Biomedical subjects

E K Asem

Publications and source records attributed to E K Asem.

At least 37 records · Page 2Linked to original sources

PubMed1999

Methodological aspects of measuring amino acid uptake in studies with porcine jejunal brush border membrane vesicles.

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.

Amino Acids↗

PubMed1998

Enzyme-linked immunosorbent assay for canine alpha 1-protease inhibitor.

OBJECTIVE: To develop and validate an ELISA for quantifying alpha 1-protease inhibitor (alpha 1-PI) in serum and fecal extracts. PROCEDURE: Affinity-purified rabbit origin canine alpha 1-PI antibodies were biotinylated and, after addition of streptavidin-horseradish peroxidase, were used as the labeled complex in a noncompetitive immunoassay. The alpha 1-PI standards were made from purified serum canine alpha 1-PI diluted in phosphate-buffered saline solution containing 5% newborn calf serum and 0.01% thimerosal. This assay was validated by determining linearity, recovery of added alpha 1-PI, detection limit, and intra- and interassay precision. Control range for serum and fecal alpha 1-PI concentration was determined for samples from 25 healthy pet dogs. RESULTS: The standard curve was linear between 5 and 50 ng/ml. Curves plotted after serial dilution of serum also were linear and ran parallel to the standard curve. Between- and within-assay coefficients of variation were 9.1 and 8.7%, respectively. The accuracy of the assay was tested by measuring the recovery of alpha 1-PI added to serum and fecal extracts and ranged from 93 to 111%. The reference range of fecal alpha 1-PI concentration was 0.023 to 5.67 (mean +/- SD, 2.0 +/- 1.82) micrograms/g and of serum alpha 1-PI was 0.901 to 1.96 (1.42 +/- 0.32) g/L. CONCLUSIONS: This ELISA had high precision, accuracy, and reproducibility for quantification of canine alpha 1-PI concentration in serum and fecal extracts. CLINICAL RELEVANCE: Specific ELISA for alpha 1-PI may be a useful test for the diagnosis of protein-losing enteropathy in dogs, and as such, will facilitate further characterization and better understanding of this canine disorder.

Animals↗

PubMed1997

Luteinizing hormone activates chloride currents in hen ovarian granulosa cells.

Luteinizing hormone (LH) induces progesterone production in hen ovarian granulosa cells, and this induction is inhibited when chloride ions are removed from the culture medium. This suggests that chloride channels may be involved in the signal transduction pathway responsible for the LH-induced progesterone production. In this report, we examined effects of LH on plasma membrane ion currents in single granulosa cells isolated from the largest preovulatory follicle (Fl) of the hen (Gallus domesticus). Using the perforated patch whole cell voltage clamp technique, we found that addition of LH rapidly activated a chloride current in these cells. This chloride current was present at all voltages tested (-90 to +50 mV), showed outward rectification and showed no obvious time or voltage dependence. Its magnitude was 3.5-fold that of the total resting membrane current measured before LH treatment. LH is known to elevate cyclic AMP in these cells. We found that addition of the cAMP analog Sp-cAMPS mimicked LH in inducing chloride currents in these cells. We conclude that LH can activate a chloride conductance in granulosa cells, and that this action may be mediated by cAMP.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1997

Influence of follicular maturation on the susceptibility of chicken ovarian granulosa cells to free oxygen radicals.

The influence of ovarian follicular maturation on lipid peroxidation-induced changes in the fluidity of plasma membrane of granulosa cells was investigated using the fluorogenic polyunsaturated fatty acid cis-parinaric acid (cPNA). An increase in lipid peroxidation results in the decrease of fluorescence intensity of cPNA. A decrease in membrane fluidity results in the decrease of fluorescence polarization of cPNA. Granulosa cells isolated from the largest (F1; mature), and third largest (F3; developing) preovulatory follicles as well as from a pool of immature small yellow follicles (SYF) of the domestic hen ovary were pre-labelled with cPNA. The cPNA-labelled cells were exposed to a free oxygen radical generating agent 2,2'azobis-(2-amidinopropane)-hydrochloride (AAPH). AAPH enhanced the rate of decrease in fluorescence intensity of cPNA-labelled cells. This effect of AAPH was greater in the more differentiated granulosa cells obtained from mature follicles than in less differentiated cells isolated from immature follicles. Similarly, the degree of change in fluorescence polarization was greater in more differentiated F1 granulosa cells than in the less differentiated ones. The analysis of fatty acid composition of phospholipids indicated that the plasma membrane of differentiated granulosa cells contained a higher proportion of the unsaturated fatty acids, oleic and linoleic acids. The fluorescence data show that the rigidity of the plasma membrane of chicken granulosa cells decreases with advancing follicular maturation presumably due to relative increase in the unsaturated fatty acid content of plasma membrane phospholipid.

