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Asterosap, an Egg Jelly Peptide, Elevate Intracellular Ca2+ and Activate the Motility of Spermatozoa

M. S. Islam
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

T. Akhter
Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4359-B39 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan

M. Matsumoto
Department of Biosciences and Informatics, Keio University, 3-14-1, Hiyoshi, Kouhoku-ku, Yokohama 223-8522, Japan

Abstract/ Executive Summary:

Components from the outer envelopes of the egg that influence the flagellar beating and acrosome reaction of spermatozoa are regulated by ion flux across the plasma membrane. Asterosap, a sperm-activating peptide from the starfish egg jelly layer, causes a transient increase in intracellular cyclic GMP (cGMP) through the activation of the asterosap receptor, a guanylyl cyclase (GC), and causes an increase in intracellular Ca²⁺. Here we describe the pathway of asterosap-induced Ca²⁺ elevation using different Ca²⁺ channel antagonists. Fluo- 4 AM, a cell permeable Ca²⁺ sensitive dye was used to determine the channel caused by the asterosap-induced Ca²⁺ elevation in spermatozoa. Different L-type Ca²⁺ channel antagonists, a non specific Ca²⁺ channel antagonist (nickel chloride), and a store-operated Ca²⁺ channel (SOC) antagonist do not show any significant response on asterosap-induced Ca²⁺ elevation, whereas KB-R7943, a selective inhibitor against Na+/Ca²⁺ exchanger (NCX) inhibited effectively. We also analyzed the flagellar movement of spermatozoa in artificial seawater (ASW) containing the asterosap at 100 nM ml⁻¹. We found that spermatozoa swam vigorously with more symmetrical flagellar movement in asterosap than in ASW and KB-R7943 significantly inhibited the flagellar movement.

Keywords: Asterosap, Flagellar motility, Intracellular Ca2+ elevation, Spermatozoa, Starfish

Location:

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Aquatic animal

Objectives:

To identify the asterosap, an egg jelly peptide, elevate intracellular ca²⁺ and activate the motility of spermatozoa

Methodology:

Starfish, A. amurensis, were collected from several locations of Japan and of Tasmania. Spermatozoa were obtained in the form of dry spermatozoa” by cutting the testes and were stored in a refrigerator until use.Pluronic F-127 and Fluo-4 AM were purchased from Sigma (St. Louis MO, USA) and Nacalai Tesque (Kyoto, Japan), respectively. Verapamil was purchased from Wako Chem. Co. (Osaka, Japan), 2-(2-(4-nitrobenzyloxy)-phenyl)-isothiurea methanosul-phonate (KB-R7943 mesylate, KB) from Tocris (Bristol, UK), and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) from Sigma and were dissolved in DMSO. The remaining reagents used were of the highest available quality. The synthetic asterosap isoform, P15 (Nishigaki et al., 1996), was used at a concentration of 1 μM. Artificial seawater (ASW) contained 430 mM NaCl, 9 mM CaCl₂, 9 mM KCl, 23 mM MgCl₂, 25 mM MgSO₄, and 10 mM EPPS (N-2-hydroxyethyl-piperazine-N’-3-propane sulphonic acid, pH 8.2). Low Ca²⁺ ASW was also treated as ASW but had 1 mM CaCl₂. Dry spermatozoa were diluted 10-fold in low Ca2+ ASW and incubated with 10 μM Fluo-4 AM plus 0.1 mM EDTA (ethylenediamine-N, N, N’, N’-tetraacetic acid), and 0.5% Pluronic F-127, and then incubated for 2 h at 16°C. Free Fluo-4 was washed with low Ca²⁺ ASW by centrifugation (1000 g for 5 min at 4°C), resuspended in the original volume of low Ca²⁺ ASW and kept on ice in the dark until use. For the measurement of intracellular Ca²⁺, 20 μl loaded spermatozoa were diluted in 1.5 ml of ASW in a round cuvette at 16°C under constant stirring. The fluorescence intensity was recorded on a spectrofluorophotometer (Shimadzu Science, Tokyo, Japan) with an excitation of 494 nm and emission of 516 nm. To examine the chemotactic activity of the spermatozoa, stored semen was diluted about 103-fold into 0.5% (w/v) polyvinylpyrrolidone in ASW and incubated for 1 min at room temperature for the induction of motility. The suspension of spermatozoa was immediately placed on a glass slide coated with BSA. All observations and recordings of movements were performed within 20 min after dilution and pictures of the movements were recorded at room temperature. Movements of the spermatozoa were observed under a phase-contrast microscope (Nikon, Optiphot) with an x20 objective lens (Olympus, BX51). The swimming trajectories were recorded after mixing with asterosap (100 nM) or/and spermatozoa incubated with 1 μM of KB for 3 min then added asterosap. A computer at 50 msec intervals for 1 msec, by using a high-speed chargecoupled device camera (HAS-200R, Ditect, Tokyo) and a video card (HAS-PCI, Ditect) were used to record the movements. The position of each spermatozoon was analyzed by using an image-analyzing program (Dip-motion 2D, Ditect).

Source of Information: Progress. Agric. 19(1) : 79-88, 2008 ISSN 1017-8139

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The pathway of asterosap-induced Ca²⁺ elevation using different Ca²⁺ channel antagonists. Fluo- 4 AM, a cell permeable Ca²⁺ sensitive dye was used to determine the channel caused by the asterosap-induced Ca²⁺ elevation in spermatozoa. Different L-type Ca²⁺ channel antagonists, a non specific Ca²⁺ channel antagonist (nickel chloride), and a store-operated Ca²⁺ channel (SOC) antagonist do not show any significant response on asterosap-induced Ca²⁺ elevation, whereas KB-R7943, a selective inhibitor against Na+/Ca²⁺ exchanger (NCX) inhibited effectively. We also analyzed the flagellar movement of spermatozoa in artificial seawater (ASW) containing the asterosap at 100 nM ml⁻¹. We found that spermatozoa swam vigorously with more symmetrical flagellar movement in asterosap than in ASW and KB-R7943 significantly inhibited the flagellar movement.

Knowledge Management: Journal