Synergistic Roles of Bone Morphogenetic Protein 15 and Growth Differentiation Factor 9 in Ovarian Function
Changning Yan,
Pei Wang,
Janet DeMayo,
Francesco J. DeMayo,
Julia A. Elvin,
Cecilia Carino,
Sarvamangala V. Prasad,
Sheri S. Skinner,
Bonnie S. Dunbar,
Jennifer L. Dube,
Anthony J. Celeste and
Martin M. Matzuk
Departments of Pathology (C.Y., M.M.M.), Molecular and Cellular
Biology (P.W., J.D., F.J.D., C.C., S.V.P., S.S.S., B.S.D., M.M.M.), and
Molecular and Human Genetics (J.A.E., M.M.M.) Baylor College of
Medicine Houston, Texas, 77030
Department of Tissue
Growth and Repair (J.L.D., A.J.C.) Genetics Institute,
Inc. Cambridge, Massachusetts 02140
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ABSTRACT
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Knockout mouse technology has been used over the
last decade to define the essential roles of ovarian-expressed genes
and uncover genetic interactions. In particular, we have used this
technology to study the function of multiple members of the
transforming growth factor-ß superfamily including inhibins,
activins, and growth differentiation factor 9 (GDF-9 or
Gdf9). Knockout mice lacking GDF-9 are infertile due to a
block in folliculogenesis at the primary follicle stage. In addition,
recombinant GDF-9 regulates multiple cumulus granulosa cell functions
in the periovulatory period including hyaluronic acid synthesis and
cumulus expansion. We have also cloned an oocyte-specific homolog of
GDF-9 from mice and humans, which is termed bone morphogenetic protein
15 (BMP-15 or Bmp15). To define the function of BMP-15 in
mice, we generated embryonic stem cells and knockout mice, which have a
null mutation in this X-linked gene. Male chimeric and
Bmp15 null mice are normal and fertile. In contrast to
Bmp15 null males and Gdf9 knockout females,
Bmp15 null females
(Bmp15-/-) are subfertile and
usually have minimal ovarian histopathological defects, but demonstrate
decreased ovulation and fertilization rates. To further decipher
possible direct or indirect genetic interactions between GDF-9 and
BMP-15, we have generated double mutant mice lacking one or both
alleles of these related homologs. Double homozygote females
(Bmp15-/-Gdf9-/-)
display oocyte loss and cysts and resemble
Gdf9-/- mutants. In contrast,
Bmp15-/-Gdf9+/-
female mice have more severe fertility defects than
Bmp15-/- females, which appear to
be due to abnormalities in ovarian folliculogenesis, cumulus cell
physiology, and fertilization. Thus, the dosage of intact
Bmp15 and Gdf9 alleles directly influences the
destiny of the oocyte during folliculogenesis and in the periovulatory
period. These studies have important implications for human fertility
control and the maintenance of fertility and normal ovarian physiology.
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INTRODUCTION
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Although important molecular events occur during all stages
of mammalian ovarian folliculogenesis, few oocyte-expressed
regulatory proteins have been identified. Our group and others have
used embryonic stem (ES) cell technology to produce mouse models with
ovarian abnormalities (for review, see Refs. 1, 2). These knockout
mouse models demonstrate defects in germ cell maintenance,
proliferation, and development [e.g. Dazla knockout mice
(3)], formation of primordial follicles [e.g. Fig
knockout mice (4)], formation of secondary follicles [e.g.
Gdf9 knockout mice (5)], formation of antral follicles
[e.g. FSHß knockout mice (6)], ovulation
[e.g. cyclooxygenase 2 (7) and progesterone receptor
knockout mice (8)], or postovulation [e.g. Mater knockout
mice (9)]. Whereas mutations in oocyte-expressed genes
(e.g. Fig
, kit receptor, and Mater) result in intrinsic
defects in the oocyte or early embryo (4, 9, 10), growth
differentiation factor 9 (GDF-9) is the only known oocyte-secreted
growth factor that is required for somatic cell function in mice
in vivo. Using Gdf9 knockout mice, we have shown
that GDF-9 is directly required for granulosa cell growth and
differentiation and indirectly for oocyte meiotic competence and
formation of a theca (5, 11, 12).
Gdf9 mRNA and GDF-9 protein are not only expressed at
the primary follicle stage but are also present in oocytes through
ovulation (13, 14, 15). Since mice lacking GDF-9 have a block at the
primary follicle stage, fail to form a theca, and eventually
demonstrate defects in meiotic competence, it was unclear from these
knockout studies whether GDF-9 also plays a role at later stages of
folliculogenesis. It was therefore important to determine the function
of GDF-9 in the periovulatory period since oocyte-secreted growth
factors had been identified to play key regulatory functions in this
period. Using recombinant mouse GDF-9, we demonstrated that GDF-9 can
regulate a diverse number of genes and processes in the periovulatory
stage, including cumulus expansion, induction of hyaluronan synthase 2,
cyclooxygenase 2, and the EP2 PGE2 receptor, and
inhibition of LH receptor and urokinase plasminogen activator (13, 15, 16). In addition, recombinant rat GDF-9 stimulates rat preantral
follicle growth and also stimulates basal estradiol production in
granulosa cells (17, 18). Thus, GDF-9 functions as a multipurpose
oocyte-secreted growth factor during the early stages of
folliculogenesis and in the periovulatory period.
Using a homology-based cloning strategy, we fortuitously cloned GDF-9
homologs from the mouse and human that we termed bone morphogenetic
protein 15 (BMP-15) (19) [also called growth differentiation factor 9B
(20)]. In addition to the 52% identity with GDF-9, BMP-15 had several
interesting features. First, Bmp15 mRNA was exclusively
expressed in oocytes in an identical pattern as Gdf9 (15, 19). Second, mouse Bmp15 and human BMP15 map to
syntenic positions on the X chromosome. Third, similar to GDF-9, BMP-15
protein lacks the mature peptide cysteine that normally forms an
intermolecular disulfide bond in the other TGFß superfamily members.
These findings suggest that BMP-15 and GDF-9 may directly interact
(i.e. form heterodimers) or functionally interact
(i.e. play redundant or antagonistic roles). Recent evidence
from studies in sheep suggests interacting roles of these proteins in
the ovary. The BMP15 gene was cloned in sheep and shown to
be mutated in Inverdale and Hanna sheep carrying
the fecundity X (FecXI and
FecXH) mutations (21). Both strains
have mutations in the mature peptide sequence. Sheep heterozygous for
these BMP15 mutations show an increased ovulation frequency
resulting in more twins and triplets. Surprisingly, sheep homozygous
for these mutations are infertile and have a block in folliculogenesis
that phenocopies the mouse Gdf9 knockout ovarian phenotype.
