Development of single blastomeres from four- and eight-cell mouse embryos fused into the enucleated half of a two-cell embryo

Single blastomeres from four- and eight-cell mouse embryos were fused into the enucleated halves of two-cell embryos, and the ability of these reconstituted embryos to develop in vitro and in vivo was examined. The proportion of these reconstituted embryos developing to blastocysts was 74% (60/81) when four-cell embryo blastomeres were used as nuclei donors and 31% (57/182) when eight-cell embryo blastomeres were used. Eight complete sets of the quadruplet-reconstituted embryos developed to blastocysts, and five live young (9%, 5/57) were obtained after transfer; however, none of the live young were clones. Although when using blastomeres from eight-cell embryos no complete set of eight developed to blastocysts, sextuplets were obtained. The blastocysts, however, failed to produce live young after transfer. In assessing the outgrowths, it was found that 43% of those derived from reconstituted embryos using blastomeres from four-cell embryos had an inner cell mass (ICM); however, outgrowths derived from reconstituted embryos using blastomeres from eight-cell embryos lacked an ICM. These results suggest that the genomes of four- and eight-cell nuclei introduced into the enucleated halves of two-cell embryos are reversed to support the development of the reconstituted embryo.     

Chimeras between parthenogenetic or androgenetic blastomeres and normal embryos: allocation to the inner cell mass and trophectoderm

A series of chimeras was generated by injecting single normal, parthenogenetic, or androgenetic blastomeres carrying transgenic markers under the zona pellucida of nontransgenic eight-cell embryos. These chimeras were cultured to the blastocyst stage and sectioned, and the transgenic component was detected by in situ hybridization. No statistically significant difference was found among the normal, parthenogenetic, and androgenetic chimeras in the number of chimeric blastocysts with a transgenic contribution to the inner cell mass (ICM), the trophectoderm, or both the ICM and trophectoderm. Since androgenetic and parthenogenetic cells were present in chimeras at a high frequency in both the ICM and trophectoderm at the blastocyst stage, but not in similar chimeras at late gastrulation, these cells must not respond normally to developmental signals subsequent to blastocyst formation and prior to late gastrulation.     

Four-cell stage mouse blastomeres have different developmental properties

Blastomeres of the early mouse embryo are thought to be equivalent in their developmental properties at least until the eight-cell stage. However, the experiments that have led to this conclusion could not have taken into account either the spatial origin of individual blastomeres or the spatial allocation and fate of their progeny. We have therefore readdressed this issue having defined cell lineages in mouse embryos undergoing different patterns of cleavage in their second division cycle. This has enabled us to identify a major group of embryos in which we can predict not only the spatial origin of each given four-cell blastomeres, but also which region of the blastocyst is most likely to be occupied by its progeny. We show that a pattern of second cleavage divisions in which a meridional division is followed by one that is equatorial or oblique allows us to identify blastomeres that differ in their fate and in their developmental properties both from each other and from their cousins. We find that one of these four-cell stage blastomeres that inherits some vegetal membrane marked in the previous cleavage cycle tends to contribute to mural trophectoderm. The progeny of its sister tend to donate cells to part of the ICM lining the blastocyst cavity and its associated trophectoderm. Chimaeras made entirely of these equatorially or obliquely derived blastomeres show developmental abnormalities in both late preimplantation and early postimplantation development. By contrast, chimaeras made from four-cell stage blastomeres from early meridional divisions develop normally. The developmental defects of chimaeras made from the most vegetal blastomeres that result from later second cleavages are the most severe and following transplantation into foster mothers they fail to develop to term. However, when such individual four-cell blastomeres are surrounded by blastomeres from random positions, they are able to contribute to all embryonic lineages. In conclusion, this study shows that while all four-cell blastomeres can have full developmental potential, they differ in their individual developmental properties according to their origin in the embryo from as early as the four-cell stage.     

Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system

The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.     

Reprograming of murine blastocoele formation

The present study shows that there is communication between reaggregated asynchronous cleavage stage blastomeres that regulates blastocoele formation. Individual blastomeres from eight-cell murine embryos were transferred to empty zonae pellucidae, intact two-cell embryos, or enucleated two-cell embryos, and were examined over a period of 75 hours for development of cavitation. It was found that the isolated blastomeres cavitated concurrently with intact control eight-cell embryos, while intact control two-cell embryos cavitated 24 hours later. However, the embryos resulting from combining a two-cell embryo and a blastomere from an eight-cell embryo cavitated at a time in between the eight- and two-cell controls.     

