Calcium inhibits ovarian steroidogenesis in the blowfly Phormia regina

Abstract

Calcium is frequently involved in the stimulation of steroidogenesis in gonads and endocrine glands, generally in association with cAMP. However, our present observations show that it has the opposite effect in the ovary of the blowfly Phormia regina. Our in vitro experiments first showed that extracellular calcium does not play a role during the stimulation of steroidogenesis in fly ovaries; indeed steroidogenesis was activated in vitro as efficiently in a medium with or without calcium, either by pharmacological compounds mimicking cAMP signaling or by active brain extracts. When calcium was experimentally introduced into biosynthetic cells by ionophores or liberated from internal stores by thapsigargin, it did not activate, but clearly inhibited both basal and acute steroidogenesis respectively in previtellogenic and in vitellogenic ovaries. Our experiments also demonstrated that calcium decreases cAMP concentrations in the ovaries of Phormia, by stimulating its degradation, without modifying its biosynthesis. Moreover, inhibitors of calcium-calmodulin phosphodiesterases (PDEs) increased steroid biosynthesis in vitro, whereas inhibitors of calciuminsensitive PDEs did not. These data thus demonstrate that, in blowfly ovaries, calcium ions inhibit cAMPstimulated steroidogenesis by activating a calmodulinsensitive (type I) PDE.

Control of ovarian steroidogenesis by insulin-like peptides in the blowfly (Phormia regina)

“…More importantly, they were found to have no toxic effect, as judged by the fact that, when used alone, they did not decrease basal steroidogenesis, this parameter being easily measurable and extremely sensitive to any entry of calcium (Manière et al 2002), which would be inevitable after cellular deteriorations. Thus, our observations corroborated that several components of the insulin-like signaling system, namely ILP receptor, PI3K and PKB, are involved in the control of ovarian steroidogenesis stimulated by bovine insulin in blowflies.…”

“…Moreover, experiments with the cAMP antagonist adenosine-3 , 5 -cyclic monophosphorothioate, Rpisomer (Rp-cAMPS), used at doses known to be effective on cAMP-stimulated biosynthesis (Manière et al 2000(Manière et al , 2002, did not decrease insulin-stimulated steroidogenesis. Amounts of ecdysteroids secreted by ovaries simultaneously treated with 10 5 M insulin and with either 10 4 M or 10 3 M Rp-cAMPS (64·2 10·4 pg (n=4) and 72·0 5·1 pg (n=6) respectively) were not significantly different from those secreted by ovaries treated with 10 5 M insulin only (63·2 8·2 pg; n=6, P.0·05), but were different from those of untreated controls (28·1 3·1 pg; n=14, P,0·05).…”

Inhibition of ovarian steroidogenesis by cyclic GMP in a fly

“…Incubations were made at 26 1 C in Grace's insect culture medium (Gibco, Life Technologies-France, CergyPontoise, France). Some experiments were also carried out using a defined saline solution without calcium (128 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 1 mM Na 2 HPO 4 , 0·34 mM KH 2 PO 4 , 26·8 mM glucose, see Manière et al 2002). Our biotests were made as previously described (Manière et al 2000): in general, one ovary from an individual pair was tested for its response to a precise pharmacological treatment, whereas the contralateral ovary was used as its corresponding untreated control.…”

“…In our previous studies on the control of steroidogenesis and vitellogenesis in our model, the blowfly Phormia regina, we have demonstrated that ovarian biosynthesis of ecdysteroids is stimulated by several (unidentified) brain factors, triggering either cAMP-dependent or cAMPindependent signaling cascades (Manière et al 2000) and is also subjected to an unusual inhibitory control by calcium (Manière et al 2002). During these investigations, we also observed that 3-isobutyl-1-methylxanthine (IBMX), a broad spectrum inhibitor of phosphodiesterases (PDEs), failed to mimic the steroidogenic effects of cAMP analogs or of forskolin, whereas some specific PDE inhibitors were able to do so (Manière et al 2002).…”

“…During these investigations, we also observed that 3-isobutyl-1-methylxanthine (IBMX), a broad spectrum inhibitor of phosphodiesterases (PDEs), failed to mimic the steroidogenic effects of cAMP analogs or of forskolin, whereas some specific PDE inhibitors were able to do so (Manière et al 2002). Attempts to elucidate this paradox have led us to the hypothesis that IBMX could have simultaneously two opposite pharmacological effects on steroid biosynthesis in blowfly ovaries, a stimulatory effect via the cAMP-signaling pathway and an inhibitory effect, possibly via cGMP involvement.…”