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A R Midgley

Publications and source records attributed to A R Midgley.

At least 37 records · Page 2Linked to original sources
PubMed1992

Dynamics of gonadotropin-releasing hormone release during a pulse.

This study examined the nature of the GnRH signal that travels down the pituitary portal vessels and causes an LH pulse. Individual GnRH pulses were described in terms of abruptness of increase and decrease, amplitude, duration, and amount of GnRH released. Pituitary portal blood was obtained at 30-sec intervals for 2.5 or 5 h from five short-term ovariectomized ewes. Jugular blood was sampled every 10 min for LH. We examined 13 GnRH pulss; each produced an LH pulse. The contour of most GnRH pulses approximated a square wave. The rising edge of the GnRH pulse was very abrupt; GnRH secretion increased as much as 50-fold within 1 min. The mean peak amount of GnRH collected during pulses (24 pg/min, range 2-66) was 70-fold greater than the interpulse baseline (0.2-0.5 pg/min). The release period was sustained an average of 5.5 min; thereafter, GnRH fell to prepulse levels within 3 min. Overall, the larger and more prolonged pulses of GnRH were associated with higher amplitude LH pulses. To assess the distortion of the GnRH signal by the collection procedure, samples were obtained in vitro using the same technique during application of 4- and 7-min square wave GnRH pulses by means of a syringe pump. Signals were carried as square-waves through the sampling operation with minimal distoration, with the exception that amplitude decreased during the collection procedure. Our findings indicate the square-wave pulses observed in vivo are an accurate description of the dynamics of GnRH release during a pulse in short-term overiectomized ewes.

Animals↗
PubMed1991

Electrochemical performance, biocompatibility, and adhesion of new polymer matrices for solid-state ion sensors.

Ammonium and potassium ion-selective membranes formulated with PVC/hydroxylated PVC, polyurethane/hydroxylated PVC, and moisture-curable silicone rubber matrices are studied in an effort to extend the lifetime of solid-state ion sensors through improved membrane adhesion. The PVC/membranes exhibit electrochemical performance equivalent to that of conventional PVC membranes in terms of slope, detection limit, and selectivity. The polyurethane- and silicone-rubber-based membranes have better adhesion to silicon nitride than do PVC or hydroxylated PVC matrices. Incorporating a silanizing reagent (silicon tetrachloride) significantly improves the adhesion of the polyurethane matrix. The use of silicon tetrachloride in membrane matrices also enhances the electrochemical stability of the interfacial potential between ion-selective polymer-matrix membranes and silver epoxy inner reference electrodes of solid-state sensors. The biocompatibility of the polymer matrices is examined via radiotracer protein adsorption studies and whole blood clotting time measurements. The polyurethane- and silicone-rubber-based membranes exhibit less overall nonspecific protein adsorption than the PVC or hydroxylated PVC matrices.

Adhesiveness↗
PubMed1990

Morphologically and functionally distinct subpopulations of steroidogenic cells in corpora lutea during pregnancy in rats.

On days 5, 10, 15 and 20 of pregnancy, rat corpora lutea (CL) were dissected and dissociated into single cell suspensions by enzyme treatments. The suspended luteal cells were allowed to sediment in a BSA gradient at 4 degrees C for 3.5 hours. Five fractions were collected from the top (Fraction (Fr.) 1) to the bottom (Fr. 5) of the gradient. Cells were incubated in serum-free DME-F12 for 20 hours with or without hCG (100 ng/ml) to test them functionally, and the accumulation of progesterone and testosterone was determined by radioimmunoassay. To assess 3,3-hydroxysteroid dehydrogenase (HSD) activity, a histochemical suspension-staining procedure was used. Cells were examined by light microscopy, and the percentage of cells containing dark blue formazan deposits and their diameters were determined in at least 40 microscopic fields. The number of cells staining for 3 beta-HSD did not vary by day 15 but decreased from 141.6 +/- 16.5 X 10(3) cells/CL on day 15 to 113.8 +/- 13.2 X 10(3) cells/CL on day 20 of pregnancy. However, 3 beta-HSD-positive cells maintained the same levels of progesterone secretion until the advent of luteolysis, then they increased in size progressively throughout pregnancy. In BSA gradients, the relatively larger 3 beta-HSD-positive cells migrated faster than the smaller 3 beta-HSD-positive cells on each day of pregnancy. The diameters of 3 beta-HSD-positive cells differed significantly in Frs. 2, 3, 4 and 5 on days 15 and 20 of pregnancy. On day 15 of pregnancy, less progesterone accumulated in wells containing 3 beta-HSD-positive cells from Fr. 2 (mean diameter; 24.96 microns) than from Fr. 3, Fr. 4 and Fr. 5 (mean diameters; 27.20, 30.79 and 31.28 microns, respectively) but the Fr. 2 cells responded more to hCG stimulation. Fr. 2 also showed a higher ratio of testosterone accumulation to progesterone accumulation than the other fractions. The response to hCG stimulation of cells in Fr. 2 tended to be higher than that in Fr. 3 on day 20 of pregnancy. These data suggest that the steroidogenic rat luteal cells are comprised of morphologically and functionally different cell types after day 15 of pregnancy. No stimulating nor inhibiting effects were observed in co-incubation of cells from Fr. 2 with cells from Fr. 3 or Fr. 4 on days 15 and 20 of pregnancy.

