Analysis Of Label-free Quantification Proteomics In Yak’s Ovary And Expression Of Related Proteins In The Reproductive System

As the major livestock for Tibetan herdsmen,yak has a poor fertility,usually one birth in two years or two births in three years.And,even there are only 4-5 calves for those with higher reproduction.Its fertility could hardly meet the demands of herdsmen and restrict the increase in yak population,as a result of relatively late sexual maturity and short estrous period of 1-2 days even in lush pasture.Besides,molecule related to the adjustment and controlling of its reproduction has been rarely studied because of the bad natural conditions and limited technology.Therefore,veterinary researchers should pay more attention to boosting its fertility through identifying the related molecular markers.In this research,in order to further study the physiological and molecular regulation mechanism about yak reproduction for the improvement of its fertility,differentially expressed proteins of ovary were screened during the follicular and luteal phase of yak based on Label-free quantitative proteomics,and then their functions and involvement of biological processes or metabolic pathways were analyzed in terms of bioinformatics.At the same time,QRT-PCR,Western blot and immunohistochemistry technology were applied to research on the localization and quantification of the collected differentially expressed proteins from ovary,uterus and fallopian tube with the aim of discovery those which affected the yak’s fertility.The main research results were as follows:(1)After the analysis of protein expression profiling of ovarian tissue during the follicular and luteal phase with the application of Label-free quantitative proteomics and the screening of differentially expressed proteins,it discovered 2867 proteins during follicular phase and 2180 proteins during luteal phase,among which there were 2011 proteins of expression during both phases,856 during follicular phase,and 169 during luteal phase.And,there were 63 differentially expressed proteins(P<0.05),among which there were 47 proteins with an increase in expression levels,and 16 proteins with a decrease during luteal phase.(2)According to the bioinformatics and Go analysis,the screened differentially expressed proteins mostly located in the cell membrane and extracellular region,and primarily acted on activity of laminin.From the perspective of COG,the identified differentially expressed proteins in ovary were analyzed,revealing the presence of proteins which were involved in translation,ribosome structure and biogenesis,RNA processing and modification,transcription,biosignaling pathway transduction,post-translational modification,protein turnover,molecular chaperone,metabolism.And,the proteins involved in biosignaling pathway transduction were frequently present,while those with the involvement of cell movement were least frequent.KEGG analysis indicated that the main involvement of differentially expressed proteins included steroid biosynthesis,chemokine signaling pathway,protein processing in endoplasmic reticulum,glycerol phospholipid metabolism,carbon metabolism,PI3K-Akt signaling pathways,PPAR signaling pathways,NOD receptors signaling pathways,purine metabolic pathways,RNA transportation pathways,pancreatic secretion metabolic pathways,RNA degradation pathways,HIF-1 signaling pathways,AMPK pathway,insulin signaling pathway,calcium signaling pathways and glutathione metabolism.(3)The expression of FSHR、LHR、StAR、Rab-8a and HSP105 in ovary、uterus and fallopian tube tissue of yak during the luteal and follicular phases was verified by QRT-PCR and Western blot.The result showed that the expression of FSHR,St AR and HSP105 were significantly more during the follicular phase than the luteal phase,while the expression levels of LHR and Rab-8a increased during the luteal phase.There was an evident decrease in expression level of FSHR in ovary、uterus and fallopian tube during follicular phase,while,an increase in LHR.What’s more,a comparison between follicular phase and luteal phase showed a significant difference.The expression level of StAR in ovarian tissue during follicular phase was 5.05 times that of luteal phase,and its’ level in womb during follicular phase was 2.38 times that of luteal phase(P<0.01).Meanwhile,its’ level in fallopian tube during follicular phase was 1.82 times that of luteal phase(P<0.05).The expression level of Rab-8a in ovary,uterus and fallopian tube during follicular phase was lower than that of luteal phase(P<0.05).The expression level of HSP105 in ovary,uterus and fallopian tube during luteal phase was lower than that of follicularl phase(P<0.05).(4)The results of immunohistochemistry showed that: In the luteal stage,expression of FSHR was mainly positive in the ovarian granulosa layer,the connective tissue of the uterine intrinsic membrane and the mucosal epithelial cells of the fallopian tube;LHR was mainly expressed in the ovarian follicular granule cell layer,the mucosal epithelial cells of the uterus and the mucosal epithelial cells of the fallopian tube;Expression of StAR was weakly positive in the granular cell layer of ovarian follicle and the mucosal epithelium of the fallopian tube,but its was not showed in the uterus;Rab-8a was weakly positive in mucosal epithelium of ovarian follicular layer cells,uterus and fallopian tube;HSP105 was mainly expressedin the mucosal epithelium of the connective tissue of the ovary and the muscular layer,uterus and fallopian tube of the arteries in the medullary region.In the follicular phase,FSHR was mainly expressed in ovarian follicle granular cell layer,uterus mucosal epithelium and mucosa lamina propria of the connective tissue and uterus gland epithelial cells.Meanwhile,the expression of tubal mucosa epithelial cells was positive;LHR was expressed mainly in the epithelial ovarian reproduction and primary follicles,uterus mucosal epithelium,connective tissue of lamina propria and uterus gland epithelial cells,as well as the tubal mucosa epithelium;StAR was mainly expressed positively in the ovarian follicular granule cell layer,the uterine gland epithelium and connective tissue of the inherent layer of the uterus mucosa,and the mucosal epithelium of the fallopian tube;Expression of Rab-8a was weakly positive in the follicular granule cell layer of ovary,the connective tissue of the inherent layer of the uterine mucosa and the mucosal epithelium of the fallopian tube;Positive expression of HSP105 was mainly showed in ovarian follicular granule cell layer,uterine gland epithelium of inherent layer of uterine mucosa and mucosal epithelium of fallopian tube.