# Influence of sexual dimorphism on satellite cell regulation and inflammatory response during skeletal muscle regeneration

Charline Jomard, Julien Gondin

► To cite this version:

Charline Jomard, Julien Gondin. Influence of sexual dimorphism on satellite cell regulation and inflammatory response during skeletal muscle regeneration. Physiological Reports, 2023, 11, 10.14814/phy2.15798. hal-04302849

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## REVIEW

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The
Physiological
Society

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Physiological Reports

# Influence of sexual dimorphism on satellite cell regulation and inflammatory response during skeletal muscle regeneration

Charline Jomard

Julien Gondin

<figure><img src="image_5.png" /></figure>

Institut NeuroMyoGène (INMG),
Physiopathologie et Génétique du
Neurone et du Muscle (PGNM),
Université Claude Bernard Lyon, Lyon,
France

### Correspondence

Julien Gondin, Institut NeuroMyoGène (INMG), Physiopathologie et Génétique du Neurone et du Muscle (PGNM), CNRS 5261—INSERM U1315—UCBL1, Faculté de Médecine et de Pharmacie, 8 Avenue Rockefeller, 69008 Lyon, France.

Email: julien.gondin@univ-lyon1.fr

### Funding information

Centre National de la Recherche
Scientifique (CNRS), Grant/Award

Number: GDR Sport et Activité
Physique

### Abstract

After injury, skeletal muscle regenerates thanks to the key role of satellite cells (SC). The regeneration process is supported and coordinated by other cell types among which immune cells. Among the mechanisms involved in skeletal muscle regeneration, a sexual dimorphism, involving sex hormones and more particularly estrogens, has been suggested. However, the role of sexual dimorphism on skeletal muscle regeneration is not fully understood, likely to the use of various experimental settings in both animals and human. This review aims at addressing how sex and estrogens regulate both the SC and the inflammatory response during skeletal muscle regeneration by considering the different experimental designs used in both animal models (i.e., ovarian hormone deficiency, estrogen replacement or supplementation, treatments with estrogen receptors agonists/ antagonists and models knockout for estrogen receptors) and human (hormone therapy replacement, pre vs. postmenopausal, menstrual cycle variation...).

### KEYWORDS

estrogens, inflammation, satellite cells, skeletal muscle

## 1 | INTRODUCTION

Muscle damage may occur after unaccustomed muscle contractions, especially when exercise involves repeated eccentric contractions at high intensities and/or over a large range of motion or isometric contractions induced by neuromuscular electrical stimulation at long muscle length (Clarkson & Hubal, 2002; Fouré & Gondin, 2021). This condition is usually defined as exercise-induced muscle damage (EIMD). In human, EIMD is usually assessed from indirect markers such as delayed onset muscle soreness (DOMS), increased levels of plasmatic enzymes (e.g., creatine kinase [CK]), a

decreased maximal joint range of motion and a long-lasting reduction of force production, the latter being considered as the best indirect marker of muscle damage (Paulsen et al., 2012; Warren et al., 1999). Histological analysis after EIMD may reveal disruptions of the myofibrillar structure (z-line streaming, increase of desmin-negative myofibers), necrosis (at least in animal models of EIMD) and cellular infiltration (Clarkson & Hubal, 2002; Crameri et al., 2007).

Upon muscle damage, satellite cells (SC, skeletal muscle stem cells), which are located under the basal lamina of myofibers, are indispensable for muscle regeneration (Lepper et al., 2011; Murphy et al., 2011; Sambasivan

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© 2023 The Authors. *Physiological Reports* published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.
et al., 2011). Indeed, SCs get activated, proliferate, com-
mit to terminal myogenic differentiation and eventually
fuse to rebuild new functional myofibers (Yin et al., 2013).
Proper muscle regeneration relies on the dynamic inter-
play between SCs and their microenvironment including
immune cells (macrophages), endothelial cells and fibro-
adipogenic progenitors (FAPs)/fibroblasts (Bentzinger
et al., 2013). Inflammatory response plays a key role in
this process (Arnold et al., 2007). Indeed, the recruited cir-
culating monocytes differentiate into pro-inflammatory
macrophages, which enhance SC proliferation before
switching their phenotypes to anti-inflammatory mac-
rophages, to promote their differentiation and fusion to
regenerate injured myofibers (Arnold et al., 2007). Endo-
thelial cells and FAPs also participate to the regeneration
process by promoting SC proliferation and extracellular
matrix remodeling after damage, respectively (Christov
et al., 2007; Lemos et al., 2015). Readers are invited to refer
to recent reviews on the cellular interactions and the mo-
lecular effectors involved in muscle regeneration (Bernard
et al., 2022; Chazaud, 2020; Panci & Chazaud, 2021).

