HDAC1-Mediated lncRNA Stimulatory Factor of Follicular Development to Inhibit the Apoptosis of Granulosa Cells and Regulate Sexual Maturity through miR-202-3p-COX1 Axis

Abnormal sexual maturity exhibits significant detrimental effects on adult health outcomes, and previous studies have indicated that targeting histone acetylation might serve as a potential therapeutic approach to regulate sexual maturity. However, the mechanisms that account for it remain to be further elucidated. Using the mouse model, we showed that Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, downregulated the protein level of Hdac1 in ovaries to promote the apoptosis of granulosa cells (GCs), and thus arrested follicular development and delayed sexual maturity. Using porcine GCs as a cell model, a novel sexual maturity-associated lncRNA, which was named as the stimulatory factor of follicular development (SFFD), transcribed from mitochondrion and mediated by HDAC1, was identified using RNA sequencing. Mechanistically, HDAC1 knockdown significantly reduced the H3K27ac level at the −953/−661 region of SFFD to epigenetically inhibit its transcription. SFFD knockdown released miR-202-3p to reduce the expression of cyclooxygenase 1 (COX1), an essential rate-limited enzyme involved in prostaglandin synthesis. This reduction inhibited the proliferation and secretion of 17β-estradiol (E2) while promoting the apoptosis of GCs. Consequently, follicular development was arrested and sexual maturity was delayed. Taken together, HDAC1 knockdown-mediated SFFD downregulation promoted the apoptosis of GCs through the miR-202-3p-COX1 axis and lead to delayed sexual maturity. Our findings reveal a novel regulatory network modulated by HDAC1, and HDAC1-mediated SFFD may be a promising new therapeutic target to treat delayed sexual maturity.


Introduction
Sexual maturity is a complex transitional phase in which adolescents acquire sexual maturation and reproductive capacity [1], and its early or late initiation seriously affects adolescent's mental and physical illnesses, such as depression [2] and polycystic ovarian syndrome (PCOS) [3].Follicular maturation and the secretion of steroid hormones closely regulate sexual maturity [4,5].An increase in follicular maturation is associated with precocious sexual maturity, while a decrease in follicular maturation is associated with delayed sexual maturity in adolescent girls [5].Previous studies have reported that delayed sexual maturity is often marked with arrested follicular development [6,7], reduced maturation rate and quality of oocytes [8][9][10], and is also the earliest manifestation of PCOS [3,11].However, the molecular therapeutic targets regulating sexual maturity remain unclear.
Histone acetylation, the most extensively studied and appreciated modification, exhibits an essential role in modulating chromatin structure to regulate gene transcription [12].Previous studies have demonstrated that the disorders of DNA methylation and histone acetylation contribute to delayed sexual maturity [13,14].Tomikawa et al. reported that 17β-estradiol (E2) increases the expression of Kiss1, a sexual maturity-activating gene, through elevating the level of histone H3 acetylation.Notably, the acetylation level in the Kiss1 promoter is remarkably higher in proestrus animals compared to diestrus animals [15].Decreased levels of H3K27ac at the IGF1 promoter in fetal ovarian granulosa cells (GCs) notably inhibit the synthesis of E2 and follicular development [16].Downregulation of histone deacetylase 3 (HDAC3) in GCs elevates the level of H3K14ac and promotes the binding of SP1 in the Areg promoter to initiate transcription and follicular maturation [17].
In GCs, GDF9 and BMP15 collaboratively recruit p300 to the AMH promoter, leading to elevated histone H3K27ac and markedly increased the expression of AMH, a natural gatekeeper of follicle growth and female reproduction [18].These observations indicate that targeting histone acetylation may serve as a potential therapeutic strategy to regulate sexual maturity in mammals.
The growth and function of GCs exhibit a dominant role in follicular maturation and sexual maturity [19,20].The proliferation and E2 secretion of GCs facilitate follicular growth [21,22], while the excessive apoptosis of GCs results in massive follicular atresia [23].Moreover, our previous study indicated that the growth of GCs participates in follicular maturation and sexual maturity [19].These findings reveal that histone acetylation may regulate the function of GCs to control sexual maturity.
To characterize the mechanisms for histone acetylation controlling sexual maturity, Trichostatin A (TSA), an HDAC inhibitor, was treated with mice to explore the effects of histone acetylation on follicular development and sexual maturity.The physiological mechanisms of porcine follicular development are more similar to those of humans than mice [24].Therefore, in our study, porcine GCs were used as a cellular model in vitro.Here, we found a novel sexual maturity-associated lncRNA, which was named as the stimulatory factor of follicular development (SFFD).Next, we aimed to explore the molecular mechanisms which histone acetylation regulated SFFD transcription and how SFFD regulated sexual maturation by affecting the functions of GCs.The regulatory mechanism and effects of the SFFD-miR-202-3p-COX1 axis on the proliferation, apoptosis, and estrogen secretion of GCs, follicular development, and sexual maturation were verified.Our results provide a new piece of evidence for HDAC1-mediated SFFD in the treatment of delayed sexual maturity.

