Effects of cytochalasin B on meiosis and development of fertilized and activated eggs of Sabellaria alveolata L. (Polychaete Annelid)

Development ◽  
1974 ◽  
Vol 31 (1) ◽  
pp. 61-74
Author(s):  
G. Peaucellier ◽  
P. Guerrier ◽  
J. Bergerard
Keyword(s):  
Cytochalasin B ◽  
Polar Body ◽  
Polar Lobe ◽  
Developmental Abnormalities ◽  
Drug Concentrations ◽  
Internal Mechanism ◽  
Polar Body Extrusion

1. Unfertilized, fertilized and activated eggs of Sabellaria alveolata were submitted to cytochalasin B concentrations ranging from 0·1 to 20 μg/ml. Their behaviour was studied either in vivo or in acetocarmine squash preparations. 2. Polar body extrusion, cytokinesis and polar lobe formation are completely inhibited by cytochalasin B concentrations as low as 0·3–0·5 μg/ml. 3. Caryotype determinations demonstrate that chromosomal meiotic and mitotic processes are not affected by the drug. Thus, polyploid embryos usually developed from fertilized eggs whilst they did not from activated ones. This is related to the contrasting behaviour of meiotic and cleavage centres. While the latter duplicates at each cycle, the former cannot replicate at the end of meiosis. This leads to an abortive monastral stage even if inhibition of polar body extrusion has provided the egg with two or four centres. These observations suggest the existence of an internal mechanism regulating the number of effective centrioles at the end of meiosis. They demonstrate also that the main cause of developmental failure in activated eggs cannot be related to ploidy. 4. Eggs treated throughout meiosis with moderate drug concentrations developed into swimming larvae. However, frequent developmental abnormalities affecting lobe dependent structures were obtained even if polar lobe formation was unimpaired. This suggests either that cytochalasin B has irreversibly affected some decisive cortical element or that previously described activating processes, which begin with polar lobe formation, are actually exerted on specific materials segregated during meiosis.

scholarly journals Intra-cytoplasmic sperm injection in a marsupial

Reproduction ◽  
2004 ◽  
Vol 128 (5) ◽  
pp. 595-605 ◽  
Author(s):  
Nadine M Richings ◽  
Geoffrey Shaw ◽  
Peter D Temple-Smith ◽  
Marilyn B Renfree
Keyword(s):  
Cell Adhesion ◽  
Polar Body ◽  
Tammar Wallaby ◽  
Mature Oocyte ◽  
Cell Stage ◽  
Intra Cytoplasmic Sperm Injection ◽  
Polar Body Extrusion ◽  
Cell Cell

Here we report the first use of intra-cytoplasmic sperm injection (ICSI) in a marsupial, the tammar wallaby (Macropus eugenii ), to achieve in vitro fertilization and cleavage. A single epididymal spermatozoon was injected into the cytoplasm of each mature oocyte collected from Graafian follicles or from the oviduct within hours of ovulation. The day after sperm injection, oocytes were assessed for the presence of pronuclei and polar body extrusion and in vitro development was monitored for up to 4 days. After ICSI, three of four (75%) follicular and four of eight (50%) tubal oocytes underwent cleavage. The cleavage pattern was similar to that previously reported for in vivo fertilized oocytes placed in culture, where development also halted at the 4- to 8-cell stage. One-third of injected oocytes completed the second cleavage division, but only a single embryo reached the 8-cell stage. The success of ICSI in the tammar wallaby provided an opportunity to examine the influence of the mucoid coat that is deposited around oocytes passing through the oviduct after fertilization. The presence of a mucoid coat in tubal oocytes did not prevent fertilization by ICSI and the oocytes cleaved in vitro to a similar stage as follicular oocytes lacking a mucoid coat. Cell–zona and cell–cell adhesion occurred in embryos from follicular oocytes, suggesting that the mucoid coat is not essential for these processes. However, blastomeres were more closely apposed in embryos from tubal oocytes and cell–cell adhesion was more pronounced, indicating that the mucoid coat may be involved in maintaining the integrity of the conceptus during cleavage.

