Further evidence that serotonin mediates the steroid-independent inhibition of luteinizing hormone secretion in anestrous ewes

Two photoperiod-controlled neuroendocrine systems appear to suppress secretion of tonic luteinizing hormone (LH) in anestrous ewes: a steroid-independent system that decreases LH pulse frequency in ovariectomized ewes and a steroid-dependent system whereby estradiol gains the capacity to suppress LH pulse frequency in anestrus. This study was designed to test the hypothesis that serotonergic neurons inhibit LH pulse frequency in ovariectomized ewes and to examine the possible interaction of this system with the steroid-dependent inhibition of LH pulse frequency in the anestrous season. In Experiment 1, i.v. injection of serotonin receptor antagonist, methysergide, significantly increased LH pulse frequency in ovariectomized ewes during the anestrous season. In Experiment 2, we examined the effects of oral administration of parachlorophenylalanine for 5 days on the synthesis of serotonin. This treatment significantly increased LH pulse frequency in ovariectomized ewes, but had no effect on the negative feedback action of estradiol. These data support the hypothesis that a serotonergic neural system mediates the steroid-independent inhibition of LH pulse frequency in anestrous ewes and suggest that this system is not absolutely essential for the functioning of the steroid-dependent system responsible for the negative feedback action during the anestrous season.     

The role of nutrition in the regulation of LH secretion during anestrus by the serotoninergic and dopaminergic systems in Mediterranean ewes treated with melatonin

The steroid-dependent inhibition of LH secretion involves dopaminergic and serotoninergic systems but it is unclear how the plane of nutrition affects this inhibition during anestrus in melatonin treated ewes. Melatonin implants (18 mg) were inserted (Day 0) into ovariectomized, estradiol treated adult Rasa Aragonesa ewes on a high (H; n = 8) or low energy diet (L; n = 6) which were applied in early anestrus (Day 29-57) and late anestrus (Day 90-104). Cyproheptadine (0.1 mg/ kg), a serotoninergic (SHT2) receptor antagonist, was administered in early and late anestrus (Day 50 and 107) followed by pimozide (0.08 mg/kg), a dopaminergic2 receptor antagonist (Day 57 and 114). The H ewes had significantly higher LH concentrations (P < 0.05) before cyproheptadine treatment in early anestrus. The H and L ewes responded in a similar way to the antagonists in both early and late anestrus, except for L ewes who had a higher LH pulse amplitude after pimozide treatment in both periods (P < 0.05). During early anestrus, cyproheptadine tended to increase (P = 0.06) LH pulse frequency in L ewes and LH concentrations in H ewes. The LH secretion also increased in L ewes after pimozide administration during early anestrus (P < 0.05 for mean LH concentrations and LH pulse frequency and amplitude). However, pimozide dramatically increased LH secretion during late anestrus (Day 114) irrespective of the plane of nutrition (P = 0.06-0.08 for LH pulse frequency and P < 0.05 for LH concentrations and pulse amplitude). In melatonin treated Mediterranean ewes, the plane of nutrition appeared to modify the effect of dopaminergic and serotoninergic systems on the steroid-dependent inhibition of LH secretion throughout anestrus.     

Effects of pentobarbital anesthesia on tonic luteinizing hormone secretion in the ewe: evidence for active inhibition of luteinizing hormone in anestrus

