1. Introduction

The integrity of mammalian sperm DNA is of vitalimportance for the paternal genetic contribution to anormal offspring. Damaged DNA in the single spermthat fertilizes a female oocyte can have a dramaticnegative impact on fetal development and health ofthe offspring throughout adult life. The introductionof the intracytoplasmic sperm injection (ICSI)procedure for fertilization has focussed increasedattention on the quality of the paternal genome[8, 10]. The ICSI procedure bypasses virtually all ofMother Nature’s checks for insuring that a highquality sperm reaches the oocyte

in vivo when anindividual sperm with the best looking morphologyand motility as seen by light microscopy and judgedby a technician is picked up with a micromanipulatorand injected into an oocyte. However, the only realconsideration for success by the paternal contributionis whether or not the integrity of the paternal genomeis sufficient for proper embryo development resultingin the birth of a healthy offspring.

In assessing semen quality, animal and human fer-tility clinics typically measure sperm density, totalcount, motility and morphology. Clinics rarelymeasure sperm DNA integrity, primarily becausethey are unaware of the availability a rapid, reliableand practical test. Our laboratory has developed andrefined such a test, the sperm chromatin structureassay (SCSA), over the last two decades. SCSA dataon thousands of semen samples from bulls [2, 3, 9],stallions [30], boars [34] and exotic cats (unpub-

lished) show the clinical value of this assay foranimal fertility assessment. Light microscopemeasurements are typically made on 100–200 spermper sample, while the SCSA can utilize a fresh(or frozen-thawed) semen sample and using thefeatures of flow cytometry (FCM), collect andanalyze data on 5000 or more cells within a few minof time.

A potential father attending an ART (assistedreproductive techniques) fertility clinic can have analiquot of his semen analyzed by SCSA prior to anyproposed IVF/ICSI procedure in order to make a finaldecision on whether or not to proceed with theclinical work. Also, the SCSA could aid in thedecision of a young male patient just diagnosed withcancer on whether his semen should be frozen andstored for future use. The chromatin quality of spermin cancer patients prior to treatment may range fromexcellent to very poor [32, 33, 35]. Therefore,patients with good chromatin quality semen wouldbe candidates for cryopreservation whereas thosewith lower quality should consider the possiblefutility and expense of storing such semen.

Data on sperm from humans, bulls and stallionsand some other mammals have established athreshold measurement that is predictive of sub/infertility. Under the low pH conditions imposed, if30% or more of the sperm have SCSA detected DNAdenaturation (COMP

αt), fertilization attempted by invivo or in vitro methods are likely to fail [16, 31, 39].The etiology of susceptibility to DNA denaturationin situ is not known, although evidence has shown a

Methods in Cell Science 22: 169–189 (2000). 2000 Kluwer Academic Publishers. Printed in the Netherlands.

Sperm chromatin structure assay is useful for fertility assessment

Donald Evenson & Lorna JostDepartment of Chemistry and Biochemistry, South Dakota State University, Brookings, South Dakota 57007-1217,USA

Abstract. The Sperm Chromatin Structure Assay(SCSA) serves as a tool for measuring clinicallyimportant properties of sperm nuclear chromatinintegrity. The assay utilizes the metachromaticfeatures of Acridine Orange (AO), a DNA probe, andthe principles of flow cytometry (FCM). SCSA dataare not well correlated with classical sperm quality

Key words: Acridine orange, Animal and human fertility, DNA denaturation, Flow cytometry, SCSA, SpermChromatin Structure Assay, Toxicology

Abbreviations: αt = alpha-t; ART = Assisted Reproductive Technology; COMPαt = Cells Outside the MainPeak of αt; FCM = Flow Cytometry; HIGRN = % High Green Fluorescence; ICSI = Intracytoplasmic SpermInjection; IVF = In Vitro Fertilization; SCSA = Sperm Chromatin Structure Assay

parameters and have been solidly shown to predictsub/infertility. This assay is ideally suited to humanand animal fertility clinics to assess male spermDNA integrity as related to fertility potential andembryo development as well as effects of reproduc-tive toxicants. A detailed description of the SCSAfollows.

correlation between susceptibility of sperm cell DNAdenaturation and the presence of nuclear DNA strandbreaks [1, 37, 45]. The origin of DNA strand breakshas in some cases been attributed to an aborted typeof sperm cell apoptosis [37].

An overview of the SCSA

This very repeatable assay measures the suscepti-bility of DNA to denaturation in situ following lowpH treatment that potentially induces the denatura-tion. This assay is an adaptation of the ‘two-step AO’procedure originally designed by Darzynkiewicz etal. [4, 5] for simultaneous measurements of DNA andRNA content in somatic cells. Whatever minuteamounts of RNA may be present in a mature spermdo not interfere with SCSA data. It is of interest, butnot understood, that this procedure denatures prota-mine associated DNA in sperm but does not denaturesomatic cell DNA associated with histones [18].

The SCSA provides statistically strong data.Single cell scattergram data from independent SCSAmeasurements of a single sample are almost identical[25], and the correlation of their SCSA values arealways near 0.99 [31].

Besides the relationship to fertility potential, theSCSA is also an excellent, well-proven technique toassess toxicant-induced sperm chromatin damage[12, 17, 19–21, 24, 28, 29] especially for rodentstudies. The SCSA parameter SDαt, is the mostsensitive measure for the detection of abnormalchromatin in mouse epididymal sperm obtained fromtestes exposed to low level X-irradiation 40 daysearlier when the sperm cells were at the stem cellstage [44]. Likewise, the assay detects DNA defects(within hours) at the molecular level that may beobserved by light microscopy only 1–3 weeks lateras metaphase chromosome breaks or embryonicallyas dead fetuses [7, 24].

Age of men appears to have some influence onSCSA data [49] as does long periods of abstinence.Very short abstinence times, e.g. several hours, donot affect SCSA data significantly [25]. Limitedevidence suggests that long-term environmentalpollution does have a negative effect [41]. Few SCSAtoxicology studies have been done on human sperm,but data have been published or preliminary datapresented through meeting abstracts showing thatexposure to environmental stresses and pollutants andcigarette smoking alters chromatin structure [26, 38,40–43, 46, 51, 53]. When comparing sperm DNAintegrity assays in our laboratory, including TUNEL[37, 45], COMET [1] and Nick Translation [37], themost practical assay for assessing DNA integrity ofa semen sample is the SCSA. From extensive studieson human, bull, stallion and boar semen samples,there is strong evidence that with the currentprotocol, COMPαt (percentage of sperm withdenatured DNA) thresholds of ≈ 0–15%, 16–29% and≥ 30% relate to high, moderate and very low fertilitypotential, respectively [31]. Unusual cases of sterilityrange up to as high as 80–90% COMPαt. Refer toTable 1. These same threshold values also appear toapply to ART clinical samples. EVEN IF the spermare purified by either swim-up techniques to increasepercent motility or density gradients to remove deadcells, limited data from our laboratory show that ifsemen samples contain ≥ 27% sperm with denaturedDNA, then NO pregnancies occurred when fertilizedby in vitro fertilization (IVF) or ICSI techniques [39].The logical question follows ‘If ≈ 30% of the spermare abnormal by the SCSA, why can’t the other 70%contribute to a normal fertilization and pregnancy?’We refer to the ‘tip of the iceberg’ phenomena, i.e.,that the physical/chemical stresses placed on thesperm are sufficient to draw out 30% of abnormalsperm as a discrete population but likely the sametype of damage exists to a lesser extent throughout

170

Table 1. Sperm Chromatin Structure Assay least squares means and standard errors (SE) by pregnancy outcome groupfor selected parameters [31]

Pregnancy outcome group n Xαt SDαt COMPαt % HIGRN

1 = PG in 3 months 73 234.6 137.9 11.2 08.95SE 004.6 004.6 00.85 00.8

3 = PG 4–12 months 40 255.1** 157.9** 15.5** 08.78SE 005.9 006 01.09 01.07% increase 008.7 014.7 38.3 –1.9

4 = No pregnancy 31 270.3*** 173.7*** 17.2*** 15.03***SE 006.7 006.8 01.24 01.22% increase 015.2 030.0 53.6 68.0

** p < 0.01; *** p < 0.001.73 of 144 couples became pregnant during the first three months. SCSA data from these 73 men served as the ‘GoldStandard’ for semen with high fertility potential. Semen from couples that became pregnant during months 4–12 hadsignificantly (p < 0.01) poorer semen quality and that from couples that did not achieve pregnancy after 12 months onthe study were of even poorer quality (p < 0.001). No couple achieved pregnancy if the COMPαt was ≥ 30%.

the whole sperm population but in a sufficientamount to cause subfertility.

The concept of alpha t (

at) and other SCSA parameters

Understanding the variables of αt is very importantfor using the full potential of this protocol. Figure 1shows two examples of a SCSA clinical report, onefrom stallion and one from human semen, and thedifferent variables of αt. The extent of DNA denat-uration is quantified by the calculated parameteralpha-t [αt = red/(red + green) fluorescence] [5]. Thisratio is calculated on each of the 5000 cells measuredin the sample and this forms an αt distribution(histogram) with its own population statisticsincluding mean channel (Xαt) and standard deviation(SDαt). Standard deviation of αt (SDαt) describes theextent of chromatin structure abnormality within apopulation and the mean of αt (Xαt) indicates shiftsand trends in the whole population of cells. Thepercent of Cells Outside the Main Population of thisdistribution (COMPαt; i.e. the percent of cells withdenatured DNA) is also derived from the αt his-togram. The actual αt values are calculated from theαt frequency histogram. Normal, native chromatinremains structurally sound and produces a narrow αt

distribution, with relatively low αt values. DNA insperm with abnormal chromatin structure exhibitsincreased red fluorescence [16, 18] usually yieldinga broader αt distribution with an increased Xαt

and SDαt and more cells with denatured DNA(%COMPαt). Mean green fluorescence reflects DNAcontent and/or degree of sperm chromatin conden-sation, of which the latter inhibits DNA stainability.COMPαt has been determined as the most importantvariable of this assay for fertility assessment whileSDαt is the most important variable for toxicologystudies.

2. Materials

A. Equipment01. Flow cytometer (Ortho Diagnostics1, Becton-

Dickinson2, Beckman Coulter Corporation3,BioRad4, Partec5) The flow cytometer laser must have 488 nmexcitation wavelength and 10–30 mW power.Fluorescence of individual cells is collectedthrough red (630 to 650nm long pass) andgreen (515–530 nm band pass) filters. Table2 gives a list of flow cytometers that havebeen used successfully with the SCSA [27].

