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Energy substrates, mitochondrial membrane potential and human preimplantation embryo division
RBMOnline - Vol 5. No 1. 39–42 Reproductive BioMedicine Online; www.rbmonline.com/Article/247 on web 9 May 2002
Articles Energy substrates, mitochondrial membrane potential and human preimplantation embryo division Martin Wilding obtained his BSc degree in Biochemistry at Imperial College, London, followed by a PhD degree from University College, London. His tutor was Michael Whitaker. He has studied total fertilization in marine animals for 7 years: the sea urchin in London for the first 3 years and then ascidian at Stazione Zoologica, Naples, Italy for the subsequent 4 years with Brian Dale. His special interests lie in fertilization and early embryo development with special reference to human fertilization. Dr Wilding is currently studying the role of maternal age and mitochondrial physiology on human embryo development at the Centre for Reproductive Biology, Naples, Italy.
Dr Martin Wilding M Wilding1,2,3, A Fiorentino1, ML De Simone1, V Infante1, L De Matteo1, M Marino1, B Dale1 for Reproductive Biology, Clinica Villa del Sole, Via Manzoni, 15, 80126 Naples, Italy 2Dipartimento Clinico di Emergenza Ostetrica, Ginecologica e Medicina della Riproduzione, Area Funzionale di Medicina della Riproduzione ed Endoscopia Ginecologica, Università degli Studi di Napoli ‘Federico II’, Naples, Italy 3Correspondence: Tel: +39–81–641689; Fax: +39–81–5479251; e-mail: [email protected]
1Centre
Abstract Carbohydrate additives to modern embryo culture media are based on three basic energy sources, glucose, pyruvate and lactate. Although the use of these substrates is almost universal, debate continues as to the roles of the individual components in the human. This is mainly due to the lack of human embryos for research and the reliance on animal model systems. In the present work, the human embryo was used to study the role of the above simple substrates in the maintenance of the mitochondrial membrane potential and cell division. The mitochondrial membrane potential was measured with fluorescence techniques. Cell division was scored as the number of blastomeres on day 3. Both the mitochondrial membrane potential and cell division were dramatically lost in the absence of energy sources. The mitochondrial membrane potential and cell division were normal in media containing all three energy sources, or in pyruvate-containing media. Both glucose and lactate individually proved poor energy sources for the maintenance of the mitochondrial membrane potential. However, cell division continued in the presence of glucose, suggesting that some energy production can continue. These data suggest that pyruvate is an absolute requirement for mitochondrial respiration and cell cleavage during human preimplantation development. The role of lactate is as yet unclear. Keywords: cell cycle, human embryo, in-vitro fertilization, JC-1, mitochondria
Introduction The cell cycle can be thought of as a type of chemical reaction where the reactants are the major cell cycle control proteins (p34cdc2 and cyclins; see Fisher, 1997) and the product is the completion of cell division after the formation of two separate cells. This reaction, although seemingly simple, can cause disastrous effects in the body (such as when cancers form), and can also determine the creation of new life (such as the division of an embryo). The reaction of the cell cycle is controlled by two influencing elements. On one hand, control elements allow the cycle to proceed in an ordered fashion (Nurse, 1990). On the other hand, the supply of energy to enable the diverse cell cycle signalling reactions (Draetta et Paper based on contribution presented at the Alpha meeting in New York, USA, September 2001.
al., 1988; Gould and Nurse, 1989) enables the cell cycle to be completed in optimal time. The intracellular energy supply in all mammalian cells is provided through the production of adenosine trisphosphate (ATP). This is formed through two metabolic pathways, one of which is mitochondria-dependent and the second independent of mitochondria. In mammalian embryos, it is not clear which pathway is preferred by developing embryos. Mitochondrial metabolism clearly occurs and human preimplantation embryo development is strongly influenced by the efficiency of mitochondrial metabolism (Van Blerkom et al., 2000; Wilding et al., 2001). However, mouse embryos have been shown to develop normally in the absence of mitochondrial-dependent
39
Articles - Energy substrates and cell division - M Wilding et al.
metabolism (Piko and Chase, 1973; Larsson et al., 1998; Li et al., 2000). These data suggest that the pathways are mutually exclusive and suggest that mammalian embryos can switch between pathways. Mammalian embryos prefer pyruvate to glucose during the first cell divisions (Brinster, 1969, 1971; Cross and Brinster, 1973; Rushmer and Brinster, 1973; Leese and Barton, 1984; Leese et al., 1986, 1993; Conaghan et al., 1993; Gardner et al., 1993; Leese, 1995; Butcher et al., 1998; Biggers, 2000; Mehta, 2001). Glucose becomes the preferred carbohydrate source during development of the blastocyst and after implantation (Leese and Barton, 1984; Leese et al., 1993; Leese, 1995; Martin and Leese, 1995; Gardner et al., 1993). In fact, the presence of glucose may be toxic to preimplantation mammalian embryos, although recent evidence has suggested otherwise (Seshagiri and Bavister, 1989; Coates et al., 1999; Biggers and McGinnis, 2001). The role of lactic acid as a carbohydrate source is unclear in preimplantation mammalian embryos, and lactate may be simply the end-product of pyruvate metabolism.
