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Phytoestrogens, plant derived estrogen like-compounds exert numerous effects on the reproductive functions of animals. The present study was designed to demonstrate if exogenous genistein infused during the breeding season into the third ventricle of the brain of ovariectomized ewes could affect the secretory activity of the GnRH/LH axis. Two-year-old ovariectomized ewes (n=8) were infused with vehicle (control, n=3) or genistein (10 microg/100 microl/h, n=5) into the third ventricle. The infusions were done from 10.00 to 14.00 h and blood samples collection was performed this day up to 20.00 h and next day from 8.00 to 10.00 h. The animals were slaughtered, thereafter. Immunoreactive (IR) GnRH neurons in the hypothalamus and LH cells in the adenohypophysis were localized by immunohistochemistry. Messenger RNA analyses were performed by nonisotope in situ hybridization using sense and anti-sense riboprobes produced from beta subunits of LH cDNA clones. Plasma LH concentrations were measured by radioimmunoassay. Immunohistochemical analysis revealed that genistein infusion affected the morphology of GnRH neurons evoking a visualization of long axons in the GnRH perikarya and visibly diminished IR GnRH stores in the median eminence. The number of IR LH cells and IR material stored in the adenohypophyses increased in genistein-infused animals, which was confirmed by statistical analysis (P<0.001). The in situ hybridization analyses showed in these ewes the increase of mRNA LHbeta hybridization signal. The changes in LH release in response to genistein infusion had a biphasic character: it decreased within 6 h after infusion and increased 24 h later. Mean concentration of LH and amplitude of pulses measured from the beginning of infusion up to end of the experiment were significantly higher (P<0.05) in genistein-infused ewes compared to vehicle-treatment. In conclusion, our data show that genistein, a phytoestrogen, may effectively modulate GnRH and LH secretion in OVX ewes by acting directly on the CNS. The biphasic character of the LH response is similar to that of estradiol during the breeding season in the ewes.

In the present investigation we have examined the ability of melatonin to modify the pulsatile LH secretion induced by treatment with a DA antagonist (sulpiride, SULP) or opioid antagonist (naloxone, NAL) in intact mid-anestrous ewes. The experimental design comprised the following treatments-in experiment 1: (1) intracerebroventricular (i.c.v.) infusion of vehicle (control I); (2) pretreatment with SULP (0.6 mg/kg subcutaneously) and then i.c.v. infusion of vehicle (SULP + veh); (3) pretreatment with SULP and then i.c.v. infusion of melatonin (SULP + MLT, 100 microg per 100 microl/h, total 400 microg). In experiment 2: (4) i.c.v. infusion of vehicle (control II); (5) i.c.v. infusion of NAL (NAL-alone, 100 microg per 100 microl/h, total 300 microg); (6) i.c.v. infusion of NAL in combination with MLT (NAL + MLT, 100 microg + 100 microg per 100 microl/h). All infusions were performed during the afternoon hours. Pretreatment with SULP induced a significant (P < 0.01) increase in LH pulse frequency, but not in mean LH concentration, compared with control I. In SULP + MLT-treated animals, the LH concentration was significantly (P < 0.01) higher during MLT infusion, but due to highly increased LH secretion in only one ewe. The significant changes in the SULP + MLT group occurred in LH pulse frequency. A few LH pulses were noted after melatonin administration compared with the number during the infusion (P < 0.05) and after vehicle infusion in the SULP + MLT group (P < 0.05). The i.c.v. infusion of NAL evoked a significant increase in the mean LH concentration (P < 0.001) and amplitude of LH pulses (P < 0.01) compared with these before the infusion. The enhanced secretion of LH was also maintained after i.c.v. infusion of NAL (P < 0.01) with a concomitant decrease in LH pulse frequency (P < 0.05). In NAL + MLT-treated ewes, mean plasma LH concentrations increased significantly during and after the infusion compared with that noted before ( P < 0.001). No difference in the amplitude of LH pulses was found in the NAL + MLT group, but this parameter was significantly higher in ewes during infusion of both drugs than during infusion of the vehicle (P < 0.01). The LH pulse frequency differed significantly (p < 0.05), increasing slightly during NAL + MLT administration and decreasing after the infusion. In conclusion, these results demonstrate that: (1) in mid-anestrous ewes EOPs, besides DA, are involved in the inhibition of the GnRH/LH axis; (2) brief administration of melatonin in long-photoperiod-inhibited ewes suppresses LH pulse frequency after the elimination of the inhibitory DA input, but seems to not affect LH release following opiate receptor blockade.

