Name: planarian ovary dissection for ultrastructural analysis and antibody staining
Uploaded: 2021-09-10T13:00:00
Description: Watch this Scientific Journal Video about Planarian Ovary Dissection for Ultrastructural Analysis and Antibody Staining at JoVE.com

The work was supported by the Howard Hughes Medical Institute (LK and ASA) and the Helen Hay Whitney Foundation (LHG). 
 
Original data underlying this manuscript can be accessed from the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-1628

1. Preparation
<ol>
	<li>Prepare worms: feed sexual planarians twice a week with organic liver paste to achieve sexual maturity. 
	NOTE: Generally, such worms are bigger than 1 cm in length and have a gonopore posterior to the pharynx opening.</li>
	<li>Prepare solutions.
	<ol>
		<li>Prepare the following reagents: 16% paraformaldehyde (PFA); 50% glutaraldehyde (GA) aqueous solution; N-acetyl-L-cysteine (NAC); 1x phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4.</l...

<ol>
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Normal eye.</a> Cell Tissue Research. 148 (2), 143-158 (1974).</li><li>Ishii, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ultrastructure+of+the+protonephridial+flame+cell+of+the+freshwater+planarian+Bdellocephala+brunnea.">The ultrastructure of the protonephridial flame cell of the freshwater planarian Bdellocephala brunnea.</a> Cell Tissue Research. 206 (3), 441-449 (1980).</li><li>Morita, M., Best, J. B., Noel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+studies+of+planarian+regeneration:+I.+Fine+structure+of+neoblasts+in+Dugesia+dorotocephala.">Electron microscopic studies of planarian regeneration: I. Fine structure of neoblasts in Dugesia dorotocephala.</a> Journal of Ultrastructure Research. 27 (1), 7-23 (1969).</li><li>Hyman, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The invertebrates: Platyhelminthes and Rhynchocoela, the acoelomate Bilateria. 2, 550 (1951).</li><li>Higuchi, S., 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=Characterization+and+categorization+of+fluorescence+activated+cell+sorted+planarian+stem+cells+by+ultrastructural+analysis.">Characterization and categorization of fluorescence activated cell sorted planarian stem cells by ultrastructural analysis.</a> Development, Growth &amp; Differentiation. 49 (7), 571-581 (2007).</li><li>Chong, T., Stary, J. M., Wang, Y., Newmark, P. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+the+planarian+reproductive+system+through+characterization+of+Schmidtea+mediterranea+CPEB+homologs.">Genetic dissection of the planarian reproductive system through characterization of Schmidtea mediterranea CPEB homologs.</a> Developmental Biology. 426 (1), 43-55 (2017).</li><li>Sato, K., 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=Identification+and+origin+of+the+germline+stem+cells+as+revealed+by+the+expression+of+nanos-related+gene+in+planarians.">Identification and origin of the germline stem cells as revealed by the expression of nanos-related gene in planarians.</a> Development, Growth &amp; Differentiation. 48 (9), 615-628 (2006).</li><li>Tharp, M. E., Collins, J. J., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lophotrochozoan-specific+nuclear+hormone+receptor+is+required+for+reproductive+system+development+in+the+planarian.">A lophotrochozoan-specific nuclear hormone receptor is required for reproductive system development in the planarian.</a> Developmental Biology. 396 (1), 150-157 (2014).</li><li>Wang, Y., Zayas, R. M., Guo, T., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nanos+function+is+essential+for+development+and+regeneration+of+planarian+germ+cells.">nanos function is essential for development and regeneration of planarian germ cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (14), 5901-5906 (2007).</li><li>Zhang, S., 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=A+nuclear+hormone+receptor+and+lipid+metabolism+axis+are+required+for+the+maintenance+and+regeneration+of+reproductive+organs.">A nuclear hormone receptor and lipid metabolism axis are required for the maintenance and regeneration of reproductive organs.</a> bioRxiv. , 279364 (2018).</li><li>Saberi, A., Jamal, A., Beets, I., Schoofs, L., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPCRs+direct+germline+development+and+somatic+gonad+function+in+planarians.">GPCRs direct germline development and somatic gonad function in planarians.</a> PLoS Biology. 14, 1002457 (2016).