A total of eight lncPrep + 96kb-KO mice were obtained from GemPharmatech Co., Ltd (cat. no. AB849013, C57BL/6J, female mice, 20–25 g, 30 day old, GemPharmatech Co., Ltd). All animals were housed at the Laboratory Animal Center of the Medical College of Nanchang University (Nanchang, China) under controlled conditions, including a stable temperature of 22±2°C, relative humidity of 50±10% and a 12-h light/dark cycle, and free access to sufficient food and water. All animal care and procedures were approved by the Experimental Animal Center at Nanchang University (approval no. NCULAE-202209280023).

Genotyping of mice was performed using PCR amplification (2 X EasyTaq PCR SuperMix(+dye), cat. no. AS111-11, TransGen Biotech Co., Ltd.) of genomic DNA extracted from tail biopsies. Genotyping was performed using the following primers: LncPrep + 96kb wild-type (WT) mice, forward 5′-GGTAATCCTAACGCCACG-3′ and reverse 5′-TACCGAGGCACAGTTTCCA-3′; lncPrep + 96kb-KO mice, forward 5′-GACACGCCTATTCATACAAGGC-3′ and reverse 5′-GCAAGCGAGTCAGATTTGGCTTAGAAG-3′. Thermocycling conditions were as follows: 94°C for 10 min, followed by 36 cycles of 94°C for 30 s, 55°C for 45 s, and 65°C for 45 s, with a final extension at 65°C for 10 min. PCR products were separated on a 2% agarose gel (cat. no. BKM-AG-100, Shenzhen Bikamei Biotechnology Co., Ltd.) with 1 X Green fluorescent nucleic acid dye (10000X) (cat. no. G8140, Beijing Solarbio Science & Technology Co., Ltd.), and visualized under UV light. Wild-type mice showed a single band at 344 bp, while heterozygous mice showed bands at 390 bp and 344 bp, and homozygous mice showed a single band at 390 bp (Fig. S1)

Six female mice aged 21 days, both WT and KO female mice, were randomly selected. The body weight of the female mice was recorded every three days for a total of five times (Fig. S2). The female mice were mated with 12 proven fertile male mice (C57BL/6J, 20–25 g, 21 day old, Changsha Tianqin Biotechnology Co., Ltd.) at a 1:1 ratio. Successful mating was confirmed by the presence of a vaginal plug. After natural delivery, the pregnancy rate and the number of offspring were recorded.

Superovulation was induced in KO and WT female mice through intraperitoneal injection of 10 IU pregnant mare serum gonadotropin (PMSG; cat. no. P9970; Beijing Solarbio Science & Technology Co., Ltd.), followed by an additional 10 IU human chorionic gonadotropin (cat. no. YZ-150555; Beijing Solarbio Science & Technology Co., Ltd.) injection 48 h later. The mice were euthanized by cervical dislocation 13 h after the second injection. Subsequently, the ampulla of the fallopian tube was dissected under a stereomicroscope to collect and count oocytes for further analysis.

The ovarian tissue were extracted from KO and WT mice and fixed in 4% paraformaldehyde (cat. no. P1110; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 24 h and then embedded in paraffin. Subsequently, 3-4-µm sections were prepared, followed by dewaxing and hydration. Using a H&E Staining Kit (cat. no. G1121; Beijing Solarbio Science & Technology Co., Ltd.), the nuclei were stained with hematoxylin for 15 sec and the cytoplasm was stained with eosin at room temperature for 30 sec. Finally, dehydration, clarification, neutral gum sealing and light microscopic observation were carried out sequentially.

Ovarian tissue sections (5 µm) were deparaffinized and stained using the Sirius-Red Staining solution (cat. no. G1472; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions at room temperature for 24 h. The slices were then immersed in xylene and alcohol and mounted with synthetic resin. Three images from different fields of view were randomly selected for each tissue section, and images were captured using a light microscope (magnification, ×10). The images were exported in JPG format with a resolution of 1,920×1,080 pixels via NDP.view2 (Hamamatsu Photonics K.K.). ImageJ (National Institutes of Health) was used to select areas and collagen fiber content was semi-quantified by calculating the ratio of staining intensity in the selected area to the total staining intensity of the ovarian tissue.

