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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02667
1 Department of Medical Biochemistry and Biophysics, Umeå University,
SE-90187, Umeå, Sweden.
2 Qilu Hospital, Shandong University, Jinan, 250012, Shandong, China.
3 Department of Molecular Biology and Umeå University, SE-90187,
Umeå, Sweden.
4 Department of Radiation Sciences, Umeå University, SE-90187, Umeå,
Sweden.
Author for correspondence (e-mail:
kui.liu{at}medchem.umu.se)
Accepted 29 September 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Oocyte, Foxo3a, PI3K pathway, Follicular development, Transgenic, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
McGee and Hsueh, 2000;
Wassarman and Albertini,
1994). In recent years, it has been suggested that oocytes may
orchestrate the development of mammalian ovarian follicles, and that the rate
of follicular development is based on a developmental program intrinsic to the
oocyte (Eppig et al., 2002;
Matzuk et al., 2002). Although
gonadotropins are essential for driving the differentiation of granulosa cell
phenotypes, the oocyte is probably the dominant factor within its range of
influence and determines the direction of differentiation and the function of
the granulosa cells associated with it
(Eppig, 2001). It is generally
believed that some intra-ovarian factors have important roles in regulating
oocyte growth and early follicular development before the action of
follicle-stimulating hormone (FSH) becomes important
(Matzuk et al., 2002;
Peters et al., 1975).
The bi-directional communication between oocytes and granulosa cells has
been shown to be essential for follicular development. The growth and meiotic
regulation of oocytes are dependent on granulosa cells; at the same time,
oocytes secrete various factors that have key roles in folliculogenesis. For
example, oocyte and follicular growth are dependent on kit ligand (Kitl -
Mouse Genome Informatics; stem cell factor, SCF) which is produced by
granulosa cells; oocyte-derived factors such as bone morphogenic protein 15
(Bmp15) and growth differentiation factor 9 (Gdf9) also have fundamental roles
in the control of granulosa cell proliferation and differentiation during
follicular development (for reviews, see
Albertini and Barrett, 2003;
Eppig, 2001;
Matzuk et al., 2002;
McGee and Hsueh, 2000;
Shimasaki et al., 2004).
Our previous report has shown that in cultured mouse and rat oocytes, Kitl
can activate the oocyte phosphatidylinositol 3-kinase (PI3K) pathway via Kit
receptor on the oocyte surface, a process that involves activation of the
growth-enhancing molecule Akt and also the suppression of the Akt substrate
Foxo3a (also known as FKHRL1), which is a transcription factor that regulates
the cell cycle and apoptosis (Reddy et
al., 2005). Our next question is what functional roles the PI3K
pathway plays in mammalian oocytes. Although the conventional Foxo3a-knockout
mice have been shown to exhibit excessive activation of primordial follicles
in a pioneer study by Castrillon et al.
(Castrillon et al., 2003), the
question of how Foxo3a influences follicular growth remains unanswered. This
involves several issues, such as whether Foxo3a functions within the ovary
itself (Brenkman and Burgering,
2003), the nature of the cells and the developmental stages in
which Foxo3a plays its role, and the identity of the pathways through which
Foxo3a regulates follicular growth. Based on previous reports and our own
data, we have proposed that intra-oocyte Foxo3a may play an important role in
the regulation of follicular activation and development
(Liu et al., 2006;
Reddy et al., 2005). To test
this hypothesis and to learn more about the functions of intra-oocyte Foxo3a
in follicular development, we generated a transgenic (Tg) mouse model where
constitutively active Foxo3a is maintained in the oocytes, mediated by the
oocyte-specific zona pellucida glycoprotein 3 (Zp3) promoter
(Epifano et al., 1995). We
found that the Tg mice showed infertility caused by retardation of oocyte
growth and follicular development, and anovulation. The expression of several
important ovarian genes, which we supposed to be responsible for the
