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.     

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

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

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.     

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.     

Social suppression in nonreproductive female Damaraland mole-rats, Cryptomys damarensis: no apparent role for endogenous opioid peptides

The role of endogenous opioid peptides (EOPs) on LH secretion was examined to investigate the neuronal mechanisms responsible for the inhibition of GnRH and the resultant infertility in nonreproductive female Damaraland mole-rats, Cryptomys damarensis. The endorphin antagonist naloxone was administered to five groups of females to determine its effect on plasma LH levels: Grouping was determined by social status, social environment, and whether the females were ovariectomized. A single injection of naloxone had no significant effect on LH secretion in either intact or hystero-ovariectomized females. Multiple injections with naloxone failed to affect basal LH concentrations but did result in a decrease in GnRH-stimulated LH secretion in ovariectomized nonreproductive and reproductive females. A significant response to a single naloxone injection following GnRH priming was obtained in both nonreproductive females and in nonreproductive females housed in the absence of the reproductive pair. These results suggest EOPs play a role in sexual function but that socially induced infertility is unlikely to be mediated through the EOP system.     

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.     

Opioidergic control of luteinizing hormone secretion in the female rabbit: influence of age on the response to naloxone

To examine the role of opioid neurons on luteinizing hormone (LH) secretion in the female rabbit, we determined LH release at timed intervals after naloxone administration to rabbits aged 25-150 days. The LH response to naloxone (10 mg/kg) was not significantly elevated until day 43 when LH rose 76-113% above basal levels at 40-80 min. In 56-day-old females the corresponding increase was 160% at 15 min and in 65- to 67-day-olds it was 154%. From 70 to 80 days of age the LH response was blunted and no significant elevations could be elicited. By contrast, naloxone-induced LH increases were again evident when rabbits were older than 100 days. At all ages no significant change in FSH concentrations was observed. In the adult females, naloxone at 2.5, 5, and 10 mg/kg caused increases in LH secretion which occasionally were high enough to induce ovulation as exemplified by elevated serum progesterone 4 days later. These data suggest that opioid peptides may be involved in the prepubertal rise in LH and in the normal inhibition of adult secretion in the female rabbit.     

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.     

Opioidergic, dopaminergic and adrenergic regulation of LH secretion in prepubertal heifers

Studies have shown inhibitory effects of endogenous opioids on LH secretion in early post-natal heifers. However, it is not clear whether these effects change during the rest of the prepubertal period or whether the inhibitory influences on the GnRH neurones are direct or by way of other neuronal systems. Two experiments were performed in heifer calves to study the developmental patterns of opioidergic, dopaminergic and adrenergic regulation of LH and the possible interactions between opioids and dopaminergic and adrenergic neuronal systems, in the regulation of LH secretion. In Expt 1 four groups each of five heifer calves were used. Blood samples were taken every 15 min for 10 h and each calf received one of the following treatments as a single injection at 4, 14, 24, 36 and 48 weeks of age: (i) naloxone (opioid antagonist, 1 mg kg(-1), i. v.); (ii) sulpiride (dopamine D2 antagonist, 0.59 mg kg(-1), s.c.); (iii) naloxone and sulpiride combined; or (iv) vehicle (control group). Treatments began after the first blood sample was taken. The design of Expt 2 was similar; a separate group of heifer calves was assigned to receive one of the following treatments as a single injection at 4, 14, 24, 36 and 48 weeks of age: (i) naloxone; (ii) phenoxybenzamine (an alpha-adrenoreceptor blocker, 0.8 mg kg(-1), i. v.); (iii) naloxone and phenoxybenzamine; (iv) or vehicle. Results from Expt 1 showed that the maximum concentration of LH and the number of calves responding to treatments with an LH pulse was higher in the first hour after treatments at 36 and 48 weeks of age in the naloxone group compared with the control or sulpiride groups (P < 0.05). These values in the naloxone group also increased over time and were greatest at 48 weeks of age (P < 0.05). In heifers given naloxone + sulpiride treatment at 36 and 48 weeks of age, maximum concentrations of LH in the first hour after treatment did not differ from the naloxone and control groups. In Expt 2, at 36 and 48 weeks of age, treatment with naloxone with or without phenoxybenzamine resulted in higher concentrations of LH than in the controls (P < 0.05). No pulses were seen over the first hour of treatment at 36 and 48 weeks of age in heifers treated with phenoxybenzamine. The 10 h periods of blood sampling at 48 weeks of age revealed that phenoxybenzamine alone suppressed LH pulse frequency and mean serum concentrations of LH compared with the control group (P < 0.05). It was concluded that a strong or more acute inhibition of LH secretion by endogenous opioids developed in mid- to late prepubertal heifers, or alternatively, that removal of opioidergic inhibition at the GnRH neurone unmasked stimulatory inputs that were greater in heifers close to first ovulation. Since sulpiride appeared to negate in part the effects of naloxone on LH release, the suppressive effects of opioids could be exerted in part through the inhibition or blocking of a stimulatory dopaminergic system. alpha-Adrenergic neuronal systems have stimulatory effects on LH release, especially during the late prepubertal period, but do not appear to mediate opioidergic inhibition of LH secretion in prepubertal heifer calves.     

