Dietary restriction-induced alterations on estrogen receptor alpha expression in regulating fertility in male Coturnix coturnix japonica: Relevance of Withania somnifera in modulation of inflammation and oxidative stress in testis.

PROBLEM: Reproductive performance of animals gets affected by nutritional restrictions which act as potential stressors leading to hormonal imbalance and testicular inflammation, the major causes of infertility. Withania somnifera (WS), well-known traditional medicinal plant, has been used as antistress and infertility treatment. Therefore, the present study looks into the ameliorative effects of WS on the reproductive and immune system of male Coturnix coturnix japonica in stressed conditions like water and food restriction focussing on the modulation in estrogen receptor alpha (ERα). METHOD OF STUDY: Biochemical estimations for oxidative stress, histological alterations, immuno-fluorescent localization of ERα, interleukin (IL)-1ß, IL-4, and interferon gamma (IFN-γ) in testicular cells were performed. RESULTS: Nutritional restriction declines endogenous estradiol, ERα in testicular cells while it elevates corticosterone leading to oxidative stress in testis thereby reducing fertility by decrease in sperm. Results indicate significant reversal in all the parameters after the administration of WS by improving testicular cell morphology, increased superoxide and catalase activity thus reducing oxidative stress. WS increases spermatogenesis and enhances expression of ERα in testicular cells in quail. Further, WS increases IL-4, decreases IL-1ß and IFN-γ expression in testis, thereby improving immune profile contrary to stressed conditions. CONCLUSION: WS stimulates HPG-axis even after stress resulting in increased endogenous estradiol which stimulates the expression of ERα in testis; increases sperm count and immunity thereby improving the reproductive performance. WS may be the best therapy against nutritional-restriction stress induced reproductive toxicity by reducing oxidative stress mediated inflammatory response via increased testicular expression of ERα in quail.

Withaferin-A attenuates diabetes mellitus induced male reproductive dysfunction mediated by ERα in brain and testes of Swiss albino mice.

Diabetes mellitus (DM) is a chronic metabolic disease, characterized by persistent hyperglycemia resulting from diminished insulin secretion or insulin resistance. The present study evaluated the ameliorative effects of Withaferin-A (WA) on DM-induced reproductive dysfunction in mice. For the same, mice were intraperitoneally injected with Streptozotocin (STZ), (40 mg/kg/day) for 5 consecutive days to induce DM. Mice were then treated with WA (8 mg/kg/day) in normal and diabetic conditions (STZ + WA). Next, blood glucose levels, oral glucose tolerance, intraperitoneal insulin tolerance, oxidative stress and reproductive parameters were estimated. For reproductive performance, immunofluorescent localization of gonadotropin-releasing hormone (GnRH-I) and estrogen receptor alpha (ERα) in the preoptic area and paraventricular nucleus region of hypothalamus and ERα in testes was performed. STZ-induced diabetes triggered reproductive dysfunctions as mediated by low GnRH-I and ERα in the brain and ERα in the testes along with declined testosterone and estradiol levels. Treatment with WA significantly reduced the blood glucose levels and enhanced glucose clearance accompanied by reduced oxidative stress in the brain, pancreas and testes as indicated by the low levels of H2O2 and MDA in diabetic mice treated with WA (STZ + WA). This study reports, for the first time, that WA can efficiently ameliorate DM-induced reproductive dysfunctions by enhancing endogenous testosterone, estrogen and increased GnRH-I and ERα in the brain and ERα in the testes of DM-induced male mice.

Withania somnifera inhibits photorefractoriness which triggers neuronal apoptosis in both pre-optic and paraventricular hypothalamic area of Coturnix coturnix japonica: involvement of oxidative stress induced p53 dependent Caspase-3 mediated low immunoreactivity of estrogen receptor alpha.

Light has a very important function in the regulation of the normal physiology including the neuroendocrine system, biological rhythms, cognitive behavior, etc. The variation in photoperiod acts as a stressor due to imbalance in endogenous hormones. Estrogen and its receptors ER alpha and beta play a vital role in the control of stress response in birds. The study investigates the estrogenic effects of a well-known medicinal plant Withania somnifera (WS), mediated by estrogen receptor alpha (ERα) in the hypothalamic pre-optic area (POA) and paraventricular nuclei (PVN). Further the study elucidates its anti-oxidants and anti-apoptotic activities in the brain of Japanese quail. To validate this hypothesis, mature male quails were exposed to long day length for 3 months and then transferred to intermediate day length to become photorefractory (PR) while controls were still continued under long daylength. Supplementation of WS root extract in PR quail increases plasma estrogen and lowers corticosterone. Further, in PR quail the variation in light downregulates immunoreactivity of ERα, oxidative stress and antioxidant enzyme activities i.e. superoxide dismutase and catalase in the brain. Neuronal apoptosis was observed in the POA and PVN of PR quail as indicated by the abundant expression of Caspase-3 and p53 which reduces after the administration of WS root extract. The neuronal population also found to decrease in PR although it increased in WS administered quails. Further, the study concluded that change in photoperiod from 3 months exposure of 16L: 8D to 13.5L: 10.5D directly activates neuronal apoptosis via expression of Caspase3 and p53 expression in the brain and increases neuronal and gonadal oxidative stress while WS root extract reverses them via enhanced estrogen and its receptor ERα expression in the hypothalamic pre-optic and PVN area of Japanese quail.