above-mentioned defects, was also studied in the Zp3-Foxo3a Tg mice.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Reagents, antibodies and immunological detection methods
The rabbit polyclonal antibodies against phospho-Smad1 (serine
463/465)/Smad5 (serine 463/465)/Smad8 (serine 426/428), Smad1, phospho-Smad2
(serine 465/467), Smad2, connexin 43, p27kip1 (p27; Cdkn1b), Akt,
phosphor-Akt (serine 473), p70S6 kinase, phospho-p70S6 kinase (threonine 389),
mammalian target of rapamycin (mTOR), phosphomTOR (serine 2448),
phospho-p44/42 mitogen activated protein kinase (MAPK) (threonine 202/tyrosine
204), and p44/42 MAPK were obtained from Cell Signaling Technologies (Beverly,
MA). Rabbit polyclonal antibody against connexin 37 was purchased from Alpha
Diagnostics International (San Antonio, TX). Rabbit polyclonal antibody
against Foxo3a (FKHRL1) was from Upstate Biotechnology (New York, NY). The
proliferating cell nuclear antigen (PCNA) Staining Kit was purchased from
Invitrogen (Sweden). Rabbit polyclonal antibody against progesterone receptor
(PR) was purchased from Dako Sweden (Solna, Sweden). Mouse monoclonal
antibodies against ß-actin, bromodeoxyuridine (BrdU) and FLAG M2-HRP, and
pregnant mare serum gonadotropin (PMSG), human chorionic gonadotropin (hCG),
and BrdU were purchased from Sigma-Aldrich (Stockholm, Sweden). Western blot
analyses were carried out according to the instructions for different
antibodies from the suppliers, and visualized using the ECL Plus Western
Blotting Detection System (Amersham Biosciences, Uppsala, Sweden).
Quantification of ovarian follicles, histological analyses and immunohistochemistry
Ovaries were fixed in 4% paraformaldehyde, dehydrated and embedded in
paraffin. To count the numbers of ovarian follicles, paraffin-embedded ovaries
were serially sectioned at 8 µm and stained with Hematoxylin for
morphological observation. Ovarian follicles at different developmental
stages, including type 3b, type 4, type 5, and types 6-7 were counted in all
sections of an ovary, based on the standards established by Pedersen and
Peters (Pedersen and Peters,
1968). In each section, follicles that contained oocytes with
clearly visible nuclei were scored as previously reported
(Johnson et al., 2004), and
the total number of follicles at any particular developmental stage was
calculated as the sum of the number of follicles from all sections of an
ovary. Judged from careful morphological analysis, the incidence of counting
the same follicle twice or missing a follicle was low. Oocyte diameters were
measured with a Zeiss AX10 microscope, and only oocytes in which the nucleoli
were clearly visible were measured.
Immunohistochemistry for detection of Foxo3a, PCNA, BrdU, p27 and PR was performed on rehydrated paraffin sections using the ABC Staining System (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer's instructions. Negative controls omitting primary antibodies were included in each experiment. For the in vivo BrdU incorporation assay, mice were injected with BrdU (100 mg per kg body weight) for 2 hours, and the ovaries were examined by immunohistochemistry using an anti-BrdU antibody as described above.
Preparation of ovarian extract
Ovaries were dissected free of fat and adhering tissues and extracts were
prepared on ice by homogenizing in a lysis buffer containing 50 mM Tris-HCl
(pH 8.0), 120 mM NaCl, 20 mM NaF, 20 mM ß-glycerophosphate, 1 mM EDTA, 6
mM EGTA (pH 8.0), 1% NP-40, 1 mM DTT, 5 mM benzamidine, 1 mM PMSF, 250 µM
sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1
µg/ml pepstatin, followed by centrifugation at 18,000 g for
20 minutes at 4°C. The supernatants were collected and protein
concentrations were measured using the bicinchoninic acid (BCA) protein assay,
and equal amounts of proteins were used for the western blot. Results of
western blot were normalized against levels of ß-actin in the
lysates.
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). Under the light
conditions used, ovulation was considered to take place at around 0200 h. To
obtain preovulatory follicles or newly formed corpora lutea, ovaries were
collected 2-4 hours before ovulation, or 36-48 hours after ovulation.
