Tunable Surface Wettability via Terahertz Electrowave Controlled Vicinal Subnanoscale Water Layer.

Achieving timely, reversible, and long-range remote tunability over surface wettability is highly demanded across diverse fields, including nanofluidic systems, drug delivery, and heterogeneous catalysis. Herein, using molecular dynamic simulations, we show, for the first time, a theoretical design of electrowetting to achieve remotely controllable surface wettability via using a terahertz wave. The key idea driving the design is the unique terahertz collective vibration identified in the vicinal subnanoscale water layer, which is absent in bulk water, enabling efficient energy transfer from the terahertz wave to the rotational motion of the vicinal subnanoscale water layer. Consequently, a frequency-specific alternating terahertz electric field near the critical strength can significantly affect the local hydrogen-bonding network of the contact water layer on the solid surface, thereby achieving tunable surface wettability.

The function of the cytoplasmic dynein light chain PTKM23 in the transport of PTSMAD2 during spermatogenesis in Portunus trituberculatus.

Cytoplasmic dynein participates in transport functions and is essential in spermatogenesis. KM23 belongs to the dynein light chain family. The TGFß signaling pathway is indispensable in spermatogenesis, and Smad2 is an important member of this pathway. We cloned PTKM23 and PTSMAD2 from Portunus trituberculatus and measured their expression during spermatogenesis. PTKM23 may be related to cell division, acrosome formation and nuclear remodeling, and PTSMAD2 may participate in regulating the expression of genes related to spermatogenesis. We assessed the localization of PTKM23 with PTDHC and α-Tubulin, and the results suggested that PTKM23 functions in intracellular transport during spermatogenesis. We knocked down PTKM23 in vivo, and the expression of p53, B-CATAENIN and CYCLIN B decreased significantly, further suggesting a role of PTKM23 in transport and cell division. The localization of PTDIC with α-Tubulin and that of PTSMAD2 with PTDHC changed after PTKM23 knockdown. We transfected PTKM23 and PTSMAD2 into HEK-293 T cells and verified their colocalization. These results indicate that PTKM23 is involved in the assembly of cytoplasmic dynein and microtubules during spermatogenesis and that PTKM23 mediates the participation of cytoplasmic dynein in the transport of PTSMAD2 during spermatogenesis. This study provides a theoretical molecular biological basis for the breeding of P. trituberculatus.

The relationship between kidney disease and mitochondria: a bibliometric study.

BACKGROUND: Due to its highly reabsorptive function, the kidney is a mitochondria-dependent organ. Research on the association between mitochondria and kidney disease has always been a serious focus of researchers, with many publications. Bibliometrics is a secondary analysis of published literature that extracts relevant information to gain insights into hotspots and trends in the field. Through bibliometric analysis, we aimed to understand the development trends and hotspots in the field of research on the association between kidney disease and mitochondria. METHOD: Three bibliometric mapping tools (Biblimetrix R Package, VOS Viewer, CiteSpace) were used to provide an overview of the literature and analyze the co-occurrence of keywords and reference citations. RESULTS: A total of 2672 relevant research articles were included. The co-occurrence network identified three clusters related to the association between mitochondria and kidney disease, including experimental methods, research mechanisms, and disease phenotypes. We found that research in this field has shifted from disease-level studies to mechanism-based studies, with the most prominent disease being diabetic nephropathy and the most prominent pathogenic mechanism being related to mitochondrial ROS production. CONCLUSION: The bibliometric analysis provided a comprehensive understanding of the progress of research on the role of mitochondria in kidney disease, enriching the review literature in this field.

Expression and Functional Analysis of the Metallothionein and Metal-Responsive Transcription Factor 1 in Phascolosoma esculenta under Zn Stress.

