First published online 24 September 2003
doi: 10.1242/dev.00707
The role of neurotrophin receptors in female germ-cell survival in mouse and human
Norah Spears1,*,
Michael D. Molinek1,
Lynne L. L. Robinson2,
Norma Fulton2,
Helen Cameron1,
Kohji Shimoda1,
Evelyn E. Telfer3,
Richard A. Anderson2 and
David J. Price1
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
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Fig. 1. Sections of ovaries from wild-type and mutant mice at P4-5 stained with
haematoxylin and eosin. Ovaries from 50% of
TrkB–/– mice from a mixed genetic background
and all mice from the congenic C57BL/6 background have grossly abnormal
ovaries that contain greatly reduced numbers of follicles. (A)
TrkB+/+,TrkC+/+ ovary, containing many
follicles. (B) TrkB–/– ovary on C57BL/6
x 129Sv background with only few follicles remaining (arrowheads). (C)
Unaffected TrkB–/– ovary on C57BL/6 x
129Sv background, which contains as many follicles as (A). (D) Ovary from
fourth generation C57BL/6 congenic TrkB–/–
mouse, which contains large areas of cell death and only a few follicles
(arrowhead). Scale bars: 20 µm.
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Fig. 2. Ovaries of TrkC–/– mice contain normal
numbers of follicles but those of TrkB–/– mice
contain few follicles. This phenomenon is found in  50% of
TrkB–/– mice on either the mixed genetic
background or the 129/Sv congenic line, but all ovaries in the C57BL/6
congenic line are affected. Scattergram of number of oocytes in ovaries of
TrkB+/+,TrkC+/+ mice (column 1);
TrkC–/– mice (column 2);
TrkB–/– mice from the original mixed genetic
background colony (column 3); TrkB–/– mice
from the 129/Sv congenic colony (column 4); and
TrkB–/– mice from the C57BL/6 colony (column
5). In column 1, TrkB+/+, TrkC+/+
mouse on C57BL/6 background is represented with a filled square and those on
129/Sv background are represented with open triangles, other mice are of the
mixed genetic background. In column 5, filled diamonds represent mice from the
seventh generation of the C57BL/6 congenic line of
TrkB–/– mice and crosses are mice from the
fourth generation.
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Fig. 3. TrkB (full length and truncated), NT4 and BDNF
are expressed in mouse ovaries at E16, P0 and P4. Full-length TrkB is
expressed at very low levels in all ovary samples. Each RT-PCR reaction was
carried out on P0 brain and on ovary at E16.5, P0 and P4. RT-PCR was carried
out for: (1) cyclophilin; (2) full-length TrkB using equivalent
amounts of mRNA (in terms of cyclophlin expression); (3) full-length TrkB
receptor with eight times more E16 ovary mRNA than brain mRNA and five times
more P0 and P4 ovary mRNA than brain mRNA (in terms of cyclophlin expression);
(4) TrkB receptor, both truncated and full length, using equivalent
amounts of mRNA (in terms of cyclophlin expression); (5) NT4, using
equivalent amounts of mRNA (in terms of cyclophlin expression); and (6)
BDNF using equivalent amounts of mRNA (in terms of cyclophlin
expression).
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Fig. 4. TrkB is expressed in germ cells of ovaries both before and after follicle
formation in the mouse. In situ hybridisation uses a probe that recognises
truncated and full-length forms of TrkB, whereas immunocytochemistry
is specific for full length TrkB. (A-D) In situ hybridisation. (A) Using
antisense probe (staining is primarily in oogonia, arrowheads) and (B) sense
probe on E16.5 ovaries. (C) Antisense probe (staining is primarily in oocytes,
arrowheads) and (D) sense probe on P4 ovaries (D). (E-H) Immunocytochemistry.
(E) On E16 ovary with primary antibody present and (F) negative control for
E16 ovary with no primary antibody. There is little full-length TrkB present
at E16, but some expression can be seen in oogonia. (G) P0 ovary with primary
antibody present and (H) P0 ovary with no primary antibody (full-length TrkB
is strongly expressed in oocytes). Scale bars: 20 µm in A-D; 50 µm in
E-H.
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Fig. 5. Photomicrographs of sections of ovarian pieces stained with haematoxylin
and eosin. (A) Uncultured ovary at P0 containing oogonia (open arrowhead) and
primordial follicles (closed arrowhead). (B) P0 ovary cultured for 7 days,
which contains many growing, primary follicles with larger oocytes
(arrowhead). (C) P0 ovary cultured for 7 days in the presence of 100 nM K252a
(which inhibits oocyte survival). The ovary contains some oocytes and large
areas of cell death. (D) P0 ovary cultured for 7 days in the presence of 40 ng
ml–1 bFGF contains many healthy follicles. (E) P0 ovary
cultured for 7 days in the presence of 100 nM K252a and 40 ng
ml–1 bFGF. The ovary has been rescued from the effect of
K252a and contains many healthy follicles. (F) Uncultured ovary from E16.5
mouse containing oogonia (arrowhead) but no follicles. (G) E16.5 ovary
cultured in control medium for 4 days contains many follicles, all of which
are at the primary, growing stage with rounded granulosa cells (arrowhead).