Samples and Chemical Treatment
Three-week-old C57BL/6J mice (36 female and 4 male) from the Guangdong medical laboratory animal center were randomly assigned to three groups: blank (n = 12), DMSO (n = 12), and TSA (n = 12) group.Mice were treated with TSA (HY-15144, MedChemExpress, purity: 99.53%, 0.5 mg/kg) and an equal volume of DMSO for three weeks.Mice were injected 3 times weekly.Among them, 8 mice in each group were randomly selected for counting the age of sexual maturity (known as the pubertal initiation and indicated by the vaginal opening [25]), and their ovaries were used for HE staining, dUTP Nick-End Labeling (TUNEL), immunofluorescence, and Western blot.Two independent researchers examined the vaginal opening of mice for the visual appearance of an opening [26].The number of follicles in each group was counted in one ovary section (the largest cross-section of each mouse ovary) with three biological replicates.The female mice in each group were mated with the 4 male mice to measure fertility (litter birth weight and number of little size).

Culture of GCs and Real-Time Quantitative PCR (qPCR)
Fresh pig ovaries were collected from a slaughterhouse and maintained in phosphatebuffered saline (PBS) containing penicillin and streptomycin during transporting.Porcine GCs were extracted from the follicular fluid of 3-5 mm follicles with a syringe and washed with PBS.Then, the GCs were cultured in DMEM containing 10% fetal bovine serum and finally incubated at 37 • C under 5% CO 2 .GCs were transfected with vectors using Lipofectamine TM 3000 Reagent (Thermo, Waltham, MA, USA).In total, 100 ng/mL GDF9 (MCE, USA) and 50 ng/mL BMP15 (MCE, USA) were used to treat GCs for 24 h.
The purified total RNA was used as a template for cDNA synthesis with the RevertAid First Strand cDNA Synthesis Kit (Thermo, USA).qPCR was conducted using the SYBR Green qRT-PCR Master with the Bio-Rad CFX96 Touch Real-Time PCR system.The procedure steps were as follows: 95 • C for 10 min, 40 cycles at 95 • C for 15 s, annealing at 60 • C for 30 s.The levels of genes were calculated with the 2 −∆∆ct method, and the primer sequences used in pig and mouse are listed in Tables 1 and 2, respectively.The process and details of RNA-seq were reported in our previous study [27].Differential screening was conducted using DESeq2 with |log2FC| > 1 and FDR < 0.05.

EdU Assay
An EdU assay was conducted to detect the proliferation of GCs using the Cell-Light TM Edu Kit (RiboBio, Guangdong, Guangzhou, China).Briefly, GCs were treated with 50 µM EdU at room temperature for 2 h, 80% acetone for 30 min, 0.5% Triton X-100 for 10 min, 1 × Apollo for 30 min, and Hoechst for 30 min.

Flow Cytometry and Caspase 3/7 Activity Assay
Flow cytometry was conducted to detect the apoptosis of GCs using the Annexin V-FITC Apoptosis Detection Kit (BioVision, Milpitas, CA, USA).Briefly, GCs were incubated with Annexin V-FITC and propidium iodide for 15 min.The Caspase 3/7 Activity Assay was also performed to detect the apoptosis of GCs using the Caspase 3/7 Activity Assay Kit (Elabscience, Wuhan, China).A total of 50 µL of GCs lysis in 96-well plates was added along with 45 µL 2xReaction buffer and 5 µL of Ac-DEVD-pNA for 2 h.An absorbance of 405 nm was detected.