02. Software for calculating the SCSA parame-ters of αt.No major commercial flow cytometer with itsown computer and software is able to calcu-late the αt variables used in this protocol.

However, list mode data can be transferredto a PC and processed with offline softwarecapable of producing all necessary calculatedparameters.a) LISTVIEW, Phoenix Flow, Inc.6

b) WINLIST, Verity Software House7

c) FCS EXPRESS, DeNovo Software8

These three software packages have beensuccessfully used to generate the requiredcalculated αt variables offline. The macrofor analyzing SCSA data using WINLISTsoftware is not provided by Verity Housebut can be obtained at no cost from ourlaboratory. It is NOT appropriate to usesoftware or hardware configurations thatdefine αt as red (> 630 nm)/total (515longpass) fluorescence; this adds theunknown component of (530 to 630 nm)wavelength data into the equation.

03. PC Computer/Flow cytometer systema) Cicero System, Cytomation9

The Cicero/Cyclops software is the bestacquisition and analysis package foron/off-line PC analysis of flow cytometricdata at this time. It allows for viewing thecalculated αt parameters in live time whilemeasuring samples. This is advantageousfor setting up the instrument and checkingthroughout the measurement session forsystem stability and alignment. The CiceroSystem can be interfaced with all knowncommercial flow cytometers. The bestoption for a large Andrology laboratory isto have their own dedicated flowcytometer rather than use a core facility.For those on a limited budget, a recondi-tioned FACScan interfaced to a CiceroSystem is about US $40,000–$60,000.This used system costs no more than agood light microscope and is well worththe investment.

04. Biological safety hood, class II laminar flowLabconco* Purifier* Class II Safety Cabinets,Cat. No.: 16107121 (not found in database)10

05. Ultracold freezer (–70 to –110 °C, i.e. RevcoElite Cat. No. 13-990-187; Revco Model No.:ULT790 9 A; freezer10 or

06. LN2 (LRS-19) tank11

07. Branson Sonifier II, Model 450 (400W @20,000 Hz; Branson #101-063-198; VWR #33995-310) VWR Scientific12 coupled to a

08. Branson Cup Horn, 1/2″ dia., stepped, solid,external thread; Branson #101-147-038;VWR #33995-32412 and linked to a

09. Masterflex peristaltic pump, Model 900-197,and head adapter #7014, Cole ParmerInstrument Company13 or equivalent.

10. Benchtop, clinical centrifuge, e.g., TJ6,Beckman14

171

172

173

Figure 1. Two standard Clinical Reports from our laboratory, showing SCSA data and calculated parameters of humanand stallion semen. Top panel: SCSA data from a man whose wife conceived several days from the time of sample col-lection. Bottom panel: SCSA data from a stallion.

Red vs. green fluorescence cytograms (A and D):1. The sperm signal is well resolved from the debris signal in both examples (A and D). 2. The shift in sperm from normal chromatin (in A, the largest, oval-shaped cluster of cells with little red fluorescence,

i.e., the Main Population) to denatured DNA (anything to the right of the normal chromatin) is at a descending 45°angle. These Cells Outside the Main Population (COMPαt) represent the percentage of cells with denatured DNA.

3. A small percentage of the sperm have increased DNA stainability (High green fluorescence) and includes all spermabove the threshold that starts at the top edge of the main sperm population up to but EXCLUDING the cells in theuppermost green fluorescence channel. All sperm cells falling within these parameters are characterized by increasedDNA stainability and ARE INCLUDED in the final % high green calculation as shown in Figure 2.

4. All sperm cell fluorescence signals falling within the cytogram region of A (and D) ARE GATED into an αt vs.total fluorescence cytogram (B and E) and four histograms [red, green and total fluorescence (not shown) and αt

(C and F)] and ARE INCLUDED in the statistics.

at vs. total fluorescence cytograms (B and E)Computer software converts raw fluorescence data (A and D) into αt vs. total fluorescence (B and E) and this allows foran easier visual interpretation of the cell types present.

SCSA statistics Two successive measurements are always done using two aliquots of the same semen sample.Values are calculated to one decimal place. A very high repeatability is common between two independently measuredsamples.

Table 2. Flow cytometers that have been used to run the SCSA [27]

Flow cytometer Configuration Company

Cytofluorograf Orthogonala Ortho DiagnosticsFACScan Orthogonala Becton Dickinson FACScaliber Orthogonala Becton DickinsonElite Orthogonala Coulter ICP22A Epiilluminationb Ortho DiagnosticsSkatron Epiilluminationc BioRadFACStar Plusd Orthogonala Becton Dickinson

a Right angles exist between laser light beam, and axes of flow and fluorescence collection. The highly condensedsperm nucleus has a much higher index of refraction than sample sheath (water) in a flow cytometer. This differential,plus the typical nonspherical shape of sperm nuclei and their orientation in the flow channel, produces an optical artifactconsisting of an asymmetric, bimodal emission of DNA dye fluorescence [36] when measured in orthogonal configura-tion flow cytometers. This optical artifact of DNA-stained sperm does not significantly interfere with SCSA data [22,23] because αt is a computer calculated ratio. Each orthogonal flow cytometer having different lens and fluidic config-urations produces different cytogram patterns, but αt data are essentially the same [27]. b Cells are confined in sheath and measured as they emerge from a tube oriented along the axis of a fluorescent micro-scope objective and make a 90° turn into a flowing stream [47, 48].c Cells are confined in sheath ejected from a nozzle onto a slide surface and is called a JOOS or ‘jet on open surface’flow chamber. As the cells flow along this surface the signal is observed using a fluorescence microscope objective [47,48].b, c The same lens is used to focus excitation light and collect fluorescence. Hydrodynamic forces orient the longer axisof each nucleus in the direction of the flow. Orientation of the plane of each nucleus in the flow stream and the end ofthe nucleus that leads into the flow channel are random. No artifact is produced because of the parallel nature of flowdirection and the optical axis that render the emitted fluorescence signals insensitive to the rotational orientation of thesperm. The Partec flow cytometer, a currently marketed epiillumination instrument, has not been used with the SCSA,but data should resemble that from the Ortho ICP.d This instrument, apparently does not resolve sperm signal from seminal debris very well.

11. Fluorescence Light Microscope16

12. Ultracentrifuge14

13. Adjustable pipetters (adjustable 0–10 µl,10–100 µl, 100–1000 µl and a nonadjustable200 µl) and tips10

14. Waterbath, Precision* General-Purpose WaterBath, with Digital Control, e.g., Precision No.51220044; 1.5 L; Model: 280s; FisherScientific No. 15-474-6510

15. Stop watch, Fisher Scientific No. 14-648-310

16. ThermoSafe Insulated Containers, PolyfoamPackers Corp., Wheeling, IL16 or equivalent

17. Hemacytometer, e.g., Bright-Line CountingChamber, Fisher Scientific #02-671-510 or

18. Microcell counting chambers, ConceptionTechnologies17

19. Lab Counter, e.g., Fisher Scientific No. 02-670-1210

20. Analytical Balance (4 place, 0.1 mg), e.g.,Denver Instrument M-120, Fisher Scientific#01-913-4310

B. Flow Cytometer Supplies01. Fluorescent Beads for alignment18, 19

02. Mini Capsule Filter, 0.45 µm, e.g., PallGelman No. 12123, Fisher Scientific No. 09-743-5610 to filter sheath water

03. Sampling tubes for flow cytometer, conicaltubes, 4.5 ml, Sarstedt, Inc. No. 57.47720

C. Glass and Plastic Supplies01. Clinical specimen jars, e.g., Fisher Scientific

No. 14-375-22110

02. Oxford adjustable, 0.20 to 0.80 ml automaticdispenser for the acid-detergent solution withglass amber bottle, Fisher Scientific No. 13-687-6510

03. Oxford adjustable, 0.80 to 3.0 ml automaticdispenser for the AO staining solution glassamber bottle, Fisher Scientific No. 13-687-6610

04. 0.22 µm filters, e.g., Corning disposablevacuum filter units No. 430946, FisherScientific No. 09-761-98B10

05. Polypropylene microtubes, 0.5 up to 1.5 ml,Sarstedt, Inc. No. 72.699, 72.69020

06. Corning #430489 cryogenic vials, internallythreaded caps, 2 ml, Fisher Scientific No.03-374-2210

07. Three 1 L capacity glass reagent bottles,calibrated if possible, e.g., Kimax SolutionBottle with PTFE stopper – Kimble No.15097-1000; Fisher Scientific No. 02-920-1D10 or 1 L capacity disposable tissue cultureflasks

08. One 500 ml capacity glass reagent bottle,calibrated if possible, e.g., Kimax SolutionBottle with PTFE stopper – Kimble No.15097-500; Fisher Scientific No. 02-920-1C10

or 500 ml capacity disposable tissue cultureflasks

09. Several 750 ml disposable tissue cultureflasks, e.g., Falcon 353045, Fisher ScientificNo. 08-772-1A10

10. Disposable gloves, Fisher Scientific10

11. Ice buckets (3) for samples and reagentbottles, Fisher Scientific10

12. Several 15 ml, flat bottom scintillation vials,Fisher Scientific10

13. Micro-spatulas, Fisher Scientific10

14. Aluminum foil15. Stir bars, Fisher Scientific10

16. Stir plate with heating capacity, FisherScientific10

D. Reagents and Chemicals – Use only the purest grade reagents01. Acridine Orange (AO) chromatographically

purified, Polysciences, Inc. No. 0453919

02. 2.0 N HCl – Use purchased 2.0 N HCl, e.g.,Sigma-Aldrich Cat. # 251-221; do not dilutefrom a more concentrated HCl solution thatis likely less pure and may be of questionablestrength

03. Triton X-10004. 5 N HCl05. Citric Acid Monohydrate (F.W. = 210.14) 06. Sodium Phosphate Dibasic (F.W. = 141.96)07. EDTA (disodium, FW = 372.24)08. NaCl09. Tris-HCl (FW = 158)10. 2 N NaOH11. NaOH pellets12. Sucrose, ultrapure13. Hank’s Balance Salt Solution, HBSS, Gibco

BRL, Life Technologies No. 14065-05622

14. Ethyl alcohol (ETOH), dehydrated, 200Proof, Pharmco23

15. Household bleach (contains ~5% sodiumhypochlorite)

All chemicals were purchased from Sigma-Aldrich unless stated otherwise.

