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.

Photoperiod-dependent regulation of inhibin in Siberian hamsters: I. Ovarian inhibin production and secretion.

Follicle-stimulating hormone (FSH) stimulates ovarian follicle development and the production of protein hormones including inhibin A and inhibin B. The inhibins are dimeric proteins (alpha-beta(A) or alpha-beta(B)) secreted by growing follicles that suppress FSH in a classical endocrine negative feedback loop. Siberian hamsters, Phodopus sungorus, exhibit seasonal variation in FSH levels. Given the role of inhibin in FSH regulation, we hypothesized that ovarian inhibin expression differs between animals reared in long (16 h light:8 h darkness) and short (6 h light:18 h darkness) photoperiods. To examine inhibin expression in animals housed under long or short photoperiods, hamster inhibin alpha-, beta(A)-, and beta(B)-subunits were cloned and used to detect and localize inhibin subunit mRNA in developing follicles. Ovarian inhibin alpha-subunit mRNA levels were significantly higher in long day-exposed (LD) than in short day-exposed (SD) hamsters. In addition, dimeric inhibin, as well as inhibin alpha-, beta(A)-, and beta(B)-subunit protein levels were higher in the LD than in the SD hamster ovaries.

Photoperiod-dependent regulation of inhibin in Siberian hamsters: II. Regulation of inhibin production and secretion by pregnant mare serum gonadotropin.

Inhibin production differs in ovaries of Siberian hamsters (Phodopus sungorus) exposed to long days (LD) or short days (SD). We believe that seasonal differences in serum follicle-stimulating hormone contribute to this difference. However, given the profound photoperiodic differences in follicle maturation, serum gonadotropins alone may not account for all of the observed differences in inhibin processing. To test this hypothesis, we challenged LD and SD female hamsters with exogenous gonadotropins. While both groups responded with increased inhibin expression, the effects were muted in ovaries of SD females and there was no evidence of ovulation in these animals. These data indicate that the ovaries of SD females are not immediately equipped to respond to gonadotropin stimulation. More generally, these data suggest that photoperiodic history affects ovarian inhibin production and secretion in response to gonadotropins.

Aging, reproduction, and the melatonin rhythm in the Siberian hamster.

The present study tested the hypothesis that responsiveness to melatonin, the presence of the melatonin rhythm in circulation, and parameters of the GnRH neuron system are sustained across the aging continuum in Siberian hamsters. Afternoon melatonin injections induced testicular atrophy in 42% of aged males compared with 100% of adult males. The proportion of aged males failing to respond to the melatonin injections was similar to the proportion that failed to undergo testicular regression upon exposure to short days. Exposure to short days induced testicular atrophy in juvenile and adult hamsters; however, regression was incomplete or absent in 43% of aged males. The nocturnal rise in melatonin was similar with regard to duration and peak amplitude, and appropriate with respect to photoperiod in 25-day-old juveniles, adult (5 months), and aged (17 months) hamsters. Neither advanced age nor timed melatonin treatments affected GnRH neuron numbers or distribution. Fertility was maintained in aged and adult males to a comparable extent with respect to latency to first litter and number of pups per litter; reproductive success was dramatically reduced in aged compared with adult females. Because melatonin rhythms accurately reflect day length information throughout the continuum from puberty to advanced age, the present evidence suggests that limitations in testis regression in response to short days or exogenous melatonin in a subset of aged males result from a reduced ability to respond to melatonin. In the wild, failure to undergo testicular regression in the presence of shortening day lengths may extend the breeding season of aged males.

Twenty-four-hour profiles of serum leptin in siberian and golden hamsters: photoperiodic and diurnal variations.

