A PLastic MIcrostrainer for HAndling MAmmalian BLastocysts

Friend (1963) has described a microstrainer for handling microscopic marine ova which permits direct transfer of batches of ova through various histological solutions without centrifugation or decanting. This strainer consists of a length of glass tubing with an electron microscope grid cemented over one end. In our laboratory, several modifications of this device have been made for use in handling mammalian ova and blastocysts. The most satisfactory model, which can be made easily, was contructed from BEEM plastic capsules (Better Equipment for Electron Microscopy, Inc., P.O. Box 132 Jerome Avenue Station, Bronx, New York 10468) and circular electron microscope grids of suitable mesh size.     

Consistent micro, macro and state-based population modelling

A population system can be modelled using a micro model focusing on the individual entities, a macro model where the entities are aggregated into compartments, or a state-based model where each possible discrete state in which the system can exist is represented. However, the concepts, building blocks, procedural mechanisms and the time handling for these approaches are very different. For the results and conclusions from studies based on micro, macro and state-based models to be consistent (contradiction-free), a number of modelling issues must be understood and appropriate modelling procedures be applied. This paper presents a uniform approach to micro, macro and state-based population modelling so that these different types of models produce consistent results and conclusions. In particular, we demonstrate the procedures (distribution, attribute and combinatorial expansions) necessary to keep these three types of models consistent. We also show that the different time handling methods usually used in micro, macro and state-based models can be regarded as different integration methods that can be applied to any of these modelling categories. The result is free choice in selecting the modelling approach and the time handling method most appropriate for the study without distorting the results and conclusions.     

Contrast in levels of metabolic enzymes in human and mouse ova

A methodology is described for analyzing single human ova for 8 or 9 different metabolic enzymes, or 4 or 5 enzymes plus as many metabolites. This overcomes an obstacle to the study of human ovum metabolism: the severe limitation of usable material. Results obtained with this methodology, applied to discarded specimens from an in vitro fertilization program, indicate that in spite of imperfections these ova can provide a valid picture of the metabolic characteristics of normal human ova. Data are presented for 17 enzymes from 8 metabolic pathways in human and mouse ova. Relative to size, 10 of the enzymes were substantially higher in human than mouse ova. Most dramatically so were 2 enzymes of fatty acid metabolism (10-fold and 15-fold), hexokinase (9-fold), and aspartate aminotransferase (19-fold). This suggests that major species differences in metabolism are present. The validity of the human data, in spite of restriction to discarded material, is supported by (1) consistency of results among most of the ova, 2] concordance between average levels with those of rare specimens that were discarded because sperm were not available, and (3) the presence of adenosine triphosphate (ATP) concentrations similar to those of normal mouse ova. Surprisingly, both human and mouse ova contain phosphocreatine at levels nearly equal of those of ATP.     

REPRODUCTION SEXUÉE D'UNE ASTÉRIE FISSIPARE,SCLERASTERIAS RICHARDI (PERRIER, 1882) / SEXUAL REPRODUCTION IN A FISSIPAROUS ASTEROID,SCLERASTERIAS RICHARDI (PERRIER, 1882)

Sclerasterias richardi, a relatively deep sea asteroid (140–200 m) from the border of the Mediterranean continental shelf, is characterized by an asexual reproduction by fissiparity concomitant with a functional sexuality.

A monthly sampling of a population from Calvi (Corsica) has allowed a study of the complete sexual cycle from 354 histologically-treated specimens.

The 218 sexually defined animals (62% males, 38% females) show strict gonochorism. In males, spermatogenesis is cyclic and sexual maturity seems to be reached before that of the females. In females, the different stages of oogenesis are well marked: oogonia and parietal oocytes disappear only at maturity. Oligolecithic oocytes (120–150 μn) show a synchronous growth.

The annual reproductive cycle is well defined in both sexes with one spawning period from mid-September to mid-October.

After spawning, a resting period (from mid-October to mid-January) occurs during which unspawned oocytes are phagocytized by more or less isolated accessory cells. These phagocytic cells have never been found in male specimens.

Each month the presence of specimens without gonads or unsexable individuals is one of the characteristics of this cycle. Their high proportion during the organization stage and after spawning can be easily explained. In March they are frequent too, owing to the infestation of gonads by Ciliates.

As shown by our samples, the bottom water temperature is nearly the same during the whole year and cannot be directly involved as the dominant exogenous variable stimulating spawning.

As a consequence of fissiparity which affects the main part of the population there is a great inter- and intra-individual variability.

