DISTRIBUTION OF MITOCHONDRIA IN BOVINE OOCYTES & DVMTL COMPETENCE IN VITRO

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The current study was developed in conjunction with previous work done in the D4R by Jayne Squirrell involving the distribution of mitochondria, in early stage hamster embryos. The hypothesis of this study is that mitochondrial distribution in bovine oocytes after in vitro maturation in suboptimal conditions is altered consistent with reduced developmental competence. The process of maturation is extremely critical, and can ultimately affect the ability of the resulting embryo to develop into a healthy blastocyst and fetus, and give rise to offspring. We would like to first investigate the distribution of mitochondria, in immature oocytes collected from the ovaries of slaughtered cattle. We will then subject oocytes to good and poor maturation medium developed in our laboratory, then stain the matured oocytes to look for mitochondrial distribution and also for tubulin and actin. We hypothesize that the distribution after maturation will reflect the developmental capability of the oocyte after in vitro fertilization and culture. This will assist us in further developing maturation medium for bovine oocytes to produce embryos with maximal developmental competence in vitro. After this first major project, we would like to investigate these same parameters with oocytes derived from transvaginal ultrasound guided oocyte retrieval. These oocytes are aspirated directly from the ovaries of living cattle, and will provide us with further information as to the optimal mitochondrial distribution. We would also like to divide the maturation period of 24 hours into 6 hour intervals and closely study the movements of mitochondria during the maturation process. We would then like to continue to investigate the effects of poor and good maturation medium on the resulting embryos, at the pronuclear, or one cell stage, at the 2 cell stage, and at the 8 cell stage. We hypothesize that the alterations in mitochondrial distribution that may occur during maturation persist in the resulting embryo and that this may be the mechanism whereby development is compromised. Lastly, we would like to examine these changes over time in a single living oocyte and embryo, using 2 photon microscopy. We believe these experiments will help us learn more about what happens to an oocyte during the critical process of maturation, and how perturbations which occur during maturation effect the development of the resulting embryo.