- Open Access
Systematic analysis of the factors that adversely affect the rate of cell accumulation in mouse embryos during their culture in vitro
© Jin and O’Neill; licensee BioMed Central Ltd. 2014
- Received: 23 January 2014
- Accepted: 1 May 2014
- Published: 8 May 2014
Retarded embryo growth is a pervasive effect of culture in vitro.
A systematic analysis of the interactions between media design, embryo culture density, oxygen tension, amino acids, trophic ligands and the genetic background of the mouse on embryo growth rates in vitro was performed.
Growth retardation of mouse zygotes was greater in 20% O2 than 5%, a sequential media design was superior to static simple media designs, but the supplementation of simple media with mixed amino acids mitigated this difference. There was a beneficial effect of communal culture in small volumes, and supplementation with a trophic ligand (Paf) further enhanced growth rates. For hybrid strain zygotes (B6CBF1) communal culture in KSOM media supplemented with amino acids, albumin and Paf under 5% O2 resulted in complete rescue of their rate of accumulation of cells and blastocyst formation. Inbred strain (C57BL6/J) zygotes, however, still showed some retardation of development under these conditions. The additional supplementation of media with another trophic ligand (IGF1) showed a further additive beneficial effect on development of inbred strain embryos but they still showed a growth deficit of ~ 23% cell number. The results show that optimising the interactions between a range of culture conditions and media design can rescue hybrid strain embryos from a retarded rate of cell proliferation caused by culture in vitro, but this was incomplete for the B6 strain.
The results indicate that the growth requirement of embryos in vitro varies depending upon their genetic background and provide models for the further genetic analysis of embryo growth.
- Reproductive techniques
- Cell hypoxia
The culture of the preimplantation embryo is central to most forms of reproductive technology and assisted reproduction. From the earliest days of development of this technology the retarded rate of development of the embryo in vitro compared to those growing in vitro has been a pervasive feature of the technology [1, 2].
Recent advances in media design have resulted in improvements in the viability of the resulting embryos, yet the developmental potential of embryos created by assisted reproductive technologies is still regarded as a major limiting factor to success. Advances include the development of sequential media systems in an attempt to more closely replicate the dynamic metabolic requirements of the embryo at different stages of development . An alternative approach has been the systematic empirical development of more optimised media, as exemplified by KSOM . The supplementation of media with mixed amino acids [5, 6] provides further benefits, and the advantage of low oxygen concentration has long been recognised [7–10]. There is also evidence for a beneficial role of communal culture of embryos [11–13] to maximise the actions of autocrine tropic ligands [14–16]. This can also be partially achieved by supplementation of media with exogenous ligands [11, 17, 18]. It is likely that there is a significant genetic component to the embryo’s capacity to develop successfully. There is much anecdotal evidence for marked differences in development in vitro of embryos from mouse strains of different genetic backgrounds. The inbred C57BL/6 J (B6) strain is particularly sensitive to culture in simple defined media, compared to the robust growth response of a hybrid strain . Of interest, the release of putative trophic ligands may also be associated to the genetic background in mice [20–22].
The last decade has seen marked improvements in media and culture system design and this has been associated with consistent improvements in outcomes of assisted reproductive technologies. Yet most studies have looked at variables in isolation and there is only limited understanding of the potential beneficial interactions between the wide range of variables affecting embryo growth rates in vitro. The aim of this study was to determine whether beneficial or deleterious interactions exist between these factors on embryo growth in vitro. The results define the range of conditions required for complete rescue of the growth rate of mouse hybrid strain embryos, and points to areas for further development for strains that are most sensitive to the effects of culture.
The use of animals was in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and was approved by the Institutional Animal Care and Ethics Committee. Inbred strain (C57BL/6 J, B6) and hybrid strain (C57BL/6 J X CBA/He, B6CBF1) mice were used in experiments. Animals were housed and bred in the Kearns Research Laboratory, St Leonards, NSW, Australia. All animals were under 12 h light: 12 h dark cycle and had access to food and water ad libitum. Six week old females had ovulation-induced by intraperitoneal injection of 5 IU equine chorionic gonadotrophin (Folligon, Intervet International, Boxmeer, The Netherlands) followed 48 h later by 5 IU human chorionic gonadotrophin (hCG, Chorulon, Intervet). Females were paired with males of proven fertility. Pregnancy was confirmed by the presence of a copulation plug the following morning (day 1). Embryos were collected at various times after hCG (h post-hCG) as shown in results.
