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BOR - Papers in Press, published online ahead of print April 14, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026104
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BIOLOGY OF REPRODUCTION 71, 697–704 (2004)
DOI: 10.1095/biolreprod.103.026104
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

In Vitro Embryo Production Efficiency in Cattle and Its Association with Oocyte Adenosine Triphosphate Content, Quantity of Mitochondrial DNA, and Mitochondrial DNA Haplogroup

M. Tamassia1,2, F. Nuttinck2, P. May-Panloup5, P. Reynier5, Y. Heyman3, G. Charpigny3, M. Stojkovic4, S. Hiendleder4, J.-P. Renard3, and S. Chastant-Maillard2

Ecole Vétérinaire d'Alfort,2 Biologie de la Reproduction, UMR 1198 INRA/ENVA, 94704 Maisons-Alfort, France INRA, Biologie du Développement et Biotechnologies,3 UMR 1198 INRA/ENVA, 78352 Jouy en Josas, France Department of Molecular Animal Breeding and Biotechnology,4 Ludwig-Maximilian-University Munich, D-85764 Oberschleissheim, Germany INSERM EMI-U 00-18,5 Laboratoire de Biochimie et Biologie Moléculaire, CHU d'Angers, 49033 Angers, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitochondria have a broad range of functions that affect reproduction, and structural as well as quantitative variation in mtDNA has been associated with gamete quality and reproductive success. To investigate the mitochondria effect on in vitro embryo production, we collected oocytes by ultrasound-guided follicular aspiration from donor cows known to differ in the developmental capacity, measured by the blastocyst formation rate, of their oocytes. To evaluate the potential effects of mtDNA and mitochondrial function on oocyte quality, the donor cows' mtDNA control region was sequenced and, after pairwise comparisons of polymorphisms, animals were grouped into two major haplogroups. The number of mtDNA molecules per oocyte was quantified by real-time PCR, and the adenosine triphosphate (ATP) content was measured in each oocyte to identify variations between haplogroups. Overall, ATP stocks in oocytes of the two haplogroups differed significantly (P < 0.05; means ± SEM) both at the germinal vesicle and metaphase II stages (2.8 ± 0.06 pmol vs. 2.6 ± 0.07 pmol and 2.9 ± 0.1 pmol vs. 2.3 ± 0.06 pmol, respectively). The proportion of development to blastocyst was significantly different between haplogroups (22.3 ± 2.1 % vs. 36.7 ± 2.9 %). The number of mtDNA molecules per oocyte was highly variable (377 327 ± 14 104, ranging from 2.0 x 103 to 1.2 x 106) but not significantly different between the two haplogroups; significant differences were observed between animals without any apparent relationship to blastocyst production. These data suggest that mitochondria and mtDNA haplogroup affect the developmental capacity of bovine oocytes in vitro.

early development, embryo, fertilization, gamete biology, oocyte development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In bovine in vitro embryo production (IVP) programs, the aptitude of the oocyte to support embryo development (i.e., oocyte quality) is routinely assessed by oocyte's morphological appearance [1–4]. However, both macroscopic appearance and the maternal origin affect oocyte quality for IVP. We have previously demonstrated that, when oocytes recovered in vivo by ovum pick-up (OPU) are submitted to the same IVP conditions, the blastocyst production rate varies according to the oocyte donor and is independent of the source of semen used [5]. This group of females, with known oocyte phenotypes for in vitro blastocyst production, provides a powerful means to further study factors affecting oocyte quality.

Because mitochondria are maternally inherited organelles and they are the main energy producer in the oocyte, it is possible to hypothesize their role in the aptitude of the oocyte to support in vitro embryo development. These organelles use the oxidative phosphorylation pathway to supply adenosine triphosphate (ATP) for all energy-requiring cellular activities. In oocytes and during the early stages of embryonic development, mitochondria are undifferentiated and generate ATP at relatively low levels when compared with the morula or blastocyst stages [6, 7]. The energy substrates (glucose, pyruvate, lactate) as well as oxygen tension and pH in the culture medium affect in vitro embryonic development through glycolysis and/or oxidative phosphorylation in mitochondria [8], revealing a clear relationship between metabolism and developmental competence. Adequate ATP reserves in oocytes and embryos are critical for normal nucleic acid and protein synthesis, and they have been suggested to be an indicator of the developmental potential of mouse [9, 10] and bovine embryos [11]. Furthermore, variations in ATP concentrations appear to be associated with embryo developmental competence in humans [6].

