Effect of culture environment on gene expression and developmental characteristics in IVF-derived embryos

It is generally accepted that mammalian preimplantation embryos are sensitive to their environment and that conditions of culture can affect future growth and developmental potential both pre- and postnatally. Evidence suggests that while culture conditions during bovine in vitro embryo production can impact somewhat on 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. In addition, evidence suggests that the period of post fertilization embryo culture is the most critical period affecting blastocyst quality assessed in terms of cryotolerance, gene expression pattern and ability to establish a pregnancy. This paper reviews the current literature, with emphasis on the bovine model, demonstrating evidence for an effect of post fertilization culture environment on embryo gene expression and quality.     

Energy metabolism in preimplantation bovine embryos derived in vitro or in vivo

This study was an investigation of metabolism during bovine preimplantation development from the oocyte up to the hatched blastocyst derived in vitro or in vivo. Metabolism was determined by estimating the consumption of radiolabeled glucose, pyruvate, or lactate during a 4-h incubation period in a closed noninvasive system with NaOH as trap for the continuous collection of CO(2). The postincubation medium was analyzed for the presence of lactate. Embryonic metabolism from the matured oocyte to the 12-cell stage was more or less constant, with pyruvate being the preferred substrate. The first marked increase in oxidation of glucose occurred between the 12- and 16-cell stage. Compaction of morula and blastocyst expansion was accompanied by significant increases in oxidation of all three energy substrates. The incorporation of glucose increased steadily 15-fold from the 1-cell to the blastocyst stage. In general, the pattern of metabolism was similar between the embryos derived in vitro and in vivo but with some distinct differences. The most apparent feature of glucose metabolism by in vitro-produced embryos was a 2-fold higher rate of aerobic glycolysis as compared to that in their in vivo counterparts. In vitro-matured oocytes produced measurable amounts of lactate, whereas in vivo-matured oocytes exhibited a significantly lower metabolic activity and did not produce any lactate. When in vivo-collected embryos were preexposed to culture conditions, lactate production increased significantly and at the hatched blastocyst stage matched that of their in vitro counterparts. In vitro-produced embryos up to the 8-cell stage oxidized significantly higher amounts of lactate and had a lower ratio of pyruvate-to-lactate oxidation than the in vivo-obtained embryos. The results of this study show that under our culture conditions, important differences exist at the biochemical level between bovine embryos produced in vitro and those generated in vivo that may well affect the developmental capacity.     

A potential role for triglyceride as an energy source during bovine oocyte maturation and early embryo development

The potential role of endogenous triglyceride in bovine oocyte maturation and preimplantation development has been investigated. Bovine immature oocytes were recovered from abattoir-derived ovaries, matured and fertilised in vitro and the zygotes grown to the blastocyst stage in SOFaaBSA. Methyl palmoxirate (MP) blocks the oxidation of fatty acids by inhibiting mitochondrial carnitine palmitoyltransferase A. The development of zygotes exposed to MP during oocyte maturation, and of zygotes exposed to MP during embryo culture has been assessed in terms of oxygen consumption by oocytes and embryos during a 4-6 hr incubation period in the presence of MP and as blastocyst formation and cell number. Immature oocytes exposed to MP during maturation had reduced capacity to form blastocysts after fertilisation; the same effect was apparent, but to a lesser extent, in zygotes exposed to MP during embryo development. Oxygen consumption values of oocytes and blastocysts in the absence of exogenous substrates were similar to those in control medium containing nutrients. MP-inhibited oxygen consumption of immature oocytes, mature oocytes, cleavage stages embryos and blastocysts by 64, 45, 12 and 13%, respectively. The data are consistent with a role for triglyceride as a key energy source during bovine oocyte maturation and potentially, during preimplantation embryo development.     

Inhibition of endoplasmic reticulum stress improves mouse embryo development

X-box binding protein-1 (XBP-1) is an important regulator of a subset of genes during endoplasmic reticulum (ER) stress. In the current study, we analyzed endogenous XBP-1 expression and localization, with a view to determining the effects of ER stress on the developmental competency of preimplantation embryos in mice. Fluorescence staining revealed that functional XBP-1 is localized on mature oocyte spindles and abundant in the nucleus at the germinal vesicle (GV) stage. However, in preimplantation embryos, XBP-1 was solely detected in the cytoplasm at the one-cell stage. The density of XBP-1 was higher in the nucleus than the cytoplasm at the two-cell, four-cell, eight-cell, morula, and blastocyst stages. Furthermore, RT-PCR analysis confirmed active XBP-1 mRNA splicing at all preimplantation embryo stages, except the one-cell stage. Tunicamycin (TM), an ER stress inducer used as a positive control, promoted an increase in the density of nuclear XBP-1 at the one-cell and two-cell stages. Similarly, culture medium supplemented with 25 mM sorbitol displayed a remarkable increase active XBP-1 expression in the nuclei of 1-cell and 2-cell embryos. Conversely, high concentrations of TM or sorbitol led to reduced nuclear XBP-1 density and significant ER stress-induced apoptosis. Tauroursodeoxycholic acid (TUDCA), a known inhibitor of ER stress, improved the rate of two-cell embryo development to blastocysts by attenuating the expression of active XBP-1 protein in the nucleus at the two-cell stage. Our data collectively suggest that endogenous XBP-1 plays a role in normal preimplantation embryonic development. Moreover, XBP-1 splicing is activated to generate a functional form in mouse preimplantation embryos during culture stress. TUDCA inhibits hyperosmolar-induced ER stress as well as ER stress-induced apoptosis during mouse preimplantation embryo development.     

