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. 2020 Mar 24;10:5335. doi: 10.1038/s41598-020-62091-y

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Human sperm SPACA6 expression. (a) Western blot using extracts of COS-7 cells transfected or not with mouse Spaca6-GFP and human SPACA6-GFP, revealed with rabbit polyclonal anti-human SPACA6, anti-Tubulin and anti-GFP antibodies. A band about 72 kDa (~36 kDa for SPACA6 + ~36 kDa for GFP) was visible only in COS-7 human SPACA6-GFP samples. In contrast, all samples were positive for Tubulin (55 kDa band) and those transfected with GFP were positive for it, demonstrating the quality of the deposited proteins and the efficiency of transfection respectively.(b) Western blot of human sperm protein extract using the anti-hSPACA6 antibody. The expected size band (~36 kDa) is only in the antibody lane. Controls with secondary antibody alone or with rabbit isotype (IgG) were negative. The corresponding full-length blots are presented in Supplementary Fig. S1. (c) Human SPACA6 distribution on sperm. Rabbit polyclonal anti-hSPACA6 revealed by a Donkey anti-rabbit Alexa 594 conjugated secondary antibody (red), localized SPACA6 on the acrosomal cap, equatorial and neck regions and midpiece of acrosomal intact sperm as attested by positive PSA-FITC conjugated (green) staining. After acrosome reaction (PSA-FITC negative), SPACA6 was remained essentially in the equatorial segment of sperm head. Nucleus was stained with Dapi (blue). AI: Acrosome Intact, AR: Acrosome Reacted, Es: Equatorial segment, Ac: Acrosomal cap, Nc: Neck, Mp: Midpiece.