Project description:Purpose: Sperm-borne RNA are particularly sensitive to degradation and methodology-induced bias, thus necessitating the use of a consistent, effective RNA extraction protocol for inter-species comparisons. To this end, we established SpermBase, an RNA expression database consisting of small and large RNA expression data obtained using consistent methodologies. Methods: Total RNA was extracted from total sperm and sperm head samples using an RNA extraction protocol that required only slight, species-specific alterations at the lysis stage. Total RNA was subjected to either RNA-Seq (large RNA) or sncRNA-Seq (small RNA). Results: By using a consistent methodology, we were able to perform a cross-species analysis on the conserved features of large and small sperm-borne RNAs. We identified conserved features in both populations of RNAs in the four mammalian species (i.e., mouse, rabbit, rat, and human) surveyed. Conclusions: Our study demonstrates an effective, near-universal approach to the study of sperm-borne RNAs, and identifies conserved characteristics in the large and small RNA populations of mammalian sperm.
Project description:Since their discovery ~three decades ago, sperm-borne RNAs, both large/small and coding/noncoding, have been reported in multiple organisms, and some have been implicated in spermatogenesis, early development, and epigenetic inheritance. Despite these advances, isolation, quantification and annotation of sperm-borne RNAs remain nontrivial. The yields and subspecies of sperm-borne RNAs isolated from sperm can vary drastically depending on the methods used, and no cross-species analyses of sperm RNA contents have ever been conducted using a standardized sperm RNA isolation protocol. To address these issues, we developed a simple RNA isolation method that is applicable to sperm of various species, thus allowing for reliable interspecies comparisons. Based on RNA-Seq analyses, we established SpermBase (www.spermbase.org), a database dedicated to sperm-borne RNA profiling of multiple species. Currently, SpermBase contains large and small RNA expression data for mouse, rat, rabbit and human total sperm and sperm heads. By analyzing large and small RNAs for conserved features, we found that many sperm-borne RNA species were conserved across all four species analyzed, and among the conserved small RNAs, sperm-borne tsRNAs and miRNAs can target a large number of genes known to be critical for early development.
Project description:Prototypical micro RNAs (miRNAs) are 21~25-base-pair RNAs that regulate differentiation, carcinogenesis and pluripotency by eliminating mRNAs or blocking their translation, processes collectively termed RNA interference (RNAi). RNAi mediated by miRNAs regulates early development in zebrafish, and mouse embryos lacking the miRNA precursor processor, Dicer, are inviable. However, the role of miRNAs during mammalian fertilization is unknown. We here show using microarrays that miRNAs are present in mouse sperm structures that enter the oocyte at fertilization. Sperm contained a broad profile of miRNAs and a subset of potential mRNA targets were expressed in fertilizable, metaphase II (mII) oocytes. Oocytes contained transcripts for the RNAinduced silencing complex (RISC) catalytic subunit, EIF2C3 (formerly AGO3). However, levels of sperm-borne miRNA (measured by quantitative PCR) were apparently low relative to those of unfertilized, mII oocytes, and fertilization did not alter the part of the mII oocyte miRNA landscape that included the most abundant sperm-borne miRNAs. Coinjection of mII oocytes with sperm heads plus anti-miRNAs - to suppress miRNA function - did not perturb pronuclear activation or preimplantation development. Contrastingly, we provide evidence that nuclear transfer by microinjection alters the miRNA profile of enucleated oocytes. These data argue that sperm-borne prototypical miRNAs play a limited role, if any, in mammalian fertilization or early preimplantation development. Keywords: miRNA profiling Seven samples were analyzed for the study.
Project description:Prototypical micro RNAs (miRNAs) are 21~25-base-pair RNAs that regulate differentiation, carcinogenesis and pluripotency by eliminating mRNAs or blocking their translation, processes collectively termed RNA interference (RNAi). RNAi mediated by miRNAs regulates early development in zebrafish, and mouse embryos lacking the miRNA precursor processor, Dicer, are inviable. However, the role of miRNAs during mammalian fertilization is unknown. We here show using microarrays that miRNAs are present in mouse sperm structures that enter the oocyte at fertilization. Sperm contained a broad profile of miRNAs and a subset of potential mRNA targets were expressed in fertilizable, metaphase II (mII) oocytes. Oocytes contained transcripts for the RNAinduced silencing complex (RISC) catalytic subunit, EIF2C3 (formerly AGO3). However, levels of sperm-borne miRNA (measured by quantitative PCR) were apparently low relative to those of unfertilized, mII oocytes, and fertilization did not alter the part of the mII oocyte miRNA landscape that included the most abundant sperm-borne miRNAs. Coinjection of mII oocytes with sperm heads plus anti-miRNAs - to suppress miRNA function - did not perturb pronuclear activation or preimplantation development. Contrastingly, we provide evidence that nuclear transfer by microinjection alters the miRNA profile of enucleated oocytes. These data argue that sperm-borne prototypical miRNAs play a limited role, if any, in mammalian fertilization or early preimplantation development. Keywords: miRNA profiling
Project description:Spermatozoa harbor a complex and environment sensitive pool of small non-coding RNAs (sncRNA)1, which influences offspring development and adult phenotypes1-7. Whether spermatozoa in the epididymis are directly susceptible to environmental cues is not fully understood8. We used two distinct paradigms of preconception acute high fat diet to dissect epididymal vs testicular contributions to the sperm sncRNA pool and offspring health. We show that epididymal spermatozoa, but not developing germ cells, are sensitive to the environment and identify mitochondrial tRNAs and their fragments (mt-tsRNA) as sperm-borne factors. In humans, mt-tsRNAs in spermatozoa correlate with BMI and paternal overweight at conception doubles offspring obesity risk and compromises metabolic health. Sperm sncRNA-seq of mice mutant for genes involved in mitochondrial function, and metabolic phenotyping of their wild-type offspring, suggest that alterations of mt-tsRNAs are downstream of mitochondrial dysfunction. Most importantly, single embryo transcriptomics of genetically hybrid two-cell embryos demonstrated sperm-to-oocyte transfer of mt-tRNAs at fertilisation and implied them in the control of early embryo transcription. Our study supports the importance of paternal health at conception for offspring metabolism, shows that mt-tRNAs are diet-induced and sperm-borne and demonstrates, for the first time in a physiological setting, father-to-offspring transfer of sperm mitochondrial RNAs at fertilization.
Project description:Purpose: to construct a library of miRNAs in bovine sperm was constructed using Illumina high‐throughput sequencing technology. Result: Unique sequences that were 18–26 nucleotides in length were mapped to specific precursors in miRBase 20.0 using BLAST. A total of 951 known miRNAs and 8 novel, highly expressed miRNA candidates were identified.