Advanced Immunostaining Approaches to Study Early Male Germ Cell Development

Overview

Journal Stem Cell Res

Publisher Elsevier

Specialty Cell Biology

Date 2018 Feb 25

PMID 29475796

Citations 10

Authors

Bryan A Niedenberger

Christopher B Geyer

Affiliations

Soon will be listed here.

Abstract

Mammalian male germ cell development takes place in the testis under the influence of a variety of somatic cells and an incompletely defined paracrine and endocrine influences. Since it is not recapitulated well in vitro, researchers studying spermatogenesis often manipulate the germline by creating transgenic or knockout mice or by administering pharmaceutical agonists/antagonists or inhibitors. The effects of these types of manipulations on germline development can often be determined following microscopic imaging, both of stained and immunostained testis sections. Here, we describe approaches for microscopic analysis of the developing male germline, provide detailed protocols for a variety of immunostaining approaches, and discuss transgenic fluorescent reporter lines for studying the early stages of spermatogenesis.

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References

Tanaka S, Nagamatsu G, Tokitake Y, Kasa M, Tam P, Matsui Y . Regulation of expression of mouse interferon-induced transmembrane protein like gene-3, Ifitm3 (mil-1, fragilis), in germ cells. Dev Dyn. 2004; 230(4):651-9. DOI: 10.1002/dvdy.20085. View

Ikami K, Tokue M, Sugimoto R, Noda C, Kobayashi S, Hara K . Hierarchical differentiation competence in response to retinoic acid ensures stem cell maintenance during mouse spermatogenesis. Development. 2015; 142(9):1582-92. PMC: 4419276. DOI: 10.1242/dev.118695. View

Shima J, McLean D, McCarrey J, Griswold M . The murine testicular transcriptome: characterizing gene expression in the testis during the progression of spermatogenesis. Biol Reprod. 2004; 71(1):319-30. DOI: 10.1095/biolreprod.103.026880. View

Pangas S, Choi Y, Ballow D, Zhao Y, Westphal H, Matzuk M . Oogenesis requires germ cell-specific transcriptional regulators Sohlh1 and Lhx8. Proc Natl Acad Sci U S A. 2006; 103(21):8090-5. PMC: 1472434. DOI: 10.1073/pnas.0601083103. View

Nakagawa T, Nabeshima Y, Yoshida S . Functional identification of the actual and potential stem cell compartments in mouse spermatogenesis. Dev Cell. 2007; 12(2):195-206. DOI: 10.1016/j.devcel.2007.01.002. View

Bellve A, Cavicchia J, Millette C, OBrien D, Bhatnagar Y, Dym M . Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. J Cell Biol. 1977; 74(1):68-85. PMC: 2109873. DOI: 10.1083/jcb.74.1.68. View

Ohbo K, Yoshida S, Ohmura M, Ohneda O, Ogawa T, Tsuchiya H . Identification and characterization of stem cells in prepubertal spermatogenesis in mice. Dev Biol. 2003; 258(1):209-25. DOI: 10.1016/s0012-1606(03)00111-8. View

Yang Q, Oatley J . Spermatogonial stem cell functions in physiological and pathological conditions. Curr Top Dev Biol. 2014; 107:235-67. DOI: 10.1016/B978-0-12-416022-4.00009-3. View

Yoshimizu T, Sugiyama N, De Felice M, Yeom Y, Ohbo K, Masuko K . Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Dev Growth Differ. 2000; 41(6):675-84. DOI: 10.1046/j.1440-169x.1999.00474.x. View

10.

Geyer C, Kiefer C, Yang T, McCarrey J . Ontogeny of a demethylation domain and its relationship to activation of tissue-specific transcription. Biol Reprod. 2004; 71(3):837-44. DOI: 10.1095/biolreprod.104.028969. View

11.

Nakagawa T, Sharma M, Nabeshima Y, Braun R, Yoshida S . Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment. Science. 2010; 328(5974):62-7. PMC: 2981100. DOI: 10.1126/science.1182868. View

12.

Nayernia K, Li M, Jaroszynski L, Khusainov R, Wulf G, Schwandt I . Stem cell based therapeutical approach of male infertility by teratocarcinoma derived germ cells. Hum Mol Genet. 2004; 13(14):1451-60. DOI: 10.1093/hmg/ddh166. View

13.

Kapuscinski J, Szer W . Interactions of 4', 6-diamidine-2-phenylindole with synthetic polynucleotides. Nucleic Acids Res. 1979; 6(11):3519-34. PMC: 327953. DOI: 10.1093/nar/6.11.3519. View

14.

John G, Gallardo T, Shirley L, Castrillon D . Foxo3 is a PI3K-dependent molecular switch controlling the initiation of oocyte growth. Dev Biol. 2008; 321(1):197-204. PMC: 2548299. DOI: 10.1016/j.ydbio.2008.06.017. View

15.

Payer B, Chuva de Sousa Lopes S, Barton S, Lee C, Saitou M, Surani M . Generation of stella-GFP transgenic mice: a novel tool to study germ cell development. Genesis. 2006; 44(2):75-83. DOI: 10.1002/gene.20187. View

16.

Kluin P, Kramer M, de Rooij D . Proliferation of spermatogonia and Sertoli cells in maturing mice. Anat Embryol (Berl). 1984; 169(1):73-8. DOI: 10.1007/BF00300588. View

17.

Losinno A, Morales A, Fernandez D, Lopez L . Peritubular myoid cells from rat seminiferous tubules contain actin and myosin filaments distributed in two independent layers. Biol Reprod. 2012; 86(5):150, 1-8. DOI: 10.1095/biolreprod.111.095158. View

18.

Ballow D, Meistrich M, Matzuk M, Rajkovic A . Sohlh1 is essential for spermatogonial differentiation. Dev Biol. 2006; 294(1):161-7. DOI: 10.1016/j.ydbio.2006.02.027. View

19.

Ohinata Y, Payer B, OCarroll D, Ancelin K, Ono Y, Sano M . Blimp1 is a critical determinant of the germ cell lineage in mice. Nature. 2005; 436(7048):207-13. DOI: 10.1038/nature03813. View

20.

Chan F, Oatley M, Kaucher A, Yang Q, Bieberich C, Shashikant C . Functional and molecular features of the Id4+ germline stem cell population in mouse testes. Genes Dev. 2014; 28(12):1351-62. PMC: 4066404. DOI: 10.1101/gad.240465.114. View