Protein synthesis in mouse embryos with experimentally produced asynchrony between chromosome replication and cell division

Summary The cleavage of fertilized mouse eggs was prevented during cytochalasin B incubation and consequently these eggs became tetraploid the following day during in vitro culture. When the eggs were cultured further in normal medium, they cleaved and gave rise to tetraploid blastocysts. Protein synthesis was analysed in these embryos at different developmental stages using two-dimensional polyacrylamide gel electrophoresis. The protein synthesis pattern of one-cell tetraploid eggs was intermediate between those of normal one- and two-cell embryos. Tetraploid two-cell embryos expressed protein sets equivalent to those of untreated four-cell embryos, and tetraploid four-cell embryos synthesized proteins similar to those of four- to eight-cell controls. At subsequent pre-implantation stages the asynchrony was no longer detectable. When fertilized eggs were cultured continuously in the presence of cytochalasin B, they became tetraploid, octoploid and more and more polyploid without cleavage occurring. The protein synthesis patterns expressed by these one-cell polyploid eggs did not resemble that of normal fertilized eggs, but were similar to those of cleaving control embryos and blastocysts of equivalent age and nuclear division. These results strongly suggest that in early mouse embryos stage-specific translation is temporally correlated with chromosome replication (karyokinesis) and independent of cell division (cytokinesis) or cell interaction.Some of these results were presented at the IX Congress of the International Society of Developmental Biologists in Basle, Switzerland, August 28–September 1, 1981     

Brain and sperm cell surface antigen (NS-4) on preimplantation mouse embryos

Antiserum prepared in rabbit against 4-day-old mouse cerebellum (anti-NS-4 serum) reacts in the complement-mediated cytotoxicity test with unfertilized and fertilized mouse eggs, cleavage stage embryos, and cells of the trophoblast and inner cell mass of the mouse blastocyst. This activity is specifically removed by absorption of antiserum with adult mouse brain and epididymal sperm but not with adult liver, spleen, kidney, and thymocytes. The antiserum reacts most strongly with cells of the trophoblast and inner cell mass and, in order of decreasing reactivity, with four- to eight-cell stage embryos, zygotes, unfertilized eggs, and two-cell stage embryos.     

Nucleolar ultrastructure in bovine nuclear transfer embryos

Nuclear transfer experiments in mammals have attempted to reprogram a donor nucleus to a state equivalent to the zygotic one. Reprogramming of the donor nucleus is, among other features, indicated by a synthesis of ribosomal RNA (rRNA). The initiation of rRNA synthesis is simultaneously reflected in nuclear morphology as a transformation of the nucleolus precursor body into a functional rRNA synthesising nucleolus with a characteristic ultrastructure. We examined nucleolar ultrastructure in bovine in vitro produced (control) embryos and in nuclear transfer embryos reconstructed from a MII phase (nonactivated) or S phase (activated) cytoplasts. Control embryos were fixed at the two-, four-, early eight- and late eight-cell stages; nuclear transfer embryos were fixed at 1 and 3 hr post fusion and at the two-, four-, and eight-cell stages. Control embryos possessed a nucleolar precursor body throughout all three cell cycles. In the eight-cell stage embryo, a primary vacuole appeared as an electron lucid area originating in the centre of the nucleolar precursor body. In nuclear transfer embryos reconstructed from nonactivated cytoplasts, the nuclear envelope was fragmented or completely broken down at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary vacuoles. A nucleolar precursor body typical for the two-cell stage control embryos was never observed. None of the reconstructed embryos of this group reached the eight-cell stage. Nuclear transfer embryos reconstructed from activated cytoplasts, in contrast, exhibited a complete nuclear envelope at all time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear transfer embryos, which was one cell cycle earlier than in control embryos. Only nuclear transfer embryos reconstructed from activated cytoplasts underwent complete remodelling of the nucleolus. The reorganisation of the donor nucleolar architecture into a functionally active nucleolus was observed as early as in the four-cell stage nuclear transfer embryo. These ultrastructural observations were correlated with our autoradiographic data on the initiation of RNA synthesis in nuclear transfer embryos.     

