Ignacio S. Alvarez

Phosphorylation of STIM1 at ERK1/2 target sites modulates store-operated calcium entry

Store-operated calcium entry (SOCE) is an important Ca2+ entry pathway that regulates many cell functions. Upon store depletion, STIM1, a transmembrane protein located in the endoplasmic reticulum (ER), aggregates and relocates close to the plasma membrane (PM) where it activates store-operated calcium channels (SOCs). Although STIM1 was early defined as a phosphoprotein, the contribution of the phosphorylation has been elusive. In the present work, STIM1 was found to be a target of extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in vitro, and we have defined the ERK1/2-phosphorylated sites on the STIM1 sequence. Using HEK293 cells stably transfected for the expression of tagged STIM1, we found that alanine substitution mutants of ERK1/2 target sites reduced SOCE significantly, suggesting that phosphorylation of these residues are required to fully accomplish SOCE. Indeed, the ERK1/2 inhibitors PD184352 and PD0325901 decreased SOCE in transfected cells. Conversely, 12-O-tetradecanoylphorbol-13-acetate, which activates ERK1/2, enhanced SOCE in cells expressing wild-type tagged STIM1, but did not potentiate Ca2+ influx in cells expressing serine to alanine mutations in ERK1/2 target sites of STIM1. Alanine substitution mutations decreased Ca2+ influx without disturbing the aggregation of STIM1 upon store depletion and without affecting the relocalization in ER–PM punctae. However, our results suggest that STIM1 phosphorylation at ERK1/2 target sites can modulate SOCE by altering STIM1 binding to SOCs, because a significant decrease in FRET efficiency was observed between alanine substitution mutants of STIM1–GFP and ORAI1–CFP.

Contribution of culture media to oxidative stress and its effect on human oocytes

The adverse effects of reactive oxygen species (ROS) on many aspects of reproduction are well documented. However, much less is known regarding the contribution of culture media to the oxidative stress of gametes during assisted reproductive techniques. This study measured the generation of ROS by culture media during IVF procedures and its effects on human oocytes. Commercially supplied culture media generated ROS at various rates, depending on the composition, whereas follicular fluid generated ROS at a much lower level. The incubation of cumulus-oocyte complexes (COC) in culture media induced marked lipid peroxidation compared with levels found in freshly retrieved COC. This plasma membrane damage, measured with the quenching of cis-parinaric acid fluorescence assay, was attenuated by supplementation of the medium with alpha-tocopherol or catalase. Moreover, there was an association between ROS production by culture medium and thiolic content consumption within the oocytes, suggesting that the intracellular reduced glutathione pool was partially depleted during in-vitro manipulation. The results show that culture medium could damage oocytes (and consequently embryo development) depending on their composition, and it is proposed that current IVF protocols could be revised in order to decrease ROS generation.

Pax2, Otx2, Gbx2 and Fgf8 expression in early otic vesicle development.

The inner ear is a suitable system to study the mechanisms involved in the specification of different functional domains during morphogenesis. Using single and double in situ hybridization (ISH) we show that three transcription factors (Otx2, Gbx2and Pax2) and a member of the fibroblast growth factor family (Fgf8) could participate in the compartmentalization of the otic vesicle and in the formation of the acoustic-vestibular ganglion.

Induction of cardiogenesis by Hensen's node and fibroblast growth factors

Abstract. The earliest events underlying cardiac induction and morphogenesis remain largely unknown. In the present study, we show that Hensens node, the organizer of the avian embryo, induces cardiogenesis. Specifically, following heterotopic transplantation, Hensens node induces ectopic host tissue that expresses two early cardiac markers (cNkx-2.5 and cNkx-2.8), as well as a ventricular marker (VMHC1), but not an atrial marker (AMHC1). Moreover, we examine the potential roles of candidate growth factors known to be secreted by Hensens node. Our results show that fibroblast growth factors (FGF-2 and FGF-4) when ectopically expressed can initiate cardiac development, inducing host tissue to express the two cardiac transcription factors cNkx-2.5 and cNkx-2.8, as well as the cardiac-restricted structural gene VMHC1, but not AMHC1. In contrast to FGFs, TGFβ family members fail to induce ectopic tissue and expression of cardiac marker genes. We also examined the effects of growth factors on the morphogenesis of the host embryos heart. Both exogenous FGFs and TGFβ family members perturb normal morphogenesis of the early cardiac tube and alter patterns of ventricular and atria gene expression in characteristic ways. Namely, exogenous FGFs expand areas expressing the ventricular marker VMHC1 at the expense of areas expressing the atrial marker AMHC1. Conversely, exogenous TGFβ1 inhibits expression of VMHC1, expanding AMHC1 expression. We show here that Hensens node and FGFs induce ectopic expression of cardiac lineage markers, and that FGF and TGFβ family members can modulate early development of the heart. Collectively, these data suggest that the organizer plays a crucial role in cardiac induction and morphogenesis, mediated in part by endogenous members of the FGF and TGFβ families.

