IGF2BP2 regulates the proliferation and migration of endometrial stromal cells through the PI3K/AKT/mTOR signaling pathway in Hu sheep

Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), a significant member of the conserved RNA-binding protein family, plays various roles in numerous physiological and pathological processes. However, the specific function of IGF2BP2 in regulating endometrial function in sheep remains largely unknown. In this study, we observed a significant upregulation in IGF2BP2 mRNA abundance in the endometrium during the luteal phase compared to the follicular phase in Hu sheep. The knockdown of IGF2BP2 resulted in accelerated cell proliferation and migration of Hu sheep endometrial stromal cells (ESCs). Moreover, RNA sequencing analysis revealed that genes with significantly altered expression in IGF2BP2 knockdown cells were predominantly enriched in endometrial receptivity-related signaling pathways, such as cytokine-cytokine receptor interaction, NOD-like receptor, PI3K-AKT, and JAK-STAT signaling pathway. Additionally, the knockdown of IGF2BP2 significantly increased the expression of matrix metalloprotein 9 (MMP9), vascular endothelial growth factor, and prolactin (PRL) in ESCs. The knockdown of IGF2BP2 was also observed to stimulate the PI3K/AKT/mTOR pathway by upregulating integrin β4 (ITGB4) expression. Notably, the downregulation of ITGB4 attenuates IGF2BP2 knockdown-induced facilitation of proliferation and migration of Hu sheep ESCs by inhibiting the PI3K/AKT/mTOR pathway. Collectively, these findings highlight the important role of IGF2BP2 in regulating endometrial function, particularly through the modulation of ESC proliferation and migration via the PI3K/AKT/mTOR pathway.

endometrial stromal cells, endometrial function, Hu sheep, IGF2BP2, PI3K/AKT/mTOR pathway IGF2BP2 was found to be ubiquitously expressed in various reproductive organs, with notably high levels of expression in the endometrium during the luteal phase (LP) in Hu sheep. Moreover, our findings suggest that IGF2BP2 regulates the PI3K/AKT/mTOR signaling pathway by modulating ITGB4 expression, thereby influencing the proliferation and migration of ESCs.

IGF2BP2, also known as IMP2, is a member of the Insulin-like growth factor 2 mRNA binding proteins family. Since its discovery in 1999 (Nielsen et al., 1999), the multifaceted roles of IGF2BP2 in various biological processes have been progressively elucidated. To date, IGF2BP2 has been shown to play vital roles in various biological systems, such as embryonic development, skeletal muscle development, tumorigenesis, and metabolism, by controlling the post-transcriptional life cycle of RNAs (Yaniv and Yisraeli, 2002; Li et al., 2012; Degrauwe et al., 2016; Dai, 2020; Wang et al., 2021). However, the role of IGF2BP2 in female reproduction remains largely unexplored. A recent study on female mice lacking IGF2BP2 revealed that the absence of IGF2BP2 results in zygote arrest at the two-cell stage, thereby reducing the fertility of these mice (Liu et al., 2019). Moreover, a genome-wide association study conducted in goats has identified an insertion/deletion site within the IGF2BP2 gene that is significantly associated with litter size in goats (Xin et al., 2021). These findings suggest that IGF2BP2 may play a role in regulating litter size in mammals. The uterus is a vital reproductive organ in domestic animals and plays a significant role in uterine receptivity, litter size, and neonatal survival (Spencer and Bazer, 2004). It is well known that normal endometrial function is essential for embryo implantation, development, and the maintenance of pregnancy (Owusu-Akyaw et al., 2019). Recent studies have highlighted the importance of N6-methyladenosine (m6A) methylation in the mammalian uterus during early pregnancy (Li et al., 2021c; Sun et al., 2023; Wan et al., 2023; Zheng et al., 2023). Dysregulation of m6A regulators, including methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5), may contribute to the development of endometriosis, ultimately resulting in infertility (Zhao et al., 2021; Kobayashi et al., 2023; Wang et al., 2023b). IGF2BP2, an m6A “reader,” has been shown to promote the progression of ovarian endometriosis by enhancing the mRNA stability of Meis homeobox 2 and GATA binding protein 6 (Zhao et al., 2022). Furthermore, it has been reported that abnormal expression of the IGF2BP2 gene in the oocytes of patients with polycystic ovary syndrome is closely associated with irregular steroid production (Haouzi et al., 2012). Although these studies have confirmed the involvement of IGF2BP2 in the development of endometriosis