followed by ovarian stimulation with exogenous FSH. Oocyte retrieval was performed 36 h after the administration of 10,000 IU β-human chorionic gonadotrophin (HCG) when two or three follicles of 18–20 mm diameter were observed by ultrasound examination, and blood 17β-oestradiol concentrations reached 150–200 pg/ml per follicle >18 mm. Oocytes were processed for standard IVF and intracytoplasmic sperm injection (ICSI) protocols using commercial IVF medium (Medicult, Copenhagen, Denmark) pre-equilibrated to 37°C and 5% CO2. Human grade 1 (i.e. two normal blastomeres, one nucleus per blastomeres, no fragmentation) two-cell preimplantation embryos were obtained on day 2 after fertilization, after the completion of embryo transfer or cryopreservation. The embryos were graded morphologically, and examined and graded during the experimental embryo checks. Day 2 embryo grading was performed 40–44 h after fertilization and day 3 checks were performed at 64–68 h after fertilization. All embryos used were scored as two-pronuclear on day 1 (16–20 h after fertilization).
The rate of mitochondrial respiration can be measured by fluorescence techniques. In this article, the mitochondrial membrane potential and rate of cell division in human embryos incubated in protein-deficient, defined media containing one or more energy substrates are measured. It is shown that the highest rate of cell division and the highest mitochondrial membrane potential is obtained with pyruvate in the culture medium. These data suggest that glucose and lactate are not essential for the initial 3 days of embryo culture in humans.
On days 2 and 3, a grade 1 rating was assigned to embryos after the observation of blastomeres of equal volume containing one nucleus per blastomere. Grade 2 embryos contained one to three defects in blastomere volume and number of nuclei per blastomeres. Grade 3 embryos had multiple defects or had arrested division. Percentage fragmentation was scored as percentage relative to the embryo volume. All day 2 embryos had no visible fragmentation as observed under the light microscope. Fragmentation on day 3 was not considered in the present data.
Materials and methods
Embryos donated for the present work were washed out of the IVF culture medium into an experimental culture medium 48–52 h after fertilization, after the embryos were released for research use. Experimental cell culture media were based on Earle’s salts (120 mmol/l NaCl, 5.4 mmol/l KCl, 0.8 mmol/l MgSO4, 2 mmol/l CaCl2, 1 mmol/l NaPO3, 1 mmol/l NaH2CO3, pH 7.4, Table 1) in which modifications were made to the energy substrate. Basic media contained salts only (Table 1). Control media contained 0.8 mmol/l pyruvate, 5 mmol/l glucose and 0.8 mmol/l lactate (Table 1). Other media contained one of the three energy substrates in the concentration defined by the control media (Table 1). Media were all equilibrated in an atmosphere of 37°C and 5% CO2 and embryos were incubated for 24 h in one of the media before the confocal analysis.
Patients Patients were attending IVF clinics for in-vitro fertilization protocols. Spare embryos not suitable for transfer or cryopreservation were donated for research after informed consent. The maternal age of patients was 30 years or less at the time of oocyte retrieval. Patients were prepared for IVF using standard ovarian stimulation protocols including downregulation of the pituitary gland with a gonadotrophinreleasing hormone (GnRH) agonist (Decapeptyl; Ipsen, Italy) Table 1. Culture media used in the experimental protocol. All media were pre-equilibrated to 37°C in an atmosphere of 5% CO2 in humidified air before use. All units are millimoles.
NaCl
40
Control Basic
Basic + pyruvate
Basic + glucose
Basic + lactate
120.0
120.0
120.0
120.0
120.0
KCl
5.4
5.4
5.4
5.4
5.4
MgSO4
0.8
0.8
0.8
0.8
0.8
CaCl2
2.0
2.0
2.0
2.0
2.0
NaPO3
1.0
1.0
1.0
1.0
1.0
NaH2CO3
1.0
1.0
1.0
1.0
1.0
Pyruvate
0.8
–
0.8
–
–
Glucose
5.0
–
–
5.0
–
Lactate
0.8
–
–
–
0.8
Fluorescence labelling and confocal microscopy The potential-sensitive fluorescence dye 5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Molecular Probes, Oregon, USA) was used to measure the activity of mitochondria (Reers et al., 1991, 1995). The dye was dissolved to a stock concentration of 0.5 mmol/l in dimethylsulphoxide and diluted into pre-equilibrated IVF medium (Medicult), using a vortex to aid the dissolution of the dye, as required. Under these conditions, the dye was found to remain dissolved for up to 1 h, permitting accurate loading of oocytes. An Olympus Fluoview (Olympus, Segrate, Italy) confocal microscope, based on an Olympus IX-70 inverted microscope, was used for all experiments. A Kr/Ar laser was used to produce the excitation laser line at 488 nm and
Articles - Energy substrates and cell division - M Wilding et al.
