Received Date : 02-Oct-2013
Revised Date : 26-Aug-2014
Accepted Date : 29-Sep-2014
Article type : Regular Paper

The activity of satellite cells and myonuclei following 8 weeks of strength training in young men with suppressed testosterone levels
${ }^{1,2}$ Thue Kvorning, ${ }^{3}$ Fawzi Kadi, ${ }^{4}$ Peter Schjerling, ${ }^{5}$ Marianne Andersen, ${ }^{5}$ Kim Brixen, ${ }^{6}$ Charlotte Suetta and ${ }^{7}$ Klavs Madsen
${ }^{1}$ Team Danmark, Idraettens Hus, Broendby, Denmark.
${ }^{2}$ Institute of Sport Science and Clinical Biomechanics, University of Southern Denmark, Denmark.
${ }^{3}$ School of Health and Medical Sciences, Örebro University, Örebro, Sweden.
${ }^{4}$ Institute of Sports Medicine, Department of Orthopedic Surgery M, Bispebjerg Hospital and Center for Healthy Aging, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
${ }^{5}$ Department of Endocrinology, Odense University Hospital, Denmark.
${ }^{6}$ Department of Clinical Physiology and Nuclear Medicine, Bispebjerg Hospital.
${ }^{7}$ Department of Food and Nutrition, and Sport Exercise, University of Gothenburg.

Corresponding author: Thue Kvorning,
Telephone: +45 51922319
Email: thkv@teamdanmark.dk
Address: Team Danmark, Idraettens Hus, Broendby Stadion 20, DK-2605 Broendby, Denmark.

Running title: training, testosterone, satellite cells and myonuclei

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1111/apha. 12404
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Abstract

AIM: To investigate how suppression of endogenous testosterone during an 8 week strength training period influences the activity of satellite cells and myonuclei.

METHODS: Twenty-two moderately trained young men participated in this randomized, placebo-controlled, and double-blinded intervention study. The participants were randomized to treatment with a GnRH-analogue, goserelin ( $\mathrm{n}=12$ ), which suppresses testosterone or placebo $(\mathrm{n}=10)$ for 12 weeks. The strength training period of 8 weeks started after 4 weeks of treatment and included exercises for all major muscles. Biopsies were obtained from the midportion of the vastus lateralis muscle.

RESULTS: Testosterone resting level in goserelin was 10-20 times lower compared with placebo and the training-induced increase in the level of testosterone was abolished in goserelin. Training increased satellite cells number in type II fibers by $20 \%$ in placebo and by $52 \%$ in goserelin ( $\mathrm{p}<0.01$ ), whereas the myonuclear number significantly increased by 12 $\%$ in type II fibers in placebo and remained unchanged in goserelin ( $\mathrm{p}<0.05$ ). No changes in satellite cells and myonuclei were seen in type I fibers in either group. Data from the microarray analysis indicated that low testosterone affects the bone morphogenetic proteins signaling, which might regulate proliferation versus differentiation of satellite cells.

CONCLUSION: Eight weeks of strength training enhances the myonuclear number in type II fibers and this is largely blocked by suppression of testosterone. The data indicate that low testosterone levels could reduce the differentiation of satellite cells to myonuclei via the bone morphogenetic proteins signaling pathway, resulting in reduced increases in lean leg mass.

Key Words: training, testosterone, GnRH-analogue, microarray, satellite cells and myonuclei

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Introduction

Strength training-induced muscle hypertrophy is initiated by a number of complex molecular events. We know that activation of satellite cells (SCs) and an increase in myonuclei are two important mechanisms in muscle hypertrophy, where the myonuclear number is increased to satisfy the demand of increased protein synthesis (Kadi & Thornell 2000, Kadi et al. 2004). Mechanisms responsible for the molecular events inducing activation, proliferation and differentiation of SCs and enhanced number of myonuclei in relation to strength training are, however, still not clear.

