Studies On The Reproduction Function Of Male Mouse Due To The Heat Stress And Involved Molecular Mechanisms

Heat stress is a general name for nonspecific responses caused by the stimuli in vivo or in vitro such as temperature change, ionizing radiation, overtiredness, starvation, starvation, new feeds, infection and intoxication. Researches have shown that the high temperature has great negative effect on the sperm quality. In this study, we first established a mild heat-treated mouse model using the intelligent lighting incubator via several different times and temperatures. Then, the results by the sperm quality detection showed that:At 31℃,34℃and 37℃, the percentage of motile, the viable-spermatozoa and the integrity acrosin in epididymis were decreased separately according with the dealing time. Meanwhile, the sperm abnormality rate showed a reverse trend, but nearly returned to normal in later stage. Among all the heat-treated group, the 37℃got the most typical phenotype. So we take it to the next step of our analysis. Meanwhile, the detection of the sperm associated enzyme activities showed that the positive rate and the activity of the Hyaluronidase (HYD), the activity of the acrosomal enzyme(ACE) and the lactate dehydrogenase-C4 in the heat-treated mice was lower significantly than the control(P<0.05 or 0.01). In contrary, the acid phosphatase(ACP)activity assay show the higher tendency than the control(P<0.05 or 0.01). The results above indicated that the activity of the HYD et al have significant positive correlation with PIA, and HYD and PIA together showed the significant negative correlation with heat stress.Further, in order to reveal the molecular mechanism underlying the sperm quality decreasing caused by the heat stress, we analyzed the role and the function of the differential expression proteins during the process of spermatogonia differentiation in the testis of the heat stressed mouse by the proteomic method. First, the mouse testicular proteome profile was mapped and compared with that of the heat treated mice using 2D electrophoresis and mass spectrometry.18 protein spots on the 2D gel (16 proteins because of one spot possibly contains two different proteins) were found differentially between the heat shocked tissues and the control. Of these spots, some repair proteins which might give some explanation to the influence of the sperm quality were found. These included the up-regulated proteins (mainly the repair proteins in DNA damage and the HSPs) and the down-regulated ones (mainly associated with the protein conformation folding, the cell cycle and the apoptosis). In order to reveal the quantitative change of the differential protein expressions before and after heat shock, we then focused on the differential expressions of Bag-1, HSP40, HSP60 and HSP70 which were found expressed differently (p<0.05) after heat shock and would be involved in the heat stress response by real-time PCR and western-blotting. We found that the expression of Bag-1 decreased in the testis tissue of rats along with the heat-treated time, and reached to the lowest on day 7. Meanwhile, the amount of HSP60 increased slowly and stably from day 1 to day 5, but sharply on day 7. Although HSP40 showed different change on different days, it is more than normal everyday, and increased to the peak on day 5, followed by a descent trend on day 7;Similarly, the expression of HSP70 increased after heat shock, and reached the maximum on day 5-7. All the above results together indicated that Hsps, as molecular chaperones, protect the function of the germ cell generally through ensuring the correct refolding of the proteins.In summary, the changes of the sperm quality and the related enzymes were detected using the constructed mouse heat-stressed model and the differential protein expressions under heat stress were revealed by the proteomics and the following analysis in this research. Our data provide scientific basis for the studies of the relationship between the sperm quality decrease under heat.stress and the testicular proteome including the heat stressed proteins. The proteins and the protein networks identified will provide important leads for pursuing a more complete understanding of the mechanism for the male reproductive toxicity under heat stress. Clearly the results will be valuable to improve the anti-heat stress ability and the sperm quality for mammals.