emission wavelengths were separated by a 530 nm dichroic mirror followed by analysis in a photomultiplier after further filtering through a 515–530 nm bandpass filter (green emission) or a 585 nm longpass filter (red emission). Laser power and photomultiplier settings were kept constant for all experiments. Oocytes were positioned with the polar body in the plane of focus where present and a single scan through the centre of the oocyte was used for the analysis. For embryos, single scans through the centre of focus of each blastomere were used for the analysis. Areas of embryo fragmentation and overlaps between blastomeres were excluded from the analysis by deselecting then with the confocal software. Images were processed by the confocal software and Adobe Photoshop. The JC-1 ratio (see Results section below) is calculated by pixel-by-pixel division of the membrane potential sensitive (red) confocal fluorescence emission by the membrane potential insensitive (green) fluorescence. Areas of embryo fragmentation and overlaps between blastomeres are excluded from the analysis by deselecting them with the confocal software. The resulting value is a mean of the individual ratio values of each pixel. This is synonymous with mitochondrial membrane potential (Reers et al., 1991, 1995) but calibrated readings are not given.
Statistics All data were plotted as mean ± SD unless stated. All plots and statistical analysis was calculated using the Sigma Plot and Sigma Stat software packages (SPSS, Erkrath, Germany) except where stated. Tukey’s one-way analysis of variance (ANOVA) was used to compare the significance of the groups to controls and α indicates the power of the test.
Results In total, 89 2-cell embryos on day 2 after fertilization were donated to the experimental protocol. This included 26 embryos in the control media, 14 embryos in medium starved Table 2. Effect of energy substrates on cell division and mitochondrial membrane potential in preimplantation human embryos. Values are mean ± SD. All embryos used for the present work were grade 1 two-cell embryos on day 2. Day 3 JC-1 ratio
Day 3 morpho- Blastomeres logical score on day 3
Controls (n = 26)
1.82 ± 0.33
1.9 ± 0.1
5.8 ± 1.6
Basic medium (n = 14)
1.11 ± 0.12a
1.7 ± 0.5
2.6 ± 1.0a
Basic + pyruvate 1.73 ± 0.28 (n = 22)
2.1 ± 0.2
6.2 ± 2.0
Basic + glucose 1.22 ± 0.11a (n = 16)
1.7 ± 0.3
3.2 ± 1.2a
1.09 ± 0.18a
2.2 ± 0.2
2.3 ± 0.3a
Basic + lactate (n = 11)
aStatistically significant P < 0.001 when compared with controls (Tukey’s oneway ANOVA, α = 1.000).
of energy substrate, 22 embryos in medium containing pyruvate, 16 embryos in medium with glucose and 11 embryos in lactose-containing medium. All embryos were donated by patients in whom the maternal age was <30 years, due to the known effects of maternal age on the basal mitochondrial activity. Embryos affected by >30% fragmentation on day 2 were also excluded from the study due to the low potential for further development of these embryos. Embryos in control media were characterized by good levels of cell division (5.8 ± 1.6 blastomeres on day 3, n = 26, see Table 2) and a high JC-1 ratio (1.82 ± 0.33, n = 26, see Table 2) 24 h after washing into the experimental medium. This was in marked contrast to embryos in basal medium with no energy source. In this media, cell division had completely arrested (2.6 ± 1.0 blastomeres on day 3, n = 14, see Table 2) and a low JC-1 ratio was observed (1.11 ± 0.12, n = 14, see Table 2). Embryos placed in media supplemented with a single energy source were characterized by intermediate results. Where pyruvate was the only energy source, a confocal ratio not significantly different from the controls was observed (1.73 ± 0.28, n = 22, see Table 2), and cell division occurred (6.2 ± 2.0 blastomeres on day 3, n = 22, see Table 2). Glucose-containing medium caused a marked reduction in JC-1 ratio (1.22 ± 0.11, n = 16, see Table 2). However, a low level of cell division occurred (3.2 ± 1.2 blastomeres on day 3, n = 16, see Table 2), suggesting that sufficient energy substrate was available to enable the completion of the cell cycle. In contrast, lactatecontaining medium caused a dramatic reduction in the JC-1 ratio (1.09 ± 0.18, n = 11, see Table 2) combined with cleavage arrest (2.3 ± 0.3 blastomeres on day 3, n = 11, see Table 2).
