Liu Y X, Peng X R, Liu H Z, Chen Y J, Ny T
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.
Biol Reprod. 1998 Aug;59(2):409-16. doi: 10.1095/biolreprod59.2.409.
The present study was designed to investigate the effect of prolactin (PRL) on plasminogen activator inhibitor-I (PAI-I) and tissue type plasminogen activator (tPA) gene expression in eCG-primed granulosa cells in vitro. At 46 h after the hormone treatment, ovaries were removed, and granulosa cells were prepared for culture. Cells were incubated for various times in serum-free medium in the presence or absence of LH and PRL alone or in combination. tPA and PAI-I activities in the media were assayed by fibrin overlay and reverse fibrin autograph, respectively. Cytoplasmic RNA from granulosa cells was prepared using the NP-40 method and was assayed for PAI-I and tPA mRNA levels. We demonstrated the following. 1) PRL increased PAI-I mRNA production in cultured granulosa cells. Inclusion of LH with PRL had a synergistic effect on increasing PAI-I mRNA levels. After 48-h culture, 3-fold increases in PAI-I mRNA levels were seen with LH in combination with PRL as compared with PRL alone. The synergistic increase in PAI-I mRNA levels occurred in a dose- and time-dependent manner. 2) The increase in PAI-I mRNA synthesis by PRL alone, or by PRL in combination with LH, was well correlated with the changes in PAI-I activity and antigen levels in the conditioned media. 3) PRL in the culture also dramatically decreased LH-induced tPA mRNA and activity in a dose- and time-dependent fashion. The decrease in the tPA activity by PRL was also correlated with an increase in the amount of PA-PAI-I complexes in the cell-conditioned media. 4) In situ hybridization of tPA and PAI-I mRNAs in the cultured granulosa cells also showed that PRL was capable of enhancing PAI-I mRNA while diminishing tPA mRNA production induced by LH. This suggests that the dose- and time-dependent decrease in the gonadotropin-induced tPA activity in the culture by the presence of PRL may be due to decreasing tPA mRNA synthesis on one hand and to neutralization of the tPA activity by the increased PAI-I activity on the other.
本研究旨在体外研究催乳素（PRL）对经eCG预处理的颗粒细胞中纤溶酶原激活物抑制剂-I（PAI-I）和组织型纤溶酶原激活物（tPA）基因表达的影响。激素处理46小时后，取出卵巢，制备颗粒细胞用于培养。细胞在无血清培养基中单独或联合存在LH和PRL的情况下孵育不同时间。分别通过纤维蛋白覆盖法和反向纤维蛋白自显影法检测培养基中tPA和PAI-I的活性。使用NP-40法制备颗粒细胞的细胞质RNA，并检测PAI-I和tPA mRNA水平。我们得到了以下结果。1）PRL增加了培养的颗粒细胞中PAI-I mRNA的产生。PRL与LH共同作用对增加PAI-I mRNA水平有协同作用。培养48小时后，与单独使用PRL相比，LH与PRL联合使用时PAI-I mRNA水平增加了3倍。PAI-I mRNA水平的协同增加呈剂量和时间依赖性。2）单独使用PRL或PRL与LH联合使用时PAI-I mRNA合成的增加与条件培养基中PAI-I活性和抗原水平的变化密切相关。3）培养中的PRL也以剂量和时间依赖性方式显著降低LH诱导的tPA mRNA和活性。PRL对tPA活性的降低也与细胞条件培养基中PA-PAI-I复合物数量的增加相关。4）培养的颗粒细胞中tPA和PAI-I mRNA的原位杂交也表明，PRL能够增强PAI-I mRNA，同时减少LH诱导的tPA mRNA产生。这表明，由于PRL的存在，培养中促性腺激素诱导的tPA活性呈剂量和时间依赖性降低，一方面可能是由于tPA mRNA合成减少，另一方面是由于PAI-I活性增加对tPA活性的中和作用。
