1. i
Effects of Changing Branched-Chain Amino Acid
and Insulin Levels In Vitro on Developing Pre-implantation
Embryos
By Tristan Demuth
Biology Bsc 2013
Supervisor: Professor Tom P. Fleming
Word Count: 9954

2. ii
Summary
If unborn babies (fetuses) are exposed to poor conditions in the uterus, they adapt
their development which can cause them to become more susceptible to diseases such as high
blood pressure and diabetes in adult life. This theory is called the ‘Developmental Theory of
Health and Disease’. The most common way that a fetus may be exposed to a poor
environment is through its mother’s diet.
My study will aid the understanding of how young embryos interact with their
environment during pregnancy. This is significant because it has been shown that if embryos
are exposed to a poor environment in the uterus, specifically during just the first few days of
pregnancy, then they are still at a greater risk of disease once they reach adult life. Therefore
it is important that the way which young embryos communicate with their environment in the
womb is better understood. A greater understanding will allow development of better medical
treatments and improve public health when women become better informed about dietary
requirements during pregnancy.
This project focussed on the effects of insulin and branched-chain amino acid levels
in the maternal environment. Branched-chain amino acids are a specific group of amino
acids, which are the building blocks of proteins. This was because, a study using mice
recently revealed that the level of both insulin and branched-chain amino acids available to
the embryo, are greatly reduced when the mother’s diet is poor (by means of a low protein
content). Therefore in this experiment, embryos were incubated in solutions containing
different levels of branched-chain amino acids and insulin. The results should show whether a
reduction in branched-chain amino acids and/or insulin is sufficient to cause the fetus to adapt
its own development. As stated above this adapted development indicates that the offspring
will be predisposed to disease once it reaches adulthood.

3. To measure whether the embryo had changed its development, the number of cells in
the embryo were analysed. For example, an increase in a specific cell type known as
iii
‘trophectoderm’ cells would indicate that the embryo had adapted its development.
The results from this experiment showed that reducing the levels of branched-chain
amino acids and insulin available to the early embryo is not sufficient to cause the embryo to
adapt its development. Therefore, further experiments are required to test other possible
methods that the early embryo may be using to detect a poor maternal environment.
Word Count: 399

4. iv
Abstract
The Developmental Origins of Health and Disease hypothesis states that challenges in
the maternal environment cause the developing fetus to undergo a predictive adaptive
response which can predispose the offspring to chronic disease in later life. The challenge
addressed here is maternal diet, specifically a low protein diet during the initial stages of
gestation, as the methods by which the maternal environment signals to the preimplantation
embryo are of great interest.
When mouse dams are fed a low protein diet during the preimplantation period it
causes a significant drop in the concentrations of branched-chain amino acids and insulin in
the maternal uterine fluid and serum. Therefore the focus of this experiment was to conclude
whether reducing the concentrations of branched-chain amino acids and insulin in vitro is
sufficient to cause the initiation of fetal programming, by culturing embryos from the 2-cell
to the late blastocyst stage in four different treatment groups. Blastocysts were analysed by
observation of developmental stage then by differential cell staining of late blastocysts
followed by fluorescence microscopy to measure cell number of trophectoderm and inner cell
mass. Statistical analysis was performed via chi-squared and Kruskal-Wallis tests.
The results from this experiment concluded that depletion of branched-chain amino
acid and insulin levels was not sufficient to initiate fetal programming by the late blastocyst
stage. A potential role for insulin levels in blastocyst nutrient sensing was observed but not a
significant one. This result means that further analysis of nutrient sensing in preimplantation
embryos is required.
Word Count: 248

5. v
Table of Contents
Title Page………………………… …………………………………………………………………………………
Summary…………………………………………………………………………………………………………….
Abstract………………………………………………………………………………………………………………
Table of Contents……………………………………………………………………………………………….
List of Abbreviations…………………………………………………………………………………………..
Acknowledgements…………………………………………………………………………………………….
1. Introduction
1.1 Developmental Origins of Health and Disease…………………………………………….
1.2 Developmental Re-programming caused by maternal under nutrition……….
1.3 Developmental Re-programming caused by maternal under nutrition
and culture conditions specifically during the preimplantation period………
1.4 Potential role for AA and insulin levels in induction of preimplantation
fetal programming………………………………………………………………………………………
1.5 Mammalian Target of Rapamycin (mTOR) regulation of cell growth
and development………………………………………………………………………………………..
1.6 Differing TE cell numbers as an indication of fetal programming…………………
1.7 Aims and objectives…………………………………………………………………………………….
2. Materials and Methods
2.1 Creation of culture media for four treatment groups………………………………….
2.2 Dissection and procuring of 2-cell stage embryos……………………………………….
2.3 Differential cell Staining
2.3.1 Differential Cell Staining Materials………………………………………………………….
2.3.2 Differential Cell Staining Protocol…………………………………………………………..
2.4 Blastocyst picture acquisition……………………………………………………………………..
2.5 Statistical analysis……………………………………………………………………………………….
3. Results
3.1 Activity of developing embryos…………………………………………………………………..
3.2 Effect of depleted branched-chain amino acids and insulin on fetal
programming…………………………………………………………………………………….....…….
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4. Discussion
4.1 Comparison with Original Hypothesis…………………………………………………………
4.2 Potential Methods used by the Preimplantation Embryo to
Communicate with the Maternal Environment after an Emb-LPD………………..
4.3 Evaluation of Experimental Techniques………………………………………………………..
4.4 Effects of In Vitro Culture……………………………………………………………………………..
4.5 Future Work…………………………………………………………………………………………………
4.6 Concluding Remarks …………………………………………………………………………………….
5. References……………………………………………………………………………………………………………

7. vii
List of Abbreviations
ACE - Angiotensin converting enzyme
Akt – Protein Kinase B
Anti-DNP – Anti Dinitrophenyl
BSA – Bovine serum albumin
CO2 – Carbon Dioxide
DNA – Deoxyribonucleic Acid
DOHaD – Developmental Origins of Health and Disease
E3.5 – Day 3.5 of embryo development
eIF# - Eukaryotic translation initiation factor
eIF4BP1 - Eukaryotic translation initiation factor binding protein 1
Emb-LPD – Low protein diet (9% caesin) fed exclusively during the preimplantation period
GR – Glucocorticoid receptor
ICM – Inner cell mass
IGF-1 – Insulin-like growth factor 1
KSOM – K Simplex optimization media
mRNA – messenger Ribonucleic acid
mTOR – Mammalian target of rapamycin
mTORC – Mammalian target of rapamycin complex
NPD – normal protein diet
PAR – Predictive adaptive response
PCR – Polymerase chain reaction
PI – Propidium Iodide
PIC – Preimplantation initiation complex
PI3K - Phosphotidylinositide 3-kinase
PPAR - Peroxisomal proliferator-activated receptor
PVP – Polyvinylpyrrolidone

8. viii
TC – Total Cells
TE – Trophectoderm
TNBS – Trinitrobenzene sulphonic acid
TSC1/2 – Tuberculosis sclerosis complex 1/2
UF – Uterine Fluid
Acknowledgements
I would first like to thank Professor Tom Fleming of the University of Southampton,
for the help and guidance he has provided throughout this project and additionally for
allowing me to use his laboratory at Southampton General Hospital to perform my
experiments. I would also like to thank Miguel Velazquez, research fellow at University of
Southampton for all the experimental training that he provided and for all the guidance given
to me throughout the course of the project.