Discussion In this study, the role of diverse energy substrates was tested in the maintenance of the mitochondrial membrane potential and the completion of the cell cycle in human embryos, by examining the effect of substrate starvation on human embryo development. Two parameters were tested, the first being the mitochondrial membrane potential, measured with confocal microscopy and the fluorescence dye JC-1, and the second was the rate of cell division. These two parameters have been shown to be correlated (Wilding et al., 2001), although the correlation is not perfect, suggesting that other factors such as cell cycle proteins also have a strong effect on cell division. In the present report, it was found that that media containing pyruvate only caused the maintenance of the JC-1 ratio achieved with control culture medium. Basic medium supplemented with glucose or lactate alone caused the JC-1 ratio to diminish to levels not significantly greater than basic medium without energy substrates. These data suggest that pyruvate but not glucose or lactate cause energy production through a mitochondria-dependent pathway. The data demonstrate that human preimplantation embryo is unable to metabolize either glucose or lactate sufficiently to maintain a reasonable mitochondrial membrane potential and hence presumably ATP production through aerobic respiration. Despite the low JC-1 ratio after incubation of embryos in glucose-containing culture medium, embryos were often found to have cleaved on day 3 after fertilization in glucosecontaining medium. In contrast, almost no cleavage occurred in lactate-containing medium. These data suggest that the
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Articles - Energy substrates and cell division - M Wilding et al.
addition of glucose does provide sufficient substrate to permit the cell cycle to continue. It is suggested that this is due to the continuation of ATP production through anaerobic respiration. These data suggest that, in the absence of amino acids and protein, pyruvate is an absolute requirement for the maintenance of the mitochondrial membrane potential and cell division during the days 2–3 of human embryo development. Neither glucose nor lactate is either metabolized through mitochondria-dependent pathways or is essential for mitochondrial respiration during this phase. However, in the absence of pyruvate, glucose appears to provide sufficient ATP for continued embryo cleavage. Whether this is sufficient to enable blastocyst development or embryo implantation has not been tested in the present data. However, the data strongly suggest that oxidative phosphorylation through the mitochondrial-dependent metabolism of pyruvate is an active pathway during the first 3 days of human preimplantation embryo development.
Acknowledgements This project was supported by grants from Serono Pharma, Rome, Italy to B Dale. We further thank the Fondazione Nuovi Orizzonti, Naples, Italy and Vincenzo Monfrecola for technical support.
References Biggers J 2000 Ethical issues and the commercialisation of embryo culture media. Reproductive BioMedicine Online 1, 74–76. Biggers J, McGinnis L 2001 Evidence that glucose is not always an inhibitor of mouse preimplantation development in vitro. Human Reproduction 16, 153–163. Brinster R 1969 Incorporation of carbon from glucose and pyruvate into the preimplantation mouse embryo. Experimental Cell Research 58, 153–158. Brinster R 1971 Oxidation of pyruvate and glucose by oocytes of the mouse and rhesus monkey. Journal of Reproduction and Fertility 24, 187–191. Butcher L, Coates A, Martin K et al. 1998 Metabolism of pyruvate by the early human embryo. Biology of Reproduction 58, 1054–1056. Coates A, Rutherford A, Hunter H, Leese H 1999 Glucose-free medium in human in vitro fertilisation and embryo transfer: a large-scale, prospective, randomised clinical trial. Fertility and Sterility 72, 229–232. Conaghan J, Handyside A, Winston R, Leese H 1993 Effects of pyruvate and glucose on the development of human preimplantation embryos in vitro. Journal of Reproduction and Fertility 99, 87–95. Cross P, Brinster R 1973 The sensitivity of one-cell mouse embryos to pyruvate and lactate. Experimental Cell Research 77, 57–62.
