Lorna K. Jost

Sperm Chromatin Structure Assay is useful for fertility assessment.

The Sperm Chromatin Structure Assay (SCSA) serves as a tool for measuring clinically important properties of sperm nuclear chromatin integrity. The assay utilizes the metachromatic features of Acridine Orange (AO), a DNA probe, and the principles of flow cytometry (FCM). SCSA data are not well correlated with classical sperm quality parameters and have been solidly shown to predict sub/infertility. This assay is ideally suited to human and animal fertility clinics to assess male sperm DNA integrity as related to fertility potential and embryo development as well as effects of reproductive toxicants. A detailed description of the SCSA follows.

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 individuals 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.

Bull sperm head morphometry related to abnormal chromatin structure and fertility.

This study investigated the relationship between morphologically abnormal sperm, sperm chromatin structure, and fertility. Semen samples were obtained from 13 bulls. The sperm chromatin structure assay (SCSA), a sensitive measure of denaturability of sperm nuclear DNA in situ following acid treatment, was performed on each sample to quantitate abnormal chromatin structure. Feulgenstained sperm head morphology was measured by computerized image analysis (ONCOR-Image): Sixteen parameters were measured for each of 200 nuclei per sample. Fertility estimates were available for nine of the bulls. The SCSA variable SD alpha t was correlated with fertility ranking (r = 0.617, P < 0.01). No correlations were seen between means of the imaging variables and SCSA variables % COMP alpha t or SD alpha t. Significant correlations (P < 0.05) were seen between SD alpha t and the variation of imaging variables eccentricity, width, and light blobs. Significant correlations (P < 0.05) were seen between % COMP alpha t and the variation of imaging variables area, perimeter, p2a, bending energy, nmac, sphericity, eccentricity, length, and width. A regression model for fertility rankings incorporating the standard deviation of the imaging variables area, bending energy, nmac, eccentricity, condensity, light blobs, and dark blobs was highly significant (r2 = 0.999, P < 0.05). These results indicate that variation of morphometry measurements is likely a sensitive biomarker related to fertility potential and abnormal chromatin structure.

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.

Sperm Chromatin Structure Assay for Fertility Assessment

The integrity of mammalian sperm DNA is of prime importance for the paternal genetic contribution to normal offspring. Damaged DNA in the single sperm that fertilizes the female egg can have a dramatic negative impact on fetal development. This comprehensive and detailed unit presents a rapid, reliable, practical test for DNA integrity based on staining with acridine orange. SCSA data have been conclusively shown to predict sub/infertility. This assay is ideally suited to human and animal fertility clinics to assess male sperm DNA integrity as related to fertility potential and embryo development. The authors, who have decades of experience in studying sperm viability, provide extensive commentary and methodological tips, making this unit the most detailed method for this test published to date. Keywords: flow cytometry; sperm chromatin structure assay; SCSA; DNA denaturation; acridine orange;animal and human fertility; toxicology The integrity of mammalian sperm DNA is of prime importance for the paternal genetic contribution to normal offspring

Normospermic Versus Teratospermic Domestic Cat Sperm Chromatin Integrity Evaluated by Flow Cytometry and Intracytoplasmic Sperm Injection

Abstract Teratospermia (>60% of morphologically abnormal spermatozoa) is well documented in felids. Even morphologically normal spermatozoa from teratospermic ejaculates have reduced ability to undergo tyrosine phosphorylation, acrosome react, and bind and penetrate oocytes compared with normospermic (<40% abnormal spermatozoa) counterparts. However, it is unknown whether fertilization deficiencies originate at a nuclear level. This study examined whether fertilization failure also was attributable to abnormal sperm chromatin, using the sperm chromatin structure assay (SCSA), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). Aliquots of unprocessed and swim-up-processed (to isolate morphologically normal spermatozoa) spermatozoa from teratospermic and normospermic domestic cats were analyzed by the flow cytometric SCSA. Swim-up-processed sperm were incubated with in vivo-matured oocytes or used for ICSI. Teratospermic ejaculates expressed more (P < 0.05) chromatin heterogeneity (abnormal chromatin structure) than their normospermic counterparts, both in unprocessed and swim-up-processed samples. Fertilization success in vitro was higher (P < 0.05) from normo- compared with teratospermic inseminates. Similar (P > 0.05) proportions of oocytes fertilized after ICSI using spermatozoa from normo- and teratospermic cats. Results reveal that teratospermia in the cat is expressed at the nuclear level as increased sperm chromatin heterogeneity, but ICSI showed that this does not apparently affect fertilization rates if the zona pellucida and oolemma can be bypassed.

