Spermatogenesis in testis primary cell cultures of the tilapia (<i>Oreochromis niloticus</i>)

Tokalov, Sergey V.; Gutzeit, Herwig O.

doi:10.1002/dvdy.20379

Cited by 29 publications

(16 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In fish, cell separation is generally performed by density-gradient centrifugation [7][8][9]; however, it is difficult to obtain highly pure stage-specific cells without contamination using this approach. The panning technique has also been used to separate adhesive and floating cells in fish [10][11][12]; this approach is useful for collecting Sertoli cells and spermatogonia from cell cultures but is not suitable for isolating different types of testicular germ cells.…”

Section: Introductionmentioning

confidence: 99%

Flow-Cytometric Isolation of Testicular Germ Cells from Rainbow Trout (Oncorhynchus mykiss) Carrying the Green Fluorescent Protein Gene Driven by Trout vasa Regulatory Regions

Yano

Suzuki

Yoshizaki

2008

Biology of Reproduction

View full text Add to dashboard Cite

There is a need to isolate different populations of spermatogenic cells to investigate the molecular events that occur during spermatogenesis. Here we developed a new method to identify and purify testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions (pvasa-GFP) at various stages of spermatogenesis. Rainbow trout piwi-like (rtili), rainbow trout scp3 (rt-scp3), and rainbow trout shippo1 (rt-shippo1) were identified as molecular markers for spermatogonia, spermatocytes, and spermatids, respectively. The testicular cells were separated into five fractions (A-E) by flow cytometry (FCM) according to their GFP intensities. Based on the molecular markers, fractions A and B were found to contain spermatogonia, while fractions C and D contained spermatocytes, and fraction E contained spermatids. We also classified the spermatogonia into type A, which contained spermatogonial stem cells (SSCs), and type B, which did not. As none of the molecular markers tested could distinguish between the two types of spermatogonia, we subjected them to a transplantation assay. The results indicated that cells with strong GFP fluorescence (fraction A) colonized the recipient gonads, while cells with weaker GFP fluorescence (fraction B) did not. As only SSCs could colonize the recipient gonads, this indicated that fraction A and fraction B contained mainly type A and type B spermatogonia, respectively. These findings confirmed that our system could identify and isolate various populations of testicular cells from rainbow trout using a combination of GFP-dependent FCM and a transplantation assay.

show abstract

Section: Introductionmentioning

confidence: 99%

Flow-Cytometric Isolation of Testicular Germ Cells from Rainbow Trout (Oncorhynchus mykiss) Carrying the Green Fluorescent Protein Gene Driven by Trout vasa Regulatory Regions

Yano

Suzuki

Yoshizaki

2008

Biology of Reproduction

View full text Add to dashboard Cite

show abstract

“…Massive cell death of Sz in primary cultures prevents a long-term culture of primary cells and spermatogenesis in vitro. A simple Percoll gradient centrifugation step enriches for those cells we are interested in and allows, for example, the analysis of the molecular control of germ cell proliferation over a period of more than 2 weeks (Tokalov and Gutzeit, 2005).…”

Section: Discussionmentioning

confidence: 99%

“…Such single cell preparations can then be analysed by LSM or by flow cytometry (see below). The depletion of haploid cells was achieved by centrifugation of the primary cells through a single-step Percoll gradient as described earlier (Tokalov and Gutzeit, 2005). The procedure was monitored by flow cytometry and aliquots of unfixed and fixed cells (PI stained) were analysed.…”

Section: Staining Pattern Of Primary Cellsmentioning

confidence: 99%

“…Cell type-specific markers or properties that allow the identification of specific cell types are a precondition for such studies. We have shown previously that flow cytometry is a powerful tool for this analysis (Song and Gutzeit, 2003;Tokalov and Gutzeit, 2005). This approach has been used previously to identify different spermatogonial cells populations using scattering parameters, total amount of DNA or intracellular lipid content (Woodruff et al,'92) and could be even more successful if specific cell types could be identified by fluorescein isothiocyanate (FITC)-labelled lectins.…”

mentioning

confidence: 96%

“…However, a comprehensive picture of the molecular signalling between soma and germ line in vertebrates has not emerged. The early events, like sex determination and the proliferation of spermatogonia (Sg), appear to be controlled by the Sertoli cells (Loir, '99;Tokalov and Gutzeit, 2005). At all stages of spermatogenic maturation, germ cells and Sertoli cells communicate through direct cell-cell contact and paracrine interactions (Zipato et al, '80;Griswold, '95;Kerr, '95;Loir, '99;Akama et al, 2002).…”

mentioning

confidence: 97%

See 2 more Smart Citations

Lectin‐binding pattern as tool to identify and enrich specific primary testis cells of the tilapia (Oreochromis niloticus) and medaka (Oryzias latipes)

