From the Department of Reproductive Medicine, University of California San Diego, School of Medicine, La Jolla, California 92093-0633
Received for publication, October 16, 2002, and in revised form, November 20, 2002
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ABSTRACT |
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Bone morphogenetic protein-15
(BMP-15) and growth and differentiation factor-9 (GDF-9) are members of
the transforming growth factor- Two structurally similar and oocyte-derived growth factors termed
growth and differentiation factor-9
(GDF-9)1 (1, 2) and bone
morphogenetic protein-15 (BMP-15, also called GDF-9B) (3, 4) have
recently been identified by adapting a homology-based PCR cloning and
in silico (data base search) cloning strategies using
conserved amino acid sequences of BMP subfamily members of the
transforming growth factor- The crucial role of GDF-9 in follicle development and female fertility
has recently been demonstrated by loss-of-function studies in mice in
which it was shown that female mice lacking GDF-9 are infertile due to
a block in folliculogenesis at the one-layer primary follicle stage
(8). In contrast, female mice lacking BMP-15 are subfertile and exhibit
only minimal ovarian histopathological defects (9). Therefore, it seems
that BMP-15 is a less critical factor for normal folliculogenesis than
GDF-9 in mice.
The recent discovery of naturally occurring mutations in the
bmp15 gene in domestic sheep termed Fecundity X
Inverdale (FecXI) and Hanna,
(FecXH) has provided a new insight into
understanding the physiological relevance of BMP-15 in female
reproduction (10). In FecXI ewes, a T-A
transition at nucleotide 92 substitutes a Val with an Asp at residue 31 of the mature protein. In FecXH ewes, a C-T
transition in nucleotide 67 of the bmp15 gene replaces a Glu
by a stop codon at amino acid residue 23 of the mature domain of
BMP-15, thus resulting in the synthesis, if any, of a very short
peptide, which is highly unlikely to be biologically active. Both
mutant ewes exhibit increased ovulation and lambing rates in the
heterozygotes, whereas the homozygous mutant ewes are infertile with a
phenotype that closely resembles that of GDF-9 knockout mice (10).
Therefore, there is a distinct difference in phenotypes between mice
and sheep having bmp15 gene mutations. A major difference between mice and sheep with homozygous mutations in the
bmp15 gene is that in BMP-15 mutant sheep, GDF-9 is
incapable of rescuing the defects in early follicular development,
whereas the progression of the ovarian follicles through the component
stages of folliculogenesis occurs quite normally in BMP-15 knockout
mice, suggesting that, in mice, GDF-9 may be sufficient to compensate
for the lack of BMP-15 in promoting folliculogenesis (8-10). There has
been speculation as to how the differences in the phenotype of the
BMP-15 mutant mice and sheep can be explained. One hypothesis is that
by virtue of species differences GDF-9 plays a dominant and necessary
role in mouse folliculogenesis with BMP-15 providing a dispensable supporting role, whereas in sheep BMP-15 is indispensable. It has been
noted that these differences may be associated with the poly
versus mono-ovulatory nature of mice and sheep, respectively (11). Here, we have developed an alternative hypothesis by focusing on
the difference in the point mutations in the bmp15 gene
observed in sheep as compared with the deletion of the entire second
exon of the bmp15 gene in knockout mice. In this hypothesis
we propose that the relative roles of intact GDF-9 and BMP-15 could be
similar in mice and sheep, but that the FecXI
and the FecXH mutations in the BMP-15 protein
may be able to affect GDF-9 biosynthesis in a dominant-negative
fashion, possibly manifested through BMP-15/GDF-9 heterodimer
formation, whereas the deletion of the entire second exon of the
bmp15 gene in the knockout mouse has no effect on regulating
GDF-9 biosynthesis; thus the differences in GDF-9 bioavailability may
account for the differences in the phenotype caused by the single point
mutations in BMP-15 in sheep versus the entire deletion of
the second exon of the bmp15 gene in mice. In the present
study, as a first step in evaluating these hypotheses, we investigate the intracellular interactions of BMP-15 (wild-type and mutant) and
GDF-9 and characterize the impact of these interactions on the
processing and secretion of these growth factors.
