Ethics
All animal treatments were approved by the Laboratory Animal Ethics Committee of Anhui Medical University (ethical approval number: LLSC20220640 and LLSC20190297). The case-control study design complied with all relevant regulations regarding the use of human study participants and was conducted in accordance to the criteria set by the Declaration of Helsinki. This was approved by the Clinical Medical Research Ethics Review Committee of the First Affiliated Hospital of Anhui Medical University (ethical approval number: PJ2023-04-12) and obtained informed consent from the donor.
Animal treatments
The C57BL/6 N mice were provided by Bei**g Vital River Laboratory Animal Technology Co., Ltd (Bei**g, China). All animals were accustomed to standard conditions (20–25 °C environment temperature, 50–60% air humidity and 12-h light/dark cycle) for a week. The purified control feed (TP23302) and 60% fat high-fat feed (TP23300) were acquired from Trophic Animal Feed High-Tech Co., Ltd (Nantong, China). In the different generations, 8 to 11 pregnant mice were obtained in each group after mating with different male mice, and the balances of sexes between pups (male: female, 3: 3) were performed on postnatal day (PND) 1. At PND28, one male mouse from each litter was selected. For euthanasia, all mice were injected intraperitoneally with 2,2,2-tribromoe-thanol (250 mg/kg), and then cervical dislocation was executed under anesthesia.
Experiment 1. The experimental design used to investigate the susceptibility to spermatogenesis disorders in the offspring of fathers stressed by multigenerational HFD is shown in Fig. 1a. Briefly, 20 F0 generation male mice were fed with HFD or normal chow (NC) from 5 weeks to 15 weeks old, and then mated with normal female mice to breed F1 generation. After mating, 10 pregnant mice were obtained in each group. Similarly, 10 F1 generation male mice (whose fathers were exposed to HFD) from different litter continued to be fed HFD for 10 weeks, and mated with normal females to breed F2 generation. Then, 10 male mice from each group of F1 and F2 generations were exposed to CdCl2 (0 or 100 mg/L) by drinking water for 10 weeks, and named NC (normal control), NCD (the offspring were treated with Cd after paternal treatment with normal chow), HFD1D (the offspring were treated with Cd after paternal exposure to one-generational HFD) or HFD2D (the offspring were treated with Cd after paternal exposure to bi-generational HFD) groups, respectively. Cd, a well-known reproductive toxicant, impairs testicular development and spermatogenesis. Population-based studies found that Cd concentrations in blood and semen were negatively correlated with sperm quality55,56. A series of animal experiments also confirmed that Cd exposure impaired testicular development and reduced sperm count in mice18,57. Here, Cd was used as an inducer of reproductive toxicity to induce spermatogenesis disorders in mice. It’s well-known that Cd exposure impairs testicular spermatogenesis mainly via the dose of internal exposure. In the present study, the dosage of 100 mg/L CdCl2 was chosen according to the internal exposure level in population studies and previous research18,58. Our pervious study showed the sera Cd concentration when mice were exposed to CdCl2 (100 mg/L) through drinking water was close to the blood Cd of patients with low semen quality and was related to Cd level of human in severe Cd-contaminated areas and smokers58,59. Meanwhile, single- and double-generations of HFD-fed fathers named HFD and HFD1 + HFD group, and their offspring named HFD1 and HFD2 group, were created. Euthanasia was performed in all groups at 15 weeks of age. The testes, epididymal sperm and serum from mice were obtained for further analysis.
Experiment 2. The experimental design used to explore the effect of WT1 overexpression on multigenerational paternal HFD-enhanced susceptibility to spermatogenesis disorders in offspring, as presented in Fig. 5a. In summary, F0 generation male mice were fed with HFD or NC from 5 weeks to 15 weeks old, and mated with normal female mice to breed F1 generation. After mating, 9 pregnant mice were obtained in each group. At PND35, 9 F1 generation mice from each group overexpressed WT1 or NC by local testicular injection of adeno-associated virus 9 (AAV9). After injection of AAV9-Wt1 or NC, mice were exposed to CdCl2 for 10 weeks, and named the NCD, NCD + WT1, HFD1D or HFD1D + WT1 group, respectively. All mice were euthanized at 15 weeks of age. Mouse epididymal sperm and testes were obtained.
