1 Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George
Square, Edinburgh EH8 9XD, UK
2 Medical Research Council Human Reproductive Sciences Unit, Centre for
Reproductive Biology, The University of Edinburgh Chancellor's Building, 49
Little France Crescent, Edinburgh EH16 4SA, UK
3 Institute of Cellular and Molecular Biology, University of Edinburgh, Darwin
Building, Kings Buildings, Edinburgh, UK
* Author for correspondence (e-mail: norah.spears{at}ed.ac.uk)
Accepted 4 July 2003
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SUMMARY |
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Key words: Trk, Oogonia, Oocyte, Survival, Human, Mouse, Neurotrophin
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Introduction |
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Primordial follicles consist of an oocyte surrounded by flattened granulosa cells, and are considered to be at a `resting' stage of development. Follicles can remain at this stage throughout the reproductive lifespan of a female. The first sign of further development of the primordial follicle is the rounding up of granulosa cells. When follicles contain primarily rounded granulosa cells, they are considered to have entered the growth phase and are termed `primary follicles'. This process first occurs shortly after birth in the mouse.
The neurotrophins are a small family of closely related peptide factors.
Nerve growth factor (NGF) was the first to be discovered; BDNF, NT3, NT4 and
NT6 have since been identified (Snider,
1994). The neurotrophins act on both high and low affinity
cell-surface receptors. Many of the effects of the neurotrophins on cell
survival and neuronal growth are mediated by high affinity glycoprotein
tyrosine receptor kinases, or Trk receptors. Trk receptors consist of an
extracellular domain, which contains the neurotrophin-binding site, a short
transmembrane segment, and an intracellular domain that encodes a tyrosine
kinase. The neurotrophins bind selectively to the high affinity Trk receptors,
which form homodimers and autophosphorylate to trigger the intracellular
cascade (Segal and Greenberg,
1996
). There are three members of the Trk receptor family: TrkA,
the receptor for NGF; TrkB, the receptor for BDNF and NT4; and TrkC, the
receptor for NT3. The functions of truncated forms of the TrkB and TrkC
receptors, which lack the intracellular tyrosine kinase domains
(Klein et al., 1990
;
Dechant, 2001
), are unclear. In
addition to the Trk receptors, all neurotrophins bind with relatively equal,
low affinity to a membrane receptor known as p75, a member of the tumour
necrosis receptor superfamily. The p75 receptor lacks tyrosine kinase
activity, but it does appear to have signalling capabilities. It might
modulate cellular responses to the neurotrophins by enhancing the sensitivity
of the Trk receptors (Hantzopoulos et al.,
1994
), whereas in the absence of Trk receptors it can induce cell
death (Friedman, 2000
).
The neurotrophins are implicated in a variety of developmental processes at
numerous neural sites. Their best-known roles are in the regulation of cell
survival. Thus, neurons that contain one or more of the Trk receptors might
require the presence of sufficient concentrations of the appropriate
neurotrophin(s) for their continued survival. They might also be involved in
the regulation of neuronal differentiation, growth and migration
(Ghosh and Greenberg, 1995;
Segal and Greenberg,
1996
).
All three Trk receptors are expressed around the time of follicle formation
in rats and humans (Dissen et al.,
1995; Anderson et al.,
2002
). In rats, expression of TrkB mRNA increases sharply
and TrkA mRNA decreases abruptly during the period of follicle
formation whereas TrkC remains constant throughout. Expression of
NT4 mRNA increases concomitantly with that of its ligand
TrkB. In humans, the expression pattern of NT4 mRNA changes
as follicles start to form, with expression, which is predominantly in oogonia
before follicle formation, switching predominantly to the somatic pregranulosa
cells around the time of follicle formation
(Anderson et al., 2002
). Thus,
the location of NT4 mRNA production moves from the germ cell to the
somatic cell just as germ cells undergo the massive wave of apoptosis.
Together, this indicates the possible involvement of TrkB signalling in
regulating germ-cell survival as follicles form.
