This study was conducted in accordance with guidelines established by the Animal Care and Use Committee of China. The experimental procedures were approved by the Animal Ethics and Welfare Committee of Sichuan Agricultural University (#SCAUAC2020-84).

The isolation and culture of primary yGCs were based on a previous report, with minor modifications (26). Briefly, the ovaries of yak heifers were collected from Qinghai Yutai Livestock Products Co., Ltd. (Xining, China) and transported to the laboratory within 3 h at a constant temperature (approximately 37°C) in phosphate-buffered saline (PBS; Solarbio, Beijing, China) containing 5% streptomycin and penicillin (Gibco, Shanghai, China). Follicular fluid was aspirated from 2 to 8 mm small follicles using a 2.5 mL sterile syringe (Honghu Taining Medical Devices Co., Ltd., Jingzhou, China), placed in a 15-mL sterile centrifuge tube (NEST Biotechnology, Wuxi, China), and centrifuged at 300 × g for 3 min to obtain the precipitate. The precipitate was resuspended in PBS (containing 2% streptomycin and penicillin), preheated at 37°C, filtered through BeyoGold™ 40 μm cell strainers (Beyotime, Shanghai, China), and centrifuged at 300 × g for 3 min to remove impurities. The final cell precipitate was resuspended in Dulbecco’s Modified Eagle’s Medium and Ham’s F12 Nutrient Mixture (DMEM/F12, Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Shanghai, China) and 1% streptomycin and penicillin. The resuspended yGC was transferred to a T25 flask (NEST) at a density of 1 × 10⁵ cells/mL and cultured in an incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C and 5% CO₂. The medium was replaced every 48 h to prevent nutrient depletion. When yGCs reached approximately 85% confluence, they were passaged at a 1:2 ratio.

The HBLV-ZsGreen-Puro vector was purchased from VectorBuilder (Guangzhou, China), and the HBLV-SV40T-3*flag-ZsGreen-Puro lentiviral expression vector was constructed and packaged. The method for establishing immortalized yGC was based on a previous report with minor modifications (27). Briefly, when the yGC reached 50% confluency, the culture medium was changed to DMEM/F12 with multiple infection numbers (MOIs) of 80 and 5 mg/L of polybrene (Sigma, St. Louis, MO, United States). After overnight infection with the lentivirus, yGCs were cultured in a fresh medium for 4 days. Subsequently, the yGCs were cultured in a medium containing 2 mg/L of puromycin for 4 days to screen for surviving cell lines, which were designated as immortalized yGCs. The medium was changed every 48 h, unless otherwise specified.

Primary (passage 1) and immortalized (passage 12) yGCs were seeded at a density of 2 × 10³ cells/well in 96-well plates (NEST) and cultured overnight. Cell viability was determined using the Cell Counting Kit 8 (CCK-8, APExBio, Houston, TX, United States) on days 0, 1, 2, 3, 4, 5, and 6, with six replicates per time point. The culture medium in each well was replaced with a mixture (100 μL) containing 90% serum-free DMEM/F12 and 10% CCK-8 solution and incubated at 37°C for 2 h. The absorbance of each well at 450 nm and 650 nm was measured using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA, United States). Three wells without cells were used as blank controls.

Primary yGCs (passages 1, 5, and 7) and immortalized (passages 7, 15, and 23) yGCs were incubated in 6-well plates (NEST) for 48 h. The medium was discarded, and the cells were washed with PBS and fixed in 4% paraformaldehyde (Beyotime) for 15 min at room temperature. Next, the cells were stained with a senescence β-galactosidase (SA-β-Gal) staining kit (Beyotime) at 37°C for 12 h, following the manufacturer’s instructions. The presence of senescent cells triggered by the activity of a specific senescence-associated β-galactosidase was identified under a light microscope by observing the resulting dark blue product.

Immunofluorescence of ovarian tissue, yGCs, and negative controls (without additional primary antibodies) was performed according to the previous reports, with minor modifications (27, 28). Briefly, fresh yak ovaries were fixed in 4% paraformaldehyde for 24 h and embedded in wax, sliced, and dewaxed before antigen retrieval. Primary (passage 1) and immortalized (passage 10) yGCs were incubated in 6-well plates for 24 h and then fixed with 4% paraformaldehyde for 15 min. The ovarian tissue and cells were sealed with 5% goat serum (Solarbio, Beijing, China) and incubated with an anti-follicle-stimulating hormone receptor (FSHR) antibody (Bioss, Beijing, China). Immortalized yGCs were incubated with an anti-SV40T-antigen antibody (Abcam, Cambridge, MA, United States) for 12 h at 4°C. The target protein was subsequently labeled with fluorescein isothiocyanate- (FITC-) or sulfo-cyanine 3 (CY3)-conjugated goat anti-rabbit IgG secondary antibodies (Bioss) for 2 h. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Solarbio, Beijing, China) for 2 min and then observed under a fluorescence microscope (Nikon, Japan).

