The effects of caloric restriction and a high-fat diet on ovarian lifespan and the expression of SIRT 1 and SIRT 6 proteins in rats Aging Clinical and Experimental Research

Background and aims: Caloric restriction (CR) extends mammals’ lifespans and suppresses ovary development. Sirtuins are involved in these mechanisms. If, and to what extent CR affects ovarian lifespan and follicle development is largely unknown. We investigated the effects of moderate and severe caloric restriction compared with a high-fat dietary regimen on ovarian follicle reserves in rats. Methods: Female Sprague-Dawley rats (n=48) randomly divided into four groups including normal control (NC), 25% caloric restriction (MCR), 45% CR (SCR) and high-fat diet (HF) were maintained on these regimens for 2 months. Results: Histological analysis showed that both the 25 and 45% CR rats had a significantly higher percentage of primordial follicles and a larger number of healthy follicles than the NC rats, whereas the HF rats did not differ significantly from the NC rats. Immunohistochemical analysis revealed that SIRT1 and SIRT6 proteins were present in the nucleus and cytoplasm of the oocytes. The 25% CR diet increased the expression of both SIRT1 and SIRT6 in the ovary, whereas the 45% CR and HF diets caused a decrease in SIRT1 expression. The level of SIRT6 protein did not change with the 45% CR diet, and it appeared slightly lower in the HF than in the NC groups. Conclusions: Caloric restriction may inhibit the transition from primordial to developing follicles and extend the entire growth phase of a follicle to preserve the reserve of germ cells. SIRT1 and SIRT6 are both associated with these effects. (Aging Clin Exp Res 2012; 24: 125-133) ©2012, Editrice Kurtis INTRODUCTION Aging is a complex biological phenomenon characterized by a gradual and progressive loss of function in diverse organs and tissues. Increased life expectancy leads to increased age-associated health issues in both sexes. Women, who generally live longer than men, will spend nearly a third of their lifetime in menopause. Furthermore, concomitant with the primary cause for the gradual depletion of oocytes, the quality of the remaining ones in females generally declines with advancing age, leading to consequences that include osteoporosis, obesity, sexual dysfunction, depression, anxiety, a higher incidence of Alzheimer’s disease and cardiovascular disease (1, 2). One of the best measures to slow aging and maintain health and function in animals is caloric restriction (CR) (3). Restricting the caloric intake of laboratory rodents markedly delays the age-related loss of many physiologic functions, including fertility in females (4). Retarding growth and adolescent maturity is a high price to pay for delaying the reproductive senescence of CR according to previous reports. Mice subjected to adult-onset CR maintained the function of the female reproductive axis into an advanced age without stunting overall growth or delaying the onset of normal sexual maturation (5). However, the mechanisms by which CR may delay reproductive aging remain to be characterized. Mice over-expressing SIRT1 exhibit some physiological properties including a delay in reproduction similar to those of mice in a CR regimen (6). Another mammalian sirtuin, which was recently implicated in the regulation of aging, is SIRT6. SIRT6-deficient mice are small, and by 23 weeks of age they develop abnormalities usually associated with aging (7). SIRT1 and SIRT6 proteins are known to be associated with longevity and are involved in repairing and controlling DNA and the response to oxidative stress. Whether SIRT1 and SIRT6 are involved in the mechanisms of caloric restriction in prolonging reproductive lifespan is unknown. The aims of our study were to determine if different levels of caloric restriction affect the ovarian follicle reserve and development, and if and in what way SIRT1 and SIRT6 are involved in how caloric restriction influences the reproductive lifespan. MATERIAL AND METHODS Animals and regimens Forty-eight female 8-week-old Sprague-Dawley rats with an average body weight of 200 g±18 g were purchased from the Animal Center of Shantou University Medical College. Body weights were recorded once a week before and during the trial. After a 14-day acclimation period, the rats were randomly divided into four groups: 1) normal controls (NC) fed ad libitum with standard rodent chow containing 4.84% fat, 7.34% fiber, 20% protein, plus