EMMA SOPs - In vitro fertilization

Equipment

Culture dishes
Media: HTF, MEM, M16, KSOM + AA, PBS-BSA
Mineral oil
Finely drawn pipettes for oocyte handling
Dissecting instruments
CO2 incubator

Method (see end of protocol for suggested timetable of events)

72 hours before collecting oocytes:

Inject 20-25 days-old females with 2.5-10 IU PMSG (carry out treatment at 6.00-7.00 pm); actual hormone dose and animal age depending on mouse strain; e.g.: 5.0 IU PMSG for C57BL/6J, 21 days-old.

48-52 hours post-PMSG injection:

Inject 20-25 days-old females with 2.5-10 IU hCG (carry out treatment at at 7.00-8.00 pm) actual hormone dose and animal age depending on mouse strain; e.g.: 5.0 IU hCG for C57BL/6J, 21 days-old. During the same day, also prepare an appropriate number 35x10 mm of petri-dishes for the IVF and embryo culture.

Three types of dishes will be needed:

Oocyte harvest dish
Sperm dispersal dish
Fertilsation/Wash/Culture dish

All dishes must be stored in the 37^oC in a CO2 incubator overnight.

Preparation of oocyte harvest dishes

Add approximately 2-3ml of the HTF (with Pen/Strep/BSA) to a numbered 35mm Petri Dish (Falcon 351008). One dish is required for every five-ten females of the donor stock.
Place the dishes in the incubator overnight at 37°C, in 5% CO2 in air to equilibrate.

Preparation of Fertilisation/Wash/Culture dishes

Prepare one 60mm Petri Dish (Falcon 353004) for every five-ten females of the oocyte donor stock.
See diagram below: Into each combined Fertilization /Wash/Culture dish, carefully pipette 5 drops of HTF (with Pen/Strep/BSA) as follows:
- 1 x 500µl for fertilization
- 4 x 150µl for washing and overnight culture
Carefully overlay the drops with Mineral Oil (Sigma Chemical Co.; Cat. No. M8410, embryo culture tested) ensuring that they do not run together.
Equilibrate the dishes overnight at 37°C, in 5% CO2 in air.

Preparation of sperm dispersal dish (for freshly harvested sperm)

Pipette 1000 µl HTF into the centre of a 60mm Petri Dish (Falcon 351016).
Overlay with Mineral oil and equilibrate overnight at 37°C, in 5% CO2 in air.

Preparation of sperm samples:

Freshly harvested sperm
1. The selected male should be at least 8 weeks old, and not have been used for mating for at least 3 days before sperm collection.
2. Sacrifice the male and dissect the cauda epididymides and vasa deferentia.
3. Place on a clean paper tissue, and remove as much adipose and vascular tissue as possible, using size 5 watchmakers' forceps under the dissecting microscope lit from above. Work quickly to prevent the material from desiccating.
4. Place the material into the sperm dispersal drop and nick the surface of the cauda epididymides with the needle of a 0.5ml insulin syringe. Gently palpate the tissue to expel the sperm.
5. Extrude the sperm from the vasa differentia by "walking" along them with two pairs of watchmakers forceps. Remove the tissue from the drop.
6. Allow the sperm to disperse into the medium for approximately 10 minutes at 37ºC in the CO2 incubator.
7. Place IVF dishes on a heated stage or hot pad, then pipette 5-10µl of the sperm suspension into each fertilization drop and return the dishes to the incubator until the oocytes are harvested.
Cryopreserved sperm
1. Using forceps, hold the cryotube in air for 30 seconds. If liquid nitrogen is present in the cryotube, wait for it to evaporate and escape by rolling the cryotube around on the bench.
2. Take special care that the cryotube is not filled with liquid nitrogen before plunging into the water bath (such tubes may explode).
3. Thaw the sperm sample rapidly by placing in a 37oC water bath.
4. Once thawed, pipette 10-20µl of the sperm suspension into each fertilization drop and return the dishes to the incubator.

Harvesting oocytes

Dissect the oviducts from three superovulated female mice and place into a preincubated dish of HTF (for superovulation methods, see the protocol for harvesting and cryopreservation of in vivo derived embryos and the table below).
Under a dissecting microscope, hold each oviduct down with forceps and gently tear the swollen ampulla with a second pair of forceps to release the cumulus masses into the HTF then remove the oviduct from the dish.
When all the cumulus masses have been extracted, gently draw them up into a wide bore pipette tip in a maximum of 30µl HTF.
Transfer the cumulus masses to a fertilisation drop (containing sperm) being careful to transfer as little HTF as possible.
Incubate the dishes at 37°C, in 5% CO2 in air for approximately 5-7 hours to allow fertilization to occur.
Repeat steps 1-5 for each fertilisation dish in succession (i.e. complete all of the steps from collecting the oviducts to returning the fertilization dishes to the incubator for one batch of females before starting the next batch). Aim to take no more than 5 minutes from collecting the oviducts to returning the fertilization drop (including oocytes) to the incubator.

Washing and culturing the fertilized oocytes

Between 5-7 hours after the cumulus masses were placed in the fertilization drop, remove all of the oocytes found in the drop and place in wash drop 1 (see diagram).
Move the oocytes from wash drop 1 to wash drop 2. Removing as much cellular debris as possible in the process.
Divide the washed oocytes approximately equally between the two culture drops (3 and 4).
Incubate overnight at 37°C, in 5% CO2 in air.
Next morning, separate the 2-cell embryos from those which have not fertilized or have degenerated in preparation for cryopreservation, embryo transfer or further in vitro culture in M16 or KSOM.

Timetable of events for IVF

Day -3 (e.g. Saturday)	Day -1 (e.g. Monday)	Day 0 (e.g. Tuesday)	Day 1 (e.g. Wednesday)
Superovulate between ten and thirty 3-4 week old females by injecting PMSG during the afternoon (6.00- 7.00pm).	Prepare dishes for oocyte harvest, fertilization/wash/ culture and sperm dispersal (is using freshly harvested sperm)	7:00am Thaw cryopreserved sperm sample, or collect and disperse freshly harvested sperm.	Morning: score the IVF success (2-cell vs others).
	Induce ovulation in the females by injecting hCG at 7:00pm.	08:00-09:00 Harvest oocytes and place into diluted sperm preparation (12-14 hours after oocyte collection).	Prepare the 2-cell embryos for cryopreservation, embryo transfer or culture.
		15:00 Wash the presumptive zygotes and place into culture drops.

This timetable assumes that the mice are exposed to 12 hours of darkness between 19:00 and 07:00.