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Original Studies |
Department of Bacteriology and Immunology, Haartman Institute (J.A., M.P.L., K.V., R.J., L.S., O.R.), Haartmaninkatu 3, University of Helsinki, FIN-00014 Helsinki; the Laboratory of Molecular Genetics, Helsinki University Central Hospital (N.H.-K.), Haartmaninkatu 4, FIN-00290 Helsinki; the Infertility Clinic, Family Federation of Finland (H.L., T.T., O.H.), Kalevankatu 16, FIN-00100 Helsinki, Finland; and the Department of Obstetrics and Gynecology, University of Helsinki (J.S., R.B.), Haartmaninkatu 2, FIN-00290, Helsinki, Finland; and the Department of Anatomy and Developmental Biology, University College London (L.D.), London, United Kingdom WC1E 6BT
Address all correspondence and requests for reprints to: Dr. J. Aaltonen, Department of Bacteriology and Immunology, Haartman Institute, P.O. Box 21, Haartmaninkatu 3, University of Helsinki, FIN-00014 Helsinki, Finland. E-mail: johanna.aaltonen{at}helsinki.fi
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Abstract |
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We conclude that 1) both GDF-9 mRNA and protein are abundantly expressed in oocytes of primary follicles in human ovary, suggesting that the GDF-9 transcript is translated at this early stage of folliculogenesis; 2) human GDF-9B is specifically expressed in gonads at low levels; and 3) the expression of GDF-9 mRNA begins slightly earlier than that of GDF-9B in the human oocytes during follicular development. Our results are consistent with the suggestion that GDF-9 and GDF-9B may regulate human folliculogenesis in a manner specific to the ovary.
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Introduction |
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T) has been identified in the FSH receptor (FSHR)
gene, which is expressed in the granulosa cells of developing follicles
(2). A similar phenotype was obtained in mice homozygous for the
targeted disruption of the FSHR gene (3). In studies using knockout
mice, several genes have been shown to be essential for normal ovarian
function (4). For example, both male and female Dazla knockout mice are
infertile due to the loss of germ cells and the complete absence of
gamete production (5). Also, mice mutated at the X chromosomal Zfx
locus demonstrate a diminished number of germ cells as well as small
animal size in both male and female mice (6). Oocytes communicate with their surrounding somatic cells via paracrine signaling that is crucial for normal ovarian function (7). Growth differentiation factor 9 (GDF-9) is the first oocyte-derived growth factor found to be indispensable for fertility. In female mice disruption of the GDF-9 gene leads to a block in follicular development at the primary one-layer follicle stage, whereas male mice are fertile (8). Structurally, GDF-9 belongs to the transforming growth factor-ß (TGFß) growth factor family (9), which is a large group of polypeptide growth factors that have multiple roles in embryogenesis and in the control of cell growth and differentiation. During mouse folliculogenesis, GDF-9 expression begins at the primary follicular stage (10). Fitzpatrick et al. have shown that GDF-9 is also expressed in several nonovarian tissues in rodents and human (11). Human GDF-9 complementary DNA (cDNA) was initially cloned from ovarian cDNA library (10), and GDF-9 has been detected by RT-PCR from ribonucleic acid (RNA) derived from human preovulatory oocytes (12). However, localization of GDF-9 expression to specific cell types in the human has not been performed. We determined here by in situ hybridization and immunohistochemical analyses the localization of the GDF-9 messenger RNA (mRNA) and protein in human ovarian tissue and compare GDF-9 expression with that of human GDF-9B, a novel GDF-9-related gene that we recently identified in the mouse (13). This novel TGFß family member is coexpressed with GDF-9 in oocytes during mouse folliculogenesis. Based on the mouse EST sequence, we cloned and characterized genomic and complementary DNA (cDNA) clones of the human ortholog of mouse GDF-9B and show here that the expression of human GDF-9B is gonad specific. We also determined by synthetic mRNA injection studies whether GDF-9 and GDF-9B share biological effects observed previously for TGFß family members that induce mesoderm in Xenopus laevis embryos (14).
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Materials and Methods |
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Human ovarian tissue samples were obtained from women (<35 yr of age) whose ovaries were removed because of cervical cancer without preoperative irradiation or as cortical biopsy material during laparoscopic surgery. Human testis samples were obtained from patients who had undergone orchidectomy due to prostate cancer. All tissue samples were collected by approval of the local ethical committee. For histological analyses the oophorectomy samples were formalin fixed and paraffin embedded, whereas ovarian biopsy and testis samples were directly embedded in OCT cryopreservation solution (Tissue-Tek, Miles, Inc., Elkhart, IN).
