Expression of testicular 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase: regulation by luteinizing hormone and forskolin in Leydig cells of adult rats.

LH is required to maintain the activity of 3 beta-hydroxysteriod dehydrogenase/delta 5----4-isomerase (3 beta HSD) in testicular Leydig cells. The objective of the present study was to determine whether LH and effectors such as forskolin, which act via the intracellular cAMP signal transduction pathway, can regulate the expression of 3 beta HSD in rat Leydig cells in vitro. Primary cultures of Leydig cells were prepared from testes of adult rats and treated with oLH, forskolin, (Bu)2cAMP, or cholera toxin. The effects of treatment on 3 beta HSD activity were measured using [3 alpha-3H]dehydroepiandrosterone as substrate. Immunoreactive 3 beta HSD was quantified by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a polyclonal antiserum against 3 beta HSD. The synthesis of 3 beta HSD was quantified after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated cellular lysates of Leydig cells radiolabeled with L-[35S]methionine. The levels of 3 beta HSD mRNA were quantified by Northern analysis and hybridization with a cDNA encoding testicular 3 beta HSD (rat type I). A cell-free protein-synthesizing system was used to test the ability of 3 beta HSD mRNA to be translated into immunoreactive 3 beta HSD. 3 beta HSD activity increased 3.5- and 5.0-fold in Leydig cell cultures treated with forskolin (1 microM) and (Bu)2cAMP (1 mM), respectively, compared with control cultures. Maximal activity was attained after 48-72 h and maintained through 120 h of treatment. The increase in 3 beta HSD activity could be accounted for quantitatively by increases in the steady state levels and the rates of synthesis of 3 beta HSD. The cellular levels of immunoreactive 3 beta HSD increased 4.0- and 7.6-fold in Leydig cells treated with forskolin and (Bu)2cAMP, respectively. Moreover, both of these effectors increased by 6- to 8-fold the levels of newly synthesized 3 beta HSD after 24-72 h of treatment. Ovine LH, forskolin, cholera toxin, and (Bu)2cAMP increased the cellular levels of 3 beta HSD mRNA in a dose-dependent manner. The magnitude of the increases ranged from 2- to 42-fold, compared with that in control cultures, after 12 h of treatment. Maximal responses were effected by 1 ng/ml ovine LH, 1 microM forskolin, 1 ng/ml cholera toxin, and 1 mM (Bu)2cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of melatonin on the in vitro secretion of progesterone and estradiol 17 beta by ovine granulosa cells.

This study was undertaken to determine the effect of melatonin on steroid hormone production by ovine granulosa and luteal cells in vitro. Granulosa and luteal cells from ovine ovaries were cultured for nine days either in D-MEM only or in the presence of melatonin (0.86, 8.6, 86 nmol/l), ovine luteinizing hormone (oLH, 2 micrograms/l) or a combination of both these hormones. Progesterone (P4) and estradiol 17 beta (E2) were determined by validated RIAs. Melatonin stimulation began at either day 1 or day 5 of culture. Melatonin (0.86 nmol/l) significantly increased (p < 0.001) progesterone secretion by granulosa cells both when administered alone and when administered in combination with oLH; the more marked response was observed in the latter case. When the stimulation began at day 5, at a more advanced degree of differentiation of the cells, higher levels of P4 were observed. Higher concentrations of melatonin did not further increase progesterone production. Melatonin alone did not have a significant effect on the production of estradiol 17 beta; neither did melatonin stimulate progesterone production in either long-term cultured luteal cells or in short-term (1-2 h) cultured luteal and granulosa cells. The results of this study document a direct effect of melatonin in stimulating granulosa cells to produce progesterone, a synergistic activity between melatonin and luteinizing hormone and a different ability of granulosa cells to secrete P4 depending on the degree of differentiation.     