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.     

Cell allocation in half- and quadruple-sized preimplantation mouse embryos

The size of preimplantation mouse embryos was experimentally manipulated in order to examine the consequences for the allocation of cells to the two primary tissues, trophectoderm and inner cell mass (ICM). Half embryos were produced by the mechanical lysis of one cell at the two-cell stage and quadruple embryos by the aggregation of four whole eight-cell embryos. Such procedures are shown not only to alter the absolute number of cells that are assigned to the trophectoderm and ICM, but also to disturb significantly the proportions of these two tissues in the blastocyst. The proportion of trophectoderm is directly related to the surface area of the morula, as is predicted by a purely epigenetic scheme for cell allocation.     

Developmental rate and allocation of transgenic cells in rabbit chimeric embryos

The objective of this study was to compare developmental capacity of rabbit chimeric embryos and the allocation of the EGFP gene expression to the embryoblast (ICM) or embryonic shield. We produced chimeric embryos (TR< >N) by synchronous transfer of two or three blastomeres at the 16-cell stage from transgenic (TR) into normal host embryos (N) at the same stage. In the control group, two to three non-transgenic blastomeres were used to produce chimeric embryos. The TR embryos were produced by microinjection of EGFP into both pronuclei of fertilized rabbit eggs. The developmental rate and allocation of EGFP-positive cells of the reconstructed chimeric embryos was controlled at blastocyst (96 h PC) and embryonic shield (day 6) stage. All chimeric embryos (120/120, 100%) developed up to blastocyst stage. Using fluorescent microscope, we detected green signal (EGFP expression). In 90 chimeric (TR< >N) embryos (75%). Average total number of cells in chimeric embryos at blastocyst stage was 175+/-13.10, of which 58+/-2.76 cells were found in the ICM area. The number of EGFP-positive cells in the ICM area was 24+/-5.02 (35%). After the transfer of 50 chimeric rabbit embryos at the 16-cell stage, 20 embryos (40%) were flushed from five recipients on day 6 of pregnancy, of which five embryos (25%) were EGFP positive at the embryonic shield stage. Our results demonstrate that transgenic blastomeres in synchronous chimeric embryos reconstructed from TR embryos have an ability to develop and colonize ICM and embryonic shield area.     

Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment

The development of nuclear-transfer oocytes and zygotes was tested in the rabbit. Metaphase II oocytes and zygotes in the early pronuclear stage were treated with a cytoskeletal inhibitor (cytochalasin D), enucleated, and subsequently fused either with single blastomeres from eight- and 16-cell stages (oocytes and zygotes) or with pronuclei-containing karyoplasts (zygotes only). Also, nonenucleated zygotes were fused with 1/8 blastomeres. Fusion was performed by means of an electric field. Development of reconstituted embryos was monitored mainly in vitro, but a certain number of embryos developed from oocytes and zygotes receiving nuclei from eight-cell stages were also transferred into pseudopregnant does. Development of nuclear-transfer oocytes was distinctly better than that of nuclear-transfer zygotes, since 16.9% and 9.5% oocytes vs. 8.1% and 3.7% zygotes carrying eight- and 16-cell nuclei, respectively, developed to the blastocyst stage. Two advanced but already dead fetuses were found after transfer of 27 four-cell embryos obtained after fusion of oocytes with 1/8 blastomeres. No implantations were observed after transfer of 25 four-cell embryos developed from enucleated zygotes receiving eight-cell nuclei. These findings indicate that, in the rabbit, some nuclei from 16-cell embryos are still capable of promoting at least preimplantation development. Comparison between the developmental abilities of oocyte- and zygote-derived nuclear-transfer embryos also suggests that the cytoplasmic environment of recipient cell is more crucial for the development of reconstituted embryos than the stage of introduced nuclei (at least up to the 16-cell stage). The majority of pronuclear exchange embryos (69.9%) and 40% of nonenucleated zygotes receiving eight-cell nuclei were able to develop to the blastocyst stage. This latter observation indicates, similarly as with mouse, a supporting role of residual pronuclei for participation of an eight-cell nucleus in the development of reconstituted zygotes.     

The effects of altering the position of cleavage planes on the process of localization of developmental potential in ctenophores.

