The effect of calcium ions on testosterone production in Leydig cells from rat testis.

Leydig-cell suspensions, prepared from rat testes, were incubated with different amounts of Ca2+ with and without added luteinizing hormone. The basal testosterone production in the absence of luteinizing hormone was unaffected by the Ca2+ concentration in the incubation medium. The luteinizing hormone-stimulated testosterone production, however, was progressively decreased in the absence of Ca2+ to one-third of that with 2.50 mM-Ca2+. This decrease in luteinizing hormone-stimulated testosterone production was independent of the different concentrations of luteinizing hormone (0-10mug/ml) used and could be restored by the addition of Ca2+ to the incubation medium. The restoration of the stimulation was achieved within 30 min after the addition of Ca2+ to the medium. Activation of cyclic AMP-dependent protein kinase by luteinizing hormone was not decreased by omission of Ca2+ from the incubation medium, suggesting that Ca2+ may be involved in steroidogenesis at a stage beyond the luteinizing hormone receptor-adenylate cyclase-protein kinase system.     

The effects of calcium ionophore A23187 on interstitial cell steroidogenesis

The present study was performed to evaluate the effects of calcium ionophore A23187 on adenosine 3',5'-monophosphate (cyclic AMP) and testosterone production in rat interstitial cells. Interstitial cells were incubated in Krebs-Ringer solution with varying amounts of luteinizing hormone, pregnenolone, or A23187. Cyclic AMP and testosterone were measured in the incubation medium after 4 h incubation. A23187 (0.01--10 microgram/ml) caused progressive increases of cyclic AMP formation (from 0.18 +/- 0.02 (S.E.) pmol/10(6) cells for the control of 0.42 +/- 0.02 pmol/10(6) cells, P less than 0.025), while testosterone production remained unaltered. When varying amounts of A23187 were added concomitantly with luteinizing hormone (5 IU/l), A23187 inhibited luteinizing hormone-induced steroidogenesis in a dose-dependent manner, but it had no effect on luteinizing hormone-induced cyclic AMP formation. When pregnenolone (10(-6) M) was added to the cells, testosterone formation increased from 1.50 +/- 0.22 to 8.46 +/- 1.65 ng/10(6) cells. A23187 (1 microgram/ml) had no discernable effect on the conversion of pregnenolone to testosterone. The main effect of increased cytosol calcium on steroidogenesis seems to be at the steps beyond adenylate cyclase-cyclic AMP. These results suggest that calcium is important for the conversion of cholesterol to pregnenolone, while the steps beyond pregnenolone are relatively independent of Ca2+.     

Molecular control of rabbit follicular testosterone production. Role of protein and RNA after stimulation with luteinizing hormone.

The time and dose dependence of the relationship between uptake of labelled precursors into protein and RNA and production of testosterone by rabbit follicles was examined. Although testosterone production was stimulated by luteinizing hormone at concentrations between 0.1 and 10 microgram/ml, the uptake of [3H]leucine into protein was significant only when the concentration of luteinizing hormone was greater than 2.5 microgram/ml. Increased production of testosterone was observed within 15 min of stimulation with luteinizing hormone whereas uptake of [3H]leucine was only significant at 90 min. Puromycin (40 microgram/ml) and cycloheximide (10 microgram/ml) in the presence of luteinizing hormone inhibited the synthesis of both testosterone and protein. However, lower concentrations of puromycin (0.1, 1 and 10 microgram/ml) and cycloheximide (1 microgram/ml) had no effect on luteinizing hormone-induced testosterone production but significantly inhibited protein synthesis by 58, 37, 31 and 71%, respectively. Actinomycin D (20, 80 and 160 microgram/ml) alone and in combination with 5 microgram luteinizing hormone/ml severely inhibited uptake of [3H]uridine into RNA without affecting testosterone production. However, with 1 microgram actinomycin/ml, testosterone production was significantly (P less than 0.01) greater than in the presence of luteinizing hormone alone. These results cast doubt on the obligatory role of RNA and protein synthesis in rabbit ovarian follicular steroidogenesis.     

