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.