Preimplantation development of in vitro-matured and in vitro-fertilized ovine zygotes: comparison between coculture on oviduct epithelial cell monolayers and culture under low oxygen atmosphere.
01 Apr 1994-Biology of Reproduction (Society for the Study of Reproduction)-Vol. 50, Iss: 4, pp 715-724
TL;DR: Two distinct culture environments are characterized, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro, and survival of the embryo under a reduced oxygen atmosphere is investigated.
Abstract: The roles of medium composition, serum source, embryo coculture, and culture under low O2 conditions on the development of in vitro-matured and in vitro-fertilized (IVMF) ovine zygotes were investigated in three separate experiments. In the first experiment, the proportion of cocultured IVMF zygotes developing to the blastocyst stage was significantly higher (38.0% vs. 3.5%; p < 0.05) than that of non-cocultured zygotes treated within three embryo culture media (TCM-199 + 10% fetal bovine serum [FBS]; bicarbonate-buffered, glucose-free synthetic oviduct fluid medium [mod-SOFM] + 10% FBS; and bicarbonate-buffered BSA-free Tyrode's salt solution [mod-TALP] + 10% FBS) under a 5% CO2 atmosphere in air. In a second experiment, a significantly higher (p < 0.05) proportion of cocultured zygotes placed in TCM-199 medium survived to the blastocyst stage (37.4% blastocysts vs. 23.4% in mod-SOFM). No significant effect of serum (FBS vs. human serum [HS]) was observed on embryonic development, but coculture was confirmed to exert a significant influence on development to the blastocyst stage. In the final experiment, survival of the embryo under a reduced oxygen (5% CO2:5% O2:90% N2) atmosphere was investigated. In contrast to results in the initial experiments, embryonic survival was significantly higher (p < 0.05) in the non-cocultured treatment groups (21.9% blastocysts vs. 0.4% for cocultured zygotes). Serum source also had a significant (p < 0.05) influence upon the development of non-cocultured zygotes: 32.3% of zygotes cultured with HS progressed to the blastocyst stage vs. 11.5% of zygotes cultured in FBS-supplemented medium. These results have characterized two distinct culture environments, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro.
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TL;DR: Coculture of cryopreserved preimplantation-stage embryos with Vero cells seems to be a useful tool to eliminate the postthaw deleterious effect of freezing and also to obtain better-quality embryos appropriate for transfer.
Abstract: Purpose:Our purpose was to evaluate the beneficial effects of long-term coculture of Vero cells on the development of frozen–thawed two-cell mouse embryos.
Methods:Two-cell mouse embryos were frozen slowly with 1,2-propandiol and sucrose as cryoprotectants and thawed rapidly, followed by stepwise dilution. Vero cells were cultured in drops of RPMI 1640 to establish monolayers. Frozen–thawed embryos were cultured alone (control) or cocultured with Vero cells. The rate of development in both groups was compared.
Results:After 4 days of culture, significantly more embryos in coculture were developed to expanded blastocysts (61 vs 37% for controls; P ≤ 0.0001). In addition, on the fifth day of cultivation, more embryos in coculture showed the potential of hatching from the zona pellucida (26 vs 7% in controls; P ≤ 0.0001). The rate of degeneration in coculture was also much lower than in controls (6 and 15%, respectively).
Conclusions:Coculture of cryopreserved preimplantation-stage embryos with Vero cells seems to be a useful tool to eliminate the postthaw deleterious effect of freezing and also to obtain better-quality embryos appropriate for transfer.
4 citations
Cites background from "Preimplantation development of in v..."
...Application of coculture and freezing is by no means a new concept but it has been used most often for embryo development through the blastocyst stage prior to freezing (10,29)....
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...Some helper cells from different origins have been shown to have the potential to overcome the embryonic block that occurs following culture of embryos at the time of genomic activation in different species: mouse (8), equine (9), ovine (10), and human (11) embryos....
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Dissertation•
01 Sep 2009
TL;DR: The development of several methods of embryo manipulation tailored to the morphology of the blastocyst is described here, which resulted in the derivation of seven lines from four di�fferent procedures and provided the tools for subsequent research.
