This method can help answer key questions in the field of reproduction. And the endocrinology. And are applicable to the study of Polycystic Ovary Syndrome or PCOS and hyperandrogenism.
The advantage of this technic is that it facilitates a consistent, uniform, long-term release of hormones in a costly effective way. JHUs treating the procedure will be Ping Xue, a technician from my laboratory. For dihydrotestosterone or DHT pellet preparation, use a razor blade to cut a 15 millimeter piece of silicone tubing.
And use a three milliliter syringe equipped with a blunted 20 gauge needle to inject two to five millimeters medical adhesive silicone into one end of the tube. Allow the tubing to dry overnight. And check for air bubbles on the sealed end.
Wearing the appropriate personal protective equipment pour DHT powder into a plastic wayboat in the flowhood. Impress the opened end of the adhesive capped tube into the powder. Using a large straightened paperclip, tamp the powder down into the tubing, until the DHT reaches the appropriate experimental depth.
Then seal the opened side of the tube with silicone overnight. The next morning, cut each sealed side to obtain a two millimeter length of silicone on both sides of the DHT and no DHT control pellets. Then store the pellets in the 50 milliliter conical tube wrapped with foil at room temperature for up to three months.
For PCOS mouse model generation equilibrate up to 20 DHT or control pellets in separate 50 milliliter conical tubes containing 30 milliliters of 0.9 percent saline for 24 hours at 37 degrees Celsius. The next day, confirm lack of response to toe pinch in an anesthetized two month old female mouse and use sterile gauze to administer sequential Povidone-iodine and 70 percent ethynol scrubs to the surgical area. Next, make a five millimeter incision through the skin near the neck.
And use a ten gauge trocar to make a 15 millimeter tunnel in the rostral direction. Use the trocar to insert a pellet dorsally into the tunnel and seal the incision with the surgical adhesive. Manually approximate the wound edges with forceps and use gentle brushing strokes to apply three thin film layers of liquid adhesive to one centimeter out from each side of the opposed wound edges.
Then place the mouse alone in a cage on a heat pad with monitoring until full recumbency. Replacing the pellet every four weeks to maintain a constant level of androgen exposure. Three days after pellet insertion, use a p10 pipette to squirt ten microliters of sterile saline into the vaginal cavity and use the pipette tip to immediately recover the saline.
Spread the vaginal cell saline sample onto a labeled slide. And allow the saline to dry at room temperature. Fix the dried slides in a container of 100 percent ethynol for at least five minutes.
Then stain the slides for one minute each in buffer B and buffer C from a dif-quick staining kit. At the end of the buffer C incubation wash the slides with tap water for three seconds and dry them at room temperature. To determine the estrous cyclicity view the slides under the ten times objective of a light microscope.
Count the days in each stage and divide by the total number of days to calculate percent time at each stage. To test the glucose tolerance, 20 days DHT insertion, fast the mice overnight. Weigh the mice the next morning, and use a glucometer and test strips to measure basil glucose level of tail vein blood.
Inject the mice with two grams per kilogram body weight of 20 percent dextrose. Then measure the glucose level of tail vein blood samples at 15, 30, 60, 90, 120 minutes post dextrose administration. To acquire female offspring from DHT inserted dames, move the female mouse to tested in the cage with a proven fertile male at day 15, following pellet insertion.
Document the dames body weight every week to determine if the mouse is pregnant. On post-natal day seven, dip a lancet tip into tattoo paste and mark each pup with a tattoo on the toe. Weigh the marked mice every seven days until 70 days of age.
Between post-natal date 12 to 16, ear punch the mice to number them according to the system as illustrated in the figure. To distinguish the sex, examine the ventral side of each tagged mouse. The females will have visible nipples.
To collect blood samples for various downstream analyses use a lancet to collect the approximate experimental volume of blood between nine and ten am by submandibular vein bleeding. And centrifuge the samples to allow serum collection. Then store the serums samples in 1.5 milliliter micro-centrifuge tubes at minus 80 degrees Celsius until their analysis.
To assess puberty in the DHT female offspring, every day after weaning at post-natal day 21, check the vaginal opening by visual inspection. And use calibers to measure the ano-genital distance. Although the absolute values of serum DHT are different between mass spectrometry and Elisa analysis, the relative folk change of DHT versus no-DHT insertion is similar from both assays and across experiments.
DHT levels are significantly increased from pre-conception through pregnancy in both DHT and no DHT mice. However, DHT levels are two-fold higher in DHT mice compared to no DHT mice at both pre-conceptional and gestational time points. Adult DHT daughters exhibit differences in reproductive function.
For example, disrupted eestrous cyclicity compared to adult no DHT daughters experiencing significantly longer times in metestrus diestrus and less time in pro-esterous and esterous. While attempting this procedure it's important to remember to incubate the pellets for 24 hours before they are used for uniform release of the hormone. Following this procedure, the reproductive and metabolic consequences of DHT exposure can also be examined.
After it's development, this technic paved the way for researchers in the field of endrochronology and maternal fetal interactions to study PCOS and the impact of low level hyperandrogenism in female mice. Don't forget that working with DHT can be extremely hazardous and the precautions such as wearing gloves and a mask and goggles, and working in a bio-safety hood, should always be taken while performing this procedure.