One candidate for SC activation could be the anabolic hormone testosterone. Strength training is a robust physiological stimulator of testosterone and within a few minutes after the onset of training, plasma testosterone starts to increase (Kraemer et al. 1990, Kraemer et al. 1991, Hakkinen & Pakarinen 1993). We know that SCs can be targets for testosterone action through the androgen receptor (Doumit et al. 1996, Sinha-Hikim et al. 2004, Chen et al. 2005). Exogenous testosterone supplementation increases skeletal muscle SCs and positive associations between testosterone, muscle hypertrophy and the number of SCs have been reported. These findings indicate that testosterone could be an important stimulus for activation and proliferation of SCs (Sinha-Hikim et al. 2003, Sinha-Hikim et al. 2004).

Muscle fibers contain hundreds of myonuclei and each myonucleus sustains the protein synthesis in a limited cell volume. During the process of muscle fiber enlargement in adult muscles, an elevation of the myonuclear number occurs in order to satisfy the need for increased protein synthesis (Kadi & Thornell 2000, Kadi et al. 2004, Petrella et al. 2008). Long-term use of anabolic steroids (e.g. testosterone) has powerful effects on muscle fiber size and myonuclear number (Kadi 2008). However, it remains to be clarified whether SCs

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and myonuclei are regulated by endogenous testosterone during the process of traininginduced skeletal muscle hypertrophy.

To study endogenous testosterone and its role in stimulating and regulating muscle hypertrophy, we performed a strength training study in which testicular production of testosterone was blocked through inhibition of pituary-secreted lutenizing hormone (LH) by administration of a GnRH analogue (goserelin). This resulted in resting levels of serum endogenous testosterone, both total and free, remained constant in the placebo group throughout the intervention period, but decreased significantly in the goserelin treated group to a level 10-20 times lower than normal. Furthermore, the placebo group responded acutely to the strength training sessions with a significantly larger training induced increase in serum testosterone compared to the goserelin group, whereas the goserelin treated group responded with a decrease in total and free testosterone (Kvorning et al. 2006, 2007). We reported that suppression of testosterone production in combination with strength training attenuates the response to the training stimulus with a less pronounced increase in lean leg mass (i.e. muscle hypertrophy) compared with strength training combined with placebo (Kvorning et al. 2006). In spite of blocked acute testosterone responses as well as very low resting levels of endogenous testosterone in the goserelin treated group, strength training resulted in a similar mRNA expression of myoD, myogenin, IGF-IEa, IGF-IEb, IGF-IEc, myostatin, and androgen receptor as observed in the placebo group, suggesting that endogenous testosterone is not involved in the direct regulation of the expression of these particular signaling mRNAs in the course of strength training-induced muscle hypertrophy (Kvorning et al. 2007). The present study used pre-existing data from our previous work, Kvorning et al. (2006 and 2007), to investigate whether the training-induced SC and myonuclear responses are influenced by the GnRH-analogue treatment (i.e. suppression of endogenous testosterone).

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Since SC responses may vary between fiber types, we studied the fiber type-specific changes in SCs and myonuclei.

The aim of the present study was to investigate how suppression of endogenous testosterone during an 8 week strength training period influences the activity of SCs and myonuclei. We hypothesized that suppression of endogenous testosterone will lead to a less pronounced increase in the number of SCs and therefore less addition of myonuclei compared to a placebo group. In addition, a microarray analysis was performed to test for other potential SC regulating mechanisms.

Material and methods

Subjects and study design
Details of this study design have been reported elsewhere (Kvorning et al. 2006, Kvorning et al. 2007). The schedule of study procedures are shown in Figure 1. Briefly, 26 subjects volunteered to participate in this randomized, placebo-controlled, and double-blinded intervention study. The subjects participated in leisure sport once or twice a week. Some of the subjects had previously strength trained for shorter periods, but nothing structured, and strength training did not exceed 1 hour/week during the last year. The study conformed to the guidelines in the Declaration of Helsinki and was approved by the local Ethical Committee (VF 20040173). All subjects were informed of the risks and purposes of the study before their written consent was obtained. The subjects were carefully matched in pairs with regard to isometric knee extension strength, BMI, and age. Within each pair, the subjects were randomized to placebo (saline) or goserelin 3.6 mg (GnRH-analogue) injections once every fourth week, three times in total. Clinical examination of the subjects was performed before the experiment and two subjects were disqualified due to exclusion criteria (metabolic

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disorders, low testosterone levels, angina pectoris, lower back disorders, prescription medication for heart or lung diseases, or any recent physical trauma). Moreover, two subjects did not complete the study due to an injury unrelated to the study and due to side-effects of the goserelin treatment (hot flushes), respectively. Therefore, 22 young men completed the study (Table 1). The subjects and investigators involved in training and testing were blinded regarding the allocation of the subjects while two investigators (MA and KB) administering the study drugs and monitoring safety parameters were aware of the allocation.

