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. 2026 Sep 14;13:1932131. doi: 10.3389/fvets.2026.1932131

Novel circNSD2 promotes proliferation and testosterone synthesis while inhibiting apoptosis in bovine Leydig cells

Xiang Meng 1, Tianxiang Meng 1, Xinqi Zhou 1, Sihua Jin 1,2, Yunhai Zhang 1,2,3, Ning Song 1,2,3,*, Hongyu Liu 1,2,3,*
PMCID: PMC13616769  PMID: 42807274

Abstract

Leydig cells (LCs) are the primary cells responsible for testosterone synthesis in male animals, and their steroidogenic function is crucial for testicular development and spermatogenesis in bulls. Circular RNAs (circRNAs) play important roles in various biological processes and reproductive development; however, their functions in bovine LCs remain largely unexplored. Our previous whole-transcriptome sequencing of calf and adult bovine testes identified a differentially expressed circRNA, circ_0027599 (designated circNSD2 after its host gene NSD2), that was highly expressed in adult testes. To investigate its function, circNSD2 was overexpressed in bovine LCs, and its effects on cell proliferation, apoptosis, and testosterone synthesis were assessed. The results showed that circNSD2 overexpression significantly enhanced cell viability, increased the proportion of cells in the S phase, and upregulated the expression of proliferation-related genes (proliferating cell nuclear antigen [PCNA] and CDK2). Concurrently, circNSD2 overexpression significantly reduced the number of apoptotic cells, downregulated the expression of pro-apoptotic genes (BAX and Caspase3), and upregulated the anti-apoptotic gene (Bcl2). Furthermore, circNSD2 overexpression promoted testosterone secretion and increased the expression of key steroidogenic genes (STAR, CYP11A1, CYP17A1, and HSD17B3). In conclusion, circNSD2 promotes proliferation, inhibits apoptosis, and enhances testosterone synthesis and secretion in bovine LCs. These findings reveal a potential regulatory role of circNSD2 in bovine testicular development and provide a theoretical foundation and molecular target for improving reproductive performance in breeding bulls.

Keywords: apoptosis, bovine Leydig cells, circNSD2, proliferation, testosterone synthesis

1. Introduction

The reproductive potential of male livestock is a critical economic trait in modern animal husbandry. The reproductive performance of breeding bulls directly determines herd productivity and the efficiency of genetic improvement, and is therefore essential for the sustainable development of the industry (1). Wandong is an indigenous yellow cattle breed in eastern Anhui, China, exhibiting desirable traits, including strong roughage tolerance and disease resistance. However, breeding bulls of this breed commonly exhibit low libido and suboptimal semen quality, which severely hinder conservation and breeding programs (2). Recent studies have reported the regulatory roles of circular RNAs (circRNAs) in male reproductive development (3), however, research on circNSD2 remains limited. Therefore, investigating the regulatory mechanisms of functional genes in testicular development is essential for enhancing the reproductive value and economic benefits of Wandong cattle (4).

The testis is the primary male reproductive organ responsible for producing male gametes and secretes androgens (5). Leydig cells (LCs), located in the interstitial compartment between the seminiferous tubules, are the main cells responsible for testosterone synthesis and secretion (6). Testosterone, a key androgen, is essential for sexual development, maintenance of reproductive function, spermatogenesis, and regulation of reproductive performance in bulls. Its synthesis involves a complex multi-step enzymatic process regulated by luteinizing hormone (LH) (7). After binding to its membrane receptor on LCs, LH activates adenylate cyclase, which converts ATP into cAMP and subsequently upregulates the expression of the steroidogenic acute regulatory protein (STAR) (8). STAR mediates cholesterol transport from the outer mitochondrial membrane to the inner mitochondrial membrane, which is the initial and rate-limiting step in testosterone synthesis (9). Subsequently, cholesterol is converted to pregnenolone by cytochrome P450 family 11 subfamily A member 1 (CYP11A1). Pregnenolone is then sequentially catalyzed by cytochrome P450 family 17 subfamily A member 1 (CYP17A1), 3β-hydroxysteroid dehydrogenase (3β-HSD), and 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) in the smooth endoplasmic reticulum, ultimately yielding testosterone (10). Among these, STAR, CYP11A1, CYP17A1, and HSD17B3 are key marker genes for testosterone synthesis, and their expression levels directly determine the testosterone-synthesizing capacity of LCs (11, 12). Abnormal expression of these genes can lead to decreased testosterone levels and impaired reproductive function.

CircRNAs are a class of closed-loop endogenous non-coding RNAs formed by the back-splicing of precursor mRNAs. They lack 5′ caps and 3′ poly(A) tails and are characterized by stable expression and resistance to degradation by exonucleases (13, 47). CircRNAs function primarily as miRNA sponges, regulators of RNA-binding proteins, or transcriptional modulators and participate in diverse biological processes, including cell proliferation, apoptosis, and differentiation (14, 46). NSD2, which encodes a histone methyltransferase, has been implicated in cell proliferation, differentiation, and tumorigenesis (15). However, the biological function of circNSD2 in LCs remains unclear. Our previous whole-transcriptome sequencing experiment revealed high circNSD2 expression in the testes of adult Wandong bulls, suggesting its potential involvement in regulating bovine LC function (16).

Therefore, this study aimed to investigate the effects of circNSD2 overexpression in bovine LCs. The findings provide a theoretical basis for further elucidating the molecular mechanisms of circNSD2, improving the reproductive performance of Wandong breeding bulls, and optimizing conservation strategies for local breeds.

