Immunohistochemical evaluation of xenografted cat ovarian tissue

Introduction and aim. Gonadal tissue cryopreservation and transplantation is a promising option for preserving reproductive potential and for conservation of biodiversity. Cryopreservation of cat ovarian tissue followed by xenotransplantation to immunodeficient mice has been also reported (1). Immunohistochemical determination of hormonal receptors and apoptosis can provide useful information on functional integrity and viability of the transplanted tissue. Our aim is the immunohistochemical evaluation of follicular development, revascularisation and sex-hormone receptor expression of cat ovarian tissue xenotransplanted to immunodeficient mice. Materials and methods. The whole procedure fulfils all the requirements of the European Animal Welfare regulations. Ovarian tissue was collected from healthy domestic cats. Serum hormone determinations were performed by commercially available ELISA tests validated for cat serum (Quanticheck, Budapest, Hungary; E2-EIA-2693, Testosteron-EIA-1559 DRG, Mountainside, USA). Ovarian cortex from each ovary was sliced into small fragments (2 mm×2 mm×1mm). One fragment was fixed in 5–8% neutral-buffered formalin and the remaining was xenotransplantated to immunodeficient mice (Balb/c nunu). Xenotransplanted tissues were removed one month later. Four µm thick sections were cut and stained with haematoxylin and eosin in order to evaluate follicular development and revascularisation. Sections were immunostained using the En Vision System with primary mouse antibody. Progesterone antibody, diluted 1:300, provided by the laboratory of the Department of Reproduction, SzIU, Budapest, Hungary. This antibody is routinely produced for the quantitative ELISA determination of canine serum progesterone (2). AE1/AE3, CD3, CD79a, Ki67 (DAKO Glostrup, Denmark) and oestrogen receptor, progesterone receptor, PCNA (Abcam, Cambridge, UK) immunohistology were performed. The association between hormonal levels and receptor expression was determined using polynomial regression analysis. The level of significance was set at p<0.05. Results. Comparison between fixed and xenotransplanted ovarian tissue after removal indicated a decrease in cytokeratin positivity of epithelial cells. T and B cell receptors and proliferation was low in both sample types. Ki67 positivity was lost during xenotransplantation, which means presence of resting (G0) cells. PCNA positivity was retained in primary follicles and oocytes. Although oestrogen receptors were present on oocytes, progesterone receptors and progesterone positivity in granulosa cells decreased in xenotransplanted tissue. Conclusions. Cat and rodents are useful experimental models for research aimed to restoration of human fertility throughout cryopreservation and xenografting of gonadal tissues. Effect of xenotransplantation on ovarian tissue and consequently on receptor expression demonstrates a modified hormonal activity. Evaluation of functional integrity and viability of xenotransplanted ovarian tissue helps to understand the subcellular effects of sexual hormones and the initial steps of programmed luteolysis in fresh and xenotransplanted ovarian tissue.