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Dynorphin A1-77 (DYN A1-17) acting in the CNS is known to affect thermoregulation, water and energy balance in the short time scale. In this study a long-term alteration of these functions induced by changes of day length in the highly photoperiodic species, the Siberian hamster (Phodopus sungorus) was studied using immunohistochemistry for DYN A1-17. We found that in the long day (LD, L:D 16 h:8 h) more brain areas express DYN A1-17 peptide than in the short day (SD, L:D 8 h:16 h) conditions. Structures of the hypothalamo-pituitary axis as well as cells of the ependyma, subcomissural organ and choroid plexus of the lateral and third brain ventricles are immunoreactive to anti-dynorphin IgG only in the LD. This might indicate a seasonal regulatory role of DYN A1-17 in physiological adaptations to severe climate changes.

A micellar electrokinetic chromatography method has been developed for simultaneous determination of melatonin and its precursors and metabolites. A 20 mM borate buffer pH 9.5 with 50 mM SDS served as the electrolyte. Tryptophan, 5-methoxyindoleacetic acid, 6-hydroxymelatonin, melatonin, serotonin, and 5-methoxytryptamine were baseline separated in less than 13 min. The limits of detection for UV detection and fluorometric detection based on native fluorescence of analytes were at the sub-ppm level. The proposed method with UV detection was applied to melatonin content control in pharmaceutical tablets with a precision expressed as RSD (n = 7) = 1.6%. For biological samples extraction with chloroform and ethyl acetate was examined. With ethyl acetate and chloroform recoveries of 87.2% and 82.1% melatonin, respectively, were obtained from plasma samples. The recovery of melatonin from spiked urine samples was 80.0% for ethyl acetate and 82.5% for chloroform. Fluorometric detection provides about two-fold improvement over UV in the detection of melatonin and minor improvements for three other analytes, but is much poorer than UV for tryptophan and 6-hydroxymelatonin in applied conditions.

The hypothalamo-pituitary-gonadal axis spontaneously activates in hibernating frogs, Rana temporaria, under constant conditions (0-4 degrees, darkness). The hypothesis that the spontaneous hibernatory activation involves other regulatory processes preparing the frogs for breeding and posthibernatory activity was tested. The serum concentrations and glandular contents of thyroid hormones (THs) were determined during hibernation. It was shown that (i) in both sexes, serum thyroxine and triiodothyronine levels significantly increased in the middle of January (week 13/14, between day 92 and 98 of hibernation); (ii) the peak of THs blood concentration was accompanied by a slow decrease of free forms of THs and the bound forms of both hormones dropped rapidly over this period; (iii) after a decrease on day 111, the low level of serum THs (but higher than before the peak) was sustained to the end of hibernation; (iv) the thyroid content of free THs significantly rose after their "surge" into the blood, reached maximal values in the middle of February, and remained at this level to the end of hibernation (last week of March); (v) in spring, after spawning, the THs levels in glands and in serum were much lower than those at the end of hibernation or were not detectable; and (vi) the results were confirmed during the second season, when the material in the period of expected elevation of serum THs was collected every other day., (Copyright 2000 Academic Press.)

Highly specific antisera and reversed-phase HPLC were used to examine the nature and distribution of gonadotropin-releasing hormone (GnRH) in the brains and plasma of three species of Rana frog previously reported to vary in the types and brain distributions of GnRH. The three species examined, Rana pipiens, Rana esculenta, and Rana ridibunda, exhibit differences in total concentration and relative concentrations of GnRH in brain areas, but all three contain the mammalian and chicken-II forms of GnRH (mGnRH and cGnRH-II). Both forms were found in the telencephalon and diencephalon, but mGnRH is consistently the most abundant form in the preoptic-hypothalamic area (e.g., ratios of mGnRH:cGnRH-II > 3:1 in hypothalamus-median eminence), whereas, cGnRH-II is the most abundant in the telencephalon and the only form measured in the cerebellum and medulla. The total content of cGnRH-II in the whole brain is about 1.5-2 times higher than that of mGnRH, due to the larger size of the areas outside the preoptic-hypothalamic area. These general patterns were the same for adults and juveniles. We found no evidence of a third form of GnRH corresponding to salmon GnRH in hypothalamus or other brain areas of R. esculenta as previously reported. These analyses also revealed the presence of both forms of GnRH in plasma draining the hypothalamic area, but the concentration of cGnRH-II is relatively higher than that in the corresponding hypothalamic tissue, suggesting differentially greater release or slower degradation of this form. These results do not support the conclusion that the ranid frogs are highly variable in the nature and distribution of GnRH in the brain, although they suggest that both forms of GnRH are potentially involved in the direct regulation of pituitary gonadotropes.