In the ewe, seasonal anestrus appears to result from two effects of inhibitory photoperiod: 1) estradiol gains the capacity to suppress luteinizing hormone (LH) pulse frequency and hence becomes a potent inhibitor of tonic LH secretion and 2) a steroid-independent decrease in LH pulse frequency occurs in ovariectomized ewes. In this study, we have obtained evidence, using pentobarbital anesthesia, that both these actions of photoperiod reflect the activation, in anestrus, of an inhibitory neural system. Administration of pentobarbital to intact anestrous ewes produced a dramatic, 3-fold increase in LH pulse frequency during the 6 h of anesthesia. In contrast, during the breeding season, pentobarbital inhibited LH pulse frequency in luteal phase animals. There was also a seasonal variation in the effects of pentobarbital in ovariectomized ewes. During the breeding season this drug again suppressed LH secretion, inhibiting both LH pulse amplitude and frequency. In anestrus, pentobarbital also suppressed pulse amplitude, but it produced a transitory increase (lasting 3 h) in pulse frequency. To account for the stimulatory actions of pentobarbital, we propose that in anestrus, but not the breeding season, LH pulse frequency is held in check by a set of estradiol-sensitive inhibitory neurons. Further, we suggest that these neurons are activated by inhibitory photoperiod and account for both the steroid-dependent and steroid-independent actions of photoperiod.     

Evidence that estrogen receptor alpha,but not beta,mediates seasonal changes in the response of the ovine retrochiasmatic area to estradiol

In ewes, anestrus results from a reduction in LH pulsatility due to an increased sensitivity of the hypothalamic estradiol negative feedback system. Considerable evidence has implicated the A15 group of dopaminergic neurons in the retrochiasmatic area in this seasonally dependent estradiol effect. Moreover, estradiol administered to the retrochiasmatic area in ovariectomized anestrous ewes inhibits LH secretion. However, A15 neurons do not appear to contain the classical estrogen receptors (ERalpha). Therefore, we tested the hypothesis that beta-estrogen receptors mediate the action of estradiol in the retrochiasmatic area by comparing the effects of estradiol and genistein, a selective ERbeta agonist. We also examined whether there are seasonal changes in response of the retrochiasmatic area to these agonists and if these effects are mediated by dopamine. To test these hypotheses, ovariectomized ewes were implanted with bilateral guide cannulae targeting the retrochiasmatic area. Crystalline agonists were administered via microimplants inserted down the cannulae. Blood samples taken before and 4 days after microimplant insertion were analyzed for LH concentrations, pulse frequency, and amplitude. Genistein treatment produced no significant change in LH levels in either season. Estradiol treatment decreased both mean LH concentrations and pulse frequency in anestrous but not breeding-season ewes. Administration of the dopamine antagonist sulpiride to ovariectomized ewes with estradiol microimplants in the retrochiasmatic area returned LH pulse frequency to levels indistinguishable from controls. From these data, we hypothesize that estradiol acts on local ERalpha-containing neurons in this area to stimulate a dopaminergic pathway that inhibits LH secretion during anestrus.     

Different neuroendocrine systems modulate pulsatile luteinizing hormone secretion in photosuppressed and photorefractory ewes.

The objective of this study was to determine whether two photoperiod regimens that induce anestrus in the ewe-short-day photorefractoriness (SDPR) and long-day photosuppression (LDPS)--act by different neuronal mechanisms. In separate experiments, ovary-intact (INTACT), ovariectomized (OVX), and ovariectomized estradiol-treated (OVX + E) ewes were subjected to three different photoperiodic regimens that resulted in reproductive quiescence: (1) exposure to long days (16L:8D), which caused photosuppression (INTACT, n = 9; OVX, n = 6; OVX + E, n = 5; (2) prolonged exposure to short days (10L:14D)), which caused photorefractoriness (INTACT, n = 10; OVX, n = 6; OVX + E, n = 5); (3) exposure to natural photoperiod, which induced seasonal anestrus (INTACT, n = 11; OVX, n = 6; OVX + E, n = 5). Effect of photoregimen was monitored by measuring progesterone or LH. Drug challenges were made after two sequential estrous cycles were missed in INTACT ewes, after mean LH concentrations dropped below 1 ng/ml in OVX + E ewes, and after LH interpulse intervals increased in OVX ewes. Effects of drug on LH pulse pattern were determined by taking blood samples at 12-min intervals for 8 h after i.v. diluent injection; then for 8 h after i.v. injection of cyproheptadine, a serotonin antagonist (3 mg/kg); and again 7 days later after i.v. injection of diluent or pimozide, a dopamine antagonist (0.25 mg/kg). Cyproheptadine had little effect except to decrease (p = 0.05) mean LH in INTACT anestrous ewes and decrease (p less than 0.01) pulse amplitude in OVX + E SDPR ewes. Pimozide did not affect LH pulse frequency in LDPS ewes. However, pimozide increased LH pulse frequency (p less than 0.005) and mean concentrations (p less than 0.005) in SDPR OVX + E ewes, whereas it suppressed LH pulse frequency (p less than 0.05) and amplitude (p less than 0.03) in SDPR INTACT and SDPR OVX ewes. The results suggest that (1) the role of the dopaminergic system differs in SDPR and LDPS ewes, and that different neuronal systems may effect SDPR and LDPS, (2) the effect of pimozide in SDPR ewes is altered by ovarian steroids, and (3) the serotonergic system has relatively little role in regulating pulsatile LH secretion in any of the three different states of anestrus.     

