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Human Reproduction Vol.21, No.11 pp. 2776–2782, 2006 doi:10.1093/humrep/del038 Advance Access publication September 1, 2006. Amino acid, ammonia and urea concentrations in human pre-ovulatory ovarian follicular fluid Marcin Józwik1,3, Maciej Józwik1, Cecilia Teng2 and Frederick C. Battaglia2 1 Department of Gynecology, Medical University of Bialystok, Bialystok, Poland and 2Division of Perinatal Medicine, University of Colorado, Aurora, CO, USA 3 To whom correspondence should be addressed at: Department of Gynecology, Medical University of Bialystok, Sklodowskiej 24 A, 15–276 Bialystok, Poland. E-mail: [email protected] BACKGROUND: This study aimed to determine amino acid (AA), ammonia and urea concentrations in human ovarian follicular fluid and to compare these concentrations with those in the circulation. METHODS: Samples of pre-ovulatory follicular fluid and peripheral venous blood were obtained from 14 IVF patients. High-performance liquid chromatography (HPLC) measurements of 25 AAs were the main outcome measures. RESULTS: There was a significant gradient of most AAs from plasma to follicular fluid, with the exception of glutamate, which demonstrated a three-fold increase in follicular fluid concentration (70.0 ± 3.80 mM) compared with plasma (23.18 ± 2.20 mM; P < 0.001). The plasma-to-follicular fluid concentration difference for glutamine (81.83 ± 9.2 mM) was greatest among all AAs. Among essential AAs, this difference was greatest for the branched-chain AAs, isoleucine, leucine and valine. Ammonia concentrations in follicular fluid and blood were 38.87 ± 2.23 and 22.11 ± 1.96 mM, respectively (P < 0.001). Urea concentration in follicular fluid was 3.37 ± 0.18 mM, a value not significantly different from plasma concentration (3.36 ± 0.22 mM; P = 0.911). CONCLUSIONS: These plasma–follicular fluid differences may reflect both the utilization of AAs and the transport characteristics of the follicular cells. There is accumulation of glutamate and ammonia in pre-ovulatory follicular fluid. The data for urea are consistent with transport by passive diffusion, with no evidence of an active urea cycle in the cells of the follicle. Key words: amino acids/ammonia/human pre-ovulatory follicular fluid/IVF patients/urea Introduction The chemical composition of extracellular fluid in Graafian follicles is a matter of importance, because this medium bathes developing oocytes and is an indicator of the secretory activity and metabolism of granulosa cells (Edwards, 1974; McNatty, 1981). The follicular fluid may also reflect the utilization of amino acids (AAs) by these cells. Interestingly, in an in vitro study, it has been shown that rabbit oocytes utilize glutamine at an appreciable rate, and this utilization is accompanied by the production of ammonia (Bae and Foote, 1975). Thus, in vivo, there could be a link between AA and ammonia concentrations in the follicular fluid. No previous study has addressed the relationships between blood plasma and follicular fluid concentrations of AAs. Furthermore, the presence of an ammonia concentration gradient from pre-ovulatory follicular fluid to peripheral venous blood has been established, suggestive of the production of ammonia by the granulosa cells and perhaps the oocyte (Józwik et al., 2001). In recent years, improved human embryo culture results and an enhanced pregnancy rate for IVF have been reported when the composition of the medium was based on the composition of human tubal fluid (Quinn et al., 1985; Quinn, 1995). One such modification of the medium composition includes inorganic salts, lactate, pyruvate and glutamine as the only AA provided in the concentration of 1 mM (Quinn, 1995). However, the AA composition of human tubal fluid (Tay et al., 1997) differs from that reported in studies providing the AA composition of human pre-ovulatory follicular fluid (Jimena et al., 1993; Nakazawa et al., 1997). This raises the question whether the event of ovulation is associated with a change in the oocyte’s AA environment. Of interest, the AA composition of rabbit embryos was similar to those of the corresponding reproductive tract fluids (Miller and Schultz, 1987). In addition, in sheep, the use of synthetic oviductal fluid containing oviductal fluid concentrations of AAs facilitated the development of blastocysts and produced embryos with improved morphology and viability compared 2776 © The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected] The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] Amino acid concentrations in human follicular fluid with the use of other non-supplemented media (Walker et al., 1996). In line with these observations, AAs at concentrations present in follicular fluid also facilitated the development of mouse embryos in vitro (Nakazawa et al., 1997). This study was designed to examine (1) whether there were relationships between human blood plasma and follicular fluid concentrations of AAs as well as (2) whether follicular fluid ammonia concentration correlated with AA concentration. For this purpose, we measured concentrations of 25 AAs in human follicular fluid in the final stage of the oocyte’s growth, shortly before ovulation, and correlated them with ammonia and urea concentrations. The results may be of interest as they may shed new light upon the nutritional environment in the direct proximity of the oocyte. In addition, the study enabled a comparison of follicular fluid AA concentrations with reported oviductal fluid AA composition. Materials and methods Patients’ stimulation protocols Fourteen randomly selected women, aged 32.2 ± 1.2 years (range 26–41 years), attending an IVF programme were studied. They were white Caucasian women in good general health and non-smokers and had no history of liver disease. All patients gave informed consent to the study, which was approved by the Ethics Committee of the Medical University of Bialystok. The protocol of ovarian stimulation as well as the details of medical management have been described elsewhere (Józwik et al., 1999c). Briefly, a short protocol of stimulation was applied. Subcutaneous injections of the GnRH agonist triptorelin acetate (Decapeptyl; Ferring, Germany) 0.1 mg starting on day 1 were followed by gonadotrophins, FSH and/or HMG administered in individual doses for every patient starting on day 3 of her cycle. The stimulation was monitored using serum estradiol (E2) concentrations, together with ultrasound measurements of follicle numbers and diameters. The induction of ovulation with HCG was performed when the leading follicle reached 18–20 mm in diameter and when the serum E2 concentration per follicle was 150–200 ng/l. Analytical methods AA and urea concentrations were determined by high-performance liquid chromatography (HPLC) using a Dionex HPLC Amino Acid Analyzer (Dionex, Sunnyvale, CA, USA), as previously described (Józwik et al., 1999a,b). Previously, frozen plasma was quickly thawed and deproteinized with a solution of 10% sulfosalicylic acid. LNorleucine was added as an internal standard, and samples were buffered with LiOH to pH 2.2 and centrifuged at 14 000 rpm at 4°C for 10 min. The supernatant fraction was filtered through a Millipore filter (pore size 0.45 μm) and was analysed by an HPLC cation exchange column (Cat. No. 0353150; 3 mm × 150 mm; 5 μM cation exchange) from Pickering Laboratories (South Mountain View, CA, USA) using lithium buffers for separation. The quantification was done by postcolumn reaction with ninhydrin. All AAs were measured spectrophotometrically at 570 nm, and proline was measured at 440 nm. Calibration was obtained using AA standards from Pickering Laboratories. All the instrument operation and data processing were controlled with Dionex A1–450 software. The same HPLC column and buffers were used for samples from all patients. Reproducibility within the column was within ±2%. Ammonia concentrations in whole blood were determined in quadruplicate with the Berthelot-indophenol method using a Wako Chemicals (Neuss, Germany) assay and read on a spectrophotometer (model DU 640; Beckman Instruments, Fullerton, CA, USA) set at 630 nm, as discussed previously (Józwik et al., 2001). The intra-assay coefficient of variation calculated on eight randomly selected samples was 5.2%. Statistical analysis Data are expressed as means ± SEM. For each AA, plasma-to-follicular fluid concentration differences and ratios were calculated. Statistical analysis was performed with Student’s t-test for paired samples (twotailed values) and Pearson’s linear correlation. The distribution of all data was first confirmed for their agreement with normal distribution using the Kolmogorov–Smirnov goodness-of-fit test. The statistical package was SPSS® 8.0 for Windows PL (SPSS, Chicago, IL, USA). P-value <0.05 was considered statistically significant. Results AA concentrations