Effect of different steroidogenic stimuli on protein phosphorylation and steroidogenesis in MA-10 mouse Leydig tumor cells.

Numerous studies have indicated that treatment of Leydig cells with gonadotropin results in increased levels of intracellular cAMP, binding of cAMP to and activation of protein kinase A, phosphorylation of proteins, synthesis of new proteins and eventually, stimulation of steroidogenesis. In addition, recent studies have indicated that protein phosphorylation is an indispensable event in the production of steroids in response to hormone stimulation in adrenal cells. Because of the important role of phosphorylation in steroidogenic regulation, we investigated the effects of human chorionic gonadotropin (hCG), dibutyryl cyclic AMP (dbcAMP), forskolin and the phorbol ester, phorbol-12-myristate 13-acetate (PMA) on protein phosphorylation in MA-10 mouse Leydig tumor cells. Cells were stimulated with different steroidogenic compounds in the presence of [32P]orthophosphoric acid for 2 h and phosphoproteins analyzed by two-dimensional polyacrylamide gel-electrophoresis (PAGE). Results demonstrated an increase in the phosphorylation of four proteins (22 kDa, pI 5.9; 24 kDa, pI 6.7 and 30 kDa, pI 6.3 and 6.5) in response to 34 ng/ml hCG, 1 mM dbcAMP and 100 microM forskolin. Conversely, treatment of cells with PMA increased the phosphorylation of only one of these proteins (30 kDa, pI 6.3). At least two of these proteins (30 kDa, pI 6.5 and 6.3) appear to be identical to proteins which we and others have shown to be synthesized in response to trophic hormone stimulation in adrenal, luteal and Leydig cells. In addition, they also appear to be identical to adrenal cell mitochondrial proteins demonstrated to be phosphorylated in response to ACTH. These data indicate that proteins similar to those phosphorylated in adrenal cells in response to ACTH are phosphorylated in hormone stimulated testicular Leydig cells and that these proteins may be involved in steroidogenic regulation.     

Regulation of progesterone secretion in human syncytiotrophoblast in culture by human chorionic gonadotropin.

In the present investigation we sought to define the specific sites in the pathway of placental progesterone biosynthesis that underlie the action of human chorionic gonadotropin (hCG). When the cells were challenged with dibutryl cAMP (dbcAMP), forskolin or isobutylmethylxanthine, they produced significantly higher amounts of progesterone which in the presence of the hCG antibody was reduced to the level of the control set of cells. Trophoblast cells cultured in serum free medium with 25-hydroxycholesterol (25-OHC) produced increased amounts of progesterone. In the presence of hCG antibody at a concentration which neutralized the secreted hCG, the steroid production was completely blocked, even when the 25-OHC was added to the medium. Also, direct quantitation of the cytochrome P450 SCC enzyme in the absence of hCG indicated a significant decrease. The exogenous addition of low density lipoproteins (LDL) increased the progesterone secretion by the trophoblast cells in culture. Neutralization of hCG by the antibodies, however, drastically reduced the LDL induced progesterone secretion, which was restored by the addition of dbcAMP to the medium. Based on these findings, we suggest a stimulatory effect of hCG on normal trophoblast cells at the level of LDL utilization and cytochrome P450 SCC enzyme. Since dbcAMP could mimic these actions of hCG, the data suggest a possible autocrine/paracrine role of hCG on the trophoblast cells. An additive effect of hCG and cAMP on progesterone secretion observed in our studies, indicate that apart from hCG, adenylate cyclase activity may also be regulated by other factors.     

