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Biology of Reproduction 61, 541-547 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Coenzyme Q10 in Submicron-Sized Dispersion Improves Development, Hatching, Cell Proliferation, and Adenosine Triphosphate Content of In Vitro-Produced Bovine Embryos1

Miodrag Stojkovica, Kirsten Westesenb, Valeri Zakhartchenkoc, Petra Stojkovica, Katja Boxhammera, and Eckhard Wolf2,a,c

a Lehrstuhl für Molekulare Tierzucht und Haustiergenetik, Ludwig-Maximilians-Universität München, 85764 Oberschleissheim, Germany b Lehrstuhl für Pharmazeutische Technologie, Institut für Pharmazie, Friedrich-Schiller-Universität, 07743 Jena, Germany c Bayerisches Forschungszentrum für Fortpflanzungsbiologie GmbH & Co KG (BFZF), 85764 Oberschleissheim, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Coenzyme Q10 (CoQ10) is an essential component of the plasma membrane ion transporter (PMIT) system and of the electron transport chain in the inner mitochondrial membrane. Because of its intrinsic functions in cell growth and energy metabolism (ATP synthesis), and its protective effects against oxidative stress, CoQ10 is a good candidate for supporting growth of cells in culture. However, because of its quinone structure, CoQ10 is extremely lipophilic and practically insoluble in water. We used a specific technology to prepare a submicron-sized dispersion of CoQ10, inhibiting re-crystallization by a stabilizer. This dispersion, which exhibits a very large specific surface area for drug dissolution, was tested as a supplement for the in vitro culture of bovine embryos in a chemically defined system. The rate of early cleavage of embryos (5- to 8-cell stages) was evaluated 66 h postinsemination (hpi) and was highest in medium supplemented with 30 or 100 µM CoQ10 (66.5 ± 0.8% and 68.7 ± 1.1%, respectively) and lowest in 10 µM CoQ10 (55.3 ± 0.8%). The proportions of oocytes developing to blastocysts by 186 hpi were 19.0 ± 0.6% and 25.2 ± 0.3% in medium supplemented with 10 µM and 30 µM CoQ10, respectively, and were significantly (p < 0.001) higher than those obtained with the equivalent amounts of stabilizer (9.9 ± 0.4% and 11.3 ± 0.4%). In the presence of 30 µM CoQ10, significantly (p < 0.001) more blastocysts hatched by 210 hpi than in the equivalent amount of stabilizer (31.8 ± 1.3 vs. 8.4 ± 2.2). Expanded blastocysts produced in the presence of 30 µM CoQ10 had significantly (p < 0.01) more inner cell mass cells and trophectoderm cells, and a significantly (p < 0.001) increased ATP content as compared to expanded blastocysts produced in the presence of the corresponding amount of stabilizer.

Our results show that noncrystalline CoQ10 in submicron-sized dispersion supports the development and viability of bovine embryos produced in a chemically defined culture system.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
After in vitro maturation (IVM) and in vitro fertilization of bovine oocytes, the next and the longest step in the production of viable transferable bovine embryos is in vitro culture. This step is still not satisfactory since cleavage, blastocyst formation, and hatching under in vitro conditions are limited. Some of the failures of culture systems that are often overlooked are those leading to increased intracellular pH [1] and disturbed function of the plasma membrane ion transporter (PMIT) system [2].

The PMIT system has NADH oxidase activity and plays a very important role in transferring electrons from cytoplasmic NADH to extracellular electron acceptors [3, 4]. In this aspect, the PMIT system serves as a backup system for the mitochondrial respiratory chain regulating cellular redox homeostasis. Twelve or more carriers are grouped into four multiprotein intramembranous complexes ([5], reviewed in [6]). One complex, which carries electrons from NADH to the small lipophilic compound ubiquinone (also termed coenzyme Q [CoQ]), is NADH-CoQ reductase. The second, succinate CoQ reductase, transfers electrons from succinate (released during its oxidation to fumarate) and flavin adenine dinucleotide in reduced form (FADH2) to CoQ. The third complex, CoQH2-cytochrome c reductase, transfers electrons from reduced CoQH2 to the water-soluble protein cytochrome c, to yield its reduced form. The fourth, cytochrome c oxidase, transfers electrons from reduced cytochrome c to O2, the ultimate electron acceptor. Three of these complexes—NADH-CoQ reductase, CoQH2-cytochrome c reductase, and cytochrome c oxidase—are sites for pumping of protons across the membrane [7].

