Cytochalasin D restores nuclear size acting on F-actin and IZUMO1 localization in low-quality spermatozoa

Abstract

In spermatozoa, the nuclear F-actin supports the acroplaxome, a subacrosomal structure involved in the correct exposure of several acrosomal membrane proteins; among them, the glycoprotein IZUMO1 is the major protein involved in sperm-oocyte fusion. Nuclear F-actin is also involved in sperm head shaping and chromosome compartmentalization. To date, few notions regarding the bivalent role of F-actin on sperm chromatin organization and IZUMO1 positioning have been reported. In our work, we characterized subcellular organization of F-actin in human high- and low-quality spermatozoa (A- and B-SPZ), respectively, showing that F-actin over-expression in sperm head of B-SPZ affected IZUMO1 localization. A correct IZUMO1 repositioning following in vitro induction of F-actin depolymerization, by cytochalasin D treatment, occurred. Interestingly, F-actin depolymerization was also associated with a correct acrosome repositioning, thus to favor a proper acrosome reaction onset, with changes in sperm nuclear size parameters and histone acetylation rate reaching high-quality conditions. In conclusion, the current work shows a key role of F-actin in the control of IZUMO1 localization as well as chromatin remodeling and acetylation events.

Keywords: F-actin; IZUMO1; chromosomes territories; histone acetylation; sperm quality.

Figure 1

F-actin and IZUMO1 characterization in human high- and low-quality spermatozoa. (A-B) Western blot analysis of (A) F-actin (F-ACT) and (B) beta-actin (ACTB) proteins in A- and B-SPZ fractions collected from normozoospermic volunteers (n=6 different samples in triplicate for each sperm fraction). Signals were quantified by densitometry analysis and normalized to Ponceau Red (Pon.S). Data were expressed in OD values as fold change and reported as mean ± SEM; **p < 0.01. (C) F-actin immunofluorescence analysis by phalloidin staining (red) in A- and B-SPZ fractions collected from normozoospermic volunteers (n=6 different samples for each sperm fraction). White arrowheads represent F-actin localization in sperm head. Nuclei were labeled with DAPI (blue). Scale bar corresponds to 4 μm. (D) Histogram showing the quantification of phalloidin fluorescence signal intensity using ImageJ software. Values were expressed as mean ± SEM, **: p<0.01 (n=6 different samples in triplicate for each sperm fraction). (E) Western blot analysis of IZUMO1 protein in A- and B-SPZ fractions collected from normozoospermic volunteers (n=6 different samples in triplicate for each sperm fraction). Signals were quantified by densitometry analysis and normalized to Ponceau Red (Pon.S). Data were expressed in OD values as fold change and reported as mean ± SEM. (F) Immunofluorescence analysis of IZUMO1 (FITC-green) in A- and B-SPZ fractions collected from normozoospermic volunteers (n=6 different samples for each sperm fraction). White arrowheads represent acrosomal IZUMO1 localization in sperm head of A-SPZ; white asterisk represents IZUMO1 localization in the neck-midpiece of B-SPZ. Nuclei were labeled with DAPI (blue), while F-actin was labeled with phalloidin (red). Scale bar corresponds to 4 μm. (G) Immunofluorescence analyses of Ab-IZUMO1 (FITC-green), rabbit IgG (FITC-green) and Ab-IZUMO1+IZUMO1-peptide (FITC-green) in A- and B-SPZ. White arrowheads represent sperm head acrosomal localizations; white asterisks represent localizations in sperm head posterior part and the apical part of midpiece. Nuclei were labeled with DAPI (blue), while F-actin was labeled with phalloidin (red). Scale bar corresponds to 4 μm. (H) Confocal z-stack 3-D reconstruction strategy showing the head (white arrowheads) vs neck-midpiece (white asterisks) localization of IZUMO-1 (green). (I) Western blot analysis of protein immunoprecipitation assay (IP) in A- and B-SPZ using IZUMO1 antibody. IZUMO1-IP was analyzed in comparison with Input protein extracts.

