1 Molecular and Cellular Graduate Program, Arizona State University, Tempe, AZ
85284-4501, USA
2 Biology Graduate Program, Arizona State University, Tempe, AZ 85284-4501,
USA
3 Minority Access to Research Careers (MARC) Program at ASU, Arizona State
University, Tempe, AZ 85284-4501, USA
4 School of Life Sciences, Box 4501, Arizona State University, Tempe, AZ
85284-4501, USA
Author for correspondence (e-mail:
nrawls{at}imap4.asu.edu)
Accepted 1 December 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Lunatic fringe, Notch, Ovary, Follicle, Meiosis, Fertility
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The interaction between Notch and its ligands is modulated by
O-linked fucose moieties that are added to the EGF repeats of the
extracellular domain. Usually fucose is unaltered when it is added to
proteins; however, on the Notch receptors fucose is modified with
N-acetylglucosamine (GlcNac) added by the Fringe proteins
(Moloney et al., 2000;
Bruckner et al., 2000
). The
Fringe proteins are Golgi-localized and belong to a large family of
ß1,3-N-acetylglucosaminyl transferases
(Moloney et al., 2000
;
Bruckner et al., 2000
;
Schwientek et al., 2002
).
Enzymes in this family have strong substrate and target specificity and
diverse functions. The only known targets of the mammalian fringe proteins are
the Notch receptors (Schwientek et al., 2000). In mammals, there are three
fringe proteins, radical (Rfng), manic (Mfng) and lunatic fringe (Lfng)
(Johnston et al., 1997
).
Modification of the extracellular domain of Notch by Lfng can potentiate or
inhibit the interaction between a particular Notch receptor-ligand pair. For
example, Lfng potentiates the interaction between Notch1 and Dll1, but
inhibits Notch1-Jagged1 interactions. However, Lfng potentiates both Dll1 and
Jagged1 mediated activation of Notch2
(Hicks et al., 2000
). Lfng and
Mfng reportedly modify different sites in the extracellular domain of Notch2
(Shimizu et al., 2001
),
indicating they may have different roles to play in regulating Notch
signaling.
In Drosophila, two Golgi localized
ß1,3-N-acetylglucosamine transferases, Fringe and Brainiac, play
important roles in oogenesis and folliculogenesis (Goode et al., 1996a; Goode
et al., 1996b; Hicks et al.,
2000; Bruckner et al.,
2000
; Munro and Freeman, 2000;
Schwientek, 2002
). Brainiac
activity is needed in the germ line for proper organization of the follicle
(Goode, 1996). Fringe, the homolog of Lfng, is necessary for specification of
the polar cells (Grammont and Irvine,
2001
). Brainiac has been demonstrated to modify glycosphingolipids
by adding GlcNac residues to mannose and galactose moieties on ceramide
(Schwientek et al., 2002
). In
mice, a null mutation of the murine homolog of brainiac demonstrated that this
protein is important for very early development, as
braniac/ embryos die prior to implantation
(Vollrath et al., 2001
). No
role for this family of proteins in mammalian folliculogenesis has been
described.
It has been demonstrated that Lfng is an important regulator of Notch signaling. For example, Lfng null mutants have segmentation defects that are similar to those seen in null mutations of Notch1 and Dll1 (Evrard et al., 1997; Zhang and Gridley, 1997). In somites, where Lfng is the only family member expressed, Notch receptors and ligands are expressed normally in Lfng/ mutants, but the Notch downstream target gene Hes5 was not detected, indicating a lack of Notch activation in the presence of ligand. However, Hes5 was expressed normally in the neural tube and developing brain of Lfng null mutants, probably due to expression of Mfng and Rfng in these tissues (Evrard et al., 1997). Interestingly, Rfng-deficient mice had no phenotype and Rfng/Lfng double null mutants had only defects associated with a lack of Lfng (Zhang et al., 2000).
Folliculogenesis is the process by which oocytes develop in response to
hormonal cues. This requires the coordination of the proliferation and
differentiation of granulosa cells and the growth and maturation of the
oocyte. Primordial follicles consist of a small oocyte surrounded by squamous
somatic cells. When recruited to develop, the granulosa cells proliferate and
become cuboidal. As these cells continue to proliferate, layers develop around
the growing oocyte. Once a follicle has several layers of cells a fluid filled
space, the antrum, will begin to form. The antrum spatially separates the two
functionally distinct granulosa cell populations, cumulus and mural. During
this time the oocyte has grown and at the time of antrum formation it becomes
competent to resume meiosis in response to luteinizing hormone (LH).
Resumption of meiosis is marked by the breakdown of the germinal vesicle
(GVB). Meiosis continues to metaphase II (MII), and oocytes are blocked at
this stage until fertilization. Studies done in mice have demonstrated that
reciprocal signaling between the oocyte and the granulosa cells is necessary
for the differentiation of the cumulus granulosa cells and meiotic maturation
of the oocyte (Rodgers et al.,
1999; Erickson and Shimasaki,
2000
; Eppig, 2001
;
Matzuk et al., 2002
).
We have previously shown that Notch2, Notch3 and Jagged2 are expressed in
granulosa cells, and Jagged1 is expressed in the oocytes of developing
mammalian follicles (Johnson et al.,
2001). Furthermore, transcripts of the Notch downstream target
genes, Hes1, Hes5, Hesr1, Hesr2 and Hesr3 also were detected
in the granulosa cells of follicle types 3b-8
(Johnson et al., 2001
),
indicating that Notch signaling was active. As all three mammalian fringe
proteins can modify the Notch receptors when expressed in the same cell
(Bruckner et al., 2000
;
Moloney et al., 2000
;
Hicks et al., 2000
), we
hypothesized that the fringe genes would also be expressed in the granulosa
cells, and furthermore, that they would have a role in regulating
folliculogenesis through Notch2 and Notch3. In this study, we demonstrate that
Lfng is expressed in the granulosa cells and theca of developing follicles
from primary to preovulatory in size. Rfng is expressed briefly in granulosa
cells of early antral follicles. Mfng is only detected in the vasculature.
Null mutations of the Notch receptors and ligands result in embryonic lethal
phenotypes (Swiatek et al.,
1994
; Conlon et al.,
1995
; Hrabé de Angelis
et al., 1997
; Jiang et al.,
1998
; Hamada et al.,
1999
; Xue et al.,
1999
; McCright et al.,
2001
). Some Lfng/ mice survive
to adulthood, therefore we examined folliculogenesis in these mutants. Female
Lfng null mutant mice have many aberrant follicles. When induced to
ovulate they released oocytes into the oviduct, but only a small percentage
could be fertilized in vitro. Examination of these oocytes demonstrated that
cumulus expansion occurred in response to exogenous hormones, but the oocytes
were not at metaphase of meiosis II, and had not completed meiotic maturation.
Mutations that block the progression of meiosis have been described, but they
are all germ-cell-specific genes. These are novel observations because the
disregulation of meiosis is caused by a change in the somatic cells. This
represents a new regulatory pathway in folliculogenesis and a new role for
Notch signaling in mammals.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Whole-mount thick section in situ hybridization (ISH)
Whole-mount ISH was done on thick sections according to Johnson et al.
(Johnson et al., 2001).