Reagents and Supplies--
Fetal bovine serum, penicillin,
streptomycin, L-glutamine, and zeocin were purchased from
Invitrogen (San Diego, CA). Anti-FLAG M2 monoclonal antibody (anti-FLAG
mAb), Anti-FLAG M2 affinity gel, and Triton X-100 were purchased from
Sigma-Aldrich Co. Anti-c-Myc polyclonal antibody (anti-Myc Ab) was from
Santa Cruz Biotechnology Inc. (Santa Cruz, CA). All restriction enzymes
were purchased from New England Biolabs Inc. (Beverly, MA).
Construction of Expression Plasmids--
Human GDF-9 cDNA
was amplified by reverse transcription-PCR (RT-PCR) from ovarian RNA
with a primer set encompassing the amino- and carboxyl termini of
prepro-GDF-9 (GenBankTM accession no. NM_005260). A PCR product of
expected size was cloned in-frame at the upstream site of the c-Myc
epitope tag of the pcDNA3.1/Zeo( Transfection and Cell Culture--
Human embryonic kidney 293T
cells (293T) were transfected with each expression plasmid using FuGENE
6 (Roche Molecular Biochemicals, Basel, Switzerland) according to the
manufacturer's instructions. Cells containing the expression plasmid
were selected under 1 mg/ml of zeocin and maintained in Dulbecco's
modified Eagle's medium (DMEM)/F12 containing 10% fetal bovine serum,
100 µg/ml penicillin, 100 µg/ml streptomycin, 2 mM
L-glutamine (PSG), and 100 µg/ml zeocin. When the cells
reached 90-100% confluency, the medium was replaced by DMEM/F12
containing PSG without zeocin. After 3-4 days of the serum-free
culture, the conditioned media (CM) were harvested.
SDS/PAGE Immunoblot Analysis and Chemical Cross-linking--
For
analysis of intracellular proteins, cells were washed with ice-cold
phosphate-buffered saline containing 1 mM
phenylmethylsulfonyl fluoride followed by lysis in radioimmune
precipitation assay buffer (Upstate Biotechnology, Lake Placid, NY)
containing Protease-Arrest (Geno Technology Inc., St Louis, MO), 1 mM EDTA, 2% SDS, and 4%
For chemical cross-linking, a non-cleavable cross-linker,
bis-sulfosuccinimidyl suberate (BS3) (Pierce, Rockford,
IL) was added to CM at a final concentration which ranged from 0.125 to
5 mM, and incubated for 2 h on ice followed by
SDS/PAGE immunoblotting analysis.
Immunoprecipitation--
Anti-FLAG monoclonal antibodies
conjugated to agarose beads (Anti-FLAG M2 affinity gel, 10 µl) and
Triton X-100 (0.1% final concentration) were added to 800 µl of CM
from BMP-15, BMP-15/GDF-9, or GDF-9 cell lines. After a 2-h incubation
at 4 °C on a rotator, proteins bound to the agarose beads were
eluted with SDS/PAGE sample buffer and subjected to SDS/PAGE
immunoblotting analysis. For immunoprecipitation of intracellular
proteins, BMP-15, BMP-15/GDF-9, or GDF-9 cells were grown to 100%
confluency in 100-mm dishes followed by lysis in a buffer containing
1% Triton X-100, Protease-Arrest and 1 mM
phenylmethylsulfonyl fluoride. Lysates were diluted 10-fold in
phosphate-buffered saline, and immunoprecipitations were carried out as
described above.
