Daily variations of endolymph composition: relationship with the otolith calcification process in trout
1 Laboratoire Réponse des Organismes aux Stress Environnementaux, UMR
INRA-UNSA 1112, Université de Nice-Sophia Antipolis, Faculté des
Sciences, BP 71, 06108 Nice Cedex 2, France
2 IFREMER, DRV, RH, Laboratoire de Sclérochronologie des Animaux
Aquatiques, BP 70, 29280 Plouzané, France
3 Centre Scientifique de Monaco, Avenue Saint-Martin, MC-98000 Monaco,
Principality of Monaco
* Author for correspondence (e-mail: payan{at}unice.fr)
Accepted 7 May 2003
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Summary |
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Key words: otolith, calcification, daynight cycle, endolymph
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Introduction |
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As with other calcified biominerals, the otolith structure displays
alternate optically dense layers (rich in organic materials) and translucent
layers (rich in minerals), forming concentric rings
(Pannella, 1980;
Morales-Nin, 1987
). In many
fish, the deposition of these two layers produces a recognisable daily
increment (Pannella, 1971
).
Several studies have suggested a close relationship between the cyclic process
of otolith calcification and photoperiod
(Taubert and Coble, 1977
;
Radtke and Dean, 1982
).
Reversal of the light:dark cycle was shown to induce an inversion of the two
layers in Tilapia nicolica
(Tanaka et al., 1981
).
However, in juvenile fish, the daily increment of the two layers was
maintained when fish were kept under constant dark or constant light,
suggesting a control by endogenous factors
(Mugiya, 1987
; Wright et al.,
1991b
,
1992
). Finally, Mugiya
(1984
) showed that there is a
seasonal reversal in the rhythm of trout otolith calcification that can be
associated with a seasonal inversion in plasma calcium cycle.
Otolith growth is an acellular process, carried out far from the saccular
epithelium, implying that calcification process is strictly dependent on
endolymph fluid chemistry. This morphological particularity provides a unique
opportunity to sample and analyse calcifying fluid in order to establish
relationships between its composition and the mineralisation process. The
composition of endolymph is characterised by a high K+
concentration, an alkaline pH, and saturated Ca2+ and
HCO3 concentrations
(Enger, 1964;
Mugiya and Takahashi, 1985
;
Kalish, 1991
;
Payan et al., 1997
;
Takagi, 2002
). The endolymph
also expressed an anticalcifying activity, probably involved in the mechanism
of regulation of calcification (Borelli et al.,
2001
, 2002). It has been
suggested that the otolith calcification process is related to the specific
composition of the endolymph (Romanek and
Gauldie, 1996
; Borelli et al.,
2001
; Takagi,
2002
), and recent studies have shown a lack of uniformity in the
spatial distribution of ionic and organic endolymphatic components that could
also be involved in the otolith formation
(Payan et al., 1999
;
Borelli et al., 2001
).
To our knowledge, there have only been two studies following plasma and
endolymph parameters during the circadian cycle. Mugiya and Takahashi
(1985) found simultaneous
variations in trout plasma and endolymph for pH, with a nocturnal maximum
(+0.4 pH unit), and total CO2 concentration, which peaked in the
daytime (+25%). More recently, simultaneous variations in total CO2
concentration in turbot plasma and endolymph have been observed
(Edeyer et al., 2000
);
however, the peak was nocturnal. In this last work, the concentrations of
protein in endolymph samples clearly decreased during the night, and the
non-uniformity described for the endolymph components in this species
(Payan et al., 1999
) was
maintained during the circadian cycle. However some important components of
endolymph that play a role in calcification, such as calcium, collagen and the
anticalcifying factor (Borelli et al.,
2001
; Payan et al.,
2002
), were not measured.
The aim of the present work was to examine ionic and organic modifications of trout endolymph composition during the day:night cycle, taking into account recent work, in order to determine limiting key factor(s) in the regulation of calcification. An integrated view of our results is presented to link the observed daily variations of endolymphatic precursors with the cyclic process of otolith calcification, which takes place at the endolymphotolith interface where the CaCO3 deposition occurs.
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Materials and methods |
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Collection of plasma, endolymph and otolith
The experiments reported in this paper complied with the Principles of
Animal Care of the National Institute of Health (publication no. 86, revised
1985) and the French laws for experiments on animals (decree no. 87-848).
Blood was sampled from the caudal vessels and the pH measured immediately; the samples were then centrifuged and the plasma kept on ice until analysis.
