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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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,

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.

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 .......

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 subce.......

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
16% paraformaldehydeElectron Microscopy Sciences15710EM grade
2% aqueous OsO4Electron Microscopy Sciences19152
50% glutaraldehydeElectron Microscopy Sciences16320EM grade
Digital MicrographGatan Inc.Version 2.33.97.1, TEM data collection
Epon resinElectron Microscopy Sciences14120Embed 812 Kit, liquid, epoxy resin
EthanolTed Pella19207Denatured
Hoechst 33342Thermo Fisher ScientificH3570
Horse serumSigmaH1138
Lead AcetateElectron Microscopy Sciences6080564
MilliQ waterreverse-osmosis treated water
N-Acetyl-L-cysteineSigmaA7250
ParafilmsigmaP7793
Prolong Diamond Antifade MountantThermo Fisher ScientificP36965
Propylene oxideElectron Microscopy Sciences75569EM grade
 Proteinase KThermo Fisher Scientific25530049
Toluidine blue OElectron Microscopy Sciences92319
Transmission Electron MicroscopeFEITecnai G2 Spirit BioTWIN
Uranyl acetateElectron Microscopy Sciences541093

References

  1. Brubacher, J. L., Vieira, A. P., Newmark, P. A. Preparation of the planarian Schmidtea mediterranea for high-resolution histology and transmission electron microscopy. Nature Protocols. 9 (3), 661-673 (2014).
  2. Carpenter, K. S., Morita, M., Best, J. B.

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PlanarianOvaryDissectionUltrastructural AnalysisAntibody StainingGerm CellsOocytesTransmission Electron MicroscopyImmunostainingRNA Interference

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