REGULATION OF EXPRESSION OF THE LIVER, MUSCLE,
BRAIN/PLATELET/PLACENTA SPECIFIC ISOFORMS OF
PHOSPHOFRUCTOKINASE IN PREIMPLANTATION MOUSE
EMBRYOS
A RESEARCH PAPER
SUBMITTED TO THE GRADUATE SCHOOL
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE
MASTER OF BIOLOGY
BY
PAIGE VERNASCO
DR. CLARE L. CHATOT
BALL STATE UNIVERSITY
MUNCIE, IN
MARCH, 2011
ABSTRACT
THESIS:
Regulation of Expression of the Liver, Muscle, and
Brain/Platelet/Placenta Isoforms of Phosphofructokinase in
Preimplantation Mouse Embryos
STUDENT:
Paige Vernasco
DEGREE:
Master of Biology
COLLEGE:
Science and Humanities
DATE:
March, 2011
PAGES:
69
Glycolysis is one of two metabolic pathways in which most mammalian cells
generate energy. Preimplantation mouse embryos do not run the glycolysis pathway until
the 8-16 cell stage. A major regulator of glycolysis is the enzyme 6-phosphofructo-1kinase (PFK). Jeff Henry’s research found that mRNA for the liver specific form of PFK
is found inconsistently at the 1-cell stage and consistently from the 2-cell stage through
the blastocyst stage and that mRNAs for muscle or brain/platelet/placenta isoforms of
PFK were not expressed at any stage. Studies suggest that PFK is not enzymatically
active in the mouse embryo until the 8-cell stage when glycolysis begins. Using specific
polyclonal anti-PFK antibodies, immunofluorescence and laser confocal microscopy, we
found that the liver specific PFK protein expression follows the same pattern as the
i mRNA expression in preimplantation mouse embryos. The muscle and brain/platelet/
placenta PFK protein antibodies stained embryos in the absence of mRNA but the
expression pattern did not follow the enzyme activity pattern with results that suggest
specificity problems or cross reactivity of the antibodies. The liver specific PFK isoform
expression showed an increase in expression throughout development with a significant
a increase of 2.3 fold in expression at the 8-16-cell transition. There was also a significant
increase of 1.9 fold in PFK-B expression from the 1-cell to the 2-cell stage corresponding
to the activation of the embryonic genome. Enzymatic assays are currently being run to
look at the activity levels throughout development.
ii INTRODUCTION
Most mammalian cells utilize organic carbon to generate energy by combined use
of 2 metabolic pathways; glycolysis, which allows cells to hydrolyze glucose into 2
readily usable molecules of pyruvate, and the tricarboxylic acid (TCA) cycle, that
converts pyruvate into energy. Glycolysis (Figure 1) is an important metabolic, cellular
function that supplies all mammals and many other species with a source of energy. Two
molecules of pyruvate are generated when one glucose sugar molecule splits during the
process of glycolysis (Figure 1).
In the presence of oxygen, mitochondria within the cell respire the pyruvate via
the TCA cycle, to generate large quantities of adenosine triphosphate, ATP, which is a
more basic and easily used intermediate source of energy for the mammalian cells. ATP
is involved in many cellular activities that involve energy in all living organisms. These
activities include active transport, protein and nucleic acid synthesis, protein activation or
deactivation, and muscle contraction (Campbell, 1996).
The enzyme 6-phosphofructo-1-kinase (PFK) is the major regulator of glycolysis
(glucose metabolism) in mammalian cells (Boscá et al., 1985; Gekakis et al., 1989). PFK
converts fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (black arrow, Figure
1). Glucose utilization by early preimplantation mouse embryos seems to be regulated by
PFK (Gardner and Leese, 1988).
1 Figure 1. The Glycolytic Pathway. This pathway converts one molecule of glucose into two
molecules of pyruvate. The black arrow shows the regulating step of the pathway where
phosphofructokinase converts fructose-6-phosphate into fructose-1,6-bisphophate. The black stars
demonstrate the important enzymes added to the PFK enzyme assays to detect the production of
NAD+ when PFK is present in the reaction. Aldolase and triose phosphate isomerase are
responsible for the conversion of fructose-1,6-bisphophate into two molecules: dihydroxyacetone
phosphate and glyceraldehyde-3-phosphate. Glyceradehyde-3-phosphate dehydrogenase is the
enzyme responsible for the conversion of glyceraldehydes-3-phosphate into 1,3bisphosphoglycerate which produces NADH + H+. (Bettelheim, Brown, March, Introduction to
General, Organic and Biochemistry, 6/e and Introduction to General, Organic and Biochemistry,
4/e. Figure 26.8.)
2 PFK is an allosteric tetramer comprised of 4 protein subunits coming from 3
different gene products, each named for the tissue in which the isoform predominates.
However, any given tissue can express more than one isoform. A letter has been assigned
to each isoform by Tsai and Kemp: PFK-muscle (A), PFK-liver (B), and PFKbrain/platelet/placenta (C) (Tsai and Kemp, 1973). In human models, however, PFK-M
and PFK-L have been assigned for muscle and liver and PFK-C, PFK-P or PFK-F has
been assigned for the brain/platelet/placenta isoform (Kahn et al., 1979; Vora et al.,
1981). For this project, the isoforms will be referred to as PFK-A, PFK-B, and PFK-C.
The three isoforms form tetramers composed of 4 protein subunits (Tsai
and Kemp, 1973) creating a holoenzyme (Foe and Kemp, 1985; Tsai and Kemp, 1973;
Tsai and Kemp, 1974). The composition of the subunits can be all one type of protein, or
a combination of multiple protein subunits. For example, 2 PFK-muscle protein subunits,
1 PFK-liver protein subunit, and 1 PFK-brain protein subunit can form a functional PFK
enzyme, or the functional PFK enzyme can be composed of 4 PFK-brain protein
subunits. Enzymatic activity of functional PFK is determined by the composition of the 4
PFK protein subunits expressed in the tetramer because of differences in substrate affinity
and allosteric regulation between the different isoform subunits (Shirmer and Evans,
1990; Tsai and Gonzales, 1975; Vora et al., 1981). Regulation of PFK and glycolytic
metabolism is important for normal, healthy development of the preimplantation mouse
embryo.
PFK is allosterically regulated. This involves many changes in its activity that are
a result of PFK structural changes. These changes occur in the presence of activator and
3 inhibitor molecules that cause increases or decreases in activity, respectively, when
bound to specific sites that are away from the active site (Lowry and Passonneau, 1966).
ATP and citrate are strong allosteric inhibitors while AMP is a strong allosteric activator
of PFK activity (Bloxham and Lardy, 1973; Foe and Kemp, 1985). In early
preimplantation embryos, it is thought that PFK activity is repressed/inhibited through the
8-16-cell transition either post-transcriptionally, post-translationally or by gene
transcription repression so the utilization of glucose is prevented (Lowry and Passonneau,
1966; Barbehenn et al., 1974; Howlett and Bolton, 1985; Schultz, 1993; Paynton and
Bachvarova, 1994). Gene repression includes the addition or deletion of regulatory
molecules to inhibit the gene from being transcribed. Regulation of PFK expression
results in glycolytic pathway repression since PFK regulates the pathway and is the main
point of the glycolysis blockade during early embryo development (Barbehenn et al.,
1974).
Developing mouse embryos do not initially run the glycolytic pathway so they
must gain energy using the TCA cycle until they reach the glycolysis transition between
the 8- and 16-cell stages. The TCA cycle is driven by maternal stores of pyruvate,
lactate, and glutamine present in the oviduct and uterine horn (Brinster, 1965; Brinster
and Thomson, 1966; Chatot et al., 1990; Leese and Barton, 1984; Martin and Leese,
1995; Wales and Whittingham, 1973). During early embryo development, allosteric
inhibitors and activators change concentrations within the embryo and its environment to
allow for PFK to function in the glycolysis pathway and remove the blockade. Although
the timing of activation of PFK and glycolysis is understood in the embryo, the identity
4 of the PFK isoform(s) and the pattern of RNA transcription and protein translation of
PFK have not been completely characterized. Since PFK is the major regulator of
glycolysis and its regulation and expression have a major impact on preimplantation
embryo development, characterizing its pattern of expression is important for both in vivo
and in vitro development and could have broader implications for human embryo
development.
Multiple preimplantation embryo studies using animal models have been useful in
understanding the sensitivity of embryos to their environment and the effects on their
growth and development, pre- and postnatally. Often, murine research can be applied to
humans because they have multiple systems and proteins in common. Murine models are
used to study many developmental concepts that can later be applied to human research.
The impact of the in vivo research proposed can be applied to in vitro mouse and human
models, and more importantly to in vitro fertilization (IVF) research and development.
The IVF success rate for an individual is between 11%-50% (The Jones Institute
for Reproductive Medicine: Eastern Virginia Medical School). According to multiple
reproductive specialists (The Jones Institute for Reproductive Medicine; Huntington
Reproductive Center Medical Group; Advanced Fertility Center of Chicago), IVF success
rates depend on many factors including the age of the woman, the normalcy of the uterus,
the semen quality, the number of embryos transferred, the adequacy of the luteal phase
after transfer, and the factor most important to this research, success or failure of
fertilization and cleavage in vitro. Embryo development in vitro causes the blastocyst
stage embryo to be retarded up to 24 hours compared with those in vivo. The retardation
5 is associated with slow cleavage rates and perturbation of activities associated with
metabolic pathways (Bowman and McLaren, 1970).
Understanding the control of expression of PFK at different developmental stages
in the murine embryo may help researchers to continue to optimize IVF conditions in
human systems and ultimately increase the success rates. Since PFK is a major regulatory
enzyme in preimplantation mouse and human embryo metabolism, it is important to
determine when the mRNA and protein are expressed during development.
Understanding details of embryo expression of this important enzyme in the mouse
embryo will allow researchers to better understand development for IVF in humans.
In our laboratory, Henry (Master’s Thesis, BSU, 2006) began the characterization
of preimplantation mouse embryo PFK gene expression by using RT-PCR to determine
the developmental timing and isoform specific expression patterns of PFK mRNA. In this
study, specific primers for each PFK isoform, PFK-A, B, and C, were designed and used
in RT-PCR to characterize pfk gene expression patterns during different stages of
preimplantation mouse embryo development using GAPDH as an internal control. A
planned contrast method was used to analyze the data based on literature that suggests
that glycolysis activation occurs at the 8-cell/morula stage transition (Brinster and
Thomson, 1966; Leese and Barton, 1984; and Gardner and Leese, 1986).