Endolymphs were collected as described by Payan et al.
(1997) between 13:00 h and
15:00 h (as day reference) and between 03:00 h and 05:00 h (as night
reference). Briefly, the fish were decapitated and the operative field washed
3 times with 150 mmol l-1 NaCl. The saccular epithelium was incised
to allow the collection of endolymph as two samples, one from the proximal
zone (the space between the macula and the otolith) and the other from the
distal zone (the space facing the opposite side of the otolith). Samples were
stored at 20°C until analysis.
Otoliths (sagittas) were washed with 1 mol l-1 NaOH, rinsed with deionised water, wiped and ground into powder. The powder was decalcified using acetic acid (maintained at pH 4). After centrifugation (10 000 g, 10 min) the soluble and insoluble fractions were separated. The supernatant, containing the soluble fraction of the organic matrix, was ultrafiltered (Amicon, Saint-Quentain, France; CO 3 kDa) at 4°C and lyophilised. This freeze-dried OM was used to generate polyclonal antibodies in a rabbit (Eurogentec, Angers, France).
Measurements
The pH was measured with a Tacussel (Villeurbane, France) pH meter
connected to a Metler Toledo electrode (InLab 423; Paris, France). Endolymph
pH was measured in situ by inserting a minielectrode (VIP, Mini
Combo, Stevenage, UK; tip diameter 450 µm) into the proximal and distal
zones through a small incision. The pH value was recorded within the 40 s
period following the incision of the saccular wall. These endolymph pH
measurements were performed in January on a batch of trout of the same size,
and measurements were done in daytime (08:00-12:00 h).
Total calcium and total CO2 concentrations were measured by spectrophotometry using kits (Sigma, Saint-Quentain, Fallavier, France). Protein concentration was measured by colorimetry using Coomassie Blue (G 250) with bovine serum albumin as standard. Collagen concentration was determined using a Sircol kit (Biocolor) with acid-soluble type I collagen as standard. In this assay, the dye reagent binds specifically to the [Gly-X-Y]n helical structure found in all collagens and analysis of Sircol dye binding versus hydroxyproline (tested by the manufacturer) showed a linear and significant correlation.
SDS-PAGE and western blotting
Electrophoresis was performed on 7.5% or 12% Trisglycine
polyacrylamide gels under reduced conditions, 5 µg protein per well,
following the method of Laemmli
(1970). Gels were run at a
constant voltage of 150 V for 1 h at 4°C. Standard proteins used were
`kaleidoscope prestained standards' (Biorad, Marne la Coquette, France):
myosin (208 kDa), ß galactosidase (132 kDa), bovine serum albumin (91
kDa), carbonic anhydrase (45.2 kDa), soybean trypsin inhibitor (35.1 kDa),
lysozyme (18 kDa) and aprotinin (7.6 kDa). Proteins were stained with silver
using the `silver stain plus kit' (Biorad). The densitometric profiles were
analysed using software developed by the Research Services branch of the
National Institute of Health (NIH).
Western blotting of endolymph proteins on 7.5% gels was done using a
nitro-cellulose membrane (Sigma; 0.45 µm pore size) and immunoblotting
performed following the method of Towbin et al.
(1979). The membrane was first
incubated with 5% milk for 1 h to block non-specific binding sites. Then, the
membrane was incubated (overnight at 4°C, antiserum diluted 1:2000) with a
rabbit antiserum raised against the soluble OM of trout otolith. After
rinsing, the membrane was incubated with peroxidase-conjugated goat
anti-rabbit globulin (Biorad). Localisation was visualised by the ECL
technique (Perkin Elmer, Courtaboeuf, France). The reactivity of the rabbit
antiserum was tested by comparison of the labelling obtained with pre-immune
and immune serum on the OM of trout by dot blots. The immune serum reacted
whereas the pre-immune serum did not (results not shown). The antiserum
(dilution 1:200) did not recognise trout plasma proteins on western blots
(results not shown).
Calculations and statistical analysis
The partial pressure of CO2 (PCO) was
calculated using the HendersonHasselbach equation
(pH=pK+log[HCO3]/d[CO2]
with a pK value for CO2/HCO3 of 5.75
and a CO2 solubility coefficient of 0.057 mmol l-1
mmHg-1 at 17°C; Albers,
1970).
