Name: fertilization of <em>xenopus oocytes</em> using the host transfer method
Uploaded: 2010-11-02T17:00:00
Description: Watch this Scientific Journal Video about Fertilization of Xenopus oocytes using the Host Transfer Method at JoVE.com

<p class="jove_content">The authors would like to thanks members of the Houston lab for critical reading of the manuscript. Research is supported by the National Institutes of Health (GM083999) awarded to D.W.H.</p>

<p class="jove_title">1. Surgical Removal of Ovary Tissue</p>
<ol class="lalpha">
	<li>Prepare a fresh batch of oocyte culture medium (OCM; Materials), between 400-800  mL depending on the size of the experiment. Adjust the pH to 7.6-7.8, until the phenol red turns a dark red color (six or so small drops of 5N NaOH). Pour out a small amount to check the pH in a separate tube since the pH electrode may contaminate the media. OCM should be stored at 18&deg;C and used within a week.</li>
	<li>Select 3-5 females and place one in ~ 2L buffered an...

<ol>
	<li>Brun, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+maturation+in+vitro:+contribution+of+the+oviduct+to+total+maturation+in+Xenopus+laevis.">Oocyte maturation in vitro: contribution of the oviduct to total maturation in Xenopus laevis.</a> <em style='font-style: italic'>Experientia</em>. <b>31</b>, 1275-1276 (1975).</li><li>Elinson, R. P., Pasceri, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+UV-sensitive+targets+in+dorsoanterior+specification+of+frog+embryos.">Two UV-sensitive targets in dorsoanterior specification of frog embryos.</a> <em style='font-style: italic'>Development</em>. <b>106</b>, 511-518 (1989).</li><li>Heasman, J., Holwill, S., Wylie, C. C., C, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+of+cultured+Xenopus+oocytes+and+use+in+studies+of+maternally+inherited+molecules.">Fertilization of cultured Xenopus oocytes and use in studies of maternally inherited molecules.</a> <em style='font-style: italic'>Methods Cell Biol</em>. <b>36</b>, 213-230 (1991).</li><li>Holwill, S., Heasman, J., Crawley, C., Wylie, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axis+and+germ+line+deficiencies+caused+by+u.v+irradiation+of+Xenopus+oocytes+cultured+in+vitro.">Axis and germ line deficiencies caused by u.v irradiation of Xenopus oocytes cultured in vitro.</a> <em style='font-style: italic'>Development</em>. <b>100</b>, 735-743 (1987).</li><li>Houston, D. W., King, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+Xdazl,+a+germ+plasm-localized+RNA,+in+the+differentiation+of+primordial+germ+cells+in+Xenopus.">A critical role for Xdazl, a germ plasm-localized RNA, in the differentiation of primordial germ cells in Xenopus.</a> <em style='font-style: italic'>Development</em>. <b>127</b>, 447-456 (2000).</li><li>Mir, A., Heasman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+mother+can+help:+studying+maternal+Wnt+signaling+by+anti-sense-mediated+depletion+of+maternal+mRNAs+and+the+host+transfer+technique.">How the mother can help: studying maternal Wnt signaling by anti-sense-mediated depletion of maternal mRNAs and the host transfer technique.</a> <em style='font-style: italic'>Methods Mol Biol</em>. <b>469</b>, 417-429 (2008).</li><li>Rugh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Experimental Embryology: Techniques and procedures</em>. , (1962).</li><li>Smith, L. D., Ecker, R. E., Subtelny, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+induction+of+physiological+maturation+in+Rana+pipiens+oocytes+removed+from+their+ovarian+follicles.">In vitro induction of physiological maturation in Rana pipiens oocytes removed from their ovarian follicles.</a> <em style='font-style: italic'>Dev Biol</em>. <b>17</b>, 627-643 (1968).</li><li>Smith, L. D., Xu, W., Varnold, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+and+oocyte+isolation.">Oogenesis and oocyte isolation.</a> <em style='font-style: italic'>Methods Cell Biol</em>. <b>36</b>, 45-60 (1991).</li><li>Woolf, T. M., Jennings, C. G., Rebagliati, M., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stability,+toxicity+and+effectiveness+of+unmodified+and+phosphorothioate+antisense+oligodeoxynucleotides+in+Xenopus+oocytes+and+embryos.">The stability, toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos.</a> <em style='font-style: italic'>Nucleic Acids Res</em>. <b>18</b>, 1763-1769 (1990).</li><li>Wylie, C., Kofron, M., Payne, C., Anderson, R., Hosobuchi, M., Joseph, E., Heasman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+beta-catenin+establishes+a+'dorsal+signal'+in+early+Xenopus+embryos.">Maternal beta-catenin establishes a 'dorsal signal' in early Xenopus embryos.</a> <em style='font-style: italic'>Development</em>. <b>122</b>, 2987-2996 (1996).</li><li>Zhang, J., Houston, D. W., King, M. L., Payne, C., Wylie, C., Heasman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+maternal+VegT+in+establishing+the+primary+germ+layers+in+Xenopus+embryos.">The role of maternal VegT in establishing the primary germ layers in Xenopus embryos.</a> <em style='font-style: italic'>Cell</em>. <b>94</b>, 515-524 (1998).</li><li>Zuck, M. V., Wylie, C. C., Heasman, J., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+mRNAs+in+Xenopus+embryos:+an+antisense+approach..">Maternal mRNAs in Xenopus embryos: an antisense approach..</a> <em style='font-style: italic'>A comparative methods approach to the study of oocytes and embryos</em>. , 341-354 (1998).</li></ol>

<p class="jove_content">A successful transfer will result in fertilization and normal cleavage of &gt; 50% of the oocytes (Figure 3). Generally between 30-60% of the transferred oocytes will survive past the neurula stages. We usually transfer 75-150 oocytes per experimental group, which will yield enough embryos for several types of analysis (in situ, RT-PCR) as well as visualizing the phenotype. Implanting substantially more oocytes does not seem to greatly increase the yield. Also, at least 30 oocytes per group should be transferred to guara...

