A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Here, we present detailed protocols for DHT pellet preparation and insertion, and for reproductive and metabolic testing. The mice used in this study were a mixed background (C57/B6, CD1, 129Sv) and were maintained with food and water ad libitum in a 14/10 h light/dark cycle at 24 °C in the Broadway Research Building animal facility at the Johns Hopkins University School of Medicine. All procedures were approved by the Johns Hopkins University Animal Care and Use Committee.

1. Create PCOS-like Mouse Model

1. DHT pellet preparation
   1. Autoclave silastic tubing to sterilize. Cut silastic tubes to a length of 15 mm with a razor blade;
   2. Seal one side by injecting medical adhesive silicone into the tube with a 20 G blunted needle attached to a 3 mL syringe. Plunger should be withdrawn from the syringe and then adhesive inserted into the reservoir. The plunger is reinserted and adhesive pushed down until it begins to emerge from the needle. The blunted needle is made by cutting the sharp tip side of 20 G needle with any strong scissors. The length of silicon in the tube should be more than 2 mm to allow for post-production trimming;
   3. Dry overnight; check for air bubbles on the sealed side. Those with no air bubbles are used for DHT pellets. Others can be used for no-DHT control pellets.
   4. Wear gloves, mask, goggles and lab coat before making DHT-pellet in a hood to avoid DHT exposure to skin.
   5. Pour DHT powder into a plastic weigh boat and press the open side of the adhesive capped tubes (produced in A1.1) into the DHT powder.
   6. DHT powder can be tamped down with a large paper clip that is straightened.  Continue until DHT reaches a height of 4 mm (or desired length). Check the length of DHT with a ruler, and seal the open side with silicon, dry overnight.
   7. Cut each sealed side to make the length of silicon 2 mm long. The total length of pellet will be 8 mm.
   8. Seal both side of empty silastic tubing as control (no-DHT) pellet.
   9. Keep pellets in a 50 mL conical tube wrapped with foil (to prevent light exposure) at room temperature until  use. The DHT pellets maintain full efficacy for at least 3 months of storage.
2. DHT insertion and replacement
   1. Up to 20 DHT pellets or control pellets are submerged separately in a 50 mL conical tube with 30 mL sterile 0.9% saline for 24 h at 37 °C for equilibration just before insertion.
   2. Prior to surgery, the surfaces and gloves are disinfected with clidox and the work surfaces are covered   with clean plastic backed absorbent paper (surgical pad).  All instruments that come in contact with animals are decontaminated prior to entering the animal facility by autoclaving.  Instruments will be allowed to dry and cool prior to use.
   3. 2-month-old female mice are used (4–5 mice/cage). Mice are injected intraperitoneally with Xylazine (3.5 mg/kg bw) and Ketamine (78.8 mg/kg bw) using an insulin syringe. Calculations for mixing and dosing anesthesia is in Table 1. 
   4. After adequate anesthesia is achieved, as measured by loss of toe reflex and slowed breathing, the mouse will be prepped for surgery.
   5. Disinfect the skin of the area with betadine using sterile gauze and clean with 70% ethanol.  The area will again be painted with betadine.
   6. Cut a hole around 5 mm length with scissors under the skin near the neck. Using a 10 G trochar, make a small tunnel (15 mm) in the rostral direction. The pellet is inserted dorsally with the trochar.
   7. The opening is then sealed with surgical adhesive. Manually approximate the wound edges with forceps and gentle brushing strokes to apply a thin film of liquid adhesive to the approximated wound edges. Buildup 3 thin layers of adhesive to ensure the adhesive is evenly distributed over the wound. The adhesive should extend 1 cm on each side of the apposed wound edges. Suture or surgical clips can also be used to close the hole. Put mice back into cage, individually housed, with a heat pad for recovery. Replace pellet every 4 weeks to maintain a constant level of androgen exposure. The original pellet will be removed and new pellet will be inserted as described in A1.15 in the similar position.
   8. Test estrous cyclicity after 3 days of DHT insertion. Estrous cycle stage is assessed by vaginal cytology.  Vaginal cells are collected for 16 consecutive days by using a p10 pipettor to squirt sterile saline (around 10 µL) into the vaginal cavity and then withdrawing the saline with the same sterile pipette tip.  Vaginal cells sloughed from vaginal wall mix with saline and are collected.
   9. Spread saline with cells onto a labeled slide. Each slide can contain six samples. Slides are labeled to note which sample is placed in each of the six locations.
   10. After the saline has completely dried out on the slide, put slides into a container with 100% ethanol to fix cells. Slides should be fixed for at least 5 min, but can remain in fixative indefinitely until further use.
   11. Put slides into staining solutions for 1 min each for buffer B and C. Wash slides with tap water and dry at room temperature.
   12. Examine cell morphology under a 10X objective light microscope.  Cell morphology to distinguish proestrus (P), estrus (E), metestrus (M) and diestrus (D) is described in references^27,28,29.
   13. Count the days in each stage and divided by the total number of days to calculate percent time at each stage.
   14. Test glucose tolerance at 21 days after DHT insertion by fasting the mice overnight (16 h) and injecting 2 g/kg body weight of 20% dextrose intraperitoneally. For example, if a mouse body weight is 25 g, this mouse will be injected with 250 µL of 20% dextrose with a 50 cc (0.5 mL) insulin syringe. Glucose levels are assessed by glucometer and test strips by sampling blood from tail vein at 0, 15, 30, 60, 90, 120 min. This is described in detail elsewhere³⁰.
3. Blood collection
   1. Collect blood at various days after DHT insertion (we limit collection volume to 100 µL for steroid and 30ul for LH/FSH assay) between 9 and 10 am by submandibular vein bleeding with lancet. This is described in¹¹.
   2. Centrifuge blood at 6,000 x g at 4 ˚C for 10 min, and collect the serum layer into another 1.5 mL tube that can be stored at -80 °C until assay.

2. Assess Reproductive Profiles of Female Offspring from Chronically DHT Inserted Dams

1. Mating
   1. The female mouse to be tested is moved into the cage with a proven fertile male (that has  previously had pups with a female mouse) at 15 days following pellet insertion. 
   2. Document dam’s body weight every week to determine if the mouse is pregnant. Dam weight increases of more than 3 g in a week indicates pregnancy.
   3. Collect blood of dams the second week after we observe increased body weight of dams greater than 3 g from the week before. 
2. Puberty assessed by vaginal opening and first estrus
   1. Check vaginal opening by visual inspection every day after pups are weaned at 21 post-natal day (PND), measure the anogenital distance and record the age of vaginal opening.
   2. Once vagina opens, collect vaginal cells daily, as described in 1.2.8–1.2.11 to check estrous cyclicity.
   3. Observe cell morphology as described 1.2.12, and the first estrus is defined as the date that all cells are cornified epithelial cells. 
3. Body weight and ear punch
   1. Dip the lancet tip into the tattoo paste and mark each pup with tattoo on the toe as   described in reference¹² at 7 PND, and weigh mice every 7 days until 70days of age.
   2. Ear punch mice to number them using system in Figure 1 between 12 to 16 PND. To distinguish sex, examine ventral side of body, females will have visible nipples.
   3. Collect blood at 21, 26, 70 PND after birth as described above in 1.3.1
4. Hormonal Assay
   1. DHT levels in blood serum are measured by both enzyme-linked immunosorbent assay (ELISA) and by liquid chromatography tandem mass spectrometry (LC-MS)^23,24,31,32. T is measured by LC-MS, or a rat/mouse ELISA that has been validated by the University of Virginia Ligand Assay Core in the Center for Research in Reproduction^23,24.