Shift and longtime light induces endometrioid adenocarcinoma via activation of PKC-α/Akt pathway in female golden hamster: Involvement of altered Aanat and Bmal1 rhythm.

Female night-workers get exposed to frequent light shifts, hence have altered circadian rhythm and are at high risk of endometrial cancer; the underlying mechanism however is still not clear. We, therefore examined the effect of long light exposure (16L:8D, LD1) and regular shift (8 h) in long nighttime (LD2) on endometrial changes of female golden hamsters. Morphometric analysis, scanning electron microscopy imaging, alcian blue staining, and cytological nuclear atypia of endometrial stromal cells confirmed the incidence of endometrial adenocarcinoma in LD2 exposed hamsters. But, less severe pathomorphological alterations were noted in uterus of LD1 exposed hamsters. Altered Aanat and Bmal1 mRNA, melatonin rhythm, downregulation of important marker gene of adenocarcinoma like Akt, 14-3-3, and PR protein expression and upregulation PKCα, pAkt-S473 and vascular epithelial growth factor (VEGF) were observed in LD2 exposed hamsters suggesting the endometrial adenocarcinoma. Further, our western blot analysis supported the immunohistochemical localization of PR, PKCα, and VEGF in uterine tissues along low progesterone. Overall, our data indicates that light shift and long light exposure potentially induced endometrioid adenocarcinoma via activation of PKC-α/Akt pathway in female hamsters. Therefore, duration of light is essential for female normal uterine function.

BPS-induced ovarian dysfunction: Protective actions of melatonin via modulation of SIRT-1/Nrf2/NFĸB and IR/PI3K/pAkt/GLUT-4 expressions in adult golden hamster.

Ever-increasing occurrence of plastic-manufacturing industries leads to environmental pollution that has been associated with declined human health and increased incidence of compromised reproductive health. Female subfertility/infertility is a complex phenomenon and environmental toxicants as well as lifestyle factors have a crucial role to play. Bisphenol S (BPS) was believed to be a "safer" replacement of bisphenol A (BPA) but recent data documented its neurotoxic, hepatotoxic, nephrotoxic, and reprotoxic attributes. Hence based on the scarcity of reports, we investigated molecular insights into BPS-induced ovarian dysfunction and protective actions of melatonin against it in adult golden hamsters, Mesocricetus auratus. Hamsters were administered with melatonin (3 mg/kg BW i.p. alternate days) and BPS (150 mg/kg BW orally every day) for 28 days. BPS treatment disrupted hypothalamo-pituitary-ovarian (HPO) axis as evident by reduced gonadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH), ovarian steroids such as estradiol (E2) and progesterone (P4), thyroid hormones namely triiodothyronine (T3) and thyroxine (T4) and melatonin levels along with their respective receptors (ERα, TRα, and MT-1) thereby reducing ovarian folliculogenesis. BPS exposure also led to ovarian oxidative stress/inflammation by increasing reactive oxygen species and metabolic disturbances. However, melatonin supplementation to BPS restored ovarian folliculogenesis/steroidogenesis as indicated by increased number of growing follicles/corpora lutea and E2/P4 levels. Further, melatonin also stimulated key redox/survival markers such as silent information regulator of transcript-1 (SIRT-1), forkhead box O-1 (FOXO-1), nuclear factor E2-related factor-2 (Nrf2), and phosphoinositide 3-kinase/protein kinase B (PI3K/pAkt) expressions along with enhanced ovarian antioxidant capacity. Moreover, melatonin treatment reduced inflammatory load including ovarian nuclear factor kappa-B (NFĸB), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) expressions, serum tumor necrosis factor α (TNFα), C-reactive protein (CRP) and nitrite-nitrate levels as well as upregulated ovarian insulin receptor (IR), glucose uptake transporter-4 (GLUT-4), connexin-43, and proliferating cell nuclear antigen (PCNA) expressions in ovary thereby ameliorating inflammatory and metabolic alterations due to BPS. In conclusion, we found severe deleterious impact of BPS on ovary while melatonin treatment protected ovarian physiology from these detrimental changes suggesting it to be a potential preemptive candidate against environmental toxicant-compromised female reproductive health.

