The Bone Morphogenetic Protein 15 Gene Is X-Linked and Expressed in Oocytes
Jennifer L. Dube,
Pei Wang,
Julia Elvin,
Karen M. Lyons,
Anthony J. Celeste and
Martin M. Matzuk
Department of Tissue Growth and Repair (J.L.D., A.J.C.)
Genetics Institute, Inc. Cambridge, Massachusetts 02140
Departments of Orthopaedic Surgery and Biological Chemistry
(K.M.L.) University of California Los Angeles School of
Medicine Los Angeles, California 90095
Departments of
Pathology (P.W., J.E., M.M.M.), Cell Biology (M.M.M.), and Molecular
and Human Genetics (J.E., M.M.M.) Baylor College of Medicine
Houston, Texas 77030
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ABSTRACT
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We have taken advantage of the sequence
relationships among the bone morphogenetic proteins (BMPs) to identify
the mouse Bmp15 and human BMP15 genes. The
392-amino acid prepropeptides encoded by these BMP genes exhibit
significant homology to each other, although the 70% identity observed
between the 125-amino acid mature peptides is considerably lower than
that seen in comparisons of other mouse and human orthologs. Both genes
share a common structural organization and encode mature peptides that
lack the cysteine residue normally involved in the formation of a
covalent dimer. In addition, mouse Bmp15 and human
BMP15 map to conserved syntenic regions on the X
chromosome. We demonstrate, through a combination of Northern blot and
in situ hybridization analyses, that mouse
Bmp15 is expressed specifically in the oocyte beginning at
the one-layer primary follicle stage and continuing through ovulation.
Interestingly, BMP-15 is most closely related to and shares a
coincident expression pattern with the mouse growth/differentiation
factor 9 (GDF-9) gene that is essential for female fertility. Our
findings will be important for defining the role of BMP-15 in
follicular development.
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INTRODUCTION
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The transforming growth factor-ß (TGFß) superfamily,
which includes the bone morphogenetic proteins (BMPs) and the
growth/differentiation factors (GDFs), is the largest family of
extracellular signaling proteins yet described. Homology-based cloning
approaches have resulted in the identification of TGFß-like factors
in such diverse species as Caenorhabditis
elegans, Drosophila melanogaster, and
Xenopus laevis and have extended the number of mammalian
members to more than 30 (1, 2, 3). These molecules, synthesized as
prepropeptides and processed into dimeric proteins, are structurally
related in their mature, carboxy-terminal region. Members of the BMP
subfamily have been shown to be involved in a wide array of cellular
processes during embryonic development. For example, BMP-2 and BMP-4
are required for cellular differentiation in early embryogenesis (4, 5), BMP-5 is required for the formation of specific skeletal structures
(6), BMP-7 is necessary for normal eye and kidney development (7), and
BMP-8a and BMP-8b are essential factors for spermatogenesis (8, 9). BMP
subfamily members have also been shown to affect adult tissue repair.
Animal models have demonstrated the ability of several BMPs to induce
the formation of bone and cartilage (10, 11) and tendon/ligament (12).
Identification of new members in this gene family is important for
continued characterization of the key factors involved in
mammalian development and physiology.
Reproductive development and function are complex processes
involving both genetically determined and physiological events.
Discerning the role that each of these growth factors plays in
vivo would lead to a better understanding of the complex and
sex-specific physiology of the mammalian reproductive system. Several
members of the TGFß superfamily, such as the inhibins and activins
(13), Müllerian inhibiting substance (14), BMP-8a (8),
BMP-8b (9), and GDF-9 (15, 16), are implicated as important regulatory
factors in mammalian reproduction. For example, we have previously
generated mice lacking this oocyte-specific growth factor using
embryonic stem cell technology (16) and found that absence of GDF-9
results in an early block in folliculogenesis at the one-layer primary
follicle stage leading to infertility. These studies have defined GDF-9
as the first oocyte-derived growth factor required for somatic cell
function in vivo.
In the present study, we report the identification of new genes,
which we have named bone morphogenetic protein 15 (BMP15
in humans, Bmp15 in
mice).1 Using the sequence
relationships of previously characterized BMPs and a degenerate PCR
approach, we first identified the mouse gene. To identify the human
sequence, low-stringency hybridization with a probe derived from a
mouse genomic clone was necessary because the two sequences were
unusually divergent. Chromosomal mapping of mouse Bmp15
and human BMP15 has identified them as X-linked genes.
