In Vitro Fertilization

The IVF protocol described here was provided by Carlisle Landel, The Jackson Laboratory, Bar Harbor, Maine 04609, and is based on methods described by Fraser and Drury (1975) and Sztein et al. (1997-2000); http://www.jax.org/resources/documents/cyro/ivf.html. A different method used by the Monash Institute is described in Comments, below.

Materials

Animals

  • Female mice
  • Male mice for fresh sperm collection or frozen sperm

Equipment

  • Centrifuge (optional)
  • Clean cage
  • Forceps, watchmaker’s #5
  • Heating stage or block at 37ºC
  • Hypodermic needles, 25- or 30-gauge, ½ inch
  • Incubator, humidified 37ºC, with 5% CO2, 95% air or 5% CO2, 5% O2, 90% N2
  • Organ culture dishes
  • Scissors, fine dissection
  • Syringe, 1-ml
  • Tissue culture dishes, 35-, 60-mm plastic center-well organ culture (e.g., BD-Falcon 35-3001, 35-3004, 35-3037)
  • Transfer pipettes consisting of mouth or hand-held pipette assembly and pulled capillary (see Chapter 4)
  • Warming pad
  • Water bath
  • Wide-bore pipette tips (e.g., Rainin HR-250W, 1000W)

Reagents

  • Human chorionic gonadotropin (hCG)
  • Human tubal fluid (HTF) medium (as described above)
  • KSOM-AA medium (see Chapter 4)
  • Paraffin oil, pre-gassed embryo-tested light (e.g., Sigma M8410)
  • Pregnant mare serum gonadotropin (PMSG)

Procedure

Day 1
Inject oocyte donors with PMSG 48 hours before hCG regardless of the light cycle in the animal room. Injections are done according to the planned oocyte collection, and insemination should be as close as possible to 13-14 hours post-hCG injection (e.g., hCG injection at 8 p.m., insemination at 9 a.m. the following morning).

Day 3

  1. Inject oocyte donors with hCG 48 hours after PMS injection.
  2. Prepare the fertilization and culture dishes, as listed below, on the afternoon of the day prior to the experiment. The medium should be covered with pre-gassed embryo-tested light mineral oil; 35-mm (e.g., BD-Falcon 35-3001) or center-well organ culture dishes (BD-Falcon 35-3037) are convenient for this step.
  • Fresh sperm dish: 1 ml of HTF.
  • Egg collection dish: 1 ml of HTF.  HTF-HEPES can also be used for this purpose.
  • Fertilization dish (1 per three females): 250 or 500 ųl of HTF.
  • Wash dish (1 per each fertilization dish): 5 drops x 250 ųl of HTF in a 60-mm dish.  It is also convenient to use the same 60-mm dish for both fertilization (central drop) and washes (surrounding drops).
  • Culture dish (1 per each fertilization dish): 5 drops x 250 ųl of KSOM-AA in a 60-mm dish.  It is also possible to use smaller drops in a 35-mm dish.

Day 4
If fresh sperm is used:

Fresh sperm must be capacitated by incubating the concentrated suspension at 37ºC for at least 1 hour (the time may vary between strains) before insemination.

  1. Sacrifice the male about 12-13 hours after the females were injected with hCG.
  2. Immediately dissect out the cauda epididymides and vasa deferentia, removing as much fat and blood vessels as possible (see Fig. 6.2A in Chapter 6). Place them in the fresh sperm dish. Mince the cauda making 507 slashes with a 30-gauge needle on a syringe. Using forceps, gently squeeze out the sperm from the vasa deferentia. Minimizing the trauma by making a single slit instead of mincing may improve the results.
  3. Gently shake the dish with the sperm suspension for 30 seconds (optional) and place the dish in the incubator for at least 1 hour for capacitation. Using an aliquot of diluted sperm suspension, assess the motility and determine the sperm concentration with a hemocytometer. It is necessary to obtain a final motile sperm concentration of 1 x 106 to 2.5 x 106 sperm/ml for fertilization.

If frozen/thawed sperm is used:
Freezing/thawing of mouse sperm (Chapter 15) results in capacitation-like changes, and therefore preincubation of frozen/thawed sperm is unnecessary (Fuller and Whittingham 1996).

  1. Remove the cryovial with frozen sperm suspension from liquid nitrogen and place it into a water bath at 37ºC until the ice crystals are melted (~2 minutes).
  2. Optional:  Centriguge the sperm sample at 735g for 4 minutes.  Carefully add 50 ųl of HTF medium and very carefully flick the tube to resuspend the sperm pellet (do not pipette)).
    Although it is beneficial for embryo development to remove any cryoprotectant, centrifugation may reduce sperm viability in some cases. This step is no longer used by The Jackson Laboratory.
  3. Evaluate the morphology and motility of the sperm and immediately use for IVF.

In Vitro Fertilization:

  1. Add an aliquot of fresh or thawed sperm to each fertilization dish using wide-bore pipette tips (usually 10 ųl, or more if the concentration is low).
  2. At 13 hours post-hCG administration, sacrifice three females. Quickly dissect out oviducts and place them into a drop of HTF medium. Tear the ampullae to release cumulus masses as described earlier (Collecting Zygotes, Protocol 4.9). Transfer cumulus masses from all three females to a single fertilization dish using a wide-bore pipette tip. Repeat until oocytes from all donors are collected. Then distribute the oocytes among all fertilization dishes.
  3. Incubate the fertilization dishes at 37ºC, 5% CO2, 95% air or 5% CO2, 5% O2, 90% N2 for 4-6 hours.
  4. Remove the dishes from the incubator, and wash the oocytes through several drops of HTF medium to remove the excess of sperm and debris. At this time, it should be possible to observe the presence of two pronuclei and the extruded second Polar body in fertilized oocytes.
  5. Although washed fertilized oocytes from nonblocking strains may be left to culture overnight in fresh HTF microdrops, it is recommended to use KSOM supplemented with amino acids for culture of embryos from all strains, especially from those exhibiting two-cell block in HTF medium. It is important to wash embryos through several drops of equilibrated KSOM-AA medium before transferring them into the final microdrop for overnight culture.


Day 5

  1. Count the two-cell stage embryos in the morning.
    Apparent two-cell stage embryos can develop overnight due to parthenogenetic activation or fragmentation of unfertilized oocytes (see Fig. 4.8, p. 200). Therefore, fertility rates based on the number of two-cell stage embryos must be interpreted with caution. Transfer two-cell-stage embryos to KSOM-AA microdrop dishes (if this was not already done the previous day) for culture to later stages. Alternatively, transfer two-cell-stage embryos to the oviduct of 0.5- dpc pseudopregnant recipient females as described in Protocol 6.3.

Comments

The use of oocytes without cumulus cells may facilitate fertilization using frozen/thawed sperm (Luis Gabriel Sanchez-Partida and Alan Trounson, Monash Institute of Reproduction and Development, Clayton, Australia, pers. comm.).
-Add 2 ųl of frozen/thawed sperm suspension to 20 ųl of equilibrated drops of fertilization medium (HTF or mT6; see Tables 14.1 and 14.2) under oil.
-Transfer 10 cumulus-denuded oocytes (see Chapter 4) into each drop and incubate as described above for 3-4 hours.
-Wash the oocytes into culture medium (e.g., KSOM) and return them to the incubator for subsequent development.