Block to development in cultured rat 1-cell embryos is overcome using medium HECM-1

Abstract

Rat pronuclear embryos were cultured in hamster embryo culture medium-1 (HECM-1) or a modified Krebs-Ringer bicarbonate solution (mKRB). Embryo cultures in HECM-1 were also challenged with low oxygen concentrations. In HECM-1, 57.9% (70/121) of the pronuclear embryos developed into the 4-cell stage after 48 h of culture. The rates of 8-cell, morula and blastocyst formation were 32.2% (39/121), 17.4% (21/121) and 9.9% (12/121), respectively. On the other hand, in mKRB, rat pronuclear embryos showed developmental blockages at the 2-cell and 4-cell stages, and never developed beyond the 4-cell stage. The rate of blastocyst formation under a low oxygen concentration was 20.1% (43/214), showing a significant difference from the value of 5.5% (11/201) obtained under a standard oxygen concentration (P less than 0.005). This is the first report of successful culture of rat pronuclear embryos to the blastocyst stage. Furthermore, it is suggested that protection from oxidation stress is a prerequisite for rat embryo development in vitro.

Development of rat one-cell embryos in a chemically defined medium: effects of glucose, phosphate and osmolarity

“…However, Brown and Whittingham (1992) (Kishi et al, 1991 Bavister, 1990), clear osmolar dependency is reported in mice with the optimum ranges of 250-280 mosmol for one-cell (Whitten, 1971) and [272][273][274][275][276][277][278][279][280] mosmol for two-cell (Biggers and Brinster, 1965;Brinster, 1965) embryos.…”

“…They obtained consistent development of hamster embryos in vitro beyond the two-cell and four-cell stages to the blastocyst stage in a modified Tyrode's medium that did not contain glucose and phosphate (Schini and Bavister, 1988;McKieman and Bavister, 1990; Bavister, 1990;Seshagiri and Bavister, 1991a). This new medium, designated as hamster embryo culture medium 1 (HECM-1; Schini and Bavister, 1988), was applied to the culture of rat (Kishi et al, 1991) and bovine (Pinyopummintr and Bavister, 1991;Kim et al, 1993) one-cell embryos in which development to the blastocyst stage was supported to a limited extent. However, it is not clear whether phosphate and glucose affect rat embryo development independently or in combi¬ nation.…”

Developmental Responses of 2-Cell Embryos to Oxygen Tension and Bovine Serum Albumin in Wistar Rats

“…In rats, early stage embryos could not be cultured before 1993, when developmental retardation, called '2-cell block', was overcome by application of hamster embryo culture medium (HECM-1) [6]. Then, Miyoshi et al modified HECM to obtain high percentages (>90%) of embryo development in vitro.…”

“…In this study, to further improve embryo culture condition, the effects of oxygen tension and bovine serum albumin (BSA), two possibly important factors for embryo culture in other mammalian species [2], were examined using Wistar rats as a model, because Wistar rats have often been used for establishing the basic techniques of embryo and gamete manipulation [6,11]. Wistar rats from two breeders, Crlj:WI and Slc:Wistar, were used to examine whether or not their effects are -Note-consistent across colonies since some differences in developmental responses have been observed among rat strains [14], and these particular breeder colonies are likely to have different genetic backgrounds [16].…”

Molecular and ultrastuctural changes of rat pre-implantation embryos during two-cell developmental arrest

“…It has been shown that uterine and oviduct surface oxygen concentrations ranged between 2 and 9 % [38]. In-vitro culture of embryos under low oxygen (5 %) as opposed to atmospheric oxygen (20 %) resulted in higher blastocyst formation rates in rats [39,40], mice [41,42], sheep and cattle [43]. Similarly, culture of embryonic or adult stem cells under low oxygen improved their growth and maintained their pluripotency [44].…”