In situ hybridization in the mouse has demonstrated that
Bmp15 is expressed exclusively in the oocyte soon after
primordial follicles are recruited, and expression is maintained
until after ovulation. Thus, BMP-15 is the second oocyte-derived
growth factor of the TGFß superfamily that may be critical for
ovarian function.
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RESULTS
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Identification of the Mouse Bmp15 Gene and cDNA
To identify novel BMP genes, we adopted a PCR approach based
on conserved amino acid sequences of the BMP/Vg-1/DPP subgroup of the
TGFß superfamily (12). Two consensus mature peptide sequences,
WQ/NDWIV/IA and NHAIV/LQT, were used to design degenerate
oligonucleotide primers. Based on the relative location of these
consensus sequences in other BMPs, we predicted that 128-bp products
would be generated by PCR. All products of this size range were
subcloned and analyzed for novel BMP-like sequences. A subclone
produced by degenerate PCR from mouse genomic DNA, designated mPC-3,
contained such a novel sequence. Oligonucleotides derived from this
sequence were used to screen a mouse strain 129SvEv genomic library and
resulted in the identification of a recombinant phage clone, ø60,
which contains sequences identical to that of mPC-3 (Fig. 1A
). DNA sequence analysis of ø60
indicated that it contained a segment of a novel gene, which we named
mouse Bmp15. Based on homology with other BMP subfamily
members, this sequence identifies a single exon that encodes the
complete mature domain and part of the propeptide region of BMP-15. A
portion of ø60 that contained 508 bp of mouse Bmp15 coding
sequence was designated probe 1031 (Fig. 1
, A and B).

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Figure 1. Genomic and cDNA Representations of Mouse
Bmp15 and Human BMP15
In each case, diagonally shaded boxes indicate
prepropeptide-encoding domains and black boxes indicate
mature- encoding domains. A, Shown on the bottom, the
structure of the mouse Bmp15 gene as determined by
overlapping phage clones and 5'-RACE. The entire protein-coding
sequence is contained within two exons, separated by a 3.5-kb intron.
5'- and 3'-UTR sequences (open boxes) indicate the
limits of the exons. Probe Pc261 is the region used to map the mouse
Bmp15 gene. B, Schematic representation of mouse
Bmp15 coding sequence and two representative clones
isolated from the mouse ovary cDNA library. The signal peptide-encoding
sequence is shaded. Probe 1031, which includes a portion of the
propeptide- and mature-encoding domains, is shown. C, Structure of the
human BMP15 gene. The entire protein-coding sequence is
contained within two exons (shown with boxes), separated
by a 4.2-kb intron. A 3-kb EcoRI fragment encoding part
of the propeptide domain and the entire mature domain was used for
chromosomal localization by FISH. B, BamHI; RI,
EcoRI; RV, EcoRV; K,
KpnI.
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Based on Northern blot analysis results (see below), we screened a
mouse ovary cDNA library with probe 1031 and identified the complete
coding sequence of mouse Bmp15. Several positively
hybridizing clones were identified; two of these, designated PC61 and
PC71, were characterized by DNA sequence analysis. These cDNA clones
defined an open reading frame of 1176 bp that encoded a 392-amino acid
BMP-15 prepropeptide. The transcriptional start site of the mouse
Bmp15 mRNA was determined by rapid amplification of cDNA
ends (5'-RACE) (Fig. 1A
). The 5'-untranslated region (UTR) and 3'-UTR
are 0.64 kb and 1.6 kb, respectively.
Probe 1031 was used to rescreen the mouse genomic library and
identified several overlapping recombinant phage clones including PW-8
and PW-9 (Fig. 1A
). Restriction endonuclease digestion, Southern blot
hybridization, and DNA sequence analysis of PW-8, PW-9, and ø60 were
used to determine the structure of the mouse Bmp15 gene. Two
exons, separated by a 3.5-kb intron, encode the entire mouse BMP-15
protein. The first exon encodes a 17-amino acid signal peptide (as
predicted with the aid of the GeneWorks computer program, Oxford
Molecular Groups, Inc., Oxford, England) and part of the
propeptide domain. The second exon encodes the remaining propeptide
region and the entire predicted 125-amino acid mature domain (Fig. 1
, A
and B).
Identification of the Human BMP15 Gene
A portion of the ø60 mouse clone was used to screen a human
genomic library under reduced stringency conditions. Two hybridizing
clones, JLDc1 and JLDc19, were characterized by DNA sequence analysis.
