The Global Relationship between Chromatin Physical Topology, Fractal Structure, and Gene Expression.

Most of what we know about gene transcription comes from the view of cells as molecular machines: focusing on the role of molecular modifications to the proteins carrying out transcriptional reactions at a loci-by-loci basis. This view ignores a critical reality: biological reactions do not happen in an empty space, but in a highly complex, interrelated, and dense nanoenvironment that profoundly influences chemical interactions. We explored the relationship between the physical nanoenvironment of chromatin and gene transcription in vitro. We analytically show that changes in the fractal dimension, D, of chromatin correspond to simultaneous increases in chromatin accessibility and compaction heterogeneity. Using these predictions, we demonstrate experimentally that nanoscopic changes to chromatin D within thirty minutes correlate with concomitant enhancement and suppression of transcription. Further, we show that the increased heterogeneity of physical structure of chromatin due to increase in fractal dimension correlates with increased heterogeneity of gene networks. These findings indicate that the higher order folding of chromatin topology may act as a molecular-pathway independent code regulating global patterns of gene expression. Since physical organization of chromatin is frequently altered in oncogenesis, this work provides evidence pairing molecular function to physical structure for processes frequently altered during tumorigenesis.

Quality and freezing qualities of first and second ejaculates collected from endangered Gulf Coast Native rams.

The Gulf Coast Native sheep, or Louisiana Native sheep, is an endangered previously feral domestic sheep population of European origin that has been under natural selection pressure for reproductive survival in their transplanted range while roaming in the southern Gulf Coast Region of the United States. This sheep population has an increased natural resistance to internal parasites, breeds year-around and has a greater percentage of live lambs as compared with other breeds of sheep raised in similar environments. To preserve the genetic diversity of this important feral sheep population, semen was collected by electro-ejaculation and subjected to cryopreservation for subsequent storage in a genome resource bank. Unrelated rams (n=5) were collected 3 days-a-week, allowing at least 2 days of rest between collections. Two ejaculates were obtained from each ram per collection day, with the second collection conducted 10min after the first ejaculation. Semen was processed using the standard Salamon cryopreservation procedure in a Tris-yolk-glycerol extender, frozen in 0.5ml plastic straws using liquid nitrogen (LN(2)) vapor and stored in LN(2). Each ejaculate was evaluated for volume, sperm concentration/ml (x10(9)/ml), number of spermatozoa/ejaculate (x10(9)), sperm progressive motility (%) for pre-cooled semen, cooled semen and semen after thawing. For the five rams, each semen variable for the first ejaculate was compared with that of the second ejaculate collected 10min later. The mean semen volume, sperm concentration and number of spermatozoa per ejaculate obtained from the first ejaculate were significantly greater (P< or =0.01) than those of the second ejaculate (comparisons being 1.62 and 1.06; 3.2 and 1.5; 5.4 and 1.8, respectively). Overall, the mean motility of pre-cooled (22 degrees Celsius), cooled (5 degrees Celsius) and frozen (-196 degrees Celsius) post-thawed spermatozoa was less (P< or =0.01) in the first ejaculate (71.5, 64.8 and 34.1%, respectively) compared with that of the second ejaculate (75, 72.4 and 44.1%, respectively). Conversely, no differences were detected in loss in the percent progressive motility of sperm from cooled sperm to post-thaw sperm from the first and second ejaculates. In summary, our findings suggest sperm collected during the second ejaculate 10min after the first ejaculate of rams survives thawing with a greater rate of progressive motility than that of the first ejaculate. The ability to collect two consecutive ejaculates in a short period by electro-ejaculation could be valuable for gamete resource banking and preserving genetic diversity of the Gulf Coast Native sheep.

Spermatozoal methylene blue reduction: an indicator of mitochondrial function and its correlation with motility.

