ALKBH5 facilitates Srgn stability via m6A demethylation to disrupt blood-testis barrier integrity in the high-fat diet mice

Tian Du; Liangyu Yao; Wenhua Wang; Zhou Li; Jie Dong; Zheng Zhou; Chengyu Shi; Xie Ge; Rujun Ma; Li Chen; Yijian Xiang; Zhaowanyue He; Lu Zheng; Yao Xu; Chun Cao; Jun Jing; Jinzhao Ma; Bing Yao

doi:10.1007/s00018-026-06189-y

. 2026 Mar 30;83(1):197. doi: 10.1007/s00018-026-06189-y

ALKBH5 facilitates Srgn stability via m6A demethylation to disrupt blood-testis barrier integrity in the high-fat diet mice

Tian Du ^1,^2,^#, Liangyu Yao ^3,^#, Wenhua Wang ^4,^#, Zhou Li ^5,^#, Jie Dong ^1,², Zheng Zhou ¹, Chengyu Shi ¹, Xie Ge ^1,², Rujun Ma ^1,², Li Chen ^1,², Yijian Xiang ⁶, Zhaowanyue He ², Lu Zheng ², Yao Xu ⁶, Chun Cao ⁷, Jun Jing ^1,^2,^✉, Jinzhao Ma ^1,^2,^4,^6,^✉, Bing Yao ^1,^2,^4,^6,^✉

PMCID: PMC13062045 PMID: 41910735

Abstract

Obesity has become an important factor affecting male fertility and is often accompanied by abnormal levels of fatty acids, especially saturated fatty acids (SFAs). Palmitic acid (PA; C16:0) is the most frequent SFA in high-fat diet (HFD) and impairs blood-testis barrier (BTB) integrity and decreases sperm quality. RNA N6-methyladenosine (m6A) is involved in lipid metabolism and affects BTB integrity and spermatogenesis. However, it is unclear whether RNA m6A modification plays a role in the BTB damage caused by PA. Herein, we unraveled that PA induced the upregulation of ALKBH5 in TM4 and primary Sertoli cells and this upregulation of ALKBH5 was involved in PA-induced BTB disruption. Conversely, suppressing Alkbh5 expression could alleviate the BTB disruption induced by PA. Moreover, we found Srgn might be a potential downstream target of Alkbh5-mediated BTB integrity suppression. Mechanistically, the Alkbh5 upregulation caused by PA disrupted BTB integrity via enhancing Srgn stability in a RNA m6A dependent manner, and Srgn impaired the integrity of the BTB by activating the NF-κB and MAPK pathways and thereby elevating the expression of Ccl2 and TNF-α in TM4 and primary Sertoli cells. Our study revealed an important role of the regulatory network of ALKBH5-m6A-Srgn in the PA-induced BTB disruption and provided potential therapeutic strategies for improving fertility potential in obese patients.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-026-06189-y.

Keywords: Blood-testis barrier, ALKBH5, N6-methyladenosine, High-fat diet, Male infertility, Spermatogenic microenvironment

Introduction

Due to changes of people’s living habits and diet structure in modern society, there is an increasing number of people with obesity or overweight in the population [1]. Obesity is known to be a major risk factor for male infertility [2]. A meta-analysis showed that total sperm count and sperm progressive motility were significantly decreased in obese men compared with the normal-weight population [3]. Hence, it is valuable to maintain the reproductive health of obese males by exploring the specific mechanism of spermatogenesis reduction in these individuals and developing potential intervention measures.

Our previous study demonstrated obesity was always accompanied by elevation of free fatty acids levels, especially saturated fatty acids (SFAs), and the palmitic acid (PA) was the major component of the serum SFAs and inhibin B (INHB), an important hormone produced by Sertoli cells, was significant decreased in the patients with severe dyszoospermia [4]. Sertoli cells are the most critical cells regulating lipid metabolism in the testis, and BTB is a barrier structure made up of Sertoli cells, including tight junctions, basal ectoplasmic specializations, desmosomes, and gap junctions, which plays an important role in maintaining the spermatogenic microenvironment [5]. Meanwhile, our previous studies have suggested PA decreased the expression of BTB-related proteins and impaired barrier function in Sertoli cells [4, 6].

N6-methyladenosine (m6A) is the most prevalent internal modification of mRNA in higher eukaryotes [7], and is involved in numerous biological processes, including lots of main processes in spermatogenesis [8–11]. Abnormalities in lipid metabolism often alter m6A modification levels of relevant tissue. For example, previous studies indicated that both ALKBH5 and FTO expression were higher in the liver of High Fat Diet (HFD) mice than in normal mice and that these demethylases play an important role in lipid metabolism [12, 13]. RNA m6A modification also has critical effects on male fertility. It has been demonstrated that ALKBH5 knock out (KO) in mice results in testicular atrophy and a decrease in sperm count and viability [8]. Furthermore, RNA m6A modification also plays an important role in Sertoli cells. A study indicated that specific knockdown of ALKBH5 in Sertoli cells injured the function of BTB via regulating Cdh2 mRNA translation in mice [14]. Nevertheless, RNA m6A modification is a dynamic and reversible process. Either downregulation or upregulation of METTL3 expression promoted colorectal cancer progression [15, 16]. We analysed the single-cell sequencing data from a comparison of testicular tissue between patients with high and low BMI [17], and found that ALKBH5 expression was elevated in Sertoli cells of patients with high BMI. This observation suggested that ALKBH5 might play a role in obesity-induced dysfunction of Sertoli cells and that the dynamically reversible process of RNA m6A modification was involved in the processes of spermatogenesis. However, there are few studies exploring the role of RNA m6A modification in HFD-induced BTB injury.

In this study, we aimed to explore the role of RNA m6A modification in Sertoli cells in HFD-induced BTB injury and identified potential therapeutic targets for men with spermatogenic dysfunction due to hyperlipidemic factors.

Materials and methods

Cellular experiment

The TM4 cell line (iCell Bioscience, Inc., Shanghai, China) was cultured in DMEM/F12 (Yuanye, Shanghai, China) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37 °C under a 5% CO₂ atmosphere. Primary Sertoli cells were isolated from the testes of 3-week-old C57BL/6 mice. The specific method was executed using a two-step enzyme digestion based on previous study [6]. The TM4 cell line and primary Sertoli cells were induced to damage using PA at concentration of 0.4 mM, which was determined by referring to estimated concentration of PA in serum and the dose commonly applied in the studies [4, 6, 18–20].

Plasmid and small interfering RNA (siRNA) transfection

pcDNA3.1 vector was used for the construction of the full-length overexpressing mouse ALKBH5, ALKBH5-H205A and Srgn plasmids, which were generated by GenePharma (Shanghai, China). Cells were transfected with plasmids or siRNAs using Lipofectamine 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer’s instructions after being seeded in a dish and cultured for 24 h. The transfection efficiency was evaluated after 24–72 h. The siRNA sequences used in this study were as follows: siAlkbh5-1: 5′ - CACCGCGAUUGGAAACAAATT- 3′, siAlkbh5-2: 5′- GCUGCAAGUUCCAGUUCATT − 3′; siSrgn-1: 5′ - GGATCTTCAGTGCAAGGTT − 3′, siSrgn-2: 5′ - GACCACAGTTTGACCTAAT − 3′, siSrgn-3: 5′ - CCAACAGATGAAAGCAATA − 3′.

