Sea urchin testicular cells evaluated by fluorescence microscopy of unfixed tissue

A procedure is presented for rapid, quantitative evaluation of cell and nuclear types present in the male gonad of the sea urchin. Vitally stained whole mounts of tissue fragments or dissociated cells are prepared, which reveal detailed 3-dimensional chromatin patterns and enough cytoplasmic features to provide reliable markers for most of the somatic and germ line cell types. Representative cellular morphologies are described. Nuclear volume changes during spermatogenesis are quantified. Spermatid nuclei contain an apparently interconnected network of heterochromatin. Regions relatively devoid of chromatin decrease in size as nuclear condensation proceeds and spherical nuclear shape is maintained. The major decrease in nuclear volume occurs prior to the late spermatid stage. The volume of the spermatozoan nucleus is achieved by the smallest late spermatid nucleus before the change from spherical to conoid morphology. The relationship of this morphological transition to sperm histone dephosphorylation is discussed.     

Interaction of the activated cytoplasmic glucocorticoid hormone receptor complex with the nuclear envelope

Highly purified activated cytoplasmic glucocorticoid hormone receptor binds with high affinity to sites in the nuclear envelope. Nuclear envelope fragments can be isolated from purified chromatin. They bind activated cytoplasmic glucocorticoid receptor with the same equilibrium constant as nuclear envelopes. The presence of envelope components in chromatin is confirmed by the virtual identity of the gel electrophoretic glycoprotein pattern of nuclear envelope, chromatin nonhistones, and nuclear envelope fragments from chromatin.     

Studies on the action and chemical nature of the nuclear factor promoting degradation of chromatin DNA

Chromatin DNA of liver and kidney, obtained by the method of Dingman & Sporn, is inaccessible in 0.14 M NaCl to pancreatic DNase and cytoplasmic DNase. Under the combined action of DNase and nuclear extract (NE) (extraction with 0.14 M NaCl) on chromatin, the DNA of the latter is intensively degraded. The action of NE is tissue-specific—liver NE has almost no effect on kidney chromatin DNA degradation. The removal of protein or RNA from NE deprives it of its ability to accelerate chromatin DNA degradation by DNases. It is assumed that the active part of NE is a complex of a protein and RNA. Here, tissue specificity is determined by both components of this complex. The biological role of the nuclear factor promoting chromatin DNA degradation is not known at present.     

A reconstituted cell-free system for the specific transfer of steroid–receptor complexes into nuclear chromatin isolated from rat ventral prostate gland

1. A system has been developed for the specific transfer of [(3)H]dihydrotestosterone-receptor complexes into prostatic chromatin in vitro. 2. Under optimum conditions the overall transfer of [(3)H]dihydrotestosterone into purified chromatin in this reconstituted system is entirely consistent with the results obtained in whole tissue both in vivo and in vitro. 3. The transfer of [(3)H]dihydrotestosterone into chromatin is tissue-specific and maximal into chromatin isolated from androgen-dependent tissues. 4. The tissue specificity is maintained at two levels: first, in the presence of specific cytoplasmic androgen-receptor proteins; secondly, by the nature and composition of the chromatin itself. 5. Evidence is presented that androgenic steroids in vivo may maintain the tissue-specific nature of chromatin in androgen-dependent tissues by the selective induction of nuclear protein synthesis. 6. The relevance of these findings to the mechanism of action of androgenic steroids is discussed.     

Meiotic competence acquisition is associated with the appearance of M-phase characteristics in growing mouse oocytes