Briefly, ovaries were fixed in 10% neutral-buffered formalin (NBF)
(Richard-Allen Scientific, Kalamazoo, MI), and embedded in paraffin wax after
stepwise dehydration in ethanol. Thirty micron (µm) sections were cut
perpendicular to the axis of entry of ovarian blood vessels. Sections were
dewaxed, rehydrated and antisense digoxigenin-labeled gene-specific RNA probes
were hybridized. Transcripts were identified using anti-digoxigenin antibody
(Roche, Indianapolis, IN) conjugated to alkaline phosphatase and the BM purple
substrate (Roche, Indianapolis, IN). Replicates were performed on sections
from at least 3 ovaries/genotype and probes were checked for specificity by
ISH on embryos.
Histology
Tissues were fixed as above and sectioned to 10 µm. Sections were
prepared by standard procedures and stained with Hematoxylin and Eosin.
Bone and cartilage preparation
The mice were skinned and eviscerated. The carcasses were placed in Alcian
Blue (Sigma A3157) for 48 hours to stain the cartilage, followed by 2% KOH for
48 hours. Skeletons were then placed in Alizarin Red (Sigma A5533) to stain
the bone for 72 hours.
Hormone treatment and isolation of OCC and oocytes
Mice were injected intraperitoneally (ip) with 5 international units (IU)
of pregnant mare's serum gonadotropin (PMSG) (Calbiochem, Carlsbad, CA) and 48
hours later, were injected ip with 5 IU of human chorionic gonadotropin (hCG)
(Calbiochem, Carlsbad, CA). Oocyte cumulus complexes (OCC) were harvested from
the oviduct 16 hours later. OCC and ovaries were collected in KSOM (Specialty
Media, La Jolla, CA) with 10% FBS. The OCC were incubated in KSOM containing
hyaluronidase (300 µg/ml) for 30 seconds, then washed in KSOM, and fixed as
described in LeMaire-Adkins et al.
(LeMaire-Adkins et al., 1997).
Briefly, oocytes were fixed in 2% formaldehyde, 1% Triton X-100, 0.1 mM PIPES,
5 mM MgCl2, 1 mM DTT and 2.5 mM EGTA in D2O (Sigma, St
Louis, MO) containing aprotinin (Sigma, St Louis, MO) and taxol (Sigma, St
Louis, MO) at 37°C. Oocytes were washed in 0.1% normal goat serum (NGS) in
PBS (GIBCO/BRL, Gaithersburg MD) and blocked at 37°C in PBS containing 10%
NGS and 0.1% Triton X-100. Oocytes were stored in this at 4°C until
staining was performed. For staining, oocytes were transferred to 1% Triton
X-100 in PBS at room temperature then incubated with a monoclonal
anti-
-tubulin (clone DM 1A, Sigma, St Louis, MO) conjugated to FITC at
a 1:50 dilution. The oocytes were washed in PBS, incubated in PBS containing 1
µg/ml Hoechst 33258 (Molecular Probes, Eugene, OR). Oocytes were washed in
PBS and mounted in glycerol with p-phenylenediamine and visualized by
confocal microscopy. Confocal analysis was done using a Leica TCS NT, final
magnification of 800 x. The FITC was visualized using an Ar laser, and
Hoechst 33258 was visualized using an UV laser.
In vitro fertilization (IVF)
OCC were collected after hormone administration as above. Sperm were
collected from the vas deferens and cauda epididymis in human tubal fluid
(HTF) and capacitated for 2 hours at 37°C. Sperm (1 x106)
were added to each OCC sample and fertilization allowed to proceed for 2 hours
at 37°C. Eggs were washed three times in sperm free KSOM and incubated at
37°C. Eggs were scored as fertilized by the presence of two pronuclei and
embryogenesis was scored daily.
Immunohistochemistry (IHC)
Sections were heated at 80°C for 30 minutes, cooled to room
temperature, followed by xylenes, rehydrated through graded alcohols to 70%
ethanol. Slides were incubated in water, then PBS, placed in 0.1 M sodium
citrate (pH 6) and epitope retrieval done in the microwave. The sections were
cooled to room temperature, rinsed in PBS, and incubated in 3%
H2O2 in 60% methanol to destroy endogenous peroxidases.
IHC was performed using the HistostainSP kit according to the manufacturer's
instructions (Zymed Labs, San Francisco, CA), and primary antibodies were
diluted according to this protocol except for the following: polyclonal
anti-c-Kit antibody (Ab-1, Calbiochem, Carlsbad, CA) was diluted 1:25, and
anti-connexin43 (Santa Cruz Biotechnology, Santa Cruz CA), 1:50. Proteins were
detected with alkaline-phosphatase-conjugated anti-rabbit secondary antibody
and exposed to colour reagent. No primary antibody controls were included in
each experiment.
Reverse transcription polymerase chain reaction (RT-PCR)
Total ovary RNA was isolated using TRIzol (Life Technologies, Gaithersburg,
MD), according to the manufacturer's directions, from 3 different
animals/genotype. For oocytes, 15 oocytes per sample were denuded using
hyaluronidase and total RNA extracted. cDNA was synthesized using Superscript
III (Invitrogen, Carlsbad CA), according to the manufacturer's protocol. For
each gene examined by semi-quantitative (sq) RT-PCR, 3 sets of samples
comprising all three genotypes and no RT controls were amplified using
-[32P]dATP (Perkin-Elmer Life and Analytical Sciences,
Boston, MA). For each gene-specific primer pair the minimum number of cycles
to the linear range was determined and used for all subsequent experiments.
All primer sets span at least one intron. Control experiments were done using
total embryo RNA. All cDNA samples were normalized using the ribosomal gene L7
(Meyuhas et al., 1990), and quantified using a Storm 860 PhosphorImager and
ImageQuant software (Molecular Dynamics, Sunnyvale, CA). To detect the
presence of transcripts in oocytes, a qualitative PCR protocol and
amplification beyond the linear range after normalization was used
(Münsterberg and Lassar,
1995
).
Kinase assays
Kinase assays were carried out as described in Svoboda et al.
(Svoboda et al., 2000). Single
eggs were transferred in 1.5 µl of KSOM into 3.5 µl of double kinase
lysis buffer (10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µM
p-nitrophenyl phosphate, 20 mM ß-glycerophosphate, 0.1 mM sodium
orthovanadate, 5 mM EGTA) and immediately frozen in liquid nitrogen, then
stored at 80°C until the assay was performed. The kinase reaction
was initiated by the addition of 5 µl of double kinase buffer (24 mM
p-nitrophenyl phosphate, 90 mM ß-glycerophosphate, 24 mM
MgCl2, 24 mM EGTA, 0.2 mM EDTA, 4.6 mM sodium orthovanadate, 4 mM
NaF, 1.6 mM dithiothreitol, 60 µg/ml aprotinin, 60 µg/ml leupeptin, 2
mg/ml polyvinyl alcohol, 2.2 mM protein kinase A inhibitor peptide (Sigma), 40
mM 3-(nmorpholino) propanesulfonic acid (MOPS), pH 7.2, 0.6 mM ATP, 2 mg/ml
histone (type III-S, Sigma), 0.5 mg/ml MBP with 500 mCi/ml
-[32P]ATP (3000 Ci/mmol) (Perkin-Elmer Life and Analytical
Sciences). To determine the background level of phosphorylation, 5 µl of
double kinase lysis buffer was added instead of egg lysate. Reactions were
incubated for 30 minutes at 30°C, and terminated by the addition of 10
µl 2 xSDS-PAGE sample buffer and boiling for 3 minutes. Following 15%
SDS-PAGE, the gel was dried and exposed to a phosphorimager screen and
quantified. The mean value of the control samples was set to one and all
others expressed as fold activity of control.
