Unusual Configurations of Endoplasmic Reticulum in Pinealocytes of the Djungarian Hamster (Phodopus sungorus)

Ultrastructural observations of the pineal gland of Phodopus sungorus revealed different morphological forms of endoplasmic reticulum. These included crystalloids composed of 200A-wide tubules with an intervening space of 200A and connected at right angles to form a rectangular lattice; complexes of undulating 500A-wide tubules in an electron-dense cytoplasmic matrix; and stacks of flattened cisternae covered with granular electron-dense material. All these structures are continuous with the surrounding endoplasmic reticulum. Frequently one structure is directly connected with another. The exact function of these structures derived from the endoplasmic reticulum is still unclear, but they can be assumed to be morphological features of augmented secretory activity.     

Afternoon injections of melatonin in the Djungarian hamster Phodopus sungorus: Long lasting sex-specific effects and influence of acute treatment on the endogenous pineal melatonin rhythm

Abstract: The effects of exogenous melatonin on gonadal function and on the endogenous melatonin profiles of male and female Djungarian hamsters were investigated. Daily afternoon subcutaneous injections of melatonin (50 μ.g) in hamsters kept in long photoperiods (LD 16: 8) led to gonadal inhibition in all the treated females (n=13) but in only 2 of 13 males within the treatment period of 8 weeks (uteri: 244±11 mg in controls vs. 79±4 mg in treated hamsters, P <0.001; ovaries: 13.6±0.6 mg in controls vs. 7.9±0.7 mg in treated hamsters, P <0.001; testes: 1,021±54 mg in controls vs. 732±100 mg in treated hamsters, P <0.05; and accessory glands: 641±38 mg in controls vs. 548±70 mg in treated hamsters, P >0.05). These results indicate that there are some circumstances under which the gonadal responses of the reproductive organs of male and female Djungarian hamsters differ. Interestingly, pineal concentrations of melatonin were found to be significantly higher in males ( P <0.01), possibly indicating a more robust endogenous supply with the hormone, whereas serum melatonin levels were not significantly different between males and females. However, the subcutaneous injection of melatonin (5, 25, and 125 μg) exerted no acute effect on the endogenous, circadian melatonin-profile, independent of dosage and sex.     

Photoperiod control of reproductive development in the male Djungarian hamster (Phodopus sungorus)

The Djungarian or Siberian hamster (Phodopus sungorus) is a seasonally breeding rodent in which maturation of gonadal function depends upon the photoperiod during rearing. It was hypothesized that the ability of short days to block testicular growth resulted from insufficient gonadotropin secretion during critical stages of development. This question was studied by measurement of serum concentrations of FSH, LH, PRL, and androgens by RIA systems validated for use in this species. Males reared from birth in long (16 h of light, 8 h of darkness) or short (10 h of light, 14 h of darkness) photoperiods were killed at 5- to 10-day intervals between 5 and 60 days of age. Regardless of photoperiod before 15 days of age, body and testes weights similarly increased. Serum concentrations of FSH and PRL gradually increased during this age period, although PRL concentrations were statistically higher in males under long days compared to those under short days. Circulating serum LH and androgen levels were high before 10 days of age, but decreased by 15-20 days of age in both photoperiods. Under long days, the period between 15 and 30 days of age was characterized by rapid body (1 g/day) and testicular (10-38 mg/day) growth, peak serum FSH concentrations (20-25 days), sustained elevation in serum LH and androgen concentrations, and further increases in serum PRL values. After 30 days of age, a reduced growth rate for body and testes occurred; serum FSH levels declined, while adult serum concentrations of LH, PRL, and androgens were attained. In contrast, hamsters exposed to short days from birth exhibited a slower rate of body and testicular growth by 20 days of age. Short days blocked peak FSH secretion and suppressed serum concentrations of LH and androgens after 20 days of age. PRL titers were significantly lower in short day compared to those in long day housed hamsters at all ages. These results support the hypothesis that the short day-induced suppression of gonadotropins and PRL secretion during development blocked gonadal maturation.     

Circadian rhythm of plasma testosterone in the male Djungarian hamster (Phodopus sungorus)

A marked circadian rhythm of plasma testosterone was found in male Djungarian hamsters. Maximal values in the evening just prior to activity onset were about 12 times higher than minimum values in the early morning. The peak of serum testosterone concentration coincides with onset of oestrus in females.     

