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J Biol Chem. 2013 Sep 27;288(39):28104-15. doi: 10.1074/jbc.M113.450981. Epub 2013 Aug 13.

Phospholipase B is activated in response to sterol removal and stimulates acrosome exocytosis in murine sperm.

Author information

1
From the Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853.

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.

KEYWORDS:

Acrosome Exocytosis; Acrosome Reaction; Capacitation; Fertilization; Membrane Lipids; Phospholipase B; Raft; Serine Protease; Sperm

PMID:
23943622
PMCID:
PMC3784722
DOI:
10.1074/jbc.M113.450981
[Indexed for MEDLINE]
Free PMC Article

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