Journal: Frontiers in Cell and Developmental Biology

Article Title: Heat shock affects the Ca 2+ /calmodulin-dependent protein kinase II dynamic during bovine sperm capacitation and acrosome reaction

doi: 10.3389/fcell.2025.1552282

Figure Lengend Snippet: CaMKII dynamic during bovine sperm capacitation. (A) Spermatozoa were evaluated after thawing (control 0 h) or after 4 h of incubation in the capacitating or non-capacitating medium. The CaMKII (a, f) and acrosome (b, g) were evaluated using fluorescence microscopy following fluorescent labeling with rabbit anti-CaMKII antibody and Pisum sativum agglutinin (FITC-PSA), respectively. DNA was labeled with Hoechst 33342 (c, h) . Merged images (CaMKII + Acrosome + DNA) are shown in (d, i) , while merged images of CaMKII with Acrosome are shown in (e, j) . During capacitation, two distinct patterns (P) of CaMKII localization were observed: P1 - High CaMKII concentration at the post-acrosomal region ( a–e ; arrow in a) and P2 - high CaMKII concentration at the acrosomal region ( f–j , arrow in f). (B) Effect of bovine sperm capacitation on CaMKII localization. Results are expressed as the mean (%) ± SME of 3 independent replicates. Lowercase letters represent comparisons between patterns (P1 versus P2) within each treatment (0 h; uncapacitated – 4 h or capacitated – 4 h). Capital letters represent comparisons for each pattern (P1 or P2) among treatments (0 h, uncapacitated – 4 h and capacitated – 4 h). Different letters indicate a significant difference ( P < 0.05).

Article Snippet: After blocking, the spermatozoa were incubated for 1 h at room temperature with the rabbit monoclonal (EP1829y) antibody anti-CaMKII (abcam, ab52476) or rabbit polyclonal antibody anti-phosphorylated (T286) CaMKII (pCaMKII; abcam, ab47565) used at a dilution of 1:100 each.

Techniques: Control, Incubation, Fluorescence, Microscopy, Labeling, Concentration Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heat shock affects the Ca 2+ /calmodulin-dependent protein kinase II dynamic during bovine sperm capacitation and acrosome reaction

doi: 10.3389/fcell.2025.1552282

Figure Lengend Snippet: Subcellular localization of phosphorylated CaMKII ( p CaMKII) during sperm capacitation. (A) Bovine sperm were evaluated after thawing (0 h) or after 4 h of incubation in the capacitating or non-capacitating medium. p CaMKII (a) and acrosome (b) were evaluated by fluorescence microscopy using rabbit anti-phosphorylated-CaMKII (T286) antibody and Pisum sativum agglutinin (FITC-PSA), respectively. The DNA was counterstained with Hoechst 33342 (c) . The merged images ( p CaMKII + Acrosome + DNA) are shown in (d) , while the merged images of p CaMKII with Acrosome are shown in (e) . During capacitation, p CaMKII is predominantly observed at the apical acrosome region in sperm with intact acrosome (arrow in a, e ). (B) Sperm capacitation effect on p CaMKII localization in bovine sperm. Results are expressed as the mean (%) ± SD of 3 independent replicates.

Article Snippet: After blocking, the spermatozoa were incubated for 1 h at room temperature with the rabbit monoclonal (EP1829y) antibody anti-CaMKII (abcam, ab52476) or rabbit polyclonal antibody anti-phosphorylated (T286) CaMKII (pCaMKII; abcam, ab47565) used at a dilution of 1:100 each.

Techniques: Incubation, Fluorescence, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heat shock affects the Ca 2+ /calmodulin-dependent protein kinase II dynamic during bovine sperm capacitation and acrosome reaction

doi: 10.3389/fcell.2025.1552282

Figure Lengend Snippet: CaMKII localization during acrosomal reaction in bovine sperm. (A) After 4 h of incubation in capacitating medium, the acrosome reaction was induced by Ca 2+ and heparin for 1 h. CaMKII (a, f, k, p) and acrosome (b, g, l, q) were evaluated by fluorescence microscopy using rabbit anti-CaMKII antibody and Pisum sativum agglutinin (FITC-PSA), respectively. The sperm nucleus was stained with Hoechst 33342 (c, h, m, r) . The merged images of CaMKII + Acrosome + DNA are shown in (d, i, n, s) , and merged images of CaMKII with acrosome are shown in (e, j, o, t) . The CaMKII observed at the acrosomal region of the spermatozoa with intact acrosome (a–e) , changes its localization to the post-acrosomal region at the beginning (f–j) and the end (k–o) of the acrosome reaction until it is no longer observed in spermatozoa with reacted acrosome (p–t) . See arrows in (a, f, k, p) for CaMKII and arrowheads in (b, g, l, q) for acrosome dynamics during the acrosome reaction. Large white square shown in “ i ” is enlargement of the corresponding small box. (B) Effect of the acrosome reaction on the CaMKII localization in bovine spermatozoa. Results are expressed as the mean (%) ± SD of 3 replicates. Lowercase letters represent comparisons of CamKII localization (acrosomal versus post-acrosomal) within each acrosomal status (intact or reacted). Capital letters represent comparisons of the same CamKII localization pattern (acrosomal or post-acrosomal) between different acrosomal status (intact versus reacted). Different letters represent a significant difference (p < 0.05).

Article Snippet: After blocking, the spermatozoa were incubated for 1 h at room temperature with the rabbit monoclonal (EP1829y) antibody anti-CaMKII (abcam, ab52476) or rabbit polyclonal antibody anti-phosphorylated (T286) CaMKII (pCaMKII; abcam, ab47565) used at a dilution of 1:100 each.

