mGCs were procured from Haixing Biosciences Co., Ltd. (cat. no. ORCM050). The cells were grown in high-glucose DMEM (cat. no. 12,100; Beijing Solarbio Science & Technology Co., Ltd.), which included 100 U/ml penicillin and 100 µg/ml streptomycin as standard components, with 10% heat-inactivated FBS (cat. no. FSS500; Shanghai ExCell Biology, Inc.). mGCs were maintained at 37°C in an atmosphere with 5% CO₂, and the culture medium was replaced every 2 days. To preserve physiological relevance, cell passage was restricted to early generations (P2-P3), thereby minimizing cellular dedifferentiation. Additionally, follicle stimulating hormone receptor (FSHR) expression levels were periodically confirmed by immunofluorescence, ≥80% of cells exhibited positive FSHR staining (Fig. S1A).

Cell coverslips were prepared by seeding 1×10⁵ cells in 1 ml of medium and incubating them overnight at 37°C with 5% CO₂ until they reached 70–80% confluency. After removing the medium, the cells were washed with ice-cold PBS and fixed using 4% paraformaldehyde (PFA) for 30 min at room temperature. Following additional washes with PBS, the cells were permeabilized with 0.5% Triton^™ X-100 for 15 min and then blocked with 10% goat serum (cat. no. S9070; Beijing Solarbio Science & Technology Co., Ltd.) for 60 min at room temperature. The coverslips were then incubated overnight at 4°C with a rabbit anti-FSHR antibody diluted to 1:200 (cat. no. 22665-1-AP; Proteintech Group, Inc.). After washing, the cells were stained with an Alexa Fluor^® 488-conjugated secondary antibody at a dilution of 1:500 (cat. no. SA00006-2; Proteintech Group, Inc.) for 2 h at room temperature. Finally, the coverslips were mounted on slides using DAPI antifade medium (cat. no. S2110; Beijing Solarbio Science & Technology Co., Ltd.), and the samples were imaged for analysis using an inverted platform (Primovert LED; ZEISS, Germany).

Recombinant lentiviruses, rLV-short hairpin (sh)RNA-Puro-mPRNP and rLV-mPRNP−3flag-ZsGreen-Puro were procured from Haixing Biosciences Co., Ltd. Empty vectors were used as negative controls (NCs). The target sequences of the PRNP shRNAs were as follows: mPRNP sh-1, 5′-GGACAACCTCATGGTGGTAGT-3′; mPRNP sh-2, 5′-GCGTCAATATCACCATCAAGC-3′; and mPRNP sh-3, 5′-GCCTATTACGACGGGAGAAGA-3′. To achieve stable silencing and OE of the PRNP gene, the cells were seeded into 6-well plates at a density of 2.5×10⁵ cells per well, and cultured overnight until they reached a confluence of 40–50%. On the subsequent day, lentiviral transfection of the cells was performed following the protocol supplied by the manufacturer. Lentiviral vectors that encode EGFP reporters were generated using a third-generation system in 293T cells (cat. no. CL-0005; Wuhan Pricella Biotechnology Co., Ltd.). The cells were transfected at 70% confluence with 3 µg pLVX-shRNA2-Puro-mPrnp or pLVX-mPrnp-3flag-ZsGreen-Puro transfer plasmid, 2 µg of psPAX2, and 1 µg of pMD2.G in a ratio of 3:2:1 using PEI in Opti-MEM. After 48 h of incubation at 37°C with 5% CO₂, fluorescence screening indicated a packaging efficiency of over 85%. To obtain stably transfected mGCs, lentiviral transduction was performed at the optimized multiplicity of infection (MOI) of 80, followed by selection with 3 µg/ml of puromycin for 72 h (cat. no. PS1224; Beijing Puxitang Biotechnology Co., Ltd.), and 1 µg/ml thereafter for culture maintenance. After 72 h of transduction, the infected mGCs were collected for subsequent experiments. Stable PrP^c-knockdown mGCs were established through lentiviral shRNA transduction. Following this, these cells were infected with a lentivirus designed to overexpress PrP^c, allowing for both the knockdown and overexpression of PrP^c within the same cellular environment.

