Department of Obstetrics & Gynaecology, The Queen's University of Belfast, Northern Ireland
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Abstract |
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Key words: apoptosis/CASA/COMET assay/mitochondria/TUNEL assay
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Introduction |
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One factor which remains of the utmost importance for ICSI is sperm DNA integrity. Good quality sperm DNA is essential for the accurate transmission of genetic material to the next generation. Poor DNA may not necessarily prevent fertilization from occurring and it has been shown that genetically damaged human spermatozoa are still able to form normal pronuclei in oocytes after ICSI (Twigg et al., 1998). Therefore, fertilization is not necessarily a barrier to damaged DNA and the first indication of a DNA problem may be the occurrence of congenital abnormalities in offspring.
Fragmentation of genomic DNA is one of the hallmarks of apoptosisthe most common form of eukaryotic cell death. Apoptosis, programmed cell death, occurs under normal physiological conditions and proceeds in two main phases. The first is a commitment phase followed by an execution phase which is characterized by a series of stereotypic changes including cell shrinkage, plasma membrane disruption, phosphatidylserine externalization and condensation and fragmentation of chromatin (Wyllie et al., 1980; Earnshaw, 1995
; Martin et al., 1995
). A characteristic feature of this process is activation of an endogenous endonuclease which generates numerous DNA strand breaks in chromatin (Wyllie et al., 1980
; Arends et al., 1990
; Compton, 1992
).
Apoptosis differs from necrosis in that the cell plays an active role in its own destruction. It has characteristic ultrastructural features (Kerr et al., 1972; Wyllie et al., 1980
) and biochemical changes (Wyllie, 1980; Williams and Smith, 1993
) and is dictated by precise genetic and molecular induction (Williams, 1991
; Boise et al., 1993
; Oltvai et al., 1993
; Williams and Smith, 1993
).
Since transcription and translation do not occur in human spermatozoa, therefore alternative theories have been proposed as to how apoptosis may occur in spermatozoa (reviewed by Sakkas et al., 1999a). One theory is that DNA fragmentation could be due to flaws in endogenous endonuclease activity resulting in DNA nicks (McPherson and Longo, 1992, 1993a
, b
; Sakkas et al., 1995
). Another is that apoptosis in spermatozoa is mediated through a cell surface protein, Fas (Suda et al., 1993
; Lee et al., 1997
).
A number of the crucial events in apoptosis commence in the mitochondria (Zamzami et al., 1995; Liu et al., 1996
; Petit et al., 1996
; Li et al., 1997
). Mitochondria ensheath the midpiece of the spermatozoa and deliver adenosine triphosphate (ATP) to the axenome where it is utilized for flagellar propulsion. These organelles are required for efficient energy metabolism, production of membrane lipids and cell growth but are also the primary determinants of cellular life or death (Arends and Wyllie, 1991
).
The involvement of mitochondria in apoptosis may centre on a number of different mechanisms (Zhuang et al., 1998a; Dinsdale et al., 1999
; Finucane et al., 1999
). These include the activation of the family of cysteine proteases known as caspases, e.g. cytochrome c and disruption of the electron transport chain leading to changes in oxidative phosphorylation and ATP synthesis (reviewed by Green and Reed, 1998). Other mechanisms include loss of mitochondrial membrane potential; variations in cellular reductionoxidation (redox) potential and involvement of pro- and anti-apoptotic family proteins, e.g. Bcl-2. Although apoptosis is not dependent on oxidative phosphorylation or the presence of mitochondrial DNA (Jacobson et al., 1993
), mitochondria have been verified as the co-ordinators of apoptosis in numerous cell systems (Frade and Michaelidis, 1997; Kroemer, 1997; Zhuang et al., 1998b; reviewed by Green and Reed, 1998)
Apoptosis can be detected in somatic cells by electrophoresis of DNA fragments to produce a characteristic `ladder' pattern (Wyllie et al., 1980) due to endonuclease cuts in the linker section of the helix. However, this kind of analysis is not possible for spermatozoa due to the presence of protamines. Other methods to detect apoptosis include in-situ end labelling involving the incorporation and detection of biotinylated nucleotides at the 3'-OH ends of DNA strand breaks (Gavrieli et al., 1992
; Wijsman et al., 1993
; Nishikawa and Sasaki, 1995
). One such assay is the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end labelling (TUNEL) assay.
