Effects of Calyculin a on the Motility and Protein Phosphorylation in Frozen-Thawed Bull Spermatozoa
Effects of Calyculin a on the Motility and Protein Phosphorylation in Frozen-Thawed Bull Spermatozoa
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DOI：
10.2108/zs210046
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发表时间：
2021-10
影响因子：
0.9
通讯作者：
Honami Ogata;Mariko Tsukamoto;Kenichi Yamashita;T. Iwamori;Hideyuki Takahashi;T. Kaneko;N. Iwamori;T. Inai;H. Iida
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文献类型：
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作者：
Honami Ogata;Mariko Tsukamoto;Kenichi Yamashita;T. Iwamori;Hideyuki Takahashi;T. Kaneko;N. Iwamori;T. Inai;H. Iida
在这项研究中，我们研究了磷酸酶抑制剂calyculin A对冷冻解冻公牛精子中运动性、蛋白质磷酸化和磷酸化（Ser/Thr）PKA底物分布的影响，这些精子实际上被大多数农民用于繁殖。数据显示，已报道对射出的新鲜精子的活动性具有积极作用的calyculin A明显降低冷冻解冻的公牛精子的活动性，即使细胞活化剂，如咖啡因，的抑制作用是剂量-免疫印迹分析显示，在暴露于咖啡因或dbcAMP的精子中未检测到从头蛋白磷酸化（一种细胞可渗透的cAMP类似物），而向培养基中加入calyculin A导致出现几种50 kDa和75 kDa的磷酸化蛋白，这表明通过cAMP依赖性PKA的活化而磷酸化的50 kDa和75 kDa蛋白，由于抑制了calyculin A-敏感的蛋白磷酸酶，未去磷酸化并在精子中积累。免疫荧光显微镜显示，calyculin A引起，单独或与咖啡因或dbcAMP，磷酸PKA底物的积累在环，虽然咖啡因或dbcAMP单独没有。本研究表明，calyculin A降低冻融公牛精子的活力，伴随着在鞭毛环的磷酸-（丝氨酸/苏氨酸）PKA底物的积累。
In this study, we examined the effects of calyculin A, a phosphatase inhibitor, on motility, protein phosphorylation, and the distribution of phospho-(Ser/Thr) PKA substrates in frozen-thawed bull spermatozoa that are actually used by most farmers for breeding. The data showed that calyculin A, which has been reported to have a positive effect on the motility of ejaculated fresh spermatozoa, distinctly decreased the motility of frozen-thawed bull spermatozoa even if a cell activator, such as caffeine, was present in the incubation medium and that the suppressive effect of calyculin A was dose-dependent and continued for at least 200 min. Immunoblot analyses revealed that de novo protein phosphorylation was not detected in spermatozoa exposed to caffeine or dbcAMP (a cell-permeable cAMP analog), while the addition of calyculin A to the medium brought about the appearance of several phosphorylated proteins at 50 kDa and 75 kDa, suggesting that 50 kDa and 75 kDa proteins, which were phosphorylated by activation of cAMP-dependent PKA, were not dephosphorylated and were accumulated in spermatozoa due to the suppression of calyculin A-sensitive protein phosphatases. Immunofluorescence microscopy revealed that calyculin A caused, alone or in conjunction with caffeine or dbcAMP, the accumulation of phospho-PKA substrates at the annulus, although caffeine or dbcAMP alone did not. This study suggested that calyculin A decreases the motility of frozen-thawed bull spermatozoa concomitant with the accumulation of phospho-(Ser/Thr) PKA substrates at the annulus of flagella.