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Identification of differentially expressed ovarian genes during primary and early secondary oocyte growth in coho salmon, Oncorhynchus kisutch.

Luckenbach JA, Iliev DB, Goetz FW, Swanson P - Reprod. Biol. Endocrinol. (2008)

Bottom Line: 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.In 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.Significant increases in transcripts for fshr, gsdf, and amh at the CA stage suggest a role of FSH and TGFbeta peptides in previtellogenic oocyte growth and puberty onset in female salmon.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Aquatic and Fishery Sciences, University of Washington, Seattle, Washington 98195, USA. jalucken@u.washington.edu

ABSTRACT

Background: 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.

Methods: Reciprocal 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.

Results: SSH 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.

Conclusion: In 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 TGFbeta peptides in previtellogenic oocyte growth and puberty onset in female salmon.

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Abundance of other ovarian transcripts during primary and early secondary oocyte growth. These include likely follicle cell transcripts. Open bars denote perinucleolus (P) stage samples, while solid bars denote cortical alveolus (CA) stage samples. Each bar represents the mean + SEM of 10 fish per stage with *P < 0.05, **P < 0.01 and ***P < 0.001 indicating statistical differences. Relative transcript levels were normalized to ef1a.
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Figure 5: Abundance of other ovarian transcripts during primary and early secondary oocyte growth. These include likely follicle cell transcripts. Open bars denote perinucleolus (P) stage samples, while solid bars denote cortical alveolus (CA) stage samples. Each bar represents the mean + SEM of 10 fish per stage with *P < 0.05, **P < 0.01 and ***P < 0.001 indicating statistical differences. Relative transcript levels were normalized to ef1a.

Mentions: A number of other genes not falling into the above cascades were differentially expressed across stages (Fig. 5). Fth3, DnaJ subfamily A member 2 (dnaja2), and cyclin E (ccne) transcripts were significantly elevated at the P stage, while retinol dehydrogenase 1 (rdh1) and coatomer protein epsilon subunit (cope), showed the opposite pattern and were higher at the CA stage. Anterior pharynx defective 1b (aph1b) was not differentially expressed based on qPCR results. Finally, three transcripts likely originating from the follicle cells, fshr, amh, and gonadal soma-derived growth factor (gsdf) were dramatically upregulated at the CA stage.


Identification of differentially expressed ovarian genes during primary and early secondary oocyte growth in coho salmon, Oncorhynchus kisutch.

Luckenbach JA, Iliev DB, Goetz FW, Swanson P - Reprod. Biol. Endocrinol. (2008)

Abundance of other ovarian transcripts during primary and early secondary oocyte growth. These include likely follicle cell transcripts. Open bars denote perinucleolus (P) stage samples, while solid bars denote cortical alveolus (CA) stage samples. Each bar represents the mean + SEM of 10 fish per stage with *P < 0.05, **P < 0.01 and ***P < 0.001 indicating statistical differences. Relative transcript levels were normalized to ef1a.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2262088&req=5

Figure 5: Abundance of other ovarian transcripts during primary and early secondary oocyte growth. These include likely follicle cell transcripts. Open bars denote perinucleolus (P) stage samples, while solid bars denote cortical alveolus (CA) stage samples. Each bar represents the mean + SEM of 10 fish per stage with *P < 0.05, **P < 0.01 and ***P < 0.001 indicating statistical differences. Relative transcript levels were normalized to ef1a.
Mentions: A number of other genes not falling into the above cascades were differentially expressed across stages (Fig. 5). Fth3, DnaJ subfamily A member 2 (dnaja2), and cyclin E (ccne) transcripts were significantly elevated at the P stage, while retinol dehydrogenase 1 (rdh1) and coatomer protein epsilon subunit (cope), showed the opposite pattern and were higher at the CA stage. Anterior pharynx defective 1b (aph1b) was not differentially expressed based on qPCR results. Finally, three transcripts likely originating from the follicle cells, fshr, amh, and gonadal soma-derived growth factor (gsdf) were dramatically upregulated at the CA stage.

Bottom Line: 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.In 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.Significant increases in transcripts for fshr, gsdf, and amh at the CA stage suggest a role of FSH and TGFbeta peptides in previtellogenic oocyte growth and puberty onset in female salmon.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Aquatic and Fishery Sciences, University of Washington, Seattle, Washington 98195, USA. jalucken@u.washington.edu

ABSTRACT

Background: 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.

Methods: Reciprocal 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.

Results: SSH 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.

Conclusion: In 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 TGFbeta peptides in previtellogenic oocyte growth and puberty onset in female salmon.

Show MeSH
Related in: MedlinePlus