Kruppel Like Factor 12 gene regulates ovarian granulosa cells proliferation and apoptosis via the PI3K/Akt signaling pathway

Kruppel Like Factor 12 gene regulates ovarian granulosa cells proliferation and apoptosis via the PI3K/Akt signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Kruppel Like Factor 12 gene regulates ovarian granulosa cells proliferation and apoptosis via the PI3K/Akt signaling pathway Jiawei Cai, Bohao Zhao, Zhiyuan Bao, Yunpeng Li, Xiaoman Han, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7203220/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Feb, 2026 Read the published version in BMC Veterinary Research → Version 1 posted 8 You are reading this latest preprint version Abstract Granulosa cells (GCs), as the largest proportion of somatic cells in mammalian ovarian follicles, play a central regulatory role in follicular development and oocyte maturation. In-depth analysis of the molecular mechanisms related to the growth and development and functional regulation of GCs is of great significance for improving the regulatory network mechanism in the field of female reproduction. Kruppel Like Factor 12 (KLF12) gene is involved in the regulation of various cellular physiological activities, but its specific regulatory function in ovarian GCs is not yet clear. In this study, the coding sequence (CDS) of KLF12 gene in New Zealand female rabbits was cloned for the first time, and the expression abundance and cellular localization of this gene in ovarian tissues of female rabbits at different reproductive stages were systematically analyzed. The results showed that KLF12 was highly expressed in the ovaries of 18-month-old female rabbits. Functional studies found that overexpression of KLF12 significantly inhibited the synthesis of Estradiol (E 2 ) and Progesterone (P) in GCs, and the expression of genes related to cell proliferation, apoptosis and cycle development also changed. Further Western blot (WB) detection confirmed that KLF12 overexpression significantly reduced the phosphorylation levels of key molecules PI3K and Akt in the PI3K/Akt signaling pathway, while KLF12 knockdown had the opposite effect on GCs function. These results reveal the molecular mechanism of KLF12 regulating the development and function of ovarian GCs in New Zealand female rabbits through PI3K/Akt signaling pathway, which not only provides a theoretical basis for improving the reproductive performance of female rabbits, but also provides a new scientific perspective for the study of the molecular mechanism of mammalian reproductive regulation. ovarian development KLF12 gene granulosa cells proliferation PI3K/Akt signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction As the core organ of female reproductive regulation, the function of ovary depends on complex cellular networks. As the largest proportion of somatic cells in mammalian ovarian follicles, GCs are not only the main functional units for the synthesis of ovarian steroid hormones (such as E 2 and P)[ 18 , 20 , 28 ], but their proliferation and apoptosis dynamics directly regulate key physiological processes such as follicular development, oocyte maturation, ovulation and luteinization[ 3 , 17 ]. Studies have shown that female fertility gradually declines with age, and its important features include: a significant increase in the apoptosis rate of GCs, an accelerated process of follicular atrophy, and a double decline in the number and quality of oocytes, eventually leading to degenerative changes in ovarian function[ 10 , 30 ]. By providing material nutritional support and suitable growth microenvironment, GCs play an irreplaceable role in oocyte maturation and follicular dynamic development. We previously established an ovarian GCs injury model through transcriptome sequencing and found that the KLF12 gene is a candidate gene significantly highly expressed during the apoptosis of ovarian GCs[ 2 ]. The Kruppel-like transcription factor family is involved in diverse cellular mechanisms, such as cell growth, differentiation, and execution of apoptosis[ 14 ]. The KLF family members, including KLF4, KLF9, KLF13, and KLF15, play pivotal regulatory role in orchestrating the ovarian function[ 19 , 25 ]. Distinct from other KLF family members, KLF12 is categorized as a zinc finger transcription factor residing within this family. KLF12 restrains the transcription of its target gene through the interaction between its N-terminal PVDLS motif (Pro-Xaa-Asp-Leu-Ser) and the C-terminal binding protein[ 21 , 24 ]. KLF12 is known to activate apoptosis in diverse cell populations, such as endometrial stromal cells[ 34 ], ovarian cancer cells[ 32 ], and bladder cancer cells[ 27 ]. Up to now, the action mechanism of the KLF12 gene in ovarian GCs has not been reported. The purpose of this study was to understand the molecular mechanism of KLF12 gene in the regulation of female rabbit reproduction. By cloning the coding sequence of KLF12 gene, analyzing its expression profile in multiple tissues of female rabbits and the cellular localization of ovarian tissues at different reproductive stages, and combining with knockdown and overexpression function experiments, the biological function of KLF12 in GCs was analyzed. The results showed that KLF12 further affected the oocyte maturation and reproductive performance of female rabbits by regulating the development process and endocrine function of GCs. The results of this study not only provide a potential molecular target for rabbit genetic breeding, but also provide a new theoretical basis for further understanding the mechanism of mammalian reproductive regulation. 2. Materials and Methods 2.1 Animal and sample collection Six healthy female New Zealand rabbits aged 6 months and 18 months were selected. The female rabbits were purchased from Guifeng Rabbit Industry Professional Cooperative of Yangzhou City and obtained the informed consent of the owner. The selected age groups represented different reproductive stages, and there was no history of infertility or reproductive disorders. Animals were reared under controlled conditions (12:12 light-dark cycle, free use of standard rabbit food and water). Animals were fasted overnight, and then Zoteil-50 (5mg/kg) was injected into the ear vein to ensure that they lost consciousness and had no pain, and then tissue samples of heart, liver, spleen, lung, kidney and ovary were collected. The specimens were promptly submerged in liquid nitrogen for instantaneous freezing and transferred to a storage environment maintained at − 80℃ for long-term preservation. The animal experiment was approved by the Animal Care and Use Committee of Yangzhou University (Approval No. 202212006). 2.2 Cell culture and transfection Rabbit immortalized GC lines constructed by our group in the early stage were used in this study[ 1 ]. The GCs were cultured in a DMEM/F12 medium supplemented with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution (Beyotime, Shanghai, China), which is a mixture of 100 units/mL penicillin and 0.1 mg/mL streptomycin. The cells were cultured in an incubator at 37℃ under 5% CO 2 . The GCs were seeded into a 24-well cell culture dish to ensure 80% cell fusion before transfection was performed using Lipofectamine™ 2000 (Invitrogen, CA, USA). Subsequent experiments were conducted 48 h after transfection. 2.3 KLF12 overexpression and knockdown To synthesize the overexpression vector for KLF12, high-grade cDNA from the rabbit ovary tissue was synthesized using the PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, China). The primers were crafted based on the rabbit KLF12 mRNA sequence (GenBank accession number: XM_051850621.1) to amplify the KLF12 CDS (primer-F: 5ʹ-gggagacccaagctggctagcATGAATATCCCTATGAAGAGGAAACC-3ʹ, primer-R: 5ʹ-tgctggatatctgcagaattcTCACACCAGCATGTGCCTCC-3ʹ). The amplified CDS was secured through polymerase chain reaction (PCR) by using the Phanta Max Super-Fidelity DNA Polymerase (Vazyme, China) and subsequently inserted into the pcDNA3.1(+) vector. To reduce KLF12 expression, a small interfering RNA (siRNA) was custom designed and synthesized by Shanghai GenePharma. Co., Ltd, and designated as siRNA-1-4. The siRNA sequences are listed in Table 1. 2.4 Hematoxylin eosin (HE) Staining Ovarian tissues of female rabbits were collected and treated with paraformaldehyde (BL539A; Biosharp, Hefei, China) was fixed for 48h, dehydrated, embedded in paraffin, sliced, dewaxed, hydrated with ethanol, and washed with distilled water. The sections were stained with hematoxylin for 8 min, and then stained with eosin for 1 min. After that, the sections were treated with xylene for permeabilization, sealed with neutral glue, and observed under an inverted microscope. 