Goyeneche Alicia A, Harmon Jacquelyn M, Telleria Carlos M
Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, 414 East Clark Street, Vermillion, 57069, USA.
Reproduction. 2006 Jan;131(1):103-11. doi: 10.1530/rep.1.00751.
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.
黄体是一种专门分泌孕酮的临时性内分泌腺。黄体的退化涉及到其产生孕酮能力的突然下降，随后是其结构退化，这与黄体细胞的凋亡有关。需要一种体外实验方法来研究在特定实验条件下黄体细胞死亡的激素调节的分子机制。在本研究中，我们研究了猿猴病毒40转化的黄体细胞，以确定它们是否能被诱导凋亡，如果可以，确定所涉及的细胞内途径。黄体细胞在有或无胎牛血清的情况下培养24或48小时。在血清饥饿条件下，黄体细胞经历生长停滞并伴有细胞死亡，这通过染料排斥法评估，并通过双色荧光细胞活力/细胞毒性测定法得到证实。接下来，我们研究血清饥饿诱导的黄体细胞死亡是否通过凋亡发生。在经历48小时血清饥饿后，用苏木精染色的细胞中观察到凋亡的形态学特征。原位3'-末端标记和基因组DNA片段化进一步证实了凋亡的性质。血清剥夺的黄体细胞的凋亡依赖于半胱天冬酶的激活。血清饥饿诱导聚（ADP-核糖）聚合酶（PARP）的切割，表明在生长因子撤出的应激下，半胱天冬酶-3已被激活。全长前半胱天冬酶-3的切割证实了这一点。最后，血清饥饿促进全长前半胱天冬酶-9的切割以及内源性Bid（Bcl-2家族中仅含BH-3的促凋亡蛋白）表达的降低这一事实表明，凋亡的内在（即线粒体）途径被激活。总之，我们已经建立了一个黄体细胞死亡的体外实验模型，该模型可用于评估激素在特定培养条件下黄体细胞凋亡中的作用，并研究黄体退化的机制。
