Effects of Nanostructures and Mouse Embryonic Stem Cells on In Vitro Morphogenesis of Rat Testicular Cords

Morphogenesis of tubular structures is a common event during embryonic development. The signals providing cells with topographical cues to define a cord axis and to form new compartments surrounded by a basement membrane are poorly understood. Male gonadal differentiation is a late event during organogenesis and continues into postnatal life. The cellular changes resemble the mechanisms during embryonic life leading to tubular structures in other organs. Testicular cord formation is dependent on and first recognized by SRY-dependent aggregation of Sertoli cells leading to the appearance of testis-specific cord-like structures. Here we explored whether testicular cells use topographical cues in the form of nanostructures to direct or stimulate cord formation and whether embryonic stem cells (ES) or soluble factors released from those cells have an impact on this process. Using primary cell cultures of immature rats we first revealed that variable nanogratings exerted effects on peritubular cells and on Sertoli cells (at less than <1000 cells/mm²) by aligning the cell bodies towards the direction of the nanogratings. After two weeks of culture testicular cells assembled into a network of cord-like structures. We revealed that Sertoli cells actively migrate towards existing clusters. Contractions of peritubular cells lead to the transformation of isolated clusters into cord-like structures. The addition of mouse ES cells or conditioned medium from ES cells accelerated this process. Our studies show that epithelial (Sertoli cell) and mesenchymal (peritubular cells) cells crosstalk and orchestrate the formation of cords in response to physical features of the underlying matrix as well as secretory factors from ES cells. We consider these data on testicular morphogenesis relevant for the better understanding of mechanisms in cord formation also in other organs which may help to create optimized in vitro tools for artificial organogenesis.     

Recombination and the Progression of Meiosis in Saccharomyces Cerevisiae

Recombination is an essential part of meiosis; in almost all organisms, including Saccharomyces cerevisiae, proper chromosome segregation and the viability of meiotic products is dependent upon normal levels of recombination. In this article we examine the kinetics of the meiotic divisions in four mutants defective in the initiation of recombination. We find that mutations in any of three Early Exchange genes (REC104, REC114 or REC102) confer a phenotype in which the reductional division occurs earlier than in an isogenic wild-type diploid. We also present data confirming previous reports that strains with a mutation in the Early Exchange gene MEI4 undergo the first division at about the same time as wild-type cells. The rec104 mutation is epistatic to the mei4 mutation for the timing of the first division. These observations suggest a possible relationship between the initiation of recombination and the timing of the reductional division. These data also allow these four Early Exchange genes examined to be distinguished in terms of their role in coordinating recombination with the reductional division.     

The Capacity of Testicular Cells of the Postnatal Rat to Reorganize into Histotypic Structures

Cell reorganization experiments in vitro were performed with dissociated rat testes at different ages of postnatal development namely, newborn, 8–10, 18–25, 35–40, and 90 days. Only newborn and juvenile rat testicular cells reassociated into testicular-like organization in rotation culture. Puberal and adult rat testicular cells show morphogenetic organization when they were deprived of germ cells by busulphan pretreatment. A factor present in testicular tissue of puberal and adult rats inhibits reorganization. The inhibitor is confined to the spermatic cell fraction in the testis.     

Non-Exponential Growth By Mammalian Cells In Culture

The concept of exponential growth by mammalian cells in culture is based upon the apparent linearity of semilogarithmic data plots. This method of graphical analysis is known to be an unreliable test of the exponential hypothesis. We have re-examined the question of growth exponentiality using the more sensitive method of Smith plots, in which specific growth rate is plotted against either time or density on transformed graphical coordinates which linearize the mathematical expression of the growth hypothesis being tested. With exponential growth, data points fall on a horizontal straight line when specific growth rate is plotted against time or density. Using both our own and literature data, we have performed Smith plot analyses on the growth of 125 different mammalian and avian cell lines. of these, only eleven exhibited an exponential phase. the remaining cell lines all had non-exponential growth patterns. the most common of these consisted of an initial period of growth acceleration followed by a later phase of deceleratory growth. A smaller number of lines exhibited deceleratory kinetics at all times after plating. We conclude that mammalian cell growth in culture is predominantly non-exponential, and that the apparent exponentiality of semilogarithmic data plots is usually a methodological artifact.     

Testicular Morphology and Cell Proliferation Kinetics of Immature Germ Cells and Sertoli Cells In Suckling Undernourished Rats

Undernutrition during suckling was induced in newborn rats by increasing the litter size to sixteen pups to be fed by one mother. Animals reared in litters of eight served as controls. Undernourished animals showed retarded body and testicular growth during a suckling period of 22 days. Sequential morphogenesis of the testis was not altered up to 15 days of age. However, certain morphological alterations in Sertoli cells and Leydig cells were observed from 15 days onwards. Cell generation cycle of spermatogonial germ cells and supporting cells (future Sertoli cells) on day 9 showed marked prolongation of DNA synthetic phase (S), unaltered post-DNA synthetic phase (G₂) and total cycle (T_c) and shortening of the pre-DNA synthetic phase (G₁) indicating a depression in DNA synthesis in undernutrition.     

