Are sirtuins markers of ovarian aging?

    Are sirtuins markers of ovarian aging? Jinjin Zhang, Li Fang, Zhiyong Lu, Jiaqiang Xiong, Meng Wu, Liangyan Shi, Aiyue Luo, Shixuan Wang PII: DOI: Reference:

S0378-1119(15)01152-X doi: 10.1016/j.gene.2015.09.043 GENE 40861

To appear in:

Gene

Received date: Accepted date:

28 July 2015 18 September 2015

Please cite this article as: Zhang, Jinjin, Fang, Li, Lu, Zhiyong, Xiong, Jiaqiang, Wu, Meng, Shi, Liangyan, Luo, Aiyue, Wang, Shixuan, Are sirtuins markers of ovarian aging?, Gene (2015), doi: 10.1016/j.gene.2015.09.043

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Are sirtuins markers of ovarian aging?

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Research paper

Jinjin Zhang a, Li Fang a, Zhiyong Lu a, Jiaqiang Xiong a, Meng Wu a, Liangyan Shi b, Aiyue

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a

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Luo a*, Shixuan Wang a*

Department of Obstetrics and Gynecology, Tongji Hospital, Huazhong University of Science

Department of Obstetrics and Gynecology, Hubei Province, Maternity and Child Health

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b

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and Technology, Wuhan, Hubei, P.R. China

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Care Hospital, Wuhan, Hubei 430030, P.R. China

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*Corresponding authors: Dr Shixuan Wang and Dr Aiyue Luo, Department of Obstetrics and Gynecology, Tongji Hospital, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan City, Hubei, People’s Republic China. Tel: +86 27 83662878 Fax: +86 27 83663698 E-mail addresses: [email protected] (S. Wang); [email protected] (A. Luo)

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ACCEPTED MANUSCRIPT E-mail addresses of all authors Jinjin Zhang: [email protected]

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Li Fang: [email protected]

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Zhiyong Lu: [email protected] Jiaqiang Xiong: [email protected] Meng Wu: [email protected]

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Liangyan Shi: [email protected]

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Aiyue Luo: [email protected]

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Shixuan Wang: [email protected]

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ACCEPTED MANUSCRIPT Highlights Sirtuins have been implicated in mammalian aging and age-related diseases.

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The relationship between ovarian function and sirtuins was explored in mouse models

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Expression levels of SIRT1, SIRT3, and SIRT6 were closely related to ovarian reserve.

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Consequently, these sirtuins may be markers of ovarian aging.

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This finding may help provide pharmacological insight into delaying ovarian aging.

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Abbreviations: AFC, antral follicle count; AMH, anti-Müllerian hormone; ANOVA, analysis of variance; CR, caloric restriction; CTX, cyclophosphamide; FOXOs, Forkhead box class O

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transcription factors; FSH, follicle-stimulating hormone; GDH, glutamate dehydrogenase; H&E, hematoxylin and eosin; HSF1, heat shock transcription factor 1; NAD, nicotinamide adenine dinucleotide; NF-κB, nuclear factor-kappaB; mTOR, mammalian target of rapamycin; NC, normal control; PBS, phosphate-buffered saline; PGC-1, peroxisome proliferator-activated receptor-gamma coactivator-1; PMSF, phenylmethanesulfonyl fluoride; POF, premature ovarian failure; RIPA, radio-immunoprecipitation assay; ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SIRT, sirtuin; SOD2, superoxide dismutase 2; UCP1, uncoupling protein 1.

