Molecular Human Reproduction, Vol. 7, No. 5, 425-429,
May 2001
© 2001 European Society of Human Reproduction and Embryology
Embryology |
Mitochondrial DNA content affects the fertilizability of human oocytes
1 INSERM EMI-U 00-18, Laboratoire de Biochimie et Biologie Moléculaire, CHU d'Angers, F-49033 Angers, 2 Laboratoire d'Histologie-Embryologie-Cytologie et Laboratoire de FIV, CHU d'Angers, F-49033 Angers and 3 Biologie de la Reproduction, Pavillon de la Mère et de l'Enfant, CHU de Nantes, B.P. 1005, F-44093 Nantes cedex 1, France
Abstract
Mitochondrial DNA content varies considerably in oocytes, even when collected from the same patient. In the present study, real-time quantitative polymerase chain reaction analysis of 113 unfertilized oocytes obtained from 43 patients revealed an average of 193 000 (range: 20 000 to 598 000) mitochondrial genomes per cell. We compared several groups of oocytes to investigate the relationship between mitochondrial DNA content and fertilizability. The average mitochondrial DNA copy number was significantly lower in cohorts suffering from fertilization failure compared to cohorts with a normal rate of fertilization. In addition, the mitochondrial copy number of oocytes from patients with fertilization failure due to unknown causes was significantly lower than that of oocytes from patients in which IVF failure was due mainly to a severe sperm defect. The lower mtDNA copy number could be due to defective cytoplasmic maturation of oocytes. We conclude that low mitochondrial DNA content, due to inadequate mitochondrial biogenesis or cytoplasmic maturation, may adversely affect oocyte fertilizability.
cytoplasmic maturation/mitochondrial biogenesis/mtDNA/oocyte/real-time PCR
Introduction
Most cases of male infertility can be treated by intracytoplasmic sperm injection (ICSI), but successful IVF is still limited by factors such as oocyte quality. Whereas the nuclear maturation of the oocyte is easily identified by the appearance of the first polar body, the cytoplasmic maturation, which is also essential for successful fertilization and early embryo development, remains difficult to evaluate. Defects in oocyte maturation, suspected of being involved in some cases of IVF failure, are sometimes difficult to overcome by ICSI (Miller et al., 1995
; Thomas et al., 1998). A spectacular growth of the oocyte takes place during follicular maturation (Wolf et al., 1995) and many morphological, metabolic and gene expression changes seem to be essential to oocyte maturation (Wassarman and Albertini, 1994; Bell et al., 1997; Gosden et al., 1997; Ji et al., 1997). Large variations in oocyte ATP have been reported and the level of ATP production is believed to be associated with the developmental competence of the embryo (Van Blerkom et al., 1995, 1998). The growth of the oocyte also involves mitochondrial biogenesis and mitochondrial DNA (mtDNA) replication (Piko and Taylor, 1987; Cummins, 1998).
Mitochondria are maternally inherited organelles that use oxidative phosphorylation to supply ATP to the cell. Each mitochondrion carries its own multicopy genome, which is a circular, double-stranded DNA molecule with 16.6 kilobases (Anderson et al., 1981). mtDNA codes for 13 essential subunits of the respiratory chain complexes that provide the main ATP supply of the cell (Wallace, 1992). Human mtDNA is derived exclusively from maternally inherited DNA (Giles et al., 1980). Paternal mitochondria are specifically eliminated after fertilization and before the 4-cell stage (Kaneda et al., 1995), probably by a mechanism involving ubiquitination of sperm mitochondria (Sutovski et al., 1999). It has been suggested that defects in sperm mitochondria may lead to male infertility (Lestienne et al., 1997; Kao et al., 1998; Ruiz-Pesini et al., 1998). However, the significance of the defective mtDNA observed in some oocytes remains unclear (Chen et al., 1995; Brenner et al., 1998; Cummins et al., 1998; Reynier et al., 1998).
Mammalian somatic cells contain several thousands of mitochondria, each of them containing 1–10 copies of mtDNA. It is currently thought that oocytes contain 100 000 to 200 000 mitochondrial genomes (Cummins, 1998). The first estimations of mouse oocyte mitochondrial content were made in the 1970s. Electron microscopy morphometric analysis has found an average of 92 500 mitochondria per mouse pronucleate oocyte (Piko and Matsumoto, 1976). Subsequently, dot blot techniques revealed an average of 119 000 mtDNA copies per cell in mature pooled mouse oocyte (Piko and Taylor, 1987). Hybridization techniques on pooled oocytes determined that bovine oocytes contained an average of 260 000 mtDNA copies per cell (Michaels et al., 1982). More recently, competitive polymerase chain reaction (PCR) (Chen et al., 1995) and real-time PCR (Steuerwald et al., 2000) have quantified the mtDNA copy number in unfertilized human oocytes obtained after IVF failure. The former found an average of 138 000 mtDNA copies in nine oocytes, and the latter found an average of 314 000 mtDNA copies in 18 oocytes, showing a surprising variation in oocyte mtDNA content.
