C57BL/6 male and female mice were obtained from Jackson Laboratories. Mice aged 4 to 6 weeks were housed in the mouse facility at the Institute of Microbial Technology (IMTECH). For CR disruption, female mice were initially placed under a 12-h light/dark (L/D) cycle. After 1 week of activity recording in the LD cycle, the mice were placed in constant darkness for 4–6 weeks to induce CR disruption. Experimental mice were 6–8 weeks old at the time of use. A total of 132 female mice were included in the study. Mice were euthanized by cervical dislocation without anesthesia. All experimental procedures were authorized by the Institutional Animal Ethics Committee and conducted in accordance with national regulatory guidelines (No. 55/1999/CPCSEA), Ministry of Environment and Forests, Government of India.

For fertility evaluation, control female and CR-disrupted female mice, with or without SR9011 treatment, were paired with normal male mice (monogamous mating). CR disruption and SR9011 administration were applied both during and after CR disruption to evaluate their prophylactic and therapeutic effects on fertility. Mating behavior was observed under a normal 12-h L/D cycle. Breeding data were generated following the method described by Handelsman et al. (24), with modifications detailed in the Materials and methods section. Pup counts and body weight measurements were recorded. Breeding data were analyzed using group-specific endpoints reflecting the cessation of breeding activity for each group. This approach was chosen to account for natural differences in reproductive performance between groups under their respective conditions.

Total RNA was extracted from the ovaries using the TRIzol method (Ambion, Invitrogen, Massachusetts, USA). Using 1 µg of RNA, complementary DNA (cDNA) was synthesized with the Verso cDNA Synthesis Kit (Thermo Fisher Scientific, Massachusetts, USA) following the manufacturer’s protocol. cDNA amplification was performed using the Dynamo ColorFlash SYBR Green Kit (Thermo Fisher Scientific). Relative fold change was calculated using the 2^−ΔΔCt method.

The primer sequences used for qRT-PCR are as follows:

Per2 Forward: 5′-CAGGCTGAGTTCCCTAGTCG-3′, Reverse: 5′-TGTGCAGTCCAGACCAGAAG-3′; Cry1 Forward: 5′-GTGGATCAGCTGGGAAGAAG-3′, Reverse: 5′-CACAGGGCAGTAGCAGTGAA-3′; Fshr Forward: 5′-TGATGTTTTCCAGGGAGCCT-3′, Reverse: 5′-CTGGCCTCAATGAGCATGAC-3′; Star Forward: 5′-TTGGGCATACTCAACAACCA-3′, Reverse: 5′-GAAACACCTTGCCCACATCT-3′; Amh Forward: 5′-GGGAGACTGGAGAACAGCAG-3′, Reverse 5′-GTCCACGGTTAGCACCAAAT-3′; Cyp11a1 Forward: 5′-CACAGACGCATCAAGCAGCAAAA-3′, Reverse: 5′-GCATTGATGAACCGCTGGGC-3′; and Actin Forward: 5′-ATTTCTGAATGGCCCAGGTC-3′, Reverse: 5′-GTCTCAAGTCAGTGTACAGGC-3′.

The ovary was homogenized, and cell lysates were prepared. Protein concentrations were determined using Bradford reagent (Sigma-Aldrich, Darmstadt, Germany). Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Darmstadt, Germany). Membranes were blocked in 1 × TBS containing 0.1% Tween 20 and 5% skim milk (Merck Millipore) prior to incubation with the following primary antibodies: rabbit anti-CYP11A1 (1:1,000, Affinity Biosciences company/manufacturer's location Cincinnati, USA), rabbit anti-STAR (1:1,000, Affinity Biosciences), rabbit anti-follicle-stimulating hormone receptor (FSHR; 1:1,000, Affinity Biosciences, Cincinnati, USA), rabbit anti-PER2 (1:1,000, Affinity Biosciences), rabbit anti-CRY1 (1:1,000, Affinity Biosciences), rabbit anti-p27 (1:1,000, Affinity Biosciences), and rabbit anti-CYCLIN D2 (1:1,000, Affinity biosciences). The PVDF membrane was then incubated with Horseradish Peroxidase (HRP)-conjugated secondary antibodies (1:2,000, Abcam, Cambridge, UK) for 1 h at room temperature (RT) and detected using chemiluminescent HRP substrate Luminata Forte (Millipore).

