Knockdown of KDM5B Leads to DNA Damage and Cell Cycle Arrest in Granulosa Cells via MTF1

KDM5B is essential for early embryo development, which is under the control of maternal factors in oocytes. Granulosa cells (GCs) play a critical role during oocyte mature. However, the role of KDM5B in GCs remains to be elucidated. In the current study, we found that KDM5B expressed highly in the ovaries and located in goat GCs. Using an RNA sequence, we identified 1353 differentially expressed genes in the KDM5B knockdown GCs, which were mainly enriched in cell cycle, cell division, DNA replication and the cellular oxidative phosphorylation regulation pathway. Moreover, we reported a decrease in the percentage of proliferated cells but an increase in the percentage of apoptotic cells in the KDM5B knockdown GCs. In addition, in the KDM5B knockdown GCs, the percentage of GCs blocked at the S phase was increased compared to the NC group, suggesting a critical role of KDM5B in the cell cycle. Moreover, in the KDM5B knockdown GCs, the reactive oxygen species level, the mitochondrial depolarization ratio, and the expression of intracellular phosphorylated histone H2AX (γH2AX) increased, suggesting that knockdown of KDM5B leads to DNA damage, primarily in the form of DNA double-strand breaks (DSBs). Interestingly, we found a down-regulation of MTF1 in the KDM5B knockdown GCs, and the level of cell proliferation, as well as the cell cycle block in the S phase, was improved. In contrast, in the group with both KDM5B knockdown and MTF1 overexpression, the level of ROS, the expression of γH2AX and the number of DNA DSB sites decreased. Taken together, our results suggest that KDM5B inhibits DNA damage and promotes the cell cycle in GCs, which might occur through the up-regulation of MTF1.


Introduction
Oocyte maturation is closely related to early embryonic development. Failure of oocyte maturation will seriously affect both female reproductive health and fertility [1]. Increasing studies have shown that abnormal oocyte maturation leads to fertilization failure, embryo development retardation, and embryo death before implantation [2]. Oocyte maturation is achieved with follicular growth and development, during which ovarian granulosa cell (GC) proliferation and apoptosis are required [3,4]. A previous study has revealed that apoptosis of GCs is linked to decreased ovarian reserve in female animals [5]. For example, in polycystic ovary syndrome patients' granulosa cells, the level of apoptosis and the expression of BAX, CAS3, CAS9 and other vital genes regulating apoptosis increased significantly [6]. Therefore, it is critical to maintain GC proliferation and apoptosis during follicular development.
During proliferation, DNA replication occurs at the S phase of interphase [7]. Every stage of the cell cycle is very important, and the normal entry and development of the S phase is crucial for cell development, tissue repair and immune function [8,9]. After a cell enters mitosis, it is prone to different types of damage, such as DNA double-strand breaks. When DNA DSBs occur during mitosis, the cell will continue to divide without DNA damage repair, so the wrong chromatids are expressed, preventing normal cell division and proliferation [10]. However, overactive or abnormal entry of the S phase can lead to replication stress, DNA damage and canceration [11]. Other studies have shown that the entry of the S phase in the daughter cell G1 phase is independent of mitogen, but this phase is very prone to DNA damage, such as DNA single-strand breaks and DNA double-strand breaks [12]. It was proven that continuous DNA DSBs induced by  inhibited Caenorhabditis elegans meiosis [13,14]. In granulosa cells, increased DNA DSBs and reduced DNA repair might contribute to ovarian aging [15]. Jose Antonio et al. found that DNA damage caused by environmental influences led the cell cycle to stop. In this way, the damaged DNA was prevented from replicating further [16]. These studies suggest that the occurrence of DNA damage may lead to the obstruction of cell cycle progression, thus leading to abnormal cell division.
It has been reported that reactive oxygen species (ROS) causes adverse changes, such as proliferation arrest, aging, apoptosis and necrosis [17]. Recently, there were research studies pointing out that chloroquine increased ROS in ovarian cancer cell lines, causing DSBs [18,19]. The primary sources of ROS were mitochondria and plasma membrane [20]. ROS oxidizes macromolecules in cells and destroys their normal function, causing cell damage or even cell death [21]. Severe oxidative stress with a higher level of ROS in the ovarian granulosa cells of patients with polycystic ovary syndrome was reported [22]. On the other hand, decreased intracellular ROS levels reduced pancreatic β-cells apoptosis in alloxan-induced larvae [23]. These and subsequent studies suggest that ROS needs to be maintained at a normal level in cells that typically divide and grow [24].
It has been pointed out that epigenetic factors play pivotal roles in DNA DSB repair. For example, histone H3K36me3 methyltransferase SETD2 could promote DNA DSB repair, thus maintaining genome stability [20]. KDM5B (also known as JARID1B/PLU1), a H3K4me2/3 histone demethylase, is also indispensable for DNA damage responses. Previous studies have demonstrated that attenuation of KDM5B alters H3K4 methylation patterns, affects DNA damage responses, increases DNA DSBs and reduces DNA damage repair ability [25]. Studies have shown that KDM5B leads to ROS overproduction by regulating SIRT3, thereby leading to oxidative stress and mitochondrial metabolism disorder [26]. Moreover, KDM5B is thought to modulate the expression of tumor-suppressing genes and oncogenes, thus affecting cancer cell proliferation [27,28]. Further research has revealed that the knockdown of KDM5B significantly inhibits the proliferation of hepatocellular carcinoma cells both in vivo and in vitro by arresting the cell cycle at the G1/S phase, partly by up-regulating P15 and P27 [29,30]. However, the function of KDM5B in GCs remained to be elucidated.
MTF1 is a metal transcriptional regulator, which encodes a transcription factor that induces the expression of metallothionein and other genes involved in metal homeostasis in response to heavy metals, such as cadmium, zinc, copper and silver [31]. It has been reported that the accumulation of zinc and copper ions induced by MTF1 leads to cell apoptosis [32]. However, to our best knowledge, there is no research pointing out the effects of KDM5B and MTF1 in goat GCs. Additionally, no research has focused on the regulatory relationship between KDM5B and MTF1.
Using small interfering RNA, RNA sequencing and rescue experiments, we investigated the expression pattern and function of KDM5B in goat granulosa cells in the present study. We found that knockdown of KDM5B caused DNA damage and S phase arrest in GCs. In contrast, overexpression of MTF1 reduced DNA DSBs and S phase arrest, suggesting that knockdown of KDM5B caused DNA damage and S phase arrest in GCs by repressing MTF1.