2.5 Fluorescence in situ hybridization The ovarian tissue was fixed with an insitu hybridization fixative for 12h and dehydrated with a series of graded alcohols. Subsequently, the samples were infiltrated with wax, embedded, and sectioned into slices. The sections were immersed in the antigen retrieval solution for 10-15min for heat-induced epitope retrieval. Then, the sections were allowed to cool down to room temperature naturally. Afterward, protease K (20µg/mL) was added to the sections dropwise for digestion at 37°C for 30min. The sections were rinsed with pure water and washed with PBS three times. The pre-hybridization solution was added then added dropwise, and the sections were incubated at 37°C for 1h. The pre-hybridization solution was then poured out, and the 5ng/µL probe-containing hybridization solution (probe synthesis sequence: 5-CY3-TGCGTCCTCCGATGAGCCTTCAGG-3, Wuhan Sevier Biotechnology Co., Ltd.) was added dropwise. Hybridization was performed at 37°C for 12h, and the hybridization solution was washed away. The nucleus was then restained with DAPI, and the antifluorescence quenching sealing agent was added dropwise after washing the sections. The sections were observed under a Nikon positive fluorescence microscope, and the images were captured. 2.6 Total RNA extraction, cDNA synthesis, and qRT-PCR After 48h of transfection, the original medium was discarded, PBS was added to wash the cells, and then 600µL of lysis buffer was added to each well, according to the manufacturer 's instructions, RNA was extracted from the cells by using the SteadyPure RNA extraction kit. (AG21024; Accurate Biotechnology (Hunan) Co., Ltd., China). The RNA was reversely transcribed into cDNA using the Evo M-MLV Mix Kit (AG11728; accurate Biotechnology (Hunan) Co., Ltd.). Gene expression levels were quantified using the SYBR® Green Premix Pro Taq HS qPCR Tracking Kit (AG11733; Accurate Biotechnology (Hunan) Co., Ltd.) on the QuantStudio® 5 system (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). The relative gene expression was measured using the 2 −∆∆Ct method[ 23 ], and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. The specific primer sequences are listed in Table 2. 2.7 Protein extraction and western blotting The cellular specimens were disrupted using RIPA buffer (Solarbio, Beijing, China) to facilitate protein extraction. The concentration of the extracted proteins was determined using an Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China). All protein samples were subsequently normalized to a density of 0.5µg/µL, and 3µL of this standardized solution was dispensed into each well. Then, the protein was automatically separated using a western blotting apparatus (ProteinSimple, CA, USA), adhering to the protocol detailed by Harris[ 8 ]. In all protein analyses, GAPDH was used as an internal reference to ensure standardization. The specific information of the antibodies used in this study is detailed in Table 3. 2.8 Cell apoptosis and proliferation assay Cellular apoptosis was analyzed using the Annexin V-FITC Apoptosis Detection Kit (Vazyme, China). Apoptosis levels were quantified using a flow cytometer (FACSAria SORP model; Becton Dickinson, USA). Following acquisition, the cytometric data were analyzed using FlowJo V10 software (FlowJo, OH, USA). Meanwhile, cell proliferation was detected using the cell Counting Kit-8 (Vazyme, China). The optical density values of 96 holes at 0, 24, 48, and 72h were measured at 450nm by using Infinite M200 pro (Tecan, Switzerland). 2.8 5-Ethynyl-2′-deoxyuridine (EdU) assay After 48 h of transfection, EdU cell proliferation assay kit (C0075S, Beyotime, Shanghai, China) was used to evaluate cell proliferation ability according to the product instructions. Immediately after the completion of the staining step, the fluorescence microscope was used to observe and take images. The EdU labeling index was calculated as the ratio of the number of EdU-positive nuclei to the total number of nuclei. 2.9 Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay (ELISA) kits for E 2 (Cat.# ml027864) and P (Cat.# ml041624) were obtained from Mlbio (Shanghai, China). According to the manufacturer's instructions, E 2 and P levels in the cell culture supernatant were measured using the ELISA kits. 2.10 Statistical analysis Statistical significance was determined by two-tailed Student 's t test or one-way analysis of variance (ANOVA) in SPSS 25.0 statistical software package (SPSS Inc., Chicago, IL, USA). All collected data were then expressed as mean ± standard error (SEM). Furthermore, the data were graphically represented using GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA). Results were considered statistically significant at P < 0.05 (*) and highly significant at P < 0.01 (**). 3. Results 3.1 Cloning and vector construction of rabbit KLF12 gene CDS sequence The CDS sequence of rabbit KLF12 gene was cloned using rabbit ovary-specific primers, with a total length of 1209 bp (Fig. 1 A). To determine the role of KLF12 in ovarian GCs, a pcDNA3.1-KLF12 vector was constructed and four siRNAs were designed to knock down KLF12 in rabbit GCs. They were transfected into GCs. The qRT-PCR and WB results unveiled that pcDNA3.1-KLF12 significantly increased KLF12 expression, and siRNA-4 had the most effective knockdown efficiency ( P < 0.01) (Fig. 1 B-D). 3.2 Tissue and spatiotemporal expression profiles of KLF12 in female rabbits of different ages The results of HE staining showed that the ovarian structure of 6-month-old female rabbits was relatively dense, with more immature follicle morphology, uniform matrix staining and close cell arrangement. However, the ovarian structure of 18-month-old female rabbits was loose, with obvious mature follicles, relatively sparse follicle distribution and loose cell arrangement (Fig. 2 A). The KLF12 gene expression levels were quantified across various developmental stages in the heart, liver, spleen, lung, kidney, and ovarian tissues obtained from the female rabbits. The KLF12 gene was expressed in different tissues of the rabbits. KLF12 expression was higher in the 18-month-old rabbits than in the 6-month-old rabbits. The KLF12 expression level was the highest in the ovarian tissues, which was significantly higher than that in the 6-month-old rabbits ( P < 0.01) (Fig. 2 B). Additionally, the results of fluorescence in situ hybridization unveiled that KLF12 gene expression was relatively higher in the GCs of the 18-month-old rabbits (Fig. 2 C). In summary, KLF12 gene expression in these tissues increased with age, which indicated that this gene plays a crucial biological function in the ovarian tissues of the female rabbits. 3.3 KLF12 inhibits E 2 and P levels in GCs In order to further explore the regulatory mechanism of KLF12 on steroid hormone synthesis, the effects of KLF12 on the secretion of E2 and P in rabbit GCs were detected. ELISA results showed that overexpression of KLF12 significantly reduced the secretion levels of E 2 and P in GCs ( P < 0.05). On the contrary, after knocking down KLF12, the content of E 2 and P increased significantly ( P < 0.05) (Fig. 3 A, B). At the same time, the expression of key genes in the steroid synthesis pathway was detected. KLF12 overexpression significantly inhibited the expression of CYP11A1, CYP19A1 and StAR genes ( P < 0.01). Similarly, the expression of CYP11A1, CYP19A1 and StAR genes was significantly increased after KLF12 knockdown ( P < 0.05) (Fig. 3 C). These results indicate that KLF12, as a negative regulator of steroid synthesis, regulates the synthesis of E 2 and P in rabbit GCs by inhibiting the expression of key enzyme genes. 3.4 KLF12 inhibits cell proliferation-related genes To further clarify the role of KLF12 gene in ovarian follicular development. We detected genes related to GCs proliferation and apoptosis. According to the data, KLF12 promoted the mRNA and protein expression of Bax, Caspase-3, Caspase-9, and P53 and inhibited the mRNA and protein expression of BCL-2 and PCNA ( P < 0.05) (Fig. 4 A, B). At the same time, we detected the expression of cell cycle key protein molecules. The mRNA and protein levels of the cell cycle regulatory molecules CCNB1, CCNA2, and CDK4 in KLF12-overexpressing GCs were significantly downregulated. By contrast, the CCNB1, CCNA2, and CDK4 e xpression levels in GCs were significantly increased after KLF12 knockdown ( P < 0.05) (Fig. 4 C, D). The above studies further showed that KLF12 negatively regulated the proliferation and apoptosis of rabbit ovarian GCs by inhibiting cell cycle progression, thus playing a key regulatory role in follicular development. 