Amidines↗

PubMed1995

Bovine ovarian follicular fluid vitronectin content is influenced by the follicle size.

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.

Journal Article↗

PubMed1995

Role of progesterone in luteinizing hormone-induced fibronectin production and deposition by chicken granulosa cells in vitro.

The role of progesterone in luteinizing hormone- (LH) induced fibronectin production and deposition by chicken ovarian granulosa cells was examined in vitro. Granulosa cells isolated from pre-ovulatory follicles of the domestic hen ovary were incubated in serum-free Medium 199, and the total amount of fibronectin (deposited, secreted into the medium and associated with cells) produced was measured by ELISA. LH increased the amount of fibronectin deposited by granulosa cells. Similarly, it increased the quantity of fibronectin secreted into the medium or associated with cells. Cyanoketone (an inhibitor of progesterone synthesis) suppressed dose dependently basal and LH-induced fibronectin deposition. Cyanketone also attenuated the total amount of fibronectin produced by control or LH-stimulated granulosa cells. Exogenous progesterone reversed the inhibitory effects of cyanoketone on basal and LH-induced fibronectin production and deposition. The non-degradable synthetic progestin R5020 stimulated fibronectin production in a dose-dependent manner. R5020 also reversed the inhibitory effects of cyanoketone on LH-induced fibronectin production and deposition. The antiprogestin, ZK 98.299, inhibited basal and LH-stimulated fibronectin production. The data demonstrate that endogenous progesterone regulates fibronectin production and deposition perhaps in an intracrine/autocrine manner. They indicate that LH-stimulated fibronectin production and deposition by chicken granulosa cells is mediated (at least in part) by progesterone.

Animals↗

PubMed1995

Intracrine role of progesterone in fibronectin production and deposition by chicken ovarian granulosa cells in vitro: effect of extracellular calcium.

The role of extracellular Ca2+ in progesterone-induced fibronectin production and deposition by hen granulosa cells was studied. Granulosa cells isolated from preovulatory follicles of the chicken ovary were incubated in Ca(2+)-deficient or Ca(2+)-containing medium 199, and the amount of total fibronectin produced (fibronectin deposited in the matrix, secreted into the medium, or associated with cells) was measured by ELISA. The quantity of fibronectin deposited as well as the total amount of fibronectin produced in the presence or absence of exogenous progesterone was suppressed in Ca(2+)-deficient medium. It required greater concentrations of exogenous progesterone to significantly increase fibronectin production in Ca(2+)-deficient medium than in Ca(2+)-replete medium. Cyanoketone, an inhibitor of progesterone synthesis, suppressed total fibronectin produced by unstimulated cells both in the absence and presence of Ca2+. However, the inhibitory effect of cyanoketone was significantly less in Ca(2+)-replete medium than in Ca(2+)-deficient medium. Exogenous progesterone reversed completely the inhibitory effects of cyanoketone. In the presence of cyanoketone, progesterone caused a greater (3-fold) increase in fibronectin production in Ca(2+)-replete medium than in the absence of Ca2+. Thapsigargin, an agent that mobilizes Ca2+ from internal stores, suppressed basal and progesterone-induced fibronectin production in the absence and presence of cyanoketone. However, the inhibitory effect of thapsigargin was significantly reduced in Ca(2+)-replete medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Intracrine role of progesterone in follicle-stimulating hormone- and cyclic adenosine 3',5'-monophosphate-induced fibronectin production and deposition by chicken granulosa cells: influence of follicular development.