Primary (passage 1) and immortalized (passage 10) yGCs were incubated in 6-well plates until 80% confluency was reached. Thereafter, the medium was replaced with fresh culture medium, and the cells were cultured for 24 h before collection. The concentrations of estradiol and progesterone in the culture medium were determined using commercially available ELISA kits (Meimian, Jiangsu, China), following the manufacturer’s instructions.

Total RNA from primary (passage 1) and immortalized (passage 10) yGCs was extracted and purified using a total RNA extraction kit (Accurate; Changsha, China). The concentration and purity of the extracted RNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Subsequently, the qualified RNA was reverse-transcribed to cDNA using a reverse transcription kit (ExonScript RT SuperMix with dsDNase; Exongen, China). The synthesized cDNA was diluted to 1:4 with enzyme-free water (Sangong, Shanghai, China). Quantitative polymerase chain reaction (qPCR) was performed using the 2 × Fast SYBR Green qPCR Master Mix Kit (Servicebio, Wuhan, China) in the Applied Biosystems^™ QuantStudio^™ 5 Real-Time PCR System (Thermo Fisher Scientific) according to the manufacturer’s instructions. The primer sequences used in the qPCR analysis are listed in Table 1, with β-actin serving as the housekeeping gene. The relative mRNA abundance of target genes was determined using the comparative cycle threshold (2^−ΔΔCt) method.

Statistical significance was determined using an independent sample t-test with SPSS v23.0 (SPSS Inc., Chicago, IL, United States), and the findings are presented as the mean and standard error. A p-value of <0.05 was considered statistically significant.

The formation of typical “paving stones” monolayer cells (Figure 1A) characterized the transition of primary yGCs into a stationary density state. The growth curve of primary yGC (Figure 1B) exhibited an S-shaped trajectory culminating in a plateau at 120 h.

Immunofluorescence analysis of yak ovaries revealed that the FSHR protein was specifically expressed in ovarian follicles (Figure 2A). Furthermore, cell immunofluorescence revealed that the isolated cultured cells were yak GCs, with over 85% of the cells in the field of view expressing the FSHR protein (Figure 2B).

SA-β-Gal staining has been widely used to evaluate cellular senescence. The results of this study demonstrated that primary yGCs from the first passage stained negatively for SA-β-Gal (Figure 3A). However, the fifth-generation primary yGCs displayed positive staining for SA-β-Gal, as indicated by the appearance of blue-colored areas within the cells (Figure 3B). Furthermore, the seventh generation primary yGCs showed distinctly positive SA-β-Gal staining, as evidenced by the midnight blue color areas within almost all cells (Figure 3C). These findings revealed that primary yGCs exhibited replicational senescence in the late passage.

Similar to the primary yGCs, the growth curve of the immortalized yGCs followed a characteristic S-shape, as shown in Figure 4A. The immortalized yGCs retained the morphological features of the primary yGCs across different generations (Figure 4B). Furthermore, negative staining for SA-β-Gal indicated that immortalized yGCs lost the replicative senescence characteristics of the primary yGCs within a limited number of generations (Figure 4C).

The immortalized yGCs, similar to the primary yGCs, showed strong immunoreactivity to the FSHR protein (Figure 5A). Chromosomal karyotype analysis was used to confirm the origin of the cell line based on cytogenetic characterization. Chromosomes were analyzed, compared, ranked, and numbered based on chromosome length, centromere position, ratio of long and short arms, and presence or absence of followers. The results revealed that the immortalized yGCs had a diploid karyotype consistent with the chromosomal characteristics of the yak species (2n = 60), including 29 pairs of autosomes and one pair of sex chromosomes (Figure 5B). The immortalized yGCs were infected with lentivirus expressing SV40 large T antigen, which was produced by co-transfecting the lentiviral vector (HBLV-SV40T-3*flag-ZsGreen-Puro) with packaging vectors (Figure 5B). The immortalized yGCs had a significantly higher abundance of SV40T mRNA than the primary yGCs (p < 0.05) (Figure 5C).

There was no difference in the concentrations of estradiol and progesterone in the culture media of primary and immortalized yGCs (Figures 6A,B). However, the mRNA expression of CYP19A1, FSHR, and HSD17B1 was significantly higher in immortalized yGCs than in primary yGCs (p < 0.05). In contrast, STAR mRNA expression was significantly higher in primary yGCs than in immortalized yGCs (p < 0.05) (Figure 6C).