Theriogenology 81 (2014) 49–55
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40th Anniversary Special Issue
The ART of studying early embryo development: Progress and challenges in ruminant embryo culture Pat Lonergan*, Trudee Fair School of Agriculture and Food Science, University College Dublin, Belﬁeld, Dublin, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2013 Received in revised form 17 September 2013 Accepted 17 September 2013
The study of preimplantation mammalian embryo development is challenging due to difﬁculties in accessing in vivo-derived embryos in large numbers at the early stages and the inability to culture embryos in vitro much beyond the blastocyst stage. Nonetheless, embryos exhibit an amazing plasticity and tolerance when it comes to adapting to the environment in which they are cultured. They are capable of developing in media ranging in composition from simple balanced salt solutions to complex systems involving serum and somatic cells. At least a proportion of the blastocysts that develop in culture are developmentally competent as evidenced by the fact that live offspring have resulted following transfer. However, several studies using animal models have shown that such embryos are sensitive to environmental conditions that can affect future pre- and postnatal growth and developmental potential. This review summarises some key aspects of early embryo development and the approaches taken to study this important window in early life. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Development In vitro culture In vivo culture Oviduct Embryo
1. Introduction The study of early mammalian embryo development is challenging for a number of reasons. First, the very early stages of development have historically been difﬁcult to access in vivo because the embryo is located in the oviduct for the initial 3 to 4 days, from where it cannot be nonsurgically recovered using standard techniques of uterine ﬂushing. This challenge can be overcome to some extent by producing embryos using IVF, although such embryos might be inferior to those derived in vivo. Furthermore, recent developments with endoscopy allow the collection of very early stage tubal embryos . Second, the post-blastocyst hatching stages of embryo development, including the crucial period of maternal recognition of pregnancy, are particularly challenging to study in vitro because conceptus elongation is a maternally-driven process and does not occur in vitro (see later in text [Section 3]). That said, the development of * Corresponding author. Tel.: þ353 1 6012147; fax: þ353 1 6288421. E-mail address: [email protected]
(P. Lonergan). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.09.021
methods to culture embryos in vitro to the blastocyst stage has opened up a vista of possibilities to explore how embryos developdfrom basic morphological studies to highly sophisticated studies investigating underlying transcriptomic and proteomic regulatory networks that control development. In this review, we focus on speciﬁc aspects of early embryo development and, although an exhaustive review of the literature is not possible, we reference other recent reviews to which the reader can refer. 2. The effect of Theriogenology on the ﬁeld of embryo culture Considering that this special issue commemorates the 40th year of Theriogenology, it is interesting to summarize the contribution that the Journal has made to the study of embryo culture. In some way, one can chart the development of technologies, and indeed personalities, in various areas of animal reproduction, including embryo culture, by ﬂicking sequentially through the pages and issues of key journals in the area. Theriogenology is one such journal. At
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the time of writing, a search on the PubMed database with the terms ‘embryo’ and ‘theriogenology’ reveals 2420 hits dating back to 1974. The ﬁrst embryo-related article appeared in issue 2 of the Journal, entitled ‘Embryo transfer in cattle - experience of twenty-four completed cases’ by Betteridge and Mitchell . A PubMed search for the terms ‘embryo culture’ and ‘Theriogenology’ reveals 888 hits, beginning in the early- to mid-80s (e.g., [3–5]). It is important to note that these early reports involved in vivoderived embryos recovered approximately 7 days after estrus. A search for ‘IVF’ and ‘Theriogenology’ yields 474 hits including some of the seminal papers that launched IVF in cattle [6,7]. It is probably fair to say that the effect of Theriogenology on the ﬁeld of embryo technologies was facilitated by the publication of the proceedings of the International Embryo Transfer Society (IETS) as the ﬁrst issue of Theriogenology from 1978 (the fourth annual meeting of what was then still a new society) to 2003 inclusive. To an extent, these two ‘institutions’ grew up together in the early years and their association guaranteed the publication of highly citable up-to-date reviews on various aspects of embryos and embryo technology. As noted by Betteridge  in his ‘A history of farm animal embryo transfer and some associated techniques’, these [IETS] proceedings ‘serve as a yardstick with which to measure changes in emphasis and intensity of activity in embryo transfer as time progresses’. 