Gene expression pattern of myosin Va during spermatogenesis of Chinese mitten crab, Eriocheir sinensis

Myosin Va is an F-actin dependent molecular motor with multiple functions that are essential for acrosome formation in mouse spermiogenesis. The spermatozoon of the crab has a complicated acrosome surrounded by a cup-shaped nucleus. In the present study, the myosin Va cDNA was cloned from the testis of the Chinese mitten crab Eriocheir sinensis using degenerate PCR and rapid amplification of cDNA ends (RACE). The myosin Va cDNA consists of a 125bp 5'-untranslated region (5' UTR), a 5331bp open reading frame (ORF) and a 590bp 3' UTR. The putative myosin Va protein contains the head domain, neck domain and tail domain. Multiple alignment and phylogenetic tree showed that E. sinensis myosin Va is more closely related to the vertebrate myosin Va than to the invertebrate myosin V. E. sinensis myosin Va was expressed in various tissues. In situ hybridization demonstrated that myosin Va mRNA is located in the entire process of spermatogenesis. Quantitative real-time PCR indicated that the expression level at the mitotic and meiotic phases is higher than the spermiogenesis phase. Taken together, our work suggests that myosin Va may function in E. sinensis spermatogenesis.     

The expression pattern of the C-terminal kinesin gene kifc1 during the spermatogenesis of Sepiella maindroni

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 2432 bp 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 structures, 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, the 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.     

Mighty Piwis defend the germline against genome intruders

Piwis are a germline-specific subclass of the Argonaute family of RNA interference (RNAi) effector proteins that are associated with a recently discovered group of small RNAs (piRNAs). Recent studies in Drosophila and zebrafish directly implicate Piwi proteins in piRNA biogenesis to maintain transposon silencing in the germline genome (Brennecke et al., 2007; Gunawardane et al., 2007; Houwing et al., 2007). This function may be conserved in mice as loss of Miwi2, a mouse Piwi homolog, leads to germline stem cell and meiotic defects correlated with increased transposon activity (Carmell et al., 2007).     

The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells

The Piwi-piRNA pathway is active in animal germ cells where its functions are required for germ cell maintenance and gamete differentiation. Piwi proteins and piRNAs have been detected outside germline tissue in multiple phyla, but activity of the pathway in mammalian somatic cells has been little explored. In particular, Piwi expression has been observed in cancer cells, but nothing is known about the piRNA partners or the function of the system in these cells. We have surveyed the expression of the three human Piwi genes, Hiwi, Hili and Hiwi2, in multiple normal tissues and cancer cell lines. We find that Hiwi2 is ubiquitously expressed; in cancer cells the protein is largely restricted to the cytoplasm and is associated with translating ribosomes. Immunoprecipitation of Hiwi2 from MDAMB231 cancer cells enriches for piRNAs that are predominantly derived from processed tRNAs and expressed genes, species which can also be found in adult human testis. Our studies indicate that a Piwi-piRNA pathway is present in human somatic cells, with an uncharacterised function linked to translation. Taking this evidence together with evidence from primitive organisms, we propose that this somatic function of the pathway predates the germline functions of the pathway in modern animals.     

Spermiogenesis in three species of cicadas (Hemiptera: Cicadidae)

Spermiogenesis in three species of cicadas representing one cicadettine (Monomatapa matoposa Boulard) and two cicadines (Diceroprocta biconica [Walker] and Kongota punctigera [Walker]) was investigated by light and electron microscopy. Although spermiogenesis was occurring in the testis of adult males of all species, earlier spermiogenic stages were observed in D. biconica only. While spermiogenesis was similar to that described for other insects, some differences were noted. For example granular material did not assemble around the centriole to form a centriolar adjunct but did accumulate in the cytoplasm of early spermatids adjacent to a region of the nuclear membrane where nuclear pores were aggregated. In late spermatids this material accumulated anterior to the mitochondrial derivatives in a developing postero‐lateral nuclear groove. While this material has been named the ‘centriolar adjunct’ by previous authors, its formation away from the centriole raises questions about its true identity. Second, during acrosome maturation an ante‐acrosomal region of cytoplasm develops. Although present in later spermatids, this region is lost in spermatozoa. Interspecific variations in chromatin condensation patterns and the number of microtubule layers encircling the spermatid nucleus during spermiogenesis were noted.     

The role of piRNAs in mouse spermatogenesis.

The Argonaute proteins, which are the direct partners of the small RNAs involved in RNA interference mechanisms, can be divided into two subfamilies, the Argonautes and the Piwis. In animals, the Argonaute subfamily binds 21-22 nucleotide small interfering RNAs (siRNAs) and microRNAs (miRNAs), which direct cleavage and translational inhibition of their target RNAs respectively. The partners of the Piwi proteins are 24-30-nucleotide small RNAs called Piwi-interacting RNAs or piRNAs. In Drosophila, Piwi proteins and piRNAs protect the genome of the germline against selfish elements. Recent studies suggest that this function is conserved in mammals.La famille des Argonautes, les partenaires directs des petits ARNs dans les mécanismes d'interférence par l'ARN, se divise en deux sous-groupes : les Argonautes et les Piwis. Chez les animaux, le sous-groupe des Argonautes se lie aux petits ARNs interférents (siARNs) et aux microARNs (miARNs) qui mesurent 21-22 nucléotides et sont responsables du clivage et de l'inhibition traductionnelle des ARNs cibles respectivement. Les protéines Piwis ont pour partenaires de petits ARNs de 24-30 nucléotides appelés Piwi-interacting RNAs ou piARNs. Chez la drosophile, les protéines Piwi et les piARNs protègent le génome de la lignée germinale contre les éléments mobiles. Des analyses récentes suggèrent que cette fonction est conservée chez les mammifères.     

