Vitamin B6 promotes the activation of primordial follicles through the PI3K/Akt signaling pathway.

Firstly, we cultured 3-dpp mouse ovaries for 4 days to examine the roles of 11 vitamins on primordial follicle activation. Compared with control, 9 vitamins, including VB1, VB2, VB3, VB5, VB6, VB9, VB12, VC, and VE, increased the number of growing follicles in a dose-dependent manner, and the most effective concentrations were 1 µM, 10 µM, 1 µM, 20 µM, 10 µM, 40 µM, 10 µM, 250 µM, and 1 µM, respectively. However, VB7 and VK had no effect on the number of growing follicles (Fig.  1 A and B; Fig. S1 and S2A). Fig. 1 Vitamins promote primordial follicle activation in cultured mouse ovaries. Three-dpp newborn mouse ovaries were cultured in the medium supplemented with different concentrations of vitamins, including VB1, VB2, VB3, VB5, VB6, VB7, VB9, VB12, VC, VE, and VK. ( A and B ) The ovaries were collected for morphological examination ( A ) and counting primordial and growing follicles ( B ) after 4 days of culture. Nuclei were stained with hematoxylin, with red arrows indicating growing follicles. ( n  = 3, and each from five ovaries; t-test). Data are presented as mean ± SD, Scale bars = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 Vitamins promote primordial follicle activation in cultured mouse ovaries. Three-dpp newborn mouse ovaries were cultured in the medium supplemented with different concentrations of vitamins, including VB1, VB2, VB3, VB5, VB6, VB7, VB9, VB12, VC, VE, and VK. ( A and B ) The ovaries were collected for morphological examination ( A ) and counting primordial and growing follicles ( B ) after 4 days of culture. Nuclei were stained with hematoxylin, with red arrows indicating growing follicles. ( n  = 3, and each from five ovaries; t-test). Data are presented as mean ± SD, Scale bars = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 Next, we selected VB6 for subsequent experiments in this study. Compared with control, VB6 treatment significantly increased the number of growing follicles, and the protein levels of DEAD-box helicase 4 (DDX4, a cytosolic marker of oocytes; Fig.  2 B). We detected the mRNA levels of oocyte-specific genes Gdf9 and Zp3 in neonatal mouse ovaries. VB6 significantly increased the mRNA levels of Gdf9 and Zp3 compared with control, suggesting that VB6 increased Gdf9 and Zp3 mRNA levels in oocytes. (Fig.  2 A). The activation of primordial follicles is accompanied by the proliferation of granulosa cells. Therefore, we assessed the effect of VB6 on cell proliferation by immunofluorescence examination of proliferation cell nuclear antigen (PCNA), Ki-67, and BrdU after 2 days of culture. Compared with control, VB6 significantly increased the proportions of PCNA-, Ki-67-, and BrdU-positive somatic cells (in primordial follicles: Ki-67: 8.59 ± 1.77% versus 13.87 ± 2.87%; PCNA: 14.85 ± 1.12% versus 25.60 ± 1.32%; BrdU: 6.23 ± 1.17% versus 11.24 ± 0.80%; in primary follicles: Ki-67: 19.83 ± 2.09% versus 23.38 ± 2.23%; PCNA: 19.56 ± 2.38% versus 37.23 ± 2.06%; BrdU: 9.72 ± 0.84% versus 15.97 ± 0.52%. Figure  2 E-F). Consistent with these, VB6 treatment significantly increased the mRNA levels of Pcna and Ki-67 , as well as the protein levels of PCNA. However, VB6 had no effect on the mRNA levels of B-cell lymphoma 2-associated X ( Bax )/B-cell lymphoma 2 ( Bcl-2 ) and Caspase-3 , the protein levels of BAX/BCL-2 and Cleaved Caspase-3, or the number of cells with Cleaved Caspase-3 positive signals (Cleaved Caspase-3: 2.78 ± 0.23% versus 2.95 ± 0.46%. Figure  2 C-G). These results indicate that VB6 promotes the activation of primordial follicles in mice in vitro. Fig. 2 VB6 promotes somatic cell proliferation in cultured mouse ovaries. Three-dpp newborn mouse ovaries were cultured in the medium supplemented without or with 10 µM VB6 for 1 day ( A - D ) or 2 days ( E - G ). ( A and B ) The mRNA levels of Gdf9 and Zp3 ( A ) and the protein levels of DDX4 ( B ) in the ovaries ( n  = 4, and each from six ovaries; t-test). ( C ) The mRNA levels of Pcna , Ki-67 , Bax/Bcl-2 , and Caspase-3 in the ovaries ( n  = 4, and each from six ovaries; t-test). ( D ) The protein levels of PCNA, BAX, BCL-2, and Cleaved Caspase-3 in the ovaries ( n  = 4, and each from six ovaries; t-test). ( E ) The immunofluorescence stain of PCNA, Ki-67, BrdU, and Cleaved Caspase-3 in the ovaries. Green staining for PCNA, Ki-67, BrdU, and Cleaved Caspase-3 was combined with DAPI (blue). ( F and G ) The proportion of somatic cells with Ki-67-, PCNA-, and BrdU-positive signals in primordial (PF) and primary (PrF) follicles. ( F ) The number of cells with Cleaved Caspase-3-positive signals ( n  = 4, and