Reviews of Reproduction (1998) 3, 7–12

Protein kinase C action at fertilization: overstated or undervalued?

Keith T. Jones

Department of Anatomy and Developmental Biology, University College, Gower Street, London WC1E 6BT, UK

At fertilization, the spermatozoon is generally held to generate two important second messen- gers, inositol trisphosphate and diacylglycerol. A similar situation arises when these signalling molecules are generated after a hormone binds to its plasma membrane receptor. This sig- nalling mechanism releases intracellular Ca2+ which causes cortical granule release and initiates meiotic resumption. This review will examine the role played at fertilization by protein kinase C which is a primary target of diacylglycerol. The pharmacological agents phorbol esters, which mimic the action of diacylglycerol, when added to mammalian oocytes induce cortical granule release and may cause meiotic resumption. However, the originally accepted mechan- ism of fertilization is now questioned with the recent finding of a soluble sperm Ca2+-releasing factor expelled directly into the oocyte cytoplasm, bypassing any membrane receptor. Therefore, it is timely to re-evaluate the role played by protein kinase C at fertilization in light of a mech- anism that may produce Ca2+ without producing diacylglycerol concomitantly. This article will examine the evidence implicating activation of protein kinase C in Ca2+ oscillations, cortical granule release and meiotic resumption. It will contend that pharmacological studies relying on the specificity of phorbol esters and other agonists, as well as inhibitors of protein kinase C, have produced conflicting interpretations of the role of this kinase at fertilization.

Protein kinase C (PKC) is a major intracellular threonine–serine in mammals that are observed over a period of several hours kinase that has been implicated in a large number of signal (Jones et al., 1995). Furthermore, sea urchin eggs are arrested in transduction processes that mediate extracellular signals into interphase, having completed meiosis, rather than at meta- intracellular events. For maximal activity PKC requires co- phase II, as is the case with mammalian oocytes. In addition, factors, most notably Ca2+ and diacylglycerol (DAG). In this the eggs of some animals, such as molluscs, do not release cor- respect phorbol esters have proved valuable pharmacological tical granules to block polyspermy. In light of these obser- tools for examining the role of PKC in physiological processes vations, and for clarity, I have focused on mammalian oocytes. because they irreversibly activate PKC through attachment to its DAG-binding domain. Protein kinase C and increasing intracellular Ca2+ One important signal transduction process is that which occurs at fertilization, where the spermatozoon triggers oocyte It may seem odd to ask whether PKC has a role in raising activation. I have examined evidence of a role for PKC in three intracellular Ca2+, when it is well known that PKC is a target major events associated with mammalian oocyte fertilization: for Ca2+ action. However, the question is raised because of (1) periodic increases in intracellular Ca2+ (Ca2+ oscillations); the studies carried out by Cuthbertson and Cobbold (1985) (2) release of cortical granules (CGs) which block polyspermy; which made the first direct measurement of sperm-induced and (3) release from meiotic arrest. The reason for listing an in- Ca2+ oscillations in mammalian (mouse) oocytes. Cuthbertson crease in Ca2+ first is that it is the primary trigger for the sub- and Cobbold found that application of 325 nmol l–1 of the phor- sequent two events. That is, by blocking changes in cytoplasmic bol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) could in- Ca2+ both release of CGs and meiotic resumption are inhibited duce Ca2+ oscillations in mouse oocytes, albeit with a delay of (Kline and Kline, 1992). This review outlines the evidence im- at least 30 min. Higher concentrations induced oscillations more plicating PKC in these three processes. quickly, but still with a delay of up to 10 min. A similar ability This review has been limited to mammalian oocytes since of TPA at comparably high concentrations to increase intra- there are subtle differences in the events associated with fertil- cellular Ca2+ concentrations has been noted in human keratino- ization in other species, such as the widely studied sea urchin, cytes, over a similar time course to that observed in oocytes which make comparisons difficult. For example, in sea urchin (Sharpe et al., 1989). eggs, but not mammalian oocytes, a membrane depolarization is The ability of TPA to induce oscillations in oocytes with a responsible for a fast block to polyspermy (Jaffe, 1976; Jaffe et al., delay has not been shown by other researchers to my knowl- 1983), a ‘respiratory burst’ occurs after egg activation (Heinecke edge. More recent studies have confirmed the original finding and Shapiro, 1989) and a cytoplasmic alkalinization occurs that there is not an immediate increase in Ca2+ after addition (Whitaker and Steinhardt, 1982; Kline and Zagray, 1995). Sea of TPA (Colonna et al., 1989; Moses and Kline, 1995). In urchin eggs also show a single Ca2+ wave, lasting 24 s (Whitaker some studies, TPA seems actually to mitigate or even inhibit and Irvine, 1984), compared with the series of Ca2+ oscillations sperm-induced oscillations. In hamster oocytes, it decreases © 1998 Journals of Reproduction and Fertility 1359-6004/98 $12.50 Downloaded from Bioscientifica.com at 09/25/2021 06:16:45AM via free access 8 K. T. Jones the frequency of oscillations when added before spermatozoa (Swann et al., 1989), whereas in human oocytes, it eventually stops the oscillations whether added before or after spermato- zoa (Sousa et al., 1996). We are left to conclude that the signal at fertilization is not PKC activation, leading to Ca2+ oscillations and then to release from meiosis. Although the initial study seemed to suggest this, high doses were required and lower doses did not affect intracellular Ca2+ immediately. In hamsters, the first Ca2+ transient occurs within 30 s after sperm–oocyte fusion (Miyazaki et al., 1986), while in mice, there is a longer delay but by only a few minutes (Lawrence et al., 1997). Therefore, the timing is wrong for PKC to be involved in the generation of Ca2+ oscil- lations. Ca2+ oscillations must be induced immediately by ap- plication of TPA. It is interesting to note that a protein kinase inhibitor, staurosporine, which is often used to inhibit PKC, also causes an increase in intracellular Ca2+ in oocytes if used at a high enough concentration (Fig. 1).