Results are expressed as means ± S.E.M. (N) and analysed statistically using STATVIEW Software (Brain Power, Inc., Berkeley, CA, USA). Comparison of the means was made by a one-way analysis of variance (ANOVA). Differences were considered significant at P<0.05.
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Results |
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Endolymph composition in day samples
Calcium and CO2 concentrations in endolymph samples are shown in
Table 2. Total calcium
concentration [Ca]tot was significantly greater (by 20%) in the
proximal samples than in the distal ones, in contrast to
[CO2]tot, which was threefold greater in the distal
samples. Direct measurements of pH within the endolymph revealed significant
differences between proximal and distal endolymph (7.38±0.047,
N=8 and 7.87±0.078, N=8, respectively,
P<0.0001; not shown). The protein concentration was approximately
threefold greater in the proximal than the distal endolymph in day
(Table 3).Collagen
concentration was only measured in pooled proximal endolymph. These results
demonstrate that the composition of endolymph surrounding the otolith is not
uniform for the components measured.
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Diurnal variation of endolymph heterogeneity
The [Ca]tot measured during the night was not significantly
different from that measured during the day
(Table 2), but the significant
difference between proximal and distal samples was lost during the night.
During the night, the [CO2]tot was significantly
increased (by 30%) in the proximal endolymph, but was unchanged in the distal
endolymph (Table 2), and
although the magnitude of the difference between the proximal and distal
endolymph was reduced during the night, it was still significant.
Protein concentrations in endolymph were followed in a preliminary
experiment and the protein levels increased after the darklight
transition, peaking approximately 5 h after the light period began
(Fig. 1). The differences
between the night and day samples in proximal endolymph was highly
significant. The results of this experiment and those obtained previously on
turbot (Edeyer et al., 2000)
were used to select the sampling periods for the main experiment. Protein
concentration decreased by 25-30% in both proximal and distal zones, but the
decrease was only significant for the proximal value
(Table 3). Although collagen
was only measured on pooled proximal endolymph, the decrease (90%) during the
night was important.
|
Qualitative diurnal variation of endolymphatic organic compounds
Protein patterns in proximal endolymph samples collected during day and
night periods were compared by gel electrophoresis
(Fig. 2A). Whatever the
collecting period, the endolymph displayed a complex protein pattern composed
mainly of major bands together with several minor bands, covering a large
range of molecular mass. After densitometric analysis, comparison between day
and night samples (Fig. 2B),
revealed that the profiles were globally similar in number and position of
most bands. While some peaks were similar in size in both samples, peak(s) of
high molecular mass were denser in the day samples whereas several bands
(approximately 65-80, 35 and 21 kDa) were denser in the night samples (arrows
in Fig. 2B).
|
Presence of organic precursors of the otolith organic matrix in the
endolymph
Western blotting of proximal endolymph, using the antiserum raised against
the soluble OM of trout otolith, revealed only two bands of estimated
molecular mass 65 kDa and 75 kDa. They were detected in samples from both day
and night (Fig. 2C), but
comparative densitometric analysis (Fig.
2D) clearly showed that both bands were more concentrated (1.6 and
4-fold, respectively) in the day sample than in the night one.
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Discussion |
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Importance of Ca2+ and CO32
endolymphatic levels in daily otolith increments
The calcification process is strictly a function of endolymph fluid
chemistry (see Introduction). Recently, Takagi
(2002) calculated the
aragonite saturation state (Sa) of the trout endolymph from direct
quantification of electrolyte concentrations and concluded that endolymph is
supersaturated with respect to aragonite. We will mainly discuss results
concerning the proximal endolymph that bathes the proximal face of the
otolith, characterised by the maximal growth rate (Panella, 1980).
Many studies have suggested that otolith calcification is related to plasma
calcium concentration (Edeyer et al.,
2000; Mugiya,
1984
; Wright et al.,
1992
). Consequently, changes in plasma calcium levels would be
expected to have a direct effect on calcium levels in the endolymph. This
hypothesis was directly confirmed by Payan et al.
(2002
) on trout during
experiments in vivo (induced hypercalcemia) and in vitro
(perfused isolated inner ear). These authors observed a tight relationship
between [Ca]tot in perfusing fluid and endolymph. Calcium diffuses
via a paracellular pathway located in the proximal area of the
saccular epithelium with a very high turnover rate of the endolymphatic
calcium pool (approximately 200% h-1). These results suggest that
rapid variations of [Ca2+] in plasma induce same variations in the
endolymph. In the present study, we confirmed that [Ca]tot in
plasma varied daily as previously noted (Mugiya,
1984
,
1987
;
Mugiya and Oka, 1991
;
Edeyer et al., 2000
;
Wright et al., 1992
). However,
although [protein] and [Ca]tot in the plasma were both
significantly increased during the night
(Table 1), it is not evident
that plasma [Ca2+] was increased.
