Hum. Reprod. Advance Access published online on June 25, 2008
Human Reproduction, doi:10.1093/humrep/den187
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Erythrocytes counteract the negative effects of female ageing on mouse preimplantation embryo development and blastocyst formation
Department of Obstetrics and Gynecology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
1 Correspondence address. E-mail: fukuhara{at}cc.hirosaki-u.ac.jp
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Abstract |
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BACKGROUND: The low developmental competence of embryos from ageing females remains an enigma; it is presumably attributable to oxidative stress. A number of antioxidant mechanisms exist in the erythrocyte and these have been investigated in other cells and tissues. However, very few studies have reported the effects of erythrocyte supplementation on developmental competence in ageing embryos.
METHODS: In Experiment 1, IVF embryos from young (7–10 weeks) mice were cultured in medium supplemented with an oxidizing agent, hypoxanthine/xanthine oxidase, in the presence and absence of erythrocytes. In Experiment 2, the development of embryos derived from young and ageing (40–50 weeks) female mice was assessed in the presence and absence of erythrocytes.
RESULTS: In Experiment 1, the presence of hypoxanthine/xanthine oxidase significantly inhibited embryo development (P < 0.0001). Erythrocyte supplementation clearly overcame the detrimental effects in a dose-related manner. In Experiment 2, in the absence of erythrocytes, developmental competence was significantly lower in embryos from ageing females than in those from young females (P < 0.01). However, in ageing females, the supplementation of erythrocytes significantly promoted the development of embryos to the blastocyst stage (51.1% versus 77.3%; P < 0.01).
CONCLUSIONS: Supplementation with erythrocytes can counteract the negative effect of maternal ageing on embryo development and blastocyst formation.
Key words: ageing/antioxidant/embryo/erythrocyte/reactive oxygen species
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Introduction |
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Female fertility declines with age. A variety of factors may contribute to this age-related decline in fertility, including reduced numbers of oocytes, poor oocyte quality and diminished endometrial receptivity. However, one of the well-recognized aspects of reproductive decline is poor oocyte quality, as seen in the significantly better pregnancy rates during ovum donation (Navot et al., 1991
). Aneuploidy (Koehler et al., 1996), mitochondrial DNA mutations (Keefe et al., 1995; Wei et al., 1998), mitochondrial dysfunction (Van Blerkom et al., 1995) and cytoskeletal alterations (Battaglia et al., 1996) have been suggested to play a part in the age-associated reduction of oocyte quality (Baird et al., 2005). In addition, reduction in rates of blastocyst formation with increasing age has been reported (Janny and Menezo, 1996; Keefe, 1997). Although many hypotheses have been suggested to account for the relationship between female age and oocyte quality, the mechanisms that underlie these changes remain unclear.
The free radical theory of ageing emphasizes that biomolecules are attacked by free radicals and that the repeated damage to biomolecules plays a major role in ageing (Harman, 1956, 1981). It is suggested that reactive oxygen species (ROS) play essential roles in the age-related decline in female fertility (Tarin, 1995, 1996). ROS may originate either directly from oocytes and embryos or from their surroundings, and are detrimental to embryo development (Goto et al., 1993). Oxidative damage may result from overproduction and/or decreased clearance of ROS by the scavenging mechanisms. Oxidative stress experimentally induced by oxidizing agents (Liu and Keefe, 2000; Liu et al., 1999, 2000), visible light or high atmospheric O2 concentrations (Kitagawa et al., 2004) has deleterious effects on the survival and nuclear and cytoplasmic maturation of oocytes and the developmental competence of the embryo. On the other hand, multiple defence mechanisms are present in both embryos and their surroundings (Guerin et al., 2001; Salmen et al., 2005). ROS scavengers counteract the disturbing effects of female ageing on oocyte quality and quantity in the mouse (Tarin et al., 2002). In embryos from ageing females, antioxidant defence mechanisms may be insufficient and oxidative stress may increase. Therefore, it is particularly important to pay careful attention to culture conditions for embryos from ageing females to protect them from oxidative stress in vitro.