RNA Extraction and RT-PCR--
RNA was extracted by guanidium
acid-isothiocyanate-phenol-chlorform methods using TriZOL®
(Invitrogen). After the treatment of RNA with DNase I (Promega,
Madison, WI), cDNA was synthesized by reverse transcriptase
(Invitrogen). PCR was performed using MgCl2 (1.5 mM), dNTP (0.2 mM), and 2.5 units of Platinum
TaqDNA polymerase (Invitrogen) under the following
conditions: 25 cycles of denaturation at 94 °C for 30 s,
annealing at 55 °C (for GDF-9 and Statistical Analysis--
All SDS/PAGE immunoblotting data
presented here are representative examples of at least three separate
experiments. Differences in the ratios of the mature/proprotein in the
CM are shown as mean ± S.E. of at least three separate
experiments. Differences between groups were analyzed for statistical
significance using analysis of variance (SPSS Standard Version 10.0.1, SPSS Inc, Chicago, IL). A p value In order to evaluate whether there are intracellular interactions
between BMP-15 and GDF-9 following translation (i.e. the formation of heterodimers), we established stable transformant 293T
cell lines expressing either BMP-15 or GDF-9 alone or both BMP-15 and
GDF-9 simultaneously. The pBudCE4.1 expression vector was used to
ensure equal copy number of BMP-15 and GDF-9 genes in the
co-transfected cell line because this vector contains two separate
cloning sites, each having an independent promoter. To verify the
expression of BMP-15 and GDF-9 in the CM of each, the CM were subjected
to SDS/PAGE immunoblotting analysis using an anti-FLAG Ab to detect
BMP-15 and an anti-Myc Ab to detect GDF-9 (Fig.
1). Both cell lines that were transfected
with phBMP-15F or with phBMP-15F/hGDF-9M secreted BMP-15 which was
detected with the anti-FLAG Ab as three bands: the proprotein detected
at ~50 kDa and two bands of the mature protein detected at 16 and 17 kDa as previously reported (12). On the other hand, the GDF-9 and the
BMP-15/GDF-9 cell lines both secreted GDF-9, with the proprotein
migrating at ~70 kDa and mature GDF-9 migrating at ~20 kDa.
superfamily. Both molecules are
closely related in their primary structures and share a nearly
identical spatiotemporal expression pattern in the oocyte during
folliculogenesis in mammals. Here we have established a series of cell
lines, which express recombinant BMP-15, GDF-9, or both, and
investigated whether they form homodimers and/or heterodimers. We
demonstrate the first evidence that both BMP-15 and GDF-9 can form
non-covalent homodimers when expressed individually, while when both
are co-expressed BMP-15/GDF-9 heterodimers are produced. Interestingly,
when GDF-9 and BMP-15 are co-expressed the processing of both
proproteins are significantly impaired as compared with that of the
singly expressed proproteins, suggesting that the proprotein
heterodimer is less susceptible to proteolytic cleavage than the
individual homodimers. Since BMP-15 mutant sheep, called Inverdale,
exhibit severe defects in ovarian function we have also established
stable transformants expressing the mutant BMP-15 (InvBMP-15) alone or together with GDF-9. Although InvBMP-15 was previously predicted to be
unable to form homodimers, we show here that it does form non-covalent
dimers; however, the processing efficiency of InvBMP-15 proprotein is
significantly lower than wild-type BMP-15. Surprisingly, when GDF-9 is
co-expressed, the processing and secretion of InvBMP-15 is abolished,
and the processing of GDF-9 is also severely impaired, suggesting that
the heterodimers of InvBMP-15/GDF-9 proproteins are not
susceptible to proteolytic cleavage and thus degrade in the cells.
Based on these findings we propose a novel hypothesis that a decrease
in GDF-9 secretion may be involved in causing infertility in homozygous
Inverdale ewes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(TGF-
) superfamily. Like other
members of the TGF-
superfamily, GDF-9 and BMP-15 are synthesized as
preproproteins comprised of a signal peptide, a prodomain and a mature
(fully processed bioactive)-domain (1, 3, 4). Members of the TGF-
superfamily possess a proteolytic cleavage site(s) between the pro and
mature domain where a specific protease binds and separates these two
domains as an important component of the post-translational processing of these molecules (5). Importantly, all of the evidence to date shows
that for the proteolytic cleavage of TGF-
superfamily member
proproteins to occur, the proproteins must first dimerize (6). A
characteristic feature of most TGF-
superfamily members is the
presence of seven conserved Cys residues in the mature region, the
fourth of which forms the intersubunit disulfide bond responsible for
the covalent linkage of the two subunits (7). BMP-15 and GDF-9 are
unique in that the fourth conserved Cys is substituted by a Ser, thus,
it is not known whether these molecules exist as monomers or form
non-covalent dimers.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) expression vector (Invitrogen).
