The Functional Roles of the Intra-Oocyte
Phosphatidylinositol 3-kinase (PI3K) Signaling in
Controlling Follicular Development in Mice
Krishna Rao Jagarlamudi
Akademisk avhandling
som med vederbörligt tillstånd av rektorsämbetet vid Umeå universitet för
avläggande av filosofie doktorsexamen
framläggs till offentligt försvar i KBC-huset, sal KB3A9,
fredagen den 30 oktober 2009, kl. 13.00.
Avhandlingen kommer att försvaras på engelska.
Fakultetsopponent
Dr. Lena Sahlin
Institutionen för kvinnors och barns hälsa
Karolinska Universitetssjukhuset, Solna, Sweden
Copyright © 2009 Krishna Rao Jagarlamudi
ISBN: 978-91-7264-827-2
Printed in Sweden by
Arkitektkopia AB, Umeå 2009
2 I have not failed. I've just found 10,000 ways that won't work."
Thomas Alva Edison (1847-1931)
3 4 The Functional Roles of the Intra-oocyte Phosphatidylinositol 3-kinase (PI3K) Signaling in
Controlling Follicular Development in Mice
Krishna Rao Jagarlamudi, Department of Medical Biochemistry and Biophysics, Umeå
University, Umeå, Sweden
Abstract
The key functions of the mammalian ovary are the production of fertilizable oocytes and the
secretion of steroid hormones. At the time of birth the human ovary is composed of basic units
termed primordial follicles. Primordial follicles are long-lived structures in the ovary and some of
them last until the woman reaches menopause. However, the intra-oocyte signaling pathways that
activate primordial follicles and early follicular development are largely unknown.
In this thesis, the functional roles that the phosphatidylinositol 3-kinase (PI3K) signaling pathway
plays in follicular development were investigated. In vivo approaches using genetically modified
mouse models were used to determine the functions of several members of the PI3K signaling
pathway in oocytes and in follicles. The function of Foxo3a, a substrate of Akt, was investigated
by expressing Foxo3a constitutively in oocytes of primary follicles. We found that continuously
active Foxo3a in mouse oocytes caused retardation of oocyte growth, resulting in arrest of
follicular development. The functions of p27kip1 (p27) were studied using p27-deficient (p27-/-)
mice. It was found that p27 suppresses follicle endowment/formation and activation, and that it
induces follicle atresia. The functions of PI3K signaling in oocytes during follicular activation
were also investigated using conditional mutant mice, by disrupting the Pten in oocytes of
primordial follicles. We found that, all primordial follicles were prematurely activated due to
overgrowth of oocytes and these follicles were depleted in young adulthood, causing premature
ovarian failure (POF). At the same time, disruption of the Pten from oocytes of primary follicles
had no effect on activation of primordial follicles, and the follicles developed and matured
normally. The results clearly show that the PI3K pathway in the mammalian oocyte plays a key
role in follicular activation through control of initiation of oocyte growth and follicular
development.
New series No. 1291
ISSN: 0346-6612
ISBN: 978-91-7264-827-2
5 6 Table of Contents
Publication List ................................................................................................................................ 9
1. Introduction of the ovary ............................................................................................................ 11
1.1. The mammalian ovary ....................................................................................................... 11
1.2. Primordial follicle formation ............................................................................................. 12
1.3. Primordial follicle activation ............................................................................................. 13
1.4. Regulation of follicular development by FSH .................................................................. 15
1.5. Oocyte secreted factors for follicular development .......................................................... 16
1.6. Granulosa cell secreted factors for follicular development ............................................... 17
1.7. Ovulation and oocyte maturation ...................................................................................... 18
1.8. Follicular atresia ................................................................................................................ 20
1.9. Corpus luteum (CL) .......................................................................................................... 22
2. The PI3K/Akt signaling pathway ............................................................................................... 23
3. Premature ovarian failure (POF) ................................................................................................ 26
4. Current knowledge on infertility treatment methods ................................................................. 27
5. Aims of the dissertation.............................................................................................................. 29
6. Results ........................................................................................................................................ 30
6.1. Paper I ............................................................................................................................... 30
6.2. Paper II .............................................................................................................................. 31
6.3. Paper III ............................................................................................................................. 31
6.4. Paper IV............................................................................................................................. 32
7. Conclusions ................................................................................................................................33
8. Acknowledgements .................................................................................................................... 34
9. Reference list .............................................................................................................................. 35
7 8 Publication List
This dissertation is based on the following papers.
I.
*Liu L., *Rajareddy S., *Reddy P., Du C., Jagarlamudi K., Shen Y., Gunnarsson D.,
Selstam G., Boman K. and Liu K. (2007) Infertility caused by retardation of follicular
development in mice with oocyte-specific expression of Foxo3a. Development 134(1),
199-209. (* Equal contributions)
II.
Rajareddy S., Reddy P., Du C., Liu L., Jagarlamudi K., Tang W.L., Shen Y., Berthet C.,
Peng. S.L., Kaldis P. and Liu K. (2007) p27kip1 (cyclin-dependent kinase inhibitor 1B)
controls ovarian development by suppressing follicle endowment and activation, and
promoting follicle atresia in mice. Molecular Endocrinology 21(9):2189-202.
III.
Reddy P., Liu L*., Adhikari D*., Jagarlamudi K*., Rajareddy S*., Shen Y., Du C.,
Tang W., Hämäläinen T., Peng SL., Lan ZJ., Cooney AJ., Huhtaniemi I. and Liu K.
(2008) Oocyte-specific deletion of Pten causes activation of the entire primordial follicle
pool. Science 319(5863):611-3. (* Equal contributions)
IV.
Jagarlamudi K., Liu L., Adhikari D., Reddy P., Idahl A., Ottander U., Lundin E. and Liu
K. Oocyte-specific deletion of Pten in mice reveals a stage-specific function of
PTEN/PI3K signaling in oocytes in controlling follicular activation. PLoS ONE
4(7):e6186.
9 10
1. Introduction of the ovary
In this dissertation, a brief introduction of the mammalian ovary is first given, including how the
ovary functions and the main events that take place in the ovary before I discuss my results and
conclusions on which this thesis is based on.
1.1. The mammalian ovary
The mammalian ovary is a heterogeneous organ containing follicles and corpora lutea at different
stages of development. The two key functions of the ovary are the release of mature oocytes for
fertilization, which is necessary for successful propagation of the species, and the secretion of
steroid hormones necessary for follicular development to prepare the reproductive tract for proper
fertilization and the establishment of pregnancy (McGee and Hsueh, 2000).
Ovarian follicles are functional units of the ovary, which start to form at the time of mid gestation
and continue until birth in humans. In particular, primordial follicles provide the source of
fertilizable ova for the entire reproductive lifespan of a woman. In order to produce mature
oocytes, primordial follicles should be recruited into the growing follicle population through
primary, secondary, and preantral stages before acquiring an antral cavity. At this stage, very few
follicles are destined to ovulate (less than 0.1% in humans), whereas most of them undergo atretic
degeneration. The growing follicles that survive reach pre-ovulatory stage under the cyclic
gonadotropin stimulation that occurs after puberty. The dominant pre-ovulatory follicle releases
the mature oocyte for fertilization in response to a pre-ovulatory surge of gonadotropins. The
remnant follicle undergoes so called luteal phase to become the corpus luteum (CL), which
further secretes steroid hormones for establishment of pregnancy in females (Elvin and Matzuk,
1998; Matzuk et al., 2002; McGee and Hsueh, 2000; Richards et al., 2002; Vanderhyden, 2002).
In addition to this, it has been recognized in recent years that the bidirectional communication
between oocytes and somatic cells plays an important role in ovarian follicular development. Oocyte
growth depends on signals, growth factors, and nutrients from granulosa cells. At the same time,
oocytes are not simply passive recipients; instead, they have essential roles in controlling the
proliferation and differentiation of granulosa cells during follicular development (Albertini and
Barrett, 2003; Eppig, 2001; Eppig et al., 2002; Matzuk et al., 2002; Richards et al., 2002;
Vanderhyden, 2002). However, compared to the well characterized events of hormone-regulated
follicular development, the intrinsic ovarian factors that regulate oocyte and follicular
development before the follicle-stimulating hormone (FSH) stage are less well understood (Elvin
and Matzuk, 1998; Peters et al., 1975). The development of ovarian follicles at different stages in
the mammalian ovary is illustrated in Fig. 1.
11
Fig. 1. Structure of a mammalian ovary (obtained from Google Images).
1.2. Primordial follicle formation
Primordial germ cells (PGCs) are the precursors of oocytes in the female germ line. The
migration, mitosis, and meiosis of PGCs at different stages in the mouse embryo will be
described here. PGCs are first discernible in the extra-embryonic mesoderm at embryonic day 7.5
(E7.5) in the mouse (Lawson and Hage, 1994). The PGCs migrate through the hindgut and dorsal
mesentery to colonize the genital ridge. Throughout their migration, PGCs undergo mitosis,
which ceases around E13.5 (Ginsburg et al., 1990). Female germ cells in the embryonic ovary
form clusters (syncytia) that are connected by intercellular bridges as a result of incomplete
cytokinesis, and through which organelles and mitochondria might be interchanged (Epifano and
Dean, 2002; Gondos, 1973; Pepling and Spradling, 1998). The entire population of PGCs then
enters prophase of meiosis I (McLaren and Southee, 1997).