To determine whether the acquisition of meiotic competence during the growth phase of oogenesis is associated with the appearance of M-phase characteristics, oocytes obtained from 13- to 30-day-old mice were evaluated by fluorescence microscopy with respect to chromatin and microtubule organization , in vitro maturation ability, and the distribution of M-phase phosphoproteins. Meiotically incompetent oocytes were distinguished from their competent counterparts in displaying elaborate interphase-like arrays of cytoplasmic microtubules and dispersed germinal vesicle chromatin. Meiotically competent oocytes were larger in size, exhibited condensation of chromatin around the nucleolus, and displayed a progressive diminution of cytoplasmic microtubules in conjunction with the appearance of multiple microtubule organizing centers. After 24 hr in culture, medium- to large-sized oocytes exhibiting perinucleolar chromatin condensation resume meiosis whereas smaller meiotically incompetent oocytes retain GVs with diffuse chromatin. Moreover, indirect immunofluorescence studies using the M-phase phosphoprotein specific monoclonal antibody MPM-2 indicate that the appearance of reactive cytoplasmic foci is directly correlated with nuclear changes characteristic of meiotically competent oocytes. Thus, the earliest transition to a meiotically competent state during oocyte growth in the immature mouse ovary is characterized by stage-specific and coordinated modifications of nuclear and cytoplasmic components.     

Small RNA species of the HeLa cell: Metabolism and subcellular localization

The small molecular weight RNAs of the HeLa cell have been located in specific subcellular fractions. SnA is located in the nucleolus and is partially bonded to nucleolar 28S RNA. SnD, the most abundant of the small nuclear RNAs, is partially released from the nucleus when the nuclear preparation is briefly warmed. SnF is released from the nuclei when chromatin is digested with the micrococcal nuclease and not when pancreatic DNAase is used. The remainder of the small nuclear species remain in the nucleus following the digestion of chromatin and are concluded to be elements of the “nuclear skeleton.” SnK is found predominantly in the cytoplasm, but migrates quantitatively to the nuclear fraction in the presence of high levels of actinomycin D. ScL is totally cytoplasmic and is partially bound to cell membranes. It is the 7S RNA found in oncornavirus virions. All the small nuclear RNAs appear initially in the cytoplasmic fraction before fixation in the nucleus. Two short-lived cytoplasmic species behave kinetically as precursors to the stable nuclear RNAs.     

The use of deoxyribonucleic acid–cellulose chromatography and isoelectric focusing for the characterization and partial purification of steroid–receptor complexes

1. Two characteristic properties of the specific high-affinity steroid-binding proteins or receptors, their ability to bind to DNA-cellulose and their relatively acidic isoelectric point, have been exploited as a means of purification. These two fundamental properties distinguish the receptors from the steroid-binding proteins in serum and the non-specific low-affinity steroid-binding proteins in hormone-responsive cells. 2. A significant degree of purification of both cytoplasmic and nuclear steroid-receptor complexes can be achieved with practical facility by these procedures. The purity of the receptor complexes is sufficient to enable studies on their possible control of metabolic processes to be investigated in the future. 3. After extensive purification the physicochemical properties of the cytoplasmic androgen-receptor complex, such as sedimentation coefficient, were unchanged. Further, the purified complex fully retained at least one of its fundamental physiological properties, namely the ability to transfer 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) into chromatin in vitro. 4. The methods may also be employed for studying the changes in the structure and properties of the receptor complexes that are an essential prerequisite for the transfer of cytoplasmic receptor complexes into nuclear chromatin. The temperature-dependence of the binding of androgen-receptor complexes into chromatin is essentially due to a major change in cytoplasmic receptor complex before its attachment to nuclear chromatin. 5. The resolution of these analytical procedures was sufficient to enable a critical comparison of the receptor proteins from different male accessory glands to be undertaken. From these studies, no substantial evidence in support of the tissue specificity of androgen receptors could be established; rather the receptors from different androgen-dependent glands were remarkably similar in physicochemical properties. 6. Although the methods were initially developed for the partial purification of androgen-receptor complexes, they are equally suitable for the prompt and extensive purification of oestrogen-receptor and progesterone-receptor complexes.     

