Yu-Cai Fu

Obesity accelerates ovarian follicle development and follicle loss in rats

OBJECTIVEnStudies have shown that excess body fat negatively affects reproductive functions in females. However, whether obesity affects the ovarian follicle development and ovarian lifespan and the underlying mechanism has not been well elucidated. The aim of the present study was to investigate the association between obesity and ovarian follicle development.nnnMETHODSnAdult female Sprague-Dawley rats (n = 36) were randomly divided into three groups: the normal control (NC) group, the caloric restriction (CR) group (fed 70% food of the NC group) and the high-fat diet (HF) group. They were maintained on these regimens for 18 weeks.nnnRESULTSnThe body weight, ovary weight and visceral fat in the HF group were significantly higher than those in the NC group and the CR group at the end of treatment. Histological analysis showed that the HF rats had significantly less number and percentage of primordial follicles, but greater number and percentage of developing and atretic follicles than the NC rats and CR rats. Western blot analysis demonstrated that the level of mTORC1 and p-S6K1 proteins significantly increased in the ovaries of HF rats, whereas that of SIRT1, SIRT6, FOXO3a and NRF-1 decreased compared to the NC rats. In contrast, the expression of mTORC1 and p-S6K1 dramatically declined, while that of SIRT1, SIRT6, FOXO3a and NRF1 increased in the ovaries of CR rats.nnnCONCLUSIONSnOur study suggests that the HF diet induced obesity may accelerate the ovarian follicle development and rate of follicle loss through activating mTOR and suppressing SIRT1 signaling, thus leading to POF, and that CR may inhibit the activation of primordial follicles, follicular development and loss, thus extending the ovarian lifespan through suppressing mTOR and activating SIRT1 signaling.

Rapamycin preserves the follicle pool reserve and prolongs the ovarian lifespan of female rats via modulating mTOR activation and sirtuin expression

To maintain the normal length of female reproductive life, the majority of primordial follicles must be maintained in a quiescent state for later use. In this study, we aimed to study the effects of rapamycin on primordial follicle development and investigate the role of mTOR and sirtuin signaling. Rats were treated every other day with an intraperitoneal injection of rapamycin (5mg/kg) or vehicle. After 10weeks of treatment, ovaries were harvested for hematoxylin and eosin (HE) staining, and analysis by immunohistochemistry and Western blotting. HE staining showed that the number and percentage of primordial follicles in the rapamycin-treated group were twice the control group (P<0.001). Immunohistochemical analysis showed that mTOR and phosphorylated-p70S6K were extensively expressed in surviving follicles with strong staining observed in the cytoplasm of the oocyte. Western blotting showed decreased expression of phosphorylated mTOR and phosphorylated p70S6K in the rapamycin-treated group, and increased the expression of both SIRT1 and SIRT6 compared to the control group (P<0.05). Taken together, these results suggest that rapamycin may inhibit the transition from primordial to developing follicles and preserve the follicle pool reserve, thus extending the ovarian lifespan of female rats via the modulation of mTOR and sirtuin signalings.

The effects of caloric restriction and a high-fat diet on ovarian lifespan and the expression of SIRT1 and SIRT6 proteins in rats

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.

SIRT1 activator (SRT1720) improves the follicle reserve and prolongs the ovarian lifespan of diet-induced obesity in female mice via activating SIRT1 and suppressing mTOR signaling

