gaae015-html.html

Cellular senescence of granulosa cells in the pathogenesis of polycystic ovary syndrome

Running title:

Cellular senescence and senolytics in PCOS

Tsurugi Tanaka, Yoko Urata, Miyuki Harada

, Chisato Kunitomi, Akari Kusamoto, Hiroshi Koike,

Zixin Xu, Nanoka Sakaguchi, Chihiro Tsuchida, Airi Komura, Ayaka Teshima, Nozomi Takahashi,

Osamu Wada-Hiraike, Yasushi Hirota, Yutaka Osuga

Department of Obstetrics and Gynecology, Faculty of Medicine, The University of Tokyo, Tokyo

113-8655, Japan

*Correspondence address:

Miyuki Harada, Department of Obstetrics and Gynecology, Faculty of

Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo, Tokyo 113-8655, Japan. Tel: +81-3-

3815-5411; Fax: +81-3-3816-2017; E-mail:

haradam-tky@umin.ac.jp

ORCID:

https://orcid.org/

0000-0003-1071-5600

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Abstract

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of

reproductive age, but its pathology has not been fully characterized and the optimal treatment

strategy remains unclear. Cellular senescence is a permanent state of cell-cycle arrest that can be

induced by multiple stresses. Senescent cells contribute to the pathogenesis of various diseases,

owing to an alteration in secretory profile, termed ‘senescence-associated secretory phenotype’

(SASP), including with respect to pro-inflammatory cytokines. Senolytics, a class of drugs that

selectively eliminate senescent cells, are now being used clinically, and a combination of dasatinib

and quercetin (DQ) has been extensively used as a senolytic. We aimed to investigate whether

cellular senescence is involved in the pathology of PCOS and whether DQ treatment has beneficial

effects in patients with PCOS. We obtained ovaries from patients with or without PCOS, and

established a mouse model of PCOS by injecting dehydroepiandrosterone. The expression of the

senescence markers p16

INK4a

, p21, p53,

H2AX, and senescence-associated

β-galactosidase

(SA-β-

gal); and the SASP-related factor interleukin (IL)-6; were significantly higher in the ovaries of

patients with PCOS and PCOS mice than in controls. To evaluate the effects of hyperandrogenism

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and DQ on cellular senescence

in vitro,

we stimulated cultured human granulosa cells (GCs) with

testosterone and treated them with DQ. The expression of markers of senescence and a SASP-

related factor was increased by testosterone, and DQ reduced this increase. DQ reduced the

expression of markers of senescence and a SASP-related factor in the ovaries of PCOS mice and

improved their morphology. These results indicate that cellular senescence occurs in PCOS.

Hyperandrogenism causes cellular senescence in GCs in PCOS and senolytic treatment reduces the

accumulation of senescent GCs and improves ovarian morphology under hyperandrogenism. Thus,

DQ might represent a novel therapy for PCOS.

Keywords:

cellular senescence, senolytic, polycystic ovary syndrome, dasatinib, quercetin, p16

INK4a

p21,

γH2AX,

senescence-associated

β-galactosidase,

senescence-associated secretory phenotype

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Introduction

Polycystic ovary syndrome (PCOS) is the most common hormonal disorder in women,

affecting 6%–10% of women of reproductive age (McCartney and Marshall, 2016). PCOS causes

~80% of cases of anovulatory infertility (Balen

et al.

, 2016), and is also characterized by

hyperandrogenemia, obesity, impaired glucose tolerance, and abnormal lipid metabolism. PCOS is

a heterogeneous condition, and according to the Rotterdam criteria, is present if two of the three

following features are present: hyperandrogenism, ovulatory dysfunction, and polycystic ovarian

morphology (Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2004).

The characteristic morphological features of polycystic ovaries are the accumulation of antral

follicles of 5–8 mm and follicular growth failure (Franks

et al.

, 2000; Franks

et al.

, 2008). Its

pathophysiology has not been well characterized, but various mechanisms, including endoplasmic

reticulum (ER) stress (Harada, 2022; Koike

et al.

, 2023), oxidative stress (Zeber-Lubecka

et al.

2023), and abnormal inflammation and immunity (Lima

et al.

, 2018) have been implicated. Women

with PCOS have high serum concentrations of pro-inflammatory molecules, such as tumor necrosis

factor

(TNF)-α,

interleukin (IL)-1, IL-6, and IL-18 (Kaya

et al.

, 2010; Alissa

et al.

, 2021; Mazloomi

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et al.

, 2023).

Although the pathogenesis of PCOS has not been fully characterized, androgen excess has

been shown to be a key component (Walters

et al.

, 2018), and findings made in a number of animal

models created through the administration of androgen are consistent with this (Elia

et al.

, 2006;

Manneras

et al.

, 2007; Corrie

et al.

, 2021). For example, dehydroepiandrosterone (DHEA)-treated

mice develop polycystic ovaries (Elia

et al.

, 2006); an estrus cycle disorder; a high circulating

luteinizing hormone (LH) concentration (Zhang H.

et al.

, 2023); and a metabolic phenotype

comprising insulin resistance, hypertriglyceridemia, and a high serum low-density lipoprotein-

cholesterol concentration (Li

et al.

, 2023). In addition, human and animal studies have shown that

the etiology of PCOS involves genetic, epigenetic, and metabolic factors; inflammatory processes;

and ER stress (Bednarska and Siejka, 2017; Harada, 2022; Koike

et al.

, 2023).

Cellular senescence is defined as a cellular state that is induced by certain insults and

physiological processes; and is characterized by prolonged and generally irreversible cell-cycle

arrest, an altered secretory profile, macromolecular damage, and altered metabolism (Gorgoulis

al.

, 2019). Cellular senescence occurs in response to cellular damage or stress (Munoz-Espin and

Serrano, 2014) associated with genotoxic drugs, oncogenic stress, oxidative stress, irradiation, or

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ER stress (Gorgoulis

et al.

, 2019). Senescent cells secrete senescence-associated secretory

phenotype (SASP) -related factors, which play a role in the crosstalk between senescent cells and

their neighbors. SASP-related factors include many immune mediators, such as

TNF-α,

IL-1α/β,

IL-6, IL-8, and tissue remodeling factors, including transforming growth factor

(TGF)-β

and matrix

metalloproteinases (Tchkonia

et al.

