Project description:The maturation-inducing hormone 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) was first identified in the amago salmon. However, although carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase (CR/20beta-HSD) was reported to convert 17alpha-hydroxyprogesterone (17alpha-P) to DHP in rainbow trout, we previously found that CR/20beta-HSD mRNA was not up-regulated in stimulated granulosa cells from masu salmon, which suggests that DHP is synthesized by a different enzyme. Accordingly, the present study aimed to identify the specific 20beta-hydroxysteroid dehydrogenase (20beta-HSD) responsible for DHP production by granulosa cells during final oocyte maturation in masu salmon. Granulosa layers were isolated from ovarian follicles at one month before ovulation and incubated with or without forskolin, which was used to mimic luteinizing hormone. Subsequent RNA-sequencing yielded ~12 million reads, with an average length of 51 bp, and 71,062 contigs of >100 bp were constructed. Of the 953 contigs that were exclusively constructed from the reads of forskolin-induced granulosa layers, tBlastx analysis identified one contig (#f103496) that matched 17beta-hydroxysteroid dehydrogenase type 12. We found that mammalian cells transfected with full-length omhsd17beta12l exhibited considerable 20beta-HSD activity, as indicated by efficient conversion of exogenous 17alpha-P to DHP. In addition, we found that omhsd17beta12l mRNA levels were consistently low in follicles during vitellogenic growth; however, the levels increased significantly during final oocyte maturation. The levels of omhsd17beta12l mRNA were also considerably increased in granulosa layers in which 20beta-HSD activity was induced by salmon pituitary extract. Therefore, we suggest that omhsd17beta12l, not CR/20beta-HSD, is the 20beta-HSD responsible for DHP production by granulosa cells in masu salmon during final oocyte maturation. Comparison of mRNA levels between control and forskolin-incubated sample.
Project description:Chinook salmon (Oncorhynchus tshawytscha) display the greatest variability of return times to freshwater of all Pacific salmon. Populations return to freshwater for spawning at many different times of year, resulting in segregated populations that may use differing molecular pathways for these large behavioral and physiological differences. Using a population of Chinook from California’s Central Valley, we sought to generate novel expressed sequences using Long Serial Analysis of Gene Expression (LongSAGE). We constructed three LongSAGE libraries from brains of samples caught in the spring and fall in freshwater and from the ocean. Using cDNA libraries from Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), we were able to assign 59% of putatively differentially expressed tags to genes. Additionally, we tested the expression levels of seven genes, indicated by LongSAGE to be putatively differentially expressed between the fall and spring, and found none significantly differentially expressed. This study is the first to apply LongSAGE to salmon and provides a framework for conducting future research on gene expression differences between Chinook salmon of different populations, as well as underlying mechanisms of differing physiology and behavior. Keywords: seasonal difference