Cryopreservation of Plagiognathops microlepis sperm.

Sperm was collected from cultured male fish and cryopreserved in 0.25 ml straws for the study of sperm cryopreservation. Different parameters were evaluated, including extender, dilution ratio, cryoprotectant type and concentration, equilibrium time, cooling height (in a two-step cooling protocol), and thawing temperature. The optimum result was obtained when the sperm was diluted at a 1:7 ratio in D-16 with 5% DMSO as a cryoprotectant, equilibrated for 20 min, held at 3 cm above liquid nitrogen for 10 min, and then stored in liquid nitrogen. After thawing in a water bath at 40 °C, the percentage of motile cells and fertilization rates of frozen-thawed sperm were 35.33 ±â€¯2.52% and 39.00 ±â€¯4.58%, respectively, while the corresponding rates for fresh sperm were 87.67 ±â€¯3.06% and 88.67 ±â€¯4.62%. We also used a programmed cooling protocol in which temperature was decreased from 4 °C to -80 °C by a rate of 30 °C/min, and then straws (0.25 ml) were placed above the surface of liquid nitrogen for 2 min before being stored in liquid nitrogen. This protocol provided a post-thaw activation rate of 36.67 ±â€¯4.77%. Further parametric optimization is required to improve the quality of frozen-thawed sperm.

Analysis of the function of KIF3A and KIF3B in the spermatogenesis in Boleophthalmus pectinirostris.

Spermatogenesis represents one of the most complicated morphological transformation procedures. During this process, the assembly and maintenance of the flagella and intracellular transport of membrane-bound organelles required KIF3A and KIF3B. Our main goal was to test KIF3A and KIF3B location during spermatogenesis of Boleophthalmus pectinirostris. We cloned complete cDNA of KIF3A/3B from the testis of B. pectinirostris by PCR and rapid amplification of cDNA ends (RACE). The predicted secondary and tertiary structures of B. pectinirostris KIF3A/3B contained three domains: (a) the head region, (b) the stalk region, and (c) the tail region. Real-time quantitative PCR (qPCR) results revealed that KIF3A and KIF3B mRNA were presented in all the tissues examined, with the highest expression seen in the testis. In situ hybridization (ISH) showed that KIF3A and KIF3B were distributed in the periphery of the nuclear in the spermatocyte and the early spermatid. In the late spermatid and mature sperm, the KIF3A and KIF3B mRNA were gradually gathered to one side where the flagella formed. Immunofluorescence (IF) showed that KIF3A, tubulin, and mitochondria were co-localized in different stages during spermiogenesis in B. pectinirostris. The temporal and spatial expression dynamics of KIF3A/3B indicate that KIF3A and KIF3B might be involved in flagellar assembly and maintenance at the mRNA and protein levels. Moreover, these proteins may transport the mitochondria resulting in flagellum formation in B. pectinirostris.

Molecular cloning and expression analysis of five heat shock protein 70 (HSP70) family members in Lateolabrax maculatus with Vibrio harveyi infection.

Heat shock proteins 70 (HSP70s) are molecular chaperones that aid in protection against environmental stress. In this study, we cloned and characterized five members of the HSP70 family (designated as HSPa1a, HSC70-1, HSC70-2, HSPa4 and HSPa14) from Lateolabrax maculatus using rapid amplification cDNA ends (RACE). Multiple sequence alignment and structural analysis revealed that all members of the HSP70 family had a conserved domain architecture, with some distinguishing features unique to each HSP70. Quantitative real-time (qPCR) analysis revealed that all members of the HSP70 family were ubiquitously and differentially expressed in all major types of tissues, including testicular tissue. This indicated that HSP70s have vital and conserved biological functions, and may also function in the development of germinal cells. The expression of mRNA of the five HSP70 family members mRNA expression was significantly increased in the head kidney, intestine and gill after Vibrio harveyi challenge, suggesting that HSP70s play an important role in the immune response.

