DILANTIN AFFECTS THE RATE OF DNA SYNTHESIS VIA CYCLIN A AND
DECREASED CONCENTRATIONS OF DNA POLYMERASE δ IN PREIMPLANTATION
MOUSE EMBRYOS
A THESIS PROPOSAL
SUBMITTED TO THE GRADUATE SCHOOL
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE
MASTER OF BIOLOGY
BY
AUTUMN RENEE TOLLIVER
DR. C.L. CHATOT
BALL STATE UNIVERSITY
MUNCIE, INDIANA
DECEMBER 2013
i
Abstract
THESIS: Dilantin Affects the Rate of DNA Synthesis via Cyclin A and Decreased
Concentrations of DNA Polymerase δ in Preimplantation Mouse Embryos.
STUDENT: Autumn R. Tolliver
DEGREE: Master of Science
COLLEGE: Sciences and Humanities
DATE: December, 2013
PAGES: 78
Dilantin (DPH) is one of the most popular anticonvulsant drugs prescribed to women
with epilepsy. DPH causes fetal hydantoin syndrome (FHS) characterized by mental
retardation and growth abnormalities such as digit hypoplasia. DPH slows cell division
in preimplantation mouse embryos, extends S phase in the second cell cycle, and
deregulates cyclin A in G1, S, and G2 in the first, second and third cell cycles compared
to NaOH controls. To determine if DPH is altering rates of DNA synthesis in
preimplantation mouse embryos, this study examined the activity of DNA polymerase
during replication in S phase of the second cell cycle in DPH treated mouse embryos
compared to NaOH treated vehicle controls. DNA synthesis was measured by following
incorporation of the thymidine analog, EdU, into DNA over time, which was detected
using a fluorescent Alexa Fluor 488 azide stain. The results showed a 31% decline in
rate of DNA synthesis in DPH treated embryos compared to controls over a 40 minute
reaction period. The initial 0-5 min reaction time had an 84.5% decline in the rate of
synthesis in DPH treated embryos. These results support the hypothesis that when
DPH alters the expression of cyclin A in the second cell cycle then DNA replication
ii
activity decreases. This reduction may lead to the abnormal growth patterns attributed
to DPH in children with FHS.
iii
Acknowledgements
I would like to sincerely thank Dr. Clare Chatot for being an outstanding mentor and
friend during the completion of my thesis. I would especially like to thank her for her
helpful criticism, patience and complete support through out this journey. I would also
like to thank my committee members, Dr. Susan McDowell and Dr. Derron Bishop, for
the skills you have taught me that gave me the ability to complete my thesis and the
support you have given me. I would like to thank Department Chair Dr. Kemuel Badger
and his staff for always being so helpful whenever it was needed. Finally I would like to
thank my family and friends for the encouragement during this project.
iv
Table of Contents
Abstract ……………………………………………………………………………………… ii
Acknowledgements………………………………………………………………………..…iv
Table of Contents ……………………………………………………………………….…...v
Table of Figures ...…………………………………………………………………………...vi
Table of Tables ...……………………………………………………………………………vii
Abbreviations ...……………………………………………………………………………..viii
Introduction ...…………………………………………………………………………………1
Literature Review ...…………………………………………………………………………..5
Research Methods .………..……………………………………………………………..…23
Results...………………………………………………………………………………..…… 29
Discussion...……………………………………………………………………..…….……..42
References...………………………………………………….………………….…………..51
Appendix ...…………………………………………………………………………...………55
v
Table of Figures
Figure 1. Chemical structure of DPH and metabolites. ……………………………… 9
Figure 2. Reactive Intermediates Formed by Bioactivation of DPH by Prostaglandin H
Synthetase………………………………………………………………………………….12
Figure 3. DNA polymerase δ catalytic subunit p125: polymerase and exonuclease
Domains. ……………………………………………………………………………………19
Figure 4. Optimization of labeling conditions and timing for DNA synthesis in 2-cell
preimplantation mouse embryos…………………………………………………….…... 32
Figure 5. Controls for auto fluorescence and background fluorescence from EdU or
Alexa Fluor azide...…………………………………………………………………….….. 34
Figure 6. NaOH treated Embryos labeled in EdU at 5, 10, 20, and 40 min to
demonstrate DNA synthesis…………………………………………………………….….37
Figure 7. DPH embryos labeled in EdU at 5, 10, 20, and 40 min to demonstrate DNA
synthesis………………………………………………………………………….………… 38
Figure 8. Average Relative Fluorescence at 0, 5, 10, 20, 40 min time points in EdU for
NaOH and DPH Treated Embryos Nuclear Fluorescence – Cytoplasmic Background..41
vi
Table of Tables
Table 1. Embryo collection times, in vivo and in culture…………………………………..3
Table 2. Preimplantation Mouse Embryo Time Course of Average Corrected Nuclear
Intensity in DPH and NaOH Treated Embryos During S Phase During the Second Cell
Cycle. ...……………………………………………………………………………………….36
Table 3. Rate of DNA synthesis NaOH and DPH treated embryos was determined by
average relative fluorescence at each time point indicative of the amount of EdU
incorporated into the DNA per minute……………………………………………………..40
vii
Abbreviations and Definitions
BSA -Bovine Serum Albumin
CDK - CYCLIN DEPENDENT KINASE- Proteins related to cell cycle which requires an
associated cyclin in order to be active. Active cdk proteins cause the cell to move
between phases of the cell cycle by adding phosphate groups to a variety of proteins.
CZB – a medium used for embryo culture.
DPH - Dilantin; also called phenytoin (PTH)
EH – Epoxide Hydrolase- enzyme that detoxifies DPH arene oxide
EPR – Electron Paramagnetic Resonance; electron spin resonance
ETYA - 5,8,11,14-eicosatetraynoic acid; an inhibitor of PHS
FSH – Fetal Hydantoin Syndrome – a cluster of human abnormalities observed in
children of women taking Dilantin.
HBSS- Hank’s Balanced Salt Solution, a buffering solution
MVM - Minute Virus of Mice; a parvovirus
PCNA - proliferating cell nuclear antigen; a component of DNA Polymerase δ
PHS – Prostaglandin H Synthetase – an enzyme that metabolizes DPH to reactive
oxidative intermediate other than the arene oxide.
TERATOGEN - A drug or other foreign agent that causes birth defects in a fetus.
viii
Introduction
Dilantin (DPH), one of the most widely prescribed anticonvulsant drugs, is the
teratogen responsible for fetal hydantoin syndrome (FHS) (Oguni and Osawa, 2004).
DPH is also known as phenytoin (PHT; a 5,5,-diphenylimidazolidinedione) (Shih et al.,
2004). For pregnant women taking DPH, there is a 3-7% risk for major fetal congenital
malformations (Tomson and Battino, 2012) representing up to a 2-fold increase above
the normal rate of congenital abnormalities in an untreated population (Congenital
Malformations Registry, Department of Health, New York State, 2002). FHS is
characterized by mental retardation and growth abnormalities such as hypoplasia
(shortening) of the digits. If mothers with epilepsy do not take an antiepileptic drug then
the fetus is at risk of death, bradycardia (low heart beat), intracranial hemorrhaging
(blood vessels of the scull that have erupted), and heart beat abnormalities (Hanson
and Smith, 1975; Oguni and Osawa, 2004).
DPH disrupts normal fetal development in a number of mammalian species
including chicken (Temiz, 2007) rabbit (Danielsson et al., 1992) and mouse embryos
(Harbison and Becker, 1969; Waclaw and Chatot, 2004, Gonzalez and Chatot, 1993,
Blosser and Chatot, 2003). In humans, a cluster of defects associated with FHS have
been characterized, including: low birth weight, irregular growth of the distal phalanges,
facial dimorphisms that include a broad flat nasal bridge, upturned nose, wide bulging
lips, and physical and mental growth retardation (Oguni and Osawa, 2004). The
mechanisms behind this teratogen are still not known but theories support that a
1
reactive intermediate of DPH and not the parent drug itself causes the irregular growth
patterns resulting in FHS (Oguni and Osawa, 2004).
Previous work has demonstrated that DPH has had an effect on preimplantation
mouse embryo cell cycle progression, cyclin A expression, and length of second cell
cycle S phase. The cell cycle consists of four stages, G1, S, G2, grouped as interphase
and M. G1 is the phase where the cell prepares for DNA replication and is called the
gap phase (Johnson and Walker, 1999). This phase is where the cell will either continue
on with the cell cycle and DNA replication or terminate the cell cycle. S phase is the
stage where DNA synthesis takes place. G2, also known as the second gap phase, is
where the cell gets ready for cell division by growing, i.e. increasing cytoplasm,
organelle growth, replicating mitochondria and checking for damaged DNA. M phase
stands for mitosis. In mitosis, replicated chromosomes from S phase are separated into
two nuclei and cytokinesis occurs forming two daughter cells. G0 is when the daughter
cells have exited the cell cycle and remain dormant until the next cell cycle (Johnson
and Walker, 1999).
The length of the each phase of the cell cycle varies. In the first cell cycle of
preimplantation mouse development, G1 is 3-8 hours long, S phase is 6 hours, and
G2/M phase is 6 hours long totaling 15-23 hours (Krishna and Generosa, 1977). In the
second cell cycle, on average, G1 is 1.3 hours, S phase is 6.1 hours and 15.4 hours for
G2/M phase for a total of 22.8 hours, although in some mouse strains the second cell
cycle can be as long as 30 hours (Sawicki et al., 1981). Table 1 indicates the normal
timing used in the Chatot lab for collection of in vivo preimplantation mouse embryos.
For the experiments in this study, embryos were collected for S phase of the second cell
2
cycle starting at 2:30 AM on day 2 of development for labeling at 3:30 AM; i.e. 27.5
hours post-fertilization.
Table 1: Embryo Collection Times, in vivo and in Culture
Cell Cycle Stage
Embryo collection time in
Embryo collection time in
vivo
vitro
(post fertilization)*
(post fertilization)*
First
G1
3 hours
First
S
12 hours
11 hours
First
G2
18 hours
18 hours
Second
G1
21 hours
26 hours
Second
S
27 hours
30 hours
Second
G2
32 hours
33 hours
Third
G1
44 hours
50 hours
Third
S
47 hours
54 hours
Third
G2
48 hours
55 hours
*Fertilization was assumed to occur at 12:00 AM midnight.
Cyclins and cyclin dependent kinases (cdks) regulate the cell cycle. Different
cyclins accumulate and are degraded throughout the cell cycle resulting in differential
expression. This process is repeated at critical functional transitions in the cell cycle
(Sherr and Roberts, 1999) with cyclin degradation occurring via the ubiquitin proteolytic
pathway (Ciechanover et. al, 2000). The cell cycle progresses based on accumulation
of specific cyclins and their interactions with specific cdks. The different concentrations
3
of cyclins and cdks determine gene expression by activating and inactivating
transcription factors (Pestell et. al, 1999). Cyclins bound to cdks are activated and
function in cell cycle regulation, including DNA synthesis regulation, DNA repair and
apoptosis and allow the cell to move to the next phase in the cell cycle. Cyclins D and E
and cdk2, cdk4, and cdk6 are found in the G1 phase allowing the progression from G1
to S. Cyclin D cdk4/6 complex works to control G1-Sphase transition by phosphorylating
retinoblastoma tumor suppressor gene (RB). When RB is not phosphorylated, it inhibits
EDF family transcription factors preventing cell cycle progression. When RB is
phosphorylated by cyclin D cdk4/6 this allows for activation of transcription including
cyclin E. Cyclin E also phosphorylates RB allowing the transition from G1-Sphase
(Pascal and Anne, 2011). Cyclin A and cdk2 are found in the G1 to S phase transition
and in S phase. Cyclin A and B play roles in the cellular progression from G2 to M
phase along with cdk1 (Sherr and Roberts, 1999).
Initial studies in the Chatot laboratory showed that DPH slowed the growth and
division of preimplantation mouse embryos in vivo and in vitro, and decreased the
crown to rump length and weight of mouse fetuses during the last five days of
development in approximately 25-35% of NSA x B6SJL/F1J embryos (Waclaw and
Chatot, 2004, Gonzalez and Chatot, 1993, Blosser and Chatot, 2003). Embryos
exposed to DPH in vivo during preimplantation only had a reduction in rate of
endochondral bone conversion from cartilage particularly in the limbs (Gonzalez and
Chatot, 1993). Work conducted in cultured preimplantation mouse embryos also
showed that therapeutic DPH concentrations of 5, 10, and 20 μg/ml slowed
development in 25-35% of embryos tested (Blosser and Chatot, 2003). The embryos
4
that were seriously affected developed only to 2-cell or 3-4 cell, while unaffected
embryos developed to the blastocyst stage. An explanation for this phenomenon could
be that DPH sensitive embryos have 2 copies of the slow allele of epoxide hydrolase
that cannot rapidly metabolize the highly reactive DPH arene oxide intermediate to a
water-soluble non-toxic form (Cheong et al., 2009). Embryos that have at least one fast
allele of epoxide hydrolase will metabolize the DPH arene oxide intermediate and
continue to grow to the blastocyst stage.
DPH treated preimplantation mouse embryos have also shown an extended S
phase in the second cell cycle in culture, a deregulation of cyclin A in G1, S, and G2 in
the first, second and third cell cycles in vivo, and a decrease in DNA polymerase δ
concentrations in G1 and S phases in vivo of 2-cell mouse embryos compared to NaOH
treated controls. Early work in the lab using bromodeoxyuridine labeling showed that the
timing of DNA synthesis in S phase of the second cell cycle in 30% of DPH treated
cultured preimplantation mouse embryos was extended for 20 hours with no apparent
exit from S phase compared to vehicle controls (Blosser and Chatot, 2003). Cyclin A
was then studied in in vivo preimplantation mouse embryos because cyclin A is the
cyclin present in S phase when DNA synthesis is occurring (Bashir et al., 2000).
Immunofluorescence experiments using an anti-cyclin A2 polyclonal antibody
demonstrated that preimplantation mouse embryos treated with DPH had a decrease in
cyclin A of 1.28 fold during S phase of second cell cycle, an increase in cyclin A
expression during G1 of the second cell cycle of 1.55 fold, and an increase in G2 of
second cell cycle of 1.39 fold compared to NaOH vehicle controls (Tolle and Chatot,
2009). This pattern does not correlate with the normal pattern of cyclin A in second cell
5
cycle preimplantation mouse embryos which was shown to be present at moderate
levels throughout cell cycles 1 and 2 but begins to transition into somatic cell patterns of
expression in the third cell cycle increasing in S phase and peaking in G2 (Waclaw and
Chatot, 2004). The data suggests that the alteration of cyclin A, extended S phase and
DNA synthesis in DPH treated preimplantation mouse embryos during second cell cycle
could cause alteration of growth rate consistent with the types of developmental delays
characteristic of FHS.
The mechanisms behind what is affecting DNA synthesis are still unknown. High
levels of cyclin A can reduce the initiation of DNA synthesis by inactivation of DNA
polymerase α (Pavlov and Shcherbakova, 2010). If DPH is increasing cyclin A in G1
then the initiation of DNA synthesis may be altered by inhibition of DNA polymerase α.
DPH’s effect causing reduced levels of cyclin A in S phase might also be inhibiting DNA
polymerase δ as it has been shown to regulate DNA elongation and proofreading in
parvovirus minute virus of mice (Bashir et al., 2000). DPH is affecting the concentration
of DNA polymerase δ that is produced in the preimplantation mouse embryos. Cornielle
and Chatot (2011) observed a 43% decrease in cytoplasmic staining, and a 36%
decrease in nuclear staining in G1, but a 44% increase in nuclear staining in late S
phase. These results might show the cause for an initial decrease in DNA synthesis and
extension of S phase in preimplantation mouse embryos (Cornielle and Chatot, 2011).
