Developmental miR-17-92 cluster and sfmbt2 miRNA cluster can’t rescue the abnormal embryonic development caused by the obstructive epididymal environment

Developmental miR-17-92 cluster and sfmbt2 miRNA cluster can’t rescue the abnormal embryonic development caused by the obstructive epididymal environment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Developmental miR-17-92 cluster and sfmbt2 miRNA cluster can’t rescue the abnormal embryonic development caused by the obstructive epididymal environment Xunwei Wu, Xiaomei He, Qian Liu, Honggang Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1814149/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background Sperms during epididymal transit acquire microRNAs(miRNAs), which are crucial for embryonic development. However, whether miRNAs transmitted from an obstructive epididymal environment affect embryonic development remains unknown. The purpose of the present study was to investigate the effects of an obstructive epididymal environment on embryonic development. Method The control group (CON) and the obstructive epididymal environment group(OEE) were treated with sham operation and vasectomy, respectively. The effects of an obstructive epididymal environment on testis, epididymis, sperm, and embryonic development were analyzed via hematoxylin and eosin staining (HE staining), sperm quality test, epididymosomes fusion, intracytoplasmic sperm injection (ICSI) and embryo transfer. Moreover, sRNA sequence, RT-qPCR, and DNA methylation were conducted to investigate the regulation of sperm miRNAs by an obstructive epididymal environment. miRNAs microinjection and embryo transfer were used to explore the impacts of sperm miRNAs on embryonic development. Results The long obstructive interval impaired the morphology of the testis and epididymis. In comparison to the CON group, the OEE group had lower sperm quality and developmental potential. Sperm miRNAs profiles were also altered in the OEE group, particularly the developmental miR-17-92 cluster and Sfmbt2 miRNA clusters; the expression of these two miRNA clusters was regulated via epididymal DNA methylation; however, the disruption of neither the miR-17-92 cluster nor the Sfmbt2 miRNA clusters into normal zygotes did not impair embryonic development. Conclusion The obstructive epididymal environment influences embryonic development and the abundance of the sperm miR-17-92 cluster and the sfmbt2 miRNA cluster, but these miRNA clusters are not the cause of abnormal embryo development. It implies that epididymis is important in early embryonic development and may play a potential role in sperm epigenome. miRNAs epididymis embryonic development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Obstructive azoospermia (OA) refers to the absence of sperm in the ejaculate due to occlusion of the male reproductive tract. It can occur in any region of the male reproductive tract (rete testis, efferent ducts, epididymis, vas deferens, and ejaculatory duct). It’s a relatively frequent disease accounting for approximately 1% of all men and 10–15% of infertile men [ 1 ] . In most OA cases, the testis and the endocrine function are normal compared to non-obstructive azoospermia [ 2 ] . The clinical treatment for OA includes microsurgical reconstruction of the reproductive tract and sperm retrieval for ICSI. The OA patients’ post-testicular sperm maturation is in an abnormal epididymal environment because of the obstructive reproductive tract; therefore, the debate over whether OA affects sperm and results in abnormal embryonic development has continued; nevertheless, no definite conclusion has been drawn regarding this issue [ 3 – 9 ] . The epididymis is an important organ for post-testicular sperm maturation, where sperm acquires proteins, sRNAs, forward ability, and fertilization capability [ 10 ] . Recently, Colin's study revealed that epididymis has an impact on embryonic development, as the caput sperm can’t support the post-implantation development of the embryo due to a lack of miRNAs transmitted from epididymis [ 11 ] . However, whether an obstructive epididymal environment affects sperm miRNAs and results in abnormal embryonic development is not clear. MiRNAs are a subset of non-coding RNAs, 19–25 nt long in size. It participates in a series of biological processes through silencing target genes, such as differentiation, proliferation, and apoptosis [ 12 , 13 ] . For decades, increasing evidence has suggested that sperm miRNAs play a pivotal role in epigenetic inheritance. It acts as a carrier of epigenetic inheritance information that transmits parents’ phenotype to the offspring, such as depression, stress, and metabolic disorders [ 14 – 17 ] . Despite miRNAs-mediated inheritance mainly focused on offspring’s long-term health, such as obesity and diabetes, the sperm miRNA was also found to affect embryonic development. Micro-34c and micro-449b has been reported to be required for the early cleavage of the embryo [ 18 , 19 ] . DROSHA and DICER are RNase III enzymes that process primary miRNAs (pri-miRNAs) and precursor miRNAs (pre-miRNAs). Conditional knockout (cKO) of DICER or DROSHA in testis disrupted miRNA synthesis, resulting in abnormal early embryo development, whereas injecting normal sperm RNA into these embryos rescued their development [ 20 ] . Furthermore, miRNAs delivered from the epididymis to sperm have supported post-implantation embryo development. [ 11 ] . However, unlike DNA methylation and histone modification, the mechanism of RNA-mediated epigenetic inheritance in embryonic development remains largely unknown [ 21 ] . The epididymis has recently received much attention because of its ability to deliver miRNAs to sperm, which is critical for embryonic development. Previous research has found that the epididymis influences the sperm miRNAs profile via epididymosomes, an extracellular vesicle secreted by the epididymis epithelium [ 22 – 24 ] ; the high-throughput sequence of sperm sRNAs from various developmental stages has confirmed this, indicating the abundance of miRNAs increasing during epididymal maturation [ 25 ] . Furthermore, studies on the epididymis have revealed that miRNAs transmitted from epididymis influence not only embryonic development but also the long-term health of the offspring [ 11 , 26 ] . However, whether miRNAs derived from the obstructive epididymal environment are involved in embryonic development remains unknown. To investigate whether an obstructive epididymal environment affects sperm miRNAs and results in abnormal embryonic development, we developed a mice model to mimic an obstructive epididymal environment by a surgical vasectomy. The current study identified that the obstructive epididymal environment reduced embryo development potential and altered sperm miRNAs profiles. Materials And Methods Animals. Male C57BL/6J were purchased from the Experiment Animal Center, Center for Disease Control and Prevention, Hubei Province (Wuhan, China), female B6D2F1 mice (C57BL/6J: 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/6J mice to create the CON and OEE groups. Male C57BL/6J 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 cauda 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 30min. 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, 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) × 10 6 /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 4ml centrifuge tube and prewarmed at 37℃. The sperm suspension was gently added to the gradient solution and centrifuge at 600g for 15min. Collected sperm precipitate was washed by G-IVF PLUS twice and resuspended by 500μl G-IVF PLUS. The sperm head was separated by a 30% power output of ultrasonic sonicator for 2min; 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 sequential centrifugation (centrifuge at 300g, 2000g, 10000g, and 100000g for 10min, 15min, 30min, and 80min, respectively). After centrifugation, the precipitate was reserved and resuspended with G-IVF PLUS to incubate with optimized sperm for 4.5h. Sperm was collected by centrifuging 600g for 15min 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) w (NSNF, Ningbo, China). Oocytes were collected 12-14h after hCG administration, and the cumulus-oocyte complexes (COC) were washed in prewarmed G-MOP PLUS medium (Vitrolife AB, Goteborg, Sweden) containing 0.4mg/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 [27, 28] . 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 H 2 O) 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 H 2 O) to lyse sperm utterly. Sperm lysis was transferred to 800μl TRIzol LS reagent (No. 10296028; Invitrogen; Thermo Fisher, Waltham, MA, USA) and blended vigorously. After placing in ice for 1h, 200μl 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. 500μl isopropanol was added to the aqueous phase and incubated at − 20°C for 30 min. The precipitate was collected by centrifuging at 12 000g 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 sequence Sperm RNA isolated from OEE and CON groups were used to build an sRNAs library. sRNAs is heavily decorated by RNA modifications that interfere with small RNA-seq library construction. The acylation and phosphorylation of the end of RNA sequence and methylation of m1A and m3C were removed. The libraries are qualified and quantified using Agilent BioAnalyzer 2100. Standard small RNA sequencing was performed on the Illumina NextSeq instrument. Real-time quantitative polymerase chain reaction (qRT-PCR) Reverse transcription and quantitative PCR (qPCR) of sperm miRNAs were using the All-in-one miRNA qRT-PCR Detection Kit (GeneCopoeia, Maryland, USA). cDNAs obtained were diluted for qPCR. All primers of miRNAs are listed in table S1. Amplifications were performed at least triplicate for each sample. Relative gene expression levels were evaluated using the 2 −ΔΔCt method and were normalized to the level of U6 mRNA. 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 table S2. 