2. Materials and methods

2.1. Ethics statement

Testis samples were collected from three healthy 3-year-old adult Wandong bulls at Fengyang Daming Agricultural Animal Husbandry Technology Development Co., Ltd. The samples were cleaned with iodophor, 70% ethanol, and PBS containing 2% penicillin/streptomycin and were immediately transported on ice to the laboratory for primary LC isolation. All animal procedures were approved by the Animal Ethics Committee of Anhui Agricultural University (SYXK2021-009).

2.2. Culture of LCs

Primary bovine LCs were isolated from the testicular tissue of the three Wandong bulls and subsequently established and cryopreserved in our laboratory, as previously described (17). The tissue was digested with collagenase type P (Merck Millipore, Darmstadt, Germany) and centrifuged at 210 ×g for 45 min. The supernatant was discarded, and the upper tissue pellet was filtered through 100- and 200-mesh steel sieves. The filtered tissue was washed with Dulbecco’s Modified Eagle Medium (DMEM)/F12 (SH30023, Hyclone, Logan, UT, USA), and centrifuged at 210 ×g for 15 min. LCs were further purified using the density gradient centrifugation. The gradient consisted of six phases of Percoll (P8370, Solarbio, Beijing, China) at concentrations of 5, 30, 40, 50, 60, and 70%. The filtered cell suspension was layered onto the gradient and centrifuged at 1,000 ×g for 30 min at 18 °C. The cell band located between the 40 and 50% Percoll layers was collected, washed with DMEM/F12 medium, and centrifuged at 210 ×g for 15 min at 34 °C. The resulting cells were resuspended in DMEM/F12 medium supplemented with 10% fetal bovine serum. When 80% confluence was reached, LCs were identified via 3β-HSD staining. Cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a 5% CO₂ incubator.

For all experiments, LCs at passages 2–4 were used. Cells were seeded in different culture plates at the following densities: 1 × 106 cells/well in 6-well plates for RNA and protein extraction and cell cycle and apoptosis analyses; 4 × 105 cells/well in 6-well plates for the 5-ethynyl-2′-deoxyuridine (EdU) assay; 5 × 103 cells/well in 96-well plates for the CCK-8 assay; and 2 × 105 cells/well in 24-well plates for testosterone measurement. After seeding, cells were cultured for 24 h until they reached 60–70% confluence prior to transfection.

2.3. Identification of circRNA

Analysis of previous whole-transcriptome sequencing data (NCBI database BioProject PRJNA760322) from calf and adult bovine testes identified a differentially expressed circRNA, novel_circ_0027599, which was named circNSD2 based on its host gene, NSD2 (16). To verify its circular structure, total RNA was treated with RNase R (Beyotime, Shanghai, China) at a final concentration of 2 U /μg at 37 °C for 20 min, and incubated at 70 °C for 10 min. After treatment, RT-PCR was performed using GAPDH as a linear control. The amplification products were analyzed by electrophoresis and then sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing to verify the back-splicing site. The full-length sequence of circNSD2 is shown in Supplementary Tables S1 and S2.

2.4. Cell transfection

The full-length circNSD2 sequence was amplified and cloned into the pEGFP-N1 vector to generate the circNSD2 overexpression plasmid (pEGFP-N1-circNSD2). The empty pEGFP-N1 vector was used as a negative control to eliminate any potential confounding effects of the vector backbone on cell function. All constructs were synthesized by Sangon Biotech (Shanghai, China) and verified by Sanger sequencing. For transfection, LCs were seeded into appropriate culture plates and cultured to 60–70% confluence. Transfections were performed using Lipofectamine 3,000 (Invitrogen, USA) according to the manufacturer’s protocol. For 6-well plates, 2.5 μg of plasmid DNA and 5 μL of Lipofectamine 3,000 were used per well. At 24 h post-transfection, cells were harvested for RNA extraction. At 48 h post-transfection, cells were collected for protein extraction, cell cycle analysis, apoptosis assays, and EdU assays, while culture supernatants were collected for testosterone quantification.

2.5. RNA extraction and quantitative RT-PCR

Reverse transcription was performed in a 20 μL reaction containing 2 μg of total RNA using TRUEscript RT Master Mix (Aidlab, China) according to the manufacturer’s instructions. RT-qPCR was performed on a CFX96 Real-Time PCR System (Bio-Rad, USA) in a 10 μL reaction composed of 5 μL 2 × SYBR Green qPCR Master Mix (Aidlab, China), 0.4 μL each of forward and reverse primers (10 μM), 1 μL cDNA template, and 3.2 μL of RNase-free water. The thermal cycling conditions were as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 30 s. Melting curve analysis was conducted from 65 °C to 95 °C in 0.5 °C increments (5 s per step) to confirm amplification specificity. All samples were analyzed in triplicate, and each experiment was independently repeated three times. GAPDH was used as the internal control, and relative gene expression levels were calculated using the 2−ΔΔCt method (18). All primer sequences are listed in Supplementary Table S3.

2.6. Western blotting

Treated LCs were collected and lysed on ice using High-Efficiency Rapid RIPA Lysis Buffer (Solarbio) to extract proteins. The protein concentration was determined using a bicinchoninic acid (BCA) assay kit. After separation by 10% SDS-PAGE, protein samples were transferred to a polyvinylidene fluoride membrane (PVDF; IPVHO0005, Merck, Germany) and blocked with 5% skim milk at room temperature (18–25 °C) for 1 h. The membranes were incubated with primary antibodies overnight at 4 °C. After three washes with TBST, the membranes were incubated with secondary antibodies at room temperature for 2 h. Finally, protein signals were detected using an ECL Chemiluminescent Substrate Kit (BL502B, Biosharp, China) and the Alliance Q9 Advanced imaging system. Grayscale values were analyzed using ImageJ (v1.8.0), with β-actin used as the internal control. The specific antibodies and dilution ratios are listed in Supplementary Table S4.

