotropins, ovariectomized ewes either were treated with progesterone (n = 5) for 3 wk or served as controls (n = 5) during the anestrous season. After treatment for 3 wk, blood samples were collected from progesterone-treated and ovariectomized ewes. After collection of blood samples, hypothalamic and hypophyseal tissues were collected from all ewes. Half of each pituitary was used to determine the content of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the number of receptors for gonadotropin-releasing hormone (GnRH). The amounts of mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were measured in the other half of each pituitary. Treatment with progesterone reduced mean serum concentrations of LH (p less than 0.001) but ot FSH (p greater than 0.05). Further, progesterone decreased (p less than 0.05) the total number of pulses of LH. We were unable to detect pulsatile release of FSH. Hypothalamic content of GnRH, number of receptors for GnRH, pituitary content of gonadotropins and mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were not affected (p greater than 0.05) by treatment with progesterone. Thus, after treatment with progesterone, serum concentrations of LH (but not FSH) are decreased. This effect, however, is not due to a decrease in the steady-state amount of mRNA for LH beta or alpha subunits.     

Seasonal changes of gonadotropin-releasing hormone secretion in the ewe.

A sustained volley of high-frequency pulses of GnRH secretion is a fundamental step in the sequence of neuroendocrine events leading to ovulation during the breeding season of sheep. In the present study, the pattern of GnRH secretion into pituitary portal blood was examined in ewes during both the breeding and anestrous seasons, with a focus on determining whether the absence of ovulation during the nonbreeding season is associated with the lack of a sustained increase in pulsatile GnRH release. During the breeding season, separate groups (n = 5) of ovary-intact ewes were sampled during the midluteal phase of the estrous cycle and following the withdrawal of progesterone (removal of progesterone implants) to synchronize onset of the follicular phase. During the nonbreeding season, another two groups (n = 5) were sampled either in the absence of hormonal treatments or following withdrawal of progesterone. Pituitary portal and jugular blood for measurement of GnRH and LH, respectively, were sampled every 10 min for 6 h during the breeding season or for 12 h in anestrus. During the breeding season, mean frequency of episodic GnRH release was 1.4 pulses/6 h in luteal-phase ewes; frequency increased to 7.8 pulses/6 h during the follicular phase (following progesterone withdrawal). In marked contrast, GnRH pulse frequency was low (mean less than 1 pulse/6 h) in both groups of anestrous ewes (untreated and following progesterone withdrawal), but GnRH pulse amplitude exceeded that in both luteal and follicular phases of the estrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of transport on pulsatile LH release in ovariectomized ewes with or without prior steroid exposure at different times of year

