[Show abstract][Hide abstract] ABSTRACT: We used single-cell gel electrophoresis (SCGE) to detect the integrity of sperm DNA of the teleost large yellow croaker, Pseudosciaena crocea, cryopreserved with Cortland solution and a range of 5% to 30% DMSO concentrations in order to test how sperm cryopreservation affected the DNA stability of nuclei. Electrophoresis was conducted for 60 min at 130 mA and 15 V. The comet images were analyzed with software CometScore 1.5, and parameters such as comet length, tail length and percentage DNA in the tail were obtained. Then the comet rate and damage coefficient were calculated. Results demonstrated that there were no significant differences in motility, comet rate and damage coefficient between fresh sperm and cryopreserved sperm stored in 5%, 10%, 15% and 20% DMSO, while the sperm cryopreserved with 25% and 30% DMSO had a lower motility, higher comet length and damage coefficients than those of fresh sperm. There was a positive correlation between comet rate of cryopreserved sperm and the concentration of DMSO. Our results demonstrate that toxicity of the cryoprotectant is the main cause of DNA damage in cryopreserved sperm nuclei.
[Show abstract][Hide abstract] ABSTRACT: The Piwi genes have important role in stem cell development, gametogenesis and RNA interference in diverse organisms. So far, most of the studies have focused on the function of Piwis in vertebrates, but their function during spermiogenesis in invertebrates still remains largely unclear. In order to investigate the function of Piwis during spermiogenesis in the crab Portunus trituberculatus, we use RT-PCR and RACE to identify three Piwis complete cDNA sequence from the total RNA of the testis in P. trituberculatus. The deduced amino acid sequences of P. trituberculatus Piwi-1, Piwi-2 and Piwi-3 showed that each contains a well-conserved PAZ domain and PIWI domain. RT-PCR analyzed the tissue expression pattern of P. trituberculatus Piwi-1, Piwi-2 and Piwi-3 in the testis, heart, muscle, hepatopancreas and gill. All of the Piwis are found in germ cells of adult testis in P. trituberculatus by in situ hybridization, suggesting that these genes may play function during spermiogenesis in this species.
[Show abstract][Hide abstract] ABSTRACT: In this study, we investigated the gene sequence and characteristic of kifc1 in Sepiella maindroni through PCR and RACE technology. Our research aimed particularly at the spatio-temporal expression pattern of kifc1 in the developmental testis through in situ hybridization. The particular role of kifc1 in the spermatogenesis of S. maindroni was our particular interest. Based on multiple protein sequence alignments of KIFC1 homologues, kifc1 gene from the testis of S. maindroni was identified, which consisted of 2432bp including a 2109 in-frame ORF corresponding to 703 continuous amino acids. The encoded polypeptide shared highest similarity with Octopus tankahkeei. Through the prediction of the secondary and tertiary structure, the motor domain of KIFC1 was conserved at the C-terminal, having putative ATP-binding and microtubule-binding motifs, while the N-terminal was more specific to bind various cargoes for cellular events. The stalk domain connecting between the C-terminal and N-terminal, determined the direction of movement. According to RT-PCR results, the kifc1 gene is not tissue-specific, commonly detected in different tissues, for example, testis, liver, stomach, muscle, caecum and gills. Through an in situ hybridization method, the expression pattern of KIFC1 protein mimics in the spermatogenesis of S. maindroni. During the primary stage of the spermatogenesis, the kifc1 mRNA signal was barely detectable. At the early spermatids, the signal started to be present. With the elongation of spermatids, the signals increased substantially. It peaked and gathered around the acrosome area when the spermatids began to transform to spindle shape. As the spermatids developed into mature sperm, the signal vanished. In summary, the expression of kfic1 at specific stages during spermiogenesis and its distribution shed light on the potential functions of this motor in major cytological transformations. The KIFC1 homologue may provide a direct shaping force to the nucleus or influence the shaping process through indirect regulation.
