Chapter 38 Flow Cytornetric Analysis of Male Germ Cell Quality DONALD P.EVENSON Olson Biochemistry Laboratories Department of Chemistry South Dakota State University Brooktngs South Dakota 57007

I. Introduction The testis of a fertile mammal is characterized by rapid proliferation of germ cells that undergo unique and complex patterns of differentiation. Testicular tissue is easily dissociated into a cellular suspension that is readily amenable to flow cytometry (FCM) studies. We describe here dual-parameter (DNA, RNA) FCM measurements of acridine orange (A0)-stained cells (Darzynkiewicz ef al., 1976; see Darzynkiewicz, Chapter 27, this volume) used to assess the ratio of testicular cell types present, providing an indicator of testicular function. Because of differential DNA stainability and amounts of RNA present, seven to eight distinct populations of cells can be resolved by this technique (Evenson et al., 1985). This measurement is very practical for animal studies but impractical for human and animal husbandry studies because of the invasive sampling procedures, although fine-needle biopsy samples are utilized by some laboratories (Thorud et al., 1981). The same A 0 staining and FCM measurement technique used for testicular biopsy samples is also a rapid and practical method for measuring abnormal cells in semen (Evenson and Melamed, 1983). In addition to normal sperm, semen may include immature germ cells that have staining characterisitics similar or identical to testicular cells. Also, the presence of somatic cells (e.g., leukocytes) in semen can readily be detected by this same protocol (Evenson and 401 METHODS IN CELL BIOLOGY, VOL. 33

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Melamed, 1983) and distinguished from morphologically similar round spermatids. Several studies have shown an interesting correlation between sperm cell chromatin structure and exposure to toxic chemicals (Evenson et a l . , 1985, 1986, 1989) and also a correlation with fertility (Ballachey et al., 1987, 1988; Evenson, 1986,1989; Evenson et al., 1980). The 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 siru. The extent of DNA denaturation following heat (Evenson, et al. 1980,1985) or acid (Evenson et al. 1980,1985) treatment is determined by measuring the metachromatic shift from green luminescence ( A 0 intercalated into double-stranded (ds) nucleic acid) to red luminescence ( A 0 associated with single-stranded (ss) DNA; Darzynkiewicz et al., 1976). 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 (Evenson et al., 1986). The FCM measurement of sperm chromatin structure as described here has been termed the sperm chromatin structure assay (SCSA) to distinguish it from other A 0 staining protocols. This protocol has also been subdivided into SCSAaCidand SCSAheatto distinguish the physical means of inducing DNA denaturation.

11. Application The primary applications of these techniques are in the fields of environmental toxicology, animal husbandry, and human fertility. The described techniques provide for rapid, objective assessment for germ cell toxins that interfere with cell division and differentiation. Evenson and colleagues have shown that exposure of mice to toxic chemicals caused changes in the relative ratio of testicular cell types, (Evenson, et al., 1985, 1986, 1989a) appearance of abnormal cell types in epididymi (Evenson et al., 1989b), and increased sensitivity to acid (Evenson et al., 1985, 1986, 1989) or heat-induced denaturation (Evenson, 1986; Evenson et al., 1980, 1985) of sperm DNA. In studies exposing mice to 10 different toxic chemicals, the dose-response curves of FCM-derived at values were very similar in shape and sensitivity to the percentage abnormal sperm head morphology curves (Evenson et a l . , 1985, 1986, 1989). Of added interest, several studies show that sperm cells arising from, stem cells exposed to stem cell-specific mutagenic chemicals maintain for at least 45 weeks chromatin structural abnormalities detectable by these FCM methods (Evenson et al., 1989).