3.5 KLF12 plays a negative role in GCs proliferation The proliferation of GCs is essential for follicular development. In order to study the regulation of KLF12 on the proliferation and apoptosis of GCs, CCK-8 activity detection and EdU proliferation labeling experiments showed that KLF12 overexpression significantly inhibited the proliferation of GCs ( P < 0.01) (Fig. 5 A, B). Flow cytometry apoptosis analysis further confirmed that the apoptosis rate of KLF12 overexpression group was significantly increased. On the contrary, KLF12 knockdown significantly promoted GCs proliferation and reduced apoptosis ( P < 0.01) (Fig. 5 C). Taken together, these findings suggest that KLF12 is a negative regulator of rabbit GCs proliferation. 3.6 KLF12 regulates the PI3K/Akt signaling pathway in GCs To delve deeper into the mechanisms underlying KLF12-regulated GC proliferation and apoptosis, we performed WB to quantify the expression levels of key molecules involved in the PI3K/Akt signaling pathway, namely PI3K, phosphorylated PI3K (p-PI3K), Akt, and phosphorylated Akt (p-Akt). The findings revealed that, the protein levels of the activated forms p-PI3K and p-Akt were notably reduced within the KLF12 overexpression group. Conversely, protein concentrations of these phosphorylated molecules, p-PI3K and p-Akt, were considerably elevated in the KLF12 knockdown group. (Fig. 6 A, B). The data imply that KLF12 overexpression has a suppressive effect on the PI3K/Akt signaling pathway, whereas KLF12 reduction or silencing leads to a notable stimulation of this pathway. To further prove the activation and inhibition effect of KLF12 on the PI3K/Akt signaling pathway of GCs, 740 Y-P (10µM) and LY294002 (10µM) were used to treat the transfected GCs. According to the findings, upon the introduction of 740 Y-P, p-PI3K and p-Akt protein levels substantially increased within the KLF12-overexpressing group. Conversely, LY294002 addition caused a pronounced decrease in p-PI3K and p-Akt protein concentrations in the cohort with KLF12 knockdown (Fig. 6 A, B). The aforementioned outcomes provide additional confirmation that KLF12 indeed acts as a crucial modulator in PI3K/Akt signaling pathway activation within GCs. 4. Discussion GCs are the largest somatic cell population in the ovary. They provide material guarantee and a microenvironment for oocyte maturation and follicular development. During follicular growth and development, only a very small number of follicles successfully develop and ovulate, whereas most of the remaining follicles undergo atresia. The key cellular event mediating this physiological process is GC proliferation and apoptosis. We previously found that KLF12, a member of the Kruppel-like factors family, was upregulated during ovarian GC apoptosis[ 2 ]. In this study, the cloning and functional analysis of the KLF12 gene coding sequence of New Zealand female rabbits were completed for the first time. It was found that KLF12 was significantly highly expressed in the ovaries of 18-month-old female rabbits, suggesting its potential role in follicular development. In addition, as the main unit of follicles, GCs are the key to ensure animal oocyte maturation and maintain normal hormone levels. The initial stage in P synthesis is the translocation of cholesterol from the exterior to the interior of the mitochondrial membrane. StAR, a pivotal component, governs steroid production. Then, CYP11A1 transforms cholesterol into pregnenolone, and mitochondrial enzymes-mediate the subsequent conversion of pregnenolone into progesterone[ 6 ]. KLF12 knockdown changes the expression of key genes involved in E 2 and P syntheses and promotes E 2 and P secretion by GCs. This indicates that KLF12 can regulate E 2 and P levels by regulating the key proteins involved in their syntheses. To regulate gene transcription, KLFs bind to the GC box or CACCC of the target gene promoter region through a highly conserved carboxyl-terminal DNA domain, thereby affecting cell proliferation, differentiation, and apoptosis[ 4 , 21 ]. KLF9 levels are meticulously controlled to preserve the cellular equilibrium, which encompasses processes such as cell proliferation, extracellular matrix formation, cell migration, and adherence[ 7 , 15 , 16 , 26 , 31 ]. KLF5 is involved in promoting the ovarian cancer stem cell-like phenotype[ 5 ]. KLF8 promotes the induction of tumorigenic breast stem cells by targeting miR-146a[ 29 ]. KLF4, KLF9, and KLF13 transcripts are expressed in ovarian cells and regulate these cells[ 19 ]. These collective results indicate that KLFs are promising therapeutic targets in modulating cell proliferation and apoptosis. Intriguingly, our study revealed that heightened KLF12 expression concurrently facilitates apoptosis induction in ovarian GCs. KLF12 gene knockdown and overexpression can regulate the expression of GC proliferation- and apoptosis-related genes Bax, BCL-2, Caspase-9, and Caspase-3, and can affect the expression of cell cycle-related proteins CCNB1, CCNA2, and CDK4. Cell cycle regulation is a highly fine-tuned process, which results in cell growth, genetic material replication, and cell division. The PI3K/Akt signaling pathway can activate Cyclin A, Cyclin B, Cyclin D, and Cyclin E, to promote GC proliferation[ 13 ]. The PI3K/Akt pathway, serving as a significant axis governing cell proliferation and apoptosis, plays a critical role in orchestrating GC growth and programmed cell death throughout follicular maturation[ 9 , 12 , 33 ]. PI3K is activated when growth factors and cytokines bind to their cell surface receptors[ 11 ]. As the main target of proto-oncogenes and PI3K, Akt regulates cell survival and proliferation[ 22 ]. It oversees cellular proliferation and protein synthesis and counteracts pro-apoptotic entities such as BAD and Caspase-9, which are pivotal elements in executing programmed cell death. Within the scope of this investigation, we observed that KLF12 overexpression notably reduced p-PI3K and p-Akt protein levels. Conversely, when KLF12 was knocked down, p-PI3K and p-Akt protein concentrations were significantly elevated. Subsequently, the transfected GCs were treated with 740 Y-P and LY294002. After 740 Y-P addition, p-PI3K and p-Akt protein levels significantly increased in the KLF12 overexpression group, whereas the protein levels of the phosphorylated molecules p-PI3K and p-Akt significantly decreased after LY294002 addition. This result again proves that KLF12 molecules exert a regulatory effect on the PI3K/Akt signaling pathway. Our study established a new molecular mechanism. Overexpression of KLF12 inhibited the secretion of E 2 and P in rabbit GCs, and also inhibited the expression of genes related to cell proliferation, apoptosis and cycle development. Mechanism studies further found that KLF12 is a key regulator of the PI3K/Akt signaling pathway. These results demonstrate that KLF12 inhibits the proliferation of GCs and promotes their apoptosis by regulating the PI3K/Akt signaling pathway, which provides a theoretical basis for improving the reproductive performance of rabbits and opens up a new perspective for the study of mammalian reproductive regulatory networks. 5. Conclusion In summary, this study completed the cloning and functional analysis of the coding sequence of KLF12 gene in New Zealand female rabbits for the first time. It was found that KLF12 was significantly highly expressed in the ovaries of 18-month-old female rabbits, suggesting its potential role in follicular development. Functional experiments showed that KLF12 overexpression significantly reduced the secretion levels of E 2 and P by inhibiting the transcriptional activity of key enzyme genes for steroidogenesis, revealing its negative feedback effect in the regulation of hormone synthesis. Mechanism studies further confirmed that overexpression of KLF12 inhibited the expression of genes related to cell proliferation, apoptosis and cycle development. In addition, KLF12 was also proved to be a key regulator of the PI3K/Akt signaling pathway in GCs. These results reveal the new function of KLF12 as a negative regulator of follicular development and the molecular mechanism of regulating the development and function of ovarian GCs in New Zealand female rabbits through the PI3K/Akt signaling pathway. The results provide a molecular basis for analyzing the mechanism of ovarian development, and also open up a new perspective for the study of mammalian reproductive regulation (Fig. 7 ). Declarations Ethics approval and consent to participate The female rabbits were purchased from Guifeng Rabbit Industry Professional Cooperative of Yangzhou City and obtained the informed consent of the owner. These animals can be used in this study. The animal experiment was approved by the Animal Care and Use Committee of Yangzhou University (Approval No. 202212006). Consent for publication Not applicable. Competing interests We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. Funding This research was funded by the China Agriculture Research System of MOF and MARA (CARS-43-A-1). Author Contribution JC BZ, YC and XW conceived and designed research. JC and ZB conducted experiments. YL and XH contributed new reagents or analytical tools. All authors read and approved the manuscript. Acknowledgements We gratefully acknowledge the members of the laboratory for their suggestions and critical reading of the manuscript. Data Availability All data generated or analyzed during this study are included in this published article and its supplementary information files. References Bao Z, Chen Y, Li J, Cai J, Zhao B, Wu X. 