L. G. Mesquita

Ooplast-mediated developmental rescue of bovine oocytes exposed to ethidium bromide

Ooplasm transfer has been used successfully to treat infertility in women with ooplasmic insufficiency and has culminated in the birth of healthy babies. To investigate whether mitochondrial dysfunction is a factor in ooplasmic insufficiency, bovine oocytes were exposed to ethidium bromide, an inhibitor of mitochondrial DNA replication and transcription, during in-vitro maturation (IVM). Exposure of immature oocytes to ethidium bromide for 24h during IVM hampered meiotic resumption and the migration of cortical granules. However, a briefer treatment with ethidium bromide during the last 4h of IVM led to partial arrest of preimplantation development without affecting oocyte maturation. Ooplasm transfer was then performed to rescue the oocytes with impaired development. In spite of this developmental hindrance, transfer of normal ooplasm into ethidium bromide-treated oocytes resulted in a complete rescue of embryonic development and the birth of heteroplasmic calves. Although this study unable to determine whether developmental rescue occurred exclusively through introduction of unaffected mitochondria into ethidium bromide-damaged oocytes, e.g. ethidium bromide may also affect other ooplasm components, these results clearly demonstrate that ooplasm transfer can completely rescue developmentally compromised oocytes, supporting the potential use of ooplasm transfer in therapeutic applications.

Serum-Starved Apoptotic Fibroblasts Reduce Blastocyst Production but Enable Development to Term after SCNT in Cattle

Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.

Effect of nitric oxide on the cyclic guanosine monophosphate (cGMP) pathway during meiosis resumption in bovine oocytes.

Nitric oxide (NO) is a chemical messenger involved in the control of oocyte maturation. It stimulates guanylate cyclase to produce cyclic guanosine monophosphate (cGMP), which in turn activates cGMP-dependent protein kinase (PKG) and some phosphodiesterases that may interfere with cAMP levels, a nucleotide also involved in meiosis resumption. The aim of this study was to determine the role played by NO on the cGMP/cAMP pathway during meiosis resumption in bovine oocytes. The effects of increasing NO generated by S-nitroso-N-acetylpenicillamine (SNAP; 10(-7)-10(-3) mol/L) and of other drugs that may affect the NO/cGMP pathway (proptoporfirin IX and 8-Br-cGMP) on meiosis resumption were investigated in bovine cumulus-oocyte complexes (COCs) matured for 9 hours in a semidefined medium (TCM199 + 3 mg/mL BSA). The COCs matured with 10(-7) mol/L SNAP associated or not with 100 μmol/L oxadiazole-one quinoxaline, a guanylate cyclase inhibitor, also had their cGMP and cAMP levels measured during the first hours of maturation (1, 3, and 6 hours). Quantitative polymerase chain reaction was performed by real-time polymerase chain reaction to determine the effects of NO on expression of genes encoding for enzymes of the NO/guanylate cyclase/cGMP and cAMP pathways during the first 9 hours of oocyte maturation. Increasing NO levels using 10(-7) mol/L SNAP resulted in lower rate of germinal vesicle breakdown (36% germinal vesicle breakdown; P < 0.05) at 9 hours IVM, whereas control group and the treatments with 10(-9) and 10(-8) mol/L SNAP showed about 70% germinal vesicle breakdown (P > 0.05). A temporary increase in cGMP levels was also observed with the same treatment (4.51 pmol/COC) at 1 hour IVM, which was superior to the control group (2.97 pmol/COC; P < 0.05) and was reversed by inhibiting guanylate cyclase activity with 100 μmol/L oxadiazole-one quinoxaline. Neither cAMP levels nor gene expression were affected by NO. These results suggest that NO acts via guanylate cyclase/cGMP and that even a temporary increase in cGMP levels leads to a delay in meiosis resumption, even when cAMP levels have declined. Nitric oxide does not act on oocyte maturation by affecting cAMP levels or the expression of genes related to the NO/guanylate cyclase/cGMP and cAMP pathways. Also, to our knowledge this is the first report to detect PKG1, PKG2, phosphodiesterase-5A, ADCY3, ADCY6, and ADCY9 transcripts in bovine oocytes.

