Abstract
Gonadal sex differentiation is an important developmental process, in which a bipotential primordial gonad undergoes two distinct pathways, i.e., testicular and ovarian differentiation, dependent on its genetic sex. Techniques of isolating fetal gonads at various developmental stages are valuable for studies on the molecular events involved in cell-fate determination, sex-specific somatic and germ-cell differentiation and structural organization. Here we describe various procedures for isolation of embryonic gonads at different developmental stages from embryos of timed-pregnant mice. The isolated fetal gonads can be used for a variety of studies, such as organ culture, gene and protein expression. As examples of applications, we describe the immunofluorescence detection of SOX9 expression in gonadal tissue sections and microRNAs profiling/expression in fetal gonads at a critical stage for sex determination.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wilhelm D, Palmer S, Koopman P (2007) Sex determination and gonadal development in mammals. Physiol Rev 87, 1–28.
Sekido R, Lovell-Badge R (2009) Sex determination and SRY: down to a wink and a nudge? Trends Genet 25, 19–29.
Taketo T, Lee CH, Zhang J, Li Y, Lee CY, Lau YF (2005) Expression of SRY proteins in both normal and sex-reversed XY fetal mouse gonads. Dev Dyn 233, 612–622.
Sekido R, Lovell-Badge R (2008) Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer. Nature 453, 930–934.
Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge R (1991) Male development of chromosomally female mice transgenic for Sry. Nature 351, 117–121.
Tomizuka K, Horikoshi K, Kitada R, Sugawara Y, Iba Y, Kojima A, Yoshitome A, Yamawaki K, Amagai M, Inoue A, Oshima T, Kakitani M (2008) R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling. Hum Mol Genet 17, 1278–1291.
Kim Y, Kobayashi A, Sekido R, DiNapoli L, Brennan J, Chaboissier MC, Poulat F, Behringer RR, Lovell-Badge R, Capel B (2006) Fgf9 and Wnt4 act as antagonistic signals to regulate mammalian sex determination. PLoS Biol 4, e187.
Uhlenhaut NH, Jakob S, Anlag K, Eisenberger T, Sekido R, Kress J, Treier AC, Klugmann C, Klasen C, Holter, NI, Riethmacher D, Schutz G, Cooney AJ, Lovell-Badge R, Treier M (2009) Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. Cell 139, 1130–1142.
Taketo TTR, Adeyemo O, Koide SS (1984) Influence of adenosine 3′,5′-cyclic monophosphate analogs on testicular organizaion of fetal mouse gonads in virtro. Biol Reprod 30, 189–198.
Capel B, Batchvarov J (2008) Preparing Recombinant Gonad Organ Cultures. Cold Spring Harb Protoc
Li Y, Yue L, Taketo T, Lau YF (2003) Protein transduction as a strategy for evaluating important factors in mammalian sex determination and differentiation. Cytogenet Genome Res 101, 237–241.
Capel B, Albrecht KH, Washburn LL, Eicher EM (1999) Migration of mesonephric cells into the mammalian gonad depends on Sry. Mech Dev 84, 127–131.
Nef S, Schaad O, Stallings NR, Cederroth CR, Pitetti JL, Schaer G, Malki S, Dubois-Dauphin M, Boizet-Bonhoure B, Descombes P, Parker KL, Vassalli JD (2005) Gene expression during sex determination reveals a robust female genetic program at the onset of ovarian development. Dev Biol 287, 361–377.
Beverdam A, Koopman P (2006) Expression profiling of purified mouse gonadal somatic cells during the critical time window of sex determination reveals novel candidate genes for human sexual dysgenesis syndromes. Hum Mol Genet 15, 417–431.
Toyooka Y, Tsunekawa N, Takahashi Y, Matsui Y, Satoh M, Noce T (2000) Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development. Mech Dev 93, 139–149.
Shah AA, Meese E, Blin N (2010) Profiling of regulatory microRNA transcriptomes in various biological processes: a review. J Appl Genet 51, 501–507.
Kim GJ, Georg I, Scherthan H, Merkenschlager M, Guillou F, Scherer G, Barrionuevo F (2010) Dicer is required for Sertoli cell function and survival. Int J Dev Biol 54, 867–875.
