Wessel GM et al. (2021), Bindin is essential for fertilization in the se...

ECB-ART-49026

Proc Natl Acad Sci U S A 2021 Aug 24;11834:. doi: 10.1073/pnas.2109636118.

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Bindin is essential for fertilization in the sea urchin.

Wessel GM , Wada Y , Yajima M , Kiyomoto M .

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Species-specific sperm-egg interactions are essential for sexual reproduction. Broadcast spawning of marine organisms is under particularly stringent conditions, since eggs released into the water column can be exposed to multiple different sperm. Bindin isolated from the sperm acrosome results in insoluble particles that cause homospecific eggs to aggregate, whereas no aggregation occurs with heterospecific eggs. Therefore, Bindin is concluded to play a critical role in fertilization, yet its function has never been tested. Here we report that Cas9-mediated inactivation of the bindin gene in a sea urchin results in perfectly normal-looking embryos, larvae, adults, and gametes in both males and females. What differed between the genotypes was that the bindin -/- sperm never fertilized an egg, functionally validating Bindin as an essential gamete interaction protein at the level of sperm-egg cell surface binding.

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gRNAs referenced: bindin gRNA1 bindin gRNA2

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Fig. 1. (A) Anti-bindin antibody reveals gene KOs in sperm. Sperm proteins were isolated, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, blotted to nitrocellulose, and challenged by antibody incubation. Anti-tubulin antibodies were used to estimate relative loading levels of sperm protein. HpBindin represent sperm isolated from different individuals of bindin gRNA/Cas9 mRNA microinjections, whereas Hp-WT and Sp-WT are from WT sperm of Hp (Hp-WT) and S. purpuratus (Sp-WT). (B–D) Immunolabeling of sperm in situ with anti-bindin antibodies. (B) S. purpuratus sperm. Note the Bindin spot at the tip of the sperm (arrow). (C) Hp-WT sperm. Note Bindin at the tip of the sperm (arrow), and also background labeling of the midpiece (asterisk). Surprisingly, any intact IgG (containing an Fc region) binds to the midpiece in Hp, but the Fab used as secondary antibodies does not. (D) Hp bindin KO sperm. Note that there is no Bindin label at the tip of the sperm (arrow), even though background labeling of the midpiece is present (asterisk). (Scale bar, 4 μm.)

Fig. 2. Scanning electron micrographs of (A and B) WT Hp sperm and eggs and (C and D) bindin−/− sperm on WT eggs. Note the binding to microvilli in B and lack thereof in D (arrow). (Scale bar in A and C, 20 μm; scale bar in B and D, 2 μm.) (E–H) Hp bindin−/− sperm do not bind to nor activate eggs. WT eggs challenged with WT sperm (E) or with bindin−/− sperm (G) after 30 min of incubation show a robust fertilization envelope (arrow in E) with WT sperm but not with bindin−/− sperm. After 2.5 h of incubation, the bindin−/− sperm still do not activate eggs (H), whereas sibling eggs challenged with WT sperm have developed to the eight-cell stage (F). Scale bar in G = 100 micrometers; scale bar in H = 50 micrometers.

References [+] :

Foltz, Sea urchin egg receptor for sperm: sequence similarity of binding domain and hsp70. 1993, Pubmed, Echinobase