Comparison of the developmental potential of 2-week-old preantral follicles derived from vitrified ovarian tissue slices, vitrified whole ovaries and vitrified/transplanted newborn mouse ovaries using the metal surface method

Background

Cryopreservation of preantral follicles or ovarian tissues would enable the storage of large numbers of primordial follicles or preantral follicles and preserves the structural integrity of somatic and reproductive cells. In the present study, we compared the developmental potential of cryopreserved two-week-old mouse preantral follicles, ovarian tissue slices, two-week-old mouse ovaries and newborn mouse ovaries using a metal plate with a high cooling rate for cooling the droplet of vitrification solution.

Methods

Groups of 2 to 4 samples (including of 14-day old preantral follicles, ovarian tissue slices, whole ovaries, and whole newborn ovaries) were exposed to 4% ethylene glycol (EG) in DPBS + 10% FBS for 15 min and then rinsed in a vitrification solution composed of 6 M ethylene glycol and 0.4 M trehalose in DPBS + 10% FBS. Equilibration in room temperature was performed for 20–30 seconds for preantral follicle and 5 min equilibration was performed in an ice bath for ovaries. The samples were dropped onto the surface of metal plate around -180°C in the volume of 2 μl and 6 μl. After thawing, the ovarian tissue was mechanically isolated for collecting the preantral follicles. The thawed newborn ovaries were transplanted under the renal capsule of recipient male mice for 14 days. Preantral follicles collected from each groups were cultured individually in 20-μl droplets of α-MEM culture medium in culture dish for 12 days. On the day 12 of culture, the cumulus-oocyte complexes (COCs) were collected for IVM and IVF. Fertilization and embryo cleavage were scored.

Results

After the vitrification of 14-day-old preantral follicles using 2 μl or 6 μl droplet onto surface of metal plate, the results indicated that no significant difference in survival rate, antral-like cavity formation, COCs collected, 2 cell embryo cleavage and blastocyst development was found in vitrification of the 2 μl and 6 μl droplet groups. As comparing 14-day old ovarian tissue (ovarian tissue slices and whole ovaries) and whole newborn ovaries vitrified in 6 μl droplet, lower success rates of antral-like cavity formation and COCs collection were found in the whole ovaries group.

Conclusion

Our results suggest that the metal plate surface vitrification method is an appropriate and convenient method for cryopreservation of mouse ovaries and preantral follicles. The droplet volume of vitrification solution in 2 μl and 6 μl can be an option.     

Synthesis of cellulose-graft-poly(N,N-dimethylamino-2-ethyl methacrylate) copolymers via homogeneous ATRP and their aggregates in aqueous media

Cellulose-graft-poly(N,N-dimethylamino-2-ethyl methacrylate) (cellulose-g-PDMAEMA) copolymers were prepared by homogeneous atom transfer radical polymerization (ATRP) under mild conditions. Cellulose macroinitiator was successfully synthesized by direct acylation of cellulose with 2-bromopropionyl bromide in a room temperature ionic liquid (RTIL), 1-allyl-3-methylimidazolium chloride. Copolymers were obtained via ATRP of N,N-dimethylamino-2-ethyl methacrylate (DMAEMA) with CuBr/pentamethyldiethylenetriamine (PMDETA) as catalyst and N,N-dimethylformamide (DMF) as solvent without homopolymer byproduct. The grafting copolymers were characterized by (1)H NMR, FT-IR, and TGA measurements. The results confirmed that PDMAEMA had been covalently bonded to cellulose backbone. Furthermore, the assemblies or aggregates formed by cellulose-g-PDMAEMA copolymers in water were studied at various concentrations, temperatures, and pH values by means of UV, DLS, TEM, and AFM. The results indicate that the copolymers had the pH- and temperature-responsive properties similar to the expected stimuli-responses by PDMAEMA. The synthetic strategy presented here could be employed in the preparation of other novel biomaterials from a variety of polysaccharides.     

Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture

Malaysia is the world’s leading producer of palm oil products that contribute US$ 7.5 billion in export revenues. Like any other agro-based industries, it generates waste that could be utilized as a source of organic nutrients for microalgae culture. Present investigation delves upon Isochrysis sp. culture in POME modified medium and its utilization as a supplement to Nanochloropsis sp. in rotifer cultures. The culture conditions were optimized using a 1 L photobioreactor (Temp: 23°C, illumination: 180 ∼ 200 μmol photons m⁻²s⁻¹, n = 6) and scaled up to 10 L outdoor system (Temp: 26–29°C, illumination: 50 ∼ 180 μmol photons m⁻²s⁻¹, n = 3). Algal growth rate in photobioreactor (μ = 0.0363 h⁻¹) was 55% higher compared to outdoor culture (μ = 0.0163 h⁻¹), but biomass production was 1.3 times higher in outdoor culture (Outdoor = 91.7 mg m⁻²d⁻¹; Photobioreactor = 69 mg m⁻²d⁻¹). Outdoor culture produced 18% higher lipid; while total fatty acids (FA) was not significantly affected by the change in culture systems as both cultures yield almost similar concentrations of fatty acids per gram of sample (photobioreactor = 119.17 mg g⁻¹; outdoor culture = 104.50 mg g⁻¹); however, outdoor cultured Isochrysis sp. had 26% more polyunsaturated fatty acids (PUFAs). Rotifers cultured in Isochrysis sp./ Nanochloropsis sp. (1:1, v/v) mixture gave similar growth rate as 100% Nanochoropsis sp. culture (μ = 0.40 d⁻¹), but had 45% higher counts of rotifers with eggs (t = 7, maximum). The Isochrysis sp. culture successfully lowered the nitrate (46%) and orthophosphate (83%) during outdoor culture.     

Common themes and variations in serine protease autotransporters

The serine protease autotransporters of the Enterobacteriaceae (SPATEs) represent a group of large-sized, multi-domain exoproteins found only in pathogenic enteric bacteria. These proteins contain a highly conserved channel-forming C-terminal domain, which functions together with YaeT/Omp85 to facilitate secretion of the passenger domain to the cell surface. The C-terminal domain also mediates autoproteolytic cleavage, which releases the passenger from the bacterial cell. The passenger folds into a characteristic parallel beta-helical stalk-like structure with an N-terminal globular domain that performs serine proteolytic activity. Here, we review and discuss recent findings that have led to a better understanding of these unique features in this virulence protein family, including their biogenesis, structural architecture, sequence variation, sub-grouping, evolution and biochemical function.     

Synergistic effects of cinnamaldehyde in combination with eugenol against wood decay fungi

The combined effects of using cinnamaldehyde with catechin, quercetin or eugenol against wood decay fungi were examined by comparing their isoeffective concentrations to that of individual compound. Among all combinations, cinnamaldehyde with eugenol revealed the strongest synergy against Laetiporus sulphureus. The synergism was due to the interference of fungal cell wall synthesis and cell wall destruction plus radical scavenging effect. Results also suggested that antioxidant with fungicidal effect might be a better candidate than pure antioxidant for the system of fungicide/antioxidant.     

Optimization of conditions for protease production by Chryseobacterium taeanense TKU001

A protease-producing bacterium was isolated and identified as Chryseobacterium taeanense TKU001. An extracellular metalloprotease with novel properties of solvent- and surfactant-stable was purified from the culture supernatant of C. taeanense TKU001 with shrimp shell wastes as the sole carbon/nitrogen source. The optimized condition for protease production was found when the culture was shaken at 37 degrees C for 3 days in 50 mL of medium containing 0.5% shrimp shell powder (SSP) (w/v), 0.1% K2HPO4, and 0.05% MgSO4.7H2O. Two extracellular proteases (FI and FII) were purified and characterized, and their molecular weights, pH and thermal stabilities were determined. The molecular masses of TKU001 protease FI and FII determined by SDS-PAGE and gel filtration were approximately 41 kDa and 75 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU001 protease FI were 8, 60 degrees C, pH 6-9, and 60 degrees C, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU001 protease FII were 7, 60 degrees C, pH 7-9, and 50 degrees C, respectively. TKU001 protease FI and FII were both inhibited completely by EDTA, indicating that the TKU001 protease FI and FII were metalloproteases. TKU001 protease FI and FII retained more than 75% of its original protease activity after preincubation for 10 days at 4 degrees C in the presence of 25% most tested organic solvents. Additionally, the TKU001 protease FI retained 79%, 80%, and 110% of its original activity in the presence of 2% Tween 20, 2% Tween 40, and 2% Triton X-100, respectively. However, at the same condition, the activity of TKU001 protease FII retained 100%, 100%, and 121% of its original activity, respectively. This is the first report of C. taeanense being able to use shrimp shell wastes as the sole carbon/nitrogen source for proteases production. The novelties of the TKU001 protease include its high stability to the solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis.     