Progonadal role of the pineal in the Djungarian hamster (Phodopus sungorus sungorus): mediation by melatonin

A progonadal role of the pineal in both adult and juvenile male Djungarian hamsters has been demonstrated which confirms and extends previous reports. In short day-raised hamsters, stimulation of testicular development was achieved by transfer to long days. This stimulatory effect of long days was blocked by pinealectomy. Gonadal stimulation could be elicited in pinealectomized juveniles by timed daily infusions of melatonin. When given for 4 or 6 h daily at a dose rate of 0.83 or 83 ng/h, melatonin infusions stimulated testicular development and body growth. Measurement of plasma FSH and PRL indicated that this pubertal response resulted at least in part from a stimulatory effect of melatonin on the secretion of these hormones. Longer duration infusions (8 or 12 h) did not stimulate testicular development or body growth, and plasma FSH and PRL levels remained low. The efficacy of the short duration infusions did not appear to depend upon the time of day of administration. These findings are in close agreement with those of a previous study which investigated the antigonadal effect of long duration melatonin infusions. The present results demonstrate that melatonin administration can mimic the progonadal effects of the pineal in this species and add further support to the hypothesis that the pineal influences the reproductive system through changes in the duration of daily melatonin production.     

Effects of timed melatonin infusions on reproductive development in the Djungarian hamster (Phodopus sungorus)

In confirmation of earlier work, daily melatonin infusions of 9- or 12-h duration inhibited testicular development in pinealectomized juvenile Djungarian hamsters, while daily infusions of 6-h duration did not prevent gonadal growth. Two other methoxyindoles (5-methoxytryptophol and 5-methoxytryptamine) had less than 4% of the activity shown by melatonin. When pinealectomized hamsters received two short duration (i.e. 3, 5, or 6 h) melatonin infusions each day, gonadal development was not inhibited if the two daily infusions were separated by a period of 2 or 3 h without melatonin treatment. Thus, the effects of separate pulses of melatonin were not additive in this paradigm. When single, short duration daily infusions of melatonin (5-h) or isoproterenol (6-h) were administered to pineal-intact hamsters, gonadal inhibition occurred only when the infusions were given at times that would lead to an expected overlap with and extension of the endogenous nocturnal melatonin peak. Significant inhibition of testicular growth could be obtained with as few as 2-4 days of melatonin treatment. These observations further support the importance of melatonin peak duration and also indicate other interesting temporal aspects of melatonin action in the Djungarian hamster.     

Urinary 6-sulphatoxymelatonin excretion reflects pineal melatonin secretion in the Djungarian hamster (Phodopus sungorus)

Abstract: To monitor pineal function in the Djungarian hamster (Phodopus sun-gorus), we measured the urinary excretion of the melatonin metabolite 6-sulpha-toxymelatonin (aMT6s) at 3-hr intervals by radioimmunoassay. Hamsters maintained in either long photoperiod (LP, LD 16: 8) or short photoperiod (SP, LD 8: 16) showed marked daily rhythms in aMT6s excretion, with elevated levels during the dark phase. In both photoperiods, we found large interindividual differ-ences, mainly in the amplitude of the signal. However, the amplitude as well as the duration of nocturnal aMT6s excretion was higher in SP than in LP. Light ex-posure at night (180 mW/m², 30 min) caused a decrease in aMT6s excretion, indicating that the pineal gland is the major source of urinary aMT6s. Moreover, there was a significant correlation between nocturnal pineal/plasma melatonin contents and 24-hr aMT6s excretion. We conclude that, measurements of aMT6s provide a valid and quantitative index of pineal melatonin synthesis in this hamster species. As an advantage in determining pineal melatonin contents, this approach will al-low noninvasive long-term studies of individual animals under varying environ-mental conditions.     

Serotonin content and melatonin production in the pineal gland of the male Djungarian hamster (Phodopus sungorus)

Diurnal variations in serum melatonin levels and pineal concentrations of serotonin (5-HT), N-acetylserotonin (NAS), 5-hydroxyindole acetic acid (5-HIAA), and melatonin were estimated in adult male Djungarian hamsters kept under long-day (LD 16:8) or short-day (LD 8:16) photoperiods. The nocturnal increase in melatonin production was accompanied by a marked drop in pineal serotonin concentrations. Serotonin levels, however, decreased approximately 4 hr before pineal melatonin increased. Correlations of the mean values for pineal serotonin and pineal melatonin indicated significant correlations at both LD 16:8 (r = -0.92, P less than 0.001) and LD 8:16 (r = -0.85, P less than 0.001). The mean levels of pineal serotonin and serum melatonin were correlated as well (LD 16:8, r = -0.91, P less than 0.001; LD 8:16, r = -0.81, P less than 0.01). The levels of pineal serotonin declined at approximately the same time as serum melatonin levels increased. These data suggest that the drop in pineal serotonin is primarily a consequence of melatonin production (as reflected by increasing serum concentrations). Consequently, pineal concentrations of melatonin may not be the best estimate of actual melatonin production, but a measure of how much melatonin is accumulated within the pineal due to high synthesis rates while the release of the hormone from the gland is limited.     

