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Sertoli Cells pp 113-128 | Cite as

Assessing Autophagy in Sertoli Cells

  • Chao Liu
  • Jehangir Khan
  • Wei LiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1748)

Abstract

Autophagy is an important cellular homeostatic process, it degrades most long-lived proteins and some organelles by lysosome to provide raw materials for the survival of the cells during nutrient or energy deprivation condition. Autophagy is active in Sertoli cells and involved in many cellular processes. However, the precise role of autophagy in Sertoli cells is still largely unknown. Thus, the assessment of autophagy in Sertoli cells should be helpful for investigating the functional roles of autophagy in Sertoli cells. This chapter describes some methods for assessing autophagy in Sertoli cells, including detection of LC3 maturation/aggregation, transmission electron microscopy, half-life assessments of long-lived proteins, immunofluorescence microscopy, and co-localization of autophagy-targeted proteins with autophagy components or lysosomal proteins.

Keywords

Autophagy Sertoli cell Transmission electron microscopy LC3 Immunoblotting Immunofluorescence 

Notes

Acknowledgments

This work was supported by National Key R&D program of China (Grant No. 2016YFA0500901), and the National Nature Science of China (Grant No. 91519317, 91649202 and 31471277).

References

  1. 1.
    Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132(1):27–42. https://doi.org/10.1016/j.cell.2007.12.018 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140(3):313–326. https://doi.org/10.1016/j.cell.2010.01.028 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Wang H et al (2014) Atg7 is required for acrosome biogenesis during spermatogenesis in mice. Cell Res 24(7):852–869. https://doi.org/10.1038/cr.2014.70 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Tang Z et al (2013) Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites. Nature 502(7470):254–257. https://doi.org/10.1038/nature12606 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147(4):728–741. https://doi.org/10.1016/j.cell.2011.10.026 CrossRefPubMedGoogle Scholar
  6. 6.
    Mizushima N, Levine B (2010) Autophagy in mammalian development and differentiation. Nat Cell Biol 12(9):823–830. https://doi.org/10.1038/ncb0910-823 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Li WW, Li J, Bao JK (2012) Microautophagy: lesser-known self-eating. Cell Mol Life Sci 69(7):1125–1136. https://doi.org/10.1007/s00018-011-0865-5 CrossRefPubMedGoogle Scholar
  8. 8.
    Cuervo AM, Wong E (2014) Chaperone-mediated autophagy: roles in disease and aging. Cell Res 24(1):92–104. https://doi.org/10.1038/cr.2013.153 CrossRefPubMedGoogle Scholar
  9. 9.
    Mizushima N (2007) Autophagy: process and function. Genes Dev 21(22):2861–2873. https://doi.org/10.1101/gad.1599207 CrossRefPubMedGoogle Scholar
  10. 10.
    Mizushima N, Yoshimori T, Ohsumi Y (2011) The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 27:107–132. https://doi.org/10.1146/annurev-cellbio-092910-154005 CrossRefPubMedGoogle Scholar
  11. 11.
    Yang Z, Klionsky DJ (2009) An overview of the molecular mechanism of autophagy. Curr Top Microbiol Immunol 335:1–32. https://doi.org/10.1007/978-3-642-00302-8_1 PubMedPubMedCentralGoogle Scholar
  12. 12.
    Yu X, Long YC, Shen HM (2015) Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy. Autophagy 11(10):1711–1728. https://doi.org/10.1080/15548627.2015.1043076 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y (2009) Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat Rev Mol Cell Biol 10(7):458–467. https://doi.org/10.1038/nrm2708 CrossRefPubMedGoogle Scholar
  14. 14.
    Carlsson SR, Simonsen A (2015) Membrane dynamics in autophagosome biogenesis. J Cell Sci 128(2):193–205. https://doi.org/10.1242/jcs.141036 CrossRefPubMedGoogle Scholar
  15. 15.
    Itakura E, Mizushima N (2010) Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy 6(6):764–776CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Karanasios E, Stapleton E, Manifava M, Kaizuka T, Mizushima N, Walker SA, Ktistakis NT (2013) Dynamic association of the ULK1 complex with omegasomes during autophagy induction. J Cell Sci 126(Pt 22):5224–5238. https://doi.org/10.1242/jcs.132415 CrossRefPubMedGoogle Scholar
  17. 17.
    Klionsky DJ et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12(1):1–222. https://doi.org/10.1080/15548627.2015.1100356 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Griswold MD (1998) The central role of Sertoli cells in spermatogenesis. Semin Cell Dev Biol 9(4):411–416. https://doi.org/10.1006/scdb.1998.0203 CrossRefPubMedGoogle Scholar
  19. 19.
    Hai Y, Hou J, Liu Y, Yang H, Li Z, He Z (2014) The roles and regulation of Sertoli cells in fate determinations of spermatogonial stem cells and spermatogenesis. Semin Cell Dev Biol 29:66–75. https://doi.org/10.1016/j.semcdb.2014.04.007 CrossRefPubMedGoogle Scholar
  20. 20.
    Ma Y, Yang HZ, Xu LM, Huang YR, Dai HL, Kang XN (2015) Testosterone regulates the autophagic clearance of androgen binding protein in rat Sertoli cells. Sci Rep 5:8894. https://doi.org/10.1038/srep08894 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Yefimova MG et al (2013) A chimerical phagocytosis model reveals the recruitment by Sertoli cells of autophagy for the degradation of ingested illegitimate substrates. Autophagy 9(5):653–666. https://doi.org/10.4161/auto.23839 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Chen Y, Zhou Y, Wang X, Qian W, Han X (2013) Microcystin-LR induces autophagy and apoptosis in rat Sertoli cells in vitro. Toxicon 76:84–93. https://doi.org/10.1016/j.toxicon.2013.09.005 CrossRefPubMedGoogle Scholar
  23. 23.
    Eid N, Ito Y, Otsuki Y (2012) Enhanced mitophagy in Sertoli cells of ethanol-treated rats: morphological evidence and clinical relevance. J Mol Histol 43(1):71–80. https://doi.org/10.1007/s10735-011-9372-0 CrossRefPubMedGoogle Scholar
  24. 24.
    Liu C et al (2016) Autophagy is required for ectoplasmic specialization assembly in sertoli cells. Autophagy 12(5):814–832. https://doi.org/10.1080/15548627.2016.1159377 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Mizushima N (2004) Methods for monitoring autophagy. Int J Biochem Cell Biol 36(12):2491–2502. https://doi.org/10.1016/j.biocel.2004.02.005 CrossRefPubMedGoogle Scholar
  26. 26.
    Tanida I, Ueno T, Kominami E (2008) LC3 and autophagy. Methods Mol Biol 445:77–88. https://doi.org/10.1007/978-1-59745-157-4_4 CrossRefPubMedGoogle Scholar
  27. 27.
    Eskelinen EL (2008) Fine structure of the autophagosome. Methods Mol Biol 445:11–28. https://doi.org/10.1007/978-1-59745-157-4_2 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Stem Cell and Reproductive Biology, Institute of ZoologyChinese Academy of SciencesBeijingPeople’s Republic of China
  2. 2.Zoology Department, Buner CampusAbdul Wali Khan University MardanMardanPakistan

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