3. Autonomy and interdependency of the embryo and reproductive tract Up to the blastocyst stage, the ruminant embryo is relatively autonomous (i.e., does not need contact with the maternal reproductive tract) as evidenced by the fact that blastocysts can be successfully developed in vitro in large numbers using IVF technology. Furthermore, based on the fact that such in vitro-derived embryos, and those recovered from superovulated donors, can establish pregnancy after transfer to the uterus of synchronized recipients, the reproductive tract does not need exposure to the embryo before Day 7 (and even up to Day 16)  for a pregnancy to be established. In contrast, the post-hatching and preimplantation conceptus is dependent on substances in the uterine lumen, termed histotroph, that are derived from the endometrium, particularly the uterine glands, for growth and development. This is demonstrated by the fact that: (1) post-hatching elongation does not occur in vitro [10,11]; and (2) the absence of uterine glands in vivo results in a failure of blastocysts to elongate [12,13]. On the maternal side, preparation of the uterine luminal epithelium for attachment of trophectoderm and implantation in all studied mammals, including ruminants, involves carefully orchestrated spatiotemporal alterations in gene expression within the endometrium. In cyclic and pregnant animals, similar changes occur in endometrial gene expression up to initiation of conceptus elongation (approximately Day 13), suggesting that the default mechanism in the uterus is to prepare for, and expect, pregnancy . Indeed, as already mentioned, it is possible to transfer an embryo to a synchronous uterus 7 days after
estrus and establish a pregnancy, as is routine in commercial bovine embryo transfer. It is only in association with maternal recognition of pregnancy, which occurs on approximately Day 16 in cattle, that signiﬁcant changes in the transcriptomic proﬁle are detectable between cyclic and pregnant endometria [14,15], when the endometrium responds to increasing interferon-tau secreted by the ﬁlamentous conceptus. 4. Interaction between the developing embryo and the oviductdtwo-way trafﬁc or a one-way street? Successful culture of the early embryo requires recapitulation of the conditions experienced during development in vivo, initially in the oviduct and then in the uterus. The oviduct is a fascinating structure. It is the site of fertilization in all domestic mammals, and has the unique ability to facilitate transport of gametes in opposite directions and in the case of the mare, apparently has the ability to distinguish between fertilized and unfertilized oocytes . During transit through the oviduct, the embryo morphologically undergoes the ﬁrst mitotic cell divisions and transcriptionally undergoes embryonic genome activation (at the eight- to 16-cell stage). Despite clear evidence of an interaction between the developing conceptus and the uterine endometrium in early pregnancy , and evidence that temporal changes occur in oviduct epithelium gene expression during the estrous cycle  reﬂecting the changing requirements of the embryo, the evidence for reciprocal cross-talk during the transit of the embryo through the oviduct is less clear. There is very convincing evidence for a positive effect of the oviduct on the quality of the early embryo. For example, short-term culture of in vitro-produced zygotes in the oviducts of sheep [18–20], cattle , rabbits , and even mice [23,24] has been shown to improve embryo quality measured in a variety of ways including morphology, gene expression, cryotolerance, and pregnancy rate after transfer. In contrast, relatively little evidence exists demonstrating an effect of the embryo on the oviduct. The limited data reporting an effect of gametes on the oviduct come from litter-bearing species, in which any effect is likely to be ampliﬁed [25–27]. We have recently characterized the transcriptome of the bovine oviduct epithelium at the initiation of embryonic genomic activation on Day 3 after estrus in pregnant and cyclic heifers (Maillo et al., unpublished). The isthmic regions, from which all eight-cell embryos and unfertilized oocytes were collected, were compared. Although large differences in gene expression were observed between the isthmus and ampulla, preliminary data suggest that the presence of an eight-cell embryo had no effect on the transcriptome of the isthmus, although a local effect at the precise position of the embryo cannot be ruled out. 5. In vitro embryo production As mentioned in the Introduction, the ability to generate large numbers of embryos in vitro at a relatively low cost has been an enormous beneﬁt to the study of early
