CDC2/SPDY transiently associates with endoplasmic reticulum exit sites during oocyte maturation

<p>Abstract</p> <p>Background</p> <p>Mammalian oocytes acquire competence to be fertilized during meiotic maturation. The protein kinase CDC2 plays a pivotal role in several key maturation events, in part through controlled changes in CDC2 localization. Although CDC2 is involved in initiation of maturation, a detailed analysis of CDC2 localization at the onset of maturation is lacking. In this study, the subcellular distribution of CDC2 and its regulatory proteins cyclin B and SPDY in combination with several organelle markers at the onset of pig oocyte maturation has been investigated.</p> <p>Results</p> <p>Our results demonstrate that CDC2 transiently associates with a single domain, identified as a cluster of endoplasmic reticulum (ER) exit sites (ERES) by the presence of SEC23, in the cortex of maturing porcine oocytes prior to germinal vesicle break down. Inhibition of meiosis resumption by forskolin treatment prevented translocation of CDC2 to this ERES cluster. Phosphorylated GM130 (P-GM130), which is a marker for fragmented Golgi, localized to ERES in almost all immature oocytes and was not affected by forskolin treatment. After removal of forskolin from the culture media, the transient translocation of CDC2 to ERES was accompanied by a transient dispersion of P-GM130 into the ER suggesting a role for CDC2 in redistributing Golgi components that have collapsed into ERES further into the ER during meiosis. Finally, we show that SPDY, rather than cyclin B, colocalizes with CDC2 at ERES, suggesting a role for the CDC2/SPDY complex in regulating the secretory pathway during oocyte maturation.</p> <p>Conclusion</p> <p>Our data demonstrate the presence of a novel structure in the cortex of porcine oocytes that comprises ERES and transiently accumulates CDC2 prior to germinal vesicle breakdown. In addition, we show that SPDY, but not cyclin B, localizes to this ERES cluster together with CDC2.</p

Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos

<p>Abstract</p> <p>Background</p> <p>In the developing embryo, total RNA abundance fluctuates caused by functional RNA degradation and zygotic genome activation. These variations in the transcriptome in early development complicate the choice of good reference genes for gene expression studies by quantitative real time polymerase chain reaction.</p> <p>Results</p> <p>In order to identify stably expressed genes for normalisation of quantitative data, within early stages of development, transcription levels were examined of 7 frequently used reference genes (<it>B2M, BACT, GAPDH, H2A, PGK1, SI8</it>, and <it>UBC</it>) at different stages of early porcine embryonic development (germinal vesicle, metaphase-2, 2-cell, 4-cell, early blastocyst, expanded blastocyst). Analysis of transcription profiling by geNorm software revealed that <it>GAPDH, PGK1, S18</it>, and <it>UBC </it>showed high stability in early porcine embryonic development, while transcription levels of <it>B2M, BACT</it>, and <it>H2A </it>were highly regulated.</p> <p>Conclusion</p> <p>Good reference genes that reflect total RNA content were identified in early embryonic development from oocyte to blastocyst. A selection of either <it>GAPDH </it>or <it>PGK1</it>, together with ribosomal protein <it>S18 </it>(<it>S18</it>), and <it>UBC </it>is proposed as reference genes, but the use of <it>B2M, BACT</it>, or <it>H2A </it>is discouraged.</p

A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis.

BACKGROUND: Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed. RESULTS: Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated. CONCLUSION: The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells

Usefulness of bovine and porcine IVM/IVF models for reproductive toxicology

Women presenting fertility problems are often helped by Assisted Reproductive Techniques (ART), such as in vitro fertilization (IVF) programs. However, in many cases the etiology of the in/subfertility remains unknown even after treatment. Although several aspects should be considered when assisting a woman with problems to conceive, a survey on the patients' exposure to contaminants would help to understand the cause of the fertility problem, as well as to follow the patient properly during IVF. Daily exposure to toxic compounds, mainly environmental and dietary ones, may result in reproductive impairment. For instance, because affects oocyte developmental competence. Many of these compounds, natural or synthetic, are endocrine disruptors or endocrine active substances that may impair reproduction. To understand the risks and the mechanism of action of such chemicals in human cells, the use of proper in vitro models is essential. The present review proposes the bovine and porcine models to evaluate toxic compounds on oocyte maturation, fertilization and embryo production in vitro. Moreover, we discuss here the species-specific differences when mice, bovine and porcine are used as models for human

Usefulness of bovine and porcine IVM/IVF models for reproductive toxicology

Women presenting fertility problems are often helped by Assisted Reproductive Techniques (ART), such as in vitro fertilization (IVF) programs. However, in many cases the etiology of the in/subfertility remains unknown even after treatment. Although several aspects should be considered when assisting a woman with problems to conceive, a survey on the patients' exposure to contaminants would help to understand the cause of the fertility problem, as well as to follow the patient properly during IVF. Daily exposure to toxic compounds, mainly environmental and dietary ones, may result in reproductive impairment. For instance, because affects oocyte developmental competence. Many of these compounds, natural or synthetic, are endocrine disruptors or endocrine active substances that may impair reproduction. To understand the risks and the mechanism of action of such chemicals in human cells, the use of proper in vitro models is essential. The present review proposes the bovine and porcine models to evaluate toxic compounds on oocyte maturation, fertilization and embryo production in vitro. Moreover, we discuss here the species-specific differences when mice, bovine and porcine are used as models for human

