Night-restricted feeding preserves the ovarian circadian rhythm of IL-17 and promotes follicular development in prepubertal and pubertal rabbits.

A total of 216 weaning female Ira rabbits (35 days of age, initial body weight 0.91 ± 0.10 kg) were raised in an open barn under natural lighting at the Qingdao Kangda Rabbit Development (Shandong, China) from April to July. The average indoor and outdoor temperatures were 17.55 ± 2.99 °C and 18.65 ± 3.50 °C, respectively, and the indoor and outdoor relative humidity were 67.16 ± 3.50% and 60.66 ± 17.17%, respectively. According to our previous study [ 35 , 36 ], rabbits with similar body weight were randomly assigned to daytime feeding (DF, n = 108) and night-restricted feeding (NRF, n = 108) groups and these rabbits were housed and fed in individual cages(three rabbits per cage) with dimensions of 0.6 m × 0.62 m × 0.3 m (length × width × height). The DF group was given ad libitum access to feed (with feed provided at 6:30 am). The NRF group had access to food only during the night (from 6:30 pm to 6:30 am, with feed provided at 6:30 pm). All rabbits were provided with water ad libitum. Both groups were provided with the same amount of feed following the commercial production recommendation. The feed formulation, nutrient composition, rabbits body weight, and feed intake from 35 to 84 days of age were detailed in our previous work [ 36 ]. At 84 days of age, ovarian tissues were collected from six healthy rabbits of each group and then immediately euthanized by cervical dislocation at 4-h intervals (daytime: 07:00 (ZT1), 11:00 (ZT5), 15:00 (ZT9); nighttime: 19:00 (ZT13), 23:00 (ZT17), 03:00 (ZT21)). Cervical dislocation was performed by a trained operator in accordance with the AVMA Guidelines for the Euthanasia of Animals: 2020 Edition. The remaining rabbits were maintained under identical husbandry conditions. Beginning at 135 days of age, female rabbits were assessed at the same time each day by a single trained observer, and sexual receptivity was evaluated based on vulvar color and edema. At 145 days of age, serum was collected from six healthy rabbits per group at each time point, specifically at 11:00 (ZT5) and 23:00 (ZT17). One side of the ovaries was snap-frozen in liquid nitrogen and stored at -80 °C, while the other side was fixed in 4% paraformaldehyde for histological examination. After blood samples were collected, serum was isolated by centrifugation from whole blood samples and stored at -80 °C until analysis. Serum concentrations of GnRH, FSH, LH, and E2 (Estradiol) were quantified using commercial radioimmunoassay kits (Beijing Kangtai Hongyuan Biotechnology, Beijing, China). The experimental procedures were performed in strict accordance with the kit instructions. For histological examination, ovaries were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm intervals through the mid-sagittal plane. Every 20 μm section was subjected to hematoxylin-eosin (HE) staining and digitized using a Leica Aperio VERSA 200 automated slide scanner (Leica Biosystems, Richmond, IL, USA) with a 20× objective. Five slices of each ovary were selected to count the number of follicles. Follicles were classified and counted according to a unified morphological criterion across all samples. To avoid double counting, only follicles with visible oocyte nuclei were counted, including primordial, primary, secondary, antral, and atretic follicles. Slides for follicle counting were randomized and coded by an independent investigator and was performed in a single-blind manner. Proliferation and apoptosis were evaluated in ovarian sections using Ki67 (1:200, 27309-1-AP, ProteinTech, Wuhan, China, rabbit) and cleaved caspase-3 antibodies (1:200, WL01992, Wanleibio, Shenyang, China, rabbit). The tissues were subsequently fixed in 4% paraformaldehyde and embedded in paraffin. The embedded tissue was sectioned at 5 μm thickness, followed by deparaffinization and hydration. The slides were subjected to antigenic recovery in sodium citrate buffer (pH 6.0) by high-pressure heating. Endogenous peroxidase activity was inhibited by 3% H 2 O 2 for 10 min followed by blocking in 0.5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h at room temperature. The slides were then incubated overnight at 4 °C with the primary antibody. Subsequently, they were incubated with HRP-conjugated Goat Anti-Rabbit IgG (1:500, ZB-2301, Golden Bridge, Beijing, China) and 3,3’-diaminobenzidine (DAB) visualization (ZLI-9018, Golden Bridge). Nuclear counterstaining was performed with hematoxylin. For quantitative analysis, Ki67-positive granulosa cells were quantified using ImageJ. Cleaved caspase-3 expression was quantified as average optical density (AOD) using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA). Total RNA was extracted from ovarian tissues using the TRIzol reagent. RNA-Seq library construction and sequencing were carried out using the MGI2000 (BGI, Guangdong, China) to generate 150 bp paired-end reads. The raw data obtained by sequencing were filtered using the filtering software SOAPnuke (v1.4.0). These filtered reads were aligned to the rabbit genome (OryCun2.0) using HISAT2 (v2.2.1), followed by assembly using Stringtie (v2.1.5). Differential gene expression analysis was performed using DESeq2 (v1.38.0). The differentially expressed genes (DEGs) were defined using the following criteria: |log2FoldChange| > 1 and adjusted P  < 0.05. These DEGs were further analyzed using DAVID Bioinformatics Resource(david.ncifcrf.gov) for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Gene set enrichment analysis (GSEA) was implemented via the R package clusterProfiler. The differential rhythmicity analysis of gene expression was performed using the dryseq () function from the R package dryR. This function fits gene expression rhythms based on a generalized harmonic regression model. Genes exhibiting rhythmically expressed patterns were selected based on the following criteria: Bayesian Information Criterion weight (BICW) ≥ 0.4 and amplitude ≥ 0.25 [ 37 ]. The ggplot2 package was used to create phase polar coordinate plots of selected differential genes. For circadian pathway annotation, the PSEA v1.1 software platform was employed to configure the temporal parameter space (0–24 h). Statistical