Cell movement and adhesion in the developing chick wing bud: studies on cultured mesenchyme cells from normal and talpid mutant embryos

1975 ◽  
Vol 18 (2) ◽  
pp. 301-313 ◽  
Author(s):  
D.A. Ede ◽  
O.P. Flint
Keyword(s):  
Cell Morphology ◽  
Average Distance ◽  
Cell Movement ◽  
Time Lapse ◽  
Limb Bud ◽  
Cell Periphery ◽  
Significant Difference ◽  
Two Parameters ◽  
Mutant Cells

Mesenchyme fragments from early wing buds of normal and talpid3 mutant chick embryos were explanted for culture in plastic Petri dishes and the behaviour of individual cells as they moved out on to the plastic surface was studied by time-lapse cine photography, followed by statistical analysis. Two parameters of cell movement were recorded: (1) the distances moved over measured 100-s intervals and (2) the length of time each cell spent at rest before moving on. The average speed of movement over the whole path tracked for each cell, inclusive of time at rest, was significantly greater in normal than talpid3 cells. There was no significant difference between normal and mutant cells in the average distance mover per 100-s step, equivalent to the speed over the whole path exclusive of time at rest, but the percentage of time spent at rest was significantly less in normal than in talpid3 cells. This difference appears to be related to a difference in cell morphology, since it was observed that the mutant cells were more flattened than normals, with very extensive ruffled membranes and short spiky microvilli all round the cell periphery. The relation of these differences in cell morphology and behaviour in vitro to the production of the characteristically fan-shaped limb bud outgrowth and altered pattern of cartilage elements in the developing mutant limb bud is discussed.

Time-lapse Phase-contrast Microphotography of Cell Populations as a Basis for Improvement of In Vitro Toxicity Assessment

1992 ◽  
Vol 20 (2) ◽  
pp. 302-306
Author(s):  
Miroslav Červinka
Keyword(s):  
Cell Proliferation ◽  
Cell Morphology ◽  
Video Recording ◽  
Toxicity Testing ◽  
Time Lapse ◽  
Time Dependent ◽  
In Vitro Toxicity ◽  
Simple Modification ◽  
Pertinent Data

Recent trends in the field of in vitro toxicology have centred around the validation of in vitro methods. The ultimate goal is to obtain pertinent data with the minimum of effort. In our laboratory, we have used toxicological methods based on the evaluation of cell morphology and cell proliferation. A method suitable for this purpose is time-lapse microcinematographic (or video) recording of cellular changes, which we used for many years. For practical in vitro toxicity testing, however, this method is far too complicated. Therefore, we have tried to develop a simple modification for the evaluation of cell morphology and cell proliferation, which would still allow for a basic time-dependent analysis. Comparison of detailed microcinematographic analysis with analysis according to our new proliferation assay is demonstrated with cisplatin as the toxicant. We believe that a time-dependent approach could improve the in vitro assessment of toxicity.

scholarly journals An In Vitro Model for Assessing Corneal Keratocyte Spreading and Migration on Aligned Fibrillar Collagen

2018 ◽  
Vol 9 (4) ◽  
pp. 54 ◽  
Author(s):  
Pouriska Kivanany ◽  
Kyle Grose ◽  
Nihan Yonet-Tanyeri ◽  
Sujal Manohar ◽  
Yukta Sunkara ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Cell Movement ◽  
Time Lapse ◽  
Collagen Fibrils ◽  
Fibrillar Collagen ◽  
Freeze Injury

Background: Corneal stromal cells (keratocytes) are responsible for developing and maintaining normal corneal structure and transparency, and for repairing the tissue after injury. Corneal keratocytes reside between highly aligned collagen lamellae in vivo. In addition to growth factors and other soluble biochemical factors, feedback from the extracellular matrix (ECM) itself has been shown to modulate corneal keratocyte behavior. Methods: In this study, we fabricate aligned collagen substrates using a microfluidics approach and assess their impact on corneal keratocyte morphology, cytoskeletal organization, and patterning after stimulation with platelet derived growth factor (PDGF) or transforming growth factor beta 1 (TGFβ). We also use time-lapse imaging to visualize the dynamic interactions between cells and fibrillar collagen during wound repopulation following an in vitro freeze injury. Results: Significant co-alignment between keratocytes and aligned collagen fibrils was detected, and the degree of cell/ECM co-alignment further increased in the presence of PDGF or TGFβ. Freeze injury produced an area of cell death without disrupting the collagen. High magnification, time-lapse differential interference contrast (DIC) imaging allowed cell movement and subcellular interactions with the underlying collagen fibrils to be directly visualized. Conclusions: With continued development, this experimental model could be an important tool for accessing how the integration of multiple biophysical and biochemical signals regulate corneal keratocyte differentiation.

