Name: lipid supplementation for longevity and gene transcriptional analysis in caenorhabditis elegans
Uploaded: 2022-12-09T13:05:00
Description: Watch this Scientific Journal Video about Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans at JoVE.com

We thank P. Svay for maintenance support. This work was supported by NIH grants R01AG045183 (MCW), R01AT009050 (MCW), R01AG062257 (MCW), DP1DK113644 (MCW), March of Dimes Foundation (MCW), Welch Foundation (MCW), HHMI investigator (M.C.W.), and NIH T32 ES027801 pre-doctoral student fellow (M.S.). Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).

Figure 1 depicts a schematic of lipid feeding using different experimental settings.
1. Preparation of lipid-conditioned bacteria
<ol>
	<li>Prepare the Bacterial dilution dietary restriction (BDR) base solution by dissolving 5.85 g of NaCl, 1.0 g of K2HPO4, and 6.0 g of KH2PO4 (see Table of Materials) in 999 mL of deionized water. Adjust the pH to 6.0 with 0.5 M KOH, and then filter th...

<ol>
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Lipid supplementation has been employed in aging research to elucidate the direct impact of certain lipid species on healthy aging6,7,23,26,27,31. However, the lipid supplementation procedure can be challenging, and any inconsistency between experiments can cause non-reproducible results. Here, the first detailed step-by-step...

