Differences In Nuclear Volume Research Articles

DATA DRIVEN AND CELL SPECIFIC DETERMINATION OF NUCLEI-ASSOCIATED ACTIN STRUCTURE

In the specialized field of cellular mechanosignaling, the intricate relationship between the cytoskeleton and nucleus is crucial for determining the behavior and fate of mesenchymal stem cells (MSCs). Existing analytical methods for filamentous actin fibers (F-actin) suffer from issues of accuracy and reproducibility. To address this, our research introduces a significant advancement in image analysis by utilizing two deep-learning segmentation Convolutional Neural Networks (CNNs) based on U-Net architecture. This algorithm precisely quantifies F-actin and offers reproducible data from 3D confocal microscopy images. Our CNNs automatically extract the 3D shape of both the nucleus and individual actin fibers, thereby enabling the geometric reconstruction of these critical cellular components. We applied this methodology to MSCs exposed to low-intensity vibration, and hypothesized that mechanical stimuli will induce perinuclear actin remodeling. Confocal Z-stack images of MSCs were captured using a 40x oil lens and a Zeiss LSM 900 microscope. Nucleus and F-actin structures were stained with Hoechst 33342 and Phalloidin, respectively. Nucleus-based images were employed to mask and isolate actin fibers within the nuclear vicinity. A specialized deep-learning segmentation model was engineered within the PyTorch framework. For model training, a selective 2% of nuclei and F-actin images were annotated. Specifically, three layers from each of six representative nuclei, among approximately 500 cross-sectional layers, were meticulously selected; a similar approach was employed for actin. The model underwent 200 epochs of training. Post-training, the deep-learning model was employed to process all collected cross-sectional images of F-actin and nuclei. A subsequent analytical step involved the scrutiny of intersecting segmented fibers between consecutive layers for geometrically accurate actin fiber reconstruction. We analyzed 102 control and 110 vibration-treated mesenchymal stem cells (MSCs) by utilizing our deep-learning-based reconstruction algorithm on confocal Z-stack images. Our data revealed an 8% decrease in nuclear height from 5.73 µm to 5.28 µm (p < 0.001) upon application of low-intensity vibration. In contrast, the two groups observed no significant difference in nuclear volume. Accompanying these changes at the nuclear level, the following changes in the F-actin cytoskeleton were observed upon LIV application. The total number of actin filaments per cell increased from 117 to 150. The average volume of individual actin fibers rose from 0.40 µm³ to 0.43 µm³. Notably, the volume of fibers at the apical nuclear surface increased from 0.35 µm³ to 0.46 µm³. While the average length of actin fibers in the apical nuclear surface remained largely unchanged due to the addition of smaller fibers in LIV groups, the LIV group had a larger number of fibers that were larger than 15µm.

Human uterine lower segment myometrial cell and nuclear volume at term: influence of maternal age.

Little is known about the cytoarchitecture of human myometrial cells in pregnancy, and whether or not this may be influenced by maternal characteristics such as age, parity and body mass index (BMI). The aim of this study was primarily to evaluate human myometrial smooth muscle cell (SMC) and nuclear volume in the third trimester of human pregnancy, and secondarily to investigate if these parameters are altered in relation to the maternal characteristics outlined above. Myometrial biopsies were obtained from 30 women undergoing elective caesarean delivery at term. One-micrometer sections were prepared for light microscopy and 100-nm sections for electron microscopy. The nucleator technique was used to assess nuclear volume from the light microscopy images. Point-counting methodology was used on transmission electron micrographs to assess the percentage of the cell volume occupied by the nucleus. Cell volume was calculated from these measurements. The euchromatin to heterochromatin (Eu/Het) ratio was determined to ascertain whether differences in nuclear volume were due to an increased range of genes being transcribed. The mean (± SEM) nuclear volume was 175 ± 10 μm(3) , the nucleus occupied 1.5 ± 0.1% of the SMC and the mean cell size was 14 047 ± 1352 μm(3) . The Eu/Het ratio was 7.54 ± 0.4. The mean volume of heterochromatin and euchromatin in the nucleus was 21.5 ± 1.7 and 149 ± 9 μm(3) , respectively. A multivariate regression analysis revealed that advanced maternal age was associated with an increase in the percentage of the cell occupied by nucleus (R(2) = 0.32, P = 0.004). There were no other significant effects of maternal age, BMI or parity on the measured parameters. These findings provide reliable volumes for human myometrial cells and their nuclei at term gestation, and show that nuclear volume fraction may be influenced by maternal age.