Sample Collection
All animal experiments were performed according to the Measures for the Administration of Laboratory Animals of Nanjing Agricultural University and approved by the Animal Care and Use Committee of Nanjing Agricultural University. Goat heart, liver, spleen, lung and kidney were cut into small pieces and stored in liquid nitrogen. Parts of the ovaries were immediately frozen at −196 • C for subsequent experiments. In contrast, the remaining parts of the ovaries were fixed in 4% formaldehyde for at least 24 h and embedded in paraffin for immunofluorescence staining.

Transfection of siRNA against KDM5B
GCs were thawed and cultured in T25 (TC Flask T25, 5 × 10 6 cells/flask) before being passed into 6-well plates (2 × 10 6 cells/well). GCs were only transfected once after thawing to ensure greater cell viability, and no other passages were permitted. At 60-70% confluence, GCs were transfected with siRNA against KDM5B (Genepharma, Shanghai, China), which was premixed with lipofectamine 3000 transfection reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. A well of cells was transfected with the universal negative control siRNA to exclude the non-specific influence of the siRNA treatment (NC). The sequences of siRNAs are shown in Table 1. The transfected cells were harvested for RNA extraction after 48 h of transfection and for protein extraction after 72 h of transfection.

Quantitative Real-Time PCR
Trizol reagent (Invitrogen, CA, USA) was used to extract total RNAs from the goat tissues and GCs. Reverse transcription was performed using a Strand cDNA Synthesis Kit with a gDNA wiper (Vazyme Biotech Co., Ltd., Nanjing, China). Following the manufacturer's instructions, all the qPCR reactions were performed in a QuantStudio 5 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using Chamq Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China). The reaction was performed as follows: annealing at 95 • C for 30 s, followed by 40 cycles at 95 • C for 10 s and at 60 • C for 30 s, and, finally, a dissociation step consisting of 95 • C for 15 s, 60 • C for 60 s, and 95 • C for 15 s. The 2 −∆∆CT method was used to quantify relative mRNA expression, and β-actin (ACTB) was used as an internal control. All experiments were repeated at least three times independently. The primer sequences are listed in Table 2.

