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Hydrothermal decomposition of permanganate, conducted in a range of pH-controlled solutions (from strongly acidic to strongly basic), is used to prepare manganese dioxides that are well-suited for use as supercapacitor electrode materials. While permanganate is thermodynamically unstable, the kinetics of its decomposition in an aqueous environment are very slow, until the temperature is raised to ∼200 °C. Although the resultant materials are relatively crystalline and have low total pore volume, their prominent meso-porosity leads to good electrochemical performance. Best behaviour is obtained for material from permanganate decomposition in 0.01 M H 2 SO 4 solution, for which composite electrodes (150 μm thick) yield ∼150 F g −1 at 5 mV s −1 in a 9 M KOH electrolyte.

Loading of bioavailable phosphorus, traditionally measured as soluble reactive phosphorus (SRP), contributes to the eutrophication of aquatic ecosystems. However, polyphosphates are also bioavailable but escape detection by the standard method used for measuring SRP. 31P nuclear magnetic resonance spectrometric analysis of sediment extracts and enzymatic assay of surface waters reveal heretofore unreported presence of pyrophosphate (Ppi) in coastal wetlands. We show that the accumulation of Ppi (the smallest chemical form of polyphosphate) in coastal wetlands is related to human impact and can occur in quantities that exceed that of SRP. We further demonstrate that Ppi is readily utilized by microbes in coastal wetland sediments in the presence of nitrogen and carbon and can serve as a reservoir of orthophosphate. Thus, Ppi accumulation in estuaries will subsidize the in situ biogeochemical phosphorus cycle. This has important ecological implications for trophic responses and estuarine productivity.

The utility of on-line liquid chromatogaphy coupled to UV diode array detection and atmospheric pressure chemical ionization mass spectrometry for the analysis of microsomal metabolites of felodipine has been investigated. A previously unreported metabolite of felodipine, extracted from male rat microsomes, has been detected using this technique. UV diode array detection has been used to characterize this metabolite as a dihydropyridine metabolite and tandem mass spectrometry data used to assign a structure.

1. The metabolism of a model xenobiotic plant metabolite S-benzyl-N-malonyl-L-cysteine (BMC) administered to rat at 10 mg/kg has been studied using a combination of radio-t.l.c. and h.p.l.c. 2. The major route of excretion for the administered 14C was via the urine (79% in 3 days). 3. The major metabolite was hippuric acid. The extent of biotransformation of BMC indicated the lability of the N-malonyl bond whose hydrolytic removal initiated a metabolic sequence which involved the action of C-S lyase to produce benzyl thiol. 4. A comparison of the findings from this study with those from experiments with N-acetyl-S-benzyl-L-cysteine and S-benzyl-L-cysteine is made to support the metabolic pathway proposed.

There are several models for the CYP2D6 active site with the characteristics of their substrates and inhibitors well defined. Imipramine possesses such characteristics and is both a substrate and an inhibitor of the CYP2D6 enzyme. Possible synthetic strategies to avoid interaction with the enzyme have been investigated, including: attenuation of basicity; and alteration of rigidity and length of the alkyl chain. Imipramine inhibited the 1'-hydroxylation of bufuralol (10 microM), an in vitro marker of CYP2D6 activity, in a CYP2D6 cell line (IC50 = 2.4 microM). Inhibitory potency was attenuated by the removal of the basic centre; imipramine N-oxide had no inhibitory effect on bufuralol 1'-hydroxylation. However, removal of this basic centre, as a strategy to decrease CYP2D6 interaction, may well have a detrimental effect on pharmacological efficacy. Both an increase and decrease in the N-N carbon chain length [2C,4C] caused a reduction in inhibitory potency. In addition, introduction of a carbonyl adjacent to the amino dibenzyl moiety into 2C, 3C and 4C compounds brought about a further reduction in inhibitory potency. These data demonstrate that changes to the molecule, distal to the basic centre, can attenuate the affinity of the molecule for CYP2D6 and are in keeping with the known characteristics of the enzyme.

