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Growing female lambs were fed diets containing 14.2% (standard) or 8.1% (protein restricted) of proteins to determine their effects on puberty and luteinizing hormone releasing hormone (LHRH), gonadotropin hormone associated peptide (GAP), luteinizing hormone (LH) hormonal system. At the end of the experiment (30-34 weeks of age), hypothalamic LHRH, GAP and pituitary LH were analysed by immunocytochemical methods using specific antibodies. Plasma LH were determined by radioimmunoassay at 21 weeks of age. It was found that lowering of the dietary proteins content decreased the concentration of basal plasma LH significantly in lambs of 21 weeks of age. None of the sheep of this group reached sexual maturity at the same time as the animals of the standard group. However, immunoreactive (ir) LHRH neuronal system of protein restricted lambs was normally developed: Numerous irLHRH perikarya, dense network of axons and abundant material stored in the nerve terminals were well visualized in the typical sites of the preoptico-septal area, hypothalamus and the median eminence (ME). Gonadotropin associated peptide (GAP) of the LHRH precursor was present in the same populations of neurons that contained LHRH in the sheep brain. The proportion of pituitary LH-cells was three fold higher in pituitaries of the nutritionally restricted group. They displayed hypertrophy and very strong immunoreaction. These results show that protein deficiency in diets of growing female sheep delays their puberty but does not impair the synthesis and processing of LHRH in the brain neurons and synthesis of LH in pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)

In the light of evidence for endogenous opioid peptide involvement in the modulation of pituitary hormone release and in the body's overall response to stress, the aim of the present study was to determine the beta-endorphin-like immunoreactivity (beta-END-LI) in the blood plasma of ewes during different phases of the estrous cycle and in anestrous ewes subjected to acute or intermittent prolonged footshocks. The highest concentration of beta-END-LI was found in the blood plasma during the luteal phase and lowest one during the follicular stage at proestrus. Nearly equal concentrations of beta-END-LI were determined at estrus and metestrus; their values were over 10 times lower than those observed in the luteal phase. Acute stress caused transient increase of beta-END-LI in the blood plasma of anestrous ewes, with a peak after 15-30 min of footshock application and changes in the molar ratio of beta-LPH to beta-endorphin. No apparent increase in mean daily levels of beta-END-LI was noted in ewes subjected to prolonged footshocking; however, on the day after stimulation beta-END-LI concentration decreased below control values. These results indicate that: 1. secretion of beta-END-LI varies during different phases of the estrous cycle, 2. acute stress is a potent activator for beta-END-LI secretion, 3. no apparent increase of beta-END-LI in the blood plasma of ewes subjected to prolonged stress concomitant with accumulation of this material in pituitary (Polkowska and Przekop, 1988) supports the idea, that prolonged stress augments the synthesis of beta-END-LI but not its release.

The effect of an infusion of beta-endorphin and naloxone into the 3rd cerebral ventricle on the blood serum concentration of cortisol in non-stressed and stressed anoestrous sheep was studied. The infusion of naloxone alone did not alter the secretion of cortisol in non-stressed animals but the response to footshock was inhibited in that the cortisol values were significantly lower (P less than 0.05) when compared with those during the infusion of saline and footshocking. This opiate antagonist also suppressed the cortisol response to an infusion of beta-endorphin (P less than 0.001 and P less than 0.05). When the sheep infused with beta-endorphin were additionally exposed to footshock there was less enhancement of the secretion of cortisol (P less than 0.001 and P less than 0.05). The depressive action of beta-endorphin upon cortisol secretion in stressed sheep is matched by corresponding observations on man and it seems that endogenous opioids may inhibit or attenuate some hormonal responses to stress.