Inhibitory action of forskolin on adenylate cyclase activity and cyclic AMP generation

In testicular Leydig cells, forskolin causes the expected stimulation of cAMP and testosterone production and potentiates gonadotropin-induced responses, when present in concentrations of 1-10 microM. In addition, when added at lower doses that did not affect cAMP generation and testosterone responses (100 nM), forskolin caused an increase in sensitivity to hormonal stimulation for all cAMP pools (extracellular, intracellular, and receptor-bound) and a 70% reduction in the ED50 for human chorionic gonadotropin (hCG) stimulation of testosterone production. Forskolin-induced increases in receptor-bound cAMP were less effective than those elicited by hCG in stimulating steroidogenesis. In contrast to the well-known stimulatory actions of forskolin, low doses of the diterpene (in the picomolar to nanomolar range) markedly inhibited the production of cAMP and testosterone. Such inhibitory actions of low-dose forskolin were prevented by preincubation of Leydig cells with pertussis toxin before addition of forskolin and/or hCG. Low concentrations of forskolin also inhibited adenylate cyclase activation by GTP and luteinizing hormone, and this effect was prevented by pretreatment of cell membranes with pertussis toxin. These studies have defined the stimulatory effects of forskolin on Leydig-cell cAMP pools, including potentiation of the hormonal increase in receptor-bound cyclic AMP by forskolin, and have provided additional evidence for the functional importance of cAMP compartmentalization during hormonal stimulation of steroidogenesis. We have also demonstrated a novel, high-affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation, an effect that appears to be mediated by the Ni guanine nucleotide regulatory subunit of adenylate cyclase.     

The response of large and small luteal cells from the pregnant rat to substrates and secretagogues

Large (greater than 22 microns) and small (12-21 microns) luteal cells from Day 8 pregnant rats were separated by elutriation after enzyme dissociation. Aliquots of cells were incubated for 4 h at 37 degrees C in Medium 199 alone (control) or with medium containing dibutyryl cyclic adenosine 3', 5'-monophosphate (cAMP) at 0.5 mM or 5 mM; rat luteinizing hormone (LH) at doses of 1, 10, 100, or 1000 ng/ml; 10 micrograms/ml 25-OH-cholesterol; or 10 ng/ml testosterone. Production of progesterone, testosterone, and estradiol was measured by radioimmunoassay. Both cell types showed a similar increase in estradiol synthesis when stimulated with LH (1 microgram/ml) or dibutyryl cAMP (5 mM); however, large luteal cells aromatized exogenous testosterone, whereas small luteal cells did not. Large luteal cells produced increased amounts of progesterone at lower doses of dibutyryl cAMP (0.5 mM) and LH (10 ng/ml), compared to small cells, which required 5 mM dibutyryl cAMP or 1 microgram/ml LH for minimal stimulation. Dibutyryl cAMP (5 mM) also resulted in an increase of testosterone release from small luteal cells. Progesterone synthesis in both cell types was enhanced by 25-OH-cholesterol. These results suggest that the two cell types differ functionally with respect to steroidogenesis during pregnancy, and that the large luteal cells appear to be the primary site of progesterone and estradiol production at this stage of pregnancy.     

Regulation of the synthesis of lutropin-induced protein in rat testis Leydig cells

The mechanism of action of lutropin on the stimulation of the synthesis of a specific lutropin-induced protein in rat testis Leydig cells was investigated. Lutropin-induced protein has a mol.wt. of approx. 21000 and is detected by labelling the Leydig-cell proteins with [³⁵S]methionine, followed by separation by polyacrylamide-gel electrophoresis and radioautography of the dried gel. The incorporation of ³⁵S into lutropin-induced protein was used as an estimate for the synthesis of the protein. Incubation of Leydig cells with dibutyryl cyclic AMP or cholera toxin also resulted in the stimulation of synthesis of the protein. Synthesis of lutropin-induced protein, when maximally stimulated with 100ng of lutropin/ml, could not be stimulated further by addition of dibutyryl cyclic AMP. Addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, further increased synthesis of the protein in the presence of a submaximal dose of lutropin (10ng/ml) but not in the absence of lutropin or with maximal amounts of lutropin (100 and 1000ng/ml). Actinomycin D prevented the effect of lutropin on the stimulation of lutropin-induced protein synthesis when added immediately or 1h after the start of the incubation, but not when added after 5–6h. This is interpreted as reflecting that, after induction of mRNA coding for lutropin-induced protein, lutropin had no influence on the synthesis of the protein in the presence of actinomycin D. Synthesis of the protein was also stimulated in vivo by injection of choriogonadotropin into rats 1 day after hypophysectomy, and the time course of this stimulation of lutropin-induced protein synthesis in vivo was similar to that obtained by incubating Leydig cells in vitro with lutropin. From these results it is concluded that stimulation of lutropin-induced protein synthesis by lutropin is most probably mediated by cyclic AMP and involves synthesis of mRNA.     

