Theriogenology 81 (2014) 321–325
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Theriogenology journal homepage: www.theriojournal.com
Localization of alkali-labile sites in donkey (Equus asinus) and stallion (Equus caballus) spermatozoa Elva I. Cortés-Gutiérrez a, *, Martha I. Dávila-Rodríguez a, Carmen LópezFernández b, José Luis Fernández c, Francisco Crespo d, Jaime Gosálvez b a
Departamento de Genética, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social (IMSS), Monterrey, Nuevo León, México Departamento de Biología, Unidad de Genética, Universidad Autónoma de Madrid (UAM), Madrid, Spain c Unidad de Genética, Unit, INIBIC, Complejo Hospitalario Universitario A Coruña, La Coruña, Spain d Servicios de Veterinaria de la Armada Española (FESCCR-Ministerio de Defensa), Ávila, Spain b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 April 2013 Received in revised form 30 September 2013 Accepted 1 October 2013
The presence of constitutive alkali-labile sites (ALS) has been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization and comet assay in spermatozoa of donkey (Equus asinus) and stallion (Equus caballus). These results were compared with those obtained using a similar experimental approach using somatic cells. The relative abundance of ALS was of the order of four times more in spermatozoa than in somatic cells. Alkali-labile sites showed a tendency to cluster localized at the equatorialdistal regions of the sperm. The amount of hybridized signal in the ALS in the sperm of donkey (Equus asinus) was 1.3 times greater than in stallion (Equus caballus), and the length of the comet tail obtained in donkey sperm was 1.6 times longer than that observed in stallion (P < 0.05); however, these differences were not appreciated in somatic cells. In conclusion, ALS localization in sperm is not a randomized event and a different pattern of ALS distribution occurs for each species. These results suggest that ALS represents a species-specific issue related to chromatin organization in sperm and somatic cells in mammalian species, and they might diverge even with very short phylogenetic distances. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Sperm DNA structure Alkali-labile sites DNA breakage detection-FISH
1. Introduction The formation of mature spermatozoa is a unique process involving a series of meiotic and mitotic changes in cytoplasmic architecture, replacement of nuclear somatic celllike histones with transition proteins, and the final addition of protamines, leading to a highly packaged chromatin [1]. This forms a chromatin fiber coiled into loops that collapse into a toroid of densely packed chromatin. In the case of human spermatozoa, each toroid corresponds to a single DNA loop domain of approximately 40 to 50 kilobases that is attached at its base to the nuclear matrix [1,2]. Sperm DNA is
* Corresponding author. Tel./fax: þ52 81 81 904035. E-mail address: [email protected] (E.I. Cortés-Gutiérrez). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.10.001
organized in a specific manner that maintains the chromatin in the nucleus in a compact and nearly paracrystalline condition that is at least six times more condensed than the chromatin in mitotic chromosomes [3,4]. Another characteristic of sperm DNA structure, as reported in human and mouse, is its extreme sensitivity to incubation in alkaline denaturing agents [5–7]. The denatured DNA motifs on the genome achieved after alkaline incubations are known as alkali-labile sites (ALS). A new procedure now exists to quantify putative DNA breaks and ALS in situ within single cells, with the added advantage that it can also be used to scan the whole genome or specific DNA sequences. This protocol, known as DNA breakage detection (DBD)-fluorescence in situ hybridization (FISH), uses sperm cells that have been embedded within an inert agarose matrix on a specifically prepared microscope
322
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slide [8]. The cells are then lysed to remove membranes and proteins and the resultant nucleoids are exposed to a controlled denaturation step using alkaline buffers. The alkali gives rise to single-strand DNA (ss-DNA) stretches starting from the 50 -30 free DNA ends or from highly sensitive DNA motifs to alkaline conditions. These ss-DNA threads can then be detected using hybridization with specific or whole-genome fluorescent DNA probes [9,10]. As DNA breaks increase in a target region, more ss-DNA are produced and more DNA probes are hybridized; this results in a more intense FISH signal as additional ss-DNA is produced. The resulting hybridized signal on the whole genome can be quantified using image analysis systems [8,9,11]. The DBD-FISH signal obtained in the absence of exogenous DNA-damaging agents reflects the background level of ALS present in a genome [10]. It is noteworthy that when a whole-genome probe is applied to somatic cells, the background DBD-FISH signal is not homogeneous, and certain chromatin regions result as more strongly labeled. In human leukocytes, the more intense background DBD-FISH areas visualized with a whole-genome probe correspond to areas containing highly repetitive five-base pair satellite DNA sequences [12]. In mouse splenocytes, the background areas correspond to repetitive DNA satellite sequences in pericentromeric regions [13], and in Chinese hamster ovary cell lines derived from Chinese hamster cells, these corresponding to pericentromeric interstitial telomeric-like DNA sequences [14]. The role of ALS in chromatin packaging is exemplified in human spermatozoa. Unlike leukocyte DNA, certain repetitive satellite DNA sequences and the whole genomes of spermatozoa are extremely alkali-sensitive, and the signals produced with a whole-genome probe used for DBD-FISH were 12.7 times more intense than the corresponding signal in peripheral blood leukocytes [15]. The aim of this study was to assess and compare the presence of constitutive ALS using a DBD-FISH protocol in sperm cells and peripheral blood leukocytes in Equus asinus and Equus caballus. In addition to gaining information about this unknown aspect of sperm chromatin organization in these species, we investigated any possible differences between closely evolutionarily and genetically related species. 