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.