E. Semen or Caudal Epididymal or TesticularAspirate Sample(s)

3. Procedures

Pre-measurement preparations

The SCSA involves four broad procedural areas, a)the pre- measurement setup, b) cell preparation andstaining, c) measuring cells by FCM and d) dataanalysis. Several detailed pre-measurement steps areneeded which include cell collection, cell handlingand flow cytometer set-up.A. Prepare SCSA Buffers and Solutions – For all

solutions, use 0.45 µm filtered, double-distilledwater (dd-H2O) 01. Acridine Orange (AO) stock solution, 1.0

mg/ml

174

a) Tare a 15 ml, flat bottom scintillation vialon a ≥ 4-place electronic balance

b) Carefully remove and transfer 3–6 mg AOpowder from the stock bottle with a micro-spatula into the vial

c) Add an exact, equivalent (w/v) amount ofdd-H2O

d) Cap the vial and wrap in aluminum foilto protect from light

e) Store at 4 °C up to several monthsPrecautions: DO NOT use a more crudepreparation of AO; failure will result. AO isa toxic chemical and highly soluble in water.Flush any remnants of AO adhering to thespatula under running tap water to disposeof, or as dictated by each individual institu-tion’s biosafety board.

02. Acid-detergent solution, pH 1.2 [0.08 N HCl,0.15M NaCl, 0.1% Triton-X 100]a) Place about 300 ml dd-H2O into a 500 ml

capacity calibrated reagent bottleb) Add 4.39 gm NaCl c) Because of the viscosity, carefully

measure 0.5 ml Triton X-100 using a widemouth pipette

d) Wipe the outside of the pipette free ofTriton-X

e) Expel with force in and out of the pipetteuntil all Triton X-100 is dispensed into thereagent bottle (or leave pipette tip in bottlewhile mixing)

f) Carefully add 20 ml 2.0 N HCl g) Bring up to 500 ml with dd-H2Oh) Carefully pH to 1.2 with 5 N HCli) Store at 4 °C up to several months

03. 0.1 M citric acid buffera) Carefully measure 21.01 g citric acid

monohydrate into a 1 liter capacity glassreagent bottle

b) Bring up to 1 L with dd-H2Oc) Stir well d) Store at 4 °C up to several months

04. 0.2 M Na2PO4 buffera) Carefully measure 28.4 g sodium phos-

phate dibasic into a 1 liter capacity glassreagent bottle and

b) Bring up to 1 L with dd-H2Oc) Stir well d) Store at 4 °C up to several months

05. Staining buffer, pH 6.0 [0.1 M citric acid, 0.2M Na2PO4, 1 mM EDTA, 0.15 M NaCl]a) Measure 370 ml 0.1 M citric acid buffer

into a 1 liter capacity glass reagent bottle.b) Add 630 ml 0.2 M Na2PO4 buffer

Note that when the 0.2 M Na2PO4 bufferis removed from the refrigerator, saltcrystals will be present. Heat in 37 °Cwater bath or on a heated stir plate untilthe salts are fully dissolved.

c) Carefully measure and add– 372 mg EDTA (disodium) – 8.77 g NaCl

d) Mix overnight on a stir plate to insure thatthe EDTA is entirely in solution.

e) Carefully pH to 6.0 with concentratedNaOH pellets. Slowly and carefully adjust the pH to 6.0using very small pieces of concentratedNaOH pellets (cut with a scalpel andhandled with a forceps). Avoid adding toomuch NaOH so that excessive HCl wouldthen be needed for correction.

f) Store at 4 °C up to several months.06. AO staining solution

a) Add 600 µl AO stock solution to each 100ml of staining buffer

b) After dispensing the 600 µl AO stocksolution into the staining buffer, rinse thepipette tip up and down several times

c) Store at 4 °C up to 2 weeks in the glassamber bottle that comes with the Oxfordadjustable, 0.80 to 3.0 ml automatic dis-penser

07. AO equilibration buffer a) Dispense 400 µl acid-detergent solution

into a flow cytometer sample tubeb) Add 1.20 ml AO staining solution.

This mixture is run through the instrumentfluidic lines for ≈15 min prior to samplemeasurement to insure that AO is equili-brated with the sample tubing. This is alsorun through the instrument between dif-ferent samples to maintain the necessaryAO equilibrium conditions and help flushthe prior sample out of the lines.

08. TNE buffer, 10X, pH 7.4a) Carefully weigh and place each of the

following into a 750 ml disposable tissueculture flask: – 9.48 g Tris-HCl– 52.6 g NaCl – 2.23 g EDTA (disodium)

b) Add 600 ml dd-H2Oc) Stir and pH to 7.4 with 2 N NaOHd) Store at 4 °C up to 1 year

09. TNE buffer, 1×, pH 7.4, working solution[0.01 M Tris-HCl, 0.15 M NaCl, 1 mMEDTA]a) Add 60 ml of 10× TNE solution in a 750

ml disposable tissue culture flask b) Adjust volume to 600 ml with dd-H2O c) Check to make sure the pH is 7.4 d) Store at 4 °C for several months

Make a fresh dilution if there is anyevidence of bacterial contamination.

10. Sucrose buffer, 10×, pH 7.5 – used for soni-cated and purified nucleia) Carefully measure and place the following

175

into a 750 ml disposable tissue cultureflask: – 9.48 g Tris-HCl – 4.467 g EDTA (disodium) – dd-H2O up to 600 ml

b) pH to 7.5 with 2 N NaOHc) Store at 4 °C up to 1 year

11. Sucrose buffer, 1×, pH 7.5, working solution[0.01 M Tris-HCl, 2 mM EDTA] – used forsonicated and purified nucleia) Add 60 ml 10× sucrose buffer to a 750 ml

disposable tissue culture flask b) Adjust volume to 600 ml with dd-H2O c) Check pH (7.5) d) Store at 4 °C up to 1 year

12. 60% (w/w) sucrose solution, pH 7.5 – usedfor sonicated and purified nucleia) Measure 360 g ultrapure sucrose into a

750 ml disposable tissue culture flask b) Add 240 ml 1× sucrose buffer.

This solution has to be stirred for hours,usually overnight, to get all of the sucroseinto solution.

c) Check pH (7.5)d) Store at 4 °C up to 1 year

13. Hank’s Balance Salt Solution B. Prepare Flow Cytometer Supplies and Solutions

01. Flow cytometer sheath fluid (0.05% Triton-X 100)a) Filter dd-H2O by gravity flow through a

0.45 µm pore size filter unitb) Add 0.5 ml Triton-X 100 to each liter of

filtered dd-H2OTriton-X 100 minimizes surface tension inthe flow cytometer fluidic lines, therebyreducing air bubble retention. If your corefacility uses a typical saline solution forsheath, this is fine for the SCSA.

02. Solution for removing AO from FCM sampletubinga) Mix

– 50 ml H2O– 50 ml household bleach (contains ~5%

sodium hypochlorite)b) Store at room temperature

This solution is passed through the flowcytometer fluidic components in contactwith AO to remove adherent AO.

03. FCM Tubing Cleansera) Mix

– 50% ETOH– 50% household bleach (contains ~5%

sodium hypochlorite)– 0.5 M NaCl (2.92 g/100 ml)

b) Store at room temperatureThis solution is used for an occasionalthorough cleaning or flushing of flowcytometer sample lines to remove adherentcell debris and any fluorochromes.

C. Cell Preparation Techniques01. Collection and Handling of Human Samples:

a) Using standard laboratory procedures andpractices and in compliance with theInstitution’s Review Board’s rules andregulations, human semen samples aretypically obtained by masturbation intoplastic clinical specimen jars preferablyafter 2–3 days abstinence. These collec-tion principles hold true for all humansemen samples.

b) Allow 30 min for semen liquefaction atroom temperature (some use 37 °C).Freshly ejaculated semen consists of alarge amount of a gel-like substance thatsperm are lodged within. Normally thismaterial ‘liquefies’ approximately 30 minafter ejaculation and frees the spermthereby providing a homogenous suspen-sion of sperm that insures a randomsampling of all sperm that are in thesample.

c) Semen can be kept for up to 5 hours atroom temperature prior to measuring/freezing without loss of quality, allowingfor collections within a medical institutionand transport to the andrology unit If transport is required outside of abuilding complex, the sample may beconveyed in an insulated box or jacketpocket to keep from freezing or on liquidice if the ambient temperature is hot.

d) Once a sample has been diluted in TNEbuffer we recommend that it is measuredor frozen immediately.

Caution: For personnel safety against poten-tial infectious agents, e.g., hepatitis and HIV,human samples are handled using disposablegloves in a biological safety cabinet.Prepared samples are preferably run on aflow cytometer with a closed flow cell asopposed to a jet-in-air system, although bio-logical hazard containment is available formost systems. The HIV virus is not as stableas DNA viruses and they may be inactivatedby the low pH, acid-detergent treatment; thishas not been tested in our laboratory.

02. Collection of Animal specimens: Many toxi-cology studies on rodents and field trials onbulls, stallions, etc. have been reported withcollection methods outlined in detail [11–13,15, 17–22, 24, 28, 34, 44].

03. Identify, Prepare and Freeze ReferenceSample Aliquots: Because αt variables arevery sensitive to small changes in chromatinstructure, studies on sperm using this protocolrequire very precise, repeat instrumentsettings for all comparative measurementswhether done on the same or different days.

