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Biomedical subjects

B L Marrone

Publications and source records attributed to B L Marrone.

At least 19 recordsLinked to original sources
PubMed1992

Hormone stimulated steroid biosynthesis in granulosa cells studied with a fluorogenic probe for cytochrome P-450SCC.

The regulation of steroidogenesis by luteinizing hormone (LH) was studied in granulosa cells during follicular development using a fluorescent reporter assay based on the metabolism of a fluorescent probe specific for cytochrome P-450SCC (cholesterol side-chain cleavage enzyme). Intact granulosa cells or mitochondria were obtained from the first (F1) second (F2) and third (F3) largest preovulatory follicles of the hen ovary and incubated with the fluorogenic substrate. Metabolism of this substrate by cytochrome P-450SCC generates the highly fluorescent resorufin anion (the fluorescent reporter). In both mitochondria and intact granulosa cells, incubated with the fluorescent substrate, an increase in resorufin fluorescence was observed and the increase was greater in samples derived from F1 than in samples from F2 or F3. In cells, LH added simultaneously with the P-450SCC substrate significantly increased resorufin fluorescence above control values in a time- and dose-dependent manner up to 2-3 h after the incubation was initiated. Forskolin and 8-bromo-cAMP also stimulated metabolism of the P-450SCC substrate significantly by 15 min. When granulosa cells were preincubated with LH before exposure to the P-450SCC substrate resorufin fluorescence was significantly attenuated compared to controls (not exposed to LH in the preincubation period). The decrease in resorufin fluorescence observed when cells were pretreated with LH, may be due to the release of cholesterol from endogenous pools and its competition with the exogenous fluorogenic for the substrate P-450SCC enzyme. In granulosa cells that were preloaded with the P-450SCC substrate, the stimulatory effect of LH treatment remained constant from 30 min to 2 h after hormone addition. The results show that this fluorescent probe can be used in a rapid assay for the continuous measurement of the acute effects of hormone agonists on cholesterol conversion to pregnenolone in steroidogenic cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Flow cytometric analysis of steroidogenic organelles in differentiating granulosa cells.

Functional and structural changes accompany the differentiation of granulosa cells during follicular development. We used flow cytometry and fluorescent dyes to characterize two organelles important to the steroidogenic process. Mitochondria, which contain the rate-limiting enzyme responsible for cholesterol conversion to pregnenolone, and lipid droplets, which store cholesterol substrate, were probed in viable hen granulosa cells during differentiation. The fluorescent dye Dio3-C5 (DiO) was used to probe mitochondrial membrane potential, indicative of mitochondrial activity and/or number, during rapid granulosa cell differentiation in a hierarchy of individual developing hen preovulatory follicles (F6, smallest, to F1, largest). Cellular DiO fluorescence, granularity, and cell size were significantly elevated with increasing maturation state. Treatment with LH significantly increased DiO fluorescence in granulosa cells from F1 but not F3. The increased mitochondrial activity/number in granulosa cells that accompanies follicular maturation and is influenced by LH may reflect, at least in part, increased activity or amount of hormone-regulated mitochondrial enzymes controlling steroidogenesis. Flow spectrofluorometry and the metachromatic lipid dye, nile red, were used to probe lipid droplets in differentiating granulosa cells from F6 to F1. There was a dramatic increase in the fluorescence component related to lipid droplets with increasing stages of follicular maturation, suggesting recruitment of lipids into droplets during the differentiation of granulosa cells into hormone-responsive steroidogenic cells. The results demonstrate the dynamic nature of the granulosa cell morphology involved in steroidogenesis during follicular development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Rapid DNA sequencing based upon single molecule detection.

We are developing a laser-based technique for the rapid sequencing of 40-kb or larger fragments of DNA at a rate of 100 to 1000 bases per second. The approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA fragment into a flowing sample stream, and detection of individual fluorescently labeled bases as they are cleaved from the DNA fragment by an exonuclease. The ability to sequence large fragments of DNA will significantly reduce the amount of subcloning and the number of overlapping sequences required to assemble megabase segments of sequence information.

