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M. P. Carrasco, G. Asbóth, S. Phaneuf, and A. López Bernal

Prostaglandin F (PGF) has regulatory (mainly luteolytic) effects in the ovary but the mechanism of action is not completely understood. Reverse transcriptase–polymerase chain reaction (RT–PCR) techniques were used to demonstrate the presence of mRNA encoding the PGF receptor (FP receptor) in human granulosa–lutein cells. Specific primers for the amplification of cDNA were designed and yielded a single product of 696 bp corresponding to the FP receptor. The identity of this product was verified by sequencing. Fluprostenol, a selective FP receptor agonist, activated phospholipase C (PLC) and increased intracellular free calcium concentration, confirming the functional activation of the receptor. We have demonstrated by Western blotting that granulosa cells express PLC-β and PLC-γ isoforms. The cells responded to pervanadate with increased PLC activity and increased tyrosine phosphorylation, demonstrating a functional PLC-γ tyrosine kinase pathway. However, fluprostenol did not provoke any detectable tyrosine phosphorylation. Moreover, the effect of fluprostenol was inhibited through protein kinase C stimulation by phorbol 12,13-dibutyrate, and was not affected when cells were treated with phenylarsine oxide, which blocks tyrosine phosphorylation. These results suggest that the FP receptor activates PLC-β rather than PLC-γ isoforms. Fluprostenol-induced activation was pertussis toxin resistant. Granulosa cells express G proteins of the Gq family (resistant to pertussis toxin) and mRNA for both Gαq and Gα11 has been identified by RT–PCR. In conclusion, human granulosa cells have a functional FP receptor the effects of which are mediated through PLC-β activation probably via Gq/11.

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G Asboth, SA Price, J Bellinger, W Ledger, DH Barlow, and AL Bernal

Granulosa cells play an essential role in follicular development and formation of corpora lutea. Many functions of granulosa-lutein cells are controlled by activation of G protein-coupled receptors and the formation of cyclic AMP (cAMP) by adenylyl cyclase. There are at least nine mammalian adenylyl cyclase isoenzymes, which show different sensitivities towards other signalling systems. The aim of this study was to identify the types of adenylyl cyclase present in human granulosa cells and to investigate its functional regulation by G proteins, calcium and the protein kinase C and A pathways. Granulosa cells were obtained from women undergoing IVF. The cells were maintained in primary culture and they consistently expressed mRNA coding for adenylyl cyclase I, III, VI, VII and IX. The signals for adenylyl cyclase V and VIII were more variable among patients and there was no signal for adenylyl cyclase II. The expression of multiple adenylyl cyclase proteins was confirmed by immunochemistry with subtype-specific antibodies. The formation of cAMP in cultured cells was stimulated many times by hCG (EC(50) value 4.2 iu ml(-1)) and by prostaglandin E(2) (PGE(2); EC(50) = 0.75 micromol l(-1)) in a concentration-dependent manner, thus confirming the presence of receptors coupled positively to G(s). The diterpene forskolin, which stimulates all isoforms of adenylyl cyclase except for adenylyl cyclase IX, increased cAMP formation to higher levels than hCG or PGE(2). The strong stimulation by forskolin indicates that adenylyl cyclase IX is unlikely to be the major source of cyclase activity in these cells. Basal and forskolin- or PGE(2)-stimulated adenylyl cyclase activity was amplified 1.5-2.0 times by phorbol-12,13-dibutyrate, indicating that protein kinase C-sensitive enzymes (for example, adenylyl cyclase types IV, V, VI or VII) may be active in the cells. In contrast, hCG-stimulated activity was inhibited (76 +/- 6%) by phorbol ester. Stimulation of G(i) with the alpha-adrenoceptor agonist clonidine inhibited hCG-induced cyclase activity. This finding indicates that adenylyl cyclase II and IV subtypes, which are stimulated by betagamma subunits released from G(i), are not predominant. Increases in intracellular free calcium concentrations by the ionophore A23187, the calcium-ATPase inhibitor thapsigargin or by fluprostenol, a selective prostanoid FP receptor agonist, which is known to open calcium channels in granulosa cells, or removal of calcium by EGTA, had no significant effects on basal or forskolin-stimulated formation of cAMP. These results indicate that subtypes adenylyl cyclase I, III and VIII, which are activated by calcium, and adenylyl cyclase V and VI, which are inhibited by calcium, are not dominant isoforms in granulosa-lutein cells. The protein kinase A inhibitor H89 had no effects on formation of cAMP; this finding rules out the involvement of adenylyl cyclase V and VI subtypes, which are subjected to negative feedback by protein kinase A. These results indicate that adenylyl cyclase VII is the dominant functional isoenzyme in human granulosa-lutein cells.

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A. López Bernal, J. Bellinger, J. M. Marshall, S. Phaneuf, G. N. Europe-Finner, G. Asbóth, and D. H. Barlow

The expression of heterotrimeric (αβγ subunits) GTP-binding regulatory proteins (G proteins) and the activation of G protein-linked receptors in human granulosa cells were investigated. The cells were obtained from stimulated follicles in women undergoing in vitro fertilization and were cultured in serum-supplemented medium. Immunoblotting with specific antibodies showed that granulosa cell membranes express αs, αi3 αi1,2, αq,11 and β subunits. Three antibodies against αo failed to detect this protein. The cells responded to hCG and to prostaglandin E2 with a dose-dependent increase in cAMP formation, confirming the functional activation of Gαs. The α2 adrenoceptor agonist, clonidine, inhibited hCG-stimulated cAMP formation and this effect was blocked with pertussis toxin, thus involving a Gi-type protein, most likely Gαi2. Oxytocin provoked an increase in formation of inositol phosphates and intracellular calcium concentration, which was partly pertussis toxin resistant, providing evidence of Gαq,11 activation. However, a significant component of the response to oxytocin could be blocked by pertussis toxin, indicating Gi-mediated phospholipase C activation (by either αi or βγ subunits). These data demonstrate the presence of G proteins in granulosa cells and suggest a complex regulation of hormonal signalling. The concentration of cAMP in these cells depended on the balance of Gαs:Gαi activation, whereas activation of the inositol phospholipid pathway and rises in intracellular calcium involved both Gq,11 and Gi pathways.