Based on previous experiments, it appears that DPH affects timing of DNA synthesis via
alterations in cyclin A in preimplantation mouse embryos and reduces DNA polymerase
δ concentrations in 2-cell mouse embryos. The current study proposes to examine if
DPH affects DNA synthesis machinery, DNA polymerase δ, resulting in slow rates of
6
DNA synthesis activity compared to controls. The results could support the hypothesis
that DPH altered expression of cyclin A in second cell cycle S phase results in a
decrease in the rate of the DNA replication. If the rate of DNA replication and synthesis
is slowed this may contribute to the growth retardation and abnormal growth patterns in
preimplantation mouse embryos and also in children with FHS.
7
Literature Review
Dilantin and its Metabolism
DPH is a known human teratogen whose mechanisms are not yet fully
understood (Denise et al., 2010). DPH teratogenic properties are credited to the
intermediate metabolites that are formed from the bioactivation of DPH (Buehler et al.,
1990). Cytochrome P-450 monooxygenase, a pathway used to excrete compounds
from the body (Sankar, 2007), metabolizes DPH into an electrophilic arene oxide
reactive intermediate (Figure 1) (Strickler et al., 1985). The epoxide is highly reactive
due to an oxygen bridge that allows it to bind to and damage nucleic acids, proteins and
other cellular macromolecules that could contribute to abnormal development. Based on
DPH arene oxide formation and its ability to form free radicals, multiple theories for the
teratogenic mechanism of DPH have been proposed. 1) DPH is metabolized to an
arene oxide. When arene oxides are increased in an organism and are not bioactivated
or inactivated, the arene oxides can bind cellular macromolecules including protein and
nucleic acids in the fetus causing abnormalities in development. 2) The co-oxidation of
DPH into free radical intermediates by prostaglandin synthetase also results in oxidative
stress, lipid peroxidation interactions, and binding to nucleotides (Denise et al., 2010).
8
Figure 1. Chemical Structure of DPH and Metabolites. Cytochrome P450
metabolizes a. phenytoin into b. phenytoin arene oxide. c. water soluble diphenyl
hydantoic acid. Epoxide hydrolase breaks down the phenytoin arene oxide to d. phydroxy Phenytoin, e. m-hydroxy phenytoin or f. phenytoin dihydrodiol. (Chatot Lab)
DPH is metabolized via cytochrome P-450 mixed function oxidases to an arene
oxide intermediate. This highly reactive intermediate containing an oxygen bridge is
then detoxified to several water soluble nontoxic products, para-hydroxy phenytoin,
meta-hydroxy phenytoin and phenytoin dihydrodiol (Figure 1). These water soluble
9
products are eliminated from the body via the kidney. The enzyme epoxide hydrolase is
responsible for metabolizing the arene oxide intermediate.
Embryos have an epoxide hydrolase gene make up that limits the ability of some
embryos to produce sufficient active epoxide hydrolase (EH) enzyme to detoxify the
DPH arene oxide intermediate (Strickler et al., 1985). The DPH arene oxide is said to be
the proximal DPH teratogen rather than the parent drug itself. The two isoforms of
epoxide hydrolase enzyme were identified by Buehler et al. (1990). Epoxide hydrolase
activity was assayed using amniocytes from 100 random pregnant women. The EH
activity level was assayed with thin layer chromatography producing results with
trimodal assortment. Based on the results, there were high, low and intermediate
enzyme activities in the fetus. Normal Mendelian inheritance was proposed by Buehler
et al. (1990) based on consistent ratios of each allele. Buehler et al. (1990) saw that
when EH activity in newborns was assayed, babies with fast/slow or fast/fast isoforms
had more positive clinical outcomes compared to newborns with slow/slow isoforms. In
the slow/slow isoforms cases, Dilantin teratogenesis was detected. (Buehler et al.,
1990).
Embryos have two copies of the epoxide hydrolase gene (Buehler et al., 1990).
Depending on the genetic competition, any embryo will either be fast-fast, fast-slow or
slow-slow. Embryos with fast-fast copies of the gene will be able to metabolize the
arene oxide intermediates of DPH quickly. Embryos with fast-slow copies have the
ability to metabolize arene oxide intermediates of DPH in fast enough fashion, reducing
the possibility of the arene oxide interacting with the macromolecules. Embryos with
slow-slow copies of the gene will not be able to metabolize the arene oxides of DPH
10
quickly enough, leading to an increased risk of damage in cells causing abnormal
growth of the fetus. Recent work conducted from blood specimens and extractable DNA
from 174 pregnancies in 155 women who used DPH during pregnancy showed that the
polymorphisms maternal Y113H and H139 of microsomal epoxide hydrolase EPHX1
gene related to the presence of craniofacial abnormalities in children exposed to DPH
during pregnancy. If the alleles stated above are absent, there is a decrease in the
presence of craniofacial abnormalities in the children of DPH treated mothers (Azzato et
al., 2010). Prenatal predictions of these alleles could change the percent of craniofacial
abnormalities in children whose mothers take DPH during pregnancy.
Parman et al. (1998) studied the bioactivation of DPH by prostaglandin H
synthases (PHS) and its initiation of reactive oxygen species (ROS) by EPR (electron
paramagnetic resonance; electron spin resonance) spectroscopy. EPR revealed that
when DPH was bioactivated by the PHS, nitrogen centered free radical and a carbon
centered free radical were generated. The carbon centered free radical and several of
its metabolic by-products are capable of binding to macromolecules or causing oxidative
stress leading to teratogenesis (Figure 2). Parman et al., (1998) also showed that PHS
was actually the component in the medium bioactivating DPH by using a PHS inhibitor
ETYA (5,8,11,14-eicosatetraynoic acid). ETYA was used because it has been shown to
inhibit embryotoxicity of DPH in embryo culture (Miranda et al., 1994).
11
Figure 2. Reactive Intermediates Formed by Bioactivation of DPH by
Prostaglandin H Synthetase. Teratogenesis can occur via the covalent binding of the
carbon-centered free radical, the alkylisocyanate or the hydroxyl free radical generated
via peroxidase and superoxide dismutase. (From Parman et al., 1998)
12
Preimplantation Mouse Embryo Development
The early mouse embryo has been the animal model of choice for the study of
DPH. The mouse unfertilized egg is ovulated from the ovary encased in a protective
glycoprotein membrane, the zona pellucida. Preimplantation mouse embryo
development begins at fertilization of the egg in the ampulla of the oviduct (Sakkas and
Vassalli, 2008). The preimplantation mouse embryo will travel down the oviduct for 4
days. When the embryo reaches the uterus, final implantation will occur. During
preimplantation, the embryo undergoes cellular division until the blastocyst stage of 1632 cells. This process of cleaving from 1 to 32 cells takes approximately 3 and half
days. During the travel to the oviduct, the embryo goes through each division, but does
not change its overall size (Nagy, 2003). The 1- cell embryo spends about 24 hours in
the cell cycle, G1 is 3-8 hours long, S phase is 6 hours, and G2/M phase is 6 hours long
(Krishna and Generosa, 1977). In the second cell cycle, G1 is 1.3 hours, S phase is 6.1
hours and 15.4 hours for G2/M phase for a total of 22.8 hours (Sawicki et al., 1978). In
the mid 2-cell stage, a change in protein synthesis is seen. In the 1-cell and early 2- cell
stage, the embryo relies primarily on maternal RNA and protein stored in the cytoplasm
to function. Towards the middle to late 2-cell stage, the embryonic genome is switched
on; this is called zygotic gene activation. This is the switch from maternal control of
development to zygotic/embryonic control of development. When the genome is
switched, the maternal RNA is degraded (Piko and Clegg, 1982). The switch to embryocontrolled development makes the second cell cycle longer than a normal cell cycle,
and can extend as long as 30 hours (Sakkas and Vassalli, 2008). During the 3-4 cell
stages, cleavage becomes asynchronous, making it difficult to distinguish the different
13
stages of the cell cycle at this point (Smith and Johnson, 1986). Once the embryo has
divided into an 8-cell, the embryo develops initiation of specific cell-cell adhesion and
compaction between the blastomeres by the expression of E-cadherins (Fleming et al.,
2001). The formations of tight junctions are formed by the proteins cingulin and ZO-1
(Stevenson et al., 1989) and gap junctions are formed by connexin (Segretain and Falk,
2004). The blastomeres that are loosely connected to each other start to flatten against
one another until the cell boundaries disappear (Gilbert, 2012) This formation of a cell
mass is called the morula stage. As the morula grows to 16 cells, the embryo
establishes the inner and outer cell populations. The inner cells will become the inner
cell mass that forms the embryo. The inner cell mass cells express Oct 4, which down
regulates the cdx2 transcription factor allowing Sox2 and Nanog to be expressed. The
outer cells will become trophoblast cells that form the embryonic portion of the placenta;
these cells express cdx2 transcription factor that down regulates Oct 4, Sox 2, and
Nanog.
At the point of trophoblast differentiation, the stage is set for blastocyst formation.
During the blastocyst stage, cavitation occurs where the trophoblast cells secrete fluid
into the internal area of the embryo creating the blastocoel; this is mediated by the
membranes of the trophoblasts. The sodium/potassium ATPase pumps in the
trophoblasts pumps Na+ into the inside of the embryo; the accumulation of Na+ brings in
water by osmosis to equilibrate the Na+ allowing the blastocoel to expand. At one end of
the trophoblast cells the inner cell mass forms the embryo proper. By this time, the
embryo has entered the uterus and is ready to hatch from the protective zona pellucida
14
and attach to the uterine lining forming the embryonic portion of the placenta (Gilbert,
2012).
Cyclin A
Although the exact mechanisms of DPH effects on preimplantation mouse
embryos are not known, it is known that DPH differentially alters the level of cyclin A
protein during the cell cycle of preimplantation mouse embryos (Tolle and Chatot,
2009). Cyclin A is composed of two subtypes, A1 and A2. Fuchimoto et al. (2001)
presented evidence by RT-PCR and immunoblotting that cyclin A1 is present in the
meiotic cell cycle of mouse unfertilized eggs and cyclin A2 is present in the mitotic cell
cycle in preimplantation mouse embryos. This was determined by using germinal
vesicle stage oocytes and preimplantation embryos from 1-cell to blastula stage.
Although both cyclin A1 and A2 mRNA was present in oocytes and embryos, cyclin A1
protein was present only in oocytes and 1-cell embryos but not present after the 1-cell
stage. Cyclin A2 protein was present in 1-cell up to blastula stage embryos. To further
investigate the regulation of cyclin A2 protein synthesis via polyadenylation and
recruitment of cyclin A2 mRNA into the translational machinery, 3'-deoxyadenosine was
used to inhibit poly(A) tail elongation. The results demonstrated an inhibitory effect of 3'deoxyadenosine in elongation of the cyclin A2 mRNA poly A tail by Northern blotting.
The use of 3’-deoxyadenosine also inhibited increases in cyclin A2 protein synthesis as
demonstrated by immunoblotting and decreased the percentage of pronuclei at the 1cell stage that incorporated bromodeoxyuridine into DNA during replication in a dose
15
dependent manner. These data support an important role for cyclin A2 in DNA
synthesis in the early mouse embryo (Fuchimoto et al., 2001).
A study conducted with the parvovirus minute virus of mice (MVM) in mouse A9
fibroblast cells showed that with the addition of recombinant cyclin A to the MVM
infected A9 cells, the cyclin A dependent cdk2 kinase activity is increased. This led to
conversion of the single stranded MVM DNA into the double stranded replicative form of
DNA supporting cyclin A’s role in DNA replication. Inhibition of cyclin A/cdk2 activity in S
phase with Ab E23 specific for cyclin A inhibited conversion to the double stranded DNA
form (Bashir et al., 1999). The study also showed by Western blot analysis that cyclin A
and cdk2 activity increase at the G1- S phase transition into S phase supporting its role
in cell cycle regulation. Cyclin A is necessary for DNA polymerase δ dependent
elongation in DNA synthesis. Studies by Tolle and Chatot (2009) showed that when
female mice were treated with DPH, cyclin A protein levels increased by 1.55 fold in G1
phase of the second cell cycle before DNA synthesis and decreased by 1.28 fold during
DNA synthesis at 2-cell stage compared to the vehicle control (Tolle and Chatot, 2009).
The change in cyclin A expression may contribute to the extended time of the second
cell cycle S phase observed by Blosser and Chatot (2003). The alteration in expression
of cyclin A due to DPH treatment may affect the DNA polymerase activity in S phase
and therefore the rate of DNA synthesis in preimplantation mouse embryos.
DNA Polymerases
DNA polymerase α
DNA polymerase α/primase structure consists of 4 subunits: a catalytic P180
subunit, a P68 subunit responsible for protein-protein interactions and p150 responsible
16
for translocation to the nucleus (Muzino et al., 1998), and primase activity subunits p55
and p48 (Hübscher et al., 2002). Studies using purified trimeric human polymerase αprimase that lacked the p68 subunit were analyzed for the initiation of DNA replication in
simian virus 40 DNA. In an enzyme assay using radiolabeled dTTPs, the results
demonstrated that p68 is necessary for the initiation of replication (Ott et al., 2002). The
function of DNA polymerase α is to initiate DNA synthesis using RNA primers at the
origin of replication on the leading strand and prime Okazaki fragments on the lagging
strand of replicating DNA (Pavlov and Scherbakova, 2010). DNA polymerase α has
usually been considered to be the enzyme that elongates the leading strand (Pavlov
and Scherbakova, 2010). DNA polymerase α initiates DNA replication in SV40 DNA in
late G1 early S phase and stops initiation of DNA replication in late S phase
(Voitenleitner et. al, 1999). Cyclin A/cdk2 modifies DNA polymerase α p68 in G2-S
phase in human cells. Voitenleitner (1999) showed by PAGE and phosphoimager
analysis that when cyclin E/cdk2 phosphorylation of the DNA polymerase α – primase
p68 subunit occurs DNA synthesis initiation is stimulated but when cyclin A/cdk2
phosphorylates the DNA polymerase α - primase p68 subunit initiation was inhibited.
This showed how DNA replication is stopped at the end of S phase when cyclin A is
increased. If cyclin A phosphorylates p68, then DNA polymerase α primase is inhibited
and the rate of DNA replication decreases (Voitenleitner et al., 1999). The increase in
cyclin A in preimplantation mouse embryos found in G1 before the start of S phase
could decrease the initiation of DNA synthesis due to limiting activity of DNA
polymerase α by cyclin A/cdk2 (Tolle and Chatot, 2009). Also the reduction in cyclin A
during S phase could prevent the termination of DNA synthesis because low levels of
17
cyclin A may not be sufficient enough to inhibit initiation allowing for DNA synthesis in
DPH treated embryos to be extended beyond the time of DNA cessation in normal
control embryos.
DNA Polymerase δ
After DNA polymerase α has initiated and primed DNA synthesis there is a pol
switch that occurs between DNA polymerase α and DNA polymerase δ. This switch
changes the replication process from initiation to elongation of DNA synthesis
(Hubscher et al., 2002). DNA polymerase δ is an enzyme consisting of subunits that
synthesize DNA, repair synthesis errors in DNA and fix damaged DNA (Pavlov and
Shcherbakova, 2010). DNA polymerase δ is made of 4 subunits. The p125 subunit is
catalytic, p55 subunit is the structural component, p66 subunit is the proliferating cell
nuclear antigen (PCNA) which is encases the DNA along with the RF-C loader
(replication factor C; loads the sliding clamp of PCNA onto DNA, necessary to DNA
polymerase δ interaction with DNA) allowing the pol switch from the initiating DNA
polymerase α to δ, and increases processivity by making strong protein interactions
including those which help with DNA repair (Maga and Hübscher, 2003). The p12
subunit is involved in protein-protein interactions (Hübscher et al., 2002). The p125
subunit is the largest and has the DNA polymerase activity and 3’-5’ exonuclease
proofreading properties and has a protein-protein interaction site for PCNA binding
(Figure 3) (Pavlov and Scherbakova, 2010). The p55 subunit is used to help stabilize
the catalytic subunit to the p66 subunit. The p66 subunit has several roles: it interacts
with polymerase δ, has a PCNA-binding motif, and regulates error-prone translesion
synthesis (Pavlov and Shcherbakova, 2010). The fourth subunit p12 is not fully
18
understood but in human enzyme experiments, it plays a role in the response to DNA
damage (Pavlov and Scherbakova, 2010).
