Journal Pre-proofs

Research paper

The Effect of different types of exercise training on diet-induced obesity in

rats, Cross-talk between cell cycle proteins and apoptosis

Javad Tolouei Azar, Aref Habibi Maleki, Sana Moshari, Mazdak Razi

PII: S0378-1119(20)30519-9
DOI: https://doi.org/10.1016/j.gene.2020.144850
Reference: GENE 144850

To appear in: Gene Gene

Received Date: 2 April 2020

Revised Date: 11 May 2020
Accepted Date: 3 June 2020

Please cite this article as: J. Tolouei Azar, A. Habibi Maleki, S. Moshari, M. Razi, The Effect of different types of
exercise training on diet-induced obesity in rats, Cross-talk between cell cycle proteins and apoptosis, Gene Gene
(2020), doi: https://doi.org/10.1016/j.gene.2020.144850

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 The Effect of different types of exercise training on diet-induced obesity in
rats, Cross-talk between cell cycle proteins and apoptosis

Javad Tolouei Azar 1;

Aref Habibi Maleki1;
Sana Moshari 2, 3;
Mazdak Razi 2;

1 Department of Exercise Physiology and Corrective Exercises, Faculty of Sport Sciences, Urmia
University, Urmia, Iran.
2 Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
3Andrology and in-vitro Fertilization Division, RASTA Research Center, West Azerbayjan Science and
Technology Park (WASTP), Urmia, Iran.

Correspondence
Javad Tolouei Azar, Department of Exercise Physiology and Corrective Exercises, Faculty of Sport
Sciences, Urmia University, Urmia, Iran.
Email: j.toloueiazar@urmia.ac.ir
TEL:+989143410949

Javad, Tolouei Azar: https://orcid.org/0000-0002-2724-8474

Aref Habibi Maleki: https://orcid.org/0000-0001-7715-0734
Sana Moshari: https://orcid.org/0000-0002-1253-0450
Mazdak Razi: https://orcid.org/0000-0002-4309-4831
Abstract:

Obesity is associated with germ cell apoptosis, spermatogenesis arrest, and testicular endocrine
suppression. The aim of the present study was to investigate the crosstalk between germ cell
apoptosis and cell cycle machinery in sedentary and obese rats after moderate-intensity
continuous (MICT), high-intensity continuous (HICT) and High-intensity interval (HIIT)
exercise trainings. Male Wistar rats (n= 25) were randomly divided into 5 groups; the control,
sedentary high-fat diet (HFD)-received (HFD-sole), MICT, HICT and HIIT-induced HFD-
received groups. The serum levels of LDL-C, HDL-C, triglyceride, and testosterone, mRNA
and protein levels of Cyclin D1, Cdk4, p21, apoptotic cell number/mm2 of testicular tissue and
testicular DNA fragmentation ratio were investigated. The obese animals in HFD-sole group
represented a significant (p<0.05) reduction in serum HDL-C and testosterone levels, Cyclin
D1, Cdk4 expressions, and exhibited a remarkable (p<0.05) increment in LDL-C, triglyceride,
p21 expression, apoptotic cell number and DNA fragmentation ratio versus control animals.
However, the animals in MICT, HICT, HIIT groups exhibited a significant (p<0.05) increment
in serum HDL-C and testosterone, Cyclin D1 and Cdk4 expressions and showed a significant
(p<0.05) reduction in serum LDL-C and triglyceride, p21 expression, apoptotic cell number
and DNA fragmentation versus the HFD-sole group. In conclusion, a crosslink between cell
cycle machinery and apoptosis of germ cells was revealed in the testicles of HFD-sole animals,
and MICT, HICT and HIIT could ameliorate the obesity-induced impairments, respectively.
This effect may be attributed to the effect of exercise training protocols on maintaining Cyclin
D1 and Cdk4 and suppressing p21 expression levels in the testicles.

Key Words: Obesity, Exercise training, Spermatogenesis, Cell cycle machinery, Apoptosis
 1. Introduction:

Today, obesity as a consequence of a high-fat diet (HFD) has become a serious public health
concern worldwide. Generally, individuals with Body Mass Index (BMI) above 30 kg m-2are
considered obese (Franceschi et al., 2014; Wang et al., 2019). Decreased physical activity and
sedentary life style have increased the prevalence of obesity in male and female individuals
over 40 years (Di Cesare et al., 2016). Obesity is known to increase the risks of several
disorders including; hypertension, diabetes, cardiovascular disease, cancer in males and
females (Liu and Ding, 2017; Wang et al., 2019). Among the obesity-induced risks, the cardio-
metabolic disorders, increasing plasma low-density lipoprotein (LDL)-cholesterol (LDL-C) and
triglyceride (TG) concentrations have been reported as the main risk-factors of mortality (Jia et
al., 2013). In addition to these reports, the adverse effect of obesity on spermatogenesis has
been demonstrated in the human and animals (Jia et al., 2018; Miao et al., 2018; Wang et al.,
2019). Inline, an excessive germ cell apoptosis through suppressing anti-apoptotic Bcl-2
expression and up-regulating pro-apoptotic proteins Bax and Fas expressions (Yan et al., 2015;
Wang et al., 2018), a significant reduction in the testicular somatic cell population (Miao et al.,
2018), consequent suppression of testosterone secretion (Katib, 2015), and progressive
oxidative stress induced by a remarkable reduction in superoxide dismutase, glutathione
peroxidase and glutathione reductase activities (Wang et al., 2019) have been revealed as main
reasons for obesity-induced infertility.

