Structural and functional characterization of Hdh-HSBP1 and its involvement in heat stress and early development in Pacific abalone, Haliotis discus hannai.

Heat shock factor binding protein 1 (HSBP1) is known to regulate the activity of heat shock factor 1 (HSF1) and the early development of organisms. To understand the involvement of HSBP1 in the heat shock response and embryonic and larval development of Pacific abalone (Haliotis discus hannai), the Hdh-HSBP1 gene was sequenced from the digestive gland (DG) tissue. The full-length sequence of Hdh-HSBP1 encompassed 738 nucleotides, encoding an 8.42 kDa protein consisting of 75 deduced amino acids. The protein contains an HSBP1 domain and a coiled-coil domain, which are conserved features in the HSBP1 protein family. Protein-protein molecular docking revealed that the coiled-coil region of Hdh-HSBP1 binds to the coiled-coil region of Hdh-HSF1. Tissue expression analysis demonstrated that the highest Hdh-HSBP1 expression occurred in the DG, whereas seasonal expression analysis revealed that this gene was most highly expressed in summer. In heat-stressed abalone, the highest expression of Hdh-HSBP1 occurred at 30 °C. Moreover, time-series analysis revealed that the expression of this gene began to increase significantly at 6 h post-heat stress, with higher expression observed at 12 h and 24 h post-heat stress. Furthermore, Hdh-HSBP1 mRNA expression showed a link to ROS production. Additionally, the expression of Hdh-HSBP1 showed significantly higher expression in the early stages of embryonic development in Pacific abalone. These results suggest that Hdh-HSBP1 plays a crucial role in the stress physiology of Pacific abalone by interacting with Hdh-HSF1, as well as its embryonic development.

Molecular and structural analysis of Hdh-MIRP3 and its impact on reproductive regulation in female Pacific abalone, Haliotis discus hannai.

Molluscan insulin-related peptides (MIRP) play a crucial role in various biological processes, including reproduction and larval development in mollusk species. To investigate the involvement of MIRP in the ovarian development of Pacific abalone (Haliotis discus hannai), the Hdh-MIRP3 was cloned from cerebral ganglion (CG). Hdh-MIRP3 cDNA was 993 bp long, encoded a 13.22 kDa peptide, comprising 118 amino acids. Fluorescence in situ hybridization confirmed the localization of Hdh-MIRP3 in the CG and ovary. Molecular docking revealed that Hdh-MIRP3 binds to the N-terminal region of Hdh-IRP-R. Tissue expression analysis showed the highest Hdh-MIRP3 expression in the CG, followed by ovarian tissue. Hdh-MIRP3 expression was significantly upregulated in the CG and ovary during the ripe stage of seasonal ovarian development and in effective accumulative temperature conditioned abalone. Furthermore, siRNA silencing of Hdh-MIRP3 significantly downregulated the expression of four reproduction-related genes, including Hdh-GnRH, Hdh-GnRH-R, Hdh-IRP-R, and Hdh-VTG in both the CG and ovary, and Hdh-MIRP3 as well. These results indicate that Hdh-MIRP3 acts as a regulator of ovarian development in Pacific abalone. Additionally, expression analysis indicated that Hdh-MIRP3 plays a role in embryonic and larval development. Overall, the present findings elucidate the role of Hdh-MIRP3 in reproductive development in female Pacific abalone.

Cloning, characterization, and spatio-temporal expression patterns of HdhSPARC and its responses to multiple stressors.