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Draetta G, Piwnica-Worms H, Morrison D et al. 1988 Human cdc2 protein kinase is a major cell cycle regulated tyrosine kinase substrate. Nature 336, 738–744. Fisher R 1997 CDKs and cyclins in transition(s). Current Opinion in Genetics and Development 7, 32–38. Gardner D, Lane M, Batt P 1993 Uptake and metabolism of pyruvate and glucose by individual sheep preattachment embryos developed in vivo. Molecular Reproduction and Development 36, 313–319. Gould K, Nurse P 1989 Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis. Nature 342, 39–45. Larsson N, Wang J, Wilhelmsson H et al. 1998 Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice. Nature Genetics 18, 231–236. Leese H 1995 Metabolic control during preimplantation mammalian embryo development. Human Reproduction Update 1, 63–72. Leese H, Barton A 1984 Pyruvate and glucose uptake by mouse ova and preimplantation embryos. Journal of Reproduction and Fertility 72, 9–13. Leese H, Hooper M, Edwards R, Ashwood-Smith M 1986 Uptake of pyruvate by early human embryos determined by a non-invasive technique. Human Reproduction 1, 181–182. Leese H, Conaghan J, Martin K, Hardy K 1993 Early human embryo metabolism. Bioessays 15, 259–264. Li K, Li Y, Shelton J et al. 2000 Cytochrome c deficiency causes embryonic lethality and attenuates stress-induced apoptosis. Cell 101, 389–399. Martin K, Leese H 1995 Role of glucose in mouse preimplantation embryo development. Molecular Reproduction and Development 40, 436–443. Mehta RH 2001 Growth of human preimplantation embryos in vitro. Reproductive BioMedicine Online 2, 113–119. Nurse P 1990 Universal control mechanism regulating control of Mphase. Nature 344, 503–508. Piko L, Chase D 1973 Role of the mitochondrial genome during early development in mice: effects of ethidium bromide and chloramphenicol. Journal of Cell Biology, 58, 357–378. Reers M, Smith T, Chen L 1991 J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30, 4480–4486. Reers M, Smiley ST, Mottola-Hartshorn C et al. 1995 Mitochondrial membrane potential monitored by JC-1 dye. Methods in Enzymology 260, 406–417. Rushmer R, Brinster R 1973 Carbon dioxide production from pyruvate and glucose by bovine oocytes. Experimental Cell Research, 82, 252–254. Seshagiri P, Bavister B 1989 Glucose inhibits development of hamster 8-cell embryos in vitro. Biology of Reproduction 40, 599–606. Van Blerkom J, Davis P, Alexander S 2000 Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organisation, ATP content and competence. Human Reproduction 15, 2621–2633. Wilding M, Dale B, Marino M et al. 2001 Mitochondrial aggregation patterns and activity in human oocytes and preimplantation embryos. Human Reproduction 16, 909–917.
Articles Energy substrates, mitochondrial membrane potential and human preimplantation embryo division Martin Wilding obtained his BSc degree in Biochemistry at Imperial College, London, followed by a PhD degree from University College, London. His tutor was Michael Whitaker. He has studied total fertilization in marine animals for 7 years: the sea urchin in London for the first 3 years and then ascidian at Stazione Zoologica, Naples, Italy for the subsequent 4 years with Brian Dale. His special interests lie in fertilization and early embryo development with special reference to human fertilization. Dr Wilding is currently studying the role of maternal age and mitochondrial physiology on human embryo development at the Centre for Reproductive Biology, Naples, Italy.
Dr Martin Wilding M Wilding1,2,3, A Fiorentino1, ML De Simone1, V Infante1, L De Matteo1, M Marino1, B Dale1 for Reproductive Biology, Clinica Villa del Sole, Via Manzoni, 15, 80126 Naples, Italy 2Dipartimento Clinico di Emergenza Ostetrica, Ginecologica e Medicina della Riproduzione, Area Funzionale di Medicina della Riproduzione ed Endoscopia Ginecologica, Università degli Studi di Napoli ‘Federico II’, Naples, Italy 3Correspondence: Tel: +39–81–641689; Fax: +39–81–5479251; e-mail: [email protected]
1Centre
Abstract Carbohydrate additives to modern embryo culture media are based on three basic energy sources, glucose, pyruvate and lactate. Although the use of these substrates is almost universal, debate continues as to the roles of the individual components in the human. This is mainly due to the lack of human embryos for research and the reliance on animal model systems. In the present work, the human embryo was used to study the role of the above simple substrates in the maintenance of the mitochondrial membrane potential and cell division. The mitochondrial membrane potential was measured with fluorescence techniques. Cell division was scored as the number of blastomeres on day 3. Both the mitochondrial membrane potential and cell division were dramatically lost in the absence of energy sources. The mitochondrial membrane potential and cell division were normal in media containing all three energy sources, or in pyruvate-containing media. Both glucose and lactate individually proved poor energy sources for the maintenance of the mitochondrial membrane potential. However, cell division continued in the presence of glucose, suggesting that some energy production can continue. These data suggest that pyruvate is an absolute requirement for mitochondrial respiration and cell cleavage during human preimplantation development. The role of lactate is as yet unclear. Keywords: cell cycle, human embryo, in-vitro fertilization, JC-1, mitochondria
Introduction The cell cycle can be thought of as a type of chemical reaction where the reactants are the major cell cycle control proteins (p34cdc2 and cyclins; see Fisher, 1997) and the product is the completion of cell division after the formation of two separate cells. This reaction, although seemingly simple, can cause disastrous effects in the body (such as when cancers form), and can also determine the creation of new life (such as the division of an embryo). The reaction of the cell cycle is controlled by two influencing elements. On one hand, control elements allow the cycle to proceed in an ordered fashion (Nurse, 1990). On the other hand, the supply of energy to enable the diverse cell cycle signalling reactions (Draetta et Paper based on contribution presented at the Alpha meeting in New York, USA, September 2001.
