Honours Project Book - Faculty of Health Sciences - University of ...

PROJECT: (Basic) Why is there haemoglobin in
cumulus cells?
R. O&G.
Project Backgound
We have recently produced microarrays of mouse
cumulus cells obtained from naturally ovulated
cumulus-oocyte complexes (COCs) and in vitro
matured COCs. A consistent finding is that mRNA for
haemoglobin is produced by cumulus cells derived
from in vivo matured COCs at high levels compared
to in vitro matured COCs. Furthermore, this
difference has been validated by RT-PCR.
Haemoglobin production by cumulus cells has never
previously been reported and is a unique
observation. This begs the question “Why would
cumulus cells require the production of
haemoglobin?” One candidate mechanism involves
the regulation of cGMP and nitric oxide, both of
which are known to be involved in the ovulatory
process. Haemoglobin is a known sequestering
molecule for nitric oxide, so we have hypothesised
that cumulus cells derived from in vivo ovulated
COCs produce haemoglobin in response to the
ovulatory process and this is lacking during in vitro
maturation. This project aims to investigate the
production of haemoglobin and determine its roles
during follicle ovulation and oocyte maturation.
Techniques will initially include RT-PCR,
immunohistochemistry and western blotting.
References
Sutton ML, Gilchrist RB and Thompson JG (2003),
Effects of in vivo and in vitro environments on
metabolism of the cumulus-oocyte complex and its
influence on oocyte developmental capacity, Hum
Reprod Update, 9 (1): 35-48
Sutton-McDowall ML, Gilchrist RB and
Thompson JG (2004) Cumulus-expansion and glucose
utilisation by bovine cumulus-oocyte complexes
during in vitro maturation: the influence of
glucosamine and follicle stimulating hormone,
Reproduction, 128 (3): 313-19
Sutton-McDowall ML, Mitchell M, Cetica P,
Dalvit G, Pantaleon M, Lane M, Gilchrist RB and
Thompson JG (2006) Glucosamine supplementation
during in vitro maturation inhibits subsequent oocyte
developmental competence, Biol Reprod, 74: 881-88.
Schelbach CJ, Kind KL, Lane M and Thompson
JG (2010) Mechanisms contributing to the reduced
developmental competence of glucosamineexposed
mouse oocytes. Reprod Fertil Dev, 22 (5):
771-9.
Sutton-McDowall ML, Gilchrist RB and
Thompson JG (2010) The pivotal role of glucose
metabolism in determining oocyte developmental
competence. Reproduction, 139(4):685-95.
PROJECT: (Basic) Lipid oxidation in cattle/sheep
oocytes. Unlocking an un-used energy source to
increase developmental competence during in vitro
maturation.
R. O&G.
Supervisors
Dr Kylie Dunning
Dr Melanie McDowall
A/Prof Jeremy Thompson
Project Backgound
Intra-oocyte ATP levels are correlated with oocyte
developmental competence post-fertilisation.
Oocytes have little capacity to produce ATP and are
dependent on oxidative phosphorylation within
mitochondria. Cattle, sheep and pig oocytes are
characterised by significant intracellular stores of
lipid. It has been calculated that there is enough
“stored energy” within these lipid stores to drive all
the necessary ATP production for early development.
However, when oocytes mature in vitro (or early
embryos are cultured in vitro), exogenous
carbohydrates and derivatives are essential for
further development, suggesting the lipid stores are
not utilised. Work conducted in the Ovarian Cell
Biology Group has found that in the mouse, the
addition of carnitine to oocyte maturation media
significantly increases lipid metabolism and ATP
production. Carnitine is a co-factor required for the
transfer of lipids, in the form of fatty acids, from the
cytosol, into mitochondria, where they undergoe �oxidation
and producte ATP. This project will
investigate the role of carnitine and lipid oxidation
within cattle/sheep oocyte culture in vitro. Skills
learnt will include oocyte maturation and embryo
production in vitro as well as metabolic assays and
fluorescence microscopy investigating mitochondrial
function.
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