scholarly journals Calcium-Free and Cytochalasin B Treatment Inhibits Blastomere Fusion in 2-Cell Stage Embryos for the Generation of Floxed Mice via Sequential Electroporation

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1088
Author(s):  
Takuro Horii ◽  
Ryosuke Kobayashi ◽  
Mika Kimura ◽  
Sumiyo Morita ◽  
Izuho Hatada

The generation of conditional knockout mice using the Cre-loxP system is advantageous for the functional analysis of genes. Flanked by two loxP sites (floxed) mice can be directly obtained from fertilized eggs by the CRISPR/Cas9 genome editing system. We previously reported that sequential knock-in (KI) of each loxP site by electroporation (EP) at the 1- and 2-cell embryonic stages increases the number of mice with floxed alleles compared with simultaneous KI. However, EP at the 2-cell stage frequently induced blastomere fusion. These fused embryos cannot develop to term because they are tetraploidized. In this study, we examined the following three conditions to inhibit blastomere fusion by EP at the 2-cell stage: (1) hypertonic treatment, (2) Calcium (Ca2+)-free treatment, and (3) actin polymerization inhibition. Hypertonic treatment of 2-cell stage embryos prevented blastomere fusion and facilitated blastocyst development; however, KI efficiency was decreased. Ca2+-free treatment and actin polymerization inhibition by cytochalasin B (CB) reduced fusion rate, and did not have negative effects on development and KI efficiency. These results suggest that Ca2+-free and CB treatment at the 2-cell stage is effective to generate floxed mice in combination with a sequential EP method.

2012 ◽  
Vol 24 (1) ◽  
pp. 200
Author(s):  
Z. Machaty ◽  
C. Wang ◽  
K. Lee

At fertilization, an acrosome-reacted sperm fuses to the plasma membrane of the oocyte. It is then internalized into the ooplasm and its head forms the male pronucleus that moves toward the female pronucleus. Experiments using cytochalasin, an inhibitor of actin polymerization, suggest that filamentous actin is necessary for spermatozoa incorporation deep into the oocyte cytoplasm. However, the exact role of actin in the process is not entirely clear and the consequences of inhibiting actin polymerization on embryo development are not known. In the present study, we investigated the effect of cytochalasin B on fertilization and subsequent embryo development in the porcine model. In vitro-matured oocytes were rinsed in fertilization medium, a modified Tris-buffered medium supplemented with 0.1% BSA and 1 mM caffeine. Groups of 30 oocytes were placed into 50-μL droplets of the medium covered with mineral oil. Fresh semen was collected from both a wild-type boar and a transgenic boar heterozygous for the green fluorescent protein (GFP), washed 3 times in Dulbecco's PBS and added at a final concentration of 5 × 105 cells mL–1 to each droplet containing the oocytes. The gametes were co-incubated for 5 h at 39°C under 5% CO2 in air. In the treatment group, 10 μg mL–1 of cytochalasin B was supplemented into the fertilization droplets; control groups received an equivalent amount of dimethyl sulfoxide, the solvent used to dissolve the inhibitor. After gamete co-incubation, potential zygotes were transferred to PZM-3 medium for culture. In experiment 1, mature oocytes were fertilized with the wild-type boar sperm. Embryos at the 6- or 8-cell stage were then collected, the blastomeres were separated and DNA from each blastomere was isolated for PCR analysis to monitor the presence of the male-specific SRY gene. In experiment 2, the GFP transgenic sperm was used for fertilization. Fertilized oocytes were cultured for 7 days and the resultant embryos were examined for GFP expression using an epifluorescence microscope. The developmental stages of the embryos were also determined by staining their nuclei with Hoechst 33342. We found that after cytochalasin B treatment, only 19.1% (21/110) of the blastomeres were positive for the SRY gene, whereas in the control group, SRY was detected in 54.5% (61/112) of the cases. In addition, in the cytochalasin B-treated group, 1.9% (7/376) of the embryos expressed GFP, as indicated by green fluorescence; this percentage was 35.4% (146/412) in the control embryos. The frequency of 2-cell and blastocyst-stage embryos was similar between the control and cytochalasin B-treated groups (84.1 vs 81.0%; and 11.6 vs 12.5%, respectively). The results indicate that the presence of cytochalasin B during fertilization effectively blocks the incorporation of the male nucleus into the newly formed zygote without affecting the developmental potential of the pre-implantation embryo.


