Retrieval of Mouse Oocytes
April 28th, 2007
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.
My name is Amanda dli. I'm from the laboratory of Dr.Paul Verana at the University of California Irvine. Today I'm gonna be showing you the procedure for oocyte retrieval.
This entails isolating the videx from the female. Once the ovex are isolated, we'll be injecting FHM media through the videx, and then cytes should be exiting from the uterus. Once this is done, we'll isolate the cytes in the media and use this for future experiments.
What you might wanna use this procedure for in addition to isolating cytes, is isolating embryos at specific time points. One reason you might wanna see this procedure in the video format is because the different parts of the oviduct can be very hard to recognize. In this video, I will be pointing out the infundibulum where you will inject the FHM media.
The dissecting tools that you'll be using are two sets of sharp forceps and one pair of sharp scissors. The ova ducts that were taken from the female will be placed in FHM media, which is in a 35 millimeter tissue culture dish. The eggs will be retrieved using a 30 millimeter blunt end needle attached to a one milliliter syringe, and the uc will be stabilized with a sharp forcep.
FHM is a holding media used to harvest the cytes. KSOM is the media used to have the cytes grow within the incubator if they become fertilized. Once the eggs and embryos have been retrieved from the oviduct, we will use a glass transfer pipette that has been pulled from a capillary tube, attaches some tubing and mouth pipet those into some holding media.
A glass concave dish is used to hold the embryos and eggs. They can be placed in each concave circle so that they're in a discreet location, which makes them easier to find. This is placed into this container so that it can be put into a CO2 incubator without using oil.
On top of the FHM media or KSOM, depending on the media, water is put in the base, the tap is closed and the liquid stays in place. 70%ethanol is used to ensure the hair does not get in away. We'll make a small incision in the abdominal region and incision in the abdominal cavity.
We'll enlarge a cut to expose the abdominal region. Once this is done to expose the uterus, we'll move the intestines by the way, and then the uterus is quite, is below. The uterus is Y shaped and they rot wildly taking part of the uterus.
When it's that is cut, the oviduct is looks like spaghetti. I'll be trying to take part of the oviduct. I'll take the UCT and part of the ovaries, which is encased in fat.
The other side is gonna be taken At this point, again, we're gonna pick up the ovary or the uterus. Pick up the uterus here, cut below our attachment point. The vid ducts are found at this point.
I'll be taking part of the OV ovary with us. This is then gonna be placed in media. The uterus is still contracting.
The uterus is made up of smooth muscle, so even though it's detached from the body, the neurons are still firing. The uterus is shown here. While the OV duct is looks like spaghetti and the ovary is above that, the OV duct is not actually attached to the ovary.
While we have only show a portion of the ovary at this point, my dominant hand is my right hand in that I hold the 30 gauge blunt end needle. I use that as a forcep to isolate the in fibula. Once that is isolated, I will inject the blunt and needle into the in fibula and inject some liquid which will flow out of the uterus.
My left hand holds sharp forceps. This is used to isolate the in fibula and hold everything in place. The am dula is a trumpet shaped isolated here.
The tip of my needle is in the in fibula, which was pointing through the layer of tissue.Okay. To ensure that I've isolated all of the embryos and eggs, I like to squeeze like a tube of toothpaste on the uterus. Once you focus on the fat pads, you look for what looks like droplets of fat that are perfectly symmetrical.
There's one right now just to the left of that long fat. That's one a single egg. So what I'm gonna do is take the mouth, pipet, pick up the egg, put it into some more holding media so that we can isolate the eggs themselves.
Today I've shown you how to isolate cytes from the female reproductive tract. In addition, I've shown you the different structures of the oviduct. In particular, the infundibulum, which is the only structure in which you can inject the FHM media.
With a me a 30 gauge needle. With this technique, I've isolated cytes and any embryos that have been present in the female reproductive track. I can use these for gene expression studies Or transgenic work.
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Overview
This article demonstrates a detailed protocol for the retrieval of oocytes and early embryos from female Peromyscus, a genus of rodents. The procedure is designed to optimize the collection of these reproductive cells for downstream applications such as gene expression studies and transgenic research.
Key Study Components
Area of Science
- Reproductive biology
- Developmental biology
- Rodent model systems
Background
- Few studies have successfully manipulated Peromyscus embryogenesis or reproductive biology.
- Optimizing oocyte and embryo retrieval is essential for advancing genetic and developmental studies in this model.
- Accurate identification of reproductive tract structures is critical for successful cell isolation.
- The Peromyscus Genetic Stock Center collaborates to refine these protocols.
Purpose of Study
- To provide a reproducible method for isolating oocytes and embryos from female Peromyscus.
- To facilitate further research in gene expression and transgenic techniques using these cells.
- To clarify anatomical landmarks and technical steps for effective cell retrieval.
Methods Used
- Dissection of the female reproductive tract to expose the uterus and oviducts.
- Use of sharp forceps and scissors for tissue isolation.
- Injection of FHM media into the infundibulum of the oviduct using a 30-gauge blunt-end needle and syringe.
- Collection of oocytes and embryos into holding media using a glass transfer pipette and mouth pipetting technique.
- Placement of retrieved cells into a glass concave dish for incubation or further analysis.
Main Results
- Successful isolation of oocytes and embryos from the oviduct and uterus of female Peromyscus.
- Clear identification of key anatomical structures, such as the infundibulum, for precise media injection.
- Demonstration of techniques to maximize retrieval efficiency, including gentle manipulation and visual cues.
- Preparation of cells for downstream applications, such as gene expression analysis or transgenic work.
Conclusions
- The described protocol enables reliable retrieval of oocytes and embryos from Peromyscus females.
- Mastery of anatomical identification and technical steps is crucial for reproducibility.
- This method supports advanced genetic and developmental studies in Peromyscus models.
