Analysis of adrenergic regulation of melatonin synthesis in Siberian hamster pineal emphasizes the role of HIOMT

Seasonal variations of environmental factors are translated into annual fluctuations in synthesis and release of melatonin, which in turn acts as a neuroendocrine messenger for the synchronization of annual functions. So far, most studies performed to understand the regulation of melatonin synthesis have used the non seasonal laboratory rat. It was demonstrated that nocturnal melatonin synthesis depends on alpha- and beta-adrenergic activation of the enzyme arylalkylamine N-acetyltransferase (AA-NAT). In this study, we investigated the mechanisms of melatonin synthesis in the Siberian hamster, a seasonal species with marked photoperiodic variation in melatonin peak duration and amplitude. A beta-adrenergic receptor agonist alone markedly stimulated AA-NAT activity and melatonin synthesis and release. An alpha-adrenergic receptor agonist, while having no effect per se, potentiated the beta-adrenergic stimulation of AA-NAT activity both in vitro and in vivo. Strikingly, the potentiation of AA-NAT activity did not result in a potentiation of melatonin synthesis, suggesting that the rate of melatonin production is limited downstream in the metabolic pathway, most probably at the level of hydroxyindole-O-methyltransferase (HIOMT). HIOMT presented a constitutively high activity that was not acutely (within hours) stimulated by beta-adrenergic agonist, but was rather up-regulated by chronic application of the agonist. This long-term beta-adrenergic regulation may explain the reported large photoperiodic variation of HIOMT activity that drives the photoperiodic variation in melatonin peak.     

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.     

Changes in social behavior of the male golden hamster accompanying photoperiodic changes in reproduction

Testicular regression and decreased serum testosterone levels result when male hamsters are placed in a short-day photoperiod, and these changes are reversed after reexposure to long-days. The present study was undertaken to determine whether these physiological changes lead to modifications in social behavior in the male hamster. Animals in a nonstimulatory short-day (LD 6:18) or a stimulatory long-day (LD 14:10) photoperiod were observed every 3 weeks for 21 weeks. After 9 weeks, half of the LD 6:18 males were returned to LD 14:10 and showed rapid testicular recrudescence. The other half remained in LD 6:18 and showed a slower rate of testicular recrudescence. Assessment of aggression was accomplished by placing an intruder in the home cage of a test animal and recording latency to attack, decisive encounters, number of attacks, and number of aggressive postures. Dominance was evaluated in a neutral arena with opponents of different weights, using the same parameters. Scent-marking was recorded in response to both bedding from an intact male and bedding from an ovariectomized female in a clean cage. Short-day animals undergoing testicular regression showed heightened levels of aggression and an upward shift in their dominance status. Alteration of the photoperiod did not affect the pattern or number of scent marks. As animals underwent testicular recrudescence, there was a return to lower levels of aggression and to a lower dominance status. Males undergoing rapid testicular recrudescence also showed the most rapid decline in aggressive behaviors. These results suggest that levels of agonistic behavior in the male hamster are inversely related to serum testosterone levels. Scent-marking appears to be unreleated to androgen levels in the male hamster and its role in hamster social behavior remains unclear.     

Schiff base protonation changes in Siberian hamster ultraviolet cone pigment photointermediates