Neutralization of CCL2 and TNF-α

Following the instructions provided in the reagent manual, the TM4 and primary Sertoli cells were treated with anti-CCL2 antibody (R&D Systems, AF-479-SP) at a concentration of 2 µg/mL and anti-TNF-α antibody (R&D Systems, AF-410-SP) at 10 ng/mL. Cells treated with Anti-IgG antibody (R&D Systems, AB-108-C) were used as the control. The cells were treated with the neutralizing antibodies for 24 h, and then processed for subsequent experiments.

Quantitative RT-PCR

Total RNA was extracted using a Total RNA Isolation Kit (CWBIO, CW0581S) and reverse-transcribed with Evo M-MLV RT Mix Tracking Kit (Accurate Biology, AG11734). Then, transcripts were amplified using AceQ^® qPCR SYBR Green Master Mix (Vazyme, Q121) by Roche LightCycler 96 Real-time PCR system (Roche Diagnostics, Basel, Switzerland). The sequences of the primers used in this study are listed in Supplementary Table 1.

Western blotting (WB) analysis

When the extraction of proteins was completed, equal protein was loaded for electrophoresis and transferred to PVDF membrane (Merck Millipore, Billerica, MA, USA). Then, the primary antibody against each specific protein and appropriate secondary antibody was incubated with the membrane. Finally, the membrane was visualized with BlodtightTM Western Chemiluminescent HRP Substrate (Bioworld, Visalia, CA, USA), and images were captured by Tanon-5200 Chemiluminescent Imaging System (Tanon Science and Technology, Co., Ltd., Shanghai, China). The main primary antibodies used included: anti-ALKBH5 (1:1000, ab195377, Abcam), anti-SRGN (1:500, sc-374657, Santa; 1:1000, A13340, Abclonal), anti-ZO-1 (1:1000, 21773-1-AP; ProteinTech), anti-Occludin (1:1000, 13409-1-AP; ProteinTech), anti-p38 (1:1000, ab170099, Abcam), anti-phospho-p38 (1:1000, ab45381, Abcam), anti-p65 (1:500, 6956 S, Cell Signaling Technology), anti-phospho-p65 (1:500, 3033 S, Cell Signaling Technology), anti-Vinculin (1:1000, 66305-1-lg, ProteinTech), anti-GAPDH (1:1000, 60004-1-lg; ProteinTech). The secondary antibodies used included: anti-rabbit IgG (1:5000, 31460, Invitrogen) and anti-mouse IgG (1:5000, 31430, Invitrogen).

Transepithelial resistance (TER) measurements

We measured TER to assess the integrity of functional cell barriers as previously described [4]. Briefly, cells were inoculated on to each Millicell Hanging Cell Culture Inserts (PET 0.4 μm, Merck Millipore, Billerica, MA, USA) with 0.5 × 10⁶ cells/cm² and PA was added in the medium 3 days later when the cell barrier formed. Then, the TER was monitored using a Millicell Electrical Resistance System (Merck Millipore).

Animal experiment

All animal experiments were conducted at the Department of Comparative Medicine, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, and approved the Ethics Committee of the Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University (approval number: 2020JLHGKJDWLS-173 of December 15th, 2020).

C57BL/6 mice were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. The control group mice were fed with standard diet (CD) (1010009, Jiangsu Xietong Pharmaceutical Bioengineering Co., Ltd.); and the HFD group mice were fed with HFD (D12492, Research Diet, USA) from week 5 onwards [21].

Constructing a mouse model with Alkbh5 knockdown in Sertoli cells

C57BL/6 male mice of uniform body weight were selected to establish Alkbh5 interference or overexpression model by infection with LV-shAlkbh5-GFP (shAlkbh5: 5′- GCUGCAAGUUCCAGUUCATT − 3′) or LV-oeALKBH5-GFP generated by Corues Biotechnology (Nanjing, China). After the mice were anaesthetized by 2, 2, 2-trimbromoethanol (Avertin) intraperitoneal injection at a dose of 0.2 ml per 10 g, a small wound was made in the midsection to expose the testis. Around 10 µl of PBS containing concentrated lentivirus (1 × 10⁸ TU/ml) was taken up with microinjector and injected into the testicles. Finally, the testis was restored into the scrotum, and the incision was carefully sutured. The mice were housed with free food and water in a 12-h light:12-h dark and 22 ± 2 °C environment for more than 35 days before used for subsequent experiment.

Assessment of BTB integrity in vivo by biotin tracer experiment

The integrity of the BTB was assessed as previously described [22]. Briefly, the testes of anesthetized mice were exposed, and 20 µl of 10 mg/ml Sulfo NHS-LC-Biotin in PBS was administered to each testicle. After 30 min, the mice were euthanised by cervical dislocation under deep anesthesia, and then the testes were removed and flash frozen in liquid nitrogen. Then, the testes were sectioned to 5-µm thickness using a cryostat microtome. Fluor-488-conjugated streptavidin was used to stain the tissues and images were acquired on a fluorescence microscope.

Transmission electron microscopy

The testis tissue was cut into approximately 1 mm³ in size and fixed with an appropriate amount of 2.5% glutaraldehyde. Then, the sections were stained with 1% uranyl acetate and 0.1% lead citrate sequentially, and furthermore observed under a transmission electron microscope.

Immunohistochemical (IHC) and immunofuorescence (IF) of testis tissue

After fixation with 4% paraformaldehyde, the testis tissue was embedded in paraffin and sliced. For IHC, sections were dewaxed in xylene and washed in ethanol. Then, the sections were heated in citric acid antigen repair buffer for 10 min for antigen repair. Subsequently, the sections were incubated in 3% hydrogen peroxide for 25 min at room temperature to block endogenous catalase. The tissue was enclosed with Bovine Serum Albumin (BSA) and incubated with primary antibody (anti-ALKBH5, 1:750, ab195377, Abcam) and horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG, 1:2000, ab205718, Abcam). Finally, the sections were stained with DAB+ SUBSTRATE BUFFER (K3468, DAKO) and counterstained nucleus with hematoxylin. For IF, after dewaxing, the sections were heated in EDTA antigen repair buffer for 10 min for antigen repair, followed by enclosing with BSA. Then, incubation of the sections with primary antibodies (anti-ALKBH5, 1:750, ab195377, Abcam) and secondary antibodies (anti-rabbit IgG, 1:2000, T2769, Thermo) was conducted. The sections were washed and another primary antibody (anti-SOX9, 1:750, ab5535, Merk) and secondary antibodies (anti-rabbit IgG, 1:2000, F2765, Thermo) was conducted. Finally, the DAPI (C1029, Beyotim) was used to stain the nuclei.

