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WO2003102199A1 - Nouvelles sequences gdf-9 et gdf-9b (bmp-15) destinees a modifier la fonction ovarienne et la vitesse d'ovulation chez des mammiferes - Google Patents

Nouvelles sequences gdf-9 et gdf-9b (bmp-15) destinees a modifier la fonction ovarienne et la vitesse d'ovulation chez des mammiferes Download PDF

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Publication number
WO2003102199A1
WO2003102199A1 PCT/NZ2003/000109 NZ0300109W WO03102199A1 WO 2003102199 A1 WO2003102199 A1 WO 2003102199A1 NZ 0300109 W NZ0300109 W NZ 0300109W WO 03102199 A1 WO03102199 A1 WO 03102199A1
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gdf
mutated
nucleic acid
dna
mammal
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PCT/NZ2003/000109
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English (en)
Inventor
George Henry Davis
Susan May Galloway
Scott Michael GREGAN
Jennifer Lee Juengel
Kenneth Pattrick Mcnatty
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Ovita Limited
HANRAHAN, James, Patrick
Mulsant, Philippe
POWELL, Richard, Patrick
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Application filed by Ovita Limited, HANRAHAN, James, Patrick, Mulsant, Philippe, POWELL, Richard, Patrick filed Critical Ovita Limited
Priority to BR0311465-1A priority Critical patent/BR0311465A/pt
Priority to AU2003235525A priority patent/AU2003235525B2/en
Priority to EP03723538A priority patent/EP1551973A4/fr
Priority to CA002490051A priority patent/CA2490051A1/fr
Priority to JP2004510436A priority patent/JP2006510345A/ja
Publication of WO2003102199A1 publication Critical patent/WO2003102199A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/18—Feminine contraceptives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to new sequences for altering mammalian ovarian function and ovulation rate.
  • the invention broadly concerns a novel mutation in the GDF-9 gene and two novel mutations in the GDF-9B gene. These mutations have been found to be involved in increasing the ovulation rate in heterozygous female mammals; or causing sterility in homozygous female mammals.
  • GDF-9 and GDF-9B also known as BMP 15 code for proteins which are expressed exclusively in the oocyte of the developing follicle, and which play an essential role in mammalian fertility.
  • GDF-9 is a member of the transfo ⁇ -ning growth factor beta (TGF ⁇ ) superfamily (McPherron and Lee, 1993) which is expressed in oocytes from the primary stage of follicular development until ovulation (McGrath et al, 1995; Laitinen et al, 1998).
  • TGF ⁇ transfo ⁇ -ning growth factor beta
  • GDF-9B is closely related to GDF-9 (Dube et al, 1998; Laitinen et al, 1998) and is expressed in mouse oocytes at the same time as GDF-9, but in human primary follicles slightly later than GDF-9.
  • GDF-9 and GDF-9B have now been shown to be expressed exclusively in the developing oocyte in humans (Aaltonen et al, 1999), rodents (Laitinen et al, 1998; Dube et al, 1998; Jaatinen et al, 1999), -ruminants (Bodensteiner et al, 1999; Bodensteiner et al, 2000; Galloway et al, 2000) and marsupials (Eckery et al, 2002).
  • sheep expression of GDF-9 can be seen in primordial follicles whereas GDF-9B is expressed in primary follicles (Bodensteiner et al, 1999; Galloway et al, 2000).
  • GDF-9 is an essential growth factor for folliculogenesis in mice.
  • Female GDF-9 knockout mice (GDF-9 -/-) are infertile due to a block in follicular development at the primary stage (Dong et al, 1996).
  • GDF-9B does not appear to be crucial for mouse folliculogenesis as knockout female mice (BMP 15 -/-) are fertile (Yan et al, 2001), even though fecundity is somewhat reduced.
  • BMP 15 -/- are fertile
  • GDF-9B is essential for folliculogenesis in sheep as those carrying two copies of naturally-occurring inactivating GDF-9B mutations are infertile due to a block in follicular development at the primary stage (Galloway et al, 2000).
  • Double knockouts of both GDF-9 and GDF-9B in mice are infertile with a similar phenotype to GDF-9 -/- mice alone, but GDF-9B knockout mice (BMP 15 -/-) with one active copy of GDF-9, have a lower fecundity than BMP 15 -/- females (Yan et al, 2001), suggesting that the relative dose of these gene products may also play a role in mice.
  • GDF-9 maps to a region of sheep chromosome 5 (Sadighi et al., 2002) which is syntenic to the map locations for GDF-9 on human chromosome 5 and mouse chromosome 11.
  • GDF-9B maps to the sheep X chromosome (Galloway et al., 2000) in a region of the chromosome syntenic to the map locations for GDF-9B on the human and mouse X chromosomes (Dube et al, 1998; Aaltonen et al, 1999).
  • GDF-9 and GDF-9B are coded as prepropeptides containing a signal peptide, a proregion and a C-terminal mature region which is the biologically active peptide. Cleavage of the mature region from the proregion is carried out by an intracellular furin-like protease, and occurs at a conserved furin protease cleavage site.
  • TGF ⁇ superfamily are biologically active as dimers, and although GDF-9 and GDF-9B do not contain the cysteine molecule responsible for covalent interchain disulphide bonding seen in other members of the family, these molecules are thought to be biologically active as dimers (Galloway et al, 2000; Yan et al., 2001). However it is unclear whether the physiologically active dimers are homodimers (GDF-9-GDF-9 and GDF-9B-GDF-9B), or heterodi ers (GDF-9- GDF-9B) or whether all three dimer forms play a role.
  • GDF-9 homodimers play a more important role in the mouse but in sheep the GDF-9B homodimers are the most bioactive (Yan et al, 2001). It is unclear whether any such difference is related to the fact that sheep are mono-ovulatory animals (maturing usually only one egg per cycle) whereas mice are poly-ovulatory.
  • GDF-9 and GDF-9B play crucial roles in controlling and maintaining fertility in mammals, and understanding the nature of their actions is essential for the development of therapies.
  • the sheep GDF-9 gene spans about 2.5 kb and contains 2 exons separated by a single 1126 bp intron (Bodensteiner et al, 1999).
  • the full length coding sequence is 1359 nucleotides long and encodes a pre-propeptide of 453 amino acid residues (Genbank accession number AF078545).
