Expression and function of cell surface extracellular matrix receptors in mouse blastocyst attachment and outgrowth.

Mouse-hatched blastocysts cultured in vitro will attach and form outgrowths of trophoblast cells on appropriate substrates, providing a model for implantation. Immediately after hatching, the surfaces of blastocysts are quiescent and are not adhesive. Over the period 24-36 h post-hatching, blastocysts cultured in serum-free medium become adhesive and attach and spread on the extracellular matrix components fibronectin, laminin, and collagen type IV in a ligand specific manner. Attachment and trophoblast outgrowth on these substrates can be inhibited by addition to the culture medium of an antibody, anti-ECMr (anti-extracellular matrix receptor), that recognizes a group of 140-kD glycoproteins similar to those of the 140-kD extracellular matrix receptor complex (integrin) recognized in avian cells by CSAT and JG22 monoclonal antibodies. Addition to the culture medium of a synthetic peptide containing the Arg-Gly-Asp tripeptide cell recognition sequence of fibronectin inhibits trophoblast outgrowth on both laminin and fibronectin. However, the presence of the peptide does not affect attachment of the blastocysts to either ligand. Immunoprecipitation of 125I surface-labeled embryos using anti-ECMr reveals that antigens recognized by this antibody are exposed on the surfaces of embryos at a time when they are spreading on the substrate, but are not detectable immediately after hatching. Immunofluorescence experiments show that both the ECMr antigens and the cytoskeletal proteins vinculin and talin are enriched on the cell processes and ventral surfaces of trophectoderm cells in embryo outgrowths, in patterns similar to those seen in fibroblasts, and consistent with their role in adhesion of the trophoblast cells to the substratum.

Immunoprecipitation of intracisternal A-particle-associated antigens from preimplantation mouse embryos.

Murine intracisternal A-particles (IAP) are endogenous retrovirus particles expressed in large numbers in certain neoplastic tissues and during early stages of normal preimplantation mouse (outbred ICR) embryogenesis. IAP-associated antigens synthesized by preimplantation mouse embryos were precipitated by a rabbit (New Zealand White) antibody prepared against IAP from murine myeloma 104E. Characterization of the embryo immunoprecipitates with the use of one- and two- dimensional polyacrylamide gel electrophoresis revealed that a group of five proteins was synthesized by two- to eight-cell stages, but only three of these (molecular weight of 67,000, 69,000, and 73,000) correlated with the morphologic expression of IAP in embryos. ONly the proteins with molecular weights of 75,000 and 77,000 were detected in morulae and blastocysts, when embryos were IAP-negative. This is the first biochemical identification of endogenous retrovirus-associated antigens in preimplantation mouse embryos.

Immunologic relatedness of intracisternal A-particles in mouse embryos and neoplastic cell lines.

Murine intracisternal A-particles (IAP) are retroviruses expressed in large numbers in certain neoplastic cells and preimplantation mouse embryos. Immuno-electron microscopic detection of IAP-associated antigens in mouse rhabdomyosarcoma cells and early mouse embryos provides the first direct evidence that IAP produced by these two sources are serologically related. In both embryos and cells, data suggest that, after synthesis, IAP core protein rapidly associates with the endoplasmic reticulum. In addition, in embryos only, data confirm earlier observations that IAP-associated antigens are expressed on the cell surface. There is no evidence that the Golgi apparatus is involved in the processing of IAP antigens in either cell type.

HIV-1 expression during early mammalian development.

OBJECTIVE: Our long-term objective is to determine the consequences of HIV-1 expression in the mammalian fetus using the mouse as a model system. This study describes HIV gene expression in micro-injected and electroporated embryos during the pre-implantation period. DESIGN: Procedures were adopted to create a non-infectious HIV system to assay HIV long terminal repeat (LTR) activation in a non-human mammal. METHODS: Two constructs (RSV promoter-HIV tat gene; HIV LTR-lac Z gene) were co-micro-injected (10 micrograms/ml each) into pronuclei of fertilized eggs or one nucleus of the two-cell stage and expression assessed after 2-5 days. The techniques of electroporation and micro-injection were compared for their ability to deliver the constructs expressed subsequently. RESULTS: We found that HIV-1 expression, as monitored by beta-galactosidase production, can occur as early as the eight-cell to blastocyst stage. The intensity of HIV expression varied from high to low; the proportion of the embryo expressing HIV varied from half to only two cells. The material was too limited to allow a determination of integration. CONCLUSIONS: The expression rate of the HIV-LTR was low, averaging slightly over 1%. The data demonstrate that the HIV-LTR can be activated in early mammalian development, even before implantation. This information is valuable to those studying HIV-transgenic mice, to those interested in factors governing HIV expression, and to those concerned about pathologies accompanying developmental expression of HIV.