2.7. Cell viability assay

Cells were seeded in 96-well plates, and cell viability was assessed at 0, 24, 48, 72, and 96 h using the CCK-8 kit according to the manufacturer’s instructions. Absorbance at 450 nm was measured using the TECAN Spark®(Switzerland).

2.8. Apoptosis and cell cycle analysis

Cells were seeded in 6-well plates at a density of 1 × 106 cells per well. After transfection, the cells were harvested, washed with PBS, and analyzed by flow cytometry using an Annexin V-FITC/PI Apoptosis Detection Kit (KeyGen, Jiangsu, China) to detect apoptosis in the LCs. The cells were treated with Annexin V-FITC and propidium iodide and incubated in the dark for 15 min. Binding buffer was then added, and apoptosis was measured using a flow cytometry (BD Accuri C6 Plus, USA). For cell cycle analysis, cells were fixed in 70% ethanol at 4 °C overnight, washed with PBS, and incubated in the dark at 37 °C with RNase A/PI solution at a ratio of 1:9 for 30 min before detection using an Accuri C6 flow cytometer. The data were analyzed using FlowJo v10.

2.9. EdU incorporation assay

Cells were seeded in 6-well plates at a density of 4 × 105 cells per well. After transfection, cell proliferation was assessed using the BeyoClick EdU Kit (C0085L Beyotime). The nuclei of proliferating cells were stained brown with EdU and observed under a phase-contrast microscope (Axio Vert, Zeiss). Quantitative analysis was performed using ImageJ software.

2.10. Enzyme-linked immunosorbent assay (ELISA)

LC culture supernatants were collected 48 h after transfection and centrifuged at 3,000 ×g for 10 min. Testosterone levels were then measured using a Bovine Testosterone ELISA Kit (Kexing, Shanghai, China), and absorbance was detected at 450 nm using a microplate reader (TECAN, Switzerland). Testosterone concentrations were calculated using a standard curve.

2.11. Data analysis

Statistical analysis was performed using GraphPad Prism v10 (GraphPad Software, USA), and an independent samples t-test was applied. p < 0.05 was considered statistically significant.

3. Results

3.1. Identification of circRNAs

Novel_circ_0027599 was identified as a 468-bp circRNA formed by back-splicing of the NSD2 gene located on chromosome 6 (Figure 1A). This circRNA is hereinafter referred to as circNSD2. To validate circNSD2, its back-splicing site was amplified using divergent primers and confirmed by Sanger sequencing (Figure 1B). RNase R is a ribonuclease that efficiently digests linear RNA molecules but not circRNAs. RNA extracted from cells was treated with or without RNase R. RT-PCR results showed that both linear GAPDH mRNA and circNSD2 were amplified in the untreated group, whereas only circNSD2 was amplified in the RNase R-treated group (Figure 1C). These results indicate that circNSD2 is a stable circRNA.

Figure 1.

Panel A displays a schematic of exons five through eight forming a circular RNA (circNSD2), with a labeled junction site. Panel B shows an agarose gel image comparing RNase R-treated and untreated samples for GAPDH and circNSD2, with molecular markers. Panel C presents a chromatogram and sequence highlighting the back-splice junction site joining exon eight and exon five, with color-coded bases and exon labels.

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Identification of the circular structure of circNSD2. (A) The reverse splicing site of circNSD2 was identified by Sanger sequencing. (B) RT-PCR analysis of RNA treated with RNase R or left untreated. (C) circNSD2 is formed by the circularization of exons 5, 6, 7, and 8 of the source gene.

3.2. circNSD2 overexpression promotes cell proliferation and cell cycle progression

After the circular structure of circNSD2 was confirmed, its function in LCs was investigated. RT-qPCR was used to assess the overexpression efficiency of the circNSD2 overexpression vector in bovine LCs. The results showed that circNSD2 expression was significantly higher in the overexpression group than in the control group (Figure 2A, p < 0.001). Subsequent RT-qPCR and western blotting showed that circNSD2 overexpression significantly increased the mRNA and protein expression levels of the proliferation-related genes proliferating cell nuclear antigen (PCNA) and CDK2 (Figures 2B,C; Supplementary Figure S1, p < 0.01). Similarly, CCK-8 and EdU assays demonstrated that the proliferative capacity of LCs was significantly enhanced after circNSD2 overexpression (Figures 2D,E, p < 0.01). Furthermore, flow cytometric analysis of the cell cycle revealed that the proportions of cells in the G0/G1 and G2/M phases did not change significantly after circNSD2 overexpression, whereas the proportion of cells in the S phase increased significantly (Figure 2F, p < 0.001).

Figure 2.

Panel A shows a bar graph comparing relative expression of circNSD2 between NC and circNSD2 groups, with circNSD2 overexpressed. Panel B contains bar graphs displaying increased PCNA and CDK2 mRNA levels in circNSD2. Panel C presents immunoblots and corresponding quantification showing elevated PCNA and CDK2 protein in circNSD2. Panel D is a line graph indicating higher cell proliferation in circNSD2 over 96 hours with significant differences at each time point. Panel E displays EdU staining images (brown nuclei) of cells for NC and circNSD2, with quantification showing increased proliferation in circNSD2. Panel F features cell cycle flow cytometry histograms for NC and circNSD2 groups and a bar graph depicting a reduced G0/G1 population and increased S and G2/M phases in circNSD2.