Among the mechanisms involved in skeletal muscle
damage and regeneration, a sexual dimorphism has been
suggested (Amelink et al., 1990; Amelink & Bär, 1986;
Deasy et al., 2007; Enns & Tiidus, 2008; Hubal & Clark-
son, 2009; Stupka et al., 2000; Tiidus & Enns, 2009; Velders
et al., 2012). The pioneering study of Amelink and col-
leagues showed that males displayed a higher resting CK
activity as compared with females (Amelink et al., 1990).
In addition, while CK activity remained unchanged in fe-
males after EIMD (Amelink & Bär, 1986), this parameter
was significantly elevated in both males and ovariecto-
mized females, illustrating that ovarian hormones might
have a protective effect on muscle damage. It has been fur-
ther suggested that hormonal regulation, and more partic-
ularly a greater estrogen production, may have a positive
impact on the regulation of SC fate and inflammatory re-
sponse (Enns & Tiidus, 2008; Liao et al., 2019).

Briefly, estrogens (i.e., estrone [E1], 17β-estradiol [E2] and estriol [E3]), a group of sex hormones, are involved in the development and regulation of the female reproductive system. Their main source of production and secretion is the granulose cells of the ovaries while other tissues (adrenal glands, liver, fat, skeletal muscle) contribute to a lesser extent to circulating estrogens. E2 is the most biologically active form of estrogen and therefore the most widely investigated. Estrogens exert their function through three different estrogen receptors (ER) (Collins et al., 2019; Enns et al., 2008; Seko et al., 2020; Velders et al., 2012): ER-α, ER-β, and G protein-coupled receptor (GPER). The biological effects of estrogens are mediated by genomic mechanisms through their binding to cytosolic ER. This leads to the translocation to the nucleus and

the binding of the estrogen-estrogen receptor complex
to estrogen response element in the promotor and en-
hancer regions of the target genes, ultimately resulting in
the transcription of estrogen responsive genes. Estrogens
can also act through non-genomic mechanisms involving
intracellular signaling pathways after GPER activation
(Arnal et al., 2017).

So far, the role of sexual dimorphism on skeletal mus-
cle regeneration has been investigated using various
experimental settings, including for instance ovarian hor-
mone deficiency, E2 replacement (i.e., when E2 is given to
ovariectomized animals) or supplementation (i.e., when
E2 is given to healthy animals), mouse models knockout
for ER or observational human studies. Although recent
reviews focused on the impact of E2 on skeletal muscle
mass, regeneration and mitochondrial function (McMil-
lin et al., 2022; Pellegrino et al., 2022), no clear distinction
was made between the above-mentioned methodologies,
which may explain some controversies in this research
field.

This review aims at addressing how sex and estro-
gens regulate both the SC and the inflammatory response
during skeletal muscle regeneration by considering the
different experimental designs used in both animal mod-
els (i.e., ovarian hormone deficiency, E2 replacement or
supplementation, treatments with estrogen receptors
agonists/antagonists and models knockout for ER) and
human (hormone therapy replacement, pre vs postmeno-
pausal, menstrual cycle variation...). Finally, we provided
a brief critical analysis of these different experimental
conditions and proposed perspectives on how future stud-
ies could most effectively evaluate the impact of sex/estro-
gen on skeletal muscle regeneration.

2 | SATELLITE CELLS

2.1 | Animal studies

The regulation of SC fate after muscle damage has been
assessed in different contexts such as ovarian hormone
deprivation (i.e., ovariectomy [OVX]) combined or not
with E2 replacement and in estrogen receptor knockout-
mice (Figure 1). On the contrary, the impact of sex (i.e.,
direct comparison between males and females) on SC fate
during muscle regeneration has been scarcely investigated
(Seko et al., 2020).