In Vivo Delivery of Antisense Oligodeoxynucleotides into Rat Kupffer Cells

Abstract

Kupffer cells play a key role in the pathogenesis of liver diseases. Liver injury is believed to result from an excessive release of cytokines and prostanoids from these cells. A targeted delivery of antisense oligonucleotides into Kupffer cells might reduce or prevent liver injury. In this report, we describe a method in which anionic liposome-encapsulated antisense phosphorothioate oligodeoxynucleotides (S-Oligos) are delivered to Kupffer cells in vivo. Delivery was assessed using an antisense S-Oligo (TJU-2749) targeted against the 3’ untranslated region of rat tumor necrosis factor-α mRNA. At 90 min post-intravenous injection, 90% of the S-Oligo was absorbed from circulation. Of this, 40% was found in the liver and 10% in spleen. Other organs, including lungs, kidneys, muscle, stomach, brain, testes and small intestine, showed only minor incorporation (<5%). Greater than 65% of the liver-associated S-Oligo was found in Kupffer cells. Relative accumulation of S-Oligo in Kupffer cells was 200-fold that of the combined body tissues. For an average injected dose of 1.2 mg antisense/Kg body weight, the intracellular concentration of the S-Oligo attained in Kupffer cells was 65 μM. These studies suggest that liposome-encapsulated delivery provides an efficient means of targeting antisense molecules to Kupffer cells in vivo.     

一条大鼠睾丸新基因的生物信息学分析及真核表达

目的：探讨大鼠睾丸组织一条新基因的生物信息学特征和真核表达。方法：构建pEGFP-N1载体的融合质粒进行真核表达,利用生物信息学手段分析基因和蛋白功能。结果：生物信息学分析表明RSA14-44的编码区序列与人类及鼠源RAS同源基因家族核酸序列达到85%以上的同源性;RSA14-44蛋白没有典型的跨膜结构域,也没有典型的N末端信号肽;与人类RhoA蛋白序列达到了89%的同源且具有Rho家族成员的GAAX盒和p-loop结构的基序特征;RSA14-44蛋白大部分氨基酸序列与Rho家族7个已知结构域高度同源;RSA14-44基因真核表达定位于细胞质。结论：RSA14-44基因真核表达定位于CHO-K1细胞质,;编码蛋白质与Rho家族同源性高,为进一步研究其生物学功能提供参考。     

Clonal In Vitro Analysis of Neurotrophin Receptor p75-Immunofluorescent Cells Reveals Phenotypic Plasticity of Primary Rat Olfactory Ensheathing Cells

Clonal in vitro analysis represents a powerful tool for studying cellular differentiation. In the present study, microscope-assisted single cell transfer was combined with immunofluorescence to establish clonal cultures of identified primary rat olfactory ensheathing cells (OECs). During development, OECs originate from the neural crest, a transient population of multipotent cells. Since only neural crest cells have been analyzed at clonal density, it remained unclear whether OECs may retain multipotent features. Neurotrophin receptor p75 (p75^NTR)-immunolabelled rat OECs were seeded at clonal density under visual control using a semiautomated cell selection and transfer device (Quixell?) and emerging clones were analyzed with regard to proliferation and antigenic expression. We demonstrate that OECs from neonatal (P1) and 7 day-old (P7) but not from adult rats formed clones in the presence of OEC- and astrocyte-conditioned media (OEC-CM, A-CM). Cloning efficiency but not in vitro growth of OECs was independent of age but increased upon treatment with OEC-CM. Interestingly, about 75 % of P1 compared to 27 % of P7 OEC clones lost p75^NTR expression during 2 weeks in vitro and acquired immunoreactivity for Thy-1. The observation that primary OECs from P1 lost expression of p75^NTR at clonal density and initiated expression of the fibroblast marker Thy-1 may suggest that their developmental potential is greater than previously anticipated. Since microscope-assisted selection of immunofluorescent cells combined with semiautomated transfer guarantees monoclonality in a single step and affords selection of cells according to fluorescent label and/or morphological criteria it may be relevant for a variety of other cell types.     

