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Effect of Inhibiting Nitric Oxide Production on Mouse Preimplantation Embryo Development and Metabolism¹

Department of Biology, University of York, York YO10 5YW, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Nitric oxide (NO) is a free radical that functions as a cell signaling molecule but at high concentrations can be toxic. It is formed from arginine, which is consumed by the mouse blastocyst, but its effect on early embryo development has been little studied. In this study, the role of NO in mouse preimplantation development has been examined in terms of developmental rate and oxidative metabolism. Zygotes were cultured in one of four media; potassium simplex optimization medium (KSOM), KSOM with amino acids (KSOMaa), KSOM without glutamine (KSOM-glut), or KSOM with 0.5 mM arginine (KSOMarg) ± L-NAME (a specific inhibitor of NO production). End points were Day 4 blastocyst rates, cell counts determined using bisbenzimide and oxygen consumption. In KSOM and KSOM-glut, the blastocyst rate was decreased by 1 mM L-NAME from 50.2% ± 3.1% and 37.4% ± 4.5% to 6% ± 3% and 0%, respectively. In KSOMaa, cavitation rates were unaltered but the blastocysts contained fewer cells (P < 0.001). Blastocysts cultured in KSOM and KSOM-glut consumed significantly more oxygen than those cultured in KSOMaa (P < 0.001 and P < 0.05, respectively). However, the addition of 0.1 mM or 1 mM L-NAME to KSOMaa significantly increased the amount of oxygen consumed (P < 0.05 and P < 0.001, respectively). The data suggest a physiological role for NO in mouse preimplantation metabolism and development. One possibility is that NO may limit oxygen consumption at the blastocyst stage at the level of mitochondrial cytochrome c oxidase.

early development, embryo, nitric oxide

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Nitric oxide (NO) is a free radical that functions as a cell signaling molecule but at high concentrations can be toxic [1]. It is produced by the action of nitric oxide synthase (NOS) on L-arginine to yield L-citrulline and NO. Four isoforms of NOS have been characterized; endothelial (eNOS), inducible (iNOS), neuronal (nNOS), and mitochondrial NOS (mtNOS), each displaying a cell-specific pattern of expression [2–4].

NO can influence signal transduction in many ways (reviewed in [5]), the pathway of choice being highly dependent on the local concentration of NO and the surrounding molecular environment [5]. For example, the binding of NO to soluble guanylate cyclase (sGC), a common target of NO, and the subsequent generation of cGMP is a clearly understood low-concentration physiological effect, as is reversible inhibition of mitochondrial respiration [6, 7]. Toxicity to NO generally occurs at high concentrations and is attributed to different mechanisms. These include reaction with superoxide anions to yield peroxynitrite, which can result in the generation of reactive species such as nitrogen dioxide and hydroxyl radicals and to irreversible mitochondrial damage [8, 9].

NO is important in reproductive processes, including embryo implantation, pregnancy, and labor [10–14]. Arginine is consumed in significant quantities by mouse and human preimplantation embryos [15, 16], suggesting that NO production might also be important in early development. In support of this proposition, Gouge et al. [17] and Chen et al. [18] found that N-omega-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NOS [19], severely inhibits development to the blastocyst stage. In addition, research using NO donors suggests that a high exogenous concentration of NO is detrimental to embryo development [20, 21].

The temporal expression patterns of nNOS, eNOS, and iNOS mRNA throughout mouse preimplantation development were recently measured using the reverse transcription polymerase chain reaction (RT-PCR) [22]. All the genes were expressed from the two-cell to the late blastocyst, except for iNOS, which was not detected at the early blastocyst stage. Gouge et al. [17] reported that iNOS and eNOS were expressed by trophectoderm cells of blastocysts from delayed-implanting mice 1 h after estrogen reactivation. These mRNA and protein expression profiles of multiple NOS isoforms further indicate the potential importance of NO in preimplantation development.

The mechanism of NO action during embryo development is unknown, but mitochondrial respiration represents a potential target. Nanomolar concentrations of exogenously added NO can inhibit cytochrome c oxidase reversibly in isolated liver mitochondria in a competitive manner with oxygen [23–25], and even at basal levels, NO can modulate cellular respiration [26, 27]. The oxygen consumption of mouse preimplantation embryos has been measured by Houghton et al. [28]. Prior to cavitation, oxygen consumption is low before increasing fourfold at the blastocyst stage. The control of oxygen consumption in the early embryo is poorly understood, but inhibition of cytochrome c oxidase by NO is a potential regulatory mechanism.