IF staining of primary Sertoli cells

The method of IF staining protocol for primary Sertoli cells was essentially similar to that for testis tissue. Briefly, the cells were fixed with 4% paraformaldehyde and then permeabilized with Triton X-100 (P0096, Beyotime), and then blocked with BSA. After washing twice with PBS, cells were incubated with primary antibodies of m6A (1:200, 202003, Synaptic Systems) at 4 °C overnight. Finally, cells were incubated with secondary antibody and the DAPI was used to stain the nuclei.

m6A IP‑qPCR

The m6A IP-qPCR test was performed according to the riboMeRIPTM m6A Transcriptome Profiling Kit instructions (C11051-1). First, total RNA (50 µg) was fragmented into approximately 100–200 nt fragments. Then, fragmented RNA was incubated with anti-m6A magnetic beads A/G. Finally, immunoprecipitated RNA was eluted and recovered and used for qPCR experiments. Primers were designed based on the predicted m6A modification sites of SRAMP(http://www.cuilab.cn/sramp/) and the sequences were listed in Supplementary Table 2.

mRNA stability assay

mRNA stability assay was carried out according to protocols published in the literature [23]. Briefly, after treatment with Actinomycin D (5 µg/mL, Sigma, #c4859) at 37 °C for 0, 4 and 8 h, approximately 2 × 10⁷ cells were taken from each sample and lysed. RT–qPCR was performed to detect the mRNA expression.

Enzyme-linked immunosorbent assay (ELISA)

Cells were processed as needed and cell supernatants were collected and analyzed for Mmp2, Il-6, Il-1β, TNF-α, Il-18, Ccl2 and Ccl3 using ELISA kits according to the manufacturer’s protocol (Mlbio, Shanghai, China).

Single-cell RNA sequencing (scRNA-seq) data source and analysis

Single cell RNA-sequencing data of 3 adult WT and 3 adult ALKBH5 KO mice testes samples were accessed from the open-access data of Hong S et al. (GSE190396) [24]. For scRNA-seq data analysis of obese population, 5 old individuals (> 60 years old) with normal BMI and 3 old individuals with higher BMI (> 30 kg/m²) data (Old+obesity group) were obtained from Nie X et al. (GSE182786) [17]. ScRNA-seq data were analyzed by Seurat program (http://satijalab.org/seurat/, R package, version 4.4). Briefly, after cell filtration, normalization, dimensionality reduction, and clustering, data from different groups were integrated based on the canonical-correlation analysis (CCA) algorithm to eliminate batch effects. Finally, cell type annotation was achieved by marker genes from previous research [25, 26]. The ALKBH5 mRNA expression matrix was extracted and visualized using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). For identification of differential expression genes (DEGs), FindMarkers function of Seurat (test.use=wilcox, min.pct = 0.1) was performed between Sertoli cells from different group.

RNA sequencing and data analysis

Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After eukaryotic mRNA was enriched by Oligo(dT) beads, fragmented into short fragments, and transcribed into cDNA by using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB#7530, New England Biolabs, Ipswich, MA, USA), the cDNA library was sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China). RNA-seq reads were mapped to the mouse genome (Mus musculus, GRCm39) and transcriptome (Ensembl, release 111) using HISAT (v2.2.4) with default parameters. For multiple correction, we utilized Benjamini–Hochberg method to control the False Discovery Rate (FDR) using sequential modified Bonferroni correction for multiple hypothesis testing [27]. And FDR < 0.05 was considered significant.

For RNA-Seq data of 4 Srgn^+/+ and Srgn^−/− groups of adult mice’s epididymal white adipose tissue (eWAT) (GSE166019) [28], the GEO query R package (v.2.66.0) was used to retrieve raw RNA data and to assign gene and group information. DEGs between the two groups were identified using DESeq2 package in R [29], with the parameter of |log2FoldChange| ≥1 and false discovery rate (FDR) < 0.05 using the Benjamini–Hochberg method.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs were performed using DAVID online analysis tool (https://davidbioinformatics.nih.gov/). DEGs and gene enrichment analyses were visualized using the ggplot2 R package (v3.5.0) [30].

Statistical analysis

Data are expressed as the mean ± standard deviation (SD) for at least three independent experiments. SPSS 22.0 (SPSS Inc., Chicago, IL, USA) was used to conduct statistical analyses and GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA) was used to plot data. Differences between the two groups were analyzed using Student’s t-test and One-way ANOVA was adopted when groups were more than three. P value < 0.05 was considered statistically significant.

Results

ALKBH5 was upregulated with PA treatment in TM4 and primary Sertoli cells

Our prior study demonstrated that HFD induced local accumulation of saturated fatty acids, especially PA, in the testis of mice [4]. Meanwhile, we analyzed the data in dataset GSE182786 and found that the expression of ALKBH5 in Sertoli cells of people with high BMI was higher than that of people with low BMI (Supplementary Fig. 1A-C). This observation implied that ALKBH5 might contribute to HFD-induced dysfunction of Sertoli cells.

In order to investigate whether PA affects the RNA m6A modification in Sertoli cells, we extracted primary Sertoli cells from the testes of 3-week-old C57BL/6 mice and identified their purity using immunofluorescence staining of SOX9, a marker of Sertoli cells (Supplementary Fig. 1D). Then, we added PA into TM4 and primary Sertoli cells and analyzed the expression of major “writers” and “erasers” involved in m6A modification by RT-qPCR. The results revealed that Alkbh5 was upregulated significantly after PA treatment in TM4 and primary Sertoli cells (Fig. 1A). Consistently, the WB results indicated that ALKBH5 expression increased in a dose-dependent manner upon PA treatment (Fig. 1B). These results indicated that PA treatment might generate the upregulation of Alkbh5 expression in mRNA and protein levels in TM4 and primary Sertoli cells. To further explore whether PA affects the level of RNA m6A modification through ALKBH5 in TM4 and primary Sertoli cells, we introduced small interfering RNAs (siRNAs) targeting ALKBH5 (siALK) or negative control (siNC) to transfect TM4 cells and found that the siALK-2 was the most efficient one to suppress Alkbh5 (Supplementary Fig. 1E), which was selected for the following study. Then, we found the total m6A modification level of mRNA in TM4 and primary Sertoli cells decreased with PA treatment, and recovered in the group of PA treatment with Alkbh5 knockdown (Fig. 1C). IF staining of m6A was performed on primary Sertoli cells and the result exhibited m6A signals decreased with PA treatment, and it was recovered in the group of PA treatment with Alkbh5 knockdown (Supplementary Fig. 1F). These results demonstrated that PA treatment led to upregulation of ALKBH5 expression thereby causing a decrease in total RNA m6A modification levels in TM4 and primary Sertoli cells, which implied that ALKBH5-mediated RNA m6A modification may be involved in dysfunction of Sertoli cells caused by PA. Then, the IHC assay was conducted to further investigate whether HFD would modify Alkbh5 expression in Sertoli cells in vivo, and the result revealed that Alkbh5 expression increased in Sertoli cells of HFD-treated mice (Supplementary Fig. 1G). In order to exhibit Sertoli cells clearly, we labeled Sertoli cells with SOX9 (a marker of Sertoli cells), and detected the expression of ALKBH5 in Sertoli cells via IF staining. The results showed that the ALKBH5 signal increased in Sertoli cells of HFD-treated mice compared with CD-treated mice (Fig. 1D). In summary, the above results indicated that ALKBH5 was involved in HFD-induced dysfunction of Sertoli cells and upregulated with PA treatment in TM4 and primary Sertoli cells.

