Elisabeth Bon

Plasma Vitellogenin Levels during the Annual Reproductive Cycle of the Female Rainbow Trout (Oncorhynchus mykiss): Establishment and Validation of an ELISA

Rainbow trout, Oncorhynchus mykiss, vitellogenin (Vtg) was purified from plasma of E2-treated male by direct anion exchange chromatography and some of its biochemical characteristics were studied. Our results demonstrated that, under SDS-PAGE conditions, rainbow trout Vtg was composed of two molecular forms of 390 and 176 kDa representing, respectively, the dimeric form and the monomeric from of the molecule. The purified Vtg was used to raise a polyclonal antibody for Vtg (anti-Vtg). Using this anti-Vtg, a competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of rainbow trout Vtg. The practical sensitivity range of this ELISA was 20-320 ng/ml (80-20% of binding) and the detection limit was 9 ng/ml. The intra- and the inter-assay coefficients of variation (at 50% of binding) were estimated at 1.8% (n = 10) and 3.9% (n = 13), respectively. This ELISA was validated by detecting changes in Vtg levels in rainbow trout at different physiological stages, as well as in 2-year-old female rainbow trout throughout the reproductive cycle.

Oenococcus oeni Genome Plasticity Is Associated with Fitness

ABSTRACT Oenococcus oeni strains are well-known for their considerable phenotypic variations in terms of tolerance to harsh wine conditions and malolactic activity. Genomic subtractive hybridization (SH) between two isolates with differing enological potentials was used to elucidate the genetic bases of this intraspecies diversity and identify novel genes involved in adaptation to wine. SH revealed 182 tester-specific fragments corresponding to 126 open reading frames (ORFs). A large proportion of the chromosome-related ORFs resembled genes involved in carbohydrate transport and metabolism, cell wall/membrane/envelope biogenesis, and replication, recombination, and repair. Six regions of genomic plasticity were identified, and their analysis suggested that both limited recombination and insertion/deletion events contributed to the vast genomic diversity observed in O. oeni. The association of selected sequences with adaptation to wine was further assessed by screening a large collection of strains using PCR. No sequences were found to be specific to highly performing (HP) strains alone. However, there was a statistically significant positive association between HP strains and the presence of eight gene sequences located on regions 2, 4, and 5. Gene expression patterns were significantly modified in HP strains, following exposure to one or more of the common stresses in wines. Regions 2 and 5 showed no traces of mobile elements and had normal GC content. In contrast, region 4 had the typical hallmarks of horizontal transfer, suggesting that the strategy of acquiring genes from other bacteria enhances the fitness of O. oeni strains.

Effects of accelerated photoperiod regimes on the reproductive cycle of the female rainbow trout: II Seasonal variations of plasma gonadotropins (GTH I and GTH II) levels correlated with ovarian follicle growth and egg size

Female rainbow trout were exposed over their second reproductive cycle to a simulated natural photoperiod (control group) and to two accelerated photoperiod regimes (S9 and S6 groups). Early spawning was achieved in both accelerated groups, coupled, however, with a reduction of mean egg size. To investigate this reduction of egg size, circulating levels of GTHs and two indices of ovarian growth (gonadosomatic index and follicle diameter) were regularly measured in association with histological studies of structural changes in ovarian follicles. Regardless of the photoperiod regime, the seasonal profile of plasma GTH I levels appeared to be multiphasic. The successive transient elevations in GTH I levels appeared to be connected with the initiation of ovarian growth and vitellogenesis and also, with the synchronization of the late stages of maturation and ovulation. In contrast, the seasonal profile of plasma GTH II levels was monophasic, with a single peak at ovulation, confirming that GTH II is not associated with ovarian growth but promotes gamete maturation and release. Our results demonstrate that the reduction of egg size cannot be due to a deficient secretion of GTH I in plasma, since GTH I levels were much higher during vitellogenesis in both accelerated groups, but rather to an alteration of ovarian follicle growth during the late stages of vitellogenesis. Finally, the early and middle stages of ovarian growth appeared to be photosensitive periods, whereas the late stages were less so, and appeared to be controlled rather by an endogenous biological clock synchronized by the photoperiod.

Vitellogenin receptors during Vitellogenesis in the rainbow trout Oncorhynchus mykiss

Rainbow trout vitellogenin receptors have been characterized by ligand blotting and Scatchard analysis. Their evolution has been studied over a reproductive cycle in a broodstock of 2-year-old females. The receptors were prepared from ovarian membrane homogenates and were solubilized using n-octyl-beta-D-glucopyranoside. The visualization of the receptor by ligand blotting using 125iodine-vitellogenin after sodium dodecyl sulfate electrophoresis revealed the existence of one major binding component corresponding to a protein of 113 kDa. The Scatchard transformation of the binding data revealed a single class of binding sites with an apparent Kd of 1.8 x 10(-8) M/L. The variations of the binding characteristics (Kd and maximum binding) were investigated during vitellogenesis. This study revealed that the Kd was not affected by oocyte growth during vitellogenesis, but was highly decreased in ovulated eggs. The receptor number increased during the same period from 35 to 860 fM per oocyte, while the receptor number per mm2 of oocyte membrane surface was doubled during the same period.

Intraspecific diversity of Oenococcus oeni strains determined by sequence analysis of target genes

Using molecular techniques and sequencing, we studied the intraspecific diversity of Oenococcus oeni, a lactic acid bacterium involved in red winemaking. A relationship between the phenotypic and genotypic characterization of 16 O. oeni strains isolated from wine with different levels of enological potential was shown. The study was based on the comparative genomic analysis by subtractive hybridization between two strains of O. oeni with opposite enological potential. The genomic sequences obtained from subtractive hybridization were amplified by polymerase chain reaction and sequenced for the 16 strains. A considerable diversity among strains of O. oeni was observed.

Characterization of an acquired dps‐containing gene island in the lactic acid bacterium Oenococcus oeni

Aims:  To identify novel actors responsible for the marked adaptation of the Oenococcus oeni species to its environment.