Metallothioneins (MTs) are non-enzymatic metal-binding proteins widely found in animals, plants, and microorganisms and are regulated by metal-responsive transcription factor 1 (MTF1). MT and MTF1 play crucial roles in detoxification, antioxidation, and anti-apoptosis. Therefore, they are key factors allowing organisms to endure the toxicity of heavy metal pollution. Phascolosoma esculenta is a marine invertebrate that inhabits intertidal zones and has a high tolerance to heavy metal stress. In this study, we cloned and identified MT and MTF1 genes from P. esculenta (designated as PeMT and PeMTF1). PeMT and PeMTF1 were widely expressed in all tissues and highly expressed in the intestine. When exposed to 16.8, 33.6, and 84 mg/L of zinc ions, the expression levels of PeMT and PeMTF1 in the intestine increased first and then decreased, peaking at 12 and 6 h, respectively, indicating that both PeMT and PeMTF1 rapidly responded to Zn stress. The recombinant pGEX-6p-1-MT protein enhanced the Zn tolerance of Escherichia coli and showed a dose-dependent ABTS free radical scavenging ability. After RNA interference (RNAi) with PeMT and 24 h of Zn stress, the oxidative stress indices (MDA content, SOD activity, and GSH content) and the apoptosis indices (Caspase 3, Caspase 8, and Caspase 9 activities) were significantly increased, implying that PeMT plays an important role in Zn detoxification, antioxidation, and anti-apoptosis. Moreover, the expression level of PeMT in the intestine was significantly decreased after RNAi with PeMTF1 and 24 h of Zn stress, which preliminarily proved that PeMTF1 has a regulatory effect on PeMT. Our data suggest that PeMT and PeMTF1 play important roles in the resistance of P. esculenta to Zn stress and are the key factors allowing P. esculenta to endure the toxicity of Zn.

Transcriptome profiling and differential expression analysis of altered immune-related genes in goldfish (Carassius auratus) infected with Aeromonas hydrophila.

Goldfish (Carassius auratus) have been employed as a model organism to investigate the innate immune system and host-pathogen interactions. A Gram-negative bacterium called Aeromonas hydrophila has been found to cause mass mortality due to infection in a wide variety of fish species in the aquatic system. In this study, damages in Bowman's capsule, inflammatory tubular (proximal and distilled convoluted) structure, and glomerular necrosis were observed in A. hydrophila-infected head kidney of goldfish. To increase the better understanding of immune mechanisms of host defense against A. hydrophila, we performed a transcriptome analysis in head kidney of goldfish at 3 and 7 days of post-infection (dpi). Comparing to the control group, 4638 and 2580 differentially expressed genes (DEGs) were observed at 3 and 7 dpi, respectively. The DEGs were subsequently enriched in multiple immune-related pathways including Protein processing in endoplasmic reticulum, Insulin signaling pathway, and NOD-like receptor signaling pathway. The expression profile of immune-related genes such as TRAIL, CCL19, VDJ recombination-activating protein 1-like, Rag-1, and STING was validated by qRT-PCR. Furthermore, the levels of immune-related enzyme (LZM, AKP, SOD, and CAT) activities were examined at 3 and 7 dpi. The knowledge gained from the current study will be helpful for better understanding of early immune response in goldfish after A. hydrophila challenge, which will aid in future research on prevention strategies in teleost.

The Effect of Prohibitins on Mitochondrial Function during Octopus tankahkeei Spermiogenesis.