3-Hydroxysteroid Dehydrogenases↗
PubMed1989

[Morphological and functional changes in 3 beta-HSD positive cells in corpora lutea during pregnancy in rats].

On days 5, 10, 15 and 20 of pregnancy, rat corpora lutea (CLs) were dissected and dissociated into single cell suspensions by enzyme treatments. To assess 3 beta-hydroxysteroid dehydrogenase (HSD) activity, a histochemical suspension-staining procedure was used. The number of cells positive for 3 beta-HSD in CL were 148.0 +/- 13.7, 130.1 +/- 25.4, 134.0 +/- 23.5 and 116.8 +/- 13.5 X 10(3) cells on days 5, 10, 15 and 20 of pregnancy, respectively. The 3 beta-HSD positive cells increased in size from 18.5 +/- 0.28 microns on day 5 to 35.7 +/- 0.50 microns on day 20 of pregnancy. The suspended luteal cells were incubated in serum-free DME-F12 for 20 hours with or without human chorionic gonadotropin (hCG, 100 ng/ml) or dibutyryl cyclic AMP (dbcAMP, 10mM) to test their functionality, and progesterone accumulation was determined by radioimmunoassay. Progesterone secretion from the 3 beta-HSD positive cells was maintained at the same levels until day 15 and decreased significantly on day 20 of pregnancy. However the response to hCG stimulation of the 3 beta-HSD positive cells decreased significantly on day 20, the 3 beta-HSD positive cells maintained the same responsiveness to dbcAMP stimulation on progesterone secretion throughout pregnancy. These data suggest that the steroidogenic rat luteal cells may be regulated by morphologically and functionally different mechanisms, and that progesterone may be secreted by at least two different pathways.

3-Hydroxysteroid Dehydrogenases↗
PubMed1988

Biphasic action of retinoids on gonadotropin receptor induction in rat granulosa cells in vitro.

Vitamin A (retinol) has been held to be uniquely essential for normal vision and reproduction, all other functions being served by its metabolite retinoic acid. The inability of retinoic acid to maintain adequate serum progesterone is implicated as the cause of fetal resorption. The availability of lipoproteins is a major limiting factor in progesterone production and the ovarian expression of lipoprotein receptors is dependent on the action of luteinizing hormone (LH). Therefore, we investigated the effects of retinol and retinoic acid on LH receptor induction by ovarian cells in an attempt to determine the basis for the reported differences in the gonadal action of these two retinoids. Our results indicate that retinoic acid (10(-10) M) and retinol (10(-8) M) each synergistically enhance the ability of follicle stimulating hormone (FSH) to induce LH-receptors and to stimulate the formation of cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone. However, at higher concentrations, both retinoids inhibited these effects of FSH. For every measured effect, retinoic acid was more potent than retinol. Since retinol is metabolized to retinoic acid in other tissues, these results suggest that retinoic acid may be the mediator of the action of retinol on the ovary and that retinol's unique effect on reproduction needs to be investigated further.

Animals↗
PubMed1987

Lack of difference between retinoic acid and retinol in stimulating progesterone production by luteinizing granulosa cells in vitro.

