Localization of DNA Sequences Tightly Associated with the Synaptonemal Complex in Compositional Fractions of the Golden Hamster Genome*

Synaptonemal complex (SC) isolated from spermatocyte nuclei after their exhaustive hydrolysis by DNase II contains DNA sequences tightly associated with it (SCAR DNA). Here, the compositional properties of a cloned family of golden hamster SCAR DNA were studied. For this purpose, 27 SCAR DNA clones were hybridized with compositionally fractionated golden hamster genomic DNA. The sequences of the SCAR DNA family were mainly localized in the GC-poor isochore families L1 and L2, which accounted for 63% of hybridization signals. The remaining 37% of signals pertained to the GC-rich isochore families H1 and H2. Thus, SCAR DNA proved to be distributed throughout the genome, irrespective of differences in density and sequence type between isochore families. Moreover, the SCAR DNA sequences containing the regions of homology with LINE/SINE repeats were found in all the isochore families. The compositional localization of SCAR DNA is in agreement with the hypothesis that the SC and SCAR DNA participate in chromatin reorganization during meiosis prophase I, which should result in the attachment of chromatin loops to the lateral elements of SC throughout its length.     

Internalization of surface HLA-DR molecules by human epidermal Langerhans cells: analysis by flow cytometry and confocal microscopy

Langerhans cells (LC) play a pivotal role in antigen processing and presentation to T cells during delayed-type hypersensitivity reaction in the skin. Antigen presentation involves the interaction between the class II molecules of MHC (HLA-DR) expressed by LC and T receptor of CD4⁺ T lymphocytes. It is now recognized that class II molecules are internalized into LC and can be associated with processed immunogenic peptides. This process involves receptor-mediated endocytosis. The aim of this study was to investigate the time-course of endocytosis of HLA-DR by freshly isolated human LC. Epidermal cells, obtained from normal skin samples, were labeled by indirect immunofluoresence using anti-HLA-DR monoclonal antibodies (MAb). The cell suspension was incubated at 37°C for different periods (15, 30, 45, 60 and 90 min) and then analyzed by flow cytometry and confocal microscopy. Flow cytometry analysis showed decreased HLA-DR molecule expression by LC after incubation at 37°C. Confocal microscopic analysis showed different strain patterns depending on the incubation time: (1)T=0, continuous peripheral staining; (2)T=15 min, patchy peripheral staining; (3)T=30 min, patches or intracellular vesicular staining; (4)T=45 min, intracellular vesicular staining; (5)T=60 min, diffuse intracellular staining; (6)T=90 min, aggregated staining. In our study model, flow cytometry provides quantitative information for the HLA-DR endocytosis, whereas confocal microscopy provides qualitative results concerning the intracellular distribution of internalized HLA-DR molecules. The use of the two complementary techniques allows us to characterize the spontaneous endocytosis of HLA-DR molecule by freshly isolated LC. Thisin vitro study model might be useful for testing the sensitizing potential of different chemical substances.Abbreviations Ab antibodies - APC antigen-presenting cells - BG Birbeck granules - DNCB 1-chloro-2,4-dinitrobenzene - DNFB 2,4-dinitrofluorobenzene - DTAF dichlorotriazinylfluorescein - FSC forward light scatter - LC Langerhans cells - LSCM laser scanning confocal microscopy - MHC major histocompatibility complex - MAb monoclonal antibodies - PFA paraformaldehyde - SSC side light scatter     

