Hum. Reprod. Advance Access originally published online on March 5, 2007
Human Reproduction 2007 22(6):1585-1596; doi:10.1093/humrep/dem030
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The expression of polymerase gamma and mitochondrial transcription factor A and the regulation of mitochondrial DNA content in mature human sperm
1 The Mitochondrial and Reproductive Genetics Group, The Medical School, University of Birmingham, Birmingham, UK 2 Center for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra, Coimbra, Portugal
3 To whom correspondence should be addressed at: The Mitochondrial and Reproductive Genetics Group, The Medical School, University of Birmingham, Birmingham B15 2TT, UK. E-mail: j.stjohn.1{at}bham.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
BACKGROUND: Human mitochondrial DNA (mtDNA) encodes 13 polypeptides of the electron transfer chain. Its replication is dependent on the nuclear-encoded polymerase gamma (POLG) and mitochondrial transcription factor A (TFAM). For POLG, only the polyglutamine tract, characterized by a series of CAG repeats, has been investigated in human sperm. However, TFAM is associated with the reduction in mtDNA content of testicular sperm. We have determined whether POLG and TFAM have functional roles in post-ejaculatory sperm mtDNA.
METHODS: Sperm samples were categorized as: normals, samples with one or two abnormal sperm parameters and oligoasthenoteratozoospermics (OATs). These were analysed by fluorescent PCR to determine the number of CAG repeats, real-time PCR for mtDNA copy number and immunocytochemistry and western blotting for patterns of expression for POLG, TFAM and the mtDNA-encoded COXI.
RESULTS: Only the OAT group presented with a significantly higher incidence of heterozygosity for CAG repeats, higher mtDNA content and a lower percentage of sperm expressing POLG and TFAM. Paradoxically, good-quality sperm had fewer mtDNA copies but significantly more sperm expressed POLG, TFAM and COXI.
CONCLUSIONS: Our data support the original findings that an association between sperm quality and POLG CAG repeats does exist. However, the biological significance of these variants in male infertility remains unclear, as these do not seem to affect mtDNA maintenance. The reduction in mtDNA content in normal samples likely reflects normal spermiogenesis, whereas increases in POLG and TFAM expression possibly compensate for the low mtDNA content, maintaining mitochondrial homeostasis.
Key words: mitochondria/mitochondrial DNA/mitochondrial transcription factor A/polymerase gamma/sperm
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Mitochondria are dynamic organelles that provide eukaryotic cells with energy. The number of mitochondria per cell is variable, depending on the cell's requirement for ATP (Moyes et al., 1998
). In mature mammalian sperm, there are between 22 and 75 mitochondria (Otani et al., 1988) per sperm localized to the midpiece, where they provide energy for several proposes, including flagellar propulsion, and thus sperm motility (reviewed in Turner, 2003). Mitochondria generate ATP through oxidative phosphorylation (OXPHOS), which takes place within the electron transfer chain (ETC) and is far more productive than anaerobic generation of ATP (Pfeiffer et al., 2001). The importance of OXPHOS-derived ATP for sperm motility has been demonstrated through classical mitochondrial inhibitor experiments (Ruiz-Pesini et al., 2000a).
The ETC is the only cellular apparatus that is encoded by both the chromosomal and mitochondrial [mitochondrial DNA (mtDNA), Anderson et al., 1981] genomes. Human mtDNA, which tends to be maternally inherited (Giles et al., 1980), is 
16.6 kb in size and encodes 13 of the polypeptides contributing to the ETC along with 22 tRNAs and 2 rRNAs (Anderson et al., 1981). Mutation, deletion or depletion to any of these genes is associated with a range of mitochondrial diseases that can both impair cellular function and/or can be lethal (Wallace, 1999).
A series of mtDNA rearrangements are associated with male infertility. For example, the maternally inherited A3243G point mutation has been identified as the cause of poor sperm motility (asthenozoospermia) in a male presenting with mtDNA disease (Spiropoulos et al., 2002). In a similar case, addition of metabolic supplements to bypass a Complex I point mutation increased sperm motility 3-fold (Folgero et al., 1993). Single large-scale mtDNA deletions have also been associated with male subfertility (Kao et al., 1995, 1998), although this association was not verified by others (Cummins et al., 1998; Reynier et al., 1998; St John et al., 2001). However, an accumulation of multiple deletions in poorer quality sperm samples has also been demonstrated (Reynier et al., 1998; St John et al., 2001), with a greater proportion of wild-type molecules being present in higher density gradient fractions than in lower density fractions (O'Connell et al., 2003).
It is evident that there is variability in the number of mtDNA molecules in different populations of human sperm with a tendency towards a greater proportion being present in poorer quality samples (Diez-Sanchez et al., 2003; May-Panloup et al., 2003). The vast range in content between these reports tends to be technique dependent, although one other study contradicts this trend but still reports significant differences between good- and poor-quality samples (Kao et al., 2004). However, these outcomes suggest that an association may exist between mtDNA copy number, sperm quality and regulation of mtDNA replication.
The replication of mtDNA is controlled by nuclear-encoded replication factors that are translocated to the mitochondria (Clayton, 1998). Both the mitochondrial-specific DNA polymerase gamma (POLG; Ropp and Copeland, 1996) and mitochondrial transcription factor A (TFAM; Fisher and Clayton, 1985; 1988) are required. POLG comprises two subunits: a catalytic subunit (POLG), with both polymerase and 3' 
5' exonuclease activity, and an accessory subunit (POLG2), which confers processivity. Mutations to POLG are associated with the accumulation of mtDNA deletions in diseases such as progressive external ophtalmoplegia (Van Goethem et al., 2001, 2003) and Parkinsonism and premature menopause (Luoma et al., 2004; Pagnamenta et al., 2006) and with mtDNA depletion syndromes such as Alper's disease (Naviaux and Nguyen, 2004; 2005; Nguyen et al., 2005; 2006).
Unlike other species, human POLG comprises a CAG-repeat region at its 5' end that gives rise to a polyglutamine tract (Ropp and Copeland, 1996). This region, usually 10 codons long (constituting the ‘common allele’), is also variable, with reports of alleles containing between 5 and 15 repeats (Rovio et al., 1999, 2001, 2004). The effect of such variability in sperm quality and function is unclear. In a wide-ranging study, a significant increase in the homozygous mutant genotype has been described in infertile men when compared with a fertile cohort (Rovio et al., 2001). Furthermore, a correlation between the lack of the common allele and idiopathic infertility (undefined fertility disorders but presenting with normal semen parameters) was identified in a Danish population (Jensen et al., 2004). However, analysis of an Italian (Krausz et al., 2004) and a French (Aknin-Seifer et al., 2005) cohort determined the frequency of the homozygous mutant genotype to be similar in both infertile and normozoospermic fertile men. Likewise, no significant differences in the frequency of each genotype were found in Italian men with low sperm counts (oligozoospermia) when compared with normozoospermics (Brusco et al., 2006). In a more recent study, the combined frequency of heterozygous and homozygous mutant POLG genotypes was higher in normozoospermic than in non-normozoospermic men (Harris et al., 2006). However, the functional significance of any of these genetic correlations has not been defined in sperm.
TFAM is a High Mobility Group (HMG) protein that activates mtDNA transcription in mammals (Parisi and Clayton, 1991; Garstka et al., 1994) and, consequently, the generation of the RNA primer necessary for the initiation of mtDNA replication (Chang et al., 1985; Xu and Clayton, 1996). Mouse knockout studies have suggested that TFAM is a regulator of mtDNA copy number with heterozygous knockouts displaying decreased mtDNA content and OXPHOS deficiency of the heart. Homozygous knockout embryos exhibited severe mtDNA depletion, loss of OXPHOS, and died post-gastrulation (Larsson et al., 1998). In the human, low levels of TFAM expression have been associated with mtDNA-depletion syndromes, such as infantile mitochondrial myopathy and encephalomyophathy disorders (Larsson et al., 1994; Poulton et al., 1994; Spelbrink et al., 1998; Siciliano et al., 2000). In human, mouse and rat, testis-specific TFAM mRNA transcripts result in down-regulation of TFAM protein levels in the mitochondria, as they either encode a nuclear protein isoform (mice: Larsson et al., 1996) or are not translated (human: Larsson et al., 1997; rat: Rantanen et al., 2001). This most likely accounts for the 10-fold reduction in mtDNA copy number that occurs during the latter stages of mammalian spermatogenesis (Hecht and Liem, 1984).