Quantifying cumulus expansion
OCC were collected from the oviducts of Lfng+/
and Lfng/ mice (n=5/genotype)
post-hormone administration. OCC were photographed at 70 x and
photomicrographs printed the same size. The widest diameter of each OCC was
measured in mm and mean diameter±s.d. was determined.
Statistical analysis
Analysis was carried out using the SAS system and the FREQ procedure. Our
data was found to be significant (P<0.0001) by Chi-square,
likelihood ratio Chi-square, Continuity-adjusted Chi-square and
Mantel-Haenszel Chi-square analysis.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In order to determine which cells in the ovary expressed Lfng,
whole-mount thick section in situ hybridization (ISH) was carried out using
antisense digoxigenin-labeled RNA probes, as described by Johnson et al.
(Johnson et al., 2001). Using
two different probes, one that included 3' untranslated sequences and
another that only included the coding region, Lfng mRNA was detected
in the granulosa cells of follicles from type 3 to those preovulatory in size
(Fig. 1A,B). Interestingly, in
small growing follicles, Lfng transcripts clearly demarcate the outer
edge of the follicle (Fig. 1B,
inset). In antral follicles, Lfng expression in the theca was evident
(Fig. 1A,B). Furthermore, this
gene was expressed in blood vessels (Fig.
1B, red arrow), so it is possible that the expression noted in the
theca is also in the vascular component. Lfng was not expressed in
oocytes, primordial or primary follicles, or corpus luteum (CL). The lack of
Lfng transcripts in oocytes was confirmed by RT-PCR performed using
total RNA from denuded germinal vesicle (GV) stage oocytes and ovulated MII
eggs (Fig. 1F).
|
Lunatic fringe-deficient ovaries have aberrant follicles
The role of Notch signaling in the ovary is unknown, and null mutants of
most of the Notch receptors and Jagged ligands have embryonic or perinatal
lethal phenotypes (Swiatek et al.,
1994; Conlon et al.,
1995
; Hrabé de Angelis
et al., 1997
; Jiang et al.,
1998
; Hamada et al.,
1999
; Xue et al.,
1999
; McCright et al.,
2001
). Lfng is an important modifier of Notch signaling
(del Barco Barrantes et al.,
1999
; Hicks et al.,
2000
; Moloney et al.,
2000
); in its absence, Notch signaling in the somites was
impaired, as determined by the lack of Hes gene expression in
Lfng/ embryos (Evrard et al., 1997).
Furthermore, Lfng null mutants have segmentation defects that are consistent
with mutations in Dll1 and Notch1 (Zhang and Gridley, 1997;
Evrard et al., 1997). Interestingly, of the two different
Lfng/ mutations, one results in complete
embryonic lethality of Lfng/ offspring
(Zhang and Gridley, 1997), whereas the other has a 25% survival rate (Evrard
et al., 1997); we studied the role of Lfng and the Notch signaling pathway in
folliculogenesis using the latter mutant.
Initially studies were carried out to determine whether female Lfng-deficient mice would mate and produce litters. Lfng null and heterozygous mice were paired with Lfng+/ male mice. All heterozygous females (n=11) mated within 6 days, as determined by the presence of a copulatory plug. There were 26 litters from heterozygous pairings; by comparison, over the same time, null females demonstrated neither copulatory plugs nor pregnancies. These observations indicated the possibility of a fertility defect. However these mice have abnormalities of their axial skeletons, including fusions of the vertebrae and kyphosis, which may make lordosis and mating impossible (Evrard et al., 1997; Zhang and Gridley, 1997) (see Fig. S1 in the supplementary material).
In order to determine whether infertility was due to either defects in ovary development or folliculogenesis in the Lfng/ mice, gross morphological and histological examination of ovaries from neonatal and adult Lfng/ and Lfng+/ mice was done. At the gross morphological level, the ovaries and reproductive tracts of 4- and 7-week-old Lfng null mice were smaller than heterozygous littermates, but no abnormalities were noted. Protuberances on the surface of the ovaries indicated the presence of developing follicles (Fig. 2A,B). Lfng null mice have spinal defects that result in a shortened body axis, so smaller organs are not unexpected. The ovarian histomorphology of neonatal Lfng null mutants was disorganized compared with heterozygous littermates (Fig. 2, compare C with D), but primordial follicles were clearly present. Ovaries from sexually mature Lfng/ mice had developing follicles of all sizes, but there were many abnormal follicles present. For example, there were polyovular follicles (Fig. 2F-H, red arrows in F). Furthermore, there were follicles that lacked a complete layer of theca developing next to the theca of other follicles or sharing theca, but not truly polyovular (Fig. 2E,F, black arrows in F). Many of these follicles appeared atretic. CL were noted, but, there were also many large lutealinized follicles with trapped oocytes (Fig. 2I,J).
|
Lfng null mice released approximately the same number of eggs with cumulus cells as controls in response to exogenous hormones, and cumulus expansion was evident (Table 1, Fig. 3C). After fertilization, 48.8% of wild-type eggs became two-cell embryos, but only 9.7% of null eggs did. Furthermore, whereas 41.9% of heterozygous embryos continued to develop to the four- to eight-cell stage, and 31.4% became blastocysts, only 2% of the null embryos became four- to eight-cell embryos, and none developed into blastocysts (Table 1). The Lfng null mutants can respond to exogenous hormone, but they had a very low fertilization rate and there may be a block in early development. A lack of fertilization and development suggests a defect in folliculogenesis.
|
|
Exogenous hormones were used to induce ovulation in Lfng null and
control littermates, and OCC were harvested from the oviduct, as described
above. Eggs were fixed and stained with anti--tubulin antibody
conjugated to FITC; the chromatin was stained with Hoechst 33258. We found
that 77.8% and 88.2% of heterozygous and wild-type eggs, respectively, were in
MII, as determined by the presence of a barrel-shaped meiotic spindle with
chromosomes on the metaphase plate and a polar body, but only 5% of
Lfng/ eggs were in MII
(P<0.0001, null compared with controls). Most of the oocytes from
Lfng/ mice were at metaphase I (MI),
anaphase/telophase I, or had multiple bodies with chromatin fragments
throughout (Fig. 3A). These
eggs were not characteristically parthenogenic, there were no obvious nuclei,
nor polar bodies, none were two cells
(Hirao and Eppig, 1997
), and
the diffuse chromatin was indicative of apoptosis. These data indicated that
in Lfng-deficient follicles, oocytes were not completing meiotic maturation
prior to induced ovulation. Interestingly, these oocytes have a normally
expanded cumulus (Fig. 3C): the
mean diameter of the OCC from Lfng heterozygous and null mice was not
different when compared (mean diameter heterozygous: 3.8±0.4 mm; null:
3.4±0.4 mm; P=0.1) (see Fig. S2 in the supplementary
material).
To further examine oocyte maturation in the Lfng null ovary, the level of
maturation promoting factor (MPF) and cytostatic factor (CSF) kinase
activities was determined. MPF is necessary for GVB, and its activation
precedes CSF activation. Mos activates MAP kinase, which is a component of CSF
and is necessary for the MII block. MPF and CSF activity both peak in MII eggs
(Zhao et al., 1991;
Verlhac et al., 1993
;
Gebauer and Richter, 1997
;
Sagata, 1997
). Eggs were
collected from oviducts post-hormone administration, and kinase activity
determined in null and control eggs (n=9, representative data in
Fig. 3B). Both MPF and CSF
kinase activity was evident in null oocytes, but it was less than in controls
(Fig. 3D). Consistently, 75% of
Lfng+/ (n=364) and 74.9% of
Lfng/ (n=355) (P=1)
oocytes, underwent GVB in vitro within 2 hours, thus null oocytes resume
meiosis normally.
