Study For Testicular Reproductive Function Effect Of Epas1 Heterozygous Deficient Mice During Prolonged Hypoxia

Research purposes:Located in Asia,the Qinghai-Tibet Plateau is the highest plateau in the world,with an average altitude of more than 4,000 meters.At high altitude,the ultraviolet ray is relatively strong,the oxygen partial pressure in the environment is reduced and the air is thin,which brings a great test to human survival.With the rapid development of economy and society,the number of people who settle down on the plateau or enter the plateau in a short period is increasing year by year,and the impact of the plateau environment on reproductive health is gradually attracting attention.According to statistics,environmental characteristics of high altitude areas have certain effects on spermatogenesis and male reproductive ability,and are positively correlated with altitude.As early as 30,000 years ago,the Tibetan population had developed a strong adaptability to the high-altitude environment.Compared with the residents of the plain area,Tibetans had higher pulmonary ventilation,lower pulmonary arterial pressure,higher oxygen saturation and relatively low hemoglobin concentration.It is found that the adaptive ability of Tibetan population is mainly due to the adaptation mutation of EPAS1 gene,which leads to the down-regulation of its mRNA expression and has been confirmed in the Epas1 heterozygous knockout mouse model.Previous studies on Epas1 heterozygosity knockout mice mainly focused on physiological diseases at high altitude,and studies on Epas1 gene in male reproductive system at high altitude were very limited.Some articles pointed out that decreased oxygen level and activation of Epas1 gene were key components of spermatogenesis.However,the reproductive ability of Epas1 heterozygous knockout mice at high altitude hypoxia has not been systematically described.Therefore,we used the testicular pathology and transcriptomic data of Epas1 heterozygous deletion male mice?Epas1+/-male mice?under long-term hypoxia to explore the reproductive mechanism of male testis at high altitude.Methods:1.Long-term hypoxia experiment of male mice:The oxygen concentration in the hypoxia chamber was set at 12%?equivalent to an altitude of 4,400 meters?.Five wild-type male mice and five Epas1^+/-male mice were selected to enter the hypoxia chamber and left after four weeks.Mice of the same type and number were placed in a constant oxygen environment for four weeks as controls.Several physiological indicators were measured,including body weight before and after entry,blood oxygen saturation,hemoglobin concentration,heart weight,spleen weight,brain weight and pulmonary artery pressure were observed after exit.2.Tissue sampling and preservation of male mice:Anatomy was performed on mice in each group,blood was taken from the inner thigh,and serum was subsequently extracted.Cardiac perfusion was performed,Samples were taken from the heart,liver,spleen,lung,kidney,brain,muscle and testicle,and then fixed in paraformaldehyde solution.Part of the liver,spleen,lung,kidney,muscle and testicle were frozen in liquid nitrogen and stored at-80?.3.Male mice testicular pathological experiment:fixed groups of mice testis after dehydration embedding made paraffin section,in order to determine the pathological changes of testis in mice to histological staining of testicular biopsy,including HE staining?wood grain and eosin staining?and PAS staining dye?glycogen?determine whether mice testicles have pathological damage,and using the TUNEL kit to detect internal sperm cells apoptosis of testis.4.ELISA was used to detect the testosterone levels of mice in each group to determine whether the partial deletion of Epasl gene affected the endocrine system of the reproductive system of mice.Immunohistochemical technique was used to determine the specific localization of Epasl protein in the testes of mice and reveal the correlation between Epasl gene and testicular pathology.5.Testicular RNA was extracted from mice in each group,and transcriptomic sequencing was carried out,and the expression profiles of Epas1^+/-male mice and wild-type male mice were compared and analyzed in both aerobic and hypoxic environments.Results:1.Compared with wild-type male mice,Epas1^+/-male mice showed better physiological adaptability under long-term hypoxia,which was reflected in the fact that Epas1^+/-male mice had less weight loss,higher oxygen saturation,relatively low hemoglobin concentration,and maintained low pulmonary artery pressure in a hypoxia environment.2.The partial deletion of Epasl gene under constant oxygen environment had a certain influence on the development of testes in mice,which was mainly manifested in the decrease in the number of round sperm and the increase in the number of tubules in the testes with abnormal proportion.3.Hypoxic environment resulted in hyperemia of testes in some wild-type male mice and decreased spermatocytes and increased number of apoptotic cells in the tubules.However,the reproductive function of Epas1^+/-male mice showed certain adaptability under hypoxia:the proportion of sperm cells of all levels was normal under hypoxia,and the number of apoptosis was not significantly different from that of normal oxygen mice.4.Hypoxic environment affected testosterone synthesis in wild-type male mice,but no significant change in testosterone concentration was observed in Epas1^+/-male mice.The Epasl protein is not clearly located in the sperm cells of the tubules.5.Analysis of testicular transcriptome data showed that Epasl gene knockout affected energy metabolism and T cell proliferation pathway in testicular environment Wild-type mice under hypoxic environment mainly affected the oxygen related molecular function and hydrogen peroxide metabolism pathway in testis.Epas1^+/-male mice under hypoxia had little difference in testicular genes from Epas1^+/-male mice under normal oxygen,so it was speculated that hypoxia had little damage to testicular function of Epas1^+/-male mice.