At the end of a nonconception estrous cycle, the sheep corpus luteum undergoes involution (luteolysis), a process thought to involve apoptotic deletion of cells. It is not yet clear which of the heterogeneous luteal cell types is involved or what mechanisms drive the apoptotic progression. We examined intact paraffin-embedded corpora lutea (in situ terminal dUTP nick end-labeling method) and found direct evidence for apoptotic deletion of cells during luteolysis, but not in healthy, nonregressing corpora lutea. We then sought to implement in vitro models to dissect apoptotic mechanisms in the constituent cells of the corpus luteum. Cells prepared using standard collagenase dispersion of corpus luteum were evaluated for evidence of apoptosis (DNA laddering) by direct agarose gel electrophoresis, a method that obviates the need for DNA extraction, so allowing examination of relatively few cells (≤ 0.5 × 106). When cells were prepared from nonregressing corpus luteum for in vitro manipulation, a population(s) of cells undergoing spontaneous apoptosis was detected. Apoptosis was inhibited by Zn2+ (5 mM), by the tyrosine phosphatase inhibitor sodium orthovanadate (100 μM), or by maintenance at 4 °C. It appears that simple collagenase digestion of intact corpus luteum removes a subset of constituent cells from their survival signal, leading to rapid initiation of endonuclease activity and apoptotic cell death. Identification of the required survival factors and their actions is being pursued to facilitate development of appropriate in vitro models for this endocrine system.Key words: corpus luteum, apoptosis.
TAMANHO DA AMOSTRA PARA ESTUDO DA PROPORÇÃO VOLUMÉTRICA DOS CONSTITUINTES DO CORPO LÚTEO BOVINO