Tokmakov AA et al. (2011), Unfertilized frog eggs die by apoptosis followi...

XB-ART-44637

BMC Cell Biol 2011 Dec 23;12:56. doi: 10.1186/1471-2121-12-56.

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Unfertilized frog eggs die by apoptosis following meiotic exit.

Tokmakov AA , Iguchi S , Iwasaki T , Fukami Y .

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A characteristic feature of frog reproduction is external fertilization accomplished outside the female's body. Mature fertilization-competent frog eggs are arrested at the meiotic metaphase II with high activity of the key meiotic regulators, maturation promoting factor (MPF) and cytostatic factor (CSF), awaiting fertilization. If the eggs are not fertilized within several hours of ovulation, they deteriorate and ultimately die by as yet unknown mechanism. Here, we report that the vast majority of naturally laid unfertilized eggs of the African clawed frog Xenopus laevis spontaneously exit metaphase arrest under various environmental conditions and degrade by a well-defined apoptotic process within 48 hours after ovulation. The main features of this process include cytochrome c release, caspase activation, ATP depletion, increase of ADP/ATP ratio, apoptotic nuclear morphology, progressive intracellular acidification, and egg swelling. Meiotic exit seems to be a prerequisite for execution of the apoptotic program, since (i) it precedes apoptosis, (ii) apoptotic events cannot be observed in the eggs maintaining high activity of MPF and CSF, and (iii) apoptosis in unfertilized frog eggs is accelerated upon early meiotic exit. The apoptotic features cannot be observed in the immature prophase-arrested oocytes, however, the maturation-inducing hormone progesterone renders oocytes susceptible to apoptosis. The study reveals that naturally laid intact frog eggs die by apoptosis if they are not fertilized. A maternal apoptotic program is evoked in frog oocytes upon maturation and executed after meiotic exit in unfertilized eggs. The meiotic exit is required for execution of the apoptotic program in eggs. The emerging anti-apoptotic role of meiotic metaphase arrest needs further investigation.

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Species referenced: Xenopus laevis
Genes referenced: casp3.2 ccnb2 cdk1 mapk1

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	Figure 1. Spontaneous meiotic exit in unfertilized Xenopus eggs. Changes in the morphology of water-deposited (a) and OR-2 buffer-deposited (b) eggs, major morphological types of the unfertilized eggs (c-h), MAPK dephosphorylation and cyclin B2 degradation (i, j), and H1 kinase activity of Cdk1 in water-deposited and buffer-deposited eggs (k) are presented. Time after egg ovulation (hours) is indicated.
	Figure 2. Features of classical apoptosis in unfertilized Xenopus eggs. Freshly squeezed eggs obtained after hCG injection were dejellied and placed into OR-2 buffer over the indicated times. Cytochrome c release (a), caspase 3 activation (b), apoptotic nuclear morphology (c), and quantification of morphology scores (d) are shown. Immunoblotting with anti-Î² tubulin (lower panel in (a)) represents the loading control. The upper panel in (b) shows blotting of egg extracts with anti-caspase 3 antibody, whereas the lower panel presents data of the fluorescent caspase 3 assay, carried out as described in "Methods". Bars in panel (b) indicate SD of five to eight measurements using eggs obtained from three different female frogs. About one hundred nuclei were observed in (c) and (d).
	Figure 3. Cell death events in unfertilized Xenopus eggs. Intracellular ATP content (a), ADP/ATP ratio (b), intracellular pH (c), and egg diameter (d) were monitored over 48 hours after ovulation. Intracellular pH in panel (c) was measured using spectrometric and fluorescent assays (closed and open circles, respectively). Bars in panel (c) represent the range of pH readings taken by two persons in double-blind trials and data in panel (d) are means Â± SD obtained by measurement of five eggs.
	Figure 4. Single-cell analysis of unfertilized Xenopus eggs. MAPK phosphorylation state (a) and caspase 3 activity (b) were determined in the single unfertilized eggs #1-10 taken at different times (12 - 48 hours) after their deposition into OR-2 buffer. Eggs #1-3 were collected within 12 hours of ovulation, eggs #4-8 - within 18-36 hours, and eggs #9-10 - at 48 hours after ovulation. The control egg E was withdrawn immediately after its deposition. Relative intracellular ATP content (c), ADP/ATP ratio (d), and egg diameter (e) were determined for the eggs #3, 4, 9, and E. Bars in (b) and (e) represent the range of duplicate measurements. The total number of eggs taken for the single-cell analysis was 39, among them, 10 eggs were collected within 12 hours of ovulation, 22 eggs - within 18-36 hours, and 7 eggs - at 48 hours after ovulation.
	Figure 5. Apoptotic degradation of ionophore-treated Xenopus eggs. Freshly squeezed dejellied eggs were placed into OR-2 buffer, treated with 1 Î¼M calcium ionophore A23187 for 5 min and monitored over indicated times. Egg morphology (a), MAPK activation state (b), cytochrome c release (c), caspase 3 activity (d), intracellular ATP content (e), ADP/ATP ratio (f), intracellular pH (g), and egg diameter (h) have been monitored. In panel (d), the data of three to six measurements in two independent experiments are shown. Bars in panel (h) represent SD of the mean obtained by measurement of three to five eggs.
	Figure 6. Stability of progesterone-treated and intact Xenopus oocytes. Defollliculated OR-2 buffer-deposited oocytes were treated with 10 Î¼M progesterone or left untreated and monitored over 72 hours. Morphology of the progesterone-treated (a) and -untreated (b) oocytes, MAPK phosphorylation state (c), and time course of GVBD (d) after progesterone addition.
	Figure 7. Apoptotic degradation of progesterone-treated Xenopus oocytes. Cytochrome c release (a), caspase 3 activation (b), intracellular pH (c), intracellular ATP content (d), ADP/ATP ratio (e), and egg diameter (f). Bars in panel (f) indicate SD of the mean obtained by measurement of five eggs.

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