Biomedical subjects

M Biggiogera

Publications and source records attributed to M Biggiogera.

At least 37 records · Page 2Linked to original sources

PubMed1997

Detection of apoptotic cells by annexin V labeling at electron microscopy.

Annexin V is a Ca(++)-binding protein which is widely used as a marker for apoptotic cells, as it binds to the phosphatidylserine residues exposed at the surface of apoptotic cells. In this paper, we describe a method for the immunogold-labeling of biotin-conjugated Annexin V, to detect apoptotic thymocytes at electron microscopy. Etoposide-treated thymocytes were reacted in tissue culture medium with biotin-conjugated Annexin V, fixed with glutaraldehyde, and processed for resin embedding; thin sections were incubated with antibiotin antibodies coupled with colloidal gold. Cytometric estimates of the apoptotic index were also performed by evaluating either the DNA content after propidium iodide staining or the light-scattering values, as well as the positivity for fluorescein isothiocyanate-conjugated anti-biotin antibodies. At electron microscopy, gold-labeling of Annexin V was located on the plasma membrane only of apoptotic thymocytes and on cytoplasmic debris, likely resulting from the typical apoptotic blebbing. Unlabeled thymocytes always showed normal, non-apoptotic nuclear morphology. The application of Annexin V labeling at electron microscopy will allow a more refined description of the morphological events occurring during apoptosis.

Animals↗

PubMed1996

Localization of heat shock proteins in mouse male germ cells: an immunoelectron microscopical study.

We have examined the intracellular distribution of heat shock proteins HSP27 and HSP90 by means of specific antibodies and ultrastructural immunocytochemistry in the normal mouse testis as well as after heat shock. In the unstressed testis, these heat shock proteins are present in the cytoplasm and to a lesser extent in the nucleus throughout spermatogenesis. They do not show preferential association with any specific cytoplasmic structures and are absent from mitochondria. They disappear from the cell nucleus at the stage of elongating spermatids. After heat shock (42 degrees C), both HSP90 and HSP27 increase and partly relocate to the nucleus. Similarly to the localization in unstressed cells, they are mainly associated with perichromatin fibrils and the nucleolus. Moreover, a remarkable increase in the frequency of perichromatin fibrils in Sertoli cells, spermatogonia, and primary spermatocytes is also observed upon heat shock. Finally, a sharp increase in the labeling of HSPs in chromatoid bodies of round spermatids occurs following hyperthermic treatment. Interestingly, these two HSPs are localized on nuclear structures which are actively involved in RNA synthesis and processing, suggesting that they may have protective functions in these processes in a tissue which is particularly sensitive to heat stresses.

Animals↗

PubMed1996

Fine structural cytochemical and immunocytochemical analysis of nucleic acids and ribonucleoprotein distribution in nuclei of pig oocytes and early preimplantation embryos.

The fine structure of pig oocytes at the germinal vesicle (GV) stage and early preimplantation embryos (one to four blastomeres) isolated at slaughter was investigated by cytochemical and immunocytochemical methods. The distribution of nucleic acids and ribonucleoproteins (RNPs) in "compact nucleoli" [denominated nucleolus-like bodies (NLB) in oocytes and nucleolus precursor bodies (NPB) in early embryos] and in intranuclear bodies or granules was investigated by staining methods preferential for nuclear RNPs or using the osmium ammine or ethidium bromide-phosphotungstic acid (EB-PTA) reactions for nucleic acids. The distributions of the Sm antigen of nucleoplasmic small nuclear RNPs (snRNPs), the methyl-3 guanosine (m3G) cap of snRNAs and the splicing factor SC-35 were detected by immunoelectron microscopy using specific antibodies. The RNP nature of both NLBs and NPBs, and of nuclear granules in oocytes and embryos, and of fibrillar strands radially projecting from NLBs was revealed. Cytochemical evidence for RNA as a component of NLBs was further provided by EB-PTA staining in combination with the enzymatic removal of RNA, or by osmium-ammine staining without previous acid hydrolysis, while the absence of DNA in NLBs was established by Feulgen-like osmium-ammine staining. In addition, autoradiography demonstrated the absence of [6-3H]thymidine incorporation into NPBs. Other autoradiographic evidence attested the accumulation of RNA in NLBs of oocytes after a 60 min in vitro pulse of [5-3H]uridine. Immunoelectron microscopy using specific antibodies revealed the occurrence of nucleoplasmic snRNPs in both NLBs and NPBs. The presence of snRNA in NLB was confirmed by means of an antibody recognizing the m3G-cap structure. Another spliceosomal component, the protein SC-35 was also detected in NLBs. Among the numerous and variable intranuclear granules occurring mostly in aggregates, the Sm antigen was clearly detected only in the interchromatin granule-type component. Some Sm labeling was occasionally seen in other categories of larger granules. No reaction was detected over any granules when using the anti-m3G-cap antibody. The aggregates consisting of large granules and a finely fibrillar component were intensely immunolabeled by the anti-SC-35 splicing factor probe. Our observations suggest that the compact nucleoli, known to be present before and after fertilization in mammals (NLBs of oocytes and NPBs of early embryos), represent nuclear structural elements containing nonnucleolar, spliceosomal components.

