| The reproductive performance of boars is the top priority in the breeding industry.Semen quality is one of the important indicators for appraising reproductive traits.Some proteins in semen are related to reproductive performance and sperm quality,which can be as biomarkers for screening high and low fertility boars.Osteopontin(OPN)is considered as an important candidate gene for screening high piglet survival rate,however,the effect of OPN on sperm fertilization ability and its mechanism are still unclear.We screened glutathione peroxidases 6(GPx6)from proteomics isobaric tags for relative and absolute quantitation(i TRAQ)results of high and low fecundity in the boar semen,and studied its effect on sperm fertility.GPxs are regarded as important protectors against oxidative stress.Some members were reported to play key roles in protecting sperm against oxidative stress,however,it is not much known about GPx6,which was a member of the GPx family.The purpose of this study was to determine the relationship between two proteins with boar reproductive performance and sperm fertility.Here,we studied the differential expression analysis of OPN and GPx6 in boar semen,studied their localization in boar reproductive organs and sperm,and discussed the influence of OPN and GPx6 on boar sperm motility,in vitro capacitation,acrosome reaction and fertilization efficiency.The main results are as follows:1.Correlation analysis results showed that OPN protein was significantly related to the number of litters born alive of Large White boars and Landrace boars,and GPx6 protein was negatively related to the reproductive traits(total number born per litter,number born alive per litter and fecundity)of Large White boars.The feasibility verification analysis of screening high-fertility boars found that OPN can be used as a potential bio-tag for screening high-fertility boars,but not GPx6.However,the results of GPx6 were consistent with i TRAQ that GPx6 was relatively highly expressed in the seminal plasma of the low fertility boar.2.Obtain of porcine OPN protein were from lentiviral packaging system and eukaryotic expression system of chinese hamster ovary cells substrain(CHO-K1).After the sperm incubated with 0.1 μg/ml porcine OPN for 3 h,it was found that porcine OPN can significantly improve the motility of sperm after capacitation.The results of immunofluorescence(IF)and western blot(WB)showed OPN protein were mainly located on the head and neck of sperm,meanwhile,the expression of this protein was increased after capacitation.Results of cyclic adenosine monophosphate(c AMP)concentrations in after capacitation sperm showed that OPN significantly increased c AMP concentration.Results showed that 0.1 μg/ml OPN could significantly increase embryo cleavage rate in vitro fertilization(IVF).3.Analysis results of real-time quantitative reverse transcription polymerase chain reaction(q RT-PCR)and WB demonstrated the presence of GPx6 were highly in epididymis and seminal plasma.GPx6 is mainly expressed in early spermatogonial stem cells and located in the neck and tail of sperm by immunohistochemistry(IHC)and IF.WB results showed that it was decreased after capacitation in sperm.After sperm incubated with anti-GPx6 for 3 h,sperm motility was significantly improved.Fluorescein isothiocyanate-conjugated pisum sativum agglutinin(FITC-PSA)results showed GPx6 significantly decreased acrosome reaction rate.Furthermore,enzyme-linked immunosorbent assay(ELISA)results showed that GPx6 significantly reduced c AMP concentration;IVF results showed that GPx6 decreased embryo cleavage rate.4.Porcine OPN monoclonal antibodies were obtained by immunizing mice with porcine OPN protein and the titer was 1: 64000.Subtype identification showed that the monoclonal antibodies were immunoglobin G1(Ig G1)and the light chain types were Kappa.The specificity of the monoclonal antibody was better than that of the polyclonal antibody by WB detection.In application,OPN monoclonal antibody can accurately detect the expression of OPN protein in porcine reproductive tissue,sperm, oocytes and cumulus cells.In summary,we found that OPN and GPx6 were both related to the reproductive performance of boar.For OPN protein,we successfully obtained porcine OPN protein and monoclonal antibody.Meanwhile,we found that OPN was attached to the sperm membrane,improved sperm motility and c AMP synthesis,and thus affected embryo cleavage rate;For GPx6 protein,it was found to be highly expressed in epididymis and seminal plasma,attached to the sperm membrane to prevent premature sperm capacitation,affect c AMP synthesis,and thus affect acrosome reaction rate.In this study,OPN protein and GPx6 protein were studied the role in selection of high-breeding boar,and improvement of sperm fertility and reproductive performance by addition in diluent.Otherwise,the mechanisms of action were analyzed.All results provide new clues to explore the functional mechanism of OPN and GPx6 on sperm. |
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