Tumor necrosis factor-a (TNF) may play a role in luteolysis by inducing cell death, yet it is not known if TNF induces apoptosis in luteal cells. The action of TNF is governed by two receptors, and the effects of TNF in the corpus luteum (CL) may be dictated by the type of receptor that dominates at any given time. The aims of the present study were 1)to assess expression of mRNA of the two types of TNF receptors, p55 and p75, during the estrous cycle and luteolysis; 2)to determine the ability of TNF combined with IFN-? to induce DNA fragmentation in luteal cells. Corpora lutea were collected on days 5 (early), 10-12 (middle), or 18 (late) of the estrous cycle, or 1, 4, 12, or 24 hours following a luteolytic dose of prostaglandin (PG) F2
(n=4 per time point) and RNA was isolated. After electrophoresis, ratios of RT-PCR product intensity of glyceraldehyde-3-phosphate dehydrogenase (GPDH) and each of the receptors were used to assess mRNA abundance. Neither the GPDH nor the p75 RT-PCR products varied at any time (P>0.05). However, p55 receptor mRNA was less abundant (P<0.05) during the early estrous cycle than at the mid and late stages. The RT-PCR product abundance for this receptor was not affected (P>0.05) by PGF2. In experiment 2, luteal cells from midcycle CL (n = 3) were treated for 2, 4, or 6 days with medium alone or TNF+IFN. Single cell gel electrophoresis and microscopic analysis was used to assess DNA damage. Fragmentation of DNA was induced (P<0.05) by 48 hours following cytokine treatment. Damage of DNA was enhanced by longer treatment with TNF+IFN (P<0.05), while controls remained free of DNA damage. From these results it is concluded that TNF with IFN induces DNA fragmentation typical of apoptosis, and that the p55 TNF receptor mRNA is expressed in the CL in a pattern that may correspond to luteal sensitivity to PGF2.
This abstract is being presented on Monday, August 2 at 3:00 PM at Todd 125.