Morphometric assessment of mature and diminished-maturity human spermatozoa: sperm regions that reflect differences in maturity. (1/298)

As part of our studies on sperm maturity and function, we examined the head, midpiece and tail of human spermatozoa using computerized morphometry in order to determine which regions reflect the differences between mature spermatozoa and spermatozoa of diminished cellular maturity. We studied 20 men, who were divided into two groups based on their lower (LCKM: 14.6 +/- 7.0%, n = 8) and higher sperm creatine kinase (CK-M) isoform ratios (HCKM: 48.0 +/- 4.3%, n = 12) in the initial semen. Using a sequential centrifugation method which relies on the lower density of immature spermatozoa with retained extra cytoplasm, we prepared three sperm fractions with progressively declining maturity, as confirmed with CK-M isoform ratio measurements. Following the sequential fractionation, we affixed the spermatozoa to glass slides, stained the midpiece and the sperm contour, and photographed 25 spermatozoa in each of the 60 fractions (1509 spermatozoa in all). The spermatozoa were then individually digitized on the Image-1 system, and the dimensions of the head, midpiece, and tail were determined. While the data showed significant differences in the midpiece and tail dimensions between the mature and diminished-maturity sperm fractions, the head dimensions were similar and did not reflect sperm maturity. We postulated that the relationship between the biochemical markers of sperm maturity and sperm morphology is based on common spermiogenic events. The data support this idea. In immature spermatozoa in which cytoplasmic extrusion, CK-M isoform expression, and tail sprouting are all diminished, the retained extra cytoplasm in the midpiece and shorter tail length contribute to the morphological variations that we identified by morphometry and considered in sperm morphology. These morphometric features, in association with fluorochrome-coupled biochemical probes, can facilitate the identification of mature spermatozoa in computer-assisted semen analysis.  (+info)

Comparative histopathology of radial artery versus internal thoracic artery and risk factors for development of intimal hyperplasia and atherosclerosis. (2/298)

BACKGROUND: In this study, we examined the comparative histopathology, morphometry, and risk factors for the development of intimal hyperplasia and atherosclerosis in the radial artery (RA) and the internal thoracic artery (ITA). METHODS AND RESULTS: Paired specimens of RAs and ITAs, obtained from 150 patients who underwent CABG, were evaluated with histopathology; 110 pairs of arteries were suitable for morphometric analysis. The severity of disease was evaluated on the basis of percentage of luminal narrowing, intimal thickness index, and intima-to-media ratio. Risk factors were determined with stepwise linear regression. Intimal hyperplasia was seen in 141 RAs (94%) and 103 ITAs (69%) (P<0.001). Atherosclerosis was seen in 5% of RAs and 0.7% of ITAs (P=0.04). Medial calcification was found only in RAs (20 of 150, 13.3%) (P<0.001). Morphometric analysis showed that compared with ITAs, RAs had a significantly higher intimal area, medial area, percentage of luminal narrowing, intimal thickness index, and intima-to-media ratio (all P<0.001) Factors found to be significant (P<0.05) predictors of the 3 severity indices of intimal hyperplasia, including atherosclerosis, in RAs were peripheral vascular disease, smoking, age, and diabetes. Risk factors for intimal hyperplasia in ITAs were age and smoking. CONCLUSIONS: The RA is more likely to have atherosclerosis, intimal hyperplasia, and medial calcification than the ITA. Morphometric analysis indices showed marked differences between the RA and the ITA. Care should be taken when selecting the RA as a conduit in CABG, particularly in patients who are elderly, diabetic, smoke, or have peripheral vascular disease.  (+info)

Association between tissue hypoxia and elevated non-protein sulphydryl concentrations in human cervical carcinoma xenografts. (3/298)

A double staining technique was developed for the simultaneous measurement of tissue hypoxia and the concentration of non-protein sulphydryls (NPSH), based on the fluorinated nitroimidazole EF5 and the fluorescent histochemical NPSH stain 1-(4-chloromercuriphenoylazo)-naphthol-2 (mercury orange). Cryostat sections of tumour tissue were examined by fluorescence image analysis, using a computer-controlled microscope stage to generate large tiled field images of the cut tumour surface. This method was applied to the human cervical squamous cell carcinoma lines ME180 and SiHa, grown as xenografts in severe combined immunodeficient (SCID) mice, in order to determine if there is a systematic relationship between tissue hypoxia and NPSH levels. Hypoxic regions of the tumours, defined by EF5 labelling, were found to show greater NPSH concentrations relative to better oxygenated regions. This is probably due to increases in glutathione, since the ME180 and SiHa xenografts contained low levels of cysteine and metallothionein; the other major cellular thiols that can bind to mercury orange. Because the effects of glutathione on radiation and chemotherapy resistance are likely to be greater under hypoxic conditions, these results have potentially important implications for the study of resistance mechanisms in solid tumours.  (+info)

