Spatial alignment of the mouse blastocyst axis across the first cleavage plane is caused by mechanical constraint rather than developmental bias among blastomeres

The embryonic-abembryonic (Em-Ab) axis of the mouse blastocyst has been found in several studies to align orthogonal to the first cleavage plane, raising the possibility that a developmental prepattern already exists at the two-cell stage. However, it is also possible that such alignment is not due to any developmental disparity between the two-cell stage blastomeres, but rather is caused by an extrinsic mechanical constraint that is conferred by an irregular shape of the zona pellucida (ZP). Here, we conducted a series of experiments to distinguish between these possibilities. We showed that the shape of the ZP at the two-cell stage varied among embryos, ranging from near spherical to ellipsoidal, and that the ZP shape did not change until the blastocyst stage. In those embryos with an ellipsoidal ZP, the Em-Ab axis tended to lie orthogonal to the first cleavage plane, while in those embryos with a near spherical ZP, there was no such relationship. The clonal boundary between the descendants of the two-cell stage blastomeres tended to lie orthogonal to the Em-Ab axis when the rotation of the embryo within the ZP was experimentally prevented, while the control embryos did not exhibit such tendency. These results support the possibility that an apparent correlation between the first cleavage plane and the blastocyst axis can be generated by the mechanical constraint from the ZP but not by a developmental prepattern. Moreover, recent reports indicate that the vegetal blastomere of the four-cell stage embryo that had undergone a specific type of second cleavages is destined to contribute to the abembryonic side of the blastocyst. However, our present study shows that in spite of such specific second cleavages, the vegetal blastomere did not preferentially give rise to the abembryonic side. This result implicates that the lineage of the four-cell stage blastomere is not restricted even when embryos undergo a specific type of second cleavages.     

Early patterning of cloned mouse embryos contributes to post-implantation development

Several research groups have suggested that the embryonic-abembryonic (Em-Ab) axis in the mouse can be predicted by the first cleavage plane of the early embryo. Currently, it is not known whether this early patterning occurs in cloned embryos produced by nuclear transfer and whether it affects development to term. In this work, the relationship between the first cleavage plane and the Em-Ab axis was determined by the labeling of one blastomere in cloned mouse embryos at the 2-cell stage, followed by ex-vivo tracking until the blastocyst stage. The results demonstrate that approximately half of the cloned blastocysts had an Em-Ab axis perpendicular to the initial cleavage plane of the 2-cell stage. These embryos were classified as "orthogonal" and the remainder as "deviant". Additionally, we report here that cloned embryos were significantly more often orthogonal than their naturally fertilized counterparts and overexpressed Sox2. Orthogonal cloned embryos demonstrated a higher rate of post-implantation embryonic development than deviant embryos, but cloned pups did not all survive. These results reveal that the angular relationship between the Em-Ab axis and the first cleavage plane can influence later development and they support the hypothesis that proper early patterning of mammalian embryos is required after nuclear transfer.     

Unbiased contribution of the first two blastomeres to mouse blastocyst development

Several recent studies have proposed a model that the organization of the mouse blastocyst is determined by the pattern of early cleavages: the plane of first cleavage divides the two-cell embryo into embryonic (Em) and abembryonic (Ab) halves, while the timing of the second cleavages specifies which blastomere becomes the Em half. This model is still controversial because of conflicting observations in various studies. Here, we investigated the possibility that the difference between mouse strains contributed to the discrepancy of the findings of different experiments regarding the relationship between the first two cleavages and the blastocyst axial pattern. First, we showed by using a lipophilic, fluorescent tracer that the plane of the first cleavage bears no consistent spatial relationship to the Em-Ab axis of the blastocyst regardless of the genotypic background. Secondly, the order of the second cleavage does not correlate with the Em-Ab polarity of the blastocyst. This was demonstrated by tracing the lineage of the early- and later-dividing two-cell stage blastomeres in the whole embryo as well as by comparing the developmental potential of isolated early- and later-dividing blastomeres and chimeras made entirely of early- or later-dividing blastomeres. These results suggest that contrary to recent studies, the differences between the early- and later-dividing blastomeres of the two-cell embryo are not functionally evident and do not define the Em-Ab polarity of the blastocyst. The significance of these findings is discussed in relation to human assisted reproduction and preimplantation genetic diagnosis.     

