Photoperiodic changes in opiate binding and their functional implications in golden hamsters

Daylength modulates gonadotropin secretion, gonadal steroid hormone feedback, sexual behavior and body weight in male golden hamsters. Endogenous opiates regulate each of these phenomena, and the ability of opiate receptor blockade to elevate serum LH secretion is photoperiod-dependent. We used in vitro autoradiography to localize and quantify effects of daylength in golden hamsters. Hamsters were exposed to stimulatory (14 h light: 10 h dark) or inhibitory (10 h light: 14 h dark) photoperiods for 10 weeks before specific [3H]naloxone binding was assessed. Short days significantly decreased binding in medial amygdala and the intercalated amygdaloid nucleus. This effect was reversed by superior cervical ganglionectomy. No significant effects of daylength were observed in other amygdaloid, hypothalamic or preoptic areas. Lesions of the medial amygdala decreased copulatory behavior, short day-induced weight loss, and anogenital chemoinvestigation but did not affect gonadal regression or other forms of chemoinvestigation. These lesions facilitated testosterone's negative feedback on luteinizing hormone in long days but did not interfere with the potentiation of negative feedback by short days.     

Refractoriness to short day lengths augments tonic and gonadotrophin-releasing hormone-stimulated lutenising hormone secretion

Siberian hamsters (Phodopus sungorus) undergo reproductive involution following exposure to short winter day lengths. Following approximately 20 weeks of exposure to short day (SD) lengths, hamsters become refractory to the inhibitory effects of SD, and reproductive competence is restored in anticipation of spring. The extent to which changes in gonadal steroid-dependent and -independent regulation of gonadotrophin secretion participate in this vernal reactivation of the gonads is not known. This experiment tested whether tonic and gonadotrophin-releasing hormone (GnRH)-stimulated regulation of lutenising hormone (LH) secretion differs between photoresponsive and photorefractory Siberian hamsters. Male hamsters born into long day (LD) lengths were castrated or subjected to a sham-castration surgery at 17 days of age, implanted s.c. with blank or testosterone-filled capsules, and housed in LD or SD thereafter. Baseline LH and LH responses to GnRH (200 ng/kg, s.c) were measured at 14 (photoresponsive) and 40 (photorefractory) weeks of age. Despite lower circulating testosterone concentrations in gonadally regressed SD hamsters on week 14, tonic LH concentrations were comparable among all groups of gonad-intact hamsters on weeks 14 and 40; however, week 14 SD hamsters exhibited significantly higher GnRH-stimulated LH responses. Tonic LH concentrations were indistinguishable among all groups of castrated hamsters bearing empty implants on week 14, but prolonged exposure to LD led to a decrease in resting LH, whereas prolonged exposure to SD resulted in an increase in LH. In castrated hamsters bearing testosterone implants, baseline LH concentrations were comparable in all groups, but GnRH treatment resulted in significantly higher LH concentrations in photorefractory (week 40, SD) hamsters relative to all other groups. The data suggest that the development of photorefractoriness in Siberian hamsters is characterised by enhanced gonadal hormone-independent stimulation of LH secretion, and diminished sensitivity to inhibitory negative-feedback effects of testosterone on LH secretion. Decreases in responsiveness of gonadotrophin secretion to gonadal hormone negative feedback may contribute to the process of photorefractoriness and assist in maintaining the growth of reproductive organs during the process of gonadal recrudescence.     

Effect of Photostimulation on Concentrations of Plasma Prolactin in Castrated Bantams (Gallus domesticus)

