| Sperm cryopreservation has become an important technology for the continuation of genetic superiority of male germplasm resources and endangered species,and has greatly contributed to the promotion of artificial insemination and in vitro fertilization techniques,but during the freezing process,spermatozoa suffer from different degrees of DNA,molecular,acrosome and plasma membrane damage,which seriously affects sperm quality and fertilization ability.Therefore,in order to explore the mechanism of sperm freeze resistance,this study took Chinese Merino(Xinjiang Military Reclamation Type)fine wool breeding rams as the experimental object,collected the semen of breeding rams,and subjected to a slow freezing procedure to identify the differential lipid changes and functions of sheep sperm before and after freezing and thawing,the membrane protein molecule FCGR1 A was screened and its fertilization function was verified.1.Lipidomics to identify lipid changes in sheep sperm before and after freezing and thawing.In this experiment,after the overall viability and motility of sheep sperm were examined,lipid histology was used to investigate the lipid changes before and after freezing and thawing of sheep sperm in the fresh sperm group(unfrozen sperm)and the frozen group(programmed freezing of sperm).The results showed that the viability and motility of spermatozoa decreased significantly after slow freezing(P < 0.05);a total of216 differential lipids were screened according to the threshold criteria of |FC| ≥ 2 and VIP ≥ 1,of which 53 were up-regulated and 163 were down-regulated,among which glycerophospholipids and fatty acids were significantly different before and after freezethawing;the differential lipids were analyzed for KEGG functional enrichment and the differential lipid metabolites were mainly enriched in The KEGG functional enrichment analysis of differential lipids revealed that the differential lipid metabolites were mainly enriched in the glycerophospholipid metabolic pathway.2.Comparison of differential metabolites of spermatozoa before and after freezethawing among different species.The literature on metabolome and lipidome studies of spermatozoa of different species after freezing and thawing were investigated and screened twice.Combining the sheep lipid metabolites identified in Experiment 1,the differential metabolites of sheep,goat,buffalo,pig and human were subjected to functional analysis after KEGG-ID transformation in five species,and the results revealed that the differential metabolites were involved in seven metabolic pathways,further analysis showed that the differentially expressed protein FCGR1 A was involved in the regulation of unsaturated fatty acid biosynthesis and citric acid cycle metabolism pathway.In order to further explore how FCGR1 A regulates the fertilization function of sheep through the above biological processes,bioinformatics analysis of FCGR1 A protein was conducted.The results showed that the nucleotide and amino acid sequences of FCGR1 A gene in different species were relatively similar,and FCGR1 A encoded 349 amino acids,which was a hydrophilic transmembrane protein localized in cell membranes.3.Study on the function of FCGR1 A in vitro fertilization of sheep.In this experiment,the sperm of the control group(FCGR1A antibody was not added during the floating process)and the experimental group(FCGR1A antibody was added during the floating process)were thawed,and after centrifugation and floating treatment,the sperm viability,motility,mitochondrial membrane potential,early apoptosis,oxidative stress(ROS and GSH),acrosome damage and fertilization ability were detected.The results showed that the test compared with the control group for sperm viability and motility showed a significant decrease(P < 0.05);the mitochondrial membrane potential of spermatozoa was not significantly different between the test and control groups(P > 0.05);the early apoptosis of spermatozoa was not significantly different between the test and control groups;no significant difference in early apoptosis between test and control sperm(P >0.05);no significant difference in sperm ROS and GSH levels between test and control(P > 0.05);the test showed a significant increase in the rate of sperm acrosome damage compared to the control group(P < 0.05).In summary,the viability and motility of spermatozoa decreased after programmed freezing,and the differential lipid protein FCGR1 A in lipidomics is involved in the glycerophospholipid metabolic pathway,and it is located in the sperm plasma membrane involved in regulating unsaturated fatty acid biosynthesis and citric acid cycle metabolic pathway,inhibition of FCGR1 A affects sperm fertilization ability. |
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