Mechanisms On Porcine Luteal Cells Apoptosis And Inhibition Of Progesterone Production Induced By Porcine Parvovirus

Porcine parvovirus?PPV?,a member of the Protoparvovirus genus of the Parvovirinae subfamily of the Parvoviridae family,is a major causative agent of reproductive failure which is characterized as infertility,early embryonic death and resorption,stillbirth,mummified fetuses,and dead fetuses in swine.Porcine parvovirus has been endemic in most parts of the world,and could cause devastating abortion in pigs without vaccination or with inappropriate vaccination.A mount of studies have reported that PPV infection causes the fetal death or weak piglet through inducing the variety of tissue damage.However,the effects of PPV infection on pregnant sow are rarely reported.The corpus luteum plays a pivotal role in establishment and maintenance of pregnancy in swine depending on the producing progesterone?P4?by the luteal cells.Previous studies have demonstrated that PPV infection cause the infertility and abortion via inducing maternal corpus luteum damage.However,the underlying molecular mechanism remains unknown.In the present study,the in vivo and in vitro porcine luteal cells were utilized as the research model to explore the roles of PPV on luteal cells apoptosis,progesterone synthesis and the underlying molecular mechanism after the PPV infection in primary and immortalized porcine luteal cell line.The results were as follows:1.Ovaries were obtained from healthy gilts at day 30 to day 50 of gestation at local abattoir.The primary luteal cells were dispersed and purified by collagenase and a discontinuous Percoll gradient.The primary porcine steriodogenic luteal cells were immortalized with pCI-neo-hTERT,and then treated with 100?g/ml G418 to obtain the monoclonal cells by a series of dilution.These monoclonal cells were further cultured and defined by progesterone assay.Finally,one clone,which did not become senescence over 50passage and exhibits positive reaction in detection of steroidogenic property,was selected and named hTERT-PLCs.RT-PCR and telomerase activity assay results showed the hTERT-PLCs continuously expressed hTERT and maintained high telomerase activity over the examined time.Karyotyping results showed that primary and hTERT-PLCs at passage 30 and 50exhibited the normal diploid number of chromosomes at passage 30 and 50.The results of3?-HSD staining and Oil-Red-O staining also demonstrated that both hTERT-PLCs and primary PLCs were steroidogenic luteal cells.Transmission electron microscope observation demonstrated that there are the gap junctions between adjacent cells,abundance of mitochondria and smooth endoplasmic reticulum in both hTERT-PLCs and primary porcine luteal cells.RT-PCR detection showed that hTERT-PLCs expressed the key genes?CYP11A1,HSD3B1,STAR and AKR1C1?and characteristic receptors?ESR1,ESR2,PGR,PRLR,and LHCGR?involved in regulating progesterone synthesis and degradation.Treatment of LH on hTERT-PLCs or primary PLCs for 24 h,the StAR and 3?-HSD expression and progesterone production?p?0.05?were increased in a dose dependent manner.In contrast,the cells treated with the PGF2?analog results showed StAR and 3?-HSD expression and progesterone production were significantly decreased?p?0.05?.Tumorigenicity analysis showed there was no malignant transformation in vitro and in vivo.2.The primary and immortalized luteal cells were infected with PPV and MTT results showed PPV-infection could reduce the cell ability?p?0.05?.The q-PCR detection showed PPV could replicate in primary and immortalized luteal cells and the copy number were significantly increased?p?0.05?in a time-dependent manner.Hoechst 33258 staining showed PPV induced nuclear condensation and fragmentation in both cells.DNA fragmentation assay showed PPV induced DNA ladder.The flow cytometer detection results demonstrated that the apoptotic ratio of primary and immortalized luteal cells were also significantly increased?p?0.05?in a time-dependent manner.Caspase activity assay results showed that PPV infection increased the caspase-9 and caspase-3 activity?p?0.05?,but the caspase-8 activity did not change?p>0.05?.Meanwhile,the pretreatment of caspase-8 specific inhibitor did not affect the caspase-3 activity?p>0.05?.Western blotting results showed that PPV-infection had no effects on Fas and FasL expression?p>0.05?,but upregulated the expression of Bax protein?p?0.05?and downregulated the expression of Bcl-2 protein?p?0.05?,resulting the increase of Bax/Bcl-2 ratio?p?0.05?.Moreover,the protein levels of cytochrome c were significantly decreased in mitochondria and increased in cytoplasm?p?0.05?.The results of