Mitochondria have the particularity of possessing their own genome. In mammals, mitochondria have retained a restricted set of independent genes in a circular genome of 16.6 kilobases encoding 13 proteins that are transcribed and translated in the mitochondrion. The remaining genes, approximately 80, involved in the respiratory chain are located within the nucleus and are governed by Mendelian rules of inheritance. Mitochondria and their DNA (mtDNA) are semiautonomous and replicate, divide, and fuse independently from somatic nuclear division [12]. Each oocyte contains a huge quantity of mitochondria in their cytoplasm [13, 14] each one containing one copy of the mtDNA. Recently, blastocyst formation has been linked to the number of mitochondria in the oocyte [15], where the average mitochondrial DNA copy number was significantly lower in cohorts of oocytes suffering from fertilization failure than cohorts with a normal fertilization. However, the influence of the quantity of mitochondria in the oocyte on blastocyst production is unknown.

Because mtDNA is exposed to free radicals generated by the electron transport chain and because its genome replicates frequently with a deficient DNA proofreading mechanism, its genome mutates rapidly [16, 17]. The control region (mtDNA-CR, also known as D-loop) is the most rapidly evolving region of the mtDNA with a mutation rate up to 20 times that of the nuclear genome [18, 19]. This results in approximately one mutation every 33 generations [19]. Specific mutations in the mtDNA-CR have been associated with the proportion of oocytes with high developmental ability recovered by OPU as well as with the rate of transferable nuclear-transfer embryos [20]. Additionally, mtDNA-CR polymorphisms allow the subdivision of animals in haplogroups that have been associated with calving rate in beef cattle [21].

Because of the uniqueness of maternal inheritance of mitochondria and their influences on reproduction, the goal of the present study was to quantify the mtDNA and ATP present in each oocyte recovered from animals previously selected for their IVP ability. In addition, it intended to characterize the sequence of the mtDNA-CR from the selected animals and compare it with family lines with reported mutations affecting in vitro embryo production.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Animals used in the present study were previously selected according to their IVP capacity [5]. They were six contemporary unrelated, herd-mate Holstein cows that received the same nutritional and health management from birth and throughout the experiment. Experimental animals will be referred to in the text as cows 1–6. All animal procedures and the experimental protocol were approved by the ENVA and the INRA committee for experimentation and ethical treatment of animals.

Ovum Pick-Up

Experimental animals had their oestrous cycles synchronized (CRESTAR method; Intervet, Angers, France) before oocyte recovery. Oocyte collection was performed twice a week (two sessions per week), starting 5 days after implant removal without any other additional hormonal treatment to stimulate follicular growth. Oocyte recovery was realized in three different series of 6, 10, and 6 wk over a period of 12 mo, for a total of 43 OPU sessions. Follicular aspiration was performed as described by Pieterse et al. [22]. In summary, cows were properly restrained and a low epidural anesthesia administered (5 ml of 2% lidocaine; Xylovet; CEVA sante animale, Libourne, France) 10 min before OPU. Ovarian follicles were identified and aspirated using an ultrasound scanner (Starvet 3; Pie Medical, Pouilly, France) equipped with a 7.5-MHz sectorial array transducer mounted on a probe, specially designed for OPU and placed in the cranial vagina. Follicular contents were collected into sterile conical tubes (one per animal) containing 10 ml of heparinized PBS (40 IU/ml) and were maintained at 37°C during the entire procedure.

