In vitro-produced bovine embryos: dealing with the warts

Bovine in vitro embryo production is an inefficient process; while maturation and fertilization proceed apparently normally, the proportion of embryos reaching the transferable (blastocyst) stage is rarely over 40% and those that do reach this stage are often compromised in quality and competence. There is considerable evidence of a significant influence of follicular origin on oocyte developmental potential and it appears that once the oocyte is removed from the follicle its developmental capacity is capped. Evidence suggests that while culture conditions during bovine in vitro embryo production can impact somewhat the developmental potential of the early embryo, the intrinsic quality of the oocyte is the key factor determining the proportion of oocytes developing to the blastocyst stage. This paper highlights some of the problems associated with in vitro production of embryos and discusses some of the ways of overcoming these problems.     

Regulation of gene expression in bovine blastocysts in response to oxygen and the iron chelator desferrioxamine

Low (2%) oxygen conditions during postcompaction culture of bovine blastocysts improve embryo quality and are associated with small increases in the expression of glucose transporter 1 (SLC2A1), anaphase promoting complex (ANAPC1), and myotrophin (MTPN), suggesting a role for oxygen in the regulation of embryo development, mediated through oxygen-sensitive gene expression. However, bovine embryos, to at least the blastocyst stage, lack detectable levels of the key regulator of oxygen-sensitive gene expression, hypoxia-inducible 1 alpha (HIF1A), while the less well-characterized HIF2 alpha protein is readily detectable. Here we report that other key HIF1 regulated genes are not significantly altered in their expression pattern in bovine blastocysts in response to reduced oxygen concentrations postcompaction-with the exception of lactate dehydrogenase A (LDHA), which was significantly increased following 2% oxygen culture. Antioxidant enzymes have been suggested as potential HIF2 target genes, but their expression was not altered following low-oxygen culture in the bovine blastocyst. The addition of desferrioxamine (an iron chelator and inducer of HIF-regulated gene expression) during postcompaction stages significantly increased SLC2A1, LDHA, inducible nitric oxide synthase (NOS2A), and MTPN gene expression in bovine blastocysts, although development to the blastocyst stage was not significantly affected. These results further suggest that expression of genes, known to be regulated by oxygen via HIF-1 in somatic cells, is not influenced by oxygen during preimplantation postcompaction bovine embryo development. Oxygen-regulated expression of LDHA and SLC2A1 in bovine blastocysts suggests that regulation of these genes may be mediated by HIF2. Furthermore, the effect of a reduced-oxygen environment on gene expression can be mimicked in vitro through the use of desferrioxamine. These results further support our data that the bovine blastocyst stage embryo is unique in its responsiveness to oxygen compared with somatic cells, in that the lack of HIF1-mediated gene expression reduces the overall response to low (physiological) oxygen environments, which appear to favor development.     

Factors influencing oocyte and embryo quality in cattle

Following in vitro maturation, approximately 90% of immature bovine oocytes will reach metaphase II and extrude the first polar body; approximately 80% will undergo fertilization and cleave, at least once, to the two-cell stage. However, only about 30-40% will ever reach the blastocyst stage. This would suggest that the post-fertilization part of the process of in vitro embryo production, the longest part, is the main period determining blastocyst yield. The experiments described in this paper clearly demonstrate that this is. in fact, not the case and that it is events further back along the developmental axis that determine the proportion of immature oocytes reaching the blastocyst stage. The results also show, however, that the post-fertilization culture period is of profound importance in determining the equality of those blastocysts that do develop, with those produced in vitro consistently being of inferior quality to their in vivo produced conterparts. The challenge for the future is to modify our conditions of post-fertilization embryo culture in an attempt to mimic those that occur naturally in vivo and in that way improve blastocyst quality.     

