The cell nucleus in early bovine and caprine preimplantation embryos: fine structural cytochemistry and immunoelectron microscopy.

Fine structural cytochemistry and immunocytochemistry were used to study nucleic acids and nuclear proteins in nuclear bodies (NB) of pronuclear and 2-cell bovine and caprine embryos on ultrathin sections of paraformaldehyde fixed and Lowicryl K4M or LR White embedded specimens. The most striking feature detected in some of these nuclear bodies (NBs) was the presence of non-nucleolar proteins known to be involved in pre-mRNA splicing. One category of such intranuclear bodies (showing a rather dense finely fibrillar composition and named here dense body-DB) contained the Sm-antigen (an antigen common to a major group of nucleoplasmic spliceosomal snRNPs). Another, more numerous category of NBs differed morphologically from the former one by a much looser composition of fibrillogranular elements (loose body-LB). Moreover, it showed the presence of the non-snRNP splicing factor SC-35, in addition to the Sm-antigen. Both categories of these nuclear bodies were distinguished clearly from the nucleolar precursor bodies (NPBs) by an absence of immunolabeling of NPB with antibodies against nuclear proteins involved in splicing. Moreover, the former NBs are not stained with silver, while NPBs already in pronuclei exhibit strong affinity to silver. In addition to the immunolabeling in prominent (approx. 0.2-2.0 microns) NBs, regularly occurring high concentration of snRNP was revealed in very small (approx. 0.05 micron), morphologically poorly defined areas (named here small snRNP-enriched areas-SSA), harboring moreover a set of nuclear proteins similar to that of the coiled body. Numerous observations of the presence of these small areas in nuclear bodies and in their close vicinity, in nucleoplasm, in proximity of the nuclear envelope and also in ooplasm suggested that they are possible carriers of certain nuclear proteins moving between nuclear bodies, nucleoplasm and cytoplasm. A functional relationship of all these embryonic subnuclear elements has not been elucidated so far but their mutual relation is suggested, since the NPBs and other nuclear bodies usually occur in a close association. Fine structural and immunoelectron microscopic observations further suggest a similarity of the nuclear bodies in the early ruminant embryo with specific intranuclear bodies ("snurposomes") known from Xenopus laevis oocytes. A new and striking feature emerging from these observations is a possible involvement of a group of nucleoplasmic proteins in a yet unknown way in the differentiation processes concomitant with early embryonic nucleologenesis.

Selective binding of human cumulus cell-secreted glycoproteins to human spermatozoa during capacitation in vitro.

The results of this study demonstrate that glycoproteins manufactured by human cumulus cells can be detected bound to human spermatozoa incubated in capacitational medium containing the labeled cumulus-cell secretions. Cumulus-cell-secreted glycoproteins were labeled with a mixture of 3H-methionine and 3H-tryptophan or with 3H-fucose, and the binding of the labeled compounds to spermatozoa was evaluated by autoradiography. The binding was highly selective, involving only approximately 1% of the samples of spermatozoa used. The results suggest that the binding of cumulus-cell-secreted glycoproteins to spermatozoa may represent a final and highly selective step in human sperm capacitation.

Genome reactivation in developing early pig embryos: an ultrastructural and autoradiographic analysis.

The onset of RNA synthesis in developing early pig embryos from 1-cell to 8-cell and morula stages was studied using high-resolution autoradiography of (5-3H)uridine incorporation. No transcriptional activity was detected in nuclei of 1- and 2-cell stage embryos with this technique. In these embryos nucleolus-like bodies (NLB) consist of sharply delineated, round, electron dense fibrillar masses. In the 4-cell stage embryos, the first uridine-3H incorporation in the nucleoplasm was detected and localized mainly near the regions of condensed chromatin. The first signs of reticulation and chromatin association were observed at the periphery of NLBs. In the next cell cycle (5- to 8-cell embryos) uridine-3H labelling was detected in the nucleoplasm and nucleoli. In these embryos, nucleoli consist of a central dense fibrillar mass without any transcriptional activity and fibrillo-granular cortex over which label was localized. The degree of functional restructure of nucleoli was variable within one blastomere or among different blastomeres, some nucleoli being more reticulated and showing more transcriptional activity than others. Fully developed nucleoli were present in early morulae. Electron dense unidentified structures described here as small dense round-shaped bodies (RDB) often surrounded by blocks of large chromatin granules were observed in intact 2-cell and alpha-amanitin treated 4-cell stage embryos. These structures did not show any transcriptional activity.

