Construction of animal models

The sequence of 22AA is NH2-GSQIYTICSQFYTPGGTGLYVD-COOH (from the 209th to 230th amino acid of occludin) (Chung et al., 2001), and 22AA was synthesized by Beijing Protein Innovation Co., Ltd., China. To prepare a 200.0 g/L solution, 22AA was dissolved in 0.9% sterile saline. Male KM mice were anesthetized with 7% chloral hydrate (0.5 mL/100 g), the scrotal skin was cut, both (left and right) testicles were exposed, and a 26-gauge needle (Becton Dickinson, Rutherford, NJ) was used for injection of 7.0 µL of 22AA solution at three different locations in each testicle. The control group was injected with 0.9% sterile saline. After injection, the testicles were sutured, and the mice were put back into the cage. Thirty-six experimental animals were divided into six groups, with six mice in each group: A, normal control group; B, 7 days (7D) after injection of 22AA; C, 17D after 22AA injection; D, 27D after 22AA injection; E, 37D after 22AA injection; and F, 47D after 22AA injection. After the mice were anesthetized by intraperitoneal injection of 7% chloral hydrate (0.5 mL/100 g) at different time points, the skin of the scrotum was removed, and one testis was removed and stored at −80 °C for western blotting (WB) and quantitative PCR (qPCR). The other testis was fixed with 4% paraformaldehyde for morphological examination. At the end of the experiment, the mice were euthanized using 0.3 mL 7% chloral hydrate (0.5 mL/100 g) according to the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals, 2013 Edition.