, 2013; Gorgoulis

et al.

, 2019). The SASP is a hallmark of

senescent cells and mediates many of their pathophysiological effects (Gorgoulis

et al.

, 2019;

Harries, 2023), such as in aging and chronic diseases including lung fibrosis (Barnes

et al.

, 2019),

osteoarthritis (Coryell

et al.

, 2021), age-related macular degeneration (Lee

et al.

, 2021),

neurodegeneration (Carreno

et al.

, 2021), chronic kidney disease (Goligorsky, 2020), and diabetes

(Palmer

et al.

, 2019). To date, no specific marker of cellular senescence has been identified

(Hernandez-Segura

et al.

, 2017), and therefore cellular senescence can only be defined using a

combination of multiple factors and features.

Two principal signaling pathways are responsible for the initiation and maintenance of

senescence-associated cell-cycle arrest: the p53–p21–retinoblastoma protein (RB) and p16

INK4a

–RB

pathways (Childs

et al.

, 2017; Gorgoulis

et al.

, 2019). Markers of cellular senescence include high

expression of p16

INK4a

and p21; high

SA-β-gal

activity; high expression of SASP-related factors,

such as IL-6, IL-8,

IL-1α,

and

TGF-β-1

(Childs

et al.

, 2017; Gorgoulis

et al.

, 2019; Harries, 2023);

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and DNA damage mediators, such as

γH2AX

(Hewitt

et al.

, 2012; Kirkland and Tchkonia, 2017).

A senolytic is an agent that induces the death of senescent cells, thereby eliminating them. Some

senolytics, including a combination of dasatinib and quercetin (DQ), are undergoing clinical trials

as treatments for Alzheimer’s disease (Gonzales

et al.

, 2023), idiopathic pulmonary fibrosis

(Nambiar

et al.

, 2023), and diabetic kidney disease (Hickson

et al.

, 2019).

In the present study, we aimed to determine whether (a) granulosa cells (GCs) show a high

level of senescence in PCOS; (b) hyperandrogenism induces cellular senescence in GCs; (c)

100

hyperandrogenism induces the secretion of a SASP-related factor by GCs; and (d) DQ ameliorates

101

PCOS in mice.

102

103

Materials and Methods

104

105

Human samples

106

Follicular fluid containing GCs was obtained from patients undergoing oocyte retrieval for

107

IVF at the University of Tokyo Hospital, Matsumoto Ladies’ Clinic, and Phoenix ART Clinic.

108

Informed written consent was obtained from all of the participants. PCOS was diagnosed according

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109

to the Rotterdam criteria (Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop

110

Group, 2004). The inclusion criteria for control patients were a normal ovulatory cycle, no

111

endocrinological abnormalities, and normal ovarian morphology, confirmed ultrasonographically.

112

The basal mRNA expression of markers of senescence in GCs was measured in samples obtained

113

from 12 control patients and 12 patients with PCOS, the characteristics of whom are shown in

114

Table 1. There were no significant differences between the groups with respect to age, BMI, serum

115

basal FSH concentration, or the number of oocytes retrieved; whereas the serum basal LH

116

concentration, LH/FSH ratio, and serum anti-Müllerian hormone (AMH) concentration of the

117

patients with PCOS were significantly higher.

118

For immunohistochemical analysis, ovaries were obtained from women undergoing

119

gynecological surgery. Informed written consent was obtained from all the participants. Normal

120

ovaries were obtained from women with regular menstrual cycles, who were not undergoing

121

hormonal treatment, and who underwent radical or extended hysterectomy as a treatment for

122

carcinoma of the uterine cervix or endometrium. Ovaries were also obtained from patients with

123

PCOS who were oligo- or anovulatory and who also underwent hysterectomy because of uterine

124

cancer. The presence of polycystic ovaries was histologically confirmed. Four control ovaries and

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four PCOS ovaries were obtained from eight different patients for analysis. There was no

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significant difference in the age of the two groups, but the BMI of the patients with PCOS was

127

higher: the median (range) ages were 38.0 years (27–46 years) and 27.0 years (24–36 years) (

128

0.1855); and the median (range) BMIs were 18.77 kg/m

(16.80–20.96 kg/m

) and 28.54 kg/m

129

(25.97–31.96 kg/m

) (

= 0.0007) for the control and PCOS groups, respectively

130

All the experimental procedures were approved by the relevant institutional review board

131

(authorization number: 3594-11). All the participants provided their written informed consent, and

132

the study was conducted in accordance with the principles of the Declaration of Helsinki.

133

134

Animal model

135

We used a well-established mouse model of DHEA-induced PCOS (Elia

et al.

, 2006; Koike

136

et al.

, 2022). Three-week-old female BALB/c mice were obtained from Japan SLC, Inc.

137

(Hamamatsu, Japan). First to characterize the accumulation of senescent cells in the ovaries of

138

PCOS mice, the animals were divided into two groups. The control group (n = 5) received daily

139

subcutaneous (s.c.) injections of sesame oil for 20 days, whereas the PCOS group (n = 5) received

140

daily s.c. injections of DHEA (60 mg/kg per day; Sigma-Aldrich, St. Louis, MO, USA) for 20 days.

141

The animals were anaesthetized with isoflurane and sacrificed by decapitation, and their ovaries

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142

were collected on day 21. Next, to determine the effects of DQ on the cellular senescence of GCs

143

of the PCOS mice, other animals were obtained and divided into three groups: a PCOS group, a

144

PCOS + vehicle group, and a PCOS + DQ group. All three groups received daily s.c. injections of

145

DHEA (60 mg/kg per day) for 20 days. The PCOS group (n = 6) received no oral treatment, the

146

PCOS + vehicle group (n = 6) was orally administered vehicle (10% polyethylene glycol 400;

147

Wako, Osaka, Japan), and the PCOS + DQ group (n = 6) was orally administered dasatinib (5

148

mg/kg per day; Sigma-Aldrich) and quercetin (50 mg/kg per day; Sigma-Aldrich) on days 1, 6, 11,

149

and 16. The ovaries of the mice were collected on day 21. Then, to determine the effect of DQ on

150

the ovarian morphology of the PCOS mice, other mice were obtained and divided into four groups;

151

a control group (n = 4), a PCOS group (n = 4), a PCOS+Vehicle group (n = 4) and a PCOS+DQ

152

group (n = 4). All group mice were treated as described above and the ovaries of the mice were

153

collected on day 21. All the animal procedures described were approved by the University of Tokyo

154

Committee on the Use and Care of Animals (approval number: P21-005) and were performed in

155

accordance with relevant guidelines and regulations, and the ARRIVE guideline 2.0 (Percie du Sert

156

et al.

