Automated scoring of multiprobe FISH in human spermatozoa.

In the case of chromosomal aneuploidy in sperm wherein the incident rate is low and a large number of cells require scoring, automated methods that rely on computer software to segment and to count fluorescence signals are particularly necessary due to countless hours spent in reading slides and to the potential for interoperator differences. The purpose of this pilot experiment was to determine whether there were significant differences in the estimates of disomy frequency produced by automated versus manual scoring of signals for chromosome X, Y, and 18 in human sperm. The frequency of X18, Y18, XX18, YY18, and XY18 were determined in four separate normozoospermic samples. Slides were hybridized using a standard sperm FISH protocol for centromere-specific probes. Between 500 and 564, DAPI positive nuclei were captured from each sample and scored using the automated system, and the same slides were scored by a trained cytogeneticist, who was blind to the purpose of the study and the automated system results. None of the estimated frequencies was significantly different between manual and automated methods, regardless of whether individual slides or pooled results across all samples were compared. To our knowledge, this is the first report examining the validity of automated cell scoring in human spermatozoa. The results from this pilot exploration of sperm FISH suggest the comparability between automated and manual methods for estimating sex chromosome disomy and provide evidence that automated laser scanning of multiprobe sperm FISH should be explored further.     

Hybridization of polyuridylic acid to human DNA immobilized onto nitrocellulose filters.

The level of deoxyadenylate (da) regions in human DNA was estimated from formation of poly(U)-poly(da) triplexes on nitrocellulose filters that were RNAase resistant. The (dA) rich sequences were determined following mild ribonuclease treatment of the poly(U)-DNA hybrids (5 mug/ml at 25 degreesC for 30 min), where as exhaustive ribonuclease treatment (5 mug/ml at 25 degrees C for 6 hr) estimated the more (dA) pure sequences. The level of (dA) rich regions was 0.39% of the DNA and for the more (dA) pure regions it was 0.07%. The (dA) regions were widely distributed throughout human DNA regardless of base composition or sequence repetition. However, a concentration of (dA) regions into main band CsC1 gradient fractions of DNA and into repeated DNA was observed.     

Male meiotic segregation of gonosomes analysed by two colour FISH in human interphase spermatozoa

Human meiotic segregation of X and Y chromosomes was simultaneously analysed by dual fluorescence in situ hybridization (FISH) on 10638 interphase spermatozoa from the same donor. A modified method for sperm decondensation ensured access of both X and Y probes to the sperm chromatin and a 99% hybridization efficiency. Expected sex ratios were obtained (49.30% haploidy X and 49.22% haploidy Y). The frequencies of meiotic II non-disjunctions for X and Y chromosomes (0.05%) were similar to those observed in sperm karyotypes after heterospecific fertilization of hamster eggs. In contrast, the frequency of XY bearing cells was significantly higher (0.42%). However, XY cells detected by FISH could either be diploid somatic cells, diploid germinal cells or hyperhaploid XY spermatozoa, the latter resulting from meiotic I non-disjunctions.     

Mechanism of human papillomavirus binding to human spermatozoa and fertilizing ability of infected spermatozoa

Human papillomaviruses (HPVs) are agents of the most common sexually transmitted diseases in females and males. Precise data about the presence, mechanism of infection and clinical significance of HPV in the male reproductive tract and especially in sperm are not available. Here we show that HPV can infect human sperm, it localizes at the equatorial region of sperm head through interaction between the HPV capsid protein L1 and syndecan-1. Sperm transfected with HPV E6/E7 genes and sperm exposed to HPV L1 capsid protein are capable to penetrate the oocyte and transfer the virus into oocytes, in which viral genes are then activated and transcribed. These data show that sperm might function as vectors for HPV transfer into the oocytes, and open new perspectives on the role of HPV infection in males and are particularly intriguing in relation to assisted reproduction techniques.     

Studies on the influence of aneuploidy of spermatozoa obtained from patients with oligozoospermia on their structure

The presence of aneuploidy in spermatozoa influences their biological characteristics, especially their ability to fertilise the ovum. The aim of the present study was to investigate if aneuploidy is accompanied by any changes in the morphology of spermatozoa in oligozoospermic patients. For this purpose, the percentage of aneuploid cells in sperm and the correlation between the specific morphological forms of spermatozoa and aneuploidy were evaluated. The study proved a negative correlation between DNA content of aneuploid and normal spermatozoa. A weak positive correlation was demonstrated between the presence of aneuploid spermatozoa and DNA content of spermatozoa with large heads. No such correlations could be detected for DNA content of the remaining morphological forms of spermatozoa. Thus, men with a lowered number of spermatozoa and/or with abnormal spermatozoal morphology should have their spermatozoal DNA content tested in order to evaluate the degree of aneuploidy, especially in cases where in vitro fertilisation is intended.     

