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Background: Gonadotropin-inhibitory hormone (GnIH, human homologue of RFRP-3) suppresses gonadotropin secretion in animal models, but its effects have not been studied in the human., Objective: We tested the hypotheses that exogenous GnIH inhibits LH secretion (i) in postmenopausal women and (ii) in men concurrently administered exogenous kisspeptin., Design: Following in vitro and in vivo preclinical studies to functionally characterize the GnIH peptide, a dose-finding study (human GnIH: 1·5-150 μg/kg/h, iv for 3 h) was undertaken, and 50 μg/kg/h selected for further evaluation. Five postmenopausal women were administered 50 μg/kg/h iv infusion for 3 h or vehicle on two separate days. Four men were administered kisspeptin-10 (0·3 μg/kg iv bolus) with simultaneous infusion of GnIH (50 μg/kg/h, iv for 3 h) or vehicle., Participants: Healthy postmenopausal women (mean age 58 ± 2 years, LH: 30·8 ± 2·9 IU/l, FSH: 78·7 ± 6·4 IU/l, oestradiol: <50 pmol/l) and men (39·8 ± 2·1 years, mean total testosterone 12·1 ± 1·8 nmol/l, LH 2·2 ± 0·2 IU/l)., Primary Outcome: Change in area under curve (AUC) of LH during GnIHvs vehicle., Results: During GnIH administration in postmenopausal women, LH secretion decreased (ΔAUC: -9·9 ± 1·8 IU/3 h) vs vehicle (ΔAUC: -0·5 ± 1·7 IU/3 h; P = 0·02). Kisspeptin-10-stimulated LH responses in men were not affected by GnIH co-administration (60-min AUC of LH 6·2 ± 0·8 IU/h with kisspeptin-10 alone, 6·3 ± 1·0 IU/h, kisspeptin-10 with GnIH, P = 0·72). Exogenous GnIH was well tolerated, with no adverse events reported., Conclusions: Gonadotropin-inhibitory hormone decreased LH secretion in postmenopausal women in this first-in-human study. Kisspeptin-stimulated LH secretion in men was not inhibited during concomitant administration of GnIH., (© 2017 John Wiley & Sons Ltd.)

Pulsatile luteinising hormone (LH) secretion is suppressed by food restriction and rapidly restored by return to ad lib. feeding concomitant with an increase in the oxidation of free fatty acids, although there is no increase in plasma leptin concentrations or body fat content in ovariectomised ewes. The ingestion of food may stimulate LH secretion by increasing availability of oxidisable metabolic substrates. Ruminal digestion is characterised by the production of volatile fatty acids and, of these, propionate is the major gluconeogenic substrate, and both glucose and propionate are oxidisable in a variety of tissues. To examine whether increases in mesenteric propionate concentrations are sufficient for restoration of pulsatile LH secretion during a period of food restriction, adult, food-restricted, hypogonadotrophic, ovariectomised ewes received mesenteric vein infusions of 5 μmol/min/kg body weight (BW) propionate or saline, whereas normal weight, ad lib.-fed ewes received mesenteric infusions of saline for 10 days. Blood samples were taken every 10 min for 5 h before the start of the 10-day infusion period, and continued throughout the first 5 h of infusion on the afternoon of day 1, and in the morning on days 2, 7 and 10. Propionate-infused, food-restricted and ad lib.-fed, saline-infused ewes showed a significantly higher LH pulse frequency compared to that of food-restricted-saline-infused ewes on postinfusion days 1 and 2 but not on days 7 and 10, and only the saline-infused, food-restricted group lost a significant amount of body weight. These results indicate that the reproductive system can respond acutely to infusion of metabolic fuels such as propionate, although a sustained recovery of pulsatile LH secretion requires more than an increase in this single metabolic substrate., (© 2011 The Authors. Journal of Neuroendocrinology © 2011 Blackwell Publishing Ltd.)

Kisspeptin is the product of the Kiss1 gene that regulates GnRH secretion. In sheep, Kiss1 mRNA-expressing cells are found in the preoptic area (POA) and arcuate nucleus (ARC), and expression is up-regulated in the caudal ARC during the periovulatory period. We hypothesized that kisspeptin neurons in the ARC are activated by estradiol-17beta prior to the preovulatory LH surge. Ovariectomized ewes were treated as follows: 1) estradiol-17beta implants (sc 2 wk) to cause tonic negative feedback; 2) vehicle (no estrogen negative or positive feedback); or 3) positive feedback/GnRH surge-inducing injection of estradiol-17beta (50 microg iv). For groups 2 and 3, brains were collected 1 h after treatment and kisspeptin/Fos immunoreactivity was examined. In the caudal and mid-ARC, the percentage of kisspeptin cells that were Fos immunoreactive increased after acute estradiol treatment (group 3) over that seen in the other two groups. Kisspeptin/Fos colocalization was also quantified in ewes during the luteal and late-follicular phase of the estrous cycle, showing a trend toward an increase in colocalization in the late-follicular phase. Kisspeptin/Fos colocalization was similar in the POA across groups in both experiments. Analysis of Kiss1 mRNA by in situ hybridization revealed an increase in expression during the late-follicular phase in the caudal ARC and POA. These data suggest kisspeptin neurons located in the caudal extent of the ARC are involved in generating the positive feedback preovulatory GnRH/LH surge in the ewe, but there may also be a role for Kiss1-expressing cells in the POA.

Stress disrupts the preovulatory luteinizing hormone (LH) surge in females, but the mechanisms are unknown. We tested the hypothesis that cortisol compromises the ability of estrogen to induce a preovulatory-like LH surge in ovariectomized ewes in both the breeding and nonbreeding season. Luteinizing hormone surges were induced in ovariectomized ewes by treatment with progesterone followed by a surge-inducing estradiol-17beta (E2) stimulus using a crossover design. The experiment was replicated in the breeding and nonbreeding seasons. Cortisol reduced the incidence of LH surges irrespective of season. Cortisol increased the latency from E2 stimulus to the onset of the surge in the breeding season only and suppressed the LH surge amplitude during both seasons (P<0.01). We conclude that cortisol can interfere with the LH surge in several ways: delay, blunt, and in extreme cases prevent the E2-induced LH surge. Furthermore, the effect of cortisol to delay the E2-induced LH surge is more pronounced in the breeding season. These results show that cortisol disrupts the positive feedback effect of E2 to trigger an LH surge and suggest the involvement of multiple mechanisms.

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.

There is strong evidence that kisspeptin acts to regulate GnRH secretion, but whether there is also a component of action on the gonadotropes is not clear. Using quantitative RT-PCR, we found that G protein-coupled receptor-54 mRNA is expressed in ovine pituitary cell fractions enriched for gonadotropes as well as in somatotropes and lactotropes. To test whether kisspeptin acts directly on the pituitary gonadotropes, we first examined LH release from primary ovine pituitary cell cultures treated with kisspeptin. We found that kisspeptin treatment increased the concentration of LH in culture media by 80%, compared with control, but only in pituitary cultures from ewes during the follicular phase of the estrous cycle. After this, we determined whether kisspeptin acts on the pituitary gland in vivo. Using GnRH-replaced ovariectomized hypothalamo-pituitary-disconnected ewes, we were not able to achieve any effect of kisspeptin on LH under steady-state conditions or during the period of an estrogen-induced LH surge. Finally, we collected hypophysial portal blood samples from ovariectomized ewes and measured kisspeptin levels. Low but detectable amounts of kisspeptin were found in portal plasma, but levels were similar in ovariectomized ewes that were untreated or given estrogen to elicit an LH surge. Thus, although we observed an effect of kisspeptin on LH release in vitro in some situations, similar findings were not obtained in vivo. Moreover, the low concentrations of kisspeptin in hypophysial portal blood and the lack of any change during the period of an estrogen-induced GnRH/LH surge suggest that action on the pituitary gland is not of major consequence in terms of LH release.

The reproductive system, including pulsatile luteinising hormone (LH) secretion, is inhibited by deficits in energy availability and restored by energy surfeits. Plasma LH, insulin, leptin, ghrelin, glucose, ketone body, and nonesterified fatty acid concentrations were measured in ovariectomised, food-restricted ewes before and after return to ad libitum feeding to determine the factors that change in time to account for the restoration of pulsatile LH secretion. At 07.00 h, blood was sampled every 10 min for 5 h from ovariectomised, hypogonadotrophic, chronically food-restricted and ad libitum-fed ewes (Fed). At 12.00 h, four of the food-restricted sheep were given ad libitum access to food (Re-Fed), while three ewes continued to be food restricted (Restricted). Sampling continued for 5 h and resumed again on the mornings of days 2, 4, and 9. A pulse of LH was seen within 1 h of re-feeding in all Re-Fed ewes, and interpulse interval (IPI) was significantly shorter in Re-Fed compared to Restricted ewes and longer than in Fed ewes during the period after re-feeding. Re-Fed LH IPI was not restored to that of Fed ewes until sometime between days 4 and 9. The first pulse occurred within minutes, whereas restoration of IPI occurred after 4-8 days. Prior to the initial LH pulses seen in Re-Fed ewes, plasma ketone bodies first fell and then rose to levels significantly above those in Restricted ewes. Significant changes in circulating insulin, ghrelin, glucose, and total ketone body concentrations, daily food intake and lean body mass preceded restoration of Re-Fed LH IPI some time between days 4 and 9, but there were no significant changes in adiposity or circulating leptin concentrations, consistent with the hypothesis that LH pulses are reinitiated by changes in the availability of oxidisable metabolic fuels and possibly insulin, but not leptin concentrations.

