Knockdown of HongrES1 expression by RNAi and its effect of sperm capacitation.

<p>(A) Northern blot and western blot analysis showed reduced expression of HongrES1 mRNA and protein in vitro 48 h after siRNAs treatment. Csi, scrambled siRNA control; Hsi1 and Hsi2, two siRNAs specifically targeting the different sites of HongrES1 sequence. 18S and GAPDH were measured as the internal control for RNA and protein loading. (B) The expression of HongrES1 was significantly suppressed by Hsi1 and Hsi2 in rat caudal tissues of epididymis on mRNA and protein levels. Data are expressed as the means±SEM (n = 7–9), **p<0.01, ***p<0.001, as compared with the scrambled siRNA control. Csi, scrambled siRNA control; Hsi1 and Hsi2, two siRNAs specifically targeting the different sites of HongrES1 sequence. 18S and GAPDH acted as a loading control respectively. (C, D) The immunofluorescence staining of sperm from cauda epididymis treated with scrambled siRNA and two specific siRNAs (C), and corresponding binding percentages obtained by flow cytometry (D). The scale bars represent 50 µm in panels. Pre, the negative control of preimmune serum. Csi, scrambled siRNA control; Hsi1 and Hsi2, two siRNAs specifically targeting the different sites of HongrES1 sequence. (E–H) The percentage of sperm motility (E), unpacitated sperm or F pattern (F), capacitated sperm or B pattern (G), and acrosome reacted sperm or AR pattern (H) after expression of HongrES1 was down-regulated. The spermatozoa of caudal epididymis from rats treated by siRNAs for 48 h were collected and incubated, CTC assay was conducted at 0 h, 1 h, 3 h of incubation in the capacitation medium. Data are expressed as the means±SEM (n = 8), **p<0.01, ***p<0.001, as compared with the scrambled siRNA control. (I) The change of protein tyrosine phosphorylation on cauda sperm proteins after HongrES1 expression was inhibited. The spermatozoa of caudal epididymis from rats treated by siRNAs for 48 h were collected and incubated. The spermatozoa total proteins were collected at 0 h, 1 h and 3 h of incubation; α-tubulin was used as loading control. Csi, scrambled siRNA control; Hsi1 and Hsi2, two siRNAs specifically targeting the different sites of HongrES1 sequence.