Relation of mammalian sperm chromatin heterogeneity to fertility.

Flow cytometry of heated sperm nuclei revealed a significant decrease in resistance to in situ denaturation of spermatozoal DNA in samples from bulls, mice, and humans of low or questionable fertility when compared with others of high fertility. Since thermal denaturation of DNA in situ depends on chromatin structure, it is assumed that changes in sperm chromatin conformation may be related to the diminished fertility. Flow cytometry of heated sperm nuclei may provide a new and independent determinant of male fertility.

Assessment of sperm quality, DNA integrity and cryopreservation protocols in men diagnosed with testicular and systemic malignancies.

Men diagnosed with malignancy are often referred for semen banking to preserve their fertility prior to cancer treatment. The chances of cancer patients for achieving future fecundity will be determined by the sperm quality including the integrity of the genomic material in the frozen samples. The objectives of this study were to compare the sperm quality and DNA integrity in men diagnosed with testicular and systemic malignancies before receiving treatment and to identify the optimum cryopreservation protocol for their samples including a remote semen collection option. In comparison with fertile donors, patients with testicular malignancies had significantly lower sperm concentration, while both testicular and systemic malignancy patients had significantly lower sperm motility and cryosurvival rates. In addition, the SCSA defined DNA fragmentation index was significantly higher in patients with testicular and systemic malignancies compared with fertile donors. It was noted that the extent of deterioration in sperm quality and DNA integrity seen in cancer patients did not reach the previously defined statistical threshold for impaired fertility. Freezing spermatozoa with the seminal plasma offers the highest protection against cryo-injury. Nevertheless, remote semen collection can still be used as it yields adequate results.

Sperm DNA damage is related to field fertility of semen from young Norwegian Red bulls.

Flow cytometry was utilised for the first time to independently measure five sperm parameters of individual spermatozoa of bull ejaculates to differentiate between outcome successes after artificial insemination (AI). These parameters included plasma membrane and acrosome integrity, mitochondrial functionality and DNA damage measured by sperm chromatin structure assay (SCSA) and terminal deoxynucleotide transferase-mediated dUTP nick end labelling (TUNEL) assays. For each parameter, results of 142 ejaculates (30 bulls) were ranked into three groups according to their flow cytometric measures: (1) ejaculates with the 25% lowest measures; (2) the 50% middle measures; and (3) the 25% highest measures. In total, 20 272 first-service inseminations (18 ;10(6) spermatozoa per AI dose) were performed, where fertility was defined as non-return within 60 days after first insemination. While plasma membrane and acrosome integrity, and mitochondrial functionality were not significantly related to fertility, data from SCSA and TUNEL assays were significantly associated with fertility. Ejaculates in SCSA group 1 had higher odds of AI success (1.07, 95% CI = 1.02-1.12), whereas those in group 3 had lower odds of AI success (0.94, 95% CI = 0.89-0.99), compared with the average odds of all three groups. Ejaculates in group 2 did not have significantly higher odds of AI success compared with the average odds. For TUNEL-positive spermatozoa, the odds of AI success was higher in group 1 compared with the average odds (1.10, 95% CI = 1.02-1.13), whereas odds of AI success in groups 2 and 3 were not significant compared with the average odds. In conclusion, despite the high number of spermatozoa per AI dose from high-quality bulls, both SCSA and TUNEL assays were valuable measures in this study for evaluating sperm quality in relation to fertility after AI.

Effects of 9,10-anthracenedione, 1,4-bis[[2-[(2-hydroxyethyl)amino]-ethyl]amino]-, diacetate on cell morphology and nucleic acids of friend leukemia cells.

Treatment of Friend leukemia cells for 18 hours with 9,10-anthracenedione, 1,4-bis[[(2-hydroxyethyl)amino]ethyl]amino]-, diacetate (ANT) at concentrations up to 1.0 microgram/ml induced significant changes in cell metabolism and structure. Alterations in cell nucleic acid content were detected in cells stained with acridine orange under conditions such that DNA and RNA contents could be measured simultaneously by flow cytometry. Cells treated for 18 hours with ANT at concentrations of 0.05-0.1 microgram/ml became partially blocked at the G2 phase. In addition, about 30% of the cells became polyploid and demonstrated diplochromosomes at the 8C level of mitosis. The nuclear chromatin of blocked cells had an altered structure as reflected by a change in sensitivity of DNA in situ to denaturation induced by low pH. All viable cells treated with ANT for 18 hours at concentrations of 0.4-1.0 microgram/ml were blocked in G2 phase. These cells had significantly more RNA than did untreated cells. Transmission electron microscopic observations of thin-sectioned cells suggested that this increased RNA content in ANT-treated cells was mostly due to an approximately 50% increased cell diameter and partly due to a disproportionate increase in nucleolar size. In addition, electron microscopy revealed that ANT caused increased chromatin condensation and granulation. The drug had no apparent effect on production of the endogenous Friend murine leukemia virus.