The use of laser scanning cytometry to assess depth of penetration of adenovirus p53 gene therapy in human xenograft biopsies. (4/298)

SCH58500 is an agent for gene therapy of cancer, consisting of a replication-deficient type 5 adenovirus (Ad5) expressing the human p53 tumor suppressor gene (Ad5/p53). An important question about the use of Ad5/p53 gene therapy is how to achieve the therapeutically effective delivery of an Ad5/p53 vector to the tumor. We wanted to determine the effective depth of penetration of an Ad5/p53 vector by dosing the vector in an experimental human xenograft/SCID model. To assess depth of penetration, we developed a novel methodology for scanning tissue sections by laser scanning cytometry (LSC). SCID mice were given intraperitoneal injections of either p53(null) SK-OV-3 human ovarian tumor cells or p53(mut) DU-145 human prostate tumor cells to establish xenograft solid tumors. Mice were then dosed once or twice at 24-hour intervals by intraperitoneal injection with SCH58500 (Ad5/p53), an adenovirus construct expressing beta-galactosidase (Ad5/beta-gal), or a buffer control. Additional groups of mice received a single intraperitoneal dose of 10 mg/kg paclitaxel either alone or coadministered with Ad5/p53. Twenty-four hours after each last dose, the human solid tumor xenograft and relevant mouse tissue were removed from each mouse for the analysis of Ad5/p53 penetration. Immunohistochemistry (IHC) for beta-galactosidase protein revealed a depth of penetration of between 1 and 10 cells from the tumor surface. In some mice, hepatocytes in the periportal regions of liver lobules were also positive, indicating systemic absorption of adenovirus from the peritoneal cavity. IHC staining for p53 and p21 proteins in SK-OV-3 solid tumor xenografts revealed similar Ad/p53 penetration. LSC was used to map and quantitate apoptosis in both tumor and liver tissue biopsies, with over 450,000 nuclei from liver tissue and 150,000 nuclei from tumor tissue being evaluated. LSC analysis demonstrated a high level of apoptosis in the tumors that had been removed from Ad5/p53-dosed mice (12.7-19.7%). This level of apoptosis was significantly higher (P < 0.05) than was observed for liver tissues taken from Ad5/p53-dosed mice (2.7-8.0%) or tumor tissues taken from either Ad5/beta-gal-dosed mice (3.0-6.4%) or buffer control-dosed mice (3.0-5.3%). Scan bit maps from the extensive LSC analyses confirmed that apoptosis was present to about the same depth (1-10 cells) as had been identified by IHC for beta-galactosidase, p53, and p21 proteins. Paclitaxel coadministered with Ad5/p53 had no effect on Ad5 penetration into solid tumors in vivo as measured by IHC for p53 or p21 protein. However, the combination therapy did cause an elevation in the number of tumor cells undergoing apoptosis.  (+info)

Image morphometric nuclear grading of intraepithelial neoplastic lesions with applications to cancer chemoprevention trials. (5/298)

A new image morphometric method of nuclear grading is described and assessed in the context of the evaluation of histological samples from ductal carcinoma in situ of the breast and cervical intraepithelial neoplasia. The method results in a continuous scaled variable, or nuclear grading scale, expressed in SD units from measured normal nuclei from breast or cervix. For a given histological preinvasive neoplastic lesion, the mean nuclear grade of measured nuclei was shown to be analogous to the histopathological nuclear grade of the same lesion assigned subjectively by the pathologist. In a chemoprevention trial of the effect of difluoromethylornithine given for 1 month to subjects with cervical intraepithelial neoplasia grade 3, pathologists could see no difference in 14 histological sections taken before and after difluoromethylornithine treatment. However, the image morphometric method detected a systematic effect of lowered mean nuclear grade and a decrease in the variability of nuclear grade expression. Twelve of 14 samples showed a lower posttreatment mean nuclear grade (P<0.05), and 13 of the 14 samples showed a decrease in the SD of their nuclear grade distributions (P<0.01). This study demonstrates the use of image morphometric nuclear grading in a chemoprevention setting. It may be very useful in supplementing the pathologist's histopathological grading by providing objective, quantitative assessments.  (+info)