ELISA
The mouse E2 (ml063198, Enzyme-linked Biotechnology, Shanghai, China), progesterone (P4) (ml001945), and androgen (ml037278) ELISA kit were used to measure serum E2, P4, and androgen levels, respectively.Porcine E2 (ml002366) was used to measure the level of E2 secreted by porcine GCs.Briefly, 100 µL of HRP was added to the wells with 50 µL of standard and samples and incubated for 60 min at 37 • C and 50 µL substrate A and B for 15 min.An OD value of 450 nm was obtained.

Lentivirus Delivery and TUNEL Assay
Three-week-old female C57BL/6J mice (n = 20) were randomly assigned into four groups: LV-NC, LV-lnc SFFD, sh-NC, and sh-lnc SFFD.Then, 1 × 10 7 TU of synthesized lentiviral vectors (LV-lnc SFFD and sh-lnc SFFD) (Dongze Biotech, Guangzhou, China) was delivered once a week for 3 weeks according to our previous work [19].The TUNEL assay was performed to detect the apoptosis of GCs in mouse ovaries using the TUNEL Apoptosis Assay Kit (Beyotime Biotech, Shanghai, China).Briefly, the ovaries in paraffin sections were treated with xylene, ethanol, protease K, and TUNEL reaction mixture, respectively.

Chromatin Immunoprecipitation (ChIP) and FISH Assay
ChIP was used to detect the H3K27ac level in the SFFD promoter region.According to the Pierce TM ChIP kit (Thermo, Waltham, MA, USA), the chromatin fragments were incubated with H3K27ac antibody (Active Motif, Carlsbad, CA, USA) and IgG antibody (12-370, Millipore, Burlington, MA, USA), and the purified DNA was used for qPCR analyses; the primers are listed in Table 3.The localization of SFFD in GCs was detected using lncRNA FISH probe mix and matching kits (RiboBio, Guangdong, China).The GCs were treated with 4% paraformaldehyde, 0.5% Triton X-100, the SFFD probe, and DAPI.

Dual-Luciferase Reporter Gene Assay
To assess the binding between SFFD and miR-202-3p, the SFFD 3 untranslated region (UTR) segment (lnc SFFD-WT) and the mutation sites of the seed sequence (lnc SFFD-MUT) were established and inserted into the pmirGLO-reporter (Promega, Madison, WI, USA) with Sac1 and Sall endonuclease.Then, lnc SFFD-WT/MUT and mimics NC/miR-202-3p mimic were co-transfected into GCs for 48 h, respectively.The luciferase activity was performed using a luciferase detection kit (Promega, Madison, WI, USA).

Amplification and Coding Ability of SFFD
The full-length amplification of SFFD was performed using the 5 /3 -RACE Kit, 2nd Generation (Roche, Basel, Switzerland).To determine the coding ability of SFFD, the GFP-WT, GFP-MUT, and Open Reading Frame of SFFD with GFP-MUT were inserted into the pcDNA3.1 vector.The primers and oligonucleotide sequences are listed in Tables S1 and S2.

Statistical Analysis
Data are exhibited as mean ± standard deviation (SD).Student's t-test was used to measure differences.p < 0.05 or p < 0.01 were considered statistically significant.

Hdac1 Is Required for Follicular Development and Sexual Maturity in Mice
To explore the role of histone acetylation on follicular development and sexual maturity, an HDAC inhibitor, TSA, was delivered into mice.Compared with DMSO (34.4 ± 1.19 d) and the blank group (34.5 ± 0.53 d), TSA significantly delayed the age of sexual maturity (40.5 ± 1.69 d, p < 0.01, n = 8) (Figure 1A,B).We found that TSA significantly raised the concentration levels of P4 (p < 0.05) and androgen (p < 0.01) but depressed the concentration level of E2 (p < 0.05), suggesting that the follicular development was blocked (Figure 1C).Moreover, the litter birth weight and litter sizes were significantly decreased at the first, second, and third parity by TSA (Figure 1D).Especially, compared to DMSO-treated mice, the ovaries of 45-day-old TSA-treated mice showed increased preantral follicles but bare corpora lutea (Figure 1E).The TUNEL assay further showed that TSA remarkably elevated the apoptosis of GCs (Figure 1F).Immunofluorescence (Figure 1G) and Western blot (Figure 1H) showed that TSA markedly decreased the protein level of Hdac1 (p < 0.01) but displayed an insignificant effect on the protein levels of Hdac2 and Hdac3.

