Comparative aspects of early lineage specification events in mammalian embryos – insights from reverse genetics studies

Within the mammalian class, formation of the blastocyst is morphologically highly conserved among different species. The molecular and cellular events during preimplantation embryo development have been studied extensively in the mouse as model organism, because multiple genetically defined strains and a plethora of reverse genetics tools are available to dissect specific gene functions and regulatory networks. However, major differences in preimplantation developmental kinetics, implantation, and placentation exist among mammalians, and recent studies in species other than mouse showed, that even regulatory mechanisms of the first lineage differentiation events and maintenance of pluripotency are not always conserved. Here, we focus on the first and the second lineage segregation in mouse and bovine embryos, when the first differentiated cell types emerge. We outline their common features and differences in the regulation of these essential events during embryonic development with a glance at further species. In addition, we show how new reverse genetics strategies aid the study of regulatory circuits in embryos of domestic species, enhancing our overall understanding of mammalian preimplantation development.     

Protein kinase Akt2/PKBβ is involved in blastomere proliferation of preimplantation mouse embryos

Activation of Akt/Protein Kinase B (PKB) by phosphatidylinositol-3-kinase (PI3K) controls several cellular functions largely studied in mammalian cells, including preimplantation embryos. We previously showed that early mouse embryos inherit active Akt from oocytes and that the intracellular localization of this enzyme at the two-cell stage depends on the T-cell leukemia/lymphoma 1 oncogenic protein, Tcl1. We have now investigated whether Akt isoforms, namely Akt1, Akt2 and Akt3, exert a specific role in blastomere proliferation during preimplantation embryo development. We show that, in contrast to other Akt family members, Akt2 enters male and female pronuclei of mouse preimplantation embryos at the late one-cell stage and thereafter maintains a nuclear localization during later embryo cleavage stages. Depleting one-cell embryos of single Akt family members by microinjecting Akt isoform-specific antibodies into wild-type zygotes, we observed that: (a) Akt2 is necessary for normal embryo progression through cleavage stages; and (b) the specific nuclear targeting of Akt2 in two-cell embryos depends on Tcl1. Our results indicate that preimplantation mouse embryos have a peculiar regulation of blastomere proliferation based on the activity of the Akt/PKB family member Akt2, which is mediated by the oncogenic protein Tcl1. Both Akt2 and Tcl1 are essential for early blastomere proliferation and embryo development.     

Altering intracellular pH disrupts development and cellular organization in preimplantation hamster embryos

In early cleavage stage hamster embryos, the inability to regulate intracellular pH (pHi) properly is associated with reduced developmental competence in vitro. The disruption of mitochondrial organization is also correlated with reduced development in vitro. To determine the relationship between pHi and the disruption of cytoplasmic organization, we examined the effects of altering pHi on hamster embryo development, mitochondrial distribution, and cytoskeletal organization. The weak base trimethylamine was used to increase pHi and was found to reduce embryo development and disrupt the perinuclear organization of mitochondria. The weak acid 5,5-dimethyl-2,4-oxazolinedione was used to decrease pH(i) and was also found to reduce development and disrupt the perinuclear organization of mitochondria. With either treatment, the microfilament organization was perturbed, but the microtubule cytoskeleton was not. However, the temporal progression of the disruption of mitochondrial distribution was more rapid in alkalinized embryos than acidified embryos, as revealed by two-photon imaging of living embryos. Additionally, the disruption of the microfilament network by the two treatments was not identical. The cytoplasmic disruptions observed were not due to acute toxicity of the compounds because embryos recovered developmentally when the treatment compounds were removed. These observations link ionic homeostasis, structural integrity and developmental competence in preimplantation hamster embryos.     

Spatial separation of parental genomes in preimplantation mouse embryos

We have used two different experimental approaches to demonstrate topological separation of parental genomes in preimplantation mouse embryos: mouse eggs fertilized with 5-bromodeoxyuridine (BrdU)-labeled sperm followed by detection of BrdU in early diploid embryos, and differential heterochromatin staining in mouse interspecific hybrid embryos. Separation of chromatin according to parental origin was preserved up to the four-cell embryo stage and then gradually disappeared. In F1 hybrid animals, genome separation was also observed in a proportion of somatic cells. Separate nuclear compartments during preimplantation development, when extreme chromatin remodelling occurs, and possibly in some differentiated cell types, may be associated with epigenetic reprogramming.     