Kisspeptins are encoded by the gene KiSS-1 and regulate gonadotrophin-releasing hormone (GnRH) and gonadotrophin secretion in various species, including humans. Here, we quantify gene expression of KiSS-1 in the arcuate nucleus (ARC) across the ovine oestrous cycle and demonstrate an increase in the caudal division of the ARC during the preovulatory period. These data strongly suggest that kisspeptins are involved in the generation of the preovulatory GnRH and luteinising hormone surge.

We aimed to determine the importance of leptin in the regulation of luteinizing hormone (LH) and growth hormone (GH) secretion in ovariectomized (OVX) ewes. Lean and fat sheep were produced by dietary manipulation over 8 months and were then fasted for 32 h. Plasma concentrations of glucose, insulin and leptin were higher in the fat group. Fasting decreased plasma concentrations of glucose and insulin and increased concentrations of nonesterified fatty acids (NEFA) in fat and lean ewes, but leptin concentrations were reduced in the fat group only. Plasma GH concentrations were higher in the lean group and LH concentrations were lower; there was no effect of fasting. These data suggested that long-term changes in plasma leptin concentrations might affect LH and GH secretion, but acute changes with fasting had no effect. OVX ewes of normal body weight were fasted for 72 h with or without intracerebroventricular (i.c.v.) infusion of leptin (4 microg/h), achieving similar metabolic effects to the 32 h fast. The 72-h fast increased LH pulse amplitude, mean GH and cortisol concentrations, but these changes were corrected towards normal by leptin treatment. Thus, leptin could attenuate fasting-induced alterations in the secretion of LH, GH and cortisol. Finally, we food-restricted OVX ewes for 4 months (lean), leading to a 20-kg reduction in body weight. Plasma concentrations of leptin and insulin were decreased, and plasma GH concentrations increased, but there was no effect on plasma concentrations of LH, glucose or NEFA. Icv infusion of leptin did not affect any endocrine or metabolic parameter in these ewes. In summary, maintenance of a lean or fat condition for a prolonged period (8 months) or an extended fasting (72 h) can affect LH and GH secretion, but short-term food restriction (4 months) affected only GH secretion and short-term fasting (32 h) had no effect on either LH or GH secretion. This is in spite of altered plasma leptin concentrations in all circumstances studied. Although leptin treatment can restore plasma concentrations of LH, GH and cortisol towards normal in sheep fasted for 72 h, some other factor(s) must signal to the brain to cause shifts in neuroendocrine function in other conditions where nutritional/metabolic status is altered.

Studies in rats suggest that neuropeptide Y (NPY) plays a stimulatory role in the generation of the preovulatory luteinizing hormone (LH) surge, via the Y1 receptor. We have investigated this issue using the oestradiol benzoate (EB)-treated ovariectomized (OVX) ewe which is a model for the preovulatory LH surge. A Y1 receptor antagonist (BIBO3304) was infused (25 microg/h) into the third cerebral ventricle (III-V) from 2 h before EB injection for 24 h, and had no effect on the ensuing LH surge. Using in situ hybridization, we then examined expression of NPY mRNA in the arcuate nucleus during the luteal, follicular and oestrous phases of the oestrous cycle, and found that levels were greatest during the luteal phase. Thus, reduced NPY synthesis might be an integral factor in the events leading to the cyclic preovulatory LH surge. This was tested by infusion of NPY (25 microg/h) into the III-V (as above). The NPY infusion delayed the LH surge until the infusion was ceased. High levels of NPY expression during the luteal phase of the oestrous cycle may be caused by progesterone. Thus, we determined whether NPY cells possess progesterone receptors (PR) and whether progesterone treatment up-regulates NPY mRNA expression in the arcuate nucleus. Immunohistochemistry for NPY and PR was performed in OVX, oestrogen-treated ewes, but no NPY cells of the arcuate nucleus were seen to colocalize PR. In situ hybridization for NPY was performed in OVX and OVX ewes treated with progesterone. There was no significant effect of progesterone treatment on NPY mRNA expression in the arcuate nucleus. We conclude that chronically elevated levels of NPY block the preovulatory surge of gonadotropin-releasing hormone/LH secretion in sheep, but high levels of NPY mRNA expression in the luteal phase of the oestrous cycle cannot be explained by an action of progesterone.

Stress responses are thought to act within the hypothalamopituitary unit to impair the reproductive system, and the sites of action may differ between sexes. The effect of isolation and restraint stress on pituitary responsiveness to GnRH in sheep was investigated, with emphasis on possible sex differences. Experiments were conducted during the breeding season and the nonbreeding season. In both experiments, 125 ng of GnRH was injected i.v. every 2 h into hypothalamopituitary disconnected, gonadectomized rams and ewes on 3 experimental days, with each day divided into two periods. During the second period on Day 2, isolation and restraint stress was imposed for 5.5 h. Plasma concentrations of LH and cortisol were measured in samples of blood collected from the jugular vein. In the second experiment (nonbreeding season), plasma concentrations of epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylglycol were also measured. In both experiments, there was no effect of isolation and restraint stress on plasma concentrations of cortisol in either sex. During the breeding season, there was no effect of isolation and restraint stress on plasma concentrations of LH in either sex. During the nonbreeding season, the amplitude of the first LH pulse after the commencement of stress was significantly reduced (P < 0.05) in rams and ewes. In the second experiment, during stress there was a significant increase (P < 0.05) in plasma concentrations of epinephrine in rams and ewes and significantly higher (P < 0.05) basal concentrations of norepinephrine in ewes than in rams. These results suggest that in sheep stress reduces responsiveness of the pituitary gland to exogenous GnRH during the nonbreeding season but not during the breeding season, possibly because of mediators of the stress response other than those of the hypothalamus-pituitary-adrenal gland axis.

There are sex differences in the response to stress and in the influence of stress on reproduction which may be due to gonadal steroids but the nature of these differences and the role of the gonads are not understood. We tested the hypotheses that sex and the presence/absence of gonads (gonadal status) will influence the cortisol response to injection of ACTH, insulin-induced hypoglycaemia and isolation/restraint stress, and that sex and gonadal status will influence the secretion of LH in response to isolation/restraint stress. Four groups of sheep were used in each of three experiments: gonad-intact rams, gonadectomised rams, gonad-intact ewes in the mid-luteal phase of the oestrous cycle and gonadectomised ewes. In Experiment 1 (n=4/group), jugular blood samples were collected every 10 min for 6 h; after 3 h, two animals in each group were injected (i.v.) with ACTH and the remaining two animals were injected (i.v.) with saline. Treatments were reversed 5 days later so that every animal received both treatments. Experiment 2 (n=4/group) used a similar schedule except that insulin was injected (i.v.) instead of ACTH. In Experiment 3 (n=5/group), blood samples were collected every 10 min for 16 h on a control day and again 2 weeks later when, after 8 h of sampling, all sheep were isolated and restrained for 8 h. Plasma cortisol was significantly (P<0.05) elevated following injection of ACTH or insulin and during isolation/restraint stress. There were no significant differences between the sexes in the cortisol response to ACTH. Rams had a greater (P<0.05) cortisol response to insulin-induced hypoglycaemia than ewes while ewes had a greater (P<0.05) cortisol response to isolation/restraint stress than rams. There was no effect of gonadal status on these parameters. Plasma LH was suppressed (P<0.05) in gonadectomised animals during isolation/restraint stress but was not affected in gonad-intact animals, and there were no differences between the sexes. Our results show that the sex that has the greater cortisol response to a stressor depends on the stressor imposed and that these sex differences are likely to be at the level of the hypothalamo-pituitary unit rather than at the adrenal gland. Since there was a sex difference in the cortisol response to isolation/restraint, the lack of a sex difference in the response of LH to this stress suggests that glucocorticoids are unlikely to be a major mediator of the stress-induced suppression of LH secretion.