The initial aim of the present study was to test whether the stress of transport suppresses LH pulsatile secretion in ewes. In a pilot experiment in the late breeding season, transport resulted in an unexpected response in three out of five transported, ovariectomized ewes pretreated with oestradiol and progesterone. Before transport, seasonal suppression of LH pulses had occurred earlier than anticipated, but LH pulsatility suddenly restarted for the period of transport. This finding was reminiscent of unexplained results obtained in ovariectomized ewes infused centrally with high doses of corticotrophin-releasing hormone after pretreatment with low doses of oestradiol with or without progesterone. Hence, an additional aim of the present study was to examine whether these latter results with corticotrophin-releasing hormone could be reproduced by increasing endogenous corticotrophin-releasing hormone secretion by transport. Subsequent experiments used groups of at least eight ovariectomized ewes at different times of the year with or without prior exposure to steroids to assess whether these unexpected observations were associated with season or the prevailing endocrine milieu. In the mid-breeding season, transport for 4 h in the absence of steroid pretreatment for 8 months reduced LH pulse frequency from 7.5 +/- 0.3 to 6.3 +/- 0.4 pulses per 4 h (P < 0.05) and LH pulse amplitude from 2.6 +/- 0.5 to 1.8 +/- 0.3 ng ml-1 (P < 0.05). Similarly, in the mid-breeding season, 34 h after the cessation of pretreatment with oestradiol and progesterone, transport suppressed LH pulse frequency from 6.1 +/- 0.4 to 5.5 +/- 0.3 pulses per 4 h (P < 0.05) with a tendency of effect on amplitude (6.2 +/- 2.7 to 2.61 +/- 0.6 ng ml-1; P = 0.07; note the large variance in the pretransport data). During mid-anoestrus, evidence of a suppressive effect of transport was only observed on LH pulse amplitude (4.7 +/- 0.6 versus 3.0 +/- 0.5 pulses per 4 h; P < 0.05) in ovariectomized ewes that had not been exposed to ovarian steroids for 4 months. Repetition of the pilot experiment with 12 ewes during the transition into anoestrus resulted in one ewe with LH pulses seasonally suppressed but increased by transport; 11 ewes had a distinct pulsatile LH pattern which was decreased by transport in six ewes. In anoestrus, there was no effect of transport on LH pulse frequency or amplitude in intact ewes, or those ovariectomized 2-3 weeks previously, with or without prior oestradiol and progesterone treatment. However, basal concentrations of cortisol were greater in anoestrus than in the breeding season, and the increment in cortisol during transport was similar in anoestrus and the breeding season but greater during the transition into anoestrus (P < 0.05). Progesterone concentrations increased from 0.31 +/- 0.02 ng ml-1 before transport to 0.48 +/- 0.05 ng ml-1 during the second hour of transport (P < 0.05). In conclusion, transport reduced LH pulse frequency and amplitude in ovariectomized ewes that had not been exposed to exogenous steroids for at least 4 months. In most animals, the previously observed increase in LH pulsatility induced by exogenous CRH was not reproduced by increasing endogenous CRH secretion by transport. However, in four ewes, transport did increase LH pulsatility, but only during the transition into anoestrus in ewes with seasonally suppressed LH profiles after withdrawal of steroid pretreatment.     

Progesterone directly inhibits pituitary luteinizing hormone secretion in an estradiol-dependent manner.

We recently demonstrated that progesterone and estradiol inhibit pituitary LH secretion in a synergistic fashion. This study examines the direct feedback of progesterone on the estradiol-primed pituitary. Nine ovariectomized (OVX) ewes underwent hypothalamic-pituitary disconnection (HPD) and were infused with 400 ng GnRH every 2 h throughout the experiment. After 7 days of infusion, estradiol was implanted s.c. Four days later, estradiol implants were exchanged for blank implants in 4 ewes and for progesterone implants in 5 ewes. These implants remained in place for another 4 days. Blood samples were collected around exogenous GnRH pulses before and 0.5 to 96 h after implant insertion and exchange. Serum LH and progesterone concentrations were determined through RIA. One month later, 4 of the HPD-OVX ewes previously implanted with steroids were reinfused with GnRH and the implantation protocol was repeated using blank implants only. In estradiol-primed ewes, progesterone significantly lowered LH secretion after 12 h of implantation and LH secretion remained inhibited while progesterone implants were in place (p less than 0.05). Removing estradiol transiently lowered LH secretion, and this effect was significant only 24 h after estradiol withdrawal (p less than 0.05). These data suggest that progesterone has a direct, estradiol-dependent inhibitory effect on pituitary LH release and that estradiol may sustain pituitary gonadotrope response to GnRH.     

