Effects of phorbol esters on steroidogenesis in small bovine luteal cells

The possible influence of an activator of protein kinase C, the tumor-promoting phorbol ester, PMA (phorbol-12-myristate-13-acetate), upon small bovine luteal cell steroidogenesis was investigated in vitro, PMA had no significant effect on basal and dibutyryl cyclic AMP (dbcAMP)-stimulated progesterone production but markedly modulated the LH-stimulated progesterone and cAMP productions. PMA potentiated the LH-stimulated cAMP accumulation whatever the dose of LH used. It also potentiated the LH-induced progesterone production in the presence of low doses of LH. Paradoxically, in the presence of maximal or submaximal effective doses of LH, PMA exerted a time- and dose-dependent inhibition of progesterone synthesis. Diacylglycerol was able to mimic the effects of PMA on LH-induced steroidogenesis. These observations suggest that the Ca2+- and phospholipid-dependent protein kinase C can modulate the regulation by LH of small bovine luteal cell steroidogenesis at a step before the synthesis of cAMP. They also suggest that the interaction between LH and its receptor is able to trigger a negative regulatory signal which would be only expressed for high doses of LH and in the presence of an activator of PKC.     

A comparison of the effects of cyclooxygenase prostanoids on progesterone production by small and large bovine luteal cells

Highly purified preparations of small and large bovine luteal cells were utilized to examine the effects of prostaglandins F2 alpha (PGF2 alpha), E2 (PGE2) and I2 (PGI2) analog on progesterone production. Corpora lutea were obtained from Holstein heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. Progesterone accumulation was determined in 1 x 10(5) small and 5 x 10(3) large cells after 2 and 4 h incubations respectively. Progesterone synthesis was increased (p less than 0.05) in the small cells by the increasing levels of PGF2 alpha, PGE2, carba-PGI2 and LH. PGF2 alpha, but not PGE2 or carba-PGI2 increased (p less than 0.05) LH-stimulated progesterone production. There was no interaction of various combinations of prostaglandins on progesterone production in the small cells. In the large cells, PGF2 alpha had no effect on basal progesterone production. However, it inhibited LH-stimulated progesterone synthesis. In contrast, PGE2 and carba-PGI2 stimulated (p less than 0.05) basal progesterone production in the large cells. In the presence of LH, high levels of carba-PGI2 inhibited (p less than 0.05) progesterone synthesis. The PGE2 and PGI2-stimulated progesterone production in the large luteal cells was also inhibited in the presence of PGF2 alpha. These data suggest all of the prostaglandins used exert a luteotropic action in the small cells. In the large cells only PGE2 and carba-PGI2 are luteotropic, while PGF2 alpha exerts a luteolytic action. The effects of the prostaglandins in the small and large luteal cells suggest that their receptors are present in both cell types.     

The lipoxygenase pathways are involved in LH-stimulated progesterone production in bovine corpus luteum.

We have examined the effects of endogenous lipoxygenase products on basal progesterone (P4) production by cultured bovine mid-luteal cells. The involvement of lipoxygenase products in the stimulatory effect of LH on luteal cAMP accumulation and P4 production was also examined. Bovine luteal cells from mid-cycle corpora lutea (CL) were exposed for 16 h to a lipoxygenase inhibitor (nordihydroguaiaretic acid: NDGA; 0.33-33 microM). For the last 4 h of incubation, the cells were exposed to LH and/or three different lipoxygenase products, 5-, 12- and 15-hydroxyeicosatetraenoic acid (HETE). NDGA inhibited P4 production by the cells in a dose-dependent manner (P < 0.05). NDGA-reduced P4 production was reversed by the addition of 12-HETE, but not 5- or 15-HETE, whereas 5-, 12- and 15-HETE alone showed no significant effect on P4 production in the intact cells. Furthermore, NDGA (33 microM) blocked the stimulatory action of LH on P4 production (P < 0.05), without changing cAMP accumulation (P > 0.1). When the cells were exposed to 5-, 12- or 15-HETE with LH and NDGA, only 15-HETE maintained the stimulatory effect of LH on P4 production in the cells (P < 0.05). These results suggest that endogenous lipoxygenase products play important roles in P4 production by bovine CL, i.e. basal P4 production is supported by 12-HETE, and LH-stimulated P4 production is partially mediated via the activation of lipoxygenase and subsequent 15-HETE formation downstream of the LH-activated cAMP-PKA-phosphorylation pathway.     