each from six ovaries; t-test. G ). In western blot results, the protein expression levels were normalized to those of GAPDH. Arrows indicate signal-positive cells. Data are presented as mean ± SD, Scale bars = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 VB6 promotes somatic cell proliferation in cultured mouse ovaries. Three-dpp newborn mouse ovaries were cultured in the medium supplemented without or with 10 µM VB6 for 1 day ( A - D ) or 2 days ( E - G ). ( A and B ) The mRNA levels of Gdf9 and Zp3 ( A ) and the protein levels of DDX4 ( B ) in the ovaries ( n  = 4, and each from six ovaries; t-test). ( C ) The mRNA levels of Pcna , Ki-67 , Bax/Bcl-2 , and Caspase-3 in the ovaries ( n  = 4, and each from six ovaries; t-test). ( D ) The protein levels of PCNA, BAX, BCL-2, and Cleaved Caspase-3 in the ovaries ( n  = 4, and each from six ovaries; t-test). ( E ) The immunofluorescence stain of PCNA, Ki-67, BrdU, and Cleaved Caspase-3 in the ovaries. Green staining for PCNA, Ki-67, BrdU, and Cleaved Caspase-3 was combined with DAPI (blue). ( F and G ) The proportion of somatic cells with Ki-67-, PCNA-, and BrdU-positive signals in primordial (PF) and primary (PrF) follicles. ( F ) The number of cells with Cleaved Caspase-3-positive signals ( n  = 4, and each from six ovaries; t-test. G ). In western blot results, the protein expression levels were normalized to those of GAPDH. Arrows indicate signal-positive cells. Data are presented as mean ± SD, Scale bars = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 The mTOR activity in pre-granulosa cells and PI3K/Akt activity in oocytes have been proven to be essential for the activation of primordial follicles. Then, we cultured 3-dpp mouse ovaries to detect the effects of VB6 on the protein levels of phosphorylated Akt (p-Akt) and phosphorylated mTOR (p-mTOR). Compared with control, VB6 significantly increased the protein levels of p-Akt and p-FOXO3a (Fig.  3 A and B) and the proportion of primordial follicle oocytes with FOXO3a nuclear export (Fig.  3 C and D). However, VB6 had no effect on the protein levels of p-mTOR. Moreover, PI3K inhibitor LY294002 blocked the VB6-induced increase of growing follicles (control: 400.00 ± 20.31; LY294002: 243.75 ± 37.98; VB6: 650.00 ± 36.74; LY294002 + VB6: 366.25 ± 17.23. Figure  3 E and F; Fig. S2C). Thus, VB6 activates mouse primordial follicles by the PI3K/Akt signaling pathway. Fig. 3 VB6 activates PI3K/Akt pathway in cultured mouse ovaries. Three-dpp newborn mouse ovaries were cultured in either standard medium (control group) or medium supplemented with 10 µM VB6 (VB6 group), or 5 mM LY294002 (LY294002 group), or a combination of 10 µM VB6 and 5 mM LY294002 (VB6 + LY294002 group) for 1 day ( A and B ), 2 days ( C and D ), or 4 days ( E and F ). ( A and B ) The protein levels of p-mTOR, p-Akt, and p-FOXO3a in the ovaries ( n  = 4, and each from six ovaries; t-test). ( C and D ) FOXO3a localization in oocytes cytoplasm (arrows) of primordial follicles ( C ) and the proportion of primordial follicle oocytes displaying FOXO3a nuclear export ( n  = 4, and each from six ovaries; t-test. D ). FOXO3a, green; DDX4, red; DAPI, blue. ( E and F ) The ovaries were collected for morphological examination ( E ) and counting primordial and growing follicles ( n  = 4, and each from four ovaries; one-way ANOVA. F ). Nuclei were stained with hematoxylin, with red arrows indicating growing follicles. In western blot results, the levels of total mTOR, Akt, and FOXO3a were used as the corresponding internal control for p-mTOR, p-Akt, and p-FOXO3a, respectively. Data are presented as mean ± SD, Scale bars = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 VB6 activates PI3K/Akt pathway in cultured mouse ovaries. Three-dpp newborn mouse ovaries were cultured in either standard medium (control group) or medium supplemented with 10 µM VB6 (VB6 group), or 5 mM LY294002 (LY294002 group), or a combination of 10 µM VB6 and 5 mM LY294002 (VB6 + LY294002 group) for 1 day ( A and B ), 2 days ( C and D ), or 4 days ( E and F ). ( A and B ) The protein levels of p-mTOR, p-Akt, and p-FOXO3a in the ovaries ( n  = 4, and each from six ovaries; t-test). ( C and D ) FOXO3a localization in oocytes cytoplasm (arrows) of primordial follicles ( C ) and the proportion of primordial follicle oocytes displaying FOXO3a nuclear export ( n  = 4, and each from six ovaries; t-test. D ). FOXO3a, green; DDX4, red; DAPI, blue. ( E and F ) The ovaries were collected for morphological examination ( E ) and counting primordial and growing follicles ( n  = 4, and each from four ovaries; one-way ANOVA. F ). Nuclei were stained with hematoxylin, with red arrows indicating