scholarly journals 273 OPTIMIZATION OF PROTOCOLS FOR ACTIVATION OF GOAT OOCYTES WITH IONOMYCIN IN COMBINATION WITH 6-DIMETHYLAMINOPURINE

2005 ◽  
Vol 17 (2) ◽  
pp. 286
Author(s):  
J.-H. Tan ◽  
G.-C. Lan ◽  
Z.-L. Chang ◽  
D. Han ◽  
Z.-B. Han ◽  
...  

The protocol of ionomycin in combination with 6-dimethylaminopurine (6-DMAP) is commonly used for activation of oocytes and reconstructed embryos of different species. Since numerous abnormalities and impaired development have been observed when oocytes are activated with 6-DMAP, this protocol needs optimization. Effects of concentration and treatment duration of both drugs on activation kinetics and parthenogenetic development of goat oocytes were examined in this study. When goat oocytes matured in vitro in TCM-199 were treated with 5 M ionomycin in PBS for different periods before exposure to 6-DMAP in CR1aa, the activation rate obtained with ionomycin treatment for 1 min (95.2%) was significantly (P < 0.05, Duncan multiple comparison test) higher than with ionomycin treatments for 3, 5, 7, or 9 min. When oocytes were treated with different concentrations of ionomycin for 1 min before exposure to 6-DMAP, activation rates obtained with 0.625, 1.25, 2.5, 5, 10, and 20 M ionomycin (87–95%) did not differ significantly but were significantly higher than that achieved with 0.3125 M ionomycin. Progressive reduction of time for 6-DMAP exposure showed that the duration of 6-DMAP treatment can be reduced to 1 h from the 2nd up to the 4th hour after ionomycin treatment, to produce activation rates greater than 85%. When oocytes were treated with different concentrations of 6-DMAP for the 3rd hour (atotal of 1 h, 3 h after the exposure to ionomycin), activation rates with 4 and 2 mM 6-DMAP (>90%) were significantly higher than those with 1and 0.5 mM. Therefore, the best protocol for goat oocyte activation would be a 1-min exposure to 2.5 M ionomycin followed by 2 mM 6-DMAP treatment for the 3rd hour. When oocytes matured in vitro for different times were stimulated with the best protocol, activation rates of the 27-, 30-, and 33-h oocytes (85, 85, and 91%) were significantly higher than those of the 24-, 26-, and 39-h oocytes. When activated oocytes were co-cultured in CR1aa with cumulus cell monolayers, the highest rates of cleavage (92%) and morulae/blastocysts (23%) were obtained with oocytes activated by the best protocol, and any increase in the intensity of ionomycin treatment and in the duration of 6-DMAP exposure impaired the development of the parthenotes. During anaphase II, chromosomes (the dyads) did not separate into two units in oocytes that were activated by long exposure to 6-DMAP, but they did in oocytes that were activated by short or no exposure to 6-DMAP; as a result, only one pronucleus developed in most of the former but two pronuclei were formed in most of the latter cases. Laser scanning confocal microscopy showed that microtubules also behaved differently in these two groups of activated oocytes. It is therefore concluded that to obtain better activation and development, goat oocytes matured in vitro for 27–33 h should be used, and these should be activated by a 1-min exposure to 2.5 M ionomycin followed by 2 mM 6-DMAP treatment for the 3rd hour. This study was supported by the “973” Project of China Sci. Technol. Ministry (No. G200016108).