2. Materials and methods 2.1. Animals Fresh spermatozoa from four different stallions (Equus caballus) and four donkeys (Equus asinus) were used in this study. The stallions were a pure Spanish breed, and the donkeys corresponded to a singular Spanish breed (Zamorano-Leones) that is at risk of extinction. The animals ranged in age from 2 to 4 years and the samples were obtained throughout the month of April during the breeding season. All of the animals belong to the Spanish Army and were localized at the Depósito de Sementales de Ávila in Spain. The animals are maintained in controlled feeding and housing conditions with regular veterinary supervision. All of them were clinically healthy at the time of ejaculate collection. Semen samples (one ejaculate per animal) were collected
using an artificial vagina, and only ejaculates with 100 106 spermatozoa per mL and 50% progressively motile sperm were included in the analysis. Somatic cells (leukocytes) were obtained from heparinized peripheral blood samples. 2.2. DNA breakage detection-fluorescence in situ hybridization DNA breakage detection-FISH in sperm and leukocytes was performed according to Fernández et al., 2005 [10]. Whole DNA probes were prepared from isolated DNA from the leukocytes and labeled using digoxigenin (Roche Diagnostics Corp., Indianapolis, IN, USA) according to the protocol described elsewhere [10]. When the DNA probes were prepared and for hybridization purposes, they were denatured at 70 C for 10 minutes and hybridized at 37 C overnight. DNA probe hybridization was performed in the donkey and stallion leukocytes and sperm cells, using similar stringency conditions for sperm cells and leukocytes. Slides were washed in 50% formamide/2X SSC (pH 7) for 5 minutes, and washed twice in 2X SSC (pH 7) for 3 minutes. A nonspecific antibody-blocking solution was applied for 5 minutes at 37 C before detection of the hybridized signal. The hybridized DNA probe was detected using 30-minute incubation with a fluorescein-labeled antidigoxigenin antibody (Roche Diagnostics Corp.). The slides were counterstained with propidium iodide (1 mg/mL) in Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA). 2.3. Localization of ALS To determine regional localization of ALS or the genome regions exhibiting highest sensitivity to alkaline denaturation, sperm was exposed to gentle protein depletion according to DBD-FISH previously used for leukocytes. In this case, dithiothreitol (DTT; 0.001 M) was added to the lysing solution for single-strand bond reduction. Alkali-labile sites in sperm head were referred to proximal (near the flagellum insertion of the sperm) or distal location (near the acrosomal region). 2.4. Comet assay The alkaline comet assay was basically similar to that conducted by Singh et al. [5]. Sperm-gel slides were prepared as described previously for the DBD-FISH test. The slides were immersed in two lysing solutions for 30 minutes each at room temperature. The first solution contained 0.4 M Tris-HCl (pH 7.5) and 1% SDS, and the second, 0.4 M Tris-HCl (pH 7.5), 1% SDS, DTT (0.001 M), and 0.05 M EDTA. The slides were washed in 1X Tris-borate EDTA (89 mM Tris, 89 mM boric acid, 2.5 mM EDTA, pH 8.3) buffer for 10 minutes and then treated with fresh alkaline solution (0.03 M NaOH, 1 M NaCl, pH 12.5) for 2.5 minutes to cleave the ALS. The slides were placed horizontally in an electrophoresis tray, which was filled with fresh alkaline electrophoresis solution (0.03 M NaOH, pH 13). Electrophoresis was conducted at 20 V for 20 minutes at room temperature. After electrophoresis, the slides were gently removed from the tray and washed with neutralizing buffer (0.4 M TrisHCl, pH 7.5) for 5 minutes. The slides were washed in
E.I. Cortés-Gutiérrez et al. / Theriogenology 81 (2014) 321–325
distilled H2O for 5 minutes and then dehydrated in a sequential series of 70%, 90%, and 100% ethanol. Finally, the slides were stained with propidium iodide (2.5 mg/mL). 2.5. Fluorescence microscopy and digital image analysis All slides were analyzed using a digital image analysis platform based on a Leica DMLB fluorescence microscope equipped with three low-pass band filters for visualization of green, red, and blue fluorescence emissions. Images were acquired, using a Leica Digital DF-35 16-bit black-and-white charge coupled device, in 16-bit TIFF format. P The integrated density (ID ¼ number of pixel at the region of interest gray level values at each pixel obtained after background subtraction) was calculated using Leica QWin imaging analysis software, version 2.1. Fifty different cells per animal were measured in each experimental approach using an identical protocol of image thresholding. Migration of the DNA at the tail comet was measured using Leica QWin software. Migration distance of the comet tail was considered as the number of pixels from the physical center of the nucleioid to the end of the tail. To perform this experiment, sperm of both species were processed on the same slide in identical alkaline DNA denaturation and electrophoretic conditions. 