Experimental manipulation of compaction of the mouse embryo alters patterns of protein phosphorylation

Compaction, occurring at the eight-cell stage of mouse development, is the process of cell flattening and polarisation by which cellular asymmetry is first established. Changes in the pattern of protein phosphorylation have been correlated with this early event of development (TL Bloom, J McConnell: Mol Reprod Dev 26:199-210, 1990). In the study reported here, groups of embryos were treated in ways known to affect particular features of compaction and were then labeled with [32P]orthophosphate; the phosphoproteins obtained were examined following electrophoresis in one and two dimensions. Four-cell embryos were treated with protein synthesis inhibitors, which advance cell flattening. This treatment resulted in only minor differences from the phosphoprotein profile of untreated four-cell embryos. Inhibition of protein synthesis at the eight-cell stage has little effect on cell flattening or polarisation. However, some phosphoproteins that are observed normally in eight-cell but not in four-cell embryos were no longer detectable if labeling took place in the presence of protein synthesis inhibitors. Eight-cell embryos incubated in phorbol 12-myristate 13-acetate, which disrupts various features of compaction, showed a relative increase in the phosphorylation of a group of phosphoprotein spots associated with the eight-cell but not with the four-cell stage. Embryos incubated in Ca2(+)-free medium, which prevents intercellular flattening and delays polarisation, showed a relative decrease in the phosphorylation of the same group of phosphoprotein spots. The behaviour of these phosphoproteins may therefore be correlated with some of the features of compaction.     

Spontaneous parthenogenesis in Mus musculus: Comparison of protein synthesis in parthenogenetic and normal preimplantation embryos

Summary In preimplantation stages of normal and spontaneously activated parthenogenetic embryos of the LT/Sv mouse strain, protein synthesis was analyzed by using two-dimensional polyacrylamide gel electrophoresis. Fertilization and parthenogenetic activation cause similar changes of polypeptide synthesis when compared with those of unfertilized eggs. The overt developmental delay of early parthenotes, which is probably due to an initial retarded activation in comparison with normal fertilization, is documented molecularly by a similar delay in their protein synthesis pattern. These differences are clearly visible at the two-cell stage but gradually disappear during further cleavage. The basic protein patterns of normal and parthenogenetic embryos are remarkably similar up to the blastocyst stage. However, quantitative differences occur in all preimplantation embryos analyzed and become more distinct at the blastocyst stage. In addition, only minor qualitative changes appear during late preimplantation. These alterations in protein synthesis may reflect at the molecular level early events in abnormal development of parthenotes. Our biochemical results are discussed in context with biological experiments rescuing parthenogenetic LT/ Sv embryos by chimera formation.     

兔核移植胚胎蛋白质变化的电泳分析

采用ＳＤＳ－聚丙烯酰胺凝胶电泳及银染法,对兔核移植胚胎蛋白质合成类型的变化进行了研究。结果表明,“原核”期核移植胚胎中有几种蛋白质与核供体桑椹胚及受体卵母细胞的蛋白质类型明显不同,与同期受精卵中蛋白质类型相似,但在某些带上也有质或量的差别。结果提示,供体核基因表达被卵母细胞质所重排,但并非很完全。本文还对提高兔核移植胚胎发育能力可能的途径进行了讨论。     

Relationship of gap junction formation to phosphorylation of connexin43 in mouse preimplantation embryos

To clarify the relationship of gap junction formation to phosphorylation of connexin43 (Cx43) in mouse preimplantation embryos, immunofluorescence and Western blot analysis were conducted. Immunofluorescence showed Cx43 positive spots first at the mid-eight-cell stage (6 hr postdivision to the eight-cell stage). The number of spots increased from 6 to 15 hr postdivision to the eight-cell stage. Western blot analysis suggested Cx43 to possibly be present in the nonphosphorylated form at the mid-four-cell stage (6 hr postdivision to the four-cell stage), and phosphorylated Cx43 to increase from the mid-eight-cell stage (6 hr post-division to the eight-cell stage) onward. Dibutyryl cAMP (dbcAMP), a protein kinase A (PKA) activator, added to the culture medium increased the phosphorylation of Cx43 and Cx43 positive spots. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, increased the phosphorylation of Cx43, but decreased Cx43 positive spots. These results suggest that the phosphorylation of Cx43, induced by different protein kinase, leads to a different effect on gap junction formation in mouse preimplantation embryos.     

Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells.

In a previous paper, we have shown that in the absence of stress, mouse embryonal carcinoma cells, like mouse early embryo multipotent cells, synthesize high levels of 89- and 70-kilodalton heat shock proteins (HSP)(O. Bensaude and M. Morange, EMBO J. 2:173-177, 1983). We report here the pattern of proteins synthesized after a short period of hyperthermia in various mouse embryonal carcinoma cell lines and early mouse embryo cells. Among the various cell lines tested, two of them, PCC4-Aza R1 and PCC7-S-1009, showed an unusual response in that stimulation of HSP synthesis was not observed in these cells after hyperthermia. However, inducibility of 68- and 105-kilodalton HSP can be restored in PCC7-S-1009 cells after in vitro differentiation triggered by retinoic acid. Similarly, in the early mouse embryo, hyperthermia does not induce the synthesis of nonconstitutive HSP at the eight-cell stage, but induction of the 68-kilodalton HSP does occur at the blastocyst stage. Such a transition in the expression of HSP has already been described for Drosophila melanogaster and sea urchin embryos and recently for mouse embryos. It may be a general property of early embryonic cells.     

Amino acid uptake and protein synthesis in preimplanatation mouse embryos

Amino acid uptake and cycloheximide inhibitable incorporation into protein are demonstrable in follicular ova, unfertilized eggs, and in mouse embryos ranging from the 1-cell to the late blastocyst stages. The rates of uptake and incorporation are low and relatively constant until the early blastocyst (day 3) stage of development when they increase 3- to 9-fold. The rate of uptake continues to increase during the fourth day (late blastocyst stage) of development, but, despite embryonic growth, incorporation into protein remains constant. By exposing embryos to leucine and lysine at different concentrations, it is possible to dissociate the processes of uptake and incorporation into protein from one another and to use the latter as a measure of protein synthesis. Taking the number of embryonic cells into account, it is postulated that the period between 8- to 16-cell stage (day 2) and the early blastocyst stage is the only one in which the synthesis of major amounts of protein based on new messenger RNA synthesis is occurring.Leucine and lysine uptake take place by a facilitated process, although lysine transport is not readily saturated. Inhibitors of energy metabolism do not significantly affect amino acid uptake, but they do inhibit protein synthesis. However, since the rate of transport is highly temperature sensitive (Q₁₀ ? 3) and leucine is accumulated against a concentration gradient, active amino acid transport appears to be present.     

Regulation of stage-specific gene expression during early mouse development: effect of cytochalasin B and aphidicolin on stage-specific protein synthesis in mouse eggs

Mechanisms regulating stage-specific translation in mouse embryos were studied by inhibitor experiments. When fertilized eggs were continuously treated with cytochalasin B, cleavage was prevented, whereas karyokinesis proceeded, resulting in protein synthesis patterns changing stage-specifically as in control embryos through preimplantation development. When fertilized eggs were continuously exposed to aphidicolin, cleavage and DNA synthesis were inhibited, thus keeping their protein synthesis at the level of fertilized eggs with few new polypeptides appearing after one day. The next day these eggs stopped translation almost completely. Stage-specific translation therefore might be controlled by nuclear replications rather than by cytoplasmic clock.     

Construction of the nuclear matrix at the transition from maternal to zygotic control of development in the mouse: an immunocytochemical study.

The nuclear matrix is thought to be responsible for DNA organization, DNA replication, RNA synthesis, and RNA processing. We have looked for the presence of nuclear matrix antigens during early mouse embryogenesis. Antibodies to peripheral and interior antigens (P1, Pl1, Pl2, and lamin B) were used to immunolocalize nuclear matrix antigens in germinal vesicle oocytes, metaphase II oocytes, zygotes, two-cell-stage embryos, and eight-cell stage embryos. All antibodies reacted with the nuclei of germinal vesicle oocytes, and two- and eight-cell-stage embryos; however, only P1 and lamin B were present at the pronuclear stage. In eggs collected at the pronuclear stage and cultured to the late two-cell stage in the presence of alpha-amanitin, the matrix morphology was altered for Pl1 and Pl2. alpha-Amanitin had no affect on the distribution of P1 or lamin B antigens. If alpha-amanitin was added 2 hr after cleavage to the two-cell stage, the normal staining pattern of Pl2 was retained. These results suggest that the presence of specific components of an internal matrix is correlated with normal genomic activity.     