Fig. 1 — Alkbh5 was upregulated with PA treatment in TM4 and primary Sertoli cells. A RT-qPCR analysis of m6A regulator genes expression in control (CTL) and PA treatment groups in TM4 and primary Sertoli cells via RT-qPCR. B WB analysis of ALKBH5 protein expression in TM4 and primary Sertoli cells treated with different concentrations of PA. C Total m6A levels of CTL, PA treatment, PA treatment with negative control (siNC) and siALKBH5 (siALK) groups in TM4 and primary Sertoli cells. D Immunofluorescence analysis of ALKBH5 and SOX9, a marker of Sertoli cells, in the testis of control diet (CD) and high-fat diet (HFD) mice groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Alkbh5 mediated PA-induced Sertoli cell barrier disruption in vitro

Our previous study indicated that PA could induce BTB disruption [4, 6]. To determine whether Alkbh5 is involved in PA-induced Sertoli cell barrier disruption, we established a stable TM4 cell line with Alkbh5 knockdown using Alkbh5-interference lentiviral vectors (LV-shALK) or negative control (LV-shNC) (Supplementary Fig. 2A). The WB and TER assay showed the BTB-related proteins (ZO1 and Occludin) and permeability barrier were decreased with PA treatment, but were restored after Alkbh5 knockdown in TM4 cell line (Fig. 2A, B). Similarly, we suppressed Alkbh5 expression in primary Sertoli cells using siRNA (Supplementary Fig. 2B), and found a decrease in BTB-related proteins expression and permeability barrier with PA treatment, but were rescued by suppressing Alkbh5 expression (Fig. 2C, D). These results suggested that Alkbh5 was involved in PA-induced BTB disruption in vitro. Conversely, we established a stable Alkbh5-overexpression TM4 cell line via Alkbh5-overexpression lentiviral vectors (LV-oeALK) or negative control (LV-oeNC), and found a downregulation of BTB-related proteins (ZO1 and Occludin) in the LV-oeALK group compared with LV-oeNC group (Supplementary Fig. 2C-D), which indicated elevated Alkbh5 expression could impair the BTB function. In summary, the above results confirmed that PA disrupted the integrity of BTB via facilitating the expression of Alkbh5 in vitro.

Fig. 2 — Alkbh5 mediated PA-induced BTB disruption in vitro. A WB analysis of the ZO-1 and Occludin proteins expression of TM4 cells in infecting negative control lentivirus (LV-shNC), PA treatment with LV-shNC and ALKBH5 interference lentivirus (LV-shALK) groups. B The TER of TM4 cells in LV-shNC, PA treatment with LV-shNC and LV-shALK groups. C WB analysis of the ZO-1 and Occludin proteins expression of primary Sertoli cells in siNC, PA treatment with siNC and siALK groups. D The TER of primary Sertoli cells in siNC, PA treatment with siNC and siALK groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Alkbh5 in Sertoli cells was involved in HFD-induced spermatogenic dysfunction and BTB disruption in vivo

To further explore whether Alkbh5 mediates HFD-induced BTB disruption in vivo, we suppressed Alkbh5 expression of HFD mice and increased the Alkbh5 expression in CD mice by microinjecting corresponding lentivirus into testicles (Supplementary Fig. 3A). IF and IHC assays demonstrated that ALKBH5 expression in Sertoli cells was either downregulated or upregulated following injection of distinct lentiviruses (Supplementary Fig. 3B and 3C). The sperm parameters were assessed by CASA and the result revealed that HFD decreased the sperm motility and concentration of mice, but knockdown of Alkbh5 in Sertoli cells alleviated this decrease (Fig. 3A). Conversely, overexpression of Alkbh5 in the Sertoli cells of CD mice reduced the sperm motility and concentration (Fig. 3B). H&E staining results confirmed that HFD induced destruction of seminiferous tubules structure, but knockdown of Alkbh5 in Sertoli cells rescued this destruction (Fig. 3C), yet overexpression of Alkbh5 in the Sertoli cells led to disruption of the seminiferous tubules in CD mice (Fig. 3D). which was consistent with the results of sperm parameters above. Besides, the BTB assay exhibited that fluorescence was detected in the lumen of the seminiferous tubules in the testes of HFD mice, but was observed only in the interstitial portion and the basal lines of the seminiferous tubules after suppressing Alkbh5 expression in Sertoli cells (Fig. 3E). Conversely, fluorescence was discovered in the lumen of the seminiferous tubules after overexpressing Alkbh5 in Sertoli cells (Fig. 3F). Finally, the cell junctions between Sertoli cells in the testes were observed by electron microscopy. And the result revealed that the cell junctions between Sertoli cells were loose, broken and structurally disordered in HFD mice, but it was improved by suppressing Alkbh5 in Sertoli cells (Fig. 3G). On the contrary, the cell junctions between Sertoli cells were injured after overexpressing Alkbh5 in Sertoli cells (Fig. 3H). To summarize, these results suggested that Alkbh5 might be involved in HFD-induced spermatogenic dysfunction and BTB disruption in vivo.

Fig. 3 — Alkbh5 in Sertoli cells was involved in HFD-induced spermatogenic dysfunction and BTB disruption in vivo. A Testis weight/body weight, sperm concentration and motility of the CD, HFD mice with LV-shNC and LV-shALK groups. B Testis weight/body weight, sperm concentration and motility of the CD mice with overexpression negative control lentivirus (LV-oeNC) and ALKBH5 overexpression lentivirus (LV-oeALK) groups. C The H&E analysis was conducted to observe the pathological variation of the testis of the CD, HFD mice with LV-shNC and LV-shALK groups. D The H&E analysis of the testis of the CD mice with LV-oeNC and LV-oeALK groups. E BTB assay detecting the integrity of the BTB of the CD, HFD mice with LV-shNC and LV-shALK groups. F BTB assay of the BTB of the CD mice with LV-oeNC and LV-oeALK groups. G The transmission electron microscopy pictures of BTB of the CD, HFD mice with LV-shNC and LV-shALK groups. H The transmission electron microscopy pictures of BTB of the CD mice with LV-oeNC and LV-oeALK groups. White arrows indicate the tight junctions. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