Ellipticine-induced changes in cell growth and nuclear morphology.

Twenty-four-hour exposure of Friend leukemia (FL) and L1210 cells to 1.0 micrograms ellipticine/ml resulted in a slowdown in cell proliferation, accumulation of cells in G2 phase, an increase in cellular RNA content, and an increase in the proportion of cells with greater than 4C (tetraploid) content of DNA in both cell lines. Removal of the drug and subsequent cell culturing in fresh medium for 24 hours led to a further increase in the proportion of cells having more than a 4C content of DNA, with no change in cell number, in FL cell cultures. Distinct micronucleation and an increase in cell size without any sign of cell division were seen in FL cells so treated. In contrast, L1210 cells after transfer to fresh medium reentered the normal cell cycle, continued to proliferate, and demonstrated only a slight increase in cells with greater than a 4C content of DNA within 24 hours of removal of the drug.

Action of dihydroxyanthraquinone on cell cycle progression and survival of a variety of cultured mammalian cells.

Dihydroxyanthraquinone, 1,4-dihydroxy-5,8-bis(( (2-[(2-hydroxyethyl)amino]ethyl)amino))-9,10-anthracenedione (NSC 279836), was observed to alter the cell cycle kinetics of a variety of mammalian cell lines as monitored by flow cytometry. Continuous exposure of Friend leukemia, L1210, and Chinese hamster cells to the drug in vitro at concentrations of 1.0 to 10 ng/ml resulted in the accumulation of cells in G2 by 24 hr in culture. When cells were exposed to dihydroxyanthraquinone for 30 min, washed free of drug, and cultured in fresh medium for 24 hr, a 10 times higher drug concentration was required to produce a G2 block identical to that observed during continuous exposure. Stimulation of human lymphocytes by phytohemagglutinin could be inhibited in a dose-dependent manner by brief pretreatment of cells with the drug. However, while previously stimulated but as yet noncycling lymphocytes were profoundly affected by much lower concentrations, proliferating lymphocytes were refractory to treatment with the drug up to a concentration of 1 microgram/ml. Exposure to the drug for 24 hr, at concentrations as low as 3.2 ng/ml, inhibited colony formation of exponentially growing Chinese hamster cells by 50%, whereas stationary culture required an 8-fold higher concentration to produce the same results. Drug concentrations in the range of 0.3 to 0.8 micrograms/ml over a period of 24 hr reduced the viability of Friend leukemia and L1210 cells by 50% as measured by trypan blue dye exclusion. In contrast, human lymphocyte viability was only mildly affected following 24 hr incubation with up to 5.0 micrograms dihydroxyanthraquinone per ml. There was a marked effect on cellular RNA content in two of the cell lines tested. Friend leukemia and L1210 cells blocked in G2 by the drug manifested a 140 and 70% increase in RNA content, respectively, when compared to control cells. In addition, though suboptimal concentrations of the drug resulted in a transient accumulation of cells in G2, optimal drug concentrations not only blocked cells in G2 but in the case of Friend leukemia and L1210 cells led to an increase in the proportion of cells with greater than 4C amounts of DNA. The results obtained with dihydroxyanthraquinone were compared to those obtained previously with a nonhydroxylated analog, anthracenedione (NCS 287513).

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.

Hydroxyurea exposure alters mouse testicular kinetics and sperm chromatin structure.

