Functional Characterization of IGF2BP1, CDC25A, and RXFP2 Genes: Implications for Ovarian Function and Reproductive Regulation in Goats

TL;DR

This study identifies three genes that are crucial for goat reproduction, showing they support cell growth and egg development, which could help improve goat farming productivity.

Contribution

The study functionally characterizes IGF2BP1, CDC25A, and RXFP2 as key regulators of granulosa cell dynamics in goats.

Findings

Overexpression of IGF2BP1 promotes granulosa cell proliferation and suppresses apoptosis.

CDC25A enhances granulosa cell proliferation, while its knockdown increases apoptosis.

RXFP2 supports follicular development, as its overexpression enhances proliferation and its knockdown impairs it.

Abstract

Goats that consistently produce twins are more valuable than those bearing single offspring, so understanding the biological basis of this difference is important for agriculture. The research focused on genes active in ovarian tissues, specifically within granulosa cells—specialized cells that surround and support developing eggs. By investigating the genes IGF2BP1, CDC25A, and RXFP2, the team found that increasing their activity promoted the proliferation and survival of granulosa cells, which are essential for follicle development. Conversely, suppressing these genes inhibited cell growth and triggered cell death. These results demonstrate that all three genes play vital roles in maintaining ovarian cell health and supporting egg development, providing insights into why some goats are more prolific. In the long term, this knowledge could inform selective breeding programs, enabling farmers to choose goats with favorable genetic traits for larger, healthier litters. This would improve the productivity and sustainability of goat farming, benefiting food production and rural livelihoods.

Goat reproductive performance is a key determinant of the productivity and economic value of goat farming, especially in meat and milk production. In a previous study, to investigate the genetic basis of prolificacy, we divided goats into groups according to their consistent reproductive performance (producing either single kids or twins) over five consecutive kidding cycles, and performed whole-genome resequencing and RNA-seq analysis on their ovarian tissues. Through integrated analysis, we identified three candidate genes—IGF2BP1 (insulin-like growth factor 2 mRNA-binding protein 1), CDC25A (cell division cycle 25A), and RXFP2 (relaxin family peptide receptor 2)—as potential key regulators of reproductive capacity. Using goat ovarian granulosa cells, we systematically assessed the impact of each gene through gain- and loss-of-function experiments. Overexpression of IGF2BP1 promoted cell proliferation and suppressed apoptosis, underscoring its role in maintaining cellular viability. Conversely, its knockdown significantly impeded growth and induced cell death. Similarly, CDC25A enhanced granulosa cell proliferation, whereas its knockdown led to marked growth impairment and increased apoptosis. Proliferation was also enhanced by RXFP2 overexpression but impaired upon its knockdown, suggesting that RXFP2 is functionally important for follicular development. Collectively, these findings establish IGF2BP1, CDC25A, and RXFP2 as fundamental regulators of granulosa cell dynamics and ovarian follicular development, providing crucial functional insights and promising targets for genetic selection to enhance reproductive efficiency in goats.

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1. Introduction

Reproductive performance is a critical determinant of productivity and economic sustainability in goat farming, particularly for meat and milk production [1]. Enhancing fertility traits is therefore a paramount objective in livestock breeding programs [2]. Among these, a comprehensive understanding of the functional mechanisms of genes is crucial for fully exploiting their potential to enhance reproductive efficiency. Therefore, investigating the regulatory functions and mechanisms of fertility-related genes can provide more targeted and effective breeding strategies to improve goat fertility. According to the Chinese Livestock and Poultry Genetic Resources Annals (Goat Volume, ISBN: 978-7-109-15881-8), Shaanbei White Cashmere Goats (SWCGs) were classified as a low-prolificacy breed during initial registration, exhibiting a kidding rate of only 105.8%. However, through decades of artificial selection and population expansion (current herd size >10 million), distinct subpopulations of multiparous does have emerged within the breed, characterized by either single-kid or multiple-kid litters per parity. This phenotypic divergence likely stems from domestication and production-oriented breeding practices that have profoundly reshaped genomic variation patterns [3]. Consequently, SWCGs represent an ideal model population for elucidating the genetic basis of reproductive trait variation—specifically, identifying candidate genes and developing high-fertility breeding lines. Integrated genomic and transcriptomic analyses of ovarian tissues from SWCGs with divergent, stable litter sizes identified three strong candidate genes: CDC25A (cell division cycle 25A), IGF2BP1 (insulin-like growth factor 2 mRNA binding protein 1), and RXFP2 (relaxin family peptide receptor 2) [3,4].