Immunofluorescence Staining
Immunofluorescence was performed as described in a previous study [33]. Briefly, the transfected GCs were passaged into a 24-well plate on round coverslips, and they were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT). Then, the cells were permeabilized using 0.5% Triton X-100 in DPBS for 20 min at RT and blocked in 5% BSA in DPBS for one hour at RT, followed by incubation with anti-KDM5B/PLU1/JaridlB antibody (1:500, Abcam, Waltham, MA, USA) at 4 • C overnight. After washing in DPBS, the cells were incubated for one hour at RT with Alexa fluor goat anti-rabbit IgG (Boster, 1:1000) in the dark. Then, the cells were washed with DPBS three times. The nuclei were stained with Hoechst 33342 for 5 min at RT. Before the coverslips were placed on a glass slide, an antifade mounting medium was dripped on the glass slide. Then, a laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) was used to observe the cells with the same condition.

Cell Proliferation Analysis
Cell proliferation was assessed using the Alexa Fluor 647-Click-iT EdU Assay Kit (KGA332-50, Beyotime Biotechnology, Shanghai, China). The transfected goat GCs were cultured in a 24-well plate on round coverslips and incubated with 100 mM EdU for 2 h. Subsequently, the cells were fixed with 4% PFA for 20 min at RT and washed with 2 mg/mL of glycine for 5 min. The cells were then permeabilized in DPBS containing 0.5% Triton X-100 for 20 min. After washing with DPBS, the nuclei were stained with Hoechst 33342 for 5 min at RT. Before the coverslips were placed on a glass slide, an antifade mounting medium was dripped on the glass slide. Finally, a laser scanning confocal microscope was used to observe the cells with the same condition.

Flow Cytometry Analysis
The cell culture fluid of the transfected GCs was collected, and the cells in the 6-well plate were digested with trypsin for 3 min. Then, the culture fluid was added to the solution to terminate the digestion. The cell suspensions were obtained twice, mixed and centrifuged at 1200-1500 r/min. After washing with DPBS, the cell suspension was centrifuged again. Finally, the single-cell suspension was mixed with DPBS and then sent to Nanjing Liangwei Biotechnology Co., Ltd. (Nanjing, China) immediately for flow cytometry analysis to detect cell apoptosis. We calculated the proportion of cells with early and later apoptosis.
The cell culture fluid of the transfected GCs was discarded, and then the cells were digested, washed and centrifuged to make a single-cell suspension with 70% alcohol and sent to Wuhan Service Co., Ltd. (Wuhan, China) for flow cytometry analysis to detect the cell cycle stage.

Western Blot Analysis
Western blot was performed as described in a previous study [33]. The goat tissues and the transfected GCs were harvested and lysed using RIPA buffer with a protease inhibitor (Thermo Pierce, Waltham, MA, USA). After 20 min of incubation on ice, the ultrasonic cell disruptor was used to disrupt the cells in order to lyse them completely. Next, protein concentrations were measured using a BCA protein assay kit (Beyotime, China). Protein was denatured using Nupage sample reduction agent (10×) and Nupage LDS sample buffer (5×) from Thermo Pierce in the United States. The supernatant was combined with the abovementioned reagents and boiled for 10 min at 70 • C. Then, 20 µg of protein was loaded onto a 12% SDS-polyacrylamide gel and transferred onto polyvinylidene fluoride (PVDF) membranes from each group (Millipore; Billerica, MA, USA). After blocking the membrane with 5% BSA for 2 h at room temperature, it was incubated overnight at 4 • C with primary antibodies (diluted according to Table 3). As the internal controls, β-actin (ACTB) was used. The membranes were washed with TBST for a minimum of one hour, followed by one-hour incubation with secondary antibodies (diluted per Table 3). Immunoblotting was visualized with enhanced ECL ultra-sensitive luminescence fluid (Thermo Pierce, USA) and exposed with Image Quant LAS 400. (Fiji film, Tokyo, Japan). The Image J 1.x (National Institutes of Health, Bethesda, MD, USA) software was used to analyze the changes in protein levels.