THE MYCOTOXIN BEAUVERICIN IMPAIRS EMBRYO DEVELOPMENT AND BLASTOCYST QUALITY IN THE JUVENILE SHEEP

Beauvericin (BEA) is a mycotoxin that disturbs nuclear and cytoplasmic in vitro maturation (IVM) of prepubertal sheep oocytes [1]. The aim of this study was to examine long-term carry-over effects of BEA exposure during IVM on embryo cleavage and blastocyst quality. Cumulus-oocyte complexes recovered from the ovaries of slaughtered prepubertal sheep (&lt;6 months) were cultured for IVM in presence of 0 (Control), 0.5, 1 and 3 M BEA [1,2]. After IVM, oocytes were subjected to in vitro fertilization and in vitro embryo culture [2]. Embryo developmental stage was assessed at day 8. Blastocysts were classified according to expansion and hatching status and their diameter was evaluated. After that, embryos were stained with Hoechst 33258 to evaluate number of nuclei and chromatin integrity, and with MitoTracker Orange CMTM Ros and 2′,7′-dichlorodihydrofluorescein diacetate to asses mitochondrial activity and ROS levels (expressed as the percentage of the signal of the control sample). Data were analyzed by the Chi-square test except for mitochondrial activity and ROS levels data compared by one-way ANOVA (significance at P&lt;0.05). Data are presented for 0.5, 1, 3 μM BEA vs Control, respectively. A total of 523 oocytes were used (n.5 replicates). The result of percentages of embryos at the 16-31 cell after oocyte exposure to BEA were 16/134, 12%, 6/134, 4.5%, 6/128, 4.7% vs 15/127, 12% (P&lt;0.05), and for morula- stage 4/134, 3%, 2/134, 1.5%, 2/128, 1.6% vs 9/127, 7% (P&lt;0.05). From these results it is possible to underline that the exposure to 1 and 3 μM BEA decreased these rates. After exposure to BEA, the 2–3 cell stages were 8/134, 6%, 15/134, 11.2%, 15/128, 12% vs 3/127, 2.4% (P&lt;0.01) but in this case 1 and 3 μM BEA increased the rate. No significant effects were found on blastocyst formation rates (3/134, 2%, 6/134, 4.5%, 4/128, 3% vs 4/127, 3%). Blastocysts derived from oocytes exposed to 1 and 3 μM BEA had not hatched after 8 days, in contrast, a few embryos obtained after exposure to 0 and 0.5 μM BEA did hatch (1/3, 0/6, 0/4, vs 1/4). Blastocyst diameter (131±34, 125±16, 125±13 vs 133±24 μm) and numbers of nuclei (79±15, 87±16, 87±9 vs 82±9) did not vary. Interestingly, increased percentages of embryos with more than 20% affected blastomeres with lobulated nuclei and/or micronuclei were observed after BEA exposure (1/3, 4/6, 3/4 vs 0/4). No effects on mitochondrial pattern and activity were detected (91±30, 103±36, 95±30 vs 100±21) whereas oocyte exposure to any BEA concentration reduced ROS generation ability (64±16, 87±23, 78±20 vs 100±23; P&lt;0.01), possibly indicating viability loss. In conclusion: exposure to BEA during maturation of sheep oocytes compromised embryo development and blastocyst quality, possibly reducing fertility. [1] Mastrorocco et al., Proc. LXXII SISVet Congress, Torino 20-22 Giugno 2018; p203; [2] Martino et al. Reprod Toxicol 2016;65:204-21

One-step automated bioprinting-based method for cumulus-oocyte complex microencapsulation for 3D in vitro maturation

Three-dimensional in vitro maturation (3D IVM) is a promising approach to improve IVM effi- ciency as it could prevent cumulus-oocyte complex (COC) flattening and preserve its struc- tural and functional integrity. Methods reported to date have low reproducibility and validation studies are limited. In this study, a bioprinting based production process for gener- ating microbeads containing a COC (COC-microbeads) was optimized and its validity tested in a large animal model (sheep). Alginate microbeads were produced and characterized for size, shape and stability under culture conditions. COC encapsulation had high efficiency and reproducibility and cumulus integrity was preserved. COC-microbeads underwent IVM, with COCs cultured in standard 2D IVM as controls. After IVM, oocytes were analyzed for nuclear chromatin configuration, bioenergetic/oxidative status and transcriptional activity of genes biomarker of mitochondrial activity (TFAM, ATP6, ATP8) and oocyte developmental competence (KHDC3, NLRP5, OOEP and TLE6). The 3D system supported oocyte nuclear maturation more efficiently than the 2D control (P&lt;0.05). Ooplasmic mitochondrial activity and reactive oxygen species (ROS) generation ability were increased (P&lt;0.05). Up-regula- tion of TFAM, ATP6 and ATP8 and down-regulation of KHDC3, NLRP5 expression were observed in 3D IVM. In conclusion, the new bioprinting method for producing COC-microbe- ads has high reproducibility and efficiency. Moreover, 3D IVM improves oocyte nuclear mat- uration and relevant parameters of oocyte cytoplasmic maturation and could be used for clinical and toxicological applications

Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos-0

<p><b>Copyright information:</b></p><p>Taken from "Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos"</p><p>http://www.biomedcentral.com/1471-213X/7/58</p><p>BMC Developmental Biology 2007;7():58-58.</p><p>Published online 31 May 2007</p><p>PMCID:PMC1896162.</p><p></p>DH 219 bp, H2A 83 bp, PGK1 126 bp, S18 149 bp, UBC 186 bp). 100 bp = 100 base pair ladder

Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos-3

<p><b>Copyright information:</b></p><p>Taken from "Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos"</p><p>http://www.biomedcentral.com/1471-213X/7/58</p><p>BMC Developmental Biology 2007;7():58-58.</p><p>Published online 31 May 2007</p><p>PMCID:PMC1896162.</p><p></p>DH 219 bp, H2A 83 bp, PGK1 126 bp, S18 149 bp, UBC 186 bp). 100 bp = 100 base pair ladder