PCDNA3.0-lncPrep + 96kb 2.2kb and PCDNA3.0-lncPrep + 96kb 2.8kb plasmids (37), and a pMigR1-POP plasmid (18) were constructed as previously described. The lncPrep + 96kb 2.2kb and 2.8kb sequences were amplified by PCR and inserted into the PCDNA 3.0 vector (cat. no. V79020, Thermo Fisher Scientific Inc.) following restriction enzyme digestion with KpnI (cat. no. R3142M; New England BioLabs, Inc.) and EcoRI (cat. no. R3101V; New England BioLabs, Inc.). A guide RNA (gRNA) targeting lncPrep + 96kb was cloned into the pSpCas9(BB)-2A-GFP vector (PX458) (cat. no. 48138, Addgene, Inc.). The PX458 plasmid was digested with BbsI and then annealed to the gRNA, and successful insertion was confirmed by Sanger sequencing. PCR primer sequences for lncPrep + 96kb 2.2kb were: Forward, 5′-TTGGTACCAGCTTGTGTATTGCTCATAT-3′ and reverse, 5′-GGAATTCTTTGCTTTTTAATTTTTATTTG-3′; and the sequences for lncPrep + 96kb 2.8kb were: Forward, 5′-AGATCATGACAGGGGCTCCT-3′ and reverse, 5′-AGCTGGCTGGTCCTCACAG-3′.

To construct the pMigR1-POP plasmid, the entire sequence of POP was amplified and digested with XhoI (cat. no. R0146V; New England BioLabs, Inc.) and EcoRI, and were subsequently cloned into the pMig plasmid vector (cat. no. MZ0431, Shanghai Qiming Biotechnology Co., Ltd.). The successful insertion was confirmed through Sanger sequencing. PCR primer sequences for POP were: Forward, 5′-CCGCTCGAGATGCTGTCCTTCCAGTACCC-3′ and reverse, 5′-CCGGAATTCTTACTGGATCCACTCGATGTT-3′.

A total of six female mice (C57BL/6J, 25–30 g, 21 day old, Changsha Tianqin Biotechnology Co., Ltd.) were injected with 5 IU PMSG. After 48 h, the mice were euthanized by cervical dislocation and their ovaries were harvested. The ovaries were then placed in petri dishes containing sterile PBS (cat. no. P1020; Beijing Solarbio Science & Technology Co., Ltd.) and follicles were mechanically punctured with an injection needle to release granulosa cells. The PBS containing granulosa cells was collected in a centrifuge tube (cat. no. UFC910096; Merck KGaA) and centrifuged at 2,500 × g for 5 min at room temperature. After discarding the supernatant, the cells were cultured in DMEM/F12 (cat. no. D6501, Beijing Solarbio Science & Technology Co.; Ltd.), 1% penicillin-streptomycin (cat. no. FG101-01, TransGen Biotech Co., Ltd.,) in a 6-well culture plate (cat. no. CFE-F1006-01; TransGen Biotech Co., Ltd.,) at 37°C in a humidified atmosphere of 5% CO₂. Once cell confluence reached 70–80%, the cells were transfected with 2 µg plasmid using FuGENE^® 6 Transfection Reagent (cat. no. E2691; Promega Corporation). After 8 h at 37°C, the medium was replaced with fresh medium. The cells underwent RNA and protein extraction 48 h post-transfection, and the effects of plasmid transfection were verified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting (WB) (Fig. S3).

RNA was extracted from granulosa cells or ovarian tissue using TransZol UP Enhanced RNA extraction kit (cat. no. ET111-01-V2; TransGen Biotech Co., Ltd.), and its concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). RT was performed using the RT Kit (cat. no. AE311; TransGen Biotech Co., Ltd.;) according to the manufacturer's protocol. RT was carried out at 42°C for 30 min, with reverse transcriptase inactivation performed at 85°C for 5 min. qPCR was conducted using the QuantiNova^® SYBR Green PCR Kit (cat. no. 208054; Qiagen GmbH). According to the kit's instructions, the amplification system was prepared and run on a PCR instrument (CFX96 Connect; Bio-Rad Laboratories, Inc.). The reaction conditions were as follows: Initial denaturation step at 95°C for 5 min, followed by 40 cycles at 60°C for 10 sec and 95°C (+0.5°C/cycle) for 15 sec, and a final step at 95°C for 15 sec. β-actin was used as an internal control for standardization, and relative quantification was calculated using the 2^−ΔΔCq method (38). Primer sequences, supplied by TransGen Biotech Co., Ltd, are listed in Table I.

Ovarian tissue or cells were lysed in RIPA buffer (cat. no. PC101; Shanghai Yamei Biomedical Technology Co., Ltd.,) supplemented with 1X alkaline phosphatase inhibitors 100X (cat. no. P1260; Beijing Solarbio Science & Technology Co., Ltd.) and 1X protease inhibitors 100X (cat. no. P6731; Beijing Solarbio Science & Technology Co., Ltd.). Protein concentration was measured using the Easy II Protein Quantitative (BCA) Kit (cat. no. DQ111-01; TransGen Biotech Co., Ltd). A total of 20 µg/lane protein samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were then transferred to PVDF membranes (pore size, 0.2 µm; cat. no. ISEQ00010; Merck KGaA). The membranes were blocked with Blocker™ BLOTTO TBS solution (cat. no. 37530; Thermo Fisher Scientific, Inc.) for 1 h at room temperature, followed by overnight incubation at 4°C with primary antibodies. After an incubation with the secondary antibodies at room temperature for 1 h, the protein bands were visualized and semi-quantified using the EasySee Western Blot Kit (cat. no. DW101-01; TransGen Biotech Co., Ltd.) and Image Lab™ software 3.0 (Bio-Rad Laboratories, Inc.).