Here, we report evidence that TrkB plays an important role in the survival
of germ cells in mouse and human ovaries around the time of follicle
formation. We have examined the ovaries of transgenic mice with a mutation in
the catalytic domain of the TrkB and TrkC receptors
(Klein et al., 1993;
Klein et al., 1994
), and show
the results of culturing fetal and neonatal mouse ovaries and fetal human
ovaries in the presence of (1) K252a, a potent inhibitor of the Trk receptors,
and (2) blocking antibodies against NT4 and BDNF.
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Materials and methods |
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Genotyping transgenic mice
For TrkB+/ x TrkB+/
offspring, sense primer 5'-TCGCGTAAAGACGGAACATGATCC and antisense primer
5'-AGACCATGATGAGTGGGTCGCC were used to amplify a 900 bp
TrkB+/+ band, and sense primer
5'-CCAGCCTCTGAGCCCAGAAAGC and antisense primer
5'-GCTGAAGGACGCAGCGACAAT were used to amplify a
TrkB/ band of 450 bp. PCR reactions for
the TrkB+/+ band and the
TrkB/ band were performed separately. For
TrkC+/ x TrkC+/
offspring, we used the sense primer 5'-CTGAAGTCACTGGCTAGAGTCTGGG
together with antisense primers for TrkC+/+
(5'-GTCCCATCTTGCTTACCCTGAGG) and TrkC/
(5'-CCAGCCTCTGAGCCCAGAAAGC), which amplified 400 bp and 500 bp bands,
respectively (Schimmang et al.,
1995
). PCR reactions for the TrkC+/+ band and
the TrkC/ band were performed
simultaneously.
Human fetal ovaries
Human fetal ovaries were obtained following medical termination of
pregnancy. Women gave written consent according to national guidelines
(Polkinghorne, 1989) and the study was approved by the Lothian
Paediatrics/Reproductive Medicine Research Ethics SubCommittee. Termination of
pregnancy was induced by treatment with mifepristone (200 mg orally) followed
48-hours later by prostaglandin E1 analogue (Gemeprost, Beacon
Pharmaceuticals, Tunbridge Wells, UK) 1 mg 3-hourly per vaginam. None of the
terminations were for reasons of fetal abnormality, and all fetuses appeared
morphologically normal. Gestational age was determined by ultrasound
examination prior to termination and confirmed by subsequent direct
measurement of foot length.
Mouse ovary cultures
Neonatal C57BL/6 x CBA/Ca mice were killed by decapitation. Fetal
ovaries were obtained from pregnant females killed by cervical dislocation
16.5 days after mating. Ovaries were removed aseptically and placed in watch
glasses containing Liebovitz L-15 dissecting medium (Gibco-BRI, Renfrew, UK)
supplemented with 0.3% (w/v) bovine serum albumin (BSA) (Fraction V, Sigma,
Poole, UK). Tissue surrounding the ovaries was removed using sterile needles.
Freshly dissected newborn ovaries were bisected using a sterile scalpel blade.
In the first cultures with K252a alone (Calbiochem, Nottingham, UK) ovaries
were subsequently halved again using fine-gauged needles. In the cultures with
K252a and bFGF (R&D Systems, Abington, UK) and in those with the anti-NT4
and anti-BDNF blocking antibodies (Sigma), ovary halves were used because the
quarter ovaries used in the earlier experiments were more difficult to handle
and process for histological analysis. Whole ovaries were used with embryonic
day 16.5 (E16.5) mice, as it was not possible to cut the E16.5 ovaries cleanly
because of fragility of the tissue. Tissue was either fixed in Bouin's for
analysis (uncultured control) or cultured. Culture pieces were placed on a
polycarbonate membrane on the base of a 96-well plate (Iwaki, Japan). Wells
contained 100 µl of pregassed medium overlaid with 100 µl of silicone
fluid (Gibco-BRL). Ovarian pieces were cultured in -MEM (Gibco-BRI)
supplemented with ascorbic acid (28 µM) and 0.3% (w/v) BSA, with additions
as detailed below. The tissue was cultured in a humidified incubator (5%
CO2, 37°C). Half of the used medium (50 µl) was exchanged
for fresh medium every other day for the duration of the culture period. Upon
fixation, ovarian pieces were washed in PBS containing polyvinyl pyrrolidone
(3 mg ml1) to remove any medium and fixed for 1.5-2 hrs in
Bouin's solution.