Receptors for retinoids in the immature rat ovary and the effects of retinol and retinoic acid on luteinizing granulosa cells were studied. Radioreceptor assay demonstrated the presence of specific cellular retinol-binding protein and cellular retinoic acid-binding protein in the ovaries of rats injected with PMSG alone or PMSG and hCG. In addition, when luteinizing granulosa cell from PMSG/hCG-injected immature rats were cultured with or without retinoic acid, the morphology, viability, number of cells in culture, and progesterone (P) accumulation were not affected by up to 10 microM retinoic acid. Beyond 10 microM, the cells began to round up, which was associated with a decrease in cell viability. Surprisingly, the deleterious concentrations of retinoic acid increased progesterone accumulation significantly higher than the medium control value. This increase in progesterone, however, was not accompanied by an increase in cAMP. When cells preincubated for 2 days with 1 microM of either retinoic acid or retinol were subsequently incubated in retinoid-free medium containing various substrates for steroidogenesis, the following results were obtained. Basal progesterone and its accumulation in response to human low density lipoprotein were significantly higher in cells preincubated with retinoids than in the control cells. However, no difference was seen in the degree of stimulation between retinol and retinoic acid pretreatments. Both 25-hydroxycholesterol, a substrate for side-chain cleavage enzyme, and pregnenolone, a substrate for 3 beta-hydroxysteroid dehydrogenase, significantly stimulated the accumulation of progesterone in cells preincubated with retinoids over the control value. Again, no appreciable difference was observed between retinol and retinoic acid pretreatments. Our results suggest that receptors for retinoids are present in gonadotropin-primed immature rat ovaries, retinoids increase luteal cell progesterone accumulation, and no difference exists between retinol and retinoic acid in their ability to increase the accumulation of progesterone by these cells.

3-Hydroxysteroid Dehydrogenases↗
PubMed1987

Monoclonal antibodies used in solid-phase and liquid-phase assays, as exemplified by progesterone assay.

Two immunoglobulins secreted by hybridoma cell lines have been systematically investigated to determine if they could be used in solid-phase assays to give results comparable with those obtained by conventional liquid-phase radioimmunoassay (RIA). The antibodies, BQ.1 and BQ.2, bind with high specificity to the steroid hormone progesterone. The affinity constants, Ka, to 125I-labeled progesterone derivatives are 1.1 X 10(11) L/mol and 9.1 X 10(9) L/mol, respectively. Progesterone inhibited the binding of radioiodinated derivatives (amides of tyramine, histamine, and tyrosine methylester with 11 alpha-progesterone hemisuccinate) equally well. For solid-phase assays, we immobilized antibody BQ.1 via Protein A to different polystyrene surfaces (about 30 pg per tube at 50% inhibition of radiolabeled tracer). Under these conditions, the performance of this antibody for the quantification of progesterone was equivalent to that obtained in RIA. For the immobilized antibody BQ.2, only 1/10 of the amount used for optimal results in RIA was required in solid-phase assays. Binding of either antibody to the antigen was undiminished after several freezing-thawing cycles. When immobilized on solid matrices, both antibodies retained up to 95% of their binding properties for one year. Thus high-affinity, high-specificity monoclonal antibodies can be obtained for haptens and, when suitably immobilized, can be used in solid-phase assays with results equal to or better than those obtained with liquid RIA.

Antibodies, Monoclonal↗
PubMed1987

Surface modification with protein A for uniform binding of monoclonal antibodies.

We describe optimal conditions for immobilization of two monoclonal antibodies to progesterone for solid-phase assays. Polystyrene surfaces are refined with Protein A to achieve uniform, reproducible, stable, and sterically accessible immobilization of immunoglobulins (IgG). To this end, we optimized the amount of immobilized Protein A, the pH of the medium for immobilization, the concentration of antibody, and the polystyrene surface. We also investigated three carriers for solid-phase assays: 12 X 75 mm polystyrene test tubes, Macrowells (Skatron, Inc.; suitable for processing with multiple pipettors), and microwell strips (Immulon II, Dynatech Inc.). Immunoglobulin does not appreciably dissociate from any of these solid matrices, even if the assay procedure takes several hours. Therefore, we postulate that more than one molecule of immobilized Protein A binds to IgG, or that there is an additional interaction between the antibody and the polymer surface.

Animals↗
PubMed1983

Preferential masking by the receptor of immunoreactive sites on the alpha subunit of human choriogonadotropin.

