Antisteroidogenic actions of hydrogen peroxide on rat Leydig cells

It has been well known that reactive oxygen species (ROS) are produced in the steroidogenic pathway and spermatozoa. H2O2, one of ROS produced by spermatozoa, appears to be a primary toxic agent. In the present study, we examined the effects of H2O2 on the basal and evoked-testosterone release from primary Leydig cells, the protein expressions of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein were also investigated. Our preparation was found to contain approximately 87% Leydig cells and very few macrophages. The results demonstrated that H2O2 (>1 x 10(-4) M) significantly inhibited the basal and hCG-stimulated testosterone release. H2O2 abolished forskolin- or 8-Br-cAMP-evoked testosterone release. In the presence of pregnenolone, progesterone, or androstenedione, the inhibitory effect of H2O2 on testosterone release was prevented. H2O2 also inhibited pregnenolone production in the presence of trilostane (an inhibitor of 3beta-hydroxysteroid dehydrogenase), therefore diminished the activity of P450scc in Leydig cells. In addition to the inhibition of hormone secretion, H2O2 also regulated steroidogenesis by diminishing protein expression of StAR. These results suggest that H2O2 acts directly on rat Leydig cells to diminish testosterone production by inhibiting P450scc activity and StAR protein expression.     

Arachidonic acid metabolism and casein secretion in lactating rabbit mammary epithelial cells: effects of inhibitors of prostaglandins and leukotrienes synthesis

The metabolism of arachidonic acid (AA) in fragments of lactating rabbit mammary glands in vitro was studied by considering the distribution of 13-[14C]AA in the cells, and the effects of inhibitors of cyclooxygenase and lipoxygenase pathway on the basal and prolactin (PRL)-stimulated casein secretion. 13-[14C]AA was incorporated in all classes of lipids and PRL increased transiently the percentage of free fatty acid after 1 and 5 min. Ten microM ETYA (5,8,11,14-Eicosatetraynoic acid), a tetrayne analogue of AA inhibited prostaglandins F2 alpha (PGF2 alpha) production but not leukotrienes B4 and C4 (LTB4 and LTC4) production and increased basal casein secretion. 10(-4) M DCHA (Docosahexaenoic acid) a competitive inhibitor of prostaglandin-synthetase inhibited PGF2 alpha production but did not affect basal nor PRL-stimulated casein secretion. Fourteen microM indomethacin inhibited PGF2 alpha and LTC4 production and PRL-stimulated casein secretion. Ten microM NdgA (nordihydroguaiaretic acid) an inhibitor of lipoxygenase pathway, inhibited LTB4 and LTC4 production, increased basal level of casein secretion and inhibited PRL-stimulated casein secretion. Hundred microM caffeic acid, an inhibitor of glutathione-S-transferase (GST), a class of enzymes implied in the transformation of LTA4 into LTC4, had the same effect that NDGA on basal and PRL-stimulated casein secretion. These findings show that inhibitors of AA metabolites alter casein secretion.     

PGE2 release is independent of upregulation of Group V phospholipase A2 during long-term stimulation of P388D1 cells with LPS

P388D1 cells release arachidonic acid (AA) and produce prostaglandin E2 (PGE2) upon long-term stimulation with lipopolysaccharide (LPS). The cytosolic Group IVA (GIVA) phospholipase A2 (PLA2) has been implicated in this pathway. LPS stimulation also results in increased expression and secretion of a secretory PLA2, specifically GV PLA2. To test whether GV PLA2 contributes to PGE2 production and whether GIVA PLA2 activation increases the expression of GV PLA2, we utilized the specific GIVA PLA2 inhibitor pyrrophenone and second generation antisense oligonucleotides (AS-ONs) designed to specifically inhibit expression and activity of GV PLA2. Treatment of P388D1 cells with antisense caused a marked decrease in basal GV PLA2 mRNA and prevented the LPS-induced increase in GV PLA2 mRNA. LPS-stimulated cells release active GV PLA2 into the medium, which is inhibited to background levels by antisense treatment. However, LPS-induced PGE2 release by antisense-treated cells and by control cells are not significantly different. Collectively, the results suggest that the upregulation of GV PLA2 during long-term LPS stimulation is not required for PGE2 production by P388D1 cells. Experiments employing pyrrophenone suggested that GIVA PLA2 is the dominant player involved in AA release, but it appears not to be involved in the regulation of LPS-induced expression of GV PLA2 or cyclooxygenase-2.     