Human ovary cultures
Ovaries were dissected free of adherent tissues using sterile technique,
bisected longitudinally and cut into 0.5 mm-thick slices. Samples of
fresh tissue were fixed for histological analysis. The remaining tissue
fragments were cultured on 0.4 µm pore Millicell CM filters (Millipore,
Bedford, MA, USA) in a 24-well plate (Transwell, Costar, High Wycombe, UK).
Medium (0.4 ml) was added to each well to just cover the tissue fragments. Any
remaining wells were partially filled with medium to maintain humidity in the
culture vessel. The medium comprised
MEM containing 3 mg
ml1 BSA, 100 IU ml1 penicillin, 100 µg
ml1 streptomycin sulphate, 0.125 µg ml1
amphotericin, 5 µg ml1 insulin, 5 µg
ml1 transferrin, 5 µg ml1 sodium
selenite, 2 mM glutamine and 2 mM pyruvate (all chemicals supplied by Sigma).
Ovaries were cultured in the presence or absence of K252a. Because K252a was
reconstituted in dimethylsulfoxide (DMSO), the equivalent amount of DMSO was
added to control wells. The cultures were maintained at 37°C in a
humidified incubator under 5% CO2 in air for 48 hours and fixed for
histological analysis at the end of the culture period.
Histological assessment of mouse ovaries
After fixation in Bouin's, ovaries or ovarian pieces were embedded in wax
and 5 µm sections cut. Every third (cultured ovarian piece) or fifth (in
vivo ovaries) section was analysed. Individual images were captured using the
Leica Q5001W digital imaging microscope (Leica Microsystems, Milton Keynes,
UK) using a 40xobjective. Healthy oocytes containing a visible germinal
vesicle were counted. In addition, in the experiment in which NT4 and BDNF
activity was inhibited, a count was made of dead and dying oocytes. In some
culture experiments, the maximum and minimum diameters of each oocyte were
measured. All analyses were carried out blind.
Histological assessment of human ovaries
Sections of tissue were analysed to determine the density of germ cells in
the ovary. Analysis was carried out blind using the Area Fraction Probe in the
Stereologer software programme (Systems Planning and Analysis Inc, Alexandria,
VA, USA) as previously described (Sharpe
et al., 2002). A 121-point graticule was used to count the number
of germ cells within a frame: only cells whose nuclei lay beneath the
intersections on the grid were counted. Between 18 and 42 frames were used on
each ovary piece, as determined by the programme. Tissue sections were at
least 20 µm apart to ensure that no cell was counted twice. Data are
presented as number of germ cells per frame.
RT-PCR
Ovaries were dissected from E16.5, postnatal day 0 (P0) and P4 C57BL/6
x CBA/Ca F1 mice, frozen in liquid nitrogen and mRNA subsequently
extracted using a Quickprep micro mRNA purification kit (Pharmacia, St.
Albans, UK). Brain tissue was collected from mice at P0. cDNA was prepared
from mRNA using random primers (Promega, Southampton, UK). Separate PCR
reactions were then carried out for cyclophilin, TrkB, NT4 and BDNF. Two
separate TrkB reactions were carried out. The first set of primers were to the
tyrosine kinase domain of the gene and, thus, recognised only full length
transcripts of TrkB, the second were to the ligand-binding domain and
recognised both full-length and truncated TrkB receptors. The following
primers were used:
In situ hybridisation
Ovaries from E16.5 and P4 C57BL/6xCBA/Ca F1 mice were fixed for 30
minutes in freshly-made 4% paraformaldehyde/PBS and embedded in wax. Sections
(6 µm) were cut and mounted on TESPA-coated slides. Slides were then
dewaxed, treated in proteinase K (20 µg ml1 for 2 minutes
at 37°C) and hybridised with digoxigenin-labelled riboprobes. The probe,
which was cloned into pBluescript (Stratagene, La Jolla, CA, USA), has been
described previously (Klein et al.,
1990) and recognised both truncated and full length TrkB. For
antisense probes, plasmids were digested with EcoRI (Roche, Lewes,
UK) and transcribed in vitro with T7 (Roche). For sense probes, plasmids were
digested with XhoI (Roche) and transcribed in vitro with T3 (Roche).
