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Laboratory Evaluation of Sperm Chromatin: TUNEL Assay See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/251406945 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay Article · January 2014 DOI: 10.1007/978-1-4419-6857-9_14 CITATIONS READS 5 1,361 2 authors: Rakesh K Sharma Ashok Agarwal Cleveland Clinic Cleveland Clinic 458 PUBLICATIONS 10,148 CITATIONS 1,829 PUBLICATIONS 31,891 CITATIONS SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: SDF testing View project Research Profile View project All content following this page was uploaded by Ashok Agarwal on 27 January 2014. The user has requested enhancement of the downloaded file. Laboratory Evaluation of Sperm Chromatin: TUNEL Assay 14 Rakesh Sharma and Ashok Agarwal Abstract Routine semen analysis is unable to assess alterations in sperm chromatin organization such as DNA damage. Because fertility is based not only on the absolute number of spermatozoa but also on their functional capability, methods for exploring sperm DNA stability and integrity are being used to evaluate fertility disorders. A large number of direct and indirect tests that measure sperm DNA damage have been developed. This chapter focuses on one of those tests – the terminal deoxytransferase mediated deoxyuri- dine triphosphate (dUTP) nick end-labeling or TUNEL assay – which is increasingly being used in many laboratories. Keywords 3PERMCHROMATINs45.%,ASSAYs3EMENANALYSISs,ABORATORYEVALUATION OFSPERMCHROMATINs4ERMINALDEOXYTRANSFERASEMEDIATEDDEOXYURIDINE triphosphate assay One of the possible causes of infertility in men terminal deoxytransferase mediated deoxyuri- with normal semen parameters is abnormal sperm dine triphosphate (dUTP) nick end-labeling DNA. Fortunately, a number of sperm function assay, which is otherwise called TUNEL. This tests are available to assess sperm DNA integrity. test identifies in situ DNA strand breaks resulting One of the most commonly used tests is the from apoptotic signaling cascades by labeling the 3c-hydroxyl (3c-OH) free ends with a fluorescent label. The fluorescence, which is proportional to the number of strand breaks, can be measured either with microscopy or with flow cytometry. R. Sharma ( ) This chapter discusses the TUNEL assay in Andrology Laboratory and Center for Reproductive detail, including clinical protocols, clinical out- Medicine, Glickman Urological and Kidney Institute, comes, and future strategies aimed at optimizing OB-GYN and Women’s Health Institute, Cleveland Clinic, Cleveland, OH, USA this test and increasing its application as the test e-mail: [email protected] of choice in clinical andrology. A. Zini and A. Agarwal (eds.), Sperm Chromatin: Biological and Clinical Applications in Male 201 Infertility and Assisted Reproduction, DOI 10.1007/978-1-4419-6857-9_14, © Springer Science+Business Media, LLC 2011 202 R. Sharma and A. Agarwal Mechanisms of Sperm DNA Damage the 3c-OH end, and the fluorescence is propor- tional to the number of DNA strand breaks When sperm DNA is damaged, infertility, mis- (Fig. 14.1). This assay can be run either as a slide- carriage, and birth defects in offspring can occur based (fluorescent microscopy) (Fig. 14.2) or [1]. The main cause of sperm DNA damage is flow-cytometry assay [16] (Fig. 14.3, Table 14.3). oxidative stress [2–5]. Oxidative stress occurs TUNEL identifies what is termed as “real” or when levels of reactive oxygen species (ROS) actual DNA damage – that is, damage that has increase, when levels of antioxidant decrease, or already occurred – as opposed to “potential” both. A number of factors can lead to oxidative damage caused by exposing sperm to denaturing stress, including infection (viral or bacterial), conditions (Table 14.4). exposure to xenobiotics, and tobacco and alcohol All of the assays shown in Table 14.1 have a consumption. strong correlation with one another. Unfortunately, DNA fragmentation may also occur during sper- none of them are able to selectively differentiate miogenesis. During this process, torsional stress clinically important DNA fragmentation from increases, as DNA is condensed and packaged into clinically insignificant fragmentation. The assays the differentiating sperm head. Endogenous endo- also cannot differentiate the DNA nicks that nucleases (topoisomerases) may induce DNA frag- occur normally (physiological) from pathologi- mentation as a way of relieving this stress [6, 7]. cal nicking, nor can they evaluate the genes that Spermatozoa are transcriptionally and transla- may be affected by DNA fragmentation. These tionally inactive and cannot undergo conventional assays, including TUNEL, can only determine programmed cell death or “regulated cell death” the amount of DNA fragmentation that occurs called “apoptosis” but are