2.1.1 | Effects of ovarian-hormone deficiency

Ovariectomy (OVX) is a surgical procedure where ova-
ries are removed, which leads to an ovarian hormonal
<figure><img src="image_6.png" /><figcaption>**FIGURE 1** Effects of sex and estrogens on satellite cell fate and skeletal muscle regeneration after injury. After injury, satellite cells (SC) get activated, proliferate, commit to terminal myogenic differentiation and eventually fuse to rebuild new functional myofibers. Ovarian hormone deficiency and estrogen receptors (ER) inhibition/KO affect the different steps of myogenesis (For the sake of clarity, OVX-induced impairment in SC self-renewal and differentiation is indicated in red, underlined font). This ultimately leads to impaired muscle regeneration with decreased myofiber cross-sectional area (CSA) and muscle mass, increased fibrosis and impaired force production. On the contrary, 17β-estradiol, though often delivered at a supraphysiological dose, and ER-agonists promote SC activation, proliferation and differentiation, leading to increased myofiber CSA, muscle mass, force and reduced fibrosis. Human studies are rare with some evidence illustrating a decline in SC content from peri to postmenopausal. The influence of both sex and menstrual cycle on SC fate and skeletal muscle regeneration remains to be carefully investigated in human.</figcaption></figure>

deficiency (i.e., including E2) (Souza et al., 2019). A consensus is emerging illustrating that OVX-induced hormonal deficiency affects the number of SCs at steady-state and after damage (Collins et al., 2019; Enns & Tiiidus, 2008; Kitajima & Ono, 2016; Seko et al., 2020; Velders et al., 2012). The total number of SCs assessed by flow cytometry (i.e., CD31⁻, CD45⁻, VCAM⁺, α7-integrin⁺) or by immunostaining (i.e., Pax7⁺ cells) was reduced in several uninjured muscles, such as *tibialis anterior*, *extensor digitorum longus*, *gastrocnemius* and *diaphragm*, after ovarian hormone deficiency lasting from 14 days to 7 months (Collins et al., 2019; Kitajima & Ono, 2016; Larson et al., 2022). On the contrary, the SC content of the slower *soleus* muscle was unaffected, suggesting that OVX could specifically affect SC number of the fast muscles (Collins et al., 2019).

The number of Pax7⁺ cells was also not affected by OVX when assessed from freshly isolated *extensor digitorum longus* myofibers (Kitajima & Ono, 2016). However, this result should be interpreted with caution as the number of isolated myofibers stained for Pax7 was not reported in this study. Interestingly, SCs obtained from isolated myofibers and cultured for 3 days showed reduced self-renewal and differentiating capacities after OVX (Kitajima & Ono, 2016). In addition, SCs from OVX mice showed a reduced capacity to transition from G0/G1 to S and G2/M phases as compared with SCs extracted from intact female mice (Larson et al., 2022). This was associated with an upregulated gene expression of *ccna2* and *p16*<sup>INK4a</sup>, known as key cell cycle regulators. OVX also resulted in a lower engraftment of SCs and a reduction in myogenic
cell differentiation and fusion when intact SCs were trans-
planted in OVX mice as compared to controls (Collins
et al., 2019). Interestingly, SCs extracted from OVX do-
nors and transplanted into control mice showed similar
engraftment than SCs obtained from control, illustrating
the influence of circulating E2 on SC regulation (Collins
et al., 2019). Finally, OVX mice showed decreased muscle
weight, reduced myofiber cross-sectional area (CSA) and
increased content of collagen type I (an index of fibrosis)
as compared with intact females after cardiotoxin (CTX)-
induced muscle damage (Kitajima & Ono, 2016). Overall,
OVX-induced alterations of both SC number and fate led
to impaired muscle regeneration.

## 2.1.2 | Effects of E2 replacement and supplementation

E2 replacement rescues the total number of SCs (Collins
et al., 2019) and the CSA of fast-type myofibers (Kita-
jima & Ono, 2016), which both decreased after 7 months
post-OVX. In vitro studies showed that isolated SCs
from intact female mice have greater proliferation, dif-
ferentiation and fusion index when cultured in the pres-
ence of E2 as compared with normal medium (Larson
et al., 2022). In the same way, male bovine SCs treated
with E2 also showed a higher number of proliferat-
ing SCs (Kamanga-Sollo et al., 2008, 2004). The effects
of E2 on SC regulation in vitro were related to IGF-1-
dependent (Chakravarthy et al., 2000; Kamanga-Sollo
et al., 2004) and -independent mechanisms (Kamanga-
Sollo et al., 2008; Pampusch et al., 2003) inasmuch as the
use of a competitive inhibitor of IGF-I binding to type-I
IGF receptor partially reduced E2-stimulated prolifera-
tion in bovine SC cultures.