Melatonin attenuates LPS-induced ovarian toxicity via modulation of SIRT-1, PI3K/pAkt, pErk1/2 and NFĸB/COX-2 expressions.

The association between inflammation and metabolic disturbances leads to various female pathophysiological conditions. Bacterial lipopolysaccharide (LPS), found in the outer membrane of gram-negative bacteria, elicits an oxidative and inflammatory response that profoundly interferes with female reproductive health. We investigated the ameliorative action of melatonin on LPS-induced ovarian pathophysiology in golden hamsters, Mesocricetus auratus. Hamsters were administered with exogenous melatonin (5 mg/kg BW) and LPS (100 µg/kg BW) intraperitoneally for 7 days. LPS treatment impaired ovarian folliculogenesis as evident by histoarchitecture (elevated number of atretic follicles, reduced number of growing follicles and corpus luteum) and steroidogenesis (decreased aromatase/ERα, estradiol and progesterone). On the other hand, LPS administration also perturbed thyroid hormone (T3 and T4) homeostasis, ovarian melatonin receptor (MT-1) expression, antioxidant potential (SOD and catalase) and concomitantly elevated nitro-oxidative stress (decreased SOD, catalase and elevated CRP, TNFα and nitrate/nitrite level) and inflammatory load (NFĸB and COX-2) which culminated into ovarian follicular apoptosis (elevated caspase-3). LPS also disrupted metabolic homeostasis as indicated by hyperinsulinemia with a simultaneous decrease in ovarian IR/GLUT-4 and glucose content. Moreover, LPS treatment decreased expressions of key markers of ovarian physiology (SIRT-1, pErk1/2, PI3K and pAkt). Melatonin co-treatment with LPS improve these detrimental changes proposing melatonin as a potent therapeutic candidate against ovarian dysfunction induced by endotoxin.

Continuous artificial light potentially disrupts central and peripheral reproductive clocks leading to altered uterine physiology and reduced pregnancy success in albino mice.

AIMS: The mechanism behind clock coordination in female reproductive disorders is poorly understood despite the known importance of coordinated and synchronized timing of central and clocks in reproductive organs. We investigated the effect of continuous artificial light (LL) on the central and peripheral reproductive clock gene (Bmal1, Clock, Per1, Per2 and Cry1) and its downstream regulators (Hgf, PR-A and HOXA10) during non-pregnancy and pregnancy phases of female mice. MAIN METHODS: Mice (n = 60) in two sets, were maintained under continuous light (LL) and natural day cycle (LD;12L: 12D) for both non-pregnant and pregnant study. Tissues from hypothalamus-containing SCN, ovary, uterus and serum were collected at different zeitgeber time points (ZT; at 4-h intervals across 24-h periods). KEY FINDINGS: LL exposure desynchronized the expressions of the clock mRNAs (Bmal1, Clock, Per1, Per2 and Cry1) in SCN, ovary, and uterus along with Hgf mRNA rhythm. LL significantly increased the thickness of endometrial tissues. Furthermore, the pregnant study revealed lower serum progesterone level during peri- and post-implantation under LL along with downregulated expression of progesterone receptor (PR) as well as progesterone dependent uterine Homeobox A-10 (Hoxa10) proteins with lowered pregnancy outcomes. SIGNIFICANCE: Our result suggests that LL disrupted the circadian coordination between central and clock genes in reproductive tissue leading to interrupted uterine physiology and altered pregnancy in mice. This led us to propose that duration of light exposure at work-places or home for females is very important in prevention of pregnancy anomalies.

Stress associated ovarian dysfunctions in a seasonal breeder Funambulus pennanti: Role of glucocorticoids and possible amelioration by melatonin.