176

These settings are obtained by using aliquotsof a single semen sample called the ‘referencesample’ which insures day to day standard-ization of the protocol. We do not refer to thisas a ‘control sample’ since that term impliesthat the sample is consistent with normalfertility. It becomes obvious that this sampleis prepared after preliminary use and trials ofthe SCSA are successful and before anymajor studies are undertaken.a) Chose a donor whose semen sample that

demonstrates heterogeneity of αt as areference sample, e.g., 10–20% COMPαt,for human, bull or stallion; 2–3% forrodents, rabbits.

b) Have this donor provide a sample.c) Determine the sperm concentration.d) Dilute with cold (4 °C) TNE buffer to a

working concentration of 1–2 × 106

cells/ml.e) Immediately place ~300 µl aliquots into

0.5 ml snap cap vials or cryovials.f) Freeze immediately without cryoprotec-

tants at –70 to –100 °C in a freezer orplunge directly into a LN2 tank (refer tothe next section on freezing spermcells).

g) When measuring samples, these referencesamples are used to set the red and greenphotomultiplier tube (PMT) voltage gainsto yield the same mean red and greenfluorescence levels (≈ 130/1000 and≈ 500/1000 channels, respectively) and αt

values (Xαt, SDαt and COMPαt) from dayto day.

h) The values established by a laboratory(preferably the same as above) should beused consistently thereafter.

i) All reference sample mean red and greenfluorescence values should fall within±5 channels of this established laboratorystandard.

j) A freshly thawed aliquot of the referencesample is run after approximately every5–10 experimental samples to insure thatthe instrument has not drifted or lost focus[22, 23]. These aliquots are used only onceand are not saved to be used an hour ortwo later even if kept at 4 °C.

k) Strict adherence to keeping the referencevalues in the range set by the flowoperator should be maintained throughoutan experiment. The precision used in ourlaboratory is necessary for doing compar-isons between samples of the same man oranimal over time.

l) Because mature, AO-stained normal mam-malian sperm have very little red fluores-cence due to lack of RNA, the red PMT

gain may need to be set high. Potentiallyresulting electronic noise in some instru-ments can usually be eliminated byincreasing the signal threshold value.

m) Most instruments are very stable overdozens of successive measurements duringa single day of experimentation.

n) However, we have noted on our instru-mentation, that the mean green fluores-cence value tends to rise very slightlythroughout the day, and a minor adjust-ment needs to be made to bring thereference signal back to the establishedvalue.

o) When the reference sample supply isbecoming depleted, it would be advanta-geous to prepare a new batch of referencesamples from the same individual donor.

p) Consistency between measurements madeover time can be maintained by enrollinga new reference sample donor and com-paring that sample with the previousreference donor’s before that previoussupply has been depleted.

q) Reference samples can be stored (–100 °Cor LN2) for years so that a donor couldprovide enough sample for hundreds ofreference aliquots.

r) Very few FCM protocols are as demandingas the SCSA for using a reference sample.

04. Freezing Sperm Cells – for samples that areto be stored before measuring by the SCSA:a) For human samples allow 30 min for

semen liquefaction at room temperatureafter ejaculation.

b) Determine the sperm concentration.c) Quickly dilute aliquots of raw semen to

1–2 × 106 sperm/ml with TNE. d) Freeze immediately and directly without

cryoprotectants in an ultracold freezer(–70 to –110 °C; 0.5–1.5 ml snap captubes) or place directly into a LN2 tank(cryovials).

e) Samples should be frozen in vials that areapproximately 1/4 larger than the semenvolume in order to reduce the air tosurface interface to minimize relatedreactive oxygen damage.

f) Keep the tubes vertical when freezing, assamples frozen at the bottom of a tube aremore easily and safely thawed in a waterbath when needed.

g) Cryoprotectants are not needed sincequick-frozen cells and those frozen witha cryoprotectant provide equivalent SCSAdata [34]. This feature is unique tomammalian sperm cells due to the highlycondensed, crystalline nature of thenuclei.

177

05. Sonication of Sperm Cells – for SCSA ofSperm Nuclei (not always used):SCSA data from sonicated vs. unsonicatedsemen have been essentially identical [25].Sonication serves to verify that potential RNAin cytoplasmic droplets is not contributing tothe red fluorescence signal of denaturedsingle-stranded DNA and is preferred overRNAse incubation since that procedure hasthe potential of introducing incubation-relatedchanges in chromatin structure from proteasedigestion of chromatin proteins [18]. Becauseof the huge tail to head ratio, rat sperm arealways sonicated.a) Test your sonicator – check and verify the

optimal time and power required for a≥ 95% head/tail separation as this variesbetween species and needs to be tested foreach sonicator.

b) Using optimal settings (our laboratoryuses 30 sec on, 30 sec off, 30 sec on; lowpower) sonicate the sample.

c) Allow the sample to sit on ice for two minbefore starting the SCSA.

d) Rat sperm are always sonicated to breakthe very large tails that tend to clog theflow cell [17, 19, 28].

e) Again, human semen samples, potentiallycontaining infectious agents such ashepatitis or HIV, must be sonicated onlyin a closed tube. Fortunately, sonication ofsemen samples is rarely needed since wehave repeatedly found no significant dif-ferences between whole cells and theirsonicated counterpart.

f) When needed, our laboratory utilizes acup horn Branson Sonifier II, where thesample temperature is kept cold by 4 °Cwater flowing through the cup horn (seeFigure 2).

g) Use a peristaltic pump to drive water (21ml/min) through approximately 3 ft ofcopper tube coil (1/4 in id) set in a 4 literflask filled with an ice water slurry.

h) Place 0.5 ml of TNE buffer containing1–2 × 106 sperm cells into a 2 ml Corningcryogenic screw cap vial.

i) The top end of this capped vial is insertedinto the bottom side of a No. 11 rubberstopper which has a 12 mm holedrilled through it that will hold the vialsecurely.

j) The rubber stopper, holding the vial, isthen placed on top of the cup horn withthe vial protruding down into the cup hornso that the bottom of the vial is just off thebottom of the cup.

k) In this particular system, human samplesare sonicated for 40 sec using 70% of

1 sec cycle bursts at a setting of 3.0 outputpower.

l) The cup horn sonicator is preferred forease of use, uniformity between samples,and safety precautions.

m) The probe sonicator is a good substitutefor all species other than human.

06. Sucrose gradient purified nuclei [18]:Purified nuclei are needed for some studiesrelated to sperm chromatin structure inparticular, measurements on nuclear – SHgroups that are important for sperm nuclearcondensation [11, 26, 30]. Nuclei must bepurified from cytoplasmic components,especially since the tail segment hasnumerous – SH groups [11, 26, 30].a) Sonicate fresh or frozen-thawed sperm.b) Mix 800 µl sonicate with 200 µl of 60%

(w/w) sucrose solution. c) Layer this over 900 µl 60% (w/w) sucrose

solution in a centrifuge tube.d) Centrifuge 7 min @ 214,000 ×g @ 4 °C.e) Resuspend pellet in 100 to 500 µl TNE

buffer depending on desired final concen-tration.

07. Ethanol-fixed sperm – rarely required:Data from ethanol-fixed sperm are similar,but never quite as good as that obtained onfresh/frozen-thawed sperm [13]. a) Centrifuge sperm out of semen (300 ×g for

10 min). b) Resuspend cells in HBSS at a concentra-

tion of about 107/ml.c) Forcefully pipette 1 ml of this suspension

into 10 ml cold (–20 °C) 80% ethanol. d) Store samples at –20 °C. e) For SCSA measurement, the sperm are

pelleted. f) Wash once in TNE buffer.g) These are ready for the SCSA.h) Always keep the sample at 4 °C.

D. Flow Cytometer Preparation and Setup01. Signal processing:

Importantly, green and red fluorescencesignals MUST be processed and displayed aspeak (or height) rather than area signals.Also, since hundreds of figures have beenpublished with the green fluorescence (FL1on some instruments) data on the Y-axis andthe red fluorescence (FL3) data on the X-axis,we highly encourage the continuation of thisorientation so that readers can make easycomparisons between manuscripts and datasets. a) Orthogonal configuration and related

signal artifacts. The highly condensed sperm nucleus hasa much higher index of refraction thansample sheath (water) in a flow cytometer.

178

This differential, coupled with the typicalnonspherical shape of sperm nuclei andtheir orientation in the flow channel,produces an optical artifact consisting ofan asymmetric, bimodal emission of DNAdye fluorescence [36] when measured inorthogonal configuration flow cytometerswhere the collection lens is situated at aright angle to both sample flow and exci-tation source. Since αt is a computer cal-culated ratio of red to total (red + green)fluorescence [5], the optical artifact ofDNA-stained sperm measured in theorthogonal instruments [36] did not sig-nificantly interfere with SCSA data [22,23] and the αt frequency histogram wasvery narrow for a normal population ofsperm. In a study on running the SCSA ondifferent types of flow cytometers withdifferent configurations of lens andfluidics, the cytogram patterns weresomewhat different between instrumentsbut the αt data were essentially the same[27].

b) Epiillumination configuration. In epiillumination flow cytometers, thesame optical lens is used to focus theexcitation light and collect the fluores-cence of the sperm nuclei as they flow inthe optical axis of the instrument. The longaxis of the nuclei is oriented in the direc-tion of the flow by hydrodynamic forces.Orientation of the plane of each nucleus inthe flow stream and the end of the nucleusthat leads into the flow channel arerandom. However, since the flow directionand the optical axis are parallel, theemitted fluorescence signals are insensi-tive to the rotational orientation of thesperm and no artifact is produced. Acurrent commercial epiillumination flowcytometer is the Partec instrument.Although we have not tried the SCSA onthis instrument, we would expect data toresemble that obtained on our OrthoICP22A, an excellent but no longer com-mercially available instrument.

02. Workstation:The SCSA procedure requires that samplesare thawed and processed in the immediatevicinity of the flow cytometer. Humansamples are thawed and prepared in a safetyhood near the flow cytometer. The followingequipment should be close for quick and easyuse. All items can be handily placed on a lab-oratory cart.a) 2–3 ice buckets containing wet ice to hold

the reagent bottles, sample tubes, and TNEbuffer.

b) Disposable gloves.c) Stop watch.d) Automatic pipetters and tips.e) Reagent bottles deeply imbedded in the ice

buckets containing wet ice.03. Define flow cytometer acquisition and

analysis parameters and regions. Use Figure3 for clarification of this section:a) Determine how to generate the necessary

calculated parameters of alpha t (αt) inyour software package. Generally,1) Total fluorescence = 0.5 (red + green)

fluorescence2) Alpha t (αt) = 0.5 (red)/total fluores-

cence b) The analysis protocol should mimic the

acquisition protocol and display the fol-lowing: 1) Two-parameter cytograms (1000 dis-

play channels in both X and Y axes) of:A) Green Y (peak) vs red X (peak)

fluorescence andB) Total (Y) fluorescence vs alpha t

(gated from A1)2) One-parameter histograms (1000 dis-

play channels) of:C) Alpha t (αt)D) Green fluorescence E) Red fluorescence andF) Total fluorescence

c) Draw the gates and statistic regions ineach cytogram or histogram 1) Draw a region 1 in cytogram A to be

used as a gate that includes all spermsignals and excludes cellular debrissignals (signals located at the origin inthe red (X) vs. green (Y) fluorescencecytograms) from the analysis. Thisregion should not include signals builtup in the highest green fluorescencechannel.For most SCSA samples, a gate is firstdrawn very near the perimeters of thecytogram boundaries to exclude thoseevents that are beyond the full channellimits (i.e. 100% of scale). Next, debristhat falls to the lower left-hand corneris dealt with in one of two ways. Withsamples having very little debris a ≈45° line is drawn just below the bottomof the sperm signal as in Figure 2. Thisline is based on the fact that cells gainred fluorescence at the expense ofgreen fluorescence at a ≈ 45° angle.Human SCSA data are often more com-plicated and in some cases ellipticalcircles e.g., Figure 1 have been used toexclude the debris signal from the data[25]. After inspection of the whole

179

sample set, a decision is made on whatbest fits the experiment. Whatevermethod is chosen, it is important to beconsistent with analysis configurations,and preferably between previous,current and future experiments.