Animals↗

PubMed1996

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.

Animals↗

PubMed1996

Osmium ammine: review of current applications to visualize DNA in electron microscopy.

This review has been collectively written. The contribution of the authors is mentioned for each part. References have been grouped at the end of the review. The objective of this review is to outline the principle of the method for electron microscopy, to emphasize the major applications and recent developments of this technique for DNA detection and finally to compare this technique with some other methods of DNA detection.

Coloring Agents↗

PubMed1995

Activation of osmium ammine by SO2-generating chemicals for EM Feulgen-type staining of DNA.

We present herein an improved method for the use of osmium ammine in a Feulgen-type reaction for specific staining of DNA at EM level and an analysis of its Schiff-type reagent behaviour. The activation of osmium ammine to a Schiff-type reagent (so far routinely performed by bubbling with gaseous SO2) can be accomplished by adding S0(2)-generating chemicals as for light microscopy. When used after HCI hydrolysis on epon or acrylate sections, activated osmium ammine behaves like a Schiff-type reagent, and the DNA staining can be selectively and completely abolished by aldehyde blocking agents. This preparation has the advantage of eliminating the use of gaseous SO2 thus rendering the technique more widely available to laboratories which cannot handle gas cylinders containing SO2. We recommend the use of osmium ammine in 8N acetic acid and 40mM sodium metabisulfite for 1 h at 37 degrees C for epon sections, and in 0.2N HCl and 0.2M metabisulfite for 30 min at room temperature for acrylate sections.

Adrenal Glands↗

PubMed1994

Physiologically inactive nucleoli contain nucleoplasmic ribonucleoproteins: immunoelectron microscopy of mouse spermatids and early embryos.

Physiologically inactivating or reactivating nucleoli represent a good model to investigate modifications of the nucleolar activity, which influence to a great extent the morphology of this organelle. We have studied the nucleoli of mouse spermatids during gradual inactivation, as well as those of early mouse preimplantation embryos, which undergo reactivation. In the 2-cell systems, inactive nucleoli are represented by homogeneously, finely fibrillar spherical bodies. With the aim of clarifying the composition of these nucleoli, we have analyzed them by means of immunoelectron microscopy using specific antibodies directed against nucleoplasmic snRNPs, hnRNPs, ribosomal proteins, or fibrillarin as well as by cytochemical methods for visualizing DNA and RNA. Our results indicate that RNA is present in zygote and 2-cell nucleolus precursor bodies (NPBs) as well as in 4- to 8-cell or more advanced embryo nucleoli, but not in inactive spermatid nucleoli. DNA is absent from inactive spermatid nucleoli and NPBs but is present within the nucleolus-associated chromatin and the nucleolonema of active nucleoli. The dense masses constituting the NPBs of the zygote and 2-cell embryos contain ribosomal proteins and fibrillarin but also hnRNPs and nucleoplasmic snRNPs. Of these, only fibrillarin is present in the spermatid residual nucleoli.

Animals↗

PubMed1994

Distribution of DNA, nuclear micro-heterogeneities and compaction of the chromatin in rabbit epididymal spermatozoa. Ultrastructural evaluation of the Feulgen-like technique using osmium ammine.

