Name: Isolation and In vitro Activation of Caenorhabditis elegans Sperm (Video) | JoVE Journal
Uploaded: 2011-01-31T19:00:00.000+00:00
Duration: 5 min 46 s
Description: Learn to isolate and activate Caenorhabditis elegans sperm in vitro using protease treatment. This protocol enables visualization of sperm morphology for studying reproductive biology and fertility.

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5 minutes

We thank Samuel Ward, Diane C. Shakes and Gregory Nelson for pioneering this technique. We thank Pavan Kadandale and Brian Geldziler for videos showing activated spermatozoa; members of the Singson lab for helpful discussions; CGC for strains and NIH for supporting us through grant (R01HD054681).

1) Enrichment of male population
<ol>
	<li>Depending on the experimental need, large numbers of males can be obtained by employing one of the following strategies:
		<ol>
			<li>large population of wild type males can be obtained by crossing 5 wild type males and 1 hermaphrodite on a small lawn of OP50 seeded at the center of NGM plate. Roughly 50% of the succeeding generation will be wild type males.</li>
			<li>him-5(e1490) or him-8(e1489) hermaphrodites throw large number of males. him-5 and him-8 males are fertile and there is no obvious defect in sperm morphology and function. So, him-5 or him-8 males can be used in lieu of wild type males for many experiments.</li>
 </ol>
 </li>
</ol>
2) Identification and isolation of males
<ol>
	<li>Examine the tail morphology of worms; the male worm's tail is rounded.</li>
	<li>Pick L4 stage males and transfer them to a NGM plate seeded with E. coli OP50 and let them grow for a day or two. Growing celibate males in the absence of hermaphrodites prevent the loss of sperm and thus large number of spermatids would be available during experimental procedure.</li>
	<li>On the day of dissection, transfer the celibate males on to a NGM plate without bacteria and let them crawl for few minutes. This step helps to remove small layer of E. coli adhered on the surface of worms. Alternatively, pipette small volume of M9 buffer (6g Na2HPO4, 3g KH2PO4, 5g NaCl, 0.25g MgSO4.7H2O per liter) in a watch glass and transfer males into the buffer.</li>
</ol> 
3) Dissection of males and in vitro activation
<ol>
	<li>Take a glass slide and mark a small circle using a pap pen. Pipette 30-50 μl of 1X Sperm Medium (50mM HEPES, 25mM KCl, 45mM NaCl, 1mM MgSO4, 5mM CaCl2, 10mM Dextrose; pH 7.8) within the circle. The hydrophobic circle helps to retain the sperm medium within its boundary.</li>
	<li>Transfer the 5-10 males on to the sperm medium.</li>
	<li>Viewing the slide under microscope, hold two syringes attached with 27 gauge needles on both of your hands.</li>
	<li>Use one needle to 'hold' the worm and use the other needle to cut the posterior end of males to release spermatids.</li>
	<li>Cutting the worm usually expels vast number of spermatids instantaneously. The spermatids are visible as minute 'granules' under the stereo microscope. However, sometimes part or most of the spermatids may be retained within the carcass. In that case, gently 'dragging' the carcass over the glass slide may facilitate the release of spermatids from the carcass.</li>
	<li>To activate the spermatids, dissect the worms in 1X Sperm Medium containing Pronase E(20μg/ml) and let them sit for 5 minutes.</li>
	<li>Do not allow the slides to become dry at any time point of the experiment. Add more 1X Sperm Medium if needed.</li>
</ol>
4) Visualization of spermtids and spermatozoa
<ol>
	<li>Gently place a coverslip on the surface of dissected sample.</li>
	<li>Find the field of spermatids under low magnification (10X)</li>
	<li>Fine details of spermatids or spermatozoa can be observed by moving to higher magnification - usually at 100X oil immersion.</li>
</ol>
5) Representative Results
An example of DIC image of spermatids isolated from male C. elegans is shown in Figure 1. Spermatids are spherical in shape and the nuclei are prominent. In vitro activated sperm are shown in Figure 2.
<img src="/files/ftp_upload/2336/2336fig1.jpg" alt="Microscope image of single-cell microorganisms; focus on static arrangement and morphology study." data-altupdatedat="1761735611000" data-alt="Figure 1"> Figure 1. DIC image of C. elegans spermatids.
<img src="/files/ftp_upload/2336/2336fig2.jpg" alt="Yeast cells; microscope image; cell morphology analysis; fermentation research; biology study." data-altupdatedat="1761735611000" data-alt="Figure 2"> Figure 2. DIC image of in vitro activated C. elegans spermatozoa.