Facilitative actions of the protein kinase-C effector system on hormonally stimulated adenosine 3',5'-monophosphate production by swine luteal cells

The exact nature of the interaction(s) between cAMP and calcium-sensitive phospholipid-dependent protein kinase-C effector pathways is not well understood in many tissues, including the ovary. In the present work we have evaluated the ability of protein kinase-C to modulate receptor-and nonreceptor-mediated cAMP generation in acute suspension cultures of swine luteal cells. Cells were exposed to LH (1 micrograms/ml), forskolin (100 microM), cholera toxin (1 microgram/ml), pertussis toxin (100 ng/ml), and/or phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] for 0-90 min. TPA had no effect on basal cAMP accumulation, but increased (P less than 0.05) LH-, forskolin-, and cholera toxin-activated cAMP formation, with maximal facilitation at 30, 45, and 60 min, respectively. This facilitative effect was robust, as it could be demonstrated in both the presence and absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM). TPA increased dose-dependent LH (0.1-1 microgram/ml)-, forskolin (3-300 microM-, and cholera toxin (0.3-10 microgram/ml)-stimulated cAMP accumulation. TPA induced a dose-dependent (0.3-30 ng/ml) increase in cAMP accumulation when incubated with the half-maximally effective (ED50) and maximally effective doses of LH (0.8 and 1 microgram/ml, respectively), forskolin (10 and 300 microM), and cholera toxin (0.2 and 3 micrograms/ml). TPA had an ED50 for this functional activation of 6.1 (67% confidence interval, 4.4-9.7) nM. The stimulatory effect of TPA could be mimicked by two synthetic diacylglycerols, 1,2-Dioctanoylglycerol and 1-oleoyl-2-acetylglycerol, but not by inactive phorbol esters. In addition, TPA augmented the stimulatory effect of pertussis toxin when combined with maximally effective doses of LH, forskolin, and cholera toxin. The stimulatory action of TPA on cAMP production was limited to endogenous cellular adenylyl cyclase. Bacterially derived adenylyl cyclase toxin isolated from Bordetella pertussis resulted in a dose-dependent increase in cAMP formation over 60 min, which was not facilitated by phorbol ester. We conclude that stimulatory coupling exists between the calcium-dependent protein kinase-C and cAMP-generating systems in swine luteal cells. This stimulatory coupling is enacted in part at the levels of both the guanine binding and the catalytic subunits of adenylyl cyclase.     

In vitro net progesterone production by human corpora lutea: effects of human chorionic gonadotropin, dibutyryl adenosine 3',5'-monophosphate, cholera toxin, and forskolin

Slices of human corpora lutea (CL) obtained at varying stages of the luteal phase from 21 women were used to study the effect of hCG on progesterone (P4) production. Slices obtained from mid- and late CL incubated with 10 IU/mL hCG exhibited a significant increase in net P4 production (P less than 0.001), whereas slices from early CL did not. Mid-CL slices were the most sensitive to hCG (4.2-fold increase in P4 production compared to 1.2-fold for early CL and 2.7-fold for late CL). To investigate the unresponsiveness of early CL to hCG, [125I]hCG binding was studied. All early CL had LH/hCG-specific receptors, and the apparent Kd for this binding was 1.95 X 10(-10) M. Dibutyryl cAMP (1 mM), cholera toxin (0.84 mM), and forskolin (50 microM) stimulated net P4 production (P less than 0.05) in slices of early CL tissue incubated in the presence of methylisobutylxanthine (0.1 mM). Cholera toxin and forskolin stimulated cAMP formation by the early CL, but hCG failed to do so. These results confirm that hCG has an age-dependent stimulatory effect on CL P4 synthesis. Our findings suggest that there is inadequate coupling of the LH/hCG receptor and adenylate cyclase in the early human CL, which explains in part the relative insensitivity of this tissue to the steroidogenic action of hCG.     

Antigonadotropic and antisteroidogenic actions of peroxide in rat granulosa cells.

Reactive oxygen species are produced in the ovary. In luteal cells, peroxide abruptly inhibits LH-sensitive cAMP and progesterone production, and may serve a role as a mediator of luteolysis by such mechanisms. The objective of the present studies was to evaluate the acute actions of peroxide in rat granulosa cells. Peroxide at concentrations in the low micromolar range produced a marked and dose-dependent inhibition of FSH-sensitive cAMP accumulation and progesterone production, and depleted cell levels of ATP within 1 min. Longer treatment with peroxide (60 min) caused complete abrogation of the actions of FSH. Peroxide-induced depletion of ATP was prevented by 3-aminobenzamide, an inhibitor of DNA repair, but maintenance of cell levels of ATP did not prevent the anti-FSH effects of peroxide. Peroxide also abrogated cAMP accumulation and progesterone production in response to LH in granulosa cells. Unlike that seen with LH, inhibition of FSH-sensitive cyclic AMP accumulation by peroxide was partially reversed with isobutylmethyl xanthine, an inhibitor of cyclic AMP phosphodiesterase. Although peroxide inhibited cAMP accumulation in response to cholera toxin, it did not inhibit this same response to forskolin, which indicates that peroxide may interfere with G-protein-dependent activation of adenylate cyclase. Peroxide inhibited steroidogenesis in response to cholera toxin, forskolin, and 8-bromo-cAMP. The marked inhibitory actions of peroxide on gonadotropic hormone action and steroidogenesis in granulosa cells raise the possibility that peroxide may mediate events associated with loss of follicular function.     