Activation of protein kinase C potentiates cyclic AMP production and stimulates steroidogenesis in differentiated ovarian granulosa cells

Gonadotropin-stimulated steroidogenesis in the differentiating ovarian granulosa cell is mediated through the activation of cAMP-dependent protein kinase, and is also modulated by calcium-dependent mechanisms. Granulosa cells contain calcium-activated, phospholipid-dependent protein kinase (C kinase), and show an increase in phosphatidylinositol turnover in response to GnRH agonist analogs. To evaluate the role of C kinase in ovarian steroidogenesis, the potent phorbol ester, TPA, and the permeant diacylglycerol, OAG, were used to activate C kinase in granulosa cells from PMSG-treated immature rats. Both TPA and OAG caused dose-dependent stimulation of progesterone production without affecting intra- or extracellular cAMP levels. However, the maximum steroid responses to these compounds were less than those stimulated by cAMP. The ED50 for TPA-stimulated progesterone production was 3 nM, which is close to the known Km for activation of C kinase. Stimulation of steroidogenesis was only observed with biologically-active phorbol esters and permeant diacylglycerols such as OAG and DOG. Exposure of granulosa cells to phospholipase C also increased progesterone production in a dose-dependent manner without changing the cAMP content. Although TPA and OAG did not increase basal cAMP production, both agents enhanced the cAMP responses stimulated by hCG and forskolin; likewise, phospholipase C alone did not change cAMP production but caused a dose-dependent increase in the cAMP responses to hCG and forskolin. These results demonstrate that activation of C kinase promotes steroidogenesis in ovarian granulosa cells, and potentiates the activation of adenylate cyclase by hCG and forskolin. Such findings support the possibility that the calcium, phospholipid-dependent enzyme could be involved in the regulation of progesterone production by hormonal ligands such as gonadotropins and GnRH.     

Role of cAMP in modulating intrafollicular progesterone levels and oocyte maturation in amphibians (Rana pipiens)

The role of cAMP in regulating follicular progesterone levels and oocyte maturation was investigated following in vitro culture of amphibian (Rana pipiens) ovarian follicles. Intrafollicular levels of cAMP were manipulated with the use of a stimulator of cAMP synthesis (forskolin) or by exogenous addition of cAMP alone or either of these in combination with an inhibitor of cAMP catabolism (3-isobutyl-1-methyl xanthine, IBMX). Follicular progesterone content was determined by RIA and oocyte maturation was assessed cytologically. In the presence of increasing doses of forskolin (0-3 microM), cAMP (0-3 mM), or dibutyryl cAMP (dbcAMP, 0-2.5 mM) increasing but low levels of progesterone were detected. Increasing doses of IBMX (0-0.09 mM) alone had no significant effect on follicular steroid content. Exogenous cAMP, dbcAMP, or IBMX (0.09 mM) suppressed hormone-induced oocyte maturation. Simultaneous exposure of follicles to increasing doses of both forskolin (0-3 microM) and IBMX (0-0.09 mM) markedly increased intrafollicular progesterone levels to those produced by frog pituitary homogenate (FPH). A marked increase in progesterone levels also occurred when follicles were exposed to exogenous cAMP (3 mM) and IBMX (0.09 mM). These results indicate that exogenous cAMP is incorporated by follicle cells and that forskolin effects are mediated through cAMP. Changes in follicular progesterone levels (increase and decrease) over time following FPH or cAMP manipulation (cAMP + IBMX or forskolin + IBMX) were essentially identical. In contrast to cAMP, cGMP was inactive in inhibiting hormone induced GVBD or stimulating follicular progesterone accumulation. Elevation of follicular and medium levels of progesterone resulting from FPH or cAMP stimulation required the presence of the somatic follicular cells. The decrease in follicular progesterone levels with prolonged culture was not associated with a corresponding increase in progesterone levels in the medium. The decrease in follicular progesterone levels appears to reflect steroid catabolism rather than loss of steroid to the culture medium. The results suggest that the level of intracellular cAMP in the follicle cells is modulated by the relative activity of the adenylate cyclase system and phosphodiesterase and that FPH can affect both components. Thus, intracellular levels of cAMP play a key role in regulating follicular progesterone levels and FPH action on the follicle cells. The steroidogenic capacity of follicle cells can be manipulated independently of FPH stimulation.     

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.     

