Caenorhabditis elegans requires sterol, usually supplied as cholesterol, but this is enzymatically modified, and different sterols can substitute. Sterol deprivation decreased brood size and adult growth in the first generation, and completely, reversibly, arrested growth as larvae in the second. After one generation of sterol deprivation, 10 ng/ml cholesterol allowed delayed laying of a few eggs, but full growth required 300 ng/ml. C. elegans synthesizes two unusual 4 ␣ -methyl sterols (4MSs), but each 4MS supported only limited growth as the sole sterol. However, addition of only 10 ng of cholesterol to 1,000 ng of 4MS restored full growth and egg-laying, suggesting that both a 4MS and an unmethylated sterol are required for development. Filipin stained sterols in only a few specific cells: the excretory gland cell, two amphid socket cells, two phasmid socket cells and, in males, spicule socket cells. Sterols were also present in the pharynx and in the intestine of feeding animals in a proximal-to-distal gradient.This non-random sterol distribution, the low concentration requirements, and the effects of 4MSs argues that sterols are unlikely to be used for bulk structural modification of cell membranes, but may be required as hormone precursors and/or developmental effectors. Dietary sterol is required by Caenorhabditis elegans (1, 2) because, like insects, C. elegans is incapable of synthesizing the four-ring sterol nucleus, but its functions remain largely unknown. The existence of sterol-based hormones in C. elegans has recently been suggested, but no hormone has yet been identified (3, 4). Cholesterol is known to be extensively metabolized by C. elegans to form several other sterols, including two unusual 4 ␣ -methyl sterols (4MSs), which are present in substantial amounts (5-8, 9). These sterols might thus be functional, instead of or in addition to cholesterol itself.Insects resemble these nematodes in requiring sterols but being unable to synthesize them. Two functions for cholesterol are known in insects: as the metabolic precursor of the molting hormone ecdysone (10), and as the moiety required for activation by covalent attachment to the morphogen protein hedgehog (11). Insect cells, unlike vertebrate cells, grow normally under sterol-free conditions, and thus do not need cholesterol in their plasma membranes (12)(13)(14).We have now characterized the sterol requirements of C. elegans in some detail. Conditions for stringent sterol deprivation were developed, and the consequences are described. The use of these conditions allowed us to investigate minimum cholesterol requirements, and the ability of other sterols to substitute. Partial and synergistic effects were found, suggesting that different sterols have diverse effects mediated by several pathways. The accumulation of sterol in the intestinal tract and in a few specific cells in C. elegans was also demonstrated by filipin staining, which stains all 3 ␤ -hydroxy sterols.These observations provide a basis for a comprehensive study of ster...

Three major long-term effects of sterol deprivation in Caenorhabditis elegans are described. 1 ) The life expectancy of sterol-deprived wild-type animals is decreased by more than 40%. Similar decreases are found in animals carrying mutations in the daf-9 , daf-12 , daf-16 , and clk-1 genes, suggesting that previously described aging pathways involving these genes are not involved in the life-extending effects of sterols. 2 ) There is a premature loss of motility, measured by response to mild touch. 3 ) There is a rapid postreproductive onset of sarcopenia (muscle wasting) as measured by total body fluorescence in a myo3::GFP -expressing strain. We also report that five sterols (the desmethylsterols cholesterol, 7-dehydrocholesterol, and lathosterol and the 4 ␣ -methyl sterols lophenol and 4 ␣ -methyl-cholesta-⌬ 8(14)-en-3 ␤ -ol) are found in significant amounts at all stages of development and aging in cholesterol-fed animals. Supplying any one of these as the sole sterol confers similar protection from the long-term effects of sterol deprivation. These findings suggest that sterols are required continuously throughout the animal's life. Supplementary key words Caenorhabditis elegansCaenorhabditis elegans cannot synthesize sterols and so requires dietary sterol, which is usually supplied in the laboratory as cholesterol (1, 2). Studies of the ability of different sterols to substitute for cholesterol have revealed the presence of at least two independent essential sterol-requiring pathways. One of these pathways can use a highly unusual class of sterols, the C4 ␣ -methyl sterols (4MS), and accounts for at least 98% of the total sterol requirement. The other pathway cannot use 4MS, but requires a C4 desmethyl sterol (desMS) (2). 4MSs were present as major components of the total sterol content in axenically grown unsynchronized (mixed age) populations of C. elegans when any of several dietary desMSs were supplied (3). C. elegans is unique among presently characterized organisms in possessing the ability to convert dietary desMSs to 4MSs, while simultaneously lacking the ability to demethylate sterols at C4 (3).The functions of sterols in C. elegans have not been characterized. Sterol deprivation results in decreased fertility in the first generation and developmental arrest with early death in the second. Development to adulthood and timing of reproduction both occur normally in the first generation of sterol deprivation, apparently supported by the utilization of stored sterol (2, 4, 5).Sterols do not appear to be required for bulk modification of plasma membrane bilayer properties, as indicated by several observations. First, only minute amounts of sterol were required: limited fertility was supported by 10 ng/ml of cholesterol, and normal growth and reproduction by ‫ف‬ 100 ng/ml, compared with 5-8 g/ml present in standard medium (2). Second, sterol-specific staining by filipin showed sterol accumulation largely in five specific cells plus the intestine, rather than uniform distribution in plasma mem...