al., 1988; Gould and Nurse, 1989) enables the cell cycle to be completed in optimal time. The intracellular energy supply in all mammalian cells is provided through the production of adenosine trisphosphate (ATP). This is formed through two metabolic pathways, one of which is mitochondria-dependent and the second independent of mitochondria. In mammalian embryos, it is not clear which pathway is preferred by developing embryos. Mitochondrial metabolism clearly occurs and human preimplantation embryo development is strongly influenced by the efficiency of mitochondrial metabolism (Van Blerkom et al., 2000; Wilding et al., 2001). However, mouse embryos have been shown to develop normally in the absence of mitochondrial-dependent
39
Articles - Energy substrates and cell division - M Wilding et al.
metabolism (Piko and Chase, 1973; Larsson et al., 1998; Li et al., 2000). These data suggest that the pathways are mutually exclusive and suggest that mammalian embryos can switch between pathways. Mammalian embryos prefer pyruvate to glucose during the first cell divisions (Brinster, 1969, 1971; Cross and Brinster, 1973; Rushmer and Brinster, 1973; Leese and Barton, 1984; Leese et al., 1986, 1993; Conaghan et al., 1993; Gardner et al., 1993; Leese, 1995; Butcher et al., 1998; Biggers, 2000; Mehta, 2001). Glucose becomes the preferred carbohydrate source during development of the blastocyst and after implantation (Leese and Barton, 1984; Leese et al., 1993; Leese, 1995; Martin and Leese, 1995; Gardner et al., 1993). In fact, the presence of glucose may be toxic to preimplantation mammalian embryos, although recent evidence has suggested otherwise (Seshagiri and Bavister, 1989; Coates et al., 1999; Biggers and McGinnis, 2001). The role of lactic acid as a carbohydrate source is unclear in preimplantation mammalian embryos, and lactate may be simply the end-product of pyruvate metabolism.
followed by ovarian stimulation with exogenous FSH. Oocyte retrieval was performed 36 h after the administration of 10,000 IU β-human chorionic gonadotrophin (HCG) when two or three follicles of 18–20 mm diameter were observed by ultrasound examination, and blood 17β-oestradiol concentrations reached 150–200 pg/ml per follicle >18 mm. Oocytes were processed for standard IVF and intracytoplasmic sperm injection (ICSI) protocols using commercial IVF medium (Medicult, Copenhagen, Denmark) pre-equilibrated to 37°C and 5% CO2. Human grade 1 (i.e. two normal blastomeres, one nucleus per blastomeres, no fragmentation) two-cell preimplantation embryos were obtained on day 2 after fertilization, after the completion of embryo transfer or cryopreservation. The embryos were graded morphologically, and examined and graded during the experimental embryo checks. Day 2 embryo grading was performed 40–44 h after fertilization and day 3 checks were performed at 64–68 h after fertilization. All embryos used were scored as two-pronuclear on day 1 (16–20 h after fertilization).
The rate of mitochondrial respiration can be measured by fluorescence techniques. In this article, the mitochondrial membrane potential and rate of cell division in human embryos incubated in protein-deficient, defined media containing one or more energy substrates are measured. It is shown that the highest rate of cell division and the highest mitochondrial membrane potential is obtained with pyruvate in the culture medium. These data suggest that glucose and lactate are not essential for the initial 3 days of embryo culture in humans.
On days 2 and 3, a grade 1 rating was assigned to embryos after the observation of blastomeres of equal volume containing one nucleus per blastomere. Grade 2 embryos contained one to three defects in blastomere volume and number of nuclei per blastomeres. Grade 3 embryos had multiple defects or had arrested division. Percentage fragmentation was scored as percentage relative to the embryo volume. All day 2 embryos had no visible fragmentation as observed under the light microscope. Fragmentation on day 3 was not considered in the present data.