Stimulation of cholesterol side-chain cleavage enzyme activity by cAMP and hCG in MA-10 Leydig tumor cells

Using a cloned Leydig tumor cell line (designated MA-10), we have studied the activity of cholesterol side-chain (CSCC) enzyme, the rate-determining step in steroidogenesis, in mitochondria isolated from cells pretreated either with human chorionic gonadotropin (hCG) or dibutyryl cyclic adenosine monophosphate (dbcAMP). Results showed a slight but significant increase in CSCC activity with treatment by cAMP (25% increase) and hCG (60% increase), as compared to mitochondria isolated from nontreated control cells. However, this stimulation of CSCC activity appears to be of limited significance when compared to the approximately 1000-fold or greater increase observed in progesterone production in the presence of hCG or dbcAMP. On the other hand, unstimulated MA-10 cells or isolated mitochondria efficiently converted 25-hydroxycholesterol and 22R-hydroxycholesterol into progesterone, and this conversion was not affected by cycloheximide. The addition of cholesterol to intact cells or to isolated mitochondria did not affect progesterone production. Our observations clearly indicate that given the proper hydroxy substrates (22R-hydroxycholesterol or 25-hydroxycholesterol), MA-10 Leydig cells are able to convert them into progesterone without any stimulation by steroidogenic stimuli, i.e. cAMP or hCG. Since MA-10 Leydig cells can efficiently convert 22R-hydroxycholesterol--an intermediate in CSCC reaction--into progesterone, these results suggest that the key regulatory step in the mechanism of trophic hormone-stimulated steroid production is the first hydroxylation step of the 3 sequential monooxygenation reactions involved in the conversion of cholesterol to pregnenolone.     

Oncogene-transformed granulosa cells as a model system for the study of steroidogenic processes

Highly steroidogenic granulosa cell lines were established by transfection of primary granulosa cells from preovulatory follicles with SV40 DNA and Ha-ras oncogene. Progesterone production in these cells was enhanced to levels comparable to normal steroidogenic cells, by prolonged (> 12 h) stimulation with 8-Br-cAMP, forskolin and cholera toxin, which elevate intracellular cAMP. The steroidogenic capacity of individual lines correlated with the expression of the ras oncogene product (p21) and the morphology of the cells. Formation of the steroid hormones was associated with de novo synthesis of the mitochondrial cytochrome P450scc system proteins. Since cholesterol import into mitochondria is essential for steroidogenesis, the expression of the peripheral benzodiazepine receptor (PBR) and the sterol carrier protein 2 was characterized in these cells. The induction of the expression of the genes coding for both proteins appeared to be mediated, at least in part, by cAMP. Stimulation of the PBR by specific agonists enhanced progesterone production in these cells. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) dramatically suppressed the cAMP-induced steroidogenesis, in spite of enhanced intracellular cAMP levels, suggesting that TPA can modify the effects of cAMP. cAMP stimulation suppressed growth of transformed cells concomitantly with induction of steroidogenesis. The transformed cells lacked receptors for the native stimulants, the gonadotropic hormones. After transfection of the cells with a lutropin (LH) receptor expression plasmid, the LH and hCG response was reconstituted. In these newly established cell lines gonadotropins were able to stimulate the formation of cAMP and progesterone in a dose-dependent manner with an ED₅₀ characteristic of the native receptor. High doses caused desensitization to gonadotropins as observed in normal cells. These newly established oncogene-transformed granulosa cell lines can serve as a useful model to study inducible steroidogenesis and the effect of oncogene expression on this process.     

Inhibition of oocyte maturation in the mouse: Participation of cAMP,steroid hormones,and a putative maturation-inhibitory factor