Sample collection Pre-ovulatory ovarian follicular fluid was collected from the women during transvaginal ultrasound-guided oocyte retrieval. Only follicular fluids macroscopically free from blood were retained for further determinations (Józwik and Wolczynski, 1998). Sampling was done when follicles ranged from 24 to 26 mm in diameter. Each follicular fluid sample represents fluid from a single follicle in each patient. No follicular fluids were pooled for analysis. The follicular fluids were collected into capped disposable polypropylene tubes. Each patient’s blood was sampled from an antecubital vein before an anaesthetic administration for the oocyte retrieval procedure. The blood samples were collected into capped disposable preheparinized plastic tubes. A high purity, high molecular-weight heparin (5000 IU/ml) in the form of a sodium salt (Polfa, Warsaw, Poland) was used. The tubes were from Life Sciences (Denver, CO, USA). Both fluids were collected in two portions. One portion was used for the ammonia determination that was performed within 10 min from obtaining the sample. The other portion was centrifuged at 2500 g at 4°C for 5 min, and the aliquots of plasma and follicular fluid were snap frozen in liquid nitrogen and stored at −44°C for AA and urea analysis. Examples of partial chromatograms from the glutamate region of the HPLC are shown in Figure 1. The highest AA concentrations in both plasma and follicular fluid were found for glutamine, alanine, glycine, valine and proline, respectively (Table I). For all essential AAs and for most non-essential AAs, there was a significant decrease in follicular fluid concentration in comparison with plasma concentration. Among essential AAs, the concentration difference was greatest for branched-chain AAs (namely isoleucine, leucine and valine) and also for lysine (Table II). For non-essential AAs, serine, arginine, glutamine, asparagine, aspartate, taurine, ornithine, tyrosine and cysteine showed a significant decrease in follicular fluid concentration. Of note, the concentration difference for glutamine (81.83 ± 9.2 μM) was greatest among all AAs, essential and non-essential (Table II). In contrast, glutamate was the only AA which was significantly increased in follicular fluid compared to plasma (70.00 ± 3.80 μM versus 23.18 ± 2.20 μM, respectively; P < 0.001). The concentration of glycine and proline remained virtually the same in both compartments (Table I). 2777 M.Józwik et al. PLASMA 0.100 0.090 0.080 0.070 GLU_NH2 μM 0.060 0.050 0.040 TAU 0.030 UREA THR SER 0.020 ASP PSER 0.010 0 0 2.50 5.00 7.50 ASPG GLU M.S. 10.00 SARC 12.50 15.00 17.50 20.00 MINUTES FOLLICULAR FLUID 0.100 0.090 0.080 0.070 μM 0.060 GLU_NH2 0.050 0.040 UREA 0.030 THR 0.020 PSER 0.010 0 0 2.50 GLU SER TAU ASP PEAM 5.00 7.50 ASPG M.S. 10.00 12.50 15.00 17.50 20.00 MINUTES Figure 1. A representative of high-performance liquid chromatography (HPLC) chromatograms depicting the peaks for neutral and acidic amino acids (AAs) and urea in plasma and follicular fluid. Note the much higher peak for glutamate in follicular fluid. Magnification 5×. The highest plasma-to-follicular fluid concentration ratios were found for aspartate, taurine and ornithine: 3.36, 3.10 and 2.09, respectively (Table II). The lowest value of the ratio (0.35) was found for glutamate. The verification of correlations between plasma and follicular fluid AA concentrations indicated that there was a significant link between follicular fluid concentration and plasma concentration for all the essential AAs, except for tryptophan, and for the nonessential AAs: alanine, glycine, arginine, citrulline, proline, glutamate, asparagine, aspartate, ornithine and cysteine (Table III). There were also a number of correlations of interest related to follicular fluid AA concentrations. Glutamine concentration in follicular fluid inversely correlated with glutamate concentration difference (P = 0.039; r = −0.5559), supporting the metabolism of glutamine to glutamate and ammonia in the cells of the follicle. 