The annual breeding cycle of ‘unimproved’ breeds of domestic chicken, including the bantam, at temperate latitudes, is terminated by decreasing daylength in autumn and is initiated in late winter, while daylengths are still short. Observations on photoperiodic birds that terminate seasonal breeding by the development of long day photorefractoriness suggest that the photoinduced pattern of prolactin secretion is associated with the pattern of gonadal growth and regression. It was predicted that, if there is a causal relationship between photoinduced changes in prolactin secretion and gonadal function in birds then, in the bantam, the pattern of prolactin secretion observed after photostimulation would not be the same as in birds terminating breeding by the development of long day photorefractoriness. Experiments were carried out on surgically castrated bantams to avoid confounding the effects of photostimulation and the stimulatory actions of testicular hormones on prolactin secretion. Transfer of photosensitive castrated bantams from 8 to 14, 16, 18 or 20 h light/day initially stimulated prolactin release and, subsequently, after 20–30 days, concentrations of plasma prolactin progressively decreased. After 148 days of photostimulation, concentrations of plasma prolactin approached but were still higher than short day controls. Transfer of photosensitive castrated bantam cockerels from 8 to 12 h light/day stimulated a slower increase in plasma prolactin that subsequently remained higher than in other photostimulated groups. A further 4 h increase in photoperiod in the birds exposed for 148 days to 12 or 16 h light/day resulted, respectively, in a transitory increase and no increase in prolactin secretion. Recovery of photosensitivity for prolactin release was observed in the birds transferred to 18 or 20 h light/day for 148 days after treatment with 8 h light/day for 35 days. Attempts to obtain an independent hormonal correlate of the prolactin responses to photostimulation by measurement of plasma luteinizing hormone (LH) were unsuccessful. The concentration of plasma LH in castrated bantams did not change in response to a change in photoperiod. These observations show that the photoinduced pattern of prolactin release in the bantam, a species which terminates seasonal breeding in response to decreasing daylength, is the same as that in birds which terminate seasonal breeding by the development of long day photorefractoriness. It is concluded that the photoinduced pattern of prolactin secretion in birds can be dissociated from the neuroendocrine mechanisms controlling the termination of seasonal breeding.     

Effect of Ovariectomy or Oestrogen Implants upon Pituitary Function in Female Hypogonadal Mice Bearing Normal Fontal Preoptic Area Grafts

The effects of ovariectomy and oestrogen feedback for 10 days upon pituitary and serum luteinizing hormone (LH) content, pituitary glycoprotein subunit messenger ribonucleic acid (mRNA) and prolactin mRNA content in normal females, female hypogonadal mice and hypothalamic grafted female hypogonadal mice, bearing a graft of normal mouse preoptic area tissue into the third ventricle, have been investigated. In normal females ovariectomy resulted in a rise in serum LH, LHβ-subunit and common α-subunit mRNAs with no significant change in pituitary LH content or follicle-stimulating hormone (FSH) β-subunit mRNA. In the hypogonadal females, preoptic area grafting resulted in an elevation in all of the above parameters into the normal range. Ovariectomy in this group resulted in a further elevation of serum LH, LHβ-subunit and α-subunit mRNAs with no change in pituitary LH content or FSHβ-subunit mRNA, which in all cases were comparable to ovariectomized normal animals. Oestrogen treatment caused a fall in pituitary LH content and the serum LH fell below the detection of the assay. LHβ-subunit and a-subunit mRNA mirrored this fall but there was no change in FSHβ-subunit hybridization. These experiments suggest that even though normal neuronal input to the gonadotrophin-releasing hormone neurons is disrupted, oestrogen-induced negative feedback can still occur in grafted female hypogonadal animals. Gonadotrophin-releasing hormone neurons are reported to lack oestrogen receptors but feedback within this graft by co-transplanting oestrogen-sensitive neurons remains a possibility, as does feedback at the level of the host median eminence where graft axons extend to the pituitary portal vessels. The similarity of the response in normal and grafted animals indicates that these actions of oestrogen may be effected predominantly at the pituitary level.     

Photoperiodic Control of the Concentration of Luteinizing Hormone, Prolactin and Testosterone in the Male Emu (Dromaius novaehollandiae), a Bird that Breeds on Short Days

The objective of this study was to establish, for a short-day breeding bird, the male emu, whether the breeding season is principally controlled by changes in photoperiod, and to investigate the endocrine mechanisms involved. Two groups of adult males were subjected to three alternating periods of 150-185 days of 14 h light/day (LD) and 10 h light/day (SD) terminating in a 360-day period of LD or SD. Transfer from LD to SD led to increases in plasma concentrations of luteinizing hormone (LH) and testosterone, after 82 +/- 8 and 73 +/- 3 (SEM) days, and an increase in prolactin concentrations after 115 +/- 12 days. Concentrations of LH and testosterone began to decrease before transfer back to LD, at a time when prolactin concentrations were approaching peak values. Transfer from LD to 360 days of SD resulted in increases in LH and testosterone concentrations, and these terminated after an increase in prolactin concentrations. After transfer from SD to 360 days of LD, plasma concentrations of LH and testosterone began to increase, after delays of 222 +/- 24 and 225 +/- 13 days, and were high at the end of the study, while prolactin values remained depressed throughout. These observations clearly show that seasonal breeding in the emu is directly controlled by changes in photoperiod. The dynamics of the hormonal responses to change of photoperiod suggest that, despite being short-day breeders, the photoregulation of breeding in emus involves mechanisms that are currently accepted for birds, rather than mechanisms that have been proposed for short-day breeding mammals. The initiation of breeding in emus is due to dissipation of photorefractoriness by short days which leads to an increase in the secretion of gonadotrophins to levels that are sufficient to support full reproductive condition. The termination of breeding, while days are still short, is due to the antigonadotrophic action of prolactin which, unusually for birds, increases while the days are still short. In conclusion, breeding activity in male emus is strongly controlled by photoperiod. Emus are short-day breeders, but the central mechanisms that regulate the secretion of reproductive hormones seem to be similar to those previously proposed for long-day breeding birds. The pattern of prolactin secretion in emus suggests an important role for this hormone in the termination of the breeding cycle.     