RNA extraction, RT-PCR and real-time PCR
Total RNA from ovaries was extracted using TRIzol (Invitrogen), following
the manufacturer's instructions. To avoid contamination with genomic DNA, each
total RNA sample was treated with RNase-free DNase I (Roche) and mRNA was
purified with the mRNA Purification Kit (Qiagen). The mRNA was reverse
transcribed (RT) using Superscript III (Invitrogen) according to the
manufacturer's instructions for PCR or real-time PCR. Real-time PCR was run
under standard conditions using iQ SYBR Green Supermix (BioRad) in the
iCycleriQ Multicolor Real-Time PCR Detection System (BioRad) according to the
manufacturer's instructions. All real-time PCR results were normalized against
levels of ß-actin mRNA in the same samples. Sequences of primers
for RT-PCR or real-time PCR for Bmp15, Gdf9 and ß-actin
are available upon request.
Synthesis of FSH receptor RNA probe and in situ hybridization
A cDNA fragment (nucleotides 805-1224) of the mouse FSH receptor cDNA
(kindly provided by Dr Ilpo Huhtaniemi, Imperial College, London, UK) was
subcloned into the pCRII-TOPO vector (Invitrogen) as a template for synthesis
of a probe for in situ hybridization. The riboprobe for in situ hybridization
was labeled with digoxigenin-labeled UTP using the Dig RNA-labeling kit (Roche
Diagnostics Scandinavia, Bromma, Sweden). In situ hybridization was performed
on 10 µm cryostat sections as previously described
(Liu et al., 1996;
Schaeren-Wiemers and Gerfin-Moser,
1993). Sense strands of the probes were used in parallel as
background controls.
Isolation of oocytes from postnatal mouse ovaries
The isolation of oocytes, separation of small oocytes from partially grown
oocytes using a cell-dispersing screen with a 25 µm opening, and the lysis
of oocytes were performed as previously described
(Reddy et al., 2005).
Statistical analysis
All experiments were repeated at least three times. For comparison of
follicle number and oocyte diameter in WT and Tg ovaries, differences between
the two groups were calculated by Student's t-test; a difference was
considered to be significant if P<0.01.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) (for details, see Fig.
2).
|
Retarded follicular development in Zp3-Foxo3a Tg mice
The severely reduced fertility in female Zp3-Foxo3a Tg mice indicates that
there may be defects in their ovarian follicular development. To test this
hypothesis, we studied the first wave of synchronized follicular development
by morphological analysis of Tg and WT ovaries at postnatal day (PD) 8, 13 and
24.
At PD8, the morphologies of Tg and WT ovaries were grossly similar, containing mostly primordial and primary follicles (Fig. 3D,E). The numbers of primary (type 3b) follicles in Tg ovaries were not significantly different from the corresponding numbers in WT ovaries (Fig. 3A). There were less secondary (type 4) follicles in the Tg ovaries than in WT ovaries, although the number of type 4 follicles in the 8-day-old ovaries was low (Fig. 3A). There were also a few type 5 follicles with 3-5 layers of granulosa cells in the ovaries of WT mice at PD8, but not in the ovaries of Tg mice of the same age (Fig. 3A). At PD13, in terms of gross morphology, the Tg ovaries appeared to be smaller than the WT ovaries (Fig. 3F,G), which was found to be caused by dramatically greater numbers of type 4 and type 5 follicles in the WT ovaries (Fig. 3B). As shown in Fig. 3H (arrows), in WT mice the first wave of follicular growth had typically reached the type 4 and 5 stages (with 2 or 3 layers of granulosa cells), whereas the majority of follicles found in the Tg mice were still at the stages of types 3b or 4 (Fig. 3I, arrows).
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Arrest of granulosa cell proliferation and reduced FSH receptor expression in Zp3-Foxo3a Tg mice
In order to determine the cause of retarded follicular growth, we attempted
to determine whether there is less proliferation or, alternatively, more
apoptosis of the granulosa cells in the Tg mice. Using 25-day-old mice, we
found that the number of apoptotic granulosa cells in Zp3-Foxo3a Tg mice and
their WT controls was similar, as measured by 3'-end labelling (data not
shown). On the other hand, there was dramatically more proliferation of
granulosa cells in WT ovaries than in Tg ovaries, as shown by
immunohistochemical staining for PCNA in ovarian sections
(Fig. 4A,B), and by in vivo
BrdU incorporation assays (Fig.