Lipids 
Lipids are small hydrophobic or amphipathic molecules soluble in organic solvents but insoluble in water1,2. Distinct lipid molecules differentiate from each other based on the number of carbons contained in their chains, location, number of double bonds, and bound structures, including glycerol or phosphates. Lipids play crucial roles within and across distinct cells to regulate organismal functions, including constituting membrane bilayers, providing energy storage, and acting as signaling molecules3,4.
First, lipids are structural components of biological membranes, including the plasma membrane and intracellular subcellular membranes that divide the internal compartments from the extracellular environment. Second, lipids are the major form of energy storage in vertebrate and invertebrate animals. Neutral lipids, including triacylglycerols, are stored for an extended period in various tissues, including in adipose tissue. In the nematode Caenorhabditis elegans, the intestine is the major metabolic fat storage organ; its function is not only involved in digestion and absorption of nutrients, but also in the process of detoxification, which resembles the activity of mammalian hepatocytes. Other fat storage tissues include the germline, in which lipids are essential for developing oocytes, and the hypodermis, which is composed of skin-like epidermal cells3,5. Third, in recent years, more evidence has suggested that lipids are powerful signaling molecules involved in intra- and extracellular signaling by directly acting on a variety of receptors, including G protein-coupled and nuclear receptors, or indirectly via membrane fluidity modulation or post-translational modifications6,7,8,9. Further studies will continue to elucidate the underlying molecular mechanisms of lipid signaling in promoting longevity and healthspan.
Model organisms are important to address specific biological questions that are too complex to study in humans. For example, the roundworm C. elegans is an excellent model for conducting genetic analysis to dissect biological processes relevant to human nutrition and disease10. The highly conserved molecular pathways relevant to human physiology, complex tissues, behavioral patterns, and abundant genetic manipulation tools make C. elegans a remarkable model organism11. For instance, C. elegans is excellent in forwarding genetic screens to identify phenotype-specific genes, as well as in genome-wide reverse genetic screens via RNA interference12.
In laboratories, the nematodes are grown on agar Petri plates seeded with a lawn of Escherichia coli bacteria, providing macronutrients such as proteins, carbohydrates, and saturated and unsaturated fatty acids as sources of energy and building blocks, and micronutrients such as co-factors and vitamins13. Similar to mammals, nematodes synthesize fatty acid molecules from both palmitic acid and stearic acid (saturated 16-carbon and 18-carbon molecules, respectively) that are sequentially desaturated and elongated to a variety of mono-unsaturated fatty acids (MUFAs) and poly-unsaturated fatty acids (PUFAs)14,15,16,17,18. Interestingly, C. elegans is capable of de novo synthesis of all the required fatty acids and core enzymes involved in fatty acid biosynthesis, desaturation, and elongation, facilitating the synthesis of long-chain PUFAs19. Different from other animal species, C. elegans can convert 18-carbon and 20-carbon &#969;-6 fatty acids into &#969;-3 fatty acids with its own &#969;-3 desaturase enzymes. Additionally, worms possess a &#916;12 desaturase that catalyzes the formation of linoleic acid (LA) from oleic acid (OA, 18:1)20,21. Most animals or plants lack both &#916;12 and &#969;-3 desaturases and thus rely on dietary intake of &#969;-6 and &#969;-3 to obtain their PUFAs, whereas C. elegans does not require dietary fatty acids22. Isolated mutants lacking functional desaturase enzymes have been used to study the functions of specific fatty acids in distinct biological processes, including reproduction, growth, longevity, and neurotransmission. The effect of individual fatty acids on specific biological pathways can be addressed using both a genetic approach and diet supplementation16,17,23. To date, lipid research has focused on characterizing genes involved in the lipid synthesis, degradation, storage, and breakdown in neurological and developmental conditions24. However, the roles of lipids in longevity regulation are just beginning to be revealed.
Lipid signaling in longevity regulation 
Lipids play crucial roles in longevity regulation by activating cellular signaling cascades in distinct tissues and cell types. Recent studies have highlighted the active roles of lipids in modulating transcription and cell-cell communication via lipid-binding proteins or recognition of membrane receptors25. Additionally, dietary lipid supplementation offers an excellent tool to dissect how lipid metabolism influences lifespan in C. elegans. Distinct MUFAs and PUFAs have been shown to promote longevity by activating transcription factors26,27.
Longevity models, including the insulin/IGF-1 signaling and the ablation of germline precursor cells, are associated with the MUFA biosynthesis pathway, and MUFA supplementation, including oleic acid, palmitoleic acid, and cis-vaccenic, is sufficient to extend C. elegans lifespan26. Although the longevity effect conferred by the MUFA administration requires further investigation, the underlying mechanism is likely to be mediated by the SKN-1/Nrf2 transcription factor, which is a key activator of oxidative stress response and longevity regulation28,29. Among MUFAs, a particular class of fatty acyl ethanolamides called N-acylethanolamines (NAEs) plays crucial roles in distinct mechanisms including inflammation, allergies, learning, memory, and energy metabolism30. Particularly, the lipid molecule known as oleoylethanolamide (OEA) has been identified as a positive regulator of longevity by promoting the translocation of the lipid-binding protein 8 (LBP-8) into the nucleus to activate the nuclear hormone receptors NHR-49 and NHR-807. Supplementation of the OEA analog KDS-5104 is sufficient to extend lifespan, and induces the expression of genes involved in oxidative stress responses and mitochondrial &#946;-oxidation7,8.