GDF-9 antibody and immunohistochemistry
To raise antibodies against GDF-9, rabbits were immunized with a peptide containing 13 amino acids corresponding to a C-terminal sequence of GDF-9 (EPDGSIAYKEYED) and an additional N-terminal cysteine residue allowing coupling to key hole limpet hemocyanin. The best immune serum, K3S1, was affinity purified using an affinity column containing the peptide coupled to epoxy-activated Sepharose 6B (Pharmacia Biotech, Uppsala, Sweden). Five-micron paraffin sections were deparaffinized, rehydrated, and treated in a microwave oven twice for 5 min each time in 0.01 mol/L citric acid monohydrate, pH 6.0. Sections were then incubated overnight at 4 C with the primary antibody diluted 1:100 in phosphate-buffered saline (PBS) containing 1% normal goat serum as a blocker. Sections were washed three times for 3 min each time in PBS and incubated for 30 min at room temperature with biotinylated goat antirabbit secondary antibody (Vector Laboratories, Inc., Burlingame, CA) diluted 1:200 in PBS containing 1% normal goat serum as a blocker. Sections were rinsed three times for 3 min each time in PBS and incubated with avidin-biotin-peroxidase complex (Vector Laboratories, Inc. at room temperature for 30 min (15). Finally, antigenic sites were visualized by using 3-amino-9-ethyl carbazole (Sigma Chemical Co., St. Louis, MO) in 50 mmol/L acetate buffer, pH 5, containing 0.03% H2O2. All experiments were controlled by incubating parallel sections without primary antibody or incubating the sections with primary antibody in the presence of the respective blocking peptide. The K3S1 antibody recognizes specifically oocyte GDF-9 in control mouse ovarian sections (our unpublished observations).
Cloning and characterization of the human GDF-9B gene
Primers were designed based on the mouse EST1 sequence (GenBank
accession no. AA422665) to amplify part of the human GDF-9B. One primer
pair (A/B, Table 1
) amplified a 320-bp
fragment from human genomic DNA and was cloned into pGEMT-Easy vector
(Promega Corp., Madison, WI) for further analyses. Total
human genomic PAC library (a gift from Pieter de Jong, Roswell Memorial
Institute, Buffalo, NY) was screened by PCR using primers A and B.
Fluorescent in situ hybridization (FISH) was performed on
metaphase chromosomes derived from human peripheral blood lymphocytes.
Identification of chromosomes was based on the banding pattern achieved
using 5-bromo-deoxyuridine incorporation (200 µg/mL) at the early
replicating phase, as previously described (16, 17). Hybridizations
with biotin 11-deoxy (d)-UTP (Sigma Chemical Co.)-labeled
human GDF-9B gene-specific clones were carried out in 50% formamide
and 10% dextran sulfate in 2 x SSC (standard saline citrate) as
described previously (17, 18, 19). The slides were stained with Hoechst
33258 (1 µg/mL), exposed to UV light for 30 min, and counterstained
with DAPI including antifading reagent (Vectachield, Vector Laboratories, Inc.). A multicolor image analysis was used for
acquisition, display, and quantification of hybridization signals of
metaphase chromosomes with the previously described system (20). The
amplified 320-bp PCR product was labeled with
[
-32P]deoxy-CTP using the Prime-a-Gene kit
(Promega Corp., Madison, WI) and hybridized onto high
density filter containing human X chromosome specific cosmid library
obtained from Resource Center of the German Human Genome Project via
Max Planck Institute for Molecular Genetics (Berlin, Germany;
http://resource.rzpd.de/cgi-resource/newlib). The GDF-9B-positive
cosmids were digested with restriction enzymes EcoRI,
BamHI, KpnI, and XbaI, and suitable
sized fragments containing the human GDF-9B by hybridization were
subcloned into pGEM7Zf(+) vector (Promega Corp.). The
cloned fragments were sequenced with an ABI PRISM 377 DNA sequencer
(Perkin Elmer Corp., PE Applied Biosystems,
Foster City, CA).
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A 5'-end cDNA representing the human GDF-9B gene was obtained by the rapid amplification of 5'-cDNA ends (RACE) technique, using the human testis Marathon-Ready cDNA kit (no. K7414–1, CLONTECH Laboratories, Inc. Palo Alto, CA). Gene-specific primer (C) was used in the first round PCR amplification with the AP1 primer in a 50-µL reaction [10 pmol of each primer, 0.2 mmol/L of each dNTP, 2.5 mmol/L MgCl2, 1 x GeneAmp PCR buffer, and 2.5 U AmpliTaq DNA polymerase (Perkin Elmer Corp. PE Applied Biosystems)] in 30 cycles with an initial standard hot start procedure: 5 cycles of 95 C for 30 s and 72 C for 3 min, 5 cycles of 95 C for 30 s and 70 C for 3 min, followed by 25 cycles of 95 C for 30 s and 68 C for 3 min. One microliter of the first round PCR reaction was amplified with nested primers AP2 and D as described above, and the 350-bp fragment of human GDF-9B obtained was directly sequenced.
Northern blot, RT-PCR, and in situ hybridization analyses
Northern blot analyses were performed as previously described
(21). As probes for filter hybridizations we used a 798-bp human GDF-9B
(primers E/F) and a 465-bp human GDF-9 (primers G/H) PCR fragment
derived from genomic DNA and subcloned into pGEMT-Easy vector
(Promega Corp.). The probes were labeled with
[-32P]deoxy-CTP as described above. RT-PCR analyses
were performed on Multiple Tissue cDNA panels I and II (K1420–1 and
K1421–1, respectively, CLONTECH Laboratories, Inc.) using
primers I and C to amplify a 340-bp cDNA fragment in a total volume of
50 µL [3 pmol of each primer, 0.08 mmol/L of each dNTP, 2.5 mmol/L
MgCl2, 1 x GeneAmp PCR buffer, and 0.75 U AmpliTaq
DNA polymerase (Perkin Elmer Corp. PE Applied Biosystems)] in 40 cycles of 95 C for 30 s, 62 C for
45 s, and 72 C for 1.5 min preceded by initial denaturation at 95
C for 5 min and followed by a final extension at 72 C for 15 min.
