Estimation Of Cell Number Research Articles

Reversible alterations in epithelial cell turnover in digestive gland of winkles ( Littorina littorea) exposed to cadmium and their implications for biomarker measurements

In marine molluscs, the epithelium of the digestive gland is composed of two cell types, namely, digestive and basophilic cells. Under normal physiological conditions digestive cells outnumber basophilic cells, but under different stress situations the composition of the epithelium changes, basophilic cells apparently replace digestive cell. Winkles, Littorina littorea, were exposed to 1.25 mg/l Cd for 20 days to provoke cell type replacement. Then, animals were depurated in clean seawater for 10 days to determine whether cell type replacement was reversible. Digestive glands were fixed in Carnoy and paraffin embedded for histological analysis. The volume densities of basophilic cells (Vv BAS) and digestive cells (Vv DIG) were calculated by stereology on hematoxylin–eosin stained sections. Vv BAS increased and Vv DIG decreased in Cd-exposed animals. After estimation of cell size and absolute cell numbers, these changes were attributed to digestive cell loss and concomitant basophilic cell hypertrophy but not to increased numbers of basophilic cells. Cell type composition and cell size almost fully returned to normal values after 10-day depuration. Accordingly, PCNA immunohistochemistry demonstrated that proliferating digestive cells were more abundant in winkles exposed to Cd and after 10-day depuration than in control specimens, suggesting that net digestive cell loss was accompanied by increased digestive cell proliferation. Thus, Cd-exposure seems to provoke an enhanced digestive cell turnover in order to cope with Cd detoxification. Intralysosomal accumulation of metals (autometallographied black silver deposits; BSD) was used as a biomarker of exposure to Cd and lysosomal structural changes as an effect biomarker to see whether cell type composition might have any effect on these endpoints. BSD formed around Cd ions, in digestive cell lysosomes of Cd-exposed winkles whereas basophilic cells appeared devoid of them. After depuration, BSD were less conspicuous. Enlarged lysosomes were observed in Cd-exposed winkles, lysosome size returning to control levels after 10-day depuration. Changes in digestive cell proliferation, digestive cell loss and basophilic cell hypertrophy did not apparently affect the biomarkers investigated herein.

A real-time PCR assay for the enumeration and detection of Zygosaccharomyces bailii from wine and fruit juices

Zygosaccharomyces bailii is a major food and beverage spoilage organism. Existing methods for its detection involve lengthy enrichment techniques and then the result does not always differentiate between Z. bailii and Saccharomyces cerevisiae. In this work, we developed a quantitative real-time PCR assay for the rapid detection of Z. bailii from fruit juices and wine even in the presence of non-target DNA. Primers were designed to the gene coding for the D1/D2 loop of the 26S ribosomal RNA subunit producing a single PCR product with a melting temperature of 83.5 °C. As few as 2 cells per ml could be detected by the assay in cranberry raspberry and apple juices and 22 cells per ml from grape juice. The assay was equally efficient in wine, detecting 6 cells per ml even in the presence of 10 7 S. cerevisiae cells. The CFU/ml as determined by plating on YM media showed excellent correlation with the CFU/ml established by the QPCR assay for all the beverages examined. Unknown samples of Z. bailii were prepared in the juices and wine and examined by QPCR. The QPCR estimated cell number was in good agreement with the cell counts obtained by plating, the exception being the cranberry raspberry juice sample. It was determined by live/dead cell counts that the Z. bailii cells were less viable in this juice thus leading to an overestimation of CFU/ml by QPCR. However, the correlation was high between QPCR and total cell count as determined by fluorescent microscopy. This assay provides a rapid and accurate method to establish the levels of the total Z. bailii population which consists of both viable and nonviable cells.

Optimized conditions for measuring lipolysis in murine primary adipocytes

The current literature on lipolysis in murine primary adipocytes is rife with experiments performed under conditions not optimized for reproducible and reliable results. Here, we present conditions for optimizing the measurement of lipolysis in murine adipocytes. We demonstrate that adenosine management is of paramount importance in evaluating the lipolytic response under basal and stimulated conditions. Also, adipocyte concentrations in the 10,000-15,000 cells per milliliter range produce a greater increase in stimulated lipolysis than higher concentrations, and the response is further enhanced by agitating the cells.