Methylene blue reduction rates (MeBRR) were evaluated spectrophotometrically to study bovine spermatozoal mitochondrial function and its relation to motility. A chemical reaction (H2SO4, methylene blue solution, and zinc powder) was used to quantify methylene blue reduction. Absorbance measurements were made for 10 min at 609 nm in a narrow band spectrophotometer. In a second experiment, fresh ejaculates were assessed for concentration and motility evaluated with phase microscopy (37 degrees C). Semen was diluted to 100 million cells/mL in a sodium citrate-glucose buffer and methylene blue. Absorbance and motility were evaluated every 30 min for 2.5 h in a water-jacketed cuvette (41 degrees C). Methylene blue reduction rates and motility decreased at each subsequent period. Methylene blue reduction rates were correlated to sperm motility. Lastly, the methylene blue reduction rate was measured with a broad band spectrophotometer and compared with motility using similar conditions. Motility estimates were made on sperm from the cuvettes. Sperm motility was correlated to methylene blue reduction rates measured spectrophotometrically.

Bovine spermatozoal head size variation and evaluation of a separation technique based on this size.

The objectives of this study were to determine the variation of head areas of normal spermatozoa attributable to breed, individual bull and ejaculate and to verify separation of X and Y chromosome-bearing spermatozoa and separation effectiveness. Spermatozoa were evaluated using video enhanced contrast microscopy combined with video intensified fluorescent microscopy and the polymerase chain reaction (PCR). In Experiment 1, spermatozoal head areas were measured from 2 ejaculates collected from bulls of 3 beef and 2 dairy breeds. No differences in head areas were found between breeds or between bulls within breeds; variation was observed among ejaculates from individual bulls across breeds. In Experiment 2, spermatozoa from 5 ejaculates were separated on individual SEPDEVICEs (Patented). Head area, fluorescent intensity and PCR of spermatozoa retained in the SEPDEVICEs suggested a separation based on size in 1 of 5 samples. Ejaculate variation in head areas affected separation efficiency.

A technique for the evaluation of sperm penetrating ability and quality of bovine semen processed in an extender made with Brackett-Oliphant medium and egg yolk.

Egg yolk-sodium citrate (EYC) semen extender was compared with an extender made of Brackett-Oliphant medium and egg yolk (BOEY). Ejaculates were divided into equal portions, processed and frozen. Semen was thawed and evaluated for quality. Additional semen was thawed, stained with Hoechst 33342 and the spermatozoa capacitated, after which they were co-incubated with zona-free hamster oocytes to determine their penetrating ability. Sperm penetration of non-compressed, unfixed oocytes was evaluated using an optical sectioning technique on a standard research microscope. Sperm penetration was considered successful if a fluorescing sperm head was observed within the living oocyte in a hanging drop of fertilization medium. There were small differences in percentage of secondary abnormalities and percentage of progressive motility immediately after thawing between spermatozoa extended in EYC or BOEY diluent. There were no differences due to by extender composition in percentage of spermatozoa with intact acrosomes or percent of progressively motile after a 3 h incubation at 37 degrees C, nor the percentage of spermatozoa with head abnormalities. While there were significant correlations between all seminal quality characteristics, no quality measurements were correlated to percentage of oocyte penetration. The new penetration evaluation method allowed for examination of the fertilized oocytes using fluorescent microscopy initially and again after re-incubation for further development.

Sex ratio variation between ejaculates within sire evaluated by polymerase chain reaction, calving, and farrowing records.

Ejaculates from sires were examined by polymerase chain reaction to determine percentage of sperm bearing the Y chromosome. Results were verified by examining the percentage of male calves per ejaculate used in artificial insemination (AI) and the percentage of male piglets per litter from a controlled mating program. Spermatozoal DNA was amplified by polymerase chain reaction with specific primers for the Y chromosome. Image analysis measured the fluorescent intensity of the 194-bp band. Ejaculates were compared with a pooled standard of spermatozoal DNA equated to a 50% Y-bearing sperm ejaculate. Calving data were obtained from information collected for the National Association of Animal Breeders for dystocia evaluation of cows bred to AI bulls. Breeding data were obtained from AI technician receipts. Calving and breeding data were merged on cow, sire, calving date, and breeding date. The percentage of males were calculated per sire, ejaculate, and herd combination. Farrowing data were evaluated for the percentage of male piglets per litter. Ejaculates within bulls contributed to variation (24 +/- 9.8% to 84 +/- 9.8%) in the percentage of sperm bearing the Y chromosome. Ejaculates from the same bull contributed to variation in the percentage of male calves (16.1 to 72.3%). Ejaculates from the same boar contributed to variation in the percentage of male piglets that ranged from 7.8 to 94.7%. These percentages and the results obtained by polymerase chain reaction analysis of ejaculates suggested that spermatozoa bearing X and Y chromosomes were unequally represented in ejaculates. The use of ejaculates screened by polymerase chain reaction could enhance production of the desired sex of calf.

