Calcitriol is involved in maintaining primordial follicle reserve through the inhibition of the PI3K/Akt signaling pathway.

First, the expression and localization of VDR were examined in ovaries from neonatal mice. The mRNA levels of Vdr were gradually decreased in mouse ovaries from 1 to 7 dpp (Fig.  1 A). We further compared the mRNA levels in oocytes and somatic cells, in which approximately 15,000 somatic cells and 120 oocytes were used in each group. Compared to somatic cells, oocytes exhibited significantly higher Vdr mRNA levels (Fig.  1 B). Immunofluorescence staining analysis in the ovaries of mice at 7 dpp also showed that VDR levels were significantly higher in oocytes (cytoplasm and nucleus) than those in primordial follicle pre-granulosa cells (Fig.  1 C, D, Fig. S1A). VDR levels were decreased in oocytes but increased in granulosa cells during the transition of primordial follicles to growing follicles (Fig.  1 C, D, Fig. S1A). Fig. 1 Calcitriol inhibits neonatal mouse primordial follicle activation in vitro. A and B The Vdr mRNA expression levels in 1, 4, 7 dpp mouse ovaries ( A ) and in 4 dpp mouse somatic cells and oocytes ( B ), n  = 4, and four ovaries were used in each repetition. C and D VDR immunofluorescence staining of 1, 4, 7 dpp mouse ovaries ( C ) and the relative fluorescence intensity analysis in oocytes and granulosa cells from 7 dpp mouse ovaries ( D ), n  = 9, and one section was used in each repetition. VDR, green; DDX4, red. E and F The ovarian morphological analysis ( E ) and follicle counts ( F ) of neonatal mouse ovaries after 4-day culture without or with 50 ng/mL calcitriol, 0.5 µM TEI-9647, or 1 µM MeTC7, n  = 4, and four ovaries were used in each repetition. The image is a representative picture from the largest section of the ovary. G and H The comparison of Gdf9 , Zp3 , Ki-67 , Pcna , Caspase-3 , and Bax/Bcl-2 mRNA expression levels ( G ) and GDF9, PCNA, and Cleaved Caspase-3 protein levels ( H ) in neonatal mouse ovaries after a 2-day culture without or with 50 ng/mL calcitriol, n  = 4, and four ovaries were used in each repetition. I-K Immunofluorescence staining for BrdU, Cleaved Caspase-3, Ki-67, and PCNA (green, I ), proportion of granulosa cells with positive signals ( J ), and number of cells with positive signals ( K ) in neonatal mouse ovaries after a 2-day culture without or with 50 ng/mL calcitriol, n  = 4, and four ovaries were used in each repetition. PF, primordial follicle; PrF, primary follicle. FOXL2, red; DAPI, blue. Hematoxylin dye showed cell nuclei. Red arrows, growing follicles; red arrowheads, primordial follicles; white arrowheads, positive signal cells. Scale bar = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 Calcitriol inhibits neonatal mouse primordial follicle activation in vitro. A and B The Vdr mRNA expression levels in 1, 4, 7 dpp mouse ovaries ( A ) and in 4 dpp mouse somatic cells and oocytes ( B ), n  = 4, and four ovaries were used in each repetition. C and D VDR immunofluorescence staining of 1, 4, 7 dpp mouse ovaries ( C ) and the relative fluorescence intensity analysis in oocytes and granulosa cells from 7 dpp mouse ovaries ( D ), n  = 9, and one section was used in each repetition. VDR, green; DDX4, red. E and F The ovarian morphological analysis ( E ) and follicle counts ( F ) of neonatal mouse ovaries after 4-day culture without or with 50 ng/mL calcitriol, 0.5 µM TEI-9647, or 1 µM MeTC7, n  = 4, and four ovaries were used in each repetition. The image is a representative picture from the largest section of the ovary. G and H The comparison of Gdf9 , Zp3 , Ki-67 , Pcna , Caspase-3 , and Bax/Bcl-2 mRNA expression levels ( G ) and GDF9, PCNA, and Cleaved Caspase-3 protein levels ( H ) in neonatal mouse ovaries after a 2-day culture without or with 50 ng/mL calcitriol, n  = 4, and four ovaries were used in each repetition. I-K Immunofluorescence staining for BrdU, Cleaved Caspase-3, Ki-67, and PCNA (green, I ), proportion of granulosa cells with positive signals ( J ), and number of cells with positive signals ( K ) in neonatal mouse ovaries after a 2-day culture without or with 50 ng/mL calcitriol, n  = 4, and four ovaries were used in each repetition. PF, primordial follicle; PrF, primary follicle. FOXL2, red; DAPI, blue. Hematoxylin dye showed cell nuclei. Red arrows, growing follicles; red arrowheads, primordial follicles; white arrowheads, positive signal cells. Scale bar = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 Then, we used VDR ligand (calcitriol) and inhibitors (TEI-9647 and MeTC7) to study their effects on primordial follicle activation [ 30 – 32 ]. Three dpp mouse ovaries were incubated without or with these drugs for 4 days. Compared with control, calcitriol decreased while TEI-9647 and MeTC7 increased the number of growing follicles in a dose-dependent manner. The optimal concentrations were 50 ng/mL, 0.5 µM and 1 µM for calcitriol, TEI-9647 and MeTC7, respectively (Fig. 1 E, F, Fig. S1B-D). Calcitriol treatment for 2 days significantly decreased zona pellucida 3 ( Zp3 ), growth differentiation factor 9 ( Gdf9 ), Ki-67 , and proliferating cell nuclear antigen ( Pcna ) mRNA levels (Fig. 1 