Mice were killed, and ovaries were collected. For immunohistochemistry, ovaries were fixed in 10% formalin at RT. For RNA analysis, ovaries were stored in RNAlater for total RNA isolation. For protein analysis, ovaries were frozen in liquid nitrogen and stored at – 80°C until use for Western blotting.

Ovarian sections were incubated overnight at 4 °C with rabbit anti-Ki67 and rabbit anti-p27 antibodies (1:100, Affinity Biosciences), followed by a 1 h incubation at RT with a biotinylated secondary antibody. After washing with PBS, sections were treated with diaminobenzidine (DAB) and H₂O₂ as a chromogen and then submerged in water to stop the reaction. Sections were counterstained with Cole’s hematoxylin for 1–2 min, air-dried, cleaned in xylene, and mounted with DPX. Images were captured using a light microscope.

Ovaries from the control and treated groups were extracted and fixed in 10% formalin for 24 h at RT. The fixed samples were then dehydrated, embedded in paraffin blocks, and sectioned using a rotary microtome. Ovary sections were cut at 6 μm thickness, stained with hematoxylin and eosin (H&E), and mounted on slides. For follicle counting, serial sections from each ovary were placed on glass slides in order and stained with H&E. The first section was selected randomly, and only follicles with clearly visible nuclei were scored. Follicles were counted in every fifth section throughout the ovary to avoid double-counting, as individual follicles span multiple consecutive sections. To estimate total follicle numbers, the counted follicles in each category (primordial, primary, secondary, antral, preovulatory, and atretic) were multiplied by the section interval 5 and section thickness (6 µm), following the approach described by Hirshfield et al. (25, 26). This systematic sampling strategy provides a representative estimate of total follicle numbers while accounting for tissue depth and section sampling. Stained sections were analyzed at different developmental stages using a Nikon ECLIPSE E 600 light microscope equipped with an E66 digital camera (Nikon, Tokyo, Japan).

Ovarian follicles were categorized into different stages based on their morphology: primordial, primary, secondary, preovulatory, ovulatory, and atretic follicles. A follicle containing an oocyte surrounded by a single layer of squamous granulosa cells was identified as a primordial follicle. Primary follicles consisted of oocytes enclosed by a single layer of cuboidal granulosa cells, whereas secondary follicles were surrounded by multiple layers of granulosa cells. Follicles with five or more granulosa cell layers were classified as antral follicles, which typically contained one or two small antral fluid spaces. Follicles exhibiting cells in the zona pellucida were categorized as atretic follicles.

Mice aged 6–8 weeks were treated intraperitoneally (i.p.) with 5 IU of PMSG (Sigma-Aldrich) followed 48 h later by 5 IU of Human Chorionic Gonadotropin (hCG) (Sigma-Aldrich) via i.p. injection. Mice were euthanized 12–16 h after hCG administration, and oocytes were collected from the oviducts. Oocytes were counted manually under a stereomicroscope.

Mice were killed after 4–6 weeks, and blood was collected by cardiac puncture into microcentrifuge tubes. After centrifugation, serum was stored at – 80°C until analysis. Hormone assays for progesterone and melatonin (Elabscience, Houston, USA) were performed on the serum samples according to the manufacturer’s instructions.

