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. 2021 Oct 29;148(21):dev199644. doi: 10.1242/dev.199644

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© 2021. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 3. — Fam71f1 but not Fam71f2 is required for the acrosome reaction. (A) IVF assay of Fam71f1-mutant males. Fam71f1-mutant spermatozoa were unable to fertilize CI or CF eggs. In contrast, the fertilization rate (percentage of two pronuclei observed 8 h after insemination) was partially rescued with ZF eggs. (B) IVF assay of Fam71f2-mutant males. The fertilization rates with CI and CF eggs were significantly lower in Fam71f2^{−9516/−9516} mice compared with Fam71f2^+/−9516 mice. In contrast, the fertilization rate was rescued with ZF eggs. (C) Immunofluorescence staining for IZUMO1 (green) of live Fam71f1-mutant spermatozoa. After the acrosome reaction (4 h incubation), IZUMO1 spread to the equatorial segment in the control. However, in Fam71f1-mutant spermatozoa, IZUMO1 localization was abnormal, although it was exposed on the surface of the acrosome. PI (red), which stains nuclei of dead spermatozoa, was used to distinguish live cells from dead cells. (D) The acrosome reaction rates of Fam71f1-mutant spermatozoa at 15 min and 4 h after incubation in capacitation medium. After 4 h incubation, the Ca²⁺ ionophore A23187 was added to induce the acrosome reaction. Live spermatozoa with IZUMO1 signal were considered acrosome reacted. After 4 h of incubation and A23187 addition, the acrosome reaction rates were significantly lower in Fam71f1^−4/−4 mice compared with Fam71f1^+/−4 mice. (E) Immunofluorescence staining for IZUMO1 of live Fam71f2-mutant spermatozoa. After the acrosome reaction (4 h incubation), IZUMO1 spread to the equatorial segment in both the control and Fam71f2-mutant spermatozoa. PI was used to distinguish live cells from dead cells. (F) The acrosome reaction rates of Fam71f2-mutant spermatozoa at 15 min and 4 h after incubation in capacitation medium. After 4 h incubation, A23187 was added to induce the acrosome reaction. Live spermatozoa with IZUMO1 signal were considered acrosome reacted. There were no significant differences in acrosome reaction rates between Fam71f2^+/−9516 and Fam71f2^{−9516/−9516} mice. Error bars represent s.d. n=3 males in each group; *P<0.05, ***P<0.001 (unpaired Student's t-test). Scale bars: 10 µm in C,E.