1 School of Medicine, Obstetrics & Gynaecology, Queen's University Belfast, Institute of Clinical Science, Grosvenor Road and 2 Regional Fertility Centre, Royal Maternity Hospital, Belfast BT12 6BJ, UK
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Abstract |
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Key words: electron transfer chain/long PCR/mitochondria/mitochondrial DNA deletions/nuclear DNA
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Introduction |
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In a previous study (Steele et al., 1999), it was shown that the nuclear (n) DNA from testicular sperm is less damaged than in epididymal sperm. As a result, we suggested that testicular sperm should be used in preference to epididymal sperm in ICSI treatments. However, the nucleus is not the only source of DNA. The mitochondrion (mt) also has its own genome. Until the introduction of ICSI, a spermatozoon with defective mtDNA was unlikely to penetrate the oocyte. This is because, in IVF or in vivo, sperm motility is one of the crucial characteristics of the successful spermatozoon and adequate motility requires functional mitochondria as the mitochondria produce the energy for movement by processes driven from their own genome.
Mutations in mtDNA have been implicated in a range of debilitating diseases (Wallace, 1993). Most are causally related to distinct neuromuscular and neurodegenerative diseases, although they have a perplexing raft of clinical symptoms. Point mutations can lead to myoclonic epilepsy with ragged red fibres (MERRF) and to mitochondrial encephalomyopathy (lactic acidosis), stroke-like episodes (MELAS). Large-scale rearrangements are associated with KearnsSayre syndrome (KSS), progressive external ophthalmoplegia (PEO) and other multi-systemic disorders (Lestienne, 1992
) such as Alzheimer's and Parkinson's diseases (Wallace et al., 1994
). More recently, mitochondrial mutations have been associated with defective sperm function, the commonest cause of male infertility (Lestienne et al., 1997
; St John et al., 1997
; Kao et al., 1998
).
MtDNA is believed to be strictly maternally inherited (Bourgeron, 2000). Sperm mitochondria are tagged with the protein ubiquitin. This tagging allows the embryo to recognize the paternal mitochondria and expel them by the 8-cell stage (Sutovsky et al., 1999
). However, paternal mtDNA has now been detected in normal blastocyst-stage human embryos (St John and de Jonge, 2000
). This new evidence suggests that they may avoid expulsion and become incorporated into the embryo at the expense of the offspring's health, both in the short and long term. Thus, it is important to know if the mtDNA in testicular sperm destined for ICSI deteriorates in the same way as nDNA, as sperm progress from the seminiferous tubules to the obstructed epididymis. In this study, we have compared the incidence and size of mitochondrial deletions in testicular and epididymal sperm from men with obstructive azoospermia by Long polymerase chain reaction (LPCR). The alkaline Comet assay has also been used to determine nDNA fragmentation, to assess if there is a relationship between nDNA and mtDNA quality in these sperm.
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Materials and methods |
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Epididymal and testicular sperm retrieval and sperm preparation (after Steele et al., 1999, 2000
)
Briefly, the spermatic cord was located and 10 ml of 0.5% bupivocaine injected around it. Five minutes later, the testis was firmly palpated to ensure numbness. The epididymis was then located and stabilized to allow a 21G butterfly needle to be passed slowly through its substance whilst continuous suction was applied from a 5 ml syringe. Once the flashback of epididymal fluid into the butterfly tubing was seen, the tubing was clamped distally and the needle removed from the epididymis. The fluid was then transferred into BiggersWhittenWhittingham (BWW; Biggers et al., 1971) media and spun at 110 g for 10 min to pellet the sperm. The supernatant was discarded and the pellet resuspended in BWW for analysis. A testicular biopsy was performed by passing a 14G Trucut biopsy needle (Baxter Healthcare Ltd, Thetford, Norfolk, UK) into the testis, advanced 1 cm and the biopsy specimen removed in the needle's specimen notch. The biopsy was then transferred into BWW medium.
Testicular sperm were retrieved from the seminiferous tubules by `milking' the tubular contents with size 5 jeweller's forceps into BWW, under a dissecting microscope. The contents of the seminiferous tubule were then centrifuged at 110 g for 10 min to remove debris and the sperm pellet was resuspended.
Determination of mtDNA mutations by LPCR
Sperm DNA isolation
A total of 1x106 cells were added to a 15 ml tube. This was centrifuged at 2000 g for 3 min to pellet the cells and the supernatant removed, leaving behind 200400 µl residual liquid. The tube was vortexed vigorously to resuspend the cells in the residual supernatant: this facilitates cell lysis. To the resuspended cells, 3.0 ml of cell lysis solution (Flowgen, Staffordshire, UK) was added and pipetted up and down to mix. Dithiothreitol (200 µl), (1 mol/l; Sigma-Aldrich Company Ltd, Poole, Dorset, UK) was added followed by 1.5 µl proteinase K (20 mg/ml; Sigma). This mix was inverted 25 times and incubated at 55°C overnight, until the cells had completely lysed.
