Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Sex hormones establish a reserve pool of adult muscle stem cells

Nature Cell Biology volume 18, pages 930–940 (2016)Cite this article

Subjects

An Erratum to this article was published on 28 September 2016

This article has been updated

Abstract

Quiescent satellite cells, known as adult muscle stem cells, possess a remarkable ability to regenerate skeletal muscle following injury throughout life. Although they mainly originate from multipotent stem/progenitor cells of the somite, the mechanism underlying the establishment of quiescent satellite cell populations is unknown. Here, we show that sex hormones induce Mind bomb 1 (Mib1) expression in myofibres at puberty, which activates Notch signalling in cycling juvenile satellite cells and causes them to be converted into adult quiescent satellite cells. Myofibres lacking Mib1 fail to send Notch signals to juvenile satellite cells, leading to impaired cell cycle exit and depletion. Our findings reveal that the hypothalamic–pituitary–gonadal axis drives Mib1 expression in the myofibre niche. Moreover, the same axis regulates the re-establishment of quiescent satellite cell populations following injury. Our data show that sex hormones establish adult quiescent satellite cell populations by regulating the myofibre niche at puberty and re-establish them during regeneration.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Notch1 activation in cycling SCs at puberty.
Figure 2: Conversion of cycling juvenile SCs into quiescent SCs by Notch signalling.
Figure 3: Defective Notch activation and precocious depletion of SCs by Mib1-null MFs.
Figure 4: Induction of Mib1 in MFs by sex hormones.
Figure 5: Establishment of quiescent SCs via AR/ER-induced Mib1-expressing MFs.
Figure 6: Establishment of adult SC populations by the HPG axis.
Figure 7: Impaired re-establishment of SC populations in skeletal muscle regeneration.
Figure 8: Requirement of sex hormones in re-establishment of quiescent adult SC populations after muscle injury.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Change history

  • 08 September 2016

    In the version of this Article originally published, in the key in Fig. 1b, the red and black data points were incorrectly labelled; the red data points should have been labelled ‘DHT’ and the black data points as 'Veh'. Furthermore, in the key in Fig. 2c, the red and black data points were also incorrectly labelled; the red data points should have been labelled 'N1ΔSC', and the black data points as 'N1WT'. These errors have been corrected in the online versions of the Article.

References

  1. Tajbakhsh, S. Skeletal muscle stem cells in developmental versus regenerative myogenesis. J. Int. Med. 266, 372–389 (2009).

    CAS  Google Scholar 

  2. Schultz, E. Satellite cell proliferative compartments in growing skeletal muscles. Dev. Biol. 175, 84–94 (1996).

    CAS  PubMed  Google Scholar 

  3. White, R. B., Bierinx, A. S., Gnocchi, V. F. & Zammit, P. S. Dynamics of muscle fibre growth during postnatal mouse development. BMC Dev. Biol. 10, 21 (2010).

    PubMed  PubMed Central  Google Scholar 

  4. Sambasivan, R. et al. Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. Development 138, 3647–3656 (2011).

    CAS  PubMed  Google Scholar 

  5. Gunther, S. et al. Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells. Cell Stem Cell 13, 590–601 (2013).

    PubMed  PubMed Central  Google Scholar 

  6. Wierman, M. E. Sex steroid effects at target tissues: mechanisms of action. Adv. Physiol. Educ. 31, 26–33 (2007).

    PubMed  Google Scholar 

  7. Sinha-Hikim, I., Taylor, W. E., Gonzalez-Cadavid, N. F., Zheng, W. & Bhasin, S. Androgen receptor in human skeletal muscle and cultured muscle satellite cells: up-regulation by androgen treatment. J. Clin. Endocrinol. Metab. 89, 5245–5255 (2004).

    CAS  PubMed  Google Scholar 

  8. Kalbe, C., Mau, M., Wollenhaupt, K. & Rehfeldt, C. Evidence for estrogen receptor alpha and beta expression in skeletal muscle of pigs. Histochem. Cell Biol. 127, 95–107 (2007).

