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.

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 (X t ,t∈ℝ2) is thresholded on a given level u, the excursion set is given by its indicator 1[u, ∞)(X t ). 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.

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.

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.

Fifty tetrathyridia of Mesocestoides corti showing various degrees of scolex fission during asexual multiplication were subjected to estimation of the total number of neurons in the central ganglia, using a ‘fractionator’ designed sampling scheme on every 10th Historesin section through the specimen. Neurons were stained with Cresyl fast violet and viewed by transmission light microscopy. Cells were only counted if a nucleolus was present. The animals were divided into groups according to their stage in the asexual reproduction process indicated by head shape and sucker number. Animals which had a narrow head with 4 suckers contained about 250 neurons. As their heads became wider, in preparation for fission, neurons were overproduced by up to 5-fold. As additional suckers were developed (8 in total), neurons were reduced to about 500. Finally, when the animals had 2 clearly discernible heads, with 4 suckers on each, the total number of neurons (per head) was again approximately 250. As the heads became wider again, neuron over-production recommenced. Exceptionally shaped animals with an X- or T-shaped single head were observed. These results suggest that there is a preparatory stage for neuronal regeneration, during which the basic population of neurons is about 250. This is followed by neuron over-production followed by a selective cell death of neurons which occurred during asexual multiplication. Another cycle for neuronal regeneration can occur before an animal completes its fission process.

The present study aimed to evaluate the effects of a post-weaning high-fat (HF) diet upon hepatic morphology in rats subjected to perinatal protein restriction. Pregnant Wistar rats were assigned to a normal-protein diet (NP; with 19 % of protein) or a low-protein (LP) diet (with 5 % of protein). At weaning, the following groups were formed: NP and NP-HF, males and females, which were fed standard chow and an HF diet, respectively. Likewise, LP rat dams originated LP and LP-HF offspring, both sexes. Euthanasia was performed at 6 months of age. Three-way ANOVA disclosed a three-factor interaction among sex, perinatal diet and HF diet in relation to body mass, retroperitoneal fat pad, liver mass:tibia length ratio, binucleation rate and hepatocyte area at 6 months old (P < 0·05). The high-fat diet intensified the effects of perinatal protein restriction concerning systolic blood pressure, genital fat pad and hepatocyte number (P < 0·05; two-way ANOVA). Furthermore, higher steatosis rates and insulin and leptin concentrations were found in males fed on the HF diet, indicating a sex–post-weaning diet interaction (P < 0·05; two-way ANOVA). Fetal programming and HF diet as a single stimulus caused mild hypertension at 3 months, an important reduction in hepatocyte number as well as stage 1 steatosis at 6 months. However, hypertension and hepatocyte number deficit were worsened and grade 2 steatosis occurred after exposure to the HF diet. All of these serve to highlight the paramount importance of intra-uterine conditions and postnatal diet quality when it comes to the pathogenesis of chronic diseases.

Our aim is to estimate the volume-weighted mean of the volumes of three-dimensional ‘particles’ (compact, not-necessarily-convex subsets) from plane sections of the particle population. The standard stereological technique is to place test lines in the plane section, and measure cubed intercept lengths with the two-dimensional particle profiles. This paper discusses more efficient estimators obtained by integrating over all possible placements of the test line. We prove that these estimators have smaller variance than the line transect estimators, and indeed are related to them by the Rao-Blackwell process. In the improved estimators, the cubed intercept length is replaced by a moment of the distance between two points in the section profile. This can be computed as a moment of the set covariance function, which in turn is computable using the fast Fourier transform. We also derive an isoperimetric-type inequality between the improved estimator and the area-weighted 3/2th moment of the profile areas. Finally, we present two practical applications to particles of silicon carbide and to synaptic boutons in brain tissue. We estimate the variance of the technique and the gain in efficiency over line transect techniques; the efficiency improvement appears to be as much as one order of magnitude.

Recently, systematic sampling on the circle and the sphere has been studied by Gual-Arnau and Cruz-Orive (2000) from a design-based point of view. In this note, it is shown that their mathematical model for the covariogram is, in a model-based statistical setting, a special case of the p-order shape model suggested by Hobolth, Pedersen and Jensen (2000) and Hobolth, Kent and Dryden (2002) for planar objects without landmarks. Benefits of this observation include an alternative variance estimator, applicable in the original problem of systematic sampling. In a wider perspective, the paper contributes to the discussion concerning design-based versus model-based stereology.

The ability to accurately quantify ovarian condition in cultured species of fish is of paramount importance in the understanding of their reproductive biology and the development of appropriate culture conditions. Here, we discuss stereological analysis, a mathematically proven technique that determines 3-dimensional topology and geometry from 2-dimensional thin sections, as a safer and more precise replacement for existing methodology.