Mitochondria are essential for spermiogenesis. Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins that act as scaffolds in the inner mitochondrial membrane. In this study, we analyzed the molecular structure and dynamic expression characteristics of Ot-PHBs, observed the colocalization of Ot-PHB1 with mitochondria and polyubiquitin, and studied the effect of phb1 knockdown on mitochondrial DNA (mtDNA) content, reactive oxygen species (ROS) levels, and apoptosis-related gene expression in spermatids. Our aim was to explore the effect of Ot-PHBs on mitochondrial function during the spermiogenesis of Octopus tankahkeei (O. tankahkeei), an economically important species in China. The predicted Ot-PHB1/PHB2 proteins contained an N-terminal transmembrane, a stomatin/prohibitin/flotillin/HflK/C (SPFH) domain (also known as the prohibitin domain), and a C-terminal coiled-coil domain. Ot-phb1/phb2 mRNA were widely expressed in the different tissues, with elevated expression in the testis. Further, Ot-PHB1 and Ot-PHB2 were highly colocalized, suggesting that they may function primarily as an Ot-PHB compiex in O. tankahkeei. Ot-PHB1 proteins were mainly expressed and localized in mitochondria during spermiogenesis, implying that their function may be localized to the mitochondria. In addition, Ot-PHB1 was colocalized with polyubiquitin during spermiogenesis, suggesting that it may be a polyubiquitin substrate that regulates mitochondrial ubiquitination during spermiogenesis to ensure mitochondrial quality. To further investigate the effect of Ot-PHBs on mitochondrial function, we knocked down Ot-phb1 and observed a decrease in mtDNA content, along with increases in ROS levels and the expressions of mitochondria-induced apoptosis-related genes bax, bcl2, and caspase-3 mRNA. These findings indicate that PHBs might influence mitochondrial function by maintaining mtDNA content and stabilizing ROS levels; in addition, PHBs might affect spermatocyte survival by regulating mitochondria-induced apoptosis during spermiogenesis in O. tankahkeei.

Optimization of Sperm Cryopreservation Formulation in Portunus trituberculatus.

Portunus trituberculatus is a very important marine economic species, and its aquaculture industry has been developing rapidly. However, the phenomenon of marine wild capture of P. trituberculatus and germplasm degradation has become increasingly serious. It is necessary to develop the artificial farming industry and carry out germplasm resource protection, for which sperm cryopreservation technology is an effective method. This research compared three methods (mesh-rubbing, trypsin digestion, and mechanical grinding) for acquiring free sperm, and the best method was mesh-rubbing. Then, the optimal cryopreservation conditions were selected, and the optimal formulation was sterile calcium-free artificial seawater, the optimal cryoprotectant was 20% glycerol, and the best equilibrium time was 15 min at 4 °C. The optimal cooling program was suspending the straws at 3.5 cm on the liquid nitrogen surface for 5 min and then storing them in liquid nitrogen. Finally, the sperm were thawed at 42 °C. However, the expression of sperm-related genes and the total enzymatic activities of frozen sperm were significantly decreased (p < 0.05), which showed that sperm cryopreservation damaged the sperm. Our study improves the sperm cryopreservation technology and the yield of aquaculture in P. trituberculatus. Additionally, the study provides a certain technical basis for the establishment of a sperm cryopreservation library of crustaceans.

Expression Dynamics Indicate Potential Roles of KIF17 for Nuclear Reshaping and Tail Formation during Spermiogenesis in Phascolosoma esculenta.

Kinesin family member17 (KIF17), a homologous dimer of the kinesin-2 protein family, has important microtubule-dependent and -independent roles in spermiogenesis. Little is known about KIF17 in the mollusk, Phascolosoma esculenta, a newly developed mariculture species in China. Here, we cloned the open reading frame of Pe-kif17 and its related gene, Pe-act, and performed bioinformatics analysis on both. Pe-KIF17 and Pe-ACT are structurally conserved, indicating that they may be functionally conserved. The expression pattern of kif17/act mRNA performed during spermiogenesis revealed their expression in diverse tissues, with the highest expression level in the coelomic fluid of P. esculenta. The expressions of Pe-kif17 and Pe-act mRNA were relatively high during the breeding season (July-September), suggesting that Pe-KIF17/ACT may be involved in spermatogenesis, particularly during spermiogenesis. Further analysis of Pe-kif17 mRNA via fluorescence in situ hybridization revealed the continuous expression of this mRNA during spermiogenesis, suggesting potential functions in this process. Immunofluorescence showed that Pe-KIF17 co-localized with α-tubulin and migrated from the perinuclear cytoplasm to one side of the spermatid, forming the sperm tail. Pe-KIF17 and Pe-ACT also colocalized. KIF17 may participate in spermiogenesis of P. esculenta, particularly in nuclear reshaping and tail formation by interacting with microtubule structures similar to the manchette. Moreover, Pe-KIF17 with Pe-ACT is also involved in nuclear reshaping and tail formation in the absence of microtubules. This study provides evidence for the role of KIF17 during spermiogenesis and provides theoretical data for studies of the reproductive biology of P. esculenta. These findings are important for spermatogenesis in mollusks.

