Interactions Between Opioid Peptides and Adrenaline-Containing Neurons Modulate Luteinizing Hormone Secretion in Male Rats

There is increasing evidence that the opioid inhibition of luteinizing hormone (LH) secretion is mediated, at least in part, by catecholaminergic mechanisms. This study determined the effects of selective manipulation of noradrenergic and adrenergic systems on the ability of opiate receptor blockade to induce the release of LH in adult male rats. Selective depletion of hypothalamic noradrenaline levels by 80% following 6-hydroxydopamine infusions into the central tegmental tract did not alter the 2- to 3-fold increase in serum LH levels following opiate receptor blockade with naloxone (2.5 mg/kg). In contrast, both selective depletion of hypothalamic adrenaline by prior treatment with the phenylethanolamine N-methyltransferase inhibitor, LY134046 (2 × 50 mg/kg) and non-selective depletion of all three catecholamines with α-methyl-p-tyrosine (250 mg/kg), abolished the naloxone-induced increase in LH. These results suggest that the inhibition of LH secretion by endogenous opioid peptides is influenced by catecholaminergic neurotransmission and further support the view that adrenaline rather than noradrenaline or dopamine is of importance in this context.     

Photoperiod Regulates the LH Response to Central Glutamatergic Stimulation in the Male Syrian Hamster

This study investigated central glutamatergic function in relation to photoperiodically-induced changes in the secretion of luteinizing hormone (LH). The experimental approach was to compare the central effects of glutamate agonists on LH secretion in reproduc-tively active hamsters kept in long days (LD) with those in photoinhibited hamsters kept in short days (SD) for 6 weeks and having regressed testes. Agonists were delivered via a cannula into the III ventricle of freely moving hamsters, and blood samples collected 10 to 15 min after the start of the infusion. A high dose (3.0 nmole) of N-methyl-D-L-aspartate (NMDA) induced significant (P<0.01) increases in serum concentrations of LH in hamsters in both photoperiods, though the NMDA-induced increase relative to endogenous LH concentrations was greater in SD than in LD. However, a lower dose of NMDA (0.3 nmole) revealed a difference in sensitivity. This dose significantly increased serum LH (P<0.05) in hamsters in SD but had no effect in those in LD. The seasonal difference in response to NMDA was compared with the response to an equimolar dose of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a non-NMDA agonist. This dose of AMPA (0.3 nmole) induced a two-fold increase (P<0.05) in serum concentrations of LH in hamsters in both photoperiods, relative to vehicle-treated controls. In a third experiment the dose-response effects of central AMPA on LH secretion were examined more closely. The sensitivity of LH secretion to stimulation with AMPA did not differ between SD- and LD-housed hamsters. Thus the photoperiod-related difference in sensitivity to stimulation with glutamate agonists is specific for NMDA receptor-mediated activation, rather than a passive reflection of differences in the capacity to secrete GnRH/LH in SD and LD photoperiods. To investigate the site of action of NMDA, the expression of the c-fos immediate-early gene, as assessed by immunocytochemistry for its protein product Fos, was used as a marker of neuronal activation, because previous studies in rodents indicate that a high proportion of GnRH neurons express c-fos at the time of the mid-cycle LH surge. NMDA induced widespread expression of c-fos in many periventricular regions including the medial preoptic area (PDA) and ventromedial hypothalamic nucleus. However, dual ICC revealed that in neither photoperiod was Fos present in GnRH-positive neurons 1 h after infusion of 3 nmole of NMDA, despite the increases in LH secretion induced by the infusion. AMPA injected icv at doses which released LH did not enhance expression of c-fos in the hypothalamus. Thus, in the male, enhanced expression of c-fos cannot be detected in GnRH neurons at the time of increased secretion of this hormone induced by glutamate agonists. In conclusion, these results show that both NMDA and non-NMDA glutamatergic pathways potentially regulate LH secretion in the Syrian hamster. The increased sensitivity to NMDA but unaltered sensitivity to AMPA in photoinhibited hamsters in SD is consistent with the view that changes in photoperiod might induce specific alterations in NMDA-mediated pathways that ultimately regulate GnRH neurosecretory activity.     