Luteinizing hormone increases inositol trisphosphate and cytosolic free Ca2+ in isolated bovine luteal cells

The present studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases intracellular cAMP, also increases "second messengers" derived from inositol phospholipid hydrolysis in isolated bovine luteal cells. In luteal cells prelabeled with 32PO4, LH provoked increases in labeling of phosphatidic acid, phosphatidylinositol, and polyphosphatidylinositol (PIP). No reductions in 32P-prelabeled PIP and PIP2 were observed in LH-treated cells. In luteal cells prelabeled with myo-[2-3H]inositol, LH provoked rapid (10-30 s) and sustained (up to 60 min) increases in the levels of inositol mono-, bis-, and trisphosphates (IP, IP2, and IP3, respectively. IP3 was formed more rapidly than IP2 or IP following LH treatment. In addition, LH increased (50%) levels of [3H]inositol phospholipids in 30-min incubations. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH. Maximal increases in IP3 occurred at 1-10 micrograms/ml of LH. Similar temporal and dose-response relationships were observed for LH-stimulated IP3 and cAMP accumulation. However, exogenous cAMP (8-bromo-cAMP, 5 mM) and forskolin (10 microM) had no effect on inositol phosphate synthesis. The initial (1 min) effects of LH on IP3 and cAMP were independent of extracellular calcium concentrations, whereas the sustained (5 min) effect of LH on IP3, but not cAMP, was dependent on a source of extracellular calcium. LH-stimulated progesterone synthesis was also dependent on the presence of extracellular calcium. LH induced rapid and concentration-dependent increases in [Ca2+]i as measured by Quin 2 fluorescence. The LH-induced increases in [Ca2+]i were maximal within 30 s (approximately 2-fold) and remained elevated for at least 10 min. In Ca2+-free media containing 2 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, LH was still able to increase [Ca2+]i, but the increase was slightly less in magnitude and of shorter duration (2-4 min). These findings demonstrate that LH can rapidly raise levels of IP3 and [Ca2+]i, as well as, cAMP in bovine luteal cells. These findings suggest that at least two second messenger systems exist to mediate the action of LH in the corpus luteum.     

Second messenger systems and progesterone secretion in the small cells of the bovine corpus luteum: effects of gonadotropins and prostaglandin F2a

The present studies were conducted to determine the effects of gonadotropins (LH and hCG) and prostaglandin F2a (PGF2a) on the production of "second messengers" and progesterone synthesis in purified preparations of bovine small luteal cells. Corpora lutea were removed from heifers during the luteal phase of the normal estrous cycle. Small luteal cells were isolated by unit-gravity sedimentation and were 95-99% pure. LH provoked rapid and sustained increases in the levels of [3H]inositol mono-, bis-, and trisphosphates (IP, IP2, IP3, respectively), cAMP and progesterone in small luteal cells. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH but had no effect on LH-stimulated cAMP or progesterone accumulation. Time course studies revealed that LH-induced increases in IP3 and cAMP occurred simultaneously and preceded the increases in progesterone secretion. Similar dose-response relationships were observed for inositol phosphate and cAMP accumulation with maximal increases observed with 1-10 micrograms/ml of LH. Progesterone accumulation was maximal at 1-10 ng/ml of LH. LH (1 microgram/ml) and hCG (20 IU/ml) provoked similar increases in inositol phosphate, cAMP and progesterone accumulation in small luteal cells. 8-Bromo-cAMP (2.5 mM) and forskolin (1 microM) increased progesterone synthesis but did not increase inositol phosphate accumulation in 30 min incubations. PGF2a (1 microM) was more effective than LH (1 microgram/ml) at stimulating increases in inositol phosphate accumulation (4.4-fold vs 2.2-fold increase for PGF2a and LH, respectively). The combined effects of LH and PGF2a on accumulation of inositol phosphates were slightly greater than the effects of PGF2a alone. In 30 min incubations, PGF2a had no effect on cAMP accumulation and provoked small increases in progesterone secretion. Additionally, PGF2a treatment had no significant effect on LH-induced cAMP or progesterone accumulation in 30 min incubations of small luteal cells. These findings provide the first evidence that gonadotropins stimulate the cAMP and IP3-diacylglycerol transmembrane signalling systems in bovine small luteal cells. PGF2a stimulated phospholipase C activity in small cells but did not reduce LH-stimulated cAMP or progesterone accumulation. These results also demonstrate that induction of functional luteolysis in vitro requires more than the activation of the phospholipase C-IP3/calcium and -diacylglycerol/protein kinase C transmembrane signalling system.     