The hypothesis that cumulus cells inhibit oocyte maturation by a cAMP-dependent process was tested (R. M. Schultz, R. Montgomery, P. F. Ward-Bailey, and J. J. Eppig (1983). Dev. Biol.95, 294–304.). Treatment of isolated cumulus cell-oocyte complexes with follicle-stimulating hormone (FSH) resulted in a dose-dependent increase in both cumulus cell cAMP levels and in the extent of inhibition of germinal vesicle breakdown (GVBD), the first morphological manifestation of oocyte maturation. Furthermore, it was found that concentrations of a membrane-permeable analog of cAMP, dibutyryl cAMP (dbcAMP), that were below those required for complete meiotic inhibition had a greater inhibitory effect on cumulus cell-enclosed oocytes than on denuded oocytes. Cumulus cell-enclosed and denuded oocytes matured at the same time in the absence of dbcAMP. Ablation of the gap junctions that couple cumulus cells to the oocyte abolished the maturation-inhibitory action of cumulus cells that was promoted either by FSH or low concentrations of dbcAMP. These results are consistent with the hypothesis that inhibition of oocyte maturation is mediated by a factor of granulosa/cumulus cell origin, other than cAMP, which requires cAMP for its activity and/or generation, and an intact intercellular coupling pathway between cumulus cells and the oocyte. A variety of steroid hormones potentiated the FSH-induced inhibition of maturation in cumulus cell-enclosed oocytes. In addition, steroid hormones inhibited maturation in denuded oocytes, but only when oocyte cAMP levels were elevated by cAMP analogs or forskolin. Steroids alone did not inhibit maturation of either cumulus cell-enclosed or denuded oocytes. Moreover, the steroids alone or in combination with FSH did not affect metabolic coupling between the cumulus cells and oocytes, nor did testosterone affect the forskolin-induced level of cAMP in denuded oocytes. Therefore, it is proposed that the oocyte is a site for the synergistic activity of steroid hormones with a cAMP-dependent process in inhibiting maturation. Results of these studies are discussed in terms of the roles of intercellular communication, cAMP, a putative maturation-inhibiting factor, and steroid hormones in the inhibition of maturation of mouse oocytes.     

Regulation of mRNA expression of 3 beta-hydroxy-5-ene steroid dehydrogenase in porcine granulosa cells in culture: a role for the protein kinase-C pathway

We studied the effect of the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA), which activates protein kinase-C, on porcine granulosa cells in culture. PMA as well as cholera toxin, forskolin, and hCG increased cAMP accumulation. PMA further augmented the elevation in cAMP accumulation induced by cholera toxin, forskolin, and hCG. In the same cell culture model, hCG induced a time-dependent increase in the 3 beta-hydroxy-5-ene steroid dehydrogenase (3 beta HSD) mRNA levels with a maximal 3-fold stimulation obtained at 8-16 h of incubation with 1 IU hCG/ml. PMA inhibited the increase in 3 beta HSD mRNA levels induced by hCG in a dose-dependent manner. The phorbol ester also inhibited the increase in 3 beta HSD mRNA levels stimulated by LH as well as cholera toxin and forskolin and the cAMP analogs (Bu)2cAMP and 8-bromo-cAMP. Activation of protein kinase-C by mezerein similarly inhibited hCG stimulation of 3 beta HSD mRNA levels. The present data indicate that activation of the protein kinase-C pathway induces generation of cAMP, but causes a near-complete inhibition of the stimulatory effects of hCG, LH, forskolin, cholera toxin, and cAMP analogs on 3 beta HSD mRNA levels in porcine granulosa cells in culture.     

Steroidogenic enzyme content and progesterone induction by cyclic adenosine 3',5'-monophosphate-generating agents and prostaglandin F2 alpha in bovine theca and granulosa cells luteinized in vitro.

In vitro luteinization of bovine granulosa (LGC) and theca (LTC) cells was achieved by culturing cells with forskolin (10 microM) and insulin (2 micrograms/ml) for 9 days. This treatment induced the presence of cytochrome P450scc and adrenodoxin in both cell types, but to substantially higher levels in LGC than in LTC. Forskolin dose-dependently stimulated the secretion of progesterone and cAMP after 3 h of incubation in both cell types although LGC were less sensitive to this stimulation than were LTC. Only LTC were responsive to LH, in accordance with their higher LH/hCG binding capacity. Both prostaglandin F2 alpha (PGF2 alpha) and phorbol 12-myristate 13-acetate (TPA) increased progesterone production during 3 h incubation of LGC and LTC, and treatment with staurosporine (a protein kinase C inhibitor) reversed this effect. Neither TPA nor PGF2 alpha alone affected cAMP levels but each acted synergistically with forskolin to increase cAMP accumulation. These results indicate that 1) elevated progesterone output from LGC is related to steroidogenic enzyme level; 2) bovine LH (up to 100 ng/ml) does not provoke a response in LGC due to their low LH/hCG binding capacity; 3) cAMP-protein kinase A and protein kinase C pathways are both involved in progesterone production by LGC and LTC, possibly by enhancing cholesterol transport.     