The known functions of these genes in other species underscore their potential role in ovarian biology. CDC25A is an unstable protein phosphatase during the transition from the G1 phase to mitosis. It primarily functions in the G1-to-S phase transition, where it inhibits apoptosis by binding to ASK1 and interrupting ASK1 oligomer formation [5]. Additionally, CDC25A has been shown to promote the transition of oocytes from G2 to M phases, thus playing a crucial role in reproductive traits [6]. IGF2BP1, a binding protein for IGF2 containing six RNA-binding domains, is considered an m6A-binding protein and has been confirmed as an important regulatory factor for early embryonic growth and development. Much of the current research focuses on its role in cancer progression [7]. Moreover, studies in livestock have demonstrated that IGF2BP1 is associated with growth and reproductive traits in species such as pigs [8], sheep [9], and goats [10]. RXFP2 is a G-protein-coupled receptor for INSL3, which has long been recognized as a regulator of testicular descent in males [11]. In females, INSL3 exerts its effects in a paracrine or autocrine manner via RXFP2, coordinating the production of the major steroid precursor androstenedione and its conversion into estrogen in granulosa cells. Additionally, in ruminants such as cattle and sheep, research on RXFP2 has largely focused on its role in horn formation [12].

Based on this evidence, we hypothesize that in goat ovaries, IGF2BP1, CDC25A, and RXFP2 regulate follicular development by modulating granulosa cell proliferation, apoptosis, and cell cycle progression. To test this hypothesis, this study aims to functionally characterize these three genes using goat ovarian granulosa cells. Through gain-of-function and loss-of-function experiments, we will systematically investigate their specific roles in regulating cell viability, proliferation, and apoptotic processes, thereby providing mechanistic insights and validating their potential as targets for marker-assisted selection to enhance reproductive efficiency in goats.

2. Materials and Methods

2.1. Tissue Sample Collection and Total RNA Extraction

Tissue samples from 12 adult SWCGs, including heart, liver, lung, kidney, skeletal muscle, and ovary, were collected for qRT-PCR analysis (based on five consecutive kidding records, the does were categorized into two groups: a single-kid (SK) group (n = 6, consistently bearing one kid per parity) and a multi-kid (MK) group (n = 6, consistently bearing twins per parity)). All animals were raised under the same standard management, feeding, and environmental conditions at the Shaanxi Cashmere Goat Engineering Research Center to minimize non-genetic variation (Yulin, Shaanxi, China) and were stored at −80 °C. Total RNA was extracted using the Trizol^®^ method (Cat: 15596018CN, Solarbio, Beijing, China), and cDNA was synthesized with the PrimeScript™ RT Reagent Kit (Cat: RR037A, Takara, Japan) following the manufacturer’s instructions.

2.2. Conserved Analysis, siRNA Synthesis, and Vector Construction

The coding sequences of goat CDC25A (NC_030829.1), RXFP2 (NC_030819.1), and IGF2BP1 (NC_030826.1) were retrieved from the NCBI database. For each gene, the full-length CDS was synthesized and cloned into a mammalian expression vector to generate the overexpression construct (Figure S1). Gene-specific small interfering RNAs (siRNAs) were designed (Table S1). Both the overexpression vectors and siRNA oligos were synthesized commercially by Sangon Biotech (Shanghai, China). The integrity of all plasmid constructs was confirmed by 1.0% agarose gel electrophoresis, and their sequence accuracy was verified by Sanger sequencing performed by the same vendor using an ABI 3730xl DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA). Plasmid DNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), with all samples having an A260/A280 ratio between 1.8 and 2.0, and diluted to a working concentration of 0.5 µg/µL in nuclease-free water for transfection.

2.3. Culturing and Treatment of Goat Ovarian Granulosa Cells

Goat ovarian granulosa cells were cultured in DME/F12 (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; ZETA life, San Francisco, CA, USA) and 1% penicillin–streptomycin (Biosharp, Hefei, China), and maintained at 37 °C in a humidified incubator with 5% CO_2_ (Forma Series II, Thermo Fisher Scientific, USA). Upon reaching 80–90% confluence, cells were passaged using 0.25% trypsin–EDTA (Solarbio, Beijing, China).