The acyclic cytosine nucleoside analog cytallene [1-(4'-hydroxy-1',2'-butadienyl)cytosine], which has both (+)- and (-)-enantiomers, was evaluated for its anti-hepatitis B virus (HBV) activity in 2.2.15 cells and was found to have potent activity against HBV DNA synthesis. The R-(-)-enantiomer was found to be the more active of the cytallene enantiomers, with a 50% inhibition concentration against HBV synthesis (HBIC50) of 0.08 microM. Its antiviral activity could be reversed by deoxycytidine (dC) and less efficiently by cytidine. Upon removal of the R-(-)-enantiomer from culture medium, the synthesis of HBV DNA could reinitiate, which suggested that the antiviral action is reversible. The R-(-)-enantiomer was also found to be more cytotoxic than the S-(+)-enantiomer. The degree of cytotoxicity varied among the cell lines, with a 50% inhibition of cell growth at greater than 10 microM. The R-(-)-enantiomer had no effect on HBV RNA synthesis and mitochondrial DNA synthesis at a concentration of 10 times or more than the HBIC50. The two enantiomers cannot be deaminated by dC deaminase, and they can be phosphorylated by cytoplasmic dC kinase. The R-(-)-enantiomer of cytallene is the first acyclic cytosine analog with potent inhibitory activity against HBV similar to those of other L-(-)-ddC analogs.

Using the method of adjusted learning, subjects learned number-noun pairs while breathing either placebo or 30% nitrous oxide. Subjects breathing nitrous oxide required more acquisition trials to attain a learning criterion than did subjects breathing placebo. Two weeks later, half of the subjects from each group were administered either placebo or nitrous oxide and were asked to recall the noun that had accompanied each number cue. Results showed that: 1) nitrous oxide inhalation can decrease the accessibility of to-be-recalled material and 2) nitrous oxide administered during the acquisition of material can paradoxically improved the recall of that material 2 weeks later. The additional number of acquisition trials subjects received during nitrous oxide inhalation could potentially account for this paradoxical enhancement of delayed recall; however, correlational analyses suggest this was not the case. No evidence for any state-dependent effects of nitrous oxide on cued recall were found.

The direct effects of adrenocortical steroids on basal and FSH-stimulated production of progesterone by ovarian granulosa cells were investigated in vitro. Granulosa cells from immature, hypophysectomized, estrogen-treated rats were cultured for 2 days in the presence or absence of FSH and/or corticoids. Treatment with FSH resulted in substantial increases in the accumulation of progesterone, whereas dexamethasone by itself was only minimally effective. However, concomitant treatment with increasing concentrations of various native and synthetic corticoids led to dose-dependent increases (200-500%) in the FSH-stimulated accumulation of progesterone. The stimulatory potencies of the corticoids tested correlated with their glucocorticoid rather than their mineralocorticoid potencies (dexamethasone greater than cortisol much greater than aldosterone). Treatment with dexamethasone also led to significant increases in the accumulation of progesterone stimulated by prostaglandin E2, cholera toxin, or dibutyryl cAMP. In the presence or absence of FSH, treatment with dexamethasone, cortisol, or corticosterone led to significant decreases in the activity of 20 alpha-hydroxysteroid dehydrogenase. In addition, treatment with dexamethasone led to significant increases in basal and FSH-stimulated activities of 3 beta-hydroxysteroid dehydrogenase. Our findings indicate that glucocorticoids synergize with FSH and other trophic agents in the accumulation of progesterone in accordance with their glucocorticoid rather than mineralocorticoid potencies and that this direct effect may be accounted for, at least in part, by the stimulation of 3 beta-hydroxysteroid dehydrogenase and the inhibition of 20 alpha-hydroxysteroid dehydrogenase.