Before the formation of primordial follicles, the syncytia undergo programmed breakdown with a
subset of oocytes within individual syncytia dying (Pepling and Spradling, 2001). This process is
associated with massive apoptosis of oocytes in the later stages of gestation (Epifano and Dean,
2002; Pru and Tilly, 2001). Studies in mice (and sheep) have revealed that during follicle
formation, somatic cells in the ovary invade clusters of germ cells, and syncytial breakdown
occurs just before primordial follicles are assembled (Epifano and Dean, 2002; Pepling and
Spradling, 2001; Byskov, 1978; Odor and Blandau, 1969). Once formed, the pool of primordial
follicles serves as the source of developing follicles and oocytes. The small ovarian follicles that
appear in mammalian ovaries are termed primordial follicles. A primordial follicle consists of an
oocyte arrested at the diplotene stage of meiosis I, which is surrounded by a few flattened somatic
cells, termed pregranulosa cells (Peters, 1969). Oocytes that are not enclosed in primordial
follicles are lost, presumably by apoptosis (Pepling and Spradling, 2001; Coucouvanis et al.,
12
1993; Pesce and De Felici, 1994). A schematic representation of events that occur during
primordial follicle formation is given in Fig. 2.
In humans, primordial follicle formation begins around the time of mid gestation, when a single
layer of flattened pregranulosa cells enclose each oocyte. This process continues until just after
birth (McGee and Hsueh, 2000; Baker, 1963). After oocytes have become enclosed within
primordial follicles, they remain arrested in the diplotene stage of meiosis I. From a peak of 6–7
million at 20 weeks of gestation, the number of oocytes falls radically so that at birth, there are
less than 1 million (Hansen et al., 2008) with approximately 300,000 to 400,000 remaining per
ovary (Block, 1953; Forabosco et al., 1991).
Once formed, the pool of primordial follicles serves as a source of developing follicles and
oocytes. During the reproductive years in humans, the decline in the number of primordial
follicles remains steady at about 1,000 follicles per month and accelerates after the age of 37,
causing ovarian aging. It is generally believed that when the available pool of primordial follicles
has become depleted, reproduction ceases and women enter menopause (Hirshfield, 1991;
Broekmans et al., 2007). Nowadays, researchers also believe that primordial follicles may die out
directly from their dormant stage (Adhikari and Liu, 2009).
1.3. Primordial follicle activation
Every primordial follicle has the potential to grow, mature, and ovulate. It has been proposed that
each primordial follicle may have three possible fates: to remain quiescent, to begin the
development but later undergo atresia, or to develop, mature, and ovulate (Greenwald, 1972). The
activation of primordial follicles is defined by a dramatic growth of the oocyte itself,
accompanied by proliferation and differentiation of the surrounding pregranulosa cells, i.e. 2–4
flattened to 10–15 cuboidal granulosa cells (McGee and Hsueh, 2000). In the mouse ovary, the
oocyte grows faster when it is surrounded by about 10 cuboidal granulosa cells (Lintern-Moore
and Moore, 1979). The critical point in humans and sheep is when there are about 15 cuboidal
granulosa cells surrounding the oocyte (Gougeon and Chainy, 1987; Cahill and Mauleon, 1981).
During activation from a primordial follicle to a primary follicle, the growth of the oocyte per se
is remarkable. For example, during follicular activation and early development, the oocyte grows
aggressively with an almost 300-fold increase in volume during this 2- to 3- week growth phase
in the mouse (Elvin and Matzuk, 1998; Wassarman and Albertini, 1994). This growth phase is
also accompanied by a 300-fold increase in RNA content (Sternlicht and Schultz, 1981) and a 38fold increase in the absolute rate of protein synthesis, as calculated per hour and per oocyte
(Schultz et al., 1979). These events are indicative of a period of robust growth of the oocyte cell,
with intense metabolic activity. Although it was reported in a previous study (Fair et al., 1997)
that transcription in oocytes is initiated at about the time of the secondary follicle stage, oocytes
of non-growing follicles to be active in RNA synthesis, with low but significant levels of RNA
polymerase activity (Lintern-Moore and Moore, 1979).
Our recent study involving deletion of Pten from the oocytes of primordial follicles also
supported the conclusions of the latter study that gene transcription is active in oocytes of
primordial follicles, since we found that deletion of the Pten from oocytes of primordial follicles
13
did cause changes in their development (Reddy et al., 2008). However, very little is known about
the mechanisms that underlie the selective activation of primordial follicles in vivo, as the
initiation of follicular growth is a protracted process characterized by the slow growth of a
substantial number of small follicles over a prolonged period of time, which makes it difficult to
study (McGee and Hsueh, 2000; Hirshfield, 1989). The mechanisms underlying follicular
activation have remained a mystery for decades, especially the question of why some primordial
follicles enter the growth phase while others remain quiescent.
Activation of primordial follicles has been successfully accomplished in vitro. Culture of fetal
ovarian cortical pieces (bovine and baboon) in serum-containing medium, but without exogenous
gonadotropins, led to successful activation of primordial follicles. Under these conditions, many
primordial follicles became activated between 12 and 24 hours from the start of culture; their
granulosa cells changed shape from flattened to cuboidal, and they began to express proliferating
cell nuclear antigen (PCNA). During 7 days of culture, the activated primary follicles and their
oocytes increased significantly in diameter (Fortune et al., 1998; Wandji et al., 1996; Wandji et
al., 1997). Similar results were also observed later with in vitro-cultured slices of ovarian cortex
from goat (Silva et al., 2004). The spontaneous activation of primordial follicles in serum-free
medium suggests that the immature follicles may be subject to inhibition of growth initiation in
vivo, or alternatively that some in vitro factors stimulate the initiation of growth (Fortune et al.,
2000). It has also been reported that primordial follicles are activated by several growth factors
such as Kit ligand (KL), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF),
and insulin in cultured rat ovaries (Nilsson and Skinner, 2004; Nilsson et al., 2002; Kezele et al.,
2002). Growth differentiation factor-9 (Gdf-9), which is secreted by oocyte, increases the
progression of primordial follicles to primary follicles in in vivo condition, indicating that Gdf-9
may be involved in follicular activation (Vitt et al., 2000). Importantly, live offspring have been
generated in vitro from primordial follicles of newborn mouse ovaries by using several growth
factors at different developmental stages (O'Brien et al., 2003).
Furthermore, the addition of FSH was found to have no effect on the development of primordial
follicles in pieces of bovine cortex (Fortune et al., 1998), suggesting that pituitary hormones are
not necessary for the initiation of growth of primordial follicles. With the help of gene-modified
mouse models, it became apparent that primordial follicles were present, and were able to
develop up to the late preantral stage, in ovaries of mice lacking the β-subunit of FSH (Kumar et
al., 1997) or lacking the FSH receptor (FSHr) (Abel et al., 2000; Dierich et al., 1998). These
models further supported the initial findings on the gonadotropin-independent nature of activation
of primordial follicles. At the same time, however, FSH has been shown to have a role in the
positive regulation of the ovarian primordial follicle reserve by enhancing the survival and overall
development of these follicles in mice (Allan et al., 2006), and a role in somatic cell
differentiation into early granulosa cells during the onset of primordial follicle development in
hamsters (Roy and Albee, 2000).
It has been reported that there are significant differences in the expression patterns of various
genes during the transition from primordial to primary follicles. However, the functional roles of
these candidate genes during the transition from primordial to primary follicles are yet to be
elucidated (Kezele et al., 2005). As primordial follicles are long-lived structures in the female
germ line, it is generally believed that primordial follicles may be under constant inhibitory
influence(s) of systemic and/or local origin, to remain dormant. It has thus been hypothesized that
14
a decrease in inhibitory influences and/or an increase in stimulatory factors might allow the
initiation of follicle growth (McGee and Hsueh, 2000; Wandji et al., 1996). Studies in mice have
revealed that many molecules involved in the formation and activation of primordial follicles.
The importance of a number of molecules at different stages of ovarian development during fetal
life and just after birth (based on studies in mice) is illustrated in Fig. 2.
Fig. 2. Schematic diagram of PGCs development, follicle formation, and follicular activation in
mice (figure obtained from Jagarlamudi et al., 2009).
1.4. Regulation of follicular development by FSH
FSH is a major component of the hypothalamic–pituitary–gonad axis, which regulates
reproductive function and ultimately the production of mature gametes for fertilization (Chappel
et al., 1983; Chappel and Howles, 1991; Ulloa-Aguirre et al., 1995). FSH is a heterodimeric
pituitary glycoprotein hormone, composed of an α-subunit and a β-subunit. Interestingly, both
subunits are encoded by two different genes. Both subunits are bound together by non-covalent
bonds and form a biologically active hormone. However, it has been reported that the β-subunit
of FSH confers specificity of the hormone (Achermann and Jameson, 1999; Huhtaniemi and
Aittomaki, 1998). The α-subunit, encoded by single gene is common to luteinizing hormone
(LH), and thyroid stimulating hormone (TSH). FSH hormone levels are controlled by
hypothalamic gonadotropin-releasing hormone (GnRH), which induces its synthesis and
secretion from the anterior pituitary gland. This process is also regulated by secretion of factors
such as inhibin, activin, and steroid hormones by granulosa cells (Huhtaniemi and Aittomaki,
1998; Layman and McDonough, 2000). The blood concentration of FSH is low until puberty. It is
well known that the preantral stages of follicular development occur independently of FSH
action. When the menstrual cycle begins, increased secretion of FSH by the pituitary rescues a
small number of follicles from atresia and they move on to further stages of development. In each
cycle, one follicle becomes dominant and ovulates, while others become atretic (Gougeon and
Lefevre, 1983). FSH acts via its receptor, which is located exclusively on granulosa cells of
healthy ovarian follicles. FSHr belongs to the family of G protein-coupled receptors and complex
15
transmembrane proteins characterized by seven hydrophobic helices with three intracellular and
three extracellular loops and an intracellular tail (Gudermann et al., 1995; Kelton et al., 1992).
