Vasudevan SR et al. (2010), The calcium-mobilizing messenger nicotinic acid...

ECB-ART-41601

J Biol Chem 2010 Jun 11;28524:18262-9. doi: 10.1074/jbc.M109.087858.

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The calcium-mobilizing messenger nicotinic acid adenine dinucleotide phosphate participates in sperm activation by mediating the acrosome reaction.

Vasudevan SR , Lewis AM , Chan JW , Machin CL , Sinha D , Galione A , Churchill GC .

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Before a sperm can fertilize an egg it must undergo a final activation step induced by the egg termed the acrosome reaction. During the acrosome reaction a lysosome-related organelle, the acrosome, fuses with the plasma membrane to release hydrolytic enzymes and expose an egg-binding protein. Because NAADP (nicotinic acid adenine dinucleotide phosphate) releases Ca(2+) from acidic lysosome-related organelles in other cell types, we investigated a possible role for NAADP in mediating the acrosome reaction. We report that NAADP binds with high affinity to permeabilized sea urchin sperm. Moreover, we used Mn(2+) quenching of luminal fura-2 and (45)Ca(2+) to directly demonstrate NAADP regulation of a cation channel on the acrosome. Additionally, we show that NAADP synthesis occurs through base exchange and is driven by an increase in Ca(2+). We propose a new model for acrosome reaction signaling in which Ca(2+) influx initiated by egg jelly stimulates NAADP synthesis and that this NAADP acts on its receptor/channel on the acrosome to release Ca(2+) to drive acrosomal exocytosis.

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Species referenced: Echinodermata
Genes referenced: LOC100887844 LOC115919910 LOC115923812 LOC576539 LOC578064 LOC581395 LOC587800 LOC762863

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	FIGURE 1. Effect of Ca2+-releasing messengers on Mn2+-mediated quenching of fura-2 trapped in the lumen of yolk platelets. Yolk platelets are lysosome-related organelles in sea urchin eggs. A, addition of 100 μm MnCl2 rapidly decreases fluorescence. The gray box highlights the area expanded in panel B. B, basal rate of fura-2 quenching is accelerated by NAADP (500 nm) and ionomycin (5 μm) but not a second addition of NAADP (500 nm) or inositol 1,4,5-trisphosphate (10 μm) or cADP-ribose (1 μm). C, the effect of a messenger is not affected by the order of addition (concentrations as in B). D, pre-treatment with a low concentration of NAADP (5 nm) abolishes the subsequent response to supramaximal NAADP (500 nm) demonstrating diagnostic self-desensitization. Other concentrations are as in panel B.
	FIGURE 2. Effect of NAADP on Mn2+-mediated quenching of fura-2 trapped in the lumen of vesicles isolated from sperm. A, separation of fractions from homogenized sperm with density-gradient centrifugation and distribution of marker enzymes. Marker enzymes identify the endoplasmic reticulum (Glc-6-Pase, glucose-6-phosphatase), lysosomes (Acid Pase, acid phosphatase), and mitochondria (Suc Dehyr, succinate dehydrogenase). Fraction density increases from left to right. B, addition of 100 μm MnCl2 decreased fura-2 fluorescence. The area highlighted in the gray box is expanded in panel B. C, basal rate of fura-2 quenching is accelerated by supramaximal NAADP (500 nm) and ionomycin (5 μm) but not inositol 1,4,5-trisphosphate (10 μm) or cADP-ribose (1 μm). D, basal rate of fura-2 quenching in the absence of any second messenger additions.
	FIGURE 3. 45Ca2+ flux in permeabilized sea urchin sperm. A, time course of 45Ca2+ accumulation in sea urchin sperm. B, effect on 45Ca2+ uptake of thapsigargin (10 μm, inhibits sarcoplasmic-endoplasmic reticulum calcium ATPase), bafilomycin (1 μm, inhibits V-H+-ATPase), GPN (50 μm, bursts lysosomes), and ionomycin (5 μm, Ca2+ ionophore). C, time course of 45Ca2+ release induced by 1 μm NAADP. D, effect of second messengers and egg jelly on 45Ca2+ release from permeabilized sea urchin sperm. For B–D, the error bars are mean ± S.E., n = 3, and filled bars or symbols are significantly different from the control (one-tailed t test, p ≤ 0.5).
	FIGURE 4. Sea urchin sperm show specific NAADP binding. NAADP competes with [32P]NAADP binding, but the related nucleotides NADP or ATP do not compete. Absolute values of radioactivity vary between experiments and with time of exposure during detection of radioactivity with a phosphorimaging device and were therefore normalized. These particular data were obtained with 0.2 nm [32P]NAADP (1000 Ci/mmol) and had a Bmax of 9.1 × 107 absorbance units with a zero of 6.4 × 107 absorbance units.
	FIGURE 5. Ca2+ stimulates NAADP synthesis in sea urchin sperm via base exchange. A, NAADP levels in sperm diluted into normal artificial sea water (n = 7). B, NAADP levels in sperm diluted into normal artificial sea water containing the Ca2+ ionophore ionomycin (5 μm) with (11 mm Ca2+) (n = 11) or (C) without (no added Ca2+ and 1 mm EGTA) extracellular Ca2+ (n = 3). D, NAADP levels in sperm diluted into normal (11 mm Ca2+) artificial sea water containing the Ca2+ ionophore ionomycin (5 μm) and the base exchange inhibitor nicotinamide (50 mm) (n = 6). E, summary of the NAADP levels following the indicated treatments. Data are the means ± S.E. of the mean based on all values after time zero from panels A–D (n = 3–11). Filled bars are significantly different from the control (p < 0.01, Dunnett's multiple comparisons test after one-way analysis of variance, p = 0.006, performed on the logged data). F, nicotinamide concentration-inhibition relationship for NAADP synthesis by base exchange monitored by addition of exogenous substrates. G and H, Michaelis-Menten plots demonstrating mixed inhibition of NAADP synthesis monitored by exogenous substrate addition.
	FIGURE 6. Signaling pathway model showing the role of NAADP in the acrosome reaction in sea urchin sperm. The fucose sulfate polymer (FSP) in the egg jelly activates the receptor of egg jelly (REJ, 1), which stimulates a plasma membrane channel resulting in a transient Ca2+ increase (2). The initial Ca2+ increase stimulates NAADP synthesis possibly by ADP-ribosyl cyclase (ARC, 3). NAADP binds to its receptor (Two-Pore Channel) on the acrosome and releases Ca2+ (4). These steps parallel the traditional pathway in which the Ca2+ increase stimulates phospholipase Cδ (PLCδ, 5) to produce inositol 1,4,5-trisphosphate that activates its receptor (6). Ca2+ release from the acrosome might trigger exocytosis directly or indirectly via store-activated Ca2+ entry (7).

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