Multiplication stimulating activity regulates ornithine decarboxylase in isolated porcine granulosa cells in vitro.

The polypeptide growth factor, multiplication stimulating activity (MSA), stimulates ornithine decarboxylase (ODC) activity in isolated porcine granulosa cells maintained under chemically defined conditions in vitro. The stimulatory action of MSA is saturable, and dose-dependent (0.1-1000 ng/ml). MSA effects are additive to those of luteinizing hormone (LH), but not those of follicle stimulating hormone (FSH). Stimulation of ODC activity by MSA requires cellular protein and RNA synthesis, and appears to be mediated independently of cyclic AMP. These observations provide the first demonstration of MSA action in the mammalian ovary.     

Effect of lutropin on phosphorylation of endogenous proteins in testis Leydig cells. Correlation with testosterone production

The effect of lutropin on phosphorylation of endogenous proteins in testis Leydig cells was investigated, by incubating purified Leydig cells with lutropin and [(32)P]P(i) followed by sodium dodecyl sulphate/polyacrylamide-slab gel electrophoresis of the [(32)P]phosphoproteins. The radioactivity of the proteins was quantified by densitometry of the radio-autograms obtained. The following results were obtained. 1. Lutropin increased the amount of (32)P incorporated into three proteins (A, B and C) with apparent mol.wts. of 14300, 57000 and 77600 respectively. 2. The increase in incorporation of (32)P into these proteins was detectable within 5min, reaching a maximum in approx. 20min. 3. The (32)P incorporated into protein B (but not proteins A and C) was significantly increased with 0.1 and 1.0ng of lutropin/ml. Incorporation of (32)P into all three proteins was significantly increased with 10ng of lutropin/ml, reaching a maximum with 100ng/ml. 4. Testosterone production was significantly increased with 1ng of lutropin/ml, and between 10 and 1000ng/ml the degree of stimulation of testosterone production and incorporation of (32)P into proteins A, B and C was similar. 5. Cyclic AMP production was significantly increased with 10ng of lutropin/ml and had not reached a maximum with 1000ng/ml. 6. In Leydig cells isolated from hypophysectomized rats 3h after injection of choriogonadotropin in vivo, phosphoproteins with the same molecular weights as proteins A, B and C were found. No further increases in incorporation of (32)P into these proteins were obtained when lutropin was added to the Leydig cells in vitro. 7. Dibutyryl cyclic AMP (but not follitropin or testosterone) also stimulated the incorporation of (32)P into proteins A, B and C in Leydig cells.     

Adenosine 3'':5''-cyclic monophosphate-dependence of protein kinase isoenzymes from mouse liver.