significance was evaluated using the Kuiper test ( P < 0.05). Total protein was extracted from ovaries using RIPA lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China) supplemented with protease and phosphatase inhibitors. Following centrifugation at 12,000 g for 10 min at 4 °C, supernatants were collected and protein concentrations determined using the BCA protein assay kit (P0010, Beyotime Biotechnology, China). Equal protein quantities (20 µg) were resolved by SDS-PAGE and electrotransferred to PVDF membranes (Millipore, Billerica, MA, USA). The membrane was blocked in 5% non-fat dry milk in TBST for 1 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies: anti-IL-17 (1:1000, MAA063RA21, Cloud-Clone Corp., Wuhan, China, mouse). After TBST washing, membranes were incubated with HRP-conjugated Goat Anti-Mouse IgG (1:8000, ZB-5305, Golden Bridge, Beijing, China) for 1 h at room temperature. Finally, proteins were determined using an ECL Plus western blotting Detection System (Tanon, China). Rabbit ovarian granulosa cells (RGCs) were obtained as a gift from Professor Xinsheng Wu of Yangzhou University [ 38 ]. RGCs were cultured in DMEM/F12 medium (11330-032, Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO₂ atmosphere. For IL-17 stimulation experiments, recombinant rabbit IL-17 (HY- P73175 , MedChemExpress, USA) was added to culture media at specified concentrations. Cells were treated with IL-17 under three experimental conditions: control group (12 h at 0 ng/mL, 12 h at 0 ng/mL), constant IL-17 group (12 h at 15 ng/mL, 12 h at 15 ng/mL), oscillatory IL-17 (12 h at 5 ng/mL, 12 h at 25 ng/mL). All cells underwent a 24-hour serum starvation treatment prior to stimulation to synchronize their biological clocks. After 2 days of IL-17 treatment, cells were collected at 4-hour intervals over a 24-hour period on day 3 to assess the effects of IL-17 treatment. RGCs were plated in 96-well plates at a density of 1 × 10⁴ cells/well. Cell viability was quantified using the Cell Counting Kit-8 assay (C0039, Beyotime, China) following the manufacturer’s instructions. For proliferation assessment, the BeyoClick™ EdU Cell Proliferation Kit (C0075S, Beyotime, China) was applied according to the standardized protocol. Briefly, cells were labeled with 10 µM EdU for 2 h at 37 °C, followed by fixation with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 (15 min, room temperature). After washing the cells three times with 3% BSA in PBS, cells were incubated with Click-iT reaction cocktail for 30 min in the dark. Cell nuclei were stained with Hoechst 33342. Fluorescent signal images were conducted using Operetta CLS High-Content Analysis System (Perkin Elmer, USA). Total RNA was extracted using TRIzol reagent (15596026CN, Thermo Fisher Scientific, USA) following the manufacturer’s protocol. For reverse transcription, 1 µg total RNA was converted to cDNA using the HiScript II Q RT SuperMix Kit (R223, Vazyme, Nanjing, China) in a 20 µL reaction system. Quantitative PCR was performed using Taq Pro Universal SYBR qPCR Master Mix (Q712, Vazyme, Nanjing, China) in the CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). Relative mRNA expression was normalized to reference gene β-actin using the 2 −ΔΔCT method. The primers used are listed in Table S1. Circadian rhythm analysis of 24-hour oscillatory patterns was performed using the JTK_Cycle algorithm, as previously described [ 39 ]. Student’s t-tests were used to compare the differences between the groups. Statistical analyses were conducted using IBM SPSS Statistics version 20.0 (Armonk, NY, USA). Graphs were generated using GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla, CA, USA).

To investigate the relationship between feeding time and ovarian circadian rhythms in rabbits, ovarian tissue samples were collected from two groups at day 84: the DF group and the NRF group. Samples were obtained every 4 h over a 24-hour period and subjected to transcriptomic analysis. Principal component analysis (PCA) revealed distinct transcriptomic profiles between the DF and NRF groups across multiple time points (Fig.  1 A). In total, 583 DF-specific and 924 NRF-specific rhythmic transcripts were identified. Notably, 33 transcripts exhibited differential rhythmicity between the two groups, suggesting regulation by feeding time, whereas 9,366 transcripts showed consistent rhythmic patterns in both groups, indicating regulation independent of feeding time (Fig.  1 B). Phase distribution analysis revealed a bimodal pattern among DF-specific rhythmic transcripts, with a primary peak between ZT10 and ZT15 and a secondary peak between ZT21 and ZT23 (Fig.  1 C). In contrast, NRF-specific transcripts exhibited a unimodal distribution, with peak expression occurring around ZT21 (Fig.  1 D). Among the transcripts with altered rhythmicity between the two groups, those in the DF group peaked predominantly at ZT7, while their NRF counterparts peaked around ZT3 (Fig.  1 E). These results indicate that feeding time significantly influences rhythmic gene expression in the ovaries. Fig. 1 Feeding time regulates circadian gene and pathway expression in rabbit ovaries at day 84. A PCA of ovarian transcriptomes in the DF and NRF groups. B Number of genes exhibiting circadian rhythmicity in the DF and NRF groups. White denotes non-rhythmic genes, identical colors indicate rhythmic genes exhibited consistent rhythmicity (identical phases and amplitudes) in DF and NRF groups. C Heatmap of unique circadian pathways and related genes in the DF group. The radial plot illustrates the distribution of peak expression phases for all rhythmic genes identified in the DF group. D Heatmap of unique circadian pathways and related genes in the NRF group. The radial plot illustrates the distribution of peak expression phases for all rhythmic genes identified in the NRF group. E Heatmap of circadian rhythmic pathways and related genes with altered rhythms in the DF and NRF groups. The radial plot illustrates the distribution of peak expression phases for all differentially rhythmic genes between the two groups. n  = 5–6 in each group at each time point Feeding time regulates circadian gene and pathway expression in rabbit ovaries at day 84. A PCA of ovarian transcriptomes in the DF and NRF groups. B Number of genes