Testicular function and pelage color have different critical daylengths in the Djungarian hamster, Phodopus sungorus sungorus

Testicular function and pelage color are regulated by photoperiod in the Djungarian hamster. To investigate the critical daylengths of these functions, adult male hamsters were exposed to one of four photoperiods: 16 h of light, 8 h of darkness (16L:8D), 14L:10D, 12L:12D, or 10L:14D. 10L:14D and 12L:12D induced the winter molt and testicular regression, in contrast to 14L:10D which induced only the latter response, and 16L:8D which maintained the summer pelage and large testes. Melatonin injections administered 4, 2, or 0 h before lights-off to hamsters exposed to 16L:8D mimicked the effects in hamsters exposed to 10:14D, 12L:12D or 14L:10D, respectively, on pelage color and testicular weight. Based on previous observations, the elevated circulating melatonin levels resulting from these injections were expected to extend the endogenous melatonin peak. Thus, this finding suggests that the duration of circadian melatonin elevation is the critical parameter determining its effect not only on the gonads, but also on the pelage. Since 14L:10D induced testicular regression but not the winter molt, this study also investigated whether circulating FSH levels, known to affect testicular function, and PRL levels, which have been shown to affect pelage color, might be affected differently by 14L:10D. Both FSH and PRL levels were found to be suppressed in 14L:10D hamsters compared to those in 16L:8D hamsters, although the interval between the initial decrease and eventual recovery was less than that in 10L:14D hamsters. Thus, the differential responses of the pelage and gonads to 14L:10D do not appear to be based on selective suppression of FSH in this photoperiod. However, different responses to 14L:10D compared to 10L:14D may be related to the shorter period of suppression of both PRL and FSH by the 14L:10D daylengths.     

Diurnal changes in the content of indoleamines, catecholamines, and methoxyindoles in the pineal gland of the Djungarian hamster (Phodopus sungorus): Effect of photoperiod

Abstract: Early investigations of the effect of sleep deprivation on plasma melatonin reported no major changes. Recently, 36 hrs of sleep deprivation was reported to elevate melatonin levels on the post-sleep deprivation night. Given these contradictions melatonin, Cortisol, prolactin, and thyroid stimulating hormone before, during, and, after sleep deprivation were examined in nine healthy young males following one night of sleep deprivation. Hormone levels at hourly intervals, for each night, were statistically analyzed by a repeated measures, two-way factorial ANOVA. ANOVA was also performed for measures of area under the curve (AUC). No significant differences were observed for melatonin levels. Cortisol was significantly higher on the sleep deprivation night presumably reflecting the aroused state accompanying being awake; however, there were several time points on the control night when it was elevated also. Prolactin was higher on the post-sleep deprivation and control nights but did not rise on the deprivation night, indicating a useful nonpolysomnographic index for discriminating overnight sleep and awake states. TSH levels showed a similar rise during the control and sleep deprivation nights, but remained flat on the post-sleep deprivation night. It appears that the pineal is insulated against feedback from changes to the level of arousal accompanying sleep and wakefulness. In comparison, Cortisol, prolactin, and TSH levels vary with these states and are, therefore, useful indices of arousal and sleep-wake.     

Antigonadal Activity of the Melatonin Analogs 2-Iodomelatonin and 2-Chloromelatonin in the Juvenile Djungarian Hamster, Phodopus sungorus campbetti

The antigonadal effects of daily (20 micrograms, s.c.) injection of melatonin and two analogs, 2-iodomelatonin and 2-chloromelatonin, were compared in juvenile Djungarian hamsters housed under long photoperiod (L:D 16:8). Melatonin, 2-iodomelatonin, and 2-chloromelatonin injected 3 h before lights off for 16 days (17-34 days of age) significantly inhibited testis growth compared to vehicle-injected hamsters. In addition, melatonin and both analogs significantly reduced body weight gain. These 2-substituted analogs appear to be melatonin agonists with a potency in vivo similar to the parent compound, melatonin.     

