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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.

Through binding with estrogen receptors, phytoestrogens, plant-derived estrogen-like compounds, affect numerous reproductive functions. It is not known whether these compounds are capable of evoking effective changes in luteinizing hormone (LH) and prolactin (PRL) secretion in ewes by acting directly within the central nervous system (CNS). The hypothesis studied was that genistein, infused for several hours into the third ventricle, could immediately affect LH and PRL secretion in ovariectomized (OVX) ewes during seasonal anestrus. Two doses of genistein, 1 microg/100 microl/h (total 4 microg, n = 7) and 10 microg/100 microl/h (total 40 microg, n = 7), were infused intracerebroventricularly from 12.00 to 16.00 h and blood samples were collected from 8.00 to 20.00 h at 10-min intervals. Randomly selected ewes were infused with a vehicle (control, n = 5). The mean plasma LH concentration in control ewes was significantly (p < 0.01) higher during infusion of the vehicle than before the infusion. It remained on an insignificantly changed level after the infusion. The frequency of LH pulses in control ewes did not differ significantly before, during, or after vehicle infusion. In ewes infused with a lower dose of genistein, plasma LH concentrations decreased significantly (p < 0.001) after the infusion, as compared with the values noted before and during genistein infusion. Only a tendency towards a decrease in LH pulse frequency occurred after infusion of a lower dose of genistein. In ewes infused with a higher dose of genistein, the plasma LH concentration decreased significantly (p < 0.01) after phytoestrogen administration as compared with the values noted before and during infusion. The frequency of LH pulses was also significantly (p < 0.01) lower after genistein administration. Because the changes in PRL secretion were more dynamic in response to genistein infusion, the statistical analysis included 2-hour periods. The mean plasma PRL concentration in control animals was significantly enhanced (p < 0.01) only during the first 2-hour period of sampling. After that it decreased and remained on an unchanged level up to the end of sampling. Similar changes in PRL secretion were observed in both experimental groups before genistein infusion. In contrast, significant (p < 0.01 to p < 0.001) increases in PRL concentration were noted regularly during and shortly after the genistein infusion in either low-dose or high-dose genistein-infused ewes, compared with the concentrations noted before genistein treatment. Plasma PRL concentrations during and after genistein infusion in both experimental groups were also significantly higher than the control (p < 0.01 to p < 0.001). The presented data demonstrate that genistein, a phytoestrogen, may effectively modulate LH and PRL secretion in OVX ewes by acting within the CNS., (Copyright 2004 S. Karger AG, Basel)

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.

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.

The relations between the circadian rhythms of melatonin and prolactin, and the effect of melatonin infused into the third ventricle or the mediobasal hypothalamus (MBH) on prolactin secretion and dopamine (DA) release were studied in anestrous ewes under increasing daylength conditions. The decreased amplitude and duration of nocturnal melatonin secretion were accompanied with changes in the daily pattern of prolactin secretion. Marked peaks of prolactin occurred after sunrise and melatonin decreases in February, as well as significant evening or nocturnal peaks under long day conditions between April and July. Melatonin infused into the third ventricle evoked an abrupt increase in the concentration of prolactin after 30 min, and the enhanced prolactin level was significantly higher than during the control infusion (range from 204 +/- 75 to 248 +/- 48 ng/ml vs. 128 +/- 68 to 149 +/- 93 ng/ml, mean +/- SD). The concentrations of DA in MBH perfusates decreased during perfusion of melatonin but to a degree similar to that noted in untreated ewes. These data suggest that short-term infusions of melatonin stimulate the secretion of prolactin in the ewe under increasing daylength conditions, and that this effect is not mediated by changes in DA release.

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.

A considerable amount of data suggest the involvement of calcium-mediated processes in the activation of the renin-angiotensin-aldosterone (RAA) cascade. To investigate the effect of calcium-channel inhibition on the RAA system, we studied 21 conscious pigs. Blood renin and aldosterone levels increased by subjecting animals to 24 hours of immobilization stress. Renin and aldosterone levels were repeatedly measured by radio-immunoassay in blood samples taken periodically over 24 hours from a chronically implanted arterial cannula. Pretreatment of the animals (N = 11) with nisoldipine, 2 x 20 mg p.o. daily for 2 days before and on the day of immobilization, transiently attenuated the stress-induced increase of plasma renin activity and completely prevented the rise of aldosterone, as compared to nontreated controls (N = 10). The finding that nisoldipine suppresses RAA activation induced by a nonpharmacologic stimulus in the conscious intact animal may have clinical implications.