The DNA status of an individual spermatozoon can be determined using a modified alkaline single cell gel electrophoresis (Comet) assay (Hughes et al., 1998; Donnelly et al., 1999
) where fragmented strands of DNA are drawn out by electrophoresis to form a comet `tail' leaving a `head' of intact DNA. Intact and damaged DNA is quantified using epifluorescence microscopy and image analysis.
The aim of the current study was to determine the nuclear DNA integrity and the level of DNA fragmentation in neat and prepared human spermatozoa using both Comet and TUNEL techniques and to determine if any correlation exists between the two methods. Functional integrity of sperm mitochondria was also determined and the results compared to those obtained for integrity of nuclear DNA.
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Materials and methods |
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Preparation of samples
Samples were prepared using a two-step discontinuous Percoll gradient (95.047.5%; Pharmacia Biotech AB, Uppsala, Sweden). Each aliquot of liquefied semen was layered on top of the gradient and centrifuged at 450 g for 12 min. The resulting sperm pellet was concentrated by centrifugation at 200 g for 6 min. The final sperm preparation was suspended in a suitable volume of Biggers, Whitten and Whittingham medium (BWW; Biggers et al., 1971) supplemented with 600 mg albutein (Alpha Therapeutic UK Ltd, Thetford, Norfolk, UK).
Determination of sperm motility parameters
Semen and post-preparation samples were analysed using 20 µm depth Microcell counting chambers (Conception Technologies Inc., La Jolla, CA, USA). Sperm motility parameters were measured at 37°C using a HamiltonThorne Integrated Visual Optical System (IVOS) Sperm Analyzer (Version 10.7; HamiltonThorne Research, Beverly, MA, USA). The settings employed for analysis were from acquisition rate (Hz) 50; minimum contrast 7; minimum size 6; low-size gate, 0.4; high-gate size, 1.6; low-intensity gate. 0.4; high intensity gate, 1.6; magnification factor, 2.04.
The motility parameters measured were: rapid progressive motility (those spermatozoa which exhibit an actual space-gain motility); straight line velocity (VSL; the straight line distance from beginning to end of a sperm track divided by the time taken); average path velocity (VAP: the average velocity of sperm movement; spermatozoa were counted as exhibiting rapid progressive motility if VAP >25 µm/s); curvilinear velocity (VCL; a measure of the total distance travelled by a given spermatozoon divided by the time elapsed); sperm head movements, i.e. the amplitude of lateral head displacement (ALH; the mean width of sperm head oscillation); and beat cross frequency (BCF; the frequency of the sperm head crossing the sperm average path).
Evaluation of sperm morphology
Sperm morphology was assessed in semen using strict criteria (Kruger et al., 1987). The sample (5 µl) was evenly spread along the length of a microscope slide which had been thoroughly cleaned with 95% v/v industrial methylated spirit (Adams Healthcare, Leeds, UK). The resulting thin smear was air-dried for 20 min before staining with a Diff-Quik staining kit (Baxter Dade Diagnostics AG, Dubingen, Switzerland). Stained slides were air-dried for 30 min and large (22x50 mm) coverslips were applied, in a fume cupboard, using Shandon synthetic toluene-based mounting medium (Shandon Inc., Pittsburgh, PA, USA). Morphological assessment was performed at x1000 magnification under oil-immersion and at least 100 spermatozoa were counted on each slide. Results were expressed as the percentage of normal spermatozoa observed on each slide.
In order to be classified as normal by strict criteria, a spermatozoa must have a smooth, oval configuration with a well-defined acrosome incorporating 4070% of the sperm head, no neck, midpiece or tail defects, and no cytoplasmic droplets more than one-half the size of the sperm head. Head defects were subdivided into amorphous, megalo, small, loose head or duplicated. Midpiece defects included all midpiece defects and cytoplasmic droplets, and tail defects included coiled and duplicated tails. Spermatozoa with borderline morphologies were counted as abnormal.
Determination of DNA integrity using a modified alkaline single cell gel electrophoresis (Comet) assay
The following procedure [adapted from Hughes et al. (1998) and Donnelly et al. (1999)] was carried out under yellow light to prevent further induced damage to DNA.
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, Poole, Dorset, UK) 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 spermatozoa 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.