Conditions influencing the cyclic AMP-dependence of protein kinase (ATP-protein phosphotransferase, EC 2.7.1.37) during the phosphorylation of histone were studied. Protein kinase from mouse liver cytosol and the two isoenzymes [PK (protein kinase) I and PK II] isolated from the cytosol by DEAE-cellulose chromatography were tested. A relation between concentration of enzyme and cyclic AMP-dependence was observed for both isoenzymes. Moderate dilution of isoenzyme PK II decreased the stimulation of the enzyme by cyclic AMP. Isoenzyme PK I could be diluted 200 times more than isoenzyme PK II before the same decrease in cyclic AMP-dependence appeared. Long-term incubation with high concentrations of histone increased the activity in the absence of cyclic AMP relative to the activity in the presence of the nucleotide. This was more pronounced for isoenzyme PK II than for isoenzyme PK I. The cyclic AMP concentration needed to give half-maximal binding of the nucleotide was the same as the cyclic AMP concentration (Ka) at which the protein kinase had 50% of its maximal activity. The close correlation between binding and activation is also found in the presence of KCl, which increased the apparent activation constant (Ka) for cyclic AMP. With increasing [KCl], a progressively higher proportion of the histone phosphorylation observed in cytosol was due to cyclic AMP-independent (casein) kinases, leading to an overestimation of the degree of activation of the cyclic AMP-dependent protein kinases present. The relative contributions of cyclic AMP-dependent and -independent kinases to histone phosphorylation at different ionic strengths was determined by use of heat-stable inhibitor and phospho-cellulose chromatography.     

Effects of age and luteinizing hormone antiserum on human chorionic gonadotropin-stimulated testosterone secretion in young male rats

The steroidogenic capacity of young male rats of different ages was studied. Two days prior to sacrifice at 5, 10, 15, 20, 25 and 30 days of age, the rats in treatment groups were given intramuscularly either human chorionic gonadotropin (HCG) at 20 I.U. twice daily/rat or luteinizing hormone (LH) antiserum (AS) at 0.25 ml twice daily/rat. Either saline or normal sheep serum (NSS) was given to control rats. The serum and testicular testosterone concentrations in the control rats averaged 0.85 +/- 0.03 ng/ml and 1.35 +/- 0.06 ng/mg testicular protein, respectively. At day-15 the serum and testicular testosterone concentrations in the HCG-treated rats had significantly increased to 9.30 +/- 0.85 ng/ml and 11.92 ng/mg of testicular protein, respectively. At the same age, the HCG-induced higher levels of serum and testicular testosterone concentrations were significantly reduced to 2.80 +/- 0.70 ng/ml and 6.02 +/- 1.00 ng/mg protein by concomitant administration of LH/AS and HCG. Our results suggest that the testosterone production in response to HCG stimulation is age-related. It was also determined that neutralization of circulating gonadotropin in LH/AS-treated rats decreased the sensitivity of Leydig cells to gonadotropin stimulation. This in vivo model should provide an excellent opportunity for the investigation of the testicular function in developing young males.     

The aging Leydig cell: 2. Two distinct populations of Leydig cells and the possible site of defective steroidogenesis

Using metrizamide gradient centrifugation two populations of Leydig cells were found in both 60-90 day-old and 24 month-old rats. Cells from both Band 2 (B2) and Band 3 (B3) responded to LH stimulation with increased cyclic AMP formation; however, only B3 cells produced significant amounts of testosterone. Cells from both B2 and B3 of the old rats synthesized less cyclic AMP and testosterone than cells from their younger counterparts. In response to LH stimulation, 0.01 - 1.0 mIU/ml, no appreciable difference of cyclic AMP formation could be detected between young and old Leydig cells. Maximal testosterone production occurred when 1 mIU/ml LH was used. Only when LH concentration was increased to 10 and 100 mIU/ml, did young Leydig cells produce significantly more cyclic AMP than old Leydig cells. After addition of 5X10(-7)M of pregnenolone or progesterone to the incubation medium, both young and old Leydig cells produced comparable amounts of testosterone. These results demonstrate no impairment of old rat Leydig cells to synthesize testosterone from pregnenolone and progesterone.     

