MATER protein expression and intracellular localization throughout folliculogenesis and preimplantation embryo development in the bovine

BACKGROUND: Mater (Maternal Antigen that Embryos Require), also known as Nalp5 (NACHT, leucine rich repeat and PYD containing 5), is an oocyte-specific maternal effect gene required for early embryonic development beyond the two-cell stage in mouse. We previously characterized the bovine orthologue MATER as an oocyte marker gene in cattle, and this gene was recently assigned to a QTL region for reproductive traits. RESULTS: Here we have analyzed gene expression during folliculogenesis and preimplantation embryo development. In situ hybridization and immunohistochemistry on bovine ovarian section revealed that both the transcript and protein are restricted to the oocyte from primary follicles onwards, and accumulate in the oocyte cytoplasm during follicle growth. In immature oocytes, cytoplasmic, and more precisely cytosolic localization of MATER was confirmed by immunohistochemistry coupled with confocal microscopy and immunogold electron microscopy. By real-time PCR, MATER messenger RNA was observed to decrease strongly during maturation, and progressively during the embryo cleavage stages; it was hardly detected in morulae and blastocysts. The protein persisted after fertilization up until the blastocyst stage, and was mostly degraded after hatching. A similar predominantly cytoplasmic localization was observed in blastomeres from embryos up to 8-cells, with an apparent concentration near the nuclear membrane. CONCLUSION: Altogether, these expression patterns are consistent with bovine MATER protein being an oocyte specific maternal effect factor as in mouse

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 (<it>RNF18</it>) and breast cancer anti-estrogen resistance 4 (<it>BCAR4</it>), whose oocyte preferential expression was not previously reported in any species, as well as Maternal embryonic leucine zipper kinase (<it>MELK</it>) and <it>STELLA</it>. We included three known oocyte marker genes (Maternal antigen that embryos require (<it>MATER</it>), Zygote arrest 1 (<it>ZAR1</it>), NACHT, leucine rich repeat and PYD containing 9 (<it>NALP9</it>)). In addition, we selected transcripts previously identified as differentially regulated during maturation, peroxiredoxin 1 and 2 (<it>PRDX1, PRDX2</it>), inhibitor of DNA binding 2 and 3 (<it>ID2</it>, <it>ID3</it>), cyclin B1 (<it>CCNB1</it>), cell division cycle 2 (<it>CDC2</it>), as well as Aurora A (<it>AURKA</it>). 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 <it>PRDX1</it>, 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 <it>MELK </it>; and the corresponding genes remained silent after major embryonic genome activation.</p> <p>Conclusion</p> <p>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.</p

Spatio-temporal expression patterns of aurora kinases a, B, and C and cytoplasmic polyadenylation-element-binding protein in bovine oocytes during meiotic maturation.

International audienceMaturation of immature bovine oocytes requires cytoplasmic polyadenylation and synthesis of a number of proteins involved in meiotic progression and metaphase-II arrest. Aurora serine-threonine kinases--localized in centrosomes, chromosomes, and midbody--regulate chromosome segregation and cytokinesis in somatic cells. In frog and mouse oocytes, Aurora A regulates polyadenylation-dependent translation of several mRNAs such as MOS and CCNB1, presumably by phosphorylating CPEB, and Aurora B phosphorylates histone H3 during meiosis. We analyzed the expression of three Aurora kinase genes--AURKA, AURKB, and AURKC--in bovine oocytes during meiosis by reverse transcription followed by quantitative real-time PCR and immunodetection. Aurora A was the most abundant form in oocytes, both at mRNA and protein levels. AURKA protein progressively accumulated in the oocyte cytoplasm during antral follicle growth and in vitro maturation. AURKB associated with metaphase chromosomes. AURKB, AURKC, and Thr-phosphorylated AURKA were detected at a contractile ring/midbody during the first polar body extrusion. CPEB, localized in oocyte cytoplasm, was hyperphosphorylated during prophase/metaphase-I transition. Most CPEB degraded in metaphase-II oocytes and remnants remained localized in a contractile ring. Roscovitine, U0126, and metformin inhibited meiotic divisions; they all induced a decrease of CCNB1 and phospho-MAPK3/1 levels and prevented CPEB degradation. However, only metformin depleted AURKA. The Aurora kinase inhibitor VX680 at 100 nmol/L did not inhibit meiosis but led to multinuclear oocytes due to the failure of the polar body extrusion. Thus, in bovine oocyte meiosis, massive destruction of CPEB accompanies metaphase-I/II transition, and Aurora kinases participate in regulating segregation of the chromosomes, maintenance of metaphase-II, and formation of the first polar body