Studies have shown that stress caused by lack of physical activity disrupts the normal pattern of glucocorticoid secretion which adversely affects the reproductive axis. We studied the effect of chronic movement restriction on ovarian responses in the Indian Palm Squirrel Funambulus pennanti, a highly active diurnal rodent. Physical restraint of squirrels induced stress that led to a significant increase in plasma cortisol, corticosterone and decreased 17ß-estradiol level leading to follicular atresia. Ovarian Reactive Oxygen Species (ROS) content, lipid peroxidation (LPO), activities of superoxide dismutase (SOD) and catalase (CAT) enzymes increased in restrained squirrels. Elevated ROS increased the oxidative load that led to ovarian cell death as evidenced by increased Bax and decreased Bcl2 expression causing further decline in Aromatase and ERα proteins. To elaborate the mechanism(s) involved in stress induced glucocorticoid mediated oxidative damages to the ovary we extended our study by exposing ovaries in vitro to the synthetic glucocorticoid dexamethasone (200 µM). We observed that glucocorticoid receptor (GR) expression was significantly increased in dexamethasone treated ovaries in vitro with a decrease in expression of Nrf2 and HO-1 proteins. Melatonin supplementation (10 nM) along with dexamethasone significantly decreased ovarian ROS production, lipid peroxidation and increased antioxidant enzyme activities by improving the expression of Nrf2 and HO-1, reinstating the cellular redox homeostasis. Therefore, it can be suggested that physical restraint induced glucocorticoid and its receptor activation interfered with the ovarian antioxidant defense mechanism. Melatonin via its receptor MT1 significantly alleviated ovarian damages acting as a cytoprotective agent.

Photoperiodic modulation of ovarian metabolic, survival, proliferation and gap junction markers in adult golden hamster, Mesocricetus auratus.

Female reproductive physiology is greatly dependent on tight regulation of metabolic and survival factors. Photoperiod regulates female reproductive rhythms but very less information exists explaining whether photoperiod could modulate thyroid hormone homeostasis, metabolic/energy parameters along with survival, proliferation and gap junction proteins in the ovary of a long-day breeder, Mesocricetus auratus. Adult female hamsters were exposed to different photoperiodic regimes i.e., critical photoperiod (CP; 12.5L:11.5D), short photoperiod (SP; 8L:16D) and long photoperiod (LP; 16L:8D) for 12 weeks. LP upregulated thyroidal and gonadal activity as apparent by histoarchitecture, thyroid hormone profile [triiodothyronine (T3), thyroxin (T4) and thyroid stimulating hormone (TSH)], luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol (E2) and progesterone (P4) levels when compared with SP exposed hamsters. Further, LP increased thyroid hormone receptor-α/deiodinase-2 (TRα/Dio-2), estrogen receptor-α (ERα)/aromatase and insulin receptor/glucose transporter-4 (IR/GLUT-4) expressions in ovary. Interestingly, ovarian sirtuin-1 (SIRT-1) expression was also upregulated under LP condition along with cell proliferation (proliferating cell nuclear antigen or PCNA), survival (B cell lymphoma-2 or Bcl-2) and gap junction (connexin-43) markers when compared to SP exposed hamsters. We also noted elevated levels of circulatory leptin, insulin along with melatonin and its receptor (MT-1) in ovary under SP condition. Thus, we suggest that photoperiod plays a vital role in regulation of thyroid and reproductive hormone homeostasis along with key metabolic and survival markers in the ovary of adult golden hamsters, M. auratus providing further insight into the regulation of female reproductive seasonality in a long-day breeder.

Melatonin induces apoptosis and cell cycle arrest in cervical cancer cells via inhibition of NF-κB pathway.