We tested the hypotheses that progesterone enhances the negative feedback actions of testosterone in rams and that this occurs through actions at the hypothalamus. In the first part of this study, blood samples were collected every 10 min for 12 h before and after 7 days of treatment (i.m.) of castrated Romney Marsh rams (n=5 per group) with vehicle, progesterone (4 mg/12 h), testosterone (4 mg/12 h) or a combination of progesterone (4 mg/12 h) and testosterone (4 mg/12 h). In the second part of this study the brains of four gonad-intact Romney Marsh rams were collected, the hypothalamus was sectioned and in situ hybridisation of mRNA for progesterone receptors conducted. After 7 days of treatment with vehicle or progesterone or testosterone alone, there were no changes in the secretion of LH. In contrast, treatment with a combination of progesterone and testosterone resulted in a significant (P<0.01, repeated measures ANOVA) decrease in mean plasma concentrations of LH, the number of LH pulses per hour and the pre-LH pulse nadir and a significant (P<0.01) increase in the inter-LH pulse interval. We found cells containing mRNA for progesterone receptors throughout the hypothalamus, including the preoptic area (where most GnRH neurons are located in sheep), the periventricular, ventromedial and arcuate nuclei and the bed nucleus of the stria terminalis. This study shows that progesterone is capable of acting centrally with testosterone to suppress the secretion of LH in castrated rams and that cells containing mRNA for progesterone receptors are located in the hypothalamus of rams in the vicinity of GnRH neurons.

In this study we examined the release of dopamine and noradrenaline in the ventromedial hypothalamus (VMH) of ovariectomized ewes during the oestrogen-induced luteinizing hormone (LH) surge by measuring their respective metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and methoxyhydroxyphenylglycol (MHPG) using microdialysis. Further we investigated whether inhibition of catecholamine synthesis in the VMH by bilateral reverse dialysis of alpha-methyl-p-tyrosine (alpha-MPT) would block the oestrogen-induced LH and/or prolactin surges. Oestradiol treatment (50 microg oestradiol benzoate) of ovariectomized ewes resulted in a biphasic LH response, significantly (p < 0.05) decreasing LH concentrations from 2.5 to 10.5 h after injection, followed by an LH surge beginning at 16 h. Prolactin concentrations were also significantly (p < 0.05) increased in oestradiol-treated ewes from 13 h. VMH DOPAC concentrations in oil-vehicle-treated animals were at the level of detection (0.02 ng/ml) in most samples over the 24-hour sampling period. In oestradiol-treated ewes, VMH DOPAC levels were initially low before and up to 8 h after oestradiol injection but then increased significantly (p < 0.05) at 10-12 h and remained elevated up to 20 h after injection. In contrast, oestradiol injection had no effect on MHPG concentrations in the VMH. Bilateral reverse microdialysis of alpha-MPT into the VMH significantly (p < 0.05) delayed the time from oestradiol injection to the onset of the LH surge, the time to peak LH concentration and attenuated the LH surge compared with reverse dialysis of Ringer solution alone. In contrast, alpha-MPT treatment had no effect upon the oestradiol-induced increase in prolactin concentrations. This study provides evidence that the VMH is an important hypothalamic site in the neuro-endocrine control of the LH surge in ewes. The results suggest that dopaminergic neurons with terminals in the VMH are part of a neuronal pathway mediating the positive feedback effects of oestradiol on gonadotropin-releasing hormone secretion and the LH surge., (Copyright 2001 S. Karger AG, Basel.)

Leptin can act as a satiety factor and exert neuroendocrine effects, but most studies have been performed in fasted animals. We aimed to determine the effect of chronic under-nutrition on the response to a 3-day intracerebroventricular infusion of leptin with regard to food intake and the secretion of pituitary hormones. Ovariectomised ewes (n=6) had a mean (+/-s.e.m. ) bodyweight of 56+/-0.8 kg on a diet available ad libitum (ad lib) or 33.4+/-1 kg on a restricted diet. The differential bodyweight was achieved by dietary means over a period of 6 months prior to the commencement of the study. Leptin (4 microg/h) or vehicle (artificial cerebrospinal fluid (aCSF)) was infused into the third cerebral ventricle for 3 days. Blood samples were taken prior to commencement and on day 3 of infusion for the assay of plasma hormone levels. The experiment was repeated one week later in a cross-over design. Food intake and metabolic status were monitored daily. The luteinising hormone (LH) pulse amplitude was lower (P<0.05) but plasma growth hormone (GH) levels were higher (P<0.05) in the food-restricted animals. Plasma levels of glucose, lactate, insulin, urea and triglycerides were similar in the two groups but non-esterified fatty acid levels were higher (P<0.01) in the animals on an ad lib diet. Leptin reduced (P<0.05) food intake only in the animals fed an ad lib diet. Leptin increased (P<0.05) the secretion of LH in the food-restricted group only and increased (P<0.05) GH irrespective of bodyweight. In conclusion, leptin does not alter food intake in animals on a restricted diet but can increase the secretion of LH in the same animals. The treatment of leptin was not sufficient to reduce plasma GH levels in the food-restricted animals, suggesting that other factors or mechanisms must be involved in the regulation of this axis.

Within the hypothalamus, neurones that express neuropeptide Y (NPY) and galanin have been implicated in the regulation of gonadotropin-releasing hormone (GnRH) and gonadotropin secretion. We aimed to determine the extent to which the expression of these two neuronal systems is linked to the seasonal reproductive cycle, and the effect of chronic oestrogen treatment. Immunohistochemical analysis was used to examine changes between the breeding season and anestrus in ovariectomized (OVX) ewes with or without oestrogen treatment (s.c. implants for 2 weeks). Serial blood sampling established plasma luteinizing hormone (LH) profiles, and the ewes were subsequently killed and the brains perfused for immunohistochemistry. In OVX ewes, the amplitude of LH pulses was greater in the nonbreeding season than in the breeding season. Oestrogen treatment caused a marked reduction in plasma LH concentrations during anestrus, but not in the breeding season. The number of cells in the arcuate nucleus/median eminence region (ARC-ME) that stained for NPY was lower in ewes killed in anestrus (September) than in ewes killed in the breeding season (May), but there was no seasonal change in the number of galanin-stained cells. Within season, oestrogen treatment did not affect NPY- or galanin-cell number. There was no effect of season or oestrogen on the area of varicose fibres/terminals for either peptide in the ARC-ME, but galanin immunostaining was more intense during the breeding season. We conclude that the amount of NPY in cell bodies of the ARC-ME is lower in ewes in the nonbreeding season; this could reflect a steroid-independent effect of photoperiod. We also conclude that the long-term negative-feedback effect of oestrogen on GnRH/LH secretion does not appear to be mediated by NPY- or galanin-containing neurones in the ewe.

Leptin is a secreted product of the adipocytes that regulates a variety of functions. The presence of the leptin receptor (LR) has been demonstrated in the endocrine and neuroendocrine tissue, but only limited information is available regarding cell-specific expression in the anterior pituitary gland. We have used double-label immunofluorescence histochemistry to study the distribution of LR-like immunoreactivity (LR-ir) in the corticotropes, somatotropes, and gonadotropes of the ovine anterior pituitary. LR-ir was found in 34% of cells in the pars distalis and 94% of the cells in the pars tuberalis. In the pars distalis, LR-ir was present in 27% of corticotropes, 69% of somatotropes, and 29% of gonadotropes. In contrast, 90% of the gonadotropes in the pars tuberalis were immunopositive for LR. There was no alteration in the number of gonadotropes containing LR-ir during the various phases of the estrous cycle (n = 3/group) in the pars distalis (luteal phase, 36%; follicular phase, 32%; and estrous phase, 32%). In conclusion, we show that, in the pars distalis, LR-ir is expressed to a greater extent in the somatotropes than in the gonadotropes or corticotropes. This is in accordance with the documented effects of leptin on pituitary GH secretion. The differential expression of LR-ir between the gonadotropes of the pars distalis and pars tuberalis probably reflects the different phenotypes of the cells in these two regions. Lower levels of LR-ir expression in gonadotropes and corticotropes of the pars distalis may suggest that leptin does not substantially influence these particular cells, at least in this species.

This study tested the hypothesis that central administration of corticotrophin-releasing hormone (CRH) and/or arginine vasopressin (AVP) will affect the secretion of LH in rams and that testosterone is necessary for these actions to occur. Plasma LH levels were measured in castrated rams during 1 h infusion of either 100 microliter vehicle/mock cerebrospinal fluid (CSF) or mock CSF containing 25 microgram CRH, 25 microgram AVP or 25 microgram of each peptide through guide cannulae into the third cerebral ventricle. These intracerebroventricular (i.c.v.) infusions were given to the castrated rams following injections (i.m.) each 12 h of oil or 8 mg testosterone propionate for 7 days. Blood samples were collected every 10 min for 4 h before i.c.v. infusion, during infusion and for 4 h following the infusion. Infusion of vehicle did not affect any endocrine parameters. In contrast, the plasma concentrations of LH and the amplitude of LH pulses were increased significantly during and following infusion of CRH, and this effect was not influenced by whether the castrated rams were treated with testosterone propionate or whether the CRH was administered in combination with AVP. Infusion of AVP alone did not affect LH secretion. The frequency of LH pulses and the plasma concentrations of FSH did not change with any of the i.c.v. treatments. The plasma concentrations of cortisol were significantly increased by CRH and AVP infusions. The plasma concentrations of cortisol achieved during and following i.c.v. infusion of CRH and AVP combined were greater than the concentrations achieved as a result of treatment with AVP alone but were similar to those with CRH. There was no effect of testosterone propionate on cortisol levels. These results show that CRH, but not AVP, is capable of acting either centrally or at the pituitary level to increase the secretion of LH in rams and these actions are not affected by testosterone. The stimulatory effects of CRH on LH secretion are to increase the amplitude of GnRH pulses and/or the responsiveness of the pituitary to the actions of GnRH with no effect on the frequency of GnRH pulses. The secretion of FSH in rams is not influenced by either CRH or AVP. The effect of CRH to increase LH pulse amplitude occurs in the face of increased cortisol levels, further reinforcing our belief that this adrenal steroid does not affect the reproductive axis in this species.

