The corpus luteum is a transient endocrine gland specializing in the production of
progesterone. The regression of the corpus luteum involves an abrupt decline in its capacity for producing
progesterone followed by its structural involution, which is associated with apoptosis of the luteal cells. An in vitro experimental approach is needed to study the molecular mechanisms underlying hormonal regulation of luteal cell death under defined experimental conditions. In this study, we investigated simian virus-40-transformed luteal cells to determine whether they can be driven to apoptosis and, if so, to define the intracellular pathway involved. Luteal cells were cultured in the presence or absence of
fetal bovine serum for 24 or 48 h. Under serum
starvation conditions, the luteal cells underwent growth arrest accompanied by cell death as evaluated by
dye exclusion, and confirmed by two-color fluorescence cell viability/cytotoxicity assay. We next studied whether serum
starvation-induced death of luteal cells occurred by apoptosis. Morphologic features of apoptosis were observed in cells stained with
hematoxylin after being subjected to serum
starvation for 48 h. The apoptotic nature was further confirmed by in situ 3'-end labeling and fragmentation of genomic
DNA. Apoptosis of serum-deprived luteal cells was dependent upon
caspase activation. Serum
starvation induced cleavage of
poly (ADP-ribose) polymerase (PARP), suggesting that
caspase-3 had been activated under the stress of withdrawal of
growth factors. This was confirmed by cleavage of full-length
procaspase-3. Finally, the fact that serum
starvation promoted the cleavage of full-length
procaspase-9 and the decrease in the expression of endogenous Bid, a BH-3-only proapoptotic
protein of the Bcl-2 family, indicates that the intrinsic (i.e., mitochondrial) pathway of apoptosis was activated. In summary, we have characterized an in vitro experimental model of luteal cell death that can be utilized to evaluate the role of
hormones in apoptosis of luteal cells under defined culture conditions, and to study the mechanism of luteal regression.