in females, the role of IGF2BP2 in regulating endometrial function in ruminants, such as sheep, has yet to be elucidated. Hu sheep is a distinctive indigenous breed in China, renowned for its suitability for family raising, tolerance of coarse forage, high prolificacy, and widespread utilization as a maternal lineage in mutton sheep crossbreeding programs (Li et al., 2022). Previous studies on the mechanisms responsible for the high prolificacy of Hu sheep have mainly focused on female reproductive organs, including the ovaries, pituitary gland, and hypothalamus (Zhang et al., 2019; Zheng et al., 2019; Yao et al., 2021), with limited consideration of the endometrial function. Recently, our research team has constructed the expression profiles of mRNAs and non-coding RNAs in the endometrium of high- and low-fertility Hu sheep during both the FP and luteal phase (LP) stages using RNA-seq techniques (Gao et al., 2020; Li et al., 2023, 2024). Interestingly, IGF2BP2 exhibited differential expression in the endometrium during the FP and LP stages in Hu sheep. It is well-established that the endometrium in mammals undergo a series of morphological and functional changes during the transition from the FP to the LP (Lessey and Young, 2019). Moreover, the intricate processes of proliferation, migration, and apoptosis in Endometrial stromal cells (ESCs), the primary cellular constituents of the endometrium, are essential for maintaining the normal physiological functions of the endometrium (Owusu-Akyaw et al., 2019; Cui et al., 2021). Thus, it is necessary to explore whether IGF2BP2 influences endometrial function by modulating the proliferation, migration, and apoptosis of ESCs in Hu sheep. In this study, we elucidate the expression pattern of IGF2BP2 in female sheep reproductive organs and investigate its role and underlying mechanisms in the function of sheep ESCs. We observed a significant increase in the expression of IGF2BP2 in the endometrium during the LP compared to the FP in Hu sheep. Furthermore, the knockdown of IGF2BP2 was found to promote the proliferation and migration of ESCs by activating the PI3K/AKT/mTOR pathway. Our results further revealed that IGF2BP2 regulates the activity of the PI3K/AKT pathway by modulating the expression of ITGB4, an upstream activator of this pathway. Taken together, this study is the first to demonstrate that IGF2BP2 regulates the proliferation and migration of Hu sheep ESCs by modulating the PI3K/AKT/mTOR pathway through ITGB4.

Animal samples All animal studies were conducted in accordance with the guidelines established by the Ethics Committee of Nanjing Agricultural University, China (SYXK2022-0031). The sheep tissue samples used in this experiment were obtained from our previous experiments (Gao et al., 2020). Briefly, sixteen pluriparous ewes with FecBBB genotypes and eight pluriparous ewes with FecBB+ genotypes were selected from Xilaiyuan sheep breeding farm, in Jiangsu, China. Based on their litter size record, all ewes were evenly divided into three groups: HBB (high prolificacy, each record of litter size = 3), LBB (low prolificacy, each record of litter size = 1), and LB + (low prolificacy, each record of litter size = 1). Ewes from each group were slaughtered at 12 h (FP) and day 9 (LP) post-estrus, respectively. Endometrium samples were collected from one side of the uterine horn. Furthermore, samples from the hypothalamus, pituitary, uterus, oviduct, and ovary were obtained from ewes in each group and preserved at − 80 °C for subsequent gene expression analysis. Cell culture ESCs were isolated from the fresh uterus of nonpregnant ewes using the methods described in our previous study (Li et al., 2021d). After purification via trypsin differential digestion, the ESCs were identified using immunofluorescence staining with anti-vimentin antibody (Johnson et al., 2003). The ESCs were cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, MA, USA) with 10% FBS (GIBCO, NY, USA), and 1% penicillin/streptomycin at 37 °C and 5% CO2. Plasmid construction, siRNA, and cell transfection The coding sequence (CDS) of ovine IGF2BP2 was synthesized and subsequently cloned into the PEX3 plasmid to generate an overexpression plasmid (oeIGF2BP2). The PEX3 plasmid (Pex3) served as a vector control for subsequent analyses. All siRNA sequences were designed and synthesized by Genepharma (Shanghai, China). ESCs were cultivated to approximately 50% confluence in growth media before transfection with siRNAs, vector control, or the plasmid construct using Lipofectamine 3,000 transfection reagent (Thermo Fisher Scientific) following the manufacturer’s guidelines. The