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.
"When it comes to early embryo tissue like in this study, such stable references are difficult to obtain. For example, neither β-actinelongation factor 1- α (ef1α)18S, actin related protein (arp) nor ubiquitin (ubi) showed stable reference gene expression in stages assayed for gene expression [Additional file 1, [25,26]. For this reason we chose not to use any of the reference genes tested, but instead apply a technique (external spiking) where external RNA (rabbit alpha hemoglobin (hba)) is added in equal amounts to each RNA sample . "
[Show abstract][Hide abstract] ABSTRACT: Zygotic transcription in fish embryos initiates around the time of gastrulation, and all prior development is initiated and controlled by maternally derived messenger RNAs. Atlantic cod egg and embryo viability is variable, and it is hypothesized that the early development depends upon the feature of these maternal RNAs. Both the length and the presence of specific motifs in the 3'UTR of maternal RNAs are believed to regulate expression and stability of the maternal transcripts. Therefore, the aim of this study was to characterize the overall composition and 3'UTR structure of the most common maternal RNAs found in cod eggs and pre-zygotic embryos.
22229 Sanger-sequences were obtained from 3'-end sequenced cDNA libraries prepared from oocyte, 1-2 cell, blastula and gastrula stages. Quantitative PCR revealed that EST copy number below 9 did not reflect the gene expression profile. Consequently genes represented by less than 9 ESTs were excluded from downstream analyses, in addition to sequences with low-quality gene hits. This provided 12764 EST sequences, encoding 257 unique genes, for further analysis. Mitochondrial transcripts accounted for 45.9-50.6% of the transcripts isolated from the maternal stages, but only 12.2% of those present at the onset of zygotic transcription. 3'UTR length was predicted in nuclear sequences with poly-A tail, which identified 191 3'UTRs. Their characteristics indicated a more complex regulation of transcripts that are abundant prior to the onset of zygotic transcription. Maternal and stable transcripts had longer 3'UTR (mean 187.1 and 208.8 bp) and more 3'UTR isoforms (45.7 and 34.6%) compared to zygotic transcripts, where 15.4% had 3'UTR isoforms and the mean 3'UTR length was 76 bp. Also, diversity and the amount of putative polyadenylation motifs were higher in both maternal and stable transcripts.
We report on the most pronounced processes in the maternally transferred cod transcriptome. Maternal stages are characterized by a rich abundance of mitochondrial transcripts. Maternal and stable transcripts display longer 3'UTRs with more variation of both polyadenylation motifs and 3'UTR isoforms. These data suggest that cod eggs possess a complex array of maternal RNAs which likely act to tightly regulate early developmental processes in the newly fertilized egg.
[Show abstract][Hide abstract] ABSTRACT: In differentiated gonochoristic species, a bipotential gonad develops into an ovary or testis during sex differentiation. Knowledge about this process is necessary to improve methods for masculinizing genetically female Atlantic cod for the subsequent purpose of producing all-female populations.
Gonads were examined histologically in juveniles from 14 to 39 mm total body length (TL). Number and size of germ cells were determined in a subset of the samples. Relevant genes were cloned, and mRNA levels determined by qPCR of amh, cyp19a1a; dax1 (nr0b2); shp (nr0b2a) and sox9b in a mixed-sex and an all-female population ranging from 12-49 mm TL.
Individuals between 14-20 mm TL could be separated in two subgroups based on gonad size and germ cell number. Ovarian cavity formation was observed in some individuals from 18-20 mm TL. The mixed sex population displayed bimodal expression patterns as regards cyp19a1a (starting at 12 mm TL) and amh (starting at 20 mm TL) mRNA levels. After approximately 30 mm TL, cyp19a1a and amh displayed a gradual increase in both sexes. No apparent, sex-dependent expression patterns were found for dax1, shp or sox9b transcripts. However, shp levels were high until the larvae reached around 35 mm TL and then dropped to low levels, while dax1 remained low until 35 mm TL, and then increased sharply.
The morphological sex differentiation in females commenced between 14-20 mm TL, and ovarian cavities were evident by 18-20 mm TL. Testis development occurred later, and was morphologically evident after 30 mm TL. This pattern was corroborated with sexually dimorphic expression patterns of cyp19a1a from 12-13 mm TL, and a male-specific increase in amh from 20 mm TL.
"Unstable expression of reference genes may be connected to the pronounced physiological and structural changes in the ovarian tissue occurring during development and, additionally, may arise from the marked differences in the RNA yield and composition in the tissue samples at various developmental stages (Mazabraud et al., 1975; Wegnez et al., 1978; Luckenbach et al., 2008). However, the latter feature has not been studied comprehensively, and the consequences connected with it have only rarely been considered in the ovarian gene expression analysis (Mittelholzer et al., 2007; Luckenbach et al., 2008; Rocha et al., 2009). "
[Show abstract][Hide abstract] ABSTRACT: The influence of changing composition and content of RNA on the results of expression profiling was studied in the group-synchronous ovaries of roach (Rutilus rutilus) over the course of their maturation. The highest yield of total RNA was detected in the primary growth and early cortical alveolus stages. The total RNA yield gradually decreased through the late cortical alveolus and late vitellogenic stages. In the primary growth and early cortical alveolus stages, total RNA was characterized by a low percentage of 18S and 28S rRNA and a high percentage of smaller-sized RNAs (tRNA, 5S and 5.8S rRNA), whereas 18S and 28S rRNA had increased by the late cortical alveolus stage and dominated by the late vitellogenic stage. The ratio of mRNA to total RNA was highest at the primary growth stage but decreased significantly in later ovarian stages. When total RNA was used for reverse transcription (RT), the shift in the mRNA/total RNA ratio influenced the results of qPCR expression profiling of several commonly used reference genes (ribosomal protein L8, elongation factor-1α, RNA polymerase-subunit B5, and β2-microglobulin) and of two target genes, gonad-type aromatase (cyp19a1a) and follistatin (fst). We conclude that the expression of target genes should be related to the mRNA pool using the same input of either mRNA to RT or cDNA to qPCR. Furthermore, gene expression was related to tissue-specific RNA yield per body mass (RNA yield x ovary mass x body mass⁻¹) thereby reflecting the massive increase in the size and cellular composition of the ovary during the reproductive cycle.
Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 02/2011; 159(2):141-9. DOI:10.1016/j.cbpa.2011.02.007 · 1.97 Impact Factor
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