Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

So it's difficult to separate the role of uterine and ovarian hormonal influences on pregnancy establishment as both are intimately linked. So this protocol allows us to directly examine the uterine contributions. While some of these techniques like ovariectomy are quite invasive, they are relatively simple to perform after practice and training, which makes them suitable for researchers that are new to mouse models.

So this method can be utilized to understand the broader regulation of the HPG axis as both the ovaries and the uterus play an important role in regulation of this axis. If you've never performed these techniques before, consult with your institute's veterinarian team for training and support. To begin, apply eye lubricant generously to an anesthetized mouse by gently dabbing it on the eye after shaving a small area at dent below the spine hunch.

Inject 0.1 milliliters of carprofen subcutaneously into the scruff of the neck. Test if the animal is anesthetized by pinching its back toe and checking for a reflex. Next, apply Betadine to the surgical site.

Then cover it with a surgical drape. Using rat-toothed forceps, pull away the skin at the hunch upwards, and make a five millimeter longitudinal incision. Then, using blunt forceps, dissect the skin away from the underlying muscle layer down to one side toward the kidney.

Identify the kidney, ovary, and ovarian fat pad through the muscle wall. Grab and lift the muscle layer with the forceps. Then, using scissors, make a 0.5 to one centimeter long incision, and pull the ovarian pad through the incision with a pair of blunt forceps.

Next, using a curved needle holder, clamp underneath the ovary and oviduct at the distal end of the uterine horn and excise the ovary with scissors. After 30 seconds, remove the clamp and dab with sterile gauze. To close the muscle wall incision, lift the top of the incision using forceps.

Then tie the incision with a surgeon's knot using silk sutures. Topically apply a few drops of bupivacaine using a one-milliliter syringe with no needle attachment, and dab the excess with sterile gauze. After similarly performing the ovariectomy on the other side, close the skin incision by pressing the two sides of the skin together and applying a couple of seven-millimeter surgical clips, leaving room for wound swelling.

Place all required equipment on a foil inside a laminar flow or Class II biosafety hood and UV sterilize for 20 minutes. Wash the silastic tubing in 100%desinol and allow it to air dry inside the hood. Once dry, cut the tubing into one-centimeter long segments using a scalpel.

Next, squeeze approximately 200 microliters of sealant into a syringe without a plunger. Replace the plunger and push out a small amount of sealant. Apply the sealant to one end of the tubing, smoothing it over with a gloved finger.

And allow the sealant to dry overnight or under UV light for 30 minutes in the hood. Next, pour progesterone powder into a sterilized Petri dish. Scoop the powder into pellets with the help of forceps.

Tap the sealed end of the pellet down the hood surface to condense the powder. Then seal the open end with the sealant. Wrap the Petri dish containing the pellets in foil to protect them from light.

About 72 hours before subcutaneous insertion, activate the pellets by incubating them in charcoal-stripped FCS. Demonstrating this procedure will be Dr.Fiona Cousins, an early career research scientist from the Hudson Institute of Medical Research. After hormone priming the animals with subcutaneous estradiol injections, prime the animals with subcutaneous progesterone pellets two days before artificial decidualization.

Before the surgery, anesthetize the mouse and test the depth of anesthesia by checking for the toe pinch reflex. Then place the mouse in a prone position, lift its tail and slowly insert a six millimeter speculum into its vagina. Place the lower body of the mouse between the first two fingers of the non-dominant hand.

Then, using the index finger, gently push the tail upward to allow for better vaginal accessibility. Transfer 20 microliters of sesame oil into a non-surgical embryo transfer tip, and insert the tip through the vagina and cervix into the uterine horn. Slowly expel the oil.

Once done, remove the speculum from the vagina. In this study, 80%of the the mice underwent successful decidualization, while 20%did not. So it's important to keep all your surgical equipment and working areas sterile and having patients when inserting the NSAID device through the cervix into the uterine horn of the mouse.

After inducing artificial decidualization, the progesterone palate can be removed to mimic menstruation and study endometrial breakdown. Using this method, we can study uterine biology and regulation independent of the influence of the ovaries. This is really exciting in the area of fertility and women's health in general.