, 2020).

157

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RNA extraction, reverse transcription, and real-time quantitative PCR

159

RNA was extracted from the whole ovaries of mice using Isogen (Nippon Gene, Tokyo,

160

Japan), then 1

μg

of each preparation was reverse transcribed using ReverTra Ace qPCR RT Master

161

Mix, with genomic DNA remover (Toyobo, Osaka, Japan), in a volume of 40

μL.

The cDNA

162

template was synthesized from human GCs using a SuperPrep Cell Lysis & RT Kit for qPCR

163

(Toyobo). To measure the expression of target mRNAs, quantitative real-time PCR was performed

164

on a Light Cycler system (Roche Diagnostics GmBH, Mannheim, Germany), and the mRNA

165

expression levels were normalized to that of glyceraldehyde-3-phosphate dehydrogenase

166

(

GAPDH

), which served as the reference gene. The primer sequences are shown in Table 2. The

167

PCR conditions were as follows: 40 cycles of 98

℃

for 10 s, 60

℃

for 10 s, and 68

℃

for 30 s.

168

169

Immunohistochemistry

170

Human and mouse ovaries were fixed in 10% v/v neutral-buffered formalin, embedded in

171

paraffin, and sectioned at a thickness of 5

μm.

Antigen retrieval was performed using Target

172

Retrieval Solution (Dako, Tokyo, Japan). Non-specific binding was blocked using peroxidase-

173

blocking solution (Dako) for 5 min at room temperature. The ovarian sections were then incubated

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with anti-p16

INK4a

(1:1,000, RRID: AB_2737282; Abcam, Cambridge, UK), anti-p21 (1:1,600,

175

RRID: AB_2077682; Proteintech Group, Tokyo, Japan), anti-p53 (1:4,000, RRID: AB_2881401;

176

Proteintech Group),

anti-γH2AX

(1:400, RRID: AB_2118009; Cell Signaling Technology, Danvers,

177

MA, USA), or anti-IL-6 (1:3,000, RRID: AB_2881543; Proteintech Group) antibodies, or isotype-

178

specific IgG

(RRID: AB_737182; Santa Cruz Biotechnology, Dallas, TX, USA) as a negative

179

control, followed by secondary antibody (Real EnVision Detection System, RRID: AB_2888627;

180

Dako). Immunohistochemistry was performed on at least three independent occasions using

181

identical samples. The stained sections were examined using a BX50 microscope (Olympus, Tokyo,

182

Japan), and ImageJ software (RRID: SCR_003073; National Institutes of Health, Bethesda, MD,

183

USA) was used for their quantitative analysis of % of positive GCs area. (Schneider

et al.

, 2012;

184

Jensen EC, 2013; Varghese F et al., 2014).

185

186

Scoring of

γH2AX

immunoreactivity

187

To assess the protein expression of

γH2AX

we used the immunoreactive score (IS), since

188

the intensity of

γH2AX

is considered to indicate the degree of DNA damage per cell (Mei

et al.

189

2015). After immunohistochemical staining with an

anti-γH2AX

antibody, 10 follicles were

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randomly selected from each of the control and PCOS groups. All of the GCs in a follicle were

191

scored according to their staining intensity (0 (negative), 1 (weak staining = light yellow), 2

192

(moderate staining = yellow brown), or 3 (strong positive = brown or black)) independently by two

193

authors. The proportions of cells with each score were multiplied by the scores, and all these were

194

added together to obtain the IS of a follicle.

195

196

Isolation and culture of human GCs

197

GCs were isolated as previously reported (Takahashi

et al.

, 2019; Kunitomi

et al.

, 2021).

198

The follicular fluid samples were centrifuged, and the pellets were resuspended in phosphate-

199

buffered saline (PBS) containing 0.2% w/v hyaluronidase (Sigma-Aldrich) and incubated at 37

℃

200

for 30 min. The suspension was then centrifuged again at 400 ×

for 30 min after being layered

201

over Ficoll-Paque (GE Healthcare, Amersham, UK). The GCs were collected from the interface

202

and washed with PBS, then cultured in 6-, 12-, or 48-well plates at densities of 5 × 10

, 5 × 10

, or

203

2 × 10

cells/well, respectively, in Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F-12;

204

Thermo Fisher Scientific, Waltham, MA, USA) containing 10% w/v fetal bovine serum (Sigma-

205

Aldrich) and antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin, and 250 ng/mL

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amphotericin B; Thermo Fisher Scientific). Prior to each experiment, the GCs were cultured for

207

3–5 days at 37

℃

in a humidified atmosphere containing 5% CO

208

209

Treatment of human GCs

210

At the beginning of the study, to determine the most appropriate concentration of

211

testosterone to use in the assessment of markers of senescence, human GCs were treated with 5, 10,

212

or 15

g/mL testosterone (Tokyo Chemical Industry Co., Tokyo, Japan) based on previous reports

213

(Azhary

et al.

, 2019) (Azhary

et al.

, 2020) for 9 h, and then the protein expression of p21 was

214

analyzed using western blotting

(Supplementary Figure S1). To evaluate the effects of testosterone

215

on the markers of senescence p16

INK4a

, p21, p53,

γH2AX,

and

SA-β-gal;

and the SASP-related

216

factor IL-6; human GCs were treated with 15

g/mL testosterone. To evaluate the effects of DQ on

217

cellular senescence, human GCs were concurrently incubated with 1 nM dasatinib, 10

M quercetin,

218

and 15

g/mL testosterone for 24 h. The optimal concentration of DQ was chosen on the basis of

219

that used in a previous study (Du

et al.