Twenty microliters of PCR reaction were analyzed on a 2% agarose gel,
Southern blotted, and hybridized with the
[-32P]deoxy-CTP-labeled 350-bp 5'-RACE fragment. The
filter was washed with 1 x SSC-0.1% SDS and exposed overnight at
room temperature. For in situ hybridization analyses, the
[-33P]UTP-labeled antisense complementary RNA probes
were in vitro transcribed from SphI-linearized
plasmids containing the GDF-9B and GDF-9 cDNAs described above.
In situ RNA analyses were carried out on 9-µm cryostat
sections as previously described (22). The slides were dipped in NTB-2
emulsion (Eastman Kodak Co., New Haven, CT) and exposed up
to 57 days.
Xenopus laevis mesoderm induction assays
A 415-bp GDF-9B cDNA encoding the mature region of GDF-9B was synthesized by RT-PCR from mouse ovarian RNA using oligonucleotides J/K, cloned into pGEM-T vector (Promega Corp.), and sequenced. This GDF-9B cDNA was flanked with a SphI site, allowing fusion to the mouse activin ßA subunit proregion (A). For generating synthetic transcripts for embryo injections, a full-length mouse GDF-9 cDNA was subcloned into pSP64T3, and the mature region of mouse GDF-9B was subcloned into pRN3-AVg1 to replace the Vg1 mature region, resulting in pRN3-AGDF-9B. A similar AGDF-9 open reading frame (ORF) containing construct was generated. Capped transcripts were synthesized from linearized templates with SP6 or T3 RNA polymerase (Promega Corp.) using the Megascript kit (Ambion, Inc. Austin, TX). The translatability of the transcripts was tested using a reticulocyte lysate-based in vitro translation kit (Promega Corp.). Samples were analyzed by SDS-PAGE for visualization of the 35S-labeled translated protein bands. X. laevis embryos were obtained as previously described (23). Embryos were injected at the four-cell stage into dorsal or ventral blastomeres with 0.02–2 ng GDF-9, AGDF-9, and AGDF-9B RNAs, and embryos were observed up to stage 40 to detect possible morphological alterations compared to the uninjected embryos. In some experiments animal caps were cut at stage 8 (midblastula stage), and these were allowed to grow in culture overnight. To control the responsiveness of the system to known dorsal and ventral mesoderm inducers, in some experiments the RNAs of activin A, AVg1, or bone morphogenetic protein-4 (BMP-4) were injected into parallel embryos (24, 25, 26).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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To study the distribution of GDF-9 transcripts in human ovary,
in situ hybridization analysis was carried out on frozen
sections of ovarian cortical tissue obtained as biopsy material samples
from women undergoing laparoscopic surgery. The results indicate that
GDF-9 mRNAs are clearly expressed in oocytes of primary follicles (Fig. 1). As these tissue samples very seldom
contained secondary or tertiary follicles, the expression of GDF-9 mRNA
could not be well studied during these later stages of follicular
development. For immunolocalization studies, we raised an antipeptide
antibody, K3S1, against a C-terminal epitope of mature GDF-9 protein
(conserved in the mouse, rat, and human sequences) to investigate
whether the GDF-9 transcript in oocytes is translated.
Immunohistochemical analyses of sections of paraffin-embedded whole
human ovaries indicated that the K3S1 antibody stains strongly oocytes
of primary follicles (Fig. 2, A and B).
No staining was observed when sections were incubated with only the
secondary antibody (Fig. 2C), and the staining seen with K3S1 was
abolished in the presence of the blocking peptide (data not shown).
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We previously identified by searching the database a 406-bp mouse EST sequence (mouse EST1, GenBank accession no. AA422665) that showed significant homology to GDF-9 (13). Based on this mouse EST sequence, several primers were designed, and one such primer pair (A/B) amplified a 320-bp fragment from human genomic DNA. The PCR fragment was sequenced and verified to contain a novel GDF-9-like sequence, designated as human GDF-9B. Genomic clones for the human GDF-9B gene were first obtained by screening a total human genomic P1 artificial chromosome (PAC) library by PCR with primers A and B. Only one PAC clone, PAC 72O6, was positive for this gene and was hybridized by FISH to chromosome Xp11.2.