growing follicles. In western blot results, the levels of total mTOR, Akt, and FOXO3a were used as the corresponding internal control for p-mTOR, p-Akt, and p-FOXO3a, respectively. Data are presented as mean ± SD, Scale bars = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 We also evaluated the effect of VB6 on mouse primordial follicle activation in vivo. Follicle counting showed that VB6 intraperitoneal injection increased the number of growing follicles after 2 days of treatment compared with control (control: 630.00 ± 18.37; VB6: 877.50 ± 32.88. Figure  4 A and B; Fig. S2B). Western blot results also showed that VB6 significantly increased the protein levels of p-Akt and p-FOXO3a after 1 day of treatment (Fig.  4 C and D), and immunofluorescence staining revealed that VB6 significantly increased the proportion of primordial follicle oocytes with FOXO3a nuclear export after 2 days of treatment compared with those of control (Fig.  4 E and F). We further evaluated whether one week of feeding adolescent mice with VB6 in water had an effect on primordial follicle activation. In the adolescent mice, oral administration with different concentrations of VB6 promoted adolescent mouse primordial follicle activation, and 75 µM VB6 significantly increased the number of primary and secondary follicles (Fig.  4 G and H; Fig. S2D). The average administration concentration was calculated to be 4.98–5.05 mg/kg/d (average 5.02 mg/kg/d). However, there was no obvious effect on the ovarian morphology, vital organs and the body weight of mice (Fig. S3A-C). These results suggest that VB6 can activate mouse primordial follicles in vivo through the PI3K/Akt pathway. Fig. 4 VB6 promotes primordial follicle activation in vivo. Three-dpp female mice were injected intraperitoneally with PBS (control) or 2.06 mg/kg VB6 twice daily for two consecutive days. Ovaries were collected 2 days ( A and B ) or 1 day ( C - F ) after the end of injection. Adolescent mice were provided with normal water or water supplemented with VB6 (50–100 µM) for one week. Ovaries were collected for immunofluorescence staining and follicle counting after the end of feeding ( G and H ). ( A and B ) The ovaries were collected for morphological examination ( A ) and counting primordial and growing follicles ( n  = 4, and each from four ovaries; t-test. B ). Nuclei were stained with hematoxylin, with red arrows indicating growing follicles. ( C and D ) The protein levels of p-mTOR, p-Akt, and p-FOXO3a in the ovaries ( n  = 4, and each from six ovaries; t-test). ( E and F ) FOXO3a localization in oocytes cytoplasm (arrows) of primordial follicles ( E ) and the proportion of primordial follicle oocytes displaying FOXO3a nuclear export ( n  = 4, and each from six ovaries; t-test. F ). FOXO3a, green; DDX4, red; DAPI, blue. ( G and H ) Ovary morphology comparison ( G ) and the number of primordial and growing follicles ( H ) in adolescent mice ( n  = 4, and each from six ovaries; t-test). DDX4, red; DAPI, blue. Arrowheads, primordial follicles; arrows, growing follicles. PF, primordial follicle; PrF, primary follicle; SF, secondary follicle; AF, antral follicle; Total, total follicle. In western blot results, the levels of total mTOR, Akt, and FOXO3a were used as the corresponding internal control for p-mTOR, p-Akt, and p-FOXO3a, respectively. Data are presented as mean ± SD, Scale bars = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 VB6 promotes primordial follicle activation in vivo. Three-dpp female mice were injected intraperitoneally with PBS (control) or 2.06 mg/kg VB6 twice daily for two consecutive days. Ovaries were collected 2 days ( A and B ) or 1 day ( C - F ) after the end of injection. Adolescent mice were provided with normal water or water supplemented with VB6 (50–100 µM) for one week. Ovaries were collected for immunofluorescence staining and follicle counting after the end of feeding ( G and H ). ( A and B ) The ovaries were collected for morphological examination ( A ) and counting primordial and growing follicles ( n  = 4, and each from four ovaries; t-test. B ). Nuclei were stained with hematoxylin, with red arrows indicating growing follicles. ( C and D ) The protein levels of p-mTOR, p-Akt, and p-FOXO3a in the ovaries ( n  = 4, and each from six ovaries; t-test). ( E and F ) FOXO3a localization in oocytes cytoplasm (arrows) of primordial follicles ( E ) and the proportion of primordial follicle oocytes displaying FOXO3a nuclear export ( n  = 4, and each from six ovaries; t-test. F ). FOXO3a, green; DDX4, red; DAPI, blue. ( G and H ) Ovary morphology comparison ( G ) and the number of primordial and growing follicles ( H ) in