Investigations on cyclophosphamide treatment during the preimplantation period. I. Differential sensitivity to maternal cyclophosphamide treatment.

To study the effect of cyclophosphamide (CPA) treatment of pregnant mice during the preimplantation period on different embryonic cells even before implantation, the cell numbers of the immunosurgically isolated inner cell mass (ICM) and the blastocyst total cell number (BTCN) were determined in late blastocysts. In animals treated at 2 P.M. on day 2 of pregnancy a dose-related reduction of the cell numbers was found for both ICM and BTCN in morphologically normal late blastocysts (day 4, 8 A.M.) at CAP doses of 20-60 mg/kg which did not induce embryonic death before implantation but did later during organogenesis. At identical CPA doses the reduction of the cell numbers of the ICM was significantly greater than that of the BTCN. These data provide evidence for a differential sensitivity of the two groups of cells in preimplantation mouse embryos (trophectoderm and ICM) to maternal CPA treatment even before implantation.

Immunosurgical studies on inner cell mass development in rat and mouse blastocysts before and during implantation in vitro.

Eighty per cent of rat blastocysts (Wistar, SW72) cultured for 96 h in NCTC-109 supplemented with fetal calf serum (FCS) hatched from the zona pellucida and developed a trophoblast giant cell lyer. Thirty seven per cent from the rat blastocysts developed an inner cell mass (ICM) which, in about 7% consisted of two germ layers (ectoderm and endoderm) compared to 84% in NMRI mice. A significantly better ICM development was obtained with cultured rat blastocysts that had hatched in vivo. Similar to the in vivo situation LDH-5 was present in rat blastocysts after implantation in NCTC-109-FCS. Differentiation of C57BL mouse blastocysts in NCTC-109-FCS proceeded as poorly as in the rat. ICM development of rat and mouse blastocysts in NCTC-109-FCS was studied in detail. ICMs of the two species were isolated immunosurgically using complement from different species, e.g. human, rat and rabbit complement, since guinea-pig complement did not lyse trophectoderm cells of rat blastocysts. All immunosurgically isolated rat ICMs degenerated within 48 h, but mouse ICMs isolated with rat or rabbit complement developed significantly better than mouse ICMs isolated with guinea-pig complement. Determinations of the blastocyst total cell number (BTCN) and of the cell number of immunosurgically isolated ICMs were performed in rat and mouse blastocysts to investigate growth kinetics of the ICM before implantation in vitro. In the mouse an exponential increase in both BTCN and cell number of the ICM was observed during the 48 h before implantation in NCTC-109-FCS and also during the 16-24 h before implantation in vivo. In the rat, doubling of the BTCN was found only during the first 24 h in NCTC-109-FCS and there was hardly any increase in the cell number of the ICM during the first 48 h in culture. ICM growth of blastocysts in NCTC-109-FCS is therefore, stimulated in the mouse before and after implantation and in the rat it is inhibited already before implantation.