Download Amino acid, ammonia and urea concentrations in human pre

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
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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.
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Submitted on October 21, 2005; resubmitted on January 4, 2006; accepted on
January 23, 2006