4C,D). In follicles of Tg mice, very few granulosa cells were
PCNA- or BrdUpositive (Fig.
4B,D, arrows) as compared with their WT controls
(Fig. 4A,C, arrows), indicating
that there was an arrest of granulosa cell proliferation.
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Reduced oocyte sizes in Zp3-Foxo3a Tg mice
We measured the sizes of oocytes in type 3b and type 4 follicles of Tg and
WT ovaries at PD8 and PD13, and found that the average diameters of oocytes in
Tg type 3b follicles (19.47±3.14 µm) and Tg type 4 follicles
(24.08±6.29 µm) were significantly less (P<0.001) than
the corresponding values for their WT counterparts (24.27±3.06 µm in
type 3b follicles and 34.36±4.21 µm in type 4 follicles)
(Fig. 5). The theoretical
average volumes of WT type 3b, WT type 4, Tg type 3b and Tg type 4 oocytes as
calculated from the average radii were 7481 µm3, 21,229
µm3, 3863 µm3 and 7307 µm3,
respectively. This indicates that oocyte growth was severely depressed in the
Zp3-Foxo3a Tg mice.
|
; Lan et al.,
2004), we measured Bmp15 and Gdf9 mRNA levels in
ovaries of 6- and 8-day-old Tg and WT ovaries, where the exogenous Foxo3a
transgene starts to be expressed in Tg ovaries although no apparent
morphological changes in follicular development are observed. We found that in
6- and 8-day-old mice, ovarian Gdf9 mRNA levels were similar in WT
and Tg ovaries (not shown). The Bmp15 mRNA levels in Tg ovaries were
approximately 14.0% and 16.3% of the levels in WT ovaries at PD6 and PD8,
respectively (Fig. 6A,B),
indicating that Bmp15 transcription may be directly regulated by
Foxo3a in the oocyte. In 15- to 17-day-old mice, where granulosa cells are
undergoing rapid proliferation in WT mice but not in the Tg mice, we found
that levels of both Bmp15 and Gdf9 mRNA in Tg ovaries were
less than 10% of those in WT ovaries (Fig.
6C,D). Even so, we consider the lower Gdf9 expression in
Tg ovaries at PD15-17 to be a consequence, but not the cause, of the retarded
oocyte growth and follicular development in the Tg mice, as Gdf9 mRNA
levels at PD6 and PD8 were similar in Tg and WT ovaries.
|
; Kaivo-Oja et al.,
2003; Moore et al.,
2003; Roh et al.,
2003), we also studied the activation states of Smads in ovaries
of 8-day-old and 15- to 17-day-old Tg and WT mice, using antibodies against
phosphorylated Smad1/5/8 (Smad1/5/8-P, also known as p-Smad1/5/8) and
phosphorylated Smad2 (Smad2-P, also known as p-Smad2). We
found that although the levels of Smad1/5/8-P, Smad1,
Smad2-P and Smad2 were similar in WT and Tg ovaries at PD8
(Fig. 6E-I, PD8), the
activation level of Smad1/5/8 (Fig.
6E, PD15-17, Tg) and the expression level of Smad1
(Fig. 6F, PD15-17, Tg) were
suppressed in 15- to 17-day-old Tg ovaries as compared with either WT controls
(Fig. 6E,F, PD15-17, WT) or Tg
ovaries at PD8 (Fig. 6E,F, PD8,
Tg). The level of activated Smad2 in Tg ovaries at PD15-17
(Fig. 6H, PD15-17, Tg) was
similar to the Smad2-P level at PD8
(Fig. 6H, PD8, Tg) and did not
show any elevation as in the WT ovaries at PD15-17
(Fig. 6H, PD15-17, WT). As the
level of Smad2 in Tg ovaries at PD15-17 was similar to that in the WT ovaries
(Fig. 6I, PD15-17), our data
indicate that the lower Smad2-P level in the Tg ovaries at PD15-17
was caused by Smad2 activation per se. These data indicate that the
downregulation of Bmp15 in Zp3-Foxo3a Tg mice may result in
suppressed activation of the Smad pathways, leading to retardation of
granulosa cell proliferation.
