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 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.

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.

Vanadium compounds induced damage of human umbilical vein endothelial cells and the protective effect of berberine.

This study was conducted to investigate the damage caused by vanadium compounds and to explore the protective effects of berberine (BBR) in human umbilical vein endothelial cells (HUVECs). BBR is a biologically active small molecule found in Coptis rhizome, a remedy used in traditional Chinese medicine to treat diabetes. BBR has also been shown to lower blood glucose in diabetic patients. MTT assay was performed to observe the influence of bis(acetylacetonato)-oxidovanadium [VO(acac)2] or sodium metavanadate (NaVO3) and BBR on viability of HUVECs. The monolayer permeability of the HUVECs was assessed by measuring the transendothelial electrical resistance (TER). The endothelial nitric oxide synthase (eNOS) activity was detected by ELISA. Flow cytometry was performed to detect the generation of reactive oxygen species (ROS). The results showed that the viability of HUVECs was decreased by treatment with vanadium compounds 50-400 µM in a concentration-dependent manner, while 0.01-1 µM BBR effectively protected HUVECs from the inhibitory effects of vanadium compounds on cell viability. Also 100 and 200 µM VO(acac)2 induced high permeability and decreased eNOS activity in HUVECs. While 0.01-1 µM BBR showed no improvement in the permeability, and failed to reverse the VO(acac)2-induced changes of eNOS activity, but BBR treatment increased the eNOS activity in control cells. The addition of 200 µM VO(acac)2 significantly induced ROS generation in HUVECs, while 0.01 or 0.1 µM BBR reversed the change of ROS. In summary, BBR has protective effects in HUVECs damage induced by vanadium compounds, which is not mediated by eNOS, but related to reduced intracellular ROS.

Multiple vitellogenin genes (vtgs) in large yellow croaker (Larimichthys crocea): molecular characterization and expression pattern analysis during ovarian development.

The large yellow croaker (Larimichthys crocea) is a marine fish that is economically important to Chinese fisheries, and its reproductive and developmental biology have been extensively investigated. However, the molecular mechanism of oogenesis in L. crocea is not clear. Here, we investigated the multiple vitellogenin (Vtg) system in large yellow croaker. Three different vtg cDNA sequences, including vtgAa, vtgAb and vtgC, were cloned, which indicate the existence of multiple Vtg proteins in large yellow croaker (Lc-Vtgs). Subsequently, the vtg cDNA sequences and predicted Vtg protein structures were analysed, and Vtg protein structures were found to be highly conserved. To research the expression of vtgs during the development of the ovaries, we examined ovarian development and oogenesis by histological analysis. Four stages of ovary development - stages II, III, IV and V - were observed and their boundaries were defined. Soon afterwards, the expression of vtgs in the liver (known as the main site of Vtg synthesis in teleosts) and ovary were analysed. The expression of vtgs was detected in the two tissues. Interestingly, in the early stages of development (stages II and III), there is little or no generation of yolk granules and the expression of vtgs in the liver is low. However, in the late stages (stages IV and V), yolk granules are generated rapidly and the expression of vtgs is significantly increased in the liver. These results support the hypothesis that the Vtgs were synthetized by the liver, and absorbed by the growing oocytes to promote oogenesis in large yellow croaker. We also detected the presence of vtg mRNA in the liver cells and oocytes by in situ hybridization, which indicated that vths were expressed both in the liver and ovaries. Importantly, we found that the distribution of vtgAa and vtgAb mRNA was close to the sites of yolk granule formation in oocytes.

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.

KIFC1 and myosin Va: two motors for acrosomal biogenesis and nuclear shaping during spermiogenesis of Portunus trituberculatus.

To investigate the molecular mechanisms underlying the spermiogenesis of the swimming crab Portunus trituberculatus, full lengths of motor proteins KIFC1 and myosin Va were cloned by rapid-amplification of cDNA ends from P. trituberculatus testes cDNA, and their respective probes and specific antibodies were used to track their localization during sperm maturation. Antisense probes were designed from the gene sequences and used to detect the mRNA levels of each gene. According to the results of fluorescence in situ hybridization (FISH), the transcription of kifc1 and myosin Va began at the mid-stage of spermatids, with the kifc1 mRNA being most active at the location where the acrosome cap was formed and the myosin Va was more concentrated in the acrosome complex. Immunofluorescence results showed that KIFC1 and myosin Va were highly expressed in each stage of spermigenesis. In the early spermatids, they were randomly dispersed in the cytoplasm together with cytoskeletons. At the mid-stage, the motors were gathered above one side of the nucleus where the acrosome would later form. In the late spermatids and mature sperm, the KIFC1 was closely distributed in the perinuclear region, indicating its role in nucleus deformation. Myosin Va was distributed in the acrosome complex until sperm maturity. This suggests myosin Va's potential role in material transportation during acrosome formation and maturation. The above results provide a preliminary illustration of the essential roles of KIFC1 and myosin Va in the spermiogenesis of the swimming crab P. trituberculatus.