Cervical cancer is the most prevalent cancer in females. Melatonin, a neurohormone has been documented as a promising therapeutic molecule for cervical cancer. However, the underlying molecular mechanism is not known. We explored the dose-dependent anti-tumor response of melatonin against cervical cancer cell lines, HeLa (HPV-18 positive) and SiHa (HPV-16 positive). The anti-cancer effect of melatonin was evaluated by MTT assay, cell imaging, colony formation, DAPI, AO/PI, LDH, Flow cytometry, scratch assay, western blot analysis and real-time PCR. Results of DAPI, AO/PI, LDH, and Annexin/PI staining revealed that melatonin induces apoptosis. The results of cell cycle analysis revealed that melatonin arrests the HeLa and SiHa cells in sub-G1 and G1 phases, respectively. Western blot analysis revealed that melatonin downregulated the expression of pro-inflammatory transcription factor, NF-κB and the expression of COX-2 protein, a key mediator in cell proliferation. In addition, melatonin downregulated the expression of an invasive marker, MMP-9, an antiapoptotic protein, Bcl-2, and upregulated the expression of pro-apoptotic protein, Bax at both transcriptional and translational levels. Overall, the results suggest that melatonin exhibited strong anti-cancer therapeutic potential against human cervical cancer cell line progression possibly through inhibition of NF-κB signalling pathway.

Retinal and extra-retinal photoreceptor responses and reproductive performance of Japanese quail (Coturnix coturnix japonica) following exposure to different photoperiodic regime.

Japanese quail is a truly photoperiodic avian species. In general long days are gonado-stimulatory and short days are gonado-inhibitory for this poultry bird. To investigate the correlation of retinal and extra-retinal photoreceptors with different photoperiodic conditions quail were divided into 2 groups and kept under long day (16L: 8D) and short day (8L: 16D) condition separately to develop photosensitivity and scotosensitivity respectively. Transfer of long day quail to intermediate day-length (13.5L: 10.5D) developed photorefractoriness (relative) and prolonged exposure to short photoperiodic conditions led the birds to develop scotorefractoriness. Increased expression of mRNA and immunosignaling of photoreceptors rhodopsin, transducin in eye and hypothalamus while decreased mRNA expression of melatonin receptors (Mel1b, Mel1c) were noted in the eyes of photosensitive (PS) and scotorefractory (SR) quail compared to photorefractory (PR) and scotosensitive (SS) birds respectively. Decreased expression of hypothalamic GnIH and melatonin receptors mRNA was observed in PS and SR birds compared to PR and SS birds respectively. Modulation of retinal and extra retinal photoreceptors leads to increased spermatogenesis as well as mRNA expression of steroidogenic genes and androgen receptor in the testis of sexually active PS and SR quail. These results led us to conclude that gonadal stimulation in PS as well as SR quail is outcome of activated retinal and extra retinal photoreceptors which lowered melatonin receptors and GnIH expression. Contrarily testicular inhibition in PR and SS is the outcome of decreased photoperception. It is suggested that decreased photoperception in SS quail increases after prolong exposure of the short day (in SR) leading to increased activity of HPG axis.

Melatonin ameliorates Bisphenol S induced testicular damages by modulating Nrf-2/HO-1 and SIRT-1/FOXO-1 expressions.

BPS has detrimental effects on human reproductive health and emerged as an environmental contaminant for global health concern. This study deals with the adverse impact of BPS exposure on testicular oxidative stress, inflammation and apoptosis in adult male golden hamster, Mesocricetus auratus and its amelioration by melatonin. BPS (75 mg/kg BW/day) exposure caused testicular impairment as evident by histological degenerative changes, declined sperm quality (viability and motility), serum levels of testosterone and melatonin with a concomitant decrease in testicular androgen receptor (AR) and melatonin receptor (MT1) expression. The BPS exposure caused marked increase in testicular oxidative load, inflammation (NF-kB/COX-2) and apoptosis (caspase-3). Melatonin (10 mg/kg BW/alternate day) pretreatment to BPS exposed hamsters resumed normal testicular histoarchitecture, sperm quality and decreased testicular oxidative load as evident by enhanced antioxidant enzymes (SOD and catalase) activities and decreased lipid peroxidation (LPO) level. Further, melatonin also stimulated the testicular antioxidant proteins Nrf-2/HO-1, SIRT-1/FOXO-1 and reduced inflammatory proteins NF-kB/COX-2 expression to counteract BPS induced testicular damages. Melatonin administration to the BPS treated hamsters resulted in increased testicular cell proliferation (PCNA), survival (Bcl-2), gap junction (connexin-43) and decreased apoptosis (caspase-3). In conclusion, our study documented the detrimental effects of BPS on testes that compromises male fertility. Further, melatonin was found as a potent molecule that rescued the BPS induced testicular damages in male golden hamster Mesocricetus auratus.