Abstract: Stem cells are unique cells that have both the capacity for self-renewal and, depending on their origin, the ability to form at least one, and sometimes many, specialised cell types of all three embryonic germ lineages -
germ cells (endoderm, mesoderm and ectoderm), extra-embryonic tissue and trophoblast. Since the derivation of the fi�rst human embryonic stem cell (hESC) line in 1998, there has been substantial interest in the potential of these cells both for regenerative medicine and cell therapy, and as disease models for monogenic disorders. Aside from the need to improve derivation efficiency and further the understanding of the basic biology of
these cells, the ability to work with hESC opens up three broad research areas. The �first is the development of clinical grade culture systems with
the aim of producing cell lines suitable for subsequent manipulation for therapy. The second is the opportunity to use these cells as a tool to study the earliest determinative events in mammalian development, such
as the origins of patterning in the mammalian embryo. The third is the use of hESCs carrying clinically relevant genetic mutations as models for disease research and therapeutic target identifi�cation.
The development of several methods of embryo manipulation tailored to the morphology of the blastocyst is described here, which resulted in the derivation of seven lines from four di�fferent procedures and provided the
tools for subsequent research. Acknowledging that each laboratory in isolation is unlikely to derive sufficient lines to draw signifi�cant conclusions regarding manipulation methodology and culture parameters, an
international collaboration was initiated with the aim of standardising the reporting of derivation and thus obtaining the maximum information from the generation of each new hESC line. To address the need for the development of clinical grade culture systems, alternative feeder cells were assessed for their suitability in hESC culture
and derivation. Modi�fied human foreskin fi�broblasts and human amniotic epithelial cells (hAECs) were investigated, as both cell types can be fully qualifi�ed and validated. Whilst both were able to support the culture of
existing lines, only the hAECs showed promise in supporting derivation. In addition, analysis of in-house and commercially available media showed
that neither were physiologically optimal for the growth of inner cell mass (ICM) cells or putative hESC, as metabolite concentrations were in excess and subsequent catabolite levels exceeded known toxic levels.
The timing and mechanisms establishing patterning and future polarity
in the mammalian embryo remains a subject of intense debate. Here,
the potential of single blastomeres to generate hESC was used as an assessment of pluripotency. The determination of the most appropriate
day for attempting derivation was performed by assessing blastomere development and pluripotent marker expression, and the predicted success
of derivation was considered in the light of division patterns. Putative
stem-like cells were visible in several cultures. Furthermore, isolated blastomeres from two-, four-, and eight-cell embryos were analysed for the
quantitative expression of multiple target genes known to be associated
with lineage formation and the stem cell state. Analysis suggested that
broad changes in gene expression were occurring with development stage.
However, no consistent grouping structure for cells within embryos was
observed, and no convincing pattern was seen when considering the individual embryo variance scores. Several approaches are discussed to
diff�erentiate between the biological and methodological variability in this experimental design.
The suitability of hESC as models for genetic disease was studied following the derivation of two lines carrying Huntington disease (HD).
Subsequent di�fferentiation using a stromal co-culture neural induction protocol resulted in the establishment of a stable, highly proliferative
cell population which was simple to culture and bank. The cells were of an astroglial phenotype, and therefore highly suited for subsequent studies regarding HD pathophysiology, as glial cells are severely aff�ected in
HD. During diff�erentiation the CAG repeat size increased from 46 to 70, showing the salient feature of somatic instability of the huntingtin gene.
Therefore this cell population provides a valuable tool in the study of
disease pathogenesis and transmission.
4 citations
Cites background from "Preimplantation development of in v..."
...Low oxygen culture has been shown to enhance in vitro preimplantation embryo development in the mouse (Karagenc et al., 2004), sheep and cow (Thompson et al., 1990), goat (Batt et al., 1991) and pig (Watson et al., 1994)....
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TL;DR: The new era of gene editing might offer innovative perspectives in sheep breeding, but the application of such novel techniques implies involvement of specialized operators and is limited by relatively high costs for embryo manipulation and molecular biology analysis.