In the present work, we have determined whether POLG CAG repeats are associated with sperm quality in a series of patients classified according to their fertility status and whether CAG-repeat content can effect POLG protein expression. We have further sought to determine whether mtDNA copy number is uniformly regulated in those patients and whether there is a relationship between mtDNA copy number and the expression of POLG and TFAM. These outcomes were then assessed in terms of frequency of a mtDNA-encoded gene and its presence in varying populations of sperm.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Chemicals
All chemicals were supplied by Sigma Chemical Company (St. Louis, MO, USA), unless stated otherwise.
Biological material
All patients were recruited from the Fertility Clinic (University Hospitals of Coimbra, Portugal) and were undergoing routine semen analysis or fertility treatment involving both in vitro fertilization and intracytoplasmatic sperm injection. They signed informed consent forms, and all human material was used in accordance with the appropriate ethical and Internal Review Board (IRB) guidelines provided by the University Hospitals of Coimbra.
Fresh semen samples were obtained by masturbation after 3–5 days of sexual abstinence, and routine seminal analysis was performed according to the World Health Organization (WHO, 1999) Guidelines. In order to isolate sperm from both seminal plasma and round cells, semen samples were prepared by density gradient centrifugation using ISolate® Sperm Separation Medium (Irvine Scientific, Santa Ana, CA, USA), according to the manufacturer's protocol. Samples were categorized on the basis of sperm concentration, motility and morphology. For morphology analysis semen smears were prepared on microscopy slides and then treated with the Diff–Quick stain set (Dade Behring Inc., Newark, USA). Sperm morphology was assessed using the strict criteria, as described in Kruger et al. (1986). Samples were categorized as: normal (normozoospermic samples, i.e. concentration >20 x 106 sperm ml–1, >50% motile sperm, >14% normal forms); 1 or 2 defects (samples with one or two abnormal parameters) or oligoosthenoteratozoospermic (OAT), i.e. samples with low sperm concentration, motility and morphology), according to WHO (1999) criteria.
Extraction of total DNA
As samples were prepared using density gradient centrifugation, we were able to eliminate the possibility of contaminating somatic cells. However, before DNA extraction, the absence of contaminating cells was confirmed by light microscopy. DNA from isolated sperm samples was extracted according to the whole blood DNA isolation protocol using the Puregene DNA Isolation Kit (Flowgen, Nottingham, UK) supplemented with 1.5 µl of 20 mg ml–1 Proteinase K and 12 µl of 1 mol l–1 DTT and incubated overnight at 55°C, as described previously (St John et al., 2001).
Molecular analysis of POLG CAG repeats
Fluorescent PCR amplification of 287 bp of the POLG gene was performed in 50 µl reactions using primers and conditions as described previously (Jensen et al., 2004). The forward primer was labelled with a fluorescent tag. Each reaction contained 5 ng of total DNA, 1 x PCR buffer (BioLine, London, UK), 1.5 mM MgCl2 (BioLine), 200 µM dNTPs (BioLine), 125 nM of each primer (see Table 1) and 2.5 U BioTaq DNA Polymerase (BioLine). The size of the DNA fragments obtained was determined using a Capillary Electrophoretic Genetic Analysis System (CEQTM 8000, Beckman Coulter Inc, Fullerton, CA, USA). Mixtures containing 0.2 µl PCR products, 30 µl CEQ sample loading solution (Beckman Coulter Inc) and 0.8 µl CEQ 400-bp DNA size standards (Beckman Coulter Inc) were analysed. For conventional PCR, non-labelled primers were used. Reaction mixtures were similar to the ones used for fluorescent PCR, except for the amount of DNA (200 ng) and the concentration of the primers (0.5 µM each). PCR products were resolved on 3% agarose gels, and product size confirmed against a 100 bp DNA ladder (Bioron, Ludwigshafen, Germany). The PCR products were excised from the agarose gels and purified using the QIAquick Gel Extraction Kit (Qiagen, London, UK) as described in the manufacturer's protocol. The purified DNA was then sequenced using the automated direct sequencing protocol (Hopgood et al., 1992) using an ABI PRISM BigDye terminator v 3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA).
|
mtDNA/Beta-Globin quantification
For mtDNA quantification, an external standard of 152 bp PCR product was generated, using primers D41 and D56 (Reynier et al., 2001
; see Table 1). Reaction conditions were as follows: initial denaturation at 94°C for 3 min, 35 cycles of 94°C for 30 sec, 53°C for 30 s and 72°C for 1 min and final extension at 72°C for 3 min. To determine the number of sperm in each sample, quantification of the housekeeping gene Beta-Globin was also carried out. An external standard of 268 bp PCR product was generated, using primers previously described (Wykes and Krawetz, 2003; see Table 1) with the following reaction conditions: initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 30 s, 56.5°C for 30 s and 72°C for 1 min and final extension at 72°C for 5 min. For both genes, the 25 µl final volume reaction mixture contained 50 ng of total DNA, 1 x PCR buffer (BioLine), 1.5 mM MgCl2 (BioLine), 200 µM dNTPs (BioLine), 0.5 µM of each primer and 2.5 U BioTaq DNA polymerase (BioLine). Standards were purified using the QIAquick Gel Extraction Kit, as described above. After spectrophotometer quantification, a series of 10-fold dilutions were prepared to generate a standard curve for each gene. It was considered that the number of molecules of double-stranded DNA present in 1 ng of a 152 bp fragment (mtDNA) was 6 x 109 molecules, and that 1 ng of a 268-bp fragment (Beta-Globin) contained 3.4 x 109 double-stranded DNA molecules. Real-time PCR was performed using a 72-well Rotorgene-3000TM 4 Channel Multiplexing System machine and analysed with version 6 software (Corbett Research, Mortlake, NSW, Australia). PCR mixtures were prepared using a CAS-1200 Robotic Liquid Handling System (Corbett Robotics, Queensland, Australia). Reaction mixtures (15 µl final volume) contained 7.5 µl 2 x SensiMix (2 x SensiMix DNA Kit, Quantace, London, UK), 0.3 µl 50 x SYBR® Green solution (2 x SensiMix DNA Kit), 330 µM each primer (see Table 1) and 2 µl of each standard (range: 2 x 10–2 – 2 x 10–8 ng µl–1) or 2.5 ng µl– 1 DNA samples solutions. Each reaction was run in triplicate both for standards and samples, as well as for negative controls. An aliquot from a positive control was used in triplicate on each occasion to determine the repeatability of each reaction. Each reaction was repeated at least once.
For mtDNA, reactions were performed as follows: initial denaturation at 95° C for 10 min, followed by 45 cycles with denaturation at 95° C for 10 s, annealing at 53° C for 10 s and extension at 72°C for 15 s. For Beta-Globin, reactions consisted of an initial denaturation at 95° C for 10 min, 45 cycles with denaturation at 95°C for 15 s, annealing at 56.5° C for 15 s and extension at 72°C for 20 s. Data were acquired in the FAM/Sybr channel during the extension phase. For each of the 45 cycles, a step of 76°C for 15 s (for mtDNA) and 81°C for 20 s (for Beta-Globin) with data acquisition was added, to exclude fluorescence generated by potential primer-dimers. Melt curve analysis was performed by ramping from 65°C to 99°C at 1°C intervals and data acquired from the FAM/Sybr channel.
Only reactions with high efficiencies (>88%) were considered, and only standard curves with a Pearson correlation coefficient of at least 0.99 were taken into account. As each sample was run in triplicate on two separate occasions, six values were considered for each sample. To correct for pipetting errors, the highest and lowest values obtained were discarded, and the remaining four were averaged (Bustin, 2000). The standard deviation and coefficient of variation (CV) were also calculated and only values with low CVs (maximum 10%) were considered reliable.