In fact, [Ca2+] depends on several factors such as
[Ca]tot, pH of the fluid, and the nature and concentration of
Ca2+-binding proteins. Concerning [Ca2+] in the
endolymph, Payan et al. (2002)
failed to measure it in situ using a Ca2+-sensitive
minielectrode. Mugiya (1966
)
measured the ultrafiltrable fraction (72%) and Takagi
(2002
) found that 47% of the
calcium of trout endolymph was ionised, thus showing that the endolymphatic
calcium was partly bound to proteins. Our results show for the first time that
in the proximal endolymph of trout, protein levels are minimal at the end of
the night, whereas [Ca]tot and pH were unchanged from daytime
values. This suggests that the Ca2+ activity in the proximal
endolymph varies, being maximum at the end of the night. Similar conclusions
were proposed by Edeyer et al.
(2000
) on turbot from the
variations detected in plasma concentration of [Ca]tot and
proteins. It must be noted that Bettencourt and Guerra
(2000
) observed that protein
and calcium levels showed discrete antiphasic variations during the day in
cephalopod endolymph. There were significantly higher protein levels in the
morning than in the evening and the opposite trend was seen for calcium. These
authors associated these variations to a daily deposition of CaCO3
on cephalopod statoliths.
Our results agree with previous reports of diurnal variations in
[CO2]tot in plasma and saccular endolymph
(Mugiya and Takahashi, 1985;
Edeyer et al., 2000
), with a
maximum at the end of the night for autumnwinter experiments (present
results; Edeyer et al., 2000
).
[CO32] in the endolymph can be calculated from
the dissolved CO2 and pH values. The pH of endolymph was calculated
using the constants of the HendersonHasselbach equation for the plasma
(see Results), assuming that the PCO in endolymph was
similar to that in plasma. In the proximal endolymph, we estimated the pH
values to be 7.39±0.047 (N=8) during the day and
7.40±0.051 (N=8) during the night. In the distal endolymph, we
estimated the pH values to be 7.84±0.078 (N=8) during the day
and 7.76±0.081 (N=8) during the night. These results clearly
suggest that the pH of endolymph remained unchanged during the day:night cycle
and that the proximal endolymph was more acidic than the distal one. Direct
measurements of endolymph pH for the day period are in accordance with these
calculations (see Results). An alkaline pH value for endolymph has been
repeatedly proposed (Mugiya and Takahashi,
1985
; Payan et al.,
1997
, 1998;
Gauldie and Romanek, 1998
;
Takagi, 2002
) but the samples
were probably more representative of distal endolymph as the distal space is
larger than the proximal space in the sacculus. On the other hand, the pH
value of 7.4 in proximal endolymph was quite unexpected and raises the
following question: is the proximal endolymph supersaturated with respect to
aragonite crystallisation? From the results of Takagi
(2002
), when the pH value
switched from 8.0 to 7.4 both the [CO32] and the
Sa should decrease by a factor of about 3, so the Sa value estimated for the
proximal endolymph would not be supersaturated but be around the unity.
In conclusion, our results show that, in the proximal endolymph bathing the
convex shape of the otolith characterised by maximal growth, the
Ca2+ and CO32 concentrations were
in-phase variations during the day:night cycle, whereas the pH remained
unchanged. The increase in both Ca2+ and
CO32 levels at the end of the night period should
increase the saturation state of the aragonite within the proximal endolymph,
thus promoting the CaCO3 deposit. Under these conditions, we
propose that the CaCO3 deposit would occur only when the
concentration thresholds of the ionic parameters are reached. Thus, the
synchronous increase of Ca2+ and CO32
in the endolymph will not be sufficient to cause an immediate deposit of
CaCO3 as previously proposed (Mugiya,
1984,
1987
;
Mugiya and Takahashi, 1985
;
Edeyer et al., 2000
). Under
our conditions this would indicate that CaCO3 deposition starts
once the solubility product of CaCO3 in the proximal endolymph is
exceeded, probably at the beginning of the day. This hypothesis is in
agreement with the results of Wright et al.
