ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2016, Vol. 47 ›› Issue (3): 484-492.doi: 10.11843/j.issn.0366-6964.2016.03.009

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The Effects of VEGF on DNMT3a Expression in Oocytes Maturation of Ovine in vitro

CAO Xin1，2，ZOU Yun-long2，CAI Yong2，ZHOU Ping3，LI Ming1，SHI Guo-qing3，KANG Xiang-tao1*   

  1. (1.College of Veterinary Medicine，Henan Agricultural University，Zhengzhou 450002，China；2.Scientific Experimental Center，Northwest University for Nationalities，Lanzhou 730070，China；3.Xinjiang Academy of Agriculture and Reclamation Science，Shihezi 832000，China)
  • Received:2015-07-15 Online:2016-03-23 Published:2016-03-23

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Abstract:

To study the effect of vascular endothelial growth factor (VEGF) on DNMT3a expression during ovine oocytes maturation in vitro. In this research, in order to test DNMT3a mRNA and protein expression of oocytes and granule cells (GCs) at different time points during cumulus-oocytes complexes (COCs) cultured in vitro, RNAi and microinjection were used to import 2 pairs of effective double chain small interfering RNA (dsRNA) fragments which were designed for VEGF receptors FLT1 and KDR/Flk-1 into COCs. The results indicated that, during the culturing process of COCs maturation in vitro, the expression level of DNMT3a mRNA of oocytes would be decreased regardless of the presence of VEGF. DNMT3a mRNA expression quantity fell sharply at the 8 h, then, it decreased slowly and tended to be similar to each other groups at the 24 h. DNMT3a mRNA without dsRNA of control group significantly declined faster than that of the other treatment groups (P＜0.01). The expression level of DNMT3amRNA of VEGF groups in oocytes showed a faster decrease than the groups without VEGF. After dsRNA fragments microinjection, interfering effect of FLT1-siRNA & KDR-siRNA group was better than that of the KDR-siRNA group and that of FLT1-siRNA group was the lowest (P＜0.01). During the maturation process, DNMT3a mRNA of GCs would be decreased regardless of the presence of VEGF, and the difference between each group were tiny. At the mid maturity of COCs, DNMT3a mRNA without dsRNA of control group decreased faster than that of the other treatment groups (P＜0.05 or P＜0.01), and the effect of RNAi on FLT1-siRNA group was the best. Whether adding VEGF or not, the protein expression level of DNMT3a in oocytes had been increasing from initial time until the 4 h, then it started to decrease at a constant speed. A noteworthy decrease of DNMT3a protein expression was then observed at the 16 h and was completely unexpressed at the 24 h. The protein expression level of DNMT3a in control group without dsRNA had the fastest decreasing speed. At the 16 h, DNMT3a protein levels of the control group, FLT1-siRNA group, KDR-siRNA group and FLT1-siRNA & KDR-siRNA group were decreased to 23.12%, 31.07%, 41.47% and 37.90% respectively with VEGF comparing to that of the 0 h. At the same time, that of the non-VEGF groups had decreased to 37.38%, 54.60%, 57.58% and 82.75%. In GCs, DNMT3a protein expression levels of groups with VEGF decreased gradually since 0 h and could not be tested at the 20 h, and that of groups without VEGF increased slightly from 0 to 4 h, then presented the average decline gradually until the 20 h where a rapid drop appeared and went completely undetected at the 24 h. In addition, dsRNA had negligible effect on DNMT3a protein expression levels, while the original DNMT3a protein expression level in the GCs were low. In conclusion, during the process of COCs maturation in vitro, VEGF could accelerate the decreasing speed of both mRNA and protein expression levels of DNMT3a. dsRNA fragments microinjection could slow down the decreasing speed of both mRNA and protein levels of DNMT3a. VEGF and dsRNA had no obvious influences on both DNMT3a mRNA and protein expression of GCs.

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