This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University Bundang Hospital (IACUC number BA-2108-325-081-01). All animal handling and treatment procedures adhered strictly to the institutional protocols established by the IACUC.

Six-week-old female bromodomain factor 1 (BDF-1) mice (Orient Bio.) were maintained in an environment with a controlled temperature of 22 ℃ and a 12-hour light/dark cycle, with free access to food and water [23,24]. Observations regarding changes in weight, appearance, and behavior were conducted two to three times daily. Euthanasia was performed if a mouse’s body weight dropped by more than 20% or if the animal demonstrated no response or movement when stimulated. Euthanasia was consistently conducted via cervical dislocation.

After a 1-week acclimatization period, 120 female BDF-1 mice were randomly assigned to one of four treatment groups (30 mice per group): (1) The Cp group received 75 mg/kg of Cp (Cp monohydrate, Cat. no. 29875, Sigma-Aldrich); (2) Everolimus (LC Laboratories) was administered at 0.75 mg/kg; (3) Rapamycin (LC Laboratories) was given at 4.0 mg/kg.

The experimental design is depicted in Table 1: (1) In the control group, normal saline (0.1 mL) was injected on days 1, 3, 5, and 13; (2) In the Cp group, normal saline (0.1 mL) was injected on days 1, 3, and 5, followed by Cp on day 13; (3) In the everolimus+Cp group, everolimus was injected on days 1, 3, and 5, with Cp on day 13; (4) In the rapamycin+Cp group, rapamycin was administered on days 1, 3, and 5, followed by Cp on day 13.

On day 20, all mice were euthanized, and their ovaries were harvested. A portion of each ovary was processed for histological examination, while the remainder was used to assess the protein expression levels of B-cell lymphoma-extra large (BCL-xL; an anti-apoptotic marker), cleaved caspase-3 (an apoptotic marker), and mTOR via Western blot analysis, using α-tubulin as an internal control.

Histological assessment was conducted according to methodologies reported in a prior study [25]. Ovarian tissue was fixed with 4% buffered paraformaldehyde for 1 day and embedded in paraffin. Embedded samples were serially sectioned at 4-µm thickness. Slides were stained with hematoxylin and eosin (Merck) and analyzed for follicle counting. Every section was examined under a light microscope at least twice by a single experienced technician (B.Y. Choi), who was blinded to the treatment groups to minimize observer bias. The average follicle count across sections was recorded. Only follicles with a clearly visible nucleus in the oocyte were counted.

Each follicle type was classified into the following categories [26]: Primordial: a single layer of flattened pre-granulosa cells; Primary: a single layer of granulosa cells, 1 or more being cuboidal cells; Secondary: two or more layers of cuboidal granulosa cells, with the antrum absent; Antral: multiple layers of cuboidal granulosa cells, with the antrum present.

Follicle quality was assessed based on integrity according to previously described criteria [27]: G1 (good quality) follicle: an intact spherical follicle and oocyte; G2 (fair quality) follicle: granulosa cells pulled away from the edge of follicles, but with an intact oocyte; G3 (poor quality) follicle: disruption and/or loss of granulosa-theca cells, with pyknotic nuclei and/or a missing oocyte.

Representative histological images of ovarian follicles are shown in Figure 1.

Western blot analysis involved collecting ovarian samples from five mice per group, with each sample run in technical duplicates across three independent experiments, yielding six measurements per marker per sample. Ovaries were rinsed with phosphate-buffered saline and lysed using cell lysis buffer (20 mM Tris-HCl at pH 8.0, 137 mM NaCl, 1% Nonidet P-40, and 10% glycerol), supplemented with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 0.025 mM N-CBZ-L-phenylalanine chloromethyl ketone, 0.025 mM N-p-tosyl-lysine chloromethyl ketone, and 0.025 mM L-1-tosylamide-2-phenyl-ethylchloromethyl ketone) for 20 minutes on ice. After centrifugation at 10,000 ×g for 15 minutes at 4 ℃, supernatants were collected, and protein concentrations were measured using a bicinchoninic acid protein assay kit (Thermo Scientific Pierce). Samples (20 µg/µL) were resolved and transferred onto polyvinylidene difluoride membranes (Millipore). Membranes were blocked and incubated overnight at 4 ℃ with primary antibodies: BCL-xL (1:100, sc-271121; Santa Cruz Biotechnology), cleaved caspase-3 (1:500, 5a1e; Cell Signaling Technology), and mTOR (1:500, orb99435; Biorbyt Ltd.). BCL-xL serves as an anti-apoptotic marker, whereas caspase-3 indicates late apoptosis. After washing three times for 15 minutes with tris-buffered saline with Tween (TBST), membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for one hour at room temperature. Following additional TBST washes, membranes were developed using ECL Prime Western Blotting detection reagent (GE Healthcare).

