Sterol sulfates in the epididymis; synthesis and possible function in the reproductive process.

Sterol sulfates are present in relatively high concentrations in the male reproductive tract. Cholesteryl sulfate is the major sterol sulfate in the human epididymis while desmosteryl sulfate is the major sterol sulfate in the hamster epididymis. While the testis is the major source of sterol sulfate in the human, the epididymis of the hamster is the source of demosteryl sulfate. This conjugate accumulates along the length of the epididymis and is taken up by the plasma membrane in the acrosomal region of the spermatozoa. Sulfotransferase activity increases along the epididymis and this is due to the actual synthesis of the enzyme. Sterol sulfates are potent and specific inhibitors of the proteolytic enzyme, acrosin. This property could provide protection against the premature release of proteolytic activity within the male reproductive tract. It is proposed that the removal of this inhibition occurs within the female tract via sulfatase activity in order to enable the acrosome reaction and ovum penetration to occur.

Aphidicolin, a specific inhibitor of DNA polymerase alpha, inhibits conversion of lanosterol to C-27 sterols in mouse L cells.

Aphidicolin, a fungal metabolite which is a specific inhibitor of DNA polymerase alpha, inhibited the incorporation of [14C]acetate into desmosterol in mouse L cells by 50% at a concentration of 8.8 microM. It had no effect on acetate metabolism into fatty acids or CO2. The site of inhibition was determined to be distal to the formation of mevalonic acid since aphidicolin also inhibited the incorporation of [14C]mevalonolactone into desmosterol but had no effect on the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) or the incorporation of [14C]acetate into total nonsaponifiable lipids. High pressure liquid chromotographic analysis of the distribution of radioactivity among the nonsaponifiable lipids formed from [14C]acetate in the presence of aphidicolin indicated an accumulation of lanosterol accompanied by a proportional decrease in radiolabeled desmosterol and two of its precursors, delta 5,7,24-cholestatrienol, and 4 alpha-methyl-delta 8,24-cholestadienol. In cells exposed to aphidicolin, lanosterol accumulation was rapid (15 min) and reversible after a 3-h exposure when cells were rinsed and fresh medium added. It was concluded that aphidicolin inhibits the conversion of lanosterol to C-27 sterols. Although the exact mechanism of this inhibition has not yet been determined, addition of aphidicolin to 20,000 X g supernatant fractions of mouse liver homogenates inhibited the incorporation of [14C]mevalonolactone into cholesterol in a concentration-dependent manner, suggesting that aphidicolin may act directly on one or more of the enzymatic steps involved in lanosterol demethylation. The ubiquitous occurrence of an aphidicolin binding site on eukaryotic DNA alpha polymerases and the inhibitory action of aphidicolin at a proposed secondary regulatory site in sterol biosynthesis (lanosterol metabolism) suggest that a naturally occurring compound may exist which can regulate both DNA replication and cholesterogenesis.

The relationship between the rate of hepatic sterol synthesis and the incorporation of [3H]water.

The true rate of sterol synthesis in liver cells was determined by measurement of the weight of desmosterol produced over a given time period during incubations in the presence of triparanol. The simultaneous presence of tritiated water (3H2O) during the incubations permitted a direct observation of the weight of tritium incorporated into a given mass of newly synthesized sterol. The incorporation of tritium per atom of sterol carbon (H/C ratio) was lower than some previously reported values and suggests that a sizeable proportion of the reducing equivalents (NADPH) required for sterol synthesis arises via the pentose phosphate pathway. The H/C ratio changed significantly with length of the incubation period. The value of the ratio was also dependent upon whether the acetyl-CoA units utilized for sterol synthesis were derived predominantly from a carbohydrate or a fatty acid source.

Source of cholesterol for the ocular lens, studied with U18666A: a cataract-producing inhibitor of lipid metabolism.

Cholesterol is the major lipid component of the ocular lens. The source of lens cholesterol during the first month of post-natal life of the rat was investigated by use of U18666A, a potent inhibitor of cholesterol biosynthesis which can also produce cataracts. Lenses from rats treated with U18666A at a level known to produce cataracts were of smaller size and accumulated total sterol at about one-half the rate of untreated controls. The lens content of phospholipid also lagged behind that of controls. Desmosterol accounted for 50-75% of the total sterol in lens of all treated rats, this paralleled the percent content of desmosterol in liver and serum of these animals. Lenses taken from 20-day-old treated rats and incubated in vitro synthesized little digitonide-precipitable sterol (DPS) from 3H2O as compared to lenses from age-matched controls. The steady state concentration of U18666A in lens was found to be 1-2 X 10(-6) M; this concentration almost completely blocked sterol synthesis in vitro when added to normal lenses. Although U18666A inhibited lens synthesis in vitro of phospholipids from 1,3-[3H]-glycerol and 32Pi, it did so only at levels much higher than those encountered in vivo. Thus, the changes seen in lens phospholipids appear secondary to the decreases in sterols. Since lenses of treated rats synthesized little if any sterol but accumulated sterol at one-half the rate of control lens, we conclude that during early post-natal development of the rat the ocular lens possesses the potential to satisfy about one-half of its sterol requirements from sources outside of the lens, perhaps from lipoproteins in aqueous humor. This conclusion is consistent with our earlier work which indicated that the rat's lens can furnish 50-100% of its total cholesterol by synthesis de novo during the first two weeks of life and less thereafter. The relationship of the inhibition of sterol synthesis to production of the U18666A-induced cataract is discussed.

