Differentiation-dependent expression of connective tissue growth factor and lysyl oxidase messenger ribonucleic acids in rat granulosa cells

Searching for novel genes involved in tissue remodeling during ovarian foll iculogenesis, we carried out differential display RT-PCR (DDRT-PCR) on RNA from gonadotropin-stimulated rat granulosa cells (GC). GC from preantral an d early antral follicles in immature rat ovaries were cultured in serum-fre e medium containing no hormone (control), recombinant human FSH (10 ng/ml), 5 alpha -dihydrotestosterone (DHT; 10(-6) M), or FSH plus DHT. Total cellu lar RNA was extracted from cells at 6, 12, 24, and 48 h of treatment for DD RT-PCR analysis, corresponding to an estimated 60% saturation of the messen ger RNA (mRNA) population. Six distinct complementary DNA clones were obtai ned that reproduced the DDRT-PCR profile on a Northern blot of the correspo nding RNA samples. Two of these clones detected transcripts that were stron gly down regulated by FSH. One corresponded to connective tissue growth fac tor (CTGF), a cysteine-rich secreted protein related to platelet-derived gr owth factor that is implicated in mitogenesis and angiogenesis, and a secon d was identical to lysyl oxidase (LO), a key participant in extracellular m atrix deposition. In detailed expression studies, Northern analysis reveale d a single, approximately 2.5-kb CTGF transcript maximally suppressed withi n 3 h of exposure to FSH with or without DHT and two LO transcripts (simila r to3.8 and similar to5.2 kb) maximally suppressed at 6 h. DHT alone did no t affect CTGF mRNA, but strongly enhanced LO mRNA relative to the control v alue. 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/e arly 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 d ifferentiation. The encoded proteins probably have roles in the regulation of tissue remodeling and extracellular matrix formation during early follic ular development.