A cytophotometric analysis of the structure of hypothalamic cell populations in the frog,Rana temporaria (L.), with special reference to seasonal changes in chromatin status

Summary We used cytophotometry after the Feulgen reaction and UV cytophotometry to measure the DNA content of quiescent cells of the hypothalamic preoptic region (HPR) of adult and juvenile frogs (Rana temporaria) that had been caught in their natural habitat in winter, spring and summer. The histone-to-DNA ratio in cell nuclei was cytophotometrically determined using a combined Feulgen, heparine and alcian-blue staining procedure. The vast majority of HPR cells studied had nuclei with a diploid DNA content. However, we observed great variability in the Feulgen-DNA content of the HPR cell population, which was not detected in the diploid standard (hepatocytes). This heterogeneity in the diploid sample of the HPR cell populations was always greater in prespawning frogs and may have been due to differences in the chromatin arrangement in nuclei. About 1% of cells had a DNA content either ranging between diploid and tetraploid levels (H2C cells) or at the tetraploid level (4C and 2C x 2 cells). The proportion of these cells was not affected by the age of the animals or the annual cycle, thus suggesting that there is no age-related increase in the mean DNA content in the frog HPR. The mean DNA contents of H2C and 4C cells were much higher than those in the standard (hepatocytes). This cannot be simply attributed to the presence of different amounts of nuclear proteins, but rather indicates that at least a certain proportion of the highest DNA contents may be due to a real extra-DNA synthesis.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Cytophotometric analysis of the cell population composition of the the preoptic area of the hypothalamus in the common frog

DNA contents in squashed cells of the adult frog hypothalamic preoptic region (HPR) were measured using the Feulgen and UV cytophotometry techniques. The histone-DNA ratio in the cell nucleus was determined by means of a combined Feulgen-heparin-Alcian blue staining procedure. The nuclei of the vast majority of HPR cells have a diploid DNA content. However, in cells of this group the mean values of DNA amount and the distribution range were always higher than those in hepatocytes used as a diploid standard. Such a heterogeneity in DNA content in the diploid part of HPR cell population could apparently suggest some differences in the nuclear chromatin arrangement to be always higher in spring before the frog spawning, and it seems to be characteristic of this type of cells. About 1 per cent of cells with hyperdiploid surplus of DNA (H2c cells) as well as of tetraploid cells (4c and 2c X 2 cells) is found in HPR in frogs sacrificed both in winter and in summer. The quota of these cells has no reference either to the frog's age or to the annual cycle. The fact that the mean DNA values in H2c and 4c cells are much higher than in the standard cannot be explained by the presence of different amounts of nuclear proteins only. It is suggested that at least some part of the highest DNA values may be due to an actual extra DNA synthesis in a small constantly existing pool of HPR cell population.     

Autoradiographic study of the proliferative activity of the preoptic recess ependyma in yearling and juvenile common frogs

Proliferative activity of the ependyma, lining the recessus praeopticus in juvenile frogs was studied with 3H-thymidine radioautography. Usually much more pronounced proliferation of ependymal cells occurred in the preoptic region in one year old frogs as compared with two year old ones. It can be concluded that in the former animals the migration of postmitotic labeled cells into the subependymal zone of the recessus preoptic area is significantly more intense. By the 30th day after multiple isotope injections some newly formed neurosecretory cells with labeled nuclei were found in the 1-5 cellular position of the recessus praeopticus subependymal zone. It is postulated that in juvenile frogs the ependyma of lateral wall of recessus praeopticus is probably a source ("cambium"), from which some young neurosecretory cells may originate.     

Determination of DNA surplus in the nuclei of some cerebellar Purkinje cells with different cytochemical methods

Cytofluorometry and cytophotometry (Feulgen staining) as well as ultraviolet cytophotometry revealed similar histograms of DNA content in squashed nuclei of rat cerebellar Purkinje cells. More than 90% diploid (2c) cells and 1% tetraploid (4c) cells were obtained. DNA content in other Purkinje cells ranged between the 2c and 4c level.     

