Determination of a new index of sexual maturity (ISM) in zebra mussel using flow cytometry: interest in ecotoxicology.

The global dynamic spread of chemical contamination through the aquatic environment calls for the development of biomarkers of interest. Reproduction is a key element to be considered because it is related to the sustainability of species. Spermatogenesis is a complex process that leads to the formation of mature germ cells, whose steps and impairments need to be finely described in ecotoxicological analyses. The physiological process has been commonly described by histological analyses of gonads in different taxa. In the present paper, we describe the development of a novel technique to characterize spermatogenesis based on the analysis of the DNA content of germ cells by flow cytometry, using a DNA-intercalating agent. This new biomarker, referred to as an index of sexual maturity, proved relevant to describe the seasonal reproductive cycle of the zebra mussel, Dreissena polymorpha (Pallas, 1771), used as a sentinel species in the biomonitoring of continental waters and sensitive to highlight the reprotoxicity of carbamazepine (an anti-epileptic pharmaceutical) tested under ecosystemic conditions (mesocosms).

A crucial role in fertility for the oyster angiotensin-converting enzyme orthologue CgACE.

Angiotensin-converting enzyme (ACE) is a highly conserved metallopeptidase. In mammals, the somatic isoform governs blood pressure whereas the germinal isoform (tACE) is required for fertility. In Ecdysozoans, ACE-like enzymes are implicated in reproduction. Despite ACE orthologues being present from bacteria to humans, their function(s) remain(s) unknown in distant organisms such as Lophotrochozoans. In silico analysis of an oyster (Crassostrea gigas) EST library suggested the presence of an ACE orthologue in molluscs. Primer walking and 5'-RACE revealed that the 1.9 kb cDNA encodes CgACE, a 632 amino acid protein displaying a conserved single active site and a putative C-terminal transmembrane anchor, thus resembling human tACE, as supported by molecular modelling. FRET activity assays and Maldi-TOF spectrometry indicated that CgACE is a functional dipeptidyl-carboxypeptidase which is active on Angiotensin I and sensitive to ACE inhibitors and chloride ion concentration. Immunocytochemistry revealed that, as its human counterpart, recombinant CgACE is synthesised as a transmembrane enzyme. RT-qPCR, in-situ hybridization and immunohistochemistry shed light on a tissue, and development stage, specific expression pattern for CgACE, which is increased in the gonad during spermatogenesis. The use of ACE inhibitors in vivo indicates that the dipeptidase activity of CgACE is crucial for the oyster fertilization. Our study demonstrates that a transmembrane active ACE is present in the oyster Crassostrea gigas, and for the first time ascribes a functional role for ACE in Lophotrochozoans. Its biological function in reproduction is conserved from molluscs to humans, a finding of particular evolutionary interest especially since oysters represent the most important aquaculture resource worldwide.

Intragonadal somatic cells (ISCs) in the male oyster Crassostrea gigas: morphology and contribution in germinal epithelium structure.

The ultrastructure of somatic cells present in gonadal tubules in male oyster Crassostrea gigas was investigated. These cells, named Intragonadal Somatic Cells (ISCs) have a great role in the organization of the germinal epithelium in the gonad. Immunological detection of α-tubulin tyrosine illustrates their association in columns from the basis to the lumen of the tubule, stabilized by numerous adhesive junctions. This somatic intragonadal organization delimited some different groups of germ cells along the tubule walls. In early stages of gonad development, numerous phagolysosomes were observed in the cytoplasm of ISCs indicating that these cells have in this species an essential role in the removal of waste sperm in the tubules. Variations of lipids droplets content in the cytoplasm of ISCs were also noticed along the spermatogenesis course. ISCs also present some mitochondria with tubullo-lamellar cristae.

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

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.