Total RNA was extracted from mGCs following the manufacturer's instructions (cat. no. UE-MN-MS-RNA-10; Suzhou UElandy Biotechnology Co., Ltd.). Quantity and purity of RNA were determined using a NanoDrop^™ 2000 spectrophotometer (Thermo Scientific). The RNA concentrations were measured at A260, while the purity was evaluated by calculating the A260/A280 ratios. Subsequently, RT was performed following the manufacturer's instructions for the RevertAid^™ Master Mix (cat. no. M1632; Thermo Fisher Scientific, Inc.). qPCR was carried out on a CFX Connect fluorescent Real-Time PCR System (Bio-Rad Laboratories, Inc.), using the Universal SYBR Green qPCR Supermix (cat. no. S2024L; US Everbright, Inc.). The thermocycling conditions were as follows: 95°C for 5 min, followed by 45 cycles of 95°C for 5 sec and 60°C for 30 sec. The PRNP primer sequences (cat. no. MQP094390; GeneCopoeia, Inc.) were as follows: PRNP forward (F), 5′-GGCCCATGATCCATTTTGGC-3′ and reverse (R), 5′-TGCTGTACTGATCCACTGGC-3′. Additionally, the following primer sequences were used for β-actin (Wuhan Servicebio Technology Co., Ltd.): β-actin F, 5′-GACTTTGTACATTGTTTTG-3′ and R, 5′-TGCACTTTTATTGGTCTCA-3′, which was selected as the internal control for normalization. The 2^−ΔΔCq method was employed for quantification of the data (20). qPCR was selected for its superior sensitivity in detecting transcript-level changes (detection threshold ≤10 copies/µl) compared with conventional PCR (21,22), with SYBR Green chemistry providing cost-effective quantification of PRNP expression dynamics.

mGCs and ovarian tissue were lysed on ice for 30 min in RIPA buffer supplemented with protease inhibitors (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.). The total protein content was determined using a BCA Protein Quantification Kit (cat. no. C0050; TargetMol Chemicals Inc.). Following protein quantification, 25 µg total protein/lane in each sample were separated via 10% SWE Rapid High Resolution Running Buffer (cat. no. G2081-1L; Wuhan Servicebio Technology Co., Ltd.). Subsequently, the separated proteins were transferred onto PVDF membranes (cat. no. IPVH00010; MilliporeSigma).

To block non-specific binding, the PVDF membranes were incubated in a solution of 5% non-fat dried milk (cat. no. S10191; Beijing Puxitang Biotechnology Co., Ltd.) in Tris-buffered saline containing 0.1% Tween 20 (TBST; cat. no. T1082; Beijing Solarbio Science & Technology Co., Ltd.) for 2 h at room temperature. After blocking, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-CD230 (Prion; cat. no. 808001; BioLegend, Inc.) and anti-β-actin at a 1:5,000 dilution (cat. no 20536-1-AP; Proteintech Group, Inc.).

After the overnight incubation, the membranes were washed three times with TBST and incubated for 2 h at room temperature with the following secondary antibodies: HRP-conjugated goat anti-rabbit IgG at a 1:1,000 dilution (SA00001-2; Proteintech Group, Inc.) and HRP-conjugated goat anti-mouse IgG at a 1:10,000 dilution (SA00001-1; Proteintech Group, Inc.).

Finally, an enhanced chemiluminescence detection reagent (cat. no. S6009L; Suzhou UE Landi Biotechnology Co., Ltd.) was used to visualize the immunoreactive bands on ChemiDoc MP (Bio-Rad Laboratories, Inc.). Densitometry of the WB bands was performed using ImageJ 1.48V software (National Institutes of Health).