Opioid modulation of LH secretion in the ewe

Administration of opioid agonists and antagonists and measurement of resulting hormone changes were used to study the possible effects of opioids on reproductive function in the ewe. Intravenous administration of the long-acting methionine-enkephalin analogue FK33-824 (250 micrograms/h for 12 h) to 3 ewes during the follicular phase of the oestrous cycle depressed episodic LH secretion. This effect was reversed by administration of the opiate antagonist naloxone (25 mg/h) in combination with the FK33-824 treatment; in fact LH secretion was enhanced by the combined regimen. Naloxone (25 mg/h for 12 h) administered alone to 3 ewes in the follicular phase also enhanced LH secretion. In 3 animals treated with FK33-824 during the follicular phase, progesterone remained basal for 14 days after treatment, suggesting that ovulation was blocked. Jugular venous infusion of naloxone (25, 50 or 100 mg/h for 8h) into 5 ewes during the early and mid-luteal phase of the cycle resulted overall in a significant increase in mean plasma LH concentrations and LH episode frequency. To investigate whether endogenous opioids suppress LH release in seasonally anoestrous sheep, naloxone was infused intravenously into mature (25, 50 or 100 mg/h for 8 h) and yearling ewes (12 . 5, 25 or 50 mg/h for 8 h) during early, mid- and late anoestrus and plasma LH concentrations were measured. In the mature ewes, there was a trend for naloxone to increase LH values during the early anoestrous period but naloxone was without effect during mid- and late anoestrus. In the yearlings, naloxone infusion consistently increased plasma LH concentrations as a result of a significant increase in LH episode frequency. These experiments indicate that endogenous opioid peptides probably modulate gonadotrophin secretion during both the follicular and luteal phases of the oestrous cycle. However, the follicular phase of the sheep cycle is of short duration, and there may be residual effects of luteal-phase progesterone during this period. Secondly, there may be an age-dependent effect of naloxone on LH secretion during seasonal anoestrus in the ewe, with opioids playing a part in the suppression of LH in young but not in mature animals.     

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.     

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.     

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.     

Modulation of luteinizing hormone and follicle-stimulating hormone in circulation by interactions between endogenous opioids and oestradiol during the peripubertal period of heifers.

The aim of this study was to determine whether the decline in oestradiol inhibition of circulating luteinizing hormone (LH) and follicle-stimulating hormone (FSH) during the peripubertal period of heifers is associated with a change in opioid modulation of LH and FSH secretion. Opioid inhibition of LH secretion was determined by response to administration of the opioid antagonist naloxone. Prepubertal heifers (403 days old) were left as intact controls, ovariectomized or ovariectomized and chronically administered oestradiol. Control heifers were used to determine time of puberty. Three weeks after ovariectomy, four doses of naloxone (0.13-0.75 mg kg-1 body weight) or saline were administered to heifers in the treatment groups in a latin square design (one dose per day). Blood samples were collected at intervals of 10 min for 2 h before and 2 h after administration of naloxone. This procedure was repeated four times at intervals of 3 weeks during the time intact control heifers were attaining puberty. All doses of naloxone induced a similar increase in concentration of serum LH within a bleeding period. During the initial bleeding period (before puberty in control heifers), administration of naloxone induced an increase in LH concentration, but the response was greater for heifers in the ovariectomized and oestradiol treated than in the ovariectomized group. At the end of the study when control heifers had attained puberty (high concentrations of progesterone indicated corpus luteum function), only heifers in the ovariectomized and oestradiol treated group responded to naloxone. Opioid inhibition of LH appeared to decline in heifers during the time control heifers were attaining puberty. Heifers in the ovariectomized group responded to naloxone at the time of administration with an increase in FSH, but FSH did not respond to naloxone at any other time. Administration of naloxone did not alter secretion of FSH in ovariectomized heifers. These results suggest that opioid neuropeptides and oestradiol are involved in regulating circulating concentrations of LH and possibly FSH during the peripubertal period. Opioid inhibition of gonadotrophin secretion appeared to decline during the peripubertal period but was still present in ovariectomized heifers treated with oestradiol after the time when age-matched control heifers had attained puberty. We conclude that opioid inhibition is important in regulating LH and FSH in circulation in heifers during the peripubertal period. However, opioids continue to be involved in regulation of circulating concentrations of LH after puberty.     