HDAC1 Downregulation Promotes the Expression of CASP3 to Induce the Apoptosis of Porcine GCs
To understand the role of HDAC1 in GCs, the porcine GCs were treated with TSA and HDAC1-siRNA.The results showed that the viability (Figure 2A,B) of GCs was significantly decreased by TSA in a dose-dependent manner at 24 h and 48 h.Although the mRNA level of HDAC1 was significantly inhibited by 0.1, 0.25, and 0.5 µM TSA in GCs (Figure 2C), 0.1 µM TSA for 24 h was selected to reduce the toxic effects on GCs.We found that TSA-mediated HDAC1 downregulation prominently reduced the mRNA levels of PCNA (p < 0.01), CDK1 (p < 0.01), CCNA1 (p < 0.05), and CCND1 (p < 0.01) related to cell proliferation (Figure 2D) and remarkably elevated the mRNA levels of CASP3 (p < 0.01), CASP8 (p < 0.05), and BAX (p < 0.01) (Figure 2E).Moreover, TSA-mediated HDAC1 downregulation markedly elevated the protein level of CASP3 (p < 0.05) (Figure 2F).Subsequently, EdU staining (Figure 2G) showed that TSA-mediated HDAC1 downregulation significantly inhibited the proliferation of GCs.Flow cytometry (Figure 2H) and Caspase 3/7 activity assay (Figure 2I) further showed that TSA-mediated HDAC1 downregulation significantly promoted the apoptosis and Caspase 3/7 activity of GCs.To further characterize the biological roles of HDAC1 in GCs, three HDAC1 small interfering RNA (HDAC1-siRNA1, HDAC1-siRNA2, and HDAC1-siRNA3) were constructed for HDAC1 knockdown.Notably, HDAC1-siRNA2 markedly decreased the mRNA (p < 0.01) and protein (p