Serum leptin concentrations were obtained from male Siberian hamsters (Phodopus sungorus) and golden hamsters (a.k.a. Syrian, Mesocricetus auratus) housed on long [light:dark (LD) 16:8] and short (LD 6:18) photoperiods for 10-11 weeks. Blood samples were collected at 45-min intervals for 24 h from individual animals using an in-dwelling atrial catheter. In Siberian hamsters, exposure to short photoperiods as compared to long photoperiods reduced body weight (32.5 +/- 1.5 vs 47.7 +/- 1.1 g) and leptin (24-h mean: 5.3 +/- 0.4 ng/ml vs 18.6 +/- 2.1 ng/ml). Although photoperiod influenced the temporal distribution of leptin in golden hamsters, the main effect of photoperiod on leptin levels in golden hamsters did not reach significance (24-h mean: 7.1 +/- 1.0 ng/ml vs 5.1 +/- 0.8 ng/ml.). Body weights of golden hamsters did not vary significantly following exposure to short photoperiod for 11 weeks (178.3 +/- 3.6 g in LD 6:18 vs 177.8 +/- 7.3 g in LD 16:8). There was no nocturnal increase in serum leptin in either species. Marked interindividual differences were apparent in individual leptin profiles. Periodogram analysis revealed that only a few animals exhibited 24-h periodicities; the presence of a significant 24-h periodicity was more common in hamsters exposed to short days. Photoperiod-associated differences in the 24-hour profile of leptin secretion may be the result of photoperiod-associated changes in feeding behavior or metabolism. A full understanding of the regulation of leptin secretion in multiple time domains may enhance our understanding of the function of leptin.

Differential regulation of pituitary gonadotropin subunit messenger ribonucleic acid levels in photostimulated Siberian hamsters.

FSH levels begin to rise 3-5 days after male Siberian hamsters are transferred from inhibitory short photoperiods to stimulatory long photoperiods. In contrast, LH levels do not increase for several weeks. This differential pattern of FSH and LH secretion represents one of the most profound in vivo examples of differential regulation of the gonadotropins. The present study was undertaken to characterize the molecular mechanisms controlling differential FSH and LH synthesis and secretion in photostimulated Siberian hamsters. First, we cloned species-specific cDNAs for the three gonadotropin subunits: the common alpha subunit and the unique FSHbeta and LHbeta subunits. All three subunits share high nucleotide and predicted amino acid sequence identity with the orthologous cDNAs from rats. We then used these new molecular probes to examine the gonadotropin subunit mRNA levels from pituitaries of short-day male hamsters transferred to long days for 2, 5, 7, 10, 15, or 20 days. Short-day (SD) and long-day (LD) controls remained in short and long days, respectively, from the time of weaning. We measured serum FSH and LH levels by RIA. FSHbeta, LHbeta, and alpha subunit mRNA levels were measured from individual pituitaries using a microlysate ribonuclease protection assay. Serum FSH and pituitary FSHbeta mRNA levels changed similarly following long-day transfer. Both were significantly elevated after five long days (2.3- and 3.6-fold, respectively; P < 0.02) and declined thereafter, but they remained above SD control values through 20 long days. Alpha subunit mRNA levels also increased significantly relative to SD control values (maximum 2-fold increase after seven long days; P < 0.03), although to a lesser extent than FSHbeta. Neither serum LH nor pituitary LHbeta mRNA levels changed significantly following long-day transfer. The results indicate that long-day-associated increases in serum FSH levels in Siberian hamsters reflect an underlying increase in pituitary FSHbeta and alpha subunit mRNA accumulation.

Photoperiodic effects on gonadotropin-releasing hormone (GnRH) content and the GnRH-immunoreactive neuronal system of male Siberian hamsters.

