An ultrastructural investigation of the early stages of oocyte differentiation in Actinia fragacea (Cnidaria; Anthozoa)

Individuals from a population of the intertidal sea anemone Actinia fragacea (Tugwell) were collected at approximately monthly intervals over an 18 month period. Samples of gonad were removed from each anemone and examined by light and electron microscopy. During late spring and early summer, large numbers of small cells were seen in the endoderm of the female gonads, lying close to the mesoglea. For convenience, these cells were classified into three types. Type I cells are 6–9 μm in diameter, with relatively very large nuclei, which may contain synaptinemal complexes, and scant cytoplasm containing few organelles. Type II cells are larger, reaching 15 μ m in diameter, with more abundant cytoplasm containing more organelles and inclusions. The nucleus is more dense, but may also contain synaptinemal complexes. Type III cells are less common. They are similar in size to Type II cells, but their nuclei contain irregular dense chromatin masses, and the nuclear envelope is incomplete or absent. The possible significance of the various cell types is discussed. It is suggested that Type I cells are oocytes at a very early stage of differentiation and that Type II cells are rather later oocytes. The status of the Type III cells is uncertain.     

The ultrastructure of the nuclei and the behaviour of the sex chromosomes of human spermatogonia

Summary The nuclear structure of human spermatogonia has been studied with electron microscopical and histochemical methods. Type B spermatogonia have chromatin clumps without any special ultrastructure and several nucleoli. Five different types of nuclear bodies, and besides, a nuclear vacuole, have been observed in type A spermatogonia. Type I bodies are typical nucleoli consisting of three regions: amorphous, fibrillar and granular. Type II, III and V are considered to be atypical nucleoli. Type IV bodies are small chromatin condensations. Type I bodies are the only ones in which RNA was demonstrated by light histochemical techniques and no PAS positive material was found inside the nuclei. The absence of any special ultrastructure in the chromatin from spermatogonia, and the small mass of the chromatin condensations, show that the human X chromosome and perhaps the Y chromosome are not heteropycnotic in the interphasic nuclei of human spermatogonia.Abbreviations Used RNA ribonucleic acid - gonia spermatogonia This work has been supported by a grant (No. 2623) of the Consejo Nacional de Investigaciones Cientificas y Tecnicas, and partially by a grant (C.M. 6522) from the Population Council.We wish to thank Professor R. E. Mancini for his suggestions during this investigation and his support for its achievement, and to Dr. J. C. Lavieri for providing the biopsies.     

Residual trophoblastic tissue as a source of highly atypical cells in the postpartum cervicovaginal smear

A cervicovaginal smear containing atypical cells, which were interpreted as dysplastic cells, was obtained from a women one-year postpartum. These cells were seen singly, in small groups and in clusters embedded in an amorphous pink matrix. They had amphophilic cytoplasm and increased nuclear/cytoplasmic ratios, as well as hyperchromatic nuclei with variably prominent nucleoli, features that are characteristic of trophoblastic cells. No evidence of dysplasia was seen on subsequent colposcopic examination or cervical biopsy. Endocervical curettage yielded fragments of exfoliated endometrium and residual trophoblastic tissue associated with a placental implantation site. Although involution of the placental site is generally complete by six to seven weeks postpartum, maternal-fetal tissue may in fact continue to be exfoliated for several months or longer after delivery. If seen on a cervicovaginal smear, these cells can be highly atypical and may be mistaken as dysplastic or malignant. The cytologic features that characterize trophoblasts and their persistence in postpartum cervicovaginal smears are discussed.     

Seasonal changes in the structure and secretory activity of the androgenic gland of Travancoriana schirnerae

This study investigated the seasonal variation in the structure and secretory activity of the androgenic gland (AG) in the freshwater crab: Travancoriana schirnerae. The androgenic gland is an elongate structure, attached to one side on the wall of the ejaculatory duct. Histological studies showed the presence of three cell types, which differ in size, shape of nuclei, and presence or absence of secretory vesicles. Type I cells are small with large nuclei whereas type II cells are large with small nuclei. Type III cells are intermediate in size and exhibited streak-like nuclei and transparent cytoplasm. Seasonal changes were discerned in the morphology, histology and secretory activity of the gland. March-June appeared to be the active season with type II cells containing secretory vesicles. The mode of release of secretion found to be holocrine. The secretory activity almost completed by July-August (the mating season) with vacuolization of type II cells. The gland remained inactive from September-December with abundance of vacuoles, scattered pycnotic nuclei, indistinct cell membranes and total cellular degeneration. January-February was the revival period with type I cell proliferation. The present study revealed that the secretory activity of the gland is in tune with the male reproductive cycle.     

