GDF9 and BMP15: Species
Difference and Synergistic
Interactions
Georgia Alice Martin
School of Paediatrics and Reproductive Health
Research Centre for Reproductive Health
Discipline of Obstetrics and Gynaecology
University of Adelaide, Adelaide
Australia
Thesis submitted to The University of Adelaide in fulfilment of the requirements
for admission to the degree of Master of Philosophy
March 2014
Abstract
GDF9 and BMP15 are two oocyte-secreted proteins which have been shown to be
essential for normal mammalian fertility. There are a number of factors which impact their
efficiency, including species difference of the proteins, GDF9/BMP15 interactions and the
presence of post-translational modifications. However, factors such as species difference and
post-translational modifications have yet to be investigated in terms of their effects on
GDF9/BMP15 interactions.
The aims of this study were to produce well purified human and mouse GDF9 and
human BMP15 to test the effects of these factors. We found clear species differences
between mouse and human GDF9 in thymidine incorporation in mouse and bovine granulosa
cells. However, not only did we find a species difference due to the species of the protein,
but also a difference due to the species of the cells on which the proteins were acting. GDF9
and BMP15 were found to interact synergistically on mouse granulosa cells, but not on
bovine cells. Human GDF9 was found to be dependent on the presence of BMP15 for
bioactivity in both species of cell however, the introduction of mouse-like residues into the
human GDF9 sequence was able to produce a protein capable of functioning independent of
BMP15 and with even higher bioactivity than wild-type mouse GDF9.
Post-translational modifications were also found to have significant effects on
synergistic GDF9/BMP15 synergistic interactions. Both GDF9 and BMP15 have previously
been shown to be phosphorylated. This appears to be more important to the correct
functioning of GDF9. Loss of GDF9 phosphorylation was found not only to decrease its
bioactivity but also to decrease its synergistic interactions with BMP15. Phosphorylation was
not found to affect BMP15 however; the loss of o-linked glycosylation decreased its ability to
synergise with GDF9. To fully assess the implications and applications of this work, there is
still a great deal of work to be done however, it is clear that for any embryological studies,
the species differences of the proteins and cells need to be carefully considered.
ii
Declaration
I certify that this work contains no material which has been accepted for the award of any
other degree or diploma in any university or other tertiary institution and, to the best of my
knowledge and belief, contains no material previously published or written by another
person, except where due reference has been made in the text.
In addition, I certify that no part of this work will, in the future, be used in a submission for
any other degree or diploma in any university or other tertiary institution without the prior
approval of the University of Adelaide and where applicable, any partner institution
responsible for the joint-award of this degree.
I give consent to this copy of my thesis, when deposited in the University Library, being made
available for loan and photocopying, subject to the provisions of the Copyright Act 1968.
I also give permission for the digital version of my thesis to be made available on the web, via
the University’s digital research repository, the Library catalogue and also through web
search engines, unless permission has been granted by the University to restrict access for a
period of time.
Georgia Alice Martin
2014
iii
Dedication
To my wonderful family and friends.
iv
Acknowledgements
Firstly I’d like to say a massive thank you to my wonderful supervisors David and Rob. You
two have been so supportive through all the ups and downs of my honours and masters
work. David, thank you for steering the project and knowing all the things about these
sometimes infuriating proteins that no one knows. Rob, thank you for being my stats guru,
helping me make my very messy thoughts into something coherent and shouting the odd
few rounds of tequila slammers.
Thao and Lesley, thank you so much for teaching me all the tricky techy things over the last
few years. It was a blast, although I really do hope I can get by never having to do another
western blot. Also a huge thank you to the wonderful Kay Govin for all of your support,
understanding and hugs.
I wouldn’t have had nearly as good a laugh doing all this as I did without all my office
buddies, Laura, Ryan, Luisa, Little Mel, Big Mel Siu, Qian, Nicole, Jacky, Dulama and Hannah. I
loved all the endless hours of gossip, CollegeHumour videos (Ryan :P) and occasionally
adding our “cupboard of attractiveness”.
Lastly, but definitely not least, I want to say a huge thank you to my family (biological and
unbiological), especially Mum, Vanya and Marek. You put up with 3+ years of me being
moody and mental but were always there with a cup of tea/cheese platter/bottle of wine
when I needed you. I also want to thank you and James for always telling me to never give up
on myself. I definitely wouldn’t have been able to do this without you xox
v
Abstracts Arising from This Thesis
TGF-β DownUnder Conference – 2011
Studies on the function of the pro-regions of BMP15 and GDF9
Georgia A. Martin, Thao Nguyen, Lesley J. Ritter, Junyan Shi, Robert B. Gilchrist and David G.
Mottershead
Research Centre for Reproductive Health,
Robinson Institute,
University of Adelaide
Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are two
oocyte secreted TGF-β superfamily proteins which have been shown to be essential for
normal mammalian fertility. Similar to other TGF-β proteins, the mature regions of BMP15
and GDF9 maintain a close association with their corresponding pro-regions after processing,
however it is not known if the BMP15/GDF9 pro-region plays a role in the bioactivity of the
corresponding mature region. The aim of this study was to produce purified forms of both
the BMP15 and GDF9 pro-mature complexes and isolated mature regions in order to gauge
the function of the pro-region. Human BMP15 and GDF9 were produced by transfecting
human embryonic kidney 293T cells with plasmids containing BMP15 or GDF9 expression
cassettes with poly-histidine and StrepII affinity tags engineered into the N-terminus of the
pro-region. The pro-mature complexes of both proteins were purified from conditioned
media using two affinity purification steps using the poly-histidine and StrepII tags. The
mature regions were isolated from the purified pro-mature complexes using an additional
step of reverse phase high performance liquid chromatography. The BMP15 and GDF9
mature regions and pro-mature complexes were then compared in mouse granulosa cell
vi
proliferation assays. The resulting data showed that the mature regions of human BMP5
and GDF9 were significantly more bioactive than the corresponding pro-mature
complexes. This suggests that the pro-regions of both human BMP15 and GDF9 are
inhibitory to the actions of the corresponding mature regions in mouse granulosa cells.
World Congress on Reproductive Biology Conference – 2011
BMP15/GDF9 Synergistic Interactions
Georgia Martin, Lesley Ritter, Junyan Shi, Robert Gilchrist and David Mottershead
Robinson Institute, Research Centre for reproductive Health
University of Adelaide, Discipline of Obstetrics and Gynaecology
BMP15 and GDF9 are two TGF-β superfamily proteins which are essential for normal
mammalian fertility. Both proteins share a similar pre-propeptide structure and exist after
proteolytic processing as a pro-mature complex. Both BMP15 and GDF9 have been shown,
among other functions, to stimulate granulosa cell proliferation. Previous studies using
unpurified preparations of recombinant mouse or ovine BMP15 and GDF9 have indicated
that these proteins display synergistic interactions. Further, recently it has been suggested
that there are species differences with regard to GDF9/BMP15 synergistic responses. The
purpose of this study was to investigate the synergistic interactions of purified recombinant
human BMP15 and GDF9. The proteins were produced by transfecting human embryonic
kidney 293T cells with plasmids containing BMP15 or GDF9 expression cassettes with a polyhistidine tag engineered into the N-terminus of the pro-region. The purified mature regions
of these proteins were obtained via a single step of His tag affinity chromatography followed
by two steps of reverse phase high performance liquid chromatography. The effects of the
vii
human BMP15 and GDF9 mature regions, both individually and together, were measured
using [3H]-thymidine incorporation in primary mouse granulosa cells. Here we present our
recent results characterising the synergistic interactions of our purified human GDF9 and
BMP15 proteins in comparison with those observed utilizing a purified commercially
available mouse GDF9.
viii
Table of Contents
Abstract ......................................................................................... ii
Declaration .................................................................................... iii
Dedication ..................................................................................... iv
Acknowledgements ........................................................................ v
Abstracts Arising from this Thesis .................................................. vi
Table of Contents .......................................................................... ix
List of Figures .............................................................................. xiii
List of Tables ................................................................................ xvi
Abbreviations ............................................................................. xvii
CHAPTER 1
Introduction: GDF9 and BMP15 ............................ 1
1.1
GDF9 and BMP15 Biological Functions ......................................................................... 2
1.2
GDF9 and BMP15 in Folliculogenesis ............................................................................ 4
1.3
GDF9 and BMP15 Structure .......................................................................................... 6
1.4
GDF9 and BMP15 Signalling ......................................................................................... 8
ix
1.5
GDF9 and BMP15 Expression and Processing ............................................................... 9
1.6
GDF9 and BMP15 Interactions .................................................................................... 10
1.6.1 Genetic Interactions ....................................................................................... 10
1.6.2 Biochemical Interactions ................................................................................ 11
1.6.3 Functional Interactions ................................................................................... 12
1.7
Significance ................................................................................................................. 13
1.8
Hypotheses and Aims ................................................................................................. 15
1.8.1 Hypothesis 1 ................................................................................................... 15
1.8.2 Hypothesis 2 ................................................................................................... 16
CHAPTER 2
2.1
Materials and Methods ...................................... 17
Proteins ...................................................................................................................... 18
2.1.1 Wild-type proteins .......................................................................................... 18
2.1.2 Mutant proteins .............................................................................................. 18
2.2
GDF9 and BMP15 Expression Plasmids ....................................................................... 19
2.3
Cell Lines and Culture Conditions ............................................................................... 19
2.4
Establishment of Stable Protein Expressing Cell Lines ................................................ 19
2.5
GDF9 and BMP15 Purification .................................................................................... 20
2.5.1 Nickel Immobilised Metal Affinity Chromatography (IMAC) Purification ....... 20
2.5.2 Reverse Phase High Performance Liquid Chromatography (rpHPLC) ............. 21
x
2.6
Colorimetric Western Blotting and Silver Staining ..................................................... 22
2.7
Fluorescence Western Blotting and Protein Quantification........................................22
2.8
Granulosa Cell Proliferation Assays ............................................................................ 23
2.9
Statistical Analyses ...................................................................................................... 24
CHAPTER 3
GDF9 and BMP15 Wild-type Proteins: Species
Difference, Synergism and Pro-region Interactions ....................... 25
3.1
Introduction ................................................................................................................ 26
3.2
Methods ...................................................................................................................... 29
3.3
Results ........................................................................................................................ 30
3.4
Discussion ................................................................................................................... 44
CHAPTER 4
GDF9 Species Specific Point Mutations ............... 48
4.1
Introduction ................................................................................................................ 49
4.2
Methods ...................................................................................................................... 49
4.3
Results ........................................................................................................................ 50
4.4
Discussion ................................................................................................................... 55
CHAPTER 5
Post-translational Modifications ......................... 56
xi
5.1
Introduction ................................................................................................................ 57
5.2
Methods ...................................................................................................................... 58
5.3
Results ........................................................................................................................ 59
5.4
Discussion ................................................................................................................... 65
CHAPTER 6
Discussion ........................................................... 67
CHAPTER 7
Summary ............................................................ 73
CHAPTER 8
References .......................................................... 75
APPENDIX 1
Protein Production and Sequences ...................... 81
7.1
pEF-IRES Vector and Expression Cassette Layout ...................................................... 82
7.2
Protein Sequences ...................................................................................................... 83
7.2.1 Wild-type proteins .......................................................................................... 83
7.2.2 Mutant Proteins .............................................................................................. 85
xii
List of Figures
Figure1.1
Oocyte/somatic cell bidirectional communications ......................................... 2
Figure 1.2
Folliculogenesis ................................................................................................. 5
Figure 1.3
TGF-β protein primary structure ....................................................................... 7
Figure 1.4
TGF-β protein tertiary structure ....................................................................... 8
Figure 1.5
Smad mediated pathways ................................................................................. 9
Figure 3.1
Schematic diagram of basic protein expression cassette ............................... 30
Figure 3.2
Silver stained SDS-PAGE gels of hGDF9 and hBMP15 purification products .. 31
Figure 3.3
rpHPLC is an effective means of separating the mature region from the proregion .............................................................................................................. 32
Figure 3.4
Silver stained gels of hGDF9 and hBMP15 final HPLC products ...................... 33
Figure 3.5
Purified wild-type GDF9 and hBMP15 ............................................................ 33
Figure 3.6
There is no difference in bioactivity between in-house prepared mGDF9 and
commercial mGDF9 mature regions ............................................................... 34
Figure 3.7
The species difference between human and mouse GDF9 is displayed with
both the mature regions and pro-mature complexes when acting on mouse
granulosa cell thymidine incorporation .......................................................... 36
Figure 3.8
Both the human mature region and pro-mature complex synergise with
hBMP15. Only the mouse pro-mature complex synergises with hBMP15...... 37
xiii
Figure 3.9
The species difference between human and mouse GDF9 is displayed with the
mature regions when acting on bovine granulosa cell thymidine
incorporation....................................................................................................39
Figure 3.10
Neither the human nor the mouse GDF9 synergise with hBMP15 on bovine
granulosa cells ................................................................................................. 40
Figure 3.11
The pro-region affects the bioactivity of mGDF9 but not hGDF9 ................... 42
Figure 3.12
The presence of the pro-region effects mGDF9 interactions with hBMP15 but
not hGDF9 interactions ................................................................................... 43
Figure 4.1
hGDF9 mutant protein western blot .............................................................. 51
Figure 4.2
Both mouse-like GDF9 mutants are more active than either human or mouse
wild-type GDF9 ................................................................................................ 52
Figure 4.3
hGDF9 mutant proteins only synergise with hBMP15 at low doses .............. 53
Figure 5.1
Western blot of GDF9 mutant proteins .......................................................... 59
Figure 5.2
hBMP15 mutant protein western blot ............................................................ 60
Figure 5.3
Eliminating o-linked glycosylation decreases BMP15’s synergistic
capabilities........................................................................................................61
Figure 5.4
Eliminating both BMP15 o-linked glycosylation and phosphorylation decreases
synergistic capabilities compared to hBMP15wt ............................................ 62
Figure 5.5
There is no additional decrease in synergistic capability due to the additional
elimination or phosphorylation compared to the elimination of o-linked
glycosylation alone .......................................................................................... 63
xiv
Figure 5.6
The conversion of Ser7 to an Ala residue decreases GDF9 bioactivity ........... 64
Figure 5.7
The conversion of Ser7 to an Ala residue reduces GDF9 synergistic capability
with BMP15 ..................................................................................................... 65
xv
List of Tables
Table 3.1
Wild-type Proteins .......................................................................................... 29
Table 4.1
GDF9 Mouse-like Mutant Proteins ................................................................. 50
Table 5.1
Post-translational Modification Mutant Proteins ........................................... 58
xvi
Abbreviations
GDF9
Growth and differentiation factor 9 (h and m
denote human or mouse GDF9)
BMP15
Bone morphogenetic protein 15
GDF9 Mut 1
Human GDF9 with a single mouse-like
Gly391Arg mutation
GDF9 Mut 2
Human GDF9 with multiple mouse-like
mutations
GDF9 S7A
GDF9 Mut 2 with phosphorylation sites
eliminated
BMP15 T277A
BMP15 with o-linked glycosylation sites
eliminated
BMP15 S/T
BMP15 with o-linked glycosylation and
phosphorylation sites eliminated.
