Effects of melatonin during IVM in defined medium on oocyte meiosis, oxidative stress, and subsequent embryo development.

Melatonin may have beneficial effects when used in oocyte maturation and embryo development culture. The effect of melatonin during IVM on meiosis resumption and progression in bovine oocytes and on expression of antioxidant enzymes, nuclear fragmentation and free radicals, as well as on embryo development were assessed. Cumulus-oocyte complexes were matured in vitro with melatonin (10(-9) and 10(-6) M), FSH (positive control), or without hormones (negative control) in defined medium. Maturation rates were evaluated at 6, 12, 18, and 24 hours. Transcripts for antioxidant enzymes (CuZnSOD, MnSOD, and glutathione peroxidase 4 (GPX4)) in oocytes and cumulus cells, nuclear fragmentation in cumulus cells (TUNEL) and reactive oxygen species levels in oocytes (carboxy-H2 difluorofluorescein diacetate) were determined at 24 hours IVM. Effect of treatments on embryo development was determined after in vitro fertilization and culture. At 12 hours, meiosis resumption rates in FSH and melatonin-treated groups were similar (69.6%-81.8%, P > 0.05). At 24 hours, most oocytes were in metaphase II, with FSH showing highest rates (90.0%, P < 0.05) compared with the other groups (51.6%-69.1%, P > 0.05). In cumulus cells, MnSOD expression was higher in FSH group (P < 0.05) whereas Cu,ZnSOD transcripts were more abundant in melatonin group (10(-6)M; P < 0.05). Nuclear fragmentation in cumulus cells was highest in controls (37.4%/10,000 cells; P < 0.05) and lower in FSH and 10(-6)M melatonin (29.4% and 25.6%/10,000 cells, respectively). Reactive oxygen species levels were lower in oocytes matured with 10(-6)M melatonin than in control and FSH groups (P < 0.05). Embryo development from oocytes matured only with melatonin was similar to those matured in complete medium (P > 0.05). In conclusion, although melatonin during IVM in a defined medium does not stimulate nuclear maturation progression it does stimulate meiosis resumption and such treated oocytes support subsequent embryo development. Melatonin also shows cytoprotective effects on cumulus-oocyte complexes.

In vitro maturation alters gene expression in bovine oocytes.

Gene expression profiling of in vivo- and in vitro-matured bovine oocytes can identify transcripts related to the developmental potential of oocytes. Nonetheless, the effects of in vitro culturing oocytes are yet to be fully understood. We tested the effects of in vitro maturation on the transcript profile of oocytes collected from Bos taurus indicus cows. We quantified the expression of 1488 genes in in vivo- and in vitro-matured oocytes. Of these, 51 genes were up-regulated, whereas 56 were down-regulated (≥2-fold) in in vivo-matured oocytes in comparison with in vitro-matured oocytes. Quantitative real-time polymerase chain reaction (PCR) of nine genes confirmed the microarray results of differential expression between in vivo- and in vitro-matured oocytes (EZR, EPN1, PSEN2, FST, IGFBP3, RBBP4, STAT3, FDPS and IRS1). We interrogated the results for enrichment of Gene Ontology categories and overlap with protein-protein interactions. The results revealed that the genes altered by in vitro maturation are mostly related to the regulation of oocyte metabolism. Additionally, analysis of protein-protein interactions uncovered two regulatory networks affected by the in vitro culture system. We propose that the differentially expressed genes are candidates for biomarkers of oocyte competence. In vitro oocyte maturation can affect the abundance of specific transcripts and are likely to deplete the developmental competence.

The interval between the emergence of pharmacologically synchronized ovarian follicular waves and ovum pickup does not significantly affect in vitro embryo production in Bos indicus, Bos taurus, and Bubalus bubalis.