Base Sequence↗
PubMed1991

Single cell endocrinology: analysis of P-450scc activity by fluorescence detection methods.

A mechanism-based, fluorogenic probe for the cytochrome P-450scc (cholesterol side chain cleavage) enzyme, rate-limiting for the conversion of cholesterol to steroid hormones, is introduced and its application to the study of enzyme activity and regulation in single steroidogenic cells by several fluorescence detection methods is demonstrated. Reaction of the probe with P-450scc gives pregnenolone and the highly fluorescent resorufin anion. Spectroscopic changes in probe fluorescence, indicative of P-450scc activity, were monitored by steady-state fluorescence spectroscopy, flow cytometry, and microspectrofluorometry. This unique probe provides a nonradiometric indicator for real-time measurement of P-450scc activity in single living cells.

Animals↗
PubMed1990

Regional pattern of cell maturation and progesterone biosynthesis in the avian granulosa cell layer.

The objective of the present study was to compare the structural and functional features of cells derived from histologically different regions of the granulosa cell layer of hen preovulatory follicles. Granulosa cells were isolated from a 0.8-1.5-cm diameter region of the granulosa layer overlying the germinal disc (GD) or from the remainder of the granulosa layer peripheral to the disc region (GP). In the first study, the isolated cells were prepared from each region of the five largest preovulatory follicles; fixed; stained with fluorescent dyes for DNA, total protein, and RNA; and analyzed by use of multiparameter flow cytometry. A greater percentage of cells from the GD region than from the GP region were in proliferative (S and G2/M) stages of the cell cycle in the four largest follicles. In addition, GD cells had lower relative protein content than GP cells in the two largest follicles. In the second study, progesterone biosynthesis in response to treatment with luteinizing hormone (LH) or forskolin was examined in granulosa cells from the GD and the GP regions of the largest preovulatory follicles. GP cells had greater responsiveness to the treatments than GD cells. In addition, conversion of 25-hydroxy-cholesterol to progesterone was greater in GP cells than in GD cells. There were no differences in cyclic adenosine 3',5'-monophosphate (cAMP) production by GD and GP cells in response to LH or forskolin or in the ability of cells from each region to convert pregnenolone substrate to progesterone via 3 beta-hydroxysteroid dehydrogenase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules.

We are developing a laser based technique for the rapid sequencing of large fragments (approximately 40 kb) of DNA based upon the detection of single, fluorescently tagged nucleotides cleaved from a single DNA fragment. We have demonstrated significant progress on several of the important steps of this technique. The projected rate of sequencing is several hundred bases per second which is orders of magnitude faster than existing methods. Once developed, this technology could be utilized by investigators for rapid sequencing of genetic material from virtually any source.

Base Sequence↗
PubMed1989

Quantitative cytochemistry of 3 beta-hydroxysteroid dehydrogenase activity in avian granulosa cells during follicular maturation.

Previous studies have shown that biosynthesis of progesterone, the major steroid product of hen granulosa cells, increases during follicular maturation. However, the contribution of individual granulosa cells to the total progesterone production of each follicle is not known. The objective of the present study was to determine the presence and relative activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in individual granulosa cells isolated from each of the five largest yolk-filled preovulatory follicles of laying hens. 3 beta-HSD cytochemistry in the presence or absence of pregnenolone substrate was performed on digitonin-permeabilized granulosa cells in suspension. The stained cells were fixed in a 70% ethanol solution until 1) the percentage of cells from each follicle that stained dark blue-indicating the presence of 3 beta-HSD activity-was determined by counting under light microscopy, and 2) the intensity of staining-indicating the relative amount of enzyme activity-was quantified using video image analysis. There were three findings. First, 100% of granulosa cells from each of the five largest preovulatory follicles stained positively for the presence of 3 beta-HSD activity. Second, the amount of 3 beta-HSD activity was normally distributed among granulosa cells in the population from each follicle. Third, as follicles matured from the fifth largest to the largest follicle, 3 beta-HSD activity increased steadily in individual cells, as indicated by increased staining intensities. The results indicate uniformity in the steroidogenic capacity of cells in the granulosa layer of hen preovulatory follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1988

Characterization of granulosa cell subpopulations from avian preovulatory follicles by multiparameter flow cytometry.