exhibiting circadian rhythmicity in the DF and NRF groups. White denotes non-rhythmic genes, identical colors indicate rhythmic genes exhibited consistent rhythmicity (identical phases and amplitudes) in DF and NRF groups. C Heatmap of unique circadian pathways and related genes in the DF group. The radial plot illustrates the distribution of peak expression phases for all rhythmic genes identified in the DF group. D Heatmap of unique circadian pathways and related genes in the NRF group. The radial plot illustrates the distribution of peak expression phases for all rhythmic genes identified in the NRF group. E Heatmap of circadian rhythmic pathways and related genes with altered rhythms in the DF and NRF groups. The radial plot illustrates the distribution of peak expression phases for all differentially rhythmic genes between the two groups. n  = 5–6 in each group at each time point To further characterize the biological processes associated with circadian genes, PSEA was performed to identify pathways significantly associated with rhythmic gene expression. In the DF group, apoptotic signaling pathway peaked during the daytime. Immune-related pathways, such as antigen processing and presentation and the NOD-like receptor signaling pathway, peaked at the night. Ovarian steroidogenesis was enriched during phase transitions (Fig.  1 C and S1A). In the NRF group, immune-related pathways (IL-17 signaling) and proliferation-associated pathways (cell cycle and DNA replication) peaked at night (Fig.  1 D and S1B). Among the pathways commonly enriched in both groups, peak phases were predominantly clustered between ZT5 and ZT10. Apoptosis signaling, steroid hormone biosynthesis, and cholesterol metabolism peaked during the day, while pathways related to the cell cycle and gonadotropin-releasing hormone secretion peaked at night (Fig. S1C). Additionally, among genes exhibiting differential rhythmicity, only three pathways, p53 signaling pathway, prion disease, and transcriptional misregulation in cancer were enriched in the DF group (Fig.  1 E and Fig. S1D). Collectively, these findings demonstrate that feeding time significantly alters the timing of rhythmic signaling pathway expression in the ovary. To investigate whether feeding time induced circadian alterations on pubertal onset in rabbits, estrous status was monitored. At 136 days of age, the estrus rate was significantly higher in the NRF group than that in the DF group (Fig.  2 A, B), suggesting that night-restricted feeding accelerates pubertal onset. Furthermore, the mean age of first estrus in the NRF group was significantly earlier than that in the DF group (Fig.  2 C). Notably, cumulative estrus rate and estrus duration did not differ between groups (Fig.  2 D-E). Serum hormone analyses at 145 days revealed no significant differences in pubertal associated reproductive hormones, including GnRH, E2, and P4 between the two groups (Fig.  2 F-H). However, distinct diurnal differences in FSH levels were observed exclusively in the NRF group and were absent in the DF group (Fig.  2 I). Fig. 2 The effects of feeding time on pubertal onset and pubertal-associated reproductive hormones. A and B The percentages of first estrus ( n  = 89–92 in each group). C Average age at first estrus ( n  = 89–92 in each group). D Cumulative percentage of first estrus ( n  = 89–92 in each group). E Estrus duration ( n  = 89–92 in each group). F - I Serum concentrations of GnRH, E2, FSH, and P4 at 145 days of age ( n  = 6 at each time point in each group). All data represent the means ± SEM. * p  < 0.05; ** p  < 0.01 The effects of feeding time on pubertal onset and pubertal-associated reproductive hormones. A and B The percentages of first estrus ( n  = 89–92 in each group). C Average age at first estrus ( n  = 89–92 in each group). D Cumulative percentage of first estrus ( n  = 89–92 in each group). E Estrus duration ( n  = 89–92 in each group). F - I Serum concentrations of GnRH, E2, FSH, and P4 at 145 days of age ( n  = 6 at each time point in each group). All data represent the means ± SEM. * p  < 0.05; ** p  < 0.01 To investigate the effects of feeding time on follicular development, histological analysis of ovarian tissues was performed (Fig.  3 A). The NRF group had a greater number of antral follicles than the DF group, indicating that NRF promotes follicular development. Notably, diurnal different in follicle numbers at different developmental stages were observed only in the NRF group: the number of primordial follicles was significantly higher at ZT17 than at ZT5, whereas the number of secondary follicles was higher at ZT5 than at ZT17 (Fig.  3 B). These findings demonstrate a diurnal difference in follicle development in the NRF group that were not observed in the DF group. Fig. 3 Effects of feeding time on follicular development, granulosa cell proliferation, and apoptosis in rabbit ovaries at day 145. A Hematoxylin–eosin staining of ovarian sections. B Number of follicles at different developmental stages in sections. C Immunohistochemical staining for Ki67 (a proliferation marker) and cleaved caspase-3 (an apoptosis marker) in ovarian tissues. D Ki67-positive rate in granulosa cells of follicles at different developmental stages. E Average optical density of cleaved caspase-3 in granulosa cells of follicles at different developmental stages. n  = 4–6 at each time point in each group. All data are presented as mean ± SEM. * p  < 0.05; ** p  < 0.01 Effects of feeding time on follicular development, granulosa cell proliferation, and apoptosis in rabbit ovaries at day 145. A Hematoxylin–eosin staining of ovarian sections. B Number of follicles at different developmental stages in sections. C Immunohistochemical staining for Ki67 (a proliferation marker) and cleaved caspase-3 (an apoptosis marker) in ovarian tissues. D Ki67-positive rate in granulosa cells of follicles at different developmental stages. E Average optical density of cleaved caspase-3 in granulosa cells of follicles at different developmental stages. n  = 4–6 at each time point in each group. All data are presented as mean ± SEM. * p  < 0.05; ** p  < 0.01 To further elucidate the molecular mechanisms underlying the effects of feeding time on follicular development, we examined the expression of the proliferation