The crosstalk between pro-apoptotic genes and the proteins involving in germ cells cycle
machinery is demonstrated previously (Zamir-Nasta et al., 2017). In line, different proteins are
involved in regulating the cell cycle process during germ cell division. Among these proteins,
the cyclins and their special kinases are known as the key regulators of germ cell development.
Indeed, three different Cyclins, named as Cyclin D1, D2, D3, are identified in mammalian
cells. Among the various members of the Cyclin family, the Cyclin D1 and Cdk4/6 are reported
to actively control the cell cycle process through spermatogenesis (Satyanarayana and Kaldis,
2009; Cheraghi et al., 2015). Thus, any disruption in the expressions of Cyclin D1 and its
kinase Cdk4 results in cell cycle arrest in different germ cell and ends with spermatogenesis
arrest (Adibnia et al., 2016). Moreover, the p21 (WAF1, CAP20, Cip1, and Sdi1), another
important protein in the cell cycle process, participates in Cyclins-dependent pathways. The
p21 is a family member of cyclin kinase inhibitors and inhibits Cyclin D1/Cdk4/6 complex
formation and through this mechanism, it stops the cycling process after severe DNA damage
(Amin et al., 2018). However, the crosslink between the proteins involving in germ cells cycle
machinery and apoptosis, especially in the obesity condition, remained unclear. Therefore, as a
preliminary aim of the current study, investigating these molecular interactions might be of
therapeutic interest for spermatogenesis impairment.

The benefits of physical activities/exercise in controlling the body metabolism and obesity
have been reported several times (Henriksen, 2002; Gaesser et al., 2011; Zouhal et al., 2013;
Ibáñez et al., 2017). Inline, exercise training is known as an efficient stimulus in controlling the
body weight gain, metabolic disorders and obesity (Andreazzi et al., 2009; Gomes et al., 2013).
Using animal models it came clear that performing the exercise training in mice (during
adulthood), remarkably ameliorated the obesity-induced damages at sperm level and
significantly improved the fertility potential (Santos et al., 2015). In addition to these findings,
the effects of moderate-intensity continuous (MICT), high-intensity continuous (HICT) and
High-intensity interval (HIIT) training exercises against obesity-induced infertility are not
completely revealed. Moreover, the effect of these exercise training protocols on cell cycle
machinery during spermatogenesis is not fully investigated in obesity condition. In this context,
the current study was performed to investigate the effect of MICT, HICT and HIIT exercise
training protocols on spermatogenesis, the expression levels of cell cycle-related proteins
(Cyclin D1, Cdk4 and p21) and apoptosis of the germ cell in the obese rats, programmed by
high fat-diet (HFD).

2. Materials and Methods:

2.1. Animals and grouping:

To perform the current study, 25 male Wistar rats weighting 180–220 g, were purchased from
the Animal Resource Center of Urmia University (ARCUU). After one-week adaptation, the
animals were divided into 5 groups (n=5/group), including control (Con), which received basic
diet, and the HFD-received groups. Next, the HFD group was subdivided into sedentary HFD-
received (HFD-sole), moderate-intensity continuous (MICT), high-intensity continuous (HICT)
and High-intensity interval (HIIT) training exercise protocols applied groups. During the
experiment, body weight was recorded twice a week and the obesity model was considered
successful when the average body weight of the HFD group was 1.4 times more than of the
Con group. The basic diet contained: 54% corn, 14% wheat bran, 13% alfalfa meal, 10% cotton
meal, 6% fish meal, 1.5% vitamin and mineral, 1% limestone, 0.3% sodium chloride and 0.2%
dicalcium phosphate (Rodent diet datasheet, 2016). The High-fat diet contained: 69.5% basic
diet, 15% pork fat, 15% sucrose and 0.5% pig bile. The standard environment conditions (12 hr
light/dark cycle) were provided to the animals. The experimental protocols were performed in
compliance with the guidelines of Urmia University Animal Care and Use Committee, and the
study was approved by the Ethics Committee for Animal Experiments
(IR.UMSU.REC.1397.485).

2.2. Exercise performance test [maximum speed test (Smax)]

The exercise training protocols were conducted based on previous published researches
(Burniston, 2009; Chavanelle et al., 2017), with the following adaptations:
The animals in the training groups participated in a special exercise program for each type, for
two weeks, 3 sessions per week, each session 10 to 15 min at a speed of 7 to 15 m/min. At the
end of the two weeks of adaptation, the rats maximal speed (Smax) was evaluated.
The rats in MICT and HICT groups were placed on the treadmill (5- channel laboratory
treadmill special for the rat; Danesh Yakhte, Iran), and warmed up during 5 min at a speed of
10 to 15 m/min. Next, the speed was increased by steps of 2 m/min, every 2 min, until the rats
were unable or unwilling to continue despite mild stimulation with a wooden cane.
The rats in the HIIT group performed an uphill performance test based on a similar protocol on
a treadmill set on a 20° incline. After maximum speed test the intensity for each exercise
modality was determined based on maximum Smax. According to previous study, 6 weeks
after exercise training, the Smax test was again performed and a new training speed was
applied in order to re-adjust training intensity (Burniston, 2009; Chavanelle et al., 2017).
 2.3. MICT and HICT protocol
The MICT and HICT exercises were considered after the S max test. Accordingly, the 5 min of
warming up to 7 to 10 m/min was considered. The MICT and HICT groups exercised at 50-
60% Smax (table 1), and at 70-75% Smax for 80 min (table 2), respectively with a zero-degree
slope. The animal in these two groups (MICT and HICT) were participated in 5 sessions of
exercise per week, for 12 weeks. At the end of each session, cooling was performed at 6 m/min
for 3 min (Burniston, 2009; Chavanelle et al., 2017).

2.4. High intensity interval training (HIIT) protocol

The Rats in the HIIT group performed a HIIT uphill training which consisted of 13 series;
alternating between 4 min at an intensity corresponding to 85–90% (20° slope) of the Smax,
which was revealed during the last uphill performance test and 2 min of active rest (20-25%
Smax). To let the animals adapt with condition and/or exercise and to reach the objectives
described above, the exercise intensity was gradually increased over the first 3 weeks at level
(0°) or 20° incline. Next, the animals exercised 12 weeks consisting of treadmill running 5 days
per week (Table 3) (Burniston, 2009; Chavanelle et al., 2017). Note: The exercise training
protocols were considered after obesity approval and the animal in all experimental groups
continued to receive HFD until the end of the experiment.

