Johnson A L, Bridgham J T, Anthony R V
Department of Biological Sciences, University of Notre Dame, Indiana 46556, USA.
Biol Reprod. 1997 Mar;56(3):581-8. doi: 10.1095/biolreprod56.3.581.
While several studies have documented the presence of plasminogen activator (PA) activity in hen ovarian follicle granulosa and theca tissues, to date it has not been possible to conclusively distinguish between the urokinase (u) and the tissue-type (t) form of the enzyme; this inability is due, in part, to lack of the cloned or characterized chicken tPA gene or gene product. Thus, the present studies were conducted to identify a partial cDNA for chicken tPA and subsequently to evaluate expression of uPA and tPA mRNA in granulosa and theca tissues in vivo and in vitro. Urokinase PA and mRNA levels were highest in prehierarchical-follicle granulosa (3- to 5- and 6- to 8-mm follicles) and theca (6- to 8-mm follicles) tissue compared to hierarchical (9-12 mm through largest preovulatory) follicles. In vitro treatment with a phorbol ester (phorbol 12-myristate, 13-acetate), but not a cAMP analogue (8-bromo-cAMP), significantly increased uPA mRNA levels in both granulosa and theca tissue from the largest and second-largest preovulatory follicles. Of special interest was the finding that levels of uPA mRNA were 10.9-fold higher in atretic compared to morphologically normal 3- to 5-mm follicles. Moreover, 4- to 8-mm-follicle granulosa cells, which spontaneously undergo apoptosis in vitro, demonstrated a rapid increase in uPA mRNA levels after 1 h of incubation (prior to the detection of oligonucleosome formation) while levels in preovulatory-follicle granulosa cells, which do not undergo spontaneous apoptosis, were not altered after 18 h of incubation. By contrast, while tPA mRNA can be identified in granulosa and theca tissues from prehierarchical and preovulatory follicles following polymerase chain reaction amplification, constitutively expressed levels of the transcript were too low to reliably measure by Northern blot analysis. These data indicate that while the chicken expresses a tPA gene that is homologous to the mammalian tPA, uPA is the predominant PA expressed in the hen ovary. In addition, the higher levels of uPA mRNA found in granulosa cells actively undergoing apoptosis and in follicles most susceptible to atresia (4-8 mm) suggest a role for this protease in mediating processes both during the early stages of programmed cell death and in the later stages of follicle atresia and resorption.
虽然多项研究已证明在母鸡卵巢卵泡颗粒层和卵泡膜组织中存在纤溶酶原激活物（PA）活性，但迄今为止，尚无法明确区分该酶的尿激酶型（u）和组织型（t）；这种情况部分归因于缺乏克隆的或已鉴定的鸡组织型纤溶酶原激活物（tPA）基因或基因产物。因此，开展了本研究以鉴定鸡tPA的部分互补DNA（cDNA），随后评估uPA和tPA mRNA在体内和体外颗粒层和卵泡膜组织中的表达。与等级卵泡（9 - 12毫米直至最大的排卵前卵泡）相比，尿激酶型PA及其mRNA水平在等级前卵泡的颗粒层（3至5毫米和6至8毫米卵泡）和卵泡膜（6至8毫米卵泡）组织中最高。用佛波酯（佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯）而非环磷酸腺苷类似物（8 - 溴 - 环磷酸腺苷）进行体外处理，可显著提高最大和第二大排卵前卵泡的颗粒层和卵泡膜组织中的uPA mRNA水平。特别值得关注的是，与形态正常的3至5毫米卵泡相比，闭锁卵泡中的uPA mRNA水平高出10.9倍。此外，在体外会自发发生凋亡的4至8毫米卵泡的颗粒细胞，在孵育1小时后（在检测到寡核苷酸小体形成之前）uPA mRNA水平迅速升高，而在不会自发发生凋亡的排卵前卵泡颗粒细胞中，孵育18小时后其水平未发生改变。相比之下，虽然在经过聚合酶链反应扩增后可在等级前和排卵前卵泡的颗粒层和卵泡膜组织中鉴定出tPA mRNA，但该转录本的组成型表达水平过低，无法通过Northern印迹分析可靠地测量。这些数据表明，虽然鸡表达与哺乳动物tPA同源的tPA基因，但uPA是母鸡卵巢中表达的主要PA。此外，在正在积极发生凋亡的颗粒细胞以及最易发生闭锁的卵泡（4 - 8毫米）中发现的较高水平的uPA mRNA表明，这种蛋白酶在程序性细胞死亡的早期阶段以及卵泡闭锁和吸收的后期阶段的介导过程中发挥作用。