The aim of the present study was to determine the optimal phase of the follicular wave to perform ovum pickup (OPU) for in vitro embryo production (IVEP) in various genetic groups. For this purpose, 27 heifers-nine Bos taurus (Holstein), nine Bos indicus (Nelore), and nine Bubalus bubalis (Mediterranean)-were maintained under the same nutritional, management, and environmental conditions. Heifers within each genetic group were submitted to six consecutive OPU trials with 14-day intersession intervals, at three different phases of the pharmacologically synchronized follicular wave (Day 1, 3, or 5 after follicular wave emergence), in a 3 × 3 crossover design. When OPU was performed at different phases of the pharmacologically synchronized follicular wave (Day 1, 3, or 5), no differences were found in the percent of oocytes recovered (70.5 ± 3.1%, 75.0 ± 3.1%, 76.0 ± 3.2%, respectively; P = 0.41) or blastocyst production rates (19.4 ± 2.9%, 16.6 ± 2.9%, 15.9 ± 2.6%, respectively; P = 0.36). Comparing genetic groups, B indicus showed a higher blastocyst rate (28.3(a) ± 2.8%; P < 0.01) than B taurus and B bubalis (14.1(b) ± 2.9% and 10.2(b) ± 2.0%, respectively). However, only B indicus heifers showed a variation in the number of visualized follicles and the total and viable oocytes along consecutive OPU sessions. In conclusion, different phases of the pharmacologically synchronized ovarian follicular wave did not affect OPU-IVEP in B indicus, B taurus, and B bubalis heifers. Additionally, B indicus heifers showed greater OPU-IVEP efficiency than did the other genetic groups, under the same management conditions.

Constitutive androstane receptor ligands modulate the anti-tumor efficacy of paclitaxel in non-small cell lung cancer cells.

BACKGROUND: Lung tumors are the leading cause of cancer deaths worldwide and paclitaxel has proven to be useful for patients with lung cancer, however, acquired resistance is a major problem. To overcome this problem, one promising option is the use of Constitutive Androstane Receptor (CAR) ligands in combination with chemotherapeutics against cancer cells. Therefore, we wish to elucidate the effects of CAR ligands on the antineoplastic efficacy of paclitaxel in lung cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: Our results from cell viability assays exposing CAR agonist or inverse-agonist to mouse and human lung cancer cells modulated the antineoplastic effect of paclitaxel. The CAR agonists increased the effect of Paclitaxel in 6 of 7 lung cancer cell lines, whereas the inverse-agonist had no effect on paclitaxel cytotoxicity. Interestingly, the mCAR agonist TCPOBOP enhanced the expression of two tumor suppressor genes, namely WT1 and MGMT, which were additively enhanced in cells treated with CAR agonist in combination with paclitaxel. Also, in silico analysis showed that both paclitaxel and CAR agonist TCPOBOP docked into the mCAR structure but not the inverse agonist androstenol. Paclitaxel per se increases the expression of CAR in cancer cells. At last, we analyzed the expression of CAR in two public independent studies from The Cancer Genome Atlas (TCGA) of Non Small Cell Lung Cancer (NSCLC). CAR is expressed in variable levels in NSCLC samples and no association with overall survival was noted. CONCLUSIONS/SIGNIFICANCE: Taken together, our results demonstrated that CAR agonists modulate the antineoplastic efficacy of paclitaxel in mouse and human cancer cell lines. This effect was probably related by the enhanced expression of two tumor suppressor genes, viz. WT1 and MGMT. Most of NSCLC cases present CAR gene expression turning it possible to speculate the use of CAR modulation by ligands along with Paclitaxel in NSCLC therapy.

Infectious bronchitis virus replication in the chicken embryo related cell line.

The susceptibility of the chicken embryo related (CER) cell line to infectious bronchitis virus (IBV M41) was characterized after five consecutive passages in CER cells. Virus replication was monitored by cytopathic effect observation, electron microscopy, indirect immunofluorescence, and reverse transcription polymerase chain reaction (RT-PCR). At 96 h post-infection (p.i.), the cytopathic effect was graded 75% by cell fusion, rounding up of cells and monolayer detachment, and the electron microscopy image characterized by coronavirus morphology. Cytoplasmic fluorescence was readily observed by from 24 h p.i. onwards, and at all times the respective viral RNA from IBV-infected monolayers was demonstrated by RT-PCR. Extra-cellular virus was measured by virus titration performed on chicken kidney cells and embryonated chicken eggs, and respective titres ranged from 4.0 to 6.0 log10 EID50/ml on embryonated chicken eggs, and from 2.0 to 6.0 log10 TCID50/ml on both CER cells and chicken kidney cells studied from 24 to 120 h p.i. These results confirmed that the M41 strain replicated well in the CER cell line.