G). Consistent with these, calcitriol significantly decreased GDF9 and PCNA protein levels, but had no effect on Cleaved Caspase-3 protein levels (Fig. 1 H). Furthermore, calcitriol significantly decreased the percentage of Ki-67- and PCNA-positive granulosa cells and the number of BrdU-positive somatic cells, but had no effect on the number of cells exhibiting Caspase-3-positive signals (Fig. 1 I-K). Thus, calcitriol inhibits mouse primordial follicle activation in vitro. We further investigated the mechanism of calcitriol in inhibiting the activation of mouse primordial follicles. Compared with the control, calcitriol significantly decreased, while TEI-9647 and MeTC7 significantly increased p-Akt protein levels in cultured neonatal mouse ovaries (Fig.  2 A). Consistent with this, calcitriol significantly decreased the Akt downstream effector p-FOXO3a protein levels (Fig.  2 A). Immunofluorescence staining analysis also revealed that, compared to the control, calcitriol significantly decreased the proportion of oocytes in primordial follicles exhibiting FOXO3a nuclear export (Fig.  2 B, C). Fig. 2 The mechanism of calcitriol on mouse primordial follicle activation. A The comparison of p-mTOR, p-Akt, and p-FOXO3a protein levels in neonatal mouse ovaries after 1-day culture without or with 50 ng/mL calcitriol, 0.5 µM TEI-9647, or 1 µM MeTC7, n  = 4, and four ovaries were used in each repetition. B , C FOXO3a localization in the cytoplasm of primordial follicle oocytes (white arrows, B ) and the proportion of primordial follicle oocytes with the nuclear export of FOXO3a ( C ), n  = 4, and five sections from one ovary were used in each repetition. D Co-IP assay showed that calcitriol increases the interaction of RXR with both VDR and p85α in neonatal cultured mouse ovaries after 1-day culture without or with 50 ng/mL calcitriol, 0.5 µM TEI-9647, or 1 µM MeTC7, n  = 4, and four ovaries were used in each repetition. Green, FOXO3a; red, DDX4. Scale bar = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 The mechanism of calcitriol on mouse primordial follicle activation. A The comparison of p-mTOR, p-Akt, and p-FOXO3a protein levels in neonatal mouse ovaries after 1-day culture without or with 50 ng/mL calcitriol, 0.5 µM TEI-9647, or 1 µM MeTC7, n  = 4, and four ovaries were used in each repetition. B , C FOXO3a localization in the cytoplasm of primordial follicle oocytes (white arrows, B ) and the proportion of primordial follicle oocytes with the nuclear export of FOXO3a ( C ), n  = 4, and five sections from one ovary were used in each repetition. D Co-IP assay showed that calcitriol increases the interaction of RXR with both VDR and p85α in neonatal cultured mouse ovaries after 1-day culture without or with 50 ng/mL calcitriol, 0.5 µM TEI-9647, or 1 µM MeTC7, n  = 4, and four ovaries were used in each repetition. Green, FOXO3a; red, DDX4. Scale bar = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 The PI3K regulatory subunit p85α and catalytic subunit p110 are essential for regulating Akt activity [ 33 ]. Calcitriol exerts its effects by interacting with RXR [ 34 , 35 ], and RXR positively regulates the PI3K/Akt pathway via its non-transcriptional function through a direct interaction with the p85α regulatory subunit [ 36 ]. Thus, we investigated the interaction between RXR, VDR and p85α in neonatal mouse ovaries. The Co-IP assay verified that calcitriol treatment increased the binding of RXR to both VDR and p85α, and enhanced the interaction between PI3K subunits p110 and p85α (Fig. 2 D). This suggests that calcitriol promotes the binding of RXR to p85α to reduce PI3K p110 subunit activity and consequently decreases p-Akt levels. RNA-seq analyses revealed significant transcriptomic alterations between the calcitriol and control groups, with 126 upregulated and 101 downregulated transcripts (Fig.  3 A). The expression changes of the representative transcripts were validated by RT-qPCR (Fig.  3 B). Furthermore, we used gene enrichment analysis. The downregulated transcripts were enriched in the PI3K/Akt pathway, developmental processes, and calcium ion binding (Fig.  3 C-E), and the upregulated transcripts were prominently associated with signaling receptor binding and steroid metabolic processes (Fig.  3 E). These results indicate that calcitriol inhibits mouse primordial follicle activation via the VDR-mediated PI3K/Akt pathway. Fig. 3 Effects of calcitriol on the transcriptome in cultured neonatal mouse ovaries. The 3 dpp mouse ovaries were cultured without or with 50 ng/mL calcitriol for 1 day. A Volcano plot illustrating differentially expressed genes between the control and calcitriol groups in the ovaries. B RT-qPCR was conducted to validate the transcriptomic changes detected by RNA-seq, n  = 4, and four ovaries were used in each repetition. C Bubble chart showing enriched gene ontology (GO) terms associated with significantly changed transcripts between control and calcitriol treatment. The x-axis represents the gene ratio, defined as the proportion of enriched genes relative to the total genes in the corresponding pathway. D Gene set enrichment analysis (GSEA) revealing enrichment of the PI3K