The Olfactory Bulbs Enhance Reproductive Hormone Secretion in Male Rats on Long or Short Photoperiod

Unlike seasonally breeding species such as the Syrian hamster, Sprague-Dawley laboratory rats do not normally respond to short photoperiod (6L18D) with reproductive regression. However, removal of the olfactory bulbs (BX) unmasks a photoperiodic response in pre-pubertal rats so that blinding or short photoperiod results in an inhibition of reproductive hormone secretion and/or a delay in pubertal development. This is apparently mediated by pineal melatonin which inhibits gonadotropin and/or prolactin secretion, but the mechanism by which BX facilitates the response to photoperiod is not clear. Experiment I was performed to determine serum levels of reproductive hormones at frequent intervals following BX and/or maintenance on short days. Twenty-three-day old male rats were BX or underwent sham BX (SH). They were thereafter maintained on a 14L:10D (long photoperiod) or 6L:18D photoperiod for the duration of the study. At 6 weeks following surgery, BX rats on either photoperiod had smaller testes than the SH groups. At week 8, the BX group on 6L:18D had smaller testes than the other three groups. There were transient reductions in serum luteinizing hormone and follicle-stimulating hormone in the BX rats on short photoperiod, but there were prolonged effects of BX decreasing prolactin levels in rats on long or short photoperiod. In SH rats, testosterone was elevated for weeks 6 through 10 of the study, and BX blocked this increase. Experiment II was performed to determine whether BX alters testosterone feedback on gonadotropin secretion. Twenty-three-day old male rats were BX or underwent SH operation and were then returned to a room on 14L10D. Six to 8 weeks later, all animals were castrated and placed on 6L18D or returned to 14L:10D. Eight weeks following castration, the rats were implanted with Silastic capsules containing 0, 10, 20 or 40mm testosterone. The post-castration increase in serum luteinizing hormone and follicle-stimulating hormone was lower in the BX than SH rats. In long photoperiod, serum luteinizing hormone and follicle-stimulating hormone were often lower in the BX rats receiving no testosterone replacement or lower doses of testosterone than in the SH group receiving similar capsules. Maintenance on short photoperiod increased the responsiveness to testosterone so that even the rats receiving low doses of testosterone had very low luteinizing hormone and follicle-stimulating hormone levels whether they were SH or BX. In summary, BX rats on long or short photoperiod had lower serum prolactin and testosterone levels than the comparable SH group and BX inhibited the post-castration increase in gonadotropin secretion. The data therefore suggest that the olfactory bulbs tonically enhance reproductive hormone secretion (especially around the time of pubertal development).     

Neuropeptide Y and Luteinizing Hormone Releasing Hormone Synergize to Stimulate the Development of Cellular Follicle-Stimulating Hormone in the Hamster Adenohypophysis

Luteinizing hormone releasing hormone (LHRH) stimulates the development of cellular FSH immunoreactivity in the perinatal hamster adenohypophysis. Because neuropeptide Y (NPY) can act directly on rat adenohypophysial cells to stimulate FSH and LH release and potentiate the stimulatory effect of LHRH on FSH and LH release, we investigated the effects of NPY alone and in combination with a low, ineffective dose of LHRH on inducing cellular FSH immunoreactivity in the neonatal hamster adenohypophysis. Neonatal female pituitary glands were grafted beneath the right renal capsules of hypophysectomized-ovariectomized adult hamster hosts with a catheter implanted in the external jugular vein. After treatment, hosts were decapitated and graft tissue was stained for FSH and LH immunoreactivity. The mean percentage of adenohypophysial cells that stained for FSH was low (2.8%) in grafts in hosts infused continuously with heparinized saline vehicle for 7 days. In other hosts, peptides were pulsed through the catheter every 12 h for 7 days. The mean percentage of FSH cells also was low after pulsing 6 ng LHRH or 2 μg NPY but increased substantially when the two peptides were pulsed simultaneously. No differences in the mean percentage of LH cells existed between any of the groups. The results demonstrate that NPY and LHRH can synergize to induce cellular FSH immunoreactivity in the neonatal female hamster.     

