Proliferating cell nuclear antigen in gonad and associated storage tissue of the Pacific oyster Crassostrea gigas: seasonal immunodetection and expression in laser microdissected tissues

Abstract

To understand the processes involved in tissue remodeling associated with the seasonal reproductive cycle of the oyster Crassostrea gigas, we used immunodetection and expression measurements of proliferating cell nuclear antigen (PCNA). The expression of the PCNA gene was measured by real-time polymerase chain reaction in the whole gonadal area compared with laser microdissected gonad and storage tissue. Results underlined the advantage of the laser microdissection approach to detect expression, mainly for early stages of spermatogenesis. In the storage tissue, PCNA expression was reduced in the gonadal tubules, but immunolabeled hemocytes and vesicular cells were detected when the storage tissue was being restored. In the gonadal tubules, the PCNA gene was more highly expressed in males than in females. As soon as spermatogenesis was initiated, PCNA expression showed a high and constant level. In females, the expression level increased gradually until the ripe stage. The immunological approach established the involvement of peritubular cells in gonadal tubule expansion during early gametogenesis. In both sexes, gonial mitosis was immunodetected throughout the reproductive cycle. In males, the occurrence of two types of spermatogonia was ascertained by differential immunolabeling, and intragonadal somatic cell proliferation was noted. As expected, immunolabeling was never observed from stage II spermatocytes to spermatozoa. In females, positively stained cells were detected from oogonia to growing oocytes with various labeled intracellular locations.

Expression of a Natural Antisense Transcript of Cg-Foxl2 during the Gonadic Differentiation of the Oyster <b><i>Crassostrea gigas</i></b>: First Demonstration in the Gonads of a Lophotrochozoa Species

“…It may play a role in key steps of the gonadic differentiation of C. gigas , (i) at the first gonadic establishment in spats of around 1-2-months of age, when primordial germ cells may differentiate into germinal stem cells and (ii) in adults for vitellogenesis in females. The role of this factor in the oyster's gonadic differentiation is still not clear for many reasons: (i) the oyster sex determination is still unknown and no molecular female-or male-specific markers are actually available; (ii) the adult gametogenesis is poorly known as no advanced study on oogenesis and only few on spermatogenesis [Franco et al, 2008[Franco et al, , 2010[Franco et al, , 2011 are available; (iii) the first gonadic development is still unknown regarding both the time-window, the factors, the cells and the mechanisms involved.…”

Cellular development of the germinal epithelium during the gametogenic cycle of the golden mussel Limnoperna fortunei (Bivalvia: Mytilidae)

“…Thus, the germinal epithelium rests upon a basement membrane that separates it from the pallial tissue. Recent studies by Franco, Heude-Berthelin, Goux, Sourdaine and Mathieu (2008), Franco et al (2010) and Franco, Kellner, Goux, Mathieu and HeudeBerthelin (2011) also recognized that bivalve gonads are formed by a continuous system of branched tubules. As seen in L. fortunei, except for the gonoduct, in both males and females, the epithelium, bordering the gonadal tubules, houses germ cells and is active in gamete production.…”

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

“…The initiation of gametogenesis in males is marked by numerous gonial mitosis directly related to the quality and the quantity of gametes produced at the end of the sexual cycle. This cellular proliferation of spermatogonia is continuous throughout the gametogenetic cycle in Pacific oyster (Franco et al 2010), although the mechanisms involved in the reinitiation of spermatogenesis and regulation of gonial mitosis are still unknown in this species. These steps of the reproductive cycle depend on the quality and the quantity of gametes produced.…”

“…Oysters are not perfectly synchronous and the development of protocols for spermatogonia enrichment first requires preliminary strategies to obtain a homogeneous sample of male oysters in stage I and II of the gametogenetic cycle. At these stages, the sex of oysters is discernible from histological slides, and spermatogonia are the majority cell type in the gonadal tubules (Franco et al 2008(Franco et al , 2010.…”

“…This procedure enables the identification of germ cell types based on DNA content and mitochondrial staining. To complement the microscope observations and flow cytometry analysis, expression of the Oyster vasa-like gene (Oyvlg), a vasa ortholog gene that is a marker of early germ cells (Fabioux et al 2004), and proliferating cell nuclear antigen (PCNA), a marker of proliferative cells (Franco et al 2010), were measured in the different cell fractions obtained. The spermatogonia and spermatocyte enrichment achieved in this way led to a preliminary trial in cell sorting.…”