Enzymatic amplification of a Y chromosome repeat in a single blastomere allows identification of the sex of preimplantation mouse embryos.

The polymerase chain reaction (PCR) technique has been adapted to identify the sex of preimplantation mouse embryos rapidly. PCR was used to amplify a specific repeated DNA sequence on the Y chromosome from a single isolated blastomere in under 12 hr. The remainder of the biopsied embryo was then transferred to a pseudopregnant female and carried to term. Using this technique, 72% of embryos can be classed as potentially either male or female. Transfers of such embryos have produced pregnancies with 8/8 fetuses (100%) being of the predicted sex. Variations of the technique have demonstrated certain limitations to the present procedure as well as indicated possible strategies for improvement of the assay. The PCR technique may have wide application in the genetic analysis of preimplantation embryos.

[Biogenic monoamines in ovum cells and preimplantation embryos of mice]. / Biogennye monoaminy v iaitsekletkakh i doimplantsionnykh zarodyshakh myshei.

Histofluorescence technique using glyoxylic acid revealed a specific fluorescence suggesting the presence of biogenic monoamines in early developmental stages of CBA x C57 Black mice. A yellow fluorescence observed in the blastomere surface from the stage of zygote up to that of four blastomere points to the presence of indole derivates. As development proceeds, the fluorescence increases and its colour becomes more and more green, which is characteristic of catecholamines. From the stage of eight blastomeres up to stage of blastocyst specific fluorescence is revealed in the cytoplasm. The inhibitors of monoamine oxidase, introduced into pregnant mice, markedly increased the specific fluorescence. An assumption is made of functional activity of biogenic monoamines in early mouse embryos.

Specification of notochord cells in the ascidian embryo analysed with a specific monoclonal antibody.

Among 40 notochord cells of an ascidian tadpole larva, 32 notochord cells originate from the anterior-vegetal blastomeres (the A4.1 pair) of an 8-cell embryo and eight cells originate from the posterior-vegetal blastomeres (the B4.1 pair), but the animal blastomeres (the a4.2 and b4.2 pairs) are not engaged in the formation of the notochord. If four pairs of cells, separated from an 8-cell embryo, were allowed to develop into quarter embryos, expression of the notochord-specific antigen was evident in the A4.1 and B4.1 quarter embryos. Embryos, in which cytokinesis had been permanently blocked at the 8-cell and later stages with cytochalasin B, were found to develop the notochord-specific antigen only in the presumptive notochord cells. These findings suggest the developmental autonomy of presumptive notochord cells in the ascidian embryo.

Diagnosis of beta-thalassaemia by DNA amplification in single blastomeres from mouse preimplantation embryos.

Mouse preimplantation embryos were accurately diagnosed as normal or mutant at the beta-major haemoglobin locus by amplification of specific DNA sequences in a single cell. A DNA sequence containing the whole of exon 3 and some 3' untranslated sequences within the beta-major haemoglobin gene was amplified in single blastomeres by means of the polymerase chain reaction (PCR). Blastomeres were removed from embryos of four to eight cells from normal BALB/c mice and from mutant (thalassaemic) BALB/c mice homozygous for a deletion of the whole beta-major haemoglobin gene. The sensitivity of the amplification procedure was enhanced by the sequential use of two sets of oligonucleotide primers for 30 cycles of amplification each, the second pair being located within the segment amplified by the first pair. The product (204 base-pairs) could be easily visualised in ethidium bromide-stained agarose gels. Stringent precautions to prevent contamination were taken, and with these precautions the PCR amplification procedure could be carried out under normal laboratory conditions. These procedures for diagnosis of genetic disease before implantation should be applicable to preimplantation diagnosis of any monogenic disorder in man for which the affected DNA sequence is known.

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.

Immunocytochemical localization of histones H2B, H3 and H4 in pronuclei and four-cell stages of porcine embryos. Preliminary results.

In this preliminary work, using pig embryos ultrastructural immunocytochemistry with polyclonal antibodies against purified histones was used to demonstrate both their localization and the time of their appearance in pronuclei, from 15 h after ovulation (pronuclear stage) to 48 h postinsemination (4-cell stage). In pronuclei, the histones H2B, H3, and H4 were located in the heterochromatin as soon as it appeared. Usually, one of the pronuclei seemed to be more heavily labelled. The chromatin facing the zone of pronuclear contact formed a bowl-shaped region in each pronucleus heavily labelled for these histones. The so-called pseudo-nucleoli were present in both pronuclei and contained H2B. In 4-cell stages, the labelling intensities of heterochromatin for H2B, H3 and H4 were equal in all the nuclei. H2B was still evident in the pseudo-nucleoli, but in a lower quantity than before. The condensed chromatin located either under the nuclear envelope or surrounding the pseudo-nucleoli was heavily labelled for H2B, H3 and H4.