Representative bands from the Western blot analysis are shown in Figure 2.

Statistical analyses were performed using R software version 2.14.2 (R Foundation for Statistical Computing). Ovarian follicle counts were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s multiple-comparison test. Normality of data distribution was verified before performing ANOVA. Protein expression levels were compared using the Kruskal-Wallis test followed by the Mann-Whitney U test. A p-value of less than 0.05 was considered statistically significant.

Detailed counts of ovarian follicles across the four groups are presented in Table 2. The median number of primordial follicles was significantly lower in the Cp group compared to the control group (8 vs. 22, p=0.001). Similarly, the everolimus+Cp group (11.5 vs. 22, p=0.001) and the rapamycin+Cp group (8.5 vs. 22, p=0.003) also showed significantly lower numbers of primordial follicles than the control group. No significant differences in primordial follicle counts were observed among the Cp group, everolimus+Cp group, and rapamycin+Cp group. When evaluating primary, secondary, and antral follicle counts across the four groups, no significant differences were noted.

Figure 3 illustrates the proportions of G1 follicles within the four groups. There were no significant differences in the proportions of G1 primordial or G1 secondary follicles among the four groups.

The Cp group and the everolimus+Cp group had significantly lower proportions of G1 primary follicles than the control group. Conversely, the proportion of G1 primary follicles in the rapamycin+Cp group was preserved at levels comparable to the control group. The proportions of G1 antral follicles were significantly lower in the Cp group, everolimus+Cp group, and rapamycin+Cp group than in the control group.

To summarize the findings regarding ovarian follicle subtype counts and proportions of G1 follicles, pre-treatment with either everolimus or rapamycin did not preserve the number of primordial follicles. However, pre-treatment with rapamycin preserved the number of G1 primary follicles.

The expression levels of BCL-xL, cleaved caspase-3, and mTOR in the four groups are displayed in Figure 4. No significant differences in the expression levels of these proteins (BCL-xL, cleaved caspase-3, and mTOR) were identified among the four groups.

Pre-treatment with rapamycin prior to Cp administration was effective in preserving the proportion of G1 primary follicles. However, based on the findings of the current study, it is difficult to conclude that rapamycin has a sufficient fertoprotective effect in attenuating Cp-induced follicular damage. Specifically, our data suggest that premedication with either everolimus or rapamycin before Cp administration did not effectively preserve either the number of primordial follicles or the proportion of G1 primordial follicles.

Recent theories, particularly the ‘burn-out theory,’ provide explanations for the ovotoxic effects of chemotherapy. Agents like Cp may initiate the growth of dormant follicles, accelerating the depletion of the follicle reserve. Although this ‘burn-out theory’ has garnered significant support, the mechanisms underlying ovarian follicle depletion and their associations with infertility and early menopause remain incompletely understood. In our study, Cp treatment reduced primordial follicle numbers without impacting counts of primary, secondary, or antral follicles, consistent with our previous research [28]. This finding suggests that Cp specifically targets primordial follicles through mechanisms inducing apoptotic cell death rather than by activating or destroying growing follicles.

However, follicle activation is not the sole factor in chemotherapy-induced ovarian damage. Chemotherapy may also trigger apoptosis or cause oxidative stress. Our understanding of the mechanisms responsible for chemotherapy-induced ovarian damage remains incomplete. Further research is necessary to understand why ovarian function impairment is more severe in some women than others.