Regulation of testicular function by insulin and transforming growth factor-beta

Hyperinsulinism is associated with disorders of androgen production in humans. We have studied the effects of insulin and insulin-like growth factor-1 on androgen production in vitro using a crude preparation of mouse Leydig cells incubated with luteinizing hormone in a serum-free medium. We found a positive correlation between testosterone production and the luteinizing hormone dose over 3 hours. Exposure of the cells for 1 hour to insulin (1 micrograms/ml) prior to the addition of luteinizing hormone significantly augmented the amount of testosterone produced in response to the gonadotropin when added after this preincubation. In contrast, prior exposure of the cells to proinsulin (30 micrograms/ml), insulin-like growth factor-1 (30 ng/ml), or epidermal growth factor-1 (1 micrograms/ml) did not influence the testosterone response to luteinizing hormone. Transforming growth factor-beta reduced the testosterone response to luteinizing hormone. Transforming growth factor-beta (1,000 pg/ml) blocked the insulin augmentation of luteinizing hormone-stimulated testosterone production. We conclude that insulin has an endocrine effect on testosterone production by mouse Leydig cells in vitro. Furthermore, the Leydig cell response to insulin is itself sensitive to interaction with transforming growth factor-beta which may operate as part of the paracrine control of Leydig cell function.     

Effect of protein-synthesis inhibitors on testosterone production in rat testis interstitial tissue and Leydig-cell preparations.

Luteinizing-hormone-stimulated testosterone biosynthesis was inhibited by cycloheximide during incubation of rat testis intersitial tissue in vitro and also by puromycin and cycloheximide during incubation of Leydig-cell preparations, but not by chloramphenicol. These results suggest that a protein regualtor(s) formed by cytoplasmic protein synthesis is involved in steroidogenesis in the rat testis. The specific effect of cycloheximide and puromycin on protein synthesis rather than on other non-specific processes is suggested by the inhibition of protein synthesis and steroidogenesis with different doses of the inhibitors and the lack of effect of cycloheximide on luteinizing-hormone-induced adenosine 3':5'-cyclic monophosphate production. Stimulation of testosterone production by luteinizing hormone during superfusion of interstitial tissue was detectable within 10-20 min and reached a maximum of 120 min, and thereafter slowly decreased. Cycloheximide added at maximum steroid production caused a rapid decrease in testosterone synthesis which followed first-order kinetics (half-life 13 min), thus indicating that the protein regulator(s) has a short half-life. No effect of cycloheximide, puromycin or chloramphenicol on testosterone production in the absence of added luteinizing hormone was found, suggesting that the basal production of testosterone is independent of protein synthesis.     

Progesterone is a mediator in the ovulatory process of the in vitro-perfused rat ovary

The role of steroids in the ovulatory process of the rat was explored in an in vitro perfusion system. Immature rat ovaries were primed with pregnant mare's serum gonadotropin (20 IU) and perfused in a recirculating perfusion system for up to 20 h. Unstimulated ovaries did not ovulate whereas the addition of luteinizing hormone (LH; 0.1 micrograms/ml) plus 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) resulted in 13.6 +/- 1.0 ovulations per treated ovary. Addition of an inhibitor of 3 beta-hydroxysteroid dehydrogenase (Compound A; 10 micrograms/ml) significantly (p less than 0.01) decreased the number of ovulations after LH plus IBMX stimulation (1.6 +/- 0.8 ovulations per treated ovary). This inhibition was reversed by the addition of progesterone, with 6.6 +/- 2.1 ovulations at approximately 100 ng/ml progesterone in the perfusion medium and 15.2 +/- 3.4 ovulations at approximately 3000 ng/ml progesterone. The addition of testosterone (10 micrograms/ml) did not reverse the inhibition of ovulations by Compound A. High levels of progesterone in the perfusion medium (greater than 3000 ng/ml) did not significantly (p greater than 0.05) increase the number of ovulations after stimulation with LH plus IBMX (20.2 +/- 4.8 ovulations), and progesterone (greater than 3000 ng/ml) was not by itself able to induce ovulations. Addition of LH plus IBMX resulted in a marked increase in the levels of progesterone, testosterone, and estradiol in the perfusion medium. The production of these steroids was almost completely inhibited by the addition of Compound A, and the levels of testosterone and estradiol were restored by the addition of high concentrations of progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine 3',5'-monophosphate-dependent protein kinase(s) in diploid and SV40 transformed human fibroblasts.

