scholarly journals Isolation and properties of cholesterol esterstorage granules from ovarian tissues

1973 ◽  
Vol 134 (2) ◽  
pp. 399-406 ◽  
Author(s):  
David T. Armstrong ◽  
Anthony P. F. Flint
Keyword(s):  
Endoplasmic Reticulum ◽  
Luteinizing Hormone ◽  
Cholesterol Ester ◽  
Microsomal Fraction ◽  
Cholesterol Esterase ◽  
Interstitial Tissue ◽  
Unesterified Cholesterol ◽  
Rat Ovary ◽  
Soluble P

Cholesterol ester-storage granules were isolated from luteinized rat ovary and rabbit ovarian interstitial tissue by centrifugal flotation and were investigated with regard to their structure and function. Cholesterol ester, protein, phospholipid and unesterified cholesterol accounted for the dry weight of granules from luteinized rat ovary. The protein and the phospholipid were resistant to removal by washing. Substrate specificities of nucleotide phosphatase and specific radioactivities of lipid-soluble P (determined after administration of [32P]Piin vivo) were the same in granules and in a microsomal fraction from the same tissue. After administration of [32P]Piin vivo, luteinizing hormone increased the specific radioactivity of lipid-soluble P in granules, mitochondria and the microsomal fraction. Since granules did not swell in hypo-osmotic media, whereas microsomal particles did, it is suggested that adherent phospholipid and protein in granule suspensions is unlikely to result from contamination with endoplasmic reticulum. Luteinizing hormone administered in vivo increased the phospholipid and unesterified cholesterol contents of isolated granules relative to their cholesterol ester content, and also tended to raise their protein content. This treatment decreased the ability of isolated granules to act as a substrate for cholesterol esterase in vitro and increased the activity of cholesterol esterase. Cycloheximide in vivo also raised the unesterified cholesterol/cholesterol ester ratio of isolated granules, and when administered with luteinizing hormone acted synergistically to bring about a further increase. These results are considered compatible with evidence obtained by microscopy which suggests that granules may be surrounded by a membrane, that they arise by pinching off from the endoplasmic reticulum, and that they shrink on trophic stimulation of the tissue.

scholarly journals Control of ovarian cholesterol ester biosynthesis

1973 ◽  
Vol 132 (2) ◽  
pp. 313-321 ◽  
Author(s):  
A. P. F. Flint ◽  
D. L. Grinwich ◽  
D. T. Armstrong
Keyword(s):  
Luteinizing Hormone ◽  
Cyclic Amp ◽  
Cholesterol Ester ◽  
Cholesterol Esterase ◽  
Interstitial Tissue ◽  
Steroid Synthesis ◽  
Precursor Pool ◽  
Ester Synthetase

1. Experimental evidence is presented for a role of progesterone and 20α-hydroxypregn-4-en-3-one as inhibitors of cholesterol ester synthetase in the acute depletion of ovarian cholesterol ester after trophic stimulation. 2. Luteinizing hormone in vitro decreased by 84% the rate of esterification of cholesterol with added [14C]oleate by slices of rabbit ovarian interstitial tissue; this effect was mimicked by cyclic AMP (adenosine 3′:5′-cyclic monophosphate) in vitro, and occurred without large changes in precursor pool sizes or membrane permeability. 3. Cyclic AMP was shown to have no direct effect on cholesterol ester synthetase or cholesterol esterase in cell-free extracts of rabbit ovarian interstitial tissue, but decreased the activity of cholesterol ester synthetase (not that of cholesterol esterase) in extracts prepared from slices previously incubated with it. 4. The inhibitory effect of cyclic AMP on esterification of cholesterol with added [14C]-oleate was prevented by both cycloheximide and aminoglutethimide phosphate (which also inhibited steroid synthesis in response to cyclic AMP). 5. Cyclic AMP raised the intracellular concentrations of progesterone and 20α-hydroxypregn-4-en-3-one in incubated slices by factors of 2.8 and 3.9 respectively. 6. Cycloheximide and aminoglutethimide phosphate administered in vivo blocked cholesterol ester depletion in response to luteinizing hormone in rats; in these ovaries cycloheximide and aminoglutethimide phosphate decreased the concentrations of progesterone and 20α-hydroxypregn-4-en-3-one and luteinizing hormone raised them. 7. Progesterone and 20α-hydroxypregn-4-en-3-one added to cell-free extracts of rabbit ovarian interstitial tissue in vitro (at concentrations comparable with those found in incubated slices) inhibited cholesterol ester synthetase by up to 85%. 8. The results are discussed with reference to the acute control of cholesterol ester concentrations in the ovary and adrenal cortex.

