microRNAs in aged sperm confer psychiatric symptoms to offspring through causing the dysfunction of estradiol signaling in early embryos

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tissue debris and somatic cells, a 40-micron cell filter and somatic cell lysate (0.1% SDS, 0.5% Triton X-100 in DEPC treated water) were used in sequence. The sperm was added with 10 mL of somatic cell lysate and incubated on ice for 40 minutes, after which the sperm be pelleted by centrifugation at 600 g for 15 minutes. After removal of suspension, the sperm pellet was resuspended and washed twice in 10 mL of PBS and then pelleted at 600 g for 15 minutes. Sperms were counted under a light microscope to determine the concentration.

Human sperm collection
Human sperm samples were donated by men who were visiting the Center of Reproductive Medicine, Jinling Hospital (Nanjing, China). All donors signed written informed consent forms. Sperm from aged donors (45-56 years) were enrolled as F0-Aged group, while sperm from young donors (22-27 years) were F0-Ctl group (Supplementary Table 1). Sperm samples were obtained from both group after 3-5 days of sexual abstinence. Sperm quality parameters, including concentration, motility and morphology, were evaluated according to World Health Organization guidelines (5th edition, 2010) to ensure that samples were normal.

sRNA library construction and sequencing
All sRNA library construction and sequencing were performed by BGI (Shenzhen, China). Briefly, sRNA libraries were constructed according to the TruSeq Small RNA Sample PreKit (Illumina). After library quality validation, raw data for each sRNA library were generated on the Illumina HiSeq 4000 platform. Sequence reads that fit any of the following standard quality control criterion parameters were removed: (i) reads with N (more than four bases whose quality score is lower than 10 or more than six bases whose quality score is lower than 13), (ii) reads with 5' primer contaminants or without a 3' primer, (iii) reads without the insert tag, (iv) reads with ploy A, and (v) reads shorter than 15 nt. The clean reads were obtained after data filtration. miRNA precursor and mature sequences, tRNA and rRNA sequences were obtained from miRBase v21, GtRNAdb and National Center for Biotechnology Information, respectively. Bowtie was used to align clean reads to these reference sequences for annotation. To annotate miRNA, only candidates with one mismatch and no more than two shifts were counted as miRNA matches. To annotate tsRNA, SPORTS 1.1 based on bowtie was used for tsRNA annotation. The total sequencing frequency of each type of sRNA in each sample was normalized to 1,000,000. Differential analysis was used by Student's t test. Significance was set at uncorrected P < 0.05 for broad pattern identification. A fold change threshold was set at > 2. The average expression level threshold was set at > 500.

RNA isolation and quantitative RT-PCR analyses
Total RNA was isolated from sperm using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. To detect miRNAs, total RNA was reverse transcribed to cDNA using miRNA 1st Strand cDNA synthesis kit (Vazyme, China) and stem-loop RT primer, and sequentially incubated at 25°C for 5 minutes, 50°C for 15 minutes and 85°C for 5 minutes. Next, real-time PCR was performed using a miRNA Universal SYBR qPCR Master Mix kit (Vazyme, China) on a Light Cycler 480 real time PCR System (Roche, Mannheim, Germany). Relative miRNA expression in sperm was normalized to U6.
To detect mRNAs, total RNA was reverse transcribed to cDNA using HiScript ® Ⅲ RT SuperMix for qPCR (+gDNA wiper) (Vazyme, China) and corresponding RT primer, and sequentially incubated at 37°C for 15 minutes, 85°C for 5 second. Next, real-time PCR was performed using a ChamQ Universal BYBR Qpcr Master Mix kit (Vazyme, China) on a Light Cycler 480 real time PCR System (Roche, Mannheim, Germany). Relative mRNA expression in embryos and N2A cells was normalized to GAPDH.
Microinjection of sperm sRNA and miRNA mimics and transfer of embryos Sperm sRNA was isolated by using mirVana TM miRNA Isolation kit (by Thermo Fisher Scientific). Synthetic miR-9-5p mimic and scrRNA were obtained from Ribobio (Guangzhou, China). Sequences of synthetic miRNA mimics were shown as follow: miR-9-5p mimic, 5'-UCUUUGGUUAUCUAGCUGUAUGA-3'; scrRNA, provided by Ribobio (Guangzhou, China). sRNA or miRNA mimic was adjusted to a concentration of 2 ng/μL, which is approximately equal to the total RNA of 10 sperm. sRNA or miRNA mimic was microinjected into the cytoplasm of normal zygotes by a Leica microinjection system. After injection, the zygotes were cultured in M16 (Sigma-Aldrich) at 37°C in 5% CO2 until to the two-cell stage embryos. Then the embryos were transferred to the oviduct of the surrogate mother (ICR background). The offspring of each group (sRNA-Aged vs. sRNA-Ctl; miR-9-5p vs. scrRNA) were screened for behavioral performances until adulthood (8-10 weeks). Approximately 300 embryos were injected for each condition, and 80 to 85% implanted embryos survived up to E3.5, 30 to 35% to E5.5, and 30 to 35% until adulthood. No difference was shown in embryo survival rate between the sRNA-Aged group and sRNA-Ctl group in each stage.
Early embryo collection and single-cell transcriptome RNA sequencing Early embryo collection sRNAs isolated from the sperm of F0-Aged and F0-Ctl mice or aged and young human donors were microinjected into zygotes of the C57Bl/6J background at a concentration of 2 ng/μL. The zygotes were then cultured in M16 medium (Sigma Aldrich) at 37°C in 5% CO2. On the second day, the 2cell embryos were transferred to potassium-supplemented simplex optimized medium (KSOM, Millipore). Embryos at the 2-cell, 4-cell, 8-cell and blastocyst stage were collected at approximately 18, 42, 76 and 96 hours after microinjection, respectively. To remove the zonapellucida, the selected embryos were transferred into an acidic solution drop (1 mL of PBS supplemented with 1 mL of 36% HCl), and all the embryos without zonapellucida were washed carefully in Dulbecco's Phosphate Buffered Saline several times to remove all the potential maternal contaminants. In addition, some 2-cell embryos were transferred to the oviduct of the surrogate mother of the ICR background. In addition, 2-cell, 4-cell, 8-cell and blastocyst embryos were pooled at each developmental stage, and total RNA was isolated from these early embryos using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions for quantitative RT-PCR analysis.