The greatest impact of the SCSA technique may be for assessment of animal and human subfertility (Evenson, 1986; Ballachey et al., 1987, 1988). Studies in this laboratory (Ballachey et al., 1987) have shown a correlation of - .58 (< .01) between bull sperm chromatin structure and fertility ratings (FR) derived from 2 X lo6 artificial insemination services and adjusted for handling, environment, cost of semen, quality of cows, and so on. It should be noted that this study measured one to eight random semen samples collected over several years of time. In contrast, a heterospermic study (Ballachey et al., 1988) measured aliquots of the same semen samples used for fertility field trials; the correlation between heterospermic index and FCM data on sperm chromatin structure was 0.94 (< .01). Data for the relationship between human sperm quality and fertility is limited. In addition to our original report (Evenson et al., 1980), studies have assessed chromatin quality following successful therapy for leukemia and testicular carcinoma (see Evenson, 1986, for review). Perhaps the most interesting cases have been studied in infertility clinics where semen quality was shown to be excellent by the standard criteria of sperm count, motility, and morphology and yet the sperm cell chromatin had a highly abnormal susceptibility to heat denaturation, leading to the speculation that sperm chromatin quality is related to clinical infertility (Evenson, 1986). All samples obtained from men of recent proven fertility have shown homogeneous A 0 staining profiles; evidence to date suggests that broadly heterogeneous profiles are consistent with subfertility.

111. Materials

A. Acridine Orange Staining Solutions 1. A 0 stock solution: Chromatographically purified A 0 (Polysciences,

Inc. Warrington, PA) is dissolved in double-distilled water to a final concentration of 1 mg/ml and kept at 4°C for several months in darkness. 2. Acid-detergent treatment solution for first step of two-step acridine orange (TSAO) staining procedure: 0.15 M NaCl, 0.1% Triton X-100 (Sigma Chemical Co.), 0.08 N HCl in double-distilled water. The solution may be stored at 4°C up to several months. 3. AO-staining solution for second step of TSAO staining procedure: Mix 370 ml of 0.1 M citric acid buffer (kept as stock at 4°C) with 630 ml 0.2 M Na2P04 buffer (kept as stock at 4°C); add 372 mg Na,,,EDTA and 8.77 g NaC1. Adjust to pH 6.0. Now, 0.6 ml of A 0 stock solution is added to each 100 ml of stain solution. The stain



solution, kept in a glass amber bottle until use, is made fresh biweekly. Other details on the staining solutions are given in Darzynkiewicz (Chapter 27, this volume).

B. Buffers and Other Materials 1. TNE buffer: 0.01 M Tris, 0.15 M NaCl, and 1 mM EDTA, pH 7.4 2. Hanks’ balanced salt solution (HBSS): GIBCO Laboratories (Grand

Island, NY) 3. RNase A: DNase-free RNase. Cooper Biomedical, Inc. (Malvern, PA). 4. Nylon filters: 53 and 153 pm mesh, 1 in. diameter (Tetko, Inc., Briarcliff Manor, NY)

IV. Cell Preparation and Staining A. Testis Biopsy A testicular biopsy sample ranging in size from 1 to 10 mm2 is placed in a Petri dish containing HBSS and resting on a plate of steel on crushed ice. The tissue is minced with a razor blade or pair of curved scissors until a cellular suspension is obtained. This suspension is filtered through a 53-pm nylon mesh filter placed between a plastic tuberculin syringe and its protective end cap (tip cut off with wire cutter). The cellular suspension is transferred to the barrel of the syringe and the plunger is used to push the cells slowly through the filter. A 0.20-ml aliquot of suspended cells (1 X 106/ml) is stained by the TSAO technique of Darzynkiewicz et al. (1976) by first admixing with 0.40 ml of the Triton X-100-acid solution already described. After 30 seconds, 1.2 ml of the A 0 staining solution containing 6 pg AO/ml is admixed, placed in the flow cytometer sample chamber, and the sample flow initiated. Flow cytometric measurement is started 3 minutes after the sample is placed on the flow cytometer. The sample is kept at 4°C throughout this procedure.

B. Sperm Samples 1.



Fresh or frozen, thawed semen is diluted with TNE buffer to 1 x lo6 cells/ml and stained by the TSAO procedure exactly as described for testicular cells. Frozen semen samples can be prepared by diluting semen





to 2 X lo6 cells/ml in TNE buffer plus 10% glycerol and placing in a -70" to -100°C freezer. For severe oligospermic samples, undiluted semen can be used directly; the acid-detergent solution used in the first step dramatically reduces any semen vicosity (Evenson and Melamed, 1983). Semen extended in milk for use in artificial insemination may also be used directly without producing apparent artifacts; nonclarified, egg yolk citrate extender causes some background noise that may occasionally present a problem. A single freezing and thawing has no effect on sperm chromatin structure (Evenson et al., 1989a) but may disrupt early spermatid germ cells; these cells may be preserved by freezing the sample with techniques used for freezing tissue culture cells. Whole sperm or nuclei isolated and purified through sucrose gradients (Evenson et al., 1985) may also be fixed in 70% ethanol or 1: 1 70% ethanol/acetone and then pelleted by centrifugation, suspended, and rehydrated in TNE buffer for 30 minutes at 4°C prior to acid treatment, staining with A 0 and measurement by FCM. The data from fixed samples are essentially similar to that obtained on fresh material (Evenson et al., 1986); however, fresh or frozen samples are preferred.