Induction of Cellular Follicle-Stimulating Hormone in the Hamster Adenohypophysis Requires Intermittent Stimulation by Luteinizing Hormone Releasing Hormone

We investigated the effectiveness of continuous vs intermittent LHRH stimulation of the neonatal female anterior pituitary gland on inducing cellular FSH immunoreactivity in the Golden Syrian hamster. Neonatal female pituitary glands were grafted beneath the right renal capsules of hypophysectomized-ovariectomized adult hosts with a catheter implanted in the external jugular vein. In experiment 1, vehicle or LHRH (6 ng/h) was infused continuously or LHRH was pulsed at 1 h (6 ng) or 12 h (72 ng) intervals through the catheters for 8 days. Hamsters were decapitated for collection of trunk blood shortly after the end of treatment, and grafts were prepared for immunocytochemical staining for LH and FSH. Anterior pituitary glands removed from neonatal (day 1) and day 9 female pups also were stained for LH and FSH. The mean percentage of adenohypophysial cells staining for LH increased from 11% in neonatal pups to mean percentages (24-28%) that were similar in day 9 pups and in all groups with grafts. The mean percentage of adenohypophysial cells staining for FSH increased from 1% in neonatal pups to percentages (16–21%) that were similar in day 9 pups and in grafts in hosts administered 6 or 72 ng LHRH pulses. By contrast, the mean percentage of FSH cells did not increase in grafts in hosts administered vehicle or LHRH by continuous infusion. Serum LH concentration was low in hosts given vehicle or LHRH by continuous infusion but elevated in hosts given 72 ng LHRH pulses and in all but one host given 6 ng LHRH pulses. Serum FSH concentration was detectable in some of the hosts given 6 or 72 ng LHRH pulses and non-detectable in the rest of the hosts. In experiment 2, the mean percentage of adenohypophysial cells staining for FSH did not increase in grafts in hosts infused with LHRH continuously at 72 ng/h compared to that observed in hosts pulsed with vehicle every h for 8 days (1.4 vs 2.0%). The data demonstrate that intermittent but not continuous stimulation of neonatal female adenohypophysial cells by LHRH is effective in inducing cellular FSH immunoreactivity. The data also strongly suggest that LHRH release is episodic during the first week after birth in the female hamster. To our knowledge, these results are the first to provide evidence suggesting that the episodic LHRH release system is functioning to exert an important biological effect in a neonatal mammal and that the mechanism for down-regulation of LHRH receptors in gonadotrophs can be activated this early in life.     

Effect of Naloxone on the Pulsatile Secretion of Luteinizing Hormone in Gonadectomized Male and Female Ferrets Before and After Oestradiol Replacement

Intravenous infusions of the opioid receptor antagonist, naloxone, caused a significant rise in luteinizing hormone (LH) pulse frequency in male and female ferrets which had been gonadectomized 10 days earlier while in breeding condition; mean LH concentrations and LH pulse amplitudes were not affected. By contrast, naloxone failed to stimulate LH pulse frequency, or other LH parameters in gonadectomized ferrets of either sex which received daily injections of a low dose of oestradiol. Our results for the ferret, in which the female is an induced ovulator, resemble those previously obtained in another induced ovulator, the rabbit. They contrast, however, with the results of numerous studies using spontaneously ovulating species in which sex steroids, if anything, facilitate the ability of naloxone to stimulate LH secretion.     

Effects of Beta-Endorphin on Pulsatile Luteinizing Hormone and Prolactin Secretion During the Follicular Phase in the Ewe

The present study was undertaken to determine the effects of the endogenous opioid ligandβ-endorphin on pulsatile luteinizing hormone (LH) secretion and plasma prolactin concentrations during the follicular phase of the ewe. Oestrous cycles were synchronized by injection of prostaglandin analogue and, commencing 13 h later, saline or β-endorphin (2, 10 or 50 μg) was injected intracerebroventricularly at hourly intervals for 3 h. Treatment with β-endorphin was followed by a significant reduction in LH pulse frequency at all doses due to almost complete cessation of pulses. There were no significant changes in LH pulse amplitude or mean LH concentrations. At the lowest dose ofβ-endorphin, LH pulses recommenced within 3 h of the last injection in all animals and pulse frequency was not significantly different from the saline-injected controls during the 3 h post-treatment period. Following treatment with 10 or 50 μg β-endorphin, LH pulse frequency remained suppressed during the 3 h post-treatment period but was not different from saline-treated controls on the following day. The time to the onset of the LH surge was not affected by intracerebroventricularβ-endorphin. Plasma prolactin concentrations were significantly increased following intracereb-roventricular injection of 10 or 50 μg β-endorphin, declining to control values soon after treatments stopped. Intravenous administration of 50 μg β-endorphin had no effect on LH but was accompanied by a small increase in prolactin concentrations. While these results indicate that hypothalamicβ-endorphin may be involved in the central control of LH and prolactin secretion, they provide no evidence for subtle modulation of LH pulse frequency by this neuropeptide.     

