Store-Operated Calcium Entry in Human Oocytes and Sensitivity to Oxidative Stress1

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 Ca(2+) 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 Ca(2+) stores through inhibition of sarco(endo)plasmic Ca(2+)-ATPase with thapsigargin triggers Ca(2+) entry in resting human oocytes. Ba(2+) and Mn(2+) influx was also stimulated following inhibition, and Ca(2+) 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 Ca(2+) 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 [Ca(2+)](i) that was dependent on the presence of extracellular Ca(2+). 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.

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

“…Intracellular Ca 2C store depletion induced by TG leads to the opening of Ca 2C channels at the PM, which is controlled by the filling state of these Ca 2C stores. Since Mn 2C is transported through SOC channels, this technique is a very convenient method to describe the existence of SOCE, as we have reported previously (Gutierrez-Martin et al 2005, Martin-Romero et al 2008.…”

“…First, we examined the occurrence of SOCE in mouse metaphase II (MII) oocytes as we reported recently for human oocytes (Martin-Romero et al 2008). SOCE was activated in mouse MII oocytes with the addition of thapsigargin (TG), a potent and specific inhibitor of the SERCA (Thastrup et al 1990).…”

“…This opening of SOC channels can be followed by employing calcium or barium addition to the extracellular medium. Barium (Ba 2C ) has been shown to enter the cell via SOC channels in other cell types (Hoth 1995, Fischer et al 1998, as well as in oocytes (Martin-Romero et al 2008), but cannot be extruded by PM calcium pumps. Since fura-2 is also sensitive to Ba 2C , this offers a useful tool with which to reveal the occurrence of SOCE.…”

“…However, the pathway for this Ca 2C entry in mammalian oocytes at fertilization remains unknown. SOCE could be one of the mechanisms regulating Ca 2C entry at fertilization, since this pathway has been observed in Xenopus oocytes (Machaca & Haun 2000), porcine oocytes (Machaty et al 2002), mouse oocytes (McGuinness et al 1996, Halet et al 2004, and recently in human oocytes (Martin-Romero et al 2008) in resting conditions. Despite the importance of this mechanism in Ca 2C signaling, the actual function of SOCE at fertilization has yet to be addressed.…”

The birth of quail chicks after intracytoplasmic sperm injection

“…3H). We employed heparin (Yue et al, 1995) and 2-aminoethoxydiphenyl borate (2-APB), a selective inhibitor of IP 3 receptor (IP 3 -R) (Martin-Romero et al, 2008) Fig. S2B).…”