169 INVASION OF MURINE ANNEXIN A1 IN BOVINE OVARIAN CORTEX TISSUE DURING SHORT-TIME XENOTRANSPLANTATION IN CONVENTIONAL AND IMMUNE DEFICIENT MICE

Abstract

In 2012, 6.7 million women worldwide were diagnosed with cancer, including young women and children. Almost 85% of the latter survive the disease. However, the necessary cytotoxic treatment reduces their reproductive potential by damaging the stock of, basically preantral, ovarian follicles. Current fertility preservation techniques try to overcome this problem. Still, the success rate is low due to a lack of optimization and standardization. Nowadays, culturing preantral follicles in vitro is not yet routinely possible. Therefore, we used an in vivo model based on xenotransplantation of bovine ovarian cortex tissue in recipient mice, as an alternative to studying human reproduction. To further characterise this model, we assessed the influence from the host on the graft at the protein level. Visualising murine annexin A1, an anti-inflammatory protein, in the transplanted bovine cortex tissue should help to elucidate the process of immunological rejection. Hereto its distribution around the present bovine follicles is measured. In total, 12 mice (6 conventional fluorescent C57BL/6-eGFP and 6 immune deficient Balb/c-Nu) were used as graft recipients. All mice were anesthetized with an intraperitoneal injection and subsequently sterilized. Small pieces (max. 9 mm³) of adult bovine ovarian cortex, retrieved from slaughterhouse ovaries, were grafted retroperitoneal. After a transplantation period of 14 (3 mice/mice strain) or 28 (3 mice/mice strain) days, the mice were killed and all grafts were successfully recovered. These were fixed (4% formaldehyde) and processed into histological paraffin slides. Murine annexin A1 (Sigma-Aldrich HPA011271) was localised using immunofluorescence through a rhodamine label (RITC filter). We measured the shortest distances between 10 annexin fluorescent signals and the basal membrane of each follicle, found in the graft. We calculated the average of these measurements, resulting in one data point per follicle. The dataset was fitted in a linear mixed model with annexin as dependent variable, and transplantation period and mice strain as fixed effects. Fluorescent signals of murine annexin A1 were found in all grafts form both mice strains, surrounding bovine follicles. Data show no interaction between mice strain and the duration of the transplantation period. The influence of the mice strain showed a trend towards significance (P = 0.08), possibly due to the immunological state of the host. To our knowledge, this is the first time an attempt has been made to characterise the host/donor interaction in xenografting procedures. We can conclude that annexin A1 from the murine host (whether immunodeficient or immunocompetent) invades the bovine graft tissue in a short period of time.