[Show abstract][Hide abstract] ABSTRACT: Although the single-cell gel electrophoresis (SCGE) alkaline comet assay can detect DNA damage quickly and with high sensitivity, it does not work well for cryopreserved sperm of some marine teleosts. Using large yellow croaker Pseudosciaena crocea sperm that were cryopreserved in increasing concentrations (5 to 30%) of DMSO, we made modifications to the classic SCGE method that included using common microscopic slides without rough surfaces, pretreatment of sperm before gel spread, and a single layer of gel. Electrophoresis conditions were 130 mA, 15 V, and 60 min, providing a high SCGE sensitivity and definition of the comet image.
The improved method is useful for detecting cryopreserved sperm DNA damage of some marine
teleosts, especially for testing the quality of genetic resources.
[Show abstract][Hide abstract] ABSTRACT: Metallothioneins (MTs) possess a unique molecular structure that provides metal-binding and redox capabilities. These capabilities include the maintenance of metal equilibria that protect against heavy metals (especially cadmium) and oxidative damage. Past studies have focused on the function of MTs in vertebrates. However, the functions of MTs during spermiogenesis in invertebrates remain unclear. In order to investigate the function of MTs during spermiogenesis in Portunus trituberculatus, we used RT-PCR and RACE to identify two MT complete cDNA sequences in the total RNA from the P. trituberculatus testis. The 450bp MT-1 cDNA consists of a 77bp 5' untranslated region, a 196bp 3' untranslated region, and a 177bp open reading frame that encodes 58 amino acids including 19 cysteines. The 581bp MT-2 cDNA consists of 73bp 5' untranslated region, a 328bp 3' untranslated region, and a 180bp open reading frame that encodes 59 amino acids including 18 cysteines. MT-1 and MT-2 of P. trituberculatus more closely resemble invertebrate (especially crab) MT homologues than vertebrate MT homologues as indicated by protein alignment comparisons and phylogenetic tree analysis. MT-1 and MT-2 were detected in the heart, testis, muscle, hepatopancreas, and gill of P. trituberculatus by tissue expression analysis. In addition, MT-1 and MT-2 are present during the entire process of spermiogenesis in P. trituberculatus as indicated by H&E staining and in situ hybridization. MT-1 and MT-2 expression levels significantly increase after cadmium (Cd) exposure as measured by real-time quantitative PCR analysis. Therefore, we suggest that MT-1 and MT-2 perform important functions in spermiogenesis and testis detoxification in P. trituberculatus.
[Show abstract][Hide abstract] ABSTRACT: Metallothionein (MT) participates in metallic homeostasis and detoxification in living animals. Previous studies have focused mainly on the functions of MT in vertebrates, but the functions of MT during spermiogenesis in invertebrates remain unclear. In order to investigate the functions of MT during spermiogenesis in the Japanese stone crab (Charybdis japonica), we identified the C. japonica MT complete cDNA sequence from the total RNA of the testis using RT-PCR and RACE. The 587bp MT cDNA contains: an 80bp 5' untranslated region, a 333bp 3' untranslated region, and a 174bp open reading frame. MT has 57 amino acids including 19 cysteines. The protein alignment between MT sequences of C. japonica and other crabs shows a high similarity and a strong identity in cysteine residues vital for the metal-binding affinity of MT. After the cadmium (Cd) exposure, the testis displays both abnormal morphology and MT mRNA expression both of which indicate a sensitive response of testis MT to Cd. Therefore, we suggest that MT is an excellent biomarker candidate for evaluating Cd pollution.