The effects of hydroxyurea (HU) on testicular cell kinetics and sperm chromatin differentiation were investigated in mice. Whole testis, minced testicular cell suspensions and caudal epididymal sperm cells were obtained at 8 and 29 days after i.p. injections containing 0, 25, 50, 100, 200, 400 and 500 mg/kg HU x 5 days. Testis weights were unaffected by 25 mg/kg HU while 500 mg/kg caused up to a 50% loss of testicular weight by 29 days. Flow cytometrically measured acridine-orange (AO) stained testicular cells revealed altered population ratios at the highest dosages at 8 days and for all dosages except 25 mg/kg HU at 29 days. At 8 days, 400-500 mg/kg HU caused a near depletion of tetraploid cells. Flow cytometry of AO stained sperm, previously treated with acid to potentially induce DNA denaturation, was used to follow the shift from normal chromatin structure to an abnormal form with increased sensitivity to DNA denaturation in situ. The extent of DNA denaturation was quantitated for each cell by the computer-derived value alpha t, alpha t = [red/(red+green) fluorescence]. The flow cytometry measures, standard deviation of alpha t (SD alpha t), mean of alpha t (X alpha t) and cells outside the main peak of alpha t (COMP alpha t), gave similar dose response curves to the sperm head morphology assay. SD alpha t was more sensitive than the X alpha t as a measure of HU-induced alteration of chromatin structure. The major conclusions reached are that HU inhibits DNA synthesis, probably by inhibiting ribonucleotide reductase, causing maturation depletion of pachytene spermatocytes and, subsequently, depletion of meiotic daughter cells and differentiated cell types leading to mature sperm. This inhibition of DNA synthesis is related to an alteration of sperm chromatin structure and abnormal sperm head morphology.

Rapid analysis of normal and abnormal cell types in human semen and testis biopsies by flow cytometry.

A simple, rapid procedure is described that quantitates RNA content and DNA content/chromatin condensation for each of many possible cell types and differentiation levels of the cells present in human semen. A fresh semen sample (1-6 hr postemission) or frozen sample (allowing samples to be accumulated and sent to a laboratory) is treated with a detergent solution, stained with acridine orange (AO), and measured by flow cytometry (FCM); approximately 10 minutes are required to measure 5,000 cells per sample and analyze the data with computer assistance. The following can be learned from a single measurement: a) the percentage of each cell type in semen including, i) mature sperm, ii) immature sperm precursor cells, representing all stages of development from spermatogonia to mature sperm, iii) somatic cells, e.g., leukocytes; b) normality/abnormality of sperm nuclear chromatin condensation. These measurements can be correlated with cell types in testis biopsies identified by two-parameter FCM measurements (RNA versus DNA) using AO as the fluorescent probe.

Rhodamine 123 alters the mitochondrial ultrastructure of cultured L1210 cells.

Exposure of exponentially growing L1210 cells in vitro to 5-10 micrograms/ml of rhodamine 123 (R123) for 16-48 hr inhibits cell proliferation and induces cell arrest in the G1A phase of the cell cycle. The cells remain viable during the arrest and resume growth after removal of R123; extended exposure to R123 is cytotoxic. Exposure to R123 results in morphological alterations in mitochondria of all cells observed; specifically, mitochondria of R123-treated cells are characterized by a distention of the intracristal spaces and a significant increase in the number of matrix granules. Gross morphological changes of mitochondria include formation of extended organelles and the appearance of doughnut-shaped structures.

Semen quality and reproductive health of young Czech men exposed to seasonal air pollution.

This study of male reproductive health in the Czech Republic resulted from community concern about potential adverse effects of air pollution. We compared young men (18 years of age) living in Teplice, a highly industrialized district with seasonally elevated levels of air pollution, to those from Prachatice, a rural district with relatively clean air. Surveys were scheduled for either late winter, after the season of higher air pollution, or at the end of summer, when pollution was low. Participation included a physical examination, donation of a semen sample, and completion of a questionnaire on health, personal habits, and exposure to solvents and metals through work or hobby. Analysis of data from 408 volunteers showed that the men from Teplice and Prachatice were similar in physical characteristics, personal habits, and work- or hobby-related exposures. Sixty-six percent (272) of these men donated a single semen sample for routine semen analysis, computer-aided sperm motion analysis, and sperm chromatin structure assay. The mean (median) sperm concentration and sperm count were 61. 2 (44.0) million/mL semen and 113.3 (81.5) million, respectively, and were not associated with district of residence or period of elevated air pollution. However, periods of elevated air pollution in Teplice were significantly associated with decrements in other semen measures including proportionately fewer motile sperm, proportionately fewer sperm with normal morphology or normal head shape, and proportionately more sperm with abnormal chromatin. These results suggest that young men may experience alterations in sperm quality after exposure to periods of elevated air pollution, without changes in sperm numbers.

Effects of methyl methanesulfonate on mouse sperm chromatin structure and testicular cell kinetics.