adolescent mice ( n  = 4, and each from six ovaries; t-test). DDX4, red; DAPI, blue. Arrowheads, primordial follicles; arrows, growing follicles. PF, primordial follicle; PrF, primary follicle; SF, secondary follicle; AF, antral follicle; Total, total follicle. In western blot results, the levels of total mTOR, Akt, and FOXO3a were used as the corresponding internal control for p-mTOR, p-Akt, and p-FOXO3a, respectively. Data are presented as mean ± SD, Scale bars = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 Finally, we explored the effect of VB6 on primordial follicles activation in human ovarian tissue. Human ovarian tissue fragments were cultured without or with VB6. Compared with control, VB6 treatment significantly increased the proportion of growing follicles (uncultured: 18.26 ± 2.38%; control: 21.86 ± 2.93%; VB6: 33.49 ± 1.90%. Figure  5 A and B;). Western blot results showed that VB6 treatment significantly increased the protein levels of p-Akt in human ovarian tissue (Fig.  5 C). These results indicate that VB6 increases PI3K/Akt activity and promotes human primordial follicle activation in vitro. Fig. 5 VB6 promotes human primordial follicle activation in vitro. Human ovarian tissue fragments were directly collected for histological analysis and protein detection (uncultured) or were cultured in either without (control) or with 10 µM VB6 (VB6 group) for 4 days, followed by an additional 2 days in standard medium. The tissues were collected after 4 days for western blotting analysis or after 6 days for follicle counting. ( A and B ) Human ovarian tissue morphology comparison ( A ) and primordial and growing follicle percentage ( n  = 3, and each from two ovarian tissue blocks; one-way ANOVA. B ). Nuclei were stained with hematoxylin. Arrowheads, primordial follicles; arrows, growing follicles. ( C ) The protein levels of p-Akt were assessed in the tissues ( n  = 3, and each from two ovarian tissue blocks; one-way ANOVA). In western blot results, the levels of total Akt were used as the corresponding internal control for p-Akt. Data are presented as mean ± SD, Scale bars = 50 μm. * p  < 0.05, ** p  < 0.01 VB6 promotes human primordial follicle activation in vitro. Human ovarian tissue fragments were directly collected for histological analysis and protein detection (uncultured) or were cultured in either without (control) or with 10 µM VB6 (VB6 group) for 4 days, followed by an additional 2 days in standard medium. The tissues were collected after 4 days for western blotting analysis or after 6 days for follicle counting. ( A and B ) Human ovarian tissue morphology comparison ( A ) and primordial and growing follicle percentage ( n  = 3, and each from two ovarian tissue blocks; one-way ANOVA. B ). Nuclei were stained with hematoxylin. Arrowheads, primordial follicles; arrows, growing follicles. ( C ) The protein levels of p-Akt were assessed in the tissues ( n  = 3, and each from two ovarian tissue blocks; one-way ANOVA). In western blot results, the levels of total Akt were used as the corresponding internal control for p-Akt. Data are presented as mean ± SD, Scale bars = 50 μm. * p  < 0.05, ** p  < 0.01

Two-month-old and adolescent ICR mice were obtained from the Guangdong Medical Laboratory Animal Center (Foshan, China). All mice were raised at 24 ℃ with free access to chow and water, and were maintained under a 12/12-hour light/dark cycle. Adult mice of female were mated with male at a 1:1 ratio to acquire newborn mice. The day of birth was counted as 0.5 days post-partum (dpp). Female mice at 3-dpp were used for collecting ovaries for culture in vitro or intraperitoneal injection. Each neonatal female mouse received intraperitoneal injection twice daily: 2 µL PBS (control) or 2 µL of 2.05 mg/kg VB6. In some experiments, adolescent female mice were treated with water (control) or water supplemented with 50, 75, and 100 µM VB6. (V108688, Aladdin, Shanghai, China). All animal procedures were approved by the Animal Care and Use Committee of South China University of Technology (approval number: 2022102, approved on December 30, 2022). Unless otherwise specified, all reagents used in this study were sourced from Sigma-Aldrich (St. Louis, Missouri, USA). A list of primary antibodies is provided in Supplementary Table S1. Three-dpp newborn mouse ovaries were isolated and washed in sterile phosphate-buffered saline (PBS). Whole ovaries were cultured on the membrane (PICMORG50, Millipore, Billerica, MA, USA) inserts in six-well culture plates (NEST, Beijing, China) at 37 ℃ with 5% CO 2 . The medium for ovary culture was Dulbecco’s Modified Eagle Medium with nutrient mixture F-12 (DMEM/F12; Thermo