Protein kinase C and release of cortical granules If PKC has a role at fertilization, it must be possible to mimic the events of fertilization by phorbol esters. There is some evi- dence to suggest that this is so for the block to polyspermy which occurs minutes after sperm–oocyte fusion. Phorbol esters, Fig. 1. The protein kinase inhibitor staurosporine causes an immedi- by activating PKC, cause some of the changes associated with ate and sustained Ca2+ increase in mouse oocytes. Staurosporine the zona after fertilization. Endo et al. (1987) showed that phor- is often used as a PKC inhibitor and can have unwanted effects bol esters added to zona-intact mouse oocytes prevent sperm if used at a high enough concentration. The ordinate axis (ratio of penetration and, therefore, fertilization. This was attributed to fluorescence at the two wavelengths used) is in effect Ca2+ concen- changes in the zona since its removal gave normal rates of pen- tration (for an explanation of Ca2+ measurement, see Jones et al., etration in oocytes treated with TPA. At a molecular level, TPA 1995). This trace is typical of three others. In parallel experiments, increased the conversion of the zona glycoprotein ZP2 to ZP2f 56 of 57 oocytes treated with staurosporine developed pronuclei. 2+ after fertilization which is believed to be responsible in part for Does staurosporine activate oocytes because of an increase in Ca the subsequent block of the zona to polyspermy. concentration or protein kinase inhibition? The author believes that probably both are involved. This illustrates that the interpretation After fertilization, the changes in ZP2 and also ZP3 are of the effects of PKC activators and inhibitors is problematic. caused by release of CGs from the oocyte. Therefore, it should be possible to induce their release using phorbol esters. Ducibella et al. (1993) measured the loss of CGs from the oocyte cytoplasm and found that, within 1 h of its addition, TPA led to there is a biochemical pathway in oocytes in which PKC acti- a decrease in the number of CGs to 25–33% of controls. Within vation leads to CG release but that this pathway is not used by 2 h of addition of TPA, similar CG loss and ZP2 conversion the spermatozoon at fertilization. occurred as in fertilized oocytes. In one study, the PKC activator 1-oleyl-2-acetyl-sn-glycerol Protein kinase C and release from meiotic arrest (OAG) was used to assess the role of PKC in mouse oocytes at fertilization (Colonna and Tatone, 1993). OAG is structurally The morphological events associated with meiotic resumption similar to the endogenously produced DAG but lacks a long include extrusion of the second polar body and formation of chain fatty acid in the number 2 position. OAG, like TPA, pronuclei. The most important biological assay for meiotic re- caused CG release but this effect was not blocked by inhibitors sumption is a decrease in maturation promoting factor (MPF) of PKC and the authors concluded that CG release was a PKC- activity, which is high in metaphase II oocytes but declines independent event. However, studies such as this are compli- rapidly after oocyte activation. MPF activity in extracts pre- cated by the specificity, or lack of it, of both activators and pared from oocytes is determined by its ability to phosphory- inhibitors. late exogenously added histone H1. The fact that phorbol esters are able to induce CG release At present, it remains disputed whether phorbol esters actu- does not show that PKC is actually activated at fertilization. ally activate mammalian oocytes (compare Gallicano et al. The most convincing evidence to date against a role for PKC in (1993) with Moore et al. (1995)). This may seem surprising the release of CGs at fertilization was presented in an elegant given the obvious criteria for activation. However, the assess- study by Ducibella and LeFevre (1997). In this they blocked CG ment of activation seems to depend on the mammal studied. release induced by phorbol esters using known PKC inhibitors For example, in polar body formation, the simplest criterion but found that these same inhibitors failed to have any effect on for activation, phorbol esters can induce second polar body the extent of CG release caused by spermatozoa. This suggests extrusion in hamster oocytes but the polar body is quickly

