Male germ cells of the Pacific oyster Crassostrea gigas: flow cytometry analysis, cell sorting and molecular expression

¹ UMR 100 Ifremer Physiologie et Ecophysiologie des Mollusques marins, IFR 146 ICORE, Université de Caen Basse Normandie, 14032 Caen Cedex, France
² Centre de Microscopie appliquée à la Biologie, IFR 146 ICORE, Université de Caen Basse Normandie, 14032 Caen Cedex, France

^a Corresponding author: clothilde.heude@unicaen.fr

Received: 25 January 2011
Accepted: 11 March 2011

Abstract

A technique was developed for dissection and isolation of male germ cells in the oyster Crassostrea gigas. This procedure can provide cells for the exploration of processes involved in the reproductive physiology of bivalves. Spermatogonia were chosen because of their essential role in spermatogenesis and the impact of gonia proliferation on reproductive effort. A non lethal method for determining sex and reproductive cycle stage was first validated in oysters. This first step was essential in order 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 each fraction was ratified by electron microscopy and by means of a 2-parameter flow cytometry procedure (DNA and mitochondrial staining). A significant enrichment in spermatogonia and spermatocytes was confirmed by the increased expression of markers of proliferative cells (proliferative cell nuclear antigen, PCNA) and early germ cells (oyster vasa-like gene). A preliminary cell sorting procedure is also reported, which was applied to fractions enriched in spermatogonia.