Chromatin condensation in hamster sperm: A flow cytometric investigation

In this study we have used acridine orange staining, as described by Evenson (1990), to follow changes in DNA packaging as they occur in hamster spermatozoa which have left the testis and are undergoing maturation in the epididymis. Measurement of the green and red fluorescent intensities of hamster sperm nuclei by flow cytometry demonstrated a decrease in acridine orange binding to DNA as sperm made their way from proximal corpus epididymis to the vas deferens. Using sperm from the cauda epididymis of the mature hamster as the standard, a method was developed for estimating the % of cells in a given sample that have matured with regard to DNA packaging. Staining with bromobimane was used to determine the extent of sulfhydryl oxidation in the nuclei. It was seen that sulfhydryl oxidation occurred mainly in the cauda epididymis whereas another process in chromatin condensation occurred earlier, during sperm passage through the caput epididymis. This earlier process could be mimicked by incubating sperm nuclei with alkaline phosphatase, suggesting that it consists of removal of phosphate in protamine. © 1994 Wiley-Liss, Inc.     

Spermatogenesis in the golden hamster: the role of c-kit.

c-kit is related to the family of transmembrane tyrosine kinase receptors. Mutations in genes for either c-kit or its ligand, Steel factor, result in infertility, but the role of c-kit/SCF system in spermatogenesis is not well understood. In this study Western blot analysis together with confocal microscopy were used to follow c-kit expression in hamsters during the first spermatogenic wave in mature animals and in old age. Three antibodies raised against different domains of c-kit were tested on Western Blot. Confocal microscopy was performed after incubation of fixed seminiferous tubules with tested antibodies followed by binding of FITC-labeled secondary antibody. Longitudinal sections of seminiferous tubule were observed by confocal microscopy to determine in which stages of spermatogenesis and in which cell types c-kit was found. C-kit bands of 80,140, and 150 kDa were observed on Western blot, indicating that c-kit is a name related to several proteins sharing some common domains. Only the band of 150 kDa correlated with positive staining of c-kit in tubules using confocal microscopy. We term this protein c-kit150T (150 kDa, testis). We demonstrated that c-kit150T appeared in differentiating hamster spermatogonia at stages VII-VIII of adult spermatogenesis and at day 13-14 during the first spermatogenic wave. It remained attached to the cell until late pachytene. This suggests that c-kit may play a role in preparing the germinal cells to enter meiosis. In order to evaluate the effect of aging on the number of germ cells, B2 spermatogonia/Sertoli cell ratio was calculated in the group of young animals (5-7 months) compared to this ratio in older ones (20-26 months). A significant decrease (P < 0.01) in the number of B2 spermatogonia in the group of old hamsters as compared to young ones was seen. The calculated value for the B2 spermatogonia/Sertoli cell ratio was 5.6 +/- 0.7 in young animals and 3.8 +/- 1.2 in the 20-26 months ones. In addition, decrease in the intensity of staining for c-kit was detected in the old hamsters. These may be the reasons for subfertility in old age and in other cases of testicular disorders.     

Msh4 and Msh5 Function in SC-Independent Chiasma Formation During the Streamlined Meiosis of Tetrahymena

ZMM proteins have been defined in budding yeast as factors that are collectively involved in the formation of interfering crossovers (COs) and synaptonemal complexes (SCs), and they are a hallmark of the predominant meiotic recombination pathway of most organisms. In addition to this so-called class I CO pathway, a minority of crossovers are formed by a class II pathway, which involves the Mus81-Mms4 endonuclease complex. This is the only CO pathway in the SC-less meiosis of the fission yeast. ZMM proteins (including SC components) were always found to be co-occurring and hence have been regarded as functionally linked. Like the fission yeast, the protist Tetrahymena thermophila does not possess a SC, and its COs are dependent on Mus81-Mms4. Here we show that the ZMM proteins Msh4 and Msh5 are required for normal chiasma formation, and we propose that they have a pro-CO function outside a canonical class I pathway in Tetrahymena. Thus, the two-pathway model is not tenable as a general rule.     

