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. Author manuscript; available in PMC: 2025 Aug 25.
Published in final edited form as: Toxicology. 2025 Jan 31;512:154068. doi: 10.1016/j.tox.2025.154068

Comparing the cannabidiol-induced transcriptomic profiles in human and mouse Sertoli cells

Yuxi Li a, Xilin Li b, Patrick Cournoyer c, Supratim Choudhuri d, Lei Guo a, Si Chen a,*
PMCID: PMC12376089  NIHMSID: NIHMS2105632  PMID: 39894194

Abstract

Cannabidiol (CBD), a major cannabinoid found in Cannabis sativa L., has been used in the treatment of seizures associated with Lennox-Gastaut syndrome, Dravet syndrome, and tuberous sclerosis complex. Recently, concerns have been raised regarding the male reproductive toxicity of CBD in animal models, such as monkeys, rats, and mice. In our previous studies, we reported that CBD inhibited cell proliferation in both primary human Sertoli cells and mouse Sertoli TM4 cells. Transcriptomic analysis revealed that in primary human Sertoli cells CBD disrupted DNA replication, cell cycle, and DNA repair, ultimately causing cellular senescence. In this study, we further investigated the molecular changes induced by CBD in mouse Sertoli TM4 cells using RNA-sequencing analyses and compared the transcriptomic profile with that of primary human Sertoli cells. Our findings demonstrated that, unlike in primary human Sertoli cells, CBD did not induce cellular senescence but caused apoptosis in mouse Sertoli TM4 cells. Through transcriptomic data analysis in mouse Sertoli TM4 cells, immune and cellular stress responses were identified. Moreover, transcriptomic comparisons revealed major differences in molecular changes induced by CBD between mouse Sertoli TM4 and primary human Sertoli cells. This suggests that primary human Sertoli cells and mouse Sertoli cells may respond differently to CBD.

Keywords: Cannabidiol, Male reproductive toxicity, mRNA-sequencing, Mouse Sertoli TM4 cells, Primary human Sertoli cells, Apoptosis

1. Introduction

Cannabidiol (CBD), a major cannabinoid present in the Cannabis sativa plant, has attracted substantial interest in recent years. Notably, CBD’s efficacy in treating several rare forms of childhood epilepsy, including Lennox-Gastaut syndrome, Dravet syndrome, and tuberous sclerosis complex, led to FDA’s approval of Epidiolex, an oral solution of CBD (FDA, 2018, 2020). The growing popularity of CBD coincides with its increased availability and widespread acceptance, stemming partly from the changing legal landscape regarding cannabis and its derivatives. In the United States, the 2018 Farm Bill removed hemp (Cannabis sativa L. containing no more than 0.3 % of delta-9-tetrahydrocannabinol), including hemp-derived CBD, from Schedule I under the Controlled Substances Act (FDA, 2019). However, the use of CBD is not risk-free. Animal studies have revealed adverse effects, including developmental issues, embryo-fetal death, central nervous system suppression, neurotoxicity, liver damage, reduced sperm production, changes in organ weights, alterations in the male reproductive system, and low blood pressure (Gingrich et al. 2023; Huestis et al. 2019). Research on CBD in humans has identified side effects such as drug-drug interactions, hepatic issues, and somnolence (Chen et al. 2024b; Huestis, 2005).

In mice, rats, and monkeys, negative effects of CBD on the male reproductive system have been observed, including changes in testis size, number of germ and Sertoli cells, fertilization rates, hormone concentrations, and sexual behavior (Carvalho et al. 2020). For example, a recent study demonstrated that orally administered 30 mg CBD/kg daily to 21-day-old male Swiss mice (34 consecutive days followed by a 35-day recovery period) led to a 76 % decrease in circulating testosterone levels, although the levels remained within the normal physiological range (Carvalho et al. 2018a). The same study also observed abnormalities in the different stages of spermatogenesis, a significant reduction in the number of Sertoli cells, and a 38 % reduction in sperm count. Additionally, abnormalities in sperm structure were observed, indicating compromised sperm quality (Carvalho et al. 2018a). The same group also reported that orally administering 15 or 30 mg CBD/kg daily to 21-day-old male Swiss mice for 34 days without a recovery period caused aberrant sexual behavior in the 15 mg/kg group, and a 30 % reduction in fertility rate and a 23 % reduction in the number of litters in the 30 mg/kg group (Carvalho et al. 2018b).

Given the findings in animal models, questions arise regarding whether CBD could cause male reproductive toxicity in humans. Sertoli and Leydig cells are two essential cell types that play important roles in spermatogenesis, which takes place in the seminiferous tubules of the testis. Sertoli cells line the seminiferous tubules and surround germ cells. These essential somatic cells are pivotal to spermatogenesis by providing critical support for the development and maintenance of sperm cells (Petersen and Soder, 2006). Leydig cells are the primary source of testosterone in males and are crucial to the maintenance of reproductive capacity and fertility (Shima, 2019). The TM3 and TM4 cell lines, originating from mouse Leydig and Sertoli cells, respectively, have served as important models for understanding testicular physiology. To understand the responses of different cells to CBD treatment and address knowledge gaps, we treated Leydig and Sertoli cells, both from humans and mice, with CBD and studied the underlying mechanisms. Our previous reports demonstrated that CBD treatment inhibited cell proliferation following 24 and 48 h of exposure in both primary human Sertoli cells and mouse Sertoli TM4 cells (Li et al. 2022). CBD treatment also induced apoptosis and caused comparable transcriptomic changes in both primary human Leydig cells and mouse Leydig TM3 cells (Li et al. 2023b; Li et al. 2024). Transcriptomic analyses with primary human Sertoli cells revealed that a 24 h CBD exposure disturbed DNA replication, cell cycle, and DNA repair signaling pathways and eventually induced cellular senescence after prolonged treatment of 6 – 12 days (Li et al. 2023c). However, the molecular mechanisms underlying CBD-induced cytotoxicity in mouse Sertoli cells are unclear. Specifically, the potential toxic effects of prolonged CBD treatment in mouse Sertoli cells are not well understood, and it is unknown whether the transcriptomic changes caused by CBD in mouse Sertoli cells are similar to those in primary human Sertoli cells. These are important questions because data on the safety of prolonged CBD consumption are limited and a better understanding of the molecular mechanisms of toxicity in both species can indicate how relevant the results from mouse models are to humans.

