Early differentiation in rainbow trout gonads was investigated by expression profiling and in situ hybridization (ISH). Expression of cyp19a1 and fst in females and sox9a1 in males were sexually dimorphic between 32 to 35 days post-fertilization (dpf). After 35 dpf, the differentiation proceeded with sexually dimorphic profiles for sox9a2, dmrt1, cyp11b2.1, amh in males and foxl2a, foxl2b, hsd3b1, inha in females. cyp17a1, cyp11a1, star, nr5a1b increased only after 40 dpf in both sexes with a slightly higher expression in females. cyp19a1 expression was localized in a cluster of somatic cells in the ventral side of female gonads, and sox9a2 and amh in somatic cells surrounding the germ cells, at 28 dpf and thereafter, both in male and female gonads. cyp11b2.1, cyp17a1, and cyp11a1 expressions were only detected in scattered somatic cells in males after 46 dpf. This confirms the early implication of cyp19a1 in trout ovarian differentiation and suggests that early testicular differentiation does not need androgen production. Developmental Dynamics

The overall understanding of the sex differentiation cascade in vertebrates is still growing slowly, probably because of the variety of vertebrate models used and the number of molecular players yet to be discovered. Finding conserved mechanisms among vertebrates should provide a better view of the key factors involved in this process. To this end, we used real-time reverse transcription-polymerase chain reaction to produce a temporal map of fluctuations in mRNA expression of 102 genes during sex differentiation and early gametogenesis in the rainbow trout (Oncorhynchus mykiss). We used these 102 temporal gene expression patterns as a basis for a hierarchical clustering analysis to find characteristic clusters of coexpressed genes. Analysis of some of these gene clusters suggested a conserved overall expression profile between the sex differentiation cascade in fish and mammals. Among these conserved molecular mechanisms, sox9, dmrt1, amh, nr5a1, nr0b1, igf1, and igf1ra are, for instance, characterized as early expressed genes involved in trout testicular differentiation as it is known or suggested in mammals. On the contrary, foxl2, fst, and lhr are characterized as early expressed genes during trout ovarian differentiation, as also found in mammals. Apart from this high conservation, our analysis suggests some potential new players, such as the fshb subunit gene, which is detected here for the first time, to our knowledge, in the female differentiating gonad of a vertebrate species and displays a specific overexpression that coincides in timing with the occurrence of first oocyte meioses, or the pax2 gene, which displays an early and testis-specific expression profile.

Operationally tolerant patients (TOL) display a higher number of blood B cells and transcriptional B cell signature. As they rarely develop an allo-immune response, they could display an abnormal B cell differentiation. We used an in vitro culture system to explore T-dependent differentiation of B cells into plasma cells. B cell phenotype, apoptosis, proliferation, cytokine, immunoglobulin production and markers of differentiation were followed in blood of these patients. Tolerant recipients show a higher frequency of CD20 þ CD24 hi CD38 hi transitional and CD20 þ CD38 lo CD24 lo na€ ıve B cells compared to patients with stable graft function, correlating with a decreased frequency of CD20 À CD38 þ CD138 þ differentiated plasma cells, suggestive of abnormal B cell differentiation. B cells from TOL proliferate normally but produce more IL-10. In addition, B cells from tolerant recipients exhibit a defective expression of factors of the end step of differentiation into plasma cells and show a higher propensity for cell death apoptosis compared to patients with stable graft function. This in vitro profile is consistent with down-regulation of B cell differentiation genes and anti-apoptotic B cell genes in these patients in vivo. These data suggest that a balance between B cells producing IL-10 and a deficiency in plasma cells may encourage an environment favorable to the tolerance maintenance.

Whereas a B cell-transcriptional profile has been recorded for operationally tolerant kidney graft patients, the role that B cells have in this tolerance has not been reported. In this study, we analyzed the role of B cells from operationally tolerant patients, healthy volunteers, and kidney transplant recipients with stable graft function on T cell suppression. Proliferation, apoptosis, and type I proinflammatory cytokine production by effector CD4 + CD25 2 T cells were measured after anti-CD3/anti-CD28 stimulation with or without autologous B cells. We report that B cells inhibit CD4 + CD25 2 effector T cell response in a dosedependent manner. This effect required B cells to interact with T-cell targets and was achieved through a granzyme B (GzmB)-dependent pathway. Tolerant recipients harbored a higher number of B cells expressing GzmB and displaying a plasma cell phenotype. Finally, GzmB + B-cell number was dependent on IL-21 production, and B cells from tolerant recipients but not from other patients positively regulated both the number of IL-21 + T cells and IL-21 production, suggesting a feedback loop in tolerant recipients that increases excessive B cell activation and allows regulation to take place. These data provide insights into the characterization of B cell-mediated immunoregulation in clinical tolerance and show a potential regulatory effect of B cells on effector T cells in blood from patients with operationally tolerant kidney grafts.

FOXL2 is a forkhead transcription factor involved in ovarian development and function. Here, we have studied the evolution and pattern of expression of the FOXL2 gene and its paralogs in fish. We found well conserved FoxL2 sequences (FoxL2a) and divergent genes, whose forkhead domains belonged to the class L2 and were shown to be paralogs of the FoxL2a sequences (named FoxL2b). In the rainbow trout, FoxL2a and FoxL2b were specifically expressed in the ovary, but displayed different temporal patterns of expression. FoxL2a expression correlated with the level of aromatase, the key enzyme in estrogen production, and an estrogen treatment used to feminize genetically male individuals elicited the up-regulation of both paralogs. Conversely, androgens or an aromatase inhibitor down-regulated FoxL2a and FoxL2b in females. We speculate that there is a direct link between estrogens and FoxL2 expression in fish, at least during the period where the identity of the gonad is sensitive to hormonal treatments.