For animal studies, a specific segment of the epididymis can be surgically removed from a killed animal and minced in TNE buffer as described before (Evenson et al., 1986) for testis biopsies. The vas deferens may also be excised, placed in a 60-mm Petri dish containing TNE buffer, and the sperm removed by pressing a blunt-shaped probe along the length of the organ. The resulting sperm suspensions are filtered through 153-pm nylon mesh before analysis by the SCSA. 3.


For measurements of sperm chromatin structure that are dependent on association of A 0 with both dsDNA and ssDNA, some, or all, samples should be sonicated and/or treated with RNase to insure against any abnormally retained RNA producing a red luminescent signal unrelated to DNA. This is considered a precaution only because little or no significant differences have been observed between sonicated, RNase-treated or untreated cells. Sperm suspended in TNE buffer in a test tube (Falcon 3033; Becton Dickinson Labware, Lincoln Park, NY) immersed in an ice-water slurry are sonicated for 30 seconds at a setting of 50 on low power (Bronwill Biosonik IV Sonicator, VWR Scientific, Inc., Minneapolis, MN), cooled for 30 seconds, and sonicated again for 30 seconds. The +-in. probe is placed just above the bottom of the tube. Optimal time and power required for sperm head-tail/cytoplasm separation varies between species.



The sonicate can be measured directly or the sperm heads can be purified if desired by centrifuging through a 60% sucrose solution. Addition of 1.5 x lo3 RNase units/ml to the sonicate and incubation for 30 minutes at room temperature (RT) apparently has no effect on the A 0 staining distribution (Evenson et al., 1985). Additional incubation may cause an increased red luminescence due to possible chromatin digestion by endogenous proteases. Whole unsonicated cells may also be incubated with RNase as before with the exception that 0.1% Triton X-100 is added to the suspension to permeate the cells.

V. Instrument Blue laser light (488 nm) excitation of AO-stained cells at a power of 2 3 5 mW is optimal. Luminescence of individual cells is measured at wavelength bands of red (> 630 nm) and green (515-530 nm). Since mature, AO-stained mammalian sperm have very little red luminescence because they lack RNA and ssDNA, the red photomultiplier tube (PMT) gain may need to be set high enough that electronic noise may result with some instruments. Ortho Diagnostics engineers changed one resistor in the two preamplifier circuit boards to reduce background noise on our Cytofluorograf I1 for studies on abnormal sperm chromatin structure. The electronic hardware or the software of the interfaced computer must have the capability to generate the at data, which is the ratio of red to total (red + green) luminescence and is a measure of the extent of acid- or heat-induced DNA denaturation in situ. Other technical points relevant to cell staining with A 0 are discussed by Darzynkiewicz (Chapter 27, this volume).



A. Testis Biopsy Figure 1 shows a typical distribution of AO-stained mouse testicular cells with respect to their green and red luminescence. Note that seven distinct cell populations can be discerned here in contrast to the four that can be distinguished by single-parameter DNA staining. This measurement is simple, rapid, and highly reproducible. The percentage total for each population, determined with computer assistance, may be related to specificity of the perturbing agent (Evenson et d., 1986).

' Please see biohazard caution following references.














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FIG. 1. FCM cytograms of two-parameter (green/DNA vs red/RNA luminescent signals) distribution of two-step acridine orange-stained mouse testicular cells. (A) Boxes 1 and 2 correspond to 4n cells, boxes 3 and 4 to 2n cells, and boxes 5-7 to In cells. Region 8 corresponds to the staining position of mature sperm. (B) Computer software enlargement of the In cell data points that provides an increased resolution of the round, elongating, and elongated spermatids.