SPARC is an extracellular Ca2+-binding, secreted glycoprotein that plays a dynamic role in the growth and development of organisms. This study aimed to describe the isolation, characterization, and expression analysis of HdhSPARC in Pacific abalone (Haliotis discus hannai) to infer its potential functional role. The isolated HdhSPARC was 1633 bp long, encoding a polypeptide of 284 amino acid residues. Structurally, the SPARC protein in abalone is comprised of three biological domains. However, the structure of this protein varied between vertebrates and invertebrates, as suggested by their distinct clustering patterns in phylogenetic analysis. In early development, HdhSPARC was variably expressed, and higher expression was found in veliger larvae. Moreover, HdhSPARC was highly expressed in juvenile abalone with rapid growth compared to their slower-growing counterparts. Among the testicular development stages, the growth stage exhibited higher HdhSPARC expression. HdhSPARC was also upregulated during muscle remodeling and shell biomineralization, as well as in response to different stressors such as heat shock, LPS, and H2O2 exposure. However, this gene was downregulated in Cd-exposed abalone. The present study first comprehensively characterized the HdhSPARC gene, and its spatio-temporal expressions were analyzed along with its responses to various stressors.

Physiological evaluation of seasonal sperm quality in a biannual spawner, Pacific abalone: Effects on in-vitro fertilization and cryotolerance.

Pacific abalone, Haliotis discus hannai, is a highly valuable gastropod mollusk commonly found in Southeast Asia. The present study aims to analyze the seminal plasma quality, sperm quality, and cryotolerance of the Pacific abalone sperm during its reproductive season. The seminal plasma quality was evaluated by analyzing biochemical and metabolite composition, enzymatic activity (superoxide dismutase, catalase, and glutathione), and lipid peroxidation (LPO) activity. The sperm quality was evaluated by analyzing motility, concentration, volume, ATP content, acrosome integrity (AI), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), DNA integrity, and fertilization potential. The cryotolerance capacity was evaluated by analyzing post-thaw motility, AI, PMI, MMP, and DNA integrity. Seminal plasma osmolarity was significantly higher (1123.3 ± 1.5 mOsmL-1) in May compared to other reproductive periods, with Cl- (516.8 ± 0.5 mM) and Na+ (460.2 ± 0.4 mM) as the dominant ions. The seminal plasma pH remained constant at 6.8 throughout the reproductive season. Improved enzymatic activity and lower LPO were detected in May or June. Sperm quality indicators were similar in May and June, except for sperm production. The fertilization potential (May: 93.0 ± 4.4%, June: 86.0 ± 7.2%) and hatching rate (May: 86.6 ± 5.78%, June: 82.3 ± 3.2%) of Pacific abalone were significantly higher in May or June than they were in other reproductive seasons. The motility (May: 50.19 ± 2.35%, June: 49.96 ± 1.60%), AI (May: 44.02 ± 3.46%, June: 42.16 ± 3.61%), PMI (May: 54.12 ± 3.29%, June: 52.82 ± 2.58%), and MMP (May: 44.02 ± 3.46%, June: 42.16 ± 3.61%) of the cryopreserved sperm were similar in May and June compared with those preserved in other reproductive seasons. The DNA integrity of the cryopreserved sperm was similar in May (80.3 ± 6.7%) or June (78.9 ± 7.4%) and had a higher cryotolerance than in other reproductive seasons. Hence, it can be suggested that May and/or June are suitable periods for sperm physiology experiments, artificial reproduction, and sperm cryopreservation of Pacific abalone.

Functional Characterization of Gonadotropin-Releasing Hormone and Corazonin Signaling Systems in Pacific Abalone: Toward Reclassification of Invertebrate Neuropeptides.