We have used different media and inhibitors in an attempt to understand the role of NO in mouse preimplantation development. Specifically, we have examined the effect of NO on blastocyst developmental rate and cell number; the origin of NO in the embryo, particularly the role of exogenous amino acids; the influence of amino acids on the sensitivity of embryos to NOS inhibition; and the effect of NO on blastocyst oxygen consumption.

	MATERIALS AND METHODS

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Embryo Production and Recovery

Virgin mice, 6–8 wk old, of the strain CBA/Ca x C57BL/6 were superovulated by intraperitoneal injection of 5 IU (0.1 ml) eCG (Folligon; Intervet, Cambridge, U.K.) between 1200 and 1400 h, followed 48 h later by 5 IU (0.1 ml) hCG (Chorulon; Intervet). Females were placed with F1 males of the same strain and the presence of a vaginal plug the following morning was taken as an indication that mating had occurred.

Mice were killed by cervical dislocation. Zygotes were retrieved from the oviducts 24 h post-hCG and cultured under oil in groups of 10–15 to the blastocyst stage in 10-µl drops of one of four media; 1) KSOM [29], 2) KSOM supplemented with a physiological concentration of amino acids [30] (KSOMaa), 3) KSOM with no glutamine (KSOM-glut), and 4) KSOM supplemented with 0.5 mM arginine (KSOMarg). Media were supplemented with L-NAME (10 µM, 500 µM, 100 µM, 1000 µM) (Sigma, Poole, Dorset, U.K.) as appropriate. The proportion of embryos reaching the blastocyst stage on Day 4 (96 h post-hCG) or Day 5 (120 h post-hCG) was recorded.

All work was carried out in accordance with the U.K. Home Office Animals (Scientific Procedures) Act, 1986.

Cell Counts

The zona pellucida was removed using acid Tyrode solution, pH 2.1, and embryos were incubated in 50 µg/ml bisbenzimide (Hoechst 33428; Sigma, U.K.) at 2–4°C for at least 1 h. The embryos were washed in absolute alcohol for 5–30 min before being mounted in a drop of glycerol on a microscope slide beneath a coverslip. The number of cells in each embryo was counted using a fluorescence microscope (Vickers, York, U.K.) with filter block A.

Oxygen Consumption Measurement

The oxygen consumption of preimplantation embryos was performed as previously described [28]. Briefly, 1 µl 1 mM pyrene (Sigma Chemical Company, Poole, Dorset, U.K.), dissolved in paraffin oil (BDH, Poole, Dorset, U.K.), was drawn into a 5-µl PCR micropipette (Laser Laboratory Systems, Southhampton, U.K.). Groups of 10–15 blastocysts were taken up in 2 µl Hepes-buffered KSOMaa and the tube sealed. Oxygen consumption was measured by following the increase in pyrene fluorescence at 340 nm over a 3- to 4-h period using a Fluovert fluorescence microscope (Leica U.K., Ltd., Milton Keynes, U.K.) with photomultiplier and photometer attachments. Two control tubes were measured concurrently; a 0% oxygen control containing 1 µl pyrene and 2 µl 1 mg/ml yeast in 60 mM glucose, pre-equilibrated overnight, and a 20% oxygen control containing 1 µl pyrene and 2 µl KSOMaa Hepes in the absence of embryos. Arbitrary fluorescence readings were converted to values for oxygen consumption in nl embryo^–1 h^–1 using a computerized mathematical model [28].

Statistical Analysis

Oxygen consumption data, expressed in nl embryo^–1 h^–1, and cell counts were analyzed by one-way analysis of variance. Differences between individual means were compared by Fisher test. When comparing developmental rates ± L-NAME, the average percentage ± SEM of embryos at each developmental stage on Day 4 was calculated and analyzed by one-way analysis of variance and Fisher tests following angular transformation. Developmental rates on Days 4 and 5 were compared using a Student t-test. The data were derived from a minimum of three replicates per treatment.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The effect of inhibiting NO production on the rate of mouse blastocyst development was determined in the presence and absence of amino acids. An average of 49.8% ± 3.4% of embryos cultured from the one-cell stage in KSOMaa reached the blastocyst stage by Day 4. The presence of 0.5 mM or 1 mM L-NAME did not affect the Day 4 blastocyst rate (Fig. 1) but significantly reduced the cell number (Fig. 2). When embryos were exposed to 0.01 mM and 0.1 mM L-NAME, the blastocyst cell count was not affected (Fig. 2).

