Introduction
Female reproductive aging is a complex process that extends from fetal life to menopause, marked by reduced ovarian function1,2. The primary change is the decline in the ovarian reserve of primordial follicles with advancing age2,3, accompanied by a decrease in oocyte quality due to cumulative DNA damage and exposure to environmental stressors4. Ovarian aging is associated with an increased rate of spontaneous abortions and embryonic abnormalities, negatively impacting fertility in older women5,6. In mice, there is also a progressive reduction in the number of primordial follicles with age, which is more evident between six and nine months of age3. From 10 months onward, the ovarian reserve becomes severely reduced, with significant accumulation of DNA damage4, resulting in a reduction of fertility and offspring survival5. The depletion of ovarian reserve with age also affects reproductive cyclicity, leading to irregular cycles and cessation of estrous in mice7,8. In women, hormonal and menstrual cycle changes also occur, culminating in menopause2,9.
Caloric restriction (CR) is known for increasing longevity in several species, from invertebrates to primates10,11,12,13. CR promotes lifespan extension due to improved metabolic health14, reduction of inflammatory status, and modulation of cellular stress response15,16,17,18. Additionally, CR increases insulin sensitivity19 and reduces adipose tissue depots20. CR is associated with a lower incidence of DNA damage, as CR improves cellular repair mechanisms21,22,23. Interestingly, CR induces different metabolic adaptations depending on the age at which intervention is started24. The positive effects of CR also quickly disappear once mice return to ad libitum feeding25,26,27. This suggests that the timing and duration of CR are crucial factors to the successful implementation of this strategy.
Beyond its impacts on longevity and metabolic health, CR influences ovarian aging. In mice, 30% CR can reduce activation of primordial follicles, thereby preserving the ovarian reserve28,29,30,31. The age of start and duration of CR seems to affect ovarian aging; however, no previous studies have compared it directly. For example, CR initiated at one month of age and maintained for three months resulted in the strongest ovarian reserve preservation29. A milder effect was observed when CR was started at two months of age and maintained for six months30, or initiated at three months of age and maintained for four months28. While mice maintained with ad libitum feeding drastically reduce fertility after 10 months of age, those subjected to CR and later fed ad libitum remain fertile beyond 20 months of age5. In this regard, ad libitum feeding after CR is crucial for evaluating the fertility, as CR mice cease estrous cycles during the intervention28 and exhibit a reduced ovulation rate30. Additionally, CR mimetics such as rapamycin can mitigate oocyte DNA damage32, reinforcing the potential of these strategies in fertility preservation. Ovarian aging is also accompanied by a persistent inflammatory microenvironment, characterized by immune cell infiltration, epigenetic alterations, and increased oxidative stress33,34. This results in fibroblast activation and excessive collagen deposition, ultimately leading to structural and functional impairment of the ovary35,36. Based on this evidence, we aimed to test the effect of long- and short-term CR in mice at different stages of the reproductive lifespan on ovarian aging.
Results
In this study, mice were divided into four groups to receive food ad libitum (AL), long-term 30% CR (from 3 to 11 months; CR/CR), early short-term 30% CR (from 3 to 7 months; CR/AL), and late short-term 30% CR (from 7 to 11 months; AL/CR) (Fig. 1). Both long (CR/CR) and short-term (CR/AL and AL/CR) CR groups had reduced body mass (Fig. 2A). However, the CR/AL group regained lost body mass upon returning to ad libitum feeding (Fig. 2A). Throughout the experimental period, the CR/CR group exhibited an average body mass reduction of 5.0%, while the AL group increased body mass in 16.7% (Fig. 2B). Most of the body mass loss in the CR/CR group occurred during the initial phase of CR, up to 7 months of age, while the AL/CR group experienced a more pronounced reduction after CR start from 7 to 11 months-of-age. Mice subjected to CR reduced intrabdominal adipose tissue mass (Fig. 2C), whereas mice returned to ad libitum feeding (CR/AL) increased adipose tissue mass to levels similar to the AL group. Although the AL/CR group lost body mass in the 7-to-11-month period, they maintained levels of abdominal fat similar to AL mice. Therefore, long-term CR resulted in the strongest reduction in intrabdominal fat, as short-term CR resulted in intrabdominal fat levels similar to the AL mice at 11 months of age, independent of the age it started. The CR/CR group exhibited a greater glucose decay rate than AL mice at 11 months of age (Fig. 2D). The CR/AL group lost the CR-induced improvement in insulin sensitivity upon resuming ad libitum feeding (Fig. 2D). Insulin sensitivity was also similar between AL/CR and AL mice, indicating both short term CR were not as effective in improving insulin sensitivity in 11-month-old mice. The glucose response curves during the ITT are shown in Supplementary Fig. 1. Therefore, the reversal of body mass change observed in the CR/AL group, along with increased intrabdominal adipose tissue and reduced insulin sensitivity, suggests that discontinuing CR may attenuate the long-term benefits of CR in female mice.