Effects of antibiotics and medium supplements on steroidogenesis in cultured cow luteal cells

Corpora lutea were removed from regularly cycling dairy cows, dissociated with collagenase and cultured for 8 or 10 days in Ham's F-12 medium. In Exp. 1 treatment with insulin, or an insulin-transferrin-selenium combination (ITS), increased progesterone production from basal levels on Day 4 of culture to 234% (P less than 0.01) above controls on Day 10. LH alone increased progesterone production 45% above controls on Day 10 (P greater than 0.05). When LH was combined with insulin or ITS, progesterone production was stimulated to an average of 1802% (P less than 0.01) above controls on Day 10 of culture. Transferrin or selenium without insulin did not allow LH to stimulate progesterone synthesis. In Exp. II, LH alone or LH plus gentamicin or penicillin-streptomycin increased progesterone production from basal levels on Day 2 steadily to an average of 468% (P less than 0.01) above controls (no antibiotics) by Day 8 of culture. The addition of amphotericin-B, alone or in combination with the other antibiotics, inhibited all LH-stimulated progesterone synthesis, but did not affect basal progesterone levels. We conclude that insulin is essential for maximal steroidogenesis in a bovine luteal cell culture system, and that LH-stimulated progesterone production is inhibited in the presence of amphotericin-B, but is not inhibited by gentamicin or penicillin-streptomycin. The elimination of amphotericin-B, coupled with the addition of insulin to the cell culture system increased the responsiveness of the cells to LH. These culture conditions represent the first report in which LH increased total progesterone production for 10 days, maintaining luteal function in a chemically-defined culture system.     

Do small and large luteal cells of the sheep interact in the production of progesterone?

Corpora lutea from cyclic ewes were dissociated by collagenase and trypsin/EGTA treatments, and enriched fractions of small and large luteal cells were prepared on gradients of Ficoll. These fractions were incubated separately or remixed before incubation. Colchicine, cytochalasin B and the calcium channel-blocker verapamil significantly reduced progesterone production by both small and large luteal cell fractions, while isoprenaline stimulated an increase in progesterone production by large luteal cell fractions only. When fractions of small and large luteal cells were remixed, no more and no less progesterone was produced than would have been predicted from equivalent fractions incubated separately. There was therefore no evidence of synergism between small and large luteal cells in the production of progesterone. Prostaglandin F-2 alpha, which can inhibit LH-stimulated progesterone production by ovine luteal tissue in vitro, had no effect on LH-stimulated progesterone production by small luteal cell fractions, but significantly inhibited that by enriched fractions of large luteal cells. Since large luteal cell fractions were contaminated with small luteal cells, which are probably responsible for the progesterone-secretory response of these fractions to LH, it was concluded that the inhibition of LH-stimulated progesterone production by small luteal cells is dependent on the presence of large luteal cells. Oxytocin added to large and small luteal cell fractions did not affect progesterone production by either fraction. It was therefore concluded that the inhibitory action of PGF-2 alpha on LH-stimulated progesterone production may require the interaction of large and small luteal cells, but that oxytocin is not likely to be an intermediary in this interaction.     

The effects of elevation and depletion of intracellular free calcium on progesterone and prostaglandin production by the primate corpus luteum.