Downloaded from Bioscientifica.com at 09/25/2021 06:16:45AM via free access Protein kinase C in oocytes 9 resorbed (Gallicano et al., 1993; Moore et al., 1995). In Syrian It might be predicted that entry into interphase is ac- hamsters, up to 50% of oocytes that extrude a polar body resorb companied by a fall in MPF activity since MPF declines rapidly them within 1 h of addition of TPA (Gallicano et al., 1993). This after metaphase (Verlhac et al., 1994). However, with respect polar body may not even be a bona fide polar body because to TPA, experimental evidence is limited to one study which cytokinesis does not take place (Moore et al., 1995) and its for- suggests that, at least within 1 h of its addition, there is no de- mation may be due to the ability of PKC activation to disrupt crease in MPF (Moore et al., 1995). If it is to play a part in the metaphase spindle, which is rapidly lost after addition of meiotic resumption, PKC must exert its effects downstream of phorbol ester, or to disrupt cytoskeletal structure (Moore et al., MPF, causing activation even in the presence of high concen- 1995). In mouse oocytes, it appears that phorbol esters do trations of MPF. Is it possible to achieve meiotic resumption not induce second polar body formation (Cuthbertson and even in the presence of high concentrations of MPF? A study Cobbold, 1985; Colonna et al., 1989; Moore et al., 1995; Moses by Bement and Capco (1991) suggested that sperm head and Kline, 1995). Although Gallicano et al. (1997a) have shown chromatin introduced into frog eggs can be induced to de- polar body formation in the CD-1 strain of mouse, the polar condense by TPA while MPF activity remains high, although it body is resorbed after a few hours, similar to the case in remains to be seen whether this is observed in general. At least hamsters. in the parthenotes generated by TPA of the CD-1 mouse, the Is there any other evidence to suggest PKC is involved in pronuclei show bromodeoxyuridine staining which suggests second polar body extrusion? PKC itself contains a pseudo- that they have entered S-phase (Moore et al., 1995). sequence that binds to its ligand-binding domain, keeping PKC It must be concluded that phorbol esters are poor activators inactive. A myristoylated, hence membrane-anchored, version of oocytes showing, at best, only some criteria of normal acti- of this pseudosequence blocks the second polar body formation vation. In comparison, the parthenogenetic agents of strontium, induced by Ca2+ ionophore and importantly spermatozoa ethanol and the calcium ionophore A23187 are regarded as acti- (Gallicano et al., 1997a), presumably by binding to activated vating agents without contention. A high proportion of oocytes PKC. This suggests that, at least in part, second polar body degenerate when treated with phorbol esters, a dramatic illus- formation is reliant on PKC activation. Alternatively, the view tration of which was shown by a study of 43 oocytes treated may be taken that loading the oocyte membrane with the with 162 nmol TPA l–1 for only 10 min in which 16 degenerated, pseudosequence interferes indiscriminately with the normal 24 formed pronuclei and three remained arrested at metaphase signalling pathway required for polar body extrusion. II (Moses and Kline, 1995). There appears to be a thin line Interpretation of this observation is difficult for two reasons. between degeneration and activation. First, there is not an appropriate control peptide lacking the It is unfortunate that much of the study of PKC at fertil- myristolylated pseudosequence effect. The pharmacological