Cytomorphology of hyaline-vascular Castleman''s disease: a diagnostic challenge

OBJECTIVE: Hyaline-vascular Castleman's disease (CD) is difficult to diagnose on fine needle aspiration and may be mistaken to be a lymphoreticular malignancy because of the presence of large cells having nuclei showing atypical features. The cytomorphological findings in three histopathologically documented cases of hyaline-vascular CD were evaluated to a set of cytomorphological criteria which could help in the identification of this condition on aspirate smears. METHODS: The Papanicolaou and Diff-Quik stained smears from three cases of histologically documented hyaline-vascular CD were reviewed by one author. After review the following cytomorphological criteria were suggested to be indicators of the lesion. (i) The presence of large oval to round cells having ill-defined cytoplasmic margins and large nuclei with irregular nuclear outlines having fine or coarse chromatin giving a crumpled tissue paper appearance. (ii) A polymorphous population of lymphoid cells predominantly of small lymphocytes in the background. The smears from these three cases were then mixed with smears from four cases of reactive lymphoid hyperplasia and three cases of Hodgkin's lymphoma. These ten cases were blindly evaluated by two other cytopathologists in order to evaluate the utility of the proposed criteria in identifying CD. RESULTS: The cytomorphological criteria seen in the methodology section were present in all the cases. These features were helpful in distinguishing CD from reactive lymphoid hyperplasias and Hodgkin's Lymphomas in all cases except one case. CONCLUSION: Although hyaline-vascular CD is a difficult diagnostic entity on aspirate material the presence of large histiocytic cells with a crumpled tissue paper appearance of the nuclei in a background of small lymphocytes are useful indicators for suspecting this lesion. However, these findings should be analysed in larger studies to determine if they could in anyway reduce the diagnostic dilemma in cases of CD.     

Relationship between computerized morphonuclear image analysis and histopathologic grading of breast cancer

A pilot study analyzed the relationship between several morphonuclear parameters and the Bloom-Richardson score for 37 invasive, not-otherwise-specified (NOS) ductal breast carcinomas. The SAM-BA 200 cell image processor and its software were used to measure the nuclear features on Feulgen-stained imprint smears. Two parameters representing the numbers of large and dense chromatin clots and two parameters describing the heterogeneity of the chromatin among nuclei in a specimen evolved in a continuous manner parallel with the Bloom-Richardson score from stages NOS-4 to NOS-8. The systematic measurement of these four parameters on a large series of breast cancers may be able to define an objective and reproducible "scale" of differentiation that could be a helpful tool for pathologists and clinicians.     

Standardization of the Feulgen reaction: the influence of chromatin condensation on the kinetics of acid hydrolysis.

The purpose of the present study was to investigate the influence of chromatin compactness on the kinetics of acid hydrolysis in the Feulgen reaction in cytology. Tissue imprints of rabbit liver, of human bronchial carcinoma and of human blood smears, fixed with alcohol, formaldehyde or with B?hm's solution with and without prior air drying, were stained with a standardized pararosanilin-Feulgen reagent. The time for hydrolysis varied between 7.5 and 120 min. The integrated optical density (IOD) of the cell nuclei was measured with an image analyzer (IBAS 2000). Cells with condensed chromatin (lymphocytes, small cell carcinoma, formaldehyde fixed cells) showed a slow increase of staining intensity and late plateau phase as compared with cells with decondensed chromatin. DNA in condensed nuclei was less susceptible to acid hydrolysis. The degree of chromatin compactness which determines the sensitivity of DNA to hydrolysis is influenced by the type of fixation, cell type and by the functional status of the cell. The conclusion is that Feulgen staining intensities of cells with different degrees of chromatin compactness cannot be compared unless measured in the respective plateau phases of the relevant hydrolysis curves which must be determined individually for each cell type.     

Fine needle aspiration of the liver. Significance of hepatocytic naked nuclei in the diagnosis of hepatocellular carcinoma

Fine needle aspiration (FNA) smears from 60 cases of histologically (34) or clinically (26) confirmed hepatocellular carcinomas were reviewed. In about 90% of the cases, the cytologic preparations contained clusters of malignant cells with variable degrees of hepatocytic features and a distinctive type of naked nuclei. These naked nuclei had features similar to those of the malignant hepatocytes, but with more evident atypia. They were numerous in about 75% of the cases and less frequent in 15%; in three cases, they were practically absent. All three cases of well-differentiated hepatocellular carcinomas presented with numerous naked nuclei with slight atypia. Flowerlike-configured naked nuclei were especially found in poorly differentiated hepatocellular tumors. Hepatocytic naked nuclei were rarely found in FNA smears from normal liver, metastases, cysts or degenerative, regenerative and inflammatory liver processes; when seen in these cases, they showed no atypia. The presence of atypical hepatocytic naked nuclei appears to be a very useful criterion for the diagnosis of hepatocellular carcinoma.     