No evidence for killer sperm or other selective interactions between human spermatozoa in ejaculates of different males in vitro. (6/298)

This study examines one of the possible mechanisms of sperm competition, i.e. the kamikaze sperm hypothesis. This hypothesis states that sperm from different males interact to incapacitate each other in a variety of ways. We used ejaculates from human donors to compare mixes of semen in vitro from the same or different males. We measured the following parameters: (i) the degree of sperm aggregation, velocity and proportion of morphologically normal sperm after 1 and 3 h incubation in undiluted semen samples, (ii) the proportion of viable sperm plus the same parameters as in (i) in 'swim-up' sperm suspensions after 1 and 3 h incubation, (iii) the degree of self and non-self sperm aggregation using fluorescent dyes to distinguish the sperm of different males, and (iv) the extent of sperm capacitation and acrosome-reacted sperm in mixtures of sperm from the same and different males. We observed very few significant changes in sperm aggregation or performance in mixtures of sperm from different males compared with mixtures from the same male and none that were consistent with previously reported findings. The incapacitation of rival sperm therefore seems an unlikely mechanism of sperm competition in humans.  (+info)

Sjogren's syndrome and MALT lymphomas of salivary glands: a DNA-cytometric and interphase-cytogenetic study. (7/298)

Few and conflicting cytogenetic data are available concerning the chromosomal constitution of (mainly gastric) extranodal marginal zone B-cell non-Hodgkin's lymphoma arising from mucosa-associated lymphoid tissue (MALT)-type lymphoma. The majority of salivary gland MALT lymphomas are thought to develop from longstanding Sjogren's syndrome/benign lymphoepithelial lesion (BLEL). We tried to achieve a better comprehension of related cytogenetic alterations by comparing DNA-ploidy and numerical chromosomal (#) aberrations, assessed by different techniques of DNA cytometry (image cytometry) and interphase cytogenetics using nonradiographic in situ hybridization (centromere specific probes for #3, 7, 12, 18) on 12 cases of BLEL, 13 low-grade MALT lymphomas (LG-MALT-L) and 4 high-grade MALT lymphomas (HG-MALT-L) of salivary gland. Both techniques were applied on tissue sections preferentially, enabling a reliable measurement of histomorphologically identified areas. No case of BLEL showed cytogenetic abnormalities. Three of 4 HG- and 2 of 13 LG-MALT-L exhibited complex chromosomal gains in nonisotopic in situ hybridization, which were reflected by DNA nondiploidy in image cytometry. In 6 of 13 LG- and lof 4 HG-MALT-L, one or two numerical chromosomal aberrations were demonstrated by nonisotopic in situ hybridization, which could not be resolved by image cytometry. In the 11 DNA-diploid LG-MALT-L, trisomies 18, 3, and 12 were found in 36, 12, and 9%, respectively. In conclusion, comparing BLEL, which showed no chromosomal aberrations, with LG- and HG-MALT-L, an increase in frequency and number of numerical aberrations and DNA nondiploidy was seen. Peritetraploid DNA nondiploidy might be characteristic for HG-MALT-L of salivary gland as it is a rare finding in MALT lymphomas of other sites. It is unclear whether the documented chromosomal aberrations in LG-MALT-L, especially increased rate of trisomy 18, indicate a pathogenic impact or merely reflect genetic instability.  (+info)

Telomere shortening is an in vivo marker of myocyte replication and aging. (8/298)

To determine whether adult cardiac myocytes are capable of multiple divisions and whether this form of growth is restricted to a subpopulation of cells that retain this capacity with age, telomere lengths were measured in myocyte nuclei isolated from the left ventricle of fetal and neonatal Fischer 344 rats and rats at 4, 12, and 27 months after birth. Two independent methodologies were used for this analysis: laser scanning cytometer and confocal microscopy. In each case, fluorescence intensity of a peptide nucleic acid probe specific for telomeric sequence was evaluated. The two techniques yielded comparable results. Telomeric shortening increased with age in a subgroup of myocytes that constituted 16% of the entire cell population. In the remaining nondividing cells, progressive accumulation of a senescent associated nuclear protein, p16(INK4), was evidenced. In conclusion, a significant fraction of myocytes divides repeatedly from birth to senescence, counteracting the continuous death of cells in the aging mammalian rat heart.  (+info)

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