Preimplantation mouse embryos synthesize membrane sterols

The total cholesterol content of preimplantation mouse embryos increases approximately threefold (to 1 pmole) during the development of a blastocyst from a fertilized egg. From the two-cell stage onwards embryos are capable of converting [³H]mevalonate into the membrane sterols lanosterol and cholesterol. However, activity of the ratelimiting enzyme in sterol synthesis, hydroxymethylglutaryl coenzyme A reductase, was only measurable in late expanded blastocysts. These estimates of cholesterol content and the amounts of ³H-sterol formed suggest that the preimplantation mouse embryo can synthesize membrane sterols from early cleavage stages onwards. Late compaction and early fluid accumulation (approx. 84 hr post-hCG) are associated with a transition from lanosterol to cholesterol synthesis. The possible relationship between this transition and changes in the properties of embryo membranes which occur at this time is discussed. The results, taken together with previous evidence for phospholipid synthesis in early embryos, demonstrate that the preimplantation mouse embryo is capable of synthesizing major membrane lipids and hence has the potential for assembling cell membranes and modulating their lipid-mediated properties.     

Intracellular pH of the preimplantation mouse embryo: Effects of extracellular pH and weak acids

Although intracellular pH (pHi), is a regulator of numerous biological processes, it has received relatively little attention with regard to the physiology of the mammalian preimplantation embryo. Interestingly, there is some controversy as to whether the early embryo can recover from an acid load. The significance of this is that two constituents of mouse embryo culture media are pyruvate and lactate. These carboxylic acids are utilised by the early mouse embryo for energy production. However, as weak acids, pyruvate and lactate may induce perturbations in the pHi and thus alter the physiology of the embryo. The aims of this study were therefore to measure the pHi of the mouse preimplantation embryo and to determine the effect of lactate on pHi at different developmental stages. The pHi was measured using the ratio-metric fluorophore carboxy-seminaphthorhodafluor-1-acetoxymethylester (SNARF-1) in conjunction with confocal microscopy. The pHi increased significantly with development from the zygote to the morula stage. Furthermore, at concentrations greater than 5 mM, lactate caused the pHi of the zygote to become significantly more acidic. It was demonstrated that facilitative transport in association with a smaller passive component was responsible for the movement of lactate into the zygote. Metabolic studies revealed that, through their acidifying effect, weak acids caused a reduction in glycolytic activity in the early embryo. In contrast, the pHi of the compacted embryo remained unchanged by the presence of lactate in the external media. Furthermore, incubation with weak acids did not affect the rate of glycolysis in the morula. These data suggest that, by the generation of a transporting epithelium at compaction, the embryo develops the ability to regulate pHi against an acid load. Mol. Reprod. Dev. 50:434–442, 1998. © 1998 Wiley-Liss, Inc.     

DNA methylation,retroviruses, and embryogenesis

By exposing preimplantation embryos to Moloney leukemia virus (M-MuLV), we have previously derived substrains of mice designated as Mov-1-Mov-13 which genetically transmit the virus from one generation to the next. In some of the substrains the inserted viral genome becomes activated at specific stages of embryogenesis and the available evidence suggests that these viral genomes are developmentally regulated. To investigate the effect of cellular differentiation on virus expression, M-MuLV was introduced either into preimplantation or postimplantation mouse embryos or into embryonal carcinoma (EC) cells. Whereas preimplantation embryos or EC cells are not permissive for virus expression, efficient replication occurred in postimplantation embryos or in differentiated cell lines. The viral genomes introduced into early embryonal cells were highly methylated and noninfcctious when analyzed in the adult. In contrast, viral genomes introduced into postimplantation embryos or into differentiated cells remained unmethylated and were infectious in a transfection assay. These results demonstrate an efficient de novo methylation activity which appears to be involved in repression of genes introduced into pluripotent embryonal cells and which is not observed in cells of the postimplantation embryo or in differentiated cells in tissue culture.     

Mitochondria in early mammalian development

The role of mitochondria as central determinants of development competence of oocytes and preimplantation stage embryos is considered in the context of the diverse activities these organelles have in normal cell function. Stage- and cell-cycle-specific mitochondrial translocations and redistributions are described with respect to mechanisms of cytoplasmic remodeling that may establish domains of autonomous regulation of mitochondrial function and activity during early development. The functions of mitochondria as intracellular signaling elements, as regulators of signaling pathways, and oxygen sensors in differentiated cells are suggested to have similar capacities during mammalian oogenesis and early embryogenesis. Questions concerning the numerical size of the oocyte mitochondrial complement, the energy required to support normal preovulatory oogenesis and preimplantation embryogenesis, and the regulation of mitochondrial activity by extrinsic and intrinsic factors are addressed with respect the potential they may have for new investigational approaches to study the origin of the differential developmental competence of human oocytes and preimplantation stage embryos.     