We have measured dopamine beta hydroxylase (DBH) immunoreactivity in the preoptic hypothalamus as an index of release of noradrenaline in the ovariectomised (OVX) ewe at the time of an oestrogen-induced surge in luteinizing hormone (LH) release. OVX ewes (n=5) were given an injection of 50 microg oestradiol benzoate (or oil), which causes a surge in the secretion of LH. Blood samples were taken and sheep were killed 16 h later. Other groups (n=3) were given oil or oestrogen and killed 6 h later. Brains were collected for immunohistochemistry and image analysis. The number of DBH-stained cells and the integrated optical density of the cells was measured throughout the A1 field of the brainstem. The DBH staining was measured in the terminal beds of the hypothalamus. There was no difference between the controls and the EB-treated OVX ewes in the number of DBH positive cells or the optical density of DBH-staining cells in the A1 field. Within the preoptic area, there was reduced (P<0.02) DBH staining in the 16 h EB-treated ewes. There was no change in the DBH staining in the paraventricular nucleus or the arcuate nucleus of the hypothalamus. These data suggest that there is release of noradrenaline in the preoptic area at the time of the E-induced GnRH/LH surge in the OVX ewe.

In this study we used an isolation/restraint stress to test the hypothesis that stress will affect the secretion of LH differently in gonadectomised rams and ewes treated with different combinations of sex steroids. Romney Marsh sheep were gonadectomised two weeks prior to these experiments. In the first experiment male and female sheep were treated with vehicle or different sex steroids for 7 days prior to the application of the isolation/restraint stress. Male sheep received either i.m. oil (control rams) or 6 mg testosterone propionate injections every 12 h. Female sheep were given empty s.c. implants (control ewes), or 2x1 cm s.c. implants containing oestradiol, or an intravaginal controlled internal drug release device containing 0.3 g progesterone, or the combination of oestradiol and progesterone. There were four animals in each group. On the day of application of the isolation/restraint stress, blood samples were collected every 10 min for 16 h for the subsequent measurement of plasma LH and cortisol concentrations. After 8 h the stress was applied for 4 h. Two weeks later, blood samples were collected for a further 16 h from the control rams and ewes, but on this day no stress was imposed. In the second experiment, separate control gonadectomised rams and ewes (n=4/group) were studied for 7 h on 3 consecutive days, when separate treatments were applied. On day 1, the animals received no treatment; on day 2, isolation/restraint stress was applied after 3 h; and on day 3, an i. v. injection of 2 microg/kg ACTH1-24 was given after 3 h. On each day, blood samples were collected every 10 min and the LH response to the i.v. injection of 500 ng GnRH administered after 5 h of sampling was measured. In Experiment 1, the secretion of LH was suppressed during isolation/restraint in all groups but the parameters of LH secretion (LH pulse frequency and amplitude) that were affected varied between groups. In control rams, LH pulse amplitude, and not frequency, was decreased during isolation/restraint whereas in rams treated with testosterone propionate the stressor reduced pulse frequency and not amplitude. In control ewes, isolation/restraint decreased LH pulse frequency but not amplitude. Isolation/restraint reduced both LH pulse frequency and amplitude in ewes treated with oestradiol, LH pulse frequency in ewes treated with progesterone and only LH pulse amplitude in ewes treated with both oestradiol and progesterone. There was no change in LH secretion during the day of no stress. Plasma concentrations of cortisol were higher during isolation/restraint than on the day of no stress. On the day of isolation/restraint maximal concentrations of cortisol were observed during the application of the stressor but there were no differences between groups in the magnitude of this response. In Experiment 2, isolation/restraint reduced the LH response to GnRH in rams but not ewes and ACTH reduced the LH response to GnRH both in rams and ewes. Our results show that the mechanism(s) by which isolation/restraint stress suppresses LH secretion in sheep is influenced by sex steroids. The predominance of particular sex steroids in the circulation may affect the extent to which stress inhibits the secretion of GnRH from the hypothalamus and/or the responsiveness of the pituitary gland to the actions of GnRH. There are also differences between the sexes in the effects of stress on LH secretion that are independent of the sex steroids.

A reduction in inhibition (disinhibition) of gonadotropin-releasing hormone (GnRH) secretion by endogenous opioid systems in the hypothalamus is thought to be permissive of the preovulatory surge of GnRH and luteinising hormone (LH). In rats, proopiomelanocortin (POMC) mRNA levels in the arcuate nucleus are reduced at the time of the LH surge, and this is thought to be part of the neural mechanism of oestrogen positive feedback. There are no studies of POMC mRNA levels at the time of the LH surge in other species. POMC mRNA levels were measured by in-situ hybridization using a 35S-labelled cRNA probe and computer-assisted grain counting in the arcuate nucleus of ovary-intact ewes (n=4) killed on day 10 of the luteal phase or 24 or 48 h into the follicular phase (experiment 1), and ewes killed on day 10 of the luteal phase or during the preovulatory LH surge (experiment 2). Grain counts per cell, the proportion of cellular area covered by grains and the number of labelled cells per section were not significantly different (P > 0.1) between animals killed in the luteal phase and animals killed during the follicular phase or during the LH surge. We conclude that in sheep, POMC mRNA levels are not reduced at the time of the preovulatory LH surge, and reduced POMC gene transcription does not appear to be part of the neural mechanism of oestrogen positive feedback in this species.

Endogenous opioid systems in the hypothalamus inhibit gonadotropin-releasing hormone (GnRH) secretion, and a reduction in this inhibitory input (disinhibition) is thought to be part of the neural mechanism of the preovulatory GnRH/luteinizing hormone (LH) surge. We showed previously that intracerebroventricular infusion of the highly specific opioid mu-receptor agonist DAMGO delayed the oestrogen-induced LH surge in ovariectomized (OVX) ewes, whereas both delta- and kappa-agonists were ineffective. The aim of the present study was to establish the anatomical site of this effect. The most likely hypothalamic sites of action are the medial preoptic area (MPOA), where most GnRH perikarya are located in sheep, and/or the median eminence (ME), where GnRH fibres terminate on hypophysial portal blood vessels. Conscious, unrestrained OVX ewes with permanent bilateral guide tubes implanted into either the MPOA or the mediobasal hypothalamus (MBH), close to the ME, were injected (i.m.) with oestradiol benzoate (EB) 50 microg (t = 0 h). In this model, EB elicits a time-delayed surge in LH secretion after 13-19 h. Jugular venous blood was sampled at half-hourly intervals from -2 to 0 h, and from 10 to 26 h. From 12 to 20 h, bilateral infusions of either the highly specific opioid mu-agonist DAMGO (40 nmol/h bilaterally) or saline were given into the MPOA or MBH at 2.5 microl/h. Guide tube placements were confirmed histologically. The mean (+/- SEM) time to the onset of the LH surge was significantly (p < 0.01) increased in the animals (n = 9) that received DAMGO infusion into the MPOA (20.5 +/- 1.4 vs. 15.7 +/- 0.6 h in the saline-infused controls). The effect was clearly apparent in 6/9 of the DAMGO-infused animals. The mean (+/- SEM) time to LH surge onset was also significantly (p < 0.01) increased in the animals (n = 8) that received DAMGO infusion into the MBH (19.7 +/- 1.2 vs. 14.3 +/- 0.5 h). In this case, the effect was clearly apparent in 4/8 of the DAMGO-infused animals. We conclude that bilateral infusion of DAMGO into either the MPOA or the MBH can delay the EB-induced LH surge in OVX ewes. These data provide further evidence for dual hypothalamic sites of opioid regulation of GnRH secretion, and are consistent with the hypothesis that disinhibition from opioid tone at both the MPOA and MBH/ME is permissive of the preovulatory GnRH/LH surge.