The cells were then lysed by removing the coverslips and immersing the slides in a Coplin jar containing freshly prepared cold lysis 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. Slides were then incubated for 30 min at 4°C with 10 mmol/l dithiothreitol (DTT; Sigma) followed by 90 min incubation at 20°C with 4 mmol/l lithium diiodosalicyclate (LIS; Sigma) (Robbins et al., 1993) in order to decondense the DNA.
The 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 with the electrophoresis buffer at 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.
The DNA fragments were then separated by electrophoresis for 10 min at 25 V (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) each for 5 min. This removed any remaining alkali and detergents which would interfere with ethidium bromide staining. The slides were then drained before being stained with 50 µl of 20 µg/ml ethidium bromide (Sigma) and covered with a large coverslip.
The slides were viewed using a Nikon E600 epifluorescence microscope which 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 Komet 3.1 software (Kinetic Imaging Ltd, Liverpool, UK).
Measurement of DNA fragmentation using the TUNEL assay
Post-Percoll sperm preparations were adjusted to 30x106/ml and 10 µl was smeared onto a glass microscope slide coated with aminopropyltriethoxysilane (APES; Sigma) and allowed to dry at room temperature for 30 min. A small area of the smear was marked out using a diamond marker.
The cells were lysed and the DNA decondensed by immersing the slides in a Coplin jar containing freshly prepared cold lysis solution for 1 h at 4°C and incubation for 30 min at 4°C with 10 mmol/l dithiothreitol followed by 90 min incubation at 20°C with 4 mmol/l lithium diiodosalicyclate, as previously described.
Detection of apoptosis
TUNEL assay was performed using the in-situ Cell Death Detection Kit (Fluorescein) from Boehringer Mannheim (Mannheim, Germany). The reagents provided in the kit were diluted using 10 mmol/l levamisole (Sigma) to eliminate any possible alkaline transferase activity. In our experience the use of undiluted reagents resulted in non-specific labelling which made analysis extremely difficult. 50 µl of terminal deoxynucleotidyl transferase (TdT) from calf thymus (EC 2.7.7.31) in storage buffer [supplied in the kit as 5x50 µl aliquots (10xconc)]. One aliquot was diluted 1 in 50 with 10 mmol/l levamisole. The label solution, consisting of nucleotide mixture in reaction buffer, was supplied in the kit as 5x550 µl aliquots (1xconc) was diluted 1 in 5 with 10 mmol/l levamisole.
Diluted TdT (25 µl) was mixed with diluted nucleotide mixture (225 µl) to provide 250 µl of TUNEL reaction mixture. This was sufficient for 10 slides (25 µl per slide). For use as a negative control, nucleotide solution without TdT was included in all experiments and consisted of 25 µl of nucleotide solution without TdT.
Following cell lysis and decondensation of DNA, slides were rinsed twice with PBS and the area around the spermatozoa was dried gently with absorbent tissue. TUNEL reaction mixture (25 µl) was added to the spermatozoa on each slide and the area covered with a small (22x22 mm) coverslip to avoid evaporative loss. The slides were incubated in a humidified chamber at 37°C for 60 min, in darkness. The coverslips were then removed and slides rinsed three times with PBS.
Total numbers of spermatozoa and percentages of cells with fragmented DNA were determined by simultaneously analysing each microscope field using both using light microscopy and fluorescence microscopy. Slides were viewed using a Nikon E600 epifluorescence microscope which was equipped with an excitation filter of 450490 nm from a 100 W mercury lamp and a barrier filter of 520 nm. At least 100 images were captured and analysed by an image analysis system using Fenestra software (Kinetic Imaging Ltd, Liverpool, UK).
Determination of functional integrity of sperm mitochondria
Functional integrity of sperm mitochondria, which may be indicative of apoptosis, was detected using an ApoalertTM Mitochondrial Membrane Sensor Kit (MitoSensor; Clontech Laboratories Inc., Palo Alto, CA, USA). This kit allows detection of changes in mitochondrial transmembrane potential which may occur during the early stages of apoptosis.
Spermatozoa were divided in aliquots into Eppendorf tubes (Sigma) and centrifuged at 200 g for 5 min to pellet the cells. The cells were then resuspended in 1 ml of MitoSensor incubation buffer, gently mixed and incubated under 5% CO2 at 37°C for 40 min in darkness. MitoSensor (1 µl) was then added followed by incubation at 37°C for a further 10 min.