The GDF-9 cDNA with the Myc tag was then released by digestion with
NotI and KpnI, subcloned into one of the multiple
cloning sites of a dual expression vector, pBudCE4.1 (Invitrogen), to
form a plasmid designated as phGDF-9M. Human BMP-15 cDNA fused with
a FLAG epitope tag was released from the previously constructed
phBMP-15-FLAG (12) by digestion with SacII and
PacI, blunt-ended with T4 DNA polymerase and subcloned into
the ScaI site of the other multiple cloning site of
phGDF-9M. The resulting plasmid, designated as phBMP-15F/hGDF-9M,
contains the full structure of human BMP-15 tagged with a FLAG epitope under the control of the cytomegalovirus promoter and human GDF-9 tagged with a c-Myc epitope under the control of the EF-1
promoter. We next performed standard PCR-based site-directed
mutagenesis using phBMP-15F/hGDF-9M to make a plasmid that co-expresses
intact GDF-9 and mutant BMP-15 having a single amino acid substitution of the Ile at position 31 of the mature region to an Asp (I31D), which
mimics the mutation found in the Inverdale ewe. This plasmid was
designated as phBMP-15FI31D/hGDF-9M. To make plasmids that
singly express BMP-15 or BMP-15I31D we released the GDF-9
cDNA from phBMP-15F/hGDF-9M or phBMP-15FI31D/hGDF-9M,
respectively. The resulting plasmids were designated as phBMP-15F or
phBMP-15FI31D, respectively. The structures of all plasmids
were confirmed by DNA sequencing.
-mercaptoethanol. Cell lysates
or CM in SDS sample buffer with 4%
-mercaptoethanol were then
subjected to SDS/PAGE-immunoblotting using 12% polyacrylamide gels.
Anti-FLAG Ab were used to detect BMP-15, and anti-Myc Ab were used to
detect GDF-9. Where indicated, integrated relative densities of
individual bands were digitized by multiplying the absorbance of the
surface areas using the software package, NIH ImageJ 1.28.
-actin) or 60 °C (for
BMP-15) for 30 s, and extension at 72 °C for 30 s.
0.05 was
considered to be statistically significant.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Production of recombinant BMP-15 and GDF-9 by
BMP-15, GDF-9, and BMP-15/GDF-9 cell lines. CM from 293T cells
stably transformed with BMP-15, GDF-9 or both were subjected to
SDS/PAGE immunoblotting analysis. Anti-FLAG Ab and anti-Myc Ab were
used to detect BMP-15 (left panel) and GDF-9 (right
panel), respectively.
To determine whether BMP-15 and GDF-9 can form homodimers we subjected
the CM to chemical cross-linking using BS3 followed by
SDS/PAGE immunoblotting analysis (Fig.
2). Cross-linking of BMP-15 revealed a
homodimer of mature BMP-15 that migrated at ~34 kDa and cross-linking
of GDF-9 revealed a homodimer of mature GDF-9, which migrated at ~40
kDa (Fig. 2, lanes 2 and 6). Both BMP-15 and
GDF-9 homodimers were also clearly detectable in CM from the
BMP-15/GDF-9 cells (Fig. 2, lanes 4 and 8).
Additionally, there were also high molecular mass complexes detectable
in cross-linked samples, which migrated at 65~180 kDa. These
molecules most likely represent complexes of the mature domains bound
to the prodomains, a phenomenon that is common in the TGF-
superfamily (13-15).
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To determine whether these cells are also capable of producing
BMP-15/GDF-9 heterodimers, we used anti-FLAG Ab conjugated to agarose
beads to co-immunoprecipitate GDF-9 with BMP-15 from CM of the various
cell lines followed by SDS/PAGE immunoblotting using anti-Myc Ab to
detect GDF-9. Following co-immunoprecipitation from the CM of
BMP-15/GDF-9 cells, mature GDF-9 was clearly detectable (Fig.
3A, lane 5),
whereas no detectable GDF-9 was immunoprecipitated from the CM of GDF-9
cells (Fig. 3A, lane 6). The ability of GDF-9 to
specifically co-immunoprecipitate with BMP-15 provides evidence that
these molecules exist as heterodimers in the CM of co-transfected cells. The presence of GDF-9 in the CM of the respective cell lines
before immunoprecipitation with anti-FLAG Ab agarose was verified (Fig.