Photic regulation of L-arginine uptake in the golden hamster retina

One of the limiting steps in the regulation of nitric oxide (NO) synthesis is the availability of its precursor, L-arginine, which depends on the presence of a specific uptake system. A characterization of the L-arginine uptake mechanism in the golden hamster retina was performed. This mechanism was stereospecific, saturable, and monophasic, with an apparent of 56.1 +/- 2.0 microM and a maximum velocity of 36.0 +/- 2.8 pmol/mg prot/min. The basic amino acids L-lysine and L-ornithine but not D-arginine or the nitric oxide synthase inhibitors, N(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine impaired L-arginine influx. Preincubation with L-lysine for 1 h prior to the transport assay significantly stimulated L-arginine uptake. Saturation studies of L-arginine uptake performed at 12.00 and 24.00 h indicated a higher value of Vmax at midnight than at midday. When the hamsters were placed under constant darkness or constant light for 48 h and killed at equivalent time points, representing subjective day and subjective night, the differences in L-arginine influx disappeared. Semiquantitative RT-PCR analysis showed that the levels of mRNAs for both CAT-1 and CAT-2B were significantly higher at midnight than at midday. L-Arginine significantly increased cGMP accumulation in a time-dependent manner, with maximal effects during the night. Based on these results, it might be presumed that hamster retinal L-arginine uptake is regulated by the photic stimulus.     

Microsatellites for diversity studies in the golden hamster (Mesocricetus auratus)

Ten polymorphic microsatellites were developed for the golden hamster (Mesocricetus auratus), a widely used model organism in biological and medical researches. All loci were used to analyse the microsatellite variability in wild golden hamsters from Syria and in a sample of domestic animals comprising different strains. Average mean expected heterozygosity (H_E) and mean allele number (A) of domestic hamsters measured 0.279 ± 0.058 and 2.6 ± 0.306, respectively, compared to 0.809 ± 0.019 and 8.3 ± 1.075 found for wild hamsters. Cross‐species application in other Mesocricetus species proved conservation of most loci throughout the genus.     

Spermatogenesis in the golden hamster during the first spermatogenic wave: a flow cytometric analysis

In the present study propidium iodide was used as a fluorescent dye to stain DNA of cells of hamster testicular origin and fluorescent intensities were analyzed by flow cytometry. We used hamster testicular cells from the first spermatogenic wave to observe the consecutive appearance of the different types of cells during puberty. At 12 days postpartum (dpp) diploid cells (including spermatogonia) predominated and some tetraploid cells were also present. Tetraploid spermatocytes increased dramatically by 21 dpp. The first haploid cells appeared at 21 dpp but substantial numbers were first present at 23 dpp. Immature haploid cells predominated at 32 dpp. Elongating condensing spermatids appeared at 34 dpp and spermatozoa began to leave the testis to enter the epididymidis at 36-38 dpp marking the end of the first round of spermatogenesis. Using acridine orange staining flow cytometry, chromatin condensation was followed by measuring fluorescence decrease from early round spermatids to spermatozoa obtained from the initial segment and from the cauda epididymides. The major portion of sperm chromatin condensation (88-90%) in the hamster occurred in the testis and only 10-12% occurred during epididymal sperm maturation. Spermatozoa in the initial segment of the epididymidis of the hamster contained a small amount of RNA that was no longer present in sperm of the cauda epididymidis, indicating that RNA was lost during epididymal sperm maturation in this species. Mol. Reprod. Dev. 55:205-211, 2000.     

Effect of neurosteroids on the retinal gabaergic system and electroretinographic activity in the golden hamster