Henry (2006) determined that PFK-B mRNA is the only isoform present in
preimplantation mouse embryos. PFK-B RNA was detected from the 2-cell through
blastocyst stages. He observed a continued increase throughout development with a
significant increase (planned contrast) in mRNA expression between the 8-cell and
6 morula transition. PFK-B mRNA was not present in the unfertilized egg and only trace
amounts were seen in 1-cell embryos. There was also a significant increase in PFK-B
expression between the UFE stage and 2-cell stage, which corresponds to zygotic gene
activation (Latham et al., 1992).
Studies suggest that PFK is not enzymatically active in the mouse embryo until the 8-cell
stage when glycolysis begins (Brinster, 1965; Brinster and Thompson, 1966). Although
Henry’s work has demonstrated that mRNA transcription of PFK is initiated by the 2-cell
stage, the translation of PFK RNA into protein may be inhibited. The goal of this thesis
project was to characterize the presence of PFK-liver (B), muscle (A) and brain (C)
protein isoform patterns throughout mouse preimplantation development stages using
indirect immunofluorescence and laser confocal microscopy. This study was designed to
determine which PFK protein isoforms are present and if the proteins are expressed at the
1-2-cell stage as soon as the mRNA is present or if the proteins are posttranscriptionally/
translationally regulated and not synthesized until the 8-cell stage when glycolysis
begins. PFK enzyme assays were also developed using fructose-1,6-bisphosphate to
generate a standard curve and to develop a positive control using mouse liver extracts to
optimize concentrations of glycylglycine buffer and ATP in the reaction. This will allow
future embryo assays to be performed to confirm the glycolysis blockade. The original
hypothesis for this study was that the liver PFK isoform, PFK-B, would be present at the
2-cell stage but would show a significant increase in protein expression at the 8-16-cell
transition and activity would be allosterically inhibited until glycolysis begins in the
preimplantation mouse embryo. 7 LITERATURE REVIEW
Preimplantation Mouse Embryo Development
The joining of the egg and sperm to form pronuclei begins the development of
the mouse embryo. This fusion creates a zygote and the 1-cell stage of development.
Relative to other non-mammalian vertebrate embryos, the mouse embryo is relatively
slow to develop. Cleavage divisions of the cells occur without an increase in size while
the embryo moves toward the uterus in the oviduct. The second cell division and each
division following until the blastocyst stage is an asynchronous division. The blastocyst
attaches to the uterine epithelial wall and then implantation occurs about 4.5 days post
fertilization which allows the uterine tissue time to prepare for the embryo (Gilbert,
2000).
Before fertilization, the male and female pronuclear genomes are transcriptionally
inactive (Pacheco-Trigon, 2002). Growing mammalian oocytes must synthesize and
accumulate maternal compounds, such as transcripts, proteins and organelles during
oogenesis (Shultz, 1993). These accumulations are essential maternal contributions
toward supporting early embryo development until the 2-cell stage.
During the preimplantation development, maternal mRNA is stored during
oogenesis and is translated to provide the developing embryo with essential proteins for
cell division and differentiation (Bachvarova et al., 1989; Shultz, 1993). The growing
8 oocytes synthesize and accumulate mRNAs, proteins, and organelles under the direction
of the maternal genome that will be critical to sustain the 1-cell embryo (Shultz, 1993).
Translation of maternal mRNA into proteins prepares the 1-cell embryo for
differentiation and division. The embryo relies largely on maternal protein and RNA
synthesized during oogenesis up to the 2-cell stage. After 24 hours, the first cell division
occurs and nucleases begin to degrade maternal mRNA and the embryo must begin
transcribing its own mRNA and translating it into protein. (Teleford et al., 1990). At this
point, many embryonic genes begin to be transcribed (Leese and Barton, 1984) and 90%
of maternal mRNA is degraded (Martin and Leese, 1995; Whales and Whittingham,
1973; Barbehenn et al., 1974).
Zygotic Genome Activation:
Before fertilization, the male and female pronuclear genomes are transcriptionally
inactive (Pacheco-Trigon, 2002). Growing mammalian oocytes must synthesize and
accumulate maternal compounds, such as transcripts, proteins and organelles during
oogenesis (Schultz, 1993). These accumulations are essential maternal contributions
toward supporting the early embryo development until the 2-cell stage. The embryonic
genome soon becomes capable of transcription at the end of the 1-cell stage to begin the
minor zygotic gene activation. The initial activation is followed by the major zygotic
genome activation (ZGA), which is a burst of translation-coupled zygotic transcription at
the 2-cell stage (Latham et al., 1992). The maternal to zygotic genome transition is
important to initiate: 1) degradation of maternal signals that allowed for oocytes and 1-
9 cell embryos remain healthy and differentiate and 2) activation of novel zygotic genes
that are required for further differentiation and growth of the embryo (Schultz, 2002).
ZGA allows the oocytes and early embryo to replace maternal transcripts and
generate novel transcripts that are required prior to implantation (Schultz, 1993). ZGA is
the first critical event in early embryo development; however, little is known about the
variety of genes that are activated and the molecular basis for ZGA. ZGA begins with the
activation of a few genes at the early G1 phase of the 2-cell stage (Conover et al., 1991;
Latham et al., 1991). By the 2-cell stage in mouse embryo development, more than 90%
of the maternally derived RNA has been degraded so the transcripts must be replaced by
zygotic transcripts some time in development (Schultz, 1993). During ZGA,
reprogramming of gene expression occurs which is likely the molecular basis for the
transformation of the differentiated oocyte into totipotent blastomeres throughout early
preimplantation development (Ma, 2001). ZGA must occur for continued development
because mRNAs common to both the oocyte and the embryo are replenished and new
genes are expressed for the first time (Bultman, 2006).
Compaction
Committed cellular assignment and differentiation begins at a stage in
development involving a process called compaction (Johnson et al., 1984). The process
of compaction refers to an embryo’s progression from loosely associated blastomeres to a
tightly packed spherical embryo (Sutherland, 1983). At the 8-cell stage of development,
the blastomere surfaces start to flatten and move closer to one another. This process
causes blastomere compaction and allows for the formation of tight junctions between the
10 external surfaces to create outer and inner environments and gap junctions between the
internal surfaces of the cells to allow for easier cellular communication that is important
for normal development (Lee et al., 1987).
Gap Junctions
The gap junctions allow the embryo to utilize cell-to-cell communication until
around the time of implantation (Kalimi and Lo, 1988) and consist of a pattern of paired
hemichannels known as connexons (Bruzzone et al., 1996). Pairs of connexons from
plasma membranes in different cells form an end-to-end alignment that allows for small
molecules to pass through intercellular channels from cell-to-cell. The junctions are
formed when the cells move to a polarized arrangement so the external blastomeres are
exposed to the maternal environment and an internal embryonic environment is created
(Kalthoff, 1996).
Gap junctions are an important cellular structure because they maintain cellular
homeostasis by allowing cell to cell communication by spanning the plasma membrane of
two adjacent cells (Makowski et al., 1977. Gap junctions are formed by connexins that
form channels. The connexins are classified according to their molecular mass (Beyer et
al., 1987). Gap junctions with different sizes of connexins have different rates of
diffusion for different molecules (reviewed by Koval, 2002). The mRNA expression
patterns of the different types of connexins vary during the mouse preimplantation
development and the proteins seem to be translated shortly after the mRNA expression.
11 There seems to be some controversy regarding whether or not gap junctions are
important to preimplantation development. Becker et al. (1995) designed an anti-Cx43
antibody and used a technique to block communication. Anti-peptide antibodies were
designed and connected to dyes to observe cell-cell communication. The blocking was
effective and prevented the dyes from transferring between blastomeres of 8-16-cell
mouse embryos. The antibody treated blastomere decompacted and did not develop
further. Becker and Davies, 1995, concluded that gap-junction communication is
essential for proper compaction and embryo development. In spite of Becker et al.
(1995), Reaume et al. (1995) developed Cx43-null homozygous mutant embryos which
seemed to develop normally. So, controversy still remains about the importance and
requirements of gap junction intercellular communication among preimplantation mouse
embryos. In her review, F. D. Houghton (2005), suggests it is possible that the
development of gap junctions are essential for maximal efficiency to in vivo stresses such
as prolonged time in the fallopian tube or early implantation into the uterus. Gap junction
formations also ensure that cavitation occurs and the trophectoderm and inner cell mass
develop (Houghton, 2005). The trophectoderm allows for selective movement of required
molecules for the inner cell mass in and out of the cells ( Hewitson & Leese, 1993).
Tight Junctions
The tight junctions form between apical surfaces of external blastomeres near the
32-cell stage of development allowing blastulation to begin (Ducibella et al., 1975).
Blastulation involves tightly compacted blastomeres that divide evenly and form 2 layers
of cells; the inner cell mass (ICM) and the trophectoderm. The ICM cells have smooth
12 membranes that express the gap junctions. The ICM is surrounded by the trophectoderm
with tight junctions that further differentiates to form cytotrophoblasts, which form the
chorionic placenta, and syncytiotrophoblasts that invade the uterine wall and cause the
formation of the decidua (Gilbert, 2000).
Embryo Energy Metabolism:
The main source of energy used by murine embryos is pyruvate (Brinster, 1965;
Brinster and Thompson, 1966). Maternal pyruvate is utilized by the 1-cell to 8-cell
embryo while a limited amount of glucose is taken up and stored as glycogen. The
glucose is needed later in development so glycogen is used as a storage reserve (Pike and
Whales, 1982). Once the embryo has grown past the 8-cell stage, the uterine fluid can no
longer provide sufficient pyruvate to meet energy needs; therefore, the embryo must
make its own energy by utilizing glucose stores (Leese and Barton, 1984; Martin and
Leese, 1995; Barbehenn et al., 1978). Previous in vitro research has shown that the
glycogen stores are used if embryos (2-cell to 8-cell stages) are starved of pyruvate or
glucose. Reintroduction of pyruvate after the embryos were starved created a block for
the formation of the PFK end product, fructose-1,6-bisphosphate. This block shows that
the presence of pyruvate inhibits glycolysis during early preimplantation development
(Barbehenn et al., 1974).
After compaction, mouse embryo metabolism makes a switch from preferentially
utilizing the tricarboxylic acid (TCA) cycle to using a combination of glycolysis and
TCA cycle (Leese and Barton, 1984; Gardner and Leese, 1988). During the 8-cell to
morula transition, the embryo begins to metabolize glucose to carry out cellular
13 functions. There is an increase in the dependency upon glycolysis from the 8-cell stage
through the blastocyst stage of development (Brinster and Thompson, 1966; Leese and
Barton, 1984).
