Search

Cross-phylum and cross-species comparative transcriptomic analyses provide an evolutionary perspective on how specific tissues use genomic information. A significant mRNA subset present in the oocytes of most vertebrates is stabilized or stored for post- LH surge use. Since transcription is arrested in the oocyte before ovulation, this RNA is important for completing maturation and sustaining embryo development until zygotic genome activation. We compared the human oocyte transcriptome with an oocyte-enriched subset of mouse, bovine and frog (Xenopus laevis) genes in order to evaluate similarities between species. Graded temperature stringency hybridization on a multi-species oocyte cDNA array was used to measure the similarity of preferentially expressed sequences to the human oocyte library. Identity analysis of 679 human orthologs compared with each identified official gene symbol found in the subtractive (somatic -oocyte) li- braries comprising our array revealed that bovine/human similarity was greater than mouse/human or frog/human similarity. However, based on protein sequence, mouse/human similarity was greater than bovine/human similarity. Among the genes over-expressed in oocytes relative to somatic tissue in Xenopus, Mus and Bos, a high level of conservation was found relative to humans, especially for genes involved in early embryonic development.

The oocyte capacity to rejuvenate a differentiated nucleus to restart the proper embryonic program has been highly conserved between vertebrate species. In view of the recent progress to induce pluripotency in somatic cells with stemness genes, we investigated the potential of oocyte genes to contribute to chromatin rearrangements in somatic cells. We selected conserved genes that are naturally expressed mainly in oocytes and that were susceptible to play a role in reprogramming during early embryogenesis. We induced their expression by transient transfection in HEK293 cells. We then assessed whether they had a global impact on epigenetic events such as histone core modifications, and also on transcription and expression of pluripotency-associated transcription factors. Nucleoplasmin 2 (NPM2), activation-induced cytidine deaminase (AICDA), and Geminin (GMNN) overexpression induced differences in histone core modifications (methylation and acetylation). AICDA and NPM2 also influenced RNA neosynthesis. NPM2, GMNN, and STELLA induced overexpression of well-known pluripotency transcription factors. Overall, AICDA, GMNN, NPM2, and STELLA influenced at least one of the aspects analyzed. Their potential could be useful in increasing the cell receptivity to pluripotency induction.

Maturation 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.

We have cloned and sequenced the bovine NALP9 cDNA, which contains a 2991 bp long open reading frame with 76% homology to its human counterpart. The putative 996 amino acids protein presents the domain organization typical of the Nacht, Leucine rich repeat and Pyrin domain containing (NALP) family that includes the maternal effect factor MATER. By reverse transcription coupled to polymerase chain reaction, we have analyzed expression of NALP9 in a panel of somatic and gonadic tissues and during in vitro preimplantation development. The transcript was detected exclusively in testis and ovary, and at a remarkably high level in the oocyte from antral follicles. NALP9 mRNA remained present after in vitro maturation and fertilization, and was detected in embryos, but transcription was not reactivated at the maternal to embryo transition. Thus NALP9 is characterized as a novel oocyte marker gene in cattle. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc.

We have cloned the bovine homologue of Mater (maternal antigen that embryos require) cDNA, potentially the first germ cell-specific maternal-effect gene in this species. The 3297 base-pair longest open reading frame encodes a putative protein of 1098 amino acids with a domain organization similar to its human counterpart. By reverse transcription coupled to polymerase chain reaction, we have analyzed the spatiotemporal expression of MATER, along with other potential markers of germ cells or oocytes: ZAR1 (zygotic arrest 1), GDF9 (growth and differentiation factor 9), BMP15 (bone morphogenetic protein 15), and VASA. In agreement with a preferential oocyte origin, MATER, ZAR1, GDF9, and BMP15 transcripts were detected in the oocyte itself at a much higher level than in the gonads, while no significant expression was detected in our panel of somatic tissues (uterus, heart, spleen, intestine, liver, lung, mammary gland, muscle). In situ hybridization confirmed oocyte-restricted expression of MATER and ZAR1 within the ovary, as early as preantral follicle stages. VASA was highly represented in the testis and the ovary, and still present in the oocyte from antral follicles. Maternal MATER, ZAR1, GDF9, and BMP15 transcripts persisted during oocyte in vitro maturation and fertilization and in preimplantation embryo until the five- to eight-cell or morula stage, but transcription was not reactivated at the time of embryonic genome activation.

International audience; The anti-Müllerian hormone type II (AMHRII) receptor is the primary receptor for anti-Müllerian hormone (AMH), a protein produced by Sertoli cells and responsible for the regression of the Müllerian duct in males. AMHRII is a membrane protein containing an N-terminal extracellular domain (ECD) that binds AMH, a transmembrane domain, and an intracellular domain with serine/threonine kinase activity. Mutations in the AMHRII gene lead to persistent Müllerian duct syndrome in human males. In this paper, we have investigated the effects of 10 AMHRII mutations, namely 4 mutations in the ECD and 6 in the intracellular domain. Molecular models of the extra- and intracellular domains are presented and provide insight into how the structure and function of eight of the mutant receptors, which are still expressed at the cell surface, are affected by their mutations. Interestingly, two soluble receptors truncated upstream of the transmembrane domain are not secreted, unless the transforming growth factor beta type II receptor signal sequence is substituted for the endogenous one. This shows that the AMHRII signal sequence is defective and suggests that AMHRII uses its transmembrane domain instead of its signal sequence to translocate to the endoplasmic reticulum, a characteristic of type III membrane proteins.

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.

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.

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.