17β-HSD Type 12-Like Is Responsible for MaturationInducing Hormone Synthesis During Oocyte Maturation in Masu Salmon

The maturation-inducing hormone 17(alpha), 20(beta)-dihydroxy-4-pregnen-3-one (DHP) was first identified in the amago salmon. Although carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase (CR/20b-HSD) was reported to convert 17 alpha-hydroxyprogesterone (17OHP) to DHP in rainbow trout, we previously found that CR/20 beta-HSD messenger RNA (mRNA) was not upregulated in stimulated granulosa cells from masu salmon, which suggested that DHP is synthesized by a different enzyme. Accordingly, the current study aimed to identify the specific 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) responsible for DHP production by granulosa cells during final oocyte maturation in masu salmon. RNA sequencing was performed on granulosa layers that were isolated from ovarian follicles at 1 month before ovulation and incubated with or without forskolin, which was used to mimic luteinizing hormone, and similar to 12 million reads were obtained, which yielded 71,062 contigs of > 100 bp. tBlastx analysis identified 1 contig (#f103496)as similar to 17 beta-hydroxysteroid dehydrogenase type 12 (hsd17 beta 12); however, because the full-length #f103496 sequence was different from hsd17 beta 12, it was termed hsd17 beta 12-like (hsd17 beta 12l). We found that mammalian cells transfected with full-length hsd171 beta 2l exhibited considerable 20b-HSD activity, as indicated by efficient conversion of exogenous 17OHP to DHP. In addition, we found that hsd17 beta 12l mRNA levels were consistently low in follicles during vitellogenic growth; however, the levels increased significantly during final oocytematuration. The levels of hsd17 beta 12lmRNA were also considerably increased in granulosa layers in which 20 beta-HSD activity was induced by salmon pituitary extract. Therefore, we suggest that hsd17 beta 12l, not CR/20 beta-HSD, is the 20 beta-HSD responsible for DHP production by granulosa cells in masu salmon during final oocytematuration.