20b-hydroxysteroid dehydrogenase and CYP19A1 are differentially expressed during maturation in Atlantic cod (Gadus morhua) C Mittelholzer, E Andersson 1 , D Consten, T Hirai 2 , Y Nagahama 3 and B Norberg Institute of Marine Research, Austevoll Research Station, N-5392 Storebø, Norway 1 Institute of Marine Research, N-5817 Bergen, Norway 2 Department of Biosciences, Teikyo University of Science and Technology, Uenohara, Japan 3 Laboratory of Reproductive Biology, National Institute for Basic Biology, 444-8585 Okazaki, Japan (Correspondence should be addressed to C Mittelholzer who is now at University of Basel, Klingelbergstrasse 60, CH-4056 Basel, Switzerland; Email: christian.mittelholzer@unibas.ch) D Consten is now at St Elisabeth Hospital, PO Box 90151, 5000 LC Tilburg, The Netherlands Abstract In order to better quantify the molecular mechanisms regulating final oocyte maturation and spawning, complete coding sequences with partially or fully untranslated regions for the steroidogenic enzymes, cytochrome P450 aromatase and 20b- hydroxysteroid dehydrogenase, were cloned from ovaries of Atlantic cod (Gadus morhua). The nucleotide and amino acid sequences showed high homologies with the corresponding sequences of other fish species, and conserved features important for functionality were identified in both predicted proteins. The sequences of the corresponding genomic loci were also determined, allowing the design of mRNA-specific quantitative PCR assays. As a reference gene for the real-time RT-PCR assays, eukaryotic elongation factor 1a was chosen, and the mRNA as well as the genomic sequence was determined. In addition, a real-time quantitative PCR assay for the 18S rRNA was adapted to be used in cod. Analysis of immature and maturing female cod from July to January respectively showed that the enzyme genes showed the expected quantitative changes associated with physiological regulation. However, mRNA for eukaryotic elongation factor 1a, and to a lesser extent even 18S rRNA, showed variable expression in these samples as well. To find accurate standards for real-time PCR in such a dynamic organ as the cod ovary is not an easy task, and several possible solutions are discussed. Journal of Molecular Endocrinology (2007) 39, 319–328 Introduction In teleosts, like in other vertebrates, puberty and reproduction are controlled by the brain–pituitary– gonadal axis. The gonadotropic hormones, follicle- stimulating hormone (FSH) and luteinizing hormone (LH), secreted from the pituitary under control of gonadotropin-releasing hormones (GnRHs) released by the brain, play a central role. They act on somatic cells in the gonads, and thereby regulate gonadal biosynthesis of steroid hormones. During final oocyte maturation, a shift in steroidogenesis occurs in ovarian follicles, mediated by changes in gene expression of steroidogenic enzymes like ovarian P450 aromatase (encoded by the cyp19a1 gene) and 20b-hydroxysteroid dehydrogenase (20b-HSD; Senthilkumaran et al. 2004). This is concomitant with a switch from predominantly FSH toward higher levels of LH, and leads to the production of primarily maturation- inducing steroid (MIS), instead of conversion of testoster- one into estradiol (E 2 ), in the ovarian follicles. Whereas MIS is produced by the action of 20b-HSD, the conversion of androgens (C19) to estrogens (C18) is catalyzed by an enzyme complex containing P450 aromatase, product of the cyp19 gene, and a flavoprotein NADPH-cytochrome P450 reductase (Simpson et al. 1994). The enzymatic activity of P450arom is thought to be the rate-limiting step in estrogen biosynthesis (Simpson et al. 1994) and changes in P450arom enzyme activity or expression of cyp19 genes have been shown to be major regulators of the gonadal production of E 2 during reproduction and development (Chang et al. 1997). Numerous reports have therefore focused on the identification and characterization of cyp19a1 genes in fish, but studies in periodic spawners, like Atlantic cod, are scarce (Van Nes et al. 2005), and to our knowledge none is dealing with events during final oocyte maturation and spawning. Sequences for the carbonyl-reductase like 20b-hsd gene are available only from a few teleost species (ayu, Tanaka et al. 2002; Japanese eel, Kazeto et al. unpublished; rainbow trout, Guan et al. 1999; Nile tilapia, Senthilk- umaran et al. 2002; and zebrafish, Wang & Ge 2002), despite high similarity scores that should facilitate 319 Journal of Molecular Endocrinology (2007) 39, 319–328 DOI: 10.1677/JME-07-0070 0952–5041/07/039–319 q 2007 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 05/06/2020 02:33:54AM via free access