Reproductive phase depedent circadian variation in the pineal biochemical constituents of Indian palm squirrel, Funambulus pennanti

In mammals, pineal gland is intimately concerned with the co-ordination of rhythm physiology. Biochemical characteristics of pineal gland in man and other mammals may provide strong, yet sometimes elusive support for the belief in functional individuality and probable importance of this tiny gland. In seasonal breeding animals, pineal gland function is very much dependent on the reproductive status. Therefore, the aim of this experiment is to note the circadian rhythmicity of different biochemical constituents of pineal gland during active and inactive phases of reproductive cycle of a seasonally breeding rodent, F. pennanti. In the present study, pineal biochemical constituents i.e. protein and cholesterol showed higher values during daytime (1400 h). The plasma melatonin level presented two peaks during active (April; at 1800 h and 0200 h) and inactive (December; at 1400 h and 0200 h) phases of reproductive cycle. The pineal protein, cholesterol and plasma melatonin values in term of basal and peak levels were higher during the reproductive inactive/pineal active phase. Therefore, pineal--also known to have antigonadotropic properties and cholesterol which appears conjugated with pineal serotonin, presented circadian rhythmicity along with the plasma level of melatonin. This rhythmicity noted in present study was dependent on the reproductive and pineal activity status, and might be regulated by the sex steroid receptor present on the pineal gland.     

Impact of photoperiodic exposures during late gestation and lactation periods on the pineal and reproductive physiology of the Indian palm squirrel, Funambulus pennanti

Studies on the maternal transfer of photoperiodic information in mammals indicate that the daily photoperiod perceived by the mother during the gestation-lactation period is communicated to the fetus either through the placenta or via the milk. However, the impact of photoperiodic exposures during gestation and lactation on the maternal pineal and reproductive physiology has not been reported for any tropical rodent. The exposure of pregnant female Indian palm squirrels (Funambulus pennanti) to constant light (24 h light:0 h dark), constant dark (0 h light:24 h dark), long daylength (14 h light:10 h dark) or short daylength (10 h light:14 h dark) during early gestation (< 30 days) resulted in the resorption of pregnancy, while during late gestation (> 30 days), it did not interfere with the maintenance of pregnancy. Alterations in photoperiodic condition during late gestation and lactation altered the postpartum recovery process. Pineal gland activity, as assessed by pineal mass, protein content and plasma melatonin, was lowest during the breeding phase, but increased gradually after parturition until the next breeding phase. During gestation and lactation, constant light, long daylength and short daylength conditions were less effective, while constant dark condition had a profound effect in depressing pineal gland activity, which subsequently advanced postpartum recovery. Hence, lactating females under constant darkness prepare themselves for next mating much earlier than females under natural daylength (12 h light:12 h dark) conditions. Therefore, photoperiodic information, mediated via the pineal gland, may be important for maintaining gestation physiology as well as postpartum recovery in female rodents.     

Profile of organ weights and plasma concentrations of melatonin, estradiol and progesterone during gestation and post-parturition periods in female Indian palm squirrel Funambulus pennanti

Todate, report about the role of pineal gland in maintaining the normal physiology of gestation is scanty. Present study is the first of its kind giving a detail profile of organ weights and plasma concentration of melatonin, estradiol and progesterone to suggest a possible role of pineal gland in maintaining normal physiology during gestation and post-parturition periods of female Indian palm squirrel F. pennanti. Inspite of, inverse pineal-gonadal/melatonin-steroids interrelationship in adult (non-pregnant) females, the present results study suggest a direct relationship of pineal gland activity with ovarian steroids especially during the gestation period. The inverse relationship of melatonin and ovarian steroids is again established after parturition and maintained throughout the life. Thus the pineal gland (activity as judged by its weight, biochemical contents i.e. protein and cholesterol and plasma melatonin level) maintained ovarian/uterine physiology and regulated plasma concentrations of estradiol and progesterone during gestation and post-parturition periods. It is suggested that the pineal gland and its hormone melatonin play an important role to maintain the normal physiology of gestation and the post-partum recovery in Indian palm squirrel F. pennanti.     