The established function of the erythrocyte is to transport oxygen to tissues and remove carbon dioxide, because of which the erythrocytes come into contact with highly toxic oxygen species during this process. Against this, a number of antioxidant mechanisms exist in the erythrocyte (Richards et al., 1998a,b). The protective effect of erythrocytes against ROS has been discussed with respect to other cells and tissues subjected to oxidative stress (Richards et al., 1998a,b). On the other hand, it has been reported (Matsuoka et al., 1995) that, in mice, small numbers of erythrocytes effectively protect cultured oocytes and embryos from oxidative stress and that the presence of erythrocytes improves the early development of embryos by their antioxidant effect. However, very few studies have reported the effects of erythrocyte supplementation on developmental competence of oocytes and embryos in vitro.
The objectives of this study were to evaluate the adverse effects of exogenously induced ROS by investigating the effect of hypoxanthine/xanthine oxidase on mouse embryo development in vitro, to examine the protective effect of erythrocyte supplementation and then to examine the effects of erythrocytes on the blastocyst development rate and the hatching rate of embryos derived from ageing female mice.
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Materials and Methods |
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Animals
Institute of Cancer Research (ICR) mice were originally purchased from central laboratories for experimental animals Japan, Inc. and subsequently bred in our laboratory. The mice were maintained on a 12 h light:12 h dark photoperiod in a temperature-controlled room at 21–23°C. All experiments were performed in accordance with the guidelines for animal experimentation of Hirosaki University. ICR mice were used at 7–10 weeks of age as young mice, and at 40–50 weeks of age as ageing mice.
Chemicals and culture media
All chemicals used in the study were purchased from Sigma Chemical Company (St Louis, MO, USA) unless otherwise stated.
The basic medium used for the culture of embryos was Quinn’s Advantage Protein Plus (QA-P+) system (Sage, Oxford, UK), and QA-P+Fertilization medium for fertilization procedures. The original formulation of QA-P+Fertilization and cleavage medium was modified by the addition of citrate, non-essential amino acids, taurine and calcium lactate. The original formulation of the QA-P+Blastocyst medium was modified by the addition of non-essential and essential amino acids, taurine, glutathione, minimum essential medium, vitamins and calcium lactate. Embryos were cultured to the blastocyst stage in QA-P+sequential media. Embryos were cultured in each well of a 4-well multidish (Nunc, Roskilde, Denmark) containing 800 µl/well of the culture medium, which was covered with mineral oil in a humidified atmosphere of 5% CO2 at 37°C.
Oocyte isolation, IVF and in vitro culture
Superovulation was induced in the female mice by i.p. injection of 5 IU pregnant mare serum gonadotrophin (Serotropin, Teikokuzouki, Tokyo, Japan) followed 48 h later by an i.p. injection of 5 IU HCG (Mochida, Tokyo, Japan). The mice were sacrificed by cervical dislocation 14 h after HCG injection and ovulated oocytes were retrieved from the oviducts. Spermatozoa from the cauda epididymis from mature ICR male mice (aged 10–12 weeks) were collected and capacitated in QA-P+Fertilization medium for 1 h at 37°C. Oocytes were then inseminated in vitro with 1.0 x 106/ml capacitated spermatozoa in medium. Five hours after insemination, oocytes were denuded of surrounding cumulus cells by repeated pipetting and then washed several times in culture medium.
The normal, fertilized embryos were then transferred to 4-well multidishes for culture. Culture was performed in 800 µl of QA-P+system medium, covered with mineral oil, in a humidified atmosphere of 5% CO2 at 37°C for 5 days. Medium was equilibrated in this atmosphere for at least 3 h before culture. The numbers of embryos that developed to the 2-cell, 4-cell, 8-cell, morula, blastocyst, hatching and hatched blastocyst stages were determined under an inverted microscope (SZX12; Olympus, Tokyo, Japan).