The percentage of cells with dysfunctional mitochondria was determined by fluorescence microscopy: at least 100 spermatozoa were counted for each sample. Slides were viewed using a Nikon E600 epifluorescence microscope which was equipped with an excitation filter of 450490 nm from a 100 W mercury lamp and a barrier filter of 520 nm. In healthy cells, MitoSensor is taken up by the mitochondria where it forms aggregates which exhibit intense red/orange fluorescence. In dysfunctional (possibly apoptotic) cells, MitoSensor cannot aggregate in the mitochondria due to alterations in membrane potential. The stain remains as a monomer in the cytoplasm where it fluoresces green.
Statistical analysis
Results were analysed using Statistica 5.0 (Statsoft of Europe, Hamburg, Germany). In view of the non-Gaussian distribution of data, the non-parametric Wilcoxon matched pairs test was employed to determine differences in DNA integrity and percentage of cells with fragmented DNA in neat and prepared samples from the same ejaculate. Stepwise linear regression was used to correlate results obtained from Comet and TUNEL assays and to correlate the percentage of spermatozoa with dysfunctional mitochondria with computer-assisted semen analysis (CASA) parameters and sperm morphology.
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Results |
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Discussion |
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The sperm chromatin structure assay (SCSA) which measures the susceptibility of DNA to heat- or acid-induced denaturation in situ has been shown to be effective in identifying fertility potential (Evenson et al., 1980). This technique has been used to demonstrate that spermatozoa with high levels of DNA fragmentation are less likely to achieve a pregnancy (Evenson et al., 1999
). Using selected threshold values for chromatin integrity, data from the SCSA allowed the prediction of seven out of 18 miscarriages where pregnancy had been achieved using spermatozoa with high levels of fragmented chromatin (Evenson et al., 1999
). In addition, it is known that a significant proportion of men who have a normal WHO semen profile and are from couples where infertility is unexplained, actually possess strand breaks in their sperm DNA (Høst et al., 1999
).
DNA fragmentation, which may be associated with apoptosis in human spermatozoa, may be connected with the cell surface protein Fas (Lee et al., 1997) which is a type I membrane protein known to mediate apoptosis (Suda et al., 1993; Krammer et al., 1994; reviewed by Sakkas et al., 1999a). Binding of the Fas ligand (FasL) or an antagonistic anti-Fas antibody to the Fas membrane protein kills the cell by apoptosis (Suda et al., 1993
). An increase in Fas positivity has been found in spermatozoa from infertile men, particularly in oligozoospermic samples (Sakkas et al., 1999b
) with as many as 50% of spermatozoa expressing high levels of Fas. This may indicate that these cells have been pre-selected to undergo apoptosis but have survived and reached the ejaculate. This may be due to errors in activation of apoptosis, a phenomenon that has been termed `abortive apoptosis' (Sakkas et al., 1999b
). Abortive apoptosis may be caused by problems in the synchronization of apoptosis and spermiogenesis that would allow spermatozoa to proceed to maturity despite the initiation of an apoptotic process (Sakkas et al., 1999a
). Thus the correlation between abnormal semen parameters and high levels of sperm DNA fragmentation and possible apoptotic-appearing spermatozoa can be explained.
It may also be feasible that DNA fragmentation in ejaculated spermatozoa is a consequence of endogenous nicks resulting from incomplete endogenous endonuclease activity which creates and ligates DNA nicks during spermiogenesis (McPherson and Longo, 1992). Chromatin packaging in spermatozoa may sanction endogenous nuclease activity to create and ligate nicks that facilitate protamination (McPherson and Longo, 1992
, 1993a
, b
). It is possible that, having initiated apoptosis, a spermatid may abort the activation of endogenous endonuclease activity and/or incorporate errors into the ligation of DNA nicks (Sakkas et al., 1999a
). Endogenous nicks in sperm DNA may, therefore, be an indication of an incomplete maturation process during spermiogenesis.
Previously, methods such as conventional and CASA were an adequate means of determining the quality of a semen sample. CASA parameters, such as progressive motility and average path velocity, have recently been shown to provide a useful indicator of the potential of spermatozoa to achieve fertilization in vitro and pregnancy (Donnelly et al., 1998). The percentage of post-preparation morphologically normal forms (determined using the Tygerberg criteria; Kruger et al., 1987) is known to be strongly correlated with the likelihood of spermatozoa achieving pregnancy following IVF (Donnelly et al., 1998
). However, techniques such as CASA and determination of sperm morphology have become redundant in recent years and the relevance of these parameters has diminished with the introduction of ICSI.