regulator activity in the control group relative to the calcitriol group. NES, normalized enrichment score. E Heatmaps revealing differential expression profiles of multiple transcripts involved in various processes between control and calcitriol treatment. Data are presented as mean ± SD of three biological independent experiments. * p  < 0.05, ** p  < 0.01 Effects of calcitriol on the transcriptome in cultured neonatal mouse ovaries. The 3 dpp mouse ovaries were cultured without or with 50 ng/mL calcitriol for 1 day. A Volcano plot illustrating differentially expressed genes between the control and calcitriol groups in the ovaries. B RT-qPCR was conducted to validate the transcriptomic changes detected by RNA-seq, n  = 4, and four ovaries were used in each repetition. C Bubble chart showing enriched gene ontology (GO) terms associated with significantly changed transcripts between control and calcitriol treatment. The x-axis represents the gene ratio, defined as the proportion of enriched genes relative to the total genes in the corresponding pathway. D Gene set enrichment analysis (GSEA) revealing enrichment of the PI3K regulator activity in the control group relative to the calcitriol group. NES, normalized enrichment score. E Heatmaps revealing differential expression profiles of multiple transcripts involved in various processes between control and calcitriol treatment. Data are presented as mean ± SD of three biological independent experiments. * p  < 0.05, ** p  < 0.01 We further investigated the function of calcitriol in mouse primordial follicle activation in vivo. Three dpp female mice received intraperitoneal injections of either physiological saline (control) or 0.05 mg/kg calcitriol (an amount equivalent to 50 ng/mL) twice daily for two consecutive days. Follicle counting analysis showed that calcitriol significantly decreased growing follicle numbers but had no effect on the primordial follicle numbers compared with control (Fig.  4 A, B). Western blot results showed that calcitriol significantly decreased p-Akt and p-FOXO3a protein levels but had no effect on p-mTOR protein levels compared with control (Fig.  4 C, D). Immunofluorescence staining analysis revealed that calcitriol significantly decreased the percentage of primordial follicle oocytes that exhibited FOXO3a nuclear export compared with control (Fig.  4 E, F). These results suggest that calcitriol inhibits primordial follicle activation in mice in vivo. Fig. 4 Effects of calcitriol on mouse primordial follicle activation in vivo. A , B The ovary morphologies ( A ) and the number of growing and primordial follicles ( B ) in neonatal mouse ovaries collected from the mice 48 h after intraperitoneal injection with physiological saline (control) or 0.05 mg/kg calcitriol twice a day for two consecutive days, n  = 4, and four ovaries were used in each repetition. The image is a representative picture from the largest section of the ovary. C , D The comparison of p-mTOR, p-Akt, and p-FOXO3a levels in neonatal mouse ovaries collected from the mice 24 h after intraperitoneal injection with physiological saline (control) or 0.05 mg/kg calcitriol twice a day for two consecutive days, n  = 4, and four ovaries were used in each repetition. E , F FOXO3a localization in primordial follicle oocyte cytoplasm (white arrows, E ) and the proportion of primordial follicle oocytes displaying FOXO3a nuclear export ( F ), n  = 4, and five sections from one ovary were used in each repetition. Hematoxylin dye showed the cell nuclei. Red, DDX4; green, FOXO3a. Red arrows, growing follicles. Scale bar = 50 μm. ** p  < 0.01 Effects of calcitriol on mouse primordial follicle activation in vivo. A , B The ovary morphologies ( A ) and the number of growing and primordial follicles ( B ) in neonatal mouse ovaries collected from the mice 48 h after intraperitoneal injection with physiological saline (control) or 0.05 mg/kg calcitriol twice a day for two consecutive days, n  = 4, and four ovaries were used in each repetition. The image is a representative picture from the largest section of the ovary. C , D The comparison of p-mTOR, p-Akt, and p-FOXO3a levels in neonatal mouse ovaries collected from the mice 24 h after intraperitoneal injection with physiological saline (control) or 0.05 mg/kg calcitriol twice a day for two consecutive days, n  = 4, and four ovaries were used in each repetition. E , F FOXO3a localization in primordial follicle oocyte cytoplasm (white arrows, E ) and the proportion of primordial follicle oocytes displaying FOXO3a nuclear export ( F ), n  = 4, and five sections from one ovary were used in each repetition. Hematoxylin dye showed the cell nuclei. Red, DDX4; green, FOXO3a. Red arrows, growing follicles. Scale bar = 50 μm. ** p  < 0.01 In the oral administration experiment, 3-week female mice were provided with drinking water containing 0–40 ng/mL VD3 for 1 week to screen the optimal concentration (Fig.  5 A). Hematoxylin and immunofluorescence staining for ovarian serial sections were used for follicle counting analysis. VD3 treatment increased primordial follicle numbers, and decreased primary and secondary follicle numbers in a dose-dependent