Fetal-Maternal Interactions in the Synepitheliochorial Placenta Using the eGFP Cloned Cattle Model

Background To investigate mechanisms of fetal-maternal cell interactions in the bovine placenta, we developed a model of transgenic enhanced Green Fluorescent Protein (t-eGFP) expressing bovine embryos produced by nuclear transfer (NT) to assess the distribution of fetal-derived products in the bovine placenta. In addition, we searched for male specific DNA in the blood of females carrying in vitro produced male embryos. Our hypothesis is that the bovine placenta is more permeable to fetal-derived products than described elsewhere. Methodology/Principal Findings Samples of placentomes, chorion, endometrium, maternal peripheral blood leukocytes and blood plasma were collected during early gestation and processed for nested-PCR for eGFP and testis-specific Y-encoded protein (TSPY), western blotting and immunohistochemistry for eGFP detection, as well as transmission electron microscopy to verify the level of interaction between maternal and fetal cells. TSPY and eGFP DNA were present in the blood of cows carrying male pregnancies at day 60 of pregnancy. Protein and mRNA of eGFP were observed in the trophoblast and uterine tissues. In the placentomes, the protein expression was weak in the syncytial regions, but intense in neighboring cells on both sides of the fetal-maternal interface. Ultrastructurally, our samples from t-eGFP expressing NT pregnancies showed to be normal, such as the presence of interdigitating structures between fetal and maternal cells. In addition, channels-like structures were present in the trophoblast cells. Conclusions/Significance Data suggested that there is a delivery of fetal contents to the maternal system on both systemic and local levels that involved nuclear acids and proteins. It not clear the mechanisms involved in the transfer of fetal-derived molecules to the maternal system. This delivery may occur through nonclassical protein secretion; throughout transtrophoblastic-like channels and/or by apoptotic processes previously described. In conclusion, the bovine synepitheliochorial placenta displays an intimate fetal-maternal interaction, similar to other placental types for instance human and mouse.

Differential gene expression and developmental competence in in vitro produced bovine embryos

The embryonic developmental block occurs at the 8-cell stage in cattle and is characterized by a lengthening of the cell cycle and an increased number of embryos that stop development. The maternal-embryonic transition arises at the same stage resulting in the transcription of many genes. Gene expression studies during this stage may contribute to the understanding of the physiological mechanisms involved in the maternal-embryonic transition. Herein we identified genes differentially expressed between embryos with high or low developmental competence to reach the blastocyst stage using differential display PCR. Embryos were analysed according to developmental kinetics: fast cleavage embryos showing 8 cells at 48 h post insemination (hpi) with high potential of development (F8), and embryos with slow cleavage presenting 4 cells at 48 hpi (S4) and 8 cells at 90 hpi (S8), both with reduced rates of development to blastocyst. The fluorescence DDPCR method was applied and allowed the recovery of 176 differentially expressed bands with similar proportion between high and low development potential groups (52% to F8 and 48% in S4 and S8 groups). A total of 27 isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the identification of 27 gene transcripts. PI3KCA and ITM2B were chosen for relative quantification of mRNA using real-time PCR and showed a kinetic and a time-related pattern of expression respectively. The observed results suggest the existence of two different embryonic genome activation mechanisms: fast-developing embryos activate genes related to embryonic development, and slow-developing embryos activate genes related to cellular survival and/or death.

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.

Effect of fiber digestibility and conservation method on feed intake and the ruminal ecosystem of growing steers

Fiber digestibility is an important factor regulating DMI in ruminants. Additionally, the ensiling process can also affect digestibility and chemical composition of the forage. The objective of this study was to investigate effects of sugarcane NDF digestibility (NDFD) and conservation method on intake, rumen kinetics, and the ruminal ecosystem of steers. Eight ruminally cannulated Nellore steers (275±22 kg BW) were used in a replicated 4×4 Latin square design with a 2×2 factorial arrangement of treatments. Two sugarcane genotypes divergent for stalk NDFD were used: IAC86-2480 with high NDFD and SP91-1049 with low NDFD. Experimental diets were formulated with 40% sugarcane, either freshly cut or as silage, and 60% concentrate on a DM basis. Each experimental period lasted for 14 d, with the last 4 d used for determination of intake, ruminal evacuation, and ruminal fluid collection. The effect of fiber digestibility on DM and NDF intake was dependent on the forage conservation method (P=0.01). High NDFD increased (P<0.01) DMI only when sugarcane was offered as silage, having no effect (P=0.41) on DMI when offered as freshly cut. Conservation method had no effect on total ruminal mass, with only a tendency (P<0.10) for greater NDF and indigestible NDF ruminal mass in steers fed the low-NDFD genotype. The NDF turnover and passage rates were greater (P<0.05) for the genotype with high NDFD but only when offered as silage. Liquid turnover rate in the rumen was greater (P=0.02) for diets containing silage, with no effect of genotype (P=0.87). There was no effect of NDFD genotype on ruminal pH (P=0.77); however, diets containing sugarcane as silage increased (P<0.01) ruminal pH. Total concentration of short chain fatty acids (P=0.05) and proportions of propionate (P=0.01) were greater for diets containing fresh sugarcane. Diets with fresh sugarcane increased the ruminal population of Streptococcus bovis (P<0.01) and Ruminococcus albus (P=0.03). The relative population of R. albus was also greater (P=0.04) for diets containing the sugarcane genotype with high NDFD. Feeding diets containing the sugarcane genotype with high NDFD increased Fibrobacter succinogenes population but only when sugarcane was fed as freshly cut (P=0.02). Using sugarcane genotypes with high NDFD can increase intake and benefit fiber-degrading bacteria in the rumen.