During the transition from the four- to the eight-cell stage in ctenophore embryos, each blastomere produces one daughter cell with the potential to form comb plate cilia and one daughter cell that does not have this potential. If the second cleavage in a two-cell embryo is blocked, at the next cleavage these embryos frequently form four blastomeres which have the configuration of the blastomeres in a normal eight-cell embryo. At this division there is also a segregation of comb plate-forming potential. By compressing a two-cell embryo in a plane perpendicular to the first plane of cleavage it is possible to produce a four-cell blastomere configuration that is identical to that produced following the inhibition of the second cleavage. However, under these circumstances the segregation of comb plate potential does not occur. These results suggest that the appropriate plane of cleavage must take place for a given cleavage cycle, in order for localizations of developmental potential to be properly positioned within blastomeres.     

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.     

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.     

Identical triplets and twins developed from isolated blastomeres of 8- and 16-cell mouse embryos supported with tetraploid blastomeres

We studied the developmental potential of single blastomeres from early cleavage mouse embryos. Eight- and sixteen-cell diploid mouse embryos were disaggregated and single blastomeres from eight-cell embryos or pairs of sister blastomeres from sixteen-cell embryos were aggregated with 4, 5 or 6 tetraploid blastomeres from 4-cell embryos. Each diploid donor embryo gave eight sister aggregates, which later were manipulated together as one group (set). The aggregates were cultured in vitro until the blastocyst stage, when they were transferred (in sets) to the oviducts of pseudopregnant recipients. Eighteen live foetuses or pups were obtained from the transfer (11.0% of transferred blastocysts) and out of those, eleven developed into fertile adults (one triplet, one pair of twins and four singletons). In all surviving adults, pups and living foetuses, only diploid cells were detected in their organs and tissues as shown by analysis of coat pigmentation and distribution of glucose phosphate isomerase isoforms. In order to explain the observed high rate of mortality of transferred blastocysts, in an accompanying experiment, the diploid and tetraploid blastomeres were labelled with different fluorochromes and then aggregated. These experiments showed the diploid cells to be present not only in the inner cell mass (ICM) but also in the trophectoderm. The low number of diploid cells and the predominance of tetraploid cells in the ICM of chimaeric blastocysts might have been responsible for high postimplantation mortality of our experimental embryos.     

Stage-specific response of preimplantation mouse embryos to W-7, a calmodulin antagonist

Involvement of calmodulin-dependent processes in preimplantation development of mouse embryos was studied with the use of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a specific antagonist of calmodulin. At 25 microM, W-7 interfered with compaction of eight-cell embryos, caused decompaction of compacted eight-cell embryos, inhibited cavitation of late morulae, and caused collapse and degeneration of blastocysts. These effects of W-7 appear to be due to specific inhibition of calmodulin-dependent processes, because W-5, a less active analogue of W-7, was less effective in interfering with development; at 25 microM, W-5 had only a slight effect on compaction and had no effect on blastocyst formation, maintenance of blastocoels, or post-blastocyst development. In addition to the developmental effects just described, W-7 inhibited cell proliferation in four-cell embryos and reduced cell numbers of morulae after treatment at the two- to eight-cell stages. There was a marked increase in embryos' sensitivity to W-7 at the late morula stage, and the sensitivity increased further as embryos developed into blastocysts; the effects of W-7 were largely reversible after treatment at the two-cell through the compacted eight-cell stages, but not after treatment at the late morula or blastocyst stage. At the blastocyst stage, inner cell mass cells appeared to be slightly more resistant to W-7 than trophectoderm cells. This differential sensitivity became more pronounced at the late blastocyst stage: after 3.5-4-h exposure of late blastocysts to 25 microM W-7, all trophectoderm cells degenerated but most of the inner cell masses survived. From these results it appears that calmodulin-dependent processes are involved in development of mouse embryos at all of the preimplantation stages examined.     

Ultrastructural localization of cytoplasmic phosphatases in preimplantation mouse embryos.