The reproductive potentiality is low: as a female emits approximatly 400–500 ova whose development produces planktotrophic larvae with a long pelagic life, it is clear that sexual reproduction is accessory in comparison with asexual reproduction by fission.     

Green fluorescent protein-based system for analysis of E-selectin-mediated adhesion

Numerous cell-based or cell-free systems for study of selectin adhesion use radiolabeled tracers. However, in addition to handling problems associated with the use of radioisotopes, these assays have difficulty relating a number of counts to a number of adherent cells. Here, we describe an assay that uses the natural fluorescence of the green fluorescent protein (GFP) to measure binding of cells to E-selectin. We elaborated an adhesion system composed of a cell monolayer expressing E-selectin ligand to which monodispersed fluorescent Chinese hamster ovary (CHO) cells expressing E-selectin are added. Due to GFP autofluorescence, adhered cells can be easily distinguished from cell monolayers by fluorescence microscopy, and adhesion can be measured by cytofluorometry. We applied this GFP-based adhesion assay to measure the adherence of a pancreatic tumor cell line and found that the binding parameters of these cells satisfy a number of E-selectin-specific criteria.     

Immunocytochemistry of cerebrospinal fluid

In order to determine how best to study cells in cerebrospinal fluid (CSF) by immunocytochemical techniques, several crucial technical variables and five immunocytochemical methods were examined. Immunocytochemical studies could be performed on either cell suspensions or smears. The method using cell suspensions was more sensitive, producing less background staining, but requiring more cells than that using smears. Among the five methods examined, indirect immunoperoxidase (IP) and indirect immunoalkaline phosphatase (IAP) were comparable in sensitivity. The peroxidase-antiperoxidase (PAP), alkaline phosphatase-antialkaline phosphatase (APAAP) and avidin-biotin complex-immunoalkaline phosphatase (ABC-AP) methods were comparable in sensitivity and were more sensitive than either the IP or IAP technique. The peroxidase methods were plagued with problems related to endogenous enzyme activity and the ABC-AP method may exhibit undesirable background staining. Therefore, the IAP method should be used for cell suspensions and the APAAP for cells on smears. In CSF specimens with a small number of cells, immunocytochemical studies should be done on smears by the APAAP method. These conclusions are supported by our experience with CSF specimens from patients with reactive and neoplastic lymphocytoses.     

Editorial note

Abstract

The histology laboratory can face many challenges when small, often critical, specimens of cultured cells are submitted for specialized immunocytochemical studies or special stains. Although clinical pathology labs often receive cell preparations, these usually contain enough cells so that pellets can be formed by centrifugation, and the pellets directly embedded and sectioned. Research labs, however, often need to submit very small samples of cells for experimental studies. We summarize here a number of techniques that currently are available and methods we have developed and/or adapted and used in our laboratory over the years. We describe the utility of multi-chambered slides for cell culture and histologic studies, multi-well cell culture plates, monolayer cell culture on specialized coated cell wells, cell well inserts, and agarose embedding techniques for small cultures of cells and for cultures that require antigen retrieval or multiple antibody localizations. Traditional double embedding techniques, such as the use of agar, are also cited.     

Techniques in the preparation of a monolayer of gynecologic cells for automated cytology. An overview

The computer-assisted microscope demands rigid specifications for specimen preparation. This paper addresses the variety of techniques developed by researchers attempting to automatically screen uterine cervical specimens. These same techniques could also be utilized for specimens from other body sites. In contrast to the easily prepared routine Papanicolaou smear, these techniques can be broken down into various steps as follows: transport media, cellular disaggregation, cell number estimation, cell separation and specimen enrichment, cellular adhesion to glass slide and cell transfer onto the slide. Variations on the theme are contrasted among specimen preparation methods utilized by prominent research groups. The plea for a simpler preparation method or, hopefully, utilization of the routine Papanicolaou smear for computer-assisted microscopy is made.     