View larger version (9K): [in this window] [in a new window]   FIG. 1. Day 4 blastocyst rate of embryos cultured in KSOMaa ± L-NAME. Values are expressed as percentage ± SEM (n = 397)

View larger version (11K): [in this window] [in a new window]   FIG. 2. Cell counts of Day 4 blastocysts cultured in KSOMaa ± L-NAME. Values are mean ± SEM. ***, P < 0.001, significantly different from control

When zygotes were cultured in KSOM, 50.2% ± 3.1% developed to the blastocyst stage by Day 4. The addition of 0.5 mM L-NAME decreased this rate to 6.1% ± 2.3% (P < 0.001), while 1 mM L-NAME prevented Day 4 blastocyst formation (Fig. 3).

View larger version (9K): [in this window] [in a new window]   FIG. 3. Day 4 blastocyst rate of embryos cultured in KSOM ± L-NAME. Values are expressed as percentage ± SEM. ***, P < 0.001, significantly different from control (n = 345)

KSOM contains a high concentration of glutamine (1 mM) as the sole amino acid. Embryos were therefore cultured in media lacking glutamine (KSOM-glut). There was no significant difference in the Day 4 blastocyst rate between KSOM-glut, KSOM, or KSOMaa (Fig. 4). However, blastocysts cultured in KSOM (38.0 ± 1.49) or KSOM-glut (37.8 ± 1.56) contained significantly fewer cells than in KSOMaa (51.8 ± 2.62) (Fig. 5). The addition of 0.5 mM or 1 mM L-NAME to KSOM-glut prevented cavitation, with most embryos arresting at or before the eight-cell and two-cell stages, respectively (Fig. 6).

View larger version (9K): [in this window] [in a new window]   FIG. 4. Day 4 blastocyst rate of embryos cultured in KSOM, KSOMaa, and KSOM-glut. Values expressed as percentage ± SEM

View larger version (8K): [in this window] [in a new window]   FIG. 5. Cell counts of Day 4 blastocysts cultured in KSOM, KSOMaa, or KSOM-glut. Values are mean ± SEM; ***, P < 0.001, significantly different from KSOMaa

View larger version (20K): [in this window] [in a new window]   FIG. 6. Day 4 development of zygotes cultured in KSOM-glut ± L-NAME. Values expressed as percentage ± SEM. Stages of development: DEG = degenerate, C8 = compacted eight-cell, M = morula, EB = early blastocyst, B = blastocyst, ExB = expanding blastocyst, FEB = fully expanded blastocyst, HB = hatching blastocyst (n = 349)

The oxygen consumption of Day 4 blastocysts cultured in KSOMaa was 0.24 ± 0.02 nl embryo^–1 h^–1. In the presence of 0.1 mM L-NAME or 1 mM L-NAME, oxygen consumption increased significantly to 0.34 ± 0.06 nl embryo^–1 h^–1 and 0.49 ± 0.015 nl embryo^–1 h^–1, respectively (Fig. 7a). When cultured in KSOM, Day 4 blastocysts consumed significantly more oxygen (0.4 ± 0.03 nl embryo^–1 h^–1; P < 0.001) than those cultured in KSOMaa (0.24 ± 0.02 nl embryo^–1 h^–1; Fig. 7b). In the presence of KSOM containing 0.5 mM arginine (KSOMarg), blastocysts consumed oxygen at a similar rate to those cultured in KSOMaa (0.19 ± 0.009 nl embryo^–1 h^–1) but at a significantly lower rate than blastocysts cultured in KSOM (P < 0.01). In KSOM-glut, blastocysts consumed oxygen at a rate of 0.32 ± 0.036 nl embryo^–1 h^–1, which was significantly greater than in KSOMaa and KSOMarg (P < 0.05 and P < 0.01, respectively) but significantly lower than in KSOM (Fig. 7b, P < 0.05).

