Effects Of Low Oxygen Tension On Vitro Maturation And Early Embryo Development In Buffalo

In vitro maturation (IVM) of oocytes and in vitro culture (IVC) of early embryos are the key technologies for producing embryos with high quality and high-efficiency. In recent years, considerable progress has been made in this area. However, the developmental competence of embryos which cultured in vitro was still lower than in vivo. The purpose of this study was to investigate the effects of low oxygen tension on in vitro maturation and the development of early embryo in buffalo. Further, the impact of hypoxia inducible factor in the pathway of glucose metabolism on oocyte maturation and early embryo development were also explored, so as to optimize the gas phase environment for in vitro culture system to improve the efficiency of IVM and IVC.Firstly, the effects of different oxygen concentration on IVM of buffalo oocytes were investigated. Meanwhile, the molecule mechanism of HIF which focused on anaerobic glycolysis were preliminary studied. A total of 1613 cumulus oocyte complexes (COCs) were divided into five groups to culture in the different concentration of oxygen (2%O2,3.5%O2,5%O2,6.5%O2 and 20%O2), respectively. The results showed that there was no significant effect on the mature rate among of buffalo COCs cultured in 5%O2,6.5%O2 and 20%O2 groups (50.73%vs 55.31%,49.58%vs 55.31%, P>0.05). However, the mature rate in the groups of 2%O2 and 3.5%O2 had a significant decrease compared with groups of 20%O2 (21.22%vs 55.31%,29.71%vs 55.31%, P<0.05). Meanwhile, the analytical results of expansion parameters of buffalo COCs showed that comparing with groups of 20%O2, the expansion parameters of buffalo COCs had a significant increase in groups of 2%O2,3.5%O2and 5%O2 (2.84 vs 2.70,2.88 vs 2.70,2.89 vs 2.70, <0O.05), but no significant difference in group of 6.5%O2(2.74 vs 2.70, P>0.05),. Then, the developmental ability of the IVF embryos from the buffalo COCs respectively cultured in 5%O2 and 20%O2 groups was examinated, and found that the developmental rates of 8-cell and the blastocyte were significantly increased in the group of embryos from COCs cultured in 5%O2 compared with the group of 20%O2 (44.88%vs 38.47%,26.81%vs 15.60%, P<0.05). Results of immunofluorescence staining on HIF1? and HIF2? of COCs showed that the expression of HIF1? and HIF2a had been detected in granulosa cells which around oocyted before FVM and after IVM in the 5%O2and 20%O2. However, only HIF1? presented in the oocytes after IVM but did not HIF2?. Furthermore, the result of real-time fluorescent Quantitative PCR analysis showed that the transcript expression level of HIF1?, HIF2?, FSHR, IGF1R, FOXO1, VEGF, VEGFR1, VEGFR2 genes, glucose metabolism-related genes (GLUT1, LDHA, G6DP) and development-related genes (HSP70, HSP90, Ptx3) in granulosa cells from the group of buffalo COCs cultured in 5%O2 was significantly higher than the group of 20%O2 (p<0.05), while the expression of Ptgs2, Bax and Bcl2 in the group of 5% O2 was significantly lower than the group of 20% O2 (p<0.05). TUNEL analysis also found that COCs cultured in 5% O2 could significantly reduce the apoptosis rate of cumulus cells compared with the group of 20% O2 during COCs IVM (21.84% vs 30.87%,p<0.05).Secondly, the effects of low oxygen concentration (5% O2) on the IVM of buffalo oocytes and subsequent embryonic development after IVF were examined, as well as the mechanism of HIF on embryonic development was also preliminary studied. The IVF embryos derived from buffalo oocytes cultured in 5% O2 were respectively cultured at different oxygen partial pressure (5% O2, 20% O2), and found that the 2-cell,4-cell,8-cell developmental rates and blastocyst rates of the IVF embryos cultured in 5% O2 (M5C5) group were significantly higher than the group of 20% O2 (M5C20) (72.38% vs 68.98%, 62.32% vs 57.18%,49.65% vs 45.23%,p<0.05), but there was no significant difference in blastocyst formation rate between the two groups (27.24% vs 26.11%, P>0.05). Results of immunofluorescence staining suggested that HIF1? presented in the blastocysts, but no HIF2?. Q-PCR analysis found that comparing the M5C20 group,the expression levels of HIF1?, IGF1R, FOXO1, GLUT1, LDHA, G6DP, HSP70, HSP90 and Bcl2 genes were up-regulated significantly in the blastocysts from the embryos cultured in M5C5 group (p<0.05), and the expression levels of Bax gene was down-regulated evidently (p<0.05). TUNEL analysis also showed that the apoptosis rate of blastocysts from the M5C5 group was significantly reduced than that of M5C20 group (3.89%vs 6.90%,p<0.05).In conclusion:(1) The HIF1? presents in the granulosa cells, oocytes and the blastocytes, but HIF2a only expresses in the granulosa cells. (2) Low oxygen concentration (5%O2) contributes to improve the oocytes maturation and early embryonic developmental ability. (3) Low oxygen concentration (5%O2) can induce the nuclear transcription factor HIF1? to express stably in granulosa cells of buffalo COCs and subsequent IVF embryos via the pathway of FSH and IGF1 which are regulated by PI3K/AKT. The stable expression of HIF1? maybe facilitates the buffalo COCs and early embryos to adapt the low oxygen environment by means of anaerobic glycolysis, then stimulate the expansion of granulosa cells and the expression of development-related genes in the COCs and embryos, moreover, inhibit the apoptosis of cells, thus improving oocytes maturation and its subsequent developmental competence.