Dietary influences over proliferating cell nuclear antigen expression in the locust midgut
1 Department of Histology and Pathology, Schools of Medicine and Sciences,
University of Navarra, E-31080 Pamplona, Spain
2 Department of Zoology and University Museum of Natural History, University
of Oxford, South Parks Road, Oxford OX1 3PS, UK
* Author for correspondence (e-mail: lmontuenga{at}unav.es)
Accepted 29 March 2004
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Summary |
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Key words: proliferating cell nuclear antigen, PCNA, Locusta migratoria, Insecta, BrdU incorporation, DNA synthesis, protein, carbohydrate, midgut, feeding behaviour, diet, nutritional balance
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Introduction |
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Little is known about cell proliferation in insects and especially about
the turnover of the insect midgut epithelial cell population. There have been
studies on isolated aspects of cell proliferation in insect neurogenesis,
muscle remodelling in metamorphosis and glandular growth
(Hegstrom and Truman, 1996;
Lee et al., 1995
;
Fahrbach et al., 1995
). Endo
(1984
), using incorporation of
tritiated thymidine, reported differences in the rate of renewal between
enterocytes and endocrine cells in the cockroach midgut. In addition,
proliferation of embryonic tissues during Drosophila development
(including the gut) has been studied by immunocytochemical techniques using
antibodies raised against proliferating cell nuclear antigen (PCNA;
Yamaguchi et al., 1991
).
PCNA is a 36-kDa polypeptide present in the nuclei and cytoplasm
(Yamaguchi et al., 1991;
Grzanka et al., 2000
) of
mitotically active (S phase) cells
(Miyachi et al., 1978
). It is
known to be involved in DNA replication through an association with DNA
polymerase
(Bravo et al.,
1987
) and in DNA repair
(Fairman, 1990
), although
there is yet no evidence for a role in other processes that require DNA
synthesis, such as DNA endoreduplication. PCNA shows a high degree of
molecular similarity throughout the animal and plant kingdoms (Sukuza et al.,
1989; Mathews et al., 1984
;
Bauer and Burgers, 1990
;
Daidoji et al., 1992
).
A number of assays have been used to assess cell proliferation levels,
including direct counting of mitotic figures, tritiated thymidine
autoradiography and bromodeoxyuridine (BrdU) immunocytochemistry
(Gratzner, 1982). Each of
these methods has disadvantages, however. Counting mitotic figures is
cumbersome and represents only one phase of the cycle. Tritiated thymidine
autoradiography and BrdU immunocytochemistry are also restricted to one phase
of the cell cycle, and these methods require previous administration of a
thymidine analogue, one of which is a radioactive substance. The
immunohistochemical assay for PCNA avoids some of these disadvantages
(Foley et al., 1991
). The PCNA
antigen is stable and can be detected by immunohistochemistry during the S
phase of cells of all tissues (Kurki et
al., 1986
; Foley et al.,
1991
). Several monoclonal antibodies have been developed and used
in immunocytochemical techniques to detect PCNA in paraffin-embedded tissues
(Ogata et al.,
1987a
,b
;
Hall et al., 1990
). This
technology has been applied mostly to mammalian tissues
(Foley et al., 1991
). PCNA has
been immunocytochemically demonstrated in only a few non-mammalian species,
including the fruit fly (Yamaguchi et al.,
1991
). A number of studies have characterised the PCNA gene in
Drosophila (mus209, NT_033778.1) and have described the
molecular interactions of PCNA and other proteins and its relevance to the
development of this insect (Yamaguchi et al.,
1990
,
1991
,
1995
,
1996
;
Yamamoto et al., 1997
).
Several cDNAs with high homology to Drosophila PCNA have also been
sequenced in other insect species such as Anopheles gambiae
(gi/31236419), Bombyx mori (gi/3334291) Spodoptera
frugiperda (gi/21717394), Hyphantria cunea (gi/21717396) and
Sarcophaga crassipalpis (gi/3334293).