水域生态学中生物稳定同位素样品采集、处理与保存

稳定同位素分析技术由于能够刻画复杂的食物网结构并追踪食物网中的能量流而成为水域生态学研究中的重要手段。但是当水生生物样品采集、处理和保存过程中存在不确定性时, 营养关系分析中的同位素结果可能会产生误导性解释。文章采用数据模拟分析和文献总结的方法, 研究了水域生态系统中样品采集、处理和保存对于稳定同位素的影响, 概括性地建议了水域生态系统中适合应用稳定同位素分析技术开展生态学研究的样品采集、处理和保存的注意事项。但今后仍需进一步评估样品采集、处理和保存对稳定同位素比值的影响效果, 确定化学动力学在水生生物样品采集、处理和保存中的作用, 以进一步完善水生生物样品的采集、处理和保存稳定同位素生态学研究规范。     

Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay

The activity of proteins is typically regulated by secondary modifications and by interactions with other partners, resulting in the formation of protein complexes whose functions depend on the participating proteins. Accordingly, it is of central importance to monitor the presence of interaction complexes as well as their localization, thus providing information about the types of cells where the proteins are located and in what sub-cellular compartment these interactions occur. Several methods for visualizing protein interactions in situ have been developed during the last decade. These methods in most cases involve genetic constructs, and they have been successfully used in assays of living cell maintained in tissue culture, but they cannot easily be implemented in studies of clinical specimens. For such samples, affinity reagents like antibodies can be used to target the interacting proteins. In this review we will describe the in situ proximity ligation assays (in situ PLA), a method that is suitable for visualizing protein interactions in both tissue sections and in vitro cell lines, and we discuss research tasks when this or other method may be selected.     

Clonal expansion of human pluripotent stem cells on gelatin-coated surface

The research of human pluripotent stem cells is important for providing the molecular basis for their future application to regenerative medicine. To date, they are usually cultured on feeder cells and passaged by partial dissociation with either enzymatic or mechanical methods, which are problematic for the research using them in the convenience and reproducibility. Here we established a new culture system that allows handling as easily as culturing feeder-free mouse ES cells. This newly developed culture system is based on the combinatorial use of ROCK inhibitor and soluble fibronectin, which enables us to expand human pluripotent stem cells from single cell dissociation on gelatin-coated surface without any feeder cells. In this new culture system, these human pluripotent stem cells can stably grow, even if in clonal density with keeping expression of stem cell markers. These cells also have abilities to differentiate into three germ layers in vivo and in vitro. Furthermore, no chromosomal abnormalities are found even after sequential passage. Therefore this system will dramatically simplify genetic engineering of these human pluripotent stem cells or defining process of their signal pathway.     

Effect of gonadotrophin dose on oocyte retrieval in superovulated BALB/c mice

Mice are commonly used animal models in reproductive and developmental research. In order to get satisfying results from such experiments, large numbers of ova must be available and this can be achieved by using various ovulation induction protocols. To obtain an optimal response from these stimulation protocols, parameters such as breeding-housing conditions of the animal strains, the best age for superovulation, and type and dose of gonadotrophins must be optimized. The aim of this study was to investigate the impact of exogenous stimulation with increasing amounts of gonadotrophins on the number and quality of oocytes/pre-embryos recovered from outbred BALB/c mice. A dose-response analysis was performed by stimulating prepubescent (21- to 25-day-old) and sexually mature (6 to 8 weeks old) female mice with hMG, which contains equal amounts of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The stimulation dose contained 5, 10, 15, 20, 25 or 30 IU of FSH/LH. The effect of increasing stimulation was assessed by monitoring the number and maturity of ova recovered from the tubes. The data were analyzed by using a one-way Anova test and student t-test. Increasing stimulation doses in the prepubescent females resulted in an increased number of ova. A maximum of 55 ova per mouse was reached when stimulating with 20 IU of FSH/LH; higher stimulation doses showed no further increase in oocyte recovery. In the prepubescent group, a maximal number of recovered mature ova was reached with 15 IU of FSH/LH. In the sexually mature female group, 20 IU of FSH/LH gave the best quantitative and qualitative results. Positive effects of copulation on the number and maturity of oocytes in all induction doses were more evident in the prepubescent females and these parameters were significantly more improved (P < 0.05) in this group when compared to the pubertal females. Our findings led to the conclusion that ovulation induction of prepubescent outbred BALB/c mice with 15 IU FSH/LH and sexually mature ones with 20 IU FSH/LH give the best results in terms of oocyte number and maturity.     

Costs of parental care in the gastropod Conus pennaceus: Age-specific changes and physical constraints

Summary Age-specific changes in the allocation of reproductive energy to protective capsules, ova and intracapsular fluid are documented for the marine gastropod Conus pennaceus. As female snails grow in shell length they produce larger capsules with thicker and stronger walls. Because large capsules contain lower densities of ova than do small ones, growing females must increase the number, as well as the size, of egg capsules they produce. As a result of this pattern of ova packaging, per ovum costs of encapsulation (parental care) increase with increasing female size and age. The data suggest that the number of embryos a capusle can support may be limited by respiratory constraints related to capsule surface area or wall thickness. As capsule size increases, surface/volume ratios decline and capsule wall thickness increases. Either of these processes should result in a reduction in net gas transport per unit of capsule contents.     

