Establishment of porcine ovarian follicles treated with t-BHP for an in vitro aging model

Ovarian aging is closely associated with low female fertility. Excessive oxidative stress can induce ovarian senescence and follicular atresia, thereby reducing reproductive performance. In this study, intact antral follicles of pigs were treated with tert-butyl hydroperoxide (t-BHP) to simulate aging stimulation conditions. The results showed that 200 μM t-BHP induced the senescence-related phenotype of antral follicles, which was time-dependent and changed significantly after 6 h of treatment. t-BHP can induce an increase in reactive oxygen species (ROS) and senescence mediated by Caspase-3, P53, Foxo1, and SOD. Senescence-associated β-galactosidase (SA-β-Gal) staining revealed an increase in the number of positive cells. Enzyme-linked immunosorbent assays (ELISA) showed that the level of reactive oxygen species increased, and that the ratio of progesterone (P4) to estradiol (E2) increased. Finally, the findings of this study showed that the relative expression levels of Caspase-3, P53, Foxo1 mRNA, and protein increased, while the relative expression levels of SOD decreased. In conclusion, treatment with 200 μM of t-BHP effectively induced follicular senescence at 6 h. Therefore, the in vitro treatment of follicles with 200 μM t-BHP for 6 h may be a feasible in vitro culture model to simulate ovarian senescence in gilts.

tissue is reduced. Cellular senescence is regularly related to age cause a lack of regenerative ability is a marker 48 of tissue senescence [4]. The number of senescent cells increases with age in multiple tissues [5][6][7].
In female mammals, aging has an extensive impact on reproductive performance. Ovarian senescence is 50 a process in which the potential of the ovaries to produce fully functional gametes is progressively weakened.

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Ovarian senescence includes the non-stop loss of follicles and a limit in the whole quantity of follicles, which 52 consequently leads to a gradual decrease in female fertility. In addition, due to a decrease in ovarian follicle 53 cells, ovarian aging leads to the failure of the ovary to produce adequate sex hormones to maintain normal 54 physiological features in animals or humans [8,9]. Therefore, the continuous decline of follicle quantity with 55 increasing age eventually leads to hormone imbalance and an irregular ovarian cycle. Additionally, aging 56 ovaries also produce poor-quality oocytes, which can lead to developmental stagnation, aneuploidy, 57 incapability to implant embryos, and abortion [10][11][12]. Granulosa cells and corpus luteum cells of follicles are 58 necessary sources of estrogen and progesterone for female physiological activities. A permanent increase in 59 the arrest of granulosa cells might also inhibit the capability of ovarian follicles to mature, mainly causing 60 atresia of immature follicles.
The reproductive physiology of sows significantly changes after aging. On one hand, fecundity decreases, 62 and the elimination rate increases. However, the decrease in dominant sows will result in serious losses in 63 production efficiency. The ovaries of pigs typically develop unexpectedly from 72-165 days of age. HE 64 staining for atresia regulation of follicles in the ovarian tissues of pigs confirmed that in the course of oocyte proliferation, spare oocytes were apoptotic. In the follicular stage, atresia of a wide range of primordial 66 follicles and dominant follicles were observed, and the total primordial follicle population confirmed constant 67 atresia [13] . Atresia of follicles at all stages can be observed in the fast follicular boom phase, with the 68 degeneration of primordial follicles being the most significant. Some studies have shown that TUNEL staining 69 can be used to examine the atresia of a large variety of primordial follicles and principal follicles in the oocyte 70 proliferation stage, and that atresia is derived from the apoptosis of follicular oocytes [14]. Atresia can occur 71 in all stages of follicle development. Atresia of the main follicles is typically prompted by the apoptosis of 72 oocytes, and some are accompanied by the simultaneous apoptosis of oocytes and follicular cells. Atresia of 73 secondary follicles was once broadly thought to be prompted by the apoptosis of granulosa cells. With the 74 rapid increase in the length of follicles and the rapid boom of follicles, atresia of follicles at all ranges becomes 75 more obvious.
With the enhanced survival of patients after treatment with chemotherapy for malignant tumors and 77 immune diseases, it has been reported that chemotherapeutic drugs such as cyclophosphamide, cisplatin, 78 tripterygium wilford, and hydrocortisone affect ovarian function and precipitate reproductive issues. There is 79 an increased frequency in the use of chemical strategies to manage diseases. Cyclophosphamide, one of 80 common chemotherapeutic drugs, has an exceptionally poisonous impact on the ovary. It works by inhibiting 81 the synthesis of DNA, RNA, and protein, inflicting gonadal injury [15]. Because of the limited technology of 82 ovarian culture in vitro and most of the chemical methods used to establish mouse models [16], aging models 83 of porcine ovaries are rare [17,18]. Most in vitro aging models are constructed using cells, and oxidative 84 damage is one of the most commonly used methods to construct aging models. Oxidative stress-inducing 85 conditions were adjusted for experimental purposes using different cells. Human skeletal myoblasts were 86 treated with 1 mmol/L hydrogen peroxide for 30 min to investigate the regulatory effect of the tocotrienol-87 rich fraction on senescence [19]. Another study reported that t-BHP could induce the aging of mouse 88 hematopoietic stem cells, and the percentage of aging cells reached 57.92% ± 4.24% after 6 h[20].