scholarly journals Activities of enzymes responsible for steroid biosynthesis and cholesterol ester metabolism in rabbit ovarian interstitial tissue and corpora lutea. A comparison of enzyme activities with flow rates

1973 ◽  
Vol 132 (2) ◽  
pp. 301-311 ◽  
Author(s):  
A. P. F. Flint ◽  
D. T. Armstrong
Keyword(s):  
Cholesterol Ester ◽  
Cholesterol Esterase ◽  
Interstitial Tissue ◽  
Corpora Lutea ◽  
Chain Cleavage ◽  
Cleavage Enzyme ◽  
Storage Granules ◽  
Ester Synthetase

A method involving the use of isolated cholesterol ester-storage granules as substrate is described for the assay of cholesterol esterase in rabbit ovarian tissues. Activities of cholesterol esterase 100–200-fold higher than those previously reported in ovarian tissues were measured by using this method. In addition to that of cholesterol esterase, activities of cholesterol ester synthetase, cholesterol side-chain cleavage enzyme and 3β-hydroxy steroid dehydrogenase were determined in rabbit ovarian interstitial tissue and corpora lutea. Activities of these enzymes are in general compatible with the flows through them measured under a variety of conditions both in vivo and in vitro. It is concluded that, in the rabbit ovarian tissues investigated, these enzymes are capable of catalysing the conversions usually attributed to them.

Studies on the rapid cholesterol-depleting and steroidogenic actions of luteinizing hormone in the rat ovary: effects of aminoglutethimide phosphate

1970 ◽  
Vol 48 (8) ◽  
pp. 881-884 ◽  
Author(s):  
Harold R. Behrman ◽  
David T. Armstrong ◽  
Roy O. Greep
Keyword(s):  
Luteinizing Hormone ◽  
Cholesterol Ester ◽  
Free Cholesterol ◽  
Tissue Concentration ◽  
Control Level ◽  
Side Chain ◽  
Tissue Levels ◽  
Chain Cleavage ◽  
Luteal Tissue ◽  
Rat Ovary

Superovulated, immature rats were administered luteinizing hormone (LH) and/or aminoglutethimide phosphate (AGP), an inhibitor of cholesterol side-chain cleavage, to ascertain whether the steroidogenic action could be separated from the cholesterol-depleting action of LH. The injection of AGP [Formula: see text] and [Formula: see text] before sacrifice significantly reduced tissue levels of progesterone and 20α-dihydroprogesterone to 25–30% of normal, and increased tissue levels of free cholesterol in the absence and presence of LH given [Formula: see text] or 4 h before sacrifice. Cholesterol ester concentration was increased twofold after [Formula: see text] AGP, whereas LH injection [Formula: see text] after AGP and 4 h before sacrifice reduced the tissue concentration to the control level but not as low as that observed when only LH was given 4 h before sacrifice (50% of control level). The cholesterol ester depletion induced by LH, even though steroidogenesis was inhibited, indicates that these may represent separate events in the action of LH on luteal tissue.

scholarly journals The activity and kinetic properties of mevalonate kinase in superovulated rat ovary

1970 ◽  
Vol 120 (1) ◽  
pp. 145-150 ◽  
Author(s):  
A. P. F. Flint
Keyword(s):  
Luteinizing Hormone ◽  
Total Activity ◽  
Partial Purification ◽  
Kinetic Properties ◽  
Mevalonate Kinase ◽  
Farnesyl Pyrophosphate ◽  
Rat Ovary ◽  
High Concentrations ◽  
Geranyl Pyrophosphate

Methods are described for the assay and partial purification of mevalonate kinase from superovulated rat ovary. The total activity of mevalonate kinase in superovulated rat ovary was 1.6±0.14units/g wet wt.; it was unchanged by the administration of luteinizing hormone in vivo. The Km of a partially purified preparation of mevalonate kinase for dl-Mevalonate was 3.6±0.5μm; its Km for MgATP2− was 120±7.7μm. The enzyme was inhibited by geranyl pyrophosphate and farnesyl pyrophosphate, but not by isopentenyl pyrophosphate or 3,3′-dimethylallyl pyrophosphate. dl-mevalonate 5-phosphate inhibited at high concentrations. With both geranyl pyrophosphate and farnesyl pyrophosphate the inhibition was competitive with respect to MgATP2−. The Ki for inhibition by geranyl pyrophosphate was 1.3±0.2μm; the Ki for inhibition by farnesyl pyrophosphate was 1.0±0.3μm. These findings are discussed with reference to the control by luteinizing hormone of steroidogenesis from acetate.

