Effect Of Chronic Social Stress On Male Mice Spermatogenesis Fuction

Background and objectives:Chronic stress is a common natural reaction which occurs when an individual could not bear the burden of various stress stimuli(stressors) which beyond his or her ada ptive capacity for a long time. From 2007 to 2010 worldwide epidemiological surveys showed that chronic stress states were widespread in the middle-aged and young population and the prevalence of psychological stress was increasing. At the same time, the global male reproductive function(especially the semen quality) is declining year by year, which may be closedly related to living habits, the factors of social environment and psychological health,and so on. We focus on the effect of chronic stress on male reproductive function. And preliminary experiments confirmed that chronic stress mainly activated the hypothalamus-pituitary-adrenal(HPA) axis which sustained releases a variety of neuroendocrine hormones, resulting in reproductive dysfunction. But the specific molecular mechanism has not yet been studied. The results of clinical researches also showed that chronic stress was one of the major risk factors of semen quality decline.HPA axis which activated by chronic stress,releases many kinds of neuroendocrine hormones, among which adrenocorticotropic hormone releasing hormone(CRH) plays a vital role. In the central nerve system,stressful events lead to the release of CRH in the parvocellular neurons of the hypothalamic paraventricular nucleus(PVN). CRH acts on adrenocorticotropic hormone releasing hormone receptor(CRHR) in the pituitary and triggers the processing and release of adrenocorticotropic hormone(ACTH). CRH also can be transported to peripheral target organs through the blood stream.Recent studies find that CRHR is also observed expressing in testis and epididymis.But the research about the function of CRH or CRHR in the male reproductive system is less.So there is a huge space of study.Nectin protein family is a kind of important cell-cell adhesion molecules, widely exists in the hippocampus, epithelial cells, testicular tissue and other parts and plays important roles in cell adhesion, migration and polarization. And intratesticular Nectin protein family mainly includes Nectin-2(located in Sertoli cells) and Nectin-3(on the membrane of spermatids head), they connect to each other and underlie Sertoli–spermatid junctions.This structure is an important anchoring junction device that provides mechanical adhesion of spermatids onto the nourishing Sertoli cells to assist movement of developing spermatids across the epithelium and to ensure proper orientation of spermatids in the epithelium so that fully developed spermatids can be released to the tubule lumen during spermiation. Nectin-2–/– and Nectin-3–/– mice have defects in the later steps of sperm development and have shown male-specific infertility.And disturbing expression of Nectin also functionally impairs spermatogenesis. So the Sertoli cells–spermatid junctions have an important role in sperm maturation.Wang, et al demonstrated that chronic stress induced Nectin-3 expression decrease in hippocampus via the CRH-CRHR1 pathway. In early preliminary experiment, we found Nectin-3 expression in testicular tissue of mice is also decreased after 25-day chronic social stress.We hypothesize that the expression change of Nectin protein family is closely related to chronic stress reaction, and Nectin protein family may become a molecular target of mouse spermatogenesis dysfunction induced by chronic stress; testicular high CRH levels induced by chronic stress increase may lead to the changes of Nectin proteins family in the testis directly or indirectly(though CRHR1).Therefore, this work will be started and in accordance with the following several aspects:(1) to build a suitable chronic stress mouse model,(2) to investigate the effect of chronic stress on male mice spermatogenesis;(3) to explore the influence of chronic stre ss in male mice testicular Nectin protein family;(4) to explore whether the changes of Nectin protein family induced by chronic stress are associated with CRH-CRHR pathways.Methods:1.Build the chronic stress animal model. Chronic social defeat stress model is established.By use of the behavior experiment(social interaction test and open field test) to evaluate whether the chronic stress model is built successfully.2.Assess changes of reproductive index. We test the weight change of the gonads and sperm quality(sperm counts, sperm deformity rate) after chronic stress.Make testis sections and observe testicular histology change after HE staining. Mice are mated with health female mice and the time period of producing offspring is recorded.3.Make sure the location and expression changes of Nectin protein family and CRHR1. The distribution of Nectin-3, Nectin-2 and CRHR1 are comfirmed by use of immunofluorescence staining. Western Blot is a great method to detect testicular Nectin-3 and Nectin-2 protein level after chronic stress or chronic stress is removed for 4 months.4.Evaluate the reproductive index and the change of the Nectin protein family after CRH intervention. The high levels of CRH during chronic stress are simulated by intraperitoneal injection of CRH. Sperm quality of male mice is detected. Use Western Blot to make sure the changes of testicular Nectin-3 and Nectin-2 expression after CRH intervention.Results:1.After 25-day chronic social defeat stress, stressed mice showed obvious anxiety-like behavior and social avoidance behavior.And they also showed weight loss, thymic atrophy and other typical symptoms of chronic stress.2.After chronic social stress, we found gonad atrophy(testis and epididymis end atrophy) and semen quality was reduced(P<0.05). Testicular histology structure was changed(testis sections showed the epithelial of seminiferous tubules were thinner). The time of producing offspring was extended, after stressed male mice were mated with health female mice.3.Immunofluorescence results showed that the location of each protein(Nectin-2, Nectin-3 and CRHR1) in testis. Nectin-2 and Nectin-3, asymmetrically localized at the Sertoli cell side and at the spermatid side of Sertoli–spermatid junctions. CRHR1 was located in Leydig cell. Nectin-3 protein level in stressed mice was significantly decreased in testis(P<0.05), but the Nectin-2 protein and CRHR1 in stressed mice were overexpressed in testis compared with control mice(P<0.05). After four months without chronic stress Nectin-3 protein level in stressed mice was no tatistical differences with control mice(P>0.05). But the Nectin-2 protein was still increased(P<0.05).4.After intraperitoneal injection of CRH, the number of sperm(from the epididymis) was decrease. Compared with control group, Nectin-3 protein level in the CRH injection group was decreased and nectin-2 protein level was increased(P<0.05).Conclusion:1.The spermatogenesis dysfunction of male mice is induced by chronic social stress.2.The Sertoli cells-spermatid junctions formed by Nectin protein family are damaged by chronic social stress, and this structure may be a molecular target of mouse spermatogenesis dysfunction induced by chronic stress.3.The increased CRH level is associated with the change of Nectin protein family.4.The changes of Nectin protein family in testis maybe the reason of spermatogenesis dysfunction induced by chronic stress and via CRH-CRHR pathways.