Intracellular Reactive Oxygen Species Detection
The ROS Assay Kit (S0033S, Beyotime Biotechnology) was used to assess ROS in the GCs. The cell culture medium was removed, and 1 mL of DCFH-DA diluted 1:1000 in a serum-free medium was added. Then, the cells were incubated for 20 min in a 37 • C incubator. Serum-free DMEM/F12 glutaMAX was used for washing the cells three times. Lastly, a laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) was used to observe the cells. ROS was detected using an excitation light of 488 nm and an emission light of 525 nm.

Mitochondrial Membrane Potential Detection
JC-1 (KGMP021, KeyGEN Biotechnology, Nanjing, China) was used to detect mitochondrial membrane potential in the GCs. The JC-1 working solution was prepared according to the instructions of KGMP021 and added to a 6-well plate. Then, the cells were incubated for 20 min in a 37 • C incubator. Lastly, a laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) was used to observe the cells. JC-1 monomers were detected using an excitation light of 460 nm and an emission light of 530 nm, and JC-1 multimers were detected using an excitation light of 525 nm and an emission light of 500 nm.

RNA Sequence Data Analysis
RNA libraries were constructed using the Smart-seq2 method. Then, cDNA was fragmented by dsDNA fragments (M0348S, NEB) by incubating at 37 • C for 30 min, and size selection was performed with the provided sample purification beads. The fragmented cDNA at 150-300 bp was used for library construction. This was followed by paired-end sequencing on an illumine Novaseq 6000 platform (LC bio, Hangzhou, China). Quality control was performed to remove adaptors and low-quality bases using fastp (v0.19.6). All reads that passed quality control were mapped to the goat genome ARS1 using HISAT2 (v2.2.1) with the default settings. Uniquely mapped reads were subsequently assembled into transcripts guided by the reference annotation using featureCounts (v2.0.1). Differential expression analysis was performed using DESeq2 (v3.11). Genes with log2 (fold change) >1 or <−1 and with p-value < 0.05 were deemed as differentially expressed genes (DEGs).

GO, KEGG and GSEA Analysis
The DEGs were annotated using the Gene Ontology (GO) database (http://www. geneontology.org/, accessed on 20 May 2022), and hypergeometric tests examined these genes' biological functions and pathways. p-values were calculated, and GO terms were considered significantly enriched when p < 0.01. The enriched pathways were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.kegg. jp, accessed on 20 May 2022), and pathways with p < 0.01 were considered significantly enriched pathways. The R (v4.2, New Zealand) programming language, including the Biocoductor software packages (v3.17, New Zealand), was mainly used for statistical analysis and data visualization. Heatmap and boxplot of coding potential score, volcano, gene expression, GO, and signal intensity were generated using R package pheatmap (v1.0.12, New Zealand) and ggplot2 (v3.3.2, New Zealand), respectively. The Gene Set Enrichment Analysis (GSEA) of all DEGs was performed using the cluster Profiler (v4.0, New Zealand) and enrichplot R package (v1.10.2, New Zealand). For detailed analysis, please see Deng et al.'s article [33].

Statistical Analysis
All experiments were repeated at least three times, and the data are presented as means ± standard error of the mean based on three independent experiments. Statistical analyses were performed with GraphPad Prism 8 (GraphPad, San Diego, CA, USA) using the independent sample student's t-test. p-values < 0.05 were considered as indicating statistically significant differences. A p-value < 0.01 indicates a more significant difference than a p-value < 0.05.

High Expression of KDM5B in Goat GCs
To investigate the expression profile of KDM5B, we performed qPCR. As shown in Figure 1A, KDM5B is highly expressed in the ovary compared to the heart, liver, spleen, lung, and kidney tissues in goats. Furthermore, the immunofluorescence staining reveals that KDM5B is localized in GCs ( Figure 1B), suggesting that KDM5B might play an important role during follicular development. We therefore isolated GCs from goat ovarian follicle and found a high expression of FSHR, one of the markers of GCs, suggesting that GCs were successfully established ( Figure 1C).