For experiments, cells were seeded in 6-well plates at a density of 3.5 × 10^5^ cells per well. After attachment (approximately 60% confluence), transfection was carried out using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, USA), following the manufacturer’s protocol, with all procedures conducted in triplicate.

2.4. Detection of Cell Proliferation and Apoptosis

Cell proliferation was assessed using the CCK-8 (Cell Counting Kit-8) assay and EdU incorporation assay. Cell viability was determined by measuring the absorbance at 450 nm using a Cell Counting Kit-8 (CCK-8; Beyotime, Shanghai, China), with a SpectraMax iD3 microplate reader (Molecular Devices, San Jose, CA, USA), following the manufacturer’s protocol. The EdU cell proliferation assay was performed according to the instructions provided in the EdU Cell Proliferation Kit (Vigorous, Shanghai, China). Fluorescent images were captured with an inverted fluorescence microscope (Eclipse Ti2, Nikon, Japan). Apoptosis was detected using the TUNEL assay. The TUNEL assay was performed using the One-step TUNEL Cell Apoptosis Detection Kit (Beyotime, Shanghai, China), and images were acquired using the same microscope system.

2.5. Western Blot Analysis

Protein extraction was performed using RIPA lysis buffer supplemented with PMSF at a 100:1 ratio (Shaanxi Zhonghui Hecai, Xi’an, China). After removing the culture medium, the lysis mixture was added to the cells and incubated on ice for 10–15 min to ensure complete lysis. Cells were then gently scraped and transferred to a centrifuge tube. Protein concentration was determined using a BCA protein assay kit (Solarbio, Beijing, China).

The protein lysate was mixed with 5× SDS-PAGE loading buffer containing DTT (Dinin Biotech, Beijing, China) at a 4:1 ratio. After thorough mixing, samples were denatured by heating at 100 °C for 10 min and stored at −80 °C until further use.

Proteins were separated on a 12.5% SDS-PAGE gel. Approximately 20 μg of protein per sample was loaded into each lane, and electrophoresis was performed until the bromophenol blue dye front reached the bottom of the gel. Proteins were subsequently transferred onto a 0.45 μm PVDF membrane (Millipore, Burlington, MA, USA). Following transfer, the membrane was blocked with 5% skim milk for 2 h at room temperature and washed three times with TBST buffer.

The membrane was incubated overnight at 4 °C with the appropriate primary antibody (Table S2), followed by three TBST washes, and then incubated with the corresponding secondary antibody for 2 h at room temperature. Protein signals were detected using a GelView9000 Lite chemiluminescence imaging system (Guangzhou Biolight Biotechnology, Guangzhou, China).

2.6. Primer Design, qRT-PCR, and Statistical Analysis

Quantitative primers for the mRNA of genes including IGF2BP1, CDC25A, and RXFP2 were designed using the NCBI Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi, accessed on 12 February 2025), with GAPDH serving as the internal reference gene (Table S3). Gene expression levels were quantified using the 2^−ΔΔCt^ method. Statistical analysis was performed using GraphPad Prism 9.5.0 software. Data were analyzed using one-way ANOVA, followed by post hoc comparisons with Dunnett’s or Tukey’s test as appropriate. Results are expressed as mean ± standard deviation (SD), with p ≤ 0.05 considered statistically significant.

3. Results

3.1. Conservation Analysis and Overexpression/Knockdown Efficiency of IGF2BP1, CDC25A, and RXFP2

The efficiency of both overexpression and knockdown for the target genes IGF2BP1, CDC25A, and RXFP2 was first confirmed in goat ovarian granulosa cells. qRT-PCR analysis revealed that transfection with the specific overexpression vectors robustly upregulated the mRNA levels of all three genes compared to the empty vector control. The results indicated that the overexpression efficiencies of CDC25A and IGF2BP1 were particularly high, with gene expression levels exceeding those in the empty vector control by more than 50-fold. Similarly, the RXFP2 overexpression vector significantly increased RXFP2 expression in goat ovarian granulosa cells. Evaluation of the interference efficiency of the siRNAs revealed that siRNAs targeting IGF2BP1, CDC25A, and RXFP2 effectively suppressed the normal expression of these genes in granulosa cells (Figure 1). These findings demonstrate that both the overexpression vectors and siRNAs for IGF2BP1, CDC25A, and RXFP2 genes are suitable for subsequent investigations into gene function.