Blastomeres arising from the first cleavage division have distinguishable fates in normal mouse development

Two independent studies have recently suggested similar models in which the embryonic and abembryonic parts of the mouse blastocyst become separated already by the first cleavage division. However, no lineage tracing studies carried out so far on early embryos provide the support for such a hypothesis. Thus, to re-examine the fate of blastomeres of the two-cell mouse embryo, we have undertaken lineage tracing studies using a non-perturbing method. We show that two-cell stage blastomeres have a strong tendency to develop into cells that comprise either the embryonic or the abembryonic parts of the blastocyst. Moreover, the two-cell stage blastomere that is first to divide will preferentially contribute its progeny to the embryonic part. Nevertheless, we find that the blastocyst embryonic-abembryonic axis is not perfectly orthogonal to the first cleavage plane, but often shows some angular displacement from it. Consequently, there is a boundary zone adjacent to the interior margin of the blastocoel that is populated by cells derived from both earlier and later dividing blastomeres. The majority of cells that inhabit this boundary region are, however, derived from the later dividing two-cell stage blastomere that contributes predominantly to the abembryonic part of the blastocyst. Thus, at the two-cell stage it is already possible to predict which cell will contribute a greater proportion of its progeny to the abembryonic part of the blastocyst (region including the blastocyst cavity) and which to the embryonic part (region containing the inner cell mass) that will give rise to the embryo proper.     

Specification of embryonic axes begins before cleavage in normal mouse development

Studies on the development of aggregated, isolated and rearranged blastomeres have engendered the view that in mammals, unlike most other animals, egg organization has no role in the genesis of asymmetries that are essential for cellular diversification and the specification of embryonic axes. Such asymmetries are assumed to arise post-zygotically through interactions between initially naive cells. However, various findings are difficult to reconcile with this view. Here, a consistent relationship between the structure of the blastocyst and the two-cell stage in the mouse has been found using a strictly non-invasive marking technique: injection of small oil drops into the substance of the zona pellicuda. This has revealed that both the embryonic-abembryonic axis of the blastocyst and its plane of bilateral symmetry are normally orthogonal to the plane of first cleavage. This relationship was also seen when denuded two-cell conceptuses were prevented from rotating during subsequent cleavage by immobilizing them in a gel. Therefore, during normal mouse development the axes of the blastocyst, which have been implicated in establishing those of the fetus, are already specified by the onset of cleavage.     

The basis and significance of pre-patterning in mammals

The second polar body (Pb) provides an enduring marker of the animal pole of the zygote, thereby revealing that the axis of bilateral symmetry of the early blastocyst is aligned with the zygote's animal-vegetal axis. That this relationship is biologically significant appeared likely when subsequent studies showed that the equator of the blastocyst tended to correspond with the plane of first cleavage. However, this cleavage plane varies both with respect to the position of the second Pb and to the distribution of components of the fertilizing sperm that continue to mark the point where it entered the egg. It also maps too variably on the blastocyst to play a causal role in early patterning. The zygote has been found transiently to exhibit bilateral symmetry before regaining an essentially spherical shape prior to first cleavage. Marking experiments indicate that the plane of bilateral symmetry of the blastocyst is aligned with, and the plane of first cleavage is typically orthogonal to, the zygote's bilateral plane. The bilateral symmetry of the zygote bears no consistent relationship either to the point of sperm entry or to the distribution of the pronuclei, and may therefore be a manifestation of intrinsic organization of the egg. Finally, the two-cell blastomere inheriting the sperm entry point has not been found to differ consistently in fate from the one that does not.     

Mechanism of First Cleavage Specification in the Mouse Egg: Is Our Body Plan Set at Day 0?