POLG, TFAM, COXI and COXVIc detection
Immunocytochemistry
Immunocytochemistry (ICC) for individual proteins was performed separately using the following primary antibodies: rabbit anti-human POLG polyclonal antibody (ab2969, Abcam, Cambridge, UK), goat anti-human TFAM polyclonal antibody (sc-19050, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human COXI monoclonal antibody (A-6403, Molecular Probes, Eugene, OR, USA) and mouse anti-human COXVIc monoclonal antibody (A-6401, Molecular Probes). For secondary staining, Alexa Fluor 488 goat anti-rabbit immunoglobulin G (IgG), Alexa Fluor 488 donkey anti goat IgG and AlexaFluor 488 goat anti-mouse IgG (Molecular Probes) antibodies were used. A general procedure was performed as described previously (Ramalho-Santos et al., 2000) except for primary antibody incubation. Samples were incubated with the antibody solubilized in the blocking solution at 1:200, 1:50, 1:500 and 1:200 dilutions for POLG, TFAM, COXI and COXVIc, respectively, overnight at 37° C. Control experiments without primary antibodies were performed. Slides were examined using a Zeiss Axiophot II microscope equipped with a triple band pass filter, and 200 sperm per coverslip were counted in at least four different fields, in order to determine the percentage of stained sperm.
Sodium dodecyl sulphate–polyarylamide gel electrophoresis and western blotting
Sperm pellets were resuspended in lysis buffer (25 mM Tris, 1M NaCl, 1% Triton, 1 mM CLAP, 0.2 mM PMSF and 0.01 mM DTT) and briefly sonicated. After 1 h, the clear supernatants obtained following centrifugation were diluted in denaturing solution (1:2 dilution) and incubated at 95°C for 5 min. Samples were run on sodium dodecyl sulphate–polyorylamide gel electrophoresis and transferred to a PVDF membrane (Roche Diagnostics Corporation, IL, USA) overnight in transferring buffer [190 mM Tris, 25 mM glycine, 20% (v/v) methanol]. Membranes were blocked with Tris buffer saline medium with 0.1% (v/v) Tween 20 (TBST) containing 5% skimmed dried milk for 45 min and incubated overnight at room temperature with the primary antibodies described above, diluted in TBST. After washing five times for 5 min each in TBST, the blots were incubated for 1 h at room temperature with the appropriate (anti-rabbit, anti-goat or anti-mouse) alkaline phosphatase conjugated antibody (Amersham Biosciences UK Limited, Buckingamshire, UK), 1:20 000 diluted in TBST. The bands were developed using the ECF system (Amersham).
Statistical analysis
Statistical analysis was carried out using SPSS for Windows (version 11, Chicago, IL, USA). Pearson 
2-test was used to compare POLG CAG variants (allele and genotype frequencies) in the different groups defined. All numerical variables were checked for normal distribution using the one-sample Kolmogorov–Smirnov test. For ICC and mtDNA copy number analysis, the different groups were compared using the one-way analysis of variance test (or Kruskal–Wallis' non-parametric test when distributions were not normal, even after logarithmic transformation). Post Hoc analyses were done using Tukey's test or Dunnet's test, depending on whether equal variances were assumed or not. Pearson's and Spearman's tests, for parametric and non-parametric data, respectively, were applied to assess the correlations of the principal semen parameters (concentration, motility and morphology) with both ICC and relative mtDNA copy number results, as well as between ICC outcomes and relative mtDNA copy numbers. P < 0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
POLG CAG repeats polymorphisms
We have analysed the CAG repeat region of the POLG gene in groups of patients attending an infertility clinic, according to the WHO (1999)
criteria. These patients were categorized as normal (sperm with normal parameters), possessing 1 or 2 defects (sperm with one or two abnormal parameters) and OAT (samples with three abnormal parameters). Samples were examined by fluorescent PCR followed by fragment analysis. These outcomes were verified by conventional PCR and DNA-sequencing. CAG repeats ranging from 9 to 13 codons were identified. After the ‘common’ 10 repeat allele, the most frequent allele consisted of 11 codons (Table 2). The frequency of the 11 CAG-repeat allele was significantly higher (P < 0.05; Table 2) in the OAT group (21.4%) compared with both the normal group (12.5%) and the 1- or 2-defects group (8.2%). Likewise, the combined frequency of 
10 alleles (i.e. 9, 11, 12 and 13) was also higher (P < 0.05; Table 2) in the OAT group (23.2%) compared with the others (16.2 and 9.7% in the normal and in the 1- or 2-defects groups, respectively).
|
Combining the two alleles for each man allowed patients to be categorized into one of the three possible genotypes: homozygous wild type (10/10), heterozygous (10/
10) and homozygous mutant (10/10). Of 135 men, 96 (71.1%) were homozygous wild type and 39 (28.9%) were heterozygous. A different distribution of the genotypes was observed in the three groups analysed (Fig. 1), with heterozygosity being higher in the OAT group (46.4%) than in the normal and in the 1- or 2- defects groups (32.5% and 19.4%, respectively; P < 0.05). However, by comparing each of the three principal sperm parameters (concentration, motility and morphology) individually, we found no differences between the genotypes (P = 0.298 for concentration and P = 0.417 for motility and morphology; data not shown).
|
Assessment of mtDNA copy number
To determine whether sperm mtDNA was distributed evenly amongst sperm of differing qualities, we have quantified the number of copies of mtDNA per sperm from 42 samples comprising the three patient groups (14 normal, 14 with 1 or 2 defects and 14 OATs). The mean number of mtDNA copies per haploid genome was determined by the ratio of mtDNA to Beta-Globin molecules through real-time PCR. The mean (±SEM) mtDNA copy number for all samples analysed was 21.6 ± 5.8 (range: 0.2–206.1). Comparing the three groups individually (Fig. 2; Table 3), it is evident that mtDNA copy number per sperm increases as sperm quality decreases. To this extent, the normal group possessed 6.8 ± 1.8 mtDNA copies per sperm and the 1- or 2-defects group 11.3 ± 4.5, whereas the OAT group possessed 46.7 ± 15.0. The OAT group was statistically different to the normal group (P < 0.01) and 1- or 2- defects group (P < 0.05). Moreover, there was a significant negative correlation between the number of mtDNA molecules per sperm and sperm concentration (R = – 0.561, P < 0.001) and morphology (R = – 0.467, P = 0.002). The same was not verified for motility, although there was a tendency towards this (R = –0.285, P = 0.067).
|
|
MtDNA copy number and POLG CAG repeats
To determine whether the POLG CAG-repeat genotype influences sperm mtDNA content, we compared 38 samples, used to estimate the average mtDNA copy number in sperm, with their respective numbers of CAG repeats. Twenty two were homozygous wild type and 16 were heterozygous for POLG CAG-repeat variants. The mean mtDNA copy number was higher in the heterozygous men (mean ± SEM: 27.0 ± 12.6; range: 0.7–206.1) compared with the homozygous wild type group (mean ± SEM: 16.8 ± 6.0; range: 0.2–100.1). However, this difference was not statistically significant (P = 0.261).
Expression and localization of POLG
To determine whether POLG persisted in mature sperm having completed spermiogenesis and thus mtDNA replication, we further analysed 93 of the sperm samples from our cohorts of normal, 1 or 2 defects and OAT men for patterns of protein expression with ICC. As anticipated, POLG localized to the midpiece, the compartment housing the mitochondria (Fig. 3A) and POLG expression was confirmed by western blotting (Fig. 3C). ICC demonstrated that most of the sperm in each of the samples analysed were stained in the midpiece (mean% ± SEM: 73.0 ± 1.6), whereas the remainder presented no staining. Interestingly, in almost all samples analysed, we observed that some sperm were also stained in a ring-like structure localized between the head and the midpiece (referred to here as the ‘ring’; Fig. 3B; mean% ± SEM: 29.3 ± 2.5). The mean percentage of sperm stained in the midpiece (mean% ± SEM: 79.3 ± 2.3 for the normal group; 72.9 ± 2.3 for the 1 or 2 defects and 62.8 ± 2.8 for the OATs) is significantly lower in the OAT group when compared with the other 2 groups (normal versus OAT, P < 0.01; 1 or 2 defects versus OAT, P < 0.05; Fig. 3D). In relation to the mean percentage of sperm stained both in the midpiece and the ring (mean% ± SEM: 35.9 ± 4.5, 30.6 ± 3.5 and 14.9 ± 3.8 in the normal, 1- or 2- defects and OAT groups, respectively), the OAT group is also significantly different from the other groups (normal versus OAT and 1 or 2 defects versus OAT, P < 0.05; Fig. 3E).