Reproduction ◽  
2006 ◽  
Vol 131 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Mark G Larman ◽  
Courtney B Sheehan ◽  
David K Gardner

Despite the success of embryo cyropreservation, routine oocyte freezing has proved elusive with only around 200 children born since the first reported birth in 1986. The reason for the poor efficiency is unclear, but evidence of zona pellucida hardening following oocyte freezing indicates that current protocols affect oocyte physiology. Here we report that two cryoprotectants commonly used in vitrification procedures, dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in intracellular calcium concentration in mouse metaphase II (MII) oocytes comparable to the initial increase triggered at fertilization. Removal of extracellular calcium from the medium failed to affect the response exacted by DMSO challenge, but significantly reduced the ethylene glycol-induced calcium increase. These results suggest that the source of the DMSO-induced calcium increase is solely from the internal calcium pool, as opposed to ethylene glycol that causes an influx of calcium across the plasma membrane from the external medium. By carrying out vitrification in calcium-free media, it was found that zona hardening is significantly reduced and subsequent fertilization and development to the two-cell stage significantly increased. Furthermore, such calcium-free treatment appears not to affect the embryo adversely, as shown by development rates to the blastocyst stage and cell number/allocation. Since zona hardening is one of the early activation events normally triggered by the sperm-induced calcium increases observed at fertilization, it is possible that other processes are negatively affected by the calcium rise caused by cryoprotectants used during oocyte freezing, which might explain the current poor efficiency of this technique.


Development ◽  
1980 ◽  
Vol 55 (1) ◽  
pp. 343-354
Author(s):  
J. R. Whittaker

This research shows that myoplasmic crescent material of the ascidian egg has both functional autonomy and functional specificity in establishing the differentiation pathway of muscle lineage cells. The cytoplasmic segregation pattern in eggs of Styela plicata was altered by compression of the embryos during third cleavage. This caused a meridional division instead of the normal equatorial third cleavage; first and second cleavages are meridional. Since eggs of S. plicata have a pronounced yellow myoplasmic crescent, one observes directly that third cleavage under compression resulted in a flat 8-cell stage with four cells containing yellow myoplasm instead of the two myoplasm-containing cells that would be formed by normal equatorial division at third cleavage. If such altered 8-cell-stage embryos were released from compression and kept from undergoing further divisions by continuous treatment with cytochalasin B, some embryos eventually developed histospecific acetylcholinesterase in three and four cells instead of in just the two muscle lineage cells found in cleavage-arrested normal 8-cell stages. The wider myoplasmic distribution effected by altering the division plane at third cleavage apparently caused a change in developmental fate of the extra cells receiving myoplasm. This meridional third cleavage also resulted in a changed nuclear lineage pattern. Two nuclei that would ordinarily be in ectodermal lineage cells after third cleavage were now associated with yellow myoplasm. Acetylcholinesterase development in these cells demonstrates that nuclear lineages are not responsible for muscle acetylcholinesterase development in the ascidian embryo.


1992 ◽  
Vol 282 (2) ◽  
pp. 393-397 ◽  
Author(s):  
J Norgauer ◽  
M Eberle ◽  
H D Lemke ◽  
K Aktories

In human neutrophils, mastoparan induced rapid F-actin polymerization which was followed by a slow and sustained depolymerization to below the initial F-actin content. Incubation of neutrophils with pertussis toxin inhibited mastoparan-stimulated actin polymerization; however it did not prevent sustained depolymerization of F-actin. Analyses of phospholipids performed in parallel revealed that mastoparan stimulated rapid formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and consumption of phosphatidylinositol 4,5-bisphosphate (PIP2). Pertussis toxin treatment blocked mastoparan-induced formation of PIP3. Furthermore, mastoparan stimulated the release of N-acetylglucosaminidase from primary granules. Cytochalasin B enhanced mastoparan-stimulated secretion. Mastoparan triggered superoxide radical production in a cytochalasin B-sensitive manner and induced complement type 3 receptor (CR3) up-regulation.


Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2310
Author(s):  
Marina Di Domenico ◽  
Melanie Jokwitz ◽  
Walter Witke ◽  
Pietro Pilo Boyl

Profilin functions have been discussed in numerous cellular processes, including actin polymerization. One puzzling aspect is the concomitant expression of more than one profilin isoform in most tissues. In neuronal precursors and in neurons, profilin 1 and profilin 2 are co-expressed, but their specific and redundant functions in brain morphogenesis are still unclear. Using a conditional knockout mouse model to inactivate both profilins in the developing CNS, we found that threshold levels of profilin are necessary for the maintenance of the neuronal stem-cell compartment and the generation of the differentiated neurons, irrespective of the specific isoform. During embryonic development, profilin 1 is more abundant than profilin 2; consequently, modulation of profilin 1 levels resulted in a more severe phenotype than depletion of profilin 2. Interestingly, the relevance of the isoforms was reversed in the postnatal brain. Morphology of mature neurons showed a stronger dependence on profilin 2, since this is the predominant isoform in neurons. Our data highlight redundant functions of profilins in neuronal precursor expansion and differentiation, as well as in the maintenance of pyramidal neuron dendritic arborization. The specific profilin isoform is less relevant; however, a threshold profilin level is essential. We propose that the common activity of profilin 1 and profilin 2 in actin dynamics is responsible for the observed compensatory effects.


Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 187-193 ◽  
Author(s):  
So Gun Hong ◽  
Goo Jang ◽  
Hyun Ju Oh ◽  
Ok Jae Koo ◽  
Jung Eun Park ◽  
...  

SummaryBrain-derived neurotrophic factor (BDNF) signalling via tyrosine kinase B receptors may play an important role in ovarian development and function. It has been reported that metformin elevates the activity of Tyrosine kinase receptors and may amplify BDNF signalling. The objective of this study was to investigate the effect of BDNF during in vitro maturation (IVM) and/or in vitro culture (IVC) (Experiment 1), and to evaluate the collaborative effect of BDNF and metformin treatment on the developmental competence of bovine in vitro fertilized (IVF) embryos (Experiment 2). In Experiment 1, BDNF, which was added to our previously established IVM systems, significantly increased the proportions of MII oocytes at both 10 ng/ml (86.7%) and 100 ng/ml (85.4%) compared with the control (64.0%). However, there was no statistically significant difference in blastocyst development between the control or BDNF-supplemented groups. In Experiment 2, in order to investigate the effect of BDNF (10 ng/ml) and/or metformin (10−5 M) per se, TCM-199 without serum and hormones was used as the control IVM medium. The BDNF (48.3%) and BDNF plus metformin (56.5%) significantly enhanced the proportions of MII oocytes compared with the control (34.4%). Although, BDNF or metformin alone had no effect in embryo development, BDNF plus metformin significantly improved early embryo development to the 8–16-cell stage compared with the control (16.5 vs. 5.5%). In conclusion, the combination of BDNF and metformin may have a collaborative effect during the IVM period. These results could further contribute to the establishment of a more efficient bovine in vitro embryo production system.


1996 ◽  
Vol 109 (13) ◽  
pp. 3089-3102 ◽  
Author(s):  
A. Paoletti ◽  
M. Moudjou ◽  
M. Paintrand ◽  
J.L. Salisbury ◽  
M. Bornens

Centrin is a member of the calcium-binding EF-hand protein superfamily present in centrosomes of widely divergent species. Investigating the cellular distribution of human centrin by both immunofluorescence and cell fractionation, we report that centrin is biochemically complex in human cells, displaying as much as ten isoforms in 2-D electrophoresis. This suggests that centrin may be subject to multiple regulations. Strikingly, more than 90% of centrin is not associated with the centrosome fraction. The centrosome-associated centrin, however, displays a specific pattern in 2-D electrophoresis and is concentrated within the distal lumen of the centrioles, where a complex structure has been previously described. This precise localization allows the resolution of centrioles at the optical level throughout the cell cycle and provides a valuable tool for monitoring centriole duplication. To get insights on centrin function, we performed injection experiments of recombinant heterologous centrin in two-cell stage frog embryos in an attempt to produce dominant negative effects. We report that green algae and human centrin delay cleavage and promote the formation of abnormal blastomeres in which the distribution of microtubule asters and of nuclei is dramatically impaired. This suggests that centrin could be involved in the centrosome reproduction cycle, in the coordination of cytoplasmic and nuclear division or in cytokinesis.