capable of exhibiting with the assumption that higher levels of DNA some of the hallmarks of apoptosis including cas- fragmentation are pathological. pase activation and phosphatidylserine exposure on the surface of the sperm. This form of apopto- sis is termed as “abortive apoptosis” [8, 9]. Sperm Measurement of DNA Damage cells are able to repair some DNA damage during in Spermatozoa by TUNEL Assay spermatogenesis, but once they mature, they lose this innate ability [10, 11]. Therefore, posttestic- DNA damage can be measured using the ular sperm are more vulnerable to DNA damage. TUNEL assay by various protocols such as the Studies show that DNA damage is lowest in tes- following: ticular sperm and that it increases in epididymal 1. Biotin-d(UTP)/avidin system. and ejaculated sperm [12–15]. 2. BrdUTP/anti-Br-dUTP-FITC system. 3. Fluorescein isothiocynate labeled (FITC) dUTP system (In Situ Cell Detection kit, Measuring Sperm DNA Damage Catalog No. 11 684 795 910, Roche with TUNEL Diagnostics GmBH, Mannheim, Germany or Roche Diagnostics, Indianapolis, IN). Sperm DNA damage can be assessed with a num- 4. Apoptosis detection kit (Apo-Direct kit; ber of techniques that measure different aspects Catalog No. 556381; BD Pharmingen, San of DNA damage (Table 14.1). Each assay has its Diego, CA). own advantages and disadvantages (Table 14.2). We describe protocol #3 and #4 because they One of the most commonly used assays is the are commonly used tests for measuring sperm TUNEL assay. The quantity of DNA 3c-OH free DNA damage in sperm. The detection of sperm ends can be assessed in spermatozoa using this DNA fragmentation by flow cytometry and epif- assay in which the terminal deoxytransferease luorescence microscopy methods will also be (TdT) enzyme incorporates a fluorescent UTP at described. 14 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay 203 Table 14.1 Basics of common sperm DNA integrity assays Basis of assay Measured parameter Direct assays TUNEL Adds labeled nucleotides to free % Cells with labeled DNA DNA ends Template independent Labels SS and DS breaks Comet Electrophoresis of single sperm cells % Sperm with long tails (tail length, DNA fragments form tail % of DNA in tail) Intact DNA stays in head Alkaline Comet Alkaline conditions, denatures all DNA Identifies both DS and SS breaks Neutral Comet Does not denature DNA Identifies DS breaks, maybe some SS breaks In situ nick translation Incorporates biotinylated dUTP at SS % Cells with incorporated dUTP DNA breaks with DNA polymerase I (fluorescent cells) Template-dependent Labels SS breaks, not DS breaks Indirect assays DNA break detection FISH Denatures nicked DNA Amount of fluorescence propor- Whole genome probes bind to tional to number of DNA breaks SS DNA SCD Individual cells immersed in garose % Sperm with small or absent halos Denatured with acid then lysed Normal sperm produce halo Acridine orange flow cytometric Mild acid treatment DFI – the percentage of sperm with assays (e.g., SCSA, SDFA) denatures DNA with SS or DS breaks a ratio of red to (red + green) Acridine orange binds to DNA fluorescence greater than the DS DNA (nondenatured) main cell population fluoresces green SS DNA (denatured) fluoresces red Flow cytometry counts thousands of cells Acridine orange test Same as above, hand-counting % Cells with red fluorescence of green and red cells DFI DNA fragmentation index; DS double-stranded; FISH fluorescence in situ hybridization; SCD sperm chromatin dispersion test; SCSA sperm chromatin structure assay; SDFA sperm DNA fragmentation assay; SS single-stranded; TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling From Zini and Sigman [17], with permission First, the semen specimen is collected: (b) The sample should be collected in private in (a) Ideally, the sample should be collected after a room near the laboratory. If not, it should a minimum of 48 h and not more than 72 h be delivered to the laboratory within 1 h of of sexual abstinence. The name of the collection. patient, period of abstinence, date, and time (c) The sample should be obtained by masturba- and place of collection should be recorded tion and ejaculated into a clean, wide-mouth on the form accompanying each semen plastic specimen cup. Lubricants should not analysis. be used to facilitate semen collection. 204 R. Sharma and A. Agarwal Table 14.2 Advantages and disadvantages of various DNA integrity assays Direct assays Pros Cons TUNEL Can perform on few sperms Thresholds not standardized Expensive equipment not required Variable assay protocols Simple and fast Not specific to oxidative damage High sensitivity Special equipment required Indicative of apoptosis (flow cytometer) Correlated with semen parameters Associated with fertility Available
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