Department of Environmental Health Sciences, School of Public Health and Health Sciences, University of Massachusetts Amherst, Amherst, MA
BioTechniques, Vol. 58, No. 6, June 2015, pp. 293–300
The unique DNA packaging of spermatozoa renders them resistant to DNA isolation techniques used for somatic cells, requiring alternative methods that are slow and labor intensive. Here we present a rapid method for isolating high-quality sperm DNA. Isolated human sperm cells were homogenized with 0.2 mm steel beads for 5 min at room temperature in the presence of guanidine thiocyanate lysis buffer supplemented with 50 mM tris(2-carboxyethyl)phosphine (TCEP). Our method yielded >90% high-quality DNA using 3 different commercially available silica-based spin columns. DNA yields did not differ between immediate isolation (2.84 ± 0.04 pg/cell) and isolation after 2 weeks of homogenate storage at room temperature (2.91 ± 0.13 pg/cell). DNA methylation analyses revealed similar methylation levels at both time points for three imprinted loci. Our protocol has many advantages: it is conducted at room temperature; lengthy proteinase K (ProK) digestions are eliminated; the reducing agent, TCEP, is odorless and stable at room temperature; nucleic acids are stabilized, allowing storage of homogenate; and it is adaptable for other mammalian species. Taken together, the benefits of our improved method have important implications for settings where sample processing constraints exist.
There is a growing interest in elucidating the role of sperm genetics and epigenetics on reproductive success and the life-course trajectory of health outcomes of subsequent generations. Recent genetic studies have shown a positive association between germline de novo mutations and paternal age (1-4). Aberrations in sperm DNA methylation of imprinted genes (5-7) and epigenome-wide dysregulation (8-10) have also been reported among men with infertility issues such as low sperm count and sperm quality. Moreover, compelling animal data indicate that the epigenome of sperm harbors a legacy of environmental exposures that can influence offspring phenotype (11-13).
Our optimized protocol for isolation of sperm DNA utilizes bead-based homogenization to facilitate sperm cell lysis in concert with an odorless reducing agent, tris(2-carboxyethyl)phosphine (TCEP), to dissociate disulfide bonds without the use of proteinase K (ProK). The procedure is conducted at room temperature, and the nucleic acids are sufficiently stabilized to allow storage of homogenate for future DNA isolation. After the homogenization step, DNA can be extracted by silica-based spin columns for a total processing time of 15–20 min.
Spermatogenesis requires extensive epigenetic reprogramming during the progression from diploid spermatogonia to haploid spermatozoa and involves stage- and testis-specific gene expression and mitotic and meiotic divisions (14, 15). Extensive reorganization of chromatin structure occurs where 90% and 99% of histones are replaced by protamines in humans and mice, respectively (16, 17). During this protamine-histone transition, tight compaction of the sperm nucleus is achieved by the oxidation of cysteine-rich residues of protamines and the subsequent formation of disulfide bridges that link protamines together (18). This nuclear compaction is necessary for sperm motility and protection of the genome from oxidation within the female reproductive tract (19). Furthermore, the protamine-bound packaging of DNA precludes transcriptional activity and has been considered a nontraditional form of epigenetic regulation unique to sperm cells (20).
Their unique DNA packaging renders spermatozoa resistant to DNA isolation techniques used for somatic cells (21, 22). The development of efficient methods for isolating DNA from mammalian sperm has been a gradual process. All existing protocols use a combination of three components to gain access to sperm DNA: (i) detergents and/or chaotropic salts to facilitate cell lysis; (ii) proteinase K (ProK) to digest nuclear proteins; and (iii) reducing agents to break disulfide bonds between protamines. One such widely adopted method for the isolation of mammalian sperm DNA uses an ionic detergent, sodium dodecyl sulfate (SDS), ProK, and either dithiothreitol (DTT) or 2-mercaptoethanol (βME). Af ter overnight incubation at 55°C, DNA is isolated by ethanol precipitation (9) or silica-based spin columns (11). Other popular approaches utilize guanidine salts, such as guanidinium thiocyanate (GTC), as the cell lysis reagent. GTC is a chaotropic agent that disrupts cell membrane and organelles by solubilizing individual molecules or cellular structures, including separating nucleic acids from associated proteins (23). In addition, it is able to denature proteins, inactivate nucleases, and enhance ProK activity (23, 24). Bahnak et al. first reported a protocol incorporating GTC in a lysis buffer along with the ionic detergent, sarkosyl, and βME (21). However, this protocol required overnight incubations and time consuming CsCl ultracentrifugation for DNA isolation. More recently, this protocol has been modified to include ProK in the lysis buffer, which significantly reduced incubation time to 2 h and replaced lengthy CsCl ultracentrifugation with isopropanol precipitation of DNA, resulting in an 80% yield of sperm DNA (24, 25).
