Here we report a quantitative analysis of human metaphase II (MII) oocytes from a 22-year-old oocyte donor, retrieved after ovarian-controlled hyperstimulation. Five surplus donor oocytes were processed for transmission electron microscopy (TEM), and a stereological analysis was used to quantify the distribution of organelles, using the point-counting technique with an adequate stereological grid. Comparisons between means of the relative volumes (Vv) occupied by organelles in the three oocyte regions, cortex (C), subcortex (SC) and inner cytoplasm (IC), followed the Kruskal–Wallis test and Mann–Whitney U-test with Bonferroni correction. Life cell imaging and TEM analysis confirmed donor oocyte nuclear maturity. Results showed that the most abundant organelles were smooth endoplasmic reticulum (SER) elements (26.8%) and mitochondria (5.49%). Significant differences between oocyte regions were found for lysosomes (P = 0.003), cortical vesicles (P = 0.002) and large SER vesicles (P = 0.009). These results were quantitatively compared with previous results using prophase I (GV) and metaphase I (MI) immature oocytes. In donor MII oocytes there was a normal presence of cortical vesicles, SER tubules, SER small, medium and large vesicles, lysosomes and mitochondria. However, donor MII oocytes displayed signs of cytoplasmic immaturity, namely the presence of dictyosomes, present in GV oocytes and rare in MI oocytes, of SER very large vesicles, characteristic of GV oocytes, and the rarity of SER tubular aggregates. Results therefore indicate that the criterion of nuclear maturity used for donor oocyte selection does not always correspond to cytoplasmic maturity, which can partially explain implantation failures with the use of donor oocytes.

Maternal protein restriction is often associated with structural and functional sequelae in offspring, particularly affecting growth and renal-cardiovascular function. However, there is little understanding as to whether hypertension and kidney disease occur because of a primary nephron deficit or whether controlling postnatal growth can result in normal renal-cardiovascular phenotypes. To investigate this, female Sprague-Dawley rats were fed either a low-protein (LP, 8.4% protein) or normal-protein (NP, 19.4% protein) diet prior to mating and until offspring were weaned at postnatal day (PN) 21. Offspring were then fed a non ‘growth’ (4.6% fat) which ensured that catch-up growth did not occur. Offspring growth was determined by weight and dual energy X-ray absorptiometry. Nephron number was determined at PN21 using the disector-fractionator method. Kidney function was measured at PN180 and PN360 using clearance methods. Blood pressure was measured at PN360 using radio-telemetry. Body weight was similar at PN1, but by PN21 LP offspring were 39% smaller than controls (Pdiet < 0.001). This difference was due to proportional changes in lean muscle, fat, and bone content. LP offspring remained smaller than NP offspring until PN360. In LP offspring, nephron number was 26% less in males and 17% less in females, than NP controls (Pdiet < 0.0004). Kidney function was similar across dietary groups and sexes at PN180 and PN360. Blood pressure was similar in LP and NP offspring at PN360. These findings suggest that remaining on a slow growth trajectory after exposure to a suboptimal intrauterine environment does not lead to the development of kidney dysfunction and hypertension.

The egg is one of the fundamental parts of the life cycle of Neoechinorhynchus buttnerae, and this stage involves the acanthor larva. It is also the infection phase for the intermediate host. Under normal conditions, the larva inside the egg can survive for months in the environment; however, information regarding this phase of life of the parasite is scarce. In addition, there is no quantitative information about the structural composition of the parasite's body from a histological point of view. Such information is essential in order to support decisions aimed at controlling infestations by these parasites in fish farming. This study aimed to present a detailed description of the stages of embryonic development of N. buttnerae eggs, as well as a stereological evaluation of the body of adult females of the parasite. Three phases of development characterized the eggs: cell division (with four stages), formation of the internal nuclear mass (with four stages) and formation of the acanthor larva (with five stages). The ovary comprised 26.61% of the volume of the animal and most of it contained eggs (21.28%), ovarian balls (3.88%) and empty spaces (1.45%). These results are of great importance and will support future studies that seek to interrupt the life cycle of this parasite.

