Introduction
Regenerative capacity of adult skeletal muscles is mediated by muscle stem cells, termed satellite cells (Lepper et al., Reference Lepper, Partridge and Fan2011; Murphy et al., Reference Murphy, Lawson, Mathew, Hutcheson and Kardon2011; Sambasivan et al., Reference Sambasivan, Yao, Kissenpfennig, Wittenberghe, Paldi, Gayraud-Morel, Guenou, Malissen, Tajbakhsh and Galy2011), which express the transcription factor Pax7 (Seale et al., Reference Seale, Sabourin, Girgis-Gabardo, Mansouri, Gruss and Rudnicki2000). Sex hormones, including androgens, are thought to play an important role in satellite cell function during muscle development and repair after injury (Kim et al., Reference Kim, Han, Seo, Park, Park, Jeong, Lee, Bae, Seong, Yum, Hann, Kwon, Seo, Choi and Kong2016). However, the molecular mechanisms by which androgen receptor (AR), a member of the nuclear receptor superfamily, might regulate satellite cells remain poorly understood.
Although Dubois described mice having satellite cell-specific knockout of AR (Dubois et al., Reference Dubois, Laurent, Sinnesael, Cielen, Helsen, Clinckemalie, Spans, Gayan-Ramirez, Deldicque, Hespel, Carmeliet, Vanderschueren and Claessens2014), they used MyoD-iCre mice in which the MyoD locus was active through satellite cell development and ARs were inactivated prenatally in satellite cell progenitors and all MyoD-positive myoblasts during development. To our knowledge, there are no studies that examined whether AR plays important roles in satellite cells during muscle regeneration using satellite cells-specific knock-out mice of AR in adults.
Objective
Here we sought to characterize the role of AR in muscle satellite cells during muscle regeneration. We tested the hypothesis that AR regulates satellite cell function during activation or differentiation in mice using two types of a tamoxifen-inducible Cre loci at Pax7, Pax7CreERT2(Fan)/+ and Pax7CreERT2(Gaka)/+, which were crossed with ARL2/Y mice to generate mice having satellite cell-specific AR knockout.
Methods
Animals
Pax7CreERT2(Fan)/+, Pax7CreERT2(Gaka)/+ and R26tdTomato/+ (Madisen et al., Reference Madisen, Zwingman, Sunkin, Oh, Zariwala, Gu, Ng, Palmiter, Hawrylycz, Jones, Lein and Zeng2009) mice were from Jackson Laboratories (Stock No: 012476, 017763 and 007908, respectively). ARL2/Y mice were previously described (Matsumoto et al., Reference Matsumoto, Takeyama, Sato and Kato2003). Tamoxifen (150 μl of 20 mg/ml; Sigma, T5648) was injected intraperitoneally on 5 consecutive days. We induced muscle injury by cardiotoxin injection (100 μl of 10 μM; Latoxan, L8102) in the left tibialis anterior (TA) muscle under anesthesia. The Ehime University Animal Experiment Committee approved the animal protocols.
Immunofluorescence and microscopy
For immunofluorescence staining (protocol: http://dx.doi.org/10.17504/protocols.io.sujeeun), muscles were frozen in liquid nitrogen-chilled isopentane. BIOREVO (Keyence) and Fiji software (https://imagej.net/Fiji) were used to photograph stained tissues and quantify Pax7+ nuclei and Myh3+ muscle fibers.
Satellite cell isolation
Hindlimb muscle satellite cells were isolated as described (Liu et al., Reference Liu, Cheung, Charville and Rando2015).
Quantitative real-time PCR
Using a RNeasy Plus Micro Kit (Qiagen, 74034) we extracted satellite cell total RNA for synthesis of cDNA with PrimeScript (Takara, RR036A). We performed duplicate qPCR using TB Green Premix Ex Taq II (Takara, RR820S) and Thermal Cycler Dice (Takara, TP850).
Statistics
Data were analyzed using Prism 8 (GraphPad Software). Welch’s t test (for 2 groups) and Bonferroni&s multiple comparisons test (for 3 groups) was used.
