Oocyte maturation,embryo development and gene expression following two different methods of bovine cumulus-oocyte complexes vitrification

Objective

To examine the maturational competence, embryo development and expression of genes involved in oocyte maturation and cumulus expansion (GDF9, BMP15, HAS2, TNFAIP6, FGF17 and FSHr) following two standard methods of bovine COCs vitrification.

Methods

Bovine cumulus-oocyte complexes (COCs) were aspirated from slaughtered ovaries and then distributed into three groups: non-vitrified COCs (control), vitrification 1 group (V1); vitrification was performed by 15% ethylene glycol (EG) and 15% DMSO in holding media (TCM-199 with 20% FCS); and vitrification 2 group (V2); vitrification was performed by 40% EG in holding media. After vitrification, COCs were warmed in two steps and cultured and then evaluated for nuclear maturation, embryo development and gene expressions.

Results

The mean (±SD) percentages of nuclear maturation and blastocyst/cleaved were higher in control group (79.5 ± 8.0 and 31.0 ± 5.1%) than the V1 (34.8 ± 9.1 and 4.4 ± 5.1%) and V2 (47.8 ± 11.7 and 7.1 ± 5.8%) groups (P < 0.05), respectively. Further, COCs in V2 group showed higher mean (±SD) percentages of cleavage compared to V1 group (31.8 ± 1.0 vs 21.7 ± 2.8%; P < 0.05). GDF9 and BMP15 expression levels were higher in COCs in the control than of the vitrification groups (P < 0.05). In addition, expression level of GDF9 and BMP15 was higher in V2 group than in V1group (P < 0.05). The expression of HAS2 and FGF17 in V1 group was lower (P < 0.05) than that of the V2 groups.

Conclusions

Expression of oocyte maturation genes was affected by vitrification procedure and conditions. Using EG alone for vitrification of bovine immature COCs, resulted in higher expression of GDF9, BMP15 and production of more in vitro matured and cleaved oocytes.

     

Anticonvulsant Effect of Cicer arietinum Seed in Animal Models of Epilepsy: Introduction of an Active Molecule with Novel Chemical Structure

Background: Cicer arietinum (Chickpea) is one of the most important harvests in the world with high nutritional value. Lack of essential oils in the seeds of Chickpea is an advantage in search for drug-like molecules with less toxicity. We evaluated anticonvulsant effect of C. arietinum in common animal models of epilepsy. Methods: Dichloromethane extract was obtained from C. arietinum seeds by percolation. Acute toxicity of the extract was assessed in mice. Protective effect of the extract was examined against tonic seizures induced by maximal electroshock (MES; 50 mA, 50 Hz, 1 s) in mice, clonic seizures induced by pentylenetetrazole (PTZ; 60 mg/kg, i.p.) in mice, and electrical kindling model of complex partial seizures in rats. The extract was fractionated by n-hexane to f1 and f2 fractions. The extract and fractions underwent phytochemical analysis by thin layer chromatography. The active anticonvulsant fraction, f1, was subjected to matrix assisted laser desorption/ionization (MALDI) mass analysis. Results: The crude extract had neither toxicity up to 7 g/kg nor protective activity in MES and kindling models. However, it significantly inhibited clonic seizures induced by PTZ. f1 fraction mimicked protective effect of the extract. Phytochemical screening revealed the presence of considerable amount of alkaloids in the extract and fractions. Moreover, a novel structural class was detected in f1 fraction. Conclusion: Finding an anticonvulsant molecule pertaining to a new structural class in the seeds of C. arietinum promises an effective and inexpensive source of antiepileptic medication. Further studies are needed to identify its mechanism of action and more clues into its structure-activity relationship. Key Words: Anticonvulsants, Cicer, Kindling, Pentylenetetrazole (PTZ)     

Partial Relief of Drought-Stressed Fennel (Foeniculum vulgare Mill.) in Response to Foliar-Applied Zinc

Few attempts have been made to explore the effect of Zn fertilization on the performance of medicinal plants in general and fennel in particular, under adverse conditions. This study was conducted to examine the efficacy of zinc(Zn) in correcting physiological responses of fennel to water deficit. Twelve field-grown fennel genotypes were subjected to two levels of irrigation(irrigation after 25%–35% and 75%–85% depletion of available soil water) and two levels of foliar-applied Zn(0 and 4 g kg~(-1)). Water deprivation had significant effects on all characteristics; i.e., in contrast to seed essential oil concentration, harvest index, and superoxide dismutase(SOD) and catalase activities, it led to decreases in the rest of the attributes. Varietal differences in response to drought were meaningful for most of the traits. Genotypic variations for dry mass and seed yield were consistent with differences among the genotypes in leaf water potential, relative water content, proline concentration, and chlorophyll concentrations. Zinc led to significant increases in all physiological attributes, including activation of antioxidant defense responses manifested in ascorbate peroxidase and SOD activities and carotenoid concentration. The Zn-effect tended to be more notable in drought-stricken fennel plants in a majority of the traits.Based on our findings, Zn could be considered an effective micronutrient for ameliorating, at least in part, the stressful effects of water deficit on fennel.     