Study of Morphokinetics in Day 3 Embryo with Implantation Potential and Effect of Sperm Cryopreservation on Embryogenesis

2017 ◽  
Vol 8 (2) ◽  
pp. 61-67
Author(s):  
Harsha K Bhadarka ◽  
Nayana H Patel ◽  
Kruti B Patel ◽  
Nilofar R Sodagar ◽  
Yuvraj D Jadeja ◽  
...  
Keyword(s):  
Time Lapse ◽  
Mean Value ◽  
Primary Objective ◽  
Sperm Cryopreservation ◽  
Cell Stage ◽  
Frozen Semen ◽  
Significant Difference ◽  
Cryopreserved Sperm ◽  
Day 3 Embryo

ABSTRACT Aim In recent past, many studies had come up with the combination of time-lapse (TL) imaging of embryo morphokinetics as a noninvasive means for improving embryo selection and in vitro fertilization (IVF) success. The primary objective of the study was to find out if there is significant variation in morphokinetics of embryos with different implantation potential and also to study the effect of sperm freezing on time points of embryogenesis events in embryos with implantation potential. Materials and methods Kinetic data and cycle outcomes were analyzed retrospectively in 142 patients who had undergone IVF/intracytoplasmic sperm injection (ICSI) cycles using semen with normal parameters and embryo transfer (ET) on day 3. For the surety of specificity of morphokinetics, only cases with single ET cycles were included in the study. Timing of specific events, from the point of ICSI, was determined using TL imaging. Kinetic markers like time to syngamy (t-pnf), t2, time to two cells (c), 3c (t3), 4c (t4), 5c (t5), 8c (t8), tMor, CC2, CC3, t5–t2, t5–t4, s1, s2, and s3 were calculated. The cleavage synchronicity from the 2–8 cell stage (CS2–8), from 4 to 8 cell stage (CS4–8), and from 2 to 4 cell stage (CS2–4) were calculated as defined elsewhere. Deoxyribonucleic acid replication time ratio (DR) was also included in the comparison. Analysis of variance test was used for comparison of the mean timing of cell division and cell cycle intervals. Results Morphokinetics t-pnf, t2, t8, CC2, S2, S3, CS2–8, CS4–8, and CS2–4 differed significantly between embryos with and without implantation potential, when embryos were developed using fresh semen, while t3, t4, t5, CC2, S2, t5–t2, CS2–4, and DR differed significantly between the embryos with and without implantation potential when frozen semen was used. No significant difference was found in mean value of any of the above-stated parameters when comparison was done between implanted embryos fertilized by either fresh or cryopreserved sperm. Conclusion Many morphokinetics parameters of embryogene­sis vary significantly between embryos with different ability to implant; therefore, the criteria developed in our IVF lab can be useful for selection of suitable embryo even at day 3 of development with more chances of implantation. Clinical significance Study indicates necessity of development of individualized selection model based on morphokinetics for every IVF lab and also confirms freezing as an important tool for fertility preservation of males as it does not affect events of embryogenesis. How to cite this article Bhadarka HK, Patel NH, Patel KB, Sodagar NR, Jadeja YD, Patel NH, Patel MN, Patel AV, Patel DH, Patel JS. Study of Morphokinetics in Day 3 Embryo with Implantation Potential and Effect of Sperm Cryopreservation on Embryogenesis. Int J Infertil Fetal Med 2017;8(2):61-67.

scholarly journals Virus-Induced Cell Motility

1998 ◽  
Vol 72 (2) ◽  
pp. 1235-1243 ◽  
Author(s):  
Christopher M. Sanderson ◽  
Michael Way ◽  
Geoffrey L. Smith
Keyword(s):  
Cell Migration ◽  
Cell Motility ◽  
Uv Light ◽  
Cell Metabolism ◽  
Cell Movement ◽  
Time Lapse ◽  
Late Gene Expression ◽  
Infected Cells ◽  
And Function

ABSTRACT Many viruses induce profound changes in cell metabolism and function. Here we show that vaccinia virus induces two distinct forms of cell movement. Virus-induced cell migration was demonstrated by an in vitro wound healing assay in which infected cells migrated independently into the wound area while uninfected cells remained relatively static. Time-lapse microscopy showed that the maximal rate of migration occurred between 9 and 12 h postinfection. Virus-induced cell migration was inhibited by preinactivation of viral particles with trioxsalen and UV light or by the addition of cycloheximide but not by addition of cytosine arabinoside or rifampin. The expression of early viral genes is therefore necessary and sufficient to induce cell migration. Following migration, infected cells developed projections up to 160 μm in length which had growth-cone-like structures and were frequently branched. Time-lapse video microscopy showed that these projections were formed by extension and condensation of lamellipodia from the cell body. Formation of extensions was dependent on late gene expression but not the production of intracellular enveloped (IEV) particles. The requirements for virus-induced cell migration and for the formation of extensions therefore differ from each other and are distinct from the polymerization of actin tails on IEV particles. These data show that poxviruses encode genes which control different aspects of cell motility and thus represent a useful model system to study and dissect cell movement.