Separate neural systems mediate the steroid-dependent and steroid-independent suppression of tonic luteinizing hormone secretion in the anestrous ewe

In the ewe, two types of seasonal fluctuations in secretion of tonic luteinizing hormone (LH) have been described: a steroid-dependent change whereby estradiol gains the capacity to suppress LH pulse frequency in anestrus, and a steroid-independent decrease in pulse frequency in ovariectomized animals during anestrus. We have proposed that the former reflects activation, in anestrus, of estradiol-sensitive catecholaminergic neurons that inhibit gonadotropin-releasing hormone (GnRH). Three results reported here support this hypothesis: dopaminergic (pimozide) and alpha-adrenergic (phenoxybenzamine) antagonists increased LH in intact anestrous ewes without altering pituitary responses to GnRH; other dopaminergic (fluphenazine) and alpha-adrenergic (dibenamine) antagonists also increased LH in anestrus; agonists for dopaminergic (apomorphine) and alpha-adrenergic (clonidine) receptors suppressed LH secretion in both seasons, suggesting that the appropriate receptors are present in breeding-season ewes. In contrast, catecholamines do not appear to mediate the steroid-independent suppression of pulse frequency; neither pimozide nor phenoxybenzamine increased LH pulse frequency in ovariectomized ewes during anestrus. When antagonists for 6 other neurotransmitter receptors (muscarinic and nicotinic cholinergic, GABAnergic, serotonergic, opioid, and beta-adrenergic) were tested in anestrus, only cyproheptadine, the serotonergic antagonist, increased pulse frequency in ovariectomized ewes. Cyproheptadine had no effect on frequency during the breeding season. On the basis of these results, we propose that the steroid-dependent and -independent actions of anestrous photoperiod occur via catecholaminergic and serotonergic neurons, respectively.     

Psychosocial stress suppresses attractivity, proceptivity and pulsatile LH secretion in the ewe

Various stressors suppress pulsatile secretion of luteinizing hormone (LH) in ewes and cortisol has been shown to be a mediator of this effect under various conditions. In contrast, little is known about the impact of stress and cortisol on sexual behavior in the ewe. Therefore, we tested the hypothesis that both psychosocial stress and stress-like levels of cortisol will reduce the level of attractivity, proceptivity and receptivity in addition to suppressing LH secretion in the ewe. In Experiment 1, a layered stress paradigm of psychosocial stress was used, consisting of isolation for 4 h with the addition of restraint, blindfold and noise of a barking dog (predator stress) at hourly intervals. This stress paradigm reduced LH pulse amplitude in ovariectomized ewes. In Experiment 2, ovariectomized ewes were artificially induced into estrus with progesterone and estradiol benzoate treatment and the layered stress paradigm was applied. LH was measured and sexual behavior was assessed using T-mazes and mating tests. Stress reduced pulsatile LH secretion, and also reduced attractivity and proceptivity of ewes but had no effect on receptivity. In Experiment 3, ewes artificially induced into estrus were infused with cortisol for 30 h. Cortisol elevated circulating plasma concentrations of cortisol, delayed the onset of estrus and resulted in increased circling behavior of ewes (i.e. moderate avoidance) during estrus and increased investigation and courtship from rams. There was no effect of cortisol on attractivity, proceptivity or receptivity during estrus. We conclude that psychosocial stress inhibits LH secretion, the ability of ewes to attract rams (attractivity) and the motivation of ewes to seek rams and initiate mating (proceptivity), but cortisol is unlikely to be the principal mediator of these effects.     

Suppression of pulsatile luteinizing hormone secretion by gonadotrophin-releasing hormone antagonist does not affect episodic progesterone secretion or corpus luteum function in ewes.