Photorefractoriness of immune function in male Siberian hamsters (Phodopus sungorus)

Short days induce multiple changes in reproductive and immune function in Siberian hamsters. Short-day reproductive inhibition in this species is regulated by an endogenous timing mechanism; after approximately 20 weeks in short days, neuroendocrine refractoriness to short-day patterns of melatonin develops, triggering spontaneous recrudescence of the reproductive system. It is unknown whether analogous mechanisms control immune function, or if photoperiodic changes in immune function are masked by prevailing photoperiod. In Experiment 1, 3 weeks of exposure to long days was not sufficient to induce long-day-like enhancement of in vitro lymphocyte proliferation in short-day adapted male Siberian hamsters. Experiment 2 tested the hypothesis that immunological photorefractoriness is induced by prolonged exposure to short days. Adult male hamsters were gonadectomized or sham-gonadectomized and housed in long (14 h light/day) or short (10 h light/day) photoperiods for 12, 32 or 40 weeks. Somatic and reproductive regression occurred after 12 weeks in short days, and spontaneous recrudescence was complete after 32-40 weeks in short days, indicative of somatic and reproductive photorefractoriness. In gonad-intact hamsters, 12 weeks of exposure to short days decreased the number of circulating granulocytes and increased the number of B-like lymphocytes. After 32 weeks in short days, these measures were restored to long-day values, indicative of photorefractoriness; castration eliminated these effects of photoperiod. In both intact and castrated hamsters, in vitro proliferation of splenic lymphocytes was inhibited by 12 weeks of exposure to short days. After 40 weeks in short days lymphocyte proliferation was restored to long-day values in intact hamsters, but remained suppressed in castrated hamsters. These results suggest that short-day-induced inhibition of lymphocyte function does not depend on gonadal regression, but that spontaneous recrudescence of this measure is dependent on gonadal recrudescence. In Experiment 3, in vitro treatment with melatonin enhanced basal proliferation of lymphocytes from male hamsters exposed to short days for 12 weeks, but had no effect on lymphocytes of photorefractory hamsters or long-day control hamsters. Lymphocytes of castrated hamsters were unresponsive to in vitro melatonin, suggesting that photoperiodic changes in gonadal hormone secretion may be required to activate mechanisms which permit differential responsiveness to melatonin depending on phase in the annual reproductive cycle. Together, these data indicate that, similar to the reproductive system, the immune system of male Siberian hamsters exhibits refractoriness to short days.     

Photoperiodic control of oestrous cycles in Syrian hamsters: mediation by the mediobasal hypothalamus

To assess whether the mediobasal hypothalamus (MBH) is necessary for photoperiodic control of oestrous cycles and prolactin secretion, we tested intact female Syrian hamsters (controls) and those that had sustained unilateral or bilateral lesions of the MBH. All hamsters displayed 4-day oestrous cycles postoperatively in the long-day photoperiod (14 h light/day); control females and those with unilateral MBH damage ceased to undergo oestrous cycles approximately 8 weeks after transfer to a short-day photocycle (10 h light/day), whereas 12 of 15 females with bilateral MBH lesions continued to generate 4-day oestrous cycles throughout 22 weeks in short days. Serum prolactin concentrations were either undetectable or low in all hamsters 8 or 14 weeks after the transfer to short-day lengths, but increased above long-day baseline values by week 22. We conclude that melatonin-binding sites in the MBH mediate suppression of oestrous cycles but not prolactin secretion by short-day lengths; recovery of prolactin secretion in females during prolonged exposure to short-day lengths reflects development of refractoriness to melatonin in a substrate distinct from the MBH. These findings suggest that separate neural pathways mediate photoperiodic control of gonadotropin and prolactin secretion in female hamsters.     