Studies on targeted disruption of the α-subunit of FSH in mice results in smaller ovary size and
limited follicular development due to a block in folliculogenesis prior to antrum formation.
Homozygous female mice are infertile due to absence of mature follicles and ovulatory defects.
Surprisingly, the pituitary gland is enlarged and TSH-β producing cells are increased due to lack
of thyroid function. As FSH, TSH, and LH share a common α-subunit, but this finding does not
explain the exclusive function of FSH in ovarian development (Kendall et al., 1995). In order to
investigate the function of FSH, researchers have used mouse models with mutations in FSH-β or
FSHr. These mutant mice have revealed that loss of FSH signaling in females results in a block in
folliculogenesis prior to antrum formation and the mice are completely sterile. Follicles at all
stages up to the preantral stage were observed in the ovaries of these mutant mice, and no mature
follicles and CL were identified. The main difference between these two models is the level of
FSH. Mice homozygous for FSH-β have low levels of FSH whereas FSH levels are elevated in
FSHr mutant mice (Dierich et al., 1998; Kumar et al., 1997). Importantly, mutations of both FSHβ and FSHr have been reported in humans that cause infertility in women (Aittomaki et al., 1996;
Matthews et al., 1993). The results from all the studies mentioned above suggest that both FSHr
and FSH action on granulosa cells is critical for development of mature follicles and ovulation.
1.5. Oocyte secreted factors for follicular development
Growth and development of the granulosa cells and oocyte compartments of the ovarian follicle
occur in a mutually dependent manner. Oocyte growth occurs from primordial follicles to
preantral follicles where the oocyte is closely associated with granulosa cells. At the same time,
the proliferation and differentiation of granulosa cells into cumulus granulosa cells and mural
granulosa cells rely on growth factors secreted by the oocyte (Albertini et al., 2001; Reddy et al.,
2008).
Oocyte secreting factors regulate folliculogenesis by modulating a broad range of activities
associated with proliferation and differentiation of granulosa cells. Gdf-9, bone morphogenetic
protein-15 (Bmp-15), and bone morphogenetic protein-6 (Bmp-6) belong to transforming growth
factor-β (TGF-β) superfamily members are synthesized and secreted by oocytes. It has been
shown that Gdf-9 is an important factor involved in primordial follicle development. Gdf-9 is not
expressed in the primordial follicles of rodents; it is expressed in the primary follicle, but is
expressed in the primordial follicles of cows, mice, and sheep (Bodensteiner et al., 1999; Elvin et
al., 2000; Jaatinen et al., 1999; McGrath et al., 1995; McNatty et al., 2001). Mice with a null
mutation in the Gdf-9 are infertile and show arrested folliculogenesis at the primary stage of
development, indicating that oocyte-derived Gdf-9 is essential for further progression of follicles
(Dong et al., 1996). Primary follicles in Gdf-9 mutant mice have abnormal granulosa cells with
increased expression of KL, and they fail to acquire a theca layer, indicating that Gdf-9 exerts a
paracrine action on the surrounding somatic cells. Oocyte growth and zona pellucida (Zp)
formation proceed normally, but these mice remain infertile due to a lack of mature follicles
(Carabatsos et al., 1998; Dong et al., 1996). Rats injected with Gdf-9 were found to show a
higher rate of primordial-to-primary follicle transition in vivo and to have a higher serum
concentration of the thecal cell marker CYP-17 (Vitt et al., 2000).
16
Another important TGF-β superfamily factor secreted by oocyte is Bmp-15. Female mice lacking
a functional Bmp-15 are sub-fertile; they produce fewer litters of smaller litter size than their
heterozygous or wild-type counterparts. Although follicular growth appears to be normal with
normal ovarian morphology, impaired cumulus granulosa cell development, reduced ovulation,
and reduced fertilization rates were observed (Su et al., 2004; Yan et al., 2001). The other family
of growth factors secreted by oocyte is the fibroblast growth factor (FGF) family, including
FGF8. In adult mouse ovaries, FGF8 is expressed specifically in oocytes (Valve et al., 1997).
Furthermore, expression of FGF receptors has been reported in granulosa cells of human, mouse,
rat, and bovine ovaries. It has been shown recently that oocyte derived Bmp-15 and FGFs
cooperate to promote glycolysis in cumulus granulosa cells (Sugiura et al., 2007).
1.6. Granulosa cell secreted factors for follicular development
In humans, FOXL2 is a single-exon gene of 2.7 kb; its product belongs to the family of wingedhelix/forkhead transcription factors. The Foxl2 is specifically expressed in the eyelids and ovaries
of mammals (Cocquet et al., 2002; Crisponi et al., 2001). In mice, Foxl2 is expressed throughout
ovarian development. It is detected mainly in somatic cells of the mouse fetal ovary, but is absent
from germ cells (Cocquet et al., 2002; Pisarska et al., 2004; Schmidt et al., 2004; Uda et al.,
2004). After birth, Foxl2 is expressed in pregranulosa cells; the expression level becomes slowly
reduced in granulosa cells of adult follicles from the primordial stage to the preantral stage.
Studies on Foxl2-deficient mice have shown that Foxl2 plays an important role in controlling the
activation of and probably also the formation of primordial follicles. In these mice, although
primordial follicles are formed normally, the pregranulosa cells do not complete their flattened to
cuboidal transition, which leads to the absence of secondary follicles. Also, there are no signs of
granulosa cell differentiation in Foxl2 mutant ovaries. These findings indicate that Foxl2 in (pre)
granulosa cells is important for the differentiation of pregranulosa cells into granulosa cells
during follicular activation (Schmidt et al., 2004; Uda et al., 2004).
Anti-müllerian hormone (AMH), also called Müllerian inhibitory substance (MIS), is a member
of the TGF-β superfamily of growth and differentiation factors. In ovaries of the rat (Baarends et
al., 1995; Hirobe et al., 1992) and mouse (Durlinger et al., 2002), AMH and its mRNA are not
found in the pregranulosa cells of primordial follicles. Once these follicles have been activated,
their granulosa cells promptly start to express AMH, which peaks in the granulosa cells of large
preantral and small antral follicles. It is believed that this typical expression pattern of AMH in
granulosa cells has two important functions. During the initial follicular recruitment, it inhibits
the activation of primordial follicles; and during cyclic recruitment, it reduces the responsiveness
of growing follicles to FSH (Durlinger et al., 1999; Durlinger et al., 2001; Durlinger et al., 2002).
Studies on AMH mutant (AMH-/-) female mice revealed that the number of growing follicles was
increased despite the fact that serum FSH levels in these animals were lower than in wild-type
controls (Durlinger et al., 1999). This finding was confirmed in an in vitro culture system of
mouse preantral follicles, in which AMH was found to inhibit FSH-induced follicular growth
(Durlinger et al., 2001). This observation was further confirmed by an in vivo experiment
showing that in immature AMH-null females, more follicles started to grow under the influence
of exogenous FSH than in their wild-type littermates (Durlinger et al., 2001). These results
confirmed the role of AMH in the cyclic recruitment of ovarian follicles in mice.
17
KL is a growth factor with both soluble and membrane-bound forms that have a wide range of
effects on various cell types (Besmer, 1991). KL produced by granulosa cells appears to act on
the oocyte to stimulate it to enlarge and initiate development (Parrott and Skinner, 1999; Kezele
and Skinner, 2003). KL was found to induce the primordial-to-primary follicle transition and also
to stimulate stromal cell and thecal cell growth (Parrott and Skinner, 2000). KL stimulates thecal
cell androgen production, but has no effect on stromal cell steroidogenesis (Parrott and Skinner,
2000). The receptor for KL is the Kit tyrosine kinase receptor (Zsebo et al., 1990). The White
Spotting (W) locus encodes Kit (Chabot et al., 1988) and the Steel (Sl) locus encodes KL (Huang
et al., 1993). In mouse embryos, KL is expressed in somatic tissues in the pathway of PGC
migration, with the highest levels in the genital ridges, whereas Kit is expressed on PGCs from
E7.5 through their migratory phase and until shortly after they have stopped proliferating in the
gonads (Besmer, 1991; Lev et al., 1994; Russell, 1979; Zhang et al., 2000). The proliferation and
survival of PGCs is supported by Kit and KL. Mutations in the Sl and W loci leads to improper
migration of PGCs, resulting in germ cell deficiency (Russell et al., 1948; Matsui et al., 1990;
Buehr et al., 1993).
1.7. Ovulation and oocyte maturation
Ovulation is defined as the rupture of a follicle at the surface of the ovary, and release of the
oocyte into the oviduct for proper fertilization under the influence of gonadotropin (Parr, 1975).
Ovulation is a complex process that is initiated by the surge of LH, and is controlled by
expression of a number of genes. LH, a member of the glycoprotein hormone family, initiates the
ovulation by acting on thecal and granulosa cells of pre-ovulatory follicles. In the ovulation
process, the ovary must undergo a series of several steps such as acquiring the ability to produce
estrogens and increase the expression of LH receptors (LHr) on thecal cells to respond to LH
(Richards, 1994). In the ovary, LH acts on pre-ovulatory follicles, which further lead both thecal
cells and granulosa cells to stimulate signaling cascades that further induce specific genes
responsible for both follicle rupture and hormone synthesis (Robker et al., 2000b).
It is well established that during ovulation, extensive tissue remodeling is the essential step.