Evaluation of different biopsy methods of blastomeres from 2-cell mouse embryos.

As an outgrowth of in-vitro fertilization and embryo transfer, detection of genetic and metabolic defects prior to implantation might be possible in the future. The objective for preimplantation diagnosis would be to sample a minimum of cell material of the conceptus for diagnosis prior to transfer. Different protocols for isolating individual blastomeres out of 2-cell mouse embryos were evaluated. 2-cell mouse embryos (from F1 hybrids C57B1 females x CBA males) were collected and the zona pellucida was removed by enzyme treatment (pronase), by exposure to Ca2+-Mg2+-free acid Tyrode (pH = 2.5) or by mechanical force. Individual blastomeres were obtained by exposure to an enzyme (pronase), to a chelating agent (EDTA-glycine mixture), to Ca2+-Mg2+-free PBS or after isolation by mechanical force. The biopsied blastomeres were then cultured in vitro as such or first replaced into a host zona pellucida. Evaluation was performed by culture in vitro up to the blastocyst stage and by transfer of embryos appearing morphologically normal into pseudopregnant foster mothers. A chromosomal study of the second mitotic division of the isolated blastomere was also performed. All isolation procedures had a negative impact on the in-vitro and in-vivo growth patterns of the isolated blastomeres. After culture in vitro to the blastocyst stage, different abnormalities could be observed: embryos lacking compaction, embryos with double blastocoelic cavities and embryos with no inner cell mass (trophoblastic vesicle). After replacement of the isolated blastomeres into a host zona pellucida, similar observations could be made. Chromosomal analysis did not reveal a clear influence of the different biopsy methods on the mitosis of the isolated blastomeres.

Spectrin and calmodulin in spreading mouse blastomeres.

The role of spectrin and its association with calmodulin in spreading mouse blastomeres was investigated. Embryonic spectrin binds 125I-calmodulin in a calcium-dependent fashion in the blot overlay technique. Double-labeling experiments show coordinate redistribution of spectrin and calmodulin in blastomeres preparing to undergo active spreading movement. At this stage cortical spectrin staining is lost from the region of cell-substrate contact and spectrin and calmodulin become concentrated in two structures closely associated with the contacted region: a group of spherical bodies located on the cytoplasmic side of the cortical layer and a subcortical ring that marks the perimeter of the contacted region. The localization pattern of spectrin and calmodulin is also coordinated with that of actin and myosin. The results suggest that spectrin plays a role in the spreading of blastomeres and that this function may involve linkage of spectrin, calmodulin, and the cortical contractile apparatus.

A possible maternal-effect mutant of Xenopus laevis I. Cytological and biochemical analyses of the unfertilized eggs and embryos.

In an effort to define the cause of the developmental arrest of offspring from a certain Xenopus female (designated as No. 65), we have examined eggs and embryos from the female both cytologically and biochemically. Light and electron microscopic observations revealed that all of the blastomeres from embryos of female No. 65 had multiple small spherical nuclei, while wild-type counterparts had a single lobulated nucleus. Two-dimensional gel electrophoretic analyses demonstrated that a major acidic protein, whose molecular weight was 38 kDa, was always found in samples from wild-type unfertilized eggs and embryos, but was not recognizable in those from female No. 65. These facts, coupled with the death of the offspring at gastrulation, suggest the possibility that female No. 65 carries a mutation of the ova-deficient type.

Fourth cleavage of sea urchin blastomeres: microtubule patterns and myosin localization in equal and unequal cell divisions.

This study traces the morphological appearance, organization, and disappearance of the cytoskeletal machinery for cell division during the fourth cell cycle of isolated sea urchin blastomeres by immunolocalization of tubulin and myosin. Mesomere-mesomeres (which divide equally) and macromere-micromeres (which divide unequally) are compared in terms of their asters (both mitotic and so-called interphase asters), spindle apparatus, and contractile ring. The results suggest that the distinctive nuclear positioning of these blastomeres is established in late interphase, that centrosomal alignment occurs in prophase, that all of the dominant astral configurations in the cell cycle belong to a single cycle of assembly-disassembly, that a second interphase-specific cycle of assembly-disassembly is confined to a diffuse cytoplasmic reticulum, and that contractile ring myosin concentrates and disperses in precise coincidence with the beginning and end of cleavage furrowing.

Preimplantation diagnosis of deficiency of hypoxanthine phosphoribosyl transferase in a mouse model for Lesch-Nyhan syndrome.