Classification of Cumulus Oocyte Complexes

Cumulus oocyte complexes (COC) were identified after rinsing and filtering the recovered follicular fluid using a 100-µm mesh with warm (37°C) PBS. Once identified, COC were immediately placed into warm M199 (Sigma, St. Quentin-Fallavier, France) with 10% fetal calf serum (FCS) (Life Technologies, Cergy, France). COCs were classified according to their morphology into the following categorical grades: i) COC grade 1, corresponding to intact immature COC with three or more layers of dense cumulus cells and homogeneously granulated cytoplasm; ii) COC grade 2 have fewer layers of compact cumulus investment or are partially denuded oocytes with a healthy cytoplasm; and iii) COC grade 3 are oocytes that are completely devoid of cumulus cells but with normal cytoplasm. A fourth category, COC grade 4, included oocytes that did not fit into the first three categories, degenerated oocytes, oocytes with irregular ooplasm (presenting dark areas), as well as COCs with abnormal or expanded cumulus investments. COCs of this grade were discarded; therefore, the results represent the analysis of oocytes quality 1–3. Oocytes from slaughterhouse ovaries were used as controls for the ATP and mtDNA quantification studies. Ovaries were collected immediately after slaughter and transported to the laboratory in a temperature-controlled container. The content of the antral follicles between 2 and 8 mm was aspirated and recovered in a conical 50-ml tube containing 10 ml of M199 medium at 39°C. Oocytes were selected and rinsed before utilization. COC-1 to -3 (using the same classification as described above) were used as controls.

In Vitro Maturation

In vitro maturation conditions are described elsewhere [23]. Briefly, the COC grades 1, 2, and 3 from each animal were matured in vitro for 22–24 h in 50-µl microdrops of M199 supplemented with 10% FCS, 10 µg/ml FSH, 10 µg/ml LH, and 1 µg/ml estradiol 17ß (Sigma) over a layer of Vero cells (Rhone-Mérieux, Lyon, France) to improve maturation of denuded oocytes [24]. One microdrop was assigned to each cow and all oocytes collected from that animal in one OPU session were matured as a group. Oocytes from all six animals were processed at the same time and matured under controlled atmospheric conditions (5% CO2 in air).

Oocyte Storage

All oocytes were frozen, either before or after maturation, and stored until analysis. Each oocyte had its cumulus cells removed mechanically by gentle pipetting either before or after maturation. After maturation, oocytes at the metaphase II (MII) stage (expanded cumulus and first polar body) were treated with hyaluronidase (1 mg/ml in PBS; Sigma) for 3–4 min before cumulus extirpation. After being rinsed three times in filtered (0.2 µm) PBS (oocytes for mtDNA quantification) or ATP buffer (oocytes used for ATP measurement; 99.0 mM NaCl, 3.1 mM KCl, 0.35 mM NaH2PO4, 21.6 mM Na-lactate, 10.0 mM Hepes, 2.0 mM CaCl2, 1.1 mM MgCl2, 25.0 mM NaHCO3, 1.0 mM Na-pyruvate, 0.1 mg/ml of gentamicin), oocytes were loaded into 500-µl tubes for freezing. Oocytes used for ATP measurements were stored in 50 µl ATP buffer and frozen in liquid nitrogen, while those used for mtDNA quantification were stored in 100 µl PBS at –21°C.

ATP Quantification

ATP concentrations were measured in denuded oocytes at the germinal vesicle (GV) or MII stage, i.e., before or after in vitro maturation. The measurement was performed using a commercial assay kit based on the luciferin-luciferase reaction (Bioluminescent Somatic Cell Assay Kit, Fl-ASC; Sigma) following the technique described by Rieger [25] and the manufacturer's recommendations. Briefly, oocytes were thawed and kept on ice. One hundred microliters of ice-cold somatic cell reagent (FL-SAR reagent) were then added to the oocyte solution and incubated for 5 min on ice. Next, 100 µl of diluted ice-cold assay mix (FL-AAM reagent; 1: 25 with ATP assay mix dilution buffer, FL-AAB reagent) was added, and the tubes were kept for an additional 5 min at room temperature in the dark. The solution was then transferred into appropriate plastic tubes fitted for the high-sensitivity (0.01 pmol) luminometer (Bioluminat Junior; Berthold, Wildbad, Germany) and luminescence was measured. To obtain simultaneous measurements of samples, a set of tubes with oocytes was measured once (M1) and again (M2) in reverse order. The geometrical mean of the measures was calculated for final use. A seven-point standard curve (0–6 pmol/tube) was determined and, at every 20 oocytes, a negative control was run. The ATP content was calculated using the formula derived from the linear regression of the standard curve.