Matrix-remodeling proteases belonging to two proteolytic systems, the plasminogen activator
(PA) system and the matrix metalloproteinase (MMP) system, have been suggested to play key
roles in tissue remodeling (Ny et al., 2002). It has also been reported that the activity and mRNA
levels of several components of the PA system such as PA inhibitor-1 (PAI-1) and tissue type PA
(tPA) are coordinately regulated by gonadotropins in a time-dependent and cell-specific manner
during ovulation, indicating that these components have a role in follicle rupture (Liu et al.,
1991). Expression of progesterone receptor (PR), a nuclear receptor transcription factor, is
induced in granulosa cells of pre-ovulatory follicles during ovulation and is essential for
induction of two different proteases named ADAMTS-1 (a disintegrin and metalloproteinase
with thrombospondin-like motif 1) and cathepsin-L (a lysosomal cysteine protease) (Robker et
al., 2000a). Mice that lack PR are infertile due to defects in ovulation. Under normal conditions,
transcription of ADAMTS-1 is induced in granulosa cells and cumulus granulosa cells by LH in
the pre-ovulatory follicles during ovulation, but is greatly reduced in PR-deficient mice (Robker
et al., 2000a). ADMTS-1 null mice have defects in ovulation due to inhibition of cleavage of
vercican, the substrate of ADMTS-1 (Russell et al., 2003). Expression of cathepsin L in the
granulosa cells of growing follicles is induced by FSH, but the highest levels of cathepsin L
18
mRNA expressed in pre-ovulatory follicles in response to LH, in a PR-dependent manner,
because cathepsin L mRNA expression is completely abolished in PR-deficient mice (Robker et
al., 2000a). All these factors in the cumulus oocyte complex (COC) and selected signaling events
within the COCs of pre-ovulatory follicles are essential for proper formation of an extracellular
matrix, which is critical for ovulation.
Oocyte maturation involves germinal vesicle breakdown (GVBD), chromatin condensation,
segregation, and the first polar body extrusion (Trounson et al., 2001). It is also defined as
resumption of meiosis from the prophase of the meiotic division, where the primary oocyte is
arrested at meiosis І. It is well known that meiotic arrest in primary oocyte is maintained by
cAMP generated by granulosa cells and transported through gap junctions. Oocyte resumes
meiosis when the intracellular concentrations of cAMP decline, an effect mediated by the
surrounding granulosa cells (Downs, 1995). In humans, when oocytes prematurely removed from
their follicular environment, rapidly resume the meiotic division due to the lack of supply of
cAMP from granulosa cells (Edwards, 1965). Maintenance of the elevated concentrations of
cAMP in the oocyte, either by inhibition of phosphodiesterase or by incorporation of cyclic
nucleotides in the culture medium, inhibits the resumption of meiosis (Tornell and Hillensjo,
1993). There was also speculation that the cAMP-dependent kinases maintain meiotic arrest via
phosphorylation of the regulatory proteins that are responsible for initiation of nuclear
maturation. It has been shown in Xenopus oocytes that high concentrations of cAMP-dependent
protein kinase A (PKA) result in phosphorylation of the p34cdc2, subunit of maturation
promoting factor (MPF), and thus inhibit activation of MPF, which is known to be responsible for
the start of resumption of meiosis (Rime et al., 1992). The oocyte generates cAMP by way of a
constitutively active heterotrimeric G protein (Gs)-linked receptor, GPR3 or GPR12, which
stimulates adenylyl cyclase (Mehlmann, 2005). cAMP maintains arrest of meiotic prophase by
way of PKA mediated phosphorylation of proteins that regulate cyclin-dependent protein kinase
(CDK) (Jones, 2008). Maintenance of germinal vesicle (GV) arrest depends on CDK1 activity
(maturation or M-phase promoting factor; cdc2), a kinase implicated in dissolution of the nuclear
envelope and condensation of chromatin (Doree and Hunt, 2002; Jones, 2004): two events
associated with entry into the first meiotic division. Key to maintaining low levels of CDK1 is
regulation of the kinase and phosphatase responsible for phosphorylation/dephosphorylation of
CDK1, respectively. Thus, during GV arrest the activity of wee1B, the kinase that negatively
affects CDK1 activity through phosphorylation is high, while the activity of cdc25B, the
phosphatase responsible for activating CDK1 is low, probably by direct phosphorylation through
PKA (Duckworth et al., 2002; Han et al., 2005; Lincoln et al., 2002). Regulation of CDK1 and
cyclin B1 also appears to be important for maintenance of GV arrest. This is because it is
possible to raise CDK1 activity by overexpressing cyclin B1, the regulatory partner of CDK1,
and in so doing induce GVBD even in the presence of high levels of cAMP (Ledan et al., 2001;
Marangos and Carroll, 2004; Reis et al., 2006). It has been shown that APCcdh1 (anaphasepromoting complex), the regulator of cyclin B1 levels in oocytes, is itself regulated during GV
arrest by association with its inhibitor Emi1, since overexpression of Emi1 leads to GVBD,
presumably inhibition of APCcdh1 allows cyclin B1 levels to accumulate (Marangos et al., 2007).
These studies suggest that mammalian CDK 1 has a physiological role in ovulation.
There is considerable evidence that phosphodiesterase type 3A (PDE3A), which hydrolyzes
cAMP, plays an important role in regulating resumption of meiosis. PDE3A is expressed in the
rodent oocyte and is responsible for the regulation of cAMP concentration in oocytes (Conti et
19
al., 2002). PDE3A null female mice are sterile because of a meiotic block at the G2–M transition
(Masciarelli et al., 2004). Recently, it was reported that Akt phosphorylates PDE3A and activates
during oocyte maturation, thus reducing the levels of cAMP (Han et al., 2006). The downstream
pathway(s) by which high cAMP levels prevent meiotic maturation is/are not clearly understood,
but a recent study has shown that cGMP produced by granulosa cells passes to the oocyte through
gap junctions and inhibits meiosis by maintaining high levels of cAMP. cGMP inhibits
hydrolysis of cAMP by phosphodiesterase PDE3A. During ovulation, LH signaling reduces
cGMP synthesis in somatic cells, and decrease in cGMP levels leads to increased activity of
PDE3A (Norris et al., 2009).
It has also been reported that the phosphatidylinositol 3-kinase (PI3K) signaling pathway plays an
important role in the meiotic maturation induced by FSH (Hoshino et al., 2004). Akt is
phosphorylated at two residues, threonine 308 (Thr308) and serine 473 (Ser473), and both
residues are necessary for its activation (Alessi et al., 1996). Inhibition of Akt phosphorylation in
both MІ and MІІ oocytes by the PI3K-specific inhibitor LY294002 results in suppressed GVBD
and polar body extrusion in mouse oocytes (Hoshino et al., 2004). Interestingly, Akt
phosphorylated (p-Akt) at Thr308 has been found to be located in pericentriolar materials (PCM)
and p-Akt at Ser473 is located in the spindles of oocytes at meiosis І and meiosis ІІ (Hoshino et
al., 2004; Kalous et al., 2006). These reports indicate that p-Akt at Thr308 and p-Akt at Ser473
may function individually in oocytes for the completion of meiosis (Hoshino and Sato, 2008;
Kalous et al., 2006). It has also been reported that microtubule organization is regulated by p-Akt
at Thr308 and second polar body extrusion by p-Akt at Ser473 in meiosis ІІ oocytes (Hoshino et
al., 2004; Hoshino and Sato, 2008; Kalous et al., 2006). Moreover, inhibition of Akt leads to
inactivation of CDK1 through Myt1, which in turn causes inhibition of resumption of meiosis. It
has also been shown that p-Akt at Thr308 in PCM triggers GVBD through activation of the
CDK1/cyclin B complex (Kalous et al., 2006).
Recent studies have suggested that LH stimulates ovarian production of epidermal growth factor
(EGF)-like factors from granulosa cells and insulin-like 3 (INSL3) from thecal cells to promote
GVBD (Kawamura et al., 2004; Park et al., 2004). EGF-like growth factors such as amphiregulin,
epiregulin, and β-cellulin stimulate expansion of cumulus granulosa cells, which is an essential
step for oocyte maturation process. Pre-ovulatory increases in LH also stimulate granulosa cells
to produce brain-derived neurotrophic factor (BDNF), which is capable of promoting first polar
body extrusion and the cytoplasmic maturation of oocytes (Kawamura et al., 2004; Park et al.,
2004). A recent study has shown that KL also stimulates oocyte maturation by inducing cyclin
B1 expression, and expression of both KL and Kit receptor was found to be increased by
injection of hCG. Treatment of COC complex with KL stimulated first polar body extrusion (Ye
et al., 2009).
1.8. Follicular atresia
The mammalian ovary consists of a finite store of germ cells, which is established during
oogenesis and used later for folliculogenesis. Most of these follicles are small, named dormant
primordial follicles and a small number of follicles are at different developmental stages. The
human ovary contains less than 1 million follicles at the time of birth (Hansen et al., 2008).
However, only about 400 oocytes are released through ovulation and the rest of the oocytes
20
undergo apoptosis (Kaipia and Hsueh, 1997). Although several pro-survival and pro-apoptotic
molecules are involved in the apoptosis of ovarian follicles, the molecular mechanisms involved
are still not completely unknown.
In humans, primordial follicle formation begins around the time of mid gestation. This process
continues until just after birth. After oocytes have become enclosed within primordial follicles,
they remain arrested in the diplotene stage of meiosis I (Baker, 1963; McGee and Hsueh, 2000).
Oocytes that are not enclosed in primordial follicles are lost, presumably by apoptosis. As a
consequence of apoptosis, the number of germ cells enclosed in primordial follicles at birth is
less than 20% in humans and less than 5% in the cow. There is solid evidence that the normal fate
of a female germ cell during oogenesis is death (Baker, 1963; Erickson, 1966). However, the
molecular mechanisms underlying oogonia death are not clearly understood.
