Date of Award

1991

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Kenneth L. White

Abstract

The transplantation of early embryonic cell nuclei into oocytes in which the maternal chromosomes have been removed is a method by which multiple offspring of the same genotype can be produced. One critical factor required for successful development of nuclear transfer derived embryos is inducing the release of the recipient ooplasm from meiotic arrest (activation). This is accomplished during fusion of the two cells. The DC electrofusion pulse used to fuse the two cells together also activates the recipient ooplasm to resume meiotic events, however, the efficiency of electrofusion pulse induced activation remains low. It is known that activation of the oocyte at fertilization is associated with an increase in intracellular calcium concentration ((Ca$\sp{2+}$) $\sb{\rm i}$). A series of experiments were designed to look at changes in (Ca$\sp{2+}$) $\sb{\rm i}$ in murine oocytes after application of a DC electrofusion pulse and its effect on oocyte activation and embryonic development. Oocytes and pronuclear embryos were subjected to the electrofusion pulse in six fusion media consisting of nonelectrolyte (0.3 M mannitol) and electrolyte (phosphate buffered saline, PBS) media containing 0.0, 0.05 or 0.9 mM Ca $\sp{2+}$. Significantly (P $<$ 0.01) higher (Ca$\sp{2+}$) $\sb{\rm i}$ levels were observed in oocytes pulsed in media containing 0.9 mM Ca$\sp{2+}$. An experiment was then designed to test the effect of the three levels of Ca$\sp{2+}$ on oocyte activation. Oocytes pulsed in media containing 0.9 mM Ca$\sp{2+}$ exhibited significantly higher rates of activation (P $<$ 0.01). When 25 $\mu$M inositol 1,4,5-triphosphate (IP3) was added to Ca$\sp{2+}$-free media, a series of (Ca$\sp{2+}$) $\sb{\rm i}$ oscillations similar to that found in fertilized oocytes, was observed. Additional experiments were designed to test the rate of fusion of 2-cell embryos subjected to the six fusion media. Significantly higher rates of fusion were obtained in PBS medium than 0.3 M mannitol (P $<$ 0.05). The fusion treatments were then applied to pronuclear stage embryos to determine the effect on in vitro development. No difference in rate of development was observed between fusion media (P $>$ 0.05). In addition, no difference in in vitro development to the blastocyst stage of pronuclear transplanted embryos was observed after fusion in 0.3 M mannitol and PBS, each containing 0.9 mM Ca$\sp{2+}$.

Pages

104

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