Abstract
The precise identity of spermatogonial stem cells—the germline stem cell of the adult testis—remains a controversial topic. Technical limitations have included the lack of specific markers and methods for lineage tracing of Asingle spermatogonia and their subsets. Immunolocalization of proteins in tissue sections has been a standard tool for the in situ identification and visualization of rare cellular subsets. However, these studies are limited by the need for faithful and reliable protein markers to define these cell types, as well as the availability of specific antibodies to these markers. Here we describe the use of a monoclonal antibody to Pax7 as a means to detect spermatogonial stem cells (SSCs) both in tissue sections and in intact seminiferous tubules. Furthermore, we describe methods for lineage tracing as an alternative method to visualize Pax7+ spermatogonial stem cells and their progeny.
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References
Oatley JM, Brinster RL (2008) Regulation of spermatogonial stem cell self-renewal in mammals. Annu Rev Cell Dev Biol 24:263–286. doi:10.1146/annurev.cellbio.24.110707.175355
de Rooij DG (2001) Proliferation and differentiation of spermatogonial stem cells. Reproduction 121(3):347–354
de Rooij DG, Russell LD (2000) All you wanted to know about spermatogonia but were afraid to ask. J Androl 21(6):776–798
Oakberg EF (1971) Spermatogonial stem-cell renewal in the mouse. Anat Rec 169(3):515–531. doi:10.1002/ar.1091690305
Yang QE, Oatley JM (2014) Spermatogonial stem cell functions in physiological and pathological conditions. Curr Top Dev Biol 107:235–267. doi:10.1016/B978-0-12-416022-4.00009-3
Brinster RL, Avarbock MR (1994) Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad Sci U S A 91(24):11303–11307
Nagano MC, Yeh JR (2013) The identity and fate decision control of spermatogonial stem cells: where is the point of no return? Curr Top Dev Biol 102:61–95. doi:10.1016/B978-0-12-416024-8.00003-9
Chan F, Oatley MJ, Kaucher AV, Yang QE, Bieberich CJ, Shashikant CS, Oatley JM (2014) Functional and molecular features of the Id4+ germline stem cell population in mouse testes. Genes Dev 28(12):1351–1362. doi:10.1101/gad.240465.114
Nakagawa T, Sharma M, Nabeshima Y, Braun RE, Yoshida S (2010) Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment. Science 328(5974):62–67, doi:science.1182868 [pii] 10.1126/science.1182868
Zhang Z, Shao S, Meistrich ML (2007) The radiation-induced block in spermatogonial differentiation is due to damage to the somatic environment, not the germ cells. J Cell Physiol 211(1):149–158. doi:10.1002/jcp.20910
Yoshida S (2012) Elucidating the identity and behavior of spermatogenic stem cells in the mouse testis. Reproduction 144(3):293–302. doi:10.1530/REP-11-0320
Nagano MC (2003) Homing efficiency and proliferation kinetics of male germ line stem cells following transplantation in mice. Biol Reprod 69(2):701–707. doi:10.1095/biolreprod.103.016352
Abid SN, Richardson TE, Powell HM, Jaichander P, Chaudhary J, Chapman KM, Hamra FK (2014) A-single spermatogonia heterogeneity and cell cycles synchronize with rat seminiferous epithelium stages VIII-IX. Biol Reprod 90(2):32. doi:10.1095/biolreprod.113.113555
Aloisio GM, Nakada Y, Saatcioglu HD, Pena CG, Baker MD, Tarnawa ED, Mukherjee J, Manjunath H, Bugde A, Sengupta AL, Amatruda JF, Cuevas I, Hamra FK, Castrillon DH (2014) PAX7 expression defines germline stem cells in the adult testis. J Clin Invest. doi:10.1172/JCI75943
Keller C, Hansen MS, Coffin CM, Capecchi MR (2004) Pax3:Fkhr interferes with embryonic Pax3 and Pax7 function: implications for alveolar rhabdomyosarcoma cell of origin. Genes Dev 18(21):2608–2613. doi:10.1101/gad.1243904