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development. There are, however, signiﬁcant challenges associated with the technology revolving around issues of oocyte quality at one end of the process and embryo quality at the other end . Highlighting the importance of this area, and in recognition of some of the inherent problems facing users of this technology, a symposium was held in Orlando, Florida in conjunction with the 32nd Annual Conference of the IETS and published in Theriogenology, entitled, ‘Realizing the promise of IVF in cattle: optimizing embryonic and fetal survival’, at which many related topics were addressed including procedures for transferring a better embryo, errors in embryonic and fetal development, recipient management, and cryopreservation . Evidence of the potential of this technology in commercial practice is reﬂected in a follow-on IETS workshop, also in Florida, in October 2012, entitled: ‘Bringing the in vitroproduced embryo to the commercial cattle producer’. In 2011, the last year for which data are available, close to 400,000 in vitro-produced bovine embryos were transferred worldwide; of those, Brazil accounted for approximately 85%, reﬂecting an enormous increase in the use of IVF technology in commercial practice there in recent years . The in vitro production of ruminant embryos is a threestep process involving in vitro oocyte maturation, in vitro fertilization, and in vitro culture. In terms of efﬁciency, in cattle, approximately 90% of immature oocytes, generally recovered from follicles at unknown stages of the estrous cycle, undergo nuclear maturation in vitro from prophase I to metaphase II (the stage at which they would be ovulated in vivo); approximately 80% undergo fertilization after insemination and cleave at least once, to the two-cell stage. However, only 30% to 40% of such oocytes reach the blastocyst stage, at which they can be transferred to a recipient or frozen for future use. Thus, the major fall-off in development occurs during the last part of the process (in vitro culture), between the two-cell and blastocyst stages, suggesting that postfertilization embryo culture is the most critical period of the process in terms of determining the blastocyst yield. However, evidence demonstrates that the quality of the oocyte is crucial in determining the proportion of immature oocytes that form blastocysts and that the postfertilization culture environment, within certain limits, does not have a major inﬂuence on the capacity of the immature oocyte to form a blastocyst . It would appear that when the oocyte has been removed from the follicle, its ability to develop to the blastocyst stage is more or less sealed and despite attempts at temporarily inhibiting resumption of meiosis to allow cytoplasmic maturation to proceed in vitro, thereby improving development or modiﬁcations of maturation media, blastocyst yields in vitro using oocytes recovered from slaughtered animals rarely exceed 40% on a consistent basis [28,31]. In contrast, there is considerable evidence that the postfertilization culture environment is critical in determining the quality of the blastocyst. For example, by culturing in vitro-produced bovine zygotes in vivo, in the ewe oviduct, it is possible to dramatically increase the quality of the resulting blastocysts, measured in terms of cryotolerance, to a level similar to that of totally in vivoproduced embryos [18–20]. Furthermore, in the reciprocal
experiment, the culture in vitro of in vivo-produced bovine zygotes resulted in blastocysts of low cryotolerance . In other words, the culture of ‘poor quality’ zygotes, produced by in vitro maturation and fertilization, in vivo leads to the production of high-quality blastocysts (albeit at low frequency) and, conversely, the culture of ‘high quality’ zygotes, produced by in vivo maturation and fertilization, in vitro leads to the production of poor quality blastocysts (albeit at high frequency). Other evidence of the effect of culture conditions on embryo quality come from an examination of the trancriptome. Many articles have reported differences in gene expression between bovine blastocysts produced in vitro or derived in vivo. Other elaborate studies have demonstrated that the developing embryos exhibit a clear temporal sensitivity to their culture environment [32,33]. This has been achieved by culturing bovine zygotes produced using IVM/IVF either in vitro in synthetic oviduct ﬂuid (SOF) or in vivo in the sheep oviduct and recovering embryos at various stages up to the blastosyst stage to pinpoint at which stage the divergence in gene expression seen at the blastocyst stage originates . An extension of this study examined the effect of culturing in vitro-produced embryos in vitro in SOF for the ﬁrst 2 to 4 days followed by