Effects of mobile phone electromagnetic fields at nonthermal SAR values on melatonin and body weight of Djungarian hamsters (Phodopus sungorus)

Abstract:  In three experiments, adult male Djungarian hamsters ( Phodopus sungorus ) were exposed 24 hr/day for 60 days to radio frequency electromagnetic fields (RF-EMF) at 383, 900, and 1800 MHz, modulated according to the TETRA (383 MHz) and GSM standards (900 and 1800 MHz), respectively. A radial waveguide system ensured a well defined and uniform exposure at whole-body averaged specific absorption rates of 80 mW/kg, which is equal to the upper limit of whole-body exposure of the general population in Germany and other countries. For each experiment, using two identical waveguides, hamsters were exposed (n = 120) and sham-exposed (n = 120) in a blind fashion. In all experiments, pineal and serum melatonin levels as well as the weights of testes, brain, kidneys, and liver were not affected. At 383 MHz, exposure resulted in a significant transient increase in body weight up to 4%, while at 900 MHz this body weight increase was more pronounced (up to 6%) and not transient. At 1800 MHz, no effect on body weight was seen. The results corroborate earlier findings which have shown no effects of RF-EMF on melatonin levels in vivo and in vitro. The data are in accordance with the hypothesis that absorbed RF energy may result in metabolic changes which eventually cause body weight increases in exposed animals. The data support the notion that metabolic effects of RF-EMFs need to be investigated in more detail in future studies.     

Melatonin controls photoperiodic changes in tanycyte vimentin and neural cell adhesion molecule expression in the Djungarian hamster (Phodopus sungorus)

The Djungarian hamster displays photoperiodic variations in gonadal size synchronized to the seasons by the nightly secretion of the pineal hormone melatonin. In short photoperiod (SP), the gonads regress in size, and circulating sex steroids levels decline. Thus, the brain is subject to seasonal variations of both melatonin and sex steroids. Tanycytes are specialized glial cells located in the ependymal lining of the third ventricle. They send processes either to the meninges or to blood vessels of the medio-basal hypothalamus. Furthermore, they are known to locally modulate GnRH release in the median eminence and to display seasonal structural changes. Seasonal changes in tanycyte morphology might be mediated either through melatonin or sex steroids. Therefore, we analyzed the effects of photoperiod, melatonin, and sex steroids 1) on tanycyte vimentin expression by immunohistochemistry and 2) on the expression of the neural cell adhesion molecule (NCAM) and polysialic acid as markers of brain plasticity. Vimentin immunostaining was reduced in tanycyte cell bodies and processes in SP. Similarly, tanycytes and their processes contained lower amounts of NCAM in SP. These changes induced by SP exposure could not be restored to long photoperiod (LP) levels by testosterone supplementation. Likewise, castration in LP did not affect tanycyte vimentin or NCAM expression. By contrast, late afternoon melatonin injections mimicking a SP-like melatonin peak in LP hamsters reduced vimentin and NCAM expression. Thus, the seasonal changes in vimentin and NCAM expression in tanycytes are regulated by melatonin independently of seasonal sex steroid changes.     

Direct suppressive effects of weak magnetic fields (50 Hz and 16 2/3 Hz) on melatonin synthesis in the pineal gland of Djungarian hamsters (Phodopus sungorus)

In many investigations performed thus far on rodents, a suppression of melatonin synthesis was observed when animals were exposed to weak magnetic fields. However, among the several issues not yet resolved is the question of whether the observed changes are caused by direct effects on the pineal gland or by indirect effects, e.g., at the level of the eyes. We, therefore, performed a series of experiments in which direct effects of weak magnetic fields were studied in isolated pineal glands of Djungarian hamsters (Phodopus sungorus). After sacrifice of animals during morning hours, pineal glands were removed and placed individually into glass chambers that were perfused with oxygenated buffer. Experiments (n = 8) lasted for 8 hr. Magnetic fields (1,623 or 50 Hz at 86 microTesla) were generated by Helmholtz coils, and were present during the entire period. In each experiment, 12 exposed, and 12 sham-exposed, pineal glands were placed individually in glass chambers, which were surrounded by identical coils. Because of the design of the coils (bifilament), and the experimental setup (black box), the experimentator was not aware which coil was activated until the results were obtained. After 3.25 hr of adaptation, melatonin production was stimulated by isoproterenol (10(-7) M) for 30 min. Eluted fractions were collected, and melatonin concentrations were measured by radioimmunoassay. Maximum melatonin production was achieved after 5-7 hr. In all experiments, maximum melatonin concentrations were lower in the exposed groups compared with the sham-exposed controls. Statistical analyses for each frequency showed significant suppressive effects at 16(2/3) Hz (P < 0.01), and 50 Hz (P < 0.00). It is concluded that the suppressive effects of magnetic fields on the synthesis of melatonin are a result of primary mechanisms at the level of the pineal gland.     