Testing procedures

The subjects underwent 3 test procedures during the study. Test 1, 2, and 3 included measurements of hormonal resting levels, and measurements of acute hormonal responses to a strength training session. These measurements were completed in succession and on a separate day. Muscle biopsies were performed on separate days in relation to Test 2 and Test 3 (Figure 1). The subjects were familiarized with the study procedures approximately 2 weeks before entering Test 1. This included measuring of anthropometrics of the subjects and a careful introduction to the testing procedures and to the strength training exercises. Subsequently, a 10 repetition maximum (RM) load was measured for all exercises in the training program, to determine the initial training load.

Treatment with goserelin
Goserelin (Zoladex®, AstraZeneca) 3.6 mg depot was injected subcutaneously in the abdomen once every fourth week, in order to reduce and maintain endogenous testosterone concentrations within castrate range. Goserelin prevents the reappearance of LHRH receptors and consequently inhibits the secretion of LH from the pituitary gland and thus testicular

production of testosterone (Cockshott 2000). All subjects received 3 injections in total, either treatment or placebo, starting right after Test 1(Figure 1).

Training

A standardized warm-up was performed before training consisting of 4 sets of squats with 20 repetitions without load and with 1 min rest between sets. Subjects from both groups trained using the same progressive strength training program. The programs were performed 3 times a week for 8 weeks and consisted of leg press, knee extension, leg curl, bench press, lat pull down, biceps curl, and elbow extension. Subjects did 4 sets of each exercise for the lower body and 3 sets of each exercise for the upper body. The strength training period consisted of 24 training sessions periodized in 3 cycles of 8 training sessions with changing training loads (6 RM - 10 RM ). The goserelin group and the placebo group increased training loads to the same extent and underwent the same training volume. All training sessions were supervised and both groups carried out the same amount of training sessions (except for 1 training session), so subjects in the goserelin group completed 23.7 training sessions on average and the placebo group completed 23.6 training sessions on average. All subjects participated in a minimum of 22 training sessions (see Kvorning et al. 2006, for details).

Muscle biopsies

In a resting condition, muscle biopsies ( 100 mg ) from the middle portion of the vastus lateralis were obtained by using the Bergström needle technique (Bergström 1962). Incisions were made through the skin and muscle fascia following the administration of local anaesthesia (2-3 ml of $1 \%$ lidocaine). Pre- and post-training biopsies were taken from the same region and depth of the muscle. The tissue was immediately freed from blood and visible connective tissue, rapidly frozen in liquid $\mathrm{N}_{2}$, and stored at $-80^{\circ} \mathrm{C}$. Biopsies were

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obtained at four time points to measure SC and myonuclei response to strength training. The first biopsy was taken in the right leg (Test 2) and served as a pre-training period biopsy (Pre). In connection with the second last strength training session (Test 3), three biopsies were taken. One biopsy was taken in the right leg before the start of the training session and served both as a post-training period biopsy and a pre-training session biopsy (Post/Pre). This biopsy was taken 48 hours after the previous strength training session. The subjects then completed the exercises and another biopsy was taken in the left leg 4 hours after completion of the strength training session (Post4h). The final biopsy was taken in the right leg 24 hours after the pre-training biopsy (Post24h). Time points for all subjects were standardized and equal from day to day. The subjects had been fasting from 24:00 hours the day before and had refrained from strenuous physical activity for 48 hours. Two hours prior to all biopsies subjects were served a standardized meal. Subjects were divided in 3 groups (e.g. light, medium and heavy body mass group) receiving different sizes of meals, containing in total $8.01 \pm 0.14 \mathrm{kcal} / \mathrm{kg}, 0.45 \pm 0.01 \mathrm{~g}$ of protein $/ \mathrm{kg}, 1.31 \pm 0.03 \mathrm{~g}$ of carbohydrate $/ \mathrm{kg}$ and $0.10 \pm$ 0.00 g of fat $/ \mathrm{kg}$.