Protein synthesis in microsurgically produced androgenetic and gynogenetic mouse embryos

Summary In order to compare paternal and maternal gene activity at the protein synthesis level during early development, androgenetic and gynogenetic mouse embryos were experimentally produced by microsurgically removing either the female or the male pronucleus from fertilized mouse eggs. The resulting haploid eggs were diploidized in a medium containing cytochalasin B and then cultured under normal conditions to the blastocyst stage. Protein synthesis was analyzed at different stages of preimplantation development using 2-dimensional polyacrylamide gel electrophoresis. Both types of uniparental embryos synthesized a similar set of proteins independent of whether the paternal or the maternal genome was present. The isodiploid embryos expressed a protein pattern that corresponded remarkably to normal embryos at the subsequent cleavage stage. This temporal change is probably due to the fact that the operated haploid eggs were kept overnight in cytochalasin B in order to allow chromosomal replication to occur without cell division, and the resulting eggs therefore corresponded to normal 2-cell embryos with respect to karyokinesis but differed as far as cytokinesis was concerned. Several 2-cell specific proteins appeared in these isodiploid eggs and, similarly, following their first cleavage some 4-cell specific proteins were detected in 2-cell androgenetic and gynogenetic embryos. The discordance between nuclear and cellular division, which was retained through the 4-cell stage, however disappeared during subsequent cleavage divisions. At the blastocyst stage, both kinds of uniparental embryos showed a similar protein pattern compared to normal embryos. Our data suggest that some stage-specific proteins are synthesized during preimplantation development and correspond to nuclear rather than cellular divisions.Some of these results were presented at the 13th Annual Meeting of the Union of Swiss Societies of Experimental Biology in Lausanne, March 1981 (Petzoldt et al. 1981)     

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

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

Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as well as embryo loss during development may occur even in cloned embryos reconstructed with nuclei from preimplantation-stage embryos, and these abnormalities are not specific to somatic cloning.     

The effect of the nucleocytoplasmic ratio on protein synthesis and expression of a stage-specific antigen in early cleaving mouse embryos

The nucleocytoplasmic ratio of fertilized mouse eggs was manipulated by removing or injecting cytoplasm by micropipette, and bisection of denuded eggs to obtain both pronuclei in one half of the eggs cytoplasm. The experimental eggs were capable of cleavage to the morula stage and, in some instances, developed to the blastocyst stage similar to unmanipulated eggs. The removal of large quantities of cytoplasm by micropipette and injecting them into a recipient egg did not provide sufficient numbers of viable eggs, whereas transfer of smaller quantities (about a quarter of the cytoplasm) was less deleterious, at least for recipient eggs. However, the alteration of the nucleocytoplasmic ratio by this method was not of the correct magnitude for the purpose of this experiment. Therefore, bisection was the preferred method whereby the nucleocytoplasmic ratio was doubled. This resulted in both pronuclei residing in one half of the egg's cytoplasm. Half eggs with one pronucleus (haploid) but retaining a nucleocytoplasmic ratio similar to unmanipulated control eggs served as additional controls for the bisection experiments. Protein synthesis was analysed by two-dimensional gel electrophoresis, showing that the 2-cell- and 4-cell-stage bisected embryos with double and normal nucleocytoplasmic ratio expressed equivalent protein synthesis patterns as control embryos of the same stage. Likewise, the stage-specific surface antigen SSEA-1 did not appear before the 6- to 8-cell stage. Also in cytoplasm transfer experiments, there was no indication that altering the nucleocytoplasmic ratio in either direction changed the timing of stage-specific gene expression. These results support the idea that stage-specific gene activity during early mouse cleavage might proceed in parallel to DNA replication cycles and is independent of the nucleocytoplasmic ratio.     