278 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND SUPERSTIMULATION ON THE NORMALITY OF BOVINE EMBRYOS PRODUCED IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 296 ◽  
Author(s):  
K. Imai ◽  
T. Somfai ◽  
M. Ohtake ◽  
Y. Inaba ◽  
Y. Aikawa ◽  
...  
Keyword(s):  
Cell Division ◽  
Development Program ◽  
Time Lapse ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Cell Stage ◽  
Follicular Wave ◽  
Significant Difference

We previously reported that follicular wave synchronization by dominant follicle removal on Day 5 and the start of a superstimulatory treatment on Day 7 after ovum pick-up (OPU) was effective to increase oocyte quality (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). The present study was designed to examine the effect of superstimulatory treatment-induced follicular wave synchronization on quality of embryos obtained by OPU and in vitro production. Japanese Black cows were reared under the same feeding and environmental conditions and 2 OPU sessions were conducted in each cow. The first OPU session was performed in 7 cows at arbitrary days of estrous cycle using a 7.5-MHz linear transducer with needle connected to an ultrasound scanner. Then, follicles larger than 8 mm in diameter were aspirated and CIDR was inserted on Day 5 (the day of first OPU session = Day 0). The cows then received 30 mg of FSH twice a day from Days 7 to 10 in decreasing doses (4, 4, 3, 3, 2, 2, 1, 1 mg per shot) by i.m. injections. Cloprostenol (PGF; 0.75 mg) was administered in the morning of Day 9. The second OPU session was performed 48 h after PGF administration (Day 11) and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Grade 1 and 2 cumulus oocyte complexes were in vitro matured, fertilized (IVF), and cultured as described by Imai et al. (2006 J. Reprod. Dev. 52, Suppl. S19-29). Some zygotes were fixed and stained to check their sperm penetration. Embryo development was monitored by time-lapse cinematography for 168 h after IVF. Cleavage pattern of embryos was classified morphologically into normal and abnormal (including those with multiple fragments, protrusions, 3 to 4 blastomeres, and uneven cell division) groups at their first cleavage. Normal penetration rate of second OPU session was significantly (P < 0.05) higher than that of the first OPU session. There were no differences in the mean percentage of total blastocyst and grade 1 blastocyst rates between the first (45.2 and 46.9%, respectively) and second (47.5 and 41.8%, respectively) OPU sessions. However, the rates of blastocysts developing from embryos that were beyond the 4-cell stage at 48 h after IVF was significantly (P < 0.05) higher after the second OPU session (81.2%) than after the first OPU session (67.4%). Furthermore, a significant difference (P < 0.05) was found in the rates of normal cleavage at the first cell division in embryos that developed to the blastocyst stage between the first and second OPU sessions (53.3% and 73.9%, respectively). These results indicate that superstimulatory treatment-induced follicular wave synchronization improved the normality of fertilization and development of cattle oocytes obtained by OPU. This work was supported by the Research and Development Program for New Bio-industry Initiatives.

153 Fertilisation of Cattle Oocytes is Linked to Novel Waves of a Ca2+ Fluorophore in Cumulus Cells

2018 ◽  
Vol 30 (1) ◽  
pp. 216
Author(s):  
H. J. McLennan ◽  
M. L. Sutton-McDowall ◽  
S. Heng ◽  
J. G. Thompson
Keyword(s):  
Xenopus Oocytes ◽  
Cumulus Cells ◽  
Time Lapse ◽  
Molecular Probes ◽  
Vitelline Membrane ◽  
Oocyte Activation ◽  
Sperm Binding ◽  
Significant Difference ◽  
The Media