9. 1
1. Introduction
1.1 The Developmental Origins of Health and Disease.
The developmental origin of health and disease (DOHaD) is a theory which states that
environmental challenges during the embryo’s early development, particularly maternal
undernutrition, trigger fetal programming events to aid fetal development. However this also
leads to an increased likelihood of several diseases in adulthood, including metabolic
syndrome, cardiovascular disease and obesity (McMillen, et al., 2008).
The DOHaD hypothesis is based on David Barker and his colleagues’ original
geographical experiments which showed that high systolic blood pressure was linked to low
birth weight (Barker, et al., 1989). From this original study, Barker proposed that challenges
in utero caused by maternal undernutrition lead to fetal programming events that allow the
fetus to successfully grow through the remaining gestational period by adapting its
metabolism to nutrient availability. However this then predisposes the offspring to suffer
from cardiovascular disease in adult life (Barker, 1993). Since then multiple epidemiological
studies have added proof of this developmental origin of disease (Kwong, et al., 2000,
Campbell, et al., 1996) and the theory has spread to include more than just increased
cardiovascular disease. For example, an embryo exposed to maternal low protein is more
likely to have increased adiposity (Watkins, et al., 2011) and increased anxiety behaviour
(Watkins, et al. 2008) once it reaches adulthood.
The DOHaD hypothesis has continued to develop since its discovery and ideas such as
the thrifty phenotype hypothesis (Hales & Barker 1992) and the predictive adaptive response
hypothesis (Gluckman, et al., 2005a) have contributed to the understanding of developmental
origins of disease. The thrifty phenotype hypothesis states that fetal malnutrition induces a
mechanism of nutritional thrift in the developing fetus, causing an immediate survival

10. advantage of the developing offspring in utero via differential organ growth (Hales & Barker
2001). Different tissues in the body have a hierarchy in relation to necessity for short term
adaptive advantages and therefore tissues such as muscle and liver show reduced growth in
response to under nutrition to preserve brain development (Wells 2011). This early altered
growth permanently affects the function of the offspring, which leads to an increased risk of
disease in adult life, for example fetal malnutrition can reduce endocrine pancreas
development that leads to less insulin production and increased insulin resistance, which
2
causes a predisposition towards type 2 diabetes (Hales & Barker 2001).
The predictive adaptive response (PAR) hypothesis builds on the thrifty hypothesis.
This hypothesis states that the fetus uses maternal nutrition to predict nutrient availability
postnatally and therefore early adaptations and fetal programming in development are aimed
at creating a benefit in adult life, rather than just an immediate benefit as seen in the thrifty
hypothesis (Gluckman & Hanson, 2004). This predisposes the offspring to adult metabolic
disease when there is a mismatch in the predicted and the actual post natal environment and
the greater the mismatch, the greater the risk of disease (Gluckman, et al., 2005b). Tests on
mouse models have proven that offspring which are malnourished in utero and then given a
rich nutritional diet postnatally, have significantly reduced life spans (Ozanne and Hales,
2004). Gluckman proposed that PARs are the reason that developing societies changing to a
resource rich environment from an impoverished environment have greatly increased
numbers of people suffering from metabolic syndrome (Gluckman, et al., 2005a).
DOHaD is a challenge to the pre-existing theory that the risk of chronic disease is
dependent on genetics and adult lifestyle. David Barker’s theory explains how a disease such
as cardiovascular disease often associated with affluence has become most common in the
poorest areas of Britain. Challenges to the fetus during critical periods of development
increase the risk of heart disease in adult life (Barker & Martyn, 1992). DOHaD doesn’t

11. discount the importance of adult lifestyle factors such as smoking and diet, instead it works
3
alongside these factors.
1.2 Developmental Re-programming Caused by Maternal Malnutrition
Studies in both humans and animals have determined that maternal malnutrition, often
undernutrition, leads to alterations in fetal programming. An early human model study in
1988 observed that fetal undernutrition during conception was linked to low fetal birth weight
and therefore this undernutrition was likely to be adversely influencing embryonic
development (Wynn & Wynn, 1988). In 1996 it was shown that fetal programming caused by
undernutrition leads to long term health issues. A low protein diet fed to mothers during late
gestation led to their offspring having significantly increased blood pressure at 40 years of
age compared to the offspring of mothers fed a control diet throughout gestation (Campbell,
et al., 1996).
In 1997 a human study showed that fetal malnutrition can lead to fetal programming
that alters the development of specific tissues. Mothers with a low protein but high
carbohydrate diet during pregnancy gave birth to offspring with significantly lower skeletal
muscle tissue and once these offspring reached adult life, they were more susceptible to
coronary heart disease and type 2 diabetes (Godfrey, et al., 1997).
Additionally, rats fed a low protein diet just prior to pregnancy and then throughout
gestation, gave birth to offspring that had increased systolic blood pressure compared to
control mice (Langley & Jackson, 1994). Several different low protein concentrations were
tested (6, 9, 12% by weight) and offspring showed that there was an inverse relationship

12. between maternal protein intake and the offspring systolic blood pressure. An experiment
using a guinea pig model showed that undernutrition (85% ad libitum intake) throughout the
mother’s pregnancy led to decreased fetal birth weight and altered adult cholesterol
homeostasis (Kind, et al., 1999). Male offspring showed an exaggerated response to
cholesterol loading, taking in around 30% more than control offspring due to altered fetal
4
programming in utero.
Animal models using rats have determined the effects of a maternal low protein
throughout gestation, showing that fetal growth is altered differently at different stages of
pregnancy. Up to day 20 of gestation, fetal growth is actually increased but from day 20
onwards growth is retarded so that by the time the offspring reaches term, they are more
likely to be low birth weight compared to control pups (Langley-Evans, et al., 1996a) which
is known to be an indicator that the offspring will be more susceptible to disease in adulthood
(Barker, et al., 1989). This growth retardation affects overall length, as well as specific
organs such as skeletal muscle and liver
1.3 Developmental Re-programming caused by challenges to the fetal environment
specifically during the preimplantation period
Fetal programming is differentially sensitive to environmental challenges depending on
the period in gestation when the fetus is exposed to the nutritional challenge. Evidence from
the 1945 Dutch hunger winter famine has shown this to be true. Children conceived during
the famine and therefore exposed only to malnutrition during conception and early
development, developed an increased glucose tolerance in adult life (De Rooij, et al., 2006).
This phenotype was not observed in offspring who were conceived prior to the famine and so

13. were only exposed to undernutrition during late fetal development. Therefore fetal
programming must be possible from very early stages of development. This is further proven
by an experiment using a rat model which showed that rat mothers fed a low protein diet only
during the first 7 days of pregnancy, resulted in the male offspring still having a significantly
Figure 1 – A) Diagrammatic representation of the
mouse blastocyst including the 2 different cell lineages
and the Zona pellucida. Modified from Rolstan &
Rossant, 2010)
B) Light Microxcope image of a mouse blastocyst.
Trophectoderm (TE), Inner Cell Mass (ICM) and
Blastocoel (BC) are labelled. (Modified fromMarikawa
and Alarcon, 2009)
5
increased risk of hypertension in adult life (Langley-Evans, et al., 1996b).
The first experimental model to test fetal malnutrition just the preimplantation period
was performed by Kwong, et al (2000) and they showed that fetal reprogramming can occur
if the challenge is presented at the
preimplantation stage alone. Mouse dams were
fed a low protein diet (9% casein), known to
cause increased blood pressure in adult offspring
when fed throughout gestation, only during the
preimplantation period and then were fed a
normal protein diet (18% casein) for the
remainder of the pregnancy. The offspring
produced still showed the increased systolic
blood pressure phenotype and had significantly
lower birthweights. Figure 1 A and B show the
differentiation between the TE and ICM cell
lineage in a mouse blastocyst and this experiment
also observed a reduction in the inner cell mass
(ICM) and trophectoderm (TE) cell numbers,
indicating a reduced mitotic index. These results
proved that fetal reprogramming was occurring
during the preimplantation period and that it
A)
B)

14. began soon after the low protein challenge. Further experimental proof that reprogramming
can occur by the blastocyst stage was shown by Watkins, et al (2008) who transferred
blastocysts collected from mothers fed a low protein diet, into mothers who were fed a
control NPD diet (prior to the transfer and afterwards for the remainder of gestation) and
observed that the offspring still produced a phenotype associated with low protein diet
6
induced adaptations.
The periconceptual period of development is highly vulnerable, as the maternal nutrient
environment provides metabolic information to the developing embryo that regulates its
progression through preimplantation development and can trigger fetal programming events
(Fleming, et al., 2012). There is increasing evidence, that human assisted reproductive
technologies which pose a challenge to the preimplantation embryo, may be linked to the
offspring having low birth weight (Schieve, et al., 2002) and being more susceptible to a
number of imprinting disorders such as beckwith-wiedeman syndrome (Gicquel, et al., 2003).
Furthermore in a follow up experiment, performed on 131 IVF children compared against
131 control offspring, Ceelen et al (2008) showed that IVF children had significantly
increased systolic and diastolic blood pressure and that this could not be explained by birth
weight or post natal environmental factors, suggesting that the altered phenotype was due to
the challenge to the preimplantation embryo of being exposed to an in vitro culture
environment.
The mechanistic understanding of why these phenotypes persevere from challenges
during the preimplantation period in humans is largely unclear however, but several animal
models are now providing more information. A mouse model experiment showed that
mothers fed Emb-LPD produced male and female offspring with significantly higher blood
pressure than control offspring (Watkins, et al, 2008). Watkins, et al., (2010) discovered that
this increased blood pressure was caused by an increased expression of angiotensin