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.

The C-terminal kinesin motor KIFC1 may participate in nuclear reshaping and flagellum formation during spermiogenesis of Larimichthys crocea.

Spermatogenesis is a highly ordered process in the differentiation of male germ cells. Nuclear morphogenesis is one of the most fundamental cellular transformations to take place during spermatogenesis. These striking transformations from spermatogonia to spermatozoa are a result of phase-specific adaption of the cytoskeleton and its association with molecular motor proteins. KIFC1 is a C-terminal kinesin motor protein that plays an essential role in acrosome formation and nuclear reshaping during spermiogenesis in mammals. To explore its functions during the same process in Larimichthys crocea, we cloned and characterized the cDNA of a mammalian KIFC1 homolog (termed lc-KIFC1) from the total RNA of the testis. The 2481 bp complete lc-KIFC1 cDNA contained a 53 bp 5' untranslated region, a 535 bp 3' untranslated region, and a 1893 bp open reading frame that encoded a special protein of 630 amino acids. The predicted lc-KIFC1 protein possesses a divergent tail region, stalk region, and conserved carboxyl motor region. Protein alignment demonstrated that lc-KIFC1 had 73.2, 49.8, 49.3, 54.6, 56.5, 53.1, and 52.1% identity with its homologs in Danio rerio, Eriocheir sinensis, Octopus tankahkeei, Gallus gallus, Xenopus laevis, Mus musculus, and Homo sapiens, respectively. Tissue expression analysis revealed that lc-kifc1 mRNA was mainly expressed in the testis. The trend of lc-kifc1 mRNA expression at different growth stages of the testis showed that the expression increased first and then decreased, in the stage IV of testis, its expression quantity achieved the highest level. In situ hybridization and immunofluorescence results showed that KIFC1 was localized around the nucleus in early spermatids. As spermatid development progressed, the signals increased substantially. These signals peaked and were concentrated at one end of the nucleus when the spermatids began to undergo dramatic changes. In the mature sperm, the signal for KIFC1 gradually became weak and was mainly localized in the tail. In summary, evaluation of the expression pattern for lc-KIFC1 at specific stages of spermiogenesis has shed light on the potential functions of this motor protein in major cytological transformations. In addition, this study may provide a model for researching the molecular mechanisms involved in spermatogenesis in other teleost species, which will lead to a better understanding of the teleost fertilization process.

The potential function of prohibitin during spermatogenesis in Chinese fire-bellied newt Cynops orientalis.

Prohibitin proteins are multifunctional proteins located mainly at the inner membrane of mitochondria expressed in universal species. They play a vital role in mitochondria's function, cell proteolysis, senescence, apoptosis and as a substrate for ubiquitination. In this study, we used PCR cloning, protein and nucleotide acids alignment, protein structure prediction, western blot, in situ hybridization and immunofluorescence to study the characteristics of the prohibitin gene and the potential role of prohibitin in spermatogenesis and spermiogenesis processes in the Chinese fire-bellied newt Cynops orientalis. First, we cloned a 1452-bp full-length cDNA from the testis of Cynops orientalis. Second, we found that the 272 amino acids of prohibitin have a SPFH family domain. Thirdly, the western blots showed high expression of prohibitin in testis while the protein size was approximately 32 kDa. Fourthly, the results of in situ hybridization and immunofluorescence experiments showed that most of the prohibitins travelled with the mitochondria's migration in Cynops orientalis. The quantities of mRNA decreased as spermiogenesis proceeded, although the signals of prohibitins existed during the whole period of spermatogenesis and spermiogenesis. In the mature germ cells, the signals of prohibitins were weak and aggregated at the end of the cell. Finally, we discovered that the Sertoli cells had a large quantity of prohibitins and we made several assumptions of prohibitins' potential roles in those cells. This is the first time that the relationship between mitochondria and prohibitin in different stages of the sperm cells in Cynops orientalis has been examined, which also revealed that Sertoli cells have abundant prohibitins.