Fisher Scientific, Waltham, MA, USA), and its components have been reported previously [ 12 ]. In the experimental groups, newborn mouse ovaries were cultured in the medium supplemented with various vitamins, including VB1 (0–10 µM), VB2 (0–100 µM), VB3 (0–1.5.5 µM), VB5 (0–30 µM), VB6 (0–20 µM), VB7 (0–40 µM), VB9 (0–50 µM), VB12 (0–20 µM), VC (0–500 µM), VE (0–20 µM), VK (0–20 µM), and/or the PI3K inhibitor LY294002 (10 µM; MedChemExpress, New Jersey, USA). VB1, VB3, VB6, VB12, and VC were prepared as stock solutions in ultrapure water, while VB2, VB5, VB7, VB9, VE, VK, and LY294002 were prepared as stock solutions in dimethyl- sulfoxide (DMSO). The concentration of DMSO is no more than 0.1% in the cultured system, and the same concentration of DMSO was also added to the corresponding control group. Ovaries were harvested at designated time points for follicle counting, immunofluorescence staining, and subsequent gene and protein analysis. The injection dosage was based on the concentration chosen from in vitro culture experiment, in which the volume ratio (mg/L) was converted to the mass ratio (mg/kg). Three-dpp female mice (body weight: 2.57 ± 0.29 g) were intraperitoneally injected either PBS (control) or 2.05 mg/kg VB6 twice daily for two consecutive days. Ovaries were collected 1 day post-final injection for immunofluorescence staining and protein analysis, and 2 days post-final injection for follicle counting. The concentration of VB6 in drinking water was determined based on the daily water intake of the mice (approximately 0.4 L/kg/d as established in the pre-experiment), with the daily dose of VB6 administered through the water equivalent to the daily injection dose of VB6. According to the calculation, we prepared VB6 solutions at concentrations of 50 µM, 75 µM, and 100 µM in water for the experiments. Thus, adolescent mice were provided either normal water or water supplemented with VB6 for one week. Meanwhile, the body weight and daily water intake of the mice were recorded. The daily VB6 dose was calculated by dividing the quantity of ingestion (mg) by the body weight (kg). Ovaries were collected for follicle counting and immunofluorescence staining at the end of feeding. Human ovarian cortical tissues were collected during laparoscopic surgery from nine patients aged 28 to 31 years (mean ± SD: 29.25 ± 1.26 years) with endometriosis at Zhongshan City People’s Hospital in Guangdong, China. Written informed consent was obtained from each patient prior to the procedure. This study adhered to the principles outlined in the Declaration of Helsinki. The collection and use of human ovarian tissue were approved by the Ethics Committee of Zhongshan City People’s Hospital. The ovarian tissues were immediately transferred to the laboratory in cold pre-equilibrated PBS with 100 IU/ml penicillin–streptomycin and 1% ITS. The tissues were cut into 1 mm³ fragments with a scalpel under aseptic conditions. Some of these fragments were reserved for protein analysis or fixed in 4% paraformaldehyde (PFA, Solarbio, Beijing, China) for serial sectioning, followed by hematoxylin staining for follicle counting (uncultured). The remaining fragments were randomly divided into two groups: one group cultured in DMEM/F-12 medium for 6 days (control), and the other group cultured in medium supplemented with 10 µM VB6 for 4 days, followed by an additional 2 days in drug-free medium. These fragments were cultured under the same conditions as described in mouse ovarian culture. The fragments were harvested for protein analysis after 4 days of culture and for follicle counting after 6 days culture. Mouse ovaries and human ovarian tissues were fixed in 4% (w/v) paraformaldehyde (PFA) solution (Solarbio, Beijing, China) for 12 h, followed by embedding in paraffin. Paraffin sections were then cut at a thickness of 5 μm using a serial cutting technique, and affixed to glass slides. Subsequently, the sections were dried at 42 ℃ for 12 h, deparaffinized, rehydrated, and stained with hematoxylin (Solarbio, Beijing, China). Follicle counting was based on the presence of a distinct oocyte nucleus, ensuring each follicle was counted only once. The classifications of primordial, growing, and atretic follicles were as described in previous work [ 23 ]. Newborn mouse ovaries were counted in every fifth section, and the total primordial follicle number in each ovary was calculated based on the formula: average follicle number per section × 5. Growing and atretic follicles were counted in serial sections of the entire ovaries. In adolescent mouse ovaries, primordial and growing follicles, including primary, secondary, and antral follicles, were