Abstract: 【Objective】The aim of present study was to identify of paraspeckle formation stages during early embryonic development in yak (Bos grunniens). Furthermore, we also determined long non-coding RNAs (LncRNAs) involved in paraspeckle formation and study effects and regulatory mechanism of their formation on the subsequent developmental ability of yak embryos.【Method】The yak embryos were produced by in vitro fertilization (IVF), DAPI staining of embryonic nuclei combined with paraspeckle protein 1 (PSPC1) mRNA detection were done by quantitative real-time fluorescence PCR (qRT-PCR) at different stages in order to confirm paraspeckle formation. PSPC1 protein in embryos was verified by immunofluorescence technique. The levels of encoding nuclear paraspeckle assembly transcript 1(NEAT1), coactivator associated arginine methyl transferase 1(CARM1) and non-POU domain containing octamer-binding protein(p54nrb) mRNAs were also detected by qRT-PCR at different stages. The mRNA level of PSPC1 in zygote was inhibited by RNA interference technology, and the developmental rate of embryos in subsequent stages was compared. The blastocyst quality was evaluated by analyzing the number of total cells, trophoblast cells(TE) and inner cell mass (ICM). B-cell lymphoma/leukemia-2(Bcl-2) and b-cell lymphoma/leukemia associated X protein (Bax) in blastocysts form in the control and PSPC1 mRNA interference groups was detected.【Result】(1) Paraspeckle could be observed in the nuclei of embryos at all different stages; however, nuclei can be more clearly seen at 2-cell stage and 4-cell stage. The PSPC1 mRNA was higher in yak embryos from 2-cell to morula stage, which were highest in embryos at 4-cell embryos and morula. The fluorescence intensity of PSPC1 protein was strongest in embryos from those stages. (2) Levels of NEAT1, CARM1 and p54nrb mRNA were higher from 2-cell to morula stage than in other stages. NEAT1 and p54nrb were found to be highest in embryos at 4-cell stage, while CARM1 was not significantly different from 2-cell to morula stage (p > 0.05). (3) The developmental rates of morula and blastocyst in PSPC1 mRNA interference group were reduced, which was more significantly reduced in morula rate. Total number of blastocyst cells in PSPC1 mRNA interference group was significantly lower than that in the control group, which was mainly caused by ICM reduction. There was no significant difference in number of TE between the two groups. (4) The levels of Bax mRNA and protein were enhanced in blastocyst form PSPC1 mRNA interference group, while the levels of Bcl-2 mRNA and protein were reduced in blastocyst; and the cell lysis was observed in ICM.【Conclusion】Results in our present study show that paraspeckle formed at 2-cell to morula stage transition in the yak embryo, and was more prominent in 4-cell stage. The expression of PSPC1, NEAT1, CRAM1 and p54nrb in the stages of paraspeckle formation were on high levels. Interference with PSPC1 mRNA in yak zygotes resulted in decreased developmental ability of subsequent embryo. The blastocyst quality was also reduced by inducing apoptosis of inner cell mass, which was also involved in the regulation of cell fate determination in early embryo development.

Key words: yak, paraspeckles, cell fate, apoptosis, LncRNAs