Synapsoarchitectonics of the septum of the cat brain

In the medial and lateral septal nuclei, 4 types of axonal terminals are distinguished. Type I contains spherical vesicles and forms asymmetric synapses on small and middle stems and spines of the dendrites; type I terminals comprise 63% in the medial nucleus of the total number of axons, and in the lateral one--52%. Type II contains polymorphic vesicles and forms symmetrical synapses on the soma and large dendrites. In the medial nucleus they comprise 6%, and in the lateral one--3%. Type III contains either clear spherical (IIIa), or polymorphic (IIIb) vesicles, as well as 1-2 vesicles with a dense core. They form axodendritic, axospine and axosomatic synapses. In the medial nucleus they comprise 25% and 3%, respectively, in the lateral one--40% and 2%. Type IV contains a great number of vesicles with a dense core. These terminals in both septal nuclei comprise 3% and do not participate in formation of active contacts.     

Innate apoptosis of human B lymphoblasts transformed by Epstein-Barr virus: modulation by cellular immortalization and senescence

B-lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus have a phenotype corresponding to activated B-lymphoblasts. Although they are widely used as models in various biological and medical studies, their innate morphological differentiation and apoptosis has been little studied. We report here that a large proportion of LCL cells spontaneously differentiate into smaller lymphoid cells which ultimately undergo apoptosis during conventional cell culture. Two distinct types of apoptosis with some intermediate types exist: type 1 apoptosis in small and medium-size cells with shrunken nuclei having heavily condensed chromatin in the whole nucleus region accompanied by relatively large internucleosomally fragmented DNA (above 2 kbp); type 2 apoptosis in large lymphoblasts with extremely lobulated nuclei having chromatin condensation beneath the nuclear membrane alone accompanied by smaller internucleosomally fragmented DNA (below 2 kbp). Type 1 apoptotic cells were far more numerous than type 2 apoptotic cells. The incidence of type 1 apoptosis was suppressed by cellular immortalization and was extremely stimulated at the end of the lifespan (crisis). These results provide essential information for us to use LCLs for various biological and medical studies including cellular immortalization, tumorigenesis and senescence.     

DNA constancy and chromatin structure in some cell nuclei ofAmphiuma

Synopsis In a series of microspectrophotometric and microphotometric investigations, it has been found that in lymphocytes of the primitive amphibianAmphiuma the very large amount of Feulgen-DNA per nucleus (that is, the amount of DNA revealed by a Feulgen hydrolysis-Schiff technique) is constant within the limits of the measuring error. The Feulgen hydrolysis time had to be reduced considerably in order to bring down the extinctions (optical densities) of these very voluminous and densely staining nuclei. Off-peak absorption of cells in smears stained in the usual way with the Feulgen-Schiff method appeared to be of no value in these experiments. The relation between the Feulgen-DNA content of lymphocyte nuclei of human andAmphiuma cells, as determined from the slope of the hydrolysis curve, appeared to be around 124, which fits well with biochemical data from the literature.Cytologically, a large part of the chromatin appeared to exist in large clumps of heterochromatin. In marginated plaques of condensed chromatin, local areas of lower density occur and are in close association with nuclear pores. The dense lamina is often very pronounced in these nuclei.     

Stereo-specific substrate recognition by phosphatidylinositol phosphate kinases is swapped by changing a single amino acid residue.

Type I and type II phosphatidylinositol phosphate (PIP) kinases generate the lipid second messenger phosphatidylinositol (PtdIns) 4,5-bisphosphate and thus play fundamental roles in the regulation of many cellular processes. Although the two kinase families are highly homologous, they phosphorylate distinct substrates and are functionally non-redundant. Type I PIP kinases phosphorylate PtdIns 4-phosphate at the D-5 hydroxyl group and are consequently PtdIns 4-phosphate 5-kinases. By contrast, type II PIP kinases are PtdIns 5-phosphate 4-kinases that phosphorylate PtdIns 5-phosphate at the D-4 position. Type I PIP kinases, in addition, also phosphorylate other phosphoinositides in vitro and in vivo and thus have the potential to generate multiple lipid second messengers. To understand how these enzymes differentiate between stereoisomeric substrates, we used a site-directed mutagenesis approach. We show that a single amino acid substitution in the activation loop, A381E in IIbeta and the corresponding mutation E362A in Ibeta, is sufficient to swap substrate specificity between these PIP kinases. In addition to its role in substrate specificity, the type I activation loop is also key in subcellular targeting. The Ibeta(E362A) mutant and other mutants with reduced PtdIns 4-phosphate binding affinity were largely cytosolic when expressed in mammalian cells in contrast to wild-type Ibeta which targets to the plasma membrane. These results clearly establish the role of the activation loop in determining both signaling specificity and plasma membrane targeting of type I PIP kinases.     