  • a pre-pro region of 318 residues includes a predicted signal sequence, and ends with the RHRR furin protease cleavage site at residues 315 - 318. Residues 319 to 453 beyond the cleavage site code for the 135 amino acid mature active peptide.
  • the amino acid sequence of the sheep GDF-9 mature peptide is 92.8 % similar to the human mature peptide and 87.1 % similar to the mouse mature peptide.
  • Sheep GDF-9B has previously been sequenced by us (Galloway et al. 2000; Genbank accession nos. AF236078, AF236079) and has a very similar gene structure to GDF-9.
  • the full length coding sequence of 1179 nucleotides is contained in two exons, separated by an intron of about 5.4 kb, and encodes a pre-propeptide of 393 amino acid residues.
  • a pre-pro region of 268 residues includes a predicted signal sequence, and ends with the RRAR furin protease cleavage site at residues 265 - 268. Residues 269 to 393 beyond the cleavage site code for a 125 amino acid mature active peptide.
  • the amino acid sequence of the sheep GDF-9B mature peptide is 78.3 % similar to the human mature peptide and 78.6 % similar to the mouse mature peptide.
  • Hanna sheep have a single C to T mutation at nucleotide 871 of the GDF-9B coding sequence (nucleotide 67 of the mature GDF-9B peptide coding region) which produces a premature stop codon in the place of a glutamic acid (Q) at amino acid residue 291 (residue 23 of the mature protein).
  • Inverdale sheep have a distinct T to A mutation at nucleotide 896 (nucleotide 92 of the mature GDF-9B peptide coding region) which substitutes valine (V) for aspartic acid (D) at residue 299 (residue 31 of the mature peptide).
  • the Cambridge breed was established at the Cambridge University farm in 1964 by screening 54 ewes selected for their high prolificacy from nine British sheep breeds. Ewes within the screened flock were subsequently selected on high litter size. Ewes with the highest ovulation rates were selected from this flock in 1984 to provide the foundation animals for the flock now maintained at Teagasc Sheep Research Centre in Ireland (Hanrahan, 1991). A progeny test of 10 Cambridge rams, descended from the flock in Ireland, gave progeny mean ovulation rates ranging from 2.1 - 4.2.
  • the Belclare breed was established in 1978 at the Belclare Research Centre of Teagasc in Ireland by crossing three populations of prolific sheep assembled by Teagasc in Ireland. These were Fingalway, High Fertility, and Lleyn sheep (Hanrahan, 1991).
  • the Fingalway was an interbred cross (from FI) of the Finnish Landrace and Galway breeds; the Lleyn is a breed native to north west Wales and selected animals were imported into Ireland in 1975 by Teagasc for the purposed of developing the Belclare breed; the High Fertility was developed in Ireland during the 1960s from ewes with exceptional litter size performance collected from farms in Ireland between 1963 and 1965.
  • a subline of the Belclare (called F700 line) was derived from Belclare sheep that had exceptionally high ovulation rates (Hanrahan 1991). Progeny of 10 Belclare rams had mean ovulation rates ranging from 1.9 - 4.2.
  • the present invention is concerned with novel mutated GDF-9 and GDF-9B gene sequences which alter mammalian ovarian function and ovulation rate.
  • the invention broadly has application in increasing or decreasing the ovulation rate, or causing sterility in a female mammal, and additionally encompasses regulation of the function of the corpeus luteum.
  • the present invention concerns a novel mutation in GDF-9 which increases ovulation rate in heterozygotes and causes sterility in homozygotes for this gene.
  • the present invention also concerns two mutations in GDF-9B. Heterozygotes for either one of these mutations have an increased ovulation rate. Mammals which are heterozygotes for both mutations in GDF-9B (where each mutation is on a separate X chromosome) are sterile.
  • the inventors have also discerned that in female mammals that are heterozygous for the mutated GDF-9 gene and heterozygous for one of the GDF-9B gene mutations, an even higher ovulation rate exists than in mammals heterozygous for one mutation in either GDF-9 or GDF-9B alone.
  • Knowledge of the mutated gene sequences can be applied to a test for identifying heterozygous or homozygous female and male mammals carrying the mutated GDF-9B and/or GDF-9 genes.
  • This knowledge of the biological function of the genes and their mutations can also be utilised to increase or decrease the ovulation rate of female mammals, or to induce sterility or reduced fertility in female mammals.
  • an increase in ovulation rate in mammals may be induced by mimicking the heterozygous state, e.g. by reducing the biological activity of GDF-9 and/or GDF-9B by aound 50%.
  • said antibodies may be administered directly in a partial or short term passive immunisation regime.
  • a decrease in ovulation rate sufficient to reduce fertility or induce sterility may be induced by mimicking the homozygous state, e.g. by reducing the biological activity of GDF-9 and/or GDF-9B to around zero.
  • This can be achieved by a frill or long term active immunisation regime whereby wild-type GDF-9 and/or GDF-9B or a functional variant or fragment thereof is administered to raise sufficient antibodies to affectively neutralise all of the endogenous GDF-9 and/or GDF-9B.
  • said antibodies may be administered directly in a full or long term passive immunisation regime. Where the effect is permanent, sterility in the mammal is induced. Where the effect is reversible or temporary, a contraceptive effect is induced.
  • Figure la shows the predicted amino acid sequence of sheep GDF9 protein. Numbers at the start of each line indicate amino acid positions of full- length unprocessed protein. Numbers in brackets indicate amino acid positions of the mature peptide.
  • the RRHR furin protease cleavage site and predicted start of the mature processed peptide is shaded in grey.
  • the filled triangle indicates the position of the single intron within the gene.
  • the open triangles indicate positions of mutations that confer amino acid substitutions but are not associated with the sterility phenotype.
  • Figure 2a shows the predicted amino acid sequence of sheep GDF9B protein. Numbers at the start of each line indicate amino acid positions of full- length unprocessed protein. Numbers in brackets indicate amino acid positions of the mature peptide.
  • the RRAR furin protease cleavage site and predicted start of the mature processed peptide is shaded in grey.
  • the filled triangle indicates the position of the single intron within the gene.
  • the open triangle indicates the position of a single Leu deletion polymorphism.