Betaine is a highly effective organic osmolyte but does not appear to be transported by established organic osmolyte transporters in mouse embryos

Betaine protects early preimplantation mouse embryos against increased osmolarity in vitro, functioning as an organic osmolyte. Betaine is effective at very low external concentrations, with half-maximal protection of 1-cell embryo development to blastocysts at approximately 50 microM, making it one of the best osmoprotectants for mouse preimplantation embryos. We performed studies designed to determine whether known high-affinity organic osmolyte transporters could account for the ability of betaine to act as an organic osmolyte in preimplantation embryos. We found no evidence in 1-cell embryos of transport by a betaine/GABA transporter (BGT1), the osmoregulated betaine transporter found in a number of cell types, as betaine and GABA did not inhibit each other's transport. Instead, all saturable GABA transport in embryos was apparently via the beta-amino acid transporter. We also found that the glycine transporter, GLY, which mediates osmoprotective transport of glycine in early preimplantation embryos, does not appear to transport betaine. Finally, increased osmolarity did not induce any detectable System A amino acid transporter activity, which is osmotically-inducible in other cells and can transport betaine. There does appear, however, to be a saturable betaine transporter in 1-cell mouse embryos, as considerable 14C-betaine transport was measured which was substantially inhibited by excess unlabeled betaine. Our data imply that betaine functions as an organic osmolyte in embryos due to its saturable transport via a mechanism distinct from known osmolyte transporters. We propose that an unidentified high-affinity betaine transporter may be expressed in early embryos and mediate transport of betaine as an organic osmolyte.     

Effect of polyamine limitation on DNA synthesis and development of mouse preimplantation embryos in vitro

In-vitro treatment of preimplantation mouse embryos with spermine and spermidine biosynthesis inhibitor, methylglyoxal-bis-(guanylhydrazone) (MGBG), arrested embryo development at the 8-cell or morula stage. In addition, the embryo DNA synthetic rate, as measured by [3H]thymidine incorporation, was strongly inhibited. The inhibition of blastocyst formation and DNA synthesis by MGBG was readily reversible by an exogenous supply of spermine and/or spermidine to the culture medium. DL-alpha-Methylornithine or DL-alpha-difluoromethylornithine (alpha-DFMO), inhibitors of putrescine biosynthesis, had no effect on embryos cultured for 1 or 2 days, but on the 3rd day embryo DNA synthesis was significantly depressed in the presence of alpha-DFMO. These observations suggest that, during early development of the preimplantation mouse embryo, spermine and spermidine are involved in regulation of embryo growth and DNA synthesis. They may also indicate a role of putrescine at a later stage of mouse embryo development.     

Intersection clock reveals a rejuvenation event during human embryogenesis

Recent research revealed a rejuvenation event during early development of mice. Here, by examining epigenetic age dynamics of human embryogenesis, we tested whether a similar event exists in humans. For this purpose, we developed an epigenetic clock method, the intersection clock, that utilizes bisulfite sequencing in a way that maximizes the use of informative CpG sites with no missing clock CpG sites in test samples and applied it to human embryo development data. We observed no changes in the predicted epigenetic age between cleavage stage and blastocyst stage embryos; however, a significant decrease was observed between blastocysts and cells representing the epiblast. Additionally, by applying the intersection clock to datasets spanning pre and postimplantation, we found no significant change in the epigenetic age during preimplantation stages; however, the epigenetic age of postimplantation samples was lower compared to the preimplantation stages. We further investigated the epigenetic age of primed (representing early postimplantation) and naïve (representing preimplantation) pluripotent stem cells and observed that in all cases the epigenetic age of primed cells was significantly lower than that of naïve cells. Together, our data suggest that human embryos are rejuvenated during early embryogenesis. Hence, the rejuvenation event is conserved between the mouse and human, and it occurs around the gastrulation stage in both species. Beyond this advance, the intersection clock opens the way for other epigenetic age studies based on human bisulfite sequencing datasets as opposed to methylation arrays.     