marker Ki67 and the apoptosis marker cleaved caspase-3 in the ovaries. Immunohistochemical analysis indicated that granulosa cells were the primary cell type exhibiting both proliferation and apoptosis (Fig.  3 C). Quantitative analysis showed significantly higher Ki67 expression in granulosa cells of primary, secondary, and antral follicles in the NRF group than in the DF group, indicating that night-restricted feeding promotes granulosa cell proliferation. Interestingly, Ki67 expression in granulosa cells of primary and secondary follicles exhibited a diurnal difference in the NRF group, with significantly higher expression at ZT17 than at ZT5 (Fig.  3 D). These findings indicate there are diurnal difference in granulosa cell proliferation in the NRF group, with higher proliferative activity at night. Immunohistochemical staining for cleaved caspase-3 revealed significantly lower expression in granulosa cells of primary and secondary follicles in the NRF group than in the DF group, suggesting that night-restricted feeding suppresses granulosa cell apoptosis. Specifically, the expression of cleaved caspase-3 in granulosa cells of primary follicles was higher at ZT17 than at ZT5 in the NRF group. In addition, cleaved caspase-3 expression in primary follicle granulosa cells at ZT5 was significantly higher in the DF group than in the NRF group (Fig.  3 E). Collectively, these findings demonstrate that night-restricted feeding promotes ovarian follicle development by enhancing granulosa cell proliferation and inhibiting apoptosis. To further elucidate the mechanisms through which feeding time influences ovarian function, RNA-seq analysis was performed on ovarian tissues. The DF group exhibited the greatest number of differentially expressed genes between ZT5 and ZT17 (Fig.  4 A). GSEA-based KEGG pathway enrichment analysis revealed significant downregulation of several biological pathways in the NRF group compared to the DF group, including prion disease, thermogenesis, spliceosome, cell cycle regulation, Parkinson disease, and oxidative phosphorylation (Fig.  4 B). Notably, ribosome biogenesis in eukaryotes and collecting duct acid secretion were upregulated in the NRF group at ZT17, whereas the tuberculosis and lipid and atherosclerosis were markedly downregulated relative to the NRF group at ZT5 (Fig.  4 C). The diurnal differences in the DF group were predominantly associated with immune-related pathways (Fig. S2A). GO enrichment analysis highlighted significant enrichment of terms such as CXCR3 chemokine receptor binding and immunoglobulin receptor activity (Fig. S2B). Both KEGG and GSEA-KEGG analyses consistently demonstrated significant enrichment of the IL-17 signaling pathway throughout diurnal difference in the DF group (Fig.  4 D and S2C). Intriguingly, IL-17 pathway associated genes were highly expressed at ZT5 but significantly suppressed at ZT17 in the DF group (Fig.  4 E). Fig. 4 The effect of feeding time on the ovarian transcriptome at day 145. A Volcano plot of differentially expressed genes ass between ZT17 and ZT5 in DF and NRF groups (screening criteria: |log2FC| > 1 and adjusted P-value < 0.05). B GSEA based on the KEGG analysis between NRF and DF groups. C GSEA based on the KEGG analysis between ZT17 and ZT5 in the NRF group. D GSEA based on the KEGG analysis between ZT17 and ZT5 in the DF group. E GSEA analysis of the IL-17 signaling pathway between ZT17 and ZT5 in the DF group. F Western blot analysis of IL-17 protein expression, with β-actin used as an internal control. All data represent the means ± SEM. n  = 4–6 at each time point in each group. * p  < 0.05; ** p  < 0.01 The effect of feeding time on the ovarian transcriptome at day 145. A Volcano plot of differentially expressed genes ass between ZT17 and ZT5 in DF and NRF groups (screening criteria: |log2FC| > 1 and adjusted P-value < 0.05). B GSEA based on the KEGG analysis between NRF and DF groups. C GSEA based on the KEGG analysis between ZT17 and ZT5 in the NRF group. D GSEA based on the KEGG analysis between ZT17 and ZT5 in the DF group. E GSEA analysis of the IL-17 signaling pathway between ZT17 and ZT5 in the DF group. F Western blot analysis of IL-17 protein expression, with β-actin used as an internal control. All data represent the means ± SEM. n  = 4–6 at each time point in each group. * p  < 0.05; ** p  < 0.01 We also examined IL-17 protein expression in ovarian tissues. Overall IL-17 protein levels did not differ significantly between the DF and NRF groups. However, IL-17 protein expression exhibited significant diurnal difference in the NRF group, with lower levels during the day and higher levels at night (Fig.  4 F). These findings suggest that diurnal difference of the IL-17 signaling pathway may be one of the mechanisms through which feeding time influences follicular development during puberty. We compared the rhythmic pathways at day 84 and found that 16 pathways exhibited unique rhythmicity in the DF group, while 29 pathways showed unique rhythmicity in the NRF group (Fig.  5 A). At day 145, 33 pathways were unique to the DF group and 7 to the NRF group. Across both developmental stages, eight pathways were consistently enriched, including NOD-like receptor signaling, prion disease, ribosome biogenesis in eukaryotes, spliceosome, proteasome, IL-17 signaling, legionellosis, and tuberculosis. Among them, the IL-17 signaling pathway exhibited time-specific differences at different ages, suggesting a potential role in mediating the effects of feeding time on ovarian circadian rhythms. Fig. 5 Pathway specificity analysis and the expression of IL-17 signaling pathway-related genes at day 84 and 145. A Venn diagrams of rhythmic pathways in the DF and NRF groups at day 84, diurnally differential pathways at day 145, and group-specific rhythmic or differential pathways at both day 84 and day 145. B The gene expression profile of IL-17 signaling pathway at day 84. C The gene expression profile of IL-17 signaling pathway at day 145. n  = 4–6 at each time point in each group. All data represent the means ± SEM. * p  < 0.05; ** p  < 0.01 Pathway specificity analysis and the expression of IL-17 signaling pathway-related genes at day 84 and 145. A Venn diagrams of rhythmic pathways in the DF and NRF groups at day 84, diurnally