Neural pathway from the olfactory bulbs regulating tonic gonadotropin secretion

Removal of the olfactory bulbs of male golden hamsters results in a marked increase in tonic gonadotropin, prolactin and testosterone secretion which counteracts inhibitory effects of manipulations such as maintenance on short photoperiod, food restriction or treatment with gonadal steroids. The bulbectomy-induced increase in hormone secretion is interpreted to reflect a tonic inhibitory influence of the olfactory bulbs. This inhibition is not dependent upon chemosensory stimulation and may be mediated by olfactory bulb fibers projecting through the lateral olfactory tract to or through the olfactory tubercle. This review will relate these studies conducted on hamsters to results in other species, such as the rat, where the olfactory bulbs enhance serum gonadotropin levels.     

Photoperiod and androgens act independently to induce spinal nucleus of the bulbocavernosus neuromuscular plasticity in the Siberian hamster,Phodopus sungorus

In the Siberian hamster, Phodopus sungorus, short-day photoperiods induce the winter phenotype, which in males includes a decrease in the production of androgens and changes in physiology to inhibit reproduction. Motoneurones of the spinal nucleus of the bulbocavernosus (SNB) and their target muscles, the bulbocavernosus and the levator ani, a neuromuscular system involved in male copulation, also display seasonal plasticity in P. sungorus. It is not known whether the plasticity seen in the SNB system of gonadally intact hamsters is due to the effects of photoperiod per se, or to the photoperiod-induced changes in androgen production. To answer this question, we castrated adult male hamsters from long days and then implanted them with capsules containing either testosterone or blanks. Half of the hamsters from each hormone condition were moved into short photoperiod (8 : 16 h light/dark cycle) while the rest were maintained under long-day conditions (15 : 9 h light/dark cycle). After 15 weeks, many measures of the SNB system, such as somata size and weight of target muscles, responded only to androgen, not to photoperiod. However, there were effects of photoperiod on the neuromuscular junctions (NMJs) that were independent of androgen status. For example, the number of synaptic zones per NMJ and the area of the NMJs were significantly increased by short days and/or testosterone treatment. The two factors exerted an additive, rather than an interactive, effect on these measures. Another striated muscle, the extensor digitorum longus, which is present in both sexes and plays no specialized role in reproduction, displayed neither an effect of androgen nor of photoperiod on fibre size or NMJ structure. These results suggest that, in addition to androgenic effects on SNB plasticity, there is also an androgen-independent effect of photoperiod on the SNB neuromuscular system.     

Photoperiodic regulation of prolactin gene expression in the Syrian hamster by a pars tuberalis-derived factor

Syrian hamsters exhibit a marked seasonal variation in prolactin secretion. The aim of this study was to analyse the nature of the photoperiodic regulation of prolactin gene expression, and to define the role of melatonin and the pars tuberalis of the anterior pituitary in this process. Pituitary prolactin gene expression, restricted to the pars distalis, was increased in hamsters maintained in long daylengths (16 h : 8 h, light : dark) compared to hamsters exposed to short daylengths (8 h : 16 h, light : dark) for 8-12 weeks. Analysis of single cells by in situ hybridization showed that photoperiod had no effect on the percentage of pars distalis cells expressing prolactin mRNA, but shifted the frequency distribution of prolactin mRNA expression per cell, such that in long photoperiods a greater proportion of cells were recruited to a higher expressing population. In vitro coculture of hamster pars tuberalis fragments increased prolactin promoter-driven luciferase activity in stably transfected GH3 cells in a dose- and duration-dependent manner. Conditioned medium from hamster and ovine pars tuberalis also activated the prolactin promoter. Furthermore, basal and forskolin-stimulated conditioned medium from hamster pars tuberalis increased prolactin mRNA expression in primary cultures of pars distalis cells. Melatonin attenuated the activity of pars tuberalis-conditioned medium but had no direct effect on either prolactin mRNA expression or secretion in pars distalis cell cultures. Finally, pars tuberalis fragments from long photoperiod hamsters stimulated prolactin gene promoter activity to a greater extent than those from short photoperiod hamsters. In conclusion, this study provides the first evidence in a seasonal mammal that the synthesis of prolactin depends on photoperiodic modulation of a pars tuberalis-derived factor. Our data support further the hypothesis that seasonal modulation of prolactin gene expression depends upon a melatonin-dependent paracrine action of the pars tuberalis on pars distalis lactotrophic cells.     

