Figure 1.

Expression of pattern recognition receptors that recognize viral nucleic acids in the testis. A, Total RNA was extracted from the testis and peritoneal macrophage (Mϕ) of 5-week-old WT mice. The expression of MDA5, RIG-I, and the indicated TLRs was examined at mRNA levels using real-time qRT-PCR. B, Protein levels of MDA5 and RIG-I. The lysates of the testis and Mϕ were analyzed via Western blot using specific antibodies. C, Localization of MDA5 and RIG-I. Immunofluorescence staining was performed in frozen testicular sections of 5-week-old mice using specific antibodies and tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary antibodies. The sections were costained with DAPI. The seminiferous tubules (ST) were delineated by dotted lines. The square areas were magnified for high (Hi) resolution (right panels). Asterisk, closed arrow, open arrow, closed arrowhead, and open arrowhead indicate the interstitial cells, acrosome, round spermatid, spermatocyte, and spermatogonium, respectively. D, Cell-specific expression of MDA5 and RIG-I. Sertoli cells (Sc), Leydig cells (Lc), germ cells (Gc), and Mϕ were isolated from 5-week-old WT mice. The mRNA levels of MDA5 and RIG-I in these cells were analyzed using real-time qRT-PCR (left panel), and their protein levels were determined using Western blot (right panel). E, Germ cell stage-specific expression of MDA5. Spermatogonia (Spg), spermatocytes (Spc), and spermatids (Spm) were isolated. The mRNA levels (left panel) and protein levels (right panel) of MDA5 in the 3 stages of germ cells were analyzed using real-time qRT-PCR and Western blot, respectively. β-Actin was used as loading control for Western blot. Images are representatives of at least 3 experiments. Data are the mean ± SEM of the 3 independent experiments. Bar, 20 μm.