2.6. Statistical analysis Statistical analysis was based on total cell number. The Student t Kruskal-Wallis test was used to investigate any differences of integrated density values among the four variables studied (sperm and leukocytes of donkey and stallion). A value of P < 0.05 was considered significant. All analyses were performed using SPSS for Windows v. 13.0 software (SPSS, Inc., Chicago, IL, USA). 3. Results When Equus somatic cells were processed within a DBD-FISH environment, the protein depletion produced with the alkaline lysing solution allowed detection of discrete quantities of ALS (Fig. 1A and B); in this case, hybridization of the DNA probe was visible as nonclustered but dispersed series of hybridized signals on each of the nuclei (yellow in Fig. 1A and B). In the sperm, the use of lysing solution, with the addition of DTT to produce disulphide bonding reduction, facilitated protein depletion in the protaminized sperm chromatin, and large numbers of ALS was detected (Fig. 1C and D). The use of DTT is imperative for ALS visualization in the sperm. Equus sperm exhibited fluorescence signal intensity four times greater than that obtained in leukocytes after correction for ploidy level. In the case of stallion, ALS showed a tendency to cluster in 92% of spermatozoa (Fig. 1F). In contrast, 90% of spermatozoa in donkey presented two discrete clusters of hybridized signal (Fig. 1E). Both signals tended to be localized at the equatorial-distal regions of the sperm. The presence of ALS in the sperm was 1.3 times higher in donkey than in stallion (compare Fig. 1C and D) (Table 1), and the differences were statistically significant (Table 1). However, the amount of ALS in leukocytes
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detected using DBD-FISH was similar for both species (Table 1) (Fig. 1A and B). The comet assay performed in alkaline conditions revealed that all spermatozoa showed the presence of a comet. In this case, the length of the comet tail obtained in donkey sperm was 1.6 times longer than that observed in stallion (P < 0.05) (Table 1, Fig. 1G and H). 4. Discussion The results obtained in this study show that ALS in the spermatozoa of equids are highly represented, especially when compared with those observed in some somatic cells. Additionally, ALS appear to be constitutively present in each cell type, but could be considered a transient chromatin structure during cell development, in which each differentiated cell would exhibit its own pattern of ALS concentration and localization [15]. Interestingly, at least in spermatozoa, they might also differ for genomes of closely related species. The molecular nature of the constitutive ALS is not well established. The classic concept of ALS includes abasic sites, apurinic or apyrimidinic, deoxyribose moieties, or masked DNA breaks that can be easily denatured by exposing them to weak alkaline conditions. However, a permanent and extremely high local concentration of DNA breaks or abasic sites in the genome should not be compatible with life. This effect can be partially attenuated with strong association with the repetitive DNA sequences observed for these DNA regions; but, in any case, this leaves the massive and variable presence of these moieties in every analyzed genome unexplained [16]. The most reasonable hypothesis to assume regarding the role of these DNA sequences would be that they are involved in structural modifications linked to the particular requirements of the chromatin organization merited at each cell. Alkali-labile sites, in addition to being constitutive DNA motifs, might behave as the expression of pre-existing DNA lesions in the DNA molecule of different etiology [10]. When ALS are not the product of directional DNA damage and are present as a constitutive part of the chromatin structure, it appears assumable that they are differentially represented in each cell; this is because the functional-structural requirements of each cell are known to be different. In particular, this is the case of the spermatozoa. However, they are ubiquitously present in all analyzed genomes irrespective of the targeted cell [10]. Thus, DBD-FISH used on intact undamaged somatic cell nuclei allowed the identification of ALS in mouse major satellite DNA at pericentromeric chromosome regions [13]. In the Chinese hamster ovary cell line, the pericentromeric chromatin contains long interstitial telomeric-related DNA sequences as long tandem repeats, and a similar situation was reported in the orthopteran Pyrgomorpha conica. In both cases, both genome domains are highly enriched in constitutive ALS [14,17]. In human somatic cells, a part of the ALS is related to the five-base pair classical satellite DNA sequence areas also located at the pericentromeric chromosome regions, and in the constitutive heterochromatic Yqh region [12]. In mature human spermatozoa, the background fluorescence intensity of the DBD-FISH signal after hybridizing with the whole-genome probe
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Fig. 1. Localization of alkali-labile sites (ALS) in somatic and sperm in donkey (Equus asinus) (upper panels) and stallion (Equus caballus) (lower panels). DNA breakage detection-fluorescence hybridization in peripheral blood leukocytes and spermatozoa of donkey (Equus asinus) (A and C, respectively) and stallion (Equus caballus) (B and D, respectively). Localization of ALS in spermatozoa of donkey (Equus asinus) (E), and stallion (Equus caballus) (F). Visualization of the ALS as a structural feature of the spermatozoa after an alkaline comet assay test, (G) Equus asinus, and (H) Equus caballus.