Role of melatonin and HSF-1\HSP-70 in modulating cold stress-induced immunosuppression in a tropical rodent- Funambulus pennanti [Volume 87, January 2020, 102456].

The neuroendocrine hormone melatonin and molecular chaperones (heat shock proteins) are evolutionarily conserved molecules that play an important role in protecting organisms from abiotic and biotic stressors. Environmental temperature and seasonality modulates immunity which impacts the overall health of animals. Most studies in relation to thermal stress are based on animals inhabiting temperate zones however, the substantial effect of climatic stress on tropical animals is less explored. Therefore, in this study we focused on the immunosuppressive effect of cold environment on a seasonally breeding tropical rodent and highlighted the importance of melatonin and HSF-1/Hsp-70 in regulating immunity. Animals were exposed to different temperatures with or without melatonin treatment. Our results suggest that, low temperature elicited cold-associated stress in animals marked by reduced body weight, decreased TLC/LC count in the blood and increased corticosterone production which was central to all immune alterations. Cold temperature also increased the oxidative stress which further induced apoptosis in the immune cells and activated stress response molecular chaperones HSF-1/HSP-70. Exogenous melatonin treatment not only ameliorated cold-induced immune suppression but also upregulated the expression of HSF-1 and HSP-70 in the immune cells thereby preventing protein unfolding and cell death. Thus, we conclude that melatonin and molecular chaperones synergistically alleviated immune suppression and could emerge as a promising combination therapy to target temperature stress in animals while boosting immunity.

Short day length enhances physiological resilience of the immune system against 2-deoxy-d-glucose-induced metabolic stress in a tropical seasonal breeder Funambulus pennanti.

Studies demonstrate the importance of metabolic resources in the regulation of reproduction and immune functions in seasonal breeders. In this regard, the restricted energy availability can be considered as an environmental variable that may act as a seasonal stressor and can lead to compromised immune functions. The present study explored the effect of photoperiodic variation in the regulation of immune function under metabolic stress condition. The T-cell-dependent immune response in a tropical seasonal breeder Funambulus pennanti was studied following the inhibition of cellular glucose utilization with 2-deoxy-d-glucose (2-DG). 2-DG treatment resulted in the suppression of general (e.g., proliferative response of lymphocytes) and antigen-specific [anti-keyhole limpet hemocyanin IgG titer and delayed-type hypersensitivity response] T-cell responses with an activation of the hypothalamic-pituitary-adrenal axis, which was evident from the increased levels of plasma corticosterone. 2-DG administration increased the production of inflammatory cytokines [interleukin (IL)-1ß and tumor necrosis factor (TNF)-α] and decreased the autocrine T-cell growth factor IL-2. The immunocompromising effect of 2-DG administration was retarded in animals exposed to short photoperiods compared with the control and long photoperiod-exposed groups. This finding suggested that short photoperiodic conditions enhanced the resilience of the immune system, possibly by diverting metabolic resources from the reproductive organs toward the immune system. In addition, melatonin may have facilitated the energy "trade-off" between reproductive and immune mechanisms, thereby providing an advantage to the seasonal breeders for their survival during stressful environmental conditions.

Photoperiodic modulation of local melatonin synthesis and its role in regulation of thymic homeostasis in Funambulus pennanti.