Differential expression of alternatively spliced transcripts of HLA-G in human preimplantation embryos and inner cell masses

It has been reported that preimplantation human embryos secrete HLA-G, and the levels may be predictive of their ability to implant. However, it is not known which of the membrane-bound (HLA-G 1-4) and soluble (HLA-G 5-6) alternatively spliced forms are present, nor the developmental stage at which they appear. Therefore, we have investigated HLA-G mRNA isoform expression on single embryos at the two-, four-, six-, and eight-cell, morula, and blastocyst stages. The percentage of embryos expressing each HLA-G isoform mRNA increased with developmental stage, but contrary to expectation, HLA-G5 mRNA was not detected in single two- to eight-cell embryos and was only expressed by 20% of morulae and blastocysts. Similarly, soluble HLA-G6 mRNA was not detected until the blastocyst stage and then in only one-third of embryos. In contrast, labeling with MEM G/9 Ab (specific for HLA-G1 and -G5) was observed in 15 of 20 two- to eight-cell embryos and 5 of 5 blastocysts. This disparity between mRNA and protein may be due to HLA-G protein remaining from maternal oocyte stores produced before embryonic genome activation and brings into question the measurement of soluble HLA-G for clinical evaluation of embryo quality. Although HLA-G is expressed in the preimplantation embryo, later it is primarily expressed in the invasive trophoblast of the placenta rather than the fetus. Therefore, we have investigated whether down-regulation of HLA-G first occurs in the inner cell mass (precursor fetal cells) of the blastocyst and, in support of this concept, have shown the absence HLA-G1 and -G5 protein and mRNA.     

Molecular differentiation of the rabbit ovum. II. During the preimplantation development of in vivo and in vitro matured oocytes

The developmental capacity of in vitro matured rabbit oocytes was assessed after transfer to inseminated, ovariectomized recipients such that fertilization and preimplantation development occurred in vivo. The results demonstrate that of the total number of transferred oocytes (1) 75% were fertilized, (2) 50% underwent cleavage, and (3) 13% developed into expanded blastocysts. By light microscopic criteria, embryos recovered at representative stages of preimplantation development were morphologically indistinguishable from embryos recovered at comparable stages from normally mated animals. Autoradiographs produced by high resolution, two-dimensional polyacrylamide gel electrophoresis demonstrated that changes in the pattern of polypeptide synthesis during the preimplantation stages were directly and entirely comparable for embryos derived either from normally mated animals or from in vivo or in vitro matured and transferred oocytes. Up to approximately the eight-cell stage, the translational patterns indicate the progressive disappearance of numerous oocyte-characteristic polypeptides from the autoradiographs as well as the appearance of some new species of polypeptides. Between the eight-cell and early blastocyst period, extensive and complex changes (qualitative and quantitative) occur in the patterns, whereas, in contrast, the phase of blastocyst growth and expansion that occurs during the latter portion of the preimplantation period is characterized by a fairly uniform and constant translational pattern.     

Survival of equine embryos co-cultured with equine oviductal epithelium from the four- to eight-cell to the blastocyst stage after transfer to synchronous recipient mares

In this study we examined the ability of equine oviductal epithelial cells (OEC) to support the development of four- to eight-cell equine embryos in vitro and investigated the ability of co-cultured embryos to continue normal development after transfer to synchronous recipient mares. Equine embryos obtained at Day 2 after ovulation were cultured with or without OEC for 5 days. Those OEC co-cultured embryos that reached the blastocyst stage and embryos recovered from the uterus at Day 7 were surgically transferred to synchronous recipient mares. Co-culture with OEC improved (P < 0.01) development of four- to eight-cell embryos to blastocysts compared to medium alone (11/15 vs 0/6) during 5 days in vitro. Embryos co-cultured with OEC were smaller (P < 0.05) and more delayed in development than Day-7 uterine blastocysts. There was no difference in the Day-30 survival rate of co-cultured blastocysts (3/8) or Day-7 uterine blastocysts (5/8) after transfer to recipient mares. These results indicate that co-culture with OEC can support development of four- to eight-cell equine embryos in vitro and that co-cultured embryos can continue normal development after transfer to recipient mares.