Probes were labelled with digoxygenin using a DIG RNA-Labeling Mix (Roche) and
then cleaned with 70% ethanol. The probe was revealed with an anti-digoxigenin
alkaline phosphotase antibody (Roche) (100 µl made up to 50 ml with
ddH2O and left overnight at 4°C). Colour detection was carried
out the following day in nitro blue tetrazolium
chloride/5-bromo-4-chloro-3-indlyl phosphate, toluidine salt (Roche), with
levamisole (Vector, Peterborough, UK). Slides were counterstained with nuclear
fast red (Vector).
Immunocytochemistry
Ovaries were fixed in Bouin's fixative for 1 hour then transferred to a 70%
ethanol/eosin solution and embedded in wax. Sections (5 µm) were cut and
mounted on electrostatically charged slides (BDH Laboratory Supplies), dried
overnight in a 60°C oven and dewaxed. Endogenous peroxidases were quenched
with a 3% hydrogen peroxidase solution in methanol for 30 minutes at room
temperature. Immunocytochemistry was performed as described
(Anderson et al., 2002).
Briefly, slides were blocked with 20% normal donkey serum (NDS; Diagnostics
Scotland, Carluke, UK) in TBS containing 5% BSA and 8 drops avidin solution
per ml (Avidin/Biotin Blocking Kit, Vector) for 30 minutes at room
temperature. Slides were blocked using biotin from the same kit in the same
way as avidin. Chicken IgY primary antibody specific to full-length TrkB was
diluted 1/10 in TBS/BSA/NDS, applied to the slides and incubated overnight at
4°C (Anti-TrkB In pAb, Promega, UK). Biotinylated donkey anti-chicken IGY
secondary antibody was diluted 1/500 in TBS/BSA/NDS, applied to the slides and
incubated at room temperature for 30 minutes, with avidin biotin horseradish
peroxidase linked complex (DAKO) applied according to the manufacturers
instructions. Bound antibody was visualised using 3,3'-diaminobenzidine
tetrahydrochloride (DAKO). Sections were counterstained with haematoxylin.
Statistics
Data from mouse in vivo ovary counts were analysed with Mann-Whitney U
tests. Total counts of mouse and human cultured ovaries were analysed with
probability values (P) of differences in oocyte numbers determined by
analysis of variance: where appropriate, paired comparisons were made using
Student's t-test. Where the data did not have a normal distribution,
a Kruskal-Wallis test was used. The Kolmagorov Smirnov test was used to
compare differences in proportions of oocytes with varying diameters in the
cultured mouse ovaries.
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Results |
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Expression of TrkB, NT4 and BDNF mRNA and protein
Ovaries were obtained from female mice at E16.5, P0 and P4. RT-PCR showed
that TrkB (both full length and truncated), NT4 and
BDNF were expressed in mice at E16.5 (prior to the start of follicle
formation), at P0 (in the middle of follicle formation) and at P4 (when
follicle formation is complete) (Fig.
3). Full-length TrkB was present at very low levels at
all times, compared to expression in a similar amount of brain tissue (as
determined by equivalent expression of cyclophilin)
(Fig. 3, lane 2), but when more
ovary tissue was used in the reaction, the presence of the full length form in
the ovary was seen clearly (Fig.
3 lane 3). By contrast, the PCR reaction that detected both
full-length and truncated forms of TrkB showed expression at high
levels in brain and in ovaries at all ages
(Fig. 3, lane 4). Thus,
truncated TrkB was expressed in the ovary at a much higher level than
full-length TrkB.
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Culture of fetal human ovaries in the presence of a Trk receptor
blocker
Ovaries from five fetuses, ranging from 13 to 16 weeks of gestation, were
cultured in 0 nM or 100 nM K252a. At these ages, ovaries contained oogonia
only (Fig. 11A). Even in
control medium, follicles did not form but oogonia survived
(Fig. 11B). In ovaries
cultured in 0 nM K252a, a mean of 78% of oogonia survived after 48 hours in
culture, whereas only 36% survived when ovaries were cultured in the presence
of 100 nM K252a (P=0.01; Fig.
11C,D).