AluI-resistant chromatin of chromosome 18: classification,frequencies and implications

The pericentric chromatin of chromosome 18 was found to be far more heterogeneous for restriction endonuclease AluI (5 ··· AG CT···3) than previously thought. The extent of such heterogeneity was characterized using 50 normal Caucasians and 5 cases of trisomy 18 or Edwards' Syndrome. The AluI-resistant chromatin can arbitrarily be classified into at least five sizes by comparison with the length of the short arm (p) of chromosome 18. They are: negative (1), small (2), medium (3), large (4) and very large (5) with incidences of 11.30%, 19.13%, 29.57%, 29.57% and 10.43%, respectively. In addition the location of the chromatin can be classified into four types depending upon the position relative to the primary constriction. For example: Type I (absent); Type II (present on p arm only); Type III (present on q arm only); Type IV (present on centromere and extending into both p and q arms). The incidences of types I, II, III, and IV were 11.30%, 62.61%, 0.87%, and 25.22%, respectively. Based on limited data, AluI-resistant chromatin was found to be predominantly large and very large in Edwards' Syndrome samples. In addition, no case with negative Alu-resistant chromatin was noted. Therefore, it is tempting to speculate that the amount of chromatin present on the centromere might play a role in non-disjunction in Edwards' Syndrome cases. Although the variation observed in the present study is continuous, the proposed classification has some important implications for future investigations.     

A whole range of fine structural criteria for immunohistochemically identified LH cells in rats

Summary Pituitaries from normal, young and adult male rats were fixed either in sublimate-formalin or in glutaraldehyde-osmium. In adjacent Paraplast sections, almost all the gonadotrophs were immunostained with both LH and FSH antisera. The rat LH and FSH antisera used were shown to be highly specific by the absorption test and by double antibody radioimmunoassay. Thin and thick adjacent Epon sections were prepared for EM and immunohistochemical examination. Cells stained with the rat LH antiserum were identified by LM, and then observed in detail by EM. On the basis of these observations we suggest that the LH cells are arranged in a sequence of basophils, i.e., Types II/III, III, III/IV and IV: Type II/III basophils are elongate with a cytoplasmic process and less vesiculated. They have morphological features of Type II (classical thyrotrophs) and also of Type III basophils. Type III basophils are oval in shape and moderately vesiculated. Both Types II/III and III basophils can be divided into two classes of cell characterized mainly by the existence of only small secretory granules (150–220 nm in diameter) (Type A) or by the coexistence of small and large (350–500 nm) (Type B). Type III/IV basophils are cells intermediate between types III and IV basophils, and moderately vesiculated with an abundance of secretory granules (150–300 nm in diameter). Type IV basophils are large, spherical or oval cells whose RER cisternae are conspicuously dilated; they contain less numerous secretory granules (150–300 nm in diameter). It is concluded that LH cells are not a single cell type, but include a wide range of subtypes.     

A study on the different types of spermatogonia in buffalo (Bubalus bubalis).

As a first step to understanding spermatogenesis in the buffalo bull the cytological details of different types of spermatogonia were determined in adult buffalo bulls. Morphological changes in the nuclear details were used as a basis for classifying the different types of spermatogonia. The type A spermatogonia had a spherical to ovoid nucleus with finely granulated chromatin, homogeneously dispersed in the nucleoplasm and having one to two nucleoli adhering to the nuclear membrane. The type A0 spermatogonia were characterized by nuclei containing moderately stained, finely granulated chromatin and a nucleolus attached to the nuclear envelope. The A1 type spermatogonia, on the other hand, have pale stained, finely granulated chromatin with the nucleolus adhering to the nuclear membrane. The nuclei of A2 type spermatogonia resembled those of type A1, but contained coarse granular chromatin dispersed in the pale nucleoplasm. The intermediate type of spermatogonia acquired a central position of the nucleolus, but the chromatin remained coarsely granulated and non-clumped. Three classes of type B (B1-B3) spermatogonia were determined on the degree of clumping of the chromatin and the central position of the nucleolus. The type B1 cells were characterized by nuclei containing a few flakes of lightly stained chromatin and a centrally located nucleolus. The type B2 cells showed comparatively more clumping of chromatin than type B1 spermatogonia, which was dispersed at random in the pale nucleoplasm and along the nuclear envelope. The type B3 spermatogonia demonstrated chromophilic chromatin dispersed in the slightly grey nucleoplasm and adhering along the nuclear membrane. Since there seems to be a succession of events following differentiation of type A1 spermatogonia till the last type B cell differentiates into resting primary spermatocytes, may intermediate stages between the presently described classes of type A (A0-A2) and type B (B1-B3) could also be located in sections of the seminiferous tubules.     