was 12.7 times higher in spermatozoa than in leukocytes [5,15,18]. Because the general high density of constitutive ALS has only been reported in spermatozoa of a limited number of species [16], it is of interest to investigate whether these chromatin modifications occur in the sperm of other mammalian genomes, in that they are probably fully related to the singular chromatin arrangement inherent in these cells. This should provide additional information about the universal but distinctive presence of expanded ALS in these types of cells. In the case analyzed here, both species presented distinctive ALS in the spermatozoa and in peripheral blood leukocytes, but with certain distinctive features for each species when the target cell was the sperm. This result indicates that even relatively short phylogenetic distances between species might result in substantial differences in chromatin organization at the spermatozoa. The sensitivity to alkali achieved in the spermatozoa in comet assay conditions pointed to a correlation with the results obtained with the DBD-FISH procedure [15]. The largest displacement of DNA at the comet tail in donkey spermatozoa would explain the slight increase of
1.3 points in the hybridized signal after DBD-FISH. This result has been found for the large, pericentromeric interstitial telomeric-repeat sequence blocks observed in Chinese hamster cell lines [14]. Alkali-labile sites behave as unpaired DNA segments that could be a consequence of torsional stress of DNA loops by strong chromatin packing and could also be the reason that they are initially abundant in the chromatin of the condensed mitotic chromosome [19]. In fact, it has been demonstrated that when subjected to constant tension greater than a critical value, the DNA molecule begins to unwind [20]. The result obtained in pig sperm appears to be an exception to the rule of dense chromatin packing and the presence of ALS, although even in this case, these can be detected with trypsin pretreatment [16]. However, it is noteworthy that only particular and highly repetitive satellite DNA sequences are related to constitutive ALS, their being very scarce or absent in other repetitive DNA sequences within the same genome; i.e., tight chromatin packing does not necessarily imply the presence of ALS, although ALS appear to be preferential in strongly condensed chromatin [16].
Table 1 Fluorescence area values and length of the comet tail in sperm cells and lymphocytes of donkey (Equus asinus) and stallion (Equus caballus) after DBD-FISH. Species
Fluorescence Analysis N
DBD-FISH (ID) Sperm (mean SD)
Comet assay (LCT) Leukocytes (mean SD)
Sperm (mean SD)
6910.7 1324.9 3467.8 754.4 102.50 10.12 3808.6 816.0 63.33 7.96a 5184.6 1095.8a P N represents the number of studied cells. ID (integrated density) ¼ ( number of pixels at the region of interest gray level values at each pixel obtained after background subtraction) segmented area of interest gray level values. Abbreviations: DBD-FISH, DNA breakage detection-fluorescence in situ hybridization; LCT, length of the comet tail. a Student t Kruskal-Wallis test: significant differences, P < 0.05. Equus assinus Equus caballus
200 200
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4.1. Conclusions Localization of hybridization signals in stallion and donkey sperm genomes is not randomly distributed and tends to be present at equivalent regions in the sperm, with the exception of there being a tendency toward ALS clustering in the case of stallion. This fact is probably informing concerning differences in the topology of the chromatin packing that might exist between both subspecies. In conclusion, the result obtained in this experiment show that ALS are represented in stallion and donkey genomes, although these exhibited differences when leukocytes and spermatozoa were compared. Differences between both species were also found when the sperm of both species were probed with DBD-FISH and comet assay. The fact that these differences were found in closely related species indicates that substantial differences in chromatin organization could be present in equivalent cells such as spermatozoa. More intense investigation using ALS as easy chromatin domains to be identified at the cellular level might provide valuable knowledge in terms of different aspects of the yet obscure sperm chromatin organization.
[7]
[8]
[9]
[10]
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[12]
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[14]
[15]
References [1] Allen MJ, Bradbury EM, Balhorn R. AFM analysis of DNA protamine complexes bound to mica. Nucleic Acids Res 1997;25:2221–6. [2] Ward WS. Chromosome organization in mammalian sperm nuclei. In: Barratt C, de Jonge C, Mortimer D, Parinaud J, editors. Genetics of human male infertility. Paris: Editions EDK; 1997. p. 205–21. [3] Ward WS, Coffey DS. DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol Reprod 1991; 44:569–74. [4] Bedford JM, Calvin HI. The occurrence and possible functional significance of -S-S- crosslinks in sperm heads, with particular reference to eutherian mammals. J Exp Zool 1974;188:137–56. [5] Singh NP, Danner DB, Tice RR, McCoy MT, Collins GD, Schneider EL. Abundant alkali-sensitive sites in DNA of human and mouse sperm. Exp Cell Res 1989;184:461–70. [6] Van Loon AA, Den Boer PJ, Van der Schans GP, Mackenbach P, Grootegoed JA, Baan RA, et al. Immunochemical detection of DNA damage induction and repair at different cellular stages of