RNase A solution (Flowgen), 15 µl was added to the cell lysate and the sample inverted 25 times to aid mixing before incubation at 37°C for 1560 min. The sample was cooled to room temperature (~22°C), and 100 µl of protein precipitate solution (Flowgen) was added to the RNase A-treated cell lysate; this was then vortexed to mix the sample. The sample was placed on ice for 5 min and then centrifuged at 2000 g for 10 min. The precipitated proteins formed a tight pellet; the supernatant containing the DNA was poured into a 1.5 ml Eppendorf tube containing 300 µl isopropanol (100%, Sigma). This was centrifuged at 2000 g for 3 min prior to mixing. The supernatant was poured off and the Eppendorf tube drained on clean absorbent paper. Ethanol, 2 ml (70%; Sigma) was added to the Eppendorf to wash the DNA pellet before being centrifuged at 2000 g for 3 min. The ethanol was poured off so as not to disturb the DNA. The Eppendorf tube was inverted and allowed to air dry for 15 min.
DNA hydration solution 400 µl (Flowgen, UK) was added to the DNA. This was heated at 65°C for 1 h, to rehydrate.
DNA calibration
Deionized and distilled water (DDW) (490 µl) was added to a 0.5 ml quartz cuvette (Sigma) and mixed with 10 µl of the hydrated DNA sample. The DNA quantity was calculated at 260 nm using a Ultrospec II, LKB Biochrom spectrophotometer.
8.7kb LPCR amplification
LPCR products were amplified in a Hybaid, TouchDown thermal cycling system (Hybaid Ltd, Middlesex, UK). LPCR using Bio-X-Act (Bioline, London, UK) was performed in 50 µl volumes. Each reaction contained 1xOptiform PCR Buffer (Bioline) 0.25 mmol/l dNTPs, 500 ng DNA template, 1.5 mmol/l MgCl2, 2 IU of Bio-X-Act (Bioline, London) and 0.5 µmol/l of each primer [D6: 5'TCT AGA GCC CAG CAC TGT AAA G 3' L strand sequence, position 82868304 and R10: 5' AGT GCA TAC CGC CAA AAG AAG A 3'-L strand sequence position 421403 (Lestienne et al., 1997)]. In brief, the steps consisted of initial denaturation at 94°C for 2 min, followed by 34 cycles of denaturation at 94°C for 10 s, annealing at 52°C for 30 s and extension at 68°C for 10 min. The `semi hot' technique was employed, i.e. the reaction tubes, with all the components present, were placed in the PCR machine at the start of the denaturation phase.
A total of 8.7 kb out of the 16.6 kb of the mitochondrial genome was amplified from whole sperm samples. This region was chosen as it encompasses the most heavily deleted region of the genome. The region amplified incorporates the following genes: complex I, the NADH dehydrogenase genes ND6, ND5, ND4, ND4L and ND3; complex III, cytochrome b; complex IV, the cytochrome c oxidase gene CO III; and complex V, the ATPase synthase genes ATPase 6.
In each set of reactions, one negative control and one positive blood control sample was run to determine whether mispriming of the multi-enzyme system had taken place. LPCR was repeated in the same samples to ensure reproducibility of the proportions of deleted mtDNA molecules and identical mutations were found.
The reaction products were electrophoresed on a 0.8% agarose, TBA (GibcoBRL, Life Technologies, 10X TAE Buffer, Scotland, UK) gel containing ethidium bromide (1 µg/ml) at 120 V for 60 min.
Determination of DNA integrity by modified alkaline single cell gel electrophoresis (Comet) assay
The following procedure (adapted from Hughes et al., 1997 and Donnelly et al., 1999
) was carried out under yellow light to prevent further induced DNA damage.
Embedding of sperm in agarose gel
Fully frosted microscope slides (Richardsons Supply Co. Ltd, London, UK) were gently heated, covered with 100 µl of 0.5% normal melting point agarose in Ca2+ and Mg2+ free phosphate buffered saline (PBS; Sigma) at <45°C and immediately covered with a large (22x50 mm) coverslip. The slides were placed in a chilled metal tray and left at 4°C for at least 30 min to allow the agarose to solidify. The coverslips were then removed and 1x105 sperm in 10 µl BWW were mixed with 75 µl of 0.5% low melting point agarose at 37°C. This cell suspension was rapidly pipetted on top of the first agarose layer, covered with a coverslip and allowed to solidify at room temperature.