The role of E2 replacement has been further high-
lighted in response to muscle damage. SC content of
both fast and slow-type muscles (i.e., white vastus, white
gastrocnemius and soleus muscles) was higher in both
males and OVX females rats treated with E2 as compared
with non-treated animals 3 days after downhill running
(Enns & Tiidus, 2008; Mangan et al., 2014, 2015; Tiidus
et al., 2005). In the same way, SC number and muscle
mass were higher and strength recovery was faster in
OVX females treated with E2 as compared with untreated
females following repeated muscle damage induced by
BaCl₂ injections (Collins et al., 2019; Larson et al., 2020).
Also, early administration of E2 (i.e., within 2 weeks post-
OVX) increases the number of SCs 3 days after treadmill
running as compared with non-treated females while late
administration (i.e., starting 11 weeks post-OVX) prevents
this beneficial effect (Mangan et al., 2015). In addition,
inhibition of the phosphatidylinositol-3 kinase (PI3K)/

Akt pathway hinders E2-induced SC proliferation both
in vitro (Kamanga-Sollo et al., 2008) and in vivo (Mangan
et al., 2014), illustrating that the influence of E2 on SC reg-
ulation are mediated, at least in part, by this intracellular
signaling system (Chakravarthy et al., 2000).

In summary, E2 supplementation/replacement in cell
culture or in OVX animals illustrated the positive role of
this hormone on SC regulation during myogenesis and
skeletal muscle regeneration.

## 2.1.3 | Modulation of ER

The pioneering study of Enns and colleagues (Enns
et al., 2008) demonstrated that downhill running induced
higher content of total (Pax7⁺), proliferating (BrdU⁺)
and differentiating (MyoD⁺) SC in OVX rats treated with
E2 as compared with untreated females. This beneficial
effect of E2 was totally prevented by the administra-
tion of a specific ER inhibitor (i.e., ICI 182780). Similar
inhibitory effects were also demonstrated in vitro on
bovine SC proliferation (Kamanga-Sollo et al., 2008).
In addition, protein levels of proliferating cell nuclear
antigen and MyoD mRNA expression, used as mark-
ers of SC activation and differentiation, were higher in
controls and in OVX animals treated with either E2 or
ER-β agonist as compared with untreated OVX or those
treated with ER-α agonist (Velders et al., 2012) 3 days
after muscle damage induced by notexin (NTX) injec-
tion. On that basis, it has been suggested that SC fate
could be mainly mediated by ER-β. However, this result
should be considered with caution since ER-β agonist
concentration was ten-fold higher than the concentra-
tion of ER-α agonist. Interestingly, ER-β whole-body
knockout (KO) females showed lower mRNA expres-
sion of Pax7, Ki67 and MyoD (i.e., markers of SC sta-
tus) as well as embryonic myosin heavy chain (i.e., a
marker of muscle regeneration) compared with either
controls or ER-α whole-body KO females at 3 days post-
damage (Velders et al., 2012). This illustrates that mus-
cle regeneration could be preferentially mediated by
ER-β. Indeed, E2 or propyl pyrazole triol (i.e., a specific
ER-α agonist) treatment in females resulted in a higher
percentage of Pax7⁺ or MyoD⁺ myofibers 3 days after
downhill running (Thomas et al., 2010).

Over the last few years, our knowledge on the role of
ER on muscle regeneration has been greatly improved
thanks to the development of mouse models specifically
ablated for ER-α or ER-β in SCs. Although not directly
quantified, ERα protein expression in whole skeletal
muscle does not seem to differ between male and female
mice (Morfoisse et al., 2018). ERα gene expression in
whole muscle was the most abundant followed by Gper
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

and *ERβ* (Baltgalvis et al., 2010; Couse et al., 1997) in intact female mice while mRNA expression and protein levels of ER are also dependent on myofiber-type composition (Baltgalvis et al., 2010; Gustafsson et al., 1984; Lemoine et al., 2002; Saartok, 1984), training status (Lemoine et al., 2002) and E2 circulating levels (Baltgalvis et al., 2010).