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ACCEPTED MANUSCRIPT ABSTRACT Sirtuins, a family of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases that

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play diverse roles in regulating metabolism, cell proliferation, and genome stability, have

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been implicated in mammalian aging and age-related diseases, including cancers, metabolic disorders, and neurodegenerative diseases. Ovarian aging is thought to be characterized by a gradual decrease in both the number of follicles and the quality of oocytes. Ovarian reserve is

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indicated by the number of primordial follicles. In this study, ovarian reserve was assessed in

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mice of different ages and mice subjected to caloric restriction (CR) and chemotherapy (2 commonly used models for ovarian aging research) by counting primordial follicles and

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determining the expression levels of SIRT1, SIRT3, and SIRT6 to explore the relationship

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between ovarian function and sirtuin expression. A gradual decline in the number of follicles

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(especially primordial follicles) was observed in aging mice and mice subjected to chemotherapy. Histological analysis showed that CR mice displayed a significantly greater

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number of primordial follicles and less atretic follicles. Western blot analysis indicated that expression levels of SIRT1, SIRT3, and SIRT6 were significantly decreased in the ovaries of aged mice and mice treated with chemotherapy, but increased in CR mice. SIRT1, SIRT3, and SIRT6 all showed a significantly positive correlation with the numbers of primordial follicles (r2 = 0.6399, P < 0.0001; r2 = 0.5445, P < 0.001; and r2 = 0.4956, P < 0.0001, respectively). These results indicate that SIRT1, SIRT3 and SIRT6 are closely related to ovarian reserve, and suggest that these sirtuins may be markers of ovarian aging.

Keywords: 4

ACCEPTED MANUSCRIPT Sirtuins Ovarian aging

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Ovarian reserve

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Chemotherapy

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Caloric restriction

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ACCEPTED MANUSCRIPT 1. Introduction

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The sirtuin (SIRT) family of nicotinamide adenine dinucleotide (NAD)-dependent

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deacetylases plays diverse roles in regulating metabolism, cell proliferation, and genome

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stability, and has been implicated in mammalian aging and age-related diseases, including cancers, metabolic disorders, and neurodegenerative diseases (Finley and Haigis, 2012;

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Firestein et al., 2008; Jiang et al., 2011; Kaplon et al., 2013; Sebastian et al., 2012). The first link between sirtuins and longevity was made16 years ago in yeasts. Many studies in diverse

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experimental models have been performed to elucidate the relationship between sirtuins,

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lifespan, and age-associated dysfunction. To date, 7 homologues (SIRT1-7) of the yeast SIR2

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gene have been identified in mammals. SIRT1 and SIRT6, which are mainly localized in cell nuclei, regulate the transcription of genes and DNA repair, while SIRT3 regulates

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mitochondrial bioenergetics. The anti-aging effects of mammalian homologues of the SIRT family (SIRT1, SIRT3, and SIRT6) are the most extensively studied members in relation to

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the effects of caloric restriction (CR) on longevity. A large body of evidence now indicates that these sirtuins suppress age-related dysfunction and increase longevity by enhancing the expression of SIRT1 or SIRT6 (Camins et al., 2010; Giblin et al., 2014; Park et al., 2013; Satoh et al., 2011). Overall, the available data point to important roles for sirtuins in promoting mammalian health, and perhaps in modulating the aging process. However, the anti-aging mechanism of sirtuins remains elusive. The ovarian aging process is parallel to that of somatic aging in that the underlying mechanisms have remarkable similarity to the mechanisms of general aging. The ovarian aging process is thought to be characterized by a gradual decrease in both the quantity and 6

ACCEPTED MANUSCRIPT quality of the oocytes in the ovarian cortex (te Velde and Pearson, 2002). Inevitably, menopause is the final step in the process of ovarian aging. The primordial follicle pool

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serves as the source of developing follicles in mammals, and reflects the lifespan of ovaries in

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mice, undergoing an inevitable decline during their lifetimes (Broekmans et al., 2007; Hansen et al., 2008; McGee and Hsueh, 2000). A decreased ovarian antral follicle count and primordial follicle pool suggest a lower ovarian reserve (Broekmans et al., 2009; Hansen,

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2013; Li et al., 2012).