Progesterone secretion has been observed to be episodic in the late luteal phase of the oestrous cycle of ewes and is apparently independent of luteinizing hormone (LH). This study investigated the effects of suppressing the pulsatile release of LH in the early or late luteal phase on the episodic secretion of progesterone. Six Scottish Blackface ewes were treated i.m. with 1 mg kg-1 body weight of a potent gonadotrophin-releasing hormone (GnRH) antagonist on either day 4 or day 11 of the luteal phase. Six ewes received saline at each time and acted as controls. Serial blood samples were collected at 10 or 15 min intervals between 0 and 8 h, 24 and 32 h, and 48 and 56 h after GnRH antagonist treatment and daily from oestrus (day 0) of the treatment cycle for 22 days. Oestrous behaviour was determined using a vasectomized ram present throughout the experiment. Progesterone secretion was episodic in both the early and late luteal phase with a frequency of between 1.6 and 3.2 pulses in 8 h. The GnRH antagonist abolished the pulsatile secretion and suppressed the basal concentrations of LH for at least 3 days after treatment. This suppression of LH, in either the early or late luteal phase, did not affect the episodic release of progesterone. Daily concentrations of progesterone in plasma showed a minimal reduction on days 11 to 14 after GnRH antagonist treatment on day 4, although this was significant (P < 0.05) only on days 11 and 13. There was no effect of treatment on day 11 on daily progesterone concentration, and the timing of luteolysis and the duration of corpus luteum function was unaffected by GnRH antagonist treatment on either day 4 or day 11. These results indicate that the episodic secretion of progesterone during the luteal phase of the oestrous cycle in ewes is independent of LH pulses and normal progesterone secretion by the corpus luteum can be maintained with minimal basal concentrations of LH.     

Changes in pulsatile LH secretion after ovariectomy in Ile-de-France ewes in two seasons

Two experiments were conducted in Ile-de-France ewes to study changes in pulsatile LH secretion in ewes ovariectomized during anoestrus or during the midluteal phase of the oestrous cycle. In Exp. 1, blood samples were taken every 20 min for 12 h the day before ovariectomy (Day 0). After ovariectomy, samples were taken every 10 min for 6 h (10 ewes per group), on Days 1, 3, 7 and 15. In Exp. 2 samples were taken every 10 min for 6 h (10 ewes per group) on Days 7, 15, 30, 60, 90, 120, 150 and 180 after ovariectomy. Further samples were taken (5 ewes per group) at 9 and 12 months after ovariectomy. There were significant interactions between season and day of sampling for the interval between LH pulses in both experiments. LH pulse frequency increased within 1 day of ovariectomy and the increase was more rapid during the breeding season. There were clear seasonal differences in pulse frequency in Exp. 2. Compared with ewes ovariectomized in anoestrus, pulse frequency was significantly higher for ewes ovariectomized in the breeding season, from Day 7 until Day 120. Once pulse frequency had increased in ewes about the time of the normal breeding season, pulse frequency remained high and subsequent seasonal changes were greatly reduced. Pulse amplitude increased immediately after ovariectomy to reach a maximum on Day 7 and there were no differences between season of ovariectomy in the initial changes in amplitude. In Exp. 2, changes in amplitude followed changes in pulse interval and there was a significant interaction between season and day of sampling. There were no significant effects of season on nadir LH concentrations which increased throughout the duration of the experiments. These results show that, in ovariectomized ewes, LH pulse frequency observed on a given day depends on time after ovariectomy, season at the time of sampling and on previous exposure of ewes to stimulatory effects of season. The direct effects of season on LH pulse frequency and seasonal changes in sensitivity to steroid feedback may contribute to control of the breeding season and their relative contributions to the beginning and end of the breeding season may differ.     

Exposure to ram wool stimulates gonadotropin-releasing hormone pulse generator activity in the female goat

In the sheep and goat, exposure of anestrous females to a conspecific male odor enhances reproductive activity. Interestingly, a previous report indicated that male goat hair stimulated pulsatile luteinizing hormone (LH) secretion in the ewe. In the present study, we addressed whether ram wool affects the gonadotropin-releasing hormone (GnRH) pulse generator activity in the female goat. Five ovariectomized (OVX) goats were chronically implanted with recording electrodes in the mediobasal hypothalamus, and manifestations of the GnRH pulse generator were monitored as characteristic increases in multiple-unit activity (MUA volleys). Wool or hair samples were collected from a mature ram, ewe and male goat, and their effects on the MUA volley were examined. The exposure to ram wool induced an MUA volley within 1 min in all five OVX goats, as did the exposure to male goat hair. The ewe wool had no effect on the timing of an MUA volley occurrence. An invariable association of MUA volleys with LH pulses in the peripheral circulation was also confirmed in two OVX goats exposed to ram wool. The present results clearly indicate that exposure to ram wool stimulates pulsatile GnRH/LH release in the female goat. Since exposure to male goat hair enhances pulsatile LH secretion in the ewe, it is likely that very similar, if not identical, molecules are contained in the male-effect pheromone in the sheep and goat.     

