Harvey M B, Leco K J, Arcellana-Panlilio M Y, Zhang X, Edwards D R, Schultz G A
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Development. 1995 Apr;121(4):1005-14. doi: 10.1242/dev.121.4.1005.
Several proteinases from different multigene families have been implicated in the uterine invasion required for establishment of pregnancy in some mammals. In this study, the expression of matrix metalloproteinase gelatinase B (MMP-9), urokinase-type plasminogen activator (uPA) and their inhibitors was investigated during early mouse embryo development. Transcripts for tissue inhibitors of metalloproteinases (TIMP-1,-2,-3) and uPA receptor were detected throughout pre- and peri-implantation development whilst MMP-9 and uPA mRNAs were first detected in peri-implantation blastocysts associated with the invasive phase of implantation. Through use of in situ hybridization, it was shown that MMP-9 transcripts were strongly expressed in the network of trophoblast giant cells at the periphery of implanting 7.5 day embryos and TIMP-3 transcripts were strongly expressed in the decidua immediately adjacent to the implanting embryo. uPA transcripts were preferentially expressed in the ectoplacental cone and its derivatives. Because these proteinases are regulated by growth factors and cytokines in other tissues, the effect of leukaemia inhibitory factor (LIF) and epidermal growth factor (EGF) on their activity was investigated. Both LIF and EGF, like the proteinases, have been implicated in peri-implantation development. Blastocysts collected on day 4 of pregnancy were cultured 2 days in TCM 199 + 10% fetal bovine serum to allow outgrowth followed by 24 hour culture in defined media containing either LIF or EGF. Conditioned media were assayed for uPA activity by a chromogenic assay and MMP activity by gelatin zymography. Both LIF and EGF stimulated uPA and MMP-9 activity in blastocyst outgrowths after 3 days of culture (day 7). Proteinase activity was assayed again at the 5th to 6th day of culture (day 9 to 10). EGF was found to have no effect whereas LIF decreased production of both proteinases. These results demonstrate that proteinase activity in early embryos can be regulated by growth factors and cytokines during the implantation process and, in particular, they demonstrate the possible involvement of LIF in establishment of the correct temporal programme of proteinase expression.
来自不同多基因家族的几种蛋白酶与某些哺乳动物建立妊娠所需的子宫侵袭有关。在本研究中，研究了基质金属蛋白酶明胶酶B（MMP-9）、尿激酶型纤溶酶原激活剂（uPA）及其抑制剂在小鼠早期胚胎发育过程中的表达。在整个植入前和植入周围发育过程中均检测到金属蛋白酶组织抑制剂（TIMP-1、-2、-3）和uPA受体的转录本，而MMP-9和uPA mRNA首次在与植入侵袭期相关的植入周围胚泡中检测到。通过原位杂交表明，MMP-9转录本在植入7.5天胚胎周边的滋养层巨细胞网络中强烈表达，而TIMP-3转录本在紧邻植入胚胎的蜕膜中强烈表达。uPA转录本优先在外胎盘锥及其衍生物中表达。由于这些蛋白酶在其他组织中受生长因子和细胞因子调节，因此研究了白血病抑制因子（LIF）和表皮生长因子（EGF）对其活性的影响。LIF和EGF与蛋白酶一样，都与植入周围发育有关。收集妊娠第4天的胚泡，在含有10%胎牛血清的TCM 199中培养2天以使其生长，然后在含有LIF或EGF的限定培养基中培养24小时。通过显色测定法检测条件培养基中的uPA活性，通过明胶酶谱法检测MMP活性。培养3天（第7天）后，LIF和EGF均刺激胚泡生长中的uPA和MMP-9活性。在培养的第5至6天（第9至10天）再次测定蛋白酶活性。发现EGF没有作用，而LIF降低了两种蛋白酶的产生。这些结果表明，在植入过程中，早期胚胎中的蛋白酶活性可受生长因子和细胞因子调节，特别是，它们证明了LIF可能参与建立蛋白酶表达的正确时间程序。