It has been established that neurosteroids can either inhibit or enhance GABA(A) receptor activity. Although GABA is the main inhibitory neurotransmitter in the mammalian retina, the effects of neurosteroids on retinal GABAergic activity have not been investigated. The aim of this work was to study the neurochemical and electroretinographic effects of neurosteroids in the golden hamster. On one hand, pregnenolone sulfate inhibited and allotetrahydrodeoxycorticosterone increased GABA-induced [36Cl]- uptake in neurosynaptosomes. On the other hand, in whole retinas, pregnenolone sulfate increased, whereas allotetrahydrodeoxycorticosterone decreased high potassium-induced [3H]GABA release. The effect of both neurosteroids on GABA release was Ca2+-dependent, as in its absence release was not altered. The intravitreal injection of pregnenolone sulfate or vigabatrin (an irreversible inhibitor of GABA degradation) significantly decreased scotopic b-wave amplitude, whereas the opposite effect was evident when bicuculline or allotetrahydrodeoxycorticosterone were injected. A protein with a molecular weight close to that of hamster adrenal cytochrome P450 side-chain cleavage (P450scc) was detected in the hamster retina. P450scc-like immunoreactivity was localized in the inner nuclear and the ganglion cell layers. These results indicate that neurosteroids significantly modulate retinal GABAergic neurotransmission and electroretinographic activity. In addition, the selective localization of P450scc suggests that neurosteroid biosynthesis might occur only in some layers of the hamster retina.     

Photic regulation of heme oxygenase activity in the golden hamster retina: involvement of dopamine

The photic regulation of heme oxygenase (HO) activity was examined in the golden hamster retina. This enzymatic activity was significantly higher at midday than at midnight. When the hamsters were placed under constant darkness for 48 h and killed at subjective day or at subjective night, the differences in HO activity disappeared. Western blot analysis showed no differences in HO levels among these time points. Dopamine significantly increased this activity in retinas excised at noon or at midnight, with a higher sensitivity at night. The effect of dopamine was reversed by SCH 23390 but not by spiperone and clozapine and it was not reproduced by quinpirole. In vitro, the increase in HO activity found in retinas incubated under light for 1 h was significantly reduced by SCH 23390. Two cAMP analogs increased HO activity and their effect, as well as the effect of dopamine was blocked by H-89, a protein kinase A (PKA) inhibitor. Tin protoporphyrin IX, an HO inhibitor, significantly decreased cGMP accumulation with maximal effects during the day. Low concentrations of bilirubin decreased retinal thiobarbituric acid substances levels (an index of lipid peroxidation) in basal conditions and after exposing retinal cells to H2O2. These results suggest that hamster retinal HO activity is regulated by the photic stimulus, probably through a dopamine/cAMP/PKA dependent pathway.     

Quantification of cell hybridoma yields with confocal microscopy and flow cytometry

The fusion of antigen presenting and cancer cells leads to the formation of hybrid cells, which are considered a potential vaccine for treating cancer. The quality assessment of hybrid cell vaccines is crucial for the introduction of this new treatment. Flow cytometry was the method used recently, since it is faster in comparison to classical microscopy. Here we describe a rapid confocal microscopy based approach to quantify hybrid cell yields. The extent of fusion rate was determined by confocal microscopy by counting dual fluorescent cells and by measuring the area of co-localized pixels. Results of both methods showed high degree of correlation. The same samples were also analyzed by flow cytometry. Fusion rates determined with both techniques showed significant correlation. In conclusion, using confocal microscopy we developed a sensitive and a rapid method to assess the yield of hybridomas in a large number of electrofused cells.     

Flow cytometry and live confocal analysis for the evaluation of the uptake and intracellular distribution of FITC-ODN into HaCaT cells

In this study, the mechanism of the internalization and the cellular distribution of 59 fluorescein conjugated PS-ODN (FITC-ODN) after transfection with different mixed lipidic vesicles/oligo complexes (lipoplexes) have been investigated. Mixed lipidic vesicles were prepared with one of the most used cationic lipid (DOTAP) and different amounts of a cholic acid (UDCA) to release the oligo into HaCaT cells. Using flow cytometry, the cellular uptake of the oligo was studied with and without different inhibitors able to block selectively the different pathways involved in the internalization mechanism. The intracellular distribution of the oligo was analyzed by confocal laser scanning microscopy (CLSM), treating the cells with the lipoplexes and directly observing without any fixing procedure. To better carry out the colocalization studies, fluorescent-labeled markers, specific for the different cellular compartments, were coincubated with 59 fluorescein-conjugated 29-mer phosphorotioate oligonucleotide (FITC-ODN). The different lipidic vesicles affect the internalization mechanism of FITC-ODN. After using the inhibitors, the uptake of complexes involved a different internalization mechanism. The live CLSM analysis demonstrated that, after 1 hour from the complex incubation, the oligo was transferred into cells and localized into the endosomes; after 24 hours, the oligo was intracellularly localized close to the nuclear structure in a punctuate pattern. However, the results from fusion experiments showed also a binding of a quite low amount of oligo with the cell membranes.     