Relationship between the annual rhythms in melatonin and immune system status in the tropical palm squirrel, Funambulus pennanti

Melatonin (MEL) regulation of seasonal variation in immunity has been studied extensively in temperate mammals. This report is the first on a tropical mammal, the Indian palm squirrel, F. pennanti. In response to the annual environmental cycle, we studied the rhythms of plasma MEL and the immune parameters of total blood leucocytes, absolute blood lymphocytes and blastogenic responses of blood, thymus and spleen lymphocytes. We found that in parallel with MEL all the immune parameters increased during the month of April onward, when natural day length, temperature, humidity and rainfall were increasing. Maximum values occurred during November (reproductively inactive phase) when the values of all the physical factors were comparatively low. Lowest values occurred during January-March (reproductively active phase) when the values of the physical factors were lowest. In order to establish a clear interrelationship between the pineal MEL and the immune system function, we manipulated these squirrels with exogenous MEL (25mg/100g B wt/day) at 1730 h during their pineal inactive phase (March) while another group was pinealectomized (Px) during November when their pineal was active. The MEL injection significantly increased all the immune parameters, while Px decreased them significantly. Hence, we suggest that MEL is immuno-enhancing for this tropical squirrel, and plays an important role in the maintenance of its immunity in accordance with the seasonal changes in environmental factors and gonadal status. (Chronobiology International, 18(1), 61-69, 2001)     

Bidirectional communication between the pineal gland and the immune system

The pineal gland is a vertebrate neuroendocrine organ converting environmental photoperiodic information into a biochemical message (melatonin) that subsequently regulates the activity of numerous target tissues after its release into the bloodstream. A phylogenetically conserved feature is increased melatonin synthesis during darkness, even though there are differences between mammals and birds in the regulation of rhythmic pinealocyte function. Membrane-bound melatonin receptors are found in many peripheral organs, including lymphoid glands and immune cells, from which melatonin receptor genes have been characterized and cloned. The expression of melatonin receptor genes within the immune system shows species and organ specificity. The pineal gland, via the rhythmical synthesis and release of melatonin, influences the development and function of the immune system, although the postreceptor signal transduction system is poorly understood. Circulating messages produced by activated immune cells are reciprocally perceived by the pineal gland and provide feedback for the regulation of pineal function. The pineal gland and the immune system are, therefore, reciprocally linked by bidirectional communication.     

Daily variation in antioxidant enzymes lipid peroxidation in thyroid and plasma level thyroxine and triiodothyronine levels of a tropical bird Perdicula asiatica during reproductively active and inactive phases

The aim of this work was to study the variations in the interference of neuroendocrine pineal gland and metabolically active thyroid gland in a tropical bird, Perdicula asiatica. Maximum pineal gland activity (pineal weight and melatonin level), minimum thyroid gland activity (weight, T3/T4 and thymidine kinase activity) along with less oxidative load (MDA level, SOD, CAT and ABTS activity) were observed during reproductively inactive phase (RIP) was observed. Further, a robust and significant rhythmicity was noted in melatonin levels during RIP and RAP, but no significant rhythmicity was noted in T4/T3 level by cosinor analysis. Overall, melatonin and thyroid circadian profile suggested that melatonin might be acting as an antioxidant molecule with time of the day effect in rescuing thyroid gland from free radical load in birds.     

Time and reproductive phase-dependent effects of exogenous melatonin on the pineal gland and ovary of a nocturnal bird, the Indian spotted owlet, Athene brama

In a tropical nocturnal bird, the Indian spotted owlet, Athene brama, the intraperitonial injection of an identical amount (20 mg/100 g b. wt/day) of exogenous melatonin (MEL) for 15 consecutive days increased the pineal weight and plasma MEL level in sexually active birds while it decreased them in inactive birds more potently when injected in the evening (18.30-19.30 h) rather than the morning (0500-0600 h). On the other hand, more efficiently than the morning hour treatment, the evening hour MEL injection decreased the ovary weight and plasma estradiol and progesterone levels both in sexually active and inactive birds, but more potently in active than inactive birds. Thus, the exogenous MEL showed the time and reproductive phase dependent effects on the pineal gland and the ovary of this nocturnal bird.     