The aim of the present work was to study the differences in the expression of PCNA in the regenerative compartment of the epithelium of the locust midgut in relation to feeding and food nutritional quality. The possible cellular and physiological implications of these differences are discussed. We also report our modification of the immunocytochemical technique to show the expression of PCNA in the epithelial cells of the locust midgut, which is likely to be useful for other insect tissues and a variety of invertebrate epithelial models.
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Materials and methods |
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Experimental procedure
Locusts were housed individually in clear plastic boxes
(17x12x6 cm) containing an aluminium perch, a water dish and a
Petri dish of food. They were kept at 30°C under a 12 h:12 h light:dark
photoregime. Two experiments were performed.
Experiment 1
Thirty newly ecdysed fifth-instar nymphs (15 males, 15 females) were kept
until day 1 or 4 on one of the five synthetic foods listed above (P%:C% 7:35,
14:28, 21:21, 28:14 or 35:7). Nymphs were taken during the light phase of day
4 as they commenced a meal during ad libitum feeding (defined as
having walked from the perch to the food dish and fed for 10 s). After
crushing the head capsule, the tip of the abdomen was cut and the head pulled
until the cervical membrane tore. Next, the entire gut was gently pulled out
of the body by the head and opened along its length with bowspring scissors
under Ringer solution (Mordue,
1969). The gut contents were removed and the tissue pinned flat
onto a piece of photographic paper (3x1 cm) in a wax dish. The dissected
and pinned gut, with its paper support, was then placed into Bouin's fluid for
24 h. Thereafter, the fixed guts were removed from the photographic paper,
washed and stored in 70% ethanol prior to embedding in paraffin.
Experiment 2
Eighty-four nymphs (42 males and 42 females) were fed one of two synthetic
foods (P%:C% 7:7, 21:21) and their guts were sampled (as described above) on
days 0 (before feeding commenced for the stadium), 1, 2, 3, 4, 6 and 8. The
mean stadium duration under the prevailing experimental conditions is
1011 days on both diets, with insects ceasing feeding by day 89
(Raubenheimer and Simpson,
1993; Zanotto et al.,
1993
).
Immunocytochemistry
For the immunocytochemical localisation of PCNA in paraffin sections (4
µm in thickness), a variant of the avidinbiotin complex (ABC)
technique of Hsu et al. (1981)
was employed. After removal of paraffin with xylol, followed by 10 min in
absolute ethanol, endogenous peroxidase was blocked by treatment with 3%
H2O2 in absolute methanol. Sections were then hydrated
through a graded series of ethanol (96%, 80%, 70%) and rinsed for 5 min in
deionized H2O to remove any remaining alcohol.
PCNA epitopes masked by fixation were revealed by antigen retrieval by heating the sections in a retrieval solution [citric buffer (CB): 0.01 mol l1 citric acid]. To obtain optimal antigen retrieval, several different antigen retrieval heating protocols using different pHs (2.311) of CB and different microwave heating times (090 min) were tested. For the microwave treatment, a glass slide rack was immersed in a plastic container with 1000 ml CB. The open container was heated in a 700 W microwave oven for 15 min. The solution was allowed to bubble.
After the microwaving protocols, slides were cooled for 15 min, rinsed in
deionized H2O and then placed in Tris-HCl buffered saline (TBS:
0.05 mol l1 Tris buffer, pH 7.4 and 0.5 mol
l1 NaCl). Non-specific binding sites were blocked with 5%
mouse normal serum in TBS, and the sections were then incubated overnight at
4°C with anti-PCNA serum (mouse anti-PCNA immunoglobulins; clone PC10;
Dakopatts, Glostrup, Denmark) diluted in TBS. Several dilutions of the primary
antiserum were tested (1:50, 1:100, 1:200, 1:400, 1:800) to obtain optimal
immunolabelling. Following treatment with the primary antiserum, sections were
rinsed in TBS (5 min) and then incubated for 30 min at room temperature with
biotinylated rabbit anti-mouse antiserum (Dako, Cambridge, UK) diluted 1:200
in TBS. After a second rinse in TBS, the sections were treated for 30 min at
room temperature with an avidinbiotin peroxidase complex (Dako) diluted
1:100 in TBS prepared 30 min in advance. The sections were then washed in TBS
and then in acetate buffer (AB: 0.1 mol 1l acetic acid, pH
6). Peroxidase activity was demonstrated by the diaminobenzidine
(DAB)/H2O2 method (Sigma Chemical Co., Madrid, Spain).