Progesterone secretion by luteinizing human granulosa cells: a possible cAMP-dependent but PKA-independent mechanism involved in its regulation

The corpus luteum formed after luteinization of follicular cells secretes progesterone under the control of luteinizing hormone (LH). Binding of LH to its G-protein-coupled receptor leads to the activation of the adenylate cyclase/ cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA) signalling pathway. The identification of a new class of cAMP-binding proteins termed 'guanine nucleotide exchange factors' (cAMP-GEFs) provides a means by which changes in cAMP could yield actions that are independent of PKA. Hence, in this study, we have explored the hypothesis that steroidogenesis in luteinizing cells is mediated in both a cAMP/PKA-dependent and cAMP-dependent, but PKA-independent, manner. Human granulosa cells were isolated from follicular aspirates of women undergoing assisted conception. Luteinizing human granulosa cells were cultured for up to 3 days in the presence of human (h)LH and the adenylate cyclase activator forskolin in the added presence or absence of increasing doses of the PKA inhibitors H89 (N-[2-(4-bromocinnamylamino)ethyl] 5-isoquinoline) and PKI (myristoylated protein kinase A inhibitor amide 14-22) or the cAMP antagonist, Rp-cAMP. Agonist-stimulated progesterone secretion was inhibited in a dose-dependent manner by the PKA inhibitors and the cAMP antagonist, with decreasing sensitivity as luteinization progressed. Pretreatment of granulosa cells for 4 h with human (h)LH reduced the effectiveness of H89 in inhibiting progesterone secretion. Under basal conditions, cAMP-GEFI expression increased progressively throughout culture, and this could be further enhanced when cells were incubated with increasing doses of LH and forskolin. Furthermore, incubation of cells in the presence of increasing concentrations of the novel cAMP-GEF-specific cAMP analogue, 8 CPT-2 ME-cAMP (8-(4-chloro-phenylthio)-2'-0-methyladenosine-3',5'-cyclic monophosphate), increased progesterone secretion in a dose-dependent manner. The results show that increases in cAMP generated by LH and forskolin, in addition to activating PKA, also induce increases in cAMP-GEFI protein expression in luteinizing human granulosa cells. In addition, activation of cAMP-GEFI results in increased progesterone secretion. Hence, increases in cAMP lead to the activation of PKA-dependent, as well as PKA-independent but cAMP-dependent (via cAMP-GEFI), signalling mechanisms. Since cAMP-GEFs have the capacity to activate the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB) signalling pathways, these may provide the potential mechanisms by which cAMP-dependent but PKA-independent progesterone synthesis is regulated.     

The actions of forskolin, cholera toxin and iloprost on casein secretion by lactating doe mammary glands

The secretagogue effects of prolactin (PRL) and of various agents acting on cAMP levels, forskolin, cholera toxin and iloprost (a stable analogue of prostaglandin I2) have been assessed in lactating doe mammary gland fragments in vitro. Forskolin (10 microM), cholera toxin (1 microgram/ml) and iloprost (10 mM) stimulated milk casein secretion. The effects of forskolin (10 microM) and cholera toxin (1 microgram/ml) were potentiated by PRL (10 micrograms/ml). Conversely, the action of iloprost (10 microM) was not amplified by PRL (10 micrograms/ml). Forskolin (10 microM) and cholera toxin (1 microgram/ml) stimulated the intracellular accumulation of cAMP. Neither PRL nor iloprost, at concentrations which stimulated casein secretion, modified the accumulation of cAMP. These results demonstrate that PRL does not act directly by any increase in intracellular cAMP levels. However, stimulating effects of forskolin and cholera toxin on casein secretion and intracellular cAMP levels suggest that various transduction signals are effective in the mammary cells.     