Modeling flux in tangential flow filtration using a reverse asymmetric membrane for Chinese hamster ovary cell clarification

Tangential flow filtration is advantageous for bioreactor clarification as the permeate stream could be introduced directly to the subsequent product capture step. However, membrane fouling coupled with high product rejection has limited its use. Here, the performance of a reverse asymmetric hollow fiber membrane where the more open pore structure faces the feed stream and the barrier layer faces the permeate stream has been investigated. The open surface contains pores up to 40 μm in diameter while the tighter barrier layer has an average pore size of 0.4 μm. Filtration of Chinese hamster ovary cell feed streams has been investigated under conditions that could be expected in fed batch operations. The performance of the reverse asymmetric membrane is compared to that of symmetric hollow fiber membranes with nominal pore sizes of 0.2 and 0.65 μm. Laser scanning confocal microscopy was used to observe the locations of particle entrapment. The throughput of the reverse asymmetric membrane is significantly greater than the symmetric membranes. The membrane stabilizes an internal high permeability cake that acts like a depth filter. This stabilized cake can remove particulate matter that would foul the barrier layer if it faced the feed stream. An empirical model has been developed to describe the variation of flux and transmembrane pressure drop during filtration using reverse asymmetric membranes. Our results suggest that using a reverse asymmetric membrane could avoid severe flux decline associated with fouling of the barrier layer during bioreactor clarification.     

Apoplastic pH in corn root gravitropism: a laser scanning confocal microscopy measurement

The ability to measure the pH of the apoplast in situ is of special interest as a test of the cell wall acidification theory. Optical sectioning of living seedlings of corn roots using the laser scanning confocal microscope (LSCM) permits us to make pH measurements in living tissue. The pH of the apoplast of corn roots was measured by this method after infiltration with CI-NERF, a pH-sensitive dye, along with Texas Red Dextran 3000, a pH-insensitive dye, as an internal standard. In the elongation zone of corn roots, the mean apoplastic pH was 4.9. Upon gravitropic stimulation, the pH on the convex side of actively bending roots was 4.5. The lowering of the apoplastic pH by 0.4 units appears to be sufficient to account for the increased growth on that side. This technique provides site-specific evidence for the acid growth theory of cell elongation. The LSCM permits measurements of the pH of living tissues, and has a sensitivity of approximately 0.2 pH units.     

Mitochondrial behavior during oogenesis in zebrafish: a confocal microscopy analysis

The behavior of mitochondria during early oogenesis remains largely unknown in zebrafish. We used three mitochondrial probes (Mito Tracker Red CMXRos, Mito Tracker Green FM, and JC-1) to stain early zebrafish oocyte mitochondria, and confocal microscopy to analyze mitochondrial aggregation and distribution. By using fluorescence recovery after photobleaching (FRAP), we traced mitochondrial movement. The microtubule assembly inhibitor nocodazole and microfilament inhibitor cytochalasin B (CB) were used to analyze the role of microtubules and microfilaments on mitochondrial movement. By using the dual emission probe, JC-1, and oxidative phosphorylation uncoupler, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), we determined the distribution of active and inactive (low-active) mitochondria. Green/red fluorescence ratios of different sublocations in different oocyte groups stained by JC-1 were detected in merged (green and red) images. Our results showed that mitochondria exhibited a unique distribution pattern in early zebrafish oocytes. They tended to aggregate into large clusters in early stage I oocytes, but in a threadlike state in latter stage I oocytes. We detected a lower density mitochondrial area and a higher density mitochondrial area on opposite sides of the germinal vesicle. The green/red fluorescence ratios in different sublocations in normal oocytes were about 1:1. This implies that active mitochondria were distributed in all sublocations. FCCP treatment caused significant increases in the ratios. CB and nocodazole treatment caused an increase of the ratios in clusters and mitochondrial cloud, but not in dispersed areas. Mitochondria in different sublocations underwent fast dynamic movement. Inhibition or disruption of microtubules or microfilaments resulted in even faster mitochondrial free movement.     