The PMIT system depends on CoQ that is located in the plasma membrane and mitochondrial membrane [5–11]. CoQ is a unique carrier for two-electron transfer within the lipid phase of the mitochondrial membrane and exists in three different forms: ubiquinone (oxidized form), semiquinone (after receiving one H+ and one electron), and the fully reduced form ubiquinol [10, 11]. Ubiquinol also scavenges free radicals generated chemically within liposomal membranes, thereby preventing peroxidative damage [12]. It has previously been shown that free radicals have detrimental effects on cell membranes [10, 13] and lead to developmental arrest [14–22] or limited cell growth of embryos [9]. New sites for CoQ function in Golgi and plasma membranes argue for a role in growth control and secretion-related membrane flow [10, 23].

The most frequent form of CoQ in mammalian cells is coenzyme Q10 (CoQ10), a quinone product with a chain of ten isoprenoid units [24]. Located in plasma and mitochondrial membranes, CoQ10 is a carrier for both hydrogen ions and electrons and is the only electron carrier in the electron transport system that is not tightly bound or covalently bound to a protein [23, 24]. On the basis of its multiple functions in cell growth and energy metabolism (synthesis of ATP), CoQ10 is a promising candidate for supporting the development of in vitro-produced (IVP) embryos when added to the culture medium. However, because of its structure, CoQ10 is extremely lipophilic, soluble in ethanol and chloroform, but practically insoluble in water [25].

Therefore, we applied a specific technology to prepare CoQ10 in a submicron-sized dispersion, thereby preventing re-crystallization in combination with the stabilizer. The reduction in particle size in combination with noncrystalline nature of the dispersed CoQ10 increases the dissolution rate and therefore the bioavailability [25]. Effects of various concentrations of submicron-sized CoQ10 on early cleavage, blastocyst development, hatching rate, numbers of inner cell mass (ICM) and trophectoderm (TE) cells, and ATP content of bovine blastocysts were investigated in a chemically defined culture system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In Vitro Production of Bovine Embryos

Unless otherwise indicated, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

In vitro maturation of oocytes Bovine embryos were produced as previously described [26]. Follicles with a diameter of 2–8 mm were aspirated from the ovaries of slaughtered cows using a 20-gauge needle and a vacuum pressure of approximately 100 mm Hg. The cumulus-oocyte complexes (COCs) were collected in a 50-ml centrifuge tube and washed twice with preincubated (39°C; 5% CO2) tissue culture medium 199 (TCM199) supplemented with 10% (v:v) heat-inactivated serum from cows at estrus (ECS). Only COCs with a complete dense cumulus and a dark, evenly granulated cytoplasm were selected for IVM. The COCs were washed in TCM199 supplemented with 10% ECS and 10 µg/ml FSH-p (Schering Pharmaceuticals, Kenilworth, NJ) and matured in this medium for 24 h at 39°C in an atmosphere of 5% CO2 in air and maximum humidity.

In vitro fertilization After maturation, the COCs were maintained in a Tyrode's albumin lactate pyruvate medium containing 6 mg/ml BSA, 10 µg/ml heparin, and frozen/thawed semen (106 spermatozoa/ml) that had been subjected to swim-up procedure [27]. COCs were maintained in this medium for 18 h under the same conditions as those used for IVM.

In vitro culture Cumulus cells were removed from presumptive zygotes by vortexing (120 sec) and by gentle pipetting. Then, groups of 30–35 presumptive zygotes were washed three times and cultured in 400 µl synthetic oviduct fluid medium [28] supplemented with 3 mg/ml polyvinyl alcohol (SOF-PVA; control group) and different concentrations of CoQ10 or stabilizer as described in the experimental design.