Protein Phosphorylation in Astrocytes Mediated by Protein Kinase C: Comparison with Phosphorylation by Cyclic AMP-Dependent Protein Kinase

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), has been found recently to transform cultured astrocytes from flat, polygonal cells into stellate-shaped, process-bearing cells. Studies were conducted to determine the effect of PMA on protein phosphorylation in astrocytes and to compare this pattern of phosphorylation with that elicited by dibutyryl cyclic AMP (dbcAMP), an activator of the cyclic AMP-dependent protein kinase which also affects astrocyte morphology. Exposure to PMA increased the amount of 32P incorporation into several phosphoproteins, including two cytosolic proteins with molecular weights of 30,000 (pI 5.5 and 5.7), an acidic 80,000 molecular weight protein (pI 4.5) present in both the cytosolic and membrane fractions, and two cytoskeletal proteins with molecular weights of 60,000 (pI 5.3) and 55,000 (pI 5.6), identified as vimentin and glial fibrillary acidic protein, respectively. Effects of PMA on protein phosphorylation were not observed in cells depleted of protein kinase C. In contrast to the effect observed with PMA, treatment with dbcAMP decreased the amount of 32P incorporation into the 80,000 protein. Like PMA, treatment with dbcAMP increased the 32P incorporation into the proteins with molecular weights of 60,000, 55,000 and 30,000, although the magnitude of this effect was different. The effect of dbcAMP on protein phosphorylation was still observed in cells depleted of protein kinase C. The results suggest that PMA, via the activation of protein kinase C, can alter the phosphorylation of a number of proteins in astrocytes, and some of these same phosphoproteins are also phosphorylated by the cyclic AMP-dependent mechanisms.     

Effects of prostaglandins, dibutyryl cAMP, LHRH, estrogens, progesterone, and potassium on output of prostaglandin F2 alpha, 13, 14-dihydro-15-keto-prostaglandin F2 alpha, hCG, estradiol, and progesterone by placental minces

In order to compare the endocrine response of placental minces to luteinizing hormone releasing hormone (LHRH) and dibutyryl cAMP (dbcAMP) and to screen for effects of potential stimulatory and inhibitory substances, the simultaneous outputs of PGF2 alpha, 13, 14-dihydro-15-keto-prostaglandin F2 alpha (PGFM), progesterone, 17 beta-estradiol, and hCG were evaluated during a 4 hour incubation in 5 placentas. The output of hCG was highest for 12-week placentas, intermediate for a 16 week placenta, and lowest for term placentas. The output of 17 beta-estradiol by 12 and 16 week placentas in the presence of 30 microM dehydroepian-drosterone sulfate (DHEAS) was greater than that by term placentas. Progesterone output was apparently independent of gestational age although some variation between 12-week placentas was demonstrated. Output of PGF2 alpha was lower in 12 and 16-week placentas than in term placentas and that of PGFM was lower in 12-week placentas than in term placentas. LHRH (100 nM) produced stimulation of PGF2 alpha output (P less than .005) and a trend toward inhibition of progesterone output (which failed to achieve statistical significance) but no stimulation of hCG under these conditions. Stimulation of the outputs of hCG (P less than .005) and PGF2 alpha (P less than .001) and inhibition of that of progesterone (P less than .005) was produced by 20 mM dbcAMP. DHEAS inhibited output of progesterone (P less than .01) and PGF2 alpha (P less than .01). There were no effects of potassium, estrogens, progesterone, or prostaglandins on output of any measured substance.     