The Functions of Pt-DIC and Pt-Lamin B in Spermatogenesis of Portunus trituberculatus.

Cytoplasmic Dynein is a multiple-subunit macromolecular motor protein involved in the transport process of cells. The Dynein intermediate chain (DIC) is one of the subunits of Dynein-1. In our previous studies, we showed that Pt-DIC may play an important role in the nuclear deformation of spermiogenesis in Portunus trituberculatus. Lamin B is essential for maintaining nuclear structure and functions. Surprisingly, Pt-Lamin B was expressed not only in the perinucleus but also in the pro-acrosome during spermiogenesis in P. trituberculatus. Studies have also shown that Dynein-1 can mediate the transport of Lamin B in mammals. Thus, to study the relationship of Pt-DIC and Pt-Lamin B in the spermatogenesis of P. trituberculatus, we knocked down the Pt-DIC gene in P. trituberculatus by RNAi. The results showed that the distribution of Pt-DIC and Pt-Lamin B in spermiogenesis was abnormal, and the colocalization was weakened. Moreover, we verified the interaction of Pt-DIC and Pt-Lamin B via coimmunoprecipitation. Therefore, our results suggested that both Pt-DIC and Pt-Lamin B were involved in the spermatogenesis of P. trituberculatus, and one of the functions of Dynein-1 is to mediate the transport of Lamin B in the spermiogenesis of P. trituberculatus.

Characterization of Copper/Zinc Superoxide Dismutase Activity on Phascolosoma esculenta (Sipuncula: Phascolosomatidea) and Its Protection from Oxidative Stress Induced by Cadmium.

Phascolosoma esculenta, an economically important species inhabiting the high tide areas of the intertidal zone, is particularly sensitive to water pollution. Considering its potential as a bioindicator, studies on the ecotoxicology of P. esculenta are imperative. The toxic effects of cadmium (Cd) were analyzed by exposing P. esculenta to different concentrations of Cd (6, 24, 96 mg/L). In this study, the changes in the antioxidative indexes of total superoxide dismutase (T-SOD), glutathione s-transferase (GST), reduced glutathione (GSH), and microscale malondialdehyde (MDA) were recorded. Copper/zinc superoxide dismutase (Cu/Zn SOD) is one of the most important free radical scavenging members. To reveal the antioxidative function of P. esculenta, an important member of the antioxidative system, designated Pe-Cu/Zn SOD, was cloned and analyzed. Phylogenic analysis revealed that Pe-Cu/Zn SOD was located in the invertebrate evolutionary branch of intracellular Cu/Zn SOD (icCu/Zn SOD). The quantitative real-time polymerase chain reaction results showed that Pe-Cu/Zn SOD messenger ribonucleic acid was widely expressed in all tissues examined. The highest expression levels in coelomic fluid after Cd exposure indicated its function in the stress response. Using a prokaryotic expression system, we obtained a Pe-Cu/Zn SOD recombinant protein, which enhanced the heavy metal tolerance of Escherichia coli. In vivo assays also confirmed that the Pe-Cu/Zn SOD recombinant protein had an antioxidative and free radical scavenging ability. A Cd toxicity experiment, in which purified Pe-Cu/Zn SOD protein was injected into the body cavities of P. esculenta, showed that the reactive oxygen species content in the coelomic fluid of the experimental group was significantly lower compared with the control group. These results suggest that Pe-Cu/Zn SOD played a role in Cd detoxification by chelating heavy metal ions and scavenging reactive oxygen free radicals, and that P. esculenta could be used as a bioindicator to evaluate heavy metal pollution.

Characterization of Mitochondrial Prohibitin in Opsariichthys bidens and Its Potential Functions in Spermatogenesis.