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spermatogenesis of the hamster after in vitro or in vivo exposure to ionizing radiation. Exp Cell Res 1991;193:303–9. Van Loon AA, Sonneveld E, Hoogerbrugge J, Van der Schans GP, Grootegoed JA, Lohman PH, et al. Induction and repair of DNA single-strand breaks and DNA base damage at different cellular stages of spermatogenesis of the hamster upon in vitro exposure to ionizing radiation. Mutat Res 1993;294:139–48. Fernández JL, Goyanes V, Gosálvez J. DNA breakage detection-FISH (DBD-FISH). In: Rautenstrauss BW, Liehr T, editors. FISH technology. Heidelberg: Springer-Verlag; 2002. p. 282–90. Fernández JL, Gosálvez J. Application of FISH to detect DNA damage: DNA breakage detection-FISH (DBD-FISH). In: Didenko V, editor. Methods in molecular biology. Totowa, NJ: Humana Press; 2002. p. 203–16. Fernández JL, Cajigal D, Gosálvez J. Simultaneous labeling of singleand double-strand DNA breaks by DNA breakage detection-FISH (DBD-FISH). Methods Mol Biol 2011;682:133–47. Fernández JL, Goyanes V, Ramiro-Díaz J, Gosálvez J. Application of FISH for in situ detection and quantification of DNA breakage. Cytogenet Cell Genet 1998;82:251–6. Fernández JL, Vázquez-Gundín F, Rivero MT, Goyanes V, Gosalvez J. Evidence of abundant constitutive alkali-labile sites in human 5 bp classical satellite DNA loci by DBD-FISH. Mutat Res 2001;47:163–8. Rivero MT, Vázquez-Gundín F, Goyanes V, Campos A, Blasco M, Gosálvez J, et al. High frequency of constitutive alkali-labile sites in mouse major satellite DNA, detected by DNA breakage detectionfluorescence in situ hybridization. Mutat Res 2001;483:43–50. Rivero MT, Mosquera A, Goyanes V, Slijepcevic P, Fernández JL. Differences in repair profiles of interstitial telomeric sites between normal and DNA double-strand break repair deficient Chinese hamster cells. Exp Cell Res 2004;295:161–72. Muriel L, Segrelles E, Goyanes V, Gosálvez J, Fernández JL. Structure of human sperm DNA and background damage, analysed by in situ enzymatic treatment and digital image analysis. Mol Hum Reprod 2004;10:203–9. Cortés Gutiérrez EI, Dávila- Rodríguez MI, López- Fernández C, Fernández JL, Gosálvez J. Alkali-labile sites in sperm cells from Sus and Ovis species. Int J Androl 2008;31:354–63. López-Fernández C, Arroyo F, Fernández JL, Gosálvez J. Interstitial telomeric sequence blocks in constitutive pericentromeric heterochromatin from Pyrgomorpha conica (Orthoptera) are enriched in constitutive alkali-labile sites. Mutat Res 2006;599:36–44. Fernández JL, Vázquez-Gundín F, Delgado A, Goyanes V, RamiroDíaz J, de la Torre J, et al. DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural features. Mutat Res 2000;453:77–82. Darzynkiewicz Z, Huang X, Okafuji M. Detection of DNA strand breaks by flow and laser scanning cytometry in studies of apoptosis and cell proliferation (DNA replication). Meth Mol Biol 2006;314: 81–93. Gore J, Bryant Z, Nollmann M, Le MU, Cozzarelli NR, Bustamante C. DNA overwinds when stretched. Nature 2006;442:836–9.
Contents lists available at ScienceDirect
Theriogenology journal homepage: www.theriojournal.com
Localization of alkali-labile sites in donkey (Equus asinus) and stallion (Equus caballus) spermatozoa Elva I. Cortés-Gutiérrez a, *, Martha I. Dávila-Rodríguez a, Carmen LópezFernández b, José Luis Fernández c, Francisco Crespo d, Jaime Gosálvez b a
Departamento de Genética, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social (IMSS), Monterrey, Nuevo León, México Departamento de Biología, Unidad de Genética, Universidad Autónoma de Madrid (UAM), Madrid, Spain c Unidad de Genética, Unit, INIBIC, Complejo Hospitalario Universitario A Coruña, La Coruña, Spain d Servicios de Veterinaria de la Armada Española (FESCCR-Ministerio de Defensa), Ávila, Spain b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 April 2013 Received in revised form 30 September 2013 Accepted 1 October 2013
The presence of constitutive alkali-labile sites (ALS) has been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization and comet assay in spermatozoa of donkey (Equus asinus) and stallion (Equus caballus). These results were compared with those obtained using a similar experimental approach using somatic cells. The relative abundance of ALS was of the order of four times more in spermatozoa than in somatic cells. Alkali-labile sites showed a tendency to cluster localized at the equatorialdistal regions of the sperm. The amount of hybridized signal in the ALS in the sperm of donkey (Equus asinus) was 1.3 times greater than in stallion (Equus caballus), and the length of the comet tail obtained in donkey sperm was 1.6 times longer than that observed in stallion (P < 0.05); however, these differences were not appreciated in somatic cells. In conclusion, ALS localization in sperm is not a randomized event and a different pattern of ALS distribution occurs for each species. These results suggest that ALS represents a species-specific issue related to chromatin organization in sperm and somatic cells in mammalian species, and they might diverge even with very short phylogenetic distances. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Sperm DNA structure Alkali-labile sites DNA breakage detection-FISH
1. Introduction The formation of mature spermatozoa is a unique process involving a series of meiotic and mitotic changes in cytoplasmic architecture, replacement of nuclear somatic celllike histones with transition proteins, and the final addition of protamines, leading to a highly packaged chromatin [1]. This forms a chromatin fiber coiled into loops that collapse into a toroid of densely packed chromatin. In the case of human spermatozoa, each toroid corresponds to a single DNA loop domain of approximately 40 to 50 kilobases that is attached at its base to the nuclear matrix [1,2]. Sperm DNA is
* Corresponding author. Tel./fax: þ52 81 81 904035. E-mail address: [email protected] (E.I. Cortés-Gutiérrez). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.10.001
organized in a specific manner that maintains the chromatin in the nucleus in a compact and nearly paracrystalline condition that is at least six times more condensed than the chromatin in mitotic chromosomes [3,4]. Another characteristic of sperm DNA structure, as reported in human and mouse, is its extreme sensitivity to incubation in alkaline denaturing agents [5–7]. The denatured DNA motifs on the genome achieved after alkaline incubations are known as alkali-labile sites (ALS). A new procedure now exists to quantify putative DNA breaks and ALS in situ within single cells, with the added advantage that it can also be used to scan the whole genome or specific DNA sequences. This protocol, known as DNA breakage detection (DBD)-fluorescence in situ hybridization (FISH), uses sperm cells that have been embedded within an inert agarose matrix on a specifically prepared microscope
