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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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Histological sections of coho salmon ovaries with perinucleolus (A) and mid-cortical alveolus stage follicles (B). Panel A shows a representative ovary from cohort 1 fish and panel B shows a representative ovary from cohort 2 fish used for subtractive hybridization and qPCR validations. The scale bar = 100 μm in each panel; n, nucleoli; ca, cortical alveoli.
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Figure 1: Histological sections of coho salmon ovaries with perinucleolus (A) and mid-cortical alveolus stage follicles (B). Panel A shows a representative ovary from cohort 1 fish and panel B shows a representative ovary from cohort 2 fish used for subtractive hybridization and qPCR validations. The scale bar = 100 μm in each panel; n, nucleoli; ca, cortical alveoli.

Mentions: Coho salmon (2004 and 2003 brood) were reared at the Northwest Fisheries Science Center (Seattle, WA) in recirculated fresh water under a simulated natural photoperiod and fed a standard ration of a commercial diet. These salmon typically spawn in December at 3 years of age and the same cohorts (2004 brood = cohort 1, 2003 brood = cohort 2) were used for all experiments. In October 2005, female salmon (N = 43 fish of cohort 1; N = 20 fish of cohort 2) were euthanized and their ovaries removed and weighed. At this time, cohort 1 fish were 0+ age (10 months old), 72–103 mm fork length (FL) and 4.5–13.3 g body mass with an ovary mass of 0.029 ± 0.001 (mean ± SEM) and gonadosomatic index (GSI) of 0.32 ± 0.01. Cohort 2 fish were 1+ age (22 months old), 194–235 mm FL and 93.5–157.8 g body mass with an ovary mass of 0.400 ± 0.021 g and GSI of 0.33 ± 0.01. A piece of ovary from each fish was fixed in Bouins for paraffin histology [21], and the remaining tissue was snap frozen for later RNA isolation. Histology revealed that in October 2005, ovaries of cohort 1 possessed primary growth follicles at the perinucleolus (P) stage containing minimal Balbiani material and no cortical alveoli (Fig. 1). Ovaries of cohort 2 contained follicles that were early to mid-cortical alveolus (CA) stage with cortical alveoli filling greater than 50% of the ooplasm. Samples were selected for SSH that represented primary growth (P stage, cohort 1) and early secondary oocyte growth (mid-CA stage, cohort 2). Salmon used for qPCR validation of the SSH results (N = 10 fish/stage) possessed ovaries that were in the same stages as those used for SSH. For the template RNA assessment (see below), fish were sampled as described above at a later time point in August 2006 (N = 5/cohort). At this time, fish from cohort 1 were 1+ age (20 months old) and follicles were generally late P stage with most oocytes showing very few cortical alveoli in the periphery of the ooplasm, while cohort 2 fish were 2+ age (30 months old) and oocytes were in the yolk granule 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)

Histological sections of coho salmon ovaries with perinucleolus (A) and mid-cortical alveolus stage follicles (B). Panel A shows a representative ovary from cohort 1 fish and panel B shows a representative ovary from cohort 2 fish used for subtractive hybridization and qPCR validations. The scale bar = 100 μm in each panel; n, nucleoli; ca, cortical alveoli.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Histological sections of coho salmon ovaries with perinucleolus (A) and mid-cortical alveolus stage follicles (B). Panel A shows a representative ovary from cohort 1 fish and panel B shows a representative ovary from cohort 2 fish used for subtractive hybridization and qPCR validations. The scale bar = 100 μm in each panel; n, nucleoli; ca, cortical alveoli.
Mentions: Coho salmon (2004 and 2003 brood) were reared at the Northwest Fisheries Science Center (Seattle, WA) in recirculated fresh water under a simulated natural photoperiod and fed a standard ration of a commercial diet. These salmon typically spawn in December at 3 years of age and the same cohorts (2004 brood = cohort 1, 2003 brood = cohort 2) were used for all experiments. In October 2005, female salmon (N = 43 fish of cohort 1; N = 20 fish of cohort 2) were euthanized and their ovaries removed and weighed. At this time, cohort 1 fish were 0+ age (10 months old), 72–103 mm fork length (FL) and 4.5–13.3 g body mass with an ovary mass of 0.029 ± 0.001 (mean ± SEM) and gonadosomatic index (GSI) of 0.32 ± 0.01. Cohort 2 fish were 1+ age (22 months old), 194–235 mm FL and 93.5–157.8 g body mass with an ovary mass of 0.400 ± 0.021 g and GSI of 0.33 ± 0.01. A piece of ovary from each fish was fixed in Bouins for paraffin histology [21], and the remaining tissue was snap frozen for later RNA isolation. Histology revealed that in October 2005, ovaries of cohort 1 possessed primary growth follicles at the perinucleolus (P) stage containing minimal Balbiani material and no cortical alveoli (Fig. 1). Ovaries of cohort 2 contained follicles that were early to mid-cortical alveolus (CA) stage with cortical alveoli filling greater than 50% of the ooplasm. Samples were selected for SSH that represented primary growth (P stage, cohort 1) and early secondary oocyte growth (mid-CA stage, cohort 2). Salmon used for qPCR validation of the SSH results (N = 10 fish/stage) possessed ovaries that were in the same stages as those used for SSH. For the template RNA assessment (see below), fish were sampled as described above at a later time point in August 2006 (N = 5/cohort). At this time, fish from cohort 1 were 1+ age (20 months old) and follicles were generally late P stage with most oocytes showing very few cortical alveoli in the periphery of the ooplasm, while cohort 2 fish were 2+ age (30 months old) and oocytes were in the yolk granule 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