INTRODUCTION: The proposed evolutionary origins and corresponding nomenclature of bilaterian gonadotropin-releasing hormone (GnRH)-related neuropeptides have changed tremendously with the aid of receptor deorphanization. However, the reclassification of the GnRH and corazonin (CRZ) signaling systems in Lophotrochozoa remains unclear. METHODS: We characterized GnRH and CRZ receptors in the mollusk Pacific abalone, Haliotis discus hannai (Hdh), by phylogenetic and gene expression analyses, bioluminescence-based reporter, Western blotting, substitution of peptide amino acids, in vivo neuropeptide injection, and RNA interference assays. RESULTS: Two Hdh CRZ-like receptors (Hdh-CRZR-A and Hdh-CRZR-B) and three Hdh GnRH-like receptors (Hdh-GnRHR1-A, Hdh-GnRHR1-B, and Hdh-GnRHR2) were identified. In phylogenetic analysis, Hdh-CRZR-A and -B grouped within the CRZ-type receptors, whereas Hdh-GnRHR1-A/-B and Hdh-GnRHR2 clustered within the GnRH/adipokinetic hormone (AKH)/CRZ-related peptide-type receptors. Hdh-CRZR-A/-B and Hdh-GnRHR1-A were activated by Hdh-CRZ (pQNYHFSNGWHA-NH2) and Hdh-GnRH (pQISFSPNWGT-NH2), respectively. Hdh-CRZR-A/-B dually coupled with the Gαq and Gαs signaling pathways, whereas Hdh-GnRHR1-A was linked only with Gαq signaling. Analysis of substituted peptides, [I2S3]Hdh-CRZ and [N2Y3H4]Hdh-GnRH, and in silico docking models revealed that the N-terminal amino acids of the peptides are critical for the selectivity of Hdh-CRZR and Hdh-GnRHR. Two precursor transcripts for Hdh-CRZ and Hdh-GnRH peptides and their receptors were mainly expressed in the neural ganglia, and their levels increased in starved abalones. Injection of Hdh-CRZ peptide into abalones decreased food consumption, whereas Hdh-CRZR knockdown increased food consumption. Moreover, Hdh-CRZ induced germinal vesicle breakdown in mature oocytes. CONCLUSION: Characterization of Hdh-CRZRs and Hdh-GnRHRs and their cognate peptides provides new insight into the evolutionary route of GnRH-related signaling systems in bilaterians.

The Neuropeptide HGAP Regulates Growth, Reproduction, Metamorphosis, Tissue Damage Repair, and Response against Starvation in Pacific Abalone.

INTRODUCTION: Neuropeptides regulate vital physiological processes in multicellular organisms, including growth, reproduction, metamorphosis, and feeding. Recent transcriptome analyses have revealed neuropeptide genes with potential roles in vertebrate and invertebrate growth and reproduction. Among these genes, haliotid growth-associated peptide (HGAP) was identified as a novel gene in abalone. METHODS: This study focused on HGAP in Pacific abalone (Haliotis discus hannai), where the complete cDNA sequence named Hdh-HGAP was identified and characterized. Samples from different experiments, such as metamorphosis, juvenile abalone growth, gonad development stages, muscle remodeling, and starvation, were collected for mRNA expression analysis. RESULTS: The sequence spans 552 bp, encoding 96 amino acids with a molecular weight of 10.96 kDa. Expression analysis revealed that Hdh-HGAP exhibited higher levels in muscle tissue. Notably, during metamorphosis, Hdh-HGAP exhibited greater expression in the trochophore, veliger, and juvenile stages than in the cell division stages. Regarding growth patterns, Hdh-HGAP was highly expressed during rapid growth compared to stunted, minimal, and normal growth. In gonadal development, Hdh-HGAP mRNA reached its highest expression level during the ripening stage, indicating a potential role in gonadal cell proliferation and maturation. The in vivo effects of GnRH on gonad development and the expression of the Hdh-HGAP neuropeptide indicate its involvement in regulating reproduction in Pacific abalone. While tissue remodeling is primarily governed by immune genes, Hdh-HGAP was also upregulated during muscle tissue remodeling. Conversely, Hdh-HGAP was downregulated during prolonged starvation. CONCLUSION: This study marks the first comprehensive exploration of the Hdh-HGAP neuropeptide gene in Pacific abalone, shedding light on its involvement in growth, reproduction, metamorphosis, tissue remodeling, and response to starvation, although regulatory mechanisms are mostly unknown.