The postnatal development of neocortical neurons and glial cells in the Göttingen minipig and the domestic pig brain

The first mathematically unbiased estimates of neocortical cell numbers are presented from the developing pig brain, including a full description of tissue processing and optimal sampling for application of the stereological optical fractionator method in this species. The postnatal development of neocortical neurons and glial cells from the experimental Göttingen minipig was compared with the postnatal development of neocortical neurons in the domestic pig. A significant postnatal development was observed in the Göttingen minipig brain for both neuronal (28%; P=0.01) and glial cells (87%; P<0.01). A corresponding postnatal development of neurons was not detected in the domestic pig brain. The reason for this strain difference is not known. The mean total number of neocortical neurons is 324 million in the adult Göttingen minipig compared with 432 million in the domestic pig. The glial-to-neuron cell ratio is around 2.2 in the adult Göttingen minipig. Based on these results, the domestic pig seems to be a more suitable model for evaluating the effects of developmental insults on human brain growth and neuronal development than the Göttingen minipig.

Design-Based Sampling and Quantitation of the Respiratory Airways

Design-based quantitation of the nasal cavity, larynx and tracheobronchial conducting airways after exposure to inhaled toxicants requires complete measurement of all respiratory airways or appropriate sampling followed by morphometric measurements. In vivo imaging (MRI or CT) of the nasal cavity, larynx and conducting airways provides anatomical detail of all the airways down to the distal airways. Since inhaled toxicants show predictable deposition patterns in the airways, identification and sampling of conducting airways becomes essential in a precise toxicological evaluation. Lengths, diameters and luminal surface areas can be directly measured on fixed specimens using a stereomicroscope. Estimates of cell numbers, extracellular matrix volumes and vessel/nerve lengths per airway or epithelial basal laminar surface are estimated stereologically. Selected airways are cut into smaller pieces using a "fractionator" for uniform sampling of the airways. Cell numbers are estimated using a "disector." Volumes are estimated using point probes, while length and surface areas are estimated by isotropically oriented sections with plane and line probes; an approach free of assumptions of shape, size or spatial orientation. True biological variance and the average sampling variance of the stereological measurement define the minimal sampling required to achieve precise estimates of the nasal cavity, larynx and conducting airways.

Estimation of Cell Number by Hemocytometry Counting

The number of mammalian cells in a defined volume of medium can be measured using a hemocytometer. Automated methods using cell-counting devices such as those produced by Coulter are desirable when large numbers of individual samples are to be counted. A hemocytometer contains two chambers, each of which when filled and coverslipped contains a total volume of 9 µL. Each chamber is ruled into nine major squares, and each square is 1 × 1 mm with a depth of 0.1 mm. Thus, when coverslipped, the volume of each square is 0.1 mm3 or 0.1 µL. Additional subdivisions of the major nine squares are not necessary for counting and can be ignored.

Measuring Morphological and Cellular Changes in Alzheimers Dementia: A Review Emphasizing Stereology

From a clinical as well as a neuropathological point of view Alzheimer's disease (AD) has been the focus of intense research for more than three decades. Most studies to identify morphometric correlates with the declining cognitive function in normal aging and AD have employed semi-quantitative methods to assess neuropathological markers such as neurofibrillary tangles, senile plaques, neuronal, or glial cell densities, and neuron sizes. To this end, many cell counting methods have employed two-dimensional designs in single sections, yielding estimates of cell numbers either as neuron densities (number of cell profiles per area) or estimates of the size distribution of neuron profiles in columns vertical to the cortical surface. This approach gives rise to difficulties in interpretation because of the three-dimensional size, shape, and orientation of the counted cells, and the effect of shrinkage artifacts. Modern stereological techniques offer a more rigorous approach for quantifying neuropathological changes associated with aging and degenerative disease. However the stereological studies also suffer from the limitations of high biological variability in AD-type neuropathology, and the relative scarcity of autopsied brains from well-studied non-demented comparison subjects. As a result, the clinicopathological associations between neuropathology and indices of cognitive performance in aging and AD are not yet firmly established. The requirement for the proper description of morphologic neuropathology of AD is clear: any macroscopic or microscopic abnormalities, are subtle and must consequently be demonstrated reproducibly in well-controlled studies. In this review we try to evaluate which, if any, of the contemporary claims for morphometric brain abnormalities in AD can be said to be well established, with special emphasis placed on human stereological post-mortal studies.