Prenatal androgens and the timing of seasonal reproductive transitions in sheep.

Both the onset of puberty in the lamb and the annual resumption of reproductive activity in adult male and female sheep are characterized by increased secretion of LH due to reduced responsiveness to steroid inhibition. However, the timing of puberty is sexually differentiated, for males undergo a reduction in sensitivity to steroid feedback at 10 wk of age, whereas females remain highly responsive to steroid inhibition until 30 wk. This sex difference is determined by androgens in utero. The present study was conducted to determine whether a sex difference exists in the timing of seasonal transitions in adult males and females. We compared serum LH in gonadectomized, estradiol-treated males (n = 7), females (n = 6), and androgenized females (n = 5) from blood samples collected twice weekly for one year. As determined by changes in the pattern of LH secretion, the onset and termination of the autumn breeding season were not different between males, females, and androgenized females (termination: 1 February +/- 4 days, mean +/- SE all groups; onset: males, 22 August +/- 4 days; females, 5 September +/- 18 days; androgenized females, 16 September +/- 10.5 days). However, there was a transient increase in LH (20 May to 23 June) in males, but not in females or androgenized females. Although no effects of prenatal testosterone were evident in the control of LH secretion in adult androgenized females, LH secretion in androgenized males was elevated throughout the nonbreeding season in 3 of 5 animals, indicating that exogenous testosterone may reduce seasonal increases in responsiveness to steroid inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opioid regulation of luteinising hormone and prolactin release in the horse—identical or independent endocrine pathways?

In order to determine if endogenous opioids regulate luteinising hormone (LH) and prolactin secretion via a common, gonadotropin-releasing hormone (GnRH) dependent pathway in the horse, effects of the opioid antagonist naloxone (300 mg) and the GnRH agonist buserelin (20 μg) on prolactin and LH secretion were investigated in stallions (n = 22), long-term castrated geldings (n = 15) and non-lactating mares during the luteal phase of the oestrous cycle (n = 16). Blood samples for determination of LH and prolactin concentrations were withdrawn at 15 min intervals for 120 min. After 60 min of sampling, animals were treated with either naloxone, buserelin or saline. In stallions, naloxone significantly increased LH as well as prolactin release (P < 0.05), indicating an opioid inhibition of both hormones, whereas in mares, naloxone stimulated only LH secretion (P < 0.05). No changes in plasma LH or prolactin concentrations after injection of naloxone were found in geldings. In all animal groups, buserelin induced a significant release of LH (P < 0.05) without affecting prolactin. We conclude that endogenous opioids inhibit LH and prolactin release in the horse but the regulation of these two hormones involves independent opioid pathways. These are activated differentially in stallions, geldings and mares. The opioid regulation of prolactin secretion is not mediated via GnRH.     

Opioidergic and nutritional involvement in the control of luteinizing hormone secretion of postpartum Rasa Aragonesa ewes lambing in the mid-breeding season

The role of endogenous opioids and nutrition on the inhibition of luteinizing hormone (LH) secretion during the postpartum period was investigated in a Spanish breed of sheep lambing in the mid-late breeding season. Two groups of adult Rasa Aragonesa ewes housed in individual pens and lambing on 30 December were fed during the suckling period to provide maintenance requirements and the production of 1.1 (M; n=8) or 0.55 (L; n=8) kg of milk per day. On days 10, 20 and 30 after lambing, the effect of a treatment with the opiate receptor antagonist naloxone (1 mg/kg at four hourly intervals) on LH secretion was assessed in half of the ewes of each group, the remaining females receiving four saline injections. After weaning, animals were fed to provide requirements for maintenance of liveweight. Blood samples were collected twice a week from day 20 postpartum until the end of March, and assayed for progesterone and prolactin. Although underfed ewes showed significantly lower mean plasma concentrations during the control period on day 20 postpartum, nutrition did not seem to modify LH secretion before naloxone or saline injections. Moreover, no differences between nutritional groups in the response to naloxone injections on pattern of LH secretion were found. In fact, naloxone treatment induced an increase of mean LH concentrations on days 10, 20 and 30 postpartum (at least, P<0.05), of LH pulse frequency on days 20 and 30 (P<0.05), and of LH pulse amplitude on days 10 and 20 (P<0.05). Underfed ewes during the postpartum period showed a slower decline in plasma prolactin levels, with significant differences on days 29, 36 and 39 after lambing (P<0.05). Only 3 M ewes ovulated before the onset of the seasonal anoestrus period. It is concluded that endogenous opioids are involved in the inhibition of LH secretion during the early suckling period of a reduced seasonality breed of sheep without any influence of nutrition on the response to naloxone treatment; however, ewes underfed before weaning failed to reactivate their cyclicity prior to the onset of the seasonal anoestrus.     