The dynamics of the steroidogenic response of perifused Leydig tumor cells to human chorionic gonadotropin, ovine luteinizing hormone, cholera toxin, and adenosine 3',5'-cyclic monophosphate

A perifusion system has been developed in which a dose-dependent response of isolated Leydig tumor cells to steroidogenic stimuli, as assessed by the rates of steroid production, can be measured as a function of time. In response to a continuous perifusion for 340 min with a saturating concentration of hCG, ovine LH (oLH), cholera toxin, or 8-Br-cAMP, there is a rapid increase in the rate of progesterone production, which reaches a maximum about 100 min after the onset of stimulus in the medium and then declines to a rate somewhat higher than basal. Thus cholera toxin and 8-Br-cAMP as well as hCG and oLH are able to desensitize the Leydig tumor cells to further stimulation by the same agent. Although a 10-min pulse of a saturating concentration of hCG yields the same steroidogenic response as that elicited by continuous perifusion with saturating hCG, a pulse of a saturating concentration of oLH yields a steroidogenic response only when oLH is maintained in the perifusate. These data establish a substantial difference in the actions of hCG and oLH. The results could be explained by the higher apparent affinity of hCG for the gonadotropin receptor, such that, upon removal of hormone from the perifusate, oLH would be more readily eluted from the receptor. These findings support the hypothesis that oLH and hCG exert their stimulatory effects only while bound to the cell surface. (Endocrinology 108: 632, 1981)     

Evidence for a role for adenosine 3',5'-monophosphate in progesterone secretion by human chorion

Experiments were performed to determine whether cells from human chorion can synthesize and release progesterone. Cells were isolated from term chorion laeve by collagenase-DNAse digestion and incubated in RPMI-1640 medium. Freshly isolated cells contained 9.9 +/- 1.1 ng progesterone/10(6) cells, and released 72.0 +/- 7.1 ng/10(6) cells X 24 h in the absence of precursors. When 25-hydroxycholesterol (25HC) served as a precursor, progesterone release into the medium was concentration and time dependent from 1-20 micrograms/ml up to 8 h. When pregnenolone served as a precursor, progesterone secretion followed Michaelis-Menten kinetics (Km = 6.7 microM; maximum velocity, 1.02 nmol/10(6) cells X h). In the presence of 25HC (20 micrograms/ml), progesterone release increased significantly on exposure to cholera toxin (1 microgram/ml), methylisobutylxanthine (0.1 mM), forskolin (0.1 mM), or (Bu)2cAMP (1 mM). Cells maintained in culture released progesterone when fetal calf serum (10%) or 25HC served as precursors. These studies show that trophoblasts from fetal membranes can synthesize and release progesterone from endogenous and exogenous precurors and support the suggestion that cAMP is an important mediator in this process.     

Cholera toxin inhibits chemotaxis by a cAMP-independent mechanism.

Cholera toxin inhibits chemotaxis of the RAW264 mouse macrophage cell line. The degree of inhibition by cholera toxin increases upon incubation with the cells, suggesting that the entry of the toxin is required for inhibition of chemotaxis. In the absence of guanine nucleotides, cholera toxin catalyzes the [32P]ADP-ribosylation of RAW264 cell membrane proteins of Mr 41,000, Mr 45,000, and a doublet of Mr 48,000-50,000. GTP increases the labeling of the Mr 45,000 protein and the Mr 48,000-50,000 doublet, and it decreases the labeling of the Mr 41,000 protein. Experiments with cholera toxin treatment of intact cells indicate that the Mr 45,000 protein is the major membrane protein ADP-ribosylated by the toxin in vivo. Cholera toxin increases cAMP levels in RAW264 cells, but increased cAMP levels do not correlate with inhibition of chemotaxis, because isoproterenol and forskolin, which also increase cAMP levels, have no effect on chemotaxis.     