EF-Hand-Binding Secreted Protein Hdh-SMP5 Regulates Shell Biomineralization and Responses to Stress in Pacific Abalone, Haliotis discus hannai.

The development of a shell is a complex calcium metabolic process involving shell matrix proteins (SMPs). In this study, we describe the isolation, characterization, and expression of SMP5 and investigate its potential regulatory role in the shell biomineralization of Pacific abalone Haliotis discus hannai. The full-length Hdh-SMP5 cDNA contains 685 bp and encodes a polypeptide of 134 amino acids. Structurally, the Hdh-SMP5 protein belongs to the EF-hand-binding superfamily, which possesses three EF-hand Ca2+-binding regions and is rich in aspartic acid. The distinct clustering patterns in the phylogenetic tree indicate that the amino acid composition and structure of this protein may vary among different SMPs. During early development, significantly higher expression was observed in the trochophore and veliger stages. Hdh-SMP5 was also upregulated during shell biomineralization in Pacific abalone. Long periods of starvation cause Hdh-SMP5 expression to decrease. Furthermore, Hdh-SMP5 expression was observed to be significantly higher under thermal stress at temperatures of 15, 30, and 25 °C for durations of 6 h, 12 h, and 48 h, respectively. Our study is the first to characterize Hdh-SMP5 comprehensively and analyze its expression to elucidate its dynamic roles in ontogenetic development, shell biomineralization, and the response to starvation and thermal stress.

Age-Dependent Growth-Related QTL Variations in Pacific Abalone, Haliotis discus hannai.

Pacific abalone is a high-value, commercially important marine invertebrate. It shows low growth as well as individual and yearly growth variation in aquaculture. Marker-assisted selection breeding could potentially resolve the problem of low and variable growth and increase genetic gain. Expression of quantitative trait loci (QTLs) for growth-related traits, viz., body weight, shell length, and shell width were analyzed at the first, second, and third year of age using an F1 cross population. A total of 37 chromosome-wide QTLs were identified in linkage groups 01, 02, 03, 04, 06, 07, 08, 10, 11, 12, and 13 at different ages. None of the QTLs detected at any one age were expressed in all three age groups. This result suggests that growth-related traits at different ages are influenced by different QTLs in each year. However, multiple-trait QTLs (where one QTL affects all three traits) were detected each year that are also age-specific. Eleven multiple-trait QTLs were detected at different ages: two QTLs in the first year; two QTLs in the second year; and seven QTLs in the third year. As abalone hatcheries use three-year-old abalone for breeding, QTL-linked markers that were detected at the third year of age could potentially be used in marker-assisted selection breeding programs.

Glutathione reductase, a biomarker of pollutant and stress in Pacific abalone.

Abalone are frequently exposed to several environmental factors including heavy metal toxicity, thermal stress, H2O2-stress, starvation, viral and bacterial infection that can induce oxidative stress. Glutathione reductase is a vital enzyme in the antioxidant defense system that catalyzes the reduction of oxidized glutathione to reduced glutathione. The present study aimed to identify and localize glutathione reductase in Pacific abalone (Hdh-GR) and assess its potential role in stress physiology, heavy metal toxicity, immune response, gonadal development, and metamorphosis. The mRNA expression of Hdh-GR was upregulated in response to thermal stress, starvation, H2O2-stress, and cadmium-exposed toxicity. The induced mRNA expression was also quantified in immune-challenged abalone. Moreover, the Hdh-GR expression was significantly higher during metamorphosis. The Hdh-GR mRNA expression showed an inverse relationship with ROS production in heat stressed Pacific abalone. These results suggest that Hdh-GR has central role in the stress physiology, immune response, gonadal development, and metamorphosis of Pacific abalone.

Gonadotropins and Sex Steroid Hormones in Captive-Reared Small Yellow Croaker (Larimichthys polyactis) and Their Role in Female Reproductive Dysfunction.