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 miRNAs inhibitors and scrambled RNA were manufactured by Sangon Biotech (Shanghai, China), listed in table S3. Synthetic RNA and scramble RNA concentration was adjusted to 2μmol/L. The microinjection needle was pulled by 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 C57BL/6J male to collect zygotes for miRNAs microinjection. MiRNAs microinjection was performed in a G-MOP PLUS medium. Synthetic miRNAs and scramble RNA as control were microinjected into the zygotes with two pronuclei by Femotojet 4i (Eppendorf AG, Germany) [29] . 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. Results The alteration in testis and epididymis of different obstructive interval Firstly, we observed the morphology of testis and epididymis of postoperative 4-, 8-, 12- week mice to determine the proper obstructive interval via HE staining. In postoperative 4-week mice, the morphology of testes and epididymis in the OEE group showed no differences compared to the CON group. In postoperative 8-week mice, only the epididymis became markedly swelling in the OEE group. However, in 12-week postoperative mice of the OEE group, plenty of atypical epididymis tubules appeared in the epididymis, with a cluster of floccules residing in the cavity. Moreover, the number of seminiferous tubules decreased considerably, and the seminiferous epithelium atrophied in the testis (Fig. 1a,b). According to the case-control trial and the OA definition [30] , we chose the postoperative 8-week OEE group mice for the subsequent study. Afterward, we weighed their epididymis and testis. Compared to the CON group, the OEE group showed a significant increase in the epididymis weight, while no difference was found in the testis weight (P<0.05, Fig. 1c,d). The obstructive epididymal environment results in poor sperm quality The sperm concentration in the OEE group increased significantly (P <0.01, Fig. 2a) compared to the CON group, as did the count of aberrant sperm (P < 0.01, Fig. 2d). DNA fragment index (DFI) was also significantly higher than the CON group (P < 0.01, Fig. 2e). The viability and forward motility of sperm decreased (P < 0.01, Fig. 2b,c). The obstructive epididymal environment results in an abnormal preimplantation embryo which can be saved by CON epididymosomes Due to the poor quality of sperm, we performed ICSI to observe preimplantation embryo development. Our results showed that the rate of morula in the OEE group decreased compared to the CON group (P < 0.05), as was the rate of the blastocyst, with no statistical significance (Fig. 3). The current study examined whether an abnormal epididymis environment caused the aberrant preimplantation embryo development; sperm from the OEE group was retrieved and incubated with epididymosomes from the CON group for subsequent ICSI. Compared to the OEE group, the rate of the morula and blastocyst from the OEE+CONE group observed a considerable increase (P<0.01 or P<0.05, Fig. 3), indicating that epididymosomes from the CON group could, at least partially, save the aberrant embryo development. The obstructive epididymal environment results in abnormal post-preimplantation embryonic development Investigating the impacts of the obstructive epididymal environment on post-implantation embryo development, 2-cell embryos of the OEE group, CON group, and OEE+CONE group were transplanted into the surrogate female ICR mice. The rate of live-born pups from the OEE group was significantly lower in comparison to the CON group (P<0.05). Though there was a slight increase in the number of live-born pups from the OEE +CONE group compared to the OEE group, the difference was not statistically significant (Fig. 4a). And no difference in weight was observed between the three groups of mice, including female and male offspring, at three weeks of age (Fig. 4b,c). The obstructive epididymal environment affects sperm sRNAs profile and downregulates developmental miRNAs To examine whether sperm miRNAs profile are altered under the epididymal environment, we examined sperm sRNA profiles by sRNA-seq. sRNA-seq showed that among the 1611 miRNAs detected, 594 were up-regulated, and 605 were down-regulated in the OEE group (Fig. 5a-b). miR-17-92 cluster and Sfmbt2 miRNA cluster were previously reported to be related to embryonic development. Loss of miR-17-92 cluster in mice led to lung hypoplasia and ventricular septal defect, and newborns died shortly after birth [31] . A bunch of miRNAs mainly expressed in the placenta is located in the intron 10 of Sfmbt2, and knockout of the intron 10 of Sfmbt2 resulted in placental malformation and fetal death [32, 33] Therefore, we examined the expression of the miR-17-92 cluster and Sfmbt2 miRNA cluster in sperm and epididymis. Compared to the CON group, the expression of the miR-17-92 cluster and Sfmbt2 miRNA cluster decreased significantly in sperm of the OEE group (P<0.05 or P<0.01, Fig. 5c), as well as epididymis (P<0.05 or P<0.001, Fig. 5d). These results demonstrated that the obstructive epididymal environment influenced the sperm miRNA profiles. The regulation of miRNAs in the epididymis To explore whether the epididymis regulated the biogenesis of miRNAs, we examined the expression of DICER and DROSHA in the epididymis. The expression of DICER and DROSHA enzymes increased in the OEE group, but only the DICER enzyme had a significant difference (P<0.05, Fig. 6a). Since the methylation of promoter-associated CpG-rich regions also regulates the expression of miRNAs, we also measured the methylation level of promoter-associated CpG-rich regions of the two miRNA clusters. And the methylation level of promoter-associated CpG-rich regions was increased in the OEE group compared to the CON group (P<0.05 or P<0.01, Fig. 6b,c). We determined the expression of the maintenance of DNA methylation enzymes DNMT1, de novo DNA methylation enzymes DNMT3a, and DNMT3b in the epididymis. The expression of DNMT3a mRNA in the OEE group increased significantly and DNMT3b mRNA decreased significantly (P<0.05 or P<0.01, Fig. 6 d). The abundance of the miR-17-92 cluster and Sfmbt2 miRNA cluster was regulated by DNA methylation. MiR-17-92 cluster and Sfmbt2 miRNA cluster do not impair preimplantation embryonic development To verify whether the downregulation of the expression of the miR-17-92 cluster and Sfmbt2 miRNA cluster would result in aberrant embryonic development, we injected the inhibitor of the miR-17-92 cluster and Sfmbt2 miRNA cluster into normal zygotes. The results showed that compared to the negative group, the downregulation of the miR-17-92 cluster and Sfmbt2 miRNA cluster didn’t impair embryonic development (Fig. 7). MiR-17-92 cluster and Sfmbt2 miRNA cluster do not impair post-implantation embryonic development We transferred the 2-cell embryos of the NC group, miR-17-92 group, and Sfmbt2 group into the female ICR surrogate mice. There was no difference in the rate of live-born pups and the weight of 3-week-old female and male offspring compared to the negative group (Fig.8). Together, these results indicated that the downregulation of the miR-17-92 cluster and Sfmbt2 miRNA cluster alone might not be sufficient to impair post-implantation embryonic development. The combination of miR-17-92 cluster and Sfmbt2 miRNA cluster microinjection does not affect post-implantation embryonic development. To see if a single miRNA cluster is not sufficient to impair embryonic development, we mixed the miR-17-92 cluster and the Sfmbt2 miRNA cluster to inject into normal zygotes and transferred 2-cell embryo to ICR surrogate mice. There was no difference in the rate of live-born pups compared to the negative group, indicating that the miR-17-92 cluster and Sfmbt2 miRNA cluster was not the cause of abnormal embryonic development (Fig.9). Discussion Our results showed the obstructive epididymal environment affected sperm quality and preimplantation or post-implantation embryonic development, as well as sperm miRNAs profile. In these differentially expressed miRNAs, miR-17-92 cluster and Sfmbt2 miRNA clusters were significantly downregulated in the epididymis by DNA methylation, which ultimately reduced the abundance of miRNAs in sperm. Though the two miRNA clusters were previously shown to be related to embryonic development, disruption of their expression in normal zygotes did not hinder embryonic development. Taken together, our study provides evidence that the obstructive epididymal environment affected embryonic development and the abundance of sperm miRNAs. Firstly, our research showed that the long obstructive interval affected the histologic morphology of the testis and epididymis. Singh’s study also confirmed these histologic changes in mice, but these changes emerged after the postoperative 4th month [ 34 ] . Nevertheless, population data showed that long obstructive intervals had no negative impacts on the function of testis [ 35 ] , which was in line with that discovered in vasectomized primates [ 36 ] . Differences between species might cause this discrepancy. In population data, some studies implied that the obstructive epididymal environment harms sperm quality. The first post-vasectomy sperm analysis for postoperative vasectomy patients revealed that the sum of motile sperm decreased sharply [ 37 ] . Quality tests for OA patients’ epididymal sperm showed DFI and aberrant sperm increased [ 5 , 38 – 40 ] . These results were confirmed by our findings; additionally, unvital sperm was found to increase in OEE group mice. Some studies reported embryo quality of OA sperm was not as good as the embryo quality of normal sperm. Loutradi’s and Yu’s studies showed that the testicular sperm from OA cases had poor embryo quality compared to donor sperm [ 8 , 41 ] . And our study showed the same result. Nonetheless, Solomon’s population study showed no difference in the embryonic development of epididymal sperm between the OA group and the donor sperm group [ 9 ] . However, we noticed that the patients’ obstructive interval, which was reported to be positively correlated with pregnancy rate, was not mentioned in their study [ 42 ] . And the obstructive interval might be the cause of the discrepancy between our study and Solomon’s. Matura sperm was transcriptionally and translationally silent due to a lack of ribosomes and highly compacted DNA, sperm RNA originated from remnants of spermatogenesis and epididymal transit. During epididymal