The reaction was intensified with nickel according to Shu et al.
(1988). The solution was made
by mixing a solution of 50 mg of DAB in 50 ml of deionized H2O,
with a second solution made up by adding 2.5 g of ammonium nickel sulphate
[diammonium nickel (II) sulphate 6-hydrate; BDH Laboratory Supplies, Dorset,
UK], 200 mg of b-D-glucose [b-D(+) glucose; Sigma Chemical Co., St
Louis, MO, USA], 40 mg of ammonium chloride (Sigma Chemical Co.) and 30 mg
ofglucose oxidase (Sigma Chemical Co.) in 50 ml of AB. Finally, the sections
were washed in distilled water, lightly counterstained with haematoxylin,
dehydrated and mounted in a mountant for microscopy (DPX).
Negative controls (omission of any of the layers of the immunocytochemical protocol and the use of non-immune mouse serum as the first layer) were performed and gave no immunocytochemical reaction. Also, the lack of antigen retrieval protocol was used as a negative control in the PCNA detection.
Quantification of the immunocytochemical reaction
Two different systems were used to assess the quantity of PCNA nuclear
immunoreactivity: (1) by counting PCNA-positive nuclei and (2) by measuring
the optical density of PCNA-like immunoreactive nuclei.
Counting PCNA-positive nuclei
Four sections of the gut of each experimental locust were randomly selected
among a total of 50 serial sections performed. An arbitrary threshold was set,
with the help of the image analysis system Visilog 4 (Noesis, Velicy, France),
in order to discriminate the PCNA-positive cells from the background. Four
fields of the appropriate midgut region were randomly selected so that
400 nuclei per locust were counted.
Positive and negative cells were counted, and the index of cells
immunostained for PCNA (PCNA-I) was calculated using the following equation:
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Measuring optical density of PCNA-like immunoreactive nuclei
The optical density (PCNA-OD) of the nuclei counted as described in the
above section was measured using the Photoshop 4.0 image software
(Lehr et al., 1997).
Bromodeoxyuridine immunocytochemistry
The BrdU labelling was carried out by abdominal injection of 100 µg BrdU
in 10 µl of saline. The midgut was dissected and fixed (following the same
fixation protocol described above) within one hour of the BrdU injection. For
the immunocytochemical localization
(Montuenga et al., 1992;
Hyatt and Beebe, 1992
), after
blocking endogenous peroxidase with H2O2, the sections
were treated with the nuclease supplied with the primary anti-BrdU-serum
(Amersham Pharmacia, Little Chalfont, UK) for one hour at room temperature.
After blocking non-specific binding sites with 5% goat normal serum, the
sections were incubated overnight with a primary anti-BrdU-serum (mouse
anti-BrdU immunoglobulins; clone RPN202; Amersham Pharmacia) diluted 1:200 in
TBS. Detection was enhanced with the Envision® (Dako) reagents. The
sections were counterstained with haematoxylin, dehydrated and mounted in
DPX.