Trans-pineal microdialysis in the Djungarian hamster (Phodopus sungorus): a tool to study seasonal changes of circadian clock activities

The Djungarian hamster is a highly seasonal small mammal. The rhythmic secretion of melatonin by the pineal gland is under control of the circadian clock, conveying the photoperiodic message to the organism. Trans-pineal microdialysis permits the in vivo study of this well-defined and precise clock output by measuring melatonin release directly in the pineal gland. The aim of this study was to adapt this method to the Djungarian hamster in order to monitor clock properties during photoperiodic changes. Male adult Djungarian hamsters were kept in a long photoperiod (LD 16:8) and melatonin release was measured hourly during the dark period for several weeks. Melatonin showed a regular secretion between ZT 17 and ZT 23.5 whereas the amplitude became stable only after the third day of perfusion. To test how quickly changes in melatonin profile can be measured, 15-min light pulses were given at different time points throughout the scotophase. Light-pulses immediately interrupted melatonin secretion at any time point during the scotophase and the temporal resolution for measurement could be reduced to 30 min. In accordance with studies in the rat, long-term effects of light on the clock could only be observed when a light pulse was administered in the second half of the night. For the first time we established a method to measure precisely a direct and reliable clock-output in a highly seasonal species which allows us now to study the circadian and seasonal properties of the clock in detail.     

Urinary 6-sulfatoxymelatonin profiles in male Djungarian hamsters (Phodopus sungorus) responding and not responding to short-day photoperiods: Possible role of elevated daytime levels

Abstract: The lack of endocrine and physiological responses of some Djungarian hamsters ( Phodopus sungorus ) to the transition from long to short photoperiods (L:D 16:8 → L:D 8:16) has been known for a long time but is not yet understood. We investigated the role of melatonin synthesis in this context because melatonin, as part of the circadian system, may play a role in non-responsiveness. In ten responding and ten non-responding male hamsters, the urinary 24 hr 6-sulfatoxymelatonin (aMT6s) profiles under L:D 8:16 and L:D 16:8 were measured. Both short day responding and non-responding hamsters showed diurnal aMT6s excretion rhythms. Whereas responders reacted to the transition L:D 16:8 → L:D 8:16 with a marked elevation of aMT6s excretion, in non-responders no adjustment of the melatonin rhythm to the change of the photoperiod was seen. Furthermore, under L:D 16:8 the daytime levels of aMT6s were significantly ( p <0.001) lower in responders compared to non-responders whereas under L: D 8: 16 these levels were higher ( p <0.01). It is speculated that high daytime levels of aMT6s under long-day photoperiods in non-responders result in down-regulation of melatonin receptors of the nucleus suprachiasmaticus, the pacemaker for the pineal gland, leading to a lack of response to the transition to short-day photoperiods.     

Interruption of nocturnal pineal melatonin synthesis in spontaneous recrudescent Djungarian hamsters (Phodopus sungorus)

The duration of nighttime synthesis of the pineal hormone melatonin is believed to determine the breeding season in many mammalian species. Hamsters exposed to short days undergo gonadal involution followed by a return to normal function, suggesting a developed insensitivity to regressive photoperiods. This recrudescence may be due to either exhaustion of the pineal or to target-desensitization. Both theories have been tested previously but failed to explain this phenomenon. We performed an experiment in the Djungarian hamster (Phodopus sungorus), a well-characterized photosensitive species with a type C melatonin pattern (prolonged peak during majority of dark phase), in an attempt to resolve this issue. Among age-matched male hamsters exposed to short days for either 16 weeks (involuted) or 38 weeks (spontaneous recrudescent), marked phase differences in diurnal pineal melatonin rhythms were observed. Furthermore, in recrudescent hamsters the melatonin pattern was divided into two parts, possibly no longer recognizable as a typical short-day rhythm.