Effects of 20,25-diazacholesterol on cholesterol synthesis in cultured chick muscle cells: a radiogas chromatographic and mass spectrometric study of the post-squalene sector.

Radiogas chromatography, used in conjunction with mass spectrometry, has been used to analyze the sterol content of cultured chick muscle cells. Seven sterols, plus lanosterol, were detected. These sterols conformed to a linear biosynthetic pathway linking lanosterol and cholesterol. The reaction sequence is: C-14 demethylation, C-4 demethylation, delta 8 leads to delta 5 double bond rearrangement, delta 24 double bond reduction. When chick cells were treated with increasing concentrations of 20,25-diazacholesterol, components of this pathway and aberrant products accumulated. These accumulations suggest that diazacholesterol affects reductases, double bond isomerases and the C-14 demethylation enzymes of sterol biosynthesis.

Dihydromevinolin, a potent hypocholesterolemic metabolite produced by Aspergillus terreus.

A new, potent hypocholesterolemic agent is produced by cultures of Aspergillus terreus. The isolation of the compound and its characterization as 4a,5-dihydromevinolin containing a trans-fused octahydro-naphthalene system are described. Comparative data for dihydromevinolin and mevinolin in three biological assays are given: in vitro inhibition of HMG-CoA reductase, inhibition of sterol synthesis in cell cultures, and inhibition of cholesterol synthesis in vivo in rats.

Stereochemical importance of fucosterol epoxide in the conversion of sitosterol into cholesterol in the silkworm Bombyx mori.

Studies on the stereochemistry of 24,28-epoxy-24-ethylcholesterol, a key intermediate in sitosterol to cholesterol conversion in insects, were undertaken. In nutritional experiments, the 24R,28S and 24S,28R stereoisomers (isofucosterol epoxides) were unable to support growth and development of silkworm larvae. Incubation of 3 alpha-3H-labeled (24R,28R)- and (24S,28S)-epoxide (fucosterol epoxides) with a cell-free preparation from silkworm guts resulted in effective conversion into desmosterol and cholesterol; (24R,28R)-epoxide was the slightly better substrate. Fucosterol incubation yielded (24R,28R)-epoxide and the 24S,28S isomer in approximately equal amounts, while slightly preferential formation of the 24R,28R isomer was observed in in vivo experiments. These results indicate that both the formation of the epoxide from fucosterol and its conversion to desmosterol proceed with a low degree of stereospecificity.

Utilization of endogenous and exogenous sources of substrate for cholesterol biosynthesis by isolated hepatocytes.

The rates of cholesterol biosynthesis in isolated rat hepatocytes were determined by using a method based on measurement of the rate of formation of desmosterol (cholesta-5,24-dien-3beta-ol), which accumulates during inhibition of cholesterogenesis by the drug triparanol. Incubation of cells from normal or 24h-starved animals in a medium containing albumin, glucose, amino acids and acetate as the only organic constituents led to an accelerating rate of sterol formation during the earlier stages of a 6h incubation period. The contribution of exogenously added acetate (initial concentration 3.34mm) to sterol synthesis in both types of cells reached an early maximum and then continually declined. Exogenously added pyruvate and lactate were more efficient sources of sterol carbon than was acetate. Exogenous glucose even at relatively high concentrations (11.1mm) was incapable of providing more than 6% of the total sterol carbon. Although the proportion of total sterol carbon supplied from exogenous acetate increased with increasing concentrations of the extracellular substrate, the rates of total sterol synthesis in both types of cell remained unchanged. Similar observations were made when lactate or pyruvate was the cholesterogenic precursor in normal cells. These studies suggest that, although exogenous substrates were capable of expanding an intracellular pool of cholesterol precursor, the normal supply of intermediary metabolites was not rate-limiting for cholesterogenesis.

5alpha-Cholesta-7,24-dien-3beta-ol as a major sterol of the male hamster reproductive tract.

Marked variations in the 3beta-hydroxysterol content of hamster spermatozoa were observed as they progress through the epididymis. Cholesterol is the major sterol of caputal spermatozoa while the concentration of precursors of cholesterol was higher than that of cholesterol in caudal spermatozoa. One of these precursors has been identified as desmosterol. A second sterol has now been identified as 5alpha-cholestra-7,24-dien-3beta-ol by GLC-MS and by NMR. Its concentration is approximately 3-fold higher than that of cholesterol. This 3beta-hydroxysterol is also found in epididymal tissue.

Specific inhibition of desmosterol synthesis by ML--236B in mouse LM cells grown in suspension in a lipid-free medium.

