We are grateful to Gurdon laboratory members for useful discussion. K.M. is a Research Fellow at Wolfson College and is supported by the Herchel Smith Postdoctoral Fellowship and the Great Britain Sasakawa Foundation. Gurdon laboratory is supported by grants from the Wellcome Trust (RG69899) and MRC to J.B.G.

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+oocyte+nucleus+in+physiological+maturation+in+Rana+pipiens.">Role of the oocyte nucleus in physiological maturation in Rana pipiens.</a> Developmental biology. 19 (3), 281-309 (1969).</li><li>Miyamoto, K., Pasque, V., Jullien, J., Gurdon, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+actin+polymerization+is+required+for+transcriptional+reprogramming+of+Oct4+by+oocytes.">Nuclear actin polymerization is required for transcriptional reprogramming of Oct4 by oocytes.</a> Genes &amp; development. 25 (9), 946-958 (2011).</li><li>Schneider, P. N., Hulstrand, A. M., Houston, D. 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(45), (2010).</li><li>Ishibashi, S., Cliffe, R., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+bi-allelic+mutation+rates+using+TALENs+in+Xenopus+tropicalis).">Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis).</a> Biology open. 1 (12), 1273-1276 (2012).</li><li>Sakane, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mutagenesis+of+multiple+and+paralogous+genes+in+Xenopus+laevis+using+two+pairs+of+transcription+activator-like+effector+nucleases.">Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases.</a> Development, growth &amp; differentiation. 56 (1), 108-114 (2014).</li><li>Nakayama, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+efficient+CRISPR/Cas9-mediated+targeted+mutagenesis+in+Xenopus+tropicalis.">Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis.</a> Genesis. 51 (12), 835-843 (2013).</li><li>Guo, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+RNA/Cas9-mediated+genome+editing+in+Xenopus+tropicalis.">Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis.</a> Development. 141 (3), 707-714 (2014).</li></ol>

The authors have nothing to disclose.

We here detail a new method to deplete maternal factors or to overexpress exogenous factors before fertilization. This system requires two microinjections, but instead skips the surgery of frogs, used in the host transfer method7,17. It is optimal to remove the frog surgery step in terms of animal care. Moreover, we do not have to take into account for the quality of host female frogs for the transfer experiments, meaning that we can get rid of one biological factor that affects the success of experiments. Therefore in this system the quality of oocytes obtained from PMSG-primed frogs is a major biological factor that is key for successful experiments. The quality of oocytes can be judged after collection of ovary or after defolliculation. If any abnormality is observed at these stages, it is optimal to collect another ovary from a new frog. Another point when you can check the oocyte quality is after oocyte maturation. If less than 80% of progesterone-treated oocytes show signs of maturation, you may not be able to obtain the enough number of embryos for further analyses. The use of enzymatic defolliculation instead of manual defolliculation is also another advantage of using this system to reduce the labor required for defolliculation. However, it is still possible that manual defolliculation may give a better development than the enzymatic treatment.
As shown in Figure 3, almost 10% of in vitro matured oocytes can reach the swimming tadpole stage. These data are collected from 13 independent experiments including those reported in 8 and new injection experiments. Host transfer method needs 75-150 oocytes in each treatment for obtaining meaningful data since 30-60% of the transferred oocytes can reach the neurula stage17. Our method normally starts with 200-300 oocytes in each experimental group since approximately 40-60% of cleaved embryos can reach the muscular response stage. These data suggest that both methods support a reasonable developmental rate. We have so far obtained 10 alive frogs from control mRNA-injected and control antisense oligo-injected oocytes using this technique, indicating that this approach supports development through metamorphosis.
Our oocyte manipulation-ICSI method provides an opportunity to test the role of maternal factors during very early embryonic development, soon after fertilization. In addition, overexpression of chromatin modifying factors before fertilization may make it possible to remodel maternal chromatin before fertilization for understanding maternal chromatin states necessary for development. This strategy may also work well with current gene editing systems such as Transcription activator-like effector nuclease (TALEN)18,19 and CRISPR/CAS920,21 since these can be expressed even before fertilization. Therefore, our new approach has a potential to be used for many applications in future.