Number of germ cells and somatic cells in human fetal ovaries during the first weeks after sex differentiation

This study presents the number of germ cells and somatic cells in human fetal ovaries during week 6 to week 9 post conception, i.e. the first weeks following sex differentiation of the gonads. One ovary with attached mesonephros from each of 11 individual legal abortions was used for estimation of cell numbers. After recovery of the fetus, the ovary-mesonephric complexes were immediately isolated, fixed and processed for histology. A stereological method was utilized to estimate the total number of oogonia in all ovaries and somatic cells in seven of them. The number of oogonia per ovary increased from approximately 26,000 in week 6 to approximately 250,000 in week 9 and somatic cells from approximately 240,000 to approximately 1.4 x 10(6). The ratio of oogonia to somatic cells tended to increase throughout the period. The concentration of oogonia was similar in the cranial (mesonephric connected) part and the caudal part of the ovaries. This is the first stereological estimation of the number of oogonia and somatic cells in human fetal ovaries, and the first estimation of germ cells and somatic cells in ovaries aged <9 weeks. The number of oogonia in week 9 is comparable to the numbers previously published based on non-stereological estimations. We found early stages of meiosis in fetal ovaries from week 9.

Developmental improvement in the regularity and packing of mouse horizontal cells: Implications for mechanisms underlying mosaic pattern formation

The present investigation has sought to determine whether the population of retinal horizontal cells undergoes an increase in the precision of its mosaic patterning during postnatal development, and if so, whether this increase is compatible with three different mechanistic accounts of retinal mosaic formation. Horizontal cells were labeled with antibodies to neurofilaments or calbindin at different developmental stages, and then visualized in retinal wholemounts. Multiple fields were sampled from each retina to determine horizontal cell density, while the X-Y coordinates of each cell in a field were determined. An estimate of total horizontal cell number was calculated for each retina, while the Voronoi domain regularity index and the packing factor were computed for each field. Two strains of mice showing a two-fold difference in the size of their horizontal cell population in maturity were sampled, C57BL/6J and A/J. Horizontal cell number in C57BL/6J was approximately twice that observed in A/J at all postnatal stages, with neither strain showing an effect of age on horizontal cell number. In both strains, however, the Voronoi domain regularity index and the packing factor were significantly lower at P-1 relative to later developmental stages. These results show that accounts of mosaic formation proposing the selective death of irregularly positioned cells, or the periodic occurrence of fate-determining events, are insufficient to establish the final patterning achieved by horizontal cells. Rather, they support the hypothesis that tangential dispersion enhances mosaic patterning during postnatal development.

Differential Regulation of Vascular Endothelial Growth Factor (VEGF-A) Secretion by Insulin and Insulin-Like Growth Factors (IGF) in Human Luteinizing Granulosa Cells (LGC) Following Controlled Ovarian Stimulation (COS) in a Polycystic Ovarian (PCOS) Population