, 2022).

220

221

Western blotting

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Human GCs were lysed in PhosphoSafe Extraction Reagent (Merck, Darmstadt, Germany)

223

and then centrifuged at 16,000 ×

for 5 min. The supernatants were collected, and their protein

224

concentrations was measured using a Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA,

225

USA). Ten

μg

of denatured protein were loaded per lane subjected to SDS-PAGE and then

226

electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories)

227

using a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories). After blocking with 5% w/v

228

skim milk prepared in Tris-buffered saline containing 0.1% v/v Tween-20 at room temperature for

229

1 h, the membranes were incubated with anti-p16

INK4a

(1:500, RRID: AB_628067; Santa Cruz

230

Biotechnology), anti-p21 (1:1,000, RRID: AB_2077682; Proteintech Group), anti-p53 (1:5,000,

231

RRID: AB_2881401; Proteintech Group),

γH2AX

(1:400, RRID: AB_2118009; Cell Signaling

232

Technology), or

anti-β-actin

(1:10,000, RRID: AB_476697; Sigma-Aldrich) antibodies overnight

233

at 4

℃

, and then with a secondary antibody (anti-rabbit antibody,

1:2000, RRID: AB_2099233; or

234

anti-mouse antibody, 1:2000, RRID: AB_330924; Cell Signaling Technology) at room temperature

235

for 1 h. Bands were visualized using ECL Plus western blotting detection reagents (GE Healthcare)

236

and imaged on an ImageQuant LAS 4000 Mini luminescent image analyzer (GE Healthcare).

β-

237

actin was used as the loading control. The intensities of the specific protein bands were quantified

238

using ImageJ software (National Institutes of Health) (Schneider

et al.

, 2012).

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239

240

SA-β-gal

staining

241

Staining for

SA-β-gal

activity was performed as previously described (Dimri

et al.

, 1995;

242

Serrano

et al.

, 1997). After treatment, GCs cultured in 6-well plates were washed with PBS (pH

243

7.5), fixed in 3% w/v formaldehyde for 15 min, washed twice, and then stained with fresh

SA-β-

244

gal staining solution (PBS (pH 6.0) containing 1 mg/mL X-gal (5-bromo-4-chloro-3-indoly

β-D-

245

glucopyranoside; Wako), 1 mM MgCl

, 5 mM potassium ferricyanide, and 5 mM potassium

246

ferrocyanide) for 3–4 h at 37

℃

in the absence of CO

. The stained cells were examined using an

247

IX70 microscope (Olympus).

248

249

Measurement of IL-6 concentration

250

The concentrations of human IL-6 in follicular fluid samples and culture media were

251

measured using ELISA kits (R & D systems, Minneapolis, MN, USA), according to the

252

manufacturer’s instructions. Absorbance was measured at 450 nm using a Synergy LX multi-mode

253

reader (BioTek, Winooski, VT, USA). The sensitivity of the assay was 0.7 pg/mL, and the intra-

254

and interassay coefficients of variation for the ELISA kit were 7.9 ± 0.8% and 6.5 ± 0.8%,

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respectively.

256

257

Histology

258

Human and mouse ovaries were fixed in 10% v/v neutral-buffered formalin, embedded in

259

paraffin, and sectioned at a thickness of 5

μm.

The sections were stained with hematoxylin and

260

eosin, and the number of atretic antral follicles were counted in every sixth section across the entire

261

ovary, as previously described (Azhary

et al.

, 2020). To ensure that each atretic antral follicle was

262

only counted once, the adjacent sections were also analyzed. Atretic follicles were identified by the

263

presence of shrunken granulosa cell walls and cumulus cells or their absence, the hyperplasia of

264

theca cells, and the presence of macrophages in the antrum (Osman, 1985; Kauffman

et al.

, 2015).

265

266

Statistical analysis

267

Statistical analyses were performed using JMP Pro 16 software (RRID: SCR_022199; SAS

268

Institute Inc., Cary, NC, USA). All data are summarized as mean ± standard error of the mean

269

(SEM) or median (range). Two groups were compared using Student’s t-test, and one-way ANOVA

270

followed by Tukey-Kramer honest significant difference test was used for multiple comparisons.

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271

< 0.05 was considered to represent statistical significance.

272

273

Results

274

275

Senescent GCs accumulate in patients with PCOS

276

To determine whether senescence is upregulated in the GCs of patients with PCOS, we

277

measured the mRNA expression of the markers of senescence p16

INK4a

, p21, and p53 in the GCs

278

of control patients and those with PCOS using quantitative real-time PCR. The mRNA expression

279

p16

INK4a

, p21,

and

p53

in isolated GCs from patients with PCOS was 2.27-, 1.68-, and 3.52-fold

280

higher (

= 0.0110, 0.0330, and 0.0012), respectively, than those in GCs from control patients

281

(Figure 1A). Subsequently, we performed immunohistochemical analysis of the ovaries from the

282

patients with or without PCOS to evaluate the protein expression of these markers. Two or three

283

antral follicles were randomly selected from each ovarian section for quantitative analysis and the

284

immunoreactivities of p16

INK4a

, p21, and p53 in the GCs of ovaries from patients with PCOS were

285

1.72-, 1.96-, and 1.78-fold higher (

= 0.0067, 0.0021, and 0.0002), respectively, than those of

286

controls (Figure 1B–E). These findings indicate that cellular senescence is induced in the GCs of

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287

patients with PCOS.

288

289

Senescent cells accumulate in the ovaries of PCOS mice

290

Next, we measured the expression of markers of senescence in the ovaries of PCOS mice.

291

Quantitative real-time PCR analysis demonstrated that the mRNA expression of

p16

INK4a

, p21,

and

292

p53

in the ovaries of PCOS mice was 1.61-, 1.68-, and 1.44-fold higher (

= 0.0319, 0.0864, and

293

0.0439), respectively, than that of control mice (Figure 2A). Immunohistochemical analysis

294

revealed that the protein expression of p16

INK4a

, p21, p53, and

γH2AX

in the GCs of PCOS mice

295

was 1.55-, 1.72-, 1.67-, and 1.43-fold higher (

= 0.0017, 0.0007, 0.0008, and 0.0003), respectively,

296

than that of controls. (Figure 2B–F). These findings indicate that cellular senescence is upregulated

297

in the GCs of PCOS mice, in accordance with the findings in women with PCOS.