The role of the phosphatidylinositol second messenger system in luteal regulation has not been extensively studied, particularly in the primate. The objectives of this study were (1) to further characterize the response of the primate CL to the calcium ionophore A23187, in terms of intracellular free calcium concentrations ([Ca2+]i) and progesterone (P) production; and (2) to assess the effects of depleting, as well as elevating, available calcium on luteal P and prostaglandin (PG) production. The response to A23187, in terms of [Ca2+]i, was measured by fura-2 fluorescence microscopy of single small and large luteal cells. A23187 significantly increased [Ca2+]i in both cell types (p less than 0.01). P production (basal and hCG-stimulated) by dispersed primate luteal cells incubated for various times (1-8 h) with and without A23187 was measured. Treatment with A23187 rapidly (within 1-2 h) attenuated (p less than 0.05) the time-dependent increase in basal and hCG-stimulated P production. Luteal P and PG production following treatment with the calcium ionophore, ionomycin, alone or in combination with additional CaCl2, was also monitored. Treatment with ionomycin (p less than 0.01) and CaCl2 (p less than 0.01) inhibited luteal P production. In contrast, treatment with ionomycin stimulated (p less than 0.01) luteal PG production. To determine the effects of Ca2+ depletion on luteal function, P and PG production by cells incubated for 2 and 8 h in the absence and presence of the Ca(2+)-chelator EGTA was measured. Luteal production of both P and PG was inhibited by 8-h treatment with EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luteinizing hormone inhibits conversion of pregnenolone to progesterone in luteal cells from rats on day 19 of pregnancy

We have previously reported that intrabursal ovarian administration of LH at the end of pregnancy in rats induces a decrease in luteal progesterone (P4) synthesis and an increase in P4 metabolism. However, whether this local luteolytic effect of LH is exerted directly on luteal cells or on other structures, such as follicular or stromal cells, to modify luteal function is unknown. The aim of the present study was to determine the effect of LH on isolated luteal cells obtained on Day 19 of pregnancy. Incubation of luteal cells with 1, 10, 100, or 1000 ng/ml of ovine LH (oLH) for 6 h did not modify basal P4 production. The addition to the culture medium of 22(R)-hydroxycholesterol (22R-HC, 10 microgram/ml), a membrane-permeable P4 precursor, or pregnenolone (10(-2) microM) induced a significant increase in P4 accumulation in the medium in relation to the control value. When luteal cells were preincubated for 2 h with oLH, a significant (p < 0.01) reduction in the 22R-HC- or pregnenolone-stimulated P4 accumulation was observed. Incubation of luteal cells with dibutyryl cAMP (1 mM, a cAMP analogue) plus isobutylmethylxanthine (1 mM, a phosphodiesterase inhibitor) also inhibited pregnenolone-stimulated P4 accumulation. Incubation with an inositol triphosphate synthesis inhibitor, neomycin (1 mM), or an inhibitor of intracellular Ca2+ mobilization, (8,9-N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate (1 mM), did not prevent the decrease in pregnenolone-stimulated P4 secretion induced by oLH. It was concluded that the luteolytic action of LH in late pregnancy is due, at least in part, to a direct action on the luteal cells and that an increase in intracellular cAMP level might mediate this effect.     

K+ channel cAMP activated in guinea pig gallbladder epithelial cells

In guinea pig gallbladder epithelial cells, an increase in intracellular cAMP levels elicits the rise of anion channel activity. We investigated by patch-clamp techniques whether K(+) channels were also activated. In a cell-attached configuration and in the presence of theophylline and forskolin or 8-Br-cAMP in the cellular incubation bath, an increase of the open probability (P(o)) values for Ca(2+)-activated K(+) channels with a single-channel conductance of about 160 pS, for inward current, was observed. The increase in P(o) of these channels was also seen in an inside-out configuration and in the presence of PKA, ATP, and cAMP, but not with cAMP alone; phosphorylation did not influence single-channel conductance. In the inside-out configuration, the opioid loperamide (10(-5) M) was able to reduce P(o) when it was present either in the microelectrode filling solution or on the cytoplasmic side. Detection in the epithelial cells by RT-PCR of the mRNA corresponding to the alpha subunit of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) indicates that this gallbladder channel could belong to the BK family. Immunohistochemistry experiments confirm that these cells express the BK alpha subunit, which is located on the apical membrane. Other K(+) channels with lower conductance (40 pS) were not activated either by 8-Br-cAMP (cell-attached) or by PKA + ATP + cAMP (inside-out). These channels were insensitive to TEA(+) and loperamide. The data demonstrate that under conditions that induce secretion, phosphorylation activates anion channels as well as Ca(2+)-dependent, loperamide-sensitive K(+) channels present on the apical membrane.     