To investigate the role of IGF-I and -II in the physiological regulation of VEGF-A production by LGC from preovulatory follicles in PCOS patients in the presence and absence of LH. A prospective, comparative, experimental study In an IRB approved protocol, LGC were collected via follicular aspiration from women (n=6) with PCOS undergoing COS for in-vitro fertilization. The diagnosis of PCOS was established using the ASRM/ESHRE consensus guidelines. The mean age of the study population was 30.2 ± 0.82 years. LGC were isolated and plated at 50,000 cells/well on fibronectin in DMEM-Ham’s F-12 medium with 5 mu;g/ml transferrin, 5 ng/ml selenium, 10 μg/ml aprotinin, 25 mu;g human LDL. Cells were incubated with 0, 1, 10, and 100 ng/ml of r-h IGF-1, IGF-II, and insulin (Sigma) in the presence or absence (control) of 100 ng/ml LH (Ares Serono). LGC were cultured for 72 hours and media were collected daily and assayed for free VEG-F (Quantikine VEGF ELISA, R&D Systems) and progesterone (P). After 3 days, wells were fixed with a Crystal Violet stain and analyzed for DNA content to estimate cell numbers. Data were normalized to DNA content and analyzed using a linear regression model, a mixed effects model, and adjusted using the Bonferroni method for multiple comparisons Exposure to IGF-I, IGF-II and insulin typically increased (p<0.05) DNA content in cell cultures, necessitating the normalization of P and VEGF levels to DNA content. The presence of 1-100 ng/ml IGFs or insulin did not alter control or LH-stimulated P levels during 3 days of culture. Under control conditions, LGCs secreted detectable levels of VEGF-A throughout culture. Addition of IGF-I, IGF-II or insulin (1-100ng/ml) caused a dose-dependent increase (p<0.05) in VEGF levels by day 2-3 of culture. In the absence of LH, physiological levels (1-10 ng/ml) of IGF-I or -II did not increase VEGF concentrations, whereas the same levels of insulin tended to increase (p=0.02) VEGF at 72 hrs. In the presence of LH, again there was no effect of 1-10 ng/ml IGF-I or II at 2-3 days, but insulin significantly (p<0.001) increased VEGF concentrations during this interval. Notably, these results are markedly different from those we reported earlier using LGC from non-PCOS patients during IVF protocols (Fertil Steril Supp 3, 80: S278, abstract P477, 2003): (1) IGFs and insulin did not markedly alter DNA content during 3-day culture, and (2) at 1-10 ng/ml, both IGF-I and -II, but not insulin, increased VEGF levels in the culture media. Exposure to both insulin and IGFs, as well as LH, for greater than 24 hours promotes VEGF-A secretion by LGC from PCOS women. Thus, both endocrine and local factors play a role in regulating VEGF production by the ovulatory, luteinizing follicle. However, there appear to be differences in responses between cells from non-PCOS and PCOS women, with the latter more sensitive to insulin and less sensitive to IGFs in terms of granulosa cell VEGF-A production, particularly in the presence of LH. Insulin and IGFs also selectively promoted cell proliferation of granulosa cells from PCOS women. Whether these characteristics reflect inherent differences in the maturing follicle, or the responses from treatment during COS in women with PCOS remains to be established.

Isotropic Fractionator: A Simple, Rapid Method for the Quantification of Total Cell and Neuron Numbers in the Brain

Stereological techniques that estimate cell numbers must be restricted to well defined structures of isotropic architecture and therefore do not apply to the whole brain or to large neural regions. We developed a novel, fast, and inexpensive method to quantify total numbers of neuronal and non-neuronal cells in the brain or any dissectable regions thereof. It consists of transforming highly anisotropic brain structures into homogeneous, isotropic suspensions of cell nuclei, which can be counted and identified immunocytochemically as neuronal or non-neuronal. Estimates of total cell, neuronal, and non-neuronal numbers can be obtained in 24 h and vary by <10% among animals. Because the estimates obtained are independent of brain volume, they can be used in comparative studies of brain-volume variation among species and in studies of phylogenesis, development, adult neurogenesis, and pathology. Applying this method to the adult rat brain, we show, for example, that it contains approximately 330 million cells, of which 200 million are neurons, and almost 70% of these are located in the cerebellum alone. Moreover, contrary to what is commonly assumed in the literature, we show that glial cells are not the majority in the rat brain.

A volumetric screening procedure for the Göttingen minipig brain

A screening procedure was developed to provide quantitative estimates of structural parameters, regional volumes and neuron number, in a neurotoxicologic study of the Göttingen minipig brain. The study material consisted of normal controls and brains collected from young minipigs which had been exposed in utero to the mitotic inhibitor methylazoxymethanol acetate (MAM). Based on stereological principles and systematic sampling techniques, volumetric data from pre-selected regions of the pig brain was obtained using Cavalieri's principles and point-counting. Secondarily, estimates of total hemispheric neocortical cell numbers were obtained from pre-selected groups to test the potential effect of MAM on neuron number. No significant differences were observed in volume of the pre-selected regions of MAM intoxicated pigs nor in estimates of total neocortical neuron number.