RNA purification

The RNA samples from Kvorning et al. (2007) were retrieved from the freezer and subjected to DNase treatment with the TURBO-DNA-free kit from Ambion (Life Technologies, Paisley, UK) according to manufactures protocol. After DNase treatment, the RNA samples were precipitated with $1 / 10$ vol. 3 M sodium acetate and 2.5 vol. $96 \%$ ethanol, washed with $70 \%$ ethanol and dissolved in RNase-free water. The RNA quality was reassessed by denaturing gel electrophoresis. Some samples were partially degraded and therefore excluded from further analyses. In consequence, the number of biopsies used were the following:

placebo group, Pre (n=7), Post/Pre (n=6) Post4h (n=7) and Post24h (n=6) and for the goserelin group, Pre (n=8), Post/Pre (n=8) Post4h (n=9) and Post24h (n=7).

Microarray

We theorized that suppressed testosterone levels could have several potential effects on mRNA levels in this study. The lack of testosterone may 1) constitutively increase/decrease the level of specific mRNA, 2) influence the increase/decrease of mRNA due to the training period or 3) influence the acute increase/decrease of mRNA after the training session. The 4 hour post samples (Post4h) will capture all three possibilities (though, the acute effect only 4 hours after training) and therefore these samples were screened for mRNA differences between the goserelin group and the placebo group by subjecting them to microarray analysis.

10 samples from the Post4h time point were analyzed on Affymetrix U133 Plus 2.0 microarrays by a microarray core facility, the RH Microarray Center (Rigshospital, Copenhagen). Briefly, total RNA was converted into double-stranded cDNA with the Superscript Choice System (Invitrogen, Carlsbad, CA, USA) using an oligo-dT primer containing a T7 RNA polymerase promoter (Genset, Evry, France). The cDNA was used as a template for an in vitro transcription reaction to synthesize biotin-labeled antisense cRNA (BioArray High Yield RNA Transcript Labeling Kit; Enzo Diagnostics, Farmingdale, NY, USA). After fragmentation at $94^{\circ} \mathrm{C}$ for 35 min in fragmentation buffer ( 40 mM Tris, 30 mM magnesium acetate, 10 mM potassium acetate), the labelled cRNA was hybridized for 16 h to Affymetrix HG-U133 Plus 2.0 (Affymetrix, Santa Clara, CA, USA). After hybridization the array was washed and stained with streptavidin phycoerythrin solution using the Fluidics

Station 450 (Affymetrix). Finally the arrays were scanned using the GeneChip Scanner 3000 (Affymetrix).

The probe data (CEL files) were analyzed with the Partek Genomic Suite v6.6 (Partek, MO, USA) using the RMA normalization method (Irizarry et al. 2003). One array (goserelin group) deviated highly from the other nine in Principal Component Analysis and was therefore excluded from the analysis. Statistical comparison of the two groups was performed and probe set with False Discovery Rate (FDR) of less than 5% selected. As this resulted in only a single probe set, a less stringent test was also performed with raw p-values less than $5 \%$ combined with a fold change (both ways) above 1.5 .

The data has been uploaded to the NCBI Gene Expression Omnibus and can be found at http://www.ncbi.nlm.nih.gov/geo/ with accession number GSE43437 (For Review (Remove in

Proof):

http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=lxehnmmmuecsyhg&acc=GSE43437)

Real-time RT-PCR

Real-time RT-PCR was performed as described in Kvorning et al. (2007), except new cDNA was synthesized based on the DNase treated RNA. Briefly, 500 ng DNase treated total RNA was converted to cDNA with OmniScript reverse transcriptase (Qiagen, CA, USA) and polydT. cDNA corresponding to 1.25 ng total RNA was used in each SYBR Green based reaction (Quantitect from Qiagen) with 100 nM of each primer (Table2) in an MX3005P real-time PCR machine (Stratagene, CA, USA). Ct values were related to a standard curve made with synthetic oligonucleotides (Ultramers, IDT, Leuven, Belgium) containing the PCR target sequence. Specificity was confirmed by melting curve analysis. GAPDH mRNA was used for

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normalization and RPLP0 mRNA for validating that GAPDH mRNA did not differ between the goserelin and placebo group (Figure 5).