15. converting enzyme (ACE) in the offspring of dams fed Emb-LPD, localised to the lungs of
males and the serum of females. ACE overexpression leads to increased angiotensin II
production which stimulates vasoconstriction and therefore causes the increase in blood
7
pressure in Emb-LPD offspring.
Fetal programming has also been associated with increased risk of glucose intolerance
and diabetes in later life (Phillips, 2002). An experiment using a rat model showed how dams
fed Emb-LPD produce offspring that have altered hepatic gene expression (Kwong, et al.,
2007). This altered gene expression resulted in a gender specific response whereby male
fetuses had increased expression of phosphoenolpyruvate carboxylase, a rate-limiting enzyme
in gluconeogenesis, and female fetuses displayed increased expression of 11B-hydroxysteroid
dehydrogenase type 1, which increases glucocorticoid production. These phenotypes lead to
both the male and female offspring having increased insulin resistance and males having
increased likelihood of diabetes because of the preimplantation low protein diet.
Epigenetic changes via histone
modification and DNA methylation
have also been observed to be altered
by maternal diet, summarised in figure
2. Specifically, changes to the
epigenome have been recorded in
response to Emb-LPD (Lillicrop, et al.,
2005). Effects on the regulation of the
glucocorticoid receptor (GR) and
peroxisomal proliferator-activated
receptor (PPAR) by a restricted protein
Figure 2 – An overview of the way in which maternal
diet can alter the epigenome of a developing fetus,
leading to an increase risk of adult disease (Lillycrop &
Burdge (2011)

16. diet were detected using methylation-sensitive PCR to measure DNA methylation and real
time PCR to show mRNA expression. The study showed that restricted protein diet offspring
had significantly reduced gene methylation and increased gene expression of both GR and
PPAR compared to control pups. The preimplantation period is a critical period of epigenetic
control, as the methylation and imprinting of fetal genes after the demethylation of parental
8
genome begins from the blastocyst stage, around E3.5 in mice (Reik & Walter, 2001).
1.4 Potential role for branched-chain amino acids and insulin levels in induction of
preimplantation fetal programming
When rat dams are fed a low protein diet exclusively during the periconceptional period
(Emb-LPD), the maternal environment for the preimplantation embryo is altered
metabolically by reduced plasma insulin and essential amino acid levels (Kwong et al., 2000).
Although it is currently unknown whether these changes in concentrations are nutrient
messages that the preimplantation embryo uses to stimulate fetal programming, evidence is
continually emerging that suggests a potential role for amino acids and insulin in this process.
Insulin receptors are first expressed in the preimplantation embryo at the compaction
stage (Harvey & Kaye, 1988) and therefore from this point insulin may play a potential role
in embryo nutrient sensing. Additionally human preimplantation embryos do not produce
their own insulin or insulin-like growth factor-1 (IGF-1) and cannot as they do not produce
the necessary mRNA transcripts until later in development (Lighten, et al., 1997), but as
stated above they do produce their own receptors for both insulin and IGF-1. Therefore
insulin could be a good indicator of predicted post natal nutrient environment as the
preimplantation embryo is dependent on the supply from the maternal environment.

17. Another reason for insulin to be a potential regulator of fetal programming is that it is a
moderator of blastocyst growth and is therefore required for optimal fetal growth (Kaye &
Gardner, 1999). In vitro experiments have shown that insulin stimulates protein synthesis in
both the TE and ICM cell lineages of the developing blastocyst but increased insulin only
causes an increase to cell proliferation in the ICM cell lineage, having no effect on the TE
(Harvey & Kaye 1990). An increase in the ICM increases the pool of cells available that go
on to form the developing offspring and therefore increased insulin aids optimal fetal growth
9
as stated above.
The fluid within the female reproductive tract also contains free amino acids (Miller &
Schultz, 1987) alongside insulin and other nutrients. Lane & Gardner (1997) performed an
experiment that showed that amino acid signalling increased blastocyst growth and showed
the importance of amino acids in the successful development of the preimplantation embryo.
They determined that, prior to the 8 cell stage embryo, exposure to increased non-essential
amino acids greatly increased the rate of cleavage but that exposure to increased essential
amino acids had no effect. However from the 8-cell stage onwards, they discovered that
embryo exposure to increased essential amino acids not only increased the rate of cleavage
throughout the remaining stages of preimplantation development, but also increased the
number of ICM cells produced in the blastocyst. Whereas increased non-essential amino
acids at this stage no longer increased cleavage but did increase blastocoel development.
In vitro experiments have also shown that amino acid availability is important at the late
blastocyst stage to aid in the development of a mature TE lineage, which is capable of
successfully invading the maternal stroma during implantation (Martin & Sutherland, 2001).
This experiment showed the inability of mouse embryos to form a cell outgrowth on
fibronectin, an accepted in vitro model of implantation (Wartiovaara, et al., 1979), when

18. cultured in a medium lacking amino acids. The correct phenotype returned when blastocysts
were switched back into a control medium. Martin & Sutherland further proved that amino
acids were stimulating successful outgrowth via an mTOR pathway, as introduction of
rapamycin to the culture medium containing amino acids blocked the successful formation of
10
a cell outgrowth.
An experiment by Eckert, et al., (2012) examined methods of induction of adverse fetal
programming and the onset of early compensatory responses by the embryo when mouse
dams are fed Emb-LPD. They discovered that when dams were fed Emb-LPD, the level of
insulin and combined levels of essential and branched-chain amino acids in the maternal
serum, were significantly depleted by the blastocyst stage (E3.5). Furthermore analysis of
maternal uterine fluid (UF) highlighted a potential role in ‘nutrient signalling’ of the
branched-chain amino acids specifically (isoleucine, leucine and valine), which were all
significantly lower in Emb-LPD UF at E.3.5 and still lower by E.4.5 when many other amino
acids had returned to levels comparable in control diet UF.
1.5 Mammalian Target of Rapamycin (mTOR) regulation of cell growth and
development.
Mammalian target of rapamycin (mTOR) is a protein kinase family involved in
regulating eukaryotic protein synthesis, the conversion of mRNA to protein, depending on the
availability of certain nutrients (Proud, 2002). The mTOR signal transduction pathway, is
used by the developing fetus, to allow it to adapt its own cell growth and development in
response to nutrient availability (Maloney & Rees, 2005).
Mammalian target of rapamycin complex 1 (mTORC1) is an mTOR signalling
protein kinase, that phosphorylates serine and threonine residues on target proteins via its

19. conserved C-terminal kinase domain (Mayer & Grummt, 2006). mTORC1 senses certain
nutrient levels such as amino acid, particularly leucine (Bruhat, et al., 2002) and energy levels
(Asnaghi, et al., 2004) and depending on the availability of these nutrients, mTORC1
regulates processes involved in cell growth and development, mainly protein translation as
stated above but a role has also been discovered in protein degradation and actin organisation
11
(Mayer, et al., 2004).
mTORC1 regulates protein translation via phosphorylation of proteins which are
repressors of translation initiation complex proteins (Hay, 2004). When there is high nutrient
availability, particularly amino acids, active mTORC1 phosphorylates and inactivates eIF-
4BP1, which is an inhibitor of the cap binding protein eIF-4E. In its active form, eIF-4E can
freely to bind to eIF-4G to help form the eIF-4F complex, and bind to the 5’ cap of target
mRNA to allow translation to occur (Proud, 2002). Active mTORC1 also phosphorylates S6
kinase 1 (S6K1), which when phosphorylated releases eIF3 which it is bound to in its basal
state. The released eIF3 is now free to bind to the 40s ribosomal subunit and bring it into
contact with the translation preinitiation complex (PIC) (Holz, et al., 2005).
Many nutrients including insulin regulate the mTORC1 activity via a Tuberculosis
sclerosis complex (TSC1 & 2) dependant pathway (Dowling, et al., 2010). Insulin binds
directly to the insulin receptor (IR) kinase which then triggers a phosphorylation cascade,
IR  PI3K  Akt/PKB  TSC1/2, this then leads to the activation of Rheb-GTPase which
activates mTORC1 (Cheng, et al., 2010). Amino acids however, activate Rheb-GTPase via a
poorly understood TSC1 independent mechanism which still activates Rheb-GTPase to active
mTORC1, potentially via the activity of the kinase Vacuolar sorting protein 34 (Vps34)
(Proud, 2007).
Eckert, et al., (2012) implicated a role for mTOR signalling in developmental
reprogramming caused by Emb-LPD. When mouse dams were fed Emb-LPD, the level of