Simultaneous enumeration of different bacteria using reverse sample genome probing technique

Quantitative reverse sample genome probing (RSGP) with λDNA as an internal standard was used to enumerate the total numbers of Rhizobium sp. CCRC 13560, Rhizobium meliloti CCRC 13516 and Bradyrhizobium sp. CCRC 13585. K λ / Kx ratios varied between the three species but also in response to the amounts of λDNA or genomic DNA used in the labeling mixture or fixed upon the membrane. Comparative enumerations of pure cultures revealed higher counts using genomic probing as compared with growth-based colony forming units (CFU; 3.4±1.7-fold higher for R. meliloti, 6.4±7.8-fold higher for Rhizobium sp. and 0.34±0.17-fold higher for Bradyrhizobium sp.). In mixed cultures, the estimated cell numbers using genomic probing were 126±172-, 85±83- and 4.0±3.4-fold higher (same respective order) than the growth-based assay. By replacing the k λ / k x ratio with k′ λ / k′ x (slope from signal intensity of differently diluted λDNA/slope from signal intensity of differently diluted target DNA× f x / f λ ), significant improvement in the accuracy of the estimation was achieved. The calculated cell numbers via the genomic probe technique were 0.99±0.13-, 1.25±0.23- and 0.18±0.11-fold higher than the respective CFUs in pure cultures of R. meliloti, Rhizobium sp. and Bradyrhizobium sp. In samples containing mixed populations, the estimated numbers from genomic probing were 1.25±0.51-, 45.9±14.8- and 0.27±0.07-fold higher than the CFU-derived cell count (same respective order).

Estimators of the precision of stereological estimates: An example based on the CA1 pyramidal cell layer of rats

Because of the complex and dynamic structure of the brain, there is perhaps no other organ system in which the application of stereological methods can contribute so much with regard to understanding normal and pathological processes. In order to design the studies in an optimal manner, with regard to the number of individuals, sections, probes, and to be able to critically evaluate the stereological studies made by others, it is important for neuroscientists to have an understanding of the precision or reproducibility of a stereological estimation procedure. This precision or reproducibility is often referred to as the coefficient of error of the estimate, which is a statistical expression for the size of the standard error of the mean of repeated estimates, relative to the mean of the estimates. Like the ‘margin of error’ associated with public opinion polls, it indicates how much the estimate would vary if it were repeated numerous times. It is difficult and time consuming to empirically derive the coefficient of error of estimates made of features observed in histological preparations. To overcome this obstacle, it is common practice to try to get a feeling for the precision of an estimate by estimating the coefficient of error itself. In this paper, we will compare and discuss the coefficient of error of estimates of volume and cell number made with different numbers of sections and probes in the CA1 pyramidal cell layer of the rat hippocampus. The conclusions drawn from this analysis indicate that, using practically feasible and anatomically sensible sampling schemes, the Gundersen–Jensen coefficient of error estimator or the ‘Split-Sample’ coefficient of error estimator can provide useful information about the precision of stereological estimates even in highly irregular brain regions and requires little work.

Sex differences in the stereological parameters of the hippocampal dentate gyrus of the guinea-pig before puberty

Studies in rats and mice have shown several sex-dependent functional and structural differences in the hippocampal region, a brain structure playing a key role in learning and memory. The aim of the present study was to establish whether sex differences exist prior to puberty in the stereological parameters of the dentate gyrus in the guinea-pig, a long-gestation rodent, whose brain is at a more advanced stage of maturation at birth than the rat and mouse. The number of granule cells and volumes of the granule cell layer, molecular layer and hilus were evaluated in Nissl-stained brains of neonatal (15–16 days old) and peripubescent (45–46 days old) guinea-pigs. Based on a pilot study, the optical disector method was preferred to the optical fractionator method to estimate cell number. For volume ( V ref) estimation with the Cavalieri principle, contour tracing was preferred to the point counting method, as the latter appeared to underestimate volumes. The results showed that neonatal males had more granule cells than females in both the dorsal and ventral dentate gyrus and a larger volume in all layers. Peripubescent males had a larger volume of the granule cell layer than females in both the dorsal and ventral dentate gyrus, more granule cells in the ventral dentate gyrus, a larger volume of the hilus in both the dorsal and ventral dentate gyrus and a larger volume of the molecular layer in the ventral dentate gyrus. The results show that sex differences are present in the guinea-pig dentate gyrus prior to puberty and go in the same direction at both investigated ages, with males exhibiting more granule cells and larger volumes than females. The widespread distribution of these sex differences suggests that in the guinea-pig, similarly to other rodents, hippocampus-dependent functions may be sexually dimorphic.

