INTERNUCLEOSOMAL DNA FRAGMENTATION IN OVINE LUTEAL TISSUE ASSOCIATED WITH LUTEOLYSIS - IN-VIVO AND IN-VITRO ANALYSES

Internucleosomal DNA fragmentation, a characteristic of apoptosis, can be visualized with agarose gel electrophoresis as discrete low-molecular-weight DNA fragments (laddering), in multiples of similar to 185 bp. CL were collected from superovulated ewes (control) or at 12 h after injection of prostaglandin F-2 alpha (PGF(2 alpha)) on various days after hCG injection. The ability of PGF(2 alpha) on Days 8, 10, 12, and 14 (n greater than or equal to 3 per day per treatment) to induce luteal cell DNA fragmentation was evaluated. DNA was isolated and visualized on agarose gels. No DNA fragmentation was observed in CL from control ewes on Days 8, 10, or 12. Internucleosomal fragmentation of DNA (indicative of apoptosis) as well as nonspecific DNA fragmentation (indicative of non-apoptotic cell death) in CL from Day 14 controls was observed in two of four animals. Additionally, this pattern of DNA fragmentation was observed in CL from ewes treated with PGF(2 alpha) on all days. Evidence of DNA fragmentation was observed in luteal tissue after dissociation, yet no fragmentation was observed in unsliced, non-dissociated CL collected from Day 10 control ewes (incubated 4 h), or in sliced, non-incubated CL. Slicing and incubation alone were sufficient to initiate DNA fragmentation. A variety of approaches were utilized to inhibit DNA fragmentation. Only the addition of zinc acetate (1 mM) in the incubation medium throughout the 4-h incubation period prevented DNA fragmentation that was initiated by slicing (p < 0.05). There appear therefore, to be one or more intraluteal factors that directly initiate DNA fragmentation associated with cell death in luteolysis. This fragmentation appears to a variable degree in Day 14 animals (at the onset of luteolysis) and can be induced by injection of animals with PGF(2 alpha).