Gamma-ray irradiation promotes premature meiosis of spontaneously differentiating testis�Cova in the testis of p53-deficient medaka (Oryzias latipes)

In this study, the roles of p53 in impaired spermatogenic male germ cells of p53-deficient medaka were investigated by analyzing histological changes, and gene expressions of 42Sp50, Oct 4 and vitellogenin (VTG2) by RT-PCR or in situ hybridization in the testes. We found that a small number of oocyte-like cells (testis–ova) differentiated spontaneously in the cysts of type A and early type B spermatogonia in the p53-deficient testes, in contrast to the wild-type (wt) testes in which testis–ova were never found. Furthermore, ionizing radiation (IR) irradiation increased the number of testis–ova in p53-deficient testes, increased testis–ova size and proceeded up to the zygotene or pachytene stages of premature meiosis within 14 days after irradiation. However, 28 days after irradiation, almost all the testis–ova were eliminated presumably by p53-independent apoptosis, and spermatogenesis was restored completely. In the wt testis, IR never induced testis–ova differentiation. This is the first study to demonstrate the pivotal role of the p53 gene in the elimination of spontaneous testis–ova in testes, and that p53 is not indispensable for the restoration of spermatogenesis in the impaired testes in which cell cycle regulation is disturbed by IR irradiation.     

Morphology and fertilizability of frozen human oocytes

Human oocytes were frozen and thawed by four methods previously used for cryopreser-vation of human embryos. Most of these oocytes were inseminated after thawing to assess their capacity to fertilize and form pronuclear ova. Their morphology was assessed by phase-contrast microscopy used in routine IVF. Twenty-three oocytes were examined by electron microscopy to critically evaluate the effects of cooling and cryopreservation and to confirm fertilization. Morphological survival was observed in more than 60% of the oocytes examined after freeze-thawing. The main features of cryoinjury were cracks in the zona pellucida, disruption of the plasma membrane and extensive disorganization of the ooplasm. Subtle changes in the cytosol of cumulus cells was also observed. Cooling to 0°C or ?6°C had little effect on cytoplasmic structure. Spindles were damaged in two frozen oocytes. Cumulus cell activity, sperm binding to the zona, sperm penetration of the zona seem to be largely unaffected by freeze-thawing. Fertilization was observed in eight oocytes after postthaw insemination and three embryos (8-cell to morula stages) were developed from pronuclear ova on further culture. Both monospermic and polyspermic fertilization were confirmed by electron microscopy and micronuclei were detected in three pronuclear ova. The genetic implications of these nuclear aberrations are discussed. These preliminary studies indicate that oocyte freezing needs to be integrated cautiously with clinical IVF by further assessment of embryos developed from frozen oocytes.     

Osmotic response of oocytes using a microscope diffusion chamber: a preliminary study comparing murine and human ova

The hydraulic conductivity (Lp) of the plasma membrane determines how cells respond to the stresses of dehydration encountered during cryopreservation. We have used a microscope diffusion chamber which allows for direct real-time observation of the dynamic osmotic response of individual cells in microvolume suspension to compare the Lp of murine and human unfertilized ova. In this system, the response of an individual cell to the induced osmotic imbalance is documented via a series of photomicrographs or videotape; from these data the Lp can be computed. Donated human preovulatory oocytes were compared with macroscopically normal human ova, 43 hr after insemination, which had failed to fertilize (Ff) and with murine ova collected 13 hr post human chorionic gonadotropin injection. The permeability coefficients were 0.65 +/- 0.43, 0.84 +/- 0.39, and 0.36 +/- 0.07 micron3/micron2/atm/min. The results suggest that it may be possible to use Ff ova for experiments to design suitable cryopreservation procedures.     