Inhibin is a gonadal protein hormone that suppresses the secretion of FSH from pituitary gonadotrophs. It has previously been characterized as a heterodimer of two dissimilar subunits (alpha, 18 kilodaltons and beta, 14 kilodaltons) the smaller of which exists in two forms (beta A and beta B) and can form dimers that stimulate the secretion of FSH. In the present work, cDNA clones encoding the inhibin alpha- and beta A-subunits have been isolated from rat ovary and characterized. The alpha-inhibin cDNA predicts a precursor protein of 366 amino acids containing the 133 amino acid mature alpha-subunit at its COOH-terminus. The beta A-inhibin cDNA predicts a precursor protein of 424 amino acids containing the 116 amino acid beta A-subunit at its COOH-terminus. Analysis of rat ovarian RNA indicates that alpha-inhibin mRNA levels are stimulated by PMSG treatment in vivo. In cultured granulosa cells, FSH also stimulates alpha-inhibin mRNA, and the FSH effect is suppressed by cotreatment with GnRH. Hybridization in situ to rat ovarian tissue demonstrates that both the alpha-inhibin and beta A-inhibin mRNAs are specifically expressed in granulosa cells of the developing follicles.

The mechanism by which GnRH inhibits ovarian progesterone production was investigated by studying the GnRH modulation of pregnenolone biosynthesis in cultured rat granulosa cells. Granulosa cells from hypophysectomized, estrogen-treated rats were incubated for 2 days with various hormones in vitro. Pregnenolone production was measured in the presence of cyanoketone, which inhibits the conversion of pregnenolone to progesterone. FSH stimulated pregnenolone production in a dose-dependent manner (ED50, 4.47 ng/ml). Concomitant treatment with GnRH resulted in a dose-dependent decrease in FSH-stimulated pregnenolone production (ID50, 6.3 x 10(-9) M; maximal decrease, approximately 50%). In contrast, treatment with high doses of GnRH alone stimulated pregnenolone production (ED50, 2.95 x 10(-8) M) reaching a maximal level of about 10% that induced by FSH. Treatment with a GnRH antagonist, [Ac-D-Phe1, D-pCl-Phe2, D-Trp3,6]GnRH, did not affect either basal or FSH-stimulated pregnenolone production, but blocked both inhibitory and stimulatory effects of GnRH. The addition of 25-hydroxycholesterol, a soluble substrate for side-chain cleavage enzymes, enhanced FSH-stimulated pregnenolone production, but failed to overcome the inhibitory action of GnRH. GnRH also inhibited progesterone production stimulated by 8-bromo-cAMP and cholera toxin. This action of GnRH, however, was not associated with an inhibition of pregnenolone biosynthesis, but appeared to be due to a preferential increase in the metabolism of progesterone to 20 alpha-hydroxypregn-4-en-3-one. Thus, in addition to the reported GnRH stimulation of progesterone metabolism to 20 alpha-hydroxypregn-4-en-3-one and the GnRH inhibition of FSH-stimulated 3 beta-hydroxysteroid dehydrogenase activity, the present results demonstrate that GnRH also inhibits FSH-stimulated pregnenolone biosynthesis, probably at the side-chain cleavage enzyme step.

We have previously shown that gonadotropin releasing hormone (GnRH) and its agonists inhibit ovarian functions by a direct action on ovarian granulosa cells in vitro. A labeled GnRH agonist, [des-Gly10, D-Ser (TBu)6, Pro9-NHEt]GnRH, was used here to examine the possibility that these inhibitory actions of GnRH were mediated through specific receptors which recognize GnRH. Ovarian membrane fractions obtained from immature, hypophysectomized diethylstilbesterol-treated rats were incubated with the 125I-GnRH agonist and specific binding was determined by a filtration assay. Stereospecific, high affinity binding was detected in the ovarian membranes; the dissociation constant for the labeled GnRH agonist was determined to be 0.84 ± 0.33 × 10−10 M and the binding capacity was calculated to be 12.9 fmol/mg protein, or 0.142 fmol/μg DNA. The binding affinity for the GnRH decapeptide was 3.3 times lower than that of the GnRH agonist whereas two GnRH partial peptides did not compete for the 125I-agonist binding. After sequential treatment with FSH, LH and prolactin to the hypophysectomized female rats, the ovarian GnRH binding capacity increased per ovary, but decreased per mg ovarian protein. Furthermore, ovarian granulosa cells were isolated and their binding capacity was determined to be 25.2 fmol/mg protein, or 0.133 fmol/μg DNA, suggesting that the granulosa cells contain GnRH binding sites. Thus, this report demonstrates the presence of stereospecific, high affinity GnRH binding sites in the rat ovarian granulosa cells.