STEROIDOGENIC EFFECTS OF LUTEINIZING HORMONE AND PROLACTIN ON THE RAT OVARY IN VIVO

1967 ◽  
Vol 38 (4) ◽  
pp. 423-430 ◽  
Author(s):  
K. YOSHINAGA ◽  
SUSAN A. GRIEVES ◽  
R. V. SHORT
Keyword(s):  
Luteinizing Hormone ◽  
Venous Blood ◽  
The Other ◽  
Progesterone Secretion ◽  
Rat Ovary

SUMMARY Progesterone and 20α-dihydroprogesterone were measured in the ovarian venous blood of rats in early pro-oestrus, late dioestrus, day 5 of pseudopregnancy, and in androgen-treated animals with persistent oestrus. Little progesterone was secreted in early pro-oestrus or persistent oestrus (0·1 μg./hr.), but the secretion rose in dioestrus (2·1 μg./hr.) and in pseudopregnancy (7 μg./hr.). The 20α-dihydroprogesterone secretion was low in the persistent oestrous group; in all the other groups it was 1·5–3 μg./hr. Treatment with luteinizing hormone increased progesterone secretion within 30 min. in pro-oestrus and persistent oestrus, had no significant effect in dioestrus, and depressed it markedly in pseudopregnancy. It appeared to have no significant effect on 20α-dihydroprogesterone secretion except during pseudopregnancy, when it was depressed. Treatment with prolactin produced no effect within 30 min.in pro-oestrous, dioestrous or pseudopregnant animals.

THE ENTRY OF ASCORBIC ACID INTO THE CORPUS LUTEUM IN VIVO AND IN VITRO AND THE EFFECT OF LUTEINIZING HORMONE

1967 ◽  
Vol 39 (1) ◽  
pp. 27-35 ◽  
Author(s):  
D. A. STANSFIELD ◽  
A. P. FLINT
Keyword(s):  
Ascorbic Acid ◽  
Exchange Rate ◽  
Luteinizing Hormone ◽  
Corpus Luteum ◽  
Plasma Ascorbic Acid ◽  
Progesterone Synthesis ◽  
Rat Ovary ◽  
Energy Dependent

SUMMARY Judged from the exchange rate between luteal and plasma ascorbic acid there appears to be no compartmentalization of ascorbic acid within the corpus luteum. Evidence is presented to show that the uptake of ascorbic acid into slices of superovulated rat ovary is an energy-dependent process which is inhibited by luteinizing hormone (LH) by means of its stimulatory effect on progesterone synthesis. The results are discussed in relation to the adrenal cortex and methods involving ascorbic acid depletion used in the assay of corticotrophin and LH.

scholarly journals Glucose metabolism in the superovulated rat ovary in vitro. Effects of luteinizing hormone and the role of glucose metabolism in steroidogenesis

1969 ◽  
Vol 112 (2) ◽  
pp. 243-254 ◽  
Author(s):  
A. P. F. Flint ◽  
R M Denton
Keyword(s):  
Carbon Dioxide ◽  
Glucose Metabolism ◽  
Luteinizing Hormone ◽  
Glucose Uptake ◽  
Hormone Treatment ◽  
Total Lipid Extract ◽  
Rat Ovary