Real-time quantitative PCR, a highly sensitive method, is the most suitable technique currently available for the quantification of DNA copy numbers in single cells. Unlike the studies based on hybridization techniques, which require pooled samples of oocytes, the sensitivity of real-time PCR allows investigation of inter-oocyte variability of mtDNA content. We have therefore used real-time PCR to quantify the mtDNA content of 113 unfertilized oocytes obtained from 43 patients undergoing IVF. To test the hypothesis of a link between the mtDNA content of oocytes and cytoplasmic maturation, and thus the fertilizability, the oocytes were classified into three groups according to the known fertilization characteristics of the patients, i.e. fertilization failure due to a severe sperm defect, idiopathic failure or normal fertilization.
Materials and methods
Oocyte samples
The ethics committee of the University Hospital of Angers approved the plan of the study we intended to perform on oocytes discarded during IVF procedures. Follicular growth was stimulated by recombinant FSH associated with a gonadotrophin-releasing hormone agonist. Ovulation was induced with human chorionic gonadotrophin and oocytes were collected by a transvaginal probe. For the purpose of IVF, oocytes were considered to be unfertilized if on the fourth day following insemination (72 h of culture), there were no pronuclei or second polar bodies. Oocytes were separated from follicular cells by washing and gentle pipetting in IVF-20 medium (IVF Scandinavian, Stockholm, Sweden). A total of 113 isolated metaphase II oocytes (identified by the presence of the first polar body) were individually collected from 43 women over a period of 1 year. Each oocyte was placed in 50 µl of IVF-20 medium. The oocytes were preserved at –20°C until DNA extraction, which was always performed within a month after collection. The average age of the patients was 33 years (range 25–42). The oocyte population collected from a given patient defined a cohort. The average number of oocytes per cohort was 2.6 (range 1–8).
The oocytes were classified into three groups. Group 1 consisted of 21 oocytes collected from five patients (average age 32 years, range 27-38) where the fertilization failure was evidently due to a severe sperm defect (insemination with <105 motile spermatozoa). We assumed that this group was relatively homogeneous, consisting mainly of mature fertilizable oocytes free from defects due to faulty cytoplasmic maturation. Group 2 consisted of 47 oocytes collected from 12 patients (average age 32 years, range 27-42) with idiopathic fertilization failure (fertilization rate 
20% with normal spermatozoa). In this group, 10 patients had complete fertilization failure. Group 3 consisted of 45 unfertilized oocytes collected from 26 patients (average age 34 years, range 25–42 years) with normal fertilization (fertilization rate >20% with normal spermatozoa).
Preparation of DNA
DNA was extracted from each oocyte by means of the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's recommendations. DNA bound specifically to glass fibres following the combined action of a chaotropic agent (guanidine), a detergent (Triton X-100) and the enzyme proteinase K. After washing, the silica-bound DNA was eluted with 400 µl of pre-warmed (72°C) elution buffer and preserved at 4°C.
Preparation of external standards
The external standard was a 158 bp PCR product. The nucleotide positions of the primers on light strand mtDNA were: D41 (3254–3277) and D56 (3126–3147). 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 Tris–HCl (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 units of GoldStar DNA polymerase (Eurogentec, Seraing, Belgium). Each of the 30 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 products were phenol-chloroform purified from low melting point agarose, precipitated in ethanol, diluted in water and quantified by spectrophotometry. Purification quality was checked by means of the absorbance 260/280 ratios, with values between 1.8 and 2.0 being considered acceptable. It was assumed that 1 ng of a 158 bp product contained 5.8x109 molecules of double-stranded DNA. Serial dilutions were then carefully made in order to assess several concentrations of a known number of templates. This was used as the external standard for real-time PCR. The serial dilutions were all conserved at –20°C in single-use aliquots.