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It is urgent to establish an in vitro aging model to study the biological function of ovarian aging because it is 90 difficult to obtain in high-parity sow samples. Therefore, in this study, an aging model of follicles in vitro was 91 established, which provided experimental data for the study of the mechanism of delaying aging on 92 reproductive physiology.

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The basal medium DMEM/Ham's F-12 (DMEM/F12) was purchased from Thermo Fisher Scientific. PBS  The ovaries of sexually mature pigs had been amassed from a neighborhood slaughterhouse in Baoding, Hebei 105 Province, and placed in ordinary saline supplemented with 100 U/mL penicillin and 100 g/mL streptomycin 106 at 37 ℃, stored in vacuum flasks, and introduced again to the laboratory as quickly as possible.

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Screening and culture of intact antral follicles 108 The ovaries were sprayed with 75% ethanol to create a sterile environment and then rinsed two to three times 109 with 37 °C saline containing antibiotics until water was clear. The ovaries were placed in a preheated bottle 110 containing saline and antibiotics. The isolated follicles without red/corpus luteum, with uniform and 111 transparent texture and dense distribution of antral follicles, were selected, and the diameter of the isolated 112 follicles was measured using a ruler. The intact follicles measuring 3-6 mm in diameter, with the follicular 113 cavity, full follicular cavity, rich blood vessels, and clear follicular fluid without dark spots were selected and 114 seeded in 24-well plates; one follicle per well, at 37 °C, 5% CO2, and incubated in a constant humidity 115 incubator. Morphological changes in the follicles were observed and recorded, such as whether the color of 116 the follicles turned gray, the wall of the blood vessels decreased, the follicular fluid showed flocculant 117 deposits, and dark spots were present on the inner wall of the follicles.

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The cells were divided into three groups according to treatment: control, time processing, and t-BHP-treated 120 groups. Pre-treatment for 24 and 36 h was used to evaluate the appropriate culture conditions for maintaining 121 healthy follicles in the current study. Follicles in the t-BHP-treated group were treated with 200 μM t-BHP 122 for 1, 2, 6, and 12 h to simulate the oxidative stress-induced senescence microenvironment.

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A total of 50 μL pattern was placed in every well and incubated at 37 ℃ for 30 min. Each properly was once 125 brought with a 50 μL enzyme-labeled reagent and incubated at 37 ℃ for 30 min. A50 μL shade developer was 126 added to every well, along with B50 μL shade developer, and the solution was mixed with mild shaking and 127 allowed to react in the dark at 37℃ for 15 min. Then, 50 μL quit answer was brought to every properly to the 128 reaction. The OD density of each well was measured at 450 nm.

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Then, 50 μL pattern and 100 μL of detection antibody were added into every well and incubated at 37 ℃ for 131 60 min. Substrates A and B (50 μL each) were added into every well and the solutions were incubated in the 132 dark at 37 ℃ for 15 min. 50 μL stop buffer were introduced into every well. The OD was measured at a 133 wavelength of 450 nm within 15 min.

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Ovarian granulosa cell acquisition 135 Granulosa cells were collected after the follicle culture was completed. The follicles were removed from the 136 24-well plate with sterile ophthalmic tweezers; the follicle membrane was cut with a sterile surgical blade and 137 follicle fluid was collected. The follicle fluid was filtered through a cell sieve and inoculated into a 6-well 138 plate, and cell growth was observed by adding 15% DMEM/F 12 complete medium. Briefly, DCFH-DA probes were directly added to serum-free medium to be diluted to the working 147 concentration (final concentration: 10 μM), and 1 mL of diluted DCFH-DA solution was added after the 148 medium in the six-well plate was discarded. The medium was incubated for 30 min at 37℃, discarded, and 149 washed with PBS twice before observation under a laser confocal microscope.

RNA extraction and Quantitative real-time PCR (qRT-PCR) analysis 151
The total RNA of the cells was extracted using TRIZOL. RNA concentration and quality were determined 152 using a spectrophotometer NanoDrop 2000 (Thermo Fisher Scientific, US). Samples with RNA concentrations 153 between 500 and 1,000 ng/mL and OD 260/280 value between 1.8 and 2.0 were selected for further analysis.

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According to the reverse transcription kit instructions, 1 mg of RNA was transformed into cDNA by reverse 155 transcription. qRT-PCR was performed using the SYBR Green method. The differential abundance of the 156 mRNA of genes relative to GAPDH was determined using the 2 -△ △ CT method. Primers were synthesized 157 using GENEWIZ (Jiangsu, China) (Table 1).

