292. Influence of the in vitro environment on rat gametes and pre-implantation embryos

Abstract

Rat in vitro fertilization (IVF) and culture (IVC) is attempted by few because of its reputation for difficulty. Currently very few functional rat in vitro systems (IVS) exist for sperm–oocyte interaction research. Successful fertilization of rat metaphase II (MII) oocytes was achieved with two different media, Enriched Krebs Ringer Bicarbonate (EKRB) (70.2%) and M16 (57.4%). Using this IVS we have shown that the rat germinal vesicle-intact (GV-i) oocyte lacks the necessary maturity to interact with capacitated caudal epididymal spermatozoa, whether zona pellucida intact (ZP-i) or free (ZP-f). Proteomic analysis of the protein profile of the oolemma from the GV-i stage through to the MII stage in oocytes is being conducted to characterize any maturational changes that may occur. In addition we provide initial evidence to suggest that an acrosome-intact spermatozoa can fuse with the oolemma of a ZP-f MII oocyte during IVF. Although high percentages of polyspermic embryos in ZP-f IVF (64.8–100%) were observed, the possibility that the rat oolemma may undergo a post-fusion block to polyspermy was implied by a small proportion of normally fertilized embryos (3.8–17.0%) in M16 supplemented with different ratios of hyperactived spermatozoa. Despite successful culture to the blastocyst stage for in vivo fertilized zygotes (33.73%) and 2-cell stage embryos (79.3%), IVF embryos have repeatedly failed to develop in culture. Two dimensional analyses of the protein profile of oocytes/embryos immediately prior to fertilization (MII oocyte–101 spots) and the maternal to zygotic transition (MZT) (zygotes–59 spots and 2-cell embryos–84 spots) has shown a difference in patterns of protein expressed. Comparison of IVF zygotes (41 spots) obtained from EKRB displayed reduced protein expression suggesting that nuclear maturation and/or MZT is not being adequately supported. These data illustrate that rat IVF and IVC require suitable media if its problematic reputation is finally to be shed.