Du Pasquier D et al. (2011), Unfertilized Xenopus eggs die by Bad-dependent ...

XB-ART-43778

PLoS One 2011 Jan 01;68:e23672. doi: 10.1371/journal.pone.0023672.

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Unfertilized Xenopus eggs die by Bad-dependent apoptosis under the control of Cdk1 and JNK.

Du Pasquier D , Dupré A , Jessus C .

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Ovulated eggs possess maternal apoptotic execution machinery that is inhibited for a limited time. The fertilized eggs switch off this time bomb whereas aged unfertilized eggs and parthenogenetically activated eggs fail to stop the timer and die. To investigate the nature of the molecular clock that triggers the egg decision of committing suicide, we introduce here Xenopus eggs as an in vivo system for studying the death of unfertilized eggs. We report that after ovulation, a number of eggs remains in the female body where they die by apoptosis. Similarly, ovulated unfertilized eggs recovered in the external medium die within 72 h. We showed that the death process depends on both cytochrome c release and caspase activation. The apoptotic machinery is turned on during meiotic maturation, before fertilization. The death pathway is independent of ERK but relies on activating Bad phosphorylation through the control of both kinases Cdk1 and JNK. In conclusion, the default fate of an unfertilized Xenopus egg is to die by a mitochondrial dependent apoptosis activated during meiotic maturation.

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Species referenced: Xenopus laevis
Genes referenced: bad bax bcl2l1 casp1 casp2 casp3.2 casp8 casp9 ccnb2 cdk1 clock fh h2bc21 kcnip4 mapk1 mapk8 mcl1 mos wwc2