The mechanism by which gonadotropin-releasing hormone (GnRH) inhibits ovarian progesterone production was investigated by studying the GnRH modulation of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)/delta 5-delta 4-isomerase activity in cultured rat granulosa cells. Ovarian granulosa cells, obtained from immature hypophysectomized estrogen-treated rats, were incubated with various hormones in vitro, and 3 beta-HSD activity was determined by measuring the conversion of radiolabeled pregnenolone to progesterone. Treatment with FSH increased the apparent maximal velocity of the enzyme by about 6-fold in a dose-dependent manner, with an ED50 value of 3.58 ng/ml. FSH treatment also resulted in an approximately 10-fold increase in the apparent Km of this enzyme (from 0.46 to 4.98 microM). In contrast, concomitant treatment with GnRH (10(-8) M) inhibited the FSH-stimulated increase in enzyme activity by about 27%. This inhibitory effect of GnRH was associated with a decrease in the apparent maximal velocity, while the apparent Km remained unchanged. Furthermore, the inhibitory effect of GnRH was observed whether the enzyme activity was expressed per mg protein or per mg DNA. Concomitant treatment with 10(-6) M of a GnRH antagonist, [D-pGlu1,D-Phe2,D-Trp3,6]GnRH, completely blocked the inhibitory effect of GnRH. In contrast to the inhibitory effect of GnRH on FSH-stimulated 3 beta-HSD activity, treatment with GnRH alone increased enzyme activity by about 40%. This was accompanied by a slight but significant stimulation of basal progestin production by GnRH in granulosa cells. The present results coupled with the observed GnRH stimulation of 20 alpha-HSD activity reported earlier suggest that GnRH inhibits the FSH stimulation of progesterone production by decreasing the conversion of pregnenolone to progesterone as well as by increasing the metabolism of progesterone.

An investigation, in volunteers, into the sensory effects of nitrous oxide and methoxyflurane showed that the subjective changes of reduction in visual awareness and heightened auditory awareness were not supported by objective measurement. Vision remained normal until the subjects became unresponsive, whilst hearing was in fact diminished. The analgesia produced by nitrous oxide was late in appearance relative to the other sensory changes and it disappeared very rapidly upon recovery. In contrast, analgesia appeared early with methoxyflurane and persisted well into the recovery period. The principal changes found were alterations in orientation in space, of intellectual attitudes, of mood and of emotional attitudes. The emotional attitudes were easily disturbed by extraneous noise or conversation and the need for a quiet approach not only to the induction of anaesthesia but also to the recovery from anaesthesia is stressed.

Twenty-three exploratory wells were drilled in Alaska in 1981. Ten oil or gas discovery wells were drilled on the North Slope. 154 development and service wells were drilled and completed in the Prudhoe Bay and Kuparuk River fields on the North Slope. Geologic and geophysical field activity increased significantly in 1981, mainly because of increased North Slope activity and for OCS sale preparation in the Bering Sea. Two OCS lease sales were held and the first NPR-A lease sale was held. The State of Alaska continued a series of scheduled state lease sales.

To evaluate the roles of plasminogen activator (PA) and PA inhibitor (PAI) in human ovulation, we obtained follicular fluid and granulosa cells from individual preovulatory follicles of patients undergoing gamete intrafallopian tube transfer. The follicular fluid samples (n = 25) were analyzed for total tissue-type PA antigen, PA enzyme activity by fibrin autography, PAI activity, PAI type 1 (PAI-1) antigen, and PAI-1 mRNA. The follicular fluid of preovulatory follicles contained low levels of total tissue-type PA antigen (less than 1 ng/mL). Fibrin autography experiments indicated little or no detectable PA activity associated with free or unbound PA. The results of the PAI activity assay and PAI-1 antigen determination support the concept of a relative abundance of PAI compared with PA. Hybridization analysis was used to measure the relative amounts of granulosa cell PAI-1 mRNA. The levels of PAI-1 mRNA correlate with follicular fluid PAI concentrations in individual follicles. Taken together, these results support the idea that there is very little free, or active, PA in follicular fluid of human preovulatory follicles, but there is an abundance of PAI. Furthermore, PAI-1 produced by the granulosa cells may represent a major form of PAI in follicular fluid.