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; Gittens et al.,
2003; Kidder and Mhawi,
2002; Simon et al.,
1997). Using western blot analysis, we found that connexin 37 was
below the detection limit in 6-day-old WT or Tg ovaries, and that connexin 43
levels were similar in Tg and WT ovaries at PD6 (not shown). In 8-day-old
ovaries, however, both connexin 37 and connexin 43 levels were considerably
lower in the Tg mice than in WT mice (Fig.
7A-C), indicating that the transcription of both connexin 37 and
connexin 43 is negatively regulated by Foxo3a in the oocyte. Markedly lower
levels of connexin 37 and connexin 43 were still seen in Tg ovaries at PD15-17
(Fig. 7D-F).
|
). As shown in Fig.
8, in ovaries of 8-day-old WT mice, p27 was found to be expressed
in the nuclei of oocytes in primordial, primary and secondary follicles
(Fig. 8, black arrows), which
is in accordance with a previous report
(Zhang et al., 1999). In
addition, p27 was found to be expressed in granulosa cells of these follicles
(Fig. 8A, yellow arrows). In
partially grown oocytes, however, as shown in follicles from a 20-day-old WT
mouse, the expression of p27 in the nuclei of oocytes was diminished
(Fig. 8B, black arrows),
whereas its expression was still seen in the granulosa cells
(Fig. 8B, yellow arrow). These
results indicate that p27 may be a negative regulator of oocyte growth. To
determine whether p27 is a possible downstream molecule of Foxo3a in mouse
oocytes, we studied the pattern of localization of p27 in oocytes of
Zp3-Foxo3a Tg mice. We found that in ovaries of 20-day-old Tg mice, the
expression of p27 was sustained in the nuclei of the oocytes
(Fig. 8C, yellow arrows),
implying that p27 is continuously active in Tg oocytes, which probably
functions to inhibit oocyte growth.
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;
Fingar et al., 2002). We
wondered whether these growth-enhancing molecules would be compromised in the
oocytes of Tg mice, which might explain their retarded oocyte growth. By
measuring phosphorylated protein levels of Akt, mTOR, p70S6 kinase and p42/44
MAPK in isolated oocytes from 15- to 17-day-old WT and Tg mice, we found that
the activation states of these molecules were not suppressed in Tg oocytes
relative to WT oocytes (Fig.
9), indicating that these basic growth-enhancing pathways are not
affected by the expression of constitutively active Foxo3a in the Tg
oocytes.
Anovulation and luteinization of unruptured follicles in adult Zp3-Foxo3a Tg mice
In adult Tg mice, most of the follicles were arrested at early stages of
follicular development, as illustrated in
Fig. 3M. Irregular estrous
cycles were observed in these mice. At the same time that there was a lack of
continuous follicular development in the Tg ovaries, a few follicles did
however develop further and reached the size of preovulatory follicles (type
8) (Fig. 10A). Even so, these
developed follicles showed no sign of cumulus oocyte complex (COC) expansion;
cumulus cells surrounding the oocyte always showed an atypical tight structure
(Fig. 10A, arrow). Indeed, CL
with trapped oocytes (Fig.