Spermatogenesis is the intricate and coordinated process by which spermatogonia develop into haploid differentiated spermatozoa. Mitochondria are essential for spermatogenesis, and prohibitin (PHB) is closely associated with mitochondrial structure and function during spermatogenesis. Although PHB has been implicated in spermatogenesis in some taxa, its roles in Opsariichthys bidens have not been determined. In this study, the expression patterns and potential functions of PHB in spermatogenesis in O. bidens were characterized using histological microscopic observations, PCR cloning, real-time quantitative PCR (qPCR), Western blotting (WB) and immunofluorescence (IF). The full-length cDNA of Ob-phb was 1500 bp encoding 271 amino acids. A sequence alignment demonstrated that the PHB protein is conserved among different animals. qPCR revealed that phb mRNA is widely distributed in O. bidens and highly expressed in the testes at stages IV and V. WB revealed that Ob-PHB is located in the mitochondria of testes. IF revealed the colocalization of PHB signals and mitochondria. Signals were detected around nuclei in spermatogonia and spermatocytes, gradually moving to the tail region during spermiogenesis, and finally aggregating in the midpiece. These results indicate that Ob-PHB was expressed in the mitochondria during spermatogenesis. In addition, this study proposed Ob-PHB may participate in the degradation of mitochondria and cell differentiation during spermatogenesis.

Mitochondrial Features and Expressions of MFN2 and DRP1 during Spermiogenesis in Phascolosoma esculenta.

Mitochondria can fuse or divide, a phenomenon known as mitochondrial dynamics, and their distribution within a cell changes according to the physiological status of the cell. However, the functions of mitochondrial dynamics during spermatogenesis in animals other than mammals and fruit flies are poorly understood. In this study, we analyzed mitochondrial distribution and morphology during spermiogenesis in Sipuncula (Phascolosoma esculenta) and investigated the expression dynamics of mitochondrial fusion-related protein MFN2 and fission-related protein DRP1 during spermiogenesis. The mitochondria, which were elliptic with abundant lamellar cristae, were mainly localized near the nucleus and distributed unilaterally in cells during most stages of spermiogenesis. Their major axis length, average diameter, cross-sectional area, and volume are significantly changed during spermiogenesis. mfn2 and drp1 mRNA and proteins were most highly expressed in coelomic fluid, a spermatid development site for male P. esculenta, and highly expressed in the breeding stage compared to in the non-breeding stage. MFN2 and DRP1 expression levels were higher in components with many spermatids than in spermatid-free components. Immunofluorescence revealed that MFN2 and DRP1 were consistently expressed and that MFN2 co-localizes with mitochondria during spermiogenesis. The results provide evidence for an important role of mitochondrial dynamics during spermiogenesis from morphology and molecular biology in P. esculenta, broadening insights into the role of mitochondrial dynamics in animal spermiogenesis.

The potential function of KIF17 in large yellow croaker (Larimichthys crocea) spermatid remodeling: molecular characterization and expression pattern during spermiogenesis.

KIF17, which belongs to the kinesin-2 protein family, plays an indispensable role in mammalian spermiogenesis. However, the role of KIF17 in fish spermatid remodeling during spermiogenesis remains poorly understood. Therefore, we aimed to study the role of KIF17 in spermatid remodeling during Larimichthys crocea (L. crocea) spermiogenesis. The kif17 cDNA sequence, 3247 bp in length, was cloned from L. crocea testis, which consisted of a 347-bp 5'-untranslated region (UTR), 413-bp 3' -UTR, and 2487-bp open reading frame. Bioinformatic analyses revealed that KIF17 obtained from L. crocea (Lc-KIF17) exhibited a high sequence identity compared with those from other teleosts and possessed the structural features of other kinesin-2 proteins. Based on structural similarity, we speculate that the role of Lc-KIF17 may be similar to that of KIF17 in other animals. Lc-kif17 mRNA was diffusely expressed in L. crocea tissues and was highly expressed in the testis, especially at stage IV testicular development. Immunofluorescence analysis revealed that Lc-KIF17 signals colocalized with ß-tubulin signals and migrated from the perinuclear cytoplasm to the side of the nucleus where the tail forms during spermiogenesis. These findings revealed that KIF17 may be involved in L. crocea spermiogenesis. In particular, KIF17 may participate in spermatid remodeling by interacting with perinuclear microtubules during L. crocea spermiogenesis. Collectively, this study contributes to an improved understanding of the mechanism underlying L. crocea spermiogenesis and provides a basis for further research on L. crocea reproduction and development.