  • the position of the [SI] and [S2] mutations associated with sterility are shaded black;
  • Figure 2b shows nucleotide substitutions of two GDF-9B sequence mutations which result in an amino acid change compared to the wild-type sequence in Irish Cambridge and F700 Belclare sheep;
  • Figure 3 a shows a table representing the genotype and phenotype of sire R830 mated to three ewes 9704, 8783 and 7810, and their six female offspring.
  • the phenotype of each animal is indicated as sterile S, fertile F or n/a (not applicable) for the male.
  • the genotype of each animal is shown as ++ (wild type female for X-linked [SI] and [S2] mutations, wild type male and female [787] autosomal mutation), +Y (wild type male for X- linked [SI] or [S2]).
  • Double copy carriers of the mutation are T/T, Sl/Sl or S2/S2. Single copy carriers are T/+, S1/+ or S2/+.
  • Sire R830 is hemizygous for X-linked [S2] as he can only carry one copy;
  • Figure 3b shows a table representing the genotypes and phenotypes within a F700 Cambridge pedigree.
  • the pedigree represents sire 962101 mated to two ewes 962158 and 976234, and their four female offspring, and sire 930142 mated to ewe 8874 and their one female offspring.
  • the phenotype of each animal is indicated as sterile S, fertile F or n/a (not applicable) for the male.
  • genotype of each animal is shown as ++ (wild type female for X-linked [SI] and [S2] mutations, wild type male and female for [787] autosomal mutations), +Y (wild type male for X- linked [SI] or [S2].
  • Double copy carriers of the mutations are T/T or SI /SI. Single copy carriers are T/+ or S1/+. Sires are hemizygous for X- linked [SI] as they can only carry one copy.
  • Figure 4 shows the nucleotide and amino acid sequences of wildtype sheep GDF-9 showing the positions of mutations in Irish Cambridge and F700 Belclare sheep. Numbers on the right indicate nucleotide position from the atg start codon. Numbers under each line indicate amino acid residue numbering from the start of the mature processed active peptide. Negative numbers indicate amino acids in the pro-region of the protein. The position of the single intron is marked by white boxes inserted into the sequence. Positions of the eight nucleotide polymorphisms are marked in bold within boxes, and named according to Table 1 using square brackets [ ]. The amino acids residues which are changed by the nucleotide polymorphisms are underlined. The taa stop codon indicates the end of the protein;
  • Figure 5 shows the nucleotide and amino acid sequences of sheep GDF-9B showing the positions of mutations in Irish Cambridge and F700 Belclare sheep. Numbers on the right indicate nucleotide position from the atg start codon. Numbers under each line indicate amino acid residue numbering from the start of the mature processed active peptide. Negative numbers indicate amino acids in the pro-region of the protein. The position of the single intron is marked by a dashed inserted into the sequence. Positions of the four nucleotide polymorphisms are marked in bold within boxes, and named according to Table 1 using square brackets [ ]. The amino acids residues which are changed by the nucleotide polymorphisms are underlined. Asterisks (***) indicate the positions of the previously reported Hanna (Gin to Stop codon) and Inverdale (Val to Asp codon) mutations. The tga stop codon indicates the end of the protein;
  • Figure 6 shows the alignment of GDF-9 and GDF-9B protein sequence with other members of the TGF ⁇ superfamily members for which structures have been determined.
  • the furin processing site is indicated as a solid gray block at the start of the sequences.
  • the mature processed protein begins at amino acid residue position 4.
  • conserved cysteine molecules involved in disulphide bonds are shown in grey shading. Numbers along the bottom provide a relative reference to amino acid position, but do not represent the real amino acid residue number of each protein because gaps have been introduce to allow alignment of conserved protein regions.
  • the asterisk * indicates the conserved cysteine that is present in most other TGF ⁇ family members except GDF9 and GDF9B, and which is responsible for the interchain disulphide bond present in most dimers.
  • Boxed letters indicate the [787] serine (S) in GDF9 which is changed to phenylalanine in the mutants (position 86 on this diagram), and the [S2] serine (S) in GDF9B which is changed to isoleucine (position 118 in this diagram);
  • Figure 8 shows the average concentrations of progesterone in plasma following synchronization of luteal regression. Ewes were administered 100 ml of KLH, GDF9 peptide or BMP 15 peptide antiplasma i.v. 4 days before synchronization with Estrumate (i.e. PGF 2 ⁇ , arrowed).
  • the present invention is directed to new naturally-occurring mutations in the sheep GDF-9 and GDF-9B genes. It is shown for the first time that mutation of the GDF-9 gene causes increased ovulation rate as well as infertility in a manner similar to inactivating mutations in GDF-9B, and that GDF-9 is also essential for maintaining normal ovarian folliculogenesis in sheep. Furthermore, it is shown for the first time in any species, that sheep which are heterozygous for both GDF-9 and GDF-9B mutations have higher ovulation rates than sheep that are heterozygous for GDF-9 or GDF-9B mutations alone. These observations are supported by genotype, phenotype and immunisation data discussed below.
  • an isolated mutated GDF-9 nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
  • the present invention also provides an isolated mutated GDF-9 polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, 4 or 6, or a functional fragment or variant thereof.
  • the present invention also provides an isolated mutated GDF-9B nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: a) SEQ ID NOs. 7, 9, 11, 13, 15 or 17; b) a sequence complementary to the molecule defined in a) c) an anti-sense sequence to any of the molecules defined in a) or b).
  • the present invention further provides an isolated mutated GDF-9B polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 8, 10, 12, 14, 16 or 18.
  • an immunisation regime which could mimic these genotypes would be useful in modulating ovulation in female mammals.
  • a regime which would reduce the activity of endogenous GDF-9B and/or GDF-9 to about one half (as in heterozygous animals whereby only 50% of active molecules are expressed) could be used to increase ovulation and enhance fertility in female mammals.
  • an immunisation regime which would reduce the activity of endogenous GDF-9 and/or GDF-9B to approximately zero (as in homozygous animals where no active molecules are expressed) could be used to induce sterility.
  • the present invention further provides an isolated GDF-9 nucleic acid molecule comprising a mutation in at least one codon associated with receptor binding and/or dimerisation.
  • the mutation preferably results in an amino acid substitution in the polypeptide encoded by the nucleic acid molecule, and said amino acid substitution is preferably present in the receptor binding domain and causes a disruption in receptor binding.
  • the amino acid substitution may be present in the dimersation domain to cause a disruption in dimerisation.