Constancy and variability in the content of DNA in cerebellar Purkinje cell nuclei

Summary A cytophotometric study of DNA content in Purkinje cells of the cerebellum of rats, cats, chicken and humans (Feulgen staining) revealed that in a certain number of cells the amount of DNA ranged between the diploid and tetraploid level (H2C cells). The incidence of H2C Purkinje cells varied among the species studied. In rats, which were studied most thoroughly, these cells amounted on average to 3%. In some rats, as well as in some cats and chickens H2C Purkinje cells were entirely absent. In the group of animals possesing H2C Purkinje cells, great interindividual differences were observed. In rats for instance, the incidence of these cells varied from 1 to 23 per cent. Topographic analyses carried out in rat and human cerebellum revealed that H2C Purkinje cells occurred more frequently in the hemispheres than in the vermis. No significant differences were found in the number of H2C Purkinje cells in healthy and Kilham-DNA-virus infected rats.Densitometric analysis of the distribution of nuclear chromatin showed that H2C Purkinje cells were richer in condensed chromatin, especially in the region of the nucleolus, which apparently contains the hyperploid surplus of DNA. It is proposed that the phenomenon of DNA hyperdiploidy arises as a result of either incomplete S-phase in some immature Purkinje cell precursors or the amplification of some DNA sequences particularly those localized in the nucleolar region.     

Ultrastructural radioautographic analysis of neurogenesis in the hypothalamus of the adult frog,Rana temporaria,with special reference to physiological regeneration of the preoptic nucleus

The localization and fine structure of proliferating cells in the hypothalamic preoptic area were studied by light-and electron-microscopic radioautography 1–2 h following single application of ³H-thymidine to adult Rana temporaria taken from their natural habitat in the spring and autumn. ³H-thymidine uptake by proliferating cells was much more pronounced in frogs caught in May/June, i.e., a month after the breeding period (labeled cells represent about 10% of the total ventricular zone cell population), compared to animals caught in mid-September, when it was very low. In both ³H-thymidine treatment groups the vast majority of labeled cells are found exclusively within the preoptic recess ventricular zone. With regard to ultrastructure, it contained proliferating cells of at least 4 types, ranging from immature forms (bipolar stem cells) to more differentiated elements (tanycyte-like ependymoblasts, classical ependymoblasts). All of them showed label over their nuclei indicating that these cells are capable of DNA synthesis and mitosis. The possible role of the preoptic recess ventricular zone as a source of precursor cells for new peptidergic neurosecretory cells, conventional neurons and glial cells in the hypothalamic preoptic area of the adult frog is discussed.     

Constancy and variability in the content of DNA in cerebellar Purkinje cell nuclei. A cytophotometric study.

A cytophotometric study of DNA content in Purkinje cells of the cerebellum of rats, cats, chicken and humans (Feulgen staining) revealed that in a certain number of cells the amount of NDA ranged between the diploid and tetraploid level (H2C cells). The incidence of H2C Purkinje cells varied among the species studied. In rats, which were studied most thoroughly, these cells amounted on average to 3%. In some rats, as well as in some cats and chickens H2C Purkinje cells were entirely absent. In the group of animals possesing H2C Purkinje cells, great interindividual differences were observed. In rats for instance, the incidence of these cells varied from 1 to 23 per cent. Topographic analyses carried out in rat and human cerebellum revealed that H2C Purkinje cells occurred more frequently in the hemispheres than in the vermis. No significant differences were found in the number of H2C Purkinje cells in healthy and Kilham-DNA-virus infected rats. Densitometric analysis of the distribution of nuclear chromatin showed that H2C Purkinje cells were richer in condensed chromatin, especially in the region of the nucleolus, which apparently contains the hyperploid surplus of DNA. It is proposed that the phenomenon of DNA hyperdiploidy arises as a result of either incomplete S-phase in some immature Purkinje cell precursors or the amplification of some DNA sequences particularly those localized in the nucleolar region.     

DNA synthesis and content in the nuclei of epidermal cells of mice during their differentiation and specialization

Synthesis and content of DNA in the nuclei of differentiating cells of mouse skin epidermis was studied by using cytomorphometric, autoradiographic and cytophotometric methods. It has been shown that the cells of the keratinoid series divide only in the basal layer and contain 2-4c DNA. Keratinocytes of the thorny layer are mostly tetraploid, 2c cells are lacking. H4c and 8c cells comprise 12% of the population. In the keratinocytes of the granular layer DNA content is somewhat lower due to nuclei break down and conversion of cells into anucleate scale. Part of the melanocytes of the basal layer also contain 4c DNA. Highly specialized element of the basal layer Merkel and Langerhans cells are polyploid. Conclusion is drawn that DNA hyper-replication by multiplication of the whole genome is part of the development program of the population.     