Characterization of mitochondrial prohibitin from Boleophthalmus pectinirostris and evaluation of its possible role in spermatogenesis.

Prohibitin (PHB) is an evolutionarily conserved mitochondrial membrane protein. It plays a vital role in cell proteolysis, senescence, and apoptosis and is associated with spermatogenesis and sperm quality control in mammals. To study the characteristics of the PHB gene and its potential roles during spermatogenesis in Boleophthalmus pectinirostris, we cloned a 1153-bp full-length cDNA from the testis of B. pectinirostris with an open reading frame of 816 bp, which encodes 272 amino acid residues. Real-time quantitative PCR (qPCR) analysis revealed the presence of phb mRNA in all the tissues examined, with higher expression levels found in the testis, kidney, intestine, and muscle tissues. We examined the localization of phb mRNA during spermatogenesis by in situ hybridization (ISH), showing that phb mRNA was distributed in the periphery of the nucleus in primary and secondary spermatocytes. In spermatid and mature sperm, the phb mRNA gradually moved toward one side, where the flagellum is formed. Immunofluorescence (IF) results showed co-localization of the PHB and mitochondria at different stages during spermatogenesis of B. pectinirostris. The signals obtained for PHB decreased as spermatogenesis proceeded; the strongest detection signal was found in secondary spermatocytes, with lower levels of staining in other stages. Additionally, in the mature germ cells, the PHB signals were weak and aggregate in the midpiece of the flagellum.

Expression analysis of HSP70 in the testis of Octopus tankahkeei under thermal stress.

The gene encoding heat shock protein 70 (HSP70) was identified in Octopus tankahkeei by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length cDNA (2471 bp) consists of a 5'-untranslated region (UTR) (89 bp), a 3'-UTR (426 bp), and an open reading frame (1956 bp) that encodes 651 amino acid residues with a predicted molecular mass of 71.8 kDa and an isoelectric point of 5.34. Based on the amino acid sequence analysis and multiple sequence alignment, this cDNA is a member of cytoplasmic hsp70 subfamily of the hsp70 family and was designated as ot-hsp70. Tissue expression analysis showed that HSP70 expression is highest in the testes when all examined organs were compared. Immunohistochemistry analysis, together with hematoxylin-eosin staining, revealed that the HSP70 protein was expressed in all spermatogenic cells, but not in fibroblasts. In addition, O. tankahkeei were heat challenged by exposure to 32 °C seawater for 2 h, then returned to 13 °C for various recovery time (0-24 h). Relative expression of ot-hsp70 mRNA in the testes was measured at different time points post-challenge by quantitative real-time PCR. A clear time-dependent mRNA expression of ot-hsp70 after thermal stress indicates that the HSP70 gene is inducible. Ultrastructural changes of the heat-stressed testis were observed by transmission electron microscopy. We suggest that HSP70 plays an important role in spermatogenesis and testis protection against thermal stress in O. tankahkeei.

Cloning, characterization and cadmium inducibility of metallothionein in the testes of the mudskipper Boleophthalmus pectinirostris.

Metallothioneins (MTs) are cysteine-rich, low molecular weight, and heavy metal-binding protein molecules. MT participates in metallic homeostasis and detoxification in living animals due to its abundant cysteine. In order to investigate the functions of MT during spermiogenesis in the mudskipper (Boleophthalmus pectinirostris), we identified the MT complete which contains: an 83bp 5' untranslated region, a 110bp 3' untranslated region, and a 183bp open reading frame. The protein alignment between MT sequences of other species shows a high similarity and a strong identity in cysteine residues vital for the metal-binding affinity of MT. The localizations of MT were mainly in the cytoplasm of germinal cells, indicating a role in spermatogenesis and testis protection. After the cadmium (Cd) exposure, the testis presents abnormal morphology and MT mRNA expression, both of which indicate a sensitive response of testis MT to Cd. Therefore, we suggest that MTs play an important role in spermatogenesis and testes protection against Cd toxicity in B. pectinirostris.

