Heat shock affects the Ca2+/calmodulin-dependent protein kinase II dynamic during bovine sperm capacitation and acrosome reaction

Abstract

Background: Heat shock during sperm capacitation affects the spermatozoa quality, resulting in increased early acrosome reaction and consequently decreasing their fertilizing capacity. Although the mechanisms involved in the regulation of sperm capacitation and acrosome reaction are not fully understood, it has been reported that Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is an important regulator of these processes. Thus, the present aimed to evaluate the effect of heat shock in the CaMKII signaling during the bovine sperm capacitation and acrosome.

Methods: Bovine spermatozoa were in vitro capacitated for 4 hours. The acrosome reaction was induced by exposure to heparin and calcium ionophore A23187 for 1 hour. Heat shock was applied by incubating spermatozoa at 41 °C with 7% CO₂, while the control group was maintained at 38.5 °C with 5% CO₂. At the end of each treatment, the localization of total CaMKII and phosphorylated CaMKII (pCaMKII), as well as acrosomal membrane integrity, were evaluated by immunofluorescence.

Results: It was observed that CaMKII and not phosphorylated CaMKII (pCaMKII) localization at the acrosome region was affected by sperm capacitation. In contrast, the localization of both, CaMKII and its phosphorylated form was affected by the acrosome reaction (p < 0.05). The acrosome membrane integrity, as well as the pCamKII localization in bovine spermatozoa, was affected by incubation time. This effect of incubation time was stronger in heated shock sperm, although it was observed only after 2 h of incubation. Heat shock also affected the acrosomal localization of pCaMKII in the acrosomal region of spermatozoa with intact acrosome.

Discussion: Taken together, the data present here show that CaMKII and pCaMKII localization is dynamic during bovine sperm capacitation and acrosome reaction and that this pattern of localization is affected by heat shock, suggesting that failure in CaMKII signaling is probably involved in the early acrosome reaction observed in heated-shock spermatozoa.

Keywords: acrosomal membrane; environment; heat stress; phosphorylation; spermatozoa.

FIGURE 1

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).

FIGURE 2

Subcellular localization of phosphorylated CaMKII (pCaMKII) 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. pCaMKII (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 (pCaMKII + Acrosome + DNA) are shown in (d), while the merged images of pCaMKII with Acrosome are shown in (e). During capacitation, pCaMKII is predominantly observed at the apical acrosome region in sperm with intact acrosome (arrow in a, e). (B) Sperm capacitation effect on pCaMKII localization in bovine sperm. Results are expressed as the mean (%) ± SD of 3 independent replicates.

FIGURE 3

CaMKII localization during acrosomal reaction in bovine sperm. (A) After 4 h of incubation in capacitating medium, the acrosome reaction was induced by Ca²⁺ 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).

FIGURE 4

Dynamics of the phosphorylated CaMKII (pCaMKII) localization during acrosome reaction in bovine sperm. (A) After 4 h of incubation in capacitating medium, the acrosome reaction was induced with Ca²⁺ and heparin for 1 h. pCaMKII (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 pCaMKII 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 pCaMKII and arrowheads in (b, g, l) for acrosome dynamics during the acrosome reaction. (B) Effect of the acrosomal reaction on the pCaMKII localization in bovine sperm. Results are expressed as the mean (%) ± SD of 3 replicates. Lowercase letters indicate comparisons of pCamKII localization within each acrosomal state (intact or reacted). Capital letters indicate comparisons of the same pCamKII localization pattern (acrosomal or post-acrosomal) between different acrosomal status (intact versus reacted). Different letters indicate a significant statistical difference (p < 0.05).

FIGURE 5

Heat shock effect on acrosome membrane integrity. (A) Representative image of bovine sperm with intact acrosome (a–c, arrow in c), partially reacted (d–f, arrow in f), and reacted (g–I, arrow in i). (B) Effect of incubation time and heat shock on acrosome membrane integrity during sperm capacitation. Data expressed as the mean (%) ± SD of 4 replicates. 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). Different letters and symbol (*) indicate a significant difference (p < 0.05).

FIGURE 6

Effect of heat shock on CaMKII localization. (A) Illustrative image of bovine sperm with pCaMKII 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 pCaMKII 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).

FIGURE 7

Heat shock effect on phosphorylated CaMKII (pCaMKII) 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. pCaMKII (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 pCaMKII + acrosome + DNA is shown in (d, i), while merged images of pCaMKII + Acrosome are shown in (e, j). During capacitation, sperm with intact acrosome have pCaMKII 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).