Techniques: Incubation, Fluorescence, Microscopy, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heat shock affects the Ca 2+ /calmodulin-dependent protein kinase II dynamic during bovine sperm capacitation and acrosome reaction

doi: 10.3389/fcell.2025.1552282

Figure Lengend Snippet: Dynamics of the phosphorylated CaMKII ( p CaMKII) localization during acrosome reaction in bovine sperm. (A) After 4 h of incubation in capacitating medium, the acrosome reaction was induced with Ca 2+ and heparin for 1 h. p CaMKII (a, f, k) and acrosome (b, g, l) were evaluated by fluorescence microscopy using anti-phosphorylated-CaMKII (T286) antibody and Pisum sativum agglutinin (FITC-PSA), respectively. DNA was stained with Hoechst 33342 (c, h, m) . The merged images of CaMKII + Acrosome + DNA are shown in (d, i, n) while merged images of CaMKII + Acrosome are shown in (e, j, o) . The p CaMKII observed at the apical region in intact acrosome (a–e) , decreases its apical localization during the acrosome reaction (f–j) , until it is no longer observed at the acrosomal region in reacted acrosome spermatozoa (k–o) . See arrows in (a, f, k) for p CaMKII and arrowheads in (b, g, l) for acrosome dynamics during the acrosome reaction. (B) Effect of the acrosomal reaction on the p CaMKII localization in bovine sperm. Results are expressed as the mean (%) ± SD of 3 replicates. Lowercase letters indicate comparisons of p CamKII localization within each acrosomal state (intact or reacted). Capital letters indicate comparisons of the same p CamKII localization pattern (acrosomal or post-acrosomal) between different acrosomal status (intact versus reacted). Different letters indicate a significant statistical difference (p < 0.05).

Article Snippet: After blocking, the spermatozoa were incubated for 1 h at room temperature with the rabbit monoclonal (EP1829y) antibody anti-CaMKII (abcam, ab52476) or rabbit polyclonal antibody anti-phosphorylated (T286) CaMKII (pCaMKII; abcam, ab47565) used at a dilution of 1:100 each.

Techniques: Incubation, Fluorescence, Microscopy, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heat shock affects the Ca 2+ /calmodulin-dependent protein kinase II dynamic during bovine sperm capacitation and acrosome reaction

doi: 10.3389/fcell.2025.1552282

Figure Lengend Snippet: Effect of heat shock on CaMKII localization. (A) Illustrative image of bovine sperm with p CaMKII localized ( a–c ; see arrow in a and c ) or not ( d–f ; see arrow in d and f ) at the apical region of the acrosome. (B) Effect of the incubation time and heat shock on p CaMKII localization during sperm capacitation. Data are expressed as the mean (%) ± SD of 4 replicates. Different letters and symbol (*) indicate a significant difference (p < 0.05). Letters indicate comparisons among the different time-point within each temperature (38.5°C or 41°C). Symbol (*) indicates a comparison of each time-point (0 h, 1 h, 2 h, 3 h or 4 h) between the temperatures (38.5°C versus 41°C). (C) Heat shock effect on CaMKII localization in bovine sperm. Representative image of bovine sperm with CaMKII localized (arrow in a) or not (arrow in b) at the acrosomal region. (D) Effect of heat shock during capacitation on CaMKII localization at the acrosomal region of bovine sperm. Data are expressed as the mean (%) ± SD of 25 cells. Different letters indicate a significant difference (p < 0.05).

Article Snippet: After blocking, the spermatozoa were incubated for 1 h at room temperature with the rabbit monoclonal (EP1829y) antibody anti-CaMKII (abcam, ab52476) or rabbit polyclonal antibody anti-phosphorylated (T286) CaMKII (pCaMKII; abcam, ab47565) used at a dilution of 1:100 each.

Techniques: Incubation, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heat shock affects the Ca 2+ /calmodulin-dependent protein kinase II dynamic during bovine sperm capacitation and acrosome reaction

doi: 10.3389/fcell.2025.1552282

Figure Lengend Snippet: Heat shock effect on phosphorylated CaMKII ( p CaMKII) localization in bovine sperm with intact acrosome during sperm capacitation. (A) Bovine sperm were evaluated after thawing (0 h) or after 4 h of incubation in the capacitating medium at 38.5°C and 41°C. p CaMKII (a, f) and acrosome (b, g) were evaluated by fluorescence microscopy using anti-phosphorylated-CaMKII (T286) antibody and Pisum sativum agglutinin (FITC-PSA), respectively. Hoechst 33342 was used to counterstain DNA (c, h) . The merged images of p CaMKII + acrosome + DNA is shown in (d, i) , while merged images of p CaMKII + Acrosome are shown in (e, j) . During capacitation, sperm with intact acrosome have p CaMKII localized ( a–e , arrow in a , e ) or not ( f–j , arrow in f , j ) at the acrosome apical region. (B) Effect of heat shock on CaMKII acrossomal localization in bovine sperm with intact acrosome or acrosome reacted following incubation at 38.5°C and 41°C for 4 h. Data are expressed as the mean (%) ± SD of 4 replicates. Different letters indicate a significant difference (p < 0.05).

Article Snippet: After blocking, the spermatozoa were incubated for 1 h at room temperature with the rabbit monoclonal (EP1829y) antibody anti-CaMKII (abcam, ab52476) or rabbit polyclonal antibody anti-phosphorylated (T286) CaMKII (pCaMKII; abcam, ab47565) used at a dilution of 1:100 each.

Techniques: Incubation, Fluorescence, Microscopy