Adenosine triphosphate synthesis, mitochondrial number and activity, and pyruvate uptake in oocytes after gonadotropin injections

Adenosine triphosphate synthesis, mitochondrial number and activity, and pyruvate uptake in oocytes after gonadotropin injections Seung Tae Lee, Ph.D.,a Seo Jin Oh, B.Sc.,a Eun Ju Lee, Ph.D.,a Ho Jae Han, Ph.D., D.V.M.,b and Jeong Mook Lim, Ph.D., D.V.M.a a Department of Food and Animal Biotechnology, Seoul National University, Seoul; and b College of Veterinary Medicine, Chonnam National University, Kwangju, Korea

Objective: To determine the effects of gonadotropin injection on the energy generation of mature oocytes. Design: Randomized prospective study. Setting: Gamete and stem cell biotechnology laboratory at Seoul National University in Korea. Animal(s): Twelve- to 15-week-old golden hamsters (Mesocricetus auratus). Intervention(s): Injections of pregnant mare serum gonadotropin (PMSG; 5 or 15 IU), of hCG (5 or 15 IU), or of PMSG and hCG (15 IU of each; PMSG ⫹ hCG group) were administered to female hamsters. Main Outcome Measure(s): Adenosine triphosphate (ATP) synthesis, mitochondrial population number and activity, and pyruvate uptake were measured. Result(s): Significant (P⬍.05) differences were found in the ATP levels; compared with the control (no injection), a dramatic increase was detected after injections of 15 IU of hCG or of 15 IU of PMSG and 15 IU of hCG. In the same treatments, the mitochondrial population (mitochondrial DNA copy number) significantly increased, whereas mitochondrial activity measured by the ratio of activated to less-activated mitochondria did not change. A significant increase in pyruvate uptake was detected after the injections of 15 IU of PMSG and 15 IU of hCG. Conclusion(s): The change in ATP synthesis activity was a major cause for the adverse effect of gonadotropins on oocyte development in the hamster. The injections of 15 IU of hCG, or of 15 IU of PMSG and 15 IU of hCG, dramatically increased the ATP level, the mitochondrial population number, and pyruvate uptake. (Fertil Steril威 2006;86(Suppl 3):1164 –9. ©2006 by American Society for Reproductive Medicine.) Key Words: Hamster oocyte, gonadotropin, ATP, mitochondria, pyruvate

Since 2002, we have been developing a gamete manipulation method using the golden hamster (Mesocricetus auratus). The golden hamster is a model animal that is used widely in medical and biotechnological experimentation, and both cyclicdependent ovarian hyperstimulation and cyclic-independent superovulation techniques have been established with the use of this animal (1, 2). In contrast to the conventional protocol of using 15 IU of pregnant mare serum gonadotropin (PMSG), these newly developed programs use reduced doses of PMSG (5 IU) and hCG (5 IU). The cyclic-dependent technique that uses PMSG alone optimizes preimplantation development (2), whereas the cyclic-independent method that uses PMSG and hCG can be used for the expanded application of superovulation treatments in animals in any estrus

Received October 6, 2005; revised and accepted January 1, 2006. Supported by a grant (SC-2170) (Lim JM) from the Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Gwachen, Korea, and by a graduate fellowship (Lee ST, Oh SJ) from the Korean Ministry of Education, Brain Korea 21 project. Reprint requests: Jeong Mook Lim, Ph.D., D.V.M., Laboratory of Gamete and Stem Cell Biotechnology, Department of Food and Animal Biotechnology, Seoul National University, Building 200-#4223, Sillim-9 Dong, Seoul 151-921, Korea (FAX: 822-874-2555; E-mail: [email protected]).

