LASP1, a Novel Protein in Spermatozoa and Acrosome Reaction

Figures

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  Figure 1. Morphometric analysis and reproductive function of spermatozoa from wild‐type (WT) and LASP1 knockout mice (Lasp1 ‐/‐ ). (A) The days between two litters (1–2 and 2–3) from 13 wild‐type (WT) and 18 Lasp ‐/‐ mated mice were counted. (B) Number of offspring from litter 2 and 3 of 15 WT and 14 Lasp ‐/‐ mice. (C) Western blot analysis of 2 Mio sperm per lane of WT, Lasp ‐/‐ and human sperm; β‐Actin served as loading control. (D) Sperm count of 16 WT and 16 Lasp ‐/‐ mice age 12–16 weeks. (E) Average head size and (F) tail length of 50 sperms from 10 WT and 10 Lasp ‐/‐ mice. (G) Time‐dependent analysis of WT and Lasp1 ‐/‐ sperm motility after sperm activation with calcium/bicarbonate/albumin. (H) Linear swimming distances of WT and Lasp1 ‐/‐ spermatozoa in the direction of an acetylcholine (Ach) gradient. A, B, D–F: Statistical differences were examined by unpaired Student's t‐test. Data are presented as mean ± SEM. G, H: Statistical differences were examined by Mann‐Whitney test. Data are presented as mean ± SEM.

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  Figure 2. LASP1 expression in human and mouse spermatozoa. Sperm were fixed in 95% ice‐cold ethanol and stained for nucleus (DAPI), actin (Phalloidin‐green) and LASP1 (#pan1418 with Cy3‐red). Immunofluorescence displays LASP1 expression in head, midpiece (body) and tail of mammalian spermatozoa—co‐localized with actin (merge). LASP1 immunostaining is absent in Lasp‐/‐ mice. Scale bar: 10 µm.

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  Figure 3. LASP1 localization before and after capacitation in mouse and human spermatozoa. Smear sperm preparations were fixed in 95% ice‐cold ethanol and stained for nuclei with DAPI (blue), actin (phalloidin‐green) and LASP1 (#pan1418 with Cy3‐red). After activation, human and mouse spermatozoa show co‐localization of LASP1 and actin at the equatorial segment (merge).

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  Figure 4. Quantification of the acrosome reaction in WT and LASP1‐deficient spermatozoa. (A) Left site: Exemplary mouse sperm acrosome staining by PNA‐488. Intact acrosome is stained in green (arrows). Nuclei are counterstained in blue (DAPI). Scale bar: 10 µM. Right site: Time‐dependent acrosome reaction in sperm of wild‐type (WT) and LASP1‐knockout (Lasp ‐/‐ ) after stimulation with calcium/bicarbonate/albumin. A total of 120–150 spermatozoa was visually analyzed for no, partial or total loss of PNA staining. Only spermatozoa with complete loss of PNA‐staining were counted. Statistical differences were examined by Mann Whitney test. Data are presented as mean ± SEM (n = 4). (B) Flow cytometric analysis of hyaluronan receptor (CD44) expression on the surface of sperm from wild‐type (WT) and Lasp1 ‐/‐ mice 30 and 60 min after activation. Statistical differences were examined by two‐way ANOVA test. Data are presented as mean ± SEM (n = 6). (C) In‐vitro‐fertilization of wild‐type oocytes with WT (n = 4) and LASP1‐deficient sperm (n = 6). For each experiment, 50–100 oocytes were incubated with 1.7–3.5 ×105 spermatozoa (ratio 1:3500), and two‐cell blastocysts were counted after 24 h. The…

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  Figure 5. Protein phosphorylation during sperm capacitation in WT and LASP1‐deficient spermatozoa. (A) Model illustrating the temporal sequence of sperm activation with respect to phosphorylation and G/F‐actin polymerization (Created in BioRender. Butt (2026) https://BioRender.com/bbxe67). (B) Time‐dependent Western blot analysis of phosphotyrosine (4G10 antibody), phospho‐MAP‐kinases (pERK, pP38) and pAKT in wild‐type (WT) and Lasp1 ‐/‐ mouse sperm without (control) and after activation (capacitation) with calcium/bicarbonate/albumin. Tubulin served as loading control. Statistical differences were examined by two‐way ANOVA test. Data are presented as mean ± SEM (n = 4). Significance p < 0.05 between groups at time point is indicated by an asterisk (*).

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  Figure 6. Male and female mice contribute to reduced fertility in Lasp ‐/‐ mice. Crossbreed mating studies were conducted by keeping the relevant pairs together for up to 30 weeks and recording (A) the days between litters and (B) the individual numbers of offsprings. Statistical differences for litters were measured by Kruskal‐Wallis test with p < 0.001 for all multiple comparisons. Statistical differences for offsprings were examined by unpaired Students t‐test. Data are presented as mean ± SEM. Number of breedings are shown in the bar chart.