Group IVA phospholipase A2 regulates testosterone biosynthesis by murine Leydig cells and is required for timely sexual maturation

In the present paper, we report that PLA2G4A (Group IVA phospholipase A2) is important in the development and function of rodent testes. Interstitial cells of rat testes had high PLA2 (phospholipase A2) activity that was very sensitive to the PLA2G4A-preferential inhibitor AACOCF3 (arachidonyl trifluoromethyl ketone). PLA2G4A protein was expressed primarily in the interstitial cells of wild-type mouse testes throughout maturation. Although Pla2g4a knockout (Pla2g4a-/-) male mice are fertile, their sexual maturation was delayed, as indicated by cauda epididymal sperm count and seminal vesicle development. Delayed function of Pla2g4a-/- mice testes was associated with histological abnormalities including disorganized architecture, swollen appearance and fewer interstitial cells. Basal secretion of testosterone was attenuated significantly and steroidogenic response to hCG (human chorionic gonadotropin) treatment was reduced in Pla2g4a-/- mice compared with their Pla2g4a+/+ littermates during the sexual maturation period. Chemical inhibition of PLA2G4A activity by AACOCF3 or pyrrophenone significantly reduced hCG-stimulated testosterone production in cultured rat interstitial cells. AACOCF3 inhibited forskolin- and cAMP analogue-stimulated testosterone production. These results provide the first evidence that PLA2G4A plays a role in male testes physiology and development. These results may have implications for the potential clinical use of PLA2G4A inhibitors.     

Thyrotropin stimulates progesterone secretion by luteal cells by activation of the cAMP/protein kinase A signaling system: a potential involvement of protein kinase C

Although the corpus luteum (CL) is not known as a target tissue for thyrotropin (TSH), this hormone increases progesterone production by porcine luteal cells cultured in vitro. In this study we investigated the optimal conditions for TSH-stimulated progesterone secretion as well as the involvement of protein kinase A (PKA) and protein kinase C (PKC) in the mechanism of TSH action on porcine luteal cells. To study the PKA and PKC signaling mechanisms, luteal cells collected from mature CL were incubated with the inhibitor of PKA and potent activators of both kinases: PKA-forskolin and PKC-phorbol ester 12-myriistate-13-acetate (PMA). The PKA inhibitor totally suppressed progesterone production in TSH alone, forskolin alone and in TSH plus forskolin-stimulated luteal cells. Forskolin increased basal (P < 0.05) and TSH-stimulated (P < 0.05) progesterone secretion and cAMP accumulation (P < 0.05). Forskolin and PMA added together to control (non-TSH-treated) luteal cells had an additive effect on progesterone production. In TSH-treated cells, the effect of PMA was statistically significant but did not show an additive effect with forskolin. Further PMA did not affect cAMP accumulation in control and TSH-treated luteal cells. Treatment of control and TSH-treated luteal cells with forskolin and PMA together showed the same increase in cAMP accumulation as with forskolin alone. This is the first demonstration that TSH acts on luteal cell steroidogenesis by activation of the cAMP/PKA second messenger system and also that the PKC signaling pathway may be involved in luteal TSH action on the corpus luteum.     