Lepper C, Conway SJ, Fan CM (2009) Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements. Nature 460(7255):627–631. doi:10.1038/nature08209
Murphy M, Kardon G (2011) Origin of vertebrate limb muscle: the role of progenitor and myoblast populations. Curr Top Dev Biol 96:1–32. doi:10.1016/B978-0-12-385940-2.00001-2
Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, Griswold MD, de Rooij DG, Braun RE (2004) Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet 36(6):647–652. doi:10.1038/ng1366
Goertz MJ, Wu Z, Gallardo TD, Hamra FK, Castrillon DH (2011) Foxo1 is required in mouse spermatogonial stem cells for their maintenance and the initiation of spermatogenesis. J Clin Invest 121(9):3456–3466. doi:10.1172/JCI57984, 57984 [pii]
Franca LR, Avelar GF, Almeida FF (2005) Spermatogenesis and sperm transit through the epididymis in mammals with emphasis on pigs. Theriogenology 63(2):300–318. doi:10.1016/j.theriogenology.2004.09.014
Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21(1):70–71
Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng LL, Palmiter RD, Hawrylycz MJ, Jones AR, Lein ES, Zeng H (2010) A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13(1):133–140. doi:10.1038/nn.2467
Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L (2007) A global double-fluorescent Cre reporter mouse. Genesis 45(9):593–605. doi:10.1002/dvg.20335
Gallardo T, Shirley L, John GB, Castrillon DH (2007) Generation of a germ cell-specific mouse transgenic Cre line, Vasa-Cre. Genesis 45(6):413–417
Jockusch H, Voigt S, Eberhard D (2003) Localization of GFP in frozen sections from unfixed mouse tissues: immobilization of a highly soluble marker protein by formaldehyde vapor. J Histochem Cytochem 51(3):401–404
Kusser KL, Randall TD (2003) Simultaneous detection of EGFP and cell surface markers by fluorescence microscopy in lymphoid tissues. J Histochem Cytochem 51(1):5–14
van den Pol AN, Ghosh PK (1998) Selective neuronal expression of green fluorescent protein with cytomegalovirus promoter reveals entire neuronal arbor in transgenic mice. J Neurosci 18(24):10640–10651
Sullivan KF, Kay SA, American Society for Cell Biology (1999) Green fluorescent proteins, vol 58, Methods in cell biology. Academic Press, San Diego, London
Joosen L, Hink MA, Gadella TW Jr, Goedhart J (2014) Effect of fixation procedures on the fluorescence lifetimes of Aequorea victoria derived fluorescent proteins. J Microsc 256(3):166–176. doi:10.1111/jmi.12168
Chapman SC, Lawson A, Macarthur WC, Wiese RJ, Loechel RH, Burgos-Trinidad M, Wakefield JK, Ramabhadran R, Mauch TJ, Schoenwolf GC (2005) Ubiquitous GFP expression in transgenic chickens using a lentiviral vector. Development 132(5):935–940. doi:10.1242/dev.01652
Kawakami A, Kimura-Kawakami M, Nomura T, Fujisawa H (1997) Distributions of PAX6 and PAX7 proteins suggest their involvement in both early and late phases of chick brain development. Mech Dev 66(1-2):119–130
Goldenthal KL, Hedman K, Chen JW, August JT, Willingham MC (1985) Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins. J Histochem Cytochem 33(8):813–820
Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA (2000) Pax7 is required for the specification of myogenic satellite cells. Cell 102(6):777–786
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Aloisio, G.M., Cuevas, I., Nakada, Y., Peña, C.G., Castrillon, D.H. (2017). Visualization and Lineage Tracing of Pax7+ Spermatogonial Stem Cells in the Mouse. In: Buszczak, M. (eds) Germline Stem Cells. Methods in Molecular Biology, vol 1463. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-4017-2_11
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DOI: https://doi.org/10.1007/978-1-4939-4017-2_11
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