Preparation of erythrocytes
Blood was collected (1.5 ml from each mouse) from ICR male mice, and we used two mice for each set of experiments. Citrate–phosphate–dextrose solution was used as an anticoagulant. Whole blood was filtered through a Sepacell RN-20 leukocyte depletion filter (Asahi Medical Co., Tokyo, Japan), followed by centrifugation at 3000g for 10 min and the plasma and buffy coat was removed. Erythrocytes were washed three times by centrifugation at 3000 g for 10 min using ice-cold Ca2+ and Mg2+-free Dulbecco’s phosphate-buffered saline (PBS). The concentration of erythrocytes was counted using an improved Neubauer haemocytometer, and the erythrocytes were resuspended in culture medium for the following experiments.
Total cell number of blastocysts
On Day 5, blastocysts were collected, fixed with 4% (w/w) paraformaldehyde in PBS and stained with Hoechst 33342 (Dojindo, Tokyo, Japan). The blastocysts were then washed three times with PBS and put on a glass slide with a coverslip. The total number of nuclei were counted under a fluorescence microscope (Axiovert 200M; Carl Zeiss Inc., Jena, Germany) with a UV filter.
Experiment 1
To investigate the effect of ROS on mouse embryos, embryos obtained from young mice were cultured in medium containing 0.5 mM hypoxanthine and 0.01 U/ml xanthine oxidase. The cleavage rate and the rate of development to the blastocyst stage were examined. The effect of erythrocytes on the in vitro development of IVF embryos was examined at haematocrit values of 0.001%, 0.01%, 0.1% and 1% (5 x 104, 5 x 105, 5 x 106 and 5 x 107 erythrocytes/ml): the numbers of mice were 23, 5, 5, 5, 10 and 8 in each group, respectively, and the total numbers of embryos were 532, 153, 171, 165, 400 and 283 in each group.
Experiment 2
To examine the effect of erythrocytes on IVF embryos from ageing mice, development rates were compared between embryos from young and ageing mice in the presence of erythrocytes (haematocrit 0.2%; 1 x 107 erythrocytes/ml) during in vitro culture. The numbers of mice used were 23, 25, 39 and 34 (young control, young supplemented erythrocytes, old control and old supplemented erythrocytes), and the total numbers of oocytes and embryos were 532, 607, 485 and 480, and 477, 542, 390 and 386 in each group, respectively.
Statistical analysis
Cleavage rate, development rate to the blastocyst stage, hatching rate and cell counts were evaluated as percentages or cell numbers and presented as mean (SEM). For statistical analysis, percentages were subjected to angular transformation. The data were analysed by one-way analysis of variance and subjected to Fisher’s protected least-significant difference analysis using StatView software (SAS Institute, Inc., Cary, NC, USA). Differences among groups were considered significant when the P-value was <0.05.
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Results |
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Experiment 1
Effects of hypoxanthine/xanthine oxidase on the development of IVF embryos
The presence of hypoxanthine/xanthine oxidase in the culture medium significantly decreased the cleavage rates of embryos compared with control (98% versus 44%, P < 0.0001). Although most embryos cultured in control medium developed to the blastocyst stage (82%), embryos exposed to hypoxanthine/xanthine oxidase were completely arrested at the 1- or 2-cell stage, suggesting that hypoxanthine/xanthine oxidase elicits potent inhibition of the development of embryos (Table I, Fig. 1).
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Effects of erythrocytes on development of IVF embryos cultured with hypoxanthine/xanthine oxidase
The addition of erythrocytes to medium supplemented with hypoxanthine/xanthine oxidase markedly improved preimplantation development (Table I). The percentages of embryos that developed to the 2-cell, 4-cell, morula and blastocyst stages increased significantly (P < 0.05) in an erythrocyte concentration-related manner (Table I, Fig. 1).
Experiment 2
Effects of female ageing on development of IVF embryos
No significant differences between young and ageing control groups were detected in the rates of development of IVF embryos to the 2-cell stage (Table II). However, the percentages of embryos that reached the 4-cell, morula, blastocyst and hatching blastocyst formation were significantly lower in the ageing control group than in the young control group (4-cell stage, 94.7% versus 84.7%, P = 0.01; morula, 88.0% versus 71.0%, P = 0.001; blastocyst, 82.0% versus 51.1%, P < 0.0001; hatching blastocyst, 49.9% versus 30.4%, P = 0.0007). The average cell number at the blastocyst stage in the ageing control group was significantly lower than that in the young control group (63.9 versus 84.9; P < 0.01), strongly indicating that female ageing is associated with deterioration in oocyte quality.