Phospholipid transfer protein (PLTP) mRNA expression is stimulated by developing embryos in the oviduct

In mammal, fertilization and early preimplantation embryo development occurs in the oviduct. Evidence is accumulating that the oviductal epithelia secrete various biomolecules to the lumen during the secretory phase of the estrus cycle to enhance embryo development. This secretory activity of the oviduct is under the regulation of steroid hormones. Observations also suggested that the gametes and embryos modulate the physiology and gene-expressing pattern of the oviduct. However, the underlying molecular changes remain elusive. We hypothesize that the developing embryos interact with the surrounding environment and affect the gene expression patterns of the oviduct, thereby modulating the oviductal secretory activity conducive to the preimplantation embryo development. To test this hypothesis, suppression subtractive hybridization (SSH) was used to compare the gene expressions in mouse oviduct containing transferred in vitro cultured preimplantation embryos with that of oviduct containing oocytes during the preimplantation period. We reported here the identification and characterization of phospholipids transfer protein (PLTP), which is highly expressed in the embryo-containing oviduct and localized at the oviductal epithelium by in situ hybridization. PLTP contains signal peptide putative for secretory function. More importantly, PLTP mRNA increases in the oviductal epithelia of pregnant, but not pseudo-pregnant mice when assayed by real-time PCR. Taken together, our data suggested that PLTP may play important role(s) during in vivo preimplantation embryo development. This molecule would be a target to delineate the mechanisms and the roles of oviductal secretory proteins on early embryonic development.     

Beta-alanine but not taurine can function as an organic osmolyte in preimplantation mouse embryos cultured from fertilized eggs

Early preimplantation mouse embryos are susceptible to the detrimental effects of increased osmolarity and, paradoxically, their in vitro development is significantly compromised by osmolarities near that of oviductal fluid. In vitro development can be restored, however, by several compounds that are accumulated by 1-cell embryos to act as organic osmolytes, providing intracellular osmotic support and cell volume regulation. Taurine, a substrate of the beta-amino acid transporter that functions as an organic osmolyte transporter in other cells, had been proposed to function as an organic osmolyte in mouse embryos. Here, however, we found that taurine is neither able to provide protection for in vitro embryo development against increased osmolarity nor is it accumulated to higher intracellular levels as osmolarity is increased, indicating that it cannot function as an organic osmolyte in early preimplantation embryos. In contrast, beta-alanine, the other major substrate of the beta-amino acid transporter, both protects against increased osmolarity and is accumulated to somewhat higher levels as osmolarity is increased, indicating that it is able to function as an organic osmolyte in embryos. However, we also found that beta-alanine is displaced from embryos by glycine-the most effective organic osmolyte in embryos previously identified-and beta-alanine does not increase protection above that afforded by glycine at concentrations near those in vivo. Thus, the beta-amino acid transporter is likely present in early preimplantation embryos to supply beta-amino acids such as taurine for purposes other than to serve as organic osmolytes.     

Laser Fusion of Mouse Embryonic Cells and Intra-Embryonic Fusion of Blastomeres without Affecting the Embryo Integrity

Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo’s integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.     

Vitrification of murine mature metaphase II oocytes perturbs DNA methylation reprogramming during preimplantation embryo development

Accurate reprogramming of DNA methylation occurring in preimplantation embryos is critical for normal development of both fetus and placenta. Environmental stresses imposed on oocytes usually cause the abnormal DNA methylation reprogramming of early embryos. However, whether oocyte vitrification alters the reprogramming of DNA methylation (5 mC) and its derivatives in mouse preimplantation embryo development remains largely unknown. Here, we found that the rate of cleavage and blastocyst formation of embryos produced by IVF of vitrified matured oocytes was significantly lower than that in control counterparts, but the quality of blastocysts was not impaired by oocyte vitrification. Additionally, although vitrification neither altered the dynamic changes of 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5 fC) before 4-cell stage nor affected the levels of 5 mC and 5-carboxylcytosine (5caC) throughout the preimplantation development, vitrification significantly reduced the levels of 5hmC and 5 fC from 8-cell stage onwards. Correspondingly, vitrification did not alter the expression patterns of Tet3 in preimplantation embryos but apparently reduced the expression levels of Tet1 in 4-cell and 8-cell embryos and increased the expression levels of Tet2 at morula stage. Taken together, these results demonstrate that oocyte vitrification perturbs DNA methylation reprogramming in mouse preimplantation embryo development.     

Reproduction in mice: Protein kinase mimics the sperm effect on preimplantation embryo development

The creation of an environment in mouse fallopian tubes that is sufficient to sustain preimplantation embryo development is known to require the participation of spermatozoa in excess of those involved in the process of fertilization. We have now found that highly purified cAMP-dependent protein kinase can substitute for spermatozoa in the facilitation of the first cleavage of mouse embryos. Both spermatozoa and purified protein kinase induce increases in fallopian phosphoproteins. It is suggested that nonfertilizing spermatozoa could exert their effects on preimplantation embryo development through the provision of protein kinase.