ALKBH5 might facilitate HFD-induced BTB disruption via Srgn in vitro and in vivo

To investigate the intrinsic mechanism of BTB destruction induced by HFD via Alkbh5, we treated primary Sertoli cells with PA to mimic high-lipid-induced damage in vitro, combined with Alkbh5 knockdown, and performed RNA-seq. Then, 187 up-regulated genes and 319 down-regulated genes were identified (|log₂(FC)|>1 and P value < 0.05, Fig. 4A, Supplementary Table 2). KEGG analysis indicated that the upregulated genes were predominantly associated with NOD−like receptor signaling pathway, cytokine−cytokine receptor interaction, toll−like receptor signaling pathway, fatty acid degradation and MAPK signaling pathway, and the downregulated genes were primarily associated with PI3K − Akt signaling pathway, NF−kappa B signaling pathway, chemokine signaling pathway, apoptosis, oxytocin signaling pathway and p53 signaling pathway (Supplementary Fig. 4A). Notably, they were predominantly enriched in fatty acid metabolism and related signaling pathways, which might be associated with PA-induced dysregulation of lipid metabolism. In parallel, a study achieved Alkbh5 knockdown in TM4 cell line without PA treatment and conducted RNA-seq analysis [14]. The KEGG analysis of these data indicated that the upregulated genes after suppressing Alkbh5 without PA treatment were primarily enriched in PI3K − Akt signaling pathway, transcriptional misregulation in cancer, autophagy and regulation of actin cytoskeleton processes, and the downregulated genes were primarily enriched in p53 signaling pathway, NF−kappa B signaling pathway, adherens junction and TNF signaling pathway (Supplementary Fig. 4B). By comparing two datasets, we observed that the DEGs of our RNA-seq data were more strongly associated with fatty acid metabolism pathways, which suggested that Alkbh5 knockdown in Sertoli cells may act as a key modulator of PA-triggered lipid metabolic disturbances. To determine how Alkbh5 is involved in HFD-induced BTB disruption, we combined the single-cell sequencing data of testes in Sertoli cells subset (WT and Alkbh5-KO) from the dataset GSE190396 with our RNA-seq data, and obtained 15 up-regulated genes and 50 down-regulated genes (Fig. 4B, Supplementary Fig. 4C and 4D). The GO analyses exhibited these genes were mainly enriched in Rho guanyl−nucleotide exchange factor activity, regulation of inflammatory response, protein binding, metal ion binding and cytoskeleton, and the KEGG analyses revealed these genes were predominantly associated with regulation of actin cytoskeleton, MAPK signaling pathway, NF−kappa B signaling pathway and tight junction (Fig. 4C), which suggested these overlapping genes might be associated with BTB function. By reviewing published articles of the 65 genes in the database, we selected eight candidate genes that might be associated with BTB function (Fig. 4D). To further identify potential downstream target gene of Alkbh5, we treated TM4 and primary Sertoli cells with PA with or without suppressing Alkbh5, and examined the expression of these candidate genes using RT-qPCR. We found that the mRNA levels of Serglycin (Srgn) were significantly increased in the PA treatment group, but it was significantly decreased after suppressing Alkbh5 in both TM4 and primary Sertoli cells (Fig. 4E, F), which suggested that Srgn might be the downstream target gene of Alkbh5 in HFD-induced BTB disruption.

Fig. 4 — ALKBH5 facilitated HFD-induced BTB disruption via Srgn. A Heatmap of the differentially expressed genes (DEGs) in PA treatment with siNC and siALK groups of primary Sertoli cells. B Combining the single-cell sequencing data of testes in Sertoli cells subset (WT and Alkbh5-KO) from the dataset GSE190396 with RNA-seq data of primary Sertoli cells (PA treatment with siNC and siALK). C GO and KEGG analysis of the 65 overlapping genes. D Heatmap of genes associated with BTB selected from 65 overlapping genes. Those marked in red are the most associated with BTB genes that have been reported. **E, F** The mRNA levels of eight genes most associated with BTB in LV-shNC or NC, PA treatment with LV-shNC or siNC and PA treatment with LV-shALK or siALK groups in TM4 and primary Sertoli cells. G WB analysis of the ZO-1, Occludin and Srgn proteins expression of TM4 cells in siNC, PA treatment with siNC and siSrgn groups. H The TER of TM4 cells in siNC, PA treatment with siNC and siSrgn groups. I WB analysis of the ZO-1, Occludin and Srgn proteins expression of primary Sertoli cells in siNC, PA treatment with siNC and siSrgn groups. J The TER of primary Sertoli cells in siNC, PA treatment with siNC and siSrgn groups. K WB analysis of the ZO-1, Occludin and Srgn proteins expression of testes in CD, HFD mice with LV-shNC and LV-shALK groups. L WB analysis of the ZO-1, Occludin and Srgn proteins expression of testes in CD mice with LV-oeNC and LV-oeALK groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Srgn mainly participates in regulating inflammatory responses, cancer progression, cell adhesion, migration and invasion [28, 31–33]. And numerous studies have reported a positive correlation between Srgn and various inflammatory factors and chemokines, including MMP2, MMP9, CCL2, CCL3, IL-6, IL-18, and TNF-α [28, 32–35], which were proven to take part in disrupting BTB [36–39]. Therefore, we suspected Srgn might be the target gene of Alkbh5 in PA-induced BTB disruption. To further explore whether Srgn was the target gene of Alkbh5 in PA-induced BTB disruption, we introduced siRNA targeting Srgn (siSrgn) or siNC to transfect TM4 cells and found that the siSrgn-2 was the most efficient one to suppress Srgn (Supplementary Fig. 4E), which was selected for the following study. Subsequently, we observed that PA treatment upregulated Alkbh5 expression in TM4 and primary Sertoli cells, and this elevation persisted even when Srgn was concurrently knocked down with PA exposure (Supplementary Fig. 4F). Meanwhile, the BTB-related proteins (ZO1 and Occludin) and permeability barrier were decreased with PA treatment, but were restored after Srgn knockdown in TM4 and primary Sertoli cells (Fig. 4G-J). Conversely, we upregulated Srgn expression in TM4 and primary Sertoli cells using Srgn overexpression plasmid. Transfection efficiency was verified by RT-qPCR in TM4 cells (Supplementary Fig. 5A). Subsequently, we found that Srgn overexpression resulted in decreased expression of BTB-related proteins (ZO1 and Occludin) (Supplementary Fig. 5B). These results illustrated that Srgn might be the downstream target gene of Alkbh5 in HFD-induced BTB disruption in vitro. The results of in vivo experiments indicated that HFD decreased the expression of BTB-related proteins (ZO1 and Occludin) and increased Srgn expression in testicular tissue, which was reversed by Alkbh5 knockdown in Sertoli cells (Fig. 4K). Comparatively, the expression of BTB-related proteins (ZO1 and Occludin) was decreased and the Srgn expression was increased with Alkbh5 overexpression in Sertoli cells in vivo (Fig. 4L). In general, the above results implied that ALKBH5 might facilitate HFD-induced BTB disruption via Srgn in vitro and in vivo.

