Rapid Isolation of Stage I Oocytes in Zebrafish Devoid of Granulosa Cells

This research is focused on establishing a process for rapidly and efficiently isolating pure stage I oocytes in zebrafish while eliminating granulosa cell contamination. The primary aim is to develop a method that facilities more precise analysis in oocyte-specific research areas, particularly in genome and epigenetic research. The oocytes of zebrafish are enveloped by a monolayer of granulosa cells.

It's difficult to sharply separate granulosa cells from oocytes due to the significant disparity in both number and volume, making it challenging to obtain pure oocyte samples for specific analysis, especially in genome-related studies. This method has several advantage over mechanical methods and the previous detection approaches. First, the improved detection buffer is gentler, helping to isolate oocytes and the granulosa cells while minimizing damage to those cells.

Plus, it allows us to get plenty of clean stage I oocytes. We hope this research helps scientists get a better look at the detail and growth of the fish oocytes, including things like epigenetics and genome structure. This method works just as well for pond loaches, and we think it could be applied to study a lot of different kind of fish.

To begin, place a euthanized female zebrafish on a table. Dab the excess water on the fish's surface with absorbent paper. With a pair of micro scissors, cut off the head of the fish along the posterior margin of the gill cover, then cut off the tail with the cloaca.

Transfer the middle trunk to a 35 millimeter-wide dish with two milliliters of L-15 medium. Now, cut the central axis of the abdomen and remove the viscera and swim bladder. Use a pair of tweezers to detach the bilateral ovaries from the abdomen.

Remove the adipose tissue, scales, and other body tissues attached to the ovary. To begin, transfer the excised ovaries of a zebrafish to a six-well plate containing two milliliters of cell dissociation solution. Incubate the ovaries at 28.5 degrees Celsius for two to three hours.

Add two to three milliliters of pre-warmed L-15 medium to the buffer to stop digestion. Next, place a 70 micrometer cell strainer into another well of the six-well plate. Add L-15 medium to the well, ensuring the medium level is higher than the strainer.

Now, use a pipette to draw the digestive medium through the cell strainer. Remove the excess medium with a pipette. Add four milliliters of fresh L-15 medium into the well and gently resuspend the oocytes.

After a couple of minutes, pipette out the supernatant. To select the oocytes for quality control, transfer them into a 35 millimeter-wide dish containing fresh L-15 medium. Observe the oocytes under a light microscope using a magnification of 10X or more.

Use a blunt injection tool to remove any cell fragments, stage I oocytes, or any other stage oocytes. For confirmation of the stage of growth, add Hoecsht 33342 to the L-15 medium containing the oocytes and incubate. Observe the oocytes with a fluorescence microscope under UV laser excitation.

Carefully pick out the oocytes that do not meet the desired criteria with a needle. The juvenile ovary showed an abundance of transparent stage one oocytes, accompanied by a smaller population of stage two oocytes. Adult fish ovaries showed a predominance of opaque late stage two to three oocytes.

The reference method resulted in numerous stained granulosa cell nuclei on the surface of the oocytes, densely enveloping the oocytes. In contrast, the modified method allowed for the separation of stage one oocytes, devoid of granulosa cell nuclei staining.