    CAS  PubMed  Google Scholar 

  9. Chambon, C. et al. Myocytic androgen receptor controls the strength but not the mass of limb muscles. Proc. Natl Acad. Sci. USA 107, 14327–14332 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Brown, D., Hikim, A. P., Kovacheva, E. L. & Sinha-Hikim, I. Mouse model of testosterone-induced muscle fiber hypertrophy: involvement of p38 mitogen-activated protein kinase-mediated Notch signaling. J. Endocrinol. 201, 129–139 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Velders, M., Schleipen, B., Fritzemeier, K. H., Zierau, O. & Diel, P. Selective estrogen receptor-beta activation stimulates skeletal muscle growth and regeneration. FASEB J. 26, 1909–1920 (2012).

    CAS  PubMed  Google Scholar 

  12. Yoon, K. J. et al. Mind bomb 1-expressing intermediate progenitors generate notch signaling to maintain radial glial cells. Neuron 58, 519–531 (2008).

    CAS  PubMed  Google Scholar 

  13. Song, R. et al. Mind bomb 1 in the lymphopoietic niches is essential for T and marginal zone B cell development. J. Exp. Med. 205, 2525–2536 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Conboy, I. M. & Rando, T. A. The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis. Dev. Cell 3, 397–409 (2002).

    CAS  PubMed  Google Scholar 

  15. Fre, S. et al. Notch signals control the fate of immature progenitor cells in the intestine. Nature 435, 964–968 (2005).

    CAS  PubMed  Google Scholar 

  16. Kopan, R. & Ilagan, M. X. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137, 216–233 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Rios, A. C., Serralbo, O., Salgado, D. & Marcelle, C. Neural crest regulates myogenesis through the transient activation of NOTCH. Nature 473, 532–535 (2011).

    CAS  PubMed  Google Scholar 

  18. Schuster-Gossler, K., Cordes, R. & Gossler, A. Premature myogenic differentiation and depletion of progenitor cells cause severe muscle hypotrophy in Delta1 mutants. Proc. Natl Acad. Sci. USA 104, 537–542 (2007).

    CAS  PubMed  Google Scholar 

  19. Vasyutina, E. et al. RBP-J (Rbpsuh) is essential to maintain muscle progenitor cells and to generate satellite cells. Proc. Natl Acad. Sci. USA 104, 4443–4448 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Mourikis, P., Gopalakrishnan, S., Sambasivan, R. & Tajbakhsh, S. Cell-autonomous Notch activity maintains the temporal specification potential of skeletal muscle stem cells. Development 139, 4536–4548 (2012).

    CAS  PubMed  Google Scholar 

  21. Brohl, D. et al. Colonization of the satellite cell niche by skeletal muscle progenitor cells depends on Notch signals. Dev. Cell 23, 469–481 (2012).

    PubMed  Google Scholar 

  22. Mourikis, P. et al. A critical requirement for notch signaling in maintenance of the quiescent skeletal muscle stem cell state. Stem Cells 30, 243–252 (2012).

    CAS  PubMed  Google Scholar 

  23. Bjornson, C. R. et al. Notch signaling is necessary to maintain quiescence in adult muscle stem cells. Stem Cells 30, 232–242 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Brack, A. S., Conboy, I. M., Conboy, M. J., Shen, J. & Rando, T. A. A temporal switch from notch to Wnt signaling in muscle stem cells is necessary for normal adult myogenesis. Cell Stem Cell 2, 50–59 (2008).

    CAS  PubMed  Google Scholar 

  25. Ebling, F. J. The neuroendocrine timing of puberty. Reproduction 129, 675–683 (2005).

    CAS  PubMed  Google Scholar 

  26. Ober, C., Loisel, D. A. & Gilad, Y. Sex-specific genetic architecture of human disease. Nat. Rev. Genet. 9, 911–922 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Safranski, T. J., Lamberson, W. R. & Keisler, D. H. Correlations among three measures of puberty in mice and relationships with estradiol concentration and ovulation. Biol. Reprod. 48, 669–673 (1993).

    CAS  PubMed  Google Scholar 

  28. Fukada, S. et al. Molecular signature of quiescent satellite cells in adult skeletal muscle. Stem Cells 25, 2448–2459 (2007).

    CAS  PubMed  Google Scholar 

  29. Liu, L. et al. Chromatin modifications as determinants of muscle stem cell quiescence and chronological aging. Cell Rep. 4, 189–204 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Koo, B. K. et al. An obligatory role of mind bomb-1 in notch signaling of mammalian development. PLoS ONE 2, e1221 (2007).

