Proceeding of the Biennial Conference of the Association for … · 2012-09-19 · Proceedings Association for Applied Animal Andrology Conference - Vancouver 2012 145 Development

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Proceeding of the Biennial Conference of the

Association for Applied Animal Andrology

July 28-29, 2012 - Vancouver, Canada

Reprinted in the IVIS website with the permission of the Association for Applied Animal Andrology - AAAA

9th Association for Applied Animal Andrology Biennial Conference will be held in 2014.

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Proceedings Association for Applied Animal Andrology Conference - Vancouver 2012

145

Development of a novel flow cytometric approach to evaluate fish sperm chromatin using fixed samples

Jill A. Jenkins*

U.S. Geological Survey, National Wetlands Research Center, Lafayette, LA, USA ______________________________________________________________________________

Abstract

The integrity of the paternal DNA is essential for the accurate transmission of genetic information, yet fertilization is not inhibited by chromatin breakage. Some methods are available for the sensitive detection of DNA damage and can be applied in studies of environmental toxicology, carcinogenesis, aging, and assisted reproduction techniques in both clinical and experimental settings. Because semen samples obtained from remote locations undergo chromatin damage prior to laboratory assessment, the present study was undertaken to evaluate treatments for effective chromatin staining in the development of a DNA fragmentation assay using fixed milt from yellow perch (Perca flavescens). Similar to the sperm chromatin structure assay (SCSA), susceptibility of nuclear DNA to acid-induced denaturation was measured by flow cytometry (FCM). Use of 10% buffered formalin for milt fixation allowed easier peak discrimination than 4% paraformaldehyde. The effects of time and temperature of incubation in 0.08 N HCl were evaluated in order to determine the ideal conditions for promoting DNA decondensation and making strand breaks more available for staining and detection by FCM. The best results were obtained with incubation at 37°C for 1 minute, followed by cold propidium iodide staining for 30 minutes. Keywords: DNA fragmentation; SCSA; Aquatic species; Chromatin compaction; Spermatogenesis ______________________________________________________________________________

1. Introduction

Cells must contend with genotoxic assaults arising from both endogenous chemical reactions, such as reactive oxygen species (ROS) generated by cellular metabolism, and exogenous industrial xenobiotics such as polychlorinated bipheyls and polycyclic aromatic hydrocarbons [1-3]. The detection of DNA damage is not only useful in studies of environmental toxicology [4], but in carcinogenesis, paternal aging, and with male genital tract infections [5-8]. The presence of fragmented DNA in mature sperm may also be due to residual apoptotic cells from the cell proliferation and differentiation mechanisms during spermatogenesis, or apoptosis occurring in late mature sperm [9]. Agents that are genotoxic for one group of living organisms are typically genotoxic for other groups due to the universality of the DNA molecule [10]. Because the effects of genotoxic agents are often tissue and cell-type specific [6;11;12], and because tissues differ in their ability to *Correspondence: U.S. Geological Survey, National Wetlands Research Center, Lafayette, LA70506, USA; E-mail address: [email protected]

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accumulate toxicants and their efficacy of DNA repair [11], techniques for measuring DNA integrity are best developed for individual cells [13] from particular tissues. Unlike the ovum and somatic cells, because of the reduced DNA repair capacity of late spermatids and sperm as compared with early spermatids and other spermatogenic cell types [14], strand breaks in nuceli transmitted through the germ cells may contribute to genetically determined diseases affecting the health of individuals in subsequent generations [15]. In fact, aberrant DNA repair in the zygote prior to the initiation of S-phase can result in a mutation in every cell in the body [7]. Structural integrity of DNA can yield superior diagnostic and prognostic information on fertility potential [16], and human semen samples containing a statistical threshold of > 30% sperm DNA fragmentation have a reduced level of pregnancy success [17]. The majority of genetic mutations in sperm are due to breakage rather than rearrangement [18], and the most deleterious type is the DNA double-strand break (DSB) [19]. However, defects in sperm chromatin (the complex of histones, non-histone proteins and DNA; [20]) do not inhibit the fertilization process [21;22]. Regardless of the cause or mechanism of strand breaks, strategies for accurate analysis of sperm DNA fragmentation are useful for clinicians, veterinarians, researchers, and natural resource managers. Immediate assessment of sperm quality parameters can be problematic due to the logistical constraints of distance between the sample collection location and the andrology research laboratory [23]. Collection of human semen from remotes sites is useful in human occupational and epidemiological reproduction studies [24], in domestic animals [25], in fish [26], and in threatened and endangered species [23;27;28]. During semen storage and handling, degraded cellular and nuclear proteins can induce autolysis, DNA strand breaks, and decondensation, whereby chromatin becomes more vulnerable to nucleases and polymerases [18]. Microbial contamination [29;30] and leukocytospermia [16] decrease parameters of sperm cell quality, including DNA integrity. In a study in which human male germ cells remained in seminal fluid at ambient temperature for 24 hours prior to freezing versus being frozen immediately, one of three DNA strand breakage assays showed significantly higher levels of breakage in the sample not immediately frozen [24]. The temperature modulations typically experienced during artificial reproduction technologies (ART) can negatively impact viability and DNA integrity more in the frozen-thawed spermatozoa samples than in the cooled samples [25]. In the current study, the goal was to develop a method by which DNA fragmentation could be measured without introducing further damage by way of analytical processing and handling, as occurs during remote collection, storage, and shipment to a laboratory. Preservation with fixatives is not typical with most assays of DNA fragmentation nor with documentation of specific genotoxins by DNA adduct formation [31]. Working with unfixed cells helps one avoid artifacts due to fixation, yet fixation is useful when material needs to be transported before analysis [32]. The three main DNA damage assays used to assess spermatozoa are the Comet assay, TUNEL assay, and sperm chromatin structure assay (SCSA) [7], the latter sometimes considered a gold standard for clinicians [22]. 2. Material and methods