Effects of dibutyryl cAMP, LHRH, and aromatase inhibitor on simultaneous outputs of prostaglandin F2 alpha, and 13,14-dihydro-15-keto-prostaglandin F2 alpha by term placental explants

Explants from term human placentas were maintained in culture with daily changes of medium. Daily output of PGF2 alpha and PGFM1 decreased during the course of the incubation. Addition of 4 micrograms/ml DHEAS or 67 micrograms/ml LDL cholesterol had no effect on output of PGF2 alpha or PGFM. Addition of 1.6, 3.2, or 6.4 micrograms/ml of LHRH to the culture plates had no effect on output of PGFM or PGF2 alpha, but LHRH increased hCG output. Dibutyryl cAMP (1mM, 2mM, and 4 mM) increased output of PGF2 alpha, PGFM, and hCG. Aromatase inhibitor decreased hCG output, but it was without effect on output of PGF2 alpha, or PGFM, Significant correlations were demonstrated between progesterone, PGFM, PGF2 alpha, and hCG, suggesting that PGF2 alpha originates in the syncytiotrophoblast cell. The ability of LHRH to stimulate output of hCG but not PGF2 alpha while dbcAMP stimulated both suggests that either PGF2 alpha and hCG arise in different cells or that LHRH does not act through cAMP.     

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.     

Assessment of luteal rescue and desensitization of macaque corpus luteum brought about by human chorionic gonadotrophin and deglycosylated human chorionic gonadotrophin treatment

The objective of the current study was to investigate the mechanism by which the corpus luteum (CL) of the monkey undergoes desensitization to luteinizing hormone following exposure to increasing concentration of human chorionic gonadotrophin (hCG) as it occurs in pregnancy. Female bonnet monkeys were injected (im) increasing doses of hCG or dghCG beginning from day 6 or 12 of the luteal phase for either 10 or 4 or 2 days. The day of oestrogen surge was considered as day ‘0’ of luteal phase. Luteal cells obtained from CL of these animals were incubated with hCG (2 and 200 pg/ml) or dbcAMP (2.5,25 and 100 M) for 3h at 37°C and progesterone secreted was estimated. Corpora lutea of normal cycling monkeys on day 10/16/22 of the luteal phase were used as controls. In addition thein vivo response to CG and deglycosylated hCG (dghCG) was assessed by determining serum steroid profiles following their administration. hCG (from 15–90 IU) but not dghCG (15-90 IU) treatment in vivo significantly (P < 0.05) elevated serum progesterone and oestradiol levels. Serum progesterone, however, could not be maintained at a elevated level by continuous treatment with hCG (from day 6–15), the progesterone level declining beyond day 13 of luteal phase. Administering low doses of hCG (15-90 IU/day) from day 6–9 or high doses (600 IU/day) on days 8 and 9 of the luteal phase resulted in significant increase (about 10-fold over corresponding control P < 0.005) in the ability of luteal cells to synthesize progesterone (incubated controls) in vitro. The luteal cells of the treated animals responded to dbcAMP (P < 0.05) but not to hCC added in vitro. The in vitro response of luteal cells to added hCG was inhibited by 0,50 and 100% if the animals were injected with low (15-90 IU) or medium (100 IU) between day 6–9 of luteal phase and high (600 IU on day 8 and 9 of luteal phase) doses of dghCG respectively; such treatment had no effect on responsivity of the cells to dbcAMP. The luteal cell responsiveness to dbcAMP in vitro was also blocked if hCG was administered for 10 days beginning day 6 of the luteal phase. Though short term hCG treatment during late luteal phase (from days 12—15) had no effect on luteal function, 10 day treatment beginning day 12 of luteal phase resulted in regain ofin vitro responsiveness to both hCG (P < 0.05) and dbcAMP (P < 0.05) suggesting that luteal rescue can occur even at this late stage. In conclusion, desensitization of the CL to hCG appears to be governed by the dose/period for which it is exposed to hCG/dghCG. That desensitization is due to receptor occupancy is brought out by the fact that (i) this can be achieved by giving a larger dose of hCG over a 2 day period instead of a lower dose of the hormone for a longer (4 to 10 days) period and (ii) the effect can largely be reproduced by using dghCG instead of hCG to block the receptor sites. It appears that to achieve desensitization to dbcAMP also it is necessary to expose the luteal cell to relatively high dose of hCG for more than 4 days     