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Pflug Arch Eur J Phy. 2019;471:1055–63. Tables Table 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.xlsx Table2.xlsx Table3.xlsx Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 05 Feb, 2026 Read the published version in BMC Veterinary Research → Version 1 posted Editorial decision: Revision requested 22 Oct, 2025 Reviews received at journal 21 Oct, 2025 Reviews received at journal 18 Oct, 2025 Reviewers agreed at journal 17 Oct, 2025 Reviewers agreed at journal 16 Oct, 2025 Reviewers invited by journal 02 Sep, 2025 Submission checks completed at journal 30 Aug, 2025 First submitted to journal 30 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7203220","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":509190952,"identity":"661ce7c5-34a2-4469-86b7-c124f8231585","order_by":0,"name":"Jiawei Cai","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Jiawei","middleName":"","lastName":"Cai","suffix":""},{"id":509190953,"identity":"1f70580c-2d03-4ea5-9b72-e50775d292e3","order_by":1,"name":"Bohao Zhao","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Bohao","middleName":"","lastName":"Zhao","suffix":""},{"id":509190954,"identity":"d12872ba-5231-4b3f-9690-dbd380a59b55","order_by":2,"name":"Zhiyuan Bao","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Zhiyuan","middleName":"","lastName":"Bao","suffix":""},{"id":509190955,"identity":"3bf8e784-d1dd-48f6-bba5-55b9665695de","order_by":3,"name":"Yunpeng Li","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yunpeng","middleName":"","lastName":"Li","suffix":""},{"id":509190956,"identity":"35c0bb11-f43b-42d7-b438-9e06bef50470","order_by":4,"name":"Xiaoman Han","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoman","middleName":"","lastName":"Han","suffix":""},{"id":509190957,"identity":"f3fa9616-d34e-4685-a1b1-43674343dd68","order_by":5,"name":"Yang Chen","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Chen","suffix":""},{"id":509190958,"identity":"27b2451d-defe-4db0-924a-4ae1bf5f43e8","order_by":6,"name":"Xinsheng Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYLCCDxDKgHgdjDNI1sLMQ5IWg/NnzKRt2+oSG9ibt0kw1NwhrEVyRo6ZdG4bW2IDz7EyCYZjzwhr4Zfg3QbUwpPYIJFjJsHYcJiwFjb+s9ukLdskEhvk3xCphZ8hd5s0Y5sB0BYeIrVIzsj/bNlzLsG4jSet2CLhGBFaDM4fS7zxo6xOtp/98MYbH2qI0AIGjGxAT4EYCURqAII/xCsdBaNgFIyCEQgAWAwyT8bXdUUAAAAASUVORK5CYII=","orcid":"","institution":"Yangzhou University","correspondingAuthor":true,"prefix":"","firstName":"Xinsheng","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2025-07-24 08:29:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7203220/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7203220/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12917-026-05336-8","type":"published","date":"2026-02-05T15:59:33+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90854524,"identity":"94701429-f316-4de9-87fc-25c373d8561d","added_by":"auto","created_at":"2025-09-09 04:15:10","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":142897,"visible":true,"origin":"","legend":"\u003cp\u003eCloning and vector construction of rabbit KLF12 gene CDS sequence. (A) Cloning of CDS region of rabbit KLF12 gene. M: DL2000 DNA Marker, 1-3: PCR product of CDS region of KLF12 gene. (B) pcDNA3.1-KLF12 significantly increased the expression of KLF12 mRNA levels in GCs. (C) siRNA-KLF12 significantly decreased the expression of KLF12 mRNA levels in GCs. (D) Knockdown and overexpression of KLF12 regulated the protein expression of KLF12. Data are presented as the mean±SEM (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). A two-tailed paired t-test was used for data analyses.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203220/v1/89cd76cc85679f99432b6bd2.jpeg"},{"id":90854075,"identity":"7c3326d2-d9a0-4eb7-bec3-5c5621924f6f","added_by":"auto","created_at":"2025-09-09 04:07:10","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":233332,"visible":true,"origin":"","legend":"\u003cp\u003eTissue and spatiotemporal expression profiles of KLF12 in female rabbits of different ages. (A) Ovarian morphology of female rabbits at different reproductive stages; Scale bar, 100μm. (B) The KLF12 gene expression level in different tissues of female rabbits at different s tages. (C) KLF12 localization in the ovaries of female rabbits. Red denotes KLF12 detected using the fluorescence in situ hybridization probe, and blue indicates the DAPI-stained nucleus. GC, granule cell; Scale bar, 20μm. Data are presented as the mean±SEM (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). A two-tailed paired t-test was used for data analyses.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203220/v1/14431510fe39b0d739ac95d2.jpeg"},{"id":90854077,"identity":"33b110f0-24dc-41a8-8a9f-96ae76f21028","added_by":"auto","created_at":"2025-09-09 04:07:10","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90157,"visible":true,"origin":"","legend":"\u003cp\u003eKLF12 inhibits E\u003csub\u003e2\u003c/sub\u003e and P levels in GCs. (A) E\u003csub\u003e2\u003c/sub\u003e levels were detected after GCs overexpressed KLF12 or after KLF12 knockdown in GCs. (B) P levels were detected after GCs overexpressed KLF12 or after KLF12 knockdown in GCs. (C) CYP11A1, CYP19A1 and StAR mRNA levels in KLF12-overexpressing GCs and GCs with KLF12 knockdown were detected through qRT-PCR. Data are presented as the mean±SEM (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). A two-tailed paired t-test was used for data analyses.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203220/v1/5d7a7afa526b4321abada86f.jpeg"},{"id":90854962,"identity":"7dc04a74-4656-441e-97f5-9b330c7a113d","added_by":"auto","created_at":"2025-09-09 04:31:10","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":285313,"visible":true,"origin":"","legend":"\u003cp\u003eKLF12 inhibits cell proliferation-related genes. (A) KLF12 regulated the expression of mRNA related to cell proliferation and apoptosis after KLF12 overexpression and knockdown in GCs. (B) KLF12 regulated the expression of protein related to cell proliferation and apoptosis after KLF12 overexpression and knockdown in GCs. (C) KLF12 regulated the expression of mRNA related to cell cycle after KLF12 overexpression and knockdown in GCs. (D) KLF12 regulated the expression of protein related to cell cycle after KLF12 overexpression and knockdown in GCs. Data are presented as the mean±SEM (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). A two-tailed paired t-test was used for data analyses.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203220/v1/cddb0393626f0705431da468.jpeg"},{"id":90854526,"identity":"d8a6332b-b591-4d96-a5c2-3fa168dd1328","added_by":"auto","created_at":"2025-09-09 04:15:10","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":480859,"visible":true,"origin":"","legend":"\u003cp\u003eKLF12 plays a negative role in GCs proliferation. (A) Cell proliferation was detected by CCK-8 assay. (B) Cell proliferation was detected by EdU method. Scale bar: 100 μm. (C) The apoptosis rate was detected by Annexin V-FITC/PI staining and flow cytometry. Data are presented as the mean±SEM (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). A two-tailed paired t-test was used for data analyses.