    PubMed  PubMed Central  Google Scholar 

  31. Lepper, C. & Fan, C. M. Inducible lineage tracing of Pax7-descendant cells reveals embryonic origin of adult satellite cells. Genesis 48, 424–436 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Yang, X. et al. Notch activation induces apoptosis in neural progenitor cells through a p53-dependent pathway. Dev. Biol. 269, 81–94 (2004).

    CAS  PubMed  Google Scholar 

  33. Murtaugh, L. C., Stanger, B. Z., Kwan, K. M. & Melton, D. A. Notch signaling controls multiple steps of pancreatic differentiation. Proc. Natl Acad. Sci. USA 100, 14920–14925 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Chakkalakal, J. V., Jones, K. M., Basson, M. A. & Brack, A. S. The aged niche disrupts muscle stem cell quiescence. Nature 490, 355–360 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Koo, B. K. et al. Mind bomb 1 is essential for generating functional Notch ligands to activate Notch. Development 132, 3459–3470 (2005).

    CAS  PubMed  Google Scholar 

  36. Yoon, M. J. et al. Mind bomb-1 is essential for intraembryonic hematopoiesis in the aortic endothelium and the subaortic patches. Mol. Cell. Biol. 28, 4794–4804 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Jeong, H. W. et al. Inactivation of Notch signaling in the renal collecting duct causes nephrogenic diabetes insipidus in mice. J. Clin. Invest. 119, 3290–3300 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Jeong, H. W., Kim, J. H., Kim, J. Y., Ha, S. J. & Kong, Y. Y. Mind bomb-1 in dendritic cells is specifically required for Notch-mediated T Helper Type 2 differentiation. PLoS ONE 7, e36359 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Kim, Y. W. et al. Defective Notch activation in microenvironment leads to myeloproliferative disease. Blood 112, 4628–4638 (2008).

    CAS  PubMed  Google Scholar 

  40. Bruning, J. C. et al. A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance. Mol. Cell 2, 559–569 (1998).

    CAS  PubMed  Google Scholar 

  41. Camarda, G. et al. A pRb-independent mechanism preserves the postmitotic state in terminally differentiated skeletal muscle cells. J. Cell Biol. 167, 417–423 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Wiik, A., Ekman, M., Johansson, O., Jansson, E. & Esbjornsson, M. Expression of both oestrogen receptor alpha and beta in human skeletal muscle tissue. Histochem. Cell Biol. 131, 181–189 (2009).

    CAS  PubMed  Google Scholar 

  43. Shiina, H. et al. Premature ovarian failure in androgen receptor-deficient mice. Proc. Natl Acad. Sci. USA 103, 224–229 (2006).

    CAS  PubMed  Google Scholar 

  44. Weiser, M. J., Wu, T. J. & Handa, R. J. Estrogen receptor-beta agonist diarylpropionitrile: biological activities of R- and S-enantiomers on behavior and hormonal response to stress. Endocrinology 150, 1817–1825 (2009).

    CAS  PubMed  Google Scholar 

  45. Sun, J. et al. Antagonists selective for estrogen receptor alpha. Endocrinology 143, 941–947 (2002).

    CAS  PubMed  Google Scholar 

  46. Carson, J. A., Lee, W. J., McClung, J. & Hand, G. A. Steroid receptor concentration in aged rat hindlimb muscle: effect of anabolic steroid administration. J. Appl. Physiol. 93, 242–250 (2002).

    CAS  PubMed  Google Scholar 

  47. Kadi, F., Bonnerud, P., Eriksson, A. & Thornell, L. E. The expression of androgen receptors in human neck and limb muscles: effects of training and self-administration of androgenic-anabolic steroids. Histochem. Cell Biol. 113, 25–29 (2000).

    CAS  PubMed  Google Scholar 

  48. Doumit, M. E., Cook, D. R. & Merkel, R. A. Testosterone up-regulates androgen receptors and decreases differentiation of porcine myogenic satellite cells in vitro. Endocrinology 137, 1385–1394 (1996).

    CAS  PubMed  Google Scholar 

  49. Bao, J., Ma, H. Y., Schuster, A., Lin, Y. M. & Yan, W. Incomplete cre-mediated excision leads to phenotypic differences between Stra8-iCre; Mov10l1(lox/lox) and Stra8-iCre; Mov10l1(lox/Delta) mice. Genesis 51, 481–490 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Maatta, J. A. et al. Inactivation of the androgen receptor in bone-forming cells leads to trabecular bone loss in adult female mice. Bonekey Rep. 2, 440 (2013).

