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G omes H., D
ias A.J.B
., M
or
aes J., C
ar
v alho C.S.P
o gullo C. 2010.
Dias
A.J.B.,
Mor
oraes
Car
arv
C.S.P.. & LLo
Glucose-6-phosphate Metabolic Preferential
Destinations in Bovine Oviduct Cells.
Scientiae Veterinariae. 38(4): 377-383.
Acta Scientiae Veterinariae. 38(4): 377-383,Acta
2010.
ORIGINAL ARTICLE
Pub. 927
ISSN 1679-9216 (Online)
Glucose-6-phosphate Metabolic Preferential Destinations in Bovine Oviduct Cells
Helga Gomes 1,2, Angelo José Burla Dias 1, Jorge Moraes 2, Carla Sobrinho Paes de Carvalho1 & Carlos
Logullo1,2
ABSTRACT
Background: The oviduct is a dynamic organ which facilitates gamete function, fertilization and embryo development. This
organ is covered by an epithelium containing ciliated and non-ciliated cells. Secretions of non-ciliated cells compose the
oviduct fluid, which will nourish the early embryo. During the period of ovulation, the oviduct exhibits an active role, where
the lumen provides an environment suitable for fertilization and the muscle layer contracts rhythmically to move the egg
toward the uterus. In this study we aimed to investigate the content of fuel metabolites and enzyme activity assays related to
the glycolytic metabolism in bovine oviduct cells such as Glucose-6-phosphate, Glycogen, Pyruvate, Hexokinase, Pyruvate
kinase, Phosphoenolpyruvate carboxykinase and Glucose-6-phosphate dehydrogenase.
Materials, Methods & Results: In this sense, we divided the oviduct in 3 identical portions (anterior, medium and posterior).
After division, each region was massaged and the lumen was flushed with 2mL Ca+2, and Mg+2 free PBS, pH 7.2. The material
obtained was centrifuged at 3.000 x g for 5 min at 4ºC. The pellet of cells were ressuspended again with 2 mL of Ca+2 and Mg+2
free PBS at pH 7.2 and sonicated 6 times at 60W. All the assays were performed in a Shimadzu U1240 spectrophotometer. The
data were normalized in terms of total protein content and the assay was ran in triplicate. In the anterior region a low activity
of hexokinase (HK) and pyruvate kinase (PK) and an accumulation of pyruvate were detected. Besides this, based on the
glucose-6-phosphate dehydrogenase (G6PDH) activity and the amount of glycogen, the glucose-6-phosphate (G6P) could be
directed to the pentose phosphate pathway, which provides protection against the toxicity of reactive oxygen species (ROS)
and supplies NADPH to be used in anabolic pathways. In the oviduct’s medium portion we observed an intense glycolytic
metabolism based on HK and PK activities and low pyruvate levels that could be exported to compose the lumen fluid. In the
oviduct’s posterior region we detected a different metabolic profile with high gluconeogenic activity due to elevated
phosphoenolpyruvate carboxykinase (PEPCK) activity glucose-6-phosphate concentration and HK activity.
Discussion: Low HK and PK activities were observed in the anterior portion, which could be due to the concentration of
pyruvate, Besides this, the (G6PDH) activity, assessed together with its substrate and the amount of glycogen, suggests that
cells from the anterior portion are working for the energetic maintenance of whole oviduct cells. The medium portion presents
a metabolism via high HK and PK activities. An increase in PEPCK activity could be considered as a source of
phosphoenolpyruvate. Accumulation of glycogen, high concentration of G6-P and (G6PDH) activity are also observed. The
profile of enzymes from glycolytic and gluconeogenic pathways and their substrate together with (G6PDH) activity and G6Phosphate lead us to believe that the preferential product produced in the posterior region is pyruvate.
Keywords: oviduct, glucose-6-phosphate, energy metabolism, glucose, glycogen.
