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Department of Reproductive and Developmental Sciences, University of Edinburgh (R.B.S., S.G.H., P.L., C.R.H.), Edinburgh, United Kingdom EH3 9ET; and Department of Gene Expression and Development, Roslin Institute (G.M., M.C.), Roslin, United Kingdom EH25 9PS
Address all correspondence and requests for reprints to: Stephen G. Hillier, Ph.D., Department of Reproductive and Developmental Sciences, University of Edinburgh, 37 Chalmers Street, Edinburgh, United Kingdom EH3 9ET. E-mail: s.hillier{at}ed.ac.uk
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), 5
-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 (
3.8 and 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.
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