manner, with the optimal concentration of 30 ng/mL (Fig.  5 B, Fig. S2A). The daily intake of VD3 was 12 µg/kg (10.6–13.4 µg/kg/d). Fig. 5 Oral administration of VD3 contributes to maintaining the primordial follicle reserve and prolonging fertility in female mice. A The timeline of the experimental scheme. B Immunofluorescence staining of the ovaries in the mice after 1 week of feeding without or with 30 ng/mL VD3. C , D The comparison of ovary morphology ( C ) and follicle analysis at different age stages ( D ) in the mice after 20 weeks of feeding with 30 ng/mL VD3, n  = 5, and four ovaries were used in each repetition. The image is a representative picture from the largest section of the ovary. E Cumulative numbers of pups per mouse after a 40-week fertility test in control and 30 ng/mL VD3 groups. The data are the average of 15 mice. Hematoxylin dye showed the cell nuclei. Red arrows, growing follicles; red arrowheads, primordial follicles. Scale bar = 50 μm. * p  < 0.05, ** p  < 0.01 Oral administration of VD3 contributes to maintaining the primordial follicle reserve and prolonging fertility in female mice. A The timeline of the experimental scheme. B Immunofluorescence staining of the ovaries in the mice after 1 week of feeding without or with 30 ng/mL VD3. C , D The comparison of ovary morphology ( C ) and follicle analysis at different age stages ( D ) in the mice after 20 weeks of feeding with 30 ng/mL VD3, n  = 5, and four ovaries were used in each repetition. The image is a representative picture from the largest section of the ovary. E Cumulative numbers of pups per mouse after a 40-week fertility test in control and 30 ng/mL VD3 groups. The data are the average of 15 mice. Hematoxylin dye showed the cell nuclei. Red arrows, growing follicles; red arrowheads, primordial follicles. Scale bar = 50 μm. * p  < 0.05, ** p  < 0.01 Next, 3-week female mice were provided with water supplemented with or without 30 ng/mL VD3 until 23 weeks, and then for fertility testing until 63 weeks (Fig.  5 A). A subset of mice was utilized for follicle counting in weeks 7, 13 and 23. The results showed that the number of primordial follicles was significantly increased, and the numbers of primary, secondary and antral follicles were significantly decreased in the ovaries of VD3 treated mice compared with the corresponding controls (Fig.  5 C, D). However, VD3 treatment had no obvious effect on vital organs and body weight (Fig. S2B-D). The fertility test results showed that the female mice ceased reproduction at 32 weeks and 36 weeks in the control group and the VD3 treatment group, respectively (Fig.  5 E). In the fertility test, the total number of pups in the VD3 group was slightly decreased compared to the control group. These results suggest that VD3 treatment contributes to maintaining the primordial follicle reserve and prolonging reproductive age in female mice. Finally, we investigated the expression characteristics of VDR and the effect of calcitriol on primordial follicle activation in human ovarian tissue. Immunofluorescence staining showed that VDR levels were significantly higher in pre-granulosa than those in oocytes (cytoplasm and nucleus) of primordial follicles (Fig.  6 A, B). VDR levels were increased in oocytes during the transition of primordial follicles to growing follicles (Fig.  6 B). Compared with control, calcitriol significantly decreased the percentage of growing follicles in 6-day cultured human ovarian fragments (Fig.  6 C, D). Additionally, calcitriol significantly decreased p-Akt and p-FOXO3a protein levels and had no effect on p-mTOR protein levels in 4-day cultured human ovarian fragments (Fig.  6 E). Compared with the uncultured group, the p-Akt and p-FOXO3a protein levels and growing follicle proportion were significantly increased in the control, indicating that culture promotes follicle growth (Fig.  6 D, E). Thus, calcitriol inhibits PI3K/Akt activity and suppresses the activation of primordial follicles in human ovarian tissue. Fig. 6 Calcitriol inhibits human primordial follicle activation in vitro. A , B VDR immunofluorescence staining of human ovarian fragments ( A ) and the relative fluorescence intensity analysis in granulosa cells and oocytes B . VDR, green; DDX4, red; DAPI, blue. C , D Comparative analysis of human ovarian fragment morphology ( C ) and quantification of growing and primordial follicle proportions ( D ) in fragments that were uncultured or cultured for 6 days without or with 50 ng/mL calcitriol, n  = 4, and one human ovarian fragment was used in each repetition. E The comparison of p-FOXO3a, p-mTOR, and p-Akt protein levels in human ovarian fragments that were uncultured or cultured for 4 days without or with 50 ng/mL calcitriol, n  = 4, and one human ovarian fragment was used in each repetition. PF, primordial follicle; PrF, primary follicle. The small dashed boxes indicate the enlarged part of the images. Hematoxylin dye showed the cell nuclei. Red arrows, growing follicles; red arrowheads, primordial follicles; white arrows, growing follicles; white arrowheads, primordial follicles. Scale bar = 50 μm. * p  < 0.05, ** p  < 0.01 Calcitriol inhibits