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Differential regulation of abundance and deadenylation of maternal transcripts during bovine oocyte maturation in vitro and in vivo.

Thélie A, Papillier P, Pennetier S, Perreau C, Traverso JM, Uzbekova S, Mermillod P, Joly C, Humblot P, Dalbiès-Tran R - BMC Dev. Biol. (2007)

Bottom Line: Throughout in vitro development, oocyte restricted transcripts were progressively degraded until the morula stage, except for MELK ; and the corresponding genes remained silent after major embryonic genome activation.Altogether, our data emphasize the extent of post-transcriptional regulation during oocyte maturation.They do not evidence a general alteration of this phenomenon after in vitro maturation as compared to in vivo maturation, but indicate that some individual messenger RNA can be affected.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, UMR85 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France. Aurore.Thelie@tours.inra.fr

ABSTRACT

Background: In bovine maturing oocytes and cleavage stage embryos, gene expression is mostly controlled at the post-transcriptional level, through degradation and deadenylation/polyadenylation. We have investigated how post transcriptional control of maternal transcripts was affected during in vitro and in vivo maturation, as a model of differential developmental competence.

Results: Using real time PCR, we have analyzed variation of maternal transcripts, in terms of abundance and polyadenylation, during in vitro or in vivo oocyte maturation and in vitro embryo development. Four genes are characterized here for the first time in bovine: ring finger protein 18 (RNF18) and breast cancer anti-estrogen resistance 4 (BCAR4), whose oocyte preferential expression was not previously reported in any species, as well as Maternal embryonic leucine zipper kinase (MELK) and STELLA. We included three known oocyte marker genes (Maternal antigen that embryos require (MATER), Zygote arrest 1 (ZAR1), NACHT, leucine rich repeat and PYD containing 9 (NALP9)). In addition, we selected transcripts previously identified as differentially regulated during maturation, peroxiredoxin 1 and 2 (PRDX1, PRDX2), inhibitor of DNA binding 2 and 3 (ID2, ID3), cyclin B1 (CCNB1), cell division cycle 2 (CDC2), as well as Aurora A (AURKA). Most transcripts underwent a moderate degradation during maturation. But they displayed sharply contrasted deadenylation patterns that account for variations observed previously by DNA array and correlated with the presence of a putative cytoplasmic polyadenylation element in their 3' untranslated region. Similar variations in abundance and polyadenylation status were observed during in vitro maturation or in vivo maturation, except for PRDX1, that appears as a marker of in vivo maturation. Throughout in vitro development, oocyte restricted transcripts were progressively degraded until the morula stage, except for MELK ; and the corresponding genes remained silent after major embryonic genome activation.

Conclusion: Altogether, our data emphasize the extent of post-transcriptional regulation during oocyte maturation. They do not evidence a general alteration of this phenomenon after in vitro maturation as compared to in vivo maturation, but indicate that some individual messenger RNA can be affected.

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Characterization of oocyte specific transcripts. (A) Expression pattern of MELK, RNF18, BCAR4 and STELLA in bovine somatic and gonadic tissues by RT-PCR. Tissues are indicated above the lanes: brain (Br), heart (He), pituitary gland (Pt), intestine (In), liver (Li), lung (Lu), muscle (Mu), spleen (Sp), uterus (Ut), testis (Te), Ovary (Ov), oocyte (Oo); no PCR substrate as negative control (-). RT-PCR for ACTB is shown as a positive control. An estimated 500-fold higher amount of substrate was used in PCR for somatic tissues and gonads as compared to the oocyte. (B) Schematic representation of STELLA transcript variants generated by alternative splicing. (C) Schematic representation of BCAR4 variants generated by alternative use of two polyadenylation signals (AAUAAA).
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Figure 1: Characterization of oocyte specific transcripts. (A) Expression pattern of MELK, RNF18, BCAR4 and STELLA in bovine somatic and gonadic tissues by RT-PCR. Tissues are indicated above the lanes: brain (Br), heart (He), pituitary gland (Pt), intestine (In), liver (Li), lung (Lu), muscle (Mu), spleen (Sp), uterus (Ut), testis (Te), Ovary (Ov), oocyte (Oo); no PCR substrate as negative control (-). RT-PCR for ACTB is shown as a positive control. An estimated 500-fold higher amount of substrate was used in PCR for somatic tissues and gonads as compared to the oocyte. (B) Schematic representation of STELLA transcript variants generated by alternative splicing. (C) Schematic representation of BCAR4 variants generated by alternative use of two polyadenylation signals (AAUAAA).

Mentions: One EST was 88% homologous to human MELK. As observed by RT-PCR, MELK is not really oocyte specific; rather, it is expressed at low level in most tissues, but strongly expressed in both the male and female gonads and in the oocyte (Fig 1A). A virtual Northern blot revealed a single 2.5 kbp cDNA (not shown). The full length 2445 bp sequence deduced from RACE (genbank accession hoEF446902) is assembled from 18 exons, including a first non coding exon that is lacking in the bovine MELK transcript that has since been predicted within the genome onto BTA8. The full-length sequence contains a 1953 nucleotide long open reading frame (ORF), encoding a predicted 650 aa/74 kD protein wse sequence is 90% and 80% identical to its human and mouse counterparts, respectively; it presents similar serine/threonine kinase domain and kinase associated domain at the N-terminus and C-terminus respectively.


