Planarian Ovary Dissection for Ultrastructural Analysis and Antibody Staining

Summary

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.

Abstract

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.

Introduction

Planarian anatomy has been examined by using TEM in many tissues^1,2,3,4,5,6. 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 using light or fluorescence microscopy^{7,8,9,10,11,12,13,14,15,16,17,18,19,20}. 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 oocytes^22,23,24. The body area containing the ovaries is more optically distinguishable (e.g., darker pigmented or lighter pigmented) in some species (e.g., Dugesia ryukyuensis^9,25) 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.

Protocol

1. Preparation

1. 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.
2. Prepare solutions.
   1. 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.
   2. Dilute 50% GA and 16% PFA with PBS to a final mixture of 2.5% GA and 2% PFA. Dilute 16% PFA with PBS to a final concentration of 4% PFA.
   3. Dissolve 5 g of NAC in 100 mL of PBS to make a 5% working solution.

2. Collecting ovaries

1. Treat sexually mature worms with 5% NAC in a dish at room temperature for 5 min.
   NOTE: Large worms may curl up. Brushes or pipette tips can be used to flatten the worms. The amount of 5% NAC used is minimal, enough to cover the surface of the Petri dish.
2. Replace NAC with 10 mL of freshly prepared 4% paraformaldehyde and fix the worms for 1 h at room temperature with occasional shaking. Start the dissection 10 min after the addition of 4% PFA, and perform the dissection in 4% PFA as the worms are being fixed.
3. 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.
4. Locate the ovaries.
   NOTE: The ovary is located right next to the nerve cord on the medial side (Figure 1A). Rely on epithelia pigmentation to locate the position of the ovary in the anterior-posterior direction. Ideally, the pigmentation of the ventral epithelia in the ovary region is lighter than the surrounding regions. In some worms, the pigmentation of the ventral epithelia does not provide enough confidence. These worms can be discarded as some may not have well-developed ovaries.
5. Validate the ovaries' positions once the putative ovaries are located by pigmentation.
   NOTE: First, the ovaries are posterior to the cephalic ganglia. Second, dark-colored testes are lateral to the dorsal surface above the ovary. Ovaries locate in front of the most anterior testis (Figure 1B). Sometimes, testis lobes are positioned at the level of or slightly anterior to the ovaries.
6. Use two surgical knives to cut posterior and anterior to the two ovaries (Figure 1C) to remove the anterior and posterior worm fragments completely. Use one knife to anchor the worm and clean the other knife used to make the cuts.
7. Cut in the middle line of the ovary fragment to separate the two ovaries. Flip the fragment to have the ventral side down.
8. Peel off the dorsal tissue to expose the gut (Figure 1D) with two pairs of pointed tweezers (type 5-SA).
   NOTE: The dome-shaped ovary is located beneath the gut branches.
9. Gently remove the gut branches sitting above the ventral tissues with the tip of the tweezers or a soft brush to expose the ovary (Figure 1E).
10. Remove the surrounding tissues to take out the ovary (Figure 1F,G).
11. Take the ovaries out and transfer them to a 1.5 mL tube to wash with 1 mL of PBS.

3. Fixation for TEM

1. Wash the ovaries in PBS for 10 min with gentle shaking. Keep the tubes upright, and let the ovaries sink to the bottom by gravity. Repeat the wash once.
   NOTE: If the ovaries do not sink, apply a gentle spin for 15 s with a mini-benchtop centrifuge (maximum speed 2000 × g).
2. Replace the PBS with 2% PFA/2.5% GA/PBS.
3. Fix the samples at room temperature for 1 h on a shaker at 40 rpm.
4. Keep the samples in fixative at 4 °C overnight on a shaker at 40 rpm.