Fiber type-specific CD56 ${ }^{+} S C$ content

Fiber type-specific $C D 56^{+} S C$ content was analyzed using immunohistochemistry as previously described (Mackey et al. 2009). SC were visualized on muscle cross-sections using a monoclonal mouse anti-CD56 antibody (1:100, cat. no. 347740; Becton Dickinson, San Jose, California, USA), using a protocol described in Mackey et al. (2009). Briefly, sections were incubated for 2 hours at $37^{\circ} \mathrm{C}$ with the primary antibody, followed by a biotinylated goat anti-mouse secondary antibody (1:200, cat. no. BA-9200; Vector Laboratories, Burlingame, California, USA) and then the Vectastain ABC reagent (cat. no. PK-6100, Vector Laboratories). For the visualization of primary antibody binding, the DAB substrate kit for peroxidase (cat. no. SK-4100, Vector Laboratories) were used. Sections were then rinsed, counterstained with Mayer’s hematoxylin, cleared and mounted using Mountex. Myonuclei were blue and satellite cells were stained brown (Figure 2). At least 150 muscle fibres per muscle biopsy were used in the analysis. Consecutive cross-sections were incubated with the monoclonal antibodies A4.951 and N2.261 (Developmental Studies Hybridoma Bank, University of Iowa) for determination of fiber type composition (Kadi et al. 1998). The number of CD56 ${ }^{+} \mathrm{SCs} /$ fiber in type I and type II fibers was evaluated on whole cross-sections, which corresponded to an average of 450 fibers. The number of myonuclei/fiber was measured on an average of 25 fibers of each fiber type as previously determined by Mackey et al. (2009).

Statistics

A two-way repeated measure ANOVA was used to test for statistical significance of differences in SC and myonuclei. The test included 1) two time points, before and after the intervention, and 2) intervention, goserelin and placebo.

All mRNA data from RT-qPCR were logtransformed before statistical analysis. Unparred ttest at Post4h was performed for comparison with array data (Table 3). Otherwise the RTqPCR data were analyzed with two-way repeated measures ANOVA (Time*Treatment) with Student-Newman-Keuls Post-hoc test and presented as Geometric Mean $\pm$ back-transformed SE (Figure 5).

All other data are presented as mean $\pm$ SE. A significance level of $\mathrm{p}<0.05$ was chosen, with trends ( $\mathrm{p}<0.1$ ) indicated in parenthesis.

Results

Satellite cells

The strength training period increased $C D 56^{+} S C$ content in type II fibers in the placebo group from 12.2 to $14.6 \%(\mathrm{p}<0.01)$ and in the goserelin group from 10.3 to $15.7 \%$ (p<0.01) (Figure 3). The number of $C D 56^{+} S C s / f i b e r$ in type I fibers was unaffected by the strength training period in both groups. Furthermore, no acute effect in satellite cell activity was seen, thus the number of satellite cells remained unchanged in muscle biopsies taken before, 4 and 24 hours following the second last strength training session.

Myonuclei

Myonuclear numbers in type I and type II fibers are shown in Figure 4. No change in the myonuclear number of type I fibers was seen in any of the groups. Strength training significantly increased the myonuclear number from $2.10 \pm 0.13 /$ fiber to $2.35 \pm 0.13 /$ fiber in

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type II fibers in the placebo group ( $\mathrm{p}<0.05$ ), whereas no changes were observed in the goserelin group.

Microarray

Using stringent criteria of FDR less than 5 percent, only a single mRNA, CHURC1, is shown to be differently expressed in the goserelin treated subjects (226736_at in Table 3). This difference was confirmed with qRT-PCR against the 3’UTR of the CHURC1 mRNA (Table 3).