culture in the sheep oviduct to the blastocyst stage and vice-versa on blastocyst cryotolerance and gene expression . Interestingly, blastocysts derived from embryos that spent the ﬁrst 2 days in vivo and the last 4 in vitro had the lowest survival rates after cryopreservation; those which spent the last 2 days only in SOF had intermediate rates of survival, and those which spent the last 4 days of culture in vivo had high rates of survival, compared with those cultured entirely in vivo; this difference was reﬂected in the expression of a small number of candidate genes . These data suggest that the period around the time of embryonic genome activation (EGA) is crucial to the establishment of a good quality embryo. Following a similar line of investigation, but using a whole transcriptome approach rather than a candidate gene approach, and transfer of early-stage embryos to homologous bovine oviducts, Gad et al.  recently examined the inﬂuence of alternative culture conditions (in vivo or in vitro) before and during the EGA event on bovine embryonic developmental rate and gene expression pattern. In agreement with previous studies , although alternative culture conditions (in vivo or in vitro) either before or after the time of EGA had no effect on the developmental rates, the origin of the oocyte seemed to determine the embryo development. Total developmental rates until Day 7 for in vitro-generated embryos, which were transferred to in vivo conditions at the four-cell or 16cell stages were 24.1% and 27.5%, respectively. In contrast, total developmental rates until Day 7 in vitro for embryos ﬂushed at the four-cell or 16-cell stage were 64.4% and 68.2%, respectively . The study provides detailed information on molecular mechanisms and pathways that are inﬂuenced by altered culture conditions during a speciﬁc embryonic developmental time point and highlight the signiﬁcant effect of in vitro culture conditions during EGA on the transcriptome proﬁle of the resulting blastocyst. Such data contribute greatly to our knowledge of the
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regulation of embryonic development, and could be used to advance new strategies to modify the in vitro culture conditions at this critical stage of development to enhance the growth of good quality embryos.
global transcriptome using microarray or RNA sequencing technology. Metabolomic proﬁling of embryo culture media might provide a useful adjunct to more traditional methods of embryo assessment including aspects of morphology, timing of cleavage, etc.
6. Embryo metabolism Our understanding of preimplantation embryo metabolism has progressed dramatically since the pioneering work of the late 1960s and 1970s and has been summarized in a recent review by Leese . The mammalian embryo undergoes dramatic changes in morphology and energy requirements as it develops from a unicellular zygote through the early cleavage divisions to form a multicellular blastocyst. Like most mammalian cells, preimplantation embryos derive their ATP predominantly by oxidative phosphorylation, initially from pyruvate, lactate, and amino acids. After morula compaction, glucose becomes an important substrate but in quantitative terms makes only a modest contribution to ATP generation. In general, embryos throughout pre-elongation development are reliant on oxidative phosphorylation via oxidation of pyruvate and amino acids for the generation of ATP for embryo development. However, there is a switch to an increased contribution of glycolysis during compaction and blastulation (for review, see [35,36]). The composition of bovine oviduct ﬂuid, in which the ﬁrst few cell divisions occur, has been relatively well characterized in recent years in terms of energy substrates, ions, and amino acids [37–41]. Glycine, alanine, and glutamate are the predominant amino acids; glycine was the most abundant at two- to three-fold higher than alanine, threeto six-fold higher than glutamate, and up to 30-fold higher than amino acids present at lowest concentrations such as asparagine, methionine, and tryptophan . Furthermore, oviductal amino acid concentrations are modulated by progesterone; nine of 20 amino acids increased after supplementation with progesterone, with glycine showing the largest increase of approximately two-fold . Knowledge on how embryos modify their culture environment in vitro can provide information on the embryo needs. Partridge and Leese  reported that individual amino acids are depleted at different rates by bovine preimplantation embryos in vitro, suggesting that amino acid requirements change during development. Amino acid depletion was higher at later developmental stages implying an increase in amino acid requirement as development progresses. Subsequently, amino acid turnover has been used to predict developmental competence of embryos from a variety of species (human [43–45]; pigs ; and cattle ). Until quite recently, approaches to studying the metabolism of preimplantation embryos were based primarily on the targeted analysis of single or selected metabolites. More recently, there has been a move toward a more comprehensive analysis of the metabolome (metabolomics), describing changes in all low molecular weight molecules present in cells or biological ﬂuids at a particular developmental stage or physiological state [48,49]. This is somewhat analogous to the study of individual candidate genes using reverse transcription polymerase chain reaction and the