counted in serial sections. For human ovarian tissues, primordial and growing follicles were counted by examining entire ovarian fragments, with only those follicles exhibiting clearly visible oocyte nuclei being included, and each follicle counted only once. We also calculated the number of follicles per area of the largest mouse ovarian sections. All sections were independently counted by two individuals for comparative analysis. After deparaffinization and hydration, these ovarian tissue sections underwent antigen retrieval in citrate buffer (pH 6.0) at temperatures ranging from 95 to 98 °C. After cooling for 2–4 h, the sections were blocked with 10% donkey serum (Sigma-Aldrich) for 1 h and then incubated with the primary antibody (Supplementary Table S1) at 4 ℃ overnight. Following washing, the sections were incubated with Alexa Fluor 488- or 555-conjugated secondary antibodies (1:200, Thermo Fisher Scientific) for 2 h at room temperature. Subsequently, 4’,6-diamidino-2-phenylindole (C1002, DAPI, 1:2000; Beyotime, Beijing, China) was applied to stain the nuclei for 5 min. The stained sections were then imaged using a confocal laser scanning microscope (LSM 800, Carl Zeiss, Germany). To assess the percentage of positive cells within primordial and primary follicles and the proportion of FOXO3a nuclear export in primordial follicle oocytes, the five largest sections of each ovary were selected for analysis. The proportion was calculated by dividing the number of cells or follicles with positive signals by the total number of cells or follicles. Meanwhile, a negative control was employed to rule out interference caused by non-specific positive signals (Fig. S3D). The mean values derived from 15 sections, obtained from three ovaries in each experiment, were considered as one independent sample data. In bromodeoxyuridine (BrdU) incorporation assay, three-dpp mouse ovaries were cultured for 2 days and then were incubated in a medium containing 10 µM BrdU (Sigma-Aldrich) for 2 h. Next, the ovaries were harvested, fixed, and sectioned into 5 μm slices. Following deparaffinization, antigen retrieval, and serum blocking, the sections were incubated with anti-BrdU antibody (Supplementary Table S1) at 4 ℃ overnight and then incubated with Alexa Fluor 488-conjugated secondary antibody (Supplementary Table S1) for 2 h at room temperature. Finally, the nuclei were counterstained with DAPI for 5 min. Mouse ovaries were cultured either without or with VB6 for 1 day and then were collected for the extraction of total RNA using the ReliaPrep™ RNA Miniprep system (Z6111, Promega, Madison, WI, USA). To synthesize cDNA, 1 µg of total RNA from each group was reverse-transcribed using the GoScript™ Reverse Transcription System (A5001, Promega, Madison, WI, USA). Data normalization was performed using ribosomal protein L19 ( Rpl19 ). The relative mRNA expression levels were determined using the 2 −ΔΔCT method. Details regarding the primers utilized are provided in Supplementary Table S2. Protein extraction from ovarian tissues was performed using lysis buffer (HY-16160, Beyotime, Beijing, China). The protein concentration was determined via a bicinchoninic acid (BCA) assay (P0012, Beyotime). Twenty micrograms of protein from each group were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked with 5% skim milk (Absin, Shanghai, China) for 1 h, followed by incubation with the primary antibodies at 4 °C overnight (Supplementary Table S1). After washing with Tris-buffered saline containing Tween 20 (TBST), the membranes were incubated with the secondary antibodies (Supplementary Table S1) at room temperature for 1 h. Protein bands were visualized using Super-Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, MA, USA), and images were captured with the Tanon 5200 Chemiluminescence Imaging System (Tanon, Shanghai, China). For band density quantification, ImageJ software (NIH Image, Bethesda, MD, USA) was employed. The levels of p-Akt, p-FOXO3a and p-mTOR were normalized to those of their respective total proteins, while other protein expression levels were normalized to those of GAPDH. Supplementary Fig. S4 presents all uncropped blots. Data were obtained from a minimum of three independent replicates. Results are presented as mean ± standard deviation (SD). Statistical analysis and graphing were performed using the GraphPad Prism software (v10.1.2, La Jolla, CA, USA). The statistical significance was analyzed either by unpaired Student’s t-test (two-group comparison) or by one-way ANOVA followed by Tukey’s Honestly Significant Difference (more than two groups). Any p-values below 0.05 were indicated as statistically significant.