2) Allow sperm signals in region 1 ofCytogram A to gate into Cytogram Band Histograms C and D.

3) Draw a gating region in cytogram Bthat sends signals on to Histograms Eand F.

4) Draw 1 region in each Histogram(C–F) covering channels 1–1000,inclusive.

5) Place a second region in Histogram Ethat denotes the denatured sperm cellsor COMPαt.

04. Flow cytometer alignment and hydrodynamicsettings: a) Check instrument for alignment using

standard fluorescent beads. b) Very importantly, run an AO equilibration

buffer (400 µl acid-detergent solution and1.20 ml AO staining solution) through theinstrument sample lines for ≈ 15 min priorto establishing instrument settings toinsure that AO is equilibrated with thesample tubing.Run this buffer through the instrumentduring its warm up time prior to alignmentand again just before measuring samplesso that the system is fully equilibrated withAO, as it does transiently adhere to thesample tubing. However, as noted ALLAO can easily be removed at the end ofthe experiment.

d) To initially set up proper hydrodynamicconditions, measure several spermsamples by the SCSA (as described insample measurement below) that have apredetermined cell count of ≈ 1.5 × 106

sperm/ml and adjust the flow rate settings(if possible) for ≈ 200 cells per s. On aFACScan, the low flow rate settingdelivers the correct flow rate.

Measurement procedures

The actual SCSA essentially consists of three mainparts – cell preparation and staining, FCM and dataanalysis. A. Cell Preparation and Staining (see Table 3).

01. Place a tube of AO equilibration buffer on theinstrument to flow through the sample lineswhile preparing the sample.

For Frozen samples:02. A single frozen sample is removed from the

freezer or cold box containing dry ice and

held by the top of the test tube and thenmostly immersed in a 37 °C water bath, justuntil the last remnant of ice disappears.

For all samples: 03. After collection or thawing, the sample is

diluted if necessary to 1–2 × 106 sperm/ml (allbuffers and staining solutions are kept oncrushed liquid ice), sonicated if necessary andthen stained.When analyzing a series of human samples itis extremely helpful to obtain the sperm countin advance of SCSA preparation so that timeis not lost determining the proper dilution.However, if a sample(s) needs to be measuredquickly for a clinical decision, then ratherthan wait for a sperm count, estimate adilution, check the flow rate and if necessary,resample and restain with the proper dilutionto attain the required flow rate of ≤ 300 cellsper s.

04. Place a 200 µl aliquot of fresh or frozen/

180

Table 3. Sperm Chromatin Structure Assay (SCSA) BasicProtocol Steps

1. Fresh or frozen semen/sperm are thawed in a 37 °Cwater bath and diluted to 1–2 × 106 sperm/ml with TNEbuffer:

0.01 M tris buffer0.15 M NaCl1 mM EDTApH 7.4

2. Mix 200 µl sperm suspension + 400 µl of:0.15 M NaCl0.08 N HCl0.1% Triton-X 100pH 1.2

3. After 30 s add 1.20 ml of:0.2 M Na2HPO4

1 mM EDTA0.15 M NaCl0.1 M citric acid6 µg AO/ml staining bufferpH 6.0

4. Measure by FCM

1. Freshly collected or thawed frozen aliquots of semenare diluted to 1–2 × 106 sperm cells per ml with TNEbuffer.

2. Two-tenths (0.20) ml aliquots of this dilution are mixedwith 0.40 ml of an acid-detergent solution, whichpunches holes in the cell membrane allowing access tothe dye for entering the nucleus.

3. Thirty seconds later, the cells are stained by adding 1.20ml acridine orange staining solution [5, 18].

4. The sample is placed on the flow cytometer and threemin after the staining procedure began, red (F≥ 630) andgreen (F515–530) fluorescence measurements are collectedon 5000 cells per sample and stored in list mode onthe interfaced computer.

thawed semen (1–2 × 106 sperm/ml) into a~ 12 × 75 mm FCM sample tube.

05. Add 400 µl of the acid-detergent, low pHbuffer with an automatic dispenser that issitting deep in the ice bucket on the labora-tory cart.This dispenser needs to be accurate and tohave a maximum capacity near the amountbeing dispensed. At the beginning of samplemeasurement and after a long time intervalbetween measurements, dispense severalvolumes from both dispensers before startingwith the samples as AO in the delivery tubemay have been damaged by light and solu-tions in the plastic delivery tubes may bewarmer than 4 °C.

06. Start a stopwatch immediately after addingthe acid-detergent buffer.

07. Exactly 30 s later admix 1.20 ml of the AOstaining solution.

B. Flow Cytometry – Sample measurement 01. Place the prepared, stained sample on the

flow cytometer sample chamber in a ≈ 30 mlbeaker containing an ice water slurry. Although it is preferable to have the samplesetting in an ice bath, the configuration ofsome FCM sample chambers may not permitthis. The sample flow is started immediatelyafter placing it in the sample holder.

02. Using the stopwatch that was started with theaddition of the acid-detergent solution, theacquisition of list mode data to computerdisk is started at 3.0 min. This allows ample time for AO equilibrationin the sample and hydrodynamic stabilizationof the sample within the fluidics, both veryimportant aspects of AO staining.

03. Check the sperm flow rate during this timeand if it is too rapid after about 2 min on theinstrument, i.e., > 300 cells/sec, a new sampleis made at the appropriate dilution.The AO staining technique was designed byDarzynkiewicz et al. [4, 5] to have a flow rateof ≈ 200 cells per s when the sample con-tained ≈ 1 × 106 cells/ml and the AO stainingsolution contained 6 µg AO/ml. Due to thestringent requirements of AO equilibriumstaining, the rate of sperm cell measurementis important and should be about 200–250cells/sec This protocol provides ≈ 2 AO mol-ecules/DNA phosphate group. If a sample’sflow rate is too high, this same sampleCANNOT be diluted with AO buffer to lowerthe concentration. Samples that run over300/sec are discarded and a new aliquot isdiluted and stained to produce that approxi-mate rate. Sample and sheath flow valvesettings of the instrument are never changedduring these measurements so the liquid flow

rate is constant. A change in sperm flow rateis then a function of sperm concentrationonly.

04. Experience tells us that PMT settings shouldbe fairly identical from day to day dependingon slight alignment differences between daysand sample runs.

05. Measure all samples at least twice in succes-sion for statistical considerations and recorddata on 5000 sperm cells per measurement. For the second measurement, sample from thesame thawed aliquot, dilute appropriately,process for the SCSA and measure.

06. Place a tube of AO equilibration buffer on thesample port of the instrument, after thesecond measurement of a sample is finished.This maintains AO equilibration conditions inthe instrument sample lines and help flush theprevious sample out of the tubing and thenstart preparing the next sample. There is noneed to run this buffer between the duplicatemeasurements of the same sample, just allowthe first prepared sample to remain runningthrough the instrument while preparing thesecond one. Many papers contain detailsabout the critical nature of AO staining forDNA measurements [5, 14, 22, 23].

07. Continue thawing, staining and measuringsamples in duplicate.

08. Figure 4 is a cartoon sketch of some of themost common SCSA populations encoun-tered.

09. When finished with SCSA measurements,AO can easily be cleansed from the lines byrinsing the system for about 10 min with asolution for removing AO from the lines(contains 50% filtered household bleach)followed by 10 min of filtered H2O andbackflushing.

10. For an occasional thorough cleansing of allcellular debris and other fluorochromes fromthe lines, rinse the system for 10 min with theFCM Tube Cleanser, followed by 10 min offiltered H2O and backflushing.

C. Data analysisOur laboratory generally runs all samples in the experiment and then analyzes them as a group.01. The following data are taken from the regions

outlined in the protocol set up and are gather-ed into a data set and statistically analyzed: a) Mean and standard deviations of region 1

from all four histograms (red green totaland alpha-t)The actual at values are taken from at

frequency histogram statistics.b) COMPαt from region 2 of the αt histogram

(a percentage)1) COMPat – placement of region 2. See

Figures 1 and 3.

181

182

Figure 2. Cartoon schematic of a cup-hornsonicator arrangement.

Figure 3. Schematic of cytograms, his-tograms and regions (both gating andstatistical) that are needed for the SCSA.A-F illustrate the standard SCSA flow cyto-metric protocol constraints and G shows theregions for calculating % high green fluores-cence (%HIGRN) where region 2 is a subset ofregion 1.

COMPαt is the most important variableof this assay for fertility assessment.The shift in sperm from normal chro-matin in Figure 1A, the largest, oval-shaped cluster of cells containing littlered fluorescence, i.e., the normal cellsor Main Population) to denatured DNA(anything to the right of the MainPopulation) is usually at a descending45° angle. These cells represent thepercentage of cells with denaturedDNA. This second region of histogramE, defining COMPαt is set using thereference sample as a guide for wherethe main population peak leaves offand the COMPαt cells start.

The computer gate definingCOMPαt is easy to set if the majorityof the cells have fluorescence valuesequal to a normal population. However,when the entire population shifts [24],COMPαt cannot be defined as ‘thecells to the right of the main popula-tion’ in that cytogram. In the 1993toxicology studies [17, 24, 28], the

computer gate had to be set accordingto the data from the ‘control’ samples(0 dose) and all cells to the right of thatline were defined as COMPαt cells. Insome cases this may equal 100%. In astudy on human samples where thereare no ‘controls’ per se, the computergate is set using ‘reference’ sampleinformation to precisely set instrumentvariables.

Figure 1 displays where COMPαt

fall in the red vs green cytogram (Aand D) and the subsequent αt histogram(C and F) with native DNA (normal,main population, area 1) and denaturedDNA (COMPαt, area 2). The conver-sion of the green vs. red fluorescencecytogram to the total vs. αt cytogramimproves the visualization of the cellpopulations into normal and abnormal.Note the small variation between thetwo measurements showing the incred-ibly high repeatability between the twomeasurements.