20. phosphorylated S6 was reduced as was the ratio of phosphorylated to total S6 protein,
indicating a reduction in mTORC1 signalling. As previously stated, in this experiment Emb-
LPD fed mothers had significantly decreased insulin and branched-chain amino acids in their
serum and UF, which are both known activators of mTOR signalling, summarised in figure 3,
and this lack of nutrient availability is likely to alter blastocyst programming via an mTORC1
12
dependant process.
Figure 3 – Summary of the mTOR signal transduction pathway in response to insulin and amino acids
as described in section 1.5. (Original figure created using information from sources referenced in
section 1.5).

21. 13
1.6 Differing TE cell numbers as an indication of fetal programming
When mice mothers are fed a reduced protein diet during the weeks leading up to mating
then the developing blastocysts that form have a reduced ICM to TE ratio (Mitchell, et al.,
2009). An experiment by Eckert, et al., (2012) replicated this result showing that after mouse
mothers are fed Emb-LPD, by E3.75 the produced blastocysts have a significantly increased
number of trophectoderm lineage cells and total cell number, but they also showed that this
increased TE cell number led to a significant increase in cell outgrowth after blastocyst
hatching. Therefore the increased proliferation of trophectoderm lineage cells may be viewed
as a compensatory response being triggered by the blastocyst stage in response to maternal
LPD. Stimulated fetal growth is known to be an indicator of an increased risk of adult disease
as specified by the DOHaD hypothesis (Watkins, et al., 2008).
1.7 Aims and Hypothesis
The work of Eckert, et al., (2012) suggested a potential role of branched-chain amino
acid and insulin availability in the nutrient signalling that leads to fetal reprograming in the
preimplantation embryo. Therefore in this project I will examine using an in vitro model
with mouse blastocysts whether low levels of these nutrients, branched-chain amino acids
and insulin, are sufficient on their own to bring about the same phenotype that has been
observed in the Emb-LPD in vivo studies (Eckert, et al., 2012: Watkins, et al., 2008) or if
there is another cause behind the observed phenotypes.
The first aim is to detect whether culture media, containing different levels of insulin and
branched-chain amino acids, is sufficient to significantly alter the rate at which

22. preimplantation embryos develop from the 2-cell stage (E1.5) through to the late blastocyst
stage (E3.75). My hypothesis is that there will be no significant change, as this has been
tested for in an ‘in vivo’ study and blastocysts from mouse dams fed Emb-LPD were not at a
significantly different stage of development compared to embryos from control NPD mothers
14
when removed at E3.5 (Eckert, et al., 2012).
The second aim is to assess whether the 4 different treatment groups of:
1 – Normal Insulin Normal BCAA 2 – Normal Insulin Low BCAA
3 – Low Insulin Normal BCAA 4 – Low Insulin Low BCAA,
cause a significant difference in the TE:ICM ratio which has been determined to be suitable
evidence that a compensatory response has been triggered in the developing embryo by the
blastocyst stage (Eckert, et al., 2012), which suggests there will be an adult predisposition to
metabolic disease in the offspring.
My hypothesis is that there will be a significant difference, at least in treatment group
4 and potentially in treatment groups 2 and 3 because Emb-LPD experiments have
consistently shown evidence of early fetal programming (Kwong, et al., 2000). In addition
there has been evidence of significantly lower insulin and branched-chain amino acids in the
uterine fluid and serum of mothers fed an Emb-LPD (Eckert, et al., 2012).
This area of research is important, to clarify the role of maternal diet as a cause of
fetal reprogramming leading to an increased susceptibility to disease in the offspring’s later
life. With a greater understanding of this area, an effective public health guideline can be
produced to improve the diets of pregnant women (Langley-Evans, et al., 1999). This greater
understanding will aid in preventing the increased rate of adult metabolic diseases observed
in modern society (Gluckman, et al., 2005a).

23. This experiment tested 4 different treatment groups and therefore 4 different culture
These culture media consisted of KSOM medium supplemented with serum insulin (1 ng/ml)
and UF amino acid concentrations (Table 1) based on the levels recorded in the serum and
UF of mouse dams fed normal protein diet (18% casein) (Eckert, et al., 2012). ‘Normal’ level
of these nutrients was 100% the value recorded by Eckert, et al., (2012) and ‘Low’ levels
were 50% of that. In the ‘Low’ BCAA media, only the branched-chain amino acid
(isoleucine, leucine and valine) concentrations were lowered to 50%, all other amino acid
15
2. Materials and Methods
2.1 Creation of Culture Media for Four Treatment Groups
media were produced;
1 – Normal Insulin Normal BCAA 2 – Normal Insulin Low BCAA
3 – Low Insulin Normal BCAA 4 – Low Insulin Low BCAA,
concentrations remained at 100% of the observed value.
Table 1 – Uterine fluid concentrations of free amino acids at 3.5 days of development from mice fed
Normal protein diet (modified from Eckert, et al., (2012).
Amino Acid Concentration (mM) Amino Acid Concentration (mM)
Alanine 3.80 Lysine 0.52
Arginine 0.16 Methionine 0.18
Asparagine 0.14 Phenylalanine 0.14
Aspartic Acid 1.83 Serine 0.97
Glutamic Acid 4.72 Taurine 14.74
Glutamine 1.42 Threonine 0.73
Glycine 2.68 Tryptophan 0.061
Histidine 0.15 Tyrosine 0.18
Isoleucine 0.21 Valine 0.46
Leucine 0.32

24. 16
2.2. Dissection and procuring of 2-cell stage embryos
Embryos were collected at the 2-cell stage (E1.5) from non-superovulated MF1
mouse dams. Dams were sacrificed via cervical dislocation, the oviducts were then removed
and placed into saline solution. Next the oviducts were transferred into H6-BSA drops in a
petri dish and 2-cell embryos were flushed out under a dissection microscope. Total collected
2-cell embryos were split equally into 4 groups, 1 group per different culture media. Embryos
were then moved via mouth pipette into 30μl drops of culture media, and incubated for 66
hours at 37oC in a 5% CO2 incubator. 66 hours is the time required in vitro for embryos to
reach the late blastocyst stage from the 2-cell stage.
2.3 Differential Cell Staining
2.3.1 Differential Cell Staining Materials
Acid tyrodes – sigma, pH2.3, heated to 37oC
Anti-DNP & H6-PVP solution – Anti-DNP stock solution (1 mg/ml in distilled water) and
H6-PVP in the ratio 20.8μl of anti-DNP to 29.2μl of H6-PVP.
Bizbenzimide (Hoechst) – sigma, 2.5 mg/ml in distilled water
Ethanol - 100% pure ethanol
Guinea Pig Complement – low tox guinea pig complement, diluted 1:10 ratio with H6-BSA
H6-Bovine Serum Albumin (BSA)
H6-Polyvinylpyrrolidone (PVP)
Propidium Iodide (PI) – sigma, 1 mg/ml in distilled water, aloquat stored at -20oC
Trinitrobenzene sulphonic acid (TNBS) – Picrysulfonic acid, diluted 1:10 in PBS