The regulation of steroidogenesis by opioid peptides in porcine theca cells

The present study was designed to investigate basal and LH-induced steroidogenesis in porcine theca cells from large follicles in response to various concentrations (1-1000 nM) of mu opioid receptor agonists (beta-endorphin, DAMGO, FK 33-824), delta receptor agonists (met-enkephalin, leu-enkephalin, DPLPE) and kappa receptor agonists (dynorphin A, dynorphin B, U 50488). Agonists of mu opioid receptors suppressed basal androstenedione (A4), testosterone (T) and oestradiol-17beta (E2) secretion and enhanced LH-induced A4 and T release by theca cells. The inhibitory effect of the agonists on E2 secretion was abolished in the presence of LH. All delta receptor agonists depressed basal progesterone (P4) output. However, the influence of these agents on LH-treated cells was negligible. Among delta receptor agonist used only leu-enkephalin and DPLPE at the lowest concentrations inhibited basal A4 release. The presence of LH in culture media changed the influence of these opioids from inhibitory to stimulatory. Similarly, DPLPE reduced T secretion by non-stimulated theca cells and enhanced T secretion of stimulated cells. All of delta agonists inhibited basal E2 secretion and unaffected its release from LH-treated theca cells. Agonists of kappa receptors inhibited basal, non-stimulated, P4 secretion and two of them (dynorphin B, U 50488) potentiated LH-induced P4 output. Basal A4 and T release remained unaffected by kappa agonist treatment, but the cells cultured in the presence of LH generally increased both androgen production in response to these opioids. Basal secretion of E2 was also suppressed by kappa agonists. This inhibitory effect was not observed when the cells were additionally treated with LH. In view of these findings we suggest that opioid peptides derived from three major opioid precursors may directly participate in the regulation of porcine theca cell steroidogenesis.     

Involvement of 5-lipoxygenase metabolites in ACTH-stimulated corticosteroidogenesis in rat adrenal glands

Various lipoxygenase (LO) products of arachidonic acid (AA) have been found to have potent biological activities and modulate physiological processes in various cells including endocrine cells. However, no studies concerning LO products in adrenocortical cells have been reported. The present study was performed to investigate LO products in rat adrenocortical cells and its role in ACTH-stimulated adrenal steroidogenesis. LO metabolites produced in ACTH-stimulated rat adrenocortical cells prelabeled with [3H]AA was analyzed by reverse phase and straight phase HPLC and two 5-LO products, 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4) were identified. ACTH-induced 5-HETE and LTB4 production in adrenal cells was dose dependently inhibited by AA861, a specific inhibitor of 5-LO. AA861 reduced ACTH-stimulated corticosteroid production without any change in cyclic AMP formation, while indomethacin did not affect both corticosteroid and cyclic AMP production. Reduced steroidogenesis by AA861 was reversed by the addition of 5-hydroperoxyeicosatetraenoic acid (5-HPETE). Also exogenously added 5-HPETE dose dependently augmented ACTH-stimulated corticosteroid production without any concomitant change in cyclic AMP production. However, 5-HETE and LTB4 had no such effect. These results indicate that 5-LO pathway is present in rat adrenocortical cells and its metabolites, most likely 5-HPETE, may play an important role in adrenal steroidogenesis.     

Regulation of lung surfactant secretion by phospholipase A2

Arachidonic acid has been shown to stimulate lung surfactant secretion from alveolar epithelial type II cells. To identify the (phospho)lipases responsible for generating arachidonic acid during lung surfactant secretion, the effects of various (phospho)lipase inhibitors on phosphatidylcholine (PC) secretion from rat alveolar type II cells were investigated. N-(p-amylcinnamoyl)anthranilic acid (ACA), a general inhibitor of phsopholipase A2 (PLA2), inhibited ATP-stimulated PC secretion in a dose-dependent manner. ACA also blocked PC secretion from type II cells stimulated by other secretagogues including phorbol 12-myristate 13-acetate, Ca2+ ionophore A23187 and terbutaline, indicating that PLA2 acts at a late step distal to the generation of second messengers. To determine which PLA2 isoform(s) is involved in lung surfactant secretion, selective inhibitors to different types of PLA2 were used to inhibit PLA2 activity in type II cells. The cytosolic PLA2 (cPLA2) inhibitor, arachidonyl trifluoromethyl ketone, was found to inhibit ATP-stimulated PC secretion, whereas the secretory PLA2 inhibitors, oleoyloxyethylphosphocholine, aristolochic acid, or p-bromophenacyl bromide, and the Ca2+-independent PLA2 inhibitors, palmitoyl trifluoromethyl ketone, or haloenol lactone suicide substrate, had no effect. In addition to PLA2, arachidonic acid is released from diacylglycerol (DAG) by DAG and monoacylglycerol lipases. The DAG lipase inhibitor, RHC-80267 also blocked ATP-stimulated PC secretion. The results suggest that both pathways for generating arachidonic acid via cPLA2 and DAG lipase may participate in lung surfactant secretion.     