322
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slide [8]. The cells are then lysed to remove membranes and proteins and the resultant nucleoids are exposed to a controlled denaturation step using alkaline buffers. The alkali gives rise to single-strand DNA (ss-DNA) stretches starting from the 50 -30 free DNA ends or from highly sensitive DNA motifs to alkaline conditions. These ss-DNA threads can then be detected using hybridization with specific or whole-genome fluorescent DNA probes [9,10]. As DNA breaks increase in a target region, more ss-DNA are produced and more DNA probes are hybridized; this results in a more intense FISH signal as additional ss-DNA is produced. The resulting hybridized signal on the whole genome can be quantified using image analysis systems [8,9,11]. The DBD-FISH signal obtained in the absence of exogenous DNA-damaging agents reflects the background level of ALS present in a genome [10]. It is noteworthy that when a whole-genome probe is applied to somatic cells, the background DBD-FISH signal is not homogeneous, and certain chromatin regions result as more strongly labeled. In human leukocytes, the more intense background DBD-FISH areas visualized with a whole-genome probe correspond to areas containing highly repetitive five-base pair satellite DNA sequences [12]. In mouse splenocytes, the background areas correspond to repetitive DNA satellite sequences in pericentromeric regions [13], and in Chinese hamster ovary cell lines derived from Chinese hamster cells, these corresponding to pericentromeric interstitial telomeric-like DNA sequences [14]. The role of ALS in chromatin packaging is exemplified in human spermatozoa. Unlike leukocyte DNA, certain repetitive satellite DNA sequences and the whole genomes of spermatozoa are extremely alkali-sensitive, and the signals produced with a whole-genome probe used for DBD-FISH were 12.7 times more intense than the corresponding signal in peripheral blood leukocytes [15]. The aim of this study was to assess and compare the presence of constitutive ALS using a DBD-FISH protocol in sperm cells and peripheral blood leukocytes in Equus asinus and Equus caballus. In addition to gaining information about this unknown aspect of sperm chromatin organization in these species, we investigated any possible differences between closely evolutionarily and genetically related species. 2. Materials and methods 2.1. Animals Fresh spermatozoa from four different stallions (Equus caballus) and four donkeys (Equus asinus) were used in this study. The stallions were a pure Spanish breed, and the donkeys corresponded to a singular Spanish breed (Zamorano-Leones) that is at risk of extinction. The animals ranged in age from 2 to 4 years and the samples were obtained throughout the month of April during the breeding season. All of the animals belong to the Spanish Army and were localized at the Depósito de Sementales de Ávila in Spain. The animals are maintained in controlled feeding and housing conditions with regular veterinary supervision. All of them were clinically healthy at the time of ejaculate collection. Semen samples (one ejaculate per animal) were collected
using an artificial vagina, and only ejaculates with 100 106 spermatozoa per mL and 50% progressively motile sperm were included in the analysis. Somatic cells (leukocytes) were obtained from heparinized peripheral blood samples. 2.2. DNA breakage detection-fluorescence in situ hybridization DNA breakage detection-FISH in sperm and leukocytes was performed according to Fernández et al., 2005 [10]. Whole DNA probes were prepared from isolated DNA from the leukocytes and labeled using digoxigenin (Roche Diagnostics Corp., Indianapolis, IN, USA) according to the protocol described elsewhere [10]. When the DNA probes were prepared and for hybridization purposes, they were denatured at 70 C for 10 minutes and hybridized at 37 C overnight. DNA probe hybridization was performed in the donkey and stallion leukocytes and sperm cells, using similar stringency conditions for sperm cells and leukocytes. Slides were washed in 50% formamide/2X SSC (pH 7) for 5 minutes, and washed twice in 2X SSC (pH 7) for 3 minutes. A nonspecific antibody-blocking solution was applied for 5 minutes at 37 C before detection of the hybridized signal. The hybridized DNA probe was detected using 30-minute incubation with a fluorescein-labeled antidigoxigenin antibody (Roche Diagnostics Corp.). The slides were counterstained with propidium iodide (1 mg/mL) in Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA). 2.3. Localization of ALS To determine regional localization of ALS or the genome regions exhibiting highest sensitivity to alkaline denaturation, sperm was exposed to gentle protein depletion according to DBD-FISH previously used for leukocytes. In this case, dithiothreitol (DTT; 0.001 M) was added to the lysing solution for single-strand bond reduction. Alkali-labile sites in sperm head were referred to proximal (near the flagellum insertion of the sperm) or distal location (near the acrosomal region). 2.4. Comet assay The alkaline comet assay was basically similar to that conducted by Singh et al. [5]. Sperm-gel slides were prepared as described previously for the DBD-FISH test. The slides were immersed in two lysing solutions for 30 minutes each at room temperature. The first solution contained 0.4 M Tris-HCl (pH 7.5) and 1% SDS, and the second, 0.4 M Tris-HCl (pH 7.5), 1% SDS, DTT (0.001 M), and 0.05 M EDTA. The slides were washed in 1X Tris-borate EDTA (89 mM Tris, 89 mM boric acid, 2.5 mM EDTA, pH 8.3) buffer for 10 minutes and then treated with fresh alkaline solution (0.03 M NaOH, 1 M NaCl, pH 12.5) for 2.5 minutes to cleave the ALS. The slides were placed horizontally in an electrophoresis tray, which was filled with fresh alkaline electrophoresis solution (0.03 M NaOH, pH 13). Electrophoresis was conducted at 20 V for 20 minutes at room temperature. After electrophoresis, the slides were gently removed from the tray and washed with neutralizing buffer (0.4 M TrisHCl, pH 7.5) for 5 minutes. The slides were washed in
