Abstract. The objective of the present study was to clarify the possible role of the zona pellucida (ZP) in early development of rat embryos and to determine the effect of glycosaminoglycans on the development of ZP-free 8-cell embryos before or after embryo transfer at the blastocyst stage. Eight-cell embryos were divided into three groups comprised of, 1) intact controls, 2) embryos with the ZP was removed with acidic solution and 3) pairs of ZP-free 8-cell embryos aggregated in a small hollow. These embryos were cultured in a chemically defined mR1ECM for 24 h. Developmental ability to the blastocyst stage and mean cell number in the blastocyst was lower in ZP-free embryos than in intact controls. When these blastocysts were transferred, the farrowing rate and efficiency of embryos developed to term were also lower in ZP-free embryos, but not in the aggregated ones. Supplementation with hyaluronan (HA; 63-250 μg/ml) or heparan sulfate proteoglycan (HS; 15 μg/ml) significantly improved blastocyst formation of ZP-free embryos and the cell number in the blastocyst by reducing the incidence of apoptosis. However, there were no beneficial effects of HA or HS on farrowing and newborn rates after transfer of the blastocysts. In conclusion, the ZP plays roles in maintaining successful development of early rat embryos at least from the 8-cell stage not only to the blastocyst stage but also to posttransfer stages. Glycosaminoglycans, such as HA or HS, appear to contribute to successful cleavage during early development to the blastocyst stage but may be insufficient to maintain the posttransfer survival of ZP-free embryos. Key words: Blastocyst, Embryo culture, Glycosaminoglycans, Rat, Zona pellucida (J. Reprod. Dev. 58: [295][296][297][298][299][300][301] 2012) M ammalian oocytes, zygotes and early-to blastocyst-stage embryos are enclosed by a thick layer of glycoproteins, called the zona pellucida, which is produced in the early stage of oogenesis. The zona is known to regulate sperm binding to the ovulated oocytes and the monospermic penetration, to prevent separation of blastomeres or aggregation of different embryos during early development, and to protect against viral, bacterial and fungous pathogens or immune cells in the female reproductive tract [1,2].Recent developments in embryo culture technology have made it possible, in many species, for cleavage embryos to develop efficiently to the blastocyst stage in a chemically defined medium with successful results to term following transfer of the blastocysts [3][4][5]. In rats, a chemically defined culture medium, rat 1-cell embryo culture medium (R1ECM), has been developed and succeeded in achieving early development of in vivo and in vitro fertilized rat zona-intact zygotes to the blastocyst stage [6,7]. Gametes and embryos of the rat, one of the standard experimental rodents, have been utilized not only for biomedical research but also for biotechnological attempts including in vitro fertilization [7][8][9], embryo culture [6,10] and manipulation of gametes...