Development of sex differences in the principal nucleus of the bed nucleus of the stria terminalis of mice: Role of Bax‐dependent cell death

Neuron number in the principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is greater in adult male mice than in females. Deletion of the proapoptotic gene, Bax, increases the number of BNSTp cells in adulthood and eliminates the sex difference in cell number. Here, we map the ontogeny of sex differences in nuclear volume and cell number in the BNSTp of neonatal mice, and evaluate the role of cell death in the development of these differences. We find that BNSTp volume and cell number do not differ between male and female wild-type mice on postnatal days P3, P5, or P7. Sex differences emerge after the first postnatal week and both measures are significantly greater in males than in females on P9 and P11. Cell death, assessed by TUNEL staining, was observed in the BNSTp of both sexes from P1-P8. Females had more TUNEL-positive cells than males from approximately P3-P6, with the maximum number of dying cells observed on P5/P6. To test whether the Bax gene is required for sexually dimorphic cell death in the BNSTp, TUNEL cells were counted on P6 in Bax -/- mice and their Bax +/+ siblings. Bax gene deletion nearly abolished TUNEL-positive cells in the BNSTp of both sexes. Together, these findings support the interpretation that the sex difference in BNSTp cell number seen in adulthood is due to Bax-dependent, sexually dimorphic cell death during the first week of life.

Cell death and sexual differentiation of the nervous system

Sex differences in nuclear volume or neuron number often are attributed to the hormonal control of cell death. In the spinal nucleus of the bulbocavernosus, the central portion of the medial preoptic nucleus, and the principal nucleus of the bed nucleus of the stria terminalis testicular hormones decrease cell death during perinatal life, resulting in a male advantage in neuron number in adulthood. Conversely, males have more dying cells during development and fewer neurons in adulthood than do females in the anteroventral periventricular nucleus of the hypothalamus. This review discusses several limitations and unresolved issues in the literature on sexually dimorphic cell death, and identifies molecular mechanisms by which gonadal steroids may control cell survival. In particular, evidence is presented for the hormonal regulation of neurotrophic factors and involvement of Bcl-2 family proteins in the determination of sex differences in neuron number.

Stereological estimates of nuclear volume in thin malignant melanomas.

Stereological estimation of nuclear volume was performed in a case control study of 72 malignant melanomas, thickness < or = 0.8 mm and Clark's level II-III. However, stereological measurements could be performed in only 57 thin melanomas due to too sparse cellularity. Thus, 21 thin metastasizing melanomas were individually compared with 33 thin non-metastasizing melanomas after individual matching of cases with one or two randomly chosen controls for site of primary tumour, tumour thickness, level of invasion, tumour regression and follow-up. Conditional logistic regression analysis showed no significant differences in nuclear volume between metastasizing and non-metastasizing thin malignant melanomas.

Sex differences in the volume of avian song control nuclei: Comparative studies and the issue of brain nucleus delineation