7. Systems of embryo culturedfrom microdrops to microﬂuidics Gandolﬁ and Moor  began their landmark article describing the successful coculture of in vivo-derived sheep embryos with oviduct epithelial cells with the sentence, ‘The culture of mammalian embryos from one-cell to the expanded blastocyst stage is an uncertain process’. It is probably fair to say that the process is a little more certain these days, especially using in vivo-derived embryos; in contrast, most IVF-derived embryos typically fail to reach the blastocyst stage, but this is likely because of poor oocyte quality rather than inadequacies of embryo culture media. Tervit and colleagues [51,52] were among the ﬁrst to report successful culture of ruminant zygotes to the blastocyst stage in vitro using SOF medium, which was based on the composition of ovine oviduct ﬂuid. It was later reported that supplementation of SOF with serum improved development of sheep embryos to the blastocyst stage . Around the same time many other groups were using coculture techniques, such as those described by Gandolﬁ and Moor  and Eyestone and First , and at the beginning of the 90s, coculture of cattle and sheep embryos was the most favored method to support embryo development in vitro . At the time, very little was known about the metabolic requirements of ruminant embryos. Coculture advanced the application of in vitro embryo production because it was now possible to produce large numbers of blastocysts for transfer or other studies. However, as pointed out by Bavister , such undeﬁned systems did not facilitate the understanding of factors that inﬂuence embryo development. The development of serum-free and somatic cell-free culture systems for ruminant embryosdso-called deﬁned and semideﬁned mediadcame from the desire to deﬁne the cellular requirements of the embryo during early development. One such medium was an adjustment of the original SOF formulation with the inclusion of nonessential and essential amino acids . Compared with other species, especially the mouse, progress in the development of systems for the culture of early embryos of domestic ruminants has mostly occurred since the 90s . Over the past 25 years, the most well studied variables aimed at improving embryo development have revolved around the chemical composition of the media used. Since the ﬁrst successful coculture of ruminant embryos (in vivo-derived sheep zygotes) to the blastocyst stage in the presence of oviduct epithelial cells , we have seen developments in the use of deﬁned media, of which the exact composition is known, sequential media, which, in theory, take into account the changing metabolic needs of the embryo, and methods of culturing embryos individually, or in groups, but in an individually identiﬁable manner using wells-in-wells , meshes [46,59], or on adhesive substances [59,60]. More recently, innovative
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ways of bar-coding individual embryos have even been reported [61,62]. New developments in culture platforms and the potential to improve assisted reproductive technologies have been comprehensively reviewed by Smith et al. . Automation of embryo assessment (using time-lapse) and production (using microﬂuidics) is achievable. As pointed out by Thompson , uptake of such technologies is slow because of cost and the conservative nature of embryologists. Microﬂuidics represents a very promising technology although it seems to have been promising for some time now and has not been taken up on any large scale [65–67]. 8. In vivo culturedaccessing the oviduct Culture of embryos in the oviducts of intermediate hosts was a crucial milestone in the development of methods to culture embryos in vitro. Although to go back to such in vivo culture methods might seem like a retrograde step, the application of technologies such as endoscopy to allow access to the oviducts of living animals has facilitated the study of early development in vivo in an unrivalled manner [1,68–70]. Eyestone et al.  were among the ﬁrst to describe the culture of one- and two-cell bovine embryos, collected from superovulated heifers, to the blastocyst stage in the ovine oviduct. As mentioned in Section 4, culture of in vitroproduced bovine embryos in the oviduct of the ewe has been shown by several authors to be a suitable environment for the development of embryos from the zygote