The Mechanism Of Vascular Endothelial Growth Factor On Promoting Ovine Oocyte Maturation In Vitro

To investigate the effect of vascular endothelial growth factor (VEGF) on the ovine oocytes maturation and the early development of embryo in vitro, a series of ten experiments were conducted with VEGF supplemented to the maturation medium,the fertilization medium and the culture medium respectively. To show the mechanism of VEGF on promoting ovine oocyte maturation in virto by evaluate cell cycle-dependent modifications in cell configuration, cell framework, the change of active MAPK/PKC/PTK in oocyte, the expression of VEGF and its receptors KDR/Flk-1 and Flt-1 in ovine oocytes undergoing in vitro maturation.1,The effect of VEGF on the ovine oocytes maturation and the early development of embryo in vitroExperimentation 1: Human recombinant VEGF165 was employed at 5 ng/ml and 10 ng/ml in maturation media(TCM199+fetal bovine serum, FBS), HSOF fertilization media and SOF culture media in this study. The results showed that the maturation rate was increased significantly (p <0.01) from 80.68% in the control(0 ng/ml VEGF) to 86.09% and 85.22% in the treatment groupⅠ(5 ng/ml VEGF)and the treatment groupⅡ(10 ng/ml VEGF) respectively. The cleavage rate was increased from 69.38% in the control group to 75.18% and 73.48% (p>0.05) in the treatment groupⅠand the treatment groupⅡrespectively. The development rates of morulae and blastocyst in the treatment groupⅠ(45.45% and 30.06%) were higher significantly (p <0.01)than that of the control group (37.61% and 23.65%). And the development rates of morulae and blastocyst in the treatment groupⅡ(40.25% and 27.80%)were lower than that of the treatment groupⅠ(P>0.05) and higher than that of the control group(P>0.05). These results indicated that maturation medium, fertilization medium and the culture medium with 5 ng/ml of recombinant human VEGF165 significantly improved the maturation and fertilization of ovine oocytes and consequently the rate of embryos development were markedly enhanced. VEGF may be one of the important regular factors in system of maturation and fertilization of ovine oocyte and the early development of ovine embryo in vitro.Experimentation 2: Human recombinant VEGF165 was employed at 5 ng/ml and 10 ng/ml in maturation media(TCM199+ bovine serum albumin, BSA), HSOF fertilization media and SOF culture media in this study. The results showed that the maturation rate was increased significantly (p <0.01) from 75.76% in the control (0 ng/ml VEGF) to 83.98% and 80.23% in the treatment groupⅠ(5 ng/ml VEGF)and the treatment groupⅡ(10 ng/ml VEGF) respectively. The cleavage rate was increased from 75.85% in the control group to 79.39%(p>0.05) in the treatment groupⅠ.The development rates of morulae and blastocyst in the treatment groupⅠ(45.03% and 23.54%) were higher significantly (p <0.01)than that of the control group (38.94% and 18.09%). And the development rates of morulae and blastocyst in the treatment groupⅡ(38.85% and 21.05%)were lower than that of the treatment groupⅠ(P>0.05) and higher than that of the control group(P>0.05). These results indicated that BSA maturation medium with 5 ng/ml of recombinant human VEGF165 significantly improved the maturation and fertilization of ovine oocytes and consequently the rate of embryos development were markedly enhanced also. VEGF may be one of the important regular factors in system of maturation and fertilization of ovine oocyte and the early development of ovine embryo in vitro.Experimentation 3: Human recombinant VEGF165 was employed at 5 ng/ml in maturation media(TCM199+ bovine serum albumin, BSA) in this study. The results showed that the fertilization rate was increased significantly (p <0.01) from 75.75% in the control (0 ng/ml VEGF) to 83.86% in the treatment group (5 ng/ml VEGF) and the polyspermy rate was decreased significantly (p <0.01)from 12.64% in the control to 7.68% in the treatment group. The no fertilization rate was decreased (p <0.05) from 11.60% in the control to 8.47% in the treatment group. These results indicated that VEGF obviously decreased the polyspermy rate and bated the phenomenon of polyspermy in the process of ovine oocytes IVF.1,The mechanism of VEGF on promoting ovine oocyte maturation in vitro1.1 The effect of VEGF on the nuclear and cytoplasmic maturation of ovine oocytes matured in vitroExperimentation 4: Human recombinant VEGF165 was employed at 5 ng/ml in maturation media (TCM199+ bovine serum albumin, BSA) in this study. Orcein staining was used to evaluate cell cycle-dependent modifications in nuclear configuration. The results showed that the percentages of oocytes displaying a normal configuration of chromosomes in metaphase II (MII) increased in the VEGF group (87.42%) compared to the control (83.89%). This result indicated that VEGF effectively improved the normal configuration of chromosomes.Experimentation 5: Human recombinant VEGF165 was employed at 5 ng/ml in maturation media (TCM199+ bovine serum albumin, BSA) in this study. Immunofluorescence and confocal microscopy were used to evaluate microtubule organizing centers (MTOCs) translocation and the temporal and spatially redistribution ofα-tubulin. The results showed that the percentages of oocytes displaying a normal distribution ofα-tubulin and chromosomes in metaphase II (MII) significantly increased in the VEGF group (77.50%) compared to the control (62.60%)(P<0.01). VEGF promoted the MTOCs domains to disappear from the cortex and stimulated assembly ofα-tubulin around chromosome domains when germinal vesicle breakdown (GVBD) was commencing. These results showed that