298

299

Testosterone induces cellular senescence in cultured human GCs

300

To determine whether the accumulation of senescent cells in the GCs of patients with PCOS

301

is associated with hyperandrogenism, we measured the protein expression of markers of senescence

302

following the treatment of cultured human GCs with testosterone. To determine the most

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303

appropriate concentration of testosterone for use in the assessment of senescence markers, human

304

GCs were treated with 5, 10, or 15,

g/mL testosterone, and the protein expression of p21 was

305

analyzed. The protein expression of p21 was increased more significantly by treatment with 15

306

g/mL testosterone (by 1.33-fold,

< 0.01; Supplementary Figure S1). Therefore, we used 15

307

g/mL testosterone treatment as a model of hyperandrogenism in cultured human GCs in

308

subsequent experiments. Testosterone stimulation significantly increased the protein expression of

309

p16

INK4a

, p21, p53, and

γH2AX

by 1.41-, 1.33-, 1.30-, and 1.43-fold (

< 0.0001, = 0.0006, 0.0003,

310

and 0.0025), respectively (Figure 3A–D). We also conducted

SA-β-gal

staining to further evaluate

311

the effect of testosterone on cellular senescence. As shown in Figure 3E,

SA-β-gal

activity was

312

increased by testosterone treatment. These results indicate that local hyperandrogenism in the ovary

313

causes the upregulation of senescence in human GCs.

314

315

DQ reduces the testosterone-induced senescence of, and secretion of

SASP

-related substances by

316

cultured human GCs

317

Next, to determine the effects of DQ on testosterone-induced senescence, we concurrently

318

incubated human GCs with testosterone and DQ for 24 h. DQ reduced the testosterone-induced

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319

increase in p16

INK4a

and p21 protein by 0.65- and 0.78-fold (

= 0.0548 and 0.0001), respectively

320

(Figure 4A,B). To further investigate the effect of DQ on cellular senescence, we assessed the

321

secretion of IL-6, which is known to be a pro-inflammatory SASP-related factor and to be

322

upregulated in the ovaries of PCOS mice (Van Deursen, 2014; Shi

et al.

, 2023). We measured the

323

concentrations of IL-6 in conditioned medium using ELISA, and found that testosterone stimulation

324

upregulates that by 1.99-fold (

= 0.0451), and concurrent incubation with DQ reduces the

325

testosterone-induced upregulation of IL-6 by 0.35-fold (

= 0.0344) (Figure 4C). Pretreatment with

326

DQ similarly reduced testosterone-induced senescence and IL-6 production of GCs (Supplementary

327

Figure S2).

328

329

DQ reduces the senescence of, and secretion of SASP-related molecules by, GCs of PCOS mice;

330

and restores their ovarian morphology

331

To determine whether DQ treatment affects the senescence of GCs

in vivo

, we administered

332

DQ to PCOS mice and evaluated the protein expression of markers of senescence and a SASP-

333

related factor in their ovaries by immunohistochemical analysis. The protein expression of p16

INK4a

334

p21, p53, and IL-6 in GCs from PCOS mice with DQ administration was reduced by 0.35-, 0.54-,

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335

0.50-, and 0.49-fold (

= 0.0012, 0.0002, 0.0001, and 0.0018), respectively,

. vehicle-treated

336

mice; and those that were vehicle-treated did not significantly differ from those that were untreated

337

(

= 0.9900, 0.6148, 0.2260, and 0.6293, respectively) (Figure 5A–E). Next, to determine the effect

338

of DQ on the ovarian morphology of PCOS mice, the numbers of atretic follicles were counted. As

339

shown in Figure 6, the number of atretic follicles in PCOS mice treated with DQ was 0.58-fold

340

lower than that in PCOS+Vehicle mice (

= 0.0128); and that of the PCOS mice was 1.57-fold

341

higher than that of control mice (

= 0.0135), which did not differ from that of the PCOS+Vehicle

342

mice (

= 0.9221). These findings suggest that DQ treatment reduces the cellular senescence of and

343

IL-6 production by GCs, and restores the ovarian morphology of PCOS mice.

344

345

Discussion

346

347

In the present study, we have made the following findings. (a) In human GCs from patients

348

with PCOS, the expression of p16

INK4a

, p21, and p53 is high at both the mRNA and protein levels.

349

(b) In PCOS mice, the ovarian mRNA expression of p16

INK4a

, p21, and p53 and the protein

350

expression of p16

INK4a

, p21, p53, and

γH2AX

of GCs are high. (c) In isolated human GCs,

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351

testosterone induces the protein expression of p16

INK4a

, p21, p53, and

γH2AX,

and increases

SA-β-

352

gal staining. (d) In isolated human GCs, the senolytic combination DQ reduces the testosterone-

353

induced protein expression of p16

INK4a

, p21, and IL-6. (e) In PCOS mice, DQ treatment reduces the

354

protein expression of p16

INK4a

, p21, p53, and IL-6 in GCs and the number of atretic follicles. These

355

results suggest that cellular senescence and the SASP-related factor IL-6 are involved in the

356

pathogenesis of PCOS.

357

We found that the expression of the cellular senescence markers p16

INK4a

, p21, p53, and

358

γH2AX

in GCs was high in patients with PCOS and PCOS mice. Cellular senescence has been

359

reported to affect ovarian function; and in mice, insulin resistance reduces the ovarian reserve by

360

causing the senescence of GCs (Gao

et al.

, 2023). Furthermore, in one previous study, an

361

association between cellular senescence and PCOS was identified; exosomal miR-424-5p derived

362

from the follicular fluid of patients with PCOS induces the senescence of COV434 and KGN cells

363

(Yuan

et al.