Nicotine and cotinine inhibit steroidogenesis in mouse Leydig cells

Cigarette smoking alters plasma testosterone concentrations in men. The objectives of this study were to determine if nicotine and cotinine, two alkaloid products of cigarettes, affect luteinizing hormone(LH)-stimulated steroidogenesis in isolated adult mouse Leydig cells. Leydig cells from adult Swiss-Webster mice were isolated by linear density gradient and incubated (95% O2, 5% CO2) in minimum essential medium at 37 C for 3 hours with LH (10 ng) and with or without nicotine or cotinine (10(-5)-10(-7) M). Both nicotine and cotinine produced dose response inhibition (P less than 0.05) of LH-stimulated testosterone production (50-70%). The addition of 8-bromo-3',5'-cyclic monophosphate (cAMP, 500 uM) stimulated steroidogenesis comparable to LH in the absence of the alkaloids, but both nicotine and cotinine significantly (P less than 0.05) reduced testosterone production in response to cAMP, suggesting that the alkaloids inhibit testosterone production in response to LH distal to the formation of cAMP. In MEM without calcium, LH-stimulated testosterone synthesis was decreased, and neither nicotine nor cotinine significantly affected steroidogenesis. The addition of a calcium ionophore in MEM with normal calcium content enhanced (P less than 0.05) the inhibitory effects of nicotine and cotinine on LH-responsive steroidogenesis. A calcium channel blocking agent, verapamil, at 10uM significantly (P less than 0.05) reversed the inhibition of LH-stimulated testosterone production produced by both alkaloids when incubated in the medium with a normal calcium concentration. These results suggest that nicotine and cotinine either affect intracellular calcium content or block the effects of calcium on steroidogenesis in mouse Leydig cells.     

Release of progesterone from perifused, highly luteinized ovarian cells of rats: effects of luteinizing hormone and bovine serum albumin

Release of progesterone from enzymatically dispersed luteal cells of superovulated rats was studied using a multi-channeled perifusion system. Cells were perifused with protein-free medium for up to 5 h. Basal release of progesterone showed a steady decline during the first h of perifusion to a stable baseline where it remained throughout the experiment. A 30-min exposure of the luteal cells to increasing amounts of luteinizing hormone (LH) stimulated a dose-dependent increase in progesterone release. Similar results were observed when luteal cells were exposed to 0.2 or 1.0 mM dibutyryl (Bu)2 cAMP for 30 min. Exposure of the cells to 0, 1, 10, and 100 ng LH/ml protein-free medium for 230 min showed increased release of progesterone, although the dispersed cells perifused with 100 ng LH/ml protein-free medium were unable to maintain the maximal levels of progesterone release. The effect of bovine serum albumin (BSA) in the perifusion medium on the basal and LH-stimulated progesterone release was examined. Low concentrations of BSA (0.05%) had no effect, but 0.5% and 2.0% BSA significantly increased the basal release of progesterone. However, the addition of 0.05% BSA to the medium resulted in an increased progesterone release in response to 10 ng LH/ml medium. These results suggest that the in vitro perifusion system maintains physiologically viable cells which are responsive to either LH or (Bu)2 cAMP for at least 5 h. The effect of protein in the perifusion medium or progesterone release was demonstrated by the addition of BSA.     