Male mice embryos deficient in hypoxanthine phosphoribosyl transferase (HPRT), derived from heterozygous (carrier) females and normal males, were diagnosed by biochemical microassay of HPRT activity in a single cell isolated from the eight-cell preimplantation embryo. The sampled embryos were transferred to recipient mothers and examined on the 14th day of gestation to confirm the accuracy of the preimplantation diagnosis. The diagnosis was sufficiently rapid that freezing of the embryos before transfer was not necessary. Of the embryos diagnosed as HPRT negative all 4 that grew into fetuses were correctly identified as HPRT-deficient males.

Fate map for the 32-cell stage of Xenopus laevis.

A complete fate map has been produced for the 32-cell stage of Xenopus laevis. Embryos with a regular cleavage pattern were selected and individual blastomeres were injected with the lineage label fluorescein-dextran-amine (FDA). The spatial location of the clones was deduced from three-dimensional (3D) reconstructions of later stages and the volume of each tissue colonized by labelled cells in each tissue was measured. The results from 107 cases were pooled to give a fate map which shows the fate of each blastomere in terms of tissue types, the composition of each tissue by blastomere, the location of each prospective region on the embryo and the fate of each blastomere in terms of spatial localization. Morphogenetic movements up to stage 10 (early gastrula) were assessed by carrying out a number of orthotopic grafts at blastula and gastrula stages using donor embryos uniformly labelled with FDA. Although there is a regular topographic projection from the 32-cell stage this varies a little between individuals because of variability of positions of cleavage planes and because of short-range cell mixing during gastrulation. The cell mixing means that the topographic projection fails for anteroposterior segments of the dorsal axial structures and it is not possible to include short segments of notochord or neural tube or individual somites on the pregastrulation fate map.

DNA replication and compaction in the cleaving embryo of the mouse.

The effects of aphidicolin, a reversible inhibitor of DNA polymerase alpha, both on replication and on development of the mouse embryo from the 2- and 4-cell stages to the compacted late 8-cell stage have been assessed. The continuous presence of aphidicolin from G1 of the 4-cell stage resulted in inhibition of DNA replication and prevention of division from 4 to 8 cells, but was without effect on the timing or incidence of cell flattening, surface polarization and cytoplasmic polarization. The continuous presence of aphidicolin from G1 of the 2-cell stage resulted in inhibition of DNA replication, division, and polarization. Some slight intercellular flattening in a few embryos did occur. If addition of aphidicolin was delayed by 10 h to early in G2 of the 2-cell stage, further rounds of replication were blocked and some embryos failed to cleave to 4-cells. Nevertheless, almost all embryos showed evidence of flattening and polarization regardless of cell number. In contrast, if aphidicolin was added in G1 of the 2-cell stage and removed after 10 h, the cells showed delayed DNA replication, little evidence of division, and no cell flattening or polarization. We conclude that DNA replication at the 2-cell stage may be essential for the components of compaction studied, but that DNA replication at the 4- and 8-cell stages is not.

Mapping the distribution of differentiation potential for intestine, muscle, and hypodermis during early development in Caenorhabditis elegans.

We have analyzed the differentiation potential of cells in early embryos of Caenorhabditis elegans by assessing the production of markers for intestinal, muscle, and hypodermal cell differentiation in cleavage-arrested blastomeres. Our results show that differentiation potential does not always segregate during cleavage in a linear fashion, i.e., a blastomere can express a differentiation potential that is absent in its parent blastomere and vice versa. Furthermore, the expression of a particular differentiation program by certain cleavage-arrested blastomeres is an exclusive event in that each cell will express only one program of differentiation, even though it may have the potential to express several.

High resolution cytochemical study of uterine epithelial cell surface of the rat at identified sites previous to blastocyst-endometrial contract.

A transmission electron microscopy study was carried out in prospective implantation sites in uterine horns of rats of the Long Evans strain. The purpose was to search for possible modifications in glycoproteins of outer coats of endometrial epithelium shortly before blastocyst-endometrial contact Shea's technique (Lanthanum-Alcian blue) was used to visualize glycoproteins. Prospective implantation sites disclosed a rather thin finely granular electrondense layer along the plasma membrane of the surface endometrial cells. In addition, there was a decrease in the number of microvilli as well as a lack of Lanthanum precipitate in junctional complexes. Sites remote from prospective implantation areas, disclosed a thicker electrondense layer of glycoproteins in cell plasma membranes including those of well developed microvilli. The junctional complexes were well depicted by the electrodense lanthanum salt precipitates. These observations suggest that a mechanism other than a more physical contact take place and induce the above changes.