Tokalov

Gutzeit

2006

J Exp Zool Pt B

View full text Add to dashboard Cite

Cell type-specific lectin binding is a useful tool for the analysis of developing systems. We describe the binding pattern of 21 different fluorescein isothiocyanate (FITC)-labelled lectins to the testis of two model teleost species, the medaka (Oryzias latipes) and the tilapia (Oreochromis niloticus). The analysis of the binding pattern was carried out on tissue sections (medaka and tilapia) and using primary culture cells (only tilapia). Lectin binding was studied by confocal microscopy and for histological analysis some sections were, in addition, stained with bodipy to gain additional information concerning the cytological organization of the cystic mode of spermatogenesis in fish. The observed differences in lectin staining of different cell types in primary cultures were quantified by flow cytometry. Only few lectins bound specifically to haploid cells while the reaction to diploid or tetraploid cells was generally stronger. However, the extracellular material around the haploid spermatids and spermatozoa in spermatocysts showed a strong staining reaction with several lectins (e.g., Phaseolus vulgaris Erythro agglutinin). The apparent differences in the cellular lectin-binding pattern can be used to identify particular cell types, to monitor their differentiation in vitro or to enrich particular cell types from heterogeneous cultures using magnetic beads coated with anti-FITC antibodies. Using the latter approach, we show that it is possible to enrich for gonial cells and at the same time deplete the preparation for haploid cells and Sertoli cells.

show abstract

Molecular cloning of Plk1 and Nek2 and their expression in mature gonads of the teleost fish Nile Tilapia (Oreochromis niloticus)

Matsuoka

Kobayashi

Kihara

et al. 2007

Molecular Reproduction Devel

View full text Add to dashboard Cite

Polo-like kinase 1 (Plk1) and NIMA-related kinase 2 (Nek2) are serine/threonine kinases that are involved in the G2/M phase transition of the cell cycle in vertebrates. Although they also play critical roles in the regulation of spermatogenesis and oogenesis, their characterization in meiosis has not been elucidated fully, particularly in teleost fish. To investigate the evolutionary significance of serine/threonine kinases in the reproductive system of fish, we cloned the cDNAs of Plk1 and Nek2 from a Nile tilapia (Oreochromis niloticus) testicular cDNA library. Tilapia Plk1 encodes a protein of 582 amino acids that shares 75% homology with human Plk1, while tilapia Nek2 encodes a putative protein of 446 amino acids that shares 70% homology with human Nek2. Analyses of tissue distribution by RT-PCR and Southern blotting revealed that Plk1 and Nek2 are strongly expressed in the ovary and testis. Northern blot analysis revealed two Nek2 transcripts in the ovary and testis with different expression patterns, which indicates the presence of two structural variants for tilapia Nek2. Moreover, the localization of Plk1 and Nek2 mRNAs in tilapia gonads was determined by in situ hybridization analysis. In the ovary, Plk1 and Nek2 were expressed predominantly in oocytes. In the testis, on the other hand, Plk1 was expressed in primary spermatocytes, while Nek2 was generally expressed in primary and secondary spermatocytes. These results suggest that Plk1 and Nek2 are key factors in the progression of meiosis in fish.

show abstract

Spermatogenesis in testis primary cell cultures of the tilapia (Oreochromis niloticus)

Abstract: Spermatogenesis in vertebrates is

Cited by 29 publications

References 36 publications

Flow-Cytometric Isolation of Testicular Germ Cells from Rainbow Trout (Oncorhynchus mykiss) Carrying the Green Fluorescent Protein Gene Driven by Trout vasa Regulatory Regions

Flow-Cytometric Isolation of Testicular Germ Cells from Rainbow Trout (Oncorhynchus mykiss) Carrying the Green Fluorescent Protein Gene Driven by Trout vasa Regulatory Regions

Lectin‐binding pattern as tool to identify and enrich specific primary testis cells of the tilapia (Oreochromis niloticus) and medaka (Oryzias latipes)

Molecular cloning of Plk1 and Nek2 and their expression in mature gonads of the teleost fish Nile Tilapia (Oreochromis niloticus)

Contact Info

Product

Resources

About