Mouse embryo collection and culture
Zygotes were collected from the reproductive tract 18 h post-hCG in Hepes-buffered human tubal fluid medium (Hepes-HTF)  and freed from their cumulus cells by brief exposure to 300 IU hyaluronidase (Sigma Chemical Company, St Louis, MO). Two-cell embryos were collected at 40 h post-hCG. After thorough washing in Hepes-HTF, embryos were cultured in human tubal fluid medium supplemented with glutamine and EDTA (GE-HTF) , KSOM , or a proprietary human ART media (Sydney IVF medium suite, Cook Australia, Limited) . All components of GE-HTF and KSOM media were tissue culture grade (Sigma). Amino acid supplementation was by the addition of 1 ml MEM amino acid solution (Sigma, cat#11130051) and 0.5 ml MEM non-essential amino acid solution (Sigma Cat#11140050) to 50 ml of media. GE-HTF and KSOM was supplemented with 3 mg bovine serum albumin (BSA)/mL (crystallised, Sigma). In some experiments media was supplemented with the embryotropins Paf (1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine) (Sigma) or IGF1 (recombinant Insulin-like Growth Factor-I from mouse, Life Technologies) . Sydney IVF media suite (sIVF) is a sequential media design with two formulations, sIVF cleavage medium (K-SICM-50) and Blastocyst medium (K-SIBM-50) and each is supplemented with human serum albumin. Zygotes were cultured in K-SICM-50 for 48 h (or after 28 h for culture of 2-cell embryos) followed by transfer to K-SIBM-50 for a further 48 h.
Embryos were cultured individually or in groups of 10 in 10 μL drops in 60-well plates (LUX 5260, Nunc, Naperville, IL) overlaid by approximately 2 mm of heavy paraffin oil (Sigma). Culture was performed at 37°C in 5% CO2 in either air (20% O2) or 5% O2, 90% N2 for the periods indicated in individual experiments.
In some experiments embryos were analyzed at a number of time points and cell counts performed to assess the rate of accumulation of cells in the embryo over time. Up to the uncompacted 8-cell stage cell counts could be readily performed by observation by dissecting microscope, from the time of compaction cell counts were performed on cohorts of embryos, which were fixed in 2% (w/v) paraformaldehyde (Sigma) in phosphate buffer saline (PBS) (Sigma) for 30 min and stained with 10 μg/mL Hoechst 33342 (Sigma) in PBS. The stained nuclei were visualised and counted. The proportion developing to morphological blastocysts and hatching blastocysts was recorded. The number of nuclei that showed normal morphology or punctuate or fragmented structure (indicative of degeneration) was recorded.
The statistical analysis was performed with SPSS for Windows (Version 19.0, SPSS Inc., Chicago, IL, USA). The cell number was quantitatively analysed by Univariate analysis of variance. In the model, cell number was the dependent variable, test treatments the independent variables and experimental replicate was incorporated in the model as a covariate. Test of main factor effects and interaction effects was performed. Difference between individual treatments was by multiple comparisons using the least significance difference test. The development rates of blastocysts and hatching blastocysts was assessed by binary logistic regression analysis.