Expression and functional analysis of cytoplasmic dynein during spermatogenesis in Portunus trituberculatus.

The mechanism of acrosome formation in the crab sperm is a hot topic in crustacean reproduction research. Dynein is a motor protein that performs microtubule-dependent retrograde transport and plays an essential role in spermatogenesis. However, whether cytoplasmic dynein participates in acrosome formation in the crab sperm remains poorly understood. In this study, we cloned the cytoplasmic dynein intermediate chain gene (Pt-DIC) from Portunus trituberculatus testis. Pt-DIC is composed of a p150glued-binding domain, a dynein light chain (DLC)-binding domain, and a dynein heavy chain (DHC)-binding domain. The Pt-DIC gene is widely expressed in different tissues, showing the highest expression in the testis, and it is expressed in different stages of spermatid development, indicating important functions in spermatogenesis. We further observed the colocalization of Pt-DIC and Pt-DHC, Pt-DHC and tubulin, and Pt-DHC and GM130, and the results indicated that cytoplasmic dynein may participate in nuclear shaping and acrosome formation via vesicle transport. In addition, we examined the colocalization of Pt-DHC and a mitochondrion (MT) tracker and that of Pt-DHC and prohibitin (PHB). The results indicated that cytoplasmic dynein participated in mitochondrial transport and mitochondrial degradation. Taken together, these results support the hypothesis that cytoplasmic dynein participates in acrosome formation, nuclear shaping, and mitochondrial transport during spermiogenesis in P. trituberculatus. This study will provide valuable guidance for the artificial fertilization and reproduction of P. trituberculatus.

Impact of chemotherapy and radiotherapy on the survival of elderly esophageal cancer patients undergoing surgery: a SEER database analysis.

BACKGROUND: Esophageal cancer (EC) is a common and lethal carcinoma; however, the effectiveness and feasibility of the chemo- and radio-therapy (CRT) for the elderly patients (≥ 70 years) with surgery have not been fully discussed. The purpose of this study was to investigate the potential effect of CRT on the prognosis. METHODS: A total of 1085 patients (534 CRT patients vs. 551 non-CRT patients) from 1998 to 2016 were collected from the Surveillance, Epidemiology, and End Results database according to the inclusion and exclusion criteria. Using the competing risk regression and survival analysis, an overall estimation of the effectiveness of CRT was performed on a well-balanced cohort via performing propensity score matching. Then, the specific impact of CRT on high- (n = 557) and low-risk (n = 528) cohorts derived from the nomogram's risk quantification for every patient were further evaluated respectively. Additionally, the advantages of the nomogram model and the conventional tumor, node, metastasis (TNM, 6th revision) staging system were compared. RESULTS: A better survival outcome was observed among patients receiving both surgery and CRT than those who underwent surgery alone (HR: 0.55, 95% CI 0.45-0.68, P < 0.001), especially for those with tumors characterized by poor differentiation, large tumor size, advanced T staging, lymphatic metastasis, and distant metastasis (HR: 0.48, 95% CI 0.39-0.59, P < 0.001), while no benefit was observed among the low-risk patients. Furthermore, the newly established nomogram model might be better than the TNM (6th revision) staging system but more data needed. CONCLUSION: Aggressive treatments, such as surgery, chemotherapy, and radiotherapy, were considered effective for selected elderly patients with EC according to the newly established nomogram model.

Molecular cloning, characterisation and expression analysis of the vitellogenin genes vtgAo1 and vtgC during ovarian development in Chinese hook snout carp Opsariichthys bidens.