Experiment 3. To investigate the effect of reduced sperm m6A level on paternal HFD-inhibited testicular WT1 expression and spermatogenesis in offspring, F0 generation male mice were treated with STM2457 by local testicular injection. As shown in Fig. 9a, F0 generation male mice were fed with HFD or NC from 5 weeks to 15 weeks old, and treated with STM2457 once a week from 10 weeks to 15 weeks old. After mating, 8–11 pregnant mice were obtained in each group. Then, 8–11 F1 generation male mice from each group were exposed to CdCl2 from 5 weeks to 10 weeks old, and named NCD, NCD + STM, HFD1D or HFD1D + STM group, respectively. Mouse serum, epididymal sperm and testes were obtained for further analysis.
Case-control study
To investigate the effect of overweight/obesity on sperm m6A levels, a case-control study was established. A total of 428 human sperm were obtained from the reproductive medicine center of the First Affiliated Hospital of Anhui Medical University with the donor’s informed consent. After removal of smoking or alcohol drinking donors, 168 sperm samples were available. Finally, 30 pairs of cases with overweight/obesity and corresponding controls were obtained by matching age. Semen specimens were centrifuged at 600 g for 10 min to remove seminal plasma. After PBS resuspension, sperm were incubated in somatic cell lysate (0.5% Triton X and 0.1% sodium dodecyl sulfate in nuclease-free water) at 4 °C for 10 min to remove somatic cells. After washing, sperm pellets were collected for m6A detection.
Cell culture
TM4 cells, the normal mouse testicular Sertoli cell lines, were from the Cell Bank of the Chinese Academy of Sciences (catalog number: GNM41). TM4 cells were cultured using DMEM/F-12 medium and supplemented with 5% horse serum, 2.5% fetal bovine serum, and 1% penicillin-streptomycin solution. To determine the dose of Cd-impaired TM4 cell growth, cells were exposed to CdCl2 at different concentrations, and cell viability was detected by the MTT assay at different time points. To investigate the effects of Cd exposure on WT1 expression and m6A modification level, TM4 cells were exposure to CdCl2 for 0–24 h. To investigate the effect of Mettl3 or Igf2bp1 siR on Cd-downregulated WT1 expression, cells were exposure to CdCl2 for 24 h after Mettl3 or Igf2bp1 siR treatment. To determine whether Cd downregulated Wt1 mRNA expression by methylating site1, cells were exposure to CdCl2 for 24 h after wild type (WT)-Wt1 or mutant (MUT)-Wt1 plasmids treatment.
Fertility test
For fertility rate, one male mouse was mated with two female mice at night, and vaginal plug were checked in the next morning. The number of fertile male mice was recorded and divided by the total number of male mice to calculate the fertility rate of each group on that day. This test was repeated twice again every 4 days, a total of 4 repetitions. For pregnant rate, female mice with vaginal plug were fed at single caged, and the pregnancy results were recorded. The litter sizes were also observed after delivery.
Glucose tolerance test (GTT)
The F0 generation male mice were intraperitoneally injected with 2.0 g/kg glucose solution after fasting (maintaining normal water intake) for 12 h. The blood glucose was then detected in the caudal vein using a glucometer, and recorded at different time points.
Local testicular injection
Local testicular injections were performed according to previous study with corresponding modification60. In brief, the mice were injected intraperitoneally with 250 mg/kg 2,2,2-tribromoe-thanol. After anesthesia, both testes of the mice were found by touch pressure. Then, 20 μL AAV9 or STM2457 were injected into the middle of each testes using a micropipette. After injection, the mice were placed flat in cage and observed for 30 min until they were fully awake and returned to normal. Among them, 1.03×1012 vg/ml AAV9-Wt1 were injected once into mice at PND35. The control was injected with vehicle. AAV9-Wt1 and vehicle were synthesized by General Biol Co., Ltd. STM2457 were injected at a dose of 50.0 mg/kg, once a week for 5 weeks. 20% SBE-β-CD was used as control. STM2457 and SBE-β-CD were purchased from MedChemExpress. The dose of STM2457 was chosen according to previous research61.