In women, menopause is the consequence of exhaustion of the pool of such follicles. Since study
of follicular development and apoptosis is difficult in humans, studies have been conducted in
mice and sheep in order to identify the factors of apoptosis. For example, in sheep, comparison of
the population of primordial follicles in ovaries of 2 and 8 year-old ewes has shown that
approximately 8 primordial follicles disappear from the reserve follicle pool every day
(Driancourt et al., 1985). This contrasts with the estimated rate of follicular activation from the
pool (two to three follicles per day) and also strongly suggests that five to six follicles in the
primordial follicle pool die every day (Driancourt et al., 1985; Reynaud and Driancourt, 2000).
Since granulosa cell apoptosis is never seen in such follicles, it may be assumed that oocyte death
is the cause of this high death rate in primordial follicles. Similar reasoning would mean that 50%
of the preantral follicles disappear during the preantral stage (Driancourt et al., 1985). In addition,
because the process of oocyte death appears to be fast, the chances of detecting a small fraction
of dying oocytes within several thousand primordial follicles and several hundred preantral
follicles, respectively, are rather limited. However, recent experiments describing the
consequences for the ovary of targeted expression of Bcl2 in mouse oocytes (resulting in
increased numbers of healthy preantral follicles and reduced numbers of atretic preantral
follicles) have provided the first possible evidence of this event (Morita et al., 1999b).
Furthermore, the observation that Bax mutant mice have increased numbers of primary, preantral
follicles also demonstrates that apoptosis is a key regulatory process of primary, preantral
follicular development (Perez et al., 1999).
Caspases are known to be involved in mediating follicular atresia during development. Inactive
caspase 3 is expressed in granulosa cells from healthy follicles whereas granulosa cells from
atretic follicles have increased concentrations of activated caspase-3 (Boone and Tsang, 1998).
Loss of caspase-3 function leads to defective atresia of maturing follicles in vivo. Furthermore,
loss of caspase-3 function leads to survival of more mature follicles but mutant female mice have
been found to possess aberrant atretic follicles containing granulosa cells (Matikainen et al.,
2001). Caspase-2 mutant females also have more primordial follicles at the time of birth, due to
defective atresia during meiosis (Bergeron et al., 1998).
21
1.9. Corpus luteum (CL)
CL is a transient endocrine organ derived from the ovulated follicle through a luteinization
process. CL is formed in the ovary through activation of the LHr in thecal cells; the pre-ovulatory
LH surge causes ovulation and rapidly initiates a program of terminal differentiation of the
ovulated follicle into a CL (Richards et al., 2002). In this process, transformation of proliferative
follicular cells into non-dividing progesterone-producing luteal cells is an important step. This
process is called luteinization. It has been reported that several key genes are involved in this
process. For example, PR, cyclooxygenase-2 (COX-2), CATT/enhancer binding protein-β
(C/EBP-β), early growth response protein-1 (Egr-1), and Nur77 are transiently induced by an LH
surge in follicular cells (Stocco et al., 2007). Mice deficient in PR, C/EBP-β, or COX-2 are
infertile because they develop pre-ovulatory follicles but fail to ovulate (Stocco et al., 2007).
COX-2 and PR mutant female mice do not ovulate even in response to exogenous hormones but
form CL containing trapped oocytes, suggesting that luteinization can occur in the absence of
these molecules (Lim et al., 1997; Lydon et al., 1996). In contrast, C/EBP beta null mice ovulate
fertilizable eggs in response to gonadotropin stimulation, but luteinization does not take place,
and the CL is not formed even when the ovaries are transplanted into normal hosts (Sterneck et
al., 1997). These data indicate that this phenotype is caused by intrinsic ovarian defect(s), and
suggest that C/EBP beta has a key role in the process of luteinization. The C/EBP beta
transcription factor, which belongs to the basic leucine zipper class of DNA binding proteins, is
rapidly induced by LH in granulosa cells in vivo (Sirois and Richards, 1993; Sterneck et al.,
1997).
The main function of the CL is to secrete progesterone, which acts on the uterus to prepare it for
implantation of the embryo. Several other functions of CL have also been demonstrated. In
rodents, luteal cells synthesize androstenedione and estradiol, but become a substantial site of
progesterone biosynthesis (Stocco et al., 2007). This is in contrast to the follicle, where
progesterone mainly serves as substrate for estradiol production, which has a negative feedback
effect on the pituitary gland to inhibit FSH release (Knobil, 1980). This prevents new ovulations
during pregnancy. The CL expresses high levels of key proteins involved in the uptake, synthesis,
and transport of cholesterol, and in the processing of cholesterol to progesterone, androgens, and
estrogens. As in humans, in rodents the CL also produces androgens and estrogens in addition to
progesterone. The major androgen produced by the ovary is the weak androgen androstenedione.
Conversion of progesterone to androstenedione is mediated by the enzyme P45017hydroxylase/C17–20 lyase (P450c17 or CYP17) (Stocco et al., 2007).
If fertilization or embryo implantation is unsuccessful or the pregnancy period is over, the CL
must be regularly eliminated from the body to allow normal reproductive function. In rodents,
regression of the CL occurs in two phases, the first of which is known as functional regression
and is associated with a marked decrease in progesterone production (Niswender et al., 1994).
The second phase, termed structural regression, occurs after the initial decline in progesterone
output. It is during this phase that the luteal cells die through programmed cell death. Like
luteinization, during the process of luteal regression the CL undergoes substantial changes in its
steroidogenic capacity, vascularization, and remodeling, resulting in a gland formed principally
by connective tissue and known as the corpus albicans (Gaytan et al., 1997; Paavola, 1979;
Stocco et al., 2007).
22
2. The PI3K/Akt signaling pathway
The PI3K/Akt pathway is a fundamental signaling pathway that consists of various signaling
molecules including kinases, phosphatases, and transcription factors; these facilitate the
fundamental regulation of cell proliferation, survival, migration, and metabolism (Blume-Jensen
and Hunter, 2001; Cantley, 2002; Stokoe, 2005). PI3Ks belong to a conserved family of lipid
kinases that phosphorylate the 3-hydroxyl group of phosphoinositides. The most wellcharacterized product of this reaction is phosphatidylinositol-3,4,5-trisphosphate (PIP3), a critical
secondary messenger that recruits Akt for activation of growth, proliferation, and survival
signaling (Cantley, 2002). PTEN (phosphatase and tensin homologue deleted chromosome on
ten), a lipid phosphatase, dephosphorylates the inositol phospholipids and thus functions as a
major regulator of PI3K. The PI3K pathway is unique, and some of the major elements of this
pathway have been found to be mutated or amplified in a broad range of cancers. For example,
deregulation of PIP3 levels, through the inactivation of lipid phosphatase PTEN or through
constitutive activation of p110-alpha lipid kinase activity, occurs in numerous human cancers (Li
et al., 1997).
Akt is one of the serine/threonine protein kinases; cellular homolog of the viral oncogene v-Akt.
There are three closely related enzymatic isoforms, Akt1 (PKBα), Akt2 (PKBβ), and Akt3 (PKBγ)
encoded by three different genes located on chromosomes 14q32, 19q13, and 1q43, respectively
(Okano et al., 2000). Each gene encodes a protein containing a PH domain at the N-terminus, a
central kinase domain, and a C-terminal regulatory domain. The PH domain of Akt preferentially
binds to PIP3. All three mammalian Akt genes are widely expressed in various tissues, but Akt1
is most abundant in the brain, heart, and lungs, whereas Akt2 is mainly expressed in skeletal
muscle and embryonic brown fat, and Akt3 is predominantly expressed in the brain, kidneys, and
embryonic heart (Altomare et al., 1995; Brodbeck et al., 1999; Coffer and Woodgett, 1991). The
interaction of the PH domain of Akt with PIP3 induces conformational changes in Akt, resulting
in exposure of its two main phosphorylation sites (Thr308 in the kinase domain and Ser473 in the
C-terminal regulatory domain); phosphorylation of both sites is required for its full activation
(Alessi et al., 1996). 3-Phosphoinositide-dependent kinase 1 (PDK1), which is thought to be
activated constitutively, phosphorylates Akt at Thr308, leading to stabilization of the the active
conformation (Blume-Jensen and Hunter, 2001).
Activated Akt protein modulates the function of numerous substrates related to the regulation of
cell proliferation, such as glycogen synthase kinase-3 (GSK-3). Akt inhibits GSK-3 activity by
direct phosphorylation of an N-terminal regulatory serine residue. The inhibition of GSK-3 by
Akt prevents the phosphorylation of the cytoplasmic signaling molecule β-catenin translocation
to the nucleus. Once in the nucleus, β-catenin combines with different transcription factors, such
as TCF/LEF-1, to induce the expression of several genes, such as that of cyclin D1, which
induces cell cycle progression through regulation of RB hyperphosphorylation and inactivation
(Cross et al., 1995). Akt enhances protein synthesis by increasing the phosphorylation of mTOR,
and the activated mTOR promotes translation of cyclin D mRNA. p70 ribosomal protein S6
kinase 1 (p70S6K1) is also activated, and eukaryotic initiation factor 4E-binding protein 1
(4EBP1) is inhibited by mTOR (Inoki et al., 2002; Tee et al., 2002). Akt is a good candidate for
mediating these PI3K-dependent cell-survival responses. It has been implicated as an antiapoptotic factor in many different cell death paradigms, including the withdrawal of extracellular
23
signaling factors, oxidative and osmotic stress, irradiation, and the treatment of cells with
chemotherapeutic drugs and ischemic shock (Downward, 1998; Franke et al., 1997). Akt
regulates the apoptotic machinery by phosphorylating and inactivating pro-apoptotic protein such
as Bad, which controls the release of cytochrome-c from mitochondria (del Peso et al., 1997). Akt
also exerts indirect control of apoptosis through regulation of transcription. Phosphorylation of
the forkhead family of transcription factors (Foxo1, Foxo3a and Foxo4) by Akt inhibits
transcription of pro-apoptotic genes such as FasL, IGFBP-1, and Bim (Datta et al., 1999;
Nicholson and Anderson, 2002). Amplifications of multiple Akt isoforms have been reported in
pancreatic, ovarian, and head and neck cancers (Engelman et al., 2006; Osaki et al., 2004).