Preparation of CoQ10 and Stabilizer Dispersion

Phospholipid Lipoid S 100 (Lipoid KG, Ludwigshafen, Germany) was melted together with CoQ10 (Kyowa Hakko Kogyo, Tokyo, Japan) at 65°C. Sodium glycocholate (NaGC) was dissolved in 2.25% (w:w) glycerol-containing distilled water (Wasserfuhr GmbH, Bonn, Germany). This mixture was heated to 65°C and mixed with the molten lipid phase. A pre-emulsion was prepared using an Ultra Turrax (IKA-Labortechnik, Staufen, Germany). This raw emulsion was processed in a heated (65°C) high-pressure homogenizer (Microfluidizer M-110S, interaction chamber: 37/7 75µ F12 Y; Microfluidics International Corp., Newton, MA) at approximately 1200 bars. The hot emulsion was sterile-filtered (0.22 µm) into sterilized vials and allowed to cool to room temperature. The stabilizer dispersion was produced the same way without adding CoQ10. The amounts of the components (CoQ10-dispersion/stabilizer dispersion) used for the preparation were as follows: CoQ10 (1.47/0 g), Lipoid S100 (0.74/1.01 g), NaGC (0.1499/0.2002 g), water with glycerol (28.14/39.71 g). Since the preparation method is not completely quantitative, the final composition of the dispersions may differ from the original one.

Two months after preparation of CoQ10, HPLC analysis (Beckman, Munich, Germany) revealed a CoQ10 concentration of approximately 3.3% (w:w) in the final preparation (column: octadecylsilane Hypersil [Shandon Scientific Ltd., Cheshire, UK] 5 µm, 250 x 4.6 mm; mobile phase: methanol:isopropanol 70:30 at 1.4 ml/min; detection: UV at 275 nm). The CoQ10-content did not change during a further 4 mo of storage (3.3%; w:w). After dilution with dust-free water to an appropriate scattering intensity, particle size analysis of the dispersions was performed by photon correlation spectroscopy (Zetaplus; Brookhaven Instruments Corp., Holtsville, NY) under an angle of 90 degrees. The effective diameter and polydispersity index were calculated as the mean of 6 measurements at 5 min each. Two months after preparation, the CoQ10-dispersion had an effective diameter of 117 ± 0.5 nm and a polydispersity of 0.15 ± 0.01. Six months after preparation, the effective diameter and the polydispersity had not changed. The effective diameter of the stabilizer dispersion was 47 ± 0.2 nm, and the polydispersity was 0.29 ± 0.01 directly after preparation as well as after 3 mo of storage. Investigation by differential scanning calorimetry (Pyris 1; Perkin Elmer, Norwalk, CT) revealed no transition due to crystalline CoQ10 upon heating the emulsion (stored for 2 or 6 mo, respectively) from 20°C to 55°C at 5.0°C/min.

Experimental Design

Effects of different concentrations of CoQ10 on cleavage, blastocyst formation, and hatching rate Presumptive zygotes were cultured in SOF-PVA (control group) or in SOF-PVA supplemented with 10 µM, 30 µM, or 100 µM of CoQ10 or with the corresponding concentrations of stabilizer under SOF-equilibrated paraffin oil (DAB 7160; Merck, Darmstadt, Germany) at 39°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Early cleavage rate (5–8 cells), total blastocyst rate, and hatching rate were evaluated 66 h postinsemination (hpi), 162 hpi, 186 hpi, and 210 hpi, respectively. Oocytes were randomly distributed over all 6 experimental groups (2 treatments x 3 doses), which were evaluated simultaneously in 5 independent experiments carried out at weekly intervals.

