To begin, prepare the surgery platform setup in a room with a bench area of 60 centimeters by 60 centimeters for the operation. Clean the surface with 70%alcohol and cover it with a disposable medical towel. Then, disinfect the area with ultraviolet radiation for 30 minutes.
Acclimate eight-week-old female C57 black/6 mouse to the housing facility for one week before surgery. Using a clean cotton swab, apply hair removal lotion to the back of the anesthetized mouse. After three to five minutes, remove the hairs using gauze and cotton swabs.
Next, clean the skin with 70%ethanol. Fix the mouse on the surgery platform with its back facing up using a rubber strip or cotton rope, and apply iodophor solution to clean the back. For ovariectomy, using a disposable scalpel, make a one-centimeter dorsal incision longitudinally from the thigh base upwards, ensuring that only the skin and subcutaneous fascia are incised and avoiding cutting into the posterior peritoneum.
Next, pull the incision to the left to visualize a white fat pad. Using microforceps and scissors, cut 0.5 centimeters along the white fat pad to expose the intraperitoneal cavity. After cutting the posterior peritoneum, using microforceps, slowly and gently remove the white fat pad from the intraperitoneal cavity.
Immediately following removal, moisten the white fat tissue with 0.9%sterile saline-soaked gauze. In the lower part of the white fat pad, identify the pink granular substances as ovaries and ensure they are connected to the uterus. Using 5O absorbable sutures, ligate the ovarian end of the uterus.
Then, remove the left ovary, avoiding direct contact between surgical instruments and the ovaries, preventing intraperitoneal implantation of ovarian tissue. After surgery, carefully place the white fat pad back into the intraperitoneal cavity. Then, using a 5-0 absorbable suture, perform an intermittent suture on the posterior peritoneum.
Clean any areas of bleeding with gauze soaked in 0.9%sterile saline. Next, pull the skin incision to the right and remove the right ovary using the same method. Perform an intermittent suturing with 4-0 non-absorbable sutures and clean any bleeding with 0.9%sterile saline-soaked gauze.
After completing both sutures, clean the wound with an iodophor solution. After 24 hours, inject meloxicam subcutaneously to relieve pain. Monitor the mouse daily to confirm proper surgical wound healing without any signs of complications.
After three days, feed the mouse with freshly prepared estradiol valerate. For FSH injection, prepare FSH solution by dissolving recombinant human FSH powder in 0.9%sterile saline to 100 international units per milliliter. According to the biological activity of recombinant FSH, administer solvent or different doses of recombinant FSH via intraperitoneal injection for two weeks.
The smear images of Papanicolaou-stained cells clearly identified the proestrus, estrus, metestrus, and diestrus stages of the estrus cycle. In contrast, the bilateral ovariectomy mice lost the estrus cycle. The ELIZA method showed a significant decrease in serum estradiol levels in bilateral ovariectomy mice compared to the sham group mice.
The endogenous estrogen synthesized in the extra gonadal tissue does not affect the stability of estrogen levels in the FSH-treated mouse model.
