Simultaneous detection of X- and Y-bearing human sperm by double fluorescence in situ hybridization

Double fluorescence in situ hybridization (FISH) was used to detect sex chromosomes in decondensed human sperm nuclei. Biotinylated X chromosome specific (TRX) and digoxigenin-labeled Y chromosome specific (HRY) probes were simultaneously hybridized to sperm preparations from 12 normal healthy donors. After the hybridization, the probes were detected immunocytochemically, using two different and independent affinity systems. Ninety-six percent of the 12,636 sperm showed fluorescent labeling, of which 47.4% were haploid X and 46.8% were haploid Y. A frequency of 0.46% of XX-bearing sperm (0.28% disomic, 0.18% diploid) and 0.38% YY-bearing sperm (0.21% disomic, 0.17% diploid) was found. The overall proportions of X- and Y-bearing sperm in the ejaculates were 47.9% and 47.2%, respectively, which was not significantly different from the expected 50:50 ratio. In addition 0.21% of cells appeared to be haploid XY-bearing sperm, 0.62% were diploid XY-bearing cells, and 0.05% of cells were considered to be tetraploid cells. The application of double FISH to human sperm using X-chromosome and Y-chromosome probes has allowed a more accurate assessment of the sex chromosal complements in sperm than single FISH method and quinacrine staining for Y-bodies.     

Dual control of heat shock response: involvement of a constitutive heat shock element-binding factor

Heat shock factor (HSF) has been implicated as the key regulatory protein in the heat shock response. Our studies on the response of rodent cells to heat shock or sodium arsenite indicate that a high level of HSF-DNA-binding activity, by itself, is not sufficient for the induction of hsp70 mRNA synthesis; furthermore, a high level of HSF binding is also not necessary for this induction. Analysis of the binding of protein factors to the heat shock element (HSE) in extracts of stressed rodent cells indicates that the regulation of heat shock response involves the heat-inducible HSF and a constitutive HSE-binding factor. Our results also suggest that overexpression of human hsp70 may decrease the level of heat-induced HSF-HSE-binding activity in rat cells.     

Serotonin and serotonin receptors in the central auditory system

Single channel currents were activated by GABA (0.5 to 5 microM) in cell-attached and inside-out patches from cells in the dentate gyrus of rat hippocampal slices. The currents reversed at the chloride equilibrium potential and were blocked by bicuculline (100 microM). Several different kinds of channel were seen: high conductance and low conductance, rectifying and "nonrectifying." Channels had multiple conductance states. The open probability (Po) of channels was greater at depolarized than at hyperpolarized potentials and the relationship between Po and potential could be fitted with a Boltzmann equation with equivalent valency (z) of 1. The combination of outward rectification and potential-dependent open probability gave very little chloride current at hyperpolarized potentials but steeply increasing current with depolarization, useful properties for a tonic inhibitory mechanism.     

The effect of bilateral thoracoplasty on lung development in fetal sheep

The relationship of breathing movements to lung development in the ovine fetus was investigated by partially removing ribs on each side of the chest and closing the deficiencies with silicone membranes at 114 days of gestation; the increase in compliance of the chest wall that resulted caused blunting of the amplitude of phasic negative pressures recorded in the trachea to less than 10 torr. Compared to sham operated controls (n = 5), the lungs of the thoracoplasty group (n = 5) at term weighed significantly (P less than 0.05) less, both wet (1.5 +/- 0.2 v. 2.3 +/- 0.1% of body weight) and dry (0.14 +/- 0.01 v. 0.18 +/- 0.01% of body weight. In addition, DNA content of the thoracoplasty group was less than that of the control group (0.47 +/- 0.05 mg v. 0.72 +/- 0.20 mg). Distensibility of the left lung with air at 40 cmH20 was less than in the thoracoplasty group than in controls (10.0 +/- 2.0 v. 18.9 +/- 3.0 ml.kg-1 body weight) but no differences were found in the concentrations of saturated phosphatidylcholine in lung tissue and lavage fluid, in DNA concentrations or in the amount of lung water (as % of wet weight of lung). It is concluded that phasic negative pressures of normal intensity are necessary for normal development of the fetal lungs.     

Biosynthesis of mitochondrial DNA. Is 8 S DNA an artifact?

Sucrose density gradient fractionation of isolated rat liver mitochondrial DNA ordinarily yields two peaks, one at 39 S, the other at 27 S. However, when these mitochondria are first incubated with a labeled DNA precursor, a labeled peak at about 8 S is also observed. Is this low molecular weight 8 S DNA merely an artifact of contamination or breakdown, or is it a functioning part of the mitochondrial genome? That it is not a nuclear contaminant is shown by: (a) the absence of nuclei or nuclear fragments in active mitochondrial preparations; (b) the insensitivity of 8 S DNA synthesis to treatment of mitochondria with DNase and RNase; (c) the ability of inner membrane preparations to synthesize this DNA; (d) the ability of atractyloside to inhibit incorporation of [3H]dATP into 8 S and 39 S or 27 S DNA equally; (e) the labeling of 8 S DNA (as well as 39 S and 27 S DNA) but not of nuclear DNA after the administration in vivo of [3H]thymidine. The evidence that 8 S DNA is not an artifact resulting from DNA breakdown during mitochondrial incubation or DNA isolation is as follows: (a) 8 S DNA can be isolated from unincubated mitochondrial; (b) 8 S DNA becomes labeled when labeled DNA precursors are administered in vivo; (c) 8 S DNA biosynthesis continues in the complete absence of labeled 39 S or 27 S DNA (whose synthesis is repressed by ethidium bromide), making it unlikely that 8 S DNA is formed from the breakdown of 39 S or 27 S DNA; (d) substitution of milder methods of DNA extraction does not decrease 8 S DNA labeling; moreover, the usual conditions of extraction, when applied to purified 39 S and 27 S DNA, do not generate 8 S DNA, nor does an additional mitochondrial washing cycle; (e) the specific radioactivity of 8 S DNA is higher than that of 39 S or 27 S DNA, making it improbable that the latter forms are precursors of 8 S DNA. Since 8 S DNA is double-stranded, it is not identical to the 7 S fragment of D loop DNA. The hypothesis that the artifactual nicking of those DNA molecules which contain opposing D loops leads to the release of double-stranded fragments was tested. The DNA which was released was predominantly (and probably completely) single-stranded. We conclude that 8 S DNA is probably not an artifact and studies are in progress on its function.