Distribution of nucleolar proteins B23 and nucleolin during mouse spermatogenesis

The intracellular distribution of nucleolar phosphoproteins B23 and nucleolin was studied during mouse spermatogenesis, a process that is characterized by a progressive reduction of nucleolar activity. Biochemical analyses of isolated germ cell fractions were performed in parallel with the in situ ultrastructural immunolocalization of these two proteins by means of specific antibodies and colloidal gold markers, and by silver staining. RNA blot experiments showed that mRNA for nucleolin progressively decreased during spermatogenesis whereas mRNA for B23 increased in amount during early spermatogenic stages. Immunoblotting confirmed that both proteins were present during early spermatogenesis up to the round spermatid stage and absent from mature sperm. Immunoelectron microscopy revealed that in spermatogonia, leptotene and pachtyene spermatocytes, and in Golgi phase spermatids, B23 and nucleolin were localized in the dense fibrillar component and granular component of the nucleolus but not in the fibrillar centers. In the dense fibrillar residue of the cap phase spermatids, labeling with anti-nucleolin but not with anti-B23 was observed. During nucleolar inactivation, neither of the two polypeptides was dispersed to the nucleoplasm. Silver salts stained the fibrillar centers and dense fibrillar component but not the granular component of the nucleolus. Our results suggest that there is no direct relationship between nucleolar activity and the occurrence of B23 and nucleolin or silver staining. Moreover, we confirm that silver staining and the presence of B23 or nucleolin are not directly related to each other.by M. Trendelenburg     

Nucleolar proteins and nuclear ultrastructure in preimplantation bovine embryos produced in vitro

The aim of the present investigation was to describe the basic cell biology of the postfertilization activation of rRNA genes using in vitro-produced bovine embryos as a model. We used immunofluorescence confocal laser scanning microscopy and transmission electron microscopy to study nucleolar development in the nuclei of embryos up to the fifth postfertilization cell cycle. During the first cell cycle (1-cell stage), fibrillarin, upstream binding factor (UBF), nucleolin (C23), and RNA polymerase I were localized to distinct foci in the pronuclei, and, ultrastructurally, compact spherical fibrillar masses were the most prominent pronuclear finding. During the second cell cycle (2-cell stage), the findings were similar except for a lack of nucleolin and RNA polymerase I labeling. During the third cell cycle (4-cell stage), fibrillarin, UBF, nucleophosmin, and nucleolin were localized to distinct foci. Ultrastructurally, spherical fibrillar masses that developed a central vacuole over the course of the cell cycle were observed. Early in the fourth cell cycle (8-cell stage), fibrillarin, nucleophosmin, and nucleolin were localized to small bodies that with time developed a central vacuole. UBF and topoisomerase I were localized to clusters of small foci. Ultrastructurally, spherical fibrillar masses with a large eccentric vacuole and later small peripheral vacuoles were seen. Late in the fourth cell cycle, nucleophosmin and nucleolin were localized to large shell-like bodies; and fibrillarin, UBF, topoisomerase I, and RNA polymerase I were localized to clusters of small foci. Ultrastructurally, a presumptive dense fibrillar component (DFC) and fibrillar centers (FCs) were observed peripherally in the vacuolated spherical fibrillar masses. Subsequently, the presumptive granular component (GC) gradually became embedded in the substance of this entity, resulting in the formation of a fibrillo-granular nucleolus. During the fifth cell cycle (16-cell stage), a spherical fibrillo-granular nucleolus developed from the start of the cell cycle. In conclusion, the nucleolar protein compartment in in vitro-produced preimplantation bovine embryos is assembled over several cell cycles. In particular, RNA polymerase I and topoisomerase I are detected for the first time late during the fourth embryonic cell cycle, which coincides with the first recognition of the DFC, FCs, and GC at the ultrastructural level.     

Electrophoretic analysis of proteins synthesized by preimplantation mouse embryos

Proteins synthesized during the preimplantation period of mouse embryogenesis were labeled with radioactive tyrosine and lysine and fractionated by electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. For interstage comparisons and comparisons of the incorporation of different amino acids at the same developmental stages, the embryos were incubated with either ³H- or ¹⁴C-labeled amino acids. The embryos were then combined, and the proteins were isolated and electrophoresed simultaneously. The data were analyzed with a dual isotope computer program and expressed in the form of ¹⁴C/³H ratios.Approximately 20–25 labeled protein components of apparent molecular weights between 25,000 and 115,000 can be defined, and 5 are most significant quantitatively. Of the latter, there are developmental increases in the rates of synthesis of 3 (with apparent molecular weights of 35,000 to 37,000, 37,000 to 41,000, and 66,000 to 70,000), a decrease in the rate of synthesis of another (53,000 to 57,000), and little change in the last (46,000 to 49,000). Developmental changes in the rates of synthesis of several other components are also demonstrated by the ¹⁴C/³H incorporation ratios. The relative amounts of the different proteins synthesized by day 3 (early blastocyst) embryos over an 8-hr period remain constant, as does the relative labeling by lysine and tyrosine at each developmental stage examined. Similarly, there is no change in the pattern of the radioactive proteins when day 2 (8–16 cell) embryos are labeled for 2 hr and then incubated for an additional 24 hr. The greatest change in the overall pattern of protein synthesis occurs quite early, between day 1 (2 cell) and day 2, and lesser changes occur at later stages. These findings are in contrast to the major change in the rate of protein synthesis which occurs after day 2.     