Synchronous Pulsatile Release of Luteinizing Hormone and Immunoreactive Beta-Endorphin from the Human Pituitary in vitro

An in vitro perifusion system employing very frequent (30 s) perifusate collectionswas utilized to investigate the relationship between pulsatile release of luteinizing hormone (LH) and immunoreactive β-endorphin (iβ-END) during individual perifusions of adult human anterior hemipituitaries. Each of six hemipituitaries released LH and iβ-END in a distinctlypulsatile fashion, with pulses occurring approximately every 3.2 min for each. Power spectral analysis revealed that pulsatile release of both LH and iβ-END occurred in a rhythmic pattern, with a periodicity of 3.1 and 3.2 min, respectively, and that the periodicity of pulsatile LH and iβ-END release was correlated within individual perifusions. Moreover, the relative amplitudes (% change) of the synchronous LH and iβ-END pulses were correlated. The effluent fractions from two of the perifusions were also assessed for thyrotropin, and it was determinedthat thyrotropin pulses were synchronized to both LH and iβ-END pulses. These studies confirm that LH and iβ-END are released from human anterior pituitaries in vitro in an intrinsically pulsatile fashion, and demonstrate that the LH and iβ-END pulses tend to occur rhythmically and in synchrony and proportion with each other. Furthermore, correlation of thyrotropin pulses to both the LH and iβ-END pulses suggests a common fundamental intrapituitary pulse generating mechanism.     

Ovarian Steroid Regulation of Pulsatile Luteinizing Hormone Release During Early Gestation in the Rat

The object of this study was to examine ovarian regulation of pulsatile luteinizing hormone (LH) secretion during early gestation. This was done primarily by analyzing pulsatile LH release in rats that were either sham ovariectomized (OVX) on Day 7 of pregnancy, implanted with empty Silastic capsules, and bled on Day 8, or OVX on Day 7, immediately implanted with Silastic capsules producing plasma levels of estradiol and/or progesterone characteristic of Day 7 to 8 of pregnancy, and bled on Day 8. In addition, the role of progesterone in regulating pulsatile LH secretion was also examined by administration of the progesterone receptor antagonist, RU486, on Day 7 and examining pulsatile LH release on Day 8 of pregnancy. OVX caused a marked increase in LH pulse amplitude and frequency within 24 h. Replacement with physiological plasma levels of estradiol or progesterone alone had no suppressive effect on this OVX-induced increase in pulsatile LH secretion. Restoration of physiological plasma levels of both estradiol and progesterone returned LH pulse amplitude to values seen in sham OVX controls, and prevented the OVX-induced increase in LH pulse frequency. The group mean LH pulse frequency tended to be less in estradiol + progesterone-treated rats than in sham OVX controls, but this difference was not statistically significant. RU486 blocked uterine progesterone receptors as evidenced by endometrial hemorrhaging. In agreement with the OVX + steroid replacement data, RU486 administration also resulted in increases in LH pulse amplitude and frequency. These data demonstrate that the frequency and amplitude of LH pulses on Day 8 of gestation are held in check by negative feedback signals coming from the ovary. Neither steroid alone exerts any suppressive influence over pulsatile LH secretion during early gestation, but both steroids acting together exert a prominent negative feedback regulation on the pulsatile LH release process.     

An Enkephalinergic Mechanism for the Hypothalamic Effect of Atrial Natriuretic Peptide to Inhibit Luteinizing Hormone Secretion

