Slee R B, Hillier S G, Largue P, Harlow C R, Miele G, Clinton M
Department of Reproductive and Developmental Sciences, University of Edinburgh, Edinburgh, United Kingdom EH3 9ET.
Endocrinology. 2001 Mar;142(3):1082-9. doi: 10.1210/endo.142.3.7990.
Searching for novel genes involved in tissue remodeling during ovarian folliculogenesis, we carried out differential display RT-PCR (DDRT-PCR) on RNA from gonadotropin-stimulated rat granulosa cells (GC). GC from preantral and early antral follicles in immature rat ovaries were cultured in serum-free medium containing no hormone (control), recombinant human FSH (10 ng/ml), 5alpha-dihydrotestosterone (DHT; 10(-6) M), or FSH plus DHT. Total cellular RNA was extracted from cells at 6, 12, 24, and 48 h of treatment for DDRT-PCR analysis, corresponding to an estimated 60% saturation of the messenger RNA (mRNA) population. Six distinct complementary DNA clones were obtained that reproduced the DDRT-PCR profile on a Northern blot of the corresponding RNA samples. Two of these clones detected transcripts that were strongly down-regulated by FSH. One corresponded to connective tissue growth factor (CTGF), a cysteine-rich secreted protein related to platelet-derived growth factor that is implicated in mitogenesis and angiogenesis, and a second was identical to lysyl oxidase (LO), a key participant in extracellular matrix deposition. In detailed expression studies, Northern analysis revealed a single, approximately 2.5-kb CTGF transcript maximally suppressed within 3 h of exposure to FSH with or without DHT and two LO transcripts ( approximately 3.8 and approximately 5.2 kb) maximally suppressed at 6 h. DHT alone did not affect CTGF mRNA, but strongly enhanced LO mRNA relative to the control value. In vivo, CTGF and LO transcripts were significantly suppressed in GC 48 h after equine CG injection (10 IU, ip) compared with untreated controls and were further reduced 12 h after administration of additional 10 IU hCG to induce luteinization. In situ hybridization confirmed GC in preantral/early antral follicles as principal sites of CTGF and LO mRNA expression. We conclude that expression of CTGF and LO mRNAs is inversely related to GC differentiation. The encoded proteins probably have roles in the regulation of tissue remodeling and extracellular matrix formation during early follicular development.
为寻找参与卵巢卵泡发生过程中组织重塑的新基因，我们对促性腺激素刺激的大鼠颗粒细胞（GC）的RNA进行了差异显示RT-PCR（DDRT-PCR）。将未成熟大鼠卵巢中窦前卵泡和早期窦状卵泡的GC培养于不含激素的无血清培养基（对照）、重组人FSH（10 ng/ml）、5α-二氢睾酮（DHT；10⁻⁶ M）或FSH加DHT中。在处理6、12、24和48小时后从细胞中提取总细胞RNA用于DDRT-PCR分析，这对应于信使RNA（mRNA）群体估计60%的饱和度。获得了六个不同的互补DNA克隆，它们在相应RNA样品的Northern印迹上重现了DDRT-PCR图谱。其中两个克隆检测到的转录本被FSH强烈下调。一个对应于结缔组织生长因子（CTGF），一种与血小板衍生生长因子相关的富含半胱氨酸的分泌蛋白，与有丝分裂和血管生成有关，另一个与赖氨酰氧化酶（LO）相同，它是细胞外基质沉积的关键参与者。在详细的表达研究中，Northern分析显示，在暴露于FSH（无论有无DHT）后3小时内，单一的约2.5 kb CTGF转录本被最大程度抑制，两个LO转录本（约3.8 kb和约5.2 kb）在6小时时被最大程度抑制。单独的DHT不影响CTGF mRNA，但相对于对照值强烈增强了LO mRNA。在体内，与未处理的对照相比，马绒毛膜促性腺激素注射（10 IU，腹腔注射）48小时后GC中的CTGF和LO转录本被显著抑制，在额外给予10 IU hCG诱导黄体化后12小时进一步降低。原位杂交证实窦前/早期窦状卵泡中的GC是CTGF和LO mRNA表达的主要部位。我们得出结论，CTGF和LO mRNA的表达与GC分化呈负相关。所编码的蛋白质可能在卵泡早期发育过程中组织重塑和细胞外基质形成的调节中发挥作用。