BackgroundThe prevalence of obesity is increasing worldwide and significantly affects fertility and reproduction in both men and women. Our recent study has shown that excess body fat accelerates ovarian follicle development and follicle loss in rats. The aim of the present study is to explore the effect of SIRT1 activator SRT1720 on the reserve of ovarian follicle pool and ovarian lifespan of obese mice and the underlying mechanism associated with SIRT1 and mTOR signaling.MethodsAdult female Kunming mice (n = 36) were randomly divided into three groups: the normal control (NC) group (n = 8), the caloric restriction (CR) group (fed 70% food of the NC group, n = 8) and the high-fat diet (HF) group (fed a rodent chow containing 20% fat, n = 20). After 4 months, the HF mice were further randomly divided into three groups: the control high-fat diet (CHF, n = 8) group (treated every day with an intraperitoneal injection of vehicle), the SRT1720 (SRT, n = 6) group (treated every other day with an intraperitoneal injection of SRT1720 (50 mg/kg)), the SRT1720 and nicotinamide (NAM, n = 6) group (treated every other day with an intraperitoneal injection of SRT1720 (50 mg/kg) and every day with an intraperitoneal injection of nicotinamide (100 mg/kg)). After 6 weeks of treatment, ovaries were harvested for histological and Western blotting analyses.ResultsThe body weight, ovary weight and visceral fat in the SRT group were significantly lower than those in the CHF group at the end of treatment. Histological analysis showed that the SRT mice had significantly greater number and percentage of primordial follicles, but lower number and percentage of corpora lutea and atretic follicles than the CHF mice and NAM mice. Western blot analysis demonstrated that the levels of SIRT1, SIRT6, FOXO3a and NRF-1 protein expression significantly increased in the ovaries of SRT mice, whereas those of mTORC1, p-mTOR, p-p70S6K, NFκB and p53 decreased compared to the CHF and NAM mice.ConclusionsOur study suggests that SRT1720 may improve the follicle pool reserve in HF diet-induced obese female mice via activating SIRT1 signaling and suppressing mTOR signaling, thus extending the ovarian lifespan.

Resveratrol, an effective regulator of ovarian development and oocyte apoptosis

Resveratrol, a phytopolyphenol compound found chiefly in grapes and wine, has been reported to have a variety of anti-inflammatory, anti-platelet, and anti-carcinogenic effects. However, little is known about the effects of resveratrol on ovarian development and oocyte apoptosis. We investigated the effects of resveratrol on ovarian development in rats with different ages [from post-natal day (PD) 1 to 15 months], as well as on oocyte apoptosis in PD1 and PD2 rat ovaries. We show that: a) ip injection of resveratrol (20 mg/kg/day) increased the percentage of unassembled follicles and the total number of oocytes in PD1 and PD2 rat ovaries. Similar results were obtained when mothers were treated with resveratrol (20 mg/kg/day) by intragastric administration from day 11, after the detection of vaginal plug, until delivery. In PD4 rat ovaries, the total number of oocytes was significantly increased in the groups treated with resveratrol. Moreover, more unassembled follicles and fewer primary follicles were present in the groups treated with resveratrol than in the controls; b) in 15-month-old rat ovaries, resveratrol increased the number of resting follicles and total oocytes, and decreased the number of developing follicles and atretic follicles; 3) the percentage of TUNEL-positive oocytes decreased in PD1 and PD2 rat ovaries after resveratrol treatment, and the number of oocytes positive for Foxo3a, Bim, and p27KIP1 in PD2 rat ovaries was lower in the resveratrol treatment group than in controls. These results suggest that resveratrol may delay oocyte nest breakdown and inhibit both the primordial-to-developing-follicle transition and apoptosis by decreasing the activation of Foxo3a, Bim, and p27KIP1, thus augmenting the resting follicle reserves, maintaining regular estrous cycles of early aged rats and delaying climacterium.

Caloric restriction promotes the reserve of follicle pool in adult female rats by inhibiting the activation of mammalian target of rapamycin signaling.

Caloric restriction (CR) is known to increase the number of primordial follicles and prolong the reproductive life span. However, how CR modulates follicular development is not well understood. In the present study, we examined the effects of CR on follicular development in rats and investigated the underlying mechanism. After 10 weeks of CR or high-fat diet, ovarian follicles at different developmental stages were examined by histological analysis. Plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estrogen (ESG) were measured, and the levels of mammalian target of rapamycin (mTOR), p70S6 kinase (p70S6K), and phosphorylated p70S6K in the ovary were detected by Western blot. The results showed that the reserve of follicle pool in CR rats was increased, accompanied by decreased level of phosphorylated p70S6K in the ovary, and decreased serum LH, FSH, and ESG levels. Taken together, these results suggest that CR may suppress ovarian follicular development and enhance the follicle pool reserve by inhibiting mTOR signaling.

Calorie restriction increases primordial follicle reserve in mature female chemotherapy-treated rats.