Development in vitro and in vivo of aggregated parthenogenetic bovine embryos

1995 ◽  
Vol 7 (5) ◽  
pp. 1073 ◽  
Author(s):  
A Boediono ◽  
S Saha ◽  
C Sumantri ◽  
T Suzuki
Keyword(s):  
Cytochalasin B ◽  
Polar Body ◽  
Cleavage Rate ◽  
Development In Vitro ◽  
Second Polar Body ◽  
Bovine Oocytes ◽  
Longitudinal Diameter ◽  
Parthenogenetic Embryos

Mature bovine oocytes were activated with 7% ethanol followed by cytochalasin B or D treatment. Most oocytes extruded a second polar body and formed one pronucleus when treated with 7% ethanol alone [35/43 (81%)]. With ethanol followed by cytochalasin B or D, overall activation frequency was 70% (309/441), with activated oocytes containing two pronuclei. The cleavage rate was not significantly different between treatment with ethanol alone and ethanol followed by 5 micrograms mL-1 cytochalasin B, but it was significantly lower than in fertilized oocytes (P < 0.01). However, the blastocyst production rate was significantly different (P < 0.01) among the treatments. The incidence of parthenogenetic embryos with normal (diploid) complements and with chromosome anomalies (2N/4N) was 68% (17/25) and 32% (8/25) respectively, and this was not affected by cryopreservation treatment. The longitudinal diameter of aggregated-four embryos cultured in vitro was greater (P < 0.01) than aggregated-two or single embryos. One of the aggregated-four parthenogenetic embryos was further cultured in vitro and developed up to Day 27 after activation, with a diameter of 2980 microns. The aggregated-four parthenogenetic embryos were transferred to five recipients. The oestrus was prolonged in three recipients and they returned to oestrus on Day 57, 62 and 67 after the previous oestrus. These results indicate that aggregating parthenogenetic embryos can prolong their survival in vitro and in vivo.

scholarly journals Postimplantation development of CB-induced triploid mouse embryos

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 81-89
Author(s):  
Anna Niemierko
Keyword(s):  
Embryo Development ◽  
Cytochalasin B ◽  
Polar Body ◽  
Mouse Embryos ◽  
Foetal Membranes ◽  
Polar Body Extrusion

By subjecting A strain eggs at the time of fertilization and polar body extrusion to 5 µg/ml cytochalasin B, digynic triploidy was produced in 80% fertilized eggs. Triplonucleate eggs were transplanted to recipients and examined between 9–1lth day of pregnancy. Development of triploid mouse embryos up to day 7 is normal and most embryos form early egg cylinder. At day 8 the embryonic part of the cylinders is under-developed and later development fails to form an embryo. Development of foetal membranes is much less affected, CB-induced triploids survive to 10th- day of pregnancy.

scholarly journals 301EFFECT OF THE MII-AGE AND ACTIVATION PROTOCOL ON THE PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 270
Author(s):  
I. Lagutina ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Cytochalasin B ◽  
Average Rate ◽  
Polar Body ◽  
Cell Number ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Average Cell ◽  
Electric Impulse ◽  
Polar Body Extrusion

The completion of porcine oocyte nuclear maturation (MII) in vitro, characterized by the time of polar body extrusion, starts at about 32h of maturation and lasts more than 12h. This leads to the simultaneous presence in the population of matured oocytes with differing abilities to be activated. We investigated age-dependent changes in pig oocyte maturation, activation and development in SOFaa in response to electric impulse (EL) in the presence of cytochalasin B (CB) and EL in combination with cycloheximide and cytochalasin B (EL+CHX+CB). Oocytes were matured in TCM 199 with 10% FCS, cysteine, LH, FSH (Pergovet, Serono, Geneva, Switzerland) for 36h and then decumulated. Matured oocytes were activated at 40 and 44h by double pulse of 30μs DC 1, 5kVcm−1 and cultured in 5μgmL−1 CB for 4h or by EL followed by incubation in 10μgmL−1 CHX+5μgmL−1 CB for 4h. According to the MII-age before activation oocytes were divided into 2 age classes: 3–7 and 7–11h after polar body extrusion. Embryos were cultured in SOFaa in 5% CO2, 5% O2 at 38.5°C. The rates of cleavage, blastocyst formation and cell number of BL on Day 7 (BLD7) were recorded. Our results showed that the average rate of maturation at 44h was 72% (n=1377). About 50% and 87% of oocytes, that eventually matured, extruded the polar body at 37 and 40h, respectively. The average cell number of BLD7 developed in SOFaa was 80±36 (n=52) and was not affected by activation protocol. Seventy-nine and 27% of BL had more than 50 and 100 cells per BL, respectively. Porcine oocytes activated by EL acquired their developmental competence gradually, achieving the highest rates of cleavage and blastocyst formation 7h after polar body extrusion. By contrast, oocytes activated by EL+CHX+CB showed their maximal developmental competence earlier (3–7h group). In conclusion, we demonstrate that electric impulse in combination with CHX+CB treatment permits earlier efficient activation of porcine oocytes (3–7h after polar body extrusion).