sequences of all siRNAs are provided in Supplementary Table S1. Immunohistochemistry and Immunofluorescence Immunohistochemistry Assay was performed as previously described (Li et al., 2021d). Briefly, tissue samples from the pituitary, ovary, oviduct, and uterus were initially embedded in paraffin, sectioned, deparaffinized, and subjected to antigen retrieval. Subsequently, the sections were blocked with 5% bovine serum albumin for a duration of 30 min and then incubated overnight at 4 °C with anti-IGF2BP2 antibody (1:800 dilution; Abclonal, A2749) or phosphate-buffered saline (PBS; Negative controls). Following three washes with PBS, the sections were incubated with the corresponding secondary antibody at room temperature for 1 h. The slides were developed with 3-3ʹ-diaminobenzidine (Product No. P0203; Beyotime, Shanghai, China), counterstained with hematoxylin, and sealed with neutral resin. Image analysis of the Immunohistochemistry was analyzed using a light microscope (Nikon, Tokyo, Japan). The immunofluorescence Assay was conducted following established procedures as detailed in prior research (Deng et al., 2021). Initially, cells were fixed, permeabilized, and blocked, and incubated before incubating overnight at 4 °C with primary antibodies, including anti-vimentin antibody (1:800 dilution; Proteintech, A2749), anti-IGF2BP2 antibody (1:800 dilution; Abclonal, A2749) or PBS (Negative controls). Subsequently, the cells underwent a 1 h incubation with the secondary antibody at room temperature, followed by incubation with 4ʹ,6ʹ-diamidino-2-phenylindole (DAPI). The images from the immunofluorescence were analyzed using a fluorescence microscope (Zeiss LSM710 META, Jena, Germany). RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) The total RNA was extracted from the tissues or cells using Trizol reagent (Thermo Fisher Scientific) following the manufacturer’s instruction. Subsequently, the isolated RNA was reverse transcribed into cDNA using reverse transcription reagent kits (Vazyme, Nanjing, China), according to the manufacturer’s instructions. qRT-PCR was performed with the qPCR SYBR Master Mix (Vazyme) and the QuantStudio 5 Real-Time PCR System (Applied Biosystems, Bedford, MA, USA) according to the manufacturer’s protocol. The fold changes in gene expression were quantified using the 2−ΔΔCt method, with ACTB serving as an endogenous control. Details of all primers used are listed in Supplementary Table S2. Western blot analysis Total proteins were extracted from the endometrium or cells using RIPA buffer (Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (Beyotime). Subsequently, the extracted protein was separated using 12% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore, MA, USA). The membranes were blocked for 1 h at room temperature with 5% skim milk and incubated overnight at 4 °C with the respective primary antibodies. After washing with Tris-buffered saline containing Tween-20, the membranes were incubated with appropriate secondary antibodies at room temperature for 1 h. Immunoblots were visualized using the Western Bright ECL kit (Biosharp, Hefei, China), and band intensities were quantified using ImageJ software (NIH, MD, USA). The primary antibodies used in this study are listed in Supplementary Table S3. Cell proliferation assay Cell proliferation was evaluated using the Alexa Fluor 647-ClickiT EdU Assay Kit (Keygen Biotech, Nanjing, China) according to the manufacturer’s instructions. Briefly, cells were treated with 50 mM EdU at 24 h post-transfection and incubated for 2 h at 37 °C. Subsequently, the cells were fixed, permeabilized, and stained with Apollo Dye Solution, followed by an incubation with DAPI. Images were captured using a confocal laser scanning microscope (Zeiss LSM710 META), and q quantitative analysis was performed using ImageJ software (NIH). Cell wound scratch assay ESCs were seeded at a density of 1 × 105 cells/mL in a 12-well plate. Upon reaching confluence, a straight-line scratch was generated in the cell monolayer using a 200 μL pipette tip. After the scratch induction, the cells were cultured in serum-free media. Images of the scratch were captured at 0 and 72 h using a microscope (Nikon) and subsequently quantitatively analyzed using ImageJ software (NIH). Flow cytometry for cell cycle and apoptosis assay For cell cycle analysis, cells were fixed in 70% cold ethanol at—20 °C overnight, and then washed with PBS. After incubation with propidium iodide/RNase A solution (Keygen Biotech) for 30 min, the cells were then analyzed using a flow cytometer (Beckman Coulter, Brea, CA, USA) to identify the different stages of