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. 2022 Jan 3;9:806982. doi: 10.3389/fcell.2021.806982

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Copyright © 2022 Binner, Kogan, Panser, Schleiffer, Deneke and Pauli.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Dcst2 levels are reduced in zebrafish spaca6 KO sperm. (A). Immunoblot of sperm samples of different genotypes probed with antibodies against zebrafish Dcst2. Zebrafish Dcst2 is predicted to be a glycoprotein, resulting in a wider area of detected signal. Tubulin protein levels of the same blot are shown as loading control. The uncropped immunoblot is shown in Supplementary Figure S2. (B). Quantification of sperm Dcst2 levels of different genotypes based on three independent immunoblots. Values were compared to tubulin levels and normalized to wild type. Statistical significance was calculated using one-way ANOVA and multiple comparisons analysis: n.s, not significant (p > 0.05; ****, p < 0.0001; *, p = 0.045). (C). Immunofluorescent images of Dcst2 protein in sperm heads of different genotypes. Upper panels: Overview images (scale bar = 5 μm). Lower panels: Higher magnification images of an individual exemplary sperm head (boxed in the upper panels) for each genotype (scale bar = 2 μm). Sperm were stained with DAPI (blue) to visualize nuclei and antibodies against zebrafish Dcst2 (green). Dcst2 localizes to distinct foci around the sperm head membrane (green arrowheads in the lower panels) in wild-type, spaca6 ^+/- and tg(spaca6)-rescued sperm. Autofluorescence of the sperm midpiece appears as a uniform signal in the Dcst2 channel (magenta arrowhead).