Depigmentation of hair shafts is a hallmark of human aging. However, it remains unclear how aging causes human hair to grey. Here, we found that a single session of hair plucking via waxing causes hair to grey in castrated mice. Moreover, this hair greying continued for several hair cycles. Given that androgen secretion decreases with age in both male and female humans, the present result suggests that this decrease may contribute to age-related hair greying. In addition, our experimental procedure may represent an effective way to generate a new mouse model of hair greying without the need for genetic engineering.
hair greyingandrogenhair pluckingcastrated micemouse modelThe author(s) declared that no grants were involved in supporting this work.Introduction
Hair greying is a common and ubiquitous feature of human aging.
1 Although the rate of hair greying varies among individuals, almost all males and females experience depigmentation of hair shafts with age. Because the color in hair shafts is provided by melanin granules, which are supplied to the trichocytes of growing hair shafts by melanocytes,
2 age-related hair greying is thought to involve some dysfunction to these processes.
3
In contrast to humans, mice do not show any obvious hair greying, probably because they have a much shorter lifespan. However, hair greying in mice is experimentally inducible by genetic manipulation
3 or ionizing radiation,
4 suggesting that hair greying in humans may also involve genetic variation and genomic damage. Very recently, a report demonstrated that strong acute stress leads to hair greying in mice through depletion of melanocyte stem cells.
5 Consistent with this, another report indicated that hair greying in humans likely occurs in parallel with psychological stressors.
6 Given that genomic damage and psychological stress are likely to accumulate with age, these factors may explain the mechanism of age-related hair greying in humans. However, the true mechanism remains unclear due to the lack of direct evidence for this hypothesis and the presence of multiple other factors related to aging that may be involved in human hair greying.
Many previous studies have demonstrated a relationship between sex hormones and hair physiology and drastic changes in the secretion of sex hormones with age.
7,8 Therefore, in this study, we focused on the causal link between sex hormone secretion and hair greying.
MethodsEthical approval
All protocols for animal experiments refer to the 3R principle and were approved by the Animal Research Committee of Yamaguchi University (approval number: 437). Animal studies were performed in compliance with the Yamaguchi University Animal Care and Use guidelines. All animals were assessed as healthy prior to commencement of experiments and handled by experienced researchers in such a way as to minimize stress. All efforts were made to ameliorate any suffering of animals.
Mice
C57BL6J mice were purchased from Japan SLC, maintained under standard laboratory conditions (room temperature: 24 ± 2°C), with a regular 12:12 light-dark cycle, and allowed
ad libitum access to food and water. Each mouse was housed singly in a standard mouse cage (W 213 mm, D 324 mm and H 131 mm) and transferred to a clean cage every two weeks. ALPHA-dri (Shepherd Specialty Papers, Inc., U.S.A.), made from alpha cellulose and having high absorbency, was used as animal bedding. Mice were acclimatized to laboratory conditions for at least 7 days before the commencement of experiments. They had never received any experimental treatment previously. Mice with abnormalities were excluded from the study. To evaluate hair regrowth, mice were anesthetized with isoflurane gas, and their dorsal hair shafts were trimmed and then completely removed by waxing.
Statistical analysis
No statistical methods were used to predetermine the sample size due to the lack of available estimation. A Fisher’s exact test was performed to compare the probability of hair greying between sham-operated and castrated males. P < 0.05 indicated statistical significance.
