Real-time imaging of gene promoter activity using an adenoviral reporter construct demonstrates transcriptional dynamics in normal anterior pituitary 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 promoter-reporter 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.

Dynamic patterns of growth hormone gene transcription in individual living pituitary cells.

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.

Temporal organization of feeding in Syrian hamsters with a genetically altered circadian period.

The variation in spontaneous meal patterning was studied in three genotypes (tau +/+, tau +/- and tau -/-) of the Syrian hamster with an altered circadian period. Feeding activity was monitored continuously in 13 individuals from each genotype in constant dim light conditions. All three genotypes had on average six feeding episodes during the circadian cycle (about 20h in homozygous tau mutants and 22h in heterozygotes compared with 24h in wild-type hamsters). Thus, homozygous tau mutant hamsters had significantly more feeding episodes per 24h than wild types, and heterozygotes were intermediate. The average duration of feeding bouts (FBs) was indistinguishable (around 30 minutes) among the three genotypes, whereas the intermeal (IM) intervals were significantly shorter for homozygote tau mutant hamsters (99 minutes), intermediate for heterozygotes (116 minutes), and the longest for wild-type hamsters (148 minutes). Thus, the meal-to-meal duration was on average 25% shorter in homozygous tau mutants (16% in heterozygous) than in wild-type hamsters. The reduction of the circadian period has a pronounced effect on short-term feeding rhythms and meal frequency in hamsters carrying the tau mutation.

Persistent synchronized oscillations in prolactin gene promoter activity in living pituitary cells.

PRL gene expression in the anterior pituitary gland responds rapidly to different hormonal signals. We have investigated the long-term timing of transcriptional activation from the PRL, GH, and cytomegalovirus promoters in response to different stimulus duration, using real-time imaging of luciferase expression in living stably transfected GH3 cells. Long-term stimulation of serum-starved cells with 50% serum induced a homogeneous rise in PRL promoter activity, with subsequent heterogeneous fluctuations in luciferase activity in individual cells. When cells were subjected to a 2-h pulse of 50% serum, followed by serum-free medium, there were long-term (approximately 50 h) synchronized, homogeneous oscillations in PRL promoter activity. This response was PRL-specific, because in GH3 cells expressing luciferase from the GH or cytomegalovirus promoters, a serum pulse elicited no oscillations in luciferase expression after an initial transient response to serum. The PRL promoter may therefore be a template for an unstable transcription complex subject to stochastic regulation, allowing an oscillatory transcriptional response to physiological signals. This suggests that precise timing and coordination of cell responses to different signal-duration may represent a novel mechanism for coordinating long-term dynamic changes in transcription in cell populations.

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.

Leptin acts on metabolism in a photoperiod-dependent manner, but has no effect on reproductive function in the seasonally breeding Siberian hamster (Phodopus sungorus).

Leptin may play a role in appetite regulation and metabolism, but its reproductive role is less clear. In photoperiodic Siberian hamsters, seasonal changes in fatness, leptin gene expression, and metabolism occur synchronously with activation or suppression of reproduction, analogous to puberty. Here, we test the hypothesis that seasonal changes in leptin secretion mediate the photoperiodic regulation of reproduction. Mature male and ovariectomized estrogen-treated female Siberian hamsters were kept in long (LD; 16 h of light, 8 h of darkness) or short days (SD; 8 h of light, 16 h of darkness) for 8 weeks, and recombinant murine leptin (15 microg/day) was infused for 2 weeks via osmotic minipumps. SD hamsters exhibited significant weight and fat losses, reduced serum leptin and food intake, and suppressed pituitary LH concentration. Leptin did not suppress food intake over the 2-week treatment on either photoperiod, but significantly reduced fat reserves in SD hamsters. Leptin had no significant effect on pituitary LH concentrations in either sex or photoperiod or on testicular size and testosterone concentrations in males. These results suggest hamsters are more responsive to leptin on SD than on LD and that effects on food intake and fat loss can be dissociated in this species. Our data suggest that leptin does not mediate photoperiodic reproductive changes.

Postnatal growth rate and gonadal development in circadian tau mutant hamsters reared in constant dim red light.

