Figure 4

mA3-/- mice have higher genital infection and more seminal plasma virus load. Age and weight-matched WT, mA3+/-, and mA3-/- male mice on C57BL/6 background (n = 5) were inoculated with LP-BM5 virus via the IP route. Eight weeks after infection, mice were sacrificed, and all male reproductive organs obtained. Samples were pooled based on genotype. Spermatozoa and seminal plasma were extracted from pooled epididymis, and spermatozoa examined for morphology prior to purification (A). Following purification, sperm cells were also examined for morphology (B). DNA was isolated from spermatozoa (C) and testes (D) and evaluated for level of infection by qPCR. Total RNA was isolated from a portion (200 μl) of cell-free seminal plasma virus preparation. Equivalent concentration of RNA was reverse transcribed, and the resulting cDNA subjected to qPCR examination of viral RNA (E). A portion of testes (F) and cell-free seminal plasma virus preparation (G) were subjected to protein extraction and Western blot with antibodies against MLV capsid (α-p30) and murine GAPDH (α-GAPDH -used to demonstrate that the cell-free seminal plasma virus preparation devoid of cellular extract). The remaining portion of cell-free seminal plasma virus preparation was used to infect splenocytes from WT C57BL/6 ex vivo for 24 hours. Infected splenocytes were used for DNA extraction and qPCR examination of level of infection (H). Total DNA was also isolated from different male reproductive organs. Following reverse transcription of RNA, the level of viral RNA in these tissues was determined by qPCR (I). Western blot and qPCR data were normalized to GAPDH. Error bars are standard error; * is significance with p value equal or less than 0.05; and ** is significance with p value equal or less than 0.01. Experiments were performed at least three different times and similar results were obtained.