2778 The correlation between arginine concentration in plasma and citrulline concentration in follicular fluid was not significant (P = 0.272; r = 0.3156), whereas the correlations between arginine concentration and ornithine and citrulline concentrations in follicular fluid were P = 0.007; r = 0.6875 and P = 0.011; r = 0.6579, respectively. Similarly, there was no significant correlation between plasma serine concentration and follicular fluid glycine concentration (P = 0.641; r = 0.1368), whereas there was a significant correlation between follicular fluid serine and glycine concentrations (P = 0.011; r = 0.6543). As the interconversion of serine to glycine is a reversible reaction (Kikuchi and Hiraga, 1982), the correlation of plasma glycine concentration to follicular fluid serine concentration was not significant (P = 0.587; r = 0.1591). Amino acid concentrations in human follicular fluid Table I. Concentrations of amino acids (AAs) in plasma and pre-ovulatory follicular fluid Table III. Relationship between amino acid (AA) concentrations in plasma and pre-ovulatory follicular fluid AA (μM) Plasma Follicular fluid P-value AA P-value Essential Ile Leu Val Lys Met Phe Thr His Trp 53.02 ± 2.19 92.81 ± 3.28 182.00 ± 5.96 146.34 ± 4.70 20.06 ± 1.04 47.36 ± 1.32 137.41 ± 8.14 77.69 ± 2.26 44.25 ± 2.21 31.27 ± 2.16 55.14 ± 3.39 144.93 ± 7.59 128.94 ± 4.13 19.56 ± 1.83 40.50 ± 1.69 129.94 ± 7.58 70.96 ± 2.32 32.19 ± 1.22 <0.001 <0.001 <0.001 <0.001 0.011 <0.001 0.009 0.003 <0.001 Essential Ile Leu Val Lys Met Phe Thr His Trp 0.000 0.000 0.000 0.003 0.029 0.018 0.000 0.005 NS 0.9358 0.9182 0.9474 0.7252 0.5819 0.6211 0.9555 0.7025 0.0474 Non-essential Ala Gly Ser P-Ser Arg Cit Pro OH-Pro Gln Glu Asn Asp Tau Orn Tyr Cys 306.48 ± 13.44 183.90 ± 7.71 113.29 ± 6.98 7.49 ± 0.95 76.15 ± 3.57 23.55 ± 1.31 156.90 ± 8.83 15.05 ± 1.39 522.54 ± 8.06 26.95 ± 3.38 42.02 ± 1.51 12.11 ± 1.24 82.21 ± 6.23 61.71 ± 4.06 38.10 ± 1.02 37.05 ± 1.49 329.18 ± 15.45 185.19 ± 7.19 79.17 ± 7.31 6.18 ± 0.53 61.37 ± 2.95 24.03 ± 1.40 157.55 ± 9.92 18.56 ± 1.73 440.72 ± 9.87 74.60 ± 5.02 37.04 ± 1.21 8.43 ± 1.12 27.50 ± 1.83 30.07 ± 1.90 34.70 ± 1.35 24.30 ± 1.88 NS NS 0.003 NS 0.002 NS NS NS <0.001 <0.001 <0.001 0.002 <0.001 <0.001 0.025 <0.001 Non-essential Ala Gly Ser P-Ser Arg Cit Pro OH-Pro Gln Glu Asn Asp Tau Orn Tyr Cys 0.030 0.020 NS NS 0.007 0.000 0.000 NS NS 0.004 0.000 0.020 NS 0.029 NS 0.045 0.5802 0.6105 0.2017 0.2722 0.6800 0.8892 0.8897 −0.2763 0.5093 0.7127 0.9127 0.6132 0.4034 0.5809 0.3818 0.5416 N = 14. Values are means ± SEM. P-values were determined by Student’s t-test for paired samples. r N = 14. P-values (two-tailed significance) and correlation coefficients (r) were determined by Pearson’s linear correlation. Ammonia and urea concentrations Table II. Amino acid (AA) plasma-to-follicular fluid concentration differences and ratios AA Essential Ile Leu Val Lys Met Phe Thr His Trp Non-essential Ala Gly Ser P-Ser Arg Cit OH-Pro Pro Gln Glu Asn Asp Tau Orn Tyr Cys Difference between plasma and follicular fluid (μM) Plasma-to-follicular fluid ratio 21.75 ± 0.78 37.67 ± 1.35 37.07 ± 2.73 16.01 ± 4.01 2.02 ± 0.97 6.83 ± 1.37 7.47 ± 2.41 7.25 ± 1.75 11.48 ± 2.73 1.74 ± 0.05 1.72 ± 0.05 1.27 ± 0.03 1.14 ± 0.03 1.15 ± 0.05 1.18 ± 0.03 1.06 ± 0.02 1.11 ± 0.03 1.40 ± 0.08 −22.70 ± 13.36 −1.30 ± 6.59 34.11 ± 9.04 1.36 ± 0.98 14.77 ± 2.67 −0.48 ± 0.64 −4.06 ± 2.44 1.07 ± 4.55 81.83 ± 9.02 −47.64 ± 3.53 4.98 ± 0.64 4.21 ± 0.98 54.72 ± 5.74 31.63 ± 3.34 3.40 ± 1.34 12.75 ± 1.65 0.94 ± 0.04 1.00 ± 0.04 1.56 ± 0.15 1.27 ± 0.16 1.25 ± 0.05 0.99 ± 0.03 0.92 ± 0.13 1.02 ± 0.03 1.19 ± 0.02 0.35 ± 0.03 1.13 ± 0.02 3.36 ± 1.89 3.10 ± 0.23 2.09 ± 0.14 1.12 ± 0.04 1.68 ± 0.18 N = 14. Values are means ± SEM. Table IV summarizes data on ammonia and urea concentrations in the two compartments. Ammonia concentration in follicular fluid (38.87 ± 2.23 μM) was significantly higher than that in blood (22.11 ± 1.96 μM; P < 0.001), whereas urea concentration in follicular fluid (3.36 ± 0.22 mM) was not significantly different from its plasma concentration (3.37 ± 0.18 mM; P = 0.911). Of interest, ammonia concentration in follicular fluid did not correlate significantly with any intrafollicular AA concentration or plasma-to-follicular fluid AA concentration difference. This was also true for ammonia follicular fluid-to-plasma concentration difference. There was no correlation between blood and follicular fluid ammonia concentrations (P = 0.369; r = 0.3411), whereas there was a highly significant correlation between plasma and follicular fluid urea concentrations (P = 0.000; r = 0.9670). Interestingly, there was no significant correlation between any ammonia-related variables and urea-related variables. Further, urea concentration in follicular fluid did not correlate significantly with any variables related to arginine. Discussion This