Cleavage in vivo and in culture in the Dasyurid marsupial Antechinus stuartii (macleay)

Cleavage in the brown marsupial mouse, Antechinus stuartii, from the zygote to the unilaminar blastocyst, was observed in vivo and in culture and in sections of embryos. The first three divisions were meridional and passed from the yolk pole to the opposite pole. Deutoplasmolysis, resulting in a distinct yolk mass, occurred during the first two divisions. Prior to the third and fourth divisions, the blastomeres elongated and flattened against the zona pellucida. The fourth division was latitudinal and resulted in two histologically distinct rings of eight blastomeres which were at first rounded and then became flattened against the zona. Further divisions and flattening of the blastomeres resulted in a complete unilaminar blastocyst by the time the blastocyst numbered 22 to 30 cells. Some expansion, causing compression of the zona and mucoid layers, occurred before completion of the blastocyst, but most expansion occurred once the blastocyst was complete. No histological differences could be detected between the blastomeres at this stage. Embryos were successfully cultured from the zygote to the rounded four-cell stage and from the flattened four-cell stage to the completed unilaminar blastocyst of around 32 cells. Total estimated cleavage times were slower in culture than in vivo, but the relative lengths of time for each division were approximately the same.     

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.     

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.     

Embryonic development in culture of two dasyurid marsupials,Sminthopsis crassicaudata (gould) and Sminthopsis macroura (spencer), during cleavage and blastocyst formation

Embryos of Sminthopsis crassicaudata and Sminthopsis macroura were cultured for up to 96 hours during cleavage and early expansion of the blastocyst in Dulbecco's modified Eagle's medium (DMEG), DMEG containing 2.76 gm/liter sodium lactate (DMEGL), DMEG containing 3.5 gm/liter galactose (DMEGAL), DMEG containing 15 ng/ml progesterone (DMEGP) or 150 ng/ml progesterone (DMEGP10), and DMEGL containing 15 ng/ml progesterone (DMEGLP). The disappearance of sperm was used to indicate the time of ovulation (day 0). Fertilized eggs were found in the uterus at the end of day 1, four-cell stages at the end of day 2, and embryos completing the fourth division by the end of day 3 in S. macroura and day 4 in S. crassicaudata. Estimated developmental times in culture were similar to those obtained in vivo. In both species, the first two divisions take about 24 hours, cleavage is arrested for 24 hours or longer at the rounded four-cell stage, and the third and fourth divisions take a further 24 hours. The blastocyst expands during the next 24 hours in which time the fifth and sixth divisions occur. It was possible to culture embryos from S. macroura but not S. crassicaudata over the four-cell stage to early expanding blastocysts. DMEGAL did not support cleavage in culture. DMEG, DMEGL, DMEGP, DMEGP10, and DMEGLP all supported culture during cleavage and early blastocyst expansion. Blastocyst expansion was slightly enhanced using media containing sodium lactate. More embryos completed the fifth division and formed expanding blastocysts in DMEG, DMEGL, and DMEGLP.     

A timetable of embryonic development, and ovarian and uterine changes during pregnancy, in the stripe-faced dunnart, Sminthopsis macroura (Marsupialia: Dasyuridae)

Aged stages (63) were available for establishment of a timetable of embryonic development of the stripe-faced dunnart. On Day 0 oocytes reaching maturity were found in the ovary. Within +/- 24 h of time 0 (time of minimum morning weight) polymorphonuclear leucocytes appeared and spermatozoa were last detected in the urine of 70% of females. Embryos were collected at intervals during pregnancy by hemihysterectomy and the embryos in the contralateral uterus either were examined at a later stage of pregnancy or allowed to develop to term. Cleavage to the unilaminar blastocyst stage with around 32 cells took 3 days with a cleavage arrest of 24 h at the 4-cell stage. Expansion of the unilaminar blastocyst occurred over the next 3 days. Primitive endoderm cells appeared on Day 6, fully bilaminar blastocysts by the end of Day 7 and trilaminar blastocysts on Day 8. Shell loss and implantation of 13-15-somite stage embryos occurred on Day 8 and organogenesis over the next 2-3 days. The gestation period was 9.5-12.0 days with most births occurring between 10.5 and 11.0 days. Major steps in embryonic development were correlated with stages in the development of the corpora lutea, which were maximal in size, and possibly in secretory activity, when the embryos were at the bilaminar blastocyst stage. Regression commenced when the embryos were at the primitive streak stage. At the time the corpora lutea were maximal the uterine epithelium reached its greatest height and the endometrium was thick and folded. Later in pregnancy villous-like projections of the epithelium formed, and the luminal epithelial cells became rounded. Two cell populations, a tier of 8 smaller cells above the yolk mass and a tier of 8 larger cells around the sides of the yolk mass appeared at the 16-cell stage. From the 16-cell stage to the blastocyst stage, with 150-200 cells, two cell populations distinguished by size, cell cycle time, cytoplasmic appearance and position relative to the yolk mass were present. The two populations were indistinguishable in blastocysts with greater than 200 and less than 2000 cells. They reappeared in blastocysts with greater than 2000 cells, as the darker cells of the embryoblast, and as the paler cells of the trophoblast. The darker cells lay in the yolky hemisphere and the paler cells in the non-yolky hemisphere.     