, 2021). However, no previous studies have demonstrated the existence of cellular

364

senescence in the ovaries of patients with PCOS or characterized the mechanism underpinning the

365

increase in cellular senescence in PCOS. In the present study, we have demonstrated that cellular

366

senescence is upregulated in the GCs of patients with PCOS or PCOS mice.

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367

We found that local hyperandrogenism induces the protein expression of the cellular

368

senescence markers p16

INK4a

, p21, p53, and

γH2AX

in, and the

SA-β-gal

activity of, human GCs.

369

These results suggest that local hyperandrogenism in PCOS induces the cellular senescence of GCs,

370

but the underlying mechanism requires to be defined. Local hyperandrogenism in PCOS may

371

directly induce the cellular senescence of GCs. It has been reported that androgen receptor

372

activation is linked to the induction of cellular senescence in some cancer cells, including thyroid

373

(Gupta

et al.

, 2023) and prostate (Mirochnik

et al.

, 2012) cancer cells. Alternatively,

374

hyperandrogenism may induce the senescence of GCs

via

the activation of ER stress. It has been

375

reported that ER stress induces the senescence of several types of cells. In alveolar epithelial cells

376

(AECs), epoxyeicosatrienoic acids, which are downstream metabolites of arachidonic acid, inhibit

377

ER stress and alleviate AEC senescence (Zhang CY.

et al.

, 2023); and in breast cancer cells, oroxin

378

A induces cellular senescence through the activation of ER stress (He

et al.

, 2016). ER stress

379

pathways are activated in the GCs of women with PCOS and androgen-administered mice

380

(Takahashi

et al.

, 2017; Jin

et al.

, 2020; Harada, 2022), and hyperandrogenism in the follicular

381

microenvironment is an activator of ER stress in human (Azhary

et al.

, 2019) and mice (Jin

et al.

382

2020) GCs.

383

We found that the senolytic combination DQ ameliorated the androgen-induced increases in the

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384

expression of cellular senescence markers and the SASP-related factor IL-6 in GCs

in vitro

and

385

vivo

. Dasatinib, which is used for the treatment of chronic myeloid leukemia, is an inhibitor of

386

several receptor tyrosine kinases (Montero

et al.

, 2011; Nieto

et al.

, 2023). Regarding its senolytic

387

potential, dasatinib reduces the number of senescent preadipocytes following exposure to ionizing

388

radiation (Zhu

et al.

, 2015) and reduces the cardiac expression of p16 in a mouse model of type 2

389

diabetes mellitus (Gu

et al.

, 2023). Quercetin is a flavonol of vegetable origin that has

390

pharmacological effects, including antioxidant, anti-inflammatory, and anti-senescence effects. It

391

has effects through the inhibition of the PI3K/AKT pathway (Olave

et al.

, 2010; Bruning, 2013).

392

Regarding its senolytic effects, quercetin eliminates the senescence of HUVEC cells (Zhu

et al.

393

2015) and radioresistant HT500 cells (Russo

et al.

, 2023); inhibits the SASP-related production of

394

IL-6, IL-8, and MMPs by nucleus pulposus cells (Shao

et al.

, 2021); and inhibits the production of

395

IL-8 by radioresistant HT500 cells (Russo

et al.

, 2023). Interestingly, DQ treatment before

396

testosterone also reduced the testosterone-induced expression of cellular senescence markers and

397

the SASP-related factor IL-6 in GCs as shown in Supplementary Figure S2. Taken together, the

398

senolytic combination DQ may ameliorate the androgen-induced cellular senescence both by

399

eliminating cellular senescence induced by testosterone and by converting the function of

400

testosterone in induction of cellular senescence.

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401

Zhu reported that the DQ combination reduces cellular senescence in tissues involved in

402

the etiology of multiple chronic disorders, because it targets multiple senescence-associated anti-

403

apoptotic pathways (Zhu

et al.

, 2015; Xu

et al.

, 2018). Moreover, Xu reported that DQ reduces

404

senescence; reduces the secretion of the pro-inflammatory cytokines IL-6, IL-8, MCP-1, PAI-1, and

405

GM-CSF by human adipose tissue; alleviates physical dysfunction; and improves the survival of

406

aged mice (Xu

et al.

, 2018). Phase I clinical trials of this oral senolytic combination have been

407

conducted in patients with Alzheimer’s disease (Gonzales

et al.

, 2023) and idiopathic pulmonary

408

fibrosis (Nambiar

et al.

, 2023), and these showed that this approach is safe, well-tolerated, and

409

feasible for patients. Another early clinical trial showed that treatment with DQ reduces senescence

410

in the adipose tissue of patients with diabetic kidney disease (Hickson

et al.

, 2020). Here, we have

411

reported for the first time that DQ acts as senolytic with respect to the pathology of PCOS and

412

reduces IL-6 secretion by GCs.

413

We have also demonstrated that the DQ treatment of PCOS mice improves their ovarian

414

morphology. There have been some previous reports regarding the effects of quercetin on ovarian

415

function and PCOS that did not evaluate cellular senescence. In a rat model of PCOS, quercetin

416

ameliorated the abnormalities in ovulation through effects on C-type natriuretic peptide (CNP) and

417

its receptor, natriuretic peptide receptor 2 (NPR2) (Zheng

et al.

, 2022). In addition, two randomized,

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418

double-blind, placebo-controlled clinical trials demonstrated that in patients with PCOS, oral

419

quercetin supplementation ameliorates adiponectin-mediated insulin resistance and hormonal

420

abnormalities (Rezvan

et al.

, 2017), improves the oocyte and embryo grade, and improves the

421

pregnancy rate (Vaez

et al.

, 2023). However, there have been no previous studies of the use of

422

quercetin as senolytic in PCOS. In the present study, the effects of DQ were determined

in vitro

423

cultured GCs, and the findings suggest that the

in vivo

effects of DQ on the ovary may be at least

424

in part direct and local. Therefore, cellular senescence may represent a novel therapeutic target in

425

PCOS, and senolytics represent potential treatments for PCOS.