Clone JLDc19 contained the complete coding sequence of human
BMP15. The genomic structure of human BMP15 was
determined by restriction endonuclease digestion, Southern blot
hybridization, and DNA sequence analysis. The 1176-bp coding region is
contained within two exons separated by 4.2 kb of intervening sequence:
the first exon encodes a predicted secretory leader sequence of 17
amino acids followed by 97 amino acids of the propeptide domain; the
second exon encodes the remaining 158 amino acids of the propeptide
domain and the entire predicted 125-amino acid mature domain (Fig. 1C
).
Comparison of Mouse and Human BMP-15
Both mouse and human BMP-15 are encoded by two exons
separated by a single intron. Cleavage at the predicted proteolytic
processing sites (21, 22), Arg-Ser-Val-Arg in mouse or Arg-Arg-Thr-Arg
in human BMP-15, produces 125-amino acid peptides. The prepropeptides
(392 amino acids) exhibit an overall identity of 63% (76% at the
nucleotide level), while the amino acid identities in the mature domain
and propeptide domains are 77% (81% nucleotide identity) and 60%
(74% nucleotide identity), respectively (Fig. 2
). There are five potential N-linked
glycosylation sites in the mouse and human BMP-15 proteins. Three of
the sites are spatially conserved between the two species. Both mouse
and human BMP-15 contain only six out of the seven cysteine residues
observed in the vast majority of proteins in the TGFß superfamily.
Interestingly, the mouse and human gene products differ in that mouse
BMP-15 has two additional cysteines upstream from the first conserved
cysteine, a spatial pattern more characteristic of TGFßs and
activins.

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Figure 2. Deduced Amino Acid Sequence of Mouse (M,
top) and Human (H, bottom) BMP-15
The proteolytic processing sites are boxed, and the
arrow indicates the predicted start of the mature
peptides. The triangles indicate the positions of the
single intron in the corresponding genomic DNA sequences. Potential
N-linked glycosylation sites are in bold. Cysteine
residues characteristic of TGFß superfamily members are boxed
and shaded; the cysteine residue thought to be involved in
intermolecular bonding is replaced by a serine residue and is
boxed. Two additional cysteines present in the
N-terminal region of the mouse BMP-15 mature peptide are
underlined. Note also the sequence divergence in the
mature peptide before the first conserved cysteine.
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Northern Blot Analysis of Mouse Bmp15
Many members of the BMP subfamily demonstrate widespread
expression in the developing embryo and adult. To define the mRNA
expression pattern of Bmp15, Northern blot analysis was
performed using total RNA from multiple adult mouse tissues. As shown
in Fig. 3
, the Bmp15 probe
detected a single transcript of approximately 3.5 kb in mouse ovarian
RNA. All other tissues examined were negative.

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Figure 3. Northern Blot Analysis of Mouse Total RNA from
Various Tissues
A mouse Bmp15 probe, generated from coding sequence,
detected a signal of approximately 3.5 kb in RNA derived from ovary but
no other tissue (top). 18S rRNA (bottom)
was used as a control for RNA loading.
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In Situ Hybridization Analysis of Mouse Bmp15
In situ hybridization analysis on mouse ovaries
revealed that Bmp15 expression was confined to the oocyte
(Fig. 4
). Bmp15 is not
expressed in oocytes of primordial (type 2) or small type 3a follicles
(23) but is first detected in oocytes of intermediate-size type 3a
follicles (23) and all type 3b follicles (i.e. >20
granulosa cells surrounding the oocyte in largest cross-section) and is
continually expressed in all oocytes of later stage follicles and
0.5-day postovulatory oocytes (Fig. 4
). The spatio-temporal pattern of
Bmp15 expression is identical to that of Gdf9
(Ref. 15 and data not shown).

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Figure 4. In Situ Hybridization Analysis of
Bmp15 mRNA in Mouse Ovaries
Brightfield/darkfield (A and C) or darkfield (B and D) analysis of
Bmp15 mRNA in adult wild-type ovaries. Similar to
Gdf9 (Ref. 15 and our unpublished data),
Bmp15 is absent in primordial and small type 3a
follicles (solid arrow, C and D) but is expressed in
oocytes of larger type 3a, type 3b, and type 4 follicles (open
arrow, C and D) and oocytes of all later stage follicles
including antral (a) follicles (A and B).
