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Aging of Xenopus tropicalis eggs leads to deadenylation of a specific set of maternal mRNAs and loss of developmental potential.

Kosubek A, Klein-Hitpass L, Rademacher K, Horsthemke B, Ryffel GU - PLoS ONE (2010)

Bottom Line: Furthermore, maternal mRNAs known to be deadenylated during egg maturation as well as after fertilization were preferentially deadenylated in aged eggs.Based on our novel finding we postulate that the imbalance of the polyadenylated maternal transcripts upon egg aging contributes to the loss of developmental potential.Based on this hypothesis the developmental consequences of downregulation of specific transcripts can be analyzed in future.

View Article: PubMed Central - PubMed

Affiliation: Institut für Zellbiologie (Tumorforschung), Universitätsklinikum Essen, Universität Duisburg-Essen, Essen, Germany.

ABSTRACT
As first shown more than 100 years ago, fertilization of an aged (overripe) egg increases the rate of malformations and embryonic loss in several vertebrates, including possibly humans as well. Since the molecular events in aging eggs may be similar in these species, we established in the frog Xenopus tropicalis a defined protocol for delayed fertilization of eggs. A three-hour delayed fertilization led to a dramatic increase in malformation and mortality. Gene expression profiling revealed that 14% of the polyadenylated maternal transcripts were downregulated upon aging. These transcripts were not degraded, but rather deadenylated as shown for specific maternal mRNAs. The affected transcripts are characterized by a relatively short 3'UTR and a paucity of cytoplasmic polyadenylation elements (CPE) and polyadenylation signals (PAS). Furthermore, maternal mRNAs known to be deadenylated during egg maturation as well as after fertilization were preferentially deadenylated in aged eggs. Taken together our analysis of aging eggs reveals that unfertilized eggs are in a dynamic state that was previously not realized. On the one hand deadenylation of transcripts that are typically deadenylated during egg maturation continues and this implies overripeness of the aged egg in the truest sense of the word. On the other hand transcripts that normally are deadenylated after fertilization loose their poly(A) in the aged egg and this implies that the egg awaiting fertilization starts processes that are normally only observed after fertilization. Based on our novel finding we postulate that the imbalance of the polyadenylated maternal transcripts upon egg aging contributes to the loss of developmental potential. Based on this hypothesis the developmental consequences of downregulation of specific transcripts can be analyzed in future.

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Poly(A) tail reduction of specific maternal mRNA in aged eggs.Poly(A) tail behavior of the indicated transcripts decreased (A) and not changed (B) upon egg aging are shown. Total mRNA from fresh (0 h) and aged (3 h) eggs was assayed by the RNA ligation-mediated poly(A) test (RL-PAT). * indicates RNaseH/oligo(dT)20 digestion prior to ligation. Control lanes: –Lig, Ligation reaction performed without RNA; -RT, ligated RNA was not reverse transcribed prior to PCR. M, DNA size marker are given in base pairs. Direct sequencing of atp5a1 and tpi1 (A lower panel) reveals the actual transcript 3′ending (indicated by arrows), which is in fresh eggs at the end of the poly(A) tail (italic As), but in aged eggs several nucleotides upstream of the former end of the RNA body (clear box). P1 is the ligated primer.
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pone-0013532-g006: Poly(A) tail reduction of specific maternal mRNA in aged eggs.Poly(A) tail behavior of the indicated transcripts decreased (A) and not changed (B) upon egg aging are shown. Total mRNA from fresh (0 h) and aged (3 h) eggs was assayed by the RNA ligation-mediated poly(A) test (RL-PAT). * indicates RNaseH/oligo(dT)20 digestion prior to ligation. Control lanes: –Lig, Ligation reaction performed without RNA; -RT, ligated RNA was not reverse transcribed prior to PCR. M, DNA size marker are given in base pairs. Direct sequencing of atp5a1 and tpi1 (A lower panel) reveals the actual transcript 3′ending (indicated by arrows), which is in fresh eggs at the end of the poly(A) tail (italic As), but in aged eggs several nucleotides upstream of the former end of the RNA body (clear box). P1 is the ligated primer.

Mentions: To show directly that the decrease in transcripts relies on a shortening of the poly(A) tail we used the RNA ligation-mediated poly(A) test (RL-PAT) [23] of decreased (Fig. 6A) and not-changed (Fig. 6B) transcripts. Aged eggs revealed a poly(A) tail shortening in all four decreased transcripts. Notably transcripts of aged eggs were shorter than transcripts of fresh eggs deadenylated by RNaseH/oligo(dT)20 treatment (Fig. 6A). Direct sequencing laid open a complete deadenylation plus a loss of seven and five nucleotides of the RNA body in atp5a1 and tpi1, respectively. In contrast, transcripts being not changed upon aging showed no transcript shortening, independent of the poly(A) length in fresh eggs (Fig. 6B).