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cycle (1). The number of blastocysts per treated animal was markedly improved using these new techniques. In our articles published elsewhere (1, 2), we described in detail the adverse effects of gonadotropins on oocyte development as manifested in cleavage failure and decreased proliferation and differentiation; injecting high doses of gonadotropins (15 IU of hCG with or without 15 IU of PMSG) also induced microfilament disorganization and aberrant distribution of cortical granules. The microfilament dynamics in oocytes require energy, and our previous findings on impaired microfilament formation after gonadotropin injections may have been attributable to an altered energy-generation system in oocytes (3). Thus, we performed further experiments to clarify the role of gonadotropin injections in the energy generation of oocytes. Using the same experimental treatments [injections of 5 or 15 IU of PMSG, of 5 or 15 IU of hCG, and of 15 IU each of PMSG and hCG (PMSG ⫹ hCG group)] as in our study published elsewhere (2), we measured the total adenosine triphosphate (ATP) synthesis after the treatments. Mitochondrial activity, the number of mitochondria, and the pyruvate uptake in oocytes, which is related to ATP synthesis, were monitored further. To manifest gonadotropin effect, the 5-IU PMSG injection that yielded the optimal rate of blastocyst formation in the

Fertility and Sterility姞 Vol. 86, Suppl 3, October 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.

0015-0282/06/$32.00 doi:10.1016/j.fertnstert.2006.01.059

cyclic-dependent method was used as the positive control treatment. MATERIALS AND METHODS Preparation for Experiments Male and female golden hamsters were purchased from Japan SLC (Shizuoka, Japan) and Harlan Sprague Dawley (Indianapolis, IN), respectively. The hamsters were bred in our laboratory under controlled lighting (14:10-hour light– dark cycle), temperature (20 –22°C), and humidity (40%– 60%). All procedures for animal management, breeding, and surgery followed the standard operating protocols of Seoul National University. The Institutional Review Board for Animal Research of the Department of Animal Science and Technology, Seoul National University, approved our research proposal and the relevant experimental procedures. The appropriate management of experimental samples, animal care and use, and quality control of the laboratory facility and equipment were maintained. Preparation of Oocytes Twelve- to 15-week-old female golden hamsters weighing 110 –130 g were used for this study. For ovarian hyperstimulation, PMSG (Folligon; Intervet International, Boxmeer, the Netherlands) was injected intraperitoneally, and ovulation was induced by intraperitoneal injection of hCG (Pregnyl; Organon, Oss, The Netherlands). Sexual behavior was monitored for signs of estrus, and vaginal smears were collected for determining the stage of the estrus cycle before treatment. Hamsters in estrus were relegated to the control group (natural cycle, without PMSG or hCG injections); those in metestrus, diestrus, and proestrus were treated with PMSG or hCG at the various experimental doses. Subsequently, oocytes were retrieved from unmated females by flushing the oviduct 15 hours after hCG injection (hCG and PMSG ⫹ hCG treatment groups), 95 hours after PMSG injection (PMSG treatment group), or 109 hours after the detection of estrus (control group). Experimental Design In this study, female hamsters were treated as follows: group 1 received no injections (control); groups 2 and 3 received injections of 5 or 15 IU of hCG, respectively, on day 4 of estrus; groups 4 and 5 received 5 or 15 IU of PMSG, respectively, on day 1 of estrus; group 6 received a 15-IU PMSG injection during all cycles except estrus plus a 15-IU hCG injection 56 hours after the PMSG injection. In experiment 1, the quantification of ATP synthesis in a single treated oocyte after gonadotropin injection was determined using the AutoLumat LB 953 Luminometer (EG&G Berthold, Bad Wildbad, Germany). In experiment 2, the changes in mitochondrial activity (on the basis of the ratio of activated to less-activated mitochondria) and the mitochondrial population number were determined by counting the mitochondrial DNA (mtDNA) copies in the ooplasm under a Fertility and Sterility姞