Differential regulation of abundance and deadenylation of maternal transcripts during bovine oocyte maturation in vitro and in vivo.

Thélie A, Papillier P, Pennetier S, Perreau C, Traverso JM, Uzbekova S, Mermillod P, Joly C, Humblot P, Dalbiès-Tran R - BMC Dev. Biol. (2007)

Characterization of oocyte specific transcripts. (A) Expression pattern of MELK, RNF18, BCAR4 and STELLA in bovine somatic and gonadic tissues by RT-PCR. Tissues are indicated above the lanes: brain (Br), heart (He), pituitary gland (Pt), intestine (In), liver (Li), lung (Lu), muscle (Mu), spleen (Sp), uterus (Ut), testis (Te), Ovary (Ov), oocyte (Oo); no PCR substrate as negative control (-). RT-PCR for ACTB is shown as a positive control. An estimated 500-fold higher amount of substrate was used in PCR for somatic tissues and gonads as compared to the oocyte. (B) Schematic representation of STELLA transcript variants generated by alternative splicing. (C) Schematic representation of BCAR4 variants generated by alternative use of two polyadenylation signals (AAUAAA).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211488&req=5

Figure 1: Characterization of oocyte specific transcripts. (A) Expression pattern of MELK, RNF18, BCAR4 and STELLA in bovine somatic and gonadic tissues by RT-PCR. Tissues are indicated above the lanes: brain (Br), heart (He), pituitary gland (Pt), intestine (In), liver (Li), lung (Lu), muscle (Mu), spleen (Sp), uterus (Ut), testis (Te), Ovary (Ov), oocyte (Oo); no PCR substrate as negative control (-). RT-PCR for ACTB is shown as a positive control. An estimated 500-fold higher amount of substrate was used in PCR for somatic tissues and gonads as compared to the oocyte. (B) Schematic representation of STELLA transcript variants generated by alternative splicing. (C) Schematic representation of BCAR4 variants generated by alternative use of two polyadenylation signals (AAUAAA).
Mentions: One EST was 88% homologous to human MELK. As observed by RT-PCR, MELK is not really oocyte specific; rather, it is expressed at low level in most tissues, but strongly expressed in both the male and female gonads and in the oocyte (Fig 1A). A virtual Northern blot revealed a single 2.5 kbp cDNA (not shown). The full length 2445 bp sequence deduced from RACE (genbank accession hoEF446902) is assembled from 18 exons, including a first non coding exon that is lacking in the bovine MELK transcript that has since been predicted within the genome onto BTA8. The full-length sequence contains a 1953 nucleotide long open reading frame (ORF), encoding a predicted 650 aa/74 kD protein wse sequence is 90% and 80% identical to its human and mouse counterparts, respectively; it presents similar serine/threonine kinase domain and kinase associated domain at the N-terminus and C-terminus respectively.

Bottom Line: Throughout in vitro development, oocyte restricted transcripts were progressively degraded until the morula stage, except for MELK ; and the corresponding genes remained silent after major embryonic genome activation.Altogether, our data emphasize the extent of post-transcriptional regulation during oocyte maturation.They do not evidence a general alteration of this phenomenon after in vitro maturation as compared to in vivo maturation, but indicate that some individual messenger RNA can be affected.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, UMR85 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France. Aurore.Thelie@tours.inra.fr

ABSTRACT

Background: In bovine maturing oocytes and cleavage stage embryos, gene expression is mostly controlled at the post-transcriptional level, through degradation and deadenylation/polyadenylation. We have investigated how post transcriptional control of maternal transcripts was affected during in vitro and in vivo maturation, as a model of differential developmental competence.

Results: Using real time PCR, we have analyzed variation of maternal transcripts, in terms of abundance and polyadenylation, during in vitro or in vivo oocyte maturation and in vitro embryo development. Four genes are characterized here for the first time in bovine: ring finger protein 18 (RNF18) and breast cancer anti-estrogen resistance 4 (BCAR4), whose oocyte preferential expression was not previously reported in any species, as well as Maternal embryonic leucine zipper kinase (MELK) and STELLA. We included three known oocyte marker genes (Maternal antigen that embryos require (MATER), Zygote arrest 1 (ZAR1), NACHT, leucine rich repeat and PYD containing 9 (NALP9)). In addition, we selected transcripts previously identified as differentially regulated during maturation, peroxiredoxin 1 and 2 (PRDX1, PRDX2), inhibitor of DNA binding 2 and 3 (ID2, ID3), cyclin B1 (CCNB1), cell division cycle 2 (CDC2), as well as Aurora A (AURKA). Most transcripts underwent a moderate degradation during maturation. But they displayed sharply contrasted deadenylation patterns that account for variations observed previously by DNA array and correlated with the presence of a putative cytoplasmic polyadenylation element in their 3' untranslated region. Similar variations in abundance and polyadenylation status were observed during in vitro maturation or in vivo maturation, except for PRDX1, that appears as a marker of in vivo maturation. Throughout in vitro development, oocyte restricted transcripts were progressively degraded until the morula stage, except for MELK ; and the corresponding genes remained silent after major embryonic genome activation.

Conclusion: Altogether, our data emphasize the extent of post-transcriptional regulation during oocyte maturation. They do not evidence a general alteration of this phenomenon after in vitro maturation as compared to in vivo maturation, but indicate that some individual messenger RNA can be affected.

Show MeSH
Related in: MedlinePlus