Following the instructions of the Annexin V/PI detection Kit (cat. no. Y6026S; Suzhou UE Landy Biotechnology Co., Ltd.), 1×10⁵ mGCs were digested with 0.25% trypsinization solution (without EDTA)(cat. no. T1350; Beijing Solarbio Science & Technology Co., Ltd.), then enzymatically neutralized and resuspended in cold staining buffer, then resuspended in a staining buffer, which was prepared by adding 5 µl each Annexin V storage solution and 5 µl PI storage solution. Subsequently, the cells were incubated in the dark at 4°C for 20 min and analyzed using a NovoCyte D3000 flow cytometer (Agilent Technologies, Inc.) Flow cytometry data were analyzed using De Novo FCS Express^™ 6 software (Agilent Technologies, Inc.). Annexin V/PI dual staining was employed to differentiate early apoptotic (Annexin V⁺/PI⁻) from late apoptotic/necrotic (Annexin V⁺/PI⁺) populations, enabling stage-specific analysis of mGC death pathways, the total percentage of apoptotic cells reflects the overall measurement of both early and late apoptotic populations.

For cell cycle analysis, 5×10⁵ mGCs were harvested by trypsinization at 72 h post-infection. The cells were then washed with cold PBS and fixed in 70% ice-cold ethanol at 4°C overnight. Next, mGCs were incubated with RNase A and PI in a 0.5 ml reaction mixture (cat. no. C6031S; Suzhou UE Landy Biotechnology Co., Ltd.) at 37°C for 30 min in a dark chamber. Cell cycle distribution was measured using a NovoCyte D3000 flow cytometer and analyzed with De Novo FCS Express^™ 6 software (Agilent Technologies, Inc.).

mGC culture medium containing 10% FBS and mouse serum collected by centrifugation at 3,000 × g for 5 min at 4°C were used to measure the concentrations of E₂ (cat. no. SEKM-0286; Beijing Solarbio Science & Technology Co., Ltd.), AMH (cat. no SEKM-0310; Beijing Solarbio Science & Technology Co., Ltd.), P4 (cat. no. SEKSM-0002; Beijing Solarbio Science & Technology Co., Ltd.), FSH (cat. no. KE1425; ImmunoWay Biotechnology Company) and luteinizing hormone (LH; cat. no. KE1421; ImmunoWay Biotechnology Company), following the manufacturer's instructions.

A total of 24 mice were used, comprising FVB-PRNP-/- and FVB wild-type (WT) mice, with each group consisting of 12 females. At the start of the experiment, the mice were 7 weeks old, and body weight was of 19.1–22.9 g (mean ± SD: 20.82±1.13 g). PRNP KO mice with an FVB genetic background were provided by Professor Wen-Quan Zou (Jiangxi Academy of Clinical Medical Sciences of the First Affiliated Hospital of Nanchang University) and Professor Li Cui (Department of Neurology, First Hospital of Jilin University). FVB WT mice, sourced from GemPharmatech Co. Ltd., were utilized as controls in the present study. All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the First Affiliated Hospital of Nanchang University (approval no. CDYFY-IACUC-202310QR030; Nanchang, China) in accordance with the National Institutes of Health Guidelines for animal welfare. To maintain optimal health and experimental integrity, both the KO and WT mice were housed in individually ventilated cages under specific pathogen-free (SPF) conditions, maintaining a temperature of 22±1°C and humidity levels of 55±10%. They were kept on a 12:12-h light/dark cycle. The animals had unrestricted access to autoclaved standard rodent chow and reverse-osmosis purified water. These carefully controlled housing conditions minimized the potential influence of external pathogens on the experimental outcomes, ensuring that any observed differences between the KO and WT groups could be more accurately attributed to the genetic modifications rather than environmental factors. Breeding pairs were formed by housing WT and KO female mice (n=6 per group) with proven-fertility WT males at 10 weeks of age under specific pathogen-free conditions. After confirming the presence of copulatory plugs, which indicated successful mating and was designated as Gestational Day 0, the dams were individually housed in ventilated cages. Inhalant anesthesia was induced with 5% isoflurane (cat. no. R510-22-16; RWD Life Science Co., Ltd.) in oxygen at 1 l/min flow rate, and maintained for 5 min until the pedal withdrawal reflex ceased and the respiratory rate stabilized (40–60 breaths/min). Cervical dislocation was performed with simultaneous isoflurane overdose to ensure humane euthanasia. Death validation included confirmation of apnea for >3 min and absence of the corneal reflex. Terminal whole blood samples (~0.3 ml) were obtained via retro-orbital venous plexus puncture within 2 min post-mortem to ensure coagulation integrity. Ovarian tissues were dissected, fixed in 4% paraformaldehyde at 4°C for 24–48 h, then processed for paraffin embedding and hematoxylin & eosin (H&E) staining.

