20β-hydroxysteroid dehydrogenase and CYP19A1 are differentially expressed during maturation in Atlantic cod (Gadus morhua)

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.

17β-HSD type 12-like is responsible for maturation-inducing hormone synthesis during oocyte maturation in masu salmon12

“…Some studies demonstrated weak 20β-HSD activity of CR/20β-HSD towards 17OHP (7,30), whilst others did not check its activity (28,29,31,32). Moreover, several reports used the gene name 20β-HSD without even using the prefix CR (29)(30)(31). In this study, we do not find support for crediting CR/20β-HSD with MIH production but instead present compelling evidence that 17β-HSD type 12-like is the enzyme responsible for DHP production in masu salmon.…”

“…Since the isolation of a cDNA encoding CR/20β-HSD from pig (4), many studies have targeted CR/20β-HSD orthologs as being responsible for maturation-inducing hormone (MIH) synthesis (7,(28)(29)(30)(31)(32). Some studies demonstrated weak 20β-HSD activity of CR/20β-HSD towards 17OHP (7,30), whilst others did not check its activity (28,29,31,32).…”

“…Some studies demonstrated weak 20β-HSD activity of CR/20β-HSD towards 17OHP (7,30), whilst others did not check its activity (28,29,31,32). Moreover, several reports used the gene name 20β-HSD without even using the prefix CR (29)(30)(31).…”

Identification of ovarian gene expression patterns during vitellogenesis in Atlantic cod (Gadus morhua)

“…All primer sets were designed to amplify 80-150 bp fragments and verified for target specificity using the NCBI G. morhua nucleotide database. Previously developed assays [49,50] for candidate genes involved in ovarian development were also used, including 20b-hydroxysteroid dehydrogenase (20b-HSD), aromatase (CYP19A1), follicle-stimulating hormone receptor (FSH-R), and luteinizing hormone receptor (LH-R). QPCR primers for cytochrome P450 side chain cleavage enzyme (CYP11A1) were developed from an Atlantic cod cDNA sequence (GenBank Accession # AY706102.1; [23]).…”

Effects of Dietary Arachidonic Acid on the Reproductive Physiology of Female Atlantic Cod (Gadus morhua L.).

“…The protocols for quantitative real-time PCR assays for Atlantic cod 20bhsd, cyp19a1a, and for the endogenous control 18s rRNA have been described previously (Mittelholzer et al, 2007). Sequences for all primers and probes are given in Supplement S3.…”