However, due to the vast number of mRNA analyzed at the same time, smaller changes may well be lost as type II errors (false negatives). To reduce this problem the microarray data were reanalyzed with less stringent criteria often used in microarray analyses (Guo et al. 2006). Using unadjusted $\mathrm{p}<0.05$ and fold change (FC) $>1.5$ a total of 36 significant hits were found (Table 3). Of course a number of those findings may be false positive, but since only a subset of the subjects was applied on microarrays, qRT-PCR on all the subjects can be used for confirmation of the findings. Fifteen of the hits were selected for confirmation. Only four, CHURC1, Gremlin1, Myostatin and IRX3, turned out to be confirmed by the PCR at Post4h. However, when including all time points, it turns out that also FHL2 and ACBP/DBI were influenced in the goserelin group (Figure 5). Additionally, ARNTL did show a trend towards an effect of suppressed testosterone. That is, of the 15 selected hits, about half of them could be confirmed by PCR.

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Discussion

In this study we report that subjects, after 8 weeks of strength training with suppressed endogenous testosterone levels, increase the amount of SCs to a similar extent as subjects with normal testosterone levels, but without any effect on the number of myonuclei. Whereas, strength training with normal testosterone levels increases SC content and myonuclear number by $20 \%$ and $12 \%$, respectively. These results are in accordance with earlier observations (Roth et al. 2001, Kadi et al. 2004, Dreyer et al. 2006, Olsen et al. 2006, Petrella et al. 2008).

Previous studies indicate that testosterone regulates SC activation, proliferation and differentiation into myonuclei by increasing the number of PCNA+SCs (proliferating cell nuclear antigen positive-SCs) (Sinha-Hikim et al. 2002, Sinha-Hikim et al. 2006). These findings support a connection between testosterone and SCs and thus, the role of SCs as mediators of the myotrophic action of testosterone on human skeletal muscle (Kadi 2008). Since testosterone may be able to regulate SCs, we speculated that suppression of testosterone production, would lead to a smaller increase in the number of SCs following strength training, a phenomenon that could explain the reduced training-induced increase in lean leg mass in the goserelin group as reported earlier (Kvorning et al. 2006). However, the present study shows that, 8 weeks of strength training increase the amount of SCs independently of the testosterone levels. On the other hand, we demonstrate that the significant increase in SCs in the goserelin group did not lead to a significant addition of myonuclei, which is seen, as expected, in the placebo group, where a significant addition of myonuclei in type II fibers occurs in parallel with a significant increase in lean leg mass (Kvorning et al. 2006). These data support the importance of myonuclear addition in muscle hypertrophy (Allen et al. 1995, Kadi & Thornell 2000, Kadi et al. 2004). Interestingly,

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Petrella and co-workers argue that if the demand for myonuclear addition was the only driving force determining the expansion rate of the SCs, the doubling of SCs in subjects considered as extreme responders in terms of muscle mass gained after a strength training period, would not be expected. In other words, the degree of SC content expansion would be expected to keep pace with the level of myonuclear addition but not to exceed it (Petrella et al. 2008). Furthermore, Petrella et al. (2008) reported that the rate of myonuclear addition lagged behind the rate of muscle hypertrophy. If we relate this to our study, it can be postulated that SC response is exaggerated whereas the process of myonuclear addition is regulated by the hypertrophic need (i.e. muscle mass driven). Mackey and co-workers further supports this by showing that resistance training induces enhancement of the SC pool without muscle hypertrophy in young men (Mackey et al. 2011). Furthermore, it seems that the increases in SC is related to the intensity of the exercise stimuli and increases in SC does not automatically increase myonuclear addition (Smith & Merry 2012, Kurosaka et al. 2012).