Mitochondrial prohibitin and its ubiquitination during crayfish Procambarus clarkii spermiogenesis.

Prohibitin (PHB), an evolutionarily conserved mitochondrial membrane protein, is associated with spermatogenesis and sperm quality control in mammals. It is identified as a substrate of ubiquitin and thus may function via a mitochondrial ubiquitin-proteasome pathway. In this study, we examined the localization of PHB during spermiogenesis of the macrura crustacean Procambarus clarkii. We traced phb mRNA's temporal and spatial expression pattern in spermiogenesis, and found its localization highly coherent with acrosome formation and nuclear shaping, two key events during crustacean spermiogenesis. We further detected the associations of PHB with mitochondria and ubiquitin using immunofluorescent staining. PHB was co-localized with mitochondria through spermiogenesis. PHB as well as mitochondria were co-localized with ubiquitin from the late stage of spermiogenesis, and the co-signals reached their peak in the mature sperm. The results raise the hypothesis that PHB is likely to function in nuclear shaping and acrosome formation in the spermiogenesis of P. clarkii. In addition, it might possess a more profound role in mediating mitochondrial ubiquitination. For the first time this study uncovers the role of PHB in the spermiogenesis of macrura crustacean species.

Characterization and expression pattern of p53 during spermatogenesis in the Chinese mitten crab Eriocheir sinensis.

p53, as a "Guardian of the Genome", plays an important role in cell cycle arrest, apoptosis, DNA repair and inhibition of angiogenesis in different tissues including testis. p53 gene and its protein perform many essential roles for mammalian spermatogenesis. To explore its functions during spermatogenesis in Eriocheir sinensis, we have cloned and sequenced the cDNA (1,218 bp) of p53 from the testis by degenerating primer PCR and rapid-amplification of cDNA ends. The protein alignment of p53 shows the conserved DNA binding domain, dimerization site and zinc binding site consisted of the predicted structures. Phylogenetic analysis revealed that p53 was more closer to Marsupenaeus japonicus and Tigriopus japonicus than other examined species. Tissue expression analysis of p53 mRNA showed p53 was distinctly expressed in accessory sexual gland, muscle, gill, heart, hepatopancreas and testis. In situ hybridization revealed that the p53 mRNA was weakly distributed around the nucleus, but stronger in the invaginated acrosomal tubule at the early stage. At the middle stage, p53 mRNA signal was increased than the early stage and the signal displayed dot-like pattern on the surface of cup-like nucleus. The signal on acrosomal cap is stronger than on the acrosomal tubule, despite acrosomal tubule signal was also distinct. At the late stage, the signal was still mainly located in acrosomal cap and acrosomal tubule. Sporadic signal were found surrounding the cup-like nucleus, but they were very weak. In the mature sperm, the signal was dramatically decreased. Even though the signal on cup-like nucleus and acrosomal tubule were distinct, they were weaker than those in middle stage. Based on these results, we concluded that p53 may play an important role in formation of acrosome biogenesis and nuclear shaping during spermiogenesis of E. sinensis.

New insights to the ubiquitin-proteasome pathway (UPP) mechanism during spermatogenesis.

Spermatogenesis is a complicated and highly ordered process which begins with the differentiation of spermatogonial stem cells and ends with the formation of mature sperm. After meiosis, several morphological changes occur during spermatogenesis. During spermatogenesis, many proteins and organelles are degraded, and the ubiquitin-proteasome pathway (UPP) plays a key role in the process which facilitates the formation of condensed sperm. UPP contains various indispensable components: ubiquitin, ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, ubiquitin ligase enzyme E3 and proteasomes. At some key stages of spermatogenesis, such as meiosis, acrosome biogenesis, and spermatozoa maturation, the ubiquitin-related components (including deubiquitination enzymes) exert positive and active functions. Generally speaking, deficient UPP will block spermatogenesis which may induce infertility at various degrees. Although ubiquitination during spermatogenesis has been widely investigated, further detailed aspects such as the mechanism of ubiquitination during the formation of midpiece and acrosome morphogenesis still remains unknown. The present review will overview current progress on ubiquitination during spermatogenesis, and will provide some suggestions for future studies on the functions of UPP components during spermatogenesis.

The SOX gene family: function and regulation in testis determination and male fertility maintenance.

