Giaccagli María Milagros, Gómez-Elías Matías Daniel, Herzfeld Jael Dafne, Marín-Briggiler Clara Isabel, Cuasnicú Patricia Sara, Cohen Débora Juana, Da Ros Vanina Gabriela
Laboratorio de Mecanismos Moleculares de la Fertilización, Instituto de Biología y Medicina Experimental (IByME-CONICET), Buenos Aires, Argentina.
Laboratorio de Biología Celular y Molecular de la Reproducción, Instituto de Biología y Medicina Experimental (IByME-CONICET), Buenos Aires, Argentina.
Front Cell Dev Biol. 2021 Oct 26;9:767161. doi: 10.3389/fcell.2021.767161. eCollection 2021.
To become fully competent to fertilize an egg, mammalian sperm undergo a series of functional changes within the female tract, known as capacitation, that require an adequate supply and management of energy. However, the contribution of each ATP generating pathway to sustain the capacitation-associated changes remains unclear. Based on this, we investigated the role of mitochondrial activity in the acquisition of sperm fertilizing ability during capacitation in mice. For this purpose, the dynamics of the mitochondrial membrane potential (MMP) was studied by flow cytometry with the probe tetramethylrhodamine ethyl ester (TMRE). We observed a time-dependent increase in MMP only in capacitated sperm as well as a specific staining with the probe in the flagellar region where mitochondria are confined. The MMP rise was prevented when sperm were exposed to the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP) or the protein kinase A (PKA) inhibitor H89 during capacitation, indicating that MMP increase is dependent on capacitation and H89-sensitive events. Results showed that whereas nearly all motile sperm were TMRE positive, immotile cells were mostly TMRE negative, supporting an association between high MMP and sperm motility. Furthermore, CCCP treatment during capacitation did not affect PKA substrate and tyrosine phosphorylations but produced a decrease in hyperactivation measured by computer assisted sperm analysis (CASA), similar to that observed after H89 exposure. In addition, CCCP inhibited the sperm fertilizing ability without affecting cumulus penetration and gamete fusion, indicating that the hyperactivation supported by mitochondrial function is needed mainly for penetration. Finally, complementary fertilization experiments further demonstrated the fundamental role of mitochondrial activity for sperm function. Altogether, our results show the physiological relevance of mitochondrial functionality for sperm fertilization competence.
为了完全具备使卵子受精的能力，哺乳动物精子在雌性生殖道内会经历一系列功能变化，即获能，这需要充足的能量供应和管理。然而，每条ATP生成途径对维持获能相关变化的贡献仍不清楚。基于此，我们研究了线粒体活性在小鼠精子获能过程中获得受精能力方面的作用。为此，我们使用四甲基罗丹明乙酯（TMRE）探针通过流式细胞术研究了线粒体膜电位（MMP）的动态变化。我们观察到只有获能精子的MMP呈时间依赖性增加，并且该探针在鞭毛区域（线粒体所在部位）有特异性染色。当精子在获能过程中暴露于线粒体解偶联剂羰基氰化物间氯苯腙（CCCP）或蛋白激酶A（PKA）抑制剂H89时，MMP的升高受到抑制，这表明MMP的增加依赖于获能和对H89敏感的事件。结果显示，几乎所有有运动能力的精子TMRE呈阳性，而无运动能力的细胞大多TMRE呈阴性，这支持了高MMP与精子运动能力之间的关联。此外，在获能过程中用CCCP处理并不影响PKA底物和酪氨酸磷酸化，但通过计算机辅助精子分析（CASA）测量发现超活化有所降低，这与H89处理后观察到的情况类似。此外，CCCP抑制了精子的受精能力，但不影响卵丘穿透和配子融合，这表明线粒体功能支持的超活化主要是为了穿透。最后，补充的受精实验进一步证明了线粒体活性对精子功能的重要作用。总之，我们的结果表明线粒体功能对于精子受精能力具有生理相关性。