Preimplantation Embryo Protein Expression Patterns
Protein expression throughout preimplantation mouse embryo development has
not been widely researched. However, the utilization of glucose and cell survival of
preimplantation embryos has been studied resulting in patterns of some protein
expression. Glucose is transferred across cell membranes down concentration gradients
via facilitative glucose transporters, GLUT proteins. This is an energy independent
process. There are 13 different facilitative glucose transporters that have been divided
into three classes: class I contains GLUT1-4; class II contains GLUT5, 7, 9, and 11; and
class III consists of GLUT6, 8, 10, 12 and HMIT (H+ coupled myo-inositol-transporter)
(Joost and Thorens, 2001; Joost et al., 2002; Wood and Trayhurn, 2003). The GLUT
family of proteins exhibit sequence homology, but their substrate specificity, kinetic
characteristics, tissue and subcellular distribution, and response to extracellular stimuli
differ.
Previous research demonstrated that facilitative glucose transporters, GLUT1,
GLUT2 and GLUT3, were responsible for glucose transport in mouse preimplantation
embryos (Hogan et al., 1991; Aghayan et al., 1992). More recently, additional GLUTs
14 have been detected in preimplantation mouse embryos (Pantaleon et al., 1997; Moley et
al., 1998b), however the function of each of the transporters remains unknown.
The first glucose transporter to be characterized in preimplantation mouse
embryos was GLUT1 (Hogan et al., 1991; Aghayan et al., 1992; Morita et al., 1992).
GLUT1 mRNA is expressed from the 1-cell stage through the blastocyst stage and shown
to be presence in the pronuclei and nucleus of cleavage stage embryos using
immunofluorescent confocal microscopy (Pantaleon et al., 2001). GLUT1 is stored
predominantly in the cytoplasm until compaction in preimplantation mouse embryos. In
mouse blastocysts, GLUT1 was expressed on the basolateral and apical surfaces of the
trophectoderm (TE) cells, intercellular membranes (Aghayan et al., 1992) and the plasma
membranes of the inner cell mass (ICM) (Pantaleon et al., 1997). Pantaleon et al.
hypothesized that GLUT1 is responsible for transporting glucose into the inner cell mass
from the embryonic extracellular space in preimplantation mouse embryo development.
GLUT2 transcripts were detected at the 8-cell/compacted morula transition
(Hogan et al., 1991), however, the GLUT2 protein was not detected until the blastocyst
stage in mouse embryos (Aghayan et al., 1992; Schultz et al., 1992). Pantaleon et al.
(1997) suspect that GLUT2 is responsible for transport of glucose to the blastocoel
cavity. However, conflicting research by Mortia et al.(1992) and Tonack et al. (2004) did
not show evidence of GLUT2 expression.
GLUT3 transcripts were detected from the 4-cell stage through the blastocyst
stage of preimplantation mouse embryo development (Pantaleon et al., 1997). The protein
begins to be detected as early as the late 4-cell stage although immunoreactivity is weak
15 and the protein is stored in cytoplasmic vesicles. GLUT3 seems to remain in the vesicles
throughout the 6-8-cell stages and is then seen in the plasma membranes in early morula.
As the embryo develops into a blastocyst, expression of GLUT3 is localized on the apical
surface of the polarized TE cells. GLUT3 is thought to function as the primary
transporter of glucose from the maternal reproductive tract into the embryo.
PFK Protein Characteristics
PFK tetrameric enzymes are allosterically activated and inhibited by a variety of
metabolites (Kemp and Foe, 1983). Many allosteric behaviors of eukaryotic PFK
isozymes have been studied (Foe and Kemp, 1985; Raushel and Cleland, 1977; Reinhart
and Lardy, 1980; Goldhammer and Hammes, 1978; Bock and Frieden, 1976; Pettigrew
and Frieden, 1979), however, little is known about the structural basis responsible for the
allosteric regulation (Simon H. Chang and Robert G. Kemp, 2002). R. A. Poorman and
others (Poorman et al., 1984; Levanon, et al., 1989; Gehnrich et al., 1988; Li et al., 1994;
Heinisch et al., 1989; Currie and Sullivan, 1994) have reported the amino acid sequence
of rabbit muscle PFK and 11 other eukaryotic ATP-dependent PFK molecules to be a
tandem duplication of bacterial PFK, establishing an evolutionary relationship between
prokaryotic and eukaryotic PFK.
The allosteric enzyme, PFK, is has multiple protein subunits. Each subunit has a
designated name, depending on the species in which it is being studied. PFK-A (muscle),
PFK-B (liver), and PFK-C (brain/platelet/placenta) are the designated names of the
16 subunits in rodents, lagomorphs, and sheep (Tsai and Kemp, 1973). PFK-M, PFK-L, and
PFK-C have been designated for the human model subunits (Kahn et al., 1979; Vora et al,
1981). Hesterberg and Lee, 1981, found that PFK enzymes can exist as monomers,
dimers, tetramers, and oligomers (16mer) using theoretical simulations, sedimentation
velocities and coefficients, and electron microscopy. Specifically, rabbit PFK-A had the
following sedimentation coefficients: monomer = 4.95S, dimer = 7.6S, tetramer = 13.5S
and oligomer = 34.0S.
Although the enzyme is able to aggregate in multiple ways, the smallest
functional arrangement of the subunits is a PFK enzyme tetramer of 340,000 Da
(Bloxham and Lardy, 1973). PFK can be comprised of 1 isoform to form a homotetramer,
or 2 or all 3 isoforms to form heterotetramers. The enzyme is then named for the tissue
type in which it predominates. Using SDS-PAGE analysis and immunoblotting with
isoform-specific antibodies, Dunaway and others determined the individual sizes of each
mammalian isoform. They determined that PFK-B is 76,000 to 80,000 Da; PFK-A is
82,000 to 85,000 Da; and PFK-C is 86,000 to 87,500 Da in size (Dunaway and Kasten,
1985; Dunaway and Kasten, 1987; Dunaway et al., 1988). These different sizes and
structures of the PFK enzyme allows for many types of allosteric regulation.
PFK tetrameric enzymes are allosterically activated and inhibited by a variety of
metabolites (Kemp and Foe, 1983). Many allosteric behaviors of eukaryotic PFK
isozymes have been studied (Foe and Kemp, 1985; Raushel and Cleland, 1977; Reinhart
and Lardy, 1980; Goldhammer and Hammes, 1978; Bock and Frieden, 1979; Pettigrew
and Frieden, 1979), however, little is known about the structural basis responsible for the
17 allosteric regulation (Chang and Kemp, 2002). Poorman and others (Poorman et al.,
1984; Levanon et al., 1989; Gehnrich et al., 1988; Li et al., 1994; Heinisch et al., 1989;
Currie and Sullivan, 1994) have reported the amino acid sequence of rabbit muscle PFK
and 11 other eukaryotic ATP-dependent PFK molecules to be a tandem duplication of
bacterial PFK, establishing an evolutionary relationship between prokaryotic and
eukaryotic PFK. This suggests that eukaryotic PFK is double the size of prokaryotic PFK
(Poorman et al., 1984). Poorman also concluded from his theory of duplication, tandem
fusion, and divergence that PFK contains 6 possible organic ligand binding sites; (1)
ATP active site, (2) fructose-6-phosphate (F6P) active site, (3) adenosine nucleotide
deinhibitor site, (4) fructose-2,6-bisphosphate activator site, (5) ATP inhibitory site, and
(6) citrate/3-phosphoglycerate inhibitory site.
Even though the allosteric regulation ligand binding sites have been determined,
the entire mammalian PFK 3-D structure is still unknown. Changes in enzyme activity as
a result of ligand interaction and correlation to enzyme structure would be possible if the
PFK 3-D structure was available. Eukaryotic amino acid residues have been used to help
identify the PFK binding substrates using sequence analysis, site-directed mutagenesis,
and chemical modifications (Valaitis et al., 1987). Valaitis and others removed 17 amino
acids from the carboxy terminus of rabbit PFK-A by proteolysis using Staphylococcus
aureus V8 protease. This removal caused a reduced sensitivity to ATP inhibition. The
native protein was inhibited with 3mM ATP, 1mM F6P at pH 7.0. The shortened protein
was inhibited with an ATP concentration at almost 10 mM. Comparing Km values under
allosteric and non-allosteric conditions demonstrated that the affinity for ATP was
18 decrease at the inhibitory site. Under allosteric conditions at pH 7.0 with 10 µM ATP,
0.33 mol of ATP bound per mol protease-treated enzyme. The native protein had 0.61
mol bound per mol of enzyme. At pH 8.0, the native and protease-treated enzymes had
similar affinities for both F6P and ATP. This similarity at pH 8.0 suggested that ATP
affinity reduction by PFK occurred at the inhibitory site, not the active site (Valaitis et al.,
1987). Native PFK-A had a sigmoidal response to increasing levels of F6P when tested
for cooperativity with a Km of 0.7 mM. However, the shortened PFK changed from a
sigmoidal to a hyperbolic curve which suggests an increased enzymatic activity with a
decreased Km of 0.17 mM.
Protein Structure
Regulation of PFK Activity
The transfer of a phosphate group from ATP to F6P via the catalytic enzyme,
PFK, is the rate limiting step in glycolysis. This transfer results in ADP and fructose-1,6bisphosphate (F16BP) production. The allosteric regulation of PFK activity is affected
and regulated by many factors. Changes in the enzyme activity is a result of changes in
the structure of the enzyme caused by the presence of activator and inhibitor molecules
that change the distance between the F6P and ATP binding sites (Lowry and Passonneau,
1966). When an allosteric inhibitor binds to PFK, the conformation of the enzyme
switches to form a low affinity for the substrate (Schirmer and Evans, 1990). Most of the
studies conducted on effector molecules of PFK have used rabbit, sheep, and rat PFK
enzyme models.
19 Enzymatic activities of PFK are tested at pH 8.0 to 8.2. Sheep, rabbit and rat
PFKs are less sensitive to allosteric interactions above pH 8.0 (Lowry and Passonneau,
1966; Parmeggiani et al., 1966; Kemp, 1971; Boscá et al., 1985). If the pH is lowered to
7.0, PFK is more sensitive to allosteric interactions between the substrates and ligands.
One example of this is when rabbit PFK was subjected to conditions of 0.1 mM F6P at
pH 6.9 and 26°C, the enzyme was inhibited by ATP concentrations of 0.5 mM and above
(Parmeggiani et al., 1966).