study provides new data on the metabolic and biochemical milieu of the human oocyte before ovulation after ovarian stimulation. So far, the information on AA concentrations in human follicular fluid has been incomplete and conflicting. Velázquez et al. (1977) described the AA composition of human follicular fluid obtained during surgery from women 2779 M.Józwik et al. Table IV. Comparison of concentrations of ammonia and urea in pre-ovulatory follicular fluid to concentrations of ammonia in whole blood and urea in plasma Substance Concentration in blood or plasma Concentration in follicular fluid P-value Difference between blood or plasma and follicular fluid Blood or plasma-to-follicular fluid ratio Ammonia (μM) Urea (mM) 22.11 ± 1.96 (whole blood) 3.37 ± 0.18 (plasma) 38.87 ± 2.23 3.36 ± 0.22 <0.001 0.911 −16.77 ± 2.42 0.015 ± 0.063 0.58 ± 0.05 1.01 ± 0.02 For ammonia, N = 9; for urea, N = 14. Values are means ± SEM. with polycystic ovaries, but no data from normal subjects were included. The reported concentrations of most AAs were higher in follicular fluid than in blood plasma. Jimena et al. (1993) studied AA concentrations in follicular fluid of follicles in various stages of growth and compared them to plasma AA concentrations. The samples of follicular fluid were obtained during oocyte retrieval by means of laparoscopy. This route of oocyte retrieval has been shown to alter follicular fluid pH and presumably other measures of oocyte integrity, as demonstrated by a significant decrease in oocyte fertilizability following laparoscopic retrieval (Daya, 1988; Imoedemhe et al., 1993). In another study investigating human serum and follicular fluid AA concentrations from IVF patients (Nakazawa et al., 1997), the collection of follicular fluid was transvaginal. Both in the Jimena and in the Nakazawa studies, data for AA concentrations in blood differ from those reported as normal values for adult humans (Hagenfeldt and Arvidsson, 1980; Schaefer et al., 1987). Jimena et al. (1993) reported that of 18 AAs studied, as many as 10 AAs had increased follicular fluid concentrations in comparison with plasma concentrations, and the increase was significant for histidine, phenylalanine and asparagine. Glutamate concentrations in follicular fluid and plasma were identical. In contrast, Nakazawa et al. (1997) reported a significant decrease in 11 AA concentrations in follicular fluid compared with serum. In their study, concentrations of glutamine and glutamate were not different between the two compartments. Moreover, Menezo et al. (1982) analysed AA content of follicular fluid obtained during a natural cycle or following administration of clomiphene and HCG. However, no direct comparison with blood plasma concentrations was made, and the data for glutamine and glutamate and for asparagine and aspartate were reported as a combined value for both AAs. The authors suggested that there did not appear to be any AA regulatory mechanisms between the follicle and the blood (Menezo et al., 1982). In this study, plasma AA concentrations are in good agreement with normal adult human values (Hagenfeldt and Arvidsson, 1980; Schaefer et al., 1987). The follicular fluid AA concentrations are at substantial variance from those reported by Jimena et al. (1993). All essential AAs and most of the non-essential AAs demonstrated significantly lower follicular fluid concentrations than those found in plasma (Table I). This may reflect the utilization of all essential AAs and most non-essential AAs by the cells located between the dense capillary network of the Graafian follicle and the follicle’s interior. Alternatively, it may reflect transport characteristics across these cells. It is likely that both factors contribute to creating this concentration gradient. As summarized in Table III, the intrafollicular concentrations of most AAs are a function of plasma concentra2780 tions. These correlations are explained by earlier research, which provided evidence that the follicular fluid is a selective transudate from plasma (Shalgi et al., 1972, 1973). Before ovulation in humans, there is a significant increase in intrafollicular blood flow of the dominant Graafian follicle (Campbell et al., 1993). Physiologically, this increase in blood flow could be an efficient mechanism in increasing the uptake of AAs by the cells of the follicle. Glutamine is involved in a variety of intracellular metabolic pathways. The completion of