Epidermal growth factor (EGF), but not fibroblast growth factor, inhibits the gonadotropin stimulation of estrogen and testosterone production by primary cultures of ovarian granulosa and testicular Leydig cells, respectively. EGF also inhibits gonadal steroidogenesis induced by cholera toxin and (Bu)2cAMP, and the inhibitory effect of EGF is not associated with cell proliferation. EGF may play an important endocrine role in the regulation of steroidogenic functions of gonadal cells.

Hypothalamic gonadotropin releasing hormone (GnRH) and its analogs have been shown to regulate progestin production by ovarian granulosa cells treated with FSH and prolactin. The present study examines the direct modulation of an ovarian progesterone metabolizing enzyme by GnRH and its analogs in vitro and in vivo . Granulosa cells from hypophyseclomized, diethylstilbestrol-treated rats were cultured with GnRH or its analogs. After 2 days, cells were homogenized and 20α-hydroxysteroid dehydrogenase (20α-OH-SDH) activity was measured using a direct enzyme assay. GnRH stimulated 20α-OH-SDH activity in a dose-dependent manner with an ED 50 of 8.95 × 10 −9 M and a GnRH aganist ([des-Gly 10 , d -Lcu 6 , (N 2 Me)Leu 7 ,Pro 9 NHEt]GnRH) was shown to be 5.6 fold more potent than GnRH. The stimulatory effect of GnRH was inhibited by concomitant treatment with a GnRH antagonist ([ d -pGlu 1 , d -Phe 2 d -Trp 3,6 ]GnRH) with a half maximal inhibitory dose ratio of 3 1 ([antagonist]/[GnRH]). Time course studies indicated that 12 h of GnRH treatment were required to stimulate a measurable increase in enzyme activity. Cycloheximide treatment inhibited the GnRH action, suggesting that GnRH induces the synthesis of 20α-OH-SDH. Furthermore, treatment with GnRH and its analogs in hypophysectomizcd rats in vivo stimulated enzyme activity without affecting the K m of the enzyme, whereas the stimulatory effect of GnRH was blocked by concomitant treatment with a potent GnRH antagonist. These studies demonstrated that GnRH and its analogs may directly regulate the synthesis of ovarian 20α-OH-SDH in vitro and in vivo .

1. The metabolic fates of 14C-phenol and its model plant conjugates 14C-phenyl glucoside and 14C-phenyl 6-O-malonyl-glucoside have been compared following equimolar oral dosing to rats (1.2mg phenol/kg).2. Rapid excretion of radioactivity in the urine (at least 80% within 24 h) was observed with each compound.3. Phenol was eliminated as expected mainly as phenyl sulphate (68%) and partly as phenyl glucuronide (12%).4. The excretion profile for phenyl malonyl-glucoside was very similar to that of phenol, with the exception that small amounts of phenyl glucoside and phenyl malonyl-glucoside were excreted.5. In contrast, a major part of the dose of phenyl glucoside was eliminated unchanged.6. The value of metabolism studies in the assessment of the toxicology of xenobiotic metabolites derived from plants is discussed.

Twelve exploratory wells were drilled in Alaskan waters in 1983. No discoveries were announced discoveries as all the wells were suspended or plugged and abandoned. Main industry interest continued to be in the North Slope and Beaufort Sea areas of the state. The most costly wildcat well in North American history was drilled unsuccessfully in the Beaufort Sea. OCS lease sales in the Norton basin and St. George basin of the Bering Sea dominated leasing activity.

