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

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), a metabolite of DDT (1,1-dichlorodiphenyltrichloroethane), is a persistent hormonally active environmental toxicant that has been found in human serum and follicular fluid. The objective of this study was to determine whether DDE can alter free calcium ion concentrations in the cytosol ([Ca(2+)](cyt)) of human granulosa cells. Changes in [Ca(2+)](cyt) in single cells loaded with Fura-2 were studied using a dynamic digital Ca(2+) imaging system. At a concentration of 100 ng/ml, DDE stimulated small elevations of [Ca(2+)](cyt) accompanied by Ca(2+) oscillations. At 1 microg DDE/ml, there was a biphasic Ca(2+) response with marked elevations of [Ca(2+)](cyt) over time. In Ca(2+)-free medium, cells showed an initial small elevation of [Ca(2+)](cyt), which was magnified after addition of Ca(2+) to the medium. Washing the cells after DDE treatment failed to remove the elevated [Ca(2+)](cyt) and oscillations, both of which were eliminated by addition of EGTA. ATP also induced [Ca(2+)](cyt) elevations and oscillations, and these effects were potentiated when DDE was added. FSH induced transient [Ca(2+)](cyt) elevations, whereas hCG caused a prolonged elevation and marked oscillations in [Ca(2+)](cyt). These results suggest that DDE at concentrations normally found in human tissues induces elevations in [Ca(2+)](cyt) in granulosa-lutein cells. Our data therefore highlight a novel mechanism through which DDE can alter endocrine homeostasis and possibly act as an endocrine toxicant.

Section: Effects Of Fsh and Hcgmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Dichlorodiphenylchloroethylene Elevates Cytosolic Calcium Concentrations and Oscillations in Primary Cultures of Human Granulosa-Lutein Cells1

Younglai

Kwan

et al. 2004

“…However, it is also expected that cellular processes activated by purinergic stimulation, and consequent release of intracellular Ca 2ϩ might have important effects on the metabolism of both the cumulus cell and oocyte compartments. It is important to mention that steroidogenesis, a principal functional role of granulosa cells, is affected by changes in the levels of both intracellular Ca 2ϩ and Cl Ϫ ions [52,53]. Also, it has been shown that intracellular Ca 2ϩ has a central role in the regulation of important processes in the oocyte [54].…”

Section: Electrical Characteristics Of Mammalian Granulosa Cells and mentioning

confidence: 99%

Ionic Currents Activated via Purinergic Receptors in the Cumulus Cell-Enclosed Mouse Oocyte1

Arellano

Martı́nez-Torres

Garay

2002

Several chemical signals synthesized in the ovary, including neurotransmitters, have been proposed to serve as regulators of folliculogenesis, however, their mechanisms of action have not been completely elucidated. Here, electrophysiological and molecular biology techniques were used to study responses generated via purinergic stimulation in cultured mouse cumulus cell-enclosed oocytes (CEOs). Application of extracellular ATP elicited depolarizing responses in CEOs. Using the voltage clamp technique by impaling oocytes with two microelectrodes, we determined that these responses were mainly due to activation of two distinct ionic currents. The first corresponded to the opening of Ca 2؉ -dependent Cl Ϫ channels (I Cl(Ca) ) and the second to the opening of Ca 2؉ -independent channels that are permeable to Na ؉ (I c؉ ). The potency order for different nucleotides (50 M) was UTP Ͼ ATP Ͼ 2meS-ATP Ͼ ADP, and ␣,␤me-ATP and adenosine were found to be inactive. Suramin (100 M) blocked the response elicited by ATP or UTP. In addition, voltage dependent K ؉ currents activated by depolarization of CEOs were characterized. All CEO ionic currents recorded from the oocyte were completely inhibited by octanol (1 mM), a gap junction blocker. Thus, purinergic responses and K ؉ currents originate mainly in the membrane of cumulus cells. Transcripts of the purinergic receptor P2Y 2 subtype were amplified by polymerase chain reaction from the cDNA of granulosa cells or cumulus cells. This study shows that P2Y 2 receptors are expressed in CEOs, and that their stimulation opens at least two different types of ion channels. Both the ion channels and the receptors seemed to be located in the cumulus cells, which transmit their corresponding electrical signals to the oocyte via gap junction channels.

“…This increase in Ca 2ϩ results in the formation of inositol triphosphate, which triggers the PKC signal cascade [33]. An increase in the uptake of extracellular Ca 2ϩ potentiates the effects of FSH and 8-Br-cAMP on transcription of the P450scc gene and progesterone synthesis in primary cultures of porcine granulosa cells [34]. Therefore, it is reasonable to speculate that the low concentrations of Cd 2ϩ used in the present study may be sufficient to mimic the effects of Ca 2ϩ , resulting in stimulation of the steroidogenic pathway and synthesis of progesterone in the JC-410 cells.…”

Section: Discussionmentioning

confidence: 99%

Cadmium Stimulates Transcription of the Cytochrome P450 Side Chain Cleavage Gene in Genetically Modified Stable Porcine Granulosa Cells1

Smida

Valderrama

Agostini³

et al. 2004

We investigated the effects of cadmium (Cd2+) on transcription of the cytochrome p450 side chain cleavage (p450scc) gene and on progesterone synthesis in stable granulosa cells. We used the stable porcine granulosa cell line, JC-410, genetically modified to express a luciferase genomic construct carrying 2320 base pairs (bp) of the p450scc gene promoter (p450scc-2320-LUC). A construct containing only the luciferase gene, pOLUC, was used as a promoterless control. At 1 microM, cadmium chloride (CdCl2) increased transient expression of p450scc-2320-LUC in JC-410 cells by 2.6-fold after 24-h incubation. A similar pattern of stimulation by CdCl2 was observed in cells transiently transfected with a luciferase genomic construct carrying 100 bp of the p450scc gene promoter p450scc-100-LUC, whereas no stimulation by CdCl2 was observed in cells transfected with pOLUC. At 0.6, 1, and 2 microM, CdCl2 stimulated the activity of the p450scc-2320-LUC promoter in a dose-related fashion by 1.58-, 3.19-, and 2.67-fold, respectively, after 24-h incubation. Northern blot analysis showed that CdCl2 at 0.1, 1, 2, and 3 microM increased p450scc mRNA levels by 3.13-, 1.38-, 1.61-, and 1.57-fold, respectively, after 24-h incubation. After 48-h incubation, CdCl2 at 0.6, 1, and 2 microM further increased p450scc mRNA levels by 3.43-, 2.08-, and 2.4-fold, respectively. At 1, 2, and 3 microM, CdCl2 inhibited progesterone synthesis to 0.48-, 0.38-, and 0.29-fold, respectively. After 48-h incubation, CdCl2 at 0.1 microM stimulated progesterone synthesis by 1.6-fold. We conclude that Cd2+ has a dual action in stable porcine granulosa cells: Low concentrations activate, whereas high concentrations inhibit, expression of the p450scc gene and progesterone synthesis. The stimulatory effect of Cd2+ appears to be mediated via a cis-acting element located 100 bp upstream of the p450scc gene transcription start site.