The Research On Ts Rna Dynamics During Porcine Spermatogenesis,Maturation And TsRNA Functions In Embryonic Development

Transfer RNAs?t RNAs?not only participate in the process of translation,but also can be processed to form t RNA derived small RNAs?tsRNAs?.tsRNAs present in different cell types executing various functions including regulation of RNA stability,translation,stress responses and cell proliferation.tsRNAs are highly present in sperm,and act as epigenetic factors to mediate offspring phenotypes.Although tsRNAs are scarce in testicular germ cells,sperm can acquire tsRNAs from epididymosomal exosome during sperm maturation in epididymis.Pigs are not only as the important meat animals but also as the animal model.The research findings in pigs possesses the model significance in animal production and medical research.At present,the dynamics of tsRNAs in porcine spermatogenesis and sperm maturation have not been reported,and the roles of sperm derived tsRNAs in embryo development remain unclear.Hence,the research focus on the two questions proposed above.STA-PUT,small RNA sequencing,embryo manipulation and single cell RNA sequencing were used to determine the tsRNA dynamics during porcine spermatogenesis,sperm maturation,and the biological functions of sperm derived tsRNAs in early embryo development.The main results are showed as follow:?1?Spermatogonia?SG?,pachytene spermatocytes?PS?and round spermatids?RS?were successfully isolated by using STA-PUT.The characteristics of morphology and marker expression were consistent with SG,PS and RS,respectively.Small RNA sequencing and analysis of spermatogonia,pachytene spermatocytes,round spermatids and ejaculating sperm showed that over 85%tsRNAs were derived from t RNA 5'end.Comparing to testicular germ cells,the percentage of tsRNA in total small RNA was upregulated.Among these tsRNA families,the relative abundance of t RNA^Gly-GCC derived small RNA?Gly-GCC?and t RNA^Glu-CTC derived small RNA?Glu-CTC?were constant in different porcine germ cells,and the abundance of t RNA^Gln-TTG derived small RNA?Gln-TTG?was significantly upregulated in sperm.?2?The caput sperm,cauda sperm and ejaculated sperm were isolated for small RNA sequencing.Analysis showed that the percentage of tsRNA in small RNAs were gradually downregulated.But the 5'end derived tsRNAs were decreased during maturation of sperm transition from caput to cauda,and were enriched in ejaculated spermatozoa.Among these tsRNA families,Gly-GCC and Glu-CTC abundance were constant during sperm maturation,but Gln-TTG firstly upregulated in epididymis and decreased after ejaculation.?3?The caput,corpus,cauda and semen derived exosomes were isolated.Analysis of transmission electron microscope,western blot and Nanosight revealed that the isolated exosomes exhibited the characteristics of exosomes.Then the exosomes were used for small RNA sequencing.Analysis revealed that the percentage of tsRNA in small RNAs gradually increased from caput to cauda but decreased in semen derived exosomes.In addition,5'end derived tsRNAs exhibited the similar pattern to the total tsRNAs.Correlation analysis revealed that tsRNAs abundance in caput,cauda and semen derived exosomes were highly correlated with that in sperm of caput,cauda and semen,separately.The motor pattern of cauda sperm could be modulated by the semen derived exosome incubation.Furthermore,semen exosome derived exosomes could also deliver tsRNAs to cauda sperm.?4?The functions of sperm derived tsRNAs were determined by microinjecting sense or antisense of tsRNAs.Results showed that overexpression or interfering of Gly-GCC and Glu-CTC had no effect on in vitro fertilized?IVF?embryo.Overexpression of Gln-TTG also had no effect on IVF embryo development,but interfering with Gln-TTG significantly decreased the first cleavage rate.different RNA concentration and RNA rescue assay were used to further demonstrate the roles of Gln-TTG in the first cleavage of IVF embryo.In addition,interfering with Gln-TTG function had no effect on parthenogenetically activated embryo,whihc further demonstrated the important roles of sperm derived Gln-TTG in first cleavage of porcine embryo.?5?IVF embryos and PA embryos of overexpression and interfering group were collected for single cell RNA sequencing.Analysis revealed that interfering with Gln-TTG functions affected the cell cycle associated genes in IVF embryo but not PA embryos.Furthermore,retrotransposons element?LTR4D?SS,LTR6?SS,L1M3b,MLT1l-int?were affected in IVF embryos but not PA embryos.Therefore,Gln-TTG could mediate cell cycle associated genes and retrotransposons element expression to regulate the first cleavage of porcine embryo.?6?Previous published small RNA sequencing data of human sperm were used for analysis.Results revealed that the expression of Gln-TTG was significantly higher in high quality human sperm comparing with low quality human sperm.The functions of Gln-TTG in human 3PN embryos were determined by microinjecting sense or antisense of tsRNAs.Results showed that human sperm derived Gln-TTGs play roles in regulation of human early embryo development.Human 4cell,8cell and blastocyst embryos were collected for single cell RNA sequencing.Analysis showed that Gln-TTGs participate in regulation of developmental associated genes,and human embryo genomic activation during the development of 4 to 8 cell.RNA-pull down and LC-MS/MS analysis revealed that Gln-TTG could interact with RNA binding protein and DNA binding to execute their functions.Together,by using small RNA sequencing,the characteristics of tsRNA dynamics in porcine spermatogenesis and sperm maturation were systematically analyzed,the correlation between cauda,semen derived exosomes and sperm tsRNAs were uncovered,and the semen derived exosomes deliver tsRNAs to cauda sperm was also demonstrated.In addition,sperm derived important roles of tsRNAs in regulation of porcine embryo development were uncovered by embryo manipulation and single cell RNA sequencing.The findings provide basis for research of tsRNAs in male fertility and early embryo development,and provide theoretical references for application of tsRNAs in porcine production.