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Chromosomal Mapping of Mouse Bmp15 and Human
BMP15
We used the Jackson Laboratories Backcross Panel to map the mouse
Bmp15 gene. As shown in Fig. 5A
, mouse Bmp15 maps adjacent
to the centromere on the X-chromosome and cosegregates with
DXMit26 and Fsc1. To confirm that the mouse
Bmp15 and human BMP15 genes were orthologs, we
performed fluorescence in situ hybridization (FISH) analysis
to localize human BMP15 to Xp11.2 (Fig. 5B
). This
chromosomal region in humans demonstrates conserved synteny with the
chromosomal position of mouse Bmp15 (24).

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Figure 5. Chromosomal Localization of Mouse
Bmp15 and Human BMP15 Genes
A, Bmp15 maps to the proximal region of the mouse X
chromosome. Using the Jackson Laboratory interspecific backcross panels
(C57BL/6JEi x SPRET/Ei)F1 x SPRET/Ei), the mouse
Bmp15 locus was placed adjacent to the centromere on the
X chromosome. The segregation of Bmp15 and flanking
genes in 94 backcross mice typed for all loci are shown at the
top of the figure. Black and white boxes
indicate C57BL/6JEi and SPRET/Ei alleles, respectively. Numbers
at the bottom of each column represent the number of backcross
progeny that has inherited a recombinant or nonrecombinant chromosome
from the (C57BL/6JEi x SPRET/Ei)F1 parent. Note that one mouse
has placed Bmp15 and DXMit26 proximal to
Pmv1 and the more distal markers. A partial linkage map
of the proximal X chromosome is shown below. B, BMP15
maps to the human X chromosome by FISH. The arrow
indicates the hybridization signals of twin dots on the human X
chromosome. Based on analysis of metaphase chromosomes isolated from a
male, the human BMP15 gene was localized to Xp11.2.
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DISCUSSION
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We used degenerate PCR and reduced stringency hybridization
conditions to identify a new member of the TGFß superfamily of
extracellular signaling proteins, BMP-15. The relatively low amino acid
sequence identity between mouse and human BMP-15 originally led us to
believe that the two sequences represented distinct genes. Similar
sequence comparisons between mouse and human versions of other
BMP-related genes typically exhibit identity of greater than 95%.
For example, human GDF-9 and mouse GDF-9 are 96% identical (see Fig. 6
), while human and mouse BMP-4 are
absolutely conserved. Data from extensive genomic screening and
Southern blot analysis suggest that the mouse Bmp15 and
human BMP15 genes described here are the closest sequences
to each other. DNA sequence analysis of the mouse Bmp15 and
human BMP15 genomic clones revealed a highly conserved gene
structure. Both genes possess a single intron at exactly the same
relative location with respect to the coding sequence and predict
primary translation products of the same length, 392 amino acids.
Chromosomal mapping of these genes to conserved syntenic regions on the
X-chromosome confirms that mouse and human BMP-15 are orthologs (25).
Interestingly, there are no known mouse or human mutations to date that
map to these regions.

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Figure 6. Dendogram of the TGF-ß Superfamily and
Comparisons of BMP-15 and GDF-9
A, The relationship of BMP15 to other members of the TGFß
superfamily. The figure was generated using the cysteine-rich
COOH-terminal polypeptides of the TGFß superfamily members, which
were aligned using the PILEUP program (Genetics Computer Group,
Madison, WI). B, The BMP-15/GDF-9 subfamily. Percent amino acid
identities are indicated and were generated as described above.
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The spatially conserved pattern of seven cysteine residues has served
as a defining criteria of the TGFß superfamily, although the
discovery of additional members has introduced some variations to that
motif. Several members, including the prototype protein TGFß, possess
extra cysteines at the amino-terminal end of the mature protein.
Probably more structurally relevant, however, are a few members (26, 27) that lack the cysteine residue shown to be responsible for
interchain disulfide bond formation (28, 29, 30). Both mouse and human
BMP-15, as well as their close relative GDF-9 (27), are missing this
structurally important cysteine residue. The three-dimensional
structure of TGFß family members lacking the dimer cysteine has not
been determined, but it is likely that their active forms are
noncovalently associated dimers. Size exclusion chromatography of
recombinant human GDF-9 and GDF-3/Vgr-2 produced in CHO cell expression
systems indicated an apparent mol wt of approximately twice that of
monomeric controls that were produced in Escherichia coli
(N. Wolfman and P. Lowden, personal communication). Furthermore,
increasing amounts of chaotrope (guanidine, urea) did not alter the
apparent mol wt, indicating a highly stable association of the two
monomeric subunits (N. Wolfman and P. Lowden, personal communication).
