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The transforming growth factors beta (TGF beta) have been implicated as important intrafollicular regulators of follicle development in the mammalian ovary. Recent studies in this laboratory have suggested that, when cultured, both porcine theca and granulosa cells secrete TGF beta, primarily TGF beta 1 (May et al., Endocrine 2:1045-1054, 1994). In this report, evidence is presented that during follicle development in vivo, theca but not granulosa cells are the source of follicular TGF beta. Although both theca and granulosa cells secreted TGF beta when attached to culture dishes, only theca cells secreted detectable levels of TGF beta when cells were cultured in serum-free medium without attachment. Granulosa cells secreted little if any TGF beta. This difference in TGF beta secretion was not found to be due to differences in preparation of the two cell types (i.e., mechanical versus enzymatic preparation). These results suggested that theca cells may be the source of TGF beta in the follicle. To ascertain whether TGF beta is actually secreted by follicles, intact hemi-follicle linings consisting of both theca and granulosa cells were cultured in moderate-term, organ explant culture. Hemi-follicle linings secreted TGF beta at a near linear rate for at least 4 days (approximately 300 pg/follicle/day for 6-8-mm-diameter follicles). The level of TGF beta secretion was directly related to the size of the follicle (p < 0.01). Immunoneutralization studies using TGF beta subtype-specific antibodies indicated that the major form of TGF beta secreted by porcine hemi-follicle linings was TGF beta 1. To further investigate the source of TGF beta during follicle development, a combination of molecular biology procedures was employed. Using 243-bp antisense and sense cRNA probes generated from a simian TGF beta 1 cDNA, we performed in situ hybridization on sections of whole porcine ovaries containing antral follicles. Expression of TGF beta 1 mRNA was localized to both theca and granulosa cells with no expression found in the stroma, suggesting that both cell types transcribe TGF beta 1. TGF beta 1 expression was evaluated additionally by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot analysis. Oligonucleotide primers were generated from the porcine TGF beta 1 cDNA sequence for RT-PCR, and the PCR product (287-bp sequence) was amplified and used for Northern analysis. RT-PCR of total RNA isolated from theca and granulosa cells indicated that both cell types expressed TGF beta 1 mRNA. This finding was confirmed via Northern blot analysis, which further indicated the presence of two TGF beta 1 mRNAs of 2.5 and 3.5 kb, consistent with previous reports of alternate splicing of the porcine TGF beta 1 gene. These data indicate that both cell types express TGF beta 1 mRNA. To further evaluate TGF beta 1 expression, we attempted to isolate TGF beta 1 protein from freshly collected theca and granulosa cells by partial purification and immunoprecipitation. Interestingly, the growth factor could be extracted...

Upon binding to its G protein-coupled transmembrane receptors, the actions of PGF2alpha on the corpus luteum are initiated by the phospholipase C/diacylglycerol-inositol 1,4,5-trisphosphate (InsP3)/Ca2+-protein kinase C (PKC) pathway. However, little is known about the downstream intracellular signaling events that can lead to transcriptional activation in response to PGF2alpha. The present study was conducted to examine the involvement of the mitogen-activated protein kinase (MAPK) signaling cascade in the corpus luteum. Three isoforms of the Raf family of oncoprotein kinases (A-Raf, B-Raf, and Raf-1 or c-Raf) were detected in bovine luteal cells. Raf-1 and B-Raf, but not A-Raf, were activated by PGF2alpha (1 microM) and the pharmacological PKC activator phorbol myristate acetate (PMA, 20 nM). Kinetic analysis revealed that PGF2alpha rapidly and transiently activated Raf-1. In vitro protein kinase assays demonstrated that activation of Raf-1 and B-Raf resulted in the phosphorylation and activation of MAPK kinase (MEK1), which subsequently phosphorylated p42mapk. As determined by hyperphosphorylation, tyrosine phosphorylation, and enzymatic activity, p42mapk and p44mapk were rapidly and transiently activated by both PGF2alpha (1 microM) and PMA (20 nM). Additionally, both PGF2alpha (1 microM) and PMA (20 nM) stimulated phosphorylation of Raf-1, MEK1, and p42mapk in 32P-labeled cells. Our data demonstrate that PGF2alpha activates the Raf/MEK1/p42/44mapk signaling cascade in bovine luteal cells and that the actions of PGF2alpha are mimicked by the PKC activator PMA. Activation of the Raf/MEK1/MAPK signaling cascade by PGF2alpha in luteal cells provides a mechanism to transduce signals initiated by PGF2alpha receptors on the cell surface into the nucleus. Activation of the Raf/MEK1/MAPK signaling cascade may be associated with transcriptional activation of luteal genes possessing activator protein-1-binding sites.

PGF2alpha triggers the demise of the corpus luteum whereby progesterone synthesis is inhibited, the luteal structure regresses, and the estrus cycle resumes. Upon binding to its heterotrimeric G-protein-coupled receptors, PGF2alpha initiates the phospholipase C/diacylglycerol and inositol-1,4,5-trisphosphate/Ca(2+)-protein kinase C (PKC) signaling pathway. More recently, we have demonstrated that PGF2alpha activates extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase signaling through a Raf-dependent mechanism in bovine luteal cells. However, the relationship between PKC and ERK activation in PGF2alpha signaling has not been clearly defined. Moreover, the signaling pathway that PGF2alpha uses to regulate gene expression is unknown. In this report, primary cultures of bovine luteal cells were used to address the role of PKC in ERK activation and the signaling pathway for induction of c-fos and c-jun messenger RNA (mRNA) expression in response to PGF2alpha. By using a PKC inhibitor and a PKC-deficient luteal cell model, we observed that phorbol ester-responsive isoforms of PKC were required for ERK phosphorylation and activation by PGF2alpha (1 microM) or phorbol 12-myristate 13-acetate (PMA) (20 nM). In PGF2alpha- and PMA-treated cells, active ERK MAP kinase was localized in the nucleus. PGF2alpha-induced ERK phosphorylation was dose-dependently inhibited by the MEK1 inhibitor PD098059 (1-50 microM). The expression of c-fos and c-jun mRNA in luteal cells was markedly increased by treatment with PGF2alpha (1 microM) or PMA (20 nM) for 30 min. We also observed that activation of ERK MAP kinase was required for the expression of c-fos and c-jun mRNA in response to PGF2alpha and PMA because it was abrogated by blocking the ERK pathway with PD098059. In addition, PGF2alpha and PMA-induced c-fos and c-jun mRNA expression was abolished in the PKC-deficient cells. Taken together, our data demonstrate that a PKC-dependent ERK MAP kinase pathway mediates the expression of c-fos and c-jun mRNA in PGF2alpha-treated bovine luteal cells.

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