Molecular characterization,sexually dimorphic expression,and functional analysis of 3′-untranslated region of vasa gene in half-smooth tongue sole (Cynoglossus semilaevis)

Vasa is a highly conserved ATP-dependent RNA helicase expressed mainly in germ cells. The vasa gene plays a crucial role in the development of germ cell lineage and has become an excellent molecular marker in identifying germ cells in teleosts. However, little is known about the structure and function of the vasa gene in flatfish. In this study, the vasa gene (Csvasa) was isolated and characterized in half-smooth tongue sole (Cynoglossus semilaevis), an economically important flatfish in China. In the obtained 6425-bp genomic sequence, 23 exons and 22 introns were identified. The Csvasa gene encodes a 663-amino acid protein, including highly conserved domains of the DEAD-box protein family. The amino acid sequence also shared a high homology with other teleosts. Csvasa expression was mainly restricted to the gonads, with little or no expression in other tissues. Real-time quantitative polymerase chain reaction analysis revealed that Csvasa expression levels decreased during embryonic and early developmental stages and increased with the primordial germ cell proliferation. A typical sexually dimorphic expression pattern of Csvasa was observed during early development and sex differentiation, suggesting that the Csvasa gene might play a differential role in the proliferation and differentiation of male and female primordial germ cells (PGCs). Csvasa mRNA expression levels in neomales were significantly lower than those in normal males and females, indicating that the Csvasa gene might be implicated in germ cell development after sex reversal by temperature treatment. In addition, medaka (Oryzias latipes) PGCs could be transiently labeled by microinjection of synthesized mRNA containing the green fluorescence protein gene and 3′-untranslated region of Csvasa, which confirmed that the Csvasa gene has the potential to be used as a visual molecular marker of germ cells and laid a foundation for manipulation of PGCs in tongue sole reproduction.     

Expression of vasa and nanos3 during primordial germ cell formation and migration in Atlantic cod (Gadus morhua L.)

Primordial germ cells (PGCs), progenitors of gametes, are specified very early in embryonic development and undergo an active migration to the site where the future gonads will form. While the developmental pattern of PGCs during embryogenesis has been documented in few model teleost fishes, there is currently no information available for any representative of Superorder Paracanthopterygii. This includes Atlantic cod (Gadus morhua), which is a historically important food fish in both fisheries and aquaculture industries. In the present study, we cloned and characterized vasa and nanos3 and used them as germ cell markers in Atlantic cod. Sequencing results showed prospective vasa and nanos3 mRNA contained the domains used to describe their respective protein family. Furthermore, phylogenetic analysis using the amino acid sequence placed Atlantic cod Vasa distinct from representatives of three other taxonomic Superorders. Atlantic cod Nanos3 was placed with other homologues from the Nanos3 subfamily. Expression of both genes was detected from the first cleavage division; both were specifically expressed in Atlantic cod PGCs from the 32-cell stage. While nanos3 expression ceased during early somitogenesis, vasa was strongly expressed throughout embryonic development. Using vasa as a marker, we described the Atlantic cod PGC migration pattern. We demonstrated that Atlantic cod PGCs migrate ventral to the trunk mesoderm. With the exception of Pacific herring (Clupea pallasii), PGCs in other described teleost fishes migrate lateral to the trunk. The results from this study are the first step toward understanding germ line formation in Atlantic cod.     

The proliferation and migration of immature germ cells in the mussel, Mytilus galloprovincialis: observation of the expression pattern in the M. galloprovincialis vasa-like gene (Myvlg) by in situ hybridization

In bivalve, the distribution of primordial germ cells can be traced from early embryogenesis to the veliger larva by the expression of the vasa ortholog. However, the distribution of germ cells from metamorphosis to maturation in bivalves has not been examined extensively. In this study, we used in situ hybridization to observe expression of the Mytilus galloprovincialis vasa-like gene (Myvlg). The distribution of germ cells was clarified in immature mussels. We observed germ cells in adult mussels during the non-reproductive and reproductive seasons. Myvlg was specifically expressed in germ cells. Gametogenesis occurs in acini surrounded by connective tissue. Myvlg expression was detected in spermatogonia, spermatocytes, oogonia, and oocytes. In the non-reproductive season, gametes were not observed in the acini, but Myvlg was expressed in germinal stem cells along the acini. The expression intensity in the non-reproductive season, however, was much weaker than that in the reproductive season. Myvlg-positive cells proliferated during the non-reproductive season. In immature mussels, a pair of germ cell clumps was distributed laterally in the connective tissue between the nephric tubules and posterior byssal retractor muscle. Germ cells were also observed along pericardium. When immature mussels grew, a pair of germ cell clumps migrated anteriorly in the connective tissue along the outer epithelium at the dorsal region of the mantle base between the mantle and gill. The number of germ cells increased significantly as the mussels grew. This is the first report to observe the proliferation and migration of germ cells in immature mussels.     