The seed production of small yellow croaker (SYC) is constrained by reproductive dysfunction in captive-reared females. Reproductive dysfunction is closely linked to endocrine reproductive mechanisms. To better understand the reproductive dysfunction in captive broodstock, functional characterization of gonadotropins (GtHs: follicle stimulating hormone ß subunit, fshß; luteinizing hormone ß subunit, lhß; and glycoprotein α subunit, gpα) and sex steroids (17ß-estradiol, E2; testosterone, T; progesterone; P) was performed using qRT-PCR, ELISA, in vivo, and in-vitro assay. The pituitary GtHs and gonadal steroids levels were significantly higher in ripen fish of both sexes. However, changes in lhß and E2 levels in females were not significant in the developing and ripen stages. Furthermore, GtHs and steroids levels were lower in females compared to males throughout the reproductive cycle. In vivo administration of gonadotropin releasing hormone analogue (GnRHa) significantly increased the expression of GtHs in both dose- and time-related manners. The lower and higher doses of GnRHa led to successful spawning in male and female SYC, respectively. Sex steroids in vitro significantly inhibited the expression of lhß in female SYC. Overall, GtHs were shown to play a vital role in final gonadal maturation, while steroids promoted negative feedback in the regulation of pituitary GtHs. Lower levels of GtHs and steroids might be key components in the reproductive dysfunction of captive-reared female SYC.

Water Hardness Improves the Antioxidant Response of Zinc-Exposed Goldfish (Carassius auratus).

Zinc (Zn), a heavy metal, is an essential element in fish; however, exposure to high concentrations causes oxidative stress. Water hardness reduces oxidative stress reactions caused by heavy metals. To confirm the effect of water hardness on oxidative stress caused by Zn, goldfish were exposed to various Zn concentrations (1.0, 2.0, and 5.0 mg/L) and water hardness (soft (S), hard (H), and very hard (V)). The activity of superoxide dismutase (SOD) and catalase (CAT) in plasma increased with 1.0, 2.0, and 5.0 mg/L of Zn, and decreased with H and V water hardness. The levels of H2O2 and lipid peroxide (LPO) increased with Zn above 1.0 mg/L and decreased with H and V of water hardness. Caspase-9 mRNA expression in the liver increased after 7 and 14 days of Zn exposure and decreased with H and V water hardness. It was confirmed that DNA damage was less dependent on H and V water hardness. Based on the results of this study, at least 1.0 mg/L Zn causes oxidative stress in goldfish, and a high level of apoptosis occurs when exposed for more than 7 days. It appears that the oxidative stress generated by Zn can be alleviated by water hardness of at least 270 mg/L CaCO3. This study provides information on the relationship between the antioxidant response caused by heavy metals and water hardness in fish.

Identification and Characterization of Hdh-FMRF2 Gene in Pacific Abalone and Its Possible Role in Reproduction and Larva Development.

FMRFamide-related peptides are neuropeptides involved in a wide range of biological processes, including reproduction and larval development. To characterize the involvement of FMRFamide in the reproduction and larval development of Pacific abalone Haliotis discus hannai, an FMRFamide cDNA (Hdh-FMRF2) was cloned from the cerebral ganglion (CG). Fluorescence in situ hybridization and qRT-PCR were performed for functional characterization. The Hdh-FMRF2 cDNA encoded 204 deduced amino acids that contained a putative signal peptide and four FaRP domains. The major population of Hdh-FMRF2 neuronal cell bodies was localized in the cortex of CG. Hdh-FMRF2 mRNA expression was significantly upregulated in CG during the mature stage of gonadal development and effective accumulative temperature (EAT) exposed abalone in both sexes. In the induced spawning event, Hdh-FMRF2 expression was significantly upregulated during spawning in males. However, no upregulation was observed in females, suggesting Hdh-FMRF2 might inhibit gamete release in female abalone. These results revealed Hdh-FMRF2 as a reproduction related peptide. Furthermore, mRNA expression in larval development suggested that this peptide was also involved in larval development during development of Pacific abalone. Collectively, this study provides evidence of possible involvement of an FMRFamide neuropeptide in the reproduction and larval development of Pacific abalone.

