In-vitro development of in-vivo produced rhesus monkey morulae and blastocysts to hatched, attached, and post-attached blastocyst stages: morphology and early secretion of chorionic gonadotrophin

Abstract

The earliest time of secretion of chorionic gonadotrophin (CG) by primate embryos and its role during preimplantation development and implantation are not clearly determined. We cultured in-vivo fertilized/developed zona-intact, morphologically normal morulae (n = 11) and early blastocysts (n = 11), freshly recovered (by non-surgical uterine flushing) on days 5 and 6 of pregnancy, respectively (day 0 = the day following LH surge), from non-superovulated naturally bred rhesus monkeys (Macaca mulatta). Embryos were cultured for a minimum of 24 days in dishes containing 1 ml of CMRL-1066 supplemented with 20% bovine fetal serum in a humidified atmosphere of 5% CO2 in air at 37 degrees C. The culture medium was changed every 48 h. The percentage of hatched blastocysts, developed from morulae and early blastocysts, was 90.9; elapsed times (mean +/- SEM) were 67.8 +/- 4.4 h (morula) and 37.8 +/- 3.6 h (blastocyst). The minimum number of Hoechst-stained cells/hatched blastocyst was 531. The mean diameter (+/- SEM) of cultured embryos increased from 180 microns at the beginning of culture to 374 +/- 28 and 450 +/- 19 microns at the fully expanded and hatched blastocyst stages, respectively. Hatched blastocysts continued to expand (maximum diameter: 1125 +/- 25 microns); after an additional 94-96 h they attached firmly to the serum-coated dishes and produced highly proliferating multinucleate trophectodermal cells, extending to a maximum diameter of 2-6 mm by 11-21 days of culture. Biologically active CG in embryo-grown, serial spent media samples was measured in a mouse Leydig cell bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)