25. 17
2.3.2 Differential Cell Staining Protocol
After the 66-hour incubation period, embryos were removed from the incubator and
observed under a microscope to record what stage of development they had progressed to.
Stages recorded were morulae, early blastocyst (blast), mid blast, late blast and hatching
blast. After this only the blastocysts which were at the late blastocyst stage were used and the
differential cell staining protocol began. The first step of which was removal of the
blastocysts’ zona pellucida via acid tyrodes. Each treatment group required 1 cavity block
containing 500μl of acid tyrodes, both the cavity block and tyrodes were pre-heated to 37oC.
Blastocysts were transferred from the incubation culture media into the acid tyrodes via
mouth pipette. They were then observed under a microscope to determine when the zona
pellucida had been fully dissolved. Once the zona was removed the blastocysts were moved
via mouth pipette to another cavity block (1 per treatment group), filled with 1ml of handling
medium containing BSA (H6-BSA), for 20 minutes, to allow the blastocyst to recover before
beginning the second step.
During the second step, blastocysts were incubated for 10 minutes at room
temperature in a 50μl drop of TNBS. To start this step, one small drop (25μl) and one large
drop (50μl) of TNBS were prepared per treatment group in a ‘Cellstar’ petri dish. Blastocysts
were moved between drops via mouth pipette, first into the small drop to wash away the H6-
BSA then into the large drop for the incubation period. After incubation blastocysts were
washed through 3, 50μl drops of H6-PVP to remove the TNBS.
Next, blastocysts were incubated for 10 minutes at room temperature in an anti-DNP
with H6-PVP solution (described in 2.3.1). Once again, a small drop (8μl) and a large drop
(40μl) of anti-DNP solution were prepared per treatment group in a Cellstar petri dish.
Blastocysts were washed through the small drop via mouth pipetting, to ensure the incubation

26. stage was in pure anti-DNP solution. After incubation, blastocysts were washed through 3,
Next, blastocysts were stained with propidium iodide (PI). 1 culture drop was
prepared per treatment group containing 50μl of guinea pig complement and 4μl of PI and
Blastocysts were incubated in these drops for 10 minutes at 37oC. After this incubation
blastocysts were washed through 3, 50μl drops H6-BSA. Finally embryos were fixed for 3
hours at 4oC in 990μl of absolute ethanol and 10μl of Bizbenzimide (Hoechst) in a 4 well
18
50μl drops of H6-PVP to remove the anti-DNP solution.
Figure 4 – An example fluorescence
microscopy image of a diffrerenctial cell
stained late blastocyst.
Trophectoderm = pink, Inner cell mass =
Blue
plate, one well per treatment group.
2.4. Blastocyst Picture Acquisition
After blastocysts were fixed, they were mounted onto slides for observation with a
fluorescence microscope. Blastocysts were first moved from the Ethanol and Hoechst
solution into absolute ethanol, for 5 minutes, to wash them. Coverslips were then washed
with methanol in preparation and then a drop of glycerol was placed in the centre for
mounting the embryos. Blastocysts were moved
from the ethanol to the glycerol drops via
mouth pipette in groups of 3-5. The coverslip
was then placed on top of the glycerol drop so
the slide could be used under the microscope.
Coverslips were examined under a
fluorescence microscope. Using the program
MetaMorph®, embryos were searched for on
the coverslip and then an image, such as the one
in figure 4, of each individual embryo was

27. captured. Images were captured at 20x magnification. Cell numbers were counted manually
using MetaMorph®, ICM cells in blue and TE cells in red, the values were then combined to
19
determine the total cell number.
2.5 Statistical Analysis
146 embryos were incubated at the 2-cell stage per treatment group. From these
embryos; 140 from treatment group 1, 142 from treatment group 2, 141 from treatment group
3 and 142 from treatment group 4 were useable for the first part of the experiment (outlined
in 2.3.2). The from these embryos, 95 from treatment group 1, 104 from treatment group 2,
96 from treatment group 3 and 101 from treatment group 4 were at the late blastocyst stage
by the end of incubation and were usable for cell number counts after the differential cell
staining protocol.
The data from the first experiment determining the stage reached in development
after 66 hour incubation period was analysed using a chi-square test. The data from the
second experiment determining relative TE and ICM cell proliferation, turned out to not be
normally distributed and no easy conversion was available, therefore this data was analysed
via non-parametric ANOVA based on ranks. A Kruskal-Wallis test was first used to
determine if any of the 4 treatment groups had produced a significantly different result from
the others and then Mann-Whitney tests would be used on individual pairs of treatment
groups to determine which group had produced a significant difference in trophectoderm cell
allocation.

28. Figure 5 – The effect of 4 different culture media containing different insulin and BCAA levels on
the number of embryos that reached each specific stage in development as a percentage of the total
embryos first cultured. The key relates to the four different treatment groups; N = normal, L = Low, I
= Insuling & BCAA = Branched chain amino acids.
20
3. Results
3.1 Activity of Embryos in Development
The first experiment carried out was to assess the activity of embryos developing in
response to incubation in each different culture medium. This was done by recording what
stage of development each embryo had reached by the end of the 66-hour incubation period
from the 2-cell stage. The data was analysed using a chi-squared test. Figure 5 shows the
number of embryos, per treatment group, that had reached each stage of development as a
percentage of the total number of embryos originally cultured (n = 146 per treatment group).
A chi squared test was performed to determine whether the activity of the embryos was
significantly altered by culture in different concentrations of BCAA and insulin. The test
determined that there was no significant difference in embryo activity, p ≠ 0.99 for total
embryo, total blastocyst and late blastocyst comparison. There was slightly more variation
when comparing the number of embryos which reached the hatching blastocyst stage but p-

29. values still showed an non-significant relationship. Therefore embryos did not develop
significantly slower or faster in response to a depleted BCAA and/or insulin environment,
Figure 6 – The effect of 4 different culture media containing different insulin and BCAA levels on
the average number of cells produced in each cell lineage of the blastocyst (Trophectoderm and Inner
Cell Mass) and the total cell number. The key relates to the four different treatment groups; N =
normal, L = Low, I = Insuling & BCAA = Branched chain amino acids.
21
when incubated from the 2-cell stage for 66 hours.
3.2 Effect of Depleted Branched-chain Amino Acids and Insulin on Fetal Programming
The second experiment assessed how changing the levels of insulin and branched-chain
amino acids, effected the distribution of cells within the two different possible cell
lineages of the late blastocyst, the trophectoderm and the inner cell mass. To measure this,
cells in the late blastocyst were differentially stained, depending on which lineage they
belonged to and then values were recorded by imaging via fluorescence microscopy. The data
was analysed using a Kruskal-Wallis non-parametric ANOVA based on ranks. Figure 2,
shows the number of cells in the trophectoderm and inner cell mass and then the total number
of cells on average in the blastocysts when cultured in each of the four different treatment

30. groups. The statistical tests performed showed that there was no significant difference in the
cell number of the trophectoderm lineage (p = 0.212), inner cell mass lineage (p = 0.580) or
total cells (TC) (p = 0.493) at the late blastocyst stage, between the 4 treatment groups.
Therefore the effect of lowering the insulin and branched-chain amino acid concentrations
and then the combined effect of lowering them both in the supplemented KSOM media had
no significant effect on the proliferation of cells in either of the two different lineages.
Furthermore there was no significant change to the total proliferation of cells within the entire
Figure 7 – The effect of 4 different culture media containing different
insulin and BCAA levels on ratio of Trophectoderm to inncer cell mass
cells formed in blastocysts. The key relates to the four different treatment
groups; N = normal, L = Low, I = Insuling & BCAA = Branched chain
amino acids.
22
blastocyst by the late blastocyst stage.
As well as examining just the number of cells present, I also calculated the TE:ICM
ratio and the ICM/TC proportion. These were both then statistically analysed, also with a
Kruskal-Wallis test. Figure 7 shows the TE:ICM ratio, on average, of blastocysts cultured in
the four different treatment groups. The Kruskal-Wallis test performed on this data showed
that there was no significant difference (p = 0.217) in the TE:ICM ratio of blastocysts
cultured in the different
treatment groups.
Therefore the depleted
insulin and branched-chain
amino acid levels
appear to have had no
significant effect on the
allocation of cells into
each specific lineage during
blastocyst development.