Bioconversion of squid pen by Lactobacillus paracasei subsp paracasei TKU010 for the production of proteases and lettuce growth enhancing biofertilizers

A protease-producing bacterium, strain TKU010, was isolated from infant vomited milk and identified as Lactobacillus paracasei subsp. paracasei. A surfactant-stable protease, purified 64-fold from the third day culture supernatant to homogeneity in an overall yield of 11%, has a molecular weight of about 49,000. The enzyme degraded casein and gelatin, but did not degrade albumin, fibrin, and elastin. The enzyme activity was increased about 1.5-fold by the addition of 5 mM Ba²⁺. However, Fe²⁺ and Cu²⁺ ions strongly inhibited the enzyme. The enzyme was maximally active at pH 10 and 60 °C and retained 94% and 71% activity in the presence of Tween 20 (2% w/v) and SDS (2 mM), respectively. The result of identification of TKU010 protease showed that nine tryptic peptides were identical to Serratia protease (serralysin) (GenBank accession number gi999638) with 35% sequence coverage. In comparison with the tryptic peptides of L. paracasei subsp. paracasei TKU012 protease, TKU010 protease possessed two additional peptides with sequences of AATTGYDAVDDLLHYHER and QTFTHEIGHALGLSHPGDYNAGEGNPTYR. The fourth day culture supernatant of TKU010 showed maximal activity of about 5-fold growth enhancing effect on lettuce weight, which was not shown with L. paracasei subsp paracasei TKU012.     

Role of glutathione peroxidase in the ontogeny of hippocampal oxidative stress and kainate seizure sensitivity in the genetically epilepsy-prone rats

Oxidative stress may contribute to epileptogenicity in genetic models of epilepsy. To address this, we examined the enzymatic activity of cytosolic Cu/Zn superoxide dismutase (SOD-1), mitochondrial Mn superoxide dismutase (SOD-2), and glutathione peroxidase (GPx) in the developing hippocampus of genetically epilepsy-prone rats (GEPR-9s). We also measured changes in the GSH/GSSG ratio, lipid peroxidation, and protein oxidation at post-natal days (PD) 7, 30, and 90, respectively. Compared with control Sprague-Dawley (SD) rats, GEPR-9s showed similar SOD-1 and SOD-2 activity but lower GPx activity. Epilepsy-prone rats also showed lower GSH/GSSG ratios than controls, and more lipid peroxidation (as measured by malondialdehyde levels) and protein oxidation (as measured by carbonyl levels). Treatment with kainic acid (KA) resulted in more pronounced seizures, less GPx activity, and lower GSH/GSSG ratios in GEPR-9s than in controls, but KA did not significantly affect SOD-1 or SOD-2 activity, suggesting that GEPR-9s do not compensate for reduced GPx activity by increasing SOD. Moreover, KA treatment resulted in significantly a lower GSH/GSSG ratio and GPx-like immunoreactivity and higher malondialdehyde and carbonyl levels in GEPR-9s than in controls. These findings were more evident in GEPR-9s at PD 90 than at PD 30, indicating that oxidative stress is age-dependent. Double-labeling immunocytochemical analysis demonstrated co-localization of GPx-immunoreactive glia-like cells and reactive astrocytes, as labeled by glial fibrillary acidic protein (GFAP). This suggests that mobilization of astroglial cells for synthesis of GPx protein is a response to KA insult, intended to decrease the neurotoxicity induced by peroxides. These responses were more pronounced in control SD rats than in GEPR-9s. Our results suggest that impairment of the GPx (including glutathione)-mediated antioxidant system contributed to epileptogenesis in GEPR-9s.     

Circular F-actin bundles and a G-actin gradient in pollen and pollen tubes of Lilium davidii

The distribution of and relationship between F-actin and G-actin were investigated in pollen grains and pollen tubes of Lilium davidii Duch. using a confocal laser scanning microscope after fluorescence and immunofluorescence labeling. Circular F-actin bundles were found to be the main form of microfilament cytoskeleton in pollen grains and pollen tubes. Consistent with cytoplasmic streaming in pollen tubes, there were no obvious F-actin bundles in the 10- to 20-microm tip region of long pollen tubes, only a few short F-actin fragments. Labeling with fluorescein isothiocyanate (FITC)-DNase I at first established the presence of a tip-focused gradient of intracellular G-actin concentration at the extreme apex of the tube, the concentration of G-actin being about twice as high in the 10- to 20-microm region of the tip as in other regions of the pollen tube. We also found that the distribution of G-actin was related negatively to that of the F-actin in pollen tubes of L. davidii. Caffeine treatment caused the G-actin tip-focused gradient to disappear, and F-actin to extend into the pollen tube tip. Based on these results, we speculate that the circular F-actin bundles may be the track for bidirectional cytoplasmic streaming in pollen tubes, and that in the pollen tube tip most of the F-actin is depolymerized into G-actin, leading to the absence of F-actin bundles in this region.