Numbers of ICM and TE cells of blastocysts Equal numbers of embryos recovered 186 hpi (fifteen expanded blastocysts per treatment group) were subjected to differential cell staining as described previously [29]. Briefly, blastocysts were washed several times in Dulbecco's phosphate-buffered saline (PBS) to remove excess protein. Blastocysts were treated with 0.5% (w:v) pronase (Protease, type XXV) in PBS for 3–5 min to dissolve the zona pellucida. Then, blastocysts were incubated in a 1:2 dilution in PBS of rabbit antiserum raised against recombinant bovine interferon tau and other trophoblastic secretions [30] for 45 min at 39°C in a humidified atmosphere of 5% CO2 in air. Subsequently, embryos were washed 5 times in PBS warmed at 39°C, and then incubated in PBS supplemented with 5% (v:v) guinea pig complement (ICN Immunobiologicals, Costa Mesa, CA) and 50 µg/ml propidium iodide for 45 min at 39°C in a humidified atmosphere of 5% CO2. After this step, blastocysts were washed again in PBS and then placed in cold absolute ethanol (Merck) containing 25 µg/ml of the fluorochrome bisbenzimide (Riedel-de Haen AG, Seelze, Hannover, Germany) for 30 min at 4°C. Finally, embryos were washed in absolute ethanol, mounted in undiluted glycerol (Merck), and squashed on a glass slide. The stained embryos were observed using a fluorescent microscope (Axiovert 135; Zeiss, Jena, Germany) with an HBO lamp under transmittance illumination and an ultraviolet excitation filter of 365 nm and a barrier filter of 420 nm. Bisbenzimide-stained vital ICM nuclei appeared blue, and nonvital TE nuclei, which were stained with both fluorochromes, gave red or pink fluorescence.

Measurement of the ATP content of blastocysts The ATP content of expanded blastocysts (10 per treatment) recovered 186 hpi after culture in the presence of 30 µM CoQ10 or stabilizer was measured using a commercial assay based on the luciferin-luciferase reaction (Bioluminescent Somatic Cell Assay Kit, Cat. no. FL-ASC, which includes FL-AAM, FL-AAS, FL-AAB, and FL-SAR; Sigma) according to the previously described technique by Rieger [31] and the manufacturer's instructions with some minor modifications. Briefly, blastocysts were washed four times in sample buffer (99.0 mM NaCl, 3.1 mM KCl, 0.35 mM NaH2PO4, 21.6 mM sodium-lactate, 10.0 mM Hepes, 2.0 mM CaCl2, 1.1 mM MgCl2, 25.0 mM NaHCO3, 1.0 mM sodium-pyruvate, 0.1 mg/ml gentamicin, and 6.3 mg/ml BSA). Blastocysts were transferred individually in 50 µl of sample buffer and placed individually into plastic tubes, which were transferred into ice water. Then, 50 µl of ice-cold somatic cell reagent (FL-SAR) was added to all tubes and held in ice water for 5 min. After this, 100 µl of ice-cold assay mix (FL-AAM) dilution (1:25 with ATP assay mix dilution buffer, FL-AAB) was added and tubes were held for 5 min at room temperature in the darkness. The ATP content of the samples was measured individually using a luminometer (Bioluminat LB 9500; Berthold, Wildbad, Germany). To measure samples at the same time, a set of tubes with blastocysts was counted once (C1) and then again (C2) in reverse order. The geometrical mean of measured counts at times C1 and C2 was calculated by square root of Cm (Cm = C1 x C2). A seven-point standard curve (0–6 pmol/tube) was routinely included in each assay. The ATP content was determined from the formula for the standard curve.

Statistical Analysis

Data were statistically evaluated by ANOVA. The model included effects of treatment (CoQ10, stabilizer), dose (10 µM, 30 µM, 100 µM), and the interaction between these factors (treatment x dose). Comparisons between groups were done using Scheffé`s test (doses within treatment) or Student's t-test with Levene's test for equality of variances (treatments within dose). Effects on the ATP content per blastocyst and the ATP content per cell were evaluated using Student's t-test. A value of p < 0.05 was considered significant. Data are presented as means and standard errors of means. Analysis was carried out using the SPSS 7.5 statistical program (SPSS Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Different Concentrations of CoQ10 on Cleavage, Blastocyst, and Hatching Rate

