Supplementation of Vascular Endothelial Growth Factor during in vitro Maturation of Porcine Cumulus Oocyte Complexes and Subsequent Developmental Competence after Parthenogenesis and in vitro Fertilization

In mammalian reproductive tract, the oviduct secretes huge number of growth factors and cytokines that create an optimal micro-environment for the initial stages of preimplantation embryos. Secretion of these growth factors is stage-specific. Among them, VEGF is a potent mitogen for vascular endothelium and stimulates vascular permeability. Apart from angiogenesis, VEGF in the oviduct may be involved in regulating the oocyte maturation and subsequent developmental process during embryo production in vitro. In experiment 1, to evaluate the effect of VEGF during IVM of porcine COC and subsequent developmental ability after PA and SCNT. The results from these experiments indicated that maturation rates among the different VEGF concentrations were not significant different. In experiment 2, total intracellular GSH concentrations of oocytes matured with VEGF (5-50 ng/ml) were increased significantly compared to a control and VEGF group (500 ng/ml). In experiment 3, the blastocyst formation rates and total cell number per blastocyst after parthenogenesis of oocytes matured with VEGF (5-50 ng/ml) were increased significantly compared to a control and VEGF group (500 ng/ml). Similarly, in experiment 4, the blastocyst formation rate and total cell number per blastocyst after SCNT and IVF of oocytes matured with VEGF (5 ng/ml) were significantly higher than that of oocytes matured without VEGF group. In experiment 5, at 10 hour after the onset of IVF, pronuclear formation rate was evaluated. Monospermy was significantly higher in VEGF-matured oocytes than in the control, and polyspermy and sperm penetration per oocyte were significantly higher in the control group than in the VEGFmatured oocytes. Supplementation with VEGF during IVM significantly improved male pronuclear formation as compared with the control. In experiment 6, type III cortical granule distribution in oocytes was more common in VEGF-matured oocytes than in the control. In conclusion, the present study suggested that supplementation of VEGF during IVM may enhance the developmental potential of porcine in vitro embryos through increase of the intracellular GSH level, higher MPN formation and increased fertilization rate as a consequence of an improved cytoplasmic maturation.

Authors:

• D. Biswas
• Sang H. Hyun

Keywords:

• angiogenesis
• GSH
• monospermy
• VEGF

References:

[1] K.S. Richter, The importance of growth factors for preimplantation
embryo development and in vitro culture, Curr. Opin. Obstet. Gynecol.,
vol. 20, pp. 292-304, 2008.
[2] A.J. Watson, Oocyte cytoplasmic maturation: A key mediator of oocyte
and embryo developmental competence, J. Anim. Sci.,vol. 85, pp. E1-
E3, 2007.
[3] H. Luo, K. Kimura, M. Aoki and M. Hirako, Effect of Vascular
Endothelial Growth Factor on Maturation, Fertilization and
Developmental Competence of Bovine Oocytes, J. Vet. Med. Sci., vol,
64, pp. 803-806, 2002.
[4] J. Tran, Z. Master, J.L. Yu, J. Rak, D.J. Dumont and R.S. Kerbel, A role
for surviving in chemoresistance of endothelial cells mediated by VEGF,
PNAS, USA, vol, 99, pp. 4349-4354, 2002.
[5] H. Funahashi, T.C. Cantley, T.T. Stumpf, S.L. Terlouw and B.N. Day,
Use of low-salt culture medium for in vitro maturation of porcine
oocytes is associated with elevated oocyte glutathione levels and
enhanced male pronuclear formation after in vitro fertilization, Biol.
Reprod., vol. 51, pp. 633-639, 1994.
[6] L. Zofia, The role of glutathione in mammalian gametes, Reproductive
Biol., vol. 5, no. 1, pp. 5-17, 2005.
[7] O.J. Koo, G. Jang, D.K. Kwon, J.T. Kang, O.S. Kwon, H.J. Park, S.K.
Kang and B.C. Lee, Electrical activation induces reactive oxygen species
in porcine embryos, Theriogenology, vol. 70, no. 7, pp. 1111-1118,
2008.
[8] P.J.Booth, S.J. Tan, R. Reipurth, P. Holm and H. Callesen,
Simplification of bovine somatic cell nuclear transfer by application of a
zona-free manipulation technique, Clon. Stem Cells, vol. 3, pp. 139-150,
2001.
[9] W.J. Jolliff and R.S. Prather, Parthenogenic development of in vitro
matured, in vivo-cultured porcine oocytes beyond blastocyst, Biol.
Reprod., vol. 56, pp. 544-548, 1997.
[10] L.R Abeydeera, In vitro fertilization and embryo development in pigs.
Reprod., vol. 58, pp. 159-173, 2001.
[11] A.C. Boquest, L.R. Abeydeera, W.H. Wang and B.N. Day, Effect of
adding reduced glutathione during insemination on the development of
porcine embryos in vitro, Theriogenology, vol. 51, pp. 1311-1319, 1999.
[12] K.A. Zuelke, S.C. Jeffay, R.M. Zucker, S.D. Perreault, Glutathione
(GSH) concentrations vary with the cell cycle in maturing hamster
oocytes, zygotes, and pre-implantation stage embryos, Mol.
Reprod.Deve., vol. 64, pp. 106-112, 2003.
[13] H.I. Calvin, K. Grosshans and E.J. Blake, Estimation and manipulation
of glutathione levels in prepuberal mouse ovaries and ova: relevance to
sperm nucleus transformation in the fertilized egg, Gamete Res., vol. 14,
pp. 265-275, 1986.
[14] SD Perreault, RR Barbee, VL Slott, Importance of glutathione in the
acquisition and maintenance of sperm nuclear decondensing activity in
maturing hamster oocytes, Deve. Biol., vol. 125, pp. 181-186, 1988.
[15] M. Yoshida, Role of glutathione in the maturation and fertilization of pig
oocytes in vitro, Mol. Reprod. Deve., vol. 35, pp. 76-81, 1993.
[16] M.H. Nasr-Esfahani and M.H. Johnson, Quantitative analysis of cellular
glutathione in early preimplantation mouse embryos developing in vivo
and in vitro, Hum.Reprod., vol. 7, pp. 1281-1290, 1992
[17] KS Viana, MC Caldas-Bussiere, SG Matta, MR Faes, CS Paes de
Carvalho, CR Quirino, Effect of sodium nitroprusside, a nitric oxide
donor, on the in vitro maturation of bovine oocytes, Anim. Reprod. Sci.,
vol. 102, pp. 217-227, 2006.