maturation, the payload of sperm miRNAs, piRNAs, and tsRNAs was in flux [ 43 ] . miRNAs profile of sperm from proximal and distal epididymal segments displayed loss and acquisition of miRNAs, and the intake of RNA includes novel miRNAs species which is not natively expressed in immature sperm [ 44 ] . RNA sequence for mouse epididymosomes revealed that epididymosomes contained more than 350 miRNAs, the majority of which were also presented in the sperm miRNAs profiles, with more than 50 miRNAs found to be expressed exclusively in the epididymal sperm and epididymosomes [ 45 ] . Our results showed that obstructive epididymal environment altered sperm miRNAs profiles, including the miR-17-92 cluster and Sfmbt2 miRNA cluster, which are reported to be related to development. MiR-17-92 cluster is made up of MiR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a-1 and is involved in the cell cycle, proliferation, apoptosis, and other processes [ 46 ] . The MiR-17-92 cluster has been linked to development. Humans with heterozygous microdeletions in the MIR17HG locus had severe skeletal abnormalities [ 47 ] . The deletion of MIR17HG in mice resulted in perinatal lethality, and germline deletion of the miR-17-92 cluster recapitulated the phenotype observed in humans, also growth restriction, cardiac defects, and lung hypoplasia [ 48 ] . Furthermore, the miR-17-92 cluster was discovered to be required for B cell development and lymphocyte homeostasis [ 31 ] . The Sfmbt2 gene is paternally expressed in the mouse placenta and is required for placental development; Sfmbt2 knockout mice failed to maintain trophoblasts [ 49 ] . The specifically deletion of the entire Sfmbt2 miRNA cluster in mice resulted in placental malformation, especially in the spongiotrophoblast layer [ 32 ] ; furthermore, deletion of H3K27me3 imprinting in the Sfmbt2 miRNA cluster enlarged mouse placentas [ 50 ] . The biogenesis of iRNAs is processed by DROSHA and DICER and the knockout of DROSHA and DICER decreased the biogenesis of a majority of miRNAs [ 51 ] . Li’s study showed the expression of the miR-17-92 cluster was positively correlated with the expression of DICER and DROSHA [ 52 ] . This is not consistent with our findings which indicated that the downregulation of miR-17-92 miRNA and Sfmbt2 miRNA cluster was unrelated to DICER and DROSHA. DNA methylation is an epigenetic mark involved in the repression of transcription. The methylation level of CpG-island at transcriptional start sites is positively correlated with transcription activity [ 53 ] . DNMT1, DNMT3a, and DNMT3b are canonical cytosine-5 DNA methyltransferases that catalyze the addition of methylation marks to genomic DNA [ 54 ] . Duaa’s study showed the hypermethylation of the miR-17-92 cluster promoter resulted in a significant downregulation of the miR-17-92 cluster in idiopathic pulmonary fibrosis patients, and the methylation level was upregulated by the DNMT1 [ 55 ] . The upregulation of DNMT3a repressed the expression of miRNAs [ 56 ] . Our study revealed that the expression of the miR-17-92 cluster and Sfmbt2 miRNA cluster were governed by DNA methylation, which DNMT3a and DNMT1 could regulate. Considering the transcriptional inertia of mature sperm, we speculated that epididymis regulated the expression of miR-17-92 and Sfmbt2 miRNA cluster and, more importantly, influenced the abundance of sperm miRNAs. The miRNAs transmitted by epididymis were found to influence embryonic development. Collins’s study suggested caput sperm led to abnormal post-implantation embryonic development due to a lack of some key miRNAs [ 11 , 57 ] (miR-880 cluster, the paralogous miR-17-92, miR-106b-25 clusters, and the miR-34b/c pair which are scarce in caput epididymis but enriched in cauda epididymis [ 22 , 23 , 44 ] ), and suppletion of these miRNAs to the embryo of caput sperm rescued the abnormal post-implantation embryonic development. Our study found that epididymosomes from the CON group can rescue embryonic development caused by the obstructive epididymal environment. However, repression of the expression of developmental miR-17-92 cluster and Sfmbt2 miRNA cluster in normal zygotes did not hinder embryonic development. Unlike Collins’s study, in which a bunch of different miRNAs were injected into zygotes and influenced embryonic development, we only injected two clusters of miRNAs. And it seems that these miRNA cluster microinjection into zygotes is insufficient to affect embryonic development. In addition, differences in embryos derived from the caput sperm and cauda sperm have been questioned, which might be resulted from the methods used in the preparation of the sperm head rather than the miRNAs [ 58 , 59 ] . Besides, we can’t rule out whether proteins transmitted from epididymosomes have an impact on embryonic development. The studies of epididymosomes proteins revealed that epididymosomes proteins are related to sperm function, such as motility, capacitation, acrosome reaction, and so on [ 60 ] . And the sperm cell contains a large number of proteins involved in regulating translation and transcription that might also be critical for early embryos [ 61 ] . Above all, the function of the epididymis and sperm RNA in early embryonic development is still not very clear and warrants further exploration. Conclusion In conclusion, our findings show that an obstructive epididymal environment influences sperm quality, embryonic development, and the abundance of sperm miRNAs profiles, indicating that the epididymis is important in early embryonic development and may play a potential role in sperm epigenome. Despite the fact that the miR-17-92 and Sfmbt2 miRNA clusters did not hinder embryonic development, our findings provide evidence to support further research into the effects of sperm miRNAs on embryonic development. Abbreviations miRNAs : microRNAs OEE : obstructive epididymal environment CON : control HE : hematoxylin and eosin ICSI : intracytoplasmic sperm injection OA : obstructive azoospermia sRNAs : small RNA pri-miRNAs : primary miRNAs pre-miRNAs : precursor miRNAs cKO : Conditional knockout DFI : DNA fragmentation index PMSG : pregnant mare serum gonadotropin hCG : human chorionic gonadotrophin COCs : cumulus-oocyte complexes RT-qPCR :Real-time quantitative polymerase chain reaction Declarations Availability of data and materials The data supporting the results of this study can be obtained from the corresponding authors upon reasonable request. Acknowledgments None Funding This work was supported by grants from the National Natural Science Foundation of China (No. 82071710). Author information Authors and Affiliations Institute of Reproductive Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China. Xunwei Wu, Xiaomei He, Qian Liu ＆ Honggang Li Contributions XW carried out part of animal experiments, analyzed the data, and wrote this manuscript. HW carried out most of the experiment and analyzed the data, and QL helped a part of the animal’s experiment. All authors have read and given their final approval of the submitted manuscript. Corresponding author Correspondence to Honggang Li Ethics declarations Ethics approval and consent to participate The study was approved by the Animal Care Ethics Committee of Huazhong university of science and technology. Consent for publication Not applicable. Competing interests The authors declare no conflicts of interest. Additional information None References Cocuzza, M., C. Alvarenga, and R. Pagani, The epidemiology and etiology of azoospermia . Clinics, 2013. 68 (S1): p. 15–26. Wosnitzer, M.S. and M. Goldstein, Obstructive azoospermia . Urol Clin North Am, 2014. 41 (1): p. 83–95. 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Xie, Rebooting the Epigenomes during Mammalian Early Embryogenesis . Stem Cell Reports, 2020. 15 (6): p. 1158–1175. Sharma, U., et al., Small RNAs Are Trafficked from the Epididymis to Developing Mammalian Sperm . Dev Cell, 2018. 46 (4): p. 481–494 e6. Sharma, U., et al., Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals . Science, 2016. 351 (6271): p. 391–396. Belleannee, C., et al., Epididymosomes convey different repertoires of microRNAs throughout the bovine epididymis . Biol Reprod, 2013. 89 (2): p. 30. Gervasi, M.G. and P.E. Visconti, Molecular changes and signaling events occurring in spermatozoa during epididymal maturation . Andrology, 2017. 5 (2): p. 204–218. Chan, J.C., et al., Reproductive tract extracellular vesicles are sufficient to transmit intergenerational stress and program neurodevelopment . Nat Commun, 2020. 11 (1): p. 1499. Yoshida, N. and A.C. Perry, Piezo-actuated mouse intracytoplasmic sperm injection (ICSI) . Nat Protoc, 2007. 2 (2): p. 296–304. Boiani, M., Intracytoplasmic sperm injection with the Eppendorf PiezoXpert®-a reliable partner for the research mouse facility . Eppendorf Application Note 238. www.eppendorf. com Ordering information … Yamanaka, Y., Microinjection of RNA into Mouse Zygotes . Cold Spring Harb Protoc, 2018. 2018 (7). Xiang, Y., et al., Long-term effect of vasectomy on spermatogenesis in men: a morphometric study . Asian J Androl, 2013. 15 (3): p. 434–6. Ventura, A., et al., Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters . Cell, 2008. 132 (5): p. 875–86. Inoue, K., et al., The Rodent-Specific MicroRNA Cluster within the Sfmbt2 Gene Is Imprinted and Essential for Placental Development . Cell Rep, 2017. 19 (5): p. 949–956. Malnou, E.C., et al., Imprinted MicroRNA Gene Clusters in the Evolution, Development, and Functions of Mammalian Placenta . Front Genet, 2018. 9 : p. 706. Singh, S.K. and S. Chakravarty, Histologic changes in the mouse testis after bilateral vasectomy . Asian J Androl, 2000. 2 (2): p. 115–20. Zhao, K., et al., Long-term safety, health and mental status in men with vasectomy . Sci Rep, 2018. 8 (1): p. 15703. Peng, B., et al., Quantitative (stereological) study of the effects of vasectomy on spermatogenesis in rhesus monkeys (Macaca mulatta) . Reproduction, 2002. 124 (6): p. 847–56. McMartin, C., et al., Can a Low Sperm Concentration without Assessing Motility Confirm Vasectomy Success? A Retrospective Descriptive Study . J Urol, 2021. 