aa
Amino acid
ACN
Aceto-nitrile
ALK5/ALK6
Activin-like kinase 5/6
ANOVA
Analysis of Variance
Arg
Arginine
BMPRII
Bone morphogenetic protein receptor type 2
CC
Cumulus cell
CHO cells
Chinese hamster ovarian cells
CM
Conditioned media
Cys
Cysteine
DMEM
Dulbecco’s modified eagle medium
DTT
Dithiothreitol
xvii
EGF
Epidermal growth factor
ERK1/2
Extracellular signal-regulated kinases 1/2
F
Furin-like processing site
FCS
Foetal calf serum
GC
Granulosa cell
Gly
Glycine
Has2
Hyaluronan synthase 2
HEK293T cells
Human Embryonic kidney 293T cells
hGL
Human granulosa luteal cells
IGF-1
Insulin-like growth factor 1
IMAC
Immobilised metal affinity chromatography
IVF
In vitro fertilisation
IVM
In vitro maturation
kD
Kilo daltons
KGN
Human granulosa-like tumour cell line
MAPK
Mitogen-activated protein kinase
mRNA
Messenger ribo nucleic acid
nFκ-β
Nuclear factor κ-β
PBS
Phosphate buffered saline
pEF-IRES
Elongation factor – internal ribosomal entry
site plasmid
PMSG
Pregnant mare serum gonadotropin
Ptgs2
Prostaglandin-endoperoxide synthase 2
rpHPLC
Reversed phase high performance liquid
chromatography
SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel
electrophoresis
Ser
Serine
xviii
SS
Signal sequence
TFA
Trifluoro-acetic acid
TGF-β
Transforming growth factor β
Thr
Threonine
xix
Chapter 1
Introduction: GDF9 and BMP15
1.1 GDF9 and BMP15 Biological Functions
Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15,
also known as GDF9B), are two oocyte-secreted factors which have been proven to be
essential for normal mammalian fertility. They have myriad functions which result in the
regulation of normal fertility. GDF9 has been shown to have anti-apoptotic effects in preantral and early antral follicles [1], as well as stimulating granulosa cell proliferation [2].
BMP15 has also displayed some ability to increase granulosa cell proliferation and is a highly
effective anti-apoptotic agent in cumulus cells [3]. The downstream actions of BMP15 have
also been found to stimulate the expression of other proteins such as EGF-like growth factors
which are essential for cumulus expansion [4](fig1.1). The functions of GDF9 and BMP15 are
key factors in oocyte developmental competence and therefore, reproductive success.
Figure 1.1: GDF9 and BMP15 are secreted from the oocyte and are part of the
bidirectional communication between the oocyte and surrounding cells. This
communication is responsible for correct cumulus and granulosa cell
proliferation etc. which results in eventual increased foetal viability. Image
adapted from Gilchrist 2004 [5]
Studies of GDF9 and BMP15 knockout animals and those with a naturally occurring mutation
have highlighted the importance of the correct regulation of these processes to reproductive
capability. Extensive work has been done in the past using GDF9 and BMP15 knockout mice
2
with varying results depending on the combination of knockout alleles. GDF9 null mice
(GDF9-/-) have been shown to be infertile due to a block in folliculogenesis at the primary
stage and a lack of granulosa cell proliferation [6]. The ovaries of some GDF9 null mice
displayed an absence of corpora lutea which suggests anovulation [6]. The oocytes from
these mice also display very poor meiotic competence and thus would even be unsuitable for
an artificial reproductive technology approach to generate offspring. BMP15 null mice
(BMP15-/-) were shown to have decreased fertility due to decreased ovulation rates [7].
BMP15 null mice also display poor cumulus complex stability and significantly reduced levels
of Has2 mRNA expression in their cumulus cells [8]. Has2 is linked with cumulus expansion.
Without correct cumulus expansion, the oocyte fails to mature properly. In fact, the oocytes
of BMP15 null mice also display retarded development [8].
While heterozygous BMP15 knockout mice (BMP15+/-) are essentially phenotypically
normal, GDF9 heterozygous mice (GDF9+/-) have an increased fertility rate [8]. This increase
in fertility is linked with an increased ovulation rate and a significant increase in Ptsg2 mRNA
expression, another gene associated with cumulus expansion which leads to increased
oocyte quality [8].
Alterations in fertility are also seen as a result of GDF9 and BMP15 mutations in
sheep. Where BMP15 heterozygous mice (BMP15+/-) have an essentially normal
reproductive phenotype, sheep which are heterozygous for BMP15 mutations (BMP15+/-)
have an increased ovulation rate and are thus more fertile than wild-type sheep [9, 10].
Further, sheep which are heterozygous for GDF9 mutations (GDF9+/-) have also been shown
to have an increased ovulation rate [10]. However, homozygotes for these mutations of
GDF9 and BMP15 (BMP15-/- and GDF9-/-) are infertile due to primary ovarian failure. It has
been shown in BMP15 (BMP15-/-) homozygotes that sterility is due to a block in
folliculogenesis at the primary stages, thus both the follicles and oocytes are unable to
mature [9]. These data suggest that both GDF9 and BMP15 are equally important to
reproduction in sheep and that GDF9 and BMP15 mutations in sheep are not necessarily
detrimental to the animals’ fertility. The phenotype arising from deletions mutations in GDF9
3
in mice and sheep, respectively, suggest that this protein may also exhibit species specific
functions.
There are also limited data on GDF9 and BMP15 mutations in humans. The
consequences of these mutations can be just as detrimental to fertility in humans as they can
in sheep or mice. There are a number of BMP15 mutations which have been found only in
women suffering from premature ovarian failure and thus are strongly correlated with the
condition [11]. BMP15 mutations have also been found to be a cause of hypergonadotrophic
ovarian failure [12]. A reduction of GDF9 gene expression is also seen in polycystic ovarian
syndrome, which can have detrimental effects [13].
1.2 GDF9 and BMP15 in Folliculogenesis
Folliculogenesis requires complex bidirectional communication between the oocyte
and the surrounding cells and GDF9 and BMP15 are highly significant elements of this
process (fig 1.2). Indeed, as seen in the previously mentioned knockout animal studies, loss
of either or both of these proteins results in some kind of dysfunction in the folliculogenesis
process although, this does not necessarily have a negative impact on fertility.
4
Figure 1.2: Schematic diagram of the folliculogenesis process. Image from Frank
2012 [14].
In mice, GDF9 seems to be the more significant of these two proteins. GDF9 deficient
mice are infertile and the granulosa cells have limited proliferative potential [15]. However, if
the oocytes of these mice are removed while the follicles are still immature, they are still
developmentally competent if subjected to in vitro maturation (IVM) [15]. If allowed to
mature in vivo, not only do the follicles not reach a proper antral stage due to severely
decreased granulosa cell proliferation, but the oocytes lose viability due to degradation. This
is caused by malformed trans-zonal processes from the cumulus cells invading the
perivitelline space and subjecting the oocyte to a form of phagocytosis [15]. GDF9 has also
5
been shown to have an anti-apoptotic effect in follicles. In an in vivo study by Orisaka et al,
GDF9 was inhibited resulting in activation of caspase 3 leading to apoptosis [1]. GDF9
inhibition also caused a severe reduction in follicle stimulating hormone (FSH) receptor
mRNA expression. These effects ameliorated by exposing the follicles to exogenous GDF9 [1].
In monovular species such as cows, sheep and humans BMP15 also has a significant
role in folliculogenesis. BMP15 null sheep (i.e. homozygous mutants) show a similar
phenotype to GDF9 mull mice [16] as do cows [17]. In the bovine model, immunising animals
against BMP15 leaves the level of granulosa cell proliferation insufficient to support follicle
growth to the large antral stage. The same is also the case in the absence of GDF9 [17].
Further, in bovine model, GDF9 has also been shown to regulate theca cell differentiation
and growth which, in turn, influences androgen production [18]. This has also been observed
in rodents [19]. BMP15 has been shown to be present throughout folliculogenesis [9, 20].
BMP15 in humans has also been shown to help prevent luteinisation in rats [21] and cumulus
cells apoptosis in bovine [3].
1.3 GDF9 and BMP15 Structure
GDF9 and BMP15 are two proteins belonging to the transforming growth factor-β
(TGF-β) super-family of proteins [22]. This is a superfamily consisting of over 30 members
including the inhibins, activins, TGF-βs and of course, the bone morphogenetic proteins and
growth differentiation factors [22]. GDF9 and BMP15 are more closely structurally related to
each other than to any other TGF-β protein, sharing a 53% homology in their pre-proprotein
structure and a 70% homology in their mature region [20, 23]. Their primary structure is
composed of a large amino terminal pro-region and a smaller, biologically active carboxy
terminal mature region which is 135aa in GDF9 [24] and 125aa in BMP15 [20, 23] (fig 1.3).
These two regions are separated by a 4aa furin processing site. After processing, the mature
regions of many TGF-β proteins maintain a close association with their pro-regions. It is
generally acknowledged that the mature region is the bioactive part of a protein. However,
in some cases it has been shown that the pro-regions of a number of TGF-β proteins
6
influence the bioactivity of the mature region. TGF-β1 and its pro-region (the β1 latency
associated peptide) remain closely, non-covalently associated after processing. This proregion inhibits the bioactivity of the mature region until the two are dissociated [25]. AntiMϋllerian hormone on the other hand has a pro-region which significantly increases the
bioactivity of the mature region [26]. GDF9 and BMP15 have also been shown, by coimmunoprecipitation to have the same close association with their pro-regions after
processing [27]. This suggests that the GDF9 and BMP15 pro-regions may play a role in their
bioactivity. It has also been suggested that the GDF9 and BMP15 pro-regions regulate the cooperative interactions of these proteins [27].
Figure 1.3: Schematic view of the primary structure of GDF9 and BMP15
After processing, TGF-β proteins exist as dimers. These can be either covalent
homodimers (two monomers of the same protein) or covalent heterodimers (two monomers
of different proteins). A distinguishing feature of the TGF-β super-family which allows these
covalent dimers to form is a characteristic cysteine ‘knot’ (fig 1.4). It is typically composed of
seven cysteines, six of which form disulphide bonds within the protein (the ‘knot’) leaving
the final cysteine free to allow the protein to form covalent dimers [28]. However, both
GDF9 and BMP15 along with a small group of other proteins such as LEFTY1 and LEFTY2, lack
this free cysteine and thus are unable to form covalent dimers [20, 24, 29]. As GDF9 and
BMP15 are only capable of forming non-covalent dimers, it means that once they have
7
dimerised, the two monomers are not constrained by a covalent bond and hence,
heterodimerisation post secretion as homodimers is at least theoretically possible.
Figure 1.4: The tertiary structure of TGF-β proteins, showing the six cysteine
molecules forming the ‘knot’, and the seventh cysteine, utilised in covalent
bonding. GDF9 and BMP15 lack this cysteine. Image from Vitt 2001 [28].
1.4 GDF9 and BMP15 Signalling
GDF9 and BMP15 utilise similar receptor complexes to activate their signalling
pathways. Both proteins require a type 1 and type 2 receptor. BMP15 utilises the activin-like
kinase 6 (ALK6) receptor as the type 1 receptor and the bone morphogenetic protein type II
receptor (BMPR-II) as its type 2 receptor [30]. BMP15 acts through the Smad 1/5/8 pathway.
GDF9 on the other hand uses the ALK5 receptor as its type 1 receptor and, like BMP15, uses
BMPRII as its type 2 receptor to activate the Smad 2/3 pathway [31-33] (fig 1.5). GDF9
signalling has also been shown to be dependent on ERK1/2 signalling [34]. Although GDF9
8
does not activate this pathway, the ERK1/2 pathway is required for Smad 3 phosphorylation,
which GDF9 requires for signalling [34].
Figure 1.5: Schematic of Smad mediated pathways. Image adapted from
Horbelt 2012 [35].
1.5 GDF9 and BMP15 Expression and Processing
GDF9 and BMP15 mRNAs are co-expressed in the oocyte. This has been shown by in
situ hybridisation studies on young mice [23]. This study also showed that GDF9 and BMP15
expression occurs at the same point in development.
9
GDF9 and BMP15 processing occurs as a result of the pre-proprotein being
proteolytically cleaved into the pro-region and mature region peptides by a furin-like
protease. Both the processed and unprocessed forms of GDF9 and BMP15 have been found
in follicular fluid [36] and oocyte conditioned media [37] however, it is interesting to note
that the predominant form is the unprocessed [36]. It has also been found that when GDF9
and BMP15 are co-expressed, there is a significant decrease in the amount of secreted
processed protein [38], although it should be pointed out that this study utilised the
HEK293T cell line and is therefore an in vitro model. As unprocessed proteins are generally
supposed to be inactive, this decrease in processing as a result of co-expression may be a
mechanism for regulating GDF9 and BMP15 functions.