Adenylate cyclase activators and inhibitors, cyclic nucleotide analogs, and phosphatidylinositol: effects on interrenal function of coho salmon (Oncorhynchus kisutch) in vitro

The role of cyclic AMP (cAMP) as mediator of ACTH action on interrenal steroidogenesis was evaluated in juvenile coho salmon (Oncorhynchus kisutch). Head kidneys (containing the interrenal cells) were incubated in the absence or presence of putative adenylate cyclase activators (forskolin and cholera toxin), ACTH combined with putative adenylate cyclase inhibitors (hydrolysis-resistant ATP analogs), dibutyryl cyclic (dbc) AMP, dbcGMP, or phosphatidylinositol. The cortisol content of the incubation medium was subsequently determined by radioimmunoassay. Forskolin markedly stimulated cortisol secretion by interrenal cells. Adenylate cyclase inhibitors depressed the steroidogenic response to ACTH. Dibutyryl cAMP, but not dbcGMP, enhanced steroid secretion. Thus, cAMP seems to be an important "second messenger" for ACTH action on salmon interrenal cells. In contrast to findings in mammalian adrenocortical cells, exogenous phosphatidylinositol and cholera toxin failed to stimulate corticosteroid secretion in salmon interrenal cells. However, it was unclear whether these negative findings were an artifact resulting from the use of kidney tissue fragments instead of isolated interrenal cells.     

Characterization of cell surface adenosine 3',5'-monophosphate-binding proteins in Y-1 mouse adrenal tumor cells

Adrenal cortical cells are known to export cAMP and have binding proteins and cAMP-dependent protein kinase activity associated with their plasma membranes. Because these properties suggest a function for extracellular cAMP, we have undertaken a search for specific cell surface receptors for this cyclic nucleotide. Y-1 mouse adrenal tumor cells actively export cAMP by an energy-dependent process. Analysis of Scatchard plots of the equilibrium binding of [3H]cAMP to these cells indicate the existence of two classes of cAMP binders: one with high affinity (ka = 2.9 X 10(9) M-1) and another with low affinity (ka = 7.0 X 10(7) M-1). The cell surface localization of these binders was established by the sensitivity of both the [3H]cAMP-binding proteins and the [32P]8-N3-cAMP photoaffinity labeled proteins of intact cells to mild trypsin digestion and by the surface distribution of a BSA-O2-monosuccinyl cAMP-gold complex revealed by electron microscopy. Analysis of radioautograms of cell surface cAMP-binding proteins from confluent monolayer tumor cells, photoaffinity labeled with [32P]8-N3-cAMP and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major 32P-labeled protein bands which were indistinguishable from the 49,000 and 55,000 mol wt regulatory subunits of the cytosolic protein kinase isoenzymes of this cell. These observations along with the demonstration of cell surface, cAMP-dependent protein kinase activity in the mouse adrenal tumor cell strongly suggest that these cAMP-binding proteins function as regulatory proteins for cell surface protein kinases.     

Prostaglandin F2 alpha-induced calcium transient in ovine large luteal cells: II. Modulation of the transient and resting cytosolic free calcium alters progesterone secretion

A previous study demonstrated that prostaglandin F2 alpha (PGF2 alpha) stimulates a transient increase in cytosolic free Ca2+ levels [( Ca2+]i) in ovine large luteal cells. In the present study, the magnitude of the PGF2 alpha (0.5 microM)-induced calcium transient in Hanks' medium (87 +/- 2 nM increase above resting levels) was reduced (P less than 0.05) but not completely eliminated in fura-2 loaded large luteal cells incubated in Ca2(+)-free or phosphate- and carbonate-free medium (10 +/- 1 nM, 32 +/- 6 nM, above resting levels; respectively). Preincubation for 2 min with 1 mM LaCl3 (calcium antagonist) eliminated the PGF2 alpha-induced calcium transient. The inhibitory effect of PGF2 alpha on secretion of progesterone was reduced in Ca2(+)-free medium or medium plus LaCl3. Resting [Ca2+]i levels and basal secretion of progesterone were both reduced (P less than 0.05) in large cells incubated in Ca2(+)-free medium (27 +/- 4 nM; 70 +/- 6% control, respectively) or with 5 microM 5,5'-dimethyl bis-(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (40 +/- 2 nM; 49 +/- 1% control; respectively). In addition, secretion of progesterone was inhibited (P less than 0.05) by conditions that increased (P less than 0.05) [Ca2+]i; that is LaCl3 ([Ca2+]i, 120 +/- 17 nM; progesterone, 82 +/- 8% control) and PGF2 alpha ([Ca2+]i, 102 +/- 10 nM; progesterone, 82 +/- 3% control). In small luteal cells, resting [Ca2+]i levels and secretion of progesterone were reduced by incubation in Ca2(+)-free Hanks ([Ca2+]i, 28 +/- 2 nM; progesterone, 71 +/- 6% control), however, neither LaCl3 nor PGF2 alpha increased [Ca2+]i levels or inhibited secretion of progesterone. The findings presented here provide evidence that extracellular as well as intracellular calcium contribute to the PGF2 alpha-induced [Ca2+]i transient in large cells. Furthermore, whereas an adequate level of [Ca2+]i is required to support progesterone production in both small and large cells, optimal progesterone production in large cells depends upon an appropriate window of [Ca2+]i.     