Cloning and characterization of calreticulin and its association with salinity stress in P. trituberculatus

Calreticulin (CRT) is a highly conserved and multifunctional endoplasmic reticulum (ER) chaperone protein and plays important roles in salinity stress response. Portunus trituberculatus is a commercially important fishery species, and water salinity conditions influence its commercial farming significantly. In order to research the function of calreticulin under salinity stress, the full-length cDNA sequence of calreticulin from P. trituberculatus (PtCRT) was firstly cloned and characterized. The complete cDNA sequence of PtCRT is 1676 bp with 1218 bp open reading frame (ORF), encoding a polypeptide of 405 amino acids. Multiple sequence alignments showed that the deduced acid amino sequences of PtCRT shared the highest homology to CRT of Fenneropenaeus chinensis (89 %). Fluorescent quantitative real-time PCR analysis indicated that PtCRT was expressed in all detected tissues and showed the highest expression level in hepatopancreas. In addition, salinity challenge significantly influenced the expression level of PtCRT in gill. Six single nucleotide polymorphisms (SNPs) were detected in cDNA sequence of PtCRT, and one SNP was associated with the salt tolerant trait. All results indicated that PtCRT plays an important role in mediating the salinity adaption of P. trituberculatus.     

Characterization of Bovidae sex-determining gene SRY

In mammals, testis determination is under the control of the sex-determining gene SRY. This Y-linked gene encodes a protein with a DNA binding domain similar to those found in high-mobility-group proteins. Here we report the cloning and sequences of the SRY genes of yak and Chinese native cattle. Our data show that SRY genes in Bovidae are less divergent, especially in the coding and 3'' regions.     

Characterization of a novel nm23 gene and its potential roles in gametogenesis in the prawn Macrobrachium rosenbergii (de Man, 1879) (Crustacea: Decapoda)

Nm23 is a family of genes encoding the nucleoside diphosphate (NDP) kinase, which functions in a wide variety of biological processes, including growth, development, differentiation and tumor metastasis. In this study, a novel nm23 gene, designated as Mrnm23, was identified from the freshwater giant prawn Macrobrachium rosenbergii. The full-length cDNA was 776 bp in length, encoding for a protein of 176 amino acids with one typical NDP kinase domain that harbored all the crucial residues for nucleotide binding and enzymatic activity. Like human novel nm23-H1B, the putative protein contained a unique 21-amino-acid NH₂-terminal extension as compared to human nm23 (nm23-H1) homologs. Further, 3 extra amino acid residues prolonged the COOH-terminus. The Mrnm23 was ubiquitously expressed in all tissues examined, including androgenic gland, gill, heart, liver, muscle, ovary, and testis. In situ hybridization to gonad sections indicated that the Mrnm23 mRNA was localized in the cytoplasm of cup-base of differentiating spermatids, in the spike of the umbrella-shaped spermatozoa and in the cytoplasm of the early previtellogenic oocytes, suggesting that the Mrnm23 has potential roles in spermiogenesis and early differentiation of oocyte.     

Ultrastructure of Spermatogenesis in the Testis of Paragonimus heterotremus

Lung fluke, Paragonimus heterotremus, is a flatworm causing pulmonary paragonimiasis in cats, dogs, and humans in Southeast Asia. We examined the ultrastructure of the testis of adult P. heterotremus with special attention to spermatogenesis and spermiogenesis using scanning and transmission electron microscopy. The full sequence of spermatogenesis and spermiogenesis, from the capsular basal lamina to the luminal surface, was demonstrated. The sequence comprises spermatogonia, spermatocytes with obvious nuclear synaptonemal complexes, spermatids, and eventual spermatozoa. Moreover, full steps of spermatid differentiation were shown which consisted of 1) early stage, 2) differentiation stage representing the flagella, intercentriolar body, basal body, striated rootlets, and electron dense nucleus of thread-like lamellar configuration, and 3) growing spermatid flagella. Detailed ultrastructure of 2 different types of spermatozoa was also shown in this study.     

Role of Vasa, Piwi, and Myc-expressing coelomic cells in gonad regeneration of the colonial tunicate, Botryllus primigenus

In the colonial tunicate, Botryllus primigenus Oka, gonads consist of indifferent germline precursor cells, the primordial testis and ovary, and mature gonads, of which the immature gonad components can be reconstructed de novo in vascular buds that arise from the common vascular system, although the mechanism is uncertain. In this study, we investigated how and what kinds of cells regenerated the gonad components. We found that few Vasa-positive cells in the hemocoel entered the growing vascular bud, where their number increased, and finally developed exclusively into female germ cells. Simultaneously, small cell aggregates consisting of Vasa(-) and Vasa(±) cells appeared de novo in the lateral body cavity of developing vascular buds. Double fluorescent in situ hybridization showed that these cell aggregates were both Piwi- and Myc-positive. They could form germline precursor cells and a primordial testis and ovary that strongly expressed Vasa. Myc knockdown by RNA interference conspicuously lowered Piwi expression and resulted in the loss of germline precursor cells without affecting Vasa(+) oocyte formation. Myc may contribute to gonad tissue formation via Piwi maintenance. When human recombinant BMP 4 was injected in the test vessel, coelomic Piwi(+) cells were induced to express Vasa in the blood. We conclude, therefore, that in vascular buds of B. primigenus, female germ cells can develop from homing Vasa(+) cells in the blood, and that other gonad components can arise from coelomic Vasa(-)/Piwi(+)/Myc(+) cells.