Real-time PCR quantification
A Roche LightCycler was used to determine the mtDNA copy number. The LightCycler is a combined microliter volume thermal-cycler and fluorometer suitable for real-time fluorescent PCR. Twenty µl PCR reactions were set up with final concentrations: 1xbuffer containing 4 mmol/l MgCl2, 0.2 mmol/l dNTPs, 0.5 µmol/l of both primers (D41 and R56), SYBR green I dye, 0.25 U Taq DNA polymerase, 0.2 µg of TaqStart antibody (Clontech, Palo Alto, CA, USA) and 10 µl of the extracted DNA or 10 µl of Standard with a known copy number. The reactions were performed in the LightCycler as follows: initial denaturing at 95°C for 2 min and 45 cycles at 95°C for 0 s (temperature transition rate of 20°C/s), 58°C for 5 s (temperature transition rate of 20°C/s), and 72°C for 13 s (temperature transition rate of 2°C/s). The SYBR green fluorescence was read at the end of each extension step (72°C). A melting curve was systematically analysed in order to check the absence of mispriming and the quality of amplifications. This consisted of SYBR green fluorescence analysis of the temperature transition between 66 and 94°C (temperature transition rate of 0.2°C/s), with continuous fluorescence readings. The rapid loss of fluorescence at a given temperature indicates the melting temperature of the PCR product. For each PCR run, a standard curve was generated using five 10-fold serial-dilutions (10–100 000 copies) of the target mtDNA PCR product with the same primers as those used for oocyte mtDNA amplification. The LightCycler software (version 3.01) generated a standard curve, which then allowed the determination of the starting copy number of mtDNA in each sample. Quantitative PCR was performed on 1/40 of the isolated oocyte total DNA, i.e. on a 10 µl sample taken from 400 µl of eluted DNA. All oocytes were tested twice. The raw data were then increased 40-fold to calculate the total mtDNA content in each oocyte.
Extraction efficiency
Extraction efficiency was evaluated by the extraction of several samples, containing known copy numbers of the standard, which were then diluted in 50 µl of IVF 20 medium under the same conditions as those used for oocyte extraction. PCR quantification was then performed under the same conditions as those used for DNA from oocytes.
Statistical analysis
The three groups of oocytes were compared using the Mann-Whitney U-test. Differences were considered significant when P < 0.05. Statistical analyses were performed with SYSTAT software, version 8.0 (SPSS Inc., Chicago, IL, USA).
Results
Extraction efficiency
Since accurate single-cell quantification is highly dependent upon high recoveries of the mtDNA templates, we performed DNA extraction with six samples containing 104 or 105 copies of the standard in the IVF medium, under the same conditions as those used for oocyte DNA extraction. We found an average of 1.08x104 copies for the 104 extracted molecules and an average of 0.93x105 copies for 105 extracted molecules. Thus, the extraction efficiency ranged from 93 to 108%, demonstrating the accuracy of this technique. The DNA binding capacity of the glass fibres was much greater than the small amount of single-cell DNA that was extracted. We may therefore suppose that nearly all the DNA molecules present were bound to the chromatography column prior to the washing and elution steps.
Reproducibility
Intra-assay precision was assessed from 10 measurements of three different oocyte DNA samples with varying levels of mtDNA content. The coefficients of variation (CV) ranged from 3.9 to 9.1% (Table I). When a DNA sample was amplified 10 times in the same experiment, the 10 kinetic curves were all grouped together, demonstrating good repeatability of the method (Figure 1). The reproducibility between runs (inter-assay) was assessed from five measurements of three different oocyte DNA samples. The CV ranged from 9.3 to 12.7% (Table II). These results indicate that the quantitative PCR has good intra-assay precision and reproducibility.
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Oocyte mtDNA quantification
The average mtDNA copy number of the 113 unfertilized oocytes was found to be 193 000 (±122 000) with a high inter-oocyte variation (20 000 to 598 000), even within individual cohorts. Since there was a great variation between different oocytes from a given cohort, we were unable to establish any correlation between the average mtDNA content and the age of the patients (r = 0.10).
In order to investigate the influence of mtDNA content on oocyte fertilizability, we compared 21 unfertilized oocytes from group 1 (fertilization failure mainly due to a serious sperm defect) with 47 unfertilized oocytes from group 2 (fertilization failure in spite of normal spermatozoa). The average mtDNA copy number of group 1, 255 000 (± 110 000), was significantly different (P < 0.0001) from that of group 2, 152 000 (±90 000) (Figure 2).
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We also compared the mtDNA content of unfertilized oocytes with regard to the fertilization rate of their cohort of origin. The average mtDNA copy number of group 2 (cohorts with fertilization failure due to unknown causes) was 152 000 (±90 000) compared with an average of 209 000 (±122 000) in oocytes from group 3 (cohorts with a normal rate of fertilization) (Figure 3
). The difference between the two groups was significant (P < 0.02). Interestingly, six out of seven oocytes containing less than 50 000 mtDNA molecules were found in group 2. However, this result was not significant. There was no correlation between the mtDNA content and the rate of fertilization in group 2 (r = 0.03) or in group 3 (r = 0.09). The comparison of group 1 with group 3 revealed no significant difference (P < 0.07).
