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1.
The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase: EC 1.1.1.34) was measured in a microsomal preparation of the granulosa of rapidly growing ovarian follicles of laying hens in the late preovulatory period (2-3 h before expected ovulation). The specific activity of the enzyme was measured in the five largest (F1-F5) preovulatory follicles, F1 being the follicle destined to ovulate first. Enzyme activity increased concomitantly with follicle size. The apparent Km of the enzyme decreased 60-80% from the smallest to the largest preovulatory follicle. There was no significant change in the Vmax during follicle development. Although our results have demonstrated the presence of HMG/CoA reductase in chicken granulosa cells and the progressive increase of its activity with follicular maturation, the quantitative significance of de-novo synthesized cholesterol as steroid hormone precursor remains to be ascertained. 相似文献
2.
LH was used to stimulate cAMP production in theca cells from the 5 largest preovulatory follicles of hens and this was related to LH-stimulated androstenedione production in the same cells. cAMP production was stimulated by LH to the same extent in theca cells from each follicle. However, LH was not effective in stimulating androstenedione production in theca cells from the largest follicle (T1), although androstenedione production was greatly increased by LH in the smaller follicles (T2-T5). Effects similar to those of LH on cAMP production were observed in response to forskolin, indicating that the intrinsic adenylate cyclase activity was similar in theca cells from each follicle. In addition, forskolin was unable to stimulate androstenedione production by T1 cells. Our results provide evidence that the levels of receptor-mediated and non-receptor-mediated cAMP production are similar in theca cells from the 5 largest follicles. We conclude that the step that restricts the ability of T1 cells to produce androgen is distal to cAMP generation. 相似文献
3.
Effect of calcium ionophore A23187 on pregnenolone metabolism to progesterone in rat granulosa cells
B K Tsang D F Mattice M Li E K Asem 《Canadian journal of physiology and pharmacology》1988,66(7):960-963
The effect of calcium ionophore A23187 on the metabolism of pregnenolone to progesterone was examined in rat granulosa cells during a 24-h culture period. Granulosa cells harvested from pregnant mare's serum gonadotropin treated immature rats were incubated in the presence and absence of the divalent cation ionophore A23187. The ionophore induced progesterone synthesis from both endogenous sterol substrate and exogenous pregnenolone in a time- and concentration-dependent manner. Pregnenolone metabolism was examined in the presence of aminoglutethimide phosphate, an inhibitor of endogenous pregnenolone production. Steroid secretion resulting from metabolism of endogenous substrate was more sensitive to A23187 in that a lower concentration of the ionophore was required to induce a significant increase than that noted for exogenous pregnenolone metabolism. In addition, progesterone production from endogenous sterol occurred 6 h earlier than the observed increase in the conversion of pregnenolone to progesterone. These results indicate that A23187 and therefore possibly enhanced calcium influx may play a significant role in the regulation of pregnenolone metabolism in granulosa cells depending on the duration of incubation. The earlier steroidogenic response from endogenous substrate may be a reflection of an acute effect of A23187 on certain steroidogenic steps proximal to pregnenolone production. 相似文献
4.
A radiochemical assay was utilized to study the inhibitory effects of clomiphene and tamoxifen on the cholesterol side-chain cleavage enzyme activity in a mitochondrial preparation of granulosa cells isolated from mature ovarian follicles of laying hens. At saturating substrate concentrations, both clomiphene and tamoxifen were able to suppress enzyme activity in a dose-related manner (IC50 1.8 X 10(-5) M). Double reciprocal plots of kinetic data show that the inhibition is mixed, exhibiting competitive kinetics at low concentrations, whereas at high concentrations, the inhibition is of a non-competitive nature. The competitive inhibition constants as determined from Dixon plots are 2 X 10(-5) M for clomiphene and 2.3 X 10(-5) M for tamoxifen. It is concluded that, in granulosa cells, clomiphene and tamoxifen directly inhibit the mitochondrial cholesterol side-chain cleavage activity. This inhibition may represent an important aspect of the mode of action of clomiphene and tamoxifen. 相似文献
5.
Fan MZ Adeola O Turek JJ Asem EK 《Physiological research / Academia Scientiarum Bohemoslovaca》1999,48(4):281-289
With L-glutamine, as a representative amino acid this study was undertaken to examine the effects of substrate concentrations on initial and equilibrium amino acid uptake and intravesicular volume determined with porcine jejunal brush border membrane vesicles prepared by Mg2+-aggregation and differential centrifugation. Transport measurements (24 degrees C) were conducted by the rapid filtration manual procedure. Glutamine uptake was shown to occur into an osmotically-active space ranging between 1.09-1.58 microl/mg protein with little non-specific membrane binding. At different concentrations (in parentheses), the duration of initial glutamine uptake in both Na+ gradient and Na+-free conditions was 10 s (0.01 mM), 15 s (0.17 mM), and 20 s (1.9 and 9.4 mM), respectively. Substrate concentrations affected the duration of initial uptake, with lower substrate concentrations giving shorter duration for initial amino acid uptake. At different substrate concentrations (in parentheses), the time required to reach equilibrium glutamine uptake was 5 min (0.01 mM), 10 min (0.17 mM), and 60 min (1.9 and 9.4 mM), respectively. Thus, substrate concentrations also affected the time required to reach equilibrium uptake. The higher the substrate concentration, the longer the incubation time needed to reach equilibrium amino acid uptake. At the glutamine concentrations of 0.01, 0.17, 1.9, and 9.4 mM, the average intravesicular volume was estimated to be 1.58+/-0.21, 1.09+/-0.28, 1.24+/-0.18, and 1.36+/-0.21 microl/mg protein, respectively. Substrate concentrations had no effect (p>0.05) on the intravesicular volume of membrane vesicles. In conclusion, in the experiments on amino acid transport kinetics measured with the rapid filtration manual procedure, the incubation time used for measuring the initial uptake rate should be determined from the time course experiments conducted at the lowest substrate concentration used, whereas the intravesicular volume can be obtained from equilibrium uptake measured at any substrate concentrations. 相似文献
6.