Induction and inhibition of meiotic maturation of amphibian (Rana dybowskii) follicular oocytes by forskolin and cAMP in vitro

Previous studies have demonstrated that direct or indirect elevation of cAMP levels in cultured amphibian ovarian follicles simultaneously stimulated production of oocyte maturation-inducing steroid (progesterone) by the follicles and inhibited oocyte maturation induced by endogenous or exogenous hormone. The duration of cAMP stimulation influenced arrest and reinitiation of oocyte meiotic maturation in ovarian follicles of Rana dybowskii. Addition of forskolin (adenylate cyclase stimulator) to cultured follicles inhibited both progesterone- and frog pituitary homogenate (FPH)-induced oocyte maturation. Similar inhibitory results were obtained when hormone-treated follicles were cultured in the continual presence of cAMP. Oocyte maturation increasingly occurred in follicular oocytes when cAMP or forskolin addition was delayed following treatment with FPH or progesterone. Transient exposure (6-8 hr) of ovarian follicles to forskolin or cAMP markedly stimulated oocyte maturation as well as accumulation of progesterone as measured by radioimmunoassay within the ovarian follicles. Forskolin was more effective than cAMP, at the dose tested, in stimulating progesterone production and accumulation by the follicles. The data demonstrate that transient manipulation (elevation) of cAMP levels in cultured follicles, without added FPH or steroid, was sufficient to initiate oocyte maturation. Results suggest that, with transient exposure to forskolin or exogenous cAMP, there is a sequential increase and decrease in endogenous cAMP levels in the somatic cells and germ cell components of the ovarian follicle. These changes appear to mediate production of maturation-inducing steroid and secondarily allow its effects on the oocyte to be expressed.     

3',5'-cyclic adenosine monophosphate as an intracellular second messenger of luteinizing hormone: application of the forskolin criteria

The role of adenosine 3',5'-cyclic monophosphate (cAMP) as an intracellular second messenger of luteinizing hormone (LH) was reinvestigated in vitro with diterpene forskolin, a highly specific activator of adenylate cyclase. Treatment of cultured testicular cells from adult hypophysectomized rats with increasing concentrations (10(7)-10(-4) M) of forskolin produced dose-dependent increments in cAMP and testosterone accumulation. Concomitant blockade of cAMP-phosphodiesterase activity with 3-isobutyl-1-methyl-xanthine (10(-4) M) resulted in significant (P less than 0.05) enhancement of the forskolin effect for all but the 10(-4) M forskolin dose. Potency evaluation as judged by half-maximal stimulation of testosterone accumulation revealed median effective doses (mean +/- SE) of 1.25 +/- 0.2 x 10(-5), 1.7 +/- 0.5 x 10(-5), and 2.5 +/- 0.4 x 10(-10) M for forskolin, N6, O2'-dibutyryl cAMP (Bt2cAMP), and human chorionic gonadotropin (hCG), respectively. Examination of the time requirements of forskolin disclosed time-dependent increments in the accumulation of extracellular cAMP and testosterone, the earliest significant (P less than 0.05) increases being noted by 6 hr of treatment. In comparison, a minimal time requirement of less than or equal to 12 hr was noted for hCG- and choleragen-stimulated androgen biosynthesis, whereas the apparent onset of action of Bt2cAMP was delayed to the 24-hr time point. Although 10(-7) M of forskolin by itself did not alter the accumulation of testosterone, its addition resulted in substantial amplification of the hCG effect, producing a 4.6-fold reduction in the median effective dose (ED50) of hCG. Moreover, concurrent treatment with this functionally inert dose of forskolin rendered steroidogenically inert doses of hCG (eg, 10(-11) or 3 x 10(-11) M) steroidogenically potent. However, combined treatment with maximally stimulatory doses of Bt2cAMP (10(-4) M) and one of several testicular cell agonists [forskolin (10(-4) M), choleragen (10(-9) M) or hCG (10(-9) M)] did not prove additive. Taken together, our findings indicate that forskolin, like LH, is capable of stimulating testicular cAMP generation as well as androgen biosynthesis and that a functionally inert low dose of forskolin can significantly amplify LH hormonal action. Inasmuch as forskolin-stimulated and forskolin-amplified hormonal action are acceptable as novel criteria of cAMP dependence, our observations provide new evidence in keeping with the notion that cAMP may be in intracellular second messenger of LH.     

