Age dependent expression of melatonin membrane receptor (MT1, MT2) and its role in regulation of nitrosative stress in tropical rodent Funambulus pennanti

Age-dependent declining level of melatonin induces free radical load and thereby deteriorates immune function. However, reports are lacking about age-dependent melatonin membrane receptor (MT1 & MT2) expression, their role in regulation of reactive nitrogen species (RNS) and eventually how they affect immunity of a tropical rodent F. pennanti. We checked MT1R, MT2R and iNOS expression in lymphoid organs of young middle and old aged squirrels. Nitrite and nitrate ion concentration (NOx) in lymphoid organs, testes and plasma, lymphocyte proliferation and IL-2 level was recorded. Age-dependent decrease in MT1 and MT2 receptor expression, lymphocyte proliferation, IL-2 level and increased RNS in lymphoid organs, testes and plasma was observed with decreased circulatory melatonin. Androgen and AR expression was increased in middle-aged while declined in old-aged squirrels. Present study suggests that age associated immunosenescence is consequence of increased RNS which might have important relationship with melatonin membrane receptors in F. pennanti.     

Age dependent expression of melatonin membrane receptor (MT1, MT2) and its role in regulation of nitrosative stress in tropical rodent Funambulus pennanti

Age-dependent declining level of melatonin induces free radical load and thereby deteriorates immune function. However, reports are lacking about age-dependent melatonin membrane receptor (MT1 & MT2) expression, their role in regulation of reactive nitrogen species (RNS) and eventually how they affect immunity of a tropical rodent F. pennanti. We checked MT1R, MT2R and iNOS expression in lymphoid organs of young middle and old aged squirrels. Nitrite and nitrate ion concentration (NOx) in lymphoid organs, testes and plasma, lymphocyte proliferation and IL-2 level was recorded. Age-dependent decrease in MT1 and MT2 receptor expression, lymphocyte proliferation, IL-2 level and increased RNS in lymphoid organs, testes and plasma was observed with decreased circulatory melatonin. Androgen and AR expression was increased in middle-aged while declined in old-aged squirrels. Present study suggests that age associated immunosenescence is consequence of increased RNS which might have important relationship with melatonin membrane receptors in F. pennanti.     

Reproductive phase dependent circadian variations of plasma melatonin, testosterone, thyroxine and corticosterone in Indian palm squirrel, Funambulus pennanti

To explain the complex mechanism of environmental influence along with internal hormonal (factors) milieu on daily variations in the circulating levels of melatonin, testosterone, thyroxine and corticosterone were analyzed with the help of inferential statistics (Cosinor rhythmometry) in a seasonally breeding tropical rodent, F. pennanti during the reproductively active (RAP) and inactive phases (RIP). Plasma melatonin, thyroxine and corticosterone levels exhibited a significant circadian oscillation during both the active and inactive phases of the annual reproductive cycle. Melatonin showed higher amplitude during RIP in the circulating plasma. Testosterone presented a peak level during evening hours (16:00 - 18:00 h) during RAP only. The phase of thyroxine was noted ∼09:76 h and ∼10.35 h during active and inactive phases, respectively. Corticosterone showed a peak level at ∼12.00 h during both phases of the reproductive cycle. Further, in this tropical rodent, the minimum difference in photoperiod (∼3 - 4 hours) and maximum variation in temperature (max. 18°C - min. 10°C during RIP and max. 45°C - min. 32°C during RAP) and humidity (85% during RIP and 35% during RAP) regulated the diurnal rhythm of circulating melatonin circadian rhythm by ∼1 hour phase advance during RIP. In conclusion, the studied hormonal rhythms may be part of an integrative system to coordinate reproduction and physiological processes successfully with environmental factors.     

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)     

Modulation of immunity in young-adult and aged squirrel, Funambulus pennanti by melatonin and p-chlorophenylalanine

Background  

Our interest was to find out whether pineal gland and their by melatonin act as modulator of immunosenescence. Parachlorophenylalanine (PCPA) – a β adrenergic blocker, is known to perform chemical pinealectomy (Px) by suppressing indirectly the substrate 5-hydroxytryptamine (5-HT) for melatonin synthesis and thereby melatonin itself. The role of PCPA, alone and in combination with melatonin was recorded in immunomodulation and free radical load in spleen of young adult and aged seasonal breeder Indian palm squirrel Funambulus pennanti.     

