The developmental miR-17–92 cluster and the Sfmbt2 miRNA cluster cannot rescue the abnormal embryonic development generated using obstructive epididymal environment-producing sperm in C57BL/6 J mice

Animals and establishment of the mice model

Male C57BL/6 J mice were purchased from the Experiment Animal Center, Center for Disease Control and Prevention, Hubei Province (Wuhan, China), female B6D2F1 mice (C57BL/6 J: BDA/2 F1 hybrid background) and ICR mice were purchased from Charles River Laboratories (Beijing, China). All mice were housed in the Laboratory Animal Center of HuaZhong university of science and technology, fed a chow diet ad libitum, and kept on a 12:12 light: dark cycle with a temperature of 22 °C and relative humidity of 42%. Sham operation and vasectomy were performed on 6-week-old male C57BL/6 J mice to create the CON and OEE groups. Male C57BL/6 J mice were anesthetized with 3.3% chloral hydrate, and the vas deferens were ligated by a suture line in the OEE group; the vas deferens in the CON group were not ligated. The CON group and the OEE group mice were euthanized in the postoperative period at 4, 8, and 12 weeks, and the epididymis and testis samples were collected and weighed. The study was approved by the Animal Care Ethics Committee of Huazhong university of science and technology.

HE staining

The epididymis and testis were fixed in 10% neutral buffered formalin and embedded with paraffin. The paraffin section was stained with hematoxylin and eosin to observe the morphology.

Sperm isolation and sperm quality test

Sperm was released from cauda epididymis in prewarmed G-IVF PLUS (Vitrolife AB, Goteborg, Sweden) at 37℃ for 30 min. After blending gently, 30 ul of sperm suspension was divided into three equal portions. One was for examining sperm concentration and motility, one was for observing sperm viability stained with eosin staining, and the last one was stained with Diff-Quick staining (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) for observing morphology. The sperm DNA fragmentation index (DFI) was determined by flow cytometry (BD bioscience, San Jose, CA). Sperm were diluted to a concentration of (1–2) × 106/ml with cold TNE buffer (Servicebio Technology, Wuhan, Hubei, China), stained with acridine orange solution (Sigma-Aldrich, Shanghai, China), and then tested with flow cytometry (BD Biosciences, Franklin Lakes, USA).

Sperm preparation for ICSI

Sperm was optimized by density gradient centrifugation using a Sydney IVF sperm gradient kit (Cook Medical, Sydney, Australia). The gradients were prepared by adding 1.5 mL of 40% solution to 1.5 mL of 80% solution in a 4 ml centrifuge tube and prewarmed at 37℃. The sperm suspension was gently added to the gradient solution and centrifuged at 600 g for 15 min. The collected sperm precipitate was washed by G-IVF PLUS twice and resuspended by 500 μl G-IVF PLUS. The sperm head was separated by a 30% power output of ultrasonic sonicator for 2 min; then, sperm head suspension was stored at -80℃.

Epididymosomes isolation and fusion

The epididymis was minced and immediately transferred to prewarmed PBS at 37℃ for 30 min after being collected from mice, and the suspension was filtered through 100 μm and 40 μm cell strainer to collect the supernatant. The filtered supernatant was performed in sequential centrifugation (centrifuge at 300 g, 2000 g, 10000 g, and 100000 g for 10 min, 15 min, 30 min, and 80 min, respectively). After centrifugation, the precipitate was reserved and resuspended with G-IVF PLUS to incubate with optimized sperm for 4.5 h. Sperm was collected by centrifuging 600 g for 15 min and prepared by sonication for injection.

ICSI and embryo transfer

Intraperitoneal injection with 8 IU pregnant mare serum gonadotropin (PMSG) (NINGBO SANSHENG, Ningbo, China) was performed in 6–8-week-old B2D6F1 female mice at 5 pm; after 48 h, intraperitoneal injection with 8 IU human chorionic gonadotrophin (hCG) (NSNF, Ningbo, China). Oocytes were collected 12-14 h after hCG administration, and the cumulus-oocyte complexes (COC) were washed in prewarmed G-MOP PLUS medium (Vitrolife AB, Goteborg, Sweden) containing 0.4 mg/ml hyaluronidase. Mature oocytes (with the first polar body) were selected for injection. ICSI microinjection needle (WPI, TW100-4, Sarasota, USA) was pulled by Flaming Micropipette Puller System P-1000(Sutter, Novato, CA, USA). ICSI was manipulated with the Eppendorf PiezoXpert®(Eppendorf AG, Germany) following standard ICSI procedures [26, 27]. The zygotes were then cultured in G1-PLUS/G2-PLUS sequential medium (Vitrolife AB, Goteborg, Sweden) at 37 °C in 5% CO2. The female ICR mice were mated with male ICR mice at 5 pm, and the female mice with vaginal plugs were chosen as surrogates. The two-cell embryos were transferred to the ampulla oviduct of female ICR mice.