426

There were some limitations to the present study. First, quercetin may act as an antioxidant,

427

and thereby reduce hyperandrogenism-induced IL-6 production through an effect on reactive

428

oxygen species, rather than through a senolytic effect. Second, although our

in vitro

findings

suggest

429

that DQ has direct and local effects in the ovaries, it might also act indirectly, through systemic

430

effects. Third, it is unclear whether DQ would be more effective than quercetin alone as a treatment

431

for PCOS. Therefore, future studies should address this.

432

In conclusion, we have demonstrated that the senescence of GCs is greater in patients and

433

PCOS mice, that testosterone induces senescence in cultured human GCs, and that DQ treatment

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464

Conflict of interest

465

The authors declare no conflict of interest.

466

467

Figure legends

468

Figure 1.

p16

INK4a

, p21, and p53 are upregulated in the granulosa cells (GCs) of patients with

469

polycystic ovary syndrome (PCOS). (A) The expression of

p16

INK4a

, p21,

and

p53

mRNAs in GCs

470

from control patients (n = 12) and those with PCOS (n = 12) was measured by quantitative real-

471

time PCR and normalized to that of glyceraldehyde 3-phosphate dehydrogenase (

GAPDH)

. (B–D)

472

Immunohistochemical analysis was performed on GCs in the antral follicles of ovaries from control

473

patients (n = 4) and those with PCOS (n = 4). Cross-sections of ovaries, stained with (B) anti-

474

p16

INK4a

, (C) anti-p21, and (D) anti-p53 antibodies, and counterstained with hematoxylin. (E)

475

Corresponding negative control. For the panels (c), two or three antral follicles were randomly

476

selected from each ovary (total 8 antral follicles per group) for quantitative analysis of

477

immunohistochemical staining. Representative images and data are shown. Scale bars: 50.0 µm.

478

Values are mean ± SEM, and the mean differences are analyzed using Student’s t-test. *

< 0.05

479

and **

< 0.01. GC, granulosa cell layer; GCs, granulosa cells; NC, negative control.

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480

481

Figure 2.

p16

INK4a

, p21, p53, and

γH2AX

are upregulated in the ovaries of polycystic ovary

482

syndrome (PCOS) mice. (A) The mRNA expression of

p16

INK4a

, p21,

and

p53

in whole ovaries

483

from control mice (n = 4) and those from PCOS mice (n = 4) was measured by quantitative real-

484

time PCR and normalized to that of glyceraldehyde 3-phosphate dehydrogenase (

GAPDH)

485

expression.

(B–E) Immunohistochemical analysis was performed on the GCs in antral follicles of

486

ovaries from control mice (n = 5) and those from PCOS mice (n = 5). Cross-sections of ovaries

487

were stained with (B) anti-p16

INK4a

, (C) anti-p21, (D) anti-p53, and (E)

anti-γH2AX

antibodies, and

488

counterstained with hematoxylin. (F) Corresponding negative control. Scale bars: 100 µm. (c) One

489

or two antral follicles were selected from each ovary (total 8 antral follicles per group) for

490

quantitative analysis of immunohistochemical staining. Values are mean ± SEM, and the mean

491

differences are analyzed using Student’s t-test. *

< 0.05 and **

< 0.01. GCs, granulosa cells; NC,

492

negative control. We repeated experiments three times using different mice, and representative data

493

are shown.

494

495

Figure 3.

Effects of testosterone on the expression of markers of senescence in cultured human

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496

granulosa cells (GCs). Human GCs were treated with testosterone (15 µg/mL). (A–D) Protein

497

expression levels of p16

INK4a

, p21, p53, and

γH2AX

in cultured human GCs, measured using

498

western blotting (n = 10).

β-actin

was used as a loading control. (b) Quantitative analysis of the

499

western blots and (a) representative images are shown. All full-size, uncropped images of western

500

blot used for analysis are shown in Supplementary Figure S3. Results are expressed relative to the

501

mean control value. Values are mean ± SEM, the mean differences are analyzed using Student’s t-

502

test. *

< 0.05 and **

< 0.01. (E)

SA-β-gal

staining of human GCs. Scale bars: 200 µm. (A-E) The

503

experiments were performed ten times using ten different women’s GCs, and representative images

504

are shown.

505

506

Figure 4.

A combination of dasatinib and quercetin (DQ) reduces the testosterone-induced

507

senescence of, and secretion of a SASP-related factor by, human granulosa cells (GCs). Human

508

GCs were treated with testosterone (15 µg/mL), dasatinib (1 nM) and quercetin (20 µM),

509

concurrently for 24 h. The protein expression of (A) p16

INK4a

and (B) p21 in cultured human GCs

510

was analyzed using western blotting (n=5).

β-actin

was used as a loading control. (b) Quantitative

511

analysis of the western blots using five different women’s GCs are expressed relative to the mean

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512

control value, and (a) representative images are shown. All full-size, uncropped images of western

513

blot used for analysis are shown in Supplementary Figure S4. (C) Concentration of IL-6 in the

514

culture medium, measured by ELISA using six different women’s GCs. Values are mean ± SEM,

515

and the mean differences were analyzed using one-way ANOVA followed by Tukey-Kramer honest

516

significant difference test. *

< 0.05 and **

< 0.01.

517

518

Figure 5.

A combination of dasatinib and quercetin (DQ) treatment reduces the senescence of, and

519

the secretion of a senescence-associated secretory phenotype (SASP)-related factor by, granulosa

520

cells in the antral follicles of polcystic ovary syndrome (PCOS) mice. The mice were divided into

521

three groups, which were subcutaneous (s.c.) injected with dehydroepiandrosterone (DHEA 60

522

mg/kg per day) (PCOS, n = 6), s.c. injected with DHEA and orally administered vehicle (10%

523

polyethylene glycol 400) (PCOS + vehicle, n = 6), or s.c. injected with DHEA and orally

524

administered DQ (D: dasatinib 5 mg/kg per day, Q: quercetin 50 mg/kg per day) (PCOS + DQ, n =

525

6). Subcutaneous injections were performed daily for 20 days, and oral administration was

526

performed on days 1, 6, 11, and 16. Ovaries were collected on day 21. Cross-sections of ovaries

527

were stained with (A) anti-p16

INK4a

, (B) anti-p21, (C) anti-p53, and (D) anti-IL-6 antibodies and

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528

counterstained with hematoxylin. (d) One or two antral follicles were randomly selected from each

529

ovary for quantitative analysis of the immunohistochemical staining (total 8 follicles per group).