Western blotting
Samples destined for western blotting were frozen in liquid nitrogen
immediately after dissection and maintained at 80°C until
processed. Extracts of locust gut tissue were prepared by thawing the tissue
on ice and homogenizing accurately weighed samples in ice-cold homogenization
buffer: 2x tricine buffer with 8% sodium dodecyl sulphate (SDS; Novex,
San Diego, CA, USA) containing 1 mmol l1 final concentration
of the protease inhibitors pefablock (Centerchem Inc., Stanford, CT, USA),
bestatin and phosphoramidon (Sigma Chemical Co., St Louis). The homogenate was
centrifuged at 100 000 g for 30 min, and the supernatant was
collected. Homogenate supernatant protein content was measured using a BCA kit
(Biorad Labs, Richmond, CA, USA) after trichloroacetic acid precipitation and
NaOH resolubilizing of the extract protein.
Protein extracts were diluted to an approximate protein concentration of 1 µg µl1, heated to 95°C for 5 min and loaded into the sample well and electrophoretically fractionated on a 412% tricine SDS-PAGE gel (Novex) at 140 V for 45 min under reducing conditions (5% ß-mercaptoethanol). Transfer blotting was accomplished in the same apparatus equipped with a titanium plate electrode insert, and proteins were affixed to a nylon membrane (Immobilon PVDF; Millipore, Billerica, MA, USA) at 30 V for 1.5 h. The membrane was blocked overnight in 5% non-fat milk-PBS, incubated for 1 h in a 1:500 dilution of mouse anti-PCNA and washed three times in 0.1% Igepal (Sigma, St Louis, MO, USA). The membrane was then exposed to biotinylated immunoglobulins (Dakopatts; 1:200 dilution) for 1 h and then to avidinbiotin peroxidase complex (Dakopatts; 1:500) for an additional hour. Peroxidase activity was revealed with the ECL + Plus chemiluminescence kit (Amersham, Arlington Heigths, IL, USA) following manufacturer's instructions. The negative control was performed by omitting the specific primary antibody.
Feulgen staining
In order to measure the relative DNA content in the nuclei of the midgut
caeca, the Feulgen reaction was applied in 14 µm sections of locust midguts
belonging to the first diet experiment (see Experimental procedure, experiment
1).
Sections were brought to water after removal of the paraffin with xylene
and hydration through a graded series of ethanol (96%, 80%, 70%). The optimum
Feulgen hydrolysis time was estimated after Kjellstrand
(1977). The maximum stain was
achieved after 1 mol l1 HCl hydrolysis for 12 min at
55°C. After a rinse in distilled water, the slides were placed in Schiff's
reagent (Merck, Darmstadt, Germany) at room temperature for 2 h 45 min. Next,
the slides were transferred to sulphurous acid for 2 min. The sulphurous step
was repeated twice (2 min each). Then the slides were washed in running water
for 1 min, dehydrated in alcohol and mounted after clearing in xylene.
All slides were treated simultaneously in the same solutions.
Quantification of the Feulgen reaction
Measurements were made using the 100x objective, and a shading
correction was employed in order to avoid optical aberrations. Mature nuclei
of the caeca are oval shaped and their major axis is perpendicular to the
epithelial layer. Only the nuclei sectioned through the medial plane parallel
to the long axis showing well-defined and sharp limits were chosen for
measurements in each field.
Scanning microdensitometry was performed to quantify the Feulgen reaction with the help of the Visilog 4 image analyser. The integrated optical density (IOD) values of the mature nuclei of the caeca in each treatment were measured in several random sections. 400 nuclei were counted in each specimen.
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Results |
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A microwave pre-treatment was needed to make the epitopes available to the anti-PCNA antibody. The optimisation of the microwave heating time clearly improved PCNA staining. Fig. 2 shows the effect of different microwave treatment times on PCNA-like immunoreactivity. In the locust midgut, a faint immunocytochemical signal was detected only after at least 20 min of microwave heating. The immunoreactive optimal signal was found after 40 min. Extended microwave pre-treatment time (>40 min) resulted in a dramatic decrease in immunoreactivity levels.
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Regional differences in the midgut
Regional differences within the midgut of Locusta appear in
relation to PCNA-like immunostaining (Fig.