1.6 GDF9 and BMP15 Interactions
The fact that GDF9 and BMP15 have such a high degree of homology in their mature
regions, that they are expressed from the same cell type (the oocyte) at the same point in
development and that they have similar functions suggests that it may be possible for them
to interact. While their expression patterns may indicate some redundancy in biological
function, it may be that not only is there some interaction, but that the presence of both
proteins may be required for their adequate functioning. GDF9 and BMP15 interactions have
in fact been demonstrated in the past by studies on three main levels, the genetic, the
biochemical and the functional. As both of these proteins are essential for normal
mammalian fertility, it is likely that GDF9 and BMP15 interactions are critical determinants of
reproductive success in mammals.
1.6.1 Genetic Interactions
GDF9 and BMP15 interactions on a genetic level can be seen as the result of
producing double knockout or double mutant animals. In mice, while a BMP15 heterozygous
knockout produces an essentially normal phenotype and a GDF9 heterozygous knockout
10
produces increased fertility, a double heterozygous knockout mouse (BMP15+/-GDF9+/-) is
sub-fertile [7]. A BMP15 null and GDF9 heterozygous mouse (BMP15-/-GDF9+/-) will also be
sub-fertile; however, this sub-fertility appears to be more severe than the BMP15-/knockout. These mice displayed abnormalities in folliculogenesis leading to a reduced
ovulation rate and abnormal cumulus physiology leading to reduced oocyte developmental
competence [7].
GDF9 and BMP15 genetic interactions are also seen in sheep. As previously
mentioned, sheep with heterozygous BMP15 or GDF9 mutations have increased fertility due
to an increased ovulation rate. However, when these genotypes are combined as a double
heterozygous mutant (BMP15+/-GDF9+/-) the ovulation rate of the sheep is significantly
increased over that of either individual phenotype [10].
1.6.2 Biochemical Interactions
On a biochemical level, GDF9 and BMP15 interactions have been shown in studies
involving chemical cross-linking and immunoprecipitation. Through chemical cross-linking it
has been shown that when GDF9 and BMP15 are individually expressed in separate cell lines,
they form homodimers [38]. However, as previously mentioned, in vivo GDF9 and BMP15 are
co-expressed. When co-expressed in cell lines, there is some evidence that when treated for
cross-linking GDF9 and BMP15 can form a heterodimer [38]. However, to date, the strongest
evidence for GDF9 and BMP15 heterodimer formation is seen through immunoprecipitation.
In the 2003 study by Liao et al, GDF9 and BMP15 were co-expressed in the same cell line. The
antibody used for immunoprecipitation was targeted at an affinity tag which had been
engineered into the BMP15 sequence. The resulting precipitate was then analysed by
western blotting with an antibody targeted at a different affinity tag engineered into the
GDF9 sequence [38]. This showed that GDF9 and BMP15 would co-immunoprecipitate,
indicating that the two proteins closely interact with each other. The BMP15 pro-region has
also been shown to co-immunoprecipitate with the GDF9 mature region [27]. This suggests
that the pro-region may have a role in dimerisation and is further indicative of GDF9 and
11
BMP15 interactions. It is unknown exactly how GDF9 and BMP15 interact in vivo; however,
as these studies indicate the formation of heterodimers when GDF9 and BMP15 are coexpressed, as they are in vivo, this is a possible scenario. However, the non-covalent nature
of their interactions allows for the possibility that that they could exist as a mixture of
homodimers and heterodimers.
1.6.3 Functional Interactions
An interesting aspect of GDF9 and BMP15 interactions to consider is synergism. In
this context, synergism is defined as two proteins working together to produce an effect
greater than the sum of their two individual effects. Synergism can occur in two ways, either
two homodimers simultaneously activating two different pathways such as with BMP2 and
activin [39] and TGF-β3 and GDF5 [40], or as a single heterodimer activating one pathway
such as the BMP2/6 heterodimer [41]. As previously mentioned, the unique structure of
GDF9 and BMP15 allows for the possibility that these proteins are capable of forming both
homodimers and heterodimers. However, it is currently unknown which configuration is the
basis of their functional interactions.
GDF9 and BMP15 functional interactions have been observed in in vitro bioassays.
The most common means of demonstrating GDF9 and BMP15 actions and interactions in
vitro are granulosa cell proliferation assays using thymidine incorporation as a marker of
proliferation. Mouse GDF9 conditioned media (containing the pro-mature complex) has been
found to significantly increase rat granulosa cell proliferation while ovine GDF9 and ovine
BMP15 do not [42]. However, when added together both mouse GDF9 and ovine BMP15 or
ovine GDF9 and BMP15 produce highly significant increases in proliferation due to a
synergistic response. This pattern was also seen in inhibin production on rat granulosa cells.
Functional interactions of GDF9 and BMP15 have also been observed as a result of
monitoring signalling pathway activation. As previously mentioned, GDF9 and BMP15
homodimers utilise the Smad 1/5/8 and Smad 2/3 pathways respectively. It is therefore
12
logical to suppose that the synergistic interactions of GDF9 and BMP15 involve at least one
of these pathways. The synergistic actions of ovine GDF9 and BMP15 conditioned media and
mouse GDF9 and BMP15 conditioned media on thymidine incorporation on rat granulosa
cells have both been found to be significantly inhibited by a Smad 2/3 pathway inhibitor [27].
No significant inhibition has been seen as a result of Smad 1/5/8 pathway inhibition [43].
This indicates that Smad 2/3 pathway activation is necessary for the GDF9 and BMP15
synergistic response. However, the Smad pathways are not the only pathways involved in the
GDF9 and BMP15 synergistic response. This response can also be inhibited by inhibiting the
nuclear factor-κβ pathway and can be partially inhibited by inhibiting p38-mitogen-activated
protein kinase (MAPK) [43]. This suggests that different receptors and pathways may be
utilised in the GDF9 and BMP15 synergistic response to those utilised during their individual
actions.
In addition to understanding which pathways are being activated during the GDF9
and BMP15 synergistic response, it is also important to know which receptors are being
utilised. A 2008 study by Edwards et al showed the effects of various type 1 and type 2
receptor inhibitors on ovine GDF9 and BMP15 synergism in rat granulosa cells. Inhibitors for
ALKs 1-7 were tested, including ALK 5 (the GDF9 type 1 receptor) and ALK 6 (the BMP15 type
1 receptor). None of these receptor inhibitors were found to significantly affect the GDF9
and BMP15 synergistic response [43]. A range of type 2 receptor inhibitors were also tested.
BMPRII was the only tested type 2 receptor found to significantly inhibit the GDF9 and
BMP15 synergistic response [27, 43]. From this study, it is clear that the BMPRII receptor is
required for GDF9 and BMP15 synergism.
1.7 Significance
The main process performed by GDF9 and BMP15 is their facilitation of the
communication between the oocyte and the surrounding somatic cells which is critical to
oocyte developmental competence.
13
Oocyte developmental competence, as well as being imperative to reproduction
itself, is also important to assist reproductive technologies such as in vitro fertilization (IVF)
and the newly emerging in vitro maturation (IVM). IVM has promise to replace IVF as it uses
immature oocytes matured in vitro, thus removing the need for the exogenous hormone
stimulation required by IVF. This makes IVM not only a much cheaper alternative to IVF, but
it circumvents the possible physiological complications associated with the hormone
treatment in IVF. However, the main disadvantage from which IVM suffers is that it results in
reduced oocyte developmental competence and has pregnancy rates 12% lower than
conventional IVF [36].
GDF9 and BMP15 have shown great promise in improving oocyte developmental
competence during IVM and bridging the gap in success rates between IVM and IVF. A 2006
study by Hussein et al. showed that partially purified ovine BMP15 and mouse GDF9 were
able to significantly increase bovine blastocyst development rate by 17% and 9%
respectively, from the control IVM conditions [44]. This was taken a step further by Yeo et al.
in 2008 using the same GDF9. The addition of this partially purified GDF9 to the IVM media
proved to significantly increase mouse blastocyst hatching and the eventual number of
implanted embryos which continued on to form foetuses. The study also noted that neither
foetal weight nor placental weight was increased by the addition of the GDF9 to the media,
and while these are not the only indicators of foetal health and future development, they
suggested that the treatment would have no adverse effects on the resulting offspring
[45].Studies into the metabolic development of offspring and more long-term development
may yield more insight into the potential effects of GDF9 treatment during IVM. While these
studies have shown some of the potential of GDF9 and BMP15, there is still a great deal
which remains unknown about these proteins, such as exactly how they interact both with
each other and their surrounding environment and how their actions differ between species.
14
1.8 Hypotheses and Aims
1.8.1 Hypothesis 1: There will be functional/ mechanistic differences between mouse and
human GDF9, reflected in the nature of their interactions with human BMP15.
Aim 1: To determine if there is a species difference in bioactivity and synergistic capabilities
between human and mouse GDF9.
-
Utilising purified preparations of the mature regions and pro-mature complexes of
human and mouse GDF9 and human BMP15 in mouse and bovine granulosa cell
proliferation assays.
Aim 2: To determine whether there is a difference in pro-region interactions between human
and mouse GDF9 and determine the function of the pro-region.
-
Utilising purified mature regions and pro-mature complexes of human and mouse
GDF9 and human BMP15 in mouse granulosa cell proliferation assays.
Aim 3: To determine whether mouse GDF9 residues can be introduced into human GDF9 to
produce a non-BMP15 dependant protein.
-
Utilising purified pro-mature complexes of two mouse-like human GDF mutant
proteins in mouse granulosa cell proliferation assays.
1.8.2 Hypothesis 2: Eliminating the post-translational modifications of GDF9 and BMP15
will affect their bioactivity and synergistic capabilities.
Aim 1: To determine the role of previously identified human GDF9 and BMP15 posttranslational modifications on their bioactivity and synergistic capabilities.
15
-
Utilising purified pro-mature complexes of human GDF9 and BMP15 wild-type and
post-translational modification knock-out mutant proteins in mouse granulosa cell
proliferation assays.
16
Chapter 2
Materials and Methods
17
2.1 Proteins:
The following tables show the proteins used throughout this thesis. Those in table 2.1.1 are
the wild-type proteins which are expected to act as they would in vivo. Those in table 2.1.2
are mutant proteins in which we have altered the amino acid sequence in order to ascertain
any changes in activity or interactions.
2.1.1 Wild-type Proteins:
Protein:
Preparations used:
Post-translational
Modifications:
Human GDF9
Pro-mature complex and
Possible phosphorylation
(hGDF9wt)
mature region
Human BMP15
Pro-mature complex and
O-linked glycosylation and
(hBMP15wt)
mature region
possible phosphorylation
Mouse GDF9
Pro-mature complex and
Possible phosphorylation
(mGDF9wt)
mature region
2.1.2 Mutant Proteins:
Protein:
Preparations used:
Post-translational
modifications:
‘Mousified’ GDF9 Mutant 1 (G391R)
Pro-mature complex
Possible phosphorylation
‘Mousified’ GDF9 Mutant 2
Pro-mature complex
Possible phosphorylation
Pro-mature complex
No possible
(S325R/G391R/S412P/K450R)
GDF9S7A
(S325R/S326A/G391R/S412P/K450R)
BMP15T277A
phosphorylation
Pro-mature complex
No o-linked glycosylation,
possible phosphorylation
BMP15S/T (S273A/T277A)
Pro-mature complex
No o-linked glycosylation
or phosphorylation
18
2.2 BMP15/GDF9 Expression Plasmids
The empty pEF-IRES plasmid was obtained from Dr. Stephen Hobbs (Institute of
Cancer Research, London, United Kingdom)(appendix 1.1 fig 1.1) [46]. Expression cassettes
coding for the proteins (appendix 1.1 fig 7.2) were cloned into these plasmids before
transfection into human embryonic kidney 293T cells (HEK293T cells). Sequences for all
proteins can be found in appendix 1.2.
2.3 Cell Lines and Culture Conditions
The Human Embryonic Kidney 293T Cells (HEK-293T cells) obtained from the
American Type Culture Collection were cultured in Dulbecco Modified Eagle Medium
(DMEM) (Invitrogen, Auckland, NZ) containing 10% foetal calf serum (FCS), 2mM Lglutamine, 100IU/mL penicillin (Sigma, St Louis, USA) and 100μg/mL streptomycin.
Puromycin selection also took place in this medium. Protein production from the 293T cells
was performed in DMEM/HAMS F12 media (Invitrogen) containing 0.1mg/mL bovine serum
albumin (Sigma), 100IU/mL penicillin, 100μg/mL streptomycin and 100μg/mL heparin
(Fragmin, Pfizer, New York, USA).
2.4 Establishment of Stable Protein-Expressing Cell Lines
293T cells were plated into 6-well plates and grown to 90-95% confluence. BMP15 or
GDF9 encoding plasmids were transfected into the cells using lipofectamine 2000
(Invitrogen) following the manufacturer’s instructions. The cells were then incubated at 37oC
in a CO2 incubator for at least 24 hours. After 24 hours the cells were trypsinised and split
into 4 wells of a 6-well plate. The cells were put under puromycin selection with wells
containing 0, 0.5, 1 and 2 μg/mL of puromycin (Invitrogen). The cells were grown until the
wells containing puromycin had grown to 90-95% confluence. The range of puromycin
dosages was to make sure the cells were not all killed by too high a dose. Those chosen to
expand were simply the cells which displayed the healthiest growth. In this case the cells
displaying puromycin resistance to 2μg/mL were trypsinised and expanded into 4 x T25 cell
19
culture flasks. These were then treated with a range of puromycin doses (e.g. 0, 2, 4, and
8μg/mL of puromycin). This process continued with increasing puromycin doses until the
GDF9 and BMP15 transfected cells had reached resistance to 120μg/mL of puromycin.
