In an effort to better understand oocyte function, we utilized two-dimensional (2D) electrophoresis and mass spectrometry to identify proteins that are differentially expressed during murine oocyte maturation. Proteins from 500 germinal vesicle (GV) and metaphase II-(MII) arrested oocytes were extracted, resolved on 2D electrophoretic gels, and stained with silver. Analysis of the gels indicated that 12 proteins appeared to be differentially expressed between the GV and MII stage. These proteins were then cored from the 2D gels and identified by mass spectrometry as: transforming acidic coiled-coil protein 3 (TACC3), heat shock protein 105 (HSP105), programmed cell death six-interacting protein (PDCD6IP), stress-inducible phosphoprotein (STI1), importin alpha2, adenylsuccinate synthase (ADDS), nudix, spindlin, lipocalin, lysozyme, translationally controlled tumor protein (TCTP), and nucleoplasmin 2 (NPM2). Interestingly, PDCD6IP, importin alpha2, spindlin, and NPM2 appear slightly larger in mass and more acidic on the MII oocyte gel compared to the GV oocyte gel, suggesting that they may be post-translationally modified during oocyte maturation. Given NPM2 is an oocyte-restricted protein, we chose to further investigate its properties during oocyte maturation and preimplantation development. Real-Time RT-PCR showed that NPM2 mRNA levels rapidly decline at fertilization. Indirect immunofluorescence analysis showed that, with the exception of cortical localization in MII-arrested oocytes, NPM2 is localized to the nucleus of both GV stage oocytes and all stages of preimplantation embryos. We then performed one-dimensional (1D) western blot analysis of mouse oocytes and preimplantation embryos and found that, as implicated by the 2D gel comparison, NPM2 undergoes a phosphatase-sensitive electrophoretic mobility shift during the GV to MII transition. The slower migrating NPM2 form is also present in pronuclear embryos but by the two-cell stage, the majority of NPM2 exists as the faster migrating form, which persists to the blastocyst stage.

Diurnal clearance phagocytosis by the retinal pigment epithelium (RPE) is a conserved efferocytosis process whose binding step is mediated by αvβ5 integrin receptors. Two related annexins, A5 (ANXA5) and A6 (ANXA6), share an αvβ5 integrin-binding motif. Here, we report that ANXA5, but not ANXA6, regulates the binding capacity for spent photoreceptor outer segment fragments or apoptotic cells by fibroblasts and RPE. Similar to αvβ5-deficient RPE, ANXA5 −/− RPE in vivo lacks the diurnal burst of phagocytosis that follows photoreceptor shedding in wild-type retina. Increasing ANXA5 in cells lacking αvβ5 or increasing αvβ5 in cells lacking ANXA5 does not affect particle binding. Association of cytosolic ANXA5 and αvβ5 integrin in RPE in culture and in vivo further supports their functional interdependence. Silencing ANXA5 is sufficient to reduce levels of αvβ5 receptors at the apical phagocytic surface of RPE cells. The effect of ANXA5 on surface αvβ5 and on particle binding requires the C-terminal ANXA5 annexin repeat but not its unique N-terminus. These results identify a novel role for ANXA5 specifically in the recognition and binding step of clearance phagocytosis, which is essential to retinal physiology. This article has an associated First Person interview with the first author of the paper.

The apical plasma membrane domain of retinal pigment epithelial (RPE) cells in the eye faces the outer segment portions of rods and cones and the inter-photoreceptor matrix in the subretinal space. Two important receptor-mediated interactions between the apical surface of the retinal pigment epithelium (RPE) and adjacent photoreceptors are adhesion ensuring outer segment alignment and diurnal phagocytosis of shed outer segment fragments contributing to outer segment renewal. Both depend on the apical distribution of the integrin family adhesion receptor αvβ5 as lack of αvβ5 in mice causes weakened retinal adhesion and asynchronous phagocytosis. With age, lack of αvβ5 leads to accumulation of harmful lipofuscin in the RPE and to vision loss. Here, we discuss three different possible mechanisms that could generate the exclusive apical distribution of αvβ5 integrin receptors in the RPE. (1) αvβ5 could be apical in the RPE because RPE attachment to neural retina generally or αvβ5 ligands specifically in the subretinal space stabilize apical but not basolateral αvβ5 surface receptors. (2) αvβ5 could be apical in the RPE because it resides in a complex with other components of the phagocytic machinery that assembles at the apical, phagocytic surface of the RPE. (3) αvβ5 could be apical due to mechanisms intrinsic to this receptor protein and specifically to its β5 integrin subunit.