Expression pattern of heat shock protein 90AB (HSP90AB) and stress-inducible protein 1 (Stip1) during spermatogenesis of mudskipper Boleophthalmus pectinirostris.

Heat shock protein 90 (HSP90), which functions as a molecular chaperone, plays an important role in reproduction and cellular defense. Among the HSP90 family, HSP90AB is an important isoform. Stress-inducible protein 1 (Stip1) acts as a co-chaperone that mediates interactions with HSP90 and appears to be a player in spermatogenesis and stress response. However, the functions of both HSP90 and Stip1 during spermatogenesis and heat stress response in Boleophthalmus pectinirostris remain unknown. We investigated mRNA expression patterns of HSP90AB and Stip1 under heat stress conditions. The results showed that mRNA levels of HSP90AB and Stip1 were significantly upregulated in the gill and liver tissues, indicating that HSP90AB and Stip1 appear to play roles in protection against heat stress. Then we cloned the complete cDNA of HSP90AB and Stip1, which have product lengths of 2546 and 2652 bp, respectively. The predicted secondary and tertiary structures of B. pectinirostris. HSP90AB and Stip1 contain conserved domains. We investigated the expression patterns of HSP90AB and Stip1 in different tissues by quantitative real-time polymerase chain reaction, HSP90AB and Stip1 were found to be ubiquitously expressed in all major tissues and exhibited varying expression levels, indicating that HSP90AB and Stip1 have conserved biological functions. HSP90AB and Stip1 were found to be strongly expressed in the testis, indicating their special roles in this organ. We also tracked the dynamic locations of germinal cells using in situ hybridization. Results from in situ hybridization and immunofluorescence localization showed that both mRNA transcripts and proteins of HSP90AB and Stip1 were ubiquitously expressed in spermatocytes, spermatids, and spermatozoa, indicating that HSP90AB and Stip1 are both involved in spermatogenesis.

Dynamic transcription and expression patterns of KIF3A and KIF3B genes during spermiogenesis in the shrimp, Palaemon carincauda.

Spermiogenesis is a highly ordered and complex process in the male germ cell differentiation. The microtubule-based motor proteins KIF3A and KIF3B are required for the progression of the stages of spermiogenesis. In this study, the main goal was to determine whether KIF3A and KIF3B have a key role in spermiogenesis in Palaemon carincauda. The complete cDNA of KIF3A/3B from the testis of P. carincauda was cloned by using PCR and rapid amplification of cDNA ends (RACE). The predicted secondary and tertiary structures of KIF3A/3B contained three domains which were the: a) head region, b) stalk region, and c) tail region. Real-time quantitative PCR (qPCR) results revealed that KIF3A and KIF3B mRNAs were obtained for all the tissues examined, with the greatest gene expression in the testis. In situ hybridization indicated the KIF3A and KIF3B mRNAs were distributed in the periphery of the nuclear in the early spermatid of spermiogenesis. In the middle and late spermatid stages, KIF3A and KIF3B mRNAs were gradually upregulated and assembled to one side where acrosome biogenesis begins. In the mature sperm, KIF3A and KIF3B mRNAs were distributed in the acrosome cap and spike. Immunofluorescence studies indicated that KIF3A, tubulin, mitochondria, and Golgi were co-localized in different stages during spermiogenesis in P. carincauda. The temporal and spatial gene expression dynamics of KIF3A/3B indicate that KIF3A and KIF3B proteins may be involved in acrosome formation and nucleus shaping. Moreover, these proteins can transport the mitochondria and Golgi that facilitate acrosome formation in P. carincauda.

Molecular cloning, characterization and expression analysis of metallothionein in the liver of the teleost Acrossocheilus fasciatus exposed to cadmium chloride.