all necessary vitamins and minerals; 2) 25% CR group fed 75% of the food consumed by the NC group; 3) 45% CR group fed 55% of the food consumed by the NC group; 4) high-fat group (HF) fed a high-fat diet containing 10% lard (wt/wt), 3% yolk (wt/wt), 1% cholesterol (wt/wt), 0.5% sodium cholate (wt/wt) and 85.5% standard rodent chow (8). This diet had a gross energy (GE) content of 17.3 MJ/kg. We recorded daily food intakes, and the food supply of the CR groups was adjusted accordingly. The NC group and HF group were continuously supplied with an excess of food. The difference in weight of food remaining in the cage hopper at successive weighing was taken as an index of food consumption. Animals were maintained on the prescribed diets for 8 weeks. All animal protocols were approved by the Institutional Animal Care and Use Committee of Shantou University Medical College. Blood samples were collected via the tail veins every three weeks. Sera were immediately isolated from blood samples by centrifugation and then stored at -20°C until analysis for cholesterol and triglycerides by an automatic biochemical analyzer (SYNCHRON LX20 Beckman Coulter). All the rats were sacrificed while they were under nembutal anesthesia. Various organs were isolated and weighed. Assessment of estrous cycles Vaginal smears were performed between 8:00 and 9:00 every morning beginning 2 weeks before caloric restriction began until the end of the study to assess the estrous cycle phase. Vaginal secretion was collected with a sterile cotton swab moistened with normal saline (NaCl, 0.9%), which was then placed on a glass slide. Unstained materials were observed under a light microscope to analyze the proportion of the 3 major cell types (epithelial cells, cornified cells, and leukocytes) (9). In this study, the appearance of cornified cells was used for the determination of estrous cycle patterns. The length of a cycle was determined as the number of consecutive days from the day of one cornified cell phase to the day of the next cornified cell phase. In this study, a 4to 5-day estrous cycle was determined to be a regular cycle, and a cycle duration of >5 days or <4 days was considered to be an irregular cycle (10). Rats displaying at least 4 consecutive regular cycles per month were defined as regular cycling rats, whereas those displaying constant estrus, intermittent regular and intermittent irregular estrous cycles were defined as irregular cycling rats. Preparation of ovary sections Two ovaries from each rat were isolated and trimmed free of fat and adhering tissues. One was kept at -80°C for Western blot analysis, and the other was fixed in 4% paraformaldehyde at 4°C overnight. Tissue sections of 6 μm were prepared for staining with hematoxylin and eosin (HE) and immunohistochemistry. Follicular classification Follicles were classified as described elsewhere (11) and as follows: primordial (an oocyte surrounded by 1 layer of flattened somatic cells), primary (an oocyte surrounded by 1 layer of cuboidal granulosa cells), secondary (2 or 3 layers of cuboidal granulosa cells with no antral space), preantral (3 or 4 layers of granulosa cells with 1 or more small independent antral spaces), and antral follicle (more than 4 layers of granulosa cells and an especially large clearly defined antral space). Follicles were classified as either healthy (intact basal lamina, oocyte with no more than 3 cytoplasmic vacuoles, intact germinal vesicle and nucleolus) or atretic (apoptotic). Antral follicles were considered atretic if they contained at least 20 apoptotic granulosa cells (defined by the appearance of apoptotic bodies in the granulosa cell layer), disorganized granulosa cells, a degenerating oocyte, or a fragmentation of the oocyte nucleus (12). The presence or absence of quiescent (primordial), developing (primary and secondary) and mature (antral) follicles, and the corpora lutea were checked on at least 7 sections per ovary, with each observed section separated by a distance of over 100 μm. Immunohistochemistry Ovarian sections were deparaffinized in xylene, hydrated through a series of ethanol and blocked with 2% bovine serum albumin (BSA). Tissue sections were incubated with primary antibody against SIRT1 (Santa Cruz, USA) or SIRT6 (Abcam, UK) (1:25 or 1:100 diluted in PBS) overnight at 4°C and with a biotinylated anti-rabbit IgG (Jackson Immuno Research, USA) for 1 h at room L.-L. Luo, X.-C. Chen, Y.-C. Fu et al. 126 Aging Clin Exp Res, Vol. 24, No. 2