Knockdown of KDM5B in Goat GCs
We next transfected siRNAs against KDM5B into the goat GCs to knockdown the expression of KDM5B. As expected, the mRNA expression of KDM5B decreases significantly in the KDM5B knockdown GCs compared to the controls (p < 0.01; Figure 1D). Furthermore, the protein level of KDM5B also significantly decreases as revealed by Western blot and immunofluorescence staining (p < 0.01; Figure 1E,F). As KDM5B demethylates H3K4me2/3, we explored changes in the H3K4me3 methylation levels. As shown in Figure 1G,H, H3K4me3 methylation levels increase significantly in the KDM5B knockdown GCs compared to the NC group, as revealed by Western blot and immunofluorescence staining. These data suggest that the expression of KDM5B has been successfully knocked down.

Compromise of Cell Cycle and DNA Replication-Related Functions in KDM5B Knockdown GCs
To determine the gene expression profiles in the KDM5B knockdown GCs, we performed RNA sequencing. Using DESeq2, we identified 1353 differentially expressed genes (DEGs). Of which, 998 genes are down-regulated, while 355 genes are up-regulated, in the KDM5B knockdown GCs compared to the NC group (Figure 2A,B). It is important to know the pathway that genes are de-repressed and down-regulated in knockdown experiments. As shown in Figure 2C, the down-regulated DEGs are mainly enriched in cell cycle, cell division, DNA replication and chromosome segregation. The KEGG analysis reveals that the down-regulated genes are enriched in cell cycle regulation, oocyte meiosis, DNA replication, and the cellular oxidative phosphorylation regulation pathway ( Figure 2D). These data suggest that KDM5B might play an important role in cell cycle processing, DNA replication and oxidative phosphorylation.

Blocked S Phase and Restrained Proliferation in KDM5B Knockdown GCs
The GSEA revealed the DEGs were enriched in cell cycle (p < 0.01; Figure 3A). We therefore examined cell cycle distribution after the knockdown of KDM5B to investigate the effect of KDM5B knockdown in cell cycle processing. In the KDM5B knockdown GCs, the percentage of cells in the G1 phase significantly decreases (p < 0.01), while the percentage of cells in the S phase significantly increases (p < 0.05) when compared to the NC group ( Figure 3B). Meanwhile, the mRNA expressions of cell cycle-related genes, such as Ccnd1 (p < 0.01), Ccnd3 (p < 0.01) and CDK4 (p < 0.01), are significantly increased in the KDM5B knockdown GCs compared to the NC group ( Figure 3C). Moreover, the protein levels of CDK6 (p < 0.01), Ccnd1 (p < 0.01) and Ccnd3 (p < 0.01) are all significantly increased ( Figure 3D) in the KDM5B knockdown GCs. These results and our RNA-seq data indicate that the knockdown of KDM5B leads to S phase block in GCs.
Then, we explored the proliferation of GCs when KDM5B was knocked down. As shown in Figure 3E, the percentage of proliferated cells significantly decreases (p < 0.01) in the KDM5B knockdown GCs compared to the NC group, suggesting the knockdown of KDM5B inhibits the proliferation of GCs in goat. To further confirm the result, we investigated the expression of marker genes of proliferation. As shown in Figure 3F, the expression of PCNA significantly decreases in the KDM5B knockdown GCs compared to the controls (p < 0.01). Consistently, the protein expression of PCNA also significantly decreases when KDM5B has been knocked down (p < 0.01; Figure 3F). Our data suggesting that the knockdown of KDM5B blocks the cell cycle to S phase and restrains proliferation in goat GCs. know the pathway that genes are de-repressed and down-regulated in knockdown exper-iments. As shown in Figure 2C, the down-regulated DEGs are mainly enriched in cell cycle, cell division, DNA replication and chromosome segregation. The KEGG analysis reveals that the down-regulated genes are enriched in cell cycle regulation, oocyte meiosis, DNA replication, and the cellular oxidative phosphorylation regulation pathway ( Figure  2D). These data suggest that KDM5B might play an important role in cell cycle processing, DNA replication and oxidative phosphorylation.

