Berger L and Wilde A (2013), Glycolytic metabolites are critical modulators ...

XB-ART-47833

PLoS One 2013 Oct 08;810:e77612. doi: 10.1371/journal.pone.0077612.

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Glycolytic metabolites are critical modulators of oocyte maturation and viability.

Berger L , Wilde A .

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The maturation of an oocyte into an egg is a key step in preparation for fertilization. In Xenopus, oocyte maturation is independent of transcription, being regulated at the level of translation and post-translational modifications of proteins. To identify factors involved in the maturation process we used two-dimensional differential gel electrophoresis to compare the proteome of oocytes and eggs. Protein abundance changes were observed in multiple cellular pathways during oocyte maturation. Most prominent was a general reduction in abundance of enzymes in the glycolytic pathway. Injection into oocytes of the glycolytic intermediates glyceraldehyde-3-phosphate, phosphoenolpyruvate and glucose-6-phosphate prevented oocyte maturation. Instead, these metabolites stimulated ROS production and subsequent apoptosis of the oocyte. In contrast, all other metabolites tested had no effect on oocyte maturation and did not induce apoptosis. These data suggest that a subset of glycolytic metabolites have the capacity to regulate oocyte viability.

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Species referenced: Xenopus laevis
Genes referenced: mapk1

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	Figure 2. Glycolytic and related metabolic pathways.Black circles denote enzymes that become more abundant during maturation and black stars denote enzymes that decreased in abundance during maturation. The asterisk marks an enzyme, phosphoglycerate mutase that had one spot increase and another decrease by the same level during maturation suggesting the protein level remained constant, but the protein became modified during oocyte maturation.
	Figure 3. G6P injection induces apoptosis in X. laevis oocytes is situ.A. The three phenotypes scored were oocyte, mature oocyte and apoptotic oocyte. B. Oocytes were injected with H2O, G6P or 6PG (intracellular concentration elevated by 1.38mM) and monitored for apoptosis 4 hours or overnight post progesterone addition. The results presented are representative of at least 3 independent experiments. C. Same as (B) except the oocytes were scored for maturation. D. Oocytes were injected of G6P solutions to elevate metabolite concentrations as indicated. Oocytes were monitored and scored for apoptosis at the indicated times. E. Oocytes were injected with metabolite, incubated in progesterone then collected and a post-mitochondrial supernatant was prepared and analyzed by Western blotting with antibodies specific for cytochrome C (cyto C) or phospho-ERK (pERK). O = uninjected stage VI oocyte. Error bars are +SEM.
	Figure 4. NADPH generating metabolites inhibit apoptosis induced by G6P.Representative experiment of oocytes injected with G6P alone or in combination with malate or 6PG (1.38 mM elevation in the intracellular concentration of each metabolite), then treated with progesterone. The oocytes were monitored for apoptosis in A, or maturation in B at the indicated time points post progesterone treatment. C. As (A) and (B) but combined analysis of at least 3 batches of oocytes from different animals. Error bars are +SEM. D. Cytoplasmic extracts prepared from the oocytes incubated in the presence or absence or progesterone (prog) were analyzed by Western blotting with antibodies specific for cytochrome C (cyto C) or phospho-ERK (pERK).
	Figure 5. Glycolytic intermediates regulate apoptosis and maturation in situ.A. Representative results from one batch of oocytes injected with the indicated glycolytic intermediates, induced with progesterone, and scored at the indicated times for apoptosis or maturation. B. As (A), but combined analysis at the 4 hour time point after progesterone treatment of at least 3 batches of oocytes from different animals. Error bars are +SEM. C. Oocytes were lysed and cytoplasmic extracts were analyzed by Western blotting with antibodies specific for phospho-ERK (pERK), or cytochrome C (cyto C). D. Representative results from one batch of X. laevis oocytes injected with GA3P or PEP at the indicated intracellular concentrations of injected metabolite. The oocytes were monitored and scored for apoptosis at the indicated time periods post progesterone addition. E. As (D), but combined analysis of at least 3 batches of oocytes from different animals at 4 hours after progesterone treatment. Error bars are +SEM.
	Figure 6. NADPH generating metabolites inhibit apoptosis induced by glycolytic intermediates.A. Representative results from one batch of oocytes injected with GA3P either alone or in combination with malate (metabolite concentration elevated by 1.38 mM). At the indicated time points post progesterone stimulation the oocytes and scored for apoptosis or maturation. B. As (A), but combined analysis at 4 hours after progesterone treatment of at least 3 batches of oocytes from different animals. Error bars are +SEM. C. Oocytes were collected and cytoplasmic extracts were prepared and analyzed by Western blotting with antibodies specific for cytochrome C (cytoC) or phospho-ERK (pERK). D. Representative results from one batch of oocytes injected with PEP alone or in combination with malate or 6PG (each metabolite concentration elevated by 1.38 mM). Oocytes were monitored and scored for apoptosis at the indicated time point post-progesterone addition. E. As (D) but combined analysis at 4 hours after progesterone treatment of at least 3 batches of oocytes from different animals scored for maturation and apoptosis. Error bars are +SEM. F. Representative results from one batch of oocytes injected with GA3P alone or in combination with malate or NADPH (each metabolite concentration elevated by 1.38 mM). Following progesterone addition the oocytes were scored for apoptosis at the indicated time points. G. As (F) but combined analysis at 4 hours after progesterone treatment of at least 3 batches of oocytes from different animals scored for maturation and apoptosis. Error bars are +SEM.
	Figure 7. Apoptosis inducing metabolites increase intracellular ROS levels.A. Oocytes were injected with different doses of G6P (elevating the intracellular metabolite concentration by 0.17-1.38 mM), malate (1.38 mM), or a combination of malate and G6P (1.38 mM each), incubated with progesterone then monitored until apoptosis was observed in the GA3P injected samples (4 hours post injection). Oocytes were lysed and a mitochondrial pellet prepared for analysis of mitochondrial ROS levels. Data compiled form at least 3 different batched of oocytes form 3 different animals. Error bars are +SEM. B. Same as (A) except that GA3P was used as the apoptosis-inducing reagent. As in (A) malate was injected to a final concentration of 1.38mM and when injected in combination with GA3P, both metabolites were injected to a concentration of 1.38mM. Same as (A) except PEP was used as the apoptosis inducing metabolite. As is (A) and (B) malate was injected to a final concentration of 1.38mM and when injected in combination with PEP, both metabolites were injected to a concentration of 1.38mM.
	Figure 1. Changes in the maturing oocyte proteome detected by 2D-DIGE.A. Region of a 2D-DIGE gel with arrows pointing to protein spots identified as EF1γ. B. Western blot probing 2D-gels separating stage VI oocyte and egg protein in the presence and absence of alkaline phosphatase treatment. Arrows point to the different EF1γ isoforms. C. A region of a 2D-DIGE gel with the protein spot areas of spots identified as enolase demonstrating changes in isoform abundance between stage VI oocytes and eggs. D. Pie chart of the relative proportion of different cellular pathways affected by changes in proteins levels during oocyte maturation as detected in 2D-DIGE experiments.

References [+] :

Brachet, Studies on maturation in Xenopus laevis oocytes. III. Energy production and requirements for protein synthesis. 1975, Pubmed, Xenbase