Human Papillomavirus Detection In Cervical Cells By In Situ Hybridization With Biotinylated Probes

We have investigated the applicability of human papillomavirus (HPV) DNA detection by in situ hybridization with biotinylated probes in epithelial cells obtained from the cervix using a cotton tip swab. We describe a simple procedure for obtaining homogeneous cell samples and good preservation of cellular structure. This is achieved by pretreatment of cells with L-cysteine before hybridization. Separate denaturation of cellular DNA and probe DNA is also necessary for satisfactory results. Both benign HPV DNA 6/11 and potentially oncogenic HPV DNA 16/18 could be identified in our series. In situ hybridization on cervical scrapes is a rapid, simple and very specific method for detecting patients infected with oncogenic HPV types.     

Progressive Increase In Polyamine Levels In 9l Cells in vitro During the Cell Cycle: Comparison Between Cells Isolated By Centrifugal Elutriation and Cells Grown In Synchrony

Centrifugal elutriation was used to separate 9L rat brain tumour cells into fractions enriched in the G₁, S, or G₂/M phases of the cell cycle. Cells enriched in early G₁, phase were recultured, grown in synchrony, and harvested periodically for analysis of their DNA distribution and polyamine content. Mathematical analysis of the DNA distributions indicated that excellent synchrony was obtained with low dissersion throughout the cell cycle. Polyamine accumulation began at the time of seeding, and intracellular levels of putrescine, spermidine, and spermine increased continuously during the cell cycle. In cells in the G₂/M phase of the cell cycle, putrescine and spermidine levels were twice as high as in cells in the G₁, phase. DNA distribution and polyamine levels were also analysed in cells taken directly from the various elutriation fractions enriched in G₁, S, or G₂/M. Because we did not obtain pure S or G₂/M populations by elutriation or by harvesting synchronized cells, a mathematical procedure—which assumed that the measured polyamine levels for any population were linearly related to the fraction of cells in the G₁, S, and G₂/M phases times the polyamine levels in these phases and that polyamine levels did not vary within these phases—was used to estimate ‘true’ phase-specific polyamine levels (levels to be expected if perfect synchrony were achieved). Estimated ‘true’ phase-specific polyamine levels calculated from the data obtained from cells either sorted by elutriation or obtained from synchronously growing cultures were very similar.     

Pattern of Dna Synthesis and Its Effect On the Classification of Cells By Flow Cytometry

A flow cytometer produces a DNA distribution which contains information about the proportions of cells in various phases of the cell cycle. In this report we show that the form assumed for the rate of DNA synthesis in a cell plays an important part in the estimation of those proportions. We compare three forms for the rate of DNA synthesis, one of which is derived from knowledge of the mechanism of DNA replication, and find that they give substantially different estimates of the proportions of cells in the G₁ and G₂+ M phases.     

Abortive Meiosis in Plasmodial Pollen Mother Cells of Helianthemum

The anthers of two garden varieties of Helianthemum containnot only normal pollen mother cells but also a large proportionof plasmodia with varying numbers of nuclei of varying nuclearcomplements. These abnormal cells probably resulted from a splitspindle associated with a failure of wall formation during severalpremeiotic mitoses. Chromosome pairing at meiosis is higherthan might be expected and in abnormal tissues such as theseit provides evidence for ‘somatic pairing’ havingtaken place during the premeiotic mitoses. Meiosis stops atthe end of the first division in the plasmodia and althoughit is completed in the normal pollen mother cells viable pollenis never produced.     

Kinesin 5B (KIF5B) Is Required for Progression through Female Meiosis and Proper Chromosomal Segregation in Mitotic Cells

The fidelity of chromosomal segregation during cell division is important to maintain chromosomal stability in order to prevent cancer and birth defects. Although several spindle-associated molecular motors have been shown to be essential for cell division, only a few chromosome arm-associated motors have been described. Here, we investigated the role of Kinesin 5b (Kif5b) during female mouse meiotic cell development and mitotic cell division. RNA interference (RNAi)-mediated silencing of Kif5b in mouse oocytes induced significant delay in germinal vesicle breakdown (GVBD) and failure in extrusion of the first polar body (PBE). In mitotic cells, knockdown of Kif5b leads to centrosome amplification and a chromosomal segregation defect. These data suggest that KIF5B is critical in suppressing chromosomal instability at the early stages of female meiotic cell development and mitotic cell division.     

Analysis of the Changes In the Proportion of Clustered Labelled Cells In Epidermis

Abstract. A new cell kinetic approach is presented from which the duration of the S and G₂+ M phases can be estimated. the technique involves an analysis of the spatial distribution of labelled cells in sections or sheets of epithelium (i.e. an analysis of clustered labelled cells). the technique is largely independent of the absolute number of labelled cells and hence is not influenced by factors which affect the absolute number of labelled cells. the technique is described and experimental data from dorsal murine skin are presented. the technique has also been simulated mathematically so that the phase durations and their variances could be estimated. the advantages of the technique are: (1) it is technically simple; (2) it provides at least two independent estimates of the phase durations; (3) unlabelled cells need not be counted (compare with LI or PLM analysis); (4) it is independent of variations in the absolute yield of labelled cells, and (5) it is applicable if the LI is low and the S phase is short (where the PLM technique tends to fail).     