confocal microscope (MRC-1024; Bio-Rad, Hemel Hempstead, Hertfordshire, United Kingdom) and by real-time PCR (Bio-Rad iCycler system; Bio-Rad, Hercules, CA), respectively. In experiment 3, the pyruvate uptake of oocytes after the gonadotropin treatments was measured by a radioimmune assay that used 14C-labeled pyruvate. Media The medium used to collect oocytes and embryos was modified hamster basic medium (mHBM)-5, consisting of 113.8 mM NaCl, 3 mM KCl, 25 mM N-2-hydroxyethylpiperazineN=-2-ethanesulfonic acid, 4.5 mM DL-sodium lactate, 1 mM CaCl2●2H2O, 2 mM MgCl2●6H2O, 1.0 mg/mL of gentamicin, and 0.1 mg/mL of polyvinyl alcohol. The basic medium used for embryo culture was protein-free, chemically defined hamster embryo culture medium-10, consisting of 113.8 mM NaCl, 3 mM KCl, 25 mM NaHCO3, 4.5 mM DL-sodium lactate, 1 mM CaCl2●2H2O, 2 mM MgCl2●6H2O, 0.01 mM asparagine, 0.01 mM aspartate, 0.01 mM cysteine, 0.01 mM glutamate, 0.2 mM glutamine, 0.01 mM glycine, 0.01 mM histidine, 0.01 mM lysine, 0.01 M proline, 0.01 mM serine, 0.5 mM taurine, 0.003 mM pantothenate, and 0.1 mg/mL polyvinyl alcohol (4). All substrate media were purchased from Sigma-Aldrich Corp. (St. Louis, MO), unless otherwise stated. The osmolarity of the prepared media was adjusted to within the range of 270 to 280 mOsm. Media were prepared every 2 weeks and were stored at 4°C until use. Collection of Oocytes Cumulus– oocyte complexes were retrieved from unmated females by flushing the oviducts at different times after gonadotropin injections. To remove cumulus cells, the collected cumulus– oocyte complexes were incubated in mHBM-5 containing 0.5 mg/mL hyaluronidase at 37.5°C for approximately 5 minutes and then were washed several times in equilibrated mHBM-5 medium. Subsequently, oocytes were randomly assigned to each treatment group. Quantification of ATP Synthesis The ATP level of oocytes assigned to each treatment group was measured by a commercial assay kit on the basis of the luciferin–luciferase reaction (Bioluminescent Somatic Cell Assay kit, FL-ASC, Sigma-Aldrich, St. Louis, MO) (5). Briefly, oocytes were thawed and placed into 100 ␮L of ice-cold somatic cell reagent (FL-SAR, Sigma-Aldrich) for 5 minutes. Oocytes were replaced in 100 ␮L of diluted icecold assay mix (FL-AAM reagent, diluted 1:25 with ATP assay mix dilution buffer, and FL-AAB reagent; both, SigmaAldrich) for an additional 5 minutes, and the solution was transferred into an AutoLumat LB 953 Luminometer (EG&G Berthold) for measuring luminescence. A sevenpoint standard curve (0 –5 pmol per tube) was determined for every 20 oocytes, and the ATP content was calculated using the formula from the linear regression of the standard curve. 1165

Quantification of mtDNA by Real-Time Polymerase Chain Reaction Primary3 software (Whitehead Institute/MIT Center for Genome Research, Stamford, CT) was used to design all specific primers used in this study. For quantification of mtDNA, polymerase chain reaction (PCR) primers for mtDNA sequences also were designed with DNA sequences for golden hamster mtDNA (6). The primer specificity was tested by running a regular PCR for 40 denaturation cycles of 95°C for 30 seconds, annealing at 60°C for 45 seconds, and extension at 72°C for 30 seconds. Primer sequences (Genbank number J01864) were 5=-TGAAACTTAGAGGACAAAGGA-3= for sense and 5=-TTAGTCCAAGCACACTTTCC-3= for antisense primers at 58°C for 203 base pairs. To prepare mtDNA in the retrieved oocytes, the zona pellucidae of single oocytes were removed using 0.5% (wt/ vol) protease, and the washed oocytes were transferred to a 1.5-mL microtube and lysed by adding 20 ␮L of lysis solution consisting of 1⫻ PCR buffer (50 mmol/L KCl and 10 mmol/L Tris, pH 8.4), 100 ␮L/mL of proteinase K, and 0.5 ␮L of Triton X-100. The extraction mixtures were incubated for 30 minutes at 55°C, followed by 5 minutes at 100°C, and then the mixtures were processed by centrifuge at 1,200 ⫻ g for 5 minutes. The supernatant was used as the template DNA for the PCR (7). A Bio-Rad iCycler system was used to determine the number of mtDNA copies. The PCR was performed in a volume of 20 ␮L, consisting of 10-␮L SYBR Supermix kit (Roche Diagnostics, Penzberg, Germany) and 10 ␮L of the extracted DNA or 10 ␮L of a standard with a known copy number. The DNA was denatured at 95°C for 2 minutes, and then amplification proceeded for 41 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 45 seconds, and extension at 72°C for 30 seconds. The fluorescence data were acquired during the extension for the reactions containing SYBR Green I, and a melting curve was systematically analyzed to detect mispriming and to determine amplification quality. For each PCR run, a standard curve was generated using seven 10-fold serial dilutions (10 –108 copies) of the target mtDNA PCR product and the same primers that were used for oocyte mtDNA amplification. The iCycler software generated a standard curve, which then allowed the determination of the initial copy number for mtDNA in each sample and the calculation of the total mtDNA content in each oocyte. Evaluating the Ratio of Activated to Less-Activated Mitochondria Using Confocal Microscopy We used the potential-sensitive fluorescent dye JC-1 (Molecular Probes, Eugene, OR) to examine the activity of mitochondria in the treated oocytes. The JC-1 was dissolved to a stock concentration of 0.5 mM in dimethyl sulfoxide (DMSO) and diluted into hamster embryo culture medium-10 medium, with vortexing (8). The oocytes were exposed to 1166