We have earlier reported from the present study that, MyoD, myogenin, IGF-IEa, IGF-IEb, IGF-IEc and myostatin showed similar expression independently of the testosterone level (Kvorning et al. 2007). These signaling genes are known to be involved, or at least partly involved, in the regulation of SCs (Adams 1998, Chen et al. 2005), suggesting that SCs would be activated quite similar in both groups independently of the level of testosterone and because of a significant increase in lean leg mass in the placebo group and the goserelin group although significantly larger in the placebo group (Kvorning et al. 2006, 2007). On the other hand, when taking the relatively short training period ( 8 weeks) into consideration, it could be argued that any possible difference in the amount of SCs has not been able to express itself in such a short period. Thus, it could be speculated that if the strength training period was longer in duration, then the number of SCs measured may have turned out

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significantly different between the placebo- and goserelin group, because of a larger demand for myonuclear addition (i.e. to satisfy the need for increased protein synthesis) in placebo versus the goserelin group.

Only few earlier studies have investigated the acute effect of a single training bout on SC. Studies on eccentric exercise have showed an increase in the number of SCs in response to a single training session, when biopsies were taken $0,2,4,6$ and 8 days after training (Crameri et al. 2004), but also when taken 24 hours after training (Dreyer et al. 2006). These results are in contrast to our findings, but the fact that both studies used training protocols characterized by strenuous eccentric muscle actions may explain the difference in SC activity. Furthermore, our acute biopsies (Post4h and Post24h) were taken in relation to the second last strength training session in the strength training period and it could be speculated that a possible response in SC activity may be blunted. Thus, when interpreting data on gene expression from a single training session without a prior training period it is important to acknowledge that this response is likely to differ markedly from the gene expression measured on a single training session with a prior training period (Cameron-Smith 2002). Thus, the Post4h biopsies, taken at the end of the 8 -week training period, was deliberately chosen to get the “true training response” rather than measuring a response, which is related to the unaccustomed insult to the muscle (Mackey et al. 2009).

A microarray analysis was performed to screen for other potential SC regulating mechanisms and if other relevant genes are regulated by testosterone. As the number of arrays was a limiting factor, we chose to directly compare the placebo and goserelin group at the 4 h timepoint (Post4h). It should also be mentioned that we did not compare to Pre-values, hence exclusively differential effects between placebo and goserelin will be captured, not common

exercise effects. Since the 4 h time point is also 52 h after the previous training bout, we should be able to capture both acute changes as well as long-term accumulated changes from the strength training period with differential response in the goserelin group versus placebo (see methods). However, even then very few changes in mRNA were found, suggesting that the testosterone effect is rather specific. Still, it should be kept in mind that this is a screening and changes might be lost due to small changes ( $<1.5$ fold) which was not picked up by the analysis, acute changes by the training session occurring between the 4 hour and 52 hour time points or changes during the early weeks of training which have disappeared after the 8 weeks. Furthermore, targets not confirmed by qRT-PCR in Table 3 are likely due to false positive in array, but could also be due to differences in target sequences in array versus PCR due to unexpected alternative splicing. Attempts to isolate overrepresented pathways using the gene list in Table 3 and “Gene set analysis” in the Partek software did not provide any significant pathways (data not shown).

Of those changed by goserelin treatment, all except CHURC1, were reduced by suppression of testosterone. This appears to be the case already before training, and ARNTL, ACBP, Gremlin1 and IRX3 seem to keep this lower level throughout the training period. In the case of FHL2, the down regulation of FHL2 induced by strength training is blunted by the lack of testosterone. However, as goserelin also reduces the FHL2 level, the end-result is that even though suppression of testosterone blunted the training session response, the level of FHL2 mRNA mimics the training session response anyway. CHURC1 is the only mRNA that is induced by the lack of testosterone.

Unfortunately, the few goserelin dependent mRNA does not provide any clear indication of how suppression of testosterone influence muscle hypertrophy. However, it is worth noting

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that CHURC1 has been shown to interfere with bone morphogenetic proteins (BMP) signaling by inducing a SMAD interaction protein, ZEB2 (Sheng et al. 2003). Furthermore, Gremlin1 is a BMP antagonist (Glister et al. 2005) and Myostatin uses the same signaling pathway as the BMP’s (Rebbapragada et al. 2003). Also, both FHL2 and IRX3 have recently been shown to interfere with the BMP signaling (Neuman et al. 2009, Gaborit et al. 2012). These findings suggest that BMP signaling is somehow involved in testosterone dependent muscle hypertrophy, although further experiments are warranted. Since BMP signaling has recently been shown to regulate proliferation versus differentiation of SCs (Friedrichs et al. 2011), this could offer an explanation for the goserelin effect, that is, blockage of the SC fusion to myotubes via the BMP pathway.