Glycolysis and gluconeogenesis both involve PFK enzyme in liver tissue. In these
tissues, PFK co-exists with fructose-2,6-bisphosphatase which catalyzes the reaction
converting fructose-1,6-bisphosphate to F6P using hydrolysis (Marcus and Chatterjee,
1981). Hosey et al. (1980) have previously discovered that the enzymatic and molecular
properties among isolated livers in genetically diabetic mice and normal mice do not
differ (Marcus and Hosey, 1980), although, isolated diabetic livers seem to exhibit a
physiologically less active form of PFK (Reinhart and Lardy, 1980) and is more sensitive
to ATP inhibition compared to control animals. A decreased affinity of the enzyme to
F6P causes the increased ATP inhibition. In these studies, the genetically diabetic mice
(C57BL/KsJ-db) are used as a model for maturity-onset diabetes in human.
Reinhart and Lardy, 1980, discovered slightly varying pH causes significant
displacements in the substrate saturation curve and the fructose-6-P saturation curves of
liver PFK are extremely dependent on pH. Marcus and Chatterjee (1981) studied liver
PFK activity in mice. The assay pH ranged from 7.3 to 8.3. For each 0.1-unit increment
increase in pH, the activity values for F6P decreased by 0.2 to 0.3 mM. Muscle and heart
20 phosphofructokinase usually exhibit allosteric properties at a pH below pH 7.5, however,
Reinhart and Lardy (1980) and Marcus and Chatterjee (1981) presented work that in the
presence of 1mM ATP, liver PFK exhibits allosteric kinetics in assay conditions with pH
as high as 8.2. This shows that regulatory properties of PFK are dependent on the
occupancy of the ATP inhibitory site (Marcus and Chatterjee, 1981). PFK-B allosteric
properties can be observed at a high pH because of the increased sensitivity to ATP
inhibition compared to PFK- muscle and heart PFK.
Allosteric Effects of ATP on PFK
Pettigrew and Frieden (1979) used enzyme activity fluorescence titrations to
characterize feed-back inhibition of PFK by direct binding measurements and determined
the existence of two MgATP binding sites. Binding of MgATP to one site results in
fluorescence enhancement and binding of MgATP to the other site results in quenching.
The substrate MgGTP only results in enhancement and supports the enhancement is
associated with binding at the active site, and quenching is associated with binding at the
inhibitory site. Low (10 µM) and high (150 µM) concentrations of MgATP enhanced
PFK fluorescence with 0.5 mM F6P at pH 6.9 and 25°C. Only the low concentration of
MgATP quenched the fluorescent activity. ATP and MgATP are competitors for the
binding sites because both can bind to the inhibitory site or the active site. MgATP does
not bind to PFK at the active site when it acts as an inhibitor, unlike MgGTP which binds
to the active site when it enhances PFK activity.
Pettigrew and Frieden (1979) showed more feed-back control of PFK activity by
determining that the affinity for MgATP at the inhibitory site was less than the catalytic
21 site of rabbit PFK-A using direct binding assays and fluorescence titrations. Rabbit PFKA has different affinities for MgATP and natural forms (free) ATP that aren’t coupled
with Mg (Lowry and Passonneau, 1966; Pettigrew and Frieden, 1979) and the affinities
are affected by changes in pH and temperature. Free ATP bound 10-fold tighter than
MgATP. Pettigrew and Frieden (1979) also determined that a 20°C decrease in
temperature from 35°C to 15°C caused an increase in PFK affinity at the inhibitory site.
This also caused the dissociation constant (Kd) to decrease from 76 µM free ATP to 2.5
µM free ATP. Likewise, a drop in pH from 8.0 to 6.0 increased the affinity and Kd
decreased from 2 mM free ATP to 0.8 µM free ATP.
The concentration of Mg present in analysis of enzyme activity may cause a skew
in the results (Lowry and Passonneau, 1966). Lowry and Passonneau also found a 10-fold
increase in the binding of free ATP compared to MgATP at the inhibitory site of PFK
from sheep brain which results in greater PFK inhibition. Pettigrew and Frieden (1979)
also reported that even though free ATP seems to be a better inhibitor of PFK, MgATP
binds to the catalytic site with a higher affinity.
The presence of allosteric ligands modulates the affinity for ATP that PFK
displays. Pettigrew and Frieden (1979) found that the addition of AMP and FBP
increased the dissociation constant, Ki, from 14 µM to 90 µM and 14 µM to 140 µM ATP
respectively. They also concluded that addition of F6P activated PFK and increased the
Ki to 150 µM. These increases in ATP concentration that were required to inhibit PFK
activity by 50% characterize the decrease at the inhibitory site in ATP affinity. Lowry
22 and Passonneau (1966) and Parmeggiani et al. (1966) have shown similar results using
fluorometric and spectrophotometric analyses.
Activators of PFK
There are several ligands responsible for decreasing the Km of PFK for ATP and
F6P as substrates which cause a decrease in ATP inhibition (Lowry and Passonneau,
1966). A F16BP concentration of 0.10 mM causes PFK activity to increase 2-fold. Other
ligands acting as activators include: ammonium ions (NH4+), potassium ions (K+),
inorganics phosphate (Pi), 3’,5’-cyclic AMP (cAMP), and ADP (Lowry and Passonneau,
1966; Kemp, 1971). Lowry and Passonneau (1966) found PFK activity could be
increased by half-maximal velocity with a non-inhibitory 0.09 mM ATP concentration
when 0.25 mM of AMP, 10 mM of Pi, or 10 mM NH4+ were introduced. In addition, 20
mM K+, 0.09 mM ATP, and 0.21 mM F6P cause PFK activity to double and AMP, Pi or
NH4+ combined with an inhibitory ATP concentration (4.3 mM) resulted in a 4-fold
increase. Addition of each of the ligands mentioned caused a 2-fold increase in binding
affinity of ATP to PFK at the catalytic site and a 2-fold decrease at the inhibitory site.
Citrate is an additional ligand that alters PFK interactions. The TCA intermediate
seems to decrease PFK affinity for F6P, increase PFK affinity for ATP binding sites and
concomitantly decreases PFK activity varying with each isoform (Lowry and Passoneau,
1966; Kemp and Krebs, 1967; Tsai and Kemp, 1974; Pettigrew and Frieden, 1979; Foe
and Kemp, 1985). Using fluorometric analysis, Pettigrew and Frieden (1979) discovered
that a 2-fold weakened substrate affinity occurred in rabbit PFK-A when 250 µM citrate
(Kd = 75 µMF6P) was added versus no addition of citrate (Kd = 35 µM F6P). In the
23 presence of 0.5 mM citrate, the affinity of rabbit PFK-A for ATP decreased from 5-15
µM to 1.5-3.0 µM. ATP must be available to form a type of synergism in PFK inhibition
for citrate to affect PFK activity. Protein fluorescence assays performed by MartínezCosta et al. (2004) suggested that citrate causes a change in protein conformation by
observing changes in fluorescence of citrate binding to PFK.
PFK Kinetics
pH plays a major role in the sensitivity levels to allosteric regulation in PFK
activity. This role has varying degrees of effects among each isoform. The isoforms are
less affected by inhibitory concentrations of ATP in an alkaline environment, but Tsai
and Kemp (1974) determined that PFK-A is the most sensitive. They determined this
effect caused by pH on PFK-A activity is due to changes caused by creatine hydryolysis,
glycolytic flux, and lactic acid levels seen in vivo in muscle tissue. For example, the
inhibitory concentration of 1.9 mM of creatine phosphate only affected PFK-A. The
sensitivity of the muscle PFK isoform also greater to glycolytic and TCA intermediates
such as citrate and phosphoenolpyruvate (PEP). PFK-B did not show sensitivity to PEP
(Kemp, 1971). Foe and Kemp (1985) investigated all 3 isoforms from rabbit for
sensitivity to citrate inhibition. When conditions were identical, PFK-A showed the
greatest affinity (Ki = 100 µM), PFK-B showed this lowest affinity (Ki > 2000 µM), and
PFK-C showed an affinity in between (Ki = 750 µM).
PFK-B is the most sensitive to fructose-2,6-bisphosphate activation (K0.5 = 8.6
nM) (Dunaway and Kasten, 1985; Ishikawa et al., 1990; Gekakis et al., 1994). PFK-A
and PFK-C reacted less than PFK-B to fructose-2,6-phosphate with activation constant
24 values of 10 nM and 9 nM respectively. PFK-B was least sensitive to AMP, ADP and
cAMP due to a greater inhibitory effect of ATP on the isoform (Kemp, 1971; Tsai and
Kemp, 1974).
Activation of PFK in Preimplantation Mouse Embryos
Physiological concentrations of allosteric regulators are extremely dynamic
throughout preimplantation mouse embryo development. Citrate levels are extremely
high in the early embryo, but they remain constant from the 2-cell stage to the morula
stage (Barbehenn et al., 1974). These high levels of citrate are present due to the
utilization of maternal pyruvate utilizing the TCA cycle. Barbehenn et al. (1974) believe
that the citrate helps to regulate PFK activity until F6P concentrations increase 4-fold
from the 2-cell stage to the morula stage and release PFK inhibition. When high
concentrations of citrate are present, there is plenty of pyruvate available from outside
sources entering the TCA cycle so the embryo does not need to utilize glucose and
metabolize it to pyruvate (Bloxham and Lardy, 1973).
Citrate causes inhibition of PFK activity and glycolysis because binding of citrate
to PFK causes a conformational change and PFK becomes incapable of transferring the
phosphate group from ATP to fructose-6-phosphate (Bloxham and Lardy, 1973). Citrate
levels remain similar between the morula and blastocyst stages, however, ATP decreases
35-60% in morula stage embryos and 30% more between the morula and blastocyst stage
transition (Barbehenn et al., 1974). These results indicate that a lower ATP concentration
is involved with activating PFK and embryo utilization of glucose rather than citrate
(Barbehenn et al., 1978).
25 RESEARCH METHODS
The current research was designed to determine whether the translation of the
PFK-liver specific protein is inhibited, even though mRNA transcription of PFK-B is
initiated by the 2-cell stage. PFK-liver specific protein isoform expression was examined
using in vivo isolated embryos at each stage of preimplantation mouse embryo
development from unfertilized eggs through blastocyst, but most importantly between the
2-cell stage and 8-cell stage, using an anti-PFK-liver specific antibody for indirect
immunofluorescence and laser confocal microscopy. Muscle and brain/platelet/placenta
PFK protein isoform expression patterns were also evaluated using similar antibody
staining. Standard curves and positive controls for enzyme assays using fructose-1,6bisphosphate and mouse liver, respectively, were developed to allow future examination
of the enzyme activity of preimplantation mouse embryo PFK.