DNA synthesis and distribution in parthenogenetic bovine embryos.

Parthenogenetically activated, in vitro-matured bovine oocytes and parthenogenotes obtained at 2 to 4 days post activation were analyzed by 3H-thymidine autoradiography for the timing of the S-phase and for distribution of newly replicated DNA, respectively. Spread pronuclear parthenogenotes revealed that the DNA synthesis in electrically stimulated oocytes commenced at 14 h post activation. At 20 to 24 h, a maximum number of labeled pronuclei was reached (25 to 38%), and DNA synthesis persisted in some parthenogenotes up to 30 h post activation. The DNA labeling detected on semi-thin sections showed that the distribution of newly synthesized DNA in the nuclei of 3- to 16-cell parthenogenotes was mostly irregular or abnormal, documenting that the apparent morphological normalcy of parthenogenotes was in contrast to the data concerning the DNA synthesis and distribution.

Nuclear fine structure and transcription in early goat embryos.

Early preimplantation goat embryos were investigated for the onset of major gene transcription by fine-structure morphology and autoradiographic detection of (5-(3)H)uridine incorporation. Complex nuclear bodies were already seen in early pronuclei but only in the 16-cell embryos did these methodologies offer clear-cut evidence of the already fully-developed nucleolar structure as well as of nucleoplasm labeling. Nucleoplasm labeling, which is supposed to detect the onset of major transcription, was absent in the pronuclear and the 2- to 4-cell embryos. The first (5-(3)H)uridine incorporation was detected in the 8-cell stage nuclei nucleoplasm, followed by nucleolar labeling. Based on this evidence, we found that in comparison with other ruminants, there is no striking difference in the timing of the shift in control from the maternal to the embryonic genome in the development of the early goat embryo.

Unimpaired fertilization with thymidine-3H-labelled spermatozoa in the mouse.

Thymidine-3H-labelled spermatozoa were obtained in the sixth week after injection of males with a single or repeated doses of thymidine-3H, in amounts of 3.5 to 50 muCi per gramme body weight. Mouse ova were fertilized in vivo or in vitro by such labelled spermatozoa. It was found by means of autoradiography that heavily labelled spermatozoa compete successfully with non-labelled ones in performing fertilization. The distribution of relative levels of activity in fertilizing spermatozoa was similar to that in the population of inseminated spermatozoa. The early development of ova fertilized by generally labelled spermatozoa (followed up to the first cleavage) was not impaired. It is concluded that spermatozoa labelled with thymidine-3H in their DNA to a level sufficient for autoradiographic detection may be probably used in the studies of the cytology of fertilization without greater precautions regarding possible radiation-induced artefacts.

[Dynamics of ultrastructural morphology of the nucleolar apparatus in bovine preimplantation embryos collected in an area of chronic irradiation]. / Dynamická ultramikromorfológia nukleolárneho aparátu preimplantacných embryí kráv z oblasti chronického rádioaktívneho oziarenia.

Ultrastructural morphology and immunoelectron microscopy of the nucleus and nucleologenesis in early preimplantation cow embryos were applied in an attempt to demonstrate a possible radiation injury to that early stage of development due to chronical irradiation of the animals in the Tchernobyl area. Mostly eight cell embryos as well as morulae were collected from superovulated cows which were previously constantly kept in zones of different levels of radioactive irradiation. In addition to the normometric status of reproductive organs in no case was it possible to detect an apparent deviation in the nuclear morphology or in the process of nucleologenesis as compared to the physiological situation (Kopecný et al., 1989b, 1991, 1996). This observation was supported by an immunoelectron microscope study of DNA association and penetration in the differentiated nucleolus in the late 8-cell stage. These observations show that the otherwise demonstrated radiation injury localized in the genome does not probably influence markedly the early events of the developing embryo and that the aberrant cytoplasmic command of the nuclear events known in other types of oocyte/early cow embryo impairment (review Kopecný and Nicmann, 1993; Kanka et al. 1991; Pavlok et al., 1993) is not seen in early embryos collected from chronically irradiated animals.