The following antibodies were used for WB: Anti-PREP/POP (1:1,000; cat. no. A00298-2; Bioworld Technology, Inc.), anti-β-actin (1:5,000; cat. no. 66009-1-Ig; Wuhan Sanying Biotechnology), anti-peroxisome proliferator activated receptor γ (PPAR-γ; 1:1,000; cat. no. BA2120; Boster Biological Technology Co., Ltd.), anti-TGF-β1 (1:500; cat. no. BA0290; Boster Biological Technology), anti-MMP2 (1:1,000; cat. no. A00286; Boster Biological Technology), Mouse anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (1:20,000; cat. no. HS101; TransGen Biotech Co., Ltd.), and Rabbit anti-Mouse IgG (H+L) Secondary Antibody, HRP (1:20,000; cat. no. HS201; TransGen Biotech Co., Ltd.).

Data were statistically analyzed using GraphPad Prism 7.00 (Dotmatics), and are presented as the mean ± SD. All experiments were repeated at least three times. Differences between two groups were evaluated using an independent samples Student's t-test. One-way ANOVA followed by Student-Newman-Keuls test was used for statistical comparisons among multiple groups. Two-way ANOVA followed by Tukey's HSD test was used to analyze the changes in body weight of mice. P<0.05 was considered to indicate a statistically significant difference.

The ovaries serve a crucial role in female reproduction and their abnormal development directly impacts fertility. To investigate the role of lncPrep + 96kb in ovarian function, the fertility of KO and WT mice were assessed. Over 6 months of female fertility testing, KO mice produced significantly fewer pups compared with WT mice (Fig. 1A). No significant differences were observed in body weight changes between the groups (Fig. S2). The number of litters born to KO mice was notably lower than the number born to WT mice (Fig. 1B), and the average litter size was also significantly reduced in the KO group (Fig. 1C). Additionally, the ovulation rate in immature superovulated mice was evaluated, and it was revealed that KO female mice ovulated fewer eggs (22±12) compared with WT female mice (41±21) (Fig. 1D, E).

Ovaries from 2-month-old WT and KO mice were subjected to H&E and Sirius-Red staining. H&E staining revealed no notable differences in follicular morphology between the two groups (Fig. 2A). However, Sirius-Red staining highlighted a marked increase in ovarian fibrosis in KO mice compared with that in WT mice (Fig. 2B). To improve the clarity of fibrosis assessment, Sirius-Red staining was performed without hematoxylin counterstaining; the results showed a marked increase in collagen deposition in KO mice (Fig. 2C). Analysis of three randomly selected Sirius-Red staining sections using ImageJ software confirmed a higher collagen fiber content in KO ovaries compared with in WT ovaries (Fig. 2D).

Cytokines such as MMP2, TGF-β1 and PPAR-γ are crucial in the development of tissue fibrosis (15,39). To further explore their roles in ovarian fibrosis, mRNA (Fig. 3A) and proteins (Fig. 3B) were extracted from the ovaries of KO and WT mice. The results revealed an upregulation of TGF-β1 expression in KO mice, whereas MMP2 and PPAR-γ expression levels were significantly downregulated.

LncPrep + 96kb has been identified as a lncRNA transcribed from a conserved non-coding sequence of the mouse POP gene, which regulates POP expression in ovarian granulosa cells (36). Our previous study demonstrated that POP serves a role in androgen-induced fibrosis in an animal model of PCOS by regulating TGF-β1 and MMP-2 expression (18). Analyses of both mRNA (Fig. 4A) and protein (Fig. 4B) levels revealed reduced POP expression in the ovaries of lncPrep + 96kb-KO mice. To further confirm this, granulosa cells were isolated, and it was confirmed that the mRNA expression levels of POP were lower in the KO group compared with those in the WT group (Fig. 4C). Conversely, POP expression was significantly increased in granulosa cells transfected with PCDNA3.0-lncPrep + 96kb 2.2kb and 2.8kb plasmids (Fig. 4D and E). These findings indicated that lncPrep + 96kb may modulate POP expression in the ovary.

To further investigate the role of in ovarian fibrosis, granulosa cells were cultured and transfected with the pMigR1-POP plasmid. The results showed that TGF-β1 expression was decreased, and MMP2 and PPAR-γ expression was increased at both the protein (Fig. 5A) and mRNA (Fig. 5B) levels. These findings suggested that POP may modulate the expression of fibrosis-related factors in granulosa cells.