the cell cycle. For cell apoptosis analysis, cells were harvested, washed in PBS, and then resuspended in ANXA5 binding buffer. Subsequently, the cells were incubated with a mixture containing 5 μL of ANXA5-FITC (ThermoFisher Scientific) and 10 μL of Propidium Iodide (50 μg/mL, Sigma-Aldrich, MO, USA) for 15 min at room temperature. Finally, the cells were analyzed by flow cytometer (Beckman Coulter), and the apoptosis rate was quantified using FlowJo software (BD Life Sciences, CA USA). Detection of CASP3 activity The CASP3 activity was assessed using a Caspase3 Activity kit (Beyotime) according to the manufacturer’s instructions. Briefly, ESCs were lysed with cold lysis buffer and then incubated with Ac-LEVD-pNA for 2 h at 37 °C. Enzyme activities were quantified colorimetrically by measuring absorbance at 405 nm and expressed as the fold change in enzyme activity. RNA-seq and bioinformatics Total RNAs were extracted from ESCs transfected with siRNA control (siCtrl) or IGF2BP2-siRNA (siIGF2BP2) using TRIzol reagent (Thermo Fisher Scientific). After assessing the quality and integrity of the RNA samples, cDNA was generated using the VAHTS® Universal V8 RNA-seq Library Prep Kit for Illumina according to the manufacturer’s instructions. For mRNA-seq, sequencing was performed on an Illumina Novaseq™ 6,000 (AZENTA LIFE SCIENCES., Ltd., Suzhou China). Clean reads were mapped to the reference Ovis aries genome sequence (Oar_v4.0) assembly using software Hisat2 (v2.2.1) with default parameters. The read counts were calculated using fragments per kilobase of exon model per million mapped fragments (FPKM). Differential expression analysis used the DESeq2 Bioconductor package, a model based on the negative binomial distribution. Genes with an absolute value of fold change > 1.5 and P value < 0.05 were deemed as differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed for functional enrichment analysis of the DEGs. The protein-protein interaction network of DEGs was constructed using the STRING online database (http://string-db.org). Cystoscope was used to identify functional modules in the interaction network, with a minimum required interaction score set at 0.4. mRNA stability analysis ESCs were transfected with the corresponding siRNA for 24 h, and then treated with 5 μg/mL actinomycin D (MCE; HY-17559, NJ, USA). The cells were collected at 0, 3, and 6 h time points for the evaluation of ITGB4 mRNA degradation using qRT-PCR. Statistical analysis All experiments were performed at least in triplicates, and the data are expressed as mean ± SEM. Data were confirmed to be normally distributed using the Kolmogorov-Smirnov goodness-of-fit test. Statistical analysis was performed using SPSS 24.0, using a two-tailed Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc analysis. For all the analyses, P < 0.05 was considered significant.

Immunohistochemistry analysis of IGF2BP2 in reproductive tissues of Hu sheep To detect the localization of IGF2BP2 protein in the reproductive tissues of Hu sheep, we performed an immunohistochemistry analysis. The IGF2BP2 protein was found to be widely distributed in various reproductive tissues of Hu sheep, including the hypophysis, pituitary, ovary, uterus, and oviduct tissues (Figure 1A). Notably, the expression of IGF2BP2 mRNA was higher in the hypophysis and uterus compared to the pituitary and ovary tissues (Figure 1B). Within the uterus tissue, the expression of IGF2BP2 was observed in various cellular components, including ESCs and endometrial epithelial cells (Figure 1A). To investigate the distribution of IGF2BP2 in ESCs, we isolated these cells from the uterus of Hu sheep. Immunofluorescence staining results showed a positive reaction for ESCs maker protein, vimentin, in the purified cells, thus confirming that most of the isolated cells are ESCs (Figure 1C). Moreover, IGF2BP2 was found to be primarily localized in the cytoplasm of ESCs (Figure 1C). This distribution is consistent with the known function of IGF2BP2 in regulating mRNA fate within the cytoplasm (Bell et al., 2013). IGF2BP2 expression level in endometrium of Hu sheep during follicular and luteal phase Subsequently, we analyzed the RNA-seq data from previous studies on endometrium tissues of Hu sheep with high- (HBB) and low- (LBB and LB+) fertility during the FP and LP stages. Our analysis revealed no significant difference in the mRNA abundance of IGF2BP2 in the endometrium tissues of Hu sheep with high- and low-fertility during both FP and LP stages (Figure 2A and B). However, the mRNA abundance of IGF2BP2 in the endometrium of both high- and low-fertility Hu sheep was significantly