【作者】 赵珺；

【导师】 任炳忠； 刘松财；

【作者基本信息】 东北师范大学 ， 动物学， 2010， 博士

【摘要】 热应激是指动物机体对外界或内部的各种刺激所产生的非特异性应答反应的总和。这些刺激包括温度变化、电离辐射、过度疲劳、饥饿、运输、饲料突变、感染以及中毒等。已有研究表明,由高温产生的动物热应激反应对精液品质有着显著的负面影响。本研究利用智能光照培养箱模拟夏日高温天气,通过不同温度、不同时间处理,成功构建了小鼠热应激动物模型。精液质量检测发现,31℃、34℃、37℃处理组的小鼠附睾内精子密度、精子活力和精子顶体完整率均随着热应激持续时间的延长而呈现出不同程度的降低,同时,精子畸形率则表现出相反的变化趋势,但是在热应激处理后期精液质量出现回升趋势。而37℃处理组的小鼠精子质量最终仍处于一个比较稳定的低水平状态。小鼠精液中酶活力检测表明,热应激小鼠精子透明质酸酶(HYD)、顶体酶(ACE)和乳酸脱氢酶(LDH)C4的活性均显著低于对照组(P<0.05或P<0.01),而酸性磷酸酶(ACP)的活性与对照组相比则显著提高(P<0.05或P<0.01)。以上结果表明HYD等酶活性与顶体完整率(PIA)等形态特征指标之间存在显著正相关,HYD等酶活性和PIA等形态特征指标与热应激程度存在显著负相关。为了探讨热应激导致小鼠精液品质下降的分子机理,本研究运用蛋白质组学的技术手段,对热应激后差异表达的蛋白质在生精障碍过程中的作用进行了分析和初步的功能研究。首先构建了小鼠睾丸蛋白质的2-DE图谱,通过对比发现,热应激小鼠睾丸组织存在明显差异点位18个,质谱鉴定得到16种蛋白。表达上调的蛋白主要集中于DNA损伤修复蛋白及热应激蛋白家族HSPs,下调的蛋白主要集中于蛋白构象折叠、细胞周期和细胞凋亡相关蛋白。我们选择了其中变化显著且和热应激有关的HSP70、HSP60、HSP40和BAG-1进一步进行了mRNA和蛋白质水平上的检测,动态分析热应激前后表达量变化规律。结果发现,热应激处理时,BAG-1表达随着热应激处理时间的增加呈现下降趋势,到第7天降为最低；HSP60的表达量1-5天呈温和增长态势,而在第7天出现激增现象；HSP40的表达程度虽然随着热持续天数的不同而出现不同变化,但表达量均有所增加,在热应激第5天达到峰值,其后在第7天有回落的趋势；而HSP70的表达随着热应激持续时间的增加呈现升高趋势,第5-7天左右达到最大值。以上研究结果表明,HSPs作为分子伴侣,通过确保蛋白质重折叠的正确性,对生殖细胞正常功能的行使起到了广谱的保护作用。本研究采用小鼠构建实验动物模型,探讨了在不同热应激条件下精液品质的变化,同时通过蛋白组学方法研究了睾丸组织在热应激条件下蛋白的差异表达规律。为阐明热应激条件下精液品质变化与热应激蛋白和睾丸组织蛋白表达规律的相关关系,为研究哺乳动物抗热应激及提高精液品质奠定了基础,并为深入研究热应激导致动物生殖性状降低的分子机理提供了依据。更多还原

【Abstract】 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.更多还原