Naofumi Miwa

S100–annexin complexes – biology of conditional association

S100 proteins and annexins both constitute groups of Ca2+‐binding proteins, each of which comprises more than 10 members. S100 proteins are small, dimeric, EF‐hand‐type Ca2+‐binding proteins that exert both intracellular and extracellular functions. Within the cells, S100 proteins regulate various reactions, including phosphorylation, in response to changes in the intracellular Ca2+ concentration. Although S100 proteins are known to be associated with many diseases, exact pathological contributions have not been proven in detail. Annexins are non‐EF‐hand‐type Ca2+‐binding proteins that exhibit Ca2+‐dependent binding to phospholipids and membranes in various tissues. Annexins bring different membranes into proximity and assist them to fuse, and therefore are believed to play a role in membrane trafficking and organization. Several S100 proteins and annexins are known to interact with each other in either a Ca2+‐dependent or Ca2+‐independent manner, and form complexes that exhibit biological activities. This review focuses on the interaction between S100 proteins and annexins, and the possible biological roles of these complexes. Recent studies have shown that S100–annexin complexes have a role in the differentiation of gonad cells and neurological disorders, such as depression. These complexes regulate the organization of membranes and vesicles, and thereby may participate in the appropriate disposition of membrane‐associated proteins, including ion channels and/or receptors.

Dicalcin Inhibits Fertilization through Its Binding to a Glycoprotein in the Egg Envelope in Xenopus laevis

Fertilization comprises oligosaccharide-mediated sperm-egg interactions, including sperm binding to an extracellular egg envelope, sperm penetration through the envelope, and fusion with an egg plasma membrane. We show that Xenopus dicalcin, an S100-like Ca2+-binding protein, present in the extracellular egg envelope (vitelline envelope (VE)), is a suppressive mediator of sperm-egg interaction. Preincubation with specific antibody greatly increased the efficiency of in vitro fertilization, whereas prior application of exogenous dicalcin substantially inhibited fertilization as well as sperm binding to an egg and in vitro sperm penetration through the VE protein layer. Dicalcin showed binding to protein cores of gp41 and gp37, constituents of VE, in a Ca2+-dependent manner and increased in vivo reactivity of VE with a lectin, Ricinus communis agglutinin I, which was accounted for by increased binding ability of gp41 to the lectin and greater exposure of gp41 to an external environment. Our findings strongly suggest that dicalcin regulates the distribution of oligosaccharides within the VE through its binding to the protein core of gp41, probably by modulating configuration of oligosaccharides on gp41 and the three-dimensional structure of VE framework, and thereby plays a pivotal role in sperm-egg interactions during fertilization.

Lack of hippocalcin causes impairment in Ras/extracellular signal-regulated kinase cascade via a Raf-mediated activation process.

Hippocalcin (Hpca) is a member of the neuronal calcium sensor protein family and is highly expressed in hippocampal neurons. Hpca‐deficient (Hpca–/–) mice display a defect in cAMP response element‐binding protein (CREB) activation associated with impaired spatial and associative memory. Here we examine the involvement of Hpca in the extracellular signal‐regulated kinase (ERK) cascade leading to CREB activation, because application of PD98059, a broad ERK cascade inhibitor, has resulted in similar levels of CREB activation in Hpca–/– hippocampus. N‐methyl‐D‐aspartate (NMDA)‐ and KCl‐induced phosphorylation of ERK was significantly attenuated in Hpca–/– hippocampal slices, as was ionomycin‐induced phosphorylation of ERK, whereas forskolin and 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) stimulation yielded indistinguishable levels of ERK phosphorylation in both wild‐type and Hpca–/– slices. In an in vitro reconstitution assay system, recombinant Hpca affected neither Raf‐1 protein kinase activity with recombinant MEK‐1 as a substrate nor MEK‐1 kinase activity with ERK2 as a substrate. Activation of Ras by NMDA and KCl stimulation of hippocampal slices showed no obvious changes between the two genotypes; however, phosphorylation of Raf‐1 was significantly lower in Hpca–/– slices. These results suggest that Hpca plays an important role in the activation of Raf conducted by Ras.