The influence of a dopaminergic antagonist, metoclopramide (MET), and an agonist, bromocriptine (BROMO), on reproductive status was examined in female frogs, Rana temporaria. MET induced advanced ovulation during hibernation, suggesting dopaminergic inhibition of gonadotropin (LH) release during this period. BROMO did not decrease plasma LH in intact females in comparison with vehicle (VEH)-treated controls (VEH: 11 +/- 6 vs BROMO: 5 +/- 4 ng/ml) or in sham-lesioned (SL) females (SL; 12 +/- 5 vs SL + BROMO: 9 +/- 8 ng/ml). However, BROMO significantly depressed the rise in plasma LH following lesioning (L) which disconnected the hypothalamus from the medium eminence-pituitary complex (L + BROMO: 29 +/- 10 vs L: 74 +/- 30 ng/ml; P < 0.002). Taken together with previous results of lesion studies, these data point to an important role of dopaminergic inhibition in the regulation of seasonal reproduction in this frog.

Pituitary and plasma luteinizing hormone (LH) levels were measured in female frogs, Rana temporaria, during and immediately after hibernation (0-4 degrees in darkness; 22-25 weeks) to study regulation of gonadotropin leading to posthibernation ovulation. Pituitary LH content began to rise progressively during the last third of hibernation (e.g., from starting levels of 5.3 +/- 0.09 micrograms/gland, 1.5 micrograms/mg between 8 and 11 weeks, to 18.6 +/- 9.7 micrograms/gland, 5.0 +/- 2.7 micrograms/mg at 19-22 weeks). Plasma LH increased in parallel, but with some delay (from 10 +/- 8 to 25 +/- 24 ng/ml). Frogs kept in light at low temperatures showed similar responses. Release of LH (rise in plasma levels with a drop in pituitary content) was observed during 32 hr immediately after termination of hibernation in association with the onset of ovulation. These data indicate the existence of regulatory mechanisms operating during hibernation under conditions of constant cold. Altogether, these and our previous results obtained after surgical deafferentiation of median eminence support the hypothesis of a progressive reduction of a CNS inhibition that results in the release of GnRH during hibernation in this frog.

The effects of lesions and deafferentations on accelerated hibernatory ovulation and on the concentration of GnRH in blood collected from the hypothalamo-pituitary area along with the blood and pituitary levels of luteinizing hormone (LH) in hibernating female frogs, Rana temporaria, were studied. The lesions to the caudal portion of the nucleus infundibularis ventralis resulted in: (1) an elevation in GnRH concentration in blood collected from the hypothalamo-pituitary area (x +/- SEM = 51 +/- 9.5 and 100 +/- 15 pg/ml in control and lesioned females, respectively), (2) an increase in plasma LH (from 11 +/- 1.3 to 14.7 +/- 2.5 ng/ml in controls to 73.1 +/- 12.0 and 74.2 +/- 15.8 ng/ml in lesioned females at 3 and 7 days), and (3) accelerated ovulations whose onset advanced as the hibernation season progressed. Histological and immunohistochemical analyses of the brains showed that GnRH seems to be stored during hibernation in the median eminence and suggested that the complete disruption of projections to the median eminence is prerequisite for accelerated ovulation. The influence of the hypothalamic inhibitory action on the release of GnRH and the way in which GnRH is transferred to the pituitary gland are discussed.

The effect of PRL on the diurnal changes in peripheral lymphocyte and granulocyte number, anti-sheep red blood cells, and natural anti-rabbit red blood cells serum agglutinins titre as well as plasma corticosterone concentration was examined in White Leghorn cockerels, immunized twice with sheep red blood cells. PRL was administered for six consecutive days at 4 or 8 h after light onset. Control birds were treated at the same times with hormone solvent alone. Immunized non-treated birds served as an additional control group. PRL injections influenced markedly the diurnal changes in all parameters examined. The effect of PRL administration on the diurnal changes in lymphocyte and granulocyte number and natural anti-rabbit red blood cells serum agglutinins consisted in elimination of the influence of solvent injections. In those cases where the solvent injections did not alter the pattern of the diurnal changes, i.e. in anti-sheep red blood cells serum agglutinins and in plasma corticosterone concentration, the pattern was modified by PRL injections. PRL administration affected also the correlations between the diurnal changes in plasma corticosterone concentration and those in lymphocyte number and anti-sheep red blood cells agglutinin titre. This suggests that the role of PRL in the regulation of the diurnal variations of immunity in chickens may be realized either directly, via its receptors in immune system or by its influence on plasma corticosterone concentration.

The effect of exogenous prolactin (PRL) on the weight and lipid content of the liver and abdominal adipose tissue as well as on the concentrations of total lipids and lipoprotein fractions (portomicrons, VLDL, LDL, HDL) in plasma was investigated in chickens. PRL or its solvent were administered at different time points (0, 4, 8, and 12 hours after light onset = HALO) during 5 consecutive days. The birds were exsanguinated at the same time of the day when they received the injections, 24 hours after the last one. At the same time plasma corticosterone (CS) concentration was determined in these chickens and also during 24 hours in intact chickens matched for age. PRL affected fat accumulation in the experimental birds and the intensity of this effect depends on the time of the day at which it was administered. PRL caused rise of the weight of the fat tissue and its lipid content when it was injected at 8 or 12 HALO. This was associated with a significant increase of total plasma lipids and with an insignificant increase in the level of lipoprotein fractions excluding portomicrons. PRL increased also (at 4 HALO) or decreased (at 8 HALO) the liver weight. Handling of the birds during vehicle injections decreased the corticosterone level in the control chickens as compared with the intact ones; PRL raised the level of CS at 0 HALO significantly. The role of PRL in the intensification of fat transport is discussed.