Embryo Isolation
The animal handling protocol for this research was approved by BSU ACUC, approval
#105054-5, through January 14, 2012. The reproductive cycle of female NSA strain mice
(Harlan Sprague Dawley, Indianapolis, IN) was primed with an initial injection of 10 IU
of pregnant mare’s serum gonadotropin (PMS) (Calbiochem, San Diego, CA) (diluted
1:10 from a 1000/IU/ml stock) between 11 a.m. and 2 p.m. After 48 hours, the female
26 mice were injected with human chorionic gonadotropin (hCG) (Sigma-Aldrich, St. Louis,
MO) (diluted 1:20 from 1000 IU/ml stock) and then mated overnight with B6SJLF1/J
males (Jackson Laboratories, Bar Harbor, ME). Lyophilized hormones (PMS and hCG)
were reconstituted in 0.9% sterile saline (0.9 g NaCl/100ml sterile H20) and diluted in
sterile tissue culture water (Sigma-Aldrich).
At various times after the hCG injection, the female mice were sacrificed by
cervical dislocation and unfertilized eggs (UFE), 1-cell, 2-cell, 8-cell, morula and
blastocyst embryos were flushed from the oviducts or uterine horns. UFEs and embryos
were flushed from the oviducts (UFE, 1-cell, and 2-cell stages) using a 30 gauge needle
and 1cc syringe, the oviducts and uterine horns (4-cell and 8-cell stages) with a blunt 30
gauge needle and a 1cc syringe, or the uterine horns (morula and blastocyst stages) with a
beveled 27 gauge needle and 1cc syringe using Hanks Balanced Salt Solution (5.37 mM
KCl; 0.441 mM KH2PO4; 0.137 M NaCl; 0.336 mM Na2HPO4; 5.55 mM glucose; 1.26
mM CaCl2; 0.492 mM MgCl2·6H2O; 0.406 mM MgSO4·7H2O) with 0.4% Bovine Serum
Albumin (BSA) (0.4 g BSA/100ml HBSS) (HBSS + BSA).
UFE (isolated without mating) and 1-cell embryos were isolated 24 hours post
hCG injection, 2-cell embryos at 48 hours post hCG injection, 4-cell embryos at 60 hours
post hCG injection, 8-cell embryos at 72 hours post hCG injection, morula stage embryos
at 84 hours post hCG injection, and blastocyst stage embryos at 96 hours post hCG
injection. UFEs and 1-cell embryos were washed in hyaluronidase (0.1% hyaluronidase
in 1.0 mM polyvinyl pyrollidine (PVP)) to help remove the cumulus cells surrounding the
embryos. The viable embryos were washed in HBSS+BSA. The embryos were fixed in
27 drops of 4% paraformaldehyde (4 g paraformaldehyde/100ml H20) for at least one hour
but no longer than 5 days at 4°C, prior to immunostaining.
Immunofluorescence Staining
Within five days, fixed embryos were washed in five drops of HBSS + 1% BSA +
1% normal goat serum (NGS). All drops used throughout the staining process were
between 25 and 50 µl. The plasma and nuclear membrane were permeabilized so
antibodies could enter the cell using 0.5% Triton X-100 in HBSS + 0.8% BSA for 30
minutes at room temperature. The embryos were blocked in HBSS + 1% BSA + 1% NGS
(blocking solution) for 1 hour at room temperature to remove any non-specific binding.
The blocking was followed by a 1 hour incubation at room temperature with a primary
antibody specific for one of the PFK protein isoforms diluted 1:50 in HBSS + 1% BSA +
1% NGS blocking solution. Purified rabbit polyclonal anti-PFK-liver IgG (Abgent,
#AP8136b, San Diego, CA) binds specifically to PFK-B isoform proteins. Purified rabbit
polyclonal PFK-muscle IgG (Abgent, #AP8137b) was the primary antibody used to
detect PFK-A and purified rabbit polyclonal PFK-brain/platelet/placenta IgG (Abgent,
#AP8135a) was the primary antibody used to detect PFK-C in the preimplantation mouse
embryos.
Following incubation with the primary antibody embryos were washed 3 times, 5
minutes each, at room temperature in HBSS + 0.1% BSA to remove non-specific binding
of the antibody, and then were incubated in Hoechst 33258 (1:100 dilution in HBSS +
BSA) for 15 minutes at room temperature to stain the nuclei of the embryo cells. The
embryos were washed again 3 times, 5 minutes each, at room temperature in HBSS +
28 0.1% BSA, and then incubated in goat anti-rabbit IgG secondary antibody conjugated to
Alexa Fluor 488 dye (Invitrogen, #A11008, Carlsbad, CA) (1:100 dilution in blocking
solution) for 1 hour at room temperature to attach to the primary antibody so fluorescence
could be seen where the proteins were present. The embryos were washed again in dilute
blocking solution, and then mounted on a poly-DL-lysine coated coverslip to help the
embryos adhere to the coverslip. The excess solution was drawn away and the coverslip
was inverted and placed onto a slide (previously cleaned with 70% ethanol) with a drop
of Vecta Shield Mounting medium (Vector Labs Inc., Burlingame, CA) to prevent
photobleaching. The coverslips were sealed with clear nail polish and stored in the dark
at 0 to -20ºC until analysis using a Zeiss Pascal microscope for laser confocal
microscopy. Identical procedures were used to stain embryos with primary antibodies
against PFK muscle and brain/platelet/placenta isoforms. The negative control was
stained with the secondary antibody in the absence of the primary antibody to see the
minimum background fluorescence. The positive control was stained with a rabbit anticellular actin polyclonal antibody to confirm successful staining because actin is known
to be expressed during preimplantation embryo development.
Quantitative Fluorescence:
Confocal laser scanning microscopy (LSM) is a widely accepted technique that is used to
scan applications that utilize fluorescence and provide three dimensional images by
combining images of individual optical slices through cells and other specimens. The
laser confocal microscope takes images through z-sectioning. The labeled slides were
viewed with a Zeiss Pascal confocal microscope using constant optimized microscope
29 settings for detector gain, amplifier offset, amplifier gain, and pinhole size (Turney et al.,
1996). The levels of the settings were optimized to the negative controls for minimum
staining. The following were the imaging parameters used:
Hoechst: Ch 2; Excitation--HFT 405/488/543; Mirror--BP 420-480
Alexa 488: Ch 2; Excitation--HFT 405/488/543; Mirror--BP 505-530
Beam Splitters: MBS: HFT 405/488/543; DBS1; Mirror
Wavelength: T1: 405 nm, 5%; T2: 488 nm, 3%
Filters: Ch2-1: BP 420-480; Ch2-2: BP 505-530
Pinhole: Ch2-1: 60 um; Ch2-2: 74 um
All embryos were imaged with the same C-Apochromat 40x/1.2 water immersion
objective lens (Zeiss). Z-sectioning and lateral resolutions were optimized according to
Nyquist sampling criteria. Embryo fluorescence intensity was determined by Zeiss image
analysis software on a scale from 0-2400 (arbitrary units) and was normalized by
subtracting the average background pixel intensity from each embryo image from the
actual average embryo pixel intensity. For a given stage of development, embryo pixel
intensities for all embryos were averaged, standard error of the mean (SEM) was
determined, data was plotted, and comparisons were drawn between stages of
development by Student’s t-test.
Fluorescent analysis of embryos at each stage and for each antibody was
replicated 2-5 times. The staining procedures were performed throughout one day without
30 any overnight incubations. There were between 5 and 20 embryos stained per primary
antibody or negative control for each replicate. The pixel intensities were acquired using
a drawing tool in the Zeiss imaging program to draw boundaries around the cells of the
embryos without including the zona pellucida. The background pixel intensities were also
acquired using the drawing tool to create boundaries around the outside of the entire
embryo to create a sample with a similar area to the embryo being measured.
Phosphofructokinase Enzyme Assays
An established PFK assay (Kemp, 1975; Ogushi et al., 1990) using mouse liver
was used to produce a standard curve for PFK enzyme assays to be performed in the
future. The livers were dissected from the mice used in the immunofluorescence
experiments. The liver was homogenized in 50 mM Tris-phosphate (pH 8.0), 30 mM
potassium fluoride, 10 mM EDTA, 0.1 mM ATP, and 0.1 mM DTT. Initially, 2 ml of
homogenizing buffer was added for every 1 g of mouse liver. After the liver was
homogenized with a teflon pestle on ice, an additional 1 ml of homogenizing buffer was
added and the sample was rehomogenized. The extract was centrifuged at 33,000 x g for
30 minutes at 4°C. The pH of the extract was adjusted to 8.5 with 1 M Tris base. The
extract was frozen at 4°C in an eppendorf tube for use in the enzyme assays.
The PFK enzyme reaction was run in the presence of 1mM Glyclyglycine, 0.1
mM EDTA, 6 mM MgCl2, 4 mM ammonium sulfate, 2 mM dithiothreitol, 0.16 mM
NADH, 1 mM ATP, 1 mM fructose-6-phosphate, 0.6 units aldolase, 0.3 units
glycerophosphate dehydrogenase, 0.3 units triose-phosphate dehydrogenase, and 10 µl
homogenized liver extract (when necessary) at 26oC. Production of NAD from NADH is
31 indicative of the amount of PFK activity present in the embryo. The production of NAD
from NADH was determined by measuring the decrease of absorbance over time at 340
nM using the Tecan M200 Infinite Multimode Plate Reader. NADH absorbs light at 340
nM. The loss of NADH absorbance upon production of NAD indicates the amount of
fructose-1,6-bisphosphate that is generated by PFK. For each mole of fructose-1,6bisphosphate, 2 moles of NAD are generated. One unit of PFK activity catalyzes the
formation of 1 µmol of fructose-1,6-bisphosphate per minute. Control reactions were
optimized to determine the length of time for the linear phase of the assay. The positive
control included assays of mouse liver extracts for PFK activity. The negative control
was run in the absence of tissue extracts. Standard curves were run by placing increasing
amounts of fructose-1,6-bisphosphate, needed to drive the NADH to NAD conversion,
into the standard assay components in the absence of liver or embryo extract. PFK assays
were replicated 3 times.
32 RESULTS
In order to better understand the initiation of glucose metabolism in
preimplantation mouse development, the expression patterns for each PFK protein
isoform, PFK-A (muscle); PFK-B (liver); and PFK-C (brain) were analyzed using
isoform specific anti-PFK antibodies, immunofluorescence staining and laser confocal
microscopy. Individual embryos were analyzed by densitometry to determine
fluorescence intensity using imaging software. Corrected mean fluorescence intensity
values are displayed representing embryo intensities minus the background intensities.