In conclusion, an 8 week strength training period significantly increased the number of SCs in type II fibers in the placebo and in the goserelin group independently of endogenous testosterone levels. But we demonstrate a significant addition of myonuclei in type II fibers in parallel with significant larger gains in lean leg mass in the placebo group compared to the goserelin group. Testosterone does not seem to be a main regulator of SCs and myonuclei. On the other hand, testosterone may influence the accretion of myonuclei to muscle fibers tentatively via the BMP signaling pathway.

Acknowledgements

First of all we would like to thank the subjects who participated in the study. Secondly, we would like to thank laboratory the technician Gitte Scheel Klemmensen, Bente Tøt, Donna Arbuckle-Lund, Kirsten Westermann, Anja Jokipii-Nielsen and Anette Riis Madsen. Furthermore, Rehannah Borup and the RH Microarray Center at Rigshospitalet for running the microarrays.

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Conflict of interest

This investigation does not involve any financial or personal relationships or other conditions that could be viewed as presenting a potential conflict of interest.

Grants

We would like to thank Anti-Doping Denmark, the Team Denmark Foundation, the Danish Medical Research Council and the Nordea Foundation for their financial support and Arla Foods for sponsoring the drinking chocolate.

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Figure 1. Overview of the study design. After completion of Test 1 the subjects were randomized in to a goserelin group ( $\mathrm{n}=12$ ) and a placebo group ( $\mathrm{n}=10$ ). Test 1, 2, and 3 included blood sampling (resting levels and acute hormonal response to a strength training session), muscle biopsies were performed at Test 2 and 3.

Figure 2. Fiber type-specific identification of satellite cells using the antibodies against CD56 (A) and A4.951 (B). Satellite cells are blue nuclei surrounded by a brown rim (A). In (B) type I fibres are stained and type II fibres are unstained.

Figure 3. Satellite cells per muscle fiber pre and post the 8 week strength training period. Goserelin group, $\mathrm{n}=11-12$; placebo group, $\mathrm{n}=9$. Values are mean $\pm \mathrm{SE}$.
** Significantly different from pre 8 weeks of strength training ( $\mathrm{p}<0.01$ ).

Figure 4. Myonuclear number per fiber type II, pre and post the 8 week strength training period. Goserelin group, $\mathrm{n}=11-12$; placebo group, $\mathrm{n}=9$. Values are mean $\pm \mathrm{SE}$.

• Significantly different from pre 8 weeks of strength training ( $\mathrm{p}<0.05$ ).

# Significant increase compared to goserelin ( $\mathrm{p}<0.05$ ).

Figure 5. RT-qPCR mRNA data. mRNA levels shown relative to mean of Pre samples for the placebo group. Placebo ( $\mathrm{n}=7,6,7,6$ ) and Goserelin ( $\mathrm{n}=8,8,9,7$ ). Values are presented as Geometric Mean $\pm$ back-transformed SE. Significant ANOVA results are shown in text (trends in parentheses). Post hoc comparisons ( $\mathrm{p}<0.05$ ) versus Pre, Pre/Post and Post4h are indicated with the letters $\mathrm{a}, \mathrm{b}$ and c , respectively, and goserelin versus placebo at specific timepoint with the letter d.

Table 1. Anthropometrics of the subjects measured before the strength training period. Values are mean $\pm$ SE. None of the parameters differed significantly between the groups.

Table 2. Primers for RT-PCR.

Table 3. Microarray and RT-qPCR comparison of the goserelin and placebo groups at Post4h. Goserelin group, $\mathrm{n}=9$; placebo group, $\mathrm{n}=7$.

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Table 1

Table 2.

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Table 3.

Figure 1

Figure 2

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Figure 3

Figure 4

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