Figure 3. DNA Polymerase δ Catalytic Subunit p125: Polymerase and
Exonuclease Domains (www.google.images)
Studies show that DNA polymerase δ has an impact on genomic stability and
that damage to DNA polymerase δ can cause strong defects in or death to the cell
(Pavlov and Shcherbakova, 2010). Venkatesan et al. (2007) studied the offspring of
DNA polymerase δ mutant Pold1/L604G and Pold1 /L604K mothers. Offspring were
analyzed to see the consequences of DNA polymerase δ damage. Genotyping of the
embryos showed that no homozygous mutant embryos survived to day 8 indicating that
the homozygous alleles of the mutation induced early lethality. The mutation rates in
mouse embryo fibroblast (MEF) derived from embryos between days 11.5 and 13 that
were heterozygous for the fold mutations showed a 5 -fold (Pold1/L604G) and 4-fold
(Pold1/L604K) increase in mutation rates compared to wild type rates. Chromosome
aberrations in MEF’s had a 17-fold increase in Pold1_/L604G cells and a 38-fold
19
increase in Pold1_/L604K cells compared to wild type cells. Lastly histopathology
analysis of the mice used showed that heterozygous mutants of Pold1_/L604G and
Pold1_/L604K had an increase in tumors of 8-16 % compared to wild type mice (this
may be a low estimate since most Pold1_/L604K mice died before tumors developed).
Overall it was evident that DNA polymerase δ functions in genomic stability and if DNA
polymerase δ is altered this can cause an increase in lethality to the embryo as well as
increase cancer occurrence.
The job of DNA polymerase δ is has been analyzed by Pavlov and Scherbakova
(2010). The traditional model of the replication fork suggests that DNA polymerase α
initiates DNA synthesis, DNA polymerase δ elongates the lagging strand by Okazaki
fragments and DNA polymerase ε elongates the leading strand. This traditional model is
questioned for 4 reasons. 1) Deletions of DNA polymerase ε are not lethal and can
survive but deletions of DNA polymerase δ are lethal in yeast. 2) The genome wide
mutation rate with a defect on DNA polymerase ε is lower than the genome wide
mutation rate with DNA polymerase δ defects. 3) When DNA polymerase δ and DNA
polymerase ε competed for error repair synthesis, the results did not show them working
on different strands. Under conditions of DNA polymerase ε mutation, this suggested
that DNA polymerase δ corrected errors that DNA polymerase ε would otherwise have
completed on the leading strand. 4) Experiments conducted in yeast with deletions of
the subunit Pol32 showed that DNA polymerase δ is responsible for the involvement of
recruitment of translesion synthesis (TLS) polymerases regardless of the strand
because if DNA polymerase only worked on the lagging strand then the deletion of
POL32 would only be working on half of the TLS on the lagging strand. The old model
20
underestimates the activity of DNA polymerase δ. The new model also known as the
“alternate fork” suggests that DNA polymerase α initiates DNA synthesis on the lagging
strand by short RNA-DNA fragments, DNA polymerase δ elongates the leading strand
and the lagging strand after DNA polymerase ε initiates the leading strand synthesis at
the origin. This ‘”alternate” fork is thought to be the normal mechanism that occurs
possibly because of the pausing or disassociation of DNA polymerase ε due to template
damage, collision with RNA polymerase, or generation of mismatched primer termini.
The alternate or new model demonstrates that DNA polymerase δ is not restricted to the
lagging strand but has the ability to and most likely copies both strands of DNA (Pavlov
and Scherbakova, 2010).
Cyclin A is active in S phase (with cdk2) and in G2 (with cdk1) (Bashir et al.,
2000). In our lab, Tolle and Chatot (2009) demonstrated that DPH lowers the cyclin A
levels by 1.28 fold in S phase of the 2-cell embryo. According to Bashir et al. (2000),
cyclin A/cdk2 binds to DNA polymerase δ during DNA replication and increases the rate
of elongation of DNA strands thus regulating the length and exit from S phase. DPH
lowered levels of cyclin A in S-phase of the second cell cycle would contribute to a
slowing in DNA elongation by DNA polymerase δ. Work in our lab by Cornielle
confirmed that DPH treatment alters the concentration of DNA polymerase δ in the
cytoplasm (43% decline) and the nucleus (36% decline) during G1 cell mouse embryos
compared to vehicle controls by immunofluorescence staining with a primary rabbit
polyclonal antibody raised against DNA polymerase δ (Cornielle and Chatot, 2011).
DNA polymerase δ levels did not rise again in DPH treated embryos until what would
have been the normal S to G2 phase transition in the cytoplasm (98% increase) and in
21
the nucleus (44% increase) of the DPH treated 2-cell preimplantation mouse embryos
compared to the vehicle control (Cornielle and Chatot, 2011). The decrease in DNA
polymerase δ may also contribute to a decreased rate of DNA elongation following DPH
treatment compared to vehicle controls. This thesis work will provide more information
about the hypothesis that DPH treatment affects the rate of DNA synthesis in second
cell cycle preimplantation mouse embryos.
Significance
The results of this thesis will continue to narrow down the mechanism behind alterations
in mammalian and fetal development caused by DPH that lead to FHS characteristics.
The epileptic mother does not always have a choice to stop using DPH during
pregnancy to prevent seizures so it is essential to understand the mechanisms behind
the effects of DPH in order to be able to effectively treat patients during pregnancy. This
knowledge could lead to preventative therapies that would allow the mother to take the
drug needed for epilepsy and still maintain a healthy fetus.
22
Research Methods
In order to study the effects of DPH on DNA synthesis rates in preimplantation
mouse embryos, embryos were isolated during the second cell cycle S phase when
DNA synthesis is occurring.
Specific Goals
The specific goals of this research project were to:
1) Successfully isolate 2-cell preimplantation mouse embryos that have been
exposed to Dilantin and the NaOH vehicle control to compare rates of DNA
synthesis using confocal microscopy.
2) Determine the correct timing of S-phase when DNA synthesis is occurring and
determine time points to study the rate of activity of DNA synthesis.
3) Use the Click-it EdU DNA synthesis assay to incorporate EdU into DNA for a
comparison of the rate of synthesis in DPH treated embryos and vehicle controls.
4) Detect EdU incorporation into DNA in the nucleus using Alexa Fluor azide 488
that demonstrates where DNA synthesis is occurring.
5) Label embryos with Click-it EdU substrate and azide at 4 points over a 40
minute time interval (5, 10, 20, and 40 minutes) in the second cell cycle of S
phase.
6) Determine the nuclear fluorescence intensity in each labeled embryo nucleus at
each time point for Dilantin treated embryos and vehicle control embryos and use
Student t-tests to compare the average fluorescent intensities at each time point.
The average intensity values will be used to plot the time course of DNA
synthesis in the DPH and NaOH treated embryos and to calculate the rate of
23
synthesis. Fold increase or decrease in rates of DNA synthesis due to DPH
treatment will be determined relative to NaOH controls.
Preimplantation Mouse Embryo Isolation
NSA female mice (Harlan, Indianapolis, IN or bred in-house) were injected
intraperitoneally (ip.) with (10IU) Pregnant Mare Serum Gonadotropin (PMS),
(Calbiochem, LaJolla, CA 367222) dissolved in 100μl of embryo culture water (59900C,
Sigma Aldrich) in order to cause follicle maturation. After 48 hours, the same females
were injected ip. with (5IU) human chorionic gonadotropin (hCG) (Cl063 Sigma Aldrich,
St. Louis, MO) dissolved in 100μl of embryo culture water (59900C, Sigma Aldrich) to
induce synchronous ovulation and were placed in mating cages with B6SJLF1/J males
(Jackson Labs, Bar Harbor, ME). Twenty-four hours post hCG injection, the females
were injected ip. with 55mg/kg of Dilantin 5,5-diphenylhydantoin (DPH) (D-4505, Sigma
Aldrich) in 0.001 N sodium hydroxide NaOH (S8045, Sigma Aldrich) or the vehicle
control 0.001 NaOH using a volume equal to 1/100 of their weight. For example, a 25gm
mouse was injected with 0.23ml of vehicle or DPH solution. The mice were then
separated from mating cages. During embryonic second cell cycle S phase, ie. 27.5
hours post fertilization with midnight during mating being considered the time for
completion of fertilization (See Table 1 above), females were sacrificed and embryos
were flushed from oviducts of the superovulated NSA females with Hanks buffered
saline solution (HBBS), [containing 12.62mM CaCl2, 4.92mM MgCl2-6H2O, 4.06mM
MgSO4-7H2O, 53.3mM KCl, 4.41mM KH2PO4, 1,379.3 mM NaCl, 3.35mM Na2HPO47H2O, 5.5 mM D-Glucose (H9269, Sigma Aldrich)] and 0.4 % bovine serum albumin
24
(BSA)(A-3311, Sigma Aldrich). (IACUC approval #91855-8 approved through 6/3/2015).
Two-cell embryos from each mouse were separated out into a single drop of culture
medium (CZB; Chatot et al., 1989) [containing 81.62mM NaCl (S5886, Sigma Aldrich),
4.83mM KCl (P-5405, Sigma Aldrich), 1.18mM KH2PO4 (P5655), 1.18mM MgSO4
7H2O (M1880), 25.12mM NaHCO3 (S5761, Sigma Aldrich), 1.70mM CaCl2 (C7902,
Sigma Aldrich), 31.30mM Na lactate (L4263, Sigma Aldrich), 0.27mM Na Pyruvate
(S8636, Sigma Aldrich), 0.11mM Ethylenediaminetetraacetic acid tetrasodium salt
dihydrate (EDTA) (E6511, Sigma Aldrich), 1mM L-glutamine (G8540, Sigma Aldrich),
5mM BSA (A-3311, Sigma Aldrich), 100mM Na penicillin G (P3032, Sigma Aldrich), and
0.70mM streptomycin (S-9137)] under oil and counted. The embryos were held at 37 °
C in 5 % CO2 in air during the isolation procedure. Random samples were achieved by
sorting equal numbers of embryos from a given mouse into each experimental drop.
DNA Replication Assay
The DNA replication assay utilized was a Click-It EdU assay kit (Invitrogen, C10086,
Invitrogen Eugene, OR). EdU, 5-ethynyl-2’-deoxyuridine is an analog of thymidine,
which is incorporated into DNA in place of thymidine when DNA synthesis is occurring.
A 10 mM stock of EdU (Invitrogen, C10086) was made with embryo culture water
(W1503, Sigma Aldrich). Embryos were pulsed in 30 μl of CZB medium + 4% BSA
containing a 1/20 dilution of EdU stock for 5, 10, 20, and 40 min. The higher level of
BSA prevents non-specific binding of EdU. During labeling, embryos were held in 37 °C
in 5% CO2, 5 % O2 and 90 % N2 which is optimal for embryo development (Chatot et al.,
1989). The time intervals were used to detect the activity over time of DNA polymerase
25
δ and DNA polymerase α by looking at DNA synthesis measured by EdU incorporation
at each time interval. EdU incorporation would be expected to increase at each time
interval starting at 5 min and begin to plateau at the point when DNA synthesis slows or
stops. At the end of each time interval, embryos were washed 3 times for 5 min in 40μl
drops of phosphate buffered saline (PBS) (BP399-500, Fisher Scientific, Pittsburg, PA)
+ 6% BSA (A-3311, Sigma Aldrich) to remove unincorporated EdU, and then fixed in 40
μl of 4% paraformaldehyde (P6148, Sigma Aldrich) at room temperature. The embryos
were moved from the fixation drop to be permeabilized in a 40μl of 2.5% Triton X-100
detergent (T-8787, Sigma Aldrich) in PBS (BP399-500, Fisher Scientific) for 2 min. The
embryos were then washed in PBS (BP399-500, Fisher Scientific)+ 6% BSA (A-3311,
Sigma Aldrich) with 2% normal goat serum (AG9023, Sigma Aldrich) 3 times for 5 min
to eliminate all of the Triton X-100 and to block non-specific binding of the azide.
Following the washes, the embryos were placed in a 40μl drop of Click-it EdU reaction
cocktail (C10086, Invitrogen) for the detection of EdU incorporated during DNA
synthesis. The reaction cocktail contained 86μl of Click-it reaction buffer, 4μl of CuSO4,
24μl of Click-it reaction buffer additive, and 10μl of a 1:1000 dilution of Alexa Fluor azide
488 which recognizes and binds to the incorporated EdU by a copper catalyzed reaction
between the alkyne on EdU and azide. The azide was diluted 1:1000 compared to the
kit instructions to reduce the non-specific binding of the azide. The embryos were
washed in 40μl drops of PBS (BP399-500, Fisher Scientific) + 6% BSA (A-3311, Sigma
Aldrich) 3 times for 5 min each to remove unincorporated azide. A portion of the
embryos were placed in a 40μl drop containing 2μg/ml Hoechst 33342 (H-3570, Sigma
Aldrich) dissolved in of PBS (BP399-500, Fisher Scientific)+ 3% BSA (A-3311, Sigma
26
Aldrich), to show presence of nuclear staining. Hoechst 33342 bound to adeninethymine base pairs of DNA is detected by UV excitation at 405 nm and emits a blue
fluorescence at 460 to 490nm. The embryos were washed in 40μl drops of PBS
(BP399-500, Fisher Scientific) + 6% BSA (A-3311, Sigma Aldrich) 3 times for 5 min to
wash out excess Hoechst. Once the embryos were washed, they were mounted in 9:1
glycerol: PBS containing 100 mg/ml 1,4-Diazabicyclo[2.2.2]octane solution (DABCO)
(290734, Sigma Aldrich) (to prevent photobleaching) on 100 µg/ml poly-L-lysine
(P4707, Sigma Aldrich) treated glass coverslips and inverted onto glass microscope
slides. Coverslips were sealed with clear nail polish and stored flat at 4° C in the dark
until observation. Negative controls included incubating embryos in EdU without azide
as well as incubating embryos in azide in the absence of EdU, which were collected at
the 40 min time point. Experiments were performed at least in triplicate with
approximately 20 embryos per time interval per replicate. Each time point yielded
between 31 and 117 embryos total of 62-234 nuclei for analysis.
Confocal Microscopy and Data Collection
The embryos were observed by confocal microscopy and nuclear fluorescence,
the indication of DNA synthesis, was detected. All embryos from each time interval
were scanned and imaged on the confocal microscope. Using optimal Nyquist sampling,
optical Z-sections of each embryo were obtained. The samples were scanned through
the LP505 filter at a wavelength of 488nm at a setting of 1.1% transmission. The beam
splitters used were the NFT 490 and the HFT405/488/543. Each image was scanned
with a Plan-Apochromat 20x/0.75 objective. Optimal scan settings were determined on
27
the brightest embryo in the 40 min EdU with azide sample that provides for minimal
background in the 40 min no EdU with azide control. The optimal scan settings for each
embryo had a scan speed of 6, 12 bit scan mode, 1.9 zoom, stack size X 1316 Y 1316,
pinhole 56µm, detector gain 700, amplifier offset -0.05, and amplifier gain was 1. For
each embryo nucleus, the average intensity area examined was 286 ± 5 µm2. Relative
incorporated fluorescence was determined and cytoplasmic background intensity in an
area of 286 ± 5 µm2 was subtracted. Nuclear presence was confirmed in some embryos
via co-localization of EdU/Azide label with Hoechst stain.
Data Analysis
At each time interval, the average nuclear intensity ± SEM was calculated for
DPH and NaOH treated embryos. The average intensity ± SEM across the different time
intervals were plotted as a straight-line plot from 0-40 min. The change in intensity per
minute (i.e. the slope of the line) was calculated for 0-5 min, 0-10 min, 0-20 min and 040 min to determine the rate of synthesis for both the DPH treated embryos and NaOH
vehicle controls. Statistical analysis was done by Student’s t test comparing differences
in mean intensities in DPH treated embryos at each time point to the vehicle controls
NaOH. Fold differences in DNA synthesis rate between DPH and NaOH treated
embryos where calculated.