Molecular structure and function studies of vertebrate ultraviolet (UV) cone visual pigments are needed to understand the molecular evolution of these photoreceptors, which uniquely contain unprotonated Schiff base linkages between the 11-cis-retinal chromophore and the opsin proteins. In this study, the Siberian hamster ultraviolet cone pigment (SHUV) was expressed and purified in an n-dodecyl-β-D-maltoside suspension for optical characterization. Time-resolved absorbance measurements, over a spectral range from 300 to 700 nm, were taken for the purified pigment at time delays from 30 ns to 4.64 s after photoexcitation using 7 ns pulses of 355 nm light. The resulting data were fit globally to a sum of exponential functions after noise reduction using singular-value decomposition. Four exponentials best fit the data with lifetimes of 1.4 μs, 210 μs, 47 ms, and 1 s. The first photointermediate species characterized here is an equilibrated mixture similar to the one formed after rhodopsin's Batho intermediate decays into equilibrium with its successor, BSI. The extremely large red shift of the SHUV Batho component relative to the pigment suggests that SHUV Batho has a protonated Schiff base and that the SHUV cone pigment itself has an unprotonated Schiff base. In contrast to SHUV Batho, the portion of the equilibrated mixture's spectrum corresponding to SHUV BSI is well fit by a model spectrum with an unprotonated Schiff base. The spectra of the next two photointermediate species revealed that they both have unprotonated Schiff bases and suggest they are analogous to rhodopsin's Lumi I and Lumi II species. After decay of SHUV Lumi II, the correspondence with rhodopsin photointermediates breaks down and the next photointermediate, presumably including the G protein-activating species, is a mixture of protonated and unprotonated Schiff base photointermediate species.     

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)     

Photic regulation of mt(1) melatonin receptors and 2-iodomelatonin binding in the rat and Siberian hamster

We have investigated the photic regulation of melatonin receptors both at the level of binding capacity and mt(1) mRNA expression in the suprachiasmatic nucleus (SCN) and the pars tuberalis (PT) of the pituitary of two species: a highly photoperiodic one, the Siberian hamster, and a nonphotoperiodic one, the Wistar rat. This study has been performed by looking at the effect of a light pulse applied during the night on the two receptor parameters. The results show that the photic regulations of mt(1) mRNA expression and receptor density are distinct from each other in both the SCN and PT of the two species studied. They also show that, depending on the species and the structure, this regulation may implicate either the circadian clock or melatonin.     

Photoperiodic regulation of FGF21 production in the Siberian hamster

This article is part of a Special Issue "Energy Balance".     

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.     

In vitro fertilization of Siberian hamster oocytes

Siberian hamsters were superovulated and various media were tested in an effort to fertilize the recovered oocytes in vitro. The highest percentage of fertilized ova was achieved by using a modified Tyrode's medium, designated MT (Bavister, J. Reprod. Fertil., 18:544-545, '69), previously formulated to fertilize Syrian hamster ova in vitro. Spermatozoa incubated in this medium in a concentrated state overnight (14 hr) and then diluted (1 hr) fertilized 39% of the ova. Similar results (40%) were obtained with this medium by adding 20% human follicular fluid to fresh concentrated sperm for 30 min and then diluting the sperm for 2-3 hr prior to the addition of ova. Ova fertilized in vitro cleaved to the two-cell stage but failed to develop any further in culture. Two-cell embryos recovered from mated hamsters and cultured did not undergo additional cleavage. Four-cell embryos collected from mated females and cultured cleaved to the six- to eight-cell stage and stopped. Techniques and media used for fertilizing large numbers of Syrian and Chinese hamster ova in vitro will have to be modified to achieve the same degree of success in the Siberian hamster.     

Testicular recrudescence in intermediate day lengths reflects loss of photoperiodic memory in Siberian hamsters

Siberian hamsters transferred from a long (16 h light/day [16 L]) to an intermediate (13.5 L) day length (DL) undergo testicular regression within 2 months followed approximately 2 months later by "spontaneous" testicular recrudescence. Recovery of gonadal function after prolonged exposure to intermediate DLs is thought to reflect development of neuroendocrine refractoriness to intermediate-duration melatonin signals. The authors tested the alternative hypothesis that testicular recrudescence in 13.5 L occurs when the "memory" for the 16-L photoperiod fades and hamsters can no longer compare the 13.5-L to the prior 16-L day length. Adult hamsters transferred from 16 L to 13.5 L that underwent testicular involution were either maintained continuously in 13.5 L for 41 weeks or given a supplementary 2-week treatment of 16 L before being returned to 13.5 L. The supplementary treatment was administered either after hamsters had been in 13.5 L for 10 weeks and had involuted testes, or after 24 weeks, when the gonads had undergone recrudescence. The authors found that 16 L treatment administered at week 10 delayed final gonadal recrudescence by approximately 12 weeks; similar 16-L treatment at week 24 induced a second gonadal regression when animals were returned to 13.5 L. The most parsimonious hypothesis to account for these findings is that gonadal recrudescence in intermediate DLs reflects fading of the "memory" for prior long DLs rather than induction of refractoriness to melatonin signals generated in intermediate DLs.     

