378 INTRACYTOPLASMIC SPERM INJECTION AND ESTABLISHMENT OF EMBRYONIC STEM CELLS IN AFRICAN GREEN MONKEYS (CERCOPITHECUS AETHIOPS)

Abstract

There has been little embryological research in African green monkeys (Cercopithecus aethiops), although Vero and Cos7 cell lines derived from the kidney of the green monkey are used as valuable resources for testing viral infections and introducing foreign genes. Embryological research would not only contribute to medical science but would also help to maintain and propagate other monkeys, including those in the wild. We examined intracytoplasmic sperm injection (ICSI) and in vitro culture of green monkey embryos and attempted to establish embryonic stem (ES) cells, which would be precious resources. We collected mature oocytes from females treated with eCG + hCG or human FSH + hCG to induce follicular growth stimulation and oocyte maturation. Of 61 oocytes from eCG-treated females and of 44 oocytes from FSH-treated females, 41 (67.2%) and 27 (61.4%) fertilized oocytes with 2 pronuclei and a second polar body were produced with ICSI, respectively. We cultured fertilized oocytes from eCG-treated and FSH-treated females in CMRL-1066 medium containing 10% fetal bovine serum (FBS), with or without a buffalo rat liver (BRL) cell monolayer. Of 28 zygotes from eCG-treated females, 9 were cultured without a monolayer, and 3 (33.3%) developed into blastocysts; none of 19 cultured with a monolayer did. Of 22 zygotes from FSH-treated females, 18 were cultured without a monolayer, and 5 (27.8%) developed into blastocysts; none of 4 cultured with a monolayer did. Thus, there were no differences in the fertilization rates and development into blastocysts between the oocytes from eCG- and FSH-treated females. On the other hand, BRL cells did not support development into blastocysts [0/23 (0%) with a monolayer vs. 8/27 (29.6%) without a monolayer; P < 0.01]. The other embryos were used for experiments such as embryo transfer. Parts of the inner cell mass (ICM), isolated by removing trophectoderm and the zona pellucida (ZP) with a 27-gauge needle (5 blastocysts) or by dissolving the ZP with an enzyme (3 blastocysts) were transferred to DMEM/F12 (1 : 1) medium containing 20% FBS or knockout serum replacement with a mitomycin C-treated STO cell or mouse embryonic fibroblast cell monolayer. Only 3 ICMs formed extended colonies; the others, including the 3 blastocysts from which the ZP was dissolved, did not. When the 3 colonies were subcultured with collagenase or divided into small clusters with a needle, 2 flat colonies, as in primate ES cells, appeared. At the next subculture, the flat colonies disappeared. However, we confirmed the formation of ES-like cell colonies. These results show that it is possible to produce blastocysts with ICSI and that ES cells may be established in African green monkeys.