S100P INCREASES PANCREATIC CANCER GROWTH AND INVASION.

Background: Pancreatic cancer is the most lethal of major cancers. Our previous studies indicated that S100P is highly expressed in pancreatic cancer when compared with normal pancreas and pancreatitis. In the current study, we investigated the role of S100P in pancreatic cancer growth and invasion. Methods: The influence of S100P on pancreatic cancer growth and invasion in vitro and in vivo was evaluated using wild-type and S100P transfected Panc1 cells, which have low endogenous levels, and wild-type and S100P silenced BxPC3 cells, which have high endogenous levels. S100P silencing was accomplished using an SiRNA. Cell proliferation and invasion were analyzed using MTS to estimate cell numbers in vitro. Tumor growth and invasion in vivo were examined using immune compromised mice. Tumor growth was compared between subcutaneous tumors formed from wild-type and S100P expressing Panc-1 cells. Tumor invasion was evaluated by bioluminescence imaging of orthotopically implanted SiRNA silenced and wild-type BxPC3 cells. Results: Expression of S100P stimulated cell proliferation of Panc1 cells in vitro by 164±25% at 96h. Reduction of S100P levels in BxPC3 cells using SiRNA decreased the proliferation rate in vitro by 129±22.5% within 96h. In vitro cell invasion studies indicated that S100P stimulated Panc1 cell invasion by 198±49% and silencing of S100P expression in BxPC3 cells decreased invasion by 221±51% of control. In vivo studies indicated that expression of S100P in Panc1 cells increased tumor volume by 511±140%. Bioluminescence imaging revealed that silencing of S100P expression in BxPC3 cells caused a 10-fold reduction in tumor growth and reduced invasion to liver by 50% (6 vs 3 animals) and to lung by 100% (1 versus 0 animals). Conclusions: These data indicate that S100P expression by pancreatic cancer cells stimulates cancer growth and invasion. Therefore, this molecule may be a useful target for the development of new therapies for this deadly tumor.

Detection and Enumeration of Sulphate-Reducing Bacteria in Estuarine Sediments by Competitive PCR

The distribution of sulphate-reducing bacteria (SRB) in the sediments of the Colne River estuary, Essex, UK covering different saline concentrations of sediment porewater was investigated by the use of quantitative competitive PCR. Here, we show that a new PCR primer set and a new quantitative method using PCR are useful tools for the detection and the enumeration of SRB in natural environments. A PCR primer set selective for the dissimilatory sulphite reductase gene (dsr) of SRB was designed. PCR amplification using the single set of dsr-specific primers resulted in PCR products of the expected size from all 27 SRB strains tested, including Gram-negative and positive species. Sixty clones derived from sediment DNA using the primers were sequenced and all were closely related with the predicted dsr of SRB. These results indicate that PCR using the newly designed primer set are useful for the selective detection of SRB from a natural sample. This primer set was used to estimate cell numbers by dsr selective competitive PCR using a competitor, which was about 20% shorter than the targeted region of dsr. This procedure was applied to sediment samples from the River Colne estuary, Essex, UK together with simultaneous measurement of in situ rates of sulphate reduction. High densities of SRB ranging from 0.2 − 5.7 × 108 cells ml− 1 wet sediment were estimated by the competitive PCR assuming that all SRB have a single copy of dsr. Using these estimates cell specific sulphate reduction rates of 10− 17 to 10− 15 mol of SO4 2 − cell− 1 day− 1 were calculated, which is within the range of, or lower than, those previously reported for pure cultures of SRB. Our results show that the newly developed competitive PCR technique targeted to dsr is a powerful tool for rapid and reproducible estimation of SRB numbers in situ and is superior to the use of culture-dependent techniques.