Evaluation of sperm count
Sperm motility and count were evaluated by an automatic sperm analyzer (Hamilton Thorne, USA). Mouse left epididymis was sectioned into slices and placed in a preheated medium. The medium was then incubated at 37 °C for 5 min until sperm was completely released. In the final step, the sperm suspension was diluted with preheated medium, and 50 μL of suspension was dropped into a slide for parameter evaluation.
Sperm isolation
To obtain sperm, fresh mouse epididymis was completely cut into pieces in preheated medium and incubated at 37 °C for 10 min until mature sperm were fully released. Then, the tissue debris were removed by 40-μm cell strainer. The sperm mixture was incubated with somatic cell lysate (0.5% Triton X and 0.1% sodium dodecyl sulfate in nuclease-free water) on ice for 40 min to clear somatic cell. Next, the mixture was centrifuged at 600 g for 5 min and the precipitates were resuspended with PBS. After washing twice, sperm pellets were collected for the methylated RNA microarray.
MTT assay
First, TM4 cells were treated with different concentrations CdCl2 for 6, 12, or 24 h after culture in 96 well plates for 12 h. Following that, the cells were treated with 5 mg/ml MTT, and were persistently cultured for 2 h in a 37 °C incubator. Prior to this, the cells were grown in DMEM/F-12 medium and supplemented with 5% horse serum, 2.5% fetal bovine serum and 1% penicillin-streptomycin solution. After aspirating the medium, DMSO was added to the culture plate. As a final step, the cellular absorbance at 570 nm was detected by a multimode reader (SYNERGY4, Biotek, USA).
Immunohistochemistry
Dewaxing, rehydrating, quenching endogenous peroxidase, and recovery of antigens were performed in testicular sections before immunostaining. The antibodies specific for SYCP3 and WT1 were subsequently incubated with sections overnight. On the second day, secondary antibody incubation, diaminobenzidine-staining, and hematoxylin-counterstaining were performed, respectively. An Olympus BX53F microscope was used to count positive cells in the testes.
Immunoblotting
The protein lysates from TM4 cells and mouse testes were obtained after homogenizing with RIPA buffer, and Immunoblotting was implemented as previously described62. Briefly, cellular and testicular protein extracts (6–30 ug) were separated by 10.0–12.5% SDS-PAGE electrophoresis and then transferred into PVDF membrane. After blocking with 5% skim milk for 1.5 h, the membranes were incubated with antibodies of rabbit anti-DDX4 (1:1000, Abcam, ab13840), mouse anti-PLZF (1:200, Santa Cruz Biotechnologies, sc-28319), mouse anti-C-KIT (1:200, Santa Cruz Biotechnologies, sc-365504), mouse anti-SYCP3 (1:200, Santa Cruz Biotechnologies, sc-74569), rabbit anti-STRA8 (1:1000, Abcam, ab49602), rabbit anti-RARα (1:1000, Cell Signaling Technology, 62294 S), rabbit anti-RBP4 (1:1000, Abcam, ab188230), rabbit anti-ALDH1A1 (1:1000, Abcam, ab52492), rabbit anti-ALDH1A2 (1:1000, Cell Signaling Technology, 83805 S), rabbit anti-WT1 (1:1000, Abcam, ab89901), rabbit anti-METTL3 (1:2000, Abcam, ab195352), rabbit anti-METTL14 (1:1000, Cell Signaling Technology, 51104 S), rabbit anti-ALKBH5 (1:1000, Abcam, ab195377), mouse anti-FTO (1:1000, Abcam, ab92821), rabbit anti-YTHDF1 (1:1000, Proteintech, 17479-1-AP), rabbit anti-YTHDF2 (1:1000, Proteintech, 24744-1-AP) and rabbit anti-IGF2BP1 (1:1000, Proteintech, 22803-1-AP) for 1–3 h at room temperature. Antibody of β-Actin (1:10000) for loading control. After washing, the corresponding secondary antibody were used to incubate membranes for 1–2 h. Finally, the imaging system Bio-Rad ChemiDoc™ MP was used to detect immunoreactive protein signal. The protein signal was quantified by Image J software (version 1.8.0).