|
Expression and localization of TFAM
As the initiation of mtDNA replication depends on transcription having occurred previously, we also investigated the expression and localization of TFAM in mature sperm. In the 47 samples analysed, the majority of sperm from each sample were stained in the midpiece (mean% ± SEM: 65.6 ± 2.5; Fig. 4A), whereas the remaining showed no staining. TFAM expression was confirmed by western blotting (Fig. 4B). Comparison between the three groups revealed that the percentage sperm stained in the midpiece decreased from the normal (mean% ± SEM: 72.8 ± 4.2; Fig. 4C) to the 1 or 2 defects (64.2 ± 3.6) to the OAT (54.9 ± 4.4) group. However, only the OAT group was significantly different from the normal group (P < 0.05).
|
Expression of mtDNA and nuclear-encoded genes of the ETC
To determine whether the expression of mtDNA-encoded genes would be influenced by mtDNA copy number, the expression of mtDNA replication factors and POLG CAG repeats, we first analysed the level of expression for COXI and a nuclear-encoded gene, COXVIc, from the same ETC complex. A total of 92 and 78 samples were analysed for COXI and COXVIc, respectively. Both antibodies stained the midpiece for the majority of sperm in each sample (mean% ± SEM: 79.0 ± 1.3 for COXI and 65.5 ± 2.5 for COXVIc; Fig. 5A and 5A'). The OAT group (mean% ± SEM: 68.5 ± 3.7) was distinct from the other two in the mean percentage sperm expressing COXI in the midpiece (84.4 ± 1.7 for normals; 79.2 ± 1.7 for the 1 or 2 defects), representing significant differences to the normal (P < 0.001) and 1- or 2-defects (P < 0.01) groups (Fig. 5C). On the other hand, the three groups were distinct from each other for COXVIc staining in the midpiece (mean% ± SEM: 75.3 ± 4.5 in the normal group; 67.0 ± 3.1 for the 1 or 2 defects and 45.5 ± 5.2 for the OAT group; normal versus OAT, P < 0.001; 1 or 2 defects versus OAT, P < 0.01; normal versus 1 or 2 defects, P < 0.05; Fig. 5C'). Differences in the expression of both COXI and COXVIc between the OAT and normal groups were also confirmed by western blotting (Fig. 5B and 5B').
|
The effects of POLG CAG repeats on POLG, TFAM, COXI and COXVIc proteins expression
To determine whether specific POLG CAG-repeat genotypes would affect expression of POLG and, as a consequence, TFAM, COXI and COXVIc, we compared the ICC outcomes between the distinct POLG CAG-repeat genotypes (homozygous wild type: n = 40 for POLG, n = 25 for TFAM, n = 41 for COXI and n = 33 for COXVIc; heterozygous: n = 17 for POLG, n = 8 for TFAM, n = 18 for COXI and n = 12 for COXVIc). Similar to the results obtained for sperm parameters, the two genotypes showed no significant differences in the mean percentage sperm expressing each of the proteins (P values range from 0.226 to 0.956; data not shown).
The correlation of individual sperm parameters with POLG, TFAM, COXI and COXVIc expression
To understand whether the expression of POLG, TFAM, COXI and COXVIc were correlated with sperm quality, we analysed the three principal semen parameters (concentration, motility and morphology) for the samples used to determine protein expression by ICC. To this extent, a positive correlation was found between the mean percentage sperm expressing POLG, TFAM, COXI and COXVIc, as determined by ICC, and each of the three principal sperm parameters (Table 4). Interestingly, the correlation between the expressions of POLG, COXI and COXVIc and sperm quality is more significant for sperm morphology (higher R values, associated with lower P values; Table 4) compared with concentration and motility.
|
POLG and TFAM expression and mtDNA copy number
As both POLG and TFAM are involved in mtDNA replication, and TFAM is believed to be a regulator of mtDNA copy number, we sought to determine the relationship between the expression of these proteins and mtDNA copy number. Paradoxically, we found a negative correlation between the percentage of sperm expressing POLG and mtDNA content (R = –0.557, P = 0.01; Table 5). However, unexpectedly, no correlation was found between TFAM expression and mtDNA copy number (P = 0.548; Table 5).
|
mtDNA copy number and COXI and COXVIc expression
As COXI is mtDNA encoded, it is expected that cells with higher mtDNA content would express more COXI. However, a negative correlation was found between mtDNA copy number and the percentage of cells expressing COXI (R = –0.566, P = 0.01; Table 5). As COXI and COXVIc are two subunits of the same complex, we anticipated that mtDNA content would influence COXVIc expression. However, no correlation was observed between mtDNA copy number and the percentage sperm expressing COXVIc (P = 0.098; Table 5).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
The long standing debate concerning the nature of the ATP generated for sperm function, i.e. on the relative importance of glycolysis versus OXPHOS, has been often confounded by possible changes throughout the lifespan of the male gamete, as well as by species-specific differences (Storey and Kayne, 1975
; for a review see Ford, 2006). However, in humans it has been well established that sperm require mitochondrial ATP generated through OXPHOS (Ruiz-Pesini et al., 1998, 2000a, 2000b). It would thus be expected that good quality sperm express a significant amount of ETC proteins when compared with low-quality (OAT) samples, and we have shown that this is the case for both mitochondrial (COXI) and nuclear-encoded proteins (COXVIc). These results imply a functional mtDNA complement, and, indeed, the analysis of samples from men with maternally inherited mtDNA mutations reveals decreased sperm motility (Folgero et al., 1993; Spiropoulos et al., 2002). Likewise, a patient with maternally inherited large-scale mtDNA deletions presented with oligozoospermia (Lestienne et al., 1997), whereas OAT patients appear to have acquired a higher number of spontaneously derived large-scale deletions (St John et al., 2001; O'Connell et al., 2003). These outcomes have been supported by the recent finding that a pathological maternally inherited mtDNA deletion was responsible for male infertility in a transmitochondrial mouse model (Nakada et al., 2006).
Paradoxically, we have found the expression of an mtDNA-encoded ETC protein (COXI) to be inversely correlated with mtDNA content, while no correlation was found for a nuclear-encoded ETC protein (COXVIc). This suggests that other factors are affecting COXVIc expression, which, at least in rat, seems to be regulated by nuclear respiratory factors 1 and 2 (NRF1- Evans and Scarpulla, 1990; NRF2- Ongwijitwat et al., 2006). However, we have also shown that the expression of ETC proteins (both mitochondrial- and nuclear-encoded) increases as sperm quality increases, whereas the opposite pattern occurs with mtDNA content. Thus, although human sperm possess a range of mtDNA molecules, OAT samples have a significantly higher content compared with either the normal group or the 1or 2 defects group. This is in accordance with previous work demonstrating that sperm from poorer quality semen samples are also indicative of higher numbers of mtDNA copy (Diez-Sanchez et al., 2003; May-Panloup et al., 2003), and implies that there are errors in the regulation of mtDNA replication in men who will ultimately present with poor quality sperm. The high mtDNA content in OAT sperm may therefore be an indicator of defective spermiogenesis and may also reflect problems in energy metabolism in both testicular cells and mature male gametes.