human primordial follicle activation in vitro. A , B VDR immunofluorescence staining of human ovarian fragments ( A ) and the relative fluorescence intensity analysis in granulosa cells and oocytes B . VDR, green; DDX4, red; DAPI, blue. C , D Comparative analysis of human ovarian fragment morphology ( C ) and quantification of growing and primordial follicle proportions ( D ) in fragments that were uncultured or cultured for 6 days without or with 50 ng/mL calcitriol, n  = 4, and one human ovarian fragment was used in each repetition. E The comparison of p-FOXO3a, p-mTOR, and p-Akt protein levels in human ovarian fragments that were uncultured or cultured for 4 days without or with 50 ng/mL calcitriol, n  = 4, and one human ovarian fragment was used in each repetition. PF, primordial follicle; PrF, primary follicle. The small dashed boxes indicate the enlarged part of the images. Hematoxylin dye showed the cell nuclei. Red arrows, growing follicles; red arrowheads, primordial follicles; white arrows, growing follicles; white arrowheads, primordial follicles. Scale bar = 50 μm. * p  < 0.05, ** p  < 0.01

ICR mice aged 3 weeks and 2 months were provided by the Guangdong Medical Laboratory Animal Center (Guangzhou, China). The animals were kept at South China University of Technology under controlled environmental conditions. Two-month-old mice were used to breed neonatal mice, and the date of birth was considered 0.5 days postpartum (dpp). In the oral administration trials, three-week-old female mice were used. All animal protocols received approval from the Institutional Animal Care and Use Committee of South China University of Technology (Approval No. 2024125), and all reagents were sourced from Sigma-Aldrich (St. Louis, MO, USA) unless specified otherwise. The collection of somatic cells and oocytes was carried out as described previously [ 12 , 25 ]. The ovaries were isolated from 4 dpp mice, and then were digested with 0.05% trypsin (Thermo Scientific, Waltham, MA, USA. 15050065) at 37 °C for 10 min. The digestion was stopped by adding 10% fetal bovine serum (FBS, Thermo Scientific. A5670701). Under a stereomicroscope, all oocytes were collected and then transferred to phosphate buffered saline (PBS) containing 0.2% bovine serum albumin (BSA) for 3–5 times until few somatic cells were observed. Somatic cells (principally pre-granulosa cells) were collected by centrifugation. In each experiment, approximately 15,000 somatic cells and 120 oocytes from 4−6 mice were obtained and stored at − 80 °C for RNA analysis. The culture procedure of neonatal mouse ovaries was carried out as described previously [ 12 , 25 ]. In summary, 3–4 ovaries isolated from 3 dpp mice were cultured on the membrane (PICMORG50, Millipore, Billerica, MA, USA) inserts in six-well culture plates (NEST, Beijing, China). Cultures were treated without or with 0-100 ng/mL calcitriol, 0–1 µM TEI-9674 (MedChemExpress, Monmouth Junction, NJ, USA), and 0–2 µM MeTC7 (MedChemExpress) for designated days. Following two days of culture, 10 µM bromodeoxyuridine (BrdU) was added to the medium for further 2 h to conduct the incorporation assay. All reagents were initially dissolved in dimethyl sulfoxide (DMSO) as stock solutions, with the control group receiving an equivalent volume of DMSO. The ovaries were cultured under the same standard conditions (37 °C, 5% CO 2 ), with the medium being refreshed every 2 days. At designated times, the ovaries were collected for analyses of gene expression, protein levels, immunofluorescence staining, and follicle counting. The extraction of total RNA from mouse ovarian tissues, somatic cells, and oocytes, and the synthesis of cDNA were performed as described previously [ 12 , 25 ]. 100–200 ng/µL RNA was obtained from each treatment, and 1 µg RNA was used for cDNA synthesis. Quantitative real-time PCR (qPCR) was performed using PerfectStart ® Green qPCR SuperMix (Trans, Beijing, China) on a LightCycler 96 system (Roche, Basel, Switzerland). The relative expression of target genes was assessed via the 2 − ΔΔCT method, with ribosomal protein L19 (Rpl19) serving as the internal control. Additionally, RNA-seq analysis was conducted on ovaries from the calcitriol and control groups after one day of culture. All qPCR primer sequences, along with the annealing temperatures and expected DNA sizes, are provided in Supplementary Table S1. Ovarian tissue specimens were fixed overnight using 4% paraformaldehyde (PFA) from Solarbio (Beijing, China). Subsequently, the fixed specimens were embedded in paraffin and serially sectioned at 5 μm in thickness. These sections were transferred to the glass slides. The slides were processed through xylene for deparaffinization and a graded alcohol series for hydration. Subsequently, tissues were subjected to hematoxylin staining (Solarbio) for tissue visualization. Mouse and human primordial and growing follicles were identified using the classification criteria defined in previous work [ 26 ], and their number was counted as described previously [ 26 ]. To prevent double counting, only follicles containing a visible oocyte nucleus were included. All assessments were independently