The Identification Of Differentially Expressed Proteins Of X And Y Sperm And The Functional Study On DPY30 In Dairy Goat

The dairy goat is a vital dairy animal for the development of milk products with Chinese characteristics.Goat milk and related milk products gradually become indispensable human food,with various characteristics,such as high nutritional value,low sensitization risk,and high similarity compared with human milk.In recent years,the dairy goat industry in China has developed rapidly with Shaanxi province-centric and Shandong province-centric.Traditional dairy goat breeding can’t meet the requirements of industrial development;therefore,the quantity and quality of animals are crucial for the development of the goat industry by promoting the modern,intensive,and technological development of dairy goat farming.New technology should be developed to conveniently,efficiently,and safely increase the female kid ratio.It would accelerate the scale of breeding dairy goats,reduce carbon dioxide emissions,improve animal welfare,and decrease production costs.It is critical for the development of dairy goats breeding and the supplement of goat milk products.In this study,high-purity dairy goat X and Y sperm were obtained by flow cytometry.Based on the separated X/Y sperm,15 differentially expressed proteins were identified by Label-free mass proteomic analysis.The crucial differentially expressed protein DPY30 was identified,so the full length of the DPY30 gene coding sequence(CDS)of dairy goats was cloned and the bioinformatic characteristics were analyzed.At the same time,temporal and tissular expression profiles of DPY30 were investigated in dairy goats.The mechanisms of retinoic acid(RA)mediated DPY30 on cell self-renewal was identified in dairy goat spermatogonia stem cell line(m GSCs-I-SB),while the effect of DPY30/p AKT/P21 on the proliferation of mice spermatogonia cell line(GC-1)was investigated.Finally,the lentiviralmediated RNA interference of DPY30 was packaged and injected into the goat to explore the effect of DPY30 on the sex ratio of kid and mouse testicular development.1.Identification of the crucial differentially expressed protein in X and Y sperm of dairy goatsX and Y sperm of dairy goats were separated by flow cytometry,and the purity was analyzed to be higher than 95 %.Subsequently,at least 556 proteins of dairy goat sperm were identified by Label-free mass proteomic analysis so that 15 differentially expressed proteins were obtained by quantitative analysis.The functions of these proteins were mainly related to sperm energy metabolism,protein processing,and cytoskeleton composition.The expression of DPY30 and DYNLL2 were up-regulated in Y sperm,confirmed by Western blotting,which was consistent with the results of proteomic analysis.The DPY30 m RNA level was the third highest among 6 differentially expressed proteins in goat testis and was the highest in various tissues of goats.The DPY30 protein level was the highest in the testis of kids,and the expression of DPY30 was lower in adult goats.Then,the expression of DPY30 in goat testis was further identified by immunofluorescence so that the signal of DPY30 was observed in male germ cells of different developmental stages,and the signal intensity decreased with cell differentiation and meiosis.DPY30 co-localized with spermatogonia stem cell self-renewal marker protein(PLZF)and differentiation marker protein(C-KIT)in goat seminiferous tubules but not with meiotic marker protein(STRA8).The complete CDS of the goat DPY30gene(300 bp)was cloned,and the amino acid sequences were highly conserved(99.6 %)among humans,pigs,cows,goats,and mice.2.DPY30 promotes goat spermatogonia cell self-renewal through the AKT signaling pathwayThe goat DPY30 recombinant adenovirus(Ad-DPY30)was constructed to obtain goat Ad-DPY30 adenovirus.DPY30 expression significantly increased with Ad-DPY30 transfection in the goat spermatogonia stem cell line(m GSCs-I-SB).Overexpression of DPY30 in m GSCs-I-SB inhibited cell pluripotency(SOX2,OCT4)and promoted cell selfrenewal(PLZF,PCNA),while the up-regulated level of SYCP3 could affect Y sperm meiosis.RA treatment attenuated cell pluripotency(SOX2,OCT4)and self-renewal(PLZF),while differentiation(C-Kit)and meiosis(STRA8,RARG)were promoted.Subsequently,DPY30 inhibited p STAT3 m,and p AMPK and stimulated p AKT,while RA induced p AMPK,and p AKT and decreased the level of p SMAD3 via reducing DPY30 expression.We found that RA inhibited DPY30 expression to decreasing self-renewal of m GSCs-I-SB via p AKT/PLZF.3.The effect of DPY30 on testicular development of mouseDPY30 deletion in GC-1 cells increased the G0/G1 cycle percentage via decreasing PCNA,CYCLIND1,and promoting P21.AKT inhibition also increased the percentage of G0/G1 cycle via decreasing PCNA,CYCLIND1,CDK2 and promoting P27,p21.The complete CDS of the mice DPY30 gene(300 bp)was cloned to overexpress DPY30.Then,DPY30 was demonstrated to promote GC-1 proliferation by stimulating the p AKT to increase P21 expression in GC-1cells.DPY30 promoted glucose uptake by AMPK attenuation induced increased glycolysis related genes.Finally,mouse testis received lentivirus PLL3.7-sh RNA M1 to knock down DPY30 while PLL3.7 was used as a positive control.At the same time,the expression of PLZF was down-regulated with the knockdown of DPY30 in mouse testis,while the expression of C-Kit and STRA8 were unchanged.The lentivirus PLL3.7-sh RNA M1 didn’t significantly affect mice testicular development and litter size.4.The DPY30 regulation of offspring sex of dairy goatsThe lentiviral-mediated RNA interference vectors PLL3.7-sh RNA C1 and C2 were constructed to select the lentiviral(C1)with higher interference efficiency(70 %).Then,the lentivirus PLL3.7-sh RNA C1 was injected into goat testis.Subsequently,DPY30 knock was confirmed to not affect the semen quality in injection goats.Then the sperm was used for artificial insemination of dairy goats.The lentivirus didn’t significantly affect goats’ pregnancy rate(70.27% ± 5.50%,70.29% ± 5.13%)and kidding rate(142.23% ± 20.46%,132.63% ± 4.37%);however,the offspring sex ratio increased in the injected group(35.40%± 6.00%,58.20% ± 5.50%).Overall,we identified the crucial differentially expressed protein DPY30 in dairy goats’ X and Y sperm.At the same time,DPY30 knock-down in the testis increased female kids’ ratio of does without affecting testicular development and reproductive performance.DPY30 promoted the self-renewal of goat spermatogonia through p AKT/PLZF,facilitated Y sperm meiosis by SYCP3,induced the proliferation of mouse spermatogonia cells through p AKT/P21,and improved glucose uptake by AMPK.In the present study,we analyzed the differentially expressed proteins in the X and Y sperm of dairy goats.We explored the sex regulation mechanism mediated by DPY30,which could provide a theoretical basis for related studies on sex control/determination and spermatogenesis in mammals.