  • the invention further provides an isolated GDF-9B nucleic acid molecule comprising a mutation in at least one codon associated with receptor binding and/or dimerisation.
  • the mutation preferably results in an amino acid substitution in the polypeptide encoded by the nucleic acid molecule, and said amino acid substitution is preferably present in the receptor binding domain and causes a disruption in receptor binding.
  • the amino acid substitution may be present in the dimerisation domain to cause disruption in dimerisation.
  • Suitable programs for ascertaining the structure of polypeptides from the amino acid sequence which can used to determine the regions of the nucleotide sequence associated with dimerisation and/or receptor binding will be known to persons skilled in the art.
  • suitable computer programs include The Modeller by Rockerfeller University and The SWISS Model developed by Swiss Protein database.
  • the present invention also provides a method of identifying a mammal which carries a mutated nucleic acid molecule encoding GDF-9B, said method comprising the steps of:
  • step v) determining whether the GDF-9B sequence DNA obtained in step v) carries a mutation associated with sterility or increased ovulation.
  • the present invention further provides a method of identifying a mammal which carries a mutated nucleic acid molecule encoding GDF-9, said method comprising the steps of:
  • probe and primers that can be used in this method also forms a part of this invention.
  • Said probes and primers may comprise a fragment of the nucleic acid molecule of the invention capable of hybridising under stringent conditions to a mutated GDF-9 or GDF-9B gene sequence.
  • Such probes and primers are also useful, in studying the structure and function of the mutated genes, and for obtaining homologues of the genes from mammals other than sheep expressing the Cambridge and Belclare phenotypes.
  • Nucleic acid probes and primers can be prepared based on nucleic acids according to the present invention or sequences complementary thereto.
  • a "probe” comprises a single stranded nucleic acid molecule having a known sequence which is attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. Such probes are used to locate and mark target DNA or RNA sequence by hybridizing to it.
  • a “primer” is a short nucleic acid, preferably DNA, 15 or more nucleotides in length, which are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, preferably a DNA polymerase.
  • Primer pairs can be used for amplification of a nucleic acid sequence, eg by the polymerase chain reaction (PCR) or other nucleic acid amplification methods well known in the art.
  • PCR-primer pairs can be derived from the sequence of a nucleic acid according to the present invention, for example, by using computer programs intended for that purpose such as Primer (Version 0.5 ® 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).
  • Probes or primers can be free in solution or covalently or noncovalently attached to a solid support by standard means.
  • stringent conditions are conditions that permit the primer pair to hybridise only to the target nucleic acid sequence to which a primer having the corresponding wild type sequence (or its complement) would bind.
  • Nucleic acid hybridization is affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridising nucleic acids, as will be readily appreciated by those skilled in the art.
  • the term "specific for (a target sequence)" indicates that the probe or primer hybridises under stringent conditions only to the target sequence in a given sample comprising the target sequence.
  • stringent hybridization conditions are 6 x SSC at 55°C.
  • the present invention provides the use of a nucleic acid molecule which is complementary to either strand of the mutated DNA of SEQ ID NOs. 11 or 17 as a marker to identify a mammal carrying a mutated nucleic acid molecule encoding GDF-9B.
  • either strand' refers to both the strand of DNA shown in the Sequence ID Number that is being referred to or its complementary strand which is not shown in the sequence listing but which can be determined therefrom.
  • the present invention provides the use of a marker as described above in a method of DNA marker assisted selection of mammals carrying mutated GDF-9B associated with either enhanced ovulation or sterility.
  • the present invention provides the use of a nucleic acid molecule which is complementary to either strand of the mutated DNA of SEQ ID NO 5 as a marker to identify a mammal carrying a mutated nucleic acid molecule encoding GDF-9.
  • the present invention further provides the use of a marker as described above in a method of DNA assisted selection of mammals carrying mutated GDF-9 either enhanced ovulation or sterility.
  • the above markers and methods of marker assisted selection are useful to identify sequence variants in individual animals that are associated with increased ovulation of that animal. Although these variants may not necessarily give rise to the increased ovulation or sterility trait directly, they will be sufficiently closely associated with it to predict the trait.
  • the methods by which these sequence variants are identified are known in the art, and include, but are not limited to, restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism AFLP, direct sequencing of DNA within or associated with the GDF-9 gene, or identification and characterisation of variable number of tandem repeats (VNTR), also known as microsatellite polymorphisms.
  • RFLP restriction fragment length polymorphism
  • VNTR variable number of tandem repeats
  • the genetic marker may have utility in DNA selection of animals having increased ovulation.
  • the present invention provides a construct or vector comprising a nucleic acid molecule substantially as described above.
  • construct refers to an artificially assembled or isolated nucleic acid molecule which includes the gene of interest.
  • a construct may include the gene or genes of interest and appropriate regulatory sequences. It should be appreciated that the inclusion of regulatory sequences in a construct is optional for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used.
  • construct includes vectors but should not be seen as being limited thereto.
  • vector as used herein encompasses both cloning and expression vectors. Vectors are often recombinant molecules containing nucleic acid molecules from several sources.
  • a "cloning vector” refers to a nucleic acid molecule originating or derived from a virus, a plasmid or a cell of a higher organism into which another exogenous (foreign) nucleic acid molecule of interest, of appropriate size can be integrated without loss of the vector's capacity for self-replication.
  • vectors can be used to introduce at least one foreign nucleic acid molecule of interest (e.g. gene of interest) into host cells, where the gene can be reproduced in large quantities.
  • an "expression vector” refers to a cloning vector which also contains the necessary regulatory sequences to allow for transcription and translation of the integrated gene of interest, so that the gene product of the gene can be expressed.
  • the cloning vector may be selected according to the host or host cell to be used.
  • Useful vectors will generally have the following characteristics: (a) the ability to self-replicate;
  • (c) desirably, carry genes for a readily selectable marker such as antibiotic resistance.
  • vectors Two major types of vector possessing these characteristics are plasmids and bacterial viruses (bacteriophages or phages).
  • plasmids plasmids and bacterial viruses (bacteriophages or phages).
  • bacterial, insect or mammalian vectors may include the following: the pUC, pBlueScript, pGEM, PGEX, pBK-CMV, lambda ZAP, lambda GEM, pEFIRES-P, pUB6/V5/His, pBCl, pADTrack-CMV, pAdenovator, pAdEasy-1, pSFV-PD, pCA3, pBABE, pPIC9, pA0815, pET and pSP series.
