Hdh-Tektin-4 Regulates Motility of Fresh and Cryopreserved Sperm in Pacific Abalone, Haliotis discus hannai

Abstract

As structural components of sperm, tektins are thought to play a fundamental role in sperm flagellar motility. In this study, Tektin-4 (Hdh-TEKT4) gene was successfully cloned and characterized from the testis tissue in Pacific abalone, Haliotis discus hannai. The full-length cDNA of Hdh-TEKT4 was 1,983 bp, with a coding region of 1,350 bp encoding 51.83 kDa putative protein of 449 deduced amino acids. Hdh-TEKT4 contains a tektin domain including a nonapeptide signature motif (RPGVDLCRD). Fluorescence in situ hybridization revealed that Hdh-TEKT4 localized in the spermatids of Pacific abalone testis. qRT-PCR analysis showed that Hdh-TEKT4 was predominantly expressed in testis tissues. Hdh-TEKT4 mRNA expression was upregulated during the fully mature testicular developmental stage in both seasonal development and EAT exposed abalone. Furthermore, mRNA expression of Hdh-TEKT4 was significantly higher in sperm with higher motility than in sperm with lower motility during peak breeding season, induced spawning activity stages, and after cryopreservation in different cryoprotectants. Taken together, these results indicate that the expression of Hdh-TEKT4 in Pacific abalone sperm might have a positive correlation with sperm motility.

Keywords: Haliotis discus hannai; Pacific abalone; Tektin-4; cryopreservation; motility; sperm.

FIGURE 1

Full-length nucleotide and deduced amino acid sequences of Hdh-TEKT4 (GenBank accession no. MZ265399). Numbers at the left and right sides indicate nucleotide and amino acid positions in the sequence, respectively. Start and stop codons are shown in bold red and underlined. Putative polyadenylations signals (AATAAA) are shown in bold blue. The tektin domain is marked with red dotted underline. A variant of conserved tektin nonapeptide signature sequence is indicated in a light black box. Predicted protein kinase C (PKC) phosphorylation sites are boxed in red. Potential casein kinase II phosphorylation sites are boxed in purple. cAMP- and cGMP-dependent protein kinase phosphorylation site is boxed in yellow. Potential N-linked glycosylation sites are boxed in green, and N-myristoylation sites are boxed in blue.

FIGURE 2

Schematic diagram of the motif detected in Hdh-TEKT4 and other TEKT4 of invertebrates and vertebrates. Distinct motifs are denoted by different colors. The motif analysis included Hdh-TEKT4 amino acid sequence and various other sequences of TEKT4: Tektin-4 of H. discus discus (ARR97156), Homo sapiens (NP_653306), Danio rerio (NP_001139162), Rattus norvegicus (BAD93477), Mus musculus (NP_082227), tektin-A1 identical to tektin-4 of H. asinina (ACJ15284), Ciona intestinalis (NP_001027644), and Strongylocentrotus purpuratus (NP_999787).

FIGURE 3

Multiple sequence alignment of deduced amino acid sequences of different tektins from H. discus hannai (MZ265399.1), H. discus discus (ARR97156.1), H. asinina (ACJ15284.1), Pecten maximus (XP_033751776.1), and Mytilus galloprovincialis (VDI53310.1). Dashes indicate gaps. Five cysteine residues conserved in the sequence alignment are shown in the black lined box. Variants of tektin nonapeptide signature sequences are boxed with a break line.

FIGURE 4

Structure prediction of Hdh-TEKT4 from amino acid sequence. (A) Apparent domain structure within Hdh-Tektin-4. Positions of some conserved motifs reported by Amos (2008), including the signature nonapeptide, are indicated in a single-letter amino acid code in the diagram with the amino acid position. (B) Three-dimensional homology modeling of Tektin-4 of Haliotis discus hannai. The model was constructed using I-TASSER online tools. Domains between the N terminus and C terminus were predicted from the secondary structure. (C) Predictions of coiled–coil segments from amino acid sequences of Hdh-TEKT4. The x-axis represents the amino acid number, and the y-axis represents the probability (0.0–1.0) that the sequence will form a coiled–coil secondary structure. The position corresponds to that of the tektin loop containing the conserved nonapeptide shown by a down-arrow.