DNA DSBs in KDM5B Knockdown GCs
The GSEA reveals the DEGs are enriched in oxidative phosphorylation (p < 0.01; Figure 4A). Moreover, genes that regulate oxidative stress, such as CAT, SOD1, GPX2 and GPX4, are elevated significantly after the knockdown of KDM5B ( Figure 4B). Consistently, the protein expression of CAT increases significantly (p < 0.01; Figure 4C). Therefore, we examined changes in ROS after the knockdown of KDM5B. As shown in Figure 4D, ROS fluorescence intensity is significantly increased in the KDM5B knockdown GCs (p < 0.01), indicating that the intracellular ROS level has distinctly increased. Meanwhile, the ratio of red/green is also significantly decreased (p < 0.01; Figure 4E), and the mRNA expressions of DNM1L (p < 0.01) and MFN2 (p < 0.05), which are related to mitochondrial function, both decrease significantly ( Figure 4F). γH2AX refers to phosphorylated H2AX, which is a specific indicator for the detection of DNA damage. A large amount of histone H2AX phosphorylates at the DNA DSB sites and forms a fluorescent-resolved focal point [34]. With the knockdown of KDM5B, the number of DNA damage sites and the expression of γH2AX increase significantly compared to the NC group (p < 0.01; Figure 4G,H). Meanwhile, the mRNA expressions of RAD511, RPA1, RPA2 and TP53BP1 significantly decrease after the knockdown of KDM5B (p < 0.01; Figure 4I). Our data suggest that KDM5B knockdown results in DNA damage by increasing ROS, the proportion of mitochondrial depolarization, and DNA DSBs in GCs.
ulated. (B) Heat map displays that the repeatability within the group is good and the difference between the groups is large. (C) Significantly enriched GO terms. (D) Significantly enriched KEGG terms.

Blocked S Phase and Restrained Proliferation in KDM5B Knockdown GCs
The GSEA revealed the DEGs were enriched in cell cycle (p < 0.01; Figure 3A). We therefore examined cell cycle distribution after the knockdown of KDM5B to investigate the effect of KDM5B knockdown in cell cycle processing. In the KDM5B knockdown GCs, the percentage of cells in the G1 phase significantly decreases (p < 0.01), while the percentage of cells in the S phase significantly increases (p < 0.05) when compared to the NC group ( Figure 3B). Meanwhile, the mRNA expressions of cell cycle-related genes, such as Ccnd1 (p < 0.01), Ccnd3 (p < 0.01) and CDK4 (p < 0.01), are significantly increased in the KDM5B knockdown GCs compared to the NC group ( Figure 3C). Moreover, the protein levels of CDK6 (p < 0.01), Ccnd1 (p < 0.01) and Ccnd3 (p < 0.01) are all significantly increased ( Figure  3D) in the KDM5B knockdown GCs. These results and our RNA-seq data indicate that the knockdown of KDM5B leads to S phase block in GCs.

Promotion of Apoptosis in KDM5B Knockdown GCs
Elevated levels of ROS lead to cell apoptosis. As shown in Figure 5A, the percentage of apoptotic cells significantly increases in the KDM5B knockdown group compared to the NC group (p < 0.01; Figure 5A). Moreover, the mRNA expressions of the pro-apoptotic factors, BAX, CAS3 and CAS9, increase significantly, whereas the expression of the anti-apoptotic factor BCL2 decreases, in the KDM5B knockdown GCs (p < 0.01; Figure 5B). Additionally, the ratio of BAX/BCL2 significantly increases (p < 0.01; Figure 5B). Meanwhile, the protein expressions of BAX, CAS3 and CAS9 increase significantly, whereas the protein expression of BCL2 decreases in the KDM5B knockdown GCs (p < 0.01; Figure 5C). The result of TUNEL also intuitively indicates that apoptotic cells are significantly increased in the KDM5B knockdown GCs ( Figure 5D). These data suggest that the knockdown of KDM5B accelerates apoptosis in goat GCs.

Rescued Cell Proliferation and Cell Cycle in KDM5B Knockdown and MTF1 Overexpression GCs
MTF1 is one of the differentially expressed genes after the knockdown of KDM5B, as revealed by RNA-seq, qPCR and Western blot (p < 0.01; Figure 6A-C). To investigate the role of MTF1, we knocked down MTF1 using siRNA (p < 0.01; Figure 6D) and found that the expression of PCNA significantly reduces (p < 0.01; Figure 6E). Furthermore, the mRNA and protein expressions of MTF1 are successfully rescued after the knockdown of KDM5B and overexpression of MTF1 in GCs ( Figure 6F,G). Interestingly, we found that the mRNA expression (p < 0.01) and protein level (p < 0.05) of PCNA increase significantly in the KDM5B knockdown and MTF1 overexpression GCs ( Figure 6H,I). The mRNA expressions of CDK6 (p < 0.01), CCDN1 (p < 0.05) and CCND3 (p < 0.01) decrease after the simultaneous knockdown of KDM5B and overexpression of MTF1 ( Figure 6H). The protein expressions of CDK4 (p < 0.01), CCDN1 (p < 0.01) and CDK6 (p < 0.01) decrease after the simultaneous knockdown of KDM5B and overexpression of MTF1 ( Figure 6I). These data suggest that KDM5B might regulate cell proliferation and cell cycle via MTF1, which needs further investigation.