Samples of GDF9 and BMP15 containing conditioned culture medium were tested for the
presence of the respective proteins by Western blotting. The commercially available human
BMP15 and mouse GDF9 mature regions were used as positive controls (R&D Systems,
Minneapolis, USA).
2.5 GDF9 and BMP15 Purification
GDF9 and BMP15 expressing cells were each expanded into 4 x T75 cell-bind tissue
culture flasks. Once the cells had grown to 80% confluence, the culture medium was
removed and the cells washed in un-supplemented DMEM. This was removed and 25mL of
supplemented DMEM/HAMS F12 added to each flask (see Methods, section 2.3). Collections
occurred every 3 days for a total of 4 collections. At this point the cells were discarded and
fresh cells expanded for further collections.
The proteins were prepared with two affinity tags, a His8 tag, and a StrepII tag. This
was initially for a two step IMAC/StrepII tag purification however, comparable protein purity
and higher yields were obtained using a single step of IMAC purification when the
pro/mature complex was being purified. When the isolated mature region was required, an
additional rpHPLC step was necessary.
2.5.1 Nickel Immobilised Metal Affinity Chromatography (IMAC) Purification
BMP15 or GDF9 containing conditioned DMEM/HAMS F12 media (200mL) was
prepared for IMAC by adding imidazole to a final concentration of 5mM, 500μL protease
inhibitor cocktail (Thermo Scientific, Rockford, USA) and 500μL phosphatase inhibitor
cocktail (Calbiochem, Alexandria, Australia) and subsequently adjusted to pH7.4. Invitrogen
Ni-NTA agarose resin (1mL) was equilibrated with 1x 10mL wash with MilliQ water and 1x
20
5mL wash with PBS/5mM imidazole. After each wash the resin was centrifuged at 900rpm
for 5 minutes and the supernatant was removed. The resin was added to the media. The
suspension was mixed on a rotary agitator at 4oC for 3 hours. The media and resin were then
centrifuged at 900rpm for 5 minutes and the supernatant removed. The resin was then
washed once with PBS/5mM Imidazole and twice with a PBS/40mM Imidazole. To elute the
pro-mature complexes, 2mL of PBS/500mM Imidazole elution buffer was incubated with the
resin at room temperature for 5 minutes with mixing occurring every minute to keep the
resin in suspension. Following this, the suspension was centrifuged at 900rpm for 2 minutes.
The supernatant was removed and stored in 2 x 1.5mL protein LoBind tubes (Eppendorf,
Hamburg, Germany). Elutions were continued in this manner for a total of six fractions. At
the end of the purification, the resin was suspended in 0.5mL of SDS-PAGE loading buffer
with 4mM DTT. The resin and the eluted fractions were stored on ice at 4 oC until the
following purification step. The presence of protein in the eluted fractions, flow-through and
resin samples was assessed by Western blotting and total protein staining (silver staining
using a silver quest staining kit from Invitrogen).
2.5.2 Reverse Phase High Performance Liquid Chromatography (rpHPLC)
The rpHPLC for both the GDF9 and BMP15 samples was carried out in conjunction
with the Adelaide Proteomics Centre using Jupiter 5u C4 300A HPLC columns with a
Widepore C4 4x3.0mm SecurityGuard Cartridge (Phenomenex, Lane Cove, Australia). The
column was first pre-treated with a solution containing a total of 1µg each of β-casein and
albumin to prevent the GDF9 and BMP15 irreversibly binding to non-specific binding sites on
the column. The column was then washed with three blank gradients of 0 - 99.9%
acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA) to remove any residual β-casein and
albumin. A dedicated column was used for either the BMP15 or GDF9 samples. A 1mL sample
of IMAC purified protein was then loaded onto the corresponding dedicated column in a
series of 4 x 250μL injections. The protein was eluted from the column using a 99.9%
21
acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA) solution at a flow rate of 0.8mL/minute.
The eluted fractions were collected according to the peaks of A280 absorbance.
2.6 Colorimetric Western Blotting and Silver Staining
IMAC purified samples were prepared for electrophoresis by mixing 30μL of the
sample with 10μL of SDS-PAGE loading buffer with 4mM DTT. For rpHPLC samples, 100μL of
the sample was completely dried down for analysis and suspended in 20μL of loading buffer
with 4mM DTT. All samples were heated at 95oC for 5 minutes and centrifuged for 1 minute
before loading onto a Mini PROTEAN TGX 4-15% gel (BIO-RAD Laboratories, Hercules, USA).
The gels were run for 40 minutes at 150V. Following SDS-PAGE, the gel went on to either
silver staining or Western blotting. Silver staining was performed using a silver quest staining
kit (Invitrogen) following the manufacturer’s instructions. For Western blotting, the gel was
electro blotted onto a nitrocellulose membrane for 75 minutes at 100V. The membrane was
then blocked for 1 hour using a 1% blocking solution. The membrane was washed twice in a
TBS-TWEEN 20 wash buffer and incubated with the primary antibody at room temperature
for 1.5 hours (GDF9 1o Ab = mAb53 [47] @ 1:5000 dilution. BMP15 1o Ab= mAb28 @ 1:5000
dilution) Following this, the membrane underwent 2 x 5 minute washes and a 1 x 15 minute
wash in wash buffer. The membrane was then incubated with the secondary antibody at
room temperature for 1 hour (2o Ab = anti mouse AP @ 1:10000 dilution). Following the
secondary antibody incubation, the membrane underwent 2 x 5 minute washes and a 1 x 15
minute wash in wash buffer. The membrane was then developed using alkaline phosphatase
substrate (Promega).
2.7 Fluorescence Western Blotting and Protein Quantification
For quantification of IMAC purified proteins, known concentrations of the
corresponding commercially available mature regions were used as references. The
fluorescent western protocol was the same as that for a colorimetric western (see section
22
2.6) until the secondary antibody stage. For GDF9 quantification, the secondary antibody was
goat-anti mouse IRDye used at a :10000 dilution, and for BMP15, goat-anti mouse IRDye
used at a 1:20000 dilution. Secondary antibody incubation was for a period of two hours.
This was followed by three 20 minute washes with wash buffer. The secondary antibody
incubation and subsequent washes were carried out in as dark an environment as possible.
The membranes were then read and protein band density measured using ImageJ version
1.44p (Rasband 1997 - 2011). ImageJ output (a measure of the pixel density of the selected
area of the membrane) was analysed for each protein band. The densities of the standard
band were then graphed along with the band densities of the proteins to be quantified.
Using the data linear equations, the concentrations were determined.
Quantification of post-HLPC mature region samples was determined using A280 absorbance
and the appropriate extinction coefficients for each protein.
In both cases, quantification was based in the concentration (in mass) of mature region only.
2.8 Granulosa Cell Proliferation Assay
This assay was based on the method used by Gilchrist et al [2]. Strain SV129 mice (2128 day old) were injected with 5IU of pregnant mare serum gonadotropin (Folligon, Intervet,
Castle Hill, Australia). The mice were killed 44-46 hours after the PMSG injection, and their
ovaries removed and put into warmed Hepes-buffered TCM-199. The ovaries were then
cleaned of any blood and the large follicles punctured with a 27 gauge needle. In the case of
bovine granulosa cell assays, cells were collected from follicular fluid aspirated from abattoir
derived ovaries. Large clumps of granulosa cells were either broken up or removed and all
cumulus-oocyte complexes and denuded oocytes were also removed. The granulosa cells
were then washed once in Hepes-buffered TCM-199 and twice in Bicarbonate-buffered TCM199 before being counted using a haemocytometer. The assay required 25,000 cells per well.
The cells were diluted to give approximately 25,000 cells per 50μL. The assay was carried out
in a 96 well plate with all treatments in triplicate. There were four wells containing only
media and cells (totaling 125μL) and three wells containing 50ng/mL of R&D rmGDF9 as the
23
negative and positive controls, respectively. Once all required wells were filled (with media,
cells and treatment totaling 125μL) the cells were cultured at 37 oC in a CO2 incubator at 96%
humidity for 18 hours. Following this culture period, the cells were exposed to a 6 hour pulse
of 15.4kBq 3H-thymidine in the same conditions as culturing. Following this pulse, the plates
were wrapped in cling film and refrigerated until harvesting. The cells were harvested onto a
filter mat using a Tomtec Cell Harvester. The filter mat was then dried and treated with
betaplate scintillent. The 3H-thymidine incorporation was assessed using a Wallac Trilux
Microbeta liquid scintillation counter. Readouts for this experiment indicate levels of
granulosa cell proliferation which, while not the only possible result of GDF9 and BMP15
treatment has been chosen for this study as a preliminary means of investigation and has
been interpreted as bioactivity.
All experimental procedures were approved by the University of Adelaide Animal Ethics
Committee (Medical) under ethics numbers M-2009-168, and M-2012-047.
2.9 Statistical Analysis
Statistical analyses were carried out using SigmaPlot 12.0. Data were log10
transformed and analysed using 3-Way ANOVA to test for synergistic interactions, and one
Way ANOVA to test between groups. All error bars represent the standard error of the mean
(SEM). Results were deemed significant where P<0.05.
24
CHAPTER 3
GDF9 and BMP15
Wild-type proteins: Species
Difference, Synergism and ProRegion Interactions
25
3.1 Introduction
3.1.1 GDF9 and BMP15 Species Differences
GDF9 and BMP15 have been shown to be essential for normal mammalian fertility
across species, however; one of the features which make these proteins unique among TGFβ super family proteins is that they display differences in expression and function between
species.
Firstly, a species difference can be seen in GDF9 and BMP15 expression ratios. In a
2012 study by Crawford and McNatty [48], it was found that polyovular species such as mice
and rats had a much higher expression level of GDF9 compared to BMP15. Conversely,
monovular species such as sheep and cows had equal or lower expression levels of GDF9
compared to BMP15. It would be predicted that this latter group would also include humans.
This reinforces the idea that GDF9 and BMP15 have different levels of importance between
monovular and polyovular species.
Evidence of a species difference can also be seen through studies involving GDF9 and
BMP15 deficient animals. These studies have largely been carried out using knockout mouse
strains or in the case of monovular animals such as sheep, with naturally occurring BMP15 or
GDF9 mutations. Studies where mice are completely deficient in GDF9 and those in which
sheep are homozygous for mutated GDF9 show that these animals are completely infertile
[6, 10]. In models where only BMP15 is deficient or mutated however, mice are only subfertile [7], while sheep are still completely infertile [10]. This suggests that while mice are
capable of maintaining some fertility in the presence of GDF9 alone, monovular species such
as sheep may rely on the interaction between GDF9 and BMP15 and therefore the presence
of both for fertility.
Aspects of a species difference, particularly in the case of GDF9 can be seen in
functional actions and interactions with BMP15. A 2005 study by McNatty et.al. showed that
mouse GDF9 (mGDF9) produced a significant increase in rat granulosa cell proliferation,
however ovine GDF9 (oGDF9) was inactive unless ovine BMP15 (oBMP15) was also present
26
[42]. Both species of GDF9 showed synergistic interaction with oBMP15 [42]. A similar
pattern was observed in bovine and ovine granulosa cells. In these cells, oGDF9 was inactive
unless oBMP15 was also present [49]. However, in these species, mGDF9 actually inhibited
proliferation when acting alone and increased proliferation when combined with oBMP15
[49].
To date, studies which have been suggestive of a species difference, particularly in
the case of GDF9, have largely been carried out using mouse, bovine and ovine proteins. As
this difference between polyovular and monovular species seems evident, for the purposes
of applications in human reproductive technologies, it would also be beneficial to investigate
whether human GDF9 is also dependant on BMP15 for bioactivity.
3.1.2 Pro-Region Interactions
Another feature of GDF9 and BMP15 is their interactions with their pro-regions. It is
generally acknowledged that the mature region is the bioactive part the protein. However, in
some cases it has been shown that the pro-regions of some TGF-β proteins influence the
bioactivity of the mature region. TGF-β1 and its pro-region (the β1 latency associated
peptide) remain closely, non-covalently associated after processing. This pro-region inhibits
the bioactivity of the mature region until the two are dissociated [25]. Anti-Mϋllerian
hormone on the other hand has a pro-region which significantly increases the bioactivity of
the mature region [26]. GDF9 and BMP15 have also been shown, by co-immunoprecipitation
to have the same close association with their pro-regions after processing [27]. This suggests
that the GDF9 and BMP15 pro-regions may play a role in their bioactivity. It has also been
suggested that the GDF9 and BMP15 pro-region regulates their co-operative interactions
[27]. However, in the case of GDF9 and BMP15, no direct comparison has been made
between the isolated mature regions and the processed pro-mature complexes. Therefore,
the impact that the pro-region has on their bioactivity has yet to be ascertained.
27
3.1.3 Protein Production and Purification
Any current studies on GDF9 and BMP15 largely require the in-house production and
purification of the proteins as they have limited commercial availability. The only available
forms are the mouse GDF9 and human BMP15 isolated mature regions. These are produced
using the respective CHO (Chinese Hamster Ovarian) derived cell lines and purified by
undisclosed procedures. For clear comparisons between species of the properties of the
GDF9 mature region and the pro-mature complex, the proteins should be produced and
purified by the same procedure to eliminate such methodological differences as sources of
any bioactivity differences.
An effective way of producing proteins is via the use of an expression plasmid
incorporating an internal ribosome entry site between an antibiotic resistance gene (such as
puromycin) and the gene for the desired protein [46]; in this case, BMP15 or GDF9, to
transfect cells and produce a stable cell line. Human embryonic kidney 293T cells (HEK293T
cells) have been shown to be an effective cell line for this process [50].