Characterization of S100A11, a suppressive factor of fertilization, in the mouse female reproductive tract

We recently found that Xenopus dicalcin, present in the extracellular egg‐coating envelope, suppresses the efficiency of fertilization in vitro through binding to envelope‐constituent glycoproteins. In the present study, we explored the mouse counterpart of Xenopus dicalcin, specifically its localization in the female reproductive tract and its action on mouse fertilization. Our homology and phylogenetic analyses using known S100 proteins showed that S100A11 is most closely related to Xenopus dicalcin. S100A11 was localized in the cytosol of luteal cells, but not in the follicle, in the mouse ovary, and also in the cytosol of the oviductal epithelial cells. In addition, our quantitative analyses revealed preferential expression of S100A11 in the ampullary region of the oviduct and at the estrus stage during the mouse estrous cycle. In the cumulus cell–oocyte complex dissected from the oviduct following ovulation, S100A11 was present in the plasma membrane of cumulus cells, but not in the zona pellucida, which is comparable with Ca2+‐dependent binding of exogenously applied S100A11 to the plasma membrane of cumulus cells. Pretreatment of the cumulus cell–oocyte complex with recombinant S100A11 substantially reduced the efficiency of in vitro fertilization, but S100A10, the next closest S100 protein to Xenopus dicalcin, had no effect. These results suggested that S100A11 is the mouse counterpart of Xenopus dicalcin, suppresses the fertilization process through its action on cumulus cells, and thereby plays a key role in fertilization success in the mouse. Mol. Reprod. Dev. 78:91–103, 2011.

Protein-Carbohydrate Interaction between Sperm and the Egg-Coating Envelope and Its Regulation by Dicalcin, a Xenopus laevis Zona Pellucida Protein-Associated Protein

Protein-carbohydrate interaction regulates multiple important processes during fertilization, an essential biological event where individual gametes undergo intercellular recognition to fuse and generate a zygote. In the mammalian female reproductive tract, sperm temporarily adhere to the oviductal epithelium via the complementary interaction between carbohydrate-binding proteins on the sperm membrane and carbohydrates on the oviductal cells. After detachment from the oviductal epithelium at the appropriate time point following ovulation, sperm migrate and occasionally bind to the extracellular matrix, called the zona pellucida (ZP), which surrounds the egg, thereafter undergoing the exocytotic acrosomal reaction to penetrate the envelope and to reach the egg plasma membrane. This sperm-ZP interaction also involves the direct interaction between sperm carbohydrate-binding proteins and carbohydrates within the ZP, most of which have been conserved across divergent species from mammals to amphibians and echinoderms. This review focuses on the carbohydrate-mediated interaction of sperm with the female reproductive tract, mainly the interaction between sperm and the ZP, and introduces the fertilization-suppressive action of dicalcin, a Xenopus laevis ZP protein-associated protein. The action of dicalcin correlates significantly with a dicalcin-dependent change in the lectin-staining pattern within the ZP, suggesting a unique role of dicalcin as an inherent protein that is capable of regulating the affinity between the lectin and oligosaccharides attached on its target glycoprotein.

Fertilization competence of the egg-coating envelope is regulated by direct interaction of dicalcin and gp41, the Xenopus laevis ZP3.

Fertilization begins with species-restricted interaction of sperm and the egg-coating envelope, which includes a three-dimensional meshwork of filaments composed of glycoproteins (called ZP proteins). Growing evidence has unveiled the molecular nature of ZP proteins; however, the structural property conferring fertilization competence to the egg-coating envelope remains unknown. Here, we show the molecular mechanism that mediates direct interaction between dicalcin, a novel fertilization-suppressive ZP protein-associated protein, and gp41, a Xenopus laevis ortholog of mammalian ZP3, and subsequently demonstrate the structural basis of the envelope for fertilization competence. The interactive regions between dicalcin and gp41 comprised six and nine amino acid residues within dicalcin and twenty-three within gp41. Synthetic peptides corresponding to these regions dramatically affected fertilization: treatment with dicalcin- or gp41-derived peptides decreased or increased fertilization rates, respectively. Prior application of these peptides caused distinct alterations in the in vivo lectin-staining pattern of the envelope as well. Transmission electron microscopy analysis revealed that the dicalcin-derived peptide induced the formation of a well-organized meshwork, whereas the gp41-derived peptide caused the formation of a significantly disorganized meshwork. These findings indicated that the fertilization competence of the egg-coating envelope is crucially regulated by the direct interaction between dicalcin and gp41.

Dicalcin, a zona pellucida protein that regulates fertilization competence of the egg coat in Xenopus laevis.

