Figure 6. MuV-induced testicular innate immune responses in vivo.

MuV (1 × 10⁷ plaque forming unit, PFU) in 10 μl of PBS was injected into one testis of five-week-old C57BL/6 mice. An equal volume of PBS alone was injected into the contralateral testis for the control (Ctrl). (A) Determination of MuV in the testis. Total RNA was extracted from the testis at 4 h after MuV infection. MuV nucleoprotein (MuV-NP) RNA was determined with RT-PCR (upper panels). MuV-NP protein was determined by Western blot (lower panels). β-Actin was used as control in RT-PCR and Western blot. (B) NF-κB and IRF3 activation. At 4 h post injection, p-p65 and total p65 (left panels), as well as p-IRF3 and total IRF3 (right panels) levels in the testicular lysates were determined with Western blot. (C) Nuclear translocation of p65 and IRF3. At 4 h post injection, the frozen sections of the testes were subjected to immunofluorescence staining to locate p65 (upper panels) and IRF3 (lower panels) using specific antibodies (Abs). The sections were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) to identify nuclei. (D) Cytokine levels. The testis was lysed by grinding in PBS at 24 h after injection. The cytokine levels in the lysates were measured with ELISA. (E) Cellular distribution of cytokines. The localization of TNF-α, CXCL10 and IFN-β was determined with immunohistochemical staining on the paraffin sections. Images represent at least three independent experiments. Scale bars, 20 μm. Arrows, arrowheads, and asterisks indicate Sertoli, interstitial, and germ cells, respectively. Data are presented as the means ± SEM of three experiments (n = 2 mice each experiment). **p < 0.01.