31. To further confirm this result, the ICM/TC parameter shown in figure 8 was
statistically analysed. If one treatment group produced a significantly lower value then
Figure 8 – The effect of 4 different culture media containing different
insulin and BCAA levels on the proportion of the total number of cells
in a blastocyst which is made up by the inner cell mass lineage The key
relates to the four different treatment groups; N = normal, L = Low, I =
Insuling & BCAA = Branched chain amino acids.
23
incubation in that
concentration of BCAA
and insulin would have
caused the blastocysts to
respond by investing
more cells into the TE
lineage compared to the
ICM lineage during
early development. A
Kruskal-Wallis test
performed on the data
in figure 8 determined
that there was no significant difference (p = 0.217) in the ICM/TC parameter between either
of the four different treatment groups. Therefore this confirmed the results from the analysis
of the TE:ICM ratio, that culturing 2-cell embryos up to the late blastocyst stage in depleted
insulin and branched-chain amino acid levels, both individually and the combined effect, had
no impact on allocation of cells to a specific cell lineage during blastocyst development.
Overall these results show that, after statistical analysis of the data acquired, the four
different culture media that were used to test the effect of depleting insulin and branched-chain
amino acid levels, did not have a significant effect on the developing blastocyst. There
was not a significant change in the total number of cells in either the TE or ICM, or in the
total number of cells by the late blastocyst stage. Furthermore, the relative allocation of cells
to the two different possible lineages, TE and ICM, was not affected by the different culture.

32. 24
4. Discussion
4.1 Comparison with Original Hypothesis
The result from the first half of the experiment confirms my first hypothesis stated in
the introduction, that depleting the levels of branched–chain amino acids and insulin had no
effect on the rate at which embryos developed from the 2-cell stage, over the 66-hour
incubation period. Statistical analysis via chi-squared tests proved that any variation observed
in figure 5 was almost solely due to random sampling variability, as the p-values were all
very high. This has been observed before in another study using in vitro culture. Velazquez,
et al., (2012) recently performed an experiment where embryos obtained at the 2-cell stage
were incubated in several different culture media containing gradually depleting
concentrations of branched-chain amino acids for 66-hours, by which time it embryos should
reach the late blastocyst stage. The embryos cultured in the lower levels of branched-chain
amino acids did not develop at a significantly different rate compared to those cultured at
normal levels, which is consistent with my results.
The results from the second part of my experiment disproved my hypothesis stated in
the introduction. The effects of reducing the branched-chain amino acids and/or insulin levels
by 50% in the different culture media turned out to not cause a significant effect on the
proliferation of cells in the developing blastocyst. Furthermore, there was no significant
effect on the allocation of cells to either the trophectoderm or the inner cell mass lineage
within the blastocyst. Therefore, despite the evidence from Eckert, et al., (2012) which
showed a potential link between reduced branched-chain amino acids and insulin and the
onset of preimplantation fetal reprogramming, this data instead suggests that no fetal
programming had occurred in the cultured embryos by the late blastocyst stage, in response
to the reduced nutrient levels compared to the control values (obtained from treatment group
1).

33. From looking at the distribution of data in figures 6-8, blastocysts incubated in the
two culture media which contained 50% lower levels of branched-chain amino acids, showed
no indication that fetal programming was occurring, which was then confirmed by the
statistical analysis. This was a big surprise as there is prior experimental evidence that
branched-chain amino acids specifically are known to be present at significantly lower
concentrations in maternal UF when dams are fed a low protein diet and embryos exposed to
a low protein diet undergo fetal programming. The results from this evidence could therefore
suggest that branched-chain amino acid levels are not part of the nutrient sensing mechanism
25
used by the preimplantation embryo when exposed to an Emb-LPD environment.
However, although not a significant difference, the data from figure 6 appears to
indicate a potential increase in the trophectoderm cell number when embryos were incubated
in the two culture media which contained 50% insulin (treatment groups 2 and 4) compared
to the control. Based on this observation I performed a Mann-Whitney test to analyse the
trophectoderm cell numbers between treatment group 2 (low insulin with normal BCAA) and
group 1 (the control containing normal insulin and normal BCAA levels). Although still not
significantly different, the result was very close as the p value came out to be, p = 0.058, just
0.008 away from a significant result. Furthermore the error bars in figure 6 are not very large,
but a large enough to conceive that this statistical test could be showing a significant result.
This is a strong indication that the depleted insulin level may have been stimulating fetal
programming and causing this slight increase in trophectoderm lineage by the late blastocyst
stage. If this were true then that would indicate that fetal programming may have occurred in
these embryos by the late blastocyst stage, as expected from my original hypothesis.
There are several points to consider about why these results came out as non-significant
despite the evidence which formed my hypothesis in the introduction, that a
reduction in branched-chain amino acids and insulin in an in vitro culture medium would be

34. sufficient to cause cause fetal reprogramming to occur by the late blastocyst stage. First of all
other factors in the initial in vivo LPD diet studies may have been causing the observed
change in blastocyst phenotypes, secondly there may have been some technical issues during
the experimental process that have led to a misleading conclusion. Furthermore the fact that a
replicate in vitro study may not be completely accurate when trying to determine the effects
of nutrient level changes in vivo, as the in vitro culture environment presents many unique
stresses of its own to the developing embryo. Finally, based on these results there are a
number of follow up studies that could be done to confirm or disprove the results from this
study that depleting the availability of branched-chain amino acids and insulin to a
developing preimplantation embryo is not sufficient to cause an onset of fetal reprogramming
26
by the late blastocyst stage of development.
4.2 Potential Methods Used by the Preimplantation Embryo to Communicate with the
Maternal Environment after an Emb-LPD
A study in 2000 was the first to show that when mouse dams are fed a low protein diet
selectively during the preimplantation period it is sufficient to cause fetal programming
events to occur that resulted in the offspring having an increased risk of cardiovascular
disease in later life (Kwong, et al., 2000). During this experiment it was noted that several
nutrient levels in the maternal serum changed significantly during this time in response to the
low protein diet, such as a decrease in insulin and essential amino acids and an increase in
glucose. This caused these nutrients to be proposed as potential nutrient messengers used by
the developing embryo to sense the low protein environment and adapt its development.

35. Since then, multiple research studies have focussed on the effects that a low protein
diet causes on the specific nutrient availability within the environment of the preimplantation
embryo. If this is known it can lead to an understanding as to what the developing embryo is
sensing specifically which causes it to adapt its fetal programming in an attempt to be better
27
suited to its environment.
My project studied the effects of lowering just branched-chain amino acid and insulin
levels on the blastocyst to undergo fetal programming because recent experimental evidence
suggested both of these nutrients may have a role in nutrient sensing by the preimplantation
embryo (Eckert, et al., 2012). However the fact that my results turned out to be non-significant
implies that an embryo low protein diet causes other significant changes to the
preimplantation uterine environment which are signalling to the preimplantation embryo and
triggering fetal programming. These could include changes to amino acid, glucose or pH
levels.
Amino Acids
An experiment performed by Eckert, et al., (2012), observed a significant decrease in
branched-chain amino acid levels in the maternal serum and also in the uterine fluid of mouse
dams fed a low protein diet exclusively during the preimplantation period of development.
However, the branched-chain amino acid levels in the maternal UF of dams fed low protein
diet, compared to control dams, were only uniquely lower than other amino acid
concentrations at E4.5, at which point mouse embryos begin the implantation process
(Stephens, et al., 1995). Before this stage, at E.3.5, other amino acids (glycine, histidine,
lysine, taurine and threonine) were also measured to be lower in the maternal UF, at trend
level ‘P < 0.1’, compared to the UF of control dams. During my experiment, depleted

36. branched-chain amino acid levels were found to have no effect on the developing embryo
when cultured to E3.75, the late blastocyst stage. This lack of response could therefore mean
that prior to E4.5, the preimplantation embryo may be responsive to amino acids, such as
those observed in the experiment by Eckert, et al., (2012) and not just the branched-chain
amino acids. At this stage the embryo may require a combined signal from multiple to amino
acids before fetal programming can be triggered. The branched-chain amino acids may play a
more significant role in nutrient sensing by E4.5, because by this point they are the only
amino acids to be at a significantly lower concentration in the UF of Emb-LPD fed dams,
28
compared to the UF of control dams.
The potential role of amino acids, other than just the branched-chain amino acids, in
nutrient sensing by the preimplantation embryo has been suggested by several other
experiments. One such experiment observed that both rat and mouse dams have significantly
decreased serum levels of threonine and phenylalanine in response to an Emb-LPD (Petrie, et
al., 2002). In another experiment where mouse dams were fed a low protein diet during the
preimplantation period, there was a significant reduction in the level of 6 different amino
acids (the three branched-chain amino acids and methionine, proline and threonine) in the
maternal serum (Kwong, et al., 2000). All of these results suggest that the developing
preimplantation embryo is sensitive to more amino acids than just the branched-chain amino
acids. This is a possible explanation as to why depleting the concentration of just the
branched-chain amino acids was not sufficient to trigger fetal reprogramming in my
experiment, as the embryo requires nutrient signals from multiple amino acids.