Received: May 2010
www.ufrgs.br/actavet
1
Accepted: July 2010
Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia (CBB) and Laboratório de Reprodução e
Melhoramento Genético Animal, Centro de Ciências e Tecnologias Agropecuárias (CCTA), Universidade Estadual do Norte Fluminense
(UENF), Avenida Alberto Lamego n. 2000, Horto, CEP 28013-602 Campos dos Goytacazes, RJ, Brazil. 2Laboratório Integrado de bioquímica
Hatisaburo Masuda, campus Macaé, Universidade Federal do Rio de Janeiro (UFRJ), Macaé, RJ. CORRESPONDANCE: C. Logullo
[[email protected] - Fax: +55 (22) 27397134].
377
G omes H., D
ias A.J.B
., M
or
aes J., C
ar
v alho C.S.P
o gullo C. 2010.
Dias
A.J.B.,
Mor
oraes
Car
arv
C.S.P.. & LLo
Destinations in Bovine Oviduct Cells.
INTRODUCTION
Oviducts are part of female reproductive tract that
lie between each ovary and the tip of the adjacent uterine
horn [5]. Two cell types constitute the epithelium of the
oviduct: ciliated and secretory cells. Ciliated cells play an
important role in the transport of oocytes, sperm and embryos,
while secretory cells produce and release specific secretory
materials [2,3]. The combination of a selective transudate of
serum [7] and of these secretions forms the oviductal fluid
[18].
In mammals, the oviduct plays the most fundamental role in the reproduction process [2]. Its importance in
normal reproductive processes such as gamete final
maturation, which occurs in the infundibulum [12], the sperm
capacitation in the isthmus region, fertilization in the ampulla
[13], and early embryo development has been emphasized
by the difficulty experienced in attempting to repeat these
events in vitro [7,12].
The current literature provides ample information
regarding energetic metabolites in cow oviduct fluid.
Nevertheless, there is little or no information available on
the concentrations of key metabolites in oviduct cells in
cattle.
Successful reproduction on cattle requires the
knowledge of reproductive processes of the cow and the
better understanding of the metabolism of cow oviduct cells
due to the role that this organ plays in early embryo support
and nutrition. This knowledge may be useful in correcting
and adapting the composition of the media used in in vitro
production of embryos.
MATERIALS AND METHODS
Oviduct collection
Cow’s oviducts were collected at a local
slaughterhouse immediately after death. They were
transported to the laboratory in vials with ice, and then
classified as to the presence or absence of corpus luteum. The
oviducts associated to ovaries with corpus luteum were
dissected and equally divided into three different portions:
anterior, medium and posterior (Figure 1).
Figure 1. Schematic representation of three regions of cow’s
oviduct.
378
Glucose-6-phosphate Metabolic Preferential
Acta Scientiae Veterinariae. 38(4): 377-383.
Cells collection
After division, each region was gently massaged
to remove cells. Then, the lumen was flushed with 2mL Ca+2,
and Mg+2 free PBS, pH 7.2. These wash-offs from oviducts
were centrifuged at 3.000 x g for 5 min at 4ºC. The pellet of
cells were ressuspended with 2 mL of Ca+2 and Mg+2 free
PBS at pH 7.2 and sonicated1 6 times (each time of 30 s with
an interval of 30 s between the times) at 60W. After
centrifugation at 10.000 x g for 30 min at 4ºC, the supernatant
was aliquoted in 1 mL and lyophilized2 and stored at -20ºC
until use.
Endogenous Glucose-6-P (G6P) content
Glucose-6-phosphate was enzymatically
quantified in 1mg/mL total protein of lyophilized cells
ressuspended with 55 mM Tris-HCl, pH 7.6 in the presence
of 5 mM MgCl2, 2mM NAD+, and 0.34 U/mL glucose-6phosphate dehydrogenase (G6PDH). The ß-NADH
production was detected in a spectrophotometer3 at 340 nm
using a molar extinction coefficient of 6.22 M-1, as described
by Moraes et al. [20]. The data were normalized in terms of
total protein content and the assay was ran in triplicate.
