Identification of a new expanding family of genes characterized by atypical LRR domains. Localization of a cluster preferentially expressed in oocyte

AbstractIn the present work, we have used the in silico subtraction methodology to identify novel oocyte-specific genes in the mouse. By this way, we have identified in silico a new family of genes composed of more than 80 members. Sequence analysis showed that these genes belong to the superfamily of leucine-rich repeat (LRR) proteins. However, LRRs of this family display some variability in length and in amino acids composition within the β-strands region, as more leucine residues are substituted by other hydrophobic amino acids as compared to canonical LRRs. Interestingly, for nine of these genes, the ESTs were represented almost exclusively in mouse egg libraries. Three of them were selected for experimental study. By RT-PCR and in situ hybridization, we confirmed their specific expression in the mouse oocyte from primary to preovulatory follicles. These three genes are localized in a cluster on mouse chromosome 4, in the vicinity of another recently discovered oocyte specific gene called oogenesin, that we also found to belong to the same family. We thus re-named this latter gene ‘oogenesin-1’, and the three genes identified here were named oogenesin-2, -3 and -4

Prevalence of burnout among Swiss cancer clinicians, paediatricians and general practitioners: who are most at risk?

Goals of work: Increasing economical and administrative constraints and changes in health-care systems constitute a risk for burnout, especially for cancer physicians. However, little is known about differences across medical specialties and the importance of work characteristics. Methods: A postal questionnaire addressing burnout, psychiatric morbidity, sociodemographics and work characteristics was administered to 180 cancer physicians, 184 paediatricians and 197 general practitioners in Switzerland. Results: A total of 371 (66%) physicians participated in the survey. Overall, one third of the respondents expressed signs indicative of psychiatric morbidity and of burnout, including high levels of emotional exhaustion (33%) and depersonalisation/cynicism (28%) and a reduced feeling of personal accomplishment (20%). Workload (>50h/week), lack of continuing education (<6h/month) and working in a public institution were significantly associated with an increased risk of burnout. After adjustment for these characteristics, general practitioners had a higher risk for emotional exhaustion (OR: 2.0, 95% CI: 1.1 to 3.6) and depersonalisation (OR: 2.7, 95% CI: 1.4 to 5.3). Conclusion: In this Swiss sample, cancer clinicians had a significant lower risk of burnout, despite a more important workload. Among possible explanations, involvement in research and teaching activities and access to continuing education may have protected the

Ultrabrza vitrifikacija u otvorenoj rastegnutoj slamci otvara nove mogućnosti za smrzavanje konjskih zametaka.

The aim of this research was to evaluate ultra rapid OPS vitrification on the embryo viability. The OPS vitrification technique is comprised of ultra rapid freezing of a small drop in which the embryo is placed. Before the thin straw was plunged into the liquid nitrogen, the embryos were treated with highly concentrated cryoprotectant (CPA) solutions as follows: 18% ethylene glycol (EG), 18% dimethyl-sulfoxide (DMSO) and 0.4 M sucrose. Surgical transfer into the recipient mares and morphologic examination of recollected embryos were used to measure the viability of transferred embryos. The research was performed on Welsh pony mares by collecting the embryos 6.75 days after ovulation. Twenty embryos were vitrified and transferred, four in each recipient mare. At day twelve, nine embryos were recollected after fl ushing of the recipient uterus (56%, 9/ 16). In one recipient mare, endometritis was detected when the uterus was fl ushed. Among the sixteen recollected embryos, seven (44%) had normal morphology and well developed embryonic vesicles. The vitrification procedure used proved to be encouraging.Svrha istraživanju bila je ustanoviti učinkovitost ultrabrze vitrifikacije u otvorenoj rastegnutoj slamci na vitalnost konjskih zametaka, prijenosom vitrificiranih pa otopljenih zametaka u sinkronizirane primateljice. Istraživanje je provedeno na stadu Welsh poni kobila. Davateljicama zametaka maternice su transcervikalno bile ispirane 6,75 dana nakon ovulacije, a zametci su nakon vitrifikacije bili pohranjeni u spremnik s tekućim dušikom. Nakon odmrzavanja, zametci su bili prenijeti u sinkronizirane primateljice. Maternice primateljica bile su ispirane petoga dana nakon prijenosa odmrznutih zametaka. Ukupno je bilo preneseno dvadeset zametaka, a ispiranjem maternica primateljica dobiveno je devet zametaka što iznosi 56% s obzirom da je u jedne primateljice ispiranjem ustanovljen endometritis. Od zametaka dobivenih ispiranjem primateljica, sedam (44%) je imalo morfološki normalno razvijene zametne mjehure. Na osnovi provedenih istraživanja zaključeno je da su rezultati ostvareni vitrifikacijom u otvorenoj rastegnutoj slamci ohrabrujući, ali bi ih s obzirom na mali broj prenijetih zametaka trebalo potvrditi na većem uzorku, posebice sa zametcima odabranoga promjera te brojem ždrebadi