During fertilization, multiple intracellular calcium (Ca2+) oscillations are initiated after sperm binding to the oocyte vitelline membrane. This Ca2+ signalling has been extensively studied in denuded mouse and Xenopus oocytes but minimally studied in larger mammals. Cows in particular are unusual, as the few studies on oocyte activation have observed fewer Ca2+ oscillations during fertilisation compared with mice. Furthermore, cattle intracytoplasmic sperm injection (ICSI) is inefficient, despite parthenogenetic activation occurring readily. We hypothesise that cumulus cells are important for cattle oocyte activation at fertilisation. Here, we assessed the behaviour of Ca2+oscillations in fertilising intact cattle cumulus–oocyte complexes (COC). Abattoir-derived cattle COC were matured and fertilised in vitro using Bovine Vitro Media Suite (IVF Vet Solutions). The COC were stained 3.5 h after insemination with the Ca2+ fluorescent probe Fluo4AM (5 μM, Molecular Probes Inc., Eugene, OR, USA) for 30 min, washed, and imaged every 5 min for 6 h in a Fluoview FV10i incubating time-lapse confocal microscope (Olympus) before being returned to culture. Embryo development was assessed at Day 8 to confirm fertilisation. Fluo4AM fluorescence intensity was assessed using FIJI ImageJ. Mean relative intensity over time was graphed for specific regions of interest and the area under graphs was calculated to quantify differences for comparison using a Mann-Whitney Test (mean ± SEM). Experiment 1 (4 reps of 10 COC) compared confirmed fertilised v. uninseminated; experiment 2 (2 reps of 10 COC) compared inseminated COC ± 10 μM BAPTA-AM (Ca2+ chelator, Sigma-Aldrich, St. Louis, MO, USA). There were distinct coordinated waves of differing Fluo4AM intensity in both the oocyte and the cumulus cells surrounding the confirmed fertilised oocytes. This contrasted to the random uncoordinated flashes of Fluo4AM fluorescence in the cumulus cells of the uninseminated oocytes. The fluorescence pattern in +BAPTA-AM COC matched the random flashes observed in the uninseminated group of experiment 1. The fluorescence in the media surrounding the COC immediately following the Fluo4AM waves spiked and then plateaued at a higher level of fluorescence. This was quantified by assessing the area under the graph for 1 h of the plateau following the fluorescence spike. There were no differences between confirmed fertilised (346.4 ± 41.62) and uninseminated groups (239.8 ± 32.08; P > 0.05), but this was affected by differences in cumulus dispersal due to the presence or absence of sperm. Experiment 2 used BAPTA-AM to block oocyte activation with sperm present in both groups and showed a significant difference between the fluorescence increase in the media of the 2 groups (–BAPTA-AM: 311.2 ± 31.57, +BAPTA-AM: 201.4 ± 26.59; P < 0.03). Although the physiological significance has yet to be determined, we have observed a novel Ca2+ wave in the cumulus cells that could be linked to oocyte activation in cattle. There was a significant increase in Fluo4AM fluorescence in the media surrounding the COC, which may indicate cumulus cells are releasing Ca2+ at the time of oocyte activation.

Regional differences in the morphology and motility of mesodermal cells from the early wing-bud of normal and talpid3 mutant chick embryos

Development ◽  
1978 ◽  
Vol 48 (1) ◽  
pp. 185-203
Author(s):  
D. A. Bell ◽  
D. A. Ede
Keyword(s):  
Regional Differences ◽  
Time Lapse ◽  
Distal Region ◽  
Limb Bud ◽  
Scanning Electron Micrographs ◽  
Normal Cells ◽  
Wing Bud ◽  
Scanning Electron

A method of culturing has been employed to compare the properties of cells migrating from small mesodermal explants taken from different regions of normal and mutant limb-buds at different stages of development. An analysis by time-lapse cinematography of the morphology and mobility of cells migrating from explants defines a distal region within the limb-bud where these properties are distinct from those of cells from more proximal regions. In the normal wing-bud distal cells subjacent to the apical ectodermal ridge possess a characteristic multipolar morphology and translocate slowly in vitro. Cells from more proximal regions tend to be bipolar and translocate more rapidly. Distal and proximal cells also probably differ in their adhesive strengths. In the mutant, talpid3, distal and proximal cells do not differ in the above properties and cells from all regions of the limb-bud are multipolar, translocate slowly and are more adhesive than normal cells. A study of light micrographs and scanning electron micrographs suggests that these regional differences are found in the limb-bud in vivo and are not merely an effect produced by the in vitro culturing system.

