Cryopreservation and development of antral follicles in cat ovarian tissue after transplantation into NOD-SCID mice

BOSCH, P.; HERNANDEZ-FONSECA, H. J.; MILLER, D. M.; WININGER, J. D.; MASSEY, J. B.; BRACKETT, B. G.

Foz do Iguacu, Brasil

Congreso; 28 Annual Congress of the International Embryo Transfer Society; 2002

International Embryo Transfer Society

Ovarian cortex cryopreservation and xenotransplantation into immuno-deficient mice represents a potential means for felid germplasm conservation. Our goal is to combine cryopreservation of ovarian tissue with subsequent transplantation to severe combined immuno-deficient (SCID) mice to, ultimately, obtain viable oocytes for procreation. Present objectives were: (1) to assess ovarian follicles after cryopreservation and transplantation into non-obese diabetic (NOD) SCID mice; and (2) to evaluate the effect of gonadotropin treatment on follicular development in the transplanted tissue. Ovaries obtained by ovariohysterectomy from a 1-year old female cat were transported to the laboratory in Leibovitz’s L-15 medium (Gibco BRL®/Life Technologies, Rockville, MD) with 10% fetal calf serum and 50 mg/ml of gentamicin (transport medium). Pieces of ovarian cortex (2x2x1 mm) were frozen 1 h after excision (Candy et al., Hum Reprod 1999;10:2334-2338), and after 4 days, thawed, washed (3x) and held in transport medium at 25°C before transplantation (time from thawing to completion of last surgery=2.5 h). One piece was placed under each kidney capsule of 4-month-old castrated male NOD SCID mice (n=4, Jackson Laboratory, Bar Harbor, ME). At surgery and on 3 consecutive days, mice were injected (ip) with recombinant human FSH (3.75 IU, Follistim™, Organon Inc., West Orange, NJ). Eight weeks after surgery, mice were randomly assigned to treated (n=2) and control (n=2) groups. Treated mice received ip injections of eCG (5 IU) and hCG (5 IU) 88 h later; controls received saline. Mice were euthanized with CO2 24 h after the last injection. Recovered tissue was serially sectioned (20-50 6-mm sections/graft) and stained with hematoxylin and eosin. Fresh ovarian tissue from the same original source was also processed for histology. Follicles were counted, measured and classified as primordial, primary, secondary or antral. Each histological section was digitalized and the surface measured using Scion Image Software (Scion Corporation, Frederick, MD). Surface and thickness measurements were used to estimate the tissue volumes (mm3). Proliferating cell nuclear antigen (PCNA) was assessed in every 10th section using a commercial kit (DAKO Corporation, Carpinteria, CA). Data consisting of ratios of resting (i.e., primordial) to growing follicles (i.e., primary, secondary and antral follicles), and comparisons of diameters of growing follicles between control and treated groups were analyzed by Chi-square and by Student’s t test, respectively. Histology revealed no evidence of necrosis or fibrosis. The grafts were well-vascularized with follicles at all stages. Numbers of follicles (11.1 follicles/0.1 mm3) were markedly reduced compared with fresh tissue (114.5 follicles/0.1 mm3). PCNA positive follicles, indicating cell proliferation, occurred in all xenografts. The treatment did not affect proportions of resting to growing follicles (5.0% vs 6.6% in treated and controls, respectively; p=0.34). Mean follicle diameters were similar for control and treated mice (primary: 0.06±0.01 vs 0.07±0.01 mm, p=0.34; secondary: 0.24±0.20 vs 0.19±0.08 mm, p=0.51; antral: 0.76±0.43 vs 0.85±0.33 mm; p=0.69). By contrast luteinization, but not ovulation, of antral follicles was observed only in treated mice. Results demonstrated that freeze-thawed cat ovarian cortex not only survives xenotransplantation but also has follicles able to grow to antral stages containing gonadotropin responsive granulosa cells.