br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion Although homologues of the β-galactosidase gene were known to exist in the Xenopus genome, and SA-β-gal activity has been detected in whole-mount Xenopus embryos by histochemical staining (Davaapil et al., 2017), no reports have been presented so far concerning experimental detection and characterization of SA-β-gal activity in Xenopus oocytes and eggs. In the present work, we investigated activity and subcellular localization of SA-β-gal in aging Xenopus oocytes and eggs using bioinformatics analysis and two different experimental detection methods. First, bioinformatics analysis confirmed the existence of several homologous β-galactosidase genes in the annotated genomes of Xenopus laevis and Xenopus tropicalis frogs and predicted the presence of their protein products in different subcellular compartments of frog oocytes (Fig. 1; Supplemental Fig. 1). Then, biochemical analysis revealed that SA-β-gal activity is abundantly present in the cytosolic and organelle fractions of Xenopus oocytes, and its enzymatic properties resemble those of the mammalian enzyme (Fig. 2). Furthermore, the microscopic detection method using a cell-permeable fluorescent substrate of the enzyme, SPiDER-β-gal, demonstrated that SA-β-gal is localized predominantly in the late large-sized acidic endosomes of Xenopus oocytes and eggs (Fig. 5, Fig. 6; Supplemental Fig. 5). Finally, both experimental methods detected the increase of SA-β-gal activity in Xenopus eggs, but not oocytes, aged in vitro over 48 h (Fig. 3, Fig. 7, Fig. 8). To our knowledge, this is the first report addressing SA-β-gal activity and localization in the aging gamete cells, such as oocytes and eggs. The endosomal localization of SA-β-gal in Xenopus oocytes and eggs comes as no surprise because the enzyme was previously identified as the lysosomal β-D-galactosidase (Lee et al., 2006). As determined by our study, the SA-β-gal-containing granules in the U 18666A receptor of Xenopus eggs had an average size of 8.9 ± 5.6 μm (Fig. 8D) and they were extensively stained with the lysosome-specific dye LysoTracker, protein-specific Coomassie-based dye, and lipid-specific dye Sudan Black B (Fig. 5, Fig. 6; Supplemental Fig. 5). These properties identify the SA-β-gal-containing fraction as a subpopulation of yolk platelets, specialized late endosomes or lysosomes that accumulate and store processed vitellogenin in frog oocytes. Yolk platelets occupy about half the volume of the Xenopus egg, and vitellogenin derivatives account for about 90% of egg protein (Gurdon and Wakefield, 1986). The endosome-like yolk platelets were reported to have unusual enzymatic composition resembling that of lysosomal compartment. Most of the enzymatic activity resides mainly in a subpopulation of large, several μm in size, yolk platelets that are less dense than the average platelets (Wall and Meleka, 1985). It was hypothesized that the accumulation of yolk proteins occurs in these organelles due to a failure of vitellogenin molecules to undergo complete digestion after they enter the lysosomal compartment. The presence of several acid glycosidases has been reported within the yolk granules of Xenopus laevis eggs (Wall and Meleka, 1985). These enzymes are thought to be involved in utilization of the stored protein during early embryogenesis. However, there were no reports presented so far concerning β-galactosidase activity in the yolk platelet fraction of Xenopus eggs. A recent study based on mass-spectrometric analysis have identified a number of plasma and lysosomal proteins in the yolk platelets of Xenopus eggs, however it failed to detect β-galactosidase protein in this compartment (Jorgensen et al., 2009). The reason for this failure may be related to the fact that the protocol of sample preparation for proteomic analysis included clarifying centrifugation of the yolk platelet extracts to pellet yolk platelet crystals. Although this step removes >99% of the vitellogenin derivatives, allowing mass-spectrometric detection of minor yolk platelet proteins, it may also remove some proteins tightly associated with the yolk core. At present, the exact location of SA-β-gal inside the yolk granules is not known. Previously, microscopic and ultrastructural studies of Xenopus laevis eggs disclosed that the yolk platelets are composed of a limiting membrane, a central crystal of vitellogenin derivatives, and an intervening superficial layer of unknown composition (Karasaki, 1963; Romano et al., 2004; Jorgensen et al., 2009). Further tests are required to reveal detailed ultrastructural composition and proteome of these organelles.