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My results suggest that NEBD of the 1-cell embryo and the mitotic divisions which lead to the formation of a momla are independent of InsPg-induced Ca^^ release. This is surprising given the observation that fertilised embryos generate Ca^^ transients at NEBD and during the first mitosis up until cleavage to the 2-cell stage (Kono et al., 1996). Moreover, NEBD in both fertilised (Kono et al., 1996; Tombes et al., 1992) and parthenogenetic (Kono et al., 1996) embryos is inhibited by the intracellular Ca^^ chelator BAPTA-AM. A possible cause of NEBD inhibition by BAPTA-AM is a non-specific effect. For example, lOpM BAPTA-AM has been shown to inhibit protein synthesis (a 2/3 decrease in leucine incorporation) in fertilised mouse eggs (Lawrence et al., 1998) and thus in pronucleate stage embryos may prevent the accumulation of MPF necessary for NEBD at the onset of mitosis. However, there is evidence to suggest that maximum levels of cyclin B are attained by as much as 2-3 hours prior to NEBD since onset of mitosis is unaffected if the protein synthesis inhibitor puromycin is added after this time (Hewlett, 1986). Therefore, inhibited MPF synthesis seems unlikely since in the Kono et al. (1996) (Kono et al., 1996) experiment BAPTA-AM was added within the 3 hours preceding NEBD.

There is, however, a more plausible explanation for inhibition of NEBD by BAPTA-AM. It should first be noted that mitotic Ca^^ transients are not always detected in some studies

(Tombes et al., 1992; Whitaker and Patel, 1990; Wilding et ah, 1996) and that, unlike fertihsed embryos, parthenogenotes do not generate detectable transients at NEBD or in mitosis (Kono et at., 1996). Despite the lack of detectable Ca^^ transients, parthenogenetic embryos develop normally, suggesting that the generation of global Ca^^ transients is not necessary for normal development. Rather than it having a non-specific effect, BAPTA-AM may be buffering localised Ca^^ transients, which are too small to be detected using conventional imaging systems, but which may be responsible for NEBD and progression through mitosis (Kao et a i, 1990; Kono et a i, 1996; Snow and Nuccitelli, 1993; Whitaker and Patel, 1990). Indeed, in one study on the sea urchin, global Ca^^ transients originating from the nuclear area were often detected just before NEBD of first mitosis. In the absence of global increases, however, confocal microscopy using Ca^^ Green-1 dextran revealed localised Ca^"^ transients in the nuclear region (Wilding et a i, 1996). The possibility that local Ca^^ transients in the nuclear region may induce NEBD and progression through mitosis is further supported by the finding that the nuclear envelope releases Ca^^ (Gerasimenko et al., 1995). These putative localised transients may be analogous to the InsPg-induced transient “puffs” (peak free Ca^^ 100-200nM) which have been evoked by photorelease of caged InsPg in Xenopus oocytes, and are thought to arise from the concerted opening of several clustered InsPgRs (Parker and Yao, 1996).

This study has shown that after downregulation by adenophostin in parthenogenetic pronucleate 1-cell embryos, 10-15% of InsPgRs remain. If local, undetectable Ca^^ transients are responsible for progression through mitosis, this study also suggests that 10- 15% of control levels of InsPgRs are sufficient to support these transients. This would permit the normal rate of development that I have seen, at least up to the morula stage, in these embryos.

InsP^Rs may concentrate at the pronuclei prior to NEBD

One explanation for how 10-15% of InsPgRs would be able to support local Ca^^ release is that receptors concentrate around the pronucleus prior to NEBD of mitosis. In embryos with depleted levels of InsPgRs, either via fertilisation or adenophostin, this may generate a limited area of receptors sufficiently dense to generate a Ca^^ gradient which would be located near the mitotic apparatus. Clustering of InsPgRs may be achieved either by (1) reorganisation of the ER by a cell cycle-dependent mechanism or by (2) a change in distribution of the InsPgRs themselves, independent of ER distribution. (1) In support of a reorganisation of the ER, there is evidence in the sea urchin for cell cycle changes in ER distribution during early development (Terasaki, 2000; Terasaki and Jaffe, 1991). The ER is uniformly distributed at interphase but gradually accumulates at the mitotic poles before NEBD and remains there during mitosis (Terasaki, 2000). (2) Alternatively, clustering may result from an aggregation of InsPgRs, independent of ER reorganisation. It has been shown that activation of AR4-2J cells with CCK stimulates receptor clustering in addition to InsPgR downregulation (Wojcikiewicz, 1995). Another study demonstrated Ca^^-

dependent InsP^R clustering in RBL-2H3 cells, which was independent of ER vésiculation (Wilson et al., 1998). Clusters were dispersed along the ER network and concentrated along the nuclear envelope (Wilson et al., 1998). Whether by (1) or (2), clustering of InsPgRs in the pronucleate egg may explain how a low number of receptors could support local Ca^"^ gradients.

Whether or not receptor clustering is stimulated in the fertihsed egg is not clear from my immunolocalisation studies. I have shown using immunocytochemistry that InsPgRs appear to be distributed fairly evenly throughout the ME oocyte before fertihsation and the pronucleate 1-ceU embryo after fertihsation, with no apparent receptor clustering taking place (Chapter 3). This data is supported by the finding that the ER of mature mouse eggs does not become disrupted during the first few Ca^"^ oscillations at fertihsation (Kline et al.,

1999). The distribution of receptors and ER throughout the cytoplasm may be beneficial to the fertihsed oocyte: global InsPg-mediated Ca^^ oscillations continue for up to 4 hours until pronuclear formation (see General Introduction), However, InsPgRs may stih increase in concentration around the pronucleus prior to NEBD in the fertihsed egg. This may not have been detected using immunocytochemistry because (1) the low resolution of antibody staining in this study may have obscured any receptor clustering at the pronuclear stage and (2) pronucleate eggs were fixed for staining 8 hours after fertihsation. Thus staining could not reveal any clustering which may occur at a later stage preceding NEBD, The idea that InsPgRs become clustered around the pronuclei is supported by preliminary work tracking the ER, which suggests that after fertihsation there is an increase in ER density around the pronuclei.

It remains to be determined whether or not there is a requirement for InsPgR clustering to generate the proposed local Ca^^ signals. Further experiments (using immunocytochemistry or a GFP-tagged InsPgR in conjunction with an ER-specific dye) are required to examine whether clustering of InsPgRs does occur in pronucleate mouse eggs.