View larger version (16K): [in this window] [in a new window]   FIG. 7. Oxygen consumption of Day 4 blastocysts cultured in (a) KSOMaa ± L-NAME and (b) KSOM, KSOMaa, KSOMarg, and KSOM-glut. Number of determinations is shown in parentheses. Values are mean ± SEM. Bars with same superscripts are significantly different; (a, b, e–g) P < 0.001, (c) P < 0.01, (d, h, i) P < 0.05

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Different culture media were used to assess the influence of NO on mouse preimplantation embryo development and metabolism in terms of blastocyst formation, cell number, and oxygen consumption. Until now, no clear role for NO in preimplantation development has emerged, largely due to different culture media being used in different studies. For example, in KSOM supplemented with 0.5 mM L-arginine (KSOMarg), 0.5 mM L-NAME was found to compromise development and reduce the percentage of embryos that cavitated [17]. In contrast, Tranguch et al. [22] found that addition of 0.25 mM L-NAME to Whitten medium (which is free of amino acids) significantly reduced the proportion of mouse embryos developing beyond the two-cell stage. We have found that the presence of 0.5 mM L-NAME in KSOM significantly reduces the Day 4 blastocyst rate from 50.2% ± 3.1% to 6.0% ± 2.3% (P < 0.001). In KSOM-glut, addition of 0.5 mM L-NAME prevents all development beyond the compacted eight-cell stage, but in KSOMaa, not even 1 mM L-NAME significantly affected the Day 4 blastocyst rate. These data highlight the importance of media constituents in the sensitivity of embryos to inhibition of NOS.

NO is produced from L-arginine, which may be derived intracellularly by conversion from other amino acids (e.g., from glutamine via citrulline) or from amino acids present in the culture medium. The entry of arginine is a function of the cationic amino acid transporters (CAT1 and CAT2). In a number of cell types, including endothelial cells [31], vascular smooth muscle cells [32], and macrophages [33], the internal concentration of arginine depends on the external concentration of other cationic amino acids that compete with arginine for entry. CAT1 and CAT2 are expressed throughout mouse preimplantation development and the activity of system b^0,+, one of the major arginine transporting systems in the mouse embryo, is upregulated 30 times during blastocyst formation [34, 35]. In other words, arginine availability at sites of NOS activity could be influenced by the concentration of cationic amino acids present in the surrounding medium and this could represent one mechanism for regulating embryo NO production.

In KSOMaa, we found that 1 mM L-NAME did not influence the Day 4 blastocyst rate but that, in KSOM, L-NAME completely inhibited blastocyst development by Day 4 (Figs. 1 and 3). With no direct exogenous source of arginine, KSOM-cultured embryos are likely to have a lower internal arginine pool than embryos cultured in KSOMaa. This may reduce competition by arginine for sites on NOS and limit the basal rate of NO production. These factors could cause enhanced sensitivity of KSOM-cultured embryos to changes in NOS activity induced by L-NAME.

Glutamine may serve as a precursor for arginine, with citrulline as an intermediate in this pathway [36]. When glutamine was removed from the culture medium (KSOM-glut), 1 mM L-NAME arrested preimplantation development in its very early stages (79.4% ± 12.1% of embryos arrested at the two-cell stage). This could again be explained in terms of internal arginine availability for NO production; in KSOM-glut, arginine for NO production can only be derived from internal pools of amino acids because all exogenous sources of amino acids have been removed.

The current data suggest that NO production is obligatory for mouse preimplantation embryo development and that amino acid supplementation may provide an exogenous source of arginine, which can be used by the embryo for NO production.

Oxygen consumption provides the best global indication of the ability of an embryo to produce ATP. Prior to cavitation, the oxygen consumption of preimplantation embryos is equivalent to a quiescent adult tissue, such as skin [28]. At the blastocyst stage, oxygen consumption significantly increases in all species studied, largely due to the energy requirement of the Na⁺, K⁺, ATPase, and protein synthesis [28, 37, 38]. The oxygen consumption of Day 4 blastocysts cultured in KSOMaa with 0.1 mM or 1 mM L-NAME was significantly greater than in those cultured without L-NAME (Fig. 7a), suggesting that NO inhibits mitochondrial respiration. In support of this proposition, it has been shown that nanomolar concentrations of NO can compete with oxygen for mitochondrial cytochrome c oxidase, resulting in a reduced oxygen consumption [23–25]. The increased oxygen consumption observed in the presence of L-NAME is therefore likely to be due to relief of NO inhibition of the electron transport chain at the level of cytochrome c oxidase.