UNLABELLEDnWe assessed the effects of calorie restriction (CR)-mediated protection against chemotherapy damage on ovarian reserve. Forty-eight female Sprague-Dawley rats were randomly divided into four groups: the normal control group (NC group, fed ad libitum), the CR group (fed with 65% food intake of the NC group), the CTX group (injected with cyclophosphamide (CTX) and fed ad libitum), and the CR+CTX group (injected with CTX and fed with 65% food intake of the NC group). Ovarian reserve was examined by vaginal smears and follicle counting. SIRT1 is a deacetylase that is activated by a variety of stressors and targets transcriptional regulators including p53, NF-κB, FOXO1, 3, and 4, and the transcriptional regulator PGC-1α. The expression level of SIRT1, p53 and FOXO3a in the ovary was measured by western blot. CR did not interfere with estrous cycling but maintained estrous cycling in CTX-treated CR rats. The number of primordial follicles in the CR rats was comparable to the NC group, and CR+CTX group rats had more primordial follicles and primary follicles than the CTX group. SIRT1 expression in the ovary was higher in the CR group compared to the control group, and p53 level was lower in the CR group than that in the NC group. There is no significant difference in the expression level of FOXO3a between the CR group and the NC group.nnnCONCLUSIONSnThese results indicate that CR can increase the ovarian follicular reserve and reduce the CTX-induced ovarian damage, and CR positive effects may be due to its intervention in the transition from primordial to primary follicle, and its reduction of oxidative stress.

Caloric restriction promotes the reproductive capacity of female rats via modulating the level of insulin-like growth factor-1 (IGF-1).

The insulin-like growth factor-1 (IGF-1) plays an important role in the regulation of reproductive function. In the present study, we examined the effects of caloric restriction (CR) on the reproductive lifespan in rats and investigated the potential role of IGF-1. After 10 weeks of treatment, we determined the distribution of the ovarian follicles at various stages and measured the plasma level of IGF-1, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estrogen (ESG). Our results show that IGF-1 level was decreased after CR and correlated with the decrease in the levels of LH, FSH and ESG. Moreover, a higher percentage of primordial follicles and surviving follicles was observed in CR rats than in control rats (P<0.05). Immunohistochemical analysis showed that IGF-1 was extensively expressed in the cytoplasm of granulosa cells in the surviving follicles at different stages but not in the atretic follicles. Taken together, these results suggest that caloric restriction promotes the reproductive capacity of female rats via modulating the level of IGF-1, which then regulate pituitary gonadotrope cells to reduce the release of LH, FSH and ESG, and modulate follicular development.

Effects of tea polyphenols on ovarian development in rats

Tea is the most consumed beverage in the world. Tea polyphenols are the major constituents of tea leaves and have shown many potential healthy benefits. However, whether tea polyphenols influence ovarian follicle assembly and development and ovarian life span is unknown. To study the effect of tea polyphenols on ovarian follicle development and oocyte apoptosis, we investigated rat ovarian development of different ages [from postnatal day (PD)1 after birth to 10 months] after treatment with tea polyphenols. Our data showed that the percentage of unassembled follicles increased in the ovaries of 1 — and 2-day-old rats which were ip injected with tea polyphenols (50 mg/kg/d) or whose mothers were treated with tea polyphenols (100 mg/kg/d) by intragastric administration from the day 11 after the detection of vaginal plug till delivery. The percentage of primordial follicles increased, while that of developing follicles decreased in the ovaries of 4- and 8-day-old rats following peritoneal injection with tea polyphenols compared with controls. The ratio of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)-positive oocytes decreased in the ovaries of neonatal rats following tea polyphenol treatment. In the 3- and 10-month-old rat ovaries, the number of primordial follicles augmented, whereas that of atretic follicles decreased after the treatment for 4 weeks. These data suggest that tea polyphenols may inhibit the transition from primordial to developing follicles, extend the entire growth phase of a follicle, and reduce dominant follicle numbers per cycle to increase the reserve of germ cells, inhibit oocyte apoptosis and follicle atresia during ovarian development from birth to early aged, and retard climacterium in rats. Drinking even a small cup of tea per day may benefit to extent the productive life of ovary.

