| Mammalian sperm capacitation is an important process to ensure the fertilization of sperm and mature oocytes,which involves biochemical and morphological changes.Sperm capacitation efficiency can effectively improve the fertilization rate and reproduction rate of mammals.At present,most studies focus on the optimization of in vitro capacitation system to promote the process of sperm capacitation.The rapid development of proteomics technology provides a new research approach for sperm capacitation.The identification of differential proteins before and after sheep sperm capacitation can obtain key proteins in the process of sperm capacitation,thus providing a theoretical basis for improving the efficiency of sheep sperm capacitation.Therefore,in this study,the fresh sperm and capacitated sperm of Dorper sheep were used as the research object.The differentially expressed proteins before and after capacitation of sheep sperm were detected and analyzed by protein spectrum technology,and the regulation of lactoferrin in sheep sperm capacitation was analyzed by further screening.The results are as follows :1.Through protein spectrum analysis,203 differential proteins were screened out,and40 differential proteins were screened out by wind ratio > 1.5 and < 0.05,and 14 proteins were significantly up-regulated(P<0.05),including spermatogenesis and centriole associated 1-like protein,globin chain and ADAM metal peptidase domain 2.26 proteins were significantly down-regulated(P <0.05),including lactoferrin,defensin,seminal vesicle protein,DnaJ,heat shock protein family(HSP40)members,etc.GO and KEGG analysis showed that the differentially expressed proteins were mainly involved in spermatogenesis and sperm capacitation.2.The expression and localization of LTF in sheep sperm before and after capacitation were detected by real-time fluorescence quantitative PCR,immunoblotting and immunofluorescence localization techniques.The results showed that LTF could be detected in fresh sheep sperm and capacitated sheep sperm,and the expression difference was significant(P<0.05).LTF was localized in the head and tail of capacitated sperm.3.Furthermore,0 μg/m L,10 μg/m L,100 μg/m L and 500 μg/m L LTF were added to the sperm capacitation solution,and their effects on sheep sperm capacitation,motility and plasma membrane integrity were analyzed.The results showed that the addition of 100μg/m L LTF in the capacitation solution significantly reduced the activity and plasma membrane integrity of sheep sperm and increased the capacitation rate of sheep sperm. |
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