At the same time, the role of PUFAs has also been linked to longevity regulation. Administration of PUFA &#969;-3 fatty acid &#945;-linolenic acid (ALA) promotes longevity by activating the NHR-49/PPAR&#945;, SKN-1/NRF transcription factors and inducing mitochondrial &#946;-oxidation31. Interestingly, peroxidated products of ALA, referred to as oxylipins, activate SKN-1/NRF, suggesting that both PUFAs and their oxidative derivatives can confer longevity benefits23. Supplementation of &#969;-6 fatty acid arachidonic acid (AA) and dihomo-&#947;-linolenic acid (DGLA) extends lifespan via autophagy activation, promoting protein quality control and resulting in the degradation of wasted and toxic protein aggregates27,32. More recently, a cell-non-autonomous signaling regulation mediated by the lipid-binding protein 3 (LBP-3) and DGLA has been shown to be crucial to promoting longevity by sending peripheral signals to neurons, suggesting a long-range role of lipid molecules in inter-tissue communication at systemic levels33. The present study reports each step to perform lipid supplementation with bacteria seeded on plates or bacterial suspension in liquid culture. These methodologies are used to assess lifespan and transcriptional analysis, employing whole body content or dissected tissues derived from a few worms. The following techniques can be adapted to a variety of nutritional studies and offer a valid tool to dissect how lipid metabolism influences longevity and healthy aging.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Pestle</td>
			<td>Genesee Scientific</td>
			<td>93-165P15</td>
			<td>For worm grinding with Trizol</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9639-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>AmfiRivert cDNA Synthesis Platinum Master Mix</td>
			<td>GenDEPOT</td>
			<td>R5600</td>
			<td>For reverse transcription from bulk worm samples</td>
		</tr>
		<tr>
			<td>Applied Biosystems QuanStudio 3 Real-Time PCR</td>
			<td>ThermoFisher</td>
			<td>A28567</td>
			<td>For qRT-PCR</td>
		</tr>
		<tr>
			<td>Benchmark Scientific StripSpin 12 Microcentrifuge</td>
			<td>Benchmark Scientific</td>
			<td>C1248</td>
			<td>For spin down PCR tubes</td>
		</tr>
		<tr>
			<td>Branson 450 Digital Sonifier, w/ 1/8&#34; tip</td>
			<td>Branson Ultrasonic Corporation</td>
			<td>100-132-888R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C298-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C8503-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D8418-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5424 R centrifuge</td>
			<td>Eppendorf</td>
			<td>22620444R</td>
			<td>For RNA extraction</td>
		</tr>
		<tr>
			<td>Eppendorf vapo protect mastercycler pro</td>
			<td>Eppendorf</td>
			<td>950030010</td>
			<td>For reverse transcription</td>
		</tr>
		<tr>
			<td>Ethanol, Absolute (200 Proof)</td>
			<td>Fisher Scientific</td>
			<td>BP2818-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Greiner Bio-One CELLSTAR, 12 W Plate</td>
			<td>Neta Scientific</td>
			<td>665180</td>
			<td>12-well plates for licuid feeding</td>
		</tr>
		<tr>
			<td>Greiner Bio-One Petri Dish, Ps, 100 x 20 mm</td>
			<td>Neta Scientific</td>
			<td>664161</td>
			<td>For bacterial LB plates and worm 10-cm NGM plates</td>
		</tr>
		<tr>
			<td>Greiner Bio-One Petri Dish, Ps, 60 x 15 mm</td>
			<td>Neta Scientific</td>
			<td>628161</td>
			<td>For worm6-cm NGM plates</td>
		</tr>
		<tr>
			<td>Invitrogen nuclease-free water</td>
			<td>ThermoFisher</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoproanol</td>
			<td>Sigma</td>
			<td>PX1835-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Levamisole hydrochloride</td>
			<td>VWR</td>
			<td>SPCML1054</td>
			<td></td>
		</tr>
		<tr>
			<td>lipl-4Tg</td>
			<td>MCW Lab</td>
			<td>N/A</td>
			<td>Transgenic C. elegans</td>
		</tr>
		<tr>
			<td>lipl-4Tg;fat-3(wa22)</td>
			<td>MCW Lab</td>
			<td>N/A</td>
			<td>Transgenic C. elegans</td>
		</tr>
		<tr>
			<td>Luria Broth Base</td>
			<td>ThermoFisher</td>
			<td>12795-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Sigma</td>
			<td>M2643-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp EnduraPlate Optical 96-Well Fast Clear Reaction Plate with Barcode</td>
			<td>ThermoFisher</td>
			<td>4483354</td>
			<td>96-well qPCR plate</td>
		</tr>
		<tr>
			<td>MicroAmp Optical Adhesive Film</td>
			<td>Applied BioSystem</td>
			<td>4311971</td>
			<td>For sealing the 96-well qPCR plate</td>
		</tr>
		<tr>
			<td>Milli-Q Advantage A10 Water Purification System</td>
			<td>Sigma</td>
			<td>Z00Q0V0WW</td>
			<td>Deionized water used to make all reagents, including buffer and cultural media, unless specified as nuclease-free water in the protocol</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>N/A</td>
			<td>C. elegans wild isolate</td>
		</tr>
		<tr>
			<td>NanoDrop ND-1000 Spectrophotometer</td>
			<td>ThermoFisher</td>
			<td>N/A</td>
			<td>For measuring RNA concentration</td>
		</tr>
		<tr>
			<td>OP50</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>N/A</td>
			<td>Bacteria used as C. elegans food</td>
		</tr>
		<tr>
			<td>Potasium phosphate dibasic trihydrate (K2HPO4&#183;3H2O)</td>
			<td>Sigma</td>
			<td>P5504-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Potasium phosphate monobasic (KH2PO4)</td>
			<td>Sigma</td>
			<td>P0662-2.5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Power SYBR Green cells-to-Ct kit</td>
			<td>ThermoFisher</td>
			<td>4402953</td>
			<td>For reverse transcription and qPCR from a few worms or worm tissue</td>
		</tr>
		<tr>
			<td>Power SYBR Green Master Mix</td>
			<td>ThermoFisher</td>
			<td>4367659</td>
			<td>For qPCR from bulk worm samples</td>
		</tr>
		<tr>
			<td>Pure Bright germicidal ultra bleach&#160;</td>
			<td>KIK International LLC.</td>
			<td>59647210143</td>
			<td>6% house bleach For worm egg preparation</td>
		</tr>
		<tr>
			<td>Pyrex spot plate with nine depressions</td>
			<td>Sigma</td>
			<td>CLS722085-18EA</td>
			<td>Watch glass for dissecting the worms</td>
		</tr>
		<tr>
			<td>RNaseZap RNase Decontamination Solution</td>
			<td>ThermoFisher</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium cloride (NaCl)</td>
			<td>Sigma</td>
			<td>S7653-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma</td>
			<td>SX0590-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic heptahydrate (Na2HPO4&#183;7H2O)</td>
			<td>Sigma</td>
			<td>S9390-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Sorvall Legend Mach 1.6R Centrifuge</td>
			<td>Thermo</td>
			<td>7500-4337</td>
			<td>For bacteria collection</td>
		</tr>
		<tr>
			<td>Thermo Sorvall ST 8 centrifuge</td>
			<td>Thermo</td>
			<td>7500-7200</td>
			<td>For worm egg preparation</td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>TheroFisher</td>
			<td>15596018</td>
			<td>RNA extraction reagent</td>
		</tr>
		<tr>
			<td>Turbo DNA-free kit</td>
			<td>ThermoFisher</td>
			<td>AM1907</td>
			<td>For removing DNA contamination in RNA extractions</td>
		</tr>
		<tr>
			<td>Vortexer 59</td>
			<td>Denville Scientific INV</td>
			<td>S7030</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Disposable Pellet Mixers and Cordless Motor</td>
			<td>VWR</td>
			<td>47747-370</td>
			<td>For worm grinding with Trizol</td>
		</tr>
		<tr>
			<td>VWR Kinetic Energy 26 Joules Mini Centrifuge C1413 V-115</td>
			<td>VWR</td>
			<td>N/A</td>
			<td>For worm collection. Discontinued model, a similar one available at VWR with Cat# 76269-064</td>
		</tr>
		<tr>
			<td>Worm picker</td>
			<td>WormStuff</td>
			<td>59-AWP</td>
			<td></td>
		</tr>
	</tbody>
</table>