to blastocyst stage and even through the early stages of elongation . Though not perfect, one advantage of this in vivo culture system is the ability to culture large numbers of embryos in a ‘near in vivo’ environment and in a costeffective manner. Although the yield of blastocysts after such in vivo culture is not superior to that after culture in vitro, the quality of the blastocysts has been shown to be signiﬁcantly improved [18,23]. Culture of bovine embryos in intermediate host oviducts of a variety of species including sheep, rabbit, and mouse, has been reviewed recently by Rizos et al. . However, heterologous transfer and culture of embryos (i.e., bovine embryos in the ovine oviduct) is never totally satisfactory from an experimental design viewpoint. Recently, endoscopy has been successfully used to access the oviducts of cattle for the in vivo culture of in vitro-matured or -fertilized embryos in the homologous oviduct [1,68–70]. Although this technique requires a signiﬁcant level of skill and experience (currently only practiced routinely by one group worldwide for the tubal transfer of embryos), it offers much promise for comparative studies of embryo development in different conditions (e.g., [34,73]). 9. Outcome measures Typical outcome measures in studies involving ruminant embryos include fertilization rate, cleavage rate (the proportion of inseminated oocytes undergoing at least one mitotic division), and blastocyst development (the proportion of inseminated oocytes reaching the blastocyst stage). Blastocyst quality is typically assessed using
noninvasive morphological criteria. Time-lapse monitoring has been used to study the kinetic characteristics of early embryo development in vitro in a variety of animal species. In cattle, the group at Louvain Le-Neuve in Belgium used time-lapse to study the development of normal and frozenthawed cow blastocysts [74,75], and later, the development of early bovine embryos , describing a lag-phase at the eight-cell stage. Holm et al.  used time-lapse to show that faster cleaving embryos are more likely to reach the blastocyst stage. Since then, other groups have used more sophisticated imaging approaches and mathematical algorithms to predict developmental outcome [78–80]. Others have used more invasive measures including staining and counting cell number, assessing cryotolerance, and examining gene expression patterns. The problem with many of these techniques is that they involve destroying the embryo. In a novel approach, El-Sayed et al.  addressed the relationship between the transcriptional proﬁle of embryos and pregnancy success based on gene expression analysis of blastocyst biopsies taken before transfer to recipients. This allowed the identiﬁcation of differentially regulated genes between embryos resulting in a calf delivery versus those not resulting in a pregnancy. This approach was subsequently applied to human embryo assessment . Information on live birth rate and offspring health is mostly restricted to humans because of the difﬁculty in capturing posttransfer data in domestic species. Such data are nonetheless critical, especially considering the evidence of long-term consequences of embryo culture on offspring health . 10. Concluding remarks Despite the challenges involved in successfully culturing embryos in vitro to a stage at which they can be transferred to a recipient with the realistic expectation of acceptable pregnancy rates, we have come a long way in our understanding of the factors regulating early embryo development and the interaction (or lack of interaction at the very early stages) between the embryo and the maternal reproductive tract. With the number of bovine embryos transferred worldwide increasing annually, there is a greater need than ever to optimize conditions of embryo culture in vitro to maximize embryo quality, cryotolerance, and ultimately, pregnancy rate after transfer. References  Besenfelder U, Havlicek V, Kuzmany A, Brem G. Endoscopic approaches to manage in vitro and in vivo embryo development: use of the bovine oviduct. Theriogenology 2010;73:768–76.  Betteridge KJ, Mitchell D. Embryo transfer in cattle - experience of twenty-four completed cases. Theriogenology 1974;1:69–82.  Allen RL, Bondioli KR, Wright Jr RW. The ability of fetal calf serum, new-born calf serum and normal steer serum to promote the in vitro development of bovine morulae. Theriogenology 1982;18:185–9.  Betterbed B, Wright Jr RW. Development of one-cell ovine embryos in two culture media under two gas atmospheres. Theriogenology 1985;23:547–53.  Voelkel SA, Amborski GF, Hill KG, Godke RA. Use of a uterine-cell monolayer culture system for micromanipulated bovine embryos. Theriogenology 1985;24:271–81.  Greve T, Bousquet D, King WA, Betteridge KJ. In vitro fertilization and cleavage of in vivo matured bovine oocytes. Theriogenology 1984;22:151–65.
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