PubMed Health⌕ Search

Biomedical subjects

D P Evenson

Publications and source records attributed to D P Evenson.

At least 19 recordsLinked to original sources
PubMed1991

Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay.

Eight monthly semen samples from 45 men not known to be exposed to industrial toxicants were measured by the flow cytometric sperm chromatin structure assay (SCSA). This assay determines susceptibility of sperm DNA to in situ, acid-induced denaturation and is quantitated by the metachromatic shift of acridine orange fluorescence from green (native DNA) to red (denatured DNA). The observed green versus red fluorescence scattergram (cytogram) patterns were generally unique between donors and homogeneous within a donor over time. Within a donor, the cytogram patterns were the same whether intact sperm cells or detached nuclei were measured. For some individuals the cytogram patterns differed for some months and then returned to the original pattern. Intraclass correlations for mean and standard deviation of alpha t [alpha t = red/(red + green) fluorescence] were higher (.67 to .90) than any classically measured semen variables, suggesting that SCSA results within an individual were more consistent than other measures. Furthermore, average within-donor CV of alpha t parameters expressed as a percent of any given individual's means was around 10%, which is significantly lower than those derived from common semen measures. The SCSA is an objective, technically sound, biologically stable, sensitive, and feasible measure of semen quality.

Chromatin↗
PubMed1991

Effects of egg yolk-citrate and milk extenders on chromatin structure and viability of cryopreserved bull sperm.

Semen from four Holstein bulls was evaluated to compare effects of four extender treatments on postthaw semen quality. Extender fractions A and B, either heated whole milk or 20% egg yolk-citrate, were combined to yield the extender treatments 1) milk and milk, 2) milk and egg yolk-citrate, 3) egg yolk-citrate and milk, and 4) egg yolk-citrate and egg yolk-citrate. Semen was evaluated at thawing and after 30, 60, 120, and 180 min of incubation at 38.5 degrees C. Flow cytometry showed that acridine orange-stained sperm were most susceptible to in situ DNA denaturation when fraction A was milk. For sperm stained with rhodamine 123, flow cytometry showed that the proportion with intact mitochondrial membrane potential was lowest of all treatments at thawing but greatest at 180-min incubation with milk and milk extender. Flow cytometry of propidium iodine-stained sperm showed greatest proportion of cell membrane intact sperm when fraction A was egg yolk-citrate. Light microscopy showed the lowest proportion of cell membrane intact sperm with milk and milk extender after eosin-aniline blue vital staining. Postthaw motility scores tended to be reduced when both extender fractions were egg yolk-citrate. Results demonstrate differential extender effects on postthaw semen quality and indicate that altering extender composition or sequence of adding extender components may improve postthaw quality of cryopreserved sperm.

Analysis of Variance↗
PubMed1990

Comparison of semen quality in young and mature Holstein bulls measured by light microscopy and flow cytometry.

Random samples of cryopreserved, milk-extended semen, collected from 20 Holstein bulls at about 14 mo of age (young) and again at about 4 yr of age (mature), were evaluated at thawing and during 3-h incubation to compare semen quality of young versus mature bulls. Evaluation by differential interference contrast microscopy showed greater proportions of cytoplasmic droplets in semen from young versus mature bulls. Mature bulls exhibited greater proportions of intact acrosomes in freshly thawed semen than did young bulls. Evaluation of sperm chromatin structure by flow cytometry after staining with acridine orange showed lower values for mature versus young bulls, indicating resistance of DNA in nuclear chromatin to acid denaturation increased with age. Correlations between ages for most sperm morphology, acrosome integrity, and flow cytometry variables were high and positive. Nonreturn rate for young bulls was positively related to morphologically normal sperm and acrosomal integrity and negatively related to flow cytometry traits. Results suggest semen quality of young bulls was related to subsequent quality as mature bulls. With flow cytometry, differences were detected between semen samples that were not evident with light microscopy.

