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Zebrafish sperm favor a positively charged Bncr surface, while medaka sperm require finger 2 N-glycosylation for compatibility. AlphaFold-predicted models of medaka Bncr (A) and zebrafish Bncr (B) (cartoon, left; surface representation depicting electrostatics, right). Amino acids that were mutated are indicated in the model as sticks and color-coded: hydrophobic (orange), positively charged (blue), polar (green). C Medaka/zebrafish IVF with medaka Bncr constructs, in which individual amino acids or combinations thereof were substituted for the corresponding amino acid(s) in zebrafish Bncr. D Plot of bias index values derived from the IVF data in (C). Bias could not be calculated for data pairs for which the fertilization rate with both sperm was equal to 0. Medians (dashed lines) and quartiles (dotted lines) are shown. E Medaka/zebrafish IVF with zebrafish Bncr constructs, in which individual amino acids or combinations thereof were substituted for the corresponding amino acid(s) in medaka Bncr. F Medaka/zebrafish IVF experiments to assess the importance of N-glycosylation in Bncr’s species specificity. IVF with zebrafish Bncr N-glycosylation site variants (left); IVF with medaka Bncr N-glycosylation site variants (right). G Medaka/zebrafish IVF experiments testing sufficiency of N-glycosylation pattern combined with specific amino acid changes for determining Bncr’s species specificity. IVF with zebrafish Bncr variants (left); IVF with medaka Bncr variants (right). H In vivo zebrafish fertilization rates of combined N-glycosylation and amino acid substitution Bncr variants. Means ± SD are indicated in (C, E–H). C, E–G: two-tailed Wilcoxon matched-pairs signed rank test with the method of Pratt; p values could not be calculated for samples in which all data points were 0. D: two-tailed Wilcoxon signed rank test vs. theoretical median of 0 with the method of Pratt.
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