10B) were regularly seen in ovaries of adult Tg mice, indicating
that Tg mice have defects in ovulation. In order to address whether the very
low number of pups born was indeed caused by defective ovulation or,
alternatively, by excess resorption of the embryos, we checked for the
presence of ova on the mornings when the female Tg mice were plugged by stud
males. Of the five female mice used, none showed any ovulated ova in their
oviducts. We also checked for the possible presence of embryos at E9.5;
however, none of the four plugged Tg mice carried any embryos. Our data thus
confirmed that the almost complete infertility was caused by defects in
follicular development and anovulation. Further analysis showed that the
anovulation phenotype in Zp3-Foxo3a Tg mice may be caused by substantially
reduced expression of PR in the large follicles of Tg mice
(Fig. 10D, arrow), as compared
with the high expression of PR in preovulatory follicles of WT mice
(Fig. 10C, arrow).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Liu et al., 2006;
Reddy et al., 2005). In the
current study, we have shown that Foxo3a is mainly expressed in the nuclei of
oocytes in primordial and early primary follicles, and its expression is
dramatically downregulated in oocytes of primary and more-developed follicles.
Based on our current understanding that the nuclear localization of Foxo3a
indicates an active state (Accili and
Arden, 2004; Arden and Biggs,
III, 2002), our data suggest that Foxo3a in oocytes may be a
factor that restrains primordial follicles from being activated, and that
downregulation of Foxo3a expression in oocytes may be a prerequisite for
oocyte growth and follicular development.
To test this hypothesis, we generated a Tg mouse model to maintain the
expression of constitutively active Foxo3a in oocytes in primary and
more-developed follicles using the Zp3 promoter
(Epifano et al., 1995). We
found that the constant expression of Foxo3a in mouse oocytes not only led to
retardation of oocyte growth and follicular development, but also to
anovulation and luteinization of unruptured follicles. These defects
subsequently cause infertility in female Tg mice. This work therefore suggests
that Foxo3a is an important intra-oocyte signaling molecule that negatively
regulates oocyte growth, follicular development and female fertility in mice.
Thus, we hypothesize that during follicular activation, the release from
transcription inhibition by Foxo3a may be of importance in initiating the
expression of Bmp15, connexin 37 and connexin 43, which in turn facilitates
the growth of oocytes and follicles.
Important studies by Castrillon et al.
(Castrillon et al., 2003) have
shown that conventional Foxo3a-knockout mice exhibit excessive activation of
primordial follicles. Nevertheless, the question remains whether Foxo3a
functions in the ovary itself, as raised by Brenkman and Burgering
(Brenkman and Burgering, 2003)
in their review appraising the work of Castrillon et al., or at what
developmental stages Foxo3a exerts its role in repressing follicular
development. Based on results from the current study, we suggest that it is
the intra-oocyte Foxo3a that plays a decisive role in controlling follicular
activation and early development. This study therefore also reinforces the
theory that oocytes carry key signals for follicular activation and
development (Albertini and Barrett,
2003; Eppig, 2001;
Matzuk et al., 2002).
Oocyte-granulosa and granulosa-granulosa gap junctions are essential for
normal folliculogenesis (Kidder and Mhawi,
2002). In the connexin multigene family, connexin 37 is crucial
for the establishment of oocyte-granulosa gap junctions, which is important
for oocyte development. In connexin 37-knockout mice, oocyte growth ceases at
a diameter of 52 µm (Carabatsos et al.,
2000), and the sole absence of connexin 37 from oocytes is in
itself sufficient to compromise both oocyte and follicular development, as has
been shown using chimeric ovaries with connexin 37-deficient oocytes and WT
granulosa cells (Gittens and Kidder,
2005). Connexin 43 is involved in establishing the gap junctions
between granulosa cells, and mouse ovarian follicles lacking connexin 43 are
arrested in the early preantral stages
(Ackert et al., 2001;
Gittens et al., 2003). In the
present study, we have provided evidence that both connexin 37 and connexin 43
are negatively regulated by Foxo3a in oocytes. We suggest that the negative
regulation of connexin 37 and connexin 43 by the Foxo3a transgene in oocytes
is one of the factors that lead to retardation of follicular development in
the Tg mice. Connexin 37 may be a downstream molecule of Foxo3a in mouse
oocytes. It is probable that in the Tg mice, the retarded oocyte growth is
caused by hindered oocyte-granulosa communication due to low connexin 37
expression that is insufficient to establish oocyte-granulosa gap junctions,
through which essential signals, nutrients, ions and other requisites for
oocyte growth are normally transported from the surrounding granulosa cells.