B . Semen Samples Measured at PMT Gain Settings Used for Testicular Cells Environmental toxins and stress may cause immature germ cells to be released from the testis prematurely. Measurement of germ cells from the epididymis, vas deferens, or ejaculated semen provides an easy determination of this potential abnormality. Figure 2 shows a two-dimensional frequency histogram distribution of AO-stained human semen samples. Figure 2A shows the typical narrow distribution for samples from fertile males. Figure 2B shows a majority of cells with staining characteristics of round spermatids and a minority of cells having characteristics of abnormal sperm, both populations being confirmed by light microscopy. Figure 2C shows a very heterogeneous population of cells ranging from near normal sperm to diploid cell staining characteristics. The primary feature of the protocol that allows complete resolution between mature sperm, elongated spermatids, and round spermatids, even with single-parameter DNAstaining measurements, is the use of acid extraction in the first step. Acid-extracted round spermatids and mature sperm have a 3.2- and 1.2fold increase of DNA stainability, respectively, relative to non-acidextracted, AO-stained cells. Similar differences are observed with other DNA dyes (see Evenson, 1989).



FIG.2. Computer-drawn two-parameter (F,,,,vs F,m) histogram distribution of AOstained human semen cells. (A) Control sample obtained from a healthy fertile donor. (B) Sample obtained from an 18-year-oldpatient with stage 111 embryonal cell testicular carcinoma, 6 months after a unilateral orchiectomy and 3 months post-VAB 6 induction chemotherapy. (C) Sample obtained from a 28-year-old patient with stage I testicular cancer with teratoma, embryonal cell carcinoma, and seminoma, 11 months after unilateral orchiectomy. No normal mature sperm were seen and diploid cells were present. From Evenson and Melamed (1983).

C. Sperm Chromatin Structure Assay (SCSA) The SCSA has proved useful for fertility assays and toxicology studies. Figure 3 shows the relation between fertility of human males and resistance of sperm nuclear DNA to acid-induced denaturation. The fluorescence distribution for DNA stainability is heterogeneous because of a known artifact (see discussion in Evenson, 1989), which has no effect on the at distribution of interest in this technique. Whereas early work (Evenson et al., 1980,1985) used heat to induce DNA denaturation in isolated nuclei, several later studies (Evenson et al., 1986) showed that acid-induced denaturation produced the same results and is the method of choice in that it is more efficient, less time-consuming, and allows for the use of smaller samples. Early studies used the mean of at (X at) values to quantitate the level of abnormality. While this may be of value, especially for human samples that typically are quite heterogeneous, most of our recent studies have utilized the variation of at as an indicator of abnormality. The standard deviation (SD) of at (SD q )obtained from computer analysis has been the most useful and most closely related to known fertility ratings (Ballachey et al., 1987, 1988). The SD at is very sensitive to small changes in chromatin structure, and studies using this parameter require very precise repeat settings of the PMT values for comparative measurements done on different days. The PMT values are set so that the K green luminescence of fertile sperm with high resistance to DNA denaturation is at about 50/100 channels and the K red luminescence is at about 15/100 channels. The most ideal situation is to measure all experimental samples at one time period; however, careful repeat settings of the red and green PMTs allow measurements of com-











FIG.3. FCM cytograms of two-parameter (green/dsDNA vs red/ssDNA luminescent signal) distribution of sperm from (A) a fertile human control and (B) a patient from an infertility clinic. The box marked COMP shows the cells outside the main population with an abnormal chromatin structure. The a, distribution shows the extent of the abnormality.

pared samples over an extended period of time. The most precise repeat settings are obtained by using aliquots of a single semen sample that demonstrates heterogeneity of a t . A semen sample is identified and then diluted with TNE buffer + 10% glycerol to a working concentration of 2 x lo6 cells/ml. Several hundred aliquots (250 pl) of this dilution are placed into small snap-cap vials and immediately frozen at -70" to - 100°C.These samples are used to set the red and green PMTs to the same at index (X and SD a t ) as used previously. We typically measure a new calibration standard after every 5 or 10 samples to ensure that instrument settings have not drifted. For instrument calibration, a similar biological standard is preferred to fluorescent beads, since it also serves as a monitor for sample staining. For comparison of fertile control samples with samples of questionable fertility, the following ratio provides an index of abnormality (AI) for the SCSA procedure: SD at subfertile(?) A1 = SD at control(s) At this time, it is difficult to define what values are incompatible with