The effect of photo-neuroendocrine system on the thymic (immune) functions is mediated by gonadal steroid and the pineal hormone melatonin. The present study explored the effect of photoperiod on the thymic melatonergic system and its role in protection of thymic T-cells from the testosterone induced seasonal oxidative stress and apoptosis. Exposure to long day-length (LD) was noted to decrease local (thymic) melatonin content and induce oxidative stress and apoptosis in the thymus. Increased peripheral level of testosterone upregulated the androgen receptor expression and, consequently reduced proliferation response of the thymocytes. Short day conditions (SD) however, reversed the effect of LD on the thymic physiology. Low level of testosterone was concomitant with diminished nitro-oxidative stress and decreased expression of redox sensitive factors (NF-κB, p53 and Bax/Bcl-2 ratio) in the thymus. SD retarded activation of caspase-3 resulting in procaspase-3 accumulation. Further, in vitro treatment of thymocytes with AR antagonist flutamide impaired the sensitivity of thymocytes to androgen and reversed the deleterious effects of testosterone on the proliferative and apoptotic responses of thymocytes. Therefore, it can be suggested that thymus derived melatonin protects thymic T-cells from testosterone induced seasonal oxidative stress, apoptosis and also acts as a potent paracrine factor for maintenance of redox status to ensure thymocyte survival.

Effect of Naltrexone on photoperiodic regulation of testicular steroidogenesis in adult golden hamster, Mesocricetus auratus.

Photoperiodic regulation of testicular steroidogenesis through modulation of MT1R expression and local melatonin content is well established. However, additional mediators besides local melatonergic system in photoperiodic control of testicular steroidogenesis in golden hamster have not been studied in detail. Endogenous opioid peptides (EOP) are known to regulate reproduction via acting at multiple levels of the hypothalamus-pituitary-gonadal (HPG) axis. The presence of ß-endorphin, a naturally occurring opioid peptide, and its receptor (µ-opioid receptor, µOR) has been reported in rat testes; however the functional significance of photoperiodic regulation µOR in testicular steroidogenesis is not clear. In the present study, we assessed the effect of Naltrexone (Nal), a µOR antagonist, in photoperiodic regulation of testicular steroidogenesis. Immunohistochemical (IHC) localization and expression of µOR along with the expression of steroidogenic markers in testes was analyzed through western blot analyses. IHC suggest immunoreactivity for µOR in Leydig cells with strong immunoreactivity under SD (short-day) condition, whereas weak immunoreactivity was observed under LD (long-day). The expression of µOR was significantly decreased following Nal administration in both the photoperiodic conditions. The localization and differential photoperiodic regulation of µOR in Leydig cells suggests its involvement in testicular steroidogenesis. Further, Nal administration significantly increased the expression of steroidogenic markers (AR, StAR, P450SCC, LH-R, 3ß-HSD and 17-HSD) and plasma testosterone concentration under SD condition as compared to SD-control. We may therefore suggest that photoperiod differentially regulates the expression of µOR which thereby mediates the inhibitory effect of melatonin on testicular steroidogenesis.

Melatonin prevents dexamethasone-induced testicular oxidative stress and germ cell apoptosis in golden hamster, Mesocricetus auratus.

This study investigated the protective effect of melatonin on dexamethasone (Dex), an extensively used anti-inflammatory and immunosuppressive synthetic glucocorticoid, induced testicular oxidative stress and germ cell apoptosis in golden hamster. Hamsters were randomly divided into four groups (n = 7): group I - control; group II - melatonin treated (10 mg kg(-1)  day(-1) ); group III - Dex treated (7 mg kg(-1)  day(-1) ) and group IV - combination of Dex and melatonin. All the injections were administered intraperitoneally for seven consecutive days. The histopathological changes, specific biochemical markers, including antioxidative enzymes, plasma melatonin level and the markers for germ cell apoptosis were evaluated. Dex administration decreased antioxidant enzyme activities (SOD, CAT, GSH-PX ), plasma melatonin level and melatonin receptor (MT1) expression with a concomitant increase in lipid peroxidation (TBARS) and altered testicular histopathology which might culminate into increased germ cell apoptosis as evident from increased Bax/Bcl-2 ratio and caspase-3 expression. However, melatonin pre-treatment enhanced enzyme activities for SOD, CAT, GSH-PX with a simultaneous decrease in Bax/Bcl-2 ratio and caspase-3 expression. Our findings clearly suggest that melatonin improved defence against Dex-induced testicular oxidative stress and prevented germ cell apoptosis, suggesting a novel combination therapeutic approach for management of male reproductive health.