Vitamins are essential trace organic compounds required for maintaining normal physiological functions in mammals [ 24 , 25 ]. In this study, VB1, VB2, VB3, VB5, VB6, VB9, VB12, VC, and VE promoted the activation of mouse primordial follicles, and VB6 activated primordial follicles in both mouse and human via the PI3K/Akt signaling pathway. VB6 promoted the activation of mouse and human primordial follicles in vitro and/or in vivo, suggesting that VB6 could promote the activation of primordial follicles in mammals. VB6 activates primordial follicles via the PI3K/Akt signaling pathway. This is consistent with previous studies that VB6 activates the PI3K/Akt pathway to improve insulin sensitivity in insulin-resistant mice and extend Rex rabbit hair follicle growth [ 26 , 27 ]. VB6 contains six interconvertible pyridine compound forms: pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM), and their phosphorylated derivatives (PLP, PNP, and PMP), by which VB6 participates in glucose metabolism [ 28 , 29 ]. The main active form of VB6 is PLP, which can activate the Akt signaling pathway to promote glucose utilization in the aging rat heart [ 30 ]. PLP restores PI3K function in PI3K RNAi flies, leading to the activation of the Akt/FOXO signaling pathway [ 31 ]. PI3K/Akt promotes FOXO3a phosphorylation and export from the primordial follicle oocyte nucleus to relieve transcriptional inhibition [ 32 ]. Thus, VB6 activates primordial follicles possibly by promoting the PI3K/Akt/FOXO3a pathway in the form of PLP. VB3 also can activate the PI3K/Akt signaling pathway, thereby enhancing osteoblast glycolysis [ 33 ]. Thus, VB3 activates mouse primordial follicles also possibly by the PI3K/Akt signaling pathway. Studies have shown that the primordial follicles maintain low levels of reactive oxygen species (ROS) and that the oocytes within the primordial follicles are very sensitive to ROS [ 34 , 35 ]. Mitochondrial metabolism in oocytes is accompanied by an increase in ROS, but excessive ROS lead to mitochondrial function damage and cause apoptosis of oocytes [ 36 , 37 ]. It has been reported that an antioxidant quercetin can protect ovarian reserve by increasing mtDNA copy number and enhancing antioxidant capacity [ 38 ]. VB6 inhibits the excessive formation of ROS in mitochondria and reduces oxidative damage to sperm, indicating that VB6 has antioxidant effects [ 39 ]. Mitochondria can take up PLP from the cytoplasm to maintain normal oxidative metabolism and reduce functional damage in skin fibroblasts [ 40 ]. However, VB6 treatment did not reduce the apoptosis of primordial follicles in our study. The antioxidant roles of VB6 in protecting and activating primordial follicles require further study. The research has shown that VC can reduce oxidative stress during ovarian aging and help preserve the follicle reserve [ 41 ]. VC and VE protect oocytes within the antral follicles from oxidative stress through antioxidant effects, thereby enhancing the developmental potential of oocytes [ 42 ]. Therefore, we speculate that VC and VE promote primordial follicle activation through their antioxidation. In our study, the doses of VB6, VC, and VE in mice were 5.02 mg/kg/d (equivalent to 20.33 mg/d in humans), 44.03 mg/kg/d (equivalent to 3.57 mg/kg/d in humans), and 0.43 mg/kg/d (equivalent to 0.03 mg/kg/d in humans), respectively. 10.00–340.00.00.00 mg/d VB6 were used to treat patients with nausea and vomiting during pregnancy (NVP), anxiety, and primary hyperoxaluria type 1 (PH1), respectively [ 43 – 45 ]. 60.00 mg/d of VC is used to treat tendinopathy and 10.00 mg/kg/d of VE is used to treat patients with cystic fibrosis [ 46 , 47 ]. In our previous studies, metallic compounds, theophylline derivatives and BBR activate primordial follicles in neonatal and adolescent mice, and also activate primordial follicles in aged/POI model mice [ 7 , 11 , 12 ]. In our present study, VB6 activates primordial follicles in neonatal and adolescent mice, and may also activate primordial follicles in aged/POI model mice. Thus, the doses of VB6, VC, and VE used for activation in our experiments are appropriate and may serve as potential oral therapeutic agents for patients with POI. However, high doses of vitamins may cause adverse reactions [ 43 , 48 , 49 ]. In conclusion, the current findings showed that vitamins activated mouse primordial follicles and VB6 activated human and mouse primordial follicles through the PI3K/Akt signaling pathway (Fig.  6 ). As safe oral medications, vitamins, especially VB6, may be a potential treatment for rescuing infertility in POI patients and aged women. Fig. 6 A schematic illustrating primordial follicle activation by VB6. VB6 enters oocytes of primordial follicles, and then activates the PI3K/Akt signaling, resulting in primordial follicle activation A schematic illustrating primordial follicle activation by VB6. VB6 enters oocytes of primordial follicles, and then activates the PI3K/Akt signaling, resulting in primordial follicle activation

