Motility initiation in herring sperm is regulated by reverse sodium-calcium exchange

Abstract

Sperm of the Pacific herring, Clupea pallasi, are unique in that they are immotile upon spawning in the environment. Herring sperm have evolved to remain motionless for up to several days after spawning, yet are still capable of fertilizing eggs. An egg chorion ligand termed "sperm motility initiation factor" (SMIF) induces motility in herring sperm and is required for fertilization. In this study, we show that SMIF induces calcium influx, sodium efflux, and a membrane depolarization in herring sperm. Sperm motility initiation by SMIF depended on decreased extracellular sodium (<350 mM) and could be induced in the absence of SMIF in very low sodium seawater. Motility initiation depended on > or =1 mM extracellular calcium. Calcium influx caused by SMIF involved both the opening of voltage-gated calcium channels and reverse sodium-calcium (Na(+)/Ca(2+)) exchange. Membrane depolarization was slightly inhibited by a calcium channel blocker and markedly inhibited by a Na(+)/Ca(2+) exchange inhibitor. Sodium efflux caused by SMIF-initiated motility was observed when using both extracellular and intracellular sodium probes. A Na(+)/Ca(2+) exchange antigen was shown to be present on the surface of the sperm, primarily over the midpiece, by using an antibody to the canine Na(+)/Ca(2+) exchanger. This antibody recognized a 120-kDa protein that comigrated with the canine myocyte Na(+)/Ca(2+) exchanger. Sperm of Pacific herring are now shown to use reverse Na(+)/Ca(2+) exchange in motility initiation. This mechanism of regulation of motility initiation may have evolved for both maintenance of immotility after spawning as well as ligand-induced motility initiation.

Figure 1

Effect of [Na⁺]_o on motility initiation. Motility was assessed as follows: 0, no motility; 1+, <25% motility; 2+, 25–50% motility; 3+, 50–75% motility; 4+, >75% motility. (A) Motility initiation in 1/2 NaF is inhibited with increasing [Na⁺]_o. At >25 mM [Na⁺]_o, motility was significantly inhibited (P < 0.05). (B) SMIF-induced motility was inhibited by increasing [Na⁺]_o. A significant reduction in motility was observed at [Na⁺]_o ≥ 350 mM (P < 0.05). Note that a higher concentration of Na⁺ was required to inhibit SMIF-induced motility vs. 1/2 NaF-induced motility.

Figure 2

Images of herring sperm motility patterns visualized by using darkfield optics and recorded with NIH IMAGE after frame averaging (8 frames per sec) through a DSP 200 image enhancement system (Dage-MTI). (A) Sperm in 1/2 FSW were immotile as indicated by discrete foci. (B) Sperm in 1/2 NaF exhibited a linear trajectory as indicated by straight lines. (C) Sperm in 1/2 FSW containing SMIF exhibited a circular pattern of motility.

Figure 3

A [Ca²⁺]_i increase occurs at sperm motility. (A) An immediate large increase in [Ca²⁺]_i (Δ = 241 nM) was observed in sperm suspended in 1/2 FSW to which SMIF (black arrow) was added. The SMIF-induced increase did not occur when Ca²⁺ was absent from the extracellular medium, but was observed upon addition of Ca²⁺ to the medium (white arrow). In the absence of SMIF, a gradual increase in [Ca²⁺]_i was observed when sperm were suspended in 1/2 NaF seawater. Note that the increase in [Ca²⁺]_i observed in 1/2 NaF (Δ = 90 nM) was lower than that for SMIF. No change in [Ca²⁺]_i was seen when 1/2 FSW was added as a control. (B) Micrographs of Fluo-3-loaded sperm collected on a laser scanning confocal microscope on a cooled stage. (Left and Center Right) Transmitted light image. (Center Left and Right) Fluo-3-labeled sperm. Fluo-3 fluorescence was adjusted by reducing the background level of fluorescence signal of sperm in 1/2 FSW and then recording changes after SMIF addition at the same instrument settings. Sperm exhibited an increase in fluorescence (increase in [Ca²⁺]_i) in response to SMIF, which was localized to the head and midpiece region of the sperm.

Figure 4

Effect of inhibitors on the SMIF-induced increase in [Ca²⁺]_i. Sperm loaded with Fluo-3 AM were preincubated with calcium channel blockers (flunarizine, 10 μM; nifedipine, 100 μM), or Na⁺/Ca²⁺ exchange inhibitors (bepridil, 50 μM; dichlorobenzamil, 10 μM; KB-R7943,10 μM). After baseline stabilization, SMIF was added to the sperm suspensions. The SMIF-induced increase in [Ca²⁺]_i was significantly inhibited by both calcium channel blockers and Na⁺/Ca²⁺ exchange inhibitors (n = 3; P < 0.05).

Figure 5

Change in [Na⁺]_i in response to SMIF. Sperm were loaded with cell permeant NaG_i and were suspended in cuvettes containing 1/2 FSW; baseline fluorescence readings were taken before SMIF was added. SMIF produced a decrease in fluorescence intensity (n = 9), signifying a depolarization event. This decrease (12%) was significantly greater (P < 0.05) than that observed when 1/2 FSW alone was added to sperm suspensions (1% decrease).

Figure 6

Efflux of Na⁺ from sperm in response to Ca²⁺. Sperm were suspended in 1/2 NaCaF containing the cell impermeant Na⁺ probe NaG_o. After establishing a baseline fluorescence, Ca²⁺ (5 mM final) was added to the medium. An increase in fluorescence occurred, signifying an efflux of Na⁺ (Δ = 0.59 mM) from sperm (n = 3). This efflux was blocked by bepridil (100% with 10 μM) and partially inhibited by flunarazine (51% with 20 μM).

Figure 7

Effect of SMIF on membrane potential. Sperm were suspended in 1/2 FSW or preincubated with 20 μM bepridil or 50 μM nifedipine for 5 min, followed by the addition of DiSC₃(5) and CCCP (not shown). Upon addition of SMIF (arrow), a depolarization was observed (increase in fluorescence intensity) which was inhibited slightly by nifedipine (6%) and more so by bepridil (67%). No change in membrane potential was observed in sperm to which 1/2 FSW was added.

Figure 8

Detection of the Na⁺/Ca²⁺ exchanger in whole sperm and in extracted sperm proteins. (A and A^′) Live sperm that were labeled with anti-Na⁺/Ca²⁺ exchange IgG exhibited label over the entire sperm with the most intense signal over the midpiece region. (Bar = 2 μm.) (B and B^′) Control sperm incubated with secondary antibody alone exhibited no fluorescence signal. (A and B) Transmitted light microscope image. (A^′ and B^′) Confocal fluorescent image. (C) Immunoblotting of Na⁺/Ca²⁺ exchange protein. Triton X-100 extracted sperm proteins (lanes 1, 3), and canine myocytes (lanes 2, 4) were visualized for protein (lanes 1, 2) after electrophoresis or were transferred to nitrocellulose and probed with anti Na⁺/Ca²⁺ exchange IgG and visualized using chemiluminescence detection (lanes 3, 4). A band at 120 kDa was observed for both extracts that corresponds to the known molecular mass of the exchanger in canine myocytes. Molecular mass standards: 126, 90, 43.5, 33.9 kDa.