Zygote arrest 1 gene in pig, cattle and human: evidence of different transcript variants in male and female germ cells

BACKGROUND: Zygote arrest 1 (ZAR1) is one of the few known oocyte-specific maternal-effect genes essential for the beginning of embryo development discovered in mice. This gene is evolutionary conserved in vertebrates and ZAR1 protein is characterized by the presence of atypical plant homeobox zing finger domain, suggesting its role in transcription regulation. This work was aimed at the study of this gene, which could be one of the key regulators of successful preimplantation development of domestic animals, in pig and cattle, as compared with human. METHODS: Screenings of somatic cell hybrid panels and in silico research were performed to characterize ZAR1 chromosome localization and sequences. Rapid amplification of cDNA ends was used to obtain full-length cDNAs. Spatio-temporal mRNA expression patterns were studied using Northern blot, reverse transcription coupled to polymerase chain reaction and in situ hybridization. RESULTS: We demonstrated that ZAR1 is a single copy gene, positioned on chromosome 8 in pig and 6 in cattle, and several variants of correspondent cDNA were cloned from oocytes. Sequence analysis of ZAR1 cDNAs evidenced numerous short inverted repeats within the coding sequences and putative Pumilio-binding and embryo-deadenylation elements within the 3'-untranslated regions, indicating the potential regulation ways. We showed that ZAR1 expressed exclusively in oocytes in pig ovary, persisted during first cleavages in embryos developed in vivo and declined sharply in morulae and blastocysts. ZAR1 mRNA was also detected in testis, and, at lower level, in hypothalamus and pituitary in both species. For the first time, ZAR1 was localized in testicular germ cells, notably in round spermatids. In addition, in pig, cattle and human only shorter ZAR1 transcript variants resulting from alternative splicing were found in testis as compared to oocyte. CONCLUSION: Our data suggest that in addition to its role in early embryo development highlighted by expression pattern of full-length transcript in oocytes and early embryos, ZAR1 could also be implicated in the regulation of meiosis and post meiotic differentiation of male and female germ cells through expression of shorter splicing variants. Species conservation of ZAR1 expression and regulation underlines the central role of this gene in early reproductive processes

Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress

Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages

Identification et caractérisation de gènes préférentiellement exprimés dans l'ovocyte bovin

Diplôme : Dr. d'Universit

Identification et caractérisation de gènes préférentiellement exprimés dans l'ovocyte bovin

Several oocyte-specific genes were shown to be essential for folliculogenesis and preimplantation embryo development in mouse. The aim of this thesis was to identify such genes in bovine. By RT-PCR, MATER, NALP9, ZARI, GDF9 and BMP15 genes were shown to be preferentially expressed in oocyte and transcription was not reactivated at the time of maternal to embryo transition. Full-length MATER and NALP9 cDNA were cloned. MATER protein was detected in oocytes from primary follicle onwards and in embryos until the blastocyst stage. In parallel, we performed suppressive subtraction hybridization between oocytes and somatic tissues. Macroarray hybridization revealed that 80% of EST were overexpressed fourfold in oocyte. By RT-PCR, 6 identified genes (including STELLA and SLBP2) and 8 novel EST were shown to be indeed preferentially expressed in oocyte and/or testis. These profiles suggest a role for the corresponding genes in gametogenesis and/or early embryonic development.Chez la souris, plusieurs gènes ovocyte-spécifiques sont essentiels pour la folliculogenèse ou le développement embryonnaire. L'objectif de la thèse était d'identifier de tels gènes chez le bovin. Par RT-PCR, nous avons montré que les gènes MATER, NALP9, ZARI, GDF9 et BMP15 sont préférentiellement exprimés dans l'ovocyte et qu'ils ne sont pas transcrits à la transition maternelle embryonnaire. La protéine MATER est détectée dans l'ovocyte dès le follicule primaire et dans l'embryon jusqu'au blastocyste. En parallèle, une banque soustraite d'EST prépondérantes dans l'ovocyte par rapport aux tissus somatiques a été générée par hybridation suppressive et soustractive. Plus de 80% des EST sont surexprimées dans l'ovocyte. L'expression préférentielle dans l'ovocyte et/ou le testicule de 6 gènes connus (dont STELLA, SLBP2) et 8 nouvelles EST a été confirmée par RT/PCR. Ces profils suggèrent que les gènes bovins étudiés jouent un rôle dans la gamétogenèse et/ou le développement précoce.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

A bovine orthologue of the maternal effect gene stella/PGC7 identified by subtractive and suppressive hybridization

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Bovine Mater-Like NALP9 Is an Oocyte Marker Gene

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Genes preferentially expressed in bovine oocytes revealed by subtractive and suppressive hybridization

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