normal fertility (Evenson, 1986), since the interpretation of at parameters is still being explored. However, from our experience of measuring thousands of sperm samples derived from a variety of mammals, some of which had known fertility potential, the evidence suggests that a broadly heterogeneous pattern is indicative of either reduced or absent fertility. ACKNOWLEDGMENTS This work was supported by USDA grants 88-37242-4039 and 86-CRCR-1-2170. It is Publication No. 2137 from South Dakota State University Experiment Station.

REFERENCES Ballachey, B. E., Hohenboken, W. D., and Evenson, D. P. (1987). Biol. Reprod. 36, 915-925. Ballachey, B. E., Saacke, R. G., and Evenson, D. P. (1988). J . Androl. 9, 109-115. Darzynkiewicz, Z., Traganos, F., Sharpless, T., and Melamed, M. R. (1976). Proc. Narl. Acad. Sci. U.S.A. 13, 2881-2884. Evenson, D. P. (1986). In “Clinical Cytometry” (M. Andreeff, ed.), pp. 350-367. New York Academy of Science, New York. Evenson, D. P. (1989). In “Flow Cytometry: Advanced Research and Clinical Applications”, (A. Yen, ed.), Vol. 1, pp. 217-246. CRC Press, Boca Raton, Florida. Evenson D. P., and Melamed, M. R. (1983). J . Hisrochem. Cytochem. 31, 248-253. Evenson, D. P., Darzynkiewicz, Z., and Melamed, M. R. (1980). Science 240, 1131-1133. Evenson, D. P., Higgins, P. H., Grueneberg, D., and Ballachey, B. (1985). Cyrornerry 6, 238-253. Evenson D. P., Baer, R. K., Jost L. K., and Gesch, R. W. (1986). Toxicol. Appl. PharmaCOI. 82, 151-163. Evenson, D., Baer, R. K., and Jost, L. K. (1989a). J. Environ. Mol. Mutagen. 14, 79-89. Evenson, D. P., Janca, F. C., Jost, L. K., Baer, R. K., and Karabinus, D. S . (1989b). J. Toxicol. Environ. Health 28, 81-98. Thorud, E., Clausen, 0. P. F., and Abyholm, T. (1981). In “Flow Cytometry IV” (0. Lareum, T. Lindmo, and E. Thorud, eds.), pp. 175-177. Universitetsforlaget, Oslo. NOTEADDEDIN PROOF. Biohazard caution: Since sonication as described above produces aerosols, we have modified our SCSA procedure for human semen which may contain infectious agents including HIV. Our current method utilizes a Branson Sonifier 11, Model 450, coupled to a Branson Cup Horn (VWR Scientific, San Francisco, CA). The cup horn allows sonication of materials in a sealed test tube. Temperature of the samDle is maintained by 4°C water flowing t h u g h the cup horn derived by using a peristaltic pump that drives water through a copper tube coil set in a flask containing an ice water slurry. For human samples, 0.5 ml of TNE buffer containing 51 x lo6 sperm are placed into a 2-ml Corning cryogenic screw cap vial (VWR Scientific). The top end of this vial is placed into a large rubber stopper with a hole drilled through it that will hold the vial securely. This rubber stopper is then placed on top of the cup horn with the tube protruding into the cup horn and the bottom of the tube just off the bottom of the cup. Provided that the tubes are always placed the same distance into the rubber stopper, this arrangement likely provides for more repeatable sonication to the sample than holding the sample by hand. We are currently using 40 seconds of sonication pulsed for 70% of 1-second cycles at a setting of 3.0 of output power. This removes 295% of heads from tails. Time and power settings may need to be varied for different sample volumes and for sperm of other species. Current work also indicates that sonication of human samples provides better results than the above RNAse procedure. All other human sampling procedures are done in a biological safety cabinet.

Flow cytometric analysis of male germ cell quality.

Chapter 38 Flow Cytornetric Analysis of Male Germ Cell Quality DONALD P.EVENSON Olson Biochemistry Laboratories Department of Chemistry South Dakota S...
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