1 INSERM EMI-U 00-18, Laboratoire de Biochimie et Biologie Moléculaire, 2 Laboratoire dHistologieEmbryologieCytologie, UF de Biologie de la Reproduction and 3 Service de Gynécologie, UF dAssistance Médicale à la Procréation, CHU dAngers, F-49033 Angers and 4 Biologie de la Reproduction, Pavillon de la Mère et de lEnfant, CHU de Nantes, BP 1005, F-44093 Nantes cedex 1, France
5 To whom correspondence should be addressed. e-mail: pareynier{at}chu-angers.fr
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Abstract |
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Key words: male infertility/mitochondrial DNA/real-time PCR/sperm
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Introduction |
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During the past few years, some evidence of mitochondrial involvement in male infertility has been found. First, male infertility, associated with asthenozoospermia (Folgero et al., 1993) or oligoasthenozoospermia (Lestienne et al., 1997
), has been reported in patients suffering from typical mtDNA diseases, involving point mutations or multiple deletions of mtDNA. Secondly, sperm have been shown to be particularly prone to develop deletions of mtDNA (Cummins, 1998
; Cummins et al., 1998
; Reynier et al., 1998
; St John et al., 2001
; OConnell et al., 2002a
,b). Some studies have shown that, in human sperm, these deletions are associated with a decline of motility and fertility (Kao et al., 1995
, 1998). Thirdly, a correlation has been found between the quality of the semen and the functionality of the respiratory chain in sperm mitochondria (Ruiz-Pesini et al., 1998
, 2000a; Hoshi et al., 2002
). Moreover, it has been shown that mtDNA point mutations, mtDNA single nucleotide polymorphisms and mtDNA haplogroups can greatly influence semen quality (Holyoake et al., 1999
, 2001; Ruiz-Pesini et al., 2000b
; Sutarno et al., 2002
). The high rate of deletions or substitutions observed in sperm could be due to impaired mitochondrial maintenance or result from the deleterious effects of oxidative stress. Recently, the nuclear encoded mitochondrial-specific DNA polymerase gamma (POLG) has been reported to be involved in male infertility (Rovio et al., 2001
). The absence of the most common allele of the POLG gene was found to be associated with a range of defects in sperm quality. The genotype of the nuclear encoded glutathione S-transferase M1, involved in reactive oxygen species detoxification, has also been associated with male infertility and mtDNA deletion (Chen et al., 2002
). Lastly, sperm treatment with extracellular ATP has been shown to induce a significant increase in the fertilizing potential of sperm, demonstrating the importance of the mitochondrial function in male fertility (Rossato et al., 1999
). In order to investigate the role of mtDNA in male infertility we have quantified sperm mtDNA by means of real-time PCR.
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Materials and methods |
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mtDNA/-globin real-time PCR quantification
The external standard used for mtDNA quantification was a 158 bp PCR product. The nucleotide positions of the primers on the light strand mtDNA (according to the Cambridge reference sequence) were: D41 (32543277) and D56 (31263147). PCR reactions were carried out under standard conditions with 100 ng of total DNA in a 50 µl volume: 1.5 mmol/l MgCl2, 75 mmol/l TrisHCl (pH 9 at 25°C), 20 mmol/l (NH4)2SO4, 0.01% Tween 20, 50 pmol of each primer, 200 µmol/l of each dNTP and 2 IU of GoldStar DNA polymerase (Eurogentec, Seraing, Belgium). Each of 35 cycles consisted of a denaturation step of 30 s at 94°C, a hybridization step of 30 s at 58°C, and an extension step of 1 min at 72°C. The PCR product was phenolchloroform purified from low melting point agarose and cloned using the Topo TA Cloning Kit (Invitogen, Life Technologies, Groningen, The Netherlands) into pCR 2.1-Topo® vector. The recombinant plasmid was purified using Qia Prep Spin Miniprep Kit (Qiagen, Courtaboeuf, France) and was quantified by spectrophotometry. Purification quality was checked by means of the 260/280 absorbance ratio, and the absence of residual bacterial DNA was checked by agarose gel electrophoresis. It was assumed that 1 µg of a 4066 bp product (vector 3908 bp and insert 158 bp) contained 2.2x1011 molecules. Serial dilutions were then made in order to assess several concentrations of a known number of templates. These serial dilutions were used as the external standard for real-time PCR. The serial dilutions were all stored at 20°C in single-use aliquots.