2.5. Sample collection and histological analyses:

Following 12 weeks, the testicular samples were dissected. The total body weight, testicular
weight and relative testicular/body weight of animals were evaluated. Then, the testicles were
fixed in Bouin's solution, undergone a routine tissue passage, embedded in paraffin and
sectioned (5 µm) by rotary microtome (LKB, UK). Next, the slides were stained by
Hematoxylin-Eosin staining dies. The stained histological slides were analyzed using a Leica
light microscope (under 200x, 400x or 1000x magnification). Accordingly, 100 seminiferous
tubules from each cross-section (n=5 section/each animal) were analyzed. The tubular
repopulation (RI, the relative ratio of spermatogonia A/B), tubular differentiation index (TDI,
the seminiferous tubules with more than 3 layers of germ cells were considered as a tubule with
positive TDI) and spermiogenesis index (SPI, the tubules with active spermiogenesis) were
analyzed and compared between groups. Seminiferous tubules were chosen according to the
same criteria of spermatogenesis stage. Moreover, the Leydig and Sertoli cells distributions per
mm2 of tissue (n= 5 cross-section/animal) were counted and compared between groups.

2.6. Serum lipid profile and testosterone level:

Following training termination, the animals (n= 5 for each group) have fasted 12–15 h prior
sample collection, and the blood sample was collected directly from the heart. After
centrifugation for 15 min at 1000 r/min, the serum samples were collected and stored at –70°C
until analysis. Total cholesterol (TC), TG, high-density lipoprotein cholesterol (HDL-C) levels
were evaluated using a UV-Vis spectrophotometer (DREL 3000 HACH). Laboratory kit
reagents (Randox Laboratory Ltd, UK) were used for all biochemical analyses. The LDL-C
was estimated according to the following formula. LDL-C = TC – HDL-C – TG/5 (Jia et al.,
2013). The serum level of testosterone was evaluated using ELISA kit (Mouse/Rat
Testosterone, Cat N: RTC001R, BioVender, Czech Republic). Finally, all biochemical data
were compared between groups.

2.7. RNA isolation and cDNA synthesis:

The testicles were stored at -70°C and used to extract total RNA (tRNA). The tRNA was
extracted based using TRIZOL reagent (Sina-Gen, Tehran, Iran). For this purpose, 0.1 gr of
frozen tissue was homogenized with TRIZOL reagent and chloroform and then, incubated on
ice for 15 min. Thereafter, the solution was centrifuged in 12000g for 5 minutes and the
aqueous part was transferred into a new microtube. Following the addition of isopropanol, the
tubes were incubated on ice for 5-10 min and then, centrifuged in 12000g for 5 min. Finally,
the supernatant was removed and washed with ethanol 70% and 50 µl DEPC-treated water was
added. By using a nanodrop spectrophotometer (260 nm and A260/280=1.8-2.0), the amount of
RNA was determined. For the cDNA synthesis, we used 1µg RNA, 1µl oligo (dT) primer, 4µl
5×reaction buffer, 1µl RNAse inhibitor, 2µl 10 mM dNTP mix, 1µl M-MuLV Reverse,
according to the manufacturer’s protocol (Fermentas, GmbH, Germany). Finally, the 25 µl
cDNA reaction mixture was used and incubated at 65°C, 5 min, followed by 60 min at 42°C,
and 5 min at 70°C to terminate the reaction.

2.8. qRT-PCR:

The PCR reaction mixtures containing: 0.5 µL (about 5–10 ng) of cDNA template, 10 µL 1X
SYBR GREEN master mix (High ROX, Noavaran Teb-Beinolmelal, Iran), and 0.5 µL (600
nM) from each reverse and forward primers of the target genes were prepared. The PCR
conditions were run as follows: general denaturation at 95 °C for 5 min, 1 cycle, followed by
45 cycles of 95 °C for 20 s; annealing temperature (60°C for cyclin D1 and cdk4, and 58°C for
p21) for 10 s; elongation: 72 °C for 1 min and 72 °C for 5 min (Cong et al., 2017; Guo et al.,
2016). The mean of triplicate target PCR threshold cycle (CT) values, was normalized by
subtracting the mean CT value of GAPDH. The relative expression level of interested mRNA

was calculated by the equation: 2−(∆∆Ct). The primers pair’s sequences for individual genes are
presented in table 4.

2.9. Immunohistochemical staining (IHC)

The histological slides were preheated by hot air oven at 60°C (25 min) and rehydrated in the
gradient series of ethanol (100%, 90%, 80% and 70%). In order to induce the antigen retrieval
process, the sodium citrate buffer (10 mM, PH=7.2) was used. The hydrogen peroxide solution
0.03% was used to block endogenous peroxidase. Next, the sections were washed with
Phosphate-buffered saline (PBS, PH=7.2), and subsequently incubated with primary antibodies
[Anti-Cyclin D1 antibody, Cat No: sc-20044, 1:300, Anti-Cdk4 antibody, Cat No: sc-23896,
1:300 dilution and Anti-p21 antibody, Cat No: sc-6246, 1:300 dilution]. The antibodies were
from Santa Cruz (USA). After 18 hr incubation at 4°C, the slides were washed and incubated
with Anti-polyvalent antibody conjugated with HRP (Horseradishperoxidase, 10 min).
Diaminobenzidine-substrate (DAB) chromogen was used to mark the positive reaction for
target proteins. Mayer’s hematoxylin was used for counterstaining the slides. To check the
staining procedure, the primary antibody was eliminated from the staining protocol and the
slides were considered as negative controls. The positive reaction for target proteins was
revealed as brown stains. The Cyclin D1+, Cdk4+ and p21+ cells (under 200x magnification,
provided by Leica light microscope) were counted per mm2 tissue and then, compared between
groups. Moreover, the mean intensity of brown reaction (representing the positive reaction for
target proteins) per mm2 of tissue was analyzed by software (Image pro-insight version 9:00,
Media Cybernetic, USA) (Moshari et al., 2017).