It is possible that null oocytes have a defect that blocks progression
through meiosis, so GV stage oocytes were collected from large follicles
post-PMSG administration and allowed to mature in vitro, as described by
LeMaire-Adkins et al. (LeMaire-Adkins et
al., 1997). Only oocytes that underwent GVB within 2 hours were
followed, in order to have a synchronous cohort. After 10, 12 and 16 hours in
culture, oocytes were fixed and stained and the stage of meiosis was
determined. Oocytes from heterozygous ovaries progressed through meiosis
comparably to previously published data
(LeMaire-Adkins et al., 1997
),
with 40.4% at MII at 10 hours, 57% at 12 hours and 93.6% at 16 hours
(Table 2). Oocytes from null
ovaries reached MI quickly (75% at 10 hours), but few progressed to MII. At
early timepoints, there were oocytes progressing through telophase/anaphase I,
but by 16 hours in culture this stage was not detected; possibly these cells
die quickly if progression to MII does not occur. We did not detect an
increase in parthenogenically activated oocytes (4.3% of control and 6.5% of
null, P=0.13).
|
To examine the effect of a lack of Lfng on the Notch pathway in the ovary, the expression of Notch receptor and ligand genes was determined by sqRT-PCR, using whole ovary RNA, and compared with the results obtained from ISH. Representative sqRT-PCR from three replicates is presented in Fig. 4. Notch2 and Jagged2 demonstrated no change in expression level (Fig. 4), and no change in their cell or follicle stage-specific pattern of expression when examined by ISH (data not shown). Notch3 had a slightly reduced level of expression by sqRT-PCR, but no change in expression was detected by ISH (Figs 4, 5). Jagged1 was expressed at greatly reduced levels in Lfng-deficient ovaries. This gene was still oocyte restricted, but was expressed only in very small follicles (Figs 4, 5). No change in the expression level of Notch2 and Jagged2, and a relatively small reduction in Notch3, is consistent with observations reported in the somites of Lfng/ embryos (Evrard et al., 1997).
|
|
Expression of other follicle genes in the Lfng/ ovary
Reciprocal signaling between the oocyte and granulosa cells is crucial for
folliculogenesis and oocyte maturation
(Gosden et al., 1997;
Erickson and Shimasaki, 2000
;
Su et al., 2002
;
Su et al., 2003
).
Lfng-deficient oocytes do not mature normally, so possibly other important
signaling pathways may be altered in this mutant. Kit ligand (KL) is expressed
in granulosa cells and the Kit receptor (previously known as c-Kit) is
expressed in oocytes, in follicles from primordial to preovulatory in size. In
antral follicles, KL is only expressed in the mural population. Signaling
through this pathway has been implicated in differentiation of the theca,
proliferation of granulosa cells and meiotic maturation (for a review, see
Rawls et al., 2001
). KL did
not change in expression, and there was no change detected in Kit receptor
expression in the Lfng null ovary when compared with controls
(Fig. 6).
|
Signaling through the FSH receptor (Fshr), which is expressed by every
granulosa cell, is necessary for normal development of the ovary and for
normal sexual maturation. Mice with null mutations in the Fshr gene (FORKO)
demonstrate hypergonadotropic hypogonadism
(Danilovich et al., 2000).
When expression of Fshr was examined by sqRT-PCR, no change was detected
(Fig. 4). Because the FORKO
mice demonstrate only primary and small preantral follicles and no CL
(Danilovich et al., 2000
),
these data are consistent. As there was no change in the expression of many
genes that are important for folliculogenesis, we conclude that the defects
are due to a lack of Lfng and decreased Notch signaling in the follicles of
these mutants.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have demonstrated that Lfng/ female mice are infertile. Furthermore, there is an interesting disconnection between cumulus expansion and GVB and completion of meiotic maturation. Observations of null oocytes indicated a lack of progression from MI to MII (Table 2; Fig. 3). These data indicate an important role for Lfng, and thereby for Notch signaling, in folliculogenesis.
Lfng deficiency results in morphological defects of follicles
The defects noted in developing follicles are unusual, and indicate that
there is a problem with the definition of borders between follicles. In mice,
early germ cells are found in cysts with interconnecting ring canals. At
embryonic day (E) 13.5 to E19.5, germ cells are found in cysts of eight cells
or more, by postnatal day 3, almost all germ cells are single oocytes in
primordial follicles (Pepling and
Spradling, 1998; Pepling and
Spradling, 2001
). In the process of becoming single oocytes, the
cysts break down through apoptosis of single germ cells. The current model
posits that this results in cysts of three, and each of these germ cells will
become a primordial follicle. The somatic cells of the ovary are important for
ring canal breakdown and formation of primordial follicles
(Pepling and Spradling, 2001
).
In Lfng-deficient ovaries there are polyovular follicles, this may indicate a
role for Notch signaling in the organization of primordial follicles.
Furthermore, the ring of Lfng transcripts that we observed around small
growing follicles (Fig. 1) may
indicate a process of early boundary formation that is not occurring correctly
in mutant follicles. Similarly, during oogenesis in Drosophila, the
polar cells are required to separate germ cell cysts and enclose them
appropriately in somatic cells. Fringe-deficient mutants had compound
follicles containing more than one egg. They also had a disorganized polar
epithelium; instead of a single layer there were multiple layers of cells
(Grammont and Irvine, 2001
).
Thus, these data point to evolutionary conservation of function for this
pathway in the development of follicles.
Signaling through the Notch pathway is necessary for completion of meiotic maturation
It is possible that the trapped oocytes in lutealinized follicles reflect a
pituitary defect. However, in the hypogonadal (hpg) mouse,
FSH and LH are not secreted by the pituitary, and there is a lack of postnatal
ovary and follicle development (Halpin et
al., 1986a; Halpin et al.,
1986b
). Grafting neural tissue or providing exogenous hormones to
hpg female mice completely reverses this phenotype; folliculogenesis
is initiated and results in MII eggs that are competent for fertilization and
embryonic development (Charlton,
1987
; Hashizume et al.,
1995
). The hpg phenotype is significantly different than
the Lfng null females in several ways. First, Lfng null mice
demonstrated postnatal ovary development and had developing, albeit abnormal,
follicles (Fig. 2). Second,
providing exogenous hormone to Lfng/ females
induced ovulation, cumulus expansion and GVB, but did not induce complete
meiotic maturation, and these eggs had a low fertilization rate (Tables
1,
2;
Fig. 3). These observations
indicate that Lfng/ mice have a defect at
the level of the follicle.