Phorbol ester stimulates progesterone production by isolated bovine luteal cells

The tumor promoter, phorbol 12-myristate-13-acetate (PMA), is known to modulate the response of several steroidogenic tissues presumably by activating a Ca++- and phospholipid-dependent protein kinase (protein kinase C). The presence of this kinase has been demonstrated in bovine corpus luteum, although its role in steroidogenesis by these cells is unknown. We report here the effects of PMA on progesterone production by the enzymically dispersed bovine luteal cells in vitro. PMA (1-50 nM) produced a dose- and time-related increase in progesterone production by the luteal cells. The maximum stimulation was achieved with 10 nM PMA. Higher concentrations of PMA led to a decline of steroidogenesis close to the basal level. A nonpromoting derivative, 4 alpha-phorbol 12,13-didecanoate had no effect. The PMA-induced stimulation of progesterone production was not associated with a change in the cAMP level. PMA added together with suboptimal doses of human CG, 8Br-cAMP, cholera toxin, or forskolin significantly increased the amount of progesterone produced. PMA as well as human CG-induced steroidogenesis was sensitive to cycloheximide inhibition. The conversion of exogenous pregnenolone or 25-hydroxycholesterol to progesterone was not altered by PMA. We conclude that PMA at nanomolar concentrations is able to stimulate progesterone production by bovine luteal cells and that the site of action of PMA is distal to the formation of cAMP but before the formation of pregnenolone. The observed effects of PMA in luteal cells are probably linked to its ability to activate protein kinase C, since a diacylglycerol could mimic the steroidogenic action of PMA.     

Selective amplification of luteinizing hormone by adenosine in rat luteal cells

Adenosine amplification of LH-stimulated cAMP accumulation in rat luteal cells is rapid and dependent on mitochondrial ATP production. The objective of the present studies was to determine if this effect of adenosine is specific for LH and to gain information on the mechanism of the ATP-dependent amplification of LH action in rat luteal cells. Adenosine significantly amplified maximum cAMP accumulation in response to LH, isoproterenol, forskolin, and cholera toxin. However, amplification of this response by adenosine was significantly greater for LH than for the other agonists. The relative order of amplification by adenosine was LH greater than isoproterenol greater than forskolin greater than cholera toxin; the relative magnitudes of amplification by adenosine were 1, 0.6, 0.2, and 0.2, respectively. Neither LH, isoproterenol, forskolin, nor cholera toxin had any effect on cellular levels of ATP, and adenosine produced a similar rate of increase and maximal levels of ATP in the presence of all agonists. Ionomycin, a calcium ionophore, inhibited LH- and cholera toxin-stimulated cAMP accumulation and produced a dose-dependent depletion of ATP. Adenosine reversed the inhibitory effect of ionomycin on LH-stimulated cAMP accumulation and cellular levels of ATP. However, adenosine did not reverse the inhibitory effect of ionomycin on cholera toxin-stimulated cAMP accumulation, although its effects on cellular ATP levels were identical to those on LH. Thus, the selective amplification of LH by adenosine is not merely a substrate effect on adenylate cyclase activity. The nature of adenylate cyclase activation by cholera toxin and forskolin and the weak amplification by adenosine of these agonists compared to that of LH indicate that the site of the ATP-dependent action of adenosine appears to be before or on the G-protein of adenylate cyclase. We suggest that adenosine, by an ATP-dependent process, either increases the availability of functional LH receptors or increases coupling between the LH receptor and adenylate cyclase.     