Chromosomal analysis of mammalian oocytes matured in vitro with various culture systems.

The objective of this study was to evaluate oocyte maturation in vitro. Ten virgin CD-1 mice were used with 3 replications for in vitro with 4 different culture media. Media were minimal essential medium (MEM) with Earl's salt, Waymouth MB 752/1 (MB 752/1), BGjb medium (BGjb), and tissue culture medium-199 (TCM-199). The oocyte chromosomes were C-banded to enable an objective analysis of the chromosome abnormality and number. There was a percentage of blockage at metaphase I (M I), in matured oocytes in all culture media. Metaphase II (M II) was reached by 70.9 to 87.3% of oocytes in 4 different culture media. The frequencies of hyperploid M II oocytes were 0.0, 1.1, 2.8 and 2.6% for TCM-199, MEM, MB 752/1 and BGjb, respectively. A small proportion of oocytes was also found to be polyploid in 4 different culture media. There was an occurrence of premature centromere separation among oocytes. It was concluded that the chromosomes of the oocytes matured in vitro were not all in the normal condition (being at M II). The media used in this study for oocyte maturation caused maturation delay (being blocked at M I), premature centromere separation, polyploidy, and aneuploidy (such as, hyperploid, hypoploid).

An unusual case of hypersomnolence syndrome.

Hypersomnolence syndrome has been well described in patients with acute lymphoblastic leukaemia (ALL) following cranial irradiation. It is most frequently seen in children. In adults it is clinically very mild. We describe a case of a particularly severe and prolonged post radiation hypersomnolence syndrome in an adult with ALL. The hypersomnolence syndrome is discussed in the context of side effects of central nervous system directed therapy in elderly patients with ALL.

Nanovid microscopy for assessing sperm membrane changes induced by in vitro capacitating and acrosomal reacting procedures.

This study was to verify the usefulness of Nanovid microscopy techniques for evaluating induced modifications in bovine spermatozoal membranes. Frozen thawed bovine sperm were labeled with 20-nm colloidal gold particles bound to concanavalin A (ConA) or heparin ligands. Sperm membrane changes were induced in vitro by capacitating and acrosome-reacting procedures. Capacitation was induced by incubation with 10 micrograms/ml of heparin for 4 hours at 37 degrees C, 5% CO2, and high humidity. Membrane changes associated with the acrosome reaction were induced by addition of lysophosphatidylcholine (100 micrograms/ml) and incubation for 15 minutes at 37 degrees C, 5% CO2, and high humidity. Gray intensity (black = 0; white = 255) of sperm (ONCELL) and background (OFFCELL) were evaluated with computer-enhanced videomicroscopy with either differential interference contrast (DIC) or Nanovid optics. A high gold concentration on a membrane region produced blacker video pictures with Nanovid microscopy. Gray intensity of video pictures of a region with little or no gold would have a gray intensity equal to or greater than that of the background, that is, toward white. Weighted least squares methods were used to analyze ONCELL data using OFFCELL as a covariate. In experiment 1, ONCELL intensities of cells labeled with ConA-gold complex were lower than those labeled with heparin-gold at the same treatment level. In experiment 2, ONCELL intensity decreased as the concentration of heparin-gold increased from 0 to 0.041 microgram/microliter heparin. ONCELL intensity significantly decreased after sperm were treated with the highest heparin-gold level and acrosome reacted.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotoxicity of gallium and indium ions compared with mercuric ion.

