Amphibian Bufo arenarum vitronectin-like protein: Its localization during oogenesis

Abstract

In the present study, we analyzed the localization of vitronectin-like protein in oocytes during oogenesis as well as in the serum and liver tissue of the amphibian Bufo arenarum. Vitronectin-like protein was purified from serum by heparin-affinity chromatography and showed the two biological properties in common with most animal vitronectins (VN): heparin binding activity and an RGD-dependent cell-spreading activity. SDS-PAGE of vitronectin-like protein revealed that it consists of two bands of 64 kDa and 72 kDa, while immunoblotting analyses showed that this protein strongly cross-reacts with two monoclonal antibodies against human VN. No immunofluorescent staining of vitronectin-like protein was observed in previtellogenic oocytes (stages I and II). In vitellogenic oocytes (stages III, IV and V) fluorescence was observed in the cortical cytoplasm localized in yolk platelets, extending concomitantly with the vitellogenic process. When we examined the yolk platelet formation pathway by immunoelectron microscopy, gold particles indicated that vitronectin-like protein was located on the yolk platelet precursors: multivesicular bodies and primordial yolk platelets. Gold particles also were seen sparsely distributed in all oocyte investing layers. The mean serum vitronectin-like protein concentration in amphibian animals was 127.8 ± 11.6 μg/ml in adult males and 181.5 ± 14.3 μg/ml in adult females. Serum vitronectin-like protein of males and females was susceptible to hormonal stimulation (17-β estradiol). These results suggest that vitronectin-like protein is stored in the yolk platelets and may be involved in the later events of amphibian development.