206 (1): p. 109–114. Jerre, E., et al., Sperm chromatin structure assay high DNA stainability sperm as a marker of early miscarriage after intracytoplasmic sperm injection . Fertil Steril, 2019. 112 (1): p. 46–53.e2. Kennedy, C., et al., Sperm chromatin structure correlates with spontaneous abortion and multiple pregnancy rates in assisted reproduction . Reprod Biomed Online, 2011. 22 (3): p. 272–6. Lin, M.H., et al., Sperm chromatin structure assay parameters are not related to fertilization rates, embryo quality, and pregnancy rates in in vitro fertilization and intracytoplasmic sperm injection, but might be related to spontaneous abortion rates . Fertil Steril, 2008. 90 (2): p. 352–9. Yu, Y., et al., Pregnancy and Neonatal Outcomes in Azoospermic Men After Intracytoplasmic Sperm Injection Using Testicular Sperm and Donor Sperm . Med Sci Monit, 2018. 24 : p. 6968–6974. Borges, E., Jr., et al., The obstructive interval predicts pregnancy rates in post-vasectomy patients undergoing ICSI with surgical sperm retrieval . Reprod Biomed Online, 2019. 39 (1): p. 134–140. Santiago, J., et al., All you need to know about sperm RNAs . Hum Reprod Update, 2021. 28 (1): p. 67–91. Nixon, B., et al., The microRNA signature of mouse spermatozoa is substantially modified during epididymal maturation . Biol Reprod, 2015. 93 (4): p. 91. Reilly, J.N., et al., Characterisation of mouse epididymosomes reveals a complex profile of microRNAs and a potential mechanism for modification of the sperm epigenome . Sci Rep, 2016. 6 : p. 31794. Mogilyansky, E. and I. Rigoutsos, The miR-17/92 cluster: a comprehensive update on its genomics, genetics, functions and increasingly important and numerous roles in health and disease . Cell Death Differ, 2013. 20 (12): p. 1603–14. Marcelis, C.L., et al., Genotype-phenotype correlations in MYCN-related Feingold syndrome . Hum Mutat, 2008. 29 (9): p. 1125–32. de Pontual, L., et al., Germline deletion of the miR-17∼92 cluster causes skeletal and growth defects in humans . Nat Genet, 2011. 43 (10): p. 1026–30. Miri, K., et al., The imprinted polycomb group gene Sfmbt2 is required for trophoblast maintenance and placenta development . Development, 2013. 140 (22): p. 4480–9. Inoue, K., et al., Loss of H3K27me3 imprinting in the Sfmbt2 miRNA cluster causes enlargement of cloned mouse placentas . Nat Commun, 2020. 11 (1): p. 2150. Treiber, T., N. Treiber, and G. Meister, Regulation of microRNA biogenesis and its crosstalk with other cellular pathways . Nat Rev Mol Cell Biol, 2019. 20 (1): p. 5–20. Li, J., et al., The role, mechanism and potentially novel biomarker of microRNA-17-92 cluster in macrosomia . Sci Rep, 2015. 5 : p. 17212. Jones, P.A., Functions of DNA methylation: islands, start sites, gene bodies and beyond . Nat Rev Genet, 2012. 13 (7): p. 484–92. Lyko, F., The DNA methyltransferase family: a versatile toolkit for epigenetic regulation . Nat Rev Genet, 2018. 19 (2): p. 81–92. Dakhlallah, D., et al., Epigenetic regulation of miR-17 ~ 92 contributes to the pathogenesis of pulmonary fibrosis . Am J Respir Crit Care Med, 2013. 187 (4): p. 397–405. Pang, Y., et al., MYC and DNMT3A-mediated DNA methylation represses microRNA-200b in triple negative breast cancer . J Cell Mol Med, 2018. 22 (12): p. 6262–6274. Conine, C.C., et al., MicroRNAs Absent in Caput Sperm Are Required for Normal Embryonic Development . Dev Cell, 2019. 50 (1): p. 7–8. Wang, Y., et al., Both Cauda and Caput Epididymal Sperm Are Capable of Supporting Full-Term Development in FVB and CD-1 Mice . Dev Cell, 2020. 55 (6): p. 675–676. Zhou, D., et al., Caput Epididymidal Mouse Sperm Support Full Development . Dev Cell, 2019. 50 (1): p. 5–6. Björkgren, I. and P. Sipilä, The impact of epididymal proteins on sperm function . Reproduction, 2019. 158 (5): p. R155-r167. Castillo, J., M. Jodar, and R. Oliva, The contribution of human sperm proteins to the development and epigenome of the preimplantation embryo . Hum Reprod Update, 2018. 24 (5): p. 535–555. Additional Declarations No competing interests reported. Supplementary Files additonalfile1.xlsx Additional file 1 additionalfile2.docx Additional file 2 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 25 Jul, 2022 Reviews received at journal 22 Jul, 2022 Reviewers agreed at journal 07 Jul, 2022 Reviews received at journal 04 Jul, 2022 Reviewers agreed at journal 04 Jul, 2022 Reviewers invited by journal 02 Jul, 2022 Editor assigned by journal 01 Jul, 2022 Submission checks completed at journal 01 Jul, 2022 First submitted to journal 01 Jul, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1814149","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":117835919,"identity":"00131dff-e371-45ca-b9ed-e2794fa08998","order_by":0,"name":"Xunwei Wu","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xunwei","middleName":"","lastName":"Wu","suffix":""},{"id":117835920,"identity":"8d1a2161-39cc-4cc1-9719-5c60ad25910b","order_by":1,"name":"Xiaomei He","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaomei","middleName":"","lastName":"He","suffix":""},{"id":117835921,"identity":"4e69c925-3587-4d15-b954-7ae9aa61d575","order_by":2,"name":"Qian Liu","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Liu","suffix":""},{"id":117835922,"identity":"58f97a5b-7d3d-4527-be3a-af966134a798","order_by":3,"name":"Honggang Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYBACxvbGBoMPP9jq7Y83EKmFuefwgcKZPXwJDGcOEKmFfUZawmcONrkEhhsJRGrhnZFjuJmBxyyPcebjjTcYamyiCWqR7HljbFxgkVbMLJ1WbMFwLC23gZAWw/YcM+MZPMcY26RzzCQYGw4T1mJ/IMf8Nw/bf8YeyTNEamHsSEsw5mFjS5whwUOsFmAgG87sYTM24AH6JYEYv8CiUs6A/fDGGx9qbAhrQQYGEgmkKIdoIVXHKBgFo2AUjAwAADscQXQKU3zMAAAAAElFTkSuQmCC","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Honggang","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2022-07-01 06:14:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1814149/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1814149/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":23721613,"identity":"e364ade4-9472-43ef-9d21-a7e8a15bd503","added_by":"auto","created_at":"2022-07-11 17:39:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21772273,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe establishment of the OEE mice model. \u003c/strong\u003e\u003c/p\u003e\u003cp\u003e(a) Histological testis sections stained with hematoxylin and eosin from postoperative 4-,8-and12-week CON and OEE group mice. (b) Histological epididymis sections stained with hematoxylin and eosin from postoperative 4-,8-and12-week CON and OEE group mice. (c) Testis weight of postoperative 8-week mice. (d) Epididymis weight of postoperative 8-week mice. **p-value \u0026lt;0.01.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1814149/v1/e8a571271ad18ca49d4d5913.png"},{"id":23720968,"identity":"d8a60f87-89d4-46f1-8eb8-fd02c0df3bd7","added_by":"auto","created_at":"2022-07-11 17:34:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":933125,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe obstructive epididymal environment resulted in poor sperm quality\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e(a–e) Comparison of sperm quality between CON and OEE group mice. (a) Sperm concentration comparison. (b) Sperm viability comparison. (c) Sperm forward motility. Comparison. (d) Sperm abnormality comparison. (e) Sperm DNA fragment index comparison. **P-value \u0026lt; 0.01.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1814149/v1/829de3684be6fb480ccfb5f6.png"},{"id":23720972,"identity":"7d1cfd78-c5c2-4776-a357-c3e031ee3819","added_by":"auto","created_at":"2022-07-11 17:34:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5727604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe obstructive epididymal environment resulted in an abnormal preimplantation embryo, which can be saved by CON epididymosomes\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e(a) Representative images of preimplantation embryonic development of CON, OEE, and OEE+CONE group mice, arrow refers to developmental arrest. (b) The histogram shows the preimplantation embryonic development rate of CON, OEE, and OEE+CONE group mice.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1814149/v1/b48c9bc4ba44f0467435ed70.png"},{"id":23720973,"identity":"d8eee7ee-59d2-415d-bcf5-58b3869ec828","added_by":"auto","created_at":"2022-07-11 17:34:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":822483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe obstructive epididymal environment resulted in abnormal post-preimplantation embryonic development\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e(a) Live-born pups of the mice of CON, OEE, and OEE+CONE group. (b) The weight of 3-week-old female or male offspring from CON, OEE, and OEE+CONE group mice. (c) Representative images of live-born pups. CONE refers to CON group epididymosomes, *p-value \u0026lt;0.05 or **p-value \u0026lt;0.01.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1814149/v1/44d5e80f3b46d68f45a780ba.png"},{"id":23720974,"identity":"00ff9222-6202-4784-9016-34c0d667ae0a","added_by":"auto","created_at":"2022-07-11 17:34:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2596323,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe obstructive epididymal environment changes sperm sRNAs profile and downregulated developmental miRNAs \u003c/strong\u003e\u003c/p\u003e\u003cp\u003e(a) Heat map of sperm miRNAs differentially expressed between the CON and OEE group. (b) Volcano plot of sperm miRNAs differentially expressed between CON and OEE groups. (c) The expression ofmiR-17-92 and \u003cem\u003eSfmbt2\u003c/em\u003e miRNA cluster in sperm. (d) The expression ofmiR-17-92 and \u003cem\u003eSfmbt2\u003c/em\u003e miRNA cluster in epididymis. T refers to the OEE group, C refers to the CON group, miR-17-92 and \u003cem\u003eSfmbt2\u003c/em\u003e respectively refer tomiR-17-92 and \u003cem\u003eSfmbt2\u003c/em\u003e miRNA cluster. *p-value \u0026lt;0.05 or **p-value \u0026lt;0.01.