Preimplantation Mouse Embryo Fluorescence Intensity
Negative and Positive Controls
Embryos at different stages of in vivo development were analyzed without a
primary antibody for negative controls (Table 1). The mean intensity for the 50 negative
control embryos was 301.74 ± 26.68 (SEM). The embryo shown in Figure 2 (A) shows a
negative control 8-cell embryo with an intermediate intensity of 308. The average area
for the negative control mouse embryos was 4711.79 µm2. Embryos from different stages
of in vivo preimplantation development were stained with an anti-actin antibody to
confirm that the embryos were staining. In Figure 2 (I) a blastocyst embryo that has been
stained with anti-actin antibody is shown.
33 PFK-B
Embryos at different stages of in vivo development were analyzed for the
presence of PFK-B specific protein isoform (Table 1). The mean PFK-B fluorescence
intensity for 17 UFEs stained with polyclonal anti-PFK-B antibody was 280.91 ± 18.96
(SEM) with a range from 175 to 427. The UFE shown in Figure 2 (B) shows an embryo
with an intermediate pixel intensity of 260. The mean intensity for the UFEs was slightly
lower than the mean negative control intensity which was 301.74. There was not a
significant difference between the negative control and the UFEs (p > 0.1 by student ttest). The average UFE area was 4342 µm2. The data is shown in Table 1.
The mean intensity for 30 1-cell mouse embryos stained with polyclonal antiPFK-B antibody was 356.57 ± 35.09, which was slightly higher than the mean negative
control intensity of 301.74. Intensities ranged from 97 to 869. Although there was a 1.3
fold increase in mean protein expression from the UFE to the 1-cell mouse embryos
stained with anti-PFK-B antibody there was not a significant difference (p > 0.1). The 1cell embryo shown in Figure 2 (C) shows an embryo with an intermediate intensity of
352. The average 1-cell embryo area was 2979 µm2. The data can be seen in Table 1.
The mean intensity for 40 2-cell mouse embryos stained with polyclonal antiPFK-B antibody was 688.25 ± 34.05 with a range from 403 to 1210. There was a 1.9 fold
increase in mean protein expression between the 1-cell and 2-cell embryo stages when
stained with anti-PFK-B antibody. There was a significant difference between the UFEs
and the 2-cell embryos (p < 0.01) and a significant difference between the 1-cell and 2cells embryos stained with the antibody (p < 0.01). Figure 2 (D) shows a 2-cell embryo
34 with an intermediate pixel intensity of 702. The average 2-cell mouse embryo area was
4052 µm2. The data is shown in Table 1.
The mean intensity for 45 4-cell mouse embryos stained with polyclonal antiPFK-B antibody was 700.16 ± 45.18 with a range from 141 to 1515. There was no
significant difference in the mean PFK protein expression from the 2-cell to 4-cell mouse
embryos stained with anti-PFK-B antibody (p > 0.1). However, there was a significant
difference between the UFEs and the 4-cell embryos. The 4-cell embryo in Figure 2 (E)
shows a 4-cell embryo with an intermediate intensity of 708. The average 4-cell mouse
embryo area was 4607 µm2. The data is shown in Table 1.
The mean intensity for 43 8-cell mouse embryos stained with polyclonal antiPFK-B antibody was 442.74 ± 33.71 with a range from 164 to 1317. There is a 1.6 fold
decrease in the mean PFK protein expression from the 4-cell to the 8-cell mouse embryos
that have been stained with anti-PFK-B antibody. There was a significant drop the in the
protein expression between the 4-cell embryo to the 8-cell embryo (p < 0.01). The 8-cell
embryo in Figure 2 (F) shows an intermediate intensity of 449. The average 8-cell mouse
embryo area was 4303 µm2. The data is shown in Table 1.
The mean intensity for 39 morula stage mouse embryos stained with polyclonal
anti-PFK-B antibody was 1035.92 ± 39.51 with a range from 317 to 1489. There was a
2.3 fold increase and a highly significant difference (p < 0.01) in the mean PFK protein
expression pattern from the 8-cell to morula stage mouse embryo stained with anti-PFKB antibody. The morula stage embryo in Figure 2 (G) shows an intermediate of 1012.
The average area for morula stage mouse embryos was 3609 µm2. The average area in
35 the morula embryos had a smaller mean area than the other stages of embryo
development. The smaller area could be a result of compaction that takes place at the late
8-cell stage throughout the morula transition. The data is shown in Table 1.
The mean intensity for 39 blastocyst mouse embryos stained with polyclonal antiPFK-B antibody was 1220.96 ± 86.77 with a range from 451 to 2764. There was a 1.2
fold increase and a significant difference (p < 0.05) in the mean PFK protein expression
from the morula stage embryos to the blastocyst embryos that were stained with antiPFK-B antibody. The blastocyst stage embryo in Figure 2 (H) shows an intermediate
intensity of 1246. The average blastocyst mouse embryo area was 6186 µm2. The data is
shown in Table 1.
Overall, there is no expression of PFK-B until the 2-cell stage in preimplantation
development. The expression increases slightly to the 4-cell stage and then there is a
slight decrease in expression at the eight cell stage. There is a large increase in expression
at the morula stage of development. The transition between the 8-16-cell stage is when
the embryo begins utilizing glycolysis. There was continued increase in expression in the
blastocyst mouse embryos. The PFK-B expression pattern in the preimplantation mouse
embryos is shown in Figure 3. There was localization of the PFK-B protein isoform
expression. PFK-B appears to be evenly distributed throughout the cytoplasm at 2-cell
and 4-cell stages but localizes more predominantly to the cortical cytoplasm at later
stages. A 3D image of a blastocyst with pixel intensity localization of PFK-B is shown
in Figure 4. PFK-B appears to be associated with large and smaller vesicles located in the
cortical cytoplasm.
36 Figure 2: Expression of PFK-B protein isoform in preimplantation mouse embryos.
Preimplantation mouse embryos at various stages were stained with rabbit antiPFK-B polyclonal antibody and goat anti-rabbit IgG conjugated to Alexafluor 488.
Embryos were imaged with a Zeiss laser confocal microscope and imaged with Zeiss
imaging software. Images are slices through the middle of the embryo. Mean
intensities are from embryo Z stacks. (A) Negative control 8-cell mouse embryo with
an intermediate intensity of 308. (B) Unfertilized egg with an intermediate pixel
intensity of 260. (C) 1-cell embryo with an intermediate pixel intensity of 352. (D) 2cell mouse embryo with an intermediate pixel intensity of 702. (E) 4-cell mouse
embryo with an intermediate pixel intensity of 708 (F) 8-cell mouse embryo with an
37 intermediate pixel intensity of 449. (G) Morula stage mouse embryo with an
intermediate pixel intensity of 1012. (H) Blastocyst stage embryo with an
intermediate pixel intensity of 1246. (I) Blastocyst mouse embryo that has been
stained with anti-actin antibody to confirm that the staining protocol was working
properly.
Table 1: Mean embryo area and pixel intensities of embryos stained for expression
of PFK- protein isoform.
n, number
of
embryos
mean
area,
µm2
mean
pixel
intensity
intensity standard
error of mean
(SEM)
negative
controls
50
4712
301.74
26.68
A
PFK-B UFE
17
4342
280.81
18.96
PFK-B 1-cell
30
2979
356.57
35.09
PFK-B 2-cell
40
4052
688.25B, C
34.05
PFK-B 4-cell
45
4607
700.16B, C
45.18
PFK-B 8-cell
43
4303
442.74B, C
33.71
B, C
PFK-B morula
39
3609
1035.92
39.51
PFK-B
blastocyst
39
6186
1220.96B, C 86.77
*A marks that there is not a significant difference between UFEs and the negative control
embryos (p > 0.1). B marks that there is a significant difference between the intensities of
the stage of embryo stained and the UFEs (p < 0.05). C marks that there is a significant
difference between intensities from the embryos in the previous stage tested (p < 0.05).
38 Figure 3: Developmental pattern of expression of PFK-B protein isoform in
preimplantation mouse embryos. (- cont) is the negative control with a mean pixel
intensity of 301.74 ± 26.68. (UFE) is the unfertilized eggs with a mean pixel intensity
of 280.91 ± 18.96. (1-c) is the 1-cell embryos with a mean pixel intensity of 356.57 ±
35.09. (2-c) is the 2-cell embryos with a mean pixel intensity of 688.25 ± 34.05. (4-c)
is the 4-cell embryos with a mean pixel intensity of 700.16 ± 45.18. (8-c) is the 8-cell
embryos with a mean pixel intensity of 442.74 ± 33.71. (mor) is the morula stage
embryos with a mean pixel intensity of 1035.92 ± 39.51. (blast) is the blastocyst
preimplantation mouse embryos with a mean pixel intensity of 1220.96 ± 86.77.
*A marks that there is not a significant difference between UFEs and the negative control
embryos (p > 0.1). B marks that there is a significant difference between the intensities of
the stage of embryo stained and the UFEs (p < 0.05). C marks that there is a significant
difference between intensities from the embryos in the previous stage that were stained
with anti-PFK-B antibody (p < 0.01).
39 Figure 4: 3D image of a blastocyst stained with anti-PFK-L to demonstrate the
localization of PFK-L protein isoform. The red arrows show examples of the
localization of the PFK-L protein.
PFK-A
Embryos at different stages of in vivo development were analyzed for the
presence of PFK-A specific protein isoform. The mean intensity for two 1-cell embryos
stained with polyclonal anti-PFK-A antibody was 366.5 ± 6.36 with a range from 362 to
371. The embryo shown in Figure 5 (B) shows the lower intensity of the two 1-cell
embryos with an intensity of 362. There is not a significant difference between the
intensities of the negative control embryos and the 1-cell embryos that were stained with
antibody (p > 0.1). The average area for the 1-cell PFK-A stained embryos was 3563.61
µm2. The data is shown in Table 2.
40 The mean intensity for five 4-cell embryos stained with polyclonal anti-PFK-A
antibody was 655.8 ± 30.29 with a range from 568 to 764. There was a significant
difference between the 4-cell embryos and negative control embryos as well as a
significant difference between the 4-cell embryos and the 1-cell embryos that were
stained with anti-PKK-A antibody (p < 0.1). The embryo shown in Figure 5 (C) shows a
4-cell embryo with an intermediate pixel intensity of 663. The average area for the 4-cell
anti-PFK-A stained embryos was 4592.23 µm2. The data is shown in Table 2.