Figure 2

F-actin depolymerization restores acrosomal IZUMO1 localization. (A) Western blot analysis of F-actin (F-ACT) in A- and B-SPZ fractions before (pre-A; pre-B) and following in vitro CYTO-D (post-A; post-B) (n=6 different samples for each experimental group in triplicate). Signals were quantified by densitometry analysis and normalized to Ponceau Red (Pon.S). Data were expressed in OD values as fold change and reported as mean ± SEM. Experimental groups with statistically significant differences (p<0.05; p<0.01) were indicated with different letters. (B) Phalloidin staining (red) and immunofluorescence analyses of IZUMO1 (FITC-green), PNA (FITC-green) and TSSK6 (FITC-green) in A- and B-SPZ fractions before (pre-A; pre-B) and following in vitro CYTO-D treatment (post-A; post-B) (n=6 different samples for each experimental group). White arrowheads represent sperm head acrosomal localizations; white asterisks represent localizations in sperm head posterior part. Nuclei were labeled with DAPI (blue), while F-actin was labeled with phalloidin (red). Scale bar corresponds to 4 μm. (C-E) Cellular counting of sperm cells having (C) typical IZUMO1 acrosomal localization, (D) typical PNA acrosomal localization and (E) typical TSSK6 posterior localization in A- and B-SPZ fractions before (pre-A; pre-B) and following in vitro CYTO-D treatment (post-A; post-B) (n=6 different samples in triplicate for each experimental group; more than 100 cells were analyzed for each sample). Data were expressed as percentage of positive cells on total and reported as mean ± SEM. Experimental groups with statistically significant differences (p<0.05; p<0.01) were indicated with different letters; the experimental groups without statistically significant differences were indicated with the same letter. Images of IZUMO1, PNA and TSSK6 anomalies in post-B were showed. (F) Transmission Electron Microscopy (TEM) experiments in A- and B-SPZ treated with CYTO-D (pre-A; pre-B; post-A; post-B). Short black arrows represent IZUMO1 localization, while long black arrows indicate acrosome position. Scale bar corresponds to 1 μm.

Figure 3

Characterization of IZUMO1 maturation during epididymal transit. (A) F-actin immunofluorescence analysis by phalloidin staining (red) in murine SPZ collected from caput and cauda epididymis (n=6 different samples for each experimental group). White arrowheads represent F-actin localization in sperm head. Nuclei were labeled with DAPI (blue). Scale bar corresponds to 4 μm. (B) Immunofluorescence analysis of IZUMO1 (FITC-green) and TSSK6 (FITC-green) in murine SPZ collected from caput and cauda epididymis (n=6 different samples for each experimental group). White arrowheads represent IZUMO1 and TSSK6 localizations in sperm head. Nuclei were labeled with DAPI (blue), while F-actin was labeled with phalloidin (red). Scale bar corresponds to 4 μm. (C) Immunofluorescence analyses of Ab-IZUMO1 (FITC-green), rabbit IgG (FITC-green) and Ab-IZUMO1+IZUMO1-peptide (FITC-green) in murine SPZ collected from caput and cauda epididymis. Nuclei were labeled with DAPI (blue) while F-Actin was labeled with phalloidin (red). Scale bar corresponds to 4 μm. (D) Western blot analysis of protein immunoprecipitation assay (IP) in caput and cauda SPZ using IZUMO1 antibody. IZUMO1-IP was analyzed in comparison with Input protein extracts.

Figure 4

Characterization of CYTO-D effects on sperm functional parameters. (A) Sperm capacitation analysis by chlortetracycline, CTC (green) staining in A- and B-SPZ before (pre-A; pre-B) and following in vitro CYTO-D treatment (post-A; post-B) (n=4 different samples for each experimental group). Scale bar corresponds to 4 μm. (B) Immunofluorescence analyses of PNA (FITC-green) and Ab-IZUMO1 (FITC-green) in A- and B-SPZ treated with CYTO-D (pre-A; pre-B; post-A; post-B) following AR induced by A23187. White arrowheads represent sperm head acrosomal localization of PNA and IZUMO1; white asterisks represent localizations in sperm neck-midpiece. Nuclei were labeled with DAPI (blue), while F-actin was labeled with phalloidin (red). Scale bar corresponds to 4 μm. (C) TEM experiments in A- and B-SPZ treated with CYTO-D (pre-A; pre-B; post-A; post-B) following AR induction. Short black arrows represent IZUMO1 localization, while long black arrows indicate acrosome position. Scale bar corresponds to 1 μm.