318 INHIBITION OF FIRST POLAR BODY EXTRUSION BY CYTOCHALASIN B DURING IN VITRO MATURATION OF PORCINE OOCYTES LEADS TO RE-ARRANGEMENT OF THE SEGREGATED CHROMOSOMES AND IMPROVEMENT IN THE QUALITY OF THE PARTHENOGENETIC BLASTOCYSTS

2006 ◽  
Vol 18 (2) ◽  
pp. 266
Author(s):  
T. Somfai ◽  
K. Kikuchi ◽  
J. Noguchi ◽  
H. Kaneko ◽  
K. Ohnuma ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Cytochalasin B ◽  
Polar Body ◽  
Metaphase Plate ◽  
Live Cells ◽  
Lower Probability ◽  
Control Group ◽  
First Polar Body ◽  
Polar Body Extrusion

Diploid parthenotes are usually obtained by the inhibition of second polar body (PB2) extrusion after activation of metaphase II (MII) oocytes. However, diploid embryos can be generated by the inhibition of the first polar body (PB1) extrusion as well, using cytochalasin B (CB) during in vitro maturation prior the activation procedure. A higher percentage of mouse embryos generated by the activation of MII oocytes and the inhibition of PB2 extrusion were proven to be homozygous than for parthenotes obtained by the latter method (Kubiak et al. 1991 Development 111, 763-769). The aim of the present study was to examine if such difference has any effect on the development of parthenogenetic embryos in vitro. Nuclear progression and in vitro embryonic development after parthenogenetic activation of porcine oocytes exposed to CB during in vitro maturation (IVM) was investigated in the present study. The tendency of nuclear maturation was similar in oocytes matured in the presence of 1 �g/mL CB (IVM-CB group) and control oocytes matured without CB after 37 h of IVM; at this time the frequency of oocytes that had reached/or passed through anaphase-I stage did not differ significantly (P < 0.05) between the IVM-CB and the control groups (61.3% and 69.9%, respectively), however, no polar body extrusion was observed in the IVM-CB group and the two lumps of homologue chromosomes remained in the oocyte and turned into two irregular sets of condensed chromosomes. By 41 h of IVM, the double sets of chromosomes re-united in 89.5% of IVM-CB oocytes and formed a single large metaphase plate, whereas 68.8% of the control oocytes had reached metaphase-II stage (MII) by this time. When IVM-CB oocytes were electrically (1.5 kV/cm for 100 �s) activated and subsequently cultured without CB, 39% of the oocytes extruded a polar body (PB) and 82.9% of them had a female pronucleus. When those oocytes with PB were cultured, the blastocyst rate of the cleaved embryos did not differ (P < 0.05) from those of the control that were stimulated at MII and subsequently treated with CB (43.3% and 48.2%, respectively). The number of blastomeres in Day 6 blastocysts was significantly higher (P < 0.05) in the IVM-CB derived embryos than in those in the control group (47.8 and 40.7, respectively); moreover, the ratio of dead blastomeres (dead cells : live cells) was higher (P < 0.05) in the control than in the IVM-CB blastocysts (0.047 and 0.031, respectively). A possible explanation for this result might be a lower frequency of homozygous genes in IVM-CB parthenotes, in which segregation of sister chromatids were promoted instead of segregation of homologous chromosomes to obtain diploid embryos. In such embryos the expression of recessive lethal, sublethal and subvital genes might have a lower probability. This work was supported by the Japanese-Hungarian bilateral scientific and technological cooperation (TET JAP-11/02).

59 IN VITRO DEVELOPMENT OF FELINE CLONED EMBRYOS RECONSTRUCTED WITH PREADIPOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 110
Author(s):  
R. Tomii ◽  
B. Ogawa ◽  
H. Nagashima
Keyword(s):  
Cytochalasin B ◽  
Polar Body ◽  
Primary Cultures ◽  
Fusion Rate ◽  
Culture Technique ◽  
Domestic Cats ◽  
Donor Cells ◽  
First Polar Body

