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. 2021 Oct 18;38(12):3175–3193. doi: 10.1007/s10815-021-02314-x

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© The Author(s) 2021

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Association of sH3 and sH3.3 with asthenozoospermic sperm. (A) The validation of sH3.S-sulfhydrated proteins of human sperm lysates were biotinylated via biotin-switch assay, enriched via the IP based on anti-biotin antibody, and detected in Western blotting analysis using anti-H3 antibody (top panel). H3 in the lysate of pooled human sperm (n=5) was immunoprecipitated via anti-H3 antibody, and subjected to biotin-switch assay. The S-sulfhydrated H3 was detected in Western blotting analysis using anti-biotin antibody (bottom panel). (B) Detection of overall S-sulfhydrated proteins in sperm with different motility. Subpopulation of sperm with high and low motility from three fertile men was separated via swim-up assay, respectively, and their protein lysates were subjected to biotin-switch assay, detected with anti-biotin antibody in Western blotting assay (left panel). As a loading control, the proteins in the sperm lysates stained with Coomassie blue after separated via SDS-PAGE (right panel). (C) Expression of sH3 and sH3.3 in sperm with different motility. The biotinylated proteins in (B) were immunoprecipitated with biotin antibody, and further analyzed with anti-H3 and anti-H3.3 antibodies in Western blotting assay. In addition, the biotinylated protein in (B) from sperm with different motility was subjected in Western blotting assay using the indicated antibodies. The experiments were replicated in three fertile individuals. Error bar denotes mean ± SEM. *P < 0.05 and **P < 0.01. (D) H3 and H3.3 were enriched via IP with their antibodies from lysates of clinical sperm samples with different progressive motility (PR%), as indicated, next subjected to biotin-switch assay. One portion of the biotinylated proteins, as a loading control, were analyzed using anti-H3 and anti-H3.3 antibodies in Western blotting assay, while another was analyzed for detection of sH3 or sH3.3 using biotin antibody in Western blotting assay. (E) Relative expression of sH3 and sH3.3 in asthenozoospermic (ASTH) sperm (N=19 for sH3; N=24 for sH3.3) compared with the normozoospermic (NORM) controls (N=16 for sH3; N=26 for sH3.3). Error bar denotes mean ± SEM. ***P < 0.001. (F) Correlations among sperm sH3 (n=35), sH3.3 (n=50), and progressive motility were analyzed by linear regression. (G) Effects of H₂O₂ and H₂Son expression of human sperm sH3.3. The cultured sperm was added with indicated concentration of H₂O₂ and NaHS, respectively, and 1 h later, subjected to analysis of sH3.3 expression. The analysis represented one of three independent experiments with almost the same results.