Noncovalent association may allow for the formation of heterodimeric
proteins in tissue settings in which more than one 6-cysteine partner
is produced.
Several members of the TGFß superfamily have been demonstrated to
exist naturally as heterodimers and in some cases have different
activities than their homodimeric counterparts (13, 31). The inhibins,
which inhibit FSH release from the pituitary (32), have not been shown
to exist in homodimeric form but, rather, they are examples of
functional heterodimers of two distinct TGFß superfamily members,
inhibin
and activin ßA or ßB. Recombinant heterodimers between
the BMP-2/4 subgroup and the BMP-5/6/7 subgroup have also been shown to
exhibit a significant increase in biological activity when compared
with homodimers assayed in the same system (33, 34, 35). With these BMPs,
increased levels of homodimers can match the activity of heterodimers
in these systems.
Female infertility in GDF-9-deficient mice is the result of arrested
follicular development at the primary follicle (type 3B) stage. This
block in folliculogenesis approximates the onset of expression of
Gdf9 and Bmp15, which are both detected in
oocytes of one-layer primary (type 3A) follicles and remain highly
expressed in the oocyte throughout the course of follicular maturation
and ovulation. Interestingly, GDF-9-deficient mice continue to
synthesize Bmp15 mRNA (our unpublished data). Thus,
based on their coincident expression pattern, one potential explanation
for the lack of phenotypic rescue by BMP-15 of the GDF-9-deficient mice
is that BMP-15 and GDF-9 are capable of forming heterodimers, and it is
the heterodimeric form, BMP-15/GDF-9, which supports follicular
maturation. Alternatively, BMP-15 and GDF-9 may only form homodimers
with distinct biological functions. Ongoing studies to generate
Bmp15 knockout mice may elucidate whether BMP-15 and GDF-9
form active heterodimers in vivo and will help us to further
define their roles in reproductive physiology.
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MATERIALS AND METHODS
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Degenerate PCR
The oligonucleotides
5'-gcggatccTGG(C/A)ANGA(C/T)TG-GAT(A/C/T)(G/A)TNGCN and
5'-gctctagaGT(C/T)TGNA (C/T)NATNGC(A/G)TG(A/G)TT-3' were used
for PCR on mouse genomic DNA to amplify BMP-related sequences as
previously described (12). The lowercase letters include
recognition sequences for the restriction endonucleases
BamHI and XbaI, respectively, which were added to
facilitate the subcloning of the expected 128-bp PCR products into
pGEM-3 (Promega, Madison, WI).
Genomic Library Screening
The oligonucleotides
5'-TCCTCGTCTCTATACCCCAAAT-TACTGTAAGGAATCTGT-3' and
5'-ATCTGTACTCGGGTATTACCCTATGGTCTCAATTCACCC-3' were kinased with
[
32P]-ATP and hybridized to duplicate nitrocellulose
replicas of 500,000 recombinants of a mouse genomic library (no.
946309, Stratagene, La Jolla, CA) in standard hybridization buffer
(SHB = 5xSSC/5x Denhardts solution/0.1% SDS/100 µg
denatured salmon sperm DNA) at 60 C overnight. The filters were washed
extensively with 5x SSC/0.1% SDS at 60 C and subjected to
autoradiography for 2 days at -80 C with intensifying screens. An
additional 600,000 recombinants of this same mouse genomic library were
screened. Duplicate nitrocellulose filters were hybridized to a DNA
fragment of genomic clone ø60 corresponding to nucleotides 11391418
of the mouse Bmp15 sequence, hereafter designated probe
1031. This probe was random primed with [
-32P]dCTP and
hybridized to nitrocellulose filters in Churchs solution [0.5
M sodium phosphate buffer (pH 5.2)/7.5% SDS] at 63 C.
Filters were washed with 0.1x Churchs solution and exposed overnight
at -80 C. Approximately 1,000,000 recombinants of a human genomic
library (Stratagene no. 945203) were screened with
[
-32P]dCTP random-primed mouse probe 1031 at 60 C in
SHB overnight. Duplicate nitrocellulose filters were washed with 2x
SSC/0.1% SDS at 60 C and exposed for 2 days at -80 C with
intensifying screens.
mRNA Isolation and cDNA Library Construction
Total RNA was extracted from various tissues of adult Swiss
Webster mice or C57BL/6/129SvEv hybrid mice using RNA STAT-60 (Leedo
Medical Laboratories, Houston, TX) as described by the
manufacturer. Poly (A)+ RNA was purified using Oligotex-d7
beads (Qiagen, Chatsworth, CA) according to the manufacturers
instructions. Mouse ovarian mRNA was used to construct a directional
(5'-EcoRI to 3'-XhoI), oligo (dT)-primed cDNA
library in the bacteriophage vector
ZAP Express using the
ZAP Express cDNA cloning kit (Stratagene).