Effects of glucose, glutamine, ethylenediaminetetraacetic acid and oxygen tension on the concentration of reactive oxygen species and on development of the mouse preimplantation embryo in vitro.

Analysis over the first 48 h of development in vitro from the one-cell stage to the early four-cell stage indicated that (i) ethylenediaminetetraacetic acid (EDTA) exerts the major beneficial effect on culture to the blastocyst stage of F1 and MF1 embryos, (ii) glutamine assists development of MF1, but not F1, embryos to the blastocyst stage and probably functions as part of a metabolic response to oxidative damage to mitochondria and (iii) exposure to glucose at some time during early cleavage is essential for full development to blastocysts. None of the culture conditions examined affected significantly the increase in concentration of reactive oxygen species in late two-cell embryos in vitro, although F1 embryos in vitro often had lower peroxide concentrations than MF1 embryos. A decline in oxygen tension from 20 to 50% had no consistent effect on culture to the blastocyst stage or production of reactive oxygen species. Aminooxyacetate, an inhibitor of transaminase activity, prevented non-blocking embryos from developing beyond G2 of the second cell cycle. It is concluded that the chelation of transitional metals provides the most effective method of overcoming the block to development in vitro.     

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.     

Developmental potential of isolated blastomeres from early murine embryos

Experiments were designed to evaluate the effect of blastomere separation on blastocoele formation and development of viable fetuses. Two-cell and four-cell murine embryos were dissociated into individual blastomeres and cultured to the blastocyst stage. For embryos of both stages, zona removal and blastomere separation reduced (P<0.05) the number of viable embryos at the onset of culture and reduced (P<0.01) the frequency of continuation of development of blastomeres to the blastocyst stage. Attempts to repeatedly split two-cell stage embryos decreased in vitro development to blastocysts. The number of cells in two-cell embryos that were cultured to blastocyst was not different for control (64.8 +/- 11.5) or for two-cell embryos cultured without the zona pellucida (60.9 +/- 10.1) but was reduced (P<0.01) for one-half embryos that were cultured to blastocysts (35.6 +/- 10.6). The cell number of blastocysts obtained from dissociated four-cell (1/4) embryos (17.4 +/- 1.4) was similarly reduced (P<0.01). In vivo development was assessed after cultured embryos were transferred to the uteri of day 3 pseudopregnant females. Zona free intact embryos (2/36, 6%) and zona free half embryos (7/36; 19%) developed less frequently (P<0.05) than intact controls (45/100). Noncultured morula briefly exposed to pronase to thin the zona had similar impaired development. Embryos with thinned zona or no zona developed less frequently (21/82, 2/72 respectively, P<0.05) than nonpronase-treated controls (50/83).     

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.     

Early patterning of the mouse embryo--contributions of sperm and egg

The first cleavage of the fertilised mouse egg divides the zygote into two cells that have a tendency to follow distinguishable fates. One divides first and contributes its progeny predominantly to the embryonic part of the blastocyst, while the other, later dividing cell, contributes mainly to the abembryonic part. We have previously observed that both the plane of this first cleavage and the subsequent order of blastomere division tend to correlate with the position of the fertilisation cone that forms after sperm entry. But does sperm entry contribute to assigning the distinguishable fates to the first two blastomeres or is their fate an intrinsic property of the egg itself? To answer this question we examined the distribution of the progeny of early blastomeres in embryos never penetrated by sperm - parthenogenetic embryos. In contrast to fertilised eggs, we found there is no tendency for the first two parthenogenetic blastomeres to follow different fates. This outcome is independent of whether parthenogenetic eggs are haploid or diploid. Also unlike fertilised eggs, the first 2-cell blastomere to divide in parthenogenetic embryo does not necessarily contribute more cells to the blastocyst. However, even when descendants of the first dividing blastomere do predominate, they show no strong predisposition to occupy the embryonic part. Thus blastomere fate does not appear to be decided by differential cell division alone. Finally, when the cortical cytoplasm at the site of sperm entry is removed, the first cleavage plane no longer tends to divide the embryo into embryonic and abembryonic parts. Together these results indicate that in normal development fertilisation contributes to setting up embryonic patterning, alongside the role of the egg.     

Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos

Inner cell mass (ICM) and trophectoderm cell lineages in preimplantation mouse embryos were studied by means of iontophoretic injection of horseradish peroxidase (HRP) as a marker. HRP was injected into single blastomeres at the 2- and 8-cell stages and into single outer blastomeres at the 16-cell and late morula (about 22- to 32-cell) stages. After injection, embryos were either examined immediately for localization of HRP (controls) or they were allowed to develop until the blastocyst stage (1 to 3.5 days of culture) and examined for the distribution of labeled cells. In control embryos, HRP was confined to one or two outer blastomeres. In embryos allowed to develop into blastocysts, HRP-labeled progeny were distributed into patches of cells, showing that there is limited intermingling of cells during preimplantation development. A substantial fraction of injected blastomeres contributed descendants to both ICM and trophectoderm (95, 58, 44, and 35% for injected 2-cell, 8-cell, 16-cell, and late morula stages, respectively). Although more than half of the outer cells injected at 16-cell and late morula stages contributed descendants only to trophectoderm (53 and 63%, respectively), some outer cells contributed also to the ICM lineage even at the late morula stage. Although the mechanism for allocation of outer cells to the inner cell lineage is unknown, our observation of adjacent labeled mural trophectoderm and presumptive endoderm cells implicated polarized cell division. This observation also suggests that mural trophectoderm and presumptive endoderm are derived from common immediate progenitors. These cells appear to separate into inner and outer layers during the fifth cleavage division. Our results demonstrate the usefulness of HRP as a cell lineage marker in mouse embryos and show that the allocation of cells to ICM or trophectoderm begins after the 2-cell stage and continues into late cleavage.     

3H-uridine incorporation in early porcine embryos.

The present study investigated the ontogeny of 3H-uridine incorporation into RNA as a measure for RNA synthesis in preimplantation porcine embryos from the two-cell stage up to the stage of the newly hatched blastocyst. A total of 568 embryos were cultured in vitro for 3 hr in medium (KRB plus lamb serum) containing 9 microM 3H-uridine. After disruption of cell membranes, RNA was isolated on DEAE cellulose filters, and the radioactivity was taken as a measure for the rate of RNA synthesis. No RNA synthesis was detected at the two-cell stage. From the four-cell to the morula stage, 3H-uridine incorporation per embryo increased about ninefold (P less than 0.001); in blastocyst stages, the increase between developmental stages was not statistically significant. Hatched blastocysts had the highest genomic activity. On a per cell basis, 3H-uridine incorporation was not different from the four-cell stage up to the zona pellucida-intact blastocyst and amounted to 0.29-0.37 fmol 3H-uridine incorporation/cell/3 hr. In hatched blastocysts, 3H-uridine incorporation per blastomere was increased (P less than 0.01 compared with younger stages) and amounted to 0.86 fmol 3H-uridine incorporation/cell/3 hr. It is concluded that 1) the rate of uridine incorporation depends on the cell stage in zona pellucida-intact porcine embryos and 2) uridine incorporation per blastomere is significantly increased in hatched blastocysts compared with earlier stages.     

Origin of the inner cell mass in mouse embryos: cell lineage analysis by microinjection

The mouse inner cell mass is established by cells that are allocated to internal positions after the 8-cell stage. We analyzed the timing of this allocation by microinjecting two cell lineage markers, horseradish peroxidase and rhodamine-conjugated dextran, into mouse blastomeres at the 8- to 32-cell stage. Prospective analysis was performed by coinjection of peroxidase and dextran, followed by 12-22 hr of culture and staining for peroxidase activity; retrospective analysis was performed by injection of peroxidase alone and localization of sister cells without further culture. Both approaches indicated that cells are allocated to internal positions during the fourth and fifth cleavage divisions, but not the sixth cleavage division, of the mouse embryo. Thus, outer cells can have inner descendants until the late morula/early blastocyst (32-cell) stage, but cells remaining outside after the fifth cleavage division are restricted to a trophectoderm fate. This information about cell lineage indicates that the previously observed totipotency of the cleaving mammalian embryo's cells is a regulative attribute that is used in normal development.     