Acrosome↗
PubMed1989

Long-term effects of triethylenemelamine exposure on mouse testis cells and sperm chromatin structure assayed by flow cytometry.

The toxic and potentially mutagenic actions of triethylenemelamine (TEM) on mouse body and testis weights, testicular cell kinetics, sperm production, sperm head morphology, and sperm chromatin structure were assessed in two experiments. The first experiment examined effects of four dose levels of TEM, assayed 1, 4, and 10 wk after toxic exposure. In the second study, effects from five dosage levels were measured at 1, 4, and 10 wk, and the highest dosage level was evaluated over 44 wk. TEM produced an expected dose related loss of spermatogenic activity and subsequent recovery as determined by dual-parameter (DNA, RNA) flow cytometry (FCM) measurements of testicular cells. Both testicular weights and caudal sperm reserves remained generally below controls after 44 wk recovery following exposure to the highest (1.0 mg/kg daily x 5) dosage. Chromatin structure alterations, defined as increased susceptibility to DNA denaturation in situ, and sperm head morphology were highly correlated (.87-.93, P less than .001) with dose and with each other. Data obtained from the sperm chromatin structure essay (SCSA) on fresh sperm was highly correlated with measurements of aliquots of the same sample collected over 44 wk, frozen, and then measured on the same day. Sperm head morphology and sperm chromatin structure remained abnormal at 44 wk for the 1.0 mg/kg TEM dosage, suggesting that the abnormalities, present long after the initial toxic response, may be a result of mutation. This study demonstrates that flow cytometry provides a unique, rapid, and efficient means to measure effects of reproductive toxins and potential mutagens.

Animals↗
PubMed1989

Flow cytometric analysis of rodent epididymal spermatozoal chromatin condensation and loss of free sulfhydryl groups.

Flow cytometric measurements were made on acridine orange (AO) and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methyl-coumarin (CPM)-stained epididymal- and vas deferens-derived spermatozoal nuclei to follow the course of chromatin condensation and oxidation of free sulfhydryl groups, respectively, during passage through mouse and rat posttesticular reproductive tracts. Alterations of mouse and rat spermatozoal chromatin during transition from a testicular elongated spermatids to epididymal caput spermatozoa resulted in a threefold loss of DNA stainability with AO. Passage of spermatozoa from the caput to corpus epididymis was accompanied by an approximate 15% loss of DNA stainability, which was maintained at that level throughout passage into the vas deferens. AO stainability of epididymal spermatozoal nuclei was generally independent of -SH group stainability. CPM stainability of rat spermatozoal nuclei free -SH groups was 83%, 18%, and 11% of caput spermatozoal values for corpus, cauda epididymis, and vas deferens, respectively. Comparable values for mice were 69%, 20%, and 18%. CPM stainability was relatively homogeneous for these mouse and rat reproductive tract regions, except mouse corpus epididymis spermatozoal nuclei stained very heterogeneously. Rat spermatozoa detained by ligature up to 7 days in the caput, corpus, and cauda epididymi had CPM staining values equal to or below those of normal vas spermatozoa, indicating that disulfide (S-S) bonding is intrinsic to the spermatozoa and is independent of the epididymal environment. These data suggest that chromatin condensation and loss of spermatozoal DNA stainability during passage from the testis to the vas deferens are independent of S-S bonding.(ABSTRACT TRUNCATED AT 250 WORDS)

Acridine Orange↗
PubMed1989

Effect of 1,3-dinitrobenzene on prepubertal, pubertal, and adult mouse spermatogenesis.