Discussion
At present, the epigenetic regulation mechanism of sexual maturity mainly focuses on DNA methylation.Lomniczi et al. found that the inhibition of DNA methylation resulted in rats failing to ovulate and a delay in sexual maturity [14].However, the mechanism by which histone acetylation regulates sexual maturity remains to be further clarified.To explore the role of histone acetylation on follicular development and sexual maturity in mice, TSA, an effective histone deacetylating inhibitor, was delivered into mice.In the present study, we found that TSA remarkably delayed the age of sexual maturity (Figure 1B) and fertility (Figure 1D) in mice.Consistent with our study, gilts that reach sexual maturity at a younger age exhibit higher fertility in their lifetime than gilts attaining sexual maturity at an older age [28].Furthermore, the ovaries of TSA-treated mice showed increased preantral follicles but fewer corpora lutea, indicating that these TSA-treated mice did not have mature follicles and had not ovulated, and they failed to reach sexual maturity (Figure 1E).The apoptosis of follicular GCs in TSA-treated mice was remarkably higher than DMSO-treated mice (Figure 1F).In addition, TSA significantly decreased the protein level of Hdac1 in mouse ovary, but Hdac2 and Hdac3 did not show significant changes (Figure 1G,H).To further verify the effect of histone acetylation on the function of GCs, we treated porcine GCs with TSA and HDAC1-siRNA, respectively.Both TSA-mediated HDAC1 knockdown and HDAC1-siRNA prominently arrested the proliferation of GCs (Figure 2G,N) and enhanced the apoptosis of GCs (Figure 2H,O).Consistent with previous studies, the HDAC inhibitor induces apoptosis and cell death [29], and the knockdown of HDAC1 reduces proliferation and enhances apoptosis in various cell types [30].It is concluded that HDAC1 is involved in sexual maturity by mediating the apoptosis of GCs.
LncRNAs are generally considered to be transcripts that have over 200 nucleotides with no protein coding ability [31].It has been demonstrated that lncRNAs sponge miR-NAs, a category of transcripts termed competing endogenous RNAs, through base-pairing interactions [32] and mediate the growth and function of GCs to participate in follicular development [33,34] and sexual maturity [35,36].For example, lncRNA FDNCR enhances the apoptosis of GCs through the miR-543-3p/DCN/TGF-β axis in sheep [37], and lncRNA Gm2044 enhances E2 synthesis through targeting miR-138-5p in mouse pre-antral GCs [38].The downregulation of lncRNA ZNF674-AS1 markedly reduces the proliferation of GCs and follicular growth [39].In the present study, the knockdown of HDAC1 remarkably decreased the expression of SFFD (Figure 3A,B).Epigenetic modifications have been reported to regulate the expression of lncRNA by affecting promoter utilization [40,41].Previous studies have indicated that HDAC1 epigenetically regulates the H3K27ac level at the gene's promoter [42,43].To explore the mechanism by which HDAC1 inhibited the expression of SFFD, we analyzed the histone acetylation of the SFFD promoter and found that HDAC1-siRNA significantly decreased the H3K27ac level in the −953/−661 region at the SFFD promoter (Figure 3I).It has been shown that HDAC1 exhibits deacetylase activity to inhibit transcription [44].H3K27ac, a marker of transcriptional activation, is reported to be regulated by HDAC1 [45] and participates in follicular maturation and ovulation [46].Consistent with our results, in BrCa cells, HDAC1 knockdown inhibits the expressions of super-enhancers associated with oncogenes through decreasing the H3K27ac level [45].Therefore, we speculate that the mechanism by which HDAC1 knockdown leads to reduced levels of H3K27ac in the SFFD promoter may be the presence of a super-enhancer in the SFFD promoter region.But the mechanism of this needs to be further explored.The level of H3K27ac is significantly induced during follicular maturation in mice [46].LncRNA ROCKI reduces the expression of MARCKS through facilitating the HDAC1-mediated removal of H3K27ac [42].These observations suggest that H3K27ac targets the −953/−661 region to epigenetically regulate the transcription of SFFD.
In the present study, SFFD was found to promote the proliferation (Figure 4I,J) and E2 secretion (Figure 4K) of GCs by regulating the expression of CCND1 and HSD17B1, respectively, and regulates the expression of CASP3 to inhibit the apoptosis of GCs (Figure 4L,N).The molecular function of lncRNA mainly depends on its subcellular localization [47].To clarify the downstream mechanism of SFFD, the localization of SFFD in GCs was explored.The results showed that SFFD existed in both the cytoplasm and nucleus, but it was more abundant in the cytoplasm (Figure 3E,F).Therefore, we hypothesized that SFFD may exert its cellular functions by acting as a sponge for miRNA.Through bioinformatics tools, miR-202-3p was predicted to be one of the potential targets of SFFD, and the target relationship between them was further verified using RNA pull-down (Figure 3J) and dual luciferase analysis (Figure 3K).MiR-202-3p, as a member of the let-7 family, is highly conserved across animal species and has been reported to increase cell apoptosis while decreasing cell proliferation [48,49].MiR-202-3p enhances apoptosis and arrests proliferation via binding CCND1 in human Sertoli cells [48].An elevated level of miR-202-3p has been observed in atretic follicles compared to chicken yellowish follicles, indicating its potential involvement in follicular atresia [50].In mouse spermatogonial stem cells, miR-202-3p has been shown to arrest the cell cycle [51].Consistent with previous studies, we revealed that miR-202-3p reduced the proliferation (Figure 5E) and E2 secretion (Figure 5F) and induced the apoptosis of GCs (Figure 5G).Furthermore, miR-202-3p partially reversed the cellular function mediated by SFFD.MiRNAs, as a vital epigenetic regulator, are evolutionarily conserved and exert an inhibitory effect on the expression of mRNA via a base-pairing interaction with the 3 UTRs of target mRNAs [52,53].In the present study, the interaction between COX1 and miR-202-3p was verified through dual luciferase analysis (Figure 3P).COX1, one of the rate-limiting enzymes of prostaglandin synthesis, exhibits a vital role in cellular proliferation [54], apoptosis [55], and follicular angiogenesis [56] and development [57].In this study, we found that COX1-siRNA partially attenuated the pro-proliferation and anti-apoptotic effects of the miR-202-3p inhibitor (Figure 5E,G).GDF9 and BMP15, the oocyte-secreting factors, had been identified to be indispensable for normal follicular development and sexual maturity [46].In our study, GDF9 + BMP15 was found to markedly promote the mRNA levels of HDAC1 and SFFD in porcine GCs (Figure 6B,C), which also provides further evidence that HDAC1-mediated SFFD is involved in follicular development and sexual maturation.To characterize the in vivo function of SFFD, SFFD overexpression and knockdown were simulated by the direct injection of lentiviral in mouse ovary.We found that SFFD knockdown notably blocked the follicular maturation (Figure 6I), significantly inhibited E2 secretion (Figure 6H), and markedly delayed the age of sexual maturity in mice (Figure 6G).These findings provide valuable insights into the therapeutic potential of SFFD in ovarian follicles and sexual maturation.