Antioxidant activity of melatonin in Chinese hamster ovarian cells: changes in cellular proliferation and differentiation

Melatonin is an endogenously generated molecule with free radical scavenging and antioxidant properties. Here, we studied the antiproliferative role of melatonin and other antioxidants on transformed Chinese hamster ovarian cells. Melatonin reduces cell proliferation in a dose- and time-dependent manner. Natural antioxidants which appear in edible plants including resveratrol and vitamin E mimicked the effect of melatonin. Flow cytometer analysis revealed that melatonin treatment reduces the number of cells in S-phase and increases cells in both G0/G1 and G2/M gaps. In addition, melatonin, as well as trolox, caused a clear morphological change by inducing the cells to become spindle shaped and fibroblast-like. Its effect is a reversible phenomenon that disappeared when melatonin was withdrawn from the culture medium. GSH levels are increased after melatonin treatment but pharmacologically blockade of GSH synthesis did not abolish melatonin's antiproliferative effect. Reduction of cell proliferation and the apparent induction of cell differentiation overlapped with melatonin's ability to change the intracellular redox state of CHO cells. We conclude that the cellular redox state may be involved in cellular transformation caused by antioxidants such as melatonin and trolox.     

Photoperiod-dependent modulation of cardiac excitation contraction coupling in the Siberian hamster

In mammals, changes in photoperiod regulate a diverse array of physiological and behavioral processes, an example of which in the Siberian hamster (Phodopus sungorus) is the expression of bouts of daily torpor following prolonged exposure to a short photoperiod. During torpor, body temperature drops dramatically; however, unlike in nonhibernating or nontorpid species, the myocardium retains the ability to contract and is resistant to the development of arrhythmias. In the present study, we sought to determine whether exposure to a short photoperiod results in alterations to cardiac excitation-contraction coupling, thus potentially enabling the heart to survive periods of low temperature during torpor. Experiments were performed on single ventricular myocytes freshly isolated from the hearts of Siberian hamsters that had been exposed to either 12 wk of short-day lengths (SD) or 12 wk of long-day lengths (LD). In SD-acclimated animals, the amplitude of the systolic Ca(2+) transient was increased (e.g., from 142 +/- 17 nmol/l in LD to 229 +/- 31 nmol/l in SD at 4 Hz; P < 0.001). The increased Ca(2+) transient amplitude in the SD-acclimated animals was not associated with any change in the shape or duration of the action potential. However, sarcoplasmic reticulum Ca(2+) content measured after current-clamp stimulation was increased in the SD-acclimated animals (at 4 Hz, 110 +/- 5 vs. 141 +/- 15 mumol/l, P < 0.05). We propose that short photoperiods reprogram the function of the cardiac sarcoplasmic reticulum, resulting in an increased Ca(2+) content, and that this may be a necessary precursor for maintenance of cardiac function during winter torpor.     

Alteration of compensatory ovarian hypertrophy in the hamster by melatonin

Hamsters were unilaterally ovariectomized on day four of the estrous cycle and the subsequent compensatory ovarian hypertrophy (COH) and compensatory follicular stimulation studied on each of the following nine days. The hypertrophy stabilized at approximately 35% by the end of the second postoperative cycle, with greater ovarian weights observed on the mid-cycle days. Melatonin injections significantly reduced follicular counts when unilateral ovariectomy was performed on either day three or day four of the estrous cycle and necropsy was on the same day of the cycle, four days later.     