The role of progesterone in FSH- and 8-bromo-cAMP (8-Br-cAMP)-induced fibronectin production by chicken ovarian granulosa cells was examined. Granulosa cells isolated from the third largest (F3; developing; 15-20 mm in diameter) preovulatory follicle and a pool of immature small yellow follicles (SYF; 6-8 mm in diameter) were incubated in serum-free medium 199, and the total amount of fibronectin produced (deposited, secreted into the medium, and associated with cells) was measured by enzyme-linked immunosorbent assay. Unstimulated F3 cells deposited greater amounts of fibronectin than unstimulated SYF cells. FSH or 8-Br-cAMP significantly increased fibronectin deposition. Similarly, both agents increased the quantity of fibronectin secreted into the medium and that associated with cells. The magnitude of FSH- and 8-Br-cAMP-enhanced fibronectin deposition or secretion into medium by SYF cells was greater than that by F3 cells. Cyanoketone (an inhibitor of progesterone synthesis) significantly suppressed basal fibronectin production by F3 cells, but not that by SYF cells. Cyanoketone completely blocked FSH- or 8-Br-cAMP-induced fibronectin production by F3 cells, but caused only a modest inhibition (nonsignificant) of agonist-induced fibronectin production by SYF cells. Exogenous progesterone completely reversed the inhibitory effects of cyanoketone on agonist-induced fibronectin production. The nondegradable synthetic progestin R5020 also reversed the inhibitory effects of cyanoketone on agonist-induced fibronectin production. The antiprogestin, ZK 98.299, inhibited basal and FSH-stimulated fibronectin production. The data demonstrate that FSH- and cAMP-stimulated fibronectin production by chicken granulosa cells is dependent (at least in part) on de novo progesterone synthesis. Furthermore, they indicate that fibronectin production and deposition by these cells are stimulated by progesterone, perhaps in an intracrine/autocrine manner, and that the role of progesterone increases with advancing stages of follicular development.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1994

Progesterone stimulates fibronectin production by chicken granulosa cells in vitro.

Experiments were conducted in vitro to examine the effect of progesterone on fibronectin production by chicken ovarian granulosa cells. Granulosa cells isolated from the largest (F1; mature) and third-largest (F3; developing) preovulatory follicles as well as form a pool of immature small yellow follicles (SYF) of the domestic chicken ovary were incubated in serum-free Medium-199 and the amounts of fibronectin and progesterone produced were quantified by enzyme-linked immunosorbent assay and radioimmunoassay respectively. The amounts of basal fibronectin and progesterone produced by granulosa cells from F1, F3 and SYF follicles increased with advancing stages of follicular development. Thus, the quantity of basal fibronectin secreted by granulosa cells was directly proportional to the amount of progesterone produced by them. Exogenously supplied progesterone increased the amount of fibronectin secreted by F1 and F3 cells in a dose-dependent manner, but its effect on SYF cells was marginal. Cyanoketone (an inhibitor of progesterone synthesis) suppressed basal fibronectin production by F1 and F3 granulosa cells and its inhibitory action was reversed by exogenous progesterone. The progesterone antagonist RU 486 also attenuated basal fibronectin production by F1 and F3 granulosa cells, but only the highest concentration affected SYF cells. The inhibitory effect of RU 486 was diminished in the presence of exogenous progesterone. These data show that progesterone regulates fibronectin production by chicken granulosa cells. They suggest that in avian granulosa cells, endogenous progesterone can stimulate fibronectin synthesis in an intracrine or autocrine manner.

Animals↗

PubMed1994

Stimulation of fibronectin production and deposition by chicken granulosa cells in vitro by epidermal growth factor and transforming growth factor alpha.

Experiments were conducted in vitro to examine the effect of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) on the production of fibronectin (deposited, secreted into medium and cell-associated) by hen granulosa cells isolated from the largest (F1; about 35 mm in diameter, mature) and third largest (F3; 15-20 mm in diameter) preovulatory follicles, as well as from a pool of immature small yellow follicles (6-8 mm in diameter). The cells were incubated in culture wells coated with type IV collagen or in wells without collagen coating, and the amounts of fibronectin produced were measured using a specific ELISA. The total amount of fibronectin produced by unstimulated cells was greatest in wells containing F1 cells and increased with time. The amount of fibronectin deposited by unstimulated cells was greatest in wells containing F1 cells and was much higher in collagen-coated wells than in uncoated wells. Both EGF and TGF-alpha increased the quantity of fibronectin deposited by granulosa cells in collagen-coated and uncoated wells. Fibronectin secreted into the medium by unstimulated cells also increased with the stage of follicular maturation and was enhanced by EGF and TGF-alpha. The quantity of cell-associated fibronectin in granulosa cells in collagen-coated and uncoated wells was also increased by these growth factors. Type IV collagen did not have any appreciable effect on the amount of fibronectin present in the incubation medium or on cell-associated fibronectin. Because of its marked effect on deposited fibronectin, there were greater total quantities of fibronectin in culture wells coated with type IV collagen. These results demonstrate that EGF and TGF-alpha stimulate the production and deposition of fibronectin by chicken granulosa cells. The results also suggest that in combination with collagen IV, these growth factors can regulate the formation of the extracellular matrix (for example, basal lamina) of the hen ovarian follicle.

