Abstract
Mammalian sperm must undergo a set of structural and functional changes collectively termed as capacitation to ensure a successful oocyte fertilization. However, capacitation can be compromised by cryopreservation procedures, which alter the proteome and longevity of sperm. To date, how the protein changes induced by cryopreservation could affect the acquisition of sperm fertilizing potential remains unexplored. The present study investigated the protein profile of ram sperm during in vitro capacitation before and after cryopreservation to elucidate the impact of cryopreservation on sperm capacitation at a molecular level. Fresh and cryopreserved ram sperm were incubated under capacitating (CAP) and non-capacitating (NC) conditions for 240 min. The sperm proteome of these four treatments was analyzed and compared at different incubation times using reverse phase liquid chromatography coupled to mass spectrometry (RP-LC-MS/MS). The comparison between fresh and cryopreserved sperm suggested that cryopreservation facilitated an apoptosis-stress response and redox process, while the comparison between sperm incubated in CAP and NC conditions showed that capacitation increased those biological processes associated with signaling, metabolism, motility, and reproductive processes. In addition, 14 proteins related to mitochondrial activity, sperm motility, oocyte recognition, signaling, spermatogenesis, and the apoptosis-stress response underwent significant changes in abundance over time when fresh and cryopreserved sperm incubated in CAP and NC conditions were compared. Our results indicate that disturbances in a ram sperm proteome after cryopreservation may alter the quality of sperm and its specific machinery to sustain capacitation under in vitro conditions.
Highlights
After ejaculation, mammalian sperm become capable of fertilization into the female reproductive tract through a time-dependent process called capacitation [1]
To the best of our knowledge, this is the first time that the proteome of fresh and cryopreserved sperm has been compared in sheep during in vitro capacitation over time
We found that the proteins identified in cryopreserved ram sperm were more involved in those biological processes associated with redox status, biological regulation, cellular component rearrangement, and apoptotic-stress responses than the proteins identified in fresh sperm
Summary
Mammalian sperm become capable of fertilization into the female reproductive tract through a time-dependent process called capacitation [1]. This process involves physical and biochemical changes, including variations in protein composition, that drive hyperactivation and acrosome reaction, two crucial steps for zona pellucida penetration and subsequent oocyte fertilization. The acquisition of sperm function and their fertilizing ability depend on post-translational modifications of existing proteins [17], interaction with proteins present in the male and female reproductive tract [18], or probably even on the synthesis of new proteins [19]
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