Establishment of Rat Models Mimicking Gender-affirming Hormone Therapies

Our research developed animal models to replicate gender-affirming hormone therapies for transgender individuals using testosterone for transgender men and estrogen with anti-androgen for transgender women. We are examining whether those undergoing these therapies are as vulnerable to environmental chemicals as the general population and if there are any long side effect of hormone treatment. This model is unique in its field since data and tools are currently available to evaluate the potentially different susceptibility to chemicals of transgender people undergoing hormone therapy and their side effects.

A significant finding from our work is the use of sex-specific hepatic CPS as a marker to evaluate the success of hormone therapies. This approach, developed for the first time in our study, provides a valuable tool to confirm the accuracy and effectiveness of the model.

Our protocol addresses the gap in understanding the long-term effects of hormone therapies. It also investigates whether daily exposure to food and environmental chemicals can interact with or alter the effects of hormone treatments over time.

In the future, we will explore if the model is suitable for long-term testing, and we will investigate additional biomarkers such as narrow behavioral tests to better assess therapy effects.

[Instructor] Begin by pinching and lifting the skin at the injection site to administer testosterone and estradiol plus cyproterone acetate dissolved in sesame oil. Insert the needle of a one milliliter syringe parallel to the animal's back. Once the needle is inside, slowly inject 100 microliters of testosterone or 200 microliters of estradiol plus cyproterone acetate, depending on the model. After two weeks of treatment, place each anesthetized rat in the supine position on a heating pad. Next, prepare a five milliliter syringe with a 21-gauge needle, ensuring that the vacuum is removed. Use fingers to locate the heart. Gently clean the thoracic wall and insert the needle carefully into the lateral thoracic wall, perpendicular to the body, at the point aligned with the flexed elbows, between ribs five and six. Once the needle reaches the heart ventricle, blood will enter the syringe by capillarity. Retract the syringe piston slowly and continuously until the blood is drawn. After the sacrifice, place each animal on the dissection table supine, with the front legs facing upward and the hind legs downward. Fix the limbs with pins, then sprinkle the animal's ventral surface with a disinfectant solution to prepare for dissection. Using scissors, cut along the midline from the pubis to the upper abdomen. Make two lateral cuts along the medial surfaces of each hind limb from the midline ventral abdominal incision and expose the abdominal viscera. In male rats, excise the testes located in the intra-abdominal scrotal sac. Press the scrotal sac to ensure the testes protrude and gently grasp them. Use pliers to hold the visceral fat, then carefully cut the testes away from the viscera. Weigh the testes before storing them in Bois solution for histopathological analysis. In female rats, grab the uterus delicately. Identify the ovaries at the ends of both uterine horns and cut them away from the surrounding visceral adipose tissue. Weigh the ovaries after excision. Next, grab the liver with forceps, ensuring it does not break. Use pliers to grasp the xiphoid process and cut the diaphragm completely with scissors. Use scissors to separate the liver from the diaphragm. Lift the xiphoid process with forceps and extract the liver from the abdominal cavity. After excision, weigh the liver and store it appropriately. Using scissors, cut perpendicularly along the neck of the animal. Remove the skin and musculature to expose the trachea, where the thyroid gland is located. Gently trim the ends of the trachea to remove the thyroid gland. After removing the testis, grab the epididymis, which is attached to the posterior margin of the testis and is contained within the scrotum. To collect semen, clean the tail of the right epididymis by removing fat and connective tissue using scissors. Separate the tail from the remaining part and place it in a Petri dish containing one milliliter of DMEM. Cut the tail with scissors and use a past pipette to gently flow the contents and facilitate release. Now, transfer the collected liquid to a 15 milliliter tube and adjust the volume to 10 milliliters with DMEM to achieve a dilution factor of one to 10. Pipette the contents to homogenize and create a suspension. After gently shaking, take 10 microliters of the suspension to load the Neubauer chamber.