2008 ◽  
Vol 20 (1) ◽  
pp. 197
Author(s):  
J. Zhu ◽  
K. H. S. Campbell

The objective of the present experiments was to examine whether strontium could activate in vitro-matured ovine oocytes. Oocytes were collected and matured as previously described (Lee and Campbell 2006 Biol. Reprod. 74, 691–698). Briefly, selected cumulus–oocyte complexes were cultured in modified TCM-199 medium supplemented with 20% sheep serum and hormones for 22–23 h, at 39°C, 5% CO2 in air. Matured oocytes were randomly divided into four groups and treated as follows: (1) cultured in 10 mm strontium + 5 μg mL–1 cytochalasin B in Ca2+-free CZB medium for 4–5 h; (2) electrically activated in Ca2+-containing medium, then cultured in 10 mm strontium + 5 μg mL–1 cytochalasin B in Ca2+-free CZB medium for 4–5 h; (3) electrically activated in Ca2+-containing medium and then cultured in SOF medium containing 5 μg mL–1 cytochalasin B for 4–5 h; and (4) electrically activated in Ca2+-free medium and then transferred into SOF medium + 5 μg mL–1 cytochalasin B for 4–5 h. This experiment was repeated three times. Activation rates based on the number of pronuclear formations/the number of oocytes cultured were 96.7% (147/152), 95.9% (116/121), 75.9% (101/133), and 43.0% (56/107) in Groups 1–4, respectively. After 7 days of culture in SOF medium, 26.8%, 33.3%, 19.6%, and 0% of oocytes in Groups 1, 2, 3, and 4 developed to the blastocyst stage, respectively. Significant differences in blastocyst rate were observed across these groups except between groups 1 and 2 (P < 0.01). However, there were no significant differences in mean number of nuclei/blastocyst across Groups 1, 2, and 3 (P > 0.05). Our results demonstrated that in vitro-matured ovine oocytes can be effectively activated with strontium alone, resulting in an activation rate of 96.7% and a blastocyst rate of 26.8% (blastocysts/oocytes). Also, a combination of strontium and electrical pulses could benefit sheep oocyte activation and embryo development to the blastocyst stage (95.9% and 33.3%, respectively). We conclude that strontium is an effective activator for sheep oocyte activation and it could be used for sheep nuclear transfer. Table 1. Parthenogenetic development of oocytes activated by SrCl2+ and electrical pulses


Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 861
Author(s):  
Jacopo Cardellini ◽  
Arianna Balestri ◽  
Costanza Montis ◽  
Debora Berti

In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake (in vitro). However, the diffraction limit of light, which limits the resolution to hundreds of nanometers, has for long time restricted the extent and quality of information and insight achievable through these techniques. The advent of super-resolution microscopic techniques, recognized with the 2014 Nobel prize in Chemistry, revolutionized the field thanks to the possibility to achieve nanometric resolution, i.e., the typical scale length of chemical and biological phenomena. Since then, fluorescence microscopy-related techniques have acquired renewed interest for the scientific community, both from the perspective of instrument/techniques development and from the perspective of the advanced scientific applications. In this contribution we will review the application of these techniques to the field of drug delivery, discussing how the latest advancements of static and dynamic methodologies have tremendously expanded the experimental opportunities for the characterization of drug delivery systems and for the understanding of their behaviour in biologically relevant environments.


2017 ◽  
Vol 21 (02) ◽  
pp. 122-127 ◽  
Author(s):  
Yunman Zheng ◽  
Sizhe Zhu ◽  
Lijun Jiang ◽  
Fengshou Wu ◽  
Chi Huang ◽  
...  

Three azobisporphyrins (Por1, Por2 and Por3) were synthesized by coupling two molecules of (4-nitrophenyl/pyridyl) porphyrins in the presence of KOH/butanol. The structures of porphyrins were confirmed by UV, IR, NMR and mass spectra and elemental analysis. With tetraphenylporphyrin (H2TPP) as a control, the singlet oxygen (1O[Formula: see text] generation of porphyrins was evaluated through 1,3-diphenylisobenzofuran (DPBF) method. The order of ability to generate 1O2 for three azobisporphyrins was Por 1 [Formula: see text]Por 2 > Por 3[Formula: see text] H2TPP. The photocytotoxicity and sub-cellular localization of azobisporphyrins over Hela cells were studied through MTT analysis and confocal laser scanning microscope, respectively. The results indicated Por 1 and Por 2 displayed the low dark-cytotoxicity, while Por 3 induced a concentration-dependent cytotoxicity to Hela cells with the concentration of porphyrins ranging from 1 to 100 [Formula: see text] M. With the light dose at 4 J/cm2, Por 3 killed more than 60% Hela cells at 2 [Formula: see text] M, indicating a high photocytoxicity. As seen from the laser scanning confocal microscopy images, Por 3 was mainly localized in cell membrane, while Por 1 and Por 2 do not displayed significant fluorescent emission in Hela cells. These results suggest the synthesized cationic azobisporphyrin could be used as a potential therapeutic agent for photodynamic therapy of cancers.


2021 ◽  
Vol 12 ◽  
Author(s):  
Ping He ◽  
Shu Li ◽  
Shengtao Xu ◽  
Huacai Fan ◽  
Yongfen Wang ◽  
...  