Scanning microscopy of disaggregated and aggregated preimplantation mouse embryos

Summary Blastomeres isolated from 8-and 16-cell embryos (that is 1/8 and 1/16) show a smooth surface at their point of contact with other blastomeres and a microvillous free surface. Microvilli reappear completely on the smooth surface of 52% of 1/8 embryos and partially on 88% of 1/16 embryos if cultured in vitro for 6 h. When 2-to 8-cell embryos are aggregated to 8-cell embryos and forced apart after 1–3 h, the contact surface of the 8-cell embryos has become smooth. Fixed 8-cell embryos are also able to induce complete disappearance of microvilli on the contact surface of a living 8-cell embryo. Embryos having more than 8 cells do not induce complete disappearance of microvilli on the contact surface of 8-cell embryos. Aggregates of late morulae do not show complete disappearance of microvilli at their contact surfaces but rather a loosening of their peripheral blastomeres.Our results show that isolated 1/8 and 1/16 embryos tend to recover from regionalization, that the process of aggregation of embryos having 8 cells or less is similar to compaction and that embryos having more than 8 cells seem to aggregate by cell sorting. The processes of compaction, adhesion and reassortment are briefly discussed. We submit that blastomere regionalization, which depends on cell to cell contact, may be the spatial basis of embryonic regulation and of the inside-outside normal differentiation of early mouse embryos.     

Tubules of the trans Golgi apparatus visualized by immunoelectron microscopy

 Tubules constitute an integral part of the Golgi apparatus and have been shown to form a complex and dynamic network at its trans side. We have studied in detail structural features of the trans Golgi network and its relationship with the cisternal stack in thin sections of Lowicryl K4M embedded human absorptive enterocytes by immunolectron microscopy. Immunoreactive sites for α1,3 N-acetylgalactosaminyltransferase and blood group A substance were detectable troughout the cisternal stack and the entire trans Golgi network. Furthermore, the entire trans Golgi network was reactive for CMPase activity. Evidence for two kinds of tubules at the trans side of the Golgi apparatus was found: tubules that laterally connect adjacent and distant cisternal stacks, and others extending from central and lateral portions of trans cisternae to form the complex and extensive trans Golgi network. Trans cisternae showed often the peeling-off phenomenon and were continuous with the trans Golgi network. Both, trans cisternae and tubules of the trans Golgi network exhibited regionally buds and vesicles with a lace-like, non clathrin coat, previously reported by others in NRK cells, which contained glycoproteins with terminal N-acetylgalactosamine residues. These buds and vesicle are therefore involved in constitutive exocytosis. Accepted: 12 January 1998     

Patterns of organelle distribution in mouse embryos during preimplantation development

We have evaluated the distribution of mitochondria and acidic organelles using, respectively, the specific vital fluorescent dyes rhodamine 123 and acridine orange during preimplantation embryonic development in the mouse. Under conditions used to visualize organelles in living embryos, staining with either dye was found to have no effect on either the rate or extent of in vitro development of five- to eight-cell embryos up to the blastocyst stage. Mitochondria were randomly distributed throughout the cytoplasm and located around nuclei in blastomeres of uncompacted embryos. During compaction, mitochondria initially reorganized to the blastomere cortex; however, these organelles were later confined to the perinuclear region in the trophectoderm (TE) of expanded blastocysts. Acidic organelles were randomly distributed in the cytoplasm of uncompacted embryos, but following compaction, they were concentrated in cortical and perinuclear locations. Moreover, in TE cells of expanded blastocysts, acidic organelles were found exclusively in a tight perinuclear pattern. Microtubules and microfilaments in TE cells were localized in fixed embryos stained with antitubulin antibodies and rhodamine phalloidin, respectively; these structures were found primarily in the cortical cytoplasm at areas of cell-cell contact and secondarily in a perinuclear location. Thus mitochondria and acidic organelles undergo stage-specific redistributions from a diffuse or cortical pattern at the eight-cell stage to a tight perinuclear localization in the TE. We conclude that the polarized distributions of some organelles and cytoskeletal proteins during compaction may not be reliable permanent markers of the mature TE.     