Nevertheless, the gene encoding for the ER-α (*Esr1*) is seven times more expressed in SCs than the ones encoding for the ER-β and GPER (*Esr2* and *Gper1*) (Collins et al., 2019; Larson et al., 2022). Thus, ablation of ER-α in SC leads to a 40%-60% reduction in SC number for females which was not rescued by E2 replacement, indicating that ER-α is involved in the maintenance of SC pool (Collins et al., 2019; Larson et al., 2022). Engraftment of SC was also lower in the absence of ER-α in donor SCs (Collins et al., 2019). Moreover, loss of ER-α in females leads to SC apoptosis as illustrated by the increased number of Pax7<sup>+</sup>TUNEL<sup>+</sup> cells, the upregulation of genes involved in cell death (e.g., *H19*, *Fndc1*, *mir378c*) together with the downregulation of genes promoting cell survival (e.g., *Dhcr24*, *Snord65*, *mir485*) (Collins et al., 2019). Finally, and despite its low mRNA expression (Collins et al., 2019), deletion of *ERβ* in SC resulted in impaired muscle regeneration in females, but not in males, after BaCl<sub>2</sub> injury (Seko et al., 2020). This was illustrated by reduced myofiber CSA and increased type I collagen content (Seko et al., 2020). These alterations were not exacerbated by OVX, indicating that E2 regulates the SC function through ER-β during muscle regeneration. ER-β also regulates SC proliferation and apoptosis while differentiation and fusion of SC seem to be independent of *ERβ* expression, even though it is not totally clear how the latter steps of myogenesis were experimentally characterized (Seko et al., 2020).

To conclude, E2 do exert a positive effect on muscle regeneration, particularly by modulating SC fate in females (Collins et al., 2019; Enns & Tiidus, 2008; Kitajima & Ono, 2016; Seko et al., 2020; Tiidus et al., 2005; Velders et al., 2012). Both ER-α and ER-β are implicated in SC function even though their respective contribution to SC fate remains to be determined. In addition, although the influence of E2 on SC fate is now well-documented in response to toxic injury, their role in EIMD remains unclear.

## 2.2 | Human studies

Contrary to animals, the effects of sexual dimorphism on SC number and fate in human are less documented (Figure 1) (Bonavaud et al., 1997; Collins et al., 2019; Fortino et al., 2022; Horwath et al., 2021; Kadi et al., 2004; Roth et al., 2000).

### 2.2.1 | Effect of sex

No sex difference was reported for ER mRNA expression, *ESR1* gene expression being 180-fold higher than *ESR2* mRNA levels in both men and women (Lemoine et al., 2003), as well as for the proportion of ERα and ERβ positive nuclei (Wiik et al., 2009).

Conflicting findings have been reported on the number of Pax7<sup>+</sup> cells in men and women at steady state. While several studies showed no difference in SC number between men and women in the *vastus lateralis* and *tibialis anterior* muscles (Binet et al., 2023; Fortino et al., 2022; Kadi et al., 2004; Roth et al., 2000), other investigations reported that men had more Pax7<sup>+</sup> SCs in type II myofibers as compared with women (Abou Sawan et al., 2021; Horwath et al., 2021). In vitro investigations showed similar proliferation and fusion rates between sexes in four different muscles (i.e., *tensor fasciae, vastus lateralis and medialis and rectus femoris*) (Bonavaud et al., 1997).

To our knowledge, only one study investigated the effects of EIMD on SC regulation between men and women (Fortino et al., 2022). Men had more Pax7<sup>+</sup> cells than women 2 days after repeated maximal voluntary eccentric contractions while the number of Pax7<sup>+</sup>/MyoD<sup>+</sup> cells was not different between sexes. However, these findings should be interpreted with caution as the extent of muscle damage was not accurately compared between men and women (e.g., using force recovery as the best indirect marker of muscle damage (Paulsen et al., 2012)). Further analysis is necessary to carefully determine the impact of sex on SC regulation after EIMD in human.

### 2.2.2 | Effect of E2 level fluctuations in pre and postmenopausal women

The expression of ER varies during the menstrual cycle (Ekenros et al., 2017; Haines et al., 2018). Indeed, both mRNA and protein levels of ERα in skeletal muscle were highest in the follicular phase when circulating estradiol levels were low (Ekenros et al., 2017; Haines et al., 2018). The authors however failed to detect *ESR2* mRNA expression and did not analyze the related protein expression so that it remains to determine whether and to what extent menstrual cycle affects ERβ protein expression (Ekenros et al., 2017). Despite similar E2 muscle concentrations, a greater skeletal muscle *CCND1* mRNA expression was observed in the mid-follicular phase as compared with the mid-luteal phase after repeated lengthening contractions. On the contrary, *MYOD1* mRNA expression similarly increased during the two phases after this exercise (Haines et al., 2018). Overall, it is unclear how and to what extent menstrual cycle affects skeletal muscle regeneration.
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

During the menopausal transition, ovarian hormone levels decrease leading to a reduction in muscle mass and strength production (Collins et al., 2019). Of interest, SC number declined in five women from peri to postmenopausal and was correlated with the reduction of E2 blood concentration (Collins et al., 2019). However, this result needs to be confirmed on a larger sample size (Collins et al., 2019) while it remains to determine how the reduced SC content associated with menopause affects skeletal muscle regeneration.