The objective of the present study was to characterize the granulosa cell populations from individual hen (Gallus domesticus) preovulatory follicles at defined stages of follicular maturation using multiparameter flow cytometry. Granulosa cells were fixed and stained with three fluorochromes that selectively bind to DNA (Hoechst 33342, blue), RNA (pyronin Y, red), or protein (fluorescein isothiocyanate, green). A flow cytometer equipped with a three-laser excitation system was used to analyze three colors of fluorescence from stained cells. Forward angle light scatter and axial light loss measurements were made on each cell to determine relative cell size. In addition, the ratios of RNA to protein and DNA to protein were measured. The major findings obtained from correlated measurements of cell cycle (DNA), protein, RNA, cell size, and ratios were: 1) the percentage of proliferating cells decreased while cell size increased during follicular maturation; 2) two subpopulations of granulosa cells were identified within each follicle based on relatively high and low protein contents; the fraction of cells in the high protein subpopulation increased, and the fraction of cells in the low protein subpopulation decreased during follicular maturation; 3) the high and low protein subpopulations also differed in cell cycle distribution, RNA content, and cell size; and 4) the distribution of cells into the two subpopulations and the degree of proliferation were influenced by stage of the ovulatory cycle, primarily in the most mature follicles. The results demonstrate the dynamic heterogeneity of the granulosa cell populations from individual ovarian follicles and show the influences of follicular maturation and stage of the ovulatory cycle on cell growth and metabolism.

Animals↗
PubMed1988

Cyclic adenosine monophosphate (cAMP) production in avian theca cells during follicular maturation.

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.

Androstenedione↗
PubMed1986

Ovarian steroidogenesis in vitro during the first month posthatching in the domestic chick: gonadotropin responsiveness and [3H]progesterone metabolism.

Ovarian steroidogenesis was examined in domestic chicks during the first month posthatching. In vitro production of androstenedione during a 4-hr incubation was enhanced in a dose-dependent manner by 0.1-100 ng/ml of chicken LH (cLH). The greatest response was observed in ovaries from Day 1 chicks (eightfold), but cLH was also effective in Day 7, 14, 21, and 28 ovaries (three- to sixfold increase). Estrogen production in response to cLH was increased significantly only in Day 1 and 7 ovaries. A similar trend in androstenedione and estrogen production was seen in response to 8-bromo cAMP, oLH, and oFSH. In an additional experiment, in vitro metabolism of [3H]progesterone by ovaries from Day 1, 7, 14, and 21 chicks was examined during a 4-hr incubation. The major metabolite, comprising 18-19% of the radioactivity, coeluted with 5 beta-pregnan-3,20-dione. This is the first report of gonadotropin-stimulated steroidogenesis and progesterone metabolism in the chick during the first month post-hatching. The results show that the responsiveness to gonadotropins decreases during this period and the profile of [3H]progesterone metabolism does not resemble that seen in adult granulosa and theca cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Inhibitory action of epidermal growth factor on progesterone biosynthesis in hen granulosa cells during short term culture: two sites of action.