Cyclic AMP-dependent protein kinases (EC 2.7.1.37; ATP:protein phosphotransferase) in the human diploid fibroblast WI-38 and an SV40-transformant WI-38-VA13-2RA (VA13) have been compared on the basis of their concentrations in cells, isoenzyme composition and susceptibility to hormonal activation. In high population density cultures, total soluble cyclic AMP-dependent kinase activities measured with histone were essentially the same in WI-38 and VA13. Two soluble protein kinase forms separated by chromatography on DEAE-cellulose were present in both cell lines. The concentration of cyclic AMP required for half-maximal activation of both enzyme forms was 10-30 nM. Overall kinase stimulation was greater for the Peak I enzymes. Kinase activation induced in the presence of 0.5 M KCl was more rapid and complete for the Peak I enzymes. Under conditions which elevated the concentration of cyclic AMP in WI-38 and VA13 cells the activities of the soluble histone kinases were increased. Incubation of the cells with either of 5.7 micronM prostaglandin E1 or 1 micronM isopropylnorepinephrine induced complete activation of the cyclic AMP-dependent histone kinases within 5 min and maintained the effect for 20 min. When intracellular cyclic AMP levels were raised by prostaglandin E1, activation of glycogen phosphorylase (assayed-AMP) suggested that this enzyme cascade involving cyclic AMP-dependent protein kinase(s) was intact and responsive in both cell lines.     

The role of protein kinase activation in the control of steroidogenesis by adrenocorticotrophic hormone in the adrenal cortex

A method has been developed for investigation of the effect of adrenocorticotrophic hormone (ACTH) on the state of activation of a cyclic AMP-dependent protein kinase within cells of the adrenal cortex. Enzyme activity was measured in terms of the quantity of (32)P transferred from [gamma-(32)P]ATP to histone under conditions in which bound cyclic AMP did not dissociate from the regulatory subunit of the protein kinase ACTH (1x10(-2)i.u./ml) caused a rapid and complete activation of the cyclic AMP-dependent protein kinase activity within 2min of hormone addition to the isolated cells. In response to a range of ACTH concentrations a sigmoid log dose-response curve for protein kinase activation was obtained, with half-maximal stimulation attained at about 1x10(-3)i.u./ml. However, some low doses of ACTH that elicited a marked (but submaximal) steroidogenic response failed to cause a clear stimulation of protein kinase activity in isolated adrenal cells. Theophylline (2mm) potentiated the effect of ACTH on protein kinase activity. The results implicate an important role for protein kinase in ACTH action on the adrenocortical cell.     

Adenosine 3′,5′-monophosphate dependent phosphorylation of ribosomes and ribosomal subunits from bovine corpus luteum

In a previous publication the purification and properties of two protein kinases (KI and KII) from a soluble fraction of bovine corpus luteum and the stimulation of the latter fraction by cyclic AMP and luteinizing hormone was reported (Menon, K.M.J. (1973) J. Biol. Chem. 248, 494–501). We have now studied the effects of cyclic AMP and luteinizing hormone on ribosomal protein phosphorylation of corpus luteum by protein kinase II. Protein kinase II catalyzed the phosphorylation of ribosomes by transfer of terminal phosphate of ATP to ribosomal proteins. Extraction with hot trichloroacetic acid and non-aqueous solvent revealed that about 80% of total radioactivity incorporated remain associated with the protein residue. Radioactivity was identified in the phosphoserine and phosphothreonine residues of polypeptides by high voltage paper electrophoresis. The extent of phosphorylation was stimulated by cyclic AMP but not by luteinizing hormone. At least 9 proteins of 80-S ribosomes and 12 proteins of the 60-S ribosomal subunit were phosphorylated in the presence of cyclic AMP as resolved by urea polyacrylamide gel electrophoresis. However, only one major and four minor bands were phosphorylated in the case of 40-S ribosomal subunit under the influence of cyclic AMP. The ribosomal protein phosphorylation catalyzed by protein kinase II is regulated by cyclic AMP whereas luteinizing hormone has no effect on ribosome phosphorylation.     