Abstract: Abstract To date, large‐scale use of multiple ovulation and embryo transfer (MOET) programmes in ovine species is limited due to unpredictable results and high costs of hormonal stimulation and treatment. Therefore, even if considered reliable, they are not fully applicable in large‐scale systems. More recently, the new prospects offered by in vitro embryo production (IVEP) through collection of oocytes post‐mortem or by repeated ovum pick‐up from live females suggested an alternative to MOET programmes and may be more extensively used, moving from the exclusive research in the laboratory to field application. The possibility to perform oocytes recovery from juvenile lambs to obtain embryos (JIVET) offers the great advantage to significantly reduce the generation interval, speeding the rate of genetic improvement. Although in the past decades several studies implemented novel protocols to enhance embryo production in sheep, the conditions of every single stage of IVEP can significantly affect embryo yield and successful transfer into the recipients. Moreover, the recent progresses on embryo production and freezing technologies might allow wider propagation of valuable genes in small ruminants populations and may be used for constitution of flocks without risks of disease. In addition, they can give a substantial contribution in preserving endangered breeds. The new era of gene editing might offer innovative perspectives in sheep breeding, but the application of such novel techniques implies involvement of specialized operators and is limited by relatively high costs for embryo manipulation and molecular biology analysis.
4 citations
TL;DR: In SOF medium both blastocysts rate and embryo transcriptome suggest no influence of feeder origin on bovine early development and no beneficial impact of co-culture systems when compared to 5% O2.
Abstract: During the last few years, several co-culture systems using either BOEC or VERO feeder cells have been developed to improve bovine embryo development and these systems give better results at high oxygen concentration (20%). In parallel, the SOF medium, used at 5% O2, has been developed to mimic the oviduct fluid. Since 2010s, the SOF medium has become popular in improving bovine embryo development and authors have started to associate this medium to co-culture systems. Nevertheless, little is known about the putative benefit of this association on early development. To address this question, we have compared embryo transcriptomes in four different culture conditions: SOF with BOEC or VERO at 20% O2, and SOF without feeders at 5% or 20% O2 Embryos have been analyzed at 16-cell and blastocyst stages. Co-culture systems did not improve the developmental rate when compared to 5% O2 Direct comparison of the two co-culture systems failed to highlight major differences in embryo transcriptome at both developmental stages. Both feeder cell types appear to regulate the same cytokines and growth factors pathways, and thus to influence embryo physiology in the same way. In blastocysts, when compared to culture in SOF at 5% O2, BOEC or VERO seems to reduce cell survival and differentiation by, at least, negatively regulating STAT3 and STAT5 pathways. Collectively, in SOF medium both blastocysts rate and embryo transcriptome suggest no influence of feeder origin on bovine early development and no beneficial impact of co-culture systems when compared to 5% O2.
4 citations
TL;DR: It was concluded that the presence of cumulus cells co-culture and bovine amniotic fluid as a protein source within culture medium may have an important role on the fertilizing capacity of spermatozoa and oocytes and normal development of pre-implanted mouse embryo later.
Abstract: Successful oocyte fertilization and normal embryonic development of mice were considered the most important diagnostic criteria for the safety of materials and tools used for human in vitro fertilization and embryo transfer (IVF-ET). Therefore, we studied the influence of cumulus cells co-culture and protein supplement within culture medium on percentages of in vitro fertilization (IVF) and normal development of early stages of mouse embryo later. Oocytes were collected and treated with hyaluronidase to remove cumulus cells. Oocytes were divided into four groups namely: Group-1: Oocytes incubated within modified Earl’s medium (MEM) supplied with 10% inactivated bovine amniotic fluid as a protein source and cumulus cells; Group-2: Oocytes incubated with MEM supplied with cumulus cells only; Group-3: Oocytes incubated with MEM supplied with 10% inactivated bovine amniotic fluid only; and Group-4: Oocytes incubated with MEM free of both protein source and cumulus cells. For IVF, 5-6 oocytes were incubated with active spermatozoa under paraffin oil for 18-20 hours at 37 ° o C in 5% CO 2 . Percentages of IVF and embryonic development were then recorded. Best results for IVF and normal embryonic development were achieved from oocytes of Group-1 when compared to the other groups. As compared to Group-1, the percentage of IVF for Group-2 and Group-3 were decreased insignificantly and significantly (P<0.002), respectively. Significant (P<0.01) reduction in the percentages of IVF and normal embryonic development were reported in Group-4 as compared to Group-1. Therefore, it was concluded that the presence of cumulus cells co-culture and bovine amniotic fluid as a protein source within culture medium may have an important role on the fertilizing capacity of spermatozoa and oocytes and normal development of pre-implanted mouse embryo later.