Calcium signaling in mouse oocyte maturation: the roles of STIM1, ORAI1 and SOCE

Calcium handling is critical for the oocyte function, since the first steps of fertilization are dependent on the appropriate Ca(2+) mobilization to originate transient spikes of the cytosolic Ca(2+) concentration. It is well known that the Ca(2+) influx from the extracellular milieu is required to maintain this signaling in mammalian oocytes. However, the regulation of the Ca(2+) channels involved in this process is still unknown in oocytes. STIM1, a key regulator of store-operated Ca(2+) entry (SOCE), relocates in the mouse oocyte shortly after sperm stimulation, suggesting that SOCE is involved in the maintenance of cytosolic Ca(2+)-spiking in the fertilized oocyte. Here, we show that there is an up-regulation of the expression of STIM1 at the germinal vesicle breakdown stage, and this expression remains steady during following maturation stages. We found that oocytes express ORAI1, a store-operated Ca(2+) channel, and that ORAI1 expression level was stable during oocyte maturation. Immature oocytes showed no Ca(2+) entry and no increase in STIM1-ORAI1 colocalization in response to the store depletion induced by thapsigargin. On the contrary, in mature oocytes, STIM1-ORAI1 colocalization is enhanced 3-fold by depletion of Ca(2+) stores, enabling the activation of store-operated calcium channels and therefore Ca(2+) entry. Finally, the correlation between SOCE activation during the maturation of oocytes and STIM1-ORAI1 colocalization strongly suggests that ORAI1 is involved in the Ca(2+) entry pathway in the mature oocyte. SOCE up-regulation in the final stage of maturation is further evidence of a major role for SOCE in fully mature oocytes, and therefore in Ca(2+) signaling at fertilization.

Store-Operated Calcium Entry in Human Oocytes and Sensitivity to Oxidative Stress

Abstract Calcium signaling is a cellular event that plays a key role at many steps of fertilization and early development. However, little is known regarding the contribution of extracellular Ca2+ influx into the cell to this signaling in gametes and early embryos. To better know the significance of calcium entry on oocyte physiology, we have evaluated the mechanism of store-operated calcium entry (SOCE) in human metaphase II (MII) oocytes and its sensitivity to oxidative stress, one of the major factors implicated in the outcome of in vitro fertilization (IVF) techniques. We show that depletion of intracellular Ca2+ stores through inhibition of sarco(endo)plasmic Ca2+-ATPase with thapsigargin triggers Ca2+ entry in resting human oocytes. Ba2+ and Mn2+ influx was also stimulated following inhibition, and Ca2+ entry was sensitive to pharmacological inhibition because the SOCE blocker 2-aminoethoxydiphenylborate (2-APB) reduced calcium and barium entry. These results support the conclusion that there is a plasma membrane mechanism responsible for the capacitative divalent cation entry in human oocytes. Moreover, the Ca2+ entry mechanism described in MII oocytes was found to be highly sensitive to oxidative stress. Hydrogen peroxide, at micromolar concentrations that could mimic culture conditions in IVF, elicited an increase of [Ca2+]i that was dependent on the presence of extracellular Ca2+. This rise was preventable by 2-APB, indicating that it was mainly due to the enhanced influx through store-operated calcium channels. In sum, our results demonstrate the occurrence of SOCE in human MII oocytes and the modification of this pathway due to oxidative stress, with possible consequences in IVF.