Results and discussion
Intact male and female C57BL6 mice did not show any visibly obvious signs of hair greying over their lifetime, probably because their lifespan was too short for depigmentation to begin in the hair shaft. Based on a previous report demonstrating that repetitive shaving or plucking accelerates hair greying,
9 we applied this method to increase the susceptibility of hair shafts to greying in response to experimental intervention. As expected, neither a single shaving nor waxing session to pluck hair shafts from the back was sufficient to induce hair greying (shaving, 16-week-old males (n = 12) and females (n = 12); plucking, 16-week-old males (n = 52) and females (n = 12)). Next, to simply and directly test the involvement of androgen in hair greying, we examined whether or not a single session of plucking hair shafts from the back caused hair to grey in castrated male mice (
Figure 1a). We found that while sham-operated mice did not show any visible signs of greying, one-third of castrated mice showed hair greying in a small area on the surface of the back (16-week-old sham-operated (n = 12) and castrated males (n = 18)). A Fisher’s exact test showed that there was a significant difference in the incidence of hair greying between sham-operated and castrated males (0 out of 12 sham-operated versus 6 out of 18 castrated males, P < 0.01). Taken together, these results suggest that loss of androgen substantially accelerates hair greying. Grey hair shafts were still present after more than 100 days post-hair plucking (
Figure 1b). Given that the length of each hair cycle in mice is about 25 days,
10 this result indicates that hair greying continued across at least four hair cycles. Therefore, just a single session of hair plucking in castrated mice caused long-lasting damage to the pigmentation of hair shafts.
Long-lasting hair greying in castrated mice subjected to a single session of hair plucking.
(a) Representative images of the back of sham-operated and castrated male mice 24 days after hair plucking. The operation and waxing were performed when mice were 5 and 16 weeks old, respectively. Hair shafts were removed from almost the entire area of the back in the image. The area containing greying hairs is enlarged two-fold and shown at the bottom.
(b) An image showing the back of a castrated mouse with greying hair 103 days after hair plucking. The animal in this image is the same as that shown in the rightmost image in (a).
Considering that androgen secretion decreases with age in both male and female humans,
8 a possible interpretation of the present findings is that this decrease contributes to age-related hair greying in humans. While it is well known that androgen is involved in the pathogenesis of androgenetic alopecia,
11 the present study is the first to show that androgen is also involved in hair greying. Further studies are required to elucidate the mechanism underlying how loss of androgen causes depigmentation of hair shafts.
A potential limitation of the present study is that our experimental results may not directly explain age-related hair greying in humans because hair greying in mice was induced by hair plucking. However, it is difficult to overcome this technical limitation in animal experiments due to the creatures’ short lifespan, thus, unavoidably requiring some artificial induction of hair greying. Another potential limitation is that we cannot currently explain why a single session of hair plucking causes hair to grey in only about 33% but not all castrated mice, and in just a small area but not the entire surface of the back.
The present method may be useful for generating a new mouse model of hair greying that differs from most previous models in that it does not require genetic manipulations. However, our experimental procedure needs to be modified to improve the success rate and increase the body surface across which hair greying is induced. It is possible that a second session of hair plucking may overcome these limitations. Successful modification of the procedure for hair greying will provide a useful mouse model for studying the mechanism and prevention of human hair greying.
Data availability
All data underlying the results are available as part of the article and no additional source data are required.
Author contributions
A.M., J.K. and N.K. performed experiments. K.N. provided a wide range of general support. M.A. conceived and supervised the project and wrote the manuscript.
Acknowledgments
We express our appreciation to Nanami Yasumune, Yuna Yamamoto, Ritsuko Matsumura and Junko Sumino for providing technical assistance.
ReferencesPanhardSLozanoILoussouarnG:
Greying of the human hair: a worldwide survey, revisiting the ‘50’ rule of thumb.Br. J. Dermatol.2012 Oct;167(4):865–873. eng.
2271603410.1111/j.1365-2133.2012.11095.xTobinDJ:
The cell biology of human hair follicle pigmentation.Pigment Cell Melanoma Res.2011 Feb;24(1):75–88. eng.
2107061210.1111/j.1755-148X.2010.00803.xO’SullivanJDBNicuCPicardM:
The biology of human hair greying.Biol. Rev. Camb. Philos. Soc.2021 Feb;96(1):107–128. eng.
3296507610.1111/brv.12648InomataKAotoTBinhNT:
Genotoxic stress abrogates renewal of melanocyte stem cells by triggering their differentiation.Cell.2009 Jun 12;137(6):1088–1099. eng.
1952451110.1016/j.cell.2009.03.037ZhangBMaSRachminI:
Hyperactivation of sympathetic nerves drives depletion of melanocyte stem cells.Nature.2020 Jan;577(7792):676–681. eng.