DNA content contributes to nuclear size control in Xenopus laevis

Cells adapt to drastic changes in genome quantity during evolution and cell division by adjusting the nuclear size to exert genomic functions. However, the mechanism by which DNA content within the nucleus contributes to controlling the nuclear size remains unclear. Here, we experimentally evaluated the effects of DNA content by utilizing cell-free Xenopus egg extracts and imaging of in vivo embryos. Upon manipulation of DNA content while maintaining cytoplasmic effects constant, both plateau size and expansion speed of the nucleus correlated highly with DNA content. We also found that nuclear expansion dynamics was altered when chromatin interaction with the nuclear envelope or chromatin condensation was manipulated while maintaining DNA content constant. Furthermore, excess membrane accumulated on the nuclear surface when the DNA content was low. These results clearly demonstrate that nuclear expansion is determined not only by cytoplasmic membrane supply but also by the physical properties of chromatin, including DNA quantity and chromatin structure within the nucleus, rather than the coding sequences themselves. In controlling the dynamics of nuclear expansion, we propose that chromatin interaction with the nuclear envelope plays a role in transmitting chromatin repulsion forces to the nuclear membrane.     

Isolation and purification of myotube and myoblast nuclei from cultures of embryonic chick skeletal muscle.

Methods are described for the preparation of purified myotubes from embryonic chick skeletal muscle cultures and the preparation of purified nuclei from both myotubes and myoblasts. Myotubes are released from the culture dish by digestion of their collagen substratum with collagenase, and purified by sucrose density gradient sedimentation. Nuclei are prepared from the isolated myotubes by controlled homogenization in Ca²⁺-free medium and sedimentation through 2.1 M sucrose. Nuclei are prepared from cultured myoblasts in a similar fashion, with the inclusion of the non-ionic detergent NP-40 in the homogenization medium and sedimentation through 2.4 M sucrose. Phase contrast microscopic examination showed that the nuclear preparations are free of visible cytoplasmic contamination, and are morphologically similar to nuclei observed in situ. Biochemical assays (protein/DNA and $\text{RNA}\text{DNA}$ ratios) confirm the purity of the nuclear preparations. Both nuclear preparations have been used to prepare purified chromatin which has spectral and chemical properties similar to those reported for chromatin purified directly from several chick tissues.     

Oogenesis: chromatin and microtubule dynamics during meiotic prophase.

Changes in the organization of germinal vesicle chromatin in mouse oocytes have been analyzed by fluorescence microscopy with respect to progressive stages of follicular development and the disposition of oocyte cytoplasmic microtubules. Four discrete patterns of chromatin organization exist in germinal vesicle (GV)-stage oocytes isolated from the ovaries of 21-25-day-old gonadotropin-primed mice. Analysis of ovarian cryosections stained with the DNA-binding fluorochrome Hoechst 33258 indicates that sequential changes in GV chromatin occur during folliculogenesis that result in the formation of a continuous perinucleolar chromatin sheath at the time of antrum formation. Specific alterations in the cytoplasmic microtubule complex of GV-stage oocytes were observed that correlate with chromatin patterns. The extensive cytoplasmic microtubule complex seen in oocytes of preantral follicles initially localizes to perinuclear areas of the ooplasm. This is followed by a progressive reduction in cytoplasmic microtubules and the appearance of prominent microtubule-organizing centers at the nuclear periphery. Coordinated nuclear and microtubular alterations also occur under in vitro conditions prior to progression of meiosis to prometaphase-1. The results are discussed with respect to the ongoing differentiation of the oocyte nucleus and the microtubule cytoskeleton during folliculogenesis in preparation for the resumption of meiosis.     

Usefulness of diagnostic criteria for aspiration cytology of hepatocellular carcinoma.