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Overexpression of circNSD2 promotes the proliferation of Leydig cells. (A,B) RT-qPCR analysis of circNSD2 overexpression efficiency and mRNA expression levels of proliferation-related genes. (C) Western blot analysis of protein expression levels of proliferation-related genes. (D) Cell viability assay using the CCK-8 assay. (E) Cell proliferation assay using EdU staining. (F) Cell cycle distribution analysis by flow cytometry.

3.3. circNSD2 overexpression inhibits apoptosis

To investigate the role of circNSD2 in LC apoptosis, the expression levels of apoptosis-related genes were examined after circNSD2 overexpression using RT-qPCR and western blotting. The results showed that circNSD2 overexpression significantly decreased the mRNA expression levels of the pro-apoptotic markers Caspase3 and BAX (p < 0.05), whereas it significantly increased the mRNA expression level of the anti-apoptotic gene Bcl2 (Figure 3A, p < 0.01). At the protein level, Caspase 3 expression was significantly reduced (p < 0.05), whereas BAX expression showed no significant change. In contrast, Bcl2 expression was significantly elevated (Figure 3B; Supplementary Figure S2, p < 0.05). Subsequently, apoptosis was detected by flow cytometry, and the results showed that circNSD2 overexpression significantly reduced the total apoptosis rate of LCs (Figure 3C, p < 0.001).

Figure 3.

Figure containing three panels displaying experimental data: Panel A is a bar graph showing increased mRNA levels of Caspase3, Bax, and Bcl2 in circNSD2 samples compared to NC, with statistically significant differences marked by asterisks. Panel B presents a western blot image and a corresponding bar graph, indicating higher protein expression of Caspase3 and Bcl2 in circNSD2 compared to NC, while Bax shows no significant protein level change. Panel C contains flow cytometry dot plots comparing NC and circNSD2 groups, with the circNSD2 group showing reduced apoptotic rate, as described by a bar graph with clear reduction and statistical significance.

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Overexpression of circNSD2 inhibits apoptosis in LCs. (A) mRNA expression levels of apoptosis-related genes were detected by RT-qPCR. (B) Protein expression levels of apoptosis-related genes were detected by Western blot. (C) Total cell apoptosis rate was detected by flow cytometry.

3.4. circNSD2 overexpression promotes testosterone synthesis

To further investigate the potential role of circNSD2 in testosterone synthesis in LCs, the expression of testosterone synthesis-related genes and testosterone secretion levels after circNSD2 overexpression was examined using RT-qPCR, western blotting, and ELISA. RT-qPCR and western blotting showed that circNSD2 overexpression significantly increased the mRNA and protein expression levels of the testosterone synthesis-related genes STAR, CYP11A1, CYP17A1, and HSD17B3 (Figures 4A,B; Supplementary Figure S3, p < 0.01). ELISA results showed that circNSD2 overexpression significantly increased testosterone concentration in the LC culture supernatant (Figure 4C, p < 0.05).

Figure 4.

Panel A contains a bar graph showing that mRNA levels of STAR, CYP11A1, CYP17A1, and HSD17B3 are significantly higher in circNSD2 compared to NC, with double asterisks denoting statistical significance. Panel B includes western blot images and a corresponding bar graph, both demonstrating higher protein levels of the same genes in circNSD2 samples versus NC, with asterisks for significance. Panel C shows a bar graph indicating higher testosterone content in the circNSD2 group than in the NC group, also statistically significant.

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Overexpression of circNSD2 promotes testosterone synthesis in LCs. (A) mRNA expression levels of genes related to testosterone synthesis were detected by RT-qPCR. (B) Protein expression levels of genes related to testosterone synthesis were detected by Western blot. (C) Testosterone levels were detected by ELISA.

4. Discussion

Testosterone biosynthesis is a complex enzymatic process in which STAR mediates cholesterol transport into the mitochondria (19). Subsequently, CYP11A1 catalyzes the conversion of cholesterol to pregnenolone, whereas CYP17A1 further converts pregnenolone to 17α-hydroxypregnenolone, which is then converted into the testosterone precursors dehydroepiandrosterone and androstenedione (20, 44). Finally, HSD17B3 converts androstenedione into testosterone (21, 45). The expression levels of these four proteins directly determine the testosterone-synthesizing capacity of LCs. Knockdown of Bmal1 reduces the expression of key testosterone synthesis-related genes in mouse LCs, including STAR, CYP11A1 and 3β-HSD, and may affect testosterone secretion and apoptosis by regulating the PI3K/AKT signaling pathway (22). In rat LCs, Ad-ADM alleviates the LPS-induced reduction in LC survival and suppresses apoptosis by inhibiting TGF-β1 production through the Hippo signaling pathway, thereby restoring steroid synthesis in vitro (23). Other studies have shown that spermidine attenuates Poly(I:C)-induced immune responses, oxidative stress, and apoptosis, alleviates the downregulation of STAR, CYP11A1, and CYP17A1, and restores testosterone production in yak LCs (24). Based on previous studies, STAR, CYP11A1, CYP17A1, and HSD17B3 were selected as key marker genes to investigate the regulatory role of circNSD2 in testosterone synthesis in bovine LCs (25). The results showed that circNSD2 overexpression significantly upregulated the mRNA and protein expression levels of these four marker genes and promoted testosterone secretion, indicating that circNSD2 positively regulates testosterone synthesis in bovine LCs.