Early cleavage, development to blastocysts, and hatching rate were significantly (p < 0.001) affected by treatment, dose, and the interaction treatment x dose (Table 1). The rates of development to 5- to 8-cell stages and blastocysts under different concentrations of CoQ10 or stabilizer in the culture medium are presented in Figure 1. The effect of CoQ10 was concentration-dependent. Increasing the CoQ10 concentration enhanced the cleavage rate of 5–8 cells (p < 0.001 at 30 µM and 100 µM CoQ10 vs. the corresponding amount of stabilizer), whereas the cleavage rates in 10 µM and 100 µM of stabilizer were similar (Fig. 1A).


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  		TABLE 1. Effects of CoQ10 and stabilizer on cleavage, blastocyst rate, hatching rate, and cell numbers of IVP bovine embryos (ANOVA).



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  		FIG. 1. Effects of CoQ10 and stabilizer on the development of IVP bovine embryos to 5- to 8-cell stages (A), to blastocysts 162 hpi (B) and 186 hpi (C), and to hatched blastocysts 210 hpi (D). Means and SEM from 5 independent experiments. The numbers of oocytes used per experimental group ranged from 354 to 406. The labels of the x-axis are the concentrations (doses) of CoQ10 or stabilizer in µM. Data were analyzed by ANOVA and comparisons of experimental groups were done using Scheffé`s test (doses within treatment) or Student's t-test (treatments within dose). Significant differences between means are indicated by asterisks (treatments within dose: ***p < 0.001) or by different superscripts (doses within treatment: a vs. b, p < 0.05; a vs. c, a vs. d, b vs. c, c vs. d, p < 0.001).

Total blastocyst rate was also affected in a concentration-dependent manner. Addition of 10 µM or 30 µM CoQ10 to SOF-PVA significantly (p < 0.001) increased total blastocyst rate at 162 hpi and 186 hpi when compared with the corresponding concentrations of stabilizer (Fig. 1, B and C). The proportion of blastocysts was significantly higher in 30 µM than in 10 µM CoQ10 at both 162 hpi (p < 0.01) and 186 hpi (p < 0.001). After culture in SOF-PVA supplemented with 100 µM CoQ10, only 7.3 ± 0.4% and 8.4 ± 0.2% blastocysts were recovered 162 hpi and 186 hpi, respectively. This is significantly (p < 0.001) lower than with 10 µM and 30 µM of CoQ10.

The presence of 30 µM CoQ10 significantly (p < 0.001) increased the hatching rate of blastocysts when compared with all concentrations of stabilizer and with the 100 µM CoQ10 group (Fig. 1D). The difference from the 10 µM CoQ10 group reached the border of statistical significance (p = 0.051).

The cleavage rate 66 hpi, blastocyst rates at 162 hpi and 186 hpi, and hatching rate of embryos cultured in SOF-PVA (control group) were 55.1 ± 1.1%, 2.8 ± 0.42%, 10.3 ± 0.9%, and 8.6 ± 0.77%, respectively, and were not significantly (p > 0.05) different from those obtained with all used concentrations of stabilizer.

Numbers of ICM and TE Cells

ANOVA revealed significant (p < 0.001) effects of treatment and dose on the numbers of ICM cells and TE cells, and on total cell numbers of blastocysts recovered at 186 hpi. However, the ratio of ICM cells : TE cells was not significantly affected by these factors (Table 1). The cell numbers of blastocysts from the different treatment groups are presented in Figure 2.



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  		FIG. 2. Effects of CoQ10 and stabilizer on the numbers of ICM cells (A), TE cells (B), total cell numbers (C), and the ICM cell:TE cell ratio (D) of blastocysts recovered 186 hpi. Means and SEM from 15 expanded blastocysts analyzed per experimental group. The labels of the x-axis are the concentrations (doses) of CoQ10 or stabilizer in µM. Data were analyzed by ANOVA, and comparisons of experimental groups were done using Scheffé`s test (doses within treatment) or Student's t-test (treatments within dose). Significant differences between means are indicated by asterisks (treatments within dose: **p < 0.01; ***p < 0.001) or by different superscripts (doses within treatment: a vs. b, b vs. d, p < 0.05; c vs.d, p < 0.001).