While the previous methods for sperm DNA isolation have progressed over time, they still have drawbacks. First the limited stability of DTT, βME, and ProK in aqueous solutions at room temperature requires fresh preparation of lysis buffers and involves long incubations ranging from 2 h to overnight at 56°C (9, 24-26). In addition, DTT and βME possess odors that may not be tolerated, especially in clinical settings. Finally, most protocols recover DNA from sperm lysate through ethanol precipitation, which increases processing time and may result in co-precipitation of proteins and/or ethanol carryover that may affect downstream applications.
Given the need for a simple and rapid protocol for sperm DNA isolation, we developed a novel approach for isolating high-quality sperm DNA. Our protocol incorporates a 5 min mechanical homogenization step in the presence of a guanidine-based lysis buffer and a thiol-free reducing agent, TCEP, to facilitate sperm cell lysis and dissociation of disulfide bonds without the use of ProK. Sperm lysate is then applied to silica-based columns for the isolation of high-quality DNA with > 90% yield. To further streamline our protocol, we use commercially-available reagents that are stable at room temperature. This method is likely to expedite genetic and epigenetic research of sperm in clinical settings as well as in other mammalian species. Methods Isolation of sperm cells
This study was approved by the Institutional Review Board at the University of Massachusetts Amherst (#2014–2337). All participants gave written informed consent and were required to have at least 48 h of abstinence prior to each donation. Five healthy male participants each donated multiple whole ejaculate samples throughout the course of the study. To remove somatic cell contamination, sperm cells were isolated using a continuous one-step 90% gradient (ART-2100 and ART-1006; Sage, Beverly, MA) per the manufacturer's protocol. Sperm pellets were washed once, re-suspended, counted on a hemocytometer using the average of eight grid areas, and visually inspected for somatic cell contamination. Isolated sperm from individuals ranged from 20 to 109 million cells. Cell lysis
Reducing agents TCEP (final concentration: 10–50 mM) (#77720; Pierce, Rockford, IL), DTT (final concentration: 150 mM) (#V3151; Promega, Madison, WI), or βME (final concentration: 2%) were added to Buffer RLT (#79216; Qiagen, Limburg, The Netherlands) to a final volume of 500 μL. Three different homogenization techniques were also evaluated: (i) sperm were pulse-vortexed in lysis buffer for 5 min, diluted 1:1 in nuclease free water, and then incubated with ProK (final concentration: 200 μg/mL) at 56°C for 2 h; (ii) sperm were pulse-vortexed in lysis buffer for 5 min, and lysates were loaded onto Qiashredder columns (#79656; Qiagen) and centrifuged for 2 min at maximum speed (≥17,000 × g); (iii) sperm cells were homogenized in the presence of lysis buffer and 0.1 g of 0.2 mm stainless steel beads (#SSB02; Next Advance, Averill Park, NY) for 5 min on a Disruptor Genie (#SI-238; Scientific Industries, Bohemia, NY). To ensure equal aliquots of sperm, resuspended sperm cells after gradient isolation were vortexed for 10 s between each aliquot as previously described (27). DNA isolation
Sperm DNA was extracted with three different commercially-available kits using modified protocols:
AllPrep DNA/RNA Mini Kit (#80204; Qiagen). Lysates were added to spin columns and centrifuged at 10,000 × g for 30 s to bind DNA. Subsequent washing steps followed the manufacturer's protocol. To elute, 50 μL of Buffer EB (preheated to 70°C) was added to the columns, which were incubated at room temperature for 3 min and then centrifuged for 1 min at maximum speed. This process was repeated twice for a total elution volume of 150 μL.