TFAM not only functions as a transcription factor but, as it activates the generation of the primer necessary for mtDNA replication, it is thought to also regulate mtDNA copy number (Ekstrand et al., 2004). To this extent, a number of investigations indicate a positive correlation between mtDNA content and TFAM levels in various mammalian cells (Larsson et al., 1994; Poulton et al., 1994; Davis et al., 1996; Li et al., 2000; Seidel-Rogol and Shadel, 2002; Joseph et al., 2004). This is perhaps further exemplified during the final stages of murine spermatogenesis. Here, as meiosis II is completed, creating a haploid chromosomal complement, mtDNA content is reduced 10-fold (Hecht et al., 1984). This is concurrent with the expression of an isoform of TFAM lacking the mitochondrial targeting sequence and thus preventing its translocation to the mitochondrion (Larsson et al., 1996). In the human, TFAM is simply down-regulated in testicular sperm (Larsson et al., 1997) most likely to match the fewer copies of mtDNA present. However, we show that not only is TFAM present in mature ejaculated human sperm but, unlike events that take place in other cell types, more TFAM is present in high-quality sperm samples that have lower mtDNA content. This is also supported by the abundance of TFAM transcripts in mature ejaculated sperm from fertile men (Zhao et al., 2006). Likewise, overexpression of TFAM in cultured human cells, with outcomes similar to TFAM knockdown, resulted in mtDNA depletion, although different mtDNA replication intermediates are involved in each of the circumstances (Pohjoismaki et al., 2006). Our findings would indicate a high transcription rate per mtDNA molecule in normal sperm, to ensure that mtDNA-encoded proteins (such as COXI) are produced in sufficient amounts, overcoming the low mtDNA content. The consistent expression of COXVIc, a nuclear-encoded ETC protein, in these same samples would suggest that this is a highly regulated process. Coupled to this is the possibility that TFAM could also be packaging mtDNA (Alam et al., 2003) in good-quality sperm to ensure that the low abundant genome is protected from free radical attack, which sperm mtDNA has a higher propensity for than chromosomal DNA (Sawyer et al., 2003; Bennetts and Aitken, 2005).
Although TFAM has a role in the initiation of mtDNA replication by activating the generation of the primer for extension by POLG (Clayton, 1998), mtDNA replication is highly dependent on this sole mtDNA polymerase being present. The role of POLG as a regulator of mtDNA copy number tends to be cell-type specific. For example, in some cultured human cells subjected to mtDNA depletion, the levels of POLG transcripts and protein have been shown to be similar to non-depleted cells, suggesting that POLG expression is constitutive, even in the complete absence of mtDNA (Davis et al., 1996). Accordingly, in a cell line derived from a patient triploid for the POLG locus (SA15q-3), the increased levels of POLG transcripts, compared with control cells, was not accompanied by an increase in mtDNA levels (Schultz et al., 1998). On the other hand, inducible expression of a dominant negative POLG in HEK293 cells resulted in mtDNA depletion, whereas this was reversible following suppression of the mutator gene (Jazayeri et al., 2003). Furthermore, POLG heterozygous knockout mice developed normally, despite a small reduction in mtDNA levels, whereas homozygous knockout embryos died between embryonic days 7.5 and 8.5 through severe mtDNA depletion (Hance et al., 2005). Interestingly, overexpression of TFAM in POLG heterozygous knockout mice resulted in elevated mtDNA levels and increasing amounts of POLG transcripts, suggesting that POLG expression can be induced when up-regulation of mtDNA replication is required (Hance et al., 2005). To this extent, our results show a negative correlation between mtDNA copy number and the percentage of sperm expressing POLG, even in the presence of TFAM. Consequently, these outcomes could be explained by the increased expression of POLG attempting to compensate for the low mtDNA content, as happens in mtDNA depleted cells (Lloyd et al., 2006). A similar mechanism occurs in early pre-implantation development of in vitro fertilized incompetent oocytes, where up-regulation of POLG and TFAM transcripts and proteins occurs in an attempt to rescue the low mtDNA levels (Spikings et al., 2007). Additionally, the low mtDNA content in normal sperm may alter the predominant mode of mtDNA replication from the strand-asynchronous asymmetric mechanism (Clayton, 1982) to the bidirectional strand-coupled mechanism, as occurs in cultured human cells recovering from transient mtDNA depletion (Holt et al., 2000). The higher levels of TFAM in normal sperm would also be consistent with this, as TFAM overexpression results in a shift to the strand-coupled replication mode in cultured human cells (Pohjoismaki et al., 2006). Such an alteration would imply an up-regulation of POLG to facilitate the strand-coupled mechanism, in which both the leading- and lagging-strands are replicated at the same time, attempting to rescue the low levels of mtDNA present. This rescue mechanism does not seem to take place in mature sperm, possibly due to other, as yet unidentified, regulatory factors intervening. In OAT sperm, the high amount of mtDNA could be due to either: (i) failure to reduce copy number or (ii) continued replication. Either option would result in the down-regulation of POLG expression or, alternatively, POLG would simply not be up-regulated due to an excessive initial amount of mtDNA, explaining why POLG expression is lower in the OAT group. In any case, the low levels of TFAM and thus COXI in OAT samples suggest that spermatogenesis is severely impaired at the mitochondrial level.
The POLG gene possesses a series of CAG repeats that can vary in number (Rovio et al., 1999). The association between the expansion of CAG repeats in different genes and certain neurodegenerative disorders is well documented (for a review see Zoghbi and Orr, 2000). However, the role of certain CAG-repeat variants in idiopathic male infertility is not completely understood as different studies reporting on the same gene have often presented discordant outcomes. This is certainly the case for the length variation of the CAG-repeat region of the androgen-receptor gene, which has been positively (Tut et al., 1997; Mengual et al., 2003) and negatively (Giwercman et al., 1998; Ferlin et al., 2004) associated with infertility. Such disparity is also certainly typical for the POLG CAG-repeat region. To this extent, two studies have demonstrated an association between the absence of the common allele and male infertility, typified by a range of sperm quality defects, (Rovio et al., 2001) and idiopathic subfertility (Jensen et al., 2004). However, this association was not identified in populations of Italian (Krausz et al., 2004; Brusco et al., 2006) and French (Aknin-Seifer et al., 2005) subfertile men. Our results do not show an association between each of the three principal semen parameters, when assessed independently, and POLG genotypes, which is in agreement with Krausz et al. (2004) and Aknin-Seifer et al. (2005). However, we found a higher frequency of the heterozygous genotype in OAT samples when compared to the normal and 1- or 2-defects groups which is similar to the outcomes of Rovio et al. (2001).
Importantly, the functional meaning of the length variation of the POLG CAG-repeat region in sperm has not yet been elucidated. As POLG alleles are segregated during spermatogenesis, samples from heterozygous individuals will present sperm with 10 CAG repeats and sperm with 10 CAG repeats. If the absence of the common allele were to affect the enzymatic properties of POLG, it is anticipated that an accumulation of mtDNA mutations and/or an alteration in mtDNA copy number would occur in heterozygous and homozygous mutant men, due to the presence of sperm with 10 repeats. However, we have found no relation between mtDNA copy number and POLG CAG repeat variants. Furthermore, it is apparent that the incidence of mtDNA nucleotide substitutions is similar for each of the POLG genotypes (Harris et al., 2006). Moreover, our results show that the number of sperm expressing POLG is not significantly different in samples from different POLG genotypes. This is equally so for TFAM, COXI and COXVIc. Perhaps most striking of all is that the deletion of the POLG CAG-repeat region in cultured human cells resulted in the continued unaffected enzymatic properties of POLG, although the expression of the protein was moderately up-regulated (Spelbrink et al., 2000). Consequently, these cumulative outcomes suggest that there is currently no direct functional association between POLG CAG-repeat variants and male infertility, with a possible exception in men with very poor sperm quality (OATs). Interestingly though, when comparing POLG CAG-repeat variants and mtDNA content between our three groups of patients, a parallelism emerges: the OAT group presents both a higher percentage of heterozygozity for the POLG CAG repeat and a higher mean mtDNA copy number. This thus suggests that the absence of the POLG ‘common allele’ in sperm may affect, in certain circumstances (not yet identified, but probably operating only in very poor sperm samples), the interaction with mtDNA or with other replication factors.