2.10.Gel electrophoresis and Western blotting:

The testicles were lysed with the ice-cold RIPA buffer (Santa cruz, sc-24948, USA). To extract
the protein contents, the lysed samples were centrifuged at 12,000 rpm, at 4°C for 15 min and
then the total protein concentrations were evaluated by Bradford assay. Next, a 25 µg of
protein content was diluted in Laemmli buffer, heated for 10 min at 95°C and thereafter, loaded
into 8% SDS-PAGE gel for electrophoresis. The separated proteins were transferred to
polyvinylidene difluoride (PVDF) membrane by electroblotting. Following incubation in
blocker, membranes were incubated in primary antibody [Anti-Cyclin D1 antibody, Cat No: sc-
20044, 1:1000, Anti-Cdk4 antibody, Cat No: sc-23896, 1:1000 dilution and Anti-p21 antibody,
Cat No: sc-6246, 1:100 dilutions] for 18 hr at 4°C. Next, the membrane was washed with
TBST and incubated in appropriate secondary antibody conjugated with HRP (horse-radish
peroxidase) for 1 hr at ambient temperature. The immunoblots were visualized by using an
enhanced chemiluminescence (ECL) and analyzed by ChemDoc system (1708265, Bio-Rad,
USA).

2.11.TUNEL staining:

In order to analyze the cell programmed death (apoptosis), the Terminal Transferase and
Biotin-16-dUTP staining was conducted. Briefly; the 6 µm histological slides were minced in
xylene (2X, for 5 min), hydrated in 100% ethanol (2X, for 3 min) and thereafter, rinsed in
distilled water. Next, the slides were digested in protein kinase K [10 mg/ml stock in 100 mM
Tris-HCl (pH 7.5), 10 mM EDTA] and finally rinsed in phosphate buffer (PBS, pH 7, 2X, 3
min). The slides were pre-incubated in TdT reaction buffer (Enzyme reagent 100 ml, label
reagent 900 ml) for 10 min. Following pre-incubation, the slides were incubated in TdT
reaction mixture (1-2 h, at37°C in humidified chamber). Next, the slides were rinsed in stop
reaction buffer (NaCl 1.75 g, Sodium citrate, Trihydrate 0.88 g, distilled water 100 ml) for 10
min, incubated in FITC-Avidin D in PBS (30 min, at room temperature), washed with PBS
(3X) and finally counterstained in DAPI. Then, the slides were rinsed in PBS and mounted
with anti-fad mounting medium (Zhang et al., 2015). The pixel-based intensity of green
reactions (representing apoptotic cells) was analyzed by software (Image pro-insight, version
9:00, Media Cybernetic, USA) and then, the mean intensities were compared between groups.
Moreover, the apoptotic cell number per mm2 of tissue was evaluated and compared.

2.12.DNA ladder test for DNA fragmentation

In addition to TUNEL staining, the DNA laddering test was conducted. The total DNA content
was extracted using a commercial kit (Sinaclon, Cat N: EX6071, Iran). For this purpose, 0.3 gr
from testicular tissue was homogenized with lysis buffer. The quality and purity of the
extracted DNA were measured with a NanoDrop-1000 spectrophotometer (Thermo Scientific,
Washington, USA). Next, a volume of 2 μg eluted DNA (15–17 μl of eluted DNA) was added
to the loading buffer (50% glycerol, 2 mm ethylenediaminetetraacetic acid and 0.40%
bromophenol blue). The DNA solution was run on a 1% agarose gel for 70 min at 70- V
constant voltage. The PST1 was also loaded as a marker for the identification of DNA amounts
(Amin et al., 2018). Gels were visualized by the Gel Doc 2000 system (ATP, Iran).

2.13.Statistical and image analyses:

All data are presented in Mean±SD and A p<0.05 was considered as statistically significant.
Differences between quantitative histological and molecular data were analyzed with One-way
ANOVA using Statistical Package for Graphpad version 7 (GraphPad Software, San Diego,
CA, USA). The photomicrographs were taken and unified by using Canon onboard camera
(Japan), and Adobe Photoshop CC 2018 (version: 19), respectively.
 3. Results:
3.1. General findings:

The obese animals in the HFD-sole group represented a remarkable (p<0.05) increment in total
body weight (marking them as obese animals), a significant reduction in testicular weight, as
well as testicular/total body weight in comparison to those in the control group. In contrast, the
MICT, HICT and HIIT groups represented no statistically significant changes in the total
testicular and testicular/total body weight ratios versus the control group (Fig. 1A). Moreover,
the total body weight of these animals was lower than that of the control animals representing
the ameliorative effect of exercise training on HFD-induced obesity.