The pineal gland of the Indian palm squirrel, Funambulus pennanti (Wroughton)

In the adult palm squirrel, F. pennanti the pineal is a club shaped, elongated structure with a connective tissue capsule. It consists of various types of pinealocytes, glial cells, neurons, nerve fibres, blood vessels and connective tissue. Two types of pinealocytes could be identified by light microscopy. They are large rounded with centrally placed nucleus, and small rounded pinealocytes. They have medium sized processes stainable with Alcian blue, periodic acid Schiff and Nissl methods. The pinealocytes are not stainable with bromophenol blue. However, they are moderately stainable with PAS, Sudan black and Baker's acid hematin. Neurons are seen either singly or in groups with axonal processes. Cystic cavities often lined by cells are a normal feature of adult squirrel pineal, and the lining cells are both pinealocytes and glial cells. Often neuronal endings are seen terminating on these lining cells. PAS positive globules were also seen inside the cysts. In some squirrel pineals, fibrous cysts with an inner core of cells are also seen. Occasionally groups of lymphocytes were also encountered in the pineal. In the fetal pineal, the cells are both larger and smaller ones and arranged in a cortex and medulla pattern and no cystic cavities are seen. The third ventricle enters the base of the pineal as pineal recess.     

Maternal transfer of melatonin alters the growth and sexual maturation of young Indian palm squirrel Funambulus pennanti

To date, the phenomenon of maternal transfer of hormones to the young is an enigma. The present study explains for the first time the maternal transfer of melatonin (MEL) to the young, affecting neonatal growth and sexual maturation. The suckling pups of MEL-treated mothers exhibited significant decreases in body, testicular, vas deferens (male pups), ovarian and uterine (female pups) weights and increases in pineal gland activity along with high plasma MEL levels. The plasma level of testosterone decreased significantly in male pups, while estradiol increased and progesterone decreased in female pups of MEL-treated mothers. These results clearly suggest that MEL could be transported from the mothers to their young postnatally via the milk in order to influence neonatal growth and sexual maturation. Our results support the earlier concept and show for the first time that MEL can be transported from the mother to the young either prenatally through the placenta or postnatally via the milk. Therefore, maternal MEL can act as a biological signal for neonatal growth and sexual maturation.     

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.     

Role of melatonin and HSF-1\HSP-70 in modulating cold stress-induced immunosuppression in a tropical rodent- Funambulus pennanti

1. Download : Download high-res image (282KB)
2. Download : Download full-size image

     

Effect of metopirone on pituitary gland of male squirrel, Funambulus pennanti (Wroughton)

1. The ACTH cell type has been demonstrated for the first time in the hypophysis of the squirrels. These are large cells with homogenous cytoplasm and are more abundant in the anteromedian zone and in the peripheral portion than elsewhere. 2. A great hypertrophy and hyperplasia of the pituitary corticotrophs were observed in the experimental group at all the intervals. 3. Metopirone treated animals showed a moderate hypertrophy and degranulation of pituitary thyrotrophs. 4. By effect of metopirone, a marked regression of pituitary interstitiotrophs and total degranulation took place by 30 days treatment. The folliculotrophs did not show alterations.     

Seasonal reproductive periodicity and thyroid fluctuations in female Indian palm squirrel, Funambulus pennanti.

Seasonal reproductive rhythmicity and concomitant thyroid fluctuations have been studied in the female Indian palm squirrel, Funambulus pennanti. The majority of the squirrels enter estrus during March and April when ovarian follicles attain maximum diameter. Ovulation starts in April and continues till June. A large number of degenerating follicles, indicating decline in ovarian functions, is a characteristic feature of the ovary in August. Minimum percent thyroid 131I uptake is recorded during May and June, i.e. at the onset of mating season. In July, which also marked the gestation period in the majority of the squirrels dissected, thyroid activity significantly increases. Gonadal regression begins in August and this is accompanied by corresponding fall in the thyroid 131I uptake. Alterations in thyroid cytoarchitecture during different months of the year are in accordance with the results obtained from radioiodine uptake studies. Fluctuations in thyroid functions during gonadal regression, recrudescence and peak gonadal activity are discussed in the present communication.     

Cytology of the hypophysis, pars anterior of the Indian five-striped palm squirrel, Funambulus pennanti (Wroughton)

By employing various staining techniques the pituitary cells of the squirrels, Funambulus pennanti, can broadly be divided into 'acidophilic', 'basophilic' and 'amphophilic' types. The acidophilic cell types include somatotrophs and lactotrophs. The basophilic cell types include thyrotrophs, folliculotrophs and interstitiotrophs while the amphophilic cells are recognized as corticotrophs. The identification of the pituitary cells and their histochemical characteristics are described at the light microscopical level. Attempt has been made to interpret the cells identified in the present study on a comparative and tinctorial basis.     