4. Sample processing for TEM

1. Wash the ovaries in PBS 3 times for 10 min each.
2. Wash the ovaries in double-distilled water (ddH₂O) 3 times for 10 min each.
3. Post-fix the ovaries in 2% aqueous OsO₄ for 1-2 h at room temperature or overnight at 4 °C.
   NOTE: The sample containers need to be sealed with parafilm during this step. Prepare 2% OsO₄ with reverse-osmosis-treated water (see the Table of Materials).
4. Rinse the samples with ddH₂O 3 times, 10 min each.
5. Pre-stain the ovaries in 2% aqueous uranyl acetate (UA) overnight at 4 °C.
   NOTE: The UA solution needs to be filtered before use; avoid light during the en bloc staining. Prepare 2% OsO₄ with reverse-osmosis-treated water (see the Table of Materials).
6. Rinse the samples in ddH₂O 4 times with gentle agitation, 10 min each.
7. Dehydrate the ovaries in a graded ethanol series (30%, 50%, 70%, 95%, and two times 100%, 10 min each solution) and then equilibrate them in two incubations (10 min) in propylene oxide.
8. For infiltration with resin, incubate the samples in 50% propylene oxide/50% liquid epoxy resin mixture overnight at 4 °C. Infiltrate the samples with 100% epoxy resin with 3 changes (1 h each change) with gentle agitation.
9. Embed the ovaries in 100% epoxy resin and polymerize the resin at 60 °C for 48 h.

5. Ultramicrotomy

1. Block trimming and sample check with a light microscope
   1. Trim the sample blocks into a pyramid shape with a razor blade.
   2. Cut sections of 1-2 μm thickness with a glass or diamond knife.
   3. Transfer the sections onto a slide with a drop of water, then heat the slide on a hot plate until the sections flatten and adhere to the slide surface during water evaporation.
   4. Cover the sections with a drop of 1% toluidine blue O and heat them on a hot plate for ~10 s. Rinse the slide with running water, then let it dry. Check the sections under a regular light microscope.
2. Ultrathin sections cutting, collection, and post-staining.
   1. Cut ultrathin sections of 50-70 nm thickness with a diamond knife. Transfer the sections from the knife water boat onto mesh or single-slot copper grids.
   2. Stain the sections in 2% UA for 8 min. Wash the grids in running ddH₂O for 30 s.
   3. Stain the sections with 1% lead solution for 6 min. Wash the sections in running ddH₂O for 1 min and air-dry.

6. Data collection and analysis

1. Collect TEM data using appropriate software, e.g., Digital Micrograph (see the Table of Materials for more details).
2. Operate the scope at 80 kV. Insert the sample grid into the scope when the vacuum is ready.
3. Turn on the beam by clicking Light on the menu. Turn the Intensity knob on the left control pad until the Auto Exposure time is ~1 s. Click on Start Acquire to get a final image.
4. Construct three-dimensional EM models using the open-source image processing, modeling, and display (IMOD) package.

7. Antibody staining

1. Wash the dissected ovaries (step 2.16) in a 1.5 mL microfuge tube with 1 mL of 1x PBS supplemented with 0.5% Triton X-100 (PBST). Place the tubes on a shaker for agitation at 40 rpm for 10 min. Let the tubes stand in a rack and the ovaries sink by gravity. Replace the wash with fresh PBST, and repeat twice.
2. Digest the tissues with 2 µg/mL of Proteinase K and 0.1% sodium dodecylsulfate for 10 min at room temperature in PBST.
3. Wash the digested ovaries in PBST three times for 10 min each wash.
4. Incubate the ovaries with 10% horse serum in PBST for 1 h at room temperature.
5. Dilute primary antibodies 1 to 100 in the blocking solution (10% horse serum in PBS with 0.5% Triton X-100). Incubate the ovaries with primary antibodies overnight at 4 °C with gentle shaking.
6. Wash the ovaries in PBST three times for 10 min each wash.
7. Incubate the ovaries with secondary antibodies overnight at 4 °C with gentle shaking. Dilute all secondary antibodies 1:300 in the blocking solution.
8. Wash the ovaries in PBST three times, ensuring that the first and third washes last for 10 min. Stain the ovarian nuclei with Hoechst 33342 at 1:300 dilution in PBST for 30 min at room temperature in the second wash.
9. Mount the ovaries onto slides.
   NOTE: Anti-fading mountant can be used to prolong the fluorescence signals.

Representative Results

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 used does not provide a clear pigmentation distinction, we recommend using a different worm.

