Abstract
Despite a strict requirement for sterol removal for sperm to undergo acrosome exocytosis (AE), the mechanisms by which changes in membrane sterols are transduced into changes in sperm fertilization competence are poorly understood. We have previously shown in live murine sperm that the plasma membrane overlying the acrosome (APM) contains several types of microdomains known as membrane rafts. When characterizing the membrane raft-associated proteomes, we identified phospholipase B (PLB), a calcium-independent enzyme exhibiting multiple activities. Here, we show that sperm surface PLB is activated in response to sterol removal. Both biochemical activity assays and immunoblots of subcellular fractions of sperm incubated with the sterol acceptor 2-hydroxypropyl-β-cyclodextrin (2-OHCD) confirmed the release of an active PLB fragment. Specific protease inhibitors prevented PLB activation, revealing a mechanistic requirement for proteolytic cleavage. Competitive inhibitors of PLB reduced the ability of sperm both to undergo AE and to fertilize oocytes in vitro, suggesting an important role in fertilization. This was reinforced by our finding that incubation either with protein concentrate released from 2-OHCD-treated sperm or with recombinant PLB peptide corresponding to the catalytic domain was able to induce AE in the absence of other stimuli. Together, these results lead us to propose a novel mechanism by which sterol removal promotes membrane fusogenicity and AE, helping confer fertilization competence. Importantly, this mechanism provides a basis for the newly emerging model of AE in which membrane fusions occur during capacitation/transit through the cumulus, prior to any physical contact between the sperm and the oocyte's zona pellucida.
Highlights
How sperm sterol removal enables acrosome exocytosis (AE) is unclear; phospholipase B (PLB) is enriched in sperm membrane rafts
This was reinforced by our finding that incubation either with protein concentrate released from 2-OHCD-treated sperm or with recombinant PLB peptide corresponding to the catalytic domain was able to induce AE in the absence of other stimuli
We demonstrate that PLB is present in sperm membranes and that its active catalytic domain is proteolytically released into the extracellular fluid in response to sterol removal
Summary
How sperm sterol removal enables acrosome exocytosis (AE) is unclear; phospholipase B (PLB) is enriched in sperm membrane rafts. This was reinforced by our finding that incubation either with protein concentrate released from 2-OHCD-treated sperm or with recombinant PLB peptide corresponding to the catalytic domain was able to induce AE in the absence of other stimuli Together, these results lead us to propose a novel mechanism by which sterol removal promotes membrane fusogenicity and AE, helping confer fertilization competence. Studies of pancreatic insufficiency in rats indirectly suggested participation of PLB in the digestion of dietary lipids [24], physiological functions of mammalian PLB have remained understudied It has been shown in a pathogenic fungus that PLB is concentrated in membrane rafts, that its activity is increased markedly upon release from those rafts, and that this activity could play an important role in adhesion and invasion of the pathogens to host cells [25]. Our results provide new insight into the molecular mechanism through which sterol removal enables AE and promotes fertilizing ability in murine sperm. (Hereafter, PLA1 and PLA2 designate calcium-independent phospholipase A1 and A2 unless otherwise noted.)
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