Carambula Silvia F, Matikainen Tiina, Lynch Maureen P, Flavell Richard A, Gonçalves Paulo B Dias, Tilly Jonathan L, Rueda Bo R
Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114, USA.
Endocrinology. 2002 Apr;143(4):1495-501. doi: 10.1210/endo.143.4.8726.
Because caspase-3 is considered a primary executioner of apoptosis and has been implicated as a mediator of luteal regression, we hypothesized that corpora lutea (CL) derived from caspase-3 null mice would exhibit a delayed onset of apoptosis during luteal regression, when compared with CL derived from wild-type (WT) mice. To test this hypothesis, ovulation was synchronized in immature (postpartum d 24-27) WT and caspase-3-deficient female littermates by exogenous gonadotropins. Individual CL were isolated by manual dissection, 30 h after ovulation, and placed in organ culture dishes in the absence of serum and growth factors. At the time of isolation (0 h) and after 24, 48, and 72 h in culture, the CL were removed and assessed for the presence of processed (active) caspase-3 enzyme and for apoptosis by multiple criteria. There was no evidence of active caspase-3 enzyme or apoptosis in either WT or caspase-3-deficient CL before culture. However, CL derived from the WT mice exhibited a time-dependent increase in the level of active caspase-3 and apoptosis during culture. By comparison, CL derived from caspase-3-deficient mice, cultured in parallel, failed to exhibit any detectable active caspase-3 and showed attenuated rates of apoptosis. To extend these findings derived from ex vivo culture experiments, ovaries were collected from WT and caspase-3 null female littermates at 2, 4, or 6 d post ovulation, and the occurrence of apoptosis within the CL was analyzed. Whereas ovaries of WT mice had only residual luteal tissue at d 6 post ovulation, ovaries collected from caspase-3-deficient mice retained many CL, at d 6 post ovulation, that were similar in size to those observed in the early luteal phase of WT mice. Importantly, there was no dramatic increase in apoptosis in CL of caspase-3-deficient mice at any time point examined post ovulation, indicating that the involution process had indeed been delayed. In contrast, the levels of progesterone declined regardless of genotype. These data provide the first direct evidence that caspase-3 is functionally required for apoptosis to proceed normally during luteal regression. However, caspase-3 is not a direct mediator of the decrease in steroidogenesis associated with luteolysis.
由于半胱天冬酶 -3被认为是细胞凋亡的主要执行者，并且被认为是黄体退化的介质，我们推测，与源自野生型（WT）小鼠的黄体相比，源自半胱天冬酶 -3基因敲除小鼠的黄体在黄体退化过程中凋亡的起始会延迟。为了验证这一假设，通过外源性促性腺激素使未成熟（产后第24 - 27天）的WT和半胱天冬酶 -3缺陷型雌性同窝小鼠的排卵同步。在排卵后30小时，通过手动解剖分离出单个黄体，并将其置于无血清和生长因子的器官培养皿中。在分离时（0小时）以及培养24、48和72小时后，取出黄体并通过多种标准评估加工后的（活性）半胱天冬酶 -3酶的存在情况以及细胞凋亡情况。在培养前，WT或半胱天冬酶 -3缺陷型黄体中均未发现活性半胱天冬酶 -3酶或细胞凋亡的证据。然而，源自WT小鼠的黄体在培养过程中活性半胱天冬酶 -3水平和细胞凋亡呈现出时间依赖性增加。相比之下，平行培养的源自半胱天冬酶 -3缺陷型小鼠的黄体未表现出任何可检测到的活性半胱天冬酶 -3，并且细胞凋亡率降低。为了扩展这些来自体外培养实验的发现，在排卵后第2、4或6天从WT和半胱天冬酶 -3基因敲除的雌性同窝小鼠收集卵巢，并分析黄体中细胞凋亡的发生情况。WT小鼠的卵巢在排卵后第6天只有残留的黄体组织，而从半胱天冬酶 -3缺陷型小鼠收集的卵巢在排卵后第6天保留了许多黄体，其大小与WT小鼠黄体早期观察到的相似。重要的是，在排卵后检查的任何时间点，半胱天冬酶 -3缺陷型小鼠黄体中的细胞凋亡均未显著增加，表明 involution 过程确实被延迟了。相比之下，无论基因型如何，孕酮水平均下降。这些数据提供了首个直接证据，表明半胱天冬酶 -3在黄体退化过程中对于细胞凋亡正常进行在功能上是必需的。然而，半胱天冬酶 -3不是与黄体溶解相关的类固醇生成减少的直接介质。