  • this list should not be seen as limiting the scope of the present invention.
  • the 293T cell system with a pEFIRES-P vector (Hobbs S et ⁇ /.,1998, Biochem Biophys Res Commun. 252: 368-72) which confers puromycin resistance may be used.
  • the aforementioned vector may be modified to change the antibiotic resistance gene to bleomycin resistance.
  • the co-expression of two genes and the selection gene can be achieved by constructing a tricistronic expression vector.
  • a corresponding stably transfected insect cell system can also be used, e.g. the S2 cell system using "DES" vector expression system; www.invitrogen.com.
  • GDF's in all tissues of transgenic animals one approach is to use the pUB6/V5-His A vector (www.invitrogen.com) to make the constructs.
  • tissue-specific expression the rat PEPCK 0.6 kb promoter for liver and kidney expression can be included in the construct by replacing the Ubi-C promoter in the pUB6/V5-His A vector with the PEPCK promoter.
  • tissue-specific expression the rat PEPCK 0.6 kb promoter for liver and kidney expression can be included in the construct by replacing the Ubi-C promoter in the pUB6/V5-His A vector with the PEPCK promoter.
  • mammary tissue another promoter system would be preferred.
  • lactoglobulin gene promoter and/or the bovine ⁇ SI casein promoter (e.g. pBCl vector, www.invitrogen.com) to drive the expression of the GDFs into milk.
  • the CMV enhanced ⁇ -actin promoter Okabe M, et al.; FEBS Letters 407: 313-319, 1997) or a modified EF1
  • ⁇ -promoter can be used also (Taboit-Dameron F, et al., Transgenic Research 8:
  • Adenoviruses, retroviruses and alphaviruses are other suitable mammalian expression systems.
  • a typical approach to those skilled in the art is that described by (TC He et al, 1998, Proc Natl Acad Sci USA. 95: 2509-14).
  • the pAd Track-CMV vector or pAdenovator vectors www.qbiogene.com
  • the construct which is then co-transformed with pAd Easy-1 adenoviral plasmid into E. coli to generate a recombinant adenoviral genome which contains a CMV-promoter driven GDF expression cassette.
  • This recombinant adenoviral genome is then transfected into 293T cells to make the virus stock.
  • Alternative methods for generating adenoviruses can also be used for the same purpose (e.g. PCA3 plasmid based gene transfer (www.microbix.com); or COS-TPC method (Miyake S et al, 1996, Proc Natl Acad Sci USA. 93: 1320-4).
  • Non-cytopathogenic Semliki Forest viruses expressing GDF's can be generated using, for example, pSFV-PD vectors as described by Lundstrom et al., Histochem Cell Biol 115: 83-91, 2001.
  • retroviral expression systems based on, for example, pBABE vectors, can be used for expressing GDF's in mammalian cells (Morgenstern, JP and Land, H, 1990; Nucleic Acids Res 18: 3587-3596).
  • Yeast cells e.g. Pichia pastoris, Saccharomyces cerevisiae
  • Yeast cells e.g. Pichia pastoris, Saccharomyces cerevisiae
  • the pPIC9 vector www.invitrogen.com
  • the vector pA0815 is a preferred candidate.
  • Echerichia coli is a standard laboratory expression system in widespread use.
  • the p ⁇ T expression system www.novagen.com
  • GDF-9 and GDF-9B can be used to express recombinant mammalian GDF-9 and GDF-9B
  • the DNA molecules of the invention may be expressed by placing them in operable linkage with suitable control sequences in a replicable expression vector.
  • Control sequences may include origins of replication, a promoter, enhancer and transcriptional terminator sequences amongst others.
  • the selection of the control sequence to be included in the expression vector is dependent on the type of host or host cell intended to be used for expressing the DNA as would be understood by a person skilled in the art.
  • operably linked or grammatical variant thereof as used herein means that the regulatory sequences necessary for expression of the gene of interest are placed in the nucleic acid molecule in the appropriate positions relative to the gene to enable expression of the gene.
  • regulatory sequences refers to certain nucleic acid sequences such as origins of replication, promoters, enhancers, polyadenylation signals, terminators and the like, that enable expression of the nucleic acid molecule of interest.
  • expression as used herein broadly refers to the process by which a nucleic acid molecule is converted by transcription and then translation into a protein.
  • gene refers to a nucleic acid molecule comprising an ordered series of nucleotides that encodes a gene product (i.e. specific protein).
  • the expression vectors useful in the present invention may contain at least one expression control sequence that is operatively linked to the DNA sequence or fragment to be expressed. The control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DNA sequence.
  • Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the glycolytic promoters of yeast acid phosphatase, e.g. Pho5, the promoters of the yeast alpha-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, and cytomegalovirus e.g. the early and late promoters of SV40, and other sequences known to control the expression of genes of prokaryotic and eukaryotic cells and their viruses or combinations thereof.
  • the lac system the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda
  • the glycolytic promoters of yeast acid phosphatase e.g. Pho5
  • the promoters of the yeast alpha-mating factors e.g. the promoters of the yeast alpha-m
  • the vectors In the construction of a vector it is also an advantage to be able to identify the bacterial clone carrying the vector incorporating the foreign DNA. Such assays include measurable colour changes, antibiotic resistance and the like.
  • the ⁇ -galactosidase gene is used, in which clones are detectable as blue or white phenotypes on X-gal plates. This facilitates selection. Once selected, the vectors may be isolated from the culture using standard procedures.
  • transformation and transfection is performed according to standard techniques appropriate to such cells.
  • the calcium treatment process (Cohen, S N Proceedings, National Academy of Science, USA 69 2110 (1972)) may be employed.
  • the calcium phosphate precipitation method of Graeme and Van Der Eb, Virology 52:546 (1978) or liposomal reagents are preferred.
  • the polypeptide encoded can be produced, often in the form of a fusion protein, by culturing the host cells.
  • the polypeptide of the invention may be detected by rapid assays as indicated above.
  • the polypeptide is then recovered and purified as necessary. Recovery and purification can be achieved using any procedures known in the art, for example by absorption onto and elution from an anion exchange resin. This method of producing a polypeptide of the invention constitutes a further aspect of the present invention.
  • Host cells transformed with the vectors or constructs of the invention also form a further aspect of the present invention.
