Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea

Abstract

The effects of the mutagenic agent ethylnitrosourea ( E m on spermatogenic function and sperm chromatin structure were studied by flow cytometry and the results compared with sperm head morphology measurements. Groups of mice received daily exposures ranging from 0 to 75 mgkg body weight x 5 days and were sacrificed 28 days later. Fresh testicular cell suspensions and epididymal sperm were stained with acridine orange (AO) and measured by flow cytometry. Sperm nuclei were isolated, fixed, rehydrated, and then either subjected to thermal stress or not prior to staining with AO. Body weights were unaffected by the chemical exposure while the testicular weights were reduced by about 50%. Two-parameter (DNA, RNA) flow cytometry measurements showed a dose-response relationship in the loss of certain cell types, particularly the elongated spermatids, from the testes of treated animals. Flow cytometric analysis of both heat-stressed and non-heatstressed nuclei showed a relationship between dosage and the coefficient of variation of at {redked + green fluorescence)] measurements of A0 stained nuclei, thereby demonstrating that alterations of chromatin structure occurred in response to ENU. Enzymatic digestions with RNAse, DNAse, and nuclease S1 suggest that the increase in red fluorescence is due to an increase of singlestranded DNA induced by heat or acid treatment of chemically altered chromatin structure. The lowest daily dosage used (5 mgkg) caused no significant changes in ratios of testicular cell types, a questionable increase in abnormal sperm head morphology and a detectable change in chromatin structure expressed as at. This report shows that our technique for assaying sperm nuclear chromatin structure appears to have the same level of sensitivity to ENU induced nuclear alterations as the sperm head morphology test.Key terms: Flow cytometry, sperm head morphology, toxicology, testis, chromatin structureThe production of sperm represents one of the most dramatic examples of cellular proliferation and differentiation in any mammalian organ system. Although many biological parameters have been related to male fertility, sperm morphology is frequently used to assay testicular function. Sperm head morphology, consistent with nuclear morphology, is genotype specific (28), and is directed by genes on both the autosomes and sex chromosomes (2,411. Heritable changes in sperm head morphology can be induced by radiation and alkylating agents and followed quantitatively through successive generations (22,38,40). Wyrobek and Bruce (42,43) developed a mouse model assay system to study the mutagenic andor carcinogenic potential of chemicals using sperm head morphology as an end point. This test is relatively rapid and is considered very useful in the assessment of potentially toxic chemicals, since 100% of known mutagens tested were correctly identified as positives (44). Thus, demonstration of elevated levels of sperm abnormalities following exposure to a physical or chemical agent raises the possibility that the agent is i...

Sperm Chromatin Structure Assay: Its Clinical Use for Detecting Sperm DNA Fragmentation in Male Infertility and Comparisons With Other Techniques

“…It showed that sperm nuclear DNA was more resistant to heat-induced denaturation in fertile compared with infertile men and bulls (Evenson et al, 1980a). Heat-induced denaturation has since been replaced with acid-induced denaturation due to the similarity of results and the greater ease of the low pH technique (Darzynkiewicz et al, 1975;Evenson et al, 1985). A collaborative study showed that SCSA parameter values of 115 men seeking infertility counseling at the andrology laboratory affiliated with the National Hospital in Oslo, Norway, were twice those of men with proven fertility .…”

“…Much of the development and establishment of the SCSA as a meaningful biological assay for sperm nuclear integrity were conducted on rodent and livestock systems to study toxicology and fertility (Evenson et al, 1985(Evenson et al, , 1989a(Evenson et al, ,b, 1993a(Evenson et al, ,b,c, 1994Ballachey et al, 1987Ballachey et al, , 1988Evenson and Jost, 1993;Sailer et al, 1995a,b;Evenson, 1999a). One study with our laboratory showed that SCSA-defined chromatin damage in bovine spermatozoa negatively affects early embryo formation from what appear to be normally fertilized eggs (Ellington et al, unpublished).…”

“…The results from these studies showed that SCSA was highly dose-responsive to toxicants, highly repeatable, and provided meaningful biological information on sperm nuclear DNA defects. The extensive data from nonhumans (Evenson et al, 1985(Evenson et al, , 1989a(Evenson et al, ,b, 1993a(Evenson et al, ,b,c, 1994Ballachey et al, 1987Ballachey et al, , 1988Evenson and Jost, 1993; Sailer et al, 1995a,b;Evenson, 1999a), and human toxicology and fertility studies (Evenson et al, 1978(Evenson et al, , 1980a(Evenson et al, ,b, 1984(Evenson et al, , 1991Evenson and Melamed, 1983;Evenson, 1999b;Larson et al, 1999Larson et al, , 2000Larson et al, , 2001 provide This work was supported in part by grant R827019 from the US Environmental Protection Agency, grants EPS-9720642 and OSR-9452894 from the National Science Foundation, and by SCSA Diagnostics, Inc. This is South Dakota Agricultural Experiment Station publication 3273 of the journal series.…”

Quantitative fluorometry of abnormal mouse sperm nuclei

“…Other investigations of chromatin in abnormally shaped spermatozoa have focused on DNA content, chromatin structure or on both (Pinkel et al, 1983;Evenson et al, 1985Evenson et al, , 1986. In most instances, these analyses involved large populations of spermatozoa without regard to their indi¬ vidual shape.…”

Comparison of Anabolic Implants on Reproductive Function, Performance and Carcass Characteristics in Weaned Beef Bulls

“…Sperm samples were analyzed by the sperm chromatin structure assay (Darzynkiewicz 1979;Ballachey et al 1987Ballachey et al , 1988. In the sperm chromatin structure assay, sperm are first treated for 30 s at an acid pH (1.2) which induces DNA denaturation in sperm with abnormal chromatin structure (Evenson et al 1985). Cells were then stained with acridine orange which, when intercalated into (1985) reported that implanting bulls with Ralgro@ at 48 d of age resulted in decreased scrotal circumference, but implanting bulls at2l5 d of age had no effect on scrotal circumference.…”