In most animals the body axis is specified in the egg. Because of their highlyregulative capacity after experimental manipulations,1-4 mammalianpreimplantation embryos have long been thought to be an exception to this rule,lacking polarity until the blastocyst stage. However, it has recently been suggested5-7 that the embryonic-abembryonic (Em-Ab) axis of the mouse blastocyst arisesperpendicular to the first cleavage plane. Considering the second polar body (2pb)as a stationary marker for the “animal pole (A-pole)” during preimplantationdevelopment,5,6 the authors concluded that the polarity of the mouse embryo isalready specified in the egg, as is the case for most non-mammalian animals.5-7However, the results of our recent time-lapse recordings have shown8 that in 50 %of the embryos the first cleavage occurs at a considerable distance from the“animal-vegetal (A-V) axis” and that the 2pb moves towards the first cleavageplane, in contrast to the previous claims. Thus, there is no predetermined axis in themouse egg. We also presented a novel model for specification of the first cleavageplane: this is defined as the plane separating the two apposing pronuclei that havemoved to the center of the egg. In this review we will elucidate the discrepancybetween the previous model and our model, and discuss the possible causes.     

Analysis of cell lineage in two- and four-cell mouse embryos

Compared with other animals, the embryos of mammals are considered to have a highly regulative mode of development. However, recent studies have provided a strong correlation between the first cleavage plane and the future axis of the blastocyst, but it is still unclear how the early axes of the preimplantation embryo reflect the future body axes that emerge after implantation. We have carried out lineage tracing during mouse embryogenesis using the Cre-loxP system, which allowed us to analyze cell fates over a long period of development. We used a transgenic mouse strain, CAG-CAT-Z as a reporter line. The descendants of the manipulated blastomere heritably express beta-galactosidase. We examined the distribution of descendants of a single blastomere in the 8.5-day embryo after labeling at the two-cell and four-cell stages. The derivatives of one blastomere in the two-cell embryo randomly mix with cells originating from the second blastomere in all cell layers examined. Thus we find cells from different blastomeres intermingled and localized randomly along the body axis. The results of labeling experiments performed in the four-cell stage embryo fall into three categories. In the first, the labeled cells were intermingled with non-labeled cells in a manner similar to that seen after labeling at the two-cell stage. In the second, labeled cells were distributed only in the extra-embryonic ectoderm layers. Finally in the third category, labeled cells were seen only in the embryo proper and the extra-embryonic mesoderm. Manipulated embryos analyzed at the blastocyst stage showed localized distribution of the descendants of a single blastomere. These results suggest that incoherent clonal growth and drastic cell mixing occurs in the early mouse embryo after the blastocyst stage. The first cell specification event, i.e., partitioning cell fate between the inner cell mass and trophectoderm, can occur between the two-cell and four-cell stage, yet the cell fate is not determined.     

Perturbations in mouse embryo development and viability caused by ammonium are more severe after exposure at the cleavage stages

The presence of ammonium in culture medium has a detrimental effect on embryo physiology and biochemistry; however, the stage at which the embryo is most sensitive to this effect is unknown. The aim of this study was to determine the exact stage at which the embryo is most vulnerable to ammonium by exposing the preimplantation embryo to 300 muM ammonium either at the precompaction stage (between the zygote and two-cell or the two-cell to eight-cell) or at the postcompaction stage (between the eight-cell and blastocyst). This study determined that exposure of embryos to ammonium at the precompaction stage from either the zygote to two-cell stage or from the two-cell to the eight-cell stage did not affect the rate of development to the blastocyst stage; however, the resultant blastocysts had decreased cell numbers and inner cell mass cells. Furthermore, these blastocysts had increased levels of cellular apoptosis and perturbed levels of Slc2a3 expression and glucose uptake. Transfer of these blastocysts revealed that, while implantation was not affected, the number of fetuses was reduced by culture with ammonium at the precompaction stage and fetal development was delayed, as observed by reduced crown-rump length and maturity. In contrast, the later stage embryo was more resistant to the negative effects of ammonium, with only Slc2a3 expression and fetal maturity affected. This raises the possibility that the later stage embryo is more able to protect itself from in vitro-derived stress and that the majority of in vitro-induced damage to mouse embryos is inflicted at the early stages of development.     