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JC-1 at 37.5°C with 5% CO2 in a humidified incubator for 1 hour and then were washed three times in mHBM-5 medium to remove surface fluorescence. Stained oocytes were placed onto a coverslip forming the base of a dish containing polyvinyl alcohol–free hamster embryo culture-10 medium. To obtain the confocal image, we used a krypton–argon mixed-gas laser. The excitation laser line was set at 488 nm, and the emission wavelengths were separated by a 530-nm diachronic mirror, with further filtering through a 515- to 530-nm band-pass (green emission) or a 585-nm long-pass (red emission) filter. Using the confocal image, the ratio of the fluorescence intensity at 527 and 590 nm was measured by the LaserFix program (Bio-Rad, Hemel Hempstead, United Kingdom). The mitochondria with high membrane potential (activated) fluoresced green, whereas the mitochondria with low membrane potential (less activated) fluoresced red. Assessment of Pyruvate Uptake To assess pyruvate uptake, the culture medium was removed by aspiration, and the oocytes retrieved after gonadotropin injections were washed twice in mHBM-5 medium. After subsequent incubation at 37°C for 30 minutes in an uptake buffer containing 10 ␮Ci/mL of 14C-labeled pyruvate, the oocytes were washed with ice-cold uptake buffer and were solubilized in 1 mL of 0.1% SDS. To determine the pyruvate uptake incorporated intracellularly, 900 ␮L was removed from each sample and counted in a liquid scintillation counter (LS 6500; Beckman Instruments Inc., Fullerton, CA). The counts in each sample were normalized with respect to protein and were corrected for zero-time uptake per oocyte. Statistical Analysis Significant effects were detected by analysis of variance using the Statistical Analysis System (SAS) program (PROC–GLM model), and comparisons among treatment groups were subsequently conducted using the least-squares or Duncan methods. A value of P⬍.05 was taken to indicate a significant difference among treatments. RESULTS Experiment 1: ATP Level in Oocytes After Gonadotropin Treatments As shown in Figure 1, a significant (P⫽.0001) treatment effect on ATP synthesis was detected. Adenosine triphosphate synthesis activity was greater in oocytes retrieved after the injection of 15 IU of hCG, with or without prior administration of 15 IU of PMSG, than in control oocytes with no injected gonadotropin (0.0560 – 0.0562 vs. 0.0538). Increasing the dose of PMSG to 15 IU in the absence of injected hCG significantly reduced the ATP level compared with that of control oocytes (0.0509 – 0.0521). Vol. 86, Suppl 3, October 2006

FIGURE 1 Quantification of ATP in oocytes retrieved from female golden hamsters with or without the injection of PMSG and/or hCG. Significant increases in ATP production were detected in oocytes after the injection of 15 IU of hCG, or of 15 IU each of PMSG and hCG, compared with the control (no injection). However, less ATP production was observed in the oocytes after the injection of 5 or 15 IU of PMSG than in the controls. Different lowercase letters indicate a significant difference (P⬍.05).

Lee. Gonadotropin and ATP production in oocyte. Fertil Steril 2006.