  • host cell refers to a cell which is capable of containing a vector or construct and supports the replication and/or expression of the vector or construct.
  • Suitable hosts cells may include E.coli, yeast or mammalian cells but should not be limited thereto.
  • the present invention also provides a cell line comprising a host cell substantially as described above.
  • Knowledge of the mutated gene sequences can be applied to a test for identifying heterozygous or homozygous female and male mammals carrying the mutated GDF-9B and/or GDF-9 genes.
  • This knowledge of the biological function of the genes and their mutations can also be utilised to increase or decrease the ovulation rate of female mammals, or to induce sterility or reduced fertility in female mammals.
  • an increase in ovulation rate in mammals may be induced by mimicking the heterozygous state whereby only half of the 'normal' amount of GDF-9 and/or GDF-9B is expressed.
  • said antibodies may be administered directly in a partial or short term passive immunisation regime.
  • a decrease in ovulation rate sufficient to reduce fertility or induce sterility may be induced by mimicking the homozygous state, whereby no active GDF-9 and/or GDF-9B is expressed.
  • This can be achieved by a full or long term active immunisation regime whereby wild-type GDF-9 and/or GDF-9B or a functional variant or fragment thereof is administered to raise sufficient antibodies to affectively neutralise all of the endogenous GDF-9 and/or GDF-9B.
  • said antibodies may be administered directly in a full or long term passive immunisation regime. Where the effect is permanent, sterility in the mammal is induced. Where the effect is reversible or temporary, a contraceptive effect is induced.
  • the present invention provides a method altering GDF-9 and/or GDF-9B bioactivity in a female mammal so as to modulate ovulation comprising the steps of either:
  • the immunisation response may be induced by administration of an antigenic composition
  • an antigenic composition comprising:
  • GDF-9B polypeptide or a functional fragment or variant of GDF-9B together with a pharmaceutically or veterinarily acceptable carrier and/or diluent to a mammal in need thereof.
  • the antigenic composition may include an adjuvant to induce a partial immunisation response and enhance ovulation.
  • An example of such an adjuvant includes DEAE-Dextran adjuvant.
  • Adjuvants such as DEAE-Dextran are selected so as to provoke a partial immune response upon administration to a mammal.
  • the antigemc composition may comprise an alternative adjuvant to induce a strong immunisation response and reduce fertility or induce sterility.
  • an adjuvant examples include Freunds adjuvant.
  • Adjuvants such as Freunds are selected so as to provoke a strong immune response upon administration to a mammal.
  • Partial or strong immunisation may be induced either actively by the administration of the aforementioned antigenic compositions, or passively, by adrninistration of antibodies raised against said antigenic composition or anti-sera comprising said antibodies.
  • Partial immunisation may also be induced by a short term administration regime, whereby the antigenic composition or antibodies thereto are administered over a short time period such as one to two months.
  • Full immunisation may be induced by a long term administration regime, whereby the antigenic composition or antibodies therein are administered over a long time period such as six months or more.
  • the type of adjuvant, antigenic composition, and administration regime are selected to induce the desired response as would be understood by a skilled worker.
  • a particularly desired response is an enhanced ovulation rate and associated increased fertility. Such a method is useful to enhance the reproductive efficiencies and/or enhance multiple ovulations from high value mammals.
  • a further desired response is a decrease in ovulation rate and associated reduced fertility.
  • a response is permanent and results in sterility of the female mammal.
  • This method provides an alternative to surgical methods that are presently used to induce sterility and is much less invasive and carries no risk of infection etc as is the case with surgery.
  • the method may be used as a method of contraception. Again such a method provides advantages over the currently used methods of contraception (i.e. administration of progesterone and/or oestrogen) which are associated with health risks.
  • the method of the present invention may be used to prevent ovulation in a female mammal, such as a race horse, greyhound etc, when the natural oestrous cycle would interfere with the performance of such a mammal. As such an effect is temporary or reversible, the mammal will not suffer any deleterious effects.
  • the present invention further contemplates inducing a desired response by administration of the mutated GDF-9B polypeptide molecules of the invention.
  • polypeptide molecules are likely to be inactive as they comprise a mutation in the receptor binding and/or dimerisation domains
  • such polypeptides when administered in sufficient amounts, may compete with the endogenous GDF-9 and/or GDF-9B binding and/or dimerisation to reduce the biological activity thereof.
  • Such a response if it results in a partial reduction of endogenous GDF-9 and/or GDF-9B activity will result in enhanced ovulation and fertility. If such administration results in a full reduction of endogenous GDF-9 and/or GDF-9B activity, the response induced will be sterility.
  • transgenic non-human mammal wherein one copy of the endogenous GDF-9 and/or GDF-9B gene has been knocked out. Such a mammal would have increased ovulation and enhanced fertility.
  • transgenic mammal may be produced by known methods (Wells et al, 1998. Reprod Fertil Dev: 10:615-26; Clark 2002, Methods Mol Biol, 180: 273-87; Cousens et al 1994, Mol Reprod Dev. 39:384-91; Chen et al 2002, Biol Reprod. 67: 1488-92; Arat et al 2001, Mol Repord Dev. 60: 20-6) and may comprise the steps of introducing to the genetic material of the mammal at least one nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
  • the present invention provides a method of modulating the ovulation rate of a female mammal comprising the steps of: a) identifying the nucleotide sequences of GDF-9 or GDF-9B carried by the female mammal;
  • the present invention provides a method for breeding a mammal having increased ovulation comprising the steps of:
  • a single copy of a mutated GDF-9 nucleotide sequence comprising: A) SEQ ID NO 5; or
  • mutated GDF-9B nucleotide sequence comprising:
  • the present invention provides a method for selecting a female mammal for breeding on the basis of possessing an increased rate of ovulation comprising the steps of identifying a female mammal possessing only a single mutated copy of: 1) a mutated GDF-9 nucleotide sequence comprising:
  • the mammal selected has both a single mutated copy of GDF-9 and GDF-9B.
  • the present invention provides a composition comprising:
  • a mutated GDF-9 polypeptide comprising an amino acid sequence selected from the group consisting of:
  • a mutated GDF-9B polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 8, 10,12,14,16 or 18
  • compositions including pharmaceutical carriers are well known in the art, and are set out in textbooks such as Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Easton, Pennsylvania, USA.