3A, lanes 2 and 3), indicating that
GDF-9 homodimers do not immunoprecipitate with anti-FLAG Ab agarose. We
also attempted to co-immunoprecipitate GDF-9 with BMP-15 from the cell
lysate of the various cell lines. In these studies we found that the GDF-9 proprotein co-immunoprecipitates with BMP-15 in the BMP-15/GDF-9 cells, but not the GDF-9 cells (Fig. 3B, lanes 5 and 6), indicating that inside the cells, the proproteins of
BMP-15 and GDF-9 form heterodimers. To further verify the existence of
the BMP-15/GDF-9 heterodimer in the CM, we subjected the CM from the
cell lines to cross-linking with BS3 followed by
co-immunoprecipitation with anti-FLAG Ab agarose and detection by
anti-Myc Ab. Again, there was no detectable GDF-9 co-immunoprecipitated
from the CM of BMP-15 cells and GDF-9 cells (Fig. 3C,
lanes 1 and 3). Co-immunoprecipitation of the
cross-linked CM from BMP-15/GDF-9 cells; however, resulted in the
detection of mature GDF-9 at ~20 kDa together with a new band at
37 kDa, which is the predicted size of the heterodimer of mature
BMP-15/GDF-9 (Fig. 3C, lane 2). The mature GDF-9
is most likely derived from noncross-linked heterodimer of BMP-15/GDF-9
under these conditions. Collectively, these data provide clear evidence
that BMP-15 and GDF-9 can heterodimerize.
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To characterize the impact of the Inverdale mutation on the production
and secretion of BMP-15, we used site-directed mutagenesis to create
recombinant human BMP-15 with an Asp at residue 31 of the mature
protein (replacing the normal Ile present in wild-type human BMP-15),
which mimics the mutation identified in the Inverdale ewe (10). It is
noteworthy that all members of the TGF- superfamily from a wide
range of species (human, sheep, mouse, rat, sea urchin, worm, cattle,
zebrafish, and chicken) contain only the conserved hydrophobic amino
acids of either Val, Leu, or Ile at the 31st or its corresponding
position of the molecules (10). The mutant BMP-15
(BMP-15I31D) was successfully produced and secreted (Fig.
4, lane 3). Moreover, cross-linking studies revealed that, in disagreement with previous predictions (10), BMP-15I31D does form homodimers, similar
to wild-type BMP-15 (Fig. 4, lane 4).
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We then investigated whether BMP-15I31D can interact with
GDF-9 in a similar fashion to wild-type BMP-15. A stable transformant cell line was established for this purpose that expresses both BMP-15I31D and GDF-9 using the same techniques as described
above. Unlike the BMP-15I31D cell line that effectively
produced and secreted BMP-15I31D (Fig.
5A, lane 2), when
BMP-15I31D was co-transfected with GDF-9, the resulting
cell lines were unable to produce and secrete detectable
BMP-15I31D into the culture media (Fig. 5A,
lane 4). Indeed, we screened 52 individual
BMP-15I31D/GDF-9 cell lines, none of which produced any
detectable amount of BMP-15I31D. In addition to the lack of
BMP-15I31D secretion from BMP-15I31D/GDF-9
cells, we also observed that the levels of mature GDF-9 secreted from
the BMP-15I31D/GDF-9 cells was lower than those of the
BMP-15/GDF-9 cells and the singly-transfected GDF-9 cells (Fig.
5A, lanes 8-10). Because BMP-15I31D
was undetectable in the CM from BMP-15I31D/GDF-9 cells, we
attempted to detect the mRNA transcripts of BMP-15I31D
and GDF-9 derived from the transfected genes by semiquantitative RT-PCR
analysis. The mRNA encoding BMP-15 or GDF-9 was not detected in
GDF-9 nor BMP-15I31D and BMP-15 cells, respectively (Fig.
5B, lanes 5-7), indicating that BMP-15 and GDF-9
mRNAs possibly transcribed from their intrinsic genes (as compared
with transfected genes) are under the detection level. However, either
or both mRNAs were clearly detected in their corresponding
transformants, and their levels were comparable among these cell lines
(Fig. 5B), demonstrating that the differences in the levels
of secreted proteins were not caused by differences in the levels of
mRNA expression.
