Figure 6.

2 Hz-EA treatment improves the sperm motility and functional CatSper channels expression in the spermatozoa, and enhances the fertility of asthenozoospermic (AZS) model rats. (A) Sperm motility including grade A and grade A+B sperm (n = 10 rats per group). (B) CatSper (from CatSper1 to CatSper4) protein abundance (n = 3-6 rats per group). (C) Representative fluorescence images from Fluo-4 loaded sperm before and after administering 30 mM NH₄Cl in different groups as indicated. Scale bar = 25 μm. (D) Representative single sperm fluorescence traces. (E) Changes in normalized [Ca²⁺]_i fluorescent signals of all tested sperm. (F) Summary plot of normalized [Ca²⁺]_i fluorescent signals of all tested sperm in response to NH₄Cl treatment (n = 26-34 spermatozoa from 6-8 rats per group). (G-I) Protein tyrosine phosphorylation (pTyr) (G), hyperactivation (H), and acrosome reaction (AR) (I) in the epididymal sperm. A summary plot for the percentage of sperm acrosome reaction (H) and representative images of sperm acrosome reaction (I) are shown (n = 4 rats per group). Asterisk indicates the sperm that has acrosome reaction (acrosome disappeared). Scale bar = 25 μm. (J-O) In vivo fertility assay for 2 Hz EA- and mock EA-treated AZS rats (n = 4-8 male rats per group). (J-K) Validation of the increased CatSper1 protein (J) and sperm motility (K) for the 2 Hz EA-treated male rats that to mate with the normal female rats. (L) The pregnancy rate of female rats that were mated with the 2 Hz EA- and mock EA-treated AZS male rats. (M-N) Representative and a summary plot for number of pups per litter. (O) 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)-(B), (G)-(H), (J)-(K), and (N)-(O); one-way ANOVA with Sidak's post-hoc test for (F). See also Figures S12-S15.