In addition to steroid synthesis, the proliferative capacity and apoptotic homeostasis of LCs are critical for maintaining testicular function (26). Impaired proliferation or excessive apoptosis can lead to LC depletion and reduced testosterone output, ultimately resulting in decreased fertility or infertility (27). PCNA is a cofactor for DNA polymerase δ and is indispensable for DNA replication and repair, and its expression level is directly correlated with the proportion of cells in the S phase (28). In rat LCs, endogenous LH deficiency significantly reduces the levels of Cyclin D3 and PCNA, which are associated with cell proliferation, while simultaneously decreasing the expression of Igf1 mRNA, which is crucial for LC proliferation and development (29). Isorhamnetin enhances the G₁/S transition mediated by the cyclin E/CDK2 complex by reducing inhibitory phosphorylation at Tyr-15 of CDK2 and upregulating CDK2 protein expression, thereby inducing cells to enter S phase. Simultaneously, it downregulates cyclin A expression, ultimately arresting cells in the S phase (30). Cyclin-dependent kinase 2 (CDK2) drives the G1/S transition by forming complexes with cyclin E and cyclin A (31). Therefore, this study selected PCNA and CDK2 as key markers for evaluating the proliferative capacity of LCs. Although this study confirmed the pro-proliferative effect of circNSD2 using CCK-8, EdU, cell cycle analysis, and PCNA/CDK2 expression, a detailed exploration of the G1/S regulatory machinery (e.g., cyclin E family members) was beyond the scope of this work. Further investigation of the interaction between circNSD2 and specific cell cycle regulators is warranted to fully elucidate the underlying molecular mechanism.

Apoptosis is another fundamental process that regulates cellular homeostasis, and the intrinsic mitochondrial apoptotic pathway is primarily regulated by the BCL2 family (32). Upon activation, the pro-apoptotic member BAX increases outer mitochondrial membrane permeability, induces cytochrome c release, and subsequently activates the caspase cascade (33). In contrast, the anti-apoptotic protein Bcl2 maintains mitochondrial integrity by binding to BAX and preventing its oligomerization (34). Caspase3 is the common final effector protease in both intrinsic and extrinsic apoptotic pathways, and its activation irreversibly leads to cell death (35). In mouse LCs, alcohol induces apoptosis by activating specific apoptotic pathways, upregulating BAX and Caspase3, and simultaneously downregulating Bcl2 (36). Selenomethionine significantly increases the Bcl2/Bax ratio in sheep LCs under heat exposure by downregulating the pro-apoptotic gene BAX and upregulating the anti-apoptotic gene Bcl2, thereby inhibiting apoptosis and effectively alleviating heat stress-induced phosphorylation-mediated activation of p38MAPK and HSPB1 in sheep LCs (37). Therefore, based on previous studies on LCs, BAX, Bcl2, and Caspase3 were selected as the key apoptotic marker genes. This study found that circNSD2 overexpression significantly reduced the mRNA levels of BAX and Caspase3, increased Bcl2 expression, and significantly reduced the overall apoptosis rate of bovine LCs. Notably, although the BAX protein levels showed a downward trend, no significant difference was observed. Owing to post-transcriptional or translational regulation, such as protein degradation, mRNA expression may not necessarily correlate with final protein expression (38). Therefore, the anti-apoptotic effect of circNSD2 is likely achieved primarily through the upregulation of Bcl2 and inhibition of Caspase3 rather than through direct downregulation of BAX protein levels.

CircRNAs are widely expressed in the male reproductive system and are involved in testis development, spermatogenesis, and regulation of germ cell function (39). By binding to let-7i, circSMC1B regulates the HMGA1/NR6A1 axis to promote the proliferation of bovine male germline stem cells and participate in bovine spermatogenesis (40). circPAN3 promotes the proliferation of immature porcine Sertoli cells and inhibits their apoptosis by regulating the expression of proliferation- and apoptosis-related genes (41). NSD2, a histone methyltransferase, participates in cell proliferation, differentiation, and tumorigenesis (42). Studies have shown that NSD2 protein is highly enriched in spermatocytes and round spermatids, and NSD2 deficiency leads to increased germ cell apoptosis, elevated sperm abnormalities, and ultimately impaired male fertility (43). In this study, circNSD2 overexpression promoted proliferation, inhibited apoptosis, upregulated testosterone synthesis-related genes, and increased testosterone secretion in bovine LCs (Figure 5), thereby advancing the functional characterization of circRNAs in these cells and broadening the known biological roles of NSD2-related non-coding RNAs. However, although this study conducted circNSD2 overexpression experiments, loss-of-function experiments are still needed to determine the function of circNSD2. In addition, the molecular mechanisms by which circNSD2 regulates LC proliferation, apoptosis, and testosterone synthesis remain to be investigated. In future studies, addressing these limitations will strengthen the understanding of circNSD2 in testicular function.

Figure 5.

Diagram illustrating the process of circNSD2 identification from testicles, its overexpression via plasmid transfection in Leydig cells, and impacts on proliferation, apoptosis, and testosterone biosynthesis through regulation of Bax, Bcl2, STAR, CYP11A1, CYP17A1, and HSD17B3.

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Schematic diagram illustrating the regulatory mechanisms of circNSD2 in LCs regarding proliferation, apoptosis, and testosterone synthesis.