The current study investigates CBD-induced phenotypic and molecular changes in mouse Sertoli TM4 cells. The outcomes are compared with observations in primary human Sertoli cells, offering insights into the differences and similarities between mouse and human Sertoli cells in response to CBD. As most in vivo studies examining CBD’s reproductive toxicity have been conducted in rodents, the current study provides useful mechanistic information for the cross-species extrapolation in risk identification.

2. Materials and methods

2.1. Test chemicals and cell cultures

CBD (Batch # NQSS1951), with a purity of 100 %, was purchased from Purisys (Athens, GA). Dimethyl sulfoxide (DMSO, Catalog # D8418), used as a solvent, was purchased from MilliporeSigma (St. Louis, MO). The identity of CBD was confirmed by in-house 1H-nuclear magnetic resonance and mass spectral analyses. CBD was solubilized in DMSO to prepare a 1000 × concentration stock solution, which was then stored at a temperature of –20°C.

Mouse Sertoli TM4 cells (ATCC® CRL-1715™), were obtained from American Type Culture Collection (ATCC, Manassas, VA). The mouse Sertoli TM4 cells were maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12) media (Catalog # D8900, MilliporeSigma), supplemented with 5 % horse serum (Catalog # 16050122, Gibco, Gaithersburg, MD), 2.5 % fetal bovine serum (FBS, Catalog # S11150, R&D systems, Minneapolis, MN), 100 units/mL penicillin, and 100 μg/mL streptomycin (Catalog # 15140163, Gibco). Cultivation occurred at 37°C in a humidified chamber with 5 % CO2, and cells were subcultured by trypsinization at two- to three-day intervals for a maximum of 10 passages. The mouse Sertoli TM4 cells used in this study were the same as those used in our previous study, where we obtained the cellular growth levels presented in Table 1 (Li et al. 2022).

Table 1.

Relative cellular growth level measured using Cell Titer-Blue assay.a.

CBD (μM) Cellular growth in mouse Sertoli TM4 cells CBD (μM) Cellular growth in primary human Sertoli cells
10 92.3 ± 8.2 %# 7 101.5 ± 3.9 %#
12.5 91.1 ± 7.6 % 8 91.7 ± 3.9 %*
15 91.6 ± 9.9 % 9 76.2 % ± 3.3 %*
17.5 84.3 ± 11.1 % 10 65.2 ± 2.1 %*
20 73.2 % ± 6.3 %*
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a

These results were obtained from our previous study (Li et al. 2022). Mouse Sertoli TM4 and primary human Sertoli cells were incubated with various concentrations of CBD for 24 h. Cytotoxicity was determined using CellTiter-Blue assy. The percentage of cellular growth was calculated from normalization to the control group.

#

significant concentration-related linear trend.

*

significantly different from the DMSO control.

2.2. Annexin V and PI staining

To distinguish between viable and apoptotic cells, an Alexa Fluor® 488 annexin V/dead cell apoptosis kit (Catalog # V13245, Thermo-Fisher Scientific, Waltham, MA) was used. Mouse Sertoli TM4 cells were seeded at a density of 1.7 × 105/well in 6-well plates (Catalog # 3506, Corning, Glendale, AZ) and exposed to CBD at designated concentrations and time points. The cells were then stained with Alexa Fluor® 488 annexin V and 1 µg/mL propidium iodide (PI) for 15 min at room temperature in the dark. The samples were immediately analyzed using a FACSCanto™ flow cytometer with FACSDiva™ software (BD Biosciences, San Jose, CA) and subsequently with FlowJo® software (FlowJo, LLC, Ashland, OR). The staining results are presented using two-dimensional dot plots. Viable cells were double-negative for annexin V and PI. Early apoptotic cells were positive for annexin V but negative for PI, while late apoptotic cells were positive for both annexin V and PI. Necrotic cells stained positively for PI due to disrupted cellular membranes.

2.3. Measurement of cellular growth

Mouse Sertoli TM4 cellular growth was monitored by measuring cumulative population doublings. Mouse Sertoli TM4 cells were seeded at a density of 3.4 × 104/well in 6-well plates; Day 0 was defined as the start of DMSO or CBD treatment. DMSO-treated cells were subcultured by trypsinization at 90–95 % confluency on Days 3 and 6. Cell numbers were determined at each subculture to calculate population doubling (PD) using the formula: PD = log(N2/N1)/log2, where N1 represents the cell number at the earlier time point and N2 represents the cell number at the subsequent time point. Cumulative population doubling (CPD) at each time point represents the total number of cell division occurrences and was calculated using the formula: CPD = PD (A) + PD (B), where PD (A) is the PD of the current time point and PD (B) is the PD of the earlier time point (Chen et al. 2013). After counting, the same number (3.4 × 104) of DMSO-treated cells were reseeded to new plates with fresh media containing DMSO.

The CBD-treated cells were kept in their initial plates, and media containing 15 or 20 µM CBD was refreshed on Day 3. The CBD treated cells were counted on Days 3 and 6. Additionally, 20 μM CBD-treated mouse Sertoli TM4 cells detached from the plates on Day 6; thus, monitoring was stopped on Day 6.

2.4. Measurement of senescence-associated β-galactosidase (SA-β-gal) activity

To determine quantitatively the SA-β-gal level, a CellEvent senescence green flow cytometry assay kit (Catalog # C10841, ThermoFisher Scientific) was used. Mouse Sertoli TM4 cells were seeded in 6-well plates and treated with CBD or DMSO for 6 days as described in the section on measurement of cellular growth. On Day 6, cells were collected, and the cell numbers in each sample were adjusted to 1 × 105. The samples were washed with 1 × phosphate-buffered saline (PBS) and resuspended in 100 µL of 4 % formaldehyde for fixation at room temperature for 10 min in the dark. Samples were then washed with 1 % BSA in 1 × PBS and resuspended in 100 µL of 1:1000 diluted staining solution. Samples were kept at 37 ºC without CO2 for 2 h. After washing and resuspending in 1 % BSA in 1 × PBS, the SA-β-gal level was analyzed using a BD FACSCanto™ II flow cytometer with a 488-nm laser. The stopping gate was set to record 10,000 events.

2.5. RNA sample collection, preparation, and mRNA-sequencing analysis

Mouse Sertoli TM4 cells were seeded at a density of 1.7 × 105/well in 6-well-plates and allowed to attach for overnight before 24 h CBD treatments. Total RNA from DMSO, 10, 12.5, 15, 17.5, or 20 µM CBD-treated mouse Sertoli TM4 cells was extracted using a RNeasy Mini kit (Catalog # 74106, Qiagen, Valencia, CA). The quality and integrity of RNA samples were measured using a NanoDrop™ 8000 (ThermoFisher Scientific), RNA 6000 Nano kits (Agilent Technologies, Santa Clara, CA), and an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA samples with the RNA integrity number > 9 were sent to LC Sciences (Houston, TX) for mRNA sequencing and bioinformatic analysis.