Plasma prolactin concentrations were determined by radioimmunoassay during oestrous cycles and around the time of oestrus in different types of primitive gilts: Vietnamese, Zlotnicka and wild-boar X domestic pig hybrids. The animals were bled without stress from an indwelling arterial catheter. The following results were obtained: (1) in all gilts the main prolactin peak was observed at Day 15 or 16 of the oestrous cycle; (2) Vietnamese and hybrid gilts showed a second smaller prolactin surge after (Day 2) or before (Day 17) oestrus; (3) base levels of prolactin during the oestrous cycle were 14.8 +/- 0.93 ng/ml (Vietnamese gilts), 13.2 +/- 1.05 ng/ml (Zlotnicka gilts) and 15.6 +/- 2.01 ng/ml (hybrid gilts). The 15-16-day prolactin peaks reached maximum values of 36.4, 43.4 and 56.5 ng/ml respectively.

LH and prolactin were measured in plasma of 5 cows before and after enucleation of the persistent corpus luteum. Two cows returned to normal oestrous cycles following enucleation and did not differ from normal, cycling cows, with respect to hormone levels. Three other ones developed follicular cysts following enucleation and had decreased levels of LH and prolaction as compared with cycling cows. On the day of enucleation the fluctuations in LH-levels were higher than either before or after enucleation.

The concentration of prostaglandins E and F in different parts of the male reproductive system of CD-1 and dwarf mice was measured by radioimmunoassay. In intact CD-1 mice, the vas deferens contained a significantly higher concentration of PGE and PGF than was found in the epididymis and in the seminal vesicles. All tissues studied had more PGE than PGF. Castration resulted in increased PG concentrations in both the epididymis and the seminal vesicles and decreased PG concentrations in the vas deferens. There was also a differential response of PGE and PGF in the epididymis of genetically sterile dwarf mice (dw/dw) were significantly higher than those observed in their normal littermates ((PLUS)). A reversed PGE/PGF ratio was found in the mates. The results indicate that testicular androgens affect the levels of PGE and PGF in the reproductive system of male mice. The physiological role of PGs in male reproductive functions has not been established, but there is a suggestion that PH have a role in controlling the transit of spermatozoa through the epididymis and vas deferens.

In a group of 31 women with myasthenia the urinary excretion of 17-ketosteroids and their fractions (11-deoxy-17-ketosteroids, dehydroisoandrosterone) and 17-hydroxycorticosterone were determined. A statistically significant decrease was observed in the excretion of these substances. Besides that, the plasma cortisol level was determined in these patients and the results obtained in the whole group were at the lower normal range of the laboratory and in 40% of cases they were significantly lower (p less than 0.01). The obtained results may suggest that adrenocortical hypofunction exists in many cases of myasthenia. The pertinent literature is reviewed.

Trials were conducted with four heifers and twelve dairy cows to study the luteotropic effect of the serum of pregnant mares administered at the rates of 2000 to 3000 units. The effect of serum gonadotropin (PMSG) on ovarian activity was compared with the ovarian activity of control animals and ovarian activity after the administration of hypophyseal hormones. The highest progesterone concentration of PMSG-treated cows ranged from 41.9 to 74.3 ng per ml of milk; in the control group this range was between 32.2 and 43.1 ng per ml and in the group of gonadotropin-treated cows between 31.7 and 44.8 ng per ml. In heifers the progesterone levels increased from 3.56 to 4.58 ng per ml of plasma within 24 hours from the administration of PMSG. After 48 hours from administration the average progesterone concentration increased to 11.02 ng per ml of plasma. The animals which did not respond to PMSG administration by the growth of follicles exhibited an increased secretion of progesterone. The administration of hypophyseal hormones in the luteal stage of sexual cycle did not exert any significant influence on progesterone concentration, but stimulated the growth of follicles.