Culture of bovine embryos in intermediate host oviducts with emphasis on the isolated mouse oviduct

The oviduct provides the optimal environment for the transport of sperm and oocyte at the earliest stages of mammalian embryo development. During the early postfertilization period, several major developmental events occur in the embryo including (i) the first cleavage division, (ii) activation of the embryonic genome, (iii) compaction of the morula, and (iv) formation of the blastocyst. Most of these events are initiated in the oviduct. The absence of assistance from the oviduct may compromise the developmental ability of the cattle embryo under in vitro culture conditions. The oviducts of several mammalian species, including rabbits, cow, sheep (in situ), and mice (organ culture), can sustain early bovine embryos and yield blastocysts of better quality compared with those of culture conditions in vitro, leading to normal pregnancy rates in recipient animals. This review focuses on the use of oviducts in vitro or in vivo as intermediate hosts for postfertilization culture environment of bovine in vitro-produced zygotes with emphasis on the mouse model.     

Gene expression regulating blastocyst formation

Development of embryos to the blastocyst stage is a critical event in the early lives of all eutherian mammalian species. Blastocyst formation is essential for implantation and is the principal morphological determinant of embryo quality prior to embryo transfer. The physiological events and roles of specific gene families that regulate blastocyst formation are subjects of intense research Recent findings have demonstrated that bovine embryos express multiple members of the Na/K-ATPase ion transporter gene family. Two members of this family have been co-localized to bovine trophectoderm, but each becomes largely confined to opposing cell membrane margins. Bovine blastocysts display a greater sensitivity to ouabain (potent inhibitor of the Na/K-ATPase) than murine blastocysts, and enzyme activity (ouabain sensitive 86Rb+ uptake) undergoes a 9-fold increase from the bovine morula to the blastocyst stage. Disruption of Na/K-ATPase gene expression by antisense oligodeoxynucleotide inhibition abolishes blastocyst formation. These results have implicated the Na/K-ATPase as a key regulator of bovine blastocyst formation and have provided insights necessary for the production of healthy bovine embryos by the application of in vitro maturation, in vitro fertilization and in vitro culture methods.     

Maternal supply of omega-3 polyunsaturated fatty acids alter mechanisms involved in oocyte and early embryo development in the mouse

Despite the well-known benefits of omega-3 (n-3) polyunsaturated fatty acid (PUFA) supplementation on human health, relatively little is known about the effect of n-3 PUFA intake on fertility. More specifically, the aim of this study was to determine how oocyte and preimplantation embryo development might be influenced by n-3 PUFA supply and to understand the possible mechanisms underlying these effects. Adult female mice were fed a control diet or a diet relatively high in the long-chain n-3 PUFAs for 4 wk, and ovulated oocytes or zygotes were collected after gonadotropin stimulation. Oocytes were examined for mitochondrial parameters (active mitochondrial distribution, mitochondrial calcium and membrane potential) and oxidative stress, and embryo developmental ability was assessed at the blastocyst stage following 1) in vitro fertilization (IVF) or 2) culture of in vivo-derived zygotes. This study demonstrated that exposure of the oocyte during maturation in the ovary to an environment high in n-3 PUFA resulted in altered mitochondrial distribution and calcium levels and increased production of reactive oxygen species. Despite normal fertilization and development in vitro following IVF, the exposure of oocytes to an environment high in n-3 PUFA during in vivo fertilization adversely affected the morphological appearance of the embryo and decreased developmental ability to the blastocyst stage. This study suggests that high maternal dietary n-3 PUFA exposure periconception reduces normal embryo development in the mouse and is associated with perturbed mitochondrial metabolism, raising questions regarding supplementation with n-3 PUFAs during this period of time.     