The library for Poly(A) RNA sequencing was prepared according to the TruSeq-stranded-mRNA sample preparation guidelines provided by Illumina (San Diego, CA). mRNA containing Poly(A) tails was isolated through two rounds of purification using oligo-(dT) magnetic beads. Subsequently, the purified poly(A) RNA was fragmented in a divalent cation buffer at an elevated temperature. The DNA library construction involves converting RNA into cDNA through reverse transcription and then ligating DNA fragments to sequencing adapters. For quality control analysis and quantification of the sequencing library, a High Sensitivity DNA Chip was used on the Agilent Technologies 2100 Bioanalyzer. Finally, paired-end sequencing of the library was conducted on Illumina’s NovaSeq 6000 system.

The bioinformatics analysis was conducted by LC Sciences as follows: to assembly transcripts, Cutadapt, a tool for adapter removal, and in-house Perl scripts were used to eliminate reads with adaptor contamination, low-quality bases, and undetermined bases. Sequence quality was then verified using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). For mapping reads to the reference genome, HISAT2, an alignment tool, was utilized to align reads to the mouse genome (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/001/635/GCF_000001635.27_GRCm39). After mapping, StringTie was used to assemble the mapped reads from each sample into transcripts. To construct a comprehensive transcriptome, transcriptomes from all samples were merged using Perl scripts and gffcompare. Differentially expressed mRNAs were identified based on a log2 (fold change) greater than 1 or less than −1, coupled with statistical significance (adjusted p-value < 0.05), using the R package DESeq2. Gene expression data were further analyzed using QIAGEN Ingenuity Pathway Analysis (IPA) to generate results for canonical pathway enrichment, upstream analysis, and network graphs.

mRNA sequencing analyses in primary human Sertoli and Leydig cells were reported in our previous studies (Li et al. 2023b; Li et al. 2023c). In this study, we re-analyze the mRNA sequencing data from primary human Sertoli cells using IPA to generate results for canonical pathway enrichment, upstream analysis, and network graphs. In addition, IPA was used to re-analyze the mRNA sequencing data from primary human Leydig cells to compare upstream regulators in CBD-treated primary human Sertoli and Leydig cells.

2.6. Statistical analyses

Data are presented as the mean ± standard deviation (S.D.) of at least three independent experiments. Analyses were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by the Dunnett’s tests for pairwise-comparisons. The difference was considered statistically significant when p was less than 0.05.

3. Results

3.1. CBD induces apoptosis in mouse Sertoli TM4 cells

Previously, we reported that CBD inhibited cell proliferation in both mouse Sertoli TM4 cells and primary human Sertoli cells at the 24-h and 48-h time points, as determined by measuring cellular growth, cell cycle, and DNA synthesis (Li et al. 2022). Subsequently, we showed that CBD induced cellular senescence in primary human Sertoli cells during prolonged CBD treatment, and that senescence was likely to be the mechanism for CBD-induced growth inhibition (Li et al. 2023c). However, the mechanisms underlying CBD-induced growth inhibition in mouse Sertoli TM4 cells remain unclear. In this study, using an Annexin V-PI staining assay (Fig. 1A), we detected a small but statistically significant increase (3.5 – 5.8 % compared to the DMSO control) in the percentage of early apoptotic cells with 15 – 25 µM CBD at 48 h, but not at 24 h (Fig. 1B–C). We also observed a small increase (2.9 – 3.5 % compared to the DMSO control) in the percentage of late apoptotic cells at 22.5 and 25 µM CBD at 48 h (Fig. 1C).

Fig. 1.

Fig. 1.

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Apoptosis is observed in mouse Sertoli TM4 cells after CBD exposure for 48 h. Mouse Sertoli TM4 cells were treated with DMSO or the indicated CBD concentrations for 24 h or 48 h. (A) Representative scatter plots show the flow cytometric results for Annexin V/PI staining after CBD treatment for 24 h and 48 h. (B & C) Quantification of Annexin V/PI staining results. Bar graphs show the mean percentage of viable, early apoptotic, or late apoptotic cells ± SD (n = 3). *, p < 0.05, significantly different from the DMSO control.

To understand the effects of longer-time exposure to CBD in mouse Sertoli TM4 cells, we monitored cell growth over a period of 6 days. As shown in Fig. 2A, DMSO-treated mouse Sertoli TM4 cells exhibited continuous growth, while treatment with 15 and 20 µM CBD resulted in cell growth arrest from Day 3–6, as evidenced by the lack of increase in cumulative population doublings. This observation is consistent with our previous report indicating that CBD arrests the cell cycle in mouse Sertoli TM4 cells (Li et al. 2022).

Fig. 2.

Fig. 2.

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CBD inhibits cellular growth and induces apoptosis after longer-term incubation in mouse Sertoli TM4 cells. Mouse Sertoli TM4 cells were treated with DMSO, 15 μM CBD, or 20 μM CBD for 6 days. (A) The line graph shows the cumulative population doubling levels (n = 3). (B) The bar graph shows the relative fluorescence intensity of SA-β-gal in mouse Sertoli TM4 cells treated with DMSO or 20 μM CBD. The bar graph represents means ± SD (n = 3). (C) Representative scatter plots show the flow cytometric results for Annexin V/PI staining after DMSO, 15 μM CBD, or 20 μM CBD treatment for 3 or 6 days. (D) Quantification of Annexin V/PI staining results. Bar graphs show the mean percentage of viable, early apoptotic, or late apoptotic cells ± SD (n = 3). *, p < 0.05, significantly different from the DMSO control.

To compare with results from primary human Sertoli cells, we monitored cellular senescence in CBD-treated mouse Sertoli TM4 cells by measuring SA-β-gal activity, an indicator of senescence. On Day 6, we did not observe an increase in SA-β-gal activity, indicating that CBD treatment did not induce cellular senescence in mouse Sertoli TM4 cells (Fig. 2B). However, using an Annexin V-PI staining assay, we found that treatment of mouse Sertoli TM4 cells with 15 and 20 µM CBD for 3 and 6 days increased the percentage of early apoptotic cells and decreased the percentage of viable cells (Fig. 2C and 2D). A significant increase in the percentage of late apoptotic cells was also observed after 6 days of treatment with 20 µM CBD (Fig. 2C and 2D). Specifically, on Day 6, in the 20 µM CBD group, viable cells decreased by 35.7 %, early apoptotic cells increased by 18.7 %, and late apoptotic cells increased by 15.8 % compared to the DMSO group (Fig. 2C and 2D). These observations demonstrate that mouse Sertoli TM4 cells undergo apoptosis, but not cellular senescence, in response to prolonged CBD treatment, which differs from the cellular response observed in primary human Sertoli cells.