H&E staining was performed in accordance with a standard protocol (23,24). Specifically, ovarian tissues were fixed in 4% paraformaldehyde (w/v in 0.1 M PBS, pH 7.4) at 4°C for a duration of 24 to 48 h, 5-µm-thick paraffin sections underwent three 5-min immersions in xylene. This was followed by a rehydration process through a graded series of ethanol concentrations: 100, 95, 80, and 70% (v/v), with each step lasting 2 min, ultimately concluding in distilled water. Next, the sections were stained with H&E (cat. no. L11021604l; Nanchang Yulu Experimental Equipment Co., Ltd.). Sections were stained with Mayer's hematoxylin at room temperature (23±2°C) for a duration of 3 min, and this was followed by staining with eosin Y alcoholic solution at the same temperature for 2 min. Ovarian follicles were quantified using systematic random sampling in every sixth serial section with a thickness of 5 µm. A manual counting method was employed, examining 30 non-overlapping fields per ovary at a magnification of 200× using a light microscope (Beijing Sunny Instruments Co., Ltd.). Follicles were identified across various stages, from primordial to antral, in accordance with the Amsterdam Consensus Criteria (25,26).

Estrous cycle staging was performed through daily vaginal smear cytology between 9:00-10:00 a.m. Vaginal lavage samples were obtained using 10 µl sterile PBS, placed onto poly-L-lysine coated slides, and fixed in 4% paraformaldehyde at 4°C for 15 min. Hematoxylin (3 min) and eosin (30 sec) staining at room temperature, adhering to standard protocols.

Data are presented as the mean ± SD based on a minimum of three independent experiments. Prior to analysis, normality was assessed using Shapiro-Wilk test (α=0.05) and the homogeneity of variance was confirmed via Brown-Forsythe test. Parametric tests were applied when assumptions were satisfied. Statistical analyses were carried out using GraphPad Prism 9.5 software (Dotmatics). For the comparison between two groups, a two-tailed unpaired Student's t-test was used, with Cohen's d effect sizes and 95% confidence intervals automatically calculated by the software. For the comparison of >2 groups, one-way ANOVA with Tukey's post hoc test (for all pairwise comparisons) or Dunnett's post hoc test (for comparisons vs. the control group) was applied, with partial η² effect sizes and family-wise error rate control at α=0.05. P<0.05 was considered to indicate a statistically significant difference.

To knock down PrP^c in mGCs, the following lentivirus-based vectors were used: mPRNP sh-1, sh-2 and sh-3. Compared with those of mGCs infected with the NC vector (sh-NC), the mRNA expression levels of PRNP in the sh-1 group exhibited no significant change (P>0.05). However, infections with sh-2 and sh-3 resulted in a marked reduction in mRNA expression (sh-2:P<0.05; Sh-3:P<0.001), respectively, confirming successful knockdown of the PRNP gene (Fig. 1A). Supporting these findings, WB analysis revealed that the gray values of the PrP^c protein bands in cells transfected with sh-2 and sh-3 were significantly reduced compared with those in the sh-NC group (P<0.05; Fig. 1B). Based on these results, sh-2 and sh-3 were selected as the most efficient shRNAs for subsequent experiments.