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Several researchers have found that sirtuins play some role in ovarian function. Zhang et al. (2013) found that rapamycin preserves the follicle pool reserve and prolongs the ovarian

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lifespan of female rats via modulating mammalian target of rapamycin (mTOR) activation

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and sirtuin expression. SIRT1 activator (SRT1720) was found to improve follicle reserve and

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prolong the ovarian lifespan of diet-induced obese female mice by activating SIRT1 and suppressing mTOR signaling (Zhou et al., 2014). Fu et al. (2014) found that SIRT3 positively

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regulates the expression of folliculogenesis and luteinization-related genes, and progesterone secretion by manipulating oxidative stress in human luteinized granulosa cells. While it is known that sirtuins are closely related to mammalian longevity and aging, do they regulate ovarian aging? This study aimed to explore the relationship between ovarian reserve and the expression of SIRT1, SIRT3, and SIRT6 in the ovaries of mice with physiological ovarian aging and mice studied in 2 experimental models ‒ a cyclophosphamide (CTX)-induced premature ovarian failure (POF) model, and a CR-induced ovarian reserve protection model.

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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Mice and treatments

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C57BL/6 mice were used in these studies and were purchased from the Beijing

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HFK Bio-Technology Company (Beijing, PR China). All of the experimental procedures involving the animals were approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The mice received

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humane care according to the Guide for the Care and Use of Laboratory Animals of the

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Chinese Academy of Sciences. All mice were adult females aged either 6 weeks, 2 months, 8 months, or 1.5 years, and weighed 16-19 g. They were fed in a specific pathogen-free (SPF)

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house at controlled temperature (25°C) and light (12 hours light, 12 hours dark) conditions.

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For the POF model, 6 adult female mice aged 6 weeks were given a single intraperitoneal

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injection of 500 L phosphate-buffered saline (PBS) or an equal volume containing CTX 120 mg/kg (Sigma-Aldrich, CAS No. 6055-19-2). Ovaries were collected 7 days after CTX

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treatment. For the CR model, 6-week old adult female mice were randomly divided into 2 groups: a normal control (NC) group (fed ad libitum) and the 25% caloric restriction (CR) group (fed with 75% of the food of the NC group). Ovaries were harvested after 8 weeks of treatment. The mice aged 2 months, 8 months, and 1.5 years were at sexual maturity, reproductive aging, and ovarian aging stages, respectively (Godsen et al., 1983; Hansen et al., 2008; Li et al., 2014). All mice were sacrificed by cervical dislocation. Their ovaries were harvested, and the right-side ovaries were snap frozen and stored at 80°C for protein extraction, while left-side ovaries were fixed in 4% paraformaldehyde at 4°C for histological sectioning. 8

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2.2. Follicle counting

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Paraffin-embedded ovaries were longitudinally and serially sectioned (4 µm) and every

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fifth section was mounted on a glass slide. Routine hematoxylin and eosin (H&E) staining was performed for histological examination. The slides were analyzed under a microscope by 2 people who were blind to the origin of the sections. Only follicles containing an oocyte

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were counted to avoid any duplication. Follicles were classified as either: primordial follicles

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(oocyte surrounded by a single layer of squamous granulosa cells); primary follicles (intact enlarged oocyte with a visible nucleus and one layer of cuboidal granulosa cells); secondary

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follicles (2 or 3 layers of cuboidal granulosa cells without an antral space); or antral follicles

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(emerging antral spaces and atretic follicles, with apoptotic bodies of granulosa cells and

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fragmentation of the oocyte nucleus) [Myers et al., 2004].

