Conditions are described that permit the quantitative extraction of chromatin proteins from the epididymal sperm of the mouse. These proteins have been isolated free of contaminating tail proteins following removal of the tails with cetyltrimethylammonium bromide (CTAB). Without this treatment, numerous acid-soluble tail proteins coextract with the nuclear proteins isolated from partially purified heads. The proteins isolated in this manner do not require prior modification with iodoacetamide and show no evidence of proteolytic degradation. In acid-urea polyacrylamide gels, 99% of the sperm protein migrates as one electrophoretic band. Evidence is presented that suggests that this single band contains two protamine-like proteins.

Accelerator mass spectrometry (AMS) is used to determine the amount of carcinogen covalently bound to mouse liver DNA (DNA adduct) following very low-level exposure to a "'C-labeled carcinogen. AMS is a highly sensitive method for counting long-lived but rare cosmogenic isotopes. While AMS is a tool of importance in the earth sciences, it has not been applied in biomedical research. The ability of AMS to assay rare isotope concentrations (10Be, 14C, 26Al, 41Ca, and 129I) in microgram amounts suggests that extension to the biomedical sciences is a natural and potentially powerful application of the technology. In this study, the relationship between exposure to low levels of 2-amino-3, 8-dimethyl[2-14C~imidazo[4,5-f]quinoxaline and formation of DNA adducts is examined to establish the dynamic range of the technique and the potential sensitivity for biological measurements, as well as to evaluate the relationship between DNA adducts and low-dose carcinogen exposure. Instrument reproducibility in this study is 2%; sensitivity is 1 adduct per 10"1 nucleotides. Formation of adducts is linearly dependent on dose down to an exposure of 500 ng per kg of body weight. With the present measurements, we demonstrate at least 1 order of magnitude Improvement over the best adduct detection sensitivity reported to date and 3-5 orders of magnitude improvement over other methods used for adduct measurement. An additional improvement of 2 orders of magnitude in sensitivity is suggested by preliminary experiments to develop bacterial hosts depleted in radiocarbon. Expanded applications involving human subjects, including clinical applications, are now expected because of the great detection sensitivity and small sample size requirements of AMS.Carcinogens covalently bound to any of the deoxynucleotide bases present in DNA (DNA adducts) have been proclaimed as markers of carcinogen exposure. The relationship between adduct formation and exposure, however, has been primarily established at high carcinogen doses and not at lower, more environmentally relevant, levels because of limitations in assay sensitivity. As a consequence, the significance of using adducts as a measure of carcinogen exposure in the human population is unknown. Currently, the most sensitive technique for adduct detection is the 32p postlabeling assay. The 32p postlabeling assay has permitted measurement of 1 adduct in 1010 nucleotides and has been used to detect carcinogen-DNA binding in occupationally exposed humans and smokers, but accurate quantitative measurement at levels <1 adduct per 107-101 nucleotides is difficult because of variability in adduct recovery (1-3). The ability of accelerator mass spectrometry (AMS) to measure concentrations of rare isotopes in 20-Asg to 1-mg samples suggested to us that its extension to the biomedical sciences was a natural and potentially powerful application of the technology (4). The great enhancement in 14C detection sensitivity available with AMS offers the distinct advantage of detecting extremely small amount...

The relative content of DNA in spermatozoa presumed to be the X- and Y-chromosome-bearing gametes from bulls, boars, rams and rabbits and the amount of DNA in spermatozoa of cockerels was determined by flow cytometry. Differences in the relative content of DNA and proportions of the presumed X- and Y-sperm populations in cryopreserved semen from Holstein, Jersey, Angus, Hereford and Brahman bulls were also determined. Spermatozoa were washed by centrifugation using a series of dimethyl sulfoxide solutions made in isotonic sodium citrate, fixed in ethanol, treated with papain and dithioerythritol to loosen the chromatin structure and remove cellular organelles, and stained quantitatively for DNA with the fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). Approximately 5000 stained sperm nuclei, which were nonviable due to the removal of other cellular organelles during the washing procedure, were measured for DNA in an epi-illumination flow cytometer. A single distinct peak for cockerel spermatozoa and two symmetrical, overlapping peaks for species with X- and Y-spermatozoa were seen. This and other evidence strongly supports the interpretation that the peaks represent the X- and Y-sperm populations. The content of DNA in sperm nuclei from cockerels, bulls, boars, rams and rabbits, as determined by fluorescence flow cytometry, corresponded to biochemical estimates of DNA per sperm cell. Analyses of the bimodal histograms by computer-fitting two Gaussian distributions to the data showed the means of the peaks differed by 3.9, 3.7, 4.1 and 3.9% for bulls, boars, rams and rabbits, respectively. In four replicate analyses of semen from 25 bulls representing 5 breeds, the average population of sperm nuclei in the Y-peaks ranged from 49.5 to 50.5% for all breeds. The X-Y peak differences did not vary within each breed, but were significantly different when the breeds were compared. Spermatozoa from Jersey bulls had larger X-Y peak differences (P less than 0.001) than spermatozoa from Holstein, Hereford, and Angus bulls; spermatozoa from Brahman bulls had smaller X-Y differences (P less than 0.004). It is suggested from the evidence obtained in these studies that flow cytometry can be used to assess the proportion of X- and Y-spermatozoa in semen of domestic animals and is thereby applicable to verification of the effectiveness of enrichment techniques for X- or Y-spermatozoa.

Various types of cells from the testes of mice and hamsters were separated according to differences in sedimentation velocity by centrifugal elutriation, a counterflow centrifugation technique. Approximately 3 times 10(8) cells, prepared from six mouse testes or from one hanster testis, were separated into 11 fractions in less than two hours as compared to the 4--5 hours required for sedimentation at unit gravity ("Staput"). Fractions enriched in elongated spermatids and spermatozoa (100%), stages 1--8 spermatids (69%) and pachytene spermatocytes (58%) were obtained from mouse testis dispersions. Similarly enriched fractions were obtained from hamster cells. A single fraction enriched in stages 1--8 spermatids (mouse) was prepared in less than 30 minutes. As many as 2 times 10(9) cells were separated in a single procedure. Spermatogenic cells exhibited no evidence of structural damage with trypan blud and phase microscopy, and recovery was essentially 100%. Centrifugal elutriation had no effect on sperm motility or on the plating efficiency of CHO cells.