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Discussion
Since French law does not allow the use of fertilized human oocytes for the evaluation of mtDNA content, we quantified the average number of mtDNA molecules in isolated unfertilized oocytes. The efficiency of DNA extraction in our study allows the assumption that the average mtDNA content in human unfertilized oocytes after IVF is ~193 000. If we exclude the presence of the first polar body, the amount of mtDNA is considerable and represents ~50% of the total DNA of the oocyte (193 000 mtDNA represent 3.4 pg of DNA, whereas the nucleus contains 3 pg of DNA per haploid genome). A similar approach (Chen et al., 1995; Steuerwald et al., 2000) found an average mtDNA copy number of 138 000 (n = 9 oocytes) and 314 000 (n = 18 oocytes) respectively. The difference in the mean mtDNA content of oocytes between these studies and ours could be explained by the difference in sample size and the large inter-individual variation. It should be noted that in our study there is a potential bias in the absolute quantification of mtDNA content due to the age of the oocytes analysed but the extended period of culture was unavoidable to ensure that only unfertilized oocytes were used. However, our results confirm the great variation of mtDNA content in oocytes reported by Chen et al. (1995) and Steurwald et al. (2000). Moreover, the mtDNA content also varies widely between oocytes belonging to a given cohort.
This large variation in mitochondrial biogenesis may reflect the variation in the level of cytoplasmic maturation of the oocytes. A large part of the increase in mitochondrial content occurs during the final stages of the maturation of the oocyte (Jansen and de Boer, 1998; Poulton et al., 1998). Indeed, it has found that immature mouse oocytes have a lower mtDNA copy number than mature oocytes (Piko and Taylor, 1987). Moreover, ovarian stimulation in IVF typically leads to cohorts of oocytes at various stages of maturation, with about a quarter of them being immature (Flood et al., 1990).
Our results suggest that mitochondrial DNA content could play a role in oocyte fertilizability. A significant reduction in mtDNA content was observed in oocytes from group 2 in comparison with oocytes from group 1 (P < 0.0001). Both groups of oocytes came from cohorts with fertilization failure (fertilization rate 20%). However, group 1 could be assumed to be composed of mature oocytes since fertilization failure was due mainly to severe sperm defects. In contrast, cytoplasmic immaturity could be suspected in oocytes from group 2 since they failed to fertilize despite insemination with normal spermatozoa with apparent nuclear maturity. This suggests that a cytoplasmic maturation defect, in the cohort inseminated with normal sperm, may be related to mitochondrial deficiency.
A second analysis also indicates a relationship between mitochondrial defects and fertilization failure. The cohorts suffering from fertilization failure (despite normal spermatozoa) had a significantly lower mtDNA copy number (P < 0.02) compared to cohorts with a normal rate of fertilization (group 3). Moreover, six out of the seven oocytes with a lower mtDNA content (<50 000) were found in cohorts with fertilization failure. This result demonstrates a significant difference in the cytoplasmic maturation level of cohorts, indicating a global mitochondrial immaturity in cohorts with fertilization failure.
It is worth noting that the average mtDNA content of group 3 (unfertilized oocytes from cohorts with a normal rate of fertilization) appeared to be lower than that of group 1 (mature oocytes, unfertilized due to defective spermatozoa), but this difference was not statistically significant (P < 0.07). The apparently lower mtDNA content in group 3 might be due to the absence of mature oocytes following successful fertilization. It would therefore be interesting to further explore the difference in mtDNA content between the two groups with larger numbers of oocytes.
In conclusion, some IVF failures could be due to inadequate oocyte maturation related to defective mitochondrial biogenesis. This hypothesis needs further confirmation in an animal model where the utilization of fertilized oocytes is possible. The measurement of mtDNA content in unfertilized oocytes, however, could be an interesting tool for the investigation of the molecular mechanisms involved in the cytoplasmic maturation of oocytes. At the clinical level, a low mtDNA copy number could account for some IVF and ICSI failures. The quantification of mtDNA in the corresponding unfertilized oocytes may help in identifying faulty cytoplasmic maturation.
Acknowledgements
We are grateful to Prof. D.Chappard for his help with the statistical analysis and Dr K.Malkani for his critical reading of the manuscript. This work was supported by a grant from the Délégation Régionale de la Recherche Clinique of the University Hospital of Angers (PHRC PL 98-01).
Notes
4 To whom correspondence should be addressed. E-mail: pareynier{at}chu-angers.fr 
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Submitted on November 8, 2000; accepted on March 9, 2001.
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