The Sox (Sry-type HMG box) genes encode a group of proteins characterized by the existence of an SRY (sex-determining region on Y chromosome) box, a 79 amino acid motif that encodes an HMG (high mobility group) domain which can bind and bend DNA, which is the only part in SRY that is conserved between species. The Sox gene family functions in many aspects in embryogenesis, including testis development, CNS neurogenesis, oligodendrocyte development, chondrogenesis, neural crest cell development and other respects. The Sox gene family was originally identified through homology with Sry. The Sry gene is the mammalian testis-determining gene. It functions to open the testis determination pathway directly and close the ovary pathway indirectly. Sry and Sox9 are the most important two genes expressed during testis determination. Besides, researchers have found that Sox8 and Sox9 have functions in the male fertility maintenance after birth. In this review, information was evaluated from mouse or from human if not mentioned otherwise.

Characterization and expression pattern of KIFC1-like kinesin gene in the testis of the Macrobrachium nipponense with discussion of its relationship with structure lamellar complex (LCx) and acroframosome (AFS).

Spermiogenesis is a developmental process undergoing continuous differentiation to drive a diploid spermatogonium towards a haploid sperm cell. This striking transformation from spermatogonium to spermatozoa is made possible by the stage-specific adaption of cytoskeleton and associated molecular motor proteins. KIFC1 is a C-terminal kinesin motor found to boast essential roles in acrosome biogenesis and nuclear reshaping during spermiogenesis in rat. To explore its functions during the same process in Macrobrachium nipponense, we have cloned and sequenced the cDNA of a mammalian KIFC1 homologue (termed mn-KIFC1) from the total RNA of the testis. The 2,296 bp mn-KIFC1 cDNA contained a 87 bp 5' untranslated region, a 211 bp 3' untranslated region and a 1,998 bp open reading frame. Protein alignment demonstrated that mn-KIFC1 had 37.7, 58.7, 38.4, 37.2, 38.9 and 37.8% identity with its homologues in Salmo salar, Eriocheir sinensis, Homo sapiens, Mus musculus, Danio rerio and Xenopus laevis respectively. The phylogenetic tree revealed that mn-KIFC1 is most related to E. Sinensis KIFC1 among the examined species. Tissue expression analysis showed the presence of mn-KIFC1 in the testis, hepatopancreas, gill, muscle and heart. In situ hybridization showed that the mn-KIFC1 mRNA was localized at the periphery of the nuclear membrane and in the proacrosomal vesicle in early and middle spermatids. In late spermatids and spermatozoa, mn-KIFC1 was expressed in the acrosome and in the spike. In situ hybridization also indicated that KIFC1 works together with lamellar complex (LCx) and acroframosome (AFS) to drive acrosome formation and cellular transformation. LCx and AFS have both been previously proved to have essential roles during spermiogenesis in M. nipponense. In conclusion, the expression of mn-kifc1 at specific stages of spermiogenesis suggests a role in cellular transformations in M. nipponense.

Transport of Acrosomal Enzymes by KIFC1 via the Acroframosomal Cytoskeleton during Spermatogenesis in Macrobrachium rosenbergii (Crustacea, Decapoda, Malacostracea).

The spermatogenesis of crustaceans includes nuclear deformation and acrosome formation. The mechanism of acrosome formation is one focus of reproductive biology. In this study, Macrobrachium rosenbergii was selected as the research object to explore the mechanism of acrosome formation. The acrosome contains a large number of acrosomal enzymes for the hydrolysis of the egg envelope. How these acrosomal enzymes are transported to the acrosomal site after synthesis is the key scientific question of this study. The acroframosome (AFS) structure of caridean sperm has been reported. We hypothesized that acrosomal enzymes may be transported along the AFS framework to the acrosome by motor proteins. To study this hypothesis, we obtained the full-length cDNA sequences of Mr-kifc1 and Mr-Acrosin from the testis of M. rosenbergii. The Mr-kifc1 and Mr-Acrosin mRNA expression levels were highest in testis. We detected the distribution of Mr-KIFC1 and its colocalization with Mr-Acrosin during spermatogenesis by immunofluorescence. The colocalization of Mr-KIFC1 and microtubule indicated that Mr-KIFC1 may participate in sperm acrosome formation and nucleus maturation. The colocalization of Mr-KIFC1 and Mr-Acrosin indicated that Mr-KIFC1 may be involved in Acrosin transport during spermiogenesis of M. rosenbergii. These results suggest that Mr-KIFC1 may be involved in acrosomal enzymes transport during spermiogenesis of M. rosenbergii.

Mesenchymal Stem Cell Therapy for Patients with Ischemic Heart Failure- Past, Present, and Future.