The Olfactory Bulbs Tonically Inhibit Serum Gonadotropin and Prolactin Levels in Male Hamsters on Long or Short Photoperiod

Bilateral removal of the olfactory bulbs (BX) inhibits the testicular regression associated with maintenance of golden hamsters on short photoperiod (10L:14D). The present study was done to examine the reproductive endocrine changes following BX of hamsters, to test whether BX increases gonadotropin secretion by enhancing the rate of metabolism of peripheral testosterone, and to determine whether BX inhibits the response to photoperiod by blocking chemosensory signals from conspecifics. BX resulted in a marked increase in tonic serum gonadotropin levels in pre-pubertal (23 days old) and adult hamsters maintained on long photoperiod (14L:10D). Maintenance on 10L14D inhibited gonadotropin secretion in BX hamsters, but this only reduced the previously elevated levels to those of the sham group on stimulatory photoperiod, and the testes therefore remained large. BX hamsters on 10L:14D had a higher post-castration increase in serum luteinizing hormone than sham-operated hamsters. Following testosterone replacement (20 mm Silastic capsules), BX animals had lower serum testosterone and higher serum follicle- stimulating hormone levels than the sham group. BX hamsters had a shorter mean half-time for disappearance of testosterone from serum following removal of the capsule, but some animals in the sham group also metabolized testosterone rapidly. Isolation in cages receiving air filtered to remove pheromonal type molecules did not affect the rate or degree of testicular regression in response to short days. We conclude that the olfactory bulbs tonically inhibit gonadotropin release in golden hamsters on long or short photoperiod. The olfactory bulbs may facilitate the negative feedback of testosterone and may inhibit testosterone metabolism, but there were also steroid-independent effects. The influence of the olfactory bulbs on the hamsters' response to short days is apparently not related to chemosensory information from other hamsters.     

Androgenic Regulation of Expression of β-Tubulin Messenger Ribonucleic Acid in Motoneurons of the Spinal Nucleus of the Bulbocavernosus

Expression of β-tubulin mRNA was examined in androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using cDNA encoding mouse β-tubulin. Hybridizable β-tubulin mRNA was localized in the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression level of β-tubulin mRNA in the SNB motoneurons, whereas the change was prevented by testosterone treatment. On the contrary, castration or testosterone treatment did not induce any changes in the expression level of β-tubulin mRNA in the androgen-insensitive motoneurons of the retrodorsolateral nucleus. These results suggest that androgen regulates the expression of β-tubulin gene in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally-induced neuronal plasticity in the SNB motoneurons.     

Absence of Photoperiodic Modulation of Gonadotrophin Secretion in HPD Rams Following Chronic Pulsatile Infusion of GnRH

Hypothalamo-pituitary disconnected Soay rams (HPD rams) were treated chronically with a pulsatile infusion of GnRH (250  ng/2  h) for 10 weeks to reactivate the pituitary-gonadal axis under long days (16L:8D; LD group n=8) and short days (8L:16D, SD group n=8). The aim was to establish whether photoperiod modulates the secretion of gonadotophins (presumably through the daily melatonin signal) by an action at the level of the pituitary gland. The treatment of the HPD rams with GnRH induced a marked, long-term increase in the peripheral blood concentrations of FSH, LH and testosterone, growth of the testes and reappearance of the androgen-dependant sexual skin colouration. There were parallel changes in the short-term, pulsatile fluctuations in the blood concentrations of LH and FSH, the pituitary responsiveness to GnRH and the testicular responsiveness to a standardized injection of oLH, as observed previously in normal intact rams during reactivation of the reproductive axis induced by a switch from long to short days. There were no significant differences in any of the reproductive parameters between the LD and SD HPD groups, although the blood concentrations of prolactin were significantly higher in the LD group, and GnRH provoked a chronic increase in the blood concentrations of prolactin, particularly in the SD group. Overall, the results support the conclusion that (i) melatonin does not act within the pituitary gland to mediate effects of photoperiod on GnRH-induced gonadotrophin secretion; and (ii) changes in prolactin secretion which result from the local action of melatonin in the pituitary gland do not affect gonadotrophin secretion in the ram.     