Mice in the ad libitum (AL) and calorie restriction (CR) groups were euthanized at seven and 11 months of age for sample collection (n = 6–8/group). A group of mice (n = 5) was euthanized at three months of age as controls. All mice were fed a standard chow for rodents and water ad libitum. For the CR group, a 30% food restriction was imposed, based on the average intake during the previous week for the control group of the same age.
Body mass variation throughout the experiment (A), change in body mass from 3 to 7 months and from 7 to 11 months of age (B), percentage of intra-abdominal adipose tissue relative to total body mass (C), and glucose decay rate during the insulin tolerance test (D). The ad libitum (AL) group had unrestricted access to food throughout the experiment; the continuous calorie restriction group (CR/CR) was subjected to CR from the beginning; the CR/AL group returned to ad libitum feeding after a period under CR; and the AL/CR group began CR at a later stage. Data are presented as mean ± standard error of the mean. Statistical analyses for panels B, C, and D were performed using one-way ANOVA followed by the Tukey post hoc test. The P-value presented in each panel indicates the overall ANOVA model P-value. Between-group differences at the P < 0.05 level are represented by different letters.
The preservation of ovarian reserve in the long-term CR was evidenced by an increased number of primordial follicles in the CR/CR group (Fig. 3A). The number of primordial follicles was not different between the short-term CR (CR/AL and AL/CR) and the AL groups, indicating short-term CR is not as effective as the long-term CR. A pronounced decline in the number of primordial follicles was observed from three to seven and from seven to 11 months in AL mice; however, it remained almost unchanged from seven to 11 months of age in the CR/CR mice. No differences were found in the numbers of transition and primary follicles (Fig. 3B and 3C). There was an age-related decline in tertiary follicle numbers in the AL group, which was not observed in the CR/CR group (Fig. 3E). Numbers of tertiary follicles were severely compromised in the AL/CR compared to CR/CR mice (Fig. 3E). Overall, CR groups tended to have a higher total number of follicles compared to the AL groups (P = 0.01).
Primordial (A), transition (B), primary (C), secondary (D), tertiary (E), and total (F) follicles per section. The ad libitum (AL) group had free access to food throughout the experiment, the continuous calorie restriction (CR/CR) group underwent continuous CR, the CR/AL group returned to ad libitum feeding after a period of CR, and the AL/CR group started CR at a later stage. The dotted line represents the average follicles/section observed for three-month-old control mice. Data are presented as mean ± standard error of the mean. Statistical analyses were performed using one-way ANOVA followed by the Tukey post hoc test. The P-value presented in each panel indicates the overall ANOVA model P-value. Between-group differences at the P < 0.05 level are represented by different letters. In panel F, no statistical significance at P < 0.05 was observed and P-values are shown to illustrate the trends (0.05 > P < 0.10) observed between groups.
Lower collagen deposition was observed in the CR/CR group compared to the AL group at 7 months of age (Fig. 4A). However, collagen deposition was not different between CR groups at 11 months of age when compared to AL mice. Macrophage infiltration was reduced in the CR/CR mice at 7 and 11 months compared to AL mice (Fig. 4B). The CR/AL group also had lower macrophage infiltration compared to AL mice, which was not observed in the AL/CR group, suggesting an early start of CR is beneficial for preventing ovarian inflammation. There were no differences in ovarian DNA damage levels, as indicated by γH2AX staining (Fig. 4C).
Ovarian collagen deposition (A), macrophage infiltration (B) and DNA damage (C) in mice. Panels show representative images of collagen (A1-A6), macrophages (B1-B6, green for CD68 and blue for DAPI), and H2AX (C1-C6, green for H2AX and blue for DAPI) staining in ovarian sections. The ad libitum (AL) group had ad libitum access to food throughout the experiment, the calorie restriction (CR)/CR group underwent continuous CR, the CR/AL group was returned to ad libitum feeding after a period of CR, and the AL/CR group started CR at a later stage. Data are presented as mean ± standard error. Statistical analyses were performed using one-way ANOVA followed by the Tukey post hoc test. The P-value presented in each panel indicates the overal ANOVA model P-value. Between-group differences at the P < 0.05 level are represented by different letters.