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203220/v1/b278bb8042ba072d0b05641b.jpeg"},{"id":90854528,"identity":"2481fbb9-fe20-4906-b9d0-f07f7a321a25","added_by":"auto","created_at":"2025-09-09 04:15:10","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":98056,"visible":true,"origin":"","legend":"\u003cp\u003eKLF12 regulates the PI3K/Akt signaling pathway in GCs. (A) WB was performed to detect the expression of key molecules in the PI3K/Akt signaling pathway following KLF12 overexpression and 740 Y-P addition. (B) WB was performed to detect the expression of key molecules in the PI3K/Akt signaling pathway after the treatment of siNRA-KLF12 and LY294002.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203220/v1/300e6e0f4c1302f8392c9096.jpeg"},{"id":90854084,"identity":"078834ed-5f9e-44c0-b5e5-de61cc2f3682","added_by":"auto","created_at":"2025-09-09 04:07:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":94184,"visible":true,"origin":"","legend":"\u003cp\u003eThe mechanism of KLF12 regulating the proliferation and apoptosis of rabbit ovarian granulosa cells.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7203220/v1/2c66aa9c009c78db3ca34437.png"},{"id":102235230,"identity":"d26f27ab-237d-4ee5-b473-c6d944e7d865","added_by":"auto","created_at":"2026-02-09 16:15:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2237814,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7203220/v1/18f3b89e-eb4e-41eb-9476-ce005e1d3167.pdf"},{"id":90854668,"identity":"ee90c01a-8882-48fa-8d22-ff5c445d13ba","added_by":"auto","created_at":"2025-09-09 04:23:10","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":9313,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7203220/v1/204dbb8f077798e71159ecf8.xlsx"},{"id":90854073,"identity":"bc1e730d-aa05-408e-a425-aaf25fe1fc3f","added_by":"auto","created_at":"2025-09-09 04:07:10","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10709,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7203220/v1/40f3971330f70af750822023.xlsx"},{"id":90854070,"identity":"e96c51ee-668e-4184-b6a4-9c6ae2180ab7","added_by":"auto","created_at":"2025-09-09 04:07:10","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10101,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7203220/v1/e852de7b3f536e584ec0fdb4.xlsx"},{"id":90854093,"identity":"3a6e7f74-9c2b-4893-8be0-5d5ebd01c288","added_by":"auto","created_at":"2025-09-09 04:07:10","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1111021,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7203220/v1/fc78521dbb05b87ee6020c0f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Kruppel Like Factor 12 gene regulates ovarian granulosa cells proliferation and apoptosis via the PI3K/Akt signaling pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAs the core organ of female reproductive regulation, the function of ovary depends on complex cellular networks. As the largest proportion of somatic cells in mammalian ovarian follicles, GCs are not only the main functional units for the synthesis of ovarian steroid hormones (such as E\u003csub\u003e2\u003c/sub\u003e and P)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], but their proliferation and apoptosis dynamics directly regulate key physiological processes such as follicular development, oocyte maturation, ovulation and luteinization[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Studies have shown that female fertility gradually declines with age, and its important features include: a significant increase in the apoptosis rate of GCs, an accelerated process of follicular atrophy, and a double decline in the number and quality of oocytes, eventually leading to degenerative changes in ovarian function[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. By providing material nutritional support and suitable growth microenvironment, GCs play an irreplaceable role in oocyte maturation and follicular dynamic development.\u003c/p\u003e\u003cp\u003eWe previously established an ovarian GCs injury model through transcriptome sequencing and found that the KLF12 gene is a candidate gene significantly highly expressed during the apoptosis of ovarian GCs[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The Kruppel-like transcription factor family is involved in diverse cellular mechanisms, such as cell growth, differentiation, and execution of apoptosis[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The KLF family members, including KLF4, KLF9, KLF13, and KLF15, play pivotal regulatory role in orchestrating the ovarian function[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Distinct from other KLF family members, KLF12 is categorized as a zinc finger transcription factor residing within this family. KLF12 restrains the transcription of its target gene through the interaction between its N-terminal PVDLS motif (Pro-Xaa-Asp-Leu-Ser) and the C-terminal binding protein[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. KLF12 is known to activate apoptosis in diverse cell populations, such as endometrial stromal cells[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], ovarian cancer cells[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and bladder cancer cells[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Up to now, the action mechanism of the KLF12 gene in ovarian GCs has not been reported.\u003c/p\u003e\u003cp\u003eThe purpose of this study was to understand the molecular mechanism of KLF12 gene in the regulation of female rabbit reproduction. By cloning the coding sequence of KLF12 gene, analyzing its expression profile in multiple tissues of female rabbits and the cellular localization of ovarian tissues at different reproductive stages, and combining with knockdown and overexpression function experiments, the biological function of KLF12 in GCs was analyzed. The results showed that KLF12 further affected the oocyte maturation and reproductive performance of female rabbits by regulating the development process and endocrine function of GCs. The results of this study not only provide a potential molecular target for rabbit genetic breeding, but also provide a new theoretical basis for further understanding the mechanism of mammalian reproductive regulation.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animal and sample collection\u003c/h2\u003e\u003cp\u003eSix healthy female New Zealand rabbits aged 6 months and 18 months were selected. The female rabbits were purchased from Guifeng Rabbit Industry Professional Cooperative of Yangzhou City and obtained the informed consent of the owner. The selected age groups represented different reproductive stages, and there was no history of infertility or reproductive disorders. Animals were reared under controlled conditions (12:12 light-dark cycle, free use of standard rabbit food and water). Animals were fasted overnight, and then Zoteil-50 (5mg/kg) was injected into the ear vein to ensure that they lost consciousness and had no pain, and then tissue samples of heart, liver, spleen, lung, kidney and ovary were collected. The specimens were promptly submerged in liquid nitrogen for instantaneous freezing and transferred to a storage environment maintained at \u0026minus;\u0026thinsp;80℃ for long-term preservation. The animal experiment was approved by the Animal Care and Use Committee of Yangzhou University (Approval No. 202212006).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Cell culture and transfection\u003c/h2\u003e\u003cp\u003eRabbit immortalized GC lines constructed by our group in the early stage were used in this study[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The GCs were cultured in a DMEM/F12 medium supplemented with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution (Beyotime, Shanghai, China), which is a mixture of 100 units/mL penicillin and 0.1 mg/mL streptomycin.