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-1814149/v1/51103f1044edebf7295ec436.png"},{"id":23721612,"identity":"68f6d0b8-d6a8-43d7-b719-93e56792a103","added_by":"auto","created_at":"2022-07-11 17:39:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":898539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe regulation of miRNAs in the epididymis\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e(a) The expression of DROSHA and DICER in epididymis between CON and OEE group. (b) The expression of DNMT1, DNMT3A, and DNMT3B in epididymis between CON and OEE groups. (c-d) the methylation level of \u003cem\u003eMIR17HG\u003c/em\u003e and \u003cem\u003eSfmbt2\u003c/em\u003e promoter-associated CpG-rich regions. *p-value \u0026lt;0.05 or **p-value \u0026lt;0.01.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-1814149/v1/03448cc19e4f34af7ea6545b.png"},{"id":23722762,"identity":"9135965f-3c08-4c2f-9e07-75dbdcad0c85","added_by":"auto","created_at":"2022-07-11 17:49:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5631166,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-17-92 cluster and Sfmbt2 miRNA cluster did not impair preimplantation embryonic development \u003c/strong\u003e\u003c/p\u003e\u003cp\u003e(a) Representative images of preimplantation embryonic development of NC, miR-17-92, and \u003cem\u003eSfmbt2\u003c/em\u003e group. (b) Histogram showing the rate of preimplantation embryonic development of NC, miR-17-92, and \u003cem\u003eSfmbt2\u003c/em\u003e group.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-1814149/v1/8bcfcec396de8e3b4ec128d8.png"},{"id":23722210,"identity":"1444b969-5d2f-4445-99df-29e16ac213e3","added_by":"auto","created_at":"2022-07-11 17:44:14","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":465729,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-17-92 cluster and Sfmbt2 miRNA cluster did not impair post-implantation embryonic development\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e(a) Live-born pups of NC, miR-17-92, and \u003cem\u003eSfmbt2\u003c/em\u003e group. (b) The weight of 3-week-old female or male offspring from NC, miR-17-92, and \u003cem\u003eSfmbt2\u003c/em\u003e group. NC refers to the negative control, miR-17-92 and \u003cem\u003eSfmbt2\u003c/em\u003e respectively refers tomiR-17-92 and \u003cem\u003eSfmbt2\u003c/em\u003e miRNA cluster.\u0026nbsp;\u003c/p\u003e","description":"","filename":"figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-1814149/v1/7f0aba302f5f7ca76ead1bfc.png"},{"id":23720975,"identity":"910075d5-8082-41e2-8fdd-a72c37060da8","added_by":"auto","created_at":"2022-07-11 17:34:14","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":7564,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe combination of miR-17-92 cluster and Sfmbt2 miRNA cluster microinjection didn’t affect post-implantation embryonic development.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-1814149/v1/e328324b8decba6586810e47.png"},{"id":23722770,"identity":"3686642b-8e10-4970-8abf-39ce5322431a","added_by":"auto","created_at":"2022-07-11 17:49:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":786537,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1814149/v1/c087899d-2637-4c55-a0e7-31e58b48696d.pdf"},{"id":23721611,"identity":"579e3ccf-9d56-4830-8842-d2240853921c","added_by":"auto","created_at":"2022-07-11 17:39:14","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":63795,"visible":true,"origin":"","legend":"\u003cp\u003e\tAdditional file 1\u003c/p\u003e","description":"","filename":"additonalfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1814149/v1/89589d8f4e82d7302cdfad4c.xlsx"},{"id":23720969,"identity":"67c5f9cf-c829-4084-8ceb-ffa6185feb54","added_by":"auto","created_at":"2022-07-11 17:34:14","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19387,"visible":true,"origin":"","legend":"\u003cp\u003e\tAdditional file 2\u003c/p\u003e","description":"","filename":"additionalfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-1814149/v1/14f396d5198ad41c9666e652.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Developmental miR-17-92 cluster and sfmbt2 miRNA cluster can’t rescue the abnormal embryonic development caused by the obstructive epididymal environment","fulltext":[{"header":"Background","content":"\u003cp\u003eObstructive azoospermia (OA) refers to the absence of sperm in the ejaculate due to occlusion of the male reproductive tract. It can occur in any region of the male reproductive tract (rete testis, efferent ducts, epididymis, vas deferens, and ejaculatory duct). It\u0026rsquo;s a relatively frequent disease accounting for approximately 1% of all men and 10\u0026ndash;15% of infertile men \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. In most OA cases, the testis and the endocrine function are normal compared to non-obstructive azoospermia\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The clinical treatment for OA includes microsurgical reconstruction of the reproductive tract and sperm retrieval for ICSI. The OA patients\u0026rsquo; post-testicular sperm maturation is in an abnormal epididymal environment because of the obstructive reproductive tract; therefore, the debate over whether OA affects sperm and results in abnormal embryonic development has continued; nevertheless, no definite conclusion has been drawn regarding this issue\u003csup\u003e[\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7 CR8\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. The epididymis is an important organ for post-testicular sperm maturation, where sperm acquires proteins, sRNAs, forward ability, and fertilization capability\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Recently, Colin's study revealed that epididymis has an impact on embryonic development, as the caput sperm can\u0026rsquo;t support the post-implantation development of the embryo due to a lack of miRNAs transmitted from epididymis\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. However, whether an obstructive epididymal environment affects sperm miRNAs and results in abnormal embryonic development is not clear.\u003c/p\u003e \u003cp\u003eMiRNAs are a subset of non-coding RNAs, 19\u0026ndash;25 nt long in size. It participates in a series of biological processes through silencing target genes, such as differentiation, proliferation, and apoptosis\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. For decades, increasing evidence has suggested that sperm miRNAs play a pivotal role in epigenetic inheritance. It acts as a carrier of epigenetic inheritance information that transmits parents\u0026rsquo; phenotype to the offspring, such as depression, stress, and metabolic disorders\u003csup\u003e[\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Despite miRNAs-mediated inheritance mainly focused on offspring\u0026rsquo;s long-term health, such as obesity and diabetes, the sperm miRNA was also found to affect embryonic development. Micro-34c and micro-449b has been reported to be required for the early cleavage of the embryo\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. DROSHA and DICER are RNase III enzymes that process primary miRNAs (pri-miRNAs) and precursor miRNAs (pre-miRNAs). Conditional knockout (cKO) of DICER or DROSHA in testis disrupted miRNA synthesis, resulting in abnormal early embryo development, whereas injecting normal sperm RNA into these embryos rescued their development\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Furthermore, miRNAs delivered from the epididymis to sperm have supported post-implantation embryo development.\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. However, unlike DNA methylation and histone modification, the mechanism of RNA-mediated epigenetic inheritance in embryonic development remains largely unknown\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe epididymis has recently received much attention because of its ability to deliver miRNAs to sperm, which is critical for embryonic development. Previous research has found that the epididymis influences the sperm miRNAs profile via epididymosomes, an extracellular vesicle secreted by the epididymis epithelium\u003csup\u003e[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e; the high-throughput sequence of sperm sRNAs from various developmental stages has confirmed this, indicating the abundance of miRNAs increasing during epididymal maturation\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Furthermore, studies on the epididymis have revealed that miRNAs transmitted from epididymis influence not only embryonic development but also the long-term health of the offspring\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. However, whether miRNAs derived from the obstructive epididymal environment are involved in embryonic development remains unknown.\u003c/p\u003e \u003cp\u003eTo investigate whether an obstructive epididymal environment affects sperm miRNAs and results in abnormal embryonic development, we developed a mice model to mimic an obstructive epididymal environment by a surgical vasectomy. The current study identified that the obstructive epididymal environment reduced embryo development potential and altered sperm miRNAs profiles.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003ch3\u003eAnimals.\u003c/h3\u003e\n\u003cp\u003eMale C57BL/6J were purchased from the Experiment Animal Center, Center for Disease Control and Prevention, Hubei Province (Wuhan, China), female B6D2F1 mice (C57BL/6J: 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 \u0026deg;C and relative humidity of 42%. Sham operation and vasectomy were performed on 6-week-old male C57BL/6J mice to create the CON and OEE groups. Male C57BL/6J 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 cauda 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.\u003c/p\u003e\n\u003ch3\u003eHE staining\u003c/h3\u003e\n\u003cp\u003eThe 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.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eSperm isolation and sperm quality test\u003c/h3\u003e\n\u003cp\u003eSperm was released from cauda epididymis in prewarmed G-IVF PLUS (Vitrolife AB, Goteborg, Sweden) at 37℃ for 30min. \u0026nbsp; 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, 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\u0026ndash;2) \u0026times; 10\u003csup\u003e6\u003c/sup\u003e/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).