We have previously presented a stereological analysis of organelle distribution in human prophase I oocytes. In the present study, using a similar stereological approach, we quantified the distribution of organelles in human metaphase I (MI) oocytes also retrieved after ovarian stimulation. Five MI oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. Kruskal–Wallis and Mann–Whitney U-tests with Bonferroni correction were used to compare the means of relative volumes (Vv) occupied by organelles. In all oocyte regions, the most abundant organelles were mitochondria and smooth endoplasmic reticulum (SER) elements. No significant differences were observed in Vv of mitochondria, dictyosomes, lysosomes, or SER small and medium vesicles, tubular aggregates and tubules. Significant differences were observed in other organelle distributions: cortical vesicles presented a higher Vv (P = 0.004) in the cortex than in the subcortex (0.96% vs 0.1%) or inner cytoplasm (0.96% vs 0.1%), vesicles with dense granular contents had a higher Vv (P = 0.005) in the cortex than in the subcortex (0.1% vs 0%), and SER large vesicles exhibited a higher Vv (P = 0.011) in the inner cytoplasm than in the subcortex (0.2% vs 0%). Future stereological analysis of metaphase II oocytes and a combined quantitative data of mature and immature oocytes, will enable a better understanding of oocyte organelle distribution during in vivo maturation. Combined with molecular approaches, this may help improve stimulation protocols and in vitro maturation methods.

In this paper, prediction models are proposed which allow the mineralogical characterization of particle systems observed by X-ray micro tomography (XMT). The models are calibrated using 2D image data obtained by a combination of scanning electron microscopy and energy dispersive X-ray spectroscopy in a planar cross-section of the XMT data. To reliably distinguish between different minerals the models are based on multidimensional distributions of certain particle characteristics describing, for example, their size, shape, and texture. These multidimensional distributions are modeled using parametric Archimedean copulas which are able to describe the correlation structure of complex multidimensional distributions with only a few parameters. Furthermore, dimension reduction of the multidimensional vectors of particle characteristics is utilized to make non-parametric approaches such as the computation of distributions via kernel density estimation viable. With the help of such distributions the proposed prediction models are able to distinguish between different types of particles among the entire XMT image.

In this paper, three-dimensional (3D) image data of ore particle systems is investigated. By combining X-ray microtomography with scanning electron microscope (SEM)-based image analysis, additional information about the mineralogical composition from certain planar sections can be gained. For the analysis of tomographic images of particle systems the extraction of single particles is essential. This is performed with a marker-based watershed algorithm and a post-processing step utilizing a neural network to reduce oversegmentation. The results are validated by comparing the 3D particle-wise segmentation empirically with 2D SEM images, which have been obtained with a different imaging process and segmentation algorithm. Finally, a stereological application is shown, in which planar SEM images are embedded into the tomographic 3D image. This allows the estimation of local X-ray attenuation coefficients, which are material-specific quantities, in the entire tomographic image.

Bisphenol A (BPA) is a chemical found in plastics that resembles oestrogen in organisms. Developmental exposure to endocrine-disrupting chemicals, such as BPA, increases the susceptibility to type 2 diabetes (T2DM) and cardiovascular diseases. Animal studies have reported a nephron deficit in offspring exposed to maternal diabetes. The aim of this study was to investigate the prenatal BPA exposure effects on nephrogenesis in a mouse model that was predisposed to T2DM. This study quantitatively evaluated the renal structural changes using stereology and histomorphometry methods. The OF1 pregnant mice were treated with a vehicle or BPA (10 or 100 μg/kg/day) during days 9–16 of gestation (early nephrogenesis). The 30-day-old offspring were sacrificed, and tissue samples were collected and prepared for histopathological and stereology studies. Glomerular abnormalities and reduced glomerular formation were observed in the BPA offspring. The kidneys of the BPA10 and BPA100 female offspring had a significantly lower glomerular number and density than those of the CONTROL female offspring. The glomerular histomorphometry revealed a significant difference between the female and male CONTROL offspring for the analysed glomerular parameters that disappeared in the BPA10 and BPA100 offspring. In addition, the kidney histopathological examination showed typical male cuboidal epithelial cells of the Bowman capsule in the female BPA offspring. Exposure to environmentally relevant doses of BPA during embryonic development altered nephrogenesis. These structural changes could be associated with an increased risk of developing cardiometabolic diseases later in life.

When a random field (Xt,t∈ℝ2) is thresholded on a given level u, the excursion set is given by its indicator 1[u, ∞)(Xt). The purpose of this work is to study functionals (as established in stochastic geometry) of these random excursion sets as, e.g. the capacity functional as well as the second moment measure of the boundary length. It extends results obtained for the one-dimensional case to the two-dimensional case, with tools borrowed from crossings theory, in particular, Rice methods, and from integral and stochastic geometry.