QIAamp DNA Mini Kit (#51304; Qiagen). Lysates were combined with equal volumes of Buffer AL and 100% ethanol, loaded onto the spin columns, and the columns were centrifuged at 6000 × g for 1 min to bind DNA. Wash and elution steps followed the manufacturer's protocol, including 3 separate 200 μL elutions to maximize yield.
Quick-gDNA MiniPrep (#D3025; Zymo, Irvine, CA). DNA/RNA Shield (#R1100; Zymo) and Quick gDNA Genomic Lysis Buffer (included in kit), were used for sperm lysis instead of Buffer RLT. Samples in the Genomic Lysis Buf fer were loaded onto the columns, while samples in DNA/RNA Shield were combined with 3 volumes of Genomic Lysis Buffer before being loaded onto spin columns. Samples were centrifuged at 10,000 × g for 1 min to bind. Wash and elution steps followed the manufacturer's protocol for a final elution volume of 100 μL.
DNA yields and quality were determined using the Nanodrop 2000 Spectrophotometer (#E112352; Thermo Scientific, Somerset, NJ). A total of 350 ng of genomic DNA (gDNA) was resolved on a 0.7% agarose gel at 100 V for 45 min, stained with 0.5 μg/mL ethidium bromide solution, and visualized on a BioDoc-It Imaging System (#97-0172-01; UVP, Upland, CA). Given that haploid cells are expected to contain 3 pg DNA per cell, DNA yields were calculated as the observed yield/expected yield based on cell count. The full protocol for DNA isolation is provided in the Supplementary Material. RNA isolation
Sperm cell lysate may be partitioned for the isolation of sperm RNA by adding 1:1 ratio of Qiazol and following the protocol of Goodrich et al. (28) starting at step 18 under section 3.1. DNA methylation analysis
DNA methylation analyses of imprinted genes were performed on Sequenom's (San Diego, CA) MassARRAY platform, which uses RNA base-specific cleavage (MassCLEAVE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for quantitative DNA methylation analyses of PCR-amplified bisulfite-converted DNA (29). Briefly, 7.5 ng of bisulfite converted DNA (#D5002, EZ DNA Methylation Kit; Zymo Research) was amplified with reverse primers containing a T7-promoter tag. Primers and PCR conditions are provided in Supplementary Table S1. After treatment with shrimp alkaline phosphatase to remove unincorporated dNTPs, amplification products were subjected to in vitro transcription and T-specific cleavage and were then analyzed by MALDI-TOF MS. The C/T changes introduced by bisulfite treatment are reflected as G/A changes on the T7-directed RNA transcript and result in a mass difference of 16 Da for each CpG dinucleotide. The level of methylation for each CpG unit was quantified using EpiTYPER (Sequenom, San Diego, CA) software and methylation across loci was calculated as the average methylation of individual CpG units. Results and discussion
To improve processing time and work flow, we exploited several areas in current protocols where considerable improvements could be achieved. Because recently published methods for sperm DNA isolation relied on user-prepared GTC-based lysis buffer and ethanol precipitation, we reasoned that commercially-available GTC lysis buffers could offer an effective alternative to user-prepared lysis buffers as well as provide optimal DNA binding conditions for silica-based spin columns, thereby avoiding ethanol precipitation. Similar to the reported yield of 80% in a recent GTC-based method with ethanol precipitation (24), treatment of sperm cells with diluted Buffer RLT, 150 mM DTT, and 200 μg/mL ProK for two hours followed by DNA isolation via AllPrep DNA columns resulted in a 79% yield (2.37 ± 10 pg/cell) (Figure 1A). As an alternative, we examined the utility of TCEP, an odorless, room-temperature stable, thiol-free reducing agent primarily used for protein biochemistry. We found that 50 mM TCEP resulted in no appreciable difference in DNA yield (2.32 ± 0.09 pg/cell) compared with 150 mM DTT (Figure 1A), indicating that TCEP is a viable alternative to thiol-based reducing agents for the isolation of sperm DNA.