An adaptation of the Feulgen procedure to visualise DNA at the ultrastructural level, using osmium ammine instead of Schiff reagent, was applied to ultrathin sections of rabbit epididymal spermatozoa, known to display increasing chromatin compaction as they progress through the epididymis. In contrast with the somatic cell nuclei of the epididymal epithelium, which display classical staining, the chromatin of spermatozoa is partly or fully destroyed during the hydrolysis step of the technique. The sperm chromatin resistance towards destruction is a function of the initial sperm nuclear compaction and of the duration of hydrolysis prior to Feulgen-like staining. For a given nucleus, the maximum staining intensity is only obtained after an optimal duration of hydrolysis. However, because of this duration and local differences in chromatin compaction, the total DNA of a section is never completely visualized. The most compact parts of the nuclei are not yet stained, when the less compact parts have already been destroyed. This gives rise to a sperm-specific pattern which corresponds to the enhancement of microheterogeneities present in all sperm nuclei and to the local depolymerisation of the nuclear material during hydrolysis. The depolymerised parts of nuclei sit over the sections, and they also bind uranyl acetate, ethidium bromide and anti-protamine antibodies. Therefore, in sperm nuclei, the Feulgen-like staining at the ultrastructural level does not reveal the true DNA distribution. However, the amount of staining can be quantified to evaluate sperm chromatin compaction.

Animals↗

PubMed1993

Immunoelectron microscope localization of snRNP, hnRNP, and ribosomal proteins in mouse spermatogenesis.

We have studied the ultrastructural distribution of heterogeneous nuclear ribonucleoproteins (hnRNPs), small nuclear ribonucleoproteins (snRNPs), and ribosomal proteins during mouse spermatogenesis and spermiogenesis by means of specific antibodies and immunocytochemistry. All the above components were detectable from primary spermatocytes until the spermatid elongation phase, when the RNA synthetic activity is known to cease. Ribosomal protein (P1/P2 and L7) labeling disappeared as early as during the acrosome phase, and nucleoli were no longer labeled even during the cap phase. The nucleoplasmic structures labeled with the different anti-nucleoplasmic RNP immunoprobes corresponded, until the acrosome phase, to those previously observed as targets of the same antibodies in the nucleoplasm of somatic cell nuclei. Clusters of interchromatin granules of spermatocyte and early spermatid nuclei exhibit some labeling for hnRNP when compared with nuclei of Sertoli cells or previously analyzed liver or tissue culture cells, where these structural constituents usually remain weakly labeled or unlabeled. In spermatids in step 10, another type of nuclear granule, resembling perichromatin granules, but occurring in aggregates, can be observed. These structural constituents were labeled with antibodies recognizing nucleoplasmic snRNP antigens and therefore suggesting a non-nucleolar origin of these granules. Finally, we have observed nucleoplasmic areas of fibrogranular material, occurring only in primary spermatocytes. These components were labeled with anti-ribosomal protein antibodies but did not contain either hnRNPs or snRNPs.

Acrosome↗

PubMed1993

Ultrastructural detection of calcium and magnesium in the chromatoid body of mouse spermatids by electron spectroscopic imaging and electron energy loss spectroscopy.

We studied at the ultrastructural level the presence of calcium and magnesium in the chromatoid body (CB) in mouse spermatids. In addition, the presence of these two cations was also examined in the nucleolus. By electron spectroscopic imaging (ESI) and electron energy loss spectroscopic (EELS) analyses on glutaraldehyde/pyroantimonate-fixed material, we showed the association of pyroantimonate precipitate granules containing calcium and/or magnesium with the CB. The granules in both active (primary spermatocytes) and inactive (spermatids) nucleoli contain mainly calcium. Our results confirm that although the pyroantimonate technique used alone suffers from a lack of specificity, it can be useful for in situ immobilization of different cations that are further resolved with methods of elemental analysis. Without pyroantimonate fixation, only very few spots containing calcium can be localized by ESI and EELS.

Animals↗

PubMed1992

Immunoelectron microscopical distribution of histones H2B and H3 and protamines in the course of mouse spermiogenesis.