The ultrastructural analysis of human oocytes at different maturation stages has only been descriptive. The aim of this study was to use a stereological approach to quantify the distribution of organelles in oocytes at prophase I (GV). Seven immature GV oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. The Kruskal–Wallis test and Mann–Whitney U-test with Bonferroni correction were used to compare the means of the relative volumes occupied by organelles in oocyte regions: cortex (C), subcortex (SC) and inner cytoplasm (IC). Here we first describe in GV oocytes very large vesicles of the smooth endoplasmic reticulum (SER), vesicles containing zona pellucida-like materials and coated vesicles. The most abundant organelles were the very large vesicles of the SER (6.9%), mitochondria (6.3%) and other SER vesicles (6.1%). Significant differences in organelle distribution were observed between ooplasm regions: cortical vesicles (C: 1.3% versus SC: 0.1%, IC: 0.1%, P = 0.001) and medium-sized vesicles containing zona pellucida-like materials (C: 0.2% versus SC: 0.02%, IC: 0%, P = 0.004) were mostly observed at the oocyte cortex, whereas mitochondria (C: 3.6% versus SC: 6.0%, IC: 7.2%, P = 0.005) were preferentially located in the subcortex and inner cytoplasm, and SER very large vesicles (IC: 10.1% versus C: 0.9%, SC: 1.67%, P = 0.001) in the oocyte inner cytoplasm. Further quantitative studies are needed in immature metaphase-I and mature metaphase-II oocytes, as well as analysis of correlations between ultrastructural and molecular data, to better understand human oocyte in vitro maturation.

Axer H, Bernstein H-G, Keiner S, Heronimus P, Sauer H, Witte OW, Bogerts B, Bär K-J. Increased neuronal cell number in the dorsal motor nucleus of the vagus in schizophrenia.

Though three-dimensional (3D) imaging gives deep insight into the inner structure of complex materials, the stereological analysis of 2D snapshots of material sections is still necessary for large-scale industrial applications for reasons related to time and cost constraints. In this paper, we propose an original framework to estimate the orientation distribution of generalized cylindrical structures from a single 2D section. Contrary to existing approaches, knowledge of the cylinder cross-section shape is not necessary. The only requirement is to know the area distribution of the cross-sections. The approach relies on minimization of a least squares criterion under linear equality and inequality constraints that can be solved with standard optimization solvers. It is evaluated on synthetic data, including simulated images, and is applied to experimental microscopy images of fibrous composite structures. The results show the relevance and capabilities of the approach though some limitations have been identified regarding sensitivity to deviations from the assumed model.

Studies of the capillary bed characterized by its length or length density are relevant in many biomedical studies. A reliable assessment of capillary length from two-dimensional (2D), thin histological sections is a rather difficult task as it requires physical cutting of such sections in randomized directions. This is often technically demanding, inefficient, or outright impossible. However, if 3D image data of the microscopic structure under investigation are available, methods of length estimation that do not require randomized physical cutting of sections may be applied. Two different rat brain regions were optically sliced by confocal microscopy and resulting 3D images processed by three types of capillary length estimation methods: (1) stereological methods based on a computer generation of isotropic uniform random virtual test probes in 3D, either in the form of spatial grids of virtual “slicer” planes or spherical probes; (2) automatic method employing a digital version of the Crofton relations using the Euler characteristic of planar sections of the binary image; and (3) interactive “tracer” method for length measurement based on a manual delineation in 3D of the axes of capillary segments. The presented methods were compared in terms of their practical applicability, efficiency, and precision.

Stereological analysis has been coupled with transmission electron microscope (TEM) orientation mapping to investigate the grain boundary character distribution in nanocrystalline copper thin films. The use of the nanosized (<5 nm) beam in the TEM for collecting spot diffraction patterns renders an order of magnitude improvement in spatial resolution compared to the analysis of electron backscatter diffraction patterns in the scanning electron microscope. Electron beam precession is used to reduce dynamical effects and increase the reliability of orientation solutions. The misorientation distribution function shows a strong misorientation texture with a peak at 60°/[111], corresponding to the Σ3 misorientation. The grain boundary plane distribution shows {111} as the most frequently occurring plane, indicating a significant population of coherent twin boundaries. This study demonstrates the use of nanoscale orientation mapping in the TEM to quantify the five-parameter grain boundary distribution in nanocrystalline materials.

For stationary fiber processes, the estimation of the directional distribution is an important task. We consider a stereological approach, assuming that the intersection points of the process with a finite number of test hyperplanes can be observed in a bounded window. The intensity of these intersection processes is proportional to the cosine transform of the directional distribution. We use the approximate inverse method to invert the cosine transform and analyze asymptotic properties of the estimator in growing windows for Poisson line processes. We show almost-sure convergence of the estimator and derive Berry–Esseen bounds, including formulae for the variance.