VEGF may improve the quality of nuclear maturation of ovine oocytes in vitro by the effects on the temporal and spatial translocation or redistribution of MTOC andα-tubulin.Experimentation 6: Human recombinant VEGF165 was employed at 5 ng/ml in maturation media (TCM199+ bovine serum albumin, BSA) in this study. Immunofluorescence and confocal microscopy were used to evaluate the temporal and spatially redistribution of cortical granules (CGs). The results showed that the rate of oocytes with CGs transfering completely in cortex was significantly higher (79.24%) in the VEGF group than in the control group (60.97%)(P<0.01). VEGF may improve the quality of cytoplasmic maturation of ovine oocytes in vitro by the effects on the temporal and spatial translocation or redistribution of CGs.1.2 The effect of VEGF on the ultrastructure change of ovine oocytes matured in vitro Experimentation 7: Human recombinant VEGF165 was employed at 5 ng/ml in maturation media (TCM199+ bovine serum albumin, BSA) in this study. The results showed that the development and change of microvilli on the surface of oocytes could be affected with VEGF in the maturation media. VEGF could promote the oocytes absorbing nutrientsubstance and the communication. VEGF may effects the temporal and spatial translocation or redistribution of CGs. There were so many mitochondria with"cap"in the oocytes cytoplasm. The development and transfer of mitochondria was effected by VEGF. There were no remarkable effects of VEGF on the accumulation of cytoplasmic lipid droplet, but VEGF could avoide the integration of many droplets to bigger ones.1.3 The effect of VEGF on the change of P44/p42 MAP kinases, Protein Kinase C and Tyrosine kinases of ovine oocytes matured in vitroExperimentation 8: In this study, VEGF was supplied at 5 ng/ml in maturation media for determine the effect of it on the change of phosphorylation P44/p42 MAP kinases (ERKs), Protein Kinase C (PKC) and Tyrosine Kinases (PTKs) activity in ovine oocyte during maturation by ELISA in vitro. The results showed that: these three protein kinase took on fluctuant changes. The levels of phosphorylation ERKs in the VEGF treatment group were higher than that of the control group, especially at 16 hours and 20 hours (P<0.05). The levels of phosphorylation PTK in the VEGF treatment group were higher than that of the control group, especially at 16 hours 21 hours and 24 hours (P<0.05). The levels of phosphorylation PKC in the VEGF treatment group were higher than that of the control group too. VEGF was strongly enhanced the level of phosphorylation ERKs and PKC activity but reduced PTKs activity during the period of ovine oocyte maturation in vitro for improving the quality of oocytes.1.4 Expression of VEGF and its receptors (Flt-1 and KDR/ Flk-1) in ovine oocytes matured in vitroExperimentation 9: Human recombinant VEGF165 was employed at 5 ng/ml in maturation media (TCM199+ bovine serum albumin, BSA) in this study. Expression of VEGF and its receptors (Flt-1 and KDR/ Flk-1) mRNA in ovine oocytes matured in vitro by RT-PCR. The results showed that VEGF mRNA was expressed in cumulus cells and oocytes without cumulus cells at 0 hour,8hours and 24 hours, but it was highest in cumulus cells of the control group and in oocytes without cumulus cells of the treatment group at the 8 hours matured in vitro. Flt-1 mRNA was expressed in cumulus cells of the control group at 0 hour and 8 hours, and was not detect in the treatment group. Relative amount of Flt-1 mRNA for cumulus cells at 8 hours of control group was higher than that of 0 hour. Meanwhile, Flt-1 mRNA was expressed in oocytes without cumulus cells at 0 hour and 8 hours both in the control group and the treatment group. The Flt-1 mRNA expression at 0 hour was highest and took second place at 8 hours of the control group, the Flt-1 mRNA expression of the treatment at 8 hours was lowest. It was not detect Flt-1 mRNA expression in cumulus cells and oocytes without cumulus cells at 24 hours maturation. KDR/ Flk-1 mRNA was expressed in cumulus cells and oocytes without cumulus cells at 0 hour,8hours and 24 hours both in the control group and the treatment group. KDR/ Flk-1 mRNA expression was highest at both 8 hours in cumulus cells and 24 hours in oocytes without cumulus cells of the control group. There were weak KDR/ Flk-1 mRNA expression in cumulus cells of the treatment group at 8 hours and 24 hours.Experimentation 10: Human recombinant VEGF165 was employed at 5 ng/ml in maturation media (TCM199+ bovine serum albumin, BSA) in this study. In the present study, ovine cumulus-oocyte complexes (COC) were matured in vitro in the treatment group with 5 ng/ml human recombinant VEGF165 in maturation media and the control group without VEGF, confocal microscopy and immunohistochemical staining methods were used to assess the location and expression of Flt-1 and KDR/ Flk-1 in the plasma membranes of cumulus cells and oocytes without cumulus cells at different maturation time (0hour,8hours and 24hours). The results showed that Flt-1 and KDR/ Flk-1 were expressed in cell plasma membranes of maturation oocytes without cumulus cells and cumulus cells at 0 hour,8hours and 24hours. Flt-1 and KDR/ Flk-1 were mainly detected in the cytoplasm close to the membrane both in immature and mature oocytes without cumulus cells. The staining for Flt-1 slightly increased in the control group compared to the treatment group in maturation oocytes without cumulus cells and cumulus cells. However, the staining for Flt-1 and KDR/ Flk-1 was slightly weaker in cell plasma membranes of oocytes without cumulus cells than in that of cumulus cells.