increased during the LP compared to the FP (Figure 2C). The qRT-PCR analysis further confirmed that the expression of IGF2BP2 in the endometrium was higher during the LP compared to the FP (Figure 2D). These results indicate that IGF2BP2 may be involved in the regulation of endometrial function in Hu sheep. IGF2BP2 affects the proliferation, migration, and apoptosis of Hu sheep ESCs To investigate the role of IGF2BP2 in regulating endometrial function in sheep, we initially cloned the coding sequence (CDS) of IGF2BP2 from ovine endometrium cDNA and subsequently conducted sequence analysis. The result showed that the IGF2BP2 CDS sequence was 1,800 bp and encoded a protein containing 599 amino acids (Figure 3A). Phylogenetic analyses revealed high homology of the IGF2BP2 gene with species such as Capra hircus, Bos taurus, Sus scrofa, and Mus musculus (Figure 3B). We then investigate the effect of IGF2BP2 expression level on the proliferation, migration, and apoptosis of Hu sheep ESCs. The knockdown of IGF2BP2 using siRNA significantly increased the rate of cell proliferation (Figure 4A). Moreover, flow cytometry analysis showed that IGF2BP2 knockdown significantly reduced the number of cells in the G1 phase, accompanied by an increase in number of cells in the S and G2 phases (Figure 4B). Consistently, the protein abundance and the mRNA levels of proliferating cell nuclear antigen (PCNA), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 6 (CDK6), and cyclin D1 (CCND1) were significantly increased upon IGF2BP2 depletion (Figure 4C and D). Meanwhile, the cell wound scratch assay showed that the knockdown of IGF2BP2 increased the migratory capacity of ESCs (Figure 4E). However, IGF2BP2 overexpression did not affect the proliferation, cell cycle, or migration of Hu sheep ESCs (Figure 5A–E). Subsequent analyses of cell apoptosis revealed that the knockdown of IGF2BP2 did not significantly affect cell apoptosis levels (Figure 6A). Consistently, the knockdown of IGF2BP2 did not notably alter the expression of pro-survival protein B-cell Lymphoma-2 (BCL2) and BCL-2-associated X (BAX), CASP3, and CASP9, or the ratio of BAX to BCL2 (Figure 6B and C). To further assess the influence of IGF2BP2 deficiency on cell apoptosis, the activity of CASP3 in ESCs was examined. The results revealed no significant difference in CASP3 activity between IGF2BP2-depleted cells and control cells (Figure 6D). In contrast, the overexpression of IGF2BP2 resulted in an obvious increase in cell apoptosis levels (Figure 6E). Moreover, the forced expression of IGF2BP2 significantly increased the expression of BAX, CASP3, and CASP9, as well as the ratio of BAX to BCL2 (Figure 6F and G). Additionally, CASP3 activity was found to be increased in IGF2BP2-overexpressing cells (Figure 6H). Taken together, these results suggest that IGF2BP2 plays an important role in regulating the proliferation and migration of Hu sheep ESCs. IGF2BP2 knockdown enhances the activity of the PI3K/AKT/mTOR pathway through the upregulation of ITGB4 expression To investigate the potential mechanisms by which IGF2BP2 influences endometrial function, RNA-seq was conducted to analyze the gene expression profile in ESCs with IGF2B2P2 knockdown. Based on the criteria of |fold change| ≥ 1.5 and P-value ≤ 0.05, a total of 184 DEGs were identified between the siIGF2BP2 and siCtrl group (Figure 7A). Of these, 90 genes were significantly upregulated, and 94 genes were significantly downregulated in IGF2BP2-depleted cells compared to control cells (Figure 7A and B, Supplementary Table S4). GO enrichment analysis showed that these DEGs were primarily involved in cytokine activity, cytokine binding, protein binding involved in heterotypic cell-cell adhesin, and immune response (Figure 7C). KEGG pathway analysis of the DEGs revealed significant enrichment of signaling pathways related to endometrial receptivity, including cytokine-cytokine receptor interaction, NOD-like receptor, PI3K-AKT, and JAK-STAT signaling pathways (Figure 7D). Notably, several marker genes of endometrial receptivity, including MMP9 (Cheng et al., 2022), vascular endothelial growth factor (Kaufmann et al., 2004), and PRL (Zhang et al., 2015), were observed to be upregulated in IGF2BP2 knockdown cells (Figure 7E). These findings suggest a potential role of IGF2BP2 in regulating endometrial function via these signaling pathways. Subsequently, the protein interactome of these DEGs was further analyzed to elucidate protein-protein interactions using the STRING database. (Figure 7F). Notably, ITGB4, recognized as a key upstream activator of the PI3K-AKT signaling pathway (Li et al., 2021b; Liu