3.2. Effects of IGF2BP1 on Cell Growth and Fate Regulation

At 24 and 48 h post-transfection, CCK-8 assays (Figure 2A,B) revealed that, compared to their respective control groups, overexpression of IGF2BP1 significantly enhanced the proliferative activity of granulosa cells at the 24 h time point, whereas the proliferative activity of cells in the si-IGF2BP1 group was significantly reduced.

Alterations in IGF2BP1 expression led to significant changes in the mRNA and protein abundance of key cell fate regulators (Figure 2C,F). Overexpression of IGF2BP1 significantly upregulated the mRNA of the proliferation-promoting gene Cyclin E and the anti-apoptotic gene Bcl2, as well as the protein levels of CDK2 and Bcl2. Concurrently, it downregulated the mRNA of the pro-apoptotic gene CytC and the protein level of Bax. Conversely, IGF2BP1 knockdown significantly suppressed both the mRNA and protein expression of the proliferation marker CDK2 and the pro-apoptotic protein Bax.

In EdU staining assays (Figure 2G), overexpression of IGF2BP1 significantly increased the number of EdU-positive cells (p < 0.01), while the proportion of EdU-positive cells was significantly decreased in the si-IGF2BP1 group compared to the knockdown control group (p < 0.01). TUNEL staining results (Figure 2H) showed that the proportion of TUNEL-positive cells in the IGF2BP1 overexpression group was significantly lower than that in the overexpression control group (p < 0.05). In contrast, the proportion of TUNEL-positive cells in the si-IGF2BP1 group was significantly higher compared to the knockdown control group (p < 0.01).

3.3. Effects of CDC25A on Cell Growth and Fate Regulation

In the CCK-8 assay (Figure 3A,B), overexpression of CDC25A significantly promoted the viability of ovarian granulosa cells at 24 h post-transfection (p < 0.05). However, no significant effect on cell viability was observed at 48 h after overexpression or following CDC25A knockdown.

Alteration of CDC25A expression significantly modulated the levels of key markers involved in cell proliferation and apoptosis (Figure 3C,D). Overexpression of CDC25A significantly increased the mRNA levels of the proliferation-promoting gene CDK2 and the anti-apoptotic gene Bcl2, while decreasing the mRNA level of the pro-apoptotic gene Bax. Conversely, CDC25A knockdown significantly reduced the mRNA expression of the proliferation-related genes Cyclin E and PCNA, suppressed the protein levels of the proliferation markers CDK2 and PCNA as well as the anti-apoptotic protein Bcl2, and increased the mRNA expression of the pro-apoptotic gene CytC.

Results from the EdU staining assay (Figure 3G) demonstrated that CDC25A significantly increased the number of EdU-positive cells (p < 0.01). TUNEL staining (Figure 3H) revealed that the proportion of TUNEL-positive cells in the CDC25A overexpression group was significantly lower than that in the control group (p < 0.05). In contrast, the si-CDC25A group exhibited a significantly increased proportion of TUNEL-positive cells (p < 0.01).

3.4. Effects of RXFP2 on Cell Growth and Fate Regulation

In the CCK-8 assay (Figure 4A,B), overexpression of RXFP2 significantly promoted the viability of ovarian granulosa cells at 24 h post-transfection (p < 0.05). However, no significant effects on cell viability were observed at 48 h after overexpression or following RXFP2 knockdown.

RXFP2 expression altered key regulators of proliferation and apoptosis at both the mRNA and protein levels (Figure 4C,D). Overexpression of RXFP2 significantly increased the mRNA levels of the proliferation-related genes Cyclin E, CDK2, and PCNA, as well as the anti-apoptotic gene Bcl2. At the protein level, it enhanced the abundance of PCNA and Bcl2, while suppressing both the mRNA and protein expression of the pro-apoptotic gene Bax. Conversely, RXFP2 knockdown significantly reduced the mRNA level of CDK2 and the protein level of PCNA.

Results from the EdU staining assay (Figure 4G) demonstrated that RXFP2 significantly increased the number of EdU-positive cells (p < 0.01). TUNEL staining (Figure 4H) revealed that neither overexpression nor knockdown of RXFP2 had a significant effect on the proportion of TUNEL-positive cells.