The influence of nuclear content on developmental competence of gaur x cattle hybrid in vitro fertilized and somatic cell nuclear transfer embryos

In nondomestic and endangered species, the use of domestic animal oocytes as recipients for exotic donor nuclei causes the normal pattern of cytoplasmic inheritance to be disrupted, resulting in the production of nuclear-cytoplasmic hybrids. Evidence suggests that conflict between nuclear and cytoplasmic control elements leads to a disruption of normal cellular processes, including metabolic function and cell division. This study investigated the effects of nuclear-cytoplasmic interactions on the developmental potential of interspecies embryos produced by in vitro fertilization and somatic cell nuclear transfer: cattle x cattle, gaur x cattle, hybrid x cattle. Cattle control and hybrid embryos were examined for development to the blastocyst stage and blastocyst quality, as determined by cell number and allocation, apoptosis incidence, and expression patterns of mitochondria-related genes. These analyses demonstrated that a 100% gaur nucleus within a domestic cattle cytoplasmic environment was not properly capable of directing embryo development in the later preimplantation stages. Poor blastocyst development accompanied by developmental delay, decreased cell numbers, and aberrant apoptotic and related gene expression profiles, all signs of disrupted cellular processes associated with mitochondrial function, were observed. Developmental potential was improved when at least a portion of the nuclear genome corresponded to the inherited cytoplasm, indicating that recognition of cytoplasmic components by the nucleus is crucial for proper cellular function and embryo development. A better understanding of the influence of the cytoplasmic environment on embryonic processes is necessary before interspecies somatic cell nuclear transfer can be considered a viable alternative for endangered species conservation.     

Gene expression of bovine embryos developing at the air-liquid interface on oviductal epithelial cells (ALI-BOEC)

We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array? on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.     

Control of oocyte maturation in cows--biological factors

Since bovine in vitro fertilization became possible in the early 80s, a lot of effort has been done to clarify the mechanisms of what seems more and more one of the crucial steps in this procedure, being oocyte maturation. Undoubtedly, many biological factors act together to prepare the immature oocyte for a successful development to a competent embryo after fertilization. Defects in oocyte maturation can possibly be caused by an inadequate nuclear or cytoplasmic maturation or even by a failure of both. There is a general agreement upon the fact that the origin of the oocyte can play an important role. Oocytes derived from very small follicles show a lower rate of maturation and lower blastocyst development with currently used maturation protocols. Parthenogenetic activation of small size follicle derived oocytes suggests that their poor development was not caused by fertilization problems but more likely by intrinsic oocyte factors. Similar developmental rates achieved through nuclear transfer and parthenogenetic activation suggests that the nucleus of the incompetent oocyte may not be the sole reason for a poor development. Another important factor appears to be the donor animal age. The younger the donor animal, the more impaired is its oocyte's developmental competence in most of the embryo IVP systems. Treatment with exogeneous gonadotropins can be beneficial in young donors on the oocyte cleavage rates but does not always increase the final blastocyst outcome. This review briefly documents some of the biological factors and their possible effects on the developmental capacities of the bovine oocyte in vitro.     

Bovine embryos release extracellular vesicles with differential miRNA signature during the compaction and blastulation stages

Pre-implantation embryos release extracellular vesicles (EVs) to extracellular environment. In this work it is hypothesized that the EVs miRNA cargo will vary during pre-implantation development due to the constant changes in gene expression that take place through this period. The concentration, size and miRNA cargo of EVs secreted by competent bovine embryos during the period from compaction to blastulation (Day 3‐7) were analyzed. For this analysis tow developmental windows were defined: W2 from 8-cells (D3) to morula (D5) and W3 from morula (D5) to blastocyst (D7). For W2, in vitro produced embryos were individually cultured in EVs-depleted medium from D3 to D5; culture media were collected and assigned to Group W2. Morulae were kept in culture up to blastocyst stage to determine the developmental competence. For W3, D5 morulae were collected and cultured individually in EVs-depleted medium up to blastocyst stage; culture media were assigned to Group W3, and blastocysts were kept in culture up to day 11 to define their competence. The mean size of EVs was similar between groups, however, EVs concentration was lower in W2. A total of 140 miRNAs were identified. From them, 79 were differentially expressed between the groups, 28 upregulated and 51 downregulated. miRNAs differentially detected between both developmental windows participate in the regulation of signaling pathways which crucial for embryonic development. It was concluded that the secretion of EVs is regulated by the developmental progress of the embryo during the pre-implantation period.