02. The final SCSA parameter of interest, %High

183

Figure 4. Cartoon of normal SCSA populations and common variations. Area 1 – The main population of cells in a sperm sample that remain as a coherent population under the conditionsimposed. The DNA content in these cells is the same haploid amount, but the cytogram cluster is elliptical in shapebecause of the optical artifact discussed in the chapter.Area 2 – Cells Outside the Main Population (COMPαt) or the cells with denatured DNA. These cells typically moveout and downward from the main population at a ≈ 45° angle, showing the increase of red fluorescence at the expenseof green fluorescence. Some samples show a continuous cluster ranging from just outside the main population to oneswith very high red and very low green values. Alternatively, there are several discrete clusters in the COMPαt popula-tion with two shown in this illustration (orange and red).Area 3 – Seminal debris consisting of broken cellular components and other particulate matter stained with AO. Thesedebris signals MUST be resolved from the sperm for correct SCSA analysis. Since the COMPαt population forms a≈ 45° slope, downward and to the right, an effective means to delineate between these cell clusters is to draw a 45°computer gate between them at the bottom edge of the main population. NOTE: This is also an active gate duringacquisition so that 5000 or more sperm signals are accumulated and any debris signals excluded. This is most importantin samples with a high ratio of debris to sperm so that the same number of sperm cells are statistically analyzed persample.Area 4 – The sperm signals appearing in area 4 (shaded light green) have a high AO stainability and are termed spermwith high green stainability (HIGRN). These sperm nuclei are likely not fully condensed thus allowing a greater acces-sibility of intercalating dyes. Previously [31] we had included events in area 5 in the % High Green; however, especiallyfor poor quality human samples (of which there were very few in that previous study [31]), we have shown that manyof these events are clumps of cellular debris. Thus, we have concluded that including only area 4 is the best solution togauge the percent of cells that are not fully condensed. Furthermore, Region 6 may also contain both cellular debris andbacteria. The % HIGRN region starts at ≈ 70–75% of the green fluorescence scale, just at the top of the main popula-tion. Area 5 – Consists of sperm and possible cellular debris with very high DNA stainability. Area 6 – Represents a semen sample with excessive bacterial and cellular debris contamination. Due to random sizeclumping of the cell particles, a straight line of signal is seen to the left of the sperm population. When present, thispopulation is gated out during acquisition in order to accumulate 5000 sperm signals. Note that this tends to beconfounded with cells in the upper high green stainability area also.Area 7 – An approximation of a sperm sample that has been compromised by excessive freeze/thaw cycles or left in athawed state for an extended time period. This population has an increased DNA stainability and shifts a bit to the right,as discussed in the text, but also telltale of this compromised state is a usually present hook at the top of the populationcluster. Again, it would be hard to calculate HIGRN in these samples.

green (HIGRN) is analyzed separately usingjust Cytogram A (see Figure 3, G and Figure4, area 4 for clarification of the following).a) Draw region 1 in Cytogram A exactly the

same as the one used before continuing toEXCLUDE the cells in the uppermostgreen fluorescence channel.

b) Draw a second region that includes every-thing above the main population up to butEXCLUDING the uppermost green fluo-rescence channel and EXCLUDING anypotential bacterial and cell debris (Figure4; area 6).

c) % HIGRN is the number of cells in region2 compared to the total cells (region 1).1) %High green fluorescence (HIGRN)

region placementThe mean green fluorescence is relatedto the condensation level of the spermchromatin and extent of restrictedaccess of DNA dyes. The spermnuclear condensation process formammals normally produces a 5-foldreduction of DNA stainability withintercalating dyes such as AO relativeto round spermatids [15]. Lack ofappropriate sperm maturation results inincreased DNA stainability. Studieshave shown that patients attending aninfertility clinic often have anincreased DNA stainability [6, 33].This can be visualized by univariateanalysis [6] as well as by the SCSAbivariate analysis. In most cases, thedefects of high DNA stainability andDNA denaturation are mutually exclu-sive and any single cell rarely has bothdefects. However, we have observed alimited number of human clinic caseswhere both defects occur within thesame sample. Figures 1 and 4 alsoillustrate how we have resolved normalvs. high green fluorescing cells and thisparameter is calculated from the red vs.green fluorescence cytogram. Althoughdiscussed in a recent paper [31] we donot have enough experience with thisparameter to be so clear cut as to whatpercent indicates the likelihood ofinfertility.

The HIGRN parameter is not asprecise as the other SCSA parameters,since a small number of cell doubletscan become included in this measure-ment. Interestingly, the inclusion ofthese doublets in the αt parameters isnot a problem since αt is ratio.

4. Discussion

A. Gating and Debris Exclusion (Refer to Figures A. 1, 3 and 4)

In the measurement of a semen sample by the SCSA,if fluorescence signals from debris (i.e., free cellularcomponents, contamination,) and instrument noiseare blended in with the sperm fluorescence signals,the data become significantly compromised. Washingthe sperm or processing through density gradientsmay help eliminate this problem, but this is not afavorable option since there is a risk of differentiallylosing cell types and the advantage of using wholesemen measurements is then compromised. In flowcytometric studies of somatic cells, debris is usuallygated from true cell signals by the use of laser lightscatter. However, the asymmetrical shape and thehigh refractive index of sperm nuclei cause largevariations of forward and right angle light scatter anddo not effectively resolve cell debris signals fromsperm cell signals. The problem is accentuated insamples derived from patients with very low spermcounts or patients on chemotherapy that may resultin extensive debris from killed cells.

In difficult situations, debris can initially be gatedout of a cytogram using green peak vs. green areasignal processing resulting in a resolution between‘true’ sperm fluorescence and debris signals. Thistrue sperm signal is then gated into the normal greenpeak vs. red peak cytogram. These are worst case sce-narios as most samples can be gated with a simple45° line as stated earlier and shown in Figure 4.

B. Sampling order

If the intent of an experiment is to determine thesmallest amount of change measurable over time orincreasing toxicant dosages, measure all experimentalsamples of a particular set at one time period, in thechronological order of sample collection; or, if in adose-response toxicology study, measure the samplewith the lowest dose moving in sequence to thehighest dose. Statisticians prefer randomly codingand measuring samples. However, a practical issueof sample analysis has to be considered here. In adose-response study, the lowest dose exposures mayhave a very low percent of altered cells while thehighest dose set may have all altered cells. If a highdose sample is measured just prior to a lower dose,a few abnormal cells, stuck to the sample tubing, maybecome released and measured with the low dosesample. Thus, recognizing that a flow cytometerrandomly and objectively measures each sample, itis recommended that a series or set of samples bemeasured in sequence, in a single time frame.However, for totally random measurements, it hasbeen shown that with careful use of the referencesamples for repeat instrument settings combined with

184

extensive flushings of the sample lines betweensamples, measurements of compared samples over anextended period of time yield nearly identical results.This includes samples that were measured fresh, thenfrozen, and the frozen/thawed samples measured upto three years later [12, 15, 24]. Samples frozen atonly –70 °C for up to a decade or more may producesome difference in stainability likely due to slow butcontinual nuclear – SH oxidation.

C. Freezing and thawing

Frozen sperm samples, thawed one time and thenimmediately refrozen, do not have significantlyaltered SCSA data relative to fresh samples.However, for long term storage, samples must stayat a constant temperature in an ultracold freezer orLN2 (–70 °C or colder). Avoid refrigerator-freezerswith automatic defrosters due to the daily rise andfall of temperatures. Be aware that removal ofsamples from an ultracold freezer into ambient airmay cause the sample to undergo a temperaturechange up to nearly 70 °C. These changes might notbe apparent to the eye if the sample does not reachthe liquid state. The strong physical forces of tem-perature changes may damage the chromatin struc-ture and cause an artifact in the SCSA data.

Figure 4, area 7, shows a tell-tale signature patternthat is consistent with a sample having been thawed(e.g. freezer failure) for a while and refrozen orrepeatedly thawed and refrozen. Our establishedlaboratory rule is that for determination of criticalslight differences, we use samples frozen and thawedonly once. When handling any frozen sample, DONOT pick up the small test tube by the body of thetube; a warm human hand will produce quick samplemicrothawing. The use of gloves or forceps or at leastgrasping the tube by the very top lip of the tube helpsalleviate this potential problem. If samples must bemanipulated, place them into a deep ice chest con-taining dry ice that will keep the box and the samplesat least dry ice cold (≈ –70 °C). Ideally, a laboratoryshould have exclusive control over their ultracoldstorage. If sharing a freezer is necessary, discuss thepotential problem with the other users. Keep sampleboxes near the bottom of the freezer so they are notsubjected to ambient air when others may keep thedoor open for extended periods of time or the topboxes are placed outside the freezer to gain accessto boxes stored below. When a group of samples areto be analyzed by FCM, embed the samples in dryice in an ≈18 in deep styrofoam box with a goodcover and place it near your workbench. Individualsamples are removed, placed into a 37 °C water bathto thaw and then processed immediately.

D. Shipping of samples

Semen samples are shipped only by Federal Express(or other reputable overnight carrier) in a highquality, insulated, commercial shipping container.Small chunks of dry ice are first placed on the bottomof the shipping container, the sample box is placednear the center of the shipping box, and then moredry ice placed on top and around the box. Ten poundsof dry ice, broken up into pieces, in an insulatedshipping container are satisfactory for Priority Oneovernight shipments from any point in the UnitedStates during any season. This amount of ice willkeep for at least 2 days; however shipments shouldbe made only Monday through Wednesday in the rareevent that the shipment is ‘miss-shipped’ or is heldup for a day due to weather-related problems.Samples from international sources are also packedwith dry ice in a styrofoam shipping container butare usually hand carried so that the package is notheld up in Customs for a long time allowing the dryice to melt. Some shipments have been made fromoverseas with the samples in 70% ethanol.Regulations differ for each species, e.g. receiving bullsemen samples from countries that have hoof-and-mouth disease. In one case the Customs officeragreed to accept the shipment provided that the spermwere sonicated, and the sucrose gradient-purifiednuclei sent in well-sealed vials containing 70%ethanol. Samples stored in LN2 can be shipped in LN2

dry shippers or quickly transferred to an insulatedshipping container with dry ice as already described.