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			<div>
			<p class="jove_title">Solutions and recipes:</p>
<p class="jove_step">OCM (Oocyte culture medium; modified from Wylie <em>et al</em>., 1996)</p>
<table border="0">
     <tbody>
          <tr>
               <td>70% Leibovitz's L-15 medium (containing l-glutamine)</td>
               <td>(Invitrogen 11415-064)</td>
          </tr>
          <tr>
               <td>0.4 mg/ml Bovine serum albumin (BSA; fraction V)</td>
               <td>(Sigma A-9418-50g)</td>
          </tr>
          <tr>
               <td>1x Penicillin-Streptomycin</td>
               <td>(Sigma P-0781)</td>
          </tr>
          <tr>
               <td colspan="2">Adjust to pH 7.6-7.8 with 5N NaOH</td>
          </tr>
          <tr>
               <td colspan="2">Make fresh weekly, store at 18&deg;C.</td>
          </tr>
     </tbody>
</table>
<p class="jove_step">10X MMR (Marc's Modified Ringer's Solution; Zuck <em>et al</em>., 1998)</p>
<table border="0">
     <tbody>
          <tr>
               <td>1M NaCl</td>
          </tr>
          <tr>
               <td>20 mM KCl</td>
          </tr>
          <tr>
               <td>20 mM CaCl<sub>2</sub></td>
          </tr>
          <tr>
               <td>10 mM MgCl<sub>2</sub></td>
          </tr>
          <tr>
               <td>150 mM HEPES</td>
          </tr>
          <tr>
               <td>adjust to pH 7.6-7.8</td>
          </tr>
          <tr>
               <td>Store at 4&deg;C</td>
          </tr>
     </tbody>
</table>
<p class="jove_step">Anesthetic solution</p>
<table border="0">
     <tbody>
          <tr>
               <td>1 g/L 3-Aminobenzoic Acid Ethyl Ester (MS222)</td>
               <td>(Sigma A-5040)</td>
          </tr>
          <tr>
               <td colspan="2">0.7 g/L sodium bicarbonate</td>
          </tr>
          <tr>
               <td colspan="2">Dissolve in Amquel-treated water</td>
          </tr>
          <tr>
               <td colspan="2">Make fresh weekly</td>
          </tr>
     </tbody>
</table>
<p class="jove_step">Progesterone stocks</p>
<table border="0">
     <tbody>
          <tr>
               <td>10 mM Progesterone (Sigma P- 0130) in 100% Ethanol</td>
          </tr>
          <tr>
               <td>Dilute to 1 mM in 100% EtOH for a 500x stock/working solution</td>
          </tr>
          <tr>
               <td>Store at -20&deg;C</td>
          </tr>
     </tbody>
</table>
<p class="jove_step">Vital dyes (modified from Heasman <em>et al</em>., 1991)</p>
<table border="0">
     <tbody>
          <tr>
               <td>Blue: 0.1% Nile Blue A</td>
               <td>(Aldrich 121479-5G),</td>
          </tr>
          <tr>
               <td>Red: 0.25% Neutral Red</td>
               <td>(Sigma N-6634),</td>
          </tr>
          <tr>
               <td>Brown: 1% Bismarck Brown</td>
               <td>(Sigma B-2759),</td>
          </tr>
          <tr>
               <td colspan="2">Green (80 &mu;l blue + 80 &mu;l brown),</td>
          </tr>
          <tr>
               <td colspan="2">Mauve (80 &mu;l blue + 80 &mu;l red).</td>
          </tr>
          <tr>
               <td colspan="2">A sixth color, Orange (80 &mu;l red+ 80 &mu;l brown), can also be used but is often hard to distinguish from brown.</td>
          </tr>
     </tbody>
</table>
<p class="jove_content">Stocks of Blue, Red and Brown are made up in deionized water in 50 ml tubes, incubated for 20 minutes with rocking and spun in a clinical centrifuge. The supernatants are aliquotted into microfuge tubes and stored at -20&deg;C.</p>		</div>
		

fertilization of <em>xenopus oocytes</em> using the host transfer method

<p class="jove_content">Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as <em>Xenopus</em> and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In <em>Xenopus</em>, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.</p>

Watch this Scientific Journal Video about Fertilization of Xenopus oocytes using the Host Transfer Method at JoVE.com

Fertilization of Xenopus oocytes using the Host Transfer Method

<p class="jove_content">Procedure for fertilizing <em>Xenopus oocytes</em> by the host transfer method.</p>

Fertilization of <em>Xenopus oocytes</em> using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

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