Expression and distribution of cell adhesion molecule uvomorulin in mouse preimplantation embryos

We have examined the synthesis and distribution of the cell adhesion molecule uvomorulin in mouse preimplantation embryos. Uvomorulin can already be detected on the cell surface of unfertilized and fertilized eggs but is not synthesized in these cells. Uvomorulin synthesis starts in late two-cell embryos and seems not to be correlated with the onset of compaction. The first signs of compaction are accompanied by a redistribution of uvomorulin on the surface of blastomeres. During compaction uvomorulin is progressively removed from the apical membrane domains of peripheral blastomeres. In compact morulae uvomorulin is no longer present on the outer surface of the embryo but is localized predominantly in membrane domains involved in cell-cell contacts of adjacent outer blastomeres. On inner blastomeres of compact morulae uvomorulin remains evenly distributed. This uvomorulin distribution once established during compaction is maintained and also found in the blastocyst: on trophectodermal cells uvomorulin localization is very similar to that in adult intestinal epithelial cells while uvomorulin remains evenly distributed on the surface of inner cell mass cells. The possible role of the redistribution of uvomorulin for the generation of trophectoderm and inner cell mass in early mouse embryos is discussed.     

Handling of actinomycin D by preimplantation mouse embryos

A protein isolated from serum is required in the cell suspension for the chemotactic response of normal mouse peritoneal macrophages to complement-derived C5a. Macrophage stimulating protein (MSP) has a molecular weight of 100000 and an isoelectric point of 7.0. It is resistant to changes in pH over a range of 1.3–11, is heat labile especially after partial purification and does not survive proteolytic enzyme attack. Binding to ConA Sepharose suggests that it contains a carbohydrate moiety. Its concentration in normal serum is very low and it is detectable only by virtue of a sensitive bioassay. An upper limit has been estimated at 75 ng/ml; the actual concentration may be considerably lower.     

Simultaneous immunoelectron microscopic visualization of protein B23 and C23 distribution in the HeLa cell nucleolus

The intranucleolar distribution of phosphoproteins B23 and C23 was visualized simultaneously by post-embedding immunoelectron microscopy in HeLa cell nucleoli, using specific antibodies. The data show that proteins B23 and C23 co-localize to the same nucleolar compartments, i.e., the dense fibrillar component and the granular component. Neither of the two antibodies is significantly associated with the fibrillar centers in these cells, although the fibrillar centers appear positive after silver staining. These findings suggest that other unidentified components must be responsible for the silver staining observed in the fibrillar centers of interphase nucleoli. The results are discussed in the light of previously reported data obtained by preembedding immunolabeling techniques and by silver staining, which both suggested a localization of protein C23 inside the fibrillar centers.     

Non-random binding of N-acetoxy-N-2-acetylaminofluorene to chromatin subunits as visualized by immunoelectron microscopy

The influence of chromatin structure on the accessibility of DNA to the model ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) was investigated by means of an immunoelectron microscopic technique developed recently. An homogeneous population of core particles or trinucleosomes from chicken erythrocytes, was submitted to electrophilic attack by N-Aco-AAF. After DNA isolation, N-2-acetylaminofluorene (AAF) binding sites were mapped upon the DNA fragments using specific antibodies as a probe. Our results indicate a non-random binding of AAF along the DNA. Our data support the results of previous studies showing a preferential binding on the linker region.     

Decompaction and recompaction of mouse preimplantation embryos

Summary Early (non-compacted) and late (compacted) 8-cell embryos were observed after few hours of culture in vitro. The former embryos underwent compaction and the latter embryos were found decompacted. Cell counting suggested that decompaction preceded fourth cleavage division of any blastomere and lasted until the blastomeres divided.About one third of mouse morulae, which had about twenty cells, were found non-compacted upon obtaining from females. After few hours of culture in vitro these embryos underwent recompaction and cavitation. Increasing the contributions of mitosis-arrested and cytokinesisarrested cells within the morulae by culture with nocodazole and cytochalasin B respectively, did not delay recompaction.The data show that periods of decompaction and recompaction alternate in preimplantation development.     

Uptake and incorporation of inositol by preimplantation mouse embryos.

The uptake of myo-inositol by preimplantation mouse embryos was investigated using [3H]myo-inositol. Uptake increased about 12-fold between one- and two-cell stages and increased again at the blastocyst stage (> 6-fold compared with the two-cell stage). Uptake at the blastocyst stage was time and temperature dependent; it was stimulated by sodium, inhibited by glucose and appeared to take place mainly via a saturable mechanism. Uptake in the presence of 6.25 mmol inositol l-1 was 1424 fmol inositol per blastocyst per h. About 10% of the [3H]inositol taken up by blastocysts during 8 h in culture was incorporated into lipid. Thin layer chromatography of the lipid showed that most of this inositol was incorporated into lipid material co-migrating with phosphatidylinositol with a small proportion co-migrating with phosphatidylinositol 4-phosphate.     

