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

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

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

In vitro Bisphenol S affects in vitro early developmental oocyte competence in ewe

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Le Bisphenol S altère la compétence ovocytaire in vitro chez la brebis

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Bisphenol S affects in vitro early developmental oocyte competence in ewes

International audienceIn the plastics industry, bisphenol S (BPS) replaces sisphenol A reported to be an oestrogen mimetic endocrine disruptor damaging oocyte meiosis and maturation (Machtinger 2014 Reprod. Biomed. Online 29, 404). Studies on fish and rodents reported that BPS affects reproduction similarly to BPA (Uzumcu 2007 Reprod. Toxicol. 23, 337; Giulivo 2016 Environ. Res. 151, 251; Ullah 2016 Chemosphere 152, 353). Bisphenol S is detected in human urine at nanomolar concentrations (Liao 2012 Environ. Sci. Technol 46, 6860) and in some laboratory supplies (tips and tubes; unpublished data). Therefore, in this study, we assessed effects of BPS at low doses during in vitro maturation (IVM) on oocyte developmental competence in ewes. Cumulus-oocyte complexes (COC) collected from ovine follicles >2 mm underwent 24-h IVM, in the absence or presence of BPS at 1, 10, and 100 nM and 1 and 10 µM (Sigma Chemical Co.). Nuclear oocyte maturation rate was evaluated by MII oocyte count after chromatin Hoechst staining [n = 3 replicates (R), 1159 oocytes]. At 6 h of IVM, BPS effects on mRNA expression of oestrogen (E2) and progesterone (P4) receptors in cumulus cells (CC) were assessed by real-time quantitative PCR. After 24 h of IVM, The effect of BPS on P4 level was assessed in spent medium by enzyme-linked immunosorbent assay (n = 6 R, 40 COC/condition). Transcript expression level and P4 concentration were analysed using nonparametric one-way ANOVA, with Tukey post hoc test (Rcmdr, R version 3.5.3). After 24 h of IVM, matured COC underwent IVF and in vitro culture (IVC) for 7 days. Cleavage and blastocyst rates were assessed on Days 2 and 7, respectively, after IVF (8 experiments, 300 COC/condition). Data were analysed using logistic regression and linear model (R version 3.5.3). Our results showed a decreased oocyte maturation rate with 10 µM BPS (76.6%, n = 171; P = 0.0008) compared with control (88%, n = 152), with no effect on cell viability. The concentration of P4 decreased with 1 µM BPS (0.02 ng mL−1 per COC) compared with control (0.034 ng mL−1 per COC; P < 0.001). At 6 h IVM, BPS had no significant effect on oestrogen receptors (ESR1, ESR2, GPER) transcripts in CC but 10 nM BPS decreased mRNA expression of P4 receptor (PR) (0.00647 ± 0.00145; P = 0,005) compared with control (0.01165 ± 0.00196). Within fertilized COC, 1 µM BPS decreased cleavage rate (47.6%, n = 152) compared with control (54.6%; P = 0.004). Among cleaved embryos, blastocyst rate decreased to 14.2% and 12.5% with 10 nM and 1 µM BPS respectively (n = 26, P = 0.046; and n = 19, P = 0.017), compared with control (21.8%, n = 44). Bisphenol S at a low dose during ovine IVM reduced COC P4 secretion, PR transcript in CC, and cleavage and blastocyst rates. Our data suggest that BPS at an environmental dose (10 nM) negatively affects early developmental oocyte competence. Studies are ongoing to investigate the effect of BPS on Day 6 embryo cell number and on the ERK1/2 signalling pathway in oocyte

Bisphenol S Impairs In Vitro Ovine Early Developmental Oocyte Competence

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An oocyte-preferential histone mRNA stem-loop-binding protein like is expressed in several mammalian species

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