Overall, the impact of sexual dimorphism on SC regulation during skeletal muscle regeneration requires additional human studies, with a careful control of menstrual cycle.

# 3 | INFLAMMATORY RESPONSE

## 3.1 | Animal studies

Animal experiments investigated the influence of either sex or E2 on the regulation of both immune cell infiltration and their inflammatory status after muscle damage (Figure 2).

### 3.1.1 | Effect of sex

Male rats showed an increase in myeloperoxidase activity (MPO), an enzyme secreted by neutrophils and macrophages, in both *soleus* and *plantaris* muscles while MPO activity remains unchanged in females 1 day after downhill running exercise (Tiidus & Bombardier, 1999). In the same way, the number of myofibers invaded by acid phosphatase⁺ cells (i.e., which stain macrophages and lysosomes) was higher and the infiltration of interstitial ER-BMDM1⁺ cell (i.e., a marker of myeloid cells) occurs earlier in males as compared with females 1 day after electrically-evoked lengthening contractions (St Pierre Schneider et al., 1999). On the contrary, no sex difference was observed for the number of leukocytes (Schneider et al., 2012) and CD11b⁺ cells (i.e., a pan marker of myeloid cells) invading damaged myofibers or interstitial spaces after electrically-evoked lengthening contractions (St Pierre Schneider et al., 1999). Similarly, the number of neutrophils (i.e., CD11b⁺/Gr-1⁺ cells) and monocytes/macrophages (i.e., CD11b⁺/Gr-1⁻ cells) as well as the protein levels of monocyte chemoattractant protein 1 and 5 were not different between sex at 3 to 7 days post-CTX injury (McHale et al., 2012). This latter study also reported that the removal of necrotic myofiber was faster and the extent of fat deposition was greater in females than in males (McHale et al., 2012).

### 3.1.2 | Effect of ovarian-hormone deficiency and E2 replacement/ supplementation

The influence of OVX-induced ovarian-hormone deficiency on inflammatory response post muscle injury has been scarcely investigated. No difference was observed for leukocyte infiltration (i.e., assessed by 7/4⁺ cells) between OVX and intact females 1 day after electrically-evoked lengthening contractions (Schneider et al., 2012) while the proportion of pro-inflammatory macrophages (i.e., F4/80⁺Ly6C⁺ cells) and the gene expression of *Il1*, *Ccl2* and *SLPI* were higher in OVX than in intact females 3 days post-CTX injury (Liao et al., 2019).

E2 supplementation in intact males (Tiidus & Bombardier, 1999) and E2 replacement in OVX females showed reduced MPO activity in lower limb muscles during the first 24 h post downhill running. In addition, E2 replacement in females reduced both neutrophil (visually identified by hematoxylin and eosin staining) (Tiidus et al., 2001) and leukocyte infiltration (i.e., His48⁺ or ED1⁺ cells) at 1 h–24 h post downhill running (Enns et al., 2008; Iqbal et al., 2008). On the contrary, leukocyte infiltration (i.e., 7/4⁺ or Ly6C/G⁺ cells) was increased in both myofibers and connective tissue after E2 replacement in response to repeated electrically-evoked lengthening contractions (Fulkerson et al., 2015). In the same way, the expression of several genes encoding chemokines (*Ccl2*, *Ccl6*, *Ccl24*, *Ccr1*, *Cxcl1* and *Cxcl5*), known to modulate neutrophil and macrophage recruitment, was upregulated during the first day post freeze injury (Le et al., 2018). This was associated with a higher proportion of neutrophils (i.e., CD11b⁺LY6G⁺ cells) in injured muscles while the percentage of macrophages (i.e., CD11b⁺CD68⁺ cells) was not affected. Treated females also displayed a faster force recovery, even though the relation between the immunomodulatory effects of E2 and the functional improvement was not directly demonstrated (Le et al., 2018).