The role of the circadian clock in the reproductive development of Syrian hamsters (Mesocricetus auratus was examined in wild type and circadian tau mutant hamsters reared from birth to 26 weeks of age under constant dim red light. Testis diameter and body weights were determined at weekly intervals in male hamsters from 4 weeks of age. In both genotypes, testicular development, subsequent regression and recrudescence exhibited a similar time course. The age at which animals displayed reproductive photosensitivity, as exhibited by testicular regression, was unrelated to circadian genotype (mean +/- SEM: 54 +/- 3 days for wild type and 59 +/- 5 days for tau mutants). In contrast, our studies revealed a significant impact of the mutation on somatic growth, such that tau mutants weighed 18% less than wild types at the end of the experiment. Our study reveals that the juvenile onset of reproductive photoperiodism in Syrian hamsters is not timed by the circadian system.

Free running circadian rhythms of melatonin, luteinizing hormone, and cortisol in Syrian hamsters bearing the circadian tau mutation.

The tau mutation of Syrian hamsters induces a robust reduction in the period of circadian activity rhythms, from 24 h (wild-type; tau++) to 22 h (heterozygote; tauS+) and 20 h (homozygous mutant, tauSS). Here, we examine the effect of this mutation on circadian rhythms of LH, melatonin, and cortisol in ovariectomized hamsters. Free running circadian rhythms were observed in all three hormones. In each genotype, endocrine rhythms were synchronized with concurrently assessed activity rhythms, suggesting a shared period around 20 h in tauSS, 22 h in tausS+, and 24 h in tau++. Phasing with respect to the activity rhythm was generally similar in tau++ and mutant genotypes. However, melatonin concentrations rose significantly earlier in tauSS than in tau++ animals. Explanted pineals from both genotypes exhibited a similar time course of response to norepinephrine administration, suggesting that the phase advance of melatonin production observed in tauSS in vivo is not a direct effect of the tau mutation within the pinealocyte. The demonstration of reduced period endocrine rhythms in the mutant genotypes extends previous behavioral studies and, together with recent work on rhythmicity in the isolated retina, suggests an ubiquitous influence of the tau mutation on the processes of circadian rhythm generation in this species.

Expression of vasoactive intestinal peptide mRNA in the suprachiasmatic nuclei of the circadian tau mutant hamster.

The tau mutation of the Syrian hamster is one of only two mutations currently known to affect the circadian system in mammals. In order to investigate the molecular mechanisms associated with this mutation, we have compared the level of expression of the mRNA for vasoactive intestinal peptide (VIP) in the suprachiasmatic nuclei (SCN) and cerebral cortex of homozygous tau mutant and wild-type animals using in situ hybridization histochemistry. We observed no significant circadian variation in this message within the SCN, cingulate cortex or parietal cortex of either genotype. Comparison between genotypes indicated a significantly higher signal for VIP mRNA in the SCN of tau animals. This phenotypic effect within the SCN raises the possibility that some of the circadian consequences of the tau mutation are associated with an increased expression of the VIP gene.

The tau mutation in the Syrian hamster alters the photoperiodic responsiveness of the gonadal axis to melatonin signal frequency.

This study investigated the role of the circadian timing system (CTS) in photoperiodic time measurement by examining the response of the tau mutant hamster to programmed infusions of melatonin. The mutation is a single Mendelian gene defect which accelerates circadian period from 24 h in the wild-type (WT) to 20 h in the homozygote. If the CTS does not contribute to the photoperiodic interpretation of the melatonin signal, then the tau mutation would not influence photoperiodic responses of pinealectomised (PX) animals to systemic infusions of melatonin (10 h) once every 20, 24 or 25 h, mimics short-daylengths and causes gonadal involution. More ( < 18 h) or less ( > 25 h) frequent signals are ineffective. In this study, taus which received melatonin (10 h) once every 16 or 20 h exhibited significant gonadal atrophy relative to saline controls, whereas infusions of melatonin every 24 or 28 h were ineffective. Serum concentrations of LH and PRL were also significantly reduced in both the 16 and 20 h, but not 24 and 28 h groups. The tau mutant hamster may therefore respond to a different and higher ranger of melatonin signal frequencies than those reported for WTs. The 4 h shift in the frequency-response function correlates with the altered circadian period and suggests that the CTS contributes to the photoperiodic interpretation of a series of melatonin signals.

A mutation of the circadian timing system (tau gene) in the seasonally breeding Syrian hamster alters the reproductive response to photoperiod change.