lipid supplementation for longevity and gene transcriptional analysis in caenorhabditis elegans

Aging is a complex process characterized by progressive physiological changes resulting from both environmental and genetic contributions. Lipids are crucial in constituting structural components of cell membranes, storing energy, and as signaling molecules. Regulation of lipid metabolism and signaling is essential to activate distinct longevity pathways. The roundworm Caenorhabditis elegans is an excellent and powerful organism to dissect the contribution of lipid metabolism and signaling in longevity regulation. Multiple research studies have described how diet supplementation of specific lipid molecules can extend C. elegans lifespan; however, minor differences in the supplementation conditions can cause reproducibility issues among scientists in different labs. Here, two detailed supplementation methods for C. elegans are reported employing lipid supplementation either with bacteria seeded on plates or bacterial suspension in liquid culture. Also provided herein are the details to perform lifespan assays with lifelong lipid supplementation and qRT-PCR analysis using a whole worm lysate or dissected tissues derived from a few worms. Using a combination of longitudinal studies and transcriptional investigations upon lipid supplementation, the feeding assays provide dependable approaches to dissect how lipids influence longevity and healthy aging. This methodology can also be adapted for various nutritional screening approaches to assess changes in a subset of transcripts using either a small number of dissected tissues or a few animals.

Validation of transcriptional changes using a few whole worms upon lipid supplementation 
To investigate whether the protocol to extract and retrotranscribe RNA into cDNA from a few whole worms is reproducible and comparable with the data from bulk worms, a long-lived worm strain overexpressing the lysosomal acid lipase lipl-4 in the intestine was employed7,8,33,35</sup...

Watch this Scientific Journal Video about Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans at JoVE.com

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

The present protocol describes lipid supplementation methods in liquid and on-plate cultures for Caenorhabditis elegans, coupled with longitudinal studies and gene transcriptional analysis from bulk or a few worms and worm tissues.

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

Aging is a complex process characterized by progressive physiological changes resulting from both environmental and genetic contributions. Lipids are ...