Northern Blot Hybridization
Total RNA (12 µg) derived from multiple mouse tissues was
electrophoresed on a 1.2% agarose/7.6% formaldehyde gel and
transferred to Hybond-N (Amersham, Arlington Heights, IL) nylon
membrane. Probe 1031 was random primed with
[
-32P]dCTP. The membrane was hybridized, washed, and
subjected to autoradiography as described (17). An 18S ribosomal RNA
cDNA probe was used for the loading control.
cDNA Library Screening and 5'-RACE
Approximately 400,000 clones of the mouse ovarian cDNA library
were hybridized to [
-32P]dCTP random-primed probe 1031
in Churchs solution at 63 C. Filters were washed with 0.1x Churchs
solution and exposed overnight at -80 C. 5'-RACE PCR was performed
using the Marathon cDNA amplification kit (CLONTECH, Palo Alto, CA)
under conditions described by the manufacturer. The oligonucleotides
used for the amplification were: 5'-GGAAAGTCCAGGGTCTGTACATGCCA-3' and
5'-CCATTGCTTCATCTCTCCTTGCCA-3'.
In Situ Hybridization
In situ hybridization was performed as
described previously (18). In brief, freshly dissected ovaries from
C57Bl/6/129SvEv hybrid mice were fixed in 4% paraformaldehyde-PBS
overnight, processed, embedded in paraffin, and cut to a 5 µm
thickness. Mouse Bmp15 probe 1031 antisense and sense
strands were generated by labeling with [
-35S]UTP
using Riboprobe T7/SP6 Combination System (Promega). Hybridization was
carried out at 5055 C with 5 x 106 cpm of each
riboprobe per slide for 16 h in 50% deionized formamide/0.3
M NaCl/20 mM Tris-HCl (pH 8.0)/5 mM
EDTA/10 mM NaPO4 (pH 8.0)/10% Dextran
sulfate/1x Denhardts/0.5 mg/ml yeast RNA. High-stringency washes
were carried out in 2x SSC/50% formamide and 0.1x SSC at 65 C.
Dehydrated sections were dipped in NTB-2 emulsion (Eastman Kodak,
Rochester, NY) and exposed for 47 days at 4 C. After the slides were
developed and fixed, they were stained with hematoxylin and mounted for
photography.
Chromosomal Mapping
Chromosomal localization of the mouse Bmp15 gene was
performed using the Jackson Laboratory Interspecific backcross DNA
panel (19) using Pc261 probe located in the 3'-UTR of the mouse
Bmp15 gene. A 3-kb EcoRI fragment from a human
BMP15 genomic clone containing a portion of exon 2 and some
intron sequence was used to chromosomally map the human
BMP15 gene by FISH as described (20). FISH was performed by
Dr. Antonio Baldini and the FISH core in the Department of Molecular
and Human Genetics at Baylor College of Medicine.
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ACKNOWLEDGMENTS
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We would like to acknowledge Ms. Susan Benard for DNA
sequencing, Dr. Antonio Baldini and the FISH core in the Department of
Molecular and Human Genetics at Baylor College of Medicine for mapping
the human BMP15 gene, and Drs. Rajendra Kumar, Vicki Rosen,
and Gary Hattersley for critical review of the manuscript. The original
identification of the Bmp15 subclone mPC-3 by degenerate PCR
was performed by K.M.L. in Dr. Brigid Hogans laboratory at Vanderbilt
University. The PCR conditions for the identification of mPC-3 were
optimized by Dr. Gerald Thomsen while in the laboratory of Dr. Doug
Melton, Harvard University.
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FOOTNOTES
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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 by a sponsored research grant from
Genetics Institute and NIH Center Grant HD-07495 (to M.M.M.). Ms. Julia
Elvin is a student in the Medical Scientist Training Program supported
by NIH Grant GM-08307.
1 The mouse Bmp15 cDNA and human
BMP15 gene sequences in this manuscript have been
deposited in GENBANK under the accession numbers AF082348,
AF082349, and AF082350. 
Received for publication June 17, 1998.
Revision received August 19, 1998.
Accepted for publication August 31, 1998.
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