\u003c/p\u003e\n\u003ch3\u003eSperm preparation for ICSI\u003c/h3\u003e\n\u003cp\u003eSperm 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 4ml centrifuge tube and prewarmed at 37℃. The sperm suspension was gently added to the gradient solution and centrifuge at 600g for 15min. Collected sperm precipitate was washed by G-IVF PLUS twice and resuspended by 500\u0026mu;l G-IVF PLUS. The sperm head was separated by a 30% power output of ultrasonic sonicator for 2min; then sperm head suspension was stored at -80℃.\u003c/p\u003e\n\u003ch3\u003eEpididymosomes isolation and fusion\u003c/h3\u003e\n\u003cp\u003eThe 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\u0026nbsp;\u0026mu;m and 40\u0026nbsp;\u0026mu;m\u0026nbsp;cell strainer to collect the supernatant. The filtered supernatant was performed sequential centrifugation (centrifuge at 300g, 2000g, 10000g, and 100000g for 10min, 15min, 30min, and 80min, respectively). After centrifugation, the precipitate was reserved and resuspended with G-IVF PLUS to incubate with optimized sperm for 4.5h. Sperm was collected by centrifuging 600g for 15min and prepared by sonication for injection.\u003c/p\u003e\n\u003ch3\u003eICSI and embryo transfer\u003c/h3\u003e\n\u003cp\u003eIntraperitoneal 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) w (NSNF, Ningbo, China). Oocytes were collected 12-14h after hCG administration, and the cumulus-oocyte complexes (COC) were washed in prewarmed G-MOP PLUS medium (Vitrolife AB, Goteborg, Sweden) containing 0.4mg/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\u0026reg;(Eppendorf AG, Germany) following standard ICSI procedures\u003csup\u003e[27, 28]\u003c/sup\u003e. The zygotes were then cultured in G1-PLUS/G2-PLUS sequential medium (Vitrolife AB, Goteborg, Sweden) at 37\u0026deg;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.\u003c/p\u003e\n\u003ch3\u003eSperm RNA extraction\u003c/h3\u003e\n\u003cp\u003eAfter oozing from the epididymis, sperm were successively filtered with 100 \u0026mu;m and 40 \u0026mu;m cell strainer to remove the tissue debris. Somatic cell lysis buffer (0.1% SDS, 0.5% Triton X in DEPC H\u003csub\u003e2\u003c/sub\u003eO) 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 H\u003csub\u003e2\u003c/sub\u003eO) to lyse sperm utterly. Sperm lysis was transferred to 800\u0026mu;l TRIzol LS reagent\u0026nbsp;(No. 10296028; Invitrogen; Thermo Fisher, Waltham, MA, USA) and\u0026nbsp;blended vigorously. After placing in ice for 1h, 200\u0026mu;l\u0026nbsp;chloroform was added and placed in ice for 10 min. Then the mixture was centrifuged at 12 000 g for 15 min at 4\u0026deg;C to collect the aqueous phase. 500\u0026mu;l\u0026nbsp;isopropanol was added to the aqueous phase and incubated at \u0026minus; 20\u0026deg;C for 30 min. The precipitate was collected by centrifuging at 12 000g for 15 min at 4\u0026deg;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).\u003c/p\u003e\n\u003ch3\u003esRNA sequence\u003c/h3\u003e\n\u003cp\u003eSperm RNA isolated from OEE and CON groups were used to build an sRNAs library. sRNAs is heavily decorated by RNA modifications that interfere with small RNA-seq library construction. The acylation and phosphorylation of the end of RNA sequence and methylation of m1A and m3C were removed. The libraries are qualified and quantified using Agilent BioAnalyzer 2100. Standard small RNA sequencing was performed on the Illumina NextSeq instrument. \u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eReal-time quantitative polymerase chain reaction (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eReverse transcription and quantitative PCR (qPCR) of sperm miRNAs were using the All-in-one miRNA qRT-PCR Detection Kit (GeneCopoeia, Maryland, USA). cDNAs obtained were diluted for qPCR. All primers of miRNAs are listed in table S1. Amplifications were performed at least triplicate for each sample. Relative gene expression levels were evaluated using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method and were normalized to the level of U6 mRNA.\u003c/p\u003e\n\u003ch3\u003eDNA methylation\u003c/h3\u003e\n\u003cp\u003eEpididymis 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 \u003cem\u003eSfmbt2\u003c/em\u003e and MIR17HG. Primer information was included in table S2. 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.\u003c/p\u003e\n\u003ch3\u003eSynthetic miRNAs microinjection\u003c/h3\u003e\n\u003cp\u003eSynthetic miRNAs inhibitors and scrambled RNA were manufactured by Sangon Biotech (Shanghai, China), listed in table S3. Synthetic RNA and scramble RNA concentration was adjusted to 2\u0026mu;mol/L. The microinjection needle was pulled by 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 C57BL/6J male to collect zygotes for miRNAs microinjection. MiRNAs microinjection was performed in a G-MOP PLUS medium. Synthetic miRNAs and scramble RNA as control were microinjected into the zygotes with two pronuclei by Femotojet 4i (Eppendorf AG, Germany)\u003csup\u003e[29]\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eStatistical analysis\u003c/h3\u003e\n\u003cp\u003eAll statistical analyses were performed using Graphpad Prism 8.0 (La Jolla, CA, USA). The experimental results are presented as mean \u0026plusmn; standard error of the mean (SEM). Data were analyzed using the t-test, Mann-Whitney analysis, or chi-square test. P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003ch3\u003eThe alteration in testis and epididymis of different obstructive interval\u003c/h3\u003e\n\u003cp\u003eFirstly, we observed the morphology of testis and epididymis of postoperative 4-, 8-, 12- week mice to determine the proper obstructive interval via HE staining. In postoperative 4-week mice, the morphology of testes and epididymis in the OEE group showed no differences compared to the CON group. In postoperative 8-week mice, only the epididymis became markedly swelling in the OEE group. However, in 12-week postoperative mice of the OEE group, plenty of atypical epididymis tubules appeared in the epididymis, with a cluster of floccules residing in the cavity. Moreover, the number of seminiferous tubules decreased considerably, and the seminiferous epithelium atrophied in the testis (Fig. 1a,b). According to the case-control trial and the OA definition\u003csup\u003e[30]\u003c/sup\u003e, we chose the postoperative 8-week OEE group mice for the subsequent study. Afterward, we weighed their epididymis and testis. Compared to the CON group, the OEE group showed a significant increase in the epididymis weight, while no difference was found in the testis weight (P\u0026lt;0.05, Fig. 1c,d).\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eThe obstructive epididymal environment results in poor sperm quality\u003c/h3\u003e\n\u003cp\u003eThe sperm concentration in the OEE group increased significantly (P \u0026lt;0.01, Fig. 2a) compared to the CON group, as did the count of aberrant sperm (P \u0026lt; 0.01, Fig. 2d). DNA fragment index\u0026nbsp;(DFI) was also significantly higher than the CON group \u0026nbsp; (P \u0026lt; 0.01, Fig. 2e). The viability and forward motility of sperm decreased (P \u0026lt; 0.01, Fig. 2b,c).\u003c/p\u003e\n\u003ch3\u003eThe obstructive epididymal environment results in an abnormal preimplantation embryo which can be saved by CON epididymosomes\u003c/h3\u003e\n\u003cp\u003eDue to the poor quality of sperm, we performed ICSI to observe preimplantation embryo development. Our results showed that the rate of morula in the OEE group decreased compared to the CON group (P \u0026lt; 0.05), as was the rate of the blastocyst, with no statistical significance (Fig. 3). The current study examined whether an abnormal epididymis environment caused the aberrant preimplantation embryo development; sperm from the OEE group was retrieved and incubated with epididymosomes from the CON group for subsequent ICSI. Compared to the OEE group, the rate of the morula and blastocyst from the OEE+CONE group observed a considerable increase (P\u0026lt;0.01 or P\u0026lt;0.05, Fig. 3),\u0026nbsp;indicating that epididymosomes from the CON group could, at least partially, save the aberrant embryo development.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eThe obstructive epididymal environment results in abnormal post-preimplantation embryonic development\u003c/h3\u003e\n\u003cp\u003eInvestigating the impacts of the obstructive epididymal environment on post-implantation embryo development, 2-cell embryos of the OEE group, CON group, and OEE+CONE group were transplanted into the surrogate female ICR mice. The rate of live-born pups from the OEE group was significantly lower in comparison to the CON group\u0026nbsp;(P\u0026lt;0.05). Though there was a slight increase in the number of live-born pups from the OEE +CONE group compared\u0026nbsp;to the OEE group, the difference was not statistically significant (Fig. 4a). And no difference in weight was observed between the three groups of mice, including female and male offspring, at three weeks of age (Fig. 4b,c).\u003c/p\u003e\n\u003ch3\u003eThe obstructive epididymal environment affects sperm sRNAs profile and downregulates developmental miRNAs\u003c/h3\u003e\n\u003cp\u003eTo examine whether sperm miRNAs profile are altered under the epididymal environment, we examined sperm sRNA profiles by sRNA-seq. sRNA-seq showed that among the 1611 miRNAs detected, 594 were up-regulated, and 605 were down-regulated in the OEE group (Fig. 5a-b). miR-17-92 cluster and \u003cem\u003eSfmbt2\u003c/em\u003e miRNA cluster were previously reported to be related to embryonic development. Loss of miR-17-92 cluster in mice led to lung hypoplasia and ventricular septal defect, and newborns died shortly after birth\u003csup\u003e[31]\u003c/sup\u003e. \u003cem\u003e\u0026nbsp;\u003c/em\u003eA bunch of miRNAs mainly expressed in the placenta is located in the intron 10 of \u003cem\u003eSfmbt2,\u003c/em\u003e and knockout of the intron 10 of \u003cem\u003eSfmbt2\u0026nbsp;\u003c/em\u003eresulted in placental malformation and fetal death\u003csup\u003e[32, 33]\u003c/sup\u003e Therefore, we examined the expression of the miR-17-92 cluster and \u003cem\u003eSfmbt2\u003c/em\u003e miRNA cluster in sperm and epididymis. Compared to the CON group, the expression of the miR-17-92 cluster and \u003cem\u003eSfmbt2\u003c/em\u003e miRNA cluster decreased significantly in sperm of the OEE group (P\u0026lt;0.05 or P\u0026lt;0.01, Fig. 5c), as well as epididymis (P\u0026lt;0.05 or P\u0026lt;0.001, Fig. 5d). These results demonstrated that the obstructive epididymal environment influenced the sperm miRNA profiles.