The mean intensity for 10 8-cell embryos stained with polyclonal anti-PFK-A
antibody was 580 ± 62.4 with a range from 328 to 736. There was significant difference
between the negative control embryos and the 8-cell embryos (p < 0.01) However, there
was not a significant difference between the 4-cell embryos and 8-cell embryos that were
stained with anti-PFK-A antibody (p > 0.1). The embryo shown in Figure 5 (D) shows an
8-cell embryos with an intermediate pixel intensity of 605. The average area for the 8-cell
anti-PFK-A stained embryos was 4042.17 µm2. The data is shown in Table 2.
The mean intensity for two morula stage embryos stained with polyclonal antiPFK-A antibody was 439 ± 86.27 with a range from 378 to 500. There was not a
significant difference between the morula stage embryo and the negative control or the 8cell embryos stained with anti-PFK-A antibody (p > 0.1). The embryo shown in Figure 5
(E) shows the high intensity morula stage embryo with a pixel intensity of 500. The
average area for the morula stage anti-PFK-A stained embryos was 3501.66 µm2. The
data is shown in Table 2.
41 The mean intensity for 14 blastocyst stage embryos stained with polyclonal antiPFK-A antibody was 718 ± 69.13 with a range from 504 to 1334. There was a significant
difference between the blastocyst embryos stained with anti-PFK-A antibody and the
negative control embryos (p < 0.01). However, there was not a difference between the
morula and blastocyst embryos that were stained with anti-PFK-A antibody (p > 0.1).
The embryo shown in Figure 5 (F) shows a blastocyst mouse embryo with an
intermediate pixel intensity of 643. The average area for the blastocyst stage anti-PFK-A
stained embryos was 6815 µm2. The data is shown in Table 2.
Overall, there is no expression of PFK-A until the 4-cell stage in preimplantation
development. There is a decrease in expression at the 8-cell stage and morula stage.
There is another increase in expression at the blastocyst stage embryo. The 4-cell
embryos, 8-cell embryos and the blastocyst embryos are significantly different than the
negative control embryos. The PFK-A expression pattern in the preimplantation mouse
embryos is shown in Figure 6.
42 Figure 5: Expression of PFK-A protein isoform in preimplantation mouse embryos.
Preimplantation mouse embryos at various stages were stained with rabbit antiPFK-A polyclonal antibody and goat anti-rabbit IgG conjugated to Alexafluor 488.
Embryos were imaged with a Zeiss laser confocal microscope and imaged with Zeiss
imaging software. Images are slices through the middle of the embryo. Mean
intensities are from embryo Z stacks. Total magnification was 400X for each image.
(A) Negative control preimplantation 8-cell mouse embryo with an intermediate
pixel intensity of 308. (B) 1-cell mouse embryo that has been stained with polyclonal
anti-PFK-A IgG antibody with an intermediate pixel intensity of 371. (C) 4-cell
mouse embryos that was stained with polyclonal anti-PFK-A IgG antibody with an
intermediate pixel intensity of 663. (D) 8-cell mouse embryo that has been stained
with polyclonal anti-PFK-A antibody with an intermediate pixel intensity of 605. (E)
Morula stage mouse embryo that has been stained with polyclonal anti-PFK-A
antibody with an intermediate pixel intensity of 500. (F) Blastocyst embryo that has
been stained with polyclonal anti-PFK-A antibody with an intermediate pixel
intensity of 643.
43 Table 2: Mean embryo areas and pixel intensities of embryos stained for expression
of PFK-A protein isoform.
1-cell
n, number
of embryos
2
mean
area, µm2
3563.61
mean pixel
intensity
366.5
intensity
standard
error of
mean (SEM)
6.36
4-cell
5
4592.23
655.8A, B
30.29
8-cell
10
4034.17
580A
62.4
morula
2
3501.66
439
86.27
blastocyst
14
6815
718A
39.13
*A marks that there is significant difference between the embryos stained with anti-PFKA antibody and the negative control embryos (p < 0.01). B marks that there is a
significant difference between intensities from the embryos in the previous stage that
were stained with anti-PFK-A antibody (p < 0.05).
44 Figure 6: Developmental pattern of expression of PFK-A protein isoform in
preimplantation mouse embryos. (- cont) is the negative control with a mean pixel
intensity of 301.74 ± 26.68. (1-c) is the 1-cell embryos with a mean pixel intensity of
366.5 ± 6.36. (4-c) is the 4-cell embryos with a mean pixel intensity of 655.8 ± 30.29.
(8-c) is the 8-cell embryos with a mean pixel intensity of 580 ± 62.4. (mor) is the
morula stage embryos with a mean pixel intensity of 439 ± 86.27. (blast) is the
blastocyst preimplantation mouse embryos with a mean pixel intensity of 718 ±
39.13.
*A marks that there is significant difference between the embryos stained with anti-PFKA antibody and the negative control embryos (p < 0.01). B marks that there is a
significant difference between intensities from the embryos in the previous stage that
were stained with anti-PFK-A antibody (p < 0.05).
45 PFK-C
Embryos at different stages of in vivo development were analyzed for the
presence of PFK-C specific protein isoform. The mean intensity for six 1-cell mouse
embryos stained with polyclonal anti-PFK-C IgG antibody was 433 ± 36.04 with a range
from 301 to 490. The 1-cell embryos stained with the anti-PFK-C antibody were not
significantly different than the negative control embryos (p > 0.1) shown in Figure 7. The
embryo shown in Figure 7 (B) shows an intermediate 1-cell embryo with a pixel intensity
of 472. The average area for the 1-cell anti-PFK-C stained embryos was 3832.66 µm2.
The data is shown in Table 3.
The mean intensity for six 2-cell mouse embryos stained with polyclonal antiPFK-C IgG antibody was 570.5 ± 73.2 with a range from 323 to 745. The 2-cell embryos
stained with anti-PFK-C antibody were significantly different than the negative control
embryos (p < 0.05) and the 2-cell embryos were not significantly different than the 1-cell
embryos stained with anti-PFK-C antibody (p > 0.1). The embryo shown in Figure 7 (C)
shows a 2-cell embryo with an intermediate pixel intensity of 588. The average area for
the 2-cell anti-PFK-C stained embryos was 4724.52 µm2. The data is shown in Table 3.
The mean intensity for three 4-cell mouse embryos stained with polyclonal antiPFK-C IgG antibody was 865.33 ± 85.58 with a range from 746 to 988. The 4-cell
embryos stained with anti-PFK-C antibody were significantly difference from both the
negative control and the 2-cell embryos that were stained with anti-PFK-C antibody (p <
0.05). The embryo shown in Figure 7 (D) shows a 4-cell embryo with an intermediate
46 intensity of 862. The average area for the 4-cell anti-PFK-C stained embryos was
7021.27 µm2. The data is shown in Table 3.
The mean intensity for fifteen 8-cell mouse embryos stained with polyclonal antiPFK-C IgG antibody was 717.07 ± 65.7 with a range from 361 to 1061. There was a
significant difference between the 8-cell embryos stained with anti-PFK-C antibody and
the negative control (p < 0.05). There was not a significant difference between 8-cell
embryos and 4-cell embryos stained with anti-PFK-C antibody (p > 0.1). The embryo
shown in Figure 7 (E) shows a 8-cell embryo with an intermediate pixel intensity of 657.
The average area for the 8-cell anti-PFK-C stained embryos was 4506.37 µm2. The data
is shown in Table 3.
The mean intensity for six morula stage mouse embryos stained with polyclonal
anti-PFK-C IgG antibody was 548.83 ± 79.8 with a range from 314 to 768. The morula
stage embryos that were stained with anti-PFK-C antibody were significantly different
from the negative control (p < 0.05). There was not a significant difference between the
morula stage mouse embryos stained with anti-PFK-C antibody and the 8-cell embryos
stained with anti-PFK-C antibody (p > 0.1). The embryo shown in Figure 7 (F) shows a
morula stage embryo with an intermediate pixel intensity of 521. The average area for the
morula stage anti-PFK-C stained embryos was 3487.27 µm2. The data is shown in Table
3.
The mean intensity for seven blastocyst mouse embryos stained with polyclonal
anti-PFK-C IgG antibody was 387.57 ± 137.53 with a range from 86 to 1109. There was
not a significant difference between morula stage embryo stained with anti-PFK-C
47 antibody or negative control embryos (p > 0.1). The embryo shown in Figure 7 (G) shows
a blastocyst mouse embryo with an intermediate pixel intensity of 355. The average area
for the blastocyst anti-PFK-C stained embryos was 6332.69 µm2. The data is shown in
Table 3.
Overall, there was no expression of PFK-A protein isoform until the 2-cell stage
embryo. There was an increase in expression, with significant differences from the
negative control, in the 2-cell embryos and 4-cell embryos. There was a decrease in
expression from the 4-cell stage embryos to the 8-cell stage embryos and a continued
decrease to the morula and blastocyst stage. The 8-cell and morula stage embryos were
significantly different than the negative controls, however, the blastocyst stage embryos
were not. The PFK-C expression pattern in the preimplantation mouse embryos is shown
in Figure 8.
48 Figure 7. Expression of PFK-C protein isoform in preimplantation mouse embryos.
Preimplantation mouse embryos at various stages were stained with rabbit antiPFK-C polyclonal antibody and goat anti-rabbit IgG conjugated to Alexafluor 488.
Embryos were imaged with a Zeiss Laser confocal microscope and imaged with
Zeiss imaging software. Images are slices through the middle of the embryo. Total
magnification was 400X for each image. (A) Negative control preimplantation 8-cell
mouse embryo with an intermediate pixel intensity of 308. (B) 1-cell mouse embryo
that has been stained with polyclonal anti-PFK-C IgG antibody. (C) 2-cell mouse
embryos that has been stained with polyclonal anti-PFK-C IgG antibody. (D) 4-cell
mouse embryo that has been stained with polyclonal anti-PFK-C IgG antibody. (E)
8-cell mouse embryo that has been stained with polyclonal anti-PFK-C IgG
49 antibody. (F) Morula stage embryo that has been stained with polyclonal anti-PFKC IgG antibody. (G) Blastocyst mouse embryo that has been stained with polyclonal
anti-PFK-C IgG antibody.
Table 3: Mean embryo areas and pixel intensities of embryos stained for expression
of PFK-C protein isoform.