The technique of somatic cell nuclear transfer (NT) in domestic cats is expected to contribute to the conservation of wildcats, for which extinction is a concern. In this study, we examined in vitro developmental ability of cloned embryos produced using the preadipocytes of domestic cats as nuclear donors. Primary cultures of preadipocytes were established as reported previously (Yagi et al. 2004 Biochem. Biophys. Res. Commun. 321, 967–974). Briefly, fat tissue (2–3 g) was excised from an adult female cat and digested using 0.1% collagenase for 1 h at 37�C followed by centrifugation. Only mature adipocytes that were floating near the surface of the supernatant were collected and placed in a 12.5-cm2 culture flask filled with DMEM containing 20% FBS. The flask was filled with medium, tightly capped, and cultured upside down for 7–10 days, so that the floating adipocytes attached to the inner ceiling surface of the flask. When firm attachment of the cells to the ceiling surface of the flask was confirmed, the flask was then inverted and culture was continued using the routine cell culture technique for adherent cells. In vivo-matured oocytes were collected from the ovaries of domestic cats superovulated by eCG and hCG. IVM oocytes were obtained by culturing cumulus–oocyte complexes from the ovaries collected at local veterinary clinics in TCM199-based medium for 24 to 30 h. In vivo-matured and IVM oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm using a bevelled pipette in the presence of 7.5 µg mL–1 cytochalasin B. The nuclei of the donor cells were transferred to enucleated in vivo-matured and IVM oocytes by means of electric fusion (300 V mm–1, 30 µs, twice). The reconstructed embryos were activated electrically (125 V mm–1, 60 µs, twice), followed by treatment with 10 µg mL–1 cycloheximide and 5 µg mL–1 cytochalasin B. The cloned embryos were cultured in vitro for 7 days in MK-1 so that their developmental ability might be examined. The fusion rate of donor cells was similar between in vivo-matured and IVM oocytes (56.8%, 21/37 v. 54.5%, 42/77). The developmental ability of NT embryos reconstructed with in vivo-matured oocytes was similar to that of NT embryos reconstructed with IVM oocytes (cleavage: 52.4%, 11/21 v. 42.9%, 18/42; development to blastocysts: 9.5%, 2/21 v. 11.9%, 5/42). The results indicate that cloned feline embryos reconstructed using preadipocytes can develop in vitro into blastocysts.

Induction of triploidy in the mouse by cytochalasin B

Development ◽  
1975 ◽  
Vol 34 (2) ◽  
pp. 279-289
Author(s):  
Anna Niemierko
Keyword(s):  
Culture Medium ◽  
Cytochalasin B ◽  
Sex Chromosome ◽  
Polar Body ◽  
Cell Number ◽  
Second Polar Body ◽  
And Control ◽  
Mouse Eggs

Mouse eggs fertilized in vivo were treated with cytochalasin B in vitro (5 μg/ml of culture medium) at he moment of extrusion of the second polar body (2·5, 3·0, 3·5 h after copulation). Cytochalasin B inhibits cytokinesis of the second maturation division, so that triploid digynic eggs are formed in over 50% of treated eggs. Triploid eggs were transplanted to the oviducts of recipients. On the 4th and 5th day of development 41·7% of transplanted eggs were recovered. All embryos recovered on the 4th day were morulae, while on the 5th day blastocysts predominated. Recovered embryos were studied for cell number and ploidy. Twenty-three of 27 embryos with analysable metaphase plates were triploid and four were diploid (the latter were found in females into which both triploid and control diploid eggs were transplanted). Sex chromosome constitution was determined in seven cases: four triploids were XXY and three were XXX.

Cytochalasin B treatment of mouse oocytes during intracytoplasmic sperm injection (ICSI) increases embryo survival without impairment of development

Zygote ◽  
2011 ◽  
Vol 20 (4) ◽  
pp. 361-369 ◽  
Author(s):  
Li-li Hu ◽  
Xing-hui Shen ◽  
Zhong Zheng ◽  
Zhen-dong Wang ◽  
Zhong-hua Liu ◽  
...  
Keyword(s):  
Survival Rate ◽  
Intracytoplasmic Sperm Injection ◽  
Cytochalasin B ◽  
Polar Body ◽  
Term Treatment ◽  
Short Term Treatment ◽  
Embryo Survival ◽  
Mouse Oocytes