Endogenous Glycogen content
The lyophilized cells from each region were
ressuspended with ultra pure water (100 µL). They were
incubated with 200 mM sodium acetate buffer, pH 4.8, 1 unit
α-amiloglucosidase4 and then incubated for 4 h at 40ºC.
Glucose produced from glycogen degradation was determined
with an enzymatic Kit for glucose dosage 5 in a
spectrophotometer 3 at 510 nm, according to the
manufacturer’s instructions. A standard curve was used with
different dilutions of glycogen, subjected to the same
treatment. The data were normalized by total protein content
and the assay was ran in triplicate.
Endogenous Pyruvate content
Lyophilized cells from each region were
ressuspended with ultra pure water (100 µL). Pyruvate was
indirectly measured by the addition of lactate dehydrogenase6.
The NADH consumption was detected in a
spectrophotometer3 at 340 nm [20] and the assay was ran in
triplicate.
Hexokinase (HK) activity assay
Hexokinase activity was determined immediately
in freshly collected cells from each oviduct portion. Cells
were sonicated and centrifuged as mentioned above.
Supernatant aliquots were incubated with 20 mM Tris-HCl,
pH 7.5 containing 6 mM MgCl2, 1mM ATP, 0.5 mM NAD+,
10 mM NaF, and the reaction was started with 2 mM glucose7.
The glucose-6-phosphate formed was measured by adding
equal volumes of 20 mM Tris-HCl pH 7.5, 6 mM MgCl2,
1unit/mL of glucose-6-phosphate dehydrogenase8 from
Leuconostoc mesenteroides and 0.3 mM ß-NAD+. The
G omes H., D
ias A.J.B
., M
or
aes J., C
ar
v alho C.S.P
o gullo C. 2010.
Dias
A.J.B.,
Mor
oraes
Car
arv
C.S.P.. & LLo
Destinations in Bovine Oviduct Cells.
production of ß-NADH was detected in a Shimadzu U1240
spectrophotometer at 340 nm using a molar extinction
coefficient of 6.22 M-1 as described by Galina & Da Silva [6]
and the assay was ran in triplicate.
Pyruvate kinase (PK) activity
Samples were prepared as described for HK activity
analysis. PK activity was measured in 20 mM Tris-HCl pH
7.5, 5 mM MgCl2, 1 mM ADP, 0.4 mM NADH, 1 unit/mL
lactate dehydrogenase and the reaction was started with 1
mM phosphoenolpyruvate9 (PEP). The ß-NADH consumption
was detected in a spectrophotometer3 at 340 nm using a molar extinction coefficient of 6.22 M-1 as described by
Worthington [27] and the assay was ran in triplicate.
Glucose-6-phosphate Metabolic Preferential
Acta Scientiae Veterinariae. 38(4): 377-383.
and approximately 6x less than anterior and posterior portion,
consecutively (Figure 2A).
Pentose-phosphate pathway in oviduct cells
The pentose-phosphate pathway was investigated
by the determination of glucose-6-phosphate dehydrogenase
profile (G6PDH) activity, which is the rate limiting step in
this pathway [26]. In the anterior portion we observed a higher
G6PDH activity (0.03 units/mg protein) in comparison to the
other portions (0.025 units/mg protein - medium and 0.02 units/
mg protein -posterior portion) (Figure 2B).
Phosphoenolpyruvate carboxykinase (PEPCK) activity
assay
Samples were prepared as described in the HK
activity section. The supernatant was incubated with 100
mM Hepes buffer pH 7.0 containing 10 mM
phosphoenolpyruvate9 (PEP), 0.2 mM NADH and 24 units of
malate dehydrogenase10. The reaction was started with 2.5
mM inoside diphosphate (IDP). The ß-NADH consumption
was monitored at 340 nm in a Shimadzu U1240
spectrophotometer3 and the physiological PEPCK activity
was determined as described by Petersen et al. [22] and the
assay was ran in triplicate.