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	Figure 1. Unlaid matured eggs, die by apoptosis in the female frog body.Xenopus females were induced to lay by hCG injection. Laying started 15 h after hormonal stimulation and was completed 24 h after. Forty hours after injection, females were dissected and dying eggs with altered pigmentation could be observed in the animal body: trapped in the ovary (A, arrows indicate dying eggs, bar = 2.4 mm), in the oviduct (B, arrow-head indicates a dying egg at the exit of the oviduct, arrow indicates a dying egg in the oviduct visible through the wall, bar = 3 mm), in the uterus (C, arrow indicates the opened uterus, bar = 5 mm) or in the cloaca (D, bar = 3 mm). Representative dying eggs are shown in (E, bar = 2.4 mm). (F) Caspase 3 activation was measured by immunoblot using an antibody against the cleaved active form of caspase 3 (casp-3) in eggs laid 15 h after hCG injection and dying eggs trapped in the genital tract 40 h after hCG injection. The ERK1 protein was immunoblotted as a loading control.
	Figure 2. Time-course of morphological changes characterizing the spontaneous death of progesterone matured oocytes.Fully-grown prophase oocytes were isolated from the ovaries and incubated in the presence (+Pg) or in the absence (−Pg) of 1 µM progesterone. (A) Oocytes originating from one female were photographed at the indicated times. (B) Time-course of death of either prophase-blocked oocytes (−Pg, white circles) or progesterone-matured oocytes (+Pg, black circles) from eight different females. Oocytes were considered as dead when their external morphology was similar to the one occurring at 48 h in panel A.
	Figure 3. Mitochondrial- and caspase dependent apoptosis is responsible for unfertilized egg death.Fully-grown prophase oocytes were isolated from the ovaries and stimulated by 1 µM progesterone. (A) Western blot analysis of cytosolic and mitochondrial fractions of unfertilized eggs was performed at various times following progesterone addition (+Pg) with antibodies against ERK1, the cleaved form of caspase 3 (casp-3), p150Glued, the Ser14-phosphorylated form of histone H2B (p-H2B), Bad, Bax, Cyt c and the cleaved form of caspase 9 (casp-9). (B) Caspase and calpain activities of unfertilized eggs were assayed at various times after progesterone stimulation with the following substrates: Suc-LY-AMC (calpain, Calp, white diamonds), Ac-YVAD-AMC (caspase 1, C1, dark squares), Z-VDVAD-AFC (caspase 2, C2, white circles), DEVD-AMC (caspase 3, C3, white squares), Z-IETD-AFC (caspase 8, C8, white triangles) and Ac-LEHD-AFC (caspase 9, C9, black circles). In parallel, the percentage of dead eggs was followed using morphological criteria. (C) Oocytes were microinjected with mRNAs encoding either GFP, GFP-XR11 or GFP-Mcl-1 fusion proteins and then incubated in the presence of 1 µM progesterone. The percentage of dead eggs was estimated 70 h later using morphological criteria. (D) Oocytes were microinjected with mRNAs encoding either GFP (control, black columns) or GFP-XR11 fusion protein (white columns). They were then incubated in the presence of 1 µM progesterone. Caspase 2 (C2), caspase 3 (C3), caspase 8 (C8) and caspase 9 (C9) activities were assayed 48 and 62 h later as described in (B). (E) Oocytes were microinjected with various caspase inhibitors: VDVAD-FMK (caspase 2, C2), DEVD-FMK (caspase 3, C3) or IETD-FMK (caspase 8, C8). Progesterone was added and the percentage of dead eggs was estimated 24 h (white columns) and 48 h (black columns) later after morphological criteria. All data shown in these panels are representative experiments repeated on oocytes and eggs from at least three different females.
	Figure 4. ERK1 is inactivated in response to apoptosis whereas Cdk1 inactivation occurs during egg aging, independently of apoptosis.Fully-grown prophase oocytes were isolated from the ovaries, stimulated by 1 µM progesterone (+Pg, time 0) or not (−Pg) and then incubated for various times. Three females were used, one in (A), another in (B) and (C) and one in (D). (A) Western blot analysis of cytosolic fractions of unfertilized eggs was performed at various times after progesterone stimulation with antibodies against ERK1, the active phosphorylated form of ERK1 (p-ERK1), Mos, Cyt c and active caspase 3 (casp-3). The percentage of eggs undergoing apoptosis after morphological analysis is indicated at the bottom, the incubation times in hours (Hrs) are indicated at the top. (B) H1 kinase activity of Cdk1 and apoptotic death were assayed in eggs at various times (in Hours, Hrs) after progesterone stimulation. (C) Western blot analysis of cytosolic fractions of unfertilized eggs was performed at various times after progesterone stimulation (+Pg) with antibodies against ERK1, cyclin B2 (Cyc B2) and the Ser10-phosphorylated form of histone H3 (p-H3). The incubation times in hours (Hrs) are indicated at the top. (D) Oocytes were microinjected with either GFP or GFP-XR11 mRNA and then stimulated by progesterone (time 0). Western blot analysis of cytosolic fractions of unfertilized eggs was performed at various times after progesterone stimulation with antibodies against ERK1, the active phosphorylated form of ERK1 (p-ERK1), cyclin B2 (Cyc B2) and Cyt c. The incubation times in hours (Hrs) are indicated at the top.
	Figure 5. Egg apoptosis involves JNK and Cdk1 activities.(A) Prophase oocytes were either incubated with a MEK inhibitor (U0126, grey columns) or injected with the p21Cip1 protein (Cip, crosshatched column) and then stimulated (Pg, white column) or not (prophase, black column) with progesterone. Egg death was monitored by following external egg morphology at the indicated times (in hours, Hrs) after progesterone addition (+Pg). (B) Prophase oocytes were stimulated with progesterone (Pg, time 0). Seven hours later, metaphase II-arrested eggs were incubated with or without the JNK inhibitor, SP600125. Egg proteins were analysed at the indicated times by immunoblot with antibodies against ERK1, the active phosphorylated form of ERK1 (p-ERK1), Cyt c, active caspase 3 (casp-3) and the active phosphorylated form of JNK (p-JNK). (C) Oocytes were induced to mature as in (B) and were then incubated with (white column) or without (black column) the JNK inhibitor, SP600125, or injected with mRNA encoding a constitutive active form of JNK (JNK-JJ, crosshatch columns). Egg death was monitored by following external egg morphology for all these three different conditions at the indicated times.
	Figure 6. Bad and JNK control egg apoptosis in a cooperative manner.(A) and (B) Prophase oocytes were microinjected with mRNAs encoding either GFP (black circles), Bax (triangles) or Bad (squares) and were stimulated (B) or not (A) with progesterone one hour later. Egg death was monitored by external egg morphology at the indicated times. (C) and (D) Oocytes were microinjected with mRNAs encoding either GFP (white circles), the JNK-JJ (black diamonds), Bad (black triangles), a dominant negative form of Bad (BAD151A, black squares) or a mix of mRNA encoding for both JNK-JJ and Bad (crosses). One hour later, oocytes were incubated with progesterone (time 0). (C) Death time-course was monitored by egg morphological changes at the indicated times. (D) Western blot analysis of egg lysates using antibodies directed against ERK1, the active phosphorylated form of JNK (p-JNK) and active caspase 3 (casp-3). Note that the JNK-JJ form migrates slower than endogenous JNK. These data are representative experiments repeated on oocytes and eggs from three different females.
	Figure 7. Egg apoptosis correlates with the phosphorylation of Ser128 of Bad.(A) Oocytes were microinjected with a mRNA encoding Bad-GFP and one hour later incubated in the presence or the absence of the JNK inhibitor, SP600125, or microinjected with p21Cip1 protein (Cip). They were then stimulated with progesterone (time 0). Egg proteins were analysed at the indicated times by immunoblot using antibodies against Bad, Ser128 (pS128), Ser112 (pS112) or Ser136 (pS136)-phosphorylated forms of Bad, and the active phosphorylated form of JNK (p-JNK). These data are representative experiments repeated on oocytes and eggs from at least three different females. (B) Regulation of apoptosis in unfertilized eggs. In the ovary, prophase oocytes are protected from apoptosis by an inhibited form of Bad phosphorylated at Ser112 and Ser136. At time of ovulation, the oocyte completes meiotic maturation. Bad becomes phosphorylated on Ser128 under the control of Cdk1 and JNK. During aging, the ovulated egg progressively accumulates increasing amounts of the Ser128 phosphorylated form of Bad that can ultimately trigger the death execution, unless fertilization occurs.

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Antignani, How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane? 2006, Pubmed