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2.3. Western blot analysis

Ovaries were homogenized in a radio-immunoprecipitation assay (RIPA) with phenylmethanesulfonyl fluoride (PMSF) and a Teflon-glass homogenizer on ice. After centrifugation (12,000 rpm for 15 min at 4°C), the supernatants were collected for protein analysis. Protein concentrations were determined by the bicinchoninic acid (BCA) Protein Assay. For each sample, 40 μg of protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes (BioTrace™ NT, USA). Tris-buffered saline with 0.1% Tween 20 (TBST) buffer containing 5% non-fat dry milk was used to block non-specific binding at room temperature for 1 hour. The membranes were then incubated 9

ACCEPTED MANUSCRIPT with a primary antibody against SIRT1 (Abcam, UK, ab110304, 1:8000 dilution), SIRT3 (Abcam, UK, ab86671, 1:500 dilution), SIRT6 (Abcam, UK, ab135566, 1:100 dilution), or

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β-actin (Sigma, USA, 1:1000 dilution) overnight at 4°C, followed by incubation with a

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horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody (Sigma,USA,A0545, A6154, 1:3000 dilution) for 30 minutes at room temperature and washed three times with TBST. Bands were visualized by the ECL Plus Western Blotting Detection System

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(Amersham, USA). Band intensities were analyzed using Quantity One software (Bio-Rad

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Laboratories Pty Ltd, USA). β-Actin protein levels were used as a control to verify equal

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2.4. Statistical analysis

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protein loading.

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All results were expressed as means ± SEM and analyzed by the SPSS® 17.0 software. A paired Student's t test or one-way analysis of variance (ANOVA) was used to compare data

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between groups, with significance defined as P < 0.05. The relationship between ovarian expression levels of sirtuin proteins (SIRT1, SIRT3, and SIRT6) and the primordial follicle numbers of the mice was further analyzed using linear regression. Correlation coefficients (r) were tested for each SIRT.

3. Results 3.1. Increased expression levels of SIRT1, SIRT3, and SIRT6 proteins in the ovaries of mice whose ovarian reserve was protected by CR CR without malnutrition has been shown to delay aging and increase longevity in diverse 10

ACCEPTED MANUSCRIPT experimental models. Some researchers have also reported that CR increases ovarian reserve. The mean number of primordial follicles in the CR group was significantly greater than in the

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normal control (NC) group (112.5 ± 12.49 vs 85.0 ± 6.55, respectively; P < 0.05) [Fig. 1C],

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and the percentage of the total number of follicles that were primordial follicles was greater in the CR group (33.2% ± 0.9% vs 26.2% ± 0.4%, respectively) [ Fig. 4A]. However, the mean numbers of primary follicles (104.6 ± 9.7 vs 102.5 ± 11.4), secondary follicles (69.8 ±

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9.6 vs 73.0 ± 8.7), and antral follicles (19.5 ± 6.3 vs 12.5 ± 0.2) were not significantly

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different between the 2 groups. For atretic follicles, the mean number and percentage in the CR group were almost half those of the NC group (26.7 ± 1.1 vs 48.6 ± 5.1, respectively; and

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7.9% ± 0.4% vs 15% ± 0.7%, respectively). These results indicate that the CR increases

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ovarian reserve and reduces follicular atresia.

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Ovarian expression levels of SIRT1, SIRT3, and SIRT6 proteins were significantly higher in CR mice in comparison with NC mice (1.41-fold, 1.47-fold, and 1.40-fold higher,

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respectively) [Fig. 1E].

3.2. Decreased expression levels of SIRT1, SIRT3, and SIRT6 proteins in the ovaries of CTX-induced POF mice whose primordial follicles were greatly consumed In mice with poor ovarian reserve induced by CTX chemotherapy, the effects of CTX on follicular development and consumption were categorized, and the numbers of follicles at each stage were counted. There were significant decreases in the mean follicle numbers at each stage in the CTX-treated group in comparison with the control group: primordial follicles (22.5 ± 3.1 vs 86.3 ± 11.0, respectively; P < 0.001); primary follicles (26.2 ± 4.3 vs 11

ACCEPTED MANUSCRIPT 93.8 ± 5.9, respectively; P < 0.001); secondary follicles (29.4 ± 2.1 vs 76.0 ± 7.9, respectively; P < 0.001); and antral follicles (6.5 ± 1.4 vs 18.2 ± 5.1, respectively; P < 0.05).