Animals↗

PubMed1994

Mechanism of transovarian transmission of Salmonella enteritidis in laying hens.

To understand the mechanism of transovarian transmission of Salmonella enteritidis in laying hens, experiments were conducted to examine the isolation of S. enteritidis from the preovulatory follicles of experimentally infected hens. Salmonella enteritidis was isolated from the preovulatory follicles in 16 birds (from follicle membrane alone in 10 birds, from the follicle yolk alone in 4 birds, and from both membrane and yolk in 2 birds). In addition, 83 S. enteritidis isolates of the major phage types prevalent in United States were tested for attachment to hen ovarian granulosa cells and HEp-2 cells. Salmonella enteritidis demonstrated three different patterns of attachment to granulosa cells, namely, local, diffuse, and aggregative; whereas, only local attachment pattern was observed on HEp-2 cells. The total number of S. enteritidis isolates that demonstrated any pattern of attachment was significantly greater on the granulosa cells than on HEp-2 cells (P < .05). Salmonella enteritidis isolates of phage Types 8 and 28 demonstrated similar patterns of attachment on granulosa cells derived from the mature and developing follicles of the hen ovary. This suggest that S. enteritidis can colonize the preovulatory follicles at different stages of development. Preincubation of bacteria with the tetrapeptide arg-gly-asp-ser, the amino acid sequence known to mediate the interaction of adhesive proteins with cells, abrogated the local attachment of bacteria to granulosa cells. These results suggest that S. enteritidis can colonize the preovulatory follicles by interacting with the ovarian granulosa cells and that adhesive proteins may be involved in this process.

Animals↗

PubMed1994

Calcium enhances Acanthamoeba polyphaga binding to extracellular matrix proteins.

PURPOSE: To characterize better the ameba-host interactions that may be involved with the pathogenesis of Acanthamoeba keratitis, the role of calcium (Ca2+) on the binding of Acanthamoeba polyphaga to extracellular matrix proteins was examined in vitro. METHODS: The binding of a metabolically labeled A. polyphaga (CDC:0187:1) isolate from a case of human keratitis to collagen type IV, laminin, and fibronectin was assessed through a range of calcium concentrations in the external fluid. Binding to collagen IV was studied in detail, with and without other divalent cations and calcium channel modulators. RESULTS: Calcium increased binding in a dose-dependent manner, with significant effects at 0.1 to 1.0 microM and near-maximal effects at 1 to 100 microM, depending upon the matrix protein. Magnesium alone had no effect on ameba binding to collagen IV but suppressed the action of calcium. Strontium enhanced ameba binding, with maximal effect at 100 microM. The calcium channel antagonists nifedipine and diltiazem-HCl and a calcium channel activator, Bay-K8644, had no effect on the action of calcium. However, the inorganic calcium antagonists, lanthanum and cobalt, suppressed the effect of calcium. CONCLUSION: Low concentrations of calcium enhance the adhesion of A. polyphaga to extracellular matrix proteins. It remains uncertain whether calcium acts intracellularly or at the cell surface.

Acanthamoeba↗

PubMed1993

Phenotypic feminization in a genetic male dog caused by nonfunctional androgen receptors.

A dog raised as a female pup did not have utero-ovarian structures when ovariohysterectomy was attempted at 6 mo of age. Three months later, the dog exhibited male-like behavior, and 2 symmetrical testicular-shaped structures arose bilateral to the vulva. Intersex cases such as this one may involve chromosomal abnormalities, defects in testicular secretions or androgen insensitivity in tissues of the sex accessories. Serum concentrations of testosterone and dihydrotestosterone were measured in the intersex and in intact normal male (control; n=7) dogs. In addition, 5alpha-reductase enzyme activity was determined in the gonadal tissue of the intersex dog and control dogs. Androgen receptors were studied in cultured fibroblasts from the genital skin of the intersex and control dogs. The cytogenetic studies identified the animal's karyotype to be 78 XY. All complete cells from the gonadal tissue showed an XY sex chromosome complement. Serum testosterone concentrations before and after challenge with hCG were not different between the intersex and control dogs (6.2 vs 5.72 +/- 1.8 ng/ml and 9.5 vs 8.69 +/- 2.39 ng/ml before and after hCG challenge, respectively). Similarly serum dihydrotestosterone concentrations were not different between the intersex and control dogs before and after challenge with hCG (158 vs 162 +/- 1.4 pg/ml and 270 vs 278.71 +/- 45.98 pg/ml before and after hCG challenge, respectively). The 5alpha-reductase enzyme activity of the gonads was not different between the intersex and control dogs (105 pmol/h/mg protein vs 110 +/- 2.4 pmol/h/mg protein). The Bmax values of binding in the control dogs fibroblast strains were 25 +/- 3.5 fmol/mg protein. However, specific binding of dihydrotestosterone was virtually undetectable in the fibroblasts cultured from the intersex dog. These results exclude chromosomal abnormalities and deficient secretion of testosterone and dihydrotestosterone as causative factors. Because fibroblasts cultured from genital skin lacked any ability to specifically bind dihydrotestosterone, it is suggested that nonfunctional androgen receptors in some tissues of the sex accessories contributed to the feminine phenotype of this particular dog.

