【作者】 王萍；

【导师】 卫恒习； 石俊松；

【作者基本信息】 华南农业大学 ， 农业硕士（专业学位）， 2018， 硕士

【摘要】 精液蛋白在维持精子受精能力和精卵结合过程中具有重要调控作用,与雄性动物的繁殖性能密切相关,可作为评价雄性动物生育力的潜在生物标记。哺乳动物精液中CRISPs蛋白家族主要包括CRISP1、CRISP2和CRISP3,在人和小鼠上这三种蛋白均参与受精过程的调控。但猪精液中CRISPs蛋白的来源、功能及其与公猪繁殖性能的相关性研究报道较少。本研究首先利用ELISA方法测定了大白猪精液中CRISP1、CRISP2和CRISP3的相对含量,并与公猪的繁殖性能指标(参配母猪分娩率、窝均产仔数、窝均活仔数、窝均键仔数及繁殖效率)进行了相关性分析。然后,利用RT-PCR、Western blot、免疫荧光和免疫组化技术确定了精液中CRISP2和CRISP3蛋白的主要分泌器官、及其组织或细胞定位情况。最后,通过在受精液中添加抗体的方法分别研究了CRISP2和CRISP3蛋白对猪体外受精效果的影响。主要试验结果如下:(1)精子蛋白CRISP1含量与其参配母猪的繁殖效率存在正相关趋势(P=0.096);精子蛋白CRISP2含量与参配母猪的窝均总仔数、窝均活仔数和窝均健仔数呈显著正相关(P<0.05),与繁殖效率存在正相关趋势(P=0.096);精子蛋白CRISP3含量与参配母猪的分娩率、繁殖效率呈显著正相关(P<0.05),与窝均总仔数存在正相关趋势(P=0.065);精清蛋白CRISP3含量与繁殖效率呈显著正相关(P<0.05),与参配母猪的窝均总仔数存在正相关趋势(P=0.062)。(2)Western blot检测发现,CRISP2蛋白主要分布在成年公猪睾丸和精子中,免疫荧光染色发现CRISP2蛋白定位于未获能精子的顶体后区、颈部及尾部,获能精子的顶体区、顶体后区、颈部及尾部。Western blot检测发现,CRISP3蛋白主要分布在成年公猪前列腺、尿道球腺、精子和精清中,免疫荧光技术检测发现,CRISP3蛋白定位于未获能精子的顶体后区及尾部,获能精子的尾部中段上端。(3)体外受精试验结果发现,在受精液中添加CRISP2抗体能显著降低体外受精卵裂率(P<0.05),而添加CRISP3抗体虽然卵裂率也有降低,但与对照组相比差异不显著(P>0.05)。上述结果表明,精液中CRISP2蛋白含量与公猪繁殖性能存在正相关关系,并可能参与受精过程调控,为建立评定精液受精能力和公猪繁殖性能的生物标记提供了参考依据。更多还原

【Abstract】 Semen proteins play an important regulatory role in maintaining sperm fertilization ability and sperm-egg binding process,and are closely related to reproductive performance of male animals,and can be used as potential biomarkers for evaluating fertility of male animals.The CRISPs protein family in mammalian semen mainly includes CRISP1,CRISP2,and CRISP3,In humans and mouse,CRISP1,CRISP2 and CRISP3 proteins play an important role in regulating fertilization.However,there are few reports on the relationship between the origin and function of CRISPs protein in pig semen and their reproductive performance.In this study,the relative contents of CRISP1,CRISP2,and CRISP3 in semen of Yorkshire were firstly determined by ELISA method,and the reproductive performance index of boars(sex fertility rate,litter size per litter,alive litter size per litter,qualified litter size per litter The correlation analysis was performed on the number of traits and breeding efficiency.Then,the main secretory organ,tissue and cell location of CRISP2 and CRISP3 proteins in semen were determined by RT-PCR,Western blot,immunofluorescence and immunohistochemistry.Finally,the effects of CRISP2 and CRISP3 proteins on in vitro fertilization of pigs were studied by adding antibodies to the fertilized fluid.The main results are as follows:(1)There was a positive correlation between the relative content of sperm protein CRISP1 and its reproductive efficiency(P=0.096);relative content of sperm protein CRISP2 and litter size,alive litter size and qualified litter size There was a significant positive correlation between the number of adolescents(P<0.05)and a positive correlation with reproductive efficiency(P=0.096).The relative content of sperm protein CRISP3 was significantly positively correlated with the tiller rate and breeding efficiency of female sows(P<0.05),positive correlation with litter size(P=0.065);relative content of semen plasma protein CRISP3 and reproduction efficiency was significantly positively correlated(P<0.05)and a positive correlation with litter size(P=0.062).(2)Western blot detection found that the CRISP2 protein was mainly distributed in the testis and sperm of boars.Immunofluorescence staining revealed that CRISP2 protein was located in the acrosomal,posterior,cervical and caudal regions of unstimulated spermatozoa.After sperm capacitation,CRISP2 was mainly distributed in the sperm head.The acrosomal,posterior acrosome,neck,and tail.The CRISP3 protein was mainly distributed in the prostate,urethral glands,sperm and spermatozoa of boars.Immunofluorescence staining revealed that CRISP3 was localized in the acrosomal and posterior regions of unstimulated sperm and CRISP3 was mainly distributed after sperm capacitation.At the upper end of the sperm midsection.(3)In vitro fertilization test results showed that adding CRISP2 antibody to the fertilized solution can significantly reduce the in vitro fertilization cleavage rate(P<0.05),while the addition of CRISP3 antibody also reduced the cleavage rate,but the difference was not significant compared with the control group.(P>0.05).The above results indicate that there is a positive correlation between CRISP2 and CRISP3 protein content in the semen and reproduction performance of the boar,and may participate in the regulation of fertilization process,providing a reference for the establishment of biomarkers for assessing sperm fertilization capacity and boar reproductive performance.更多还原