This study was conceived to evaluate seasonal changes in the lipid reserves of the digestive gland of the carnivorous intertidal caenogastropod Nucella lapillus, using a stereological approach. Volume density of lipid droplets in the digestive gland, digestive gland weight and volume were assessed in animals collected in March, June, September and December on the Portuguese coast. Gonad development was evaluated to detect any relationship between lipid content in the digestive gland and the reproductive cycle. The quantitative light microscopic analysis demonstrates that lipid droplets are a major component of the digestive gland. In males, the digestive gland and its lipid reserves were quite stable without significant variations throughout the year. In females, the percentage of digestive gland volume occupied by lipid droplets was higher in June and December, coinciding with the highest values of digestive gland volume. Due to the conjugation of these two factors, in June and December the total amount of lipids in the digestive gland was substantially increased in females. In both males and females a relationship between the development status of the gonad and the lipid reserves of the digestive gland was not evident. However, significant differences in the digestive gland lipid reserves were detected between males and females in June and December, pointing to a sex related effect on lipid reserves. To evaluate the use of lipid reserves as energy source in N. lapillus, the consumption of digestive gland lipids was followed during a starvation experiment.

This study aimed to investigate myocardial injuries in mice infected with Schistosoma mansoni and fed a high-fat chow. Sections of myocardial tissue from S. mansoni-infected mice, and controls that had been killed 9 and 17 weeks post-infection, were stained with H&E and Picrosirius red. Histopathological examination, stereological design-based method (optical disector) and morphometry (vessels, cardiomyocytes and an amount of collagen) were used. Data were analysed using two-way ANOVA. Regardless of time of infection, myocardial tissue from the infected mice fed high-fat chow showed myocarditis characterized by a higher number of inflammatory foci, several areas displaying coagulation of cardiac fibres, a greater loss of cardiomyocytes and fibroblast proliferation than in the standard chow control. Comparing infected mice from acute and chronic infections, a higher cardiomyocyte hyperplasia (P < 0·0001) and higher amounts of collagen (P < 0·05) were observed than in standard chow control. In addition, all animals fed high-fat chow showed lower numerical density and total number of cardiomyocytes (P < 0·05), thicker vessel walls and narrowed luminal intramyocardial vessels (P > 0·05) than in the standard chow control. Altogether the data supported the view that a double burden has a synergistic deleterious effect on the myocardial tissue.

The large variability in kidney morphology among fish makes it difficult to build a “universal” model on its function and structure. Therefore, a morphological study of brown trout trunk kidney was performed, considering potential seasonal and sex effects. Three-year-old specimens of both sexes were collected at four stages of their reproductive cycle. Kidney was processed for light and electron microscopy. The relative volumes of renal components, such as renal corpuscles and different nephron tubules, were estimated by stereological methods. Qualitatively, the general nephron structure of brown trout was similar to that described for other glomerular teleost species. Quantitatively, however, differences in the relative volume of some renal components were detected between sexes and among seasons. Particularly, highest values of vacuolized tubules and new growing tubules were observed after spawning, being more relevant in females. Despite seasonal changes, more linear correlations were found between those parameters and the reno-somatic index than the gonado-somatic index. Thus, we verified that some brown trout renal components undergo sex dependent seasonal variations, suggesting a morphological adaptation of the components to accomplish physiological needs. These findings constitute a baseline for launching studies to know which factors govern the morphological variations and their functional consequences.

Background: The orbitofrontal cortex has been implicated as a key component in depression by several imaging studies. This study aims to examine morphometrically glial cell and neuronal density and neuronal volume in the orbitofrontal cortex of late-life major depression patients.

Methods: Post mortem tissue from 13 patients with major depression and 11 matched controls was obtained and analyzed using the optical disector and nucleator methods.

Results: No changes were found in glial cell, pyramidal or non-pyramidal neuron density, or in non-pyramidal and pyramidal neuron volume in the orbitofrontal cortex.

Conclusions: Based on previous findings, this study suggests variability in morphological changes within the orbitofrontal cortex, as well as the prefrontal cortex as a whole.

As a major morphological feature in establishing the form of the nervous system, it is recognized that neurons are initially overproduced, then naturally occurring cell death reduces the neuron number to the functional requirement. However, the mechanisms controlling the selective elimination of certain neurons during a general phase of cell death are not fully understood. One event that seems to be pivotal is the establishment of neural connections, the degree of which may be influential regarding the fate of specific neurons. However, little quantitative evidence is available to either support or refute this theory. In this current study, a Stereological measurement of gap junction per neuron was carried out within the invertebrate model system of the tapeworm metacestode Mesocestoides corti, which has previously been shown to overproduce neurons during the asexual reproduction stage of its life-cycle. Novel Stereological estimation methods with ‘ vertical sections’ indicated that prior to asexual division the cerebral ganglion possessed approximately 268 neurons, each with a gap junction surface area of 250 μm2. As division progressed, the neuron number increased to approximately 700, while the total surface area of gap junction remained statistically unchanged. As a result the surface area of gap junction per neuron decreased to 106 μm2, less than half that in the undividing stage. These results provide the first non-biased quantitative data regarding changes in the mean surface area of gap junction per neuron in a developing cerebral ganglion.