<ol><li>Kimble, J., Crittenden, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Controls+of+germline+stem+cells%2C+entry+into+meiosis%2C+and+the+Sperm%2FOocyte+decision+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Annual+Review+of+Cell+and+Developmental+Biology%22%5BJournal%5D)">Controls of germline stem cells, entry into meiosis, and the Sperm/Oocyte decision in Caenorhabditis elegans.</a> Annual Review of Cell and Developmental Biology. 23, 405-433 (2007).</li>
<li>Lhernault, S. W., Roberts, T. M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLhernault%2C+SW+%22Methods+in+Cell+Biology%22">Methods in Cell Biology</a>. , Vol. 48 273-301 (1995).</li>
<li>Hodgkin, J., Horvitz, H., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nondisjunction+Mutants+of+the+Nematode+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Nondisjunction Mutants of the Nematode Caenorhabditis elegans.</a> Genetics. 91, 67-94 (1979).</li>
<li>Altun, Z. F., Hall, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Introduction+to+male+C.+elegans+anatomy%5BTitle%5D)+AND+%22WormAtlas%22%5BJournal%5D)">Introduction to male C. elegans anatomy.</a> WormAtlas. , ColdSpring Harbor Laboratory Press. Cold Spring Harbor, New York. (2008).</li>
<li>Shakes, D., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Initiation+of+spermiogenesis+in+C.+elegans%3A+a+pharmacological+and+genetic+analysis%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis.</a> Dev Biol. 134, 189-200 (1989).</li>
<li>Nelson, G. A., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vesicle+fusion%2C+pseudopod+extension+and+amoeboid+motility+are+induced+in+nematode+spermatids+by+the+ionophore+monensin%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Vesicle fusion, pseudopod extension and amoeboid motility are induced in nematode spermatids by the ionophore monensin.</a> Cell. 19, 457-464 (1980).</li>
<li>L'Hernault, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+genetics+and+cell+biology+of+spermatogenesis+in+the+nematode+C.+elegans%5BTitle%5D)+AND+%22Molecular+and+Cellular+Endocrinology%22%5BJournal%5D)">The genetics and cell biology of spermatogenesis in the nematode C. elegans.</a> Molecular and Cellular Endocrinology. 306, 59-65 (2009).</li>
<li>Chatterjee, I., Richmond, A., Putiri, E., Shakes, D., Singson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Caenorhabditis+elegans+spe-38+gene+encodes+a+novel+four-pass+integral+membrane+protein+required+for+sperm+function+at+fertilization%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The Caenorhabditis elegans spe-38 gene encodes a novel four-pass integral membrane protein required for sperm function at fertilization.</a> Development. 132, 2795-2808 (2005).</li>
<li>Xu, X., Sternberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+C.+elegans+sperm+TRP+protein+required+for+sperm-egg+interactions+during+fertilization%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A C. elegans sperm TRP protein required for sperm-egg interactions during fertilization.</a> Cell. 114, 285-297 (2003).</li>
<li>Nelson, G., Roberts, T., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caenorhabditis+elegans+spermatozoan+locomotion%3A+amoeboid+movement+with+almost+no+actin%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Caenorhabditis elegans spermatozoan locomotion: amoeboid movement with almost no actin.</a> J Cell Biol. 92, 121-131 (1982).</li>
<li>Machaca, K., DeFelice, L. J., L'Hernault, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+chloride+channel+localizes+to+Caenorhabditis+elegans+spermatids+and+chloride+channel+blockers+induce+spermatid+differentiation%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">A novel chloride channel localizes to Caenorhabditis elegans spermatids and chloride channel blockers induce spermatid differentiation.</a> Dev Biol. 176, 1-16 (1996).</li>
<li>LaMunyon, C., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+the+viability+of+mutant+and+manipulated+sperm+by+artificial+insemination+of+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Assessing the viability of mutant and manipulated sperm by artificial insemination of Caenorhabditis elegans.</a> Genetics. 138, 689-692 (1994).</li>
</ol>

No conflicts of interest declared.

In addition to analyzing Spe mutants, this protocol has other important applications, such as analyzing sperm morphology with aging. Spermatids and spermatozoa isolated using this protocol can be used in other downstream experiments such as, immunostaining of wild type and mutant sperm8, 9, motility of sperm on slides10, physiological measurements9, 11, or even artificial insemination12.