Lysing of cells and decondensation of DNA
The coverslips were removed and the slides immersed in a Coplin jar containing freshly prepared cold lysing solution [2.5 mol/l NaCl, 100 mmol/l Na2EDTA, 10 mmol/l Tris; pH 10, with 1% Triton X-100 (Sigma) added just before use] for 1 h at 4°C. Subsequently, slides were incubated for 30 min at 4°C with 10 mmol/l DTT followed by a 90 min incubation at 20°C with 4 mmol/l lithium diiodosalicyclate (LIS; Sigma, Robbins et al., 1993).
Unwinding of DNA
Slides were removed from the lysis solution, DTT + LIS and carefully drained of any remaining liquid. A horizontal gel electrophoresis tank was filled with fresh alkaline electrophoresis solution (300 mmol/l NaOH, 1 mmol/l EDTA, pH 13.0: Sigma) at 1215°C. The slides were placed into this tank side by side, with the agarose end facing the anode, and covered with electrophoresis buffer to a level of ~0.25 cm above the slide surface. The slides were left in this high pH buffer for 20 min to allow DNA in the cells to unwind.
Separation of DNA fragments by electrophoresis
Electrophoresis was conducted for 10 min at 25V (0.714 V/cm) adjusted to 300 mA by raising or lowering the buffer level in the tank. After electrophoresis, the slides were drained, placed on a tray and flooded with three changes of neutralization buffer (0.4 mol/l Tris; pH 7.5; Sigma) for 5 min each. This removes any remaining alkali and detergents that would interfere with ethidium bromide staining. The slides were then drained and stained with 50 µl of 20 µg/ml ethidium bromide (Sigma) and covered with a large coverslip.
Image analysis
The slides were viewed using a Nikon E600 epifluorescence microscope that was equipped with an excitation filter of 515560 nm from a 100 W mercury lamp and a barrier filter of 590 nm. Fifty images were captured and analysed by an image analysis system using the programme Komet 3.1 (Kinetic Imaging Ltd, Liverpool, UK).
Statistical analysis
MtDNA and nDNA results were analysed using Statistica 5.0 (Statsoft of Europe, Hamburg, Germany). The non-parametric Wilcoxon test was employed to determine the differences between testicular and epididymal sperm. One-way analysis of variance (ANOVA) was used to determine the differences between the number of mitochondrial deletions detected (total number of bands detected from LPCR amplification/patient) and the mean size of the deletions (total deletion size divided by deletion number/patient) between testicular and epididymal sperm.
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Results |
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Nuclear DNA fragmentation of testicular and epididymal sperm
The degree of DNA fragmentation in testicular sperm (16%, 2111%) was, as previously reported (Steele et al., 1999), significantly lower (P < 0.01) than that of epididymal sperm (26%, 3121%) (Figure 4
).
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Discussion |
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Since ICSI facilitates fertilization by sperm that would be rejected in the natural process, it is important to assess the quality of paternal genetic material and to establish criteria by which to choose appropriate cohorts of sperm. It has been previously demonstrated (and confirmed in this study) that testicular sperm nDNA is significantly less fragmented than nDNA from proximal epididymal sperm in men with obstructive azoospermia (Steele et al., 1999). As a result, it was suggested that testicular sperm should be used in preference to epididymal sperm for ICSI. In agreement, Palmero et al. reported higher fertilization and pregnancy rates with testicular than with epididymal sperm from men with acquired obstructions (Palmero et al., 1999
). Conversely, Meniru et al. found no differences in pregnancy rates when using epididymal or ejaculated sperm for ICSI (Meniru et al., 1998
). Clinically, testicular sperm extraction (TESE) has not been as popular as percutaneous epididymal sperm aspiration (Silber et al., 1995
) because of problems with sample processing and poor sperm yield. However, using our technique of TESE by a Trucut needle followed by milking of the seminiferous tubules, both high yields of sperm (5x1035x105) and patient acceptability are achieved (Steele et al., 2000
).
This is the first report of mtDNA deletions and their relationship to nDNA in testicular and epididymal sperm from the same patients. The importance of healthy mitochondria is increasingly recognized in sperm (St John et al., 1997, 2001
) and, conversely, defective mitochondria have been associated with male infertility (Cummins et al., 1994
, 1998
; Cummins, 1997
). Mitochondria are crucial organelles in sperm. They are uniquely localized in the midpiece in order to provide energy quickly and effectively for sperm motility. In conjunction with glycolysis, mitochondria facilitate the spermatozoon's rigorous demands for energy (Wallace et al., 1994
) by oxidative phosphorylation (OXPHOS) via the electron transport chain (ETC). This ETC is made up of subunits that have been synthesized by the mtDNA except complex II, which is encoded in the nucleus. Primers were used in the present study (see Materials and methods) to probe the region located between the D loop and Cox II gene, since this region contains the complexes involved in oxidative phosphorylation such as cytochrome C oxidase, ATPase, NADH dehydrogenase, cytochrome b and up to nine transfer RNAs. Therefore, large or multiple deletions in this region would indicate major disruption to the ETC. This would be expected to culminate in diminished motility.