Low concentrations mono-butyl phthalate stimulates steroidogenesis by facilitating steroidogenic acute regulatory protein expression in mouse Leydig tumor cells (MLTC-1)

Di-n-butyl phthalate (DBP) is one of the most dominant phthalate esters and is widely distributed environmental contaminant. Although previous studies have demonstrated that DBP led to a variety of male reproductive abnormalities similar to those caused by androgen receptor antagonists, DBP and its active metabolite, mono-butyl phthalate (MBP), have been demonstrated no affinity for the androgen receptor, but rather exert anti-androgenic effect by altering testosterone biosynthesis. Furthermore, all these results were obtained from very high administrations of DBP or MBP. The purpose of this study was to determine the onset and the site of action of relatively low concentration of MBP on steroidogenesis in vitro. The mouse Leydig tumor cells (MLTC-1) was employed as a cellular model to investigate the effect of MBP on steroidogenesis. Various concentrations of MBP (1, 10, 100 and 1000nmol/l) and its solvent dimethyl sulfoxide (DMSO) were added to the medium for 24h followed by stimulation of some compounds such as human chorionic gonadotrophin (hCG), cholera toxin (CT), forskolin, cAMP analog 8-Br-cAMP, 22(R)-hydroxycholesterol (22R-HC) and pregnenolone. Progesterone in the medium and amounts of intracellular cAMP were measured by RIA. Expression of steroidogenic acute regulatory protein (StAR) was monitored by real-time PCR and Western blotting. The results revealed that the increases of progesterone production in the presence of hCG, CT, forskolin and 8-Br-cAMP were augmented by MBP. In contrast, the levels of intracellular cAMP exhibited no statistical significance when MLTC-1 cells were treated as above. These results implied that the site in the steroid biosynthesis pathway affected by MBP occurs after PKA activation in MLTC-1 cells. Moreover, supplementing the medium with 22R-HC and pregnenolone as progesterone precursors for P450 side chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD), respectively, resulted in no rise in progesterone production, making clear that MBP did not influence the P450scc and 3beta-HSD but on the rate-limiting step, cholesterol transportation into mitochondria. In fact, the above results were confirmed by the upgraded StAR expression in MBP-treated cells. These data support that MBP promotes steroid hormone production by facilitating StAR expression in MLTC-1 cells.     

Development of fetal rat ovaries responsiveness to LH during organ culture.

The responsiveness of fetal neonatal rat ovaries to LH was investigated in vitro using three complementary approaches. First, steroid production was assessed after culture. In control media, detectable levels of estrogens (estradiol and estrone) and progesterone were only observed from day 6 postpartum and during the second week of life respectively. In the presence of LH (100 ng/ml) ovaries produced both estrogens and progesterone from day 4 postpartum and the response to LH was enhanced with IBMX supplementation in the medium. Second, 3 beta-HSD activity was measured with either LH or (Bu)2 cAMP (1mM). Irrespective to the time-period studies (Bu)2 cAMP stimulated this enzyme whereas the stimulation with LH occurred only from day 5 postpartum Third, specific hCG binding was assessed and we found that it occurred only on days 7 and 10. However, when fetal ovaries were pretreated for 48 h with (Bu)2 cAMP, a specific hCG binding could be detected and progesterone production was enhanced in response to LH. An effect of the nucleotide via a stimulation of the neuraminidase activity did not seem to be involved. Lastly treatment of 18-day-old fetal ovaries with cholera toxin (10nM) or forskolin (1 microM) was found to stimulate progesterone production and VIP (0.1 to 1 microM) stimulated both the 3-HSD activity and the estradiol production. These data suggest that the absence of steroidogenic response to LH before day 4 postpartum could be explained by the absence of receptors, though the LH transmembrane signal-transduction system is functional in fetal ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)