Twenty-seven exploratory wells were drilled in Alaska in 1978. One oil discovery was announced at the Point Thomson area on the North Slope and 9 suspended wells near the Prudhoe Bay field and the Kuparuk River field may have found oil. Two apparent gas discoveries were announced on the west side of Cook Inlet but the first well drilled in the Lower Cook Inlet OCS area was dry. Four wells drilled in the Gulf of Alaska OCS area were dry. Fifty-three development wells were drilled and all were successful; 7 service wells were completed. Geologic-geophysical field activity by both industry and government consisted of 95.89 crew-months, a decrease of 21% compared to 1977. This decrease affected all of the major basins of the state except the North Slope, which had a 13% increase from the previous year. Federal acreage under lease decreased more than 50%. State lease acreage declined slightly. As the Prudhoe Bay field reached its potential, the year's oil production showed an increase of 261% and gas production showed an increase of 160%. The State of Alaska announced a 5-year leasing schedule that may stimulate exploration, but the federal land situation remains unresolved until the 17(d) (2) question is decided by Congress.

Twenty-four exploratory wells were drilled in Alaska in 1982. There were no announced discoveries; all wells were suspended or plugged and abandoned. Geologic and geophysical activity increased substantially as preparations for OCS sales increased. OCS sale 71 dominated the exploration news in Alaska. More than $2 billion was spent by industry in this Beaufort Sea sale.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dioxin, produces a diverse set of biological responses which, in some cases, reflects the altered expression of specific genes. An intracellular receptor protein binds TCDD saturably and with high affinity and mediates several of TCDD's biological effects. In mouse hepatoma cells, TCDD induces aryl hydrocarbon hydroxylase activity by activating the transcription of the cytochrome P1-450 gene. Studies of receptor-defective variant cells indicate that the activation of cytochrome P1-450 gene transcription requires functional TCDD receptors. Analysis of the DNA that flanks the 5′-end of the mouse cytochrome P1-450 gene reveals at least three control regions: a promoter, an inhibitory element, and a dioxin-responsive element (DRE). Therefore, expression of the cytochrome P1-450 gene represents a balance between negative and positive control. The DRE contains two discrete, non-overlapping DNA domains that respond to TCDD. Each TCDD-responsive domain acts independently of the other, each requires TCDD receptors for function, and each has the properties of a transcriptional enhancer. For example, the function of the DREs is relatively independent of both their location and their orientation with respect to the promoter. Together, the DREs and the TCDD-receptor complex constitute a dioxin-responsive enhancer system. Exposure of cells to TCDD results in the protection of a specific DNA domain from exonuclease digestion. This protection requires TCDD receptors. The protected domain maps to a DRE. This observation implies that the TCDD-receptor complex interacts with the DRE to activate the transcription of the cytochrome P1-450 gene.

The direct modulation of 20α-hydroxysteroid dehydrogenase (20α-OH-SDH) by regulatory agents of luteal functions was examined using in vitro cultures of ovarian granulose cells from immature, hypophysectomized, estrogen-treated rats. Without FSH priming, treatment with isoproterenol, PRL, or prostaglandin F2α (PGF2α) resulted in minimal modulation of the 20α-OH-SDH activity of granulosa cells. In contrast, after in vitro FSH priming, these hormones or agents caused marked changes in 20α-OH-SDH activity. Isoproterenol treatment inhibited 20α-OH-SDH activity in a dose-dependent manner (ED50, 3.4 × 10-10 M; maximal inhibition, 62% decrease). Similar inhibition of 20α-OH-SDH activity was observed after treatment with epinephrine, norepinephrine, and a β2-adrenergic agonist (terbutalino sulfate), whereas a β1 -adrenergic agonist (dobutamine) was less effective. Furthermore, the inhibitory effect of epinephrine was blocked by concomitant treatment with a β2- antagonist (IPS 339) but not by a β1-antagonist (pract...