### 3.1.3 | Modulation of ER

A specific ER inhibition using ICI 182780 had no impact on the attenuation of leukocyte infiltration (i.e., His48⁺ or ED1⁺ cells) induced by E2 treatment in females 1 day after downhill running (Enns et al., 2008). Also, mean fluorescent intensity of CD11b⁺ and F4/80⁺ cells was higher in both OVX mice and females treated with a selective ER modulator (i.e., 4-Hydroxytamoxifen) as compared with untreated animals 3–7 days after CTX injury
<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>FIG URE 2 Effects of sex and estrogens on the inflammatory response during skeletal muscle regeneration. After injury, the production of cytokines/chemokines by the damaged myofibres induces the recruitment of immune cells such as neutrophils and macrophages in order to promote skeletal muscle regeneration. In both animal and human studies, the role of sex/ovarian hormones on the inflammatory response after injury is still controversial mainly due to the use of different markers to characterize immune cell infiltration and different models of muscle damage. MPO, myeloperoxidase activity.</figcaption></figure>

(Liao et al., 2019). However, the biological effect of this
increase on skeletal muscle regeneration remains unclear.
Treatment with ER-α and ER-β agonists suppressed the
elevation of both gene and protein expression of TNF-α
(i.e., a pro-inflammatory cytokine) induced by OVX 1 day
after NTX injury in females (Velders et al., 2012). In addi-
tion, mRNA expression of *Ccl2* and *Il10* was upregulated
by ER-β agonist 1-3 days post-NTX while these markers
were unaffected by ER-α agonist (Velders et al., 2012). The
same study also showed that mRNA expression of *Tnf* was
not different between injured muscles of WT, ER-α KO
and ER-β KO females at a later timepoint (i.e., 3 days post-
NTX) while *Ccl2* mRNA expression was lower in injured
muscle of ER-β KO mice as compared with ER-α KO and
WT animals.

To conclude, there is so far no consensus on the role
of ovarian hormone deficiency and E2 replacement/sup-
plementation/modulation on the inflammatory process
during skeletal muscle regeneration.

3.2 | Human studies

Only a few human studies investigated the influence of
sex or E2 on the regulation of the inflammatory process
(Figure 2).

3.2.1 | Effect of sex

The presence of radiolabeled Tc-99m neutrophils (quantified by invivo imaging) was greater in women as compared with men 2h after repeated lengthening contractions (MacIntyre et al., 2000). On the contrary, and despite the same extent of muscle damage (i.e., quantified by z-line streaming) and similar increase in CK activity, men showed a higher number of leucocytes in muscle tissue (i.e., cells positive for leucocyte common antigen) and circulating granulocytes as compared with women 2 days after lengthening contractions (Stupka
et al., 2000). Also, the mRNA expression of *CXCL8* and *CD11* was similarly upregulated in men and women while a trend toward a greater increase of *CCL2* gene expression was found in men as compared with women 2 days after repeated lengthening contractions (Fortino et al., 2022). The number of macrophages (i.e., CD68⁺ cells) increased similarly while the number of leukocytes (i.e., cells positive for MPO) remains unchanged in men and women 1 day after a single bout of lengthening contractions leading to the same extent of muscle damage (i.e., quantified by z-line streaming and torque recovery). However, when the same damaging exercise was repeated 5–6 weeks later, women displayed an increase of MPO⁺ cells while this parameter remains unchanged in men (Stupka et al., 2001), suggesting a faster inflammatory response in women as compared with men.

Overall, the influence of sex on the inflammatory process is still controversial in humans.

### 3.2.2 | Effect of E2 supplementation in men and postmenopausal women

E2 supplementation in men attenuated the infiltration of leukocytes (i.e., MPO⁺ cells) but did not protect against macrophage infiltration (i.e., CD68⁺ cells) 2 days after lengthening contractions (MacNeil et al., 2011). However, E2 supplementation also resulted in a reduction of circulating testosterone concentrations so that these two hormones could have contributed to the lower recruitment of leukocytes in the injured muscle (MacNeil et al., 2011). Interestingly, postmenopausal women using hormonal