The appearance and localization of the cytoplasmic phosphatases [acid phosphatase (AcPase) as a marker of lysosomes, TPPase as a marker of the Golgi apparatus, and NDPase (IDPase) as enzymatic marker of the endoplasmic reticulum (ER)] were cytochemically studied on the ultrastructural level in secondary oocytes and in preimplantation mouse embryos. The detectable AcPase activity, located on the inner surface of the membrane delimiting some cytoplasmic vacuoles (lysosomes and autophagic vacuoles), appears at the eight-cell stage and grows pregressively stronger up to the blastocyst stage. Golgi-associated reaction for TPPase was detectable in oocytes, dropped in one-cell embryos and became negative in the two-cell embryos. The reaction for TPPase and IDPase was present in plasma membranes of oocytes and early embryos and appeared in the delimiting membrane of some cytoplasmic vesicles in eight-cell embryos. Some activity of IDPase was found in small segments of the ER at the morula and blastocyst stage. The observed results suggest that the lysosomes are the first organelles in early embryos showing activity of the marker enzymes of the phosphatase type, while the activity of other marker enzymes is mainly concentrated in the plasma membrane of blastomeres. It cannot be excluded, however, that positive reaction for TPPase and IDPase in the plasma membrane results from nonspecific action of other phosphatases.     

Effects of tunicamycin upon glycoprotein synthesis and development of early mouse embryos

Tunicamycin, an antimetabolite which inhibits the N-glycosylation of proteins, does not block the initial cleavages of mouse embryos, even at relatively high concentrations. However, it can interfere with compaction and blastocyst formation. Although tunicamycin treatment from the two-cell or eight-cell stage can cause developmental arrest prior to hatching from the zona pellucida, much higher (sublethal) concentrations of the antimetabolite added at the morula or blastocyst stage do not specifically affect hatching of blastocysts, their attachment to the substratum, or outgrowth of trophoblast cells. The consequence of continuous exposure of embryos to moderate amounts (0.05 to 0.1 μg/ml) of tunicamycin through peri-implantation stages is death of trophoblast cells with little effect upon the cells of the inner cell mass (ICM). The latter give rise to apparently normal early endoderm cells in the presence of the antimetabolite. The incorporation of leucine, mannose, and fucose into acid-insoluble material by ICM cells is only minimally inhibited by tunicamycin. On the other hand, the antimetabolite causes a severe inhibition of incorporation of not only mannose, but also leucine, into acid-insoluble material in trophoblast cells. Thus, trophoblast cells resemble transformed cells by their extreme sensitivity to tunicamycin.     

Full-term development of rat after transfer of nuclei from two-cell stage embryos

Cloning technology would allow targeted genetic alterations in the rat, a species which is yet unaccessible for such studies due to the lack of germline-competent embryonic stem cells. The present study was performed to examine the developmental ability of reconstructed rat embryos after transfer of nuclei from early preimplantation stages. We observed that single blastomeres from two-cell embryos and zygotes reconstructed by pronuclei exchange can develop in vitro until morula/blastocyst stage. When karyoplasts from blastomeres were used for the reconstruction of embryos, highest in vitro cleavage rates were obtained with nuclei in an early phase of the cell cycle transferred into enucleated preactivated oocytes or zygotes. However, further in vitro development of reconstructed embryos produced from blastomere nuclei was arrested at early cleavage stages under all conditions tested in this study. In contrast, immediate transfer to foster mothers of reconstructed embryos with nuclei from two-cell embryos at an early stage of the cell cycle in preactivated enucleated oocytes resulted in live newborn rats, with a general efficiency of 0.4%-2.2%. The genetic origin of the cloned offspring was verified by using donor nuclei from embryos of Black Hooded Wistar rats and transgenic rats carrying an ubiquitously expressed green fluorescent protein transgene. Thus, we report for the first time the production of live cloned rats using nuclei from two-cell embryos.     

Intercellular communication in in vivo- and in vitro-produced bovine embryos.