5. Conclusion

This study provides preliminary evidence for the positive regulatory role of circNSD2 in the proliferation, apoptosis, and testosterone synthesis of bovine LCs. These findings provide a theoretical foundation for further investigation of the molecular mechanisms of circNSD2 in bull reproductive regulation and identify a new candidate target for improving bull reproductive performance and optimizing reproductive management.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. The work was supported by the National Natural Science Foundation of China (31101696), Key Research and Development Program of Anhui Province (2023n06020055), and the Project of Beef Cattle Revitalization of Anhui Provincial Science and Technology Department (202513b10050019, 202513b10050014).

Footnotes

Edited by: Ling Yang, Hebei University of Engineering, China

Reviewed by: Wangsheng Zhao, Southwest University of Science and Technology, China

Buyue Niu, Northeast Agricultural University, China

Data availability statement

The transcriptome data presented in the study have been deposited in the NCBI BioProject repository, accession number PRJNA760322. The data are publicly available at: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA760322.

Ethics statement

The animal studies were approved by the Animal Ethics Committee of Anhui Agricultural University (SYXK2021-009). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

Author contributions

XM: Investigation, Data curation, Formal analysis, Writing – original draft, Methodology. TM: Writing – original draft, Visualization, Formal analysis, Data curation. XZ: Formal analysis, Visualization, Writing – original draft. SJ: Investigation, Validation, Writing – review & editing. YZ: Resources, Supervision, Writing – review & editing. NS: Writing – original draft, Investigation, Resources, Project administration. HL: Conceptualization, Writing – review & editing, Project administration, Supervision.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that Generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

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Supplementary material

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets.2026.1932131/full#supplementary-material

Data_Sheet_1.pdf (972.9KB, pdf)
Table_1.DOCX (20.1KB, DOCX)