Regulation by estradiol of hypothalamic somatostatin gene expression: possible involvement of somatostatin in the control of luteinizing hormone secretion in the ewe

In the ewe, the mediobasal hypothalamus (MBH) is the primary central site for estradiol to generate the preovulatory GnRH/LH surges and sexual behavior. This area contains numerous neurons expressing the estradiol receptor alpha, distributed in the ventromedial nucleus (VMN) and the infundibular nucleus (IN). A large proportion of these neurons express somatostatin, making this neuropeptide a potential candidate for transmission of the estradiol signal to the GnRH neurons located in the preoptic area. We tested this hypothesis using ovariectomized ewes that had been subjected to an artificial estrous cycle. In the first experiment, 22 h after progesterone removal, ewes received estradiol (treated ewes) or empty implants (control ewes) for 4 h and then were killed. Using in situ hybridization, we showed that this short estradiol treatment increased the somatostatin mRNA amount by about 50% in the VMN and 42% in the IN. In the second experiment, preovulatory estradiol signal was replaced by somatostatin intracerebroventricular (ICV) administration. This treatment abolished LH pulsatility and dramatically decreased the mean basal level of LH secretion while it did not affect the mean plasma GH concentration. We demonstrated that an increase in somatostatin mRNA occurs at the time of the negative feedback effect of estradiol on LH secretion during the early stage of the GnRH surge induction. As ICV somatostatin administration inhibits the pulsatile LH secretion by acting on the central nervous system, we suggest that somatostatin synthesized in the MBH could be involved in the estradiol negative feedback before the onset of the preovulatory surge.     

Effects of breed, ovarian steroids and season on the pulsatile secretion of LH in ovariectomized ewes

The effects of season and of oestradiol and progesterone on the tonic secretion of LH were studied in ovariectomized Merino and Suffolk ewes, two breeds which differ markedly in the seasonal pattern of their reproductive activity. In the absence of exogenous steroids, the frequency of LH pulses was lower and the amplitude of the pulses was higher in anoestrus than in the breeding season for Merino and Suffolk ewes 30 days after ovariectomy. In long-term (190 days) ovariectomized ewes, this seasonal change in LH secretion was observed in Suffolk ewes only. During seasonal anoestrus, treatment of ewes with subcutaneous oestradiol-17 beta implants (3, 6 or 12 mm in length) decreased the frequency of LH pulses in a dose-dependent manner, with Suffolk ewes being far more sensitive to the inhibitory effects of oestradiol than Merino ewes. The lowest dose of oestradiol (3 mm) had no effect on the secretion of LH in Merino ewes, but reduced secretion in Suffolk ewes. Treatment of ewes with the highest dose of oestradiol (12 mm) completely abolished LH pulses in Suffolk ewes, whereas infrequent pulses remained evident in Merino ewes. During the breeding season, oestradiol alone had no effect on the pulsatile release of LH in either breed, but in combination with progesterone there was a significant reduction in LH pulse frequency. Progesterone effectively decreased LH secretion in both breeds in both seasons. It was concluded that differences between breeds in the 'depth' of anoestrus could be related to differences in the sensitivity of the hypothalamus to both negative feedback by oestradiol and the direct effects of photoperiod.     