To obtain the coding sequence and characterize the exon-intron
structure of the human GDF-9B gene, two approaches, RACE and genomic
cloning, were used in parallel. We screened a human X
chromosome-specific arrayed cosmid library by hybridization using the
above-mentioned PCR fragment as a probe and obtained a total of three
cosmid clones (cos1, cos2, and cos3) for subcloning and sequencing
(Fig. 3A). By double digests, using
various restriction enzymes, Southern blotting, and hybridization
analyses, we were able to identify overlapping subclones of these
cosmids that represented human GDF-9B gene (Fig. 3A). 5'-RACE was
carried out using gene-specific primers designed to the 5'-end of the
exon 2 and human testis cDNA as a template. We obtained a 350-bp PCR
fragment that represented part of the both exons compared with the
genomic sequence, and thus unraveled the exon-intron boundaries of the
GDF-9B gene (Fig. 3, A and B). The ATG codon encoding the first
methione and the upstream 5'-untranslated region sequence, including an
in-frame STOP codon 18 codons upstream of the first ATG codon, were
deduced from the genomic sequence. A purine is present in position -3
upstream of the putative translation initiator ATG, consistent with
Kozak’s consensus sequence (27). We sequenced a total of about 6-kb
genomic DNA that contained two exons, separated by a 4643-bp intron,
and some 5'- and 3'-untranslated region sequences flanking the protein
coding regions (EMBL accession no. AJ132405; Fig. 3A). The first 109
amino acids are encoded by the exon 1 and the remaining 283 amino acids
by the exon 2, showing similar exon-intron structure as GDF-9. The
exon-intron boundaries follow the GT-AG rule (Fig. 3B) (28). The human
GDF-9B ORF contains an 18-amino acid signal peptide predicted using the
Signal P program (Signal P V1.1 server at http://genome.cbs.
dtu.dk/services/SignalP/) (29), followed by a 249-amino acid proregion
and a 125-amino acid mature region that is likely to be released
proteolytically from the proregion.
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The expression of human GDF-9B was studied initially by Northern
blot hybridization analysis using mRNAs derived from several human
tissues [heart, brain, placenta, lung, liver, skeletal muscle, kidney,
pancreas, spleen, thymus, prostate, testis, ovary, small intestine,
colon (mucosal lining), and peripheral blood leukocytes]. However,
GDF-9B was not detected in any of these tissues, whereas GDF-9 mRNA
expression could be seen in ovary and testis (data not shown),
consistent with previously reported data (11). As GDF-9B transcripts
were clearly less abundantly expressed than GDF-9 mRNAs, we chose
RT-PCR analysis as a more sensitive method to screen the tissue
distribution of human GDF-9B mRNAs. RT-PCR followed by Southern
blotting and hybridization with a 5'-RACE fragment as a probe indicated
that GDF-9B is expressed only in ovary and testis in the human (Fig. 4A). Glyceraldehyde-3-phosphate
dehydrogenase gene expression as a control was uniformly observed in
all tissues analyzed (data not shown).
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To study in more detail the distribution of GDF-9B
transcripts in human ovary, in situ hybridization analysis
was carried out on ovarian sections obtained as biopsy samples from
women undergoing laparoscopic surgery. GDF-9B expression was detected
in the oocytes of large primary follicles (Fig. 4B
), whereas no
hybridization could be seen in small primary follicles in which GDF-9
transcripts and protein were clearly present. Compared to GDF-9
expression, GDF-9B transcription appears to begin slightly later during
human folliculogenesis. Although GDF-9B transcripts were detected in
human testis by RT-PCR experiments, we could not localize GDF-9B
transcripts to a specific cell type in human testis by in
situ hybridization (data not shown).
GDF-9 and GDF-9B do not induce mesoderm in X. laevis embryos
To assess the signaling pathways used by GDF-9 and GDF-9B, we used a X. laevis assay in which a number of members of the TGFß family induce mesoderm during early embryogenesis. For example, whereas injection of activin mRNA induces dorsal mesoderm, e.g. muscle (26), injection of BMP-4 mRNA induces ventral mesoderm, e.g. blood (24). These different activities result from activation of different receptors, which transmit their signals by activating either the Smad1 (BMP-4) or Smad2 (activin) transducing molecules (14). Injection of synthetic mRNA for GDF-9 did not have an effect on embryonic development at any of the concentrations tested, whereas in parallel injections, activin and BMP-4 induced dorsal and ventral mesoderms, respectively. One explanation for this lack of activity is that GDF-9 is not efficiently processed into bioactive forms, as previously demonstrated for X. laevis Vg1 (25). Activated Vg1 was produced by fusing the COOH-terminal domain to the proregion of either BMP-4 (BVg1) or activin (AVg1) and was shown to have potent mesoderm-inducing activity (25, 30, 31). We therefore injected AGDF-9 and AGDF-9B synthetic RNA into X. laevis embryos, and again, no effect on embryogenesis was detected, whereas AVg1 efficiently induced dorsal mesoderm in control embryos. Control experiments confirmed that all of the synthetic RNAs used were efficiently translated in a reticulocyte lysate in vitro translation system.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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The human GDF-9B gene belongs to the TGFß family of growth factors and is most closely related to GDF-9. The exon-intron structure of GDF-9B is similar to that of GDF-9; two exons are separated by a single intron in both genes, the intron being 4.6 kb in GDF-9B and 1.6 kb in human GDF-9. The partial testicular cDNA of human GDF-9B obtained here in parallel with the genomic sequence unequivocally reveals the exonic structure of human GDF-9B gene. The human GDF-9B gene maps to chromosome Xp11.2, in contrast to the autosomal GDF-9 (chromosome 5, Lawrence Berkeley National Laboratory, P1 clone 1076B9, GenBank accession no. AC004500; and our FISH data not shown). During the preparation of this manuscript, a human polypeptide sequence deduced from a genomic DNA clone and named BMP-15 was reported (32). This sequence shows identity with GDF-9B, except for three amino acids in the proregion of the predicted protein encoded by exon 1. However, no data on the human BMP-15 transcripts were presented in that paper.