Exposure of prepubertal, pubertal, and adult mice to 0, 8, 16, 32, 40, or 48 mg 1,3-dinitrobenzene (m-DNB)/kg body weight and measuring responses 1-25 d posttreatment (dpt) demonstrated significant effects on testicular function only at 48 mg/kg dosage. m-DNB had no effect on body or testis weights with the exception of reduced adult mouse testis weights at 22 dpt with 48 mg/kg (p less than .05). None of the exposures resulted in detectable levels of germinal epithelial cells in the ductus epididymis. Exposure of prepubertal and pubertal mice to m-DNB caused only minimal nonsignificant changes in the relative percent of testicular cell types present up to 25 dpt. The adult mice testicular cell type ratios, in particular the round and elongating spermatid populations, changed significantly at doses of 48 mg/kg. Also, a reduction in the percent tetraploid cells occurred at d 1, suggesting these cells may be a primary target of m-DNB action. Caput and caudal sperm from mice exposed to m-DNB prior to puberty did not demonstrate an increased susceptibility to DNA denaturation when analyzed by the sperm chromatin structure assay. However, in pubertal mice, m-DNB exposure further exaggerated the abnormal chromatin structure that normally characterizes sperm during the onset of sperm production. In adult mice, 48 mg/kg resulted in increased susceptibility to DNA denaturation of caput sperm chromatin at 11 dpt (p less than .05) and in caudal sperm at 22 dpt (p less than .01). The abnormal chromatin structure of cauda sperm from adult mice was highly correlated with sperm head morphology abnormalities (ABN; 0.82 to 0.95, p less than .01, 11 and 22 dpt, respectively), but showed lower correlations with dose (0.60 to 0.79, p less than .01, 11 and 22 dpt, respectively). For pubertal mice, a positive relationship was also observed between the variation of sperm chromatin structure abnormalities and ABN. The effect of m-DNB on testicular function in prepubertal and pubertal mice appear to be less pronounced than in adult mice. Furthermore, following exposure to the same dosage, the effect of m-DNB is less severe in adult mice than that observed for adult rats as reported in the companion paper.

Animals↗
PubMed1989

Flow cytometric analysis of effects of 1,3-dinitrobenzene on rat spermatogenesis.

Exposure of 100-d old rats to 1,3-dinitrobenzene (m-DNB) at dosages up to 48 mg/kg resulted in disruption of spermatogenesis as measured by flow cytometry (FCM) of acridine orange-stained sperm and testis cells. One day (d 1) after a single exposure to 48 mg/kg m-DNB, FCM measurements of caput epididymal fluid cells demonstrated the presence of testicular germinal epithelial cells apparently sloughed off into the epididymis. Also, at d 1 after the same exposure, a decrease in pachytene spermatocytes was observed. By d 16 after exposure to 32 or 48 mg/kg, testicular damage was evidenced by an alteration of cell type ratios in FCM-analyzed populations of testicular cells. Extensive recovery of cell type ratios occurred by d 32. At d 16, dosages of 32 and 48 mg/kg caused alterations of sperm chromatin structure as determined by the flow cytometric sperm chromatin structure assay (SCSA); 48 mg/kg caused alterations at both d 16 and d 32. Exposure to m-DNB caused a dose response increase in percent sperm head morphology abnormalities (%ABN) assessed in cauda epididymal and vas sperm. A slightly higher correlation existed between dose and SCSA alpha t values (d 16, .78; p less than .01) than between dose and %ABN (d 16, .70; p less than .01). Also, a higher correlation existed between standard deviation of alpha t (SD alpha t) values and %ABN (.97; p less than .01) than between dose and %ABN (.70; p less than .01). This study demonstrated rapid and unique FCM procedures originally derived for reproductive toxicology studies in mice to be equally useful for studies in rats.

Animals↗
PubMed1988

In vivo and in vitro effects of hydralazine on cellular growth, differentiation, and chromatin structure.