Enzyme-linked immunosorbent assay (ELISA)
The quantity of retinoic acid (RA) in mouse testes and retinol in mouse serum were measured using the mouse RA-ELISA kit (MyBiosource, MBS706971) and the mouse retinol-ELISA kit (CUSABIO, CSB-E07891m), respectively. Assays were implemented according to the manufacturer’s specifications. To detect testicular RA, 50 mg of testes was accurately weighed and homogenized in 900 ml ice PBS with protease inhibitor. After centrifugation at 6000 g for 15 min, the supernatant was collected for RA detection. The concentration of testicular RA was presented as ng/g.
m6A-mRNA&lncRNA epitranscriptomic microarray
Sperm total RNA was initially extracted with TRIzol reagent, and quantified using the NanoDrop ND-1000. The methylated RNA epitranscriptomic microarray were performed at Aksomics (Shanghai, China). Briefly, the total RNAs were immunoprecipitated with anti-m6A antibody. The modified RNAs were eluted from the immunoprecipitated magnetic beads as the IP. The unmodified RNAs were recovered from the supernatant as Sup. After Sup and IP RNAs labeling with Cy3 and Cy5 respectively, the cRNAs were combined together and hybridized onto Arraystar Mouse mRNA&lncRNA Epitranscriptomic Microarray. Then, the arrays were scanned in two-color channels by an Agilent Scanner G2505C. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze the collected chip fluorescence signals. The raw signal strength of IP and Sup was normalized using the mean value of Log2-converted spik-in RNA fluorescence intensity. After standardization, the difference in m6A methylation levels between the two groups was calculated using a T-test statistical model. FC (Fold Change) >1.5 and P-value < 0.05 were set as screening thresholds for differential expression.
RNA sequencing
Total RNA of mouse testes was extracted, and quality inspections were performed. The sequencing library was constructed with 2.0 μg total RNA from each sample at Aksomics (Shanghai, China). In brief, mRNA enrichment was performed using NEB Next® Poly(A) mRNA Magnetic Isolation Module kit. The library from enriched RNA was constructed by KAPA Stranded RNA-Seq Library Prep Kit (Illumina) and inspected by Agilent 2100 Bioanalyzer. Then, the final quantification of library was conducted by qPCR. After denaturing with 0.1 M NaOH, 8 pM single stranded DNA was amplified in situ at the NovaSeq S4 Reagent Kit. The end of generated fragment was sequenced using Illumina NovaSeq 6000 sequencer for 150 cycles. After StringTie comparison of the sequencing results to the known transcriptome, the transcriptional abundances at Gene Level and Transcript Level were calculated using Ballgown. The unit of expression quantity was expressed by FPKM (Fragments Per Kilobase of gene/transcript model per Million mapped fragments). A gene or transcript with an expression value of FPKM > 0.5 was considered to be effectively expressed in the group. Finally, Ballgown software was used for statistical analysis by the F-test statistical model, FC > 1.2 and P-value < 0.05 were set as the screening threshold for differential expression.
Isolation of total RNA and real-time RT-PCR
The cellular and testicular total RNA were initially extracted with TRIzol reagent (Invitrogen). The cDNA from 2 μg total RNA was obtained using AMV reverse transcriptase (Promega). The mRNAs expression of targeted genes was then detected by the Lightcycler®480 real-time fluorescence quantitative PCR machine (Roche, Switzerland). The primer sequences in this study are presented in Supplementary Table 1. 18 S as the internal parameter. The expression of targeted mRNAs was analyzed by comparative the CT method.