3). The midgut caeca, located at the foregutmidgut
junction, and the ampullae through which the Malpighian tubules drain, at the
midguthindgut junction, are the two regions in which the immunostaining
for PCNA (PCNA-OD) was most intense and the PCNA index (PCNA-I) was highest.
In our quantitative studies, midgut caeca and ampullae showed statistically
significant differences (see below) when compared with the other regions
(Fig. 3).
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Effect of age during the stadium, nutrient content of food and sex on PCNA-I
The diet supplied and the age within the stadium have a strong influence on
both the PCNA-I and PCNA-OD levels (Fig.
4). The more balanced diet (21:21) elicited the higher PCNA-I and
PCNA-OD values, and these fell as the diets became progressively unbalanced in
their P:C ratio (diets 7:35 or 35:7). Additionally, locusts showed higher
PCNA-I and PCNA-OD values on day 4 after ecdysis than on day 1. When PCNA-I
values were included in an analysis of variance (ANOVA), the two factors
(diets and day) showed statistical differences
(Fig. 4A; d.f.=4, 50,
F=4.984, P=0.002 for the effect of diet and d.f.=1, 50,
F=29.736, P<0.000 for the effect of age/day within the
stadium), and no interaction was detected between them (d.f.=4, 50,
F=0.529, P=0.715). Similarly, PCNA-OD was related to locust
age and the nutritional balance of their food
(Fig. 4B; d.f.=1, 50,
F=10.125, P=0.003 for the effect of day during the stadium,
and d.f.=4, 50, F=6.953, P<0.000 for diet composition).
The highest PCNA-OD values were found in insects fed a balanced diet (21:21)
and on day 4 [note that PCNA-OD is expressed as a numerical value from 0
(white, representing no labelling) to 255 (black, indicating strongest
labelling)].
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These results suggest that the values of PCNA-I and PCNA-OD, and thus the
proliferative index of the midgut epithelium, are related to the nutritional
balance of the insect. The total percentage of protein and digestible
carbohydrate was constant at 42% in all the diets used, so that the total
amount of nutrients does not account for the differences found between diets.
Therefore, these could be due to the ratio of protein to carbohydrate in the
food and/or to the concentration of one or other macronutrient. If the first
hypothesis is true, food 7:7, which has the same P:C ratio as the 21:21 food,
albeit with both macronutrients diluted threefold, should give similar levels
of PCNA-I and PCNA-OD to those observed with the 21:21 diet. No significant
differences were found between diets 7:7 and 21:21 (d.f.=1, 51,
F=0.077, P=0.783 for PCNA-I and d.f.=1, 51,
F=0.655, P=0.423 for PCNA-OD), suggesting that the
differences observed are rather related to the ratio of protein to
carbohydrate in the food. In addition, diet 21:21, which is known from earlier
works to be close to the optimal ratio and concentration of protein and
carbohydrate for fifth-instar locusts (19% P:23% C;
Simpson and Raubenheimer,
1993; Chambers et al.,
1995
), elicits the highest PCNA-I and PCNA-OD values in our
experiment, while diets 7:35 and 35:7, which are distant in P:C ratio from the
optimum 19:23, induce the lowest values. The data of our experiments (day 1
and day 4) were regressed against a geometric estimation of the protein and
carbohydrate nutritional quality (see
Chambers et al., 1995
), namely
the distance in nutrient space of each of the two macronutrients from their
optimal percentage in diet 19:23 (see
Zudaire et al., 1998a
). The
regression was significant on both days 1 and 4 [d.f.=2, 27, F=4.43,
P=0.021, r2=0.23 for PCNA-I on day 1
(Fig. 5A, with distance to
optimal diet shown as relative categories) and d.f.=2, 27, F=4.48,
P=0.021, r2=0.25 for PCNA-I on day 4
(Fig. 5B) and d.f.=2, 27,
F=21.524, P<0.000 for PCNA-OD on day 1
(Fig. 5A) and d.f.=2, 27,
F=3.868, P=0.033 for PCNA-OD on day 4
(Fig. 5B)], further supporting
the hypothesis that both PCNA-I and PCNA-OD are dependent on the P:C ratio of
the food.