The effects of hydralazine (1-hydrazinophthalazine), an antihypertensive drug, on mammalian cell growth, viability, and differentiation were assessed using Friend leukemia cells, Chinese hamster ovary cells, human lymphocytes, and rat lymphocytes, testicular germ cells, and epididymal sperm. Cultured cells in exponential phase growth were more susceptible to hydralazine cytotoxicity than stationary phase (G0) cells. Growth inhibition was associated with a dose-related slowdown of cell progression through S phase and was observed prior to a decrease of cell viability. At high drug concentrations, progression in all phases of the cell cycle was partially or totally inhibited. Hydralazine did not have an effect on the proliferation and differentiation of testicular germ cells in spontaneously hypertensive rats receiving 0-90 mg/kg/day (up to 20 times the dose used in humans) of hydralazine for a 12-week period. Hydralazine-exposed, histone-containing somatic cells and protamine-containing sperm cells failed to show any alterations in stainability with a DNA-intercalating dye nor in the susceptibility of nuclear DNA to undergo acid-induced denaturation in situ. The data suggest that hydralazine causes a dose-related suppression of mammalian cell growth with S phase appearing to be the most susceptible to hydralazine cytotoxicity. Furthermore, the interaction of hydralazine with chromatin at concentrations leading to antigenicity did not inhibit DNA staining with the intercalating dye acridine orange, suggesting that the drug does not competitively intercalate at a detectable level. Association of hydralazine with chromatin did not cause a detectable level of stabilization or destabilization of the DNA to denaturation in situ.

Animals↗
PubMed1987

Effects of the fungicide methyl-benzimidazol-2-yl carbamate (MBC) on mouse germ cells as determined by flow cytometry.

Dual-parameter (DNA, RNA) flow cytometry (FCM) measurements were made on testicular and epididymal sperm cells isolated from mice exposed by oral gavage to 0, 250, 500, or 1000 mg/kg X 5 d of the fungicide methylbenzimidazol-2-yl carbamate (MBC), which is known to bind with tubulin subunits and inhibit polymerization and microtubule formation. Effects of exposure to MBC were measured at 7, 24, and 39 d posttreatment. MBC had no effect on body weights, but testis weights and sperm parameters were altered, with few exceptions, only at the highest exposure level. Testis weights were reduced by about 25% at 7 and 24 d after exposure; recovery was observed by 39 d after treatment. FCM measurements of testicular cells showed relative percentages of certain testicular populations (round, elongating, and elongated spermatids) were different from the control pattern 7 and 24 d after treatment. The mean percent of cauda epididymal sperm head morphology abnormalities and the susceptibility of the nuclear DNA to denaturation were both elevated at 7, 24, and 39 d after exposure to 1000 mg/kg. The level of denaturation was determined by FCM measurements of the metachromatic shift in acridine orange (AO) stained sperm nuclei from green (native DNA) to red (single-stranded DNA) fluorescence and quantitated by the expression alpha t[red/(red + green] fluorescence. These data demonstrate that spermatogenesis is sensitive to high-dose MBC exposure resulting in an altered ratio of testicular cell types present, abnormal sperm head morphology, and an altered sperm chromatin structure.

Administration, Oral↗
PubMed1987

Heterogeneity of sperm nuclear chromatin structure and its relationship to bull fertility.

The relationship between sperm nuclear chromatin structure and fertility was evaluated in two groups of Holstein bulls: Group 1, 49 mature bulls, and Group 2, 18 young bulls. Fertility ratings had been estimated for Group 1 and nonreturn rates were known for Group 2. Semen samples were measured by the sperm chromatin structure assay (SCSA): sperm were treated to induce partial in situ DNA denaturation, stained with acridine orange, and evaluated by flow cytometry. Acridine orange intercalated into double-stranded DNA emits green fluorescence upon excitation with 488 nm light, and red fluorescence when associated with single-stranded DNA. An index of DNA denaturation per cell is provided by alpha-t [alpha t = red/(red + green) fluorescence]. The standard deviation (SD alpha t), coefficient of variation (CV alpha t) and proportion of cells outside the main population (COMP alpha t) of the alpha t distribution quantify the extent of denaturation for a sample. Intraclass correlations of the alpha t values were high (greater than or equal to 0.70), based on four collections obtained over several years from Group 1 bulls. Negative correlations were obtained between fertility ratings and both SD alpha t (-0.58, p less than 0.01) and COMP alpha t (-0.40, p less than 0.01) in Group 1, and between nonreturn rates and both SD alpha t (-0.65, p less than 0.01) and COMP alpha t (-0.53, p less than 0.05) in Group 2. These data suggest that the SCSA will be of value for identification of low fertility sires and poor quality semen samples.

