PARENT SESSION
SLIDE SESSION 21: FOLLICLE DEVELOPMENT: INPUTS TO FUNCTIONAL MATURATION
Chairs: Jeff May, Jerry Menon, Kari Doyle (Trainee)
Univ Ottawa-Lamoureaux 122
1:30 PM-3:30 PM

HORMONE-DEPENDENT, PARACRINE-MEDIATED TRANSCRIPTIONAL REGULATION OF TGF-1 EXPRESSION IN THE PORCINE OVARIAN FOLLICLE.

May, Jeffrey¹, Mau, Yun-Hwa¹, ¹

ABSTRACT- Although strongly implicated in mammalian folliculogenesis, neither the role of TGF-1 nor the regulation of its expression have been well established. We have previously reported that FSH and LH are ineffective in stimulating TGF-1 secretion by cultured porcine granulosa (GC) and theca cells (TC), respectively. However, FSH markedly stimulates TGF-1 secretion by intact hemi-follicle linings maintained in explant culture, a system which retains the anatomical association of TC and GC. This suggests that follicular TGF-1 expression is regulated via an interactive mechanism involving GC and TC. To investigate this further, GC and TC were co-cultured and TGF-1 mRNA expression in response to FSH was compared to culture of either cell type alone. Cells (12 million for GC or TC cultures and 6 million each for co-cultures per 25 cm² flask) were attached for 2 days in medium (Ham's F-12:DMEM, 1:1, + 1 g/ml insulin) containing 5% fetal calf serum. Cells were then cultured for up to 3 days in medium +/- hFSH or pLH (50 ng/ml), or 1mM cAMP. Following this, total RNA was prepared and quantitated via UV spectrometry. TGF-1 mRNA was assessed via Northern analysis using 30 g total RNA and a random-prime-labeled 218 bp porcine TGF-1 cDNA. The membrane was then stripped and re-probed using a labeled 1800 bp human actin cDNA. Intensities of TGF-1 and actin mRNAs were determined via image analysis and the TGF-1 mRNA normalized to actin. As shown previously for TGF-1 secretion, neither FSH nor LH stimulated TGF-1 mRNA expression in GC or TC cultures. In contrast, FSH induced an approximate 2-fold increase in TGF-1 mRNA expression in co-cultures (p<0.001). Similarly, cAMP induced TGF-1 mRNA expression in co-cultures. Temporal analysis of FSH-stimulated TGF-1 mRNA expression indicated that the effect became manifest after 48 hr of culture. These results suggest that either FSH stimulates TC TGF-1 expression through a GC intermediate or that TC generate a product which synergizes with FSH to induce TGF-1 mRNA in GC. [Support; NIH Grant HD37971 and The Women's Research Inst.]

KEY WORDS: follicle, TGF-1, ovary, FSH action