Mice subjected to long term CR remained in diestrus for the majority of the evaluated period, which was reversed by returning to AL feeding (Fig. 5A). The number of estrous was also reduced in CR/CR mice and returned to the AL group levels after returning to AL feeding (Fig. 5B). This indicates full restoration of ovarian cycles upon return to AL feeding.
Female cyclicity throughout the experiment, as observed through A percentage of time spent in each estrous cycle stage, and B number of estrous in the 12-day interval evaluated. The ad libitum (AL) group had ad libitum access to food throughout the experiment, the calorie restriction (CR)/CR group underwent continuous CR, and the CR/AL group was returned to ad libitum feeding after a period of CR. Data is presented as mean ± standard error of the mean. Statistical analyses were performed using one-way ANOVA followed by the Tukey post hoc test. The P-value presented in panel B indicates the overall ANOVA model P-value. Between-group differences at the P < 0.05 level are represented by different letters.
Discussion
In this study, we demonstrated that both long and short-term CR influence metabolic and ovarian aging parameters in female mice, with distinct effects depending on the duration and timing of the intervention. Long-term CR resulted in significant preservation of the primordial follicle pool. However, short-term CR, regardless of the age of onset, did not result in primordial follicle pool preservation. Additionally, changes in collagen deposition and macrophage infiltration indicate that remodeling of the ovarian microenvironment may represent one of the mechanisms through which CR modulates reproductive aging, which are dependent on the age of onset of CR. These findings highlight that the benefits of CR on ovarian reserve can be retained even if its metabolic effects are rapidly lost following the return to ad libitum feeding.
CR-induced weight loss leads to a reduction in adipose tissue, resulting in favorable metabolic adaptations37. Since CR modulates adipose tissue, it plays a key role in lowering inflammation38, thereby improving insulin sensitivity24. We observed that CR reduced body mass gain and adipose tissue depots, which was reflected in nearly a twofold increase in glucose clearance rate in the CR/CR compared to AL mice. Notably, mice subjected to long-term CR exhibited adipose tissue mass and glucose decay rate at 11 months of age similar to 7-month-old mice. This indicates that CR can reduce aging-related metabolic impairment even in relatively young female mice. The reversal of CR adaptations upon returning to ad libitum feeding (CR/AL) reinforces the notion that the metabolic benefits of CR depend on the continuity of the intervention26,27. The rapid increase of body and adipose tissue mass after returning to ad libitum feeding, accompanied by decreased insulin sensitivity, indicates a rebound effect likely mediated by adaptive mechanisms developed during CR10,12. Therefore, our data further reinforces the notion that CR only confers metabolic benefits when applied continuously, even in young and middle-aged female mice. Although the late start CR (AL/CR) promoted body mass loss, it did not change abdominal fat mass and insulin sensitivity compared to AL mice. This suggests that both short-term CR regimens were not as effective as long-term CR in promoting beneficial metabolic adaptations.
In the reproductive context, we observed that mice on long-term CR preserved the primordial follicle pool compared to AL mice. The short-term CR/AL and AL/CR did not affect the ovarian reserve compared to AL mice, regardless of the age of CR onset. CR has been shown to preserve the ovarian reserve at the 10%30 and 30% restriction levels5,20,28,29,30. We highlight that in the CR/CR mice, the primordial follicle reserve remained almost unchanged between seven and 11 months of age, indicating that CR during this period was able to attenuate the decline in ovarian reserve typically observed3. This positive effect may be related to the improved metabolic parameters induced by CR in the long-term, such as decreased fat mas and improved insulin sensitivity. Insulin resistance, often associated with hypercaloric diets and aging, may contribute to ovarian reserve decline, as treatments that improve insulin sensitivity, such as metformin, can preserve the ovarian reserve39. The reduction in adipose tissue in CR mice can also contribute to the positive effects of CR, as previous studies observed that obese rats can have accelerated depletion of the ovarian reserve40. Furthermore, other mechanisms may be involved in the preservation of ovarian reserve by CR, such as inhibition of the ovarian mTOR signaling pathway31. Regarding the short-term CR/AL and AL/CR groups, the number of primordial follicles was not different from the AL mice. This is corroborated by the observation that both short-term CR regimens affected intrabdominal adipose tissue mass and insulin sensitivity compared to AL mice. Our findings can have translational implications, as a later age at menopause is associated with greater longevity in women41. In mouse, transplantation of young ovaries to old mice can increase longevity42. Therefore, future studies should focus on the benefits of CR ovaries in enhancing longevity in females.