Delay on the in vitro kinetic development of prepubertal ovine embryos

In the present study we characterize the developmental potential of prepubertal and adult ovine oocytes, analyzing the developmental speed to two-cell and blastocyst stages and its relationship with hatching from the zona pellucida, development after vitrification and the number and allocation of inner mass and trophoblastic cells. Prepubertal and adult ovine oocytes were matured and fertilized in vitro and first cleavage rates at 22, 26 and 32 h were recorded. Cleaved oocytes were cultured and blastocyst production was assessed at 6-9 days post-fertilization (dpf). Blastocysts from the two sources obtained on different days were divided into two groups: the first was vitrified, warmed and cultured in vitro to evaluate re-expansion of the blastocoelic cavity; blastocysts of the second were cultured separately to allow for hatching and count of trophoblastic and inner mass cells of hatched blastocysts by differential staining. We observed a significantly lower rate (P < 0.01) of cleaved prepubertal oocytes at 22 and 26 h after fertilization while it was higher (P<0.01) at 32 h than in the adult ones. Adult blastocyst production was significantly lower (P < 0.01) in prepubertal than in adult groups and began on the seventh dpf, later (P < 0.01) than in the adult group, where they appeared on the sixth dpf. Prepubertal blastocysts hatched at a lower rate than the adult ones (P < 0.01) and in both experimental groups faster blastocysts showed a higher (P < 0.01) hatching rate. Similarly, prepubertal derived blastocysts showed lower viability after vitrification (P < 0.01) compared to the adult counterparts, and in particular slower embryos had reduced viability after vitrification compared to the fastest (P < 0.01). Cell number was not different between blastocysts of both groups obtained at 6 and 7 dpf, which were higher (P < 0.01) than those obtained at 8 and 9 dpf. The ICM/trophoblast cell ratio was similar in 6- and 7-day obtained blastocyst and increased (P < 0.01) in those obtained 1 or 2 days later. These findings show that differences in kinetic development between prepubertal and adult derived embryos reflect differences in developmental capacity of the oocytes from which they derive and could be indicative of embryo quality.     

Quantitative changes in cytoskeletal beta- and gamma-actin mRNAs and apparent absence of sarcomeric actin gene transcripts in early mouse embryos

Actin is known to be synthesized both during oogenesis and in cleavage-stage embryos in mice. Cytoskeletal beta-actin appears to be the major component, followed by gamma-actin, but the synthesis of alpha-actin has also been inferred from protein electrophoretic patterns. We have studied the expression of cytoskeletal (beta- and gamma-) and sarcomeric (alpha-cardiac and alpha-skeletal) actin genes at the level of the individual mRNAs in blot hybridization experiments using isoform-specific RNA probes. The results show that there are about 2 x 10(4) beta-actin mRNA molecules in the fully grown oocyte; this number drops to about one-half in the egg and less than one-tenth in the late two-cell embryo but increases rapidly during cleavage to about 3 x 10(5) molecules in the late blastocyst. The amount of gamma-actin mRNA is similar to that of beta-actin in oocytes and eggs but only about 40% as much in late blastocysts, indicating a differential accumulation of these mRNAs during cleavage. The developmental pattern of beta- and gamma-actin mRNA provides a striking example of the transition from maternal to embryonic control that occurs at the two-cell stage and involves the elimination of most or all of the maternal actin mRNA. There was no detectable alpha-cardiac or alpha-skeletal mRNA (i.e., less than 1,000 molecules per embryo) at any stage from oocyte to late blastocyst, suggesting that the sarcomeric actin genes are silent during preimplantation development.     

Allocation of cells in mouse blastocyst is not determined by the order of cleavage of the first two blastomeres

The second cleavage of the mouse embryo is asynchronous. Some recent investigators have proposed that the sequence of division of blastomeres in two-cell embryos may predict the ultimate location of the descendants of these blastomeres within the blastocyst. To verify this model, we tracked the cells derived from two-cell stage blastomeres using tetramethylrhodamine-conjugated dextran as a lineage tracer. In the first variant of the experiment, we labeled one of two blastomeres in two-cell embryos and subsequently recorded which blastomere cleaved first. In the second variant of the experiment, fluorescent dextran was injected at the three-cell stage into the blastomere that had not yet cleaved. Subsequently, the fate of the progeny of labeled and unlabeled blastomeres was followed up to the blastocyst stage. Our results suggest that allocation of cells into the embryonic and abembryonic parts of the blastocyst is not determined by the order of cleavage of the first two blastomeres.     

