To determine whether p38MAPK contributed some feature of apoptosis, we treated eggs with the specific p38MAPK inhibitor SB203580 just before the onset of blebbing (10.5 h after 1-MA treatment). As shown in , SB203580 did not block the blebbing. Apoptotic body formation, however, was severely inhibited in the SB203580-treated eggs, exhibiting an almost rounded morphology (, b and c, arrows) with remaining small protrusions (, b and c, arrowheads). The small protrusions were separated spontaneously from the rounded egg, and finally they degraded and released apoptotic body-like particles (, arrowhead). The egg still exhibited a rounded morphology even at 20 h after 1-MA treatment (, arrow). These results indicate that p38MAPK may contribute to apoptotic body formation.
In this study, we found that MAPK has both positive and negative functions in the induction of starfish egg apoptosis. During the MAPK-dependent period (~8 h after 1-MA treatment), inactivation of MAPK blocked apoptosis, indicating that it gives the death-activating signal. Conversely, after the MAPK-dependent period but before blebbing (~8-10 h after 1-MA treatment), inactivation of MAPK resulted in caspase-3 activation, causing apoptosis. Moreover, p38MAPK, which is generally considered as a death factor (Xia et al., 1995
; Kummer et al., 1997
) was activated immediately after the inactivation of MAPK. Thus, starfish MAPK gives the death-suppressing signal after the MAPK-dependent period.
During the MAPK-dependent period, starfish eggs are likely to develop competence to die, as reported in mammalian sympathetic neurons. After nerve growth factor deprivation, apoptosis of sympathetic neurons requires the activation of two events: a protein synthesis dependent, Bax-dependent release of mitochondrial cytochrome c
and protein synthesis-independent, Bax-independent development of competence. Unlike most cells, cytosolic cytochrome c
is not sufficient to induce cell death in nerve growth factor-maintained sympathetic neurons but can do so in the neurons that have developed competence (Deshmukh and Johnson, 1998
). It is suggested that development of competence may be the result of the loss of function of one or more members of the inhibitor of apoptosis family blocking caspases (Deshmukh et al., 2002
). Because cytoplasmic microinjection of cytochrome c
into the starfish eggs during the MAPK-dependent period did not accelerate apoptosis (our unpublished data), continuous MAPK activity during the MAPK-dependent period may contribute to development of competence for cytochrome c
to induce cell death.
Usually in mammalian apoptosis, the MAPK signaling pathway promotes cell survival by a dual mechanism comprising the posttranslational modification to inhibit a component of the cell death machinery and the increased transcription of prosurvival genes. Because inactivation of MAPK after the MAPK-dependent period resulted in caspase-3 activation in starfish eggs (within 1 h after U0126 treatment; ), MAPK may regulate the apoptotic machinery directly or indirectly, presumably via phosphorylation. It is reported that egg extracts prepared from the frog Xenopus laevis
initiate and execute a full apoptotic program in vitro when egg extracts are “aged” on the bench (Newmeyer et al., 1994
). Interestingly, activation of the Mos/MEK/MAPK pathway inhibits postcytochrome c
release apoptotic events in Xenopus
extracts in the absence of new mRNA/protein synthesis (Tashker et al., 2002
). Because cytoplasmic microinjection of cytochrome c
into the starfish eggs did not accelerate apoptosis as described above, starfish MAPK may also inhibit the postcytochrome c
release event in starfish eggs.
In mammals, two proapoptotic Bcl-2-family proteins, Bad and Bim, are involved in apoptosis after withdrawal of survival factors. MAP kinase-activated kinase Rsk phosphorylates the proapoptotic protein BAD. Phosphorylated BAD is inactivated, and thus active Rsk prevents apoptosis by inhibiting BAD (Bonni et al., 1999
; Shimamura et al., 2000
). The MAPK pathway-dependent phosphorylation of Bim targets Bim for degradation by the proteasome pathway (Ley et al., 2003
). In the absence of survival factors, dephosphorylated Bim inhibits antiapoptotic proteins such as Bcl-2 and render the cells more susceptible to apoptogenic stimuli (Terradillos et al., 2002
). It is possible that starfish MAPK inactivates starfish Bad and Bim and suppresses apoptosis after the MAPK-dependent period. Spontaneous inactivation of MAPK then occurs, causing caspase-3 activation. In Drosophila
, proapoptotic protein Hid induces apoptosis by blocking a caspase inhibitor, Diap1 (Bergmann et al., 2002
). It is known that activation of the Ras/MAPK pathway inhibits Hid-induced apoptosis (Kurada and White, 1998
), and phosphorylation of Hid by MAPK is thought to inactivate Hid (Bergmann et al., 1998
). Starfish MAPK may phosphorylate and inhibit a Hid-like protein in eggs.