We observed an increased number of tertiary follicles in long-term CR/CR mice, likely reflecting the preservation of the ovarian reserve and greater availability of follicles for recruitment and maturation. In addition, CR may reduce tertiary follicle atresia through modulation of metabolic pathways such as AMPK, sirtuins31, leptin, and insulin43. We observed a significant decline in tertiary follicles between three and 11 months in AL mice, and the late CR start could not preserve the tertiary follicle population. This suggests that CR may impair the development of antral follicles. Despite the clear preservation of the ovarian reserve in the prolonged CR group, these mice did not display regular estrous cycles and remained acyclic for most of the period evaluated. This highlights a critical aspect of reproductive longevity promoted by CR, while follicle populations are preserved, estrous cycles and ovulation are impaired. This dysfunction is likely due to central inhibition of the reproductive axis, as chronic energy deficiency reduces hypothalamic GnRH pulsatility, mediated by hormonal signals such as decreased leptin and IGF-1, and increased NPY and ghrelin44. These neuroendocrine changes are well-established mechanisms through which CR suppresses ovulation, even in the presence of mature follicles. Therefore, our findings are consistent with the notion that CR-induced reproductive suppression is a centrally mediated adaptive response to perceived energetic stress. Even with a high number of tertiary follicles, the absence of regular estrous cycles indicates that ovulatory function is impaired45. In contrast, females returned to AL feeding (CR/AL) and resumed cyclicity similarly to AL-fed mice. In the context of assisted reproduction, women undergoing in vitro fertilization (IVF) may benefit from strategies that preserve ovarian reserve even at the cost of temporary suppression of cyclicity46. However, for those seeking natural conception, regular ovulation is needed47. Therefore, our findings suggest that dietary interventions such as CR should be carefully tailored according to reproductive goals and further investigated in clinical studies in reproductive-age women aiming for conception.
In addition to preserving the ovarian reserve, we observed that CR has relevant effects on the ovarian microenvironment, particularly concerning fibrosis and inflammation. Macrophage infiltration was reduced in the CR/CR group at both seven and 11 months, as well as in the CR/AL group, compared to AL mice. However, this effect was not observed in the AL/CR group, indicating that late onset CR may not be enough to reverse the established inflammatory environment. The CR/CR group had lower collagen deposition at 7 months compared to 7 and 11-month-old AL mice. This suggests that early-onset CR may delay extracellular matrix accumulation associated with ovarian aging. However, at 11 months, collagen deposition levels in CR/CR, AL/CR, and CR/AL groups were similar to those observed in AL-fed animals at 7 and 11 months. These findings suggest that CR’s ability to attenuate fibrosis is more evident at earlier stages of reproductive life. Ovarian fibrosis and inflammation are key features of reproductive aging and are associated with follicular dysfunction, decreased ovulation rates, and premature ovarian failure36,48. Evidence suggests that the cumulative number of ovulations over a lifetime contributes to extensive tissue remodeling, promoting progressive collagen deposition and immune cell infiltration in the stroma49. In this context, our findings support the hypothesis that the maintenance of estrous cycles under AL feeding contributes to the progression of inflammatory and fibrotic processes, whereas CR, particularly when initiated at an early age, better preserves the ovarian tissue environment. The reduction in inflammation observed in the CR/AL group, but not in the AL/CR group, further supports the idea that the age of exposure to CR is a critical factor for beneficial effects on the local immune response. Additionally, there is an age-related increase in multinucleated giant cells (MNGC) in the ovary50. These cells are evident in mouse ovaries already at 9 and 12 months of age and express high levels of CD68 similar to macrophages50. Therefore, our findings may also indicate this increased age-related accumulation of CD68+ ovarian MNGC, which are hallmarks of ovarian aging50. Finally, we did not observe differences in DNA damage levels, assessed by γH2AX immunolocalization. We previously also did not detect any changes in γH2AX in young mice subjected to CR30. It is possible that, at this relatively young age, genomic damage remains minimal and that structural and inflammatory changes precede alterations related to DNA integrity.