Reciprocal signaling between the oocyte and the somatic granulosa cells is
necessary for meiotic maturation of the oocyte and differentiation of the
cumulus cells. Work by several groups has demonstrated that these events are
regulated by the interplay of several different pathways. For example,
recombinant Gdf9 can induce the expression of has2 and cox2,
and cumulus expansion, but not GVB (Elvin et al., 1999;
Su et al., 2002;
Su et al., 2003
). Furthermore,
gonadotropin induced activation of ERK1/2 in granulosa cells is necessary for
cumulus expansion and meiotic maturation, and yet both of these events also
require a signal from the oocyte (Su et
al., 2002
; Su et al.,
2003
). This suggests that there is some signal that is switched on
or off in oocytes during the final stages of folliculogenesis that must
activate a signal(s) from the cumulus cells to the oocyte that is necessary
for GVB and meiotic maturation (Su et al.,
2003
). It is possible that activation of Notch, in the granulosa
cells, by Jagged1 in the oocyte, regulates some of these signals. This idea is
further supported by the observation that oocytes from preantral follicles
cultured with cumulus cells can activate ERK1/2, but that cumulus expansion
does not occur, whereas fully grown oocytes can activate ERK1/2 and induce
expression of cox2 and cumulus expansion when cultured with cumulus
cells (Vanderhyden et al.,
1990
; Joyce et al.,
2001
). Normally, expression of Jagged1 in the oocyte is
downregulated as follicles develop an antrum
(Johnson et al., 2001
). This
is the point at which the oocyte is acquiring the competence to resume meiosis
and cumulus cells would begin differentiating. But, in oocytes from
Lfng null mutants, the expression of Jagged1 is restricted to very
small growing follicles, indicating a change in the reciprocal signaling
between the germ and somatic cells (Fig.
5).
The meiosis defect and trapped oocytes reflect alterations in the granulosa
cells caused by a lack of Notch signalling, which leads to temporal changes
in, or a lack of, some signal(s) from these cells. As Notch signaling can
regulate lineage decisions, it is possible that the defects observed are due
to alterations in mural and cumulus cell populations in Lfng-deficient
follicles. In the absence of normal Notch signalling, the granulosa cells
could become predominantly cumulus at the expense of mural cells, as expansion
occurs in response to hormone. Alternatively, it could be a temporal change in
cell differentiation: a lack of Notch signaling may allow for differentiation
of the cumulus lineage in small growing follicles instead of early antral
follicles. This could alter the timing of a crucial signal, and the oocyte may
be unable to appropriately detect or respond to it. This would be consistent
with the noted loss of Jagged1 expression in small growing instead of early
antral follicles that we observed (Figs
4,
5). This is also consistent
with data from other systems, changes in lineage choice mediated by Lfng
alteration of Notch signaling has been demonstrated in lymphoid progenitors.
Transgenic expression of Lfng inhibited Notch1 activation and caused the cells
to choose the B lineage at the expense of the T lineage
(Koch et al., 2001). Future
studies will be necessary to determine whether there are changes in the mural
and cumulus populations.
We determined that oocytes from Lfng-deficient ovaries resume meiosis in
response to exogenous hormones, but do not complete it. MPF and CSF kinase
activity were decreased with respect to control oocytes. However, there is
enough MPF activity for meiosis to resume
(Fig. 3,
Table 2). There are no data
that demonstrate that a reduction of CSF activity will block progression of
meiosis. On the contrary, mos null oocytes do not arrest at MII, they
continue to divide and activate parthenogenically. Mos is a kinase that is
necessary for MAPK and CSF activity in mouse oocytes
(O'Keefe et al., 1989;
Zhao et al.,
1991
;Verlhac et al.,
1993
; Verlhac et al.,
1996
; Colledge et al.,
1994
; Hashimoto et al.,
1994
). Other germ-cell-specific genes also have roles in
progression through meiosis. For example, cks2, a Cdk1 binding
protein, is necessary for entry to anaphase I, and
cks2/ oocytes do not progress past MI
(Spruck et al., 2003
). There
are many genes necessary for progression of meiosis that participate in
synapsis and recombination (Hunt and
Hassold, 2002
). Importantly, in all cases these are
oocyte-specific genes, Lfng is not expressed in the oocyte
(Fig. 1) it is expressed in
granulosa cells, as are Notch2 and Notch3, its targets. Our data indicate a
fundamental change in the signaling between somatic granulosa cells and
oocytes in Lfng-deficient follicles. Disregulation of meiosis through a
somatic-cell-specific signal is a novel observation. These data reinforce the
importance of reciprocal signaling between the granulosa cells and the oocyte
during folliculogenesis. Altered Notch signaling results in the loss of normal
meiotic maturation, but not cumulus expansion and GVB. Collectively, these
data demonstrate that Lfng and the Notch signaling pathway play important
roles in mammalian folliculogenesis.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/4/817/DC1
* Present address: Vincent Center for Reproductive Biology, Department of
Obstetrics and Gynecology, Massachusetts General Hospital, Harvard Medical
School, Room 6607, Building 149, 149 13th Street, Charlestown, MA 02129,
USA
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ackert, C. L., Gittens, J. E. I., O'Brien, M. J., Eppig, J. J. and Kidder, G. M. (2001). Intercellular communication via Connexin43 gap junctions is required for ovarian folliculogenesis in the mouse. Dev. Biol. 233,258 -270.[CrossRef][Medline]
Anderson, E. and Albertini, D. F. (1976). Gap junctions between the oocyte and companion follicle cells in the mammalian ovary. J. Cell Biol. 71,680 -686.[Abstract]
Artavanis-Tsakonis, S., Rand, M. D. and Lake, R. J.
(1999). Notch signaling: cell fate control and signal integration
in development. Science
284,770
-776.
Bettenhausen, B., Hrabe de Angelis, M., Guenet, J. L. and Gosler, A. (1995). Transient and restricted expression during mouse embryogenesis of Dll1, a murine gene closely related to Drosophila Delta. Development 21,2407 -2418.
Bruckner, K., Peruz, L., Clausen, H. and Cohen, S. (2000). Glycosyltransferase activity of Fringe modulates Notch-Delta interactions. Nature 406,411 -415.[CrossRef][Medline]
Charlton, H. M. (1987). Neural grafts and the restoration of pituitary and gonadal function in hypogonadal (HPG) mice. Ann. Endocrinol. 48,378 -384.[Medline]
Chin, M. T., Maemura, K., Fukumoto, S., Jain, M. K., Layne, M.
D., Watanabe, M., Hsieh, C. M. and Lee, M. E. (2000).
Cardiovascular basic helix loop helix factor 1, a novel transcriptional
repressor expressed preferentially in the developing and adult cardiovascular
system. J. Biol. Chem.
275,6381
-6387.
Cohen, B., Bashirullah, A., Dagnino, L., Campbell, C., Fisher, W. W., Leow, C. C., Whiting, E., Ryan, D., Zinyk, D., Boulianne, G. et al. (1997). Fringe boundaries coincide with Notch-dependent patterning centres in mammals and alter Notch-dependent development in Drosophila. Nat. Genet. 16,283 -288.[Medline]
Colledge, W. H., Carlton, M. B. L., Udy, G. B. and Evans, M. J. (1994). Disruption of c-mos causes parthenogenetic development of unfertilized mouse eggs. Nature 370,65 -67.[CrossRef][Medline]
Conlon, R. A., Reaume, A. G. and Rossant, J.
(1995). Notch1, is required for the coordinate segmentation of
somites. Development
121,1533
-1545.
Danilovich, N., Babu, P. S., Xing, W., Gerdes, M.,
Krishnamurthy, H. and Sairam, M. R. (2000). Estrogen
deficiency, obesity, and skeletal abnormalities in follicle-stimulating
hormone receptor knockout (FORKO) female mice.