Figure 5

Characterization of IZUMO1/F-actin interaction in human sperm head. (A) Separation of A- and B-SPZ before (pre-A; pre-B) and following in vitro CYTO-D treatment (post-A; post-B) into head- and tail-enriched fractions. Representative images of sperm showing head and tail-enriched fractions, as indicated. Scale bar corresponds to 50 μm. (B) Western blot analysis of IZUMO1 protein in head- and tail-enriched protein fractions of A- and B-SPZ before (pre-A; pre-B) and following in vitro CYTO-D treatment (post-A; post-B) (n=3 different samples for each experimental group in triplicate) using IZUMO1 antibody. Signals were normalized to Ponceau Red (Pon.S). (C) F-actin immunofluorescence analysis by phalloidin staining (red) in head-enriched fractions of A- and B-SPZ before (pre-A; pre-B) and following in vitro CYTO-D treatment (post-A; post-B) (n=6 different samples for each experimental group). Nuclei were labeled with DAPI (blue). Scale bar corresponds to 4 μm. (D) Histogram showing the quantification of phalloidin fluorescence signal intensity using ImageJ software. Values were expressed as mean ± SEM, **: p<0.01 (n=6 different samples in triplicate for each sperm fraction). (E) Western blot analysis of protein immunoprecipitation assay (IP) in head-enriched fractions of A- and B-SPZ before (pre-A; pre-B) and following in vitro CYTO-D treatment (post-A; post-B) using IZUMO1 antibody. IZUMO1-IP was analyzed in comparison to Input protein extracts. (F-G) Signal quantification of (F) F-actin (F-ACT) and (G) IZUMO1 of Input protein extracts western blot analyses in head-enriched fractions of A- and B-SPZ before (pre-A; pre-B) and following in vitro CYTO-D treatment (post-A; post-B). Signals were quantified by densitometry analysis and normalized to Ponceau Red (Pon.S). Data were expressed in OD values as fold change and reported as mean ± SEM; experimental groups with statistically significant differences (p<0.05; p<0.01) were indicated with different letters; the experimental groups without statistically significant differences were indicated with the same letter.

Figure 6

F-actin depolymerization restores nuclear size of low-quality spermatozoa. (A) Nuclear area analysis in A- and B-SPZ fractions before (pre-A; pre-B) and following in vitro CYTO-D treatment (post-A; post-B) (n=3 individuals for each experimental group; with more than 500 cells were analyzed per individual); graphical representation of nuclear consensus was obtained with NMAS software. (B) Frequency of linear and radial positioning within the sperm nucleus of territory of six different chromosomes territories in A- and B-SPZ fractions before (pre-A; pre-B) and following in vitro CYTO-D treatment (post-A; post-B) (n=4 individuals for each experimental group; with more than 400 nuclei were analyzed per individual); (C-D) Representative size of the territory and intertelomeric distance of chromosome 9 in sperm nuclei from A- and B-SPZ fractions before (pre-A; pre-B) and following in vitro CYTO-D treatment (post-A; post-B); (E) Heatmaps of chromocenter position in sperm nuclei from A- and B-SPZ fractions before (pre-A; pre-B) and following in vitro CYTO-D treatment (post-A; post-B) by NMAS.

Figure 7

F-actin depolymerization restores histone acetylation grade of low-quality spermatozoa. (A-C) Western blot analysis of H4K5Ac (A), H4K8Ac (B) and H4K12Ac (C) in A- and B-SPZ fractions (n=5 different samples for each experimental group in triplicate). H4ac signals were quantified by densitometry analysis and normalized to total histone H3. Data were expressed in OD values and reported as mean ± SEM; **p < 0.01. (D) CMA3 and Aniline blue staining of A- and B-SPZ. Percentage values were reported as mean ± SEM; **p<0.01. Scale bar corresponds to 10 μm. (E) Immunofluorescence analysis of H4K5Ac (red), H4K8Ac (red) and H4K12Ac (red) in A- and B-SPZ fractions (n=6 samples for each experimental group). Nuclei were labeled with DAPI (blue); Scale bar correspond to 3 μm. (F) Immunofluorescence analysis of H4K5Ac (red), H4K8Ac (red) and H4K12Ac (red) in A- and B-SPZ fractions before (pre-A; pre-B) and following in vitro CYTO-D treatment (post-A; post-B) (n=6 samples for each experimental group); nuclei were labeled with DAPI (blue). Scale bar correspond to 3 μm. (G-I) Histogram showing the quantification of H4K5Ac (G), H4K8Ac (H) and H4K12Ac (I) signal intensity using ImageJ software. Values were expressed as mean ± SEM. Experimental groups with statistically significant differences (p<0.01) were indicated with different letters; the experimental groups without statistically significant differences were indicated with the same letter.