References

  • 1.Xie X, Shi L, Zhong Z, Wang Z, Pan D, Hou G, et al. Danzhou chicken: a unique genetic resource revealed by genome-wide resequencing data. Poult Sci. (2024) 103:103960. doi: 10.1016/j.psj.2024.103960, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Liu H, Khan IM, Liu Y, Khan NM, Ji K, Yin H, et al. A comprehensive sequencing analysis of testis-born miRNAs in immature and mature indigenous wandong cattle (Bos taurus). Genes. (2022) 13:2185. doi: 10.3390/genes13122185, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Sun Y, Wang Y, Li Y, Akhtar F, Wang C, Zhang Q. Identification of circular RNAs of testis and caput epididymis and prediction of their potential functional roles in donkeys. Genes. (2022) 14:66–75. doi: 10.3390/genes14010066, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Xu R, Pan M, Yin L, Zhang Y, Tang Y, Lu S, et al. C-type natriuretic peptide pre-treatment improves maturation rate of goat oocytes by maintaining transzonal projections, spindle morphology, and mitochondrial function. Animals. (2023) 13:3880. doi: 10.3390/ani13243880, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Li H, Wang X-R, Hu Y-F, Xiong Y-W, Zhu H-L, Huang Y-C, et al. Advances in immunology of male reproductive toxicity induced by common environmental pollutants. Environ Int. (2024) 190:108898. doi: 10.1016/j.envint.2024.108898, [DOI] [PubMed] [Google Scholar]
  • 6.Huang H, Zhang W, Zhang J, Zhao A, Jiang H. Epigenome editing based on CRISPR/dCas9p300 facilitates trans differentiation of human fibroblasts into Leydig-like cells. Exp Cell Res. (2023) 425:113551. doi: 10.1016/j.yexcr.2023.113551, [DOI] [PubMed] [Google Scholar]
  • 7.Kirkpatrick ZA, Melin VE, Hrubec TC. Quaternary ammonium compound exposure causes infertility by altering endocrine signaling and gametogenesis. Reprod Toxicol. (2025) 132:108817. doi: 10.1016/j.reprotox.2024.108817, [DOI] [PubMed] [Google Scholar]
  • 8.Zhang Q, Zou P, Zhan H, Zhang M, Zhang L, Ge R-S, et al. Dihydrolipoamide dehydrogenase and cAMP are associated with cadmium-mediated Leydig cell damage. Toxicol Lett. (2011) 205:183–9. doi: 10.1016/j.toxlet.2011.06.003, [DOI] [PubMed] [Google Scholar]
  • 9.Yang C-K, Wu RS-C, Wu C-H, Lin TR-Y, Tsai H-D. Sleep deprivation enhances peripheral serotonin secretion to regulate the large follicle steroidogenesis of rats. Taiwan J Obstet Gynecol. (2015) 54:260–9. doi: 10.1016/j.tjog.2014.10.007, [DOI] [PubMed] [Google Scholar]
  • 10.Sheweita SA, El-dafrawi YA, El-ghalid OA, Ghoneim AA, Wahid A. Antioxidants (selenium and garlic) alleviated the adverse effects of tramadol on the reproductive system and oxidative stress markers in male rabbits. Sci Rep. (2022) 12:13958. doi: 10.1038/s41598-022-16862-4, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Liu G-L, Yu F, Dai D-Z, Zhang G-L, Zhang C, Dai Y. Endoplasmic reticulum stress mediating downregulated StAR and 3-beta-HSD and low plasma testosterone caused by hypoxia is attenuated by CPU86017-RS and nifedipine. J Biomed Sci. (2012) 19:4–14. doi: 10.1186/1423-0127-19-4, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Xie T, Hu G, Dong B, Yan Y, Liu M, Yao X, et al. Roscovitine protects murine Leydig cells from lipopolysaccharide-induced inflammation. Exp Ther Med. (2017) 13:2169–76. doi: 10.3892/etm.2017.4239, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Lu L, Wang F, Chen J, Zhao C, Guo S, Dong D, et al. CircTENM3 inhibites tumor progression via the miR-558/RUNX3 axis in prostate cancer. J Transl Med. (2023) 21:850–62. doi: 10.1186/s12967-023-04708-0, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Lyu Y, Tan B, Li L, Liang R, Lei K, Wang K, et al. A novel protein encoded by circUBE4B promotes progression of esophageal squamous cell carcinoma by augmenting MAPK/ERK signaling. Cell Death Dis. (2023) 14:346–59. doi: 10.1038/s41419-023-05865-2, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zhang L, Zha X. Recent advances in nuclear receptor-binding SET domain 2 (NSD2) inhibitors: an update and perspectives. Eur J Med Chem. (2023) 250:115232. doi: 10.1016/j.ejmech.2023.115232, [DOI] [PubMed] [Google Scholar]
  • 16.Khan IM, Liu H, Zhuang J, Khan NM, Zhang D, Chen J, et al. Circular RNA expression and regulation profiling in testicular tissues of immature and mature wandong cattle (Bos taurus). Front Genet. (2021) 12:685541. doi: 10.3389/fgene.2021.685541, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Wang X, Zhou X, Tu T, Cui S, Meng X, Zhang Y, et al. PRM1 modulates proliferation, apoptosis, and testosterone synthesis in bovine Leydig cells. Anim Reprod Sci. (2025) 281:107991. doi: 10.1016/j.anireprosci.2025.107991, [DOI] [PubMed] [Google Scholar]
  • 18.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. (2001) 25:402–8. doi: 10.1006/meth.2001.1262, [DOI] [PubMed] [Google Scholar]
  • 19.Hu Y, Dong C, Chen M, Lu J, Han X, Qiu L, et al. Low-dose monobutyl phthalate stimulates steroidogenesis through steroidogenic acute regulatory protein regulated by SF-1, GATA-4 and C/EBP-beta in mouse Leydig tumor cells. Reprod Biol Endocrinol. (2013) 11:72–81. doi: 10.1186/1477-7827-11-72, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Luan X, Liu D, Cao Z, Luo L, Liu M, Gao M, et al. Transcriptome profiling identifies differentially expressed genes in Huoyan goose ovaries between the laying period and ceased period. PLoS One. (2014) 9:e113211. doi: 10.1371/journal.pone.0113211, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Carmichael SL, Shaw GM, Lammer EJ. Environmental and genetic contributors to hypospadias: a review of the epidemiologic evidence. Birth Defects Res, Part A. (2012) 94:499–510. doi: 10.1002/bdra.23021, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Ding H, Zhao J, Liu H, Wang J, Lu W. BMAL1 knockdown promoted apoptosis and reduced testosterone secretion in TM3 Leydig cell line. Gene. (2020) 747:144672. doi: 10.1016/j.gene.2020.144672, [DOI] [PubMed] [Google Scholar]
  • 23.Luo Y, Zhu X, Li M, Zhou J, Yang Z, Tong T, et al. Anti-apoptotic effect of adrenomedullin gene delivery on Leydig cells by suppressing TGF-β1 via the hippo signaling pathway. Reprod Toxicol. (2023) 119:108418. doi: 10.1016/j.reprotox.2023.108418, [DOI] [PubMed] [Google Scholar]
  • 24.Tang Y, Li H, Zeng Y, Yang C, Zhang R, Lund AK, et al. Spermidine as a potential protective agents against poly(I:C)-induced immune response, oxidative stress, apoptosis, and testosterone decrease in yak Leydig cells. Int J Mol Sci. (2025) 26:2753. doi: 10.3390/ijms26062753, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Tao S, Yao Z, Li H, Wang Y, Qiao X, Yu Y, et al. Exposure to 4-nonylphenol compromises Leydig cell development in pubertal male mice. Ecotoxicol Environ Saf. (2023) 266:115612. doi: 10.1016/j.ecoenv.2023.115612, [DOI] [PubMed] [Google Scholar]