Evaluation of cellulose acetate/nitrate filters for measuring the motility of dog spermatozoa

A modified chemotaxis chamber was used to evaluate cellulose acetate/nitrate filters for the measurement of dog sperm motility. The distance travelled into the filters was affected by filter pore size, incubation time and sperm concentration. After storage of spermatozoa at 37 degrees C the distance penetrated into the filters reflected the deterioration of the sample, but was not as sensitive a measure as the mean sperm velocity or the percentage of motile spermatozoa.     

Cyclic-AMP receptors in the human spermatozoa membrane.

The binding properties of cyclic-AMP to human spermatozoa were studied. Incubation of whole human spermatozoa and of head and tail fractions with ¹⁴C-cyclic AMP induced the binding of 7.8^± 0.86 pmoles of the nucleotide per 5 × 10⁷ sperm cells showing an intrinsic association constant of 15 × 10⁻⁸ M. No significant inhibition of cyclic-AMP binding was caused by the addition of one hundred fold excess of AMP. However, when the excess AMP reached a thousand fold a slight but significant reduction (10%) was observed. These values were not modified by using sperm homogenates instead of intact sperm cells, which suggested that cyclic-AMP binding is to surface membranes and not to protein released from broken or damaged sperm. Binding was found to be unrelated with pH, between 6 to 8, but depended on the temperature of the incubation medium, showing a maximum at 20°C. The blocking of membrane sulfhydryl groups significantly inhibited (48%) cyclic-AMP binding to sperm cells.     

Electron microscope studies of human spermatozoa.

Semen of 40 healthy fertile men aged between 22--44 years was investigated. A triple morphological investigation of the semen was performed in each person. For the ultramicroscopic evaluation the semen specimens were selected according to the quantative criterion of normospermic. The ultrastructure of spermatozoon was described in comphiance with its division into head, neck and tail. The substructure of the axial fibre was separately analyzed. The physiological role of individual organelles of normal spermatozoon in fertilization was discussed.     

Chromosome abnormalities in human arrested preimplantation embryos: a multiple-probe FISH study.

Numerical chromosome abnormalities were studied in single blastomeres from arrested or otherwise morphologically abnormal human preimplantation embryos. A 6-h FISH procedure with fluorochrome-labeled DNA probes was developed to determine numerical abnormalities of chromosomes X, Y, and 18. The three chromosomes were stained and detected simultaneously in 571 blastomeres from 131 embryos. Successful analysis including biopsy, fixation, and FISH analysis was achieved in 86.5% of all blastomeres. The procedure described here offers a reliable alternative to sexing of embryos by PCR and allows simultaneous ploidy assessment. For the three chromosomes tested, numerical aberrations were found in 56.5% of the embryos. Most abnormal embryos were polyploid or mosaics, and 6.1% were aneuploid for gonosomes or chromosome 18. Extrapolation of these results to all human chromosomes suggests that the majority of abnormally developing and arrested human embryos carry numerical chromosome abnormalities.     

A kinetic study of the participation of zinc in human spermatozoa metabolism

Incubation of washed human spermatozoa in the presence of 6 mM concentrations of EDTA, histidine and cysteine induces a release of about 75% of the zinc bound to the cells. No zinc is released by human spermatozoa when incubation is done in the absence of the mentioned reagents. No detectable amounts of calcium or magnesium were found to be released by the sperm cells under any of the experimental conditions tested. Zinc release induced by the presence of EDTA, histidine and cysteine is accompanied: 1) by a significant increase in oxygen uptake, both under basal conditions and in the presence of some substrates (glucose, pyrubate and succinate) and 2) by a significant increase in motility. This increase was greater with cysteine than with histidine, and greater with the latter than with EDTA. This behavior of human spermatozoa resembles that previously described for invertebrate spermatozoa and is related to the regulation of energy metabolism and probably to sperm capacitation.     

Application of semi-continuous culture on membrane filters for the study of activated sludge bacteria

Bacterial population of activated sludge flocs from the petrochemical wastewater purification plant was studied by semi-continuous culture on membrane filters. Almost all (90%) of bacterial cells in the flocs were metabolically active. Only a small fraction of bacteria in the flocs was able to use phenol or ethylene glycol as the sole source of carbon. At higher concentrations of these compounds growth of the bacteria was strongly retarded.