GraphPad Prism software was used for all statistical analyses. Data are presented as either the mean ± standard error of the mean (SEM) for measurements reflecting precision across images or replicates, or as the mean ± standard deviation (SD) for measurements reflecting biological variability across animals. All experiments were performed in triplicate. Graphs were prepared with GraphPad Prism. Statistical differences between groups were assessed using a two-tailed unpaired t-test with Welch’s correction. Differences were considered statistically significant at ^*p < 0.05 or ^*p = 0.05, ^**p < 0.01, ^***p < 0.001, or ^****p  < 0.0001.

The mouse is a valuable animal model for studying mammalian reproductive physiology due to its short reproductive cycle, high breeding efficiency, and small size with low maintenance costs. We generated a CR-disrupted female mouse model to study fertility. To evaluate reproductive performance, CR-disrupted female mice, treated with or without SR9011, were bred with normal male mice, while control female mice were bred with normal male mice (control pair). Breeding experiments were conducted to assess both the prophylactic and therapeutic efficacy of SR9011 in restoring fertility following CR disruption. For evaluation of the prophylactic effect, mice received SR9011 treatment during the period of CR disruption. We observed that the live litter size was larger in control pairs, with more frequent breeding cycles (Supplementary Figure S1A), compared with CR-disrupted mouse pairs (Figure 1A; Supplementary Figure S1B), indicating that CR disruption adversely affects reproductive performance. In SR9011-treated pairs, live litter size was higher, and breeding cycles were more consistent than in pairs subjected to CR disruption alone (Figure 1B). Similarly, when evaluating the therapeutic efficacy of SR9011, breeding efficiency was significantly higher in control pairs than in CR-disrupted pairs (Supplementary Figures S1C, D). The breeding efficiency of the CR-disrupted pairs treated with SR9011 showed a marked improvement compared with CR disruption alone (Figures 1C, D), suggesting that SR9011 restores fertility impaired by CR disruption (Figure 1D). Pup body weight was higher in control pairs than in CR-disrupted pairs; however, no significant difference in pup weight was observed between CR-disrupted pairs treated with SR9011 and untreated CR-disrupted pairs.

We also recorded additional reproductive metrics. Mating latency, defined as the time from pairing to first successful litter, was prolonged in CR-disrupted females; control pairs produced their first litter approximately 52 days after pairing, whereas CR-disrupted pairs required ~ 90 days. Gestational/interlitter intervals were also extended in CR-disrupted mice (mean: 51.8 days) compared with controls (32.9 days) (Supplementary Figures S1A, B), indicating delayed reproductive cycling under circadian disruption. While evaluating the prophylactic effect of SR9011, CR-disrupted females and CR + SR9011 groups were paired for mating simultaneously (Figures 1A, B). CR-disrupted females produced their first litter approximately 30 days after mating, with a mean interlitter (gestational) interval of 34.3 days. Notably, CR + SR9011-treated females also produced pups at ~ 30 days but exhibited a shorter mean interlitter interval of 31 days, indicating improved reproductive efficiency following Rev-erbα activation.

In the therapeutic setting, control females produced their first litter at approximately 44 days, with a mean interlitter interval of 43.1 days. CR-disrupted females produced their first pups at ~ 31 days but exhibited a prolonged mean interlitter interval of 49.2 days. In contrast, CR + SR9011-treated females produced pups at ~ 44 days, with a reduced mean interlitter interval of 47.1 days compared with CR-disrupted mice, suggesting partial restoration of reproductive timing.

Although the pregnancy rate per estrous cycle was not specifically recorded, the overall pregnancy rate was reduced in CR-disrupted mice. Neonatal mortality was recorded separately across five breeding cages per condition but was not included in the cumulative litter plots.

Under CR disruption, neonatal mortality was elevated. In the preventive cohort, CR-disrupted pairs exhibited 12 neonatal deaths, whereas CR-disrupted pairs treated with SR9011 showed eight neonatal deaths; no neonatal deaths were observed in control pairs. Similarly, in the therapeutic cohort, CR-disrupted mice showed nine neonatal deaths, while CR + SR9011-treated mice exhibited six neonatal deaths, with no mortality observed in controls. Mortality of the female breeding pairs was also observed, further indicating the physiological stress associated with circadian disruption.