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Discussion |
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In the original mixed-genetic background colony of
TrkB/ mice (C57BL/6 x 129/Sv),
50% of ovaries contained reduced populations of oocytes, whereas the
remainder appeared normal. Examination of congenic strains of
TrkB/ mice based on either 129/Sv or C57BL/6
lines of mice showed that, as with the original colony,
50% of the
ovaries of the 129/Sv congenic TrkB/ mice
had a normal complement of follicles, but that ovaries of all
TrkB/ C57BL/6 congenic mice had greatly
reduced numbers of follicles. By contrast, the ovaries of all
TrkC/ mice were normal. These findings
indicate that TrkB is an important factor in oocyte survival. The fact that,
on certain backgrounds, its loss does not always have a significant effect
indicates that other factors are also likely to be involved in oocyte
survival. The efficacy of these factors might vary with background, being low
in C57BL/6 and high in 129Sv mice. Similar background effects have been found
in the development of transgenic
3 Connexin mice
(Gong et al., 1999
).
TrkB/ offspring have retarded development in general and die within the first 10 days of birth. The paucity of oocytes in TrkB/ mice was not caused by this general retardation because 50% of mice on the original mixed background and of the 129/Sv congenic mice had normal ovaries but retarded general development. In addition, TrkC/ mice exhibited similarly retarded development but their ovaries were unaffected.
Ovaries were cultured either prior to (human), or during and shortly after
(mouse) follicle formation, in the presence or absence of K252a. K252a is an
indole carbazole and a potent, specific inhibitor of the intracellular
protein-kinase domain of the Trk receptors
(Tapley et al., 1992). K252a
dramatically inhibited oocyte survival in newborn mouse ovaries in culture,
inducing a loss of 85% of germ cells. In other systems, K252a is reported to
block the activity of Trk receptors but not other tyrosine kinase receptors at
the doses used here (Tapley et al.,
1992
). Evidence for this in the ovary was obtained by adding bFGF,
which acts via a non-Trk tyrosine kinase receptor, to cultures containing
K252a. bFGF rescued ovaries from the effects of K252a, indicating that K252a
did not block bFGF receptors. These findings also demonstrate that ovarian
cells are responsive to bFGF. It is conceivable that bFGF is another survival
factor for oocytes acting in concert with neurotrophins.
RT-PCR showed that mRNA encoding NT4 and BDNF are both present throughout
the period of follicle formation in the mouse. We have already shown this is
the case in humans, where production of NT4 mRNA moves from a
germ-cell to a somatic-cell location as follicles form
(Anderson et al., 2002).
Although NT4 expression increases in rat follicles as they start to
form (Dissen et al., 1995
), the
fertility of NT4/ mice appears normal
(Conover et al., 1995
). It is
not possible to examine the fertility of
BDNF/ mice because, like
TrkB/ mice, they die shortly after birth
(Conover et al., 1995
).
The combined inhibition of NT4 and BDNF activities in culture using blocking antibodies lowered oocyte survival, but blocking either ligand alone had no detectable effect. Dead and dying oocytes were rare in control ovaries, but significantly increased in all treated ovaries, irrespective of whether blocking antibodies were added singly or in combination. The lack of a significant effect of blocking either BDNF or NT4 alone on oocyte survival indicates that the ligands are able to compensate for each other to a large extent. The combined addition of anti-BDNF and anti-NT4 was less effective than the addition of K252a, probably because the antibodies were less able to penetrate the tissue. This would explain the variation in the extent of oocyte loss from region to region within the treated ovaries. A less likely explanation is that other, as yet unidentified, ligand(s) are involved.
Newborn ovary cultures supported development of follicles from the
primordial to the primary stage. By contrast, culture of E16.5 mouse ovaries
supported formation of follicles from oogonia such that follicles formed
directly at the primary stage, bypassing the primordial stage of development.
K252a had no effect in this system, indicating that Trk receptors do not play
a role in the survival of primary follicles. We conclude, therefore, that
K252a inhibits the survival of primordial follicles in newborn ovary cultures.
Thus, Trk receptors appear to play a role in the survival of follicles at the
primordial but not the primary stage of development. Although we found no
indication that neurotrophins affect primary follicle survival, they do appear
to influence follicle function at that stage:
NGF/ mice have fewer follicles leaving the
primordial follicle pool and undergoing growth initiation
(Dissen et al., 2001), whereas
early postnatal rat ovaries cultured with NGF have increased numbers of
follicle stimulating hormone receptors
(Romero et al., 2002
).