et al., 2022), exhibited a significant upregulation in IGF2BP2-depleted cells (Figure 7F). qRT-PCR and Western blot analyses further confirmed that IGF2BP2 knockdown significantly increased ITGB4 expression (Figure 8A-C). However, IGF2BP2 knockdown did not influence the mRNA stability of ITGB4 (Figure 8D). Additionally, METTL3 knockdown did not influence ITGB4 expression (Figure 8E and F), suggesting that the regulation of IGTB4 expression by IGF2BP2 is likely independent of m6A-mediated post-transcriptional regulation. Furthermore, IGF2BP2 knockdown was observed to activate the PI3K-AKT-mTOR pathway, as evidenced by changes in the phosphorylation levels of PI3K, AKT, and mTOR proteins in ESCs (Figure 8G and H). Taken together, these findings suggest that IGF2BP2 may regulate the PI3K/AKT/mTOR pathway by modulating ITGB4 expression in Hu sheep ESCs. ITGB4 knockdown attenuates the IGF2BP2 knockdown-induced facilitation of proliferation and migration of Hu sheep ESCs by inhibiting the PI3K/AKT/mTOR pathway Considering the critical role of the PI3K/AKT/mTOR pathway in cell growth and proliferation, and the regulatory effect of IGF2BP2 on ITGB4 expression, we investigated whether ITGB4 is involved in IGF2BP2-mediated proliferation and migration of Hu sheep ESCs. Our results showed that ITGB4 knockdown effectively counteracted the upregulation of ITGB4 expression induced by IGF2BP2 knockdown (Figure 9A and B). This was accompanied by a reduction in the phosphorylation levels of PI3K, AKT, and mTOR proteins in the IGF2BP2-depleted cells (Figure 9A and B). Moreover, upon ITGB4 knockdown, the growth rate of IGF2BP2-depleted ESCs was restored to levels comparable to those of control cells (Figure 9C). Consistent with these observations, ITGB4 knockdown also attenuated the upregulation of PCNA and CCND1 in IGF2BP2-depleted cells (Figure 9D and E). Furthermore, ITGB4 knockdown similarly alleviated the enhancement of cell migration in IGF2BP2-depleted cells (Figure 9F). Collectively, our findings suggest that IGF2BP2 modulates the proliferation and migration of ESCs by regulating the PI3K/AKT/mTOR pathway through ITGB4.

Embryonic implantation is the process of establishing an interaction between the implantable blastocyst and the recipient uterus, which is regulated by a multitude of genes at the maternal-fetal interface (Wang and Dey, 2006). The endometrium, serving as the primary site for implantation, plays a vital role in maintaining its normal physiological functions to ensure successful embryonic implantation (Aghajanova et al., 2008; Oghbaei et al., 2022). The function of the endometrium is governed by complex transcriptional regulatory networks, involving a diverse array of genes and epigenetic factors (Retis-Resendiz et al., 2021). Recently, several studies have demonstrated that m6A modification in the uterus plays a crucial role in embryonic implantation (Xue et al., 2021; Shen et al., 2023). However, the role of IGF2BP2, a m6A “reader” protein, in modulating endometrial function has not been reported. In this study, we observed that IGF2BP2 was ubiquitously expressed in various reproductive organs, with notably high expression in the endometrium during the LP in Hu sheep. Furthermore, our findings demonstrated that IGF2BP2 regulates the PI3K/AKT/mTOR pathway by modulating ITGB4 expression, thereby influencing the proliferation and migration of ESCs. IGF2BP2, a member of the conserved RNA-binding protein family, has been implicated in various biological processes through its role in RNA processing. Unlike other family members such as IGF2BP1 and IGF2BP3, which exhibit tissue-specific expression patterns, IGF2BP2 is expressed not only in the gonads but also in a wide variety of tissues (Hammer et al., 2005; Christiansen et al., 2009; Bell et al., 2013). Moreover, IGF2BP2 has been found to be highly expressed in various types of reproductive tract-related cancers, such as endometrial carcinoma and ovarian cancer (Zhang et al., 2011; Liu et al., 2014; Dai et al., 2017). However, the potential role of IGF2BP2 in mammalian productive systems remains to be elucidated. A recent genome-wide selection signature analysis of Dazu black goats identified IGF2BP2 as a candidate gene associated with litter size in goats (E et al., 2019). Furthermore, it was discovered that the mRNA level of IGF2BP2 in the ovaries of single-lamb group goats was significantly higher than that of multi-lamb group goats (Xin et al., 2021). Notably, the deficiency of IGF2BP2 resulted from results in impaired primordial germ cell development in zebrafish (Li et al., 2021a). In our study, we observed that IGF2BP2 was