Oviductal Extracellular Vesicles Enhance Porcine In Vitro Embryo Development by Modulating the Embryonic Transcriptome

Oviductal extracellular vesicles (oEVs) have been identified as important components of the oviductal fluid (OF) and have been pointed to as key modulators of gamete/embryo-maternal interactions. Here, we determined the functional impact of oEVs on embryo development and the embryonic transcriptome in porcine. Experiment 1 examined the effect of oEVs and OF on embryo development. In vitro-produced embryos were cultured with oEVs or OF for 2 or 7 days using an in vitro sequential system or without supplementation (control). Experiment 2 analyzed transcriptomic alterations of EV-treated embryos versus control and the oEVs RNA cargo by RNA-sequencing. Two days of EV treatment enhanced embryo development over time when compared to other treatments. Different RNA expression profiles between embryos treated with EVs for two or seven days and untreated controls were obtained, with 54 and 59 differentially expressed (DE) genes and six and seven DE miRNAs, respectively. In oEV RNA cargo, 12,998 RNAs and 163 miRNAs were identified. Integrative analyses pointed to specific oEV components that might act as modulators of the embryonic transcriptome, such as S100A11, ANXA2 or miR-21-5p. Overall, the findings suggested that oEVs could be a potential strategy to improve porcine IVP outcomes, particularly by using two days of EV treatment

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

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

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

Characterization of oviduct epithelial spheroids for the study of embryo-maternal communication in cattle

Most in vitro models of oviduct epithelial cells (OEC) used thus far to gain insights into embryo-maternal communication induce cell dedifferentiation or are technically challenging. Moreover, although the presence of developing embryos has been shown to alter gene expression in OEC, the effect of embryos on OEC physiology remains largely unknown. Here, we propose a model based on bovine oviduct epithelial spheroids (OES) with specific shape and diameter (100-200 μm) criteria. The aims of this study were to i) determine the appropriate culture conditions of bovine OES cultured in suspension by evaluating their morphology, total cell number, viability, and activity of ciliated cells; ii) monitor gene expression in OES at the time of their formation (day 0) and over the 10 days of culture; and iii) test whether the vicinity of developing embryos affects OES quality criteria. On day 10, the proportions of vesicle-shaped OES (V-OES) were higher in M199/500 (500 μl of HEPES-buffered TCM-199) and synthetic oviduct fluid (SOF)/25 (25-μL droplet of SOF medium under mineral oil) than in M199/25 (25-μL droplet of M199 under mineral oil). The proportion of viable cells in V-OES was not affected by culture conditions and remained high (>80%) through day 10. The total number of cells per V-OES decreased over time except in SOF/25, while the proportions of ciliated cells increased over time in M199/500 but decreased in M199/25 and SOF/25. The movement amplitude of OES in suspension decreased over time under all culture conditions. Moreover, the gene expression of ANXA1, ESR1, HSPA8, and HSPA1A in OES remained stable during culture, while that of PGR and OVGP1 decreased from day 0 to day 10. Last, the co-culture of developing embryos with OES in SOF/25 increased the rates of blastocysts on days 7 and 8 compared to embryos cultured alone, and increased the proportion of V-OES compared to OES cultured alone. In conclusion, M199/500 and SOF/25 provided the optimal conditions for the long-time culture of OES. The supporting effect of OES on embryo development and of developing embryos on OES morphology was evidenced for the first time. Altogether, these results point OES as an easy-to-use, standardizable, and physiological model to study embryo-maternal interactions in cattle

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