Patricia G. Calarco

Cell Surface Changes during Preimplantation Development in the Mouse

Scanning electron microscopy reveals microvilli on all preimplantation stages, indicates that their number and length may be dependent on embryo size, and provides examples of regional alterations in their number. Cellular adherence, as evidence by interactions of microvilli, migration of cellular processes, and junctional complexes, increases during development and is accompanied by changes in the shapes of cells and embryos. Cell surfaces bordering the blastocoel differ markedly from the outer cell surfaces of the embryo.

Inner Cell Allocation in the Mouse Morula: The Role of Oriented Division during Fourth Cleavage

Two populations of blastomeres become positionally distinct during fourth cleavage in the mouse embryo; the inner cells become enclosed within the embryo and the outer cells form the enclosing layer. The segregation of these two cell populations is important for later development, because it represents the initial step in the divergence of placental and fetal lineages. The mechanism by which the inner cells become allocated has been thought to involve the oriented division of polarized 8-cell blastomeres, but this has never been examined in the intact embryo. By using the technique of time-lapse cinemicrography, we have been able for the first time to directly examine the division planes of 8-cell blastomeres during fourth cleavage, and find that there are three, rather than two, major division plane orientations; anticlinal (perpendicular to the outer surface of the blastomere), periclinal (parallel to the outer surface of the blastomere), and oblique (at an angle between the other two). The observed frequencies of each type of division plane orientation provide evidence that the inner cells of the morula must derive from oriented division of 8-cell blastomeres, in accordance with the polarization hypothesis. Analysis of fourth cleavage division plane orientation with respect to either lineage or division order reveals that it is not associated with lineage from either the 2- or the 4-cell stage, but has a slight statistical association with fourth cleavage division order. The lack of association between division plane orientation and lineage supports the prediction that packing patterns and intercellular interactions within the 8-cell embryo during compaction play a role in determining fourth cleavage division plane orientation and thus, the positional fate of the daughter 16-cell blastomeres.

Centrosome precursors in the acentriolar mouse oocyte.

Centrioles disappear from the mouse oocyte during early oogenesis. However, it has been known for some time that multiple structures known as microtubule organizing centers (MTOC) form the spindle poles during meiosis as well as the mitotic poles during early cleavage. The objective of this study was to identify and describe the structures which exist prior to the appearance of the multiple MTOC associated with meiotic division. Reported here for the first time is a description of the unique structures which exist before the onset of oocyte maturation, their location, and microtubule (MT) nucleating ability. Correlative confocal, immuno‐, and electron microscopic studies of mouse oocytes released from ovaries directly into 2% paraformaldehyde (time zero) show two large γ‐tubulin‐positive structures in the cortex, averaging 10 μm in diameter. The present work is the first to demonstrate that although these structures contain γ‐tubulin, they do not resemble MTOC morphologically nor do they appear to nucleate MT. They are termed here multivesicular aggregates (MVA), and ultrastructural analysis reveals that they contain a variety of vesicular structures including many ring structures of approximately 25 nm. At the onset of maturation, these two MVA migrate toward the GV breaking into smaller units, only some of which mature into MTOC and nucleate MT. These correlative microscopic studies support the conclusion that MVA are centrosomal precursors, but with a unique ultrastructure. The ultrastructural organization of MVA may explain the cryptic function of MTOC in the prematuration environment of the dictyate oocyte. Microsc. Res. Tech. 49:428–434, 2000.

THE EXPRESSION OF INTRACISTERNAL A PARTICLE GENES IN THE PREIMPLANTATION MOUSE EMBRYO

Intracisternal A particles (IAP), murine endogenous retrovirus, make up 0.3% of the mouse genome. They are expressed in some normal tissues, certain transformed cell lines, and show stage-specific patterns of expression in early embryos. We have used peptide-specific antisera and the polymerase chain reaction to explore type-specific expression of these IAP during preimplantation development. In this paper we show that the IAP core protein, p73, characteristic of type IIAP, is present throughout preimplantation development while the gag-pol fusion protein p120, characteristic of the variant type I delta 1, is synthesized and expressed only from the 8-cell stage onward. Type IIAP RNA is present at all stages and appearance of p120 at the 8-cell stage could represent new transcription or translation from a preexisting I delta 1 message. The presence of type II IAP RNA varies according to stage, with two sizes of type II transcripts present at all stages except the 2-cell stage at which time only the smaller of the two transcripts can be detected. The reappearance of the larger type II transcript subsequent to the 2-cell stage implies new transcription of this type II subspecies. The presence of type I, II, and p73 in the unfertilized egg strongly suggests maternal inheritance from the oocyte.

The effects of anti-embryo sera and their localization on the cell surface during mouse preimplantation development

Abstract We have developed two rabbit antisera, one against mouse blastocysts and a second against mouse placentae. After absorption of these antisera with adult mouse tissues and extensive dialysis, results from indirect immunofluorescence, cytotoxicity, and culturing experiments lead us to two major conclusions. First, anti-blastocyst serum detects a group of cell surface molecules whose expression is embryo-specific, stage-specific, and whose unaltered presence is required for preimplantation development in vitro. Second, anti-placenta serum detects a different group of cell surface molecules that are present before fertilization and become segregated to the syncytiotrophoblast, appear unessential for preimplantation development in vitro, and may function in trophoblast differentiation, and/or in protection of the fetus from maternal immunologic attack.

Characterization and localization of a mouse egg cortical granule antigen prior to and following fertilization or egg activation.

