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. 2023 Feb 14;120(8):e2207263120. doi: 10.1073/pnas.2207263120

Fig. 3.

Fig. 3.

Infertility of 1700029I15Rik Knockout Males Is Attributed to Impaired Sperm–Egg Interaction. (A) In vitro fertilization (IVF) analysis of sperm fertilizing ability using wild-type ZP-free eggs. (B and C) In vivo fertility test of wild-type and 1700029I15Rik–/– males. Eggs were harvested from superovulated B6D2F1 female mice that had copulated with wild-type or 1700029I15Rik knockout males. Sperm in the perivitelline space (yellow arrowheads) and pronuclei in the fertilized eggs (yellow asterisks) were visualized by Hoechst 33342. (D and E) In vitro analysis of sperm–egg binding. Spermatozoa pre-incubated in the Toyoda, Yokoyama, Hoshi (TYH) medium were probed with an anti-EQTN antibody and an Alexa Fluor™ 488-conjugated secondary antibody to reveal the acrosomal status. The acrosome-intact and acrosome-reacted sperm are marked by solid and hollow arrowheads, respectively. Sperm heads were stained with Hoechst 33342. (F and G) In vitro analysis of sperm–egg fusion using Hoechst 33342-preloaded ZP-free eggs. Yellow arrows indicate the fused sperm heads carrying the Hoechst dye transferred from the eggs. (H) Coimmunostaining of IZUMO1 (green) and 1700029I15Rik (red) in wild-type testis cryosections. Cell nuclei were visualized by Hoechst 33342.