In mammals, the primordial follicle reservoir, established prenatally or shortly after birth, serves as the sole source of oocytes throughout the female reproductive lifespan. In each wave, a few primordial follicles are activated to produce mature oocytes for female reproduction, while the majority of primordial follicles remain in a dormant state [ 1 , 2 ]. Enhanced glycolysis in pre-granulosa cells activates the mammalian target of rapamycin (mTOR) pathway, which promotes the expression of the proto-oncogene receptor tyrosine kinase ligand (KITL). KITL binds to KIT, thereby activating the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway in oocytes [ 3 , 4 ]. Subsequently, forkhead box O3a (FOXO3a) is phosphorylated and translocated from the nucleus to the cytoplasm, resulting in the activation of primordial follicles [ 5 ]. There are multiple factors that can lead to premature ovarian insufficiency (POI) [ 6 ]. Patients with POI usually have a small number of residual primordial follicles that are difficult to be activated under physiological conditions [ 7 ]. On the other hand, fertility in older women decreases precipitously as their primordial follicle reserve dramatically decreases [ 8 ]. Various therapeutics are tried to activate primordial follicles, such as in vitro activation (IVA), intraovarian human platelet-rich plasma (hPRP) injection, and stem cell therapy [ 9 , 10 ]. However, these strategies are hindered by surgical injuries and low success rates. Therefore, it is necessary to develop effective and safe treatment strategies to save fertility in POI patients [ 11 ]. In previous studies, we identified certain drugs as potential therapeutic candidates for POI patients. For instance, theophylline derivatives and berberine (BBR) are found to activate primordial follicles in both mice and humans via the oocyte-specific PI3K/Akt signaling pathway, resulting in an increased number of ovulated oocytes in aged mice [ 7 , 11 ]. Additionally, the metal compounds can activate primordial follicles, and oral administration of ZnSO 4 is also demonstrated to rescue infertility in aged mice [ 12 ]. It is necessary to screen a broader range of pharmacological agents for the therapy of heterogeneous POI patients. Vitamins are classified into fat-soluble and water-soluble components. The fat-soluble vitamins, including vitamin A, D, E, and K, involve immune regulation, vision protection, and mental health [ 13 ]. The water-soluble vitamins, including B vitamins and vitamin C, involve normal growth, cellular metabolism, and immune function [ 14 ]. B vitamins maintain reproductive function by promoting energy metabolism and enhancing cellular function [ 15 , 16 ]. VB1 alleviates maternal high-fat diet (HFD)-induced impairment in primordial follicle formation [ 17 ]. VB6 can promote the maturation of bovine oocytes by enhancing metabolic pathways and reducing oxidative stress [ 18 , 19 ]. and alleviate aflatoxin B1-induced impairment of mouse testis by activating the phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway [ 20 ]. VB9 supplementation has a positive impact on human ovarian reserve [ 21 ]. It is well known that PI3K/Akt pathway in oocytes plays an important role in regulating primordial follicle activation [ 22 ]. Therefore, we explored the influence of vitamins on the activation of primordial follicles, especially the potential mechanism of VB6.

Supplementary Material 1. Supplementary Material 1. Supplementary Material 2. Supplementary Material 2. Supplementary Material 3. Supplementary Material 3. Supplementary Material 4. Supplementary Material 4. Supplementary Material 5. Supplementary Material 5. Supplementary Material 6. Supplementary Material 6. Supplementary Material 7. Supplementary Material 7. Supplementary Material 8. Supplementary Material 8.