The expression of MT1 and MT2 melatonin receptor mRNA in several rat tissues

The mechanisms that mediate the various effects of melatonin in mammalian tissues are not always known. Therefore, the aim of this study was to investigate whether MT(1) and MT(2) melatonin receptors are expressed in certain tissues of the rat. The expression of MT(1) and MT(2) melatonin receptor mRNA was determined using a real-time quantitative RT-PCR method. In addition, we examined whether mRNA for either subtype of receptor shows any difference in the expression between midnight and noon, similar to the changes in melatonin concentrations in plasma and tissue samples. MT(1) and MT(2) melatonin receptor mRNAs were found in the rat hypothalamus, retina and small intestine. We also showed a low expression of MT(2) mRNA in the rat liver and heart SA node. In the heart apex and the Harderian gland, no appearance of either of the receptor mRNAs was detectable. A significant difference in the expression of MT(1) mRNA between day and night was found in the hypothalamus. In conclusion, our findings suggest that at least some effects of melatonin are mediated through membrane MT(1) and MT(2) receptors in the hypothalamus, the retina and the small intestine. Down-regulation of receptors might be one reason for the difference in the hypothalamic MT(1) melatonin receptor mRNA expression between midnight and noon. In the liver and the heart SA node, the physiological significance of possible MT(2) receptors remains unclear. According to our negative midnight and noon results in the Harderian gland and heart apex melatonin may exert its effect on these tissues by a non-receptor mechanism.     

Gonado-modulatory effects of high humidity is pineal dependent in Indian palm squirrel Funambulus pennanti (Wroughton).

F. pennanti presented a clear biphasic pineal mediated seasonal sexual cycle. This sexual cycle was essentially characterised by a very short period of sexual quiescence with an arrest of spermatogenesis during October-November. A small but clear decrease in sexual activity was also observed during March-April. This decrease in sexual activity, however, had no quantifiable effect on spermatogenesis. Sexual recrudescence was observed from December-January. The testes remained sexually active from January till September. Almost an inverse relationship was observed between pineal and testicular weight. Pinealectomy, however, prevented naturally induced gonadal regression during both the periods, i.e. September-November and February-April. Exposure of animals to high RH (80 +/- 4%) during sexually active phase induced a steep regression in testicular weight of sham-operated animals even in the presence of gonad stimulatory long photoperiod (16L:8D) and high temperature (40 degrees +/- 5 degrees C) while exposure of animals to moderate RH (65 +/- 5%) during sexual regression phase partially prevented testicular regression even in presence of inhibitory short photoperiod (11L:13D) and normal environmental temperature (30 degrees +/- 5 degrees C). Pinealectomized animals, neither exhibited testicular regression in February-March nor had involuted testes in September-October, thus, suggesting that the effect of humidity is mediated via the pineal gland.     

Pineal modulation of thymus and immune function in a seasonally breeding tropical rodent, Funambulus pennanti

The immune system driven by cytokines is now known to be influenced by various other endocrine glands and its hormones. Results of the present study indicate a bidirectional relation between the pineal-thymus axis and the immune system status of an Indian tropical rodent, Funambulus pennanti, during winter months (reproductive inactive phase), when it faces maximum challenges from nature. Pinealectomy during the reproductive inactive phase inhibited thymus and spleen functions, which resulted in significant changes in leukocyte and lymphocyte counts and T-cell-mediated immune function (measured in terms of delayed-type hypersensitivity response to oxazolone). Blastogenic responses of lymphoid cells (thymocytes, splenocytes, and lymph node cells) also decreased following ablation of the pineal gland. To check the definite role of the pineal gland we injected melatonin into pinealectomized squirrels, and the suppressed immune function was significantly restored. Neuroendocrine control of the pineal gland on the histocompatible tissues in this seasonal breeder, F. pennanti, suggests an adaptive mechanism of the immune system for survival in the tropical zone. J. Exp. Zool. 289:90-98, 2001.     

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.     

Age- and sex-dependent effect of exogenous melatonin on expression pattern of melatonin receptor (MT1 and MT2) proteins in spleen of mice

Spleen is an important lymphoid organ which exerts immune activities throughout the life in mammals. In this study, we investigated the age- and sex-dependent effect of exogenous melatonin on expression pattern of MT1 and MT2 melatonin receptor proteins in spleen of laboratory Swiss albino mice in three different age-groups – 2, 4, and 8 months. The melatonin receptor expression patterns were studied by immunohistochemical localization and Western blot analysis. Immunohistochemical study showed reactivity of MT1 and MT2 melatonin receptors in spleen of both male and female mice. Exogenous melatonin significantly showed age- and sex-dependent expression pattern of MT1 receptor protein, while MT2 receptors showed only age-dependent differential expression patterns in both male and female mice. Therefore, this study may suggest that exogenous melatonin is modulating MT1 and MT2 receptor protein expression pattern in age- and sex-dependent manner in spleen of mice.