Nuclear Migration in the Drosophila Oocyte

This technique makes it possible to observe long biological processes in dissected Drosophila egg chambers, such as oocyte nuclear migration, using live imaging microscopy. This protocol describes how to maintain the dissected egg chambers alive for 12 hours to perform live imaging microscopy. To begin, pipette 200 microliters of Schneider Medium and supplement with 30 microliters of 10 milligrams per milliliter insulin, then add four microliters of heat inactivated fetal calf serum.

Use a pipette tip to apply a small amount of silicone grease all around the hole on the underside of the punctured slide. Position a coverslip, and use the wide end of a pipette tip to apply pressure on the coverslip to flatten the silicone grease for creating a silicone ring interior to the slide. Transfer the anesthetized female flies to the dissecting well containing 150 microliters of the imaging medium.

For dissecting the female, use the forceps to grab the thorax and pinch the dorsal abdomen cuticle with a second pair of forceps. Carefully remove the uterus, oviduct, and muscle sheath. Isolate and detach the pair of ovaries visible upon the cuticle opening.

Place a drop of 10 to 15 microliters of the imaging medium on the ovaries, and transfer one ovary to the imaging chamber. For separating the ovarioles, use the needle to hold the posterior end of the ovary. Use another needle to carefully pull the germarium for teasing the ovarioles apart.

Hold the sheath with one needle and pull the ovariole through the larger chambers with another needle to remove the remaining muscle sheath on the egg chambers. Allow the unsheathed ovariole to sink and contact the coverslip. Remove the late stages and rest of the ovaries from the microchamber using forceps.

Use needles to carefully distance the ovarioles to facilitate the acquisition. Cut a small 10 by 10 millimeter square of the permeable membrane. Carefully apply the membrane on the top of the imaging medium to expel any air bubbles.

Then hermetically seal the chamber with a thin layer of halocarbon oil around the well on the contour of the membrane. Place the imaging setup on the slide holder of the inverted microscope and use a 40X objective. The migration of the oocyte nucleus of a wild type egg chamber was captured using live imaging microscopy.

The nucleus reaches either the anterior plasma membrane or the lateral plasma membrane before sliding along the trajectories to reach its final position at the intersection of the two plasma membranes. Membrane deformities, disorganized follicular cells, stunted cells, and shrunken nuclei are the first signs of dying egg chambers. The degenerated oocyte before eight hours of imaging is not usually detected.

However, rare cases of early degeneration are due to problems during the dissection or mounting steps. Damaging the egg chambers compromises their survival. So delicate hand precise dissection and preparation of the microchamber are paramount.

This procedure allowed us to visualize that the oocyte nucleus can take three different trajectories during its migration and different organelles regulate these trajectories within the oocyte.