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 gamma-tubulin-positive structures in the cortex, averaging 10 microm in diameter. The present work is the first to demonstrate that although these structures contain gamma-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.

Fertilization of the mouse oocyte.

This paper presents morphological data on mouse oocyte maturation and fertilization, reviews evidence supporting the existence of a sperm receptor, and suggests future directions for this line of research. We used scanning electron microscopy to examine oocytes under a variety of conditions. The surfaces of mature mouse oocytes are seen to be similar whether maturation occurs in vivo or in vitro. Capacitated sperm (both acrosome-intact and acrosome-reacted) are observed to interact with the microvilli of the oocyte surface. Little is known about oocyte surface proteins that mediate fertilization in mammals. Data of ours and others show that enzyme treatment of live unfertilized eggs interferes with sperm binding. Enzyme treatment (trypsin, chymotrypsin treatment, or pronase) reduces the number of bound sperm, suggesting removal of a surface protein involved in fertilization. Trypsin treatment also causes some lengthening of surface microvilli in a belt surrounding the metaphase II region. After metabolic labeling, proteins of zona-free unfertilized eggs can be identified by SDS-PAGE and autoradiography. Comparison of 1-D gels from untreated and enzyme-treated eggs show the nearly complete disappearance of proteins of 263, 170, 137, 97, and 87 kD after digestion; an increase in a 66 kD protein after trypsin or chymotrypsin; and a major new band of 20 kD after chymotrypsin treatment. Fertilized eggs show the loss of a 255-265 kD band among other changes. Proteins of 97 kD and 87 kD were seen previously by surface labeling (Johnson and Calarco, 1980b), and our 97 kD and 66 kD bands are similar in molecular weight to those identified by Boldt et al. (1989). Taken together, these data identify a few candidate proteins for the role of sperm receptor on the egg surface. Future work should focus on identification of the surface protein(s) which functions physiologically in fertilization by developing fertilization-blocking antibodies. Relatedness to other mammalian sperm receptors and identification of the genes involved would provide valuable information to our understanding of fertilization and to the problems of infertility and contraception.

Intracisternal A particles in preimplantation embryos of feral mice (Mus musculus).

In embryos of feral mice, intracisternal A particles (IAP) are not observed in zygotes, but large numbers are seen budding into the endoplasmic reticulum at the 2-cell stage. In subsequent cleavage stages very few IAP are present. Organelles and developmental changes in organelles during the preimplantation period are characteristic of those described previously in embryos of laboratory mice.

Polarization of mitochondria in the unfertilized mouse oocyte.

Maturation of an immature oocyte into one capable of being fertilized involves tightly choreographed movements of chromosomes and organelles. The localization of mitochondria during maturation was studied in live mouse oocytes by confocal laser scanning microscopy (CLSM). Mitochondria were labeled with rhodamine 123 or Mitotracker (Molecular Probes, Eugene, OR) both of which are cell permeant and accumulate in mitochondria; acridine orange was used to mark chromatin. Prior to maturation, oocytes appeared to be radially symmetrical with no evident polarity; fully mature oocytes exhibited obvious polarity marked by the position of the metaphase II spindle in the cortex. CLSM revealed several interesting features of mitochondrial distribution: 1) A cortical clump of mitochondria was seen approximately 30-45 degrees to one side of the metaphase II spindle and marked the region of polar body I extrusion. 2) Large foci of mitochondria (7-14 microM) were frequently found around the central region of the mature oocyte, while the central region often exhibited markedly fewer mitochondria. 3) Small mitochondrial foci (3 microM) in the cortex and near the GV characterized several oocytes which failed to mature. 4) Non-spindle-associated mitochondria were not uniformly distributed in the mature oocyte but were concentrated in the hemisphere containing the metaphase II spindle. 5) The distal margins of this mitochondrial hemisphere were sharply demarcated at the cortex. These findings should help us understand organelle localization during mammalian oocyte maturation, and may give insights into possible causes of infertility and into early events of preimplantation development.

Ultrastructural observations of lethal yellow (Ay/Ay) mouse embryos.

