We believe that studies of in vitro fertilization with one sperm and one oocyte provide a better model for studying fertilization that those in which tens of thousands of heterogeneous sperm are added to a few oocytes. The experiments detailed in this proposal concern fertilization of ova by exposure to one or a few sperm rather than the large number of sperm that have been used in most in vitro fertilization studies. During the first year, supplementary sperm from the perivitelline space of rabbit zygotes will be used for in vitro fertilization of: (1) cumulus-intact rabbit oocytes, (2) cumulus-free rabbit oocytes, (3) zona-free rabbit oocytes, (4) zona-free hamster oocytes, and (5) zona-free hamster oocytes injected into evacuated rabbit zonae containing supplementary sperm. Capacitated rabbit sperm, collected from rabbit uteri, will be used as controls. Ova will be placed with sperm in 2-3 ul drops of Brackett's medium; one-half of these drops will contain 10 mM caffeine. Ova will be fixed and stained 3 and 6 hour later and examined for decondensing sperm heads and male pronuclei. In addition, ova from some treatments will be transferred to recipients to demonstrate normal embryonic development. During the second year, various types of mouse and rabbit sperm (supplementary, acrosome-reacted, capacitated, ejaculated, epididymal, sonicated) will be injected, one at a time, into the perivitelline space of mouse, rabbit and hamster oocytes using a micropipette 5-8 um in diameter. After culture for 3 or 6 hour, half of the ova will be stained to facilitate observation of sperm heads in the ooplasm. The remaining ova will be cultured and/or transferred to determine whether embryonic development is normal. During the third year, the types of sperm mentioned above will be injected singly into the ooplasm of mouse, rabbit, or hamster oocytes. Resulting ova will be fixed and stained, cultulred, or transferred as described above. The results of these experiments will increase our understanding of sperm-oocytle interactions in fertilization and may lead to a means to control fertility at this level.
| Fleming, A D; Cummins, J M; Kuehl, T J et al. (1986) Normal development of hamster and rabbit eggs fertilized by spermatozoa labelled with the fluorescent thiol alkylating agent, monobromobimane. J Exp Zool 237:383-90 |
