In vitro grown sheep preantral follicles yield oocytes with normal nuclear-epigenetic maturation

Background

Assisted reproductive technologies allow to utilize a limited number of fully grown oocytes despite the presence in the ovary of a large pool of meiotically incompetent gametes potentially able to produce live births. In vitro folliculogenesis could be useful to recruit these oocytes by promoting their growth and differentiation.

Methodology/Principal Findings

In vitro folliculogenesis was performed starting from sheep preantral (PA) follicles to evaluate oocyte nuclear/epigenetic maturation. Chromatin configuration, quantification of global DNA methylation, and epigenetic remodelling enzymes were evaluated with immunocytochemistry, telomere elongation was assessed with the Q-FISH technique, while the DNA methylation status at the DMRs of maternally IGF2R and BEGAIN, and paternally H19 methylated imprinted genes was determined by bisulfite sequencing and COBRA. Specifically, 70% of PA underwent early antrum (EA) differentiation and supported in culture oocyte global DNA methylation, telomere elongation, TERT and Dnmt3a redistribution thus mimicking the physiological events that involve the oocyte during the transition from secondary to tertiary follicle. Dnmt1 anticipated cytoplasmic translocation in in vitro grown oocytes did not impair global and single gene DNA methylation. Indeed, the in vitro grown oocytes acquired a methylation profile of IGF2R and BEGAIN compatible with the follicle/oocyte stage reached, and maintained an unmethylated status of H19. In addition, the percentage of oocytes displaying a condensed chromatin configuration resulted lower in in vitro grown oocytes, however, their ability to undergo meiosis and early embryo development after IVF and parthenogenetic activation was similar to that recorded in EA follicle in vivo grown oocytes.

Conclusions/Significance

In conclusion, the in vitro folliculogenesis was able to support the intracellular/nuclear mechanisms leading the oocytes to acquire a meiotic and developmental competence. Thus, the in vitro culture may increase the availability of fertilizable oocytes in sheep, and become an in vitro translational model to investigate the mechanisms governing nuclear/epigenetic oocyte maturation.     

Enhanced anandamide degradation is associated with neuronal apoptosis induced by the HIV-1 coat glycoprotein gp120 in the rat neocortex

Human immunodeficiency virus type-1 coat glycoprotein gp120 causes delayed apoptosis in rat brain neocortex. Here, we investigated the possible role of the endocannabinoid system in this process. It is shown that gp120 causes a time-dependent increase in the activity and immunoreactivity of the anandamide (AEA)-hydrolyzing enzyme fatty acid amide hydrolase (FAAH), paralleled by increased activity of the AEA membrane transporter and decreased endogenous levels of AEA. The AEA-synthesizing phospholipase D and the AEA-binding receptors were not affected by gp120. None of the changes induced by gp120 in the cortex were induced by bovine serum albumin, nor were they observed in the hippocampus of the same animals. Also, the activity of 5-lipoxygenase, which generates AEA derivatives able to inhibit FAAH, decreased down to approximately 25% of the control activity upon gp120 treatment, due to reduced protein level ( approximately 45%). In addition, the FAAH inhibitor methyl-arachidonoyl fluorophosphonate significantly reduced gp120-induced apoptosis in rat brain neocortex, whereas selective blockers of AEA membrane transporter or of AEA-binding receptors were ineffective. Taken together, these results suggest that gp120, by activating FAAH, decreases endogenous levels of AEA, and the latter effect seems instrumental in the execution of delayed neuronal apoptosis in the brain neocortex of rats.     

Investigation of Adhesion and Mechanical Properties of Human Glioma Cells by Single Cell Force Spectroscopy and Atomic Force Microscopy

Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma -HG- and Gasc for low-grade glioma -LG-) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics.     

Female sex pheromone of Sesamia cretica: chemical and behavioural evidence for a three-component blend

The female‐produced sex pheromone of the Durra stem borer, Sesamia cretica (Lederer) (Lepidoptera: Noctuidae), had been previously characterized as a 75:25 blend of (Z)‐9‐tetradecenol (Z9‐14:OH) and (Z)‐9‐tetradecenyl acetate (Z9‐14:OAc) based on field trapping experiments. The low attraction of this blend in the field led us to further investigate the sex pheromone of this pest. Coupled gas chromatography with electroantennographic detection (GC‐EAD) analysis of female pheromone gland extracts consistently revealed three EAD‐active compounds. According to their GC retention times, mass spectra, and comparative EAG analyses with authentic standards, two of these compounds were found to be the previously reported components Z9‐14:OH and Z9‐14:OAc, whereas a third compound was identified as (Z)‐11‐hexadecenol (Z11‐16:OH). In wind tunnel experiments, the highest male responses were elicited by ratios of Z9‐14:OH, Z9‐14:OAc, and Z11‐16:OH, ranging from 90:1:9 to 90:5:5. In field tests, the 90:1:9 ratio of the blend loaded onto rubber septum dispensers was significantly more effective than single‐component, two‐component, and any other ratio of the three‐component blend. The greater effectiveness of this blend resulted in a more accurate detection of S. cretica flight activity compared with the previously reported two‐component blend.     