Proliferation of glial precursors during the early development of the chick optic nerve

SummaryThe mitotic patterns and cytoarchitecture of the optic stalk were studied in the chick embryo during the period of formation of the optic cup until the elimination of the stalk lumen. Cell proliferation in the superficial regions of the stalk ventral wall is described. Superficial cell proliferation, whose beginning coincides with penetration of the earliest optic fibers, gives rise to an early glioblast plate located internally with respect to the marginal lamina of ganglion cell axon fascicles. The early glioblasts are transformed into marginal glioblasts, which undergo radial mitosis. Radially orented division seems to favour glioblast penetration towards more internal zones of the stalk. Thus the marginal glioblasts are transformed into inner glioblasts, which continue to proliferate. Cell death in the ventral wall of the optic stalk is in close topographical relation with extracellular spaces which loosen up the consistency of the stalk tissue, favouring invasion of the ventral stalk by optic fibers and the addition of new glioblasts by proliferation of preexisting cells.

Relocalization of STIM1 in mouse oocytes at fertilization: early involvement of store-operated calcium entry

Calcium waves represent one of the most important intracellular signaling events in oocytes at fertilization required for the exit from metaphase arrest and the resumption of the cell cycle. The molecular mechanism ruling this signaling has been described in terms of the contribution of intracellular calcium stores to calcium spikes. In this work, we considered the possible contribution of store-operated calcium entry (SOCE) to this signaling, by studying the localization of the protein STIM1 in oocytes. STIM1 has been suggested to play a key role in the recruitment and activation of plasma membrane calcium channels, and we show here that mature mouse oocytes express this protein distributed in discrete clusters throughout their periphery in resting cells, colocalizing with the endoplasmic reticulum marker calreticulin. However, immunolocalization of the endogenous STIM1 showed considerable redistribution over larger areas or patches covering the entire periphery of the oocyte during Ca(2+) store depletion induced with thapsigargin or ionomycin. Furthermore, pharmacological activation of endogenous phospholipase C induced a similar pattern of redistribution of STIM1 in the oocyte. Finally, fertilization of mouse oocytes revealed a significant and rapid relocalization of STIM1, similar to that found after pharmacological Ca(2+) store depletion. This particular relocalization supports a role for STIM1 and SOCE in the calcium signaling during early stages of fertilization.

Patterns of neurepithelial cell rearrangement during avian neurulation are determined prior to notochordal inductive interactions

In the epiblast of elongating primitive-streak-stage avian embryos, MHP cells--short wedge-shaped neurepithelial cells contained within the median hinge point of the bending neural plate--arise from the midline prenodal and nodal area, whereas L cells--tall spindle-shaped neurepithelial cells constituting the lateral neural plate--arise from paired areas flanking the cranial primitive streak. These characteristic differences in neurepithelial cell shape are acquired as a result of inductive interactions with the notochord. Both MHP and L cells undergo extensive rearrangement (intercalation) during shaping and bending of the neural plate, but their pattern of rearrangement differs. MHP cells intercalate with other MHP cells and the population always spans the midline, whereas L cells intercalate with other L cells, remaining in bulk lateral to the midline. The following experiment was performed to establish whether these distinctive rearrangement patterns are determined prior to notochordal inductive interactions. Quail prospective MHP and L cells were transplanted isochronically and heterotopically to chick host blastoderms at stages prior to formation of the notochord (to wit, prospective MHP cells were transplanted into prospective L cell territory and vice versa) and the distribution, fate, and morphological characteristics of grafted cells were determined in chimeras collected 24 hr later. Our results demonstrate that heterotopic MHP and L cells do not adopt the rearrangement pattern characteristic of their new site; rather, they change their position so that grafted MHP cells intermix with MHP cells of the host and grafted L cells intermix with L cells of the host. Thus, patterns of neurepithelial cell rearrangement are determined prior to notochordal inductive interactions. When and how this determination occurs are topics for further studies.

Role of cell rearrangement in axial morphogenesis.

Publisher Summary The chapter discusses the role of cell rearrangement in processes as diverse as chick neurulation, archenteron formation, elongation in sea urchin gastrulation, epiboly, and convergent extension in Xenopus gastrulation and epiboly in teleost fish gastrulation. Cell rearrangement is merely one of a number of the fundamental cell behaviors having morphogenetic consequences. The extensive cell rearrangements occurring during gastrulation and neurulation lead to the formation of the axial and associated rudiment. It is highly unlikely that any single, particular morphogenetic movement is driven by only one cell behavior. Rather, various behaviors typically act in concert. One possible interaction among three cell behaviors—change in cell shape, change in cell position, and change in cell number—is cell division during shaping and bending of the avian neural plate. All three of these behaviors are involved in neurulation.