  • the compounds and compositions of the invention may be administered by any suitable route, and the person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated. Dosage will be at the discretion of the attendant physician or veterinarian, and will depend on the nature and state of the condition to be treated, the age and general state of health of the subject to be treated, the route of administration, and any previous treatment which may have been administered.
  • the carrier or diluent, and other excipients will depend on the route of administration, and again the person skilled in the art will readily be able to determine the most suitable formulation for each particular case.
  • polypeptide of the present invention may be administered directly to a female mammal.
  • protein, or polypeptide refers to a protein encoded by the nucleic acid molecule of the invention, including fragments, mutations and homologues having the same biological activity i.e. ovulation modulating activity.
  • the protein or polypeptide of the invention can be isolated from a natural source, produced by the expression of a recombinant nucleic acid molecule, or chemically synthesized.
  • peptide analogues which include but are not limited to the following:
  • peptide and peptide analogue includes compounds made up of units which have an amino and carboxy terminus separated in a 1, 2, 1, 3, 1, 4 or larger substitution pattern.
  • Statine-like isosteres hydroxyethylene isosteres, reduced amide bond isosteres, thioamide isosteres, urea isosteres, carbamate isosteres, thioether isosteres, vinyl isosteres and other amide bond isosteres known to the art are also useful for the purposes of the invention.
  • a "common” amino acid is a L-amino acid selected from the group consisting of glycine, leucine, isoleucine, valine, alanine, phenylalanine, tyrosine, tryptophan, aspartate, asparagine, glutamate, glutamine, cysteine, methionine, arginine, lysine, proline, serine, threonine and histidine. These are referred to herein by their conventional three-letter or one-letter abbreviations.
  • An "uncommon” amino acid includes, but is not restricted to, one selected from the group consisting of D-amino acids, homo-am ⁇ no acids, N-alkyl amino acids, dehydroamino acids, aromatic amino acids (other than phenylalanine, tyrosine and tryptophan), ortho-, meta- or para-aminobenzoic acid, o-rmthine, citrulline, norleucine, ⁇ -glutamic acid, aminobutyric acid (Abu), and ⁇ - ⁇ disubstituted amino acids.
  • the present invention provides a method of modifying the function of the corpus luteum by administering supplementary GDF-9 or GDF-9 B, or analogues thereof, or GDF-9 or GDF-9B antagonists to female mammals.
  • the present invention also encompasses ligands directed to the polypeptides of the invention.
  • ligand refers to any molecule which can bind to another molecule such as a polypeptides or peptide, and should be taken to include, but not be limited to, antibodies, cell surface receptors or phage display molecules.
  • antibody encompasses fragments or analogues of antibodies which retain the ability to bind to a polypeptide of the invention, including but not limited to Fr, F (ab) 2 fragments, ScFv molecules and the like.
  • the antibody may be polyclonal but is preferably monoclonal.
  • the ligand may be a phage display molecule.
  • the invention also includes adenovirus-based gene therapy techniques for expressing GDF-9B and GDF-9/GDF-9B in cell cultures, organ cultures and whole experimental animals for manipulating ovarian follicular protein synthesis or production.
  • nucleic acid molecule may be a RNA, cRNA, genomic DNA or cDNA molecule, and may be single or double- stranded.
  • the nucleic acid molecule may also optionally comprise one or more synthetic non-natural or altered nucleotide bases, or combinations thereof.
  • analogue refers to a compound which has a biological function with improved characteristics over the native compounds (e.g. such an analogue may have a longer half-life than the native compound.)
  • antagonist refers to a compound which inhibits the effect of another compound.
  • the antagonist could refer to a purified antibody, a sera or serum containing an antibody or a plasma or pool of plasma containing an antibody that would neutralise GDF-9 or GDF-9B.
  • partial immunisation refers to immunisation of an animal either active or passive of sufficient antigen/antibody to allow for instigation of an immune response to be mounted against the antigen; but the degree of antigen/antibody administered and/or the means of administration are such that insufficient antibodies are produced by the immunised animal to effectively neutralise all the antigen of interest.
  • full immune response refers to the immune response of animal which has been fully immunised i.e. the response mounted by the immunised animal results in production of sufficient antibodies to effectively neutralise all the antigen of interest.
  • introducing when used in the context of inserting a nucleic acid molecule into a cell, means “transfection” or “transformation” or “fransduction” and includes reference to any method for incorporation or transfer of a nucleic acid molecule into a eukaryotic or prokaryotic cell for expression or replication thereof (for example this may include but should not be limited to insertion of a nucleic acid into: a chromosome, mitochondrial DNA, an autonomous replicon (eg. a plasmid).
  • fransduction refers to the process of transferring genetic information from a nucleic acid molecule from one cell to another by way of a viral vector.
  • transfection refers to the uptake, incorporation, and expression of recombinant DNA by eukaryotic cells.
  • transformation refers to a process by which the genetic material carried by an individual cell is altered by incoiporation of exogenous DNA into its genome.
  • variant refers to nucleotide and polypeptide sequences wherein the nucleotide or amino acid sequence exhibits substantially 60% or greater homology with the nucleotide or amino acid sequence of the Figures, preferably 75% homology and most preferably 90-95% homology to the sequences of the present invention. — as assessed by GAP or BESTFIT (nucleotides and peptides), or BLASTP (peptides) or BLAST X (nucleotides).
  • the variant may result from modification of the native nucleotide or amino acid sequence by such modifications as insertion, substitution or deletion of one or more nucleotides or amino acids or it may be a naturally-occurring variant.
  • variant also includes homologous sequences which hybridise to the sequences of the invention under standard hybridisation conditions defined as 2 x SSC at 65°C, or preferably under stringent hybridisation conditions defined as 6 x SSC at 55°C, provided that the variant is capable modulating the ovulation rate of a female mammal or altering ovarian function.
  • nucleotide sequence of the native DNA is altered appropriately. This alteration can be effected by synthesis of the DNA or by modification of the native DNA, for example, by site-specific or cassette mutagenesis.
  • site-specific primer directed mutagenesis is employed, using techniques standard in the art.
  • a “fragment” of a nucleic acid is a portion of the nucleic acid that is less than full length, and comprises at least a minimum sequence capable of hybridizing specifically with a nucleic acid molecule according to the invention, or a sequence complementary thereto, under stringent conditions as defined below.
