In vitro development of haploid mouse embryos produced by bisection of one-cell fertilized eggs

Development ◽  
1977 ◽  
Vol 38 (1) ◽  
pp. 187-202
Author(s):  
Andrzej K. Tarkowski
Keyword(s):  
X Chromosome ◽  
Culture Conditions ◽  
Extrinsic Factors ◽  
Cell Stage ◽  
Glass Needle ◽  
Androgenetic Embryos ◽  
Haploid Embryos ◽  
Vitro Development ◽  
Further Development

F1(CBA × C57BL'10) mouse eggs originating from spontaneous or induced ovulation and fertilized by CBA-T6T6 or PO spermatozoa were bisected with a glass needle into halves each containing a pronucleus. This technique offers a unique opportunity of producing both androgenetic and gynogenetic haploid embryos from one egg. Out of 600 operated eggs, in 406 (67·7%) both halves survived. During 96 h of culture in vitro the fragments were inspected once daily and finally examined in air-dried preparations. Eighty-seven per cent of halves underwent first cleavage but their further development was to a large extent affected by extrinsic factors connected with experimental procedure (mainly by suboptimal and variable culture conditions) and by the origin of eggs (those from spontaneous ovulation being superior). For this reason developmental capabilities of egg halves were assessed in a selected group of pairs in which at least one partner reached the stage of four or more blastomeres. The observed ratio between pairs with both or only one sister embryo developing successfully suggests that androgenetic embryos carrying Y rather than X chromosome can cleave twice but do not survive beyond 4-cell stage. None of the metaphase plates from older embryos contained a Y chromosome. These observations imply that the X chromosome is genetically active during early cleavage and that a full haploid set is required for preimplantation development to be completed. Formation of blastocysts varied from batch to batch, with an average of 12·8% and maximal incidence of 29·5% . In 34 pairs both fragments developed beyond the 4-cell stage but in only one case did both form blastocysts. Haploid blastocysts were composed of 27 cells on average which was about a half of the number of cells in control diploid zona-free whole eggs. Ten out of 51 embryos with metaphase plates proved to be haploid/diploid mosaics.

Evolution in vitro du contenu en ADN nucléaire et de la ploïdie des embryons polliniques du Datura innoxia

1981 ◽  
Vol 59 (4) ◽  
pp. 508-517 ◽  
Author(s):  
Brigitte S. Sangwan-Norreel
Keyword(s):  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Datura Innoxia ◽  
Cell Numbers ◽  
Irregular Structures ◽  
Three Stages ◽  
Androgenetic Embryos ◽  
Haploid Embryos ◽  
Vitro Development

Nuclear DNA content was estimated by densitometry in three stages of in vitro development of Datura innoxia Mill. embryos (3–25 cells) obtained from pollen. At the same time, the sizes, structure and arrangements of nuclei in young embryos were examined and chromosome numbers of adult embryos were determined. Results showed that: (i) Embryos originating from pollen were haploid, diploid, aneuploid, or myxoploid. Proportions of each ploidy level varied with cell numbers in embryos, and with ages of cultures, (ii) Young diploid embryos were very regular and had dense nuclei of one type only. Haploid embryos were less regular; they had one or two types of nuclei whose texture was often looser than that of diploid embryo nuclei. Aneuploid and myxoploid embryos had very irregular structures, (iii) Initiation of androgenetic embryos was spread out in time from the 2nd to the 12th day of culture. Embryos showed different nuclear features if they were initiated at the beginning, in the middle, or at the end of the culture period. Embryos initiated early were mostly haploid and the proportion of abnormal ploidy embryos was low during the 1st week of culture. In Datura innoxia, therefore, embryos must be taken after 1 week of culture to obtain the population which is most favourable to further genetic breeding. [Journal translation]

scholarly journals HDAC inhibition decreases XIST expression on female IVP bovine blastocysts

Reproduction ◽  
2013 ◽  
Vol 145 (1) ◽  
pp. 9-17 ◽  
Author(s):  
Clara Slade Oliveira ◽  
Naiara Zoccal Saraiva ◽  
Maria Helena Coelho Cruz ◽  
Bruna Mazeti ◽  
Leticia Zoccolaro Oliveira ◽  
...  
Keyword(s):  
Gene Expression ◽  
X Chromosome ◽  
Trichostatin A ◽  
Culture Conditions ◽  
Histone Deacetylation ◽  
Cell Stage ◽  
Initial Development ◽  
Histone Deacetylase Inhibition ◽  
Hoechst Staining