Peripheral melatonin modulates seasonal immunity and reproduction of Indian tropical male bird Perdicula asiatica

Seasonal changes in pineal function are well coordinated with seasonal reproductive activity of tropical birds. Further, immunomodulatory property of melatonin is well documented in seasonally breeding animals. Present study elucidates the interaction of peripheral melatonin with seasonal pattern of immunity and reproduction in Indian tropical male bird Perdicula asiatica. Significant seasonal changes were noted in pineal, testicular and immune function(s) of this avian species. Maximum pineal activity along with high immune status was noted during winter month while maximum testicular activity with low immune status was noted in summer. During summer month's long photoperiod suppressed pineal activity and high circulating testosterone suppressed immune parameters, while in winter short photoperiod elevated pineal activity and high circulating melatonin maintained high immune status and suppressed gonadal activity. Therefore, seasonal levels of melatonin act like a major temporal synchronizer to maintain not only the seasonal reproduction but also immune adaptability of this avian species.     

A reproductive phase-dependent effect of dietary L-tryptophan on pineal gland and gonad of a nocturnal bird, Indian spotted owlet Athene brama

Unlike other temperate owls, Indian spotted owlet Athene brama possesses a well-developed pineal gland that secrets moderate amount of hydroxy- (serotonin) and methoxy- (melatonin) indoles in circulation. However, in this study, we have reported the response of this endocrine gland to exogenous L-Tryptophan (precursor of the above indoles), and also its effect on gonads of this nocturnal bird. During breeding phase or pineal inactive phase (March), oral treatment of L-Trp (0.5 mg/100 g Bwt/day) significantly increased the pineal gland wt and plasma melatonin (MEL) level, while decreased the gonadal wt and plasma sex steroids levels (estradiol and progesterone in female and testosterone in male). Interestingly, during reproductively quiescent phase or pineal active phase (August), similar amount of L-Trp significantly decreased the plasma MEL level, while increased the above sex steroid levels in plasma. Finally, the results show a clear reproductive phase-dependent inverse effect of L-Trp on pineal gland and gonads for both sexes of the spotted owlets, and suggest that the therapeutic use of this amino acid would be a great advantage for controlling the reproduction of these economically important birds.     

Annual reproductive synchronization in ovary and pineal gland function of female short-nosed fruit bat, Cynopterus sphinx

We studied the annual correlation of ovarian activity and pineal gland in relation with seasonal variation and gestation of a tropical zone short-nosed fruit bat Cynopterus sphinx. Female bats showed bimodal polyestry (February/March and September/October) in their reproductive cycle. Plasma estradiol concentration ran parallel with ovarian activity and had an inverse relation with pineal mass and peripheral melatonin concentration. Due to the delayed embryonic development in the uterus (October-March) of female bats, interestingly, the uterine activity did not show a parallel relation with ovarian activity and estradiol level. Further, compared with normal non-pregnant females, melatonin level was high during gestation and delayed embryonic development phase. This suggests that the reproductive synchrony and annual variation in ovarian activity of this nocturnal flying mammal differ from other common tropical mammals. The delayed embryonic development in bats might be an adaptive strategy for the unfavorable conditions of the seasons and might be regulated by high peripheral estradiol and melatonin concentration.     

The role of a thymus-pineal axis in an immune mechanism of aging

The Immune Theory of Aging cannot explain the cause of immune decline. It is hypothesized that the pineal gland acting in utero and during neonatal life in altricial mammals serves as a component of the immune system. Evidence in support of the presence of a thymus-pineal axis is presented. It is postulated that the pineal gland carries a considerable burden of immunological defense during maturation of the thymus, and also acts in the programming of the immune system. By relating thymus and immune function to the pineal and its known role as a neuroendocrine transducer for the entrainment and control of biorhythms, a consilence is developed between the role of the immune system in senescence and the pineal function in biorhythmicity. The relationships developed thus permit an extension of the immune theory as regards causative mechanisms.     