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.

Zilpaterol hydrochloride improves beef yield, changes palatability traits, and increases calpain-calpastatin gene expression in Nellore heifers

This research aimed to evaluate the effects of the beta-agonist zilpaterol hydrochloride (ZH) on carcass traits, subprimal yield, meat quality, palatability traits, and gene expression in Nellore heifers. Zilpaterol increased Longissimus lumborum area and did not change back fat thickness, meat color, and cooking loss. Heifers fed ZH had greater hindquarter weight and carcass percentage. Muscles from hindquarter were heavier for animals fed ZH. Forequarter (% of carcass) decreased and brisket did not change with ZH supplementation. There were no differences between treatments for steak aroma, beef flavor, and off-flavor. However, tenderness and juiciness were reduced by ZH, depending on postmortem aging. Zilpaterol increased Calpain-1, Calpain-2, and calpastatin mRNA expression, with no effect of day of slaughter or ZH×Day interaction. In conclusion, ZH supplementation improved hypertrophy, meat production, and debone yield in Nellore heifers, which led to decreased tenderness and to increased mRNA expression in the calpain-calpastatin system.

Effects of long-term in vitro culturing of transgenic bovine donor fibroblasts on cell viability and in vitro developmental potential after nuclear transfer

Genetically modified animals have numerous applications, ranging from basic research to livestock production and agriculture. Recent progress in animal cloning by nuclear transfer has made possible the production of transgenic animals using previously genetically modified cell lineages. However, to produce such lineages, an additional time for in vitro culturing and great manipulation is needed. Herein, we aimed to characterize different aspects of genetically modified cells compared to control cells, and we also analyzed the development rate of embryos produced by nuclear transfer by using them as nuclei donors after short or long periods of in vitro culturing (early versus late passages). We hypothesized that the genetic material inserted in the genome of these cells, associated with the prolonged time in culture, ultimately alters cell growth physiology and cell viability, which leads to impaired nuclei reprogramming potential and consequent reduction in the production of cloned blastocysts. Fetal fibroblasts expressing the enhanced Green Fluorescent Protein gene (eGFP) cultured for different periods in vitro were analyzed with respect to chromosomal numeric abnormalities, nuclear DNA fragmentation, the ratio of BAX and BCL2 gene transcripts, and the intensity of mitochondrial membrane potential, and they were then used as nuclei donors for somatic cell nuclear transfer (SCNT). Early passages were defined as fewer than 11 passages, and late passages were 18th passage (18thp) to 21stp. No differences were observed in the percentage of cells with chromosomal abnormalities or in the mitochondrial membrane potential analysis. eGFP cells in late passages and control cells in early passages were not different regarding DNA fragmentation; however, control cells in late passages presented higher fragmentation (P < 0.05). The Bax and Bcl2 gene expression ratio in control and transgenic cells presented different patterns regarding cell conditions during culture. For SCNT experiments, no difference was observed between groups reconstructed with early or late-passage cells when fusion (63.1% and 49%), cleavage (67.7% and 69.9%), eight-cell embryo (36.4% and 44.4%) and blastocyst (21.6% and 20.8%) rates were compared. In conclusion, culture behavior was different between control and eGFP cells. However, when different in vitro culturing periods were compared, long-term cultured transgenic fetal fibroblasts remained competent for blastocyst production when used as nuclei donors in the nuclear transfer technique, a feature needed for the genetic manipulation of cell culture experiments aiming for transgenic animal production.