Results
Nearly all Pax7+ cells in control uninjured TA muscle stained positively with AR antibodies, indicating AR expression by satellite cells (Figure 1A). In Pax7CreERT2(Fan)/+;ARL2/Y mice having conditional AR deletion, AR mRNA levels in FACS-isolated satellite cells were decreased relative to control (Pax7+/+;ARL2/Y) mice (Figure 1B).
Surprisingly, upon muscle injury, the number of Pax7+ cells decreased by similar amounts in Pax7CreERT2(Fan)/+ control mice and Pax7CreERT2(Fan)/+;ARL2/Y mice at 5 dpi (Figure 1C-E), while Pax7CreERT2(Fan)/+ control mice showed slightly delayed muscle regeneration at 14 dpi (Figure 1F-H).
Pax7CreERT2(Gaka)/+;ARL2/Y;R26tdTomato/+ mice having different Cre-mediated AR deletions in satellite cells (Figure 2A) had similar numbers of Pax7+ cells at 5 dpi as the control (Figure 2B-D). Muscle regeneration in terms of Myh3+ fiber numbers was similar for the control and mutant mice at 14 dpi (Figure 2E-G). Together, these results suggest that AR expressed by satellite cells is not involved in muscle regeneration.
Discussion
The Pax7CreERT2(Fan)/+ regeneration phenotype is consistent with previous findings (Mademtzoglou et al., Reference Mademtzoglou, Asakura, Borok, Alonso-Martin, Mourikis, Kodaka, Mohan, Asakura and Relaix2018) and likely related to expression of only one Pax7 allele after CreERT2 insertion (Lepper et al., Reference Lepper, Conway and Fan2009). Leaky Cre recombinase activation and downregulation of Hey1, which is required to maintain satellite cells, in Pax7CreERT2(Fan)/+ mice not treated with tamoxifen (Noguchi et al., Reference Noguchi, Nakamura, Hino, Nogami, Tsuji, Sato, Zhang, Tsujikawa, Tanaka, Izawa, Okada, Doi, Kokubo, Harada, Uezumi, Gessler, Ohkawa and Fukada2019) could affect satellite cells during muscle regeneration.
The muscle regeneration activity seen in satellite cell-specific AR knockout mice despite effective AR deletion suggests that AR does not function in muscle regeneration. However, a phenotype that was not detectable in our assays might be present.
Conclusions
Our muscle regeneration experiments using Pax7CreERT2(Fan)/+ mice suggest that these Cre mice, especially gene Knockout/Knockin mice, must be used carefully and in combination with other Cre mice to avoid the problem of “pseudo phenotypes” generated by the Cre cassette or Cre insertion at the target locus. Our data for Pax7CreERT2(Gaka)/+;ARL2/Y;R26tdTomato/+ mice demonstrated that AR expression by satellite cells is not necessary for muscle regeneration in adults.
Acknowledgements
We thank S. Nakanishi and A. Nishio for technical support and K. Kameda at the Division of Analytical Bio-Medicine in the Advanced Research Support Center (ADRES) of Ehime University for flow cytometry support.
Author Contributions
HS and YI designed the study and wrote the manuscript. HS performed the experiments and analyzed the data. TS, MK, and SF provided materials.
Funding Information
This work was supported in part by MEXT/JSPS KAKENHI (H.S., 18H06439, 19K19947), (Y.I., 17H6427, 19H03786); The Nakatomi Foundation (Y.I.), Takeda Science Foundation (Y.I.), the Osaka Medical Research Foundation For Intractable Diseases (H.S., H30 2-18); and the Ehime University Grant-in-Aid Research Empowerment Program (H.S., H30 Start-up Support).
Data Availability Statements
The data that support the findings of this study are openly available in protocols.io at http://dx.doi.org/10.17504/protocols.io.sujeeun.
Conflicts of Interest:
Hiroshi Sakai, Takahiko Sato, Motoi Kanagawa, So-ichiro Fukada and Yuuki Imai have no conflicts of interest to declare.
Comments
Comments to the Author: This manuscript reports a study to investigate the role of the androgen receptor on muscle satellite cell regeneration. Performing a mouse study they find that the androgen receptor does not have a role in this process. The work seems to have been performed in suitable way and I think this is suitable for publication in Experimental Results.