3.2. Transcriptomic profile of CBD-treated mouse Sertoli TM4 cells

To explore the cytotoxic effects of CBD at the molecular level, mRNA-sequencing analysis was performed on mouse Sertoli TM4 cells exposed to 10 – 20 µM CBD for 24 h. The resulting cellular growth level was previously reported (Li et al. 2022) and is also displayed in Table 1. CBD treatment at 10 – 17.5 µM did not cause a significant inhibition of cellular growth, while 20 µM CBD treatment caused a mild (< 30 %), but statistically significant, cellular growth inhibition. We chose these treatment conditions (10 – 20 µM CBD), which caused modest toxicity responses, to study the transcriptomic profile.

The total number of differentially expressed genes (DEGs) in mouse Sertoli TM4 cells is shown in Fig. 3A. There was a noticeable increase in the total number of DEGs as the concentration of CBD increased. CBD treatment at 10 – 17.5 µM resulted in 23–383 DEGs. The number of DEGs increased to 908 with CBD treatment at 20 µM. Of 908 DEGs, 605 genes were upregulated, and 303 genes were downregulated (Fig. 3A). A Venn diagram in Fig. 3B illustrates the overlap of DEGs across different concentrations of CBD treatment. We observed that the gene changes were consistent across the various CBD concentrations, as the genes affected at lower concentrations were also affected at higher concentrations. We summarized the expression level changes of 23 common genes in Fig. 3C, all of which were upregulated in a concentration-dependent manner. Many of these genes are involved in immune responses, such as antiviral defense, or are modulated by interferon. Notable examples with large fold changes in gene expression include interferon-induced protein 44 (Ifi44), ubiquitin specific peptidase 18 (Usp18), interferon-induced protein with tetratricopeptide repeats 3 (Ifit3), and 2′−5′-oligoadenylate synthetase 3 (Oas3).

Fig. 3.

Fig. 3.

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Transcriptomic profile of CBD-treated mouse Sertoli TM4 cells. (A) Number of DEGs in mouse Sertoli TM4 cells treated with 10 – 20 μM CBD for 24 h. Blue bars indicate the number of down-regulated genes and red bars represent the number of up-regulated genes. (B) Venn diagrams show the number of DEGs from CBD-treated mouse Sertoli TM4 cells. Numbers in each section represent the numbers of DEGs at different CBD concentrations. Venn diagrams were created using InteractiVenn: http://www.interactivenn.net/. The dark orange-colored center area in the Venn diagrams denotes the number of overlapped DEGs across all concentrations tested. (C) Heatmap showing the changes of expression level of 23 common genes in mouse Sertoli TM4 cells upon treated with 10 – 20 μM CBD.

3.3. Changes in top 20 enriched pathways in CBD-treated mouse Sertoli TM4 cells compared to the same pathways in primary human Sertoli cells

Pathway enrichment analysis using DEGs revealed that the top 20 canonical pathways induced by 10 – 20 µM CBD treatment in mouse Sertoli TM4 cells showed a concentration-dependent pattern (Fig. 4, left side). These pathways may contribute to the upstream mechanisms of CBD-induced cytotoxicity or reflect outcomes of CBD-induced phenotypes in mouse Sertoli TM4 cells. Notably, immune responses, such as “Interferon alpha/beta signaling” (Rank #1), “Interferon gamma signaling” (Rank #3), and “ISGylation signaling pathway” (Rank #4) were among the top-ranked pathways.

Fig. 4.

Fig. 4.

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Comparison of pathway enrichment in CBD-treated mouse Sertoli TM4 cells with primary human Sertoli cells using IPA. Left side shows heatmap visualization of top 20 canonical pathways changed in a concentration-dependent manner in mouse Sertoli TM4 cells, based on DEGs from 10 – 20 μM CBD treatment. Right side shows corresponding changes in the same pathways in CBD-treated primary human Sertoli cells. Blue represents down-regulated pathways and orange represents up-regulated pathways. A z-score threshold of ± 2 was used to infer significant up-regulation or down-regulation. When the absolute z-score is below 2, the alterations in gene expression within the pathway do not strongly indicate a distinct pattern.

The enrichment of pathways such as “Role of hypercytokinemia/hyperchemokinemia in the pathogenesis of influenza” (Rank #2) and “Pathogen-induced cytokine storm signaling pathway” (Rank #7) suggests that CBD might induce a cytokine storm, an excessive inflammatory response. Furthermore, pathways, such as “Pyroptosis signaling pathway” (Rank #12) indicate CBD’s potential to induce cell death through inflammatory processes in mouse Sertoli TM4 cells.

The “Coordinated Lysosomal Expression and Regulation (CLEAR) signaling pathway” (Rank #19) plays a role in lysosomal function and regulates processes, such as autophagy, exocytosis, endocytosis, phagocytosis, and immune responses (Palmieri et al. 2011). CBD-induced enrichment of the CLEAR signaling pathway in mouse Sertoli TM4 cells suggests that CBD may affect lysosomal activity, potentially aiding in managing cytotoxic stress.

To compare further CBD-induced molecular responses between mouse and human Sertoli cells, the mRNA-sequencing data of mouse Sertoli TM4 cells were compared with those previously obtained by our lab from primary human Sertoli cells (Li et al. 2023c). Specifically, the changes in top 20 canonical pathways in mouse Sertoli TM4 cells were compared with the changes in the same pathways in primary human Sertoli cells treated with 7 – 10 µM CBD for 24 h using the same IPA method (Fig. 4, left side vs. right side). CBD-induced inhibition of cellular growth level in primary human Sertoli cells was previously reported (Li et al. 2022) and is displayed in Table 1. Treatment of primary human Sertoli cells with 7 µM CBD did not cause significant cellular growth inhibition, while treatment of 8 – 10 µM CBD led to statistically significant cellular growth inhibition ranging from 8.3 % to 34.8 % (Table 1).

