Normalizing Gene Expression Levels in Mouse Fetal Germ Cells

Overview

Journal Biol Reprod

Publisher Oxford University Press

Specialty Reproductive Medicine

Date 2009 May 1

PMID 19403927

Citations 23

Authors

Jocelyn A van den Bergen

Denise C Miles

Andrew H Sinclair

Patrick S Western

Affiliations

Soon will be listed here.

Abstract

Real-time PCR has become a popular method to analyze transcription of genes that are developmentally regulated during organogenesis of the testes and ovaries. However, the heterogenous cell populations and commitment to strikingly different developmental pathways of the germ and somatic cells in these organs complicate analysis of this process. The selection of suitable reference genes for quantifying gene expression in this system is essential, but to date it has not been sufficiently addressed. To rectify this problem, we have used fluorescence-activated cell sorting to purify germ cells from mouse fetal testes and ovaries and examined 16 common housekeeping genes for their suitability as reference genes. In pure populations of germ cells isolated from Embryonic Day 12.5 (E12.5) to E15.5 male and female gonads, Mapk1 and Sdha were identified as the most stable reference genes. Analysis of the heterogenous fraction of gonadal somatic cells revealed that Canx and Top1 were stable in both sexes, whereas a comparative analysis of germ and somatic cell populations identified Canx and Mapk1 as suitable reference genes through these developmental stages. Application of these reference genes to quantification of gene expression in developing gonads revealed that past assays, which employed nonverified reference genes, have in some cases provided misleading gene expression profiles. This study has identified suitable reference genes to directly compare expression profiles of genes expressed in germ and somatic cells of male and female fetal gonads. Application of these reference genes to expression analysis in fetal germ and somatic cells provides a more accurate system in which to profile gene expression in these tissues.

Citing Articles

Single-nucleus multiomics reveals the gene regulatory networks underlying sex determination of murine primordial germ cells.

Alexander A, Rodriguez K, Chen Y, Amato C, Estermann M, Nicol B Elife. 2025; 13.

PMID: 40063068 PMC: 11893106. DOI: 10.7554/eLife.96591.

Single-nucleus multiomics reveals the gene-regulatory networks underlying sex determination of murine primordial germ cells.

Alexander A, Rodriguez K, Chen Y, Amato C, Estermann M, Nicol B bioRxiv. 2024; .

PMID: 39386556 PMC: 11463670. DOI: 10.1101/2024.02.19.581036.

VEGF-dependent testicular vascularisation involves MEK1/2 signalling and the essential angiogenesis factors, SOX7 and SOX17.

Blucher R, Lim R, Ritchie M, Western P BMC Biol. 2024; 22(1):222.

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The Impact of Activin A on Fetal Gonocytes: Chronic Versus Acute Exposure Outcomes.

Moody S, Whiley P, Western P, Loveland K Front Endocrinol (Lausanne). 2022; 13:896747.

PMID: 35721752 PMC: 9205402. DOI: 10.3389/fendo.2022.896747.

Identification of qPCR reference genes suitable for normalising gene expression in the developing mouse embryo.

Hildyard J, Wells D, Piercy R Wellcome Open Res. 2022; 6:197.

PMID: 35509373 PMC: 9024131. DOI: 10.12688/wellcomeopenres.16972.2.

References

Karge 3rd W, Schaefer E, Ordovas J . Quantification of mRNA by polymerase chain reaction (PCR) using an internal standard and a nonradioactive detection method. Methods Mol Biol. 1999; 110:43-61. DOI: 10.1385/1-59259-582-0:43. View

Koubova J, Menke D, Zhou Q, Capel B, Griswold M, Page D . Retinoic acid regulates sex-specific timing of meiotic initiation in mice. Proc Natl Acad Sci U S A. 2006; 103(8):2474-9. PMC: 1413806. DOI: 10.1073/pnas.0510813103. View

Kuijk E, du Puy L, van Tol H, Haagsman H, Colenbrander B, Roelen B . Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos. BMC Dev Biol. 2007; 7:58. PMC: 1896162. DOI: 10.1186/1471-213X-7-58. View