Vitellogenesis is essential for oocyte maturation. Vitellogenin (Vtg), a yolk precursor protein, plays an important role in oogenesis and vitellogenesis. Chinese hook snout carp Opsariichthys bidens is an economically important freshwater fish in China whose reproductive and developmental biology are not well understood. In this study, we undertook histological analysis to examine ovary development and oogenesis in O. bidens. The ovaries were divided into Stages II-V and oocytes were divided into perinuclear oocytes, cortical alveoli oocytes, vitellogenic oocytes and mature oocytes. Full-length cDNA sequences were cloned of two vtg genes from the liver of O. bidens, namely Ob-vtgAo1 and Ob-vtgC. Ob-vtgAo1 and Ob-vtgC cDNA are made up of 4136 and 4392 bases respectively and encode proteins containing 1335 and 1250 amino acids respectively. Ob-vtgAo1 contains three yolk protein domains: lipovitellin heavy chain (LvH), phosvitin (Pv) and lipovitellin light chain (LvL), whereas Ob-VtgC contains LvH and LvL, which are incomplete Vtgs. Ob-vtgAo1 and Ob-vtgC mRNA expression was significantly higher in the liver of O. bidens than in all other tissues. In oocytes of Stage II-III ovaries, yolk granules are almost absent and ovarian and hepatic Ob-vtgAo1 and Ob-vtgC expression is low. At Stage IV, the oocyte is filled with yolk granules and ovarian and hepatic Ob-vtgAo1 and Ob-vtgC expression is significantly increased. Collectively, these findings help us better understand vitellogenesis in O. bidens.

The effects of extenders, cryoprotectants and conditions in two-step cooling method on Varicorhinus barbatulus sperm.

In this study, we developed an optimal cryopreservation procedure for Varicorhinus barbatulus sperm. To this end, we optimized (1) the types and dilution ratios of extenders; (2) types and final concentration of cryoprotectants; and (3) freezing conditions, including equilibration time, height above the surface of liquid nitrogen (LN), and the cooling times in the two-step cooling method. The optimum result was obtained when the sperm was diluted at a 1:9 ratio in D-17 with 10% methanol, equilibrated at 4 °C for 10 min, held at 7 cm above LN for 2 min, and finally stored in LN. After storage for 12 h in LN, the sperm was thawed in a water bath at 40 °C for 6s, the post-thaw sperm motility was 66.10 ± 7.12%, while the corresponding rate for fresh sperm was 87.08 ± 2.38%. Using computer-assisted sperm analysis, we found a significant decrease in the motility parameters of post-thaw sperm, especially the parameters related to velocity. To evaluate the effects of cryopreservation on the structural integrity of sperm, transmission electron microscopy and scanning electron microscopy were employed, which showed the defects in frozen sperm, including: abnormal heads, damaged plasma membranes, broken tails, and the disappearance of the mitochondrial internal crest. In addition, we determined the mitochondrial membrane potential to assess the functional integrity of frozen sperm. Our results showed a decrease in the mitochondrial function of frozen sperm. This procedure could be used alongside cryopreservation of V. barbatulus and supports its commercial-scale production and species conservation.

Effects of acute low temperature stress on the hormones and gene expression of glucocorticoid receptor of large yellow croaker Larimichthys crocea.

The neuroendocrine system of fish responds to low temperature via regulating hormones. To explore the adaptability of Larimichthys crocea to low temperature, the levels of the plasma cortisol, thyroid stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4), total cholesterol (TC), and glucose were determined after exposure to low temperature and during subsequent rewarming. Furthermore, the mRNA expression of the glucocorticoid receptor (GR) gene was analyzed under the stress. We found that the levels of the plasma cortisol, TSH, T3, glucose, and TC increased under the low temperature stress, suggesting that elevated hormones may be conducive to promoting the mobilization of the glucose and lipid in L. crocea exposed to low temperature. During the rewarming period, the plasma cortisol level decreased, whereas the T3 level was still significantly higher than that in the control group. Notably, the plasma T4 level was unaffected by the temperature changes. Furthermore, the sequence alignment and phylogenetic tree analysis revealed that the GR protein of L. crocea had high homology and a similar protein structure with those from other teleosts. Under the low temperature stress, the GR mRNA expression increased in the brain and head kidney, whereas it basically returned to the control level following rewarming. These findings revealed the changes of the hormones and the potential function of the GR gene in L. crocea following exposure to low temperature, providing some insights into breeding low temperature-resistant varieties of L. crocea.