Blastocysts produced in the presence of 30 µM CoQ10 exhibited the greatest number of ICM cells (p < 0.01 as compared to the corresponding concentration of stabilizer). The numbers of ICM cells in blastocysts from 100 µM CoQ10 were significantly lower than those in 30 µM CoQ10 (p < 0.001), while those in 10 µM CoQ10 tended to be lower (Fig. 2A). A similar effect was seen for the numbers of TE cells (Fig. 2B) and total cell numbers of blastocysts (Fig. 2C). The ratio of ICM cells:TE cells was similar in all experimental groups (Fig. 2D).

ATP Content

The mean of the total ATP content per expanded blastocyst recovered 186 hpi after culture in the presence of 30 µM CoQ10 was significantly (p < 0.001) higher than that of expanded blastocysts produced in the corresponding concentration of stabilizer (118.6 ± 6.0 x 10-14 mol vs. 72.6 ± 4.7 x 10-14 mol; Fig. 3A). The mean ATP content per cell of blastocysts from the 30 µM CoQ10 group was also increased (0.87 ± 0.04 x 10-14 mol vs. 0.75 ± 0.05 x 10-14 mol), but this difference reached only the border of statistical significance (p = 0.065; Fig. 3B).



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  		FIG. 3. Effects of CoQ10 and stabilizer on the ATP content of expanded blastocysts recovered 186 hpi (A) and on the ATP content per cell of expanded blastocysts (B). Means and SEM from 10 expanded blastocysts analyzed per experimental group. Means were compared using Student's t-test.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of our study demonstrate that CoQ10 increases, in a concentration-dependent manner, the proportion of oocytes developing to 5- to 8-cell and blastocyst stages in vitro. The concentration of CoQ in human blood serum ranges from 0.7 to 1.2 µM [7]. In our study, positive effects of CoQ10 were observed at a much higher concentration. However, it must be taken into account that in serum CoQ is associated with very low-density lipoproteins [4, 7]. Although 30 and 100 µM CoQ10 were found to be optimum for early cleavage (5–8 cells), the highest proportion of blastocysts was obtained with 30 µM CoQ10, with 100 µM CoQ10 significantly reducing the blastocyst rate compared to 30 µM CoQ10. This is most likely due to a negative effect of high concentrations of CoQ10 on later stages of bovine embryos, since such a decrease in the proportion of blastocysts was not seen in the corresponding stabilizer group.

CoQ10 has also been suggested to play an important role in the control of cell proliferation and growth [5–10]. Extraction of CoQ from pig liver cell membranes resulted in inhibition of trans-plasma membrane electron transport and cell growth, and addition of CoQ10 restored these activities [6]. In our study, addition of 30 µM colloidally dispersed, noncrystalline CoQ10 to the culture medium increased the numbers of ICM cells and TE cells.

When added to cultured myocardial cells of mouse fetuses, CoQ10 stimulated the formation of ATP [32], which is in line with the fact that CoQ10 plays an important role in the synthesis of ATP. In our study, the presence of CoQ10 markedly increased the ATP content of expanded blastocysts. The mean ATP content of expanded blastocysts recovered in the presence of stabilizer (72.6 x 10-14 mol) in our study was in the same range as the mean amount of ATP in bovine expanded blastocysts (75.0 x 10-14 mol) recovered on Day 7 after coculture with bovine oviduct cells and measured by the same technique [31]. On the other hand, the difference in ATP content per cell of blastocysts from the CoQ10 and stabilizer treatment groups reached only the border of significance (p = 0.065). In mice, it has previously been shown that the total amount of mitochondrial DNA does not change during preimplantation development and is not altered during in vitro culture of the 1-cell embryo to the blastocyst stage [33]. During bovine oogenesis, rapid expansion of cytoplasmic volume is accompanied by a proportional increase in the number of mitochondria, but this number does not further increase during embryonic development up to the blastocyst stage (reviewed in [34, 35]). Accordingly, Thompson et al. [36] found the ATP production per cell of bovine blastocysts to be lower than that per cell of a 16-cell embryo. In our study, the increase in the number of cells per blastocyst in the 30 µM CoQ10 treatment group may have caused a decrease in the number of mitochondria per cell when compared to the corresponding stabilizer group. Nevertheless, the ATP content per cell of blastocysts produced in the presence of CoQ10 tended to increase, suggesting a possible stimulation of ATP production per mitochondrion. It has previously been shown that CoQ10 is characterized by a high diffusion coefficient [37] and that exogenous administration of CoQ10 to rats leads to accumulation of CoQ10 in liver and spleen [38]. Exogenous administration of CoQ10 in cardioplegic solution significantly increased myocardial stores of ATP [39].