The acute effect of epidermal growth factor (EGF) on progesterone biosynthesis by hen granulosa cells in short term culture was investigated. Pretreatment of cells for 5 h with EGF at concentrations of 1000-4000 ng/ml inhibited LH-stimulated progesterone production by 54%. Shorter EGF pretreatment times of 1 and 3 h caused 25% and 35% inhibition of LH-stimulated progesterone production, respectively. In additional experiments, EGF was found to inhibit progesterone production in response to 8-bromo-cAMP (1 mM) and forskolin (100 microM) by 34% and 35%, respectively. EGF had no effect on the conversion of 25-hydroxy-cholesterol or pregnenolone to progesterone, indicating that one site at which EGF inhibits progesterone biosynthesis is distal to cAMP generation, but before the side-chain cleavage step. EGF also inhibited LH-stimulated cAMP production by 32%, but had no effect on forskolin-stimulated cAMP accumulation. This indicated that there was a second site of EGF action in these cells, probably at the level of LH receptor coupling to the adenylate cyclase. Nerve growth factor (4000 ng/ml) had no effect on progesterone production, but fibroblast growth factor (4000 ng/ml) facilitated LH-stimulated progesterone production. The results demonstrate that the acute inhibitory effect of EGF on LH-stimulated progesterone biosynthesis in hen granulosa cells is due to its action at two sites: one at a site before the production of cAMP and the other at a step beyond cAMP generation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Steroidogenesis in ovarian cells of the Japanese quail (Coturnix coturnix japonica).

The influence of follicular maturation on steroidogenesis and steroid metabolism by isolated Japanese quail granulosa and theca cells was examined. When stimulated with LH, granulosa cells of the largest follicle (F1) responded with a sixfold increase over unstimulated progesterone levels, whereas progesterone production in cells of F3 less than doubled even when maximally stimulated. Forskolin stimulated progesterone synthesis in both F1 and F3 granulosa cells, but its effect was less pronounced than that of LH. Furthermore, F1 cells metabolized 25-hydroxycholesterol to a greater extent than did F3 cells. There was no appreciable metabolism of [3H]progesterone by granulosa cells. Theca cells from the smaller follicles (F3-F5) responded to LH stimulation with greater estrogen and androstenedione production than theca cells from F1. [3H]Progesterone was metabolized mainly to androstenedione in theca cells. Thus, the overall pattern of in vitro steroidogenesis in quail granulosa cells is similar to that described for the chicken and turkey even though the quantitative differences in the steroidogenic capacity between developing and mature follicles are more striking in the quail. Furthermore, although the LH-stimulated androstenedione and estrogen production appears similar in developing quail and chicken theca cells, the profile of [3H]progesterone metabolism is different in quail theca cells from that found previously in chicken theca cells.

1-Methyl-3-isobutylxanthine↗
PubMed1979

Intrahypothalamic implants of progesterone inhibit lordosis behavior in ovariectomized, estrogen-treated rats.

Intracranial implants of crystalline progesterone (P) were used to examine in site of action of P's facilitatory and inhibitory effects on lordosis behavior in the ovariectomized, estradiol-benzoate (EB)-primed RAT. P implanted in the medial basal hypothalamus (MBH) 1 h prior to subcutaneous (s.c.) EB injection inhibited lordosis in response to a systemic P injection 44 h after EB (concurrent inhibition). P implanted in the MBH did not facilitate lordosis when implanted 44 h after EB injection, but this same implant of P inhibited lordosis in response to P injection 68 h after EB (sequential inhibition). Cholesterol (Chol) implants in the MBH did not inhibit lordosis behavior in either the concurrent or the sequential inhibition experimental paradigms. The results indicate that the MBH is an important site of P inhibition of sexual receptivity in the rat.

Animals↗
PubMed1977

Role of catechol estrogens in activation of lordosis in female rats and guinea pigs.

In a variety of experiments, we tested the effectiveness of the 2-hydroxylated estrogen in facilitating sexual receptivity. A single injection of 2-hydroxy-estradiol-17beta (2-OHE2) to ovariectomized rats or 2-hydroxy-estrone (2-OHE1) to ovariectomized guinea pigs was ineffective in priming animals for facilitation of sexual receptivity even when a subsequent injection of progesterone was administered. The only facilitatory effect of catechol estrogens on lordosis that was demonstrated in this study occurred when 2-OHE2 was injected in combination with E2 and a subsequent injection of progesterone was given to rats. These results suggest a cooperatively between catechol estrogen and E2, but they also indicate that catechol estrogens, by themselves, do not play a crucial role in mediating sexual receptivity in rodents.

Animals↗