Thus, BMP-15 appears to be the second known oocyte-secreted growth
factor that is critical for ovarian function.
In this report, we used the previously isolated mouse Bmp15
gene sequences (19) to create male and female mice with a null mutation
in the X-linked Bmp15 gene. These Bmp15 null
female mice are viable but display reproductive defects. In addition,
we have intercrossed these Bmp15 null mice with mice
carrying a mutation in the autosomal Gdf9 gene to uncover
genetic interactions. These knockout mouse models have helped us define
the important roles of BMP-15 and GDF-9 in oocyte-somatic cell
interactions during folliculogenesis and in the periovulatory
period.
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RESULTS
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Targeted Mutation of the Bmp15 Gene in ES Cells and Generation of
Bmp15 Null Mutant Mice
We had previously isolated the mouse Bmp15 gene from a
129SvEv genomic library and shown that it was composed of two exons
with a 3.5-kb intron. Bmp15 exon 1 encodes the 17-amino acid
signal peptide and 91 amino acids of the propeptide, whereas exon 2
encodes the remaining 159 amino acids of the propeptide and the
125-amino acid mature domain. To generate a mutant allele in the
Bmp15 gene in ES cells, we used the 129SvEv genomic
sequences to construct a targeting vector to delete exon 2 (Fig. 1A
). Recombination of this targeting
vector and the endogenous Bmp15 locus was anticipated to
yield a null allele because no mature (active) BMP-15 could be
synthesized if the gene sequences encoding it were deleted. A similar
strategy had been employed to generate a null allele in the
Gdf9 locus (5).

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Figure 1. Construct to Generate a Bmp15 Mutant
Allele in ES Cells and Generation of Bmp15 Mutant Mice
A, The Bmp15 replacement targeting vector to delete exon
2 is shown. The targeting vector was electroporated into hprt-deficient
AB2.1 ES cells to produce the Bmp15 recombinant mutant
allele. The mutant allele can be distinguished from the wild-type
allele using 5'- or 3'-probes as shown. B, Southern blot analysis of
tail DNA derived from seven offspring from a litter from a mating of
XBmp15tm1Y and
XBmp15tm1X parents. Genomic DNA was
digested with PstI and analyzed as described previously
(23 ) using the 3'-probe (top panel). The probe detects a
13.0-kb wild-type band in lanes derived from wild-type (+/Y) XY males
and heterozygous (+/-) XBmp15tm1X females
and a 9.2-kb recombinant mutant band in lanes derived from null
XBmp15tm1Y (-/Y) males, heterozygous
females, and null
XBmp15tm1XBmp15tm1
(-/-) females. Southern blot analysis of the same DNA as in the
top panel using an exon 2 probe (bottom
panel) detects the 13.0-kb wild-type allele in DNA derived from
the XY or XBmp15tm1X offspring but not in
the null males or females confirming the null status of the
Bmp15tm1Zuk allele.
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The Bmp15 targeting vector was electroporated into AB2.1 ES
cells (XY), and 13 of 92 (14%) of the ES cell clones analyzed were
targeted at the Bmp15 locus and thereby null. Two of these
ES cell lines were used to produce chimeric male mice that were fertile
and transmitted the X-linked Bmp15 null allele
(Bmp15tm1Zuk or
Bmp15- or
XBmp15tm1) to females.
XBmp15tm1X (heterozygous or
Bmp15+/-) females were used to
generate XBmp15tm1Y (null) males that were
intercrossed with heterozygous females to produce
XBmp15tm1XBmp15tm1
(null or Bmp15-/-) females (Fig. 1B
, top). Using Southern blot analysis of DNA, a
Bmp15 exon 2 probe demonstrated a lack of a hybridizable
signal in the DNA derived from XBmp15tm1Y
and
XBmp15tm1XBmp15tm1
mice but not XBmp15tm1X or XY mice (Fig. 1B
, bottom). This confirmed that the
Bmp15tm1Zuk allele is a null allele.
Fertility Analysis of Bmp15 Mutant Mice
Chimeric males, null males, heterozygous females, and null females
were all viable and failed to demonstrate any gross developmental
defects. In addition, male chimeric and Bmp15 null males
were fertile, and Bmp15 null males had normal testis size
[87.58 ± 2.42 ng/testis (n = 18)] compared with wild-type
controls [87.27 ± 2.53 ng/testis (n = 11)]. The viability
of Bmp15 mutant mice and the fertility of the chimeric and
null males are consistent with the limited adult ovary-specific
expression of Bmp15 mRNA (19).
To determine whether Bmp15 plays a key ovarian function in
females, heterozygous and homozygous mutant females were mated to
males. In contrast to GDF-9, which is absolutely required for fertility
in females, Bmp15 homozygous mutants (C57/129 hybrid
background) were subfertile when bred over a 1-yr period (Table 1
). When the Bmp15 mutation
was maintained on a 129SvEv inbred background strain (in which females
are normally less fertile), the Bmp15 homozygous null
females displayed a consistent (although more severe) subfertility
compared with the Bmp15 heterozygotes (Table 1
). Both the
number of pups per litter and the number of litters per month were
reduced for the Bmp15-/- females
from either hybrid or inbred genetic backgrounds. Thus, BMP-15 plays an
important role in female reproduction in mice but is not as essential
as GDF-9 or its sheep ortholog.
Ovarian and Oocyte Physiology
To determine the cause of the subfertility in the
Bmp15-/- female mice, ovaries were
analyzed both morphologically and histologically at various time
points. In contrast to Gdf9-/-
ovaries, which were extremely small (5),
Bmp15-/- ovaries were often grossly
indistinguishable from either
Bmp15+/- or wild-type ovaries.
Histological analysis of these mutant ovaries confirmed our gross
impressions. In general, Bmp15-/-
ovaries at all ages up through 1 yr demonstrated all stages of follicle
development and multiple corpora lutea and appeared indistinguishable
from control ovaries (Fig. 2
). These
findings would be consistent with the ability of
Bmp15-/- mice to become pregnant.
However, occasional Bmp15-/- ovaries
at different ages demonstrated very few follicles and had an increased
number of zona pellucida (ZP) remnants (data not shown; see below).
Single rare Bmp15-/- and
Bmp15+/- mice had unilateral or
bilateral cysts at 1 yr of age. These findings have never been seen in
wild-type mice in our laboratory.