SummaryIntracytoplasmic sperm injection (ICSI) is a technique commonly used in clinical and research settings. In mouse oocytes, conventional ICSI has a poor survival rate caused by a high level of lysis. Cytochalasin B (CB) is a toxic microfilament-inhibiting agent that is known to relax the cytoskeleton and enhance the flexibility of oocytes. CB has been used widely in nuclear transfer experiments to improve the success rate of the micromanipulation, however information describing the use of CB in ICSI is limited. Here, we demonstrated that the addition of 5 μg/ml CB to the manipulation medium of ICSI procedure significantly improved the survival rate of the ICSI embryos (80.74% vs. 89.50%, p < 0.05), and that there was no harm for the in vitro or in vivo development. The birth rates and birth weights were not significantly different between the CB-treated and -untreated groups. Interestingly, the microfilaments of the ICSI embryos were almost undetectable immediately after CB treatment; however, they gradually re-appeared and had fully recovered to the normal level 2 h later. Moreover, CB did not disturb spindle rotation, second polar body formation or pronuclei migration, and had no effect on the microtubules. We thus conclude that ICSI manipulation in CB-containing medium results in significantly improved survival rate of mouse ICSI embryos, and that short-term treatment with CB during ICSI manipulation does not have adverse effects on the development of ICSI embryos.

Methodological Approaches to Evaluate Fetal Drug Exposure

2019 ◽  
Vol 25 (5) ◽  
pp. 496-504 ◽  
Author(s):  
Naïm Bouazza ◽  
Frantz Foissac ◽  
Déborah Hirt ◽  
Saïk Urien ◽  
Sihem Benaboud ◽  
...  
Keyword(s):  
Cord Blood ◽  
Drug Exposure ◽  
Placental Transfer ◽  
Ex Vivo ◽  
Pharmacokinetic Analysis ◽  
Population Pharmacokinetic ◽  
Pbpk Models ◽  
Drug Concentrations ◽  
Fetal Drug Exposure

Background: Drug prescriptions are usual during pregnancy, however, women and their fetuses still remain an orphan population with regard to drugs efficacy and safety. Most xenobiotics diffuse through the placenta and some of them can alter fetus development resulting in structural abnormalities, growth or functional deficiencies. Methods: To summarize the different methodologies developed towards the prediction of fetal drug exposure. Results: Neonatal cord blood concentration is the most specific measurement of the transplacental drug transfer at the end of pregnancy. Using the cord blood and mother drug concentrations altogether, drug exchanges between the mother and fetus can be modeled and quantified via a population pharmacokinetic analysis. Thereafter, it is possible to estimate the fetus exposure and the fetus-to-mother exposure ratio. However, the prediction of placental transfer before any administration to pregnant women is desirable. Animal studies remain difficult to interpret due to structural and functional inter-species placenta differences. The ex-vivo perfusion of the human placental cotyledon is the method of reference to study the human placental transfer of drugs because it is thought to mimic the functional placental tissue. However, extrapolation of data to in vivo situation remains difficult. Some research groups have extensively worked on physiologically based models (PBPK) to predict fetal drug exposure and showed very encouraging results. Conclusion: PBPK models appeared to be a very promising tool in order to predict fetal drug exposure in-silico. However, these models mainly picture the end of pregnancy and knowledge regarding both, development of the placental permeability and transporters is strongly needed.

scholarly journals Drug Delivery by Ultrasound-Responsive Nanocarriers for Cancer Treatment

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1135
Author(s):  
Kristin Entzian ◽  
Achim Aigner
Keyword(s):  
Drug Delivery ◽  
Biological Effects ◽  
Invasive Technique ◽  
Stimuli Responsive ◽  
Comprehensive Overview ◽  
Drug Concentrations ◽  
Therapeutic Outcomes ◽  
Cost Efficient ◽  
Safety Considerations

Conventional cancer chemotherapies often exhibit insufficient therapeutic outcomes and dose-limiting toxicity. Therefore, there is a need for novel therapeutics and formulations with higher efficacy, improved safety, and more favorable toxicological profiles. This has promoted the development of nanomedicines, including systems for drug delivery, but also for imaging and diagnostics. Nanoparticles loaded with drugs can be designed to overcome several biological barriers to improving efficiency and reducing toxicity. In addition, stimuli-responsive nanocarriers are able to release their payload on demand at the tumor tissue site, preventing premature drug loss. This review focuses on ultrasound-triggered drug delivery by nanocarriers as a versatile, cost-efficient, non-invasive technique for improving tissue specificity and tissue penetration, and for achieving high drug concentrations at their intended site of action. It highlights aspects relevant for ultrasound-mediated drug delivery, including ultrasound parameters and resulting biological effects. Then, concepts in ultrasound-mediated drug delivery are introduced and a comprehensive overview of several types of nanoparticles used for this purpose is given. This includes an in-depth compilation of the literature on the various in vivo ultrasound-responsive drug delivery systems. Finally, toxicological and safety considerations regarding ultrasound-mediated drug delivery with nanocarriers are discussed.

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