    PubMed  PubMed Central  Google Scholar 

  51. Dupont, S. et al. Effect of single and compound knockouts of estrogen receptors alphaup (ERα) and β (ERβ) on mouse reproductive phenotypes. Development 127, 4277–4291 (2000).

    CAS  PubMed  Google Scholar 

  52. Edelstein, M. C., Gordon, K., Williams, R. F., Danforth, D. R. & Hodgen, G. D. Single dose long-term suppression of testosterone secretion by a gonadotropin-releasing hormone antagonist (Antide) in male monkeys. Contraception 42, 209–214 (1990).

    CAS  PubMed  Google Scholar 

  53. Cattanach, B. M., Iddon, C. A., Charlton, H. M., Chiappa, S. A. & Fink, G. Gonadotrophin-releasing hormone deficiency in a mutant mouse with hypogonadism. Nature 269, 338–340 (1977).

    CAS  PubMed  Google Scholar 

  54. Shea, K. L. et al. Sprouty1 regulates reversible quiescence of a self-renewing adult muscle stem cell pool during regeneration. Cell Stem Cell 6, 117–129 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Kuang, S., Kuroda, K., Le Grand, F. & Rudnicki, M. A. Asymmetric self-renewal and commitment of satellite stem cells in muscle. Cell 129, 999–1010 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Yoo, Y. E. & Ko, C. P. Dihydrotestosterone ameliorates degeneration in muscle, axons and motoneurons and improves motor function in amyotrophic lateral sclerosis model mice. PLoS ONE 7, e37258 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Neal, A., Boldrin, L. & Morgan, J. E. The satellite cell in male and female, developing and adult mouse muscle: distinct stem cells for growth and regeneration. PLoS ONE 7, e37950 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Valdez, D. J. et al. Seasonal changes in plasma levels of sex hormones in the greater Rhea (Rhea americana), a South American ratite with a complex mating system. PLoS ONE 9, e97334 (2014).

    PubMed  PubMed Central  Google Scholar 

  59. Zenclussen, M. L., Casalis, P. A., Jensen, F., Woidacki, K. & Zenclussen, A. C. Hormonal fluctuations during the estrous cycle modulate heme oxygenase-1 expression in the uterus. Front. Endocrinol. 5, 32 (2014).

    Google Scholar 

  60. Lucas, L. A. & Eleftheriou, B. E. Circadian variation in concentrations of testosterone in the plasma of male mice: a difference between BALB/cBy and C57BL/6By inbred strains. J. Endocrinol. 87, 37–46 (1980).

    CAS  PubMed  Google Scholar 

  61. Ohkawa, Y., Mallappa, C., Vallaster, C. S. & Imbalzano, A. N. Isolation of nuclei from skeletal muscle satellite cells and myofibers for use in chromatin immunoprecipitation assays. Methods Mol. Biol. 798, 517–530 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Liu, L., Cheung, T. H., Charville, G. W. & Rando, T. A. Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nat. Protoc. 10, 1612–1624 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Van Uytfanghe, K. et al. Evaluation of a candidate reference measurement procedure for serum free testosterone based on ultrafiltration and isotope dilution-gas chromatography-mass spectrometry. Clin. Chem. 50, 2101–2110 (2004).

    CAS  PubMed  Google Scholar 

  64. Xu, X., Keefer, L. K., Ziegler, R. G. & Veenstra, T. D. A liquid chromatography-mass spectrometry method for the quantitative analysis of urinary endogenous estrogen metabolites. Nat. Protoc. 2, 1350–1355 (2007).

    CAS  PubMed  Google Scholar 

  65. Moon, J. Y., Kim, K. J., Moon, M. H., Chung, B. C. & Choi, M. H. A novel GC-MS method in urinary estrogen analysis from postmenopausal women with osteoporosis. J. Lipid Res. 52, 1595–1603 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Roubinian, J. R., Talal, N., Greenspan, J. S., Goodman, J. R. & Siiteri, P. K. Effect of castration and sex hormone treatment on survival, anti-nucleic acid antibodies, and glomerulonephritis in NZB/NZW F1 mice. J. Exp. Med. 147, 1568–1583 (1978).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank S. Kato and Y. Imai for providing Arf/y mice, J.-S. Kang for suggesting useful methods for this study, and S.-K. Lee for helpful comments for manuscript preparation. This work was supported by grants from the Basic Science Research Program (NRF-2014R1A2A1A10052675), the Bio and Medical Technology Development Program (NRF-2011-0019269) and the Korea Mouse Phenotyping Project (NRF-2014M3A9D5A01073930) of the Ministry of Science, ICT and Future of the National Research Foundation, funded by the Korean government, and by the SNU-Yonsei Research Cooperation Program through Seoul National University.