158APOPTOSIS IN IN VITRO PRODUCED BOVINE EMBRYOS ACCORDING TO DEVELOPMENTAL KINETICS

Apoptosis has been previously reported in embryos during late pre-implantation development. Fast-developing embryos are known to present higher developmental competence. The aim of the present work was to evaluate the quality of in vitro-produced bovine embryos with fast (8-cells at 48 hours post-insemination (hpi) and slow (8-cells at 90hpi) cleavage and study the correlation of this phenotype with programmed cell death occurrences. Embryos were produced from immature oocytes obtained from slaughtered cow ovaries, after maturation and fertilization, presumed zygotes were cultured in CR2 medium with 10% FCS, together with granulosa cells under 5% CO2 atmosphere. The number of nuclei in the inner cell mass and trophectoderm (ICM/TE), as well as the number of nuclei with fragmented DNA, were estimated by applying differential staining and TUNEL, respectively; data were analyzed by ANOVA (JMP—SAS Institute). To test the expression of apoptosis regulating genes, a pool of fifty 8-cell embryos from each group (fast and slow) were collected. After RNA extraction and reverse transcriptase reaction, cDNA was amplified with Bax and Bcl2 primers, individually. Results indicated, as expected, higher quality in fast-cleaving embryos, estimated by the number of ICM nuclei (20.8±1.4 and 15.6±2.1—P≤0.05); however, the number of TE didn’t show significant differences (54.9±2.4 and 53.2±3.8); the same was observed for total cell number (75.7±2.8 and 68.8±4.4). The frequency of blastocyst TUNEL-positive nuclei as an estimate of total cell number was significantly larger in the slow group when compared to the rapid development group (19.0±2.5% and 8.5±1.4%, respectively, P≤0.05). The greater proportion of morphologic abnormal nuclei in both groups was located in the ICM, and may explain the lower number of ICM nuclei in slow developing embryos. Hence, embryos of slow development show TUNEL-positive blastomeres at the 8-cell stage, but no fragmented nuclei were observed in embryos at 48hpi. Bax and Bcl2 cDNA amplification showed that both mRNAs were constitutively present at the 8-cell stage in both groups. It can be concluded that in vitro-produced bovine blastocysts, with slow development to the 8-cell stage, present lower quality compared with fast development homologues, estimated by mean number of ICM nuclei, as well as nuclei fragmentation in blastomeres (TUNEL-positive). There is a difference in fragmented nuclei proportion between both groups at the 8-cell stage, but this result may be biased by the numbers of hours in culture. It was possible to demonstrate the presence of mRNA for pro (Bax) and anti-apoptotic (Bcl2) genes in slow- and fast-developing embryos at the 8-cell stage, and the future determination of the ratio between these two transcripts may allow the evaluation of the participation of pre-transcriptional regulation of these genes on the induction of DNA fragmentation. Financial support: Grant 99/12351-3 FAPESP São Paulo, Brazil.

Insulin and Insulin-Like Growth Factors (IGFs) promote Vascular Endothelial Growth Factor (VEGF-A) secretion by human luteinizing granulosa cells following Controlled Ovarian Stimulation (COS)