Annual Variations of in vitro GNRH Release by Hypothalamic Explants in Intact and Castrated Male Mink: Relations with LH, FSH and Testosterone Circulating Serum Levels

In mink, a short-day breeder, testis growth begins in autumn (November), reaches a maximum in February, before matings occur, and decreases from March to very low volumes during spring and summer. To study the effects of season and testosterone feedback on gonadotrophin and GnRH secretion, the annual variations of LH, FSH, testosterone and GnRH were studied in intact and castrated mink. As portal blood sampling raised serious difficulties, an in vitro static incubation system was used for studying GnRH variations. In intact mink, serum LH concentrations did not vary significantly throughout the year, whereas FSH concentrations increased significantly between September and November then decreased to a minimum in January. Testosterone values rose significantly from November to a maximum from January to March, decreased very rapidly thereafter. Castration in November resulted in a significant increase in LH and FSH concentrations which remained higher than the values measured in intact males throughout the year. In long-term castrated mink, FSH concentrations did not fluctuate during the year, whereas LH concentrations showed an annual variation, with high values in April and August. For the study of in vitro GnRH liberation, medio-basal hypothalamic explants were incubated in Krebs-Ringer phosphate buffer for 3 periods of 15 min, and stimulated with copper complexed equimolarly with histidine (Cu/His, 200 pM) and prostaglandin E₂ (PGE₂, 10 μM). After Cu/His, the release of GnRH was 1 to 4 fold the basal release; after PGE₂, the increase was 4–7 fold the basal release. The basal release of GnRH increased significantly between September and October to reach a maximum in November, decreased significantly in December to a minimum in February then increased progressively from May. The release of GnRH stimulated by Cu/His and PGE₂ showed the same seasonal variation as the basal release. Castration 8 days before the sacrifice did not alter the release of GnRH, except in December: the release stimulated by PGE₂ was then higher in intact than in castrated mink. Taken together, these results indicate that, with an in vitro static incubation system, it is possible to study the annual variations of GnRH release and to correlate these variations with those of serum gonadotrophin and testosterone concentrations. The synthesis and release of GnRH increased slightly from May, under long days, then more rapidly from September, resulting in an increased secretion of FSH in October, responsible for testis recrudescence. The annual pattern of basal and stimulated GnRH release was similar in intact and castrated mink, suggesting a direct effect of the season on the hypothalamus, rather than a negative feedback effect of the testis; however, testosterone seemed to feedback mainly at the pituitary level.     

Photoperiod Affects the Ability of Testosterone to Alter Proopiomelanocortin mRNA, but not Luteinizing Hormone-Releasing Hormone mRNA, Levels in Male Sheep

This study tested the hypothesis that photoperiod affects the ability of testosterone to reduce proopiomelanocortin (POMC) mRNA levels in the arcuate nucleus and luteinizing hormone-releasing hormone (LHRH) mRNA levels in both the preoptic area (POA) or medial basal hypothalamus (MBH). Twenty castrated male sheep were assigned to one of four treatment groups (i): short days (SD;
n=5) (ii), short days with testosterone (SD+T; n=5) (iii), long days (LD; n=5), or (iii) long days with testosterone (LD+T; n=5). Blood samples were collected twice weekly for the last 3 weeks of photoperiod treatment and assessed for LH to validate the response to photoperiod. After evaluating LH levels, one animal each from the LD+T and SD+T groups was excluded from the analyses. Mean concentrations of LH were lower (P<0.01) in the LD+T group than in the other treatment groups, which did not differ (P>0.10) from each other. Neither POA nor MBH LHRH mRNA levels were affected (P>0.10) by treatment. Conversely, POMC mRNA levels were suppressed (P<0.01) in the LD+T males compared with the other treatment groups which did not differ (P>0.10) from each other. These observations suggest that photoperiod specific, testosterone-induced alterations in LHRH mRNA levels are not a mechanism whereby testosterone suppresses LHRH release, and that increased beta-endorphin synthesis and release do not mediate testosterone-induced seasonal suppression of LHRH release.     

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.     