\u003c/p\u003e\u003cp\u003eThe cells were cultured in an incubator at 37℃ under 5% CO\u003csub\u003e2\u003c/sub\u003e. The GCs were seeded into a 24-well cell culture dish to ensure 80% cell fusion before transfection was performed using Lipofectamine\u0026trade; 2000 (Invitrogen, CA, USA). Subsequent experiments were conducted 48 h after transfection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 KLF12 overexpression and knockdown\u003c/h2\u003e\u003cp\u003eTo synthesize the overexpression vector for KLF12, high-grade cDNA from the rabbit ovary tissue was synthesized using the PrimeScript\u0026trade; 1st Strand cDNA Synthesis Kit (Takara, China). The primers were crafted based on the rabbit KLF12 mRNA sequence (GenBank accession number: XM_051850621.1) to amplify the KLF12 CDS (primer-F: 5ʹ-gggagacccaagctggctagcATGAATATCCCTATGAAGAGGAAACC-3ʹ, primer-R: 5ʹ-tgctggatatctgcagaattcTCACACCAGCATGTGCCTCC-3ʹ). The amplified CDS was secured through polymerase chain reaction (PCR) by using the Phanta Max Super-Fidelity DNA Polymerase (Vazyme, China) and subsequently inserted into the pcDNA3.1(+) vector. To reduce KLF12 expression, a small interfering RNA (siRNA) was custom designed and synthesized by Shanghai GenePharma. Co., Ltd, and designated as siRNA-1-4. The siRNA sequences are listed in Table\u0026nbsp;1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Hematoxylin eosin (HE) Staining\u003c/h2\u003e\u003cp\u003eOvarian tissues of female rabbits were collected and treated with paraformaldehyde (BL539A; Biosharp, Hefei, China) was fixed for 48h, dehydrated, embedded in paraffin, sliced, dewaxed, hydrated with ethanol, and washed with distilled water. The sections were stained with hematoxylin for 8 min, and then stained with eosin for 1 min. After that, the sections were treated with xylene for permeabilization, sealed with neutral glue, and observed under an inverted microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Fluorescence in situ hybridization\u003c/h2\u003e\u003cp\u003eThe ovarian tissue was fixed with an insitu hybridization fixative for 12h and dehydrated with a series of graded alcohols. Subsequently, the samples were infiltrated with wax, embedded, and sectioned into slices. The sections were immersed in the antigen retrieval solution for 10-15min for heat-induced epitope retrieval. Then, the sections were allowed to cool down to room temperature naturally. Afterward, protease K (20\u0026micro;g/mL) was added to the sections dropwise for digestion at 37\u0026deg;C for 30min. The sections were rinsed with pure water and washed with PBS three times. The pre-hybridization solution was added then added dropwise, and the sections were incubated at 37\u0026deg;C for 1h. The pre-hybridization solution was then poured out, and the 5ng/\u0026micro;L probe-containing hybridization solution (probe synthesis sequence: 5-CY3-TGCGTCCTCCGATGAGCCTTCAGG-3, Wuhan Sevier Biotechnology Co., Ltd.) was added dropwise. Hybridization was performed at 37\u0026deg;C for 12h, and the hybridization solution was washed away. The nucleus was then restained with DAPI, and the antifluorescence quenching sealing agent was added dropwise after washing the sections. The sections were observed under a Nikon positive fluorescence microscope, and the images were captured.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Total RNA extraction, cDNA synthesis, and qRT-PCR\u003c/h2\u003e\u003cp\u003eAfter 48h of transfection, the original medium was discarded, PBS was added to wash the cells, and then 600\u0026micro;L of lysis buffer was added to each well, according to the manufacturer 's instructions, RNA was extracted from the cells by using the SteadyPure RNA extraction kit. (AG21024; Accurate Biotechnology (Hunan) Co., Ltd., China). The RNA was reversely transcribed into cDNA using the Evo M-MLV Mix Kit (AG11728; accurate Biotechnology (Hunan) Co., Ltd.). Gene expression levels were quantified using the SYBR\u0026reg; Green Premix Pro Taq HS qPCR Tracking Kit (AG11733; Accurate Biotechnology (Hunan) Co., Ltd.) on the QuantStudio\u0026reg; 5 system (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). The relative gene expression was measured using the 2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e method[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. The specific primer sequences are listed in Table\u0026nbsp;2.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Protein extraction and western blotting\u003c/h2\u003e\u003cp\u003eThe cellular specimens were disrupted using RIPA buffer (Solarbio, Beijing, China) to facilitate protein extraction. The concentration of the extracted proteins was determined using an Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China). All protein samples were subsequently normalized to a density of 0.5\u0026micro;g/\u0026micro;L, and 3\u0026micro;L of this standardized solution was dispensed into each well. Then, the protein was automatically separated using a western blotting apparatus (ProteinSimple, CA, USA), adhering to the protocol detailed by Harris[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In all protein analyses, GAPDH was used as an internal reference to ensure standardization. The specific information of the antibodies used in this study is detailed in Table\u0026nbsp;3.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Cell apoptosis and proliferation assay\u003c/h2\u003e\u003cp\u003eCellular apoptosis was analyzed using the Annexin V-FITC Apoptosis Detection Kit (Vazyme, China). Apoptosis levels were quantified using a flow cytometer (FACSAria SORP model; Becton Dickinson, USA). Following acquisition, the cytometric data were analyzed using FlowJo V10 software (FlowJo, OH, USA). Meanwhile, cell proliferation was detected using the cell Counting Kit-8 (Vazyme, China). The optical density values of 96 holes at 0, 24, 48, and 72h were measured at 450nm by using Infinite M200 pro (Tecan, Switzerland).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.8 5-Ethynyl-2\u0026prime;-deoxyuridine (EdU) assay\u003c/h2\u003e\u003cp\u003eAfter 48 h of transfection, EdU cell proliferation assay kit (C0075S, Beyotime, Shanghai, China) was used to evaluate cell proliferation ability according to the product instructions. Immediately after the completion of the staining step, the fluorescence microscope was used to observe and take images. The EdU labeling index was calculated as the ratio of the number of EdU-positive nuclei to the total number of nuclei.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Enzyme-linked immunosorbent assay\u003c/h2\u003e\u003cp\u003eEnzyme-linked immunosorbent assay (ELISA) kits for E\u003csub\u003e2\u003c/sub\u003e (Cat.# ml027864) and P (Cat.# ml041624) were obtained from Mlbio (Shanghai, China). According to the manufacturer's instructions, E\u003csub\u003e2\u003c/sub\u003e and P levels in the cell culture supernatant were measured using the ELISA kits.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e\u003cp\u003eStatistical significance was determined by two-tailed Student 's t test or one-way analysis of variance (ANOVA) in SPSS 25.0 statistical software package (SPSS Inc., Chicago, IL, USA). All collected data were then expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SEM). Furthermore, the data were graphically represented using GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA). Results were considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*) and highly significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Cloning and vector construction of rabbit KLF12 gene CDS sequence\u003c/h2\u003e\u003cp\u003eThe CDS sequence of rabbit KLF12 gene was cloned using rabbit ovary-specific primers, with a total length of 1209 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To determine the role of KLF12 in ovarian GCs, a pcDNA3.1-KLF12 vector was constructed and four siRNAs were designed to knock down KLF12 in rabbit GCs. They were transfected into GCs. The qRT-PCR and WB results unveiled that pcDNA3.1-KLF12 significantly increased KLF12 expression, and siRNA-4 had the most effective knockdown efficiency (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Tissue and spatiotemporal expression profiles of KLF12 in female rabbits of different ages\u003c/h2\u003e\u003cp\u003eThe results of HE staining showed that the ovarian structure of 6-month-old female rabbits was relatively dense, with more immature follicle morphology, uniform matrix staining and close cell arrangement. However, the ovarian structure of 18-month-old female rabbits was loose, with obvious mature follicles, relatively sparse follicle distribution and loose cell arrangement (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The KLF12 gene expression levels were quantified across various developmental stages in the heart, liver, spleen, lung, kidney, and ovarian tissues obtained from the female rabbits. The KLF12 gene was expressed in different tissues of the rabbits. KLF12 expression was higher in the 18-month-old rabbits than in the 6-month-old rabbits. The KLF12 expression level was the highest in the ovarian tissues, which was significantly higher than that in the 6-month-old rabbits (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Additionally, the results of fluorescence in situ hybridization unveiled that KLF12 gene expression was relatively higher in the GCs of the 18-month-old rabbits (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In summary, KLF12 gene expression in these tissues increased with age, which indicated that this gene plays a crucial biological function in the ovarian tissues of the female rabbits.