Day 4 blastocysts cultured in KSOMaa or KSOMarg had a significantly lower oxygen consumption than those cultured in KSOM (Fig. 7b; P < 0.01). As discussed above, embryos cultured in KSOMaa (i.e., with a mixture of amino acids) may produce higher levels of NO than those cultured in KSOM due to increased internal availability of arginine. Theoretically, the same concept could apply to embryos cultured in KSOMarg medium containing 0.5 mM arginine. This potentially greater availability of NO may inhibit cytochrome c oxidase and cause the reduction in oxygen consumption observed (see Fig. 8). Embryos cultured in KSOM-glut displayed an oxygen consumption significantly greater than that of KSOMaa- or KSOMarg-derived embryos (P < 0.05) but significantly less than that of KSOM embryos (P < 0.05). If, as we have suggested, blastocysts cultured in KSOM-glut have a lower basal level of NO production than KSOM-cultured embryos, further relief of cytochrome c oxidase inhibition and a greater oxygen consumption would be predicted. However, this was not found to be the case. Because glutamine is a source of nucleotide and nucleoside precursors in the embryo, the lower level of oxygen consumed by embryos cultured in KSOM-glut may possibly reflect a reduction in protein synthesis and/or cell number.

View larger version (36K): [in this window] [in a new window]   FIG. 8. Potential mechanisms for the interaction between NO production and oxygen consumption of mouse blastocysts cultured in different media. CAA = Cationic amino acids other than arginine; Gln = glutamine; Arg = arginine; CAT = cationic amino acid transporter; NOS = nitric oxide synthase

Using respiratory chain inhibitors and uncouplers, Trimarchi et al. [39] found that the proportion of oxygen consumption that was the result of oxidative phosphorylation (i.e., the mitochondrial component) at the blastocyst stage was 70%, confirming the work of Benos and Balaban [40], who suggested that 50%–70% of all oxygen consumed by blastocyst-stage embryos was utilized for mitochondrial ATP formation. Our results show that Day 4 blastocysts cultured in KSOMaa have an oxygen consumption approximately 50% lower than those cultured in KSOM. We have suggested that embryos cultured in KSOMaa produce more NO due to the increased availability of exogenous arginine. In addition, we propose that the mitochondrial component of oxygen consumption described by Trimarchi et al. [39] is a target for NO and that embryos cultured in a physiological concentration of amino acids undergo basal inhibition of oxidative phosphorylation via NO. Relief of this inhibition by addition of L-NAME leads to blastocysts having a higher oxygen consumption but lower cell number, suggesting that they are of lower developmental competence. Relief of this inhibition by culture in conditions where production of NO by the embryo is likely to be lower (i.e., in KSOM only or KSOM-glut) also results in blastocysts with a higher oxygen consumption and lower cell number. These data are in contrast with previous work suggesting that blastocysts with a higher oxygen consumption have a greater developmental potential [39, 41, 42]. However, the different culture media used makes direct comparison with these studies difficult. It has been hypothesized that embryos with a lower amino acid turnover, and hence a quiet metabolism, are developmentally more competent [43], as shown in terms of amino acid profiling, in the human [15]. We now propose that blastocysts with a higher developmental potential also have a reduced oxygen consumption, a characteristic that may be mediated by NO.

	ACKNOWLEDGMENTS

 
The authors thank the staff of the animal house for maintaining the animals used in this study.

	FOOTNOTES

 
¹ Supported by a Wellcome Trust Research Career Development Fellowship to F.D.H.

² Correspondence: R.C. Manser, Department of Biology (Area 3), University of York, PO Box 373, York YO10 5YW, United Kingdom. FAX: 0044 1904 328505; rcm3{at}york.ac.uk

Received: 20 November 2003.

First decision: 12 December 2003.

Accepted: 19 March 2004.

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