3196969910.1038/s41586-020-1935-3PMC7184936RosenbergAMRausserSRenJ:
Quantitative mapping of human hair greying and reversal in relation to life stress.elife.2021 Jun;10:10. Epub 2021/06/23. eng.
3415597410.7554/eLife.67437PMC8219384OhnemusUUenalanMInzunzaJ:
The hair follicle as an estrogen target and source.Endocr. Rev.2006 Oct;27(6):677–706. eng.
1687767510.1210/er.2006-0020LambertsSWBeldAWvan denLelyAJvan der:
The endocrinology of aging.Science.1997 Oct 17;278(5337):419–424. eng.
933429310.1126/science.278.5337.419EndouMAokiHKobayashiT:
Prevention of hair graying by factors that promote the growth and differentiation of melanocytes.J. Dermatol.2014;41(8):716–723. eng.
2509915710.1111/1346-8138.12570Müller-RöverSHandjiskiBVeenCvan der:
A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.J. Invest. Dermatol.2001 Jul;117(1):3–15. eng.
1144274410.1046/j.0022-202x.2001.01377.xHeilmann-HeimbachSHochfeldLMHenneSK:
Hormonal regulation in male androgenetic alopecia-Sex hormones and beyond: Evidence from recent genetic studies.Exp. Dermatol.2020 Sep;29(9):814–827. eng.
3294613410.1111/exd.1413010.5256/f1000research.137979.r191777Reviewer response for version 1ChoiSekyu1Referee
Pohang University of Science and Technology, Pohang, South Korea
Competing interests: No competing interests were disclosed.
The authors showed plucking a small area of the back skin in castrated male mice can lead to hair greying, whereas the entire back skin area did not exhibit the same effect. However, this report's scientific credibility is deficient and it needs to be addressed in the following points.
The Methods sections should encompass the details of mouse castration procedure.
Testosterone levels should be measured in both sham-operated and castrated male mice.
Statistical analysis, accompanied by mouse images, should be integrated into Figure 1.
Authors should provide the results of plucking across the entire skin area of castrated mice in main figures.
Is the work clearly and accurately presented and does it cite the current literature?
Partly
If applicable, is the statistical analysis and its interpretation appropriate?
No
Are all the source data underlying the results available to ensure full reproducibility?
No source data required
Is the study design appropriate and is the work technically sound?
No
Are the conclusions drawn adequately supported by the results?
No
Are sufficient details of methods and analysis provided to allow replication by others?
I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.
10.5256/f1000research.137979.r161200Reviewer response for version 1AdachiShungo1Referee
Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan
Competing interests: No competing interests were disclosed.
This manuscript by Matsumoto et al. describes that wax depilatory treatment of castrated male mice can induce graying of newly growing body hair. Since graying does not occur in untreated mice, the authors suggest that it may be due to sex hormone effect. The results are valuable not only because it provides new insights into graying research, but also it provides an experimental model for graying. However, several concerns need to be addressed before this paper can be accepted for publication.
1. Please provide more details about the hair removal method in the METHOD section.
2. Since normal epilation does not cause graying in male castrated mice, it is likely that the artificial epilation process itself is also responsible for graying. Please provide experimental results as to whether the chemical stimulation of Wax or the physical stimulation of wiping the hairs (or both) is responsible for graying.
3. Since graying does not occur in female mice, it is possible that it is caused by sex hormones other than androgens, please provide a discussion in the text as to why graying does not occur in females. Or, In order to confirm whether the induction of graying is due to the effect of androgens, please provide an experiment result to see if gray hairs develop when male castrated mice are administered androgens.
4. Since graying after epilation does not occur in all castrated male mice and the shape of the areas varies, it seems likely that graying is related to the hair growth cycle. Please discuss that in the discussion section.
Is the work clearly and accurately presented and does it cite the current literature?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Is the study design appropriate and is the work technically sound?
Partly
Are the conclusions drawn adequately supported by the results?
Partly
Are sufficient details of methods and analysis provided to allow replication by others?
Partly
Reviewer Expertise:
cell biology, molecular biology, proteomics,
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.