ization has been limited to pharmacological manipulation that agent okadaic acid, which is used as a phosphatase 1 and 2A relies heavily on phorbol esters. These agents are not specific inhibitor, will also block polar body extrusion during oocyte for binding to PKC (Ahmed et al., 1993; Wilkinson and Hallam, maturation by interfering with cytoskeletal structure (Alexandre 1994; Kazanietz et al., 1995) and, therefore, their use should be et al., 1991). Therefore, it cannot be certain that the myristoyl- examined critically. ated pseudosequence is acting in a precise, targeted manner. There are alternative methods for assaying PKC for activity Second, Ducibella and LeFevre (1997) examined this myristoyl- at fertilization. The first is to use the addition of a known ated pseudosequence over a similar dose range to the one used substrate and radiolabelled ATP. The MARCKS (myristoylated by Gallicano et al. (1997a) and found it to be highly toxic. What alanine-rich C-kinase substrate) protein is a suitable substrate, is clear is that extrusion of the second polar body is the default being readily phosphorylated by PKC. After fertilization, there pathway that will occur in the absence of any hindrance. What is an increase in MARCKS phosphorylation (Gallicano et al., is unclear is how either activation of PKC through phorbol 1997a) which may be abolished by a bisindolylmaleimide. On ester or inhibition through myristoylated pseudosequence will the basis of their similarity to the indolocarbazole stauro- lead to a block in polar body extrusion. sporine, bisindolylmaleimides have been developed as specific In relation to pronucleus formation, there are studies that PKC inhibitors. Bisindolylmaleimides interfere with the ATP- have found high rates of TPA-induced activation of mouse binding domain of kinases, and have specificity for PKC over oocytes as assessed by pronucleus formation (Cuthbertson and other kinases. The crucial questions here are: (1) is MARCKS Cobbold, 1985; Colonna et al., 1989; Moses and Kline, 1995), protein phosphorylated by other fertilization-associated kin- whereas Moore et al.(1995) found the formation of pronuclei to ases; and (2) would these kinases be similarly inhibited by bis- be dependent on the mouse strain used, with oocytes from indolylmaleimide? There is more evidence to suggest that an the CD-1 strain but not those from the CF-1 strain showing increase in PKC activity occurs at fertilization since activation readily discernible pronuclei. It seems that mice such as MF1 of PKC is often associated with a translocation to the plasma (Cuthbertson and Cobbold, 1985) and CD-1 (Colonna et al., membrane from the cytoplasm. Such a transition can be ob- 1989; Moses and Kline, 1995) show pronucleus formation in re- served in hamster oocytes using a fluorescent PKC binding sponse to phorbol esters, while CF-1 do not (Moore et al, 1995); agent Rim-1 (rhodamine conjugated indolylmaleimide) after + 2+ all are outbred strains. In inbred C57BL/6JLac CBA/CaLac F1 treatment with Ca ionophore (Gallicano et al., 1995, 1997a) hybrid mice, there is no readily discernible second polar body and with spermatozoa (Gallicano et al., 1997a). These data or pronuclei formed by application of TPA (K. T. Jones, un- suggest, albeit with the abovementioned reservations, that published). It must be concluded that in some strains of mouse activation of PKC can be observed at fertilization. Whether phorbol esters do induce pronucleus formation but this is not a PKC is actually driving specific events such as polar body universal phenomenon. extrusion is not clear.