Aging↗
PubMed1987

Semen cryopreservation and artificial insemination for Hodgkin's disease.

Seventy-nine men with Hodgkin's disease were treated with chemotherapy protocols at Memorial Sloan-Kettering Cancer Center and had pretreatment semen analysis performed at the area semen bank. The patients were evaluated to determine: the quality of pretreatment semen, the effect of treatment on spermatogenesis, and the success rate of artificial insemination after semen cryopreservation. Pretreatment sperm concentration, fresh motility, fresh progression, postthaw motility and postthaw progression were all significantly decreased in men with Hodgkin's disease compared with normal controls. Posttreatment semen analysis in 44 men showed azoospermia in 80%, sperm concentration, less than or equal to 10 X 10(6)/mL in 11%, and sperm concentration greater than 10 X 10(6)/mL in 9%. Eleven couples attempted artificial insemination using cryopreserved semen, thus far resulting in three pregnancies. Semen cryopreservation and artificial insemination offer a partial solution to posttreatment azoospermia in this population, but further methods are needed to minimize gonadal toxicity without compromising therapy for Hodgkin's disease.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1986

Toxicity of thiotepa on mouse spermatogenesis as determined by dual-parameter flow cytometry.

Multiparameter flow cytometry (FCM) measurements were made on acridine orange (AO)-stained mouse testicular cells and epididymal sperm cells to determine the effects of varying dosages of thiotepa (0-5 mg/kg ip daily X 5 days) on spermatogenesis at 7, 28, and 67 days after the last exposure (ALE). FCM multiparameter measurements included DNA stainability vs RNA content, peak amplitude vs integrated area of DNA fluorescent signal, and double-stranded DNA vs single-stranded DNA. Thiotepa exhibited dramatic damaging effects on the kinetics and/or cell kill of seven testicular cell types measured by dual-parameter flow cytometry. At 7 days ALE, one 4N cell type, likely the pachytene spermatocyte, was absent from the testes, and another was reduced by about 70%. By 28 days ALE, most of the germ cells were absent from the seminiferous tubules, and by 67 days ALE the testes were undergoing recovery of spermatogenesis with only half of the seminiferous tubules repopulated after treatment with 5.0 mg/kg. The dual parameters of DNA stainability vs RNA content provided better resolution of testicular cell types into distinct populations than the peak vs area processing of the green fluorescent signal of AO-stained cells. Dosage of thiotepa was significantly related to percentage of sperm head morphological abnormalities assayed by light microscopy. Utilizing the metachromatic properties of acridine orange, FCM measurements of the amount of single-stranded DNA induced within acid-stressed whole sperm or heat-stressed nuclei detected alterations of chromatin structure at the same minimal effective dose required to increase abnormal sperm head morphology. Epididymal sperm isolated from mice exposed to some concentrations of thiotepa had an increased percentage of free heads and tails. DNA in free heads denatured in situ to a greater extent than DNA in intact sperm.

Animals↗
PubMed1986

Mouse testicular and sperm cell development characterized from birth to adulthood by dual parameter flow cytometry.