To date, for GDF9 and BMP15 it has been either the isolated mature regions or
partially purified pro-mature complexes (using whole conditioned media [42, 49] or
hydrophobic interaction chromatography [44]) that have been used in studies. To obtain
purified pro-mature complexes in a native form, the proteins must be purified under nondenaturing conditions which are unlikely to disrupt the pro-mature complex. Such a
purification can be performed using different affinity tags. A dual tagging strategy as
suggested by Cass et al [51] using both an 8 histidine tag (His-tag) and a StrepII tag allows for
two affinity purification steps resulting in a more highly purified protein preparation than
one resulting from a single affinity purification step. However, we have found that a single
step of immobilized metal affinity chromatography is sufficient to produce a purified protein.
The position of the tags on the protein is also of importance to the final product. Tagging the
mature region of a TGF-β superfamily protein can render the protein biologically inactive by
interfering with protein folding or ligand/receptor interactions [50, 52, 53]. Placing the tags
at the amino terminus of the pro-region is less likely to interfere with folding and will also
28
allow the pro-mature complex to be purified rather than just the mature region. From these
pro-mature complexes, the mature regions can be easily obtained by using reverse phase
high performance liquid chromatography [50].
This chapter will show the production and purification of human and mouse wild-type
GDF9 mature regions and pro-mature complexes as well as the human BMP15 wild-type
mature region and pro-mature complex.
Also explored in this chapter will be the species differences between human and
mouse GDF9 through their individual actions and their ability to synergise with human
BMP15 on cells derived from different species. There will also be a direct comparison of
GDF9 mature regions and pro-mature complexes to investigate the role of the pro-region.
3.2 Materials and Methods
All protocols (protein production and purification, and granulosa cell proliferation
assays) are described in Chapter 2. The proteins used in this chapter have been purified by
IMAC to obtain a purified pro-mature complex and by rpHPLC to obtain a purified mature
region. All bioassay data is presented in fold changes relative to a negative control of
untreated cells. Results were deemed significant where P<0.05.
Table 3.1: Wild-type Proteins
Protein:
Preparations used:
Post-translational
Modifications:
Human GDF9
Pro-mature complex and
(hGDF9wt)
mature region
Human BMP15
Pro-mature complex and
O-linked glycosylation and
(hBMP15wt)
mature region
possible phosphorylation
29
Possible phosphorylation
Mouse GDF9
Pro-mature complex and
(mGDF9wt)
mature region
Possible phosphorylation
3.3 Results
3.3.1 GDF9 and BMP15 Purification
The proteins used in this chapter are the hGDF9, mGDF9 and hBMP15 pro-mature
complexes and mature regions. The expression cassettes for all these proteins (fig 3.1)
contain His8 and StrepII affinity tags engineered into the N-terminus of the pro-region to be
used for purification.
Figure 3.1: Schematic diagram of basic protein expression cassettes. All proteins
used in this study are based on the same construct. This contains His8 and StrepII
affinity tags engineered into the N-terminus of the pro-regions for purification as well
as the rat serum albumin signal sequence (SS).
The IMAC process is an effective means of purifying these proteins. Figure 3.2 shows
the results of this process in purifying and concentrating the proteins. The initial conditioned
media sample (fig 3.2 lanes 1, 3, and 5) shows that the protein of interest is so dilute in these
samples that it cannot be seen in the presence of the extraneous proteins. Lane 2, 4, and 6 in
figure 3.2 show the first eluted fractions of the IMAC. This shows that the purification has
removed the majority of the extraneous proteins to allow the GDF9 and BMP15 to be seen
30
on a total protein stain (fig 3.2). Indeed, at this point, the major species are the processed
pro-region and mature region. In the case of mouse GDF9 there is also a significant amount
of unprocessed protein present.
Figure 3.2: Silver stained SDS-PAGE gels of GDF9 and hBMP15 IMAC purification
products: 1. hGDF9 conditioned media. 2. hGDF9 purified pro-mature complex. 3.
mGDF9 conditioned media. 4. mGDF9 purified pro-mature complex. 5. hBMP15
conditioned media. 6. hBMP15 purified pro-mature complex.
The HPLC step proved very effective in separating the mature region from the proregion. While each region of each of the proteins (hGDF9 and hBMP15) had a different
retention time on the HPLC column (fig 3.3); the peaks show that each resolved enough to
effectively separate the mature region of each protein. Figure 3.4 shows the successful
separation of the mature region and pro-region via a silver stained SDS-PAGE gel. The
proteins were also analysed using western blotting with antibodies specific to mature region
sequences of GDF9 or BMP15. Figure 5 shows western blots of the final proteins used in
bioassays. For each of the three proteins used in this chapter, these are the pro-mature
complex (fig 3.5 a1 and b1) and the isolated mature regions (fig 3.5 a2 and b2).
31
DAD1 A, Sig=280,4 Ref=450,80 (E:\10-039\GDF9_I14.D)
25.526
mAU
1
50
25.958
40
30
31.517
26.301
31.131
2
20
25.052
10
0
22
24
26
28
30
32
34
min
a) hGDF9
DAD1 A, Sig=280,4 Ref=450,80 (E:\10-039\BMP15_I4.D)
26.116
mAU
2
70
60
25.697
50
1
30
29.647
40
26.563
20
28.348
30.530
10
0
22
24
26
28
30
32
34
b) hBMP15
Figure 3.3: rpHPLC is an effective means of separating the mature region from
the pro-region. Chromatograms denoting the HPLC purification of a) hGDF9, b)
hBMP15. Proteins in the sample measured using A280 absorbance which shows
peaks containing 1: the mature region and 2: the pro-region. The numbering of
peaks is arbitrary. The parts of each protein elute at different times due to
differences in the hydrophobic nature of each protein. The numbers above each
peak indicate the exact elution time of each peak.
32
min
Figure 3.4: Silver stained gels of hBMP15 and hGDF9 final HPLC products: The GDF9
and hBMP15 mature regions are successfully separated from their pro-regions by a
single step of HPLC.
Figure 3.5: Purified Wild-type GDF9 and BMP15 A: Human GDF9 Lane 1:
hGDF9 pro-mature complex, containing the unprocessed pro-mature protein
(around 60kD) and the mature region (between 15 and 20kD). Lane 2: The
isolated hGDF9 mature region (at 15-20kD).The remaining immune-reactive
elements in this sample are impurities in the BSA carrier. B: Mouse GDF9 promature complex (lane 1) and isolated mature region (lane 2) C: hBMP15 promature complex (lane 1) and isolated mature region (lane 2). GDF9 antibody =
mAb53 [47]. BMP15 antibody = mAb28 [52]. These are mature region specific
antibodies. Therefore, the processed pro-region does not appear in these
western blots as it does on silver stained gels.
One obstacle faced was the low expression of mGDF9 compared to hGDF9. Due to this, it
was difficult to produce sufficient amounts of the mGDF9 mature region. However, when the
33
bioactivity of the in-house produced mGDF9 mature region was compared with that of the
commercially available mGDF9 mature region, it was found that there was no difference (fig
3.6). This was despite the fact that they are produced from different cells lines (HEK293T
cells as opposed to CHO cells, respectively). For this reason, it was decided to use the
commercially available mGDF9 mature region for further experiments.
Figure 3.6: There is no significant difference in bioactivity between the inhouse prepared mGDF9 and commercial mGDF9 mature regions. While there
was a trend towards the in-house mGDF9 being less bioactive, this difference
was not significant. Mouse granulosa cells (P=0.062) n=2. Negative control of
untreated granulosa cells.
34
3.3.2 GDF9 Species Difference
Prior to all bioassays both the mature regions and pro-mature complexes of each protein
were quantified relative to commercial standards of known concentration (R&D Systems
mGDF9 and hBMP15 mature regions). All proteins were quantified so that the mature
regions were at equivalent concentrations. This quantification was carried out via
fluorescence western blotting and subsequent comparison of mature region band density.
Mouse Granulosa Cell 3H-thymidine Incorporation
Both the mature regions and pro-mature complexes of mouse and human GDF9 were
compared using 3H-thymidine incorporation as a marker of SV129 mouse granulosa cell
proliferation (fig 3.7). These results showed that neither the mature region nor the promature complex of hGDF9 showed bioactivity when acting in isolation. However, there is a
significant increase in granulosa cell proliferation when hGDF9 is combined with hBMP15 (fig
3.8). This produces a synergistic interaction and occurs with both the mature regions and
pro-mature complexes.
Mouse GDF9 on the other hand shows bioactivity in isolation as both the mature
region and pro-mature complex. Mouse GDF9 also shows a synergistic response with
hBMP15 although, this only occurs in the case of the pro-mature complexes (fig 3.8). The
mature region of mGDF9 does not show a statistically significant synergistic interaction with
hBMP15.
It is also interesting to note that in the case of pro-mature complex synergism in both
species of protein (and the mGDF9 pro-mature complex alone) there is a peak in activity
after which, the response decreases.
35
Figure 3.7: The species difference between human and mouse GDF9 is
displayed with both the mature regions and pro-mature complexes when
acting on mouse granulosa cell thymidine incorporation. Mature Regions:
While the hGDF9wt mature region is not active on mouse granulosa cells
(P=0.139), mGDF9wt mature region is active (P<0.001). There is a significant
difference in activity between these two proteins (P<0.001). n= 4. Analysed by
Kruskall Wallis One Way ANOVA on Ranks with post-hoc Tukey analysis. Promature Complexes: While hGDF9wt is not active on mouse granulosa cells
(P=1.00), mGDF9wt is active (P<0.001). There is a significant difference in the
bioactivity of these two proteins (P<0.001). n=4. Analysed by Kruskall Wallis
One Way ANOVA on Ranks. * denotes significant bioactivity.
36
Figure 3.8: Both human GDF9 mature regions and pro-mature complexes synergise with
human BMP15. Only the mouse GDF9 pro-mature complex synergises with human BMP15.
Mature Regions: The human GDF9 mature region shows a significant synergistic interaction
with the human BMP15 mature region (P<0.001). The mouse GDF9 mature region shows no
synergistic interaction with the human BMP15 mature region (P=0.275). n=4. Interactions
analysed by Three Way ANOVA. Pro-mature Complexes: Both the human and mouse GDF9
pro-mature complexes show synergistic interactions with the human BP15 pro-mature
complex (P<0.001 and P=0.006 respectively). n=4. Interactions analysed by Three Way
ANOVA. * denotes synergistic response
37
Bovine Granulosa Cells 3H-thymidine Incorporation
Similar to the pattern observed in mouse granulosa cells, the hGDF9 mature region
and pro-mature complex does not show significant increases in bioactivity when acting in
isolation (fig 3.9). When combined with hBMP15 however (mature region only) there is a
significant increase in proliferation (fig 3.10). However, this is an additive increase, not a
synergistic interaction. There is no significant increase in proliferation due to the
combination of hGDF9 and hBMP15 pro-mature complexes.
Mouse GDF9 is independently active in bovine granulosa cells as the mature region
but not as the pro-mature complex (fig 3.9). Similar to hGDF9, mGDF9 does not display
synergism with hBMP15 as either the mature region or pro-mature complex (fig 3.10).
38
Figure 3.9: The species difference between human and mouse GDF9 is
displayed with the mature regions when acting on bovine granulosa cell
thymidine incorporation. Mature Regions: There is a significant difference in
bioactivity between human and mouse GDF9 mature regions (P<0.001). n=4.
Analysed by Kruskall-Wallis One Way ANOVA on Ranks. Pro-mature
Complexes: There is not a significant difference between the bioactivity of
human and mouse GDF9 pro-mature complexes (P=0.158). n=4. Analysed by
Kruskall-Wallis One Way ANOVA on Ranks. * denotes significant differences
from other samples in dosage group.
39
Figure 3.10: Neither human nor mouse GDF9 synergises with human BMP15
on bovine granulosa cells. Mature Regions: No significant interaction
between hGDF9 and hBMP15 (P=0.051) Three way ANOVA, however, there is
a significant increase in proliferation as a result of the combination of
hGDF9wt and hBMP15wt. This is an additive effect, not a synergistic effect.
Pro-mature Complexes: There is not a significant interaction between either
the human GDF9 or human BMP15 pro-mature complexes (P=0.098) or the
mouse GDF9 and human BMP15 pro-mature complexes (P=0.127). n=4.
Analysed by Three Way ANOVA.
40
3.3.3 Pro-region Interactions
As the previous results have demonstrated, hGDF9 in either the mature or promature forms is inactive unless in the presence of hBMP15. Apropos of this, there is no
difference in bioactivity between the mature region and pro-mature complex of hGDF9 (fig
3.11). The bioactivity of mGDF9 however, is significantly decreased by the presence of the
pro-region although this is only evident at higher doses (fig 3.11).
In the case of synergistic interactions, the pro-region does not significantly affect the
actions of hGDF9/hBMP15 (fig 3.12). However, with mGDF9/hBMP15, as with isolated
mGDF9, the pro-region decreases the response (fig 3.12).
41
Figure 3.11: The pro-region affects the bioactivity of mGDF9 but not hGDF9.
The presence of the hGDF9 pro-region has no observable effects on bioactivity
(P=0.099) however, the presence of the mGDF9 pro-region results in a
significant decrease in bioactivity at higher doses (P<0.037).n=4 * denotes
significant reduction in bioactivity.
42
Figure 3.12: The presence of the pro-region affects mGDF9 interactions with
hBMP15 but not hGDF9 interactions. The presence of the pro-region has no
effects on the synergistic effect between hGDF9 and hBMP15 (P=0.817). The
synergistic interaction between mGDF9 and hBMP15 however, is significantly
decreased by the presence of the pro-region (P<0.013).n=4 *denotes
significant reduction in bioactivity from other samples in dosage group.