Anterior pituitary gland contents of LH and LH beta- and alpha-subunit mRNAs, and circulating concentrations of LH and testosterone, were determined in bulls treated with the LH-releasing hormone (LHRH) agonist deslorelin. Brahman (Bos indicus) bulls (14-month-old) were allocated to two groups and received the following: Control (n = 5), no treatment; Deslorelin (n = 4), four deslorelin implants (approximately 200 micrograms total deslorelin/day) for 36 d. Plasma concentrations of LH were higher in bulls treated with deslorelin on Day 1, had returned to typical levels by Day 8, and did not differ for control bulls and bulls treated with deslorelin from Day 8 to Day 29. Pituitary content of LH on Day 36 was reduced (P < 0.001) in bulls treated with deslorelin (33 +/- 4 ng/mg) compared with control bulls (553 +/- 142 ng/mg). Relative pituitary content of LH beta-subunit mRNA was also reduced on Day 36 in bulls treated with deslorelin (Control, 0.65 +/- 0.10; Deslorelin, 0.22 +/- 0.04; P = 0.003). However, alpha-subunit mRNA relative content did not differ (Control, 0.73 +/- 0.15; Deslorelin, 1.06 +/- 0.12; P > 0.05). Plasma concentrations of testosterone were increased over the period of the experiment in the bulls treated with deslorelin compared with control bulls. This is the first demonstration of reduced pituitary content of LH beta-subunit mRNA and LH, and unaltered content of alpha-subunit mRNA, in bulls treated with LHRH agonist. This was associated with apparently typical plasma concentrations of LH and elevated plasma testosterone. The anterior pituitary in bulls treated with LHRH agonist, therefore, undergoes classical desensitization and downregulation, but plasma LH and testosterone are not suppressed.

Several peptidases have been postulated to degrade the hypothalamic peptide gonadotropin-releasing hormone (GnRH), but it is not known if such enzymes contribute significantly to the delivery of GnRH to the pituitary in vivo. Furthermore, the activity of GnRH-inactivating peptidases may vary in different reproductive states, such as across the estrous cycle. In the present study, specific fluorescent substrates were used to measure the activity of the two major GnRH-degrading enzymes, prolyl endopeptidase (PEP) and endopeptidase 3.4.24.15 (EP 24.15), in soluble extracts of the median eminentes (ME) of ewes during different phases of the estrous cycle. Levels of EP 24.15 and PEP activity in the ME did not vary significantly across the cycle, although PEP activity was lowest at the time of the preovulatory luteinizing hormone (LH) surge. However, a statistically significant decline in PEP activity (18%, P = 0.02) was observed in the ME of OVX ewes in which a surge was induced by estrogen when compared to oil-treated OVX controls, suggesting a possible negative regulation of PEP activity by this steroid. The effect of intracerebroventricular (i.c.v.) infusion of several peptidase inhibitors on the pulsatile release of LH in the conscious OVX ewe was also examined. No consistent changes in the pattern of LH release were observed with i.c.v. infusion of the EP 24.15 inhibitor N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP-AAY-pAB) or the angiotensin-converting enzyme (ACE) inhibitor captopril. Similarly, administration of the prolyl endopeptidase inhibitor bacitracin, or a more specific inhibitor of this enzyme, Z-Proprolinal (ZPP), did not alter LH release patterns. The results did not demonstrate a major role for changes in the activity of EP 24.15, PEP, or ACE in altering the pattern of GnRH secretion, but a minor reduction in PEP levels may occur at the time of the estrogen-induced LH surge.

It has previously been shown in the ewe that luteinizing hormone (LH)-containing secretory granules become polarized to the side of the gonadotrope nearest to a vascular sinusoid during the preovulatory period. We have used laser scanning confocal microscopy to monitor the migration of LH and follicle stimulating hormone (FSH)-containing secretory granules in the gonadotrope of the ewe. Ovariectomized (OVX) ewes (n = 4/group) were given either 50 micrograms of estradiol benzoate (EB) or oil and were killed 16 h later for collection of the pituitary glands for immunohistochemistry and confocal microscopy. Pituitary sections were simultaneously immunolabelled for LH and FSH and were visualized on the same sections using fluorescent markers. All cells that contained LH also contained FSH and vice versa. The results showed that LH-containing granules were uniformly distributed throughout the cytoplasm in 83% of gonadotropes from controls, while in the remaining 17% the granules were predominantly located adjacent to the plasma membrane. Following EB treatment, LH-containing secretory granules were distributed around the plasma membrane in 84% of immunopositive gonadotropes; FSH remained uniformly distributed throughout the cytoplasm. We conclude that estrogen causes the movement of LH-containing granules to the plasma membrane in gonadotropes but does not influence the distribution of FSH-containing granules. Rather than being polarized to one side of the cell, the LH-containing granules were distributed around the periphery of the cell.

A reduction in endogenous opioid inhibition (disinhibition) of GnRH secretion is thought to be permissive for the preovulatory GnRH/LH surge. There are no published studies of the effects of highly specific opioid receptor agonists on the LH surge in any species, and the relative importance of the opioid receptor subtypes mu, delta and kappa in the mechanism of disinhibition is unknown. In sheep, attempts to block the LH surge with opiates have been largely unsuccessful, and there is little evidence for reduced opioid inhibition during the GnRH/LH surge. The opioid receptor subtypes regulating PRL secretion in sheep are also unknown. Conscious, ovariectomized ewes with permanent third ventricular cannulae were injected with estradiol benzoate (EB) 50 micrograms or oil im (t = 0 h). In this model, EB elicits a time-delayed surge in LH secretion after 13-18 h. Jugular venous blood was sampled at half hourly intervals between-2 and 0 h and 10 and 26 h. From 12-20 h, infusions were made into the third ventricle of either the highly specific mu-agonist DAGO (10, 20 or 40 nmol/h), the delta-agonist DPDPE (40 nmol/h), the kappa-agonist U50488 (40 nmol/h) or saline (vehicle). In oil-treated animals (n = 4-6), DAGO infusion at 20 and 40 nmol/h reduced plasma LH whereas DPDPE or U50488 had no effect. In EB-treated animals (n = 6), DAGO (40 nmol/h) delayed the LH surge (mean +/- SEM time to surge onset 21.4 +/- 0.3 h vs. 14.0 +/- 0.4 h in controls, P < 0.0001). DAGO at 10 nmol/h did not alter surge onset and at 20 nmol/h had variable effects. DPDPE or U50488 did not affect LH surge timing or amplitude. All doses of DAGO increased plasma PRL, whereas DPDPE and U50488 had no effect. We conclude that, in ovariectomized ewes, activation of opioid mu-receptors, but not delta- or kappa-receptors, inhibits GnRH secretion, can block the estrogen-induced GnRH/LH surge and increases PRL secretion. The results are consistent with the disinhibition hypothesis.

The objective in this study was to characterize direct effects of the LHRH agonist, deslorelin, on anterior pituitary gland function in male cattle in the absence of gonadal feedback. Castrated bulls (steers), 30 mo old, were allocated to four groups: group 1, control, no treatment (n = 8); group 2, five deslorelin implants (approximately 250 micrograms total deslorelin/day) for 42 days (n = 8); group 3, control+ LHRH (50 micrograms i.m.) at weekly intervals (n = 3); group 4, five deslorelin implants+LHRH as for group 3 (n = 3). Plasma LH was similar (p > 0.05) for steers in groups 1 and 2 on Day 0 and lower (p < 0.05) for steers in group 2 on Day 4, and continued to decrease to Day 41 (group 1, 1.71 +/- 0.20 ng/ml [mean +/- SEM]; group 2, 0.38 +/- 0.03 ng/ml [p < 0.001]). Mean plasma concentrations of FSH were similar (p > 0.05) for steers in groups 1 and 2 on Day 0 and lower (p < 0.05) for steers in group 2 on Day 7, and declined to Day 41 (group 1, 43.5 +/- 3.9 ng/ml; group 2, 17.5 +/- 1.5 ng/ml [p < 0.001]). Steers in group 3 showed increases in plasma LH after injection of LHRH on all occasions, while steers in group 4 did not show increases in plasma LH from Day 14 onward. Mean relative pituitary contents (arbitrary units) of LH beta- and FSH beta-subunit mRNAs were reduced on Day 42 in steers treated with deslorelin (LH beta: groups 1 and 3, 1.56 +/- 0.27; groups 2 and 4, 0.08 +/- 0.01 [p < 0.001]; FSH beta: groups 1 and 3, 1.01 +/- 0.08; groups 2 and 4, 0.34 +/- 0.07 [p < 0.001]). However, alpha-subunit mRNA was similar for control steers and steers treated with deslorelin (groups 1 and 3, 1.00 +/- 0.11; groups 2 and 4, 0.86 +/- 0.12 [p > 0.1]). Pituitary content of LH, but not FSH, was reduced in steers treated with deslorelin. In summary, steers treated with deslorelin showed desensitization to natural LHRH, and this was associated with reduced pituitary contents of LH and FSH beta-subunit mRNAs, a reduction in pituitary content of LH, and decreases in plasma concentrations of LH and FSH. This demonstrated, for the first time, a direct action of LHRH agonist on LH and FSH beta-subunit gene expression in cattle, independent of gonadal feedback. Also, there was a differential effect of treatment with deslorelin on gonadotropin alpha- and beta-subunit mRNA contents in the anterior pituitary.

