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

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

Le développement folliculaire ovarien et l’ovulation

National audienceLe développement des follicules ovariens, ou folliculogenèse, commence au démarrage de la croissance du follicule primordial et se termine à l’ovulation, qui permet la libération d’un ovocyte fécondable dans les voies génitales femelles. L’ovulation ne se produit que si les caractéristiques endogènes (développementales, hormonales, métaboliques) de l’individu et son environnement (saison, nutrition, interactions sociales) le permettent. Pour tous les mammifères, la folliculogenèse et l’ovulation sont sous le contrôle direct du système hypothalamo-hypophysaire, qui intègre les facteurs endogènes et exogènes et les traduit par des modifications de sécrétion des gonadotropines FSH et LH. Ces processus sont aussi régulés par de très nombreux autres facteurs, dont l’importance relative varie au cours de la folliculogenèse. L’objectif de ce chapitre est de présenter l’état des connaissances sur le développement folliculaire ovarien et l’ovulation chez les mammifères, en intégrant le rôle respectif de l’ovocyte et du follicule

Identification, characterization and metagenome analysis of oocyte-specific genes organized in clusters in the mouse genome

<p>Abstract</p> <p>Background</p> <p>Genes specifically expressed in the oocyte play key roles in oogenesis, ovarian folliculogenesis, fertilization and/or early embryonic development. In an attempt to identify novel oocyte-specific genes in the mouse, we have used an <it>in silico </it>subtraction methodology, and we have focused our attention on genes that are organized in genomic clusters.</p> <p>Results</p> <p>In the present work, five clusters have been studied: a cluster of thirteen genes characterized by an F-box domain localized on chromosome 9, a cluster of six genes related to T-cell leukaemia/lymphoma protein 1 (Tcl1) on chromosome 12, a cluster composed of a SPErm-associated glutamate (E)-Rich (Speer) protein expressed in the oocyte in the vicinity of four unknown genes specifically expressed in the testis on chromosome 14, a cluster composed of the oocyte secreted protein-1 (Oosp-1) gene and two Oosp-related genes on chromosome 19, all three being characterized by a partial N-terminal zona pellucida-like domain, and another small cluster of two genes on chromosome 19 as well, composed of a TWIK-Related spinal cord K+ channel encoding-gene, and an unknown gene predicted <it>in silico </it>to be testis-specific. The specificity of expression was confirmed by RT-PCR and in situ hybridization for eight and five of them, respectively. Finally, we showed by comparing all of the isolated and clustered oocyte-specific genes identified so far in the mouse genome, that the oocyte-specific clusters are significantly closer to telomeres than isolated oocyte-specific genes are.</p> <p>Conclusion</p> <p>We have studied five clusters of genes specifically expressed in female, some of them being also expressed in male germ-cells. Moreover, contrarily to non-clustered oocyte-specific genes, those that are organized in clusters tend to map near chromosome ends, suggesting that this specific near-telomere position of oocyte-clusters in rodents could constitute an evolutionary advantage. Understanding the biological benefits of such an organization as well as the mechanisms leading to a specific oocyte expression in these clusters now requires further investigation.</p

Transcriptional Regulatory Properties of Epstein-Barr Virus Nuclear Antigen 3C Are Conserved in Simian Lymphocryptoviruses

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) is a large transcriptional regulator essential for EBV-mediated immortalization of B lymphocytes. We previously identified interactions between EBNA-3C and two cellular transcription factors, Jκ and Spi proteins, through which EBNA-3C regulates transcription. To better understand the contribution of these interactions to EBNA-3C function and EBV latency, we examined whether they are conserved in the homologous proteins of nonhuman primate lymphocryptoviruses (LCVs), which bear a strong genetic and biological similarity to EBV. The homologue of EBNA-3C encoded by the LCV that infects baboons (BaLCV) was found to be only 35% identical in sequence to its EBV counterpart. Of particular significance, this homology localized predominantly to the N-terminal half of the molecule, which encompasses the domains in EBNA-3C that interact with Jκ and Spi proteins. Like EBNA-3C, both BaLCV and rhesus macaque LCV (RhLCV) 3C proteins bound to Jκ and repressed transcription mediated by EBNA-2 through its interaction with Jκ. Both nonhuman primate 3C proteins were also able to activate transcription mediated by the Spi proteins in the presence of EBNA-2. Like EBNA-3C, a domain encompassing the putative basic leucine zipper motif of the BaLCV-3C protein directly interacted with both Spi-1 and Spi-B. Surprisingly, a recently identified motif in EBNA-3C that mediates repression was not identifiable in the BaLCV-3C protein. Finally, although the C terminus of BaLCV-3C bears minimal homology to EBNA-3C, it nonetheless contains a C-terminal domain rich in glutamine and proline that was able to function as a potent transcriptional activation domain, as does the C terminus of EBNA-3C. The conservation of these functional motifs despite poor overall homology among the LCV 3C proteins strongly suggests that the interactions of EBNA-3C with Jκ and Spi do indeed play significant roles in the life cycle of EBV