meiosis in the maturing oocyte requires the presence of nucleic acid bases, and they can be synthesized by human cells from glutamine and glutamate (Salzman et al., 1958). Bae and Foote (1975) demonstrated that glutamine in medium, but not proline, was able to stimulate rabbit oocyte maturation. Glucose, pyruvate and lactate were found to be beneficial but not indispensable for this process. In cattle oocytes, the metabolism of glutamine and glycine has been shown to increase with oocyte maturation (Rieger and Loskutoff, 1994). In preimplantation mouse embryos, glutamine uptake and utilization have been reported (Chatot et al., 1990). Glutamine may also be a vital source of energy for the oocyte, like for other cells (Zielke et al., 1984). In this study, glutamine was found to have the largest plasmato-follicular fluid concentration difference among all AAs. To our knowledge, this is the first such demonstration based on determinations done on samples obtained in humans in vivo. This study strongly suggests the presence of active metabolism of some AAs from plasma to follicular fluid, one of the most intriguing findings being glutamate production. This would need further studies to confirm their utilization. However, the large glutamine concentration gradient paralleled by the increased intrafollicular concentration of glutamate and ammonia supports the possibility of the utilization of glutamine via glutaminase. Glutaminase should be present within the granulosa cells/oocyte complex. It is present in 1cell-stage mouse embryos (Chatot et al., 1997). The high follicular glutamate concentration may reflect active transport of glutamate out of the granulosa cells, against a concentration gradient. Thus, besides ammonia, glutamate is another lowmolecular-weight nitrogen-containing compound that accumulates in follicular fluid. The three-fold increase of intrafollicular glutamate concentration as compared to plasma may reflect the role of glutamate as a readily available carrier of the amino group for transamination reactions. As indicated by the large concentration difference, other AAs with a large uptake within the follicle were the branchedchain AAs, isoleucine, leucine and valine. Of interest, in the placenta, the utilization of branched-chain AAs is a major source of ammonia production (Józwik et al., 1999b). It is Amino acid concentrations in human follicular fluid thought that the amino group produced from transamination reactions involving branched-chain AAs may be used for glutamate synthesis (Józwik et al., 1999b). In the rat model, intraperitoneal injection of 14C-leucine and 35S-methionine resulted in uptake and prolonged radioactivity of both AA isotopes in follicular fluid (Yatvin and Leathem, 1964). Cross and Brinster (1974) demonstrated the incorporation of 14C-leucine into mouse oocytes. Interestingly, the activity of leucine aminopeptidase has been detected in human follicular fluid (Caucig et al., 1971). As for other AAs, a Na+-independent transport system for methionine has been shown in mouse oocytes (Holmberg and Johnson, 1979). Haghighat and Van Winkle (1990) demonstrated the uptake of several AAs in mouse oocytes. Transport of alanine and lysine was enhanced in follicular cells, whereas transport of leucine was not. Uptake of glycine occurred by a Na+-dependent transport system, presumably system Gly. As for ammonia, its intrafollicular concentration and the concentration difference between blood and follicular fluid found in this study (Table IV) are similar to those previously reported (Józwik et al., 2001). The lack of correlation of intrafollicular ammonia concentration with AA concentration points to a complex, perhaps multi-step, metabolism of ammonia within the follicle. In contrast to marked differences in ammonia concentration between the two compartments, the follicular fluid urea concentration was approximately equal to the plasma value. This observation suggests negligible urea metabolism within the follicle. Human studies with tritium-labelled water as a tracer have demonstrated a high permeability of the barrier between the vascular compartment and the follicular fluid to water and a rapid exchange of water occurring by diffusion (Peckham and Kiekhofer, 1959). From this study, a strong correlation between plasma and follicular fluid concentration of urea indicates that there is passive diffusion of urea from body fluids to follicular fluid. This observation