The comparison showed 15 out of 20 pathways that significantly changed in mouse Sertoli TM4 cells also were altered in primary human Sertoli cells, with 6 pathways showing a consistent trend (an absolute Z score greater than 2 indicates significance) (Fig. 4). These pathways were “Role of Hypercytokinemia/hyperchemokinemia in the pathogenesis of influenza”, “Pathogen induced cytokine storm signaling pathway”, “Response of EIF2AK1 (eIF2α kinase, also called heme-regulated inhibitor, HRI) to heme deficiency”, “Platelet homeostasis”, “Neutrophil degranulation”, and “CLEAR Signaling Pathway”.

3.4. Changes in the top 20 enriched pathways in CBD-treated primary human Sertoli cells compared to the same pathways in mouse Sertoli TM4 cells

As shown in Fig. 5 (left side), analysis of DEGs using the IPA method in primary human Sertoli cells treated with 7 – 10 µM CBD revealed enrichment in pathways related to cell cycle regulation and DNA metabolism, consistent with our previously reported enriched pathways using KEGG pathway database (Li et al. 2023c). IPA offers an advantage over KEGG by indicating the direction (upregulation or downregulation) of gene/pathway regulation, whereas annotation by KEGG database only reflects relevancy without specifying directionality. Notably, pathways such as “Cell cycle checkpoints” (Rank #1), “Mitotic prometaphase” (Rank #2), and “Mitotic metaphase and anaphase” (Rank #3) were prominently downregulated. The suppression of pathways within the same category, such as “Mitotic G2-G2/M phases” (Rank #9), “Mitotic G1 phase and G1/S transition” (Rank #10), and “Mitotic prophase” (Rank #17), indicates CBD’s negative impact on the precise control of cell division. In addition, the downregulation of “Synthesis of DNA” (Rank #4) and related processes such as “Activation of the pre-replicative complex” (Rank #11) and “DNA replication pre--Initiation” (Rank #14) suggests that CBD may inhibit DNA replication at an early stage. Our previous study confirmed CBD’s ability to halt cell cycle progression and DNA replication activity (Li et al. 2022). Furthermore, the downregulation of “Homologous recombination repair or single strand annealing” (Rank #5) indicates CBD’s potential negative regulation of DNA repair processes dependent on homologous recombination repair and single strand annealing.

Fig. 5.

Fig. 5.

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Comparison of pathway enrichment in CBD-treated primary human Sertoli cells with mouse Sertoli TM4 cells using IPA. Left side shows heatmap visualization of top 20 canonical pathways changed in a concentration-dependent manner in primary human Sertoli cells, based on DEGs from 7 – 10 μM CBD treatment. Right side shows corresponding changes in the same pathways in CBD-treated mouse Sertoli TM4 cells. Blue represents down-regulated pathways and orange represents up-regulated pathways. A z-score threshold of ± 2 is used to infer significant up-regulation or down-regulation.

We then evaluated whether similar pathway changes in CBD-treated primary human Sertoli cells could be found in mouse Sertoli TM4 cells, and the results are shown in Fig. 5, left side vs. right side. Interestingly, most of the top canonical pathways that changed in primary human Sertoli cells did not exhibit similar changes in mouse Sertoli TM4 cells (Fig. 5). Among all the 20 pathways, only one pathway, “Response of EIF2AK1 to heme deficiency”, showed the same trend of changes in both primary human Sertoli cells and mouse Sertoli TM4 cells (an absolute Z score > 2). The deregulated pathways related to cell cycle regulation and DNA metabolism in primary human Sertoli cells did not significantly change in mouse Sertoli TM4 cells. Taken together, little similarity was found in CBD-induced top canonical pathway changes in primary human Sertoli cells when analyzing the same pathways in mouse Sertoli TM4 cells.

3.5. Comparison of the genes involved in male reproductive function in mouse Sertoli TM4 and primary human Sertoli cells

In addition to canonical pathways, our analyses focused on genes known to be crucial for Sertoli cell and male reproductive function. As shown in Table 2, in primary human Sertoli cells treated with 10 µM CBD, the expression of androgen receptor (AR), Wilms’ tumor 1 (WT1), and SRY-box transcription factor 9 (SOX9) genes decreased by more than 2-fold; and steroid 5-alpha reductase 1 (SRD5A1), steroidogenic acute regulatory protein (STAR), and transferrin receptor (TFRC) also significantly decreased, though less than 2-fold. Clusterin (CLU) increased by approximately 2-fold. In mouse Sertoli TM4 cells treated with 20 µM CBD, Sox9 decreased by more than 2-fold, while Srd5a1, Star, Clu, and Tfrc showed significant decreases with fold changes of less than 2-fold.

Table 2.

Gene expressions that relevant to the function of Sertoli cells.

Primary human Sertoli cells Mouse Sertoli TM4 cells
Gene name Base mean* of control Base mean of 10 µM CBD Log2 fold change# Adjusted p value Gene name Base mean* of control Base mean of 20 µM CBD Log2 fold change# Adjusted p value
LHCGR 0 0 NA NA Lhcgr NA NA NA NA
CYP11A1 17.79 13.02 −0.45 0.50 Cyp11a1 2.61 0 −0.024 NA
HSD3B1 0 0 NA NA Hsd3b1 NA NA NA NA
CYP17A1 0 0 NA NA Cyp17a1 NA NA NA NA
HSD17B3 0 0 NA NA Hsd17b3 NA NA NA NA
SRD5A1 1144.88 919.23 −0.32 2.42E—05 Srd5a1 1358.20 699.66 −0.90 1.61E—17
AKR1C4 0 0 NA NA AKR1C4 NA NA NA NA
STAR 109.53 64.21 −0.77 1.64E—03 Star 168.45 118.76 −0.41 2.71E—02
INSL3 0 0 NA NA Insl3 1.86 11.42 0.12 0.27
AR 35.55 15.66 −1.19 5.96E—03 Ar 1748.07 1578.09 −0.14 0.20
FSHR 0 0 NA NA Fshr NA NA NA NA
CLU 705.48 1397.10 0.99 1.30E—32 Clu 44.01 16.50 −0.51 2.48E—02
TGFB1 9475.36 9462.78 0.00 0.98 Tgfb1 2093.51 1954.46 −0.10 0.21
TFRC 16835.42 12631.42 −0.41 1.82E—45 Tfrc 13721.71 10185.63 −0.42 3.53E—14
GATA1 0 0 NA NA Gata1 NA NA NA NA
WT1 3038.70 1067.08 −1.51 7.16E—118 Wt1 7.3 14.94 0.12 0.42
CYP19A1 0 0.43 1.34 NA Cyp19a1 NA NA NA NA
SOX9 875.90 427.65 −1.04 3.04E—30 Sox9 1299.60 499.48 −1.32 1.48E—54
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*

base mean from DESeq2 represents the average of normalized count values divided by size factors, calculated across all samples.