Molecular Cloning and Functional Characterization of Catalase in Stress Physiology, Innate Immunity, Testicular Development, Metamorphosis, and Cryopreserved Sperm of Pacific Abalone.

Catalase is a crucial enzyme of the antioxidant defense system responsible for the maintenance of cellular redox homeostasis. The aim of the present study was to evaluate the molecular regulation of catalase (Hdh-CAT) in stress physiology, innate immunity, testicular development, metamorphosis, and cryopreserved sperm of Pacific abalone. Hdh-CAT gene was cloned from the digestive gland (DG) of Pacific abalone. The 2894 bp sequence of Hdh-CAT had an open reading frame of 1506 bp encoding 501 deduced amino acids. Fluorescence in situ hybridization confirmed Hdh-CAT localization in the digestive tubules of the DG. Hdh-CAT was induced by different types of stress including thermal stress, H2O2 induction, and starvation. Immune challenges with Vibrio, lipopolysaccharides, and polyinosinic-polycytidylic acid sodium salt also upregulated Hdh-CAT mRNA expression and catalase activity. Hdh-CAT responded to cadmium induced-toxicity by increasing mRNA expression and catalase activity. Elevated seasonal temperature also altered Hdh-CAT mRNA expression. Hdh-CAT mRNA expression was relatively higher at the trochophore larvae stage of metamorphosis. Cryopreserved sperm showed significantly lower Hdh-CAT mRNA expression levels compared with fresh sperm. Hdh-CAT mRNA expression showed a relationship with the production of ROS. These results suggest that Hdh-CAT might play a role in stress physiology, innate immunity, testicular development, metamorphosis, and sperm cryo-tolerance of Pacific abalone.

Synthetic microfiber exposure negatively affects reproductive parameters in male medaka (Oryzias latipes).

Microplastics not only accumulate in the bodies of fishes and cause damage to the organs, but also cause many other problems, such as reduced reproductive capacity, by acting directly or indirectly on the hypothalamus-pituitary-gonad axis (HPG axis). In this study, we investigated the changes in HPG axis-related genes in male medaka (Oryzias latipes) exposed to fiber-type microplastics. We confirmed the progression of vitellogenesis, a sign of endocrine disruption, in male fish. In the microfiber-exposed group, microfiber accumulation was confirmed in the gills and intestines. One week after exposure to two different concentrations of microfibers (500 and 1,000 fibers/L), the fish showed increased expression of gonadotropin-releasing hormone (GnRH) and luteinizing hormone receptor (LH-R) mRNA. From day 10 of exposure to the microfibers, there was an increase in the expression of the gonadotropin-inhibitory hormone (GnIH) mRNA and a decrease in the expression of GnRH and LH-R mRNA. There was an increase in the cytochrome P450 aromatase (CYP19a) mRNA expression and plasma estradiol (E2) concentration in the 1,000 fibers/L exposure group. High vitellogenin (VTG) mRNA expression was confirmed seven days after exposure in the 1,000 fibers/L group, which was consistent with the VTG mRNA expression signals detected in the liver using in situ hybridization. These results suggest that microfiber ingestion may cause short-term endocrinal disruption of the HPG axis in male medaka, which in turn may interfere with their normal maturation process.

Characterization and Expression Analysis of Mollusk-like Growth Factor: A Secreted Protein Involved in Pacific Abalone Embryonic and Larval Development.