Role of mitogen-activated protein kinase-mediated cytosolic phospholipase A2 activation in arachidonic acid metabolism in human eosinophils.

The objective of this investigation was to determine the role of secretory and cytosolic isoforms of phospholipase A(2) (PLA(2)) in the induction of arachidonic acid (AA) and leukotriene synthesis in human eosinophils and the mechanism of PLA(2) activation by mitogen-activated protein kinase (MAPK) isoforms in this process. Pharmacological activation of eosinophils with fMLP caused increased AA release in a concentration (EC(50) = 8.5 nM)- and time-dependent (t(1/2) = 3.5 min) manner. Both fMLP-induced AA release and leukotriene C(4) (LTC(4)) secretion were inhibited concentration dependently by arachidonic trifluoromethyl ketone, a cytosolic PLA(2) (cPLA(2)) inhibitor; however, inhibition of neither the 14-kDa secretory phospholipase A(2) by 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propanephosphonic acid nor cytosolic Ca(2+)-independent phospholipase A(2) inhibition by bromoenol lactone blocked hydrolysis of AA or subsequent leukotriene synthesis. Pretreatment of eosinophils with a mitogen-activated protein/extracellular signal-regulated protein kinase (ERK) kinase inhibitor, U0126, or a p38 MAPK inhibitor, SB203580, suppressed both AA production and LTC(4) release. fMLP induced phosphorylation of MAPK isoforms, ERK1/2 and p38, which were evident after 30 s, maximal at 1-5 min, and declined thereafter. fMLP stimulation also increased cPLA(2) activity in eosinophils, which was inhibited completely by 30 microM arachidonic trifluoromethyl ketone. Preincubation of eosinophils with U0126 or SB203580 blocked fMLP-enhanced cPLA(2) activity. Furthermore, inhibition of Ras, an upstream GTP-binding protein of ERK, also suppressed fMLP-stimulated AA release. These findings demonstrate that cPLA(2) activation causes AA hydrolysis and LTC(4) secretion. We also find that cPLA(2) activation caused by fMLP occurs subsequent to and is dependent upon ERK1/2 and p38 MAPK activation. Other PLA(2) isoforms native to human eosinophils possess no significant activity in the stimulated production of AA or LTC(4).     

Oxytocin and vasopressin have no effect on progesterone production and cyclic AMP accumulation by rat luteal cells in vitro

Enzymically dispersed luteal cells obtained from PMSG-hCG-treated immature pseudopregnant rats were incubated with oxytocin and vasopressin. In response to increasing doses of hCG the rat luteal cells produced progesterone and accumulated intracellular cAMP in a dose-dependent manner. A neuropeptide GnRH agonist (4 X 10(-6) M) produced a significant inhibition of hCG-stimulated progesterone production and of accumulation of intracellular cAMP. However, neither the basal nor the hCG-stimulated rate of progesterone production and level of intracellular cAMP was affected by the neurohypophysial peptides tested. Therefore, it is concluded that oxytocin and vasopressin do not have a direct action on steroidogenesis by rat luteal cells.     