High-resolution autoradiographic studies of comparative nucleologenesis and genome reactivation during early embryogenesis in pig, man and cattle.

Using high-resolution autoradiography, simultaneous studies of ultrastructure and nucleic acid dynamics were performed during nucleologenesis in early porcine, human and bovine embryos. In contrast to the early genome activation known to occur during the second cell cycle in the mouse, the onset of rRNA synthesis detected by (5-3H) uridine incorporation in the nucleolar compartment is delayed by cleavage of one cell cycle (to the third cell cycle) in the early pig embryo and by two cell cycles (to the fourth cell cycle) in human and cattle embryos. Extranucleolar RNA synthesis, as detected by nucleoplasm labelling, generally started shortly before rRNA synthesis. The timing of nucleolar labelling was well correlated with the penetration of embryonic DNA into the nucleolus precursor body and with nucleolus structure differentiation. Ultrastructural and/or autoradiographic techniques are suggested for the study of the onset of embryonic transcription, e.g. in embryos "reconstructed" by micromanipulation.

Developmental control of the human male pronucleus by ooplasmic factors.

The nature of oocyte cytoplasmic factors controlling the development of the male pronucleus was investigated by inseminating human, zona-free oocytes at metaphase of the 1st and 2nd meiotic division. Oocytes at metaphase of the 2nd meiotic division could support the full structural and functional development of male pronuclei, whereas the vast majority of those at metaphase of the 1st meiotic division failed to do so. This suggests that oocyte cytoplasmic factors required for male pronuclear formation do not develop fully until the oocyte reaches the 2nd meiotic metaphase. When increasing numbers of spermatozoa entered one oocyte, the transformation of sperm nuclei into pronuclei was impaired progressively. The later stages of pronuclear development were particularly sensitive to polyspermy. These factors should be taken into consideration in the development of techniques of micromanipulation-assisted insemination.

Nucleic acid synthesis and development of human male pronucleus.

Polyspermically penetrated human zona-free eggs prepared from oocytes that had failed to be fertilized in an in-vitro fertilization programme were used. The pronuclear synthetic activity was evaluated by high-resolution autoradiography and correlated with the development of pronuclear structure. Incorporation of [3H]-thymidine, signalling the occurrence of a DNA synthetic phase, was only detected in structurally fully developed pronuclei previously shown to appear no sooner than 12 h after gamete union. However, [3H]adenosine was incorporated into very early pronuclei which had not yet completed the development of their nuclear envelopes and which first appeared about 4 h after sperm-egg fusion. In the absence of DNA synthesis (shown by the lack of thymidine incorporation), this early adenosine incorporation apparently reflects an early pronuclear RNA synthesis. Taken together, these results indicate that nucleic acid synthesis in human male pronuclei is tightly bound to the development of a corresponding pronuclear structure and that DNA synthesis, beginning about 12 h after fertilization, is preceded by a slight but evident RNA synthesis taking place during an early stage of human male pronuclear formation.

Development of human male pronucleus: ultrastructure and timing.

The process of human male pronuclear formation was studied using an experimental model based on in vitro inseminated human zona-free eggs prepared from oocytes that failed to fertilize in a clinical in vitro fertilization program. The main ultrastructural changes in penetrated sperm nuclei transforming pronuclei were used to define four stages of pronuclear development. The first two stages, representing partial (Stage 1) and total (Stage 2) sperm chromatin decondensation, appeared as early as 1 hr after mixing of gametes. This rapid initial phase was followed by a more lengthy array of events leading to transformation of decondensed sperm nuclei into fully developed male pronuclei (Stages 3 and 4). Stage 3 was characterized by reformation of the nuclear envelope, reorganization of chromatin, and the assembly of nucleolar precursors. It was not completed until 12 hr after in vitro insemination when fully developed male pronuclei (Stage 4) were first observed. In some eggs pronuclei did not reach Stage 4 at all. The results of this study provide a morphological background for further research into molecular aspects of human male pronuclear development and its regulation.

