Iniesta-Cuerda María, Nevoral Jan, Krapf Dario, Garde Julián, Soler-Valls Ana Josefa, Yeste Marc
Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, 17003, Girona, Spain.
Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, 17003, Girona, Spain.
Biol Res. 2025 May 29;58(1):30. doi: 10.1186/s40659-025-00613-6.
Protein acetylation has emerged as essential for sperm function, attracting considerable attention recently. Acetylation, typically mediated by lysine acetyltransferases, involves attaching an acetyl group from acetyl-coenzyme A to lysine residues in proteins. Under alkaline conditions, however, acetylation can occur with minimal enzymatic involvement, primarily due to an elevated pH. As sperm migrate towards the ampulla, they experience increasing intracellular pH (pHi) while undergoing two crucial processes for fertilization: capacitation and the acrosome reaction (AR). Whereas the involvement of acetylating enzymes in these events has been partially investigated, the potential for non-enzymatic acetylation driven by the pHi alkalinization remains unknown.
This study examined protein acetylation (acLys) levels in sperm incubated under capacitating conditions at pH 7.2 and pH 9.0, the latter condition potentially promoting non-enzymatic acetylation. To more precisely investigate the occurrence of non-enzymatic acetylation events, acetyltransferase activity was selectively attenuated using a specific cocktail of inhibitors. The functional implications of these conditions were assessed by examining key fertilization-related sperm attributes, including motility during capacitation and the ability to initiate the AR. Results demonstrated that alkaline conditions elevated basal acLys levels even with reduced acetyltransferase activity (P < 0.05), indicative of non-enzymatic acetylation. α-tubulin, particularly in the midpiece of the sperm flagellum, was identified as a specific target of this modification, correlating with diminished motility during capacitation. Following the AR, acLys levels in the head and midpiece decreased (P < 0.05) under conditions promoting non-enzymatic acetylation, accompanied by reductions in intracellular and acrosomal pH. In contrast, acLys levels and pH in the sperm head incubated under standard capacitating conditions (pH 7.2) remained stable. Sperm exposed to conditions conducive to non-enzymatic acetylation exhibited an impaired ability to trigger the AR (P < 0.05) compared to those maintained at pH 7.2. Notably, diminished acetylase activity emerged as a key factor impairing the maintenance of intracellular and acrosomal pH levels attained during capacitation, even under a pH of 9.0.
This study provides novel evidence for the occurrence of non-enzymatic acetylation in sperm, linked to the modulation of α-tubulin acetylation levels and motility during capacitation. Additionally, it suggests that acetyltransferase activity may play a crucial role in regulating intracellular and acrosomal pH levels in capacitated sperm, facilitating the AR.
蛋白质乙酰化已被证明对精子功能至关重要，最近引起了广泛关注。乙酰化通常由赖氨酸乙酰转移酶介导，涉及将乙酰辅酶A中的乙酰基连接到蛋白质中的赖氨酸残基上。然而，在碱性条件下，乙酰化可以在极少酶参与的情况下发生，主要是由于pH值升高。当精子向壶腹迁移时，它们在经历受精的两个关键过程（获能和顶体反应（AR））的同时，细胞内pH值（pHi）会升高。虽然乙酰化酶在这些事件中的作用已得到部分研究，但由pHi碱化驱动的非酶促乙酰化的可能性仍然未知。
本研究检测了在pH 7.2和pH 9.0的获能条件下孵育的精子中的蛋白质乙酰化（acLys）水平，后者条件可能促进非酶促乙酰化。为了更精确地研究非酶促乙酰化事件的发生，使用特定的抑制剂混合物选择性地减弱了乙酰转移酶的活性。通过检测与受精相关的关键精子属性，包括获能期间的活力和引发AR的能力，评估了这些条件的功能影响。结果表明，即使乙酰转移酶活性降低，碱性条件也会提高基础acLys水平（P < 0.05），表明存在非酶促乙酰化。α-微管蛋白，特别是在精子鞭毛的中段，被确定为这种修饰的特定靶点，与获能期间活力的降低相关。在AR之后，在促进非酶促乙酰化的条件下，头部和中段的acLys水平下降（P < 0.05），同时细胞内和顶体pH值降低。相比之下，在标准获能条件（pH 7.2）下孵育的精子头部的acLys水平和pH值保持稳定。与维持在pH 7.2的精子相比，暴露于有利于非酶促乙酰化条件下的精子引发AR的能力受损（P < 0.05）。值得注意的是，乙酰化酶活性降低成为损害获能期间达到的细胞内和顶体pH水平维持的关键因素，即使在pH 9.0的情况下也是如此。
本研究为精子中发生非酶促乙酰化提供了新证据，这与获能期间α-微管蛋白乙酰化水平的调节和活力有关。此外，它表明乙酰转移酶活性可能在调节获能精子的细胞内和顶体pH水平、促进AR方面发挥关键作用。