The tau mutation is a semi-dominant autosomal mutation which, in homozygotes, accelerates the period of the circadian activity cycle by approximately 4 h. In mammals, the circadian system contributes to seasonal photoperiodic time measurement by generating a repeated daily melatonin signal during the hours of darkness. Our earlier studies suggest an altered response to the melatonin signal in tau mutants. This study investigated whether tau and wild-type hamsters exhibit a differential response to photoperiod change. Reproductively active animals were maintained on stimulatory photoperiods of 16 h light (16L) per 24 h (wild-type) or 12L per 20 h (tau) before being exposed to an increase in night-length to 9, 10, 11, 12 or 14 h for 84 cycles. Wild-types exhibited testicular atrophy at 13L:11Dark (13L:11D), with full regression at photoperiods of 12L:12D. Taus exhibited complete regression at photoschedules comprising 10 h darkness or more per 20-h cycle. Plasma prolactin concentrations were decreased following exposure to at least 9 and 10 h darkness in taus and wild-types, respectively. Thus, the tau genotype may exhibit a different critical night-length with respect to both the gonadal and prolactin axes, of approximately 1-2 h shorter than wild-type genotypes. These data support the hypothesis that the circadian tau mutation has altered the basis of photoperiodic time measurement, perhaps by altering the generation and/or interpretation of the melatonin signal.

Gonadal responses of the male tau mutant Syrian hamster to short-day-like programmed infusions of melatonin.

Changes in the duration of the nocturnal peak of the circadian rhythm of melatonin secretion by the pineal gland mediate pohotoperiodic control of reproductive cycles in mammals. The present study examined whether the tau mutation of the circadian clock of the Syrian hamster altered the ability of the animals to exhibit photoperiodic, gonadal responses to timed infusions of exogenous melatonin. Pinealectomized tau mutant and wild-type hamsters received s.c. infusions of vehicle or melatonin solution (50 ng/h) for either 8 h or 6.67 h (i.e., 83% of 8 h, a reduction by the same proportion as circadian period is shortened by the mutation, 20 h vs. 24 h) once every 20 h for 6 wk. Testicular weight and serum levels of LH and prolactin were measured to assess the effects of the infusions on the reproductive axis. Both genotypes exhibited significant gonadal regression to the 8 h and 6.67 h melatonin signals, versus saline controls, although in taus the magnitude of the response to 6.67 h melatonin was significantly greater and more consistent than in wild-types. Compared to saline-infused hamsters, animals that received infusions of melatonin of either duration showed significantly reduced serum levels of LH and prolactin. There was no significant difference between the two genotypes in the observed endocrine responses to melatonin infusions. These preliminary data demonstrate that tau mutant hamsters can respond to programmed systemic infusions of melatonin in a manner comparable to that observed in wild-type hamsters, and therefore, that the tau mutation of the circadian clock has not impaired the ability of the hamster to measure photoperiodic time, as represented by melatonin.

Kallidin stimulates prolactin secretion from individual rat anterior pituitary cells.

Kinins are localized within the adenohypophysis where they have been shown to stimulate the release of pituitary hormones. In the present study we have investigated the effect of [Lys]-bradykinin (kallidin) on prolactin secretion at the single cell level from cultured male rat anterior pituitary cells. This was assessed by use of a reverse haemolytic plaque assay which permits quantitative evaluation of the proportion of all pituitary cells which are secreting prolactin, and the amount of prolactin secreted per lactotroph (plaque area). The rate of plaque development was used as an index of the rate of hormone secretion in time-course studies. Kallidin induced a dose-dependent increase in both the percentage of plaque-forming cells and the median plaque area during the first 2 and 3 h of incubation respectively. The threshold concentration of kallidin was 10 nmol/l. After 4 h of kallidin stimulation there was no difference between treated and control monolayers with respect to median plaque area and the total secretion index. Although recruitment of additional cells into the secretory pool cannot be excluded, this seems unlikely since at 3 and 4 h little or no difference was observed in the number of plaque-forming cells. The data suggest that initially kallidin accelerated the rate of prolactin secretion primarily by inducing an increase in the number of cells secreting prolactin, and subsequently by increasing the amount of hormone secreted per lactotroph. The results presented here are consistent with the proposed role of the kallikrein-kinin system in the paracrine or autocrine control of prolactin release from the pituitary gland.

Interruption of parturition in rats by morphine: a result of inhibition of oxytocin secretion.

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.