BMP4 plays a role in apoptosis during human preimplantation development

Comprehensive understanding of lineage differentiation and apoptosis processes is important to increase our knowledge of human preimplantation development in vitro. We know that BMP signaling is important for different processes during mammalian development. In mouse preimplantation embryos, BMP signaling has been shown to play a role in the differentiation into extra‐embryonic trophectoderm (TE) and primitive endoderm (PE). In this study, we aimed to investigate the effect of bone morphogenetic protein 4 (BMP4) supplementation on human preimplantation embryos cultured in vitro. The BMP4 treatment impaired human blastocyst formation. No differences in the expression of the early lineage markers NANOG, CDX2, GATA3, and GATA6 were found between BMP4‐treated embryos and controls. Instead, BMP4 supplementation triggered apoptosis in the human blastocyst. We focused on P53, which is known to play a major role in the apoptosis. In BMP4‐treated embryos, the P53 responsive gene expression was not altered; however, the P53 deacetylase SIRT1 was downregulated and acetylated P53 was increased in mitochondria. Altogether, our findings suggest that BMP4 plays a role in the apoptosis during human preimplantation development.     

Chronology of apoptosis in bovine embryos produced in vivo and in vitro

The postimplantation developmental potential of embryos can be affected by various forms of cell death, such as apoptosis, at preimplantation stages. However, correct assessment of apoptosis is needed for adequate inference of the developmental significance of this process. This study is the first to investigate the independent chronological occurrence of apoptotic changes in nuclear morphology and DNA degradation (detected by the TUNEL reaction) and incidences of nuclei displaying these features at various preimplantation stages of bovine embryos produced both in vivo and in vitro. Different elements of apoptosis were observed at various developmental stages and appeared to be differentially affected by in vitro production. Nuclear condensation was observed from the 6-cell stage in vitro and the 8-cell stage in vivo, whereas the TUNEL reaction was first observed at the 6-cell stage in vitro and the 21-cell stage in vivo. Morphological signs of other forms of cell death were also observed in normally developing embryos produced both in vivo and in vitro. The onset of apoptosis seems to be developmentally regulated in a stage-specific manner, but discrete features of the apoptotic process may be differentially regulated and independently modulated by the mode of embryo production. Significant differences in indices of various apoptotic features were not evident between in vivo- and in vitro-produced embryos at the morula stage, but such differences could be observed at the blastocyst stage, where in vitro production was associated with a higher degree of apoptosis in the inner cell mass.     

Quantification of basigin mRNA in mouse oocytes and preimplantation embryos by competitive RT-PCR

Basigin is a member of the immunoglobulin superfamily and a key molecule related to mouse blastocyst implantation. Whether preimplantation mouse embryos express basigin mRNA is still unknown. The aim of this study was to use a quantitative competitive polymerase chain reaction to assess quantitatively the levels of basigin mRNA in mouse oocyte and preimplantation embryos. Basigin mRNA was detected in the oocyte and all the stages of preimplantation embryos. The levels of basigin mRNA were 0.0606 +/- 0.0282 in the oocyte, 0.0102 +/- 0.0036 in the zygote, 0.0007 +/- 0.0003 in the 2-cell embryo, 0.0031 +/- 0.0017 in the 4-cell embryo, 0.0084 +/- 0.0024 in the 8-cell embryo, 0.0537 +/- 0.0121 in the morula and 0.0392 +/- 0.0161 attomoles in the blastocyst, respectively. The levels of basigin mRNA in the oocyte, morula and blastocyst were significantly higher than those in the zygote and embryos at the 2-cell, 4-cell and 8-cell stages. The high level of basigin expression in the blastocyst may play a role during embryo implantation.     

Epigenetic disorders and altered gene expression after use of Assisted Reproductive Technologies in domestic cattle

The use of Assisted Reproductive Technologies (ARTs) in modern cattle breeding is an important tool for improving the production of dairy and beef cattle. A frequently employed ART in the cattle industry is in vitro production of embryos. However, bovine in vitro produced embryos differ greatly from their in vivo produced counterparts in many facets, including developmental competence. The lower developmental capacity of these embryos could be due to the stress to which the gametes and/or embryos are exposed during in vitro embryo production, specifically ovarian hormonal stimulation, follicular aspiration, oocyte in vitro maturation in hormone supplemented medium, sperm handling, gamete cryopreservation, and culture of embryos. The negative effects of some ARTs on embryo development could, at least partially, be explained by disruption of the physiological epigenetic profile of the gametes and/or embryos. Here, we review the current literature with regard to the putative link between ARTs used in bovine reproduction and epigenetic disorders and changes in the expression profile of embryonic genes. Information on the relationship between reproductive biotechnologies and epigenetic disorders and aberrant gene expression in bovine embryos is limited and novel approaches are needed to explore ways in which ARTs can be improved to avoid epigenetic disorders.