During initial development, both X chromosomes are active in females, and one of them must be silenced at the appropriate time in order to dosage compensate their gene expression levels to male counterparts. Silencing involves epigenetic mechanisms, including histone deacetylation. Major X chromosome inactivation (XCI) in bovine occurs between hatching and implantation, althoughin vitroculture conditions might disrupt the silencing process, increasing or decreasing X-linked gene expression. In this study, we aimed to address the roles of histone deacetylase inhibition by trichostatin A (TSA) on female preimplantation development. We tested the hypothesis that by enhancing histone acetylation, TSA would increase the percentage of embryos achieving 16-cell stage, reducing percentage of embryos blocked at 8-cell stage, and interfere with XCI in IVF embryos. We noticed that after TSA treatment, acetylation levels in individual blastomeres of 8–16 cell embryos were increased twofold on treated embryos, and the same was detected for blastocysts. Changes among blastomere levels within the same embryo were diminished on TSA group, as low-acetylated blastomeres were no longer detected. The percentage of embryos that reached the 5th cleavage cycle 118 h after IVF, analyzed by Hoechst staining, remained unaltered after TSA treatment. Then, we assessedXISTandG6PDexpression in individual female bovine blastocysts by quantitative real-time PCR. Even thoughG6PDexpression remained unaltered after TSA exposure,XISTexpression was eightfold decreased, and we also detected a major decrease in the percentage of blastocysts expressing detectableXISTlevels after TSA treatment. Based on these results, we conclude that HDAC is involved on XCI process in bovine embryos, and its inhibition might delay X chromosome silencing and attenuate aberrantXISTexpression described for IVF embryos.

Expression of the HSP 70.1 gene, a landmark of early zygotic activity in the mouse embryo, is restricted to the first burst of transcription

Development ◽  
1995 ◽  
Vol 121 (1) ◽  
pp. 113-122 ◽  
Author(s):  
E. Christians ◽  
E. Campion ◽  
E.M. Thompson ◽  
J.P. Renard
Keyword(s):  
Dna Replication ◽  
Mouse Embryo ◽  
Culture Conditions ◽  
Hsp 70 ◽  
Cell Stage ◽  
Embryonic Genome ◽  
Genome Activation ◽  
Alpha Amanitin ◽  
Zygotic Genome

Activation of the mouse embryonic genome at the 2-cell stage is characterized by the synthesis of several alpha-amanitin-sensitive polypeptides, some of which belong to the multigenic hsp 70 family. In the present work we show that a member of this family, the HSP 70.1 gene, is highly transcribed at the onset of zygotic genome activation. Transcription of this gene began as early as the 1-cell stage. Expression of the gene continued through the early 2-cell stage but was repressed before the completion of the second round of DNA replication. During this period we observed that the level of transcription was modulated by in vitro culture conditions. The coincidence of repression of HSP70.1 transcription with the second round of DNA replication was not found for other transcription-dependent polypeptides synthesized at the 2-cell stage.

scholarly journals Bioreactors as physiologicallike in vitro models

2020 ◽  
Author(s):  
Sara Mantero ◽  
Federica Boschetti
Keyword(s):  
Culture Conditions ◽  
In Vitro Models ◽  
In Vivo Models ◽  
In Vitro Development ◽  
Culture Cells ◽  
Vitro Development ◽  
Tissue Models ◽  
Mechanical Stretching

Bioreactors are powerful tools for in vitro development of engineered substitutes through controlled biological, physical, and mechanical culture conditions: bioreactor technology allows a closer in vitro replication of native tissues. One of bioreactors applications is the design of in vitro 3D tissue models as a bridge between 2D and in vivo models, allowing the application of 3R (replacement, reduction, refinement) principle. To this aim, bioreactors can be used to culture cells seeded on engineered scaffolds under in vivo-like conditions. Another key use of bioreactors is for perfusion decellularization of tissues and organs to be used as scaffolds. This contribution describes a dynamic stretching. bioreactor, imposing a mechanical stretching to the cultured constructs, allowing the development of skeletal muscle engineered constructs, and a decellularization bioreactor, designed for decellularization of blood vessels.

