Collecting Zygotes and Removing Cumulus Cells with Hyaluronidase

Materials

Animals

  • Pregnant female mice sacrificed humanely

Equipment

  • Embryo-handling pipette consisting of mouth or hand-held pipette assembly and pulled capillary
  • Forceps
  • Forceps, watchmaker’s #5, two pairs
  • Microdrop culture dishes
  • Needles, 26-gauge (optional)
  • Organ culture dish (Falcon 3037) (optional)
  • Petri dishes, 35-mm, or embryological watch glasses
  • Scissors, fine
  • Stereomicroscope with transmitted and reflected or fiber optics (optional)
  •      illumination (preferably a ground-glass stage) with 20x and 40x magnification

Procedure

  1. Open the abdominal cavity as described above.  Grasp the upper end of one of the uterine horns with fine forceps and gently pull the uterus, oviduct, ovary, and fat pad taut and away from the body cavity. This will reveal a fine membrane (the mesometrium), which connects the reproductive tract to the body wall and carries a prominent blood vessel.  Poke a hole in the membrane close to the oviduct with the closed tips of a pair of forceps or scissors.
  2. Pull the oviduct, ovary, and fat pad taut with fine forceps and cut between the oviduct and ovary with fine scissors.  Do not be afraid to go close to the oviduct.  Reposition the forceps and cut the uterus near the oviduct.
  3. Transfer the oviduct and attached segment of uterus to a 35-mm Petri dish or embryological watch glass containing M2 medium at room temperature.  Oviducts from several mice can be collected in the same dish.
  4. Newly ovulated oocytes, surrounded by cumulus cells, are found in the upper part of the oviduct (ampulla), which at this time (12 hours postovulation) is much enlarged.  The fimbriated end of the oviduct (infundibulum) is also swollen during ovulation and can easily be located under 20x magnification in the stereomicroscope.
  5. Transfer one oviduct at a time into another 35-mm Petri dish containing hyaluronidase solution in M2 medium (~0.3 mg/ml) at room temperature (or 37ºC) and view through the stereomicroscope at 20x or 40x magnification.
  6. Use one pair of watchmaker’s forceps to grasp the oviduct next to the swollen infundibulum and hold it firmly on the bottom of the dish.  Use another pair of watchmaker’s forceps or a 26-gauge needle to tear the oviduct close to where the zygotes are located, releasing the clutch of cumulus cells.  If the zygotes do not flow out by themselves, use the forceps to push them out by gently squeezing the oviduct.  If they stick to the outside of the oviduct, allow the oviduct to sit for several minutes in the hyaluronidase solution.  Zygotes will be released as the digestion removes the sticky cumulus cells. If the zygotes stick to the forceps, simply lift the forceps out of the Petri dish and they will be retained by the surface tension of the medium and will fall back to the bottom of the dish.
  7. Allow the zygotes to incubate in the hyaluronidase solution for several minutes until the cumulus cells fall off. If necessary, pipette them up and down a few times, but do not leave them in the hyaluronidase solution for more than a few minutes after the cumulus cells are shed, because this may be harmful.  Although ~0.3 mg/ml solution of hyaluronidase is usually recommended, more concentrated hyaluronidase solutions of 0.5-1 mg/ml that require a shorter incubation time of less than a minute may also be used.
  8. Use pipettes to pick up the zygotes and transfer them to a Petri dish containing several drops of fresh M2 medium to rinse off the hyaluronidase solution, cumulus cells, and debris.  Then transfer the zygotes to a microdrop culture dish, rinse through several drops of equilibrated medium, and keep at 37ºC, 5% CO2 until needed.  An organ culture center will dish (Falcon 35-3037) with equilibrated embryo culture medium may be used as an alternative for short-term culture, i.e., before microinjection.