Cell interactions in the developing somite: in vitro comparisons between amputated (am/am) and normal mouse embryos

Development ◽  
1982 ◽  
Vol 67 (1) ◽  
pp. 113-125
Author(s):  
O. P. Flint ◽  
D. A. Ede
Keyword(s):  
Limb Development ◽  
Mutant Mouse ◽  
Time Lapse ◽  
Cell Contact ◽  
Adhesive Properties ◽  
Cell Behaviour ◽  
Homozygous Mutant Mouse ◽  
Mutant Cells

Facial, axial and limb development are all abnormal in the homozygous mutant mouse embryo (amputated). An interpretation of cell behaviour in vivo based on sectioned material which may explain these abnormalities has been previously suggested. In this study, somite cells cultured in vitro were found to behave exactly as predicted in this interpretation: they clump together, forming extensive areas of cell contact, and this has a profound effect on their mobility as measured by time-lapse cinemicrography. The similarity of cell behaviour in vitro and in vivo under two distinct sets of environmental conditions suggests that the abnormal cell behaviour is intrinsic to the cell, and directly linked to the mutation. The more extensive areas of cell contact formed between mutant cells suggests that the mutation changes the adhesive properties of the cell surface, but it cannot be excluded that the cells' motile apparatus is also affected.

P–651 Strict embryo-endometrial synchrony does not contribute to the successful pregnancy during vitrified-warmed embryo transfer with hormone replacement cycles

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
T Takahashi ◽  
K Ota
Keyword(s):  
Embryo Transfer ◽  
Endometrial Thickness ◽  
Hormone Replacement ◽  
Time Lapse ◽  
Successful Pregnancy ◽  
Logistic Analysis ◽  
Significant Difference ◽  
The Mean ◽  
Multivariate Logistic Analysis

Abstract Study question Does strict embryo-endometrium synchronization relate to pregnancy during vitrified-warmed embryo transfer (ET) with hormone replacement (HRT) cycles? Summary answer A 12-hour delay in the embryo-endometrial synchrony was acceptable, and this delay was not an independent predictor of pregnancy during vitrified-warmed ET with HRT cycles. What is known already Embryo-endometrium synchrony is considered to be necessary for successful pregnancy in both fresh and frozen-thawed cycles. Until now, the date of ET has been determined by the synchronization of the embryo developmental stage and the endometrium on a daily basis. To date, with the advent of the time-lapse incubator, it is possible to observe the embryo development from fertilization over time and to calculate the exact time from fertilization of the transferred embryo. However, there are very few studies on the extent to which increases the accuracy of synchronization between embryo and endometrium contributes to a successful pregnancy. Study design, size, duration This retrospective cohort study included 319 consecutive cycles during vitrified-warmed ET with HRT between August 2016 and August 2018. This study was conducted in an academically affiliated private practice. Participants/materials, setting, methods We analyzed 319 vitrified-warmed single-blastocyst transfer cycles. All frozen expanded blastocysts were inseminated by intracytoplasmic sperm injection (ICSI) and cultured in a time-lapse incubator. We calculated time for the in vitro culture of the embryo after ICSI (t1) and time for progesterone-priming (t2) up to ET. The difference between t1 and t2 (delta-t) was used as an indicator of embryo-endometrium synchrony. We examined the relationship between delta-t and treatment outcomes using multivariate logistic analysis. Main results and the role of chance The mean patient’s age at oocyte retrieval was 35.7 (SD 4.3). The number of pregnant cycles was 157 in all treatment cycles (pregnancy rate, 49.2%). The mean value of delta-t was 9.9 h (SD 2.6) in all cycles. There was no significant difference of delta-t in pregnant (mean, SD: 10.0 h, 2.8 h) and non-pregnant cycles (mean, SD: 10.0 h, 2.3 h). Treatment cycles were classified according to the quartile of delta-t, and we examined the percentages of pregnant cycles in each group. There were no significant differences in pregnancy rates among the groups (p = 0.75). On multivariate logistic analysis, patient’s age (adjusted odds ratio [aOR]: 0.94, 95% confidence interval [CI]: 0.89–0.99), previous treatment cycles (aOR: 0.74, 95% CI: 0.56–0.99), endometrial thickness at ET (aOR: 1.19, 1.04–1.36), and good quality blastocysts (&gt;3BB according to Gardner’s classification) at vitrification (aOR: 2.49, 95% CI: 1.23–5.05) were independent predictive factors for pregnancy. On the other hand, delta-t did not contribute to pregnancy (aOR: 1.00, 95% CI: 0.99–1.00). Limitations, reasons for caution We did not examine the effects of embryo-endometrium synchrony during vitrified-warmed ET in a natural cycle. Therefore, careful interpretation of the significance of embryo-endometrium synchrony during the vitrified-warmed ET should be taken. Wider implications of the findings: We showed the embryo-endometrium synchrony did not contribute to the pregnancy during vitrified-warmed ET with HRT cycles. These results cast doubt on the existence of an optimal implantation window by changing the timing of ET with the results of gene expression testing of the endometrium. Trial registration number Not applicable

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