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3 KLF12 inhibits E\u003csub\u003e2\u003c/sub\u003e and P levels in GCs\u003c/h2\u003e\u003cp\u003eIn order to further explore the regulatory mechanism of KLF12 on steroid hormone synthesis, the effects of KLF12 on the secretion of E2 and P in rabbit GCs were detected. ELISA results showed that overexpression of KLF12 significantly reduced the secretion levels of E\u003csub\u003e2\u003c/sub\u003e and P in GCs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On the contrary, after knocking down KLF12, the content of E\u003csub\u003e2\u003c/sub\u003e and P increased significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). At the same time, the expression of key genes in the steroid synthesis pathway was detected. KLF12 overexpression significantly inhibited the expression of CYP11A1, CYP19A1 and StAR genes (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, the expression of CYP11A1, CYP19A1 and StAR genes was significantly increased after KLF12 knockdown (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These results indicate that KLF12, as a negative regulator of steroid synthesis, regulates the synthesis of E\u003csub\u003e2\u003c/sub\u003e and P in rabbit GCs by inhibiting the expression of key enzyme genes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4 KLF12 inhibits cell proliferation-related genes\u003c/h2\u003e\u003cp\u003eTo further clarify the role of KLF12 gene in ovarian follicular development. We detected genes related to GCs proliferation and apoptosis. According to the data, KLF12 promoted the mRNA and protein expression of Bax, Caspase-3, Caspase-9, and P53 and inhibited the mRNA and protein expression of BCL-2 and PCNA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B).\u003c/p\u003e\u003cp\u003eAt the same time, we detected the expression of cell cycle key protein molecules. The mRNA and protein levels of the cell cycle regulatory molecules CCNB1, CCNA2, and CDK4 in KLF12-overexpressing GCs were significantly downregulated. By contrast, the CCNB1, CCNA2, and CDK4 e xpression levels in GCs were significantly increased after KLF12 knockdown (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D).\u003c/p\u003e\u003cp\u003eThe above studies further showed that KLF12 negatively regulated the proliferation and apoptosis of rabbit ovarian GCs by inhibiting cell cycle progression, thus playing a key regulatory role in follicular development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.5 KLF12 plays a negative role in GCs proliferation\u003c/h2\u003e\u003cp\u003eThe proliferation of GCs is essential for follicular development. In order to study the regulation of KLF12 on the proliferation and apoptosis of GCs, CCK-8 activity detection and EdU proliferation labeling experiments showed that KLF12 overexpression significantly inhibited the proliferation of GCs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Flow cytometry apoptosis analysis further confirmed that the apoptosis rate of KLF12 overexpression group was significantly increased. On the contrary, KLF12 knockdown significantly promoted GCs proliferation and reduced apoptosis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Taken together, these findings suggest that KLF12 is a negative regulator of rabbit GCs proliferation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.6 KLF12 regulates the PI3K/Akt signaling pathway in GCs\u003c/h2\u003e\u003cp\u003eTo delve deeper into the mechanisms underlying KLF12-regulated GC proliferation and apoptosis, we performed WB to quantify the expression levels of key molecules involved in the PI3K/Akt signaling pathway, namely PI3K, phosphorylated PI3K (p-PI3K), Akt, and phosphorylated Akt (p-Akt). The findings revealed that, the protein levels of the activated forms p-PI3K and p-Akt were notably reduced within the KLF12 overexpression group. Conversely, protein concentrations of these phosphorylated molecules, p-PI3K and p-Akt, were considerably elevated in the KLF12 knockdown group. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). The data imply that KLF12 overexpression has a suppressive effect on the PI3K/Akt signaling pathway, whereas KLF12 reduction or silencing leads to a notable stimulation of this pathway.\u003c/p\u003e\u003cp\u003eTo further prove the activation and inhibition effect of KLF12 on the PI3K/Akt signaling pathway of GCs, 740 Y-P (10\u0026micro;M) and LY294002 (10\u0026micro;M) were used to treat the transfected GCs. According to the findings, upon the introduction of 740 Y-P, p-PI3K and p-Akt protein levels substantially increased within the KLF12-overexpressing group. Conversely, LY294002 addition caused a pronounced decrease in p-PI3K and p-Akt protein concentrations in the cohort with KLF12 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B).\u003c/p\u003e\u003cp\u003eThe aforementioned outcomes provide additional confirmation that KLF12 indeed acts as a crucial modulator in PI3K/Akt signaling pathway activation within GCs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eGCs are the largest somatic cell population in the ovary. They provide material guarantee and a microenvironment for oocyte maturation and follicular development. During follicular growth and development, only a very small number of follicles successfully develop and ovulate, whereas most of the remaining follicles undergo atresia. The key cellular event mediating this physiological process is GC proliferation and apoptosis. We previously found that KLF12, a member of the Kruppel-like factors family, was upregulated during ovarian GC apoptosis[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In this study, the cloning and functional analysis of the KLF12 gene coding sequence of New Zealand female rabbits were completed for the first time. It was found that KLF12 was significantly highly expressed in the ovaries of 18-month-old female rabbits, suggesting its potential role in follicular development.\u003c/p\u003e\u003cp\u003eIn addition, as the main unit of follicles, GCs are the key to ensure animal oocyte maturation and maintain normal hormone levels. The initial stage in P synthesis is the translocation of cholesterol from the exterior to the interior of the mitochondrial membrane. StAR, a pivotal component, governs steroid production. Then, CYP11A1 transforms cholesterol into pregnenolone, and mitochondrial enzymes-mediate the subsequent conversion of pregnenolone into progesterone[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. KLF12 knockdown changes the expression of key genes involved in E\u003csub\u003e2\u003c/sub\u003e and P syntheses and promotes E\u003csub\u003e2\u003c/sub\u003e and P secretion by GCs. This indicates that KLF12 can regulate E\u003csub\u003e2\u003c/sub\u003e and P levels by regulating the key proteins involved in their syntheses.\u003c/p\u003e\u003cp\u003eTo regulate gene transcription, KLFs bind to the GC box or CACCC of the target gene promoter region through a highly conserved carboxyl-terminal DNA domain, thereby affecting cell proliferation, differentiation, and apoptosis[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. KLF9 levels are meticulously controlled to preserve the cellular equilibrium, which encompasses processes such as cell proliferation, extracellular matrix formation, cell migration, and adherence[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. KLF5 is involved in promoting the ovarian cancer stem cell-like phenotype[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. KLF8 promotes the induction of tumorigenic breast stem cells by targeting miR-146a[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. KLF4, KLF9, and KLF13 transcripts are expressed in ovarian cells and regulate these cells[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These collective results indicate that KLFs are promising therapeutic targets in modulating cell proliferation and apoptosis. Intriguingly, our study revealed that heightened KLF12 expression concurrently facilitates apoptosis induction in ovarian GCs. KLF12 gene knockdown and overexpression can regulate the expression of GC proliferation- and apoptosis-related genes Bax, BCL-2, Caspase-9, and Caspase-3, and can affect the expression of cell cycle-related proteins CCNB1, CCNA2, and CDK4. Cell cycle regulation is a highly fine-tuned process, which results in cell growth, genetic material replication, and cell division. The PI3K/Akt signaling pathway can activate Cyclin A, Cyclin B, Cyclin D, and Cyclin E, to promote GC proliferation[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe PI3K/Akt pathway, serving