Next, trim away unrelated tissues in a step-by-step fashion (Figure 1C-E). Once the ovary is exposed (Figure 1E), the surrounding tissues can be trimmed away. The collected ovaries contain multiple somatic cell types and maintain ovary integrity and can be used for both ultrastructural analysis (Figure 2, left panel) and immunofluorescence staining (Figure 2, right panel)²¹. The method presented here provides details for TEM analysis and immunofluorescence analysis. The conditions can be adjusted as per individual antibody recommendations for antibody staining.

Figure 1: Locating ovaries with pigmentation patterns. (A) Ventral side of a sexually mature planarian. (B) Dorsal side of a sexually mature planarian. (C) Ventral side of the fragment with ovaries, after removing the anterior and posterior tissues. (D) The fragment after cutting in the midline of (C) and removing the dorsal half of the tissues; arrowhead: gut branch on the ventral half of the worm. (E) Fragment from (D) after removing the gut and other tissues sitting above the ovary. (F) The isolated ovary with oviduct attached. (G) Image from (F) after contrast and brightness adjustment. Red dashed lines: ovary and oviduct (or ventral nerve cord). Red arrows: ovaries. Blue dashed lines: outline of testes. (A-F) Images of the field view under a dissection scope without adjustment of contrast or brightness. A: Scale bar = 1 mm. Please click here to view a larger version of this figure.

Figure 2: Representative results. (Left) A transmission electron microscopic image of one oocyte. Magenta: nuclear envelope vesicles. Scale bar = 5 µm. (Right) A confocal image of one ovary. Magenta: nuclei stained with Hoechst 33342. Green: anti-histone H3. Scale bar = 20 µm. Please click here to view a larger version of this figure.

Discussion

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 µm to 50 µm. 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 subcellular resolution.

These procedures require fixation, which limits its application to PFA-based experiments. Other non-PFA fixation methods (e.g., methanol) may also allow for ovary dissection. If the given worm does not develop a reasonably sized ovary In gene perturbation experiments, traditional whole worm methods will likely be favored.

The most critical step in the protocol is to locate the ovaries properly and perform fine dissection. A sexually mature planarian with well-developed ovaries is expected to have lighter pigmentation on the ventral epithelium right beneath the ovaries. The area can range from 0.2 to 1 mm in size. The percentage of success in locating the ovary also relies on the culturing conditions of the worms. For an actively maintained, newly regenerated or matured stock, the percentage is high (~100%). The percentage can be low (10-50%) for a stock with worms of variable sizes or health conditions.The percentage of worms with matured ovaries in an experimental population must be evaluated before planning an RNAi experiment.

In summary, the most important strength of this method is to enable a comprehensive analysis of oocyte biology at cellular and sub-cellular resolution. Combined with RNAi-based gene expression perturbations^26,27,28, we expect that this method will allow for oocyte functional studies, including studies into oocyte regulatory mechanisms and other diverse oocyte biological processes.

Materials

Name	Company	Catalog Number	Comments
16% paraformaldehyde	Electron Microscopy Sciences	15710	EM grade
2% aqueous OsO4	Electron Microscopy Sciences	19152
50% glutaraldehyde	Electron Microscopy Sciences	16320	EM grade
Digital Micrograph	Gatan Inc.		Version 2.33.97.1, TEM data collection
Epon resin	Electron Microscopy Sciences	14120	Embed 812 Kit, liquid, epoxy resin
Ethanol	Ted Pella	19207	Denatured
Hoechst 33342	Thermo Fisher Scientific	H3570
Horse serum	Sigma	H1138
Lead Acetate	Electron Microscopy Sciences	6080564
MilliQ water			reverse-osmosis treated water
N-Acetyl-L-cysteine	Sigma	A7250
Parafilm	sigma	P7793
Prolong Diamond Antifade Mountant	Thermo Fisher Scientific	P36965
Propylene oxide	Electron Microscopy Sciences	75569	EM grade
Proteinase K	Thermo Fisher Scientific	25530049
Toluidine blue O	Electron Microscopy Sciences	92319
Transmission Electron Microscope	FEI		Tecnai G2 Spirit BioTWIN
Uranyl acetate	Electron Microscopy Sciences	541093