Glucose-6-phosphate dehydrogenase (G6PDH) activity
assay
Samples were prepared as described above for
enzyme activities. The supernatant was incubated in 55 mM
Tris-HCl, pH 7.8 containing 3.3 mM MgCl2, 2 mM NAD+, 0.3
mM glucose-6-phosphate11. The ß-NADH production was
detected in a spectrophotometer3 at 340 nm using a molar
extinction coefficient of 6.22 M-1 as described by Worthington
[27] and the assay was ran in triplicate.
Total protein content
Total protein content was quantified according to
Bradford [4] using bovine serum albumin as standard. Three
samples were analyzed for each experimental point.
Statistical analysis
The bioactivities were analysed by analysis of
variance (ANOVA) between groups using Tukey test (P < 0.05)
for means.
RESULTS
Figure 2. G6-Phosphate destinations - A) G6-Phosphate
concentration at each oviduct portion. B) GG6-P
dehydrogenase (G6PDH) activity at each oviduct portion. C)
Glycogen content at different oviducts regions. The
bioactivity was analyzed by analysis of variance (ANOVA)
between groups using Tukey test (P < 0.05) for means.
Glucose-6-phosphate distribution in oviduct cells
Glucose-6-phosphate (G6P) quantification
demonstrated that the cells from the anterior portion present
approximately 356 nM. The cells from medium portion exhibited
almost 2-fold this value (680 nM). On the other hand,
endogenous G6P in the cells from the posterior portion is 3x
Glycogen content
The medium portion exhibited higher glycogen level
(79 µg/mg total protein). In anterior portion we observed a
concentration 20% lower than that exhibited by the medium
portion. In turn, in the posterior portion we observed a
concentration about twice as low as that of the medium
segment (30 µg/mg total protein).
379
G omes H., D
ias A.J.B
., M
or
aes J., C
ar
v alho C.S.P
o gullo C. 2010.
Dias
A.J.B.,
Mor
oraes
Car
arv
C.S.P.. & LLo
Destinations in Bovine Oviduct Cells.
Glycolysis in oviduct cells
The study of the glycolytic pathway in cow oviduct
was evaluated from measurements of the activity at the initial
(Hexokinase - HK) and final (Pyruvate kinase - PK) glycolysis
steps. HK (Figure 3A) and PK activities (Figure 3B) in the
anterior portion was about 0.02 units/mg protein and 0.14 units/
mg protein, and correlated with the rise in pyruvate levels
(44.4 nM/µg protein)(Figure 3C). We observed HK and PK
activities 4 times higher in the oviduct medium portion (0.24
units/mg protein and 1.38 units/mg protein, respectively) (Figures 3 A & B). In the same segment we observed a smaller
pyruvate concentration (10 nM/µg protein) (Figure 3C). In the
posterior portion, HK activity was similar to that observed in
the anterior segment (0.02 units/mg protein) and PK activity
was 1.02 units/mg protein, representing a decrease in 26% as
compared to the level in the medium portion (Figures 3 A & B).
Pyruvate content in this piece was around 55% higher than
that measured in the medium section (15.5 nM/µg protein)
(Figure 3C).
Glucose-6-phosphate Metabolic Preferential
Acta Scientiae Veterinariae. 38(4): 377-383.
Gluconeogenesis in oviduct cells
The evaluation of gluconeogenesis in the oviduct
was carried out using the activity of the key enzyme
phosphoenolpyruvate carboxykinase (PEPCK) (Figure 4A).
We could observe a low PEPCK activity in the anterior region,
when compared with the other portions (1.5 x higher in
medium and 2.5 x higher in posterior segments, respectively)
(Figure 4A).
Figure 4. Gluconeogenesis - A) PEPCK activity on oviduct
cells. The PEPCK activity was analyzed by analysis of
variance (ANOVA) between groups using Tukey test (P < 0.05)
for means.