28
Results
DPH treated preimplantation embryos have been shown to have a lengthened S
phase in the second cell cycle of development as well as changed relative
concentrations of DNA synthesis specific proteins, DNA polymerase δ and cyclin A. This
current research studies the effect of DPH on rates of DNA synthesis during S phase of
the second cell cycle. The rate of DNA synthesis was assayed using Click-it EdU
fluorescence DNA labeling of nuclei in the second cell cycle S phase. Relative
fluorescence is indicated by incorporation of EdU, in place of deoxythymidine, into DNA
by DNA polymerases δ and α. With this assay, it is not possible to distinguish synthesis
using DNA polymerase δ vs. α although according to current models, DNA polymerase
δ should represent the vast majority of DNA synthesis occurring (Pavlov and
Shcherbakova, 2010).
Assay Optimization
All optimization experiments were conducted on NaOH vehicle control embryos.
The first optimization experiments began using manufacturer suggested standard kit
conditions, including 30 min incubation in EdU and full concentration Alexa Fluor azide.
Isolation of embryos started at 4:00 AM. This time point was selected because of the
history of previous experiments and estimated timing of the cell cycle. The zona
pellucida was kept on some embryos and removed in other embryos to see if the zona
affected the specific binding of the EdU and Alexa Fluor azide. The result at 4:00 AM for
all embryos with and without the zona and with and without EdU was nonspecific
binding and no distinct nuclei, although nuclear shadows are visible (Figure 4 A). This
experiment showed that the azide was nonspecifically binding to almost everything in
29
the cell and that this may not represent the optimal time to detect DNA synthesis in S
phase because of the lack of distinct nuclear staining.
Optimization experiment # 2 tested for DNA synthesis starting with a 5:00-6:00
AM isolation, incubation at 6:30 AM in EdU for 30 min, and stained with 1:1000 Alexa
Fluor azide (Figure 4 B). The time was moved to 5:00-6:00 AM because of the previous
results using late S phase in Cornielle and Chatot’s (2011) work which occurred around
6 AM. During the 5:00-6:00 AM experiments, EdU was diluted 1:10 to attempt to
eliminate nonspecific binding of the EdU. The Alexa Fluor azide was also diluted 1:1000
in order to minimize background staining. Normal goat serum at 2% (AG9023, Sigma
Aldrich) was added to the PBS (BP399-500, Fisher Scientific) + 3% BSA (A-3311,
Sigma Aldrich) to see if nonspecific binding of the azide could be eliminated by adding
protein to washes. In order to wash out excess EdU, the embryos were washed 3 times
instead of 2 times with PBS (BP399-500, Fisher Scientific) + 3% BSA (A-3311, Sigma
Aldrich). The CZB used for holding embryos contained 0.4% BSA (A-3311, Sigma
Aldrich). The results of 1:10 EdU and diluted azide once again resulted in significant
background (Figure 4 B) and no distinct nuclear staining.
The next set of experiments for optimization was a time course to determine the
start and end of S phase. The isolation times were 1:00 AM (yielded no embryos for
analysis), 3:00 AM, 5:00 AM, and 7:00 AM; the labeling time was approximately 1 hour
after each isolation (Figure 4 C, D, E, F, G, and H). Invitrogen was contacted about
reducing the background. The company suggested increasing the protein in the labeling
and the washes, as the kit is designed for use with cells cultured in 5-10% serum. The
BSA was increased in the CZB from .4% to 4% in the holding drops prior labeling and in
30
the labeling solution. The BSA in the PBS was increased from 3% to 6% for the washes.
To determine the location of the nucleus, the embryos were also stained in Hoechst
33342. The 3:00 AM isolation showed the brightest presence of nuclear staining from
the Hoechst (Figure 4 C) and incorporation of EdU indicated by azide staining (Figure 4
D) meaning DNA synthesis was occurring. The 5:00 AM isolation also showed that DNA
synthesis was occurring by the presence of nuclear staining with the Hoechst (Figure 4
E) and incorporation of EdU detected with the azide (Figure 4 F). The 7:00 AM isolation
had nuclear staining with Hoechst showing the presence of DNA (Figure 4 G) but had
no EdU staining (Figure 4 H) meaning DNA synthesis had stopped by the 7:00 AM
isolation point. In the time trials, isolation at 3:00 AM demonstrated the strongest
nuclear fluorescence indicating the best time to label DNA synthesis (Figure 4 I). The
background was greatly decreased with the increase in protein (Figure 4 C, D, E, F, G,
and H). The control with azide and no EdU showed the lack of nonspecific binding to the
embryo (Figure 4 I). The time trial determined the approximate timing of the second cell
cycle S Phase. Based on optimization results all subsequent experiments started with
embryo isolation at 2:30 A.M.
C
31
A
B
C
D
E
F
G
A
H
A
I
A
Figure 4. Optimization of Labeling Conditions and Timing for DNA Synthesis in 2cell Preimplantation Mouse Embryos. A. 4:00 AM labeling in EdU for 30 min stained
with full Alexa Fluor azide 488. B. 5:00-6:00 AM labeling in EdU for 30 min stained with
1:1000 Alexa Fluor azide. The white arrows in A and B are indicating nuclear shadows.
C. 3:00 AM labeling in EdU for 30 min stained with 1:1000 Alexa Fluor azide and
counter stained with Hoechst 33342, images on the Hoechst channel only. D. 3:00 AM
labeling in EdU for 30 min stained with 1:1000 Alexa Fluor azide and counter stained
with Hoechst 33342, images on the Alexa Fluor channel only. E. 5:00 AM EdU 30 min
stained with 1:1000 Alexa Fluor azide and counter stained with Hoechst 33342, images
on the Hoechst channel only. F. 5:00 AM labeling in EdU for 30 min stained with 1:1000
Alexa Fluor azide and counter stained with Hoechst 33342 on the Alexa Fluor channel
32
only. G. 7:00 AM labeling in EdU for 30 min stained with 1:1000 Alexa Fluor azide and
counter stained with Hoechst 33342, images on the Alexa channel only. H. 7:00 AM
labeling in EdU 30 min stained with 1:1000 Alexa Fluor azide and counter stained with
Hoechst 33342, images on the Alexa Fluor channel only. I. No EdU stained with 1:1000
Alexa Fluor azide only. All images were collected using 20X Plan-Aprochromat
20X/0.75 and were exported from a single slice in the Z stack that demonstrated the
middle of the nuclear region.
Analysis of Controls
Following optimization, it was important to demonstrate that labeling in EdU
alone or azide alone caused little to no background. Embryos with EdU but no Alexa
Fluor Azide were labeled for 40 min. Embryos that were not labeled with EdU sat in a
holding drop of CZB + 4% BSA for 40 min until fixation. NaOH control with EdU no azide
showed that EdU was not detected without azide and that the embryo was not
autofluorescing (Figure 5 A). NaOH control with no EdU and only Alexa Fluor azide
showed that the azide was not binding to the nuclei nonspecifically and that cytoplasmic
background was relatively low (Figure 5 B). DPH treated controls with EdU but no azide
also showed that EdU was not detected without azide and that the embryo was not auto
fluorescing (Figure 5 C). DPH treated controls with no EdU and only Alexa Fluor azide
likewise showed that the azide was not binding to the nuclei nonspecifically (Figure 5
D). However embryos labeled with both EdU and azide had cytoplasmic and
presumably nuclear background that was variable. Therefore cytoplasmic background
fluorescence intensity was determined for an area comparable to the nucleus, and was
33
subtracted from the nuclear fluorescence intensity in all experimental embryos to correct
for non-specific binding.
A
B
C
D
Figure 5. Controls for Autofluorescence and Background Fluorescence from EdU
or Alexa Fluor Azide. Controls were isolated simultaneously with all experiments. A.
NaOH injected 2-cell embryo labeled in EdU for 40 min but no staining with Alexa Fluor
azide following EdU. B. NaOH injected 2-cell embryo with no EdU labeling but stained
with Alexa Fluor Azide 488. C. DPH treated 2-cell embryo labeled in EdU for 40 min but
no Alexa Fluor azide staining after EdU. D. DPH treated 2-cell embryo with no EdU
labeling but stained with Alexa Fluor azide 488. The images were collected using 20X
Plan-Aprochromat 20X/0.75 and were exported from a single slice in the Z stack that
demonstrated the middle of the nuclear region.
Analysis of NaOH Treated Embryos
NaOH vehicle control treated embryos labeled with EdU for 5 min showed the
presence of DNA synthesis by the staining of the nuclei (Figure 6 A). The nuclear
staining was relatively dim in the 5 min samples because EdU only had 5 min to
incorporate into DNA. In support of these observations, the average relative corrected
background fluorescence of the 5 min NaOH vehicle controls value was 488.45 ± 72.02
(mean value ± standard error of the mean; SEM). The total background corrected
34
nuclear fluorescence values for 5 min NaOH ranged from -83 to 1791 per nucleus; N=
90 nuclei in 45 embryos (Table 2, Appendix 1).
NaOH vehicle control treated embryos labeled with EdU for 10 min showed a
more intense presence of DNA synthesis by the brighter staining of the nuclei (Figure 6
B). In support of these observations the average relative background corrected nuclear
fluorescence in the 10 min NaOH vehicle controls rose to 435.53 ± 62.86 (Table 2). The
total fluorescence values for 10 min NaOH ranged from 75 to 1195; N=94 nuclei in 47
embryos (Table 2, Appendix 1).
NaOH vehicle control treated embryos labeled in EdU for 20 min showed the
highest accumulated labeled DNA by having the brightest staining of the nuclei (Figure
6 C). In support of these observations, the average relative background corrected
nuclear fluorescence of the 20 min NaOH vehicle controls rose to 508.39 ± 92.87 (Table
2). The total fluorescence values for 20 min NaOH ranged from 221 to 1008; N= 62
nuclei in 31 embryos (Table 2, Appendix 1).
NaOH vehicle control treated embryos labeled in EdU for 40 min decreased in
accumulated DNA labeling demonstrated by the lack of intensity of the nuclei (Figure 6
C). In support of these observations, the average relative background corrected
fluorescence of the 40 min NaOH vehicle controls decreased to 302.51 ± 29.95 (Table
1). The total fluorescence values for 40 min NaOH ranged from -80 to 1028; N=208
nuclei from 104 embryos (Table 2, Appendix 1).
All embryos were placed in EdU for 5, 10, 20, or 40 min. Photographic images
were analyzed by measuring the relative fluorescence intensity over time of the nuclei
using Zeiss Pascal Densitometry software. The fluorescence intensity at each time
35
point was measured by subtracting the cytoplasmic background intensity from the
nuclear intensity. The fluorescence intensity of nuclei minus the cytoplasmic
background was averaged for each time point.
Table 2. Preimplantation Mouse Embryo Time Course of Average Corrected
Nuclear Intensity in DPH and NaOH Treated Embryos During S Phase of the
Second Cell Cycle.
Time In
NaOH
DPH
Fold
EDU
Nuclear Intensity-
Nuclear Intensity-
Difference
Cytoplasm Intensity
Cytoplasm Intensity
Mean ± SEM (N)
Mean ± SEM (N)
5 Min
488.45 ± 72.02 (90) a
75.78 ± 9.78 (118) a
6.45
10 Min
435.53 ± 62.86 (94) b
353.26 ± 36.06 (190) b
1.23
20 Min
508.39 ± 89.87 (62) c
287.84 ± 26.96 (234) c
1.77
40 Min
302.51 ± 29.95 (208) d
208.51 ± 22.75 (166) d
1.45
a
5 min DPH samples are statistically significantly different from NaOH control samples by
student t-test; P= 3.82831 X 10 -7
b
10 min DPH and NaOH samples are not statistically different by student t-test; P=0.083879126
c
20 min DPH samples are statistically significantly different from NaOH control samples by
student t-test; P=3.13066 X10 -6
d
40 min DPH samples are statistically significantly different from NaOH control samples by
student t-test; P=0.001488326
36
AA
B
C
D
Figure 6. NaOH Treated Embryos Labeled in EdU at 5, 10, 20, and 40 min to
Demonstrate DNA Synthesis. All samples were isolated at 2:30 AM and stained with
1:1000 Alexa Fluor azide. A. Embryo labeled in EdU for 5 min. B. Embryo labeled in
EdU for 10 min. C. Embryo labeled in EdU for 20 min. D. Embryo labeled in EdU for 40.
The light green halo around the embryos in B and C is the zona pellucida. The images
were collected using 20X Plan-Aprochromat 20X/. 75 and were exported from a single
slice in the Z stack that demonstrated the middle of the nuclear region and are
representative of the mean relative fluorescence intensity values of each time point.
Analysis of DPH Treated Embryos
DPH treated embryos labeled in EdU for 5 min showed minimal nuclear
fluorescence (Figure 7 A). In support of these observations the average relative
background corrected fluorescence of the 5 min DPH treated embryos was 75.78 ± 9.78
(mean value ± Standard Error of the Mean; SEM). The total fluorescence values for 5
min DPH ranged from -43 to 170; N=118 nuclei from 59 embryos (Table 2, Appendix 1).
DPH treated embryos labeled in EdU for 10 min showed an increase in nuclear
fluorescence (Figure 7 B). In support of these observations the average relative
background corrected fluorescence of the 10 min DPH treated embryos was 353.26 ±
37
36.06 (Table 2). The total fluorescence values for 10 min DPH ranged from 12 to 867;
N=190 nuclei from 95 embryos (Table 2, Appendix 1).
DPH treated embryos labeled in EdU for 20 min showed a decrease in nuclear
fluorescence intensity. In support of these observations, the average relative
background corrected fluorescence of the 20 min DPH treated embryos was 287.84±
26.96 (Table 2). The total fluorescence values for 20 min DPH ranged from 105 to 1233;
N=234 nuclei from 117 embryos (Table 2, Appendix 1).
DPH treated embryos labeled in EdU for 40 min showed an even larger decrease
in nuclear fluorescence compared to the DPH 20 min sample (Figure 7 C and D). In
support of these observations, the average relative background corrected fluorescence
of the 40 min DPH treated embryos was 208.51 ± 22.75 (Table 2). The total
fluorescence values for 40 min DPH ranged from 16 to 548; N=166 nuclei from 83
embryos (Table 1, Appendix 1).
A
B
C
A
D
Figure 7. DPH Embryos Labeled in EdU at 5, 10, 20, and 40 min to Demonstrate
DNA Synthesis. All samples were isolated at 2:30 AM and stained with 1:1000 Alexa
Fluor azide. A. Embryos labeled in EdU for 5 min. B. Embryos labeled in EdU for 10
min. C. Embryos labeled in EdU for 20 min. D. Embryos labeled in EdU for 40 min. The
light green halo around the embryos in B and C is the zona pellucida. The images were
38
collected using 20X Plan-Aprochromat 20X/0.75 and were exported from a single slice
in the Z stack that demonstrated the middle of the nuclear region and are representative
of the mean relative fluorescence intensities for each sample.
Comparison of NaOH and DPH Treated Embryos
Levels of relative EdU fluorescence for DPH and NaOH treated embryos were
compared at each time point on a linear plot (Figure 8). The rate of synthesis was
determined by calculating the amount of EdU incorporated into the DNA that is being
synthesized over time, i.e. determining the slope of the line. The rate of synthesis in the
NaOH treated embryos over the full 40 min time period was 7.56 relative EdU units
incorporated per min compared to the DPH rate of synthesis over 40 min which was
5.21 relative EdU units incorporated per min. This represents a 1.45 fold difference and
a 31% decrease in DPH treated embryos (Table 3, Figure 8).
Due to the general decline in EdU incorporation at 40 min for both NaOH and
DPH treated embryos, the rate of synthesis was analyzed over several different time
intervals. From 0-5 min the greatest fold difference, at 6.45 (Table 3), occurred between
the DPH and NaOH treated embryos. The DPH rate of synthesis was 15.16 relative
EdU units incorporated per min, an 84.5% decrease compared to the NaOH rate of
synthesis of 97.69 relative EdU units incorporated per min (Table 3, Figure 8). The 0-10
min rate of synthesis was 43.55 relative EdU units incorporated per min in NaOH
treated embryos compared to only 35.33 relative EdU units incorporated per min in DPH
treated samples, representing a fold difference of 1.23 (Table 3, Figure 8) and a 19%
decline in synthesis in DPH treated embryos compared to the NaOH treated controls.