Previous studies have demonstrated a hypothalamic site of action of atrial natriuretic peptide (ANP) to inhibit luteinizing hormone and an opioid mechanism has been suggested. We have identified the paraventricular nucleus as at least one locus of action of ANP since site-specific injection of 0.1 nmol, bilaterally into this nucleus, but not the medial preoptic nuclei or arcuate nucleus, resulted in a significant inhibition of plasma levels in conscious, ovariectomized rats 90 and 120 min later. This paraventricular site of action suggested an involvement of endogenous enkephalins. The stable enkephalin analog, [D-Pen^2.5]enkephalin, when injected into the third ventricle in a dose of 1.0 nmol, produced a significant and transient inhibition of luteinizing hormone secretion similar to that seen following injection of 2.0 nmol ANP. Pretreatment of the rats 15 min before peptide injection with the selective 5 opioid antagonist naltrindole (50 μg/2 μl saline) completely prevented the luteinizing hormone-inhibiting effects of both ANP and the enkephalin analog, δ opioid blockade failed to prevent the prolactin-inhibiting effect of ANP but did reverse the prolactin-stimulating effect of [D-Pen^2.5]enkephalin. Our results suggest a paraventricular nucleus site of action of ANP in addition to probable effects in the median eminence and indicate the possible enkephalinergic mediation of the peptide's ability to inhibit luteinizing hormone secretion.     

Effect of Hypothalamic Deafferentation on the Pulsatile Secretion of Luteinizing Hormone in Ovariectomized Lactating Rats*

The pulsatile luteinizing hormone (LH) secretion in ovariectomized lactating rats bearing complete (CD), anterior (AD), anterolateral (ALD), posterior (PD), or roof (RD) deafferentation of the hypothalamus was determined. All lactating rats were ovariectomized on Day 2 of lactation (Day 0, day of parturition). The deafferentation of nerve fibres to the mediobasal hypothalamus was performed on Day 6 or 7 of lactation. Twenty-four h after the surgery, blood samples were taken through the indwelling atrial catheter every 6 min for 3 h. Plasma concentrations of LH and prolactin (PRL) were measured by radioimmunoassay. The loss of LH pulses associated with lactation was still apparent following AD, PD and sham-deafferentation (SD); pulsatile LH secretion was, however, present in rats with CD, ALD and RD despite continued suckling. The only significant difference in plasma PRL concentrations among the various groups was a reduction in the PRL level in rats with RD in comparison to those with SD. We conclude that the neural signal responsible for the inhibition of pulsatile LH release by suckling is conveyed through the dorsal part of the hypothalamus and PRL does not mediate the suppression of LH pulses in mid-lactation.     

GnRH mRNA Increases with Puberty in the Male Syrian Hamster Brain

Puberty in the male Syrian hamster (Mesocricetus auratus) is characterized by decreased responsiveness to testosterone mediated negative feedback, but the neural mechanism for this change remains elusive. We hypothesized that decreased inhibition of the gonadotropin-releasing hormone (GnRH) system results in increased neurosecretory activity, which includes an increase in GnRH gene expression. This study examined GnRH mRNA in male hamsters before and after puberty, and sought to determine if any increase in mRNA was specific to particular subpopulations of GnRH neurones. Brains were collected from 21-day-old prepubertal males (n = 5) and 56-day-old postpubertal males (n = 5). Alternate 10 microm coronal sections from fresh-frozen brains were collected throughout the septo-hypothalamic region, and 25% of those sections were processed for in-situ hybridization histochemistry using an 35S-riboprobe complementary to hamster GnRH. No differences were observed in the number of GnRH mRNA expressing cells in any region, but in the diagonal band of Broca (DBB)/organum vasculosum of the lamina terminalis (OVLT) there was a significant increase in labelling intensity (defined as area of the cell occupied by silver grains) in postpubertal males. A second analysis compared the frequency distributions of cells based on labelling intensity between prepubertal and postpubertal males. This analysis revealed significant differences between the two frequency distributions in all areas analysed (DBB/OVLT, medial septum (MS), and preoptic area (POA)). Furthermore, examining the distribution of cells in these regions revealed a shift to the right in the postpubertal population of cells, which indicated an increased number of GnRH neurones with greater labelling intensity. These data clearly demonstrate increased GnRH mRNA during puberty. Furthermore, they suggest that the previous observation of brain region specific pubertal decreases in GnRH-immunoreactivity only within the DBB/OVLT and MS but not the POA are not due to differential levels of GnRH gene expression, but could indicate increased release from these neurones during puberty.     