#

log2 fold change indicates the magnitude of change in gene expression between the treatment and control groups. Gene name abbrevations: LHCGR, luteinizing hormone/choriogonadotropin receptor; CYP11A1, cytochrome P450 family 11 subfamily A member 1; HSD3B1, hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1; CYP17A1, cytochrome P450 family 17 subfamily A member 1; HSD17B3, hydroxysteroid 17-beta dehydrogenase 3; SRD5A1, steroid 5 alpha-reductase 1; AKR1C4, aldo-keto reductase family 1 member C4; STAR, steroidogenic acute regulatory protein; INSL3, insulin like 3; AR, androgen receptor; FSHR, follicle stimulating hormone receptor; CLU, clusterin; TGFB1, transforming growth factor beta 1; TFRC, transferrin receptor; GATA1, GATA binding protein 1; WT1, Wilms tumor 1; CYP19A1, cytochrome P450 family 19 subfamily A member 1; SOX9, SRY-box transcription factor 9.

3.6. Comparison of the upstream regulators and networks in mouse Sertoli TM4 and primary human Sertoli cells

In addition to pathway enrichment, we summarized the upstream regulators induced by CBD in a concentration-dependent manner. The top 25 predicted upstream regulators in mouse Sertoli TM4 and primary human Sertoli cells are shown in Fig. 6A and 6B, respectively. IFNG (IFN-γ) was the only common top upstream regulator identified in both mouse Sertoli TM4 cells and primary human Sertoli cells treated with CBD (Fig. 6A and 6B). The IFNG gene encodes a cytokine predominantly produced by T cells and natural killer cells, critical for innate and adaptive immunity against viral and intracellular bacterial infections, as well as for tumor control (Alspach et al. 2019). The predicted activation of IFNG in both human and mouse Sertoli cells suggests that CBD modulates immune responses in these cells.

Fig. 6.

Fig. 6.

Open in a new tab

Prediction of upstream regulators in CBD-treated mouse Sertoli TM4 cells and primary human Sertoli cells using IPA. Heatmap visualization of top 25 predicted upstream regulators in (A, left side) mouse Sertoli TM4 cells and (B, left side) primary human Sertoli cells from DGEs across various CBD treatments. Right sides in A and B show corresponding changes in the same upstream regulators in CBD-treated primary human Sertoli cells (A, right side) and mouse Sertoli TM4 cells (B, right side). An overlapped top upstream regulator between mouse Sertoli TM4 cells and primary human Sertoli cells is underlined. Blue represents down-regulation and orange represents up-regulation.

Despite differences in upstream regulators between mouse and primary human Sertoli cells, the top upstream regulators in primary human Sertoli cells show more similarity to those found in primary human Leydig cells. Here, using IPA, we re-analyzed our published transcriptomic data from primary human Leydig cells treated with 10 – 20 µM CBD for 24 h (Li et al. 2023b). Among the top 25 upstream regulators in primary human Sertoli cells, 11 were in common (Supplemental figure 1): nuclear protein 1 (NUPR1), T-box transcription factor 2 (TBX2), neurotrophic receptor tyrosine kinase 1 (NTRK1), Egfr long non-coding downstream RNA (ELDR), member RAS oncogene family like 6 (RABL6), cytoskeleton associated protein 2 like (CKAP2L), estrogen receptor 1 (ESR1), cyclin dependent kinase inhibitor 2 A (CDKN2A), E2F transcription factor family (E2F), TNF receptor super-family member 9 (TNFRSF9), and membrane associated guanylate kinase, WW and PDZ domain containing 1 (MAGI1), with similar predicted directional changes. The disparity in expression of upstream regulators between Sertoli cells derived from mice and humans is greater than that observed between primary human Sertoli and Leydig cells, indicating that CBD induces a species-specific response.

Network analysis revealed that treatment with 20 µM CBD in mouse Sertoli TM4 cells caused up-regulation of IFN signaling (Fig. 7A). In primary human Sertoli cells, network analysis emphasized that 10 µM CBD induced “Senescence of cells”, involving key factors such as E2F transcription factor 2 (E2F2), E2F3, forkhead box M1 (FOXM1), dual specificity tyrosine phosphorylation regulated kinase 1 A (DYRK1A), and helicase (HELLS) (Fig. 7B). This confirmed our previous observation that CBD induced senescence in primary human Sertoli cells (Li et al. 2023c). Overall, the upstream regulators and networks between CBD-treated mouse and human Sertoli cells show low similarity, indicating that Sertoli cells exhibit a unique species-specific reaction to CBD treatment.

Fig. 7.

Fig. 7.

Open in a new tab

Network prediction of biological processes in CBD-treated mouse Sertoli TM4 cells and primary human Sertoli cells using IPA. (A) Graphic summary of biological processes in mouse Sertoli TM4 cells treated with 20 μM CBD for 24 h. (B) Graphic summary of biological processes in primary human Sertoli cells treated with 10 μM CBD for 24 h. Blue indicates inhibition, and orange indicates activation.

4. Discussion

In this study, we observed apoptosis in mouse Sertoli TM4 cells following prolonged CBD exposure, which was not evident in our previous study when the treatment period was limited to 24 h. In our previous study, we reported that a 24-h CBD exposure of both mouse Sertoli TM4 and primary human Sertoli cells led to inhibition of cell proliferation, cell cycle arrest, and suppression of DNA synthesis. However, we did not observe a significant increase in activation of caspase 3/7, which would be indicative of the induction of apoptosis (Li et al. 2022). In this study, extending the exposure time to 48 h resulted in apoptosis in mouse Sertoli TM4 cells (Fig. 1), which was more profound at longer exposure times of 3 and 6 days (Fig. 2). This phenotype differs from that observed in primary human Sertoli cells, where a 6-day exposure to CBD led to cellular senescence. Furthermore, we explored the molecular changes induced by CBD in mouse Sertoli TM4 cells through mRNA sequencing and compared these changes with those in primary human Sertoli cells. We focused on pathways with a concentration-response relationship at the transcriptomic level and found that CBD induced cellular stress-related pathways in mouse Sertoli TM4 cells. The transcriptomic data revealed that the top enriched canonical pathways involve a complex interplay of immune response, inflammation, and stress response mechanisms (Fig. 4). These pathways may underlie the molecular basis of CBD-induced cytotoxicity in mouse Sertoli TM4 cells.