Two goals of research on neural sex differences are to establish the behavioral function of such sex differences and to identify precisely what features differ between males and females. Comparative studies of sex differences in the volume of brain nuclei within the songbird vocal control circuit provide one way to address these goals. Informative comparisons can be either inter-specific or intra-specific. Inter-specific comparisons of species within the songbird suborder allow one to establish how species variation in the degree to which there is a sex difference in nuclear volume relates to species variation in the degree to which there is a sex difference in vocal behavior. Intraspecific comparisons of sex differences in nuclear volume involve the comparison of a variety of histochemical methods to define nuclei and describe a nucleus within a species. Sex differences in nuclear volume have now been measured for at least some song control nuclei in 10 different passerine species. In species with more complex male than female song, the volume of key song control nuclei is on average larger in males than in females. However, future studies will require more refined measures of vocal behavior and perceptual abilities to make more precise correlations between brain and behavior. In European starlings ( Sturnus vulgaris), the volume of the vocal control nucleus, area X was found to be on average 1.95 times bigger in males than in females based on Nissl stained sections. Variation in neurotransmitter receptor density as determined by quantitative receptor autoradiography can also be used to define clearly the boundaries of a nucleus. When the boundaries of area X in male and female starlings were defined based on variation in muscarinic cholinergic and α 2-adrenergic receptor densities, volumetric estimates were obtained that are nearly identical to those obtained with the use of Nissl stains. Intra-specific comparisons of this sort extend our knowledge concerning the neurochemical basis of sex differences in nuclear volume. The wide application of this method would greatly increase our understanding of neural sex differences.

Relations between Nucleolar Morphometric Parameters and Pre-rRNA Synthesis in Animal and Plant Cells

In order to study if there are differences between cells of the same tissue with one and two nucleoli, nuclear and nucleolar volume, density of tritiated uridine incorporation, amount of DNA per nucleus and intensity of cytoplasmic basophilia were measured in mononucleolated and binucleolated rat epithelial endometrial cells, in onion root meristematic cells and in chick embryo matrix cells of the central nervous system, neuroblasts and neurons. No significant differences in nuclear volume, density of tritiated uridine incorporation and amount of DNA per nucleus were found between cells of the same type with diverse numbers of nucleoli. Binucleolated endometrial cells, matrix cells, and root meristematic cells have biphasic distributions of nucleolar volumes. One peak of this distribution roughly coincides with the nucleolar volume of mononucleolated cells, the other peak corresponds almost to double the volume. As the density of uridine incorporation is the same irrespective of the nucleolar number and volume, the cells with larger nucleolar volumes have higher pre-rRNA synthesis. These cells also have higher amounts of ribosomes in the cytoplasm, as revealed by the photometric study of basophilia. It is concluded that in this population of cells the ribosomal production is regulated to a higher steady equilibrium than in the general population. This difference is not due to polyploidism or to the increased DNA content of G2 phase cells. Binucleolated neuroblasts and neurons have nucleolar volumes similar to those of mononucleolated ones.

EVIDENCE FOR SIGNIFICANT DIFFERENCES IN NUCLEAR DNA CONTENT BETWEEN TWO GEOGRAPHICALLY ISOLATED CLONES OF ARMILLARIA MELLEA

Several collections of Armillaria mellea (Vahl: Fr.) Kummer basidiocarps were screened for relative nuclear DNA content using microspectrophotometric techniques. One collection from the Pacific Northwest showed relative DNA values approximately 30% higher than a reference collection from College Park, Maryland. Corresponding differences in nuclear volumes were also observed. It is postulated that the Washington isolate may represent a stable aneuploid or polyploid clone of the species.

Evidence for Significant Differences in Nuclear DNA Content Between Two Geographically Isolated Clones of Armillaria mellea

Several collections of Armillaria mellea (Vahl: Fr.) Kummer basidiocarps were screened for relative nuclear DNA content using microspectrophotometric techniques. One collection from the Pacific Northwest showed relative DNA values approximately 30% higher than a reference collection from College Park, Maryland. Corresponding differences in nuclear volumes were also observed. It is postulated that the Washington isolate may represent a stable aneuploid or polyploid clone of the species.

Sexual difference in nuclear volume and its ontogeny in the rat amygdala

Nuclear volume of the medial and lateral nuclei of the amygdala was investigated in male and female rats. The volume of the medial nucleus in the adult male rat was significantly greater than that of the female rat. Treatment of female rats with estrogen for the first 30 days of postnatal life increased the nuclear size, and the volume became comparable to that of the males. Although there was no significant difference in volume of this nucleus between male and female rats from days 1 (day of birth) to 11, the difference became evident at day 21, and thereafter it persisted. In contrast, the nuclear volume of the lateral nucleus was not different between the two sexes. Estrogen treatment did not influence the volume of the lateral nucleus in the female animals. These data indicate that the nuclear volume is sexually different in the medial nucleus of the amygdala, and its differentiation occurs during the early postnatal period under the influence of the organizational action of sex steroids.