Growth factors are mostly secreted proteins that play key roles in an organism's biophysical processes through binding to specific receptors on the cell surface. The mollusk-like growth factor (MLGF) is a novel cell signaling protein in the adenosine deaminase-related growth factor (ADGF) subfamily. In this study, the MLGF gene was cloned and characterized from the digestive gland tissue of Pacific abalone and designated as Hdh-MLGF. The transcribed full-length sequence of Hdh-MLGF was 1829 bp long with a 1566 bp open reading frame (ORF) encoding 521 amino acids. The deduced amino acid sequence contained a putative signal peptide and two conserved adenosine deaminase domains responsible for regulating molecular function. Fluorescence in situ hybridization localized Hdh-MLGF in the submucosa layer of digestive tubules in the digestive gland. The mRNA expression analysis indicated that Hdh-MLGF expression was restricted to the digestive gland in the adult Pacific abalone. However, Hdh-MLGF mRNA expressions were observed in all stages of embryonic and larval development, suggesting Hdh-MLGF might be involved in the Pacific abalone embryonic and larval development. This is the first study describing Hdh-MLGF and its involvement in the Pacific abalone embryonic and larval development.

Saccharides Influence Sperm Quality and Expressions of Motility and Fertilization-Associated Genes in Cryopreserved Sperm of Pacific Abalone, Haliotis discus hannai.

Pacific abalone, Haliotis discus hannai, is a highly commercial seafood in Southeast Asia. The present study aimed to determine the influence of saccharides and vitamins on post-thaw sperm quality, ATP content, fertilization capacity, hatching capacity, and mRNA content of motility and fertilization-associated genes of Pacific abalone. Sperm cryopreserved using saccharides improved the post-thaw sperm quality including motility, acrosome integrity (AI), plasma membrane integrity (PMI), and mitochondrial membrane potential (MMP). However, vitamins (l-ascorbic acid) did not result in any significant improvement in sperm quality. Sperm cryopreserved using saccharides also improved ATP content, DNA integrity, and mRNA content of motility and fertilization-associated genes of post-thaw sperm than sperm cryopreserved without saccharides. Among sperm cryopreserved using different saccharides, post-thaw sperm quality indicators (except PMI) and mRNA content of motility and fertilization-associated genes did not show significant differences between sperm cryopreserved using 3% sucrose (S) combined with 8% dimethyl sulfoxide (DMSO) and sperm cryopreserved using 1% glucose (G) combined with 8% ethylene glycol (EG). However, sperm cryopreserved using 3% S + 8% DMSO showed higher post-thaw sperm quality (motility: 58.4 ± 2.9%, AI: 57.1 ± 3.2%, PMI: 65.3 ± 3.3%, and MMP: 59.1 ± 3.2%), ATP content (48.4 ± 1.8 nmol/ml), and % DNA in tail (2.09 ± 0.20%) than sperm cryopreserved using other saccharides. When sperms were cryopreserved using 3% S + 8% DMSO, the mRNA content of motility (heat shock protein 70, HSP70; heat shock protein 90, HSP90; protein kinase A, PKA-C; axonemal protein 66.0, Axpp66.0; and tektin-4) and fertilization-associated (sperm protein 18 kDa, SP18 kDa) genes were higher than in sperm cryopreserved using other saccharides. However, changes in the mRNA contents of these genes were insignificant between sperm cryopreserved using 3% S + 8% DMSO and 1% G + 8% EG. Taken together, these results indicate that cryopreservation using 3% S + 8% DMSO can improve post-thaw sperm quality and mRNA contents better than other examined cryoprotectants. The present study suggests that 3% S + 8% DMSO is a suitable cryoprotectant for sperm cryopreservation and molecular conservation of this valuable species.

Functional Characterization of Three GnRH Isoforms in Small Yellow Croaker Larimichthys polyactis Maintained in Captivity: Special Emphasis on Reproductive Dysfunction.