Effects of methyl methanesulfonate (MMS) on mouse testicular cell kinetics and sperm chromatin structure were determined flow cytometrically. Mice were exposed to a single ip injection of saline containing 0 or 150 mg/kg MMS. Relative ratios of 1N, 2N and 4N testicular cells were not affected until 22 days postexposure. Ratios of 1N cell types were altered from 13 to 22 days and were near normal by 25 days. This study revealed an MMS induced alteration of chromatin structure in testicular, elongated spermatids by the sperm chromatin structure assay (SCSA), a flow cytometric measure of the susceptibility of acridine orange stained sperm DNA to denaturation in situ. The SCSA also detected alterations in cauda sperm chromatin structure at 3 days, which was 8 days prior to alterations in sperm head morphology, indicating the increased sensitivity of the SCSA. SCSA data were practically similar whether measuring either fresh or frozen/thawed sperm, or whether measured by two different types of flow cytometers: a) laser driven, orthogonal optical axis; or b) low cost mercury arc lamp system with epiillumination. The data support the model of Sega and Owens [Mutat Res 111:227-244:1983] that MMS alkylates cysteine-SH groups in sperm protamines, thereby destabilizing sperm chromatin structure and leading to broken chromosomes and mutations.

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.

Effects of X-irradiation on mouse testicular cells and sperm chromatin structure.

The testicular regions of male mice were exposed to x-ray doses ranging from 0 to 400 rads. Forty days after exposure the mice were killed and the testes and cauda epididymal sperm removed surgically. Flow cytometric measurements of acridine orange stained testicular samples indicated a repopulation of testicular cell types following x-ray killing of stem cells. Cauda epididymal sperm were analyzed by the sperm chromatin structure assay (SCSA), a flow cytometric measurement of the susceptibility of the sperm nuclear DNA to in situ acid denaturation. The SCSA detected increased susceptibility to DNA denaturation in situ after 12.5 rads of x-ray exposure, with significant increases following 25 rads. Abnormal sperm head morphology was not significantly increased until the testes were exposed to 60 rads of x-rays. These data suggest that the SCSA is currently the most sensitive, non-invasive method of detecting x-ray damage to testicular stem spermatogonia.

Coculture of human sperm with bovine oviduct epithelial cells decreases sperm chromatin structural changes seen during culture in media alone.

OBJECTIVE: To compare sperm chromatin structural changes seen in media only culture or in coculture with bovine oviduct epithelial cells. DESIGN: Three freshly ejaculated and three cryopreserved sperm samples in media culture or in oviduct epithelial cell coculture. Sperm in each treatment were evaluated by the sperm chromatin structure assay during a 72-hour time course. SETTING: An academic research laboratory. PATIENT(S): Normospermic donors with children. INTERVENTION(S): Semen collection through masturbation after 48 hours of abstinence. MAIN OUTCOME MEASURE(S): The sperm chromatin structure assay using flow cytometry to detect the susceptibility of sperm in either treatment to denaturation of DNA in situ. RESULT(S): The sperm chromatin structure assay data differed for sperm type (fresh or cryopreserved), over time, and between treatments within 6 hours of culture. In oviduct epithelial cell coculture, fresh sperm chromatin structure assay values for fresh sperm were stable, whereas in control medium higher chromatin degeneration levels were seen by 10 hours. For cryopreserved sperm, chromatin degeneration had increased by 1 hour postthaw in both treatments, although levels were higher in the control treatment thereafter. CONCLUSION(S): Sperm chromatin structural changes occur over time in culture. Such changes were observed within 2 hours for cryopreserved sperm. Coculture of sperm with oviduct epithelial cells results in a stabilizing effect for sperm against chromatin changes.

Higher-quality human sperm in a sample selectively attach to oviduct (fallopian tube) epithelial cells in vitro.

OBJECTIVE: To determine if sperm attachment to oviduct epithelial cells (OEC) in vitro is selective for higher quality sperm and if the system requires homologous species OEC. DESIGN: Controlled prospective study with outcomes assayed by a technician blind to sperm treatment groups. SETTING: An academic research laboratory. PATIENT(S): Experiment 1: normospermic donors with children (4 donors, 7 ejaculates). Experiment 2: cryopreserved donor samples (4 donors). INTERVENTION(S): Semen collection by masturbation after 48 hours of abstinence. MAIN OUTCOME MEASURE(S): Experiment 1: sperm assays of motility, morphology, membrane integrity, and capacitation status. Experiment 2: sperm chromatin (DNA) integrity and condensation. RESULT(S): Experiment 1: sperm not attaching to OEC had lower motility, more membrane disruptions, and more acrosome reactions than did control sperm. This selectivity was equivalent for sperm in coculture with all OEC types. Experiment 2: sperm attached to OEC had fewer abnormalities in chromatin structure compared with sperm that were not attached. CONCLUSION(S): Selective attachment of functionally superior sperm to OEC is likely important during sperm reservoir formation in vivo and may be exploitable in vitro as a method to isolate high-quality sperm for clinical procedures. Such a system does not require human origin OEC.