We also demonstrated in this study that U0126 treatment resulted in the activation of p38MAPK. This result strongly suggested that inactivation of MAPK acts upstream of p38MAPK activation. In addition, just after GVBD, MAPK is activated (Pelech et al., 1988
) by a newly synthesized Mos (Tachibana et al., 2000
), and p38MAPK was inactivated just about the same time (; Morrison et al., 2000
). Activation of MAPK may inhibit p38MAPK activation in starfish eggs.
Because activation of p38MAPK occurred spontaneously even in the eggs injected with caspase-3 inhibitor Ac-DEVDCHO (), p38MAPK does not act downstream of caspase-3. Further studies are required to determine whether p38MAPK acts upstream of caspase-3.
Regulation of actin dynamics is one of the functions of the p38MAPK pathway. After activation by p38MAPK, MAP kinase-activated protein kinase-2 phosphorylates HSP27, a protein that can modulate actin polymerization (Huot et al., 1998
; Landry and Huot, 1999
). Interestingly, apoptotic body formation is regulated by actin polymerization in starfish eggs (Sasaki and Chiba, 2001
), and the p38MAPK-specific inhibitor SB203580 antagonized apoptotic body formation. Thus, it is likely that p38MAPK in starfish also contributes to actin polymerization.
We and others had demonstrated that postmeiotic starfish eggs undergo apoptosis, if they were not fertilized (Sasaki and Chiba, 2001
; Yüce and Sadler, 2001
). It was also reported that unfertilized ovulated murine oocytes cultured in vitro spontaneously undergo apoptosis (Takase et al., 1995
; Fujino et al., 1996
; Perez et al., 1999
). Thus, in some species of animal, the default fate of the ovulated eggs is death by apoptosis. To understand normal development, it is important to know how eggs undergo apoptosis and how apoptosis is suppressed after fertilization. Starfish is a good model for studying postmeiotic egg apoptosis, because it is easy to obtain a large quantity of homogenous eggs that synchronously undergo apoptosis in vitro.
Moreover, apoptosis is a widespread event in oogenesis (reviewed by Matova and Cooley, 2001
). The role of apoptosis in the female gamete life cycle has been most extensively studied in mammals, despite the difficulty in obtaining and culturing sufficient quantities of the oocytes and eggs. In mammals, more than two-thirds of the potential germ cell pool (oogonia and oocytes) is lost through apoptosis by the time of birth (reviewed by Morita and Tilly, 1999
), and >99% of the postnatal oocytes, which are not ovulated, also undergo apoptosis. Although morphological changes during mammalian oocyte death are well studied, little is known about the molecular mechanisms responsible for initiating or executing oocyte apoptosis. It may be possible that studies of starfish egg apoptosis may lend insights into apoptotic events in mammals. Also, in the nematode Caenorhabditis elegans
, more than one-half of the hermaphrodite germ cells in adult gonad are eliminated through apoptosis, when they are about to exit the pachytene stage of meiotic prophase I. Because the oocytes of mutants in the ras/MAP kinase pathway fail to exit the pachytene stage of meiosis I and fail to die, the ras/MAP kinase pathway might directly regulate the cell death machinery, or might indirectly affect germ cell apoptosis by promoting the progression of pachytene stage cells, which are resistant to apoptosis, to a later differentiation stage that is more sensitive to proapoptotic signals (Gumienny et al., 1999
). Although the molecular mechanisms of pro- and antiapoptotic effects of MAPK in nematode as well as starfish are still unclear, similar apoptotic pathways may be shared.
The time after ovulation during which mammalian eggs can give rise to developmentally competent embryo is short. Under in vivo conditions, ovulated mouse eggs exhibit maximum ability to fertilize for only 4-6 h (Lewis and Wright, 1935
). Oocytes that are fertilized after this optimal period exhibit severely compromised developmental success that often culminates in fragmentation of blastomeres and embryonic death (Marston and Chang, 1964
). Also, in starfish, optimal development occurs when maturing oocytes are fertilized between GVBD and first polar body emission. Fertilization of eggs after completion of meiosis usually results in polyspermy (Fujimori and Hirai, 1979
). Therefore, it is apparent that postovulatory processes occurring in the egg have a significant effect on development. It is tempting to speculate that with increasing time of postovulation, the ability of fertilization to inhibit apoptotic process is reduced, resulting in abnormal development.
The target molecules of MAPK and p38MAPK for apoptotic cell death as well as cell survival have to be identified to further understand the molecular mechanism of determining the egg fate, i.e., whether the cells undergo development or apoptosis.