The use of mercury in dental amalgam restorations has become the subject of political controversy despite its long history of safe clinical use, and alternative materials based on gallium and indium rather than mercury have been developed. The biological safety of these metals must be evaluated, as part of their assessment as mercury substitutes. The cytotoxicities of mercury (II) nitrate, gallium (III) nitrate, and indium (III) nitrate were assessed at concentrations between 0.001 mmol/L and 1.0 mmol/L, using L929 mouse fibroblasts and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and scanning electron microscopy. The mitochondrial dehydrogenase activity at each metal ion concentration as a percentage of the control was calculated from the absorbance values. The 50% inhibition concentration of mercury (II) nitrate was 0.35 mmol/L for cells in the rapid-growth phase and at confluence; gallium (III) nitrate and indium (III) nitrate did not significantly inhibit dehydrogenase activity in either the growing or confluent phase. Gallium and indium ions were not significantly toxic under the conditions of this assay.

Chromosome and sperm size of Holsteins with and without bovine leukocyte adhesion deficiency.

The objective was to evaluate bull differences in chromosomal and spermatozoal areas related to the occurrence of the bovine leukocyte adhesion deficiency syndrome. Lymphocyte chromosomes from 30 Holstein bulls and 2 Holstein heifers were measured using image analysis and computer-enhanced video-microscopy. Spermatozoal head areas from 29 of the 30 bulls were measured. Autosomal rank was based on decreasing area. Average total autosomal areas were not the same across bulls. One group of bulls had significantly smaller average chromosomal areas than the others; this group carried bovine leukocyte adhesion deficiency syndrome. Area measures of spermatozoal heads showed that bulls with bovine leukocyte adhesion deficiency syndrome had significantly larger head areas than normal bulls. Lymphocyte chromosomes from 3 cattle that were homozygous for bovine leukocyte adhesion deficiency syndrome were significantly smaller than chromosomes from syndrome heterozygotes. Carrier identification was improved by the use of autosomal and sperm area measurements in addition to pedigree evaluation.

Pure red cell aplasia: further evidence of T cell clonal disorder.

We report a patient with pure red cell aplasia and type I autoimmune polyglandular syndrome who also had an expansion of suppressor T lymphocytes in the peripheral blood. Southern blotting of DNA from these cells suggest T cell receptor (TCR) gamma gene rearrangement. We confirmed true clonality of this by amplification of the gene rearrangement using the polymerase chain reaction and subsequent analysis of the product by gene cloning and DNA sequencing.

One-year clinical evaluation of three dentine bonding agents.

One hundred and twenty-five composite restorations were placed in non-undercut, non-enamel-etched Class V abrasion lesions in 20 patients using the dentine bonding agents Scotchbond 2, Tripton, and Prisma Universal Bond 2. Patients were recalled at approximately 3 months, 6 months, and 1 year. After 1 year the cumulative per cent survival of restorations was Scotchbond 2, 8 per cent; Scotchbond 2 with dentine roughening, 16 per cent; Tripton, 16 per cent; Prisma Universal Bond 2, 75 per cent.

Evaluating acrosome reaction steps with brightfield and differential interference contrast microscopy techniques.

Acrosomal integrity of bovine spermatozoa was evaluated using naphthol yellow S-erythrosine B stain with bright-field microscopy, .2% glutaraldehyde fixation with Nomarski optics, and live wet smears with Nomarski optics to compare the evaluation techniques. Four ejaculates per two Holstein bulls were enriched through a Percoll gradient, capacitated with heparin, and acrosome-reacted with lysophosphatidylcholine to prepare spermatozoa for in vitro fertilization. Spermatozoa samples were taken after each preparatory step, and percentage of intact acrosomes was evaluated with the stain, .2% glutaraldehyde, and live wet smear. Acrosomal integrity decreased with each preparatory step across all techniques. The decrease in integrity from heparin to lysophosphatidylcholine indicated that all methods detected the acrosome reaction. Technique means across preparation steps showed no differences. Correlations between microscopy techniques were high. The live wet smear used no fixation methods. Both the naphthol yellow S-erythrosine B stain and the .2% glutaraldehyde techniques employed fixation steps that may have introduced artifacts that could influence the data. The live wet smear evaluated by Nomarski optics allowed a comparable evaluation of the acrosome-reacted bovine spermatozoa. Comparing brightfield with differential interference contrast microscopy for detecting the acrosome reaction in bovine spermatozoa showed that both methods were equally accurate.