Experiment 2: Activity and Number of Mitochondria in Oocytes After Gonadotropin Treatments No combination of gonadotropin injections significantly changed the total mitochondrial activity of treated oocytes. No significant differences in mitochondrial activity were detected among treatments (1.021–1.182; Fig. 2) and the ratio of activated to less-activated mitochondria did not change after the treatments. As shown in Figure 3, however, more mitochondria were counted in the group injected with 15 IU of hCG, or 15 IU of hCG and 15 IU of PMSG, than in the control oocytes (367,249 –561,230 vs. 19,929 mtDNA). The number of mitochondria in oocytes retrieved after the injection of 5 IU of hCG, 5 IU of PMSG, or 15 IU of PMSG was not significantly different from that in the control oocytes.

DISCUSSION The results of this study demonstrate that exposing intrafollicular oocytes to exogenous gonadotropins alters the energy generation system in oocytes. The total ATP level in a single treated oocyte dramatically increased after the injection of 15 IU of hCG, with or without prior administration of 15 IU of PMSG. However, this increase may not have resulted from an increase in mitochondrial activity because the ratio of activated to less-activated mitochondria in an individual oocyte did not differ among treatments. In contrast, 15 IU of hCG, or 15 IU of PMSG and 15 IU of hCG, induced a sharp increase in the number of mitochondria. The uptake of pyruvate, which is the energy substrate in mitochondria, also increased with the same treatments. Several studies have investigated the cellular and physiological effects of gonadotropins (5, 9 –16). In the golden hamster, we recently reported (1, 2) that the administration of high doses of PMSG and/or hCG, which are conventionally applied for ovarian hyperstimulation and ovulation induction, was detrimental to embryogenesis. Cell cleavage requires a great deal of energy, and ATP generation is a prerequisite for mobilizing the microfilament system for cell diakinesis, followed by blastomere cleavage (17). Our preliminary transmission electron microscopy study (1) demonstrated that a combined dose of 15 IU of PMSG and 15 IU of hCG can alter the mitochondrial distribution, and a mitochondria-less region was produced in the ooplasm. On the basis of these previous findings, we postulated that go-

FIGURE 2 Mitochondrial activity of oocytes retrieved from female golden hamsters with or without the injection of PMSG and/or hCG. Activity was evaluated by measuring the ratio of activated to less-activated mitochondria in a confocal image with JC-1 staining. There was no significant (P⫽.2209) difference among the treatment groups.

Experiment 3: Pyruvate Uptake An increase in pyruvate uptake (1.74 –1.71 vs. 1.39) was detected after injection of 15 IU of hCG, or of 15 IU of hCG and 15 IU of PMSG, compared with uptake in control oocytes, but the difference was statistically significant only between the control and the PMSG ⫹ hCG group. The difference between the control and the group injected with 15 IU of hCG tended toward significance (P⫽.067; Fig. 4). Fertility and Sterility姞

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FIGURE 3 Comparison of mtDNA number in oocytes retrieved from female golden hamsters with or without the injection of PMSG and/or hCG. Data are represented by box and whisker plots: the box indicates the range between the 25 and 75 percentiles, and the solid and dotted lines in the box show the median and mean values, respectively. The whiskers represent the distribution of values, and the black dots show the minimal and maximal numbers. A significant increase in the number of mtDNA was detected after the injection of 15 IU of hCG, or of 15 IU of PMSG and 15 IU of hCG, compared with the control. abP⬍.05.

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nadotropin injections changed mitochondrial function and morphology. In the present study, laser-scanned confocal microscopy demonstrated that there was no change in mitochondrial activity after the treatments, despite an increase in ATP synthesis. The results of counting the number of mtDNA copies clearly showed that the increase in ATP synthesis in oocytes exposed to 15 hCG, or 15 IU of PMSG and 15 IU of hCG, resulted instead from an increase in the mitochondrial population. Thus, it is possible that an undesirable combination or too high a dose of gonadotropins induced excessive generation of ATP, which resulted in the loss of developmental competence in maturing oocytes. This postulate is supported by the previous finding (18) that surplus ATP stimulates highly cytotoxic reactive oxygen species. However, pyruvate, in addition to being used for amino acid and fatty acid synthesis during oogenesis, is the sole substrate for generating ATP in the mitochondria via the Krebs cycle (19). The apparent increase in pyruvate uptake may imply an excessive generation of reactive oxygen species in the ooplasm via promoting ATP synthesis (18). An 18- to 28-fold increase in the number of mitochondria was detected after the injection of 15 IU of hCG, or 15 IU of 1168