(H) Ovary at P0, an equivalent age for the cultured ovary shown in G, with
follicles at the primordial, resting stage with flattened granulosa cells
(arrowhead). Scale bars: 20 µm.
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Fig. 6. K252a reduces oocyte survival in newborn mouse ovaries in culture, but does
not affect the distribution of oocyte diameters in surviving follicles. (A)
The total number of follicles in uncultured day 0 ovaries and ovaries cultured
in 0 nM, 50 nM and 100 nM K252a. Asterisks indicate significant difference
compared with control culture (D7 0 nM), P<0.005. (B) The
proportion of follicles containing oocytes of various diameters in uncultured
day 0 ovaries. (C) The proportion of follicles containing oocytes of various
diameters in ovaries cultured in 0 nM, 50 nM and 100 nM K252a. Line between B
and C shows the follicle stage that corresponds to different diameters of
oocytes.
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Fig. 7. bFGF rescues cultured newborn mouse ovaries from the effect of K252a.
Histogram of the total number of follicles in ovaries cultured in control
conditions and in 100 nM K252a, 50 ng ml–1 bFGF, and K252a
plus bFGF. Asterisk indicates significant difference compared with untreated
control culture, P<0.05.
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Fig. 8. Blocking the effects of BDNF and NT4 in culture decreases germ-cell
survival and increases germ-cell death. (A,B) The density of healthy oocytes
(A), and dying and dead oocytes (B) in ovaries cultured in control medium or
in medium containing 10 µg ml–1 anti-BDNF, 10 µg
ml–1 anti-NT4, and 10 µg ml–1 each of
anti-BDNF and of anti-NT4. Asterisk indicates significant difference compared
to untreated control culture, P<0.05.
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Fig. 9. Photomicrographs of sections of ovarian pieces stained with haematoxylin
and eosin. (A-D) Ovaries were cultured for 7 days in control medium (A),
medium containing 10 µg ml–1 anti-BDNF (B), medium
containing 10 µg ml–1 anti-NT4 (C) and medium containing
10 µg ml–1 each of anti-BDNF and of anti-NT4 (D). Areas
around the edge of ovaries cultured in the presence of antibodies had few if
any germ cells. These ovaries also often contained dying/dead oocytes (white
arrowheads).
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Fig. 10. Very few primordial follicles form in E16.5 fetal ovaries in culture and
K252a has no effect on oocyte survival in this culture system. (Ai) The
proportion of oogonia and oocytes in E16.5 ovaries cultured for four days and
in P0 ovary without culture. (Aii) Further classification of oocytes into
those contained in primordial and primary follicles, in E16.5 ovaries cultured
for four days and in P0 ovary without culture. (B) The diameters of oocytes in
E16.5 ovaries cultured in the presence or absence of 100 nM K252a.
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Fig. 11. Effect of K252a on human ovaries in culture. (A-C) Photomicrographs of
human fetal ovaries at 13-weeks gestation. (A) Uncultured ovary containing
oogonia. (B) Ovary cultured in the absence of K252a with many oogonia
surviving. (C) Ovary cultured in the presence of 100 nM K252a with few oogonia
surviving (arrowhead). Scale bars: 20 µm. (D) Density of germ cells in
uncultured ovaries, and ovaries cultured in the presence of 0 nM and 100 nM
K252a. The number of oogonia was quantified by random stage microscopy. Data
are the number of oogonia (mean±s.e.m.) per 121-point grid. A total of
18-42 grids per treatment were counted in each of five experiments. Asterisks
indicate significant difference compared with control culture (0 nM K252a),
P<0.01.
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Fig. 12. The possible effect of neurotrophins on folliculogenesis. Oogonia and
pregranulosa cells associate into primordial follicles, with flattened
granulosa cells, (a `resting' stage of development). Oocytes with too few
granulosa cells die at this point. Surviving oocytes are contained in
follicles that have enough granulosa cells to provide the oocyte with
sufficient neurotrophins. Follicles that leave the primordial stage and enter
the primary stage exhibit rounding up of granulosa cells and undergo oocyte
growth. Trk receptors have no effect on primary follicle survival.
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© The Company of Biologists Ltd 2003