Dual parameter flow cytometry was used to investigate cellular changes in male germinal tissue during normal postpartum maturation in B6C3F1/J mice. Animals were killed at 2-day intervals from 2 to 42 days postpartum and at 48, 64, 72, 93 and 100 days postpartum. Testicular, cauda epididymis and vas deferens cell suspensions were stained with the metachromatic fluorochrome acridine orange and measured by flow cytometry for red and green fluorescence levels after excitation by blue laser light. Intensities of red and green fluorescence reflect amounts of single- and double-strand nucleic acid sites available for acridine orange staining, respectively, and were used to classify cells on the basis of ploidy level, RNA content, and chromatin structure, as defined by susceptibility to acid denaturation of DNA in situ. Sperm from cauda epididymis and vas deferens were examined by light microscopy to determine frequency of abnormal sperm head morphology. Fluorescence data derived from acridine orange-stained testicular cells quantified the sequential changes in 1) proportions of haploid, diploid and tetraploid cell types during the first round of spermatogenesis, and 2) proportions of round, elongating, and elongated spermatids during the first round of spermiogenesis. Ratios of the three major testicular populations (haploid, diploid, and tetraploid) reached adult levels by 48 days postpartum. Sperm cells were first detected in the cauda epididymis and vas deferens on 30 and 36 days postpartum, respectively. Early sperm populations, compared to adult sperm, exhibited up to 89% abnormalities in sperm head morphology that correlated with significant levels of abnormal chromatin structure. Percentage of sperm head abnormalities and chromatin structure in the cauda epididymis and vas deferens approached normal adult levels by 42 and 48 days postpartum, respectively.

Acridine Orange↗
PubMed1986

Sperm head morphology and nuclear chromatin structure evaluated by flow cytometry in a diallel cross in mice.

Genetic factors affecting spermatogenesis, sperm morphology, and chromatin structure in mice were estimated using a diallel cross of the inbred lines C3H/HeJ, C57BL/6J, DBA/2J, and BALB/cByJ. Flow cytometry of acridine orange stained cells was used to evaluate proportions of testicular tetraploid, diploid, and haploid cells and nuclear chromatin structure of sperm, measured by resistance of chromatin to in situ acid denaturation, and quantified by the ratio of double- to single-stranded DNA (alpha t). Percent morphologically abnormal sperm was scored by light microscopy. Heterosis, line, maternal, and reciprocal effects, and general and specific combining abilities were estimated for body and testis weights, testicular cell proportions, sperm alpha t values, and percent abnormal sperm. Heterosis was important for testis weight, alpha t values, and percent abnormal sperm. Inbreds varied in body and testicular weights, alpha t values, and percent abnormal sperm. Significant maternal effects were noted for several traits but could be due to sex-linked (X or Y) factors, since maternal and sex-linked effects were confounded. Although a high positive correlation existed between alpha t values and percent abnormal sperm, the proportion of sperm with altered chromatin structure, measured by FCM, was generally much lower than proportion of morphologically abnormal cells.

Animals↗
PubMed1986

Flow cytometry evaluation of testicular and sperm cells obtained from bulls implanted with zeranol.

Flow cytometry of testicular and sperm cells was used to evaluate effects of pre-weaning zeranol implants on spermatogenesis. Forty five Angus-Simmental bulls were randomly assigned to three treatment groups of 15 bulls each: no implant, one implant at 30 d of age and two implants, one at 30 and the second at 120 d of age. Prior to slaughter at approximately 15 mo, semen was collected from 30 bulls, 10 of each group. Following slaughter, testes were weighed, and testicular biopsies and vas deferens sperm obtained from the same 30 bulls. Testicular and sperm cells were stained with acridine orange and measured by flow cytometry. Proportions of testicular haploid, diploid and tetraploid cells were determined by relative amounts of green (DNA) and red (RNA) fluorescence. Treatment of sperm at low pH prior to acridine orange staining potentially induces partial denaturation of DNA, detectable by the metachromatic shift from green (native DNA) to red (single-stranded DNA) fluorescence. The effect of this shift was quantified by alpha-t [alpha t = red/red + green) fluorescence]. Nonimplanted bulls had heavier (P less than .01) testicular weights than treated bulls. The proportion of haploid cells was greater (P less than .02) and diploid cells less (P less than .03) in testes of nonimplanted bulls. Sperm from implanted bulls had altered chromatin structure, indicated by higher (P less than .05) alpha t values. Flow cytometry is an effective means for detecting changes in testicular cell subpopulations and chromatin structure of sperm.

Animals↗