Abstract
BackgroundThe aim of this study was to identify differentially expressed ovarian genes during primary and early secondary oocyte growth in coho salmon, a semelparous teleost that exhibits synchronous follicle development.MethodsReciprocal suppression subtractive hybridization (SSH) libraries were generated from ovaries with perinucleolus (P) or cortical alveolus (CA) stage follicles and selected genes were assessed with quantitative PCR (qPCR). An assessment of changes in RNA composition during oocyte growth and its relationship to transcript levels was also conducted.ResultsSSH revealed several differentially expressed genes during early oogenesis, some which will not likely be utilized until 1–3 years later in salmon. Zona pellucida glycoprotein (zp) genes, vitellogenin receptor (vldlr) isoforms, cathepsin B (ctsba), cyclin E (ccne), a DnaJ transcript (dnaja2), and a ferritin subunit (fth3) were significantly elevated at the P stage, while a C-type lectin, retinol dehydrogenase (rdh1), and a coatomer protein subunit (cope) were upregulated at the CA stage. Putative follicle cell transcripts such as anti-Müllerian hormone (amh), lipoprotein lipase (lpl), apolipoprotein E (apoe), gonadal soma-derived growth factor (gsdf) and follicle-stimulating hormone receptor (fshr) also increased significantly at the CA stage. The analysis of RNA composition during oocyte growth showed that the total RNA yield and proportion of messenger RNA relative to non-polyadenylated RNAs declined as oogenesis progressed. This influenced apparent transcript levels depending on the type of RNA template used and normalization method.ConclusionIn coho salmon, which exhibit a dramatic change in oocyte size and RNA composition during oogenesis, use of messenger RNA as template and normalization of qPCR data to a housekeeping gene, ef1a, yielded results that best reflected transcript abundance within the ovarian follicle. Synthesis of zp transcripts and proteins involved in yolk incorporation and processing occurred during primary growth, while increased expression of a CA component and genes related to lipid incorporation occurred concomitant with the appearance of CA, but prior to lipid accumulation. Significant increases in transcripts for fshr, gsdf, and amh at the CA stage suggest a role of FSH and TGFβ peptides in previtellogenic oocyte growth and puberty onset in female salmon.
Highlights
The aim of this study was to identify differentially expressed ovarian genes during primary and early secondary oocyte growth in coho salmon, a semelparous teleost that exhibits synchronous follicle development
As first noted by Goetz and colleagues [30] these findings suggest that it is necessary to enrich for these cell layers prior to suppression subtractive hybridization (SSH) to increase the likelihood of revealing rare transcripts of the granulosa or theca/interstitial cells
Major gene families represented in the SSH libraries included zp genes, lipoprotein receptors, yolk proteases, and cortical alveolus (CA) components, most of which appear to be derived from the oocyte
Summary
The aim of this study was to identify differentially expressed ovarian genes during primary and early secondary oocyte growth in coho salmon, a semelparous teleost that exhibits synchronous follicle development. Genes involved in sex differentiation and early gametogenesis [14,15,16], and final oocyte maturation [17] have received considerable attention, while other studies have focused on specific gene families such as TGFβ superfamily members [18], zona pellucida glycoproteins [19], and vitellogenin receptor (very low density lipoprotein receptor, vldlr) [20]. Through these studies highly expressed ovarian genes, such as zona pellucida glycoprotein (zp) genes and egg lectins have been revealed. Relatively few ovarian genes have been profiled in fish and little is known about temporal gene expression during oocyte growth
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