Using quantitative real-time PCR (qRT-PCR), we assessed the relative messenger RNA (mRNA) levels of Rev-erbα, Period (Per2), and Cryptochrome (Cry1) in the ovaries of both control and CR-disrupted mice (Supplementary Figure S2). Compared with controls, the expression of Rev-erbα, Per2, and Cry1 was decreased in the ovaries of CR-disrupted mice, indicating that circadian disruption adversely affects ovarian clock gene expression. β-Actin was used as an internal control for normalization.

Steroidogenesis is a coordinated process regulated by signals from ovarian cells and is essential for steroid hormone production, which supports follicle development, oocyte maturation, and ovulation. Genes involved in steroidogenesis include steroidogenic acute regulatory protein (Star), cytochrome P450 side-chain cleavage enzyme (Cyp11a1), and anti-Müllerian hormone (Amh). FSHR regulates folliculogenesis; PER2 and CRY1 regulate CR; CYCLIN D2 promotes cell division; and p27 inhibits follicular cell proliferation. AMH is produced by granulosa cells of developing follicles and plays a role in controlling estrogen secretion. AMH levels are closely associated with the number of antral follicles and serve as a reliable indicator of ovarian reserve (27). To investigate the potential prophylactic and therapeutic effects of SR9011 (Figures 2, 3) on ovarian steroidogenesis, follicular development, clock gene expression, and cell division in mice with disrupted CR, we measured the relative mRNA levels of Amh, Star, Cyp11a1, Per2, Cry1, and FSHR in control mice, CR-disrupted mice, and CR-disrupted mice treated with SR9011. These measurements were then correlated with the state of follicle development. mRNA expression of these genes was decreased in the ovaries of CR-disrupted mice compared with controls, indicating compromised follicular health and disrupted regulation of clock genes. In contrast, treatment with SR9011 in CR-disrupted mice elevated/restored the expression of genes associated with steroidogenesis, folliculogenesis, and cell division (Figures 2A, 3A). However, therapeutic treatment did not significantly restore the expression of clock genes; other genes, such as Amh, CYP11A1, Star, and FSHR, were rescued by SR9011 treatment (Figure 3A). A subtle change was observed in Per2 and Cry1 expression at the mRNA level, whereas significant changes were observed at the protein level (Figures 3B, C).

Ovaries from CR-disrupted mice exhibited elevated p27 and reduced FSHR and CYCLIN D2 expression, indicative of impaired cell proliferation and increased follicular atresia, a trend confirmed by Western blot analyses (Figures 2B, C, 3B, C). Notably, SR9011 treatment decreased p27 levels and increased the expression of CYP11A1, STAR, CRY1, PER2, FSHR, and CYCLIN D2 compared with CR-disrupted mice, suggesting restoration of cell proliferation, steroidogenesis, and follicle development.

We next performed H&E staining to examine ovarian follicles at different developmental stages in the ovaries of control mice, CR-disrupted mice, and CR-disrupted mice treated with SR9011. We assessed both the prophylactic and therapeutic effects of SR9011 on follicular development and atresia (Figures 4A, B). CR-disrupted mouse ovaries exhibited fewer healthy follicles and a significantly higher rate of follicular atresia (atretic follicles) compared with control ovaries, which contained healthy and Graffian follicles at all developmental stages. Treatment with SR9011 reduced the number of atretic follicles and increased the presence of healthy follicles in CR-disrupted ovaries, indicating that SR9011 restores follicle quality impaired by CR disruption.