The suspended growth of LM cells in a lipid-free chemically defined medium was almost completely inhibited in the presence of 0.1 microgram/ml of ML-236B, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis in mammalian cells. This inhibition was effectively counteracted by adding a small amount of either mevalonate or cholesterol (dispersed in delipidated calf serum) to culture medium. The synthesis of desmosterol, the end product of sterol biosynthesis in LM cells, from [14C]acetate in cultured cells was highly sensitive to ML-236B, being inhibited 35 and 60% at its concentrations of 0.1 and 1 ng/ml, respectively, while the incorporation of [3H]mevalonate into desmosterol was not affected by ML-236B at concentrations up to 0.1 microgram/ml. Synthesis of fatty acids, phospholipids, triglycerides and macromolecules like DNA, RNA and protein were not suppressed by 10 microgram/ml of ML-236B. Desmosterol content of LM cells was reduced by treatment with ML-236B. These results indicate that ML-236B inhibited cell growth via specific interference in the pathway of sterol biosynthesis, presumably on the step catalyzed by HMG-CoA reductase.

Measurement of the absolute rates of cholesterol biosynthesis in isolated rat liver cells.

Triparanol [2-(4-chlorophenyl)-1-(4-diethylaminoethoxyphenyl)-1-p-tolylethanol] at a concentration of 2 micronm has no effect on the overall conversion of [2=14C]acetate into C27 sterols by isolated liver cells. In the presence of triparanol, however, the formation of radioactive cholesterol is inhibited by 85-90% and the balance of radioactivity appears in the C27 sterol desmosterol (cholesta-5,24-dien-3beta-ol). The very small weights of desmosterol which accumulate under these conditions were, as a routine, quantitatively converted into the heptafluorobutyrate 3-enol ester of cholesta-4,24-dien-3-one. This derivative has a high electron-capturing capability, a property that enables extremely small quantities (less than 0.25pmol) of the material to be accurately measured by gas chromatography with electron-capture detection. Measurements of the mass and specific radioactivity of the newly biosynthesized desmosterol formed in the presence of triparanol provides an accurate assessment of the amount of cholesterol that would be synthesized by the liver cells in the absence of the drug.

Desmosterol in human and experimental brain tumors in tissue culture.

Desmosterol, a possible chemical indicator of brain tumors, was detected in cells of neurogenic, nitrosourea-induced rat tumors (neurinomas and gliomas, C6 cell line) and in human astrocytomas grown in lipid-poor media. A further increase in the amount of cell desmosterol was obtained when triparanol was added to media containing delipidized serum. Cholesterol was replaced almost completely by desmosterol in tumor cells grown in media containing nontoxic levels of 20,25-diazacholesterol. Desmosterol did not accumulate when these inhibitors of desmosterol-reductase were added to culture media containing cholesterol and other lipids (whole fetal calf serum). The results demonstrate that tumors of the nervous system grown in tissue culture are capable of sterol synthesis, and indicate that a central mechanism of cholesterol synthesis is operative in these cells, which may be related to the availability of exogenous cholesterol. It is concluded that these findings are relevant to clinical studies on the use of cholesterol inhibitors as tools for the detection of brain tumor activity.

Sterol synthesis by myelinating cultures of mouse spinal cord.

We studied the control of sterol synthesis during myelination of central nervous system in culture. Explants of fetal mouse spinal cord were cultured for 3-30 days in vitro (DIV). Myelination was visible by electron micriscopy at 3 DIV, and by bright-field light microscopy beginning at 6 DIV. All explants were heavily myelinated by 15 DIV. Specific activity of 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNP) increased rapidly until 15 DIV. The rate of incorporation of [1-14C]acetate into sterol was greatest at 12 DIV, more than double that at 4 and 22 DIV. Specific activity of the mevalonate synthesizing enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) was greatest at 11 DIV, threefold greater than at 6 and 30 DIV. Previous studies in fibroblasts and other non-neural tissues had shown that deprivation of exogenous lipid caused a rapid increase in HMG CoA reductase activity. In contrast, when the spinal cord explants were incubated for 24 h in a lipid-deficient medium, there was either a marked decrease in specific activity of HMG CoA reductase (at 6, 15 and 30 DIV), or no change in enzyme activity (at 11 DIV).

Sterol biosynthesis by the sea urchin Echinus esculentus.

1. The 4-demethyl sterols of Echinus esculentus consisted of cholesterol as the major component, with lower concentrations of nine other C(26), C(27), C(28) and C(29) Delta(5) sterols. 2. [2-(14)C]Mevalonic acid was readily incorporated by the urchin into squalene, lanosterol and desmosterol but only to a small extent into cholesterol. 3. [26-(14)C]Desmosterol did not appear to be reduced to give cholesterol, but conversion of 5alpha-[2-(3)H(2)]lanost-8-en-3beta-ol into cholesterol was observed. 4. No C-24 dealkylation of [4-(14)C]sitosterol or metabolism of [4-(14)C]cholesterol could be detected.