43
3.4 Discussion
3.4.1 Protein Production and Purification
Despite the significance of GDF9 and BMP15 to mammalian fertility and their
potential applications, the very limited commercial availability of these proteins make their
production and purification an essential first step for most studies of this nature. To do this,
the use of the pEFIRES plasmid [46] and transfection into HEK293T cells [50] have been
shown to be effective strategies, as also was the case in this study. Difficulties did however
arise in the production of mGDF9 as its yield was significantly less than that of hGDF9. This
has been seen in other studies where human and mouse GDF9 were being produced [47].
This may be due to the increased potency of mGDF9 and the fact that HEK293T cells are a
GDF9 responsive cell line. The reduced production levels of a fully processed mGDF9 may be
a way of the cells regulating any autocrine/paracrine effects of the GDF9. This low level of
mGDF9 production led to the use of the commercially available mGDF9 mature region for
bioassays after we had ascertained that there was no difference in bioactivity between it and
the mGDF9 mature region we produced.
The majority of the previous studies on GDF9 and BMP15 have used the proteins in
either whole conditioned media [49] or in preparations partially purified by methods such as
hydrophobic interaction chromatography [44]. While this procedure can be effective to a
certain extent, it is still a reasonably non-specific purification method. Higher degrees of
purification can be achieved using affinity tags in conjunction with immobilized metal affinity
chromatography (IMAC) [51]. Using affinity tags allows fewer non-specific proteins to make it
through the purification process. While Cass suggests a dual tagging, double purification
strategy [51], we have found that a single IMAC step is sufficient to produce a pro-mature
complex of greater than 90% purity as assessed by silver staining of SDS-PAGE gels. The
position of any affinity tags is also of great importance to protein purification. It has been
found that placing tags on the C-terminus of the mature region render the protein
biologically inactive [50, 54]. However the positioning of a his-tag on the N-terminus of the
pro-region has been proven to be a highly effective means of obtaining bioactive pro-mature
44
complexes of GDF9 and BMP15 as well as allowing for the separation of an untagged,
bioactive mature region by a further step of reverse phase high performance liquid
chromatography [50, 54] as also seen in this study.
3.4.2 Species Difference
The presence of correct dosages of GDF9 and BMP15 is unquestionably necessary for
normal mammalian fertility. However, it is evident that the importance and exact actions of
these proteins differs between species. For practical purposes within a laboratory setting,
these differences depend not only on the species of origin of the protein but also on the type
and species of the responding cell being tested.
Here, largely a granulosa cell model was used however; the results can be
extrapolated to explain results from other studies. For example, it has been shown in mice
that GDF9 deficiency leads to complete infertility due to a block in folliculogenesis which may
be due in part to a lack of correct granulosa cell proliferation [6]. A BMP15 deficiency on the
other hand results in only sub-fertility [7]. This may be due to the fact that mGDF9 is able to
act alone without the requisite presence of BMP15. It has been made clear in previous
studies using mouse granulosa cell proliferation as a marker for bioactivity that mGDF9 is
highly active on these cells and will also synergise with hBMP15 [50, 55].
There is a discrepancy between the results presented in this chapter and those in the
literature. It has previously been seen that the hGDF9 mature region is active on an
adrenocortical cell line (in Smad 2/3 activation) while the pro-mature complex is inactive
[47]. The human GDF9 mature region has also shown bioactivity on rat granulosa cells, as
well as hGL and KGN cells [50]. In this study however we observed no bioactivity in either the
hGDF9 mature region or pro-mature complex on any of the two cell types tested. We found
that both forms of hGDF9 were dependent on the presence of hBMP15 for bioactivity. Aside
from the differences in assays and cell types used, the major difference was that in the
experiment where the hGDF9 mature region showed activity the doses used were much
45
higher than those used here [47, 50]. The comparatively low doses in these experiments
were chosen as synergistic interactions were of particular interest and this range has been
shown to be the ideal range for BMP15/GDF9 synergism [55]. However, when tested at
higher does (up to 400ng/mL) the hGDF9 produced in this study still did not show any
bioactivity by itself. A possible explanation for this may be levels of post-translational
modifications. It has previously been reported that hGDF9 is phosphorylated, and that the
elimination of this modification renders the protein inactive [56]. In the previous study by
Simpson et al, where significant hGDF9 activity was seen (at doses over 200ng/mL), the initial
expression levels of hGDF9 were much lower than those in this study [47]. It is possible that
the high expression levels of hGDF9 within the host HEK293T cells in this study lead to a
subsequent reduction or elimination of hGDF9 phosphorylation as the post-translational
machinery of the host cells may have difficulty in coping with the high expression levels.
The effects of GDF9 on bovine granulosa cells have also been previously investigated
however; this was using whole conditioned media [49]. These experiments were conducted
using mouse and ovine GDF9 and ovine BMP15 and measured thymidine incorporation, and
inhibin and progesterone production on bovine and ovine granulosa cells [49]. In these
experiments, as in ours, GDF9 and BMP15 did not show any synergistic interaction in
granulosa cell proliferation. While both proteins have been proven to be important in species
such as sheep and cows, it is clear that if there is any synergistic interaction in these species,
it is not observable in the way in which the assays have been carried out in our case.
From the present study, and others mentioned thus far, it is clear that human GDF9 is
incapable of signalling without the presence of human BMP15 in a granulosa cell
proliferation model. Mouse GDF9 on the other hand, is capable of signalling without any aid.
These findings are particularly reinforced by a 2013 study by Peng et al where human GDF9
was not found to produce any increase in Ptx3, Has2 or Ptgs2 expression in mouse granulosa
cells unless in the presence of human BMP15 [57]. As would be expected and concurrent
with our results, mouse GDF9 did not require the addition of human BMP15 to produce a
significant response. The Peng study did not however, show whether the synergistic
46
interactions resulting from the combination of GDF9 and BMP15 were the result of two
homodimers or a heterodimer [58]. Our study could not determine this either.
Heterodimerisation is however, a logical means of GDF9 and BMP15 synergism but this
would be a avenue of further study.
3.4.3 Pro-region Interactions
In previous studies it has been difficult to determine the function of the GDF9 and
BMP15 pro-regions as either only pro-mature complexes or mature regions have been used,
or where both forms have been used they are not directly compared as they are either in
different assays or on different cell lines. As seen in the results so far, the activity patterns
observed with GDF9 and BMP15 are greatly impacted by the type of assay and species of
cell. However, it has recently been seen that the human GDF9 pro-mature complex is latent
but that removing the mature region results in a bioactive protein at high doses [47]. While
this was not a direct comparison of the mature region and pro-mature complex, it does point
towards the pro-region having an inhibitory function. In this study, while the comparison
between the human mature region and pro-mature complex did not result in any observable
differences in this dose range, we were able to see the effect of the pro-region when
comparing the two different forms of the proteins in synergism experiments. Here, we found
that rather than conferring latency on the BMP15 and GDF9 mature regions, the pro-region
only partially inhibits their actions. The same is true of the mouse GDF9 pro-region although
the effects of this can also be seen when comparing the two different forms of mouse GDF9
alone.
47
Chapter 4
GDF9 Species Specific Point
Mutations
48
4.1 Introduction:
GDF9 Species Difference
The experimental work in this chapter was carried out using pro-mature complexes
only as this would give a clearer understanding of what may happen in vivo. Despite the fact
that the hGDF9wt pro-mature complex is inactive by itself, [47, 50], it shows clear bioactivity
in the presence of hBMP15 (see Chapter 3).
The mouse GDF9 pro-mature complex is active alone or in the presence of BMP15
(see Chapter 3). Mouse and human GDF9 share 90% homology in their mature region,
however; it has been observed that even a single point mutation is sufficient to have
dramatic effects. This had been observed in Belclare and Thoka sheep, where point
mutations in GDF9 resulted in sterility and are predicted to disrupt the protein interactions
with receptors [10, 59].
In a 2012 study by Simpson et al, it was found that introducing a single Gly 391Arg
point mutation into human GDF9 (one of the sequence differences between human and
mouse GDF9) was sufficient to activate the pro-mature complex, and to increase the
bioactivity of the isolated mature region 8 fold [47]. However, the bioactivity of the mutated
hGDF9 mature region was still 4.5 fold less than that of the mouse GDF9 mature region [47].
It is possible that introducing further mouse-like mutations into the human GDF9 sequence
may increase its bioactivity to that of mouse GDF9, and in this chapter, this possibility is
investigated. Several more amino acid changes were chosen for a second mutant hGDF9
which, as with the Simpson GDF9 mutant, are in the receptor binding region of the protein.
4.2 Materials and Methods
All protocols for protein production/purification, western blotting, and granulosa cell
proliferation assays are described in Chapter 2. The proteins used in this chapter have been
purified by IMAC to obtain a purified pro-mature complex. All bioassay data is presented in
49
fold changes relative to a negative control of untreated cells. Results were deemed
significant where P<0.05.
Table 4.1: GDF9 Mouse-like Mutant Proteins
Protein
hGDF9wt
mGDF9wt
hBMP15wt
GDF9 Mutant 1
Sequence Change
Wild-type
Wild-type
Wild-type
Gly391Arg
Region
Pro-mature complex
Pro-mature complex
Pro-mature complex
Pro-mature complex
GDF9 Mutant 2
Gly391Arg/ Ser325Arg/ Pro-mature complex
Ser412Pro/Lys450Arg
Predicted Effects
An active human
GDF9
hGDF9
with
potentially greater
bioactivity
than
391
Gly Arg.
4.3 Results
Mutant Proteins
The proteins used in this chapter are all purified pro-mature complexes. These are
the wild-type hGDF9, hGDF9 mutant 1 and mutant 2 (human GDF9 with mouse-like
mutations) (fig 4.1, table 4.1).
50
Figure 4.1: hGDF9 Mutant protein western blot The GDF9 proteins used in
this chapter have been successfully purified using Ni2+ IMAC and contain both
the mature region and the pro-region.1o Ab = mAb 53 [47] 1/5000. 2o Ab =
anti-mouse AP 1/10000.
Prior to all bioassays both the mature regions and pro-mature complexes of each protein
were quantified relative to commercial standards of known concentration (R&D Systems
mGDF9 and hBMP15 mature regions). All proteins were quantified so that the mature
regions were at equivalent concentrations. This quantification was carried out via
fluorescence western blotting and subsequent comparison of mature region band density.
Mouse-like hGDF9 Mutant Proteins
As was seen in the previous chapter, the hGDF9 pro-mature complex is inactive
unless in the presence of BMP15. This can again be seen here as the wild-type human and
mouse GDF9 are compared to the two mouse-like hGDF9 mutant proteins. It can be clearly
seen at all doses that the hGDF9 mutant proteins have significant bioactivity and are able to
51
function independently of BMP15. It is also of note that both mutants are also more active
than the wild-type mouse GDF9 (fig 4.2).
hGDF9 ‘Mouse-like Mutants
Figure 4.2: Both mouse-like hGDF9 mutants are more active than either
human or mouse GDF9 wild-type. Both of the mouse-like hGDF9 mutant
proteins are significantly more active than hGDF9 wild-type (P<0.001 One way
ANOVA on Ranks). They are also more active than mGDF9 wild-type (P<0.001
One way ANOVA).n=5. Different letters denote statistical differences within
dosage groups.
In the case of synergistic interactions, the bioactivity of the mutant GDF9 proteins is
such that the interaction with BMP15 is only additive at high doses however; both mutant
proteins produce synergistic responses with BMP15 at lower doses (fig 4.3 c and d). This is in
contrast to hGDF9 and mGDF9 which produce synergistic responses at all tested doses (fig
4.3 a and b).
52
A
B
53
C
D
Figure 4.3: hGDF9 mutant proteins only synergise with hBMP15 at low
doses. A: There is a statistically significant interaction between hGDF9 and
hBMP15 IMAC. (P = <0.001). B: There is a statistically significant interaction
between mGDF9 and hBMP15 IMAC. (P = 0.006). C: There is not a statistically
significant interaction between GDF9 Mutant 1 and hBMP15 IMAC. (P =
0.160). D: There is not a statistically significant interaction between GDF9
Mutant 2 and hBMP15 IMAC. (P = 0.146). In the latter two groups, synergism
only occurs at low doses. (All analysed using three-way ANOVAs with post-hoc
Holm-Sidak tests).n=4
54
4.4 Discussion:
Mouse-like GDF9 Mutant Proteins
Both forms of the hGDF9 mouse-like mutants produce non-BMP15 dependent,
bioactive hGDF9 pro-mature complexes.
In the Simpson study, it was found that a single Gly391Arg point mutation (our mutant
1) was sufficient to render the human protein bioactive. However, it was still less bioactive
than the mouse GDF9 protein [47]. Hence, the necessity for our hGDF9 mutant 2 protein
with additional mouse-like mutations. Contrary to Simpson et al, in the present study, it was
found that this single mutation produced a protein which almost as bioactive as the mouse
GDF9 wild-type protein. (GDF9 mutant – 55 fold increase, mGDF9 wild type – 60 fold
increase)
55
Chapter 5
Post-translational Modifications
56
5.1 Introduction
GDF9 and BMP15 Post-translational Modifications
Post-translational modifications are another aspect (in addition to the amino acid
sequence) of a protein’s structure which are important for bioactivity and function. For
example, for proteins such as human chorionic gonadotrophin, modifications such as olinked glycosylation have been shown to regulate levels of secretion and bioactivity [60].
As produced by HEK293T cells, the hBMP15 mature region appears as two bands
when analysed by SDS-PAGE. These appear at 16kD and 17kD and were found, in a 2008
study to have different post-translational modifications [61]. The 16kD band of hBMP15 was
found to be phosphorylated at Ser6 and the 17kD band was found to be o-glycosylated at
Thr10 [61].
GDF9 has also been found to be phosphorylated (also as produced by HEK293T cells).