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Figure 2. Histological Analysis of Bmp15
Mutant Ovaries
A, Ovary from a 4-month-old Bmp15-/- 129
mouse showing normal follicular development and corpora lutea (CL). B,
Ovary from a 6-month-old Bmp15+/- 129 mouse
showing normal follicular development and corpora lutea except there is
a single follicle with two oocytes (black arrows) and
accumulation of ZP remnants (yellow arrows). C,
High-power magnification of a follicle with two oocytes from a
4-month-old Bmp15-/- 129 ovary. Additional
examples of multiple oocytes in a single follicle were seen in double
mutant mice (see below). D, Ovary from a 1-yr-old
Bmp15-/- 129 mouse showing fairly normal
follicular development and corpora lutea except for the accumulation of
PAS-positive material (arrows) and ZP remnants in the
interstitium. There is also a reduction in the number of oocytes and
follicles compared with younger mice. E, Follicle with a small, trapped
denuded oocyte (arrow) from a superovulated
Bmp15+/- C57/129 mouse. F, Follicle with a
large, trapped denuded oocyte (arrow) from a
superovulated Bmp15-/- C57/129 mouse. All
sections were stained with PAS/hematoxylin; for the scale
bars, white lines represent 100 µm;
black lines represent 200 µm.
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Since Bmp15-/- ovarian histology was
fairly normal, we subjected the
Bmp15+/- and
Bmp15-/- mice (C57/129 hybrid
background) to a pharmacological superovulation protocol and determined
the number of eggs that were ovulated. "Normal" numbers of eggs
were released by the Bmp15+/- females
(36.4 ± 3.7 eggs; n = 26 females), but
Bmp15-/- females ovulated about
two-thirds the number of oocytes as the controls (24.1 ± 3.0
eggs; n = 21 females). To uncover the cause of the reduced
ovulations, we collected and analyzed ovaries from some of these
superovulated mice. Whereas it was extremely difficult to find any
oocytes inside follicles after the PMSG and hCG treatment in the case
of the wild-type or Bmp15+/- females
(Fig. 2E
), Bmp15-/- ovaries
occasionally had denuded oocytes that were larger than normal and had
few cumulus cells surrounding the oocytes (Fig. 2F
). These findings
suggested that follicular recruitment and the number of preovulatory
follicles were likely normal in the
Bmp15-/- mice but that a percentage
of the oocytes were trapped within follicles and were not released. A
similar phenotype is also seen for the EP2 PGE2
receptor knockout mice (22) whereas an absolute failure to ovulate is
seen with cyclooxygenase 2 and progesterone receptor knockout mice (7, 8). In addition to these ovulation defects,
Bmp15-/- mice also demonstrated a
reduction of oocytes that could develop to embryos although
Bmp15+/- oocytes also appeared to
have some reduction compared with wild-type mice (Table 2
; see below).
Analysis of Gdf9 and Bmp15 mRNA in Mutant Mice
One possibility for the defects in the Gdf9 knockout
mice is that there is altered regulation of Bmp15 mRNA
whereas the less severe phenotype of the Bmp15 knockout mice
could be due to increased compensation of GDF-9. To determine whether
there were changes in the transcriptional regulation of
Bmp15 mRNA in Gdf9 knockout ovaries or vice
versa, we performed Northern blot analysis of RNA derived from
control or mutant ovaries. Gdf9 mRNA expression was
identical in control and Bmp15-/- ovaries
(Fig. 3
). In contrast, Bmp15
mRNA levels were higher in the Gdf9-/- ovary
samples compared with control ovaries (Fig. 3
). This relative increase
in Bmp15 mRNA was due to a dramatic increase in oocytes/unit
volume in the Gdf9-/- ovaries (5).
We have confirmed this by showing that four other oocyte-specific genes
also show a relative increase in mRNA levels in the
Gdf9-/- ovaries compared with
wild-type ovaries (C. Yang, P. Wang, and M. M. Matzuk, unpublished
data). Thus, the abnormalities in the Gdf9 knockout ovaries
are not due to transcriptional inactivation of the Bmp15
gene nor is there a compensatory increase of Gdf9 mRNA in
the Bmp15 knockout ovaries.

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Figure 3. Northern Blot Analysis of Gdf9 and
Bmp15 in Control and Mutant Ovaries
Analysis of the expression of Bmp15 mRNA in
Gdf9-/- ovaries and controls (top
left). Analysis of the expression of Gdf9 mRNA
in Bmp15-/- ovaries and controls
(top right). Each blot was subsequently analyzed for
expression of GAPDH mRNA as a control for RNA loading and integrity
(bottom panels).
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Characterization of
Bmp15-/-Gdf9+/-
Double Mutant Mice
To study possible genetic, physical, and functional
interactions between Bmp15 and Gdf9, we
intercrossed mice carrying the Gdf9 null mutation (5) with
the Bmp15 null mutants generated herein. Initial studies
focused on comparing the breeding of female mice homozygous for the
Bmp15 mutation and heterozygous for the Gdf9
mutation
(Bmp15-/-Gdf9+/-)
to either Gdf9+/- or
Bmp15-/- or
Bmp15+/- mutant mice. Whereas
Gdf9+/- mice appear to produce more
offspring per litter than Bmp15+/-
mice, similar litters per month were seen (Table 1
). In contrast, the
Bmp15-/-Gdf9+/-
females on a C57/129 background that gave birth produced even fewer
pups per litter (3.67 ± 0.22) compared with the
Bmp15-/- females (4.93 ±
0.25). Furthermore, the number of litters per month was further reduced
to approximately half of the controls, and four females were completely
infertile. We also saw an increased incidence of death in these
breeding
Bmp15-/-Gdf9+/-
females (4 of 21 breeding females) but not in
Bmp15-/-Gdf9+/-
females that were caged without males; we attribute these deaths
to a failure to deliver singlets and subsequent intrauterine infection.
Recent breeding of 129 inbred
Bmp15-/-Gdf9+/-
females over a 6-month period failed to produce any offspring in
contrast to the breeding of 129 inbred
Bmp15+/-Gdf9+/- females
over a 4- to 6-month period, which yielded 64 litters (0.73
litters/month) with an average litter size of 4.39 (Table 1
). These
findings for the 129 inbred
Bmp15-/-Gdf9+/-
females are even more dramatic than the
Bmp15-/-Gdf9+/-
hybrid strain data and 129 inbred
Bmp15-/- female mice that breed
poorly. Thus, by reducing the dosage of these two related
oocyte-expressed proteins, we have uncovered important genetic
interactions.