Al-Majali AM Asem EK Lamar CH Robinson JP Freeman MJ Saeed AM 《FEMS immunology and medical microbiology》2000,28(2):97-104
Enterotoxigenic Escherichia coli (ETEC) induces severe diarrhea in newborn calves through the elaboration of heat-stable enterotoxin (STa). We investigated the distribution and characteristics of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from anterior jejunum, posterior jejunum, ileum and colon of newborn calves. We found that density of the receptors and their affinity to STa were higher on enterocytes and BBMVs that were derived from the ileum than enterocytes and BBMVs prepared from other segments of the calf intestine. This study suggests that, in newborn calves, the ileum is the major part of the intestinal tract that is affected in the course of secretory diarrhea caused by STa-producing ETEC strains. 相似文献
7.
The effects of low Ca2+ on ion currents in hen ovariangranulosa cells were examined. A fast activating and inactivatingtransient outward current (TOC) and a slowly activating outward current (SOC) could be observed. In the presence of normal Ca2+concentration (2.5 mM) and with a holding potential of 80 mV, SOC wasactivated in all cells with command pulses more positive than 20 mV.In 2.5 mM Ca2+, TOC appeared in 10% of cells at thecommand pulse of +80 mV and in 60-85% of cells at +100 to +120mV. In low-Ca2+ solution and command potential of +80 mV(holding potential of 80 mV), the amplitude of TOC was enhanced incells that expressed it in normal Ca2+, and TOC appeared in43% of the cells that did not express it initially in normalCa2+. At both normal and low Ca2+ levels, TOCdecreased as the holding potential became more positive. TOC wasreduced in Cl-deficient solution and in the presence of5-nitro-2-(3-phenylpropylamino)benzoic acid, a Cl channelblocker. These findings suggest that chicken granulosa cells express aCa2+-inactivated TOC carried by Cl. Thiscurrent may serve as a signal for some of the reduced metabolic functions of granulosa cells associated with Ca2+ deficiency. 相似文献
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9.
The influence of follicular maturation on progesterone production by collagenase-dispersed hen granulosa cells was measured in short-term incubations. Granulosa cells of the largest follicle (F1) produced up to ten times more progesterone than cells from smaller follicles (F3-F5), not only in response to luteinizing hormone (LH), but also when stimulated by exogenous cyclic AMP or forskolin, both of which raise intracellular cyclic AMP levels by nonreceptor-mediated mechanisms. Moreover, when granulosa cell progesterone synthesis was stimulated by incorporating 25-hydroxy-cholesterol into the incubation medium, an identical pattern was obtained. This could be attributed to a corresponding increase in the specific activity of the mitochondrial cholesterol side-chain cleavage enzyme (20,22 desmolase). An increase in the apparent Vmax was observed without a change in the apparent Km values. Pregnenolone substrate at concentrations which raised progesterone production to levels similar to those observed in response to LH stimulation was utilized equally by granulosa cells of mature and developing follicles. However, at high pregnenolone concentrations, granulosa cells of mature follicles converted significantly more of the precursor to progesterone. Assay of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) showed that the enzyme has two Kms: a low Km present in cells of both mature and developing follicles, and a high Km found only in granulosa cells of more mature follicles. It is concluded that LH-promoted progesterone synthesis in granulosa cells of developing chicken follicles is restricted not so much by the availability of receptors and the competence of the adenylate cyclase/cyclic AMP system, but by the activity of key enzymes, principally the cholesterol-20,22 desmolase. 相似文献
10.
Vitronectin was quantified in the follicular fluid aspirated from bovine follicles with diameters of 3 to 15 mm (as determined by ultrasonography) using a specific enzyme-linked immunosorbent assay (ELISA) validated for bovine vitronectin. The primary antibody was raised in rabbit against vitronectin purified from bovine plasma. Vitronectin quantified in serial dilutions of bovine plasma and ovarian follicular fluid was highly correlated with the volume of each sample assayed. In addition, known amounts of purified bovine vitronectin added to samples of plasma or follicular fluid were accurately recovered. Follicular fluid concentrations of vitronectin were positively correlated with the follicle diameter (r = 0.8; P < 0.01). These data indicate that bovine follicular fluid concentration of vitronectin is influenced by the stage of follicular development. 相似文献