The corpus luteum formed after luteinization of follicular cells secretes progesterone under the control of luteinizing hormone (LH). Binding of LH to its G-protein-coupled receptor leads to the activation of the adenylate cyclase/ cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA) signalling pathway. The identification of a new class of cAMP-binding proteins termed ‘guanine nucleotide exchange factors’ (cAMP-GEFs) provides a means by which changes in cAMP could yield actions that are independent of PKA. Hence, in this study, we have explored the hypothesis that steroidogenesis in luteinizing cells is mediated in both a cAMP/PKA-dependent and cAMP-dependent, but PKA-independent, manner. Human granulosa cells were isolated from follicular aspirates of women undergoing assisted conception. Luteinizing human granulosa cells were cultured for up to 3 days in the presence of human (h)LH and the adenylate cyclase activator forskolin in the added presence or absence of increasing doses of the PKA inhibitors H89 (N-[2-(4-bromocinnamylamino)ethyl] 5-isoquinoline) and PKI (myristoylated protein kinase A inhibitor amide 14–22) or the cAMP antagonist, Rp-cAMP. Agonist-stimulated progesterone secretion was inhibited in a dose-dependent manner by the PKA inhibitors and the cAMP antagonist, with decreasing sensitivity as luteinization progressed. Pretreatment of granulosa cells for 4 h with human (h)LH reduced the effectiveness of H89 in inhibiting progester-one secretion. Under basal conditions, cAMP-GEFI expression increased progressively throughout culture, and this could be further enhanced when cells were incubated with increasing doses of LH and forskolin. Furthermore, incubation of cells in the presence of increasing concentrations of the novel cAMP-GEF-specific cAMP analogue, 8 CPT-2 ME-cAMP (8-(4-chloro-phenylthio)-2′-0-methyladenosine-3′,5′-cyclic monophosphate), increased progesterone secretion in a dose-dependent manner. The results show that increases in cAMP generated by LH and forskolin, in addition to activating PKA, also induce increases in cAMP-GEFI protein expression in luteinizing human granulosa cells. In addition, activation of cAMP-GEFI results in increased progesterone secretion. Hence, increases in cAMP lead to the activation of PKA-dependent, as well as PKA-independent but cAMP-dependent (via cAMP-GEFI), signalling mechanisms. Since cAMP-GEFs have the capacity to activate the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB) signalling pathways, these may provide the potential mechanisms by which cAMP-dependent but PKA-independent progesterone synthesis is regulated.

Luteinization of follicular granulosa cells leads to an increase in progesterone secretion that is regulated by luteinizing hormone (LH). LH acts mainly by elevating intracellular cyclic 3′,5′-adenosine monophosphate (cAMP) and activating cAMP-dependent protein kinase (PKA). In this study, we have examined the role of PKA in relation to progesterone output by luteinizing human granulosa cells. Human granulosa cells were obtained by percoll gradient centrifugation of follicular aspirates of patients undergoing oocyte retrieval for assisted conception. Cells were cultured in serum-supplemented medium for up to 3 days in the presence and/or absence of human (h)LH and other cAMP-elevating agents. Spent medium was assayed for cAMP and progesterone content by specific RIA. Cell lysates were collected and assessed for PKA regulatory (R)IIα/catalytic (C)α expression by Western blotting. Although basal progesterone secretion increased progressively throughout culture, cAMP levels remained unchanged. Under basal conditions, PKA RIIα/Cα expression appeared to increase throughout the 3-day culture period. In the presence of hLH and other cAMP-elevating agents, progesterone secretion increased in a dose-dependent manner coincident with an increase in cAMP. However, despite the increase in both progesterone secretion and cAMP accumulation, there was a dose-dependent decrease in both PKA RIIα and Cα expression. Thus, data presented in this study show that increases in progesterone secretion in luteinizing human granulosa cells can be dissociated from increases in PKA expression. This notion implies that progesterone secretion may be regulated by PKA-dependent as well as PKA-independent mechanisms.

Polyunsaturated fatty acids derived from the diet are incorporated into cell membranes where they act as precursors for prostaglandin (PG) synthesis. Linoleic acid (LA; 18:2 n-6) is a major constituent of plant oils and its consumption in Westernized populations is increasing. This study investigated the influence of LA on PG production by the uterus and placenta. Pregnant ewes were fed a control or an LA-enriched diet. Oxytocin (OT) was injected on day 45 (early) or day 133 (late) of gestation to measure the release of 13,14-dihydro-15-keto PGF_2α (PGFM). Ewes were killed on day 46 or day 138 for collection of uterine intercaruncular endometrium and fetal allantochorion. Basal and stimulated PG release from explant cultures was assessed before and after in vitro treatment with OT, lipopolysaccharide (LPS), dexamethasone (DEX) or calcium ionophore (CaI). Expression of cyclooxygenase (COX)-1 and COX-2 was determined by Western blot in endometrium of late-gestation ewes. Circulating PGFM levels in vivo did not differ according to diet but there were highly significant differences in the release of PGs in vitro. Basal production of PGF_2αand PGE₂ by the endometrium and of PGE₂ by the allantochorion were all higher in tissues from LA-supplemented ewes. Endometrial tissues produced more PG following OT and CaI treatment, whereas DEX inhibited production of both PGs at both stages of gestation. In allantochorion collected at day 46 LPS did not significantly alter PGE₂ release and DEX increased output, whereas at day 138 LPS was stimulatory but DEX was inhibitory. These data show that a high-LA diet can significantly increase the ability of both endometrium and placental tissues to produce PGs in vitro. This effect of diet may only become apparent after a sustained period of PG release, so was not seen following the brief pulse caused by OT treatment in vivo. As COX protein levels were unaltered, the main influence was likely to be via conversion of LA to arachidonic acid, providing an increased supply of precursor. These results support previous studies which suggest that alterations in dietary polyunsaturated fatty acids may influence the time of labour.