In ICSI, spermatozoa can be used irrespective of motility and morphology. New tests such as Comet and TUNEL assays to determine sperm DNA integrity are therefore necessary. The reaction based on the terminal transferase assay is thought to allow the identification of apoptotic cells. It has been shown to distinguish DNA strand breaks in apoptotic cells from those in necrotic cells or from primary DNA strand breaks induced by ionizing radiation or DNA topoisomerase inhibitors (Gorczyca et al., 1993a,b
). The TUNEL assay is specific for double-strand breaks and does not detect single-stranded nicks (Didier et al., 1996
; Sakaki et al., 1997
; Shinoda et al., 1998
). Previous studies using the TUNEL assay (Gorczyca et al., 1993a
,c
; Sailer et al., 1995
) have shown a significant correlation between the percentage of spermatozoa which are susceptible to DNA denaturation in situ and the percentage of sperm labelling for strand breaks.
Recent research has suggested that spermatozoa may be able to undergo apoptosis as measured by the terminal uridine nick end labelling for the detection of free ends of DNA (Gorczyca et al., 1993c; Aravindan et al., 1997
; D'Cruz et al., 1998a
,b
; Lopes et al., 1998
). However, none of these studies demonstrated that sperm DNA was fragmented into nucleosomal fragments. Nonetheless, it is possible that sperm DNA is not cleaved into the 200 base pair fragments associated with apoptosis in somatic cells. Digestion of larger DNA regions would lead to destruction of chromosomes and result in DNA fragments between 20 and 100 kb (McCarthy and Ward, 1999
).
The Comet assay, which can detect DNA single- and double-strand breaks in situ has also been found to be relevant for the characterization of apoptotic cells. It is a simple, sensitive, reliable and reproducible method for the detection of DNA damage (Hughes et al., 1997). The ability of the Comet assay to quantify DNA strand breaks and alkali labile sites has been well documented (reviewed by McKelvey-Martin et al., 1993; Fairburn et al., 1995; Collins et al., 1997). It is also considered to be an extremely sensitive method for the detection and quantification of apoptotic DNA fragmentation in individual cells using an HL-60 cell line (Poe and O'Neill, 1997
). This sensitive technique can detect DNA fragmentation occurring in the apoptotic process as early as exposure of phosphatidylserine residues on the outer cell surface of lymphoid cells (Florent et al., 1999
).
The Comet assay has also been found to be more sensitive than standard DNA flow cytometry for the detection of early DNA fragmentation events occurring during apoptosis in Chinese hamster ovary (CHO) cells (Godard et al., 1999a). Accurate detection and quantification of later stages of apoptosis have been detected by omitting the electrophoresis step of the Comet assay protocol, a technique referred to as the `halo assay' (Godard et al., 1999a
,b
). The tail moment measurement from the comet assay is considered to be sufficient to distinguish between apoptotic and necrotic DNA damage in human lymphoid cells (Fairburn et al., 1996
).
Apoptosis may also be detected by the use of annexin V binding assays. Annexin V binds to phosphatidylserine (PS) residues. PS normally occurs on the inner cell membrane, but an early biochemical feature of apoptosis is the occurrence of PS on the outer surface, due to plasma membrane disruption. Comet, TUNEL and annexin V assays have been used to detect staurosporine-induced apoptosis in CHO cells (Godard et al., 1999b). Annexin V staining was observed after 1 h of treatment while the detection of DNA fragmentation using the Comet assay occurred after 3 h of treatment and after 6 h of treatment for the TUNEL assay (Godard et al., 1999b
).
The results from this study show that there was a significant correlation between DNA fragmentation quantified using the Comet and TUNEL assays. This is in agreement with previous research which found that when spermatozoa from aliquots of the same semen sample were analysed using Comet, TUNEL and SCSA assays, significant correlations between these techniques were observed (Aravindan et al., 1997).
