| The aim of present study was to investigate the effects of follicular granular cells (GCs) mitochondrial transplant (MIT) on the developmental potential of buffalo oocytes, in order to improve the quality of buffalo oocyte and the development potential of buffalo embryo.Firstly, the method of isolation and culture for GCs was explored and the mitochondrial activity of GCs was also detected. The results showed that the viability of GCs obtained from the follicular fluid was about60%. As to the culture medium, the growth rate and growth density of buffalo GCs culturing in DMEM medium were better than those of TCM-199and DMEM/F12culture medium. The GCs began to proliferate after cultured for24h, and reached a proliferation peak during cultured for3～5d. Karyotype analysis displayed that beyond85%of GCs had normal karyotype in the1,3,5,7passage, which did not show significant difference among these passages. The apoptosis ratio of the5(8.26±0.19%) and7passage (9.35±0.47%) of GCs was higher as comparing with the1passage (7.67±0.23%)(P<0.05). Moreover, the activity of the mitochondria extracted from different passage of GCs displayed a decreased trend with the increase of the passages in vitro culture.Secondly, the relationship between the quality, developmental potential of buffalo oocyte and its copy number of mitochondrial DNA (mtDNA) were investigated. The buffalo oocytes were divided into grade1,2and3according their quality. The results of real-time fluorescent quantitative PCR (QRT-PCR) analysis showed that the average mtDNA copy number of buffalo oocytes in the grade1group was evidently higher than that of oocytes in the grade2and grade3groups (202101±34432vs118483±17028,39177±7938, P<0.01), and it was also significantly higher in the grade2group than that of the grade3group (118483±17028vs39177±7938, P<0.01). Furthermore, the cleavage rate and blastocyst development rate of embryos derived from oocytes in the grade1group were significantly higher than those of the grade2and grade3groups after parthenogenetic activation (P<0.01), and the rates of cleavage and blastocyst of embryos from oocytes in the grade2group were remarkably higher than that of the grade3group (P<0.01).Additionally, the effect of MIT on the developmental potential of buffalo oocytes was examined. The results showed that the exogenetic mitochondria labeled with Mito-Tracker fluorescent probe distributed into each of blastomeres after MIT, but the fluorescent intensity gradually became to weak with the embryonic development, and there were still some faint fluorescent signal could be observed in the morula embryos. Furthermore, more parthenogenetic embryos could development to blastocyst stage in the group of grade2oocytes after MIT (27.3%vs17.4%,7.84%; P<0.05), however, there was no significant difference in the blastocyst rate of the group of grade3oocytes after MIT. The A Ψm of buffalo embryo presented an increased trend during development of preimplantation embryo, and the ΔΨm of embryo derived from MIT group in the2-cell,4-cell,8-cell, morula, and blastocyst stage was significantly higher than that of the control group (P<0.05).These results indicated that (1) the model of buffalo GCs in vitro culture was preliminarily established, and the1-3generations of buffalo GCs were more suitable cells for extracting mitochondria.(2) The mtDNA copy number of buffalo oocyte is positively related with the quality of oocyte and its subsequent developmental ability.(3) The exogenous mitochondria could survive and participate in the process of early embryonic development.(4) MIT is propitious to improve the developmental ability of the grade2buffalo oocytes. |
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