Effect of Antiprogesterone RU486 on VEGF Expression and Blood Vessel Remodeling on Ovarian Follicles before Ovulation

Background

The success of ovarian follicle growth and ovulation is strictly related to the development of an adequate blood vessel network required to sustain the proliferative and endocrine functions of the follicular cells. Even if the Vascular Endothelial Growth Factor (VEGF) drives angiogenesis before ovulation, the local role exerted by Progesterone (P₄) remains to be clarified, in particular when its concentration rapidly increases before ovulation.

Aim

This in vivo study was designed to clarify the effect promoted by a P₄ receptor antagonist, RU486, on VEGF expression and follicular angiogenesis before ovulation, in particular, during the transition from pre to periovulatory follicles induced by human Chorionic Gonadotropins (hCG) administration.

Material and Methods

Preovulatory follicle growth and ovulation were pharmacologically induced in prepubertal gilts by combining equine Chorionic Gonadotropins (eCG) and hCG used in the presence or absence of RU486. The effects on VEGF expression were analyzed using biochemical and immunohistochemical studies, either on granulosa or on theca layers of follicles isolated few hours before ovulation. This angiogenic factor was also correlated to follicular morphology and to blood vessels architecture.

Results and Conclusions

VEGF production, blood vessel network and follicle remodeling were impaired by RU486 treatment, even if the cause-effect correlation remains to be clarified. The P₄ antagonist strongly down-regulated theca VEGF expression, thus, preventing most of the angiogenic follicle response induced by hCG. RU486-treated follicles displayed a reduced vascular area, a lower rate of endothelial cell proliferation and a reduced recruitment of perivascular mural cells. These data provide important insights on the biological role of RU486 and, indirectly, on steroid hormones during periovulatory follicular phase. In addition, an in vivo model is proposed to evaluate how periovulatory follicular angiogenesis may affect the functionality of the corpus luteum (CL) and the success of pregnancy.     

Probiotics ameliorate recurrent Th1-mediated murine colitis by inducing IL-10 and IL-10-dependent TGF-beta-bearing regulatory cells

Recent studies of murine models of mucosal inflammation suggest that, whereas some kinds of bacterial microflora are inducers of disease, others, known as probiotics, prevent disease. In the present study, we analyzed the regulatory cytokine and cell response to probiotic (VSL#3) administration in the context of the Th1 T cell colitis induced by trinitrobenzene sulfonic acid treatment of SJL/J mice. Daily administration of probiotics for 3 wk to mice during a remission period between a first and second course of colitis induced by trinitrobenzene sulfonic acid, resulted in a milder form of recurrent colitis than observed in mice administered PBS during this same period. This protective effect was attributable to effects on the lamina propria mononuclear cell (LPMC) population, because it could be transferred by LPMC from probiotic-treated mice to naive mice. Probiotic administration was associated with an early increase in the production of IL-10 and an increased number of regulatory CD4+ T cells bearing surface TGF-beta in the form of latency-associated protein (LAP) (LAP+ T cells). The latter were dependent on the IL-10 production because administration of anti-IL-10R mAb blocked their appearance. Finally, the LAP+ T cells were essential to the protective effect of probiotics because administration of anti-IL-10R or anti-TGF-beta at the initiation of recurrent colitis induction or depletion of LAP+ T cells from LPMC abolished the latter's capacity to transfer protection to naive recipients. These studies show that probiotic (VSL#3) administration during a remission period ameliorates the severity of recurrent colitis by inducing an immunoregulatory response involving TGF-beta-bearing regulatory cells.     

Cholesterol-dependent modulation of the toxicity of HIV-1 coat protein gp120 in human neuroblastoma cells

The human immunodeficiency virus type 1 (HIV-1) coat glycoprotein gp120 binds to its (co)receptors and orchestrates cell entry by the direct fusion of viral and target cell membranes. Here, we modulated membrane fluidity of human neuroblastoma CHP100 cells by modulating their cholesterol content, and investigated the ability of gp120 to induce cell death in comparison with the untreated cells. We show that in normal CHP100 cells gp120 induces necrosis by: (i) increased cyclooxygenase and 5-lipoxygenase activity, and metabolites generated thereof (prostaglandin E2 and leukotriene B4, respectively); (ii) increased membrane lipoperoxidation; and (iii) increased mitochondrial uncoupling. These events were triggered by a rapid increase in intracellular calcium, and in cholesterol-depleted cells engaged CXCR4 chemokine receptors. The intracellular calcium chelator EGTA-AM protected CHP100 cells almost completely against the toxic effects of gp120. However, gp120-induced necrosis and related biochemical changes were negligible in cholesterol-enriched, and significantly enhanced in cholesterol-depleted, CHP100 cells exposed to the viral glycoprotein under the same experimental conditions. Taken together, these results suggest that membrane fluidity may control the neurotoxic effects of HIV-1 glycoprotein gp120.