</li><li>Steiner, J. K., Tasaki, J., Rouhana, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+defects+caused+by+Smed-boule+RNA-interference+reveal+that+egg+capsule+deposition+occurs+independently+of+fertilization,+ovulation,+mating,+or+the+presence+of+gametes+in+planarian+flatworms.">Germline defects caused by Smed-boule RNA-interference reveal that egg capsule deposition occurs independently of fertilization, ovulation, mating, or the presence of gametes in planarian flatworms.</a> PLoS Genetics. 12, 1006030 (2016).</li><li>Iyer, H., Issigonis, M., Sharma, P. P., Extavour, C. G., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+premeiotic+function+for+boule+in+the+planarian+Schmidtea+mediterranea.">A premeiotic function for boule in the planarian Schmidtea mediterranea.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (25), 3509-3518 (2016).</li><li>Rouhana, L., Vieira, A. P., Roberts-Galbraith, R. H., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRMT5+and+the+role+of+symmetrical+dimethylarginine+in+chromatoid+bodies+of+planarian+stem+cells.">PRMT5 and the role of symmetrical dimethylarginine in chromatoid bodies of planarian stem cells.</a> Development. 139 (6), 1083-1094 (2012).</li><li>Wang, Y., Stary, J. M., Wilhelm, J. E., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+genomic+screen+in+planarians+identifies+novel+regulators+of+germ+cell+development.">A functional genomic screen in planarians identifies novel regulators of germ cell development.</a> Genes &amp; Development. 24 (18), 2081-2092 (2010).</li><li>Guo, L., 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=Subcellular+analyses+of+planarian+meiosis+implicates+a+novel,+double-membraned+vesiculation+process+in+nuclear+envelope+breakdown.">Subcellular analyses of planarian meiosis implicates a novel, double-membraned vesiculation process in nuclear envelope breakdown.</a> bioRxiv. , 620609 (2019).</li><li>Grudniewska, M., 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=Transcriptional+signatures+of+somatic+neoblasts+and+germline+cells+in+Macrostomum+lignano.">Transcriptional signatures of somatic neoblasts and germline cells in Macrostomum lignano.</a> Elife. 5, 20607 (2016).</li><li>Wudarski, J., 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=The+free-living+flatworm+Macrostomum+lignano.">The free-living flatworm Macrostomum lignano.</a> Evodevo. 11, 5 (2020).</li><li>Wudarski, J., 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+transgenesis+and+annotated+genome+sequence+of+the+regenerative+flatworm+model+Macrostomum+lignano.">Efficient transgenesis and annotated genome sequence of the regenerative flatworm model Macrostomum lignano.</a> Nature Communications. 8 (1), 2120 (2017).</li><li>Maezawa, T., Sekii, K., Ishikawa, M., Okamoto, H., Kobayashi, K., Kobayashi, K., Kitano, T., Iwao, Y., Kondo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Reproductive and Developmental Strategies: The Continuity of Life. , 175-201 (2018).</li><li>Newmark, P. A., Reddien, P. W., Cebria, F., Sanchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ingestion+of+bacterially+expressed+double-stranded+RNA+inhibits+gene+expression+in+planarians.">Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 11861-11865 (2003).</li><li>Orii, H., Mochii, M., Watanabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+"soaking+method"+for+RNA+interference+in+the+planarian+Dugesia+japonica.">A simple "soaking method" for RNA interference in the planarian Dugesia japonica.</a> Development Genes and Evolution. 213 (3), 138-141 (2003).</li><li>Rouhana, L., 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=RNA+interference+by+feeding+in+vitro-synthesized+double-stranded+RNA+to+planarians:+methodology+and+dynamics.">RNA interference by feeding in vitro-synthesized double-stranded RNA to planarians: methodology and dynamics.</a> Developmental Dynamics. 242 (6), 718-730 (2013).</li></ol>

These fixation-based procedures for dissecting planarian ovaries will facilitate the understanding of oocyte meiosis as well as ovary development and regeneration. The sizes of the oocytes and their somatic supportive cells can range from 20 &#181;m to 50 &#181;m. Dissection-based methods will provide accessibility to intact single-ovary cells that sectioning or whole-mount-based methods cannot achieve. This protocol will facilitate the study of intact planarian ovary anatomy and oocyte cell biology at cellular and subce...