Author information

Authors and Affiliations

  1. School of Biological Sciences, Seoul National University, Seoul 151-747, South Korea

    Ji-Hoon Kim, Gi-Chan Han, Ji-Yun Seo, Inkuk Park, Wookjin Park, Hyun-Woo Jeong, Sung-hwan Bae, Jinwoo Seong, Min-Kyu Yum, Sang-Hyeon Hann, Daekwan Seo & Young-Yun Kong

  2. Materials and Life Science Research Division, Korea Institute of Science and Technology, Seoul 136-791, South Korea

    Su Hyeon Lee & Man Ho Choi

  3. Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-752, South Korea

    Young-Guen Kwon

  4. Center for RNA Research Institute for Basic Science, Seoul 151-742, South Korea

    Daekwan Seo

Authors
  1. Ji-Hoon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gi-Chan Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ji-Yun Seo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Inkuk Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wookjin Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hyun-Woo Jeong
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Su Hyeon Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sung-hwan Bae
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jinwoo Seong
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Min-Kyu Yum
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Sang-Hyeon Hann
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Young-Guen Kwon
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Daekwan Seo
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Man Ho Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Young-Yun Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.-H.K. designed the study, performed the experiments, analysed the data, and wrote the manuscript. G.-C.H., J.-Y.S., I.K.P., W.J.P., H.-W.J., S.H.B., J.W.S., M.-K.Y. and S.H.H. conducted the experiments. S.H.L. and M.H.C. measured the serum E2 levels using LC–MS/MS and provided helpful comments. Y.-G.K. provided useful materials. D.K.S. performed statistical and bioinformatical analysis. Y.-Y.K. designed the research and wrote the manuscript.

Corresponding author

Correspondence to Young-Yun Kong.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Conversion of cycling satellite cells (SCs) into quiescent SCs by dihydrotestosterone (DHT).

(a–c) Ten-day-old mice subcutaneously (s.c.) injected with corn oil (Veh) or DHT. (a) Quantification of Pax7+Ki67+ and Pax7+Ki67− cells per 100 myofibers (MFs). n = 5 animals for each group; data are mean ± s.d.; Bootstrap t-test; ∗P < 0.05. Statistical source data for a is provided in Supplementary Table 2. (b,c) Representative IHC staining for Pax7, p57 (b) MyoD and Ki67 (c) in tibialis anterior (TA) muscles. Scale bar, 25 μm. (d,e) Microarray analysis of Veh or DHT-injected 10-day-old mice s.c. injected with Veh or DHT [GSE64454]. TA muscles were isolated 24 h after the injection. The significantly upregulated genes identified from an independent dataset are depicted as a Venn diagram (d). Selected microarray datasets: (1) Adult SCs in uninjured muscle compared to SCs in injured muscle [GSE471771], (2) quiescent SCs compared to activated SCs in culture conditions [GSE34832], and (3) TA muscles of 10-day-old male mice injected with DHT compared to Veh. Venn diagrams display the number of common genes obtained from the gene expression dataset. Integrative analysis (e). In total 1,031 genes were found to be significantly increased (>2-fold) or decreased (<-2-fold) in Veh and DHT-treated mice. These datasets were used as input for pathway analysis with the Ingenuity pathway analysis (IPA) software (Ingenuity Systems, http://www.ingenuity.com, Redwood City, CA, USA). The graph with the most significant P-values, and the highest-scoring functional pathways are shown. (f) The pathway analysis of Notch signalling and cell cycle-related genes that are expressed at a substantially higher or lower level in DHT-injected TA muscles than in Veh-injected TA muscles. The pathway analysis via IPA software is depicted as containing the majority of Notch signalling-regulated genes. The analysis was performed with the Ingenuity software package. Red and green circles represent upregulated and downregulated genes, respectively. (g) mRNA expressions of Ar, Mib1, Notch target genes (Hes1, Hey1, HeyL), cell cycle inhibitors (p21, p27, p57), Sprouty-1 (Spry1), and Cdc20 in TA muscles after 24 h of DHT treatment. n = 3 animals for each group; data are mean ± s.d.; Two-sample t-test; ∗P < 0.05. (h) Relative expression levels of Notch target genes in myogenic cells (MCs) at the indicated ages. (i) IHC staining of Pax7 and Nicd in TA muscles at the indicated ages. Arrows and arrowheads indicate Nicd−Pax7+ and Nicd+Pax7+ cells, respectively; scale bar, 25 μm.

