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, 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.
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 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.
1. Preparation
2. Collecting ovaries
3. Fixation for TEM
4. Sample processing for TEM
5. Ultramicrotomy
6. Data collection and analysis
7. Antibody staining
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.
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 perturbations26,27,28, we expect that this method will allow for oocyte functional studies, including studies into oocyte regulatory mechanisms and other diverse oocyte biological processes.
The authors have nothing to disclose.
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
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 |
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