Testosterone directly induces progesterone production and interacts with physiological concentrations of LH to increase granulosa cell progesterone production in laying hens (Gallus domesticus)

Blocking testosterone action with immunization or with a specific antagonist blocks the preovulatory surge of progesterone and ovulation in laying hens. Thus, testosterone may stimulate progesterone production in a paracrine fashion within the ovary. To test this hypothesis, we evaluated the effects of testosterone and its interaction with LH on the production of progesterone by granulosa cells in culture. Hen granulosa cells obtained from preovulatory follicles were cultured in 96 well plates. The effects of testosterone (0-100ng/ml) and/or LH (0-100ng/ml) were evaluated. LH-stimulated progesterone production in a dose response manner up to 10ng/ml (p<0.01). Testosterone, up to 10ng/ml, increased progesterone production in a dose response manner in the absence of LH and at all doses of LH up to 1ng/ml (p<0.001). However, at supraphysiological concentrations of LH (10 and 100ng/ml) there was no further increase in progesterone production caused by testosterone (p>0.05). Finally, the addition of 2-hydroxyflutamide (0-1000mug/ml) to hen granulosa cells cultured with 10ng/ml of testosterone reduced progesterone production in a dose response manner (p<0.001). In conclusion, testosterone stimulates progesterone production in preovulatory follicle granulosa cells and interacts with physiological concentrations of LH to increase progesterone production. In addition, testosterone stimulation on granulosa cells is specific since the testosterone antagonist decreased testosterone stimulatory action.     

Adenosine 3'5'-m onophosphate dependent phosphorylation of ribosomes and ribosomal subunits from bovine corpus luteum.

In a previous publication the purification and properties of two protein kinases (KI and KII) from a soluble fraction of bovine corpus luteum and the stimulation of the latter fol. Chem. 248,494-501). We have now studied the effects oc cyclic AMP and luteinizing hormone on ribosomal protein phosphorylation of corpus luteum by protein kinase II. Protein kinase II catalyzed the phosphorylation of ribosomes by transfer of terminal phosphate of ATP to ribosomal proteinsmextraction with hot trichloroacetic acid and non-aqueous solvent revealed that about 80% of total radioactivity incorporated remain associated with the protein residue. Radioactivity was identified in the phosphoserine and phosphothreonine residues of polypeptides by high voltage paper electrophoresis; The extent of phosphorylation was stimulated by cyclic AMP but not by luteinizing hormonemat least 9 proteins of 80-S ribosomes and 12 proteins of the 60-S ribosomal subunit were phosphorylated in the presence of cyclic AMP as resolved by urea polyacrylamide gel electrophoresis. However, only one major and four minor bands were phosphorylated in the ase of 40-S ribosomal subunit under the influence of cyclic AMP. The ribosomal protein phosphorylation catalyzed by protein kinase II is regulated by cyclic AMP wherease luteinizing hormone has no effect on ribosome phosphorylation.     

Dual effects of staurosporine on arachidonic acid metabolism in rat peritoneal macrophages

Staurosporine is a microbial anti-fungal alkaloid having a most potent inhibitory activity on protein kinase C and is recently found as a non-12-O-tetradecanoylphorbol-13-acetate (non-TPA)-type tumor promoter of mouse skin, although tumor promotion induced by a TPA-type tumor promoter teleocidin is suppressed by staurosporine. When rat peritoneal macrophages were incubated in the medium containing various concentrations of staurosporine, prostaglandin E2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were stimulated at concentrations of 1 and 10 ng/ml. But higher concentrations of staurosporine such as 100 and 1000 ng/ml showed no stimulative effect on prostaglandin E2 production although cytoplasmic free calcium levels were increased in a dose-dependent manner. Staurosporine-induced stimulation of prostaglandin E2 production was inhibited by treatment with cycloheximide, suggesting that a certain protein synthesis is prerequisite for the stimulation of arahcidonic acid metabolism. At higher concentrations (100 and 1000 ng/ml), staurosporine inhibited TPA-type tumor promoter (TPA, teleocidin and aplysiatoxin)-induced stimulation of arachidonic acid metabolism probably due to the inhibition of protein kinases. Tumor promotion activity and anti-tumor promotion activity of staurosporine might be explained by the fact that the lower concentrations of staurosporine stimulate arachidonic acid metabolism and the higher concentrations of staurosporine inhibit the tumor promoter-induced arachidonic acid metabolism, respectively.