ALKBH5 increased the stability of Srgn-mRNA in a m6A dependent manner

Moreover, we aimed to elucidate the potential molecular mechanism ALKBH5 regulating Srgn. Previous studies have reported that Alkbh5 served as m6A demethylation in several biological processes [40–42]. We predicted the m6A modification site of Srgn via SRAMP (http://www.cuilab.cn/sramp), a sequence-based m6A modification site predictor, and observed Srgn mRNA carrying eight potential m6A modification sites and primers were designed for the highest-scoring site (Fig. 5A). Then, gene-specific m6A-qPCR was performed to test the m6A modification of Srgn-mRNA, and the result reflected that PA treatment downregulated the m6A enrichment of Srgn in TM4 and primary Sertoli cells, but it was elevated after suppressing Alkbh5 (Fig. 5B), which suggested that Alkbh5 might regulate Srgn expression via RNA m6A demethylation. The above results demonstrated that PA treatment induced upregulation of Alkbh5 in TM4 and primary Sertoli cells, which in turn downregulated the m6A enrichment of Srgn and elevated its mRNA expression. In addition, it has been demonstrated that Alkbh5 regulated gene expression by affecting multiple events, such as pre-mRNA processing, mRNA decay and translation, in RNA metabolism via m6A demethylation [43–45]. Hence, we inferred Alkbh5 might regulate Srgn expression by affecting Srgn mRNA stability via m6A demethylation. To further confirm our hypothesis, we measured the mRNA levels of Srgn after Actinomycin D treatment using qPCR and found that PA treatment increased the half-life of Srgn, but it was reversed with suppressing Alkbh5 in TM4 cells (Fig. 5C). These results suggested that Alkbh5 increased the stability of Srgn mRNA in a m6A dependent manner. Previous studies have demonstrated that mutating amino acid 204 of Alkbh5 may result in a deficiency of ALKBH5 demethylase activity [46]. To clarify the role of ALKBH5-m6A in PA-induced BTB disruption, we constructed a plasmid of ALKBH5 H204A mutant and transfected TM4 and primary Sertoli cells. The results indicated that mutation of ALKBH5 plasmid overexpressed Alkbh5 mRNA and protein similarly to the wild-type (WT) (Fig. 5D-F). The BTB-related proteins (ZO1 and Occludin) were elevated in mutant group compared with oe-ALKBH5 group (Fig. 5G). These data suggested that PA-induced BTB disruption via Alkbh5 was mainly dependent on its demethylase activity.

Fig. 5 — ALKBH5 increased the stability of Srgn-mRNA in a m6A dependent manner. A SRAMP predicted that there may be 8 potential m6A modification sites on Srgn mRNA, of which 3 sites have the potential to be highly modified. B The relative levels of m6A of Srgn mRNA were measured by MeRIP-qPCR from TM4 and primary Sertoli cells LV-shNC or NC, PA treatment with LV-shNC or siNC and PA treatment with LV-shALK or siALK groups. C Srgn mRNA levels were analyzed by RT-qPCR assay of TM4 cells in LV-shNC, PA treatment with LV-shNC and LV-shALK groups after actinomycin D treatment for 0, 4, and 8 h. D Schematic diagram of ALKBH5 H204A. E, F MRNA and protein level of ALKBH5 in overexpression negative control (oeNC), overexpression ALKBH5 (oeALK) and overexpression ALKBH5 H204A (oeALK-H204A) groups of TM4 and primary Sertoli cells. G WB analysis of the ZO-1, Occludin and Srgn proteins expression of TM4 and primary Sertoli cells in oeNC, oeALK, and oeALK-H204A groups. H Heatmap of the DEGs in Srgn^+/+ and Srgn^−/− groups of eWAT in dataset GSE166019. **I, J** ELISA analysis of Mmp2, Ccl2, Ccl3, Il-1β, Il-6, Il-18 and TNF-α of TM4 and primary Sertoli cells in siNC, PA treatment with siNC and siSrgn groups. K Schematic diagram elucidating the network of ALKBH5-m6A-Srgn was involved in the PA-induced BTB disruption. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

What’s more, in order to explore how Srgn affects the function of BTB, we analyzed the DEGs in Srgn^+/+ versus Srgn^−/− epididymal white adipose tissue (eWAT) published in dataset GSE166019 and found that Srgn was positively correlated with inflammatory factors and chemokines, e.g., Mmp9, Mmp2, Ccl2, Il2rb, etc., and negatively correlated with the BTB-related proteins, e.g., Tjp1, Cldn5, Cldn11, Ocln, etc. (Fig. 5H). Meanwhile, numerous studies also suggested that Srgn was positively correlated with MMP2, MMP9, CCL2, CCL3, IL-6, IL-18, TNF-α, IL-1β and other inflammatory factors and chemokines [28, 32–35], which have been demonstrated to be involved in HFD-induced BTB disruption [6, 47–50]. Therefore, ELISAs analysis of these indicators was performed and the results indicated that the expression levels of Ccl2 and TNF-α were increased with PA treatment, which could be reversed after suppressing Srgn in both TM4 and primary Sertoli cells (Fig. 5I, J). Furthermore, it has been demonstrated that increased Srgn expression upregulates phosphorylated p65 and p38, leading to the activation of the NF-κB and MAPK pathway and ultimately enhancing the secretion of inflammatory factors and chemokines [34, 51]. To explore the detailed mechanism by which Srgn regulated Ccl2 and TNF-α in TM4 and primary Sertoli cells, we performed WB analysis to assess the levels of phospho-p65 and phospho-p38 in TM4 and primary Sertoli cells following treatment with either PA alone or PA with siSrgn, and the results revealed that the phosphorylation levels of p65 and p38 were significantly increased upon PA treatment, which was attenuated by siSrgn co-treatment (Supplementary Fig. 5C). Conversely, we transfected TM4 and primary Sertoli cells with a plasmid to overexpress Srgn, and WB analysis revealed that Srgn overexpression led to an increase in the levels of phosphorylated p65 and p38 (Supplementary Fig. 5D). To this end, to delineate the roles of Ccl2 and TNF-α in PA-induced BTB injury, TM4 and primary Sertoli cells exposed to PA were subsequently treated with neutralizing antibodies against Ccl2 and TNF-α, with corresponding IgG antibodies serving as controls. The WB assay showed the BTB-related proteins (ZO1 and Occludin) and permeability barrier were decreased with PA treatment, but were restored after being treated with neutralizing antibodies against Ccl2 and TNF-α in TM4 and primary Sertoli cells (Supplementary Fig. 5E). These findings suggested a mechanism whereby PA-induced Srgn upregulation in TM4 and primary Sertoli cells could enhance Ccl2 and TNF-α secretion, potentially via activation of the NF-κB and MAPK pathways, hence facilitating the BTB disruption. To summarize, our study suggested that the regulatory network of ALKBH5-m6A-Srgn axis played a significant role in the HFD-induced BTB disruption (Fig. 5K).

