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Abstract
The ability to fertilise an egg is acquired by the mammalian sperm during the complex biochemical process called capacitation. Capacitation is accompanied by the production of reactive oxygen species (ROS), but the mechanism of redox regulation during capacitation has not been elucidated. This study aimed to verify whether capacitation coincides with reversible oxidative post-translational modifications of proteins (oxPTMs). Flow cytometry, fluorescence microscopy and Western blot analyses were used to verify the sperm capacitation process. A fluorescent gel-based redox proteomic approach allowed us to observe changes in the level of reversible oxPTMs manifested by the reduction or oxidation of susceptible cysteines in sperm proteins. Sperm capacitation was accompanied with redox modifications of 48 protein spots corresponding to 22 proteins involved in the production of ROS (SOD, DLD), playing a role in downstream redox signal transfer (GAPDHS and GST) related to the cAMP/PKA pathway (ROPN1L, SPA17), acrosome exocytosis (ACRB, sperm acrosome associated protein 9, IZUMO4), actin polymerisation (CAPZB) and hyperactivation (TUBB4B, TUB1A). The results demonstrated that sperm capacitation is accompanied by altered levels of oxPTMs of a group of redox responsive proteins, filling gaps in our knowledge concerning sperm capacitation.
Highlights
Mammalian sperm capacitation is a complex process that occurs in the oviduct and is essential for the fertilisation of a mature oocyte
Our results showed that the percentage of reactive oxygen species (ROS)-positive sperm increased during capacitation, but ROS production was at a relatively low level throughout the incubation period, not exceeding 31% after 6 h (Figure 5B), which is consistent with previous reports on the generation of small amounts of ROS required for proper redox signalling during sperm capacitation [24,26]
Our research provides evidence that redox modifications of proteins accompany bull sperm capacitation
Summary
Mammalian sperm capacitation is a complex process that occurs in the oviduct and is essential for the fertilisation of a mature oocyte. Biochemical changes in the plasma membrane and other subcellular compartments have been associated with sperm capacitation [1]. At the early stages of capacitation, a series of cellular events occurs. These include, among others, the production of cAMP by the adenylyl cyclase (AC), the activation of calcium channels, reactive oxygen species (ROS) generation, cholesterol efflux from the plasma membrane, the alkalinisation of sperm plasma and the activation of protein kinases [2]. It is well known that mammalian sperm capacitation is an oxidative process [3,4]
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