Flatworm stem cells and the germ line: developmental and evolutionary implications of macvasa expression in Macrostomum lignano

We have isolated and identified the vasa homologue macvasa, expressed in testes, ovaries, eggs and somatic stem cells of the flatworm Macrostomum lignano. Molecular tools such as in situ hybridization and RNA interference were developed for M. lignano to study gene expression and function. Macvasa expression was followed during postembryonic development, regeneration and in starvation experiments. We were able to follow gonad formation in juveniles and the reformation of gonads from stem cells after amputation by in situ hybridization and a specific Macvasa antibody. Expression of macvasa in the germ cells was highly affected by feeding conditions and correlated with the decrease and regrowth of the gonads. RNA interference showed specific down-regulation of macvasa mRNA and protein. The absence of Macvasa did not influence gonad formation and stem cell proliferation. Our results corroborate the exclusive nature of the flatworm stem cell system but challenge the concept of a solely postembryonic specification of the germ line in Platyhelminthes. We address the transition of somatic stem cells to germ cells and speculate on Macrostomum as a system to unravel the mechanisms of preformation or epigenesis in the evolution of germ line specification from somatic stem cells.     

GAL4 enhancer traps that can be used to drive gene expression in developing Drosophila spermatocytes

The Drosophila testis has proven to be a valuable model organ for investigation of germline stem cell (GSC) maintenance and differentiation as well as elucidation of the genetic programs that regulate differentiation of daughter spermatogonia. Development of germ cell specific GAL4 driver transgenes has facilitated investigation of gene function in GSCs and spermatogonia but specific GAL4 tools are not available for analysis of postmitotic spermatogonial differentiation into spermatocytes. We have screened publically available pGT1 strains, a GAL4‐encoding gene trap collection, to identify lines that can drive gene expression in late spermatogonia and early spermatocytes. While we were unable to identify any germline‐specific drivers, we did identify an insertion in the chiffon locus, which drove expression specifically in early spermatocytes within the germline along with the somatic cyst cells of the testis. genesis 50:914–920, 2012. © 2012 Wiley Periodicals, Inc.     

Light and electron microscopic analyses of Vasa expression in adult germ cells of the fish medaka

Germ cells of diverse animal species have a unique membrane-less organelle called germ plasm (GP). GP is usually associated with mitochondria and contains RNA binding proteins and mRNAs of germ genes such as vasa. GP has been described as the mitochondrial cloud (MC), intermitochondrial cement (IC) and chromatoid body (CB). The mechanism underlying varying GP structures has remained incompletely understood. Here we report the analysis of GP through light and electron microscopy by using Vasa as a marker in adult male germ cells of the fish medaka (Oryzias latipes). Immunofluorescence light microscopy revealed germ cell-specific Vasa expression. Vasa is the most abundant in mitotic germ cells (oogonia and spermatogonia) and reduced in meiotic germ cells. Vasa in round spermatids exist as a spherical structure reminiscent of CB. Nanogold immunoelectron microscopy revealed subcellular Vasa redistribution in male germ cells. Vasa in spermatogonia concentrates in small areas of the cytoplasm and is surrounded by mitochondria, which is reminiscent of MC. Vasa is intermixed with mitochondria to form IC in primary spermatocytes, appears as the free cement (FC) via separation from mitochondria in secondary spermatocyte and becomes condensed in CB at the caudal pole of round spermatids. During spermatid morphogenesis, Vasa redistributes and forms a second CB that is a ring-like structure surrounding the dense fiber of the flagellum in the midpiece. These structures resemble those described for GP in various species. Thus, Vasa identifies GP and adopts varying structures via dynamic reorganization at different stages of germ cell development.     

Vasa Identifies Germ Cells and Critical Stages of Oogenesis in the Asian Seabass

Germ cells produce sperm and eggs for reproduction and fertility. The Asian seabass (Lates calcarifer), a protandrous marine fish, undergoes male-female sex reversal and thus offers an excellent model to study the role of germ cells in sex differentiation and sex reversal. Here we report the cloning and expression of vasa as a first germ cell marker in this organism. A 2241-bp cDNA was cloned by PCR using degenerate primers of conserved sequences and gene-specific primers. This cDNA contains a polyadenylation signal and a full open reading frame for 645 amino acid residues, which was designated as Lcvasa for the seabass vasa, as its predicted protein is homologous to Vasa proteins. The Lcvasa RNA is maternally supplied and specific to gonads in adulthood. By chromogenic and fluorescent in situ hybridization we revealed germ cell-specific Lcvasa expression in both the testis and ovary. Importantly, Lcvasa shows dynamic patterns of temporospatial expression and subcellular distribution during gametogenesis. At different stages of oogenesis, for example, Lcvasa undergoes nuclear-cytoplasmic redistribution and becomes concentrated preferentially in the Balbiani body of stage-II~III oocytes. Thus, the vasa RNA identifies both female and male germ cells in the Asian seabass, and its expression and distribution delineate critical stages of gametogenesis.     