Stimulation of Luteinizing Hormone-Beta Messenger Ribonucleic Acid and Post-Translational Modification of Luteinizing Hormone Isoforms by Second Messengers Mediating the Action of Gonadotrophin-Releasing Hormone

Several second messenger systems have been implicated in mediating the action of gonadotrophin-releasing hormone on the pituitary gonadotrophs and numerous studies have shown that activation of these systems induces luteinizing hormone (LH) secretion. However, it is not known how gonadotrophin-releasing hormone or the second messenger systems induce de novo LH biosynthesis and post-translational modification of the hormone. In these experiments hemipituitary glands have been perifused with drugs which activate second messengers or stimulate protein kinase C directly. The LH secretory responses have been correlated with measurements of common a and LHβ mRNA and the molecular species of LH which were present in the pituitary perifusate after exposure to the drugs. Gonadotrophin-releasing hormone (50 ng/ml, 42 nM), with and without the presence of extracellular Ca²⁺, the Ca²⁺ ionophore, A23187 (10 μM), and phorbol 12-myristate (1 μM) all stimulated an increase in LHβ mRNA compared with controls and the appearance of a different isoform of LH to that found stored in and released from the unstimulated pituitary gland. Phospholipase C was without effect on LHβ mRNA levels and showed minimal efficacy in inducing the appearance of the different LH isoform.     

Naloxone does not Affect the Luteinizing Hormone-Releasing Hormone-Induced Inhibition of Luteinizing Hormone Secretion in Sheep

Injection of luteinizing hormone-releasing hormone (21 pmol) into the third cerebral ventricle of long-term ovariectomized ewes caused a marked inhibition of luteinizing hormone secretion. Mean luteinizing hormone levels and luteinizing hormone pulse frequency were reduced significantly when compared with the control responses to saline (50 μl). A notable characteristic of the response was the delayed and sustained nature of the luteinizing hormone-releasing hormone-induced inhibition. In the presence of the opioid antagonist naloxone (4 ± 25 mg iv), the central administration of luteinizing hormone-releasing hormone still produced a marked inhibition of luteinizing hormone secretion. Again, mean luteinizing hormone levels and luteinizing hormone pulse frequency were reduced significantly. When naloxone was injected iv, there was a significant rise in mean luteinizing hormone levels as a consequence of an increase in pulse frequency (in four out of five ewes) and a significant increase in luteinizing hormone pulse amplitude.
In conclusion, these data suggest that central opioid pathways sensitive to blockade by naloxone are not involved in the luteinizing hormone-releasing hormone-induced inhibition of luteinizing hormone release. Furthermore, in the long-term ovariectomized ewe, endogenous opioid peptides exert a tonic inhibitory influence on luteinizing hormone-releasing hormone/luteinizing hormone secretion.     

Long-Term Treatments with Morphine and Naloxone have Sex-Differentiated Effects on Luteinizing Hormone Secretion in Chronically Catheterized Fetal Pigs

Short- (one bolus injection) and long-term (repeated injections over a period of at least 7 days) effects of drugs on pituitary function in pig fetuses were studied to investigate the influence of morphine and naloxone on luteinizing hormone secretion in the chronically catheterized pig fetus between days 102 and 110 of gestation (term: 113±1 SD day). Both substances were intravenously administered in two doses: 0.1 mg and 1 mg/fetus. Repeated injections at 2-day intervals enabled us to study short- as well as long-term effects. Morphine acutely inhibited luteinizing hormone secretion both in male and female fetuses. Long-term treatment with morphine at both doses caused an inhibition of basal luteinizing hormone (levels before treatment on each day) in females (0.1 mg: r=-0.60, P<0.001; 1 mg: r=-0.45, P<0.05) while male fetuses were unaffected. Naloxone did not have any short-term effects, either in females or in males. In the long-term study, however, naloxone at 1 mg dose decreased basal luteinizing hormone levels in male fetuses (r=-0.55, P<0.01), whereas in females no effect was evident. Co-administration of 0.1 mg naloxone +0.1 mg morphine abolished the long-term inhibitory effect observed in females when 0.1 mg morphine alone was given. These results indicate that the link between opioids and the luteinizing hormone system is functional in the pig from at least 2 weeks before birth. Furthermore, there is a sex difference in the long-term effects of both morphine and naloxone. The origin of the apparently paradoxical long-term effect of naloxone in male fetuses remains unclear.     