The regulation of steroidogenesis is different in the two types of ovine luteal cells

Ovine luteal tissue contains two distinct steroidogenic cell types, small (8-20 microns) and large (greater than 20 microns), which differ based on morphological and biochemical criteria. Unstimulated small cells secrete low levels of progesterone, respond to LH or dibutyryl cAMP (dbcAMP) with enhanced secretion of progesterone, and contain most of the receptors for LH. The unstimulated large cells, conversely, secrete high levels of progesterone, have few, if any, receptors for LH, and do not respond to LH or dbcAMP with increased progesterone secretion. The lack of response to dbcAMP by large cells was investigated. Large cells incubated in the presence of cholesterol, ram serum, or 25-hydroxycholesterol did not demonstrate substrate limitation. Hormone-independent stimulation of adenylate cyclase by cholera toxin or forskolin resulted in increased adenylate cyclase activities (P less than 0.01), cAMP accumulation (P less than 0.05), and the binding of endogenous cAMP (P less than 0.05) by type I cAMP-dependent protein kinase in both small and large cells. These treatments were accompanied by enhanced secretion of progesterone (P less than 0.05) in small cells. In contrast, large cells did not respond with an increase in progesterone secretion under these conditions. These observations suggest that the high rate of secretion of progesterone in unstimulated large cells is not regulated by cAMP.     

Prostaglandin F2 alpha, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: effects of oxytocin, luteinizing hormone, prostaglandin F2 alpha and estradiol-17 beta

Bovine luteal cells from Days 4, 8, 14 and 18 of the estrous cycle were incubated for 2 h (1 x 10(5) cells/ml) in serum-free media with one or a combination of treatments [control (no hormone), prostaglandin F2 alpha (PGF), oxytocin (OT), estradiol-17 beta (E) or luteinizing hormone (LH)]. Luteal cell conditioned media were then assayed by RIA for progesterone (P), PGF, and OT. Basal secretion of PGF on Days 4, 8, 14 and 18 was 173.8 +/- 66.2, 111.1 +/- 37.8, 57.7 +/- 15.4 and 124.3 +/- 29.9 pg/ml, respectively. Basal release of OT and P was greater on Day 4 (P less than 0.01) than on Day 8, 14 and 18 (OT: 17.5 +/- 2.6 versus 5.6 +/- 0.7, 6.0 +/- 1.4 and 3.1 +/- 0.4 pg/ml; P: 138.9 +/- 19.5 versus 23.2 +/- 7.5, 35.4 +/- 6.5 and 43.6 +/- 8.1 ng/ml, respectively). Oxytocin increased (P less than 0.01) PGF release by luteal cells compared with control cultures irrespective of day of estrous cycle. Estradiol-17 beta stimulated (P less than 0.05) PGF secretion on Days 8, 14 and 18, and LH increased (P less than 0.01) PGF production only on Day 14. Prostaglandin F2 alpha, E and LH had no effect on OT release by luteal cells from any day. Luteinizing hormone alone or in combination with PGF, OT or E increased (P less than 0.01) P secretion by cells from Days 8, 14 and 18. However on Day 8, a combination of PGF + OT and PGF + E decreased (P less than 0.05) LH-stimulated P secretion. These data demonstrate that OT stimulates PGF secretion by bovine luteal cells in vitro. In addition, LH and E also stimulate PGF release but effects may vary with stage of estrous cycle.     

Regulation of the corpus luteum by protein kinase C. II. Inhibition of lipoprotein-stimulated steroidogenesis by prostaglandin F2 alpha

This study was designed to examine the antisteroidogenic action of prostaglandin (PG) F2 alpha on ovine luteal cells in vitro. Purified populations of large and small steroidogenic luteal cells were treated with lipoproteins, luteinizing hormone (LH), and/or PGF2 alpha. To investigate the involvement of the protein kinase C (PKC) pathway in hormone action, luteal cells were made PKC-deficient by treatment for 12 h with 1 microM phorbol-12-myristate-13-acetate. Progesterone production by nonstimulated large and LH-stimulated small luteal cells was significantly increased by treatment with high- and low-density lipoprotein (HDL, 5-fold increase; LDL, 2-fold increase). PGF2 alpha inhibited (p less than 0.0001) progesterone production by HDL-stimulated large luteal cells in a dose-dependent manner, with 60 nM causing maximal inhibition. No effect of PGF2 alpha (20nM-20 microM) was found on production of progesterone by HDL-stimulated, PKC-deficient large cells or by LH- and HDL-stimulated small luteal cells. These results suggest that PGF2 alpha has a direct antisteroidogenic effect on the large luteal cell that is mediated through the PKC second messenger pathway.     

