Impact Of Pseudorabies Virus On Function Of Porcine Ovarian Granulosa Cells And Its Mechanism

A variety of virus can directly infect ovary,placenta,embryo or fetus,or indirectly induce high fever or high levels of pro-inflammatory cytokines,resulting in reproductive disorders in sows,such as ovarian dysfunction,abortion,stillbirth or intrauterine growth retardation.Pseudorabies（PR）is an acute and febrile infectious disease caused by pseudorabies virus（PRV）,which is widely prevalent in the world.PRV causes severe reproductive failure in sows via semen infection,vertical transmission of placenta or intrauterine infection.Although it has been confirmed that PRV can infect porcine ovarian tissue,the mechanism of the virus breaking through the"Blood-follicle barrier"and resulting in reproductive failure in sow are still unclear.Ovarian granulosa cells（GCs）,the largest cell population in ovary,play an important role in the process of follicular development.It is unclear whether PRV can infect the porcine GCs and impact the cell function,causing reproductive dysfunction in sows.Therefore,in the present study,we isolated the primary porcine ovarian GCs and studied the impact of PRV on cell proliferation,apoptosis and hormone secretion;meanwhile,we also preliminary explored its related mechanism.Firstly,the isolated porcine primary ovarian GCs were identified by indirect immunofluorescence assay（IFA）.The result showed that the isolated GCs were FSHR positive staining,and the purity meets the need of the following experiments.There was no infection of PRV and PCV2 in the isolated GCs.We used different titers of the PRV,propagated in PK-15 cells with a titer of 10^-5TCID₅₀/0.1 m L,to infect the primary ovarian GCs,and obvious cytopathic effects（CPE）could be observed under microscope in titer-dependent and time-dependent manner.The expression of PRV g E significantly increased at 24 h post infection（P<0.01）.The pathogen PRV mainly located in the nucleus of GCs.MTS and flow cytometry assays showed that PRV infection significantly inhibited cell proliferation of GCs（P<0.05）,and arrested the cells in S phase.The expression of the proliferative protein Ki67 was decreased by IFA and RT-q PCR assays（P<0.05）.Levels of S phase-related genes including CDK2,CCNE1,CCNA1 and CCNB1 were also down-regulated after PRV infection（P<0.05）.In addition,the level of ERK1/2 protein was down-regulated,and the phosphorylation form was increased in response to PRV infection（P<0.05）.The effect of PRV on apoptosis of porcine ovarian GCs was further detected by TUNEL staining,flow cytometry and Western blot.The results showed that PRV significantly induced the apoptosis of the GCs（P<0.05）,with up-regulation of proapoptotic proteins Bax,caspase-9 and cleaved-caspase-9,and caspase-3 proteins（P<0.05）.Finally,we detected the effect of PRV infection on secretion of reproductive endocrine hormones including estrogen（E₂）,progesterone（P₄）,activin A（ACT-A）,inhibin B（INH-B）,and follistatin（FS）in ovarian GCs.The results showed that PRV inhibited the secretion of E₂and P4,and increased the levels of INH-B and FS in GCs（P<0.05）.The expressions of key genes involving the hormone synthase were down-regulated,including CYP19A1,17β-HSD,3β-HSD,St AR and CYP11A1（P<0.05）.Collectively,this study demonstrates that PRV can affect cell proliferation and process of cell cycle,induce apoptosis of the porcine ovarian GCs as well the secretions of the reproductive endocrine hormones.Regulation of PRV on the functions of GCs might be mediated through MAPK or mitochondrial pathway.