DISCUSSION
Figure 3. Glycolysis pathway - A) HK activity on oviduct
regions; B) PK activity at different portions. C) Pyruvate
content at anterior, medium and posterior portions. The
bioactivity was analyzed by analysis of variance (ANOVA)
between groups using Tukey test (P < 0.05) for means.
380
In mammals, the fertilization and early embryo
development occur inside the maternal organ named oviduct
[11]. It has been recognized as a reproductive organ responsible
for creating a microenvironment that facilitates ovum transport
and maturation, sperm capacitation and fertilization [23-25]. It
has been suggested that this organ is responsible for the
supply of all the nutrients necessary for the early embryo
development [10,12].
In this article we investigated the metabolism of
glucose-6-phosphate in the bovine oviduct cells from three
equally divided segments. By dividing the oviduct in three
parts (Figure 1) we were able to observe that the cells from
each segment of bovine oviduct exhibit distinct profiles of
G6P content consumption (Figure 2A). One previous study,
using co-culture systems, suggested regional differences
associated with the physiological support provided by the
epithelial cells from oviduct [1].
Glycolysis is the metabolic pathway that converts
glucose into pyruvate . The first step in glycolysis is glucose
phosphorylation by a family of enzymes called hexokinases,
in order to form glucose-6-phosphate (G6P). Pyruvate kinase
catalyzes glycolysis’ final reaction, which generates pyruvate
and ATP. The glycolytic pathway is regulated both by enzyme
activity and metabolites/substrate availability.
We established as anterior portion the oviduct
region that includes the anatomical segment named
infundibulum (Figure 1). The free end of infundibulum is
called fimbria, and is able to capture the oocyte during
ovulation. Pickup of oocytes is achieved by a massage process,
done through muscular contractions, on the surface of the
ovary [8]. Once captured from the surface of the ovary, the
G omes H., D
ias A.J.B
., M
or
aes J., C
ar
v alho C.S.P
o gullo C. 2010.
Dias
A.J.B.,
Mor
oraes
Car
arv
C.S.P.. & LLo
Destinations in Bovine Oviduct Cells.
transport of the oocyte in the oviduct is achieved by ciliary
beating of the oviductal epithelial cells and by contraction
of the oviductal smooth muscle [13]. The massaging and
gamete transport events require energy from the cells along
the oviduct. We suggest that the accumulation of pyruvate in
this portion (Figure 3C) could support the energy demands
from oviduct cells on the moment of ovulation, when the
tissue spends intense energy capturing the oocyte and
transporting it until the site of fertilization. The availability
of the appropriate substrate is in turn controlling PK activity,
as its activity is low in this segment. Furthermore, the level of
G6P (Figure 2A) suggests that this metabolite could be
generated from another source, besides HK, since its activity
is low. Classically glycogen degradation could be useful as
an alternative source of G6P. Our results also indicate that
G6P generated in the oviduct’s anterior portion by glycogen
degradation could be preferably mobilized to supply the
pentose phosphate pathway. It is supported by the level of
G6P (Figure 2A) and the high G6PDH activity (Figure 2B) on
this segment. The pentose phosphate pathway is a process
that serves to generate NADPH and the synthesis of pentose
(5-carbon backbone) sugars [9]. NADPH is used for anabolic
pathways, such as lipid synthesis and also provides
metabolites involved in protection against the toxicity of
ROS (reactive oxygen species ) [14,20]. Previous studies
demonstrated the presence of antioxidant enzymes in oviduct
fluid as a way to protect the developing embryo [16,19,21].