Cissus quadrangularis (Hadjod) Inhibits RANKL-Induced Osteoclastogenesis and Augments Bone Health in an Estrogen-Deficient Preclinical Model of Osteoporosis Via Modulating the Host Osteoimmune System.

Osteoporosis is a systemic skeletal disease characterised by low bone mineral density (BMD), degeneration of bone micro-architecture, and impaired bone strength. Cissus quadrangularis (CQ), popularly known as Hadjod (bone setter) in Hindi, is a traditional medicinal herb exhibiting osteoprotective potential in various bone diseases, especially osteoporosis and fractures. However, the cellular mechanisms underpinning its direct effect on bone health through altering the host immune system have never been elucidated. In the present study, we interrogated the osteoprotective and immunoporotic (the osteoprotective potential of CQ via modulating the host immune system) potential of CQ in preventing inflammatory bone loss under oestrogen-deficient conditions. The current study outlines the CQ's osteoprotective potential under both ex vivo and in vivo (ovariectomized) conditions. Our ex vivo data demonstrated that, in a dose-dependent manner CQ, suppresses the RANKL-induced osteoclastogenesis (p < 0.001) as well as inhibiting the osteoclast functional activity (p < 0.001) in mouse bone marrow cells (BMCs). Our in vivo µ-CT and flow cytometry data further showed that CQ administration improves bone health and preserves bone micro-architecture by markedly raising the proportion of anti-osteoclastogenic immune cells, such as Th1 (p < 0.05), Th2 (p < 0.05), Tregs (p < 0.05), and Bregs (p < 0.01), while concurrently lowering the osteoclastogenic Th17 cells in bone marrow, mesenteric lymph nodes, Peyer's patches, and spleen in comparison to the control group. Serum cytokine analysis further supported the osteoprotective and immunoporotic potential of CQ, showing a significant increase in the levels of anti-osteoclastogenic cytokines (p < 0.05) (IFN-γ, IL-4, and IL-10) and a concurrent decrease in the levels of osteoclastogenic cytokines (p < 0.05) (TNF-α, IL-6, and IL-17). In conclusion, our data for the first time delineates the novel cellular and immunological mechanism of the osteoprotective potential of CQ under postmenopausal osteoporotic conditions.

Stress and steroid interaction modulates expression of estrogen receptor alpha in the brain, pituitary, and testes of immature Gallus gallus domesticus.

In nature, food availability stimulates hypothalamo-pituitary-gonadal (HPG) axis while its scarcity induces stress, which further stimulates hypothalamo-pituitary-adrenal (HPA) axis producing a detrimental effect on the avian reproductive physiology. The present experiment was designed to examine the interaction of stress like food restriction and estradiol on male reproductive physiology with special emphasis on estrogen receptor alpha (ERα) as these play crucial role in reproduction. To achieve this, 60 day old White Leghorn immature cockrels were taken and divided into four groups (n = 8 per group). One group was provided with food and water ad libitum. Second group was food restricted (FR) for 9 h/day after 5 days, third and fourth were administered with estradiol benzoate (EB 0.5 mg/100g/day) for 12 days. Fourth group was FR for 9 h/day after 5 days of EB treatment till last day of experiment (EB + FR). Immunofluorescent localization of ERα was principally in the pre-optic area and paraventricular nuclei of hypothalamus and in anterior pituitary gland. ERα expression was highly reduced (from 40 AU to 20 AU) after FR in testis but it increased (50 AU) after EB administration, EB + FR reflects a diminishing pattern in the increment after EB. FR decreased plasma estradiol while EB increased it. Increased plasma corticosterone, hydrogen peroxide, malondialdehyde, and decreased anti-oxidant enzymes in brain and testis of all groups indicate oxidative stress in the HPG axis. The increased ERα after EB and a decrease with FR and EB + FR support their reproductive function. Estrogen and its receptor alpha are responsible for maintaining epithelial morphology but FR along with EB administration modulates the testicular development by significantly decreasing its size (p<.0001) and seminiferous tubules (p<.0001) and no sperm formation via highly reduced expression of ir-ERα in HPG axis. Our findings led us to conclude that stress like FR and estradiol induces testicular regression immature male chickens by modulating ir-ERα expression in the HPG axis thereby resulting in reduction in reproductive physiology.

Photoperiod dependent expression of estrogen receptor alpha in testes of Japanese quail: Involvement of Withania somnifera in apoptosis amelioration.

