Interference of pseudorabies virus infection on functions of porcine granulosa cells via apoptosis modulated by MAPK signaling pathways

Abstract

Background: Pseudorabies virus (PRV) is one of the major viral pathogens leading to reproductive disorders in swine. However, little is known about the effects of PRV infection on porcine reproductive system. Ovarian granulosa cells are somatic cells surrounding oocytes in ovary and required for folliculogenesis. The present study aimed to investigate the interference of PRV on functions of porcine ovarian granulosa cells in vitro.

Methods: Primary granulosa cells were isolated from porcine ovaries. To investigate the PRV infectivity, transmission electron microscopy (TEM) was used to check the presence of viral particles, and the expression of viral gE gene was detected by quantitative real-time PCR (qPCR) in PRV-inoculated cells. After PRV infection, cell viability was detected by MTS assay, Ki67 for proliferative status was determined by immunofluorescence assay (IFA), cell cycle and apoptosis were detected by flow cytometry, and progesterone (P₄) and estradiol (E₂) were determined by radioimmunoassay. The checkpoint genes of cell cycle and apoptosis-related proteins were studied by qPCR and western blotting.

Results: Virus particles were observed in the nucleus and cytoplasm of PRV-infected granulosa cells by TEM imaging, and the expression of viral gE gene increased in a time-dependent manner post infection. PRV infection inhibited cell viability and blocked cell cycle at S phase in porcine granulosa cells, accompanied by decreases in expression of Ki67 protein and checkpoint genes related to S phase. Radioimmunoassay revealed decreased levels in P₄ and E₂, and the expressions of key steroidogenic enzymes were also down-regulated post PRV-infection. In addition, PRV induced apoptosis with an increase in Bax expression and activation of caspase 9, and the phosphorylation of JNK, ERK and p38 MAPKs were significantly up-regulated in porcine ovarian granulosa cells post PRV infection.

Conclusions: The data indicate that PRV causes infection on porcine ovarian granulosa cells and interferes the cell functions through apoptosis, and the MAPK signaling pathway is involved in the viral pathogenesis.

Keywords: Apoptosis; MAPK signaling pathway; Ovarian granulosa cells; Pseudorabies virus; Steroidogenesis.

Fig. 1

PRV infectivity in porcine ovarian granulosa cells. Granulosa cells were infected with PRV suspension (a titer of 50 TCID₅₀) at 37 °C for 1.5 h to allow viral attachment, then collected for viral nucleic acid analysis or for transmission electron microscopy analysis. (A) Nucleic acid level of PRV gE in porcine granulosa cells infected with PRV at different time points. Statistical analysis was compared with the 0 h group; data represented as mean ± SEM (n = 3); * P < 0.05, **P < 0.01. (B) Transmission electron microscopy (TEM) analysis. Porcine ovarian granulosa cells inoculated with PRV (50 TCID₅₀) was observed at 24 h post infection. A large amount of mature viral particles (dark color, about 200 nm in size) were observed, adhering to nuclear membrane of the PRV-infected cell, and immature viral particles presented in the cytoplasm; scale bars were 500 nm (left panel) and 100 nm (right panel), respectively. Yellow arrows indicate mature viral particle in dark color, about 200 nm in size, and red arrows indicate immature viral particle in light color, about 100 nm in size. N: nucleus; M: mitochondria

Fig. 2

PRV infection inhibited cell viability and Ki67 level in porcine ovarian granulosa cells. Porcine granulosa cells were infected with PRV suspension (5, 10 and 50 TCID₅₀) for the indicated time points. (A) Cell viability was detected by MTS assay. Data represented as mean ± SEM (n = 3); Asterisks indicate a significant difference compared to the Mock group. **P < 0.01. (B) PRV infection (50 TCID₅₀) caused obvious cytopathic effects in granulosa cells at 24 h post infection. (C) Expression of Ki67 in granulosa cells following PRV infection at 36 h by IFA. (D) Quantification of the fluorescence signal by Image J. Data show as mean ± SEM (n = 3); * P < 0.05, ** P < 0.01

Fig. 3

PRV induced S phase cell cycle arrest in porcine granulosa cells. (A) Proportions of porcine granulosa cells in each phrase were analyzed by flow cytometry. (B) S phase-related genes were detected by RT-qPCR assay after PRV infection. Data were mean ± SEM (n = 3). Significant differences between Mock- and PRV-infection are indicated by ** P < 0.01

Fig. 4

PRV infection impaired steroidogenesis in porcine ovarian granulosa cells. Porcine granulosa cells were infected with PRV at titers 5, 10 and 50 TCID₅₀ for 1.5 h and then cultured for 36 h, steroid hormones in the cell culture medium were measured by radioimmunoassay, and the expression of steroidogenesis-related enzymes in cells were detected by qPCR. (A) Progesterone (P₄) level. (B) Estradiol (E₂) level. (C) Expression of CYP19A1, 17β-HSD, CYP11A1, 3β-HSD and StAR mRNA. Expression of the target genes were normalized to the corresponding GAPDH level. Data were mean ± SEM (n = 3). Significant differences between Mock- and PRV-infection are indicated by * P < 0.05, ** P < 0.01

Fig. 5

Effect of PRV on apoptosis of porcine granulosa cells. Apoptosis level was measured in porcine granulosa cells infected with PRV at titers 5, 10, 50 TCID₅₀ or Mock using flow cytometry. (A) Representative image of flow cytometry-based analysis. (B) Quantitative analysis of the apoptotic cells stained as Annexin V-FITC positive. (C and D) Expression of apoptosis-related proteins including cleaved-caspase 9, caspase 9, Bax and Bcl-2 was determined by Western blotting. Data were shown as mean ± SEM values of three independent experiments. Significant differences between Mock- and PRV-infection are indicated by * P < 0.05, ** P < 0.01

Fig. 6

PRV infection activated the MAPK signaling in porcine granulosa cells. The expression of three major MAPKs family members-ERK, p38 and JNK was detected in porcine granulosa cells following PRV infection with PRV at titers 5, 10, 50 TCID₅₀ by Western blot analysis. Representative blot images and quantification for p-p38/p38 (A), p-JNK/JNK (B) and p-ERK/ERK (C). Data were shown as mean ± SEM values of three independent experiments. Significant differences between Mock- and PRV-infection are indicated by * P < 0.05, ** P < 0.01