Retrieval of Mouse Oocytes

Summary

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

Abstract

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology. Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system. A primary goal has been to optimize oocyte/early embryo retrieval.

Protocol

Embryo/Oocyte retrieval

1. At least two hours prior to retrieval of embryos/oocytes, KSOM is placed in the 37ºC CO₂ incubator and FHM must be thawed from -20°. A glass dish containing three concave shallow wells is used to house drops of KSOM in the incubator. To prevent dehydration, the KSOM and dish are placed in a lidded container that has water at the bottom. This apparatus prevents evaporation without the use of mineral oil.
   
   
2. The oviducts from an ovulating female are placed in FHM media. To ensure orientation and an intact infindibulum, a small section of the ovary is cut along with the oviduct and small section of the uterus. A blunt 30 gauge needle is used to expel embryos/oocytes from the oviduct by inserting the blunt needle into the infindibulum. The infindibulum must be used to retrieve the embryos since the oviduct tubing is too small for a needle to pass. In the event that the infindibulum is destroyed, the oviduct must be either sliced open or embryos squeezed out.
   
   
3. Embryos/oocytes are found in the FHM media by focusing the microscope on the fat droplets which have sunk to the bottom of the Petri dish. Embryo/oocyte isolation can be achieved at 20x as this provides a larger viewing area than high magnification. Embryo/oocytes look like round fat droplets that have a clear ring around it. Mouth pipetting, using a pulled glass capillary, is used to move embryos/oocytes to KSOM for incubation and further manipulation.

Procedure for Oocyte Retrieval

Preparation

1. Thaw FHM and prepare kSOM.
2. A few hours earlier, place kSOM in egg retrieval container and place in tip box with water in the bottom, place in CO₂ incubator.
3. Bring cages to the lab and kill the harvesting females.
4. Cut out the oviduct being careful to cut some of the ovary and some of the uterus to ensure that you do not cut off part of oviduct.
5. Place in FHM media in small Petri dish.
6. Pour a couple more FHM media Petri dishes to be used for flushing.

 

Flushing

1. Use a flushing needle (30 gauge blunt) connected to a 1ml needle with spiral end.
2. Take one oviduct at a time and using needle, insert into infundibulum found near the part of ovary left attached. If the infundibulum is destroyed and cannot be used, then take two forceps and hold one end of oviduct. With the other forcep, gently squeeze, like a toothpaste tube, to eject any embryo's.
   
   Note: This will create a large amount of mess in the Petri dish, as a lot of tissue will come with it, so you may want to use a different Petri dish afterwards.
   
   
3. To see embryos, focus microscope on fat pieces and look for round "fat piece" with clear circle around it.
   
   Note: The embryos have the same look as the fat, so it may be hard to find. The embryos sink to the bottom, so look there.
   
   
4. Do vaginal smears of recipient females. At this stage, they should be pseudopregnant.

Discussion

In this article, we demonstrate the retrieval of oocytes from ovulation-induced (superovulated) Peromyscus. The use of superovulation (as opposed to natural estrous cycle) is common due to the much greater resulting numbers. However, caution must be taken in interpreting results utilizing such oocytes/embryos, as both the hormones used to induce ovulation and culturing may have effects.

When estrous has been confirmed, females are euthenized and oviducts recovered along with a small porti...

Materials

Name	Company	Catalog Number	Comments
Flushing needle	Tool	Zephyrtronics	ZT5-130-L	30 G blunt
Forceps (2)	Tool	Roboz Surgical Instruments Co.	RS-5060
Peromyscus	Animal	Peromyscus Genetic Stock Center
Scissors	Tool	Roboz Surgical Instruments Co.	RS-5880
FHM HEPES Buffered Media	Reagent	Specialty Media	MR-025-d
KSOM Embryo Culture Medium (5x 10 mL)	Reagent	Specialty Media	MR-020P-5F
Mouth Pipette	Tool			Pulled from glass capillaries and attached to rubber tubing
Oocyte holding dish	Tool			Glass container with concave divets