The medium portion presented an intense
glycolytic metabolism via high HK (Figure 3A) and PK (Figure 3B) activities. This observation was corroborated by the
detection of low concentrations of pyruvate in this segment
(Figure 3C). As we mentioned before, the oviduct was equally
divided for the measurements. This portion of oviduct was
Glucose-6-phosphate Metabolic Preferential
Acta Scientiae Veterinariae. 38(4): 377-383.
divided so as to include the region that is described as the
presumptuous fertilization site [10]. The early bovine embryo
floats during the first 2 weeks after fertilization and depends
on the nutrients provided by oviduct and uterine fluids for its
development, growth, and ultimately, its survival [10]. From
the zygote to the 8- to 16-cell stage, pyruvate and lactate are
the preferred energy sources [17]. Pyruvate was shown to
play an important role in oocyte maturation, as described by
Krisher & Bavister [15]. Pyruvate, glutamine, and glycine
metabolism in the tricarboxylic acid cycle increases during
oocyte maturation, suggesting that oxidative metabolism is
the mechanism responsible for energy production during
bovine oocyte maturation [15]. Based on previous
physiological observations and pyruvate concentration, we
suggest that oviduct cells secrete this metabolite into the lumen,
to constitute oviduct fluid [10,15].
The oviduct’s posterior portion presented high
PEPCK activity levels (Figure 4A), which could be a result of
a decrease in glucose-6-phosphate concentration and of
lowered HK activity. The main function described for this
oviduct’s segment is the maintenance of embryo survival
and the posterior transfer to uterus. Based on this fact we
hypothesize that this segment exhibits an intense metabolic
energetic demand.
In summary, we observed low HK and PK activities
in the anterior oviduct region (Figure 5A), and this could be
due to the accumulation of pyruvate in the anterior segment.
Besides this, based on G6PDH activity and on the amount of
glycogen, G6P is being directed to the pentose phosphate
pathway, which could provide protection against the toxicity
of ROS and supply NADPH to be used on anabolic pathways
[20]. In the oviduct’s medium portion (Figure 5B) we observed
an intense glycolytic metabolism based on HK and PK
Figure 5. Schematic representation of glucose metabolism control in cow’s oviduct regions.
381
G omes H., D
ias A.J.B
., M
or
aes J., C
ar
v alho C.S.P
o gullo C. 2010.
Dias
A.J.B.,
Mor
oraes
Car
arv
C.S.P.. & LLo
Destinations in Bovine Oviduct Cells.
activities and low pyruvate levels that could be exported to
compose the lumen fluid. In the oviduct’s posterior region
(Figure 5C) we detected a different metabolic profile with
high gluconeogenic activity due to elevated PEPCK activity,
low glucose-6-phosphate concentration and HK activity.
These observations are schematically organized in Figure 5
and show how our hypothesis applies to different oviduct
portions.
Acknowledgements. This work was supported by grants
from Fundação Carlos Chagas Filho de Amparo à Pesquisa
do Estado do Rio de Janeiro (FAPERJ), Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
and Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq).
Glucose-6-phosphate Metabolic Preferential
Acta Scientiae Veterinariae. 38(4): 377-383.
SOURCES AND MANUFACTURERS
1
Equipment to Disrupt cells, Sonic Dismembrator 60-Fisher
Scientific ®.
2
Centrifugal Vacuum Concentrator System - Speed Vac
Plus® SC 110 A (Savant).
3
Equipment with a Spectrum mode that range of 190nm to
1100nm
Shimadzu
UV-VIS
mini-1240®
spectrophotometer.
4
α-amyloglucosidase from Aspergillus niger, Sigma, code
A 7420, Sigma Chemicals.
5
Glucox®, enzymatic kit for glucose determination, Doles
inc., Goiânia, Go, Brazil.
6
Lactate dehydrogenase, cat. Code 9001-60-9, Sigma
Aldrich.
7
Glucose, cat. Code G8270, Sigma Aldrich.
8
glucose-6-phosphate dehydrogenase from Leuconostoc
mesenteroides, cat. Code G5885, Sigma Chemicals.
9
Phosphoenolpyruvate, cat. Code 79418 Sigma Aldrich.
10
Malate dehydrogenase, Malic Dehydrogenase from
porcine heart, cat. Code M2634, Sigma Aldrich.
11
Glucose-6-phosphate, cat. Code G7250, Sigma Aldrich.
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Glucose-6-phosphate Metabolic Preferential
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