Supplementary Figure 2 Notch activation converts cycling SCs to quiescent SCs.

(a,b) Relative muscle mass normalized to body weight (a) and mRNA expression in MCs (b). n = 3 animals for each group; data are mean ± s.d.; Two-sample t-test; ∗P < 0.05. Relative muscle masses to body weights were comparable. (c) IHC staining of Pax7. Scale bar, 50 μm. (d,e) Seven-day-old N1WT and N1OE/SC mice were intraperitoneally (i.p.) injected daily with tamoxifen for 3 days. Nicd and nuclear GFP proteins are expressed in the Pax7+ cells of N1OE/SC mice. (d) IHC staining from 10-day-old N1WT and N1OE/SC TA muscles. Arrows and arrowheads indicate Pax7+GFP−p57− and Pax7+GFP+p57+ cells, respectively; scale bar, 25 μm. (e) Quantification of Pax7, GFP, and p57 as in d; n = 3 animals for each group; data are mean ± s.d.

Supplementary Figure 3 Analysis of the hindlimb muscles lacking Mib1 in myofibers.

(a–c) The expression of Mib1 (a; n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; ∗P < 0.05), IHC staining for Mib1 (b; scale bar, 25 μm), and myonuclei number (c; n = 5 animals for each group; Data are mean ± s.d.; Poisson’s general linear model regression; N.S. not significant) in MFs from 8-week-old Mib1WT and Mib1ΔMFextensor digitorum longus (EDL) muscles. Myonuclei numbers in Mib1WT and Mib1ΔMF MFs were comparable. Statistical source data for a and c is provided in Supplementary Table 2. (d) Morphometric quantification of cross-sectional area validated in Laminin-stained TA muscles, which is comparable in Mib1WT and Mib1ΔMF mice;n = 5 animals for each group; data are mean ± s.d.; χ2 test. (e) IHC staining for MyoD and Ki67 in TA muscles from Mib1WT and Mib1ΔMF mice at the indicated ages. Arrows indicate MyoD+Ki67+ cells; scale bar, 25 μm. (f) Quantification of MyoD+ cells per 100 MFs in TA muscles of Mib1WT and Mib1ΔMF mice at indicated ages. n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; ∗P < 0.05. (g,h) IHC staining (g; scale bar, 25 μm) and quantification (h; n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; N.D. not detected) for Pax7 and MyoG in TA muscles from 4- and 12-week-old Mib1WT mice. The Pax7+/MyoG+ cells were observed only in 4-week-old. Statistical source data for h is provided in Supplementary Table 2. (i) Immunoblotting of Myogenin in SCs isolated from hindlimb muscles of 4-week-old Mib1WT and Mib1ΔMF mice. Numbers represent the relative signal intensity of the designated antibody measured by densitometry. Unprocessed original scans of blots are shown in Supplementary Fig. 9. (j,k) IHC staining for Pax7 (j), Myogenin (k) and activated Caspase3 in TA muscles from 4-week-old Mib1WT and Mib1ΔMF mice. Scale bar, 25 μm. There was no significant increase of apoptotic cells in Mib1ΔMF mice. (l) mRNA expressions of Pax7, Notch target genes, differentiation markers, and stem cell markers in SCs purified from 4-week-old Mib1WT and Mib1ΔMF mice. n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; ∗P < 0.05.

Supplementary Figure 4 MF-specific Mib1 ablation affects precocious depletion of SCs in hindlimb muscles and defective muscle regeneration.