The mtDNA Sequencing of the Control Region

DNA was extracted from white blood cells and mtDNA control region sequenced after PCR amplification. Amplification was performed in 30 cycles as follows: one single denaturation step at 94°C for 90 sec followed by 35 cycles at 94°C (30 sec), 60°C (90 sec), and 72°C (90 sec). Reactions were performed using 0.5 U of TAQ-polymerase (QBIOGEN, France), 250 µM dNTP (Invitrogen, Cergy-Pontoise, France) 1.5 mM MgCl2, 12 pM of each primer (named mtDA and mtDB, Table 1) [26] and 2% formamide [27]. Sequence data were obtained after purification of PCR products (GENECLEAN Turbo for PCR Kit; Qbiogene, Carlsbad, CA) using the PCR primers mtDA and mtDB and four additional primers (named mtDAP, mtDBP, mtC, mtCP; Table 1). Primers were designed to overlap each other, creating sequencing triplicate of the amplified CR sequence. A consensus sequence was obtained from the overlapping sequences using the Pretty function of the Accelerys software (Web-based sequence analysis, version 2.1). Results were compared with the sequences of the control regions from bovine families with documented embryo production [20] in the GenBank (accession numbers AF386912 and AF386913) using the Pileup(+) from the same software package. The sequence obtained was used to create a dendrogram based on the differences found in the mtDNA control region, creating haplogroups.


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  		TABLE 1. Primers used for amplification and sequencing of the mtDNA control region

The mtDNA Real-Time PCR Quantification

DNA was extracted from each oocyte with the High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany) according to the manufacturer's recommendations. Quantification of mtDNA was then performed using a real-time polymerase chain reaction (PCR) method. Briefly, a Roche LightCycler was used to determine the mtDNA copy number using LightCycler-Faststart DNA master SYBR Green 1 kit (Roche). Twenty liters of PCR reaction mixtures were prepared as follows: 1x buffer containing 4 mM MgCl2, 0.2 mM dNTPs, 0.5 µM of both primers (DC3 and RC1; Table 1), SYBR green I dye, 0.25 U hot start Taq DNA polymerase, and 10 µl of the extracted DNA or 10 µl of standard with a known copy number. The external standard used for mtDNA quantification was the corresponding 189-base-pair PCR product cloned into PCR 2.1-TOPO vector (Invitrogen, Life Technologies, Groningen, Netherlands). The PCR reactions were performed with an initial denaturation at 95°C for 7 min and 40 cycles at 95°C for 1 sec, 56°C for 5 sec, and 72°C for 13 sec. 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 and 94°C) was analyzed to check the specificity of the PCR product. For each run, a standard curve was generated using five 10-fold serial-dilutions (10–100 000 copies) of the external standard. This curve allowed the determination of the starting copy number of mtDNA in each sample.

Statistical Analysis

The mean number of oocytes recovered was compared between haplogroups and between animals using ANOVA. A retrospective analysis of in vitro blastocyst production at Day 8 after in vitro fertilisation [5] was performed to evaluate the haplogroup effect on blastocyst rate. ATP contents as well as the quantities of mtDNA were compared using ANOVA followed by a Bonferroni post hoc test using SPSS software (SPSS, Chicago, IL). Due to the large intra-animal variations found in the mtDNA copy numbers, the coefficient of variation (CV; SD/mean) was calculated to allow a better estimation of interanimal variation. Results are reported as means ± SEM (or SD when indicated) with a significance level of 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of the mtDNA Control Region

The CR sequencing of all six cows revealed 21 mutations sites, with 13 of them differing from those published by Bruggerhoff et al. [20] (Fig. 1) conferring six different genotypes. The only insertion was in a cytosine run at the 3' end of the CR region prone to slippage. The other mutation sites are transitions, including one at position 191 in the stem site preceding the D-loop. At position 350, a C/T transition was identified in all six animals. Pairwise comparison of the genotypes of the animals selected for this study with those of families with different aptitude for embryo production by multiple ovulation embryo transfer (MOET) and nuclear transfer cloning [20] generated a dendrogram (Fig. 2). This dendrogram divides the animals in two major haplogroups, and this information was used in the statistical analyses. Haplogroup H-146 is composed of cows 1, 4, and 6 and family line AF386912, while haplogroup H-235 included family line AF386913 and cows 2, 3, and 5.