Transcriptional profiles of Caenorhabditis elegans grown on unmethylated sterols (desMSs) or on 4a-methylsterols (4MSs) were compared using microarrays. Thirty-four genes were upregulated and 2 were downregulated .2-fold by growth on 4MSs, including 13 cuticle collagen (col ) genes, 1 cuticulin gene (cut-1), 2 groundhog-like (grl ) genes, and 1 groundhog gene (grd-4 ); col-36 and grl-20 were increased 12-and 19-fold, respectively. Fifteen of these 17 genes have been assigned to metabolic mountain 17, suggesting coordinate 4MS-mediated regulation of expression. Quantitative RT-PCR was performed on 27-51 h old animals grown on cholesterol (a desMS) or lophenol (a 4MS). col-36 and grl-20 showed similar cyclic peaks of expression in cholesterol and similar alterations in lophenol, suggesting coregulation. Of six additional grl genes, only grl-3 was upregulated on lophenol; the rest were downregulated. Cyclicity of expression was lost or altered in all six. Nuclear receptor genes nhr-23, nhr-25, nhr-41, and daf-12 all showed cyclic expression in cholesterol and significant downregulation in lophenol by RT-PCR. Expression of the insulin-like receptor daf-2 was lower in lophenol, whereas that of its major downstream target daf-16 was higher. Thus, major changes in gene expression accompany growth on 4MSs, but with surprisingly little effect on normal growth and development.-Merris, M., T. Wang, P. Soteropoulos, and J. Lenard. Differential gene expression of Caenorhabditis elegans grown on unmethylated sterols or 4a-methylsterols. J. Lipid Res. 2007Res. . 48: 1159Res. -1166 Supplementary key words transcription & reverse transcription poly-Dietary sterol is required by Caenorhabditis elegans because this animal is incapable of synthesizing the four ring sterol nucleus (1, 2). Sterol deprivation results in the pleiotropic arrest of growth and development at whatever stage the animal is occupying when the (often considerable) store of maternally supplied sterol is exhausted (1-3). A difficulty in cuticle production and shedding has frequently been reported in sterol-deprived animals (4-6), but it is not clear whether this is a direct effect of sterol depletion or a secondary consequence of arrested growth.The question of how sterols are used by C. elegans has been the focus of extensive research. Sterols are apparently not essential components of plasma membranes, the way cholesterol is in vertebrate cells, as shown by the following findings. 1) Minute amounts of sterols, insufficient to modify plasma membrane properties globally, are required (3). 2) Sterols accumulate in a few specific cells rather than being distributed uniformly in cellular plasma membranes (3, 7).3) The enantiomer of cholesterol (possessing the opposite stereochemistry at all asymmetric carbons) does not substitute for cholesterol in supporting growth, although sterol-lipid interactions in bilayers are generally not enantiospecific (8). 4) The sterol requirement can be met by 4a-methylsterols (4MSs) containing 1% or less unmethylated ster...