Xenopus laevis is a widely used, powerful model organism to study development1. This is because Xenopus eggs are unusually large (approximately 1.2-1.4 mm, in comparison to mammalian counterparts being about 0.1 mm) and abundant. Eggs contain maternally synthesized and stored components, which are enough to drive embryonic development until mid-blastula transition (MBT, occurring at the stage 8-8.5 with 4,000-8,000 cells). MBT is accompanied by zygotic genome activation, which then produces embryonic gene products that direct further development.
Numerous studies aimed to identify maternal factors important for development. Many studies rely on the injection of antisense oligonucleotides (oligos) including morpholino oligonucleotides into fertilized embryos, in which case degradation of maternal proteins can be observed at the gastrula stage2-4. Alternatively, mRNAs are injected to fertilized embryos to disturb gene functions or to trace fates of overexpressed proteins. However, the injection into the one-cell stage embryos normally does not affect expression levels of maternal proteins at the very early embryonic stages before MBT.
Heasman and Wylie established the host transfer method to overcome this issue5. In their method, manually defolliculated oocytes are injected with antisense oligos and transferred into host females6. Proteins are downregulated before fertilization so that roles of downregulated proteins in early embryonic development can be examined. This maternal depletion method led to the identification of several unique developmental roles of maternal proteins, as reviewed in 7.
In this report, we detail our recently developed method, in which maternal depletion or overexpression of mRNA before fertilization is achieved without manual defolliculation and host transfer8. Manual defolliculation requires a lot of time and host transfer often needs skillful techniques and the specific license for animal surgery, thus hampering the frequent use of maternal depletion method. Defolliculation and host transfer are substituted by enzymatic defolliculation 9,10 and intracytoplasmic sperm injection (ICSI)11-13, respectively. ICSI to eggs was originally used to produce transgenic frogs 12. Sperm and embryonic nuclei were also transplanted into in vitro matured amphibian oocytes 11,14,15. Here, we show our step-by-step method to inject sperm to in vitro matured oocytes that were pre-injected with antisense oligos or mRNA.

<table border="0" cellpadding="0" cellspacing="0" width="589">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:87px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pregnant Mare Serum Gonadotropin (PMSG)&#160;</td>
			<td style="width:87px;">MSD Animal Health</td>
			<td style="width:119px;">Vm 01708/4309</td>
			<td>PMSG-Intervet 5000iu powder and solvent for solution for injection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethyl 3-aminobenzoate methanesulfonate salt (MS222)</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">A5040</td>
			<td>For anesthesia</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Liberase TM Research Grade</td>
			<td style="width:87px;">Roche</td>
			<td style="width:119px;">05 401 127 001</td>
			<td>For oocyte defolliculation. Store at -20oC</td>
		</tr>
		<tr height="72">
			<td height="72" style="height:72px;width:216px;">Drummond Nanoject</td>
			<td style="width:87px;">Drummond Scientific Company</td>
			<td style="width:119px;">3-000-205/206</td>
			<td>For microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">glass capillary&#160;</td>
			<td style="width:87px;">Alpha laboratories</td>
			<td style="width:119px;">7&#8221; Drummond #3-000-203-G/XL</td>
			<td>For microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropipette Puller&#160;</td>
			<td style="width:87px;">SUTTER Instrument</td>
			<td style="width:119px;">Model D-97</td>
			<td>For microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UltraPure Agarose</td>
			<td style="width:87px;">Invitrogen</td>
			<td style="width:119px;">16500-500</td>
			<td>For invitro maturation and ICSI</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">14 ml ultra-clear centrifuge tube&#160;</td>
			<td style="width:87px;">Beckman Coulter United Kingdom Ltd</td>
			<td style="width:119px;">344060</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">OptiPrep Density Gradient Medium (Iodixanol)</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">D1556</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Digitonin</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">D141</td>
			<td>Cell permeabilization reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shaker</td>
			<td style="width:87px;">Hybaid&#160;</td>
			<td style="width:119px;">HB-SHK-1</td>
			<td>For oocyte defolliculation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscope (Stereo zoom microscope)</td>
			<td style="width:87px;">ZEISS</td>
			<td style="width:119px;">Semi SV6</td>
			<td>For oocyte collection and microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 &#956;m pore filter</td>
			<td style="width:87px;">CellTrics</td>
			<td style="width:119px;">04-0042-2317</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ultracentrifuge</td>
			<td style="width:87px;">Beckman Coulter</td>
			<td style="width:119px;">Optima L-100XP</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">50 ml centrifuge tube</td>
			<td style="width:87px;">Cellstar Greiner Bio-One</td>
			<td style="width:119px;">227261 or 210261</td>
			<td>Oocyte collection and defolliculation reaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml centrifuge tube</td>
			<td style="width:87px;">FALCON</td>
			<td style="width:119px;">352097</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">90 mm Petri dish&#160;</td>
			<td style="width:87px;">Thermo Scientific</td>
			<td style="width:119px;">101VR20/C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Easy-Grip Cell Culture Dish, 60x15 mm</td>
			<td style="width:87px;">FALCON</td>
			<td style="width:119px;">353004</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Easy-Grip Cell Culture Dish, 35x15 mm</td>
			<td style="width:87px;">FALCON</td>
			<td style="width:119px;">351008</td>
			<td></td>
		</tr>
	</tbody>
</table>