Cloning and Characterization of a Rice Field Eel vasa-Like Gene cDNA and Its Expression in Gonads During Natural Sex Transformation

The vasa (vas)-related gene encodes an RNA helicase protein member of the DEAD-box family and plays key roles in germ-cell formation in higher metazoans. Using degenerate PCR and RACE, we cloned the vasa gene of the rice field eel (Monopterus albus), which is homologous to the Drosophila vasa gene. We named it ma-vas (Monopterus albus vas). Ma-vas encodes a protein of 618 amino acids, which contains all of the known characteristics of vasa homologs. RT-PCR analysis revealed that ma-vas was exclusively expressed in the gonads of the female, intersex, and male. During gonadal natural sex reversal, ma-vas is expressed in oocytes at all stages of oogenesis, in degenerating oocytes of ovotestis, and in spermatogonia and spermatocytes at early stages. The vasa positive signal was also observed in the peripheral layer of late ovary. It was not found, however, in that layer of the testis. Alkaline phosphatase (AKP) staining on the ovary and testis also indicated that some cells had differentiational potential in the peripheral layer of the ovary, suggesting that spermatogonia might arise from cells with AKP and vasa-positive staining in the peripheral layer of the female gonad.     

Stem cells in reproductive strategy of asexually reproducing invertebrates

Original and literature data supporting the evolutionary conservation of the morphofunctional organization of totipotent cells of germ and stem lineages in metazoan animals are reviewed. We studied stem cells of the colonial rhizocephalans, Peltogasterella gracilis, Polyascus polygenea and Thylacoplethus isaevae, the turbellarian Dugesia tigrina, the colonial hydroid Obelia longissima, and cultured embryonic stem cells of mouse. The typical germinal granules of germ plasm, selective expression of the activity of alkaline phosphatase and of proliferating cell nuclear antigen (PCNA), which are known as markers of stem and primary germ cells of vertebrates, and the specific expression of the protein product of the vasa gene in cells of rhizocephalans, which is a marker of cells of germ and stem lineages of various metazoans, specified the stem cells of invertebrates of such different taxa. The self-renewing pool of totipotent stem cells is the cellular basis of the reproductive strategy, including sexual and asexual reproduction; such cells share morphofunctional features of embryonic stem and germline cells of Metazoa.     

The Transcriptional Cell Atlas of Testis Development in Sheep at Pre-Sexual Maturity

Sheep testes undergo a dramatic rate of development with structural changes during pre-sexual maturity, including the proliferation and maturation of somatic niche cells and the initiation of spermatogenesis. To explore this complex process, 12,843 testicular cells from three males at pre-sexual maturity (three-month-old) were sequenced using the 10× Genomics Chromium^TM single-cell RNA-seq (scRNA-seq) technology. Nine testicular somatic cell types (Sertoli cells, myoid cells, monocytes, macrophages, Leydig cells, dendritic cells, endothelial cells, smooth muscle cells, and leukocytes) and an unknown cell cluster were observed. In particular, five male germ cell types (including two types of undifferentiated spermatogonia (A_pale and A_dark), primary spermatocytes, secondary spermatocytes, and sperm cells) were identified. Interestingly, A_pale and A_dark were found to be two distinct states of undifferentiated spermatogonia. Further analysis identified specific marker genes, including UCHL1, DDX4, SOHLH1, KITLG, and PCNA, in the germ cells at different states of differentiation. The study revealed significant changes in germline stem cells at pre-sexual maturation, paving the way to explore the candidate factors and pathways for the regulation of germ and somatic cells, and to provide us with opportunities for the establishment of livestock stem cell breeding programs.     

SpolvlgA is a DDX3/PL10-related DEAD-box RNA helicase expressed in blastomeres and embryonic cells in planarian embryonic development

Planarian flatworms have an impressive regenerative power. Although their embryonic development is still poorly studied and is highly derived it still displays some simple characteristics. We have identified SpolvlgA, a Schmidtea polychroa homolog of the DDX3/PL10 DEAD-box RNA helicase DjvlgA from the planarian species Dugesia japonica. This gene has been previously described as being expressed in planarian adult stem cells (neoblasts), as well as the germ line. Here we present the expression pattern of SpolvlgA in developing embryos of S. polychroa and show that it is expressed from the first cleavage rounds in blastomere cells and blastomere-derived embryonic cells. These cells are undifferentiated cells that engage in a massive wave of differentiation during stage 5 of development. SpolvlgA expression highlights this wave of differentiation, where nearly all previous structures are substituted by blastomere-derived embryonic cells. In late stages of development SpolvlgA is expressed in most proliferating and differentiating cells. Thus, SpolvlgA is a gene expressed in planarian embryos from the first stages of development and a good marker for the zygote-derived cell lineage in these embryos. Expression in adult worms is also monitored and is found in the planarian germ line, where it is showed to be expressed in spermatogonia, spermatocytes and differentiating spermatids.