OBJECTIVE: To establish new criteria for cytodiagnosis of hepatocellular carcinoma by comparing cytologic findings of hepatocellular carcinoma with those of liver cirrhosis. STUDY DESIGN: Review of cytologic findings of hepatocellular carcinoma on preoperative aspiration biopsy of 31 lesions from 27 patients who underwent surgical resection and comparison of these findings with those of liver cirrhosis in 17 patients. RESULTS: In the 11 lesions of moderately and poorly differentiated hepatocellular carcinoma, significant cytologic findings included monotonous and abundant cytoplasm, thick cytoplasm, increased nuclear/cytoplasmic ratio, irregular nuclear contours, increased chromatin density, intranuclear vacuoles and naked nuclei. In the 20 lesions demonstrating well-differentiated hepatocellular carcinoma, significant cytologic findings included monotonous and scant cytoplasm, well-defined cytoplasmic borders, thick cytoplasm, eccentric nuclei, increased nuclear/cytoplasmic ratio, thick nuclear membranes and increased chromatin density. We established the criteria for moderately and poorly differentiated hepatocellular carcinoma as including three cytologic parameters: increased nuclear/cytoplasmic ratio, irregular nuclear contours and increased chromatin density. We also established the criteria for well-differentiated hepatocellular carcinoma as including six cytologic parameters: monotonous cytoplasm, scant cytoplasm, well-defined cytoplasmic borders, thick cytoplasm, eccentric nuclei and increased nuclear/cytoplasmic ratio. For all 31 hepatocellular carcinoma lesions, including 27 lesions that were < or = 2 cm in diameter, both sensitivity and specificity were 100% by concurrently employing both criteria. CONCLUSION: The new criteria for cytodiagnosis we established were useful for differentiating hepatocellular carcinoma from liver cirrhosis. In particular, our criteria ensured appropriate diagnostic accuracy for well-differentiated hepatocellular carcinoma.     

A major 62-kD intranuclear matrix polypeptide is a component of metaphase chromosomes

We have isolated and partially characterized a major intranuclear matrix polypeptide from rat liver. This polypeptide, which is reversibly stabilized into the intranuclear matrix under conditions which promote intermolecular disulfide bond formation, has a Mr of 62,000 and pI of 6.8-7.2 as determined by two-dimensional IEF/SDS-PAGE. A chicken polyclonal antiserum was raised against the polypeptide purified from two-dimensional polyacrylamide gels. Affinity-purified anti-62-kD IgG was prepared and used to immunolocalize this polypeptide in rat liver tissue hepatocytes. In interphase hepatocytes the 62-kD antigen is localized in small, discrete patches within the nucleus consistent with the distribution of chromatin. The staining is most prominent at the nuclear periphery and somewhat less dense in the nuclear interior. Nucleoli and cytoplasm are devoid of staining. During mitosis the 62-kD antigen localizes to the condensed chromosomes with no apparent staining of cytoplasmic areas. The chromosomal staining during mitosis is uniform with no suggestion of the patching seen in interphase nuclei. Fractionation and immunoblotting studies using rat hepatoma tissue culture cells blocked in metaphase with colcemid confirm the chromosomal localization of this 62-kD intranuclear protein during mitosis. The 62-kD polypeptide fractionates completely with metaphase chromosome scaffolds generated by sequential treatment of isolated chromosomes with DNAse I and 1.6 M NaCl, suggesting that this major 62-kD intranuclear protein may be involved in maintaining metaphase chromosomal architecture.     

Repair of O6-ethylguanine DNA lesions in isolated cell nuclei. Presence of the activity in the chromatin proteins

Cell nuclei prepared from rat liver were alkylated in vitro with ethylnitrosourea; the nuclear DNA was found to lose O6-ethylguanine and 7-ethylguanine during a subsequent incubation at 37 degrees C. The rate of O6-ethylguanine loss is comparable to that observed in vivo, indicating that no cytoplasmic component is needed for the repair; no free O6-ethylguanine was found in the incubation medium of the ethylated nuclei. The rate of 7-ethylguanine loss is higher than the spontaneous depurination in vitro and an amount of free 7-ethylguanine equivalent to that lost by the nuclear DNA was found in the incubation medium; these results suggest that this DNA lesion is excised by a DNA glycosylase. The proteins of the chromatin prepared from the isolated nuclei induced the disappearance of O6-ethylguanine from an added ethylated DNA. No free O6-ethylguanine was released indicating that the repair is not catalyzed by a DNA glycosylase; no oligonucleotides enriched in O6-ethylguanine were released either, indicating that the disappearance of O6-ethylguanine from DNA is not the result of the cooperative action of a specific endonuclease and an exonuclease. Activities capable of removing O6-ethylguanine from DNA were found in other cell compartments; most of it, however, is in the nucleus where the main location is chromatin. A pretreatment of the rats with daily low doses of diethylnitrosamine during 3 or 4 weeks increased 2-3-times the repair activity of the chromatin proteins.     