A new plant pathogenic sterile white basidiomycete from Australia

A sterile white fungus was isolated from the healthy looking roots of buffalo grass (Stenotaphrum secundatum) grown on cleared bush land in Perth, Western Australia. The fungal strain was pathogenic on 12 plant species screened under the greenhouse conditions. The clamp connections and dolipore septa indicated that the isolate was a Basidiomycete. Mycelial features, growth rate at different temperatures, as well as pathogenicity patterns of this sterile white basidiomycete (SWB) were distinctly different from those of a strain with a similar morphology, ATCC 28344, previously described as a pathogen in Florida and Georgia (USA). All attempts to induce sporulation failed. The isolates were also compared using the nucleotide sequence analysis of the ribosomal DNA array. Approximately 1 kbp of the 5 end of the large subunit ribosomal RNA gene, complete sequences of the small subunit ribosomal RNA gene and the entire ITS region (including ITS1, ITS2 and 5.8S gene) were sequenced for the purpose. The obtained sequences were compared with the homologous regions of other genera of Agaricales available in GenBank. Relatively low sequence similarities between the American and Australian strains, as well as the phylogenetic analysis of the studied regions has suggested that these two fungi belong to different genera. Interesting results were achieved in the case of the large subunit ribosomal DNA since this region has been widely studied for taxonomy of Basidiomycetes. The Australian strain 3034 appeared to be closely related to the genus Campanella and the American SWB was identified as belonging to the genus Marasmius, possibly to M. graminum. Our data suggest that the Australian strain is a novel pathogen, and is different from the American SWB isolates described to date.     

The effect of starvation stress on the protein profiles in Flexibacter chinensis

Background: Analysis of many proteins produced during the transition into the stationary phase and under stress conditions (including starvation stress) demonstrated that a number of novel proteins were induced in common to each stress and could be the reason for cross-protection in bacterial cells. It is necessary to investigate the synthesis of these proteins during different stress conditions. Methods: The changes in protein profile of Flexibacter chinensis at various stages of the starvation process and the other stresses were investigated using two-dimensional gel electrophoresis which has proven to be a powerful tool for investigation of the changes in protein profiles under such conditions. Results: Most starvation proteins were synthesized during the early stationary phase and many of these proteins remained during long-term starvation. Some of these proteins were transiently synthesized. The sequencing result of one of the proteins showed that there was a 62.5% identity in 8 amino acids overlapped with the 5' residue of a 10 kDa chaperon protein which is known to be involved in the starvation stress response in other organisms. Conclusion: There are many proteins synthesized in common with many stresses in Flexibacter chinensis. Some of these proteins must play a major role in the stability of the cell under different stresses.     

The effect of wild type P53 gene transfer on growth properties and tumorigenicity of PANC-1 tumor cell line

BACKGROUND: The p53 protein function is essential for the maintenance of the nontumorigenic cell phenotype. Pancreatic tumor cells show a very high frequency of p53 mutation. To determine if restoration of wild type p53 function can be used to eliminate the tumorigenic phenotype in these cells, pancreatic tumor cell lines, PANC-1 and HTB80, differing in p53 status were stably transfected with exogenous wild type p53 gene. METHODS: The transfection was performed using Polybrene/DMSO-Assisted Gene Transfer method. The wild type p53 gene integration into genomic DNA was detected by Southern blot and PCR. Furthermore, the expression of wild type p53 protein was detected in selected clones by immunohistochemistry and Western blot. RESULTS: While HTB80 cell line failed to produce a stable p53 expressing clone, the PANC-1 cells produced stable lines. Following characterization of clones, the growth rate and tumorigenicity of PANC-1 wild type p53 clones were compared to the control cells. Our data showed that the expression of wild type p53 decreased the growth rate of PANC-1 cells. It was also observed that the expression of wild type p53 in PANC-1 cells suppressed its potential for tumor formation in nude mice, completely, while the parental line leads to the formation of a relatively large tumor. CONCLUSION: Our results suggest that gene therapy based on restoration of wild type p53 protein function in pancreatic tumor cells with high amount of mutant p53 is a feasible option in pancreatic cancer treatment.     