297 PRODUCTION OF CHIMERIC PORCINE FETUSES BY AGGREGATION METHOD USING PARTHENOGENETIC EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 296
Author(s):  
K. Nakano ◽  
M. Watanabe ◽  
H. Matsunari ◽  
T. Matsuda ◽  
K. Honda ◽  
...  
Keyword(s):  
Cell Mass ◽  
Ips Cells ◽  
Large Animal ◽  
Aggregation Method ◽  
Cell Stage ◽  
In Vitro Development ◽  
Donor Cells ◽  
Vitro Development ◽  
Parthenogenetic Embryos

Porcine induced pluripotent stem (iPS) cells are considered to be an invaluable research tool in translational research with pigs as a large animal model. Pluripotency of the iPS cells needs to be verified by their competence to contribute to chimera formation. The aim of the present study is to establish feasible system to create chimeric pig fetuses using parthenogenetic embryos. In Experiment 1, inner cell mass (ICM) was isolated by immunosurgery from Day 6 blastocysts obtained by parthenogenetic activation of in vitro matured (IVM) oocytes. Isolated ICM were used as the donor cells after staining with fluorescent carbocyanine dye (DiI). Using parthenogenetic morulae or 4- to 8-cell embryos as the host embryos, chimeric embryos were prepared by injection or aggregation method. Injection of ICM was performed by micromanipulation: a single ICM was directly injected into the centre portion of the host morulae. In the aggregation method, a single ICM was aggregated with blastomeres isolated from 2 host embryos at the morula or 4- to 8-cell stage in a micro-well (400 µm diameter, 300 µm deep). The chimeric embryos were cultured in PZM-5 (Yoshioka et al. 2008) for 2 to 3 days to examine development to blastocysts and incorporation of donor ICM cells into the resultant blastocysts ICM (ICM chimerism). In Experiment 2, donor blastomeres isolated from a parthenogenetic morula or 4- to 8-cell embryo were stained by DiI and aggregated with a parthenogenetic host embryo at the morula or 4- to 8-cell stage, and the in vitro development to the blastocyst stage and the ICM chimerism were examined. In Experiment 3, ICM isolated from IVF blastocysts harboring humanized Kusabira-Orange (huKO) gene were used as donor cells. Donor ICM were aggregated with the host embryos at the morula or 4- to 8-cell stage, and the resultant blastocysts were transferred to 4 recipient gilts to collect fetuses on Day 18. Results of Experiments 1 and 2 are summarised in Table 1. Combination of the donor ICM and host morulae yielded high rates of blastocyst formation (~95%) and ICM chimerism (~85%), regardless of the method used (injection or aggregation). Transfer of 73 blastocysts developed from host morulae to 2 recipients (Experiment 3) gave rise to 25 (34.2%) fetuses, of which 6 (24.0%) were confirmed to be chimeric by their clear orange fluorescence and immunostaining by anti-huKO antibody. Of 22 (40.7%) fetuses obtained after transfer of 54 blastocysts derived from 4- to 8-cell host embryos to 2 recipients, 3 (13.6%) were chimeric. Contribution of the donor cells in the tissues of the chimeric fetuses measured by image analysis software (ImageJ, NIH, Bethesda, MD, USA) ranged between 16.1 and 65.2%. These results demonstrate that the aggregation method using parthenogenetic host embryos is an efficient means to produce chimeric pig fetuses, and thereby feasible for verification of pluripotent cells such as iPS cells. Table 1.In vitro development of injected or aggregated porcine embryos

scholarly journals Effect of Different Culture Conditions on In vitro Development and Quality of Buffalo Embryos

2018 ◽  
Vol 46 (1) ◽  
pp. 70-78
Author(s):  
Fatma Ibrahim ◽  
Hassan Mansour ◽  
Faten Labib ◽  
Hussein Amer ◽  
Magdy Badr
Keyword(s):  
Culture Conditions ◽  
In Vitro Development ◽  
Vitro Development

scholarly journals Biochemical Compositions of Follicular Fluid and the Effects of Culture Conditions on the In Vitro Development of Pig Oocytes

2002 ◽  
Vol 15 (10) ◽  
pp. 1403-1411 ◽  
Author(s):  
Wei-Tung Huang ◽  
She-Ghi Lu ◽  
Pin-Chi Tang ◽  
Shinn-Chih Wu ◽  
San-Pao Cheng ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Culture Conditions ◽  
In Vitro Development ◽  
Biochemical Compositions ◽  
Vitro Development