In a 2008 study by McMahon et al, the phosphorylation of both BMP15 and GDF9 were
examined [56]. It was found that neither protein had bioactivity when they were
dephosphorylated and that these dephosphorylated forms were capable of inhibiting the
bioactivity of their phosphorylated counterparts [56]. Interestingly, dephosphorylated GDF9
was able to inhibit the bioactivity of BMP15 and vice versa. These dephosphorylated forms
were not able to produce a synergistic response. The dephosphorylated forms of the
proteins were still able to bind to their receptors, leading the researchers to conclude that
they exerted their inhibitory effects by competing with the wild-type proteins for their
receptors [56].
While all evidence points to GDF9 and BMP15 post-translational modifications having
a regulatory effect on their bioactivity, this area is still relatively unexplored, particularly in
the case of BMP15 o-glycosylation. It is also unknown whether these particular GDF9 and
BMP15 post-translational modifications occur in vivo.
57
The experiments in this chapter will explore the effect on bioactivity of eliminating
the possibility for post-translational modifications of hBMP15 and hGDF9, focussing on the
pro-mature complexes and their synergistic capabilities with wild-type proteins.
5.2 Materials and Methods
All protocols for protein production/purification, western blotting, and granulosa cell
proliferation assays are described in Chapter 2. The proteins used in this chapter have been
purified by IMAC to obtain a purified pro-mature complex. All bioassay data is presented in
fold changes relative to a negative control of untreated cells. All bioassays in this chapter
were carried out on mouse granulosa cells. Results were deemed significant where P<0.05.
Table 5.1 Post-translational Modification Mutant Proteins
Protein
hGDF9wt
GDF9 Mutant 2
Sequence Change
Region
Wild-type
Pro-mature complex
391
325
Gly Arg/ Ser Arg/ Pro-mature complex
Ser412Pro/ Lys450Arg
GDF9 S7A
hBMP15wt
Gly391Arg/ Ser325Arg/ Pro-mature complex
Ser412Pro/
Lys450Arg/Ser326Ala
Wild-type
Pro-mature complex
BMP15T277A
Thr277Ala
Pro-mature complex
BMP15 S/T
Ser273Ala/ Thr277Ala
Pro-mature complex
58
Potential Effects
Human GDF9 with
measurable
bioactivity
and
potential
phosphorylation
Nonphosphorylatable
GDF9 mutatnt 2
Potential o-linked
glycosylation
and
phosphorylation
Un-glycosylated
hBMP15
BMP15T277A which
cannot
be
phosphorylated
5.3 Results
In this chapter, purified pro-mature complexes of two GDF9 mutants were used.
These were the “mouse-like” GDF9 mutant 2(see Chapter 4), and GDF9S7A (nonphosphorylatable GDF9 mutant 2, both sequences can be found in appendix 1).
Figure 5.1 Western blot of GDF9 Mutant Proteins: Colourimetric western blot
of GDF9 mutant 2 and GDF9S7A pro-mature complexes. 1o Ab = mAb53 [47]
(identifies mature region sequence), 2o Ab = anti-mouse AP.
The BMP15 proteins are also all used as the purified pro-mature complex. These are
the hBMP15 wild-type, BMP15T277A (BMP15 with a mutated o-linked glycosylation site) and
BMP15S/T, a further mutant of BMP15T277A which cannot be phosphorylated. Figure 5.2
shows a western blot of these proteins. In the case of BMP15T277A and BMP15S/T, there is
no mature region bar visible at 17kD, showing that these proteins are not o-linked
glycosylated.
59
Figure 5.2 hBMP15 Mutant protein western blot. The BMP15 proteins used in
this chapter have also been purified using Ni2+ IMAC. Note that the T277A and
S/T mutants do not have a mature region bar appearing at 17kD as the wildtype protein does. 1o Ab = mAb28 [52](identifies mature region sequence), 2o
Ab = anti-mouse AP.
Prior to all bioassays both the mature regions and pro-mature complexes of each protein
were quantified relative to commercial standards of known concentration (R&D Systems
mGDF9 and hBMP15 mature regions). All proteins were quantified so that the mature
regions were at equivalent concentrations. This quantification was carried out via
fluorescence western blotting and subsequent comparison of mature region band density.
The effect of phosphorylation and o-linked glycosylation on hBMP15
While there is no difference in the individual bioactivity of hBM15 as a result of
elimination of o-linked glycosylation, this mutation does cause a significant decrease in
60
synergistic capabilities (fig 5.3). Human BMP15 lacking in both glycosylation and
phosphorylation is also no different in individual bioactivity to the wild-type hBMP15 and
also results in a comparable reduction in synergistic capability (fig 5.4). The additional loss of
phosphorylation does not cause any additional decrease in synergistic capability. There is no
difference between the synergistic responses initiated by either hBMP15 mutant protein (fig
5.5).
Figure 5.3: Eliminating o-linked glycosylation decreases BMP15’s synergistic
capability. There is no significant difference in bioactivity between hBMP15wt
and BMP15T277A on mouse granulosa cells (P=0.779. One Way ANOVA). Both
hBMP15wt and BMP15T277A interact synergistically with hGDF9wt (P<0.001
Three Way ANOVA with post-hoc Holm-Sidak test) however, the synergistic
response produced by the combination of hGDF9wt and BMP15T277A is
significantly less than that produced by the wild-type proteins. (n=5). *
denotes synergistic response. # denotes significant difference in synergistic
response.
61
Figure 5.4: Eliminating o-linked glycosylation and phosphorylation decreases
synergistic capability compared to hBMP15wt. There is no significant
difference in bioactivity between hBMP15wt and BMP15S/T on mouse
granulosa cells (P=0.627, One way ANOVA). There is a synergistic interaction
between hGDF9wt and hBMP15wt (P<0.001, Three way ANOVA with post-hoc
Holm-Sidak test) and also between hGDF9wt and BMP15S/T (P<0.001, Three
way ANOVA with post-hoc Holm-Sidak test). The synergistic response
produced by the BMP15S/T is significantly less than that produced by the
hBMP15wt (P<0.001, One way ANOVA on rank with post-hoc Tukey test).n=4.
* denotes synergistic response. # denotes significant difference in synergistic
response.
There is some difference in the fold changes between figures 5.3 and 5.4. This
is due the wellbeing of the mouse colony at the time of the experiments.
When the mice are under stress, the cells do not give a significant a
proliferative response but can still be used.
62
Figure 5.5: There is no additional decrease in synergistic capability due to the
additional elimination of BMP15 phosphorylation compared to the elimination of olinked glycosylation alone. Both BMP15T277A and BMP15S/T synergise with
hGDF9wt on mouse granulosa cells (P<0.001, Three way ANOVA with post hoc HolmSidak test). There is no difference between these synergistic responses (P=0.988, One
way ANOVA). * denotes synergistic response.
The effect of phosphorylation on hGDF9 bioactivity.
Human GDF9 mutant 2 was used as a control when testing for the importance of Ser7
and hence potential phosphorylation of this amino acid, since a bioactive GDF9 protein was
required to assess any differences in individual bioactivity as a result of eliminating posttranslational modifications. The GDF9S7A mutant has all the mutations of the hGDF9 mutant
2 and in addition, the phosphorylation site (Ser7) has been mutated to an alanine residue to
prevent this modification. As can be seen in figure 5.6, the elimination of hGDF9
phosphorylation causes a decrease in bioactivity. This difference becomes more evident as
63
dosage increases. While the un-phosphorylated GDF9 mutant is still capable of synergistic
interactions with hBMP15, the response is significantly reduced from that of the
phosphorylated protein (fig 5.7).
Figure 5.6: The conversion of Ser7 to an Ala residue decreases GDF9
bioactivity. While there is no significant difference between GDF9 Mutant 2
and GDF9S7A on mouse granulosa cells (P=0.070, One way ANOVA) at low
doses, a significant difference is apparent at 50ng/mL (P<0.05, One Way
ANOVA).n=4. * denotes significant difference within dosage group.
64
Figure 5.7: The conversion of Ser7 to an Ala residue reduces GDF9 synergistic
capability with BMP15. There is a synergistic interaction between GDF9
Mutant 2 and hBMP15wt on mouse granulosa cells (P=0.03, Three way
ANOVA with post-hoc Holm-Sidak test) and also between GDF9S7A and
hBMP15wt (P<0.001, Three way ANOVA with post-hoc Holm-Sidak test). The
synergistic interaction between GDF9S7A and hBMP15wt is significantly less
than that between GDF9 Mutant 2 and hBMP15wt (P=0.037, One way ANOVA
on ranks with post-hoc Tukey test). * denotes synergistic response. # denotes
significant difference in synergistic response.
5.4 .Discussion:
The HEK293T hBMP15wt produced for these studies appeared as two mature region
bands at 16kD and 17kD, consistent with previous studies [61]. These bands have been
shown to be phosphorylated and o-glycosylated respectively. The previous studies did not
65
investigate the effects of these post-translational modifications. In the present study, it was
found that neither post-translational modification was observed to alter the individual
bioactivity of hBMP15. A previous study by McMahon et al found that removing BMP15
phosphorylation eliminated the bioactivity of the protein, however in this study; this was not
possible to determine as mouse granulosa cells were used on which hBMP15 has very little
bioactivity. In the case of synergistic interactions however, the removal of post-translational
modifications did have a significant effect on BMP15’s synergistic capabilities with hGDF9.
Eliminating either glycosylation alone or both glycosylation and phosphorylation was found
to significantly decrease hBMP15’s synergistic interactions with hGDF9. The fact that the
additional elimination of phosphorylation did not further decrease the synergistic response
indicates that o-linked glycosylation may be more important to the bioactivity of hBMP15
than phosphorylation and may aid in either receptor binding or effective dimer formation. A
better way to examine BMP15 may be using Smad 1/5/8 activation. However, this is
problematic when using mouse granulosa cells as the Smad 1/5/8 reporter plasmids
transfect poorly. Using this method would also make looking at synergistic interactions
difficult as the main pathway activated during this response is the Smad 2/3 pathway [55]
Phosphorylation is also found on the GDF9 protein. And it has previously been
reported that removing GDF9 phosphorylation eliminates GDF9 bioactivity [62]. In this study,
the main interest was in the effect of phosphorylation of human GDF9, however, as we have
previously shown, the human GDF9 pro-mature complex is inactive. Therefore, we used our
GDF9 mutant 2 protein as a control and a mutant 2 incapable of phosphorylation as we
required a protein which had individual bioactivity. From the experiments on the GDF9s
alone we found that rather than eliminating bioactivity, the removal of phosphorylation
merely inhibited it. We also observed that the removal of the potential to be phosphorylated
decreased synergism with BMP15. This has shown that while GDF9 phosphorylation may
enhance GDF9 bioactivity and ability to synergise with BMP15, it is not absolutely necessary
for these actions.
66
Chapter 6
Discussion
67
In our society, there is an increasing need for understanding of reproduction and
fertility and the use of reproductive technology. There are a number of reasons for this, most
notably, people are starting families at older ages when fertility has naturally declined, the
prevalence of conditions such as polycystic ovarian syndrome (PCOS) and premature ovarian
failure, as well as the artificial reproductive technologies (ART) applications in the livestock
industry. In vitro fertilisation (IVF) has been the most commonly used ART in these instances
however; the potential consequences and high cost of IVF do not make it a viable option in
some cases. The cost of ART to Medicare in Australia has more than doubled in the last 10
years and is expected to continue to rise [63] and the use of hormone stimulation leads to
ovarian hyperstimulation in 15-20% of patients and in its more severe form can be fatal [64].
In vitro maturation (IVM) can be used as a way around these limitations. Here, immature
oocytes are removed from the ovaries and matured in vitro. This removes the need for
hormone treatment however; the success rates are lower than in IVF. This is due to
compromised oocyte developmental competence. For normal levels of developmental
competence the full process of folliculogenesis and correct cumulus-oocyte communications
are required. However, these are compromised in IVM.
GDF9 and BMP15 are two TGF-β superfamily proteins which have been shown to be
essential to the process of folliculogenesis and for normal mammalian fertility. Since GDF9
and BMP15 were discovered, they have primarily been studied as homodimers to gauge their
individual functions. Some of these functions include preventing apoptosis in early antral
follicles and cumulus cells [1, 3], and increasing granulosa cell proliferation [2]. However, as
research into GDF9 and BMP15 progressed, a number of factors suggested that they signal in
a co-operative manner. These were the fact that they are both produced in the oocyte, that
this expression occurs at similar stages of development [23], and that they have closely
linked functions.
Some of the earliest studies on GDF9 and BMP15 were genetic studies involving
knockout (KO) mice and other animals such as sheep which had inactivating GDF9 or BMP15
mutations. For example, in KO mice, a GDF9 deficiency leads to infertility [6]. The same is
68
true of sheep with inactivating GDF9 mutations [10]. However, things become a little less
straight forward in the case of BMP15. BMP15 deficient mice are only sub-fertile while sheep
are completely infertile [7, 8, 10]. Generally it would be expected that knocking out or
inactivating a single allele of a protein would simply decrease its effect. However, an unusual
feature of GDF9 and BMP15 is that knocking out a single allele actually increases fertility [810]. And in the case of a double heterozygous sheep, there is a dramatic increase in fertility
due to an increased ovulation rate [10]. In polyovular species such as mice and rats, there is a
very high level of GDF9 expressed compared to BMP15, while in monovular species such as
sheep and cows (humans would also be expected to fit into this category), the expression
levels of GDF9 and BMP15 are more equal [48]. These findings are in line with what was
previously known about the relative importance of BMP15 for fertility in rodents compared
to other mammals.
More recent studies have shown co-operative interactions between GDF9 and BMP15
being exhibited, largely through their biological functions. A large number of these studies
have been carried out using their functions on granulosa cells. McNatty et al found that GDF9
and BMP15 interact in mouse granulosa cells in proliferation, and bovine inhibin production
and progesterone production [42, 49]. We found similar results where both mouse and
human GDF9 produced a synergistic interaction with human BMP15 on mouse granulosa cell
proliferation but not on bovine cells.
