This protocol is an excerpt from Singaravelu et al, <a href="https://www.jove.com/video/2336/isolation-and-in-vitro-activation-of-caenorhabditis-elegans-sperm">Isolation and&#160;In vitro&#160;Activation of&#160;Caenorhabditis elegans&#160;Sperm</a>, J. Vis. Exp. (2011).

1.&#160;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.&#160;Identification and isolation of males
<ol>
	<li>Examine the tail morphology of worms; the male worm&#39;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>
</ol>

enriching for males: maintaining a stock of male c. elegans

Source: Singaravelu, G., et al. <a href="https://www.jove.com/video/2336/isolation-and-in-vitro-activation-of-caenorhabditis-elegans-sperm">Isolation and In vitro Activation of Caenorhabditis elegans Sperm</a>. J. Vis. Exp. (2011).
Nematodes can be propagated easily in the lab--in part because hermaphrodites can reproduce by self-fertilization.&#160; However, genetic crosses and other experiments may require a large number of males.&#160; This video describes a method to enrich for male C. elegans.

Enriching for Males: Maintaining a Stock of Male C. elegans

Source: Singaravelu, G., et al. Isolation and In vitro Activation of Caenorhabditis elegans Sperm. J. Vis. Exp. (2011).
Nematodes can be propagated easily ...

- C. elegans occur in two sexes: hermaphrodites, which have two X chromosomes, and males, which have only one X chromosome referred to as XO or X null. Hermaphrodites produce both oocytes and sperm and can reproduce by self- fertilization. Nearly all of their gametes carry an X chromosome, so offspring that result from self- fertilization are mostly hermaphrodites. Rarely, the X chromosome is lost during gametogenesis, resulting in male offspring only about 0.1 to 0.2% of the time. This is not a practical way to collect enough males for any experiment or genetic cross that we need them for. Instead, we can make a hermaphrodite with a male resulting in about half the offspring being male. When carrying out this genetic cross, be sure to include more males than hermaphrodites to ensure efficient mating. In the following example, we will set up a mating plate and isolate male worms in preparation to collect and study spermatids in vitro. - First, obtain a large number of males by crossing five wildtype males and one hermaphrodite on a small lawn of E. coli OP50 bacteria seeded at the center of a nematode growth medium or NGM plate. Roughly 50% of the next generation will be wildtype males. To collect L4 stage males, examine developmental stage and tail morphology under a dissecting microscope. An L4 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 7 to 10 L4 stage males per experiment and transfer them to NGM plate seeded with OP50 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 spermatids will be available during the experimental procedure.

enriching-for-males-maintaining-a-stock-of-male-c-elegans

Research

JoVE Encyclopedia of Experiments

Biology

Caenorhabditis elegans (worm)

Genetics

Nematodes can be propagated easily in the lab--in part because hermaphrodites can reproduce by self-fertilization.&#160; However, genetic crosses and other experiments may require a large number of males.&#160; This video describes a method to enrich for male C. elegans.

Enriching for Males: Maintaining a Stock of Male C. elegans

En este artículo

Overview

Protocolo