Lectin-induced blockage of developmental processes in preimplantation mouse embryos in vitro

This study attempts to assess the developmental importance of cell surface glycoconjugates of preimplantation mouse embryos. This was done by incubating early embryos in various lectins and analyzing subsequent development. If specific cell surface glycoconjugates (lectin receptors) are linked to specific developmental processes, such as cell division, compaction, and blastocyst formation, then different lectins should block these different developmental processes. The results show that wheat-germ agglutinin (WGA; N-acetyl-D-glucosamine-specific) at 50 μg/ml prevents the cell division of four-cell embryos. However, this effect of WGA occurs only in embryos with intact zonae pellucidae. Concanavalin A (Con A; α-D-glucose and α-D-mannose-specific) treatment, 20 μg/ml, of four-cell or early eight-cell embryos prevents compaction, the first major change in cell shape in early mouse embryogenesis. Divalent succinly Con A does not affect development, suggesting that the Con A effect is due to crosslinking of cell surface glycoconjugates. Exposure of four-cell or early eight-cell embryos to 10 μg/ml Lotus Tetragonolobus puprureas agglutinin (LTA; α-L-fucose-specific) or 25 μg/ml Limulus polyphemus agglutinin (LPA; sialic acid-specific) allows compaction or development to the morula stage, but blocks blastocyst formation. All lectins tested retard cell division to some extent. Late morulae and early blastocysts are more resistant than earlier stages to all of the lectins studied. This study demonstrates that very low concentrations of these lectins affect different developmental processes, presumably based upon their sugar specificities.     

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.     

Timetable of in vivo embryonic development in the grey short-tailed opossum (Monodelphis domestica)

The timing of development was examined in 496 embryos from female Monodelphis domestica, collected at known time intervals after video recorded mating. Ovulation occurred approximately 20 hr (day 1) after mating, and fertilization was observed by 24 hr. Transport through the oviducts was rapid, and pronuclear stage embryos were recovered from the uterus as early as 24 hr after mating. Second cleavage had occurred by 55 hr after mating. Three-celled embryos were among those collected on day 3 after mating, indicating that asynchronous cleavage of blastomeres can occur from the two-cell stage. The four-cell stage persisted for approximately 24 hr, and embryos that had undergone third cleavage were first recovered 74 hr after mating. Embryos that had undergone fourth to fifth cleavage were found 96–100 hr (4 days) after mating and complete unilaminar blastocysts by 5.5 days after mating. Primary endoderm formed from an already distinct embryonic area of the unilaminar blastocyst early on day 7 after mating. Formation of the bilaminar blastocyst was completed rapidly, on day 7 after mating. The primitive streak appeared on day 10 after mating, and organogenesis rapidly ensued on a timetable similar to that reported for Didelphis virginiana (McCrady, 1938). Close contact with the maternal circulation was established on day 11 and by day 12 maternal and embryonic tissues could not be separated without damage. The length of the gestation period from fertilization to birth was approximately 13.5 days. These observations provide the basis for further embryological cellular and molecular studies of this species as a laboratory model for marsupial development.© 1994 Wiley-Liss, Inc.     

Synchronization of cell division in eight-cell bovine embryos produced in vitro: effects of aphidicolin

To date, methods for synchronizing the cell division of ungulate embryos without reducing their developmental potential have not been reliable or simple. The overall objective of this study was to determine the reliability of aphidicolin, a powerful inhibitor of eukaryotic DNA synthesis, to arrest and synchronize blastomere division in cleavage-stage bovine embryos and to assess its reversibility and toxicity in vitro. Eight-cell stage embryos obtained at 58 h post insemination were treated with several concentrations of aphidicolin for 12 h. Treated embryos were assessed for cleavage arrest, chromatin morphology and DNA synthesis; scored for blastocyst formation and hatching rate; and fixed for determination of the number of nuclei. Complete arrest of cell division was observed at aphidicolin concentrations of 1.4 microM and above. At these concentrations, no morphological alteration to interphase chromatin was observed in treated embryos compared with the controls. Removal of aphidicolin led to at least a 4-h delay before resumption of DNA synthesis and cleavage. The ability of treated embryos to reach the blastocyst stage in vitro, the hatching rate and the number of cells per blastocyst were significantly reduced compared with the control group. Since the ability of treated embryos to develop to the blastocyst stage was significantly reduced even at the minimal effective dosage, it is concluded that aphidicolin is unlikely to provide suitable cell cycle synchronization without damage to the embryos.     