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  		FIG. 1. Complete mtDNA CR sequence. Previously described mutations (PDM) and different mutation sites (NMS) are punctually identified. The stem-loop structure, the control of the L-strand transcription and the D-loop strand termination site (term), as well as the sequence element (TAS-A) associated with the bovine D-loop strand terminations are marked. The conserved sequence block (CSB) 1 and 2+3, the promoters for the H- and L-strand transcription (HSP and LSP), and the origin of the H-strand replication (OH) are also identified. The mtDNA sequences for each cow have been submitted to the GenBank



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  		FIG. 2. Multiple alignment dendrogram based on mutations in the mtDNA control region; oocyte donor cows are compared with family lines with known mtDNA control region mutations previously associated with oocyte production by OPU and in vitro embryo production by somatic cell nuclear transfer cloning

The mtDNA control regions sequences for each cow was submitted to GenBank and can be found by the accession numbers AY495575, AY495576, AY495577, AY495578, AY495579, and AY495580.

Oocyte Recovery

Nine hundred and fifty-one oocytes were recovered from 2488 follicles (mean of 9.6 ± 0.2 follicles/animal) with cow 2 producing significantly more oocytes (5.3 ± 0.4 oocytes/ session) than the other animals (3.3 ± 0.4, 2.8 ± 0.3, 3.8 ± 0.3, 3.2 ± 0.3, and 3.7 ± 0.3 oocytes/session for cows 1, 3, 4, 5, and 6, respectively). Haplogroup did not have any effect on oocyte recovery, with 3.6 ± 0.2 and 3.8 ± 0.2 oocytes/session for H-146 and H-235, respectively.

Oocyte ATP Reserve

Mean ATP concentration was 2.5 ± 0.05 pmol/oocyte at the GV stage (coefficient of variation = 32.0%; n = 256) and 2.4 ± 0.05 pmol/oocyte at the MII stage (coefficient of variation = 33.3%; n = 245). Although no difference in overall ATP was observed at the two stages of maturation, comparisons of ATP (before and after maturation) between haplogroups and between animals shows significant variations. Changes in ATP stored between GV and MII shows that cow 6 and control group increased their ATP stores (P < 0.05) at MII, while a decrease was observed for cows 1, 2, 3, and 5 (P < 0.05 for cows 5 and 3); ATP stored in oocytes from cow 4 remained virtually unchanged (Table 2). Haplogroup 146 and H-235 differed in ATP contents at both GV and MII stages (Table 3).


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  		TABLE 2. ATP content and blastocyst production; animals are ranked by blastocyst rate and haplogroup


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  		TABLE 3. The mtDNA and ATP contents of oocytes for each haplogroup (mean ± SEM).*

Mean ATP in oocytes recovered by OPU at GV (2.7 ± 0.05 pmol/oocyte, coefficient of variation = 25.2%, n = 204) was significantly higher than ATP from slaughterhouse oocytes (1.5 ± 0.07 pmol/oocyte, coefficient of variation = 32.6%, n = 52). This difference (P < 0.05) persisted at the MII stage (2.5 ± 0.06 pmol/oocyte, coefficient of variation = 31.7%, n = 159 and 2.0 ± 0.06 pmol/oocyte, coefficient of variation = 27.0%, n = 86 for OPU and slaughterhouse ovaries, respectively). OPU oocytes showed a significant decrease in ATP stocks after maturation (2.7 ± 0.05 to 2.5 ± 0.06 pmol/oocyte), while control oocytes had a significant increase (1.5 ± 0.07 to 2.0 ± 0.06 pmol/ oocyte) in ATP stocked in the cytoplasm (Table 2).

ATP concentrations were not statistically different between different morphological quality COC at the GV stage (COC-1: 2.6 ± 0.07 pmol/oocyte, n = 119; COC-2: 2.4 ± 0.08 pmol/oocyte, n = 99; COC-3: 2.4 ± 0.1 pmol/oocyte, n = 38) or at the MII stage (COC-1: 2.4 ± 0.08 pmol/ oocyte, n = 76; COC-2: 2.4 ± 0.07 pmol/oocyte, n = 128; COC-3: 2.2 ± 0.1 pmol/oocyte, n = 41).

