Abstract
Sperm capacitation, one of the key events during successful fertilization, is associated with extensive structural and functional sperm remodeling, beginning with the modification of protein composition within the sperm plasma membrane. The ubiquitin-proteasome system (UPS), a multiprotein complex responsible for protein degradation and turnover, participates in capacitation events. Previous studies showed that capacitation-induced shedding of the seminal plasma proteins such as SPINK2, AQN1, and DQH from the sperm surface is regulated by UPS. Alterations in the sperm surface protein composition also relate to the porcine β-microseminoprotein (MSMB/PSP94), seminal plasma protein known as immunoglobulin-binding factor, and motility inhibitor. MSMB was detected in the acrosomal region as well as the flagellum of ejaculated boar spermatozoa, while the signal disappeared from the acrosomal region after in vitro capacitation (IVC). The involvement of UPS in the MSMB degradation during sperm IVC was studied using proteasomal interference and ubiquitin-activating enzyme (E1) inhibiting conditions by image-based flow cytometry and Western blot detection. Our results showed no accumulation of porcine MSMB either under proteasomal inhibition or under E1 inhibiting conditions. In addition, the immunoprecipitation study did not detect any ubiquitination of sperm MSMB nor was MSMB detected in the affinity-purified fraction containing ubiquitinated sperm proteins. Based on our results, we conclude that UPS does not appear to be the regulatory mechanism in the case of MSMB and opening new questions for further studies. Thus, the capacitation-induced processing of seminal plasma proteins on the sperm surface may be more complex than previously thought, employing multiple proteolytic systems in a non-redundant manner.
Highlights
To acquire fertilizing ability, mammalian spermatozoa undergo extensive post-testicular maturation and sub-cellular, molecular changes
The degradation of porcine β-microseminoprotein (MSMB) under proteasomal inhibition and E1 inhibiting conditions during in vitro sperm capacitation was studied by flow cytometric analysis and Western blot detection
We performed indirect immunofluorescence staining to monitor the MSMB localization in boar spermatozoa and changes in the anti-MSMB antibody labeling on sperm after in vitro capacitation (IVC)
Summary
Mammalian spermatozoa undergo extensive post-testicular maturation and sub-cellular, molecular changes. Seminal plasma proteins are involved in the process of capacitation, functioning as decapacitation factors that maintain ejaculated spermatozoa viability within the female reproductive system. These proteins bind to the sperm surface during ejaculation and are involved in the formation of the oviductal sperm reservoir and zona pellucida binding [3,4]. Many changes occur in the sperm plasma membrane and the removal of decapacitating factors leading to the rearrangement of sperm surface proteins [2,5] One of these proteins undergoing such changes during capacitation is a β-microseminoprotein (MSMB), known as prostatic secretory protein (PSP94), immunoglobulin-binding factor [6], sperm motility inhibitor [7], or prostatic inhibin peptide [8]. In a more recent study, sperm MSMB has been found to associate with CRISPs (cysteine-rich secretory proteins) [17] implicated in gamete binding and fusion [18]
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