Our studies for determining the tissue distribution of human GDF-9B/BMP-15 transcripts were problematic, because GDF-9B/BMP-15 transcripts were not detectable by Northern blot analysis in any tissue studied, even when using a sensitive, single stranded, DNA probe (data not shown). However, using RT-PCR followed by Southern blotting and hybridization, we were able to show that GDF-9B/BMP-15 is expressed in both ovary and testis. Our inability to detect human GDF-9B/BMP-15 transcripts on Northern blots can be explained by the fact that in the human ovary samples, most RNA species are derived from the stromal cells, and only a small fraction of mRNAs is of oocyte origin. No expression of GDF-9B/BMP-15 transcripts was observed in extragonadal tissues. Therefore, the expression of GDF-9B/BMP-15 seems to be even more gonad specific than GDF-9 expression, as GDF-9 transcripts are present also in human pituitary, uterus, and bone marrow in addition to gonads (11). The expression level of human GDF-9B/BMP-15 in gonads is apparently not as strong as that of GDF-9. The low abundance of GDF-9B/BMP-15 transcripts is also reflected by the number of ESTs identified for this gene in public databases; no human ESTs and only two mouse ESTs encoding for GDF-9B/BMP-15 were recognized. In contrast, a number of human and mouse ESTs representing GDF-9 were found in sequence databases. To localize human GDF-9 and GDF-9B/BMP-15 transcripts more specifically in the gonads, in situ hybridizations on tissue sections were performed. In human ovary, GDF-9 transcripts were already observed in small primary stage follicles, whereas the expression of GDF-9B/BMP-15 was confined to the late primary stage follicles, and no signal was observed in small primary or primordial follicles. Thus, it seems that GDF-9 expression precedes that of GDF-9B/BMP-15 during human folliculogenesis. As our ovarian biopsy samples represent the cortical region of the ovary, and they rarely contain follicles larger than the primary stage, the expression of these genes could not be studied in secondary or antral follicular stages. The expression patterns of these genes suggest that GDF-9 and GDF-9B/BMP-15 might be involved in the regulation of human ovarian function.
The significance of GDF-9 and GDF-9B/BMP-15 in human fertility is not yet known, but GDF-9 knockout mouse studies suggest that GDF-9 is likely to be important in human early folliculogenesis also. The infertility of GDF-9-deficient mice indicates that GDF-9B/BMP-15, which is still expressed in oocytes in these mice (32), is not able to rescue the biological function of GDF-9. Similar gene deletion studies may shed light on the biological function of GDF-9B/BMP-15 as well. We here evaluated using the X. laevis embryo model, whether GDF-9 and GDF-9B/BMP-15 share functional features with mesoderm-inducing members of the TGFß gene family. Our results suggest that GDF-9 and GDF-9B/BMP-15 may not activate the receptor system used by either activin or BMP-4, and that all of the necessary components of the GDF-9 and GDF-9B/BMP-15 receptor signaling system are not likely to be present in the early X. laevis embryo. These experiments suggest that GDF-9 and GDF-9B/BMP-15 may exhibit ovary-specific effects and underline the importance of studying their biology in ovarian organ and cell culture systems. The first such studies recently performed in a rat model by Hayashi et al. showed that recombinant rat GDF-9 enhances the growth and differentiation of cultured early ovarian follicles (33). Similar approaches need to be taken with GDF-9B/BMP-15 to understand its biological function during folliculogenesis. As a genetic approach to evaluate the role of these oocyte genes in human ovarian failure, we have initiated mutation-screening efforts of GDF-9 and GDF-9B/BMP-15 genes in a series of Finnish ovarian dysgenesis (XXGD) patients who do not have a mutation in the FSHR gene (2). Interestingly, a recent study by Zinn et al. (34) provides evidence that certain components of the Turner syndrome phenotype, including ovarian dysfunction, map to Xp11.2-p22.1 by deletion mapping of nonmosaic Turner patients. As GDF-9B/BMP-15 is expressed in the oocytes, it is possible that GDF-9B/BMP-15 might contribute to ovarian dysgenesis in the Turner syndrome phenotype.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Acknowledgments |
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Footnotes |
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2 The first two authors contributed equally to this work.
3 European Molecular Biology Organization fellow.
Received March 19, 1999.
Revised May 4, 1999.
Accepted May 11, 1999.