Different actions of noradrenaline and nitric oxide on the output of prostaglandins and progesterone in cultured bovine luteal cells

The effects of noradrenaline (NA) and nitric oxide (NO) on prostaglandins (PGs) and progesterone (P4) secretion during the development of the bovine corpus luteum (CL) were investigated. Bovine luteal cells of early and mid-cycle CL were cultured for 20 to 24 h in medium containing 10% calf serum, washed, and treated with NA or nitrergic agents for an additional 16 h in a serum-free medium. NA (10(-5) M) stimulated P4 from early and mid-cycle CL by 238% and 154% (P < 0.01), respectively. Moreover, although NA induced a twofold increase in PGE2 secretion (P < 0.01) in both examined periods, the effect of NA on PGF2alpha secretion was approximately 1.5 times higher (P < 0.05) in early than in mid-cycle CL. Two NO synthase inhibitors, L-NAME and L-NOARG (both 10(-4) M), stimulated P4 secretion only in mid-luteal cells (P < 0.01), although they did not affect the cells from early CL. Although a NO donor, S-NAP (10(-4) M) inhibited P4 secretion from mid-cycle luteal cells (P < 0.05), it strongly stimulated PGE2 in both examined phases (P < 0.001). On the other hand, the output of PGF2alpha was stimulated by S-NAP only in the cells of the mid-cycle CL (P < 0.01). The overall results suggest that adrenergic and nitrergic agents play opposite roles in the regulation of bovine CL functions. Whereas NA may play a supporting role in luteal development, NO may participate in the functional regression of the bovine CL by inhibiting steroidogenesis.     

Na~+、K~+离子通道阻滞剂对离体大鼠黄体细胞孕酮生成的影响

本工作用二种离子通道阻断剂四乙胺(TEA)和河豚毒素(TTX)来研究 Na~+、K~+通道的改变对大鼠黄体细胞孕酮生成的影响。10~(-3)mol/L 的 TEA 或 TTX 均使孕酮分泌量显著增加,而这种促进效应可被酪氨酸(Tyr)完全阻断。Tyr 对 TEA 或 TTX 与 hCG 联合所引起的孕酮分泌也有抑制作用。上述实验说明跨黄体细胞内外的 K~+和 Na~+浓度差与孕酮分泌有关。     

Extracellular ATP-mediated phospholipase A(2) activation in rat thyroid FRTL-5 cells: regulation by a G(i)/G(o) protein, Ca(2+), and mitogen-activated protein kinase

We investigated the mechanism of phospholipase A(2) (PLA(2)) activation in response to the P2 receptor agonist ATP in rat thyroid FRTL-5 cells. The PLA(2) activity was determined by measuring the release of [(3)H]-arachidonic acid (AA) from prelabeled cells. ATP evoked a dose- and time-dependent AA release. This release was totally inhibited by pertussis toxin (PTX) treatment, indicating the involvement of a G(i)/G(o) protein. The AA release was also diminished by chelating extracellular Ca(2+) with EGTA or by inhibiting influx of Ca(2+) using Ni(2+). Although the activation of protein kinase C (PKC) by 12-phorbol 13-myristate acetate (PMA) alone did not induce any AA release, the ATP-evoked AA release was significantly reduced when PKC was inhibited by GF109203X or by a long incubation with PMA to downregulate PKC. Both the ATP-evoked AA release and the mitogen-activated protein kinase (MAP kinase) phosphorylation were decreased by the MAP kinase kinase (MEK) inhibitor PD98059. Furthermore, the ATP-evoked MAP kinase phosphorylation was also inhibited by GF109203X and by downregulation of PKC, suggesting a PKC-mediated activation of MAP kinase. Inhibiting Src-like kinases by PP1 attenuated both the MAP kinase phosphorylation and the AA release. These results suggest that these kinases are involved in the regulation of MAP kinase and PLA(2) activation. Elevation of intracellular cAMP by TSH or by dBucAMP did not induce a phosphorylation of MAP kinase. Furthermore, neither the ATP-evoked AA release nor the MAP kinase phosphorylation were attenuated by TSH or dBucAMP. Taken together, our results suggest that ATP regulates the activation of PLA(2) by a G(i)/G(o) protein-dependent mechanism. Moreover, Ca(2+), PKC, MAP kinase, and Src-like kinases are also involved in this regulatory process.     