Changes in susceptibility of bovine sperm to in situ DNA denaturation during prolonged incubation at ambient temperature under conditions of exposure to reactive oxygen species and nuclease inhibitor.

Sperm were incubated for up to 9 days in the presence or absence of exogenous hydrogen peroxide, phenylalanine, catalase and aurintricarboxylic acid to assess the influence of reactive oxygen species and inhibition of deoxyribonucleases on sperm chromatin stability. The assessment of sperm DNA susceptibility to in situ acid denaturation by the sperm chromatin structure assay did not detect any difference in chromatin stability between sperm incubated for 9 days under aerobic and anaerobic conditions in a diluent called 14G. Exposure to exogenous hydrogen peroxide under both aerobic and anaerobic conditions and to phenylalanine under aerobic conditions (which produces hydrogen peroxide by a reaction catalysed by the aromatic amino acid oxidase present in sperm) was detrimental to sperm chromatin stability, increasing its DNA susceptibility to in situ acid denaturation over the incubation time. This effect was eliminated if catalase was present in the diluent. Inclusion of the general deoxyribonuclease inhibitor aurintricarboxylic acid in the diluent severely decreased sperm chromatin stability under both aerobic and anaerobic conditions. Aurintricarboxylic acid was mildly cytotoxic, as revealed by viability assessment, under aerobic, but not under anaerobic, incubation conditions. Exogenous hydrogen peroxide, either directly added to the diluent or generated through the enzymatic oxidation of phenylalanine, was detrimental to sperm motility and the integrity of the plasma membrane.

Chapter 10 Sperm Chromatin Structure Assay: DNA Denaturability

Publisher Summary Studies over the past decade have proven the usefulness of measurements of the integrity of sperm chromatin structure. The flow cytometry (FCM) measurement of chromatin structure is based on the principle that abnormal sperm chromatin has a greater susceptibility to physical induction of partial DNA denaturation in situ . The extent of DNA denaturation following heat or acid treatment is determined by measuring the metachromatic shift from green fluorescence [Acridine orange (AO) intercalated into double-stranded nucleic acid] to red fluorescence. Apparently acid conditions that cause partial denaturation of protamine-complexed DNA in sperm with abnormal chromatin structure do not cause denaturation of histone-complexed somatic cell DNA. The FCM measurement of sperm chromatin structure has been termed the sperm chromatin structure assay (SCSA) to distinguish it from other AO staining protocols. This protocol was formerly divided into SCSA acid and SCSA heat to distinguish the physical means of inducing DNA denaturation. The two methods give essentially the same results but the SCSA acid method is much easier to use and is the method of choice.

Glutathione depletion potentiates ethyl methanesulfonate-induced and damage to sperm chromatin structure

Male rats were treated with phorone at dosages previously shown to reduce glutathione in rodent reproductive tracts, followed by a single challenge with ethyl methanesulfonate, a known mutagenic and clastogenic agent. Epididymal sperm collected 8 and 15 days after exposure from phorone pretreated animals had a significantly greater alteration of sperm chromatin structure, defined as an increased susceptibility to DNA denaturation in situ, relative to sperm obtained from animals injected with saline alone or saline+EMS (50, 100, 150, or 200 mg/kg bw). These data support the hypothesis that ethyl methanesulfonate-induced alkylation of developing sperm chromatin protamines causes a significant stress on chromatin structure leading to increased DNA damage. This is the first report showing that glutathione depletion potentiates EMS-induced chromatin structural alterations that are likely related to dominant lethal mutations.

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.