2.1. Fixation, DNA decondensation, and staining methods The mechanism of fixation is dependent on the cell and the fixative. Formaldehyde crosslinks protein and DNA; paraformaldehyde is a polymerized form. Combining fixatives with detergents



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improves the speed and uniformity of reagent penetration [33]. The nonionic detergent Triton X-100 helps release nuclei for DNA analysis by FCM [32]. Preparing unclumped cells for single cell analysis is necessary for FCM, so cells are vortexed while adding fixative. Sperm nuclear basic protein types, protamines and histones, influence compaction and vary among animals [20]. These proteins influence availability of binding by external compounds. In assay development, heat and/or fixative use influences nuclear sensitivity, DNA decondensation, and DNA denaturation at strand breaks. A low pH, nonionic detergent solution improves dye uptake, and induces partial denaturation of the DNA in the chromatin [34;35]. Propidium iodide (PI) is a bright fluorophore with low non-specific binding, and it intercalates between nucleic acid base pairs strongly, but non-covalently. Incubation with isotonic solutions and with the stain higher than the stoichiometric level assures complete saturation of DNA without fluorescence shifting of peaks. Any remnant RNA in isolated germ cell nuclei has been ruled out as adding fluorescence above that of DNA [4], although RNAse (1 mg/mL, equivalent to 50 75 Kunitz units/mL) is suggested for use with PI staining to eliminate RNA fluorescence artifacts for other cell types [32]. Cells are stained at a selected temperature in the dark for at least 15 minutes and then filtered through 50 µm mesh before analysis.

2.2. Preliminary experiments and flow cytometer data analysis In proof-of-concept experiments, common carp (Cyprinus carpio) sperm exposed to hydrogen peroxide and stained either immediately or following fixation with 1:1 by volume 10% buffered formalin showed increased coefficient of variation (CV) and nuclei outside the main population (NOMP; [23]). Greater dispersion of DNA is indicated by increased flow cytometric CV [32], or widening of the main histogram peak. The NOMP metric (Figure 1) is a construct similar to the flow cytometric display of the proportion of cells outside the main population (COMP) produced by SCSA from fresh cells [34]. The DNA fragmentation results, analyzed in concert with other sperm quality parameters of the endangered razorback sucker (Xyrauchen texanus), were the basis for proposing a quality control threshold for disregarding use of samples containing >10% damaged nuclei prior to ART [23]. 2.3. Evaluation of yellow perch sperm chromatin As part of a larger investigation of the potential impacts of increased Chesapeake Bay urbanization on the reproductive health of yellow perch (Perca flavescens) (YP), sperm quality parameters were evaluated [36]. Briefly, adult YP were collected during spawning runs in 2008 (3/5-3/12) and 2009 (3/09-3/11). For analysis of DNA fragmentation in 2008, aliquots of milt were fixed 1:1 (v/v) in 10% buffered formalin, whereas in 2009 samples were fixed in 4% paraformaldehyde. To assess the relative numbers of cells in early or late spermatogenic stages of maturation [37,38], a fragment of the testis (< 0.10 g) was minced, maintained in the buffer solution for 10 minutes, and an equal volume of fixative was added as above. All tubes were stored at 4ºC until analysis. Prior to analysis, milt and testicular samples were centrifuged (10 minutes, 3400 rcf) and pellets were resuspended with Hank’s Balanced Salt Solution (HBSS); 200 µl aliquots of this suspension were prepared for evaluation of acid incubation with different tempteratures and times.