Arachidonic acid is involved in the regulation of hCG induced steroidogenesis in rat Leydig cells

Phospholipase C (PLC), an enzyme involved in the hydrolysis of membrane phospholipid- phosphatidylinositol-bisphosphate to inositol triphosphate and diacylglycerol, and Phorbol 12, myristate 13, acetate (PMA), a tumor promoting agent, could significantly stimulate testosterone (T) secretion from Leydig cells. Arachidonic acid (AA) stimulated T secretion by about 2 fold. The steroidogenic effect of PLC and AA was biphasic. At low concentrations both PLC and AA (100 mU and 12.5 microM, respectively) augmented hCG induced T secretion, while at higher concentrations (PLC: 500 mU and AA: 200 microM) they inhibited steroid production. AA also had a biphasic effect on hCG induced cyclic AMP secretion. 5, 8, 11, 14 Eicosatetraynoic acid (ETYA), a general inhibitor of AA metabolism, and Nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway of AA metabolism, inhibited hCG induced T secretion while indomethacin, an inhibitor of cyclo-oxygenase pathway, had no effect on hCG induced T secretion. We conclude from these data that AA plays a role in the regulation of hCG induced steroidogenic responses in rat Leydig cells and that the metabolite(s) of AA that are involved are not cyclooxygenase products.     

High-affinity cholecystokinin type A receptor/cytosolic phospholipase A2 pathways mediate Ca2+ oscillations via a positive feedback regulation by calmodulin kinase in pancreatic acini.

In rat pancreatic acini, we previously demonstrated that depending on the agonist used, activation of cholecystokinin type A (CCKA) receptor (CCK-AR) results in the differential involvement of the cytosolic phospholipase A2 (cPLA2), phospholipase Cbeta1 (PLCbeta1) and Src/protein tyrosine kinase (PTK) pathways. The high-affinity CCK-AR appears to be coupled to the Gbeta/cPLA2/arachidonic acid (AA) cascade in mediating Ca2+ oscillations. The low-affinity CCK-AR is coupled to both the Galphaq/11/PLCbeta1/inositol 1,4,5-trisphosphate (IP3) to evoke intracellular Ca2+ release and the Src/PTK pathway to mediate extracellular Ca2+ influx. The objectives of this study were to provide evidence that cPLA2 is present in pancreatic acini and to evaluate the possibility that its activation results in Ca2+ oscillations and amylase secretion. Furthermore, we investigated the mechanism of Ca2+ oscillations mediated by the high-affinity CCK-AR. In rat pancreatic acini, immunoprecipitation studies using an anti-cPLA2 monoclonal antibody, demonstrated a cPLA2 band at the location of 110 kDa. A selective inhibitor of cPLA2, AACOCF3 (100 microM), inhibited production of AA metabolites, Ca2+ oscillations and amylase secretion elicited by the high-affinity CCK-AR agonist, CCK-OPE (10-1000 nM). In addition, through the repetitive release of intracellular Ca2+, CCK-OPE enhanced phosphotransferase activities of Ca2+/calmodulin-dependent protein kinase type IV (CaMK IV), which were inhibited by AACOCF3. The CaMK inhibitor, K252-a (1-3 microM), also abolished basal and CCK-OPE-stimulated CaMK IV activities. The CaM inhibitor, W-7 (100 microM), and K252-a inhibited Ca2+ oscillations and amylase secretion evoked by CCK-OPE without affecting the AA formation. Therefore, it appears that Ca2+ oscillations elicited by the high-affinity CCK-AR/Gbeta/cPLA2/AA pathway activate CaMK IV. Activated CaMK, in turn, regulates Ca2+ oscillations through a positive feedback mechanism to mediate pancreatic exocytosis.     