Progression throughout All Stages of Meiosis from the Early Prophase of Newt Primary Spermatocytes in vitro

Stages of prophase of living primary spermatocytes were determined by use of Rose culture chambers (1). Dissociated primary spermatocytes were cultured at low cell-density in a collagen matrix at 22°C or 27°C and the percentages of cells which had progressed from various stages in prophase through meiosis to various advanced stages were measured. In a standard medium (Leibovitz-15 + 10% fetal bovine serum), more than 70% of the primary spermatocytes at stages beyond the pachytene stage could advance to round spermatids with flagella within a few days at 22°C. The percentages of cells that progressed from stages before the late zygotene stage were less, but at least 13 % of leptotene cells reached metaphase I within a week at 22°C. The percentage of cells that progressed was slightly lower at 27°C than at 22°C: 6.3 and 4.3 days were required for progress from leptotene to metaphase I at 22°C and 27C, respectively. Fetal bovine serum was not indispensable for progression through meiosis. Moreover, 0.5–5.0 μg/ml ovine follicle stimulating hormone (NIAMDD-o-FSH-13), 0.01–1.0 μg/ml 5α-dihydrotestosterone and 1.0 μg/ml testosterone propionate had no significant effect in increasing the percentage of cell progression at 22°C.     

Effect of Mitochondrial Inhibitors On Metaphase-Telophase Progression and Nuclear Membrane Formation In Chinese Hamster Cells

Chinese hamster Don cells in log-phase were exposed to Colcemid during the G₂ period with and without a combination of divalent cation chelators and mitochondrial inhibitors. Isolated metaphase cells were incubated as follows: (i) without Colcemid but with other agents and the progression was monitored from metaphase (M) to telophase (Tel) and to cell division; (ii) with Colcemid and other agents and the rate of micronuclei formation in the absence of anaphase was studied. Both EDTA and EGTA accelerated the progression from M to Tel, but did not affect the overall rate of cell division. Chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, blocked the effect of the chelators and also retarded the progression. an inhibitor of mitochondrial respiration, Antimycin A (AA), also retarded the progression in the absence of the chelators and prevented the promoting effect of the chelators. A stimulator of ATPase for ATP breakdown. 2,4-dinitrophenol (DNP), accelerated the M to Tel progression. Chloramphenicol (CAP) and AA, as well as DNP, appeared to have little effect on the formation of micronuclei in the presence of Colcemid. EGTA, which affects cell surface Ca²⁺, stimulated the formation of micronuclei. This study indicates that Ca²⁺ ions and mitochondrial function are involved in the regulation of a certain segment of mitosis beyond metaphase, with Ca²⁺ sequestration in the mitochondria and chelation of Ca²⁺ by EGTA as dominant factors.     

In vitro Effects of Plasma Acid on Proliferation of Rat Brain Endothelial Cells

Journal of Evolutionary Biochemistry and Physiology - The proliferative activity of brain microvascular endothelial cells is regulated by a wide range of factors: regulatory molecules, toxic...     

G-CSF Prevents Progression of Diabetic Nephropathy in Rat

Background

The protective effects of granulocyte colony-stimulating factor (G-CSF) have been demonstrated in a variety of renal disease models. However, the influence of G-CSF on diabetic nephropathy (DN) remains to be examined. In this study, we investigated the effect of G-CSF on DN and its possible mechanisms in a rat model.

Methods

Otsuka Long-Evans Tokushima Fatty (OLETF) rats with early DN were administered G-CSF or saline intraperitoneally. Urine albumin creatinine ratio (UACR), creatinine clearance, mesangial matrix expansion, glomerular basement membrane (GBM) thickness, and podocyte foot process width (FPW) were measured. The levels of interleukin (IL)-1β, transforming growth factor (TGF)-β1, and type IV collagen genes expression in kidney tissue were also evaluated. To elucidate the mechanisms underlying G-CSF effects, we also assessed the expression of G-CSF receptor (G-CSFR) in glomeruli as well as mobilization of bone marrow (BM) cells to glomeruli using sex-mismatched BM transplantation.

Results

After four weeks of treatment, UACR was lower in the G-CSF treatment group than in the saline group (p<0.05), as were mesangial matrix expansion, GBM thickness, and FPW (p<0.05). In addition, the expression of TGF-β1 and type IV collagen and IL-1β levels was lower in the G-CSF treatment group (p<0.05). G-CSFR was not present in glomerular cells, and G-CSF treatment increased the number of BM-derived cells in glomeruli (p<0.05).

Conclusions

G-CSF can prevent the progression of DN in OLETF rats and its effects may be due to mobilization of BM cells rather than being a direct effect.