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Changes in PCNA-I with insect age and influence of food eaten
In the second experiment, which investigated the effect of insect age
during the fifth-instar in more detail, PCNA-I increased significantly from
day 0 to day 3 after ecdysis, on which we found the maximum value
(Fig. 6), and then decreased
until day 8. To confirm the results found in the previous experiment, data
were compared between days 1 and 4. As expected, strong differences were found
in PCNA-I between the two days (d.f.=6, 52, F=6.247,
P<0.000 for PCNA-I and d.f.=6, 52, F=11.021,
P=0.000 for PCNA-OD), while no difference was found between insects
fed diets 7:7 and 21:21 (d.f.=1, 51, F=0.077, P=0.783 for
PCNA-I and d.f.=1, 51, F=0.585, P=0.484 for PCNA-OD).
|
Fig. 6 also shows the amount
of food eaten on each day. As expected from earlier work
(Raubenheimer and Simpson,
1993), the quantity of food eaten by fifth-instar locusts differed
with both age and diet. Intake reached a peak at around day 4 and declined
thereafter, while consumption was higher on 7:7 than 21:21 diet, indicating
compensatory feeding. When the PCNA-I values for diet 7:7 and 21:21 were
regressed against the quantity of food eaten by the locust on each day,
significant linear regressions were obtained
(Fig. 7A,B) for both diets
(d.f.=1, 4, F=19.17, P=0.0119 for diet 7:7 and d.f.=1, 4,
F=11.16, P=0.0288 for diet 21:21). In order to compare
statistically the slope and distance between both regression lines, we
included diet as a factor in an analysis of covariance (ANCOVA) with the mass
of food eaten as a covariate. The regression lines were parallel (d.f.=1, 43,
F=5.03, P>0.05) but differed in intercept (d.f.=1, 43,
F=11.05, P=0.010). As expected, both variables (PCNA-I and
amount of diet eaten) were significantly correlated (d.f.=1, 43,
F=12.12, P=0.008). Similar regression lines were obtained
for PCNA-OD (Fig. 7A; d.f.=1,
43, F=9.2421, P=0.0384 for diet 7:7;
Fig. 7B; d.f.=1, 43,
F=4.569, P=0.0994).
|
Effect of the quality of the food on DNA content
To further understand the significance of the differences observed in PCNA
levels, we carried out microdensitometric DNA measurements on the
undifferentiated nuclei (UN) of the regenerative nests and the mature nuclei
(MN) of the differentiated cells of the caeca of the locusts fed on different
synthetic foods. Although in the present work we have used an indirect
technique to calculate the DNA content of the nucleus, discrete peaks of
integrated optical density (IOD) were obtained
(Fig. 8). Several
subpopulations showing different IOD peaks (different DNA content) were
clearly distinguished. A marked difference was found between UN and MN. The UN
showed lower IOD values when compared with the MN. The MN of the locusts that
were fed the near optimal 21:21 diet showed the highest IOD values, including
several peaks (at 1600, 1800 and 2100 arbitrary IOD units). The rest of the
diets resulted in lower IOD values and only displayed one or two discrete
peaks (at 1000 and 1200 IOD units). MN of animals fed with diets 7:35 and
35:7, the most distant from the optimal diet, had the lowest IOD values (at
1000 IOD units; occasionally a peak at 1200 IOD units was observed). Locusts
fed diet 28:14 showed two different peaks at 1000 and 1200 IOD units,
supporting the presence of two nuclear subpopulations. Diet 14:28 provided a
single peak at 1200 IOD units, although another less conspicuous peak could be
present at 1000 IOD units. In summary, locusts fed on diets with a P:C ratio
close to the optimal 19:23 show higher IOD values than locusts fed on more
unbalanced diets, supporting a correlation between the geometric distance to
the optimal diet and the amount of DNA.