Three groups of females of R. esculenta were kept under constant temperature and photoperiod conditions (L:D = 12:12). One group consisted of intact frogs, while the remainder were given saline or prolactin (PRL) injections 6 hours after light onset. After 5 days of such treatment examination was made every 6 hours over a 24-hour period of lipid content in the liver, fat bodies (FBs) and ovaries and the level and composition of plasma lipids. Both the experimental handling and PRL treatment caused in animals a decrease in weight of the liver and mobilization of lipid metabolism, inducing a shift in lipids and a change in their distribution within the organs examined. In animals injected with saline lipids were transported chiefly to FBs, whereas in frogs given PRL the whole, combined lipid content in these organs decreased considerably, suggesting that they had been transported to the tissues and that their catabolism had been intensified. It is suggested also that PRL has sparing effect on the ovarian lipid pool. In both groups, especially in frogs given PRL, there was a marked increase in plasma lipid concentration and the contents in percentages of its different classes. Increase in concentration of phospholipids (3X) and cholesterol (10X) in animals given PRL is particularly distinct. Both experimental handling and PRL administration affected the pattern of diurnal fluctuations in the lipid content of the organs examined.

The purpose of this experiment was to elucidate whether the effect of exogenous prolactin (PRL) on immunity parameters of White Leghorn chickens varies during the day. The experiment was carried out on cockerels kept after hatching during 6 weeks under L:D = 12:12 conditions. During 5 consecutive days cockerels were injected with bovine PRL (150 micrograms per bird daily) or its solvent at different time points, i.e., at 0, 4, 8 or 12 HALO (Hours After Light Onset). The birds were sacrificed 24 hours after the last injection at the same time point when the injections were given. It was found that given at 4 HALO prolactin raised, whereas at 8 HALO it decreased the peripheral lymphocyte number. PRL had no effect on granulocyte number, natural anti-rabbit red blood cells (anti-RRBC) agglutinin titre and serum lysozyme activity. Administered at 0 and 12 HALO PRL tended to decrease the thymus and bursa of Fabricius weight. This different PRL effect on peripheral lymphocytes in chickens during the circadian period may be one of the causes of varying effect of this hormone on immunity (stimulatory or suppressive) described in literature. The role of PRL in regulation of immunity is discussed in relation to the possibility of PRL receptors occurrence on chickens lymphocytes.

In our previous experiments it was found that lesions placed in the infundibular hypothalamus induced an advanced ovulation in hibernating frogs, Rana temporaria. It was suggested that this premature ovulation was the effect of gonadotropin-releasing hormone (GnRH) due to the injury of some hypothalamic area inhibiting its release or its action on the pituitary gonadotrophs. To investigate this hypothesis, the following experiments were undertaken: (1) an attempt to induce ovulation with exogenous GnRH in hibernating frogs; (2) an attempt to inhibit ovulation with anti-GnRH serum in preovulatory hibernating animals nonlesioned and with lesions of the infundibular hypothalamus; and (3) administration of bromocriptine in hibernating animals to assess whether this substance, as an agonist of dopamine, possesses an inhibitory action on the ovulation. The following results were obtained: (1) lesions of the infundibular hypothalamus in hibernating preovulatory females caused an advanced ovulation during hibernation; (2) the exogenous GnRH administered to preovulatory females induced ovulation during hibernation; (3) the anti-GnRH serum injected into hibernating preovulatory lesioned females inhibited preterm ovulation during, but not after, hibernation; (4) the immunoneutralization of endogenous GnRH in nonlesioned females resulted in an inhibition of the posthibernatory ovulation; (5) bromocriptine inhibited posthibernatory ovulation in nonlesioned hibernating animals. Thus, the results of these experiments support the suggestion that induction of accelerated ovulation in lesioned hibernating animals involved the releasing action of GnRH. This action of GnRH seemed to be facilitated by the ablation of inhibitory dopaminergic function of hypothalamus in lesioned animals.

A selective staining by the indirect immunofluorescence method has been used for prolactin cells (PRL-cells) in the pituitary gland of Vipera berus (L.). The PRL-cells showed significant seasonal changes as judged by their morphological features, their number and localization in the pars distails. In the functional cycle of the cells 4 periods could be distinguished: 1 -- spring (March --May), when the release of the hormone as well as its increasing accumulation were observed; 2 -- early summer (June -- July), when the accumulation of the hormone in the gland reached the maximum and prevailed the release; 3 -- late summer (August), when was observed a rapid PRL release with a simultaneous exhaustion of cell reserves: 4 -- autumn (September), was characterized by small PRL content in the cells and its inconsiderable release.