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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.)

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.