Sperm RNA extraction

After oozing from the epididymis, sperm were successively filtered with 100 μm and 40 μm cell strainer to remove the tissue debris. Somatic cell lysis buffer (0.1% SDS, 0.5% Triton X in DEPC H2O) was used to wipe out somatic cell contamination and then treated with sperm lysis solution (61.1% Guanidine-HCl, 3.5%EDTA, 1.5%tris, 20%Tween-20, 5%Triton in DEPC H2O) to lyse sperm utterly. Sperm lysis was transferred to 800 μl TRIzol LS reagent (No. 10296028; Invitrogen; Thermo Fisher, Waltham, MA, USA) and blended vigorously. After placing in ice for 1 h, 200 μl chloroform was added and placed in ice for 10 min. Then the mixture was centrifuged at 12 000 g for 15 min at 4 °C to collect the aqueous phase. 500 μl isopropanol was added to the aqueous phase and incubated at − 20 °C for 30 min. The precipitate was collected by centrifuging at 12 000 g for 15 min at 4 °C and washed with 75% ethanol. The final products were resuspended with RNase-free water, and the RNA concentration was measured by Nanodrop 2000(Thermo, San Jose, CA, USA).

sRNA sequencing

Sperm RNA isolated from OEE and CON groups were used to build a library of sRNAs. Agarose electrophoresis was used to test the integrity of total RNA samples. Total RNA samples were first pretreated with NEBNext® Multiplex Small RNA Library Prep Set (Illumina, USA) for library construction. Some RNA modifications that could interfere with library construction were removed as follows: Deacetylation of 3'-aminoacyl (charged) to 3'-OH for 3' adaptor ligation, removal of 3'-(2',3')-cyclic phosphate to 3'-OH for 3' adaptor ligation, phosphorylation of 5'-OH to 5'-P for 5'-adaptor ligation, and demethylation of m1A and m3C for efficient reverse transcription. The Agilent 2100 Bioanalyzer was used to quantify the completed libraries. The sequencing run was performed on the NextSeq system using NextSeq 500/550 V2 kit. Sequencing was carried out by running 50 cycles. Image analysis and base calling were performed using Solexa pipeline v1.8 (Off-Line Base Caller software, v1.8). FastQC examined sequencing quality, and trimmed reads are aligned, allowing for 1 mismatch only to the mature tRNA sequences, then reads that do not map are aligned, allowing for 1 mismatch only to precursor tRNA sequences with bowtie software. The remaining reads are aligned, allowing for 1 mismatch only to miRNA reference sequences with miRDeep2. The differentially expressed tRFs & tiRNAs and miRNAs were screened based on the count value with R package edgeR and performed in R. All steps in the library construction, sequencing, and bioinformatics analysis of sRNAs were performed by KangChen Bio-tech (Shanghai, China).

Real-time quantitative polymerase chain reaction (qRT-PCR)

QPCR of sperm miRNAs were using the All-in-one miRNA qRT-PCR Detection Kit (GeneCopoeia, Maryland, USA). Reverse transcription and qPCR of genes were respectively using HiScript II Q RT SuperMix for qPCR (Vazyme, Nanjing, China) and ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). cDNAs obtained were diluted for qPCR. All primers of miRNAs and genes are listed in Supplementary Table 1. Amplifications were performed at least triplicate for each sample. Relative gene expression levels were evaluated using the 2−ΔΔCt method and normalized to U6 Small nuclear RNA (snRNA) level or beta-actin (Actb).

DNA methylation

Epididymis DNA was extracted using TIANamp Genomic DNA Kit (TIANGEN, Beijing, China). Genomic DNA (500 ng) was bisulfite-treated using the EZ-DNA methylation kit (Zymo, Irvine, CA). The interested DNA sequence was amplified using SYBR Premix TaqTM qPCR Reagent Kit (TAKARA, Beijing, China) with the bisulfite-treated DNA as the template. Interesting sequences were located in the promoter region of Sfmbt2 and MIR17HG. Primer information was included in Supplementary Table 2. Agarose electrophoresis was performed to purify and collect amplified cDNA. The methylation level was detected by absolute quantification qPCR using purified cDNA as a standard sample.

Synthetic miRNAs microinjection

Synthetic miRNA inhibitors and scrambled RNA were manufactured by Sangon Biotech (Shanghai, China), listed in Supplementary Table 3. Equal amounts of miRNA inhibitors were mixed and adjusted to a concentration of 2 ng/μl. The microinjection needle was pulled using borosilicate glass (Sutter, Novato, CA, USA) in Flaming Micropipette Puller System P-1000. Superovulation was conducted in the female B2D6F1 mice. The female B2D6F1 mice were mated with male C57BL/6 J mice to collect zygotes for miRNAs microinjection. MiRNAs microinjection was performed in a G-MOP PLUS medium. Synthetic miRNA inhibitors and scramble RNA as control were microinjected into the zygotes with two pronuclei by Femtojet 4i (Eppendorf AG, Germany) [28].

Statistical analysis

All statistical analyses were performed using Graphpad Prism 8.0 (La Jolla, CA, USA). The experimental results are presented as mean ± standard error of the mean (SEM). Data were analyzed using the t-test, Mann–Whitney analysis, or chi-square test. P < 0.05 was considered statistically significant.

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