Overall ATP stocks in the two haplogroups (all cows in each haplogroup combined) differed significantly both at the GV stage (2.8 ± 0.06 pmol/oocyte, n = 101; and 2.6 ± 0.07 pmol/oocyte, n = 103, for H-146 and H-235, respectively) and at the MII stage (2.9 ± 0.1 pmol/oocyte, n = 76; and 2.3 ± 0.06 pmol/oocyte, n = 83, for H-146 and H-235, respectively; Table 3).

Quantification of mtDNA

On average, each of the oocytes analyzed carried 377 327 ± 256 607 copies of mtDNA (mean ± SD; Table 4). Although the two haplogroups did not differ significantly in mtDNA (Table 3), there was a significant variation among animals within haplogroup H-235 (Table 4). No effect of oocyte quality was observed in mtDNA copy numbers. COC-1 and 2 had 379 117 ± 14 980 copies/oocyte (n = 282) and were not different from COC-3 (367 022 ± 40 938 copies/oocyte, n = 49). Nevertheless, mtDNA show a nonsignificant trend toward a lower quantity of mtDNA as oocyte quality decreases. However, a very large intra-animal variation in mtDNA copy number was observed (range from 2 000 to 1 ;200 000 copies/oocyte), which was shown by the large coefficient of variation values (Table 4). Quantities of mtDNA did not differ between oocytes recovered by OPU (382 938 ± 15 341 copies/oocyte) or from slaughterhouse ovaries (337 639 ± 34 304 copies/oocyte; P > 0.05).


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  		TABLE 4. mtDNA content and blastocyst production; animals are ranked by blastocyst rate and haplogroup.*


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The objective of this study was to examine whether oocyte ATP and mtDNA contents vary between cows and if these parameters could explain the oocyte donor effect observed on blastocyst production [5]. Furthermore, it was also intended to observe whether polymorphisms in the mtDNA-CR of these selected animals matched those that have previously been linked to in vitro fertility [20].

Mitochondrial DNA sequencing analysis has been widely used in evolutionary studies as a molecular clock to identify common female ancestor. Cattle lineages have been characterized based on mtDNA-CR mutations [28, 29], and many studies have suggested that maternal lineage affects production and reproduction traits of cattle [30–33]. Schutz et al. found that polymorphisms in the CR was associated with the number of days open in dairy cattle [34], while more recently, mtDNA polymorphisms have been associated with calving rate in beef cattle [21]. Sequencing of the mtDNA-CR in this study revealed that the six animals that were selected for having oocytes with different abilities for IVP had six different mtDNA-CR genotypes. This enabled, after pairwise alignments, the separation of these animals into two haplogroups, and analysis of blastocyst production showed that animals in H-146 had significantly lower blastocyst rate at Day 8 than H-235 (22.3% ± 2.1% and 36.7% ± 2.9%, respectively). Further statistical analysis showed that animals in H-235 had a higher blastocyst rate at Days 6 and 7 (P < 0.05, no difference detected at Day 8) than animals in H-146, indicating a difference in kinetics of blastocyst formation. This suggests the existence of differences in embryo quality between the two haplogroups, as early blastocyst formation is a sign of embryo quality [35]. However, we cannot confirm this observation because blastocyst quality was not evaluated.