FIGURE 5

Phylogenetic tree constructed by the bootstrap neighbor-joining method after clustalW alignment based on amino acid residues of different isoforms of tektins. Numbers at the nodes indicate bootstrap probability. GenBank accession numbers of sequences used to construct the phylogenetic tree are as follows: Tektin-1 of H. sapiens (NP_444515.1), Bos taurus (NP_001069251.1), D. rerio (XP_009290074.1), Takifugu rubripes (XP_011607045.1), C. intestinalis (XP_002130466.1), Aplysia californica (XP_005109632.1), Biomphalaria glabrata (XP_013064715.1), Crassostrea gigas (XP_011416301.1), Mizuhopecten yessoensis (OWF41001.1); tektin-2 or tektin-B1 of H. sapiens (NP_055281.2), B. taurus (NP_001033192.1), Rattus norvegicus (NP_001011977.1), M. musculus (NP_036032.2), Xenopus tropicalis (NP_001007884.1), D. rerio (NP_001017432.3), T. rubripes (XP_003969284.2), Aedes aegypti (XP_001657769.1), C. intestinalis (NP_001027645.1), A. californica (XP_005098539.1), B. glabrata (XP_013074221.1), M. yessoensis (OWF46183.1), S. purpuratus (NP_999789.1); tektin-3 of H. sapiens (NP_114104.1), Pan troglodytes (NP_001233472.1), B. taurus (NP_001092489.1), D. rerio (XP_701169.2), Oreochromis niloticus (XP_003453437.1), T. rubripes (XP_003961064.1), C. intestinalis (XP_002129626.1), A. californica (XP_012939623.1), B. glabrata (XP_013095021.1); tektin-4 or tektin-A1 of H. sapiens (NP_653306.1), R. norvegicus (BAD93477.1), M. musculus (NP_082227.1), Sus scrofa (AFM37359.1), B. taurus (NP_001033158.1), D. rerio (NP_001139162.1), T. rubripes (XP_003972094.2), C. intestinalis (NP_001027644.1), A. californica (XP_005092496.1), B. glabrata (XP_013090287.1), H. discus hannai (MZ265399.1), H. discus discus (ARR97156.1), H. asinina (ACJ15284.1), M. galloprovincialis (VDI53310.1), M. yessoensis (OWF46480.1), C. gigas (XP_011416098.2), C. virginica (XP_022318607.1), S. purpuratus (NP_999787.1); tektin-5 of H. sapiens (NP_653275.1), R. norvegicus (NP_001014246.1), Equus caballus (XP_023472090.1), Chrysemys picta bellii (XP_005306767.1), and Gallus gallus (XP_414934.3). The scale bar indicates 0.2 units of the expected fraction of amino acid substitutions (1.0 unit = 100 PAMs).

FIGURE 6

Confocal laser scanning microscopic observation after fluorescence in situ hybridization (FISH) of Hdh-TEKT4 mRNA in fully mature testis tissue of Pacific abalone. (A) Single confocal optical sections showing a positive signal of Hdh-TEKT4 mRNA (green) when hybridized with the anti-sense probe. (B) Nuclei are counterstained with DAPI (blue). (C) Merged image of (A,B). Scale bar: 50 μm.

FIGURE 7

Relative mRNA expression levels (2^–ΔΔCT) of Hdh-TEKT4 (mean ± SEM) in different tissues of Pacific abalone detected by qRT-PCR. Different letters above the bars indicated significant differences (p < 0.05) among organs. CG, cerebral ganglion; PPG, pleuropedal ganglion; OV, ovary; TE, testis; HCY, hemocyte; HRT, heart; CT, cephalic tentacle; PT, pleuropedal tentacle; GIL, gill; DG, digestive gland; MNT, mantle; MUS, muscle.

FIGURE 8

Expression levels of Hdh-TEKT4 mRNA in the testis of different testicular developmental stages of Pacific abalone. Different letters above the bars indicate significant differences (p < 0.05) among developmental stages. DS, degenerative stage; AS, active stage; RS, ripening stage; SS, spent stage.

FIGURE 9

Expression levels of Hdh-TEKT4 mRNA in the testis of different °C-days of Pacific abalone exposed to effective accumulative temperature during broodstock conditioning. Different letters above the bars indicate significant differences (p < 0.05) among °C-days. EAT 00, EAT 0°C-days; EAT 500, EAT 500°C-days; EAT 1000, EAT 1000°C-days; EAT 1500, EAT °C-days.

FIGURE 10

Expression levels of Hdh-TEKT4 mRNA and percent sperm motility in sperm of Pacific abalone during different months of peak breeding seasons. Different letters above the bars indicate significant differences (p < 0.05) among months of peak breeding season.

FIGURE 11

Expression levels of Hdh-TEKT4 mRNA in sperm of Pacific abalone and percent sperm motility during different steps of induced spawning activities. Different letters above the bars indicate significant differences (p < 0.05) among steps of induced spawning. IC, initial control; HI, heat-induced; UV, UV-irradiated water-induced; DS, during spawning, AS, after spawning.

FIGURE 12

Expression levels of Hdh-TEKT4 mRNA and percent sperm motility in fresh sperm of Pacific abalone and sperm cryopreserved in different cryoprotectants. Different letters above the bars indicate significant differences (p < 0.05) among different cryopreserved sperm. Fresh, fresh sperm; 8% DMSO, sperm cryopreserved in 8% DMSO; 8% EG, sperm cryopreserved in 8% ethylene glycol; 6% propanol glycol, sperm cryopreserved in 6% PG; 3% GLY, sperm cryopreserved in 3% glycerol; 2% MeOH, sperm cryopreserved in 2% methanol.