The specific dosage of Cp employed to induce ovarian dysfunction in mice varies depending on study objectives. Common dosages to induce such effects typically range from 75 to 150 mg/kg. A 75 mg/kg dosage is considered standard, whereas 150 mg/kg is frequently categorized as a sterilizing dose [21]. Our previous study demonstrated that administering Cp at 50 mg/kg did not reduce primordial follicle counts, whereas 75 mg/kg significantly decreased these counts [28]. Therefore, we selected 75 mg/kg as the lowest effective dose for the current experiment.

Everolimus, marketed as Certican (Novartis Pharma AG), is primarily indicated for preventing organ rejection after transplantation. Tablet formulations include dosages of 0.25, 0.5, 0.75, and 1.0 mg. Specific mouse dosages vary according to study objectives.

In a previous study [21], mice receiving Cp at 75 mg/kg weekly for 3 weeks were concurrently treated with everolimus orally at 2.5 mg/kg daily. This combined treatment doubled primordial follicle counts compared to mice treated solely with Cp. Everolimus effectively preserved ovarian reserves, serum AMH levels, and fertility outcomes. In ovariectomized mice, intraperitoneal injections of everolimus at 1 mg/kg/day for 4 weeks prevented bone loss [29]. In a breast cancer study, a daily oral dose of 0.75 mg/kg was used [30].

In this study, we administered everolimus at 0.75 mg/kg on days 1, 3, and 5, with Cp injected on day 13, aiming to achieve protective effects using a minimal dosage. However, the dose and duration may have been insufficient. Future studies should explore daily dosing of everolimus at 1 to 2.5 mg/kg over extended periods (e.g., 2 to 4 weeks) or co-administration with Cp to improve follicular protection.

In a previous study [31], rapamycin at 5 mg/kg administered intraperitoneally daily for 19 days effectively prevented follicular activation and preserved ovarian reserves in mice with phosphatase and tensin homolog deleted on chromosome ten (PTEN) mutations. In another study [22], mice received daily intraperitoneal injections of rapamycin at various doses (5, 10, or 20 mg/kg) for either 15 or 30 days. A dose of 5 mg/kg significantly affected folliculogenesis. Rapamycin dosages used in lifespan extension studies for mice ranged from 2 to 4 mg/kg [32,33].

We selected a rapamycin dosage of 4 mg/kg, injected on days 1, 3, and 5, followed by Cp administration on day 13. It should be noted that the shorter duration and lower dosage might have been inadequate to achieve the desired protective effects. Future studies should consider daily dosing of rapamycin at 4 to 5 mg/kg, potentially co-administered with Cp, to assess whether prolonged exposure enhances protection. Dose-response studies and pharmacokinetic analyses could further elucidate optimal regimens.

Ovarian expression levels of proteins, including BCL-xL (an anti-apoptotic marker), caspase-3 (an apoptotic marker), and mTOR, were comparable across all four groups. The similar BCL-xL levels between control and Cp groups align with our previous findings [28], suggesting that BCL-xL may not play a prominent role in Cp-induced apoptosis.

Caspase-3, a primary downstream effector enzyme during late apoptosis, was previously reported to increase following Cp administration [28,34]; however, our study did not detect increased caspase-3 expression in the Cp group. This discrepancy could result from differences in experimental conditions, such as dosing, treatment duration, and analytical methods. Additionally, apoptosis is tightly regulated, and caspase-3 activation occurs specifically at the terminal phase. Our study might have captured a time point where caspase-3 activation was either not yet apparent or had already subsided. Further investigations, including time-course studies or additional apoptosis markers, could clarify the dynamics of caspase-3 activation.

Despite expectations that pre-treatment with mTOR inhibitors such as everolimus or rapamycin would increase ovarian mTOR levels, no changes in mTOR protein expression were observed after treatment, consistent with previous research [35]. In our study, Cp-treated mice exhibited increased phosphorylation of mTOR without changes in total mTOR levels.

Several limitations should be acknowledged. We utilized a single fixed dose of everolimus and rapamycin over a limited duration. Future studies might benefit from co-administering mTOR inhibitors simultaneously with anticancer drugs, rather than solely as pre-treatment. Evaluating different dosages and durations of everolimus and rapamycin could offer deeper insights. Furthermore, the limited number of markers evaluated constrained the assessment of underlying drug mechanisms. Evaluating phosphorylation of proteins within the PI3K/AKT/mTOR pathway would enable a deeper understanding of our findings.