Methylation of ribosomal cistrons in diploid and tetraploid Odontophrynus americanus (Amphibia,Anura)

Odontophrynus americanus (Amphibia, Anura) genomic DNA from diploid and tetraploid specimens was treated with restriction enzymes sensitive to cytosine and adenine methylation (5 meC and 6 meA). In both diploids and tetraploids a high proportion of the total DNA was not cleaved by 5 meC-sensitive enzymes as observed on agarose gels stained with ethidium bromide. The DNAs were transferred to nitrocellulose filters and hybridized with cloned fragments containing sequences of Xenopus laevis 28S and 18S ribosomal DNA (rDNA). A high level of methylation of the ribosomal repeat units was revealed by 5 meC-sensitive enzymes in blood, liver, kidney and testis tissues. Adenine was methylated to a lesser degree and similarly in the rDNA from both germinative and somatic tissues. Comparison of the results obtained with DNA of diploids and tetraploids showed that methylation of ribosomal genes was increased in tetraploid genomes of adult frogs, but exact quantitative determinations could not be performed by this methodology. Cloning of the 28S region of the rDNA repeat unit was performed in the gtWESC vector. Restriction patterns obtained with methylation-sensitive enzymes using diploid and tetraploid derived clones confirmed the high level of methylation of the corresponding region of the ribosomal repeat unit in genomic DNAs. The implications of these results in the regulation of expression of the ribosomal genes in diploids and tetraploids are discussed.     

Atypical Mesothelial Cells Associated With Eosinophilic Pleural Effusions: Nuclear Dna Content and Immunocytochemical Staining Reaction With Epithelial Markers

The nuclear DNA contents of atypical mesothelial cells from five patients who had an eosinophilic pleural effusion (EPE) were studied by the use of DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) DNA staining. Analysis of the nuclear DNA content revealed a polyploid pattern, with a major peak in the tetraploid region. Using an immunocytochemical technique, the atypical mesothelial cells showed a positive reaction for cytokeratin. In contrast carcinoembryonic antigen (CEA) was always negative in these cells. It is suggested that the atypical mesothelial cells with EPE had a higher rate of proliferation than did the normal mesothelial cells.     

DNA profiles in dysplasia and carcinoma of the human esophagus

The nuclear DNA content was microspectrophotometrically measured in 16 resected esophagi having dysplasia, carcinoma in situ and/or early invasive squamous carcinoma. First, the epithelial thickness in (1) normal squamous esophageal epithelium and (2) dysplasia-carcinoma in situ areas was divided into three equal compartments (i.e., basal-parabasal, intermediate and superficial) in five cases. In the normal epithelium, while some of the nuclei in basal-parabasal normal squamous cells had elevated DNA values (corresponding to the natural DNA replication in these cells), intermediate and superficial (nonreplicating) normal squamous cells showed a more definite clustering about the 2c value. In the nonnormal epithelium, the percentage of cells with DNA levels exceeding the normal tetraploid value was highest for the intermediate zone. Therefore, in all 16 cases, normal intermediate cells were measured as internal controls, against which the DNA levels of cells in the intermediate compartment in the areas of dysplasia and/or carcinoma in situ were compared. In areas of dysplasia, two different DNA patterns were observed: one clustering around the normal diploid region and the other with aneuploid values. While the former corresponded to some of the lesions considered by conventional histologic examination to be slight and moderate dysplasias, the aneuploid pattern corresponded to the remaining slight and moderate dysplasias as well as to the severe dysplasias. The possibility that "diploid dysplasias" are reactive (i.e., nonneoplastic) lesions due to chronic inflammation or are "dormant" nonprogressive dysplasias, while aneuploid dysplasias are more aggressive lesions, seems to be substantiated by the fact that all areas with carcinoma in situ or with microinvasive squamous carcinoma had aneuploid nuclei.     

Studies of nuclei separated by zonal centrifugation from liver of rats treated with thioacetamide

1. The effects of the inclusion of thioacetamide in the diet on the properties of rat liver nuclei were studied both in adolescent rats, in which the parenchymal cells contain diploid nuclei, and in young adult rats, with a high proportion of tetraploid nuclei. 2. These investigations included a survey of the sedimentation properties of the nuclei, the nuclear volumes, content of DNA, RNA and protein, the incorporation in vivo of [(3)H]thymidine into DNA and [(14)C]orotate into RNA, and measurements of the activity of RNA polymerase and ribonuclease. These studies were conducted on nuclei fractionated by zonal centrifugation. 3. In both groups of animals, exposure to thioacetamide produced large numbers of nuclei that were abnormal in their chemical composition and enzymic activity. The changes were complex as regards both the types of nuclei that were affected and in their variation with time. 4. In adolescent rats two waves of synthesis of DNA and RNA were observed, one at 3 days and the other after 2 weeks of treatment. The first decline in the incorporations into both DNA and RNA coincided with a decrease in the pool sizes of some of the precursors. The activity of RNA polymerase was not substantially altered. A marked increase in the content of protein was observed before the first wave of synthesis. The normal progressive increase in tetraploid nuclei was prevented. 5. In young adult rats two waves of DNA synthesis were detected. Each was preceded by a large increase in the amount of protein per nucleus but was not accompanied by increased RNA synthesis. After 4 weeks of treatment, the diploid stromal nuclei appeared mainly unaffected and large numbers of tetraploid nuclei with a greatly increased quantity of protein were observed.     