Circadian Rhythms of Melatonin and Sex Steroids in a Nocturnal Bird,Indian Spotted Owlet Athene brama During Reproductively Active and Inactive Phases

The epiphyseal neurohormone melatonin (MEL) exhibits circadian cyclicity, as noted extensively in diurnal vertebrates although very little information is available regarding nocturnal species. We have studied the MEL rhythmicity with 24-hour periodicity in a tropical nocturnal bird, Indian spotted owlet Athene brama, which possesses a well-developed pineal organ. We performed our study during two crucial reproductive phases (active and inactive), when the pineal gland activity in owlet exists in inverse states, i.e., inactive and active respectively. Independent of sex, the circadian rhythm of plasma MEL in owlets showed a two-peak cyclicity with a smaller peak at around 1400 h and the higher one at about 0200 h, while the lowest value was found at 1000 h. The night (0200 h) peak activity of plasma MEL in owlet has a resemblance with the earlier findings in diurnal birds and strongly suggests that independent of species habit the peak activity of MEL is invariably dark dependent. However, the daytime peak of MEL may be due to the daytime hiding nature of this nocturnal bird. Interestingly, it was also noted that the hours of peak activity of MEL (1400 and 0200 h) were the same during both of the reproductive phases, though the environmental day length was longer and ambient temperature was higher during the reproductively inactive phase. During daytime these birds hide in a dark burrow where, in general, the intensities of light and temperature are less, and the amplitude of variations of these factors is not prominent. Hence, the seasonal variations in these oscillatory components may not have affected the entrainment of the owlet pineal oscillator, which regulates the daily MEL rhythm in a similar pattern during both the studied phases. On the other hand, a single circadian peak (around 1000 h) circadian cyclicity of gonadal steroids (i.e., testosterone in the male and estradiol and progesterone in the female) showed an inverse relationship with plasma MEL. Possibly, MEL regulates the daily steroidogenic status in owlets by an inhibitory influence.     

Antigonadotropic activity of pineal gland of the Indian palm squirrel Funambulus pennanti

Bioassay of the pineal extract of F. pennanti was performed in immature female mice which was previously sensitized with human chorionic gonadotrophin. Reduction of ovarian and uterine weights indicated an antigonadotropic nature of the pineal gland of this animal.     

Melatonin blocks dexamethasone-induced immunosuppression in a seasonally breeding rodent Indian palm squirrel, Funambulus pennanti

In vivo effect of dexamethasone and melatonin on immunomodulation has been investigated by studying the lymphocyte proliferation to the mitogen Con A from various lymphoid tissues including bone marrow cells of a seasonally breeding rodent adult male F. pennanti during reproductively inactive phase (October to December). During this phase, animal faces the maximum challenges of the nature (hypothermic stress, scarcity of food and shelter). Dexamethasone treatment (60 microg/day/squirrel) for 60 consecutive days significantly decreased the thymus and spleen activity. The lymphoid tissues mass, total leukocyte, lymphocyte count of peripheral blood, bone marrow and T-cell mediated immune function was also significantly suppressed following the dexamethasone treatment but treatment of melatonin (25 microg/squirrel/day) along with dexamethasone significantly restored the suppressed immune status in squirrels. Further, histological study of the thymus showed profound changes in the cellularity with a depletion of thymocytes in the cortex region of thymic lobules and increased in connective tissues and spindle cells. Melatonin treatment alone increased thymocytes density in thymic cortex, clearly suggesting that melatonin counteracted the experimentally induced immune stress by dexamethasone. Therefore, in nature during reproductively inactive phase of the squirrel a high level of melatonin was noted, that is required to combat nature's stress, which might have increased the internal level of corticoids.     