Does the seasonal increase in estradiol negative feedback prevent luteinizing hormone surges in anestrous ewes by suppressing hypothalamic gonadotropin-releasing hormone pulse frequency?

To test the hypothesis that the anestrous increase in estradiol negative feedback prevents estrous cycles by suppressing hypothalamic gonadotropin-releasing hormone (GnRH) pulse frequency, a variety of regimens of increasing GnRH pulse frequency were administered to anestrous ewes for 3 days. A luteinizing hormone (LH) surge was induced in 45 of 46 ewes regardless of amplitude or frequency of GnRH pulses, but only 19 had luteal phases. Estradiol administration induced LH surges in 6 of 6 ewes, only 3 having luteal phases. Anestrous luteal phase progesterone profiles were similar in incidence, time course, and amplitude to those of the first luteal phases of the breeding season, which in turn had lower progesterone maxima than late breeding season luteal phases. In the remaining ewes, progesterone increased briefly or not at all, the increases being similar to the transient rises in progesterone occurring in most ewes at the onset of the breeding season. These results demonstrate that increasing GnRH pulse frequency induces LH surges in anestrus and that the subsequent events are similar to those at the beginning of the breeding season. Finally, they support the hypothesis that the negative feedback action of estradiol prevents cycles in anestrus by suppressing the frequency of the hypothalamic pulse generator.     

Hypothalamic sites of catecholamine inhibition of luteinizing hormone in the anestrous ewe.

Norepinephrine (NE) and dopamine (DA) actively inhibit the release of LH in anestrous ewes. This can be detected as an increase in LH pulse frequency following i.v. injection of NE and DA antagonists. The objective of this study was to determine the sites of these inhibitory actions in the ovine hypothalamus by using local administrations of the NE antagonist, phenoxybenzamine (PBZ), or the DA antagonist, pimozide (PIM), into specific hypothalamic areas. Each neurotransmitter antagonist was administered via a chronically implanted steel guide tube into either the preoptic area (POA), retrochiasmatic area (RCh), or the median eminence region (ME). Blood samples were taken every 15 min for 2 h before and 4 h during implantation of these drugs and were analyzed for LH and prolactin by RIA. Control (no treatment) samples were obtained similarly from the same animals on another day. Placement of PBZ into the POA significantly increased LH pulse frequency and mean LH concentrations over control values whereas PIM did not. In contrast, PIM significantly increased LH pulse frequency and mean LH concentrations when placed in the ME or in the RCh, but PBZ did not. No effects of PIM on prolactin concentrations were detected. These results suggest that an NE neural system operates in the POA and that a DA system acts in the medial basal hypothalamus (RCh or ME) to suppress GnRH pulse frequency in the ovary-intact anestrous ewe.     

