| Stress is an inevitable phenomenon in this modern world.Stressful situations can lead to many physiological and psychological alterations.Disturbance of homeostasis is called stress and homeostasis is challenged by a stimulus is termed as stressor.Generally there are three types of stressors physical,psychological and metabolic.In research stressors are often used in a mixed type,such as restraint stress(RS)is a combination of physical and mental stressors,limiting movement and separating the animal from its group.A large variety of behavioral,physiological and neuroendocrine alterations happened to adaptation with stress and for a quick recovery.Stress has been reported to influence the animal reproduction adversely.Stress causes obvious suppression of spermatogenesis with decrease in size and weight of testes,and all the phases of cell division and maturity of spermatogenesis were inhibited.Intratesticular lipid accumulation is increased by stress.Serious degenerative alterations were also seen on spermatogenesis as a result of stress.A first sign of stress is quick increase in glucocorticoid and decrease in testosterone concentration in males is considered in the fall of steroidogenesis.Rutin is a plant pigment(flavonoid)found in buckwheat,passion flower,apples and green tea.It has been reported to exhibit various pharmacological properties including antioxidant,anticarcinogenic,cytoprotective,antiplatelet,antithrombotic,vasoprotective,anti-inflammatory and cardioprotective effects.The strong antioxidative capacity of rutin has been proven by numerous studies,particularly for excellent scavenging activity.Thyroid hormones play an important role in several mammalian tissues in regulating development,differentiation,and metabolism.Target of thyroid hormone action is mammalian testis and testicular functions are affected by distressed thyroid function.Hypothyroidism is a disease of ineffective or low thyroid hormone leads to a metabolic disturbance.Adverse effects of hypothyroidism on spermatogenesis and testicular histology has been established.Restraint stress and hypothyroidism impair testicular function both in animal and human models.As stress is increasing gradually in our daily life,there is more chance of the happening of both stress and hypothyroidism together.However,the harmful effects of this combination on testis have not been studied.The present study was carried out to investigate the potential role of rutin and thyroid hormone against the effect of restraint stress on spermatogenesis in testes of adult mice.1.Protective effects of rutin on endocrine,histoarchitecture,histopathology and spermatogenesis against restraint stress in testes of adult miceIn this study 50 adult male mice were divided into five groups consisting 10 mice each group;Control,Restraint Stress(RS),RS-DMSO,RS-20mg and RS-200mg.Mice were restrained in a conical tube for 4 hour daily and inject rutin intraperitoneally to each mouse for 15 consecutive days.The results showed that the daily water and feeds consumption of the control group has no change were found during the whole experiment.Stress groups RS,RS-DMSO and RS-20mg significantly but RS-200mg insignificantly reduced the feed and water consumption as compared to the control group.Healthy mice of control group showed body weight gain day by day during the whole experimental period but stressed groups RS,RS-DMSO and RS-20mg showed reduction significantly but RS-200mg insignificantly in body weight.In addition,stressed groups RS,RS-DMSO and RS-20mg exhibited significantly but RS-200mg insignificantly decrease in testes and epididymis weight as compared to control group.Adrenal glands weight was increased significantly in stressed groups RS,RS-DMSO and RS-20mg but insignificantly in RS-200mg group as compared to control group.Thymus and visceral fats were decreased significantly in stressed groups RS,RS-DMSO and RS-20mg but insignificantly in RS-200mg group as compared to control group.Serum concentrations of Testosterone,T3 and T4 were decreased significantly in stressed groups RS,RS-DMSO and RS-20mg but insignificantly in RS-200mg group as compared to the control group.Epinephrine serum concentration was increased significantly in stressed groups RS,RS-DMSO and RS-20mg but insignificantly in RS-200mg group as compared to the control group.Stressed groups RS,RS-DMSO and RS-20mg are showing broken seminiferous tubules,few spermatozoa in lumen and less population of leydig cells between the interstitial spaces.On the other hand,control and RS-200mg groups showing no damage to seminiferous tubules and normal arrangement of germ cells from spermatogonia to spermatozoa and cluster of spermatozoa in the lumen and leydig cells between the interstitial spaces.We also analyzed the histopathological changes at different stages of the seminiferous tubule at diverse stages in the cycle of spermatogenesis.We observed that spermiation was arrest in stage ?-? and degenerated population of round spermatids were sighted in the lumen of stressed groups RS,RS-DMSO and RS-20mg.Missing cells were found in stage of ?-? and lumen size was increased in stages of ?-?,? and X of stressed groups RS,RS-DMSO.Residual bodies were more found in stages of ?-?,?-? and degenerative cells were observed in stage ? of stressed group of RS-20mg.Vacuoles were found in stages ?