PTEN is a tumor suppressor gene that negatively regulates the PI3K signaling pathway by
dephosphorylating PIP3, the product of PI3K. It is frequently affected by germline and somatic
mutations in human cancers. Since PTEN dephosphorylates the phosphoinositide products of
PI3K that trigger the activation of Akt, inactivating mutations or a loss of PTEN expression lead
to increased levels of PI3K products in cells, followed by increased cell proliferation and
resistance to apoptosis (Cantley and Neel, 1999). In addition, PTEN mutations in human result in
a rare hereditary syndrome referred to as Cowden syndrome (CS), which is associated with a
higher risk of the development of malignant tumors (Simpson and Parsons, 2001).
Disruption of the Pten in mouse models has confirmed that Pten is a tumor suppressor. Pten+/mice have been found to develop tumors in several organs, including breast cancer, partially
resembling the spectrum of neoplasia observed in CS patients. Pten-/- mice die during
embryogenesis, before mid gestation at E6.5–9.5 due to developmental defects (Di Cristofano et
al., 1998; Lesche et al., 2002; Podsypanina et al., 1999; Stambolic et al., 2000; Suzuki et al.,
1998). Cre-loxP technology has been introduced to study the function of the Pten in specific cells
or organs.
It has been reported that both FSH and LH mediate pre-ovulatory follicle development, oocyte
maturation, and expansion of cumulus cells by inducing a complex pattern of gene expression in
granulosa cells. FSH promotes rapid activation of the PI3K pathway in these cells, resulting in
the phosphorylation of the downstream kinase Akt (Alam et al., 2004; Gonzalez-Robayna et al.,
2000). A recent study has shown that targeted deletion of the Pten gene from granulosa cells
leads to increased survival of pre-ovulatory follicles during the ovulation period, resulting in
release of more oocytes for fertilization (Fan et al., 2008). Moreover, the mice were completely
fertile and produced healthy CL. These data show that PI3K/Akt signaling in granulosa cells
upon the action of FSH plays an important role in both fertilization and maintenance of
pregnancy.
In mammals, Foxo3a is one of the important transcription factors under the control of Akt, which
modulates the apoptotic activity of the cell (Accili and Arden, 2004; Arden and Biggs, III, 2002;
Brunet et al., 1999; Nakae et al., 1999). In the absence of environmental signals or growth
factors, Foxo members were found to be localized in the nucleus, but upon phosphorylation they
moved to the cytoplasm (Arden and Biggs, III, 2002). Work in our laboratory has shown that in
cultured mouse and rat oocytes, KL can activate the oocyte PI3K pathway via Kit receptor on the
oocyte surface, a process that involves activation of the growth-enhancing molecule Akt and also
suppression of the Akt substrate Foxo3a (Reddy et al., 2005). Recent study has shown that
deletion of Foxo3a leads to overactivation of the primordial follicle pool. Foxo3a-deficient mice
24
are fertile for a short time and become infertile due to depletion of all functional follicles
(Castrillon et al., 2003). Another recent study has provided important evidence that Foxo3a in
oocytes plays a major role in activation of primordial follicles via the PI3K/Akt signaling
pathway (John et al., 2008).
The cell cycle involves a set of events that can lead to cell proliferation, senescence, or apoptosis.
Cells progress through the various phases of the cell cycle through interaction of different cyclins
with their respective CDK subunits. The CDKs are a family of serine/threonine protein kinases
that are activated at specific points of the cell cycle (Vermeulen et al., 2003). CDKs were first
identified by virtue of the fact that mutations of these proteins in yeast affected their growth rates
(Nurse, 1975; Nurse and Thuriaux, 1980). Among the most important CDKs that regulate the cell
cycle are CDK4 and CDK6 (which are structurally related) and CDK2. Following mitogenic
stimuli, quiescent cells enter the cell cycle and upregulate the D and then E type cyclins during
G1 (Koff et al., 1991; Lew et al., 1991). The D-type cyclins then associate with CDK4 and
CDK6, while cyclin E associates with CDK2. Once assembled, the cyclin-CDK complexes enter
the nucleus where they are phosphorylated by a CDK-activating kinase. These activated
complexes then phosphorylate additional proteins including various members of the
retinoblastoma family, such as pRB (RB1), p107 (RBL1), and p130 (RBL2). Phosphorylation of
pRB prevents its binding to E2F transcription factors, enabling them to activate the expression of
genes that regulate entry into the S phase (Abukhdeir and Park, 2008). Additionally, cyclin-CDK
complexes have been shown to control centrosome duplication (Lacey et al., 1999) and histone
gene transcription (Ewen, 2000), which are also key mediators of cell-cycle progression.
Inhibitors of cyclin-CDKs can modulate the cell cycle by preventing or limiting cyclin-CDKs
from phosphorylating their normal substrates. p27kip1(p27), a member of the Cip/Kip family of
CDK inhibitors, is a negative regulator of cell-cycle progression and is a tumor suppressor
(Kaldis, 2007). Proteins of the KIP class are recognised as inhibitors of cyclin-E-CDK and
cyclin-A-CDK complexes, although there are many reports that demonstrate that KIP class
proteins can also interact with cyclin-D-associated CDKs. When complexed with their respective
cyclin-binding partner, p21 and p27 are able to block the kinase activity of CDKs. p27 is located
in the nucleus in G0 and early in G1, and appears transiently in the cytoplasm at the G1/S
transition. p27 also plays a role in mid-G1, in the assembly and nuclear import of D-type cyclinCDK complexes (LaBaer et al., 1997). As cells enter the cell cycle and S phase, p27 levels
decrease and the kinase activity of CDKs increases. For example, p27 binds to CDK2 (or to
CDK1, and to other CDKs) and potently inhibits CDK2 kinase activity.
It has been reported that the PI3K signaling pathway plays an important role in oocytes to initiate
growth and follicular development. Functions of a few members of the PI3K signaling pathway
are shown in Fig. 3.
25
Fig. 3. The PI3K signaling pathway. Upon ligand binding, activation of receptor tyrosine kinases
(RTKs) initiates receptor dimerization, and activates the PI3K activity. PI3K produces PIP3,
which activates PDK1 and Akt. Akt regulates multiple cellular functions including metabolism,
protein translation, cell cycle progression, anti-apoptosis, tumor growth, and angiogenesis
through different downstream targets. The importance of a few molecules in this pathway has
been studied in this thesis. See the Results section (figure obtained from Adhikari and Liu, 2009).
3. Premature ovarian failure (POF)
POF is defined as a primary ovarian defect characterized by absence of menarche, or arrested
folliculogenesis/premature depletion of ovarian follicles before the age of 40 years. POF is not a
rare condition; it affects approximately one in 10,000 women by the age of 20 (0.01%), one in
1,000 women by the age of 30 (0.1%), and one in 100 women by the age of 40 (1%) (Coulam et
al., 1986). The etiology of most of the cases is idiopathic; however, heterogeneous circumstances
such as autoimmunity; specific genetics; metabolic, infectious, or environmental insults; or
iatrogenic causes have been associated with POF (Beck-Peccoz and Persani, 2006; Rebar, 2008).
In theory, there may be two major reasons for the development of POF: (1) failure to acquire an
adequate number of initial primordial follicles, which normally takes place during fetal life, and
26
(2) excessive clearance of primordial follicles, and suppressed activation and further development
of primordial follicles.
It has been well recognized that absence of or structural abnormalities involving the Xchromosome commonly lead to POF, which suggests that genes on the X-chromosome are
necessary for normal ovarian function (Simpson and Rajkovic, 1999). Both alleles of intact Xchromosomes are necessary for maintenance of oocytes. It has been shown that the gonads of
45,X (monosomy) fetuses contain normal numbers of oocytes at 20–24 weeks of fetal age, but the
oocytes subsequently undergo accelerated atresia so that there are usually no oocytes left at the
time of birth (Singh and Carr, 1966). In Turner’s syndrome, where only one X-chromosome is
present ovarian follicles are completely degenerated by birth (Loughlin et al., 1991). Although
pathogenesis may be clear, the precise genetic mechanism is not known.
The BMP15 gene, which encodes an oocyte-specific growth differentiation factor belonging to
the TGF-β superfamily, has also been associated with POF and primary amenorrhea, and maps to
the short arm of the X-chromosome (Dube et al., 1998). Another study reported two sisters with
POF who were heterozygous for BMP15 (Di Pasquale et al., 2004). A few other authors have also
reported heterozygous variants of BMP15 in POF patients (Dixit et al., 2006; Laissue et al.,
2006). Another important X-chromosome gene, Zinc finger transcription factor (Zfx), is located
on the Xp22.1-Xp21.3 short arm region and is transcribed in all tissues. Although Zfx mutant
variants were not found in human samples, murine studies have shown that deletion of the Zfx
results in a 50% reduction in the number of PGCs at E11.5, which leads to reduced fertility and
shortened reproductive lifespan (Luoh et al., 1997). Several other genes located on the Xchromosome long arm region, such as DIAPH2, DACH2, and POF1B have been implicated in
POF. Mutations in autosomal genes such as FSHβ, FSHr, LHβ, LHr, Cyp17, and Cyp19 result in
follicle dysfunction and ultimately lead to POF (Simpson, 2008).