Thyroid hormones mediate steroid-independent seasonal changes in luteinizing hormone pulsatility in the ewe

Thyroid hormones permit the increase in response to estradiol negative feedback in ewes at the transition to anestrus. In this study, we tested whether the thyroid hormones are also required for steroid-independent seasonal changes in pulsatile LH secretion. In experiment 1, Suffolk ewes were ovariectomized and thyroidectomized (THX) or ovariectomized only (controls) in late November. LH pulse frequency and amplitude were measured for 4 h in December, April, May, June, and August. Pulse frequency was also measured in the presence of estradiol-containing implants during the breeding (December) and early anestrus (March) seasons. As expected, in the presence of estradiol, pulse frequency declined between December and March in control but not THX ewes. In the absence of estradiol, a seasonal decline in frequency and an increase in amplitude occurred in control ewes, concurrent with lengthening photoperiod. A similar trend was seen in THX ewes, but the seasonal changes were lower in magnitude and not significant. In experiment 2, the same protocol was used (pulse measurements in December, May, and June) with a larger THX group size (n = 7). Results were similar to those of experiment 1 for controls. In THX ewes, pulse frequency did not change over time and was significantly elevated relative to that of controls during the summer. Pulse amplitude in THX ewes tended to increase during summer and did not differ from pulse amplitudes in control ewes. These results demonstrate that thyroid hormones are required for steroid-independent cycles in LH pulse frequency; however, some seasonal changes in amplitude still occur in the absence of thyroid hormones. This finding contrasts with the changes in estradiol negative feedback at the transition to anestrus, which are entirely thyroid hormone dependent.     

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)     

Steroid feedback inhibition of pulsatile secretion of gonadotropin-releasing hormone in the ewe

The long-term negative feedback effects of sustained elevations in circulating estradiol and progesterone on the pulsatile secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) were evaluated in the ewe following ovariectomy during the mid-late anestrous and early breeding seasons. GnRH secretion was monitored in serial samples of hypophyseal portal blood. Steroids were administered from the time of ovariectomy by s.c. Silastic implants, which maintained plasma concentrations of estradiol and progesterone at levels resembling those that circulate during the mid-luteal phase of the estrous cycle; control ewes did not receive steroidal replacement. Analysis of hormonal pulse patterns in serial samples during 6-h periods on Days 8-10 after ovariectomy disclosed discrete, concurrent pulses of GnRH in hypothalamo-hypophyseal portal blood and LH in peripheral blood of untreated ovariectomized ewes. These pulses occurred every 97 min on the average. Treatment with either estradiol or progesterone greatly diminished or abolished detectable pulsatile secretion of GnRH and LH, infrequent pulses being evident in only 3 of 19 steroid-treated ewes. No major seasonal difference was observed in GnRH or LH pulse patterns in any group of ewes. Our findings in the ovariectomized ewe provide direct support for the conclusion that the negative-feedback effects of estradiol and progesterone on gonadotropin secretion in the ewe include an action on the brain and a consequent inhibition of pulsatile GnRH secretion.     

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.     

Postpubertal maturation of endogenous opioid regulation of luteinizing hormone secretion in the female sheep

This study investigated whether the role of endogenous opioid peptides in the suppression of LH secretion during seasonal anestrus in the sheep changes with age. The experimental approach was to determine the effect of blockade of opioid receptors with naloxone on LH secretion at different times of year within the anestrous season, and to compare responses between seasonally anestrous sheep of different ages. Sheep, all past the normal age of puberty, were ovariectomized before the study and treated s.c. with estradiol implants to provide a fixed estradiol feedback signal. One-year-old females responded to naloxone with a rapid increase in LH pulse frequency in the early (April) and late (August) phases of their first anestrous season. This response was similar to that previously found in prepubertal female sheep. Only 5 of the 8 females responded to the same naloxone challenge in mid anestrus (June), suggesting that the contribution of opioid pathways to the inhibition of LH secretion at this time of year is not necessarily the same as that in early and late anestrus. None of the older anestrous sheep (greater than or equal to 2 yr) responded to naloxone in June, indicating age-related changes in the role of endogenous opioid mechanisms in the inhibition of LH secretion. Ovary-intact mature sheep did not respond to naloxone, in contrast to our previous observations in intact prepubertal females. We infer that the neural mechanisms underlying the superficially similar hypogonadotropic states that occur during the prepubertal period, first anestrous season, and later anestrous seasons are not identical.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Influence of day length and endocrine status on luteinizing hormone secretion in intact and ovariectomized adult ferrets

