Solea senegalensis vasa transcripts: molecular characterisation, tissue distribution and developmental expression profiles

2013 ◽  
Vol 25 (4) ◽  
pp. 646 ◽  
Author(s):  
Tiziana Pacchiarini ◽  
Ismael Cross ◽  
Ricardo B. Leite ◽  
Paulo Gavaia ◽  
Juan B. Ortiz-Delgado ◽  
...  
Keyword(s):  
Germ Cell ◽  
Expression Profiles ◽  
Primordial Germ Cell ◽  
In Situ Hybridisation ◽  
Developmental Expression ◽  
Solea Senegalensis ◽  
Senegalese Sole ◽  
Gonad Differentiation ◽  
Vasa Gene ◽  
Germinal Cells

The Vasa protein is an RNA helicase belonging the DEAD (Asp-Glu-Ala-Asp)-box family. The crucial role played by the vasa gene in the germ-cell lineage of both vertebrates and invertebrates has made this gene a useful molecular marker for germinal cells and a useful tool in surrogate broodstock production using primordial germ cell transplantation. With the aim of establishing a novel approach to improving Solea senegalensis broodstock management, the vasa gene in this species was characterised. Four S. senegalensis vasa transcripts were isolated: Ssvasa1, Ssvasa2, Ssvasa3 and Ssvasa4. Their phylogenetic relationship with other vasa homologues was determined confirming the high degree of conservation of this helicase throughout evolution. Our qPCR results showed that S. senegalensis vasa transcripts are prevalently expressed in gonads, with ovary-specific expression for Ssvasa3 and Ssvasa4. During embryonic and larval development, a switch between the longest and the shortest transcripts was observed. While Ssvasa1 and Ssvasa2 were maternally supplied, Ssvasa3 and Ssvasa4 depended on the de novo expression program of the growing juveniles, suggesting that vasa mRNA could be involved in Senegalese sole gonad differentiation. In situ hybridisation and immunohistochemical analysis performed in 150-days after hatching (DAH) larvae showed vasa product expression in the germinal region of early gonads. In our work we demonstrated the usefulness of Ssvasa mRNAs as molecular markers for primordial germ cells and germinal cells during embryonic development, larval ontogenesis and gonad differentiation. Furthermore, our results confirmed the potential of vasa to help investigate germinal cell biotechnology for Senegalese sole reproduction.

Development of interspecies testicular germ-cell transplantation in flatfish

2014 ◽  
Vol 26 (5) ◽  
pp. 690 ◽  
Author(s):  
Tiziana Pacchiarini ◽  
Carmen Sarasquete ◽  
Elsa Cabrita
Keyword(s):  
Germ Cell ◽  
In Situ Hybridisation ◽  
Survival Rates ◽  
Immunohistochemical Analysis ◽  
High Survival ◽  
Senegalese Sole ◽  
Dapi Staining ◽  
Testicular Germ Cell ◽  
Histological Techniques ◽  
Vasa Gene

Interspecific testicular germ cell (TGC) transplantation was investigated in two commercial flatfish species. Testes from donor species (Senegalese sole) were evaluated using classical histological techniques (haematoxylin–eosin staining and haematoxylin–light green–orange G–acid fuchsine staining), in situ hybridisation and immunohistochemical analysis. Both Ssvasa1–2 mRNAs and SsVasa protein allowed the characterisation of TGCs, confirming the usefulness of the vasa gene in the detection of Senegalese sole TGCs. Xenogenic transplants were carried out using TGCs from one-year-old Senegalese sole into turbot larvae. Propidium iodide–SYBR-14 and 4′,6′-diamidino-2-phenylindole (DAPI) staining showed that 87.98% of the extracted testicular cells were viable for microinjection and that 15.63% of the total recovered cells were spermatogonia. The vasa gene was characterised in turbot recipients using cDNA cloning. Smvasa mRNA was confirmed as a germ cell-specific molecular marker in this species. Smvasa expression analysis during turbot ontogeny was carried out before Senegalese sole TGC transplants into turbot larvae. Turbot larvae at 18 days after hatching (DAH) proved to be susceptible to manipulation procedures. High survival rates (83.75 ± 15.90 – 100%) were obtained for turbot larvae at 27, 34 and 42 DAH. These data highlight the huge potential of this species for transplantation studies. Quantitative PCR was employed to detect Senegalese sole vasa mRNAs (Ssvasa1–2) in the recipient turbot larvae. The Ssvasa mRNAs showed a significant increase in relative expression in 42-DAH microinjected larvae three weeks after treatment, showing the proliferation of Senegalese sole spermatogonia in transplanted turbot larvae.