In the PrP^c OE group, the mRNA expression levels of PRNP were significantly increased compared with those in the OE-NC group (P<0.001; Fig. 1C), indicating that the OE vector effectively increased the transcription of the PRNP gene. Correspondingly, the expression levels of the PrP^c protein in the OE group was significantly increased compared with that in the OE-NC group (P<0.01; Fig. 1D), aligning with the observed trend in mRNA expression levels.

Flow cytometry analysis was carried out to assess cell cycle distribution in mGCs. The results revealed no significant differences in the proportions of cells in the G₁, S and G₂ phases among the sh-2, sh-3 and sh-NC groups (P>0.05; Fig. 2A). Likewise, no notable changes were observed in the distribution of cells across these phases in the OE group compared with OE-NC (P>0.05; Fig. 2B).

Analysis of apoptosis using the Annexin V/PI double staining method by flow cytometry revealed that, under normal culture conditions, the apoptosis rate of mGCs was consistent between the sh-NC and OE-NC groups. Moreover, sh-2, sh-3 and sh-NC did not exhibit any significant differences in the proportion of apoptotic cells (P<0.05; Fig. 2C). Similarly, the OE group demonstrated no notable variation in the proportion of apoptotic cells compared with OE-NC (P<0.05; Fig. 2D).

Ovaries from WT and KO mice were isolated and WB was carried out to assess PrP^c protein expression levels. The results indicated that PrP^c levels in the ovaries of KO mice were reduced compared with those in the WT group (Fig. 4A) confirming the effectiveness of the KO.

At 7 weeks of age, ovarian tissue was collected, and the wet weight index of ovaries was measured. The WT group had an ovarian weight of 0.390±0.08 mg/g, while the KO group exhibited a weight of 0.389±0.02 mg/g, with no statistically significant difference between the groups (P>0.05; Fig. 4B).

H&E staining revealed the pathological and morphological characteristics of the ovaries in each group (Fig. 4C). Although the KO group exhibited a slight reduction in the number of primordial and primary follicles, this difference was not statistically significant (P>0.05; Fig. 4D). Similarly, there were no significant differences in the numbers of secondary, antral or atretic follicles between the two groups (P>0.05; Fig. 4D).

FSH levels in KO mice were significantly increased at 6.64±0.68 ng/ml compared with 4.52±1.18 ng/ml in the WT group (P<0.01; Fig. 5A). By contrast, E₂ levels were not significantly different between the two groups (P>0.05; Fig. 5B). Similarly, AMH and LH levels were also not significantly different between WT and KO mice (P>0.05; Fig. 5C and D).

WT and KO mice exhibited a standard estrous cycle, typically spanning 4–5 days per cycle and encompassing four distinct phases: Proestrus, estrus, metestrus and diestrus (Fig. 6A). In the proestrus stage (P), the smears are primarily made up of nucleated epithelial cells, which are characterized by their polygonal shape, large spherical nuclei, and abundant cytoplasm. As the cycle progresses to estrus (E), there is a notable increase in keratinized epithelial cells, which display a distinct eosinophilic staining in their cytoplasm. This is followed by metestrus (M), where the number of keratinized cells begins to decline, and nucleated epithelial cells become more present, accompanied by a few clusters of leukocytes. Finally, in the diestrus stage, the cytological composition shifts to predominantly leukocytes, with few scattered nucleated epithelial cells present (27). Each stage in the WT mice was clearly defined, with smooth and consistent transitions between phases. Similarly, KO mice exhibited no notable alterations in cycle duration, with neither a marked prolongation nor a noticeable shortening of the cycle observed (Fig. 6B).

No statistically significant differences were observed in the litter intervals between the two groups (P>0.05; Fig. 6C). The average litter size remained consistent at 8–10 pups, and the mothers exhibited normal nurturing abilities. The offspring survival rate was high, with no notable differences detected between the groups (P>0.05; Fig. 6D).