Defolliculation of Oocytes
Zebrafish were killed by decapitation and ovaries were extracted and immediately
placed in 5 mL L15 media in 6cm x 1.5cm Petri dishes. Samples were gently pipetted to
separate oocytes so that they could be divided according to stage of development.
Oocytes were separated as stage I-II, stage III or stage IV using a stereomicroscope, and
placed in separate petri dishes for defolliculation treatment.
60% L15 media was removed from the Stage I-II, Stage III and Stage IV samples and
replaced with 5mL 0.001% collagenase (Sigma-Aldrich) in Ca2+-free Ringer’s solution
(116mM NaCl, 2.9mM KCl, 5mM HEPES, pH 7.2). All samples were shaken at 60 RPM
for one hour and fifteen minutes. Additionally, the samples were gently pipetted for three
minutes during each ten minute period. The stage III and stage IV oocytes were pipetted
with a 9” disposable Pasteur pipette (Fisher Scientific), while the stage I-II sample was
pipetted using a pipette with a smaller (40 µm) opening. The smaller pipette was made by
heating a pipette tip over a Bunsen burner and stretching the glass to create a narrower
tip. During the one hour and fifteen minute time period, free stage IV follicular layer was
verified under a stereomicroscope and collected by pipette after each pipetting period.
After the one hour and fifteen minute time period, the follicular cells were carefully
removed from Ringer’s solution using a pipette while viewing underneath a
stereomicroscope to ensure that no oocytes were collected. The follicular cells were
placed in separate 1.5 mL microcentrifuge tubes and centrifuged for 1 minute at 13000
RPM (Eppendorf Centrifuge 5415D) to collect the follicle cells. The supernatant was
then discarded.
The stage I-II, stage III and stage IV oocytes collected above were washed twice in
Ringer’s solution to remove any left over follicle cells. Next, they were stained by the
addition of 1 µL of propidium iodide (BRAND) in 3 mL of Ringer’s solution into the
petri dishes for a period of 15 minutes. The samples were washed twice in Ringer’s
solution and the oocytes were examined under the fluorescent inverted microscope
(Axiovert 200M, Zeiss) to verify the removal of the follicular layer of cells from the
oocytes. Stage I-II, stage III and stage IV completely denuded oocytes (100% removal of
follicular layer) were selected off and placed into new 1.5 mL microcentrifuge tubes.