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From the immunoblot data of the CM we also noted that there were variations in the relative levels of the proproteins secreted from the different cell lines, causing differences in the ratios of the mature/proprotein in the CM (Fig. 5C). Specifically, the ratios of the mature/proprotein of BMP-15I31D or BMP-15 in the CM of the BMP-15I31D cells or the BMP-15/GDF-9 cells, respectively, were significantly lower than that of the BMP-15 cells (Fig. 5C, left panel). This difference is most likely due to the decreased ability of the endopeptidases to cleave the proproteins to release the corresponding mature BMP-15. Additionally the cleavage of GDF-9 in the BMP-15/GDF-9 cells was also significantly impaired as compared with the singly transfected GDF-9 cells (Fig. 5C, right panel). When GDF-9 was co-transfected with BMP-15I31D the cleavage of GDF-9 was severely impaired, to levels which were ~90% lower than GDF-9 cells.
Since it appeared that the I31D mutation in BMP-15 affects the cleavage
of the BMP-15I31D as well as GDF-9 proproteins, we further
investigated whether the component proproteins and mature proteins were
also altered in the lysates of respective cell lines (Fig.
6). BMP-15 proproteins and mature
proteins were clearly detectable in the lysates from the BMP-15 cells,
BMP-15I31D cells and the BMP-15/GDF-9 cells. In contrast,
mature BMP-15I31D was not detectable in the
BMP-15I31D/GDF-9 cells yet the proprotein was clearly
detected (Fig. 6, lane 4). Additionally, the cleavage of
GDF-9 is nearly abolished in the BMP-15I31D/GDF-9 cells
(Fig. 6, lane 9), suggesting that the inhibition of the
cleavage of the GDF-9 proprotein may be the mechanism that accounts for
the low levels of mature GDF-9 secreted by the
BMP-15I31D/GDF-9 cells.
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DISCUSSION |
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BMP-15 and GDF-9 form a unique subset of the TGF- superfamily
defined by their high amino acid homology, oocyte-specific expression
pattern and the fact that they both lack the fourth Cys residue of the
seven Cys residues that are typically conserved in almost all members
of the TGF-
superfamily (1, 2, 4, 7). Because the forth Cys is
responsible for intersubunit disulfide bond formation, there has been
an argument as to whether BMP-15 and GDF-9 exist as monomers or
homodimers. It has been predicted that GDF-9 is a monomer based on its
molecular weight determined by SDS-PAGE under non-reducing conditions
(15). However, since non-covalently linked dimers migrate as monomers
in SDS-PAGE, even under non-reducing conditions, the stoichiometry of
GDF-9 still remained unproven. In the present study, we demonstrate that both BMP-15 and GDF-9 do indeed form non-covalent homodimers. Furthermore, because of the high similarity in the structure of BMP-15
and GDF-9 as well as their nearly-identical spatiotemporal expression
patterns in the oocyte, it has been speculated that BMP-15 and GDF-9
may form heterodimers (9). In this regard, the present study clearly
demonstrates that BMP-15 and GDF-9 can also heterodimerize. The
functional significance of the BMP-15/GDF-9 heterodimers remains to be
elucidated, however, this finding reminds us of our previous discovery
of activin-A (16, 17) and activin-AB (18) and of the discovery of
activin-B by Nakamura et al. (19). Our current studies show
that, like the activins, BMP-15 and GDF-9 form both homo- and
heterodimers. At present we are unaware of the relative amounts of the
BMP-15 and GDF-9 homodimers and heterodimers that are produced and
secreted from the oocyte in vivo. To answer these questions
specific assays for these molecules, similar to the specific assays
developed for inhibins A and B utilizing two-site enzyme-linked
immunosorbent methods (20, 21), need to be established.