Melatonin regulates splenocytes proliferation via IP3-dependent intracellular Ca2+ release in seasonally breeding bird, Perdicula asiatica.

Melatonin plays an important role in the immune regulation of birds. Both endogenous and exogenous melatonin modulates lymphocyte proliferation via its specific membrane receptors, Mel(1a), Mel(1b) and Mel(1c), though the mechanisms behind this process are poorly understood. We investigated the differences in melatonin membrane receptor Mel(1a), Mel(1b) and Mel(1c) expression by western blot and reverse transcription reaction and the in vitro effect of melatonin on the intracellular Ca(2+) concentration ([Ca2+]i) in splenocytes of the Indian Jungle Bush Quail, Perdicula asiatica. We used a non-selective melatonin receptor antagonist for Mel(1a) and Mel(1b), luzindole, and the selective Mel(1b) blocker, 4P-PDOT to check the specific role of melatonin receptor on ([Ca2+]i). The expression of Mel(1a), Mel(1b) and Mel(1c) receptors mRNA and protein was upregulated by melatonin (10(-7) M) with a significant high rise in ([Ca2+]i), which was differentially blocked by supplementation of antagonist, luzindole (10(-7) M) and 4P-PDOT (10(-7) M). Furthermore, we noted in vitro effect of melatonin and 2-aminoethoxydiphenyl borate (2-APB), a cell-permeable antagonist of inositol 1, 4, 5-trisphosphate (IP3) receptor to check the rise in ([Ca2+]i) through the IP3 pathway. Significantly low ([Ca2+]i) was noted in melatonin and 2-APB pretreated splenocytes when compared with splenocytes where 2-APB was absent. Thus, our data suggest that melatonin through its membrane receptor induced the elevation of ([Ca2+]i) via IP(3)-dependent pathway for splenocyte proliferation in P. asiatica.

Nycthemeral variation in melatonin receptor expression in the lymphoid organs of a tropical seasonal breeder Funambulus pennanti.

Seasonal variations in immune functions point toward the involvement of melatonin in its regulation. These chronobiotic effects are exerted by receptors present on the immunocompetent cells. The present study investigated daily/nycthemeral variation in expression of melatonin receptor subtypes MT1/MT2 in the lymphoid organs (spleen/thymus) of a tropical squirrel, Funambulus pennanti. The receptor expression was noted every 4 h interval over 24 h under natural light-dark cycle, during two seasons and was correlated with peripheral level of melatonin. The MT1/MT2 receptor expression displayed higher levels at the time of dusk (light-dark transition; 1800 hours), while plasma melatonin was still low compared to the preceding time point. The receptors were downregulated during the nighttime with a minimum expression at 0200 hours. Thymus, during the long day length, showed a tissue-specific pattern of receptor expression with a minimum expression at 0600 hours. Results suggest that photoperiod by modulation of melatonin level inversely regulates the receptor expression. The observations imply that there exists a temporal window of sensitivity in the target organs to the melatonin signal that is regulated by modulation of melatonin receptor expression which might be involved in mediating the photoperiodic effects of melatonin in the control of seasonal immune physiology.

Daily variation in melatonin level, antioxidant activity and general immune response of peripheral blood mononuclear cells and lymphoid tissues of Indian goat Capra hircus during summer and winter.