Planarian anatomy has been examined by using TEM in many tissues1,2,3,4,5,6. However, little attention has been given to ovaries or oocytes. The paucity of oocyte literature is partly due to the difficulty accessing these cells, leaving the biology of planarian oocytes largely unexplored. Molecular tools have uncovered many regulatory mechanisms of ovary development in the planarians&#160;using light or fluorescence microscopy7,8,9,10,11,12,13,14,15,16,17,18,19,20. All these experiments were performed on whole worms or histological sections of whole worms. The antibody staining and in situ hybridization protocols on whole worms involve extensive bleaching, washing, and tissue clearing steps, which are time-consuming and will take several days.
The overall goal of the method described here is to provide accessibility to intact, dissected planarian ovaries and oocytes, which will remove the necessity of bleaching or histological sectioning and shorten the time for washing and tissue clearing in antibody staining and in situ hybridization. The dissected ovaries will also improve probe or antibody penetration and increase imaging depth and quality for light and electron microscopes. Accessibility to the dissected ovaries and oocytes allows cell biology research at cellular and subcellular resolution with whole intact oocytes. A recent study on dissected planarian ovaries characterized planarian oocyte meiosis for the first time with TEM and confocal microscopy21. The work provided a comprehensive description of a new phenomenon during meiosis called nuclear envelope vesiculation.
Here, we present the detailed procedures in the dissection of planarian ovaries. A fixation step was sufficient to preserve the ovary cell structure for dissection and downstream manipulation (i.e., processing for TEM and light microscope analysis). Given their similarity in body plans and tissue architecture, this protocol should also be broadly informative for studying oocytes and their nuclei in several other Platyhelminthes species (e.g., the genus of Dugesia or Polycelis). This protocol is likely irrelevant for Macrostomum lignano for their small sizes and almost transparent body architecture, which will allow for direct observation of the ovary and oocytes22,23,24. The body area containing the ovaries is more optically distinguishable (e.g., darker pigmented or lighter pigmented) in some species (e.g., Dugesia ryukyuensis9,25) than S. mediterranea. Studies in these species can rely less on the guidelines for locating the ovaries in S. mediterranea presented here but take advantage of the fixation and dissection conditions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16% paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>EM grade</td>
		</tr>
		<tr>
			<td>2% aqueous OsO4</td>
			<td>Electron Microscopy Sciences</td>
			<td>19152</td>
			<td></td>
		</tr>
		<tr>
			<td>50% glutaraldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>16320</td>
			<td>EM grade</td>
		</tr>
		<tr>
			<td>Digital Micrograph</td>
			<td>Gatan Inc.</td>
			<td></td>
			<td>Version 2.33.97.1, TEM data collection</td>
		</tr>
		<tr>
			<td>Epon resin</td>
			<td>Electron Microscopy Sciences</td>
			<td>14120</td>
			<td>Embed 812 Kit, liquid, epoxy resin</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Ted Pella</td>
			<td>19207</td>
			<td>Denatured</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Sigma</td>
			<td>H1138</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead Acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>6080564</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliQ water</td>
			<td></td>
			<td></td>
			<td>reverse-osmosis treated water</td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma</td>
			<td>A7250</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>sigma</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Diamond Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36965</td>
			<td></td>
		</tr>
		<tr>
			<td>Propylene oxide</td>
			<td>Electron Microscopy Sciences</td>
			<td>75569</td>
			<td>EM grade</td>
		</tr>
		<tr>
			<td>&#160;Proteinase K</td>
			<td>Thermo Fisher Scientific</td>
			<td>25530049</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine blue O</td>
			<td>Electron Microscopy Sciences</td>
			<td>92319</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>FEI</td>
			<td></td>
			<td>Tecnai G2 Spirit BioTWIN</td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>541093</td>
			<td></td>
		</tr>
	</tbody>
</table>