In the current study DNA integrity of prepared spermatozoa, determined using both Comet and TUNEL assays, was found to be significantly greater than that of semen. Further, the percentage of spermatozoa with fragmented DNA and the degree of DNA fragmentation in prepared spermatozoa was significantly less than in semen. Semen is likely to contain a number of dead or dying spermatozoa with abnormal morphology which will be selected out by the sperm preparation procedure. These spermatozoa will be detected by both the Comet and TUNEL assays and will contribute to the higher percentage of DNA damage detected in semen compared to prepared spermatozoa using both techniques. Therefore, it appears that in addition to isolating cells with the best motility and morphology, sperm preparation by density centrifugation also eliminates dead and dying cells and isolates the spermatozoa with best DNA integrity for use in assisted reproduction techniques.
Our findings are in contrast to results from a recent study where DNA integrity of prepared spermatozoa was significantly impaired in comparison with spermatozoa in semen when evaluated by flow cytometry analysis of acridine orange-treated spermatozoa (Zini et al., 1999). The authors quote previous research by Aitken and Clarkson (1988) that spermatozoa were exposed to oxidative stress during the centrifugation steps of the preparation process as a possible explanation of their results. However, the centrifugal force employed in the preparation of the spermatozoa was considerably greater than that used in our current study (600 g compared to 400 and 250 g). Further, spermatozoa were centrifuged at this high speed for a longer time period than that used in our protocol (20 min compared to 12 min) and this may have contributed to the damage that was observed in DNA of prepared spermatozoa.
In addition to measuring apoptosis in nuclear DNA, the current study also measured functional integrity of sperm mitochondria. This allowed the detection of changes in mitochondrial transmembrane potential, which may occur during the early stages of apoptosis, in the organelles that are believed to be the primary determinants of cellular life or death. An ApoAlert mitochondrial membrane sensor kit was employed which contained the mitochondrial probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). This is a reliable fluorescent probe to assess mitochondrial membrane potential changes in intact cells (Salvioli et al., 1997). The ability of this stain to discriminate between mitochondria exhibiting high membrane potential from those having low to medium membrane potential provides a rigorous estimate of metabolic function (Garner et al., 1997
; Salvioli et al., 1997
).
Our results have shown that the percentage of cells with dysfunctional mitochondria, a possible indicator of apoptosis, in prepared spermatozoa was significantly lower than that for semen. Prepared spermatozoa also had a small percentage of cells where the mitochondria stained both red and green in the same cell. This may be an indication that these spermatozoa are going through a transition stage or may be in the very early stages of apoptosis; it may also be a result of non-specific staining by MitoSensor. In both semen and prepared spermatozoa the incidence of injury was greater in mitochondria than in nuclear DNA. This confirms that this damage, which may be due to apoptosis, is recognizable at an earlier level in mitochondria and is not being detected until a later stage in nuclear DNA.
There was a negative correlation between the percentage of spermatozoa with dysfunctional mitochondria and sperm progressive motility in both semen and prepared spermatozoa. There was also a strong negative correlation between CASA parameters and the percentage of spermatozoa with dysfunctional mitochondria in both sample groups. This indicates that sperm motility is associated with and possibly reliant on healthy mitochondria and that mitochondrial damage may result in a reduction in sperm movement. This provides a possible explanation for poor sperm motility in asthenozoospermic samples.
Our results do not show any significant relationship between the percentage of spermatozoa with dysfunctional, possibly apoptotic, mitochondria and morphology of the semen sample. Therefore it appears that damage, which may be indicative of the early stages of apoptosis, is detectable in mitochondria when there are no other indications such as significant changes in sperm morphology.
In conclusion, this study has demonstrated that DNA fragmentation, a possible consequence of an apoptotic mechanism, can be detected in nuclear DNA of ejaculated spermatozoa using both Comet and TUNEL assays. It may also be the case that sperm mitochondria undergo apoptosis and a mitochondrial marker appears to be a more sensitive and immediate indicator of this process. The exact mechanism of programmed cell death in spermatozoa remains to be elucidated. Apoptosis may be a means of eliminating potentially harmful mutations to prevent the transmission of these abnormalities to the next generation. However, with the extensive use of ICSI as a treatment for male factor infertility, much more research is required to further investigate apoptosis in ejaculated spermatozoa and determine the consequences of these gametes contributing their damaged DNA to subsequent generations.
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Acknowledgments |
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Notes |
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References |
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Submitted on January 13, 2000; accepted on April 1, 2000.