37. 29
Insulin
In my project, there appeared to be some impact when depleting the level of insulin in
the culture media. The Mann-Whitney test performed in section 4.1 suggests there may have
been a response to the 50% decrease in insulin concentration via an increase in the number of
trophectoderm cells produced by the late blastocyst stage. Although not a significant
difference, the data in figures 6-8 appears to indicate a potential impact on fetal programming
caused by the reduction in insulin levels, as the trophectoderm cell number, total cell number
and TE:ICM ratio is consistently highest in treatment groups 2 and 4. However these results
were not significant and therefore it is not safe to assume that the insulin was directly
responsible for this variation and it was not simply due to random sampling variability.
Insulin may not be involved in nutrient signalling by the preimplantation embryo, previously
a reduction in insulin levels caused by a low protein diet has been observed in several
experiments (Kwong, et al., 2000: Eckert, et al., 2012) but, the reduction has always been
observed in the maternal serum and not in the UF, to which the blastocyst is directly exposed.
Glucose
Additionally, a low protein diet causes changes to the maternal environment that
were not examined in my project. The rodent preimplantation embryo is known to be
sensitive to glucose; a maternal hyperglycaemic state has been observed to significantly
lower the expression of facilitated glucose transporters in the developing blastocyst (Moley,
et al., 1999), in attempt by the blastocyst to adapt to a predicted high glucose environment in
adult life. Furthermore, an in vitro culture experiment proved that when rat preimplantation
embryos were incubated in a high glucose medium (17mM) it caused, irreversible inhibition
of the TE and ICM lineage growth and an increase in apotosis within the blastocyst.

38. Going back to the original paper examining a low protein diet specifically during the
preimplantation period, Kwong, et al., (2000), a significantly increased glucose content in the
maternal serum was observed by day four of gestation, in response to an Emb-LPD. This
result has since been replicated; mouse dams fed a low protein diet during the
preimplantation period were recorded as having significantly higher glucose in the maternal
serum by day 3.5 in development, around the time of blastocyst formation (Eckert, et al.,
2012). Therefore these results suggest that a hyperglycaemic environment surrounding the
developing blastocyst was likely contributing to the fetal reprogramming that was observed in
response to the low protein diet. This fetal programming led to increased likelihood of adult
30
diseases such as hypertension in these offspring.
However, other experiments have observed that high glucose concentrations in the
maternal environment caused by Emb-LPD have little affect once the embryo reaches later
stages of gestation (Fernandez-Twinn, et al., 2003: Kwong, et al., 2007), therefore further
experimentation is required to fully understand the effect of glucose concentration on fetal
development.
4.3 Evaluation of experimental techniques
I believe that the method I used during this experiment was well designed and suitable
to produce an accurate result. The differential cell staining protocol has been previously
tested and used on blastocysts by researchers at the University of Southampton (Velazquez, et
al., 2012) and therefore it was a reliable method to measure TE and ICM lineage and
blastocyst total cell numbers. Additionally, the concentrations of amino acids used for the

39. ‘normal’ level used in the different treatment groups, were discovered by an in vivo
experiment which directly measured the concentration of every amino acid in the uterine
fluid of mouse dams which had been fed a normal protein diet (Eckert, et al., 2012).
Similarly, the concentration of insulin used for the ‘normal’ level in the different treatment
groups was measured in the same in vivo experiment (Eckert, et al., 2012), in the serum of
dams fed a normal protein diet. Furthermore a 50% decrease from the normal values was a
sufficient reduction, therefore if any change was observed it could be reliably assumed to be
31
due to the nutrient reduction.
However, there are several technical issues that arose during the experimental process
which potentially could have contributed to the second half of the experiment disagreeing
with my hypothesis and coming to the conclusion that lowering the concentration of
branched-chain amino acids and insulin was not sufficient to trigger fetal programming in the
developing. First of all, towards the beginning of the data collection period, when I was still
not fully experienced at performing the staining protocol, some embryos from each treatment
group were lost due to errors whilst using the mouth pipette. This improved after the first few
repeats as I became increasingly proficient at the experimental procedure and no more
embryos were lost, however there is potential that some of those lost embryos from the early
repeats could have shown a fetal programming phenotype and may have contributed to a
significant result in the end.
Additionally, for the initial repeats of the differential cell staining protocol, during the
final step blastocysts were moved directly from the H6-BSA drops in the previous step, into
the ethanol and bizbenzimide stain solution for 3 hour incubation, with no interim washing
step. A problem arose whereby some of the blastocysts were attaching to the base of the four
well plate and when these blastocysts had to be moved prior to mounting onto a microscope
slide, some of the cells in the trophectoderm layer were being ripped away from the rest of

40. the blastocyst and remaining attached to the base of the plate. This would lead to a poor
image being captured with an incorrect number of trophectoderm cells being recorded. To
resolve this issue, embryos were first placed into a washing well containing 990μl of absolute
ethanol and 10μl of bizbenzimide to completely remove any H6-BSA from the previous step,
which had been responsible for the blastocysts attaching previously. Furthermore when
embryos were then moved from this washing well into their final staining solution, some air
bubbles were blown in via the mouth pipette so that the blastocysts did not sink straight to the
bottom. These changes to the method eradicated the problem of the blastocysts attaching to
the four well plates and images taken during fluorescence microscopy became consistently
accurate. However, this problem in the initial repeats meant that cell counts from the
blastocysts who had attached during the first few repeats had to be removed from the final
statistical analysis. If these blastocysts had undergone fetal programming it may have
32
contributed to a significant result in the end, but this cannot be known for sure.
Finally there may have been issues with pH levels during the experimental
procedure. pH regulation is an important part of cell homeostasis for all mammalian cells
(Phillips, et al., 2000). To maintain homeostasis, mouse embryos use a HCO3
-/Cl- transporter
to relieve alkalosis (Zhao, et al., 1995) and two different transporters to relieve acidosis, an
Na+/H+ antiporter (Steeves, et al., 2001) and an Na+,HCO3
–/Cl– exchanger (Phillips, et al.,
2000). When mouse embryos are subjected to low pH during the preimplantation period,
often caused in vivo by an increase in urea within the maternal UF, the developing
blastocysts show a significant decrease in cell proliferation and increase in apoptosis
(Bystriansky, et al., 2012).
In my experiment the pH of the final culture media was not specifically tested, however the
pH of the KSOM media was known to be ~pH 7.4. Although unlikely, there is potential that
contamination could have caused the culture media to become a low enough pH that it could

41. have caused a challenge to the developing blastocyst which led them to have a reduced cell
proliferation that led to an insignificant result. Furthermore, during the experiment at the end
of the incubation stage, blastocysts were submitted to acid tyrodes to dissolve the zona
pellucida. If the embryos were left in too long during this step then the low pH challenge
could have caused an increase in trophectoderm apoptosis. However this is again very
unlikely as the challenge would only have been very brief and furthermore steps in the
protocol were in place to prevent embryo elongated exposure to the acid tyrodes. Only 2-3
embryos at a time were moved into the tyrodes so that it would be easy to observe when the
33
zona pellucida had fully dissolved
4.4 Effects of In Vitro Culture
An in vitro model was required for this experiment as it is the only possible method
that allowed me to observe the effects of specifically reducing just branched-chain amino
acid and insulin concentrations on developing blastocysts. An in vivo model can only change
the diet fed to dams and observe its effects, it cannot be used to alter specific aspects of
uterine fluid composition. Therefore the use of an in vitro model was appropriate for this
experiment. However, placing preimplantation embryos into an in vitro culture environment
is known to induce a number of cellular stresses on the embryo (Chandrakanthan, et al.,
2006). For example, in vitro culture has been seen to induce oxidative stress via stimulating
the embryo to produce a significantly increased amount of reactive oxygen species (Nasr-
Esfahani & Johnson, 1991) and also cause metabolic imbalance where the developing
embryos don’t use enough endogenous resources if cultured in vitro (Leese 2002).