3.2. Histological alterations:

To assess the effects of HFD-received obesity and exercise training protocols on the testicular
somatic cells, the Sertoli cell and Leydig cell distribution per mm2 of tissue was examined. The
mean distribution of Sertoli and Leydig cells per mm2 of tissue was decreased in HFD-sole
group (versus control animals, p<0.05). Meanwhile, the HFD-received animals in MICT, HICT
and HIIT groups represented significantly (p<0.05) higher Sertoli cells and Leydig cell
compared to those in the HDF-sole group (Fig. 1B). The cross-sections of obese animals in the
HFD-sole group represented spermatogenesis and spermiogenesis failure and the germ cell
dissociation, which were not revealed in the control group (Fig. 1C). Moreover, the percentages
of seminiferous tubules with the negative repopulation, tubular differentiation and
spermiogenesis indices were increased in the HFD-sole group when compared to those in the
control group (Fig. 1D). In contrast, the animals in the MICT, HICT and HIIT groups showed
an ameliorated repopulation, tubular differentiation and spermiogenesis indices compared to
obese animals in the HDF-sole group.
 3.3. Exercise training ameliorated the serum Lipid profile and testosterone
concentration:

The obese animals in the HFD-sole group represented a remarkable (p<0.05) increment in the
serum LDL-C, reduction in HDL-C and enhancement in TG versus the control group.
Meanwhile, the situation was reversed in MICT, HICT and HIIT groups (Fig. 2A, 2B, 2C).
Accordingly, the animals in MICT, HICT and HIIT groups represented a remarkable (p<0.05)
decrement in serum LDL-C and TG, and showed a significant (p<0.05) increment in HDL-C
versus the obese animals in the HFD-sole group. To assess the effect of HFD and different
exercise training protocols on the testicular endocrine potential, the serum level of testosterone
was examined and then, compared between all groups. Observations revealed a significant
(p<0.05) reduction in the serum testosterone concentration in the HFD-sole group, which was
significantly (p<0.05) up-regulated in the MICT, HICT and HIIT groups. No significant
difference was revealed between the different exercise training groups with each other (Fig.
2D).

3.4. Exercise training significantly up-regulated the Cyclin D1 and Cdk4

expressions:

The cyclin D1 and cdk4 expression levels were investigated by qRT-PCR, IHC and western
blotting techniques. The obese animals in the HFD-sole group represented a significant
(p<0.05) reduction in the cyclin D1 mRNA (Fig. 3A), Cyclin D1+ cells/mm2 of tissue (Fig. 4A,
4B), pixel-based intensity (Fig. 4C) and protein content (Fig. 4D) compared to the control
group. Meanwhile, the HDF-induced animals in MICT, HICT and HIIT groups represented an
up-regulated expression of Cyclin D1 protein and cyclin D1 mRNA levels versus the obese
animals in the HFD-sole group. Moreover, the mRNA of cdk4 (Fig. 3B), Cdk4+ cells/mm2 of
tissue (Fig. 5A, 5B), pixel-based intensity of Cdk4 (Fig. 5C) and protein content (Fig. 5D) were
decreased in the obese animals (HFD-sole group) versus the control group (p<0.05). However,
the HDF-induced animals in MICT, HICT and HIIT groups represented a remarkable (p<0.05)
enhancement in cdk4 expression.
 3.5. Exercise training diminished the P21 expression

The obese animals in the HDF-sole group represented a significant (p<0.05) increment in the
mRNA level of p21 (Fig. 3C), p21+ cells/mm2 of tissue (Fig. 6A, 6B), pixel-based intensity
(Fig. 6C) and protein content (Fig. 6D) in comparison to the control animals. In contrast, the
animals in MICT, HICT and HIIT groups represented a remarkable (p<0.05) reduction in p21
expression level compared to the sedentary obese animals in the HDF-sole group.

3.6. Exercise training ameliorated the DNA fragmentation and apoptosis index

The obese animals in the HDF-sole group represented a significant (p<0.05) increment in
apoptotic cells/mm2 of tissue compared to those in the control group. However, the HFD-
induced animals in MICT, HICT and HIIT groups represented a remarkable (p<0.05) reduction
in apoptotic cells number/mm2 of tissue versus obese animals in HDF-sole group. The pixel-
based intensity of green fluorescent reaction (presenting apoptotic cells) was analyzed and the
animals in MICT, HICT and HIIT groups showed diminished reaction versus obese animals in
the HDF-sole group (Fig. 7A, 7B, 7C). Comparing different exercise training protocols with
each other showed that the HIIT training exhibits higher TUNEL positive cells versus LICT
and MICT groups, while it was lower when compared with those animals in the sedentary
obese animals in the HFD-sole group. In addition to TUNEL staining, the DNA ladder test was
conducted. In line with the findings, which revealed in TUNEL staining, the HFD-sole group
showed a severe DNA fragmentation, and the HDF-induced animals in MICT, HICT and HIIT
groups represented an ameliorated DNA fragmentation (Fig. 7D).

4. Discussion:

Obesity is a medical condition, which refers to excessive accumulation of body fat, which
exerts a negative impact on general health, reduces life expectancy and enhances the health
risks and/or problems. A shift in the diet toward high-calorie junk food, containing excess fat
and sugars with low fiber content results in ‘energy imbalance’, which is known as the main
cause of obesity in the human population. Moreover, decreased physical activity and/or
sedentary lifestyle are known as other factors in inducing obesity. Here in the current study, we
found that HFD-induced obesity in rats significantly reduces the testicular/total weight ratio,
results in a severe spermatogenesis failure, alters the serum lipid profile, suppresses testicular
endocrine status, deregulates the cell cycle machinery system by suppressing the Cyclin D1
and Cdk4 expression, and up-regulating the p21 expression in testicles. Finally, it triggers
invasive germ and somatic cell apoptosis and DNA fragmentation in testicular tissue. On the
other hand, we showed that MICT, HICT and HIIT exercise training protocols, with no
statistical difference with each other, ameliorate the obesity-induced impairments, both at
histological and molecular levels.