Connexin 43, which is expressed by granulosa cells, is also regulated by
oocyte Foxo3a, probably via an indirect pathway. The remarkable reduction in
connexin 43 expression observed in Tg ovaries at PD8 seems to be a reason for
the arrest in granulosa cell proliferation, because at this stage the
Zp3 promoter is just becoming active and primary (type 3b) follicles
are the dominant follicle types in both WT and Tg ovaries. However, the
mechanisms by which Foxo3a in oocytes regulates connexin 37 and connexin 43
expression in follicles are not currently known.
The two TGF-ß family members produced by oocytes, Bmp15 and Gdf9, have
fundamental roles in the paracrine signaling between oocytes and granulosa
cells that controls follicular development
(Dong et al., 1996;
Elvin et al., 1999b;
Elvin et al., 1999a;
Otsuka and Shimasaki, 2002;
Vitt et al., 2000a;
Vitt et al., 2000b).
Investigation into the mechanisms by which Bmp15 and Gdf9 influence female
fertility remains one of the most active areas of research. Previous studies
have indicated that these two molecules activate the Smad pathways in
granulosa cells (for reviews, see
Hashimoto et al., 2005;
Liao et al., 2004;
Mazerbourg et al., 2004;
McNatty et al., 2005;
Moore and Shimasaki, 2005;
Pangas and Matzuk, 2004;
Shimasaki et al., 2004). Our
current data provide novel information that Foxo3a in oocytes may be an
upstream regulator of Bmp15. It is possible that intra-oocyte Foxo3a
suppresses the activation of ovarian Smad pathways by negatively regulating
the production of Bmp15, thereby inhibiting the proliferation and
differentiation of surrounding granulosa cells. We propose that intra-oocyte
Foxo3a may negatively regulate the transcription of Bmp15 directly,
based on sequence data showing that several Foxo3a-binding sites are present
in the Bmp15 promoter (not shown). Thus, with the occurrence of
follicular activation, the emergence of Bmp15 expression in primary follicles
(Shimasaki et al., 2004) may
be triggered by the downregulation of Foxo3a in oocytes.
At PD6 and PD8, the levels of Gdf9 mRNA were found to be similar
in Tg and WT mice, indicating that Gdf9 is not directly regulated by
Foxo3a in oocytes. The lower Gdf9 mRNA levels in 15- to 17-day-old Tg
mice is probably caused by the retardation of oocyte growth in
development-arrested follicles in these mice. This hypothesis is supported by
a report that in Gdf9-knockout mice, although follicular development is
arrested at the primary (type 3b) stage
(Dong et al., 1996), the
oocytes generally grow larger, leading to a mismatch in oocyte:follicle size
(Carabatsos et al., 1998). This
phenomenon is opposite to the situation in our Tg mice where smaller oocyte
and follicle sizes were seen due to retarded oocyte growth.
It has been reported that female Bmp15-knockout mice are subfertile, with
reduced ovulation rate but with minimal histopathological defects in the ovary
(Yan et al., 2001). In
Bmp15-/- Gdf9+/- double-mutant (DM) mice, defects in COC
expansion were observed and the DM oocytes did not support expansion of the
oocytectomized WT cumulus cells, indicating that secretion of both Bmp15 and
Gdf9 by oocytes is important for supporting the process of cumulus expansion
(Su et al., 2004). In the
current study, the cumulus expansion defect observed in our Zp3-Foxo3a Tg mice
is comparable to what was observed in Bmp15-/- Gdf9+/-
DM mice (Su et al., 2004). On
the other hand, in our Tg mice, cumulus cells surrounding the oocyte always
showed a tight structure, which is the opposite situation to that with the
Bmp15-/- Gdf9+/- DM cumulus cells, which are loosely
attached and readily fall off the oocyte
(Yan et al., 2001). Thus, our
Tg mouse model cannot be considered to be identical to Bmp15-/-
mice or Bmp15-/- Gdf9+/- DM mice, as a low level of
Bmp15 mRNA was still found to be expressed in oocytes, and the levels
of Gdf9 mRNA were similar in primary/secondary follicles of WT and Tg
mice at PD8. It is not clear yet whether or not Gdf9 levels are lower in the
few `preovulatory' oocytes in the Tg mice, and further studies will be
required to investigate how Foxo3a in oocytes may regulate Gdf9
transcription in preovulatory follicles, which may in turn regulate cumulus
expansion prior to ovulation. We believe that the defect in ovulation in our
Tg mice is caused by multiple molecular mechanisms that are regulated by
Foxo3a in oocytes.