We have tested the hypothesis that treatment of castrated rams (wethers) with human recombinant follistatin-288 (FS-288) suppresses plasma concentrations of FSH but has no effect on plasma concentrations of LH. Wethers were given an i.v. injection of vehicle or 50 micrograms FS-288 followed by a 12-h i.v. infusion of vehicle or 800 micrograms FS-288, respectively. This dose and treatment regimen was identical to that used in a previous study in which wethers were treated with vehicle or human recombinant inhibin A (hr-inhibin). Human recombinant follistatin significantly (p < 0.05) suppressed plasma concentrations of FSH, but vehicle had no effect. The maximal suppression of FSH occurred 12-15 h after the start of treatment, at which time the plasma concentrations were reduced by 20.6%. The reduction in plasma concentrations of FSH caused by FS-288 was about 2.6-fold less than that observed after the same treatment with hr-inhibin. FS-288 had no effect on plasma concentrations of LH; this result was similar to the findings we obtained with hr-inhibin. These data suggest that follistatin is a less potent negative regulator of FSH secretion than inhibin and that follistatin is not a significant regulator of the secretion of LH in rams.

Neuropeptide Y1-36 (NPY1-36) acts through Y1 and Y2 receptors while the C-terminal NPY fragments NPY18-36 and N-acetyl[Leu28,31]pNPY24-36 act only through the Y2 receptor. We have investigated the effects of intracerebroventricular (i.c.v.) administration of NPY1-36, NPY18-36 and N-acetyl[Leu28,31]pNPY24-36 on LH secretion in the ovariectomised (OVX) ewe. These peptides were administered into a lateral ventricle (LV) or the third ventricle (3V) of OVX ewes during the non-breeding and breeding seasons. Microinjections of NPY were also made into the preoptic area (POA) during both seasons to investigate the effects of NPY at the level of the GnRH cell bodies. Tamed sheep were fitted with 19 gauge guide tubes into the LV, 3V or the septo-preoptic area (POA). Jugular venous blood samples were taken every 10 min for 3 h. Sheep were then given NPY1-36 (10 micrograms), NPY18-36 (100 micrograms) or saline vehicle into the LV; N-acetyl[Leu28,31]pNPY24-36 (100 micrograms), NPY1-36 (10 micrograms or 100 micrograms), NPY18-36 (10 micrograms or 100 micrograms) or saline vehicle into the 3V, or NPY1-36 (1 microgram, 5 micrograms, 10 micrograms) into the POA. Blood sampling continued for a further 3 h. LH was measured in plasma by radioimmunoassay. LV or 3V injection of 10 micrograms NPY1-36 caused a small but significant (P < 0.025) increase in the interval from the last pre-injection pulse of LH to the first post-injection LH pulse during the breeding season. Other LH pulse parameters were not significantly affected. NPY18-36 did not produce any significant change in LH pulsatility when injected into the LV, and neither peptide had any effect on plasma prolactin or GH levels. There was a significant (P < 0.01) reduction in LH pulse frequency after 3V injection of 10 micrograms and 100 micrograms NPY and 100 micrograms NPY18-36. Pulse amplitude was reduced by 3V administration of the Y2 agonist, N-acetyl[Leu28-31]pNPY24-36 and 100 micrograms NPY18-36. When the amplitude of the first post-injection LH pulse was analysed, 10 micrograms NPY also had a significant (P < 0.05) suppressive effect. During the non-breeding season, 100 micrograms NPY1-36 (but not 10 micrograms) decreased (P < 0.01) LH pulse frequency. LH pulse amplitude was significantly (P < 0.01) decreased by 100 micrograms NPY18-36. Doses of 10 micrograms NPY1-36 and 100 micrograms NPY18-36 had greater inhibitory effects on pulse frequency during the breeding season but the suppressive effect of 100 micrograms NPY was similar between seasons. Microinjections of NPY into the POA decreased (P < 0.01) average plasma LH levels during the non-breeding season at a dose of 10 micrograms but did not significantly affect pulse frequency or amplitude. We conclude that a substantial component of the inhibitory action of NPY on LH secretion in the absence of steroids is mediated by the Y2 receptor. This inhibition is probably exerted by way of a presynaptic action on GnRH terminals in the median eminence as NPY does not modulate the frequency or amplitude of LH pulses at the level of the GnRH cell bodies in the POA.

Experiments were performed to test the hypothesis that there is a negative feedback 'clamp' of ovarian hormones on the hypothalamus and pituitary gland during the follicular phase of the oestrous cycle that limits the secretion of GnRH and LH. GnRH secretion was monitored by sampling the hypophysial portal blood of ewes during the luteal phase of the oestrous cycle and either 24 h or 48 h after the induction of luteolysis by the injection of cloprostenol, a prostaglandin analogue. There was an increase in GnRH pulse frequency in the transition from the luteal to the follicular phase of the cycle. A reduction in the amplitude of GnRH pulses did not occur until 48 h after cloprostenol, suggestive of negative feedback at the level of the hypothalamus that is more profound in the latter part of the follicular phase. The responsivity of the pituitary gland to GnRH was monitored in ewes during the luteal phase of the oestrous cycle and 24 h or 48 h after cloprostenol. Injections of 250 ng or 1000 ng GnRH were given (i.v.) to ewes that had been anaesthetised to suppress endogenous secretion of GnRH and LH. Using the lower dose, the responses 48 h after cloprostenol were not significantly different from those in the luteal phase. With the higher dose of GnRH, a significant (P < 0.05) increase in mean responsivity was seen 48 h after cloprostenol. There was, however, a marked variation in response, with some ewes showing profound increases in LH secretion in response to GnRH and others showing responses that were similar to those obtained during the luteal phase of the cycle. These data are interpreted to mean that the secretion of LH is 'clamped' during the follicular phase of the oestrous cycle and the 'clamp' is only released near the time of the preovulatory LH surge. To test whether or not a rise in GnRH input to the pituitary gland could over-ride the 'clamp' on the pituitary secretion of LH in the late follicular phase of the cycle, sheep were treated 40 h after cloprostenol with either a bolus injection of 500 ng GnRH or four pulses of 125 ng GnRH given at 10-min intervals. These treatments caused small elevations in LH secretion but did not always cause preovulatory LH surges. In some cases, a small rise in LH secretion was induced by GnRH treatments and levels of LH in plasma returned to baseline with the preovulatory LH surge occurring a few hours later.(ABSTRACT TRUNCATED AT 400 WORDS)

We have investigated the effects of gamma-amino butyric acid (GABA) agonists and antagonists to GABAA and GABAB receptors microinjected (1 microliter) into the medial preoptic area (MPOA) on LH secretion in ovariectomized (OVX) ewes with or without estrogen (E) treatment, during the nonbreeding season. Guide tubes (19 gauge) were placed into the MPOA of OVX ewes and injections of 1 microgram or 10 micrograms of the GABAA agonist muscimol or the GABAA antagonist bicuculline, and 10 micrograms of the GABAB agonist baclofen or the GABAB antagonist phaclofen were made into conscious animals. Jugular venous blood was collected at 10-min intervals for 3 h, the injection of GABA drug or vehicle was given and samples collected for a further 3 h. The plasma samples were assayed for LH. On completion of the experiments the brains were sectioned to locate the sites of injection. Thirty-four ewes were used, of which 30 had correct guide tube placement. In OVX sheep, both muscimol and bicuculline injection caused suppression of plasma LH concentrations, with a cessation of pulsatile release in many instances. Injection with baclofen or phaclofen into these sheep had no effect on LH secretion. When OVX sheep were treated with 0.5-cm Silastic implants (sc) of E for at least 7 days, there was a variable response to muscimol and bicuculline injection, depending on the degree of suppression of LH secretion by E. When E had little effect on plasma LH levels, muscimol and bicuculline both suppressed LH secretion in a similar manner to that observed in OVX ewes. When E treatment fully suppressed plasma LH levels, muscimol and bicuculline both stimulated LH secretion. This stimulation often took the form of a sustained, nonpulsatile secretion of LH. Baclofen injection into OVX ewes treated with E increased mean plasma LH levels through an increase in pulse amplitude, although there was only an effect on the amplitude of the first pulse after injection. Conversely, phaclofen injection in OVX ewes treated with E resulted in a reduction in LH pulse amplitude. These results provide evidence for a seasonal shift in the regulation of GnRH secretion by GABAergic neurons in the MPOA of the ewe. In contrast to the breeding season, where there was no effect of GABAB ligands, these appear to function in non-breeding season, this may be part of the mechanism for the seasonal shift in the negative-feedback effect of E on LH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

There have been a number of studies of GnRH secretion into the hypophysial portal blood at the time of the estrogen-induced LH surge, but the exact pattern of secretion at the onset of the positive feedback event remains a point of some dispute. In the present study, GnRH concentrations in portal plasma and jugular venous LH concentrations were measured in samples taken at 2.5-min intervals from five ovariectomized control ewes and seven ovariectomized ewes that were treated with 50 micrograms estradiol benzoate (im) to cause a LH surge. The frequency of GnRH pulses was calculated using a modification of previous criteria because of the frequent sampling. It was found that GnRH pulse frequency increased during the LH surge, being greatest during the "leading edge" of the surge. Perhaps more importantly, the data show that variable patterns of GnRH secretion occur at the start of the LH surge and during the surge, suggesting that no single pattern can be identified as an initiation signal of the positive feedback event. These data are consistent with other results that have been obtained in this laboratory in cyclic animals.