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distilled H2O for 5 minutes and then dehydrated in a sequential series of 70%, 90%, and 100% ethanol. Finally, the slides were stained with propidium iodide (2.5 mg/mL). 2.5. Fluorescence microscopy and digital image analysis All slides were analyzed using a digital image analysis platform based on a Leica DMLB fluorescence microscope equipped with three low-pass band filters for visualization of green, red, and blue fluorescence emissions. Images were acquired, using a Leica Digital DF-35 16-bit black-and-white charge coupled device, in 16-bit TIFF format. P The integrated density (ID ¼ number of pixel at the region of interest gray level values at each pixel obtained after background subtraction) was calculated using Leica QWin imaging analysis software, version 2.1. Fifty different cells per animal were measured in each experimental approach using an identical protocol of image thresholding. Migration of the DNA at the tail comet was measured using Leica QWin software. Migration distance of the comet tail was considered as the number of pixels from the physical center of the nucleioid to the end of the tail. To perform this experiment, sperm of both species were processed on the same slide in identical alkaline DNA denaturation and electrophoretic conditions. 2.6. Statistical analysis Statistical analysis was based on total cell number. The Student t Kruskal-Wallis test was used to investigate any differences of integrated density values among the four variables studied (sperm and leukocytes of donkey and stallion). A value of P < 0.05 was considered significant. All analyses were performed using SPSS for Windows v. 13.0 software (SPSS, Inc., Chicago, IL, USA). 3. Results When Equus somatic cells were processed within a DBD-FISH environment, the protein depletion produced with the alkaline lysing solution allowed detection of discrete quantities of ALS (Fig. 1A and B); in this case, hybridization of the DNA probe was visible as nonclustered but dispersed series of hybridized signals on each of the nuclei (yellow in Fig. 1A and B). In the sperm, the use of lysing solution, with the addition of DTT to produce disulphide bonding reduction, facilitated protein depletion in the protaminized sperm chromatin, and large numbers of ALS was detected (Fig. 1C and D). The use of DTT is imperative for ALS visualization in the sperm. Equus sperm exhibited fluorescence signal intensity four times greater than that obtained in leukocytes after correction for ploidy level. In the case of stallion, ALS showed a tendency to cluster in 92% of spermatozoa (Fig. 1F). In contrast, 90% of spermatozoa in donkey presented two discrete clusters of hybridized signal (Fig. 1E). Both signals tended to be localized at the equatorial-distal regions of the sperm. The presence of ALS in the sperm was 1.3 times higher in donkey than in stallion (compare Fig. 1C and D) (Table 1), and the differences were statistically significant (Table 1). However, the amount of ALS in leukocytes
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detected using DBD-FISH was similar for both species (Table 1) (Fig. 1A and B). The comet assay performed in alkaline conditions revealed that all spermatozoa showed the presence of a comet. In this case, the length of the comet tail obtained in donkey sperm was 1.6 times longer than that observed in stallion (P < 0.05) (Table 1, Fig. 1G and H). 4. Discussion The results obtained in this study show that ALS in the spermatozoa of equids are highly represented, especially when compared with those observed in some somatic cells. Additionally, ALS appear to be constitutively present in each cell type, but could be considered a transient chromatin structure during cell development, in which each differentiated cell would exhibit its own pattern of ALS concentration and localization [15]. Interestingly, at least in spermatozoa, they might also differ for genomes of closely related species. The molecular nature of the constitutive ALS is not well established. The classic concept of ALS includes abasic sites, apurinic or apyrimidinic, deoxyribose moieties, or masked DNA breaks that can be easily denatured by exposing them to weak alkaline conditions. However, a permanent and extremely high local concentration of DNA breaks or abasic sites in the genome should not be compatible with life. This effect can be partially attenuated with strong association with the repetitive DNA sequences observed for these DNA regions; but, in any case, this leaves the massive and variable presence of these moieties in every analyzed genome unexplained [16]. The most reasonable hypothesis to assume regarding the role of these DNA sequences would be that they are involved in structural modifications linked to the particular requirements of the chromatin organization merited at each cell. Alkali-labile sites, in addition to being constitutive DNA motifs, might behave as the expression of pre-existing DNA lesions in the DNA molecule of different etiology [10]. When ALS are not the product of directional DNA damage and are present as a constitutive part of the chromatin structure, it appears assumable that they are differentially represented in each cell; this is because the functional-structural requirements of each cell are known to be different. In particular, this is the case of the spermatozoa. However, they are ubiquitously present in