Aging of Xenopus tropicalis eggs leads to deadenylation of a specific set of maternal mRNAs and loss of developmental potential.

Kosubek A, Klein-Hitpass L, Rademacher K, Horsthemke B, Ryffel GU - PLoS ONE (2010)

Poly(A) tail reduction of specific maternal mRNA in aged eggs.Poly(A) tail behavior of the indicated transcripts decreased (A) and not changed (B) upon egg aging are shown. Total mRNA from fresh (0 h) and aged (3 h) eggs was assayed by the RNA ligation-mediated poly(A) test (RL-PAT). * indicates RNaseH/oligo(dT)20 digestion prior to ligation. Control lanes: –Lig, Ligation reaction performed without RNA; -RT, ligated RNA was not reverse transcribed prior to PCR. M, DNA size marker are given in base pairs. Direct sequencing of atp5a1 and tpi1 (A lower panel) reveals the actual transcript 3′ending (indicated by arrows), which is in fresh eggs at the end of the poly(A) tail (italic As), but in aged eggs several nucleotides upstream of the former end of the RNA body (clear box). P1 is the ligated primer.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2962626&req=5

pone-0013532-g006: Poly(A) tail reduction of specific maternal mRNA in aged eggs.Poly(A) tail behavior of the indicated transcripts decreased (A) and not changed (B) upon egg aging are shown. Total mRNA from fresh (0 h) and aged (3 h) eggs was assayed by the RNA ligation-mediated poly(A) test (RL-PAT). * indicates RNaseH/oligo(dT)20 digestion prior to ligation. Control lanes: –Lig, Ligation reaction performed without RNA; -RT, ligated RNA was not reverse transcribed prior to PCR. M, DNA size marker are given in base pairs. Direct sequencing of atp5a1 and tpi1 (A lower panel) reveals the actual transcript 3′ending (indicated by arrows), which is in fresh eggs at the end of the poly(A) tail (italic As), but in aged eggs several nucleotides upstream of the former end of the RNA body (clear box). P1 is the ligated primer.
Mentions: To show directly that the decrease in transcripts relies on a shortening of the poly(A) tail we used the RNA ligation-mediated poly(A) test (RL-PAT) [23] of decreased (Fig. 6A) and not-changed (Fig. 6B) transcripts. Aged eggs revealed a poly(A) tail shortening in all four decreased transcripts. Notably transcripts of aged eggs were shorter than transcripts of fresh eggs deadenylated by RNaseH/oligo(dT)20 treatment (Fig. 6A). Direct sequencing laid open a complete deadenylation plus a loss of seven and five nucleotides of the RNA body in atp5a1 and tpi1, respectively. In contrast, transcripts being not changed upon aging showed no transcript shortening, independent of the poly(A) length in fresh eggs (Fig. 6B).

Bottom Line: Furthermore, maternal mRNAs known to be deadenylated during egg maturation as well as after fertilization were preferentially deadenylated in aged eggs.Based on our novel finding we postulate that the imbalance of the polyadenylated maternal transcripts upon egg aging contributes to the loss of developmental potential.Based on this hypothesis the developmental consequences of downregulation of specific transcripts can be analyzed in future.

View Article: PubMed Central - PubMed

Affiliation: Institut für Zellbiologie (Tumorforschung), Universitätsklinikum Essen, Universität Duisburg-Essen, Essen, Germany.

ABSTRACT
As first shown more than 100 years ago, fertilization of an aged (overripe) egg increases the rate of malformations and embryonic loss in several vertebrates, including possibly humans as well. Since the molecular events in aging eggs may be similar in these species, we established in the frog Xenopus tropicalis a defined protocol for delayed fertilization of eggs. A three-hour delayed fertilization led to a dramatic increase in malformation and mortality. Gene expression profiling revealed that 14% of the polyadenylated maternal transcripts were downregulated upon aging. These transcripts were not degraded, but rather deadenylated as shown for specific maternal mRNAs. The affected transcripts are characterized by a relatively short 3'UTR and a paucity of cytoplasmic polyadenylation elements (CPE) and polyadenylation signals (PAS). Furthermore, maternal mRNAs known to be deadenylated during egg maturation as well as after fertilization were preferentially deadenylated in aged eggs. Taken together our analysis of aging eggs reveals that unfertilized eggs are in a dynamic state that was previously not realized. On the one hand deadenylation of transcripts that are typically deadenylated during egg maturation continues and this implies overripeness of the aged egg in the truest sense of the word. On the other hand transcripts that normally are deadenylated after fertilization loose their poly(A) in the aged egg and this implies that the egg awaiting fertilization starts processes that are normally only observed after fertilization. Based on our novel finding we postulate that the imbalance of the polyadenylated maternal transcripts upon egg aging contributes to the loss of developmental potential. Based on this hypothesis the developmental consequences of downregulation of specific transcripts can be analyzed in future.

Show MeSH
Related in: MedlinePlus