The recommended dose of CBD for treating epilepsy patients is 5–20 mg/kg body weight per day, equivalent to 375–1500 mg/day for a 75 kg adult. In a pharmacokinetics study with healthy volunteers taking 1500 mg of CBD daily (750 mg twice per day) for seven consecutive days, plasma Cmax values for CBD were 0.9 μM on the morning of Day 1, 2.3 μM in the afternoon of Day 1, and 1.1 μM on the morning of Day 7 (Taylor et al. 2018). Following a high-fat breakfast, the plasma Cmax of CBD increased 5-fold compared to a fasted state (Taylor et al. 2018). Additionally, liver diseases may alter the Cmax; for example, individuals with hepatic impairment showed increased CBD Cmax proportional to the severity of liver dysfunction (Taylor et al. 2019). While CBD’s tissue distribution and accumulation in patients remain unclear, a study using physiologically based pharmacokinetic (PBPK) modeling predicted that after a 30 mg oral dose, the liver Cmax could reach as high as 16.9 times the plasma Cmax (Liu and Sprando, 2023). However, whether CBD can accumulate in the testes or what the potential testicular Cmax might be is still unknown. In this study, CBD treatment lasted up to 6 days, at which point most CBD-treated cells exhibited apoptotic death. It is important to consider that this exposure period is notably shorter than the typical duration of CBD use, whether as an unregulated supplement or prescribed medication. Therefore, given the potential for CBD accumulation in the testes and prolonged used contributing to a higher Cmax, the concentrations used in this study (7 – 10 μM in primary human Sertoli cells and 10 – 20 μM in mouse Sertoli TM4 cells) are considered biologically relevant as they approached human plasma concentrations.

In primary human Sertoli cells, the top canonical pathway changes were related to the downregulation of cell cycle related pathways and DNA synthesis. When comparing the top canonical pathways, we found that the top changed pathways in mouse Sertoli TM4 cells shared some similarities with those in primary human Sertoli cells; however, the top changed pathways in primary human Sertoli cells were unique since most of them were absent in mouse Sertoli TM4 cells. Given the differences in major cellular responses and molecular mechanisms towards CBD in mouse and human Sertoli cells, it is possible that the toxicity mechanisms might differ between species.

In mouse Sertoli TM4 cells, the most affected pathways were primarily related to immune and stress responses. For instance, interferons (IFNs) are cytokines produced by cells in response to pathogens, triggering immune defenses (Platanias, 2005). The IFN system, including IFN genes and their activated targets, serves as a broad alarm for cellular stress, including DNA damage (Nallar and Kalvakolanu, 2014). IFN-α and IFN-β activate the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, regulating genes involved in antiviral and inflammatory responses (Ivashkiv and Donlin, 2014). IFN-γ enhances immune cell activity (Alspach et al. 2019). ISGylation, induced by type I IFNs, such as IFN-α and IFN-β, is part of the cell’s antiviral defenses (Zhang et al. 2021). In the context of CBD treatment, upregulation of IFN signaling pathways suggests activation of immune responses in mouse Sertoli TM4 cells, possibly due to CBD-induced cellular stress. In addition, the activation of cytokine storm signaling pathway and CLEAR signaling pathway, both upregulated in CBD-treated mouse Sertoli TM4 cells, is associated with cytotoxic and immune responses. The cytokine storm pathways involve an overwhelming release of cytokines and chemokines, which, can cause tissue damage if dysregulated (Karki and Kanneganti, 2021). Both preclinical and clinical evidence link cytokine storms to programmed cell death, including inflammasome-dependent pyroptosis, apoptosis, and necroptosis (Karki and Kanneganti, 2021). Pyroptosis, an inflammation-associated form of cell death, involves cellular membrane rupture and release of pro-inflammatory cytokines (Man et al. 2017). The presence of this pathway suggests that higher concentrations of CBD (15 – 20 µM CBD) may induce inflammation and subsequent cell death in mouse Sertoli TM4 cells, consistent with the observed increase in early and late apoptotic cells. Similarly, pathways such as “Platelet hoemeostasis” and “Neutrophil degranulation” are related to inflammatory response (Herter et al. 2014; Lacy, 2006). The “Response of EIF2AK1 to heme deficiency” pathway is another affected pathway. It responds to heme deficiency and unfolded cytosolic proteins. EIF2AK1 phosphorylates eukaryotic initiation factor-2α (eIF2α) and enhances the translation of activating transcription factor 4 (ATF4) mRNA to induce stress response genes and inhibit general protein synthesis (Chen and London, 1995; Chen and Zhang, 2019). Besides heme deficiency, EIF2AK1 responds to various stress conditions, such as oxidative stress, osmotic shock, and heat shock (Donnelly et al., 2013; Oyadomari and Mori, 2004). Unresolved cellular stress can ultimately result in apoptosis regulated by DNA Damage Inducible Transcript 3 (DDIT3) (Oyadomari and Mori, 2004). This highlights CBD’s potential role in inducing cellular stress responses in mouse Sertoli TM4 cells. The impact of CBD on the inflammatory and immune responses at the gene expression level has been previously documented in another cell type, HepaRG cells. In a transcriptomics study conducted with liver cells, Li et al. reported that CBD treatment of human HepaRG cells, commonly used for studying metabolism and toxicity, resulted in the suppression of many genes linked to immune function and inflammatory response, with the most sensitive pathways being immune-related (Li et al. 2023a). Further analysis and explanation are needed to understand why immune modification is prominent in HepaRG cells (Guo et al. 2024; Li et al. 2023a; Marion et al. 2010) and mouse Sertoli TM4 cells (our current report).

Previous studies in immune cell types indeed suggest that the immune system is a target for CBD. For example, CBD suppressed cytokine production and induced apoptosis in a human leukemia cell line, HL-60 (3.2 – 26 µM CBD), and primary human monocytic cells (1 – 16 µM CBD) (Nichols and Kaplan, 2020). CBD also induced apoptosis in human T cells, such as Jurkat and MOLT-4 T cells (Nichols and Kaplan, 2020). CBD has also been reported to have immune enhancing effects, such as the stimulation of neutrophil degranulation, which results from the increased intracellular calcium (Nichols and Kaplan, 2020). Moreover, enhanced expression of the cytokine IFN-ɣ due to CBD treatment has been reported (Chen et al. 2012; Kozela et al. 2016). In contrast, some studies indicated IFN-ɣ is an important target of suppression by CBD in immune cell contexts (Nichols and Kaplan, 2020). In our study, CBD treatment upregulated the neutrophil degranulation pathway in mouse Sertoli TM4 cells (Fig. 3C) and upregulated IFN-ɣ gene in both human and mouse Sertoli cells (Fig. 6), which is consistent with some previous findings. The direction of the immune response may vary depending on cell type and cell fate. Further investigations are needed to clarify these mechanisms.

