RAT HEPATOMA REUBER H-35 CELLS PRODUCE FACTORS THAT PROMOTE THE HATCHING OF MOUSE EMBRYOS CULTURED IN-VITRO

The effects of coculture and conditioned medium of rat hepatoma Reuber H-35 cells on the subsequent in vitro development and hatching of mou se 2-cell embryos were examined. The hatching of embryos obtained from CD-1 mice was accelerated by coculture with Reuber H-35 cells in the presence of 3 mg/ml BSA. The promoting effect on complete hatching fro m zona pellucida was evident even in cell-conditioned medium containin g 60 mu g/ml BSA. In the presence of 60 (mu g/ml BSA, more than 20% of embryos completely hatched, whereas none hatched in the control cultu re. The promoting activity was also found in both the M(r) < 10 000 an d the M(r) > 10 000 subfractions of the conditioned medium separated b y ultrafiltration. The cell number per blastocyst was increased to 1.1 - to 1.3 times the control by culturing embryos from the 2-cell stage with the conditioned medium or its subfractions. The effective target of promoting factors for complete hatching was after the morula stage, and blastocysts hatched completely even when incubated in conditioned medium for 6 h. Inhibitors of DNA polymerase alpha, protein synthesis , and protein kinase partially reduced (40-90% inhibition) the promoti ng effect of the conditioned medium. On the other hand, protease inhib itors showed no effect. In a caseinolytic assay, protease activity was undetectable in the conditioned medium. Incubating the I-125-labeled proteins derived from the M(r) > 10 000 fraction with blastocysts reve aled that at least 9 proteins with apparent molecular masses of 76, 60 , 49, 38, 34, 31, 24, 22, and 18 kDa specifically bound to or accumula ted in the embryos. Moreover, reverse-transcriptase polymerase chain r eaction showed that Reuber H-35 cells expressed mRNAs for epidermal gr owth factor, transforming growth factors alpha and beta 1, and stem ce ll factor. These results indicated that embryonic development and the process of zona hatching was accelerated by factors synthesized by Reu ber H-35 cells. This and other studies demonstrated that Reuber H-35 c ells exert positive (later than 2-cell stage) and negative (at 2-cell stage) effects upon the development of mouse embryos at different embr yonic stages. These factors will serve as valuable tools to clarify th e proliferating and differentiating mechanisms of the preimplantation embryo.