| Determination of weight range of female donors that respond best to superovulation protocols
||Scales, dissecting kit, incubator, microscope
||Administer varying regimens of equine chorionic gonadotropin (eCG) followed by human chorionic gonadotropin (hCG)
|Syringes and needles
||Oocytes collected and scored
||Dissecting kit, incubator, microscope
||Number of unfertilized and 2-cell oocytes; percentage of fertilized oocytes
- Dissecting kit
- Speed vac drying system (Savant SpeedVac Plus, ThermoFisher Scientific, Houston TX)
- Freezer (-20°C) (Fisher Scientific, Houston TX)
- Freezer (-80°C) (Thermo-Forma, Marietta OH)
- 35-mm culture dishes (Fisher Scientific)
- 1.5-mL microfuge tubes (Fisher Scientific)
- Equine chorionic gonadotropin (eCG) (aka Pregnant mare serum gonadotropin) (National Hormone and Peptide Program (Harbor-UCLA Medical Center, Torrance CA)
- Human chorionic gonadotropin (hCG) (Sigma, St. Louis MO)
- Bacteriostatic sodium chloride (APP Pharmaceuticals, Schaumburg IL)
- Bacteriostatic water (Hospira, Lake Forest IL)
- Sterile saline
- M2 media (Sigma-Aldrich)
- Hyaluronidase (Sigma-Aldrich)
- KSOM mouse embryo medium (Millipore, Billerica MA)
- Mineral oil (Sigma-Aldrich)
Procedure: Determination of optimal weight range by strain for superovulation protocol
- Initial experiments for each strain involve determination of the weight range of female donors that respond best to the superovulation protocols used in the Parker-Thornburg lab.
- Online vendor weight tables are compared to estimate the weight range that corresponds to 3-, 4-, 5-, and 6-wk-old mice for each strain.
- eCG is routinely prepared by adding 1 mL 0.9% bacteriostatic sodium chloride to the original vial to obtain a stock solution of 2000 U/mL which is stored at -20°C until thawed for further dilution. 900 µL is added to 35.1 mL sterile saline to achieve a concentration of 50 U/mL; 1.2 mL of this solution is aliquoted into multiple microfuge tubes and stored at -80°C.
- hCG is routinely prepared by resuspending in bacteriostatic water to 2000 U/mL, aliquoted into 1.5-mL microfuge tubes at 25 µL per tube, lyophilized to dryness in a speed vac, and stored at -80°C. For use, 1 mL of 0.9% bacteriostatic sterile saline is added to the microfuge tube to make a 1 mL solution of 50 U/mL.
- Each donor mouse is injected (i.p.) with 5 IU eCG (0.1 mL). 5 IU hCG is injected i.p. into female mice 47 to 49 h after the eCG injection. Mice are manually restrained for the injections. Donor mice are then mated 1:1 immediately after the hCG injection to proven stud males of the same strain of optimal mating age (3 to 8 mo).
- The following day, donors are euthanized by cervical dislocation, weighed, oviducts collected, and oocytes pooled by donor weight and strain (except for BALB/cAnNCr which are collected and scored for each individual donor, see below).
- For all strains except BALB/cAnNCr, oviducts are collected from all female mice in a weight group and placed into 2 mL M2 media in a culture dish. Each oviduct is then moved to a dish containing 2 mL M2 media and 75 µL hyaluronidase (10 mg/mL) where ampulla are torn open to release the oocytes.
- After all oviducts for a group have been processed, all oocytes are collected and placed into a 100-µL drop of KSOM under embryo-tested mineral oil that has been equilibrated to 37°C at 5% CO2.
- Oocytes are allowed to incubate for 24h, after which the drop is scored for the number of unfertilized and 2-cell oocytes.
- For BALB/cAnNCr, oviducts from individual female donors are opened in a 50-µL drop of KSOM and incubated overnight at 37°C at 5% CO2 prior to scoring as described above.
- The most responsive weight range is chosen based initially on the range that gives the highest number of fertilized oocytes, using the highest number of total oocytes as the second criterion.
- B6(Cg)-Tyrc-2J/J: 4 wks of age, 10-13.7 g
- B6D2F1Hsd: 3 wks of age, 6-9.9 g
- BALB/cAnNCr: 4 wks of age, 10.1-14.8 g
- C57BL/6NHsd: 4 wks of age, 10.5-14.2 g
- Crl:CD-1(ICR): 6 wks, 23.5+ g
- FVB/NHsd: 5 wks of age, 14.5-16.4 g
Procedure: Superovulation dosage and timing
- Upon receipt from the vendor or after weaning, female mice are allowed to acclimate for 2 d prior to weighing.
- Female mice begin the superovulation protocol 2-3 days after achieving the correct weight (as determined above).
- An eCG dose at 2.5 IU, 5 IU, or 5 + 5 IU (1 wk interval) is administered as described above. hCG at a 5 IU dose is injected 47 to 49 h later into each donor. Note that B6(Cg)-Tyrc-2J/J donors are injected with 2.5 + 2.5 IU eCG, not 5+ 5 IU. Data for 2.5 + 2.5 IU are available in Luo et al., 2011.
- Donors are immediately mated to proven stud males of the same strain.
- The following day, donors are euthanized by cervical dislocation, oviducts collected, and oocytes processed as described above. Scores from individual mice (not pooled) are collected.
- Total number of oocytes per female for each dose
- Total number of fertilized oocytes per female for each dose
- Percentage of total oocytes that are fertilized per female for each dose
Data presented are means +/- SE