western blotting and RT-PCR detection showed PPV-infection could increase p53 mRNA and protein expression levels in both primary and immortalized steroidogenic luteal cells?p?0.05?.Pretreatment with p53 inhibitor or siRNA could significantly inhibit PPV-induced Bax protein expression?p?0.05?.Furthermore,PPV infection also induced the phosphorylation of p38 in both primary and immortalized luteal cells and p38 specific inhibitor could significantly inhibit PPV-induced p38 phosphorylation,decreas the total p53 and nuclear p53 protein expression levels?p?0.05?.The flow cytometer detection results also demonstrated that p38specific inhibitor pretreatment significantly inhibited PPV-induced luteal cells apoptosis?p?0.05?.3.The primary and immortalized luteal cells were infected with PPV and radioimmunoassay results showed that PPV decreased progesterone production in cell culture medium?p?0.05?.Western blotting and Real time-PCR results demonstrated that PPV could inhibit the expression of progesterone synthesis relative protein and emzays StAR,3?-HSD and P450scc expression?p?0.05?.In addition,pretreatment of Bax,p53 and p38 specific inhibitors could inhibit PPV-induced downregulation of progesterone production?p?0.05?.4.The thirty-six gilts were equally divided into control and PPV infection groups.The PPV-infected groups were exposed intranasally to 10 ml(10⁷ TCID₅₀/ml)of PPV stocks at Day 22 of gestation and designated as 0 day post-infection?0 dp.i.?.Blood was collected from ear vein at 7,14,21,28 and 35 dp.i.and the serum was harvested to monitor the pregnancy condition.After PPV infection,the PPV antibody levels became positively.The radioimmunoassay results showed that the circulating progesterone levels of PPV infection group were significantly lower than that of control group at 14 dp.i.?p?0.05?.The ovaries were collected from the control and PPV infection groups at 21,28 and 35 dp.i.The hybridization in situ results showed that PPV were mainly replicated in luteal cells and the amount of virus were increased in a time-dependent manner in the corpus luteum.TUNEL and indirectly IF results the apoptotic cells were increased in a time-dependent manner after PPV infection and the apoptotic cells were mainly luteal cells.Histopathology observation showed that PPV infection caused the obvious histopathologic changes of corpus luteum,including edema,part of luteal cells vacuolar degeneration,and it became more obviously depend on the infection time.Transmission electron microscope observation demonstrated that PPV infection induced the luteal cells showed condensed nuclei,reduced cytoplasmic volume,organelle lysis and apoptotic bodies at 35 dp.i..Western blotting results showed that PPV infection increased the activated caspase-9 and caspase-3 levels and Bax expression levels,but decreased Bcl-2 expression levels?p?0.05?.Meanwhile,p53 and phosphorylated p38 protein levels were also upregulated in corpus luteum?p?0.05?.The total RNA and protein in corpus luteum were extracted,Real time-PCR and Western blotting results showed that StAR,3?-HSD and P450scc mRNA and protein levels were downregulated in corpus luteum?p?0.05?.In summary,we established an immortalized porcine luteal cell line which had the genetic stability and kept the main biological characteristics and function of primary porcine luteual cells.PPV could replicate in luteum cells after the PPV infection in primary and immortalized porcine luteal cells.Hoechst 33258 staining,DNA fragment analysis and flow cytometer detection results showed that PPV infection induced luteal cells apoptosis.The further study demonstrated PPV induced luteal cells apoptosis via activating p38,p53 and mitochondrial pathway.Moreover,PPV also inhibited progesterone synthesis through downregulating the expression level of StAR,3?-HSD and P450scc in primary and immortalized steroidogenic luteal cells.The pretreatment of Bax,p53 and p38 specific inhibitors could reverse PPV-induced downregulation of progesterone production in primary and immortalized steroidogenic luteal cells.In PPV infected gilts,TUNEL staining results showed that the positive cells were main luteal cells.Meanwhile,PPV-induced in vivo luteal cells apoptosis via activating p38,p53 and mitochondria pathway.In addition,the serum progesterone levels,StAR,3?-HSD and P450scc expression levels in corpus luteum were decreased,which were coincide with the in vitro study.Our results demonstrated that PPV infection could induce luteal cells apoptosis and inhibit luteal cells progesterone production and provided the new view and theoretical basis on PPV-induced porcine luteal cells apoptosis and progesterone production.