As preliminary data, we evaluated the total body weight to approve the obesity in the rat
models. The animals in the HFD-received group exhibited a significant increment in the total
body weight, representing successful induction of the experimental obesity. Next, we tried to
see the effect of MICT, HICT and HIIT on obesity. Observations revealed a remarkable
reduction in the total body weight of the exercise-trained obese rats versus the HFD-sole
animals. As a second marker for obesity, the serum levels of the LDL-C, HDL-C and
triglyceride were assessed. Observations revealed a remarkable increment in the LDL-C and
triglyceride levels as well as a significant reduction in the HDL-C level in the HFD-sole group.
However, the animals in MICT, HICT and HIIT groups represented a decreased LDL-C and
triglyceride levels as well as increased HDL-C compared to the obese animals in the HFD-sole
group. It is well known that the cellular population of the testicular tissue determines the
testicular weight, and on the other hand, testosterone plays an important role in the developing
spermatogenesis to maintain the testicular cell population (Zamir-Nasta et al., 2017; Amin et
al., 2018). Therefore, we assessed the testicular/body weight ratio and serum testosterone level
and compared between groups. We showed that HFD-induced experimental obesity decreased
the testicular/body weight and reduced the serum testosterone level. In contrast, MICT, HICT
and HIIT exercises ameliorated the testicular weight gain, albeit by maintaining the testicular
cell population.

To show why and how obesity suppresses spermatogenesis, several previous studies
investigated different pathways such as; oxidative stress (Jia et al., 2018), cytokines
overexpression (Cabler et al., 2010), epigenetic disorders (Craig et al., 2017), intrinsic and
extrinsic apoptosis pathways (Yan et al., 2015) and metabolic impairments (Oliveira et al.,
2017). Here in the current study, we focused on the crosstalk between the cell cycle machinery
and apoptosis as a newly revealed molecular process in the spermatogenesis process.
Accordingly, the mRNA and protein expressions of Cyclin D1, Cdk4, and p21, as well as the
apoptosis ratio were analyzed in the HFD-induced obesity. At the same time, the ameliorative
effects of MICT, HICT, and HIIT exercise training protocols against these markers were
investigated. Indeed, the Cyclin D1/Cdk4, as well as the Cyclin D1/Cdk6 interactions, regulate
the spermatogonia proliferation mainly at the G1/S phases (Zamir-Nasta et al., 2017). It has
been shown that any reduction in the Cyclin D1 and/or Cdk4 expressions inhibits the Cyclin
D1/Cdk4 complex formation and consequently stops the cell cycle process in the developing
germ cells (Amin et al., 2018). Our results showed that HFD-induced obesity significantly
diminished the Cyclin D1 and Cdk4 expressions (both at mRNA and protein levels) when
compared to the control group. However, the MICT, HICT and HIIT exercise training
protocols could significantly up-regulate the Cyclin D1 and Cdk4 expressions compared to the
sedentary obese animals in the HFD-sole group. Similar results, but not in testicular tissue, are
shown by other studies. Accordingly, Fyfe and co-workers (2018) included a resistance
training (RT) insole and with MICT and HIIT to show the possible changes in the muscular
ribosome biogenesis-related signaling through the cyclin D1 and cdk4 interactions (Fyfe et al.,
2018). They showed that RT up-regulates the mTORC1 and biogenesis-related signaling in
human skeletal muscle compared to the concurrent training, suggesting the effect of MICT and
HIIT on cyclin D1 and cdk4 expression levels in muscular cells. The high-load resistance
exercise is shown to increase the Cyclin D1 protein expression in the skeletal muscle by
stimulating/targeting the translation of cyclin D1 mRNA (Figueiredo et al., 2016). Liu and co-
workers (2018), showed that physical activities promote the endogenous neural stem cells
proliferation via up-regulating the Cyclin D1 and Cdk4 expressions (Liu et al., 2018). In line
with these reports, for the first time here in the current study, we showed that MICT, HICT and
HIIT exercise training protocols significantly up-regulated the Cyclin D1 and Cdk4 expression
levels (mRNA and protein), and via this mechanism, they ameliorated the detrimental effect of
obesity against the germ cell proliferation.

In addition to the Cyclin D1/Cdk4-induced role in the germ cell proliferation system, the
crosstalk between kinase inhibitor protein p21 and the cell cycle machinery should not be
ignored. Accordingly, in the case of severe DNA damage, the p53 expression increases and
consequently stimulates the p21 protein overexpression. In turn, the force-expression of the
p21 stops the cell cycle progression through interaction with Cdk4 and generating the
p21/Cdk4 complex (Sham et al., 2011; Nicolai et al., 2015; Zamir-Nasta et al., 2017). As a
result of p21/Cdk4 interaction, the Cyclin D1 protein remains free. Thus, the p21/Cdk4
complex generation, in association with inactive free Cyclin D1 protein, stops the cell cycle
development and lets the cells to modify and/or repair the DNA damage. Otherwise, the
situation ends with the apoptosis (Cheraghi et al., 2015; Zamir-Nasta et al., 2017; Amin et al.,
2018). Here in the current study, we revealed that HFD-induced obesity enhanced the p21
expression compared to those in the control group, and MICT, HICT and HIIT significantly
reduced the p21 expression compared to the obese animals in the HFD-sole group. Moreover,
the animals in the HFD-sole group represented a significant increment in the apoptotic cell
population, which was significantly controlled in the MICT, HICT and HIIT animals.
Considering these findings, we can come close to this fact that the exercise training protocols,
in addition to their effect on Cyclin D1 and Cdk4 expressions, maintain germ cell proliferation
and differentiation by regulating the p21 expression. It means that following a significant
reduction in the p21 expression in MICT, HICT, and HIIT groups, the Cdk4 remained free and
interacted with Cyclin D1 to generate a Cyclin D1/Cdk4 complex. However, the findings of
TUNEL staining, pixel-based analyses, and DNA laddering testes showed that all types of
exercise training protocols could significantly decrease the apoptosis and DNA fragmentation
ratios. Meanwhile, the animals in the HIIT group represented higher apoptosis and DNA
fragmentation ratios compared to the LICT and MICT groups, and lower ratios versus the
HFD-sole animals. Apart from the exercise training protocols-induced effect on p21 and cell
cycle signaling system, the last part of results suggests that HIIT, insole, can induce adverse
effects on spermatogenesis may be by up-regulating the apoptosis ratio, while it inhibits the
apoptosis through regulating p21, Cyclin D1 and Cdk4 expressions when considered with
obesity. Thus minding the role of p21 in the induction of apoptosis, we can suggest that
diminished p21 expression (in MICT, HICT and HIIT groups) indirectly inhibited the apoptosis
process by reducing the p21/Cdk4 interaction.
 5. Conclusion:

Based on our findings, the HFD-induced obesity (at least in the rat model and in the current
study design) negatively affects the cell cycle machinery by suppressing the Cyclin D1/Cdk4
complex generation, inducing severe DNA fragmentation and p21 force-expression. On the
other hand, the MICT, HICT and HIIT exercise training, albeit with no statistically significant
differences, can ameliorate/diminish the obesity-induced spermatogenesis failure by
maintaining the Cyclin D1 and Cdk4 expression, reducing DNA damage and consequently
suppressing the p21 expression. However, more experimental studies are needed to make the
clear effect of different types of exercise training protocols against obesity-induced sub and
complete infertility disorders.

6. Study Limitation:

The glucose tolerance (GTT) and insulin tolerance (ITT) tests were not considered in the
current study.

7. Acknowledgment:

The authors wish to thank the Department of Exercise Physiology and Corrective Exercises,
Faculty of Sport Sciences, Urmia University, and Departments of Basic Sciences and Faculty
of Veterinary Medicine, Urmia University for scientific supports. Moreover, they would like to
appreciate Miss. Selda Zeynali for her laboratory helps. Finally, the authors deeply appreciate
RASTA scientific research laboratory for scientific and laboratory helps.
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Abbreviations list

MICT, moderate-intensity continuous ;HICT, high-intensity continuous; HIIT, High-intensity

interval; HFD, High-fat diet; LDL, Low-density lipoprotein; HDL, High-density lipoprotein;
cdk, Cyclin dependent kinase, BMI, Body Mass Index; TG, triglyceride; Smax, maximum
speed test; TDI, Tubular differentation index; SPI, spermiogenesis index; RI, the relative ratio
of spermatogonia A/B; CT, threshold cycle; IHC, Immunohistochemical staining; PBS,
Phosphate-buffered saline; HRP, Horseradishperoxidase; DAB, Diaminobenzidine; RIPA,
Radioimmunoprecipitation assay buffer; SDS-PAGE, Sodium dodecyl-sulfate polyacrylamide
gel electrophoresis; PVDF, polyvinylidene difluoride; ECL, enhanced chemiluminescence;
TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure legends:
Fig. 1:

Fig. 1: (A) Mean changes in total body (a Vs b: P=0.002; a Vs c: P=0.002; b Vs c: P=0.001),

testicular (a Vs b: P=0.001) and testicular/total body weights (a Vs b: P=0.001) in different
groups, (B) Mean number of Leydig (a Vs b: P=0.002; a Vs c: P=0.002; b Vs c: P=0.01) and
Sertoli cells (a Vs b: P=0.001; a Vs c: P=0.02; b Vs c: P=0.001) in different groups, (C) cross-
sections of seminiferous tubules in different groups; Note germ cells dissociation (arrows),
arrested spermatogenesis (head arrows) and spermiogenesis (thick arrow) in high fat diet
(HFD)-sole group, which represented in higher magnifications, (D) percentages of
seminiferous tubules with negative repopulation (RI) (a Vs b: P=0.001), differentiaton (TDI) (a
Vs b: P=0.001; a Vs c: P=0.002; b Vs c: P=0.001) and spermiogenesis (SPI) (a Vs b: P=0.001)
indices in different groups; All data are presented in Mean±SD (n=6). Note: MICT:moderate-
intensity continuous training; HICT:high-intensity continuous training; HIIT:High-intensity
interval training.
Fig. 2:

Fig. 2: Serum lipid and testosterone profiles: (A) low density lipoprotein cholesterol (LDL-C)
(a Vs b: P=0.0001; a Vs c: P=0.03; b Vs c: P=0.001), (B) high density lipoprotein cholesterol
(HDL-C) (a Vs b: P=0.001; a Vs c: P=0.002; b Vs c: P=0.01), (C) triglyceride (TG) (a Vs b:
P=0.001; a Vs c: P=0.01; b Vs c: P=0.0001) and (D) testosterone (a Vs b: P=0.001; a Vs c:
P=0.002; b Vs c: P=0.002) in different groups; All data are presented in Mean±SD, (n=6).
Note: MICT:moderate-intensity continuous training; HICT:high-intensity continuous training;
HIIT:High-intensity interval training.
Fig. 3:

Fig. 3: qRT-PCR results for fold changes of (A) cyclin D1 (a Vs b: P=0.0001; a Vs c: P=0.01;
b Vs c: P=0.001), (B) cdk4 (a Vs b: P=0.0001; a Vs c: P=0.002; b Vs c: P=0.002) and (C) p21
(a Vs b: P=0.001; a Vs c: P=0.02; b Vs c: P=0.002) mRNA in different groups; All data are
presented in Mean±SD, (n=6). Note: MICT:moderate-intensity continuous training;
HICT:high-intensity continuous training; HIIT:High-intensity interval training.
Fig. 4:
Fig. 4: (A) Immunohistochemical staining of cyclin D1 (Arrows), (B) cyclin D1+ cells per mm2
of testicular tissue (a Vs b: P=0.0001; a Vs c: P=0.02; b Vs c: P=0.02), (C) software analysis
for cyclin D1 positive reaction by pixel-based intensity ratio (a Vs b: P=0.0001; a Vs c:
P=0.02; b Vs c: P=0.01), (D) relative expression of cyclin D1 protein in different groups (a Vs
b: P=0.0001; a Vs c: P=0.01; b Vs c: P=0.02), which assessed by western blotting; All data are
presented in Mean±SD, (n=6). Note: MICT:moderate-intensity continuous training;
HICT:high-intensity continuous training; HIIT:High-intensity interval training.
Fig. 5:

Fig. 5:(A) Immunohistochemical staining of cdk4 (Arrows), (B) cdk4+ cells per mm2 of
testicular tissue (a Vs b: P=0.001; a Vs c: P=0.03; b Vs c: P=0.01), (C) software analysis for
cdk4 positive reaction by pixel-based intensity ratio (a Vs b: P=0.001; a Vs c: P=0.03; b Vs c:
P=0.01), (D) relative expression of cdk4 (a Vs b: P=0.001; a Vs c: P=0.03; b Vs c: P=0.01)
protein in different groups, which assessed by western blotting; All data are presented in
Mean±SD, (n=6).Note: MICT:moderate-intensity continuous training; HICT:high-intensity
continuous training; HIIT:High-intensity interval training.
Fig. 6:

Fig. 6:(A) Immunohistochemical staining of p21 (Arrows), (B) p21+ cells per mm2 of testicular
tissue (a Vs b: P=0.001; a Vs c: P=0.03; b Vs c: P=0.01), (C) software analysis for p21 positive
reaction by pixel-based intensity ratio (a Vs b: P=0.001; a Vs c: P=0.03; a Vs d: P=0.02; b Vs
c: P=0.01; b Vs d: P=0.02; c Vs d: P=0.03), (D) relative expression of p21 protein (a Vs b:
P=0.001; a Vs c: P=0.03; b Vs c: P=0.01) in different groups, which assessed by western
blotting; All data are presented in Mean±SD, (n=6). Note: MICT:moderate-intensity
continuous training; HICT:high-intensity continuous training; HIIT:High-intensity interval
training.

Fig. 7:

Fig. 7: (A) TUNEL staining; note apoptotic cells (arrows) in the high-fat diet (HFD)-sole
group, which are significantly decreased in MICT, HICT and HIIT groups, (B) software
analyses of pixel-based intensityfor apoptotic cells in 3000 µm×3000 µm of tissue, (C) mean
 number of apoptotic cells per mm2 of tissue in different groups; All data are presented in
Mean±SD, (n=6, a Vs b: P=0.0001; a Vs c: p=0.001, a Vs d: P=0.002; a Vs e: P=0.001; b Vs c:
p=0.001; b Vs d: P=0.002; b Vs e: P=0.002; c Vs d: P=0.03; c Vs e: P=0.02; d Vs e: P=0.03).
Note: MICT:moderate-intensity continuous training; HICT:high-intensity continuous training;
HIIT:High-intensity interval training.

Table 1. MICT protocol (5 sessions per week)

Week 1 2 3 4 5 6 7 8 9 10 11 12
Intensity 50-60 50-60 50-60 50-60 50-60 50-60 50-60 50-60 50-60 50-60 50-60 50-60
(Smax) % % % % % % % % % % % %

Time
15-20 20-30 30-40 40-50 50-60 60-65 65-70 70-75 75-80 80 80 80
(min)

Table 2. HICT protocol (5 sessions per week)

Week 1 2 3 4 5 6 7 8 9 10 11 12
Intensity 70-75 70-75 70-75 70-75 70-75 70-75 70-75 70-75 70-75 70-75 70-75 70-75
(Smax) % % % % % % % % % % % %

Time
15-20 20-30 30-40 40-50 50-60 60-65 65-70 70-75 75-80 80 80 80
(min)

Table 3. HIIT protocol (5 sessions per week)

training No. of series Duration of each series Active recovery Duration of recovery Slop
weeks intensity(Smax) (repetition) (min) intensity between series (%)
(Smax) (min)

1 85–90 % 3 4 20–25 % 2 5-10

2 85–90 % 5 4 20–25 % 2 10-15
3 85–90 % 7 4 20–25 % 2 15-20
4 85–90 % 9 4 20–25 % 2 20
5 85–90 % 10 4 20–25 % 2 20
6 85–90 % 11 4 20–25 % 2 20
7 85–90 % 12 4 20–25 % 2 20
8 85–90 % 12 4 20–25 % 2 20
9 85–90 % 13 4 20–25 % 2 20
10 85–90 % 13 4 20–25 % 2 20
11 85–90 % 13 4 20–25 % 2 20
12 85–90 % 13 4 20–25 % 2 20

Table 4: List of primers for qRT-PCR

Name Primer Product size (bp)

Cyclin D1 Forward: 5'-GAGACCATTCCCCTGACTGC-3' 79

Reverse: 5'-CCATTTGCAGCAACTCCTCG-3'
Cdk4 Forward: 5'-GGAGGCCTTTGAACATCCCA-3' 182
Reverse: 5'-ACTGGCGCATCAGATCCTTA-3'
P21 Forward: 5'-TGTTCCACACAGGAGCAAAG-3' 175
Reverse: 5'-AACACGCTCCCAGACGTAGT-3'
GAPDH Forward: 5'-ACTTTGGCATCGTGGAAGGG-3' 264
Reverse: 5'-ACTTGGCAGGTTTCTCCAGG-3'

CRediT author statement

Javad Tolouei Azar: Conceptualization, Methodology, Writing- Original draft preparation,

Aref Habibi Maleki: Data curation, Methodology, Sana Moshari: Visualization,
Investigation, software. Mazdak Razi: Supervision: Software, Writing- Reviewing and
Editing.
HFD suppressed cell cycle machinery and induced apoptosis in testicular tissue.

MICT, HICT and HIIT up-regulated cyclin D1 and cdk4 levels in HFD testicular tissue.

MICT, HICT and HIIT down-regulated p21 expression in HFD testicular tissue.

MICT, HICT and HIIT significantly diminished the HFD-induced apoptosis in testes.