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

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 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.

Keywords

gametogenesisgerm linemitochondrial stainingDNA contentVasaproliferative cell nuclear antigenMolluscaCrassostrea gigas
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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References

Badariotti, F., Kypriotou, M., Lelong, C., Dubos, M.-P., Renard, E., Galera, P., Favrel, P., 2006, The phylogenetically conserved molluscan chitinase-like protein 1 (Cg-Clp1), homologue of human HC-gp39, stimulates proliferation and regulates synthesis of extracellular matrix components of mammalian chondrocytes. J. Biol. Chem. 281, 29583–29596. CrossRefGoogle Scholar
Bellvé, A.R., Cavicchia, J.C., Millette, C.F., O’Brien, D.A., Bhatnagar, Y.M., Dym,, M., 1977, Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. J. Cell. Biol. 74, 68–85. CrossRefGoogle ScholarPubMed
Berthelin, C., Kellner, K., Mathieu, M., 2000, Histological characterization and glucose incorporation into glycogen of the Pacific oyster Crassostrea gigas storage cells. Mar. Biotechnol. 2, 136–145. Google ScholarPubMed
Celeghini, E. C., de Arruda, R. P., de Andrade, A. F., Nascimento, J., Raphael, C. F., 2007, Practical techniques for bovine sperm simultaneous fluorimetric assessment of plasma, acrosomal and mitochondrial membranes. Reprod. Domest. Anim. 42, 479–488. CrossRefGoogle Scholar
Com, E., Evrard, B., Roepstorff, P., Aubry, F., Pineau, C., 2003, New insights into the rat spermatogonial proteome: identification of 156 additional proteins. Mol. Cell. Proteomics 2, 248–261. CrossRefGoogle ScholarPubMed
Fabioux, C., Pouvreau, S., LeRoux, F., Huvet, A., 2004, The oyster vasa-like gene: a specific marker of the germline in Crassostrea gigas. Biochem. Biophys. Res. Com. 315, 897–904. CrossRefGoogle ScholarPubMed
Fabioux, C., Corporeau, C., Quillien, V., Favrel, P., Huvet, A., 2009, In vivo RNA interference in oyster-vasa silencing inhibits germ cell development. FEBS J. 276, 2566–2573. CrossRefGoogle Scholar
Franco, A., Heu de Berthelin, C., Goux, D., Sourdaine, P., Mathieu, M., 2008, Fine structure of the early stages of spermatogenesis in the Pacific oyster, Crassostrea gigas (Mollusca, Bivalvia). Tissue Cell 40, 251–260. CrossRefGoogle Scholar
Franco, A., Jouaux, A., Mathieu, M., Sourdaine, P., Lelong, C., Kellner, K., Heu de Berthelin, C., 2010, Proliferating cell nuclear antigen in gonad and associated storage tissue of the Pacific oyster Crassostrea gigas: seasonal immunodetection and expression in laser microdissected tissues. Cell Tissue Res. 340, 201–210. CrossRefGoogle Scholar
Hanquet-Dufour, A.C., Kellner, K., Heude, C., Naimi, A., Mathieu, M., Poncet, J.M., 2006, Cryopreservation of Crassostrea gigas vesicular cells: viability and metabolic activity. Cryobiology 53, 28–36. CrossRefGoogle ScholarPubMed
Heude-Berthelin, C., Laisney, J., Espinosa, J., Martin, O., Hernandez, G., Mathieu, M., Kellner, K., 2001, Storage and reproductive strategy in Crassostrea gigas of two different growing area (Normandy and Atlantic shore, France). Invertebr. Reprod. Dev. 40, 79–86. CrossRefGoogle Scholar
Jouaux, A., Heude-Berthelin, C., Sourdaine, P., Mathieu, M., Kellner, K., 2010, Gametogenic stages in triploid oysters Crassostrea gigas: Irregular locking of gonial proliferation and subsequent reproductive effort. J. Exp. Mar. Biol. Ecol. 395, 162–170. CrossRefGoogle Scholar