39
The 0-20 min time interval still demonstrated that NaOH treated embryos had a higher
rate of synthesis. In support of this, NaOH controls had a 25.42 relative rate of synthesis
while DPH treated embryos had 14.39 relative rate of synthesis representing a fold
difference of 1.77 (Table 3, Figure 8). This is a 43% decline in the DNA synthesis rate in
DPH treated embryos in comparison to NaOH controls. Overall the highest rate of
synthesis for the NaOH controls reactions occurred within the first 5 min of the reaction
after which it plateaued until a general decline after 40 min. The DPH treated samples,
the maximum rate of synthesis occurred in the 5-10 min time interval when the rate of
synthesis was at 55.5 relative EdU units per min. DPH samples demonstrated a general
decline in synthesis rate before the 20 min time point which was earlier than in the
controls.
Table 3. Rate of DNA Synthesis* in NaOH and DPH Treated Embryos.
Time
NaOH Rate of
DPH Rate of Synthesis
Fold Difference
Synthesis EdU
EdU incorporated/min
(% DPH decline)
incorporated/min
0-5 min
97.69
15.16
6.45 (84.5%)
0-10 min
43.55
35.33
1.23 (19%)
0-20 min
25.42
14.39
1.77 (43%)
0-40 min
7.56
5.21
1.45 (31%)
*Rate of synthesis is determined by calculating the slope between any 2 time points
based on relative nuclear fluorescence indicative of the amount of EdU incorporated
into DNA per minute.
40
Figure 8. Corrected Average Relative Fluorescence at 0, 5, 10, 20, 40 min Time
Points in EdU for NaOH and DPH Treated Embryos. Relative fluorescence units
equal nuclear fluorescence – cytoplasmic background. Data from NaOH treated
embryos are seen in cyan and data from DPH treated embryos are seen in red with
SEM bars in black.
41
Discussion
Rate of synthesis of NaOH and DPH treated embryos was determined by
examining the corrected average relative nuclear fluorescence after 5, 10, 20, and 40
min of labeling with EdU. The corrected relative nuclear fluorescence is proportional to
the amount of EdU incorporated into DNA in place of deoxythymidine at each time point.
The EdU was detected with Alexa Fluor azide 488 and analyzed by confocal
microscopy.
DPH decreases the overall rate of DNA synthesis compared to NaOH treated
mouse embryo controls in S phase of the second cell cycle, indicating that DPH alters
DNA synthesis machinery during this phase. Although this experiment did support that
DNA synthesis is hindered by DPH, the exact mechanism is still not known. There are
several possible explanations for this decrease.
First, the decrease in synthesis could be from the decrease in concentration of
DNA polymerase δ found by Cornielle and Chatot (2011). The decreased amount of
DNA polymerase δ could decrease the amount of EdU incorporated into DNA over time.
Cornielle and Chatot (2011) saw that fluorescence levels of DNA polymerase δ in NaOH
treated embryos was 57% higher than DPH treated embryos in G1 of the second cell
cycle. The decrease in amount of DNA polymerase δ could have resulted in decrease of
incorporation of EdU particularly at the later time points in this experiment. However,
the 84.5 % decline in rate of synthesis at the 0-5 min time point could not totally be
accounted for by this drop in enzyme concentration even if levels of DNA polymerase δ
were on the rise at the time in S phase where the current measurement occurred.
Cornielle and Chatot (2011) also observed a 44% increase in mean nuclear
42
fluorescence of DNA polymerase δ in late S/early G2 phase of the second cell cycle in
the DPH treated embryos compared to the NaOH treated embryos. The increase in
DNA polymerase δ in the nucleus at this late time would extend the actual S phase
timing in DPH treated embryos beyond that of the NaOH controls where the nuclear
levels of the δ polymerase had declined as the cell was exiting S phase. According to
Pavlov and Scherbakova’s (2010) new model for DNA synthesis, DNA polymerase δ is
not only responsible for elongation of the lagging strand but can also replace DNA
polymerase alpha and epsilon on the leading strand. This would suggest that the
majority of EdU incorporation measured, as DNA synthesis would be due to DNA
polymerase δ activity and support the hypothesis that the drop in DNA polymerase δ
levels is responsible for a significant portion of the reduced rate of synthesis.
Secondly, since DPH rate of synthesis is lower in DPH treated embryos in the S
phase of the second cell cycle compared to NaOH treated embryos it supports that DPH
arene oxide or other ROS is causing damage to DNA polymerase δ suppressing it from
elongation and perhaps targeting it for degradation. If DNA polymerase δ is being
damaged by DPH arene oxide, the 3’-5’ exonuclease domain could be decreasing the
accuracy of proofreading during replication. The decrease in proofreading could slow
the processivity of the enzyme and lead to an increase in mismatches in nucleotides.
The increase in rate of errors in DPH treated subjects could be a reason for slowed
growth and deformities in FHS. Studies show that DNA polymerase δ has an impact on
genomic stability and that damage to DNA polymerase δ can cause strong defects in or
death to the cell (Pavlov and Shcherbakova, 2010). Data support that the DPH arene
oxide intermediate or a reactive oxygen species (ROS) could be damaging existing
43
DNA polymerase δ decreasing its activity and its ability to elongate the leading and
lagging strands (Pavlov and Shcherbakova 2010).
The question of whether that the DPH arene oxide intermediate or other ROS
could also be damaging the DNA polymerase α activity as well has not been studied.
DNA polymerase α initiates DNA synthesis at the replication fork by synthesizing
RNA/DNA primers and priming Okazaki fragments for DNA polymerase δ to elongate on
the lagging strand (Hübscher et al. 2002). This current research does not demonstrate
exactly what is slowing the rate of synthesis in DPH treated embryos, but if DNA
polymerase δ concentrations are affected then DNA polymerase α concentrations could
be as well. If this is the case then initiation of DNA synthesis would also be decreased
further adding to the DPH induced decline in rate of synthesis.
Lastly, the decrease in rate of synthesis in DPH treated embryos compared to
NaOH also correlates with data that significantly high levels of cyclin A in DPH treated
embryos existed in G1 and decreased in S phase of the second cell cycle ( Tolle and
Chatot , 2009). This study demonstrated a 1.55 fold increase of cyclin A in G1 which
could additionally contribute to the 6.45 fold difference (84.5% decline) in the rate of
synthesis at 0-5 min in DPH treated embryos compared to NaOH control. This
phenomenon is significant because high levels of cyclin A inhibit DNA polymerase αdependent initiation by inhibitory phosphorylation of the p68 subunit of DNA polymerase
δ (Voitenleitner et al., 1999). Tolle and Chatot (2009) also observed a 1.28 fold
decrease in cyclin A in DPH treated embryos compared to NaOH controls this
correlates with the 1.45 fold difference (31% decline) in rate of overall synthesis (0-40
minutes) in DPH treated embryos compared to NaOH treated embryos. This
44
phenomenon is also significant because cyclin A and its associated kinase activate
DNA polymerase δ- dependent elongation machinery (Bashir et al., 2000). If DPH is
increasing the levels of cyclin A in G1 of the second cell cycle, then this could prevent
DNA synthesis initiation causing a lag in the rate of DNA synthesis initiation and some
leading and lagging strand synthesis because cyclin A has an inhibiting effect on DNA
polymerase α. The decrease in cyclin A in S phase of the second cell cycle could also
be contributing to the decrease in synthesis rate due to the lack of sufficient cyclin A to
promote leading and lagging strand elongation by DNA polymerase δ.
The largest effect of DPH on rate of synthesis was in the 0-5 min reaction. The
accumulation of label continued at 10 min in both the DPH and NaOH treated embryos
however, the rate of synthesis plateaued in NaOH controls at 20 minutes and began to
decline by the 40 min time point. This plateau and drop in synthesis levels occurred by
the 20 minute time point in DPH treated samples. This drop in EdU incorporated was
unexpected because incorporation of EdU into DNA should have been continuous over
the 40 minutes time course. The drop in EdU incorporation could have been due to the
increased amount of protein added to CZB medium to reduce non-specific background.
High protein levels in medium for preimplantation mouse embryos can cause impaired
development before the 8-cell stage (Gardner, 1998). The increase in protein in the
medium over the 3 hours time course of the experiment could have damaged the overall
health of the embryos leading to a decline in DNA synthesis at 40 minutes in the
reactions. Another possible reason for this decline is the depletion of one of more of the
limiting components for the reaction. However, this kit was designed to allow for at least
30 minute reaction times so it is not unreasonable to assume that at 40 minutes, the
45
EdU would still be in high enough concentration to be in excess in the reaction.
However, since the concentration of EdU was reduced by a factor of 10 compared to
the kit instructions, it is possible that by 40 minutes, this had been depleted to the point
where rate of incorporation into DNA was decreased.
It is also important to note that not all embryos were affected by or sensitive to
DPH treatment possibly due to the genotype of epoxide hydrolase possessed by each
individual embryo; i.e. fast-fast, fast-slow or slow-slow (Buehler et al., 1990). In this
experiment an average of 27% of embryos seem to be affected by DPH per experiment
in 5 min of EdU, 27.5% of embryos in 10 min, 26.7% of embryos per experiment in 20
min EdU, and 23.3% of embryos per experiment in 40 min EdU (the percentages were
an average of each DPH experiment for each time point). To determine this number,
each DPH embryo nuclear-cytoplasmic intensity value was compared to the average
nuclear-cytoplasmic background intensity of the NaOH treated embryos at the
appropriate time interval. If the DPH embryo intensity was below the average for the
NaOH controls this was counted as affected by or sensitive to DPH. This correlates with
the Mendelian inheritance of about 25% of embryos having the slow-slow epoxide
hydrolase genotype, indicating about 25% of embryos were affected by DPH and
agrees with data from embryo culture experiment run by Blosser and Chatot (2003).
Future Experiments
This experiment demonstrated a decrease in rate of synthesis in DPH treated
embryos compared to NaOH treated embryos. Questions concerning the exact cause
for the decreased rate of DNA synthesis still remain unanswered. 1). Does DPH affect
46
DNA polymerase α protein levels as well as DNA polymerase δ protein levels? 2). If
DPH is affecting the protein levels of DNA polymerase α and δ then is DPH affecting the
mRNA levels for these proteins as well? 3). Are protein and mRNA levels for the α
and/or δ polymerases being affected by DPH arene oxide or a DPH generated ROS that
is causing the irreversible damage? 4). Lastly is the DPH related decrease in activity of
DNA synthesis due to specific affects on DNA polymerase α, δ, or both? These
questions not answered in this current experiment should be studied in future
experiments.
1). Immunofluorescence and confocal microscopy could be used to determine if
DPH is affecting not only DNA polymerase δ levels in G1 and S phase of the second
cell cycle, but is also affecting DNA polymerase α levels. Isolated embryos from S
phase of the second cell cycle would be stained with a primary antibody against DNA
polymerase α, followed by a secondary antibody conjugated with an Alexa Fluor 488,
and imaged on the confocal microscope. Considering DPH affects DNA polymerase
levels in G1 and S phase of the second cell cycle (Cornielle and Chatot, 2010) it is
assumed DNA polymerase α may also be affected by DPH. The decrease in DNA
polymerase α would correlate with the decrease in rate of synthesis because the less
DNA polymerase α then the less DNA replication initiation would occur (Pavlov and
Scherbakova, 2010).
2). If DPH is affecting both DNA polymerase α and δ protein levels then mRNA
levels may be affected as well. RT-PCR experiments would need to be run in both DPH
and NaOH treated 2-cell embryo extracts in both G1 and S phases. If the DPH is
affecting the protein levels of DNA polymerase α and δ it would be assumed that DPH is
47
affecting the mRNA needed to produce the proteins. A decrease in mRNA would
decrease the rate of synthesis because less protein would be made and DNA
polymerase α could not initiate DNA synthesis and DNA polymerase δ could not
elongate leading or lagging strand DNA replication efficiently (Pavlov and Scherbakova,
2010).
3). If protein levels of DNA polymerase α or δ were affected by DPH and causing
the decreased rate of DNA synthesis, experiments could be designed to determine if the
DPH arene oxide or an ROS is causing damage directly to the protein. Arene oxides are
highly reactive intermediates with oxygen bridges that are capable of forming stable
covalent adducts to macromolecules as could the carbon centered ROS generated by
PHS. Embryos would be incubated in CZB culture with radioactive 14C-DPH to see if
radioactivity associates with DNA polymerase α, δ or cyclin A suggesting an adduct of
the drug was covalently bound to the protein. Following labeling, embryo extracts would
be immunoprecipitated using an antibody against the DNA polymerase α, δ or cyclin A
proteins, immunoblotted with a different primary antibody against each protein, and
autoradiographed to determine if radioactivity was associated with any of the specific
proteins bands. The expected outcome would be that DPH arene oxide is binding to the
polymerase proteins prohibiting them from their roles in DNA replication and perhaps
targeting them for degradation. If cyclin A is altered it could not activate DNA
polymerase δ (Voitenleitner et al., 1997), which could also decrease DNA synthesis
rates. If cyclin A, DNA polymerase α and δ showed signs interaction with the radioactive
DPH this would shed light on the overall decrease in DNA synthesis in DPH treated
embryos and could possibly explain the some of the characteristics of FHS mainly due
48
to possible decrease in proofreading properties of DNA polymerase δ (Venkatesan et
al., 2007).
4). Finally, it is important to determine if DPH is decreasing DNA synthesis in S
phase of the second cell cycle via a decrease in activity of DNA polymerase α vs. δ. It
is difficult to separate the activity of the two enzymes from one another. One possible
experiment that could address this issue is the use of siRNA technology in a short term
embryo culture experiment to knock out either DNA polymerase α or δ at any one time.
Following siRNA treatment, embryos would be subjected to an EdU DNA synthesis
assay to determine the rate of synthesis in the absence of DNA polymerase α and then
alternatively in the absence of DNA polymerase δ using both DPH and NaOH treated
embryos. Alternatively, the determination of activity of each polymerase enzyme
independently could also be determined by using a radioactive deoxythymidine DNA
polymerase assay specific for DNA polymerase α and δ in embryo extracts. This is
possible because each enzyme has an optimal buffer preference for DNA synthesis in
vitro. Syvaoja and Linn (1989) demonstrated that DNA polymerase δ from HeLa cells
worked optimally in DNA labeling experiments using a 50 mM Hepes-KOH buffer while
DNA polymerase α was optimal in 50 mM Tris-HCl without potassium. Each experiment
would compare the rate of DNA synthesis by 3H-thymidine incorporation into DNA over
time in DPH treated and NaOH treated embryos. This would distinguish affects on the
activity of DNA polymerase α separate from DNA polymerase δ. Since cyclin A is
increased in G1 of the second cell cycle (Tolle and Chatot, 2009) and cyclin A is
responsible for inhibiting DNA polymerase α initiation (Voitenleitner et al. 1997), DNA
polymerase α activity could be decreased slowing down the initiation of DNA replication
49
(Otto et, al,. 2002). Cyclin A was also decreased in S phase of the second cell cycle
(Tolle and Chatot, 2009). According to Bashir et al. (2000), cyclin A/cdk2 binds to DNA
polymerase δ during DNA replication and increases the rate of elongation of DNA
strands thus regulating the length of and exit from S phase, so a decline in cyclin A
could also decrease the activity of DNA polymerase δ.
These future experiments would help to shed additional light on the cause(s) of
the DPH induced decrease in DNA synthesis in S phase of the second cell cycle;
particularly focusing on the affect DPH has on DNA replication. The mechanisms behind
the decrease in DNA synthesis could lead to a better understanding of how DPH causes
defects in FHS as a whole.
50
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Waclaw, R. R. and C. L. Chatot. 2004. Patterns of expression of cyclins A, B1, D, E and
cdk 2 in preimplantation mouse embryos. Zygote 12:19-30.