This study tested the hypothesis that inhibin is a major negative feedback regulator of FSH secretion but has minimal effects on LH secretion in rams. In experiment 1, castrated rams (wethers) were given either vehicle or human recombinant inhibin A (hr-inhibin) as three s.c. or three i.v. 50-micrograms injections 6 h apart or as one 50-micrograms i.v. injection followed by 100-micrograms infusion over 12 h. Human recombinant inhibin suppressed plasma FSH while the vehicle had no effect. The greatest suppression in plasma FSH was achieved following i.v. administration of hr-inhibin given either by repeated injection or by infusion. In experiment 2, wethers were given vehicle or a 50-micrograms i.v. injection followed by 800-micrograms infusion of hr-inhibin over 12 h. Infusion of hr-inhibin suppressed plasma FSH with a maximal suppression of 53.3% occurring between 15 and 24 h after the start of treatment. During this period, the plasma concentrations of FSH and inhibin were in the range of values for intact rams. Human recombinant inhibin did not influence plasma LH in either experiment. This study demonstrated that physiological treatment with inhibin, in the absence of testosterone, has the capacity to suppress plasma concentrations of FSH in wethers to the levels found in intact rams.

We have investigated the effects of agonists and antagonists to gamma-aminobutyric acid-A (GABA-A) and GABA-B receptors microinjected (1 microliters) into the preoptic area (POA) on LH in ovariectomized (OVX) ewes with or without estrogen (E) treatment during the breeding season. Guide tubes were placed into the preoptic area of OVX ewes using lateral ventriculograms for localization of the target area. Doses of 10 micrograms of the GABA agonists muscimol (GABA-A) and baclofen (GABA-B) or the GABA antagonists bicuculline (GABA-A) and phaclofen (GABA-B) were injected into the POA of tame conscious animals. Jugular venous blood was collected at 10-min intervals for 3 h, the injection of GABA drug or vehicle was given, and samples were collected for a further 3 h. The plasma samples were assayed for LH. On completion of the experiments, the brains were sectioned to locate the site of injection. In the first year, 17 ewes were used, of which 16 had correct guide tube placement. In OVX sheep, both muscimol and bicuculline injections caused suppression of plasma LH concentrations, with a cessation of pulses in many instances. When OVX sheep were treated with 1.0-cm Silastic implants (sc) of E for at least 7 days, there was a decrease in LH interpulse interval. In these sheep, muscimol and bicuculline injections had effects on plasma LH concentrations similar to those in OVX sheep. In addition, bicuculline was injected into the POA of OVX ewes treated with 2.0-cm E implants. Despite E treatment resulting in reduced plasma LH levels, due to an increase in the interpulse interval, bicuculline injection further suppressed plasma LH levels. Neither baclofen nor phaclofen injection had any effect on plasma LH secretion in either OVX ewes or OVX ewes given 1.0-cm E implants. In the second year, eight sheep were used, all of which had correct guide tube placement. These sheep were treated with 2.0-cm E implants and injected with phaclofen. Phaclofen had no effect on LH secretion. These results suggest that GnRH secretion is regulated by GA-BAergic neurons at the level of the GnRH cell bodies in the POA and that during the breeding season, this is effected by GABA-A and not GABA-B receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Since it has become possible to sample hypophysial portal blood from sheep without totally compromising pituitary function, several important features of the secretion of hypothalamic hormones have been elucidated. The secretion of gonadotropin-releasing hormone (GnRH) has been detailed most thoroughly with the important observation that each pulsatile discharge of luteinizing hormone (LH) is the direct result of a large secretory episode of GnRH from the hypothalamus. There is high fidelity in the GnRH relationship in terms of frequency and amplitude. During the LH surge, additional factors such as an alteration in the degree of enzymic degradation of GnRH may be important physiological mechanisms. The secretion of factors that control the release of growth hormone (GH), prolactin and adrenocorticotropic hormone (ACTH) have also been studied. Hypothalamic factors controlling GH and ACTH release do not bear such an explicit relationship to the secretory episodes of pituitary hormone as seen with the GnRH/LH axis. The factor involved in the acute stress-induced release of prolactin has not yet been identified in sheep.

To determine whether Leydig cells produce inhibin in the ram, Leydig cells were stimulated by administering human chorionic gonadotrophin (hCG) or raising the levels of endogenous LH by an injection of gonadotrophin releasing hormone (GnRH). Plasma concentrations of testosterone increased in the 72 h after either a single injection (P less than 0.05) or two injections (P less than 0.01) of hCG. Plasma concentrations of inhibin were not significantly influenced by either one or two injections of hCG. Administration of GnRH (1 microgram) caused an 11-fold increase in plasma concentrations of LH but did not influence concentrations of inhibin in either the jugular or testicular veins (pampiniform plexus). In contrast, concentrations of testosterone were increased by about fourfold in both jugular (P less than 0.01) and testicular (P less than 0.05) veins. The concentrations of inhibin in the testicular vein were 1.3-fold higher than in the peripheral plasma (P less than 0.05) both before and following treatment with GnRH whereas the concentrations of testosterone were 18- to 21-fold greater than in peripheral concentrations. In view of the difference in concentrations of inhibin between testicular and jugular veins, in a further experiment a sample was taken from the jugular vein, a vein located in the tunica vasculosa of the testis (testicular vein) and from a vein (spermatic vein) and lymph vessels located in the spermatic cord. The mean (+/- S.E.M.) concentrations of inhibin were highest in the testicular lymph (45.93 +/- 4.21 micrograms/l; P less than 0.001) compared with the peripheral (4.14 +/- 0.31 micrograms/l), spermatic (8.0 +/- 1.17 micrograms/l) or testicular (6.4 +/- 0.82 micrograms/l) plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

The patterns of gonadotrophin-releasing hormone (GnRH) input to the pituitary gland that affect the expression of a positive-feedback event by oestrogen on LH secretion were investigated in ovariectomized ewes with hypothalamo-pituitary disconnection (HPD). In experiment 1, ovariectomized HPD ewes were given hourly i.v. pulses of 250 ng GnRH and an i.m. injection of 50 micrograms oestradiol benzoate (OB). The ewes were given a bolus pulse of 2.25 micrograms GnRH 16 h after injection of OB, followed by half-hourly pulses of 250 ng GnRH for 14 h (treatment A). The LH surge response was significantly (P less than 0.05) greater in these ewes compared with that in ewes given a continuous infusion of GnRH (250 ng/h) after the OB injection, followed by a continuous infusion of 500 ng GnRH/h after the bolus pulse of GnRH (treatment B). When no GnRH was administered after the OB injection, except for the bolus pulse of GnRH (treatment C), the surge response was significantly (P less than 0.05) reduced compared with that in treatment A, and was reduced compared with treatment B. These data suggest that GnRH pulses are important in the generation of the OB-induced LH surge, but that a baseline secretory component can prime the pituitary to some extent. In experiment 2, a doubling of the continuous infusion dose of GnRH used in treatment B to 500 ng/h before the bolus pulse of GnRH and to 1 micrograms/h afterwards (treatment D) gave a similar response compared with treatment A, suggesting that if the baseline input of GnRH is of sufficient magnitude, it can overcome the lack of pulsatile input. In experiment 3, halving the GnRH pulse amplitude used in treatment A from 250 to 125 ng (treatment E) did not reduce the LH surge response, implying that when the GnRH input is in a pulsatile mode, the amplitude of GnRH pulses is less important than the pulsatile nature per se. In experiment 4, removal of GnRH input after the bolus pulse of GnRH (treatment F) significantly (P less than 0.05) reduced the surge response compared with when pulses were maintained (treatment A), indicating that GnRH input is still required once the LH surge has been initiated. Collectively, these experiments show that several forms of GnRH delivery, both pulsatile and baseline, can result in the full expression of a positive-feedback response in ovariectomized ewes treated with oestrogen.

Plasma concentrations of in-vitro biological and immunological LH were measured throughout the LH surge in cyclic ewes and in ovariectomized ewes treated i.m. with oestradiol benzoate. Both activities increased in parallel during the LH surge in both groups, although the ratio of biological to immunological activities (B/I ratio) was highest at the peak of the LH surge. The two activities were highly correlated (r = 0.86-0.92), with similar slopes from their regression analysis for the cyclic and ovariectomized groups (1.15 and 1.16 respectively). However, the intercepts of the regression lines did not pass through the origin, but intersected the y (radioimmunoassay) axis, suggesting that these serum samples contained immunoactivity not associated with LH bioactivity. In conclusion, an increase in the LH B/I ratio was observed during the LH surge in oestrogen-treated ovariectomized ewes and in cyclic ewes. This increase was not attributable to a change in the relationship between these two LH activities during the LH surge, but rather to the detection of bioinactive immunoactive material in plasma of unknown composition.