In conclusion, the biological significance of POLG CAG-repeat variants in male infertility remains unclear, and the heterozygous genotype seems to be associated with very poor-quality sperm (OATs). Indeed, our data, to an extent, support the original findings that an association between sperm quality and POLG CAG repeats does exist. However, these variants do not directly affect POLG expression, nor mtDNA maintenance. Furthermore, it is evident that high-quality sperm have very low levels of mtDNA genomes compared with other fully differentiated somatic cells (Moyes et al., 1998). The possible biological significance of this finding could be to facilitate the elimination of paternal mtDNA following fertilization and thus ensure maternal inheritance of this genome (Larsson et al., 1997). This work also suggests that POLG, possibly in conjunction with other factors, may play a role in regulating mtDNA copy in human sperm. Unlike TFAM, the presence of POLG seems to (negatively) predict mtDNA copy number, and POLG levels are up-regulated in quality sperm, in an attempt to rescue the low copy number status. This could itself result in saturation and subsequent inhibition of replication. An alternative hypothesis is that high levels of TFAM interacting with mtDNA prevent the polymerase from accessing the mtDNA genome, stressing male gametogenesis as a unique system to study mtDNA replication. Thus, TFAM could play a dual role in sperm, both in regulating mtDNA copy number and in ensuring that appropriate amounts of mtDNA-encoded ETC proteins are available for ATP production and sperm function.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
We thank T. Almeida-Santos and P. Henriques for technical assistance; C. Faro for assistance with Capillary Electrophoretic Genetic Analysis; J. Facucho, E. Bowles and E. Spiking for help with real-time PCR; M.J. Cardoso and A.P. Sousa for help with immunocytochemistry and western blotting. A.A is supported by Fundação para a Ciência e a Tecnologia (FCT) Portugal (SFRH/BD/12665/2003). The work of JRS was supported by FCT (POCTI/CVT/49102/2002), by Instituto de Investigação Interdisciplinar, University of Coimbra (III/BIO/50/2005) and that of JSJ by the British Heart Foundation (PG/04/117).
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Aknin-Seifer IE, Touraine RL, Lejeune H, Jimenez C, Chouteau J, Siffroi JP, McElreavey K, Bienvenu T, Patrat C, Levy R. (2005) Is the CAG repeat of mitochondrial DNA polymerase gamma (POLG) associated with male infertility? A multi-centre French study. Hum Reprod 20:736–740.
Alam TI, Kanki T, Muta T, Ukaji K, Abe Y, Nakayama H, Takio K, Hamasaki N, Kang D. (2003) Human mitochondrial DNA is packaged with TFAM. Nucleic Acids Res 31:1640–1645.
Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, et al. (1981) Sequence and organization of the human mitochondrial genome. Nature 290:457–465.[CrossRef][Medline]
Bennetts LE and Aitken RJ. (2005) A comparative study of oxidative DNA damage in mammalian spermatozoa. Mol Reprod Dev 71:77–87.[CrossRef][Web of Science][Medline]
Brusco A, Michielotto C, Gatta V, Foresta C, Matullo G, Zeviani M, Ferrari G, Dragone E, Calabrese G, Rossato M, et al. (2006) The polymorphic polyglutamine repeat in the mitochondrial DNA polymerase gamma gene is not associated with oligozoospermia. J Endocrinol Invest 29:1–4.[Web of Science][Medline]
Bustin SA. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193.[Abstract]
Chang DD, Hauswirth WW, Clayton DA. (1985) Replication priming and transcription initiate from precisely the same site in mouse mitochondrial DNA. EMBO J 4:1559–1567.[Web of Science][Medline]
Clayton DA. (1982) Replication of animal mitochondrial DNA. Cell 28:693–705.[CrossRef][Web of Science][Medline]
Clayton DA. (1998) Nuclear-mitochondrial intergenomic communication. Biofactors 7:203–205.[Medline]
Cummins JM, Jequier AM, Martin R, Mehmet D, Goldblatt J. (1998) Semen levels of mitochondrial DNA deletions in men attending an infertility clinic do not correlate with phenotype. Int J Androl 21:47–52.[CrossRef][Web of Science][Medline]
Davis AF, Ropp PA, Clayton DA, Copeland WC. (1996) Mitochondrial DNA polymerase gamma is expressed and translated in the absence of mitochondrial DNA maintenance and replication. Nucleic Acids Res 24:2753–2759.
Diez-Sanchez C, Ruiz-Pesini E, Lapena AC, Montoya J, Perez-Martos A, Enriquez JA, Lopez-Perez MJ. (2003) Mitochondrial DNA content of human spermatozoa. Biol Reprod 68:180–185.
Ekstrand MI, Falkenberg M, Rantanen A, Park CB, Gaspari M, Hultenby K, Rustin P, Gustafsson CM, Larsson NG. (2004) Mitochondrial transcription factor A regulates mtDNA copy number in mammals. Hum Mol Genet 13:935–944.
Evans MJ and Scarpulla RC. (1990) NRF-1: a trans-activator of nuclear-encoded respiratory genes in animal cells. Genes Dev 4:1023–1034.
Ferlin A, Bartoloni L, Rizzo G, Roverato A, Garolla A, Foresta C. (2004) Androgen receptor gene CAG and GGC repeat lengths in idiopathic male infertility. Mol Hum Reprod 10:417–421.
Fisher RP and Clayton DA. (1985) A transcription factor required for promoter recognition by human mitochondrial RNA polymerase. Accurate initiation at the heavy- and light-strand promoters dissected and reconstituted in vitro. J Biol Chem 260:11330–11338.
Fisher RP and Clayton DA. (1988) Purification and characterization of human mitochondrial transcription factor 1. Mol Cell Biol 8:3496–3509.
Folgero T, Bertheussen K, Lindal S, Torbergsen T, Oian P. (1993) Mitochondrial disease and reduced sperm motility. Hum Reprod 8:1863–1868.
Ford WC. (2006) Glycolysis and sperm motility: does a spoonful of sugar help the flagellum go round? Hum Reprod Update 12:269–274.
Garstka HL, Facke M, Escribano JR, Wiesner RJ. (1994) Stoichiometry of mitochondrial transcripts and regulation of gene expression by mitochondrial transcription factor A. Biochem Biophys Res Commun 200:619–626.[CrossRef][Web of Science][Medline]
Giles RE, Blanc H, Cann HM, Wallace DC. (1980) Maternal inheritance of human mitochondrial DNA. Proc Natl Acad Sci USA 77:6715–6719.
Giwercman YL, Xu C, Arver S, Pousette A, Reneland R. (1998) No association between the androgen receptor gene CAG repeat and impaired sperm production in Swedish men. Clin Genet 54:435–436.[Web of Science][Medline]
Hance N, Ekstrand MI, Trifunovic A. (2005) Mitochondrial DNA polymerase gamma is essential for mammalian embryogenesis. Hum Mol Genet 14:1775–1783.
Harris TP, Gomas KP, Weir F, Holyoake AJ, McHugh P, Wu M, Sin Y, Sin IL, Sin FY. (2006) Molecular analysis of polymerase gamma gene and mitochondrial polymorphism in fertile and subfertile men. Int J Androl 29:421–433.[CrossRef][Web of Science][Medline]
Hecht NB and Liem H. (1984) Mitochondrial DNA is synthesized during meiosis and spermiogenesis in the mouse. Exp Cell Res 154:293–298.[CrossRef][Web of Science][Medline]
Hecht NB, Liem H, Kleene KC, Distel RJ, Ho SM. (1984) Maternal inheritance of the mouse mitochondrial genome is not mediated by a loss or gross alteration of the paternal mitochondrial DNA or by methylation of the oocyte mitochondrial DNA. Dev Biol 102:452–461.[CrossRef][Web of Science][Medline]
Holt IJ, Lorimer HE, Jacobs HT. (2000) Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA. Cell 100:515–524.[CrossRef][Web of Science][Medline]
Hopgood R, Sullivan KM, Gill P. (1992) Strategies for automated sequencing of human mitochondrial DNA directly from PCR products. Biotechniques 13:82–92.[Web of Science][Medline]
Jazayeri M, Andreyev A, Will Y, Ward M, Anderson CM, Clevenger W. (2003) Inducible expression of a dominant negative DNA polymerase-gamma depletes mitochondrial DNA and produces a rho0 phenotype. J Biol Chem 278:9823–9830.
Jensen M, Leffers H, Petersen JH, Nyboe Andersen A, Jorgensen N, Carlsen E, Jensen TK, Skakkebaek NE, Rajpert-De Meyts E. (2004) Frequent polymorphism of the mitochondrial DNA polymerase gamma gene (POLG) in patients with normal spermiograms and unexplained subfertility. Hum Reprod 19:65–70.