Cyclic AMP stimulates luteinizing-hormone (lutropin) exocytosis in permeabilized sheep anterior-pituitary cells. Synergism with protein kinase C and calcium.

Sheep anterior-pituitary cells permeabilized with Staphylococcus aureus alpha-toxin were used to investigate the role of cyclic AMP (cAMP) in exocytosis of luteinizing hormone (lutropin, LH) under conditions where the intracellular free Ca2+ concentration ([Ca2+]free) is clamped by Ca2+ buffers. At resting [Ca2+]free (pCa 7), cAMP rapidly stimulated LH exocytosis (within 5 min) and continued to stimulate exocytosis for at least 30 min. When cAMP breakdown was inhibited by 3-isobutyl-1-methylxanthine (IBMX), the concentration giving half-maximal response (EC50) for cAMP-stimulated exocytosis was 10 microM. cAMP-stimulated exocytosis required millimolar concentrations of MgATP, as has been found with Ca2(+)- and phorbol-ester-stimulated LH exocytosis. cAMP caused a modest enhancement of Ca2(+)-stimulated LH exocytosis by decreasing in the EC50 for Ca2+ from pCa 5.6 to pCa 5.9, but had little effect on the maximal LH response to Ca2+. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) dramatically enhanced cAMP-stimulated LH exocytosis by both increasing the maximal effect 5-7-fold and decreasing the EC50 for cAMP to 3 microM. This synergism between cAMP and PMA was further augmented by increasing the [Ca2+]free. Gonadotropin-releasing hormone (gonadoliberin, GnRH) stimulated cAMP production in intact pituitary cells. Since GnRH stimulation is reported to activate PKC and increase the intracellular [Ca2+]free, our results suggest that a synergistic interaction of the cAMP, PKC and Ca2+ second-messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis.     

Influence of agents that affect intracellular calcium regulation on progesterone secretion in small and large luteal cells of the sheep

Enriched fractions of small and large luteal cells were incubated for 2 h with 1 or 10 microM calcium ionophore, A23187: unstimulated secretion of progesterone and viability in small cells were not affected but these measures were decreased (P less than 0.01) for unstimulated large cells and were significantly correlated (P less than 0.05). This effect in large cells was independent of extracellular calcium. Therefore, incubations of the two cell types were made in the presence of increasing concentrations of a protein kinase C activator, phorbol 12-myristate 13-acetate (TPA). Secretion of progesterone and viability were not augmented in unstimulated small cells, but TPA prevented (P less than 0.05) the full stimulation of secretion of progesterone by LH. Secretion of progesterone in unstimulated large cells was inhibited (P less than 0.01) by TPA (100 nM and 10 microM), although viability was unaffected. The non-tumour promoting phorbol ester, 4 alpha-phorbol didecanoate, had no effect on large cells. Extracellular calcium was not required for the observed effect of TPA. Sphingosine, an agent inhibitory to protein kinase C activity, inhibited (P less than 0.01) secretion of progesterone in small and large cells, and also reduced (P less than 0.01) cell viability. These values were significantly correlated (P less than 0.05) in both cell types. The above observations suggest that protein kinase C may invoke negative regulation on progesterone production in unstimulated large and hormone-stimulated small luteal cells of sheep. Since sphingosine significantly reduced viability in small and large cells and ionophore selectively inhibited viability in large cells, the ability of these agents to influence calcium-mediated intracellular regulation of steroidogenesis is still uncertain.     