While this study presents interesting findings, several limitations should be acknowledged. First, our use of a single mouse strain and a relatively small sample size per group may limit the generalizability of our results and further statistically significant discoveries. Furthermore, this study did not evaluate the mechanistic pathways underlying the effects of CR on primordial follicle activation. Future investigations should therefore evaluate key signaling pathways, such as mTOR/FOXO3a, in the ovaries of CR mice. Additionally, while our cyclicity analysis suggests reduced ovulation, direct quantification is necessary in future studies comparing the CR and AL groups. Finally, the observed changes in tertiary follicle numbers warrant further investigation into markers of atresia and apoptosis to determine the fate of these follicles in CR mice.
Together, our findings demonstrate that CR reduces several hallmarks of ovarian aging, especially when initiated at an early age and maintained over the long term. Primordial follicle preservation was evident in the long-term CR initiated at a younger age. In contrast, the available data did not support a protective effect of short-term CR on ovarian reserve. Changes in collagen deposition and macrophage infiltration suggest that remodeling of the ovarian microenvironment may represent one of the mechanisms through which CR modulates reproductive aging, which are dependent on the age of onset of CR. Despite beneficial effects, CR impaired cyclicity and ovulation, suggesting a trade-off associated with reproductive longevity.
Methods
Animals and interventions
This study was approved by the Ethics Committee in Animal Experimentation of the Universidade Federal de Pelotas (number 033054/2022-23). Virgin female C57BL/6 mice at three (n = 34) and seven (n = 20) months of age were maintained under controlled conditions (22 ± 2 °C, cycles of 12 h light/12 h dark and 40–60% humidity). Mice were housed in groups of three mice per cage and randomly allocated into four groups: control group receiving food ad libitum (AL) (n = 13), 30% CR from 3 to 11 months of age (CR/CR) (n = 9), 30% CR from 3 to 7 months of age (CR/AL) (n = 8) and 30% CR from 7 to 11 months of age (AL/CR) (n = 10). A group of mice (n = 5) was euthanized at 3 months of age as controls. Mice in the AL and CR/CR groups were also euthanized at 7 and 11 months of age for sample collection (n = 6–8/group). All mice were fed a standard chow for rodents (Nuvilab® CR-1; 5% fat and 23% protein) and water ad libitum. For the CR group, a 30% food restriction was imposed, adjusted based on the average intake during the previous week for the control group of the same age. CR females started with a 10% CR, 20% CR in the next week, and from the third week forward, a 30% CR. The food intake and body mass data were collected weekly during the experiment.
To synchronize the estrous cycle stage, mice were administered an i.p. injection of 5 IU of equine chorionic gonadotropin (eCG), followed 48 hours later by an i.p. injection of 5 IU of human chorionic gonadotropin (hCG). Twelve hours after the hCG injection, mice were anesthetized with isoflurane and submitted to cardiac puncture exsanguination, followed by cervical dislocation after a 12-hour fasting. Mice were dissected, and the ovaries were collected in 4% buffered formaldehyde solution for histological analysis. During dissection, intra-abdominal fat was removed and weighed.
Insulin tolerance test
The insulin tolerance test (ITT) was performed a week before euthanasia in mice aged seven and 11 months of age (n = 5/group). For this, insulin at 0.5 IU/kg of body weight was administered i.p. after a 12-hour fasting period, ensuring that AL and CR mice had the same interval from their last meal. Blood was collected through a small incision at the tip of the tail at 0, 15, 30, and 60 min after insulin injection, and glucose levels were measured with a glucometer (AccuChek Activ, Roche Diagnostics®, USA). The glucose decay rate (KITT) was calculated by converting glucose values to percentage, considering the glucose level at 0 min as 100% for each mouse, then applying the formula (T0 – T15)/15. KITT was expressed as a percentage of glucose decay per minute.
Vaginal cytology
For cytological analysis, vaginal cells were collected with a micropipette using saline solution for 12 consecutive days at 11 months of age in AL, CR/CR, and CR/AL mice. The material collected was smeared on a slide and stained with fast panoptic (Laborclin, Pinhais, PR, Brazil) for visualization under a microscope (Nikon Eclipse E200, Nikon Corporation, Japan). The stages of the estrous cycle were defined by the cell types present in the slide as previously described51. Estrous cycle duration was defined from the interval between two consecutive estrous.