Joseph AM, Rungi AA, Robinson BH, Hood DA. (2004) Compensatory responses of protein import and transcription factor expression in mitochondrial DNA defects. Am J Physiol Cell Physiol 286:C867–875.
Kao S, Chao HT, Wei YH. (1995) Mitochondrial deoxyribonucleic acid 4977-bp deletion is associated with diminished fertility and motility of human sperm. Biol Reprod 52:729–736.[Abstract]
Kao SH, Chao HT, Wei YH. (1998) Multiple deletions of mitochondrial DNA are associated with the decline of motility and fertility of human spermatozoa. Mol Hum Reprod 4:657–666.
Kao SH, Chao HT, Liu HW, Liao TL, Wei YH. (2004) Sperm mitochondrial DNA depletion in men with asthenospermia. Fertil Steril 82:66–73.[CrossRef][Web of Science][Medline]
Krausz C, Guarducci E, Becherini L, Degl'Innocenti S, Gerace L, Balercia G, Forti G. (2004) The clinical significance of the POLG gene polymorphism in male infertility. J Clin Endocrinol Metab 89:4292–4297.
Kruger TF, Menkveld R, Stander FS, Lombard CJ, Van der Merwe JP, van Zyl JA, Smith K. (1986) Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril 46:1118–1123.[Web of Science][Medline]
Larsson NG, Oldfors A, Holme E, Clayton DA. (1994) Low levels of mitochondrial transcription factor A in mitochondrial DNA depletion. Biochem Biophys Res Commun 200:1374–1381.[CrossRef][Web of Science][Medline]
Larsson NG, Garman JD, Oldfors A, Barsh GS, Clayton DA. (1996) A single mouse gene encodes the mitochondrial transcription factor A and a testis-specific nuclear HMG-box protein. Nat Genet 13:296–302.[CrossRef][Web of Science][Medline]
Larsson NG, Oldfors A, Garman JD, Barsh GS, Clayton DA. (1997) Down-regulation of mitochondrial transcription factor A during spermatogenesis in humans. Hum Mol Genet 6:185–191.
Larsson NG, Wang J, Wilhelmsson H, Oldfors A, Rustin P, Lewandoski M, Barsh GS, Clayton DA. (1998) Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice. Nat Genet 18:231–236.[CrossRef][Web of Science][Medline]
Lestienne P, Reynier P, Chretien MF, Penisson-Besnier I, Malthiery Y, Rohmer V. (1997) Oligoasthenospermia associated with multiple mitochondrial DNA rearrangements. Mol Hum Reprod 3:811–814.
Li H, Wang J, Wilhelmsson H, Hansson A, Thoren P, Duffy J, Rustin P, Larsson NG. (2000) Genetic modification of survival in tissue-specific knockout mice with mitochondrial cardiomyopathy. Proc Natl Acad Sci USA 97:3467–3472.
Lloyd RE, Lee JH, Alberio R, Bowles EJ, Ramalho-Santos J, Campbell KH, St John JC. (2006) Aberrant nucleo-cytoplasmic cross-talk results in donor cell mtDNA persistence in cloned embryos. Genetics 172:2515–2527.
Luoma P, Melberg A, Rinne JO, Kaukonen JA, Nupponen NN, Chalmers RM, Oldfors A, Rautakorpi I, Peltonen L, Majamaa K, et al. (2004) Parkinsonism, premature menopause, and mitochondrial DNA polymerase gamma mutations: clinical and molecular genetic study. Lancet 364:875–882.[CrossRef][Web of Science][Medline]
May-Panloup P, Chretien MF, Savagner F, Vasseur C, Jean M, Malthiery Y, Reynier P. (2003) Increased sperm mitochondrial DNA content in male infertility. Hum Reprod 18:550–556.
Mengual L, Oriola J, Ascaso C, Ballesca JL, Oliva R. (2003) An increased CAG repeat length in the androgen receptor gene in azoospermic ICSI candidates. J Androl 24:279–284.
Moyes CD, Battersby BJ, Leary SC. (1998) Regulation of muscle mitochondrial design. J Exp Biol 201:299–307.[Web of Science][Medline]
Nakada K, Sato A, Yoshida K, Morita T, Tanaka H, Inoue S, Yonekawa H, Hayashi J. (2006) Mitochondria-related male infertility. Proc Natl Acad Sci USA 103:15148–15153.
Naviaux RK and Nguyen KV. (2004) POLG mutations associated with Alpers' syndrome and mitochondrial DNA depletion. Ann Neurol 55:706–712.[CrossRef][Web of Science][Medline]
Naviaux RK and Nguyen KV. (2005) POLG mutations associated with Alpers syndrome and mitochondrial DNA depletion. Ann Neurol 58:491.[CrossRef][Web of Science][Medline]
Nguyen KV, Ostergaard E, Ravn SH, Balslev T, Danielsen ER, Vardag A, McKiernan PJ, Gray G, Naviaux RK. (2005) POLG mutations in Alpers syndrome. Neurology 65:1493–1495.
Nguyen KV, Sharief FS, Chan SS, Copeland WC, Naviaux RK. (2006) Molecular diagnosis of Alpers syndrome. J Hepatol 45:108–116.[CrossRef][Web of Science][Medline]
O'Connell M, McClure N, Powell LA, Steele EK, Lewis SE. (2003) Differences in mitochondrial and nuclear DNA status of high-density and low-density sperm fractions after density centrifugation preparation. Fertil Steril 79 (Suppl 1):754–762.
Ongwijitwat S, Liang HL, Graboyes EM, Wong-Riley MT. (2006) Nuclear respiratory factor 2 senses changing cellular energy demands and its silencing down-regulates cytochrome oxidase and other target gene mRNAs. Gene 374:39–49.[CrossRef][Web of Science][Medline]
Otani H, Tanaka O, Kasai K, Yoshioka T. (1988) Development of mitochondrial helical sheath in the middle piece of the mouse spermatid tail: regular dispositions and synchronized changes. Anat Rec 222:26–33.[CrossRef][Medline]
Pagnamenta AT, Taanman JW, Wilson CJ, Anderson NE, Marotta R, Duncan AJ, Bitner-Glindzicz M, Taylor RW, Laskowski A, Thorburn DR, et al. (2006) Dominant inheritance of premature ovarian failure associated with mutant mitochondrial DNA polymerase gamma. Hum Reprod 10:2467–2473.
Parisi MA and Clayton DA. (1991) Similarity of human mitochondrial transcription factor 1 to high mobility group proteins. Science 252:965–969.
Pfeiffer T, Schuster S, Bonhoeffer S. (2001) Cooperation and competition in the evolution of ATP-producing pathways. Science 292:504–507.
Pohjoismaki JL, Wanrooij S, Hyvarinen AK, Goffart S, Holt IJ, Spelbrink JN, Jacobs HT. (2006) Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the mode of mitochondrial DNA replication in cultured human cells. Nucleic Acids Res 34:5815–5828.
Poulton J, Morten K, Freeman-Emmerson C, Potter C, Sewry C, Dubowitz V, Kidd H, Stephenson J, Whitehouse W, Hansen FJ, et al. (1994) Deficiency of the human mitochondrial transcription factor h-mtTFA in infantile mitochondrial myopathy is associated with mtDNA depletion. Hum Mol Genet 3:1763–1769.
Ramalho-Santos J, Moreno RD, Sutovsky P, Chan AW, Hewitson L, Wessel GM, Simerly CR, Schatten G. (2000) SNAREs in mammalian sperm: possible implications for fertilization. Dev Biol 223:54–69.[CrossRef][Web of Science][Medline]
Rantanen A, Jansson M, Oldfors A, Larsson NG. (2001) Downregulation of Tfam and mtDNA copy number during mammalian spermatogenesis. Mamm Genome 12:787–792.[CrossRef][Web of Science][Medline]
Reynier P, Chretien MF, Savagner F, Larcher G, Rohmer V, Barriere P, Malthiery Y. (1998) Long PCR analysis of human gamete mtDNA suggests defective mitochondrial maintenance in spermatozoa and supports the bottleneck theory for oocytes. Biochem Biophys Res Commun 252:373–377.[CrossRef][Web of Science][Medline]
Reynier P, May-Panloup P, Chretien MF, Morgan CJ, Jean M, Savagner F, Barriere P, Malthiery Y. (2001) Mitochondrial DNA content affects the fertilizability of human oocytes. Mol Hum Reprod 7:425–429.
