Advertisement

Assessing Autophagy in the Leydig Cells

  • Hui Gao
  • Chao Liu
  • Wei Li
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1854)

Abstract

Autophagy is an important intracellular degradation system which is implicated in many physiological and pathological processes. During autophagy, cytosolic constituents such as organelles and macromolecules are engulfed by autophagosome, and then they fuse with lysosomes for degradation and recycle of the engulfed components within the autolysosome to maintain cellular homeostasis. In male testis, the Leydig cells provide the major source of testosterone production. Autophagy is extremely active in Leydig cells and is involved in the steroid production. However, the precise role of autophagy in Leydig cells is still largely unknown. Thus, a comprehensive measurement of autophagic activity with different methods would shed light on our knowledge about the functional role of autophagy in regulating male reproductive physiology. In this chapter, we describe the morphological, cellular, and biochemical methods to monitor autophagy in Leydig cells.

Keywords

Autophagy Immunoblotting Immunofluorescence LC3 Leydig cell Transmission electron microscopy 

Notes

Acknowledgements

This work was supported by the National Nature Science of China (Grant No. 91649202 and 31471277) and National key R&D program of China (Grant No. 2016YFA0500901). Hui Gao and Chao Liu contributed equally to this work.

References

  1. 1.
    Yang Z, Klionsky DJ (2010) Eaten alive: a history of macroautophagy. Nat Cell Biol 12(9):814–822CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    De Duve C, Wattiaux R (1966) Functions of lysosomes. Annu Rev Physiol 28:435–492CrossRefPubMedGoogle Scholar
  3. 3.
    Kim I, Rodriguez-Enriquez S, Lemasters JJ (2007) Selective degradation of mitochondria by mitophagy. Arch Biochem Biophys 462(2):245–253CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Singh R et al (2009) Autophagy regulates lipid metabolism. Nature 458(7242):1131–1135CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Ravikumar B et al (2010) Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev 90(4):1383–1435CrossRefGoogle Scholar
  6. 6.
    Mizushima N, Yoshimori T, Ohsumi Y (2011) The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 27:107–132CrossRefPubMedGoogle Scholar
  7. 7.
    Jiang P, Mizushima N (2015) LC3- and p62-based biochemical methods for the analysis of autophagy progression in mammalian cells. Methods 75:13–18CrossRefPubMedGoogle Scholar
  8. 8.
    Skinner MK (1991) Cell-cell interactions in the testis. Endocr Rev 12(1):45–77CrossRefPubMedGoogle Scholar
  9. 9.
    Sharpe RM (1987) Testosterone and spermatogenesis. J Endocrinol 113(1):1–2CrossRefPubMedGoogle Scholar
  10. 10.
    Haider SG (2004) Cell biology of Leydig cells in the testis. Int Rev Cytol 233:181–241CrossRefPubMedGoogle Scholar
  11. 11.
    Habert R, Lejeune H, Saez JM (2001) Origin, differentiation and regulation of fetal and adult Leydig cells. Mol Cell Endocrinol 179(1–2):47–74CrossRefPubMedGoogle Scholar
  12. 12.
    Kuopio T, Paranko J, Pelliniemi LJ (1989) Basement membrane and epithelial features of fetal-type Leydig cells in rat and human testis. Differentiation 40(3):198–206CrossRefPubMedGoogle Scholar
  13. 13.
    Risbridger GP, de Kretser DM (1986) Percoll-gradient separation of Leydig cells from postnatal rat testes. J Reprod Fertil 76(1):331–338CrossRefPubMedGoogle Scholar
  14. 14.
    Mendis-Handagama SM, Ariyaratne HB (2001) Differentiation of the adult Leydig cell population in the postnatal testis. Biol Reprod 65(3):660–671CrossRefPubMedGoogle Scholar
  15. 15.
    Sharma RS, Pal PC, Rajalakshmi M (2006) Isolation and culture of Leydig cells from adult rats. Indian J Clin Biochem 21(1):27–33CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Tang XM, Clermont Y, Hermo L (1988) Origin and fate of autophagosomes in Leydig cells of normal adult rats. J Androl 9(4):284–293CrossRefPubMedGoogle Scholar
  17. 17.
    Yi J, Tang XM (1995) Functional implication of autophagy in steroid-secreting cells of the rat. Anat Rec 242(2):137–146CrossRefPubMedGoogle Scholar
  18. 18.
    Kuma A, Matsui M, Mizushima N (2007) LC3, an autophagosome marker, can be incorporated into protein aggregates independent of autophagy: caution in the interpretation of LC3 localization. Autophagy 3(4):323–328CrossRefPubMedGoogle Scholar
  19. 19.
    Kabeya Y et al (2004) LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J Cell Sci 117(Pt 13):2805–2812CrossRefPubMedGoogle Scholar
  20. 20.
    Kabeya Y et al (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19(21):5720–5728CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Mizushima N, Yoshimori T (2007) How to interpret LC3 immunoblotting. Autophagy 3(6):542–545CrossRefPubMedGoogle Scholar
  22. 22.
    Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140(3):313–326CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Mizushima N (2004) Methods for monitoring autophagy. Int J Biochem Cell Biol 36(12):2491–2502CrossRefPubMedGoogle Scholar
  24. 24.
    Mizushima N (2009) Methods for monitoring autophagy using GFP-LC3 transgenic mice. Methods Enzymol 452:13–23CrossRefPubMedGoogle Scholar
  25. 25.
    Zhang L et al (2007) Method for real-time monitoring of protein degradation at the single cell level. BioTechniques 42(4):446, 448, 450Google Scholar
  26. 26.
    Yewdell JW, Lacsina JR, Rechsteiner MC, Nicchitta CV (2011) Out with the old, in with the new? Comparing methods for measuring protein degradation. Cell Biol Int 35(5):457–462CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Stem Cell and Reproductive BiologyInstitute of Zoology, Chinese Academy of SciencesBeijingPeople’s Republic of China
  2. 2.University of Chinese Academy of SciencesBeijingPeople’s Republic of China

Personalised recommendations