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However, the mean number of atretic follicles was significantly increased in the CTX-treated

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group in comparison with the control group (75.2 ± 4.3 vs 46.5 ± 6.3, respectively; P < 0.001)[ Fig. 2C]. The percentage of the total number of follicles that were primordial follicles in the CTX-treated group was less than that of the control group (14.1% ± 0.7% vs 26.9% ±

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0.5%, respectively) [ Fig. 4A].

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Ovarian expression levels of SIRT1, SIRT3, and SIRT6 proteins were significantly lower in CTX-treated mice in comparison with the control group (0.11-fold, 0.74-fold, and

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0.51-fold lower, respectively) [Fig. 2E].

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3.3. Decreases in SIRT1, SIRT3 and SIRT6 protein expressions and ovarian reserve with

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As ovarian function is known to be diminished with chronological age, mice at 3 different stages were investigated to explore ovarian reserve and sirtuin protein expression levels. Mice aged 2 months, 8 months and 1.5 years were at sexual maturity, reproductive aging, and ovarian aging stages, respectively. The mean numbers of primordial follicles and the percentages of the total number of follicles that were primordial follicles were significantly decreased with aging (95.0 ± 8.1 vs 63.8 ± 4.2 vs 33.5 ± 6.3, respectively [Fig. 3D]; and 28.0% ± 0.7% vs 25.1% ± 0.7% vs 19.31% ± 0.8%, respectively) [Fig. 4A]. The mean number of primary follicles was also significantly diminished with age (102.1 ± 3.9 vs 73.2 ± 7.1 vs 53.8 ± 9.1, respectively, in the 3 age groups). While the mean number of 12

ACCEPTED MANUSCRIPT secondary follicles in the 8-month old group (50.5 ± 8.2) was also significantly decreased in comparison with 2-month old group (80.16 ± 10.44), there was no significant difference

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between the 8-month and 1.5-year old groups (50.5  8.2 vs 47.3 ± 7.9, respectively). In

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contrast, the mean number of atretic follicles was significantly increased in the 1.5-year old group in comparison with the 2-month and 8-month old groups, but there was no significant difference between the 2-month and 8-month old groups. Thus, with increasing age, ovarian

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reserve showed a clear downward trend, with the number of primordial follicles and

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growing follicles at each stage decreased, and the number of atretic follicles increased. Ovarian expression levels of SIRT1, SIRT3, and SIRT6 proteins were significantly

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decreased with chronological age. Compared with the 2-month old group, expression levels

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of ovarian SIRT1, SIRT3, and SIRT6 proteins were significantly lower in the 8-month old

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group (0.51-fold, 0.73-fold, and 0.54-fold lower, respectively) and in the 1.5-year old group

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(0.19-fold, 0.24-fold, and 0.36-fold lower, respectively) [Fig 3F].

3.4. Correlation of SIRT1, SIRT3, and SIRT6 protein expressions levels with the size of the primordial follicle pool The size of the primordial follicle pool represents the ovarian reserve. To elucidate the association of sirtuins with ovarian reserve, the relationship between primordial follicle numbers and expression levels of sirtuins was examined. Using linear regression analysis, SIRT1 (r2 = 0.6399, P < 0.0001), SIRT3 (r2 = 0.5445, P < 0.001), and SIRT6 (r2 = 0.4956, P < 0.0001) were all found to positively correlated with the number of primordial follicles (Fig. 4B,C,D). 13

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4. Discussion

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The ovary exhibits an accelerated rate of aging compared with that of other body systems, and ovarian aging is characterized by gradual declines in ovarian follicle quantity and quality. In mammals, the ovaries are endowed with a limited number of follicles after birth, and the

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follicle number decreases markedly with age as most of the follicles undergo atresia.