Assembly of the nucleolar precursor bodies in human male pronuclei is correlated with an early RNA synthetic activity.

The nucleolar precursor bodies (NPBs) are electrondense, homogeneous, and finely filamentous intranuclear structures occurring in mammalian zygotes during the early postfertilization development and subsequently transforming into nucleoli. In this study we addressed the question if the assembly of the NPBs is correlated with adenosine incorporation into the pro-nuclear chromatin, an event previously reported to precede the beginning of the pronuclear DNA synthetic phase and suggested to reflect an early pronuclear RNA synthesis. The degree of polyspermy was manipulated artificially, and the effects of the resulting changes in the nucleocytoplasmic ratio on adenosine incorporation and the NPB assembly were analyzed using a quantitative autoradiographic examination. The NPB assembly occurred only in pronuclei which incorporated [3H]-adenosine. This [3H]adenosine labeling of pronuclei was not due to DNA replication, but it could be removed by treatment of egg sections with hot trichloracetic acid before autoradiography, a method which quantitatively extracts nucleic acids. With increasing nucleocytoplasmic ratio, the capacity of developing pronuclei to incorporate adenosine was lost progressively and this phenomenon was closely correlated with impairment of the NPB assembly. These findings suggest that an early pronuclear RNA synthesis is required for the development of the NPBs.

Late preovulatory synthesis of proteoglycans by the human oocyte and cumulus cells and their secretion into the oocyte-cumulus-complex extracellular matrices.

Light- and electron-microscope autoradiography using 3H-glucosamine and 3H-fucose as precursors was employed to investigate proteoglycan synthesis and secretion by late preovulatory human oocytes and cumulus cells. Both the oocyte and cumulus cells were found to be important cellular sources supplying proteoglycans to the oocyte-cumulus-complex extracellular matrices, i.e., the zona pellucida and the cumulus intercellular matrix. Both the oocyte and cumulus cells were shown to secrete labelled proteoglycans into the zona pellucida. Labelled proteoglycans were also detected in the cumulus intercellular matrix. Chase experiments revealed the labelled molecules to be relatively closely associated with both the zona pellucida and the cumulus intercellular matrix. Staining with chromic acid and phosphotungstic acid showed proteoglycan material to penetrate from the cumulus intercellular matrix into pores of the zona pellucida. This material is thought to be a structural equivalent of the newly synthesized proteoglycans secreted by cumulus cells and migrating into the zona pellucida (as detected by autoradiography). It is concluded that newly synthesized proteoglycans secreted by the oocyte and cumulus cells in the late preovulatory period are a component of the microenvironment in which fertilization takes place.

Extranucleolar genome reactivation: topochemical studies on early bovine embryo. A review.

A review is presented of fine-structure autoradiographic [incorporation of (5-3H)uridine] and immunoelectron microscope (localization of small nuclear ribonucleoproteins--snRNPs) data on the onset of extranucleolar transcription in early preimplantation bovine embryo. First incorporation (5-3H) uridine into blastomere nuclei nucleoplasm occurs, in the early cow embryo, during the 8-cell stage. Both the degree of chromatin condensation as well as the intensity of labeling increase as the fourth cycle of the embryonic blastomeres progress. However, neither the level of extranucleolar chromatin condensation nor the degree of labeling usually detected in somatic cells have been observed during this stage but occurred only in later stages of embryo development. The association of the label with the periphery of the condensed chromatin demonstrates, as in other cell types the sites of newly synthesized hnRNA. A further insight into the functional microarchitectural changes of the early cow embryo nuclei in relation to the onset of transcription has been obtained by immunoelectron microscope studies on Lowicryl K4M embedded material. Using this technique, the pattern of the contrast in the preparations following uranyl/lead staining was comparable to that obtained by the Bernhard's (1969) regressive staining for preferential nuclear-RNP visualization. In this way, the coordinate appearance of perichromatin fibrils on the borderline of the already condensed chromatin was evident during the 8-cell stage. Especially in the "in vitro" produced embryos the condensed chromatin occurred in marked blocks with a clearly defined perichromatin region in the thin sections. In the same nuclear area Sm-antigen (associated with a group of snRNPs) has concentrated and localized to perichromatin fibrils.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic study of mouse spermatozoan arginine-rich nuclear protein in fertilization.

