Existing zebrafish embryonic stem (ES) cell lines are derived and maintained using feeder layers. We describe here the derivation and long-term culture of an ES cell-like line derived from zebrafish blastomeres without the use of feeder cells. This line, designated as ZES1, has been maintained for more than 800 days in defined Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, zebrafish embryo extract, trout serum, and human basic fibroblast growth factor. ZES1 cells possessed a morphology typical of ES cells, being round or polygonal in shape with a large nucleus and sparse cytoplasm and were mostly diploid. The cells formed individual colonies consisting of tightly packed cells that stained positively for alkaline phosphatase. ZES1 cells also formed embryoid bodies when transferred onto uncoated wells. The pluripotent nature of ZES1 cells was confirmed when they could be induced to differentiate in vitro into several cell types, through low- or high-density culture conditions. Treatment with retinoic acid also induced the differentiation of ZES1 cells into primarily neuronal cells. Using immunostaining and real-time polymerase chain reaction, we showed that Sox2, a known pluripotent marker in mammalian ES cells, was also present in ZES1 cells. Chimera experiments revealed that fluorescent-labeled ZES1 cells microinjected into zebrafish blastulas participated in the formation of all three germ layers. Using GFP-labeled ZES1 cells, chimera germline transmission was also demonstrated at the F1 generation. In conclusion, ZES1 cells possess both in vitro and in vivo pluripotency characteristics, indicating that nonmammalian ES cells can be readily derived and maintained for a long term under feeder-free culture conditions.
Accumulation of reactive oxygen species leads to oxidative stress condition that can accelerate ovarian aging. Ovarian aging caused a reduction in plasma estradiol levels, quality of embryo and eventually will lead to infertility. Tocotrienol has been proven to possess antioxidant properties by protecting the cellular membrane from free radicals damage. Therefore, the aim of this study was to determine the effect of tocotrienol supplementation on the plasma estradiol levels, quality and development of embryos in aging mice. Female mice (Mus musculus) used in this study were divided into six groups. Six weeks old mice (young group) were used as negative control while eight months old mice (aging group) were used as age-matched (positive control) group. Group 1 (6 months old mice) were given corn oil as control, group 2, 3 and 4 (6 months old mice) were supplemented orally for two months with tocotrienol (TCT) at the dose of 90, 120 and 150 mg/kg body weight (BW), respectively. Subsequently, after two months the mice were superovulated, euthanized and 2- cell stage embryos were harvested and cultured in vitro to monitor the embryonic development. Plasma was analysed using enzyme-like immunosorbent assay. The results of this study showed that there was no significant correlation between plasma estradiol levels and the quality of embryo between young and aging group. Similarly, no significant change on plasma estradiol levels were noted in all TCT supplemented groups as compared to its vehicle control. On the other hand, there was a significant reduction on the percentage of normal embryo in all aging groups including TCT supplemented groups as compared to young group. Conversely, TCT supplementation at the dose of 150 mg/kg BW was able to increase the percentage of embryos that developed to blastocyst stage as compared to control. This finding proposed that TCT supplementations for two months are not able to cause a significant change in plasma estradiol levels and quality of embryo but it can delay the consequence of aging in embryonic development.