as a significant axis governing cell proliferation and apoptosis, plays a critical role in orchestrating GC growth and programmed cell death throughout follicular maturation[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. PI3K is activated when growth factors and cytokines bind to their cell surface receptors[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. As the main target of proto-oncogenes and PI3K, Akt regulates cell survival and proliferation[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. It oversees cellular proliferation and protein synthesis and counteracts pro-apoptotic entities such as BAD and Caspase-9, which are pivotal elements in executing programmed cell death. Within the scope of this investigation, we observed that KLF12 overexpression notably reduced p-PI3K and p-Akt protein levels. Conversely, when KLF12 was knocked down, p-PI3K and p-Akt protein concentrations were significantly elevated. Subsequently, the transfected GCs were treated with 740 Y-P and LY294002. After 740 Y-P addition, p-PI3K and p-Akt protein levels significantly increased in the KLF12 overexpression group, whereas the protein levels of the phosphorylated molecules p-PI3K and p-Akt significantly decreased after LY294002 addition. This result again proves that KLF12 molecules exert a regulatory effect on the PI3K/Akt signaling pathway.\u003c/p\u003e\u003cp\u003eOur study established a new molecular mechanism. Overexpression of KLF12 inhibited the secretion of E\u003csub\u003e2\u003c/sub\u003e and P in rabbit GCs, and also inhibited the expression of genes related to cell proliferation, apoptosis and cycle development. Mechanism studies further found that KLF12 is a key regulator of the PI3K/Akt signaling pathway. These results demonstrate that KLF12 inhibits the proliferation of GCs and promotes their apoptosis by regulating the PI3K/Akt signaling pathway, which provides a theoretical basis for improving the reproductive performance of rabbits and opens up a new perspective for the study of mammalian reproductive regulatory networks.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, this study completed the cloning and functional analysis of the coding sequence of KLF12 gene in New Zealand female rabbits for the first time. It was found that KLF12 was significantly highly expressed in the ovaries of 18-month-old female rabbits, suggesting its potential role in follicular development. Functional experiments showed that KLF12 overexpression significantly reduced the secretion levels of E\u003csub\u003e2\u003c/sub\u003e and P by inhibiting the transcriptional activity of key enzyme genes for steroidogenesis, revealing its negative feedback effect in the regulation of hormone synthesis. Mechanism studies further confirmed that overexpression of KLF12 inhibited the expression of genes related to cell proliferation, apoptosis and cycle development. In addition, KLF12 was also proved to be a key regulator of the PI3K/Akt signaling pathway in GCs. These results reveal the new function of KLF12 as a negative regulator of follicular development and the molecular mechanism of regulating the development and function of ovarian GCs in New Zealand female rabbits through the PI3K/Akt signaling pathway. The results provide a molecular basis for analyzing the mechanism of ovarian development, and also open up a new perspective for the study of mammalian reproductive regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eThe female rabbits were purchased from Guifeng Rabbit Industry Professional Cooperative of Yangzhou City and obtained the informed consent of the owner. These animals can be used in this study. The animal experiment was approved by the Animal Care and Use Committee of Yangzhou University (Approval No. 202212006).\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eWe certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis research was funded by the China Agriculture Research System of MOF and MARA (CARS-43-A-1).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eJC BZ, YC and XW conceived and designed research. JC and ZB conducted experiments. YL and XH contributed new reagents or analytical tools. All authors read and approved the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe gratefully acknowledge the members of the laboratory for their suggestions and critical reading of the manuscript.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBao Z, Chen Y, Li J, Cai J, Zhao B, Wu X. Characterization and establishment of an immortalized rabbit ovary granulosa cell line for reproductive biology experiments. Vitro Cell Dev Biol Anim. 2024;60:209\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCai J, Li Y, Zhao B, Bao Z, Li J, Sun S, et al. 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Pflug Arch Eur J Phy. 2019;471:1055\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ovarian development, KLF12 gene, granulosa cells proliferation, PI3K/Akt signaling pathway","lastPublishedDoi":"10.21203/rs.3.rs-7203220/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7203220/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGranulosa cells (GCs), as the largest proportion of somatic cells in mammalian ovarian follicles, play a central regulatory role in follicular development and oocyte maturation. In-depth analysis of the molecular mechanisms related to the growth and development and functional regulation of GCs is of great significance for improving the regulatory network mechanism in the field of female reproduction. Kruppel Like Factor 12 (KLF12) gene is involved in the regulation of various cellular physiological activities, but its specific regulatory function in ovarian GCs is not yet clear.\u003c/p\u003e\u003cp\u003eIn this study, the coding sequence (CDS) of KLF12 gene in New Zealand female rabbits was cloned for the first time, and the expression abundance and cellular localization of this gene in ovarian tissues of female rabbits at different reproductive stages were systematically analyzed. The results showed that KLF12 was highly expressed in the ovaries of 18-month-old female rabbits. Functional studies found that overexpression of KLF12 significantly inhibited the synthesis of Estradiol (E\u003csub\u003e2\u003c/sub\u003e) and Progesterone (P) in GCs, and the expression of genes related to cell proliferation, apoptosis and cycle development also changed. Further Western blot (WB) detection confirmed that KLF12 overexpression significantly reduced the phosphorylation levels of key molecules PI3K and Akt in the PI3K/Akt signaling pathway, while KLF12 knockdown had the opposite effect on GCs function. These results reveal the molecular mechanism of KLF12 regulating the development and function of ovarian GCs in New Zealand female rabbits through PI3K/Akt signaling pathway, which not only provides a theoretical basis for improving the reproductive performance of female rabbits, but also provides a new scientific perspective for the study of the molecular mechanism of mammalian reproductive regulation.\u003c/p\u003e","manuscriptTitle":"Kruppel Like Factor 12 gene regulates ovarian granulosa cells proliferation and apoptosis via the PI3K/Akt signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 04:07:05","doi":"10.21203/rs.3.rs-7203220/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-22T04:30:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T10:31:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-18T18:16:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124134182958502583626521650366771104448","date":"2025-10-17T07:35:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31843491149465859126151148440341556344","date":"2025-10-16T06:33:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-02T14:57:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-30T06:37:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2025-08-30T06:34:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"96fe9aa0-5e8a-4035-b600-bed932283553","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:11:56+00:00","versionOfRecord":{"articleIdentity":"rs-7203220","link":"https://doi.org/10.1186/s12917-026-05336-8","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2026-02-05 15:59:33","publishedOnDateReadable":"February 5th, 2026"},"versionCreatedAt":"2025-09-09 04:07:05","video":"","vorDoi":"10.1186/s12917-026-05336-8","vorDoiUrl":"https://doi.org/10.1186/s12917-026-05336-8","workflowStages":[]},"version":"v1","identity":"rs-7203220","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7203220","identity":"rs-7203220","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}