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The number of the primordial follicles represents the ovarian reserve. At birth, approximately 1 to 2 million primordial follicles remain in the human ovary and by the onset

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of puberty, at least 300,000 to 400,000 primordial follicles remain (Block 1953; te Velde and

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Pearson, 2002). During the reproductive years, the number of primordial follicles decreases at

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a steady rate of approximately 1000 follicles per month and drops below 1000 at menopause (Alviggi et al., 2009; Faddy et al., 1992; te Velde et al., 1998). Mice aged 13 to 14 months

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have only 10% of the ovarian follicles that were present at 4 to 5 months of age (Li et al., 1983). In agreement with previous studies (Hansen et al., 2008; Qu et al., 2000), we found that a gradual decline in the numbers of follicles, especially primordial follicles, occurred in aging mice. Currently available data suggest that CR without malnutrition is the only nongenetic and the most robust approach to delay aging and extend the maximum lifespan of diverse experimental models, including yeasts, worms, flies, mice, and nonhuman primates (Colman et al., 2009; Fontana et al., 2010; Mattison et al., 2012). CR-mediated activation of SIRT1, SIRT3, and SIRT6 may be the mechanism underlying the CR-induced aging delay and 14

ACCEPTED MANUSCRIPT longevity (Canto and Auwerx, 2009; Corbi et al., 2012; Guarente, 2013; Morris et al., 2011; Wang, 2014). CR has been suggested to increase ovarian reserve by some researchers

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(Maduro, 2012; Xiang et al., 2012). Our results support previous studies in showing that the

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mean number of primordial follicles after CR was significantly greater than that observed in the control group (112. 5 ± 12.49 vs 85.0 ± 6.55, respectively; P < 0.05). Chemotherapy is known to induce premature ovarian failure (POF) and the pool of

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resting primordial follicles and the number of growing follicles is decreased after

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chemotherapy (Clowse et al., 2011; Kalich-Philosoph et al., 2013; Marder et al., 2012). Current findings suggest that CTX-induced loss of ovarian reserve is due to acceleration of

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primordial follicle activation, which results in a ‘burnout’ effect and follicle depletion

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(Kalich-Philosoph et al., 2013). In the present study, a significant decline in follicles,

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especially primordial follicles, was observed in mice with CTX-induced POF, with the mean number around one-fourth of that observed in the control group (22.5  3.1 vs 86.3 ± 11.0,

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respectively).

Whereas our findings suggest that CR may benefit ovarian reserve in mice, chemotherapy and chronological age have the opposite effect, significantly diminishing the size of the primordial follicle pool. As indicated above, sirtuins have been implicated in mammalian aging and various age-related diseases, including cancers, metabolic disorders, and neurodegenerative diseases (Finley and Haigis, 2012; Firestein et al., 2008; Jiang et al., 2011; Kaplon et al., 2013; Sebastian et al., 2012). In the present study, expression levels of SIRT1, SIRT3, and SIRT6 proteins in ovaries were all up-regulated under CR conditions, but down-regulated after chemotherapy and with increasing age. We also found that SIRT1, 15

ACCEPTED MANUSCRIPT SIRT3, and SIRT6 were all positively correlated with the size of the primordial follicle pool. Thus, expression levels of SIRT1, SIRT3 and SIRT6 may represent ovarian reserve as well as

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anti-Müllerian hormone (AMH), antral follicle counts (AFC), and follicle-stimulating

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hormone (FSH) levels in the clinical assessment of ovarian function.

The mechanisms of the anti-aging and lifespan prolonging effect of sirtuins have been studied by many researchers. SIRT1, which is evolutionarily the closest protein to the yeast

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Sir2, has been found predominantly in cell nuclei. An early insight into the mechanism

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whereby Sir2 could increase the replicative lifespan of yeasts indicated that it inhibits ribosomal DNA (rDNA) recombination as well as extrachromosomal rDNA circle formation

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(Kennedy et al., 1997). SIRT1 regulates cell biology, metabolism, and fate at different levels

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through the deacetylation of histones and other cellular factors such as p53, nuclear

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factor-kappaB (NF-κB), heat shock transcription factor 1 (HSF1), Forkhead box class O transcription factors (FOXOs), and peroxisome proliferator-activated receptor-gamma