To further understand the subfertility defects of the
Bmp15-/-Gdf9+/-
females, we analyzed the ovaries histologically. Similar to the
Bmp15-/- ovaries, normal
folliculogenesis and corpora lutea could be observed in a minority of
Bmp15-/-Gdf9+/-
ovaries up through 1 yr (Fig. 4
, A and
F). However, abnormalities were observed in five of nine ovaries from
6- to 7-month-old mice, three of five 9-month-old mice, and 12 of
fourteen 11- to 12-month-old mice. These abnormalities included
decreased numbers of late-stage follicles, increased oocyte loss, and
increased ZP remnants, accumulation of periodic acid Schiff
(PAS)-positive material in the interstitium, follicles with multiple
oocytes, and absence of corpora lutea (Fig. 4
, B and C). This
progressed to the point where there were very few oocytes and follicles
in some ovaries by 1 yr of age (Fig. 4G
). Thus, these findings suggest
that BMP-15 and GDF-9 play synergistic roles in oocyte survival and
folliculogenesis.

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Figure 4. Histological Analysis of
Bmp15-/-Gdf9+/-
Ovaries and Gdf9+/- Control Ovary
AF,
Bmp15-/-Gdf9+/-
ovaries at 6 months (AC), 4 months (D and E), 1 yr (F), and 10 months
(G). Normal folliculogenesis and corpora lutea (CL) are seen in two of
these ovaries (A and F). Abnormalities in folliculogenesis, a decrease
in number of corpora lutea, interstitial cell proliferation (I),
increased oocyte loss, and multiple ZP remnants (arrows)
are seen in the other sections from independent mice (B, C, and G).
Follicles with multiple oocytes are also seen (D and E). In panel D,
two small oocytes are seen (top right) as well as two
other oocytes of differing sizes (bottom left)
surrounded by a complete layer of granulosa cells and basement
membrane. In panel E, three similar size oocytes are surrounded by a
single granulosa cell layer. H and I, Treatment of
Gdf9+/- (H) and
Bmp15-/-Gdf9+/-
(I) immature mice with PMSG (48 h) and hCG (8 h) revealed normal
cumulus expansion of the granulosa cells in the control (H,
arrow) but an absence of cumulus cells around a large
oocyte (arrow) in the
Bmp15-/-Gdf9+/-
mutant (I). Sections AG were stained with PAS/hematoxylin. Sections H
and I were stained with toluidine blue. For the scale
bars, the red lines represent 50 µm, the
white lines represent 100 µm, and the black
lines represent 200 µm. The diameter of the "fixed"
oocyte in panel I is 65 µm.
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To follow up on some of the above findings, we performed
immunohistological analysis of the ovaries that contained prominent
PAS-positive (magenta-colored) structures, which resembled ZP after
oocyte degeneration. These structures are a prominent finding in the
GDF-9-deficient mice. Ovaries from five independent mice presented in
Figs. 4
and 7
were analyzed using antibodies specific for ZP protein 1
(ZP1), 2 (ZP2), or 3 (ZP3) or a nonspecific antibody. Antibodies to
ZP1, ZP2, or ZP3 stained the ZP around intact oocytes and also detected
the prominent remnants centrally (Fig. 5
, AC), confirming that these are remnants of ZP after the oocyte had
disappeared. The nonspecific antibody failed to detect any signal in
the ovaries (Fig. 5D
) confirming the specificity of the anti- ZP
antibodies. These ZP remnants appear to be quite stable after oocyte
loss (see below).

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Figure 7. Morphological and Histological Analysis of
Bmp15-/-Gdf9-/-
Ovaries (AH)
A, Morphological analysis of large bilateral cysts (1 cm and 1.7 cm)
attached to the uterine horns of a 1-yr-old
Bmp15-/-Gdf9-/-
mouse. B, Ovary from a 5-month-old mouse showing primary (type 3)
follicles around the periphery and follicular nests that resemble small corpora lutea
(arrows) in the center. The phenotype of this ovary
resembles the findings seen in Gdf9-/-
ovaries (5 12 ). C, Ovary from a 4-month-old mouse showing increased
magenta-colored ZP remnants (arrows)
throughout the center, a sign of increased oocyte turnover. D, Ovary
from a 9-month-old showing few oocytes and a further accumulation of ZP
remnants (arrows). E, Ovary from a 9-month-old mouse
showing a central cyst, few oocytes, and increased ZP remnants. F,
Ovary from a 1-yr-old mouse showing absence of oocytes. G and H, Low
power (G) and high power (H) views of an ovary from a 4-month-old mouse
showing multiple follicles (arrows) that resemble
seminiferous tubules (T) with Sertoli-like cells. There is also
interstitial (I) cell proliferation. All ovaries are derived from
C57/129 hybrid mice. For the scale bars, the red
line represents 50 µm, and the black lines
represent 200 µm.
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Figure 5. Immunohistological Analysis of ZP Proteins
The
Bmp15-/-Gdf9+/-
ovary shown in Fig. 4B was analyzed with polyclonal antibodies to ZP1
(A), ZP2 (B), ZP3 (C), and a nonspecific (control) antibody (D). Both
ZP around intact oocytes around the periphery and the ZP remnants
centrally after oocyte loss are detected (dark staining)
with all three antibodies but not the nonspecific antibody. The
sections were counterstained with hematoxylin. The black scale
bars represent 200 µm.
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It was also a surprise to find follicles that contained multiple
oocytes (Fig. 4
, D and E). To quantitatively study these findings, we
analyzed multiple adjacent sections (810 sections per pair of
ovaries) of 129SvEv inbred ovaries from
Bmp15-/-Gdf9+/-
vs.
Bmp15+/-Gdf9+/-
mice (littermate controls). Whereas only two of eight
Bmp15+/-Gdf9+/-
mice contained follicles with multiple oocytes (two follicles total),
four of eight
Bmp15-/-Gdf9+/-
mice contained multiple oocytes (eight follicles total). Interestingly,
two
Bmp15-/-Gdf9+/-
mice and one
Bmp15+/-Gdf9+/-
mouse had follicles with three oocytes in the follicle (Fig. 4E
). These
findings are rare for wild-type inbred 129SvEv mice. Thus, the dosage
of these ligands appears to somehow alter the development of the
granulosa cell layers around individual oocytes, allowing the formation
of these double and triple oocyte follicles.