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(a) (b)

PC-PLC PI-PLC PC PIP2 + SF IP3 DAG DAG 2+ 2+ + Ca + + Ca + + + PKC PKC

Fig. 2. Protein kinase C (PKC) activation at fertilization. (a) This is the more familiar mechanism in which sperm–oocyte interaction is seen in terms of a ligand–receptor interaction, activating a phosphatidylinositol (PIP2)-specific phospholipase C (PI-PLC) to generate inositol tris- 2+ 2+ phosphate (IP3), which releases Ca from an intracellular store, and diacylglycerol (DAG), which activates PKC. The released Ca acts as a cofactor to activate PKC fully. The author prefers (b). In this model the sperm Ca2+-releasing factor (SF) acts independent of a membrane receptor to release Ca2+. The increase in cytoplasmic Ca2+ can then generate DAG through phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) as shown or alternatively through PI-PLC or phospholipase D.

not have a DAG-binding domain and, therefore, are not acti- The protein kinase C family vated by phorbol esters. PKC exists as a family of isoforms. However, there is no estab- lished evidence as yet about which members of the family are Signal transduction at fertilization present in oocytes. The family comprises three groups charac- terized by their cofactor requirement and has been discussed in If the view is taken that the spermatozoon acts as a giant hor- several recent excellent reviews (for example, Nishizuka, 1995; mone molecule locking onto oolemma receptors that initiate Jaken, 1996). Briefly, there are conventional isoforms of PKC that the train of Ca2+ oscillations in oocytes, then this mechanism is require DAG for activation and are sensitive to Ca2+. Classically, mediated by either a G-protein-coupled or tyrosine kinase- activation of phospholipase C has been implicated in pro- linked membrane receptor. In this model, PKC has a clearly de- ducing these second messengers both directly for DAG and fined place (see Fig. 2a). However, there is little good evidence 2+ 2+ indirectly through inositol trisphosphate (IP3) for Ca . Other to suggest that IP3 can mimic exactly the Ca oscillations at fer- PKCs have been identified, which belong to the novel and atypi- tilization, which is central to this model (Swann, 1994; Jones and cal families, that make their activity either Ca2+-independent, or Whittingham, 1996). Recent evidence suggests that a sperm- DAG- and Ca2+-independent, respectively. These isoforms re- derived Ca2+-releasing factor is introduced directly into the quire other cofactors for activation, such as ceramide, arachi- ooplasm (Parrington et al., 1996). This may be a way of acti- donic acid and phosphatidylinositides (Nakanishi et al., 1993; vating intracellular Ca2+ release channels independent of known Muller et al., 1995). It appears that single members of the family receptor activators, such as IP3. This topic is outside the scope can be involved in cellular processes (Otte and Moon, 1992; of the present review but has been debated elsewhere (for Watanabe et al., 1992; Borner et al., 1995) and their differing example, Swann and Lai, 1997) and does show that PKC may cofactor requirements underlie distinct functions. be activated indirectly, through an increase in intracellular Ca2+ It is often assumed that oocytes, like somatic cells, express at (Fig. 2b). This may occur by a number of routes that all have least one of the conventional isoforms of PKC. This assumption Ca2+ sensitivity: hydrolysis of phosphatidylcholine (PC) by PC- is wrong in at least one mouse strain in which only one isoform dependent phospholipase C (PC-PLC; Billah and Anthes, 1990) of the novel PKC (PKC-delta) and one isoform of the atypical and by phospholipase D (Anthes et al., 1989; Halenda and PKC (PKC-lambda) is present (Gangeswaran and Jones, 1997). Rehm 1990), and hydrolysis of phosphatidylinositol (PI) by PI- With respect to oocytes, it is also of note that atypical PKCs do dependent phospholipase (PI-PLC; Whitaker and Irvine, 1984;