Discussion

At present, infertility and sterility have become a major health problem for people around the world, and male infertility has become a rising reason [52, 53]. A meta-analysis suggested that obesity negatively affected male spermatogenesis [54]. Obesity is often accompanied by abnormal levels of fatty acids, especially saturated fatty acids (SFAs) and PA is the most frequent SFA [55]. Our previous studies have suggested that PA could induce BTB disruption, thus affecting sperm parameters [4, 6]. M6A modification is the most abundant modification of mRNA in eukaryotes, and a dynamic and reversible biological process that plays a key role in various metabolic processes, e.g., mRNA splicing, translation, transportation and degradation [56, 57]. Abnormal lipid metabolism often affects m6A modification [12, 13], and RNA m6A modification also has an evident impact on male fertility. The deletion of ALKBH5 in male mice disrupted the apoptosis of spermatocytes during meiosis, leading to impaired fertility [8]. Moreover, ALKBH5 and METTL14 regulate autophagy in Leydig cells in the form of m6A modification, thereby regulating testosterone synthesis and indirectly affecting spermatogenesis [58]. Meanwhile, m6A modifications also played a role in Sertoli cells and influenced BTB function. For instance, knockdown of ALKBH5 in mice Sertoli cells disrupted the BTB integrity [14]. In this study, we confirmed that HFD feeding increased ALKBH5 expression in Sertoli cells, and that this upregulation contributes to HFD-induced BTB disruption. Meanwhile, we analyzed published data and found that ALKBH5 expression tended to increase in Sertoli cells in the high BMI group, which also suggested that the increase of ALKBH5 in Sertoli cells might play a role in the spermatogenetic dysfunction of high BMI males. The above results indicated both upregulation and downregulation of Alkbh5 in Sertoli cells impaired BTB function under different conditions, suggesting that the m6A modification of RNA was a dynamic equilibrium process, and the breakdown of the equilibrium would lead to some changes in biological functions.

Srgn is the dominant proteoglycan in immune cells, and main proteoglycan in platelets, T cells, macrophages and mast cells [59], which can interact with many inflammatory mediators and plays an important role in the process of inflammation [59, 60]. Research findings demonstrated that Srgn played an important role in the inflammatory response of adipose tissue, and the mRNA expression of Srgn in adipose tissue of obese human and mice was increased, and accompanied by many immune cell marker genes elevated [28]. Moreover, Srgn deficiency downregulated the expression of Il1b, Infg and Il12b in adipose tissue, and it had a correlation with S100a9, Ccl2, and IL6 [28]. At the same time, Srgn played an important role in tumor invasion and migration. For example, Srgn up-regulated the expression of MMP9 and MMP2 metalloproteinases by activating the ERK pathway, and promoted the invasion and metastasis of esophageal squamous cell carcinoma [35]. Another study has revealed that Srgn regulated the metastasis of triple-negative breast cancer by increasing the expression and secretion of transforming growth factor-β2 (TGFβ2) by activating CD44/CREB1 signal transduction [61]. In addition, the expression levels of IL-1β, TNF-α, and CCL3 were decreased in nucleus pulposus cells transfected with si-Srgn [34]. Notably, these genes and signaling pathways related to Srgn, such as metalloproteinases, Ccl2, inflammatory genes, Wnt/β-catenin pathway, are known regulators of the expression of TJ-related proteins [6, 38, 48–50]. What’s more, we analyzed the DEGs in Srgn^+/+ versus Srgn^−/− eWAT and found that Srgn was negatively correlated with the BTB-related proteins. In this study, we identified that PA treatment upregulated Srgn, which in turn activated the NF-κB and MAPK signaling pathways in TM4 and primary Sertoli cells. This activation drives the expression of Ccl2 and TNF-α, ultimately disrupting the BTB integrity. Furthermore, we demonstrated that Srgn was regulated by Alkbh5 in a m6A-dependent manner in the process of PA-induced BTB disruption, and it was the first time Srgn had been found to be modulated by RNA m6A modifications.

Interestingly, it has been reported that Srgn mRNA expression increases in human adipose tissue with obesity and is reduced upon weight loss [28], which suggested that Srgn expression in vivo was dynamically reversible. At the same time, another study indicated that switching from HFD to CD ameliorated BTB integrity and improved fertility potential in obese male mice [62]. Therefore, we supposed that the improvement of BTB integrity and fertility potential in obese mice by switching from HFD to CD might be due to the change of Srgn expression, which might be a valuable research direction in future.

Spermatogenesis is a complex biological process, and we focused solely on the effects of RNA m6A alterations in Sertoli cells in this study. However, RNA m6A modification in germ cells also plays a critical role in male fertility, and our work did not address how HFD influenced m6A modification of germ cells and thereby affected male fertility, which demanded systematic exploration in further studies. Meanwhile, due to the difficulty of obtaining human Sertoli cells, our study was limited to animal and cellular experiments and the role of m6A modification in obese-induced low sperm concentration needs to be further explored. Moreover, Alkbh5 knockout and knock-in specifically in Sertoli cells mice were required to be constructed to determine the role of the Alkbh5-m6A-Srgn axis in the disruption of BTB in vivo. Additionally, given that ALKBH5 functions as an m6A demethylase targeting a wide array of transcripts, it is plausible that the disruption of BTB integrity observed upon ALKBH5 modulation results from the combined dysregulation of a set of m6A-modified transcripts. Although our data establish a significant functional link within the ALKBH5-Srgn axis, contributions from other m6A-modified transcripts in the overall phenotype cannot be ruled out. Therefore, future studies employing a more comprehensive analysis of global m6A dynamics will be required to delineate the complete network of ALKBH5 targets involved in HFD-mediated BTB impairment.

Conclusion

We found HFD upregulated Alkbh5 expression in Sertoli cells. This upregulation increased Srgn expression in an m6A-dependent manner, and both molecules are crucial for the disruption of BTB. Our study also revealed the Alkbh5-m6A-Srgn axis might play a significant role in HFD-induced BTB disruption by activating the NF-κB and MAPK pathways and thereby promoting the expression of Ccl2 and TNF-α. Importantly, inhibition of Alkbh5 or Srgn could alleviate the BTB disruption induced by HFD, highlighting this pathway as a promising therapeutic target for improving the sperm quality of obese people.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1^{(18.8KB, docx)}

Supplementary Material 2^{(306.4KB, csv)}

Supplementary Material 3^{(2MB, docx)}

Supplementary Material 4^{(6.2MB, xlsx)}

Acknowledgements

We thank the other members of Prof. Yao’s laboratory for their discussion and help.

Author contributions

B.Y., J.M. and J.J. contributed to the conception and design of the study. T.D., Y.L., W.W. and Z.L. analyzed the data and drafted the manuscript J.D., Z.Z., C.S., X.G., R.M., L.C., Y.X., Z.H., L.Z., Y.X. and C.C. participated in performing the experiments and were involved in data acquisition and analysis.