differential pathways at day 145, and group-specific rhythmic or differential pathways at both day 84 and day 145. B The gene expression profile of IL-17 signaling pathway at day 84. C The gene expression profile of IL-17 signaling pathway at day 145. n  = 4–6 at each time point in each group. All data represent the means ± SEM. * p  < 0.05; ** p  < 0.01 Further analysis of genes related to the IL-17 signaling pathway showed that at day 84, HSP90AA1 , HSP90AB1 , HSP90B1 , TAB3 , and USP25 exhibited rhythmic expression in the NRF group, whereas no such rhythmicity was observed in the DF group (Fig.  5 B). Notably, the expression of HSP90AA1 and HSP90AB1 was significantly higher at ZT21 in the NRF group than in the DF group. Additionally, TAB3 expression in the NRF group was significantly higher during ZT13-21 than during ZT1-9. These results indicate that night-restricted feeding during the prepubertal stage induces circadian rhythmicity of IL-17 signaling pathway genes and enhances their expression at night. At day 145, the expression of genes involved in the IL-17 signaling pathway, including MAPK10 , CXCL10 , IFNG , and JUN showed significantly higher at ZT5 than at ZT17 in the DF group. Similar diurnal expression patterns were observed for IFNG , JUN , IL1B , and FOSB in the NRF group (Fig.  5 C). Although overall transcript levels of IL-17 signaling pathway genes did not significantly differ between the DF and NRF groups, the NRF group exhibited a diurnal difference in IL-17 protein expression, with lower levels during the day and higher levels at night. In contrast, the DF group exhibited no significant diurnal difference. The diurnal difference of protein level in the NRF group was consistent with the transcript level observed at day 84. These results suggest that feeding time influences the diurnal differences of IL-17 signaling during pubertal development, potentially linking feeding time to ovarian circadian rhythms and follicular development. To investigate the rhythmic effects of IL-17 on rabbit ovarian granulosa cell proliferation, RGCs were treated with different concentrations of IL-17, and cell proliferation ability was assessed by CCK-8 assay. 25 ng/mL IL-17 significantly enhanced granulosa cell proliferation at 24 h, 48 h, and 72 h, establishing this concentration as optimal for subsequent experiments (Fig.  6 A). Based on ovarian IL-17 protein expression patterns (Fig.  4 F), we simulated the NRF group with oscillatory IL-17 addition and the DF group with constant IL-17 addition. The CCK-8 assay revealed a significant increase in cell viability in the oscillatory IL-17 group compared to the control group at 24 h, 48 h, 60 h, and 72 h (Fig.  6 B), indicating enhanced cell viability. Furthermore, EdU assays demonstrated a significantly increased proportion of EDU-positive cells in the oscillatory IL-17 group compared to both the con and constant IL-17 groups (Fig.  6 C). Collectively, these findings indicate that oscillatory IL-17 treatment promotes granulosa cell proliferation, which may suggest a novel target for rabbit follicular development. Fig. 6 The effect of rhythmic IL-17 on granulosa cell proliferation and circadian gene expression. A The effect of different concentrations of IL-17 on the proliferation of granulosa cell proliferation was assessed using the CCK-8 assay ( n  = 5 in each group). B The proliferation of granulosa cells following IL-17 addition was assessed using the CCK-8 assay ( n  = 5 in each group). C Representative fluorescence images and quantitative analysis of an EdU assay for granulosa cells proliferation rate ( n  = 3 in each group). Red fluorescence indicates proliferating cells, while blue fluorescence represents the nuclei of all cells. Scale bar = 50 μm. D - F Effect of IL-17 addition on the circadian expression of clock genes BMAL1 , CLOCK , and PER2 in granulosa cells. n  = 3 at each time point in each group. G - I Effect of IL-17 addition on the circadian expression of proliferation-related genes CCNB1 , CCND1 , and WEE1 in granulosa cells. n  = 3 at each time point in each group. J Effect of IL-17 addition on amplitude and phase. Groups: Con (12 h at 0 ng/mL, 12 h at 0 ng/mL); Constant IL-17 (12 h at 15 ng/mL, 12 h at 15 ng/mL); Oscillatory IL-17 (12 h at 5 ng/mL, 12 h at 25 ng/mL). All data represent the means ± SEM. * p  < 0.05; ** p  < 0.01. The red * indicates the significance between constant IL-17 and oscillatory IL-17, and the blue * indicates the significance between con and oscillatory IL-17 The effect of rhythmic IL-17 on granulosa cell proliferation and circadian gene expression. A The effect of different concentrations of IL-17 on the proliferation of granulosa cell proliferation was assessed using the CCK-8 assay ( n  = 5 in each group). B The proliferation of granulosa cells following IL-17 addition was assessed using the CCK-8 assay ( n  = 5 in each group). C Representative fluorescence images and quantitative analysis of an EdU assay for granulosa cells proliferation rate ( n  = 3 in each group). Red fluorescence indicates proliferating cells, while blue fluorescence represents the nuclei of all cells. Scale bar = 50 μm. D - F Effect of IL-17 addition on the circadian expression of clock genes BMAL1 , CLOCK , and PER2 in granulosa cells. n  = 3 at each time point in each group. G - I Effect of IL-17 addition on the circadian expression of proliferation-related genes CCNB1 , CCND1 , and WEE1 in granulosa cells. n  = 3 at each time point in each group. J Effect of IL-17 addition on amplitude and phase. Groups: Con (12 h at 0 ng/mL, 12 h at 0 ng/mL); Constant IL-17 (12 h at 15 ng/mL, 12 h at 15 ng/mL); Oscillatory IL-17 (12 h at 5 ng/mL, 12 h at 25 ng/mL). All data represent the means ± SEM. * p  < 0.05; ** p  < 0.01. The red * indicates the significance between constant IL-17 and oscillatory IL-17, and the blue * indicates the significance between con and oscillatory IL-17 To investigate the effects of rhythmic IL-17 signaling on granulosa cell circadian regulation, we conducted gene expression at 4-hour intervals, focusing on core clock and proliferation-related genes. The expression of the core clock genes BMAL1 , CLOCK , and PER2 followed circadian oscillation in both constant and oscillatory IL-17 treatment groups compared to controls (Fig.  