The purpose of this study was to examine the pituitary-ovarian relationship of both estrous and anestrous female ferrets. The endocrine status of the animals was induced by manipulating photoperiod: females in estrus were housed in long days (16L:8D); females in anestrus were housed in short days (8L:16D). For studies of intact animals in both photoperiods, plasma luteinizing hormone (LH) levels were quantified in blood samples collected from adult ferrets at 5-min intervals over a 24-h period. Similar groups of females (estrous and anestrous) were ovariectomized (while remaining in their assigned photoperiods) and blood samples were collected at 5-min intervals for 4-h periods on Days 1, 2, 4, 10, 17, and 35 after ovariectomy. Intact, estrous females exhibited continuously low or undetectable levels of LH with no evidence of episodic secretion. Ovariectomy of these estrous animals resulted in rapid onset (within 24 h) of episodic LH secretion, with pulses occurring in excess of 1 pulse/h. No substantial further change in frequency or amplitude of pulses occurred in these females from 1 to 35 days postovariectomy. In contrast, intact anestrous ferrets exhibited clear episodic LH secretion at a frequency of about 0.4 pulses/h. Removal of ovaries from these females caused no change in LH secretion for 24-48 h, after which LH pulses gradually increased in frequency. By 18 days after ovariectomy, LH patterns were indistinguishable among ovariectomized females in long and short days. These studies suggest a major site of ovarian negative feedback on LH secretion during anestrus is the hypothalamus, whereas the site of the ovarian feedback in estrous females is not yet evident.     

Effects of applying gamma-aminobutyric acid(B) drugs into the medial basal hypothalamus on basal luteinizing hormone concentrations and on luteinizing hormone surges in the female sheep

Prior investigations have shown that localized infusion by microdialysis of gamma-aminobutyric acid(B) (GABA(B)) agonists into the medial basal hypothalamus of male sheep rapidly increases GnRH and LH pulse amplitude. The objectives of these studies were to determine if infusion of GABA(B) agonists SKF 97541 or baclofen into the medial basal hypothalamus of female sheep would affect basal LH secretion and if infusion of a potent antagonist would alter expression of LH surges induced by injection of estrogen. Infusion of either SKF 97541 (10 or 40 microM) or baclofen (1 mM) into estrogen-treated ovariectomized ewes did not alter basal LH secretory patterns, whereas both drugs significantly elevated mean LH and LH pulse amplitude in ovariectomized ewes during the nonbreeding season. Infusion of the antagonist CGP 52432 (250 or 500 microM) did not affect expression of estrogen-induced LH surges in ovariectomized ewes. These observations support the concept that GABA(B) receptors in the medial basal hypothalamus regulate basal LH secretion but do not regulate the surge mode of LH secretion in the female sheep.     

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.     