42. Furthermore, experimental evidence has shown mouse embryos placed into in vitro
culture produce a reduced number of TE lineage cells compared to in vivo embryos
(Giritharan, et al., 2012). This was an issue for my experiment as for part of my results I was
trying to record an increase in TE lineage cell number to represent the onset of fetal
programming by the blastocyst stage. Therefore it may have been the case that the embryos
being incubated in culture during this experiment were repressing the proliferation of the TE
lineage and could not increase TE proliferation properly in response to the nutrient challenges
34
in the different treatment groups
If it were possible to alter the concentrations of branched-chain amino acids and
insulin in the uterine fluid in vivo, without changing the concentration of any other nutrient,
then the blastocysts could be observed without having to consider the potential effects caused
by the stress of being in an in vitro culture. These hypothetical in vivo conditions may have
produced a phenotype associated with fetal reprogramming as was expected from the
literature.
4.5 Future Work
From the results of my project, there are a number of follow up experiments I would
suggest to first confirm these results and secondly examine more possibilities of how a low
protein diet limited to the preimplantation period signals to the fetus to initiate fetal
programming, causing the fetus to adapt to the predicted post-natal environment. It
First of all there are several experiments possible to confirm the results of this
experiment, that reducing the concentration of branched-chain amino acids and insulin by

43. 50% in the direct embryo environment is not sufficient to induce fetal programming in the
preimplantation embryo by the late blastocyst stage. In this experiment I used the
proliferation of the TE lineage, the TE:ICM ratio and the ICM/TC parameter to indicate
whether fetal programming had occurred, however there are other methods of ensuring that
fetal programming has occurred by the late blastocyst stage (E3.75). Therefore I would repeat
the experiment with the same 4 treatment groups and the same protocol for incubation, but
instead of performing differential cell staining at the end, the analysis of whether fetal
programming had been initiated would be done by either detecting the level of mTOR
signalling within the blastocysts, measuring blastocyst outgrowth formation on suitable
surface such as fibronectin and reinserting embryos into mothers fed a normal protein diet to
35
see if the offspring still showed an enhanced growth phenotype.
mTOR signalling
Both insulin and branched-chain amino acids are known to activate mTORC1
signalling (Bruhat, et al., 2002: Dowling, et al., 2010). When mouse dams are fed a low
protein diet specifically during the pre-implantation period, the capacity for mTOR signalling
is reduced in the blastocysts due to a significant reduction in the level of phosphorylated S6
(a downstream target of mTORC1) compared to blastocysts from dams fed a normal protein
diet (Eckert, et al., 2012). Therefore, mTORC1 activity of the blastocysts from the four
different treatment groups of my experiment would be analysed using the protocol used by
Eckert, et al., (2012). This would confirm that a reduction in insulin and/or branched-chain
amino acid levels is sufficient to cause a reduction in mTORC1 signalling and therefore is
responsible for the reduction in mTORC1 signalling observed in blastocysts in vivo when
dams are fed Emb-LPD. A reduction in mTORC1 signalling would suggest an occurrence of

44. fetal programming by the blastocysts stage, because the mTORC1 signalling pathway acts as
36
a sensor for maternal nutrient levels in the developing blastocyst (Eckert, et al., 2012).
Blastocyst Outgrowth Formation
A predicative adaptive response by the developing blastocyst in response to a reduced
nutrient environment is an increase in placental development to ensure that the developing
fetus can obtain sufficient nutrients throughout gestation and maintain necessary fetal growth
(Watkins, et al., 2008). When mouse dams were fed a low protein diet during the
preimplantation period, blastocysts were extracted at E3.5 and allowed to hatch, attach and
initiate spreading in an in vitro setting (Eckert, et al., 2012). The blastocysts from dams
which were fed a low protein diet displayed an increased capacity for trophoblast spreading
after attachment compared to blastocysts from dams fed a normal protein diet.
Therefore in a repeat of my experiment, to confirm whether embryos are undergoing
fetal programming in response to the depletion in BCAA and insulin levels, a set of
blastocysts from each treatment group would be cultured in media containing ‘normal’
nutrient levels for a further 96 hours after the initial 66 hour incubation, to allow outgrowth
formation (Velazquez, et al., 2012). If the embryos from a particular treatment group show a
significant increase in trophoblast outgrowth it would indicate fetal programming had
occurred by the end of the initial incubation period in response to the decreased concentration
of BCAA and/or insulin.

45. 37
Restricted intrauterine growth
In response to a low protein diet fed exclusively during the preimplantation period,
fetal growth is accelerated later on in gestation (Watkins, et al., 2008). Mouse embryos
transferred at the blastocyst stage from dams fed Emb-LPD into dams fed a NPD, maintain
the enhanced growth phenotype later on in pregnancy (Watkins, et al., 2008), which proves
that fetal programming has occurred by the blastocyst stage. Therefore, to determine whether
depleting branched-chain amino acid and insulin levels is sufficient to trigger fetal
programming, embryos from the 4 treatment groups would be transferred after 66 hours of
incubation from the 2-cell to the late blastocyst stage, into dams which have been fed a NPD
and will continue to be fed NPD throughout the remainder of gestation. At Day 17 of
gestation, offspring would be collected, weighed and compared to determine whether the
growth rates had been significantly different between embryos from the different treatment
groups. A significantly increased weight compared to the control offspring would indicate
that fetal programming had occurred by the late blastocyst stage in response to reduced
BCAA and insulin levels.
After these three different experiments are completed, the results of my project, that
depleting branched-chain amino acids and/or insulin alone is not sufficient to cause fetal
programming by the late blastocyst stage, will either be confirmed or contradictory results
will instead indicate a role for insulin and branched-chain amino acid levels in nutrient
sensing by the preimplantation embryo.
If these proposed future experiments were to confirm the results from my project, then
additional future research should be focussed on the effect of altering the concentrations of
other nutrients, such as glucose, in vitro on triggering fetal programming. Additionally, the

46. maternal environment after an Emb-LPD should be further investigated to determine other
possible causes of the resulting fetal programming observed in developing offspring. This
will further improve scientific understanding of how the preimplantation embryo
38
communicates with its environment during development.
4.6 Concluding remarks
The results from this project have shown that depleting the concentration of branched-chain
amino acids and/or insulin by 50% from the values found in the UF and serum of dams
fed a normal protein diet, is not sufficient to change the rate at which embryos develop and is
also not sufficient to trigger fetal programming in preimplantation embryos by the late
blastocyst stage. A possible role for insulin was observed, but it was not a significant
relationship and so further experimentation is required to prove whether insulin truly was
having an effect of the onset of fetal programming.
These results can improve the scientific understanding of how the preimplantation
embryo communicates with its environment in utero. They indicate that branched-chain
amino acids and insulin levels may not play as significant a role in nutrient sensing by the
blastocyst stage as the current literature suggests. Therefore, the focus of research may need
to change away from assessing the effect had by branched-chain amino acid concentrations
and insulin levels, to investigating additional methods by which the preimplantation embryo
may be communicating with its environment when the mother has had low protein
environment, or other forms of poor maternal nutrition, before interactions between the
preimplantation embryo and its environment can be fully understood.

47. Ultimately a greater understanding of how maternal diet triggers fetal programming is
required before advancements in accurate preventative methods can be made to reduce the
chance of fetal programming occurring in pregnant women. Additionally this understanding
will also help prevent embryos undergoing fetal programming during in vitro culture. If the
specific nutrient imbalances which can cause embryos to trigger fetal programming are
identified, it can lead to an improvement in culture media used during assisted reproductive
39
technologies such as in vitro fertilisation.

48. 40
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