Characterization of adenylyl cyclases in cultured human granulosa cells

Abstract

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 dominan...

Distribution of Adenylyl Cyclases in the Rat Ovary by Immunofluorescence Microscopy

“…No ACI expression was observed in thecal cells. The observed expression of ACI, ACIV, and ACVIII in this study is consistent with that seen in cultured human granulosa cells obtained from antral follicles (Asbóth et al, 2001). However, unlike in the cultured human granulosa cells (Asbóth et al, 2001), where no staining for ACII was observed, we observed specific ACII staining in granulosa cells, which appear to increase in intensity in response to eCG administration.…”

“…The observed expression of ACI, ACIV, and ACVIII in this study is consistent with that seen in cultured human granulosa cells obtained from antral follicles (Asbóth et al, 2001). However, unlike in the cultured human granulosa cells (Asbóth et al, 2001), where no staining for ACII was observed, we observed specific ACII staining in granulosa cells, which appear to increase in intensity in response to eCG administration. As the stimulation of ACII is primarily regulated by the simultaneous presence of both G sα and G βγ subunits (Gao and Gilman, 1991), G protein‐coupled receptor agonists might play an important role in the regulation of this enzyme in granulosa and luteal cells.…”

“…Perhaps, the observed variations in ACII and ACIII expression between human and rat could be due to differences between species, differences in methodology, or both. One other possibility is that during cell culture for 48 hr (Asbóth et al, 2001), the expression of these enzymes could have been affected. ACIII is primarily associated with the thecal cells and in the smooth muscle cells of the blood vessels.…”

“…Existence of multiple isoforms of ACs has been previously shown in human granulosa cells (Asbóth et al, 2001), CL of the cow (Mamluk, et al, 1999), and cumulus cells (Lastro et al, 2006). Transcripts for various ACs in the oocytes and cumulus cells and the expression of ACIII protein have been detected in Bouin's‐fixed and paraffin‐embedded rodent ovaries (Horner et al, 2003).…”

Adenylyl cyclase isoforms in rat testis and spermatozoa from the cauda epididymidis

“…The presence of multiple tmAC transcripts in the testis is not surprising, given the heterogeneous cell population, and is consistent with findings in other tissues. For example, in the brain (Mons et al 1998), nephron (Bek et al 2001), lung (Jourdan et al 2001) and myometrium and cultured granulosa cells (Price et al 2000;Asboth 2001), multiple tmAC are localized in distinct regions and cells. This suggests that the synthesis of cAMP in the testis is multiply regulated, as it is in other tissues.…”