Effects of Pinealectomy and Exogenous Melatonin on the Thyroid Gland Activity Varies in Relation to Reproductive Status of Male Spotted Munia (Lonchura punctulata; Aves,Passeriformes)

The purpose of this investigation was to explore whether the pineal organ and its hormone melatonin has any influence on the activity of thyroid glands, if so, how that relates to the reproductive status of a hitherto unstudied seasonally breeding wild bird. Accordingly, an identical experimental regimen was followed with adult male spotted munia (Lonchura punctulata; Passeriformes) during both its gametogenically active (August-September) and inactive (March-April) phases of the annual reproductive cycle. In either case, the levels of circulating thyroid hormones (both T₃ and T₄) and cellular characteristics of thyroid glands in groups of birds were studied following surgical removal of the pineal gland and/or daily afternoon administration of melatonin (10 μg/ 100 g body weight/ day for 30 days). The results of the same experimental schedule were found to be different depending on the sexual status of the concerned birds. During the breeding phase, pinealectomy (Px) induced significantly decreased values of T₃ and increased for T₄ along with hypertrophy of the thyroid follicular cells (TFC). The changes were reversed in melatonin treated Px birds. An increased amount of T₃ and decreased concentration of serum T₄ were also observed in melatonin injected intact birds. Conversely, the responses of TFC and of thyroid hormones in blood to either Px, or Px with melatonin, or to melatonin alone in intact munias during their inactive reproductive phase were just opposite to those noted during the breeding phase. The results of the present study suggest an influence of the pineal upon the thyroid in spotted munia. Moreover, it appears that this influence is carried out by melatonin, the action of which is reversible in relation with the gametogenic status of the concerned avian species.     

Circadian Rhythms of Melatonin and Sex Steroids in a Nocturnal Bird, Indian Spotted Owlet Athene brama During Reproductively Active and Inactive Phases

The epiphyseal neurohormone melatonin (MEL) exhibits circadian cyclicity, as noted extensively in diurnal vertebrates although very little information is available regarding nocturnal species. We have studied the MEL rhythmicity with 24-hour periodicity in a tropical nocturnal bird, Indian spotted owlet Athene brama, which possesses a well-developed pineal organ. We performed our study during two crucial reproductive phases (active and inactive), when the pineal gland activity in owlet exists in inverse states, i.e., inactive and active respectively. Independent of sex, the circadian rhythm of plasma MEL in owlets showed a two-peak cyclicity with a smaller peak at around 1400 h and the higher one at about 0200 h, while the lowest value was found at 1000 h. The night (0200 h) peak activity of plasma MEL in owlet has a resemblance with the earlier findings in diurnal birds and strongly suggests that independent of species habit the peak activity of MEL is invariably dark dependent. However, the daytime peak of MEL may be due to the daytime hiding nature of this nocturnal bird. Interestingly, it was also noted that the hours of peak activity of MEL (1400 and 0200 h) were the same during both of the reproductive phases, though the environmental day length was longer and ambient temperature was higher during the reproductively inactive phase. During daytime these birds hide in a dark burrow where, in general, the intensities of light and temperature are less, and the amplitude of variations of these factors is not prominent. Hence, the seasonal variations in these oscillatory components may not have affected the entrainment of the owlet pineal oscillator, which regulates the daily MEL rhythm in a similar pattern during both the studied phases. On the other hand, a single circadian peak (around 1000 h) circadian cyclicity of gonadal steroids (i.e., testosterone in the male and estradiol and progesterone in the female) showed an inverse relationship with plasma MEL. Possibly, MEL regulates the daily steroidogenic status in owlets by an inhibitory influence.     

Melatonin in immunity: comparative aspects

Pineal gland, by the diurnal rhythm of synthesis and release of its principal hormone, melatonin (MEL), is involved in reciprocal relationships between neuroendocrine and immune systems, responsible for keeping internal homeostasis in vertebrate animals. In this paper the experimental data, indicating that both strategic (developmental, thus antigen independent) and emergency (evoked by antigenic activation of the mature immune system) levels of interactions between pineal gland and immune system, operate in mammals and birds, are reviewed. The cells and organs of immune system using membrane receptors as well as nuclear orphan receptors perceive MEL message. Effects exerted by MEL on immune parameters are different, and depend on several factors, including dose and way of MEL application, species, sex, age of animal, its immune system maturation, way of immune system activation, and parameter examined, as well as the season, circadian rhythm of both immunity and pineal gland function, stressful conditions, accompanying experimental procedure, etc. In turn, lymphoid organ-derived hormones and cytokines, soluble factors secreted by activated immune cells act as messages understood by the pineal gland, closing the regulatory loop of the bi-directional functional connections between both systems.     