Decreased DNA Damage in KDM5B Knockdown and MTF1 Overexpression GCs
The expression of γH2AX significantly increases after the knockdown of MTF1 (p < 0.01; Figure 7A). Through the rescue experiments, we validated that the expression of γH2AX increases significantly (p < 0.01) and decreases after the simultaneous knockdown of KDM5B and overexpression of MTF1 ( Figure 7B). Then, we detected the level of ROS, and the results indicate that the fluorescence intensity of the knockdown KDM5B group is significantly higher than control group (p < 0.01) and decreases after the simultaneous knockdown of KDM5B and overexpression of MTF1 ( Figure 7C,D). The results of the IF intuitively show that DNA damage sites and fluorescence intensity of γH2AX significantly increase after the knockdown of KDM5B (p < 0.01). DNA damage sites and fluorescence intensity of γH2AX significantly reduce after the simultaneous knockdown of KDM5B and overexpression of MTF1 ( Figure 7E,F). The mRNA expression of RAD511, RPA1, RPA2 and TP53BP1 significantly decrease after the knockdown of KDM5B and fall back after the simultaneous knockdown of KDM5B and overexpression of MTF1 ( Figure 7G). factors, BAX, CAS3 and CAS9, increase significantly, whereas the expression apoptotic factor BCL2 decreases, in the KDM5B knockdown GCs (p < 0.01; Additionally, the ratio of BAX/BCL2 significantly increases (p < 0.01; Figure  while, the protein expressions of BAX, CAS3 and CAS9 increase significantly, w protein expression of BCL2 decreases in the KDM5B knockdown GCs (p < 0.01; The result of TUNEL also intuitively indicates that apoptotic cells are signi creased in the KDM5B knockdown GCs ( Figure 5D). These data suggest that down of KDM5B accelerates apoptosis in goat GCs.

Decreased DNA Damage in KDM5B Knockdown and MTF1 Overexpression GCs
The expression of γH2AX significantly increases after the knockdown of MTF1 (p < 0.01; Figure 7A). Through the rescue experiments, we validated that the expression of γH2AX increases significantly (p < 0.01) and decreases after the simultaneous knockdown of KDM5B and overexpression of MTF1 ( Figure 7B). Then, we detected the level of ROS, cantly increase after the knockdown of KDM5B (p < 0.01). DNA damage sites and fluorescence intensity of γH2AX significantly reduce after the simultaneous knockdown of KDM5B and overexpression of MTF1 ( Figure 7E,F). The mRNA expression of RAD511, RPA1, RPA2 and TP53BP1 significantly decrease after the knockdown of KDM5B and fall back after the simultaneous knockdown of KDM5B and overexpression of MTF1 ( Figure  7G).