Pulsatile LH secretion and ovarian follicular wave emergence and growth in anestrous ewes

The objective of this study was to determine if pulsatile LH secretion was needed for ovarian follicular wave emergence and growth in the anestrous ewe. In Experiment 1, ewes were either large or small (10 × 0.47 or 5 × 0.47 cm, respectively; n = 5/group) sc implants releasing estradiol-17 beta for 10 d (Day 0 = day of implant insertion), to suppress pulsed LH secretion, but not FSH secretion. Five sham-operated control ewes received no implants. In Experiment 2, 12 ewes received large estradiol-releasing implants for 12 d (Day 0 = day of implant insertion); six were given GnRH (200 ng IV) every 4 h for the last 6 d that the implants were in place (to reinitiate pulsed LH secretion) whereas six Control ewes were given saline. Ovarian ultrasonography and blood sampling were done daily; blood samples were also taken every 12 min for 6 h on Days 5 and 9, and on Days 6 and 12 of the treatment period in Experiments 1 and 2, respectively. Treatment with estradiol blocked pulsatile LH secretion (P < 0.001). In Experiment 1, implant treatment halted follicular wave emergence between Days 2 and 10. In Experiment 2, follicular waves were suppressed during treatment with estradiol, but resumed following GnRH treatment. In both experiments, the range of peaks in serum FSH concentrations that preceded and triggered follicular wave emergence was almost the same as control ewes and those given estradiol implants alone or with GnRH; mean concentrations did not differ (P < 0.05). We concluded that some level of pulsatile LH secretion was required for the emergence of follicular waves that were triggered by peaks in serum FSH concentrations in the anestrous ewe.     

Pituitary receptors for gonadotropin-releasing hormone in relation to changes in pituitary and plasma gonadotropins in ovariectomized hypothalamo/pituitary-disconnected ewes. II. A marked rise in receptor number during the acute feedback effects of estradiol

Ovariectomized (OVX), hypothalamo/pituitary-disconnected (HPD) ewes were used to ascertain the short-term effects of estradiol on the number of gonadotropin-releasing hormone (GnRH) receptors in the pituitary gland. The time course of the study was such that measurements were made during the period of short-term negative feedback and positive feedback. Groups of 4 OVX-HPD ewes were given 250-ng pulses of GnRH each hour and an i.m. injection of oil (Group 1) or 50 micrograms estradiol benzoate in oil (Groups 2-4). Blood samples were collected from each ewe prior to treatment with estradiol or oil and again immediately before slaughter. Groups 2, 3, and 4 were killed 6, 16, and 20 h, respectively, after administration of estradiol. Amplitudes of luteinizing hormone (LH) pulses and average plasma concentrations of LH were reduced 6 h after estradiol treatment. Sixteen and 20 h after injection, the average plasma LH levels were elevated, but pulse amplitudes were similar to preinjection values. The number of GnRH receptors was significantly (p less than 0.01) increased within 6 h of estrogen treatment and further increased 16 and 20 h after treatment. Pituitary content of LH was similar in all groups. These data indicate that the number of GnRH receptors in the pituitary gland of ewes can be acutely influenced by a direct effect of estradiol. However, the magnitude and direction of the change in receptors number does not account for the changes in pituitary responsiveness to GnRH, suggesting estradiol also modifies post-receptor mechanisms that influence secretion of LH.     

Gonadotropin-releasing hormone secretion during the postpartum anestrous period of the ewe