Ropp PA and Copeland WC. (1996) Cloning and characterization of the human mitochondrial DNA polymerase, DNA polymerase gamma. Genomics 36:449–458.[CrossRef][Web of Science][Medline]
Rovio A, Tiranti V, Bednarz AL, Suomalainen A, Spelbrink JN, Lecrenier N, Melberg A, Zeviani M, Poulton J, Foury F, et al. (1999) Analysis of the trinucleotide CAG repeat from the human mitochondrial DNA polymerase gene in healthy and diseased individuals. Eur J Hum Genet 7:140–146.[CrossRef][Web of Science][Medline]
Rovio AT, Marchington DR, Donat S, Schuppe HC, Abel J, Fritsche E, Elliott DJ, Laippala P, Ahola AL, McNay D, et al. (2001) Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility. Nat Genet 29:261–262.[CrossRef][Web of Science][Medline]
Rovio AT, Abel J, Ahola AL, Andres AM, Bertranpetit J, Blancher A, Bontrop RE, Chemnick LG, Cooke HJ, Cummins JM, et al. (2004) A prevalent POLG CAG microsatellite length allele in humans and African great apes. Mamm Genome 15:492–502.[CrossRef][Web of Science][Medline]
Ruiz-Pesini E, Diez C, Lapena AC, Perez-Martos A, Montoya J, Alvarez E, Arenas J, Lopez-Perez MJ. (1998) Correlation of sperm motility with mitochondrial enzymatic activities. Clin Chem 44:1616–1620.
Ruiz-Pesini E, Lapena AC, Diez-Sanchez C, Perez-Martos A, Montoya J, Alvarez E, Diaz M, Urries A, Montoro L, Lopez-Perez MJ, et al. (2000a) Human mtDNA haplogroups associated with high or reduced spermatozoa motility. Am J Hum Genet 67:682–696.[CrossRef][Web of Science][Medline]
Ruiz-Pesini E, Lapena AC, Diez C, Alvarez E, Enriquez JA, Lopez-Perez MJ. (2000b) Seminal quality correlates with mitochondrial functionality. Clin Chim Acta 300:97–105.[CrossRef][Web of Science][Medline]
Sawyer DE, Mercer BG, Wiklendt AM, Aitken RJ. (2003) Quantitative analysis of gene-specific DNA damage in human spermatozoa. Mutat Res 529:21–34.[Web of Science][Medline]
Schultz RA, Swoap SJ, McDaniel LD, Zhang B, Koon EC, Garry DJ, Li K, Williams RS. (1998) Differential expression of mitochondrial DNA replication factors in mammalian tissues. J Biol Chem 273:3447–3451.
Seidel-Rogol BL and Shadel GS. (2002) Modulation of mitochondrial transcription in response to mtDNA depletion and repletion in HeLa cells. Nucleic Acids Res 30:1929–1934.
Siciliano G, Mancuso M, Pasquali L, Manca ML, Tessa A, Iudice A. (2000) Abnormal levels of human mitochondrial transcription factor A in skeletal muscle in mitochondrial encephalomyopathies. Neurol Sci 21:S985–987.[CrossRef][Web of Science][Medline]
Spelbrink JN, Van Galen MJ, Zwart R, Bakker HD, Rovio A, Jacobs HT, Van den Bogert C. (1998) Familial mitochondrial DNA depletion in liver: haplotype analysis of candidate genes. Hum Genet 102:327–331.[CrossRef][Web of Science][Medline]
Spelbrink JN, Toivonen JM, Hakkaart GA, Kurkela JM, Cooper HM, Lehtinen SK, Lecrenier N, Back JW, Speijer D, Foury F, et al. (2000) In vivo functional analysis of the human mitochondrial DNA polymerase POLG expressed in cultured human cells. J Biol Chem 275:24818–24828.
Spikings EC, Alderson J, St John JC. (2007) Regulated mitochondrial DNA replication during oocyte maturation is essential for successful porcine embryonic development. Biol Reprod 76:327–335.
Spiropoulos J, Turnbull DM, Chinnery PF. (2002) Can mitochondrial DNA mutations cause sperm dysfunction? Mol Hum Reprod 8:719–721.
St John JC, Jokhi RP, Barratt CL. (2001) Men with oligoasthenoteratozoospermia harbour higher numbers of multiple mitochondrial DNA deletions in their spermatozoa, but individual deletions are not indicative of overall aetiology. Mol Hum Reprod 7:103–111.
Storey BT and Kayne FJ. (1975) Energy metabolism of spermatozoa. V. The Embden-Myerhof pathway of glycolysis: activities of pathway enzymes in hypotonically treated rabbit epididymal spermatozoa. Fertil Steril 26:1257–1265.[Web of Science][Medline]
Turner RM. (2003) Tales from the tail: what do we really know about sperm motility? J Androl 24:790–803.
Tut TG, Ghadessy FJ, Trifiro MA, Pinsky L, Yong EL. (1997) Long polyglutamine tracts in the androgen receptor are associated with reduced trans-activation, impaired sperm production, and male infertility. J Clin Endocrinol Metab 82:3777–3782.
Van Goethem G, Dermaut B, Lofgren A, Martin JJ, Van Broeckhoven C. (2001) Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions. Nat Genet. 28:211–212.
Van Goethem G, Martin JJ, Dermaut B, Lofgren A, Wibail A, Ververken D, Tack P, Dehaene I, Van Zandijcke M, Moonen M, et al. (2003) Recessive POLG mutations presenting with sensory and ataxic neuropathy in compound heterozygote patients with progressive external ophthalmoplegia. Neuromuscul Disord 13:133–142.[CrossRef][Web of Science][Medline]
Wallace DC. (1999) Mitochondrial diseases in man and mouse. Science 283:1482–1488.
World Health Organization. (1999) WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. 4th edn (Cambridge University Press, Cambridge, UK).
Wykes SM and Krawetz SA. (2003) The structural organization of sperm chromatin. J Biol Chem 278:29471–29477.
Xu B and Clayton DA. (1996) RNA-DNA hybrid formation at the human mitochondrial heavy-strand origin ceases at replication start sites: an implication for RNA-DNA hybrids serving as primers. EMBO J 15:3135–3143.[Web of Science][Medline]
Zhao Y, Li Q, Yao C, Wang Z, Zhou Y, Wang Y, Liu L, Wang Y, Wang L, Qiao Z. (2006) Characterization and quantification of mRNA transcripts in ejaculated spermatozoa of fertile men by serial analysis of gene expression. Hum Reprod 21:1583–1590.
Zoghbi HY and Orr HT. (2000) Glutamine repeats and neurodegeneration. Annu Rev Neurosci 23:217–247.[CrossRef][Web of Science][Medline]
Submitted on November 10, 2006; resubmitted on January 14, 2007; accepted on January 18, 2007.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Ramalho-Santos, S. Varum, S. Amaral, P. C. Mota, A. P. Sousa, and A. Amaral Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells Hum. Reprod. Update, September 1, 2009; 15(5): 553 - 572. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Blomberg Jensen, H. Leffers, J. H. Petersen, G. Daugaard, N. E. Skakkebaek, and E. Rajpert-De Meyts Association of the polymorphism of the CAG repeat in the mitochondrial DNA polymerase gamma gene (POLG) with testicular germ-cell cancer Ann. Onc., November 1, 2008; 19(11): 1910 - 1914. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Cavallini, A. Crippa, M. C. Magli, N. Cavallini, A. P. Ferraretti, and L. Gianaroli A Study to Sustain the Hypothesis of the Multiple Genesis of Oligoasthenoteratospermia in Human Idiopathic Infertile Males Biol Reprod, October 1, 2008; 79(4): 667 - 673. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. Wolff and N. J. Gemmell Estimating Mitochondrial DNA Content of Chinook Salmon Spermatozoa Using Quantitative Real-Time Polymerase Chain Reaction Biol Reprod, August 1, 2008; 79(2): 247 - 252. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||