Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

All animals and procedures used in this study were handled according to Washington State University (WSU) Animal Care and Use Committee guidelines and in compliance with WSU-approved animal protocols #4702 and #4735.

1. Mice

1. Use standard C57BL/6J adult male mice or other strains of interest. Maintain animals under a temperature- and humidity-controlled environment, with ad libitum access of water and food.
   1. Use male breeders ranging from 8 weeks old to 10 months old. Use only proven breeders in the experiments.
2. Use adult female mice with a tissue selective deletion of reproductive tract estrogen receptor α (encoded by Esr1 gene) in the epithelial cells¹⁰ (Wnt7a^Cre/+;Esr1^f/f, called knockout or "KO") and control littermates (Esr1^f/f, called "CTRL") in this study.
   1. Use 8 week-old females. Ages of females varied depending on the experiment of interest.
3. Determine the stages of the estrous cycle in the morning at 08:00h. Use only females at the estrus stage of the cycle. For detailed methods of vaginal smearing and cytological analysis, refer to the previously published procedures¹¹.

2. Mating and Assessing Copulatory Plugs

1. At the day of estrus (16:00h), house the female in a male's cage.
2. The next morning (08:00h), separate the female from the male mouse.
3. Use an adaptation of a previously published method¹² to access the presence or absence of a vaginal or copulatory plug as an indication of mating.
4. Hold the female mouse from the base of the tail to briefly lift the hind limbs.
5. Find the copulatory plug by the presence of an off-white seminal coagulum at the vaginal opening using a toothpick. If the copulatory plug is present, the toothpick will not be able to be inserted into the vaginal canal.

3. Preparation Before Tissue Collection

1. Prepare 10 mL of Leibovitz's L-15 media, supplemented with 1% fetal bovine serum (called L15 media + 1% FBS) in a 15 mL tube. Incubate the media at 37 °C for 15 min in a waterbath.
2. In order to preserve the viability of the tissue and sperm for downstream applications, aliquot 2 mL of pre-warmed L15 media + 1% FBS in a 2 mL microcentrifuge tube for tissue collection.
3. Warm the stage of the stereomicroscope to 37 °C. Set the heat-block to 37 °C and place an empty 1.5 mL microcentrifuge tube in the block.
4. If the downstream applications require imaging of sperm motility, warm the glass slide to 37 °C.

4. Tissue Collection

1. Bring pre-warmed L15 media + 1% FBS aliquot to the tissue collection area.
2. Euthanize the female (~08:30h) using carbon dioxide asphyxiation followed by cervical dislocation.
   NOTE: This is approximately 8 h after mating, assuming that mating takes place at midnight.
3. Place the animal in a supine position to expose the abdominal area.
4. Spray 70% alcohol on the abdomen near hind limbs.
5. Use the dissecting scissors to make a small cut (~1 cm) on the skin of the lower abdomen.
6. Use the dominant hand to pull the tail (and hind limbs if necessary) while non-dominant hand pulls the skin in the opposite direction (toward the head) to expose the abdominal muscle. This method is used to minimize the amount of hair contamination to the visceral organs.
7. Cut the abdominal muscle in a V-shape at the base of the abdomen, adjacent to the vagina, to expose the visceral organs.
8. Move intestines and abdominal fat to the side to expose the reproductive tract.
9. Trim off bladder and other tissues above the vaginal area.
10. Cut pubic symphysis apart to gain access to the vaginal tissue.
11. Lift the vaginal tissue at the vaginal opening and using scissors to separate the vaginal canal from the rectal tissue.
12. Carefully trim off the mesenteric fat from both sides of the uterus using spring scissors.
    CAUTION: The uterus is very fragile with the semen inside. Be careful not to puncture the uterus, or else the semen will be lost.
13. Lift the lower reproductive tract and cut the ovarian fat pad off of both sides.
14. Transfer the whole reproductive tract into the tube of pre-warmed Leibovitz's L-15 media + 1% FBS.

5. Measurement of Semen Liquefaction (Viscosity) from Uterine Content Collection

1. On the heated stage of the stereomicroscope, transfer the tissues into a 35 mm sterile tissue culture dish filled with 3.5 mL pre-warmed L-15 media + 1% FBS to clean the tissues of blood and other contaminants.
   CAUTION: While washing, grab the tissues by the fat, not the uterus. At this point, the semen is expected be intact inside the uterus.
2. Blot the tissues of excess media using laboratory wipes.
3. Hold the tissue at the vaginal end while placing the ovarian ends in the pre-heated microcentrifuge tube. While holding the vaginal end, cut the uterine horns at the base of the uterus, allowing the uterine horns to fall into the microcentrifuge tube.
4. Let the semen flow out of the uterine horns into the tube. Use toothless forceps to gently squeeze the uterine horns from the ovarian end to the vaginal end, expelling out leftover semen.
5. Discard the tissues in biohazard bag and incinerate.
6. Briefly spin down the semen in a minicentrifuge (~1-2 s).
7. Lay the tube, parallel to the heated stage – semen will not spill out.
8. Have the timer ready and set to 'count up'.
9. Insert a 25 µL capillary tube into the semen sample at a 180° angle (parallel to the tube wall). This is to minimize the variability between each investigator from holding the capillary tube at different angles.
10. Press the timer as soon as the capillary tube makes contact with the semen.
11. Record the time (s) it takes for the semen to reach the 25 µL line.
12. When the measurement is finished, immediately put the tube containing semen back in the dry heat block for further functional analysis, such as video imaging of sperm motility.
13. Disinfect the capillary tube containing semen by immersing the tube in 10% bleach solution for 20 min and discard the capillary tube in the sharps container.

6. Video Imaging of Sperm Motility

1. Turn on the microscope and video imaging software.
2. Pipette 20 µL of the remaining semen onto the pre-warmed glass slide, while the glass slide is placed on the heated stage.
3. Cover with a glass coverslip.
4. Place the slide coverslip side down on the microscope.
5. Use 20X objective lens to find area of interest.
6. Change to 100X objective lens for video imaging.
7. Record the video at 12 frames/s (fps) for 30 s.
8. Randomly record the videos in at least 3 different areas per animal.
9. Perform the imaging quickly and minimize light exposure to the slide in order to preserve sperm motility and viability.
   NOTE: This imaging procedure should be done within 2-3 min.
10. Perform the data analysis using ImageJ software as previously described⁸.