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Nakanishi et al., 1985). For both PI-PLC and PC-PLC, DAG is The author expresses many thanks to John Carroll and Karl Swann produced directly, whereas for PLD, the product is phosphat- for critical reading of this review. idic acid, which is dephosphorylated to DAG by a phospho- hydrolase (Billah and Anthes, 1990). PKC activation can be seen in sea urchin eggs at fertilization References (Heinecke et al., 1990; Olds et al., 1995). In sea urchin eggs, the Key references identified by asterisks. switching on of phospholipase C through non-hydrolysable Ahmed S, Lee J, Kozma R, Best A, Monfries C and Lim L (1993) A novel G-proteins causes, as expected, both PKC activation and an in- functional target for tumor-promoting phobol esters and lysophospha- tidic acid. The p21rac-GTPase activating protein n-chimaerin Journal of 2+ crease in Ca (Crossley et al., 1991). PKC activation is assayed Biological Chemistry 268 10 709–10 712 by its ability to switch on the Na–H antiporter responsible for Alexandre H, Van-Cauwenberge A, Tsukitani Y and Mulnard J (1991) causing alkalinization of sea urchin eggs at fertilization. When Pleiotropic effect of okadaic acid on maturing mouse oocytes Development an increase in intracellular Ca2+ is buffered by intracellular Ca2+ 112 971–980 2+ Anthes JC, Eckel S, Siegel MI, Egan RW and Billah MM (1989) chelating agents, the increase in Ca is blocked. In such Phospholipase D in homogenates from HL-60 granulocytes: implications buffered eggs after microinjection of non-hydrolysable G- of calcium and G protein control Biochemical and Biophysical Research proteins, the Na–H antiporter is still able to switch on because Communications 163 657–664 DAG is produced and PKC is activated (as in Fig. 2a). However, Bement WM and Capco DG (1991) Parallel pathways of cell cycle control in buffered eggs that are fertilized, there is no alkalinization, during Xenopus egg activation Proceedings of the National Academy of Sciences USA 88 5172–5176 even though eggs become polyspermic; therefore, there is no Berra E, Diaz-Meco MT, Lozano J, Frutos S, Municio MM, Sanchez P, activation of PKC. This suggests that there is a pathway in Sanz L and Moscat J (1995) Evidence for a role of MEK and MAPK during sea urchin eggs for DAG, through PKC, to switch on the Na–H signal transduction by protein kinase C zeta EMBO Journal 14 6157–6163 exchanger but, at fertilization, the spermatozoon does not pro- Billah MM and Anthes JC (1990) The regulation and cellular functions of 2+ phosphatidylcholine hydrolysis Biochemical Journal 269 281–291 duce DAG until there is an increase in Ca concentration. In Borner C, Ueffing M, Jaken S, Parker PJ and Weinstein IB (1995) Two addition, there is evidence to suggest that the spermatozoon, at closely related isoforms of protein kinase C produce reciprocal effects on least in part, uses a Ca2+–calmodulin dependent kinase to switch the growth of rat fibroblasts. Possible molecular mechanisms Journal of on the exchanger (Shen, 1989). It may be that, in both mam- Biological Chemistry 270 78–86 malian oocytes and sea urchin eggs, PKC is activated at fertil- Colonna R and Tatone C (1993) Protein kinase C-dependent and indepen- dent events in mouse egg activation Zygote 1 243–256 2+ ization as a consequence of the increase in Ca owing to Colonna R, Tatone C, Malgaroli A, Eusebi F and Mangia F (1989) Effects of release from intracellular stores (Fig. 2b), rather than because of protein kinase C stimulation and free Ca2+ rise in mammalian egg acti- 24 the bifurcating production of IP3 and DAG by the fertilizing vation Gamete Research 171–183 spermatozoon (Fig. 2a). Crossley I, Whalley T and Whitaker M (1991) Guanosine 5’-thiotriphosphate may stimulate phosphoinositide messenger production in sea urchin eggs by a different route than the