Effect of angiopoietin-2 and vascular endothelial growth factor on function and neovascularization of xenografted bovine ovarian tissue

Abstract

Objectives: As fertility preservation options for oncological female patients, ovarian tissue (OT) cryopreservation and transplantation have been used to restore their fertility and as a result, over 30 deliveries were recorded by these techniques. Even though such successes, it is difficult to prevent ischemic injury which occurs right after transplantation. As a result, a massive follicle loss and stromal cell destruction arise in the graft. Thus, it is necessary to shorten the ischemic period and promote neo-vascularization to overcome the ischemic injury. The aim of this study was to assess the impact of angiogenic factors, especially angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) on bovine OT after xenotransplantation (XT).

Methods: A total of 6 bovine ovaries were transported from the local slaughter house to our laboratory within 2 hours. The OT cortex was separated and sliced into 5 x 5 x 1 mm3 size strips, and the OT strips were vitrified-warmed or remained fresh for further procedure. For the XT experiment using bovine OT, 9-week-old BALB/c nude mice (n=30) were ovariectomized, and subcutaneously xenografted with fresh (XT-Fresh), or vitrified-warmed OTs. Before XT with vitrified OTs, the nude mice were randomly subdivided into 4 groups and intraperitoneally injected with saline (XT-Vitri), 500 ng Ang-2 (XT-Ang-2), 200 ng VEGF (XT-VEGF) and a combination of 500 ng Ang-2 and 200 ng VEGF (XT-Combined) before 18 hours and 30 minutes of XT. After 7 or 28 days of XT, the grafts were retrieved, and follicle normality, density, angiogenesis, and fibrosis of grafted OT were evaluated with histological evaluation, CD31 immunostaining, and Masson's trichrome staining, respectively.

Results: Improvement of primordial follicle (PF) normality was found in angiogenic factor treated groups when compared to the XT-Vitri group 7 and 28 days after grafting. Growing follicle (GF) normality was also significantly increased among angiogenic factor treated groups on day 7. On day 28, only the XT-VEGF and the XT-Combined group showed significantly increased GF normality when compared to the XT-Vitri group. With regards of follicle density, the PFs were significantly well preserved in the XT-Combined group on day 7 when compared with XT-Vitri group, which was comparable to the result of the XT-Fresh group. For the angiogenic factor treated groups, the mean numbers of GF were remarkably increased which was comparable to the XT-Fresh group on day 28. Among the 5 XT groups, the highest microvessel density was observed in the XT-Combined group on day 7 and 28. The most extensive fibrosis was detected in the XT-Vitri group on day 7 and 28, and was reduced in angiogenic factor treated groups, but this difference did not reach the statistical significance.

Conclusions: In this study, well preserved PF and GF, and remarkably improved microvessel densities were appeared in the angiogenic factor treated groups

especially in the combination of Ang-2 and VEGF treated groups. Thus, we propose that the angiogenic factors have beneficial effects on OT transplantation outcome and help to overcome the ischemic damage during the early grafting process. However further studies are necessary to clarify exact mechanisms of Ang-2 and VEGF effects in OT grafts.