General labeling of ripe mouse spermatozoa was obtained two weeks after six injections of L-arginine-5-3H monohydrochloride, administered within two days. Besides tail labeling a very intensive nuclear labeling was obtained. It is presumed that this nuclear labeling represents almost exclusively tritiated arginine incorporation into basic nuclear protein. Mouse oocytes were fertilized in vitro with such labeled spermatozoa. It was found that the nuclear label is lost very early during male pronucleus formation, probably concomitantly with sperm chromatin decondensation. This might indicate total loss of the basic spermatozoal nuclear protein before male genome activation.

Incorporation of Arginine-3H into chromatin of mouse eggs shortly after sperm penetration.

Mouse oocytes were fertilized in vitro in a complete cultivation medium enriched by L-arginine-5(3)H monohydrochloride. The oocytes were isolated four hours after insemination. The incorporation of this precursor, as detected by means of autoradiography, was significantly higher in swollen sperm heads and female chromosomes at anaphase/telophase of the second meiotic division than the incorporation into ooplasm.

An autoradiographic study of macromolecular syntheses in the epithelium of the ductus epididymidis in the mouse. II. Incorporation of L-fucose-1-3H.

Glycoprotein dynamism in the mouse epididymis was studied by means ofhistoautoradiography after injection of L-fucose-1-3H. The label was detected, at thirty minutes p.i., in the area occupied by Golgi apparatus in the epithelial cells. At 4 h p.i. the label was already present inthe lumen of ductus epididymidis. At this time interval, the luminal labelling was highest in the initial segment of the epididymis and decreased against the more distal segments considerably. At ten days p.i. very high labelling was detected in the luminal contents in the terminal segment of the ductus epididymidis and in ductus deferens, the labelling in the proximal segments of the epididymis being much lower. These observations suggested a wave of labelled glycoprotein in epididymal plasma passing through the epididymis after a fucose pulse. Higher labelling was detected in so-called "clrial was seen in epididymal and uterine spermatozoa, mostly in sperm tail region.

The nature of the 'nucleolus precursor body' in early preimplantation embryos: a review of fine-structure cytochemical, immunocytochemical and autoradiographic data related to nucleolar function.

In mammals, the restoration of rRNA transcription after fertilisation is accompanied by a gradual differentiation of the nucleolar structure by a process called embryonic nucleogenesis. During cleavage, the nucleolar components appear sterically related to a class of nuclear bodies already detectable in pronuclei. These structures, due to their apparent function as centres of nucleolus formation, have been designated nucleolus precursor bodies (NPBs). It was found recently not only that the size and morphology of the NPBs differ among mammalian species, but that the pattern of embryonic nucleologenesis and even the molecular composition of different NPB compartments vary from one species to another. Accordingly we assumed that at least two definitely different types of NPBs exist, namely the mouse-type NPB and cow-type NPB. In the mouse-type NPB, the original compact material of the NPB remains detectable in the early functional nucleolus. This NPB core does not contain DNA or typical Ag-NOR nucleolar proteins. At the onset of rRNA transcription, the nucleolonema is formed at the periphery of the NPB. The cow-type NPB shows a homogeneous distribution of typical nucleolar proteins throughout its body from the pronucleolar to the early 8-cell stage. At the beginning of rRNA transcription, the cow-type NPB is penetrated by perinucleolar DNA and rRNA synthesis is detectable deep inside the nucleolus. In this case, the entire NPB is readily transformed into a typical nucleolus. These processes are recognisable using fine-structure analysis of preimplantation mammalian embryos. For this reason this approach is often used as a method of evaluating the state of experimental embryos; in such studies, the species differences must be taken into account.