6 D-F). Notably, BMAL1 and CLOCK expression significantly higher in the oscillatory IL-17 group than in the constant IL-17 group at CT1 and CT17. Additionally, in the oscillating IL-17 group at CT1-9, the expression of BMAL1 and CLOCK was significantly higher in the oscillatory IL-17 group than in the constant IL-17 group (Fig.  6 D-E). These results indicate that rhythmic IL-17 promotes clock gene oscillation in granulosa cells. We also examined the expression of cell cycle-related genes and found that WEE1 exhibited a similar expression pattern to BMAL1 and CLOCK , with significantly higher expression in the oscillatory IL-17 group at CT1 and CT17 (Fig.  6 I). Furthermore, the amplitudes of BMAL1 and CLOCK differed between the oscillatory IL-17 and constant IL-17 groups, while WEE1 showed changes in both amplitude and phase (Fig.  6 J). In contrast, rhythmic IL-17 stimulation did not affect apoptosis-related gene expression patterns (Fig. S4). Together, these findings suggest that rhythmic IL-17 enhances granulosa cell proliferation by core clock genes ( BMAL1 , CLOCK ) and the cell cycle regulator WEE1 .

Approximately 17.5% of individuals of reproductive age worldwide experience infertility according to WHO [ 1 ]. Lifestyle factors such as shift work and disordered eating are closely linked to menstrual irregularities, premature ovarian insufficiency, and polycystic ovary syndrome (PCOS) [ 2 – 4 ]. While these associations have been extensively studied in adults, puberty represents a critical developmental window that is particularly sensitive to lifestyle influences [ 5 ]. Precocious puberty has become a growing global public health concern because early puberty is associated with an increased risk of PCOS, premature menopause, type 2 diabetes, and psychological disorders [ 6 – 10 ]. Conversely, delayed puberty is associated with reduced fertility and an increased risk of infertility [ 11 , 12 ]. Optimizing the timing of puberty onset is a critical factor in improving reproductive efficiency and economic profitability in livestock industries. The appropriate timing of pubertal onset improves reproductive performance, including a younger age at first calving and an extended reproductive lifespan, thereby enhancing lifetime productivity in beef cattle and gilts [ 13 , 14 ]. Although the roles of energy intake and nutritional composition in the initiation of puberty have been extensively studied, the potential regulatory influence of eating time has not been adequately explored. Feeding time, as a key zeitgeber for peripheral tissues, can entrain their circadian rhythms. Misalignment between feeding time and the light-dark cycle can disrupt the peripheral biological clock in mice, resulting in metabolic disturbances [ 15 ]. Follicular growth in ovaries exhibits pronounced circadian rhythms that are tightly synchronized with endocrine signalling and the light-dark cycle [ 16 ]. The circadian clock genes regulate granulosa cell proliferation and steroidogenesis, ensuring the synchronization of ovarian function with hypothalamic-pituitary signalling under the influence of gonadotropins [ 17 – 19 ]. Previous studies have investigated the relationship between dietary components and ovarian circadian rhythms [ 20 ]. In contrast, research on feeding time has primarily focused on its effects on ovarian physiology. Restricting food intake to the inactive phase has been shown to reduce the number of corpora lutea in post-adolescent female rats [ 21 ]. However, the mechanisms by which feeding time modulates ovarian circadian rhythms to influence follicular development remain unclear. Disrupted feeding times can impair the circadian rhythmicity of metabolic hormones, such as insulin and leptin [ 22 , 23 ], and abnormalities in these hormones are associated with impaired ovarian development, increased granulosa cell apoptosis, and ovulatory dysfunction [ 24 , 25 ]. Beyond metabolic pathways, disrupted feeding times may induce gut microbiota dysbiosis and compromise epithelial barrier integrity, thereby facilitating the translocation of pro-inflammatory bacterial products, such as lipopolysaccharide (LPS), into the circulation and triggering low-grade systemic inflammation [ 26 ]. LPS can activate TLR4 signaling, stimulating granulosa cells to produce inflammatory cytokines such as IL-6 and IL-8, thereby impairing oocyte meiotic maturation [ 27 ]. Moreover, circadian oscillations of gut microbe–derived metabolites, such as short-chain fatty acids and bile acids, are under feeding time [ 28 , 29 ]. Propionate can promote follicular development and inhibiting granulosa cell apoptosis via the PI3K/AKT signaling pathway [ 30 ], whereas dysregulated bile acid metabolism may impair follicular development, ovulation, and steroidogenesis by reducing IL-22 level [ 31 ]. In patients with PCOS, reduced IL-22 levels are associated with granulosa cell mitochondrial dysfunction and impaired steroid hormone synthesis, whereas E. coli Nissle 1917 can increase IL-22 levels and improve mitochondrial function [ 32 ]. However, it remains unclear whether alterations in feeding time regulate ovarian rhythmicity by modulating circadian rhythms in metabolic and inflammatory signaling. Rabbits are nocturnal animals. However, it is quite common to adopt a daytime feeding regimen in rabbit production in accordance with the working hours of the staff. This misalignment between eating behavior and the light-dark cycle may lead to disrupt the circadian rhythmicity of the gut microbiota and short-chain fatty acids in rabbits [ 33 ]. It remains unclear whether feeding time influences pubertal follicular development by modulating circadian rhythm–related pathways in the ovary. The rabbit is a model for research in embryology and reproductive developmental biology, with significant physiological similarities to humans in areas such as fertilization, embryonic development, and hormonal regulation [ 34 ]. In this study, we performed ovarian transcriptome sequencing at two developmental stages, prepuberty (84 days of age) and puberty (145 days of age), to characterize the effects of feeding time on follicular development, and to determine whether these effects are mediated by circadian rhythm–related signaling pathways involved in pubertal follicular development.