scholarly journals Zebrafish vasa RNA but Not Its Protein Is a Component of the Germ Plasm and Segregates Asymmetrically before Germline Specification

2000 ◽  
Vol 149 (4) ◽  
pp. 875-888 ◽  
Author(s):  
Holger Knaut ◽  
Francisco Pelegri ◽  
Kerstin Bohmann ◽  
Heinz Schwarz ◽  
Christiane Nüsslein-Volhard
Keyword(s):  
Germ Cell ◽  
Germ Plasm ◽  
Primordial Germ Cell ◽  
Subcellular Structure ◽  
Actin Cortex ◽  
Maternal Effect Gene ◽  
Close Proximity ◽  
Hybrid Fish ◽  
Vasa Gene ◽  
Effect Gene

Work in different organisms revealed that the vasa gene product is essential for germline specification. Here, we describe the asymmetric segregation of zebrafish vasa RNA, which distinguishes germ cell precursors from somatic cells in cleavage stage embryos. At the late blastula (sphere) stage, vasa mRNA segregation changes from asymmetric to symmetric, a process that precedes primordial germ cell proliferation and perinuclear localization of Vasa protein. Analysis of hybrid fish between Danio rerio and Danio feegradei demonstrates that zygotic vasa transcription is initiated shortly after the loss of unequal vasa mRNA segregation. Blocking DNA replication indicates that the change in vasa RNA segregation is dependent on a maternal program. Asymmetric segregation is impaired in embryos mutant for the maternal effect gene nebel. Furthermore, ultrastructural analysis of vasa RNA particles reveals that vasa RNA, but not Vasa protein, localizes to a subcellular structure that resembles nuage, a germ plasm organelle. The structure is initially associated with the actin cortex, and subsequent aggregation is inhibited by actin depolymerization. Later, the structure is found in close proximity of microtubules. We previously showed that its translocation to the distal furrows is microtubule dependent. We propose that vasa RNA but not Vasa protein is a component of the zebrafish germ plasm. Triggered by maternal signals, the pattern of germ plasm segregation changes, which results in the expression of primordial germ cell–specific genes such as vasa and, consequently, in germline fate commitment.

Identification of two arylalkylamine N-acetyltranferase 1 genes with different developmental expression profiles in the flatfish Solea senegalensis

2011 ◽  
Vol 51 (4) ◽  
pp. 434-444 ◽  
Author(s):  
Esther Isorna ◽  
María Aliaga-Guerrero ◽  
Abdeslam El M’Rabet ◽  
Arianna Servili ◽  
Jack Falcón ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Developmental Expression ◽  
Solea Senegalensis

scholarly journals Germ cell-less like-2 protein is a new component of outer dense fibers in rat sperm flagella

Reproduction ◽  
2007 ◽  
Vol 134 (6) ◽  
pp. 749-756 ◽  
Author(s):  
Emi Murayama ◽  
Michiko Katoh ◽  
Akina Kanebayashi ◽  
Takane Kaneko ◽  
Yosaburo Shibata ◽  
...  
Keyword(s):  
Amino Acid ◽  
Germ Cell ◽  
Expression Profiles ◽  
Primordial Germ Cell ◽  
Amino Acid Level ◽  
Laser Scanning Microscopy ◽  
Organ Specificity ◽  
Immunogold Electron Microscopy ◽  
Testis Development ◽  
Outer Dense Fibers

We have analyzed the expression profiles of ten genes in terms of testis development and organ specificity in rat, which were selected from 215 round spermatid-specific transcripts listed in a database. Out of the ten genes, we directed our attention to one gene, a germ cell-less like-2 gene (gcl-2), a homolog ofDrosophilagcl gene (gcl), which is a component of the germ plasma and required for primordial germ cell formation. Rat genome contains duplicate rat gcl-2 (rgcl-2) genes,rgcl-2Aandrgcl-2B, both of which are located at Xq13. RT-PCR analysis showed that the expression of the two genes was up-regulated during testis development and that they were predominantly expressed in the testis. Bothrgcl-2Aandrgcl-2Bencode a protein of 498 amino acid residues, showing 90.56% identity at the amino acid level. Confocal laser scanning microscopy revealed that rgcl-2 protein was synthesized in the cytoplasm of elongating spermatids and at least a part of it was integrated into the middle piece of spermatozoa during spermiogenesis. Immunogold electron microscopy uncovered that rgcl-2 was localized at the abaxial (convex) surface of outer dense fibers (ODF) of rat sperm flagella. Therefore, we concluded that rgcl-2 is a new component of ODF in sperm flagella.