Videomicroscopic comparison of bull sperm and leukocyte chromosome areas as related to gender.

Chromosomal areas from metaphase spreads of male bovine leukocytes were digitized and sex chromosomes identified using videomicroscopy. Autosomal areas were ranked in descending order within a cell and assigned to two categories based on alternating rank. X and Y chromosome areas were assigned to respective categories. Areas were divided by 4 to make their sum equivalent to sperm DNA content. Data were analyzed before and after inclusion of sex chromosomal areas. Before X and Y inclusion, rank contributed to difference in chromosomal areas. Rank by category interaction and category effects did not contribute to area variation. After X and Y inclusion, area variation was due to rank by category interaction, rank, and category. Differences between sums of chromosomal areas across categories was 3.57%. Head areas of morphologically normal sperm with intact acrosomes were digitized using the same optics as chromosomal areas. Sum of corrected chromosomal areas per category was used in discriminant analysis to assign sperm head areas to two categories with .5 prior probabilities. Assignment resulted in 1037 sperm in one category and 1177 in the other. Difference between largest sperm head area classes across categories was 3.2%. Discrimination of sperm head areas, based on sum of chromosomal area and measured with computerized videomicroscopy, may be used to evaluate sex of bovine spermatozoa.

Effect of feeding gossypol in cottonseed meal on growth, semen quality, and spermatogenesis of yearling Holstein bulls.

Yearling Holstein bulls were fed a corn silage ration supplemented with either cottonseed meal with gossypol or soybean meal in two trials to evaluate the effect of feeding gossypol on reproductive characteristics. In Trial 1, roughage to concentrate ratio was 88:12 and was fed for 60 d. In Trial 2, roughage to concentrate ratio was 50:50 and was fed for 42 d. Cottonseed meal concentrate had 3.03 g total gossypol/kg DM. Cottonseed meal concentrate was fed to provide 6 and 30 mg total gossypol/kg BW per d in Trials 1 and 2. Ejaculates were collected twice weekly via artificial vagina and critiqued for quantity and quality before and after thawing and after postthaw incubation. Leptotene spermatocytes to Sertoli cell ratio in stage 1 tubules was used to evaluate spermatogenesis. Growth characteristics and tissue total gossypol concentrations were also evaluated. No gossypol was found in plasma taken before, during, or after Trial 1 or from body organs or plasma taken during or after Trial 2. No signs of gossypol toxicity were observed, and growth characteristics were similar on both rations. Gossypol in cottonseed meal fed at low to moderate concentrations was not deleterious to seminal quantity or quality, and spermatogenesis was unaffected by treatment.

Effect of prostaglandin F(2)alpha at insemination on sperm cell numbers and pregnancy rate in beef cattle.

Fourteen cycling, nonlactating, multiparous beef cows were artificially inseminated (AI) 10 to 12 h after the onset of natural estrus. One unit of frozen-thawed semen containing 100 x 10(6) total sperm cells was deposited into the body of the uterus. Immediately after AI, alternating cows were injected i.m. with either 25 mg (5 ml) of prostaglandin F(2)alpha (PGF) or 5 ml of 0.9% saline-benzyl alcohol control solution. Cows were slaughtered 16 +/- 1 h post AI, oviducts were retrieved, segmented into thirds (upper, middle and lower) and flushed with 1 ml of 0.2% gluteraldehyde in phosphate buffered saline. The number of sperm cells was counted using a phase contrast microscope. There were no right or left side effects (P=0.61) on the number of sperm cells recovered per oviduct within cow (389 vs 553; average SEM = 219). PGF had no effect (P=0.77) on the number of sperm cells recovered per oviduct (642 vs 300; average SEM = 231 for PGF and control females, respectively). More sperm cells were recovered from the lower third segment (P<0.05) compared with the middle and upper segments. Ovulation tended to affect (P=0.10) the number of sperm cells recovered per oviduct (742 vs 200; average SEM = 231). Additionally, 114 beef females (68 Angus x Hereford heifers and 46 Chianina crossbred postpartum suckled cows) were treated as described above following AI at natural estrus with 20 x 10(6) motile sperm cells. Pregnancy rates did not differ significantly in heifers (70.6 vs 58.8%) or in Chianina cows (34.8 vs 52.2%) for control and PGF-treated females, respectively. Overall, pregnancy rates were identical between control and PGF-treated females at 56.1%. In this study, PGF treatment immediately following AI in beef cattle had no effect on the number of sperm cells in the oviducts or on the pregnancy rate.

