Mann J S, Kindy M S, Edwards D R, Curry T E
Department of Anatomy and Neurobiology, University of Kentucky, Lexington 40536.
Endocrinology. 1991 Apr;128(4):1825-32. doi: 10.1210/endo-128-4-1825.
Metalloproteinases, such as collagenase or gelatinase, and their associated inhibitors appear to control connective tissue remodeling during follicular rupture. We examined the regulation of metalloproteinase inhibitor activity by various treatments in cultured rat granulosa cells. Granulosa cells were harvested from immature PMSG-primed rats and cultured with LH, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), cAMP, or forskolin. Inhibitor activity was measured in the medium. Increasing concentrations of either LH (0.1-1000 ng/ml) or TPA (2.5-100 nM) resulted in a dose-dependent increase in metalloproteinase inhibitor activity (2.9- and 2.4-fold increases above control, respectively). There was also a time-dependent induction of inhibitor activity in cells incubated in the presence of LH (100 ng/ml) for 6, 12, 18, or 24 h. Forskolin (0.1 mM) or cAMP (1 mM) treatment increased inhibitor activity 2.8- and 1.6-fold above that in control cultures. LH and TPA treatment in combination resulted in an additive increase in inhibitor activity compared to LH or TPA treatment alone. This finding suggested that the granulosa cell inhibitor activity might be induced through separate intracellular pathways. The inhibitor present in conditioned medium was isolated by chromatographic separation on a Sepharose 6B mol wt exclusion column. The inhibitor present was approximately 28,000 mol wt, which is consistent with the size of tissue inhibitor of metalloproteinase (TIMP). In addition to the granulosa cell experiments, changes in ovarian mRNA levels for TIMP were determined. There was a preovulatory increase in TIMP mRNA from whole rat ovaries, with the highest levels detected 12 h after hCG administration. The present study establishes that metalloproteinase inhibitor activity from rat granulosa cells is induced through separate pathways: a LH-cAMP-dependent protein kinase-A pathway and a cAMP-independent protein kinase-C pathway. Furthermore, a TIMP-like protein is observed in granulosa cell-conditioned medium, while TIMP mRNA is present in rat ovaries and increases before ovulation, suggesting that the granulosa cell metalloproteinase inhibitor is TIMP. We propose that TIMP acts in part to control the site and extent of follicular connective tissue remodeling associated with ovulation.
金属蛋白酶，如胶原酶或明胶酶，及其相关抑制剂似乎在卵泡破裂过程中控制着结缔组织重塑。我们研究了在培养的大鼠颗粒细胞中各种处理对金属蛋白酶抑制剂活性的调节作用。从用孕马血清促性腺激素（PMSG）预处理的未成熟大鼠中获取颗粒细胞，并分别用促黄体生成素（LH）、佛波酯12 - O - 十四酰佛波醇13 - 乙酸酯（TPA）、环磷酸腺苷（cAMP）或福斯高林进行培养。在培养基中测量抑制剂活性。LH（0.1 - 1000 ng/ml）或TPA（2.5 - 100 nM）浓度的增加导致金属蛋白酶抑制剂活性呈剂量依赖性增加（分别比对照增加2.9倍和2.4倍）。在含有100 ng/ml LH的情况下孵育6、12、18或24小时的细胞中，抑制剂活性也呈时间依赖性诱导。福斯高林（0.1 mM）或cAMP（1 mM）处理使抑制剂活性比对照培养物中增加2.8倍和增加1.6倍。与单独的LH或TPA处理相比，LH和TPA联合处理导致抑制剂活性呈累加性增加。这一发现表明颗粒细胞抑制剂活性可能通过不同的细胞内途径诱导产生。通过在琼脂糖6B分子量排阻柱上进行色谱分离，从条件培养基中分离出存在的抑制剂。所存在的抑制剂分子量约为28,000，这与金属蛋白酶组织抑制剂（TIMP）的大小一致。除了颗粒细胞实验外，还测定了卵巢中TIMP的mRNA水平变化。从整个大鼠卵巢中发现，TIMP mRNA在排卵前增加，在注射人绒毛膜促性腺激素（hCG）后12小时检测到最高水平。本研究证实，大鼠颗粒细胞中的金属蛋白酶抑制剂活性是通过不同途径诱导产生的：一条LH - cAMP依赖性蛋白激酶 - A途径和一条cAMP非依赖性蛋白激酶 - C途径。此外，在颗粒细胞条件培养基中观察到一种TIMP样蛋白，而TIMP mRNA存在于大鼠卵巢中且在排卵前增加，这表明颗粒细胞金属蛋白酶抑制剂是TIMP。我们认为TIMP部分作用是控制与排卵相关的卵泡结缔组织重塑的部位和程度。