<table><caption>TABLE 1 Limitations of the current experimental conditions and alternative approaches to most effectively evaluate the impact of sex/estrogen on skeletal muscle regeneration.</caption><thead><tr><td>Experimental design</td><td>Limitations</td><td>Alternative approaches</td></tr></thead><tbody><tr><td>Ovariectomy</td><td>Ovariectomy disturbs whole body homeostasis. Several hormones are altered following removal of the ovaries</td><td>Use of specific ER knockout-mice. This would also allow to keep intact the ovarian cycle of females</td></tr><tr><td>E2 replacement/ supplementation</td><td>Use of supraphysiological E2 doses (i.e., ranging from 100 to 200 pg/mL)</td><td>Use of physiological E2 doses (i.e., 10–60 pg/mL). Consider the natural cyclic variations of E2 during the ovarian cycle (Byers et al., 2012; McLean et al., 2012)</td></tr><tr><td>ER agonists treatment</td><td>ER-β agonist concentration was ten-fold higher than the concentration of ER-α agonist (Velders et al., 2012)</td><td>Use similar concentrations for treatment with ER-β agonist and ER-α agonist</td></tr><tr><td>Kinetics of skeletal muscle regeneration</td><td>Often limited to a single (and early) timepoint post-injury</td><td>Consider multiple timepoints that cover the different key steps of myogenesis and inflammation</td></tr><tr><td>Cell culture</td><td>Assessment of SC fate in culture media with fetal bovine serum or chick embryo extracts, which contain growth factors and steroids such as E2 (Kitajima & Ono, 2016; Seko et al., 2020). Use of phenol red, as an indicator of bacterial contaminations, which is also known as a selective modulator of ER-β due to its structure very close to that of non-steroidal E2 (Berthois et al., 1986; Welshons et al., 1988)</td><td>Use phenol-free medium and charcoal stripped serum to avoid interactions between medium components and estrogens (Larson et al., 2022)</td></tr><tr><td>Characterization of cell types/status</td><td>Lack of specificity of markers used to characterize leukocyte infiltration</td><td>Use of specific markers for immunostaining Omic approaches (single-cell/single-nucleus RNA seq, spatial transcriptomics) to identify a subpopulation of myogenic/inflammatory cells and/or specific transcriptomic signature</td></tr><tr><td>Model of muscle damage</td><td>Use of different models (i.e., downhill running, electrically-evoked or voluntary lengthening contractions, CTX, NTX) leading to different magnitude of muscle injury (from minor to severe) Muscle damage was usually quantified with indirect markers such as blood parameters (i.e., CK activity), which do not reflect the extent of the damage</td><td>Control the magnitude of muscle injury using force measurements considered as the best indirect marker of muscle damage (Clarkson & Hubal, 2002; Fouré & Gondin, 2021) Use of a standardized model of muscle injury allowing the modulation of muscle damage severity (Bernard et al., 2023)</td></tr></tbody></table>
therapy displayed a lower increase in *IL6*, *IL8*, *IL15*, and *TNFA* mRNA expression and in CK activity as compared with women not using hormonal therapy after repeated lengthening contraction (Dieli-Conwright et al., 2009). However, it is unclear how and to what extent hormonal therapy really improves muscle regeneration in humans through the modulation of the inflammatory response inasmuch as the investigations were limited to the first 4h postexercise. Further studies are therefore needed to define the role and mechanisms of action of sex and E2 on muscle inflammation.

## 4 | METHODOLOGICAL CONSIDERATIONS AND PERSPECTIVES

The substantial differences in the study design in both animal and human studies often make difficult the interpretation of the role of sexual dimorphism on skeletal muscle regeneration. The limitations of these different experimental conditions as well as the alternative approaches to most effectively evaluate the impact of sex/estrogen on skeletal muscle regeneration are summarized in Table 1.

## 5 | CONCLUSIONS

Growing evidence is emerging regarding a sexual dimorphism in SC regulation after muscle damage, illustrating a positive effect of E2 on muscle regeneration. Data are however still conflicting on the impact of sex and E2 on the inflammatory response. Additional studies are therefore warranted to improve our understanding of the influence of sex and E2 on the modulation of the inflammatory process. Animal investigations should allow to understand how E2 influence the interactions between SCs and immune cells in the regulation of muscle regeneration, a key process which has been so far mainly described in males (Arnold et al., 2007; Mounier et al., 2013). In addition, studies should carefully control the influence of ovarian cycle variation in female sex hormones, hormonal contraception as well as the natural drop of E2 in post-menopausal women on the regulation of both SC and immune cells after skeletal muscle injury. This would be of utmost interest for improving the management of muscle regeneration in healthy women and in pathological conditions associated with E2 deficiency/modulation (e.g., sarcopenia, hormone therapy for breast cancer...).

### AUTHOR CONTRIBUTIONS

CJ and JG contributed to the conception and design of this review. CJ and JG wrote the draft manuscript. All authors

contributed to and revised the final manuscript. All authors read and approved the final manuscript.

### ETHICS STATEMENT

None.

### FUNDING INFORMATION

This work was supported by a PhD fellowship from GDR CNRS Sport et Activité Physique to CJ.

### ORCID

Julien Gondin https://orcid.org/0000-0002-3108-605X

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Jomard, C., & Gondin, J. (2023). Influence of sexual dimorphism on satellite cell regulation and inflammatory response during skeletal muscle regeneration. *Physiological Reports*, **11**, e15798. https://doi.org/10.14814/phy2.15798