Single-cell transcriptome RNA library construction
Single 8-cell-stage embryos (E2.0) and blastocyst-stage embryos (E3.5) were collected and lysed to release all RNAs. Single-cell transcriptome RNA sequencing was performed by Anoroad (Beijing, China). After base composition and quality tests were passed, the sequence of adapters, high content of unknown bases (unknown bases more than 10%), and low-quality reads were removed. The clean reads were used for bioinformatics analysis.

miRNA target prediction and cell transfection assay
The target genes of miRNAs were predicted by TargetScan and RNAhybrid. The detailed binding sites between miRNAs and target gene was visualized as a diagram. N2A cells were cultured in Dulbecco's modified Eagle's media (DMEM, Gibco) with 10% fetal bovine serum (Gibco). For cell transfection assay, N2A cells were seeded onto 6-well plates and allowed to grow to 70% confluence, and each well was transfected with 100 pmol of miRNA mimics or scrRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Total RNA and protein were isolated 24 or 48 hours after transfection.

Western blot analysis
Protein samples from cells were extracted by RIPA buffer. After centrifuging at 12,000 g for 10 minutes at 4°C, the supernatant was kept for western blot analysis. BCA method was used to quantify protein concentration. The following antibodies were used: ERα (1:200; SANTA CRUZ BIOTECHNOLOGY, sc-8005), anti-G-protein coupled receptor 30 (1:250; abcam, ab39742), βactin-HRP (1:1000; Cell Signaling Technology, 12620S). The data were quantified using ImageJ software (NIH, Bethesda, MD), and the relative protein expression was normalized to the level of β-actin.

Immunofluorescence
Embryos were collected and fixed in 4% paraformaldehyde for 30 minutes at room temperature. After being washed with PBST (containing 0.1% Triton X-100 and 0.1% Tween 20), they were incubated by PBS including 1% BSA and 0.1% Tween 20 for 60 minutes. Embryos were incubated overnight at 4°C with the following primary antibodies: Anti-G-protein coupled receptor 30 (1:200, abcam), β-Actin (D6A8) Rabbit mAb (HRP Conjugate). After a complete wash in PBS, the embryos were incubated in the appropriate secondary antibodies for 2 hours at room temperature in the dark. The embryos were washed and mounted in Fluoromount-G mounting medium (SouthernBiotech, Birmingham, AL). The micrographs were taken by inverted laser scanning confocal microscope (TCS SP8, Leica). Digital images were recorded and analyzed by LAS X Viewer software (Leica).

Statistical analysis
Statistical analyses were performed using the Prism v.8.0 (GraphPad Software Inc.). The D'Agostino & Pearson normality test was used to measure data distribution. Two-tailed unpaired Student's t test was conducted to compare two groups. Data are presented as the means ± standard errors of the means (SEMs). Differences are considered statistically significant at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001. (f-g) Three-chamber social interaction test. Heat map showing the amount of time spent in the threechamber arena at the sociability and social novelty preference portion of the tests, total time spent sniffing the stranger mouse 1 and empty, and total time spent sniffing the stranger mouse 1 and mouse 2 (n = 12-17 per group). (h) Behavioral performances in forced swimming test (n = 18-20 per group) and sucrose preference test (n = 17-18 per group).  Supplementary Fig. S5. Annotation and classification of the sRNA species in the sperm derived from F0-Aged and F0-Ctl. Sperm sRNA fragments of 10-50 bp were isolated from the gel and processed for sRNA deep sequencing. The sequence reads were annotated and classified through alignment with known sRNA libraries. The y-axis shows the reads relative to per million total reads. Classification of the sRNA species revealed that rsRNA, miRNA, piRNA and tsRNA were the most abundant sRNAs in sperm, which accounted for 46.35%, 28.10%, 5.05% and 3.69% in the sperm of F0-Aged and 51.95%, 23.63%, 5.00% and 7.62% in the sperm of F0-Ctl, respectively. Length distribution analysis showed a dominant distribution of miRNAs at 22 bp and tsRNA at 31 bp and a widespread distribution of rsRNA at 15-45 bp and piRNA at 15-32 bp. Unanno MG: matched to the reference genome but not annotated. Unanno UMG: unmatched to the reference genome and not annotated.  male  47  Aged  40  male  45  Aged  41  male  50  Aged  42  male  49  Aged  43  male  51  Aged  44  male  46  Aged  45  male  45  Aged  46 male 53 Aged