Increased yield of direct seeded rice (Oryza sativa L.) by foliar fertilization through multi-component fertilizers

Foliar application of fertilizers can guarantee the availability of nutrients to rice for obtaining higher yield. Rice responds favorably to macro- and micronutrients and the tolerance to salinity hazards improves by decreasing the N/S ratio. In this study, results showed that nutrient concentrations (g L^?1) for rice are: nitrogen (N) 108.0, phosphorous (P₂O₅) 6, potassium (K₂O) 81.0, calcium (CaO) 15.0, and magnesium (MgO) 6 g L^?1; and for iron (Fe), manganese (Mn), zinc (Zn), cupper (Cu), boron (B), molybdenum (Mo) and silicon (Si) the recommended concentrations are 0.6, 0.45, 0.21, 0.06, 0.09, 0.0002 and 0.004 g L^?1, respectively. A significant increase was recorded in number of panicles m^?2, 1000 grain weight, biological yield and grain yield with foliar application of nutrients. Five foliar applications of nutrients resulted in maximum number of panicles m^?2, grains panicle^?1, 1000 grain weight and biological yield. It is concluded that five foliar applications of balanced amounts of fertilizers at the seedling stage (two sprays), tillering (single spray) and at panicle initiation and panicle differentiation (two sprays) helped in enhancing yield and yield components of rice. In this research, five foliar applications produced the smallest damaging effects of blast (Pyricularia oryzae) in rice.     

Co-inoculations of arbuscular mycorrhizal fungi and rhizobia under salinity in alfalfa

Alfalfa (Medicago sativa L.) is cultivated in arid and semi-arid regions where salinity is one of the main limiting factors for its production. Thus, this experiment was conducted to evaluate the efficacy of arbuscular mycorrhizal fungus (AMF), Glomus mosseae, alfalfa rhizobia Sinorhizobium meliloti (R) seed inoculation in the development of salinity tolerance of different alfalfa cultivars (Rehnani, Pioneer and Bami) under a variety of salinity levels. The results revealed that under non-stress condition, root mycorrhizal infection, nodulation (the number and weight of nodules per plant), potassium (K), calcium (Ca), phosphorus (P), zinc (Zn), copper (Cu) and magnesium (Mg) contents of the root and shoot, the value of the K/Na ratio, protein [calculated from the nitrogen (N) content] and proline contents of the shoot and the alfalfa yield were found to be the highest while Na contents of the root and shoot were seen to be the lowest when seeds were double inoculated followed by mycorrhizae, rhizobium and control treatments, respectively. Similarly, under salinity condition, the greatest amounts of mycorrhizal infection, nodulation, root and shoot P contents, the value of K/Na ratio, the shoot proline content and the root Ca content were enhanced with the least amount of leaf Na content related to the cases of seeds which were double inoculated, followed by mycorrhizae, rhizobium and control treatments respectively. The results suggested that inoculation of alfalfa seed with AMF or R, especially double inoculation, causes a considerable increase in alfalfa yield under both saline and non-saline conditions by increasing colonization, nodulation and nutrient uptake.     

Influence of Metallic Elements Exuded from Wood Based Fly Ash on Treatment of TMP Effluent北大核心CSCD

In the process of thermomechanical pulping(TMP) effluent treatment using wood based fly ash (FA) as reactant, inorganic metallic ions can exude into the effluent from FA complex. In order to determine the metallic elements in FA would flocculate and remove the organics in the effluent under the alkaline condition, the removal behaviors of organic material were investigated by means of simulation with different kind of metallic ions addition at various dosage levels and adjusting pH value of TMP effluent, respectively. It concluded that the metallic ions combined with organics precipitate simultaneously under the alkaline condition by charge neutralization and H-bond to form large particles. The removal rates increased with the increment dosage of Mg2+ and Fe3+, while the Ca2+ and Al3+ would not be affected by their dosage under the alkaline condition. When the pH values were 10 and 12, respectively, the removal capability of Ca2+ and Mg2+ was maximum at pH value 12, while Al3+ achieved peak removal rate at pH 10. The removal efficiency from Fe3+ kept constant between pH value 10 and 12. © 2018, Editorial Board of «Chemistry and Industry of Forest Products». All right reserved.