Bacillus spp. is effective biocontrol agents for Fusarium wilt of banana (FWB), tropical race 4 (TR4). This study explores the colonization by Bacillus subtilis, Bacillus velezensis, and Bacillus amyloliquefaciens of host banana plants and elucidates the mechanism of antagonistic TR4 biocontrol. The authors selected one B. subtilis strain, three B. velezensis strains, and three B. amyloliquefaciens strains that are proven to significantly inhibit TR4 in vitro, optimized the genetic transformation conditions and explored their colonization process in banana plants. The results showed that we successfully constructed an optimized fluorescent electro-transformation system (OD600 of bacteria concentration=0.7, plasmid concentration=50ng/μl, plasmid volume=2μl, transformation voltage=1.8kV, and transformation capacitance=400Ω) of TR4-inhibitory Bacillus spp. strains. The red fluorescent protein (RFP)-labeled strains were shown to have high stability with a plasmid-retention frequency above 98%, where bacterial growth rates and TR4 inhibition are unaffected by fluorescent plasmid insertion. In vivo colonizing observation by Laser Scanning Confocal Microscopy (LSCM) and Scanning Electron Microscopy (SEM) showed that Bacillus spp. can colonize the internal cells of banana plantlets roots. Further, fluorescent observation by LSCM showed these RFP-labeled bacteria exhibit chemotaxis (chemotaxis ratio was 1.85±0.04) toward green fluorescent protein (GFP)-labeled TR4 hyphae in banana plants. We conclude that B. subtilis, B. velezensis, and B. amyloliquefaciens can successfully colonize banana plants and interact with TR4. Monitoring its dynamic interaction with TR4 and its biocontrol mechanism is under further study.


1991 ◽  
Vol 69 (11) ◽  
pp. 2560-2573 ◽  
Author(s):  
Young H. Kwon ◽  
Harvey C. Hoch ◽  
James R. Aist

Urediospore germlings of Uromyces appendiculatus sense topographical signals inherent in host stomata over which they develop appressoria. The site of signal recognition by the fungus is invariably within the apical 6 μm of the germling. The organization of the germling apex before and during initiation of appressorium formation was examined by both electron microscopy and laser scanning confocal microscopy. Most notable were changes in the distribution and organization of the microtubules and apical vesicles. Microtubules were oriented parallel to the inductive ridge, along which expansion of the germling apex occurred. In germlings that contacted artificial inductive topographies in vitro, the distribution of apical vesicles in germlings did not change within the first 3–4 min. After 6–8 min of contact, most apical vesicles became repositioned near the ridge. The apical cluster in developing appressoria was closely associated with that population of cytoplasmic microtubules that developed parallel to the inductive ridge. Coincident with growth of the germling apex over an inductive ridge was a physical deformation of the cell by the ridge, indenting both the cell wall and plasma membrane. Key words: bean rust, cytoskeleton, infection structure, Spitzenkörper, urediospore.


2009 ◽  
Vol 29 (12) ◽  
pp. 1879-1884 ◽  
Author(s):  
Christoph M Zehendner ◽  
Heiko J Luhmann ◽  
Christoph RW Kuhlmann

The blood–brain barrier (BBB) closely interacts with the neuronal parenchyma in vivo. To replicate this interdependence in vitro, we established a murine coculture model composed of brain endothelial cell (BEC) monolayers with cortical organotypic slice cultures. The morphology of cell types, expression of tight junctions, formation of reactive oxygen species, caspase-3 activity in BECs, and alterations of electrical resistance under physiologic and pathophysiological conditions were investigated. This new BBB model allows the application of techniques such as laser scanning confocal microscopy, immunohistochemistry, fluorescent live cell imaging, and electrical cell substrate impedance sensing in real time for studying the dynamics of BBB function under defined conditions.


2007 ◽  
Vol 19 (1) ◽  
pp. 146
Author(s):  
D. J. Kwon ◽  
C. K. Park ◽  
B. K. Yang ◽  
C. I. Kim ◽  
H. T. Cheong