The Photoperiodic Response in Syrian Hamster Depends upon a Melatonin-Driven Circadian Rhythm of Sensitivity to Melatonin

The pineal gland, via the daily pattern of melatonin (MEL) secretion, is directly involved in the conduction of photoperiodic information. The duration of MEL secretion is proportional to the duration of the dark period and, whatever the photoperiod is, MEL synthesis occurs 3 or 4 h after the dark onset in Syrian hamsters. In order to determine the relative importance of the duration or the coincidence hypothesis, a daily infusion protocol was used in sexually active pinealectomized hamsters. Long duration of MEL infusion (10 h) completely inhibit testes whereas short duration infusion (5 h) had no effect. When the animals were infused twice within 2 h 30 min separated by 3 h, they presented a complete gonadal atrophy, similar to the one observed with the 10 h infusion. Measurement of plasma MEL during the infusion and seperation periods revealed that MEL reached physiological nighttime values during the infusion period and fell to daytime values 1 h after the end of an infusion period. Thus, the results could not be due to a time additive action of the two MEL pulses. An intermediate response was observed when the 2 signals were applied across the light/dark transition. Gonadal regression did not occur when the 2 periods of infusion were separated by 5 h 30 min. The efficiency of this type of infusion was not dependent on the ambiant photoperiod since similar results were obtained in long and short photoperiods. The infusion was also as effective during the day as well as during the night. These results suggest that there is a rhythm of sensitivity to MEL, based on the coincidence hypotheses, that are important for transmission of photoperiodic information. This rhythm of sensitivity to MEL seems to be entrained by MEL itself, since the efficiency of the two pulses of MEL is not dependent of time of application and/or of photoperiod.     

Hypoglycaemia-Induced Inhibition of Pulsatile Luteinizing Hormone Secretion in Female Rats: Role of Oestradiol, EndogenousOpioids and the Adrenal Medulla

Oestradiol (E₂) has been shown to exacerbate the inhibitory effect of hypoglycaemic stress on gonadotrophin-releasing hormone pulse generator (GnRH) activity in primates. The mechanism by which this is mediated is not yet known. We therefore aimed to establish whether there is a sensitizing influence of E₂ on the suppression of LH pulsatility in response to hypoglycaemia in the female rat, thus providing a more amenable model in which to study this phenomenon. In ovariectomized Wistar rats with E₂ replacement, insulin-induced hypoglycaemia (0.5  U/kg iv) resulted in an interruption of pulsatile LH secretion. Induction of the same degree of hypoglycaemia in ovariectomized rats without E₂ replacement was without effect on LH pulsatility. Naloxone administration prevented the hypoglycaemia-induced inhibition of LH pulses. Because hypoglycaemia is a potent activator of the sympathetic nervous system, we also tested the hypothesis that the adrenal medulla is involved in this suppression of LH pulses in the rat. Adrenomedullectomy completely prevented this inhibitory response to hypoglycaemic stress. These data are consistent with the hypothesis that E₂ sensitizes the GnRH pulse generator to the inhibitory influences of hypoglycaemic stress in the rat. Furthermore, a clear role for both endogenous opioid peptides and the adrenal medulla in the stress-induced suppression of LH pulsatility is identified.     

Luteinizing Hormone-Releasing Hormone Neurons which are R-etrogradely Labeled After Peripheral Fluoro-Gold Administration in the Male Ferret

This study identified luteinizing hormone-releasing hormone (LHRH)-producing neurons which have access to fenestrated capillaries in prepubertal male European ferrets. Fluoro-Gold was injected intraperitoneally to retrogradely label neurons with terminals outside the blood-brain barrier. LHRH neurons were identified by immunofluorescence using a secondary antibody tagged with tetramethylrhodamine isothiocyanate. Cell bodies which demonstrated both tetramethylrhodamine isothiocyanate and Fluoro-Gold fluorescence were defined as LHRH-producing neurons with axon terminals in regions containing fenestrated capillaries. The total number and neuroanatomical distribution of immunopositive (LHRH +) cells concurred with previous studies in the ferret in which cell bodies were diffusely distributed from rostral forebrain through caudal diencephalon, with approximately 70% of the LHRH + cell bodies located in retrochiasmatic hypothalamus. In the present study, an average of 59.8% of all LHRH+ neuronal perikarya also contained Fluoro-Gold. The majority of Fluoro-Gold filled LHRH+ neurons demonstrated only faint to moderate amounts of Fluoro-Gold when compared to other Fluoro-Gold filled neurosecretory neurons. This limited uptake of Fluoro-Gold may be due to a relative inactivity of LHRH neurons projecting outside the blood-brain barrier. Double-labeled LHRH + neurons were dispersed throughout the entire population of LHRH+ cell bodies and no apparent nuclear groups of double-labeled neurons were found. This observation suggests that the LHRH+ neurons responsible for neurosecretion into the median eminence coexist with the LHRH+ neurons responsible for intracerebral neurotransmission or neuromodulation. One distinguishable population of LHRH + neurons was consistently observed in all the brains. Only 26% of total LHRH+ perikarya within the caudal arcuate nucleus contained Fluoro-Gold, while at least 50% of LHRH+ neurons in other structures, including the rostral arcuate nucleus, contained Fluoro-Gold. Thus, in the prepubertal male ferret, the majority of LHRH cell bodies located in the caudal arcuate nucleus may be differentially regulated and/or involved in non-neuroendocrine functions.     