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Discussion |
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The antigen retrieval process applied in this study is based on heating of
the sections rinsed in a dilute solution of citric acid in a microwave oven.
Similar heating protocols using heavy metal solutions are described by Shi et
al. (1991) as an
immunohistochemical enhancement method for formalin-fixed, paraffin-embedded
tissues. The mechanism concerning microwave oven recovery of antigens is not
yet fully understood, but in the case of formalin-fixed tissues it is possible
that cross-linking of proteins caused by formaldehyde may be altered by
microwave heating (Shi et al.,
1991
). In the present study, we have found that antigen retrieval
by heating in a microwave oven also works successfully for Bouin's-fixed
tissues. The citrate-based solutions used in our study were formulated
following other protocols in which citrate buffers were used
(Evers and Uylings, 1994
;
Brown and Chirala, 1995
). We
show that the antigen retrieval process is dependent on the microwave heating
time and the pH of the citric buffer. Our data suggest that the specific
parameters in relation to buffer composition and heating have to be adjusted
from the protocol used for neutral, formalin-fixed tissues because of the
particular composition and fixation characteristics of Bouin's fluid. Our
results are in agreement with those of Evers and Uylings
(1994
) who found that pH and
microwave heating time of the solution was of great importance for retrieval
of antigen in formalin-fixed, paraffin-embedded tissues. They also found that
these parameters may vary when different antibodies are used
(Evers and Uylings, 1994
). We
have found that the optimal conditions for PCNA retrieval, using clone PC10,
in Bouin's-fixed, paraffin-embedded tissues of insects are pH=6.0 and 40 min
ofmicrowave heating at 700 W. Clone PC10 has been successfully used in many
studies of cell proliferation in mammals
(Hall et al., 1990
;
Preziosi et al., 1995
;
Start et al., 1992
). It has
also been reported that clone PC10 gives the best results in PCNA detection in
fish tissues (Ortego et al.,
1994
). The only study on immunological PCNA detection in insect
tissue sections to our knowledge also used clone PC10 as the primary antibody
(Yamaguchi et al., 1991
).
Our data show PCNA-like immunoreactivity throughout the different regions
of the midgut exclusively restricted to the cells within the nests, supporting
the proliferative nature of these areas. Western blot analysis further
confirmed the specificity of the immunoreaction. In agreement with previous
studies in insects (Ng et al.,
1990), PCNA-like immunoreactivity in locusts appears as a single
band of
30 kDa. Our densitometric data suggest profound differences not
only in the number of PCNA-like immunoreactive cells (noted as PCNA-I in the
text) but also in the levels of PCNA-like immunostaining (PCNA-OD) in the
cells of locusts belonging to different experimental groups or located within
different regions of the midgut. The midgut caeca has the highest levels of
PCNA, followed by the regenerative cells in the ampullae at the
midguthindgut junction. In addition, the mature nuclei of locusts fed
on a balanced diet (21:21, closer to the optimal 19:23) show high PCNA-I and
higher DNA contents than in those locusts fed on unbalanced diets, and values
of both variables fell progressively as the diets became more unbalanced.