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eThe regulation of miRNAs in the epididymis\u003c/h3\u003e\n\u003cp\u003eTo explore whether the epididymis regulated the biogenesis of miRNAs, we examined the expression of DICER and DROSHA in the epididymis. The expression of DICER and DROSHA enzymes increased in the OEE group, but only the DICER enzyme had a significant difference (P\u0026lt;0.05, Fig. 6a). Since the methylation of promoter-associated CpG-rich regions also regulates the expression of miRNAs, we also measured the methylation level of promoter-associated CpG-rich regions of the two miRNA clusters. And the methylation level of promoter-associated CpG-rich regions was increased in the OEE group compared to the CON group (P\u0026lt;0.05 or P\u0026lt;0.01, Fig. 6b,c).\u0026nbsp;We determined the expression of the maintenance of DNA methylation enzymes DNMT1, de novo DNA methylation enzymes DNMT3a, and DNMT3b in the epididymis. The expression of DNMT3a mRNA in the OEE group increased significantly and DNMT3b mRNA decreased significantly (P\u0026lt;0.05 or P\u0026lt;0.01, Fig. 6 d). The abundance of the miR-17-92 cluster and Sfmbt2 miRNA cluster was regulated by DNA methylation.\u003c/p\u003e\n\u003ch3\u003eMiR-17-92 cluster and \u003cem\u003eSfmbt2\u003c/em\u003e miRNA cluster do not impair preimplantation embryonic development\u003c/h3\u003e\n\u003cp\u003eTo verify whether the downregulation of the expression of the miR-17-92 cluster and \u003cem\u003eSfmbt2\u003c/em\u003e miRNA cluster would result in aberrant embryonic development, we injected the inhibitor of the miR-17-92 cluster and \u003cem\u003eSfmbt2\u0026nbsp;\u003c/em\u003emiRNA cluster into normal zygotes. The results showed that compared to the negative group, the downregulation of the miR-17-92 cluster and \u003cem\u003eSfmbt2\u003c/em\u003e miRNA cluster didn\u0026rsquo;t impair embryonic development (Fig. 7).\u003c/p\u003e\n\u003ch3\u003eMiR-17-92 cluster and Sfmbt2 miRNA cluster\u0026nbsp;do not impair post-implantation embryonic development\u003c/h3\u003e\n\u003cp\u003eWe transferred the 2-cell embryos of the NC group, miR-17-92 group, and Sfmbt2 group into the female ICR surrogate mice. There was no difference in the rate of live-born pups and the weight of 3-week-old female and male offspring compared to the negative group \u0026nbsp;(Fig.8). \u0026nbsp;Together, these results indicated that the downregulation of the miR-17-92 cluster and Sfmbt2 miRNA cluster alone might not be sufficient to impair post-implantation embryonic development.\u003c/p\u003e\n\u003ch3\u003eThe combination of miR-17-92 cluster and Sfmbt2 miRNA cluster microinjection does not affect post-implantation embryonic development.\u003c/h3\u003e\n\u003cp\u003eTo see if a single miRNA cluster is not sufficient to impair embryonic development, we mixed the miR-17-92 cluster and the Sfmbt2 miRNA cluster to inject into normal zygotes and transferred 2-cell embryo to ICR surrogate mice. There was no difference in the rate of live-born pups compared to the negative group, indicating that the miR-17-92 cluster and Sfmbt2 miRNA cluster was not the cause of abnormal embryonic development (Fig.9).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur results showed the obstructive epididymal environment affected sperm quality and preimplantation or post-implantation embryonic development, as well as sperm miRNAs profile. In these differentially expressed miRNAs, miR-17-92 cluster and Sfmbt2 miRNA clusters were significantly downregulated in the epididymis by DNA methylation, which ultimately reduced the abundance of miRNAs in sperm. Though the two miRNA clusters were previously shown to be related to embryonic development, disruption of their expression in normal zygotes did not hinder embryonic development. Taken together, our study provides evidence that the obstructive epididymal environment affected embryonic development and the abundance of sperm miRNAs.\u003c/p\u003e \u003cp\u003eFirstly, our research showed that the long obstructive interval affected the histologic morphology of the testis and epididymis. Singh\u0026rsquo;s study also confirmed these histologic changes in mice, but these changes emerged after the postoperative 4th month\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, population data showed that long obstructive intervals had no negative impacts on the function of testis \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, which was in line with that discovered in vasectomized primates\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Differences between species might cause this discrepancy.\u003c/p\u003e \u003cp\u003eIn population data, some studies implied that the obstructive epididymal environment harms sperm quality. The first post-vasectomy sperm analysis for postoperative vasectomy patients revealed that the sum of motile sperm decreased sharply\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Quality tests for OA patients\u0026rsquo; epididymal sperm showed DFI and aberrant sperm increased\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. These results were confirmed by our findings; additionally, unvital sperm was found to increase in OEE group mice. Some studies reported embryo quality of OA sperm was not as good as the embryo quality of normal sperm. Loutradi\u0026rsquo;s and Yu\u0026rsquo;s studies showed that the testicular sperm from OA cases had poor embryo quality compared to donor sperm\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. And our study showed the same result. Nonetheless, Solomon\u0026rsquo;s population study showed no difference in the embryonic development of epididymal sperm between the OA group and the donor sperm group\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. However, we noticed that the patients\u0026rsquo; obstructive interval, which was reported to be positively correlated with pregnancy rate, was not mentioned in their study \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. And the obstructive interval might be the cause of the discrepancy between our study and Solomon\u0026rsquo;s.\u003c/p\u003e \u003cp\u003eMatura sperm was transcriptionally and translationally silent due to a lack of ribosomes and highly compacted DNA, sperm RNA originated from remnants of spermatogenesis and epididymal transit. During epididymal maturation, the payload of sperm miRNAs, piRNAs, and tsRNAs was in flux \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. miRNAs profile of sperm from proximal and distal epididymal segments displayed loss and acquisition of miRNAs, and the intake of RNA includes novel miRNAs species which is not natively expressed in immature sperm\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. RNA sequence for mouse epididymosomes revealed that epididymosomes contained more than 350 miRNAs, the majority of which were also presented in the sperm miRNAs profiles, with more than 50 miRNAs found to be expressed exclusively in the epididymal sperm and epididymosomes\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Our results showed that obstructive epididymal environment altered sperm miRNAs profiles, including the miR-17-92 cluster and \u003cem\u003eSfmbt2\u003c/em\u003e miRNA cluster, which are reported to be related to development. MiR-17-92 cluster is made up of MiR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a-1 and is involved in the cell cycle, proliferation, apoptosis, and other processes\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. The MiR-17-92 cluster has been linked to development. Humans with heterozygous microdeletions in the MIR17HG locus had severe skeletal abnormalities\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. The deletion of MIR17HG in mice resulted in perinatal lethality, and germline deletion of the miR-17-92 cluster recapitulated the phenotype observed in humans, also growth restriction, cardiac defects, and lung hypoplasia\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. Furthermore, the miR-17-92 cluster was discovered to be required for B cell development and lymphocyte homeostasis\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. The Sfmbt2 gene is paternally expressed in the mouse placenta and is required for placental development; Sfmbt2 knockout mice failed to maintain trophoblasts\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. The specifically deletion of the entire \u003cem\u003eSfmbt2\u003c/em\u003e miRNA cluster in mice resulted in placental malformation, especially in the spongiotrophoblast layer\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e; furthermore, deletion of H3K27me3 imprinting in the Sfmbt2 miRNA cluster enlarged mouse placentas\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. The biogenesis of iRNAs is processed by DROSHA and DICER and the knockout of DROSHA and DICER decreased the biogenesis of a majority of miRNAs\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. Li\u0026rsquo;s study showed the expression of the miR-17-92 cluster was positively correlated with the expression of DICER and DROSHA\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. This is not consistent with our findings which indicated that the downregulation of miR-17-92 miRNA and \u003cem\u003eSfmbt2\u003c/em\u003e miRNA cluster was unrelated to DICER and DROSHA. DNA methylation is an epigenetic mark involved in the repression of transcription. The methylation level of CpG-island at transcriptional start sites is positively correlated with transcription activity\u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. DNMT1, DNMT3a, and DNMT3b are canonical cytosine-5 DNA methyltransferases that catalyze the addition of methylation marks to genomic DNA\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. Duaa\u0026rsquo;s study showed the hypermethylation of the miR-17-92 cluster promoter resulted in a significant downregulation of the miR-17-92 cluster in idiopathic pulmonary fibrosis patients, and the methylation level was upregulated by the DNMT1\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. The upregulation of DNMT3a repressed the expression of miRNAs\u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e. Our study revealed that the expression of the miR-17-92 cluster and Sfmbt2 miRNA cluster were governed by DNA methylation, which DNMT3a and DNMT1 could regulate. Considering the transcriptional inertia of mature sperm, we speculated that epididymis regulated the expression of miR-17-92 and \u003cem\u003eSfmbt2\u003c/em\u003e miRNA cluster and, more importantly, influenced the abundance of sperm miRNAs.