Analysis of the microenvironment and cryopreservation of embryos on their development

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Microtechniques and highly sensitive methods such as enzymatic cycling, micro-Western analysis, reverse transcription polymerase chain reaction and so on were employed to study these processes. Low hexokinase activity and high activities of enzymes in the phosphate pathway were characteristic of immature oocytes. During maturation, the activities of hexokinase and phosphofructokinase increased significantly. These changes were used to analyze involvement of epidermal growth factor (EGF) and prostaglandins (PG) in oocyte maturation EGF is shown to stimulate maturation by increasing PG production in granulosa cells. Electro-physiologically, the sensitivity of oocyte to inositol triphosphate increased and Ca^<2+> release system developed during maturation. Progesterone production of oocyte and embryos are shown by enzymatic cycling and other methods using radiometry. This hormone produced by embryos themselves may play a role in embryonic development in intracrine fashion. There is 100-fold increase in glucose uptake from oocyte to blastocyst in mice. A switch in substrate preferende of the embryo from pyruvate to glycose during preimplantation development may be explained by increases in the activity of hexokinase and expression of glycose transporter, GLUT1. Hexokinase activities determined by NADP cycling increased 20-fold while expression of GLUT1 assessed by micro-Western method 10-fold. GLUT1 expression was also analyzed by RT-PCR,which indicated that the expression is regulated at transcription level. There is a delay in the develomental changes in glucose uptake, hexokinase activity and GLUT1 expression when the embryos are developed in vitro. It is of interest to note that EGF stimulates these developmental changes in vitro. In conclusion, expanding knowledge in the process of oocyte maturation and embryonic development may serve not only as significant clinical implications for in vitro fertilization techniques but as breakthroughs in gynecology.