This study confirms the well established observation that defined media causes a marked retardation of the accumulation of cells in the preimplantation embryo during culture in vitro. This retardation was greatest when culture commenced at the early zygote stage. As expected, culture at an oxygen tension of 5% resulted in less retardation than 20%. Culture in a sequential media design (Sydney IVF media) supported faster development than static media designs (GE-HTF or KSOM), but the addition of mixed amino acids to the static media overcame this difference. In the presence of amino acids no advantage of sequential media design was detected. The benefit of group culture (in hybrid strains) indicated a requirement for autocrine trophic support, and the addition of exogenous trophic ligands provided further additive benefits to growth. The uniformly poor development of B6 embryos (whether cultured individually or communally) and the benefits of exogenous tropic ligands, indicate that embryos of this strain may have a profound deficiency of production of autocrine trophic ligands in vitro. It has been shown that the release of the autocrine embryotrophic ligand (Paf) is under genetic control  and this might also apply to other autocrine trophic ligands. The well characterised high level of sensitivity of B6 strain embryos to culture in vitro  has been shown to be associated with the loss of P53 regulation by trophic ligands in vitro . This study does not define whether the retarded rates of accumulation of cells within embryos was a result of a reduced rate of cell-cycle progression or an increased rate of cell death (by apoptosis or necrosis) or both. These are important mechanistic questions for future analysis.
A key finding of the study was that while all the variables tested could in isolation provide a growth benefit, none were by themselves able to completely compensate for the retarded cell accumulation rate occurring in vitro. By contrast, the combination of all these beneficial treatments had additive effects so that under the best conditions tested the rate of cell accumulation in hybrid strain preimplantation embryos was not different from the rate observed in the reproductive tract. This suggests that the ionic and nutritional composition of media such as KSOM supplemented with amino acids meet the minimum requires of embryos for optimal cell-cycle progression, provided that they are in a relatively hypoxic environment and received appropriate levels of tropic stimulation, achieved by a combination of communal culture in small media volumes and the addition of exogenous ligands, including Paf and IGF1. It is noteworthy that IGF1 caused a quadratic dose–response with a beneficial effect on cell accumulation at the lowest concentration but this was reversed at the highest concentrations. This is consistent with our earlier observation  of the effect of IGF1 on the development of outbred strain embryos, but in that case the dose response was shifted to the right with optimal development occurring at concentrations up to 50 ng/mL and its reversal at 3 μg/mL. A similar quadratic dose-dependent effect has been observed for the effects of Paf on embryos in vitro . It is also important to consider in future if the shape of this dose–response curve is influenced by the co-treatment of embryos with Paf. An important point to be considered is that these potent biological mediators were provided by weight rather than known biological potency. It is probable that such commercial preparations show considerable variability in biological activity between suppliers and batches. An important requirement prior to use of biologicals as part of routine media formulation is to define appropriate standard measures of biological potency and to define the conditions required for stable storage and use of such labile reagents. Failure to do so will result in great variability in outcomes.
While these conditions completely rescued the cell-cycle rate in hybrid strain embryos, it is important to recognise that embryos of this strain are known to be relatively robust in culture and to show a smaller loss of viability than some other strains. So this rescue may not be sufficient to meet the requirements of all embryos in a genetically diverse population that can be expected in a clinical environment. Inbred strains, such as B6, by contrast are recognised for their greater sensitivity to culture and a corresponding loss of viability and developmental potential after culture. This study shows that the culture conditions that were capable of complete rescue of growth parameters of hybrid strain embryos, while providing a large improvement in the growth of B6 embryos, were not sufficient to completely rescue these embryos. The addition of an extra trophic ligand (IGF1) caused further improvement and this may indicate that further screening of a range of target ligands may allow discovery a set of conditions capable of completely rescuing cell accumulation rates across a range of genetic backgrounds.
The tropic ligands have important roles as survival signals, stimulants of cell-cycle progression [27, 28] and may be essential for activation for correct patterns of transcription from the early embryo at the time of embryonic genome activation . The narrow effective dose range of trophic ligands (and their adverse effects at supraphysiological levels), however, indicates that caution is required in the clinical use of such agents . There should be no rush to the clinic with their use without appropriate thorough investigation of their effectiveness and safety. This study shows that the use of culture-sensitive mouse strains, such as B6, may be valuable tools for further identification of the conditions and treatments required for the ongoing optimisation of embryo culture techniques.
The work was supported by grants from the Australian National Health and Medical Research Council to CO. We thank Cook Medical Australia for the gift of sIVF media.
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