We have followed the fine structural distribution of two nucleosomal core histones, H2B and H3, and of protamines in the course of mouse spermiogenesis by means of specific antibodies and ultrastructural immunocytochemistry. Our results demonstrate that the nuclear labeling density of histone H2B decreases during steps 6-8 and then increases again in step 9-10 spermatids, while the labeling for histone H3 is constant throughout this period. In step 12 spermatids, the anti-H2B antibody labels mainly the central area of the nucleus. The first signs of protamine labeling are present in step 12 spermatids, where the gold grains can be found over the periphery of the nucleus. Later on, protamine labeling constantly increases and, by the end of spermiogenesis, the whole nucleus is labeled. We suggest that the morphological and structural differences between the central area and the periphery of mouse spermatids are, at least partly, due to a difference in the protein moiety associated with DNA. The central area, which is peculiar to the mouse and has been previously considered as a focus of chromatin condensation, represents, however, the last nuclear region containing histones and consequently the last area where the substitution of histones by protamines takes place.

Animals↗

PubMed1992

Migration of centromere proteins in rabbit spermatids.

Human autoantibodies were used to localize centromere proteins by immunoelectron microscopy, immunofluorescence, and confocal microscopy in isolated cells and in cryosections of rabbit testis. A computer-assisted three-dimensional reconstruction of the positions and sizes of fluorescent spots allowed us to follow their movements during the different phases of spermiogenesis. In very young spermatids, the centromeres were distributed within a space separated from both the external nuclear limits and the nuclear core. They moved towards the nuclear center in cap phase spermatids, where they clustered into a few large centromeric masses. In preelongating spermatids, the immunolabeled proteins were dispersed within an equatorial area, where they formed one large mass. In late spermatids, the mass moved towards the posterior part of the nucleus, and, in the spermatozoon, the two basal knobs located at the base of the nuclei were the only strongly immunolabeled structures, while no labeling of the main part of the nucleus was observed. Since the number of centromeres remained close to the number of chromosomes until the cap phase of spermatid differentiation, we hypothesize that the labeling of young spermatids corresponds to centrometric proteins associated with their specific DNA counterparts, while the centromere proteins, possibly detached from their DNA loci, were released from nuclei of old spermatids in the same way as are histones and transition proteins.

Animals↗

PubMed1992

Electron spectroscopic imaging and X-ray microanalysis of phosphorus in mouse sperm chromatin.

The influence of HCl hydrolysis on DNA detection in a Feulgen-type reaction using osmium ammine has been analyzed at the electron microscopic level by means of electron spectroscopic imaging, electron energy loss spectroscopy and X-ray microanalysis in energy dispersive spectroscopy. Both the stained DNA and the phosphorus mapping for a given hydrolysis condition were studied in parallel on the same nucleus. We have found that the pattern of osmium ammine-stained DNA and phosphorus imaging can be superimposed for a short hydrolysis time. After long HCl treatment, DNA is barely detectable by osmium ammine while phosphorus is still present in the thin sections. Taking into account the fact that the cells are embedded in a plastic resin, it is reasonable to think that in this case DNA depolymerization does not completely correspond to DNA loss. An incomplete loss of this highly denatured and depolymerized DNA from the plastic sections will explain both the presence of phosphorus and the poor stainability with a Schiff-type reagent.

Animals↗

PubMed1991

The striated body: a new structure in the sperm head of the rabbit spermatozoon.

The striated body, a new structure lying in the perinuclear substance of rabbit spermatozoa, is described. It is composed of protein cords displaying a perpendicular periodic arrangement of dark and light bands. It appears in late spermatids, immediately prior to spermiation and is still present in epididymal spermatozoa, mostly, but not exclusively close to the perforatorium. The nature of this component has been analysed using a series of cytochemical techniques, as well as various antibodies and immunocytochemistry. The absence of RNA and of DNA favours a nonviral origin for this structure. The striated body is devoid of actin, differing in that respect from the rest of the perinuclear substance.

Animals↗

PubMed1991

Immunoelectron microscopic localization of small nuclear ribonucleoproteins during bovine early embryogenesis.