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 R. B. Gilchrist, L. J. Ritter, S. Myllymaa, N. Kaivo-Oja, R. A. Dragovic, T. E. Hickey, O. Ritvos, and D. G. Mottershead Molecular basis of oocyte-paracrine signalling that promotes granulosa cell proliferation J. Cell Sci., September 15, 2006; 119(18): 3811 - 3821. [Abstract] [Full Text] [PDF]
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T. Forges, P. Monnier-Barbarino, B. Leheup, and P. Jouvet Pathophysiology of impaired ovarian function in galactosaemia Hum. Reprod. Update, September 1, 2006; 12(5): 573 - 584. [Abstract] [Full Text] [PDF]
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S. Mazerbourg and A. J.W. Hsueh Genomic analyses facilitate identification of receptors and signalling pathways for growth differentiation factor 9 and related orphan bone morphogenetic protein/growth differentiation factor ligands Hum. Reprod. Update, July 1, 2006; 12(4): 373 - 383. [Abstract] [Full Text] [PDF]
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I. B Carlsson, M. P E Laitinen, J. E Scott, H. Louhio, L. Velentzis, T. Tuuri, J. Aaltonen, O. Ritvos, R. M L Winston, and O. Hovatta Kit ligand and c-Kit are expressed during early human ovarian follicular development and their interaction is required for the survival of follicles in long-term culture. Reproduction, April 1, 2006; 131(4): 641 - 649. [Abstract] [Full Text] [PDF]
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C. Wang and S. K. Roy Expression of Growth Differentiation Factor 9 in the Oocytes Is Essential for the Development of Primordial Follicles in the Hamster Ovary Endocrinology, April 1, 2006; 147(4): 1725 - 1734. [Abstract] [Full Text] [PDF]
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B. K. Campbell, C. J. H. Souza, A. J. Skinner, R. Webb, and D. T. Baird Enhanced Response of Granulosa and Theca Cells from Sheep Carriers of the FecB Mutation in Vitro to Gonadotropins and Bone Morphogenic Protein-2, -4, and -6 Endocrinology, April 1, 2006; 147(4): 1608 - 1620. [Abstract] [Full Text] [PDF]
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B. C Jayawardana, T. Shimizu, H. Nishimoto, E. Kaneko, M. Tetsuka, and A. Miyamoto Hormonal regulation of expression of growth differentiation factor-9 receptor type I and II genes in the bovine ovarian follicle. Reproduction, March 1, 2006; 131(3): 545 - 553. [Abstract] [Full Text] [PDF]
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E. Clelland, G. Kohli, R. K. Campbell, S. Sharma, S. Shimasaki, and C. Peng Bone Morphogenetic Protein-15 in the Zebrafish Ovary: Complementary Deoxyribonucleic Acid Cloning, Genomic Organization, Tissue Distribution, and Role in Oocyte Maturation Endocrinology, January 1, 2006; 147(1): 201 - 209. [Abstract] [Full Text] [PDF]
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M. K. Skinner Regulation of primordial follicle assembly and development Hum. Reprod. Update, September 1, 2005; 11(5): 461 - 471. [Abstract] [Full Text] [PDF]
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D. Goswami and G. S. Conway Premature ovarian failure Hum. Reprod. Update, July 1, 2005; 11(4): 391 - 410. [Abstract] [Full Text] [PDF]
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P.A. Johnson, M.J. Dickens, T.R. Kent, and J.R. Giles Expression and Function of Growth Differentiation Factor-9 in an Oviparous Species, Gallus domesticus Biol Reprod, May 1, 2005; 72(5): 1095 - 1100. [Abstract] [Full Text] [PDF]
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J.L. Juengel and K.P. McNatty The role of proteins of the transforming growth factor-{beta} superfamily in the intraovarian regulation of follicular development Hum. Reprod. Update, March 1, 2005; 11(2): 144 - 161. [Abstract] [Full Text] [PDF]
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N. Kaivo-Oja, D. G. Mottershead, S. Mazerbourg, S. Myllymaa, S. Duprat, R. B. Gilchrist, N. P. Groome, A. J. Hsueh, and O. Ritvos Adenoviral Gene Transfer Allows Smad-Responsive Gene Promoter Analyses and Delineation of Type I Receptor Usage of Transforming Growth Factor-{beta} Family Ligands in Cultured Human Granulosa Luteal Cells J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 271 - 278. [Abstract] [Full Text] [PDF]
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L.J. McKenzie, S.A. Pangas, S.A. Carson, E. Kovanci, P. Cisneros, J.E. Buster, P. Amato, and M.M. Matzuk Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF Hum. Reprod., December 1, 2004; 19(12): 2869 - 2874. [Abstract] [Full Text] [PDF]
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G. A. R. Maciel, E. C. Baracat, J. A. Benda, S. M. Markham, K. Hensinger, R. J. Chang, and G. F. Erickson Stockpiling of Transitional and Classic Primary Follicles in Ovaries of Women with Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5321 - 5327. [Abstract] [Full Text] [PDF]