Ay/Ay embroys were identified by the presence of large exluded blastomeres (Pedersen, 1974) and examined cytologically and ultrastructurally. Cell organelles, inclusions and junctions in the excluded blastomeres were compared with those of non-excluded cells of Ay/Ay embryos and control embryos. Excluded blastomeres always had the fine structural characteristics of earlier developmental stages and may have arrested at the 4- to 8-cell stage of slightly later. Interior cells (inner cell mass) were observed in all mutant blastocysts. Non-excluded cells of Ay/Ay embryos were normal until degenerative changes appear in the late blastocyst stage. The mode of action of the +Ay gene was not determined, but evidence from this study and others indicate that the effects of +Ay gene action occur over a wide range of time in early cleavage and implantation.

Ultrastructural observations of preimplantation stages of the sheep.

Sheep embryos were examined with the electron microscope in order to characterize their organelles and the changes that occur during the preimplantation period. The first sign of differentiation of trophoblast cells was the appearance of junctions between external cells at the 16-cell stage. Nucleoli developed a granular component suggesting the synthesis of ribosomal RNA at the 16-cell stage also. Centrioles were seen as early as the 8-cell stage. Intracytoplasmic vesicles were present in large numbers in all cleavage stages but disappeared at blastulation. Mitochondria progressed from a very electron-dense hook- or U-shaped form with a few cristae to a cylindrical or spherical form of light density with many transverse cristae. Microvilli were not seen until the blastocyst stage and then only on the exterior surface of the trophoblast cells. Crystalloid or virus inclusions were not observed. It was concluded that the fine structure and developmental changes in the early sheep embryo are very similar to those of other mammalian species.

Establishment and maintenance of a regionalized glycoprotein distribution during early mouse development.

The regionalization of the cell membranes of the mouse embryo into apical and basolateral zones has been studied using antibodies to a pair of glycoproteins expressed during the two-cell to early blastocyst stage. These antigens are found on the outer, free surface and in the underlying cortical cytoplasm, but are not detectable at areas of cell contact. In the early blastocyst stage, antigen also appears at the free surfaces of cells bordering the blastocoel. Antigen regionalization is also reestablished after experimental manipulation and appears to be a direct consequence of cell contact. Thus, blastomeres examined 4 hr after dissociation from four- and eight-cell stage embryos express antigen in cortical areas underlying newly exposed surfaces and new sites of contact between embryos in multiple-embryo aggregates lose detectable antigen within 2 to 4 hr of the formation of the contacts. Microfilaments are involved in controlling the regional expression of these glycoproteins. Incubation of embryos from the two-cell stage in medium containing cytochalasin B interferes with antigen targeting, resulting in abnormal expression of the antigens both on the surface and in the cytoplasm of the embryos. Cytochalasin B treatment of later stage embryos results in an uneven distribution of the antigen in cortical cytoplasm and prevents the complete removal of antigen from new sites of cell contact in multiple-embryo aggregates. The presence of nocodozole, which inhibits the polymerization of microtubules, had no detectable effect on the expression of the antigens. Interference with the glycosylation of these proteins, by incubation of embryos in the presence of tunicamycin, did not alter the regionalized pattern of expression.

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.

Developmental regulation of integrin expression at the time of implantation in the mouse embryo.

The trophectoderm layer of the mouse blastocyst differentiates at the late blastocyst stage to form the invasive trophoblast that mediates implantation of the embryo into the uterine wall. The first sign that trophoblast cells have developed an invasion-specific cell behavior appears about 10-15 hours after the embryo hatches from the zona pellucida, when the quiescent, non-adherent trophectoderm cells initiate protrusive activity and become adhesive to extracellular matrix. Our previous findings that trophoblast outgrowth on extracellular-matrix-coated substrata involves the integrin family of adhesion receptors (Sutherland, A. E., Calarco, P. G. and Damsky, C. H., 1988, J. Cell Biol. 106, 1331-1348), suggested that the onset of trophoblast adhesive and migratory behavior at the time of implantation may be due to changes in expression or distribution of integrin receptors. We have thus examined the mRNA and protein expression of individual integrin subunits during pre- and periimplantation development (E0-E7.5). A basic repertoire of integrins, including receptors for fibronectin (alpha 5 beta 1), laminin (alpha 6B beta 1) and vitronectin (alpha v beta 3), was expressed continuously throughout this period, whereas the expression of five other integrin subunits was developmentally regulated. The mRNA for three of these (alpha 2, alpha 6A and alpha 7) was first detected in the late blastocyst, coincident with endoderm differentiation and development of attachment competence. The mRNA for another (alpha 1) was not detected until after trophoblast outgrowth had begun, suggesting that its expression may be induced by contact with matrix. At E7.5, three of the temporally regulated integrins (alpha 1, apha 6A, alpha 7), all of which can form receptors for laminin, were detected only in the ectoplacental cone (differentiating trophoblast), and may thus play specific roles in trophoblast adhesion and/or differentiation. Because laminin expression is upregulated in decidualized uterine stroma in response to the implanting embryo, we examined trophoblast-laminin interactions, using laminin fragments and integrin antibodies to determine which integrin receptors were involved. Trophoblast cells attached and spread on both the E8 and P1' fragments of laminin; however, the P1' binding site was cryptic in intact laminin. Interaction with P1' was RGD- and alpha v beta 3-dependent, whereas outgrowth on E8 was RGD-independent and not inhibited by antibodies to the laminin receptor alpha 6 beta 1, suggesting that alpha 7 beta 1 is the major trophoblast integrin E8 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Alkaline phosphatase activity in the preimplantation mouse embryo.