On the morphology of the GOLGI apparatus and its relation to glucose-6-phosphate dehydrogenase in neurosecretory nuclei of rats unrler normal and thirsting conditions (Application of karyometry)

Histochemical studies on the distribution of TPPase and G6PD in SON and PVN of rats have been conducted by using the new TPPase method, karyometry, and statistics under normal and thirsting conditions. All differences in nuclear volume among the 4 groups classified by the TPPase and G6PD reactions were significant after thirsting for 2 and 4 days, respectively. The intimate parallel relationship was found statistically between the GA shape and G6PD activity in neurons of both nuclei. These results may substantiate the usefulness of classification of neurons of both nuclei. These results may substantiate the usefulness of classification of neurons by the TPPase method on the LM level. They also suggest strongly that the GA shape may directly indicate its synthesizing activity, and that the complicate GA shape may correspond to the phase of vigorous synthesizing activity in the neuron. Both nuclei included some neurons whose GA appeared intact under activation. This suggests that secretory cycles of individual cells may proceed asynchromously under thirsting as well as normal conditions. The unique peak revealed by the curves of mean nuclear volumes of GA Type IV and G6PD. strongly positive groups only in PVN must be representative of phenomenon of exhaustion of PVN.

A study of DNA and mitotic activity in the vegetative apex of Douglas fir during the annual growth cycle

Vegetative apices of Pseudotsuga menziesii (Mirb.) Franco were studied throughout the annual growth cycle. When observations based on anatomy, histochemistry, and external morphology are combined, the growth cycle of the vegetative apex should be subdivided into five stages: (1) dormancy, (2) early bud-scale initiation, (3) late bud-scale initiation and rapid apical enlargement, (4) early, rapid leaf initiation, and (5) late, slow leaf initiation. The same cytohistological zonation pattern is present in vegetative apices throughout the growth cycle and usually consists of apical, peripheral, and rib meristem zones. During dormancy, early bud-scale initiation, and early leaf initiation, the apical zone is separated into apical initials and central mother cells based on nuclear characteristics and mitotic activity. Cytohistological zonation is supported by constant differences in nuclear volume, mitotic activity, and DNA content between zones. The peripheral zone is mitotically more active than the apical zone; however, the apical zone is not quiescent. Zones vary in size, prominence, and mitotic activity, which often relates to a particular developmental stage of the apex. The dormant apex has no mitotic activity, and cytohistological zonation is present but not distinct. Zonation increases throughout the first half of the growing season, reaches a maximum during late bud-scale and early leaf initiation, and decreases as dormancy approaches. In general, increases in nuclear volume, percentage of nuclei at 4C, and average DNA content per nucleus correlate with increases in the prominence of zonation. Zonation did not result from different zones being "held" at certain C levels of DNA. Although nuclear volume was used in calculating the DNA content, the DNA level often varied independently of volume. Mitotic activity and dormancy appear to be related to carbohydrate levels within the bud and subtending shoot. The period of most prominent zonation is also the period of most active primordial initiation, largest apical size, and the time when new axillary shoots become determined in their pathway of development.

Cell count correction factors for the quantitative histological analysis of the germinal epithelium of the ram.

AbstractThe relationship between the Sertoli cell counts, some measurable parameters of the seminiferous tubules and the dimensions of the Sertoli cell nuclei were examined in both normal and degenerate testes. Highly significant differences were found between rams in the numbers and in the dimensions and volumes of the Sertoli cell nuclei and in the diameters of the seminiferous tubules. The Sertoli cell counts were corrected for the differences in nuclear volume by dividing the count by the calculated nuclear volume. The Sertoli cell counts were corrected for changes in the area of the seminiferous tubule wall by multiplying the count by the square of the tubule radius. Correction for either nuclear volume or tubule area alone increased the differences between the Sertoli cell counts, but a simultaneous correction for both factors equalized the counts so that there were no significant differences between rams in the number of Sertoli cells per tubule cross‐section. It was concluded that, in normal and degenerate ram testes, counts of the Sertoli cell nuclei in the seminiferous tubule cross‐sections, when corrected for differences in nuclear volume, would provide a valid basis for comparing volume‐corrected counts of spermatogenic cells in different animals and in different treatments.