1. Superovulated rat ovary slices from rats treated with 20μg. of luteininzing hormone/100g. body wt. 2hr. before death and from control animals have been incubated in vitro. Output of Δ4-3-oxo steroids (0·2μmole/g. wet wt./hr. in control tissue) was linear for 4hr., and was increased by approx. 70% in slices from luteinizing hormone-treated rats. Rate of oxygen consumption (90·0±4·6μmoles/g. wet wt./hr.) was linear for 3hr. and unaltered by luteinizing hormone treatment or addition of glucose (1mg./ml.) to the medium. 2. In slices from control animals, steady-state rate of glucose uptake was 78·0±2·9μg. atoms of carbon/g. wet wt./hr.; steady-state rates of lactate output, pyruvate output and incorporation of [U−14C]-glucose carbon atoms into carbon dioxide and total lipid extract were 60·7±0·9, 2·4±0·1, 18·0±1·1 and 0·7±0·1μg. atom of carbon/g. wet wt./hr. and accounted for 104·5±1·9% of the glucose uptake. In slices from luteinizing hormone-treated rats, glucose uptake and outputs of lactate, pyruvate and [14C]carbon dioxide were increased by approx. 25%, and 108·4±3·2% of the glucose uptake could be accounted for. 3. The total lipid extract was separated by thin-layer chromatography and saponification. Of the 14C incorporated into this fraction during incubation with [U−14C]glucose 97% was found in the fractions containing glyceride glycerol and less than 3% in the fractions containing sterols, steroids or fatty acids. Appreciable quantities of 14C were incorporated into these lipid fractions from [1−14C]acetate. 4. From a consideration of the tissue glycogen content, the specific activities of [14C]lactate and glucose 6-phosphate (C-1) derived from [1−14C]-, [6−14C]- and [U−14C]-glucose, and the ratio of [14C]carbon dioxide yields from [1−14C]glucose and [6−14C]glucose, it was concluded that there was no appreciable glycogenolysis or flow through the pentose phosphate cycle. 5. In ovary slices from both control and luteinizing hormone-treated animals, glucose in vitro raised the incorporation rate of 14C from [1−14C]acetate into sterols and steroids. Luteinizing hormone in vivo stimulated the incorporation rate in vitro but only in the presence of glucose. 6. In slices incubated in medium containing [3H]water, [14C]sorbitol and glucose (1mg./ml.), the total water space (865±7·1μl./g.) and the extracellular water space (581±22μl./g.) were unchanged by luteinizing hormone treatment in vivo but the glucose space was raised from 540±23·6μl./g. to 639±31·3μl./g. 7. Luteinizing hormone treatment was found to lower the tissue concentration of the hexose monophosphates and to increase the total activity of hexokinase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and possibly of phosphofructokinase. 8. The kinetic properties of a partially purified preparation of phosphofructokinase were found to be qualitatively similar to those from other mammalian tissues. 9. The results are discussed with reference to both the role of glucose metabolism in steroidogenesis and the mechanism by which luteinizing hormone increases the rate of glucose uptake.

An Ultra-Structural Study of Human Melanoma in Vivo and Vitro

1976 ◽  
Vol 34 ◽  
pp. 258-259
Author(s):  
James R. Gaylor ◽  
Fredda Schafer ◽  
Robert E. Nordquist
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Protein Aggregation ◽  
Structural Study ◽  
Human Melanoma ◽  
Protein Matrix ◽  
Early Form ◽  
Endoplasmic Reticulum Protein ◽  
Mitochondrial Origin

Several theories on the origin of the melanosome exist. These include the Golgi origin theory, in which a tyrosinase-rich protein is "packaged" by the Golgi apparatus, thus forming the early form of the melanosome. A second theory postulates a mitochondrial origin of melanosomes. Its author contends that the melanosome is a modified mitochondria which acquires melanin during its development. A third theory states that a pre-melanosome is formed in the smooth or rough endoplasmic reticulum. Protein aggregation is suggested by one author as a possible source of the melanosome. This fourth theory postulates that the melanosome originates when the protein products of several genetic loci aggregate in the cytoplasm of the melanocyte. It is this protein matrix on which the melanin is deposited. It was with these theories in mind that this project was undertaken.

ÉTUDES IN VIVO SUR LE MÉTABOLISME DES ANDROGÉNES APRES ADMINISTRATION DE STÉROÏDES INHIBITEURS DE L'OVULATION

1965 ◽  
Vol 50 (1) ◽  
pp. 131-144 ◽  
Author(s):  
P. Mauvais-Jarvis ◽  
M. F. Jayle ◽  
J. Decourt ◽  
J. Louchart ◽  
J. Truffert
Keyword(s):  
Luteinizing Hormone ◽  
Urinary Excretion ◽  
Normal Subjects ◽  
Hormone Activity ◽  
Testosterone Production ◽  
Normal Men ◽  
Ethinyl Oestradiol

ABSTRACT Normal subjects and hirsute women with micropolycystic ovaries were treated with ethinyl-oestrenol + 3-methoxy-ethinyl-oestradiol (Lyndiol®), in view of studying the action of this compound on the production of androgens and on the urinary excretion of their metabolites. In normal men, the production of testosterone and the excretion of androsterone and aetiocholanolone are suppressed, whereas the excretion of other 17-ketosteroids and the production of dehydroepiandrosterone sulphate are unchanged. Moreover, the luteinizing hormone activity (LH) in plasma is depressed. It seems that the preparation inhibits specifically the testicular androgen production, by suppressing the hypothalamo-hypophyseal control of LH. Testosterone production and urinary 17-ketosteroid excretion are modified in the same way in women with Stein-Leventhal's syndrome. Physiopathological and therapeutical implications which come from these results are discussed.

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