(a) IHC staining for Pax7 and Ki67 in TA muscles from Mib1WT and Mib1ΔMF mice at the indicated ages. Arrows and arrowheads indicate Ki67+Pax7+ and Ki67−Pax7+ cells, respectively; scale bar, 50 μm. (b) p21, p27 and Spry1 levels in the MCs from Mib1WT and Mib1ΔMF hindlimb muscles. n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; ∗P < 0.05. (c,d) Optic (c; scale bar, 0.2 cm) and hematoxylin and eosin-stained (d; scale bar, 25 μm) images of BaCl2-injured TA muscles in 8-week-old mice. (e) IHC stained images for Laminin in TA muscles 5 or 10 days after BaCl2 injury. Scale bar, 25 μm. (f) Flow cytometric analysis of SC populations in 8-week-old ContWT (Mib1WT and N1WT), Mib1ΔMF, and N1ΔSC mice. n = 5 animals for each group; data are mean ± s.d.; Tukey’s pairwise comparison test; ∗∗P < 0.01. Controls are designated as the WT of comparing mutant mice. Statistical source data for f is provided in Supplementary Table 2.

Supplementary Figure 5 DHT and E2 independently drive the conversion of cycling SCs into quiescent SCs.

(a–d) The expression of Mib1 (a,c), androgen receptor (Ar, b), and estrogen receptor β (Esr2, d) in TA muscles. Ten-day-old Mib1WT and Mib1ΔMF mice were s.c. injected with Veh, DHT, or E2, and TA muscles were isolated 3 days after injection. Both DHT and E2 treatment induced the expression of the target genes Ar and Esr2, respectively, in Mib1WT and Mib1ΔMF TA muscles. n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; ∗P < 0.05. Statistical source data for a–d is provided in Supplementary Table 2. (e–h) IHC staining of Pax7 (e,f), MyoD (g,h) and Ki67 in TA muscles from 10-day-old mice injected with Veh, DHT (e,g) or E2 (f,h). TA muscles were isolated 3 days after injection. Most Pax7+ cells were still Ki67+ and the number of cycling MyoD+ cells was maintained by either DHT or E2 treatment in Mib1ΔMF TA muscles, while Pax7+ cells were Ki67− and the MyoD+ cells significantly decreased in Mib1WT TA muscles. Arrows and arrowheads indicate Ki67+ and Ki67− cells, respectively; scale bar, 20 μm. (i,j) Quantification of Pax7 and Ki67 in 10-day-old TA muscles treated with Veh, DHT (i) or E2 (j) as in e,f. n = 3 animals for each group; data are mean ± s.d.; Poisson’s general linear model regression; ∗P < 0.05; N.S. not significant. Statistical source data for i,j is provided in Supplementary Table 2.

Supplementary Figure 6 E2 normally induces Mib1 in MFs lacking Ar.

(a,b) The Cre-mediated recombination was confirmed by PCR in ArΔMF mice at the indicated ages in the shown organs (a) and in myofiber but not in satellite cells (b). Unprocessed original scans of blots are shown in Supplementary Fig. 10. (c) The gross morphology in levator ani (LA) muscles from 4-week-old ArΔMF mice. Scale bars, 0.5 cm. (d,e) IHC staining (d; scale bars, 20 μm), and quantification (e; n = 3 animals for each group; data are mean ± s.d.; Bootstrap t-test; ∗P < 0.05) for Pax7, Ki67, and Laminin in TA muscles from 10-day-old ArWT (upper) and ArΔMF (bottom) mice injected with Veh or E2. TA muscles were isolated from hindlimb muscles 3 days after treatment. Arrows and arrowheads indicate Ki67+Pax7+ and Ki67−Pax7+ cells, respectively. Statistical source data for e is provided in Supplementary Table 2. (f,g) Muscle mass (f; n = 3 animals for each group; data are mean ± s.d.; two-sample t-test; ∗P < 0.05), myonuclei numbers in MFs (g; n = 3 animals for each group; data are mean ± s.d.; Poisson’s general linear model regression; N.S. not significant) of 12-week-old ContWT and ArΔMFEsr2−/− mice. Muscle mass, myonuclei numbers were comparable. Statistical source data for f and g is provided in Supplementary Table 2. (h) Mib1 levels in 12-week-old EDL muscles from indicated genotypes. n = 3 animals for each group; data are mean ± s.d.; Tukey’s pairwise comparison test; ∗P < 0.05. (i–k) IHC staining for Mib1 (i; scale bar, 100 μm), the expression of Ar and Esr2 (j) in MFs, and the mRNA expression of Notch target genes in MCs (k). n = 3 animals for each group; data are mean ± s.d.; two-sample t-test; ∗P < 0.05. Statistical source data for j and k is provided in Supplementary Table 2. The MFs were isolated from hindlimb muscles of 12-week-old ContWT and ArΔMFEsr2−/− mice.