3 citations
References
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TL;DR: In this article, a general class of regression models for ordinal data is developed and discussed, which utilize the ordinal nature of the data by describing various modes of stochastic ordering and this eliminates the need for assigning scores or otherwise assuming cardinality instead of ordinality.
Abstract: SUMMARY A general class of regression models for ordinal data is developed and discussed. These models utilize the ordinal nature of the data by describing various modes of stochastic ordering and this eliminates the need for assigning scores or otherwise assuming cardinality instead of ordinality. Two models in particular, the proportional odds and the proportional hazards models are likely to be most useful in practice because of the simplicity of their interpretation. These linear models are shown to be multivariate extensions of generalized linear models. Extensions to non-linear models are discussed and it is shown that even here the method of iteratively reweighted least squares converges to the maximum likelihood estimate, a property which greatly simplifies the necessary computation. Applications are discussed with the aid of examples.
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TL;DR: A procedure to obtain high and repeatable fertilization frequencies for bovine in vitro fertilization (IVF) with frozen-thawed sperm was developed and Heparin was the most important factor in increasing IVF frequencies.
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TL;DR: The Two Faces of Oxygen Molecular oxygen is both benign and malign and the superoxide dismutases, by catalytically scavenging 0;, provide a defense against it and against any reactive radical species which can be derived from it.
Abstract: The Two Faces of Oxygen Molecular oxygen is both benign and malign On the one hand it provides enormous advantages and on the other it imposes a universal toxicity This toxicity is largely due to the intermediates of oxygen reduction, ie 0;, H202, and OH·, and any organism that avails itself of the benefits of oxygen does so at the cost of maintaining an elaborate system of defenses against these intermediates We will here concern ourselves with the superoxide dismutases which, by catalytically scavenging 0;, provide a defense against it and against any reactive radical species which can be derived from it
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TL;DR: The in-vitro development of 1-cell embryos beyond the 2-cell stage in response to the removal of glucose and the addition of glutamine to the culture medium suggests that glucose may block some essential metabolic process, and that glutamine may be a preferred energy substrate during early development for these mouse embryos.
Abstract: One-cell CF-1 x B6SJLF1/J embryos, which usually exhibit a 2-cell block to development in vitro, have been cultured to the blastocyst stage using CZB medium and a glucose washing procedure. CZB medium is a further modification of modified BMOC-2 containing an increased lactate/pyruvate ratio of 116, 1 mM-glutamine and 0.1 mM-EDTA but lacking glucose. Continuous culture of one-cell embryos in CZB medium allowed 83% of embryos to develop beyond the 2-cell stage of which 63% were morulae at 72 h of culture, but blastocysts did not develop. However, washing embryos into CZB medium containing glucose after 48 h of culture (3-4-cell stage) was sufficient to allow development to proceed, with 48% of embryos reaching the blastocyst stage by 96 h of culture. Exposure of embryos to glucose was only necessary from the 3-4-cell stage through the early morula stage since washing back into medium CZB without glucose at 72 h of culture still promoted the development of 50% of embryos to the blastocyst stage. The presence of glucose in this medium for the first 48 h of culture (1-cell to 4-cell stage) was detrimental to embryo development. Glutamine, however, exerted a beneficial effect on embryo development from the 1-cell to the 4-cell stage although its presence was not required for development to proceed during the final 48 h of culture. Blastocysts which developed under optimum conditions contained an average of 33.7 total cells. The in-vitro development of 1-cell embryos beyond the 2-cell stage in response to the removal of glucose and the addition of glutamine to the culture medium suggests that glucose may block some essential metabolic process, and that glutamine may be a preferred energy substrate during early development for these mouse embryos.
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TL;DR: This communication describes the successful culture of one-cell to eight-cell sheep ova and one- cell and eight- cell cattle ova to the morula and blastocyst stages and reports a high embryo survival after transfer of cultured Ova to recipient animals.
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1,009 citations