Glycosaminoglycans Improves Early Development of Zona-free 8-cell Rat Embryos to Blastocysts in a Chemically Defined Medium, but Not the Pregnancy Rate Following Transfer of the Blastocysts

Abstract

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...

Advances in Genome Editing and Application to the Generation of Genetically Modified Rat Models

“…These modifications include NHEJ-mediated indels (Kaneko et al, 2014;Hashimoto and Takemoto, 2015;Kaneko and Mashimo, 2015;Qin et al, 2015;Hashimoto et al, 2016;Wang et al, 2016;Remy et al, 2017), large segment deletions (Hashimoto et al, 2016;Wang et al, 2016), conditional KO (Miyasaka et al, 2018), double-KO (Teixeira et al, 2018), HDR-mediated precise nucleotide substitutions (Kaneko and Mashimo, 2015;Qin et al, 2015;Wang et al, 2016) or short sequence insertions using ssODNs (typically < 200 bp) (Hashimoto and Takemoto, 2015;Chen et al, 2016;Wang et al, 2016;Remy et al, 2017) and lsDNA (from 600 bp to 1.5 kb) (Miyasaka et al, 2018). In some studies, electroporation was done in mouse zygotes that were denuded of the zona pellucida (ZP) by a Tyrod's acid treatment (Qin et al, 2015;Chen et al, 2016;Wang et al, 2016), without affecting the early development unlike data reported in rats (Okuyama and Funahashi, 2012). Electroporation also can be applied to mouse and rat frozen zygotes for efficient introduction of CRISPR RNP complexes, without affecting embryo viability or development (Nakagawa et al, 2018;Kaneko and Nakagawa, 2020).…”

Generation of gene-edited rats by delivery of CRISPR/Cas9 protein and donor DNA into intact zygotes using electroporation

“…To overcome this physical barrier, the ZP is weakened either by a chemical treatment (Tyrod’s acid) or by application of a strong electric field to create pores. Although the weakening of the ZP allows to better control the electroporation efficiency, many studies have shown that the early developmental potential of zona-free embryos is often compromised, particularly in rats 9 .…”