Wessel GM et al. (2021), Sperm lacking Bindin are infertile but are othe...

ECB-ART-50712

Sci Rep 2021 Nov 03;111:21583. doi: 10.1038/s41598-021-00570-6.

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Sperm lacking Bindin are infertile but are otherwise indistinguishable from wildtype sperm.

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

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Cell-cell fusion is limited to only a few cell types in the body of most organisms and sperm and eggs are paradigmatic in this process. The specialized cellular mechanism of fertilization includes the timely exposure of gamete-specific interaction proteins by the sperm as it approaches the egg. Bindin in sea urchin sperm is one such gamete interaction protein and it enables species-specific interaction with a homotypic egg. We recently showed that Bindin is essential for fertilization by use of Cas9 targeted gene inactivation in the sea urchin, Hemicentrotus pulcherrimus. Here we show phenotypic details of Bindin-minus sperm. Sperm lacking Bindin do not bind to nor fertilize eggs at even high concentrations, yet they otherwise have wildtype morphology and function. These features include head shape, tail length and beating frequency, an acrosomal vesicle, a nuclear fossa, and they undergo an acrosomal reaction. The only phenotypic differences between wildtype and Bindin-minus sperm identified is that Bindin-minus sperm have a slightly shorter head, likely as a result of an acrosome lacking Bindin. These data, and the observation that Bindin-minus embryos develop normally and metamorphose into normal functioning adults, support the contention that Bindin functions are limited to species-specific sperm-egg interactions. We conclude that the evolutionary divergence of Bindin is not constrained by any other biological roles.

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gRNAs referenced: bindin gRNA1 bindin gRNA2

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	Figure 1. A wildtype adult H. pulcherrimus seen from the dorsal (aboral) surface (A). Bindin-null adults (B and C) are indistinguishable from the wildtype adults, and throughout their developmental progression to this point (not shown). Wildtype sperm activate eggs within moments of exposure, resulting in formation of the fertilization envelop (D). In contrast, Bindin-null sperm never activate an egg, even after prolonged exposure (E). See also Video S1 in the Supplemental Material. Scale bar in C = 1 cm for A–C; Scale bar in E = 40 µm for D and E.
	Figure 2. Fertilization assays using increasing sperm concentrations (from 100 sperm to over 10 million sperm per milliliter). Eggs of a single female were used for sperm from each male (A–D) throughout these assays. Two independent cycles of experiments were performed and one cycle of the dataset is shown. Bindin #2 represents sperm from an adult whose embryos were injected with Cas9 and gRNAs, yet no mutations were detectable in this animal. (Male B2 in Table #1).
	Figure 3. Phenotypic comparison of Wildtype and bindin—KO sperm. Flagellar beat frequency and tail length are nearly identical between the wildtype and Bindin-null sperm. (A) Average values of tail beat frequency. (B) Head dimensions of individual animal values. N shows number of batches measured and n shows number of sperm measured. The bindin-KO sperm batches used for this measurement were B1, B3, and B4 of Table 1. (C) Summary of quantitative sperm morphometry. Statistical significance: Tail length p > 0.05, flagellar beat p > 0.05, head length p < 0.005, head width p > 0.05, ratio of head length to head width p < 0.005.
	Figure 4. (A) Swimming sperm of wildtype (left) and Bindin-null (right) sperm from Hemicentrotus pulcherrimus. Image contrast has been enhanced using Image-J (ver.1.52a). Note that sperm from the Bindin-null sperm has similar head and tail morphometrics. (B) Flagellar profile from 45 successive traces of sperm. Note subtle variations between samples though essential profiles are consistent. Scale bars: 10 µm.
	Figure 5. TEM of Wildtype and Bindin-null sperm. Note the acrosome of the wildtype sperm (white arrow) with densely packed Bindin. Three representative sperm from the bindin-KO adult (Bindin #3) still have an acrosomal vesicle, but it appears less dense, and vacuolated. Note the horseshoe shaped fossa (asterisks) within the sperm nucleus even in the sperm lacking bindin. Scale bar = 0.5 µm.
	Figure 6. TEM of Wildtype and bindin-KO sperm following exposure to egg jelly that stimulates the acrosome reaction. Note the acrosomal process (white arrow) in both wildtype and bindin-KO. Scale bar = 0.5 µm.

References [+] :

Brinkman, Easy quantitative assessment of genome editing by sequence trace decomposition. 2014, Pubmed