NEUTRALIZATION OF TUMOR-NECROSIS-FACTOR-ALPHA (TNF-ALPHA) ACTION ON CELL-PROLIFERATION IN RAT BLASTOCYSTS BY ANTISENSE OLIGODEOXYRIBONUCLEOTIDES DIRECTED AGAINST TNF-ALPHA P60 RECEPTOR

Antisense oligodeoxyribonucleotide inhibition of gene expression was u sed to test whether the p60 form of the tumor necrosis factor alpha (T NF alpha) receptor (Rp60) is responsible for mediating the negative ef fect of TNF alpha on the development of rat blastocysts in vitro. The antisense oligonucleotide was designed to overlap the translation init iation codon of the TNF alpha Rp60 mRNA. Preliminary experiments showe d that concentrations of oligonucleotides above 10 mu M in the culture medium were embryotoxic over 24 h. When used at nontoxic concentratio ns (8 mu M), antisense oligonucleotides specifically decreased the abu ndance of intact TNF alpha Rp60 transcripts by 80% within 3 h of expos ure. In contrast to results with control embryos, mRNA for the second form of TNF alpha receptor, TNF alpha Rp80, was detected in blastocyst s exposed to antisense oligonucleotides to TNF alpha Rp60. Antisense o ligonucleotides to TNF alpha Rp60 blocked the 25-30% decrease in cell proliferation induced by 50 ng/ml TNF alpha added to a standard cultur e medium and by 5 ng/ml TNF alpha added to a medium that had been cond itioned by rat uterine cells. Sense oligonucleotides had no such prote ctive effect. Because uterine cells from diabetic rats secrete higher levels of TNF alpha than those from control rats, antisense oligonucle otides were also tested in a medium that had been conditioned by diabe tic uterine cells (cell-secreted TNF alpha concentration was 50 pg/ml in this medium, and no exogenous TNF alpha was added). Addition of ant isense oligonucleotides to TNF alpha Rp60 improved the quality of this medium with respect to cell proliferation but failed to correct the h igh frequency of dead cells observed in the blastocysts.