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PMSG and 15 IU of hCG, whereas only a 4% increase in ATP generation and a 23%–25% increase in pyruvate uptake were observed. These results indicate that a severe imbalance occurs between the supply of the energy substrate and mitochondrial activity after the gonadotropin injections. This imbalance probably causes a pyruvate deficiency in the mitochondria of oocytes exposed to high doses of gonadotropin, which may be another cause of developmental retardation of oocytes after fertilization. Compared with no treatment, the injection of 5 IU of PMSG did not alter mitochondrial function and pyruvate uptake. These results imply that low dose (ⱕ5 IU) of PMSG on day 1 of estrus reduced intracytoplasmic ATP level of oocytes without altering mitochondrial function. Therefore, the decrease in ATP level after the injection of 5 IU of PMSG may be a result of changes in extra-mitochondrial metabolism (20). However, such decrease in intracytoplasmic ATP may have subtle influence on developmental competence of oocytes (21, 22), because embryo cleavage and blastocyst formation were not decreased after the injection of 5 IU of PMSG compared with after no injection. From different viewpoint, the in vitro embryo culture system used in our experiment fully supported the blastocyst formation of embryos having reduced level of intracytoplasmic ATP. In conclusion, exposing oocytes to excessive quantities of hCG or combined doses of PMSG and hCG may cause ATP production at a detrimental level or may induce a substrate deficiency that inhibits mitochondrial generation of ATP. On the basis of the results indicating increased pyruvate uptake

FIGURE 4 Quantification of pyruvate uptake in oocytes retrieved from female golden hamsters with or without the injection of PMSG and/or hCG. Compared with the control (no injection), a significant increase in uptake was detected in oocytes retrieved after the injection of 15 IU of PMSG and 15 IU of hCG. *P⬍.05 vs. control.

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after the treatments, the activation of the glycolytic pathway after the gonadotropin treatments may induce an in vitro developmental block of oocytes (23). Our findings contribute not only to elucidating the adverse effects of exogenous gonadotropins but also to developing a more efficient strategy for reproductive biotechnology in model animals. Furthermore, our study can be applied to the model of human assisted reproductive technology. To better understand the effects of gonadotropins, we must examine the activation of oocyte metabolism from various viewpoints, such as through analyses of the aerobic and anaerobic glycolysis pathways and calcium mobilization in oocytes. REFERENCES 1. Lee ST, Kim TM, Cho MY, Moon SY, Han JY, Lim JM. Development of a hamster superovulation program and adverse effects of gonadotropins on microfilament formation during oocyte development. Fertil Steril 2005;83:1264 –74. 2. Lee ST, Han HJ, Oh SJ, Lee EJ, Han JY, Lim JM. Influence of ovarian hyperstimulation and ovulation induction on the cytoskeletal dynamics and developmental competence of oocytes. Mol Reprod Dev 2006;73: 10022–33. 3. Kuhne W, Besselmann M, Noll T, Muhs A, Watanabe H, Piper HM. Disintegration of cytoskeletal structure of actin filaments in energydepleted endothelial cells. Am J Physiol 1993;264:H1599 – 608. 4. Ludwig TE, Squirrell JM, Palmenberge AC, Bavister BD. Relationship between development, metabolism, and mitochondrial organization in 2-cell hamster embryos in the presence of low levels of phosphate. Biol Reprod 2001;65:1648 –54. 5. Ma S, Kalousek DK, Yuen BH, Moon YS. Investigation of effects of pregnant mare serum gonadotropin (PMSG) on the chromosomal complement of CD-1 mouse embryos. J Assist Reprod Genet 1997;14: 162–9. 6. Richard B, Donald TD. The 3=-terminal sequences of the small ribosomal RNA from hamster mitochondria. Nucleic Acids Res 1980;8: 4927– 41. 7. Wan QH, Qian KX, Fang SG. A simple DNA extraction and rapid specific identification technique for single cells and early embryos of two breeds of Bos Taurus. Anim Reprod Sci 2003;77:1–9. 8. Wilding M, Dale B, Marino M, di Matteo L, Alviggi C, Pisaturo ML, et al. Mitochondrial aggregation patterns and activity in human oocytes and preimplantation embryos. Hum Reprod 2001;16:909 –17.

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