Journal Article↗

PubMed1993

Bovine serum albumin enhances calcium currents in chicken granulosa cells.

The effect of bovine serum albumin (BSA) on Ca2+ currents in chicken granulosa cells was examined using both the nystatin-perforated and the conventional whole cell patch clamp techniques. Under voltage-clamp conditions, depolarizing voltage steps evoked inward Ca2+ currents with both methods. The time- and voltage-dependence of Ca2+ currents measured with the perforated patch technique was similar to those obtained with conventional whole cell recording. Commercially prepared BSA and essentially fatty acid free BSA both rapidly enhanced the amplitude of Ca2+ currents. However, the fatty acid free BSA was more potent, and its potency was greatly reduced by incubation with saturating concentrations of oleic acid. These data show that BSA, a common constituent of incubation media, can influence ion channels in the plasma membrane of granulosa cells.

Animals↗

PubMed1993

Influence of follicular maturation on the deposition of fibronectin by chicken granulosa cells in vitro--effect of 8-bromo cyclic AMP.

Fibronectin deposition by avian granulosa cells and the effect of 8-bromo cAMP (8-br-cAMP) on this process was investigated in vitro. Granulosa cells obtained from the first (F1) and third (F3) largest preovulatory follicles and from small yellow follicles (SYF) were incubated in serum free medium 199 for 12 or 24 hr with and without 8-br-cAMP. Fibronectin deposited in incubation wells secreted in medium or associated with cells was measured by ELISA. Basal fibronectin deposition increased with time and was greatest for F1 cells. Within 12 hr, 8-br-cAMP enhanced fibronectin deposition dose-dependently by F3 and SYF cells, but not by F1 cells. The amount of fibronectin deposition caused by 8-br-cAMP (which was greater after 24 hr incubation) was 0.10- to 0.16-fold for F1 cells; 0.20- to 0.81-fold for F3 cells and more than 30-fold for SYF cells. Fibronectin secreted in the medium by unstimulated cells was also greatest in F1 cells. 8-bromo cAMP stimulated fibronectin secretion in medium dose-dependently by F3 and SYF cells, however, it had only a marginal stimulatory effect on this process in F1 cells. Cell-associated fibronectin was not increased significantly by 8-br-cAMP in F1, F3 or SYF cells. Total (deposited plus medium plus cell associated) fibronectin production was elevated in dose- and time-dependent manner by 8-br-cAMP in F3 and SYF cells, but only at high concentrations in F1 cells. Like deposited fibronectin, the relative effect of 8-br-cAMP on fibronectin secreted in the medium and on total fibronectin production was greatest in immature SYF cells. These results demonstrate that fibronectin synthesized and deposited by unstimulated chicken granulosa cells in vitro increase concomitantly with follicular maturation and cellular differentiation, and the relative change in fibronectin production caused by 8-br-cAMP was greatest in immature SYF cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1993

Evidence for the presence of fatty acid binding protein in avian granulosa cells.