Semen quality characteristics of dairy goats.

Semen was collected, processed, and frozen from five dairy bucks for 2 successive yr for use in quality classification and evaluation for inclusion in artificial insemination programs. Semen was evaluated for volume, initial, postthaw and 37 degrees C incubated percent progressive motility, percent postthaw 3-h 37 degrees C incubated intact acrosomes, autoagglutination, whey-induced agglutination, and percent primary, secondary, and tertiary abnormalities. Significant high correlations were found between: percent progressive motility and percent intact acrosomes, percent intact acrosomes and percent autoagglutination, and percent autoagglutination and percent whey agglutination. Means of the postthaw quality parameters, percent progressive motility, percent intact acrosomes, and percent primary and secondary abnormalities were used to categorize ejaculates within each incubation time (0 and 2 h). At 0 h, 25 ejaculates were classified as high quality and 11 were low quality. Using 2-h data, 19 ejaculates were classified as high quality and 17 as low. Inclusion of both agglutination parameters in the 2-h data analysis resulted in 13 ejaculates categorized as high and 23 as low quality. Based on assessment with techniques used in bovine artificial insemination programs, semen quality of goat semen could be used to discriminate between acceptable or unacceptable ejaculates. Based on recommended sperm numbers per inseminate and average ejaculate characteristics the low number of marketable units per ejaculate would make incorporation of goats into existing artificial insemination programs prohibitive.

The prevalence of neuropsychiatric disorders in a nursing home population.

The prevalence of neuropsychiatric disorders and other medical illnesses was investigated in 65 nursing home residents. The authors found neuropsychiatric disorders to be present in 94% of the sample. The neuropathologic causes of these syndromes were found to be more diverse than in previous studies. The most frequent causes were degenerative, vascular, and toxic. The most common psychiatric syndromes that resulted from these neuropathologic disorders were dementia syndrome (72%), organic personality syndrome (14%), and organic psychotic disorders (12%). The most common behavioral problems, agitation and aggression, most likely reflected the high prevalence of frontal lobe damage and affected 48% of the sample. Other non-neuropsychiatric medical problems were significantly less common. While only 4% of the sample had no neuropsychiatric diagnosis, 39% had no other non-neuropsychiatric diagnosis. These results suggest that the nursing home is not used as a referral source for chronic medical conditions in general but almost exclusively for the care of chronic neuropsychiatric disorders.

Use of linear semen quality score for classification and decision making in evaluation of individual ejaculates of Holstein bulls.

Four hundred seven ejaculates from 15 Holstein bulls collected from December 1984 to June 1985 were evaluated postthaw for viability characteristics (percent progressive motility at 0 h and after 3 h at 37 degrees C incubation, percent intact acrosomal membrane after 3 h at 37 degrees C incubation) and abnormal morphological characteristics [percent head (primary), midpiece, and tail (secondary) abnormalities]. Weighting coefficients for combining viability and abnormality characteristics were generated from between-bull and within-bull variance and covariance matrices. Two hundred ninety-eight additional ejaculates collected from July 1985 to February 1986 were added. Linear quality scores for 705 ejaculates (24 bulls) were the sum of the product of each quality characteristic and weighting coefficients. Univariate analysis yielded significant bull effects for viability and abnormality characteristics and linear quality score. Significant correlations existed between all seminal quality characteristics except primary and secondary abnormalities. A t test with preassigned critical value was used to evaluate each ejaculate to determine rejection from the population. Percent of ejaculates rejected was lower when linear quality score was used than when five independent tests were used. Use of linear quality score to critique semen based on each ejaculate's innate quality could compensate for the loss of bull fertility estimates from declining number of technician-based AI programs.