[Show abstract][Hide abstract] ABSTRACT: KIF3B is known for maintaining and assembling cilia and flagellum. To date, the function of KIF3B and its relationship with KIF3A during spermiogenesis in the cephalopod Octopus tankahkeei remains unknown. In the present study, we characterized a gene encoding a homologue of rat KIF3B in the O. tankahkeei testis and examined its temporal and spatial expression pattern during spermiogenesis. The cDNA of KIF3B was obtained with degenerate and RACE PCR and the distribution pattern of ot-kif3b were observed with RT-PCR. The morphological development during spermiogenesis was illustrated by histological and transmission electron microscopy and mRNA expression of ot-kif3b was observed by in situ hybridization. The 2,365 nucleotides cDNA consisted of a 102 bp 5' untranslated region (UTR), a 2,208 bp open reading frame (ORF) encoding a protein of 736 amino acids, and a 55 bp 3' UTR. Multiple alignments revealed that the putative Ot-KIF3B shared 68, 68, 69, 68, and 67% identity with that of Homo sapiens, Mus musculus, Gallus gallus, Danio rerio, and Xenopus laevis, respectively, along with high identities with Ot-KIF3A in fundamental structures. Ot-kif3b transcripts appeared gradually in early spermatids, increased in intermediate spermatids and maximized in drastically remodeled and final spermatids. The kif3b gene is identified and its expression pattern is demonstrated for the first time in O. tankahkeei. Compared to ot-kif3a reported by our laboratory before, our data suggested that the putative heterodimeric motor proteins Ot-KIF3A/B may be involved in intraspermatic transport and might contribute to structural changes during spermiogenesis.
[Show abstract][Hide abstract] ABSTRACT: The egg envelope is an essential structure occurring during oogenesis. It plays an important role during the process of fertilization in the large yellow croaker Pseudosciaena crocea. Elucidation of egg envelope formation helps us to understand fertilization mechanisms in teleosts. In the present work, we studied the formation of egg envelope in P. crocea by light microscopy, as well as by transmission and scanning electron microscopy. Four layers exist outside the oocyte plasmalemma, i.e., theca cell layer, basal membrane, granulosa cell layer and zona radiata. According to our observation, zona radiata is a multilaminar structure just like the same structure reported in teleosts, but the origin of this structure is a little different. Before it is formed, a peripheral space filled with different density of vesicles is the place where zona radiata is formed. Zona radiata (Z1) is secreted only by oocyte itself, it belongs to the primary envelope; zona radiata 2 (Z2) and zona radiata 3 (Z3) belong to the secondary envelope, because the two layers are formed after granulosa cells appear, and microvilli participate this process. It is very interesting that Z2 and Z3 are situated between Z1 and the granulosa cell first, but they translocate to the other side of Z1. This microanatomy difference may due to the participation of microvilli. The new finding about egg envelope formation in P. crocea will help us to do further investigation on fertilization mechanisms and will make artificial breeding possible which may contribute to the resource recovery of this species.
[Show abstract][Hide abstract] ABSTRACT: Nuclear morphogenesis is one of the most fundamental cellular transformations taking place during spermatogenesis. In rodents, a microtubule-based perinuclear structure, the manchette, and a C-terminal kinesin motor KIFC1 are believed to play crucial roles in this process. Spermatogenesis in Octopus tankahkeei is a good model system to explore whether evolution has created a cephalopod prototype of mammalian manchette-based and KIFC1-dependent sperm nuclear shaping machinery.
We detected the presence of a KIFC1-like protein in the testis, muscle, and liver of O. tankahkeei by Western Blot. Then we tracked its dynamic localization in spermatic cells at various stages using Immunofluorescence and Immunogold Electron Microscopy. The KIFC1-like protein was not expressed at early stages of spermatogenesis when no significant morphological changes occur, began to be present in early spermatid, localized around and in the nucleus of intermediate and late spermatids where the nucleus was dramatically elongated and compressed, and concentrated at one end of final spermatid. Furthermore, distribution of the motor protein during nuclear elongation and condensation overlapped with that of the cephalopod counterpart of manchette at a significant level.
The results support the assumption that the protein is actively involved in sperm nuclear morphogenesis in O. tankahkeei possibly through bridging the manchette-like perinuclear microtubules to the nucleus and assisting in the nucleocytoplasmic trafficking of specific cargoes. This study represents the first description of the role of a motor protein in sperm nuclear shaping in cephalopod.