Similarity of Domain Organization of Proteins in Phylogenetically Distant Organisms as a Basis of Meiosis Conservatism

The cytological mechanism of meiosis is very conservative in all eukaryotes. Some meiosis-specific structural proteins of yeast, nematode Caenorhabditis elegans, Drosophila, and mammals, which play identical roles in cells during meiosis, do not have homology of the primary structure, but their domain organization and conformation are similar. The enzymes of meiotic recombination in yeast and plants have similar epitopes. These facts suggest that the similarity of the higher level of organization of the meiosis-specific proteins allows these proteins to form similar subcellular structures and produce similar cytological picture of meiosis and similar functions of these subcellular structures. Finally, this leads to a conservative scheme of meiosis in evolutionally distant eukaryotes.     

Complement increases in experimental Leishmania donovani infection of the golden hamster

Complement (C') levels in experimental visceral leishmaniasis were studied in the golden hamster. Using the total haemolytic assay of C' and the haemolytic diffusion plate assay for factor B, it was observed that the total haemolytic C' as well as factor B levels increased markedly starting one week after infection and remained high throughout most of the infection. A decline in C' level was noted toward the terminal stage of infection. The reason for the persistent high level of C' noted in this study was discussed.     

Thermosensitivity of the preoptic region and the spinal cord in the golden hamster

1. 1.|The effects of thermal stimulation of the preoptic region (POAH) and the spinal cord on non-shivering thermogenesis (NST) and shivering were studied in euthermic golden hamsters.

2. 2.|Shivering intensity is suppressed by heating the POAH but is independent of spinal cord temperature. Therefore, NST in the interscapular brown adipose tissue does not suppress shivering.

3. 3.|NST is inhibited by heating of the POAH as well as of spinal cord. It is discussed that the control of NST by two different central thermosensitive areas is significant for thermoregulation during exercise.

Changes in the distribution of intramembranous particles and filipin-reactive membrane sterols during in vitro capacitation of golden hamster spermatozoa

Membrane alterations accompanying in vitro capacitation of hamster spermatozoa were examined using the freeze-fracture technique with or without use of filipin, a sterol-binding probe. In the spermatozoa prior to or at 10 min after start of incubation in capacitating medium, large (about 11 nm) and small (8–9 nm) intramembranous particles (IMPs) were present in the periacrosomal region of the sperm plasma membrane (PAPM). Filipin sterol complexes (FSCs) were densely (about 500/μ²) distributed in the PAPM prior to incubation. The density of FSCs in the PAPM was reduced by 70–80% of the original density by 2 hr of incubation. At the same time, small patches of IMP-free areas appeared in the plasma membrane above the equatorial and middle segments of the acrosome. By the end of 3 hr of incubation, the majority of small IMPs had disappeared from the PAPM. Remaining large and small IMPs tended to aggregate in the PAPM. During incubation in capacitation medium, “cords,” or linear arrangements of closely packed IMPs, appeared near the posterior ring of the sperm head. These observations strongly suggest that the acrosome reaction of the hamster spermatozoa is preceded by the removal (deletion) of filipin-reactive sterols (FRSs) and the disappearance of small IMPs from the lipid bilayer of PAPM.