In vivo bovine embryos were obtained by nonsurgical flushing of uterine horns of cows submitted to superovulatory treatment, while in vitro embryos were generated from oocytes collected from slaughtered donors. Lucifer Yellow injected into single blastomeres did not diffuse into neighboring cells until the morula stage in in vivo embryos and the blastocyst stage in in vitro embryos. In both cases diffusion was limited to a few cells. In contrast, diffusion was extensive in microsurgically isolated inner cell mass (ICM) but absent in the trophectoderm (TE). At the blastocyst stage, diffusion was always more extensive in in vivo than in in vitro embryos. Ultrastructural analyses confirmed these functional observations, and gap junction-like structures were observed at the blastocyst stage. These structures were diffuse in the ICM of in vivo embryos, scarce in the ICM of in vitro embryos and in the TE of in vivo embryos, and not observed in the TE of in vitro embryos. Blastomeres at all stages of development from the 2-cell stage to the blastocyst stage in in vitro embryos and at the morula and blastocyst stage in in vivo embryos were electrically coupled, and the junctional conductance (Gj) decreased in in vitro embryos from 4.18 +/- 1.70 nS (2-cell stage) to 0.37 +/- 0.12 nS (blastocyst stage). At each developmental stage, in vivo embryos showed a significantly (P < 0. 05) higher Gj than in vitro-produced embryos. Moreover, a significantly (P < 0.01) higher Gj was found in isolated ICM than in the respective blastocyst in both in vivo- and in vitro-produced embryos (3.5 +/- 1.4 vs. 0.7 +/- 0.3 and 2.6 +/- 1.6 vs. 0.37 +/- 0. 12 nS, respectively). The electrical coupling in absence of dye coupling in the early bovine embryo agrees with observations for embryos from other phyla. The late and reduced expression of intercellular communicative devices in in vitro-produced embryos may be one of the factors explaining their developmental low efficiency.     

Lipid-rich blastomeres in the two-cell stage of porcine parthenotes show bias toward contributing to the embryonic part

This study was designed to determine the fate of the blastomeres in two-cell porcine parthenotes that display uneven size (larger vs. smaller) or cytoplasmic brightness (darker vs. brighter) during development to the blastocyst stage. For the non-invasive tracing of cell lineage, lipophilic fluorescence dye DiI (red) and DiD (blue) was randomly microinjected into each of two different blastomeres in each embryo. In blastocysts derived from the two-cell parthenotes with unevenly-sized blastomeres, no biased contribution was found in the progeny of either blastomere. However, in the blastocysts derived from the two-cell parthenote having different cytoplasmic brightnesses, the progeny of darker (more lipid-rich cytoplasm) blastomeres were more than two-fold more likely to form the embryonic part (43.6%; 17/39) than they were to form the abembryonic part (17.9%; 7/39), while the contribution of brighter blastomeres (less lipid) was just the opposite. The expressions of four marker genes involved in lineage allocation (Cdx2, Tead4, Oct4 and Carm1) were also analyzed in darker and brighter blastomeres of two-cell parthenotes using quantitative RT-PCR. The expression of Carm1 that encodes arginine methyltransferase 1 and that promotes inner cell mass (ICM) differentiation was significantly higher (P<0.05) in darker blastomeres. The ICM marker Oct4 also tended to be more highly expressed in the darker blastomeres, while Cdx2 and the TE marker Tead4 showed comparably higher expressions in the brighter blastomeres. However, in all cases, the marginal differences in the expression levels of Oct4, Cdx2 and Tead4 were not statistically significant (P>0.05). Our findings indicate that expression of genes related to early differentiation, especially Carm1, are partially associated with lipid droplet distribution in the two-cell porcine parthenote and may lead to biased embryonal axis formation.     

Chemical enhancement in embryo development and stem cell derivation from single blastomeres

Several chemicals targeting the mitogen-activated protein (MAP) kinase signaling pathway, which play an important role in regulating cell growth and differentiation, have shown enhancing effects on the development of the inner cell mass (ICM) and the derivation of ES cells. However, investigation of such chemicals on early embryonic development and the establishment of ES cell lines has not been elucidated. This study was aimed to determine if ACTH, MAP2K1 inhibitor [MAP2K1 (I)], and MAPK14 inhibitor [MAPK14 (I)] could enhance the development of the ICM in preimplantation mouse embryos and blastocyst outgrowths, and the establishment of ES cell lines from blastomeres of early embryos. We have demonstrated that both MAP2K1 (I) and MAPK14 (I) delay early embryo development and inhibit the development of embryos from early blastomeres. On the other hand, ACTH had a positive effect on embryos derived from early blastomeres. As a result, 17 ES cell lines were established. Among these ES cell lines, nine and five ES cell lines were established from single blastomeres of two-cell embryos with and without the supplement of ACTH, respectively. In addition to two-cell isolated blastomeres, three ES cell lines were established from blastomeres of four-cell embryos only with the supplement of ACTH. Our results suggest that ACTH can enhance the derivation of ES cells from single blastomere-derived embryos.