ABSTRACT
This study is the first demonstration of successful post-thawing development to reproduction stage of diploid cryopreserved larvae in an aquatic invertebrate. Survival, growth and reproductive performances were studied in juvenile and adult Pacific oysters grown from cryopreserved embryos. Cryopreservation was performed at three early stages: trochophore (13±2 hours post fertilization: hpf), early D-larvae (24±2 hpf) and late D-larvae (43±2 hpf). From the beginning (88 days) at the end of the ongrowing phase (195 days), no mortality was recorded and mean body weights did not differ between the thawed oysters and the control. At the end of the growing-out phase (982 days), survival of the oysters cryopreserved at 13±2 hpf and at 43±2 hpf was significantly higher (P<0.001) than those of the control (non cryopreserved larvae). Only the batches cryopreserved at 24±2 hpf showed lower survival than the control. Reproductive integrity of the mature oysters, formely cryopreserved at 13±2 hpf and 24±2 hpf, was estimated by the sperm movement and the larval development of their offspring in 13 crosses gamete pools (five males and five females in each pool). In all but two crosses out of 13 tested (P<0.001), development rates of the offspring were not significantly different between frozen and unfrozen parents. In all, the growth and reproductive performances of oysters formerly cryopreserved at larval stages are close to those of controls. Furthermore, these performances did not differ between the three initial larval stages of cryopreservation. The utility of larvae cryopreservation is discussed and compared with the cryopreservation of gametes as a technique for selection programs and shellfish cryobanking.
Subject(s)
Crassostrea/physiology , Invertebrates/physiology , Larva/physiology , Ostreidae/physiology , Reproduction/physiology , Animals , Body Weight/physiology , Crassostrea/drug effects , Cryopreservation/methods , Cryoprotective Agents/pharmacology , Female , Fertilization/physiology , Freezing , Invertebrates/drug effects , Larva/drug effects , Male , Ostreidae/drug effects , Reproduction/drug effects , Sperm Motility/physiologyABSTRACT
A Foxl2 ortholog has been identified in a lophotrochozoa, the pacific oyster, which is a successive irregular hermaphrodite mollusc. Its cDNA has been called Cg-Foxl2 (Crassostrea gigas Foxl2) and the deduced protein sequence is 367aa long. This sequence contains the conserved domain Forkhead box and its gene is devoid of intron at least in the first 926 bp of the cDNA, as found for Foxl2 factors. Real time PCR and in situ hybridization have shown a gonadic male and female Cg-Foxl2 expression which increases during the adult gametogenetic cycle for both sexes, but with a significant increase occurring earlier in females than in males. In females this increase corresponds to the vitellogenetic stage. During development, a peak of Cg-DMl (a potential factor of the male gonadic differentiation) and Oyvlg (a germ cell marker) expression and a significant decrease of Cg-Foxl2 expression were observed after metamorphosis in 1-1.5-month-old spats, a period of development when primordial germ cells may differentiate into germinal stem cells during the first gonadic establishment.
Subject(s)
Crassostrea/growth & development , Crassostrea/genetics , Forkhead Transcription Factors/genetics , Gametogenesis/genetics , Gene Expression Regulation, Developmental , Amino Acid Sequence , Animals , Biomarkers/metabolism , Cloning, Molecular , Crassostrea/cytology , Female , Forkhead Transcription Factors/chemistry , Forkhead Transcription Factors/metabolism , Humans , Male , Molecular Sequence Data , Ovary/cytology , Ovary/growth & development , Ovary/metabolism , Phylogeny , Sequence Analysis, DNA , Sex Determination ProcessesABSTRACT
The Pacific oyster Crassostrea gigas is a successive not systematic protandric hermaphrodite. Searching for an ortholog to Dmrt1, a conserved sex determinism factor, we have identified the first complete cDNA of a DM factor in Lophotrochozoa which we have called Cg-DMl (Crassostrea gigas DMRT-like). It is 359aa long, with the DM domain common to all the family factors, and one DMA domain specific to members such as Dmrt4 and Dmrt5. Its gene presents one intron of 598 bp. Real time PCR and in situ hybridization have shown that Cg-DMl was expressed in both sexes, with a significantly higher expression in male than in female gonads at the end of the adult gametogenetic cycle and that a significant peak of expression was observed in spat between 1 and 2 months of age. These results suggest that Cg-DMl may be involved in the development of the gonad and may constitute preliminary clues for future work in order to better understand DM protein evolution.