Daily variation in circulatory melatonin level, during different seasons, has been reported to influence immune system and free-radical scavenging capacity in mammals, including human beings. Similar studies have not been carried out on small ruminant viz. goats that are susceptible to opportunistic infections, increased oxidative load and sickness during free-grazing activity and frequent exposure to agro-chemicals. Therefore, daily variation in immune status, antioxidant enzyme activity and its possible correlation with circulatory melatonin level during two different seasons, summer (long day) and winter (short day) were studied in the Indian goat, Capra hircus. The clinically important immune parameters, such as total leukocyte count, % lymphocyte count and % stimulation ratio of T-lymphocytes presented a day/night rhythm prominently in the winter. The oxidative load in terms of malonedialdehyde was always low during night while antioxidant enzymes superoxide dismutase, catalase and total antioxidant status were high during nighttime (1800 to 0600 hrs). Interestingly, the studied parameters were significantly higher during the winter in both the sexes. Rhythmometric analyses showed prominent rhythmicity in above parameters. The data presented strong positive correlation between high levels of nighttime melatonin levels and immune parameters during winter. It suggests that melatonin possesses immunoenhacing as well as antioxidative property during winter. This might be a necessity for maintenance of physiological harmony in goats to protect them from winter stress.

Daily variations in plasma melatonin and melatonin receptor (MT1), PER1 and CRY1 expression in suprachiasmatic nuclei of tropical squirrel, Funambulus pennanti.

The suprachiasmatic nucleus (SCN) plays a major role in photoperiodic regulation of seasonal functions by modulating the melatonin signal. To date no report exists regarding the role of the ambient photoperiod in the regulation of melatonin receptor MT1 and clock gene (PER1 and CRY1) expression in the SCN of any tropical rodent that experiences the least variation in the photoperiod. We noted the expression of MT1, PER1 and CRY1 in the SCN of a tropical squirrel, Funambulus pennanti, along with the plasma level of melatonin over 24 h during the reproductively active (summer) and inactive (winter) phases. The seasonal day length affected the peripheral melatonin, which was inversely related with the MT1 expression in the SCN. The timing for peak expression of PER1 was the same in both phases, while the decline in PER1 expression was delayed by 4 h during the inactive phase. The CRY1 peak advanced by 4 h during the active phase, while the interval between the peak and decline of CRY1 remained the same in both phases. It can be suggested that seasonally changing melatonin levels modulate MT1 expression dynamics in the SCN, altering its functional state, and gate SCN molecular "clock" gene profiles through changes in PER/CRY expression. Such a regulation is important for photo-physiological adaptation (reproduction/immunity) in seasonal breeders.

Seasonal plasticity in immunocompetent cytokines (IL-2, IL-6, and TNF-α), myeloid progenitor cell (CFU-GM) proliferation, and LPS-induced oxido-inflammatory aberrations in a tropical rodent Funambulus pennanti: role of melatonin.

In seasonal breeders, photoperiods regulate the levels of circulatory melatonin, a well-known immunomodulator and an antioxidant. Melatonin is known to play a complex physiological role in maintaining the immune homeostasis by affecting cytokine production in immunocompetent cells. In this study, we have quantified seasonal and temporal variations in immunocompetent cytokines-IL-2, IL-6, and TNF-α-and circulatory corticosterone along with in- vitro proliferation of bone marrow-derived granulocyte macrophage-colony forming unit (CFU-GM) progenitor cells of a tropical seasonal breeder Funambulus pennanti (northern palm squirrel). Transient variations in antioxidant status of seasonal breeders might be due to the fluctuations associated with immunity and inflammation. Further, to establish a direct immunomodulatory effect of photoperiod, we recorded the LPS-induced oxidative and inflammatory responses of squirrels by housing them in artificial photoperiodic chambers mimicking summer and winter seasons respectively. We observed a marked variation in cytokines level, melatonin, and corticosterone , and CFU-GM cell proliferation during summer and winter seasons. High Peripheral melatonin levels directly correlated with cytokine IL-2 levels, and inversely correlated with TNF-α, and circulatory corticosterone level. LPS-challenged squirrels housed in short photoperiod (10L:14D; equivalent to winter days) showed a marked reduction in the components of the inflammatory cascade, CRP, TNF-α, IL-6, NOx, NF-κB, Cox-2, and PGES, with an overall improvement in antioxidant status when compared to squirrels maintained under a long photoperiod (16L:8D; equivalent to summer days). Our results underline the impact of seasonality, photoperiod, and melatonin in maintaining an intrinsic redox-immune homeostasis which helps the animal to withstand environmental stresses.