Here, we have found that there is a significantly higher proportion of men with homoplasmic wild type (WT) mtDNA in their testicular sperm (45%) than in their epididymal sperm (16%). We have also observed similar findings (M.O'Connell, unpublished results) in the sperm of fertile men. Although there is much less damage than in epididymal sperm, it is surprising to see such a high incidence of abnormality in sperm still in their testicular environment. These levels of deletions are much higher than those commonly observed in somatic cells (Cooper et al., 1992; Lestienne, 1992
: Liu et al., 1998
). It may be that in the evolution of the species, if the paternal mtDNA is to be expelled from the embryo at an early stage (Sutovsky et al., 1999
), some of the mechanisms that are present in the oocyte to protect its mtDNA have become obsolete in its male counterpart during sperm differentiation (Reynier et al., 1998
), allowing mtDNA mutations to accumulate in sperm from both fertile and infertile men from spermiogenesis to ejaculation. However, the importance of non-mutated mtDNA in sperm cannot be disregarded. Recent findings (St John and de Jonge, 2000
; St John et al., 2000
) contrast with a previous study (Sutovsky et al., 1999
), and provide compelling evidence for the persistence of paternal mtDNA well beyond the 8 cell stage and into the blastocyst stage of normal and abnormal embryos created by IVF and ICSI techniques. While it is reassuring to note from some studies (Torroni et al., 1998
; Danan et al., 1999
) that the ICSI procedure did not alter the uniparental pattern of mtDNA inheritance, these studies were performed on children conceived using mature, i.e. ejaculated sperm. As yet, there are no studies to confirm that mechanisms for paternal mtDNA expulsion are not compromised when immature sperm are used.
It was found in this study that epididymal sperm have mtDNA with extensive damage in the form of multiple, large scale deletions. These sperm had been aspirated from the proximal epididymis in order to retrieve the healthiest sperm from this source. We know that even the proximal epididymis will contain substantial proportions of dead and dying sperm (Steele et al., 1999). However, sperm from the distal portions of the chronically obstructed epididymis are completely immotile (Silber et al., 1995
) as a result of either the activity of proteolytic enzymes such as procathepsin L, a lysosomal precursor of a highly active protease secreted in the distal caput of the epididymis (Dacheux et al., 1998
), or senescence. Their immotility may also be caused by this extensive and irreparable mtDNA damage. Mitochondrial DNA is also affected by a range of conditions from temperature fluctuations to transit times and enzyme additions such as hydrolysing enzymes; hyaluronidase and acrosin from dead and dying sperm acrosomes (Cummins et al., 1994
). As the numbers of degenerating sperm in the obstructed epididymis are increased compared with the normal epididymis, it may be that the epididymal fluid is unable to protect against the elevated concentrations of enzymes released from the acrosomes of these degenerating sperm (Moore, 1996
).
In addition to this hostile environment, the mtDNA of these epididymal sperm, like all sperm, lacks the protection of histones or DNA binding proteins (Shoffner and Wallace, 1994) and has only a very basic repair mechanism (Croteau et al., 1999
). It replicates rapidly, without a significant proofreading system, and has a mutation rate 10100 times higher than that of nDNA (Kao et al., 1995
, 1998
). All these characteristics make it susceptible to damage. The mitochondrial genome also differs from its nuclear counterpart in that it consists only of exons with no introns between genes. Therefore, every point mutation, or deletion, has the capacity to affect mitochondrial function.
To our knowledge, this is also the first study to demonstrate a relationship between nDNA and mtDNA in testicular and epididymal sperm. In somatic cells, there is a bi-directional flow of information between nucleus and mitochondrion. The genomes interact in the synthesis and assembly of mitochondrial proteins, particularly for OXPHOS (Poyton and McEwen, 1996). However, in post-spermiogenetic sperm where the nuclear genome is largely inactive, there is little evidence of cross-talk. Here we observed a close relationship between both the number and size of mtDNA deletions and nDNA fragmentation in sperm, suggesting that some interaction may still exist between the genomes so that malfunction of one is observed in the other. Alternatively, the assault on both mtDNA and nDNA may be so great that both respond similarly to their environment despite differences in how each genome is protected because in each, their defence is inadequate.
In conclusion, these data support previous work showing that mtDNA, as well as nDNA is of better quality in testicular sperm than in epididymal sperm. This confirms our belief that testicular sperm should be used in preference to epididymal sperm to treat men with obstructive azoospermia.
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Acknowledgements |
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Notes |
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Submitted on December 21, 2000; resubmitted on December 19, 2001; accepted on January 23, 2002.