Sex hormone-binding globulins characterization and gonadal gene expression during sex differentiation in the rainbow trout, Oncorhynchus mykiss.

Sex hormone-binding globulin (SHBG) binds androgens and estrogens in the blood of many vertebrates, including teleost fish. In mammals, SHBG is synthetized in the liver and secreted into the blood. In fish, shbga also exhibits a hepatic expression. In salmonids, in which the gene has been duplicated, the recently discovered shbgb gene exhibits a predominantly ovarian expression. The present work aimed at gaining new insight into shbgb gene structure and expression during gonadal sex differentiation, a steroid-sensitive process, and Shbgb protein structure and binding characteristics; specifically, rainbow trout (Oncorhynchus mykiss) shbgb was analyzed. shbgb structure was analyzed in silico while expression was characterized during gonadal sex differentiation using all-male and all-female populations. We observed that shbgb gene and cognate-protein structures are similar to homologs previously described in zebrafish and mammals. The shbgb gene is predominantly expressed in differentiating female gonads, with increased expression around the end of ovarian differentiation. In the ovary, shbgb mRNA was detected in a subset of somatic cells surrounding the ovarian lamellae. Furthermore, Shbgb binds steroids with a higher selectivity than Shbga, exhibiting a higher affinity for estradiol compared to Shbga. In conclusion, Shbgb binding characteristics are clearly different from those of Shbga. Shbgb is expressed in the differentiating ovary during a period when the synthesis and action of testosterone and estradiol must be tightly regulated. This strongly suggests that Shbgb participates in the regulation of steroid metabolism and/or mediation, that is, needed during early gonadal development in rainbow trout.

Ovary-predominant wnt4 expression during gonadal differentiation is not conserved in the rainbow trout (Oncorhynchus mykiss).

The Wnt/ß-catenin pathway is crucial for ovarian differentiation in mammals, and WNT4 is an important protein that regulates this process. While the role of Wnt4 in gonadal differentiation is relatively well characterized in mammals, little is known regarding its role in teleost fish. Therefore, we investigated the potential activity of wnt4 in gonadal differentiation in rainbow trout (Oncorhynchus mykiss), focusing on the teleost and salmonid gene duplications. Phylogenetic and synteny analyses demonstrated that teleost fish possess two wnt4 genes, wnt4a and wnt4b, as a consequence of the teleost-specific whole-genome duplication (3R). In rainbow trout, we also identified an additional wnt4 gene, which is a wnt4a paralog that likely resulted from the salmonid-specific whole-genome duplication (4R). These two Wnt4a proteins (Wnt4a1 and Wnt4a2) share a high identity (>80%) with other vertebrate Wnt4 proteins, whereas Wnt4b is clearly more divergent (60% identity). During embryogenesis and adulthood, the wnt4a1/2 transcripts were expressed in various tissues, including the ovaries and testes. In contrast, wnt4b expression was restricted to the nervous system, suggesting a sub- or a neo-functionalization of this divergent paralog. During early gonadal differentiation in both males and females, the wnt4a1/2 transcripts were detected in the somatic cells surrounding the germ cells, with a slight sexual dimorphism in favor of males. These results demonstrate that, unlike mammals, rainbow trout do not display an ovary-predominant wnt4 expression profile during early gonadal differentiation.

The duplicated rainbow trout (Oncorhynchus mykiss) T-box transcription factors 1, tbx1a and tbx1b, are up-regulated during testicular development.

Tbx1 is a member of the T-box transcription factor gene family involved in embryogenesis and organogenesis. Recently, within a pan-genomic screen using rainbow trout (Oncorhynchus mykiss) cDNA microarrays, we identified a tbx1 homolog with testicular over-expression during sex differentiation. Here, we characterized two very similar rainbow trout tbx1 paralogs, tbx1a and tbx1b. In adult tissues, tbx1a expression is restricted to the gonads, with high expression in the testis, while tbx1b is more widely expressed in gonads, gills, brains, muscle, and skin. During gonadal differentiation, both genes are differentially expressed in favor of testis formation shortly after hatching. These genes are expressed in somatic cells surrounding germ cells of the differentiating testis, while no or only weak expression was observed in the differentiating ovary. tbx1a and tbx1b were also both down-regulated in the differentiating testis during feminization with estrogens and up-regulated in the differentiating ovary during masculinization with an aromatase inhibitor. These results suggest that tbx1a and tbx1b are probably involved in the regulation of testicular differentiation in rainbow trout. Since Tbx1 is known to interact with the retinoic acid (RA) signaling pathway, we also examined the effect of RA on the rainbow trout tbx1 expression pattern. Expression of tbx1a and tbx1b was down-regulated in RA-treated male gonads, suggesting that tbx1 interacts with the RA signaling pathway and thus could be involved in the control of rainbow trout gonadal differentiation.

Neurodevelopment genes in lampreys reveal trends for forebrain evolution in craniates.

The forebrain is the brain region which has undergone the most dramatic changes through vertebrate evolution. Analyses conducted in lampreys are essential to gain insight into the broad ancestral characteristics of the forebrain at the dawn of vertebrates, and to understand the molecular basis for the diversifications that have taken place in cyclostomes and gnathostomes following their splitting. Here, we report the embryonic expression patterns of 43 lamprey genes, coding for transcription factors or signaling molecules known to be involved in cell proliferation, stemcellness, neurogenesis, patterning and regionalization in the developing forebrain. Systematic expression patterns comparisons with model organisms highlight conservations likely to reflect shared features present in the vertebrate ancestors. They also point to changes in signaling systems -pathways which control the growth and patterning of the neuroepithelium-, which may have been crucial in the evolution of forebrain anatomy at the origin of vertebrates.

The L84F polymorphism in the O6-Methylguanine-DNA-Methyltransferase (MGMT) gene is associated with increased hypoxanthine phosphoribosyltransferase (HPRT) mutant frequency in lymphocytes of tobacco smokers.

OBJECTIVES: O-methylguanine-DNA-methyltransferase (MGMT) is a crucial DNA repair protein that removes DNA adducts formed by alkylating mutagens. Several coding single nucleotide polymorphisms (cSNPs) in the MGMT gene have been reported. Their biological significance, however, is not known. METHODS: We used a newly modified cloning HPRT mutant lymphocyte assay to test the hypothesis that inheritance of the L84F and I143V coding single nucleotide polymorphism in the MGMT gene is associated with increases in HPRT mutant frequency in lymphocytes of individuals exposed to alkylating agents. In addition, we expanded and sequenced 109 mutant clones to test the hypothesis that the mutation spectrum would shift to a larger percentage of base substitutions and G-->A transition mutations in cells with L84F and I143 V coding single nucleotide polymorphisms. RESULTS: We observed no significant effect for the I143 V coding single nucleotide polymorphism on mutant frequency. In contrast, we observed a significant increase in mutant frequency (P<0.01) in lymphocytes from smokers with the 84F coding single nucleotide polymorphism compared with smokers homozygous for the referent L84 wild-type allele. A multiple regression analysis indicated that the mutant frequency increased significantly as a function of the 84F coding single nucleotide polymorphism and smoking, according to the model; mutant frequency (x10)=0.90+0.618 (84F polymorphism)+0.46 (smoking) with R=0.22. Mutation spectra analysis revealed an apparent increase, which was short of statistical significance (P=0.08), in base substitutions in cells with the 84F polymorphism. CONCLUSIONS: These new data suggest that the 84F coding single nucleotide polymorphism may alter the phenotype of the MGMT protein, resulting in suboptimal repair of O-methylguanine lesions after exposure to alkylating agents.