Quantification of m6A modifications
Total RNA in mouse testes and sperm was extracted by TRIzol reagent. The m6A level of total RNA was detected using the EpiQuik m6A RNA Methylation Quantification Kit (Epigentek). A total of 200 ng of RNA or positive controls were added separately to the assay wells, along with the captured antibody solution, the detected antibody solution, the enhanced solution, and the developer solution diluted to a suitable concentration. The absorbance was read at 450 nm, and the m6A levels of the RNA sample were calculated based on the standard curve.
MeRIP-qPCR
Sperm total RNA was initially extracted with TRIzol reagent, and mRNA was enriched with Arraystar Seq-Star TM poly (A) mRNA isolation kit. The interrupted mRNA (1/10 of which was left as the input control) was mixed with 2 μg anti-m6A rabbit polyclonal antibody and IgG Dynabeads into a 500 μL IP reaction system (50 mM Tris-HCl, pH 7.4, 0.1% NP-40, 150 mM NaCl and 40 U/ml RNase inhibitor), and incubated at 4 °C for 2 h. After washing and elution, the binding RNA was leached by TRIzol16,63. Afterward, reverse transcription and amplification were performed. The SRAMP website (http://www.cuilab.cn/sramp/) predicted that Wt1 mRNA has four highly credible m6A-modified sites. The corresponding primer sequences are shown in Supplementary Table 2. The different site m6A level of Wt1 mRNA was quantified by RT-qPCR, which was calculated with the following formula: Site m6A level (%) = 2Ct(Input)-Ct(MeRIP) × Fd × 100%. Among them, Fd is the input dilution factor.
RNA interference
RNA interferences were performed following the approach of our previous study18. Briefly, mouse Mettl3 or Igf2bp1 specific small interference RNA (siR, GenePharma) were mixed with Lipofectamine 3000 for 15 min in serum-free medium. The mixtures were added to the culture medium for 6 h to transfect TM4 cells. Ather this, the medium was replaced with fresh medium and TM4 cells were cultured for 42 h. The sequence of Mettl3 siRNA was 5′-GUCAGUAUCUUGGGCAAAUTT-3′ (forward) and 5′-AUUUGCCCAA GAUACUGACTT-3′ (reverse). The sequence of Igf2bp1 siRNA was 5′- GUCCCAAGGAGGAAGUAAATT-3′ (forward) and 5′- UUUACUUCCUCCUUGGGACTT-3′ (reverse).
RNA stability assay
Actinomycin D (5 μg/mL) inhibited transcription in TM4 cells. The mRNA expression of Wt1 was detected by qRT-PCR after 0, 3, and 6 h of Cd treatment.
RNA immunoprecipitation
Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) was used to implement RNA immunoprecipitation. After lysing with complete RIP lysis buffer, TM4 cells were incubated with magnetic beads coated with antibodies against IgG or IGF2BP1 for 4 h at room temperature. Then, the complexes were hatched with proteinase K digestion buffer to extract immunoprecipitated-RNA. qPCR was used to determine the relative interaction between Wt1 and IGF2BP1.
Luciferase reporter assay
The Wt1 CDS sequence containing the mutated motif (GAC to GTC) or the wild-type m6A motif was inserted into the pmirGLO-REPORT luciferase reporter vector (Promega). Then, plasmid transfections were conducted in TM4 cells with or without Cd treatment. The firefly and Renilla luciferase activities were detected by a dual-luciferase reporter assay system (Promega).
Chemicals and reagents
Detailed information on chemical reagents and primary antibodies are listed in Supplementary Table 3.
Statistical analysis
All statistical analyses were conducted by SPSS23.0 software, and data were expressed as means ± SEM. One-way ANOVA was used to compare means in multiple groups. Mean comparisons between the two groups were performed using two-tailed Student’s t-test. Repeated-measures ANOVA was applied to analyze the GTT data. Two-way ANOVA was applied to analyze RNA stability assay and mouse body weight data. The data were graphically presented by GraphPad Prism 8.0 or R (version 3.6.0) processing. Experimental design flowchart (Figs. 1a, 5a, 9a and Supplementary Fig. 8a) and graphical abstract (Fig. 10g) were generated using PowerPoint software (office 2019). P < 0.05 meant the difference was statistically significant.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