The effects of gonadotropin-releasing hormone (GnRH) and its potent agonist [des-Gly10, D-Leu6-N alpha Me) Leu7, Pro9,NHEt-GnRH (GnRH-A)] on ovarian luteal functions maintained by PRL were studied in vivo and in vitro. Hypophysectomized, diethylstilbestrol-treated female rats were primed with FSH for 2 days, followed by an ovulating dose of LH or hCG. Two days later, ovarian luteal functions were maintained by daily injections of 250 microgram PRL for 3 days. PRL treatment increased the serum progesterone level from 13.0 +/- 0.5 to 298 +/- 24 ng/ml and increased the ovarian hCG-binding capacity from 5.8 +/- 1.3 to 584 +/- 86 ng bound hCG/ovary. In contrast, concomitant treatment with GnRH or GnRH-A resulted in dose-dependent decreases in the PRL-induced increase of serum progesterone and ovarian LH/hCG receptor content. GnRH at 100 microgram/day caused a 60% decrease in serum progesterone and an 80% decrease in ovarian LH receptor content, whereas GnRH-A was effective at a 1-microgram dose level. Neither GnRH nor GnRH-A affected the binding affinity (Kd) of ovarian LH receptor. The direct inhibitory effects of GnRH and GnRH-A upon granulosa-luteal cell function were also tested in vitro. FSH treatment for 2 days induced functional LH and PRL receptors in cultured PRL, increased (by approximately 3-fold) progesterone production by these granulosa-luteal cells, whereas concomitant treatment with GnRH-A inhibited progesterone production in a dose-dependent manner. Thus, these studes demonstrated that GnRH and GnRH-A exert direct inhibition on ovarian luteal functions by decreasing LH receptor and progesterone production in vivo as well as inhibiting progesterone production by cultured granulosa-luteal cells in vitro.

Studies with rat ovarian cells indicate that proteolytic enzymes, such as plasminogen activator (PA), play a role in the tissue remodeling that occurs before ovulation. In the rat, gonadotropins appear to increase granulosa cell tissue-type plasminogen activator (t-PA) content by increasing the cellular concentration of t-PA mRNA as well as by modulating the activity of a specific PA inhibitor (PAI). We obtained granulosa cells from preovulatory follicles of women undergoing either in vitro fertilization or gamete intra-fallopian tube transfer in order to evaluate the roles of PA and PAI in human ovulation. Samples of granulosa cell total RNA were hybridized with probes for t-PA, urokinase-type PA, PAI type 1 (PAI-1), or inhibin A-chain (as a control). Northern analyses revealed that the RNA of granulosa cells from preovulatory follicles contained little or no detectable PA mRNA. In contrast, two species of PAI mRNA were detected in relative abundance. The signal intensity produced by the PAI-1 probe varied by about 8-fold among patient samples, suggesting that PAI-1 may be useful as a marker of follicular maturation and differentiation. These results demonstrate that human granulosa cells collected immediately before ovulation contain PA inhibitor mRNA, yet have little or no PA mRNA.

The influence of triphenylethylene antiestrogens on FSH-stimulated production of estrogen, progesterone, and 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P) was examined in cultured rat granulosa cells. The cells were cultured with increasing concentrations of FSH, with or without diethylstilbestrol (DES) or various antiestrogens. After 3 days, medium progesterone and 20 alpha-OH-P contents were determined. Cells were reincubated for an additional 8 h with delta 4-androstene-3,17-dione, and estrogen formation was measured. Steroid production was negligible by cultures treated with DES or antiestrogens alone. FSH treatment increased estrogen and progestin production, while the addition of DES (10(-7) M) further enhanced FSH-stimulated steroidogenesis. Likewise, treatment with 10(-6) M of the antiestrogens tamoxifen (Tam), nafoxidine (Naf), or clomiphene citrate (CC) also enhanced FSH-stimulated aromatase activity. In contrast, the antiestrogens each inhibited FSH stimulation of progesterone and 20 alpha-OH-P production (greater than 73% inhibition at 300 ng/ml FSH). Increasing concentrations (3 X 10(-8)-10(-6) M) of the antiestrogens augmented the stimulatory effect of FSH (10 ng/ml) on estrogen production in a dose-related manner (CC greater than Tam greater than Naf). Similar doses of these antiestrogens inhibited the stimulatory effect of FSH (300 ng/ml) on progesterone and 20 alpha-OH-P production (Tam greater than CC greater than Naf). The observed inhibition of progestin production is associated with decreases in FSH-stimulated pregnenolone biosynthesis in antiestrogen-treated cells incubated with 10(-6) M cyanoketone. Furthermore, the antiestrogens inhibited the binding of [3H]estradiol to ovarian estrogen receptors with binding affinity constants of 364, 437, and 2144 nM for CC, Tam, and Naf, respectively. Thus, antiestrogens exert disparate actions on granulosa cell estrogen and progestin biosyntheses. Like estrogens, CC, Tam, and Naf enhance FSH-stimulated aromatase activity with potencies comparable with their abilities to interact with ovarian estrogen receptors. However, unlike estrogens, the antiestrogens inhibit FSH-stimulated progestin biosynthesis, partially via suppression of pregnenolone biosynthesis. The present granulosa cell culture system provides a valuable model for elucidating the disparate actions of estrogens and antiestrogens on ovarian steroidogenesis.

