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Male germ cells of the Pacific oyster Crassostreagigas: flow cytometry analysis, cell sorting and molecularexpression

Published online by Cambridge University Press:  14 June 2011

Alban Franco ,
Kristell Kellner ,
Michel Mathieu ,
Christophe Lelong ,
Didier Goux  and
Clothilde Heude Berthelin
Alban Franco
Affiliation:
UMR 100 Ifremer Physiologie et Ecophysiologie des Mollusques marins, IFR 146 ICORE, Université de Caen Basse Normandie, 14032 Caen Cedex, France
Kristell Kellner
Affiliation:
UMR 100 Ifremer Physiologie et Ecophysiologie des Mollusques marins, IFR 146 ICORE, Université de Caen Basse Normandie, 14032 Caen Cedex, France
Michel Mathieu
Affiliation:
UMR 100 Ifremer Physiologie et Ecophysiologie des Mollusques marins, IFR 146 ICORE, Université de Caen Basse Normandie, 14032 Caen Cedex, France
Christophe Lelong
Affiliation:
UMR 100 Ifremer Physiologie et Ecophysiologie des Mollusques marins, IFR 146 ICORE, Université de Caen Basse Normandie, 14032 Caen Cedex, France
Didier Goux
Affiliation:
Centre de Microscopie appliquée à la Biologie, IFR 146 ICORE, Université de Caen Basse Normandie, 14032 Caen Cedex, France
Clothilde Heude Berthelin*
Affiliation:
UMR 100 Ifremer Physiologie et Ecophysiologie des Mollusques marins, IFR 146 ICORE, Université de Caen Basse Normandie, 14032 Caen Cedex, France
*
aCorresponding author:clothilde.heude@unicaen.fr
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Abstract

A technique was developed for dissection and isolation of male germ cells in the oysterCrassostrea gigas. This procedure can provide cells for the explorationof processes involved in the reproductive physiology of bivalves. Spermatogonia werechosen because of their essential role in spermatogenesis and the impact of goniaproliferation on reproductive effort. A non lethal method for determining sex andreproductive cycle stage was first validated in oysters. This first step was essential inorder to constitute a homogeneous pool of oysters at the same stages of gametogenesis.Germ cell fractions were then obtained from a density gradient, and enrichment of eachfraction was ratified by electron microscopy and by means of a 2-parameter flow cytometryprocedure (DNA and mitochondrial staining). A significant enrichment in spermatogonia andspermatocytes was confirmed by the increased expression of markers of proliferative cells(proliferative cell nuclear antigen, PCNA) and early germ cells (oyster vasa-like gene). Apreliminary cell sorting procedure is also reported, which was applied to fractionsenriched in spermatogonia.

Keywords

gametogenesisgerm linemitochondrial stainingDNA contentVasaproliferative cell nuclear antigenMolluscaCrassostrea gigas

Information

Type
Research Article
Information
Aquatic Living Resources , Volume 24 , Issue 3: Physiomar10 , July 2011 , pp. 237 - 245
Copyright
© EDP Sciences, IFREMER, IRD 2011

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