This protocol describes two lipid supplementation strategies with a combination of longitudinal and transcriptional analysis in the model organism, C.elegans. The technique describes how to examine transcriptional changes using few worms or dissected worm tissues and how the liquid feeding for a bug population could be coupled with hydro techniques. Individuals performing this technique must practice tissue dissection because of the short time window to perform the experiment to achieve adequate transcriptional analysis.To begin, collect OP50 bacteria by centrifuging the overnight-grown culture at 4, 000 G for 10 minutes at room temperature. Discard the supernatant. Suspend the bacterial pellet in 20 milliliters of bacterial dilution, dietary restriction or BDR base by vortexing and again, repeat the centrifugation for 10 minutes.After discarding the supernatant, re-suspend the bacterial pellet in the BDR medium to prepare a 20 X BDR bacterial stock. Dilute the bacterial stock to 5X using a BDR medium before use. Using ethanol or dimethyl sulphoxide as the solvent, prepare a lipid stock solution in a new autoclaved glass vial and fill the glass vial with argon or nitrogen to prevent oxidation.Transfer the lipid stock solution to the BDR bacteria solution to achieve the desired final concentration in feeding conditions. Mix it thoroughly by vortexing for 20 seconds. Prepare the vehicle controlled by mixing the same volumes of filtered ethanol or dimethyl sulphoxide with BDR bacteria.Perform lipid supplementation and liquid culture by transferring the desired amount of lipid or vehicle control to each well of a 12 well plate with three to four replicates for each feeding condition. Mix the synchronized Caenorrhabditis elegans worm suspension with 5X BDR bacteria in a one-to-one ratio to achieve a final concentration of 1, 500 worms per milliliter and 2.5X for bacteria. Then transfer two milliliters of the worm bacteria mixture to each well of the 12 well plate.When done wrap the 12 well plate with foil and shake the plate in a 20 degree Celsius incubator at 100 RPMM for the desired incubation length. For lipid supplementation on plates, seed one milliliter of the lipid conditioned bacteria onto the center of a 10 centimeter nematode growth medium or NGM agar plate. Ensure that the final working solution is vortexed multiple times between seeding the two plates when working with a high number of plates.Dry the plate in the dark inside a biosafety hood. Transfer 3000 worms from the synchronized worm culture to the 10 centimeter plate. Dry the plate in a biosafety hood until worms that were swimming on the lipid supplemented plates, start crawling.Once dried, incubate the lipid conditioned plate in a 20 degree Celsius incubator for the desired length of time and if using polyunsaturated lipids protect the plate from light. Prepare 20 microliters of final lysis solution for each sample by mixing 0.2 microliters of DNase I with 19.8 microliters of lysis solution in a PCR tube and mix it well by pipetting up and down five times. To extract the RNA from the small number of whole worms, remove the bacteria by transferring 15 to 20 worms from the bacterial lawn to a freshly unseeded NGM plate.Transfer 15 to 20 worms from the unseeded NGM plate to the PCR tube containing 20 microliters of the freshly prepared final lysis solution with the minimum number of bacteria and incubate the lysis reaction for five minutes at room temperature. Once the incubation is over, keep the sample in a nice cold water bath and probe sonicate the worms four times, five seconds each time with 30%amplitude and pulse for 15 seconds between each time of sonication. Incubate the tube at room temperature for five minutes before adding two microliters of stop solution into the lysis reaction and mix it by gentle tapping.Incubate the reaction for two minutes at room temperature and then set the tube on ice. To extract RNA from the worm tissue, pick around 20 worms from the bacterial lawn and place them into a fresh unseeded NGM plate to remove the bacteria from the worms. Transfer 20 worms with as few bacteria as possible to a watch glass containing 500 microliters of M9 solution containing four micromolar levamisole.When the worms are immobilized, dissect the germline or intestine using a 25 gauge needle that can be attached to the one milliliter syringe. Using an autoclaved glass pipette, transfer the dissected tissues into the PCR tube and settle the tube on ice for two minutes allowing the deposition of the material at the bottom. Remove the supernatant from the PCR tube before adding 20 microliters of final lysis solution and mix it well by tapping.After incubating the lysis reaction for five minutes, add two microliters of stop solution and tap the tube multiple times. Incubate the tube for two minutes before proceeding to the reverse transcription. In the validation study, RNA extracted from a large population and a few worms showed similar induction of the neuropeptide processing genes, egl-3 and egl-21.It suggests that the RNA extraction from a few worms can be a valid alternative to standard CDNA synthesis techniques from large populations. In the dissected intestine of the lipl-4 transgene worms, supplemented with dihomo-gamma-linolenic acid, or DGLA, the neuronal neuropeptide processing genes were not induced. DGLA supplementation at different concentrations rescued lifespan extension in the worms upon fat-3 knockdown.Worms with lipid supplementation may also be processed for RNA sequencing, proteomics, metabolomics and behavioral studies to identify additional phenotypes under these specific conditions. This methodology can be applied to aging research, lipid biology and any other research area that aims to establish a relationship between a certain phenotype and a nutritional factor or metabolite.

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Biology

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...