Conclusions
In conclusion, our findings demonstrate that HDAC1 knockdown markedly reduces the H3K27ac level of the −953/−661 region to epigenetically inhibit the transcription of SFFD.SFFD functions by targeting miR-202-3p to elevate the expression of COX1, enhances proliferation and E2 secretion, and inhibits the apoptosis of GCs to regulate follicular development and sexual maturity (Figure 7).Collectively, our results suggest that SFFD mediated by HDAC1 may serve as a potential molecular therapeutic approach for sexual maturity.

Conclusions
In conclusion, our findings demonstrate that HDAC1 knockdown markedly reduces the H3K27ac level of the -953/-661 region to epigenetically inhibit the transcription of SFFD.SFFD functions by targeting miR-202-3p to elevate the expression of COX1, enhances proliferation and E2 secretion, and inhibits the apoptosis of GCs to regulate follicular development and sexual maturity (Figure 7).Collectively, our results suggest that SFFD mediated by HDAC1 may serve as a potential molecular therapeutic approach for sexual maturity.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells12232734/s1,Table S1: Primers used for vector construction; Table S2: Oligonucleotide sequences in this study; The uncropped western blot images.

20 Figure 1 .
Figure 1.Downregulation of Hdac1 blocks follicular development and sexual maturity in mice.(A) The percentage of mouse estrus was assessed in the blank, DMSO, and TSA group (n = 8).(B) The age of mouse puberty was counted in the blank, DMSO, and TSA group (n = 8).(C) Effects of TSA on the concentrations of E2, P4, and androgen in the serum of mice.(D) Effects of TSA on the li er birth weight and number of li er size in the 1st to 3rd parities of mice.(E-H) Effects of TSA on the follicular development, apoptosis of follicular GCs, and levels of Hdac1, Hdac2, and Hdac3 in mice.Black arrows represent preantral follicles, red arrows represent antral follicles, and CL represents corpus luteum.* represents p < 0.05, ** represents p < 0.01.

Figure 1 .
Figure 1.Downregulation of Hdac1 blocks follicular development and sexual maturity in mice.(A) The percentage of mouse estrus was assessed in the blank, DMSO, and TSA group (n = 8).(B) The age of mouse puberty was counted in the blank, DMSO, and TSA group (n = 8).(C) Effects of TSA on the concentrations of E2, P4, and androgen in the serum of mice.(D) Effects of TSA on the litter birth weight and number of litter size in the 1st to 3rd parities of mice.(E-H) Effects of TSA on the follicular development, apoptosis of follicular GCs, and levels of Hdac1, Hdac2, and Hdac3 in mice.Black arrows represent preantral follicles, red arrows represent antral follicles, and CL represents corpus luteum.* represents p < 0.05, ** represents p < 0.01.

Figure 2 .
Figure 2. HDAC1 regulates the proliferation and apoptosis of GCs.(A) Effects TSA treatment at concentration of 0.01, 0.1, 0.25, 0.5, and 1 µM for 24 and 48 h on the viability of porcine GCs.(B) Micrographs of GCs treated with 0.1 and 0.25 µM TSA at 24 and 48 h.(C) Effects of 0.01, 0.1, 0.25, 0.5, and 1 µM TSA treatment on the expression of HDAC1 for 24 and 48 h.(D-F) The mRNA and protein levels of cell cycle and cell apoptosis-related genes in GCs treated with TSA.(G,H) The

Figure 2 . 20 Figure 3 .Figure 3 .
Figure 2. HDAC1 regulates the proliferation and apoptosis of GCs.(A) Effects TSA treatment at concentration of 0.01, 0.1, 0.25, 0.5, and 1 µM for 24 and 48 h on the viability of porcine GCs.(B) Micrographs of GCs treated with 0.1 and 0.25 µM TSA at 24 and 48 h.(C) Effects of 0.01, 0.1, 0.25, 0.5, and 1 µM TSA treatment on the expression of HDAC1 for 24 and 48 h.(D-F) The mRNA and protein levels of cell cycle and cell apoptosis-related genes in GCs treated with TSA.