all analyzed genomes irrespective of the targeted cell [10]. Thus, DBD-FISH used on intact undamaged somatic cell nuclei allowed the identification of ALS in mouse major satellite DNA at pericentromeric chromosome regions [13]. In the Chinese hamster ovary cell line, the pericentromeric chromatin contains long interstitial telomeric-related DNA sequences as long tandem repeats, and a similar situation was reported in the orthopteran Pyrgomorpha conica. In both cases, both genome domains are highly enriched in constitutive ALS [14,17]. In human somatic cells, a part of the ALS is related to the five-base pair classical satellite DNA sequence areas also located at the pericentromeric chromosome regions, and in the constitutive heterochromatic Yqh region [12]. In mature human spermatozoa, the background fluorescence intensity of the DBD-FISH signal after hybridizing with the whole-genome probe
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Fig. 1. Localization of alkali-labile sites (ALS) in somatic and sperm in donkey (Equus asinus) (upper panels) and stallion (Equus caballus) (lower panels). DNA breakage detection-fluorescence hybridization in peripheral blood leukocytes and spermatozoa of donkey (Equus asinus) (A and C, respectively) and stallion (Equus caballus) (B and D, respectively). Localization of ALS in spermatozoa of donkey (Equus asinus) (E), and stallion (Equus caballus) (F). Visualization of the ALS as a structural feature of the spermatozoa after an alkaline comet assay test, (G) Equus asinus, and (H) Equus caballus.
was 12.7 times higher in spermatozoa than in leukocytes [5,15,18]. Because the general high density of constitutive ALS has only been reported in spermatozoa of a limited number of species [16], it is of interest to investigate whether these chromatin modifications occur in the sperm of other mammalian genomes, in that they are probably fully related to the singular chromatin arrangement inherent in these cells. This should provide additional information about the universal but distinctive presence of expanded ALS in these types of cells. In the case analyzed here, both species presented distinctive ALS in the spermatozoa and in peripheral blood leukocytes, but with certain distinctive features for each species when the target cell was the sperm. This result indicates that even relatively short phylogenetic distances between species might result in substantial differences in chromatin organization at the spermatozoa. The sensitivity to alkali achieved in the spermatozoa in comet assay conditions pointed to a correlation with the results obtained with the DBD-FISH procedure [15]. The largest displacement of DNA at the comet tail in donkey spermatozoa would explain the slight increase of
1.3 points in the hybridized signal after DBD-FISH. This result has been found for the large, pericentromeric interstitial telomeric-repeat sequence blocks observed in Chinese hamster cell lines [14]. Alkali-labile sites behave as unpaired DNA segments that could be a consequence of torsional stress of DNA loops by strong chromatin packing and could also be the reason that they are initially abundant in the chromatin of the condensed mitotic chromosome [19]. In fact, it has been demonstrated that when subjected to constant tension greater than a critical value, the DNA molecule begins to unwind [20]. The result obtained in pig sperm appears to be an exception to the rule of dense chromatin packing and the presence of ALS, although even in this case, these can be detected with trypsin pretreatment [16]. However, it is noteworthy that only particular and highly repetitive satellite DNA sequences are related to constitutive ALS, their being very scarce or absent in other repetitive DNA sequences within the same genome; i.e., tight chromatin packing does not necessarily imply the presence of ALS, although ALS appear to be preferential in strongly condensed chromatin [16].
Table 1 Fluorescence area values and length of the comet tail in sperm cells and lymphocytes of donkey (Equus asinus) and stallion (Equus caballus) after DBD-FISH. Species
Fluorescence Analysis N
DBD-FISH (ID) Sperm (mean SD)
Comet assay (LCT) Leukocytes (mean SD)
Sperm (mean SD)
6910.7 1324.9 3467.8 754.4 102.50 10.12 3808.6 816.0 63.33 7.96a 5184.6 1095.8a P N represents the number of studied cells. ID (integrated density) ¼ ( number of pixels at the region of interest gray level values at each pixel obtained after background subtraction) segmented area of interest gray level values. Abbreviations: DBD-FISH, DNA breakage detection-fluorescence in situ hybridization; LCT, length of the comet tail. a Student t Kruskal-Wallis test: significant differences, P < 0.05. Equus assinus Equus caballus
200 200
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4.1. Conclusions Localization of hybridization signals in stallion and donkey sperm genomes is not randomly distributed and tends to be present at equivalent regions in the sperm, with the exception of there being a tendency toward ALS clustering in the case of stallion. This fact is probably informing concerning differences in the topology of the chromatin packing that might exist between both subspecies. In conclusion, the result obtained in this experiment show that ALS are represented in stallion and donkey genomes, although these exhibited differences when leukocytes and spermatozoa were compared. Differences between both species were also found when the sperm of both species were probed with DBD-FISH and comet assay. The fact that these differences were found in closely related species indicates that substantial differences in chromatin organization could be present in equivalent cells such as spermatozoa. More intense investigation using ALS as easy chromatin domains to be identified at the cellular level might provide valuable knowledge in terms of different aspects of the yet obscure sperm chromatin organization.
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