An increase in episodic release of LH is putatively the initial event leading to the onset of postpartum ovarian cyclicity in ewes. This experiment was conducted to determine the relationship between hypothalamic release of GnRH and onset of pulsatile secretion of LH during postpartum anestrus. Control ewes (n = 7) were monitored during the postpartum period to determine when normal estrous cycles resumed. In controls, the mean interval from parturition to the first postpartum estrus as indicated by a rise in serum progesterone greater than 1 ng/mg was 25.8 +/- 0.6 days. Additional ewes (n = 4-5) at 3, 7, 14, and 21 days postpartum (+/- 1 day) were surgically fitted with cannula for collection of hypophyseal-portal blood. Hypophyseal-portal and jugular blood samples were collected over a 6- to 7-h period at 10-min intervals. The number of GnRH pulses/6 h increased (p less than 0.05) from Day 3 postpartum (2.2 +/- 0.5) to Days 7 and 14 (3.6 +/- 0.2 and 3.9 +/- 0.4, respectively). A further increase (p less than 0.05) in GnRH pulse frequency was observed at Day 21 postpartum (6.4 +/- 0.4 pulses/6 h). Changes in pulsatile LH release paralleled changes observed in pulsatile GnRH release over Days 3, 7, 14, and 21 postpartum (0.83 +/- 0.3, 2.8 +/- 0.4, 2.9 +/- 0.6, and 4.0 +/- 1.1 pulses/6 h, respectively). GnRH pulse amplitude was higher at Day 21 than at Days 3, 7, or 14 postpartum. These findings suggest that an increase in the frequency of GnRH release promotes the onset of pulsatile LH release during postpartum anestrus in ewes.     

FACTORS AFFECTING THE SECRETION OF LUTEINIZING HORMONE IN THE EWE

(1) Luteinizing hormone (LH) is secreted as discrete pulses throughout all stages of the reproductive cycle of the ewe, including pre-pubertal, seasonal and lactational anoestrus, and the luteal and follicular phases of the oestrous cycle. Secretion is probably also pulsatile during the preovulatory surge of LH. (2) The secretion of LH is affected by the ovarian steroids, oestradiol and progesterone, both of which act principally to reduce the frequency of the pulses. During the luteal phase the two steroids act synergistically to exert this effect, and during anoestrus oestradiol acts independently of progesterone. Androstenedione secreted by the ovary apparently has no role in the control of LH secretion. (3) The amplitude of the pulses may also be affected by the steroids but there are conflicting reports on these effects, some showing that amplitude is lowered by the presence of oestrogen and others showing increases in amplitude in the presence of oestrogen and progesterone. (4) The secretion of LH pulses is affected by photoperiod, social environment and nutrition. Under the influence of decreasing day-length, oestradiol alone cannot reduce the frequency of pulses and the ewe experiences oestrous cycles. When day-length is increasing, the hypothalamus becomes more responsive to oestradiol which reduces the frequency of the pulses. (5) A hypothetical pheromone secreted by rams can increase the frequency of the LH pulses in anoestrous ewes and thereby induce ovulation, possibly by inhibiting the negative feedback exerted by oestradiol. (6) The relationships between nutrition and reproduction are poorly understood, but it seems likely that the effects of nutrition are mediated partly through the hypothalamus and its control of the secretion of LH pulses. (7) The pulses of LH secreted by the anterior pituitary gland are evoked by pulses of GnRH secreted by the hypothalamus. The location of the centre controlling the GnRH pulses and the neurotransmitter involved are not known.     

Gonadotrophin release in ovariectomized ewes fed different amounts of coumestrol

Three experiments were conducted to study changes in pulsatile secretion of LH and FSH during the breeding season or anoestrus in ovariectomized Ile-de-France ewes fed different amounts of the phyto-oestrogen coumestrol. In Exp. 1, conducted during the breeding season, ewes (3-4 per group) were fed lucerne supplying 4, 18 or 30 mg coumestrol per ewe per day for 15 days. Experiments 2 and 3 were conducted during seasonal anoestrus. In Exp. 2, ewes (4 per group) were fed lucerne supplying coumestrol concentrations ranging from 4 to 38 mg/ewe/day for 15 days. In Exp. 3, ewes (10 per group) were fed lucerne supplying 14 or 125 mg coumestrol/ewe/day for 15 days. During the breeding season, an increased concentration of coumestrol in the diet significantly decreased the amplitude of LH pulses. There were no effects on LH pulse frequency or on FSH concentrations. During seasonal anoestrus, there were no significant effects on LH pulse frequency, or amplitude and no significant effect on FSH concentration. These results show that high concentrations of coumestrol in lucerne diets would not explain seasonal variation in LH pulse frequency in ovariectomized ewes. However, lucerne diets with increased coumestrol concentrations can influence LH release during the breeding season.     