4. Current knowledge on infertility treatment methods
The common feature in POF patients is hormonal imbalance due to the nonfunctional ovaries, which
adversely affects both the physical and mental well-being of these women (Goswami and Conway,
2005; Conway et al., 1996; Beck-Peccoz and Persani, 2006). At the same time, infertility caused by
POF is also a major issue in young women. In this regard, preservation of fertility in POF patients, or
rescue from future infertility due to post-cancer treatment, is important. There are few ways available
nowadays of rescuing infertility.
Cryopreservation of embryos after in vitro fertilization (IVF) is an established method for
preservation of fertility. It requires approximately two weeks of ovarian stimulation with daily
injections of FSH from the onset of menses for the development of follicles. Follicle development
is monitored by serial ultrasounds and blood tests. At the appropriate time, an injection of hCG is
administered to trigger ovulation, and oocytes are subsequently collected by ultrasound-guided
transvaginal needle aspiration. Oocytes are fertilized in vitro and cryopreserved after fertilization
(Lee et al., 2006). This approach is mainly useful for those who have normal and functioning
ovaries. Embryo cryopreservation is, however, not applicable to prepubertal girls and women
with hormone-sensitive tumors, because ovarian stimulation is associated with breast or
27
endometrial cancer (Pena et al., 2002; Chen et al., 2003). It is also not suitable for those whose
ovaries fail to respond to gonadotropins.
The other alternative is mature oocyte collection without hormone stimulation; unfortunately, the
mature oocyte yield is significantly less this way. Alternative hormonal stimulants such as
letrozole or tamoxifen have been used to reduce the potential risk of estrogen mediated cancers in
women with hormone-sensitive tumors (Oktay et al., 2005; Oktay, 2005). Unfertilized oocytes
retrieved during a stimulated IVF cycle can be frozen (Coticchio et al., 2001). Unfortunately,
oocytes are more sensitive to the freeze-thaw process, and correspondingly, the live birth rates
are significantly less. The overall live birth rate per cryopreserved oocyte is approximately 2%,
which is much lower than the corresponding rate using fresh oocytes (Coticchio et al., 2004;
Fabbri et al., 2000; Gosden, 2005). This method is suitable for patients whose partner is
unavailable at the time and for patients whose ovaries fail to produce hormones for maintenance
of the pregnancy. Oocyte cryopreservation technology should be improved, and much higher
success rates must be achieved to rescue fertility in POF or cancer patients.
It is known that many women who are diagnosed as having POF still have small follicles in their
ovaries (Massin et al., 2004; Massin et al., 2008). Cryopreservation of ovarian cortical slices, which
contain large numbers of primordial follicles, interested many researchers because of the great
potential to preserve the fertility of any woman. It is theoretically possible to use these follicles
for the purpose of in vitro maturation (IVM) (Oktay et al., 1997). In prepubertal girls who suffer
from POF, ovarian cryopreservation is the only fertility preserving option as ovarian stimulation
and collection of mature oocytes or obtaining fertilized embryos is not feasible (Roberts and
Oktay, 2005; Hovatta, 2004). Even in post-pubertal POF patients, their ovaries may still be rich
in primordial follicles (Marhhom and Cohen, 2007). Collection of ovarian cortical tissues is
independent of the stage of the menstrual cycle, and does not require hormonal hyperstimulation.
Using this technique, hundreds of primordial follicles can be cryopreserved in a single procedure.
With our current knowledge of molecular mechanisms underlying follicular activation,
development of small-molecular compounds to inhibit the functions of PTEN, p27, or Foxo3a
and thereby increasing the rate of primordial follicle activation and survival in in vitro-cultured
follicles or ovarian slices would be beneficial for the culture of primordial follicles. Foxo3a, for
example, is a transcription factor that can be difficult to inhibit or enhance; but PTEN, other
phosphatases, and kinases are enzymes, and development of compounds that regulate their
activities should be feasible (Reddy et al., 2008). Thus, small-molecule inhibitors of PTEN may
be potential drugs for stimulation of both activation and survival of immature follicles.
Recently, IVM of primordial follicles in cryopreserved ovarian tissue has become an active field
of research. While fertilizable eggs and live pups have already been obtained from cultured
primordial follicles in mice (Eppig and O'Brien, 1996; O'Brien et al., 2003), similar success in
humans still requires an experimental breakthrough. By improving the culture conditions, human
primordial follicles and primary follicles in thin slices of ovarian tissue have been cultured, where
they have been able to develop into secondary follicles and occasionally into early antral follicles
within 3–4 weeks (however, some degree of progressive atresia) (Hovatta, 2004). In particular,
the mechanisms underlying the apoptosis of developing follicles at any given developmental
stage are poorly defined. The interactions between granulosa cells and oocytes of developing
follicles during follicular development must be investigated in depth at the molecular level in
order to improve the IVM of primordial follicles.
28
5. Aims of the dissertation
The main aim of this dissertation was to identify the genes that are responsible for primordial
follicle activation and the genes that keep primordial follicles in dormant stage thoughout
reproductive life. More specifically, we would like to:
1. Investigate the role of Foxo3a in the oocyte nucleus by using a Foxo3a transgenic (Tg)
mouse model.
2. Identify the function of p27 in follicle formation, activation, and atresia.
3. Study the function and to determine the importance of the intra-oocyte PI3K/Akt
signaling cascade in the development of primordial and primary follicles.
29
6. Results
6.1. Paper I
Infertility caused by retardation of follicular development in mice with oocyte-specific
expression of Foxo3a
Primordial follicle activation in mammals takes place when primordial follicles are recruited
from the follicle pool, when the oocyte grows rapidly, and when the surrounding granulosa cells
become cuboidal and proliferative. It has been reported earlier that disruption of the Foxo3a
leads to overactivation of primordial follicles, which then results in early depletion of all growing
follicles in the mouse ovary (Castrillon et al., 2003). Studies from our laboratory also showed that
Foxo3a is phosphorylated upon activation of the PI3K signaling cascade in oocytes stimulated by
KL, which is known to be secreted by granulosa cells (Reddy et al., 2005). Since Foxo3a has an
important role in primordial follicles and oocytes, the interesting question is how Foxo3a
regulates follicular development and oocyte attrition in the later stages of follicular development.
To study the role of Fox3a, we generated a Foxo3a transgenic (Tg) mouse model where Foxo3a
is constitutively expressed under the control of the zona pellucida glycoprotein 3 (Zp3) promoter
in oocytes of primary follicles, in order to determine whether intra-oocyte Foxo3a, influences
follicular development and female fertility. We observed Foxo3a expression in oocytes of
primordial follicles and, surprisingly, Foxo3a expression was completely absent from the oocytes
of primary follicles in wild-type ovaries. We found that the fertility of Zp3-Foxo3a Tg mice was
severely reduced due to the absence of mature follicles. Our morphological studies and follicle
counting revealed that there was retarded follicular development in Tg mice, and ovary size was
smaller than in wild-type controls. Proliferation of granulosa cells that surround the oocyte was
found to be severely impaired in the Zp3-Foxo3a Tg mouse ovary.
It is well known that the oocyte-secreted factors Gdf-9 and Bmp-15 have role in granulosa cell
proliferation during follicular development. To determine the target molecules of Foxo3a in
oocytes that control granulosa cell proliferation, we first measured mRNA levels of the oocyte
secretion factors Gdf-9 and Bmp-15 by quantitative real-time PCR at different developmental
stages of the ovary. In Tg ovaries at postnatal day (PD) 6, 8, and 15–17, we found that mRNA
levels of Bmp-15 were dramatically reduced, whereas Gdf9 mRNA levels were normal at PD6
and PD8 in Tg ovaries, but dramatically reduced in PD15-17 ovaries. This result indicates that
Foxo3a regulates the expression of oocyte-secreted factors for follicle growth. Furthermore, we
also found that activation of the Smad pathway, which is necessary for communication between
oocyte and surrounding granulosa cells, is suppressed in Tg ovaries. To determine whether a
disrupted Smad pathway in Tg ovaries would affect oocyte growth, we measured the size of
oocytes at PD8 and PD13. As expected, we observed reduced oocyte size in Tg ovaries. p27,
which is known to inhibit the cell cycle, is continuously expressed in Tg oocytes. It is possible
that Foxo3a modulates p27 expression in oocytes, depending on the stage of the follicle.
Although there were a few mature follicles in Tg ovaries, very few of them reached the preovulatory stage, but the oocyte is trapped in the developing CL; no PR expression was observed
in Tg ovaries. Taken together, our data show that the downregulation of Bmp-15 expression and
constant activation of p27 may result in suppressed activation of the Smad pathway, leading to
30
retardation of granulosa cell proliferation and reduced oocyte size, which are responsible for the
lack of mature ovarian follicles in Zp3-Foxo3a Tg mice.
6.2. Paper II
p27kip1 (cyclin-dependent kinase inhibitor 1B) controls ovarian development by
suppressing follicle endowment and activation, and by promoting follicle atresia in mice
As we found constant expression of p27 in oocytes of Zp3-Foxo3a Tg ovaries, we set out to study
the role of p27 and how both Foxo3a and p27 regulate follicular development in adult mouse
ovaries by using both p27-deficient (p27-/-) and Foxo3a-deficient (Foxo3a-/-) mice. We checked
expression of p27 in wild-type ovaries at E14.5. p27 expression was observed in somatic cells.