Glia-pinealocyte network: the paracrine modulation of melatonin synthesis by tumor necrosis factor (TNF)

The pineal gland, a circumventricular organ, plays an integrative role in defense responses. The injury-induced suppression of the pineal gland hormone, melatonin, which is triggered by darkness, allows the mounting of innate immune responses. We have previously shown that cultured pineal glands, which express toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1), produce TNF when challenged with lipopolysaccharide (LPS). Here our aim was to evaluate which cells present in the pineal gland, astrocytes, microglia or pinealocytes produced TNF, in order to understand the interaction between pineal activity, melatonin production and immune function. Cultured pineal glands or pinealocytes were stimulated with LPS. TNF content was measured using an enzyme-linked immunosorbent assay. TLR4 and TNFR1 expression were analyzed by confocal microscopy. Microglial morphology was analyzed by immunohistochemistry. In the present study, we show that although the main cell types of the pineal gland (pinealocytes, astrocytes and microglia) express TLR4, the production of TNF induced by LPS is mediated by microglia. This effect is due to activation of the nuclear factor kappa B (NF-kB) pathway. In addition, we observed that LPS activates microglia and modulates the expression of TNFR1 in pinealocytes. As TNF has been shown to amplify and prolong inflammatory responses, its production by pineal microglia suggests a glia-pinealocyte network that regulates melatonin output. The current study demonstrates the molecular and cellular basis for understanding how melatonin synthesis is regulated during an innate immune response, thus our results reinforce the role of the pineal gland as sensor of immune status.     

Pineal gland of a nocturnal bird, Indian spotted owlet, Athene brama: morphological and endocrine observations

It has been reported that owls (Strigiformes) do not have a pineal gland. However, our light microscopy study revealed an intermediate form of tubulofollicular and solid-type large pineal gland in a tropical owlet, Athene brama. The epithelial cells forming follicles (6-8) in the distal region and the solid cluster of parenchymal cells of different diameters in the proximal region anteriorly tapered with a long cylindrical stalk and continued into commissural organs and choroid plexus. The intrapineal localization of perivascular nerve fibers and blood vessels clearly explained the sympathetic innervation as well as vascularization of this neuroendocrine gland. Further, electron microscopy revealed a developed intracellular structure of the pinealocytes with a large number of mitochondria, Golgi bodies, and granular as well as clear vesicles in the process terminals. The evidence of intrapinealocyte lipid droplets and dense bodies and a moderate amount of melatonin in plasma (ranging from 100-365 pg/mL) during different reproductive phases finally proved a defined secretory activity of the gland in this tropical, nocturnal bird.     

Melatonin mediates seasonal adjustments in immune function.

In addition to seasonal changes in reproductive function, seasonal changes in immune function are mediated by the pineal hormone, melatonin. Melatonin affects immune function both indirectly, acting through other hormones, and directly by acting on components of the immune system. Melatonin also affects tumorigenesis and tumor development. We hypothesize that many of the indirect effects of melatonin on immune function are mediated through glucocorticoids, and appear to be part of an integrated series of adaptations to manage energy. Direct effects of melatonin on immune function appear to be mediated by melatonin receptors on lymphatic tissue or on immune cells in circulation. Winter is energetically demanding and stressful; thermoregulatory demands typically increase when food availability decreases. Individuals would enjoy a survival advantage if seasonally recurring stressors could be anticipated and countered by bolstering immune function. To summarize, melatonin may be part of an integrative system to coordinate reproductive, immunologic and other physiological processes to cope successfully with energetic stressors during winter.