Despite profound photoperiodic differences in circulating gonadotropin levels, consistent differences in the GnRH system have not been observed in Siberian hamsters (Phodopus sungorus) housed chronically in short or long days. During the transition from short to long days, however, male hamsters exhibit a transient increase in the number of cells expressing prepro-GnRH mRNA on the morning of the second long day. Here, we present two experiments designed to examine whether or not this change in mRNA level is associated with changes in GnRH protein synthesis. In the first experiment, we used RIA to measure GnRH content in preoptic area-mediobasal hypothalamic homogenates. We observed a significant increase in GnRH protein levels on the morning of the second long day relative to short- and long-day controls. The latter two groups did not differ from one another. In the second experiment, we used immunocytochemistry to quantify GnRH cell number in the various treatment groups. GnRH-immunoreactive (-ir) cell number did not increase significantly after long-day transfer relative to that in short-day controls; however, both of these groups had significantly more GnRH-ir neurons than long-day controls. We hypothesize that during the transition from short to long days, male Siberian hamsters experience a transient increase in GnRH production in a stable population of neurons. When GnRH secretion subsequently increases on long days, peptide content within neuronal cell bodies declines, leading to a decrease in the number of immunoreactive neurons detected. The rapid response of the hypothalamo-pituitary-gonadal axis in Siberian hamsters to a change in day length defines a narrow temporal window in which to identify the physiological, cellular, and molecular mechanisms mediating the photoperiodic regulation of reproduction.

Circadian rhythms and depression: effects of exercise in an animal model.

There is a clear link between altered circadian rhythms and depressive disorders, although the nature of this relationship is unknown. In addition, exercise affects both mood and alters clock function. To investigate the relationship between circadian rhythms, depression, and exercise, 3-wk-old mice housed on a 12:12-h light-dark cycle were exposed to chronic stress (CS) for 6 wk before being placed into constant darkness (DD). One-half of both the control and stressed mice were given access to a running wheel. Stressed mice consumed significantly less of a 2% sucrose solution during CS and exhibited a significant increase in immobility in the forced swim test 3 wk after the termination of stress relative to control mice. These effects were more pronounced in mice without running wheels. Stressed mice also exhibited altered percent distribution of total activity and increased fragmentation of daily activity rhythms during CS relative to control mice. Alterations in percent distribution were more pronounced in animals without running wheels. No activity rhythm changes were seen in DD, and there were no differences in light-induced phase shifts between stressed and control mice. These results suggest that CS causes long-term depressive-like symptoms but does not have long-lasting effects on activity rhythms. These changes were more pronounced in mice without running wheels, suggesting that exercise may protect against the harmful effects of stress.

Rapid photoperiod-induced increase in detectable GnRH mRNA-containing cells in Siberian hamster.

To determine whether changes in gonadotropin-releasing hormone (GnRH) neurons are early indicators of photostimulation, Siberian hamsters were placed in short days (6:18-h light-dark) at 3 (experiment 1) or 6 (experiment 2) wk of age where they were held for 3 (experiment 1) or 4 (experiment 2) wk. Hamsters were then moved to long photoperiod (16:8-h light-dark). In experiment 1, brains were collected 1-21 days after transfer from short to long days. In experiment 2, brains were collected only on the second morning of long day exposure. Long and short day controls were included in both experiments. Cells containing GnRH mRNA, as visualized by in situ hybridization, were counted. As expected, there were no differences in the number of detectable GnRH mRNA-containing cells among animals chronically exposed to long or short photoperiods. However, on the second morning after transfer from short to long photoperiod, a positive shift in the distribution of GnRH mRNA-containing cells occurred relative to the respective controls in the two experiments. Increases in follicle-stimulating hormone secretion and gonadal growth occurred days later. In conclusion, a rapid but transient increase in the distribution of detectable GnRH mRNA-containing cells is an early step in the photostimulation of the hypothalamic-pituitary-gonadal axis.

Testicular growth and locomotor activity of Siberian hamsters from short-day-responsive and short-day-nonresponsive lineages.