DNA damage in patients with untreated cancer as measured by the sperm chromatin structure assay.

OBJECTIVE: To examine the relationship between sperm chromatin defects, evaluated by sperm chromatin structure assay (SCSA) and semen characteristics in cryopreserved semen specimens from patients diagnosed with various types of cancer. DESIGN: Prospective study. SETTING: Andrology laboratory at a tertiary care hospital. PATIENT(S): Cryopreserved semen samples from 12 healthy fertile men and 37 men diagnosed with cancer: testicular cancer (n = 20), Hodgkin's disease (n = 11), non-Hodgkin's disease (n = 4), and other neoplasm (n = 2). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The shift of green (native DNA) to red (denatured, single-stranded DNA) fluorescence in acridine orange-stained nuclei was measured and quantified using the expression alpha(t)(red fluorescence/[red + green fluorescence] per cell). Sperm DNA damage was correlated with classical semen characteristics. RESULT(S): Cancer patients as a group had significantly higher DNA damage when compared with controls. Specimens with high COMPalpha(t) values (percentage of sperm with denatured DNA) were present in all groups of cancer patients. No meaningful correlation was seen between the extent of DNA damage and classical semen measures. CONCLUSION(S): DNA damage in spermatozoa is prevalent in the majority of cancer patients. SCSA provides important information about the biochemical integrity of sperm DNA in men with cancer before their treatment.

Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay.

Sperm from four mammalian species were analyzed by the sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase assay (TdTA) using flow cytometry. The SCSA quantitates the susceptibility of sperm nuclear DNA to in situ acid denaturation, while the TdTA quantitates the presence of endogenous DNA strand breaks in sperm nuclear chromatin. Correlations were seen between the percentage of sperm cells showing susceptibility to in situ acid denaturation and the percentage of cells showing the presence of DNA strand breaks for humans (r = 0.56, P = 0.004), rams (r = 0.84, P < 0.001), bulls (r = 0.78, P < 0.001), and stallions (r = 0.65, P < 0.001). No significant differences were seen when using fresh or frozen samples for either assay. These results suggest that sperm cells that are more susceptible to in situ DNA denaturation may have a greater number of accessible endogenous DNA strand breaks. We hypothesize that the DNA strand breaks produced in the normal transition from a somatic cell histone complex to a protamine complex are not ligated properly, resulting in residual DNA strand breaks and altered chromatin structure. Alternatively, altered chromatin structure could lead to the accessibility of the endogenous DNA strand breaks.

The sperm chromatin structure assay. Relationship with alternate tests of semen quality and heterospermic performance of bulls.

Data obtained by the sperm chromatin structure assay (SCSA) on spermatozoa from nine bulls were correlated with fertility, measured by heterospermic performance (-0.94, P less than 0.01) and by alternate tests of sperm quality, including motility, acrosome integrity, Sephadex filtration and morphology of spermatozoa (all significant at P less than 0.05 to P less than 0.01). The SCSA uses flow cytometry to determine the susceptibility of nuclear DNA to low pH-induced denaturation in situ as measured by the ratio of acridine orange binding to double- or single-stranded DNA. The error associated with multiple SCSA measurements was relatively low. The primary finding is that the assay of chromatin structure stability performed on killed spermatozoa was as highly correlated with the heterospermic performance of semen as the best of the classical tests for semen quality. The SCSA may therefore be a highly useful technique for evaluation of sperm quality.

Studies on sperm chromatin structure alterations and cytogenetic damage of mouse sperm following in vitro incubation. Studies on in vitro-incubated mouse sperm.

Mouse epididymal sperm incubated in Tyrode's T6 fertilization media were analyzed over time for chromosome damage by two methods. First, cytogenetic analysis was done on paternal pronuclei metaphase chromosomes. After 6 hours incubation 11% of the cells demonstrated chromosome structural abnormalities. Secondly, sperm nuclei were measured by the sperm chromatin structure assay, which is a measure of the susceptibility of sperm DNA to the nuclei demonstrated an increased susceptibility to DNA denaturation, reaching near 100% by 48 hours. Changes in chromatin structure at the molecular level may lead to chromosome breaks seen in pronuclear chromosomes.