manipulation and in vitro maturation of xenopus laevis oocytes, followed by intracytoplasmic sperm injection, to study embryonic development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Embryonic development of ICSI embryos using in vitro matured oocytes was examined (Figure 3A). Maturation rates of GV oocytes to the MII stage are variable and largely depends on the oocyte quality. In good experiments, almost 100% of GV oocytes respond to progesterone and show signs of oocyte maturation, finally becoming eggs at the MII stage. All matured oocytes were subjected to ICSI and about 25% of injected eggs cleaved (Figure 3A, n = 13 for control scrambled oligo-injected oocytes and n = 7 for no oligo-injected oocytes). Approximately 60% or 80% of cleaved embryos, produced from control oligo-injected oocytes or non-injected oocyte, respectively, reached the blastula/gastrula stage. Among the blastula/gastrula embryos, approximately half of embryos in oligo-injected samples were of good quality (almost no signs of abnormal cleavage and apoptosis) whereas 82% of blastula/gastrula embryos were of good quality in non-injected samples (Figure 3A). Finally, 41% and 11% of cleaved embryos in oligo-injected samples reached the muscular response and the swimming tadpole stages, respectively, while 60% and 29% of cleaved embryos in non-injected samples did (Figure 3A). These ICSI embryos are the mixture of normal and abnormal embryos (Figure 3B). Some of them undergo metamorphosis and develop to mature frogs8. These results suggest that injection of antisense oligonucleotides into GV oocytes, followed by IVM and ICSI, allows efficient early embryonic development. Although the injection of antisense oligos itself decreases embryonic development, we are still able to obtain enough embryos for many experimental purposes such as checking developmental rates, RT-PCR, western blot and so on.
<img alt="Figure 1" src="/files/ftp_upload/52496/52496fig1highres.jpg" /> 
Figure 1: Typical examples of Xenopus laevis oocytes of good quality or bad quality for in vitro maturation experiments. (A) An example of bad quality oocytes. For example, oocytes showing patchy pigmentation are not used for subsequent experiments. Each Xenopus laevis oocyte is approximately 1.2-1.4 mm in diameter. (B) A partially defolliculated oocyte. The right half of the oocyte is covered by follicle cells, which can be discerned by the presence of blood vessels. An arrow shows an area free of follicle cells and into which the injection needle is injected. (C) An example of good quality oocytes, which are equally sized and show evenly pigmented animal hemispheres with clear contrast between the animal hemisphere and the vegetal hemisphere.
<img alt="Figure 2" src="/files/ftp_upload/52496/52496fig2highres.jpg" /> 
Figure 2: Xenopus laevis oocytes before and after maturation. After oocyte maturation, clear white spots appear at the top of animal hemispheres and follicle cells are peeled off. Each Xenopus laevis oocyte is approximately 1 mm in diameter.
<img alt="Figure 3" src="/files/ftp_upload/52496/52496fig3highres.jpg" /> 
Figure 3: Development of ICSI embryos, produced from in vitro matured oocytes. (A) Development of ICSI embryos to each stage is summarized. Oocytes were injected with control scrambled antisense oligonucleotides (control oligo injection) or without injection (non injection), followed by IVM and ICSI. The corresponding number of embryos at each stage is shown above the bars. Mean &#177; SEM is shown. N = 4-13 independent experiments/different females. (B) Examples of surviving ICSI embryos. These tailbud stage embryos were produced in one experiment, in which approximately 200 oocytes were used as a starting material.

Watch this Scientific Journal Video about Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development at JoVE.com

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

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22.0K visualizações.  University of Cambridge. We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.