The effect of ionizing radiation on the reproductive capacity of forest trees.

In a pine-oak forest exposed to chronic gamma radiation the pine trees were more radiosensitive than the oaks. Low-level radiation accumulated over a period of years was lethal for many of the trees. Radiation damage was evident in pollen production, seed formation, and seedling behavior. Megasporogenesis evaluations indicated chromosome aberrations and delayed fertilization occurred at levels as low as 2.8 R/20-hr day. In general, seed production was compatible with vegetative survival despite the greater sensitivity of the meiotic cycle. Seeds stored in cones of pine trees that had been killed by radiation were able to germinate and produced apparently normal seedlings. The differential response between oaks and pines was strongly correlated with differences in nuclear volume; both species have a chromosome number of 2N = 24. Seed irradiation studies revealed that varying the physiological condition could change LD50 values by a factor of ten. Seed radiosensitivity was not greatly different between pines and oaks despite the greater sensitivity of the mature pines trees. Studies of male bud irradiation prior to pollen formation revealed this stage as one of the most radiosensitive. Pollen irradiation gave results generally similar to seed irradiation in terms of tolerance. The ability of pollen to fertilize the egg cell was destroyed at levels greater than 16 kR, although the pollen demonstrated some germination potential at levels up to 800 kR.

The relative sensitivity of the soaked seeds of nine gymnosperm species to gamma radiation

Seeds of nine gymnosperm species, soaked in water to full imbibition, were irradiated with 137Cs gamma rays in two separate experiments. Species studied were Larix laricina, Picea abies, Picea glauca, Picea mariana, Pinus banksiana, Pinus contorta, Pinus resinosa, Pinus sylvestris and Thuja occidentalis. In the first experiment exposures of 150, 300, 600, 1200, 2400, 4800, 9600, 19,200 and 38,400 R were used and the seedlings were grown for 36 days after initial germination in perlite saturated with a complete nutrient solution. In the second experiment exposures of 150, 300, 450, 600, 900, 1200, 1800, 2400, 3600, 4800, 7200, 9600, 14,400 and 19,200 R were employed and the seedlings were grown in sand for 75 days after initial germination. Endpoints scored in the first experiment were germination, survival, cotyledon length, and plant dry weight. In the second experiment germination, survival, hypocotyl length, cotyledon length, shoot length, root length, shoot dry weight, and root dry weight were scored. Seed radiosensitivity varied by a factor of 3·80–7·33 in the first experiment and from 2·67 to 5·70 in the second experiment, depending on the endpoint used. Picea abies, Picea glauca and Pinus sylvestris were the most sensitive species in both experiments and Pinus banksiana the most resistant. The average D 50 (exposure resulting in 50 per cent inhibition) for all endpoints in Experiment I varied from 2·64 kR to 9·71 kR; that in Experiment II from 2·30 kR to 10·56 kR for the nine species studied. Comparison of the results with previous reports for individual species indicated that relative radiosensitivity of seed of plant species must be investigated with all species of interest being identical in radiation exposure conditions, seed moisture content and post-irradiation storage. Germination and growing conditions optimal for each species are suggested. The more than four-fold average differences in seed radiosensitivity were found to be unrelated to differences in nuclear volume, chromosome volume, and DNA content previously reported for the same populations of these species. The relative seed sensitivity pattern showed no relation to previously reported predictions of seedling and mature plant sensitivities for the same species.