Materials and methods
Embryos donated for the present work were washed out of the IVF culture medium into an experimental culture medium 48–52 h after fertilization, after the embryos were released for research use. Experimental cell culture media were based on Earle’s salts (120 mmol/l NaCl, 5.4 mmol/l KCl, 0.8 mmol/l MgSO4, 2 mmol/l CaCl2, 1 mmol/l NaPO3, 1 mmol/l NaH2CO3, pH 7.4, Table 1) in which modifications were made to the energy substrate. Basic media contained salts only (Table 1). Control media contained 0.8 mmol/l pyruvate, 5 mmol/l glucose and 0.8 mmol/l lactate (Table 1). Other media contained one of the three energy substrates in the concentration defined by the control media (Table 1). Media were all equilibrated in an atmosphere of 37°C and 5% CO2 and embryos were incubated for 24 h in one of the media before the confocal analysis.
Patients Patients were attending IVF clinics for in-vitro fertilization protocols. Spare embryos not suitable for transfer or cryopreservation were donated for research after informed consent. The maternal age of patients was 30 years or less at the time of oocyte retrieval. Patients were prepared for IVF using standard ovarian stimulation protocols including downregulation of the pituitary gland with a gonadotrophinreleasing hormone (GnRH) agonist (Decapeptyl; Ipsen, Italy) Table 1. Culture media used in the experimental protocol. All media were pre-equilibrated to 37°C in an atmosphere of 5% CO2 in humidified air before use. All units are millimoles.
NaCl
40
Control Basic
Basic + pyruvate
Basic + glucose
Basic + lactate
120.0
120.0
120.0
120.0
120.0
KCl
5.4
5.4
5.4
5.4
5.4
MgSO4
0.8
0.8
0.8
0.8
0.8
CaCl2
2.0
2.0
2.0
2.0
2.0
NaPO3
1.0
1.0
1.0
1.0
1.0
NaH2CO3
1.0
1.0
1.0
1.0
1.0
Pyruvate
0.8
–
0.8
–
–
Glucose
5.0
–
–
5.0
–
Lactate
0.8
–
–
–
0.8
Fluorescence labelling and confocal microscopy The potential-sensitive fluorescence dye 5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Molecular Probes, Oregon, USA) was used to measure the activity of mitochondria (Reers et al., 1991, 1995). The dye was dissolved to a stock concentration of 0.5 mmol/l in dimethylsulphoxide and diluted into pre-equilibrated IVF medium (Medicult), using a vortex to aid the dissolution of the dye, as required. Under these conditions, the dye was found to remain dissolved for up to 1 h, permitting accurate loading of oocytes. An Olympus Fluoview (Olympus, Segrate, Italy) confocal microscope, based on an Olympus IX-70 inverted microscope, was used for all experiments. A Kr/Ar laser was used to produce the excitation laser line at 488 nm and
Articles - Energy substrates and cell division - M Wilding et al.
emission wavelengths were separated by a 530 nm dichroic mirror followed by analysis in a photomultiplier after further filtering through a 515–530 nm bandpass filter (green emission) or a 585 nm longpass filter (red emission). Laser power and photomultiplier settings were kept constant for all experiments. Oocytes were positioned with the polar body in the plane of focus where present and a single scan through the centre of the oocyte was used for the analysis. For embryos, single scans through the centre of focus of each blastomere were used for the analysis. Areas of embryo fragmentation and overlaps between blastomeres were excluded from the analysis by deselecting then with the confocal software. Images were processed by the confocal software and Adobe Photoshop. The JC-1 ratio (see Results section below) is calculated by pixel-by-pixel division of the membrane potential sensitive (red) confocal fluorescence emission by the membrane potential insensitive (green) fluorescence. Areas of embryo fragmentation and overlaps between blastomeres are excluded from the analysis by deselecting them with the confocal software. The resulting value is a mean of the individual ratio values of each pixel. This is synonymous with mitochondrial membrane potential (Reers et al., 1991, 1995) but calibrated readings are not given.
Statistics All data were plotted as mean ± SD unless stated. All plots and statistical analysis was calculated using the Sigma Plot and Sigma Stat software packages (SPSS, Erkrath, Germany) except where stated. Tukey’s one-way analysis of variance (ANOVA) was used to compare the significance of the groups to controls and α indicates the power of the test.