Since folliculogenesis was relatively normal in the
Bmp15-/-Gdf9+/-
ovaries at early time points, we analyzed the ability of the
Bmp15-/-Gdf9+/-
oocytes to be pharmacologically released and fertilized in
vivo. Whereas the
Bmp15-/-Gdf9+/-
mice had high numbers of oocytes released (Table 2
), only 13.8% of
these oocytes developed to embryos. Analysis of eggs from
Bmp15-/-Gdf9+/-
females that were subjected to PMSG/hCG treatment but were not mated
with males revealed the likely cause of the
Bmp15-/-Gdf9+/-
defects. Normally, cumulus cell-egg complexes from wild-type and
Gdf9+/- mice demonstrate a resilient
adhesion of cumulus cells and eggs upon removal of the complexes from
the oviduct (Fig. 6A
). In contrast,
cumulus cells fail to adhere to eggs isolated from the oviducts of
Bmp15-/-Gdf9+/-
mutant mice (Fig. 6B
). A similar finding was also seen for many of the
cumulus cell-egg complexes isolated from
Bmp15-/- mice. Furthermore,
treatment of immature
Bmp15-/-Gdf9+/-
mice with PMSG for 48 h and subsequent analysis of their ovaries
8 h after hCG injection revealed the presence of some follicles in
which cumulus expansion had not occurred or examples of large denuded
oocytes (oocytes that were completely lacking cumulus cells) (Fig. 4I
).
This finding was in contrast to
Gdf9+/- mice (Fig. 4H
). Analysis of
individual sections of ovaries from wild-type or
Gdf9+/- ovaries revealed 25
preovulatory follicles in which cumulus expansion appeared normal and
29 oocytes of antral follicles that were appropriate in size (11).
However, analysis of Bmp15-/- or
Bmp15-/-Gdf9+/-
ovaries revealed 11 of 19 antral follicles where absence of cumulus
expansion had occurred or where oocytes were larger than normal in
size. In addition to these findings, there was one case in which the
cumulus granulosa cells were seen invading the ZP. These findings were
not unique to pharmacologically treated mice but were also observed in
seven of ten 11- to 12-month-old
Bmp15-/-Gdf9+/-
mice in which denuded oocytes in antral follicles (total of seven
oocytes with cumulus cell defect) and oocytes trapped in corpora lutea
(total of eight trapped oocytes) were observed. Six of six
Bmp15+/- mice of the same age failed
to demonstrate these defects. We believe that these findings are part
of the variation in the cumulus cell adhesion/oocyte-cumulus cell
interaction phenotype. Thus, these studies demonstrate that BMP-15 and
GDF-9 play functionally redundant roles in cumulus expansion and
maintenance of a cohesive interaction between cumulus cells and oocytes
or eggs that influences subsequent fertility.

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Figure 6. Eggs Isolated from
Gdf9+/- (A) and
Bmp15-/-Gdf9+/-
(B) Mutant Mice
Eggs isolated from the oviducts of Gdf9+/-
females after PMSG and hCG stimulation were embedded in a resilient
three-dimensional extracellular matrix which contained cumulus cells
(A). In contrast, the cumulus cells of
Bmp15-/-Gdf9+/-
mice were loosely attached to the oocyte and readily fell off the
oocyte (B). Eggs were isolated from C57/129 hybrid strain mice.
|
|
Production of Mice Null for Both Bmp15 and Gdf9
One possible reason for the early block in folliculogenesis in the
Gdf9 knockout mice could be due to persistent (unopposed)
BMP-15 protein levels since the Bmp15 mRNA continued to be
expressed in these mice. Thus, to ensure that the Gdf9
knockout phenotype was due to absence of GDF-9 and not secondary to
unopposed BMP-15, mice lacking both BMP-15 and GDF-9 (i.e.
Bmp15-/-Gdf9-/-)
mice were generated. At early time points, the ovaries of these
Bmp15-/-Gdf9-/-
mice resembled the ovaries of
Gdf9-/- mice and demonstrated a
block at the type 3B primary (one-layer) follicle stage (Fig. 7B
). Furthermore, similar to
Gdf9-/- mice, unilateral and
bilateral cysts were grossly (Fig. 7A
) and microscopically (Fig. 7E
)
present. Only one cyst per ovary was present in these double mutant
mice as in the Gdf9-/- mice.
At later time points, there were a number of unique abnormalities found
in the
Bmp15-/-Gdf9-/-
ovaries. These abnormalities included increased loss of oocytes,
increased ZP remnants, complete absence of oocytes, and proliferation
of cells in the interstitium (sometimes PAS-positive) (Fig. 7
, CF),
and a rare mouse that displayed a transformation of the granulosa cells
into Sertoli-like cells (Fig. 7
, G and H) similar to mice lacking
inhibin
(23) or estrogen receptors
and ß (24). Interestingly,
accumulation of ZP remnants are seen after loss of oocytes in the
Gdf9-/- ovaries (5) but are not seen
after loss of oocytes in animal models that have blocks at later stages
such as FSHß, cyclooxygenase 2, or progesterone receptor knockouts
(6, 7, 8). It is not clear whether there are altered proteolytic
processing defects in the ovaries of mice that have blocks at the
primary follicle stage vs. blocks at later stages. Thus,
unopposed BMP-15 is not the cause of the Gdf9 knockout
phenotype but BMP-15 appears to play some additional roles along with
GDF-9 in oocyte survival.
 |
DISCUSSION
|
---|
We have used knockout mouse technology and genetic intercrosses to
define the functions of BMP-15 and its interactions with GDF-9. Our
studies have uncovered periovulatory functions of BMP-15 in
females and confirm our in vitro studies (13, 15, 16) by
showing that GDF-9 also plays a key role in this period. We have
demonstrated that hybrid and inbred Bmp15 knockout mice are
subfertile due to reduced ovulation and fertilization;
Bmp15-/-Gdf9+/-
mice demonstrate further defects in early follicle development compared
with Bmp15-/- or
Gdf9+/- mice. Previous studies had
demonstrated an important role of oocyte-secreted proteins in cumulus
expansion, hyaluronic acid synthesis signaling through the
PGE2 receptor EP2, and suppression of urokinase
plasminogen activator and LH receptor (25). Furthermore, cumulus-oocyte
complexes are known to synthesize progesterone and
PGE2. Using recombinant GDF-9, we have shown that
GDF-9 can perform all of the functions of the oocyte-secreted
protein(s) and also stimulate PGE2 through an
induction of cyclooxygenase 2 in cumulus cells and progesterone via
induction of PGE2 and the EP2 receptor. Our
in vivo studies confirm these in vitro analyses;
oocytes from
Bmp15-/-Gdf9+/-
fail to demonstrate a stable "adherence" of cumulus cells likely
due to a reduction of key periovulatory factors (e.g.
hyaluronic acid and PGE2).
