This study assessed pronuclear formation, the chromosomal constitution, and the developmental capacity of bovine zygotes formed by intracytoplasmic injection of oocytes with sperm, treated or not with dithiothreitol (DTT). Oocytes were matured in vitro for 22-24 h and then centrifuged so that sperm, prepared by swim-up in the presence or absence of 5 mM DTT, could be injected into the cleared area of the ooplasm. Injected oocytes were activated by treatment with 5 microM ionomycin (5 min) and, after a 3-h interval, with 1.9 mM 6-dimethylaminopurine (DMAP) for 3 h. They were then cocultured with bovine oviductal epithelial cells in M199. Sperm treatment resulted in a significantly higher proportion of male pronucleus formation 16 h after injection (40% vs. 11%; p < 0.0001) and a significantly higher rate of blastocyst development (24% vs. 10%; p < 0.005). Sixty-one percent of blastocysts produced with treated sperm were diploid. Of 12 blastocysts produced with treated sperm and sexed by a polymerase chain reaction, 4 were male and 7 female, and in one a definite diagnosis could not be made. Embryo transfer (2 embryos per heifer) resulted in pregnancies in 6 of 16 recipients at Day 49, but none was carried to term. These results show that the efficiency of bovine intracytoplasmic sperm injection can be improved by sperm pretreatment with DTT and by oocyte activation with ionomycin plus DMAP, although the developmental capacity of the resulting embryos remains limited.

The active metabolite of leflunomide, A77 1726 inhibits the proliferation of a variety of mammalian cell lines in culture. Epidermal growth factor (EGF)-dependent proliferation is inhibited by A77 1726 at an effective dose of 30-40 ,uM. A77 1726 appears to directly inhibit the EGF receptor tyrosine-specific kinase activity both in intact ceils and purified EGF receptors at the same effective dose. These data suggest that leflunomide inhibits cellular proliferation by the inhibition of tyrosine-specific kinase activities.Epidermal growth factor receptor; Tyrosine-specific kinase; Leflunomide; Human tbreskin fibroblast cell

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