Bannister SC, Tizard ML, Doran TJ, Sinclair AH, Smith CA (2009) Sexually dimorphic microRNA expression during chicken embryonic gonadal development. Biol Reprod 81, 165–176.
O’Day E, Lal A (2010) MicroRNAs and their target gene networks in breast cancer. Breast Cancer Res 12, 201.
Liu L, Chen L, Xu Y, Li R, Du X (2010) microRNA-195 promotes apoptosis and suppresses tumorigenicity of human colorectal cancer cells. Biochem Biophys Res Commun 400, 236–240.
Ghosh T, Soni K, Scaria V, Halimani M, Bhattacharjee C, Pillai B (2008) MicroRNA-mediated up-regulation of an alternatively polyadenylated variant of the mouse cytoplasmic {beta}-actin gene. Nucleic Acids Res 36, 6318–6332.
Giraud-Triboult K, Rochon-Beaucourt C, Nissan X, Champon B, Aubert S, Pietu G (2011) Combined mRNA and microRNA profiling reveals that miR-148a and miR-20b control human mesenchymal stem cell phenotype via EPAS1. Physiol Genomics 43,77–86.
Castilla MA, Moreno-Bueno G, Romero-Perez L, De Vijver KV, Biscuola M, Lopez-Garcia MA, Prat J, Matias-Guiu X, Cano A, Oliva E, Palacios J (2011) Micro-RNA signature of the epithelial-mesenchymal transition in endometrial carcinosarcoma. J Pathol 223, 72–80.
Iliopoulos D, Lindahl-Allen M, Polytarchou C, Hirsch HA, Tsichlis PN, Struhl K (2010) Loss of miR-200 inhibition of Suz12 leads to polycomb-mediated repression required for the formation and maintenance of cancer stem cells. Mol Cell 39, 761–772.
Jung YH, Gupta MK, Shin JY, Uhm SJ, Lee HT (2010) MicroRNA signature in testes-derived male germ-line stem cells. Mol Hum Reprod 16, 804–810.
Callis TE, Pandya K, Seok HY, Tang RH, Tatsuguchi M, Huang ZP, Chen JF, Deng Z, Gunn B, Shumate J, Willis MS, Selzman CH, Wang DZ (2009) MicroRNA-208a is a regulator of cardiac hypertrophy and conduction in mice. J Clin Invest 119, 2772–2786.
van Rooij E, Quiat, Johnson BA, Sutherland LB, Qi X, Richardson JA, Kelm RJ Jr, Olson EN (2009) A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance. Dev Cell 17, 662–673.
Bouma GJ, Washburn LL, Albrecht KH, Eicher EM (2007) Correct dosage of Fog2 and Gata4 transcription factors is critical for fetal testis development in mice. Proc Natl Acad Sci USA 104, 14994–14999.
Cheng LC, Pastrana E, Tavazoie M, Doetsch F (2009) miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche. Nat Neurosci 12, 399–408.
Gao FB (2010) Context-dependent functions of specific microRNAs in neuronal development. Neural Dev 5, 25.
Farrell BC, Power EM, Dermott KW (2011) Developmentally regulated expression of Sox9 and microRNAs 124,128 and 23 in neuroepithelial stem cells in the developing spinal cord. Int J Dev Neurosci 29, 31–36.
Sirotkin AV, Laukova M, Ovcharenko D, Brenaut P, Mlyncek M (2009) Identification of microRNAs controlling human ovarian cell proliferation and apoptosis. J Cell Physiol 223, 49–56.
Acknowledgments
This work was partially supported by an NIH grant to Y-FC Lau. Y-FC Lau is a Research Career Scientist in the Department of Veterans Affairs.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Li, Y., Taketo, T., Lau, YF.C. (2012). Isolation of Fetal Gonads from Embryos of Timed-Pregnant Mice for Morphological and Molecular Studies. In: Chan, WY., Blomberg, L. (eds) Germline Development. Methods in Molecular Biology, vol 825. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-436-0_1
Download citation
DOI: https://doi.org/10.1007/978-1-61779-436-0_1
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-61779-435-3
Online ISBN: 978-1-61779-436-0
eBook Packages: Springer Protocols