MicroRNA Signaling in Embryo Development

Expression of microRNAs (miRNAs) is essential for embryonic development and serves important roles in gametogenesis. miRNAs are secreted into the extracellular environment by the embryo during the preimplantation stage of development. Several cell types secrete miRNAs into biological fluids in the extracellular environment. These fluid-derived miRNAs have been shown to circulate the body. Stable transport is dependent on proper packaging of the miRNAs into extracellular vesicles (EVs), including exosomes. These vesicles, which also contain RNA, DNA and proteins, are on the forefront of research on cell-to-cell communication. Interestingly, EVs have been identified in many reproductive fluids, such as uterine fluid, where their miRNA content is proposed to serve as a mechanism of crosstalk between the mother and conceptus. Here, we review the role of miRNAs in molecular signaling and discuss their transport during early embryo development and implantation

Knockdown of <i>CDKN1C (p57^kip2)</i> and <i>PHLDA2</i> Results in Developmental Changes in Bovine Pre-implantation Embryos

<div><p>Imprinted genes have been implicated in early embryonic, placental, and neonatal development and alterations in expression levels of these genes can lead to growth abnormalities and embryonic lethality. However, little is known about the functions of bovine imprinted genes during the pre-implantation period. Therefore, the objective of this study was to assess the influence of altered expression of imprinted genes on developmental progress of embryos using small interfering RNA (siRNA). Expression levels of 18 imprinted genes (<i>MAGEL2</i>, <i>UBE3A</i>, <i>IGF2R, NAP1L5, TSSC4, PEG3, NDN, CDKN1C, PHLDA2, MKRN3, USP29, NNAT, PEG10</i>, <i>RTL1, IGF2, H19</i>, <i>MIM1,</i> and <i>XIST</i>) were compared between embryos reaching the blastocyst stage and growth-arrested embryos (degenerates) using quantitative real-time PCR (qRT-PCR). Ten genes were found to be differentially expressed between blastocysts and degenerates. The <i>CDKN1C</i> gene showed the highest upregulation in blastocysts whereas <i>PHLDA2</i> was highly expressed in degenerates. To assess whether the observed differential gene expression was causative or resultant of embryo degeneration, these genes were selected for functional analysis using siRNA. Injection of siRNA specific to <i>PHLDA2</i> into one-cell zygotes resulted in a substantial increase in blastocyst development, whereas injection of <i>CDKN1C</i>-specific siRNA resulted in a 45% reduction (P = 0.0006) in blastocyst development. RNA-Seq analysis of <i>CDKN1C</i>-siRNA-injected vs. non-injected embryos revealed 51 differentially expressed genes with functions in apoptosis, lipid metabolism, differentiation, and cell cycle regulation. Gene ontology analysis revealed nine pathways related to cell signaling, metabolism, and nucleic acid processing. Overall, our results show that proper expression levels of the imprinted genes <i>CDKN1C</i> and <i>PHLDA2</i> are critical for embryo development, which suggests that these genes can be used as markers for normal blastocyst formation.</p></div

Mechanisms of decompensation and organ failure in cirrhosis: From peripheral arterial vasodilation to systemic inflammation hypothesis

The peripheral arterial vasodilation hypothesis has been most influential in the field of cirrhosis and its complications. It has given rise to hundreds of pathophysiological studies in experimental and human cirrhosis and is the theoretical basis of life-saving treatments. It is undisputed that splanchnic arterial vasodilation contributes to portal hypertension and is the basis for manifestations such as ascites and hepatorenal syndrome, but the body of research generated by the hypothesis has revealed gaps in the original pathophysiological interpretation of these complications. The expansion of our knowledge on the mechanisms regulating vascular tone, inflammation and the host-microbiota interaction require a broader approach to advanced cirrhosis encompassing the whole spectrum of its manifestations. Indeed, multiorgan dysfunction and failure likely result from a complex interplay where the systemic spread of bacterial products represents the primary event. The consequent activation of the host innate immune response triggers endothelial molecular mechanisms responsible for arterial vasodilation, and also jeopardizes organ integrity with a storm of pro-inflammatory cytokines and reactive oxygen and nitrogen species. Thus, the picture of advanced cirrhosis could be seen as the result of an inflammatory syndrome in contradiction with a simple hemodynamic disturbance

Development rates for <i>CDKN1C</i> siRNA-injected embryos and control embryo groups.

1<p>Numbers represent average of six biological replicates.</p><p>Differing superscripts within a column denotes statistically significant differences (P<0.05).</p

DNA methylation of <i>PHLDA2</i> in IVF embryos.

<p>Exons of <i>PHLDA2</i> are indicated by grey boxes and the direction of transcription is indicated by arrowheads. Vertical bars below the gene model represent CpG sites near and within <i>PHLDA2</i>. The CpG island overlapping with <i>PHLDA2</i> is shown as an open rectangular box. For methylation analysis, each line connecting circles represents a single clone. Filled circles indicate methylated CpGs while open circles represent unmethylated CpGs. Clones derived from blastocysts and degenerate embryos are shown in red and black respectively. CpG sites tested for differential methylation are marked by arrows. Significant differential methylation of CpG sites are indicated by p values.</p

qRT-PCR results for control and <i>CDKN1C</i> siRNA injections.

<p>All samples were normalized to <i>GAPDH</i> using the ΔΔCt method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069490#pone.0069490-Vandesompele1" target="_blank">[60]</a>.</p

Mean+S.E.M. for fold difference of degenerative relative to blastocyst embryo pools.

<p>Each bar represents the values across four sets of embryo pools (n = 20 embryos per pool, two sires used). All samples were done in quadruplicates and normalized to <i>GAPDH</i> tested in the same cDNA samples. Expression calculations were done using the 2^−ΔΔCt method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069490#pone.0069490-Vandesompele1" target="_blank">[60]</a>. Bars above the ”0″ represent genes that were up-regulated in degenerative embryos while bars below the ”0″ indicate down-regulated genes. * P<0.05.</p

Bortezomib Prevents Acute Doxorubicin Ovarian Insult and Follicle Demise, Improving the Fertility Window and Pup Birth Weight in Mice

<div><p>Increasing numbers of female patients survive cancer, but succumb to primary ovarian insufficiency after chemotherapy. We tested the hypothesis that Bortezomib (Bort) protects ovaries from doxorubicin (DXR) chemotherapy by treating female mice with Bort 1 hour prior to DXR. By preventing DXR accumulation in the ovary, Bort attenuated DXR-induced DNA damage in all ovarian cell types, subsequent γH2AFX phosphorylation, and resulting apoptosis in preantral follicles. Bort pretreatment extended the number of litters per mouse, improved litter size and increased pup weight following DXR treatment, thus increasing the duration of post-chemotherapy fertility and improving pup health. As a promising prophylactic ovoprotective agent, Bort does not interfere with cancer treatment, and is currently used as a chemotherapy adjuvant. Bort-based chemoprotection may preserve ovarian function in a non-invasive manner that avoids surgical ovarian preservation, thus diminishing the health complications of premature menopause following cancer treatment.</p></div