The prevalence of Heart Failure (HF) has increased over time. Ischemic heart failure accounts for 50% of HF, which results from ischemic coronary heart diseases such as Myocardial Infarction (MI). Conventionally, reduction of cardiac load and revascularization partially increase cardiomyocyte survival and preserve cardiac functions. Nevertheless, how to improve cardiomyocyte rescue and prevent HF progression remain as challenges. Mesenchymal Stem Cells (MSCs) are multipotent stem cells that give rise to various lineages. The administration of MSCs promotes cardiomyocyte survival and improves cardiac functions in animal models of MI and patients with ischemic cardiomyopathy. However, after injection, MSCs persist for a very short time, indicating that the prolonged protective effects of MSCs on cardiomyocytes may be mediated by paracrine functions of MSCs, such as exosomes. In this review, we focus on MSC-derived exosomes in cardiomyocyte protection to facilitate future applications of exosomes in HF treatment.

Scavenging reactive oxygen species is a potential strategy to protect Larimichthys crocea against environmental hypoxia by mitigating oxidative stress.

The large yellow croaker (Larimichthys crocea), which is an economically important mariculture fish in China, is often exposed to environmental hypoxia. Reactive oxygen species (ROS) homeostasis is essential for the maintenance of normal physiological conditions in an organism. Direct evidence that environmental hypoxia leads to ROS overproduction is scarce in marine fish. Furthermore, the sources of ROS overproduction in marine fish under hypoxic stress are poorly known. In this study, we investigated the effects of hypoxia on redox homeostasis in L. crocea and the impact of impaired redox homeostasis on fish. We first confirmed that hypoxia drove ROS production mainly via the mitochondrial electron transport chain and NADPH oxidase complex pathways in L. crocea and its cell line (large yellow croaker fry (LYCF) cells). We subsequently detected a marked increase in the antioxidant systems of the fish. However, imbalance between the pro-oxidation and antioxidation systems ultimately led to excessive ROS and oxidative stress. Cell viability showed a remarkable decrease while oxidative indicators, such as malondialdehyde, protein carbonylation, and 8-hydroxy-2 deoxyguanosine, showed a significant increase after hypoxia, accompanied by tissue damage. N-acetylcysteine (NAC) reduced ROS levels, alleviated oxidative damage, and improved cell viability in vitro. Appropriate uptake of ROS scavengers (e.g., NAC and elamipretide Szeto-Schiller-31) and inhibitors (e.g., apocynin, diphenylene iodonium, and 5-hydroxydecanoate) may be effective at overcoming hypoxic toxicity. Our findings highlight previously unstudied strategies of hypoxic toxicity resistance in marine fish.

Hif-1α/Hsf1/Hsp70 signaling pathway regulates redox homeostasis and apoptosis in large yellow croaker ( Larimichthys crocea) under environmental hypoxia.

Oxygen is an essential molecule for animal respiration, growth, and survival. Unlike in terrestrial environments, contamination and climate change have led to the frequent occurrence of hypoxia in aquatic environments, thus impacting aquatic animal survival. However, the adaptative mechanisms underlying fish responses to environmental hypoxia remain largely unknown. Here, we used large yellow croaker ( Larimichthys crocea) and large yellow croaker fry (LYCF) cells to investigate the roles of the Hif-1α/Hsf1/Hsp70 signaling pathway in the regulation of cellular redox homeostasis, and apoptosis. We confirmed that hypoxia induced the expression of Hif-1α, Hsf1, and Hsp70 in vivo and in vitro. Genetic Hsp70 knockdown/overexpression indicated that Hsp70 was required for maintaining redox homeostasis and resisting oxidative stress in LYCF cells under hypoxic stress. Hsp70 inhibited caspase-dependent intrinsic apoptosis by maintaining normal mitochondrial membrane potential, enhancing Bcl-2 mRNA and protein expression, inhibiting Bax and caspase3 mRNA expression, and suppressing caspase-3 and caspase-9 activation. Hsp70 suppressed caspase-independent intrinsic apoptosis by inhibiting nuclear translocation of apoptosis-inducing factor (AIF) and disturbed extrinsic apoptosis by inactivating caspase-8. Genetic knockdown/overexpression of Hif-1α and dual-luciferase reporter assay indicated that Hif-1α activated the Hsf1 DNA promoter and enhanced Hsf1 mRNA transcription. Hsf1 enhanced Hsp70 mRNA transcription in a similar manner. In summary, the Hif-1α/Hsf1/Hsp70 signaling pathway plays an important role in regulating redox homeostasis and anti-apoptosis in L. crocea under hypoxic stress.