Cloning, Functional Characterization and Response to Cadmium Stress of the Thioredoxin-like Protein 1 Gene from Phascolosoma esculenta.

Cadmium (Cd) is a heavy metal toxicant and is widely distributed in aquatic environments. It can cause excessive production of reactive oxygen species (ROS) in the organism, which in turn leads to a series of oxidative damages. Thioredoxin (Trx), a highly conserved disulfide reductase, plays an important role in maintaining the intracellular redox homeostasis in eukaryotes and prokaryotes. Phascolosoma esculenta is an edible marine worm, an invertebrate that is extensively found on the mudflats of coastal China. To explore the molecular response of Trx in mudflat organisms under Cd stress, we identified a new Trx isoform (Trx-like protein 1 gene) from P. esculenta for the first time, designated as PeTrxl. Molecular and structural characterization, as well as multiple sequence and phylogenetic tree analysis, demonstrated that PeTrxl belongs to the Trx superfamily. PeTrxl transcripts were found to be ubiquitous in all tissues, and the highest expression level occurred in the coelomic fluid. Exposure to three sublethal concentrations of Cd resulted in the upregulation and then downregulation of PeTrxl expression levels over time in coelomic fluid of P. esculenta. The significant elevation of PeTrxl expression after 12 and 24 h of Cd exposure at 6 and 96 mg/L, respectively, might reflect its important role in the resistance to Cd stress. Recombinant PeTrxl (rPeTrxl) showed prominent dose-dependent insulin-reducing and ABTS free radical-scavenging abilities. After exposure to 96 mg/L Cd for 24 h, the ROS level increased significantly in the coelomic fluid, suggesting that Cd induced oxidative stress in P. esculenta. Furthermore, the injection of rPeTrxl during Cd exposure significantly reduced the ROS in the coelomic fluid. Our data suggest that PeTrxl has significant antioxidant capacity and can protect P. esculenta from Cd-induced oxidative stress.

Characterisation, expression and possible functions of prohibitin during spermatogenesis in the silver pomfret Pampus argenteus.

Mitochondria play an important role in spermatogenesis, and some mitochondrial proteins are specifically related to this process. In this study we investigated the cytological characteristics of spermatogenic cells, including mitochondrial dynamics, during spermatogenesis in Pampus argenteus. In addition, we characterised the mitochondria-related protein prohibitin (PHB), which has been reported to play roles in mitochondrial dynamics and animal fertility. The full-length cDNA of the P. argenteus phb gene (Pa-phb) is 1687bp, including a 102-bp 5'-untranslated region (UTR), a 772-bp 3'-UTR and an 813-bp open reading frame encoding 271 amino acids. The predicted P. argenteus PHB protein (Pa-PHB) contains three functional domains (a transmembrane domain, an SPFH domain (the conserved region of stomatins, prohibitins, flotillins and HflK/C) and a coiled-coil domain) and exhibits high similarity with its homologue in other animals. The Pa-phb gene was widely expressed in all tissues examined, especially the liver and heart. We primarily focused on Pa-phb expression during spermatogenesis after observing the cytological features of male germ cells, and found that Pa-phb transcripts were detected throughout the course of development of male germ cells. Notably, we observed colocalised signals of Pa-PHB and mitochondria, which were distributed in the cytoplasm around the nucleus in spermatogonia, spermatocytes and early spermatids, tended to move to one side of the cell in middle spermatids and, finally, were colocalised in the sperm midpiece. These observations indicate that Pa-PHB is primarily localised in mitochondria during spermatogenesis, indicating that it has a role in mitochondria. Based on the results of this and previous studies regarding the essential roles of PHB in mitochondria and spermatogenesis in animals, we propose a functional model for PHB during spermatogenesis, including possible roles in the proliferation of spermatogonia and in the regulation of mitochondrial morphology and function in spermatogenic cells.