不同倍性鱼的血细胞和精子DNA含量比较

我们以前的研究表明, 以红鲫 (2n=100) 为母本及湘江野鲤 (2n=100) 为父本的杂交后代的F1-F2 为二倍体 (2n= 100)。在二倍体 F2 个体中, 存在能分别产生二倍体卵子和二倍体精子的雌、雄个体, 二倍体卵子和二倍体精子结合, 形成了两性可育的四倍体鱼 (F3)。目前四倍体鲫鲤已连续繁殖了 12 代 (F3-F14), 形成了一个遗传性状稳定的四倍体鱼群体 (4n= 200) (Liu et al.,2001; 孙远东等, 2003)。雌性四倍体鲫鲤产生的二倍体卵子经紫外线照射的散鳞镜鲤精子激活后,无须染色体加倍处理, 可发育为全雌性二倍体雌核发育后代 (G1) (2n=10…     

Changes in pancreatic acinar cell nuclear number and DNA content during aging in the rat

Pancreatic acinar cells from rats 5 to 658 days (94 weeks) of age were isolated by enzymatic dissociation and stained with the DNA specific fluorochrome Hoechst 33258. The nuclear DNA content and the incidence of binucleation were estimated in these cells. Total pancreatic weight, RNA, protein and DNA, and the incorporation of 3H-thymidine into pancreatic acinar cell DNA were also estimated in similar animals as measures of pancreatic growth. From 5 to 17 days after birth, 95% of the cells were mononucleate diploid and 5% were binucleate diploid; but during the period of rapid pancreatic growth over the following 39 days, acinar cells became increasingly binucleate. By 56 days after birth, 64% of cells were binucleate with a diploid DNA content per nucleus; and the incidence of binucleation then remained constant. At 28 days of age, 4% of mononucleate cells were tetraploid, increasing to 6% at 658 days of age. At this time 3% of binucleate cells contained dual tetraploid nuclei. There is thus a rapid development towards diploid binucleate acinar cells in the growing, postnatal pancreas; and in the adult pancreas a small proportion of these cells develop tetraploid nuclei.     

Ploidy distribution and DNA content variations of <Emphasis Type="Italic">Lonicera caerulea</Emphasis> (Caprifoliaceae) in Japan

Ploidy level and geographical distribution were investigated in Japanese Lonicera caerulea L. Flow cytometric analysis revealed the presence of DNA diploid and DNA tetraploid plants in Japan. Chromosome observation confirmed that diploid and tetraploid plants showed 2n = 2x = 18 and 2n = 4x = 36, respectively. The DNA diploid populations were found only in lowland mires, Betsukai, Bekanbeushi, Kushiro and Kiritappu located in eastern Hokkaido. On the other hand, DNA tetraploid populations were distributed in a wide area of Hokkaido, and mainland of Japan. The habitats of DNA tetraploid plants were lowland to alpine region. The DNA content measurement with flow cytometry revealed significant differences in the relative DNA contents among DNA tetraploid populations. The relative DNA content within DNA tetraploid populations varied 1.157-fold at maximum, and might correlate with altitude indicating that DNA contents were smaller as altitude increases. The wide area of distribution in various environments of DNA tetraploid plants suggested the adaptability of the tetraploid plants. Although diploid and tetraploid populations were found, no triploid was detected, indicating crossing difficulty between diploid and tetraploid as confirmed by crossing experiment.     

Nuclear DNA in histologic sections from squamous-cell carcinoma and adenocarcinoma of the lung

The nuclear DNA content in morphologically identified tumor cells was analyzed in 4-micron histologic sections from 58 patients with lung carcinoma who survived for at least five years. Thirty-three of the carcinomas were invasive squamous bronchial carcinomas and 25 were pulmonary adenocarcinomas. In all squamous carcinomas, the majority of tumor cells were found to exhibit DNA values exceeding the normal tetraploid and/or diploid region. In contrast, some of the pulmonary adenocarcinomas were found to be composed of a majority of tumor cells with DNA values in the normal diploid region. The results indicate that invasive squamous bronchial carcinomas, in general, are tumors with aneuploid DNA patterns indicative of a high malignant potential and that malignancy grading based on DNA measurements does not add any significant prognostic information to that obtained by morphologic diagnosis.     