Endocrinology 141,4295
-4308.
del Barco Barrantes, I., Elia, A. J., Wünsch, K., Hrabe De Angelis, M., Mak, T. W., Rossant, J., Conlon, R. A., Gossler, A. and de la Pompa, J. L. (1999). Interaction between Notch signalling and Lunatic fringe during somite boundary formation in the mouse. Curr. Biol. 9,470 -480.[CrossRef][Medline]
Dunwoodie, S. L., Henrique, D., Harrison, S. M. and Beddington, R. S. (1997). Mouse Dll3: a novel divergent Delta gene which may complement the function of other Delta homologues during early pattern formation in the mouse embryo. Development 16,3065 -3076.
Eppig, J. J. (2001). Oocyte control of ovarian
follicular development and function in mammals.
Reproduction 122,829
-838.
Erickson, G. F. and Shimasaki, S. (2000). The role of the oocyte in folliculogenesis. Trends Endocrinol. Med. 11,193 -198.[CrossRef]
Evrard, Y. A., Lun, Y., Aulehla, A., Gan, L. and Johnson, R. L. (1998). Lunatic fringe is an essential mediator of somite segmentation and patterning. Nature 394,377 -381.[CrossRef][Medline]
Franco Del Amo, F., Smith, D. E., Swiatek, P. J.,
Gendron-Maguire, M., Greenspan, R. J., McMahon, A. P. and Gridley, T.
(1992). Expression pattern of Motch, a mouse homolog of
Drosophila Notch, suggests an important role in early postimplantation mouse
development. Development
115,737
-744.
Gebauer, F. and Richter, J. D. (1997). Synthesis and function of Mos: the control switch of vertebrate oocyte meiosis. BioEssays 19,23 -28.[Medline]
Gilula, N. B., Epstein, M. L. and Beers, W. H. (1978). Cell-to-cell communication and ovulation: a study of the cumulus-oocyte complex. J. Cell Biol. 78, 58-75.[Abstract]
Goode S., Morgan, M., Liang, Y. P. and Mahowald, A. P. (1996). Brainiac encodes a novel, putative secreted protein that cooperates with Grk TGF alpha in the genesis of the follicular epithelium. Dev. Biol. 178,35 -50.[CrossRef][Medline]
Goode, S., Melnick, M., Chou, T.-B. and Perrimon, N.
(1996). The neurogenic genes egghead and brainiac define a novel
signaling pathway essential for epithelial morphogenesis during Drosophila
oogenesis. Development
122,3863
-3879.
Gosden, R., Krapez, J. and Briggs, D. (1997). Growth and development of the mammalian oocyte. BioEssays 19,875 -882.[Medline]
Grammont, M. and Irvine, K. D. (2001). Fringe and Notch specify polar cell fate during Drosophila oogenesis. Development 128,2243 -2253.[Medline]
Halpin, D. M., Charlton, H. M. and Faddy, M. J. (1986a). Effects of gonadotrophin deficiency on follicular development in hypogonadal (hpg) mice. J. Reprod. Fertil. 78,119 -125.[Medline]
Halpin, D. M., Jones, A., Fink, G. and Charlton, H. M. (1986b). Postnatal ovarian follicle development in hypogonadal (hpg) and normal mice and associated changes in the hypothalamic-pituitary ovarian axis. J. Reprod. Fertil. 77,287 -296.[Medline]
Hamada, Y., Kadokawa, Y., Okabe, M., Ikawa, M., Coleman, J. R.
and Tsujimoto, Y. (1999). Mutation in ankyrin repeats of the
mouse Notch2 gene induces early embryonic lethality.
Development 126,3415
-3424.
Hashimoto, N., Watanabe, N., Fauruta, Y., Tamemoto, H., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. and Aizawa, S. (1994). Parthenogenetic activation of oocytes in c-mos-deficient mice. Nature 370, 68-71.[CrossRef][Medline]
Hashizume, K., Tsujii, H. and Rokutanda, M. (1995). Effects of gonadotropin administration on follicular growth and in vitro fertilization in female hypogonadal mice. Exp. Anim. 44,241 -244.[CrossRef][Medline]
Hicks, C., Johnston, S. H., diSibio, G., Collazo, A., Vogt, T. F. and Weinmaster, G. (2000). Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2. Nat. Cell Biol. 2,515 -520.[CrossRef][Medline]
Hirao, Y. and Eppig, J. J. (1997). Parthenogenetic development of Mos-deficient mouse oocytes. Mol. Reprod. Devel. 48,391 -396.[CrossRef][Medline]
Hojo, M., Ohtsuka, T., Hashimoto, N., Gradwohl, G., Guillemot,
F. and Kageyama, R. (2000). Glial cell fate specification
modulated by the bHLH gene Hes5 in mouse retina.
Development 127,2515
-2522.
Hrabe de Angelis, M., McIntyre, J., II and Gossler, A. (1997). Maintenance of somite borders in mice requires the Delta homologue Dll1. Nature 386,717 -721.[CrossRef][Medline]
Hsieh, J. J. D., Henkel, T., Salmon, P., Robey, E., Peterson, M. G. and Hayward, S. D. (1996). Truncated mammalian Notch1 activates CBF1/RBP-Jk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2. Mol. Cell. Biol. 16,952 -959.[Abstract]
Hunt, P. A. and Hassold, T. J. (2002). Sex
matters in meiosis. Science
296,2181
-2183.
Ishii, Y., Nakamura, S. and Osumi, N. (2000). Demarcation of early mammalian cortical development by differential expression of fringe genes. Brain Res. Dev. Brain Res. 7, 307-320.
Jarriault, S., Brou, C., Logeat, F., Schroeter, E. H., Kopan, R. T. and Israel, A. (1995). Signaling downstream of activated mammalian Notch. Nature 377,355 -358.[CrossRef][Medline]
Jarriault, S., Le Bail, O., Hirsinger, E., Pourquie, O., Logeat,
F., Strong, C. F., Brou, C., Seidah, N. G. and Israel, A.
(1998). Delta-1 activation of notch-1 signaling results in HES-1
transactivation. Mol. Cell. Biol.
18,7423
-7431.
Jaleco, A. C., Neves, H., Hooijberg, E., Gameiro, P., Clode, N.,
Haury, M., Henrique, D. and Parreira, L. (2001). Differential
effects of Notch ligands Delta-1 and Jagged-1 in human lymphoid
differentiation. J. Exp. Med.
194,991
-1002.
Jiang, R., Lan, Y., Chapman, H. D., Shawber, C., Norton, C. R.,
Serreze, D. V., Weinmaster, G. and Gridley, T. (1998).
Defects in limb, craniofacial, and thymic development in Jagged2 mutant mice.
Genes Dev. 12,1046
-1057.
Johnson, J., Espinoza, T., McGaughey, R. W., Rawls, A. and Wilson-Rawls, J. (2001). Notch pathway genes are expressed in mammalian ovarian follicles. Mech. Dev. 109,355 -361.[CrossRef][Medline]
Johnston, S. H., Rauskolb, C., Wilson, R., Prabhakaran, B.,
Irvine, K. D. and Vogt, T. F. (1997). A family of mammalian
Fringe genes implicated in boundary determination and the Notch pathway.
Development 124,2245
-2254.
Joyce, I. M., Pendola, F. L., O'Brien, M. and Eppig, J. J.
(2001). Regulation of prostaglandin-endoperoxide synthase 2
messenger ribonucleic acid expression in mouse granulosa cells during
ovulation. Endocrinology
142,3187
-3197.
