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

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.

FIGURE 1.

Characterization of PLB in murine germ cells. PLB localized to the acrosome in round spermatids (A). The presence of PLB in a cell lysate from whole murine sperm was verified by immunoblot, showing a specific protein band around 165 kDa (B). Localization of PLB in the APM macrodomain of the plasma membrane was determined in sperm fixed with a protocol shown to leave the plasma membrane intact (11). In this experiment, sperm with intact membranes were negative for PNA staining but showed signal for PLB in the APM and, to varying degrees, in the midpiece. Fixed sperm were briefly sonicated to permeabilize the membranes, revealing PNA fluorescence (C). RT-PCR of RNA collected from mouse testes at various days of postnatal development revealed that expression of PLB mRNA was first faintly detectable on day 17 (D), temporally consistent with the appearance of round spermatids. The nature of PLB linkage to membranes was evaluated by Triton X-114 (TX-114) phase separation and PI-PLC cleavage assay (E). PLB and testisin (TESP5) were tightly associated with membranes, appearing in the detergent (Det) fraction of a Triton X-114 phase separation assay. However, partitioning of PLB to the sperm pellet (SP) in a PI-PLC cleavage assay showed that PLB behaved as a transmembrane protein, compared with the GPI-linked TESP5 (42). DIC, differential interference contrast.

FIGURE 2.

PLA₂ activity in subcellular fractions. Murine sperm were incubated for 1 h under non-capacitating conditions (NC) or capacitating conditions (CP, which included bicarbonate and 2-OHCD) and then fractionated into pellet, released, membrane, and cytosol fractions, as indicated. Sperm taken at the start of the experiment that were not incubated under any condition were processed as a control (No incubation). The assay was performed with a fluorescent PLA₂ substrate in the presence of EGTA, a Ca²⁺ chelator, to confirm the Ca²⁺-independent nature of the enzyme activity. *, p < 0.05; **, p < 0.005 in the comparison between non-capacitating and capacitating conditions.

FIGURE 3.

Regulation of PLB activation by sterol removal. The released fractions from sperm incubated under different conditions (non-capacitating (NC), with bicarbonate (BC), with 2-OHCD, or with bicarbonate + 2-OHCD) were utilized for fluorescent Ca²⁺-independent PLA₁ and PLA₂ assays (A, *, p < 0.05 between non-capacitating and 2-OHCD; **, p < 0.05 between bicarbonate and bicarbonate + 2-OHCD). Immunoblot and gel-based extraction for the presence of PLB were performed on the fractions released from sperm with or without 2-OHCD treatment (B). We then sectioned the gel into 17 pieces corresponding with different molecular weight ranges and assayed for PLA₂ enzyme activity in the different sections. The results showed Ca²⁺-independent PLA₂ enzyme activity in the section containing the 50 kDa immunoreactive band. Immunostaining for PLB was performed on sperm treated with or without 2-OHCD (C). Sperm were fixed using methods to preserve plasma membrane integrity. The patterns of PLB localization were categorized according to the following criteria: no signal (Lost), plasma membrane overlying the acrosome (APM), apical acrosome (AA), and diffuse (Diff). *, p < 0.05. Error bars, S.E.

FIGURE 4.

Selective inhibition of PLB activation. Capacitation was initiated in the presence of several protease inhibitors (benzamidine (Ben), trypsin inhibitor (TI), leupeptin (Leu), PMSF, and Dec-RVKR-CMK (FI; a furin-specific inhibitor)). The released fractions were collected and utilized for Ca²⁺-independent PLA₁ and PLA₂ activity assays. The addition of PMSF or Dec-RVKR-CMK inhibited the increase of both activities, whereas benzamidine, trypsin inhibitor, and leupeptin did not. Induction of AE in sperm capacitated in the presence of PMSF or Dec-RVKR-CMK and stimulated with P4 was evaluated (inset). Columns with different letters are significantly different (p < 0.05). Error bars, S.E.

FIGURE 5.

Roles of PLB in AE and fertilization. Sperm were incubated under non-capacitating (NC) or capacitating (CP) conditions, in the presence of 0–100 μm dl-carnitine, a competitive PLB inhibitor, and then stimulated with P4. The status of the acrosome was examined using Coomassie Blue staining. We found that P4-induced AE was inhibited at concentrations of 50–100 μm (A, top). Columns with different letters are significantly different (p < 0.05), whereas columns with the same letter are not significantly different. Multiple letters above a column reflect the comparative statistical relationships between that column and all other columns. Dose-dependent inhibition of Ca²⁺-independent PLA₂ activity was observed in the presence of dl-carnitine (A, bottom). The impact of PLB inhibition on fertilization competence was examined by IVF. Sperm were preincubated for capacitation and then utilized for insemination in the presence (+) or absence (−) of 50 μm dl-carnitine. Fertilization success was halved when the inhibitor was present during both capacitation and fertilization (B). *, p < 0.05. Error bars, S.E.

FIGURE 6.

Supernatants from sperm that underwent sterol removal increased AE in other sperm. Sperm were initially incubated under non-capacitating (NC) or capacitating (CP) conditions for 40 min and then incubated for 20 min in the presence of proteins released from sperm that were themselves incubated under non-capacitating conditions or in the presence of 2-OHCD. The status of the acrosome was examined before or after P4 stimulation for 20 min. For non-capacitating sperm, the incubation was continued for 80 min total. Columns with different letters are significantly different (p < 0.05), whereas columns with the same letter are not significantly different. Multiple letters above a column reflect the comparative statistical relationships between that column and all other columns. Error bars, S.E.

FIGURE 7.

Stimulation of AE by recombinant PLB peptides. Constructs of the consensus catalytic domain of PLB (Met³⁶⁶–Ser⁷¹¹) were made with a N-PLB. Immunoblots were performed on the following fractions: cell lysates (lane 1), flow-through (lane 2), elution (lane 3), and elution from control cell lysates (lane 4). The N-PLB construct was verified using immunoblots to confirm immunoreactivity to anti-His antibody (A) and molecular size. Sperm were capacitated for 40 min and then incubated with P4, 5 μg of N-PLB, or 5 μg of protein from control cells for 20 min (B). A total of at least 200 sperm were assessed for evaluation of acrosome status for each tested condition, and a total of seven trials were performed. The results showed that N-PLB stimulated AE in capacitated sperm, whereas the control protein did not. *, p < 0.05.

FIGURE 8.

A working hypothesis for the regulation and the function of sperm PLB. Schematic diagram of the APM and the underlying outer acrosomal membrane (OAM). The APM possesses numerous membrane rafts enriched in PLB (sterols are shown in yellow). Sterol acceptors, such as HDLs or albumin (pink and orange and then white when having bound a sterol), initiate reorganization of rafts (1), which allows PLB (blue) to contact proteases (brown) (2). PLB undergoes proteolytic cleavage, releasing a catalytic, active fragment (free blue box with concave side) (3). The active fragment hydrolyzes phospholipids of the outer bilayer (4). The resultant changes in membrane curvature and fluidity facilitate the formation of stalk structures (5) and fusion pores (6). These point fusion events would occur during capacitation as a late event leading toward AE.