Silver Methenamine Staining for Scanning Electron Microscopy of Bone Sections Containing Biomaterials

Sections of tissue containing orthopedic materials are currently used to study the compatibility of those materials and to perform electron probe microanalysis at the material-tissue interface. Identification of the cells in contact with the material by Scanning electron microscopy (SEM) is of interest. We have developed a method for staining cells and tissue structures embedded in polymethyl methacrylate with silver methenamine once the sections have been obtained. Sections were prepared by grinding, and the silver methenamine was applied after oxidation with periodic acid. The procedure was carried out in a microwave oven. Backscatter SEM showed staining of the cell nucleus membrane, chromatin, the nuclear organizers, and the chromosomes of dividing cells. The cytoplasm and the cytoplasmic membrane were also stained. Collagen fibers of the extracellular matrix and the mineralized matrix of bone were labeled. Material particles in the macrophages were easily recognizable and Energy-Dispersive Spectrometer were not impaired by the presence of silver in the preparation.     

Interactions of alkaline phosphatase and the cell wall of Pseudomonas aeruginosa

Spheroplasts prepared by lysozyme treatment of cells of Pseudomonas aeruginosa, suspended in 20% sucrose or 0.2 m MgCl(2), were examined in detail. Preparation of spheroplasts in the presence of 0.2 m Mg(2+) released periplasmic alkaline phosphatase, whereas preparation in the presence of 20% sucrose did not, even though untreated cells released phosphatase when suspended in sucrose in the absence of lysozyme. Biochemical characterizations of the sucrose-lysozyme preparations indicated that lysozyme mediated a reassociation of the released phosphatase with the spheroplasts. In addition, the enzyme released from whole cells suspended in 20% sucrose (which represents 20 to 40% of the cell-bound phosphatase) reassociates with the cells in the presence of lysozyme. Electron microscopic examinations of various preparations revealed that phosphatase released in sucrose reassociated with the external cell wall layers in the presence of lysozyme, that sucrose-lysozyme prepared spheroplasts did not dissociate phosphatase which remained in the periplasm of sucrose-washed cells, and that phosphatase was never observed to be associated with the cytoplasmic membrane. A model to account for the binding of P. aeruginosa alkaline phosphatase to the internal portion of the tripartite layer of the cell wall rather than to the cytoplasmic membrane or peptidoglycan layer is presented.     

The number of globin gene sequences in "cytoplasmic" DNA fragments.

DNA was isolated from the cytoplasm of primary cultures of mouse foetal liver cells. The proportion of globin genes was determined by two methods of cDNA-DNA hybridisation in globin complementary DNA excess. The proportion was similar in ‘cytoplasmic’ DNA and nuclear DNA. This argues against an informational role for this class of ‘cytoplasmic’ DNA, which has all of the characteristics of nuclear DNA arising from nucleosomes derived from chromatin.     

Formation of the sea urchin male pronucleus in vitro: Membrane-independent chromatin decondensation and nuclear envelope-dependent nuclear swelling

We demonstrate that complete sea urchin male pronuclear development in vitro is a two-step process involving membrane-independent chromatin decondensation and nuclear envelope-dependent pronuclear swelling. In the absence of cytoplasmic membrane vesicles (MVs), permeabilized sperm chromatin decondenses into a spherical nucleus of ≈4 μm in diameter. Pronuclear swelling to ≈7 μm requires an intact nuclear envelope, and the degree of swelling is limited by the amount of MVs assembled on the chromatin. Furthermore, after a nuclear envelope is formed, swelling can occur in the absence of additional cytoplasmic MVs. Nuclear swelling also requires ATP hydrolysis, Ca²⁺ and cytosolic factors, some of which are sensitive to heat and to the sulfhy-dryl alkylating agent, N-ethylmaleimide. The requirement for a nuclear envelope and the rate of pronuclear swelling are consistent with previous in vivo observations. © 1995 wiley-Liss, Inc.