The present study was conducted to control nuclear remodeling types by treatment with caffeine or vanadate, and to examine the microtubule distribution of nuclear transfer embryos (NTs) after nuclear remodeling control and the mitotic spindle assembly and its morphological changes during the first mitosis of NTs in the pig. Enucleated oocytes were treated with 5 mM caffeine or 0.5 mM sodium orthovanadate (vanadate) for 2.5 or 0.5 h to increase or decrease MPF activity before injection of fetal fibroblast cells. Reconstituted eggs were fused by an electric stimulation (ES, 1.5 kV cm-1), activated by a combination of 2 pulses of ES (1.0 kV cm-1), and cultured for 3 h with 2 mM 6-dimethylaminopurine (6-DMAP) at 1 h after fusion treatment. Some matured oocytes were also treated by the same chemicals before parthenogenetic activation under the same conditions as NTs, and cultured in vitro to evaluate the effects of these chemicals on embryo development. NTs and parthenogenetic embryos were cultured in PZM-3 for 20 h or 6 days at 39�C, 5% CO2 in air, respectively. Nuclear remodeling types of NTs were examined at 1 h after fusion (before activation) by the whole-mount method. At least 3 replicates for each experiment were performed. Microtubules and DNA of NTs that were fixed at 1 h or 20 h after fusion were detected by indirect immunocytochemical technique. Images were captured using laser scanning confocal microscopy. Caffeine and vanadate did not affect the development to the blastocyst stage of porcine parthenogenetic embryos. When a nucleus was exposed to oocyte cytoplasm treated with caffeine, premature chromosome condensation (PCC) occurred at a higher rate (82/98, 83.7%) compared to control (42/73, 57.5%) and vanadate-treated (11/91, 12.1%) groups (P &lt; 0.05). The proportion of embryos that did not undergo nuclear envelope breakdown (NEBD) was higher in the vanadate treatment group (87.9%) compared to the caffeine and control groups (16.3 and 42.5%, respectively; P &lt; 0.05). The frequency of embryos showing a γ-tubulin only and both γ- and β-tubulins were 3.9–9.4% and 21.9–34.6%, respectively, in NTs (total 87 embryos) at 1 h after fusion regardless of caffeine and vanadate treatments. In the majority of NTs (61.5–68.6%), microtubules were not observed. At 20 h after fusion, the frequency of the embryos undergoing normal mitosis was similar in the control (17/45, 37.8%) and caffeine (19/43, 44.2%) groups, but it was significantly lower in the vanadate group (7/37, 18.9%; P &lt; 0.05). The present study demonstrates that the nuclear remodeling type of NTs can be controlled by treatment with MPF regulators, caffeine and vanadate, and such treatment is not related to the microtubule distribution in porcine NTs. The finding, however, that the vanadate can delay the mitotic progression of porcine NTs at the first cell cycle may be due to the lack of NEBD and PCC. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD;KRF-2005-042-F00030).


Zygote ◽  
1996 ◽  
Vol 4 (2) ◽  
pp. 145-149 ◽  
Author(s):  
Nam-Hyung Kim ◽  
Billy N. Day ◽  
Hoon Taek Lee ◽  
Kil-Saeng Chung

SummaryIn this study we imaged integral changes in microfilament assembly and cortical granule distribution, and examined effects of microfilament inhibitor on the cortical granule distribution during oocyte maturation, parthenogenetic activation and in vitro fertilisation in the pig. The microfilament assembly and cortical granule distribution were imaged with fluorescent-labelled lectin and rhodamine-labelled phalloidin under laser scanning confocal microscopy. At the germinal vesicle stage, cortical granule organelles were located around the cell cortex and were present as a relatively wide area on the oolemma. Microfilaments were also observed in a wide uniform area around the cell cortex. Following germinal vesicle breakdown, microfilaments concentrated in the condensed chromatin and cortical granules were observed in the cortex. Treatment with cytochalasin B inhibited microfilament polymerisation and prevented movement of cortical granules to the cortex. Cortical granule exudation following sperm penetration was evenly distributed in the entire perivitelline space. These results suggest that the microfilament assembly is involved in the distribution, movement and exocytosis of cortical granules during maturation and fertilisation.


2007 ◽  
Vol 59 (2) ◽  
pp. 280-287 ◽  
Author(s):  
F. Perecin ◽  
S.C. Méo ◽  
C.L.V. Leal ◽  
J.M. Garcia

The efficiency of bohemine and roscovitine in combination with ionomycin on parthenogenetic activation and initial embryonic development of bovine oocytes was studied. Two experiments were performed: in the first, different concentrations (0, 50, 75 or 100µM) and different exposure periods (2, 4 or 6 hours) to bohemine or roscovitine were tested for activation rates of in vitro matured (IVM) bovine oocytes, which were pre-exposed to ionomycin. The best treatments, 75µM bohemine and 50µM roscovitine, both for 6h, were used in the second experiment, in which IVM bovine oocytes were exposed to ionomycin, followed or not by bohemine or roscovitine treatment, and evaluated for nuclear status, activation rate and blastocyst development were assessed. The combined treatments (ionomycin + cyclin-dependent kinases inhibitors - CDKIs) showed better results for activation rates (77.3%) and initial embryonic development (35.2%) than the single ionomycin treatment (69.4% for activation and 21.9% for development); and also lead to a more uniform activation (nearly 90% single pronucleus development). The results showed that CDKIs improve the effects of ionomycin on parthenogenetic activation and blastocyst development in bovine oocytes and could help to achieve more efficient activation protocols, increasing the developmental competence of embryos obtained by reproductive biotechniques.


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