Funding

The study received funding from the National Key Research and Development Program of China (grant no. 2024YFC2706800), the National Natural Science Foundation of China (grant no. 82471651, U22A20277, 32571018, 82274651, 82571858, and 82301869), the China Postdoctoral Science Foundation grant (2023M734294), Jiangsu Provincial Medical Key Discipline Cultivation Unit (JSDW202215), Jiangsu Funding Program for Excellent Postdoctoral Talent (2022ZB759), Nanjing Drum Tower Hospital Youth Cultivation Fund (2025-JCYJ-QP-033), the Foundation of Nanjing Jinling Hospital (2024JJZX171) and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX23_1946).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Consent for publication

Not applicable.

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Tian Du, Liangyu Yao, Wenhua Wang and Zhou Li contributed equally to this work.

Contributor Information

Jun Jing, Email: jingjun36@163.com.

Jinzhao Ma, Email: majinzhao07@163.com.

Bing Yao, Email: yaobing@nju.edu.cn.

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Associated Data

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

Supplementary Materials

Supplementary Material 1^{(18.8KB, docx)}

Supplementary Material 2^{(306.4KB, csv)}

Supplementary Material 3^{(2MB, docx)}

Supplementary Material 4^{(6.2MB, xlsx)}

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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[CR42] 42.Hu Y, Gong C, Li Z, Liu J, Chen Y, Huang Y, Luo Q, Wang S, Hou Y, Yang S et al (2022) Demethylase ALKBH5 suppresses invasion of gastric cancer via PKMYT1 m6A modification. Mol Cancer 21(1):34 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR43] 43.Qu J, Yan H, Hou Y, Cao W, Liu Y, Zhang E, He J, Cai Z (2022) RNA demethylase ALKBH5 in cancer: from mechanisms to therapeutic potential. J Hematol Oncol 15(1):8 [DOI] [PMC free article] [PubMed] [Google Scholar]

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[CR45] 45.Wang T, Kong S, Tao M, Ju S (2020) The potential role of RNA N6-methyladenosine in Cancer progression. Mol Cancer 19(1):88 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR46] 46.Zhang S, Zhao BS, Zhou A, Lin K, Zheng S, Lu Z, Chen Y, Sulman EP, Xie K, Bögler O et al (2017) m(6)A Demethylase ALKBH5 Maintains Tumorigenicity of Glioblastoma Stem-like Cells by Sustaining FOXM1 Expression and Cell Proliferation Program. Cancer Cell 31(4):591–606e596 [DOI] [PMC free article] [PubMed] [Google Scholar]

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[CR48] 48.Inoue T, Aoyama-Ishikawa M, Uemura M, Kohama K, Fujisaki N, Murakami H, Yamada T, Hirata J (2023) The role of death receptor signaling pathways in mouse Sertoli cell avoidance of apoptosis during LPS- and IL-18-induced inflammatory conditions. J Reprod Immunol 158:103970 [DOI] [PubMed] [Google Scholar]

[CR49] 49.Wu H, Jiang X, Gao Y, Liu W, Wang F, Gong M, Chen R, Yu X, Zhang W, Gao B et al (2019) Mumps virus infection disrupts blood-testis barrier through the induction of TNF-α in Sertoli cells. FASEB journal: official publication Federation Am Soc Experimental Biology 33(11):12528–12540 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR50] 50.Falvo S, Minucci S, Santillo A, Senese R, Chieffi Baccari G, Venditti M (2023) A short-term high-fat diet alters rat testicular activity and blood-testis barrier integrity through the SIRT1/NRF2/MAPKs signaling pathways. Front Endocrinol (Lausanne) 14:1274035 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR51] 51.Manou D, Bouris P, Kletsas D, Götte M, Greve B, Moustakas A, Karamanos NK, Theocharis AD (2020) Serglycin activates pro-tumorigenic signaling and controls glioblastoma cell stemness, differentiation and invasive potential. Matrix Biol Plus 6–7:100033 [DOI] [PMC free article] [PubMed]

[CR52] 52.Vander Borght M, Wyns C (2018) Fertility and infertility: Definition and epidemiology. Clin Biochem 62:2–10 [DOI] [PubMed] [Google Scholar]

[CR53] 53.Eisenberg ML, Esteves SC, Lamb DJ, Hotaling JM, Giwercman A, Hwang K, Cheng YS (2023) Male infertility. Nat reviews Disease primers 9(1):49 [DOI] [PubMed] [Google Scholar]

[CR54] 54.Peel A, Saini A, Deluao JC, McPherson NO (2023) Sperm DNA damage: The possible link between obesity and male infertility, an update of the current literature. Andrology 11(8):1635–1652 [DOI] [PubMed] [Google Scholar]

[CR55] 55.Bermúdez-Cardona J, Velásquez-Rodríguez C (2016) Profile of Free Fatty Acids and Fractions of Phospholipids, Cholesterol Esters and Triglycerides in Serum of Obese Youth with and without Metabolic Syndrome. Nutrients 8(2):54 [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

ALKBH5 facilitates Srgn stability via m6A demethylation to disrupt blood-testis barrier integrity in the high-fat diet mice

Tian Du

Liangyu Yao

Wenhua Wang

Zhou Li

Jie Dong

Zheng Zhou

Chengyu Shi

Xie Ge

Rujun Ma

Li Chen

Yijian Xiang

Zhaowanyue He

Lu Zheng

Yao Xu

Chun Cao

Jun Jing

Jinzhao Ma

Bing Yao

Abstract

Supplementary Information

Introduction

Materials and methods

Cellular experiment

Plasmid and small interfering RNA (siRNA) transfection

Neutralization of CCL2 and TNF-α

Quantitative RT-PCR

Western blotting (WB) analysis

Transepithelial resistance (TER) measurements

Animal experiment

Constructing a mouse model with Alkbh5 knockdown in Sertoli cells

Assessment of BTB integrity in vivo by biotin tracer experiment

Transmission electron microscopy

Immunohistochemical (IHC) and immunofuorescence (IF) of testis tissue

IF staining of primary Sertoli cells

m6A IP‑qPCR

mRNA stability assay

Enzyme-linked immunosorbent assay (ELISA)

Single-cell RNA sequencing (scRNA-seq) data source and analysis

RNA sequencing and data analysis

Statistical analysis

Results

ALKBH5 was upregulated with PA treatment in TM4 and primary Sertoli cells

Fig. 1.

Alkbh5 mediated PA-induced Sertoli cell barrier disruption in vitro

Fig. 2.

Alkbh5 in Sertoli cells was involved in HFD-induced spermatogenic dysfunction and BTB disruption in vivo

Fig. 3.

ALKBH5 might facilitate HFD-induced BTB disruption via Srgn in vitro and in vivo

Fig. 4.

ALKBH5 increased the stability of Srgn-mRNA in a m6A dependent manner

Fig. 5.

Discussion

Conclusion

Supplementary Information

Acknowledgements

Author contributions

Funding

Data availability

Declarations

Consent for publication

Conflict of interest

Footnotes

Contributor Information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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