The reduced number of follicles in the ovaries of CR-disrupted mice suggests potential deficiencies in ovarian cell proliferation. To further evaluate the effects of SR9011 on follicular cell growth and division, we examined the expression of MKI67 (Ki67) and CDKN1B (p27) by immunohistochemistry in ovaries from control mice, CR-disrupted mice, and CR-disrupted mice treated with SR9011. Female mice received SR9011 (100 mg/kg) either during or after circadian disruption to assess the prophylactic (Figures 5A–D) and therapeutic effects (Figures 5E–H) of the ligand. Ovaries from the CR-disrupted mice exhibited a marked reduction in Ki67 expression, accompanied by increased expression of p27 in follicular cells (Figures 5A–H). Interestingly, SR9011 treatment restored follicle growth and promoted follicular cell proliferation and division, as evidenced by increased Ki67 expression and decreased p27 expression.

Quantification of ovarian follicles at different developmental stages is a key indicator of folliculogenesis. Figure 6 illustrates the observed primordial, primary, secondary, antral, preovulatory, and atretic follicles in the ovaries of control mice, CR-disrupted mice, and CR-disrupted mice treated with SR9011. As shown in Figures 6A, C, CR disruption affects both the quality and quantity of follicles (primordial, primary, secondary, antral, and preovulatory) and increases the number of atretic (degenerate) follicles. In contrast, SR9011 treatment, in both the prophylactic and therapeutic modes, improved follicle quality and increased follicle counts compared with CR disruption alone. Representative images are shown in Figures 6B, D.

Circadian misalignment might have an impact on hormonal shifts during pregnancy, although the precise mechanisms underlying this phenomenon remain largely unclear. Pregnancy is closely linked to patterns of hormone secretion (28) as well as to the regulation of the sleep–wake cycle. Progesterone, a crucial hormone for maintaining pregnancy, reaches its peak levels around gestation days 15–17 in mice (29, 30). The sleep–wake cycle represents the most overt circadian rhythm. Melatonin, because of its rhythmic and cyclical release, plays a key role as a physiological regulator of the sleep–wake cycles in diurnal species, including humans. Melatonin is also synthesized in peripheral reproductive tissues, including cumulus oophorus, granulosa cells, and oocytes. Melatonin is a potent antioxidant that protects oocytes from oxidative stress, particularly during ovulation (31). Disruption of circadian rhythms can influence the secretion of this hormone (31). In this study, we measured serum levels of progesterone and melatonin in three distinct groups: control mice, CR-disrupted mice, and CR-disrupted mice treated with SR9011, to evaluate the impact of CR disruption and the efficacy of SR9011 in both prophylactic (Figures 7A, B) and therapeutic settings (Figures 7C, D). Compared with control mice, CR-disrupted mice exhibited reduced levels of both progesterone and melatonin. Treatment with SR9011 in CR-disrupted mice restored the levels of these hormones (Figures 7A–D).

To determine whether infertility was due to a defect in Rev-erbα or other secondary changes, we performed a superovulation experiment following a standard protocol. Mice were divided into four groups, with three groups undergoing superovulation induction. The fourth group was treated with SR8278 (14 mg/kg) on alternate days for 4 weeks. Forty-eight hours after the last SR8278 injection, superovulation was performed in the three relevant groups, except for the CR-disrupted group. The number of oocytes released into the oviduct was counted in the control (superovulated), CR (without superovulation), CR (superovulated), and CR + SR8278 (superovulated) groups.

As shown in Figure 7E, control females ovulated an average of 26 ± 1.82 oocytes following superovulation. In contrast, CR-disrupted females without superovulation released an average of 2.25 ± 0.95 oocytes, while CR-disrupted females undergoing superovulation released 11.25 ± 2.62 oocytes. These results indicate that oocyte retrieval in CR-disrupted females occurs primarily after exogenous gonadotropin administration. Interestingly, CR-disrupted mice treated with SR8278 released only 3 ± 0.81 oocytes following superovulation, a number comparable to CR-disrupted females without superovulation. This finding suggests that inhibiting Rev-erbα impairs oocyte retrieval, even when superovulation is performed.