verified by two observers. Mouse ovarian sections from different treatments were prepared as above. Human ovarian sections were obtained from an 8-year-old girl with benign hematologic diseases as described before [ 27 ]. Immunofluorescence staining was carried out in the same way as before [ 12 , 25 ]. Briefly, ovarian tissue sections were subjected to antigen retrieval in 0.01% sodium citrate buffer, blocked with donkey serum, and incubated overnight with primary antibodies, followed by a one-hour incubation with corresponding secondary antibodies. Following nuclear staining with 4’,6-diamidino-2-phenylindole (DAPI; Beyotime, Beijing, China). The stained sections were imaged using a confocal laser scanning microscope (LSM 800, Carl Zeiss, Germany). The relative fluorescence intensity was determined using Zeiss Zen 3.0 software (Carl Zeiss) by dividing the cell fluorescence intensity by the background fluorescence intensity. To quantify the expression, the five largest sections from each ovary were selected for analysis. A granulosa cell was considered positive if it showed co-localization of PCNA/FOXL2 or Ki-67/FOXL2. Additionally, granulosa cells showing BrdU or Caspase-3 signal were also considered positive. The five largest sections of each ovary were used to analyze the proportion of positive cells and FOXO3a nuclear export. The average value of three ovaries was used as one repetition. In each group, total protein was extracted from six neonatal mouse ovaries and several human tissue fragments. Western blot was carried out in the same way as before [ 12 , 25 ]. The protein concentration was determined via a bicinchoninic acid (BCA) assay (P0012, Beyotime). A total of 20 µg protein (10 µl from a 2 µg/µL sample) was separated on 10% SDS-polyacrylamide gels (Vazyme, Nanjing, China. E303-01). Subsequently, the separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking with 5% skim milk (Beyotime, Beijing, China, P0216) for one hour, the membranes were incubated overnight at 4 °C with 1:1000 primary antibodies as detailed in Supplementary Table S2. Next, the membranes underwent an incubation with 1:5000 secondary antibodies of anti-mouse or anti-rabbit (ZSGB-BIO, Beijing, China) for one hour at room temperature. Target proteins were detected using the Super-Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, MA, USA), and chemiluminescence signals were captured with a Tanon 5200 Imaging System (Tanon, Shanghai, China). Quantitative analysis of band intensity was performed with ImageJ software (NIH Image, Bethesda, MD, USA). Protein levels were normalized to the corresponding unphosphorylated forms or GAPDH. Supplementary Figure S3 showed the uncropped blots. After 24 hours of culture, the neonatal mouse ovaries were collected and extracted using RIPA buffer (Thermo Scientific). The immunoprecipitation was conducted following the manufacturer’s instructions of Dynabeads™ Protein A immunoprecipitation kit (Thermo Scientific). For co-immunoprecipitation (Co-IP) assays, the protein samples were first incubated with beads coated with normal rabbit IgG to pre-clear nonspecific bindings. Subsequently, approximately 400 µg of total protein was incubated with 1.5 mg of antibody-coated Dynabeads slurry (50 µL from a 30 mg/mL stock) at 4 °C with rotation overnight. Subsequently, the beads were washed with washing buffer and denaturing elution was performed. The eluted protein samples (20 µg) and the reserved input control (20 µg, approximately 5%) were subjected to western blot and incubated with corresponding antibodies. In the intraperitoneal injection experiment, 3 dpp female mice were administered DMSO (control) or 0.05 mg/kg calcitriol via intraperitoneal injection twice daily on two consecutive days. Each volume was no more than 5 µL per mouse. A single dose of calcitriol for injection equals its cultured concentration of 50 ng/mL, in which the unit from a volumetric (mg/L) to a body-weight-based (mg/kg) ratio. The ovaries were collected at 24 hours post-injection for immunofluorescence staining and protein detection, and at 48 hours for follicle counting. For the oral administration experiment, 3-week (adolescent) female mice received water containing 0–40 ng/mL calcitriol precursor VD3. The concentration of VD3 in the drinking water was determined according to the daily water consumption of the mice [ 28 , 29 ] and the effective dose of calcitriol. The ovaries were harvested after 1, 4, 10, and 20 weeks for total ovarian follicle counting. To determine the effects on fertility, the female mice without or with 20 weeks of VD3 treatment were further fed with drug-free water for mating with fertile adult male mice until 63 weeks. The number of neonatal mice was analyzed every two weeks to plot a reproductive curve. The ovarian tissues from five cancer patients (23–43 years old) were cryopreserved for fertility preservation, and the residual ovarian cortex was collected for culture. The patient information were provided in Supplementary Table S3. All patients provided written informed consent before undergoing endometriosis laparoscopy at The Sixth Affiliated Hospital of Sun Yat-sen University. This study