Development ◽  
1990 ◽  
Vol 110 (4) ◽  
pp. 1121-1132
Author(s):  
M.H. Kaufman ◽  
S. Webb

Despite the fact that a variety of experimental techniques have been devised over the years to induce tetraploid mammalian embryonic development, success rates to date have been limited. Apart from the early study by Snow, who obtained development to term of a limited number of cytochalasin B-induced tetraploid mouse embryos, no other researchers have achieved development of tetraploid embryos beyond the early postimplantation period. We now report advanced postimplantation development of tetraploid mouse embryos following electrofusion of blastomeres at the 2-cell stage, and subsequent transfer of these 1-cell ‘fused’ embryos to appropriate recipients. Cytogenetic analysis of the extraembryonic membranes of all of the postimplantation embryos encountered in the present study has provided an unequivocal means of confirming their tetraploid chromosome constitution. A preliminary morphological and histological analysis of the tetraploid embryos obtained by this technique has revealed that characteristic craniofacial abnormalities particularly involving the forebrain and eyes were consistently observed, and these features were often associated with abnormalities of the vertebral axis and heart. The most advanced viable embryo in this series was recovered on the 15th day of gestation, and its morphological features suggest that it was developmentally equivalent to a normal embryo of about 13.5-14 days p.c.


2019 ◽  
Vol 34 (8) ◽  
pp. 1439-1449 ◽  
Author(s):  
J Barberet ◽  
C Bruno ◽  
E Valot ◽  
C Antunes-Nunes ◽  
L Jonval ◽  
...  

AbstractSTUDY QUESTIONCan time-lapse imaging systems make it possible to identify novel early non-invasive biomarkers to predict live birth?SUMMARY ANSWERFrom mostly high-grade embryos, out of 35 morphometric, morphologic and morphokinetic variables, only pronuclei (PN) position at time of PN juxtaposition and the absence of multinucleated blastomeres at the 2-cell stage (MNB2cell), were potentially associated with live birth.WHAT IS KNOWN ALREADYPrevious studies indicate that some kinetic markers may be predictive of blastocyst development and embryonic implantation. Certain teams have suggested including some of them in decisional algorithms for embryo transfers.STUDY DESIGN, SIZE, DURATIONUsing a time-lapse incubator (EmbryoScope, Unisense FertiliTech), we retrospectively explored the associations between the morphometric, morphologic and morphokinetic parameters of oocytes, zygotes and embryos, and their associations with live birth. This study assessed 232 embryos from single embryo transfers after ICSI cycles performed between January 2014 and December 2017.PARTICIPANTS/MATERIALS, SETTING, METHODSThe morphometric, morphologic and morphokinetic parameters (18, 4 and 13, respectively) of oocytes, zygotes and early embryos were studied retrospectively. The associations between these parameters were examined using a Spearman’s correlation, Mann–Whitney or chi-squared test as appropriate. We examined whether these parameters were associated with outcomes in univariate and multivariate logistic regression analyses.MAIN RESULTS AND THE ROLE OF CHANCECentral PN juxtaposition was associated with a 2-fold increase in the odds of live birth (OR = 2.20; 95% CI, [1.26–3.89]; P = 0.006), while the presence of MNB2cell was associated with half the odds of live birth (OR = 0.51; 95% CI, [0.27–0.95]; P = 0.035). These two parameters were independent of embryo kinetics. The 33 remaining parameters had no significant association with the capacity of transferred embryos to develop to term.LIMITATIONS, REASONS FOR CAUTIONEven though the population size was relatively small, our analyses were based on homogeneous cycles, i.e. young women whose transferred embryos were found to be high-grade according to conventional morphology evaluation. In addition, our conclusions were established from a specific, highly selected population, so other study populations, such as women in an older age bracket, may yield different results. Finally, because we assessed day 2/3 transfers, our findings cannot be generalized to embryos cultured up to the blastocyst stage.WIDER IMPLICATIONS OF THE FINDINGSIt would be interesting to explore, prospectively, whether PN localisation is a relevant measure to predict embryo development when added into further algorithms and whether this parameter could be suitable for use in other IVF clinics. Further studies are needed, notably to explore the added value of timing evaluation in cohorts of embryos with low or intermediate morphology grade, as well as in other maternal populations (i.e. older women).STUDY FUNDING/COMPETING INTEREST(S)No external funding was used for this study. P. Sagot received funding from the following commercial companies: Merck Serono, Finox Biotech, Ferring, MSD France SAS, Teva Sante ́ SAS, Allergan France, Gedeon Richter France, Effik S.A., Karl Storz Endoscopie France, GE Medical Systems SCS, Laboratoires Genevrier, H.A.C. Pharma and Ipsen.All the authors confirm that none of this funding was used to support the research in this study. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the journal policies on sharing data and materials.


2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.


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