530

Values are mean ± SEM, and the mean differences were analyzed using one-way ANOVA followed

531

by Tukey-Kramer honest significant difference test. **

< 0.01. (E) Corresponding negative control.

532

Scale bars: 100 µm. n.s., not significant; GCs, granulosa cells; NC, negative control. We repeated

533

experiments three times using different mice, and representative images and data are shown.

534

535

Figure 6.

A combination of dasatinib and quercetin (DQ) treatment restores the ovarian

536

morphology of polycystic ovary syndrome (PCOS) mice. To evaluate the ovarian morphology, the

537

mice were divided into four groups; control (n = 4), PCOS (n = 4), PCOS + vehicle (n = 4) and

538

PCOS + DQ (n = 4). Control mice were subcutaneous (s.c.) injected with sesame oil, PCOS mice

539

were s.c. injected with dehydroepiandrosterone (DHEA 60 mg/1 kg per day), PCOS + vehicle mice

540

were s.c. injected with DHEA and orally administered vehicle (10% polyethylene glycol 400), and

541

PCOS + DQ mice were s.c. injected with DHEA and orally administered DQ (D: dasatinib 5 mg/kg

542

per day, Q: quercetin 50 mg/kg per day) . Subcutaneous injection was performed daily for 20 days

543

and oral administration was performed on days 1, 6, 11, and 16. Ovaries were collected on day 21.

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544

Cross-sections of ovaries were stained with hematoxylin and eosin. (A)

★

indicate atretic follicles.

545

Scale bars: 500 µm. (B) The number of atretic follicles per ovary (four ovaries per group). Values

546

are mean ± SEM, and the mean differences were analyzed using one-way ANOVA followed by

547

Tukey-Kramer honest significant difference test. n.s., not significant; *

< 0.05. We repeated

548

experiments three times using different mice, and representative images and data are shown.

549

550

References

551

552

Alissa EM, Algarni SA, Khaffji AJ, and Al Mansouri NM. Role of inflammatory markers in

553

polycystic ovaries syndrome: In relation to insulin resistance.

J Obstet Gynaecol Res. 2021

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47(4): 1409-1415.

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Azhary JMK, Harada M, Kunitomi C, Kusamoto A, Takahashi N, Nose E, Oi N, Wada-Hiraike O,

556

Urata Y, Hirata T, et al. Androgens Increase Accumulation of Advanced Glycation End

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Table 1. Characteristics of the patients who provided follicular fluid samples

Control (n = 12)

PCOS (n = 12)

-value

Age (years)

34.0 (26–40)

35.0 (24–39)

0.2534

BMI (kg/m

)

22.2 (19.7–25.4)

21.5 (18.1–26.4)

0.8127

LH (mIU/mL)

4.8 (2.1–5.6)

8.8 (5.9–15.4)

0.0003

FSH (mIU/mL)

7.2 (5.3–8.3)

6.5 (4.9–8.4)

0.0898

LH/FSH ratio

0.64 (0.32–0.91)

1.34 (0.87–2.23)

<0.0001

AMH (ng/mL)

2.56 (1.70–6.73)

8.27 (5.44–24.63)

0.0017

The number of

oocytes retrieved

11.5 (6–28)

10.5 (1–22)

0.3072

Data are presented as median (range). All

-values are based on Student’s t-test. BMI, body mass

index; LH, luteinizing hormone; FSH, follicle-stimulating hormone; AMH, anti-Müllerian hormone.

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Table 2. List of primers used for quantitative real-time PCR

Gene

Forward

(5′

–

3′)

Reverse

(5′

–

3′)

Accession No.

human p16

INK4a

GGGTTTTCGTGGTTCACATCC CTAGACGCTGGCTCCTCAGTA

NM_058195

human p21

TGTCCGTCAGAACCCATGC

AAAGTCGAAGTTCCATCGCTC

NM_078467

human p53

CCCAAGCAATGGATGATTTGA GGCATTCTGGGAGCTTCATC

NM_000546

human GAPDH TGGACCTGACCTGCCGTCTA

CTGCTTCACCACCTTCTTGA

NM_002046

mouse p16

INK4a

CGCAGGTTCTTGGTCACTGT

TGTTCACGAAAGCCAGAGCG

NM_009877

mouse p21

CACAGCTCAGTGGACTGGAA ACCCTAGACCCACAATGCAG NM_001111099

mouse p53

GGCAGACTTTTCGCCACAG

CAGGCACAAACACGAACCTC

NM_011640

mouse Gapdh

TCCACCACCCTGTTGCTGTA

ACCACAGTCCATGCCATCAC

NM_008084

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25 –
20 –

15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –
20 –
15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –
20 –
15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –
20 –
15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –
20 –
15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –
20 –

15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –
20 –

15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –
20 –

15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –
20 –

15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –
20 –

15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –

20 –

15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –
20 –
15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –
20 –

15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –

20 –

15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

– p53

(53kDa)

– p53

(53kDa)

– p53

(53kDa)

Testosterone (−) (+)

(−) (+)

– p53

(53kDa)

– p53

(53kDa)

– β-actin

(42kDa)

– β-actin

(42kDa)

– β-actin

(42kDa)

– β-actin

(42kDa)

– β-actin

(42kDa)

25 –

20 –

15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –

20 –

15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –
20 –

15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –
20 –

15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

– β-actin

(42kDa)

– β-actin

(42kDa)

– β-actin

(42kDa)

– β-actin

(42kDa)

– β-actin

(42kDa)

– p53

(53kDa)

– p53

(53kDa)

– p53

(53kDa)

– p53

(53kDa)

– p53

(53kDa)

25 –

20 –

15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

25 –

20 –

15 –

37 –

50 –

100 –

75 –

250 –

150 –

10 –

Testosterone (−) (+)

(−) (+)

(kDa)

Page 62 of 65

http://molehr.oxfordjournals.org/

Draft Manuscript Submitted to MHR for Peer Review

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