The first cleavage furrow demarcates the dorsal-ventral axis in Xenopus embryos

To determine the relationship between the first cleavage furrow and the dorsal-ventral axis of the Xenopus embryo, a heritable intracellular marker was injected into one blastomere at the two-cell stage. Embryos were selected in which the cleavage furrow bisected the crescent-shaped region of pale pigmentation or in which it formed 45-90 degrees from this region. This region, which is located in the animal hemisphere of the Xenopus embryo, meets the criteria of the grey crescent as defined in other amphibian species. At tailbud stages the interface between the labeled and unlabeled halves was always coincident with the midsagittal plane. This correlation shows that the first cleavage furrow demarcates the dorsal-ventral axis. The labeling pattern was the same whether the first cleavage furrow bisected the region of pale pigmentation or whether it formed 90 degrees from it. However, when this region was bisected (70% of embryos) it always was located on the dorsal side of the embryo. Thus the region of pale pigmentation indicates the dorsal side of the embryo only when it is bisected by the first cleavage furrow.     

Heat shock induces apoptosis related gene expression and apoptosis in porcine parthenotes developing in vitro

Successful in vitro development of embryos is dependent upon maintenance of cellular function in the embryonic microenvironment. However, the molecular aspects involved in the thermoprotection of embryos, against heat and cold stress it is not clear. The aim of this study was to determine the effects of heat and cold shock on the viability and development of porcine diploid parthenotes developing in vitro. Exposure of two-cell stage embryos to 41 degrees C did not affect further cleavage. However, prolonged heat shock, greater than 12h, reduced the percentage of blastocysts that developed from two-cell stage parthenotes, as well as the total number of nuclei in the blastocysts that formed. Furthermore, the degree of apoptosis was increased (P<0.05) in these blastocyst stage parthenotes. In contrast, exposure of two-cell parthenotes to cold (30 degrees C) for 24h did not affect the cleavage rates, development to blastocyst, nor the total cell numbers per blastocyst. Real time PCR revealed that quantitative expression of the Bcl-xL gene was not different, but amounts of HSP 70.2, Bak, and Caspase 3mRNA were significantly increased in the heat shocked embryos, as compared with untreated controls. These results suggest that porcine embryos are more tolerant to cold shock than to heat shock. Heat stress seems to induce apoptosis related gene expression in porcine parthenotes developing in vitro, which results in diminished parthenote viability.     

Production of identical twin and triplet mice by nuclear transplantation

Transplantation of a single nucleus from two- or four-cell embryos into one of the enucleated blastomeres of a two-cell embryo resulted in successful production of identical triplet and twin mice. The proportion of reconstituted embryos that developed in blastocysts was 71% (84/118) when four-cell embryos were used as donors of nuclei; 10 sets of quadruplet and nine sets each of triplet and twin blastocysts were obtained by this technique. After transfer to recipients, 30% (18/61) developed to term, and one set of identical triplet and four sets of identical twin mice were obtained. When two-cell embryos were used as donors of nuclei, 79 (95%) sets of twin embryos developed to blastocysts. Of 38 twin blastocysts transferred to recipients, 21 sets (55%) developed to term as identical twin mice. These results demonstrate that the enucleated two-cell embryo develops in vitro after transfer of a nucleus from a two- or four-cell embryo and the resultant blastocyst has high potential for development to term after transfer to a recipient.     

