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. 2023 Nov 20;300(1):105486. doi: 10.1016/j.jbc.2023.105486

Figure 6.

Figure 6

The role of tACE in sperm physiological functions.A, the total number of sperm per cauda in WT and CKO mice. B, measurement of sperm length using image-J software. Each dot represents a sperm. C, measurement of sperm motility. Sperm motility video was shown in Video S1. D, measurement of sperm acrosin activity. C and D, groups of samples were treated for 12 h with 10 mM GW9662 as indicated (n = 12/group). E and F, measurement of sperm motility (E), and acrosin activity (F) ± treated for 12 h with either 10 μM ramipril or 100 μM losartan (n = 9/group). G–I, measurement of ATP production, motility, and acrosin activity. Groups of WT mice were treated with the drugs i.p. (1 dose/day) for 5 days before sperm isolation as follows: bradykinin 2 receptor (B2R) antagonist HOE-140 at 100 μg/kg/day, neurokinin 1 (NK1) receptor antagonist L-733060 at 20 mg/kg/day, and POP inhibitor KYP-2047 at 10 mg/kg/day (n = 6/group). The drugs were continued during the experiment (10 μM HOE-140, 10 μM L-733060 and 50 μM KYP-2047). J, In vitro fertilization rate of CKO and WT sperm. Left: representative image of embryos at different stages (Pronuclear, 2-cell, 4-cell, and Morulae). The scale bar represents 10 μm. Right: the fertilization rate was calculated as the number of two cell embryos divided by the number of total oocytes. K, measurement of in vivo fertilization rate. Table shows pregnancy rates, embryos per female mice, and total number of embryos at E14.0 after artificial insemination. G and H, groups of mice were treated with 40 mg/l ramipril or 600 mg/l losartan in drinking water for one week before sperm isolation. For PPARγ blockade, isolated sperm were incubated with 10 mM GW9662 for 12 h A–H, data are presented as means ± SEM. A one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons, and data are presented as means ± SEM. NS, no significance. ∗∗p < 0.01 and ∗∗∗p < 0.001. CKO, C-domain KO; POP, prolyl oligopeptidase; PPARγ, peroxisome proliferators–activated receptor gamma; tACE, testis angiotensin-converting enzyme.