Inhibition of DNA replication in preimplantation mouse embryos by aphidicolin

The regulation of trophectoderm differentiation in mouse embryos was studied by inhibiting DNA synthesis with aphidicolin, a specific inhibitor of DNA polymerase alpha. Embryos were exposed to aphidicolin (0.5 micrograms/ml) for 16 h at various preimplantation stages and scored for their ability to form a blastocyst and develop beyond the blastocyst stage. Embryos were most sensitive to aphidicolin at the late 4-cell stage and became progressively less sensitive as they developed. Aphidicolin inhibited blastocyst formation by 70%, 100%, 77%, and 24% after treatment at the 2-cell, 4-cell, noncompacted 8-cell, and compacted 8-cell stages, respectively. Although the inhibitory effect of aphidicolin on blastocyst formation decreased markedly as 8-cell embryos underwent compaction, developmental capacity beyond the blastocyst stage was poor after treatment of either noncompacted or compacted 8-cell embryos. Treatment at the morula and early blastocyst stages was less harmful to embryos than treatment at earlier stages but reduced the number of trophoblast outgrowths by interfering with hatching. Autoradiographic analysis showed that during aphidicolin treatment, incorporation of 3H-thymidine was inhibited over 90% at all stages examined, indicating an inhibition of DNA synthesis. Because inhibition of blastocyst formation by aphidicolin decreased at the compacted 8-cell stage, we suggest that approximately the first half of the fourth DNA replication cycle is critical for subsequent blastocyst formation. Furthermore, the poor further development of blastocysts formed after aphidicolin treatment of compacted 8-cell embryos suggests that the DNA replication requirements for initial trophectoderm differentiation are distinct from requirements for further development of blastocysts in vitro.     

Synthesis of H-2 antigens by preimplantation mouse embryos

An enzyme-linked immunosorbent assay (ELISA), which utilized anti-H-2 monoclonal antibody, was used to detect H-2 antigens on preimplantation mouse embryos. All embryonic stages studied, including unfertilized eggs and 1-cell, 2-cell, 8-cell, and blastocyst-stage embryos, showed the presence of H-2 antigens. To prove that the H-2 antigens were not cytophilically adsorbed to the embryos, blastocysts were treated with papain to strip off the H-2 antigens, and then the embryos were further incubated to allow the H-2 antigens to regenerate. After a 3-h incubation time, 60% of the H-2 antigens on the embryos had reappeared, proving that the H-2 antigens were synthesized by the embryos themselves.     

Glycine transport in mouse eggs and preimplantation embryos

In pre-compaction embryos glycine was taken up by the glycine-specific gly-system, which is concentrative, weakly exchangeable and dependent on Na+. After compaction glycine uptake increased, apparently due to the expression of the A-transport system and its reactivity with glycine. Studies of the metabolic fate of carbon from glycine indicated conversion to serine and alanine. These changes are interpreted to show that glycine could provide carbon for intermediary energy metabolism, resulting in CO2, as well as for macromolecular synthesis.     

Glutamine uptake and utilization by preimplantation mouse embryos in CZB medium

At least 71% of CF1 x B6SJLF1/J embryos developed from the 1-cell stage to the blastocyst stage in an optimum glutamine concentration of 1 mM, as long as glucose was present after the first 48 h of culture. Blastocysts raised under these conditions had significantly more cells than did blastocysts raised in CZB medium alone (glutamine present, glucose absent). Embryos raised in vivo accumulated 170-200 fmol glutamine/embryo/h at the unfertilized egg and 1-cell stages with a decline to 145 fmol/embryo/h at the 2-cell stage, followed by sharp increases to 400 and 850 fmol/embryo/h at the 8-cell and blastocyst stages. The presence or absence of glucose in the labelling medium had no effect on glutamine uptake by these embryos. Embryos raised in vitro accumulated 2-3 times more glutamine at stages comparable to those of embryos raised in vivo. In all cases in which 1-cell to blastocyst development in vitro was successful, glucose was present in the culture medium and the incremental uptake of glutamine between the 8-cell stage and the blastocyst stage was approximately 2-fold. This was also the increment for in-vivo raised embryos. When glucose was not present after the first 48 h, the 8-cell to blastocyst glutamine increment was not significant, and development into blastocysts was reduced. The results also show that glutamine can be used as an energy source for the generation of CO2 through the TCA cycle by all stages of preimplantation mouse development, whether raised in vivo or in vitro from the 1-cell stage. Two-cell embryos raised in vivo converted as much as 70% of the glutamine uptake into CO2, consistent with an important role for glutamine in the very earliest stages of preimplantation development. Cultured blastocysts appeared to convert less glutamine and the presence of glucose in the culture medium seemed to inhibit this conversion.