Regulation and the modification of axial properties in partial embryos of the nemertean, Cerebratulus lacteus

 Embryos acquire axial properties (e.g., the animal-vegetal, dorsoventral and bilateral axes) at various times over the course of their normal developmental programs. In the spiral-cleaving nemertean, Cerebratulus lacteus, lineage tracing studies have shown that the dorsoventral axis is set up prior to the first cleavage division; however, blastomeres isolated at the two-cell stage will regulate to form apparently perfect, miniature pilidium larvae. We have examined the nature of axial specification in this organism by determining whether partial embryos retain the original embryonic/larval axial properties of the intact embryo, or whether new axial relationships are generated as a consequence of the regulatory process. Single blastomeres in two-cell stage embryos were injected with lineage tracer, and were then bisected along the second cleavage plane at the four-cell stage. Thus, the relationship between the plane of the first cleavage division and various developmental axes could be followed throughout development in the ”half-embryos”. While some embryo fragments appear to retain their original animal-vegetal and dorsoventral axes, many fragments generate novel axial properties. These results indicate that axial properties set up and used during normal development in C. lacteus can be completely reorganized during the course of regulation. While certain embryonic axes, such as the animal-vegetal and dorsoventral axes, appear to be set up prior to first cleavage, these axes and associated cell fates are not irreversibly fixed until later stages of development in normal intact embryos. In C. lacteus, the process whereby these properties are ultimately determined is apparently controlled by complex sets of cell-cell interactions. Received: 11 October 1996 / Accepted: 21 February 1997     

Effect of oocyte maturation medium on in vitro development of in vitro fertilized bovine embryos.

The objective of this study was to examine the effects of different culture media used for maturation of bovine oocytes on in vitro embryo development following in vitro fertilization. Oocytes were aspirated from 2-5 mm follicles of ovaries collected at a local abattoir. The oocyte-cumulus complexes (OCCs) were cultured for 23-25 h in one of seven commercially available media supplemented with 6 mg/ml bovine serum albumin (BSA), 0.25 mM pyruvate, 10 micrograms/ml luteinizing hormone (LH), 0.5 microgram/ml follicle-stimulating hormone (FSH), and 1 microgram/ml estradiol. After maturation for 23-25 h, all eggs were subjected to the same in vitro fertilization protocol using modified TALP medium and subsequently cultured in the same serum-free embryo culture medium (HECM-1/BSA) for 8 days, after which embryo development was assessed. Five media (SFRE, MEM alpha, TCM199, MEM alpha/+, RPMI:MEM alpha) better supported normal oocyte maturation as determined by embryo development to the two-cell (76-82%), morula/blastocyst (25-32%), and blastocyst (12-19%) stages. Oocytes that were matured in Waymouth's medium MB 752/l or Ham's F-12 had a significantly reduced incidence of cleavage to the two-cell stage (52% and 37%, respectively), which was not attributed to failure of fertilization. Of the eggs that did cleave to the two-cell stage in these two media, 27% and 9% developed to morulae/blastocysts but only 6% and 3%, respectively, developed into blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrofusion of two-cell bovine embryos for the production of tetraploid blastocysts in vitro

Tetraploid bovine blastocysts were produced experimentally by electrofusion of in vitro matured and fertilized, zona-enclosed two-cell embryos (33-35 hr after initiation of sperm-egg incubation) using three fusion protocols. Field strengths of 1.0, 1.4, and 2.4 kV/cm were tested and the rate of fusion, subsequent cleavage, and blastocyst development were measured for each. High rates of fusion (76.5% +/- 2.8%), cleavage (72.5% +/- 7.4%) and blastocyst development (56.1% +/- 6.4%) were achieved with the application of 1. 4 kV/cm as a single 100-microseconds pulse. Embryos were scored 30 and 60 min after stimulation for fusion. No time effect for fusion, cleavage, or blastocyst development was observed. Chromosome preparations of day 7 blastocysts revealed 12.5% of fused embryos were tetraploid. This is a significant increase from that found in nonfused embryos where spontaneous tetraploidy did not occur. An electrical stimulus of 1.0 kV/cm applied as two 50-microseconds pulses produced significantly less one-cell embryos (64.2% +/- 3.0%) compared to 1.4 kV/cm while cleavage (79.9% +/- 3.4) and blastocyst development (44.6% +/- 4.0%) were not different from that for unexposed control embryos (89.5% +/- 2.3% and 57.2% +/- 3.2%, respectively). Embryos fused at 2.4 kV/cm applied as a single 30-microseconds pulse (69.7% +/- 5.7%) showed significantly lower cleavage (72.1% +/- 3.7%) and blastocyst rates (40.2% +/- 4.6%) compared to the unexposed control.