125I-Labeled human choriogonadotropin (125I-hCG) bound to rat ovarian receptor was solubilized in Triton X-100. By using increasing concentrations of nine different antisera specific for the individual subunits of human choriogonadotropin (hCG), free 125I-hCG or 125I-hCG-receptor complex was precipitated by double-antibody technique. The ability of any antiserum to bind to the hormone-specific beta subunit was not affected by hCG binding to receptor, suggesting that this subunit is not directly involved with the receptor in the final state of the hormone-receptor complex. In contrast, every antiserum specific for the alpha subunit was dramatically inhibited in binding to the solubilized 125I-hCG-receptor complex. These results suggest that the alpha subunit directly interacts with the receptor, thereby masking immunoreactive sites normally available on the free hormone. Because a number of reports describe binding activity of high concentrations of immunopurified beta subunits of hCG, we propose a two-step model for the binding of hCG to receptor and postulate separate and distinct roles for the subunits. We propose that the binding of hCG to the receptor involves a specific low-affinity initial interaction of the beta subunit with the receptor that activates a second site for the high-affinity binding of alpha subunit and stabilization of the hormone-receptor complex.

Animals↗
PubMed1983

Developmental changes in the ovarian follicular basal lamina detected by immunofluorescence and electron microscopy.

Alterations in the basal lamina (BL) of developing follicles were studied by immunofluorescent microscopy using antibodies against type IV collagen, laminin, and fibronectin, and by electron microscopy. Ovarian development was induced in immature rats by sequential administration of estradiol, follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). A continuous BL was observed in healthy follicles treated with estradiol and FSH. As determined by immunofluorescence, laminin, type IV collagen, and fibronectin were restricted to the BL and the theca but not to the granulosa. When follicles were allowed to undergo atresia or induced to ovulate with hCG, the BL became fragmented. This was confirmed by electron microscopy of healthy, atretic, and luteinizing follicles which showed that in healthy follicles the BL was continuous, whereas in both atretic and luteinizing follicles, it was fragmented. Atresia was also associated with the penetration of thecal cells into the follicles. These observations indicate that the intact BL present in healthy follicles undergo extensive changes during atresia and ovulation.

Animals↗
PubMed1981

Bleb formation in granulosa cells of rat pre-ovulatory follicles in vivo and in vitro.

To determine if hormone-induced events leading to ovulation an granulosa cell luteinization might be associated with changes in the surface configuration of granulosa cells we have studied the morphology of granulosa cells from the preovulatory follicles both in vivo and in vitro. In vivo, granulosa cells in follicles from rats primed with estradiol and FSH developed bulbous protrusions termed blebs in response to injected hCG. The blebs were restricted to the adluminal granulosa cells which possess the least number of receptors for hCG. When granulosa cells from follicles of rats primed with estradiol and FSH were cultured in vitro, in the absence of serum, approximately 10% of the cells formed blebs. In the presence of 10% rat or fetal calf serum, nearly 90% of the cells formed blebs by 18 hr. Serum-induced bleb formation was prevented by 1 mM dibutyryl cycle-AMP plus 0.5 mM methyl isobutyl xanthine and by cytochalasin B (25 mug/ml), while 0.1 muM colchicine had no effect. Fibronectin at 25 mug/ml increased bleb formation three-fold over control values in serum-free medium. When hCG was included in serum containing medium, the majority of the cells remained smooth without any blebs. Thus, in contrast to its action in vivo, hCG inhibited the formation of blebs in vitro. When the cells incubated in the presence of dbcAMP plus methyl isobutyl xanthine in serum-containing medium, none of the cells formed blebs. One explanation for the seemingly opposite actions of hCG in vivo and in vitro is that hCG might act to alter the permeability of the pre-ovulatory follicles, and thereby allow the admission of serum. The admitted serum component(s) could then induce the formation of blebs on receptor-deficient adluminal cells that did not have elevated cAMP concentrations. The results suggest that fibronectin and/or other serum components, act to induce microfilament-dependent, cAMP-inhibited bleb formation on granulosa cells in vivo and in vitro.

Animals↗
PubMed1981

Differential processing of the two subunits of human choriogonadotropin (hCG) by granulosa cells. I. Preparation and characterization of selectively labeled hCG.