Objective: To investigate the role of insulin-related growth factors and luteinizing hormone (LH) in the physiological regulation of VEGF-A production by luteinized granulosa cells (LGC) from preovulatory follicles in humans. Design: Prospective, comparative, experimental study. Materials and Methods: LGC were obtained from women (n=6), with IRB approval, by follicular aspiration after undergoing COS for in-vitro fertilization. Subjects (33.5 ± 1.2 years, mean age ± SEM) had regular menstrual cycles with no evidence of PCOS or other endocrinopathies. LGC were isolated and plated at 50,000 cells/well on fibronectin in DMEM-Ham’s F-12 medium with 5 μg/ml transferrin, 5 ng/ml selenium, 10 μg/ml aprotinin and 25 μg/ml human LDL. Cells were incubated in the presence and absence of 100 ng/ml recombinant human (r-h) LH (Ares Serono) and 0, 1, 10, 100 ng/ml of r-h IGF-1, IGF-II or insulin (Sigma). Media were collected daily and assayed for free VEGF-A (Quantikine VEGF ELISA, R&D Systems). After 3 days (d), cells were fixed, Crystal Violet stained and analyzed for DNA content to estimate cell numbers. Data were analyzed by two-way repeated ANOVA, using a randomized block design. Results: In the absence of LH or insulin-related growth factors, LGCs secreted low but detectable levels of VEGF-A through d 3 of culture (179–304 pg/ml on d 3). However, exposure to IGF-I or -II resulted in a dose-related increase in VEGF-A levels by d 1 (e.g., 568 vs 183 pg/ml or 453 vs 278 pg/ml,100 ng/ml IGF-I or-II vs controls, respectively; p<0.05), which was sustained through d 3 (1001 vs 179 pg/ml or 701 vs 255 pg/ml, p<0.05). Conversely, the addition of insulin (100 ng/ml) did not increase VEGF-A levels until d 2 (737 vs 436 pg/ml, p<0.05). Although LH alone did not increase VEGF-A levels above controls on d 1–3, the combination of LH and insulin-related growth factors increased VEGF-A levels to a greater extent than IGFs alone at all doses (1181 vs 717 or 800 vs 567 pg/ml, LH + 10 ng/ml IGF-I or -II vs IGF-I or -II alone, respectively, on d 2; p<0.05). The synergistic effect of LH with IGF-I/-II was evident earlier on d 2 and at lower doses (10 ng/ml), compared to insulin and LH, which was not significant until d 3. There was no change in DNA content following incubation for 3 days in the presence of IGF-I, -II, insulin or LH when compared to controls. However, DNA content decreased (p<0.05) in the presence of LH + IGF-II at the maximum dose. Conclusion: Exposure to both insulin-related growth factors and LH is required for optimal VEGF-A secretion by human LGC. Thus, local (IGFs) as well as endocrine (LH, insulin) factors may combine to regulate VEGF-A production, and thus angiogenesis and vascular permeability, in the primate ovulatory follicle and corpus luteum. Evidence of insulin/IGFs, as well as LH, regulation of VEGF-A production supports further studies on their role in regulating angiogenic factors during COS cycles in women with endocrinopathies, such as PCOS. Supported by: NICHD U54 HD18185, RR 00163.

Estimation of absolute microglial cell numbers in mouse fascia dentata using unbiased and efficient stereological cell counting principles

Stereology offers a set of unbiased principles to obtain precise estimates of total cell numbers in a defined region. In terms of microglia, which in the traumatized and diseased CNS is an extremely dynamic cell population, the strength of stereology is that the resultant estimate is unaffected by shrinkage or expansion of the tissue. The optical fractionator technique is very efficient but requires relatively thick sections (e.g., > or =20 microm after coverslipping) and the unequivocal identification of labeled cells throughout the section thickness. We have adapted our protocol for Mac-1 immunohistochemical visualization of microglial cells in thick (70 microm) vibratome sections for stereological counting within the murine hippocampus, and we have compared the staining results with other selective microglial markers: the histochemical demonstration of nucleotide diphosphatase (NDPase) activity and the tomato lectin histochemistry. The protocol gives sections of high quality with a final mean section thickness of >20 microm (h=22.3 microm +/- 0.64 microm), and with excellent rendition of Mac-1+ microglia through the entire height of the section. The NDPase staining gives an excellent visualization of microglia, although with this thickness, the intensity of the staining is too high to distinguish single cells. Lectin histochemistry does not visualize microglia throughout the section and, accordingly, is not suited for the optical fractionator. The mean total number of Mac-1+ microglial cells in the unilateral dentate gyrus of the normal young adult male C57BL/6 mouse was estimated to be 12,300 (coefficient of variation (CV)=0.13) with a mean coefficient of error (CE) of 0.06. The perspective of estimating microglial cell numbers using stereology is to establish a solid basis for studying the dynamics of the microglial cell population in the developing and in the injured, diseased and normal adult CNS.