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Effects of erythrocytes on development of IVF embryos from young and ageing mice
In embryos from young mice, no significant differences were observed in the rate of development to the 2-cell, 4-cell, morula, blastocyst and hatching blastocyst stages between the erythrocyte-supplemented group and the control. The total number of cells in blastocysts derived from IVF embryos cultured in medium with erythrocytes was lower than that in control blastocysts (73.0 versus 84.9; P = 0.0003).
In ageing mice, on the other hand, the addition of erythrocytes promoted the development of embryos to the 4-cell stage (84.7% for control versus 91.9%; P = 0.03), morula stage (71.0% versus 84.2%; P = 0.006), blastocyst stage (51.1% versus 77.3%; P < 0.0001) and hatching blastocyst stage (30.4% versus 44.4%; P = 0.002). For ageing mice, the development rates of embryos cultured with erythrocytes improved to a similar extent as those of embryos from young mice, suggesting that erythrocytes counteracted the negative effects of ageing on the development of embryos from ageing females. The total number of cells in blastocysts derived from IVF embryos and cultured in medium with erythrocytes was significantly higher compared with the control (63.9 versus 72.4; P = 0.015).
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Discussion |
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The results of the present study demonstrate clearly that hypoxanthine/xanthine oxidase, a potent oxidizing agent, inhibits the development of mouse preimplantation embryos and that erythrocytes completely overcome the inhibitory effects of the oxidizing agent.
Early mammalian embryos are susceptible to damage caused by ROS. ROS, up to a certain concentration, play important roles in the normal development of oocytes and embryos; however, once the ROS level exceeds a certain level, they cause oxidative stress and become toxic. It has been shown that embryo development is retarded or even arrested by exposure to media with high oxygen tension (Kitagawa et al., 2004). Under high oxygen tension, excessive ROS, such as hydrogen peroxide and superoxide radicals, are produced (Goto et al., 1993) and these ROS are considered to react with extremely high rate constants with amino acids, phospholipids, nucleotides and organic acids (Orsi and Leese, 2001), and therefore cause serious damage to embryos (Fujitani et al., 1997; Agarwal et al., 2003). On the other hand, it has been shown that ROS are produced in cultured embryos exposed to atmospheric oxygen tension, and that addition of free radical scavengers can ameliorate the 2-cell block in mouse embryo culture (Legge and Sellens, 1991). Since then, many reports have appeared describing the search for appropriate agents that can overcome the detrimental effects of free radicals (Liu et al., 1999). For example, several of these antioxidants, such as superoxide dismutase (SOD), catalase, cysteine and vitamin E (Olson and Seidel, 2000; Orsi and Leese, 2001; Wang et al., 2002; Ali et al., 2003), have been tested. Thus far, however, the effects of these chemicals have not been consistent, probably because of the difference in the animal models or culture medium composition; the exact reason remains unclear (Bavister, 1995; Orsi and Leese, 2001).
Erythrocytes, on the other hand, have an antioxidant mechanism in addition to the transport of oxygen. Therefore, it is of interest to study the effect of erythrocytes on embryo development in vitro. It is reported that erythrocytes alleviate post-ischaemic reperfusion injury in the liver (Motoyama et al., 2000) and the heart (Nohl et al., 1981), and that co-culture of human umbilical vein endothelial cells and erythrocytes removes the ROS load (Richards et al., 1998a,b). Erythrocytes are not only easily available, but they are also superior to other antioxidant agents. This is because erythrocytes contain a group of scavenging enzymes, such as SOD, catalase and glutathione, as well as haemoglobin, which works as a nitric oxide scavenger and suppresses ROS generation. Indeed, it has been reported that the addition of haemoglobin improves embryo development to the blastocyst stage and significantly increases the number of cells per blastocyst (Park et al., 2000, 2001; Kim et al., 2006). Moreover, combined administration of SOD or catalase with haemoglobin has been reported to increase the survival and fertilization rates of cryopreserved mouse oocytes (Dinara et al., 2001), strongly supporting the view that erythrocytes are the most appropriate agents for regulating ROS levels. In fact, as shown in Table I, we found that erythrocyte supplementation clearly overcame the detrimental effects of hypoxanthine/xanthine oxidase in a concentration-related manner. It has been reported that the addition of erythrocytes to the culture medium overcomes the 2-cell block in mouse IVF embryos and improves embryo development (Matsuoka et al., 1995; Musoh et al., 2002). The results of the present study support these data.