375 UNEXPECTED SEVERE ABNORMALITIES IN MOUSE ROSI OFFSPRING

2007 ◽  
Vol 19 (1) ◽  
pp. 303
Author(s):  
P. N. Moreira ◽  
R. Fernández-González ◽  
M. Pérez-Crespo ◽  
P. Bermejo ◽  
J. D. Hourcade ◽  
...  
Keyword(s):  
Testicular Tumor ◽  
Round Spermatid ◽  
Sperm Cells ◽  
Healthy Children ◽  
Clinical Value ◽  
Cell Stage ◽  
Infertile Men ◽  
Vitro Development ◽  
Round Spermatid Injection

Live offspring resulting from round spermatid injection (ROSI) was first accomplished in the mouse, but similar success has been obtained in rat, hamster, rabbit, mastomys, pig, monkey, and human. ROSI has received clinical attention because some infertile men have no spermatozoa or just a very few in their testes, and these are difficult to harvest and are frequently dead and deformed. Although some clinicians were able to generate healthy children by ROSI, others could not (reviewed in Yanagimachi 2004 Reprod. Biomed. Online 9). The clinical value of ROSI has been widely debated. It remains unclear if post-meiotic and pre-fertilization modifications of sperm cells are necessary to ensure normal development. In order to answer this question, we decided to study and compare mouse offspring generated by ROSI and intracytoplasmic sperm injection (ICSI). ROSI and ICSI with fresh sperm cells were carried out in the B6D2 mouse strain as described (Marh et al. 2003 Biol. Reprod. 69, 169–176; Moreira et al. 2005 Hum. Reprod. 20, 3313–3317). In vitro-produced embryos were transferred at the 2-cell stage into Day 1 pseudopregnant females. As shown in Table 1 oocyte survival after injection was significantly higher (z-test, P < 0.05) with ICSI (91%) than with ROSI (68%). The proportion of live offspring obtained by ICSI was also significantly higher (26% vs. 6%; z-test, P < 0.05). Moreover, fertilization with spermatozoa produced healthy offspring more efficiently than with round spermatids. Out of 30 live offspring generated by ROSI, 6 (20%) presented severe abnormalities during their first 6–8 weeks of age. One ROSI animal presented an abnormally swollen skull (hydroencephaly) with a very thin and soft cranial wall. Another developed a subcutaneous engrossment of the forehead, producing a crest-like appearance. Three others presented deviations in their vertebral columns (hyperkyphosis and scoliosis), and recently a testicular tumor was detected in another animal. These types of malformations were not observed in the control offspring. In our experience, very rarely are they observed after ICSI or in naturally mated animals. To our knowledge, and although the risks of the ROSI procedure have been extensively highlighted in human and other species, the phenotypic abnormalities observed in this study have never been reported. Presently, we keep monitoring these animals as they age, as part of an ambitious plan that is also intended to characterize and understand the origin of the possible phenotypic consequences of the ROSI procedure. Table 1. In vitro development and development to term of B6D2 mouse embryos generated by ROSI or ICSI

273 EFFECT OF IONOMYCIN ASSOCIATED WITH ROSCOVITINE, DEHYDROLEUCODINE, CYCLOHEXIMIDE, OR ETHANOL ON HAPLOID ACTIVATION OF BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 225
Author(s):  
M. Suvá ◽  
N. G. Canel ◽  
D. F. Salamone
Keyword(s):  
Chromosome Number ◽  
Polar Body ◽  
Routine Procedure ◽  
In Vitro Development ◽  
Second Polar Body ◽  
N 93 ◽  
Haploid Embryos ◽  
Vitro Development ◽  
Bovine Oocytes