planarian ovary dissection for ultrastructural analysis and antibody staining

Accessibility to germ cells allows the study of germ cell development, meiosis, and recombination. The sexual biotype of the freshwater planarian, Schmidtea mediterranea, is a powerful invertebrate model to study the epigenetic specification of germ cells. Unlike the large number of testis and male germ cells, planarian oocytes are relatively difficult to locate and examine, as there are only two ovaries, each with 5-20 oocytes. Deeper localization within the planarian body and lack of protective epithelial tissues also make it challenging to dissect planarian ovaries directly. 
This protocol uses a brief fixation step to facilitate the localization and dissection of planarian ovaries for downstream analysis to overcome these difficulties. The dissected ovary is compatible for ultrastructural examination by transmission electron microscopy (TEM) and antibody immunostaining. The dissection technique outlined in this protocol also allows for gene perturbation experiments, in which the ovaries are examined under different RNA interference (RNAi) conditions. Direct access to the intact germ cells in the ovary achieved by this protocol will greatly improve the imaging depth and quality and allow cellular and subcellular interrogation of oocyte biology.

The method presented here has been described by Guo et al.21. The key to successful dissection is to identify the ovary pigmentation and position guides correctly. The strategy of the method is to move from broad positions to a specific location. First, to achieve this, rely on dorsal and ventral pigmentation patterns (Figure 1A,B). Ventral pigmentation, where the ovaries reside, will turn white after 5% NAC treatment and 4% PFA fixation. If the worm ...

Watch this Scientific Journal Video about Planarian Ovary Dissection for Ultrastructural Analysis and Antibody Staining at JoVE.com

Planarian Ovary Dissection for Ultrastructural Analysis and Antibody Staining

This protocol presents steps taken to dissect ovaries in the freshwater planarians, Schmidtea mediterranea. The dissected ovaries are compatible for antibody immunostaining and ultrastructural analysis with transmission electron microscopy to study the cell biology of the oocytes and somatic cells, providing an imaging depth and quality that were previously inaccessible.

Accessibility to germ cells allows the study of germ cell development, meiosis, and recombination. The sexual biotype of the freshwater planarian, ...

The goal of this method is to dissect the ovaries of the planarian Schmidtea mediterranea for cellular and subcellular analysis of oocyte biology with TEM or antibody staining. This protocol uses a brief fixation step to facilitate the localization and dissection of planarian ovaries, which will provide imaging depth and quality that are previously inaccessible. The biggest struggle for someone who has never performed this technique before will be to localize the ovaries after fixation.Getting familiar with the anatomy of sexual planarians before the experiment will be helpful. Prepare for the experiment by feeding sexual planarians with organic liver paste twice a week to achieve sexual maturity. Treat sexually mature worms with 5%N-acetyl-L-cysteine, or NAC, in a dish at room temperature for five minutes.Replace NAC with 10 milliliters of freshly prepared 4%paraformaldehyde, and fix the worms for one hour at room temperature with occasional shaking. Start the dissection 10 minutes after adding 4%paraformaldehyde as the worms are being fixed. Observe the epithelial pigmentation to locate the ventral nerve cords, which appear as two lines with lighter pigmentation running from the anterior cephalic ganglia toward the tail.Locate the putative ovaries by observing the pigmentation and validate their position. Using two surgical knives, make cuts posterior and anterior to the ovaries to remove the anterior and posterior worm fragments completely. Use one knife to anchor the worm and the other knife for the dissection.Clean the knife used to make the cuts. To separate the two ovaries, cut in the middle line of the ovary fragment, then flip the fragment ventral side down. Use two pairs of type 5-SA pointed tweezers to peel off the dorsal tissue to expose the gut.Gently remove the gut branches sitting above the ventral tissues with the tip of the tweezers or a soft brush to expose the ovary. Remove the surrounding tissues to take out the ovary. Take the ovaries out and transfer them to a 1.5 milliliter tube to wash with 1 milliliter of PBS.The dorsal and ventral pigmentation patterns were used to locate the position of the ovaries. Excessive tissue was trimmed away in a step-by-step fashion. Once the ovary was exposed, the surrounding tissue was trimmed away.Ovaries containing multiple somatic cell types were used for both ultrastructural analysis and immunofluorescence staining. The dissected ovaries allow for high quality imaging of the cellular and subcellular anatomy of the oocytes. This procedure should allow for RNA interference experiments to examine molecular regulations of oocyte biology.This technique led to the discovery of a new phenomenon in the flatworm oocytes that the nuclear envelope breaks down into vesicles encapsulating nuclear proteins instead of remaining intact, aka closed mitosis, or breaking into pieces, aka open mitosis.

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