A Roche LightCycler was used to determine the mtDNA copy number using LightCycler-Faststart DNA master SYBR Green 1 kit (Roche). A total of 20 µl PCR reaction mixtures were prepared as follows: 1xbuffer containing 4 mmol/l MgCl2, 0.2 mmol/l dNTP, 0.5 µmol/l of both primers (D41 and R56), SYBR green I dye, 0.25 IU hot start Taq DNA polymerase and 10 µl of the extracted DNA or 10 µl of Standard with a known copy number. The reactions were performed as follows: initial denaturing at 95°C for 7 min and 40 cycles at 95°C for 1 s, 58°C for 5 s, and 72°C for 13 s. The SYBR green fluorescence was read at the end of each extension step (72°C). A melting curve (loss of fluorescence at a given temperature between 66°C and 94°C) was analysed in order to check the specificity of the PCR product. For each run, a standard curve (log of the initial template copy number on the abscissa, and the cycle number at the crossing point on the ordinates) was generated using five 10-fold serial dilutions (10100 000 copies) of the external standard (Figure 1). This curve allowed the determination of the starting copy number of mtDNA in each sample. All samples were tested twice.
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The calculation of mtDNA/-globin ratio was performed taking into account the volume of the extracted DNA used as a template for PCR amplification (10/200 µl for mtDNA PCR and 2/200 µl for
-globin PCR).
Statistical analysis
The various groups of sperm were compared using the non-parametric MannWhitney, KruskalWallis and Wilcoxon U-tests and the Students t-test with Systat software, version 8.0 (SPSS Inc., Chicago, IL, USA). Differences were considered significant when P < 0.05.
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Results |
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Discussion |
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The motile sperm extracted from normal sperm samples were found to contain only 1.4 mtDNA molecules on average. This means that the majority of sperm mitochondria are almost totally devoid of mtDNA, and that many sperm probably do not contain any mtDNA at all. It has been shown that the paternal mtDNA, found in the early embryonic stages, is rapidly eliminated, and we suggest that the lack of mtDNA content in some sperm with the best fertilizing ability may also explain why the mitochondrial genome is not paternally transmitted. The functionality of the respiratory chain must therefore be temporally maintained in mature sperm until fertilization, despite the quasi-absence of mtDNA in their mitochondria. Indeed, it has been shown that mtRNA transcripts remain highly stable, and that the translation of mtRNA into subunits of the respiratory chain continues actively in the mitochondria of sperm, despite the complete absence of mtDNA replication (Rantanen and Larsson, 2000).
Point mutations, deletions or haplogroups of mtDNA could be involved in male infertility. In the present report we show that highly significant mtDNA amplification was found in abnormal sperm, highlighting the multiple implications of mitochondria in male infertility. First, the mtDNA content of motile sperm was found to be up to 28 times higher in sperm samples of poor quality than in normal sperm samples. Secondly, sperm collected from the 40% layers (which contain a majority of abnormal sperm) were found to have an mtDNA content up to 70-fold greater than that of sperm collected from the 100% layers (which contain sperm with the best fertilizing ability). This mtDNA amplification could have two main causes. One could be a feedback process operating to compensate low respiratory chain activity, thus leading to an increase of mtDNA. Indeed, such compensatory processes of increased mitochondrial biogenesis are frequently observed in mitochondrial pathology. Another cause of mtDNA amplification could be the abnormal differentiation and maturation of sperm in infertile patients. In fact, mtDNA amplification was more significant in sperm samples with at least two abnormal WHO criteria, suggesting a global disorder of spermatogenesis. We therefore postulate that insufficient mtDNA copy number reduction may occur when the maturation of sperm is perturbed.
In conclusion, our study shows that sperm mtDNA is strongly amplified in sperm samples from infertile patients presenting abnormal sperm characteristics. This raises the question about the risks involved in the use of a number of methods of assisted procreation, such as ICSI and round spermatid injection. These techniques involve the treatment of extreme cases of male infertility by means of sperm that may be abnormal, and immature sperm or sperm precursors (such as spermatids and spermatocytes) obtained from testicular biopsies, all of which have a higher mtDNA content than mature sperm. Furthermore it has been shown that oocytes from aged women or from women with ovarian deficiencies may have defective mtDNA genetic filters (St John et al., 1997). Although the first studies on mtDNA inheritance after ICSI suggest that human embryos eliminate the mtDNA of the injected sperm (Danan et al., 1999
), one study has recently shown that abnormal paternal mtDNA transmission may not be uncommon when poor-quality gametes are used (St John et al., 2000b
). Thus, at present, it cannot be precluded that the intracytoplasmic injection of sperm with amplified mtDNA into defective oocytes may impair the mechanism of elimination of paternal mtDNA, thereby further jeopardizing embryonic development and mitochondrial inheritance.
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Acknowledgements |
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References |
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Submitted on September 9, 2002; accepted on November 11, 2002.