It is well recognized that during the biosynthetic processing of
TGF- superfamily members, the proproteins have to first dimerize in
order for the proteolytic separation of the mature bioactive protein
and the pro-domain to occur (6). There is evidence that for some
members of the TGF-
superfamily, low efficiency of this processing
may serve as a possible mechanism of governing the biological activity
of these molecules in vivo. For example, the proprotein of
Vg-1 is localized to vegetal blastomeres in Xenopus embryos,
and is abundant in vegetal cells, but the processed mature form is not
readily produced and secreted (22, 23). Interestingly, the fusion of
the prodomain of BMP-2 with the mature-domain of Vg-1 has been shown to
promote production of mature Vg-1 (24). Thus, the prodomain may
determine the rate and efficiency of dimer formation and proteolytic
cleavage. An intriguing finding in the present study was that when
BMP-15 was co-expressed with GDF-9, the processing of GDF-9 as well as
BMP-15 proproteins was significantly impaired (Fig. 5C).
Since the processing of both proproteins were efficient when they were
expressed individually, these findings strongly suggest that GDF-9 and
BMP-15 proproteins form heterodimers that are less susceptible to the
proteolytic cleavage and probably undergo degradation more readily than
the homodimeric proproteins of GDF-9 and BMP-15. The more drastic
effect of defective processing of BMP-15 and GDF-9 was observed when
BMP-15I31D was co-expressed with GDF-9. In these
conditions, although the BMP-15I31D proprotein was clearly
detectable in the cell lysates, the mature BMP-15I31D
protein was undetectable in both the CM and the cell lysates. Additionally, the processing of GDF-9 and the levels of mature GDF-9 in
the CM of BMP-15I31D/GDF-9 cells was markedly lower than
that of the BMP-15/GDF-9 cells. Thus, it seems likely that the
heterodimers comprised of BMP-15I31D and GDF-9 proproteins
severely resist being processed by endoproteases and are rapidly
degraded inside the cells. Given that the expression patterns of these
two factors in the oocyte are nearly identical, we speculate that the
levels of both mature BMP-15I31D and GDF-9 proteins
secreted from the oocytes of homozygous Inverdale ewes is markedly low.
It has been shown that BMP-15 and GDF-9 mRNA expression levels in
the oocyte of Inverdale ewes are indistinguishable from those in wild
type ewes (10, 25). Our present data, however, predict that the
mutation affects the post-translational processing of the proproteins
of both BMP-15I31D and GDF-9 and subsequent secretion of
the mature molecules by the oocyte, which could not be evaluated by
in situ hybridization of the transcripts as reported in the
previous studies (10, 25).
Based on our previous findings on the in vitro biological effects of recombinant BMP-15, we have presented a model by which the levels of bioactive BMP-15 may account for the divergent phenotypes of the heterozygous and homozygous Inverdale ewes (12, 26, 27). In the heterozygotes of Inverdale ewes, the reduced level of bioactive BMP-15 results in higher levels of follicle-stimulating hormone (FSH) receptor in the granulosa cells which in turn leads to more developing healthy follicles with more luteinizing hormone (LH) receptors, resulting in precocious follicle growth and increased ovulation at earlier stages of follicular development. In the homozygotes, the entire absence of BMP-15-induced mitosis of and kit ligand expression by granulosa cells may cause the cessation of follicular development at the primary stage. This hypothesis based on the biological functions of BMP-15 determined by in vitro granulosa cell culture has strongly been supported by the facts observed in the ovaries of Inverdale ewes that, in the heterozygotes: (i) there are more healthy estrogenic follicles, (ii) the number of granulosa cells in these developing follicles is significantly smaller, (iii) these granulosa cells have a higher mean LH responsiveness at smaller follicle stages, and (iv) the corpora lutea are smaller than normal (28). In contrast, follicle development in the homozygotes is arrested at the primary follicle stage and thus they are infertile (29).