Mitotic division of differentiated neurosecretory cells in the hypothalamus of adult amphibians

By means of multiple 3H-thymidine administration to adult frogs, a direct electron microscopic radioautographic evidence is first by presented regarding the possibility of an in vivo mitotic division of differentiated hypothalamic peptidergic neurosecretory cells. The absence of any signs of cytoplasmic dedifferentiation, as well as a poor development of spindle microtubules in this cell may be suggestive of a polyploidizing, possibly acytokinetic mitotic process occurring in such kind of cells.     

Accessory nuclei of the primary erythroid cells of chick embryos: the kinetics of their formation and the relationship to the synthesis of extra DNA

Kinetics of formation of accessory nuclei (AN), synthesis and accumulation of DNA in AN and main nuclei (MN) of the chick embryo primary erythroid cells (PEC) (2-7 days of development) and interdependence of these processes were studied using cytomorphology, autoradiography and differential cytophotometry. The earlier data on the existence of AN in primary erythrocytes (EC) were confirmed, and it was shown for the first time that the formation of AN accompanied the primary erythropoiesis from the very beginning of the proerythroblast (PEB) stage. The number of AN-bearing cells increases as erythropoiesis proceeds to reach the value of 50% PEC. The AN are formed in diploid, tetraploid, as well as in binuclear cells. The DNA content in AN may vary from 1-2 to 80% of the 2c value and has a tendency of increasing during erythropoiesis, while that in MN is always 2c or multiple thereof. Formed at early stages of PEC proliferation as pieces of segregated Feulgen-positive material, the AN are preserved during mitosis, taking no part in mitotic rearrangements of the chromosomes and being eventually involved in one of the daughter cells. Clusters of metaphase, anaphase and telophase chromosomes contain strictly 4c or 2c + 2c DNA. DNA synthesis in MN and AN may proceed in parallel and independently. It is frequently observed in AN of terminally differentiated PEC-reticulocytes (RC) and EC, whose MN no longer incorporate 3H-thymidine.(ABSTRACT TRUNCATED AT 250 WORDS)     

The hypogonadotropic state of the prepubertal male rhesus monkey (Macaca mulatta) is not associated with a decrease in hypothalamic gonadotropin-releasing hormone content

Hypothalamic contents of gonadotropin-releasing hormone (GnRH) in neonatally orchidectomized infant, juvenile, and adult monkeys were measured by a radioimmunoassay (RIA) and by an in vivo bioassay that utilized luteinizing hormone (LH) secretion in estrogen- and progesterone-treated ovariectomized rats. The results of the bioassay provided no evidence to suggest that hypothalamic GnRH content in juvenile monkeys (mean = 83 ng/hypothalamus; n = 3) was less than that in infants (mean = 54 ng/hypothalamus; n = 4) and adults (mean = 36 ng/hypothalamus; n = 3). A similar developmental pattern in hypothalamic GnRH content was also observed when the decapeptide was measured by RIA. In striking contrast to the maintenance of hypothalamic GnRH content throughout postnatal development, pituitary gonadotropin contents and serum gonadotropin concentrations were markedly reduced in juvenile monkeys.     

Demographic dynamics of the afro-tropical pig-nosed frog, <Emphasis Type="Italic">Hemisus marmoratus</Emphasis>: effects of climate and predation on survival and recruitment

We studied the population ecology of the West African pig-nosed frog, Hemisus marmoratus, to understand the relative contributions of adult survival and recruitment to population growth rate in savannah frogs using mark-recapture modelling. We marked a total of 821 adult frogs over 6 years and recaptured 74 at least once between years. Between-year adult survival was sex-specific and varied between 0.06 and 0.53 for males and 0.07–0.41 for females. Adult survival was significantly associated with annual rainfall and is cause for concern if rainfall declines further in the study region as predicted by changes in the global climate. There was a significant interaction between rainfall and sex with dry weather having a stronger negative effect on males than females. Pig-nosed frogs experienced boom and bust years with a single decline more dramatic than increases. Recruitment (in situ and immigration; 0.67–0.88) was substantially more important than adult survival (0.12–0.33) in determining realised population growth. In situ recruitment was highly variable between years with 1–36% of eggs and tadpoles released by females into the pond surviving to metamorphosis. Years of low tadpole survival were associated with high numbers of predatory tortoises. Thus, like other pond-breeding anurans, pig-nosed frogs showed highly variable juvenile recruitment, low adult survival and density-independent effects on population growth by predators and weather.