Young of short-day-responsive (SDR/Y) and short-day-nonresponsive (SDNR/Y) Siberian hamsters were reared in a long (16L:8D) photoperiod. At weaning, males from both lineages were maintained in 16L:8D or transferred to a short (12L:12D) photoperiod; body weight and testis size were measured every 7-10 days until 45 or 140 days of age. Contrary to expected results, juvenile SDNR/Ys responded to the inhibitory effects of the shorter photoperiods with decreased body weight and inhibited testicular growth. Under the long photoperiods, SDR/Y and SDNR/Y animals exhibited accelerated body weight gains and testicular growth from 18 to 69 days of age. Beginning at 69 days of age, however, SDNR/Ys exhibited a transient decrease in testis size that troughed at 86 days of age; testis growth subsequently resumed. Consistent with their ability to respond to short photoperiods, SDNR/Y young, in a second experiment, showed locomotor activity patterns similar to those of SDR/Ys in short days (9L:15D). It was only in adult SDNR/Ys that the delayed onset of activity characteristic of nonresponders was apparent. These results indicate that the circadian system of nonresponders changes with age, rendering animals nonresponsive to short photoperiods. The period of transient testicular decline observed in SDNR/Ys housed in 16L:8D may mark a critical period in the onset of these changes; additional studies are required to test this hypothesis.

Maternal transfer of photoperiodic information in Siberian hamsters. V. Effects of melatonin implants are dependent on photoperiod.

Photoperiodic information is transferred from female Siberian hamsters to their fetuses during gestation. Although maternal melatonin is known to be essential for the transfer of prenatal photoperiodic information, its specific role is not well defined. The duration of the daily melatonin signal, expressed as an elevation of serum melatonin levels in the maternal circulation, has been hypothesized to convey day length information to the fetus. If this hypothesis is valid, it predicts that identical maternal melatonin signals should affect the fetuses identically, regardless of the prenatal photoperiod. To test this hypothesis, adult females received melatonin in beeswax or beeswax alone. They were paired with males and housed in photoperiods of 12L:12D or 16L:8D. On the day of parturition, mother and young were transferred to constant light (LL). Young males were killed on Day 28 of life, and weights of testes were determined. Prenatal treatment with beeswax alone did not affect the nature of the signal transferred from mother to fetus; young gestated in 12L:12D and reared in LL developed small testes, while those gestated in 16L:8D had large testes. On the other hand, the effect of the prenatal melatonin treatment on postnatal testicular development in LL was inversely dependent on the prenatal photoperiod: testicular growth was stimulated in young gestated in 12L:12D, but inhibited in young gestated in 16L:8D. To verify that the melatonin pellets produced equivalent serum melatonin levels in adult females in 12L:12D and 16L:8D, unmated adult females were killed 6-10 wk after receiving melatonin pellets. Serum levels were elevated in both groups throughout the day and night.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal transfer of photoperiodic information in Siberian hamsters. IV. Peripubertal reproductive development in the absence of maternal photoperiodic signals during gestation.

Peripubertal reproductive development of Siberian hamsters is influenced by photoperiodic information received during gestation; the maternal pineal is important for this process. We observed that in the absence of the maternal pineal, the fetus appears to receive no information about gestational photoperiods. This is not the equivalent of receipt of a long-day signal by the fetus. Pinealectomized and sham-operated pregnant females were exposed to photoperiods of 12L:12D, 14L:10D, 16L:8D, or constant light (LL); young were reared from birth to 28 days of age in LL or 14L:10D. Regardless of the gestational photoperiod, LL-reared male young born to pinealectomized dams had smaller testes than LL-reared young of pineal-intact dams exposed to 16L:8D while pregnant. Thus, pinealectomy did not result in transmission of a long-day signal, nor did young born of pinealectomized dams receive short- or intermediate-day signals. Unlike young of pineal-intact females exposed to 12L:12D or 14L:10D while pregnant, young born of pinealectomized dams had small testes when reared in 14L:10D, irrespective of gestational photoperiod. Uterine weights of female young presented similar patterns of responses. In a second experiment, adult females were entrained to 12L:12D, 14L:10D, or 16L:8D for 3 wk prior to pinealectomy to determine if the effect of maternal pinealectomy would be altered. Entrainment to the new photoperiod prior to surgery did not alter the effects of maternal pinealectomy.