Regulation of the content and phosphorylation of RII by adenosine 3',5'-monophosphate, follicle-stimulating hormone, and estradiol in cultured granulosa cells

The mechanisms by which FSH and cAMP induce receptors for LH (RLH) and increase progesterone (P) production in estradiol (E)-primed ovarian granulosa cells remain unclear, but may involve increases in the regulatory subunit of cAMP-dependent protein kinase II (RII) and the phosphorylation of specific cellular proteins. To examine the relationship of these events, primary cultures of granulosa cells (10(6) cells/ml) from E-treated (1.5 mg/day for 3 days) immature female rats were incubated with 10 nM E with or without FSH (25 ng/ml) for 0-120 h. The cytosolic content of RII was analyzed by four techniques: 1) immunoblotting using an antibody to bovine heart RII; 2) photoaffinity labeling with [32P]8-azido-cAMP; 3) phosphorylation with [gamma-32P]ATP with or without 2 microM cAMP or with the catalytic subunit of cAMP-dependent protein kinase; and 4) phosphorylation of intact cells with [32P] orthophosphate. All approaches revealed a time-dependent 5- to 6-fold increase in RII content in granulosa cells cultured for 48 h with E and FSH compared to that in cells treated with E alone. The content of RI, the regulatory subunit of protein kinase type I, remained low throughout the culture period regardless of hormone treatment. Granulosa cells were also cultured with E (10 nM) and 8-bromo-cAMP (8-Br-cAMP; 0.25-3 mM) or forskolin (0.5-100 microM), agents that increase intracellular cAMP, for 48 or 72 h. The cytosolic content and phosphorylation of RII were increased by culturing granulosa cells in E and 8-Br-cAMP (1 mM) or forskolin (50 microM) for 48 h. The increase in RII was associated with a FSH-mediated increase in the content and phosphorylation of other cAMP-dependent phosphoproteins. The increases in RII and cAMP-dependent phosphoproteins were associated with specific alterations in granulosa cell function: a FSH-mediated rise in 1) RLH [59.3 +/- 7.4 cpm/micrograms DNA (without FSH) to 1171.5 +/- 157 cpm/micrograms DNA (with FSH]) and 2) P accumulation in the medium [0.05 +/- 0.03 ng/ml (without FSH) to 25.3 +/- 4.6 ng/ml (with FSH]) at 48 h. A dose-dependent increase in the RLH and P accumulation in the medium was observed at 48 h of culture with E and 8-Br-cAMP or E and forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Requirement for ERK1/2 activation in the regulation of progesterone production in human granulosa-lutein cells is stimulus specific

This study was conducted to determine whether the ERK1/2 family of MAPKs can be modulated by physiological regulators of the human corpus luteum, and whether this activation is important for progesterone secretion in human granulosa-lutein (hGL) cells. Human LH (hLH), hCG, and agents that indirectly elevate cAMP [cholera toxin, forskolin, (Bu)(2)cAMP], time- and dose-dependently activated ERK1/2 in hGL cells. ERK1/2 activation was reduced by preincubation with PKA inhibitors, including myristoylated PKI, suggesting that cAMP mediates ERK1/2 activation. Two structurally distinct inhibitors of MAPK kinase (MEK), PD 98059 and U 0126, abrogated hLH/hCG-induced ERK1/2 activation, but had no effect on hLH-, hCG-, or 22R-hydroxycholesterol-stimulated progesterone secretion. In contrast, both inhibitors blocked cholera toxin-, forskolin-, and (Bu)(2)cAMP-induced ERK1/2 phosphorylation concomitant with a reduction in progesterone secretion. The known luteotropin, PGE(2), promoted MEK- and cAMP-dependent activation of ERK1/2, and inhibitors of either MEK or PKA decreased PGE(2)-induced progesterone synthesis. Our findings demonstrate that the requirement for ERK1/2 activation as a regulator of progesterone synthesis in hGL cells is stimulus dependent, and that the MEK inhibitor-sensitive step is distal to cAMP generation, but proximal to the conversion of cholesterol to pregnenolone.     

