The purpose of this project is to investigate mammalian fertilization and embryonic development using microsurgery. Specifically, we intent to bypass the normal process of fertilization by direct microinjection of spermatozoa into unfertilized oocytes. The experiments are intended to elucidate which, if any, of the sperm:egg interactions characteristis of normal fertilization are necessary for successful embryogenesis. The results should increase our understanding of the fertilization process, and facilitate the development of techniques for restoring fertility in infertile males and diagnosing the cause of infertility. We anticipate that the knowledge gained from these efforts will improve the management of the reproductive process. The animal model to be used is the laboratory mouse. We will mocroinject normal, capacitated sperm from male mice into unfertilized mouse oocytes. Previous work with microinjection strongly indicates that such """"""""microinjection zygotes"""""""" have the potential for full development to term. However, in all prior studies, interspecies hybrids with limited developmental potential were produced. We will produce normal mouse zygotes with this technique, reimplant the embryos into pseudopregnant females, and allow development to proceed. We will thereby assess the importance of the physiologic process of sperm:egg fusion to subsequent development. In every normale jaculate, numerous morphologically abnormal and/or immotile sperm are present. We will microinject these as well. Cleaving embryos will then be karyotyed in order to determine if sperm abnormalities result from faulty chromosome segregation during meiosis. We will also reimplant some of these embryos to determine if they can develop to term, and thus if such a procedure could be used on infertile males with high numbers of abmormal sperm. This procedure will also be used to attempt to rescue male mice which are genetically sterile but which produce large numbers of postmeiotic sperm, and we will inject proximal epididymal and testicular spermatids to learn if epididmal maturation and capacitation are required for sperm to support embryonic development. Finally, we will test whether a diploid zygote resulting from dispermic fertilization can develop normally. This will be done by microinjecting a second sperm into fertilized eggs and withdrawing the female pronucleus. These studies will tell us if the zygote is capable of forming 2 male pronuclei and still continuing development. Success here would result in a model for transmission of heterozygous phenotypes directly from father to offspring.
| Gordon, J W (1988) Use of micromanipulation for increasing the efficiency of mammalian fertilization in vitro. Ann N Y Acad Sci 541:601-13 |
| Talansky, B E; Barg, P E; Gordon, J W (1987) Ion pump ATPase inhibitors block the fertilization of zona-free mouse oocytes by acrosome-reacted spermatozoa. J Reprod Fertil 79:447-55 |
| Barg, P E; Wahrman, M Z; Talansky, B E et al. (1986) Capacitated, acrosome reacted but immotile sperm, when microinjected under the mouse zona pellucida, will not fertilize the oocyte. J Exp Zool 237:365-74 |