The alpha- and beta-subunits of hCG were radioiodinated and recombined with unlabeled complementary subunits. The resultant recombined hormones, selectively labeled in either the alpha- or beta-subunit, were separated from unrecombined subunit by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, extracted with Triton X-100, and characterized by binding analysis. The estimates of maximum binding (active fraction) of the two resultant selectively labeled, recombined hCG preparations, determined with excess receptor were 0.41 and 0.59. These values are similar to those obtained when hCG is labeled as an intact molecule. The specific activities of the recombined preparations were estimated by four different methods, and the resulting values were used in combination with the active fraction estimates to determine the concentrations of active free and bound hormone. Binding analyses were run using varying concentrations of both labeled and unlabeled hormone. Estimates of the equilibrium dissociation binding constant (Kd) and receptor capacity were calculated in three different ways. The mean estimates of capacity (52.6 and 52.7 fmol/mg tissue) and Kd (66.6 and 65.7 pM) for the two preparations were indistinguishable. Additionally, these values were similar to values reported previously for hCG radioiodinated as an intact molecule. The availability of well characterized, selectively labeled hCG preparations provides new tools for studying the mechanism of action and the target cell processing of the subunits of this hormone.

Animals↗
PubMed1981

Differential processing of the two subunits of human choriogonadotropin by granulosa cells. II. In vivo studies.

Using a well characterized preparation of hCG, consisting of a mixture of hCG labeled in the alpha-subunit with 125I and hCG labeled in the beta-subunit with 131I (see preceding paper), the hormone-specific preferential retention by granulosa cells in vivo of the radiolabel originally associated with the beta-subunit of hCG has been confirmed and extended. Additional studies have shown that this retention is peculiar to the granulosa cells. Luteal and interstitial/thecal elements of the ovary failed to show preferential accumulation of the label originally associated with the beta-subunit. Measurement of both radioactivities in crude subfractions of the ovarian tissues revealed that granulosa cells retain the excess of beta-subunit label in a plasma membrane/vesicular component. No such preferential retention of label was seen in any of the subfractions obtained from luteal or interstitial/thecal tissues. The radiolabeled components associated with the granulosa cells were shown to be mainly macromolecular by their precipitability with 13% trichloroacetic acid. Luteal tissue degraded the components associated with each label more rapidly than granulosa cells. In contrast, interstitial/thecal tissue degraded very little of the bound labeled components. The differential processing of individual hCG subunits by granulosa cells was shown not to result from different kinetics of binding of serum-borne hormone by two methods. Thus, changes over time in the ability of circulating hormone to bind to LH receptor in vitro were shown not to be a function of the hCG subunit having the label. Moreover, blockade of further radiolabel uptake by injection of a large excess of unlabeled hCG 30 min after radiolabel administration did not alter the rise in the ratio of beta-subunit label to alpha-subunit label normally observed in granulosa cells. The ability of kidney tissue to accumulate and metabolize hCG also varied with the physiological state. Within the limitations of following the radioiodides added to proteins rather than the peptides themselves, these studies demonstrate that differences exist in the metabolism of hCG by the various target cells of the ovary and that changes in processing occur during luteinization.

Animals↗
PubMed1980

Differential processing of subunits of human chorionic gonadotropin by granulosa cells in vivo.

The question of whether the two subunits of the glycoprotein hormone human chorionic gonadotropin (hCG) are handled separately or as a unit by target cells was addressed by using a dual-labeling procedure. The individual alpha and beta subunits of hCG were labeled with 131I or 125I, recombined, chromatographed, and injected intravenously into hormonally primed immature rats. The ovaries of these rats contained large numbers of antral follicles, the granulosa cells of which possess high concentrations of receptor for hCG. Comparisons of the distribution of the different radioisotopic labels in various tissues over time indicated the activity associated with the beta subunit was preferentially retained by the ovarian granulosa cells in a hormone-specific manner, while the activity associated with the alpha subunit was preferentially lost. This contrasted with other tissues, including other ovarian cells with receptor for hCG, in which both radiolabels were either handled nondifferentially or handled differentially in a hormonally nonspecific manner.

Adrenal Glands↗
PubMed1979

Rapid changes in the synthesis of specific ovarian granulosa cell proteins induced by human choriogonadotropin.

Total ovarian granulosa cell proteins were labeled in vitro with [35S]methionine and analyzed by two-dimensional polyacrylamide gel electrophoresis. Of the 100-150 distinct proteins resolved by this method, 1-2% demonstrated an observable change in their synthesis during the early phases of luteinization after hormone administration. Two specific proteins first appeared as early as 30 min after treatment with human choriogonadotropin, while four additional new proteins became apparent at 3 hr. These changes in the synthesis of specific proteins occurred far in advance of the morphological changes associated with luteinization, which occur 24-72 hr later, and thus may play an important role in this process of hormonally induced cell differentiation in the rat ovary.

Animals↗