The p27 protein is a Cdk inhibitor that suppresses cell growth. After being
phosphorylated, p27 shuttles from the nucleus to the cytoplasm, whereby its
inhibitory effects can be abolished
(Cunningham et al., 2004;
Shin et al., 2005;
Viglietto et al., 2002). Our
data from the current study demonstrate that in Zp3-Foxo3a Tg mice, p27
expression is maintained in the nuclei of oocytes, indicating that Foxo3a in
oocytes prevents the shuttling of p27 from the nucleus to the cytoplasm, or
prevents the downregulation of p27 expression, thereby maintaining the
growth-inhibitory function of p27 in oocytes. This notion is supported by
previous studies with other cell types showing that Foxo3a can enhance the
expression of p27 (Chandramohan et al.,
2004; Dijkers et al.,
2000). Moreover, our unpublished data also suggest that p27 in
primary oocytes may participate in the suppression of primordial follicle
activation, because in p27-knockout mice primordial follicles were prematurely
activated. The underlying mechanism of how Foxo3a may regulate p27 levels and
regulate the localization of p27 in mouse oocytes is being investigated in our
laboratory.
Another interesting finding from the current study is that in the Tg mice,
expression of the Foxo3a transgene in oocytes resulted in luteinization of
unruptured `preovulatory' follicles, with oocytes being trapped in the CL. In
spite of the possible regulation through altered production of Bmp15 and Gdf9,
further analyses have shown that the anovulation phenotype in our Tg mice may
be caused by the dramatically reduced expression of PR in large follicles. PR
is a nuclear receptor transcription factor that is induced in the granulosa
cells of preovulatory follicles in response to the surge of luteinizing
hormone. PR has been shown to be essential for ovulation, as mice lacking this
molecule fail to ovulate and are infertile
(Robker et al., 2000). The
assumption that the considerably reduced PR levels may cause anovulation in
the Tg mice is supported by our finding that cathepsin L, a protease that
facilitates ovulation, was also downregulated in granulosa cells of large
follicles in the Tg mice (not shown), a phenomenon similar to that observed in
PR-knockout mice (Robker et al.,
2000). At this stage of the study, however, it is not clear
whether Foxo3a produced by oocytes can directly regulate the expression of PR
in preovulatory granulosa cells.
Based on the results of the current study and from our previous report that
Kitl can induce the phosphorylation/suppression of Foxo3a via the activation
of PI3K/Akt in cultured mouse and rat oocytes
(Reddy et al., 2005), we
suggest that a well-balanced activation of the intra-oocyte PI3K pathway is of
importance in controlling the rates of oocyte growth and follicular
development (Liu, 2006;
Liu et al., 2006). In other
words, a deregulated PI3K pathway in the oocyte may lead to disturbed
follicular development and impaired fertility, as is the case in the
Zp3-Foxo3a Tg mice. In addition, as Foxo3a is a transcription factor, other
downstream genes and related regulation mechanisms in oocytes remain to be
elucidated. Additional Foxo3a-independent pathways in oocytes, such as the
mTOR-p70S6 kinase-mediated cascades and the MAPK pathway, are still just as
active in the Tg oocytes, indicating that there exist overlapping signaling
mechanisms that account for the rapid oocyte growth during follicular
activation and early development.
In summary, our study has revealed the functional roles of intraoocyte Foxo3a in the regulation of oocyte growth, follicular development and ovulation. The findings from the current study may provide some useful information in the search for oocyte-borne genetic aberrations that lead to defects in follicular development and ovulation in human diseases, such as premature ovarian failure.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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