Fertilization is a highly coordinated process whereby sperm interact with the egg-coating envelope (called the zona pellucida, ZP) in a taxon-restricted manner, Fertilization triggers the resumption of the cell cycle of the egg, ultimately leading to generation of a new organism that contains hereditary information of the parents. The complete sperm-ZP interaction comprises sperm recognition of the ZP, the acrosome reaction, penetration of the ZP, and fusion with the egg. Recent evidence suggests that these processes involve oligosaccharides associated with a ZP constituent (termed ZP protein), the polypeptide backbone of a ZP protein, and/or the proper three-dimensional filamentous structure of the ZP. However, a detailed description of the molecular mechanisms involved in sperm-ZP interaction remains elusive. Recently, I found that dicalcin, a novel ZP protein-associated protein, suppresses fertilization through its association with gp41, the frog counterpart of the mammalian ZPC protein. This review focuses on molecular aspects of sperm-ZP interaction and describes the fertilization-suppressive function of dicalcin and associated molecular mechanisms. The amount of dicalcin in the ZP significantly correlates with alteration of the lectin-staining pattern within the ZP and the orientation pattern of ZP filaments, which may assist in elucidating the complex molecular mechanisms that underlie sperm-ZP interaction.

Aging of Oocyte-Coating Structures and Dicalcin

Abstract: Maternal fertility decreases with aging, possibly owing to qualitative changes in the egg itself and the egg-housing condition of the female reproductive tract. Since sperm first interact with the egg-coating structures, age-dependent alterations of egg-coating structures may be related to reduced fertility in aged female mammals. Quantitative genetic and biochemical studies of specimens prepared from aged mammals have revealed altered expression patterns of antioxidant- and apoptosis-related proteins in cumulus cells, and a substantial decrease in the amount of ZP-constituent glycoproteins. Histochemical studies have demonstrated an increase in the number of apoptotic cumulus cells and significant alteration in the appearance of ZP with irregular plaques in aged specimens. Biophysical studies have shown that both susceptibility to digestion with protease and mechanical stiffness are reduced in the ZP aged in vitro. We recently characterized a novel suppressive factor of fertilization, dicalcin, in the cumulus-oocyte complex. The expression level of dicalcin in the cultured normal human fibroblasts increases with the passage of time, which implies an age-dependent increase in its expression in the normal female reproductive tract. The potential increase in dicalcin expression with aging would represent a qualitative change of the egg-coating structure, augment its inhibitory role on fertilization, thereby causing decreased fertility in aged female mammals.

Structural and rheological properties conferring fertilization competence to Xenopus egg-coating envelope

The extracellular egg-coating envelope that comprises a meshwork of filaments polymerized by glycoproteins plays a pivotal role in species-selective sperm recognition and subsequent fertilization; however, the structural and rheological properties conferring fertilization competence to the egg-coating envelope remain poorly unveiled. Here we show several nanoscale-structural and viscoelastic properties of the egg-coat using the transmission electron microscopy and the quartz crystal microbalance experiments, following clamp of the egg-coat at either fertilization-competent or -incompetent statuses by short-term pretreatment with synthetic peptides. Individual filament of approximately 4.8 nm diameter crossed one another, forming several types of intersections. Higher competence-inducing treatment changed the proportion of V-, Y-, and T-type intersections, and induced more randomly deflected angles at intersections. Incompetence-inducing treatment increased the median of a Gaussian distribution of filament lengths that had a peak of 10–20 nm under control conditions; furthermore, this treatment created bumps in the 30–40 and 50–60 nm windows. Quartz crystal microbalance study revealed that viscoelasticity of the competent VE suspension was lower than that of incompetent VE, indicating that viscoelastic property required for successful fertilization resides within a specific range. These findings indicated that the architecture of the egg-coat is capable of rapid and dynamic remodeling, which determines fertilization efficiency.

Immunohistochemical Characterization of S100A6 in the Murine Ovary

S100 proteins comprise a large family of Ca2+-binding proteins and exhibit a variety of intra- and extracellular functions. Despite our growing knowledge about the biology of S100 proteins in some tissues such as brain and smooth muscle, little is known about S100 proteins in the normal mammalian reproductive tissue. In the present study, we investigated the distribution pattern of S100A6 (alternatively named calcyclin) in the murine ovary by immunohistochemical study using specific antibody. S100A6 was localized substantially in the cytoplasm of luteal cells, with concomitant expression of S100A11, another S100 protein, but not in the other type of cells such as oocytes, follicle epithelial cells (granulosa cells), and cells of stroma including theca interna cells in the murine ovary. S100A6-immunoreactive corpora lutea (CLs) were divided into two types: homogeneously and heterogeneously stained CLs, and possibly they may represent differentiating and mature CL, respectively. Our regression analysis revealed that expression level of S100A6 positively correlated with that of cytochrome P450 11A, a steroidogenic enzyme in the heterogeously stained CL. These results suggested that S100A6 may contribute to differentiation of steroidogenic activity of luteal cells in a synergistic manner with S100A11 by facilitating some shared functions.