PLoS ONE 12/2010; 5(12):e15616. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: KIF3A is a subunit of the heterotrimeric Kinesin-II motor which achieves fame for its pivotal roles in the assembly and maintenance of cilia and flagella and in intracellular transport of membrane bound organelles and protein complexes in various tissues. Its intimacy to the cell's antenna, namely the primary cilia, makes it also involved in some signaling transduction pathways. To test the idea that KIF3A functions during spermiogenesis of the octopod Octopus tankahkeei, we hereby identified a gene (designated as ot-kif3a) encoding a protein apparently homologous to a group of KIF3As, from the testis of this organism. The full-length ot-kif3a comprised a 344bp 5' untranslated region, a 2241bp open reading frame and a 147bp 3' untranslated region. The putative protein consisted of 746 amino acid residues with a calculated molecular weight of 85kDa and a predicted isoelectric point of 6.36. It shared an overall sequence identity of 69%, 69%, 69% and 67% to KIF3A from Homo sapiens, Rattus norvegicus, Mus musculus and Danio rerio, respectively. Tissue distribution profile analysis unraveled its presence in all the tissues examined. In situ hybridization of mRNA in spermionenic cells demonstrated that ot-kif3a was expressed moderately at the beginning of spermiogenesis. The abundance of transcripts increased in intermediate spermatid and peaked in drastically remodeling and final spermatids. In mature sperm, the message was still visible in the head and tail. The temporal and spatial expression dynamics of ot-kif3a during spermiogenesis supports the possibility that the putative motor protein, OtKIF3A, participates in the major cytological events during this differentiation program and is vital for the acquisition of the final cellular phenotype.
Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 11/2010; 157(3):237-45. · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Spermiogenesis in Octopus tankahkeei involves striking cellular reorganization to generate a mature spermatozoon. This process may require spermatid-specific adaptation of cytoskeleton and associated molecular motor proteins. KIFC1 is a C-terminal kinesin motor with important roles in acrosome biogenesis and nuclear reshaping during spermiogenesis in rat. Here, we have cloned and characterized the gene encoding a homologue of rat KIFC1, termed as ot-kifc1, from the testis of O. tankahkeei. The 2229 bp complete cDNA contains a 75 bp 5'-untranslated region, a 1992 bp open reading frame and a 162 bp 3'-untranslated region. The deduced protein shares an overall identity of 40%, 41%, 39% and 41% with its counterpart from human, rat, mouse and African clawed frog, respectively. Tissue expression analysis revealed ot-kifc1 was expressed in testis, gill and hepatopancreas, but not in other tissues examined. In situ hybridization result showed the ot-kifc1 message was hardly detectable in early spermatid, concentrated at the tail region of intermediate spermatid, abundant in spermatid undergoing dramatic elongation and compression, enriched at one end in late spermatids and disappeared in mature sperm. In conclusion, the expression of ot-kifc1 at specific stages of spermiogenesis suggests a role for this motor in major cytological transformations.
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 03/2010; 156(3):174-82. · 1.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The octopod sperm is unique especially in two aspects: the screw-shaped acrosome and its inner layered substructure (striation). The present study aims to investigate morphological changes of Octopus tankahkeei spermatozoa during the acrosome reaction (AR) and to pursue functions of the internal substructures revealed by inducing AR with the calcium ionophore A23187. Gradual changes of the spermatozoa were traced using fluorescence and electron microscopy. The AR process included the bulging, vesiculation, and dehiscence of the plasma membrane around the acrosome and the nucleus, as well as the vesiculation of the mitochondrial sheath. Membrane vesiculation outside the nucleus has never been reported in the order Octopoda. The rigid screw and the inner striation of the acrosome remained intact surmounting the nucleus, suggesting that these two structures have potential functions during fertilization. In addition, the detachment of the sperm head and the tail was commonly observed in this study, both in intact and acrosome-reacted sperm. Fluorescence microscopy revealed that the detached mitochondrial sheath usually gave weaker and more dispersive signals than the joint ones. This phenomenon implied that the intense energy release might promote the detachment of the mitochondrial sheath.