Photoperiodic regulation of prolactin release in male hamsters with hypothalamic knife cuts

Horizontal knife cuts placed dorsal to the paraventricular nucleus (PVN) of the hypothalamus prevent testicular regression in hamsters kept in short days. We examined the effects of these cuts on the photoperiodic modulation of the postcastration rise in gonadotropins, as well as on the release of prolactin in castrated and gonadally intact animals. The cuts blocked the inhibitory effects of short daylengths on the postcastration rise in circulating levels of gonadotropins. However, the cuts did not prevent the reduction in prolactin levels induced by short daylengths in castrated and gonadally intact animals. We conclude that dorsal connections of the PVN are not required for transduction of photoperiodic information used to regulate prolactin release. The knife cuts may remove tonic inhibitory influences on the release of follicle-stimulating hormone and luteinizing hormone, and thus produce elevated gonadotropin levels that mask the effects of nonstimulatory photoperiods on testicular size.     

Short day lengths enhance skin immune responses in gonadectomised Siberian hamsters

In Siberian hamsters and other photoperiodic rodents, exposure to short photoperiods simultaneously inhibits gonadal hormone secretion and enhances some measures of immune function. The present study tested whether gonadal hormones mediate the effects of short days on skin immune function (delayed-type hypersensitivity reactions) in male Siberian hamsters. The magnitude of delayed-type hypersensitivity reactions was greater in hamsters exposed to short days relative to those in long days. Comparable effects of photoperiod were obtained in castrated hamsters bearing empty or testosterone-filled implants. The data suggest that contemporary gonadal hormone secretion is neither necessary, nor sufficient to mediate the effects of short photoperiods on skin immune function.     

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.     

'Hamster-like' Cycles in Testicular Size in the Absence of Gonadotrophin Secretion in HPD Rams Exposed to Long-term Changes in Photoperiod and Treatment with Melatonin

Long term changes in the secretion of FSH, LH and testosterone, and the size of the testis were measured in groups of hypothalamo-pituitary disconnected Soay rams (HPD rams, n=8) and control Soay rams (HPD-sham operated and unoperated, total n=8) while exposed to an artificial lighting regimen of alternating 16-weekly periods of long days (16L:8D) and short days (8L:16D), and when treated with a constant-release implant of melatonin given under long days (total study: 136 weeks). Short term provocation tests using NMDA (glutamate receptor agonist), GnRH and LH were used to assess functionality of the hypothalamus, pituitary gland and testis, respectively. Control rams expressed normal photoperiod-induced cycles in the reproductive axis. Blood concentrations of FSH, LH and testosterone were significantly increased under short days, and decreased under long days with parallel changes in testicular diameter. Treatment with implants of melatonin under long days mimicked the effect of short days and induced rapid reactivation of the reproductive axis. In the HPD rams the blood concentrations of FSH, LH and testosterone declined immediately after the HPD surgery and values remained close to the lower limit of detection of the radioimmunoassays throughout the experiment. LH pulses were absent in the HPD rams and NMDA failed to induce LH secretion consistent with functional disconnection of the pituitary gland from the hypothalamus. The testes regressed to a significantly smaller size in the HPD rams compared with controls even at the nadir of the sexual cycle (testis diameter: 30.2±0.7 vs 41.3±0.8 mm, HPD vs control rams, respectively). A low amplitude cycle in testicular diameter (peak to nadir: 5.0±0.7 mm) persisted in the HPD rams with a temporal pattern opposite to the controls (growth under long days instead of short days; ‘hamster-like’). In the HPD rams, the treatment with melatonin blocked the long day-associated increase in testicular size, without effects on FSH, LH and testosterone secretion, pituitary responsiveness to GnRH (LH increment) or testicular responsiveness to LH (testosterone increment). This was in contrast to the cyclical changes in all parameters induced by melatonin in the control rams. At post-mortem, the reproductive tract in HPD rams was markedly regressed compared with the controls. The efficiency of spermatogenesis was reduced with few germ cells maturing beyond primary spermatocytes. Immunocytochemical staining, however, revealed the maintenance of androgen receptor expression in Sertoli cells, peritubular cells and Leydig cells, and steroid activity as measured by 17α-hydroxylase expression in Leydig cells. Overall, the absence of photoperiod-induced changes in gonadotrophin secretion in the HPD rams illustrates the dependence on regulation by the hypothalamus, presumably through the secretion of GnRH. The residual cycle in the size of the testes in the HPD rams was closely correlated with the photoperiod-induced changes in prolactin secretion which persisted in these animals (summary of previous published data included). The combined results support the view that melatonin acts in the hypothalamus to mediate effects of photoperiod on gonadotrophin secretion and in the pituitary gland to mediate effects on prolactin secretion (dual site hypothesis), and that FSH, LH and prolactin act synergistically to regulate the long-term cycle in testicular activity in the ram.