<table><tbody><tr><td>Tuberculin syringe</td><td>BD Biosciences</td><td>309623</td><td>Syringe: 1 ml, needle:&lt;br/&gt;27G X &amp;#189; in</td></tr><tr><td>Pap pen</td><td>Zymed Laboratories, Inc.</td><td>00-8888</td><td></td></tr><tr><td>VWR VistaVision HistoBond Microscope Slides</td><td>VWR international</td><td>16004-406</td><td>Dimension:&lt;br/&gt;75X25X1mm</td></tr><tr><td>Cover glass</td><td>VWR international</td><td>48366045</td><td></td></tr><tr><td>Protease</td><td>Sigma-Aldrich</td><td>P-6911</td><td></td></tr></tbody></table>

isolation and in vitro activation of caenorhabditis elegans sperm

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.

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 ...

The overall goal of this procedure is to visualize sperm morphology. This is accomplished by first isolating males for dissection one day in advance. The second step of the procedure is to dissect the males to release sperm.The third step of the procedure is to visualize sperm morphology. The final step of the procedure is to prepare sperm for downstream applications. Ultimately, results can be obtained that show sperm morphology and the ability to activate through treatment with in vitro activators.The sperm isolation and activation protocol was first developed over 30 years ago by Sam Ward, Diane Shakes, and Gregory Nelson. We currently use this procedure to characterize mutations that alter sperm function and fertility in C elegance. The overall goal of this procedure is to visualize CL elgan a OID sperm morphology.This is accomplished by isolating males one day prior to dissection in order to keep them celibate. The second step involves dissecting the males in order to release round non modal spermatids. The third step is to visualize spermatid activation in vitro to the mature AME sperm morphology.The procedure will be demonstrated by three postdoctoral fellows in the lab, Dr.Matt Marchello, Dr and Chatterjee, and Dr.Guna SLU To isolate sperm. First, obtain a large number of males by crossing five wild type males and one hermaphrodite on a small lawn of e coli. OP 50 bacteria seated at the center of an nematode growth medium or NGM plate.Roughly 50%of the next generation will be wild type males to collect. L four stage males examine developmental stage and tail morphology under a dissecting microscope. An L four stage worm is picked based on tail morphology.A male worm has a rounded tail while a hermaphrodite has a pointed tail. Using a platinum wire, pick seven to 10 L four stage males per experiment and transfer them to an NGM plate seeded with OP 50 and let them grow for a day or two. Growing celibate males in the absence of hermaphrodites prevents the loss of sperm, and thus a large number of sperms will be available during the experimental procedure.Prior to beginning the dissection, prepare one liter of M nine buffer using six grams of DI sodium hydrogen phosphate, three grams of mono potassium phosphate, five grams of sodium chloride 0.25 grams of magnesium sulfate, hep to hydrate per liter, and five milliliters of sperm medium For sperm activation, M nine buffer and sperm media are prepared from autoclave or filter sterilized stock solutions. On the day of dissection, transfer the celibate males onto an NGM plate without bacteria and let them crawl for a few minutes. This step helps to remove the small layer of e coli adhered on the surface of the worms.Alternatively, pipette a small volume of M nine buffer in a watch glass and transfer males into the buffer. Next, using a pat pen, Mark A.Small circle on a glass slide pipe at 30 to 50 microliters of one x sperm medium within the circle. The hydrophobic circle of the pat pen helps to retain the sperm medium within the boundary.Transfer five to 10 males onto the sperm medium am using a dissecting scope. Hold one or two syringes depending on comfort level. Attached with 27 gauge needles on both of your hands, use one needle to hold the worm and use the other needle to cut the posterior end of the males to release sids.The released spermatids are visible as minute granules under the stereo microscope. However, sometimes part or most of the spermatids may be retained within the carcass. In that case, gently dragging the carcass over the glass slide may facilitate the release of spermatids from the carcass.To visualize activation of the spermatids, dissect the worms in one x sperm medium containing P pronase E.Under a dissecting microscope, let the worm sit for five minutes. Activated sperm will have pseudopods. Gently place a cover slip on the surface of the dissected sample.Find the field of spermatids under 100 x magnification. Spermatids have a round shape. An example of a DIC image of spermatids isolated from male C elegance is shown in figure one.Spermatids are spherical in shape and the nuclei are prominent. In vitro activated sperm are shown in figure two, spermatozoa have a prominent pseudopod. 80 to 95%of spermatids are converted to spermatozoa If needed.The final step of the procedure can be to prepare sperm for downstream applications. For example, sperm can be fixed for immunofluorescence. Ultimately, the results obtained can show the ability to activate sperm in vitro and assay sperm morphology.

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Learn to isolate and activate Caenorhabditis elegans sperm in vitro using protease treatment. This protocol enables visualization of sperm morphology for studying reproductive biology and fertility.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

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