Bmp15-/-Gdf9+/-
mice also have some of the features of the progesterone receptor and
cyclooxygenase 2 knockout mice [i.e. failure to release
oocytes and the presence of oocytes trapped in corpora lutea (7, 8)],
further confirming the in vivo relationship of these
factors. Since Gdf9+/- mice do not
demonstrate these defects and some
Bmp15-/- mice demonstrate some of
these defects, these findings also indicate an important role of BMP-15
in at least some of these processes.
Gdf9-/- and
Bmp15-/-Gdf9-/-
mice develop unilateral and bilateral cysts with high frequency (Ref. 5
and the current study). These cysts can become very large (Fig. 7
), and
analysis of the cystic ovaries of these mice or after regression of the
cysts demonstrates a dramatic reduction in the number of oocytes
(i.e. the cysts lead to decreased oocyte survival). We
believe that these findings in our knockout mice have important
implications in humans. Polycystic ovarian syndrome (PCOS) is a major
cause of reduced fertility in women, and our findings suggest that the
presence of cysts in the ovaries of these women could likewise lead to
increased oocyte loss through direct structural destruction or via
indirect growth factor/hormonal effects.
Unlike Bmp15+/- mice,
BMP15 heterozygous mutant sheep demonstrate increased
fertility (i.e., increased twins and triplets), suggesting
that the BMP-15 propeptide sequences (present as the sheep mutations
are in the mature peptide encoding region) are acting in a dominant
negative fashion. This BMP-15 propeptide may be somehow interfering
with GDF-9 homodimer, BMP-15 homodimer, and/or GDF-9/BMP-15 heterodimer
formation. For example, the BMP-15 propeptide may preferentially bind
to a wild-type GDF-9 propeptide monomer to cause decreased BMP-15/GDF-9
heterodimers and shift the equilibrium toward increased BMP-15
homodimers.
Interestingly, sheep homozygous for null mutations in the
BMP15 gene do not phenocopy Bmp15 knockout mice
but instead resemble Gdf9-/- mice
(i.e. homozygotes are infertile due to a block at the
primary follicle stage). How might one explain these findings? Based on
the available animal models and in vitro studies, two
different models could be evoked to explain the functions of GDF-9 and
BMP-15 in sheep vs. mice (Fig. 8
). In model A (the mouse model), GDF-9
homodimers would be the most bioactive and play the major function.
This model is based on our findings from
Gdf9-/- mice (5, 11, 12), which
display an early block in folliculogenesis, and also on the present
study on the Bmp15-/- mice (which
have a defect in late folliculogenesis and ovulation). Furthermore,
mouse GDF-9 homodimers, but not mouse BMP-15 homodimers, are active in
our mouse in vitro bioassays (13, 16). A similar situation
has been shown recently by our group in the case of the activin ßA
and ßB monomers (26); using a gene "knockin" strategy, we
demonstrated that activin ßB (which shows 63% amino acid identity in
the mature peptide sequence) can replace activin ßA for some, but not
all, functions, demonstrating that ßB is less bioactive than ßA.
However, in model B (the sheep model), BMP-15 homodimers would be
postulated to be the most bioactive compared with either GDF-9
homodimers or BMP-15/GDF-9 heterodimers. This model would explain how
the sheep BMP15 homozygous mutant phenotype mimics the
Gdf9-/- mouse ovarian phenotype.
Interestingly, mouse and sheep GDF-9 proteins are highly conserved
whereas mouse and sheep BMP-15 proteins have diverged greatly (78%
amino acid identity). Thus, it is possible that this protein divergence
has altered the biopotency of these proteins, allowing BMP15 to become
the more essential protein in sheep. Although these models assume
signaling of the ligands through the same receptor, we cannot rule out
evolutionary divergence of a common GDF-9 or BMP-15 receptor (or
multiple receptors) to permit higher affinity interaction of the sheep
receptor with BMP-15 homodimers to explain the in vivo
findings. However, recent studies demonstrate that recombinant human
BMP-15 stimulates in vitro rat granulosa cell proliferation
and decreases FSH-induced progesterone production (27). This finding
suggests that the BMP-15 receptors are conserved between species.
Future structure-function and receptor binding studies should help us
clarify the active forms of these ligands in the different mammalian
species and determine whether BMP-15, GDF-9, or both are essential for
human fertility.

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|
Figure 8. Models for the Differential Functions of BMP-15 and
GDF-9 in Mammals
A, In mice, GDF-9 homodimers appear to be the major signaling protein,
whereas BMP-15 homodimers and BMP-15/GDF-9 heterodimers appear to play
synergistic roles in the ovary. B, In sheep, BMP-15 homodimers play a
major role although we cannot rule out significant roles of GDF-9
homodimers or BMP-15/GDF-9 heterodimers. In sheep, a model in which
BMP-15/GDF-9 heterodimers are the active form could also explain the
findings. We have shown that mouse and human BMP-15/GDF-9 heterodimers
can form in vitro (N. Wolfman, unpublished data;
P. Wang and M. M. Matzuk, unpublished data). Other members of the
TGFß superfamily can also form heterodimers that are more potent than
the respective homodimers [e.g. heterodimers between
the BMP-2/4 subgroup and the BMP-5/6/7 subgroup (36 37 38 )].
|
|
 |
MATERIALS AND METHODS
|
---|
ES Cell Technology and Southern Blot Analysis
More than 20 kb of genomic sequence encompassing the two-exon
mouse Bmp15 gene were isolated from a 129SvEv genomic
library (19). These genomic sequences were used to construct a
targeting vector to mutate the Bmp15 gene in ES cells. The
targeting vector contained 2.6 kb of Bmp15 intron 1
sequence, a positive selectable marker (the PGK-hprt expression
cassette), 5.0 kb of sequence 3' to the coding portion of exon 2, and a
negative selectable marker [the MC1-tk (thymidine kinase) expression
cassette (Fig. 1A
)]. Linearized vector was electroporated into the
hprt-negative AB2.1 ES cell line, cell clones were selected in HAT
(hypoxanthine, aminopterine, and thymidine) and FIAU [1-(2'-
deoxy-2'fluoro-ß-D-arabinofuranosyl)-5-iodouracil],
and DNA from the clones analyzed by Southern blot and targeted ES cell
clones were expanded and injected into blastocysts as described
previously (23, 28, 29). Fourteen percent of the ES cell clones were
targeted at the Bmp15 locus (data not shown), and two of
these ES cell clones (Bmp15-79-D7 and
Bmp15-79-F7), which were injected into blastocysts, produced
male chimeras that successfully transmitted the mutant
Bmp15tm1Zuk allele to F1 female
offspring. F1 and F2 female heterozygotes and
F2 null male offspring were intercrossed to
produce Bmp15 null homozygotes. Chimeras were either bred to
C57BL6/J females to produce 129SvEv/C57BL6/J hybrid mice or to 129SvEv
females to produce 129SvEv inbred mice. Southern blot analysis was used
for genotype analysis of all Bmp15 mutant offspring as shown
(Fig. 1B
) and all Gdf9 mutant offspring as described
(5).
