FIGURE 5.

Combination of sperm energy restriction and recovery (SER) treatment and A23187 (transient treatment) rescues sperm fertilization capacity and embryo development of infertile CatSper1 KO mice. Sperm from CatSper1 KO mice were incubated in four conditions as detailed in the section “Materials and Methods.” Briefly, the experimental conditions applied were: (1) capacitating conditions in the presence of glucose and pyruvate (CAP); (2) A23187 transient treatment (A23187 WASH); (3) capacitating conditions in the absence of glucose and pyruvate followed by replenishment of energy substrates (SER); and (4) SER treatment followed by A23187 transient treatment (SER + A23187 WASH). Sperm were used for IVF of cumulus-enclosed CD1 female oocytes. After 18 h, two-cell embryos were transferred to KSOM media and further incubated for a total of 3.5 days until blastocyst stage. (A) Percentage of fertilization calculated as percentage of oocytes that reached two-cell embryo stage. (B) Percentage of blastocyst development calculated as percentage of the two-cell embryos that reached 3.5-day blastocyst stage. (C) Representative image of 3.5-day blastocysts formation from combined SER treatment followed by A23187 transient treatment. Arrows indicate blastocysts hatching. (D) Results showing total 3.5-day blastocysts obtained from either SER-treated alone, or SER + A23187-treated sperm, that were NSET-transferred to pseudo-pregnant female recipients (n = 3), and the number of pups born after 21 days of gestation. In each panel, significant differences are indicated as ^∗∗∗p < 0.001.