Haploid activation of bovine oocytes after ICSI is a routine procedure. However, embryos frequently contain an abnormal chromosome set as a result of the drugs employed. We compared the efficiency of ionomycin (Io) followed by roscovitine (ROSC), cycloheximide (CHX), ethanol, and dehydroleucodine (DhL) to induce haploid parthenogenetic activation in bovine. Pronuclear (PN) formation, second polar body (2PB) extrusion, embryo development, and ploidy of blastocysts were evaluated. To this aim, COC were aspirated from slaughtered ovaries and IVM for 22 h. Oocytes were activated with 5 µM of Io for 4 min and then randomly allocated into 1 of the following treatments: 25 or 50 µM ROSC or 10 µg mL–1 of CHX for 5 h; 15 or 30 µM DhL for 3 h; or 5 min of exposure to 7% ethanol 4 h post-Io. Controls were Io followed by (1) 3 h in TCM-199 and 3 h in 1.9 mM 6-DMAP (Io-3h-DMAP) and (2) 3 h of exposure to 1.9 mM 6-DMAP (Io-DMAP). Oocytes were cultured in SOF medium. The PN formation and 2PB extrusion were assessed by 5 µg mL–1 of propidium iodide oocyte staining, 17 h after Io. Cleavage, morulae, and blastocyst stages were evaluated at Days 2, 5, and 8 of in vitro development, respectively. Chromosome number of blastocysts was evaluated by Giemsa staining. Data were analysed by Fisher's test (P < 0.05). Rates of 2PB extrusion were 75, 61.1, 60, 56.3, 54.6, and 42.9% for 15 µM DhL (n = 23), 50 µM ROSC (n = 22), Io-3h-DMAP (n = 9), CHX (n = 17), 25 µM ROSC (n = 22), and ethanol (n = 22), respectively, with no differences between groups. A PN was observed in over 81% of the oocytes activated with ethanol, 25 µM ROSC, CHX, 50 µM ROSC, and 15 µM DhL. Lower percentages of 2PB extrusion and PN formation were observed for 30 µM DhL (n = 22; 6.3 and 0%, respectively). The highest cleavage rates were 83.2% for 25 µM ROSC (n = 185), not differing from 78% in Io-DMAP (n = 159). Cleavage rates for 50 µM ROSC (n = 185), CHX (n = 143), and ethanol (n = 74; 80.5, 80.4 and 67.6%, respectively) were not different from Io-3h-DMAP (n = 78; 71.8%) and Io-DMAP. Cleavage rates for 15 µM DhL (n = 70) and 30 µM DhL (n = 93) were the lowest (48.6 and 25.8%). Blastocyst rates were the highest for CHX and 50 µM ROSC, not differing from Io-3h-DMAP (21.7 and 10.8 v. 18%). Very few or no blastocysts were obtained with ethanol, 25 µM ROSC, 30 µM DhL, and 15 µM DhL (4.1, 3.8, 1.1, and 0%, respectively), although ethanol was not different from Io-3h-DMAP. Chromosome number analysis showed that ethanol (n = 2) and CHX (n = 2) resulted in a higher percentage of haploid embryos (50% each), followed by 50 µM ROSC (n = 8), 25 µM ROSC (n = 3), and Io-3h-DMAP (n = 8; 37.5, 33.3% and 12.5%, respectively), although they were not different. Remaining embryos were diploid, aneuployd, or mixoployd. In conclusion, DhL and ROSC proved to be as effective as CHX or ethanol regarding 2PB extrusion and resulting ploidy, defining features when activating oocytes in ART, suggesting they could be efficiently used in bovine to assist ICSI.

Preimplantation development of microsurgically obtained haploid and homozygous diploid mouse embryos and effects of pretreatment with Cytochalasin B on enucleated eggs

Development ◽  
1980 ◽  
Vol 60 (1) ◽  
pp. 153-161
Author(s):  
Jacek A. Modliński
Keyword(s):  
Survival Rate ◽  
Cytochalasin B ◽  
Single Case ◽  
Cell Stage ◽  
Morula Stage ◽  
Homozygous Diploid ◽  
Haploid Embryos ◽  
Early Developmental

Haploid embryos were obtained by microsurgical removal of one pronucleus, followed by doubling of the haploid chromosome set with Cytochalasin B (CB), either at the first or second mitosis. This procedure provides a source of fully homozygous diploid embryos, which were grown in vitro or in vivo. The effect of CB treatment before and during operation on the course of enucleation and further development of embryos was studied. Out of 81 eggs made diploid at 2-cell stage and transplanted into the oviducts of immature or pseudopregnant recipients 27 morulae and blastocysts were recovered, but not a single case of implantation occurred by the eighth or ninth day of development. After 72–80 h of in vitro culture, most of the homozygous embryos were morulae but after an additional 24 h the majority of them transformed into blastocysts. The rate of development of homozygotes was markedly better than that of haploids, which progressed beyond morula stage. The immediate survival rate of operated eggs was dependent on whether or not the eggs were pre-incubated and the enucleation was performed in the presence of CB. In the former case the immediate survival rate was nearly twice as high as in the absence of CB, but more of the treated eggs underwent fragmentation and early developmental arrest.

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