Small nuclear ribonucleoproteins (snRNPs) were localized using human autoimmune or monoclonal anti-snRNP antibodies and ultrastructural immunocytochemistry in early preimplantation bovine embryos before and at transcription onset. Bovine cleavage stages up to 16-cell embryos were obtained either by culture of in vitro-fertilized ovarian oocytes or by isolation at slaughter of embryos fertilized and developed in vivo. Before transcription onset, up to the four-cell stage, diffuse labeling of nucleoplasm was detected, whereas cytoplasm labeling remained low. At the transcription onset, labeling of all eight-cell embryo nuclei was markedly concentrated at the borderline of already formed, condensed chromatin aggregates, where it was associated mainly with perichromatin fibrils. The condensed chromatin blocks revealed by several different staining methods were more prominent than is the case in most somatic cells. The degree of chromatin condensation and the pattern of its distribution differed between in vivo- and in vitro-produced eight-cell embryos: the in vitro embryos showed a higher degree of chromatin condensation and a peripheral distribution of chromatin blocks. A relation of this observation to the developmental potential of cow embryos is suggested. In two- and four-cell embryos, intensive labeling was seen in interchromatin granules, which, in their turn, were often seen in close proximity to the nucleolus precursor bodies, or in the four-cell stage were interconnected to them. No labeling was ever seen, using antibodies specific for the snRNP Sm antigen, in nucleolar precursor bodies during embryonic nucleologenesis nor in the resulting nucleoli. There was some incidental labeling of the large central vacuole appearing at the beginning of the nucleolus precursor body transformation, testifying the nucleoplasmic origin of this entity.

Animals↗

PubMed1991

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.

Animals↗

PubMed1991

A cytochemical and immunocytochemical study of DNA distribution in spermatid nuclei of mouse, rabbit, and bull.

DNA distribution in mouse, rabbit and bull spermatids was analyzed by electron microscopy, after using a Feulgen-like HCl-osmium ammine procedure, and after immunocytochemistry with anti-DNA antibodies. In addition, nucleic acids were visualized with the intercalating dye ethidium bromide and phosphotungstic acid. The parts of DNA displaying a beta helix configuration (possibly A-T rich parts) were identified by epifluorescence microscopy after staining with Hoechst 33258. In all 3 species, young spermatid nuclei were seen to have large areas poor in DNA, as well as DNA-rich areas, which were mostly concentrated into a peripheral layer close to the acrosome and into one or several masses, displaying species-specific locations. These DNA-rich areas were stained with Hoechst 33258. Elongating spermatid nucleic contained homogeneously distributed DNA, and this was evident following both immunocytochemistry and nucleic acid histochemistry in all 3 species. However, the distribution appeared more heterogeneous after the Feulgen-like procedure, and was accompanied by a disappearance of Hoechst-fluorescence. In fully elongated spermatids, all nuclear areas stained with Hoechst 33258, while the 3 other techniques labeled either all or species-specific parts of the condensed chromatin. The reasons for these variable reactions are discussed in terms of technique specificities, DNA configuration and nucleoprotein moiety replacements.

Animals↗

PubMed1990

Immunoelectron microscopical visualization of ribonucleoproteins in the chromatoid body of mouse spermatids.

The chromatoid body (CB), a cytoplasmic organelle present only in germ cell line, was studied at the electron microscopic level in mouse spermatids using cytochemical techniques and specific antibodies directed against sn-RNPs, hnRNPs, and ribosomal proteins. We found that specific staining for DNA as well as the use of monoclonal anti-DNA antibodies show a complete absence of DNA in the CB. The CB remains stained, however, after the application of the ethidium bromide-PTA technique, suggesting the presence of RNA within this organelle. snRNP as well as hnRNP proteins are demonstrated within the CB by means of specific monoclonal or polyclonal antibodies, especially during earlier spermiogenic stages. Monoclonal antibodies directed against the large ribosomal subunit proteins P1/P2 detect these antigens on the CB essentially along the internal threads of dense fibrillar material. Our findings suggest that the CB may function as a source of mRNA and/or of its partially processed precursors during the late stages of spermiogenesis, when the spermatid nucleus becomes gradually inactive.

Animals↗