E. Counseling physician / patients or managers of E. animal breeding stations

SCSA data measured on any one day is only a‘snapshot’ of the quality of sperm that have justundergone about two months of maturation in thereproductive tract. The data do not say what theejaculated sample may have been several days orweeks prior to or after the current sample. A wholevariety of conditions can profoundly affect spermchromatin quality, ranging from diseases, high fever,medications, and mentally and physically stressingsituations. Thus, if a sample is of poor chromatinquality, e.g., ≥ 30 COMPαt, and the outcome is ofgreat relevance, e.g., the sale of a high priced stallionor bull, or the question of alternative methods forhuman conception, then the counselor/consultantshould carefully inquire about any previous stressfulsituations and suggest a repeat measurement in thefuture. If the quality is still poor, another samplecould be obtained some months later, but if poor, andprovided no environmental/drug/chemical exposureis known, then the condition is perhaps inherent andthe likelihood of parenting very poor.

SCSA data only predict subfertility/infertility andnot fertility because infertility may be due to many

185

factors, e.g., poor motility, acrosome quality, whilethe chromatin quality may be excellent. We have yetto find a semen sample that has ≥ 30% COMP andpregnancy occurring within a short time of a SCSAmeasurement. We have cases however, where all theclassical parameters are normal but the SCSA resultsare very poor and the likely cause of infertility. Thus,in our clinical reports, we make note only of whetherthe sample, based mostly on the percent of cells withDNA denaturation and percent with high DNA stain-ability, is COMPATIBLE OR INCOMPATIBLE witha good or poor fertility potential.

F. A note on the light microscope AO method

Tejada et al. [50] attempted to simplify the flowcytometric SCSA technique that we published in1980 to a light microscope method . However, dueto the strict demands of AO equilibrium staining con-ditions, the light microscope method can, at best, givea rather crude estimate of sperm chromatin integrity.The repeatability of this assay has been found to bevery poor and the data do not agree well with theflow cytometer SCSA data [52]. We do not recom-mend the use of this adaptation.

G. Unusual sample cases

1. Low concentration and/or ‘azoospermic samples’– An undiluted semen sample with a low spermcount may produce a very low flow rate, e.g., 10sperm/s. These measurements are valid, but it maybe best to count only a thousand cells or toconcentrate the sperm. Remember that 103 spermare still 5–10 times the number analyzed forclassical parameters with a light microscope. Alsoa sample with a 10 cells/s flow rate may well havebeen diagnosed in an andrology laboratory asazoospermic. Thus, the SCSA is more sensitivefor detecting and evaluating the quality of a nearazoospermic sample than classical light micro-scopic methods. However, in this case DO NOTincrease the flow rate by turning up the samplepressure valve. Practically, this increases thediameter of the sample stream, decreases thesensitivity of the focus and the AO staining char-acteristics are not the same as with the regularsample stream size.

2. Viscous samples – Semen samples undergo whatis termed ‘liquefaction’ within about 30 min afterejaculation. Freshly ejaculated semen consists ofa large amount of a gel-like substance thatsperm are lodged within. Normally this material‘liquefies’ and frees the sperm providing ahomogenous suspension of sperm that insures arandom sampling of all sperm types that may existin the sample. In cases where normal liquefac-tion does not occur, it is difficult to retrieve spermfrom the sample, and the measured sperm may not

be representative of the whole sample population.In near azoospermic samples, the semen is notdiluted and the sample is viscous by definition.This viscosity is greatly reduced however by theinitial acid-detergent solution and can then behandled as any other sample.

3. Bacterially infected samples – Semen with sig-nificant bacterial infection can often be detectedby the SCSA [25]. The bacterial population isusually represented by a long thin population tothe left of and parallel to the main SCSA spermpopulation as shown in Figure 4, area 6. Semensamples obtained before, during and after anti-biotic treatment have shown a decrease and thenloss of this population. This same area of thecytogram may also include broken cellular debris,e.g., from leukocytes.

4. Samples that have been frozen and thawed severaltimes – OR if long periods of time have elapsedbetween thawing and refreezing, an atypical fluorescence pattern appears (see Figure 4, area7). Sometimes a group of samples that wereprocessed and stored together show this phenom-enon. These samples have likely been compro-mised by a freezer failure or human error offailing to return the samples back to the freezerimmediately.

Acknowledgements

This research is based upon work supported by theEnvironmental Protection Agency Grant No. 827019,National Science Foundation EPSCoR Grant OSR9452894 and South Dakota Futures Funds. This isSouth Dakota Agricultural Experiment StationPublication No. 3182 of the journal series.

Notes on suppliers

01. Ortho Diagnostics (no longer commercially available– Becton Dickinson now owns the flow cytometers)

02. Becton Dickinson Immunocytometry Systems (BDIS),2350 Qume Drive, San Jose, CA 95131-1807, USA;Phone: (800) 448-BDIS (2347); Fax: (408) 954-BDIS(2347); www.bdfacs.com/support/

03. Beckman Coulter, Inc., 4300 N. Harbor Boulevard,P.O. Box 3100, Fullerton, CA 92834-3100, USA;Phone: (714) 871-4848; Fax: (714) 773-8283;www.coulter.com/Coulter/Cytometry/Systems.asp

04. BioRad: BioRad Laboratories, 2000 Alfred Nobel Dr.,Hercules, CA 94547, USA

05. Partec GmbH, Otto-Hahn-Str.32, D-48161 Münster,Germany; Phone: +49 2534 8008-0; Fax: +49 25348008-90; http://www.partec.de

06. Phoenix Flow Systems, Inc., 11575 Sorrento ValleyRoad #208, San Diego, CA 92121, USA; Phone:(800) 886-FLOW (3569); (619) 453-5095; Fax: (619)259-5268; E-mail: webmaster@phnxflow.com

07. Verity Software House, Inc., PO Box 247, Topsham,

186

ME 04086, USA; Phone: (207) 729-6767; Fax: (207)729-5443; verity@vhs.com

08. DeNovo Software, 64 McClintock Crescent,Thornhill, Ontario L4J 2T1, Canada; Phone:(905) 738-9442; Fax: (905) 738-5126; E-mail:info@DeNovoSoftware.com

09. Cytomation, 4850 Innovative Drive, Fort Collins, CO80525, USA; Phone: (970) 226-2200; Fax: (970) 226-0107; www.cytomation.com

10. Fisher Scientific, Pittsburgh, PA, USA; Phone: ( 412)490-8300, (800) 766-7000; www.fishersci.com

11. Taylor-Wharton, PO Box 8316, Camp Hill, PA 17001-8316 USA; Phone: (717) 763-5060; Fax: (717) 763-5061; E-mail:twsales@taylor-wharton.com; http://www.taylorwharton.com

12. VWR Scientific; Corporate Headquarters 1310Goshen Parkway, West Chester, PA 19380, USA;Orders: (800) 932-5000; Web Orders:www.vwrsp.com; Phone: (610) 431-1700, (800) 932-5000; Fax: (610) 429-9340; http://www.vwrsp.com

13. Cole Parmer Instrument Company, 625 East BunkerCourt, Vernon Hills, IL 60061-1844, USA; Phone:(800) 323-4340; Fax: (847) 247-2929;http://www.coleparmer.com or equivalent.

14. Beckman Coulter, Inc., http://www.coulter.com/Beckman/biorsrch/prodinfo/cntrifug/allegraintro.asp

15. Nikon USA, Clinical Products Information, e.g.Model: Eclipse E1000; Phone: (888)-645-6652,http://www.nikon.com

16. Polyfoam Packers Corporation, Wheeling, IL, USA;Phone: (888) 765-9362; www.polyfoam.com/insul_containers.html; E-mail: info@polyfoam.com

17. Conception Technologies, 6835 Flanders Drive, Suite500, San Diego, CA 92121, USA; Phone: (800) 995-8081; Fax: (619) 824-0891

18. Spherotech, Inc., 1840 Industrial Drive, Suite 270,Libertyville, IL 60048-9817, USA; Phone: (847) 6808922; Fax:(847) 680-8927; http://www.spherotech.com

19. Polysciences, Inc., 400 Valley Road, Warrington, PA18976, USA; Phone: (800) 523-2575; Fax: (800) 343-3291; http://www.polysciences.com

20. Sarstedt, Inc., PO Box 468, Newton, NC 28658, USA;Phone: (704) 465-4000; Fax (704) 465-4003

21. Sigma-Aldrich, PO Box 14508, 3050 Spruce St., St.Louis, MO 63178, USA; Phone: (800) 325-3010; Fax:(800) 325-5052; http://www.sigma-aldrich.com

22. Gibco BRL, Life Technologies, PO Box 79464,Baltimore, MD 21279, USA; Fax: 1-800-352-1468

23. Pharmco Products, Inc., 58 Vale Road, Brookfield, CT06804, USA; phone: (800) 243-5360 or (203) 740-3471

References

01. Aravindan GR, Bjordahl J, Jost LK, Evenson DP(1997). Susceptibility of human sperm to in situ DNAdenaturation is strongly correlated with DNA strandbreaks identified by single-cell electrophoresis. ExpCell Res 236: 231–237.

02. Ballachey BE, Hohenboken WD, Evenson DP (1987).Heterogeneity of sperm nuclear chromatin structureand its relationship to fertility of bulls. Biol Reprod36: 915–925.

03. Ballachey BE, Saacke RG, Evenson DP (1988). Thesperm chromatin structure assay: Relationship withalternate tests of sperm quality and heterospermicperformance of bulls. J Androl 9: 109–115.

04. Darzynkiewicz Z, Juan G (1997). Differential Stainingof DNA and RNA. Current Protocols in Cytometry7.3.

05. Darzynkiewicz Z, Traganos F, Sharpless T, MelamedMR (1975). Thermal denaturation of DNA in situ asstudied by Acridine Orange staining and automatedcytofluorometry. Exp Cell Res 90: 411–428.

06. Engh E, Clausen OPF, Scholberg A, Tollefsrud A,Purvis K (1992). Relationship between sperm qualityand chromatin condensation measured by sperm DNAfluorescence using flow cytometry. Int J Androl 15:407-415.

07. Estop AM, Munne S, Jost LK, Evenson DP (1993).Alterations in sperm chromatin structure correlateswith cytogenetic damage of mouse sperm followingin vitro incubation. J Androl 14: 282–288.

08. Evenson DP (1999). Alterations and damage of spermchromatin structure and early embryonic failure. In:Jannsen R, Mortimer D (eds), Towards ReproductiveCertainty: Fertility and Genetics Beyond 1999 (pp313–329). Parthenon Publishing Group Ltd,NY/London, .

09. Evenson DP (1999). Loss of livestock breeding effi-ciency due to uncompensable sperm nuclear defects.Reproduction, Fertility and Development 11: 1–15.