The result of the present study, on the other hand, demonstrates that erythrocyte supplementation improved the development of embryos from aged mice. It has been well documented that developmental competence is low in embryos from ageing individuals compared with those from young individuals (Janny and Menezo, 1996). Oocytes from young females and embryos cultured with an oxidizing agent show cytoskeletal and chromosomal aberrations (Tarin, 1996) and growth arrest (Liu et al., 1999). These morphological changes are similar to those seen in oocytes and embryos from ageing mice. Therefore, one of the possible mechanisms for the low fecundity of aged animals is presumably attributable to increased ROS levels resulting from mitochondrial dysfunction (Tarin, 1995; Wilding et al., 2001; Thouas et al., 2005) and to decreased antioxidant mechanisms (Friedman et al., 1997; Van Blerkom et al., 1997; Carbone et al., 2003). It may be easier for the ROS level to reach a harmful level in ageing oocytes and embryos than in young oocytes and embryos, leading to a lower potential for embryo development and more frequent developmental arrest in vitro. This suggests that the culture environment for oocytes and embryos from aged animals needs more attention (Thouas et al., 2005). Accordingly, attempts have been made to reduce the level of ROS in oocytes, embryos and their immediate environment in order to improve embryo development. Tarin et al. (1998, 2002) have shown that oral administration of antioxidant neutralizes the disturbing effects of ageing on the segregation of chromosomes during the first meiotic division and the distribution of chromosomes in the metaphase II spindle. However, whether in vitro supplementation with antioxidants counteracts the negative effects of female ageing has not been well investigated. To our knowledge, this is the first report that has demonstrated that embryos from ageing mice show reduced growth in vitro, and that the presence of erythrocytes could improve embryo development.
As shown in Table II, addition of erythrocytes to the culture media of IVF embryos did not accelerate the development of embryos from young mice, suggesting that the addition of erythrocytes is only useful for embryos from ageing mice. However, the reason as to why the addition of erythrocytes did not have a significant effect on embryo development in the young group remains unclear. We conjecture that the scavenging constituents that originally existed in the sequential medium that we used eliminated the harmful ROS generated in vitro. Similar results, with no significant difference, have been reported for other studies in which SOD or its equivalent was added and a medium containing other scavenging constituents, such as EDTA, was used (Orsi and Leese, 2001; Ali et al., 2003). We consider erythrocytes to be effective in the ageing mice because the amount of ROS generated overwhelmed the amount that the scavenging constituents alone were able to eliminate.
Although quite a few studies have reported that the rates of development to the blastocyst stage are low in embryos from ageing females compared with those from young females, some of these studies have also reported no significant difference in implantation rate between ageing and young groups when the blastocyst is obtained in vitro (Pantos et al., 1999; Shapiro et al., 2002). However, precise inspection to check for chromosomal aberration in addition to implantation and pregnancy rates is required for blastocysts obtained by adding erythrocytes to ageing embryos. Further studies are required to find out whether this method works similarly in other species, including humans, and the mechanism should also be investigated. When the mechanism is clarified, the addition of erythrocytes can be used to rescue embryos with low potential from ageing individuals, in which development may arrest in the normal in vitro environment. Increasing numbers of women are delaying childbearing until the end of the third and sometimes the fourth decade of life, and therefore there is a strong need for the clinical application of this technique.
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Acknowledgements |
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We are grateful to Mr M. Kaneko at Asahi Medical Co., Ltd. for the generous donation of leukocyte depletion filters. We wish to thank Mr. F. Kojima for technical assistance and PhD. S. Watanabe for the advice and expertise.
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References |
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Submitted on December 2, 2006; resubmitted on April 15, 2008; accepted on April 21, 2008.
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