Alkaline phosphatase (AP) activity has been assayed in frozen sections of preimplantation mouse embryos by an azo-dye cytochemical method. The results indicate that during preimplantation mouse development AP activity is first expressed between the 8- and 16-cell stages and develops in all cells by the late morula stage. During blastocyst formation AP activity is lost or greatly reduced in trophoblast cells while activity is maintained in the inner cell mass.

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.

Expression of syndecan, a putative low affinity fibroblast growth factor receptor, in the early mouse embryo.

Syndecan is an integral membrane proteoglycan that binds cells to several interstitial extracellular matrix components and binds to basic fibroblast-growth factor (bFGF) thus promoting bFGF association with its high-affinity receptor. We find that syndecan expression undergoes striking spatial and temporal changes during the period from the early cleavage through the late gastrula stages in the mouse embryo. Syndecan is detected initially at the 4-cell stage. Between the 4-cell and late morula stages, syndecan is present intracellularly and on the external surfaces of the blastomeres but is absent from regions of cell-cell contact. At the blastocyst stage, syndecan is first detected at cell-cell boundaries throughout the embryo and then, at the time of endoderm segregation, becomes restricted to the first site of matrix accumulation within the embryo, the interface between the primitive ectoderm and primitive endoderm. During gastrulation, syndecan is distributed uniformly on the basolateral cell surfaces of the embryonic ectoderm and definitive embryonic endoderm, but is expressed with an anteroposterior asymmetry on the surface of embryonic mesoderm cells, suggesting that it contributes to the process of mesoderm specification. In the extraembryonic region, syndecan is not detectable on most cells of the central core of the ectoplacental cone, but is strongly expressed by cells undergoing trophoblast giant cell differentiation and remains prominent on differentiated giant cells, suggesting a role in placental development. Immunoprecipitation studies indicate that the size of the syndecan core protein, although larger than that found in adult tissues (75 versus 69 x 10(3) Mr), does not change during peri-implantation development. The size distribution of the intact proteoglycan does change, however, indicating developmental alterations in its glycosaminoglycan composition. These results indicate potential roles for syndecan in epithelial organization of the embryonic ectoderm, in differential axial patterning of the embryonic mesoderm and in trophoblast giant cell function.

Mouse egg integrin alpha 6 beta 1 functions as a sperm receptor.

Binding between sperm and egg plasma membranes is an essential step in fertilization. Whereas fertilin, a mammalian sperm surface protein, is involved in this crucial interaction, sperm receptors on the egg plasma membrane have not been identified. Because fertilin contains a predicted integrin ligand domain, we investigated the expression and function of integrin subunits in unfertilized mouse eggs. Polymerase chain reactions detected mRNAs for alpha 5, alpha 6, alpha v, beta 1, beta 3, and beta 5. Immunofluorescence revealed alpha 6 beta 1 and alpha v beta 3 on the plasma membrane. GoH3, a function-blocking anti-alpha 6 monoclonal antibody, abolished sperm binding, but a nonfunction-blocking anti-alpha 6 monoclonal antibody, a function-blocking anti-alpha v beta 3 polyclonal antibody, and an RGD peptide had no effect. Somatic cells bound sperm avidly, but only if they expressed alpha 6 beta 1. A peptide analog of the fertilin integrin ligand domain inhibited sperm binding to eggs and alpha 6 beta 1+ cells and diminished GoH3 staining of eggs. Our results indicate a novel role for the integrin alpha 6 beta 1 as a cell-cell adhesion receptor that mediates sperm-egg binding.