Sequencing Degraded DNA from Non-Destructively Sampled Museum Specimens for RAD-Tagging and Low-Coverage Shotgun Phylogenetics

Ancient and archival DNA samples are valuable resources for the study of diverse historical processes. In particular, museum specimens provide access to biotas distant in time and space, and can provide insights into ecological and evolutionary changes over time. However, archival specimens are difficult to handle; they are often fragile and irreplaceable, and typically contain only short segments of denatured DNA. Here we present a set of tools for processing such samples for state-of-the-art genetic analysis. First, we report a protocol for minimally destructive DNA extraction of insect museum specimens, which produced sequenceable DNA from all of the samples assayed. The 11 specimens analyzed had fragmented DNA, rarely exceeding 100 bp in length, and could not be amplified by conventional PCR targeting the mitochondrial cytochrome oxidase I gene. Our approach made these samples amenable to analysis with commonly used next-generation sequencing-based molecular analytic tools, including RAD-tagging and shotgun genome re-sequencing. First, we used museum ant specimens from three species, each with its own reference genome, for RAD-tag mapping. Were able to use the degraded DNA sequences, which were sequenced in full, to identify duplicate reads and filter them prior to base calling. Second, we re-sequenced six Hawaiian Drosophila species, with millions of years of divergence, but with only a single available reference genome. Despite a shallow coverage of 0.37±0.42 per base, we could recover a sufficient number of overlapping SNPs to fully resolve the species tree, which was consistent with earlier karyotypic studies, and previous molecular studies, at least in the regions of the tree that these studies could resolve. Although developed for use with degraded DNA, all of these techniques are readily applicable to more recent tissue, and are suitable for liquid handling automation.     

The effects of low temperature on fertilized rabbit ova in vitro,and the normal development of ova kept at low temperature for several days

Fertilized rabbit ova at the 2-blastomere stage kept in rabbit serum were stored at low temperatures for various lengths of time. They were then cultured at 38 degrees C. for about 24 hours to determine their viability. A number of the viable ova were finally transplanted into recipient does. It was found that rapid cooling of ova to 5 degrees or to 0 degrees C. was more harmful to the subsequent viability of ova than slow cooling. Rapid cooling was not more lethal to the ova than slow cooling, but did prevent their future normal cleavage. There was no difference between those ova cooled rapidly or slowly to 10 degrees C. It was concluded that temperature shock has an adverse effect on ova, especially at the lower temperatures, though temperature shock can be remedied by acclimatization (slow cooling). Thus, the physiological significance of temperature shock would seem to be broadened. The optimal temperature for the storage of ova was investigated. It was found that 10 degrees C. was the best temperature; at this temperature viable ova were obtained after storage for 144 to 168 hours. At 0 degrees , 5 degrees , or 15 degrees C. the ova were viable for 96 to 120 hours, while at 22-24 degrees C., only for 24 to 48 hours. The percentage of dead ova was low at a favorable temperature, increasing only at the end of the storage period. At an unfavorable temperature, however, the rate of death increased steadily from beginning to end of storage. The percentage of abnormally cleaved ova (arrested cleavage and fragmentation) remained at a low level at first at a favorable temperature, but then increased just before or during death of the ova. A critical time for the viability, the abnormal cleavage, and the death of ova was characteristic of each temperature. About 24 to 28 per cent of the viable ova remaining after being stored at 0-15 degrees C. for 2 to 4 days and cultured at 38 degrees C. for 24 hours were capable of development into normal young. The compatibility of serum and ova, the absence of a correlation between the viability of the ova and the source of the fertilizing spermatozoa, and the fertilization of superovulated ova (i.e., the percentage of fertile does in follicular phase and in luteal phase, the percentage of unfertilized ova and of fertilized ova at different stages, the percentage of does that had produced a normal number of ova or had produced a large number of ova, etc.), are reported. The possibility of a more efficient utilization of the germ cells of valuable animals by means of the present techniques, and the possibility of a new approach to the experimental investigation of mammalian genetics and development, have been mentioned.     

Use of whole genome amplification to rescue DNA from plasma samples

While DNA of good quality and sufficient amount can be obtained easily from whole blood, buccal swabs, surgical specimens, or cell lines, these DNA-rich sources are not always available. This is particularly the case in studies for which biological specimens were collected when genotyping assays were not widely available. In those studies, serum or plasma is often the only source of DNA. Newly developed whole genome amplification (WGA) methods, based on phi29 polymerase, may play a significant role in recovering DNA in such instances. We tested a total of 528 plasma samples kept in storage at -40 degrees C for approximately 10 years for 8 single nucleotide polymorphisms (SNPs) using the 5' exonuclease (TaqMan) assay. These specimens yielded undetectable levels of DNA following extraction with an affinity column but produced an average 52.7 microg (standard deviation of 31.2 microg) of DNA when column-extracted DNA was used as a template for WGA. This increased the genotyping success rate from 54% to 93%. There were only 3 disagreements out of 364 paired genotyping results for pre- and post-WGA DNAs, indicating an error rate of 0.82%. These results are encouraging for expanding the use of poor DNA resources in genotyping studies.