Ovarian steroid hormone involvement in endogenous opioid modulation of LH secretion in mature ewes during the breeding and non-breeding seasons

The opioid antagonist WIN-44441-3 (WIN-3, Sterling-Winthrop) caused significant increases in LH secretion in ovariectomized ewes treated with progesterone but not in ovariectomized animals treated with oestradiol-17 beta. In the non-breeding season, plasma LH concentrations in ovariectomized ewes without steroid therapy, given oestradiol-17 beta or oestradiol-17 beta and progesterone together were not affected by treatment with WIN-3 on Day 6 after ovariectomy (there was a significant increase in LH as a result of WIN-3 treatment 13 days after ovariectomy in sheep given no steroid therapy). However, WIN-3 treatment of ovariectomized sheep given progesterone resulted in a significant increase in plasma LH. WIN-3 was ineffective when given to intact ewes treated with progesterone during the non-breeding season. With ovariectomized sheep during the breeding season there was again no response to WIN-3 at 6 days after ovariectomy in sheep given oestradiol-17 beta, but significant LH elevations in animals given no steroid, those given progesterone and those given progesterone + oestradiol-17 beta. The lack of an LH response to WIN-3 in ovariectomized sheep treated with oestradiol-17 beta did not result from a reduced pituitary response to GnRH since such animals responded normally to exogenous GnRH treatment. Overall, these results are consistent with the idea that, irrespective of the time of year, progesterone exerts negative feedback upon LH release at least in part through an opioidergic mechanism, whereas oestradiol-17 beta exerts negative feedback through steps unlikely to involve opioids. Progesterone can override the effect of oestradiol-17 beta during the breeding season only. Further, there appears to be a steroid-independent opioid involvement in LH suppression, operating at both times of year.     

Effects of ovariectomy and estradiol replacement on naloxone induced LH secretion in the female rabbit

The effects of an opioid antagonist, naloxone, on the secretion of gonadotrophins were investigated in the long term ovariectomized rabbit. In the intact and acutely ovariectomized rabbit (2 days p.o.) naloxone at 10 mg/kg induced an increase of 260-300% in LH secretion at 40 min post-injection. From days 33-66 post-surgery naloxone at 10 mg/kg caused significant elevations in LH release even when animals were treated with estradiol benzoate 24 h previously. By contrast, treatment with oestradiol benzoate 3 h before naloxone abolished the LH increase. An LH surge could be elicited in these rabbits with GnRH treatment. These studies indicated that long term ovariectomy in the female rabbit does not completely remove the opioid control of GnRH release and that the LH response to naloxone is influenced by circulating estradiol levels.     

Opioidergic control of gonadotrophin secretion in the prepubertal gilt during restricted feeding and realimentation

Prepubertal gilts, having undergone a 7-day period of feed restriction to a maintenance ration, were allocated to one of 4 treatments; restricted feeding at 09:00 and 17:00 h for an 8th day both with (Group RN) and without (Group R) administration of the opioid antagonist naloxone hydrochloride (1 mg.kg-1 at 09:30 h followed by 0.5 mg.kg-1 at hourly intervals for 7 h), or feed to appetite with (Group ALN) and without (Group AL) naloxone administration. Gilts were bled at 10-min intervals on Day 8 from morning to evening feed and plasma LH, FSH and prolactin concentrations were measured by radioimmunoassay. Compared with Group R gilts, Group AL gilts exhibited significantly (P less than or equal to 0.05) higher mean and maximum LH concentrations and pulsatility, lower prolactin concentrations (P less than 0.05) but no significant difference in FSH secretion. Naloxone significantly depressed the increase in LH after re-feeding (Group ALN) (P less than 0.05). Once again there were no significant effects on FSH secretion. Naloxone also significantly depressed prolactin secretion in feed-restricted gilts (P less than 0.05). These results confirm that re-feeding of feed-restricted prepubertal gilts stimulates an immediate increase in LH secretion and that this elevation is not mediated via a suppression of inhibitory endogenous opioidergic tone. Rather, naloxone treatment appeared to expose a latent inhibition of LH secretion. The control of LH secretion is distinct from that of FSH in this model.