The mode of zona pellucida denudation and blastocyst proliferation and attachment was investigated in Wistar rats during day 5 of pregnancy and in ovariectomized pregnant animals given 5-0 mg progesterone daily until the 10th day, when 0-2 mug oestradiol was injected together with progesterone. During day 5 of pregnancy, when only 5 empty zonae were detected from 32 animals, zona denudation occurred by lysis, which took 6 h. Zona denudation occurred by shedding in the ovariectomized pregnant

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... mals; 190 empty zonae were detected from 40 animals. Oestradiol administration to the experimental animals caused lysis of the shed zonae 24-30 h after oestradiol treatment. Air-dried preparations of the embryos showed that the blastomeres proliferated exponentially from a mean of 27-52 +/- 4-0 cells per embryo at 12.00 h on day 5 of pregnancy to 53-96 %/- 2-58 cells per embryo at 22.00 h, with a cell doubling time of 10 h in normal animals. In the ovariectomized animals, blastocysts were arrested at about the 100-cell stage on the 10th day. Oestradiol administration to the experimental animals did not induce mitosis; the embryos implanted without further cell division. The number of free embryos recovered declined from a mean of 12-0 per animal at 12.00 h to 4-25 per animal at 22.00 h on day 5 of pregnancy. The number of embryos recovered from the ovariectomized animals varied between one and 11 per animal. The results show that cell division continued in embryos within ovariectomized pregnant animals in the absence of oestrogen, until about the 100-cell stage. There was no further cell division before nidation when oestradiol was administered. The empty zonae were, however, lysed between 24 and 30 h after oestradiol treatment. The zona lytic factor appears to be oestradiol dependent and of maternal origin. A causal relationship exists between zona lysis and embryo attachment, which is independent of the number of cells per blastocyst.