Calcium Ions Positively Modulate Follicle-Stimulating Hormone- and Exogenous Cyclic 3′,5′-Adenosine Monophosphate-Driven Transcription of the P450scc Gene in Porcine Granulosa Cells*

Abstract

Given the evident modulation of FSH-induced steroidogenesis by Ca2+ in granulosa cells, we here test the hypothesis that Ca2+ controls expression of the enzymatically rate-limiting cytochrome P450(scc) (CYP11A) gene. To test this postulate, we quantitated the ability of Ca2+ to regulate: 1) transcriptional activity of a transiently transfected luciferase reporter gene driven by a 2.32-kb 5'-upstream fragment of the porcine P450(scc) gene promoter region; and 2) accumulation of endogenous P450(scc) transcripts in primary monolayer cultures of porcine granulosa cells. To this end, granulosa cells were stimulated for 4 h with FSH (15 ng/ml, NIDDK-oFSH-20) or 8-Bromo-cAMP (8 Br-cAMP, 1 mM) in serum-free medium containing either 1.8 mM Ca2- or no added Ca2+ with 100 microM EGTA or 100 microM CoCl2. In the presence of extracellular Ca2+, FSH and 8 Br-cAMP stimulated expression of the transfected P450(scc) promoter-reporter fusion construct by 5.6 +/- 1.1 and 3.6 +/- 0.67-fold, respectively over Ca2+-containing unstimulated control (P < or = 0.04, n = 5-6 experiments). The foregoing two agonists augmented 4-h progesterone production by cultured granulosa cells by 1.8 +/- 0.11 and 1.6 +/- 0.16-fold, respectively (P < or = 0.001 for FSH and P < or = 0.01 for 8 Br-cAMP). FSH and 8 Br-cAMP also significantly elevated endogenous P450(scc) transcript levels as measured by homologous solution-hybridization RNase protection assay; i.e. by 3.1 +/- 0.49 and 2.9 +/- 0.45-fold, respectively (P < or = 0.001). In Ca2+-free/EGTA-supplemented medium, basal luciferase reporter-gene activity and endogenous P450(scc) messenger RNA accumulation in granulosa cells declined to 34 +/- 12% and 78 +/- 12%, respectively, of corresponding values in control (unstimulated Ca2+-containing) cultures. Extracellular Ca2+ deprivation inhibited the stimulatory effect of FSH (and 8 Br-cAMP) on P450(scc) promoter-luciferase reporter expression to 58 +/- 30% (and 58 +/- 23%), and restrained endogenous P450(scc) message accumulation to 86 +/- 15% (and 96 +/- 18%) of the value in Ca2+-containing control. Extracellular Ca2+ withdrawal suppressed FSH (and 8 Br-cAMP)-driven progesterone production over 4 h to basal levels but did not alter FSH-stimulated cAMP accumulation by granulosa cells. Ca2+-deprived cells exposed to serum-containing media regained P450(scc) responsiveness to both agonists. Antagonism of cellular uptake of Ca2+ and other divalent cations via administration of cobalt chloride (100 microM) inhibited FSH and 8 Br-cAMP's stimulation of endogenous (but not exogenous promoter-driven) P450(scc) gene expression. In contrast, granulosa-cell concentrations of messenger RNA's encoding sterol-carrier protein-2 (SCP-2) and the low density lipoprotein receptor were not altered by Ca2+ withdrawal. In summary, uptake of extracellular Ca2+ by porcine granulosa cells significantly potentiates transactivation of the endogenously expressed and exogenously transfected P450(scc) gene by FSH and 8 Br-cAMP. The agonistic impact of Ca2+ on P450(scc) promoter activi...

Effect of basal lamina of ovarian follicle on T- and L-type Ca2+currents in differentiated granulosa cells

“…Earlier studies showed that progesterone synthesis in granulosa cells of several species was reduced in Ca 2ϩ -deficient incubation media (6,22,61,63), and a recent study demonstrated that the abundance of messenger RNA of cytochrome P450scc (the enzyme that catalyzes the metabolism of cholesterol to pregnenolone) in porcine granulosa cells was reduced in Ca 2ϩ -deficient medium (37). Therefore, one of the mechanisms by which Ca 2ϩ modulates steroid hormone synthesis is the increase in the transactivation of the cytochrome P450scc gene (37).…”

“…Earlier studies showed that progesterone synthesis in granulosa cells of several species was reduced in Ca 2ϩ -deficient incubation media (6,22,61,63), and a recent study demonstrated that the abundance of messenger RNA of cytochrome P450scc (the enzyme that catalyzes the metabolism of cholesterol to pregnenolone) in porcine granulosa cells was reduced in Ca 2ϩ -deficient medium (37). Therefore, one of the mechanisms by which Ca 2ϩ modulates steroid hormone synthesis is the increase in the transactivation of the cytochrome P450scc gene (37). Because, in granulosa cells, the suppression of uptake of extracellular Ca 2ϩ resulted in the attenuation of progesterone production (6,22,61,63) and transcription of the cytochrome P450scc gene, and because basal lamina suppressed inward Ca 2ϩ current in differentiated granulosa cells (in the present study), it would be expected that basal lamina would decrease progesterone production in differentiated chicken granulosa cells.…”

Multiple facets of follicle-stimulating hormone receptor function

“…In the first scenario, cAMP appears to increase intracellular calcium by promoting calcium mobilization from intracellular stores, an effect that could be mediated by cAMP stimulation of phospholipase-Ce, whereas in the second, different G proteins (e.g., G aq/11 protein) or the b/c heterodimer of G as might be involved [41]. Further, in some models, cAMP-independent Ca 2+ influx requires the presence of extracellular calcium [59,61]. In this vein, it has been recently shown that FSH-promoted Ca 2+ influx in rat Sertoli cells is mediated by a distinct G ah /PLC-d1 pathway and that this effect is dependent on intracellular PLC-d1-mediated generation of inositol 1,4,5 triphosphate (IP3) but independent of intracellular Ca 2+ release [44].…”

“…In this vein, it has been recently shown that FSH-promoted Ca 2+ influx in rat Sertoli cells is mediated by a distinct G ah /PLC-d1 pathway and that this effect is dependent on intracellular PLC-d1-mediated generation of inositol 1,4,5 triphosphate (IP3) but independent of intracellular Ca 2+ release [44]. Whatever the mechanism(s) involved in FSH-dependent Ca 2+ influx, Ca 2+ -mediated signaling seems to be important for functional differentiation of granulosa cells as suggested by recent data showing that intracellular Ca 2+ positively modulates FSH-driven transactivation of the cytochrome P450 SCC gene (cyp11A) in porcine granulosa cells [61], that Ca 2+ /calmodulindependent protein kinase IV (CaMK IV) is involved in basal cyp11A gene expression in cultured granulosa-lutein cells [62], and that fertility is markedly reduced in CaMK IV-deficient female mice [63].…”

Expression of Osteopontin (OPN) mRNA in Bovine Ovarian Follicles and Corpora Lutea

“…Based on its high content in sialic acid and acidic amino acids, a marked calcium‐binding capacity of the OPN molecule has been predicted and it indeed has been shown to bind approximately 50 atoms of calcium per molecule (Chen et al 1992) and may act as a chelator of extracellular Ca 2+ and thereby influence calcium homeostasis within the ovary. A tightly controlled calcium flux between the extra‐ and intracellular space is critical for nearly all aspects of normal ovarian function, especially for processes like steroidogenesis, follicular atresia and luteolysis (Jayes et al 2000; Makrigiannakis et al 2000; Niswender et al 2000).…”