Study On Screening Of Proteins Related To Male Infertility Of Cattleyak

The male infertility of cattleyak(Cattle×yak) due to spermatogenic arrest greatly restricts the effective utilization of this hybrid in yak breeding. Although much work has been done to investigate the mechanisms of spermatogenic arrest of cattleyak, there is no information regarding the differences of protein composition between cattleyak and yak testis, Therefore, screening the proteins responsible for spermatogenic arrest of cattleyak on testis proteomics level and further study on these proteins have theoretical significance in biology of reproduction. Comparative investigation of testis proteomes between cattleyak and yak using iTRAQ proteomics identified 256 differentially expressed proteins, in which a large number of the cattleyak predominant proteins were identified to act in response to various stresses(especially inflammatory stresses), enhance cell adhesion and impeded germ cell migration. In contrast, Down-regulated proteins in cattleyak were associated with defects in various metabolic processes and cellular processes during spermatogenesis. This study has found that a large number of dispensable proteins are related to spermatogenic arrest, and these proteins will provide important information and pointcut for the further study on the molecular mechanisms of spermatogenic arrest.The main results were as follows:1.Based on the comparative histology of testis from cattleyak and yak, we suggest that the atrophy of seminiferous tubules in cattleyak testis may be caused by cellular apoptosis inside of the tubules and Leydig cell populations outside of the tubules may not been involved in spermatogenic arrest. In yak testis, abundant spermatogenic cells distributed throughout from the basement membrane to the lumen of seminiferous tubule, and round spermatids or lengthened sperms appeared in the lumen. However, only monolayer of spermatogenic cells clung to the basement membrane in cattleyak testis, very fewer of spermatocytes and scarcely no round spermatids or lengthened sperms were present in the lumen of seminiferous tubule.2.The top four up-regulated proteins in cattleyak testis compared to yak were Pentraxin-related protein PTX3 precursor, S-100-related calcium-binding protein, Histone H1.0 and Osteoglycin,which showed the fold change values of 3.8,3.5,3.1 and 2.9, respectively. In contrast,MHC class II,Ropporin-1,SNRPF protein and Protamine 2 were the top four down-regulated proteins in cattleyak testis compared to yak with fold change values of-6.9,-4.7,-4.5 and-4.2, respectively.3.Dozens of proteins possibly associated with spermatogenic arrest of cattleyak were identified by comparative iTRAQ proteomics, in which a large number of the cattleyak predominant proteins may act in response to various stresses(especially inflammatory stresses), enhance cell adhesion and impeded germ cell migration. Higher expression of ICAM1, Microfibril-associated glycoprotein 4(MFAP4), Insulin-like growth factor-binding protein 7 precursor(IGFBP7), Dermatopontin(DPT), integrin alpha 9(ITGA9) and Integrin beta 1(ITGB1) may enhance cell adhesion and impede the process of detachment of germ cells from the basement membrane and the migration towards seminiferous tubule lumen.4.Some differentially abundant proteins may be associated with metabolic defects during spermatogenesis. The biogenesis of Piwi-interacting RNAs(piRNAs) depends upon the endonuclease activity of PIWI proteins, and piRNAs in complex with PIWI proteins act as an innate immune system to maintain germline DNA integrity, GPX4 is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids strongly expressed in the mitochondria of testis and spermatozoa. The depletion of GPX4 in spermatocytes caused severe abnormalities in spermatozoa,which may be one of the causes of male infertility in mice and human, It is noteworthy that the downregulations of mitochondrial cytochrome Bc1 complex subunit 2, 7 and 9 were involved in the pathways of Alzheimer's disease and oxidative phosphorylation, which may lead to mitochondrial dysfunction and germ cell apoptosis in cattleyak testis. PSME4 is a broadly expressed nuclear protein and loss of PSME4 led to a marked reduction in male fertility due to defects in spermatogenesis observed in meiotic spermatocytes and during the maturation of post meiotic haploid spermatids. To sum up, down-regulation of the proteins involved in various metabolic processes may be the potential causes of spermatogenic arrest in cattleyak.5.We mapped the 465 the differentially abundant proteins to the reference pathways in KEGG database to identify significantly enriched metabolic pathways or signal transduction pathways. In total, 15 significantly enriched pathways were obtained(p?0.05), in which the top listed five pathways were ECM-receptor interaction(p=0.0003843598), Protein digestion and absorption(p=0.0007844198), Alzheimer's disease(p=0.002146642), Ribosome(p=0.004046523) and Pentose phosphate pathway(p=0.006084241). The upregulations of integrins and their ligands in the ECM(extracellular matrix) involved in ECM-receptor interaction pathway may help the germ cells to endure pulling forces without being ripped out of the ECM. Combining analysis of the Alzheimer's diseaseand oxidative phosphorylation pathways indicated that downregulation of such proteins as the subunits of mitochondrial cytochrome Bc1 complex may lead to mitochondrial dysfunction and cell death in cattleyak testis.6.The isobaric tag for relative and absolute quantification(iTRAQ) was employed in this work to identify differences between the testicular proteomes at different development stages(P1-10 months, P2-12 months and P3-14 months) of premature cattleyak. The proteomic analysis identified 318 proteins differentially expressed at P2 stage and 327 at P3 stage compared with P1, respectively. The GO annotations of the differentially expressed proteins at P2 compared with P1 did not indicate significant discrepancies from those at P3 compared with P1, which could be attributed to spermatogenic arrest of cattleyak during their premature development. On the other hand, 56 differentially expressed proteins were found to be commonly existed at P2 and P3 stages compared with P1. Gene ontology analysis revealed that most of the differently expressed proteins were involved in molecular function of the catalytic activity, transporter activity, oxidoreductase activity and protein binding. Further analysis indicated that the differently expressed proteins including Heparin-binding proteins(HBPs), Tudor domain containing 1, Heat shock-related 70 kDa protein 2(HSPA2), Estradiol 17-beta-dehydrogenase11(17HSDXI), 2,4-dienoyl-CoA reductase and Peroxiredoxin 2(Prx2) were possibly associated with testis development and spermatogenesis, which could be selected as candidate proteins in future study to reveal the mechanisms of cattleyak infertility.