The Research On The Mechanism Of Heat-stress Induced Testicular Germ Cell Injury

The efficient and ongoing production of spermatozoa in testes is the basis of male fertility.Spermatogenesis is a complex physiological process,which is highly sensitive to temperature.Generally,the temperature of the testis in the scrotum is 2-4 ℃ lower than that of the core body.However,in recent years,testicular heat stress caused by environment,pathology,occupation and unhealthy lifestyle has become an important precipitating factor of male reproductive diseases and infertility.According to statistics,arise of 1 ℃ entails a 14% decline in spermatogenesis.Increased scrotal temperature generates testicular heat stress and later causes testicular atrophy and spermatogenic arrest,resulting in a lower sperm yield and therefore impaired male fertility.Previous studies have shown that single heat shock or cryptorchidism can cause seminiferous epithelium damage and germ cell death.However,the lack of understanding of its occurrence and molecular mechanism is adverse to prevention and treatment of heat stress-induced seminiferous epithelium damage.By employing a mouse model of testicular hyperthermia(39/42 ℃,20 min),we systematically investigated the process of seminiferous epithelium damage and the testicular response to heat stress as well as the occurrence of autophagy,apoptosis and oxidative stress at testicular and cell levels.Importantly,the effect of melatonin on the prevention and treatment of heat-induced testicular damage was evaluated.Based on the Surgery-induced cryptorchidism model,the molecular mechanism of inhibition of spermatogenesis by cryptorchidism was explored by single cell sequencing.Results showed that:1.The low-dose(39 ℃)treatment can not cause significant testicular damage,but subcellular structure and molecular biology shows that low dose heat stress can induce autophagy in germ cells;the high-dose(42 ℃)treatment of testes will cause significant testicular damage.It was found that high-dose heat stress induced intense and sustained oxidative stress reaction of testis and degeneration and vacuolization of seminiferous epithelium.For heat-induced testicular damage,most spermatocyte apoptosis,while the round spermatid generated the multinucleated cell and exfoliated from seminiferous epithelium,the tight connection between Sertoli cells would also be destroyed.2.The molecular changes in testis treated with different dosages of heat stress were significantly different.The JNK MAPK pathway in testis was activated for a short time after low-dose heat treatment,autophagy was initiated downstream,and HSPA2 expression was increased in cytoplasm.While,the JNK MAPK and p38 in testis were intense and sustained increased after high-dose heat stress,which induced cell apoptosis.Meanwhile,we observed that the 42℃ treatment triggered loss or translocation of HSPA2 into nuclei,which correlates with cell apoptosis.3.Pre-treatment of melatonin can effectively reduce the proportion of injured seminiferous tubules(from 94.22 ± 3.76% to 32.1 ± 2.88%),the number of apoptotic cells in the injured tubules(64.89 ± 5.13 to 17.34 ± 3.87 cells per TUNEL positive tubule)and testicular oxidative stress.Western blot analysis showed that melatonin,by inhibiting the apoptosis-related JNK and p38 MAPK signaling pathways,protects the testis against heat-induced damage.The results reveal that pre-treatment of melatonin could prevent the heat-induced damage of seminiferous epithelium.4.Post-treatment of melatonin can effectively inhibit the multinucleation of round spermatids in seminiferous tubules after heat stress.Moreover,treatment with melatonin for 14 consecutive days can accelerate the recovery of eminiferous epithelium.With Melatonin application,the number of abnormal tubules is significantly less and the recovery of testicular index is significantly better,compared with the natural recovery group.Furthermore,melatonin treatment could protect the location and expression-levels of the tight junction protein between Sertoli cells,maintain the integrity of the blood-testis barrier,and enable the spermatogenic epithelium to recovery quickly after the damage.These results indicate that melatonin can effectively treat the heat-induced damage of seminiferous epithelium.5.Based on the surgical-cryptorchidism model,we found that the cryptorchidism can lead to spermatogenic arrest,accumulation of undifferentiated spermatogonia and the germ cells arrest in the spermatogonial stage.Single cell sequencing of testicular cells showed that the proportion of peritubular myoid cells and endothelial cells in the whole testis increased to 330 times of that in the normal testis.The growth factor,GDNF,secreted by these two kinds of cells can promote the proliferation and inhibit the differentiation of spermatogonial stem cells,which may inhibit the differentiation of spermatogonial stem cell after cryptorchidism.At the same time,the differentiated spermatogonia could’t normally express the genes related to spermatogenesis,which induces them get into meiosis failure.To sum up,we systematically probed the testicular response to heat stress,the occurrence of autophagy,apoptosis and oxidative stress in the testis and,more importantly,the utility of melatonin pre/post-treatment as a preventative measure for the heat-induced testicular insults.Single cell sequencing showed that cryptorchidism heat stress could induce the transcriptome change of spermatogonia,inhibit the differentiation of spermatogonial seal,and cause spermatogenic arrest at spermatogonial stage,resulting in male sterility.Since testicular hyperthermia perturbs spermatogenesis and leads to inferior sperm quality,gaining more insights into the response to testicular heat stress and the underlying molecular mechanisms could contribute to development of tailored therapies for heat-induced male subfertility.Furthermore,the proof-of-concept that melatonin can protect the testis against heat stress suggests the potential use of this indolamine as a therapeutic drug for sub/infertile men incurred by various testicular hyperthermia factors.