1-cell
n, number
of embryos
6
mean
area, µm2
3832.66
mean pixel
intensity
433
intensity
standard
error of
mean (SEM)
36.04
2-cell
6
4724.52
570.5A
73.2
4-cell
3
7021.27
865.33A, B
85.58
8-cell
15
4506.37
717.07A
65.7
morula
6
3487.27
548.83A
79.8
blastocyst
7
6332.69
387.57
137.53
*A marks that there is significant difference between the embryos stained with anti-PFKA antibody and the negative control embryos (p < 0.01). B marks that there is a
significant difference between intensities from the embryos in the previous stage that
were stained with anti-PFK-A antibody (p < 0.05).
50 Figure 8: Developmental pattern of expression of PFK-C protein isoform in
preimplantation mouse embryos. (- cont) is the negative control with a mean pixel
intensity of 301.74 ± 26.68. (1-c) is the 1-cell embryos with a mean pixel intensity of
433 ± 36.04. (2-c) is the 2-cell embryos with a mean pixel intensity of 570.5 ± 73.2. (4c) is the 4-cell embryos with a mean pixel intensity of 865.33 ± 85.58. (8-c) is the 8cell embryos with a mean pixel intensity of 717.07 ± 65.7. (mor) is the morula stage
embryos with a mean pixel intensity of 548.83 ± 79.8. (blast) is the blastocyst
preimplantation mouse embryos with a mean pixel intensity of 387.57 ± 137.53.
*A marks that there is significant difference between the embryos stained with anti-PFKC antibody and the negative control embryos (p < 0.01). B marks that there is a
significant difference between intensities from the embryos in the previous stage that
were stained with anti-PFK-C antibody (p < 0.05).
51 DISCUSSION
Preimplantation mouse embryos were isolated to analyze the protein expression
pattern of three PFK isoforms, PFK-A, B and C, throughout preimplantation
development. Henry’s research (Master’s Thesis) showed that mRNA expression of PFKA and PFK-C was not present in any stage of preimplantation development, however,
PFK-B isoform was present throughout preimplantation development with a significant
increase in expression at the 8-16-cell transition with a continued increase in expression
through the blastocyst stage. With the mRNA expression present before glycolysis begins
at the 8-cell stage, immunofluorescent staining and laser confocal microscopy were used
to determine whether the protein translation was suppressed until the 8-16-cell stages or
if the protein was translated at the 1-2-cell stage and allosterically inhibited until needed
at the 8-16-cell transition.
PFK-A and PFK-C
Jeff Henry’s research determined that PFK-A and PFK-C, muscle and
brain/platelet/placenta
isoforms,
mRNA
expression
did
not
exist
throughout
preimplantation mouse development; however, the current study demonstrated some
isoform specific PFK protein expression of both PFK-A and C at some stages of
52 development. These results suggest that there may be some cross reactivity with PFK-B
or something else, or a low specificity for the isoforms.
The mRNA was not present in any stage of preimplantation mouse embryo
development for PFK-A isoforms, although some protein expression occurred throughout
development. There was not a significant difference in expression PFK-A protein isoform
expression in negative controls and 1-cell mouse embryos. There was, however, a
significant increase in the expression of PFK-A protein isoform in 4-cell mouse embryos
stained with polyclonal anti-PFK-A antibody compared to the 1-cell embryos and
negative controls. 8-cell embryos stained with anti-PFK-A antibody showed a significant
difference from the negative control but there was not a significant increase compared to
the 4-cell embryos stained with the antibody. Morula staged embryos did not show a
significant difference when compared to negative controls or the 8-cells embryos stained
with polyclonal anti-PFK-A antibody. The blastocyst stage embryos stained with antiPFK-A showed a significant increase between negative control embryos but there was not
a significant increase between the blastocyst embryos and the morula embryos stained
with the antibody.
Henry’s research showed that there was not any PFK-A mRNA expression at any
stage of embryo development although further research suggests that PFK-A protein
isoform expression was significantly different (p < 0.05) than the negative control
embryos in three of five stages that were tested.
The mRNA for PFK-C isoforms also was not present in any stage of
preimplantation mouse embryo development, although some protein expression occurred
53 throughout development. The 1-cell embryos stained with polyclonal anti-PFK-C
antibody were not significant different than the negative controls which is consistent with
Henry’s research, however, the 2-cell embryos were significantly different (p < 0.05)
than the negative control embryos but not significantly different than the 1-cell embryos
that were stained with the anti-PFK-C antibody. The 4-cell embryos stained with the antiPFK-C antibody were significantly different (p < 0.05) than the negative control embryos
and the 2-cell embryos that were stained with the anti-PFK-C antibody. The anti-PFK-C
stained 8-cell embryos were significantly different than the negative control embryos but
not significantly different from the 4-cell embryos.
The specificity of staining with the PFK-A and C antibodies was questioned since
in some cases, there was a decrease in protein expression when the activity of the enzyme
increases. The PFK-A and PFK-C antibodies were generated in rabbits using a synthetic
peptide of human PFK. The sequence of amino acids is about 75% homologous to mouse
but it was never tested in mice (Abgent Technical Service Representative, personal
communication with C. Chatot). This could be a reason for the differences in the pattern
of mRNA expression of PFK-A and PFK-C and the pattern of protein expression of PFKA and PFK-C. There may have also been cross reactivity with the PFK-B isoform but this
is unlikely as the patterns of staining do not resemble that of PFK-B. A Western blot
would be a good method for confirming the specificity of the PFK-A and PFK-C
antibodies for the two isoforms.
The number of embryos stained with PFK-A and PFK-C protein specific
antibodies among developmental stages was extremely low (n=2 to n=15). This did not
54 allow for a large number of stained embryos pixel intensity to be averaged to find a mean
and standard error. The ranges for preimplantation mouse embryos stained with antiPFK-B protein specific antibody were quite large for most stages which was not possible
with the other two isoform specific antibodies with the low n.
PFK-B
PFK-B protein expression confirms Jeff Henry’s PFK-B mRNA expression
pattern. There was little if any expression in the UFE and 1-cell embryos with a gradual
increase until the 8-cell stage where the expression decreased. The PFK-B protein
expression showed a significant increase at the 8-16-cell transition. Since the PFK-B
isoform is present before the 8-cell stage when glycolysis begins, the protein is being
translated and synthesized as the message appears.
There is a continued increase in PFK-B expression between the morula and
blastocyst stage of the mouse embryo with the peak expression at the blastocyst stage
because the embryo makes a complete switch from only pyruvate oxidation to
predominately glycolytic based aerobic metabolism (Leese and Barton, 1984). Although
there is aerobic metabolism, the TCA cycle still functions but uses glucose to supply
pyruvate instead of relying on maternal sources of pyruvate. Martin and Leese (1995)
supported this by providing evidence that the mouse embryo decreased its uptake of
external pyruvate throughout preimplantation development with the lowest uptake at the
blastocyst stage because of the switch in metabolism. Other studies showed that glucose
55 uptake is the highest at the blastocyst stage when glucose is converted to glycogen to
create stores in preparation for the anoxic uterine environment (Pike and Wales, 1982;
Leese and Barton, 1984).
The protein expression of PFK-B in preimplantation mouse embryos prior to the
embryo utilizing glycolysis suggests that there is activity suppression of the protein rather
than translational control. If translational control was occurring, the protein would not be
present until it would be utilized at the 8-16-cell transition when glycolysis serves as an
additional type of embryo metabolism. Increased levels of citrate may be responsible for
the activity suppression. Enzyme assays will be used to confirm the activity suppression.
During the morula and blastocyst stages of preimplantation mouse development,
the PFK-B protein expression seems to be localized to vesicles in the embryo (Figure 4).
Another protein that seems to be a localized protein is caveolin. Caveolin is a protein
within the subdomain of the plasma membrane, caveolae, along with cholesterol and
glycosphingolipids (Anderson, 1998; Smart et al., 1999). Caveolae are oligomers of
caveolin protein isoforms about 50-100 nm that are used in lipid transport (Anderson,
1998; Engelman et al., 1999; Engelman et al., 1999(2); Fivaz, 1999; Glenney and Soppet,
1992) and as signal transduction complexes (Anderson, 1998; Bilderback et al., 1999;
Feron et al., 1998) such as G protein subunits (Engelman et al., 1998, Engleman et al.,
1998(2)), G protein-coupled receptors (Bickel et al., 1997; Bretscher et al., 1997), cellular
adhesion proteins (Fivaz et al., 1999), insulin receptor (Fiedler et al., 1993), and receptor
tyrosine kinases (Bilderback et al., 1997; Engelman et al., 1998). Vallejo and Hardin
(2004) observed that glycolysis was inhibited when caveolae were disrupted. This
56 discovery led them to use confocal microscopy to look at colocalization of caveolin-1 and
PFK in vascular smooth muscle cells. Cellular colocalization of caveolin-1 and PFK was
significant (0.68 ≥ R2 ≤ 0.77). They concluded that compartmentation of glycolysis was
in part due to the colocalization of PFK and caveolin-1. Other studies have reported that
caveolae contain proteins, such as PFK, that are involved in glucose metabolism (Scherer
and Lisanti, 1997; Lloyd and Hardin, 2001). Glycolytic enzymes bind to F-actin (Babbit
et al., 1997; Damke et al., 1994; Dietzen et al., 1995) and microtubules (Cao et al., 1998;
Das et al., 1999; Dobrwosky et al., 1995) and are found to be involved in coupling to
membrane ion channels with the plasma membrane (Brown and Rose, 1992; CasacciaBonnefil et al., 1996; Couet et al., 1997; Engelman et al., 1998). These studies combined
suggest that the glycolytic enzymes and intermediate metabolites do not mix freely within
the cytoplasm, but rather are localized on organelles of the cytoarchitecture. The
glycolytic enzymes near the surface of the plasma membrane play important roles in
cellular processes involving ATP derived from glycolysis. These include ATP-sensitive
potassium channels (Harder et al., 1998; Henley et al., 1998) and Na-K-ATPase activity
which are particularly important for mouse embryo development (Bist et al., 1997;
Fiedling et al., 1997; Fiedling and Fiedling, 1995; Galbiati, 1998). The activity of the NaK-ATPase in the basolateral membrane is responsible for the accumulation of Na+ and Clwhich control fluid movement between epithelial layers (Barcroft et al., 2002) by
controlling absorptive, secretory and ion concentrating capacity during blastocoel
formation. Membrane localization, shown in Figures 2 and 4, of PFK at the plasma
membrane may be due to the important role the enzyme plays in ATP-dependent
processes.
57 Future experiments to confirm activity of suppression at earlier stages will include
enzyme assays. These experiments will need to find the best way to optimize the number
of embryos and compare the blastocyst stage and with other stages of development.
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