Results In total, 89 2-cell embryos on day 2 after fertilization were donated to the experimental protocol. This included 26 embryos in the control media, 14 embryos in medium starved Table 2. Effect of energy substrates on cell division and mitochondrial membrane potential in preimplantation human embryos. Values are mean ± SD. All embryos used for the present work were grade 1 two-cell embryos on day 2. Day 3 JC-1 ratio
Day 3 morpho- Blastomeres logical score on day 3
Controls (n = 26)
1.82 ± 0.33
1.9 ± 0.1
5.8 ± 1.6
Basic medium (n = 14)
1.11 ± 0.12a
1.7 ± 0.5
2.6 ± 1.0a
Basic + pyruvate 1.73 ± 0.28 (n = 22)
2.1 ± 0.2
6.2 ± 2.0
Basic + glucose 1.22 ± 0.11a (n = 16)
1.7 ± 0.3
3.2 ± 1.2a
1.09 ± 0.18a
2.2 ± 0.2
2.3 ± 0.3a
Basic + lactate (n = 11)
aStatistically significant P < 0.001 when compared with controls (Tukey’s oneway ANOVA, α = 1.000).
of energy substrate, 22 embryos in medium containing pyruvate, 16 embryos in medium with glucose and 11 embryos in lactose-containing medium. All embryos were donated by patients in whom the maternal age was <30 years, due to the known effects of maternal age on the basal mitochondrial activity. Embryos affected by >30% fragmentation on day 2 were also excluded from the study due to the low potential for further development of these embryos. Embryos in control media were characterized by good levels of cell division (5.8 ± 1.6 blastomeres on day 3, n = 26, see Table 2) and a high JC-1 ratio (1.82 ± 0.33, n = 26, see Table 2) 24 h after washing into the experimental medium. This was in marked contrast to embryos in basal medium with no energy source. In this media, cell division had completely arrested (2.6 ± 1.0 blastomeres on day 3, n = 14, see Table 2) and a low JC-1 ratio was observed (1.11 ± 0.12, n = 14, see Table 2). Embryos placed in media supplemented with a single energy source were characterized by intermediate results. Where pyruvate was the only energy source, a confocal ratio not significantly different from the controls was observed (1.73 ± 0.28, n = 22, see Table 2), and cell division occurred (6.2 ± 2.0 blastomeres on day 3, n = 22, see Table 2). Glucose-containing medium caused a marked reduction in JC-1 ratio (1.22 ± 0.11, n = 16, see Table 2). However, a low level of cell division occurred (3.2 ± 1.2 blastomeres on day 3, n = 16, see Table 2), suggesting that sufficient energy substrate was available to enable the completion of the cell cycle. In contrast, lactatecontaining medium caused a dramatic reduction in the JC-1 ratio (1.09 ± 0.18, n = 11, see Table 2) combined with cleavage arrest (2.3 ± 0.3 blastomeres on day 3, n = 11, see Table 2).
Discussion In this study, the role of diverse energy substrates was tested in the maintenance of the mitochondrial membrane potential and the completion of the cell cycle in human embryos, by examining the effect of substrate starvation on human embryo development. Two parameters were tested, the first being the mitochondrial membrane potential, measured with confocal microscopy and the fluorescence dye JC-1, and the second was the rate of cell division. These two parameters have been shown to be correlated (Wilding et al., 2001), although the correlation is not perfect, suggesting that other factors such as cell cycle proteins also have a strong effect on cell division. In the present report, it was found that that media containing pyruvate only caused the maintenance of the JC-1 ratio achieved with control culture medium. Basic medium supplemented with glucose or lactate alone caused the JC-1 ratio to diminish to levels not significantly greater than basic medium without energy substrates. These data suggest that pyruvate but not glucose or lactate cause energy production through a mitochondria-dependent pathway. The data demonstrate that human preimplantation embryo is unable to metabolize either glucose or lactate sufficiently to maintain a reasonable mitochondrial membrane potential and hence presumably ATP production through aerobic respiration. Despite the low JC-1 ratio after incubation of embryos in glucose-containing culture medium, embryos were often found to have cleaved on day 3 after fertilization in glucosecontaining medium. In contrast, almost no cleavage occurred in lactate-containing medium. These data suggest that the
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Articles - Energy substrates and cell division - M Wilding et al.
addition of glucose does provide sufficient substrate to permit the cell cycle to continue. It is suggested that this is due to the continuation of ATP production through anaerobic respiration. These data suggest that, in the absence of amino acids and protein, pyruvate is an absolute requirement for the maintenance of the mitochondrial membrane potential and cell division during the days 2–3 of human embryo development. Neither glucose nor lactate is either metabolized through mitochondria-dependent pathways or is essential for mitochondrial respiration during this phase. However, in the absence of pyruvate, glucose appears to provide sufficient ATP for continued embryo cleavage. Whether this is sufficient to enable blastocyst development or embryo implantation has not been tested in the present data. However, the data strongly suggest that oxidative phosphorylation through the mitochondrial-dependent metabolism of pyruvate is an active pathway during the first 3 days of human preimplantation embryo development.
Acknowledgements This project was supported by grants from Serono Pharma, Rome, Italy to B Dale. We further thank the Fondazione Nuovi Orizzonti, Naples, Italy and Vincenzo Monfrecola for technical support.
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