Abstract
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 in vitro sperm fertilizing ability without affecting cumulus penetration and gamete fusion, indicating that the hyperactivation supported by mitochondrial function is needed mainly for zona pellucida penetration. Finally, complementary in vivo 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.
Highlights
Mammalian fertilization is a complex process that involves different sequential interactions between the spermatozoon and the egg
The fact that at time = 20 min no differences in membrane potential (MMP) values were observed between incubations with or without bovine serum albumin (BSA), and that the time-dependent increase in MMP dynamics along capacitation was observed even in the presence of BSA (Figure 1D), argues against the possibility that the difference in TMRE between non-capacitating and capacitating conditions is only caused by a different dye solubility or loading due to the presence of BSA
Mouse sperm mitochondrial activity was studied in depth in order to evaluate its dynamics during capacitation and its role for the acquisition of sperm fertilizing ability
Summary
Mammalian fertilization is a complex process that involves different sequential interactions between the spermatozoon and the egg. As this interplay occurs in the oviduct, sperm must reach it from the semen deposit site in the vagina or uterus, depending on the species (Yanagimachi, 1994) During this transport, sperm experience a series of functional and structural modifications collectively known as capacitation (Chang, 1951; Austin, 1952). Sperm experience a series of functional and structural modifications collectively known as capacitation (Chang, 1951; Austin, 1952) These changes are molecularly triggered by the entry of HCO3− and Ca2+, which activate protein kinase A (PKA)-dependent signaling cascades leading to phosphorylation of proteins, increase in intracellular pH and hyperpolarization of the plasma membrane potential (reviewed in Puga Molina et al, 2018). The acquisition of the capacitation status is mandatory for the cells to become fertilization competent
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