These two haplogroups were then compared (based on polymorphisms in the mtDNA control region) with the two family lines AF386912 and AF386913 described in Germany as having different in vitro fertility [20]. In contrast with the findings reported by Bruggerhoff et al. [20], we observed no difference in the number of oocytes collected or variation in oocyte morphological quality between haplogroups. The different experimental design employed by Bruggerhoff et al. (e.g., paternally related individuals in two haplotype groups, each group derived from a single founder female and thus with completely identical mtDNA type), in addition to OPU frequency, equipment, and the oocyte grading system used, may account for this difference in results. Mitochondrial DNA phenotypes are strongly dependent on the nuclear genetic background [36–38], and the different breeds employed by Bruggerhoff et al. (Simmental) and in the present study (Holstein) could explain some of the observed differences. However, comparison of embryo production by MOET [20] shows that family line AF386912 produced roughly three times fewer embryos than AF386913; those results are complimentary to ours. It is nevertheless striking that an mtDNA haplotype that was previously associated with superior cytoplasm for embryo production in somatic cell nuclear transfer (SCNT) was assigned to haplogroup H-146 of the present study, as this haplogroup yielded a significantly lower blastocyst rate in IVP. Conversely, an mtDNA haplotype previously associated with inferior cytoplasm in SCNT was assigned to our haplogroup H-235, which yielded a significantly higher blastocyst rate in IVP. This points to fundamental differences in cytoplasmic factors important for SCNT and IVP embryo production. The ability to produce embryos in vitro from oocytes with defined mtDNA polymorphisms may in the future serve as a tool for the selection of oocyte donor animals superior for IVP. We acknowledge that a small number of animals were used in this experiment and that a more comprehensive study is needed. It will be performed as more animals with different phenotypes for IVP are identified.

In this study, we explored the relationship between in vitro embryo developmental competence not only with specific mtDNA mutations but also with mtDNA oocyte content. During oocyte growth, as the cytoplasm increases in volume, so does the number of mitochondria [39, 40], with a mature oocyte containing on average 1.6 x 105 and 3.1 x 105 mtDNA molecules in mouse and human oocytes, respectively [13]. However, the absolute quantity of mtDNA copies in a human oocyte is quite variable. Some authors published mean values of 1.9 x 105 copies/oocyte [15] while others 8.0 x 105 copies/oocyte [14]. The present results showed a mean 3.7 x 105 mtDNA per bovine oocyte and a very large intra-animal variation. Other authors have also reported a large variation between oocytes from the same individual [13, 15]. The mean quantity of mtDNA copies per oocyte reported here in the cow is not very different from the 2.6 x 105 reported by Michaels et al. [41], but if we consider the mean quantity of mtDNA in oocytes from each cow individually, the difference varied between 1.2- to 2.1-fold.

The quantity of mitochondria in the oocyte affects its ability to produce ATP [42], to escape atresia [43], and to support embryo development [15]. However, our data did not provide any evidence that the quantity of mitochondria influences embryo production. This is confirmed by the observation that, despite a significant difference in blastocyst formation rate, the two haplogroups had equivalent numbers of mtDNA per oocyte. Our data show that oocyte morphological quality is a poor indicator of the quantity of mitochondria in the cytoplasm. A trend toward a lower quantity of mtDNA in oocytes of poorer quality was observed, but it was not statistically significant. Additionally, the best and the worst blastocyst producer had virtually the same mean number of mtDNA copies, and the animal with the highest numbers of mitochondria (cow 3) only had an average embryo production rate. The similar quantity of mtDNA between oocytes from OPU and slaughterhouse indicate that the time elapsed between slaughter and oocyte collection does not affect the quantity of mtDNA. Nevertheless, a very large variation in the number of mtDNA per oocyte was evidenced between animals; this large variation demands further investigation to permit us to correlate it with the also large intra-animal variation usually observed for in vitro blastocyst production. To observe a definitive effect of the quantity of mitochondria on IVP, taking into consideration the large interoocyte intra-animal variation, one should perform mtDNA quantification and IVF on the same oocyte. This is practically impossible using the techniques described above. The real significance of the number of mitochondria in the oocyte, on bovine in vitro embryo production, remains to be proven.