fertilizing sperm Cell Regulation 2 121–133 Conclusions Cuthbertson KS and Cobbold PH (1985) Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca2+ Nature 316 It could be argued that the role of PKC in mammalian oocytes 541–542 has been overstated. There is little general agreement that it is *Ducibella T and LeFevre L (1997) Study of protein kinase C antagonists on cortical granule exocytosis and cell-cycle resumption in fertilized mouse involved in any of the three processes associated with mam- eggs Molecular Reproduction and Development 46 216–226 malian oocyte fertilization: namely, Ca2+ oscillations, meiotic Ducibella T, Kurasawa S, Duffy P, Kopf GS and Schultz RM (1993) resumption and CG release. It has been suggested that PKC Regulation of the polyspermy block in the mouse egg: maturation- plays a pivotal role at fertilization and in the development of dependent differences in cortical granule exocytosis and zona pellucida modifications induced by inositol 1,4,5-trisphosphate and an activator of the embryo (Gallicano et al., 1997b). Although there is good protein kinase C Biology of Reproduction 48 1251–1257 evidence to implicate PKC activation at fertilization, this re- Endo Y, Schultz RM and Kopf GS (1987) Effects of phorbol esters and a di- view would argue against it necessarily driving fertilization- acylglycerol on mouse eggs: inhibition of fertilization and modification of associated events. However, individual members of the PKC the zona pellucida Developmental Biology 119 199–209 family do appear to be important in many aspects of somatic Gallicano GI, Schwarz SM, McGaughey RW and Capco DG (1993) Protein kinase C, a pivotal regulator of hamster egg activation, functions after cell signalling, such as growth and differentiation, and this may elevation of intracellular free calcium Developmental Biology 156 94–106 also be the case in oocytes. For example, PKC may mediate Gallicano GI, McGaughey RW and Capco DG (1995) Protein kinase M, the metaphase II arrest since members of the PKC family have been cytosolic counterpart of protein kinase C, remodels the internal cyto- implicated in growth arrest in somatic cells (Watanabe et al., skeleton of the mammalian egg during activation Developmental Biology 167 482–501 1992; Mischak et al., 1993). There are interactions of PKC with Gallicano GI, McGaughey RW and Capco DG (1997a) Activation of protein MAP kinase (Berra et al., 1995), one of the components of cyto- kinase C after fertilization is required for remodeling the mouse egg into static factor that helps maintain MPF integrity during meta- the zygote Molecular Reproduction and Development 46 587–601 phase arrest. Gallicano GI, Yousef MC and Capco DG (1997b) PKC – a pivotal regulator Many of the processes that PKC could be involved in have of early development BioEssays 19 29–36 Gangeswaran R and Jones KT (1997) Unique protein kinase C profile in mouse been overlooked because of the restricted signal transduction oocytes: lack of calcium-dependent conventional isoforms suggested by pathway PKC has been forced into during oocyte activation rtPCR and western blotting Federation of European Biochemistry Society by the overuse of phorbol- and bisindolylmaleimide-based Letters 412 309–312 pharmacology. The first step must be to clarify which members Halenda SP and Rehm AG (1990) Evidence for the calcium-dependent acti- vation of phospholipase D in thrombin-stimulated human erythroleu- of the family are present in oocytes and then to pursue their kaemia cells Biochemical Journal 267 479–483 activity not only at fertilization but also during previous matu- Heinecke JW and Shapiro BM (1989) Respiratory burst oxidase of fertil- ration and subsequent embryonic development. ization Proceedings of the National Academy of Sciences USA 86 1259–1263

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