Porcine Relaxin Affects the Release of Luteinizing Hormone in Rats

The effects of intravenous injection of porcine relaxin on the pulsatile secretion of luteinizing hormone (LH) were investigated in conscious rats. In untreated, ovariectomized animals, relaxin at doses 2.5 to 10/μg/rat caused a dose-dependent suppression of pulsatile release of LH. At 5μg relaxin, pulses were suppressed for approximately 60 min and there was a significant (P<0.05) fall in mean plasma LH levels. Pulses returned with the same frequency as the pretreatment period but amplitude and nadir of these pulses were significantly (P<0.05) reduced at doses >2.5μg/rat. In ovariectomized rats pretreated with either estradiol or progesterone alone, relaxin did not alter plasma LH levels. In contrast, injection of 5μg relaxin in rats primed with a combination of estradiol and progesterone caused a 90% increase in circulating LH levels. Intracerebroventricular infusion of a specific angiotensin II antagonist blocked the inhibitory effect of relaxin on LH release in untreated, ovariectomized rats and negated the stimulatory effect of relaxin on LH release in estradiol-progesterone-primed, ovariectomized rats. The results demonstrate that acute injections of porcine relaxin in ovariectomized rats suppress the pulsatile release of LH. This effect is blocked when the central angiotensinergic system is compromised suggesting that relaxin might act through the central angiotensin system. The findings are in agreement with other studies that indicate relaxin activates the central angiotensin system. It is also possible that relaxin may act at the level of the adenohypophysis to alter secretion of LH but data in the present study suggest that this may not be a significant site of relaxin action.     

Changes in Feeding Activity, Plasma Luteinizing Hormone, and Testes Weight in Ring Doves Following Hypothalamic Injections of Prolactin

Microinjections of ovine prolactin were administered unilaterally to the ventromedial hypothalamic nucleus and the preoptic-suprachiasmatic region in adult male ring doves in an attempt to determine the site(s) at which intracranial injections of prolactin act to alter feeding behaviour and gonadotropin secretion in this species. Food intake and body weight were measured daily during a 6-day pretreatment period and the 5-day treatment period that immediately followed. During the treatment period, birds received twice daily injections (0.5 μl) of either 2.5 ng ovine prolactin or saline vehicle. An additional group of birds with cannulae in the ventromedial nucleus were given twice daily injections of 25 ng ovine prolactin. Although food consumption was unaffected by low dose prolactin treatment, birds given 25 ng prolactin injections into the ventromedial nucleus showed a significant augmentation in food intake. Injections of 25 ng prolactin into the preoptic area also increased feeding; however, the magnitude of this hyperphagic response, as expressed relative to pretreatment levels, was less than that observed following prolactin injection into the ventromedial nucleus. No differences were observed between prolactin-treated and vehicle-treated birds in either cannulation group when testes weights and plasma luteinizing hormone concentrations were compared at the end of the treatment period. However, the possibility that prolactin influenced changes in luteinizing hormone and testes weight relative to baseline values could not be assessed due to constraints imposed by the experimental paradigm used. These results suggest that prolactin-sensitive neurons in the ventromedial hypothalamic region and the preoptic area are potential sites of prolactin action in promoting hyperphagia in ring doves. However, the role of these sites in mediating prolactin-induced suppression of gonadotropin secretion in this species remains to be clarified.