Mitochondrial activity is responsible for ATP production and energy accumulation during oogenesis, which is a crucial factor for successful development [6, 44, 45]. Oocyte ATP contents have been suggested to be an indicator for the developmental potential of human, mouse, and bovine oocytes [6, 11, 46, 47]. Although oocyte mitochondria are small, round to oval in shape, microscopically dense, and contain a few underdeveloped cristae, which are inherent characteristics of cells with low metabolic rate [7, 48], they can produce enough ATP to sustain embryo development. ATP equilibrium (production vs. consumption) was studied by comparing ATP stocks at GV and MII stages. Our results show that the ATP stored in the oocyte was not influenced by oocyte quality (either at GV or MII), in opposition to the results observed by Stojkovic et al. [11], where a significant difference between COC-1 and COC-3 was found. The reason for this difference could be related to the differences in the grading systems used. At MII, the results of the two studies are in agreement and both studies observed an increase in ATP stocks in oocytes from slaughterhouses. The ATP reserves in slaughterhouse oocytes at the GV stage were significantly smaller than oocytes from OPU. This is probably due to the period of nutrient deprivation to which they were submitted between slaughter and arrival in the laboratory. However, ATP reserves were restored to OPU levels after in vitro maturation. This significant increase after in vitro maturation indicates that mitochondria are functional and support embryo development at a rate of 45.7% (blastocyst rate with oocytes collected at the same time as those used for ATP measurement in our IVF laboratory, data not shown). Nevertheless, it is known that mouse oocytes can sustain maturation with very low net ATP reserves [6]. The ATP levels observed in the present experiment on slaughterhouse oocytes were similar to those reported by others, either before or after in vitro maturation [6, 11]. An interesting phenomenon was observed for ATP levels at the two maturation stages studied in the two haplogroups. Haplogroup H-235, with a significantly higher blastocyst rate, had significantly higher stores of ATP at the GV stage than at the MII stage, indicating that ATP utilization is beyond the oocyte capacity to replenish its reserves, while in haplogroup H-146, these values were not statistically different. The two haplogroups were also different in their ATP reserves at MII stage, with more ATP in the haplogroup with higher embryo production. We also showed that ATP reserves behaved differently between cows; ATP contents increased, decreased, or remained constant between the GV and MII stages. This demonstrates that the oocyte donor influenced the ATP contents of oocytes at GV and MII. Cow 5 (52.4% blastocyst rate) and cow 6 (20.3% blastocyst rate) had the same levels of ATP at GV but there was a marked difference at MII. This raises questions about mitochondrial function. It is unclear if the observed increase in ATP for cow 6 is related to production of ATP that surpasses consumption or due to decreased utilization of ATP by a metabolically compromised oocyte. The fact that embryonic metabolism remains constant in normally developing oocytes until the 12- to 16-cell stage [49] suggests that, on the one hand, oocytes from this animal are not consuming enough ATP, resulting in positive net ATP production; on the other hand, oocytes from cow 6 had significantly more mtDNA than the average oocyte (33% more mtDNA than cow 5). Cow 4, with a blastocyst rate of only 12.4%, had the same level of ATP before and after maturation. This could be due to a perfect equilibrium between production and consumption of ATP or due to a metabolically and developmentally incompetent oocyte. Future studies using differential staining of mitochondria can help in the interpretation of this finding.

In the present study, we showed that genetic variation in the mtDNA-CR successfully generated haplogroups with different phenotypes for in vitro embryo production. Haplogroups also differed in their ATP levels at GV and MII. Oocyte ATP reserves and oocyte energy metabolism were distinctly different between animals; the significance of this variation is still unclear but should be clarified as more animals, selected for blastocyst production, become available. A maternal family line study could confirm the repeatability of the results reported here. It is clear that oocytes that grew in follicles large enough to be aspirated by OPU at a 3- to 4-day interval have enough mitochondria to sustain development. Variations in in vitro embryo production, if related to the oocytes' mitochondria population, would mainly be due to factors other than inadequate oocyte mitochondrial load. Nevertheless, a large intra-animal variation in the quantity of mtDNA was observed.


   ACKNOWLEDGMENTS
 
The authors thank Dr. A. Ponter for his critical reading of the manuscript and comments. We thank also P. Stojkovic for her help with the ATP quantifications and for her kindness. Additionally, the authors gratefully acknowledge the help provided by the OPU technical team at the INRA experimental station of Bressonvilliers. We would like to thank Y. Lavergne, C. Richard, and V. Gelin for their help during this study. Without their expertise, this work would have been handicapped.


   FOOTNOTES
 
1 Correspondence: M. Tamassia, 40 av. D'Italie 12.3, 75013 Paris, France. FAX: 33 1 43967130; mtamassia{at}vet-alfort.fr Back

Received: 2 December 2003.

First decision: 19 December 2003.

Accepted: 9 April 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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