54
Appendix A
EXP #/Treatment/Embryos
5 Min NaOH
EXP1 NAOH 5 M #1
EXP1 NAOH 5 M #2
EXP1 NAOH 5 M #3
EXP1 NAOH 5 M #4
EXP1 NAOH 5 M #5
EXP1 NAOH 5 M #6
EXP1 NAOH 5 M #7
EXP2 NAOH 5M #1
EXP2 NAOH 5M #2
EXP2 NAOH 5M #3
EXP2 NAOH 5M #4
EXP2 NAOH 5M #5
EXP2 NAOH 5M #6
EXP2 NAOH 5M #7
EXP2 NAOH 5M #8
EXP2 NAOH 5M #9
EXP2 NAOH 5M #10
EXP2 NAOH 5M #11
EXP3 NaOH 5M #1
EXP3 NaOH 5M #2
Nuclear
Intensity
Standard
Dev.
Area
260.35
308.43
216.16
237.88
309.09
281.42
248.12
253.53
203.27
212.68
223.54
221.26
277.06
310.23
1045.12
900.13
1311.66
1168.87
2018.97
2153.03
3787.05
3542.96
1706.29
1645.34
3353.43
3333.69
1661.1
1724.64
587.5
514.59
2389.37
2370.93
2016.9
1735.33
868.07
752.29
1747.22
1765.4
1059.59
170.9
188.15
158.99
168.28
188.52
175.27
168.13
166.78
150.88
153.54
157.85
156.9
175.52
190.23
358.79
340.4
146.16
375.88
510.65
552.45
362.76
470.5
463.17
479.48
538.46
610.52
466.07
486.27
260.26
244.51
603.48
590.65
585.34
496.51
316.28
298.89
449.63
434.61
337.35
55
285.97
285.97
287.39
289.24
290.73
291.83
290.37
290.37
290.37
290.37
290.37
290.37
287.23
287.23
294.81
291.34
291.15
291.15
290.15
290.15
285.77
285.77
286
286
282.18
282.18
289.08
284.51
283.54
283.54
286.87
286.67
286.91
285.22
285.16
285.16
287.75
287.04
286.91
Cytoplasm N-C
Intensity
Intensity
183.01
221.66
151.36
172.89
197.71
168.97
174.51
180.14
141.37
158.39
178.21
159.1
212.27
228.75
615.36
566.97
735.18
291.15
1297.06
1014.84
1990.38
2456.55
898.13
1368.52
2119.91
1541.86
796.51
798.53
419.69
387.96
1422.95
1283.16
1127.07
814.72
332.7
477.68
932.68
1191.5
786.31
77.34
86.77
64.8
64.99
111.38
112.45
73.61
73.39
61.9
54.29
45.33
62.16
64.79
81.48
429.76
333.16
576.48
877.72
721.91
1138.19
1796.67
1086.41
808.16
276.82
1233.52
1791.83
864.59
926.11
167.81
126.63
966.42
1087.77
889.83
920.61
535.37
274.61
814.54
573.9
273.28
EXP3 NaOH 5M #3
EXP3 NaOH 5M #4
EXPP8 NAOH 5M #1
1528.5
1588.64
2597.12
1637.92
1671.98
835.04
750.35
401.33
405.05
496.11
408.82
410.07
303.2
281.56
286.13
288.53
288.53
288.17
288.17
282.28
282.28
2829.84
2685.22
1447.91
1577.52
573.7
769.71
2273.1
1872.75
1693.96
1530.6
2749.78
2402.69
810.7
672.94
813.65
767.32
618.76
824.14
1013.45
861.4
688.29
636.01
371.18
318.66
412.69
361.23
1338.64
1134.59
1042.95
993.93
1109.49
895.76
1102.9
864.6
854
591.44
594.7
477.69
511.34
254.98
308.61
730.54
519.79
553.07
465.73
592.94
560.54
346.73
303.92
392.09
367.8
308.97
373.93
379.33
347.67
322.76
303.74
215.22
201.4
215.94
199.42
411.87
365.59
350.28
340.75
355.87
314.93
358.03
321.01
320.62
290.6
289.69
287.91
282.7
283.12
283.77
291.02
285.39
285.58
281.6
281.6
281.6
285.32
285.32
289.05
287.68
289.79
289.79
290.99
288.24
284.7
289.14
289.05
286.84
288.98
288.98
290.47
288.49
288.49
288.49
288.3
290.47
286.03
288.07
290.02
1091.51
436.99
1260.26
328.38
2043.65
553.47
1497.45
140.47
1185.33
486.65
918.57
-83.53
855.22
-104.87
Avg N-C
484.4421739
10 MIN NaOH
EXP2 NAOH 10M #1
EXP2 NAOH 10M #2
EXP2 NAOH 10M #3
EXP2 NAOH 10M #4
EXP2 NAOH 10M #5
EXP2 NAOH 10M #6
EXP2 NAOH 10M #7
EXP2 NAOH 10M #8
EXP2 NAOH 10M #9
EXP2 NAOH 10M #10
EXP2 NAOH 10M #11
EXP2 NAOH 10M #12
EXP2 NAOH 10M #13
EXP3 NAOH 10M #1
EXP3 NAOH 10M #2
EXP3 NAOH 10M #3
EXP3 NAOH 10M #4
EXP3 NAOH 10M #5
56
1829.93
1666.34
404.74
963.02
418.43
417.49
1077.51
1115.43
1077.6
921.61
1938.77
1652.9
415.17
297.56
683.17
691.92
496.59
586.43
422.94
318.69
417.76
361.4
275.13
212.47
312.9
181.41
765.91
885.48
726.65
596.84
770.66
638.96
779.33
558.72
720.15
999.91
1018.88
1043.17
614.5
155.27
352.22
1195.59
757.32
616.36
608.99
811.01
749.79
395.53
375.38
130.48
75.4
122.17
237.71
590.51
542.71
270.53
274.61
96.05
106.19
99.79
179.82
572.73
249.11
316.3
397.09
338.83
256.8
323.57
305.88
133.85
EXP3 NAOH 10M #6
EXP3 NAOH 10M #7
EXP4 NaOH 10 M #1
EXP4 NaOH 10 M #2
EXP4 NaOH 10 M #3
EXP4 NaOH 10 M #4
881.03
1009.46
588.45
993.05
899.79
3276.1
3338.06
2992.54
3009.43
3481.16
3576.88
3907.48
3993.48
328.73
353.46
285.38
339.79
317.14
516.15
501.83
551.78
533.76
481.2
456.47
284.11
205.2
287.62
291.02
290.76
288.66
288.98
285.94
279.3
285.32
291.15
291.96
283.31
285.06
282.92
697.4
629.49
400.61
647.23
740.05
2786.57
2986.56
2364.71
2278.13
2702.59
3004.18
3542.59
3575.56
Avg N-C
183.63
379.97
187.84
345.82
159.74
489.53
351.5
627.83
731.3
778.57
572.7
364.89
417.92
435.526875
1296.83
965.54
1490.24
1504.27
1600.64
1446.95
696.56
578.45
822.63
1160.78
732.34
667.95
1831
1824.28
2260.69
2103.85
1699.79
1740.75
1846.54
1782.6
1721.88
1673.15
1230.75
1744.47
2743.95
2624.31
1624.73
1606.29
1933.1
1710.81
389.5
392.81
544.05
521.01
496.11
465.8
284.32
250.09
334.63
397.78
282.93
269.61
479.89
484.58
513.59
487.79
454.38
469.87
471.51
457.97
438.64
432.61
365.63
439.1
540.57
536.4
442.87
422.25
479.67
440.91
286.68
286.68
287.13
280.36
288.11
288.79
289.14
289.14
288.04
288.04
286.68
286.68
291.64
288.4
288.95
291
288.2
288.24
289.01
286.62
287.94
289.21
289.82
290.73
287.59
287.59
290.76
291.05
291.55
287.65
691.38
674.03
663.66
745.84
720.56
586.62
372.65
340.7
338.19
485.57
440.33
327.35
1467
1413.38
1500.66
1608.35
1214.63
1386.29
1442.15
1284.33
1342.96
1314.47
865.01
1490.71
1735.26
1859.07
1403.24
1244.42
1557.94
1271.98
605.45
291.51
826.58
758.43
880.08
860.33
323.91
237.75
484.44
675.21
292.01
340.6
364
410.9
760.03
495.5
485.16
354.46
404.39
498.27
378.92
358.68
365.74
253.76
1008.69
765.24
221.49
361.87
375.16
438.83
20 Min NaOh
EXP1 NaOH 20 M #1
EXP2 NaOH 20M #1
EXP2 NAOH 20M #2
EXP2 NaOH 20M #3
EXP2 NaOH 20M #4
EXP2 NaOH 20M #5
EXP3 NaOH 20M #1
EXP3 NaOH 20M #2
EXP3 NaOH 20M #3
EXP3 NaOH 20M #4
EXP3 NaOH 20M #5
EXP3 NaOH 20M #6
EXP3 NaOH 20M #7
EXP3 NaOH 20M #8
EXP3 NaOH 20M #9
57
EXP4 NaOH 20M #1
2701.49
2974.93
652.22
658.67
285.42
280.62
1255.38
1421.02
899.69
1016.68
1491.79
1162.92
1061.06
1141.35
979.51
1418.77
1551.67
1621.67
1638.66
1587.27
1258.19
1684.17
1453.72
1835.57
2305.34
2233.41
1811.47
1823.93
1666.09
1167.07
1604.53
1433.8
1526.35
1424.12
968.71
965.39
1987.58
1844.08
1263.13
1468.86
1561.18
1311.4
1414
1714.24
1650.83
866.98
360.51
414.1
305.61
326
386.58
345.69
330.96
344.5
318.96
382.67
394.4
405.98
406.48
402.2
365.3
417.06
385.73
429.33
476.44
472.41
434.17
428.69
406.5
341.62
404.06
378
386.53
381.88
311.45
318.91
447.85
434.3
357.24
386.26
403.48
364.83
383.62
514.54
484.74
297.46
280.34
285.48
283.89
287
285.97
282.83
280.66
282.37
285.03
288.62
288.27
289.21
286.49
288.11
284.38
289.5
288.17
288.01
284.54
284.54
289.89
289.89
289.69
289.89
290.6
289.6
290
283.89
292.89
290.89
290.08
280.53
288.82
286.23
289.79
289.41
291.34
288.24
287.2
280.2
1980.99
720.5
2304.49
670.44
Avg N-C
508.3853125
40 Min NaOH
EXP1 NAOH 40M #1
EXP1 NAOH 40M #2
EXP1 NAOH 40M #3
EXP1 NAOH 40M #4
EXP1 NAOH 40M #5
EXP1 NAOH 40M #6
EXP1 NAOH 40M #7
EXP1 NAOH 40M #8
EXP1 NAOH 40M #9
EXP1 NAOH 40M #10
EXP1 NAOH 40M #11
EXP1 NAOH 40M #12
EXP1 NAOH 40M #13
EXP1 NAOH 40M #14
EXP1 NAOH 40M #15
EXP1 NAOH 40M #16
EXP1 NAOH 40M #17
EXP1 NAOH 40M #18
EXP1 NAOH 40M #19
EXP1 NAOH 40M #20
EXP1 NAOH 40M #21
58
1251.7
960.97
980.35
1035.29
1417.39
1128.8
896.12
742.15
826.19
1321.32
1439.41
1437.38
1480.78
1480.61
982.47
1582.35
1065.02
1745.48
2146.17
2117.5
1450.03
1583.82
1572.07
1137.13
1538.89
1326.71
1359.71
1037
923.42
871.68
1813.31
1646.01
936.58
1425.4
1286.34
1241.71
1385.82
1430.23
831.84
709.94
3.68
460.05
-80.66
-18.61
74.4
34.12
164.94
399.2
153.32
97.45
112.26
184.29
157.88
106.66
275.72
101.82
388.7
90.09
159.17
115.91
361.44
240.11
94.02
29.94
65.64
107.09
166.64
387.12
45.29
93.71
174.27
198.07
326.55
43.46
274.84
69.69
28.18
284.01
818.99
157.04
EXP1 NAOH 40M #22
EXP 2 NaOH 40M#1
EXP 2 NaOH 40M#2
EXP 2 NaOH 40M#4
EXP 2 NaOH 40M#5
EXP 2 NaOH 40M#6
EXP 2 NaOH 40M#7
EXP 2 NaOH 40M#8
EXP 2 NaOH 40M#9
EXP 2 NaOH 40M#10
EXP 2 NaOH 40M#11
EXP 2 NaOH 40M#12
EXP 2 NaOH 40M#13
EXP 3 NaOH 40M #1
EXP 3 NaOH 40M #2
EXP 3 NaOH 40M #3
EXP 3 NaOH 40M #4
EXP 3 NaOH 40M #5
EXP 3 NaOH 40M #6
EXP 3 NaOH 40M #7
EXP 3 NaOH 40M #8
EXP 3 NaOH 40M #9
EXP 4 EDU 40MIN #1
599.74
1055.64
951.15
637.67
670.43
1658.99
1937.11
561.65
531.75
853.69
838.48
1111.05
992.96
831.28
830.72
714.62
752.71
731.67
799.8
776.6
755.49
841.2
737.48
783.06
706.58
781.13
797.56
1540.19
1596.85
1690.14
1600.6
986.29
874.93
963.99
942.3
1482.74
1312.18
1473.52
1420.7
910.99
1100.35
694.67
713.78
942.73
949.14
1108.18
253.57
358.16
347.33
245.59
270.43
492.96
520.01
252.57
241.86
354.77
338.04
370.78
347.91
305.65
304.44
279.31
298.5
284.34
296.69
297.65
288.01
311.68
289.31
294.04
278.27
294.86
297.86
424.23
425.46
427.69
432.14
333.93
312.34
334.55
325.8
436.82
414.31
434.4
424
343.93
378.66
295.32
297.12
327.15
331.04
484.15
59
280.2
288.17
279.88
288.17
291.86
276.9
285.32
283.77
283.77
290.63
285.81
291.28
286.45
287.94
290.63
288.1
289.76
286.97
290.47
285.74
287.53
287.88
290.08
287.65
288.4
286.23
288.49
290
288.17
288.69
291.38
285.32
285.32
289.66
288.33
287.04
287.81
288.85
288.56
287.52
290.44
286.19
280.4
289.53
285.29
287.33
580.38
550.72
492.85
599.16
609.78
813.81
926.01
493.01
453.71
653.16
660.07
898.91
776.71
618.51
593.63
540.67
538.09
606.2
610.7
604.57
585.31
635.71
500.89
662.62
603.8
583.56
623.68
1314.78
1198.16
1122.4
1029.91
827.19
731.54
767.35
792.48
999.91
770.08
931.25
840.59
784.97
770.69
555.94
595.77
766.64
783.91
741.62
19.36
504.92
458.3
38.51
60.65
845.18
1011.1
68.64
78.04
200.53
178.41
212.14
216.25
212.77
237.09
173.95
214.62
125.47
189.1
172.03
170.18
205.49
236.59
120.44
102.78
197.57
173.88
225.41
398.69
567.74
570.69
159.1
143.39
196.64
149.82
482.83
542.1
542.27
580.11
126.02
329.66
138.73
118.01
176.09
165.23
366.56
EXP 4 EDU 40MIN #2
EXP 4 EDU 40MIN #3
EXP 4 EDU 40MIN #4
EXP 4 EDU 40MIN #5
EXP 4 EDU 40MIN #6
EXP 4 EDU 40MIN #7
EXP 4 EDU 40MIN #8
EXP 4 EDU 40MIN #9
1063.62
3360.12
3288.99
3273.02
3262.35
3490.39
3479.88
3434.24
3508.3
3592.36
3580.34
3445.25
3423.11
3455.91
3389.62
2710.87
3041.9
420.94
575.16
569.48
634.28
632.08
546.46
533.32
526.6
515.79
509.5
520.34
580.51
585.74
559.52
590.32
716.07
674.19
287.98
291.83
290.73
288.62
288.98
291.18
287.72
286.52
290.66