Metallothionein (MT) has a characteristic molecular structure with a cysteine-rich content. This unique structure provides metal-binding and redox capabilities and promoting metal homeostasis and detoxification in living animals. In order to evaluate the effects of cadmium (Cd) on hepatic MT expression in the liver of Acrossocheilus fasciatus, we obtained the complete cDNA of the A. fasciatus liver MT for the first time. The MT cDNA contains a 605-bp sequence, which codes for 60 amino acids. Protein alignment showed that the similarity between MT protein sequences of A. fasciatus and those of other vertebrates (especially teleosts) was very high and a cysteine residue structure was also conserved. MT was detected in the liver, kidney, gill, testis, muscle, spleen, heart and brain tissues of A. fasciatus by tissue-specific expression analysis. After Cd exposure, Cd/hemoglobin saturation assay, immunohistochemistry and reverse-transcription quantitative PCR (RT-qPCR) were used to describe MT expression in liver tissue. These techniques indicate a sensitive response by liver MT to Cd exposure. The results suggest that A. fasciatus MT may play an important role in the detoxification processes in the liver, and also would be a useful biomarker for monitoring metal pollution in aquatic environments. In addition, A. fasciatus could be regarded as one candidate for a model species for bony fishes in ecotoxicology.

Glutathione peroxidase 1 expression, malondialdehyde levels and histological alterations in the liver of Acrossocheilus fasciatus exposed to cadmium chloride.

Cadmium (Cd) is known as a widespread pollutant in aquatic environment. The accumulation of reactive oxygen species (ROS) is attributed to Cd exposure, which may affect the growth, development and physiological metabolism of aquatic organisms. In response to these unfavorable damages, antioxidant systems have been developed to protect against oxidative stress. In this study, we investigated the expression pattern of glutathione peroxidase 1 genes (GPx-1a and GPx-1b) in the liver of Acrossocheilus fasciatus after Cd administration. Total RNA extraction, reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were performed in order to clone the A. fasciatus GPx-1a and GPx-1b full-length cDNA sequences and partial fragment of ß-actin cDNA from the liver for the first time. Tissue-specific expression analysis proved that GPx-1 genes were widely expressed in the liver, kidney, gill, testis, muscle, spleen, heart and brain. The changes of GPx-1 mRNA and malondialdehyde (MDA) levels in the liver treated with Cd were measured. In addition, the acute toxic effects of Cd on the microstructure of the liver were studied using light microscopy. These results suggest that GPx-1, MDA and liver histology which represent molecular, biochemical and histological levels, can be used as potential biomarkers to monitor Cd pollution. The overall findings also highlight the potential use of those three bio-indicators combined together as a multi-level tool (molecular, biochemical and histological levels) when monitoring Cd contamination and other possible exogenetic pollutants in aquatic environment.

Molecular cloning, expression pattern, and chemical analysis of heat shock protein 70 (HSP70) in the mudskipper Boleophthalmus pectinirostris: Evidence for its role in regulating spermatogenesis.

Heat shock protein 70 (HSP70) is molecular chaperone that is important for reproductive biological processes. In this study, a full length HSP70 from the mudskipper (Boleophthalmus pectinirostris) was characterized. It was found to contain: a 108 bp 5'-untranslated region, a 208 bp 3'-untranslated region, and a 1953 bp open reading frame, which encodes a protein of 650 amino acids with a theoretical molecular weight of 71.1 kDa and an isoelectric point of 5.17. RT-PCR analysis revealed that HSP70 was ubiquitously expressed in all major tissues with differential expression levels. This suggests that HSP70 has vital and conserved biological functions. HSP70 was localized mainly in the cytoplasm of germinal cells, indicating an important role of this protein during spermatogenesis. In response to heat stress, the testes presented abnormal morphology in connective tissues, in which HSP70 immunoreactivity was not observed. HSP70 mRNA expression in the gill, liver, and testes was significantly increased, which suggests that HSP70 plays an important role in protection against heat stress.