Figure 7.

CatSper1 knockdown abolishes the therapeutic effect of 2 Hz-EA treatment on asthenozoospermic (AZS) model rats. (A-B) CatSper1 protein abundance in both testis tissues (A) and epididymal sperm (B) in 2 Hz EA-treated AZS rats with locally delivering LV-shCatSper1 (knockdown, KD) or empty viral vector (control, Ctl) (n = 3-4 rats per group). (C-D) Sperm motility including grade A and grade A+B sperm in rats of EA+KD and EA+Ctl groups (n = 9-10 rats per group). (E) Representative fluorescence images from Fluo-4 loaded sperm before and after administering 30 mM NH₄Cl in different groups as indicated. Scale bar = 50 μm. (F) Representative single sperm fluorescence traces. (G) Changes in normalized [Ca²⁺]_i fluorescent signals of all tested sperm. (H) Summary plot of normalized [Ca²⁺]_i fluorescent signals of all tested sperm in response to NH₄Cl treatment (n = 28-33 spermatozoa from 4 rats per group). (I-L) Protein tyrosine phosphorylation (pTyr), hyperactivation, and acrosome reaction (AR) in the epididymal sperm of rats in EA+KD and EA+Ctl groups. A summary plot for the percentage of sperm acrosome reaction (K) and representative images of sperm acrosome reaction (L) are shown (n = 5-6 rats per group). Asterisk indicates the sperm that has acrosome reaction (acrosome disappeared). Scale bar = 25 μm. (M-P) In vivo fertility assay of rats in EA+KD and EA+Ctl groups (n = 4-6 male rats per group). (M) Summary plot of the pregnancy rate of female rats that were mated with 2 Hz EA-treated, CatSper1 KD (or vector control) AZS rats. (N-O) Representative and a summary plot for number of pups per litter. (P) Days to birth of the pup. All data are presented as mean ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Unpaired t test for (A)-(D), (I)-(K), and (O)-(P); one-way ANOVA with Sidak's post-hoc test for (H). See also Figures S16-S18.