Fish reproduction is regulated by the brain-pituitary-gonad (BPG) axis where the gonadotropin-releasing hormone (GnRH) plays a central role. Seed production of small yellow croaker (Larimichthys polyactis) is performed using captive-reared broodstock known to undergo reproductive dysfunction, which is connected to endocrinological dysfunction. To determine the endocrinological mechanism of GnRHs in the BPG axis of small yellow croaker, full-length sequences of three GnRH isoforms encoding sbGnRH (GnRH1), cGnRH-II (GnRH2), and sGnRH (GnRH3) were cloned and characterized from brain tissue. qRT-PCR, in vivo, and in vitro experiments were performed for functional characterization. The mRNA expression of GnRH1 in the brain and gonadotropin subunits (GPα, FSHß, and LHß) in the pituitary were significantly higher at the ripen stage during gonadal development and GnRH1 at spawning stage during spawning events. Expression of both GnRH1 and GtH subunits was significantly lower in females than males. GtH subunits were induced at higher concentrations of GnRH1 in vivo and in vitro. Sex-steroids significantly inhibited the GnRH1 expression in vitro in a dose-dependent manner. Taken together, results indicated that GnRH1 plays a key role in gonadal maturation and sex-steroids induced negative feedback in the regulation of GnRH. A lower level of GnRH1 and GtHs might be responsible for reproductive dysfunction in a female small yellow croaker.

Antioxidant Activity and Oxidative Stress-Oriented Apoptosis Pathway in Saccharides Supplemented Cryopreserved Sperm of Pacific Abalone, Haliotis discus hannai.

The Pacific abalone Haliotis discus hannai is a highly commercialized seafood in Southeast Asia. The aim of the present study was to determine the antioxidant activity and oxidative stress-oriented apoptosis pathway in saccharides supplemented cryopreserved sperm of Pacific abalone. Cryopreserved sperm showed impaired antioxidant defenses due to the reduced mRNA abundance of antioxidant genes (CAT, Cu/Zn-SOD, Mn-SOD, GPx, GR, and BCL-2), apoptosis inhibitor (HSP70, and HSP90) gene, and enzymatic antioxidant activity compared to fresh sperm. Such impaired antioxidant defenses caused an increase in the mRNA expression of apoptosis genes (Bax, and Caspase-3), finally leading to apoptosis. The impaired antioxidant defense also increased O2•- production and lipid peroxidation (MDA) levels, which further accelerated apoptosis. Considering all the experimental findings, an apoptosis pathway of cryopreserved sperm has been adopted for the first time. Specifically, sperm cryopreserved using 3% sucrose combined with 8% dimethyl sulfoxide (DMSO) showed improved mRNA stability, enzymatic activity, and DNA integrity with reduced O2•- production and MDA levels compared to sperm cryopreserved with the other types of examined cryoprotectants (8% ethylene glycol + 1% glucose, 6% propylene glycol + 2% glucose, 2% glycerol + 3% glucose, and 2% methanol + 4% trehalose). The present study suggests that 3% sucrose combined with 8% DMSO is suitable to cryopreserve the sperm of this valuable species for molecular conservation.

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

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.

Toxicity response to benzo[α]pyrene exposure: Modulation of immune parameters of the bay scallop, Argopectenirradians.

Bay scallops were exposed to four BaP concentrations (0.5, 1.0, 10 and 50 µg/L) for 72 h to elucidate their immune response. Immune parameters were evaluated by measuring nitric oxide (NO) levels in hemolymph. Additionally, we measured peptidoglycan recognition proteins (PGRP), fibrinogen-domain-containing protein (FReDC1), metallothionein (MT), and heat shock protein (HSP) 70 mRNA expression in digestive diverticula. NO as well as FReDC1 and MT expression in each BaP group increased significantly over time except for the BaP 0.5 group. The PGRP and HSP70 mRNA expression in the BaP 50 group increased in the range 6-24 h and then decreased. In situ hybridization also confirmed that there was higher MT mRNA expression in the BaP 50 group than in the control group at 72 h. Our results suggest that higher levels of BaP dampened scallop immune responses, while simultaneously reducing their ability to cope with oxidative stress and DNA damage. BaP exposure can be considered a potential immune inducer in bay scallop.