The neurotrophins and their receptors also play a role in later ovarian
function. TrkA and NGF are involved in the regulation of ovulation
(Dissen et al., 1996) and BDNF
might be involved in oocyte maturation in antral follicles
(Seifer et al., 2002
). In
addition, there is recent evidence that neurotrophins play a role in testis
development. TrkA/ and
TrkC/ male foetuses have reduced numbers of
germ cells and impaired seminiferous tubule development compared to wild-type
mice (Cupp et al., 2002
).
Similarly, in human fetal testes, Trk-receptor signalling is involved in the
regulation of germ cells and peritubular cells
(Robinson et al., 2003
).
TrkB was present in mouse ovaries throughout the period of follicle
formation, with mRNA (both truncated and full length) and protein (full length
only) located primarily in the germ cells. Immunocytochemistry of full-length
TrkB was strongest in the oocytes of P0 ovaries. It is likely that the effect
of K252a on germ-cell survival shown here is caused by inhibition of TrkB
function. It is less likely that it results from either inhibition of TrkC
receptors, given the absence of any ovarian phenotype in
TrkC/ mice, or inhibition of TrkA receptors,
because expression of these virtually disappears as follicles form in the
rodent (Dissen et al., 1995).
Our data do not exclude a role of TrkA and TrkC in oocyte survival, because
these receptors might become more important in the absence of TrkB signalling.
This could be investigated in double mutants.
Truncated TrkB mRNA appeared to be more abundant than full-length
TrkB mRNA in ovaries from E16.5 to P4. This pattern appears to be
common in non-neuronal tissues (Wetmore
and Olson, 1995) but the reason is unclear. The truncated
receptors lack a tyrosine-kinase domain, and their effects are not well
understood. However, the effects of mutations and antagonists on follicle
survival described here are caused by interference with the low abundance,
full-length Trk receptors. TrkB/ mice have a
mutation in the tyrosine-kinase domain. Whereas full length TrkB is not
expressed in these mice, examining the head of newborn mice showed that levels
of truncated TrkB receptors remained unchanged
(Klein et al., 1993
).
Similarly, K252a blocks the tyrosine-kinase function of the Trk receptors, but
does not interfere with potential activity of Trk receptors outwith the
tyrosine-kinase domain. The role of the truncated receptors in the ovary
remains to be determined.
It is unclear why some follicles survive in
TrkB/ mice. In only two instances, both in
C57BL/6 congenic mice, were ovaries with no follicles found. In all other
cases, some follicles survived. Lundy et al.
(Lundy et al., 1999) showed
that primordial follicles contain a variable number of somatic cells, with the
number of granulosa cells in sheep primordial follicles ranging from three to
52. It has been suggested that oocytes contained in primordial follicles
require a particular number of associated granulosa cells for their continued
survival (Sawyer et al.,
2002
). It is possible that an oocyte needs sufficient granulosa
cells to sequester sufficient neurotrophins for its continued survival.
Surviving oocytes in ovaries of TrkB/ mice
would, therefore, be the ones that have a sufficient number of granulosa cells
attached (Fig. 12). In these
instances, the increased numbers of granulosa cells might produce an increased
amount of either neurotrophins (which might signal through other Trk
receptors) or other factors that might compensate for a lack of neurotrophin
signalling (possibly bFGF).
|
In conclusion, the data demonstrate that TrkB and its ligands are present
in mouse ovaries as follicles form, as we have previously shown in human
ovaries (Anderson et al.,
2002). Our studies of mutant mice indicate that TrkB plays an
important role in oocyte survival. Culture of mouse and human ovaries with a
potent inhibitor of all Trk receptors, K252a, decreases germ-cell survival.
This effect appears to be specific to oogonia and primordial follicles and
does not occur in primary follicles. Blocking NT4 and BDNF also decreased
germ-cell survival. Together, our results point to a role of the TrkB receptor
and its ligands in the regulation of germ-cell survival at the oogonial and
primordial follicle stage in mammalian ovaries. This pathway is therefore
crucial to the determination of female reproductive lifespan.
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ACKNOWLEDGMENTS |
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