Nuclear DNA content, cytomorphologic features and clinical characteristics of small cell carcinoma of the lung

The relationship between the cytomorphologic features, the nuclear DNA patterns and the clinical prognosis of patients with small cell carcinoma of the lung was studied. In cases in the long-survival group (greater than or equal to 24 months), bronchial brushing smears contained a relatively high frequency of nuclei with large, irregular shapes and finely granular chromatin patterns, in comparison with patients in the short-survival group (less than or equal to 9 months); the correlation was not statistically significant, however. The incidence of cells with round or oval nuclei and finely granular chromatin patterns was higher in patients whose cells had hyperdiploid DNA patterns than for patients whose cells had near-diploid patterns; again, the difference was not statistically significant. Patients whose tumor cells had hyperdiploid DNA patterns had significantly shorter survival times than did patients whose tumor cells had near-diploid patterns. These results indicate that (1) judging the nuclear DNA pattern from the cytomorphologic features of small cell carcinoma is unreliable and (2) the nuclear DNA patterns are related to the clinical prognosis of patients with small cell carcinoma of the lung.     

Analysis of nuclear chromatin distribution in cervical glandular abnormalities

OBJECTIVE: To measure the intensity of hematoxylin staining for the analysis of chromatin distribution and to define a clear set of standards. STUDY DESIGN: Cervical smears obtained from 12 patients with glandular lesions, 5 with squamous lesions and 3 without cervical lesions were used for NIH image analysis (public domain NIH image program developed at the U.S. National Institute of Health, available through the Internet by anonymous ftp from zippy.nimh.nih.gov or on floppy disk from the National Technical Information Service, Springfield, Virginia). In addition, the same cervical smears were restained with propidium iodide, and the DNA content in the nuclei was compared with that with hematoxylin staining. RESULTS: Chromatin distribution was categorized into 3 patterns. Pattern A was that for which the highest staining density was localized in the periphery of the nucleus, while in pattern C it was localized in the center of the nucleus. Pattern B was the intermediate type between patterns A and C. In patients with adenocarcinoma, pattern B was predominant; pattern C was also relatively frequent in this group. In atypical glandular cells observed in patients with squamous lesions, patterns A and B were predominant and pattern C rarely seen. Analysis of DNA content in the nucleus revealed that nuclei showing pattern B contained significantly higher quantities of DNA than those showing pattern A. CONCLUSION: Nuclear chromatin distribution seems to correlate well with DNA content, and analysis of the chromatin distribution pattern is helpful for the diagnosis of cervical glandular neoplasia.     

Influence of smear preparation and fixatives on the DNA ploidy and the morphonuclear features of the MXT mammary tumor and normal tissues in the mouse

We compared cytomorphonuclear parameters--related to DNA histogram and chromatin distribution--on MXT mouse mammary tumor and murine normal cells from fresh squash smears or from deparaffinized tissue smears fixed in several fluids. We used the SAMBA 200 cell image processor with software allowing for the discrimination of parameters computed on Feulgen-stained nuclei. The spectrophotometric results--assessed by integrated optical density values--indicate that the nuclei from deparaffinized tissue fixed in Bouin's fluid are around 50% less stained than those fixed in formalin or ethanol-formalin-acetic acid (EFA). The fresh smears of nuclei fixed in formalin contain a less well-defined and more homogeneous chromatin than after Bouin's or EFA fixation. This has led to the conclusion that morphonuclear parameter comparisons performed on tissues differently processed or from different origins present severe limitations.     

Comparative cytologic features of adenocarcinoma in situ of the uterine cervix

The cytologic findings in 30 cases of adenocarcinoma in situ (AIS) and related lesions of the cervix were compared with those in 13 cases of cervical invasive adenocarcinoma and 8 cases of cervical nonneoplastic conditions that mimicked AIS cytologically. Although there was considerable overlap, the presence of large cells with irregular nuclei and uneven chromatin distribution in smears containing no normal endocervical cells helped to distinguish invasive adenocarcinoma from AIS. The presence of "feathering," rosettes, mitotic figures and very crowded nuclei with scant cytoplasm and without cilia helped distinguish AIS from benign conditions.