(1992
), who undoubtedly showed
that light led to 45Ca deposition into trout otolith. However,
supersaturation of the endolymph calcium carbonate cannot alone be the cause
of the aragonite precipitation, being necessary but not sufficient to
precipitate aragonite. Indeed, the organic matrix plays a major role in the
calcification process and could regulate the timing of CaCO3
precipitation.
Importance of organic precursors levels in daily otolith
increments
Our results revealed a strong variation in protein concentration in trout
proximal endolymph during the circadian cycle, with maximum levels during the
daytime. This confirms the results of Edeyer et al.
(2000) on turbot, the only
difference being the level of these variations (4.8 g l-1
i.e. 30%, and 2.5 g l-1 i.e. 60%, in trout and turbot,
respectively, for proximal endolymph at night compared with daytime). However,
as in turbot (Edeyer et al.,
2000
), otolith matrix formation cannot reasonably be responsible
for the decrease of the endolymphatic protein concentration during the night.
Indeed, fewer than 1 per 1000 proteins present in the endolymph is
incorporated daily during otolith increment
(Borelli et al., 2001
).
One major point of interest from our study concerns the tenfold variation
of collagen concentration in the proximal endolymph through the daily cycle.
The daily collagen increase corresponds to 16 µg/saccule, whereas daily
incorporation into the otolith matrix accounts for only 11 ng
(Borelli et al., 2001). Thus,
as for other proteins, matrix formation cannot explain the magnitude of the
variation in collagen concentration observed in the proximal endolymph. All
collagens are synthesised and secreted into the extracellular space in the
form of soluble precursors, called procollagens
(Hulmes, 2002
). Then,
proteolytic processing of N- and C-terminal propeptides by specific
procollagen N- and C-proteinases leads to the production of mature collagen
molecules, which spontaneously assemble into fibrils
(Kadler et al., 1996
).
Therefore, it is probable that the molecular mechanisms determining the
three-dimensional architecture of otolith involve the presence of complex
enzymatic activities within the endolymph rather than a simple association of
organic components previously synthesised and secreted by the saccular
epithelium. Furthermore, in-phase diurnal fluctuations of collagen and
non-collagenous proteins, both being present in the organic matrix of the
otolith (Borelli et al., 2001
),
strongly support the hypothesis that they may be linked to the cyclic process
of the otolith calcification.
To our knowledge, only two studies have presented the results of endolymph
electrophoresis (Takagi and Takahashi,
1999; Borelli et al.,
2001
), and both were done on day samples. The procedures,
particularly the staining, were different, which could explain why more
components were detected in the present study. This is the first time that a
comparison of endolymphs at day and at night has been done, but the variations
in the composition of endolymph protein appear complex, as both main and minor
bands are involved. Whether or not these unknown molecules are involved in the
formation of the otolith matrix is a focus point for further work.
There has been only one study showing that OM immunoreactive material is
present in the endolymph (Takagi and
Takahashi, 1999). However, although their antiserum was also
raised against the soluble fraction of the organic matrix of the otolith of
trout, the reactivity on the endolymph revealed molecules of different
molecular mass. The molecule recognised in the previous work clearly had a
molecular mass of more than 94 kDa, whereas we observed two bands (of
approximately 75 and 65 kDa). Possible explanations for their discrepancy
include the procedure used to obtain the soluble OM, the immune response of
the rabbit to the material injected, and/or the fact that the sample tested in
the present work was a pool of proximal endolymphs. The results show for the
first time that there are differences in the labelling of these molecules in
endolymph samples obtained during the day and at night, suggesting that they
could be precursors involved in the diurnal deposit of organic material onto
the otolith. The nature of the two protein molecules is still unknown, but 75
kDa is the molecular mass of the collagen monomer. Complementary studies are
necessary to determine the exact nature of these molecules.
Our study shows that both ionic and organic precursors present within the endolymph may act not only as substrates for otolith formation but also as regulatory parameters: the levels of Ca2+ and CO32 influence the aragonite saturation state, similar to the way that levels of non-collagenous and collagenous proteins may trigger the formation of the organic matrix. Thus, as shown in Fig. 3, we propose that the bilayer rings of otolith growth during the day:night cycle correspond to antiphasic mechanisms: organic matrix deposition starts at the end of the day, when the concentration of organic precursors is maximum in the endolymph, whereas CaCO3 deposition starts once the solubility product of CaCO3 is exceeded (i.e. at the beginning of the day).
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