Hatching of expanded blastocysts was also stimulated by the presence of 30 µM CoQ10, which could be due to several mechanisms: 1) CoQ10 and electron transport automatically activate the proton antiport [9], which is important for ion changes and fluid distribution in the blastocyst; 2) by stimulating cell proliferation and increasing the ATP content, CoQ10 could accelerate formation of the blastocoel cavity and consequently the hatching process, which in bovine embryos depends on the physical force of blastocoel expansion and repeated collapsing and re-expansion [40]; and 3) an ionic imbalance that exists in embryos cultured in conventional media [41] could be corrected by CoQ10.

To clarify how much CoQ10 was present in the culture, media supplemented with 10 µM, 30 µM, and 100 µM CoQ10 were incubated for 8 days at 39°C with an equal amount of paraffin oil. Subsequently, the media were assayed for CoQ10 by HPLC. The concentrations measured were 6.6 µM, 16.7 µM, and 44.8 µM, respectively, demonstrating that a substantial amount of CoQ10 was still in the medium, but about 50% of the initial concentration was lost by degradation or transfer to the paraffin phase. Added as powder or dissolved in ethanol to the culture medium, CoQ10 was not able to increase development of bovine embryos (data not shown). In other studies, CoQ10 dissolved in ethanol and added to serum-free culture medium has been reported to stimulate the growth of human adenocarcinoma (HeLa) cells and mouse fibroblast (BALB/3T3) cells [7], and to prevent vanilloid-induced apoptosis in human lymphoblastoid cells [3]. On the other hand, exposure to ethanol resulted in increased superoxide anion generation, increased lipid peroxidation, and excessive cell death in mouse embryos [42] and in neural crest cells [43]. These harmful effects and the teratogenicity of ethanol are mediated, at least in part, by free radical damage [42, 43]. To avoid deleterious effects of ethanol on bovine embryos, we supplemented culture medium with CoQ10 nanoparticles in which crystallization was prevented. The technology applied in our study offers a general route of supplementing media with poorly water-soluble compounds, which may be necessary to optimize chemically defined culture systems.

In summary, our data demonstrate that supplementation of chemically defined culture medium with submicron-sized noncrystalline CoQ10 has positive effects on early cleavage, blastocyst formation, cell proliferation, hatching, and ATP content of IVP bovine embryos. These effects may be due—at least in part—to an improved function of the PMIT system and improved intracellular redox state. The molecular mechanisms of action of CoQ10 on IVP embryos deserve further investigation.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Heike Bunjes and Gunhild Hammer, University of Jena, for the preparation and characterization of coenzyme Q10 and stabilizer dispersion.


   FOOTNOTES
 
1 This study was supported by the Bayerische Forschungsstiftung (76/93) and by the DFG-WO 685/2-1; WO 685/3-1. Back

2 Correspondence: Eckhard Wolf, Lehrstuhl für Molekulare Tierzucht und Haustiergenetik, Hackerstr. 27, 85764 Oberschleissheim, Germany. FAX: 49 89 74017 368; ewolf{at}lmb.uni-muenchen.de Back

Accepted: March 19, 1999.

Received: November 20, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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