Expression profile of developmental competence gene markers in comparison with prostaglandin F2α synthesis and action in the early- and late-cleaved pre-implantation bovine embryos.

The kinetics of early cleavage stages can affect embryo quality. The bovine model of early- and late-cleaved embryos has been described in the literature and is deemed a useful tool in the field of oocyte developmental competence studies. The expression of genes demonstrating developmental potential differs between early- and late-cleaved embryos. Previously, we demonstrated that prostaglandin F2α synthase (PGFS) and prostaglandin F2α receptor (PTGFR) expression depend on the developmental stage and embryo quality. In the present study, we used the same model to determine the mRNA expression profile of developmentally important genes (IGF1R, IGF2R, PLAC8, OCT4, SOX2) in early, expanded and hatched blastocysts obtained from the early- and late-cleaved group of embryos, as well as to correlate the transcription levels of these embryonic gene markers with the transcription levels of PGFS and PTGFR. The mRNA expression of PGFS, PTGFR and factors described as gene markers of embryonic implantation ability and developmental competence genes was determined by real-time PCR. The obtained results were analysed using statistical software GraphPad prism 6.05. During the course of our analyses, we observed that the transcript abundance of most analysed genes tends to be higher in the late-rather than in the early cleaved group of embryos, as well as in B and/or C grade embryos rather than in A grade embryos. On the other hand, for the early cleaved group of blastocysts with cavity, we detected higher PLAC8 mRNA expression for grade A embryos compared with grade C embryos. It suggests that the mRNA expression level of genes depends on the quality of embryos but differs according to various factors including the method of production or culture method. Moreover, numerous correlations between analysed gene markers and PGF2α synthase and PGF2α receptor suggest that PGF2α plays a role in the crucial steps of bovine embryo development.

Role of IL-36 Cytokines in the Regulation of Angiogenesis Potential of Trophoblast Cells.

IL-36 cytokines (the agonists IL-36α, IL-36ß, IL-36γ, and the antagonist IL-36Ra) are expressed in the mouse uterus and associated with maternal immune response during pregnancy. Here, we characterize the expression of IL-36 members in human primary trophoblast cells (PTC) and trophoblastic cell lines (HTR-8/SVneo and JEG-3) and upon treatment with bacterial and viral components. Effects of recombinant IL-36 on the migration capacity of trophoblastic cells, their ability to interact with endothelial cells and the induction of angiogenic factors and miRNAs (angiomiRNAs) were examined. Constitutive protein expression of IL-36 (α, ß, and γ) and their receptor (IL-36R) was found in all cell types. In PTC, transcripts for all IL-36 subtypes were found, whereas in trophoblastic cell lines only for IL36G and IL36RN. A synthetic analog of double-stranded RNA (poly I:C) and lipopolysaccharide (LPS) induced the expression of IL-36 members in a cell-specific and time-dependent manner. In HTR-8/SVneo cells, IL-36 cytokines increased cell migration and their capacity to interact with endothelial cells. VEGFA and PGF mRNA and protein, as well as the angiomiRNAs miR-146a-3p and miR-141-5p were upregulated as IL-36 response in PTC and HTR-8/SVneo cells. In conclusion, IL-36 cytokines are modulated by microbial components and regulate trophoblast migration and interaction with endothelial cells. Therefore, a fundamental role of these cytokines in the placentation process and in response to infections may be expected.

Waterborne fluoxetine disrupts the reproductive axis in sexually mature male goldfish, Carassius auratus.

Fluoxetine (FLX) is a pharmaceutical acting as a selective serotonin reuptake inhibitor and is used to treat depression in humans. Fluoxetine and the major active metabolite norfluoxetine (NFLX) are released to aquatic systems via sewage-treatment effluents. They have been found to bioconcentrate in wild fish, raising concerns over potential endocrine disrupting effects. The objective of this study was to determine effects of waterborne FLX, including environmental concentrations, on the reproductive axis in sexually mature male goldfish. We initially cloned the goldfish serotonin transporter to investigate tissue and temporal expression of the serotonin transporter, the FLX target, in order to determine target tissues and sensitive exposure windows. Sexually mature male goldfish, which showed the highest levels of serotonin transporter expression in the neuroendocrine brain, were exposed to FLX at 0.54µg/L and 54µg/L in a 14-d exposure before receiving vehicle or sex pheromone stimulus consisting of either 4.3nM 17,20ß-dihydroxy-4-pregnene-3-one (17,20P) or 3nM prostaglandin F2(α) (PGF2(α)). Reproductive endpoints assessed included gonadosomatic index, milt volume, and blood levels of the sex steroids testosterone and estradiol. Neuroendocrine function was investigated by measuring blood levels of luteinizing hormone, growth hormone, pituitary gene expression of luteinizing hormone, growth hormone and follicle-stimulating hormone and neuroendocrine brain expression of isotocin and vasotocin. To investigate changes at the gonadal level of the reproductive axis, testicular gene expression of the gonadotropin receptors, both the luteinizing hormone receptor and the follicle-stimulating hormone receptor, were measured as well as expression of the growth hormone receptor. To investigate potential impacts on spermatogenesis, testicular gene expression of the spermatogenesis marker vasa was measured and histological samples of testis were analyzed qualitatively. Estrogen indices were measured by expression and activity analysis of gonadal aromatase, as well as liver expression analysis of the estrogenic marker, esr1. After 14d, basal milt volume significantly decreased at 54µg/L FLX while pheromone-stimulated milt volume decreased at 0.54µg/L and 54µg/L FLX. Fluoxetine (54µg/L) inhibited both basal and pheromone-stimulated testosterone levels. Significant concentration-dependent reductions in follicle-stimulating hormone and isotocin expression were observed with FLX in the 17,20P- and PGF2(α)-stimulated groups, respectively. Estradiol levels and expression of esr1 concentration-dependently increased with FLX. This study demonstrates that FLX disrupts reproductive physiology of male fish at environmentally relevant concentrations, and potential mechanisms are discussed.

Mapping of the genes encoding mouse prostaglandin D, E, and F and prostacyclin receptors.

Prostaglandins and prostacyclin are metabolites of arachidonic acid and exert a variety of actions to maintain local homeostasis in the body. Their actions are mediated by cell surface receptors specific to the respective ligands. Using a panel of interspecific back-cross mice, we have mapped the prostaglandin D receptor gene (Ptgdr), prostaglandin E receptor subtype EP(1) gene (Ptgerepl), prostaglandin F receptor gene (Ptgfr), and prostacyclin receptor gene (Ptgir). Ptgdr mapped to proximal Chr 14, Ptgfr mapped to distal Chr 3, Ptgerepl mapped to middle Chr 8, and Ptgir mapped to proximal Chr 7.