Early embryo development in the Siberian hamster (Phodopus sungorus)

Development of preimplantation embryos of the Siberian hamster (Phodopus sungorus) in vivo and in vitro was examined. The timing of early development in vivo was found to be slower than that reported for the golden hamster. Progression through the cleavage stages, cavitation, and hatching from the zona pellucida occurred later, with blastocyst formation beginning on the afternoon of day 4 and uterine attachment occurring early on day 5. In vitro, morulae, and early blastocysts collected on day 4 and cultured in serum-containing medium formed expanded blastocysts and some began to hatch from the zona pellucida. With extended culture, blastocysts attached and formed trophoblast outgrowths. Outgrowth was characterized by an initial migration of small cells from the blastocyst, followed by formation of a sheet of trophoblast giant cells. Differences in the morphology of outgrowth between the hamster and mouse suggest that further comparative studies with the Siberian hamster may be useful.     

Reception of photoperiodic information by fetal Siberian hamsters: role of the mother's pineal gland

The rate of reproductive development in juvenile male Siberian hamsters is strongly influenced by daylength (photoperiod). Recent studies indicate that reception of photoperiodic cues begins during fetal life. The present experiments yielded a further demonstration that developing male Siberian hamsters receive information about the photoperiod to which their mother is exposed during pregnancy. The possibility that photoperiodic information is transmitted from mother to young after birth was investigated by cross-fostering young gestated on 12L and 16L to mothers from the other photoperiod. Litters were cross-fostered on the day of birth and then were transferred, along with their foster mothers, to 14L. We found no influence of the mother after birth, indicating that transmission of photoperiodic information from mother to young must occur during gestation. To determine if the pineal gland of the mother is required for this response, adult females were pinealectomized or sham-operated and paired with intact males in 12L, 14L, or 16L. After parturition parents and offspring were exposed to 14L. The influence of prenatal photoperiod on postnatal testicular development in 14L was blocked by pinealectomy of the mother. Postnatal testicular development was retarded in offspring that experienced a photoperiod transfer from either 15L to 14L or 8L to 12L at birth. In contrast, the inhibitory effect of a transfer from 16L to 14L at birth was abolished when juvenile males were exposed to a single long photoperiod (16.3 h light) at age 17-21 days and then were returned to 14L.     

Mitochondrial metabolism during daily torpor in the dwarf Siberian hamster: role of active regulated changes and passive thermal effects

During daily torpor in the dwarf Siberian hamster, Phodopus sungorus, metabolic rate is reduced by 65% compared with the basal rate, but the mechanisms involved are contentious. We examined liver mitochondrial respiration to determine the possible role of active regulated changes and passive thermal effects in the reduction of metabolic rate. When assayed at 37 degrees C, state 3 (phosphorylating) respiration, but not state 4 (nonphosphorylating) respiration, was significantly lower during torpor compared with normothermia, suggesting that active regulated changes occur during daily torpor. Using top-down elasticity analysis, we determined that these active changes in torpor included a reduced substrate oxidation capacity and an increased proton conductance of the inner mitochondrial membrane. At 15 degrees C, mitochondrial respiration was at least 75% lower than at 37 degrees C, but there was no difference between normothermia and torpor. This implies that the active regulated changes are likely more important for reducing respiration at high temperatures (i.e., during entrance) and/or have effects other than reducing respiration at low temperatures. The decrease in respiration from 37 degrees C to 15 degrees C resulted predominantly from a considerable reduction of substrate oxidation capacity in both torpid and normothermic animals. Temperature-dependent changes in proton leak and phosphorylation kinetics depended on metabolic state; proton leakiness increased in torpid animals but decreased in normothermic animals, whereas phosphorylation activity decreased in torpid animals but increased in normothermic animals. Overall, we have shown that both active and passive changes to oxidative phosphorylation occur during daily torpor in this species, contributing to reduced metabolic rate.