Gene Primer sequences (5'-3')
Annealing Temperature(°C)  Data are shown as the mean ± standard error of the mean (SEM) from three independent experiments. The 170 experimental data were sorted using Excel 2007 software, and the diagram was drawn using Prism 9. Statistical 171 significance was determined using the SPSS software version 26.0 (SPSS V26.0). One-way analysis of 172 variance (ANOVA) was performed, and pairwise comparisons were performed using the LSD method.

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Statistical significance was set at p < 0.05.

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Quality identification of porcine antral follicles 176 To verify the accuracy of judging the morphology of porcine isolated follicles, healthy follicles were selected 177 according to size (3-5 mm in diameter) (Fig 1A), and the follicles of the three groups were cultured and 178 observed under a microscope. deposits appeared in the cytoplasm (Fig 1B). Granulosa cells in the parietal layer of the follicles were stained 187 with SA-β-Gal to observe cellular senescence. It was found that the cells in the control group were short and 188 round with close intercellular connections, while the staining of positive cells was deepened and the 189 intercellular connection was not closed; the cells were partially deformed (Fig 1C), and the ratio of positive 190 cells was found to not be significantly higher at 24 h than in the control group, and significantly higher at 36 191 h ( Fig 1D).

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The results fluorescence staining showed that ROS in the 24 h group was not significantly higher than 193 that in the control group, but significantly increased after 36 h (Fig 1E, F). The results of hormone level measurement showed that the concentration of E 2 decreased significantly and 201 the concentration of P 4 increased significantly after 36 h, but there was no significant difference between the 202 two groups at 24 h. The P 4 /E 2 ratio increased significantly with an increase in follicular culture time (Table   203 3). This shows that atresia did not occur in the follicles during in vitro culture. Based on the above results, it 204 can be confirmed that 24 h of in vitro culture of follicles can be used in follow-up experiments. Different letters indicate statistically significant differences (P < 0.05).

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The optimal time of follicular senescence induced by t-BHP

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To verify whether t-BHP treatment at 200 μM for 1 h, 2 h, 6 h and 12 h induced follicular senescence, we did 209 further experiments with the following treatments: a control group without t-BHP and, and treatment with 200 210 μM sodium butyl peroxide for 1 h, 2 h, 6 h and 12 h, respectively. After treatment, the follicles were cultured 211 in drug-free medium for 24 h, and granulosa cells were collected and cultured until they adhered to the wall.

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The follicular phenotype showed intact follicular walls, visible blood vessels, and a clear follicular cavity 213 1 and 2 h after culture (Fig 2A). In the 6-h group, follicular blood vessels decreased, and a small number of 214 dark spots appeared in the follicular cavity (Fig 2A). However, at 12 h, the blood vessels in the wall of the 215 follicle disappeared, and the color of the follicle gradually became gray, with flocculent deposits in the lumen.

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The granulosa cells in the parietal layer broke off into clumps, which were significantly different from those 217 in the control group (Fig 2A). index, and most of the cells were senescent, with a positive staining rate of more than 80% in five random 224 fields (Fig 2B, C).

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The results of ROS fluorescence staining showed that there was weak ROS staining in the control group, 226 and staining was greater in the 1-h and 2-h group, but there was no significant difference between the two 227 groups. The expression levels of ROS in the 1-h and 2-h groups were higher than those in the control group, 228 and the ROS level increased significantly after 6 h and 12 h. Although the differences between groups were 229 not significant, they all increased significantly over time, with strong fluorescence staining of granulosa cells 230 and poorer cell status in the 12-h group (Fig 2D, E). Under the experimental conditions, the optimal time to 231 induce follicular senescence was 6 h with 200 μM t-BHP. The progesterone-to-estrogen ratio increased with time after t-BHP-induced follicular senescence.

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Progesterone can be synthesized in granulosa and membrane cells of follicles, and a low concentration of 242 progesterone contributes to the formation of the LH peak, thus inducing ovulation in mature follicles. Estrogen 243 stimulates granulosa cell proliferation and prevents apoptosis. The results of hormone level determination 244 showed that the concentration of E 2 decreased with an increase in treatment time, and the change in P 4 245 concentration was the opposite; the ratio of P 4 /E 2 was higher than that of the control group (Table 4). Different letters indicate statistically significant differences (P < 0.05).

Effect of 6-h treatment with t-BHP on the expression of genes and proteins in
249 porcine follicles 250 The gene expression levels of Foxo1, P53, Caspase-3, and SOD were determined by qRT-PCR. Normalized 251 to GAPDH, the gene expression of Foxo1, P53, and Caspase-3 was markedly increased by t-BHP treatment.

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The protein expression of Foxo1, P53, Caspase-3, and SOD was determined by western blot analysis. As  were also at normal levels, thus establishing that oxidative stress stimulation of follicles within 24 h of in vitro 284 culture induces senescence.

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The accumulation of oxidative stress has long-term effects on follicular failure [28,29]. Follicles were We would like to thank Dr. Junjie Li for helping with proofreading the manuscript.