Surprisingly, more primordial follicles were observed in p27 mutant ovaries than in wild-type
ovaries at PD1. It is possible that deletion of p27 may enhance the survival of oogonia. Here, we
demonstrated that p27 suppresses follicle endowment/formation and activation, and induces the
follicle atresia that occurs prior to sexual maturity. The ovaries in p27-deficient mice were much
larger than control wild-type ovaries at all stages of follicular development, due to overactivation
of primordial follicles and less apoptosis. The over activated follicles in p27-/- ovaries are
depleted in early adulthood, causing POF, and no primordial follicles were seen in 12-week-old
p27 mutant ovaries. To determine the major cause of the accelerated rate of follicular endowment
in p27-/- mice, we measured the kinase activities associated with CDKs and cyclins in newborn
(PD0) p27+/+ and p27-/- ovaries, which are in the developmental stage that precedes follicle
formation, and found that CDK2 and cyclin A activities were elevated in newborn p27-/- ovaries
relative to those in PD0 p27+/+ ovaries. These results show that the increased kinase activity of
the CDK2-cyclin A complex may be one of the forces that drive the accelerated follicle formation
and endowment. Furthermore, in PD18 ovaries there was an overall increase in kinase activities
associated with CDK2, CDC2, cyclin A, and cyclin B1, but not with cyclin E1, indicating that the
kinase activities of CDK2/CDC2-cyclin A/B1 complexes may also be involved in preventing the
death of ovarian follicles in p27-/- ovaries prior to sexual maturity. Also, we found that the
caspase-dependent apoptotic pathway was dramatically downregulated in p27-/- ovaries relative to
that in p27+/+ ovaries. To determine the relationship between Foxo3a and p27, we checked
Foxo3a expression in p27-/- oocytes and normal levels were found, suggesting that these
molecules have independent roles in follicular development. Collectively, our data indicate that
the caspase-dependent apoptosis pathway is suppressed in p27-/- ovaries, which leads to an
elevated follicular survival rate before sexual maturity. Thus, p27 may induce follicle atresia
prior to sexual maturity through activation of the caspase-dependent apoptotic pathways.
6.3. Paper III
Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool
Activation of primordial follicles is an important step during follicular development. To
determine the mechanism(s) responsible for follicle activation, we investigated the role of the
PI3K/Akt signaling pathway in mouse oocytes by conditionally deleting the Pten by transgenic
mice expressing the Gdf-9 promoter-mediated improved Cre recombinase (GCre+), which is
specifically expressed in oocytes of primordial and further developed follicles. PTEN protein
31
expression was completely abolished in growing oocytes (from the primary stage) in
PtenloxP/loxP;GCre+ ovaries.
We found that the PtenloxP/loxP;GCre+ females become infertile in young adulthood (12–13
weeks). To investigate the reasons for infertility in PtenloxP/loxP;GCre+ mice, we looked into the
morphology and follicle counts at different stages of development of the ovary, and found that
the entire pool of primordial follicles is activated, which leads to POF in young adulthood. In
PtenloxP/loxP;GCre+ ovaries at PD23, no primordial follicles were observed and all follicles were
depleted by 16 weeks. The ovary size in PtenloxP/loxP;GCre+ was very large at PD35 due to
activation of all primordial follicles and small ovaries were observed at 16 weeks due to a lack of
functional follicles. As expected, there were significantly higher levels of FSH and LH in the sera
of PtenloxP/loxP;GCre+ mice than in the sera of control PtenloxP/loxP mice due to depletion of
follicles.
To determine whether the PI3K/Akt pathway is activated in oocytes of
PtenloxP/loxP;GCre+ovaries, we found elevated activation of expression of Akt, which led to
upregulation of both expression and activation of ribosomal protein S6 (rpS6) in oocytes. The
results thus show that the mammalian oocyte serves as the center of programming of the
occurrence of follicular activation, and that the PI3K pathway of the oocyte governs follicular
activation by control of initiation of oocyte growth.
6.4. Paper IV
Oocyte-specific deletion of Pten in mice reveals a stage-specific function of PTEN/PI3K
signaling in oocytes in controlling follicular activation
In Paper III it was shown that primordial follicle activation relies on the PI3K signaling pathway
in oocytes. Primordial follicles are overactivated in PtenloxP/loxP; GCre+ ovaries due to
accelerated PI3K/Akt signaling. Due to activation of all primordial follicles at an early age, all
the follicles deteriorate by apoptosis. Surprisingly, such mutant mice show normal ovulation and
normal litter size in young adulthood, before their ovarian follicles are depleted. In this paper we
studied the importance of PI3K signaling cascade in growing follicles and ovulated oocytes.
To determine the role of PI3K/Akt signaling in oocytes of primary and developing follicles, we
conditionally deleted the Pten from the oocytes of primary and further developed follicles by
using Tg mice carrying Zp-3 promoter mediated-Cre recombinase (Zp3-Cre+). We found that
elevation of PI3K/Akt signaling in oocytes of primary and further developed follicles does not
affect the pool of primordial follicles, as primordial follicles were clearly seen in adult ovaries.
Follicles at all stages of development were found in PtenloxP/loxP;Zp3-Cre+ ovaries whereas no
primordial follicles were seen in PtenloxP/loxP;GCre+ ovaries. Importantly, all types of follicles
were observed at the age of 16 weeks, whereas no follicles were present in PtenloxP/loxP;GCre+
ovaries. PtenloxP/loxP;Zp3-Cre+ females are completely fertile, with normal litter size and CL,
indicating that all the major events activation, oocyte growth, follicular development, ovulation,
and fertilization are normal. As fertility is normal in these mice, GVBD was checked to see
whether an accelerated PI3K cascade would have any effect on it, but no alterations in GVBD
were observed. Zygotes from Pten mutant mice were collected for counting, but the number of
32
zygotes in mutant mice was almost equal to that in wild-type mice. Our data indicate that PTEN
is mainly essential for regulation of follicular activation in a stage-specific fashion.
7. Conclusions
Members of the PI3K family are critical for cell growth and survival upon stimulation with
mitogenic signals in many somatic tissues. In this way, these signaling kinases influence several
cellular activities such as proliferation, motility, and survival. It has been shown that the PI3K
signaling cascade is essential for germ cell survival during fetal life. PGCs are known as
precursor germ cells of oocytes in the mammalian ovary. Defects in the PI3K signaling cascade
lead to apoptosis of all germ cells in fetal life, and the adult ovary is empty of oocytes (Russell et
al., 1948; Matsui et al., 1990; Buehr et al., 1993; Morita et al., 1999a). This thesis has focused
mainly on the role of PI3K signaling molecules in follicular activation and development.
Foxo3a is one of the important transcription factors exclusively localized in the oocyte nucleus of
primordial follicles. Inhibition of Foxo3a by Akt through the PI3K signaling pathway in
primordial follicles allows them to develop into primary follicles and further developmental
stages. Our results have shown that constant activation of Foxo3a or impaired inhibition of
Foxo3a results in impaired folliculogenesis in mice. Immature follicles are unable to reach the
ovulation stage due to impaired growth of oocytes and defects in the proliferation of surrounding
granulosa cells. The conclusion from our study is that Foxo3a is specifically expressed in
primordial oocytes and this expression disappears in growing follicles to allow them to develop
further.
p27 is known to inhibit the progression of the cell cycle in somatic cells. Our results show that
p27 has two important functions in the mammalian ovary during follicular endowment,
activation, and development. One is to maintain follicular formation/endowment at the time of
birth by regulation of somatic cells and activation of primordial follicles. The other important
function is to maintain the balance between apoptosis and survival of follicles before sexual
maturity by regulating the caspase cascade.
The PI3K signaling pathway is known to activate many cellular activities in cells. PTEN is a
important negative regulator of the PI3K/Akt pathway, which is essential for balancing cell
growth and proliferation. Our results show that primordial follicle activation is controlled by the
activation status of PI3K in oocytes. Loss of Pten in oocytes of primordial follicles results in
constant activation of PI3K, which then leads to activation of all primordial follicles. However,
loss of Pten in oocytes of primary and developing follicles does not affect the events that occur in
the later stages of follicular development and, importantly, fertilization and implantation. The
main conclusion of the study is that the PI3K signaling pathway is essential for controlling
primordial follicle activation.
33
8. Acknowledgements
I would like to thank everyone at the Department of Medical Biochemistry and Biophysics for the
support during my 4 years stay and for creating a good scientific environment. In particular, I
would like to thank:
Kui Liu, my supervisor, for your great help all the time and always keeping your door open and
taking time for discussion. I am grateful for your unending support during my study. Jinan Li,
my co supervisor for constructive discussions in group meetings.
Pradeep Reddy: For creating a nice working environment in the lab and sharing ideas.
Deepak: For all your help in experiments and being a good friend.
Other members of the group: Wenjing, Naga Raju and former members, Raja, Shawn, Shen,
Shasi, Edvin for helping me a lot in animal house with mouse work.
Tor Ny and Malgorzata: For all help with presentations and interesting discussions in our regular
group meetings in the beginning of my studies. Thanks to all members of Tor’s and Malgorzata’s
groups. Especially, Rima, for making such a good environment in the office.
Ingrid and Clas: for helping me with lot of paper work.
Lola: for helping with buffer preparations.
Lena and the staff at BISAM: for helping with the mice.
Ravi, Vidya, Gaurav, Renu, Pari, Sharvani, Munender, Dinesh, Ruksana, Sanjay, and Satish
(NL group): for lot of fun, friendship and helping me all the time. I enjoyed a lot in our trips
especially Narvic (mid night sun….). Especially Dinesh, oh my god, lot of fun in our every
gathering (u know what I mean). I miss you terribly (already).
Kiran, Srinu, Bahmaiah, Suman, Siri, Murali, Venky, Prashanth (IKSU boys??): For company
at IKSU. Especially, Badminton with Kiran, never reached more than 5 points. Thanks to all my
Indian friends (especially Karunakar, Aaron, Pramod, Kumar…) in Umeå for the help and fun
during my stay.
Many many thanks to my parents, brothers and sisters for their trust and support. Above all, I am
most grateful to my Uncle Venkata Subba rao Jagarlamudi and aunt Rajini Jagarlamudi for
their immense support during all phases and in all aspects in my life.
34
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