PKCepsilon and an increase in intracellular calcium concentration are necessary for PGF2alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells

The hypotheses that PKCepsilon is necessary for: 1) PGF2alpha to inhibit LH-stimulated progesterone (P4) secretion, and 2) for the expression of key prostaglandin synthesizing/metabolizing enzymes were tested in bovine luteal cells in which PKCepsilon expression had been ablated using a validated siRNA protocol. Steroidogenic cells from Day -6 bovine corpus luteum (CL) were isolated and transfected to reduce PKCepsilon expression after 48, 72 and 96 h. A third tested hypothesis was that an increase in intracellular calcium concentration ([Ca(2+)]i) is the cellular mechanism through which PGF2alpha inhibits luteal progesterone. The hypothesis was tested with two pharmacological agents. In the first test, the dose-dependent effects on raising the [Ca(2+)]i with the ionophore, A23187, on basal and LH-stimulated P4 secretion in cells collected from early (Day -4) and mid-cycle (Day -10) bovine CL was examined. In the second test, the ability of PGF2alpha to inhibit LH-stimulated P4 secretion in Day-10 luteal cells was examined under conditions in which an elevation in [Ca(2+)]i had been buffered by means of the intracellular calcium chelator, Bapta-AM.     

Serotonin-induced stimulation of progesterone production by cow luteal cells in vitro

The addition of acetylcholine or histamine (10(-7) to 10(-4) M), gamma-aminobutyric acid, a dopamine agonist, and melatonin (10(-7) to 10(-5) M) did not alter basal or LH-stimulated progesterone production (P greater than 0.05). The addition of the specific beta 2-adrenergic agonist terbutaline and salbutamol did not significantly elevate progesterone production. Treatment of luteal cells with serotonin (5-HT), 10(-6) to 10(-4) M, increased the production of progesterone (P less than 0.05). This stimulated production was inhibited by the addition of mianserin (10(-5) M, a 5-HT antagonist; P less than 0.05). Isoproterenol (10(-7) to 10(-4) M) also resulted in significant increases in progesterone production (P less than 0.05). The combined treatments of 5-HT + LH, isoproterenol + LH, or isoproterenol + 5-HT did not result in a further increase in progesterone above that observed in response to LH or isoproterenol alone (P greater than 0.05). The isoproterenol-induced progesterone production could not be blocked by butoxamine (10(-5) M, a beta 2-antagonist), or practolol (10(-5) M, a beta 1-antagonist), but was inhibited by propranolol (10(-5) M, a general beta-antagonist; P less than 0.05). The response to isoproterenol was unaffected by mianserin (10(-5) M). These results demonstrate a possible role for 5-HT in the regulation of steroidogenesis by the corpus luteum of the cow. Furthermore, these results suggest that serotonin-induced progesterone production is a receptor-mediated event.     

Developmental change in the ability of estradiol to suppress testosterone secretion by the testis of the rat

An intratesticular site of action has been proposed for the ability of estradiol (E2) to suppress testosterone secretion. Because testicular testosterone and E2 secretion as well as E2 receptors change during development, a physiologic role for E2 is possible. The present experiments compared the testes from 12-day-old and adult rats for the capacity of in vivo estradiol treatment to change in vitro androgen secretion in response to luteinizing hormone (LH) and dibutyryl cyclic AMP (Bt2cAMP). After 5 days in vivo treatment, in vitro responsiveness was estimated by radioimmunoassay (RIA) measurement of androgen secretion elicited by various doses of NIAMDD-LH-24 or 1.0 mM Bt2cAMP. Five days of E2 alone (500 ng/g BW s.c. once daily) markedly inhibited basal, LH-stimulated and Bt2cAMP-stimulated androgen production at both ages. Similar treatment of infant rats with LH (100 ng NIAMDD-LH-24/g BW) caused an increase in basal and LH-stimulated androgen secretion in vitro, but had no effect on the response to Bt2cAMP. The same pretreatment of adults with LH had no effect on basal, but inhibited LH- or Bt2cAMP-stimulated androgen secretion. Combined treatment of infants with E2 and LH for 5 days had no effect on basal or maximally stimulated androgen production; the in vitro response to submaximal stimulation with LH was significantly inhibited. Combined E2/LH treatment of adults significantly decreased the basal production of androgens and the response to LH or Bt2cAMP. These results suggest a major difference between the response to E2 of the Leydig cells from the rats of the two ages tested.(ABSTRACT TRUNCATED AT 250 WORDS)