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Figure 1. Multiparametric flow cytometric data plots of a fixed milt sample of a yellow perch (Perca flavescens) captured from a Chesapeake Bay tributary (Allen’s Fresh Creek) and treated with the optimized protocol for detection of DNA fragmentation. Samples were treated with acid solution at 37°C for 1 minute then stained with propidium iodide. (A) Doublet discrimination mode allowed for gating out aggregates (outside of rectangular box) and analysis of single nuclei. (B) The main population of nuclei occurs within the gates in the density plot; DNA fragmentation is indicated by nuclei outside the main population (NOMP; 14% of nuclei). (C) The corresponding histogram width (CV) also reflects the integrity of DNA.



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Samples were incubated at room temperature (approximately 22oC), 37oC, or 100oC for 1, 2.5, or 5 minutes in pure HBSS or HBSS with acid (Triton X-100 at 0.1% by volume, 0.15M sodium chloride, and 0.08N hydrochloric acid). After incubation, 600 uL of cold staining solution (PI at 0.1 mg/mL, 37 mM citric acid, 0.15 M sodium chloride, 126 mM sodium phosphate dibasic, and 1 mMEDTA at pH 6.0) was added immediately. Tubes were vortexed and maintained for 30 min on ice (to stop decondensation) in the dark. Stained nuclei were filtered through 30-µm nylon mesh (Small Parts, Miami Lakes, FL) prior to FCM. Flow cytometer calibration was performed by using DNA QC Particles (Becton Dickinson Immunocytometry Systems; BDIS). Sperm nuclei at 1 x 106 per mL were analyzed in duplicate at < 300 sperm per second; 10,000 events were recorded per replicate using a 1024-channel FL2 filter after excitation at 488 nm. Cytograms were generated by plotting forward scatter (FSC-H) (size) versus side scatter (SSC) using CellQuest (BDIS). Aggregates were excluded from the analysis using gating procedures and doublet discrimination mode with FL-2 width (FL2W) versus FL2 area (FL2A). Histograms and dot plots were analyzed with CellQuest and FlowJo (FlowJo Flow Cytometry Analysis Software, Ashland, OR), respectively.

3. Results

For each replicate, nuclear size (FSC-H) versus scatter dot plots, FL2W versus FL2A dot plots for gating non-aggregates for further analysis (Figure 1A), FL2A versus size density plots (Figure 1B), and FL2A cytograms of DNA dispersion (Fig. 1C) were produced. These multiparameter displays allowed assessment of relative levels of intact DNA [39;40]. The CVs were calculated at the full width of the histogram peak at half the maximum height [32]. Beyond the CV, NOMP provided a second fragmentation indicator, whereby CV has been shown to correlate with NOMP in a controlled experiment [23;38]. Incubation at 37oC during acid treatment was preferable to room temperature because of noted peak development and separation. The smaller, more compacted nuclei represented by the peak on the left noted at room temperature after 5 minutes of incubation (Figure 2A-B) was more pronounced after incubation at 37°C for 1 minute (Figure 2C-D). Incubation at 37°C without acid for 1 minute did not produce adequate DNA decondensation as indicated by the primary peak at a low channel number and DNA trailing to the origin (Figure 2E-F); similar results were observed with incubation for 2.5 minutes. Incubation at 37°C for 1 minute without acid treatment was selected as the processing method for negative control samples. In testing for the optimal positive control temperature and time at which denaturation and optimal staining could be still be detected without uncontrolled denaturation, the acid solution for 1 minute at 100°C (Figures 3B and F) was selected rather than the 2.5 minute treatment (Figures 3D and H). At 100°C without acid for both 1 and 2.5 minutes, in samples from an urbanized (Figures 3E and G) and relatively non-developed site (Figures 3A and C), no appreciative difference was noted. However, with acid treatment before staining, incubation for 1 minute allowed for peak appearance on a measureable and comparable scale (Figures 3B and F), but incubation for 2.5 minutes resulted in accelerated breakage, as DNA crowded the origin (Figures 3D and H). The sample from the more urbanized site treated for 1 minute at 100°C (Figure 3F) showed more DNA fragmentation than the sample from the less urbanized site as shown by DNA trailing to the left of the main peak (Figure 3B).



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Figure 2. Milt collected from yellow perch (Perca flavescens) from the Chesapeake Bay tributary Mattawoman Creek (MA) with (A-D) and without (E-F) acid treatment. Acid treatment for 1 minute at 37°C (C and D) was preferable for discriminating subpopulations as compared with no acid treatments (E and F) or treatment at a lower temperature (A and B).