It has been well established that PCNA is a good marker of proliferative
cells and that PCNA is a required element for DNA synthesis
(Miyachi et al., 1978;
Celis et al., 1987
;
Mathews et al., 1984
;
Bravo et al., 1987
). Therefore,
several cellular activities, such as DNA amplification, repair of damaged DNA,
cycles of endopolyploidy, mitotic cell division or DNA excision repair
(Shivji et al., 1992
) could
be, at least in theory, associated with the DNA synthesis that we have
observed in the cells of the locust regenerative nests. By immunocytochemical
techniques we show an association between BrdU incorporation and PCNA-like
immunostaining in the cells of the regenerative nests, supporting the DNA
replicative nature of the ongoing processes in these cells. Although
important, this association between BrdU incorporation and PCNA does not
discriminate between mitosis and the other above-cited processes involving DNA
synthesis. However, the fact that we found only a small number of mitotic
figures in all the 15 µm Feulgen-stained slides examined (data not shown)
makes it unlikely that the DNA synthesis shown by PCNA and BrdU
immunocytochemistry is exclusively associated with the S phase of the mitotic
cell cycle. On the other hand, DNA amplification processes (i.e. extra
replications of certain genes or DNA sequences) have been reported in a number
of insects (Nagl, 1978
). Our
results on DNA measurements suggest that the nuclei of the caecal cells
undergo a `maturation' process from an undifferentiated status (low DNA
content) within the nidi to a `mature' form (higher DNA content) when they
move away from the regenerative nests while growing in size and
differentiation. The differences in PCNA-like immunoreactivity observed among
locusts fed on different diets, together with the concomitant variations in
the DNA content and the low number of mitotic figures in the regenerative
nests, support a process of differential DNA amplification and probably cycles
of endopolyploidy (or endoreduplication) involving genes necessary for the
correct completion of the feeding or moulting cycle for each particular diet.
This is in agreement with Nagl's hypothesis
(Nagl, 1978
) that
endopolyploidy and polyteny might be strategies by which, through the increase
of the number of DNA templates, insect cells have a high synthetic capacity
for selected proteins. This reasoning also provides the basis to understand
the significance of the high proliferative index found in the caeca and the
ampullae. It seems that ampullae may have an important role as a hitherto
unappreciated `endocrine organ' in the locust midgut
(Montuenga et al., 1996
). The
higher PCNA staining found in these regions could be related to a higher
metabolic activity required for their absorptive (in the case of caeca) and
secretory (in the case of ampullae) functions.
As expected from earlier work (e.g.
Raubenheimer and Simpson,
1993; Simpson et al.,
1988
), we found that the highest intake of food occurred near
mid-stadium. This was the case in locusts fed both diets 7:7 and 21:21. Also,
as shown previously (Raubenheimer and
Simpson, 1993
; Zanotto et al.,
1993
), the quantity of 7:7 diet eaten was greater than that of
diet 21:21, indicating compensatory feeding for nutrient dilution. The PCNA-I
and PCNA-OD levels in both groups (locusts fed 7:7 vs 21:21) were
statistically similar and there was a highly significant correlation between
the quantity of food eaten and PCNA-I values in both groups. Hence, these data
strongly suggest that PCNA-I and PCNA-OD levels (and as a consequence DNA
synthesis) reflect the nutritional status of the insect and that locusts fed
on diet 7:7 were able to reach the same level of DNA synthesis as those fed on
a 21:21 diet by altering the amount of food eaten. However, the same was not
true for those insects fed the other diets (7:35, 14:28, 28:14 and 35:7). This
is because the P:C ratio of these latter foods differed from the optimal
(19:23), forcing insects to consume too much of one nutrient relative to the
other. Such interference between nutrients leads to animals having to make a
compromise between the metabolic and other costs of overeating one nutrient
and undereating the other (Raubenheimer
and Simpson, 1993
; Simpson and
Raubenheimer, 1993
). Consequently, our results showed a
correlation between the levels of PCNA expression and of nuclear DNA content
and the ratio of protein and carbohydrates in the diet, with values falling as
foods became more unbalanced in their ratio of P to C.
It will be of great interest to see whether the effect of nutritional status is due to a general stimulation of metabolic activity by ingested nutrients, whether it is differentially stimulated by specific nutrient groups and/or whether it involves regulation of particular DNA synthetic pathways. We demonstrated that the synthesis and release of some but not all regulatory peptides present in the diffuse endocrine system of the locust midgut is also related to the quality of the food, suggesting that selected synthetic pathways in the midgut epithelial cells are specifically upregulated by protein and carbohydrate contents in the diet. Further genetic and molecular studies will be necessary to better understand these mechanisms.
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