Figure 4 .
Figure 4. SFFD as ceRNA for miR-202-3p-COX1 regulates the proliferation and apoptosis of porcine GCs.(A-D) Effects of SFFD overexpression and miR-202-3p mimic on the mRNA and protein levels of genes related to cell cycle, E2 secretion, and apoptosis.(E-H) Effects of SFFD knockdown and miR-202-3p inhibitor on the mRNA and protein levels of genes related to cell cycle, E2 secretion, and apoptosis.(I) Effects of SFFD overexpression and miR-202-3p mimic on the proliferation of GCs.(J) Effects of SFFD knockdown and miR-202-3p inhibitor on the proliferation of GCs.(K) Effects of SFFD overexpression with miR-202-3p mimic and SFFD knockdown with miR-202-3p inhibitor on the E2 secretion of GCs.(L,M) Effects of SFFD overexpression and miR-202-3p mimic on the apoptosis and Caspase 3/7 activity of GCs.(N,O) Effects of SFFD knockdown and miR-202-3p inhibitor on the apoptosis and Caspase 3/7 activity of GCs.* indicates p < 0.05, ** indicates p < 0.01.

Figure 4 .
Figure 4. SFFD as ceRNA for miR-202-3p-COX1 regulates the proliferation and apoptosis of porcine GCs.(A-D) Effects of SFFD overexpression and miR-202-3p mimic on the mRNA and protein levels of genes related to cell cycle, E2 secretion, and apoptosis.(E-H) Effects of SFFD knockdown and miR-202-3p inhibitor on the mRNA and protein levels of genes related to cell cycle, E2 secretion, and apoptosis.(I) Effects of SFFD overexpression and miR-202-3p mimic on the proliferation of GCs.(J) Effects of SFFD knockdown and miR-202-3p inhibitor on the proliferation of GCs.(K) Effects of SFFD overexpression with miR-202-3p mimic and SFFD knockdown with miR-202-3p inhibitor on the E2 secretion of GCs.(L,M) Effects of SFFD overexpression and miR-202-3p mimic on the apoptosis and Caspase 3/7 activity of GCs.(N,O) Effects of SFFD knockdown and miR-202-3p inhibitor on the apoptosis and Caspase 3/7 activity of GCs.* indicates p < 0.05, ** indicates p < 0.01.

Figure 7 .
Figure 7.The mechanistic scheme of HDAC1-mediated H3K27ac level of SFFD to impede follicular development and sexual maturity.

Figure 7 .
Figure 7.The mechanistic scheme of HDAC1-mediated H3K27ac level of SFFD to impede follicular development and sexual maturity.

Author Contributions:
Data curation, X.Z., Y.H. and H.Q.; formal analysis, X.P.; funding acquisition, X.Z., X.Y. and J.L.; investigation, Y.Z.; methodology, X.Z., Y.H. and H.Q.; writing-original draft, X.Z.; writing-review and editing, Z.Z., X.Y. and J.L.All authors have read and agreed to the published version of the manuscript.Funding: This work was supported by the National Natural Science Foundation of China (31902131 and 32072694), the earmarked fund for China Agriculture Research System (CARS-35), the Guangdong Basic and Applied Basic Research Foundation (2023A1515030054, 2023A1515010364 and 2022A1515012490), the Key R&D Program of Guangdong Province Project (2022B0202090002), and the Breed Industry Innovation Park of Guangdong Xiaoerhua Pig (2022-4408X1-43010402-0019).Institutional Review Board Statement: All experiments in the present study were conducted according to Regulations for the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, China, revised in March 2017) and approved by the Animal Care and Use Committee of South China Agricultural University with approval number: SYXK 2019-0136.Informed Consent Statement: Not applicable.

Table 1 .
Primers used for qPCR in pig.

Table 2 .
Primers used for qPCR in mouse.

Table 3 .
Primers used for ChIP-PCR.