284.06
285.94
288.66
288.53
287.49
286.45
289.17
289.21
724.21
339.41
2370.39
989.73
2752.01
536.98
2418.89
854.13
2320.87
941.48
2686.77
803.62
2626.01
853.87
2852.76
581.48
2815.03
693.27
2883.13
709.23
2976.98
603.36
2726.71
718.54
2395.1
1028.01
2807.07
648.84
2782.81
606.81
2392.75
318.12
2459.4
582.5
Avg N-C
302.5140777
1243.18
1207.59
1452.18
1449.11
1129.92
1144.62
1255.38
1205.29
1368.84
1267.97
1230.19
1215.52
1126.45
1239.49
1486.21
1540.15
382.41
363.88
415.69
414.3
363.09
364.97
381.68
380.43
395.88
379.07
365.26
366.13
360.7
370.84
417.11
415.54
287.88
287.88
289.47
291.42
293.42
293.42
292.01
288.85
285.03
288.33
287.26
286.52
287.91
288.75
286.71
288.69
1157.04
1070.73
1259.56
1251.81
960.47
950.65
1114.88
1158.8
1162.82
931.06
1123.65
1142.29
1023.95
1127.29
1295.13
1371.85
Avg N-C
86.14
136.86
192.62
197.3
169.45
193.97
140.5
46.49
206.02
336.91
106.54
73.23
102.5
112.2
191.08
168.3
153.756875
554.29
505.98
617.78
586.79
579.45
523.37
247.37
233.61
259.66
256.82
253.63
241.98
287.75
284.06
289.79
291.28
290.24
287.2
466.82
498.34
545.23
487.72
500.89
437.79
87.47
7.64
72.55
99.07
78.56
85.58
No EDU NAOH
EXP 3 NAOH NO EDU 40MIN #1
EXP 3 NAOH NO EDU 40MIN #2
EXP 3 NAOH NO EDU 40MIN #3
EXP 3 NAOH NO EDU 40MIN #4
EXP 3 NAOH NO EDU 40MIN #5
EXP 3 NAOH NO EDU 40MIN #6
EXP 3 NAOH NO EDU 40MIN #7
EXP 3 NAOH NO EDU 40MIN #8
5 Min DPH
EXP 6 DPH EDU 5M #1
EXP 6 DPH EDU 5M #2
EXP 6 DPH EDU 5M #3
60
EXP 6 DPH EDU 5M #4
EXP 7 DPH EDU 5M #1
EXP 7 DPH EDU 5M #2
EXP 7 DPH EDU 5M #3
EXP 7 DPH EDU 5M #4
EXP 7 DPH EDU 5M #5
EXP 7 DPH EDU 5M #6
EXP 7 DPH EDU 5M #7
EXP 7 DPH EDU 5M #8
EXP 7 DPH EDU 5M #9
EXP 7 DPH EDU 5M #10
EXP 7 DPH EDU 5M #11
EXP 7 DPH EDU 5M #12
EXP 7 DPH EDU 5M #13
EXP 7 DPH EDU 5M #14
EXP 7 DPH EDU 5M #15
EXP 7 DPH EDU 5M #16
EXP 7 DPH EDU 5M #17
EXP 7 DPH EDU 5M #18
EXP 7 DPH EDU 5M #19
EXP 7 DPH EDU 5M #20
EXP 5 DPH EDU 5M #1
EXP 5 DPH EDU 5M #2
490.89
530.6
540.6
540.75
478.37
509.47
438.57
472.7
517.86
466.19
633.49
601.1
565.82
487.1
548.79
561.53
598.66
618.25
579.28
601.08
615.77
652.45
518.16
540.95
650.87
609.07
577.03
582.28
643
628.31
649.96
623.38
643.93
654.57
534.1
537.75
625.72
557.48
550.74
532.22
613.7
645.08
454.85
446.81
644.86
501.39
238.53
241.89
256.62
250.01
231.06
238.03
226.51
232.99
245.88
227.4
263.05
259.51
254.65
235.6
252.33
252.9
256.95
263.35
250.82
256.91
263.71
268.45
241.16
244.37
269.9
256.52
253.04
250.5
266.61
261.81
266.52
256.66
263.57
267.47
240.99
242.22
271.7
259.6
246.94
242.65
256.18
263.97
229.79
225.27
266.18
235.42
61
289.47
291.73
282.47
290.7
290.76
283.6
284.02
289.05
290.79
290.79
281.95
284.87
286
281.37
284.74
286.1
283.9
284.58
283.9
283.9
290.18
291.02
289.5
289.65
290.41
285.22
279.88
283.09
288.88
286.56
282.37
285.87
291.28
289.34
289.82
284.38
289.56
289.67
285.64
282.96
283.65
282.96
284.87
291.86
290.28
290.08
418.85
446.3
425.64
459.2
423.4
471.37
424.63
384.4
430.93
420.01
574.85
506.28
469.42
415.35
465.8
455.42
513.32
601.39
497.56
562.33
465.7
696.19
510.74
498.27
533.72
530.45
508.5
555.42
500.36
613.07
558.79
543.92
634.66
613.61
419.5
531.99
455.03
450.71
469.55
419.25
607.37
571.42
352.82
312.95
521.46
409.52
72.04
84.3
114.96
81.55
54.97
38.1
13.94
88.3
86.93
46.18
58.64
94.82
96.4
71.75
82.99
106.11
85.34
16.86
81.72
38.75
150.07
-43.74
7.42
42.68
117.15
78.62
68.53
26.86
142.64
15.24
91.17
79.46
9.27
40.96
114.6
5.76
170.69
106.77
81.19
112.97
6.33
73.66
102.03
133.86
123.4
91.87
EXP 5 DPH EDU 5M #3
EXP 5 DPH EDU 5M #4
EXP 5 DPH EDU 5M #5
EXP 5 DPH EDU 5M #6
482.25
449.56
484.26
421.5
323.53
263.76
467.78
465.97
232.93
218.73
228.83
218.9
191.51
170.85
225.83
228.26
285.94
288.3
285.48
286.62
290.5
291.25
288.2
289.56
423.54
344.52
431.81
276.84
242.32
157.01
379.76
351.29
Avg N-C
58.71
105.04
52.45
144.66
81.21
106.75
88.02
114.68
75.775
777.93
684.83
827.36
590.17
571.61
571.68
663.07
733.82
711.01
850.32
828.02
644.74
586.58
932.84
1085.57
1108.56
801.91
620.16
923.87
1171.93
1231.98
783.25
1218.23
1209.61
872.89
784.9
1190.01
659.02
577.07
512.12
484.29
934.95
969.17
724.45
1090.32
291.07
271.74
303.64
252.76
250.93
247.55
268.34
287.96
281.2
307.67
305.33
265.37
252.4
315.9
344.23
348.86
297.15
264.38
324.42
358.43
363.09
298.67
366.75
369.34
311.32
278.84
252.89
270.83
252.81
236.25
230.98
334.86
325.42
280.95
354.44
286.91
287.72
289.69
288.53
289.76
285.64
283.64
287.78
285.61
289.76
287.56
283.28
287.85
288.79
285.97
290.41
289.73
288.29
284.25
285.48
288.01
282.02
285.51
287.52
288.3
288.3
288.59
290.05
285.61
289.17
289.17
287.62
281.66
283.77
288.07
531.83
491.81
589.65
391.12
458.39
458.59
528.74
559.58
514.88
525.2
608.48
381.08
407.5
739.61
747.62
839.4
556.77
465.78
580.5
754.65
843.94
520.81
923.32
826
621.88
678.81
827.05
450.85
464.97
301.58
334.99
603.36
619.62
537.55
667.2
246.1
193.02
237.71
199.05
113.22
113.09
134.33
174.24
196.13
325.12
219.54
263.66
179.08
193.23
337.95
269.16
245.14
154.38
343.37
417.28
388.04
262.44
294.91
383.61
251.01
106.09
362.96
208.17
112.1
210.54
149.3
331.59
349.55
186.9
423.12
20 Min DPH
EXP 5 DPH EDU 20M #1
EXP 5 DPH EDU 20M #2
EXP 5 DPH EDU 20M #3
EXP 5 DPH EDU 20M #4
EXP 5 DPH EDU 20M #5
EXP 5 DPH EDU 20M #6
EXP 5 DPH EDU 20M #7
EXP 5 DPH EDU 20M #8
EXP 5 DPH EDU 20M #9
EXP 5 DPH EDU 20M #10
EXP 5 DPH EDU 20M #11
EXP 5 DPH EDU 20M #12
EXP 5 DPH EDU 20M #13
EXP 5 DPH EDU 20M #14
EXP 5 DPH EDU 20M #15
EXP 5 DPH EDU 20M #16
EXP 5 DPH EDU 20M #17
EXP 5 DPH EDU 20M #18
EXP 5 DPH EDU 20M #19
EXP 5 DPH EDU 20M #20
EXP 5 DPH EDU 20M #21
EXP 5 DPH EDU 20M #22
62
EXP 5 DPH EDU 20M #23
EXP 5 DPH EDU 20M #24
EXP 5 DPH EDU 20M #25
EXP 5 DPH EDU 20M #26
EXP 5 DPH EDU 20M #27
EXP 5 DPH EDU 20M #28
EXP 6 DPH EDU 20M #1
EXP 6 DPH EDU 20M #2
EXP 6 DPH EDU 20M #3
EXP 6 DPH EDU 20M #4
EXP 6 DPH EDU 20M #5
EXP 6 DPH EDU 20M #6
EXP 6 DPH EDU 20M #7
EXP 6 DPH EDU 20M #8
EXP 6 DPH EDU 20M #9
EXP 6 DPH EDU 20M #10
EXP 6 DPH EDU 20M #11
EXP 7 DPH EDU 20M #1
EXP 7 DPH EDU 20M #2
EXP 7 DPH EDU 20M #3
EXP 7 DPH EDU 20M #4
EXP 7 DPH EDU 20M #5
EXP 7 DPH EDU 20M #6
EXP 7 DPH EDU 20M #7
1300.93
1302.29
1346.25
1218.27
724.53
1159.06
951.94
933.56
1093.93
1475.05
1598.95
1212.18
1173.5
979.01
919.29
981.05
919.52
1068.59
1086.54
1181.02
1250.29
942.87
872.3
957.41
1013.75
1069.94
1001.3
1391.43
1236.42
1327.16
1194.73
1164.15
1141.61
1738.93
1826.18
1737.59
1740.17
1754.85
1706.23
1662.94
1718.89
1458
1456.23
1413.74
1497.14
1525.35
380.11
377.39
380.58
372.78
282.34
357.35
323.62
325.49
344.28
402.79
420.26
387.72
372.31
335.15
337.52
349.31
335.54
360.69
362.97
366.28
380.29
353.52
336.06
356.12
366.44
370.41
357.53
411.72
405.36
409.7
384.8
399.74
390.23
482.25
479.31
466.57
464.12
487.13
479.51
440.29
447.38
417.14
412.46
397.55
428.17
428.65
63
287.49
295.36
288.43
284.25
284.25
287.36
286.54
286.99
285.03
292.15
292.15
292.58
292.64
288.72
289.98
290.41
292.02
290.83
290.28
294.26
291.41
291.83
288.04
288.53
289.63
291.15
287.39
286.62
281.89
288.17
292.64
291.64
290.57
285
289.92
290.54
287.81
283.41
290.18
292.51
293.09
286.75
283.64
287.78
286.75
286.68
921.23
1059.58
112.83
972.6
618.56
709.91
579.5
661.39
833.05
1025.85
1015.06
915.76
934.84
682.19
706.93
792.74
708.3
828.01
775.23
931.06
997.93
731.35
679.59
779.69
747.95
940.22
849.89
1184.13
1001.28
1192.66
1047.04
868.53
863.92
1368.02
1427.83
1586.98
1291.42
1262.93
1334.15
1362.08
1412.58
1298.03
1223.2
1012.26
1144.09
1219.38
379.7
242.71
1233.42
245.67
105.97
449.15
372.44
272.17
260.88
449.2
583.89
296.42
238.66
296.82
212.36
188.31
211.22
240.58
311.31
249.96
252.36
211.52
192.71
177.72
265.8
129.72
151.41
207.3
235.14
134.5
147.69
295.62
277.69
370.91
398.35
150.61
448.75
491.92
372.08
300.86
306.31
159.97
233.03
401.48
353.05
305.97
EXP 7 DPH EDU 20M #8
EXP 7 DPH EDU 20M #9
EXP 7 DPH EDU 20M #10
EXP 7 DPH EDU 20M #11
EXP 7 DPH EDU 20M #12
EXP 7 DPH EDU 20M #13
EXP 7 DPH EDU 20M #14
EXP 7 DPH EDU 20M #15
EXP 7 DPH EDU 20M #16
EXP 7 DPH EDU 20M #17
EXP 7 DPH EDU 20M #18
EXP 7 DPH EDU 20M #19
EXP 7 DPH EDU 20M #20
EXP 7 DPH EDU 20M #21
EXP 7 DPH EDU 20M #22
EXP 7 DPH EDU 20M #23
EXP 7 DPH EDU 20M #24
1575.23
1483.53
1497.69
1367.41
1407.29
1476.93
1324.32
1512.17
1458.99
1305.58
1464.82
1498.87
1477.37
1538.7
1489.06
1599.22
1639.29
1478.13
1483.63
1511.94
1562.64
1539.66
1549.66
1439.05
1482.23
1558.24
1557.13
1526.41
1445.74
1516.59
1416.8
1554.44
1572.96
1451.35
439.06
404.73
412.17
408.08
418.32
417.24
338.01
439.15
417.25
396.3
405.7
435.34
429.05
431.94
426.63
426.8
434.4
424.34
421.09
414.1
421.03
442.9
440.7
413.83
430.05
441.3
431.48
433.31
422.86
444.79
420.1
426.65
422.7
414.35
289.79
287.98
287.78
291.41
286.56
283.64
288.33
282.92
286.55
284.22
288.3
287.04
290.05
288.2
283.12
291.8
284.67
290.89
291.6
288.04
287.78
289.08
284.38
289.79
291.7
287.46
283.67
285.61
289.53
288.46
285.68
286.07
287.36
285.71
444.89
450.36
629.92
497.26
504.41
482.31
464.307
442.84
225.71
229.43
262.3
235.92
237.04
230.85
227.64
221.53
292.71
291.67
285.51
285.45
287.17
289.37
285.45
285.65
1291.81
283.42
1087.6
395.93
1137.24
360.45
1047.72
319.69
1104.68
302.61
1208.15
268.78
929.42
394.9
1028.87
483.3
1121.94
337.05
1194.59
110.99
1126.09
338.73
1169.18
329.69
1101.66
375.71
1032.37
506.33
1107.91
381.15
1205.06
394.16
1310.08
329.21
1057.08
421.05
1122.7
360.93
1274.47
237.47
1290.06
272.58
1203.16
336.5
1169.52
380.14
1328.7
110.35
1379.98
102.25
1282.86
275.38
1126.39
430.74
1217.75
308.66
1122.55
323.19
1241.44
275.15
1244
172.8
1316.39
238.05
1244.76
328.2
1223.66
227.69
Avg N-C
287.8406087
10 Min DPH
EXP 5 DPH EDU 10 MIN #1
EXP 5 DPH EDU 10 MIN #2
EXP 5 DPH EDU 10 MIN #3
EXP 5 DPH EDU 10 MIN #4
64
321.75
331.28
471.45
422.04
480.83
383.42
421.05
383.54
123.14
119.08
158.47
75.22
23.58
98.89
43.257
59.3
EXP 5 DPH EDU 10 MIN #5
EXP 5 DPH EDU 10 MIN #6
EXP 6 DPH EDU 10 MIN #1
EXP 6 DPH EDU 10 MIN #2
EXP 6 DPH EDU 10 MIN #3
EXP 6 DPH EDU 10 MIN #4
EXP 6 DPH EDU 10 MIN #5
EXP 6 DPH EDU 10 MIN #6
EXP 6 DPH EDU 10 MIN #7
EXP 6 DPH EDU 10 MIN #8
EXP 6 DPH EDU 10 MIN #9
EXP 6 DPH EDU 10 MIN #10
EXP 6 DPH EDU 10 MIN #11