Circadian rhythm disruption caused by improper diet times can lead to reproductive disorders in adult women, including menstrual irregularities, ovulatory dysfunction, and infertility [ 40 , 41 ]. However, whether such disruptions affect pubertal follicular development through ovarian circadian regulation remains poorly understood. In this study, night-restricted feeding altered the diurnal expression rhythms of IL-17 signaling pathway at day 84. Notably, feeding time significantly modulated the diurnal different of IL-17 signaling at day 145. Night-restricted feeding advanced pubertal onset, promoted follicular development, and enhanced granulosa cell proliferation. In vitro, rhythmic IL-17 treatment increased the rhythmic expression of core clock genes and promoted cell proliferation in granulosa cells. Collectively, our findings indicate that feeding time can regulate ovarian rhythmicity and pubertal development, which are linked to the rhythmic IL-17 signaling pathway. Feeding time acts as a key zeitgeber for peripheral clocks and modulates circadian rhythms in organs such as the liver, adipose tissue, and skeletal muscle [ 42 – 44 ]. Within the reproductive axis, feeding time can influence expression of clock genes in the hypothalamus [ 45 ]. Our study found that night-restricted feeding significantly induced circadian expression of ovarian pathways related to metabolism and proliferation at day 84, including purine metabolism, DNA replication, and cell cycle. Consistent with previous studies showing the impact of feeding time on metabolic pathways in the liver and adipose tissue [ 46 , 47 ], our findings also demonstrate its effect on ovarian pathways related to metabolism and proliferation. In our study, daytime feeding showed circadian enrichment of pathways such as ovarian steroidogenesis, proteoglycans in cancer, and antigen processing and presentation. This pattern suggests that the rhythmic regulation of steroidogenesis, inflammation, and apoptosis may be disrupted, potentially impairing normal follicular development. During antral follicle development, FSH stimulates CYP19A1 expression in granulosa cells, promoting E2 synthesis. Estrogen then acts synergistically with FSH to enhance granulosa cell proliferation and differentiation, thereby supporting follicular growth and maturation [ 48 ]. Ovarian inflammation can activate the TGF-β signaling pathway, leading to ovarian fibrosis, which reduces follicle numbers, promotes follicular atresia, and markedly lowers pregnancy rates [ 49 ]. At day 145, night-restricted feeding caused diurnal differences in high-energy metabolic pathways, including thermogenesis, oxidative phosphorylation, and the cell cycle. These pathways are essential for meeting the energy demands of granulosa cells. In contrast, daytime feeding triggered the activation of several immune and inflammation related pathways, such as T cell receptor signaling, Th17 cell differentiation, and the IL-17 signaling pathway. This may indicate a state of cellular stress, immune activation, or tissue remodeling in the ovary. Mistimed feeding in mice can disrupt immune rhythms and impair inflammatory defenses [ 50 , 51 ]. Studies have shown that abnormal proportions of CD8⁺ T cells disrupt immune balance within follicular fluid, impair granulosa cell proliferation, and accelerate the decline of ovarian reserve function [ 52 ]. Night-restricted feeding modulates the circadian expression pathways related to proliferation, metabolism, and inflammation. These effects may support normal follicular development and timely progression through puberty. Several pathways affected by feeding time are closely associated with metabolism and energy requirements. However, our previous study showed that the NRF group exhibited lower food intake, body weight, and body fat content than the DF group [ 53 ], whereas reduced adiposity is generally considered to delay pubertal onset and impair follicular development [ 54 , 55 ]. However, nighttime feeding was associated with earlier pubertal onset and accelerated follicular development, suggesting that although differences in energy intake and body composition may influence puberty and follicular development, but feeding time induced alterations in ovarian circadian rhythms may play a more pivotal role. Our study found that night-restricted feeding significantly accelerated follicular development in female rabbits during puberty, with increased granulosa cells proliferation and reduced apoptosis, suggesting that NRF may promote follicular growth and maturation by modulating granulosa cell. In our study, the total estrus rate in both groups exceeded 90% by approximately day 140, which is consistent with physiological developmental stages. According to classical developmental timelines, approximately one-third of female rabbits attain mating and ovulatory capacity by 14 weeks (approximately day 98), and this proportion increases to about two-thirds by 17 and 20 weeks [ 56 ]. The formation of primordial follicles in rabbits begins in the second postnatal week, and the emergence of antral follicles and the occurrence of follicular atresia occurs around 12 weeks of age (day 84), marking the completion of the first wave of follicular development [ 57 ]. We also found that night-restricted feeding increased the number of antral follicles. The formation of antral follicles is a key that follicular development has reached the ovulatory stage, and an increase in their number usually reflects activation of the hypothalamic-pituitary-gonadal (HPG) axis [ 58 ]. Impaired granulosa cell proliferation and increased apoptosis can suppress normal follicular development [ 59 ]. In addition, mice exhibiting precocious puberty show a significant increase in antral follicle numbers [ 60 ], suggesting a close association between antral follicle accumulation and early pubertal onset. Moreover, restricting food intake to the inactive (light) phase in mice has been shown to significantly reduce ovulation frequency [ 61 ], highlighting feeding rhythms as an important external cue in the regulation of reproductive function. Our study demonstrated