\u003c/p\u003e \u003cp\u003eThe miRNAs transmitted by epididymis were found to influence embryonic development. Collins\u0026rsquo;s study suggested caput sperm led to abnormal post-implantation embryonic development due to a lack of some key miRNAs\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e (miR-880 cluster, the paralogous miR-17-92, miR-106b-25 clusters, and the miR-34b/c pair which are scarce in caput epididymis but enriched in cauda epididymis\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e), and suppletion of these miRNAs to the embryo of caput sperm rescued the abnormal post-implantation embryonic development. Our study found that epididymosomes from the CON group can rescue embryonic development caused by the obstructive epididymal environment. However, repression of the expression of developmental miR-17-92 cluster and \u003cem\u003eSfmbt2\u003c/em\u003e miRNA cluster in normal zygotes did not hinder embryonic development. Unlike Collins\u0026rsquo;s study, in which a bunch of different miRNAs were injected into zygotes and influenced embryonic development, we only injected two clusters of miRNAs. And it seems that these miRNA cluster microinjection into zygotes is insufficient to affect embryonic development. In addition, differences in embryos derived from the caput sperm and cauda sperm have been questioned, which might be resulted from the methods used in the preparation of the sperm head rather than the miRNAs\u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e. Besides, we can\u0026rsquo;t rule out whether proteins transmitted from epididymosomes have an impact on embryonic development. The studies of epididymosomes proteins revealed that epididymosomes proteins are related to sperm function, such as motility, capacitation, acrosome reaction, and so on\u003csup\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e. And the sperm cell contains a large number of proteins involved in regulating translation and transcription that might also be critical for early embryos\u003csup\u003e[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e. Above all, the function of the epididymis and sperm RNA in early embryonic development is still not very clear and warrants further exploration.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our findings show that an obstructive epididymal environment influences sperm quality, embryonic development, and the abundance of sperm miRNAs profiles, indicating that the epididymis is important in early embryonic development and may play a potential role in sperm epigenome. Despite the fact that the miR-17-92 and Sfmbt2 miRNA clusters did not hinder embryonic development, our findings provide evidence to support further research into the effects of sperm miRNAs on embryonic development.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003emiRNAs\u003c/strong\u003e: microRNAs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOEE\u003c/strong\u003e: obstructive epididymal environment\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCON\u003c/strong\u003e: control\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHE\u003c/strong\u003e: hematoxylin and eosin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eICSI\u003c/strong\u003e: intracytoplasmic sperm injection\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOA\u003c/strong\u003e: obstructive azoospermia\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003esRNAs\u003c/strong\u003e: small RNA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003epri-miRNAs\u003c/strong\u003e: primary miRNAs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003epre-miRNAs\u003c/strong\u003e: precursor miRNAs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ecKO\u003c/strong\u003e: Conditional knockout\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDFI\u003c/strong\u003e: DNA fragmentation index\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePMSG\u003c/strong\u003e: pregnant mare serum gonadotropin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ehCG\u003c/strong\u003e: human chorionic gonadotrophin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOCs\u003c/strong\u003e: cumulus-oocyte complexes\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-qPCR\u003c/strong\u003e:Real-time quantitative polymerase chain reaction\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe data supporting the results of this study can be obtained from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (No. 82071710).\u003c/p\u003e\n\u003ch2\u003eAuthor information\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInstitute of Reproductive Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People\u0026apos;s Republic of China.\u003c/p\u003e\n\u003cp\u003eXunwei Wu, Xiaomei He, Qian Liu ＆\u0026nbsp;Honggang Li\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXW carried out part of animal experiments, analyzed the data, and wrote this manuscript. HW carried out most of the experiment and analyzed the data, and QL helped a part of the animal\u0026rsquo;s experiment. All authors have read and given their final approval of the submitted manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Honggang Li\u003c/p\u003e\n\u003ch2\u003eEthics declarations\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Animal Care Ethics Committee of Huazhong university of science and technology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003ch2\u003eAdditional information\u003c/h2\u003e\n\u003cp\u003eNone\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCocuzza, M., C. 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Hum Reprod Update, 2018. \u003cb\u003e24\u003c/b\u003e(5): p.\u0026nbsp;535\u0026ndash;555.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"miRNAs, epididymis, embryonic development","lastPublishedDoi":"10.21203/rs.3.rs-1814149/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1814149/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSperms during epididymal transit acquire microRNAs(miRNAs), which are crucial for embryonic development. However, whether miRNAs transmitted from an obstructive epididymal environment affect embryonic development remains unknown. The purpose of the present study was to investigate the effects of an obstructive epididymal environment on embryonic development.\u003c/p\u003e\u003ch2\u003eMethod\u003c/h2\u003e \u003cp\u003eThe control group (CON) and the obstructive epididymal environment group(OEE) were treated with sham operation and vasectomy, respectively. The effects of an obstructive epididymal environment on testis, epididymis, sperm, and embryonic development were analyzed via hematoxylin and eosin staining (HE staining), sperm quality test, epididymosomes fusion, intracytoplasmic sperm injection (ICSI) and embryo transfer. Moreover, sRNA sequence, RT-qPCR, and DNA methylation were conducted to investigate the regulation of sperm miRNAs by an obstructive epididymal environment. miRNAs microinjection and embryo transfer were used to explore the impacts of sperm miRNAs on embryonic development.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe long obstructive interval impaired the morphology of the testis and epididymis. In comparison to the CON group, the OEE group had lower sperm quality and developmental potential. Sperm miRNAs profiles were also altered in the OEE group, particularly the developmental miR-17-92 cluster and Sfmbt2 miRNA clusters; the expression of these two miRNA clusters was regulated via epididymal DNA methylation; however, the disruption of neither the miR-17-92 cluster nor the Sfmbt2 miRNA clusters into normal zygotes did not impair embryonic development.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe obstructive epididymal environment influences embryonic development and the abundance of the sperm miR-17-92 cluster and the sfmbt2 miRNA cluster, but these miRNA clusters are not the cause of abnormal embryo development. It implies that epididymis is important in early embryonic development and may play a potential role in sperm epigenome.\u003c/p\u003e","manuscriptTitle":"Developmental miR-17-92 cluster and sfmbt2 miRNA cluster can’t rescue the abnormal embryonic development caused by the obstructive epididymal environment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-11 17:34:11","doi":"10.21203/rs.3.rs-1814149/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-07-25T04:38:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-07-22T09:05:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"06e88288-ba1a-4f61-beda-42ec4d6feb1e","date":"2022-07-07T08:35:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-07-04T12:40:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8c6483ac-7c9a-447c-a96d-9a57645c4513","date":"2022-07-04T07:48:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-07-02T05:08:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-07-01T10:30:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-07-01T10:30:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Reproductive Biology and Endocrinology","date":"2022-07-01T06:07:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a05eb28d-8d6f-42fe-894d-261d41ff6a8f","owner":[],"postedDate":"July 11th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-10-16T08:14:09+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-11 17:34:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1814149","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1814149","identity":"rs-1814149","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}