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Figure 3. Milt collected from yellow perch (Perca flavescens) Chesapeake Bay tributaries Mattawoman Creek (MA) (less urbanized) (A-D) and South River (SO) (E-H) treated at 100°C for 1 or 2.5 minutes with or without (NA) acid treatment prior to staining with propidium iodide. With no acid treatment, heat alone for different times did not notably shift peak fluorescence (A, C, E, G). With acid treatment for 2.5 minutes at 100°C (D and H), DNA was overly denatured and stained peaks shifted too far to the left, disallowing clear peak measurement. With acid treatment for 1 minute at 100°C (B and F), the level of DNA denaturation was sufficient to allow peak allowed peak discrimination. Peak shift to the left was more pronounced in the more urbanized sample (F) with more fragmentation (arrowhead) as compared with the milt from the less urbanized site (B).



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Figure 4. Testicular tissue collected from two yellow perch (Perca flavescens) Chesapeake Bay tributaries Mattawoman Creek (MA) (less urbanized) (A, C, E) and South River (SO) (B, D, F) incubated at 37°C for 1, 2.5, or 5 minutes with acid solution and stained with propidium iodide prior to flow cytometric analysis. Two prominent haploid nuclei peaks were noted after incubation for 1 minute for samples from both sites (A-B). For the MA sample, the peak on the left, representing more condensed nuclei, was reduced with incubation for 2.5 and 5 minutes (C and E). For the SO sample, the left peak height was also reduced with incubation for 2.5 minutes (D) and the peak had all but disappeared with incubation for 5 minutes (F). The widths of the primary peaks of SO samples were wider, indicating more fragmentation.



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In testing for the optimal incubation time for the 37°C acid treatment, the presence of the haploid peaks was more apparent with 1 minute incubation, but lessened with 2.5 and 5 minutes of incubation. The height of the left histogram peak in a sample from a less urbanized site was equivalent at the latter times, in the sample from the urbanized area (Figure 4). This, as well as the comparative CVs of the peaks, indicated more strands breaks in the sample from the South River as compared with Mattawoman Creek. Incubation for 1 minute allowed for the maximal amount of haploid nuclei to be displayed. Density plots allowed discrimination of the two apparent haploid populations (Figure 5).

Figure 5. Testicular tissue collected from two yellow perch (Perca flavescens) Chesapeake Bay tributaries Mattawoman Creek (less urbanized) (A, B) and South River (C, D), treated at 37°C for 1 minute with an acid solution and stained with propidium iodide prior to flow cytometry analysis. On the density plots (top) and histograms (bottom), arrows point to haploid nuclei with more condensed DNA (sperm), arrowheads indicate haploid nuclei with less condensed DNA (spermatids), and unlabeled populations represent diploid nuclei (spermatogonia, spermatocytes and somatic cells). The total percentage of sperm in A and B is 23.5% and in C and D it is 12.2%. The primary peak CV in B is 5.29 and in D it is 7.18, indicating more fragmentation. Similarly, NOMP is 8.78% in A and 9.24% in C.



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4. Discussion

Flow cytometry and image analysis of human testicular tissue have been demonstrated to be quantitative means of assessing spermatogenesis [37;41]. As in this study with YP testis and in a study of western mosquitofish (Gambusia affinis) [38], the characteristic patterns of the fine needle aspirates revealed the relative numbers of haploid (1C), diploid (2C), and tetraploid (4C) cells (Figures 4 and 5) that correlated well with standard histologic technique. Because spermatids undergo a final differentiation period during which the DNA is maximally compacted, this stage is also reflective of gamete maturity. The average percentage of haploid nuclei with maximally condensed DNA from all haploid nuclei for all individuals per site can be calculated, as well as the percent of males per site which demonstrated maximally condensed DNA (i.e. sperm). A suggested metric for YP spermatogenic staging is the percentage of haploid nuclei out of the total haploid plus diploid events. Buffered formalin was the prefered fixative selected for both DNA integrity and spermatogenic staging analyses compared to the corresponding paraformaldehyde-fixed samples providing fewer significant differences among sites [36], apparently because DNA strand breaks were less available for staining after heat and acid treatments. Because milt and semen samples obtained from remote locations undergo chromatin damage prior to laboratory assessment, the present study demonstrated that milt or testicular tissue could be preserved with buffered formalin. Furthermore, as with SCSA, the susceptibility of nuclear DNA to acid-induced denaturation was measured by FCM in a comparative analysis of both DNA fragmentation and chromatin compaction. The specific method defined for YP would need to be modified for optimization for other species. The rationale for final choice of heat and acid conditions allowed for maximal stain binding at strand breaks and for suitable peak discrimination. Disclaimer

Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government. References

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Proceeding of the Biennial Conference of the Association for … · 2012-09-19 · Proceedings Association for Applied Animal Andrology Conference - Vancouver 2012 145 Development