S.N. Banerjee: &lt;i&gt;Pinus resinosa&lt;/i&gt;の発育中の

Ovules ofPinus resinosafrom 1 and 2 year-old cones were collected periodically during July to August in 1963 and 1964. The embryo-gametophyte complex containing embryos at different stages of development were irradiated by a 60Co source with doses from 0.25 to 2 kilorads. The effects of radiation on the embryo and the gametophytes were determined on the basis of abnormal differentiation and abortion of embryos and the cytological anomaly in the gametophytic cells. There was less abnormal differentiation in the embryos irradiated at stage I in comparison to other stages whereas the percentage of aborted embryos was a maximum in stage I. A possible explanation of the varying radiation effect was attempted on the basis of the average nuclear volume of embryonic cells at each stage of development. In stage I, the average nuclear volume was increased about 11 to 14-fold over those in the other stages with a concomitant peak in the percentage of aborted embryos. In contrast, higher radiation effect in root apices than that in the shoot apices could not be explained through a difference in nuclear volume. The changes in the average nuclear volume in the embryonic cells at various stages of differentiation could not be explained on the basis of an increase in the DNA content alone.

The radiosensitivity of gymnosperms. I. The effect of dormancy on the response of Pinus strobus seedlings to acute gamma irradiation

Seedlings of Pinus strobus (white pine) were exposed for 16.5 hr to Co 60 gamma radiation at dosages from 50 to 1000 r during a period of dormancy or a period of active growth. The effect on growth was determined by measurement of needle lengths. The data show the dormant plants to be considerably more resistant than the actively growing plants. Measurements of interphase nuclear volumes in apical shoot meristems made at various times during the year show a large volume during stages of active growth and much smaller volumes during dormancy. The data strongly suggest that the differences in sensitivity of dormant and actively growing plants may be largely the result of differences in nuclear volumes of these stages.

CONTROL OF NUCLEAR PROCESSES BY AUXIN IN AXILLARY BUDS OF TRADESCANTIA PALUDOSA

Regulatory effects of auxin on mitosis and DNA synthesis were studied in lateral bud apices of Tradescnntia paludosa. In axillary buds inhibited by the terminal bud, mitoses were absent in a group of cells constituting the initials of tunica and corpus within the apex. The DNA (Feulgen) content of inter-phasic nuclei in this "zone of inhibition" was uniform and no greater than that characteristic of diploid telophase (2C), despite differences in nuclear volume. In subapical bud tissues, mitoses occurred at a low rate. Upon removal of the vegetative shoot tip, this mitotic rate was increased and the previously inhibited nuclei within the apes underwent DNA doubling to 4C, then entered prophase. These results indicate that the interphase synthesis of at least one chromosomal substance (DNA) is under auxin control. Evidence is presented that mitosis is not directly inhibited by this hormone. Naphthalene acetic acid was found to substitute for natural auxin in the inhibition of DNA synthesis.

Nuclear Isotope Shift in the Spectra of HgH+and HgD+

The structure of the lines in the band spectra of Hg${\mathrm{H}}^{+}$ and Hg${\mathrm{D}}^{+}$, excited in a hollow cathode tube, is studied with Fabry-Perot etalons. The lines are found to be broadened, with traces of structure, an effect caused by the isotopic constitution of mercury. The structure shows that there is no anomalous behavior of components belonging to the odd Hg isotopes due to hyperfine splitting, but the half-widths of the lines differ widely from the expected ones. From the measured half-widths the shifts between the consecutive even isotopes of Hg are evaluated and then the differences between these and the values obtained from the formulas for the normal vibrational and rotational isotope effect calculated. These additional shifts show the same behavior as the shifts found some years ago in HgH and HgD and therefore must be explained as nuclear isotope shifts, that is, shifts connected with differences in nuclear volume of the different isotopes of Hg. For the lowest vibrational bands the shifts are of the same sign as in HgH, but decrease with the vibrational energy of both upper and lower states, changing sign for sufficiently high vibrational energies and probably tending in the limit of dissociation to the shifts found in the corresponding quadrupole line 2815A of Hg II. Consideration of the data on the nuclear isotope shifts in HgH, Hg${\mathrm{H}}^{+}$, Hg I and Hg II permits some conclusions about the deformation of the electron clouds in Hg and ${\mathrm{Hg}}^{+}$ caused by the attachment of a hydrogen atom, and a rough calculation of the nuclear shifts in the molecular levels of HgH and Hg${\mathrm{H}}^{+}$.