Maternal programming of the fetal brain dictates the response of juvenile Siberian hamsters to photoperiod: dissecting the information transfer system.

Development of neuroendocrine mechanisms regulating reproduction results from the interaction between genetic and environmental factors. For the developing fetus, the environment includes the maternal system. Our work with Siberian hamsters examines mechanisms by which prenatal photoperiods influence neuroendocrine functions regulating postnatal reproductive development. The maternal system has two effects on the young: 1) to program a reproductive growth pattern in the young and 2) to influence the ability of the young to respond to photoperiods encountered after weaning. Three paradigms have been used to study the role of the pineal hormone melatonin in this process. Injection of pregnant females with melatonin or removing melatonin has demonstrated that the maternal pineal is required for the transference of photoperiodic information to fetuses. However, when pregnant females receive continuous release implants of melatonin the effects of melatonin on fetuses are dependent on gestational photoperiod, suggesting that while melatonin is necessary it is not the only component in the mechanism for the transference of photoperiodic information to fetuses.

Maternal transfer of photoperiodic information in Siberian hamsters. III. Melatonin injections program postnatal reproductive development expressed in constant light.

In Siberian hamsters, transference of photoperiodic information from dam to fetus influences pubertal testicular development of the young when reared either in constant light (LL) or postnatal photoperiods of intermediate length (i.e. 14L:10D). The effects of short photoperiods during gestation can be mimicked by administering melatonin to pregnant females. This experiment examined whether there exists a daily pattern of sensitivity to melatonin when it is administered to pineal-intact pregnant females housed on a long photoperiod. Groups of pregnant and lactating females received melatonin at each hour of the day. The young were not treated with exogenous melatonin. At the approximate time of maturation of their endogenous pineal melatonin rhythm (Day 15), the young were placed in LL to suppress pineal melatonin secretion. Young males were killed at 28 days of age. Afternoon (1200 h-2000 h) and late night (0400 h) injections of melatonin into females caused their male young to develop as though gestation occurred on a short photoperiod. Melatonin injections at other times were ineffective. The daily pattern of effectiveness of exogenous melatonin administration to pregnant females resembles that observed in adult males of this and other hamster species and is consistent with the hypothesis that a daily rhythm in sensitivity to melatonin is involved in the transduction of photoperiodic signals.

Maternal transfer of photoperiodic information in Siberian hamsters. II. The nature of the maternal signal, time of signal transfer, and the effect of the maternal signal on peripubertal reproductive development in the absence of photoperiodic input.

Peripubertal reproductive development of Siberian hamsters is controlled by maternally derived photoperiodic information and the ambient photoperiod present after weaning. Previous experiments suggested that the maternally derived information is transferred during gestation, not during lactation. Development was examined in several photoperiods following manipulation of gestational and lactational photoperiods; development was influenced by the gestational, but not lactational, photoperiod. Second, effects of the gestational photoperiod were observed in young reared in constant light (LL) from Day 15. Depriving the young of ambient photoperiodic information after Day 15 allows a more direct assessment of the signal received from their dams. Finally, melatonin injections to long-day dams, at certain times of day, caused transmission of a short-day signal to young, as evidenced by their development in LL and light-dark cycles. Thus, a maternally derived signal that is dependent on melatonin influences reproductive development of the young during gestation; the maternally directed pattern of development can subsequently be modified by the youngs' own response to ambient photoperiods after weaning.

Age, photoperiodic responses, and pineal function in meadow voles, Microtus pennsylvanicus.