-? of stressed groups RS,RS-DMSO and RS-20mg as compared to the control group.On the other hand,RS-200mg group showed normal histology at different stages of the seminiferous tubule at diverse stages in the cycle of spermatogenesis as compared to the control group.Thus,our results showed that,restraint stress severely damage the testicular histoarchitecture,histopathology and spermatogenesis and disturbed the testicular endocrine function and rutin recovered it.2.Protective effects of rutin on caspase-dependent pathway of apoptosis against restraint stress in testes of adult miceThe present study was carried out to investigate the protective role of rutin against the effect of restraint stress on expression of PARP1 and casapase3 dependent apoptosis in testis of mice.Normal PARP1 protein expression was found on spermatogonia,primary spermatocytes,spermatid,leydig cells and sertoli cell nucleus of the control group.The level of PARP1 expression was significantly increased in stressed groups RS,RS-DMSO and RS-20mg but RS-200mg group showed normal expression like control as compared to stressed groups.The intensity of Cleaved PARP1 immunostaining was predominant on round and elongated spermatid but also in a perinuclear distribution of spermatogonia,primary spermatocytes,Leydig cells and Sertoli cell nucleus of stressed groups RS and RS-DMSO.On the other hand,Cleaved PARP1 expression in RS-20mg was less but RS-200mg and control groups showed no expression as compared to stressed groups RS and RS-DMSO.Caspase-3 expression was specifically detected in germ cells and Leydig cells.Immunostaining of Caspase-3 was predominantly observed in primary spermatocytes and to a lesser extent the more mature germ cells of control group.Expression of Caspase-3 was considerably increased in spermatogonia,primary spermatocytes,round spermatid,Leydig cells and Sertoli cell nucleus of stressed groups RS,RS-DMSO and RS-20mg as compared to the control group but RS-200mg group expression was like a control group.Active(cleaved)Caspase-3 immunostaining was found exclusively in postmeiotic germ cells particularly in round and elongated spermatids but also in a perinuclear distribution of spermatogonia,primary spermatocytes,Leydig cells and Sertoli cell nucleus of stressed groups RS,RS-DMSO and RS-20mg as compared to control group which did not show any expression of cleaved Caspase-3.Rutin groups RS-20mg showed less expression and RS-200mg showed no expression of cleaved Caspase-3 on germ cells as compared to stressed groups RS and RS-DMSO.Our study indicated that Rutin could decrease levels and prevent cleavage of PARP1 and Caspase-3 and increase antioxidative capacity of mouse testes exposed to restraint stress,suggesting that Rutin exerts some protective functions on restraint stress-induced testicular damage and apoptosis.3.Protective effects of rutin on sperm count,motility,viability and sperm morphology against restraint stress in testes of adult miceRestraint stress decreased the sperm count,motility&viability and increased the incidences of abnormal sperms.Stressed groups RS,RS-DMSO and RS-20mg significantly but RS-200mg insignificantly decreased the sperm count,motility and viability as compared to the control group.Rutin dose 20mg recovered the sperm count,motility and viability but not significantly in RS-20mg group.On other hand,200mg significantly recovered the sperm count,motility and viability in RS-200mg group as compared to the control group.Morphologically,normal sperms were mostly observed in the control and RS-200mg groups.Abnormal sperms were mainly found in stressed groups RS,RS-DMSO and RS-20mg.Rutin dose 20mg little recovered the sperm morphology in RS-20mg group as compared to the control group.But Rutin dose 200mg significantly recovered the sperm morphology in RS-200mg group as compared to the control group.The results suggested rutin has a remarkable potency to donate electron to reactive free radicals by converting them into more stable species and quenching the free radical chain reaction.The potent antioxidant activity of rutin is mainly due to the presence of phenolic rings and free hydroxyl groups in the chemical structure.These free hydroxyl groups could donate hydrogen to prevent further oxidation.Thus,rutin exerts an antioxidant effect on lipid membranes to protect the sperms against stress.This effect is attributed to its anti-free radical properties,mainly directed at the superoxide and hydroxyl radicals,which are highly reactive species involved in the initiation of the lipid peroxidation chain in the sperm.4.Impaired growth performance and testicular cells apoptosis following restraint stress in adult hypothyroid miceRestraint stress and hypothyroidism impair animal testicular functions.The harmful effects of this combination on testes have not been studied.Twenty-four adult male mice were divided into four groups:control,restraint stress(RS),hypothyroid(hypo-T)and RS+hypo-T.The results indicated that the feed and water index,and body weight exhibited a reduction in all experimental groups compared to the control.In addition,a significant reduction in testis weights was observed.Serum concentration of T3 was markedly decreased in the hypo-T and RS+ hypo-T mice,but no marked changes found in serum concentrations of T3 between the RS and control groups.Serum concentrations of T4 and testosterone were prominently diminished,but the TSH levels in the RS,hypo-T and RS+hypo-T mice distinctly augmented compared to the control mice.Histological observations of the testis from different experimental groups exhibited considerable interstitial edema,broken basement membrane and increased interstitial spaces compared with the control group.Seminiferous tubules were also morphologically shrinkage and deformed in RS+hypo-T group compared to control group.Obvious suppression of spermatogenesis by restraint stress,the degenerative population of round spermatids was markedly increased due to apoptosis in the lumen of RS group as compared to the control group.Moreover,Leydig cells,blood vessels,Sertoli cells,primary and secondary spermatocytes are absent in large numbers and exposing apoptosis in RS+hypo-T mice than RS and hypo-T mice as compared to the control mice.Restraint stress and hypothyroidism together have adverse effects on male fertility based on increasing of the apoptotic process. |
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