The species differences concerning GDF9 and BMP15 also extend to the species of
the protein as well as the species of the responding cell. In this case, it was between human
and mouse GDF9. This particular species difference has been highlighted in a study by
Simpson et al. This study showed that the human GDF9 pro-mature complex was latent but
that the mature region was active [47]. We also found that the human GDF9 pro-mature
complex was inactive but did not find the mature region to be active. We did however, find
that the human GDF9 pro-mature complex produced a synergistic response when in
combination with BMP15. As the relative expression levels of GDF9 and BMP15 would
indicate, in humans, GDF9 and BMP15 are likely to be co-expressed at similar levels. Taking
69
these two results together we can see that human GDF9 is not latent per se, but dependant
on the presence of BMP15 for bioactivity.
To further investigate the nature of GDF9 species differences it was useful to turn
again to the results of the Simpson study. Here, mouse and human GDF9 were compared and
a single mouse residue was selected from a number of others to create a mutant form of
human GDF9. As previously mentioned, when acting individually, human GDF9 is latent
whereas mouse GDF9 is active. This previous study found that substituting a single amino
acid in the human GDF9 mature region sequence to that in the mouse GDF9 sequence was
sufficient to confer bioactivity on the human GDF9 pro-mature complex [47]. However, in
this study it was found that this mutant was still not as active as mouse GDF9. This led us to
produce a second human GDF9 mutant with three more mutations in its sequence. In our
study, both this mutant and the single Gly-Arg mutant were found to be more bioactive than
both mouse and human GDF9. As the majority of the sequence changes to the human GDF9
were in the receptor binding region of the protein, it is likely that receptor binding affinity is
one of the factors responsible for the GDF9 species difference observed between human and
mouse GDF9. The discovery of this species difference makes GDF9 (as well as BMP15) unique
within the TGF-β superfamily, and has shown that results from studies using one species of
GDF9 cannot necessarily be extrapolated to other species.
Another element of GDF9 and BMP15 which may account for this species difference
is their interactions with their pro-regions. It has been shown that both GDF9 and BMP15
maintain a close association with their pro-regions after processing [27]. A paper by Watson
et al showed that the GDF9 pro-region strongly binds to heparin sepharose, an interaction
which can be extrapolated to binding to heparin sulphate proteoglycans in the cumulus
extracellular matrix [65]. We can hypothesise that as GDF9 is secreted from the oocyte, it will
bind to these proteoglycans, sequestering the protein. In order to activate the signalling
cascade in the target cells, the GDF9 mature region would need to dissociate from the proregion. From the increased bioactivity of mouse GDF9 over human GDF9, mouse GDF9 seems
to have a lower affinity for its pro-region. This would leave the, mature region free to readily
70
bind to its receptors. The higher affinity human GDF9 would have for its pro-region would
leave much lower concentrations of the mature region free in the extracellular matrix to bind
with the receptors.
This would explain its reliance on the additional presence of BMP15 for bioactivity, as
well as the fact that the presence of the pro-region does not affect the human GDF9
synergistic interactions with BMP15. This is currently highly speculative however, and would
need further study to prove or otherwise.
71
There are many potential applications for GDF9 and BMP15. Given the genetic studies
on these proteins, it is clear that modulating their expression levels can both increase and
decrease fertility. This has potential applications in contraception and increasing fertility for
ART. However, before these applications can be realised, there is a great deal more which
needs to be known about GDF9 and BMP15. This study is a further step towards
understanding their roles in fertility, particularly the causes of the GDF9 species difference.
We have also discovered that these proteins are very complex and unusual in their actions
and interactions. This means that any applications need to be carefully tailored to each
species and may not be applicable in all.
72
Chapter 7
Summary
73
Chapter 3

The synergistic interaction between GDF9 and BMP15 differs depending on the type
and species of cell on which the proteins are acting.

Human GDF9 requires the presence of human BMP15 for signalling in a mouse
granulosa cell proliferation model.

Mouse GDF9 can signal independently of human BMP15.

The presence of the corresponding pro-region partially decreases the signalling
effects of GDF9 and BMP15.
Chapter 4

The addition of a mouse-like point mutation into the human GDF9 sequence
produces a protein which can act independently of human BMP15.
Chapter 5

The elimination of human BMP15 o-linked glycosylation decreases its synergistic
capabilities.

The additional elimination of BMP15 phosphorylation does not further decrease its
synergistic capabilities, suggesting that o-linked glycosylation may be more important
to human BMP15 than phosphorylation.

The conversion of Ser7 to an Ala residue decreases GDF9 bioactivity and synergistic
capability.
74
Chapter 8
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80
Appendix 1
Protein Production and Sequences
81
7.1 EF-IRES Vector and Expression Cassette Layout
Figure 7.1.1 Expression Plasmid Construct. Plasmid contains pEF-1α – human elongation
factor 1α promoter. BMP15/GDF9 coding sequences. IRES – internal ribosome entry site
(based on [46])
Figure 7.1.2 BMP15/GDF9 Expression Cassette. Showing SS – signal sequence, F – furin
processing site, His and StrepII affinity tags on the N-terminus of the pro-region.
82
7.2 – Protein Sequences
Human BMP15wt Engineered Construct
tgagaattcaagatctgccaccatgaagtgggtaacctttctcctcctcctcttcatctcc
E N S R S A T M K W V T F L L L L F I S
ggttctgccttttctcaccaccatcaccaccaccatcatggaggtcagtggagccacccg
G S A F S H H H H H H H H G G Q W S H P
cagttcgagaaaggaggtgaacacagggcccaaatggcagaaggagggcagtcctctatt
Q F E K G G E H R A Q M A E G G Q S S I
gcccttctggctgaggcccctactttgcccctgattgaggagctgctagaagaatcccct
A L L A E A P T L P L I E E L L E E S P
ggcgaacagccaaggaagccccggctcctagggcattcactgcggtacatgctggagttg
G E Q P R K P R L L G H S L R Y M L E L
taccggcgttcagctgactcgcatgggcaccctagagagaaccgcaccattggggccacc
Y R R S A D S H G H P R E N R T I G A T
atggtgaggctggtgaagcccttgaccagtgtggcaaggcctcacagaggtacctggcat
M V R L V K P L T S V A R P H R G T W H
atacagatcctgggctttcctctcagaccaaaccgaggactataccaactagttagagcc
I Q I L G F P L R P N R G L Y Q L V R A
actgtggtttaccgccatcatctccaactaactcgcttcaatctctcctgccatgtggag
T V V Y R H H L Q L T R F N L S C H V E
ccctgggtgcagaaaaacccaaccaaccacttcccttcctcagaaggagattcctcaaaa
P W V Q K N P T N H F P S S E G D S S K
ccttccctgatgtctaacgcttggaaagagatggatatcacacaacttgttcagcaaagg
P S L M S N A W K E M D I T Q L V Q Q R
ttctggaataacaagggacacaggatcctacgactccgttttatgtgtcagcagcaaaaa
F W N N K G H R I L R L R F M C Q Q Q K
gatagtggtggtcttgagctctggcatggcacttcatccttggacattgccttcttgtta
D S G G L E L W H G T S S L D I A F L L
ctctatttcaatgatactcataaaagcattcggaaggctaaatttcttcccaggggcatg
L Y F N D T H K S I R K A K F L P R G M
gaggagttcatggaaagggaatctcttctccggcgccgcagacaagcagatggtatctca
E E F M E R E S L L R R R R Q A D G I S
gctgaggttactgcctcttcctcaaaacatagcgggcctgaaaataaccagtgttccctc
A E V T A S S S K H S G P E N N Q C S L
caccctttccaaatcagcttccgccagctgggttgggatcactggatcattgctccccct
H P F Q I S F R Q L G W D H W I I A P P
ttctacaccccaaactactgtaaaggaacttgtctccgagtactacgcgatggtctcaat
F Y T P N Y C K G T C L R V L R D G L N
tcccccaatcacgccattattcagaaccttatcaatcagttggtggaccagagtgtcccc
S P N H A I I Q N L I N Q L V D Q S V P
cggccctcctgtgtcccgtataagtatgttccaattagtgtccttatgattgaggcaaat
R P S C V P Y K Y V P I S V L M I E A N
gggagtattttgtacaaggagtatgagggtatgattgctgagtcttgtacatgcagataa
G S I L Y K E Y E G M I A E S C T C R tgagcggccgcagaattcagt
- A A A E F S
His. tag highlighted in yellow, StrepII tag highlighted in blue, Furin processing site highlighted
in red.
83
Human GDF9wt Engineered Construct
tgagaattcaggatccgccaccatgaagtgggtaacctttctcctcctcctcttcatctcc
E N S G S A T M K W V T F L L L L F I S
ggttctgccttttctcaccaccatcaccaccaccatcatggaggtcagtggagccacccg
G S A F S H H H H H H H H G G Q W S H P
cagttcgagaaaggaggtagccttggttctcaggcttctgggggagaagctcagattgct
Q F E K G G S L G S Q A S G G E A Q I A
gctagtgctgagttggaatctggggctatgccttggtccttgctgcagcatatagatgag
A S A E L E S G A M P W S L L Q H I D E
agagacagagctggcctccttcccgcgcttttcaaagttctatctgttgggcgaggtggg
R D R A G L L P A L F K V L S V G R G G
tcacctaggctgcagccagactccagagctttgcactacatgaagaagctctataagaca
S P R L Q P D S R A L H Y M K K L Y K T
tatgctaccaaggaagggattcctaaatccaatagaagtcacctctacaacactgttcgg
Y A T K E G I P K S N R S H L Y N T V R
ctcttcaccccctgtacccggcacaagcaggctcctggagaccaggtaacaggaatcctt
L F T P C T R H K Q A P G D Q V T G I L
ccatcagtggaactgctatttaacctggatcgcattactaccgttgaacacttactcaag
P S V E L L F N L D R I T T V E H L L K
tcagtcttgctgtacaatatcaacaactcagtttctttttcctctgctgtcaaatgtgtg
S V L L Y N I N N S V S F S S A V K C V
tgcaatctaatgataaaggagccaaagtcttctagcaggactctcggcagagctccatac
C N L M I K E P K S S S R T L G R A P Y
tcatttacctttaactcacagtttgaatttggaaagaaacacaaatggattcagattgat
S F T F N S Q F E F G K K H K W I Q I D
gtgaccagcctccttcaacctttagtggcctccaacaagagaagtattcacatgtctata
V T S L L Q P L V A S N K R S I H M S I
aattttacttgcatgaaagaccagctggagcatccttcagcacagaatggtttgtttaac
N F T C M K D Q L E H P S A Q N G L F N
atgactctggtgtccccctcactgatcttatatttgaatgacacaagtgctcaggcttat
M T L V S P S L I L Y L N D T S A Q A Y
cacagctggtattcccttcactataaaaggaggccttcccagggtcctgaccaggagaga
H S W Y S L H Y K R R P S Q G P D Q E R
agtctgtctgcctatcctgtgggagaagaggctgctgaggatgggagatcttcccatcac
S L S A Y P V G E E A A E D G R S S H H
cggcgccgcagaggtcaggaaactgtcagttctgaattgaagaagcccttgggcccagct
R R R R G Q E T V S S E L K K P L G P A
tccttcaatctgagtgaatacttcagacaatttcttcttccccaaaatgagtgtgagctc
S F N L S E Y F R Q F L L P Q N E C E L
catgactttagacttagctttagtcagctgaagtgggacaactggattgtggctccgcac
H D F R L S F S Q L K W D N W I V A P H
aggtacaaccctcgatactgtaaaggggactgtccaagggcagttggacatcggtatggc
R Y N P R Y C K G D C P R A V G H R Y G
tctccagttcacaccatggtacagaacatcatctatgagaagctggactcctcagtgcca
S P V H T M V Q N I I Y E K L D S S V P
agaccgtcatgtgtacctgccaaatacagccccttgagtgttttgaccattgagcccgat
R P S C V P A K Y S P L S V L T I E P D
ggctcaattgcctataaagagtacgaagatatgatagctacaaagtgcacctgtcgttaa
G S I A Y K E Y E D M I A T K C T C R tgagcggccgcagaattcagt
- A A A E F S
His. tag highlighted in yellow, StrepII tag highlighted in blue, Furin processing site highlighted
in red.
84
Mousification of hGDF9 (mature region sequences)
RXXR (processing site)
▼
mus
GQKAIRSEAK GPLLTASFNL SEYFKQFLFP QNECELHDFR LSFSQLKWDN
homo
GQETVSSELK KPLGPASFNL SEYFRQFLLP QNECELHDFR LSFSQLKWDN
mut1
GQETVSSELK KPLGPASFNL SEYFRQFLLP QNECELHDFR LSFSQLKWDN
mut2
GQETVRSELK KPLGPASFNL SEYFRQFLLP QNECELHDFR LSFSQLKWDN
*
mus
WIVAPHRYNP RYCKGDCPRA VRHRYGSPVH TMVQNIIYEK LDPSVPRPSC
homo
WIVAPHRYNP RYCKGDCPRA VGHRYGSPVH TMVQNIIYEK LDSSVPRPSC
mut1
WIVAPHRYNP RYCKGDCPRA VRHRYGSPVH TMVQNIIYEK LDSSVPRPSC
mut2
WIVAPHRYNP RYCKGDCPRA VRHRYGSPVH TMVQNIIYEK LDPSVPRPSC
mus
VPGKYSPLSV LTIEPDGSIA YKEYEDMIAT RCTCR
homo
VPAKYSPLSV LTIEPDGSIA YKEYEDMIAT KCTCR
mut1
VPAKYSPLSV LTIEPDGSIA YKEYEDMIAT KCTCR
mut2
VPAKYSPLSV LTIEPDGSIA YKEYEDMIAT RCTCR
*Simpson et al., 2012 Endocrinology 153: 1301-1310. Activation of latent human GDF9 by a single
residue change (Gly 391 Arg) in the mature domain. [47]
85