is further supported by the finding that urea in follicular fluid did not demonstrate any significant correlation with arginine-related variables. Oxidation of arginine to citrulline with the concomitant nitric oxide generation is a possibility. In our study, arginine and ornithine demonstrated a significant decrease in their concentrations in comparison with plasma concentrations, whereas citrulline did not. The ability of human granulosa cells to synthesize nitric oxide has been established (Van Voorhis et al., 1994). Interestingly, the nitric oxide metabolism was investigated in human follicular fluid (Sugino et al., 1996) where, in large ovarian follicles, the follicular fluid concentration of arginine was 27.3 ± 1.6 μM, and of citrulline 81.0 ± 5.9 μM, data which are somewhat in contrast to concentrations found in the present study (Table I). Whether the human oocyte produces nitric oxide is not known, but there is now evidence for good substrate availability for this process. In this study, there was a significant gradient of serine from plasma to follicular fluid, whereas the follicular fluid glycine concentration was virtually unchanged from that of plasma. The data suggest that the pre-ovulatory ovarian follicle may be equipped with both serine hydroxymethyltransferase and glycine-cleavage enzyme system operating in the direction from serine to glycine. The conversion of one molecule of serine to two molecules of glycine requires the use of a molecule of ammonia (Kikuchi and Hiraga, 1982). One possibility of such intracellular serine and glycine utilization is the synthesis of glutathione or γ-glutamyl-cysteinylglycine. Serine can be the precursor of both cysteine and glycine (Michal, 1999), which, together with glutamate, form glutathione. This tripeptide serves as an important intracellular redox buffer and is necessary for the γ-glutamyl cycle. The γ-glutamyl cycle is an active transport system for transporting AAs from the outside of the cell to its interior. Interestingly, the activity of γglutamyl transpeptidase, the key membrane-bound enzyme for the cycle (Diamondstone, 1982), has been detected in human follicular fluid (Menezo et al., 1982). It is of clinical relevance to compare pre-ovulatory follicular fluid AA composition with tubal fluid composition. In their elegant study, Tay et al. (1997) reported on the concentration of 17 AAs in tubal fluid of perfused human Fallopian tubes obtained surgically in various phases of the ovarian cycle. For 11 AAs, the tubal fluid concentration was lower than the follicular fluid in our study, whereas for leucine, arginine, glutamate, asparagine, aspartate and tyrosine, it was elevated. These observations suggest that the event of ovulation substantially changes the AA environment of the oocyte. Appropriate composition and concentration of nutrients in culture media may be critical for oocyte and embryo viability, influencing the IVF outcome. Specifically, the AA composition and concentration of media remains under debate (Devreker and Englert, 2000). The effect of human follicular fluid AA composition on murine embryo development has been found to be more effective and safer for embryo culture than that of other media in use (Nakazawa et al., 1997). Our data provide further information for preparing oocyte culture medium whose composition mimics the physiological environment to which mature human oocytes are exposed. Acknowledgements During this study, Dr Marcin Józwik was recipient of a postdoctoral research fellowship from the Fulbright Commission to the Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, USA. Supported in part by: the State Committee for Scientific Research, Warsaw, Poland, Grant 4 P05E 08616; Research Program 3–29639 of the Medical University of Bialystok; NIH Grant 5 RO1 HD034837–8 and GCRC Grant MO1 RR000069, General Clinical Research Centers Program, National Centers for Research Resources, NIH. References Bae I-H and Foote RH (1975) Carbohydrate and amino acid requirements and ammonia production of rabbit follicular oocytes matured in vitro. Exp Cell Res 91,113–118. Campbell S, Bourne TH, Waterstone J, Reynolds KM, Crayford TJ, Jurkovic D, Okokon EV and Collins WP (1993) Transvaginal color blood flow imaging of the periovulatory follicle. Fertil Steril 60,433–438. 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Submitted on October 21, 2005; resubmitted on January 4, 2006; accepted on January 23, 2006