for the use of Laboratory Animals of IBISS, University of Belgrade (no. 01321). At the age of 15 months, animals have been randomly divided into two groups: one particular was bilaterally orchidectomized (Orx, n = 12) via the scrotal route. Animals had been intramuscularly injected with ketamine anesthesia (15 mg/kg body mass; Richter Pharma, Austria), 150 min before orchidectomy. The scrotal region was shaved and cleaned with all the antiseptic resolution (Octenisept, Schuelke Mayr GmbH, Norderstedt, Germany). Working with a sterile scalpel, scrotum and lamina parietalis had been incised within the middle. Due to the fact rats have open inguinal canals, testicles were forced in to the scrotum in the abdomen as necessary. This was performed by exerting gentle stress towards the scrotum inside the caudal abdomen with fingers. Subsequent, the testicular content (each testicles, two epididymides, vasa deferentia, and the testicular blood vessels) was gently exteriorized. Vasa deferentia and blood vessels were ligated with an absorbable surgical suture, along with the testicles and epididymides wereInt. J. Mol. Sci. 2022, 23,three ofremoved applying scissors. The remaining PLK4 Formulation tissue was placed back inside the scrotal sac employing blunt forceps. The scrotal skin was not sutured. Just after orchidectomy, the animals were housed individually and kept beneath close observation for around 24 h right after the surgery. Thinking about healing and PAR2 web bleeding, no adverse impacts were observed. The second group (SO; n = 6) was sham-operated, in which testicles have been exposed but not removed. Two weeks following the surgery, the remedy begun: 1 group of animals was subcutaneously treated with five (200 IU) of cholecalciferol (Orx + Vit. D3 ; Sigma Aldrich, Germany; n = six)/kg b.m. day-to-day, dissolved in sterile olive oil, when two manage groups, orchidectomized (Orx; n = six) and SO, received the exact same quantity of automobile alone for three weeks. 2.2. Sample Collection and Processing Animals had been decapitated devoid of anesthesia to prevent the feasible effects of anesthesia on serum hormone results. Blood was collected from the trunk, plus the serum stored at -70 C. Soon after decapitation, the thyroids from each animal had been excised and weighed. The relative organ weights were calculated from the ratio on the measured organ weight and physique mass for every animal. For histology, the thyroids were fixed in Bouin’s option for 48 h and dehydrated in rising concentrations of ethanol and xylene. Just after embedding in Histowax (Histolab Product Ab, Sweden), tissue blocks have been serially sectioned at five thickness on a rotary microtome (RM 2125RT Leica Microsystems, Germany). Tissue slices have been subjected to hematoxylin and eosin (H E) staining and immunohistochemistry. 2.three. Transmission Electron Microscopy (TEM) For transmission electron microscopy (TEM), a single thyroid lobe was removed from two randomly selected animals per group, sliced in four glutaraldehyde solution in one hundred mM phosphate buffer, pH 7.4, for 24 h at four C, and further processed as previously described [30]. In brief, post fixation was carried out with 1 OsO4 for 1 h at 4 C, and counterstaining with uranyl acetate. Samples have been dehydrated via a graded series of ethanol and embedded in Araldite resin. A Leica EM UC7 ultramicrotome (Leica, Germany) with a Diatome ultra 45 diamond knife (Diatome, Switzerland) was made use of for cutting ultrathin sections of thyroid tissue at a thickness of 70 nm. Grids with ultrathin sections were stained with uranyl acetate and lead citrate and examined below a Morgagni 268 (FEI C