Interestingly, in primary human Sertoli cells, the upregulated pathways, including “Cholesterol biosynthesis” (Rank #12), “Superpathway of cholesterol biosynthesis” (Rank #13), and “Activation of gene expression by SREBF (sterol regulatory element-binding proteins, SREBP)” (Rank #16), suggest that CBD may positively influence the regulation of genes involved in lipid metabolism and cholesterol synthesis (Fig. 5). The “Activation of gene expression by SREBF” is an important mechanism in maintaining cellular lipid homeostasis, as it responds to changes in lipid levels by upregulating genes required for the synthesis and uptake of cholesterol, fatty acids, and triglycerides (Eberle et al. 2004). These processes are essential for the structure and function of cell membranes and for the synthesis of steroid hormones. However, in mouse Sertoli TM4 cells, the “Cholesterol biosynthesis” and “Superpathway of cholesterol biosynthesis” pathways were significantly downregulated (Fig. 5), suggesting that the role of CBD in regulating cholesterol synthesis differs between the two species.

A previous study in mice found that CBD treatment caused a decrease of Sertoli cell number (Carvalho et al. 2018a). Our observation that CBD induced apoptosis in mouse Sertoli TM4 cells provides a plausible explanation for the phenotype observed in mice. The underlying mechanism of apoptosis in mouse Sertoli TM4 cells can be related to immune response or inflammation, according to our transcriptomic analysis. The ability of CBD to induce apoptosis has been widely reported previously in cancer cell lines and immune cells through other mechanisms, such as interacting with PPARγ receptor (Ramer et al. 2013) and p53 pathways (Jeon et al. 2023; Wang et al. 2023), reactive oxygen species production (Wu et al. 2008), endoplasmic reticulum stress (Chen et al. 2024a; Shrivastava et al. 2011), and mitochondrial dysfunction (Jeong et al. 2019). Sertoli cell injury has been previously reported to be associated with the disruption of the blood-testis barrier, infiltration of macrophages, and germ cell apoptosis (Murphy and Richburg, 2014).

In addition to apoptosis, our transcriptomic data (Table 2) show a significant decrease (adjusted p value less than 0.05) in the gene expression of functional markers important for male physiology, including AR, WT1, SOX9, SRD5A1, STAR, and TFRC in primary human Sertoli cells. Similarly, we observed CBD-induced decreases in the gene expression of Sox9, Srd5a1, Star, Clu, and Tfrc in mouse Sertoli TM4 cells. AR plays a central role in responding to androgen signaling, which is essential for regulating Sertoli cell maturation, Sertoli-Sertoli and Sertoli-germ cell junctions, and germ cell proliferation and differentiation (Edelsztein and Rey, 2019). The WT1 gene encodes a transcription factor that regulates genes involved in Sertoli cell differentiation and function, which are essential for overall testis development and spermatogenesis (Chen et al. 2017; Wang et al. 2019; Wen et al. 2016). Our previous study confirmed that CBD treatment decreases WT1 protein levels in both mouse Sertoli TM4 cells and primary human Sertoli cells (Li et al. 2022). Furthermore, SOX9 is a critical transcription factor necessary for the differentiation and maintenance of Sertoli cells during fetal development and throughout adulthood (Barrionuevo et al. 2016; Jakob and Lovell-Badge, 2011). The reduction of AR, WT1, and SOX9 in primary human Sertoli cells could compromise their normal functions, such as sperm production and adult testis maintenance (Barrionuevo et al. 2016; Edelsztein and Rey, 2019; Jakob and Lovell-Badge, 2011). Similarly, the decrease in Sox9 expression in mouse Sertoli TM4 cells may impair their functionality.

STAR is involved in testosterone biosynthesis by facilitating cholesterol transport from the outer to the inner mitochondrial membrane (Manna et al. 2016). While Leydig cells are the primary site of testosterone synthesis in the testes, STAR is also expressed in mammalian Sertoli cells, where it participates in steroidogenesis (Gregory and DePhilip, 1998; Ren et al. 2022). STAR expression correlates with testosterone secretion. SRD5A1, a gene responsive to testosterone, is expressed in Sertoli cells (Ren et al. 2022). SRD5A1 primarily modulates androgen signaling by converting testosterone to dihydrotestosterone, which binds to androgen receptors, influencing spermatogenesis and other male reproductive functions (Wei et al. 2020). The absence of SRD5A1 can lead to abnormal androgen levels (Ren et al. 2022). TFRC contributes to iron homeostasis regulation, and its knockdown has been linked to spermatocyte apoptosis in neonatal mice testis (Gao et al. 2021). Although further verification and experiments are necessary, our transcriptomic results suggest that the consistent decreases in these functional marker genes may contribute to dysregulated Sertoli cells function in both mice and humans.

In summary, our study demonstrates that CBD induces apoptosis in mouse Sertoli TM4 cells. Transcriptomic analysis revealed both similarities and differences in molecular changes associated with CBD exposure between mouse Sertoli TM4 cells and primary human Sertoli cells. The distinct molecular responses to CBD between cell types potentially influence their cytotoxic outcomes. Nevertheless, our study provides insights into the molecular mechanisms underlying CBD-induced cytotoxicity in both human and mouse Sertoli cells, highlighting potential adverse reproductive consequences associated with the use of CBD.

Supplementary Material

Supplement Figure 1

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at 10.1016/j.tox.2025.154068.

Acknowledgments

This work was supported in part by the Human Foods Program. We thank Drs. Frederick A. Beland and Volodymyr Tryndyak for their critical review of this manuscript.

Footnotes

CRediT authorship contribution statement

Chen Si: Writing – review & editing, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Li Xilin: Writing – review & editing, Methodology, Investigation. Li Yuxi: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Choudhuri Supratim: Writing – review & editing, Funding acquisition, Conceptualization. Cournoyer Patrick: Writing – review & editing, Funding acquisition, Conceptualization. Guo Lei: Writing – review & editing, Data curation, Conceptualization.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

Disclaimer

This article reflects the views of the authors and does not necessarily reflect those of the U.S. Food and Drug Administration. Any mention of commercial products is for clarification only and is not intended as approval, endorsement, or recommendation.

Data availability

Data will be made available on request.

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Associated Data

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Supplementary Materials

Supplement Figure 1
NIHMS2105632-supplement-Supplement_Figure_1.pptx (53.2KB, pptx)

Data Availability Statement

Data will be made available on request.

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