Light plays important function in the regulation of reproduction in vertebrates including birds. The prolonged long day length exposure causes reproductively inactive state or photorefractoriness in many avian species including Japanese quail. Withania somnifera (WS) is a medicinal plant known to have beneficial effects on stress and infertility. The study investigates the physiological effect of WS on the light-induced stress in quail mediated by estrogen receptor alpha. Quails were exposed to long day length for three months and then transferred into intermediate day length to make them photorefractory (PR) while controls under natural day length. Administration of Withania somnifera root extract (WSRE) in PR quail induces estrogen and decreases corticosterone in male Japanese quail. Immunoreactivity of ERα decreased in testis of PR quail and increased after oral administration of WSRE compared to control. Expression of ir-Caspase-3 and ir-p53 in the testis increased in PR while decreased in PR + WS. Histologically, seminiferous tubules size decreased in PR whereas increased in PR + WS quails. Scanning electron microscopic study reveals sperms in clusters with proper head and tail in control. In PR quails sperms were few and distorted while WSRE improved the sperm morphology. From the study, it is concluded that during photorefractoriness gonadal regression occurs due to testicular apoptosis which causes stress. WSRE helps to overcome stress and improve reproductive performance via increase in expression of ir-ERα during PR condition. Further, the stress ameliorating effect of WSRE in reducing apoptosis mediated by ir-Caspase-3 and ir-p53 in the testes is clearly evident in Japanese quail.

Effect of estrogen and stress on estrogen receptor 1 in the HPG axis of immature male Gallus gallus domesticus: Involvement of anti-oxidant system.

Estrogen plays a key role in the regulation of reproductive behavior and control of the neuroendocrine system in both males and females. However, excessive quantity of exogenous estrogen produces a deleterious effect on the male reproductive system. To elucidate the mechanism by which estrogen modulates its receptor alpha (ESR1) in immature chicken during stress the study has been undertaken. The experiment investigated the physiological changes in the abundance of ESR1 in brain, pituitary and testes of immature male chickens after stress like water restriction. Twenty four immature male chickens were randomly assigned into four groups. The control group was provided with food and water ad libitum, second was water restricted 9 h each day for seven days (WR), third was treated with estradiol benzoate (EB) and fourth group was treated with EB followed by water restriction during last seven days of treatment (EB + WR). EB was administered at a dose of 0.5 mg/100 g/day for 12 days. EB administration as well as WR increases both the H2O2 and Malondialdehyde levels indicating oxidative stress in brain as well as in testis. Plasma corticosterone significantly increased in all groups while estradiol significantly decreased after water restriction. ESR1 protein was detected by immuno-fluorescence predominantly in the pre-optic area of the hypothalamus, pituitary and testes after EB administration. EB administration increases ESR1 proteins abundantly in the Sertoli cells, Leydig cells, spermatogonia and spermatids while WR decreases it. The decline in ESR1 proteins after EB administration during stress appears to be mediated by interaction of estrogen with hypothalamo-pituitary-adrenal (HPA) axis. Therefore, the findings substantiate the fact that WR and EB treatment increase the stress and alter the anti-oxidant enzymes via its receptor ESR1 in the brain, pituitary and testis of immature chicks. Moreover, these findings highlight the effect of estradiol in male chicks causing stress which is disrupting the normal physiological feedback mechanism in hormone release and the expression of receptor ESR1 along the hypothalamo-pituitary-gonadal (HPG) axis.

Water and Food restriction decreases immunoreactivity of oestrogen receptor alpha and antioxidant activity in testes of sexually mature Coturnix coturnix japonica.

Food and water are closely associated with reproductive willingness in vertebrates. These are important for animals and their non-availability act as stressors which decrease sex steroid secretion suppressing reproductive behaviour. Oestrogen plays a crucial role in reproduction via its receptors alpha (ERα) and beta (ERß). This study tested the hypothesis that ERα in testes of male Japanese quail is regulated during water and food deprivations. The present study reveals that both water and food deprivations cause oxidative stress and subsequently decrease catalase and superoxide dismutase activity, while these increase malondialdehyde and hydrogen peroxide. Both deprivations reduce plasma oestradiol whereas elevate corticosterone level. The immunofluorescent localization of ERα in the testes occurs predominantly in the seminiferous tubules of control while reduces after both food and water deprivations. All types of spermatogenic cells were seen in control testis, while after water and food deprivations size of seminiferous tubules and spermatogenic cells population decreased. Scanning electron microscopic study exhibited fully mature sperms in clusters with head and elongated flagellum, whereas after water deprivation maximum sperms were distorted, scattered with highly reduced head. On food deprivation, only few sperms were seen with head and tail. Thus, taking into account the localization of ERα in testis, it is obvious that oestrogens produced locally are involved in regulation of spermatogenesis and spermiogenesis during stress.