Seasonal Siberian hamsters lose fat reserves, decrease body weight and leptin concentrations, and suppress reproduction on short-day photoperiod (SD). Chronic leptin infusion at physiological doses caused body weight and fat loss in SD animals but was ineffective in long-day (LD) hamsters. Using ovariectomized estrogen-treated females, we tested the hypothesis that responsiveness to leptin is regulated by photoperiod. On SD, hypothalamic neuropeptide Y, agouti-related peptide, and cocaine- and amphetamine-regulated transcript gene expression in the arcuate nucleus did not exhibit significant changes, and despite SD-induced fat loss, the catabolic peptide proopiomelanocortin was down-regulated. Food restriction of LD-housed animals caused significant reduction of fat reserves and serum leptin concentrations to SD levels, suppressed serum gonadotropins, and induced increased anabolic (neuropeptide Y, agouti-related peptide) and decreased catabolic (proopiomelanocortin, cocaine- and amphetamine-regulated transcript) gene expression in the arcuate nucleus. Leptin infusion in food-restricted animals had no effect on fat reserves or gonadotropins and did not modulate neuropeptide gene expression. Also, leptin treatment did not blunt the refeeding responses or weight and fat gain in LD-housed food-restricted animals. In conclusion, our results strongly suggest that hypothalamic responses to leptin are regulated primarily by photoperiod, rather than seasonal changes in fat reserves, sex steroids, or leptin concentrations.

Oxytocin secretion is inhibited by opioids, and oxytocin is important in parturition. The effects on parturition of morphine, a relatively selective mu-opioid receptor agonist, were studied in the rat. Morphine or vehicle with or without the opiate antagonist naloxone were administered immediately after the birth of the second pup and the subsequent course of parturition was recorded in a total of 80 rats. Both s.c. morphine (10 mg/kg) and intracerebroventricular (i.c.v.) morphine (18 micrograms through a previously implanted cannula) interrupted parturition, delaying the birth of the sixth pup after treatment to 187.3 +/- 35.9 (S.E.M.) min and 195.4 +/- 19.5 min respectively, compared with 46.4 +/- 3.7 and 66.1 +/- 17.5 min after vehicle alone. The dose of morphine given i.c.v. had no effect when given s.c. Naloxone given concurrently prevented the effects of morphine. Eventually the rate of parturition in the morphine-treated groups recovered. Perinatal pup mortality rate was not increased when morphine was given to the mothers, but it did inhibit the expression of normal intrapartum maternal behaviour. Pup mortality was increased 48 h post partum by morphine given during parturition, and it reduced the proportion of rats with normal maternal behaviour 24 h post partum. Morphine did not affect spontaneous or oxytocin-stimulated contractile activity of the parturient uterus in vitro. The concentration of oxytocin in trunk blood plasma was decreased 40 min after i.c.v. morphine (24.3 +/- 3.9 vs 39.3 +/- 6.5 pmol/l in controls), as was vasopressin (7.2 +/- 1.5 vs 19.7 +/- 4.5 pmol/l in controls). Intravenous infusion of oxytocin (2-5 mU/min for 144.3 +/- 8.2 min; total infused 448.5 +/- 61.9 mU) after i.c.v. morphine re-started parturition; all pups were born to these rats (mean time to pup 6, 110.3 +/- 12.7 min) before the i.v. vehicle-infused rats given i.c.v. morphine re-started (mean time to pup 6, 406.3 +/- 125.2 min). It is concluded that morphine given during parturition acts centrally through opioid receptors to inhibit oxytocin secretion, and impairs the expression of maternal behaviour. Reversal of the effects of morphine on parturition by i.v. oxytocin demonstrates the important role of oxytocin in fetus ejection and expulsion.

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.

Most mammals use changing annual day-length cycles to regulate pineal melatonin secretion and thereby drive many physiological rhythms including reproduction, metabolism, immune function, and pelage. Prolonged exposure to short winter day lengths results in refractoriness, a spontaneous reversion to long-day physiological status. Despite its critical role in the timing of seasonal rhythms, refractoriness remains poorly understood. The aim of this study was therefore to describe cellular and molecular mechanisms driving the seasonal secretion of a key hormone, prolactin, in refractory Syrian hamsters. We used recently developed single cell hybridization and reporter assays to show that this process is initiated by timed reactivation of endocrine signaling from the pars tuberalis (PT) region of the pituitary gland, a well-defined melatonin target site, causing renewed activation of prolactin gene expression. This timed signaling is independent of per1 clock gene expression in the suprachiasmatic nuclei and PT and of melatonin secretion, which continue to track day length. Within the PT, there is also a continued short day-like profile of ICER expression, suggesting that the change in hormone secretion is independent of cAMP signaling. Our data thus identify the PT as a key anatomical structure involved in endogenous seasonal timing mechanisms, which breaks from prevailing day length-induced gene expression.