Kellner, K., Heude-Berthelin, C., Mathieu, M., 2002, Immunocytochemical demonstration of glucagon-like peptides in Mytilus edulis cerebral ganglia and an in vitro effect of vertebrate glucagon on glycogen metabolism. Tissue Cell 34, 109–116. CrossRefGoogle Scholar
Labreuche, Y. C., Lambert, P., Soudant, V., Boulo, A., Huvet, J., Nicolas, L., 2006, Cellular and molecular hemocyte responses of the Pacific oyster, Crassostrea gigas, following bacterial infection with Vibrio aestuarianus strain 01/32. Microbes Infect. 8, 2715–2724. CrossRefGoogle ScholarPubMed
Le Quéré, H., Herpin, A., Huvet, A., Lelong, C., Favrel, P., 2009, Structural and functional characterizations of an Activin type II receptor orthologue from the Pacific oyster Crassostrea gigas. Gene 436, 101–107. CrossRefGoogle ScholarPubMed
Lelong, C., Badariotti, F., Le Quéré, H., Rodet, F., Dubos, M.P., Favrel, P., 2007., Cg-TGF-beta, a TGF-beta/activin homologue in the Pacific oyster Crassostrea gigas, is involved in immunity against Gram-negative microbial infection. Dev. Comp. Immunol. 31, 30–38. CrossRefGoogle ScholarPubMed
Loir, M., 1994, In vitro approach to the control of spermatogonia proliferation in the trout. Mol. Cell. Endocrinol. 102, 141–150. CrossRefGoogle Scholar
Loir, M., 1999, Spermatogonia of rainbow trout: I. Morphological characterization, mitotic activity, and survival in primary cultures of testicular cells. Mol. Reprod. Dev. 53, 422–433. 3.0.CO;2-V>CrossRefGoogle ScholarPubMed
Loppion, G., Crespel, A., Martinez, A.S.S., Auvray, P., Sourdaine, P., 2008, Study of the potential spermatogonial stem cell compartment in dogfish testis, Scyliorhinus canicula L. Cell Tissue Res. 332, 533–542. CrossRefGoogle Scholar
Mann, R., 1979, Some biochemical and physiological aspect of growth and gametogenesis in Crassostrea gigas and Ostrea edulis at sustained elevated temperatures. J. Mar. Biol. Assoc. UK 59, 95–100. CrossRefGoogle Scholar
Misamore, M. J., Stein, K. K., Lynn, J.W., 2006, Sperm incorporation and pronuclear development during fertilization in the freshwater bivalve Dreissena polymorpha. Mol. Reprod. Dev. 73, 1140–1148. CrossRefGoogle ScholarPubMed
Parapanov, R., Nusslé, S., Hausser, J., Vogel, P., 2008, Histological description of seminiferous epithelium and cycle length of spermatogenesis in the water shrew Neomys fodiens (Mammalia: Soricidae). Anim. Reprod. Sci. 107, 148–160. CrossRefGoogle Scholar
Petit, J.M., Ratinaud, M.H., Cordelli, E., Spano, M., Julien, R., 1995, Mouse testis cell sorting according to DNA and mitochondrial changes during spermatogenesis. Cytometry 19, 304–312. CrossRefGoogle ScholarPubMed
Ruiz, C., Martinez, D., Mosquera, G., Abad, M., Sanchez, J.L., 1992, Seasonal variations in condition, reproductive activity and biochemical composition of the flat oyster, Ostrea edulis, from San Cibran (Galicia, Spain). Mar. Biol. 112, 67–74. CrossRefGoogle Scholar
Silandre, D., Delalande, C., Durand, P., Carreau, S., 2007, Three promoters PII, PI.f, and PI.tr direct the expression of aromatase (cyp19) gene in male rat germ cells. J. Mol. Endocrinol. 39, 169–181 CrossRefGoogle ScholarPubMed
Suter, L., Koch, E., Bechter, R., Bobadilla, M., 1997, Three-parameter flow cytometric analysis of rat spermatogenesis. Cytometry 27, 161–168. 3.0.CO;2-J>CrossRefGoogle Scholar