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T. Shimizu, Y. Miyahayashi, M. Yokoo, Y. Hoshino, H. Sasada, and E. Sato Molecular cloning of porcine growth differentiation factor 9 (GDF-9) cDNA and its role in early folliculogenesis: direct ovarian injection of GDF-9 gene fragments promotes early folliculogenesis Reproduction, November 1, 2004; 128(5): 537 - 543. [Abstract] [Full Text] [PDF]
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K P McNatty, L G Moore, N L Hudson, L D Quirke, S B Lawrence, K Reader, J P Hanrahan, P Smith, N P Groome, M Laitinen, et al. The oocyte and its role in regulating ovulation rate: a new paradigm in reproductive biology Reproduction, October 1, 2004; 128(4): 379 - 386. [Abstract] [Full Text] [PDF]
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S. Pennetier, S. Uzbekova, C. Perreau, P. Papillier, P. Mermillod, and R. Dalbies-Tran Spatio-Temporal Expression of the Germ Cell Marker Genes MATER, ZAR1, GDF9, BMP15,andVASA in Adult Bovine Tissues, Oocytes, and Preimplantation Embryos Biol Reprod, October 1, 2004; 71(4): 1359 - 1366. [Abstract] [Full Text] [PDF]
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R. Prochazka, L. Nemcova, E. Nagyova, and J. Kanka Expression of Growth Differentiation Factor 9 Messenger RNA in Porcine Growing and Preovulatory Ovarian Follicles Biol Reprod, October 1, 2004; 71(4): 1290 - 1295. [Abstract] [Full Text] [PDF]
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J. P. Hanrahan, S. M. Gregan, P. Mulsant, M. Mullen, G. H. Davis, R. Powell, and S. M. Galloway Mutations in the Genes for Oocyte-Derived Growth Factors GDF9 and BMP15 Are Associated with Both Increased Ovulation Rate and Sterility in Cambridge and Belclare Sheep (Ovis aries) Biol Reprod, April 1, 2004; 70(4): 900 - 909. [Abstract] [Full Text] [PDF]
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S. Mazerbourg, C. Klein, J. Roh, N. Kaivo-Oja, D. G. Mottershead, O. Korchynskyi, O. Ritvos, and A. J. W. Hsueh Growth Differentiation Factor-9 Signaling Is Mediated by the Type I Receptor, Activin Receptor-Like Kinase 5 Mol. Endocrinol., March 1, 2004; 18(3): 653 - 665. [Abstract] [Full Text] [PDF]
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J. Wang and S. K. Roy Growth Differentiation Factor-9 and Stem Cell Factor Promote Primordial Follicle Formation in the Hamster: Modulation by Follicle-Stimulating Hormone Biol Reprod, March 1, 2004; 70(3): 577 - 585. [Abstract] [Full Text] [PDF]
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D. M. Duffy Growth Differentiation Factor-9 Is Expressed by the Primate Follicle Throughout the Periovulatory Interval Biol Reprod, August 1, 2003; 69(2): 725 - 732. [Abstract] [Full Text] [PDF]
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C. Glister, N. P. Groome, and P. G. Knight Oocyte-Mediated Suppression of Follicle-Stimulating Hormone- and Insulin-Like Growth Factor-Induced Secretion of Steroids and Inhibin-Related Proteins by Bovine Granulosa Cells In Vitro: Possible Role of Transforming Growth Factor {alpha} Biol Reprod, March 1, 2003; 68(3): 758 - 765. [Abstract] [Full Text] [PDF]
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B. Mandon-Pepin, A. Oustry-Vaiman, B. Vigier, F. Piumi, E. Cribiu, and C. Cotinot Expression Profiles and Chromosomal Localization of Genes Controlling Meiosis and Follicular Development in the Sheep Ovary Biol Reprod, March 1, 2003; 68(3): 985 - 995. [Abstract] [Full Text] [PDF]
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E. Wiater and W. Vale Inhibin Is an Antagonist of Bone Morphogenetic Protein Signaling J. Biol. Chem., February 28, 2003; 278(10): 7934 - 7941. [Abstract] [Full Text] [PDF]
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N. Kaivo-Oja, J. Bondestam, M. Kamarainen, J. Koskimies, U. Vitt, M. Cranfield, K. Vuojolainen, J. P. Kallio, V. M. Olkkonen, M. Hayashi, et al. Growth Differentiation Factor-9 Induces Smad2 Activation and Inhibin B Production in Cultured Human Granulosa-Luteal Cells J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 755 - 762. [Abstract] [Full Text] [PDF]
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J.-S. Roh, J. Bondestam, S. Mazerbourg, N. Kaivo-Oja, N. Groome, O. Ritvos, and A. J. W. Hsueh Growth Differentiation Factor-9 Stimulates Inhibin Production and Activates Smad2 in Cultured Rat Granulosa Cells Endocrinology, January 1, 2003; 144(1): 172 - 178. [Abstract] [Full Text] [PDF]
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R. A. Taft, J. M. Denegre, F. L. Pendola, and J. J. Eppig Identification of Genes Encoding Mouse Oocyte Secretory and Transmembrane Proteins by a Signal Sequence Trap Biol Reprod, September 1, 2002; 67(3): 953 - 960. [Abstract] [Full Text] [PDF]
