Immunohistochemical evaluation of xenografted cat ovaries

Gonadal tissue cryopreservation and transplantation is a promising option for preserving reproductive potential and for conservation of biodiversity. 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. 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 piece of sample was fixed in 5–8% neutral-buffered formalin and the remaining was xenotransplantated to immunodeficient mice (Balb/c nunu). Xenotransplanted ovaries 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, was 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. AE1/AE3, CD3, CD79a, Ki67 (DAKO Glostrup, Denmark) and oestrogen receptor, progesterone receptor, PCNA (Abcam, Cambridge, UK) immunohistology were performed. Comparisons between hormonal concentrations and immunohistochemistry were determined using Pearson Product Moment correlation analysis. Comparison of 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.