ubiquitously expressed in the reproductive tissues of Hu sheep, particularly in the uterus. Furthermore, the expression of IGF2BP2 was notably higher in endometrial tissue during the LP than in endometrial tissue during the FP, indicating a potential role for IGF2BP2 in the regulation of endometrial remodeling. Endometrial receptivity is a critical determinant of fertility in mammals. Abnormalities in the structure and function of the endometrium significantly contribute to infertility (Munro, 2019). It is well-established that the architecture and functionality of the endometrium are influenced by various factors, including the cellular activity of ESCs. In this study, we observed that the knockdown of IGF2BP2 stimulated the proliferation and migration of ESCs in Hu sheep, aligning with previous research indicating that IGF2BP2 acts as a negative regulator of osteoblast proliferation (Zhou et al., 2023). Cell proliferation is a consequence of cell cycle progression, which is tightly controlled by specific cyclins and cyclin-dependent kinase complexes, such as CDK2, CDK6, CCND1, and CCND2 (Bertoli et al., 2013). Previous studies have reported that the silencing of IGF2BP2 enhances cell viability and promotes cell cycle progression by regulating the expression of cyclin-dependent kinase genes in human bronchial epithelial cells (Xiao et al., 2022). Similarly, an upregulation in the expression of CDK2, CDK6, and CCND1 was observed in IGF2BP2 knockout cells, which may elucidate the underlying mechanism of accelerated cell proliferation in the absence of IGF2BP2. A recent study reported significant transcriptomic changes in human ESCs during embryo invasion, suggesting a potential alteration in ESCs functions during this crucial phase (Han et al., 2021). However, abnormalities in ESCs have the potential to precipitate pregnancy failure (Qiu et al., 2023). In the present study, we found that IGF2BP2 deficiency resulted in the differential expression of 184 genes in ESCs. Notably, most of these DEGs were associated with immune response and cytokine receptor interactions, both of which play important roles in the process of embryo implantation (Lin et al., 2015; Moreno-Moya et al., 2016). Furthermore, it was observed that the knockdown of IGF2BP2 upregulates specific marker genes of endometrial receptivity, such as MMP9, vascular endothelial growth factor, and PRL (Massimiani et al., 2020). These findings suggest that IGF2BP2 may influence the endometrial receptivity by modulating the cellar function of ESCs. Among these DEGs, the ITGB4 gene aroused our attention due to its important role in the transduction of the PI3K/AKT signaling pathway, which is involved in the development of endometrial receptivity (Li et al., 2021b; Liu et al., 2022; Wang et al., 2023a). Previous studies have reported that the inhibition of PI3K and its downstream signaling pathways leads to a decrease in the proliferation, migration, and invasion capabilities of ESCs (Zhou et al., 2021). In this study, it was observed that the knockdown of IGF2BP2 resulted in an upregulation of ITGB4 expression, thereby enhancing the activity of the PI3K/AKT/mTOR pathway in ESCs. Interestingly, the downregulation of ITGB4 attenuates the IGF2BP2 knockdown-induced facilitation of proliferation and migration of Hu sheep ESCs, presumably through the inhibition of the PI3K/AKT/mTOR pathway. This suggests that IGF2BP2 may regulate the proliferation and migration of ESCs via the ITGB4-PI3K/AKT/mTOR pathway. Furthermore, we observed that the knockdown of IGF2BP2 did not significantly impact the ITGB4 mRNA stability, and Mettl3 knockdown did not induce obvious changes in ITGB4 expression, suggesting that the regulation of expression ITGB4 by IGF2BP2 may not rely on m6A-mediated post-transcriptional regulation. Considering that IGF2BP2 acts as both an m6A recognition protein and an RNA-binding protein involved in the localization, stability, and translation of specific RNAs (Dai et al., 2011, 2017), it is plausible that IGF2BP2 indirectly modulates ITGB4 expression by regulating various types of RNAs, including microRNAs, messenger RNAs, and long non-coding RNAs (Yang et al., 2022; Yu et al., 2023). Although these findings have demonstrated the regulatory impact of IGF2BP2 on the proliferation and migration of ESCs, the mechanisms by which IGF2BP2 modulates ITGB4 expression, and the specific m6A-modified genes involved in the IGF2BP2-mediated regulation of endometrial function are still unclear. More studies will be needed to address and clarify these unresolved issues to enhance our understanding of the function of IGF2BP2 in endometrial function.