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coactivator-1 (PGC-1). Previous studies have demonstrated that under CR condition, increased endogenous SIRT1 down-regulates the expression of p53 (but not FOXO3a), reduces follicle apoptosis or atresia, and maintains the ovarian reserve (Xiang et al., 2012). The apoptosis signaling pathway is also involved in chemotherapy-induced ovarian damage in rats (Huang et al., 2009). Deacetylation of p53 by SIRT1 decreases p53-mediated apoptosis induced by DNA damage or oxidative stress (Luo et al., 2001; Vaziri et al., 2001). CTX can reduce SIRT1 expression and up-regulate the expression of p53, and it participates in the ovarian failure process (Xiang et al., 2012). SIRT1 activator (SRT1720) treatment may promote the ovarian lifespan of high-fat diet-induced obese female mice by suppressing the 16

ACCEPTED MANUSCRIPT activation of primordial follicles, follicle maturation, and atresia via activation of SIRT1 signaling and suppression of mTOR signaling (Zhou et al., 2014).

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SIRT3, which is found in the mitochondrial matrix, is directly linked to longevity and is

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highly expressed in long-lifespan individuals (Bellizzi et al., 2005). Functional studies have shown that SIRT3, as the major mitochondrial deacetylase, regulates mitochondrial biogenesis. Its targets, which include PGC-1α and uncoupling protein 1 (UCP1) have

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important roles in metabolic homeostasis (Kong et al., 2010; Shi et al., 2005). More recently,

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it has become apparent that SIRT3 influences oxidative stress defence by protecting cells from reactive oxygen species (ROS) through post-translational regulation of superoxide

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dismutase 2 (SOD2) via deactylation in response to oxidative stress (Chen et al., 2011). Older

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age has been found to be associated with increased ROS and decreased antioxidant levels in

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oocytes, cumulus cells, and follicular fluid (Hammadeh et al., 2008).CTX-induced cell and tissue injury also occurs through modulation of oxidative stress, while CR may slow

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accumulation of the oxidative damage that is thought to be one of the causes of aging (Bhattachharjee et al., 2015; He et ak., 2015). SIRT3 may also play a positive role in folliculogenesis and luteinization processes in granulosa cells, possibly by sensing and regulating the generation of ROS (Fu et al., 2014). A recent study has suggested that SIRT3 post-translational modification of mitochondrial enzymes in human granulosa and cumulus cells may regulate glutamate dehydrogenase (GDH) activity, which may be a causative factor in the decline of oocyte viability in women with reduced ovarian reserve and advanced maternal age (Pacella-Ince et al., 2014). SIRT6, a nuclear protein, is involved in genomic DNA stability and repair and is possibly 17

ACCEPTED MANUSCRIPT linked to cellular metabolism and aging. Through specific histone H3 lysine 56 (H3K56) deacetylase activity, SIRT6 is able to participate in the regulation of DNA stabilization and

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repair (Michishita et al., 2008; Yang et al., 2014). In addition, SIRT6 has been found to

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interact with NF-κB and to deacetylate histone H3 lysine 9 (H3K9) at NF-κB target gene promoters, leading to inactivation of many of the transcriptional programs observed in aged tissues (Kawahara et al., 2009). The injury effect of CTX also occurs through modulation of

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DNA damage, repair and stability (Bhattacharjee et al., 2015). CR may delay mitochondrial

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aging and attenuate the age-related phenotype by reducing ROS-induced mitochondrial DNA (mtDNA) dysfunction (Christiakov et al., 2014). Faulty DNA repair is suggested to be linked

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to ovarian aging in mice and humans (Couzin-Frankel, 2013). SIRT1 and SIRT6 may be

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involved in the mechanism by which CR inhibits the transition from primordial to developing

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follicles, extends the entire growth phase of a follicle to preserve the reserve of germ cells, and delays age-related ovarian aging (Luo et al. 2012).

