Effect Of Sperm-borne RNAs On The Early Development Of Bovine Embryos

Recent studies have shown that some sperm RNAs are brought into oocytes in the process of fertilization and they can remain stable until the period of embryonic genome activation, which suggest that the RNAs brought in process of fertilization get involved in the development of early embryos, playing an important role in this stage. Due to the special outer membrane structure, low RNA content and other characteristics, being not able to extract high-quality sperm RNAs has been one of the obstacles in further study of sperm RNAs. The purpose of this experiment was to optimize the method of extracting bovine sperm RNAs and do a preliminary study of the effect of sperm-bovine RNAs on the early development of somatic cloned embryos.This experiment optimized the traditional method using TRIzol to extract RNAs, and the main improvements are: adding an appropriate amount of lysis buffer、increasing cracking temperature(65℃) and extending the time of isopropanol processed(2-3 hours or even overnight). Compared with the traditional method, this method significantly improved the total RNA yield(2,800 ng VS 29,000ng) and purity(OD260 / 280 1.60 VS 1.88).In order to explore the influence of sperm RNA on early embryonic development, the isolation RNAs of high purity and a high concentration extracted by electric fusion method were transferred into the nuclear complex, analyzing the effect of sperm RNAs on development of transgenic cloned bovine embryos. The results show that it had not significant difference of total cell number in blastocyst between treated group and control group(104.45±5.24 VS 102.36±3.21,P>0.05) after injecting sperm RNAs, but the cleavage rate(86.3%±1.23% VS 75.2%±1.57%,P<0.05) and the blastocyst rate(35.8%±0.87% VS 28.6%±1.10%,P<0.05)of cattle cloned embryos increased significantly, while the apoptosis rate(2.8%±0.23% VS 5.8%±0.11%,P<0.05) decreased significantly. The expression of development-related pluripotent genes OCT4, SOX2 in blastocysts was significantly higher(P <0.05).This experiment optimized the method of bovine sperm RNA isolaton,the result of theelectrofusion experiment showed sperm RNAs has an important influence on somatic cell nuclear transfer,which laid groundwork for further exploring regulation of early embryo development by sperm RNAs and improving the efficiency of somatic cell nuclear transfer.