10. Evenson DP (1997). Sperm nuclear DNA strandbreaks and altered chromatin structure: Are thereconcerns for natural fertility and assisted fertility inthe andrology lab? Moving Beyond Boundaries:Clinical Andrology in the 21st Century. AndrologyLaboratory Workshop. Postgraduate Course.Baltimore, MD.

11. Evenson DP, Baer RK, Jost LK (1989). Flow cyto-metric analysis of rodent epididymal spermatozoalchromatin condensation and loss of free sulfhydrylgroups. Mol Reprod and Devel 1: 283–288.

12. Evenson DP, Baer RK, Jost LK (1989). Long termeffects of triethylenemelamine exposure on mousetestis cells and sperm chromatin structure assayed byflow cytometry. Env and Mol Mutag 14: 79–89.

13. Evenson DP, Baer RK, Jost LK, Gesch RW (1986).Toxicity of thiotepa on mouse spermatogenesis asdetermined by dual parameter flow cytometry. Toxand Applied Pharmacol 82: 151–163.

14. Evenson DP, Darzynkiewicz Z (1990). Acridineorange induced precipitation of mouse testicular spermcell DNA reveals new patterns of chromatin structure.Exp Cell Res 187: 328–334.

15. Evenson DP, Darzynkiewicz Z, Jost L, Janca F,Ballachey B (1986). Changes in accessibility of DNAto various fluorochromes during spermatogenesis.Cytometry 7: 45–53.

16. Evenson DP, Darzynkiewicz Z, Melamed MR (1980).Relation of mammalian sperm chromatin hetero-geneity to fertility. Science 240: 1131–1133.

17. Evenson DP, Emerick RJ, Jost LK, Kayongo-Male H,Stewart SR (1993). Zinc-Silicon interactions influ-encing sperm chromatin integrity and testicular celldevelopment in the rat as measured by flow cytom-etry. J Anim Sci 71: 955–962.

187

18. Evenson DP, Higgins PH, Grueneberg D, BallacheyB (1985). Flow cytometric analysis of mouse sper-matogenic function following exposure to ethylni-trosourea. Cytometry 6: 238–253.

19. Evenson DP, Janca FC, Jost LK, Baer RK, KarabinusDS (1989). Flow cytometric analysis of effects of 1,3-Dinitrobenzene on rat spermatogenesis. J Tox and EnvHealth 28: 81–98.

20. Evenson DP, Janca FC, Baer RK, Jost LK, KarabinusDS (1989). Effect of 1,3-Dinitrobenzene on prepu-bertal, pubertal and adult mouse spermatogenesis. JTox and Env Health 28: 67–80.

21. Evenson DP, Jost LK (1993). Hydroxyurea exposurealters mouse testicular kinetics and sperm chromatinstructure. Cell Prolif 26: 147–159.

22. Evenson DP, Jost LK (1994). Sperm chromatinstructure assay: DNA denaturability. In:Darzynkiewicz Z, Robinson JP, Crissman HA (eds),Methods in Cell Biology, Vol 42: Flow Cytometry(pp159-176). Acad Press, Inc, Orlando, FL

23. Evenson DP, Jost LK (2000) Sperm Chromatinstructure assay for fertility assessment. In: Robinson

JP (ed), Protocols in Cytometry, Unit 7.13. John Wileyand Sons (in press).

24. Evenson DP, Jost LK, Baer RK (1993). Effects ofmethyl methanesulfonate on mouse sperm chromatinstructure and testicular cell kinetics. Env and MolMutag 21: 144–153.

25. Evenson DP, Jost L, Baer R, Turner T, Schrader S(1991). Individuality of DNA denaturation patternsin human sperm as measured by the sperm chromatinstructure assay. Reprod Tox 5: 115–125.

26. Evenson DP, Jost LK, Corzett M, Balhorn R (2000).Effect of elevated body temperature on human spermchromatin structure. J Androl 21(5): 739–746.

27. Evenson D, Jost L, Gandour D, Rhodes L, Stanton B,Clausen OP, De Angelis P, Coico R, Daley A, BeckerK, Yopp T (1995). Comparative sperm chromatinstructure assay measurements on epiilluminationand orthogonal axes flow cytometers. Cytometry 19:295–303.

28. Evenson DP, Jost LK, Gandy JG (1993). Glutathionedepletion potentiates ethyl methanesulfonate-inducedsusceptibility of rat sperm DNA denaturation in situ.Reprod Tox 7: 297–304.

29. Evenson D, Jost L, Sailer B (1995). Flow cytometryof sperm chromatin structure as related to toxicologyand fertility. Proceedings of Seventh InternationalSpermatology Symposium, Cairns, Australia(October).

30. Evenson DP, Jost LK, Varner DD (2000). Stallionsperm nuclear protamine – SH status and suscepti-bility to DNA denaturation are not strongly correlated.J Fert Reprod Suppl 52: (in press).

31. Evenson DP, Jost LK, Zinaman MJ, Clegg E, PurvisK, de Angelis P, Clausen OP (1999). Utility of thesperm chromatin structure assay (SCSA) as a diag-nostic and prognostic tool in the human fertility clinic.Hum Reprod 14: 1039–1049.

32. Evenson DP, Klein FA, Whitmore WF, Melamed MR(1984). Flow cytometric evaluation of sperm frompatients with testicular carcinoma. J Urol 132:1220–1225.

33. Evenson DP, Melamed MR (1983). Rapid analysis of

normal and abnormal cell types in human semenand testis biopsies by flow cytometry. J HistochemCytochem 31: 248–253.

34. Evenson DP, Thompson L, Jost L (1994). Flowcytometric evaluation of boar semen by the spermchromatin structure assay as related to cryopreserva-tion and fertility. Therio 41: 637–651.

35. Fossa SD, De Angelis P, Kraggerud SM, Evenson D,Theodorsen L, Claussen OP (1997). Prediction ofpost-treatment spermatogenesis in patients withtesticular cancer by flow cytometric sperm chromatinstructure assay. Commun in Clin Cyt 30: 192–196.

36. Gledhill BL, Lake S, Steinmetz LL, Gray JW,Crawford JR, Dean PN, VanDilla MA (1976). Flowmicrofluorometric analysis of sperm DNA content:Effect of cell shape on the fluorescence distribution.J Cell Phys 87: 367–376.

37. Gorzcyca W, Gong J, Darzynkiewicz Z (1993).Detection of DNA strand breaks in individualapoptotic cells by the in situ terminal deoxy-nucleotidyl transferase and nick translation assays.Can Res 53: 945–951.

38. Grajewski B, Cox C, Schrader SM, Murray WE,Edwards RM, Turner TW, Smith JM, Shekar SS,Evenson DP, Simon SD, Conover DL (2000). Semenquality and hormone levels among radiofrequencyheat sealer operators. J Occup Env Med (Oct) (inpress).

39. Larson K, DeJonge C, Barnes A, Jost L, Evenson D(2000). Relationship between assisted reproductivetechniques (ART) outcome and status of chromatinintegrity as measured by the Sperm ChromatinStructure Assay (SCSA). Human Reprod 15(8):1717–1722.

40. Lemasters GK, Olsen DM, Yiin JH, Lockey JE,Shukla R, Selevan SG, Schrader SM, Toth GP,Evenson DP, Huszar GB (1999). Male reproductiveeffects of solvent and fuel exposure during aircraftmaintenance. Reprod Tox 13: 155–166.

41. Perreault SD, Rubes J, Robbins WA, Evenson DP,Selevan SG (2000). Evaluation of aneuploidy andDNA damage in human spermatozoa: application infield studies. Andologia 32: (in press).

42. Potts RJ, Newbury CJ, Smith G, Notarianni LJ,Jefferies TM (1999). Sperm chromatin damageassociated with male smoking. Mut Res 423:103–111.

43. Rubes J, Lowe X, Moore D, Perreault S, Slott V,Evenson D, Selevan S, Wyrobek AJ (1998). Cigarette-smoking lifestyle is associated with increased spermdisomy in teenage men. Fert Steril 70: 715–723.

44. Sailer BL, Jost LK, Erickson KR, Tajiran MA,Evenson DP (1995a). Effects of X-ray irradiation onmouse testicular cells and sperm chromatin structure.Env and Mol Mutag 25: 23–30.

45. Sailer BL, Jost LK, Evenson DP (1995b). Mammaliansperm DNA susceptibility to in situ denaturationassociated with the presence of DNA strand breaksas measured by the Terminal DeoxynucleotidylTransferase Assay. J Androl 16: 80–87.

46. Selevan SG, Borkovec L, Slott VL, Zudova Z, RubesJ, Evenson DP, Perreault SD (2000). Semen qualityand reproductive health of young Czech men exposedto seasonal air pollution (submitted).

188

47. Shapiro HM (1985). Practical Flow Cytometry (p 36).Alan R Liss, Inc, NY.

48. Shapiro HM (1988). Practical Flow Cytometry, 2ndEd (pp 67–68, 205–207). Alan R Liss, Inc, NY.

49. Spanò M, Kolstad H, Lars’en SB, Cordelli E, Leter G,Giwercman A, Bonde JP, Asclepios 1998 The applic-ability of the flow cytometric sperm chromatinstructure assay in epidemiological studies. HumanReprod 13: 2495–2505.

50. Tejada RI, Mitchell JC, Norman A, Marik JJ,Friedman S (1984). A test for the practical evalua-tion of male fertility by acridine orange (AO) fluo-rescence. Fertil Steril 42: 87–91.

51. Vine MF, Hulka BS, Everson RB, Evenson D (2000).An assessment of DNA damage in the sperm ofsmokers and nonsmokers using the sperm chromatinstructure assay. Cancer Epidemiology, Biomarkersand Prevention (submitted).

52. Weiss JL, Larson KL, Marshall DM, Jost LK,

Evenson DP (2000). A comparison of the acridiineorange microscope test (AOT) and the sperm chro-matin structure assay (SCSA) for assessing spermchromatin integrity (submitted)

53. Wyrobek AJ, Schrader SM, Perrault SD, Fenster L,Huszar G, Katz DF, Osorio AM, Sublet V, EvensonD (1997). Assessment of reproductive disorders andbirth defects in communities near hazardous chemicalsites. III. Guidelines for field studies of male repro-ductive disorders. Reprod Tox 11: 243–259.

Address for correspondence: Donald Evenson, PhD,Department of Chemistry and Biochemistry, Box 2170ASC 136, South Dakota State University, Brookings, SouthDakota 57007-1217 USAPhone: (605) 688-5474; Fax: (605) 688-6295E-mail: Donald_Evenson@sdstate.edu

189