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Overall, these findings suggest that SIRT1, SIRT3 and SIRT6 are key members of the sirtuin family that exert a wide range of actions in the regulation of cellular physiology and aging. However, their biological roles in the mouse ovary are still unclear, and need further exploration. Our results provide insight into the molecular functions of SIRT1, SIRT3, and SIRT6 in mouse ovaries under CR conditions, chronological aging, and chemotherapy-associated injury. The expression of these sirtuins was positively correlated with ovarian reserve, suggesting that they may be potential markers of ovarian aging. This may help provide pharmacological insight into delaying ovarian aging, and may lead to the development of target molecules to treat the serious problem of premature ovarian failure. 18

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Acknowledgements

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Editorial assistance with the manuscript was provided by Content Ed Net, Shanghai Co.

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Ltd.

Funding

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Funding for this study was received from the National Natural Science Foundation of

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China (No. 81370469; 81300453) and the Program of International Science and Technology

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Conflict of interest

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Corporation of China (No. 2013DFA31400).

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The authors declare no conflicts of interest.

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Authors’ contributions

Shixuan Wang and Aiyue Luo designed study, revised drafts and reviewed the final manuscript. Jinjin Zhang wrote the first draft of the manuscript. Li Fang, Meng Wu, Liangyan Shi were involved in the acquisition of data. Zhiyong Lu and Jiaqiang Xiong were involved in analysis and interpretation of the data.

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Fig. 1. Expression levels of SIRT1, SIRT3, and SIRT6 proteins in the ovaries, and follicle

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numbers at each stage in caloric restricted (CR) mice. (A) Representative histological images

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of the ovaries of CR mice and normal control (NC) group mice. (B) The CR group exhibited more primordial follicles as shown by the red arrows. (C, D) The mean number of follicles at

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each stage in the NC group and the CR group. (E) Expression levels of SIRT1, SIRT3, and SIRT6 proteins in the ovaries of mice in the CR and NC groups tested by western blot.

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β-Actin was used as an internal control. Data are expressed as means ± SEM. *indicates a

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significant difference from the NC group (*P < 0.05; **P < 0.01). Scale bars, 50 m.

Fig. 2. Expression levels of SIRT1, SIRT3, and SIRT6 proteins in the ovaries, and follicle

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numbers at each stage in mice with premature ovarian failure (POF) induced by cyclophosphamide (CTX) chemotherapy. (A, B) Representative histological images of the

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ovaries of CTX-treated and control group mice. (C, D) The number of follicles at each stage in the CTX-treated group and the control (Con) group. (E) Expression levels of SIRT1, SIRT3, and SIRT6 proteins in the ovaries of mice in the CTX-treated group and the control (Con) group tested by western blot. *indicates a significant difference from the control group (*P < 0.05; **P < 0.01). Scale bars, 50 m.

Fig. 3. Expression levels of SIRT1, SIRT3, and SIRT6 proteins in the ovaries, and follicle numbers at each stage in mice aged 2 months, 8 months and 1.5 years. (A, B, C) Representative histological images of ovaries at different ages. (D, E) Mean numbers of 29

ACCEPTED MANUSCRIPT ovarian follicles at each stage in the 3 age groups. (F) Expression levels of SIRT1, SIRT3, and SIRT6 proteins in the ovaries of mice in the different age groups. *indicates a significant

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difference from the 2-month group or the 8-month old group (*P < 0.05; **P < 0.01; ***P <

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0.001). Scale bars, 50 m.

Fig. 4. Relationship between the expressions levels of sirtuins and primordial follicle

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numbers in mouse ovaries. (A) The proportion of the total number of follicles that were

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primordial follicles in the ovaries of mice in the various study groups. (B, C, D) Relationship between the expression levels of SIRT1, SIRT3, and SIRT6 proteins and the size of the

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< 0.0001, respectively).

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primordial follicle pool (r2 = 0.6399, P < 0.0001; r2 = 0.5445, P < 0.0001; and r2 = 0.0.4956, P

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