Kimble, J. and Simpson, P. (1997). The lin-12/Notch signaling pathway and its regulation. Annu. Rev. Cell. Dev. Biol. 13,333 -361.[CrossRef][Medline]
Koch, U., Lacombe, T. A., Holland, D., Bowman, J. L., Cohen, B. L., Egan, S. E. and Guidos, C. J. (2001). Subversion of the T/B lineage decision in the thymus by Lunatic Fringe mediated inhibition of Notch-1. Immunity 15,225 -236.[Medline]
Kokubo, H., Lun, Y. and Johnson, R. L. (1999). Identification and expression of a novel family of bHLH cDNAs related to Drosophila Hairy and Enhancer of Split. Biochem. Biophys. Res. Commun. 260,459 -465.[CrossRef][Medline]
Kopan, R., Schroeter, E. H., Weintraub, H. and Nye, J. S.
(1996). Signal transduction by activated mNotch: importance of
proteolytic processing and its regulation by the extracellular domain.
Proc. Natl. Acad. Sci. USA
93,1683
-1688.
Lanford, P. J., Lan, Y., Jiang, R., Lindsell, C., Weinmaster, G., Gridley, T. and Kelley, M. W. (1999). Notch signaling pathway mediates hair cell development in mammalian cochlea. Nat. Genet. 21,289 -292.[CrossRef][Medline]
Lardelli, M. and Lendahl, U. (1993). MotchA and MotchB-Two mouse Notch homologues coexpressed in a wide variety of tissues. Exp. Cell Res. 204,364 -372.[CrossRef][Medline]
Lardelli, M., Dahlstrand, J. and Lendahl, U. (1994). The novel Notch homologue mouse Notch3 lacks specific epidermal growth factor-repeats and is expressed in proliferating neuroepithelium. Mech. Dev. 46,123 -136.[CrossRef][Medline]
Leimeister, C., Externbrink, A., Klamt, B. and Gessler, M. (1999). Hey genes: a novel subfamily of hairy- and Enhancer of split related genes specifically expressed during mouse embryogenesis. Mech. Dev. 85,173 -177.[CrossRef][Medline]
LeMaire-Adkins, R., Radke, K. and Hunt, P. A.
(1997). Lack of checkpoint control at the metaphase/anaphase
transition: A mechanism of meiotic nondisjunction in mammalian females.
J. Cell Biol. 139,1611
-1619.
Lewis, J. (1998). Notch signaling and the control of cell fate choices in vertebrates. Semin. Cell Dev. Biol. 9,583 -589.[CrossRef][Medline]
Lindsell, C. E., Shawber, C. J., Boulter, J. and Weinmaster, G. (1995). Jagged: a mammalian ligand that activates Notch1. Cell 80,909 -917.[Medline]
Lindsell, C. E., Boulter, J., diSibio, G., Gossler, A. and Weinmaster, G. (1996). Expression patterns of Jagged, Delta1, Notch1, Notch2, and Notch3 genes identify ligand-receptor pairs that may function in neural development. Mol. Cell. Neurosci. 8,14 -27.[CrossRef][Medline]
Logeat, F., Bessia, C., Brou, C., LeBail, O., Jarriault, S.,
Seiday, N. and Israel, A. (1998). The Notch 1 receptor is
cleaved constitutively by a furin-like convertase. Proc. Natl.
Acad. Sci. USA 95,8108
-8112.
Maier, M. M. and Gessler, M. (2000). Comparative analysis of the human and mouse Hey1 promoter Hey genes are new Notch target genes. Biochem. Biophys. Res. Commun. 275,652 -660.[CrossRef][Medline]
Matzuk, M. M., Burns, K. H., Viveiros, M. M. and Eppig, J.
J. (2002). Intercellular communication in the mammalian
ovary: oocytes carry the conversation. Science
296,2178
-2180.
McCright, B., Gao, X., Shen, L., Lozier, J., Lan, Y., Maguire,
M., Herzlinger, D., Weinmaster, G., Jiang, R. and Gridley, T.
(2001). Defects in development of the kidney, heart and eye
vasculature in mice homozygous for a hypomorphic Notch2 mutation.
Development 128,491
-502.
Meyuhas, O. and Klein, A. (1990). The mouse
ribosomal protein L7 gene. Its primary structure and functional analysis of
the promoter. J. Biol. Chem.
265,11465
-11473.
Moloney, D. J., Panin, V. M., Johnston, S. H., Chen, J., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K. D., Haltiwanger R. S. and Vogt, T. F. (2000). Fringe is a glycosyltransferase that modifies Notch. Nature 406,369 -375.[CrossRef][Medline]
Morrison, S. J., Perez, S. E., Qiao, Z., Verdi, J. M., Hicks, C., Weinmaster, G. and Anderson, D. J. (2000). Transient Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells. Cell 101,499 -510.[Medline]
Münsterberg, A. E. and Lassar, A. B.
(1995). Combinatorial signals from the neural tube, floor plate
and notochord induce myogenic bHLH gene expression in the somite.
Development 121,651
-660.
Nakagawa, O., Nakagawa, M., Richardson, J. A., Olson, E. N. and Srivasatava, D. (1999). HRT1, HRT2, and HRT3: a new subclass of bHLH transcription factors marking specific cardiac, somitic, and pharyngeal arch segments. Dev. Biol. 26, 72-84.
Nakagawa, O., McFadden, D. G., Nakagawa, M., Yanagisawa, H., Hu, T., Srivastava, D. and Olson, E. N. (2000). Members of the HRT family of basic helix-loop-helix proteins act as transcriptional repressors downstream of Notch signaling. Proc. Natl. Acad. Sci. USA 25,13655 -13660.[CrossRef]
O'Keefe, S. J., Wolfes, J., Kiessling, A. A. and Cooper, G. M. (1989). Microinjection of antisense c-mos oligonucleotides prevents meiosis II in the maturing mouse egg. Proc. Natl. Acad. Sci. USA 86,7038 -7042.[Abstract]
Ohtsuka, T., Ishibiashi, M., Gradwohl, F., Nakanishi, S.,
Guillemot, F. and Kageyama, R. (1999). Hes1 and Hes5 as Notch
effectors in mammalian neuronal differentiation. Embo
J. 18,2196
-2207.
Pepling, M. E. and Spradling, A. C. (1998).
Female mouse germ cells form synchronously dividing cysts.
Development 125,3323
-3328.
Pepling, M. E. and Spradling, A. C. (2001). Mouse ovarian germ cell cysts undergo programmed breakdown to form primordial follicles. Dev. Biol. 234,339 -351.[CrossRef][Medline]
Pederson, T. and Peters, H. (1968). Proposal for the classification of oocytes and follicles in the mouse ovary. J. Reprod. Fertil. 17,555 -557.[Medline]
Piddini, E. and Vincent, J.-P. (2003). Modulation of developmental signals by endocytosis:different means and many ends. Curr. Opin. Cell Biol. 15,474 -481.[CrossRef][Medline]
Rand, M. D., Grimm, L. M., Artavanis-Tsakonis, S., Patriub, V.,
Blacklow, S. C., Sklar, J. and Aster, J. C. (2000). Calcium
depletion dissociates and activates heterodimeric notch receptors.
Mol. Cell. Biol. 20,1825
-1835.
Rawls, A., McGaughey, R. W. and Wilson-Rawls, J. (2001). Developmental history of the mammalian oocyte: Insight from mouse mutations. Front. Biosci. 6,d1173 -d1185.[Medline]
Robker, R. L. and Richards, J. S. (1998).
Hormone-induced proliferation and differentiation of granulosa cells: a
coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1.