Effect of intratesticular administration of TRH or anti-TRH antiserum on function of rat testis

The effect of intratesticular administration of thyrotropin-releasing hormone (TRH) and anti-TRH antiserum on steroidogenesis was studied in immature and adult rats. In 9-day-old animals local administration of the neuropeptide resulted in an increase in basal testosterone secretion in vitro. Similar treatment of 15-day-old rats suppressed hCG-stimulated testosterone secretion with no change in basal testosterone production. In both immature groups the treatment did not affect serum testosterone concentration. By contrast, in adults TRH decreased serum testosterone level, but did not influence basal and hCG-stimulated testosterone secretion. Both in immature and adult rats, the changes in steroidogenesis were evident 1 hour posttreatment. Five days after the administration of anti-TRH antiserum into the remaining testis of immature rats subjected to hemicastration just prior to the antiserum treatment, the alterations in steroidogenesis were opposite to those detected after treatment with TRH. In 9-day-old rats the antiserum suppressed steroidogenesis, while in 15-day-old animals it stimulated testosterone secretion. The results suggest that testicular TRH might exert a local action on testicular steroidogenesis, and the effect is age-dependent.     

Na^＋,K^＋离子通道阻滞剂对离体大鼠黄体细胞...

Two ionic channel blockers, TEA and TTX, were used in the present investigation to test whether blocking of Na+, K+ ion channels would affect the production of progesterone by corpus luteum (CL) cells of rat. Both TEA (10(-3) mol/L) and TTX (10(-3) mol/L) increased progesterone production significantly after treatment. This effect of the blockers could be completely inhibited by tyrosine (Tyr). In addition, Tyr was capable of reversing the combined effect of TEA, TTX and hCG on progesterone secretion. It is suggested that the concentration gradient of Na+ and K+ across the CL cell membrane in experiments mentioned above is implicated in steroidogenesis.     

Prostacyclin and steroidogenesis in goat ovarian cell types in vitro

Granulosa, theca and corpus luteum cells of the goat ovary were isolated and incubated separately for 6 hours, with or without various modulators. Arachidonic acid (AA, 10 ng to 100 micrograms/ml), the precursor for prostaglandin synthesis, produced a dose-dependent increase in progesterone (P4) and estradiol-17 beta (E2) production by all the cell types. Prostaglandin synthetase inhibitors, aspirin (10(-6)-10(-3)M) and indomethacin (100 ng-1 mg/ml), produced a dose-dependent decrease in arachidonic acid-stimulated (100 micrograms/ml) steroid production. Prostacyclin synthetase stimulators, trapidil (1.6 micrograms- 1 mg/ml) and dipyridamole (10(-6)-10(-3)M), when added alone or along with AA, did not affect steroid production. Up to 100 micrograms/ml of U-51605 (9,11-azoprosta-5,13-dienoic acid), a prostacyclin synthetase inhibitor, did not inhibit basal or AA-stimulated steroid production. Prostacyclin (PGI2) and its stable analog 6 beta PGI1 (0.01-10 micrograms/ml) produced a dose-dependent increase in P4 and E2 production in all the three cell types. Increase at 1 and 10 micrograms/ml was significant in all cases. 6-keto-PGE1 (an active metabolite of PGI2 in certain systems) produced an increase in steroid production which was significant in theca at greater than or equal to 1 microgram/ml concentrations but had no significant effect on granulosa and corpus luteum cells at any dose level. 6-keto-PGF1 alpha (stable metabolite of PGI2) was without effect in the present system. The lack of effect of PGI2 at lower concentrations was not altered by either differentiation of the cells with FSH and testosterone or addition of steroid precursors, testosterone and pregnenolone. The present results indicate that AA-stimulated steroid production in the goat ovarian cell type is mediated by prostaglandins other than PGI2 though PGI2 itself can positively modulate the steroid production.