The hormonal modulation of prolactin (PRL)-binding capacity of rat granulosa cells was studied. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 2 days in a serum-free medium in the presence of various hormones. FSH treatment in vitro stimulated granulosa cell PRL-binding capacity by approximately 4-6-fold in a dose-dependent manner. Concomitant treatment with 10-8 M GnRH inhibited the FSH-induced increase in PRL-binding capacity by 64%. In contrast, the inhibitory effect of GnRH was blocked by concomitant treatment with 10-6 M of a GNRH antagonists, [D-pGlu1, D-Phe2, D-Trp3,6]GnRH. PRL-binding capacity was also increased (approximately 2-fold) by in vitro treatment with cholera toxin (10 microgram/ml). In granulosa cells pre-treated with FSH in vitro for 2 days, hCG treatment for 2 additional days stimulated PRL-binding capacity in a dose-dependent manner (approximately 2-fold). Likewise, treatment with LH (100 ng/ml) also stimulated PRL-binding capacity by approximately 2-fold. These in vitro studies demonstrated that gonadotropins (FSH, LH and hCG) directly enhanced PRL binding by granulosa cells, whereas GnRH inhibited FSH action.

Possible influences of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) upon gonadal steroidogenesis were investigated in vitro. Granulosa cells from hypophysectomized, estrogen-treated rats were cultured for 2 days in medium containing 0.1 microM androstenedione. Follicle-stimulating hormone (FSH) treatment increased estrogen, progesterone, and 20 alpha-hydroxypregn-4-en-3-one production. Concomitant TPA treatment inhibited FSH-stimulated estrogen production by up to 95% [concentration that induced 50% inhibition of steroid production (ED50), 1.1 ng/ml]. TPA also inhibited FSH-stimulated progesterone (ED50, approximately 0.6 ng/ml) and 20 alpha-hydroxypregn-4-en-3-one (ED50, approximately 1.1 ng/ml) production. N6O2'-dibutyryl cyclic adenosine 3':5'-monophosphate increased steroidogenesis; however, cotreatment with TPA blocked progestin but not estrogen production. The TPA inhibition of progestin biosynthesis was accompanied by decreases in FSH-stimulated pregnenolone biosynthesis and 3 beta-hydroxysteroid dehydrogenase activity without decreasing the activity of 20 alpha-hydroxysteroid dehydrogenase. In primary cultures of rat testicular cells, human chorionic gonadotropin treatment increased testosterone production 44-fold, whereas concomitant treatment with TPA inhibited testosterone production by up to 86% (ED50, 10 ng/ml). Cholera toxin and N6O2'-dibutyryl cyclic adenosine 3':5'-monophosphate also increased testosterone production, while the actions of these agents were decreased by TPA. The TPA suppression of testosterone production was associated with a decrease in accumulation of 17 alpha-hydroxyprogesterone and androstenedione and an increase in progesterone production, suggesting a specific inhibition of 17 alpha-hydroxylase and 17,20-lyase activities. These results demonstrate the inhibitory effects of a tumor promoter upon gonadotropin-stimulated steroidogenesis by cultured rat granulosa and Leydig cells through specific regulation of steroidogenic enzymes. Additional studies may assist in further elucidation of cellular mechanisms associated with carcinogenesis and steroidogenesis.