Supplementary Figure 7 Surgically and pharmacologically disturbed hypothalamic and gonadal axis impair the establishment of SC population.

(a) A schematic diagram for Nal-Lys gonadotropin releasing-hormone antagonist (Antide) injection or orchiectomy (Orx). (b,c) Serum testosterone levels measured by ELISA (b) and estradiol levels measured by LC-MS/MS (c) at indicated ages. n = 3 animals for each group; data are mean ± s.d.; Bootstrap t-test; ∗P < 0.05. (d,e) The expression of Ar and Esr2 in MFs (d) and Notch target genes in SCs (e) from 4-week-old sham and Orx mice. n = 3 animals for each group; data are mean ± s.d.; two-sample t-test; ∗P < 0.05. (f) IHC staining of Pax7 and Ki67 in TA muscles from 4-week-old sham and Orx mice. Scale bar, 50 μm. Quantification data is presented in Fig. 6b left subfigure. (g,h) The expression of Ar and Esr2 in MFs (g) and Notch target genes in SCs (h) from 4-week-old Veh and Antide-treated mice. n = 3 animals for each group; data are mean ± s.d.; Two-sample t-test; ∗P < 0.05. (i) IHC staining of Pax7 and Ki67 in TA muscles from 4-week-old Veh and Antide-treated mice. Quantification data is presented in Fig. 6b left subfigure. Arrows and arrowheads indicate Pax7+Ki67+ and Pax7+Ki67− cells, respectively; scale bar, 50 μm.

Supplementary Figure 8 Genetically disturbed hypothalamic and gonadal axis impairs the establishment of SC population.

(a,c) Gross morphology of seminal vesicles, testes and LA muscles (a; scale bar, 0.2 cm), muscle mass (b; n = 3 animals for each group; data are mean ± s.d.; Two-sample t-test; *P < 0.05) and myonuclei number of EDL muscles (c; n = 3 animals for each group; data are mean ± s.d.; Poisson’s general linear model regression; N.S. not significant) in 8-week-old Gnrh1+/+ and Gnrh1hpg/hpg mice. Muscle mass and myonuclei numbers were comparable in Gnrh1+/+ and Gnrh1hpg/hpg mice. (d,e) The expression of Ar and Esr2 in MFs (d) and Notch target genes in SCs (e). n = 3 animals for each group; data are mean ± s.d.; two-sample t-test; ∗P < 0.05; ∗∗P < 0.01. (f) IHC staining (f) for Pax7 and Laminin in TA muscles. MFs, SCs, and TA muscles were isolated from 8-week-old Gnrh1+/+ and Gnrh1hpg/hpg mice. Scale bar, 50 μm. (g) Flow cytometric analysis of mononuclear cells from 8-week-old control (sum data of Sham, Veh, and Gnrh1+/+), Orx, Antide-treated, and Gnrh1hpg/hpg hindlimb muscles. n = 3 animals for each group; data are mean ± s.d.; Bootstrap t-test; ∗P < 0.05. (h) IHC staining of Pax7 in Vcam-1+Sca-1− cells sorted from mononuclear cells of Gnrh1+/+and Gnrh1hpg/hpg mice. Scale bar, 50 μm. (i) The representative chromatograms of E2 measurements by LC-MS/MS. n = 3 animals for each group; Data are mean ± s.d.

Supplementary Figure 9 Unprocessed original scans of blots.

Uncropped images of scanned western blots shown in Figures and Supplementary Figures are provided.

Supplementary information

Supplementary Information

Supplementary Information (PDF 3344 kb)

Supplementary Table 1

Supplementary Information (XLSX 19 kb)

Supplementary Table 2

Supplementary Information (XLSX 60 kb)

Supplementary Table 3

Supplementary Information (XLSX 13 kb)

Supplementary Table 4

Supplementary Information (XLSX 14 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, JH., Han, GC., Seo, JY. et al. Sex hormones establish a reserve pool of adult muscle stem cells. Nat Cell Biol 18, 930–940 (2016). https://doi.org/10.1038/ncb3401

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3401

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing