Stimulation of progesterone secretion by cultured human granulosa cells with melatonin and catecholamines

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)LHreduced the effectiveness of H89 in inhibiting progesterone 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 cAMPdependent (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 PKAindependent progesterone synthesis is regulated.

“…Human granulosa cells were isolated as previously described (Webley et al 1988, Abayasekara et al 1993). …”

Section: Isolation Of Human Granulosa Cellsmentioning

confidence: 99%

Progesterone secretion by luteinizing human granulosa cells: a possible cAMP-dependent but PKA-independent mechanism involved in its regulation

Chin¹,

Abayasekara²

2004

“…Granulosa cells were isolated from follicular aspirates using 60% (v/v) Percoll as previously described (Webley et al 1988). For each individual experiment, cells from multiple follicles (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), all from the same patient, were pooled.…”

Section: Isolation and Preparation Of Human Granulosa Cellsmentioning

confidence: 99%

Stimulation of progesterone production in human granulosa-lutein cells by lipoproteins: evidence for cholesterol-independent actions of high-density lipoproteins

Ragoobir

Abayasekara²,

Bruckdorfer³

et al. 2002

Low-density lipoproteins (LDL) have been consistently reported to stimulate ovarian steroidogenesis, apparently by the provision of cholesterol as a steroidogenic substrate. Recent studies suggest that high-density lipoproteins (HDL) can also deliver cholesterol to support progesterone synthesis in human granulosa-lutein cells. Therefore, this study investigated the contributions of (i) cholesterol delivery, (ii) cyclic AMP and (iii) protein kinase C (PKC) in the steroidogenic responses of human granulosa-lutein cells to HDL and LDL. Over a 24-h treatment incubation, HDL stimulated a larger increase in progesterone output than did LDL at equivalent cholesterol concentrations. Moreover, at equal protein concentrations (100 µg protein/ml), HDL doubled progesterone production by cells co-treated with a maximally effective concentration of 22R-hydroxycholesterol, whereas LDL had no effect on the progesterone response to this membrane-permeable sterol. These observations indicate that the progesterone response to HDL is not solely due to the delivery of cholesterol as a steroidogenic substrate. Over 24 h, the stimulation of progesterone synthesis by HDL was additive with the response to a maximally effective concentration of dibutyryl-cAMP, but was unaffected by the downregulation of PKC activity (by chronic pre-treatment with a tumour-promoting phorbol ester). We have concluded that HDL appears to stimulate progesterone production in human granulosa-lutein cells by a mechanism not solely reliant on cholesterol delivery.

“…On arrival, ovarian cells and contaminating blood cells were precipitated from the supernatant follicular fluid and follicular flushing medium (EBSS) by centrifugation at 250 g for 10 min at 4 C. After resuspension in PBS, human granulosa cells were partially purified on 60% (v/v) Percoll as described previously (Webley et al 1988). The number of granulosa cells obtained from a given patient using this protocol ranged between 1·4 and 7·6 10 6 cells.…”

Section: Isolation Of Human Granulosa-lutein Cellsmentioning

confidence: 99%

Relationship between the production of prostaglandins and progesterone by luteinizing human granulosa cells

Fowkes

Chandras²,

Chin³

et al. 2001

Luteinizing granulosa cells synthesize high concentrations of progesterone, prostaglandin (PG) E 2 and PGF 2 . The objective of this study was to explore the relationship between prostaglandin and progesterone output from human granulosa cells as they undergo functional luteinization in culture. Granulosa cells were partially purified from ovarian follicular aspirates and cultured at a density of 10 5 cells/ml in serum-supplemented DMEM:Ham's F 12 medium for 0, 1 or 2 days. Cells were then switched to serum-free medium for 24 h before measuring hormone concentrations in this spent medium by specific radioimmunoassays. Over the first 3 days in culture, PGF 2 and PGE 2 production declined progressively by up to 82 3% coincident with a 55 11% increase in progesterone output. In subsequent experiments, cells were treated for 24 h on the second day of culture with either 0·01 to 10 µM meclofenamic acid or with 10 µM and 100 µM aminoglutethimide. Meclofenamic acid inhibited synthesis of PGF 2 and PGE 2 by up to 70 9% and 64 7% respectively without affecting progesterone output. Likewise, 100 µM aminoglutethimide inhibited progesterone production by 62 6% without affecting concentrations of either PGF 2 or PGE 2 . We have concluded that the progressive decline in prostaglandin production and the rise in progesterone output from luteinizing human granulosa cells occur independently of each other.