In conclusion, our findings demonstrate that IGF2BP2 modulates the PI3K/AKT/mTOR pathway through the regulation of ITGB4 expression, thereby influencing the proliferation and migration of ESCs. These findings highlight the important role of IGF2BP2 in the regulation of cellular activity of ESCs, providing novel insights into the molecular mechanisms responsible for maintaining endometrial function in Hu sheep. Supplementary Material Acknowledgments We appreciate Mingtian Deng and Zhifei Liu for their general assistance. We also thank the High-Performance Computing Platform of the Bioinformatics Center, Nanjing Agricultural University, for their data analysis support. This study was supported by the National Natural Science Foundation of China (32072728; 32261143734), and Fundamental Research Funds for the Central Universities (KYQN2023004). Glossary Abbreviations - ALKBH5 alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 - BAX BCL-2-associated X - CCND1 cyclin D1 - BCL2 pro-survival protein B-cell Lymphoma-2 - CDK2 cyclin-dependent kinase 2 - CDK6 cyclin-dependent kinase 6 - CDS coding sequence - DAPI 4ʹ,6ʹ-diamidino-2-phenylindole - DEG differentially expressed gene - ESC endometrial stromal cell - FP follicular phase - GO Gene Ontology - IGF2BP2 Insulin-like growth factor 2 mRNA binding protein 2 - ITGB4 integrin β4 - KEGG Kyoto Encyclopedia of Genes and Genomes - LP luteal phase - m6A N6-methyladenosine - METTL3 methyltransferase-like 3 - METTL14 methyltransferase-like 14 - MMP9 matrix metalloprotein 9 - PBS phosphate-buffered saline - PCNA proliferating cell nuclear antigen - PRL prolactin - qRT-PCR quantitative real-time polymerase chain reaction - VEGF vascular endothelial growth factor Contributor Information Kaiping Deng, Hu Sheep Academy, Nanjing Agricultural University, Nanjing, 210095, China; Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, 210095, China. Xiaodan Li, Hu Sheep Academy, Nanjing Agricultural University, Nanjing, 210095, China; Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, 210095, China. Zhipeng Liu, Sanya Institute of Nanjing Agricultural University, Nanjing Agricultural University, Nanjing, 210095, China. Yalong Su, Hu Sheep Academy, Nanjing Agricultural University, Nanjing, 210095, China; Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, 210095, China. Xuan Sun, Hu Sheep Academy, Nanjing Agricultural University, Nanjing, 210095, China; Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, 210095, China. Wurilege Wei, Hu Sheep Academy, Nanjing Agricultural University, Nanjing, 210095, China; Inner Mongolia Academy of Agricultural & Animal Husbandry Sciences, Hohhot, 010000, China . Yixuan Fan, Hu Sheep Academy, Nanjing Agricultural University, Nanjing, 210095, China; Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, 210095, China. Yanli Zhang, Hu Sheep Academy, Nanjing Agricultural University, Nanjing, 210095, China; Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, 210095, China. Feng Wang, Hu Sheep Academy, Nanjing Agricultural University, Nanjing, 210095, China; Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, 210095, China. Conflict of interest statement No potential conflict of interest was reported by the author(s). Author contributions Feng Wang and Kaiping Deng were responsible for designing the experimental protocol and acquiring funds. Kaiping Deng and Xiaodan Li were responsible for performing the experiments and writing the manuscript. 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