Histological analysis
The ovaries were removed from the 4% buffered formaldehyde, dehydrated in alcohol, cleared in xylol, and embedded in paraffin. The paraffin-embedded ovaries were sequentially cut into a 5-μm thickness in a semi-automatic microtome (RM2245, Leica Biosystems Newcastle Ltd., Newcastle Upon Tyne, UK). All ovaries were serially cut, and one of every six sections was selected and placed on a standard histological slide. Intermediate sections were collected for immunofluorescence and picrosirius red staining analyses. The slides, after drying at 56 °C for 24 h, were stained with hematoxylin-eosin and mounted with coverslips and synthetic resin (Entellan®, Sigma-Aldrich, St. Louis, MO, USA). Images of the ovarian sections were captured by a digital camera (Moticam 5.0, Motic®, Hong Kong, China) coupled to a microscope (Nikon Eclipse E200, Nikon, Tokyo, Japan). Only follicles with a clearly visible oocyte nucleus were counted. The final number of follicles is divided by the number of sections counted to establish the average number of follicles per section. Follicle classification was based on previously established protocols52.
Picrosirius red staining
Collagen analysis was performed using the Picrosirius Red (PSR) dye, which is specific for collagen fibers type I and III36. After preparing the PSR solution, slides were deparaffinized by immersion in decreasing concentrations of xylene and ethyl alcohol and stained with the PSR dye. Slides were immersed in a PSR staining solution prepared by dissolving Sirius Red F3BA (Direct Red 80, Sigma-Aldrich, St. Louis, MO, USA) in a saturated aqueous solution of picric acid (Sigma-Aldrich, St. Louis, MO, USA) at 0.1% w/v. All slides were processed simultaneously to minimize variation in staining intensity by the automated sample preparation system (ST5020, Leica Biosystems, Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, UK). After drying, the slides were mounted with a coverslip in 40% TBS glycerol. Images of the ovarian sections were captured by a digital camera (Moticam 5.0 Motic®, Hong Kong, China) coupled to a microscope (Nikon Eclipse E200, Nikon Corporation, Japan), using a 10× objective and quantified using the Image J Software (NIH, Bethesda, MD, USA)36,53.
Immunofluorescence
For immunofluorescence analysis, ovarian samples were deparaffinized with xylene and rehydrated with graded alcohols. The primary monoclonal anti-γH2AX phospho S139 antibody (#9718, Cell Signaling, Danvers, MA, USA) was used at a final dilution of 1:500 and the anti-CD68 antibody (ab125212, Abcam, Cambridge, UK) was used at 1:400 dilution. Antigen retrieval was performed in humid heat for 3 minutes after reaching the boiling point in a citrate solution at pH 6.0. Tissue sections were blocked with BSA and goat serum. The slides were incubated overnight in a humid chamber at 4°C with the primary antibody, followed by 1-hour incubation with the secondary antibody Alexa Fluor® 488 (ab150077, Abcam, Cambridge, UK) and 3 minutes with DAPI (Invitrogen, Carlsbad, USA). The slides were mounted with a drop of mounting medium (Fluoroshield, Sigma-Aldrich, St. Louis, USA). Follicle images were captured using a confocal laser scanning microscope (Leica TCS SP8, Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, UK). ImageJ® software (NIH, Bethesda, MD, USA) was used to quantify γH2AX fluorescence intensity as the ratio of stained area to section area54, and CD68 as the number of positive cells per area55. Measurements were averaged from three sections per mouse (n = 4–7 mice/group).
Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0 software. The one-way ANOVA statistical test was used for analysis of continuous variables (body mass gain, adipose tissue mass, follicle count, and KITT) with the Tukey post-hoc test to compare individual means. The repeated measures ANOVA test was used for the analysis of body mass, and glucose levels during ITT, also using the Tukey post-hoc test to compare individual means. P values lower than 0.05 were considered significant, and between 0.05 and 0.10 as a trend. The results are presented as mean ± standard error of the mean.
Data availability
Data will be available upon request from the corresponding author.
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Acknowledgements
Authors are thankful for the funding provided by Coordenação de Aperfeiçamento de Pessoal de Nivel Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Fundação de Amparo a Pesquisa no Estado do Rio Grande do Sul (FAPERGS). This project has been made possible in part by grant number 1023 from the Global Consortium for Reproductive Longevity & Equality (GCRLE).
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Prosczek, J.B., Hense, J.D., Garcia, D.N. et al. Timing and duration of calorie restriction determine its impact on ovarian aging in female mice. npj Aging 11, 87 (2025). https://doi.org/10.1038/s41514-025-00276-3
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DOI: https://doi.org/10.1038/s41514-025-00276-3