Associated editor's comment: Calcium signalling in human oocytes and embryos: two-store model revival

Associate editor’s comment on the article ‘Calcium-binding proteins and calcium-release channels in human maturing oocytes, pronuclear zygotes and early preimplantation embryos’ by H. Balakier et al. Jan Tesarik

Since the first observations showing that sperm-induced activation of mouse oocytes is mediated by sustained oscillations of intracellular free calcium concentration (Cuthbertson and Cobbold, 1985Go), similar patterns of sperm-induced calcium signals have been observed in various other mammalian species (Jones, 1998Go; review). Sperm-induced calcium oscillations were also observed in human oocytes, both after conventional IVF (Taylor et al., 1993Go) and after ICSI (Tesarik et al., 1994Go; Tesarik and Sousa, 1994Go).

The rapid expansion of clinical applications of ICSI in the early-to-mid 1990s raised considerable concern about the safety of this technique, which prompted research into the differences in the form of sperm-induced calcium signals generated in conventionally-fertilized and ICSI-treated oocytes. It was noted that human oocytes recently activated by sperm, regardless of the mode of sperm delivery, display a particular pattern of spatial propagation of calcium signals during each of the sequential spikes of the sperm-induced calcium oscillation period; this pattern is characterized by a very rapid increase in intracellular free calcium concentration in the oocyte periphery, followed by a much slower but greater and longer calcium increase in the rest of the oocyte cytoplasm (Tesarik et al., 1995Go). This spatial pattern of calcium oscillations is different from those reported in other mammalian (Miyazaki et al., 1986Go; Sato et al., 1999Go) and non-mammalian (Kyozuka et al., 1998Go) species.

To explain this particular pattern of sperm-induced calcium oscillations in human oocytes, it was suggested that the periphery of mature human oocytes and the rest of the oocyte cytoplasm are equiped with calcium stores with a different threshold for calcium-induced calcium release (CICR) and calcium binding capacity (Tesarik and Sousa, 1996Go). This model was further corroborated by the observation of non-random distribution of ryanodine-sensitive and ryanodine-insensitive calcium stores in mature human oocytes, the former being found throughout the oocyte cytoplasm and the latter being restricted to a narrow band around the oocyte periphery (Sousa et al., 1996Go). In spite of these reports, the existence of functionally relevant ryanodine-sensitive calcium stores in human oocytes and embryos was questioned by many authors who argued with earlier observations obtained in hamster oocytes whose calcium oscillation mechanism appears to be entirely dependent on the presence of calcium stores gated by inositol trisphosphate (InsP3) receptors (Miyazaki et al., 1992Go; Parys et al., 1992Go).

In this issue Balakier et al. present the first direct evidence for the presence and non-random distribution of both InsP3-sensitive and ryanodine-sensitive calcium stores in human oocytes and early preimplantation embryos (Balakier et al., 2002Go). The predominance of InsP3 receptor-2 in the cell cortex and the diffuse distribution of ryanodine receptors throughout the entire cytoplasm of metaphase II human oocytes, demonstrated in the paper by Balakier et al. fits well with the model of InsP3-sensitive and ryanodine-sensitive calcium store distribution derived from confocal microscopy analysis of sperm-induced calcium rises in human oocytes (Tesarik and Sousa, 1996Go). Moreover, the observations by Balakier et al. suggest that each of the two types of calcium stores present in human oocytes are associated with a different calcium binding protein; the distribution of calreticulin and calsequenstrin with the oocytes is shown to match that of InsP3 and ryanodine receptors respectively. This is a new and important aspect of the two-store model of calcium oscillations in human oocytes which help the understanding of previous observations on the spatial patterns of sperm-induced calcium waves (Tesarik et al., 1995Go). In fact, each of the sequential calcium discharges during the steady-state phase of sperm-induced calcium oscillations was shown to be started from stores located in the oocyte periphery. This early discharge is likely to be mediated by a calcium-induced calcium release (CICR) mechanism when the concentration of intracellular free calcium, which shows a slow and progressive increase in the period between two sequential calcium spikes, attains a threshold at which CICR is activated. This limited calcium discharge, initially restricted to the oocyte periphery, can thus act as a detonator for generalized, massive calcium discharge from stores located throughout the oocyte cytoplasm. In the context of these earlier observations, the data published in this issue by Balakier et al. suggest that the peripheral stores with the relatively low threshold for CICR and low calcium binding capacity are gated by the InsP3 receptor and their principal calcium binding protein is calreticulin, whereas the higher threshold stores that are responsible for the bulk of calcium release during each spike are gated by ryanodine receptor and contain calsequestrin as the main calcium binding protein (Balakier et al., 2002Go).

What is the reason for the relatively low threshold of the peripheral calcium stores in recently fertilized human oocytes? Following the observation that calcium oscillations induced by the thiol reagent thimerosal do not show the periphery-to-centre calcium wave propagation typical of the sperm-induced calcium oscillations (Tesarik et al., 1995Go), it is very probable that the locally restricted decrease in CICR threshold in the periphery of recently fertilized human oocytes is related to the action of a factor released from sperm. The most recently suggested candidate for the sperm-specific trigger of calcium oscillations in oocytes is a sperm-specific phospholipase C (Saunders et al., 2002Go). By releasing InsP3 from phospholipids the sperm-delivered phospholipase C may thus selectively decrease the CICR threshold for the InsP3-sensitive stores in the periphery of human oocytes without influencing the CICR threshold in the rest of the oocyte’s calcium stores. This particular calcium store ‘tuning’ appears to be vital to prolonged continuation of calcium oscillations because a simultaneous decrease in the CICR threshold of ryanodine sensitive stores inhibits sperm-induced calcium oscillations in human oocytes (Sousa et al., 1996Go).

The maintenance of differential calcium store sensitivity to CICR during sperm-induced calcium oscillations appears to require the continuous action of sperm factor. A recent study comparing the sensitization of the calcium release mechanism obtained by the injection of the whole sperm or soluble sperm extract to the cytoplasm of mouse oocytes has suggested that the stability of sperm factor in the oocyte cytoplasm depends on its association with intracellular membranes (Gordo et al., 2002Go). Both the mode of sperm factor delivery to the oocyte and the functional status of oocyte organelles may thus condition the form and duration of fertilization-induced calcium signals.

In the context of the above considerations, the paper by Balakier et al. opens new perspectives in the study of the mechanisms underlying human oocyte activation (Balakier et al., 2002Go). This research is needed in view of the ever increasing use of micromanipulation-assisted fertilization techniques, including those using defective or immature spermatozoa or sperm precursor cells in which the content of oocyte-activating sperm factor or its physical association with protective intracellular structures may be abnormal. A deeper insight into the mechanism of oocyte activation will also be required for the investigation and development of new assisted reproduction techniques potentially applicable in the future, such as oocyte maturation and cryopreservation and the use of haploidized somatic cells for embryo creation.


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