This study tested a hypothesis that the enhancement of the prolactin (PRL) concentration within the central nervous system (CNS) disturbs pulsatile luteinizing hormone (LH) and growth hormone (GH) secretion in rams that are in the natural breeding season. A 3h long intracerebroventricular (icv.) infusion of ovine PRL (50 microg/100 microl/h) was made in six rams during the daily period characterized by low PRL secretion in this species (from 12:00 to 15:00 h); the other six animals received control infusions during the same time. Blood samples were collected from 9:00 to 18:00 h at 10 min intervals. A clear daily pattern of LH secretion was shown in control animals, with the lowest concentration at noon and an increasing basal level around the time of sunset (P < 0.001). No significant changes in LH concentration occurred in PRL-infused animals and the concentration noted after infusion of PRL was significantly (P < 0.05) lower than after the control infusion. The frequency of LH pulses tended to decrease in rams after PRL treatment. The changes in LH secretion clearly carried over to the secretion of testosterone in the rams of both groups. The GH concentrations changed throughout the experiment in both groups of rams, being higher after the infusions (P < 0.001). However, the mean GH concentration and GH pulse amplitude noted after PRL infusion were significantly lower (P < 0.001 and P < 0.05, respectively) from those recorded in the control. The continued fall in PRL secretion observed in rams following PRL infusion (P < 0.05 to P < 0.001) indicates a high degree of effectiveness of exogenous PRL at the level of the CNS. In conclusion, maintenance of an elevated PRL concentration within the CNS leads to disturbances in the neuroendocrine mechanisms responsible for pulsatile LH and GH secretion in sexually active rams.

This study tested a hypothesis that an acute enhancement of prolactin concentration within the central nervous system (CNS) would affect the LH secretion in ewes, depending on the level of endogenous estrogens in the organism. A 3-h long intracerebroventricular (icv.) infusion of ovine prolactin was made in late follicular-phase ewes, experiment 1, and in ovariectomized (OVX) ewes (experiment 2). No significant differences were found in mean LH concentrations and LH peak number before, during and after prolactin administration (50 microg/100 microl/h) in intact cyclic ewes. No diurnal rhythm in LH was detected in prolactin-infused ewes. From the two doses of prolactin used in OVX ewes (25 and 50 microg/100 microl/h) only the lower dose suppressed significantly the mean plasma LH concentration after the infusion, compared to those noted before (P < 0.01) and during (P < 0.001) prolactin treatment. Prolactin had no effect on LH pulse frequency in OVX ewes, however, a tendency to decrease in LH peak number was observed after administration of a lower dose. Plasma prolactin levels decreased significantly (P < 0.01 and P < 0.001) after the icv. infusion in all groups, indicating a high degree of effectiveness for exogenous prolactin at the level of the CNS., (Copyright 2002 Elsevier Science B.V.)

The nocturnal secretion of pineal melatonin provides information to vertebrates on changes in day length under the circumstances in which they live. In sheep, which are seasonal breeders, the secretion of melatonin is also a signal to the neural structures controlling the secretion of gonadotropins from the pituitary gland to drive their activity in accordance with the season of the year. The sites and mechanisms of melatonin action on GnRH/LH secretion has been the subject of intensive studies in the last decade. This article briefly reviews the most important discoveries and methods used in this research, which has led to a better understanding of the role of melatonin in the modulation of hypothalamo-pituitary-gonadotropic axis activity in sheep. The identification of melatonin receptors within the central nervous system and in the pars tuberalis of the pituitary gland, as well as the use of specific techniques of micro-implantation and micro-infusion were crucial in this aspect.

This study was conducted to find out whether daily LH secretion in ewes may be modulated by melatonin during the breeding season, when the secretion of both hormones is raised. Patterns of plasma LH were determined in luteal-phase ewes infused intracerebroventricularly (icv.) with Ringer-Locke solution (control) and with melatonin (100 microg/100 microl/h). Response in LH secretion to melatonin was also defined in ovariectomized (OVX) ewes without and after estradiol treatment (OVX+E2). Basal LH concentrations by themselves did not differ significantly before, during and after both control and melatonin infusions in intact, luteal-phase ewes. However, single significant (P<0.05) increases in LH concentration were noted during the early dark phase in the control and 1h after start of infusion in melatonin treated ewes. In both OVX and OVX+E2 ewes, melatonin decreased significantly (P<0.01, P<0.05, respectively) mean plasma LH concentrations as compared to the levels noted before the infusions. In OVX+E2 ewes, a single significant (P<0.05) increase in LH occurred 1h after start of melatonin treatment, similarly as in luteal-phase ewes. No significant differences in the frequencies of LH pulses before, during and after melatonin infusion were found in all treatments groups. In conclusion, melatonin may exert a modulatory effect on daily LH secretion in ewes during the breeding season, stimulating the release of this gonadotropin in the presence of estradiol feedback and inhibiting it during steroid deprivation. Thus, estradiol seems to be positively linked with the action of melatonin on reproductive activity in ewes.