scholarly journals Comparison of the MicroRNA Expression Profiles of Male and Female Avian Primordial Germ Cell Lines

2018 ◽  
Vol 2018 ◽  
pp. 1-17 ◽  
Author(s):  
Bence Lázár ◽  
Mahek Anand ◽  
Roland Tóth ◽  
Eszter Patakiné Várkonyi ◽  
Krisztina Liptói ◽  
...  
Keyword(s):  
Germ Cell ◽  
Cell Lines ◽  
Germ Cells ◽  
Expression Profiles ◽  
Dominant Role ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Proliferation Rate ◽  
Wide Range ◽  
Significant Difference

Primordial germ cells (PGCs) are the precursors of adult germ cells, and among the embryonic stem-like cells in the bird embryo, only they can transmit the genetic information to the next generation. Despite the wide range of applications, very little is known about the mechanism that governs primordial germ cell self-renewal and differentiation. As a first step, we compared 12 newly established chicken PGC lines derived from two different chicken breeds, performing CCK-8 proliferation assay. All of the lines were derived from individual embryos. A significant difference was found among the lines. As microRNAs have been proved to play a key role in the maintenance of pluripotency and the cell cycle regulation of stem cells, we continued with a complex miRNA analysis. We could discover miRNAs expressing differently in PGC lines with high proliferation rate, compared to PGC lines with low proliferation rate. We found that gga-miR-2127 expresses differently in female and male cell lines. The microarray analysis also revealed high expression level of the gga-miR-302b-3p strand (member of the miR-302/367 cluster) in slowly proliferating PGC lines compared to the gga-miR-302b-5p strand. We confirmed that the inhibition of miR-302b-5p significantly increases the doubling time of the examined PGC lines. In conclusion, we found that gga-miR-181-5p, gga-miR-2127, and members of the gga-miR-302/367 cluster have a dominant role in the regulation of avian primordial germ cell proliferation.

216. c-kit Expression study: timing of onset in rodent testis and irradiated rat testis model

2005 ◽  
Vol 17 (9) ◽  
pp. 83
Author(s):  
S. Mithra Prabhu ◽  
M. L. Meistrich ◽  
S. Mendis ◽  
E. A. McLaughlin ◽  
K. L. Loveland
Keyword(s):  
Protein Expression ◽  
Germ Cell ◽  
Somatic Cell ◽  
Messenger Rna ◽  
Cell Function ◽  
Primordial Germ Cell ◽  
In Situ Hybridisation ◽  
Hormone Treatment ◽  
Normal Male ◽  
Rat Testis

Primordial germ cell and spermatogonial cell function is essential for normal male fertility. These cells require Sertoli–germ cell interactions, specifically somatic cell-derived stem cell factor (SCF) that acts through the c-kit receptor to govern primordial germ cell migration in the foetus, spermatogonial differentiation during puberty and adulthood, and Leydig cell steroidogenesis. We performed a comprehensive study of the c-kit mRNA expression profile in the pre- and post-pubertal mouse and rat testes by in situ hybridisation. Expression of c-kit mRNA was first visualised in germ cells after birth, with the levels concordant with the number and appearance of the differentiated spermatogonial subtypes in both the rat and the mouse. We also studied c-kit expression in the irradiated adult rat testis, in which only undifferentiated spermatogonia are present. After treatment with Cetrorelix, GnRH antagonist (3 days, 1, 2 and 4 weeks) germ cell maturation is re-initiated. Expression of c-kit messenger RNA was observed in the undifferentiated spermatogonia in both untreated and treated testes sections. In contrast, c-kit protein expression was undetectable until 4 weeks of hormone treatment. This suggests that c-kit mRNA and protein expression are differentially regulated and that protein expression relates to somatic cell function.

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