However, given that gdf9-null mice are infertile because of an arrest of follicle development at the primary stage, similar to the phenotype of Inverdale homozygotes, yet bmp15-null mice are fertile with only minimal ovarian defects, it is also possible that the decrease in the secretion of mature GDF-9, which would be predicted based on our present data, would be sufficient to cause infertility. Our present findings that the Inverdale mutation in BMP-15 inhibits the processing and secretion of GDF-9 support this possibility. According to this model, the deletion of the entire second exon (containing most of the proprotein domain) of the bmp15 gene in the BMP-15 knockout mouse (9), may prevent the interactions of BMP-15 and GDF-9 proproteins; thus GDF-9 processing and secretion would remain unaffected, resulting in the lack of a severely disrupted ovarian phenotype. To further determine whether the phenotypes caused by the BMP-15 mutations differ due to the indispensability of either BMP-15 or GDF-9 in these two species, or if indeed the phenotype of the homozygous Inverdale ewe is primarily determined by the decrease in GDF-9 processing and secretion, a line of transgenic mice with the Inverdale mutation in the bmp15 gene would need to be generated, which is currently in progress in our laboratory. Studies on BMP-15 with the Hanna mutation may also provide insight as to whether the reduction of GDF-9 is the primary cause of infertility in BMP-15 mutant sheep. Since the phenotype of the Hanna ewe is indistinguishable from the Inverdale ewe (30), if GDF-9 is a primary factor then it would be expected that FecXH-BMP-15 would also inhibit GDF-9 processing. Because the premature stop codon in FecXH-BMP-15 leaves the entire prodomain and a portion of the mature domain of BMP-15 intact, it is likely that FecXH-BMP-15 proprotein is also capable of forming heterodimers with GDF-9 proprotein, and subsequently decreasing GDF-9 processing through a similar mechanism as Inverdale BMP-15.
An intriguing similar example of a point mutation in one TGF-
superfamily member causing the inhibition of the secretion of another
TGF-
superfamily member has been reported by Thomas et
al. (31) in which they identified a causative point mutation in
the gene encoding cartilage-derived morphogenetic protein-1 (CDMP-1,
also called as GDF-5) in chondrodysplasia Grebe type (CGT) in humans.
CGT is an autosomal recessive disorder characterized by severe
abnormality of the limbs and limb joints. The mutation substitutes a
Tyr at amino acid 400 (counting from the amino terminus of the
preproprotein) for the first of seven highly conserved Cys residues
(C400Y) in the mature-domain of CDMP-1. Because of this mutation,
CDMP-1C400Y proprotein is not processed to separate the
pro-domain and mature-domain, which prevents the secretion of CDMP-1
from the cells. Of particular interest is that CDMP-1C400Y
can form heterodimers with structurally related molecules such as
BMP-2, BMP-3 and BMP-7 when they are co-expressed by the same cells.
Intriguingly, the resulting heterodimer proproteins of CDMP-1C400Y and BMPs are not secreted, thus,
CDMP-1C400Y acts as a dominant negative regulator of the
associated intact BMPs by preventing their processing and secretion
(31).
In summary we present the first evidence that BMP-15 and GDF-9 can form
homo- and heterodimers and that the intracellular interaction of these
molecules can affect the processing and secretion of the mature
proteins. Importantly we show that BMP-15I31D is secreted
and can form homodimers when singly expressed, but when it is
co-expressed with GDF-9, the processing and secretion of
BMP-15I31D is abolished and the processing and secretion of
GDF-9 is also severely impaired. These findings suggest that levels of
bioactive GDF-9 in homozygous Inverdale sheep may be markedly low,
thus, insufficient GDF-9 could be the cause, at least in part, of
infertility in homozygous Inverdale ewes. The present study, therefore,
serves an important paradigm for the potential impact of how a mutation in one gene can influence related genes at the level of
post-translational processing.
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ACKNOWLEDGEMENT |
---|
We thank Mei Wang for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant RO1 HD41494 and the NICHD, National Institutes of Health through cooperative agreement (U54HD12303) as part of Specialized Cooperative Centers Program in Reproduction Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by National Institutes of Health Fellowship Grant F32 HD41320.
§ Supported by a fellowship grant from the Lalor Foundation.
¶ To whom correspondence should be addressed: Dept. of Reproductive Medicine, University of California at San Diego, School of Medicine, 9500 Gilman Dr., La Jolla, CA 92093-0633. E-mail address: sshimasaki@ucsd.edu.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M210598200
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ABBREVIATIONS |
---|
The abbreviations used are:
GDF-9, growth and
differentiation factor-9;
Ab, polyclonal antibody;
BMP-15, bone
morphogenetic protein-15;
BS3, bis-sulfosuccinimidyl
suberate;
CDMP-1, cartilage-derived morphogenetic protein-1;
CM, conditioned media;
RT-PCR, reverse transcription-polymerase chain
reaction;
TGF-, transforming growth factor-
;
mAb, monoclonal
antibody.
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