This study tested the hypothesis that the dopaminergic and opioidergic systems are not involved in the short-term stimulatory action of melatonin (MLT) on the secretion of prolactin in anestrous ewes. Thus, MLT should stimulate prolactin release after blockade of either dopamine (DA) or opiate receptors with specific antagonists at the level of the pituitary gland and central nervous system (CNS), respectively. During afternoon intracerebroventricular (icv.) infusion of MLT, the mean plasma prolactin concentration increased significantly (P < 0.001) as compared with the concentrations noted before and during the infusion of the vehicle (veh.). As a result of subcutaneous (s.c.) injection of sulpiride (SULP, DA antagonist), an increase in plasma prolactin concentration was observed, followed by a gradual decrease during the icv. infusion of the vehicle. MLT infused icv. significantly increased (P < 0.001) the secretion of prolactin in SULP + MLT-treated ewes, as compared with the concentration of prolactin noted during infusion of the vehicle in SULP + veh.-treated ewes. Naloxone (NAL, opioid antagonist) infused icv. did not significantly affect the secretion of prolactin, however, a significant (P < 0.01) increase in the concentration was observed after the infusion. In MLT + NAL-treated ewes, the plasma prolactin concentration increased significantly (P < 0.001) during the infusion, as compared with the concentration noted before and that in NAL-alone infused ewes. These results demonstrate that melatonin stimulates prolactin release after the pharmacological exclusion of the dopaminergic input with the DA antagonist sulpiride and also despite the presence of DA activity in the hypothalamus after NAL treatment. Secondly, endogenous opioid peptides are not a major component of this melatonin action.

The aim of this study was to investigate whether melatonin might modulate the daily prolactin secretion in the ewe during a period of ovarian activity and, if so, whether this modulatory action of melatonin was related to the presence of estradiol in the organism. Ewes in the late follicular and luteal phase, as well as overiectomized ewes without (OVX) and after 7 days of estradiol injections (OVX+E2) were examined. Melatonin was infused into the third brain ventricle (100 microgram/100 microliter/h) from 14.00 to 18.00 h. The concentration of prolactin increased significantly during the infusion of melatonin in late follicular-phase ewes, but not in luteal-phase ewes, as compared to the concentration before the infusion: range from 204.0 +/- 31.7 to 272.2 +/- 50.1 ng/ml vs. range from 68.2 +/- 31.8 to 94.7 +/- 33.1 ng/ml (mean +/- SEM, n = 4, p < 0.01) and to the concentration noted during control infusions: range from 130.0 +/- 58.0 to 179.3 +/- 55.6 ng/ml (mean +/- SEM, n = 4, p < 0.05). In ovariectomized ewes, the concentration of prolactin during infusion of melatonin increased significantly, unrelated to the presence of estradiol, as compared to the concentration before infusion: range from 136.7 +/- 20.3 to 260.0 +/- 11.6 ng/ml vs. range from 41.6 +/- 2.6 to 152.3 +/- 14.6 ng/ml in OVX ewes (mean +/- SEM, n = 4, p < 0.01) and range from 161.5 +/- 66.5 to 250.2 +/- 24.3 ng/ml vs. range from 61.2 +/- 1.7 to 159.2 +/- 43.3 ng/ml in OVX+E2 ewes (mean +/- SEM, n = 4, p < 0.01). Concentrations during infusion of melatonin in OVX and OVX+E2 ewes were also significantly higher than during the control infusions: range from 7.2 +/- 1.7 to 22.2 +/- 4.1 ng/ml (mean +/- SEM, n = 4, p < 0.001). These results indicate that melatonin may affect the daily secretion of prolactin in ewes during the breeding season, and suggest that the variable response of prolactin to the melatonin signal in intact and ovariectomized ewes relates to the interaction between both ovarian steroids - estradiol and progesterone - and the prolactin-releasing factor.

Secretion of all the pituitary hormones undergoes marked circadian and seasonal changes. The rhythmicity of these changes is controlled by the circadian pacemaker system and the pineal gland transmitting daylength information to the neuroendocrine axis via the secretion of melatonin. This article presents data on the effects of the short-term melatonin administration into the third brain ventricle on prolactin, beta-endorphin and luteotropin secretion in ewes kept under the increasing and decreasing daylength conditions. Additional emphasis is given to dopamine and LHRH release in the mediobasal hypothalamus under the melatonin treatment by the push-pull method. The long-term and short-term actions of melatonin on the hormonal status in ewes is also discussed.