1. Day 0, 1 pm: Female mice 4-5 weeks of age, 0.1 ml PMSG i.p. (pregnant mare serum gonadotropin 50 IU/ml PBS).
2. Day 2, 11 am: (46 hours later*): 0.1 ml HCG i.p. (human chorionic gonadotropin 50 IU/ml PBS).
3. Each superovulated female may be placed immediately with a single fertile male.
4. Day 3, early am: check copulation plugs (P 0.5). (Unmate nonplugged females. Rest for 7 days any males that plugged.)
5. Euthanize plugged females (CO₂ narcosis followed by thoracotomy). Dissect oviducts. flush oocytes/cumulus to 3 cm dish with 300 µg hyaluronidase/ml M2.
6. Wash serially through 3 dishes of M2 (w/HEPES) or M16 (w/o HEPES) medium. Store in 37C incubator until use.
   
7. Reimplant into both oviducts of anesthetized, naturally cycling recipient female mice (see detailed surgical protocol) mated the previous evening to vasectomized Swiss Webster males (purchased post-vasectomy from Taconic Farms).

* "It is said" that 3-4 week old females give optimal egg yield by the above protocol. However, we have also had good results with 6-10 week old females.

This protocol is designed for a 6 am-6 pm light cycle. Eggs produced display easily injectable pronuclei from at least 10 am to 3 pm, although this can vary somewhat from day-to-day. By delaying the administration of PMSG and HCG to 7 pm and 5 pm, respectively, and using essentially the same light/dark cycle, we have obtained eggs that are microinjectable from 6 pm to 10 pm.

Although the time of administration of PMSG can be varied, HCG should always be given 46 hr later (i.e. prior to endogenous gonadotropin release) for optimal egg yield.

* Proper timing of hormone injections wrt each other and wrt the light/dark cycle are critical for optimal superovulation. Do not alter these parameters unless you are specifically evaluating the effects of doing so.