EXP 6 DPH EDU 10 MIN #12
EXP 6 DPH EDU 10 MIN #13
EXP 6 DPH EDU 10 MIN #14
EXP 6 DPH EDU 10 MIN #15
EXP 6 DPH EDU 10 MIN #16
EXP 7 DPH EDU 10 MIN #1
EXP 7 DPH EDU 10 MIN #2
EXP 7 DPH EDU 10 MIN #3
EXP 7 DPH EDU 10 MIN #4
EXP 7 DPH EDU 10 MIN #5
295.09
235.01
466.95
458.43
822.66
735.11
942.07
905.97
873.67
928.69
834.79
716.83
897.51
901.37
843.95
787.07
761.39
747.08
981.03
1008.2
1185.33
1091.51
865.13
819.27
516.12
478.91
564.75
636.15
693.71
666.93
541.79
479.57
529.4
425.94
890.16
942.08
2723.13
2747.53
2710.75
2716.53
2789.57
2782.8
2357.51
2240.46
2340.45
2493.29
180.28
158.39
227.5
216.66
306.62
292.61
334.12
310.68
313.397
320.44
305.06
284.97
327.39
322.8
308.79
293.68
293.49
288.12
336.57
349.72
368.63
351.06
323.64
305.3
238.11
228.55
252.1
270.94
280.05
272.73
247.23
236.96
244.78
230.29
308.16
315.19
574.11
561.25
540.09
538
559.26
539.7
510.96
529.55
531.33
535.18
65
288.17
286.91
289.05
287.78
290.02
288.69
292.67
290.79
286.42
289.17
289.17
291.44
287.3
288.65
291.57
291.83
288.49
288.63
285.06
284.51
285.51
290.5
290.92
288.53
285.35
282.63
292.32
288.88
290.05
291.53
285.74
285.81
289.5
292.77
292.45
289.47
292.61
287.78
292.77
288.88
285.58
283.99
292.9
287.91
292.28
286.87
257.7
174.12
396.38
425.71
672.44
585.48
918.17
692.96
767.71
737.92
700.89
601.75
751.45
631.09
566.2
543.02
651.5
632.62
671.9
651.42
882.15
814.03
595.94
698.59
401.7
440.31
471.82
524.1
558.53
430.34
463.06
467.27
506.27
335.82
811.19
783.27
1855.34
2144.21
1914.99
2289.56
2204.26
2155.83
1685.1
1811.8
1641.43
1877.58
37.39
60.89
70.57
32.72
150.22
149.63
23.9
213.01
105.96
190.77
133.9
115.08
146.06
270.28
277.75
244.05
109.89
114.46
309.13
356.78
303.18
277.48
269.19
120.68
114.42
38.6
92.93
112.05
135.18
236.59
78.73
12.3
23.13
90.12
78.97
158.81
867.79
603.32
795.76
426.97
585.31
626.97
672.41
428.66
699.02
615.71
EXP 7 DPH EDU 10 MIN #6
EXP 7 DPH EDU 10 MIN #7
EXP 7 DPH EDU 10 MIN #8
EXP 7 DPH EDU 10 MIN #9
EXP 7 DPH EDU 10 MIN #10
EXP 7 DPH EDU 10 MIN #11
EXP 7 DPH EDU 10 MIN #11
EXP 7 DPH EDU 10 MIN #12
EXP 7 DPH EDU 10 MIN #13
EXP 7 DPH EDU 10 MIN #14
EXP 7 DPH EDU 10 MIN #15
EXP 7 DPH EDU 10 MIN #16
EXP 7 DPH EDU 10 MIN #17
EXP 7 DPH EDU 10 MIN #18
EXP 7 DPH EDU 10 MIN #19
EXP 7 DPH EDU 10 MIN #20
EXP 7 DPH EDU 10 MIN #21
EXP 7 DPH EDU 10 MIN #22
EXP 7 DPH EDU 10 MIN #23
EXP 7 DPH EDU 10 MIN #24
EXP 7 DPH EDU 10 MIN #25
2182.99
2326.4
2367.97
2250.62
2440.65
2421.73
2407.8
2382.61
2587.11
2483.5
2495.92
2440.46
2219.95
2302.02
2197.81
2291.59
2262.33
2313.24
2303.42
2195.98
2058.93
2147.18
2257.98
2258.65
2161.13
1911.09
2451.43
2434.53
1968.62
1998.07
2107.14
2129.03
2010.97
2012.05
2173.27
2170.91
2319.25
2375.46
2205.32
2177.74
2401.81
2335.33
500.67 289.73
525.75 287.26
532.04 291.05
506.51 292.33
541.76 290.79
562.46 289.63
546.11 282.66
520.22 280.66
549.35 291.86
532.62 289.92
563.42 290.24
546.33 288.66
524.24 283.34
519.34 284.74
512.38 291.73
516.15 287.75
526.1 2878.82
517.13 290.31
528.99 285.06
512.91 286.87
493.98 289.53
506.03 287.07
521.39 286.45
539.08 284.74
512.49 292.77
489.94 282.79
539.99 280.33
539.11 282.28
479.07 291.41
490.27 283.77
957.77 285.55
493.65
287.3
479.9 286.68
491.81
283.7
521.18 290.41
516.55 290.37
506.59
291.9
518.33 292.09
495.31 288.88
520.25 289.11
552.9 285.29
537.33 284.87
40 min DPH
66
1796.04
386.95
1912.88
413.52
1666.58
701.39
1753.71
496.91
1775.81
664.84
1799.72
622.01
1894.54
513.26
1864.22
518.39
1927.53
659.58
2209.12
274.38
1834.08
661.84
1875.92
564.54
1731.34
488.61
1698.99
603.03
1682.37
515.44
1664.96
626.63
1932.98
329.35
1754.7
558.54
1766.26
537.16
1810.11
385.87
1382.53
676.4
1634.02
513.16
1741.66
516.32
1571.15
687.5
1562.64
598.49
1458.25
452.84
1753.38
698.05
1799.52
635.01
1821.75
146.87
1587.99
410.08
1639.36
467.78
1624.27
504.76
1660.34
350.63
1603.45
408.6
1686.56
486.71
1597.29
573.62
1664.12
655.13
1982.31
393.15
1930.06
275.26
1698.75
478.99
1790.01
611.8
1762.99
572.34
Avg N-C
353.2644479
EXP 5 DPH EDU 40 MIN #1
EXP 5 DPH EDU 40 MIN #2
EXP 5 DPH EDU 40 MIN #3
EXP 5 DPH EDU 40 MIN #4
EXP 5 DPH EDU 40 MIN #5
EXP 5 DPH EDU 40 MIN #6
EXP 5 DPH EDU 40 MIN #7
EXP 5 DPH EDU 40 MIN #8
EXP 5 DPH EDU 40 MIN #9
EXP 5 DPH EDU 40 MIN #10
EXP 6 DPH EDU 40 MIN #1
EXP 6 DPH EDU 40 MIN #2
EXP 6 DPH EDU 40 MIN #3
EXP 6 DPH EDU 40 MIN #4
EXP 6 DPH EDU 40 MIN #5
EXP 6 DPH EDU 40 MIN #6
EXP 6 DPH EDU 40 MIN #7
EXP 6 DPH EDU 40 MIN #8
EXP 6 DPH EDU 40 MIN #9
EXP 6 DPH EDU 40 MIN #10
EXP 6 DPH EDU 40 MIN #11
EXP 6 DPH EDU 40 MIN #12
EXP 6 DPH EDU 40 MIN #13
1029.65
1073.29
1229.07
1246.83
949.11
1137.51
983.77
958.72
903.41
1004.06
898.19
1225.29
1359.06
1253.35
781.7
1057.47
806.03
847.29
1819.08
1922.65
941.92
853.4
511.21
560.6
1155.33
1032.75
1039.75
866.55
769.47
791.66
570.28
582.54
1006.13
958.45
931.3
816.71
1249.13
1080.05
536.71
573.68
906.86
925.88
929.63
928.11
1765.93
1685.49
332.91
343.34
370.19
371.4
320.71
345.71
320.23
318.7
313.4
324.4
309.89
364.06
684.33
368.63
29.45
328.2
299.5
307.09
446.86
456.78
335.73
314.79
248.89
261.67
365.93
345.6
352.91
320.17
303.78
301.2
252.48
254.73
332.47
323.54
326.14
302.95
379.89
348.65
249.64
262.17
333.95
333.19
324.58
324.45
428.54
420.53
67
287.52
285.16
292.74
289.24
289.01
287.72
290.02
291.64
291.64
287.43
283.51
283.51
292.48
285.09
284.22
291.67
287.39
291.28
287.2
285.64
287.36
286.36
286.84
288.66
288.62
288.11
288.53
290.21
285.29
291.18
289.24
291.96
285.45
291.12
285.13
291.99
285.68
291.44
285.68
285.48
288.72
281.56
288.4
291.28
290.96
290.96
896.12
956.57
970.89
1060.56
678.67
973.8
846.16
812.26
670.15
968.35
804.35
1103.96
940.91
1000.89
739.98
943.75
550.02
786.67
1455.25
1667.97
740.55
722.52
428.95
460.29
934.71
933.8
827.49
838.25
650.67
702.18
452.27
565.76
810.88
704.4
853.11
765.48
908.46
832.9
440.53
469.66
804.89
789.12
839.87
781.33
1596.47
1505.76
133.53
116.72
258.18
186.27
270.44
163.71
137.61
146.46
233.26
35.71
93.84
121.33
418.15
252.46
41.72
113.72
256.01
60.62
363.83
254.68
201.37
130.88
82.26
100.31
220.62
98.95
212.26
28.3
118.8
89.48
118.01
16.78
195.25
254.05
78.19
51.23
340.67
247.15
96.18
104.02
101.97
136.76
89.76
146.78
169.46
179.73
EXP 6 DPH EDU 40 MIN #14
EXP 7 DPH EDU 40 MIN #1
EXP 7 DPH EDU 40 MIN #2
EXP 7 DPH EDU 40 MIN #3
EXP 7 DPH EDU 40 MIN #4
EXP 7 DPH EDU 40 MIN #5
EXP 7 DPH EDU 40 MIN #6
EXP 7 DPH EDU 40 MIN #7
EXP 7 DPH EDU 40 MIN #8
EXP 7 DPH EDU 40 MIN #9
EXP 7 DPH EDU 40 MIN #10
EXP 7 DPH EDU 40 MIN #11
EXP 7 DPH EDU 40 MIN #12
EXP 7 DPH EDU 40 MIN #13
EXP 7 DPH EDU 40 MIN #14
EXP 7 DPH EDU 40 MIN #15
EXP 7 DPH EDU 40 MIN #16
EXP 7 DPH EDU 40 MIN #17
EXP 7 DPH EDU 40 MIN #18
1261.49
1075.54
2830.78
2658.32
1957.66
1884.75
1878.19
1933.84
1520
1430.04
1532.96
1546.85
1429.69
1545.49
1631.32
1567.04
1771.55
1629.41
1781.96
1655.94
1103.39
1173.21
1116.12
1048.73
1235.37
1209.75
1184.08
1166.17
1208.04
1087.8
1225.14
1488.31
1457.94
1460.2
1361.4
1360.46
1386.63
387.06
344.61
573.72
611.45
525
514.53
524.02
518.5
443.06
418.96
436.96
431.55
417.2
435.87
440.12
444.41
455.64
439.19
475.02
456.32
362.66
375.11
367.96
351.47
394.33
381.49
374.82
374.68
380.25
355.62
380.65
417.81
433.76
430.49
406.6
400.21
410.54
284.58
284.58
287.26
282.86
283.86
287.04
288.49
290.31
289.95
288.95
285.19
291.8
292.51
286.26
290.57
289.66
289.14
289.43
287.3
282.96
290.57
288.17
289.3
284.22
284.28
289.63
287
285.61
280.01
288.17
291.31
284.63
284.09
283.05
286.49
209.86
285.45
920.66
971.11
2282.72
2222.61
1646.83
1465.49
1521.27
1463.8
1171.36
1231.65
1244.39
1193.3
1355.16
1235.72
1336.96
1397.62
1511.62
1223.56
1378.66
1414.74
884.71
912.53
1066.96
970.29
1033.45
1055.72
914.93
912.19
1044.26
970.46
1188.26
1201.58
1238.97
1111.63
1136.44
944.7
980.39
Avg N-C
340.83
104.43
548.06
435.71
310.83
419.26
356.92
470.04
348.64
198.39
288.57
353.55
74.53
309.77
294.36
169.42
259.93
405.85
403.3
241.2
218.68
260.68
49.16
78.44
201.92
154.03
269.15
253.98
163.78
117.34
36.88
286.73
218.97
348.57
224.96
415.76
406.24
208.51
284.25
1202.96
48.65
DPH No Edu + Azide
EXP 5 DPH NO EDU + Azide #1
EXP 6 DPH NO EDU + Azide #1
no flourecence at all
1251.61
381.01
68
EXP 6 DPH NO EDU + Azide #2
EXP 6 DPH NO EDU + Azide #3
EXP & DPH NO EDU + Azide #1
EXP & DPH NO EDU + Azide #2
EXP & DPH NO EDU + Azide #3
EXP & DPH NO EDU + Azide #4
EXP & DPH NO EDU + Azide #5
EXP & DPH NO EDU + Azide #6
EXP & DPH NO EDU + Azide #7
EXP & DPH NO EDU + Azide #8
EXP & DPH NO EDU + Azide #9
EXP & DPH NO EDU + Azide #10
EXP & DPH NO EDU + Azide #11
1398.17
777.43
710.24
873.72
893.39
978.16
1031.72
821.69
801.44
626.84
535.94
590.74
625.66
1239.9
1211.05
1159.16
1137.04
987.85
967.38
531.56
530.77
629.82
603.45
1114.76
1134.05
576.15
572.26
402.65
293.9
279.32
314.26
327.37
326.44
338.34
301.24
295.09
261.81
241.7
256.33
264.91
362.74
357.91
350.34
350.57
320.83
327.86
243.2
245.22
264.23
262.62
344.19
350.05
255.3
255.08
284.35
289.14
290.11
288.62
284.02
280.53
287
288.98
280.55
282.6
284.67
286.65
285.74
291.9
281.69
281.43
281.47
286.03
285.84
280.66
280.94
287.13
287.67
282.37
282.37
282.37
282.37
1210.69
187.48
731.68
45.75
623.17
87.07
823.5
50.22
809.3
84.09
844.64
133.52
993.09
38.63
690.76
130.93
690.96
110.48
559.91
66.93
487.48
48.46
504.27
86.47
411.88
213.78
1178.69
61.21
1040.08
170.97
1062.74
96.42
1002.58
134.46
807.17
180.68
802.84
164.54
475.85
55.71
472.12
58.65
546.34
83.48
499.85
103.6
941.94
172.82
908.21
225.84
464.97
111.18
455
117.26
Avg N-C
109.6171429
1003.7
1075.03
337.92
348.87
287.13
281.43
948.84
962.24
54.86
112.79
515.76
484.37
239.66
230.66
280.11
291.47
464.34
424.32
51.42
60.05
1095.05
1070.51
359.84
356.23
289.34
286.78
847.1
921.45
247.95
149.06
707.01
678.6
281.6
282.12
285.45
280.9
646.44
619.83
60.57
58.77
305.31
288.25
186.76
178.87
285.42
290.47
267.02
266.28
38.29
21.97
DPH NO EDU
EXP 7 DPH 40 min EDU NO Azide
#1
EXP 7 DPH 40 min EDU NO Azide
#2
EXP 7 DPH 40 min EDU NO Azide
#3
EXP 7 DPH 40 min EDU NO Azide
#4
EXP 7 DPH 40 min EDU NO Azide
#5
69
EXP 7DPH 40 min EDU NO Azide
#7
278.61
315.56
173.73
187.88
70
287.85
288.83
290.55
237.6
Avg N-C
-11.94
77.96
76.8125