Ovariectomized ewes were treated with 100 IU insulin, iv, which caused reductions in blood sugar and plasma LH concentrations. The effect was prevented by the infusion (iv) of glucose, suggesting that neuroglycopenia and not a direct action of insulin was the cause of reduced LH secretion. An iv infusion of naloxone (40 mg/h for 2 h), which commenced 25 min before the insulin injection, blocked the inhibitory effect of insulin on LH secretion, but it did not prevent the decrease in plasma glucose concentrations. In this treatment group and in a group treated only with naloxone, the opioid antagonist significantly stimulated LH secretion. To determine whether CRF might be involved in the insulin-induced decrease in LH secretion, 50 micrograms CRF were injected into ovariectomized sheep. Despite producing very high circulating concentrations of CRF within 2 min of injection and the stimulation of cortisol secretion during most of the 4-h posttreatment period, plasma LH levels were not affected. In addition, the intracerebroventricular administration of 10 micrograms CRF or 10 micrograms CRF plus 10 micrograms arginine vasopressin (AVP) did not affect LH secretion. These observations suggest that insulin-induced hypoglycemia decreased LH secretion by neuroglycopenia. This may involve an opioidergic mechanism, but does not involve activation of the hypothalamo-pituitary-adrenocortical axis.

A heterologous RIA for ovine inhibin was developed which was sufficiently sensitive and specific to describe the peripheral concentrations of immunoreactive inhibin (iINH) during the estrous cycle of the ewe and to examine the effects of cautery of ovarian follicles on concentrations of iINH in ovarian and jugular venous plasma. Parallel logit-log dose-response lines were observed among ovine follicular fluid, ewe plasma, and pure native ovine (31 kDa) and bovine (31 kDa) inhibin. iINH could not be detected in ovariectomized ewe plasma, and there was no apparent cross-reactivity with a variety of structurally related and unrelated hormones and peptides, except a monomeric form of the alpha-subunit of INH, iINH in follicular fluid was 10(4)-fold higher than that in ovarian venous plasma, which was 3-fold higher than that in peripheral plasma. Cautery of the follicles resulted in a 35% reduction in iINH and an 81% reduction in estrogen concentrations in the ovarian vein within 10 min. During the estrous cycle, iINH and FSH were inversely related in samples taken over 30 h in the luteal phase (r = -0.69; P less than 0.001) and in the pre- and postovulatory phases (r = -0.45; P less than 0.001). iINH and LH were not related in the luteal phase, but were weakly positively correlated in the follicular phase (r = 0.31; P less than 0.01). iINH and estrogen concentrations in the follicular phase were also weakly correlated (r = 0.30; P less than 0.001). Furthermore, iINH concentrations rose in the follicular phase and decreased within 3-6 h of the preovulatory surges of LH and FSH, reaching a nadir around the time of the second rise in FSH 24-48 h later. It is concluded that 1) large antral follicles are a major source of peripheral iINH during the ovine estrous cycle; 2) iINH levels increase in the follicular phase with the growth of the dominant follicle and may be inhibited by the preovulatory surge of gonadotropin; 3) the fall in inhibin after the LH surge may be responsible for the second rise in FSH; and 4) the inverse relationship between FSH and iINH is consistent with the hypothesis that inhibin is involved in the feedback regulation of FSH.

The levels of plasma LH and FSH were measured in serial blood samples taken at 15-min intervals for 6 h from ewes that had remained fertile after grazing oestrogenic pasture (clover-fertile ewes), from ewes that were permanently affected by clover disease (clover-infertile ewes) and from normal ewes. Two flocks of ewes from different locations were studied. In flock 1, tonic LH secretion (total area under the curve of LH concentration versus time, 1 area unit = 1 ng ml-1 x 1 h) was significantly (P < 0.05) greater in clover-infertile ewes (10.4 area units) during anoestrus than in ewes that had remained fertile after prolonged grazing of oestrogenic clover (5.4 area units). Tonic LH and FSH secretions during the bleeding season and FSH secretion during anoestrus were not significantly different. In flock 2, LH levels during the breeding season were significantly (P < 0.05) elevated in clover-infertile ewes (10.9 area units) compared to normal ewes (5.4 area units) that had never grazed oestrogenic clover. LH secretion in clover-infertile ewes (7.8 area units) was intermediate to that found in infertile and control ewes. Concentrations of FSH, progesterone and ovarian vein oestradiol-17 beta (E2) during the breeding season were similar in the three groups. In another experiment, the positive feedback release of LH following administration of E2 (12.5, 25 or 50 micrograms per ewe) was measured in anoestrous ewes of flock 2. Significantly (P < 0.01) more clover-infertile ewes demonstrated a positive feedback effect than control ewes when given 12.5 micrograms E2 but not when given higher doses. The elevation of LH secretion in permanently affected clover-infertile ewes is inconsistent with the hypothesis that the hypothalamo-pituitary axis of these ewes is less responsive to the negative feedback effect of oestrogen. Furthermore, the patency of the positive feedback loop is consistent with the ability to ovulate.

Experiments were conducted in ovariectomized ewes after hypothalamo-pituitary disconnection (HPD) to examine LH and FSH secretion during constant infusion of gonadotrophin-releasing hormone (GnRH) or physiological saline and to determine whether or not a constant GnRH background enhances or diminishes pituitary responsiveness to GnRH pulses. Whereas pulsatile GnRH infusions maintained LH and FSH secretion, constant infusions (125 or 250 ng/h) led to the complete cessation of LH secretion and reduced FSH secretion. The rate of decline of plasma FSH concentrations was significantly (P less than 0.01) greater in animals receiving 250 ng GnRH/h than in saline-treated animals, whereas that in animals receiving 125 ng/h was not significantly different. When GnRH pulses were administered during constant GnRH infusion, the plasma LH pulse amplitudes were similar to those seen without the GnRH background. These data show that, in ovariectomized-HPD ewes FSH secretion does not require GnRH pulses and may merely reflect ongoing FSH synthesis and a constant low background of GnRH does not affect pituitary responsiveness to GnRH pulses.

The relationship between pituitary responsiveness to exogenous LH-RH, return of oestradiol-17 beta-induced positive-feedback effect on LH release and the resumption of oestrous cycles were studied in Merino ewes lambing in the breeding season. The mean (+/- s.e.m.) increase in plasma LH 2 h after 50 micrograms LH-RH i.v. was reduced (P < 0.001) in ewes tested in the last 4 weeks of pregnancy (8.6 +/- 2.1 ng/ml, N = 29) but was similar (P > 0.05) for non-parturient dioestrous ewes (N = 31) and for anoestrous ewes tested around 14 (N = 50) and 28 (N = 51) days post partum (30 +/- 3.9, 37.8 +/- 4.0, 39. 0 +/- 3.2 ng/ml, respectively). Oestradiol-positive feedback was absent in 68.6% (35/51) and 54% (27/50) of ewes tested around 14 and 28 days post partum, respectively. Neither LH-RH responsiveness nor the presence of oestradiol-postive feedback was related to the incidence of 61.9%, 166/268) or interval to (62 +/- 2.2 days, N = 99) first oestrus post partum. We suggest that these endocrine responses do not represent factor limiting the resumption of ovarian cyclicity post partum.

Plasma luteinizing hormone (LH) concentrations were measured in normal (control) Corriedale X Merino (comeback) ewes and in clover-infertile comeback ewes which had grazed oestrogenic Yarloop clover (Trifolium subterraneum L. cv. Yarloop) for more than 4 years. Plasma LH concentrations were measured in samples taken at 20-min intervals for 6 h during the dioestrous stage of the oestrous cycle in the breeding season (BS) and during the anoestrous season (AS). In the control ewes during BS, transitory elevation in plasma LH concentration (pulses) occurred, reflecting secretory episodes, with a frequency of one per 5.2 h. This frequency fell to one per 16.5 h during the anoestrous season. In clover-infertile ewes, LH pulses occurred with a frequency of one per 4.5 h during BS and one per 4.9 h during AS (difference not significant). In the controls, plasma LH levels were higher (P less than 0.05) during BS (mean +/- s.d. = 1.2 +/- 0.4 ng/ml, n = 9) than in AS (0.7 +/- 0.3 ng/ml, n = 5). In the clover-infertile ewes, plasma LH levels in BS (1.3 +/- 0.6 ng/ml, n = 12) were similar to those of controls. During AS, plasma LH levels in the clover-infertile ewes (1.0 +/- 0.6 ng/ml, n = 10) remained similar to their BS levels, being significantly (P less than 0.05) higher than LH levels in the controls at this time. These studies indicate that the higher plasma concentrations of LH which have been reported in clover-infertile ewes arise from more frequent LH pulses. Furthermore, in contrast to normal ewes, average plasma LH, reflecting pulse frequency, is not reduced in AS. This supports the view that ingestion of phytooestrogens affects neural centres involved in regulating LH secretion.