that nighttime-restricted feeding significantly advanced pubertal onset in rabbits, potentially linked to enhanced circadian fluctuations of gonadotropins. NRF leads to diurnal variations in FSH. This pattern is similar to the rhythmic secretion of FSH observed in mice [ 62 , 63 ]. Feeding time influences the rhythms of insulin [ 64 ] and thyroid hormones [ 65 ], and this study suggests that follicle-stimulating hormone is similarly regulated, thereby impacting pubertal follicular development and reproductive function. In particular, FSH stimulates the growth of antral follicles and the secretion of estrogen, providing essential hormonal support for the preovulatory LH surge [ 66 ]. In addition, granulosa cell proliferation and apoptosis are key processes in follicular development. We also found that nighttime feeding induces diurnal differences in granulosa cell proliferation. Moreover, a circadian pattern of granulosa cell proliferation was observed for the first time: proliferation was lower during the day and higher at night. This pattern is similar to the circadian proliferation rhythms observed in other tissues, such as adipocytes, intestinal cells, and pancreatic β-cells [ 7 , 67 , 68 ]. These results further suggest that granulosa cells may exhibit circadian rhythmicity in proliferation, and this rhythmic variation may be one of the key mechanisms through which night-restricted feeding promotes follicular development. Under normal physiological conditions, inflammation plays a crucial role in follicular development and ovulation. Granulosa cells promote ovulation by producing prostaglandins and other inflammatory mediators [ 69 ]. Our study shows that nighttime feeding at 84 days of age maintains the circadian rhythm of the IL-17 signaling pathway, and at 145 days of age, nighttime feeding results in diurnal differences in IL-17 protein levels. We propose that IL-17 functions as a critical mediator linking feeding time, circadian regulation, and follicular development. Abnormal elevations of IL-17 in follicular fluid and serum have been closely associated with reproductive disorders, including PCOS, endometriosis, and OHSS [ 70 ]. Persistently elevated levels of IL-17 may disrupt immune homeostasis within the follicular microenvironment, impairing granulosa cell proliferation, differentiation, and hormone secretion, thereby inhibiting follicular development and normal ovulation. Notably, sustained IL-17 elevation in the follicular fluid of patients with PCOS is considered a key mechanism contributing to follicular arrest and ovulatory dysfunction [ 71 ]. Furthermore, IL-17 itself exhibits circadian rhythmicity [ 72 ], and maternal circadian disruption has been shown to induce aberrant IL-17 expression, resulting in impaired ovarian follicular development and reduced oocyte quality [ 73 ]. Our study reveals that NRF, by regulating the circadian fluctuations of IL-17, may promote normal follicular development, while the dysregulation of IL-17 in the DF group could be a key factor contributing to delayed follicular development. Our study found that rhythmic IL-17 treatment promotes the proliferation of rabbit ovarian granulosa cells, which is associated with the rhythmic expression of BMAL1 , CLOCK , and WEE1 . Previous research has shown that IL-17 stimulates the proliferation of human mesenchymal stem cells [ 74 ]. In mouse liver, the CLOCK-BMAL1 heterodimer drives the rhythmic expression of WEE1 , with peak levels at CT8 [ 75 ]. Mechanistic studies further reveal that knockdown of BMAL1 or CLOCK significantly reduces WEE1 expression and induces G2/M phase arrest through upregulation of the cell cycle inhibitor p21 [ 76 ]. Based on this evidence, we propose that rhythmic IL-17 promotes granulosa cell proliferation and cell cycle progression by modulating oscillations of BMAL1 / CLOCK and WEE1 , thereby serving as a key mediator linking feeding time to the circadian regulation of ovarian follicular development. Feeding time may regulate IL-17 signaling and its impact on ovarian function through multiple pathways. First, feeding time controls the rhythmic secretion of IL-17 by γδ17 T cells in adipose tissue [ 72 ], with this peripherally derived IL-17 potentially influencing expression levels and rhythmicity within the ovary. Second, gut microbiota and their metabolites modulate host immune responses, particularly affecting IL-17 production by Th17 cells, thereby contributing to immune homeostasis and the regulation of local inflammation [ 77 ]. For example, in experimental autoimmune encephalomyelitis (EAE), colonization of segmented filamentous bacteria (SFB) in the gut markedly promotes intestinal IL-17 expression and induces accumulation of IL-17 A⁺ CD4⁺ T cells in the central nervous system, exacerbating neuroinflammation [ 78 ]. This reveals that gut microbiota can mediate immune regulation in distant tissues via IL-17 signaling. A similar mechanism is observed in the testes, where gut microbiota-induced Th17 cell activation of IL-17 signaling impairs male reproductive function [ 79 ]. Collectively, these findings suggest that gut-derived IL-17 may play a key role in ovarian immune regulation. In summary, feeding time may influence ovarian IL-17 signaling by modulating the rhythmic expression of IL-17 in both adipose tissue and gut microbiota.

In conclusion, our study demonstrates that misalignment between feeding time and the light-dark cycle enhances the rhythmicity of the IL-17 signaling pathway in growing rabbits. Rhythmic IL-17 promotes granulosa cell proliferation, which may accelerate follicular development, reprogram ovarian developmental rhythms, and lead to an earlier onset of puberty. Collectively, these findings provide new insights into protecting pubertal reproductive health and preventing reproductive disorders through lifestyle modification, while also offer important implications for optimizing feeding management during animal growth stage, thereby extending reproductive lifespan and improving breeding efficiency in livestock.

Supplementary Material 1. Supplementary Material 1. Supplementary Material 2. Supplementary Material 2.