4. Discussion

This study focuses on investigating the functions of the IGF2BP1, CDC25A, and RXFP2 genes, which were selected through a multi-omics strategy. The aim is to explore the roles of these genes in the goat ovary, elucidate the regulatory mechanisms of their expression, and provide a reference for understanding the regulatory mechanisms behind goat fertility and expanding the application of marker-assisted selection techniques.

The decision to utilize ovarian granulosa cells as the primary experimental model was grounded in their indispensable role in folliculogenesis and female fertility [13]. Granulosa cells are not merely structural components of the follicle but are dynamic regulators that directly govern oocyte development, steroid hormone synthesis, and follicular selection for ovulation [14,15,16]. Their proliferation and apoptosis rates are fundamental determinants of follicular atresia or survival, thereby ultimately controlling the ovulation rate and litter size [17]. Consequently, investigating the functions of IGF2BP1, CDC25A, and RXFP2 within this specific cell type provides the most direct and biologically relevant insights into the molecular mechanisms governing ovarian function and overall reproductive performance in goats.

An important observation from our functional assays was the time-dependent effect on cell viability following manipulation of CDC25A and RXFP2, where significant effects at 24 h were attenuated by 48 h. While a decline in transfection efficiency is a technical consideration, our data confirming sustained mRNA-level perturbation at 48 h makes this an unlikely sole explanation. Instead, we propose that this transient phenotype likely reflects the activation of intrinsic cellular compensatory or feedback mechanisms. Both CDC25A, as a cell cycle phosphatase, and RXFP2, as a G-protein-coupled receptor, are integral nodes within highly regulated, homeostatic signaling networks. Acute perturbation of such nodes is known to trigger adaptive responses—such as upregulation of parallel pathways, feedback inhibition, or receptor desensitization—which can restore cellular equilibrium over time. This interpretation underscores the resilience of follicular development and refines our understanding of these genes’ roles. Consequently, we define CDC25A and RXFP2 not as sustained monolithic regulators, but as critical early-phase regulators or key initiators of the granulosa cell response that dictates the initial (24 h) viability outcome.

Regarding the CDC25A gene, it is a pivotal regulator of the cell cycle and plays significant roles not only in cell cycle control and cancer regulation [18], but also in cell proliferation and hair follicle development [19]. Our results demonstrate that CDC25A promotes the proliferation of ovarian granulosa cells; this conclusion is supported by CCK-8 assays, EdU staining, and qRT-PCR analysis, and aligns with prior research. CDC25A primarily functions to remove inhibitory phosphorylations from cyclin-dependent kinases (CDKs), thereby regulating the cell cycle and promoting proliferation [18]. However, CDK activity is regulated not only by phosphorylation and dephosphorylation but also through protein–protein interactions and other precise mechanisms [20]. Additionally, CDC25A’s function is influenced by three E2F transcription factor binding sites in its promoter region [21] and its own phosphorylation modifications [22]. Therefore, the regulation of cell proliferation by CDC25A involves a complex network of regulatory mechanisms, and simply over-expressing CDC25A may not be sufficient to modulate these processes. Regarding the promotion of apoptosis following CDC25A knockdown in granulosa cells, our findings indicate that CDC25A expression positively regulates granulosa cell survival and plays a non-negligible role in reproductive processes, confirming our previous transcriptome sequencing results. This effect may be attributed to increased expression of apoptotic proteins resulting from reduced CDC25A expression [23]. However, the complex regulatory mechanisms of CDC25A, especially its interaction with apoptosis signal-regulating kinase 1 (ASK1) [5], complicate a direct interpretation. Given that CDC25A is a key regulator of the cell cycle, its role in apoptosis needs to be considered within the broader context of cell cycle regulation. Future studies on CDC25A should integrate its phosphorylation modifications and interactions with ASK1 to gain a more comprehensive understanding. Therefore, CDC25A functions as an early key regulator coordinating proliferation and survival decisions in granulosa cells.