The effect of repeated and prolonged stress-stimuli applied during 3 days on the plasma prolactin concentration in sheep, in anoestrous, prooestrous and pregnant animals were studied. The following results were obtained: Repeated and prolonged weak electric stimuli applied in the form of footshocks in sheep during 3 days elicited a rise of mesor plasma concentration. The magnitude and the onset of this rise was not associated and dependent on the physiological state of animals. The mesor values of plasma prolactin concentration in all groups of animals except prooestrous ewes on the day following stimulation returned to their prestressing levels. Repeated and prolonged stimuli disturbed circadian rhythm of prolactin secretion existing only in prooestrous ewes, i.e. they caused its disappearance on the days during stimulation and its reappearance on the day following stimulation but at an accelerated and inverse time of the day to that found before stimulation. The induced rise of plasma prolactin concentration in anoestrous and pregnant ewes on the days during electric stimulation showed, according to Cosinor analysis, all features characteristic for acrophase of the circadian rhythm. This phenomenon may be considered as induced acrophase in stress condition.

A study was performed to examine the effect of the intermittent long-term (20 min/h daily during 3 consecutive days) electric stress (footshocks) on the hypothalamic LHRH in anoestrous ewe. On the third day, immediately after the last stimulation, the animals were decapitated and the hypothalami and pituitaries were taken for analysis of LHRH and LH by immunocytochemistry; LHRH was also assayed by radioimmunoassay. In the stressed animals the concentration of immunoreactive LHRH (IR-LHRH) and LHRH analysed by radioimmunoassay (RI-LHRH) increased significantly in the median eminence (ME) and the medial preoptic area (MPOA) when compared with the controls. Additionally, IR-LHRH perikarya appeared in the MPOA of stressed animals, whereas they were not seen in the controls. RI-LHRH in the medial basal hypothalamus (MBH) of stressed ewes was markedly lower than in control ones. No significant differences in RI-LHRH concentration were found in the septum between stressed and control ewes. The present study clearly indicates that prolonged stressful stimuli modify LHRH concentration in the discrete areas of the hypothalamus of the anoestrous ewe. It is suggested that these changes are attributed mainly to an inhibition of LHRH release from the ME and a suppression of LHRH transport along neural fibres within the hypothalamus.

Two different stressors footshocking and immobilization applied for 3 days induced in sheep differential behavioural and hormonal responses in cortisol and prolactin secretion. Immobilization drastically disturbed rumen motoric activity (significantly attenuated its contractions), caused loss of appetite and severe general depression. Footshocking did not induce any of these symptoms. Immobilization induced 2-3 fold higher rise of plasma cortisol mesors on the days of stressing as compared to footshocking. On the poststressing days after footshocking as well as after immobilization plasma cortisol mesors fell to 50% of the prestimulatory values. The rise of plasma prolactin mesors in footshocked and in immobilized animals on the days of stressing was nearly of the same magnitude. However, a significant difference in the response of prolactin secretion between footshocked and immobilized animals occurred in the circadian rhythmicity of the hormone secretion. Footshocking induced circadian rhythmicity with characteristic acrophases, while immobilization did not induce the circadian pattern of prolactin secretion. An attempt of interpretation of the described phenomena has been undertaken.

The effect of 6-hydroxydopamine (6-OHDA) infusion into the third cerebral ventricle on the secretion pattern of prolactin in anoestrous sheep, subjected to repeated and prolonged stress-stimuli was followed. On the 12-15 th day after the infusion of 6-OHDA into the third cerebral ventricle and after having tested the effect of this infusion on plasma prolactin concentration, the animals were subjected to mild electric footshocks 9 hrs daily (9 a.m.-6 p.m.) for 3 consecutive days. Infusion of 6-OHDA induced a very high rise of plasma prolactin values by about 3 times as compared to those found before infusion (from 118.2 +/- 39.5 to 338.9 +/- 81.7 ng/ml mesor 24 h). On the other hand, the response of the animals pretreated with 6-OHDA to electric stimulation was characterized by the lack of changes in plasma prolactin concentration; their plasma hormone concentrations were neither raised during stimulation nor decreased after the stimulation and were maintained during all these days nearly at the same level as after the infusion of 6-OHDA. Central mechanisms which could account for the so high and prolonged elevation of plasma prolactin concentration after the infusion of 6-OHDA and for the lack of its rise under conditions of prolonged stress are discussed.