In many seasonally breeding rodents, reproduction and metabolism are activated by long summer days (LD) and inhibited by short winter days (SD). After several months of SD, animals become refractory to this inhibitory photoperiod and spontaneously revert to LD-like physiology. The suprachiasmatic nuclei (SCN) house the primary circadian oscillator in mammals. Seasonal changes in photic input to this structure control many annual physiological rhythms via SCN-regulated pineal melatonin secretion, which provides an internal endocrine signal representing photoperiod. We compared LD- and SD-housed animals and show that the waveform of SCN expression for three circadian clock genes (Per1, Per2, and Cry2) is modified by photoperiod. In SD-refractory (SD-R) animals, SCN and melatonin rhythms remain locked to SD, reflecting ambient photoperiod, despite LD-like physiology. In peripheral oscillators, Per1 and Dbp rhythms are also modified by photoperiod but, in contrast to the SCN, revert to LD-like, high-amplitude rhythms in SD-R animals. Our data suggest that circadian oscillators in peripheral organs participate in photoperiodic time measurement in seasonal mammals; however, circadian oscillators operate differently in the SCN. The clear dissociation between SCN and peripheral oscillators in refractory animals implicates intermediate factor(s), not directly driven by the SCN or melatonin, in entrainment of peripheral clocks.

Real-time imaging of the GH gene promoter linked to luciferase in living pituitary cells has revealed surprising heterogeneity and variety of dynamic patterns of gene expression. Cells treated with either forskolin or thyroid hormone generated a consistent and characteristic temporal response from cell populations, but detailed analysis of individual cells revealed different patterns. Approximately 25-26% of cells displayed no response, 25-33% of cells exhibited a sustained progressive rise in luciferase activity, and 41-50% showed a transient phasic, or oscillatory response, after given stimuli. In cells treated consecutively with the two stimuli, the population response to the second stimulus was augmented. Single-cell analysis revealed that this was partly due to an increased number of cells responding, but also that the prevalence of response patterns changed: cells that responded to an initial stimulus were more likely to respond subsequently in a progressive sustained manner. In conclusion, these studies have indicated that GH promoter activity in individual living pituitary cells is unstable and possibly stochastic, with dynamic variations from hour to hour. The prevalence of different temporal patterns of response to hormonal stimulation among a population of cells is altered by the endocrine history of those cells.

Although analysis of luciferase activity using luminescence imaging has provided new insights into the dynamic regulation of gene expression in living tissues, studies in vitro have relied on stably transfected clonal cell lines, limiting the choice of cell type and species, or DNA microinjection, which is arduous and highly selective. We report here the first use of a recombinant adenovirus in which the firefly luciferase reporter gene was regulated by the prolactin gene promoter, to study temporal dynamics of promoter activity. This vector was used to infect the pituitary GH3 cell line, and also primary cultures of Syrian hamster pituitary cells. We show that adenovirally transduced cells retained normal regulation of the promoterreporter transgene by appropriate signals. Furthermore, microscopic imaging studies indicated that both clonal and primary pituitary cells were transduced efficiently, giving readily detectable luminescence signals in real-time over long periods. Finally, analysis of single-cell expression patterns indicated that prolactin promoter activity was highly dynamic with pulses in gene expression, revealing that the transcriptional instability seen in clonal cells is a feature of normal pituitary cells. Adenoviral vectors offer a valuable tool for studies of gene regulation where conventional transgenesis and clonal cell lines are not available.

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 de®ne 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 GH 3 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 ®rst 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.Photoperiod has a signi®cant effect on the secretion of prolactin by the anterior pituitary in seasonal mammals (1, 2. ). In photoperiodic species, such as hamsters, prolactin secretion is signi®cantly suppressed by short daylengths (SD) (3±5. ) and stimulated by long daylengths (LD). These changes are accompanied by signi®cant alterations in pituitary gene expression (6. ). In vitro studies in hamsters have demonstrated that prolactin release from pituitary fragments is also greatly reduced in short photoperiods (7±9. ). The pineal gland and the hormone melatonin provide a crucial endocrine transducer for photoperiod change in seasonal mammals (10. ), and a large number of studies have demonstrated that anterior pituitary hormone secretion is critically sensitive to melatonin signal duration (11. ). In pinealectomized hamsters, imposition of a daily melatonin signal mimicking short-day durations results in profound suppression of prolactin secretion (12, 13. ).