We tested whether juvenile males of Microtus pennsylvanicus were more sensitive than adults to the suppressive effects of short photoperiods. Voles were transferred to short photoperiods (10L:14D) at 20 or 80 d of age, and 60 d later (i.e. at 80 or 140 d) the animals were killed at intervals throughout the day and night. Pineal glands were collected for measurement of melatonin, and the testes were weighed. There were no differences in paired testicular weights of 80 and 140 d old animals held on long days (median testicular weights: 1,953 and 1,843 mg). In contrast, median testicular weights of voles held on short days were 504 and 1,112 mg, respectively, at 80 and 140 d of age; the testicular weights of both groups were significantly different from their age-matched controls (P less than .001, two-sample t-tests on log transformed data). The responses of the two age groups were compared by normalizing the individual values by the mean and variance of the respective long-day controls. This comparison suggests that the responsiveness to photoperiod decreases as the animals age (t-test, P = .01). Duration and amplitude of the nocturnal rise in pineal melatonin content were similar in differently aged animals. In two experiments, voles were injected daily with melatonin from 20 to 80 or 80 to 140 d of age. Melatonin-injected animals had smaller testes than did saline-injected controls (ANOVA: P = .01), and injections were more effective in the afternoon than in the morning (P = .01). Comparison of the effectiveness of short day and melatonin injections in juvenile and adult voles suggests that while short days inhibited testicular development of young animals more than it induced regression of adults, this decrease in responsiveness may involve factors other than alterations in the nocturnal pattern of melatonin production.

Pituitary gonadotropin content in gonadectomized rats. Immunoassay measurements influenced by extraction solvent and testosterone replacement.

Increasing interest in events regulating gonadotropin synthesis and secretion led to the reexamination, by radioimmunoassay (RIA), of the pituitary content of luteinizing hormone (LH) and follicle stimulating hormone (FSH). In a preliminary experiment, the importance of the composition of the solvent used to homogenize pituitaries on the detection of LH and FSH was demonstrated by RIA. Experiment 1 demonstrated that a drop in pituitary FSH occurred acutely following gonadectomy in males, but not in females. Experiment 2 tested whether this decline resulted from loss of testosterone or inhibin. Males were castrated and treated daily with oil or testosterone propionate, porcine follicular fluid, porcine serum, or testosterone plus follicular fluid for 2 to 14 days. Castration lowered pituitary FSH at 2, 4, and 6 days. Follicular fluid suppressed serum FSH, but not LH, and did not prevent the fall in pituitary FSH. Testosterone blocked the rise of serum LH and FSH and prevented the decline in pituitary FSH.

Cross-fostering of voles demonstrates in utero effect of photoperiod.

Postweaning body growth and reproductive tract weight of montane voles raised from birth in 14 h light/day are modulated by the photoperiod to which the voles' mothers were exposed while pregnant. This effect could result from factors acting in utero or during lactation, as a result of a change in photoperiod experienced by the mother on the day she gave birth. To distinguish between these hypotheses, male voles exposed to short or long photoperiods during gestation were raised by foster mothers that had been exposed to different photoperiods while pregnant. The differences in body weight, total length, and reproductive tract weight between voles at 74 days of age can be attributed to factors acting in utero. The effects of the gestational photoperiod are not manifested in the patterns of growth until after weaning.

Growth and reproductive development of male Microtus montanus is affected by the prenatal photoperiod.

Rates of growth and sexual maturation of microtine rodents vary in response to photoperiod. Previous work with Microtus montanus has shown that the photoperiod present prior to weaning influences how voles will respond to photoperiods seen following weaning. The data presented demonstrate that information about the photoperiod seen by the mother during pregnancy influences the postweaning development of male M. montanus. Adult M. montanus were paired in photoperiods consisting of 8, 14 or 16 h light/day. Their litters were conceived and born in these photoperiods. On the day of birth the litters were recorded and retained in the gestation photoperiod (Groups C8, C14 and C16) or transferred to the 14-h photoperiod (Groups E8 and E16). The growth of males was followed from weaning until 74 days of age, at which time the voles were sacrificed and their reproductive organs weighed. There were no differences in body weight or length between groups at 18 days of age. At 74 days of age the development of the voles could be ranked in the following sequence: C8 less than E16 less than C14 less than E8 less than C16.