protocol was approved by the Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-sen University. The research was conducted in accordance with the ethical principles of the Declaration of Helsinki. The ovarian fragments were transported to the lab using precooled PBS that contained penicillin–streptomycin. Under aseptic conditions, the non-pathological ovarian tissues were dissected into small cubes of approximately 1 mm³ for western blot and follicle counting as described above. Some fragments were cultured in medium without or with 50 ng/mL calcitriol. These fragments were then collected for protein expression after a 4-day culture and for follicle counting after a 6-day culture, under the same conditions as those for mouse ovaries. The data were collected from the experiments with no less than three repetitions. GraphPad Prism (v8.0.1, La Jolla, CA, USA) was employed for statistical analysis and figure generation. Data are presented as the mean ± standard deviation (SD). Differences between groups were assessed using a two-tailed unpaired Student’s t-test, with a p -value of less than 0.05 considered statistically significant.

As an essential fat-soluble vitamin in humans, VD3 is critical for reproductive function via its active form, calcitriol [ 37 ]. In the present study, calcitriol receptor VDR was expressed in primordial follicle oocytes. Calcitriol inhibited primordial follicle activation in both mice and humans through the VDR-mediated PI3K/Akt signaling pathway. Furthermore, oral administration of VD3 maintained the primordial follicle reserve and extended the reproductive age in female mice. These findings support the role of calcitriol as a regulator of primordial follicle activation. VDR was expressed in both primordial follicle pre-granulosa cells and oocytes in mouse and human ovaries, and the levels decreased in oocytes but gradually increased in granulosa cells during primordial follicle activation and growth. A previous study in rhesus macaques reported that VDR is predominantly localized in granulosa cells and oocytes, and that its levels increase in granulosa cells during the transition from primordial to growing follicles, which is consistent with our findings [ 18 ]. The VDR ligand calcitriol inhibited, and VDR inhibitors promoted the activation of primordial follicles within ovaries from neonatal mice through ovarian culture and intraperitoneal injection. Calcitriol also inhibited the activation of human primordial follicles. These findings indicate that calcitriol and VDR are crucial for maintaining the dormancy of primordial follicles. In addition to the nucleus, VDR was also expressed in the cytoplasm of mouse and human primordial follicle oocytes. Research in rhesus macaques has confirmed that VDR is expressed in both the cytoplasm and nucleus of primordial follicle oocytes [ 18 ]. The VDR ligand calcitriol decreased p-Akt levels in both mouse ovaries and human ovarian tissues. RNA-seq analysis also revealed that the downregulated transcripts in calcitriol-treated ovaries were associated with the PI3K/Akt pathway. This is consistent with previous studies showing that calcitriol can inhibit the PI3K/Akt signaling pathway, thereby suppressing the proliferation of cancer cells [ 38 – 40 ]. Furthermore, calcitriol promoted the formation of the VDR-RXR-p85α complex and inhibited the activity of p110, thereby suppressing Akt activity. Consistent with previous findings, the p85α subunit inhibits the catalytic activity of p110, resulting in reduced phosphorylation of Akt in human endometrial cancer cells [ 41 ]. Oocyte PI3K/Akt signaling is a key regulator of primordial follicle activation. Therefore, calcitriol maintains the dormancy of primordial follicles by suppressing the PI3K/Akt signaling pathway in primordial follicle oocytes. Notably, human life expectancy has increased from 45 to 85 years over the past 150 years, but the time of reproductive aging has remained relatively unchanged [ 42 ]. On the other hand, delayed marriage and childbearing further shorten the effective reproductive period for women. In the present study, we demonstrated that the oral administration of VD3 extended the reproductive age by delaying primordial follicle depletion. In addition, the oral dose of VD3 used for mice was 12 µg/kg/d (an amount equivalent to 0.96 µg/kg/d for humans). A daily dose of 4000 IU VD3 (an amount equivalent to approximately 1.66 µg/kg/d) has been established as a safe long-term threshold with no overall toxicity [ 43 ]. Therefore, we propose that VD3 may represent a promising novel strategy to preserve ovarian reserve by mitigating primordial follicle depletion, thereby extending the female reproductive age. On the other hand, VDR inhibitors may provide a potential therapeutic strategy for patients with POI. In conclusion, calcitriol inhibited primordial follicle activation in both mice and humans via the VDR-mediated PI3K/Akt signaling pathway. Oral administration of VD3 maintained the primordial follicle reserve and extended the reproductive age in female mice. Considering its efficacy and safety, VD3 may prolong the female reproductive age by preserving the ovarian reserve.

