January 1, 2017;
Global decay of mRNA is a hallmark of apoptosis in aging Xenopus eggs.
Cytoplasmic mRNAs are specifically degraded in somatic cells as a part of early apoptotic response. However, no reports have been presented so far concerning mRNA fate in apoptotic gametes. In the present study, we analyzed the content of various cytoplasmic mRNAs in aging oocytes and eggs of the African clawed frog, Xenopus laevis. To circumvent large gene expression variation among the individual oocytes and eggs, single-cell monitoring of transcript levels has been implemented, using multiple cytoplasmic collections and reverse transcriptase quantitative PCR. It was found that numerous cytoplasmic mRNAs, coding for proteins classified in different functional types, are robustly degraded in apoptotic Xenopus eggs, but not in aging oocytes. mRNA degradation becomes evident in the eggs after meiotic exit at the time of cytochrome c release. A strong correlation between the length of PCR amplicon and specific transcript content was observed, suggesting endonucleolytic cleavage
of mRNA. In addition, it was found that mRNA deadenylation also contributes to apoptotic mRNA degradation. Altogether, these findings indicate that the global decay of mRNA represents a hallmark of apoptosis in aging Xenopus eggs. To our knowledge, this is the first description of mRNA degradation in apoptotic gamete
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Figure 1. Variability of transcript contents in Xenopus oocytes. (A) Expression levels of 18 genes measured in a single Xenopus oocyte. (B) Standard deviation of gene expression in the oocyte population determined by analyzing expression levels in 16 to 22 individual oocytes. (C) Sampling design of gene expression monitoring in single aging Xenopus oocytes and eggs. Cytoplasmic collections were made from individual oocytes (O1, O2, O3, O4) before PG treatment, then from the eggs derived from these oocytes at 12, 24, 36 and 48 hours after PG addition (E1–12, E2–24, E3–36, E4–48, respectively). Cytoplasmic samples were also taken from a control untreated oocyte at the corresponding times (Oc-0, Oc-12, Oc-24, Oc-36, Oc-48). In panel (A), mRNA levels were measured in 2 to 4 replicates. SDs of replicate measurements are small and not visible in the plot.
Figure 2. Changes of specific transcript contents in the oocytes after PG administration. In panels (A), (B), (C), specific transcript levels measured in a single Xenopus oocyte at 12, 24 and 36 hours after PG administration were referred to those measured in the same oocyte before PG treatment. Difference between the cycle threshold values of the 2 qPCR measurements (ΔCq) was plotted on the y-axis. (D) Changes of specific transcript levels were evaluated in a control oocyte after 48-hour incubation in the absence of PG. In panels (B) and (C) random hexamer (red bars) or oligo(dT) (blue bars) primers were used to generate first strand cDNA by reverse transcription. Transcript levels were measured in 2 to 4 replicates. SDs of replicate measurements are small and not visible in the panels.
Figure 3. Correlations of ΔCq with amplicon length and position obtained with random hexamer RT primers. The results of pairwise correlation analysis between the decrease in transcript level and amplicon distance from the 5′ end, (A) and (D), amplicon distance from the 3′ end, (B) and (E), and amplicon length, (C) and (F), are presented. The decrease in transcript contents was determined for 11 transcripts at 24 hours after PG administration in panels (A), (B), (C) and for 6 transcripts at 36 hours in panels (D), (E), (F), as described in the legend to Fig. 2. First strand cDNA was generated using random hexamer primers. Calculated values of the pairwise correlation coefficient (r) and one-tailed probability test (p) are indicated in the panels.
Figure 4. Correlation between Cq and amplicon length in mcl1 gene transcript observed in apoptotic eggs. Positions and sequences of 5′ qPCR primers targeting qPCR amplicons of different length in mcl1 gene transcript are presented in panels (A) and (B), respectively. First strand cDNA was generated with random hexamer primers. The results of pairwise correlation analysis between Cq and amplicon length are shown in panel (C). Calculated values of the pairwise correlation coefficient (r) and one-tailed probability test (p) are indicated in panel (C).
Figure 5. Correlations of ΔCq with amplicon length and position obtained using oligo(dT) RT primers. Panel (A) presents values of the pairwise correlation coefficient (big fonts) and one-tailed probability test (small fonts) determined at 24 and 36 hours after PG administration in the experiments using the oligo(dT) RT primer. Panel (B) specifies the region of detectable endonucleolytic degradation in the used experimental design. The results of pairwise correlation analysis between ΔCq and combined length of amplicon plus its distance from the 3′ mRNA end determined for 11 transcripts at 24 hours and for 6 transcripts at 36 hours after PG administration are presented in panels (C) and (D), respectively. Calculated values of the pairwise correlation coefficient (r) and one-tailed probability test (p) are indicated in the panels.
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