Bowles J, Knight D, Smith C, Wilhelm D, Richman J, Mamiya S . Retinoid signaling determines germ cell fate in mice. Science. 2006; 312(5773):596-600. DOI: 10.1126/science.1125691. View

Mclaren A . Primordial germ cells in the mouse. Dev Biol. 2003; 262(1):1-15. DOI: 10.1016/s0012-1606(03)00214-8. View

Yao H, Matzuk M, Jorgez C, Menke D, Page D, Swain A . Follistatin operates downstream of Wnt4 in mammalian ovary organogenesis. Dev Dyn. 2004; 230(2):210-5. PMC: 4046253. DOI: 10.1002/dvdy.20042. View

Wilhelm D, Palmer S, Koopman P . Sex determination and gonadal development in mammals. Physiol Rev. 2007; 87(1):1-28. DOI: 10.1152/physrev.00009.2006. View

Smith R, Brown B, Ikonomi P, Schechter A . Exogenous reference RNA for normalization of real-time quantitative PCR. Biotechniques. 2003; 34(1):88-91. DOI: 10.2144/03341st05. View

Loffler K, Zarkower D, Koopman P . Etiology of ovarian failure in blepharophimosis ptosis epicanthus inversus syndrome: FOXL2 is a conserved, early-acting gene in vertebrate ovarian development. Endocrinology. 2003; 144(7):3237-43. DOI: 10.1210/en.2002-0095. View

10.

Radonic A, Thulke S, Mackay I, Landt O, Siegert W, Nitsche A . Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 2004; 313(4):856-62. DOI: 10.1016/j.bbrc.2003.11.177. View

11.

Parma P, Radi O, Vidal V, Chaboissier M, Dellambra E, Valentini S . R-spondin1 is essential in sex determination, skin differentiation and malignancy. Nat Genet. 2006; 38(11):1304-9. DOI: 10.1038/ng1907. View

12.

Goossens K, Van Poucke M, Van Soom A, Vandesompele J, Van Zeveren A, Peelman L . Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos. BMC Dev Biol. 2005; 5:27. PMC: 1315359. DOI: 10.1186/1471-213X-5-27. View

13.

Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge R . Male development of chromosomally female mice transgenic for Sry. Nature. 1991; 351(6322):117-21. DOI: 10.1038/351117a0. View

14.

Maldonado-Saldivia J, van den Bergen J, Krouskos M, Gilchrist M, Lee C, Li R . Dppa2 and Dppa4 are closely linked SAP motif genes restricted to pluripotent cells and the germ line. Stem Cells. 2006; 25(1):19-28. DOI: 10.1634/stemcells.2006-0269. View

15.

Sinclair A, Berta P, Palmer M, Hawkins J, GRIFFITHS B, Smith M . A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature. 1990; 346(6281):240-4. DOI: 10.1038/346240a0. View

16.

Andersen C, Jensen J, Orntoft T . Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004; 64(15):5245-50. DOI: 10.1158/0008-5472.CAN-04-0496. View

17.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

18.

Jordan B, Shen J, Olaso R, Ingraham H, Vilain E . Wnt4 overexpression disrupts normal testicular vasculature and inhibits testosterone synthesis by repressing steroidogenic factor 1/beta-catenin synergy. Proc Natl Acad Sci U S A. 2003; 100(19):10866-71. PMC: 196894. DOI: 10.1073/pnas.1834480100. View

19.

Szabo P, Hubner K, Scholer H, Mann J . Allele-specific expression of imprinted genes in mouse migratory primordial germ cells. Mech Dev. 2002; 115(1-2):157-60. DOI: 10.1016/s0925-4773(02)00087-4. View

20.

Best D, Sahlender D, Walther N, Peden A, Adams I . Sdmg1 is a conserved transmembrane protein associated with germ cell sex determination and germline-soma interactions in mice. Development. 2008; 135(8):1415-25. PMC: 2584365. DOI: 10.1242/dev.019497. View