Background: Spermatogenesis is regulated by a complex network of intercellular communication processes. Extracellular vesicles (EVs) are one of the important mediators in intercellular communication. Previous reports have demonstrated the involvement of EVs from the epididymis and prostate in sperm maturation and function. However, the presence of EVs in the testis and their potential involvement in spermatogenesis has not been explored. Here, we have established a testis dissociation protocol that allows the isolation and characterization of testicular EVs. Results: We show that testicular EVs are specifically and efficiently taken up by somatic cells and germ cells, including the spermatozoa in the interstitial space and the seminiferous tubule compartments. We profiled the proteome of testicular EVs and probed the cell types that release them, revealing the potential contributions from the Leydig cells and testicular macrophages. Moreover, we sequenced the small RNA cargoes of testicular EVs and identified sets of small non-coding RNAs that were overlooked in the testis transcriptome. Selected miRNA candidates in testicular EVs were found in sperm RNA payload and demonstrated specific resistance towards ribonuclease A independent of the vesicle membrane. Small molecule inhibition of EV secretion perturbed spermatogenesis via inter-compartmental communication. Conclusions: Together, our study provides a valuable resource on the repertoire of cargoes carried by testicular EVs and uncovers a physiological function of testicular EVs in inter-compartmental communication associated to spermatogenesis.

Apart from the paternal half of the genetic material, the male gamete carries assorted epigenetic marks for optimal fertilization and the developmental trajectory for the early embryo. Recent works showed dynamic changes in small noncoding RNA (sncRNA) in spermatozoa as they transit through the testicular environment to the epididymal segments. Studies demonstrated the changes to be mediated by epididymosomes during the transit through the adluminal duct in the epididymis, and the changes in sperm sncRNA content stemmed from environmental insults significantly altering the early embryo development and predisposing the offspring to metabolic disorders. Here, we review the current knowledge on the establishment of the sperm sncRNA transcriptome and their role in male-factor infertility, evidence of altered offspring health in response to the paternal life experiences through sperm sncRNA species and, finally, their implications in assisted reproductive technology in terms of epigenetic inheritance.

• CTR1-overexpressing HepG2 and ZFL cell lines were created. • CTR1 overexpression in both HepG2 and ZFL cells increased Cd2+ uptake and toxicity. • CTR1 knockdown in HepG2 cells decreased Cd2+ uptake and toxicity. • CTR1 plays a significant role in Cd2+ uptake and toxicity.

Study question: Does high-fat diet alter the cargoes of testicular extracellular vesicles (tEVs) and thus modulate the sperm epigenome? Summary answer: The properties and small RNA cargoes of tEVs and the sperm epigenome were significantly altered in mice fed with a high-fat diet. What is known already: High-fat diet is known to alter spermatogenesis and sperm quality. Recent studies showed that the undesirable metabolic traits can be inherited to the next generation via paternal epigenetic inheritance. Hitherto, it has been shown that the extracellular vesicle, an important intercellular communication pathway, secreted by the epididymis conveys small RNA cargoes to sperms and mediate paternal epigenetic inheritance of metabolic traits. Surprisingly, although the sperm are first being produced in the testes, the potential contributions of testicular EVs (tEVs) in the sperm epigenome remain unexplored. Study design, size, duration: It is a proof-of-concept study using mice as an experimental model. Thirty mice were raised for nine months, high-fat diet (HFD) and chow diet (SD) were treated on each half of the subject group starting from the sixth week until they were euthanized. Participants/materials, setting, methods: The study is conducted under laboratory settings. Sperm and tEVs were obtained from mice fed with HFD or SD. The uptake of tEVs by sperm was monitored by flow cytometry analysis using fluorescence-labelled tEVs. Physical properties of testicular EVs were examined by the transmission electron microscope. The small RNA cargoes were investigated by small RNA sequencing. The sperm epigenome was examined by real-time-qPCR. Main results and the role of chance: Our results showed that sperm efficiently took up the tEVs in a dose-dependent manner, without compromising the sperm motility. Size of tEVs in HFD-fed mice (320.5 ± 99.83 nm) was significantly greater than that of SD-fed mice(251.9 ± 81.01 nm). RNA sequencing revealed a decrease in the percentage of miRNA in HFD tEVs. Eight miRNAs were differentially expressed in HFD tEVs. Among them, real-time PCR results confirmed that miR–34b and c levels were significantly up-regulated in HFD tEVs, with a log2[Fold-change] of 0.46613 and 0.42935 respectively. Unexpectedly, the levels of both miR–34b and c were similar in HFD and SD epididymis, and were both down-regulated by about 2-fold in matured sperm of HFD-fed mice. To investigate the cause of discrepancy, we carried out flow cytometry analysis to measure the absorption efficiency of tEVs, which revealed a notable decrease in absorption efficiency of HFD tEVs (70.235 ± 4.864%) by sperms compared to that of SD tEVs (79.350 ± 4.012%). Limitations, reasons for caution: Cauda sperm was used in the profiling of sperm epigenome where the contributions from the epididymosomes have not been compared. The study was conducted using mice models such that discrepancy may occur when applying to humans. Wider implications of the findings: We revealed the alteration of tEVs in HFD-fed mice which may underlie the perturbation of spermatogenesis in HFD condition. We demonstrated the efficient uptake of tEVs by sperm which may be developed as a tool for the engineering of the sperm epigenome. Trial registration number: Not applicable