Rapamycin prolongs female reproductive lifespan

The pool of ovarian primordial follicles is established during embryonic development in humans, or after birth in rodents, and serves as the source of developing follicles and fertilizable ova for the entire length of female reproductive life.1 Once the pool of primordial follicles has been exhausted, menopause occurs in women around 50 y old. With modern increases in longevity, more than one-third of a woman’s life is now spent after menopause, which is characterized by a loss of fertility and increased risk for cardiovascular disease, osteoporosis, and cognitive dysfunction. To maintain the normal length of female reproductive life, the majority of primordial follicles must be maintained in a quiescent state for later use. Therefore, the activation of primordial follicles is a critical process for female reproductive lifespan, but the mechanism is poorly understood. n nAlthough it has been well established that the development of primordial follicles is regulated by locally produced autocrine, endocrine, and paracrine signals, exactly how the primordial follicle integrates these signals to maintain dormant or to be activated to develop is unclear. However, recent studies have revealed that mammalian target of rapamycin (mTOR) pathway, which serves to integrate such signals, involves the control of primordial follicle activation and development.2,3 mTOR is a ubiquitous, evolutionarily conserved serine/threonine kinase that regulates cell growth and proliferation in response to stress, nutrients, and growth factors. mTOR functions as part of 2 multiprotein complexes, mTOR complex 1 (mTORC1) and mTORC2.4 Rapamycin inhibits mTORC1 by binding the FK506-binding protein FKBP12, which then interacts physically with the complex and decreases activity. The upstream of mTORC1, the TSC1/TSC2 protein complex, suppresses the activation of mTORC1 through a GTPase-activating protein domain located in TSC2. The major downstream effectors of mTOR known so far are S6K1 and 4E-BP1, both regulators of protein translation. Deletion of Tsc1 or Tsc2 in mouse oocytes caused premature activation of all primordial follicles around the time of puberty, and eventually led to premature ovarian failure (POF) in early adulthood. Inhibition of mTOR signaling by rapamycin effectively reversed the overactivation of primordial follicles in mutant mouse ovaries.2,3 These findings indicate that mTOR signaling is involved in controling the activation of primordial follicles. Furthermore, rapamycin could suppress mouse granulosa cell proliferation and follicle development in vitro, and resulted in reduced numbers of ovulated eggs in vivo.5,6 n nBased on these data, we speculate that inhibition of mTOR signaling by rapamycin may suppress the activation of ovarian primordial follicles in adult mammals, and thus preserve the follicle pool reserve and prolong the female reproductive lifespan. In our current study, adult female rats fed a standard diet ad libitum were treated every other day with an intraperitoneal injection of rapamycin (5 mg/kg) for 10 weeks. Our results showed that inhibition of mTOR signaling by rapamycin resulted in a 2-fold increase in the number of primordial follicles in the rapamycin-treated rats compared with the control rats, indicating that the activation of primordial follicle was suppressed. Furthermore, the numbers of antral and atretic follicles and corpora lutea were significantly decreased, suggesting the inhibitory effects also on the development of follicles at different stages to maturation and atresia. These data indicate that rapamycin can effectively suppress the activation of ovarian primordial follicles in adult animals via inhibition of mTOR signaling, thus preserving the follicle pool reserve and prolonging the female reproductive lifespan. n nHowever, some of the normal reproductive functions were also suppressed. The rapamycin-treated rats had irregular estrous cycles and failed to become impregnated during the mating trial. This could be explained by the fact that there were not sufficient antral follicles and corpora lutea in the rapamycin-treated rats to produce a certain amount of estrogen and progestogen to maintain these normal functions. Moreover, Yu et al. demonstrated that rapamycin inhibited ovulation in mice.6 n nIn addition, it is worth noting that rapamycin induced a decrease of food intake by ~33.9%. The rapamycin-treated rats were lean and smaller than the control rats, although their calorie intake per gram of body weight was comparable to that of the control rats at the end of treatment. Meanwhile, the protein expression of SIRT1 and SIRT6 was significantly increased in the rapamycin-treated rat ovaries. Sirtuins are key regulators in the mechanism by which CR extends lifespan of various organisms from yeast to mammals.7 The upregulated expression of SIRT1 and SIRT6 and increased follicle pool reserve were also found in CR rats in our previous study.8 How rapamycin induces the upregulation of SIRT signaling is unclear. Since rapamycin-treated rats have a similar phenotype to CR rats, we hypothesize that rapamycin may induce a CR condition, and thus upregulate SIRT signaling, or inhibition of mTOR signaling by rapamycin may lead to the activation of SIRT signaling via unknown pathways, thus mimicing CR effects. This warrants further studies. n nIn summary, our data indicate that rapamycin treatment has beneficial effects on the reserve of ovarian follicle pool and female reproductive lifespan in animals. We optimistically consider that rapamycin or its derivatives could be used as an effective drug for preventing POF and delaying the onset of menopause in obese or even healthy women in the future.