The existence of fatty acid binding protein in chicken ovarian granulosa cells was investigated using a fluorescent assay based on the ability of cis-parinaric acid to fluoresce as a result of its interaction with proteins. Cytosolic fractions were prepared from granulosa cells obtained from the first (F1), second (F2), and third (F3) largest preovulatory follicles as well as from a pool of small yellow follicles of the chicken ovary, and each fraction was incubated with different concentrations of cis-parinaric acid. Total fluorescence increased (but fluorescence per milligram protein decreased) as the quantity of cytosolic protein from all follicle types was increased. Cis-parinaric acid fluorescence (which was also dose-dependent) was observed within 2 sec and increased with time reaching a plateau at about 10 sec. It was greater (p < 0.001) in the cytosolic fraction from F3 and small yellow follicle groups compared with F2 and F1 groups. Arachidonic, palmitic, oleic and stearic acids attenuated the fluorescence in dose-dependent fashion in cytosol from all follicle types presumably by displacing cis-parinaric acid from protein. Whereas arachidonic acid was most effective in suppressing fluorescence in fractions from F1 follicles, arachidonic acid lost part of its suppressive potency in fractions from F3 or small yellow follicles, and in these fractions palmitic acid was the most potent suppressor of fluorescence. These results indicate the existence of fatty acid binding protein-like activity in chicken granulosa cells and demonstrate that its concentration is greatest in the immature follicles. This protein could play an important role in the transport of fatty acids within granulosa cells.

Analysis of Variance↗

PubMed1993

Follicle-stimulating hormone-enhanced fibronectin production by chicken granulosa cells is influenced by follicular development.

Experiments were conducted in vitro to examine the effect of ovine follicle-stimulating hormone (FSH) on various fractions of fibronectin (deposited, secreted in medium, and cell-associated) resulting from culture (12 or 24 h) of chicken granulosa cells isolated from the largest (F1) and third largest (F3) preovulatory follicles as well as from a pool of immature small yellow follicles (SYF). Fibronectin in each fraction was quantified with a specific ELISA. The amount of fibronectin deposited in culture wells containing unstimulated cells increased with time and was greatest in wells containing F1 cells. Follicle-stimulating hormone increased the quantity of fibronectin deposited within 12 h by F3 and SYF cells, but not by F1 cells. The magnitude of FSH-enhanced fibronectin deposition was greatest in cells derived from immature SYF. Fibronectin secreted in the medium by unstimulated cells also increased with the stage of follicular maturation. Follicle-stimulating hormone increased the amount of fibronectin secreted in the medium by F3 and SYF cells. The quantity of fibronectin associated with cells was increased by FSH in all cell types. Total (deposited plus medium plus cell-associated) fibronectin production was elevated in a dose- and time-dependent manner when FSH was added to F3 and SYF granulosa cells, but the gonadotropin was without effect in F1 cells. The magnitude of FSH stimulation (fold increase) of total fibronectin production was calculated as a multiple of the unstimulated (control) value for each cell type. The relative change in total fibronectin production resulting from addition of FSH (after 24 h incubation) was .02- to .24-fold in F1 cells as compared with .33- to .92-fold in F3 cells and 2.75- to 4.38-fold in SYF cells. These results indicate that FSH stimulates fibronectin production by chicken granulosa cells, and this stimulatory effect decreases as the follicle matures.

Animals↗

PubMed1993

Chicken gonadotropin-releasing hormones enhance soluble and insoluble fibronectin production by granulosa cells of the domestic fowl in vitro.

Experiments were conducted in vitro to examine the effect of two chicken gonadotropin-releasing hormones, cGnRH-I ([Gln8]-GnRH) and cGnRH-II ([His5,Trp7,Tyr8]-GnRH), on fibronectin (soluble and insoluble) production by chicken granulosa cells isolated from the largest (F1; about 35 mm in diameter), and third largest (F3; 15 to 20 mm in diameter) preovulatory follicles as well as from a pool of immature small yellow follicles (SYF; 6 to 8 mm in diameter). The amounts of soluble fibronectin (fibronectin secreted into the incubation medium) and insoluble fibronectin (fibronectin associated with cells plus fibronectin attached to culture substratum) were quantified with a specific ELISA. Fibronectin secreted into the incubation medium (soluble fibronectin) by unstimulated cells increased with advanced stages of follicular maturation. Addition of both cGnRH-I and -II increased the amount of fibronectin secreted into the incubation medium by all follicular cell types. The amount of insoluble fibronectin in culture wells that contained unstimulated cells also increased with advanced stages of follicle development. Both cGnRH-I and -II increased the quantity of insoluble fibronectin by granulosa cells from all follicle types. Total (soluble plus insoluble) fibronectin production was elevated when cGnRH-I or -II was added to F1, F3, and SYF granulosa cells. The magnitude of cGnRH-I or -II stimulation (percentage increase) of soluble, insoluble, or total fibronectin production was calculated as a multiple of the unstimulated (control) value for each follicle type, and they were greatest in cells derived from developing and immature follicles. These results indicate that homologous cGnRH-I and -II are capable of directly modulating the physiology of the avian ovary.

Animals↗