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E. E. Nilsson and M. K. Skinner Growth and Differentiation Factor-9 Stimulates Progression of Early Primary but Not Primordial Rat Ovarian Follicle Development Biol Reprod, September 1, 2002; 67(3): 1018 - 1024. [Abstract] [Full Text] [PDF]
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U. A. Vitt, S. Mazerbourg, C. Klein, and A. J.W. Hsueh Bone Morphogenetic Protein Receptor Type II Is a Receptor for Growth Differentiation Factor-9 Biol Reprod, August 1, 2002; 67(2): 473 - 480. [Abstract] [Full Text] [PDF]
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N. Yamamoto, L. K. Christenson, J. M. MCAllister, and J. F. Strauss III Growth Differentiation Factor-9 Inhibits 3'5'-Adenosine Monophosphate-Stimulated Steroidogenesis in Human Granulosa and Theca Cells J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2849 - 2856. [Abstract] [Full Text] [PDF]
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R. Jaatinen, J. Bondestam, T. Raivio, K. Hilden, L. Dunkel, N. Groome, and O. Ritvos Activation of the Bone Morphogenetic Protein Signaling Pathway Induces Inhibin {beta}B-Subunit mRNA and Secreted Inhibin B Levels in Cultured Human Granulosa-Luteal Cells J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1254 - 1261. [Abstract] [Full Text] [PDF]
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F. L. Teixeira Filho, E. C. Baracat, T. H. Lee, C. S. Suh, M. Matsui, R. J. Chang, S. Shimasaki, and G. F. Erickson Aberrant Expression of Growth Differentiation Factor-9 in Oocytes of Women with Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1337 - 1344. [Abstract] [Full Text] [PDF]
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J. G. Hreinsson, J. E. Scott, C. Rasmussen, M. L. Swahn, A. J. W. Hsueh, and O. Hovatta Growth Differentiation Factor-9 Promotes the Growth, Development, and Survival of Human Ovarian Follicles in Organ Culture J. Clin. Endocrinol. Metab., January 1, 2002; 87(1): 316 - 321. [Abstract] [Full Text] [PDF]
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 P. Mulsant, F. Lecerf, S. Fabre, L. Schibler, P. Monget, I. Lanneluc, C. Pisselet, J. Riquet, D. Monniaux, I. Callebaut, et al. Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Merino ewes PNAS, April 24, 2001; 98(9): 5104 - 5109. [Abstract] [Full Text] [PDF]
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T. Wilson, X.-Y. Wu, J. L. Juengel, I. K. Ross, J. M. Lumsden, E. A. Lord, K. G. Dodds, G. A. Walling, J. C. McEwan, A. R. O'Connell, et al. Highly Prolific Booroola Sheep Have a Mutation in the Intracellular Kinase Domain of Bone Morphogenetic Protein IB Receptor (ALK-6) That Is Expressed in Both Oocytes and Granulosa Cells Biol Reprod, April 1, 2001; 64(4): 1225 - 1235. [Abstract] [Full Text]
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 A. R. Zinn The X Chromosome and the Ovary Reproductive Sciences, January 1, 2001; 8(1_suppl): S34 - S36. [Abstract] [PDF]
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B. S. Dunbar, S. Prasad, C. Carino, and S. M. Skinner The Ovary as an Immune Target Reproductive Sciences, January 1, 2001; 8(1_suppl): S43 - S48. [Abstract] [PDF]
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U. A. Vitt, E. A. McGee, M. Hayashi, and A. J. W. Hsueh In Vivo Treatment with GDF-9 Stimulates Primordial and Primary Follicle Progression and Theca Cell Marker CYP17 in Ovaries of Immature Rats Endocrinology, October 1, 2000; 141(10): 3814 - 3820. [Abstract] [Full Text] [PDF]
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H. Chong, S. A. Pangas, D. J. Bernard, E. Wang, J. Gitch, W. Chen, L. B. Draper, E. T. Cox, and T. K. Woodruff Structure and Expression of a Membrane Component of the Inhibin Receptor System Endocrinology, July 1, 2000; 141(7): 2600 - 2607. [Abstract] [Full Text] [PDF]
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 E. A. McGee and A. J. W. Hsueh Initial and Cyclic Recruitment of Ovarian Follicles Endocr. Rev., April 1, 2000; 21(2): 200 - 214. [Abstract] [Full Text]
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U.A. Vitt, M. Hayashi, C. Klein, and A.J.W. Hsueh 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, February 1, 2000; 62(2): 370 - 377. [Abstract] [Full Text]
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F. Otsuka, Z. Yao, T.-h. Lee, S. Yamamoto, G. F. Erickson, and S. Shimasaki Bone Morphogenetic Protein-15. IDENTIFICATION OF TARGET CELLS AND BIOLOGICAL FUNCTIONS J. Biol. Chem., December 8, 2000; 275(50): 39523 - 39528. [Abstract] [Full Text] [PDF]
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F. Otsuka, S. Yamamoto, G. F. Erickson, and S. Shimasaki Bone Morphogenetic Protein-15 Inhibits Follicle-stimulating Hormone (FSH) Action by Suppressing FSH Receptor Expression J. Biol. Chem., March 30, 2001; 276(14): 11387 - 11392. [Abstract] [Full Text] [PDF]
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