Breeding Experiments
Bmp15 heterozygous and homozygous mutant females from
both hybrid (129SvEv/C57BL6J) and inbred (129SvEv) genetic backgrounds
were bred to males of the same genetic backgrounds at 6 weeks of age
and breeding was continued for up to 1 yr. To generate the
Bmp15/Gdf9 double mutant mice, mice carrying the
Gdf9tm1Zuk mutation (5) on either
hybrid or 129SvEv inbred genetic backgrounds were bred to
Bmp15 mutant mice of similar genetic backgrounds. Breeding
of the double mutants was also initiated at 6 weeks of age.
Immunohistological Analysis
Immunohistological analysis was carried out using
epitope-selected antibodies purified from antisera of rabbits immunized
with porcine ZP proteins as previously described (30, 31). To select
ZP1-, 2-, and 3-specific antibodies that would recognize mouse ZP
proteins, we used an epitope selection method to enhance for
cross-species ZP epitopes. Antibodies recognizing all three porcine ZP
proteins were epitope selected using human ZP proteins made from cDNAs
expressed using the baculovirus expression system (32, 33). Briefly,
antibodies were incubated with each of the three human ZP proteins that
had been isolated from SF9 insect cell lines. The individual proteins
were transferred to polyvinylidenefluoride (PVDF) membrane and
incubated with antiserum. Nonspecific antibodies were washed from the
membrane and ZP-specific antibodies were eluted with 200 mM
glycine buffer, pH 2.7, which was neutralized to pH 7. Antibody
specificity to each of the ZP proteins was demonstrated by SDS-PAGE and
immunoblot analysis as previously described (32, 34, 35).
Other Methods
RNA isolation, Northern blot analysis, histological analysis,
pharmacological superovulation, and statistical methods were performed
as described previously (5, 6, 12, 13, 19) and have been described
briefly in the body of the text or the figure legends.
 |
ACKNOWLEDGMENTS
|
---|
We thank Ms. Shirley Baker for aid in manuscript preparation and
Dr. Hua Chang for help with the figures.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Martin M. Matzuk, M.D., Ph.D., Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: mmatzuk{at}bcm.tmc.edu
These studies were supported in part by the NIH Specialized Cooperative
Centers Program in Reproduction Research (Grant HD-07495) and NIH Grant
HD-33438 (to M.M.M.).
Received for publication January 5, 2001.
Revision received February 23, 2001.
Accepted for publication March 16, 2001.
 |
REFERENCES
|
---|
-
Elvin JA, Matzuk MM 1998 Mouse models of
ovarian failure. Rev Reprod 3:183195[Abstract/Free Full Text]
-
Elvin JA, Matzuk MM 2001 Control of ovarian function. In:
Matzuk MM, Brown CW, Kumar TR (eds) Transgenics in Endocrinology. The
Humana Press, Totowa, NJ, in press
-
Ruggiu M, Speed R, Taggart M, McKay SJ, Kilanowski F,
Saunders P, Dorin J, Cooke HJ 1997 The mouse Dazla gene
encodes a cytoplasmic protein essential for gametogenesis. Nature 389:7376[CrossRef][Medline]
-
Soyal SM, Amleh A, Dean J 2000 FIG(
), a germ cell-specific
transcription factor required for ovarian follicle formation.
Development 127:464554[Abstract/Free Full Text]
-
Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM 1996 Growth differentiation factor-9 is required during early ovarian
folliculogenesis. Nature 383:531535[CrossRef][Medline]
-
Kumar TR, Wang Y, Lu N, Matzuk MM 1997 Follicle stimulating
hormone is required for ovarian follicle maturation but not male
fertility. Nat Genet 15:201204[Medline]
-
Dinchuk JE, Car BD, Focht RJ, Johnston JJ, Jaffee BD,
Covington MB, Contel NR, Eng VM, Collins RJ, Czerniak PM, Gorry SA,
Trzaskos JM 1995 Renal abnormalities and an altered inflammatory
response in mice lacking cyclooxygenase II. Nature 378:406409[CrossRef][Medline]
-
Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery
CA, Shyamala G, Conneely OM, OMalley BW 1995 Mice lacking
progesterone receptor exhibit pleiotropic reproductive abnormalities.
Genes Dev 9:22662278[Abstract]
-
Tong ZB, Gold L, Pfeifer KE, Dorward H, Lee E, Bondy CA, Dean
J, Nelson LM 2000 Mater, a maternal effect gene required for early
embryonic development in mice. Nat Genet 26:267268[CrossRef][Medline]
-
Besmer P, Manova K, Duttlinger R, Huang EJ, Packer A, Gyssler
C, Bachvarova RF 1993 The kit-ligand (steel-factor) and its
receptor c-kit/W: pleiotropic roles in gametogenesis and
melanogenesis. Development 119S: 125137
-
Carabatsos MJ, Elvin JA, Matzuk MM, Albertini DF 1998 Characterization of oocyte and follicle development in growth
differentiation factor-9-deficient mice. Dev Biol 203:373384[CrossRef]
-
Elvin JA, Yan C, Wang P, Nishimori K, Matzuk MM 1999 Molecular
characterization of the follicle defects in the growth differentiation
factor-9-deficient ovary. Mol Endocrinol 13:10181034[Abstract/Free Full Text]
-
Elvin JA, Clark AT, Wang P, Wolfman NM, Matzuk MM 1999 Paracrine actions of growth differentiation factor-9 in the mammalian
ovary. Mol Endocrinol 13:10351048[Abstract/Free Full Text]
-
McGrath SA, Esquela AF, Lee S-J 1995 Oocyte-specific
expression of growth/differentiation factor-9. Mol Endocrinol 9:131136[Abstract]
-
Elvin JA, Yan C, Matzuk MM 2000 Oocyte-expressed TGF-ß
superfamily members in female fertility. Mol Cell Endocrinol 159:15[CrossRef][Medline]
-
Elvin JA, Yan C, Matzuk MM 2000 Growth differentiation
factor-9 stimulates progesterone synthesis in granulosa cells via a
prostaglandin E2/EP2 receptor pathway. Proc Natl Acad Sci USA 97:1028810293[Abstract/Free Full Text]
-
Hayashi M, McGee EA, Min G, Klein C, Rose UM, vanDuin M, Hsueh
AJW 1999 Recombinant growth differentiation factor-9 (GDF-9) enhances
growth and differentiation of cultured early ovarian follicles.
