| Goose is a herbivorous poultry,resistant to rough feeding,highly adaptable and its meat is rich in nutrients,which is very popular among farmers and consumers.However,due to the strong broodiness behaviour of geese,the egg-laying period has been shortened,resulting in greatly reduced egg-laying performance,which seriously hinders the development of the goose industry.Studies have shown that ovarian degeneration and follicular atresia usually occur during broody period in geese,which may be related to autophagy,apoptosis and imbalanced ROS levels of granulosa cells（GCs）.Therefore,exploring the intrinsic regulate role of apoptosis of GCs during broody period will help to elucidate the physiological mechanisms of broody and control the occurrence of broodiness behaviour.Numerous studies have shown that protein phosphorylation modifications are generally involved in the regulation of a large number of biological processes such as cell cycle,signal transduction,differentiation,proliferation and metabolism,but whether protein phosphorylation can mediate the programmed death of GCs to regulate broody behavior of geese is still unclear.In view of this,this study was conducted to investigate the role of protein phosphorylation in regulating the programmed death of goose follicle GCs in the broodiness behavior of Zhedong geese.The results of this study will not only enrich the theoretical understanding of the physiological mechanism of broody in geese,but also provide new inspiration to improve the egg-laying performance of geese.1.Morphological changes and antioxidant levels in the ovaries of Zhedong geese during laying period and broody periodIn order to observe the morphological changes of the ovaries of Zhedong geese during the laying period and broody period,anatomical method was used to reveal the ovarian morphology.The results showed that the volume,length and width of the ovaries in the broody period were significantly smaller than those in the laying period（P<0.05）,the ovarian cortex and medulla were wrinkled,the follicles were severely degenerated,there were no preovulatory follicles,the follicles at all levels of development were broken,there were different degrees of collapse and deformation There was a decrease in elasticity.Ultramicroscopic observation showed that the level of apoptosis in the ovary was significantly higher（P<0.05）at the broody period.In addition,the antioxidant capacity in the serum and ovarian tissues of Zhedong white geese was examined by enzyme-linked immunosorbent assay（ELISA）,and the results showed that the serum GSH-PX activity was significantly higher（P<0.05）,SOD and CAT activities were also increased,GSH-PX,SOD and CAT activities were significantly increased in ovarian tissues during the broody period compared to the laying period,and the levels of MDA,H2O2 and GSH in serum and ovarian tissues were significantly elevated（P<0.05）;the results indicated that there was significant oxidative stress in the serum and ovarian tissues of geese from the laying period to the broody period,and it was accompanied by elevated levels of apoptosis,follicular atresia and ovarian degeneration.2.Changes in ovarian protein phosphorylation modification during the laying period and broody period in Zhedong white geeseIn order to investigate the changes of phosphorylation modification of ovarian proteins during egg-laying and broody periods in Zhedong white geese,TMT labeling technique combined with liquid chromatography-tandem mass spectrometry（LCMS/MS）were used to screen for differential phosphorylation modification of proteins.Compared to the laying period,a total of 578 up-regulated phosphorylation-modified proteins（involving 1093 loci）including FOXO1,CDC5,OPTN,etc.and 281 downregulated phosphorylation-modified proteins（involving 519 loci）including ERK,SLK,PKN2,etc were identified.,which contained 139 phosphorylation-modified motif elements.The analysis of the KEGG pathway showed that these differential protein phosphorylations were mainly involved in the broodiness behaviour of geese through apoptosis,adhesion junctions and other signaling pathways.3.Goose ERK phosphorylation mediates the MEK/ERK signaling pathway to regulate the programmed cell death of GCsIn order to verify the role of ERK protein phosphorylation in regulating programmed cell death of GCs,the WES system was used to quantify the changes of ERK protein phosphorylation modification levels in the ovaries of Zhedong white geese during the laying period and broody period.The results showed that compared to the laying period,there was no significant change in the ERK protein levels in ovarian tissues during the broody period,while the phosphorylation levels of ERK protein decreased significantly（P<0.05）.In order to verify the effects of oxidative stress on ERK protein levels and phosphorylation levels in GCs,an oxidative stress model was constructed by treating ovarian GCs with 100 μmol/L H2O2 for 12 h.It was found that ERK protein levels did not change significantly under oxidative stress conditions,while ERK protein phosphorylation levels were significantly reduced（P<0.05）.Bioinformatics predicted that the the upstream kinase of ERK was MEK,and MEK mRNA expression was significantly down regulated in the ovarian tissue of Zhedong white goose at the broody period.When overexpression of the kinase MEK the phosphorylation level of ERK was significantly increased,and the expression of BCL2 which is a downstream gene of ERK in MEK/ERK signaling pathway was significantly increased,and the cell activity also increased at the same time;while knockdown of MEK showed the opposite trend.These results suggested that ERK phosphorylation could mediate the MEK/ERK signaling pathway to inhibit programmed cell death of GCs.In summary,there was significant oxidative stress in the serum and ovarian tissues of goose during the broody period,and it was accompanied by an increase in the levels of apoptosis,follicular atresia and ovarian degeneration.Besides,a total of 859 differentially phosphorylated proteins including ERK were identified to be associated with the broodiness behaviour of goose.Moverover,ERK phosphorylation could mediate the MEK/ERK signaling pathway to inhibit programmed cell death of GCs and participate in the regulation of broodiness behaviour of geese. |
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