Discussion
KDM5B is essential for cell proliferation, cell fate and genome stability [35]. However, its function in goat GCs remains unclear. In the present study, we found that knockdown of KDM5B led to an increase in DNA DSBs, cell apoptosis and cell cycle arrest. Additionally, we inferred that KDM5B affected cell cycle and DNA damage via MTF1. Our findings laid the groundwork for future research into the role of KDM5B in follicular development.
KDM5B is highly expressed in the ovary of goats and plays a vital role in DNA DSBs. Increased intracellular ROS levels result in DNA DSBs, affecting DNA replication and cell proliferation [36]. Increased reactive oxygen species in ovarian cancer cells have been found to cause DNA DSBs [37]. Previous research has demonstrated that attenuation of KDM5B altered H3K4 methylation patterns during the genome activation phase of early pig embryos, disrupted embryonic genome activation, altered DNA damage responses, increased DNA DSBs and decreased DNA damage repair capacity, which corresponded to the outcomes of our experiment [24,38]. Our results indicated that the knockdown of KDM5B led to an increase in ROS levels and DNA DSB sites, indicating that DNA damage indeed occurred. It was further confirmed that the sequencing results were affected by the reliability of DNA replication-related functions. We also demonstrated that these DNA DSBs were caused by MTF1 by simultaneously inhibiting KDM5B and overexpressing MTF1. Similar to previous findings, MTF1 can affect intracellular ROS levels by regulating zinc redox, thereby resulting in cell damage [39]. Through the detection of DNA damage repair marker genes, we found that after the KDM5B knockdown, the expressions of RAD511, RPA1, RPA2 and TP53BP1 significantly decreased. These results mean that knockdown of KDM5B leads to reduced DNA damage repair ability and decreased DNA damage repair level. However, this damage was repaired after the overexpression of MTF1. Severe oxidative stress and increased mitochondrial depolarization both lead to cell apoptosis [40][41][42]. Previous research has indicated that KDM5B plays an essential role in the apoptosis of cancer cells [43]. Yang et al. found that LET-7i could target KDM5B to induce apoptosis in esophageal cancer cells [44]. Our study proved that KDM5B accelerated GCs apoptosis. Combined with the previous results, we speculated that this apoptosis was caused by increased intracellular ROS and the occurrence of DNA DSBs.
DNA DSBs are an essential factor affecting DNA replication [45,46]. Because the S phase is the phase of DNA replication, we suspect that cell cycle progression is also affected after the knockdown of KDM5B. Wang et al. demonstrated that KDM5B is frequently up-regulated in HCC specimens and cell lines. Knockdown of KDM5B significantly inhibits cellular proliferation of HCC cells and arrests cell cycle progression at the G1 phase [47]. The present study revealed that the knockdown of KDM5B blocked cell division in the S phase by regulating relative genes. The cell cycle phases arrested in our experiments were different from previous experiments. We hypothesized that the effect mechanism of KDM5B on the cell cycle in GCs was different from that in HCCs. Our experiments also confirmed that KDM5B affected the cell cycle in goat GCs. The elevation of related genes caused many cells to turn from the G1 phase to the S phase. DNA DSBs hindered DNA replication and the cell cycle was arrested in the S phase [48,49]. Through the rescue experiments, we could speculate that the effect of KDM5B on the cell cycle was achieved through MTF1.
The cell cycle determines the fate of cell proliferation to some extent [50,51]. Previous research showed that KDM5B blocked the proliferation of gastric cancer cells. It dramatically promoted gastric cancer cell xenograft formation and growth and promoted gastric cancer cell metastasis in a liver metastasis model [52]. Our results also demonstrated that the knockdown of KDM5B induced a decrease in cell proliferation in goat GCs. In conclusion, the knockdown of KDM5B affected cell cycle progress, standard division and proliferation. Our results provide new insights into the functional mechanisms of KDM5B in granulosa cells.
We found that KDM5B affects MTF1 expression to ensure normal cell proliferation and cell cycle, maintain DNA replication and reduce DNA damage, primarily in DNA DSBs. However, the mechanism of KDM5B and MTF1 interaction remains unknown. We intend to use CHIP experiments in the future to confirm how KDM5B affects MTF1 expression. This way, the mechanism of KDM5B on GCs' cell cycle arrest and DNA damage will be better understood.

Conclusions
In conclusion, our results suggest that KDM5B plays a crucial role in goat GCs, as evidenced by its ability to induce DNA DSBs, block the S phase of the cell cycle, restrain cell proliferation and hasten cell apoptosis when it is knocked down. Through the simultaneous knockdown and overexpression of KDM5B and MTF1, we discovered that KDM5B induces DNA DSBs and arrests the cell cycle. These findings have laid the groundwork for future research into the effect and influence of KDM5B on GCs and follicular development. Additionally, this study provides a new reference for the regulatory relationship between KDM5B and MTF1.
Author Contributions: Y.Y. was responsible for the performance of the whole experiment and writing of the manuscript. Y.C. and L.L. contributed to sample collection. J.G., K.D. and Z.C. analyzed the experimental data. F.W. provided guidance for the design of the experiment. M.D. participated in the design of the entire experiment and the revision of the paper. All authors have read and agreed to the published version of the manuscript.