Effects Of Stress Induced By Fear On Reproductive Endocrine Secretion And Apoptosis Of Spermatogenic Cell In Male Rats

Stress was non-specificity general reaction when environmental agent from exterior andinterior stimulated organism. The homeostasis of organism will be challenge and the disease will be occur when the organism was stressed for longtime .Soldiers who were subjected to stimuli of war environment will be deprived of coping ability and will suffer from psychonosema. This stimuli is called combat stress reaction. This status developed to latter will result in post-traumatic stress disorder.In our present study, the stressed rat model was established and the effect on reproduction was analyzed. Results indicated that, comparing with the control group, the rat body weight, the weigh of testis and epididymis, the number of spermatozoon were not changed significantly in acute stress group( P >0.05), the testicle structure is also not changed. 3β-HSD expression in testis of acute stress rat was reduced 7 %. In chronic stress group, comparing with the control group, the rat body weight, the weigh of testis and epididymis and the number of spermatozoon were remarkable decreased (P< 0.05), the structure of somniferous tubule became thin, spermatogenesis' cell became less in Lumina and 3β—HSD expressed in rat testis was reduced 19.6%. Accordingly, the function of generative organ will be degrading when stress time was prolonging.The level of follicle-stimulating hormone(FSH), luteum hormone (LH) and testosterone (T) in rat from the stressed group and control group had been detected by enzyme linked immunosorbent assay(ELISA). Compared with the control, the variation of FSH[(2.7±0.4) ng/ml vs.(2.6±0.2)ng/ml], LH[(3.1±0.3)ng/ml vs.(2.8±0.6)ng/ml]and T[(3.9±0.5) ng/ml vs.( 2.7±0.2) ng/ml]in acute stressed group are not significant.( P > 0.05). The variation of FSH [ (1.9±0.5) ng/ml vs. ( (2.3±0.4) ng/ml], LH [ (2.9±0.3) ng/ml vs.( 3.0±0.5) ng/ml] and T[ (1.9±1.1) ng/ml vs.( 2.9±1.3) ng/ml ]in subacute stressed group are also not significant.( P > 0.05). Interestingly, in chronic stress group, the difference of LH levels[ (2.1±0.4) ng/ml vs.( 3.0±0.8) ng/ml] (P< 0.05) and of T levels [ (0.6±0.3) ng/ml vs.( 3.0±0.4) ng/ml] are significant (P<0.001). The variation of FSH [ (1.6±0.5 ) ng/ml vs. (2.2±0.3) ng/ml] are not significant. ( P > 0.05). These results demonstrated that stress is the one of important reasons to affect reproductive endocrine function in male rat.Annexin5 was one of binding protein with phospholipids. It located on Sertoli cel and Leydig cell. Compared with the control group, the result of immunohistochemistry showed that the Annexin5 located in Leydig cell, Sertoli cell and the head of mature sperm in rat testis. Western blotting showed that the expression of Annexin 5 in testis of the acute stress rat was reduced 10.8%. In chronic stress group, comparing with the control group, the result of immunohistochemistry showed that Annexin5 in the head of mature sperm was tapered . In stressed group for 1 week and 2 weeks, Annexin5 was not shown its distribution in the head of mature sperm. In stressed group for 3 weeks and 4 weeks Annexin5 was relocating at the head of mature sperm and the mode of Annexin5 distribution in Leydig cell and Sertoli cell was not changed significantly. Western blotting showed that the expression of Annexin5 in rat testis was reduced 17.4%. Compared with control group,the expression of Annexin5 mRNA was decreased in stressed group, and it was difference between the subacute stressed group and control group( P< 0.05 ) . As stress time prolonged, the expression of Annexin5 mRNA increased slowly in the chronic stress group. These data demonstrated that stress alter normal distribute pattern of Annexin5 in rat testis.Eventually, the level of Caspase-3 mRNA ,Bax /Bcl-2 mRNA expression in rat testis were examined by real-time RT-PCR. Compared with the control group, the result of RT-PCR show that the expression of Caspase-3 mRNA is not changed significantly in the acute stressed group(3.85±0.84) vs. (3.30±0.74) (P>0.05),but there are significant variation of caspase-3 mRNA in the subacute stressed group[ (3.67±0.34) vs. ( 5.34±1.13) ] and the chronic stressed group[ (4.08±0.6) vs. (5.88±0.31) ] (P<0.05) . The ratio of Bax/Bcl-2 mRNA in the acute stressed group is not difference with control group (1.61±1.01) vs. ( 1.15±0.29 )(P>0.05) .Compared with control group ,the ratio of Bax /Bcl-2 mRNA is difference in the subacuted group[(1.60±0.42)vs. (2.68±0.86) ]and in chronic group[ (2.99±0.76) vs. (3.88±0.60) ] (P<0.001) .Compared with control group, the result of TUNEL showed the apoptotic index of germ cells is not difference in the acute stressed group[ (1.50±0.58) vs. (3.75±0.50) ],but difference in the subacute stressed group[(1.50±0.58) vs. (10.50±1.29) ]and in the chronic stressed group[ (1.50±0.58) vs. (21.50±1.73) ] (P<0.001). These results suggested that stress induced by fear play an important role in spurring germ cells apoptosis and Caspase-3, Bax and Bcl-2 were involved in this process.Stress by fear, as one set of psychological stress, disrupt testicular structure and reproductive internal secretion and spur spermatogenic cell apoptosis in rodent's animal. These results imply on that it is important to concern about psychic health and diminish stress by fear to maintain reproductive function.