In vitro development and cell allocation of porcine blastocysts derived by aggregation of in vitro fertilized embryos

In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to those of their in vivo counterparts. The objective of this study was to increase developmental competence and to gain an understanding of cell allocation in blastocysts derived from the aggregation of four-cell stage porcine embryos produced in vitro. After removal of the zona pellucida, two (2x) and three (3x) four-cell stage embryos were aggregated by co-culturing them in aggregation plates. Five days after aggregation, the developmental ability and the number of cells in the aggregated embryos were determined. The percentage of blastocysts was higher (P < 0.05) in both the 2x and 3x aggregated embryos (66.6% and 72.0%, respectively) compared to that of the 1x embryos and the intact controls (43.1% and 36.4%, respectively). The total cell number of blastocysts also increased in aggregated embryos compared to that of intact controls (2.6-fold for 2x and 3.4-fold for 3x) (P < 0.05). The cells of two differentially stained embryos were started to mix at 72 hr after aggregation. In vitro-fertilized porcine aggregates (2x) were developed to blastocyst with a random distribution of cells from each embryo. The mRNA levels for the oct-4, bcl-xL and connexin 43 genes were higher (P < 0.05) and bak gene were lower (P < 0.05) in both the 2x and 3x aggregated embryos than the intact controls. Therefore, the aggregation of the four-cell stage embryos could be used to improve the quality of porcine preimplantation stage embryos produced in vitro.     

Effect of Initiation Time of Hydrostatic Pressure Shock on Chromosome Set Doubling of Tetraploidization in Turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis>

The objective of the study was to clarify the effects of initiation time on chromosome set doubling induced by hydrostatic pressure shock through nuclear phase fluorescent microscopy in turbot Scophthalmus maximus. The ratio of developmentally delayed embryo and chromosome counting was used to assess induction efficiency. For the embryos subjected to a pressure of 67.5 MPa for 6 min at prometaphase (A group), chromosomes recovered to the pre-treatment condition after 11-min recovering. The first nuclear division and cytokinesis proceeded normally. During the second cell cycle, chromosomes did not enter into metaphase after prometaphase, but spread around for about 13 min, then assembled together and formed a large nucleus without anaphase separation; the second nuclear division and cytokinesis was inhibited. The ratio of developmentally delayed embryo showed that the second mitosis of 78% A group embryo was inhibited. The result of chromosome counting showed that the tetraploidization rate of A group was 72%. For the embryos subjected to a pressure of 67.5 MPa for 6 min at anaphase (B group), chromosomes recovered to the pre-treatment condition after about 31-min recovering. Afterwards, one telophase nucleus formed without anaphase separation; the first nuclear division was inhibited. The time of the first cleavage furrow occurrence of B group embryos delayed 27 min compared with that of A group embryos. With the first cytokinesis proceeding normally, 81.3% B group embryos were at two-cell stage around the middle of the second cell cycle after treatment. Those embryos were one of the two blastomeres containing DNA and the other without DNA. The first nuclear division of those embryos was inhibited. During the third cell cycle after treatment, 65.2% of those abovementioned embryos were at four-cell stage, cytokinesis occurred in both blastomeres, and nuclear division only occurred in the blastomere containing DNA. Of those abovementioned embryos, 14.0% were at three-cell stage and cytokinesis only occurred in the blastomere containing DNA. The result of chromosome counting showed that the tetraploidization rate of B group was only 7%. To summarize what had been mentioned above, mechanisms on chromosome set doubling of tetraploid induction would be different with different initiation time of hydrostatic pressure treatment. Chromosome set doubling was mainly due to inhibition of the second mitosis when hydrostatic pressure treatment was performed at prometaphase. Otherwise, chromosome set doubling was mainly due to inhibition of the first nuclear division when hydrostatic pressure treatment was performed at anaphase. Induction efficiency of tetraploidization resulted from inhibition of the second cleavage was higher than which resulted from inhibition of the first nuclear division. This study was the first to reveal biological mechanisms on the two viewpoints of chromosome set doubling through effect of initiation time of hydrostatic pressure treatment on chromosome set doubling in tetraploid induction.