Endocrinology 140:12361244[Abstract/Free Full Text]
-
Vitt UA, Hayashi M, Klein C, Hsueh AJ 2000 Growth
differentiation factor-9 stimulates proliferation but suppresses the
follicle-stimulating hormone-induced differentiation of cultured
granulosa cells from small antral and preovulatory rat follicles. Biol
Reprod 62:370377[Abstract/Free Full Text]
-
Dube JL, Wang P, Elvin J, Lyons KM, Celeste AJ, Matzuk MM 1998 The bone morphogenetic protein 15 gene is X-linked and expressed in
oocytes. Mol Endocrinol 12:18091817[Abstract/Free Full Text]
-
Laitinen M, Vuojolainen K, Jaatinen R, Ketola I, Aaltonen J,
Lehtonen E, Heikinheimo M, Ritvos O 1998 A novel growth differentiation
factor-9 (GDF-9) related factor is co-expressed with GDF-9 in mouse
oocytes during folliculogenesis. Mech Dev 78:135140[CrossRef][Medline]
-
Galloway SM, McNatty KP, Cambridge LM, Laitinen MP, Juengel
JL, Jokiranta TS, McLaren RJ, Luiro K, Dodds KG, Montgomery GW, Beattie
AE, Davis GH, Ritvos O 2000 Mutations in an oocyte-derived growth
factor gene (BMP15) cause increased ovulation rate and infertility in a
dosage-sensitive manner. Nat Genet 25:279283[CrossRef][Medline]
-
Hizaki H, Segi E, Sugimoto Y, Hirose M, Saji T, Ushikubi F,
Matsuoka T, Noda Y, Tanaka T, Yoshida N, Narumiya S, Ichikawa A 1999 Abortive expansion of the cumulus and impaired fertility in mice
lacking the prostaglandin E receptor subtype EP(2). Proc Natl Acad Sci
USA 96:1050110506[Abstract/Free Full Text]
-
Matzuk MM, Finegold MJ, Su J-GJ, Hsueh AJW, Bradley A 1992
-Inhibin is a tumor-suppressor gene with gonadal
specificity in mice. Nature 360:313319[CrossRef][Medline]
-
Couse JF, Hewitt SC, Bunch DO, Sar M, Walker VR, Davis BJ,
Korach KS 1999 Postnatal sex reversal of the ovaries in mice lacking
estrogen receptors
and ß. Science 286:23282331[Abstract/Free Full Text]
-
Eppig JJ, Chesnel F, Hirao Y, OBrien MJ, Pendola FL,
Watanabe S, Wigglesworth K 1997 Oocyte control of granulosa cell
development: how and why. Hum Reprod 12:127132[Abstract]
-
Brown CW, Houston-Hawkins DE, Woodruff TK, Matzuk MM 2000 Insertion of inhbb into the inhba locus rescues the inhba-null
phenotype and reveals new activin functions. Nat Genet 25:453457[CrossRef][Medline]
-
Otsuka F, Yao Z, Lee T, Yamamoto S, Erickson GF, Shimasaki S 2000 Bone morphogenetic protein-15. Identification of target cells and
biological functions. J Biol Chem 275:3952339528[Abstract/Free Full Text]
-
Bradley A 1987 Production and analysis of chimaeric mice. In:
Robinson EJ (ed) Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach. IRL, Oxford. U.K., pp 113151
-
Ramirez-Solis R, Rivera-Perez J, Wallace JD, Wims M, Zheng H,
Bradley A 1992 Genomic DNA microextraction: a method to screen numerous
samples. Anal Biochem 201:331335[Medline]
-
Wood DM, Dunbar BS 1981 Direct detection of two cross-reactive
antigens between porcine and rabbit zonae pellucidae by
radioimmunoassay and immunoelectrophoresis. J Exp Zool 217:423433[Medline]
-
Wood DM, Liu C, Dunbar BS 1981 Effect of alloimmunization and
heteroimmunization with zonae pellucidae on fertility in rabbits. Biol
Reprod 25:439450[Medline]
-
Prasad SV, Mujtaba S, Lee VH, Dunbar BS 1995 Immunogenicity
enhancement of recombinant rabbit 55-kilodalton zona pellucida protein
expressed using the baculovirus expression system. Biol Reprod 52:11671178[Abstract]
-
Prasad SV, Wilkins B, Skinner SM, Dunbar BS 1996 Evaluating
zona pellucida structure and function using antibodies to rabbit 55 kDa
ZP protein expressed in baculovirus expression system. Mol Reprod Dev 43:519529[CrossRef][Medline]
-
Drell DW, Dunbar BS 1984 Monoclonal antibodies to rabbit and
pig zonae pellucidae distinguish species- specific and shared
antigenic determinants. Biol Reprod 30:445457[Abstract]
-
Drell DW, Wood DM, Bundman D, Dunbar BS 1984 Immunological
comparison of antibodies to porcine zonae pellucidae in rats and
rabbits. Biol Reprod 30:435444[Abstract]
-
Israel DI, Nove J, Kerns KM, Kaufman RJ, Rosen V, Cox KA,
Wozney JM 1996 Heterodimeric bone morphogenetic proteins show enhanced
activity in vitro and in vivo. Growth Factors 13:291300[Medline]
-
Aono A, Hazama M, Notoya K, Taketomi S, Yamasaki H, Tsukuda R,
Sasaki S, Fujisawa Y 1995 Potent ectopic bone-inducing activity of bone
morphogenetic protein-4/7 heterodimer. Biochem Biophys Res Commun 210:670677[CrossRef][Medline]
-
Kusumoto K, Bessho K, Fujimura K, Akioka J, Ogawa Y, Iizuka T 1997 Comparison of ectopic osteoinduction in vivo by recombinant human
BMP-2 and recombinant Xenopus BMP-4/7 heterodimer. Biochem
Biophys Res Commun 239:575579[CrossRef][Medline]
-
Kumar TR, Wiseman AL, Kala G, Kala SV, Matzuk MM, Lieberman MW 2000 Reproductive defects in
-glutamyl transpeptidase-deficient
mice. Endocrinology 141:42704277[Abstract/Free Full Text]
-
Nishimori K, Young LJ, Guo Q, Wang Z, Insel TR, Matzuk MM 1996 Oxytocin is required for nursing but is not essential for parturition
or reproductive behavior. Proc Natl Acad Sci USA 93:1169911704[Abstract/Free Full Text]