Mol. Endocrinol. 12,924
-940.
Rodgers, R. J., Lavranos, T. C., van Wezel, I. L. and Irving-Rodgers, H. F. (1999). Development of the ovarian follicular epithelium. Mol. Cell. Endocrinol. 151,171 -179.[CrossRef][Medline]
Sagata, N. (1997). What does Mos do in oocytes and somatic cells? BioEssays 19, 13-21.[Medline]
Schroeter, E. H., Kisslinger, J. A. and Kopan, R. (1996). Notch1 signaling requires ligand induced proteolytic release of the intracellular domain. Nature 393,382 -386.[CrossRef]
Schwientek, T., Keck, B., Levery, S. B., Jensen, M. A.,
Pedersen, J. W., Wandall, H. H., Stroud, M., Cohen, S. M., Amado, M. and
Clausen, H. (2002). The Drosophila gene
brainiac encodes a glycosyltransferase putatively involved in
glycosphingolipid Synthesis. J. Biol. Chem.
277,32421
-32429.
Shawber, C., Boulter, J., Lindsell, C. E. and Weinmaster, G. (1996). Jagged2: a serrate-like gene expressed during rat embryogenesis. Dev. Biol. 180,370 -376.[CrossRef][Medline]
Shimizu, K., Chiba, S., Saito, T., Kimano, K., Takahashi, T. and
Hirai, H. (2001). Manic fringe and lunatic fringe modify
different sites of the Notch2 extracellular region, resulting in different
signaling modulation. J. Biol. Chem.
276,25753
-25758.
Shutter, J. R., Scully, S., Fan, W., Richards, W. G.,
Kitajewski, J., Deblander, G. A., Kintner, C. R. and Stark, K. L.
(2000). Dll4, a novel Notch ligand expressed in arterial
endothelium. Genes Dev.
14,1313
-1318.
Spruck, C. H., de Miguel, M. P., Smith, A. P. L., Ryan, A.,
Stein, P., Schultz, R. M., Lincoln, A. J., Donovan, P. J. and Reed, S. I.
(2003). Requirement of Cks2 for the first
metaphase/anaphase transition of mammalian meiosis.
Science 300,647
-650.
Struhl, G. and Adachi, A. (1998). Nuclear access and action of Notch in vivo. Cell 93,649 -660.[Medline]
Su, Y.-Q., Wigglesworth, K., Pendola, F. L., O'Brien M. J. and
Eppig, J. J. (2002). Mitogen-activated protein kinase (MAPK)
activity in cumulus cells is essential for gonadotropin-induced oocyte meiotic
resumption and cumulus expansion in the mouse.
Endocrinology 143,2221
-2232.
Su, Y.-Q., Denegre, J. M., Wigglesworth, K., Pendola, F. L., O'Brien, M. J. and Eppig, J. J. (2003). Oocyte-dependent activation of mitogen-activated protein kinase (ERK1/2) in cumulus cells is required for the maturation of the mouse oocyte-cumulus cell complex. Dev. Biol. 263,126 -138.[CrossRef][Medline]
Svoboda, P., Stein, P., Hayashi, H. and Schultz, R. M.
(2000). Selective reduction of dormant maternal mRNAs in mouse
oocytes by RNA interference. Development
127,4147
-4156.
Swiatek, P. J., Lindsell, C. E., Franco del Amo, F., Weinmaster, G. and Gridley, T. (1994). Notch1 is essential for postimplantation development in mice. Genes Dev. 8, 707-719.[Abstract]
Tamura, K., Taniguchi, Y., Minoguchi, S., Sakai, T., Tin, Furukawa, T. and Honjo, T. (1995). Physical interaction of a novel domain of the notch receptor with the RBP-Jk transcription factor. Curr. Biol. 5,1416 -1423.[Medline]
Uyttendaele, H., Marazzi, G., Wu, G., Yan, Q., Sassoon, D. and
Kitajewski, J. (1996). Notch4/int-3, a mammary
proto-oncogene, is an endothelial cell-specific mammalian Notch gene.
Development 122,2251
-2259.
Vanderhyden, B. C., Caron, P. J., Buccione, R. and Eppig, J. J. (1990). Developmental pattern of the secretion of cumulus-expansion enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation. Dev. Biol. 140,307 -317.[Medline]
Verlhac, M.-H., De Pennart, H., Maro, B., Cobb, M. H. and Clarke, H. J. (1993). MAP kinase becomes stably activated at metaphase and is associated with microtubule-organizing centers during meiotic maturation of mouse oocytes. Dev. Biol. 158,330 -340.[CrossRef][Medline]
Verlhac, M.-H., Kubiak, J. Z., Weber, M., Geraud, G., Colledge,
W. H., Evans, M. J. and Maro, B. (1996). Mos is required for
MAP kinase activation and is involved in microtubule organization during
meiotic maturation in the mouse. Development
122,815
-822.
Vollrath, B., Pudney, J., Asa, S., Leder, P. and Fitzgerald,
K. (2001). Isolation of a murine homologue of the
Drosophila neuralized gene, a gene required for axonemal integrity in
spermatozoa and terminal maturation of the mammary gland. Mol.
Cell. Biol. 21,7481
-7494.
Wharton, K. A., Johansen, K. M., Xu, T. and Artavanis-Tsakonis, S. (1985). Nucleotide sequence from the neurogenic locus Notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell 43,567 -581.[Medline]
Weinmaster, G., Roberts, V. J. and Lemke, G. (1991). A homolog of Drosophila Notch expressed during mammalian development. Development 113,199 -205.[Abstract]
Weinmaster, G., Roberts, V. J. and Lemke, G.
(1992). Notch2: a second mammalian Notch gene.
Development 116,931
-941.
Weinmaster, G. (2000). Notch signal transduction: a real RIP and more. Curr. Opin. Gen. Dev. 10,363 -369.[CrossRef][Medline]
Williams R., Lendahl, U. and Lardelli, M. (1995). Complementary and combinatorial patterns of Notch gene family expression during early mouse development. Mech. Dev. 53,357 -368.[CrossRef][Medline]
Xue, Y., Gao, X., Lindsell, C. E., Norton, C. R., Chang, B.,
Hicks, C., Gendron-Maguire, M., Rand, E. B., Weinmaster, G. and Gridley,
T. (1999). Embryonic lethality and vascular defects in mice
lacking the Notch ligand Jagged1. Hum. Mol. Genet.
8, 723-730.
Zhang, N. A. and Gridley, T. (1998). Defects in somite formation in lunatic fringe deficient mice. Nature 394,374 -377.[CrossRef][Medline]
Zhang, N., Norton, C. R. and Gridley, T. (2002). Segmentation defects of Notch pathway mutants and absence of a synergistic phenotype in lunatic fringe/radical fringe double mutant mice. Genesis 33,21 -28.[CrossRef][Medline]
Zhao, X., Singh, B. and Batton, B. E. (1991). The role of c-mos proto-oncoprotein in mammalian meiotic maturation. Oncogene 6,43 -49.[Medline]
Zhong, T. P., Rosenber, M., Mohideen, M. A., Weinstein, B. and
Fishman, M. C. (2000). Gridlock, an HLH gene required for
assembly of the aorta in zebrafish. Science
287,1820
-1824.
Zine, A., Van De Water, T. R. and de Ribaupierre, F.
(2000). Notch signaling regulates the pattern of auditory hair
cell differentiation in mammals. Development
127,3373
-3383.