Regulation of steroidogenesis in jc-410, a stable cell line of porcine granulosa origin

We investigated the regulation of steroidogenesis in a cell line of porcine granulosa origin, JC-410. Cells responded to the protein kinase-A activators, cholera toxin and forskolin, with increased accumulation of intracellular cAMP. Histochemically, cells were shown to contain 3beta-HSD, the enzyme which converts pregnenolone to progesterone. The JC-410 cells produced progesterone and responded to the protein kinase-A activators with an increase in progesterone synthesis. Progesterone levels were also increased by 25-hydroxycholesterol, pregnenolone, estradiol and androstenedione. Follicle-stimulating hormone and luteinizing hormone had no effect on cAMP or progesterone accumulation. Androstenedione was required for the synthesis of estradiol by JC-410 cells. Steady-state levels of mRNA for the steroidogenic enzymes 3beta-HSD and P450scc were increased by treatment with cholera toxin, whereas P450arom was not changed. These cells express the steroidogenic enzymes genes in a similar fashion to primary cultures of porcine granulosa cells. The JC-410 cells may represent a valuable model to study second messenger regulation and the molecular mechanisms involved in steroidogenesis in granulosa cells.     

Identification, characterization, and hormonal regulation of 3', 5'-cyclic adenosine monophosphate-dependent protein kinases in rat Sertoli cells.

Recent studies have disclosed multiple isoforms of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA) at the protein and messenger RNA (mRNA) levels. The purpose of the present study was to identify, characterize, and quantify individual R subunits in rat Sertoli cells both at the mRNA and protein levels. Unstimulated Sertoli cells contain high levels of R (approximately 9.2 +/- 0.8 pmol/mg protein) and C (approximately 7.3 +/- 0.7 pmol/mg protein). Stimulation with (Bt)2cAMP (0.1 mM) for 24 and 48 h revealed a time-dependent increase in [3H]cAMP-binding activity. During the same time period the catalytic activity remained relatively constant, resulting in an increase in the R/C ratio from approximately 1.3 to 3.0. Using diethylaminoethyl cellulose chromatography, 8-N3-[32P]cAMP photoaffinity labeling, autophosphorylation by gamma-[32P]ATP, and specific antibodies, we show that unstimulated Sertoli cells contain approximately 75% RI alpha, 25% RII alpha, and very low levels of RII beta. Stimulation of Sertoli cells with (Bt)2cAMP (0.1 mM, 48 h) was associated with a 2.1-fold increase in RI alpha (6.6-14 pmol/mg) and a 10- to 20-fold increase in RII beta (less than 0.1-1.1 pmol/mg), with little or no change in RII alpha (1.9-2.3 pmol/mg). Treatment with cAMP was associated with a slight increase in RI/RII ratio (3.3-4.1). mRNA levels for RII beta increased 30- to 50-fold after (Bt)2cAMP stimulation, whereas only minor changes in mRNA levels for RI alpha, RII alpha, and C alpha were observed (1.5- to 2.0-fold). mRNA levels for RI beta, C beta, and C gamma were not detected in either unstimulated or in cAMP-stimulated Sertoli cells. It is concluded that chronic treatment with cAMP changes the relative proportion of R subunits of PKA in a manner reflecting the changing levels in respective mRNAs. Furthermore, such treatment is associated with the appearance of a new PKA R subunit (RII beta), which is absent in untreated Sertoli cells.     

Steroid secretion by ovarian cells of the Japanese quail (Coturnix coturnix japonica)

Mixed cell preparations (theca plus granulosa) prepared from the hierarchy of follicles of quails ovaries were incubated under defined conditions with or without the addition of ovine luteinizing hormone (oLH), ovine follicle stimulating hormone (oFSH), theophylline, cycloheximide, or dibutyryl cyclic adenine monophosphate (db cAMP); or in the presence of androstenedione or testosterone as aromatizable substrate. Steroids secreted into the medium during the 4-hr incubation period were assayed by radioimmunoassay. Cells from the largest follicles (F1) secreted predominantly progesterone, were stimulated by LH and db cAMP, and the response was potentiated by theophylline, but FSH had no stimulatory effect. The F1 cells showed increasing basal and LH-stimulated responses between 18 and 12 hr before the next expected oviposition. Cells from the smaller follicles (F3 and F4) secreted predominantly estrogens, and were stimulated by FSH but not by db cAMP and only to a small extent by theophylline. Addition of androstenedione (10(-7) M) or testosterone (10(-7) M) enhanced estrogen secretion, which was further raised by the simultaneous addition of FSH. These results confirm previous reports on the sites of steroid secretion within quail follicles and suggest that while the action of LH on the cells from F1 follicles may be mediated in part through the adenylate cyclase system, the action of FSH on the smaller follicles may be substantially independent of cAMP.