Immunological approaches were used to characterize an antigen that is present within the cortical granules of mouse oocytes and eggs. Immunoelectron microscopy shows a specific localization of the antigen to the cortical granules in the cortex of mouse oocytes and eggs. Following in vitro fertilization, the antigen is present in the perivitelline space and is associated with the zona pellucida. No cortical granules and very little antigen are detected in the two-cell embryo. This antiserum detects a protein of Mr = 75,000 (p75) following immunostaining of egg proteins on Western blots, or immunoprecipitation of metabolically labeled oocyte proteins or radio-iodinated egg proteins. p75 is also present in exudates obtained from A23187-treated eggs, as detected by either radio-iodination of the released egg proteins, or maturation and ionophore activation of metabolically labeled oocytes. Two-dimensional gel electrophoresis of radio-iodinated egg proteins reveals four species of p75 with pIs between 4.9 and 5.3, whereas only the most basic form of p75 is detected in metabolically labeled oocytes. Multiple forms of the radio-iodinated p75 are present in the exudate of ionophore-treated eggs. p75 displays a greater electrophoretic mobility under nonreducing conditions, indicating the presence of intramolecular disulfide bonds, a common characteristic of secreted proteins. We conclude that p75 is synthesized in oocytes, modified and packaged into cortical granules, and released from eggs following fertilization or activation.

Immunological characterization of embryonic cell surface antigens recognized by antiblastocyst serum

Abstract Stage-specific cell surface antigens expressed during mouse preimplantation development and detected by a rabbit antiserum prepared against mouse blastocysts (A-BL2) have been characterized by serological and biochemical techniques. Immunofluorescence, immunoradiolabeling, and complement-mediated cytotoxicity assays reveal the expression of A-BL2 surface antigens beginning at the 4-cell stage and reaching a maximum at the 8-cell to morula stages. At earlier times in development A-BL2 antigens are not detectable, and there is a decline in expression at the blastocyst stage. No antibody reactivity is detected against adult mouse tissues or teratocarcinoma cell lines. The presence of A-BL2 antibodies during in vitro embryo culture interferes with normal development. Treatment of embryos with β-N-acetylglucosaminidase, but not other glycosidases, proteases, or lipases, results in a quantitative decrease in the binding of A-BL2 antibodies to surface antigens. Immunoprecipitation and electrophoretic analyses of A-BL2 antigens demonstrate specific antibody activity against a pair of embryonic glycoproteins of 65,000 to 70,000 daltons which can be metabolically labeled with 35S-methionine and 3H-glucosamine. Tunicamycin treatment alters the form of the A-BL2 immunoprecipitate to a single 60,000-dalton protein.

Biochemical heterogeneity, migration, and pre-fertilization release of mouse oocyte cortical granules.

BackgroundOocyte cortical granules are important in the fertilization of numerous species including mammals. Relatively little is known about the composition, migration, and pre-fertilization release of mammalian oocyte cortical granules.ResultsResults obtained with confocal scanning laser microscopy indicated that mouse oocytes have at least two populations of cortical granules, one that bound both the lectin LCA and the antibody ABL2 and one that bound only LCA. Both types of granules were synthesized at the same time during oocyte maturation suggesting that the ABL2 antigen is targeted to specific granules by a sorting sequence. The distribution of both populations of cortical granules was then studied during the germinal vesicle to metaphase II transition. As the oocytes entered metaphase I, the first cortical granule free domain, which was devoid of both populations of cortical granules, formed over the spindle. During first polar body extrusion, a subpopulation of LCA-binding granules became concentrated in the cleavage furrow and underwent exocytosis prior to fertilization. Granules that bound ABL2 were not exocytosed at this time. Much of the LCA-binding exudate from the release at the cleavage furrow was retained in the perivitelline space near the region of exocytosis and was deduced to contain at least three polypeptides with approximate molecular weights of 90, 62, and 56 kDa. A second cortical granule free domain developed following pre-fertilization exocytosis and subsequently continued to increase in area as both, LCA and LCA/ ABL2-binding granules near the spindle became redistributed toward the equator of the oocyte. The pre-fertilization release of cortical granules did not affect binding of sperm to the overlying zona pellucida.ConclusionsOur data show that mouse oocytes contain at least two populations of cortical granules and that a subset of LCA-binding cortical granules is released at a specific time (during extrusion of the first polar body) and place (around the cleavage furrow) prior to fertilization. The observations indicate that the functions of the cortical granules are more complex than previously realized and include events occurring prior to gamete membrane fusion.

Electrophoretic Analysis of Cell Surface Proteins of Preimplantation Mouse Embryos

Abstract Cell surface proteins expressed during mouse preimplantation development have been labeled by lactoperoxidase-catalyzed iodination and analyzed by SDS-polyacrylamide gel electrophoresis. Results reveal that embryonic surface proteins are labeled at all stages tested and that the major qualitative changes in surface protein composition occur following fertilization and coincident with blastocyst formation.

Stage-specific embryonic antigens detected by an antiserum against mouse blastocysts.

Abstract Stage-specific cell surface antigens expressed during preimplantation mouse development and detected by a rabbit antiserum prepared against mouse blastocysts (A-BL2) have been characterized by immunoprecipitation and gel electrophoresis. Antibody activity is shown to be directed primarily against a pair of possibly related proteins of 65,000 to 70,000 daltons. These proteins are stage specific, being synthesized only between the 2-cell and morula stages of mouse preimplantation development. Two-dimensional gel analysis shows the A-BL2 immunoprecipitate to consist of at least three pairs of proteins showing charge heterogeneity on isoelectric focusing. This report is the first biochemical characterization of stage-specific embryonic antigens synthesized during murine preimplantation development.