As for the IGF2BP1 gene, it has recently emerged as a prominent molecule in RNA methylation and cancer research [24], with roles identified in both cell proliferation and apoptosis. Its function has become a hot topic in regulating growth and reproduction in various livestock species. In this study, we investigated the proliferative capacity of granulosa cells following IGF2BP1 overexpression or knockdown using EdU staining and qRT-PCR. Both methods yielded consistent results, demonstrating that high IGF2BP1 expression promoted cell proliferation, whereas knockdown of IGF2BP1 expression suppressed proliferation. These findings align with previous studies, which have shown that IGF2BP1 is significantly overexpressed in various cancers, promoting cancer cell proliferation [25], and also directly participates in regulating cell proliferation factors such as c-MYC and MKI76 in normal somatic cells [26,27]. It is noteworthy that while the literature suggests IGF2BP1 can modulate granulosa cell function through m6A-dependent stabilization of specific transcripts like MDM2 [28], the present study did not experimentally validate this particular mechanism. Our conclusions are firmly based on the observed phenotypic changes and corresponding shifts in marker gene expression. Additionally, we examined apoptosis in granulosa cells after IGF2BP1 overexpression and knockdown using TUNEL staining and qRT-PCR. Both assays indicated that knockdown IGF2BP1 expression promotes apoptosis. Collectively, these results demonstrate that IGF2BP1 serves as a positive regulator of reproductive processes in goats, confirming our earlier transcriptomic analysis. Although similar phenotypes have been reported in other studies, the underlying mechanisms remain incompletely understood. One hypothesis is that reduced IGF2BP1 activity could block the phosphorylation of anti-apoptotic factors (ERK, JNK, and p38) [29], while another suggests that IGF2BP1 may drive the translation of cell apoptosis inhibitor 1 (cIAP1) [30]. Furthermore, emerging evidence from miRNA and lncRNA research has revealed their critical regulatory roles in animal growth, development, and reproduction. For instance, miR-484 overexpression has been shown to aggravate granulosa cell dysfunction and intensify ovarian oxidative stress injury [31], while lncRNA ST6GALNAC3 suppresses cashmere goat hair follicle development by modulating the chi-miR-24-3p/ID4 axis in dermal fibroblasts [32]. Currently, additional studies have focused on the complex regulation of IGF2BP1 by ncRNA [33,34].

The function of RXFP2 is broad, with much research in livestock focusing on its role in the development of bovine and ovine horns and testicular descent. While its role in directly suppressing apoptosis under our experimental conditions requires further investigation, its clear proliferative effect underscores its importance. Recent studies, however, suggest that RXFP2 in the goat ovary specifically interacts with INSL3 in the corpus luteum and various ovarian cell types, implicating RXFP2 in the regulation of pregnancy in goats [35]. This highlights the potential role of RXFP2 in female reproduction. In this study, we evaluated the proliferative capacity of granulosa cells following RXFP2 overexpression or knockdown using EdU staining and qRT-PCR assays. The results demonstrated that RXFP2 overexpression significantly promotes granulosa cell proliferation. Recent research into the co-evolution of prolificacy and thermo-hygric adaptation in ruminants revealed co-localization of BMPR1B (reproduction regulator) and RXFP2 (environmental adaptor) genes [36]. This genomic arrangement facilitates synergistic coregulation of reproductive phenotypes by environmental adaptation genes. Alternatively, RXFP2 functions as a receptor within the INSL3 ligand-receptor pathway, which exhibits spatiotemporal specificity through predominant activity in discrete ovarian cell populations during specific developmental stages. We acknowledge that the proposed model of an epigenetic cascade mechanism underlying environmental adaptation, which might explain RXFP2′s strong association with prolificacy in genome-wide studies, remains a speculative hypothesis aimed at reconciling population genetics data with functional inquiry. This study does not provide direct evidence for such a cascade.

Collectively, this study demonstrates that IGF2BP1, CDC25A, and RXFP2 are fundamental early-phase regulators of granulosa cell dynamics, each contributing uniquely to the balance between proliferation and apoptosis. These findings provide crucial functional validation of candidate genes identified from genomic studies and highlight promising targets for genetic selection aimed at enhancing goat prolificacy. Future work should explore their roles in other ovarian cell types, their in vivo interactions, and the specific compensatory mechanisms that modulate their temporal effects.

5. Conclusions

In this study, we investigated the roles of IGF2BP1, CDC25A, and RXFP2 genes in goat ovarian granulosa cells to elucidate their effects on reproductive performance in goats. The results demonstrated that overexpression of these genes enhanced cellular viability and the expression of pro-proliferation marker genes, while their interference suppressed proliferation. Collectively, these findings provide insights into the regulatory functions of IGF2BP1, CDC25A, and RXFP2 in ovarian cells, and further lay the groundwork for understanding goat fertility and improving marker-assisted breeding techniques.