  • 26.Adegoke EO, Rahman MS, Pang M-G. Bisphenols threaten male reproductive health via testicular cells. Front Endocrinol. (2020) 11:624–41. doi: 10.3389/fendo.2020.00624, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Surampudi P, Chang I, Lue Y, Doumit T, Jia Y, Atienza V, et al. Humanin protects against chemotherapy-induced stage-specific male germ cell apoptosis in rats. Andrology. (2015) 3:582–9. doi: 10.1111/andr.12036, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Radia A-M, Yaser A-M, Ma X, Zhang J, Yang C, Dong Q, et al. Specific siRNA targeting receptor for advanced glycation end products (RAGE) decreases proliferation in human breast cancer cell lines. Int J Mol Sci. (2013) 14:7959–78. doi: 10.3390/ijms14047959, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Sriraman V, Rao VS, Sairam MR, Rao AJ. Effect of deprival of LH on Leydig cell proliferation: involvement of PCNA, cyclin D3 and IGF-1. Mol Cell Endocrinol. (2000) 162:113–20. doi: 10.1016/S0303-7207(00)00201-X, [DOI] [PubMed] [Google Scholar]
  • 30.Wang J-L, Quan Q, Ji R, Guo X-Y, Zhang J-M, Li X, et al. Isorhamnetin suppresses PANC-1 pancreatic cancer cell proliferation through S phase arrest. Biomed Pharmacother. (2018) 108:925–33. doi: 10.1016/j.biopha.2018.09.105, [DOI] [PubMed] [Google Scholar]
  • 31.Cho JG, Choi J-S, Lee J-H, Cho M-G, Kim H-S, Noh H-D, et al. MED28 over-expression shortens the cell cycle and induces genomic instability. Int J Mol Sci. (2019) 20:1746. doi: 10.3390/ijms20071746, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Lucantoni F, Lindner AU, O’Donovan N, Düssmann H, Prehn JHM. Systems modeling accurately predicts responses to genotoxic agents and their synergism with BCL-2 inhibitors in triple negative breast cancer cells. Cell Death Dis. (2018) 9:42–54. doi: 10.1038/s41419-017-0039-y, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Xu F, Xu Y, Xiong J-H, Zhang J-H, Wu J, Luo J, et al. AOC1 contributes to tumor progression by promoting the AKT and EMT pathways in gastric cancer. Cancer Manag Res. (2020) 12:1789–98. doi: 10.2147/CMAR.S225229, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Shi W-Y, Che C-Y, Liu L. Human interleukin 23 receptor induces cell apoptosis in mammalian cells by intrinsic mitochondrial pathway associated with the down-regulation of RAS/mitogen-activated protein kinase and signal transducers and activators of transcription factor 3 signaling pathways. Int J Mol Sci. (2013) 14:24656–69. doi: 10.3390/ijms141224656, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Slee EA, Adrain C, Martin SJ. Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J Biol Chem. (2001) 276:7320–6. doi: 10.1074/jbc.M008363200, [DOI] [PubMed] [Google Scholar]
  • 36.Jang M-H, Shin M-C, Shin H-S, Kim K-H, Park H-J, Kim E-H, et al. Alcohol induces apoptosis in TM3 mouse Leydig cells via bax-dependent caspase-3 activation. Eur J Pharmacol. (2002) 449:39–45. doi: 10.1016/S0014-2999(02)01973-8, [DOI] [PubMed] [Google Scholar]
  • 37.Xu Y, Xia Y, Zhao J, Yu H, Zhang Y, Mao D. p38MAPK/HSPB1 is involved in the regulatory effects of selenomethionine on the apoptosis, viability and testosterone secretion of sheep Leydig cells exposed to heat. J Biochem Mol Toxicol. (2024) 38:e23826. doi: 10.1002/jbt.23826, [DOI] [PubMed] [Google Scholar]
  • 38.Yang Y, Luo D, Gao W, Wang Q, Yao W, Xue D, et al. Combination analysis of ferroptosis and immune status predicts patients survival in breast invasive ductal carcinoma. Biomolecules. (2023) 13:147–67. doi: 10.3390/biom13010147, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Li J, Shi B, Wang C, Shao C, Liu X, Zhang D. Comprehensive CircRNA profiling and selection of key CircRNAs reveal the potential regulatory roles of CircRNAs throughout ovarian development and maturation in cynoglossus semilaevis. Biology. (2021) 10:830–50. doi: 10.3390/biology10090830, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Gao Y, Li C, Jafari H, Yang G, Wang Z, Lei C, et al. CircRNA profiling reveals circSMC1B that promotes bovine male germline stem cells proliferation and apoptosis via sponging let-7i. BMC Genomics. (2025) 26:398–414. doi: 10.1186/s12864-025-11556-3, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Shi H, Yan Z, Du H, Zhang B, Gun S. CircRNA profiling reveals the regulatory role of circPAN3 in Hezuo boars sertoli cell growth. BMC Genomics. (2024) 25:1258. doi: 10.1186/s12864-024-11170-9, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Li X, Song D, Chen Y, Huang C, Liu A, Wu Q, et al. NSD2 methylates AROS to promote SIRT1 activation and regulates fatty acid metabolism-mediated cancer radiotherapy. Cell Rep. (2023) 42:113126. doi: 10.1016/j.celrep.2023.113126, [DOI] [PubMed] [Google Scholar]
  • 43.Li Z, Zhang X, Xie S, Liu X, Fei C, Huang X, et al. H3K36me2 methyltransferase NSD2 orchestrates epigenetic reprogramming during spermatogenesis. Nucleic Acids Res. (2022) 50:6786–800. doi: 10.1093/nar/gkac533, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Lao Y, Ouyang H, Huang X, Huang Y. Effect of bacterial endotoxin lipopolysaccharide treatment on duck Leydig cells. Anim Reprod. (2019) 16:871–9. doi: 10.21451/1984-3143-AR2019-0002, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Yuan L, Wang H, Yang H, Chen S, Yang D, Zhang Y. Expression of the NSE, SP, NFH and DβH in normal and cryptorchid testes of Bactrian camel. Anim Reprod. (2022) 19:e20210087. doi: 10.1590/1984-3143-ar2021-0087, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Chen S, Chen Z, Li Z, Li S, Wen Z, Cao L, et al. Tumor-associated macrophages promote cholangiocarcinoma progression via exosomal circ_0020256. Cell Death Dis. (2022) 13:94–105. doi: 10.1038/s41419-022-04534-0, [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Bian D, Wu Y, Song G. Novel circular RNA, hsa_circ_0025039 promotes cell growth, invasion and glucose metabolism in malignant melanoma via the miR-198/CDK4 axis. Biomed Pharmacother. (2018) 108:165–76. doi: 10.1016/j.biopha.2018.08.152, [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data_Sheet_1.pdf (972.9KB, pdf)
Table_1.DOCX (20.1KB, DOCX)

Data Availability Statement

The transcriptome data presented in the study have been deposited in the NCBI BioProject repository, accession number PRJNA760322. The data are publicly available at: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA760322.

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