Evidence for a direct negative effect of estradiol at the level of the pituitary gland in sheep

The purpose of this experiment was to determine if pituitary stores of LH could be replenished by administration of GnRH when circulating concentrations of both progesterone and estradiol-17 beta (estradiol) were present at levels observed during late gestation. Ten ovariectomized (OVX) ewes were administered estradiol and progesterone via Silastic implants for 69 days. One group of 5 steroid-treated OVX ewes was given GnRH for an additional 42 days (250 ng once every 4 h). Steroid treatment alone reduced (p less than 0.01) the amount of LH in the anterior pituitary gland by 77%. Pulsatile administration of GnRH to steroid-treated ewes resulted in a further decrease (p less than 0.01) in pituitary content of LH. Compared to the OVX ewes, concentrations of mRNAs for alpha- and LH beta-subunits were depressed (p less than 0.01) in all steroid-treated ewes, whether or not they received GnRH. The ability of the dosage of GnRH used to induce release of LH was examined by collecting blood samples for analysis of LH at 15 days and 42 days after GnRH treatment was initiated. Two of 5 and 3 of 5 steroid-treated ewes that received pulses of GnRH responded with increased serum concentrations of LH after GnRH administration during the first and second bleedings, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opiatergic inhibition of pulsatile luteinizing hormone release during the menstrual cycle of rhesus macaques

The endogenous opioid peptides (EOPs) may inhibit the rate of hypothalamic gonadotropin-releasing hormone (GnRH) release and hence the frequency of pulsatile luteinizing hormone (LH) release, particularly in the luteal phase of the menstrual cycle. Our objectives were to compare the effects of an opiate antagonist, naloxone (NAL), on the patterns of LH, estradiol-17 beta (E2), and progesterone (P4) secretion during the follicular and luteal phases of the macaque menstrual cycle. Plasma levels of E2, P4, and bioactive LH were measured in serial, 15-min blood samples during 8-hr infusions of NAL (2 mg/hr) or saline, either on Days 5 or 6 of the follicular phase (FN and FS, n = 5 and 4, respectively) or on Days 8, 9, or 10 of the luteal phase (LN and LS, n = 5 each) of a menstrual cycle. The pulsatile parameters of each hormone were determined by PULSAR analysis and the correspondence of steroid pulses with those of LH were analyzed for each cycle stage in each animal. As expected, LH mean levels and pulse frequencies in LS monkeys were only about one-third of those values in FS animals. NAL had no effects on pulsatile LH, E2, or P4 release during the follicular phase. In contrast, luteal phase NAL infusions increased both LH mean levels and pulse frequencies to values which were indistinguishable from those in FS animals. LH pulse amplitudes did not differ among the four groups. Mean levels and pulse frequencies of P4 secretion in LS monkeys were about 4- and 14-fold greater than those values in FS animals. Mean levels and pulse amplitudes of P4 release in LN animals were greater than those values in all other groups. LH and E2 pulses were not closely correlated in follicular phase animals, and this pulse association was not altered by NAL. In FS monkeys, LH and P4 pulses were not correlated; however, NAL increased this LH-p4 pulse correspondence. LH and P4 pulses were closely correlated in luteal phase animals and this association was not affected by NAL. Our data suggest that the EOPs inhibit the frequency of pulsatile LH secretion in the presence of luteal phase levels of P4. During the midfollicular phase when LH pulses occur every 60 to 90 min, the opioid antagonist NAL alters neither the pulsatile pattern of LH release nor E2 secretion, but NAL may directly affect P4-secreting cells.