Ovary Retrieval and Transplantation
All animal experiments were approved by the Institutional Animal Care and Use Committee. Immature 15-day-old Wistar rats were killed using CO2 and subsequent cervical dislocation, and ovaries were collected. The ovaries were cleaned from fat in L-15 Leibovitz medium (GibcoBRL, Invitrogen Corporation, Paisley, Scotland, UK) supplemented with 0.1% BSA and antibiotics (penicillin/streptomycin; 100 IU/ml) at room temperature. Several cleaned ovaries were fixed for morphological evaluation and served as controls. Ovaries were transplanted into CD-1 female nude mice, 6-10 wk old, within 20-30 min after collection. Briefly, mice were anesthetized by intraperitoneal administration of 75 mg/kg Ketaset (ketamine; Fort Dodge Laboratories, Fort Dodge, IA) and 3 mg/kg XYL-M (xylazine; VMD, Arendonk, Belgium) followed by a subcutaneous addition of approximately half of the initial dose in order to prolong the duration of anesthesia. Ovarian fragments were transplanted either subcutaneously at the hind limb above the gluteus superficialis muscle or in the muscle itself.
In the subcutaneous transplantations, the graft was inserted through a 1cm full-thickness dermal incision and placed about 3 cm away from the incision using tweezers. The incision was sealed with Super Glue Gel (ethyl-2-cyanoacrylate; Loctite, Cleveland, OH). Intramuscular grafts were inserted through a cut along the muscle fibers. The muscle was sutured using silk thread in order to allow detection of transplantation site after the animal was killed (anesthesia overdose). zoloft 100mg