In female mammals, the primordial follicle reserve is established perinatally and serves as a sustained source of oocytes for reproduction [ 1 ]. The primordial follicle consists of a single layer of flattened pre-granulosa cells surrounding a relatively quiescent oocyte [ 2 ]. The flat pre-granulosa cells differentiate into cuboidal cells, and the oocyte increases in diameter upon activation of primordial follicles [ 3 ]. The mammalian target of rapamycin (mTOR) promotes the expression of the receptor tyrosine kinase KIT ligand (KITL) in pre-granulosa cells [ 4 ]. The binding of KITL to its receptor KIT then activates the PI3K/Akt pathway in oocytes, resulting in the phosphorylation and nuclear export of FOXO3a [ 2 , 5 ]. Oocyte PI3K/Akt signaling also promotes the dissociation of the Balbiani body (B-body), leading to the release and translation of stored mRNAs required for primordial follicle activation [ 6 ]. Once primordial follicles are excessively activated, their depletion is accelerated, ultimately resulting in premature ovarian insufficiency (POI) [ 7 ]. Therefore, strict regulation of primordial activation are key determinants of female reproductive age. Primordial follicle activation is subject to precise regulation. In pre-granulosa cells, the downstream effector of transforming growth factor-beta (TGF-β), Sma- and Mad-related protein family member 3 (SMAD3), inhibits cell cycle gene transcription and stabilizes the p27 complex to delay primordial follicle activation [ 8 ]. Histone deacetylase 6 (HDAC6) also participates in maintaining primordial follicle dormancy by inhibiting mTOR activity [ 9 , 10 ]. In contrast, glycolysis triggers primordial follicle activation by inhibiting AMP-activated protein kinase (AMPK) to relieve the suppression of mTOR [ 4 ], and bone morphogenetic protein 4 (BMP4) activates primordial follicles by the SMAD1/5/8 signaling pathway to upregulate KITL expression [ 11 ]. In primordial follicle oocytes, the highly expressed phosphodiesterases (PDEs) maintain primordial follicle dormancy by degrading cyclic adenosine monophosphate (cAMP) and attenuating PKA-mediated PI3K/Akt signaling [ 12 ]. On the contrary, cell division cycle 42 (CDC42) binds to the PI3K subunit p110β and inhibits PTEN expression, initiating primordial follicle activation [ 13 ]. Sirtuin-1 (SIRT1) directly enhances the transcriptional expression of PI3K/Akt and then promotes primordial follicle activation [ 14 ]. Vitamin D (VD), a crucial nutrient and hormone precursor, primarily exists in two main forms: VD2 (the chemical name is ergocalciferol) in plants and VD3 (the chemical name is cholecalciferol) in humans and animals [ 15 ]. In humans, the primary source of vitamin D3 is 7-dehydrocholesterol in the skin, which is converted upon exposure to UV light [ 16 ]. VD3 can also be obtained from fish and animal livers. Following hepatic and renal metabolism, VD3 is converted into its biologically active form, calcitriol [ 17 ]. Calcitriol mediates its action through the specific vitamin D receptor (VDR), which exists in both the cell nucleus and cytoplasm [ 18 ]. VDR is widely distributed across various organ systems throughout the body [ 19 ]. Calcitriol promotes the heterodimerization of VDR with the retinoid X receptor (RXR), thus facilitating its downstream functions [ 20 ]. In addition to its role in regulating calcium and phosphorus metabolism to maintain skeletal homeostasis, the calcitriol-VDR signaling pathway is critical for immune regulation, cell proliferation and differentiation, and metabolic homeostasis [ 21 ]. In the female reproductive system, VDR is found in the uterus, ovaries, reproductive tract, and placenta [ 22 ]. VD3 insufficiency in humans can impair reproductive function by disrupting follicular development and embryo implantation [ 23 ]. In the ovaries of VDR knockout mice, follicles can only develop to the secondary stage but cannot progress to the antral stage [ 24 ]. It is reported that calcitriol could inhibit Akt phosphorylation induced by lipopolysaccharide (LPS) in human umbilical vein endothelial cells (HUVECs) [ 21 ]. Therefore, we explored the role and mechanism of calcitriol and its receptor in primordial follicle activation.

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