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.
Embryo/Oocyte retrieval
Procedure for Oocyte Retrieval
Preparation
Flushing
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 portion of ovary. The small region of ovary is used as a marker for the location of the infindibulum. The infindibulum sits next to the ovary, often times projecting through a thin layer of tissue. On the other side of this tissue, the rest of the oviduct is coiled and eventually leads into the vagina. It is best to push fluid through the infindibulum to extract the oocytes. This direction is necessary due to a one way valve located at the vagina-oviduct interface. The oocytes may be harmed if they are projected backwards through the valve.
A 30-gaugle blunt end needle is inserted into the infindibulum. The blunt end needle will fit the infindibulum opening, but will not fit into the oviduct tubing. Therefore, care must be taken to ensure the infindibulum is not damaged during dissection or oocyte retrieval. If the infindibulum is damaged, the oviduct tubing can be squeezed via forceps to allow any trapped oocytes to exit.
Assuming the female was in estrous, the reproductive organs will have a red appearance due to increased blood supply and appear swollen. We observed differences in the two deer mouse species tested: P. polionotus had much fattier ovaries and oviducts than P. maniculatus, making oocyte retrieval more difficult.
Once the oocytes have removed from the oviduct, they may be observed by light microscopy at low magnification. Viewing the oocytes at a lower magnification allows the user to scan the Petri dish quickly. The microscope should be focused on the bottom layer of fat granules. The oocytes will look like perfectly round fat granules with a clear ring around the outside. To aid in detecting the oocytes, try adjusting the light projecting through the Petri dish. In addition, if the Petri dish is lightly shaken oocytes will not move compared to the fat droplets. These hints may help the novice researcher to locate the oocytes; however, it is likely that several trials will be necessary before one is comfortable with the procedure.
The isolated oocytes may be used in a variety of experiments including production of stem cell lines, production of chimeras, or genetic manipulations. While we have found considerable variation in the parameters and timing required to induce ovulation, the general techniques for retrieval should be applicable to many rodent species (deer mice are ~ 30 million years diverged from both laboratory mice and rats). We are particularly hopeful that this will benefit those working in novel systems where such techniques are not well-established.
I would like to thank Mike Dewey from the Peromyscus Genetic Stock Center for his help, patience and knowledge.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Flushing needle | Tool | Zephyrtronics | ZT5-130-L | 30 gauge blunt |
Forceps (2) | Tool | Roboz | RS-5060 | |
Peromyscus | Animal | Peromyscus Genetic Stock Center | ||
Scissors | Tool | Roboz | RS-5880 | |
FHM HEPES Buffered Media | Reagent | Specialty Media | MR-025-d | |
KSOM Embryo Culture Medium (5×10 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 |