Kisspeptin Signaling Is Required for the Luteinizing Hormone Response in Anestrous Ewes following the Introduction of Males

The introduction of a novel male stimulates the hypothalamic-pituitary-gonadal axis of female sheep during seasonal anestrus, leading to the resumption of follicle maturation and ovulation. How this pheromone cue activates pulsatile secretion of gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH) is unknown. We hypothesised that pheromones activate kisspeptin neurons, the product of which is critical for the stimulation of GnRH neurons and fertility. During the non-breeding season, female sheep were exposed to novel males and blood samples collected for analysis of plasma LH profiles. Females without exposure to males served as controls. In addition, one hour before male exposure, a kisspeptin antagonist (P-271) or vehicle was infused into the lateral ventricle and continued for the entire period of male exposure. Introduction of a male led to elevated mean LH levels, due to increased LH pulse amplitude and pulse frequency in females, when compared to females not exposed to a male. Infusion of P-271 abolished this effect of male exposure. Brains were collected after the male effect stimulus and we observed an increase in the percentage of kisspeptin neurons co-expressing Fos, by immunohistochemistry. In addition, the per-cell expression of Kiss1 mRNA was increased in the rostral and mid (but not the caudal) arcuate nucleus (ARC) after male exposure in both aCSF and P-271 treated ewes, but the per-cell content of neurokinin B mRNA was decreased. There was also a generalized increase in Fos positive cells in the rostral and mid ARC as well as the ventromedial hypothalamus of females exposed to males. We conclude that introduction of male sheep to seasonally anestrous female sheep activates kisspeptin neurons and other cells in the hypothalamus, leading to increased GnRH/LH secretion.     

Pituitary receptors for gonadotropin-releasing hormone in relation to changes in pituitary and plasma luteinizing hormone in ovariectomized-hypothalamo pituitary disconnected ewes. I. Effect of changing frequency of gonadotropin-releasing hormone pulses

Studies were undertaken to determine if changes in the amplitude of luteinizing hormone (LH) pulses that occur in response to changes in the frequency of gonadotropin-releasing hormone (GnRH) pulses are due to an alteration in the number of GnRH receptors. Ewes were ovariectomized (OVX) and the hypothalamus was disconnected from the pituitary (HPD). Ewes were then given pulses of GnRH at a frequency of 1/h or 1/3 h. Two control groups were included: OVX ewes not subjected to HPD, and HPD ewes that were not OVX. At the end of one week of treatment, blood samples were collected to determine the amplitude of LH pulses. The treated ewes were killed just before the next scheduled pulse of GnRH, and the content of LH and number of GnRH receptors were measured in each pituitary. The amplitude of LH pulses was highly correlated with the amount of LH in the pituitary gland (r = 0.71, p less than 0.01), and both LH content and pulse amplitude (mean + SEM) were higher in ewes receiving GnRH once per 3 h (189.7 +/- 39.3 microgram/pituitary, 10.3 +/- 1.1 ng/ml, respectively) than in ewes receiving GnRH once per h (77.8 +/- 11.4 microgram/pituitary, 5.2 +/- 1.3 ng/ml). The pituitary content of LH was highest in the OVX ewes (260.2 +/- 57.4 micrograms/pituitary) and lowest in the nonpulsed HPD ewes (61.7 +/- 51.2 micrograms/pituitary). The number of GnRH receptors was similar in all groups, and was not correlated with any other variable.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The role of GABA(A) and GABA(B) receptors in the control of GnRH release in anestrous ewes

The paper reviews data concerning the involvement of GABA(A) and GABA(B) receptors in the control of GnRH secretion in anestrous ewes. Generally, GABA influences the GnRH release through GABA(A) and GABA(B) receptors located on perikaria of the GnRH neurons in the preoptic area (MPOA) or through the influence on beta-endorphinergic and catecholaminergic systems activity in MPOA and in ventromedial-infundibular region of the hypothalamus (VEN/NI). Stimulation of GABA(A) receptors in VEN/NI and MPOA attenuates GnRH release, while activation of GABA(B) receptors in MPOA decreases GnRH secretion, and in VEN/NI increases concentration of GnRH. The different neural mechanisms could be involved in this process: direct ligand action on the GABA(A) and GABA(B) receptors located on GnRH cells and axon terminals or indirect effect through the changes in the beta-endorphinergic and catecholaminergic systems activity in these structures of the brain.