[Show abstract][Hide abstract] ABSTRACT: Spermatogenesis is a complicated process during which spermatogonia undergo proliferation and divisions leading, after a series of dramatic changes, to the production of mature spermatozoa. Many molecular motors are involved in this process. KIFC1, a C-terminal kinesin motor, participates in acrosome biogenesis and nuclear shaping. We report here the expression profile of KIFC1 during spermatogenesis in the Chinese mitten crab, Eriocheir sinensis. KIFC1 mainly localizes around the nucleus but is also present within the nucleus of the spermatogonium and spermatocyte. At the early spermatid stage, KIFC1 begins to be distributed on the nuclear membrane at the region where the proacrosomal vesicle is located. By the late spermatid stage, KIFC1 is found on the acrosome. Immunocytochemical and ultrastructural analyses have shown that KIFC1 localizes on the perforatorium, which is composed of an apical cap and an acrosomal tubule. We demonstrate that, during spermatogenesis in E. sinensis, KIFC1 probably plays important roles in the biogenesis of the acrosome and in its maintenance. KIFC1 may also be essential for the eversion of the acrosome during fertilization.
Cell and Tissue Research 08/2009; 337(1):113-23. · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ultrastructure of mature spermatozoa of Estellarca olivacea was studied by transmission electron microscopy and its phylogenetic implications are discussed for the first time in this paper. The mature spermatozoon is composed of a head which contains a cone-shaped acrosome, a round nucleus and a tail region. The subacrosomal space is less electron dense which contains a homogeneous material. No axial rod and a basal plate were observed in subacrosomal space. No anterior invagination exists in the nucleus, but an inverted shallow V-shaped posterior invagination is visible. Nuclear lacunae could be seen clearly although the nucleus is highly condensed. Within the mid-piece of the spermatozoon there exist five spherical mitochondria while the long whip-like end portion is composed of an axoneme with the typical 9+2 structure. The spermatozoon of Estellarca olivacea is a product of the evolution of the reproductive system of the family Arcidae. Whether the particular acrosome, subacrosomal space, or the highly condensed nucleus might be adaptations of high fertilization rate in the particular environment of this species is discussed.
[Show abstract][Hide abstract] ABSTRACT: The ultrastructure of mature spermatozoa of the giant clam bivalve Scapharca broughtoni was investigated by transmission electron microscopy for the first time. The mature spermatozoon consists of a head which is composed of a cone-shaped acrosome, a round nucleus, and a tail region. A subacrosomal space contains an axial rod and a basal plate, the latter lying between the acrosome and the nucleus. Although the nucleus lacks an anterior invagination, an inverted shallow V-shaped posterior invagination is present within the nucleus. Within the middle portion of the spermatozoon lie five spherical mitochondria while the long whip-like end portion is composed of an axoneme with the typical 9+2 structure. Our conclusion is that the spermatozoon of S. broughtoni is of the type I anacrosomal "aquasperm", and the morphology of acrosome and nucleus are an adaptation to external fertilization.
[Show abstract][Hide abstract] ABSTRACT: In this paper, DMSO was used as cryoprotectant for cryopreservation of Sparusmacrocephalus spermatozoa in 0.5 mL straws. Detection of DNA damage in response to a cryopreservation process in Sparus macrocephalus spermatozoa was also carried out. The results demonstrated that there were no significant differences between frozen-thawed sperm conserved by Cortland solution diluted with 5%, 10%, 15%, 20% DMSO and fresh sperm in motility. The best motility of frozen-thawed sperm were obtained when DMSO concentration was 10%, and the activation rate, moving time, living time and fertilization rate of frozen-thawed sperm were 92.91±1.25%, 39.90±2.70 min, 53.82±2.84 min and 89.35±1.99% respectively. However, a significant drop in sperm motility and fertilization rate was observed in sperm cryopreserved with 25% and 30% DMSO. The comet rate and damage coefficient of frozen-thawed sperm conserved with 5%, 10%, 15%, 20% DMSO was similar to fresh sperm, but at 25% and 30% were significantly differed to fresh sperm. In fact, there was a positive correlation between comet rate of frozen-thawed sperm and concentration of DMSO in protocol. The majority of sperm with DNA damage within the nucleus were slightly and mildly damaged, while minorities were heavily damaged. Few were totally damaged, and only occurred under the conditions of 25% and 30% DMSO. Our analysis suggests that high concentration of DMSO is the main factor that causes the DNA damage in frozen-thawed sperm nucleus.
Zoological Research (ISSN: 0254-5853) Vol 30 Num 2.