20 -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.
- [Show abstract] [Hide abstract]
ABSTRACT: Vertebrate oocytes undergo dramatic changes during development as they accumulate many RNA transcripts, glycoproteins, and yolk proteins, necessary to ensure proper fertilization and embryogenesis. Oogenesis in teleosts often requires several years for completion, but very little is known about the early developmental stages. Recently, two-stage gene expression comparisons were made during oocyte growth in coho salmon (Oncorhynchus kisutch) and Atlantic cod (Gadus morhua), but more broad-scale, comprehensive assessments have not been conducted. The objectives of the present study were to characterize the gene expression patterns throughout oocyte growth in cod and compare them to changes previously identified in coho salmon. A quantitative PCR survey was conducted using 50 genes at six ovarian stages, ranging from the onset of primary growth (oocyte differentiation) to late vitellogenesis. Most expression patterns could be grouped into three major clusters, consisting of oocyte-derived (cluster 1) and likely follicle cell (clusters 2 and 3) genes. Oocyte genes were elevated during primary growth, while many follicle cell transcripts were abundant during oocyte differentiation and vitellogenesis. Few expression changes identified in coho salmon were evident in cod, which is likely due to differences in reproductive strategies. These results demonstrate that dynamic changes in gene expression occur during oocyte growth in teleost fish.Comparative Biochemistry and Physiology Part D Genomics and Proteomics 11/2013; 9C:1-10. · 2.88 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: A stable supply of viable eggs and embryos is crucial for successful farming of Atlantic cod. Stress during broodstock rearing can have negative effects on offspring, but little is known about the molecular mechanisms that cause abnormal development. Maternally transferred mRNAs have been shown to be essential for normal development, and stress may therefore influence their expression and the subsequent embryonic development. We investigated if mimicked stress in cod females affects mRNA concentrations in eggs/embryos, and if this can be linked to viability of embryos. Three weeks before peak spawning, 20 fish were intraperitoneally implanted with either cortisol-containing or cortisol-free (sham) osmotic pumps. At peak spawning all individuals were stripped and eggs were fertilized and incubated until hatching. Samples were collected from unfertilized eggs and embryos for analysis of gene expression (microarray), viability, steroids and vitellogenin. Plasma concentration of cortisol (ng/ml) in treated females was significantly higher at spawning (127.1±20.9) than that of sham control (11.3±6.7). This difference was also reflected in eggs and embryos. Percent fertilization, asymmetric cell division and hatching were not affected. However, numerous genes were differentially expressed in eggs and embryos in response to elevated cortisol, especially in maternal (oocyte and blastula) stages. Among these differentially expressed genes, some were found to be linked to cytogenesis (stxbp6, fbxw2, capn12, thbs4, sytl2, coro1c, sel1l3), induction of mesodermal fate (fgfrl1) and import of the glucocorticoid receptor to the cell nucleus (ipo7). Gene ontology overrepresentation analysis on the whole set of differentially expressed genes at maternal stages (539 genes) revealed enriched activity in membrane associated regions, which largely corresponds to cytogenesis related processes. These results suggest that despite no visible phenotypic effects in early embryos, broodstock stress affects the egg/embryonic transcriptome, especially in relation to cytogenesis. Furthermore, effects related to egg/embryo phenotypes are difficult to measure at early stages of development, and instead might become apparent at later life stages.General and Comparative Endocrinology 05/2013; · 2.82 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The molecular mechanisms underlying oogenesis and maternally controlled embryogenesis in fish are not fully understood, especially in marine species. Our aim was to study the egg and embryo transcriptome during oogenesis and early embryogenesis in Atlantic cod. Follicles from oogenesis stages (pre-, early-, and late-vitellogenic), ovulated eggs, and two embryonic stages (blastula, gastrula) were collected from broodstock fish and fertilized eggs. Gene expression profiles were measured in a 44K oligo microarray consisting of 23,000 cod genes. Hundreds of differentially expressed genes (DEGs) were identified in the follicle stages investigated, implicating a continuous accumulation and degradation of polyadenylated transcripts throughout oogenesis. Very few DEGs were identified from ovulated egg to blastula, showing a more stable maternal RNA pool in early embryonic stages. The highest induction of expression was observed between blastula and gastrula, signifying the onset of zygotic transcription. During early vitellogenesis, several of the most upregulated genes are linked to nervous system signaling, suggesting increasing requirements for ovarian synaptic signaling to stimulate the rapid growth of oocytes. Highly upregulated genes during late vitellogenesis are linked to protein processing, fat metabolism, osmoregulation, and arrested meiosis. One of the genes with the highest upregulation in the ovulated egg is involved in oxidative phosphorylation, reflecting increased energy requirements during fertilization and the first rapid cell divisions of early embryogenesis. In conclusion, this study provides a large-scale presentation of the Atlantic cod's maternally controlled transcriptome in ovarian follicles through oogenesis, ovulated eggs, and early embryos. Mol. Reprod. Dev. © 2014 Wiley Periodicals, Inc.Molecular Reproduction and Development 03/2014; · 2.81 Impact Factor