  • a “fragment” of a polypeptide is a portion of the polypeptide which is less than full length, but which still retains the biological function of either; increasing or decreasing the ovulation rate of a mammal, causing sterility in a mammal; or altering the regulation of the corpus luteum.
  • a fragment according to the invention has at least one of the biological activities of the nucleic acid or polypeptide of the invention.
  • the biological activity of a fragment of the GDF-9 sequence of the present invention encompass only those mutations which will increase the ovulation rate in female mammals heterozygous for the mutation.
  • isolated means substantially separated or purified away from contaminating sequences in the cell or organism in which the nucleic acid naturally occurs and includes nucleic acids purified by standard purification techniques as well as nucleic acids prepared by recombinant technology, including PCR technology, and those chemically synthesised.
  • modulation of ovulation means increasing or decreasing the rate of ovulation compared to the endogenous rate observed in an untreated animal.
  • hybridization or grammatical variants thereof means the process of joining two complementary strands of DNA or one each of DNA and RNA to form a double stranded molecule.
  • the term "specific for (a target sequence)" indicates that the probe or primer hybridizes under stringent conditions only to the target sequence in a given sample comprising the target sequence.
  • Ovulation rate data were analysed by least squares procedures with the individual animal as the experimental unit using the GLM procedure of SAS.
  • the factors in the models were ewe, age, year of record, and the number of copies (0 or 1) of each of the mutations described below.
  • the sheep GDF-9 and GDF-9B genes were amplified using the polymerase chain reaction (PCR) with primers designed from published sheep sequences (sheep genomic GDF-9B exon 1, AF236078; sheep genomic GDF-9B exon 2, AF236079; sheep genomic GDF-9 exon 1 and 2, AF078545).
  • the PCR primers used were as follows:
  • GDF-9B exon 1 B13 5'-ACTGCTGCCTTGTCCCAC-3'
  • GDF-9B exon 2 B25 5'-CAGTTTGTACTGAGCAGGTC-3'
  • GDF-9 exon 1 Gl : 5'-GAATTGAACCTAGCCCACCCAC-3'
  • GDF-9 exon 2 G5: 5'-ATCCCACCCTGACGTTTAAGGC-3 '
  • the resulting PCR products were sequenced on an ABI 373 sequencer.
  • SSCP single stranded conformational polymorphism
  • GDF-9B genotypes were determined by analysis of three nucleotide fragments which spanned most of exon 2. Fragments analysed by SSCP were:
  • exon 2 primer 9B-359 5'-CGC TTT GCT CTT GTT CCC TCT-3 '
  • primer 9B-691 5'-CCT CAC TAC CTC TTG GCT GCT-3' 273 bp
  • exon 2 primer 9B-664 5'-GGG TTC TAC GAC TCC GCT TC-3 '
  • exon 2 primer 9B-915 5'-CAT GAT GGG CCT GAA AGT AAC-3'
  • primer 9B-1205 5'-GGC AAT CAT ACC CTC ATA CTC C-3'
  • Primers were designed from nucleotide sequence Genbank Accession number AF236079 and primer names correspond to nucleotide position within that sequence.
  • GDF-9 genotypes were determined by analysis of five fragments which spanned exon 1, part of the intron and most of exon 2. Fragments analysed by SSCP were:
  • exon 1 primer G9-1734 5'-GAA GAC TGG TAT GGG GAA ATG-3'
  • exon 2 primer G9-3270 5'-TGG CAT TAC TGT TGG ATT GTT TT-3'
  • primer G9-3546 5'-CAA GAG GAG CCG TCA CAT CA-3'
  • exon 2 primer G9-3543 5'-GAT TGA TGT GAC GGC TCC TCT-3'
  • primer G9-3728 5'-GGG AAT GCC ACC TGT GAA AAG-3'
  • exon 2 primer G9-3939 5'-TCT TTT TCC CCA GAA TGA ATG T-3'
  • primer G9-4140 5'-CAC AGG ATG GTC TTG GCA CT-3' Primers were designed from nucleotide sequence Genbank Accession number AF078545 and primer names correspond to nucleotide position within that sequence.
  • Amplification was carried out for 30 cycles in a 40 ⁇ L reaction mixture, using 150 ng of genomic DNA, with 1.5 mM or 3 mM magnesium and an annealing temperature of 55 to 58° C. PCR fragments were analysed by SSCP in polyacrylamide gels with overnight migration at 9-15 V/cm, 15°C.
  • the [El] polymorphism identified in GDF-9 exon 1 produced a G to A nucleotide change which disrupts a Hha I restriction enzyme cleavage site (GCGC to GCAC) at nucleotide 260 of the 462 bp PCR fragment produced by primers G9-1734 and G9-2175 above.
  • Digestion was carried out using 9 ⁇ l of PCR product and 3 U Hha I in 15 ⁇ l final volume, for 6 h at 37° C. Restriction digestion of the PCR product from wildtype animals with Hha I resulted in cleavage of the 462 bp product (at two internal Hha I sites) into fragments of 52 bp, 156 bp and 254 bp.
  • DNA fragments containing the A nucleotide are not cleaved at this site and fragment sizes of 52 bp and 410 bp are seen.
  • Animals heterozygous for the mutation have fragments of all four sizes (52 bp, 156 bp, 254 bp and 410 bp).
  • SNPs single nucleotide polymorphisms
  • GDF-9 and GDF-9B The remaining single nucleotide polymorphisms (SNPs) in GDF-9 and GDF-9B identified by sequencing did not affect common restriction endonuclease cleavage sites.
  • PCR was carried out using primers with single mismatches in order to deliberately generate products that contained restriction enzyme sites. Assays were designed so that digestion with the appropriate restriction enzyme cleaved either PCR products from wild-type animals or PCR products from animals containing the SNP, as specified below. The resulting band shift was resolved on a high percentage agarose gel.
  • the primer sequences and PCR conditions for each assay were as follows. The mismatch created in the appropriate primer to generate the restriction enzyme cleavage site is underlined.
  • the primers used for the GDF-9 [324] nucleotide change amplify a 161 bp PCR product were:
  • the annealing temperature was 63°C.
  • the non-wildtype strand was cleaved by restriction enzyme Sfu I.
  • the primers used for the GDF-9 [714] nucleotide change amplify a 158 bp PCR product were:
  • the annealing temperature was 63°C.
  • the non-wildtype strand was cleaved by restriction enzyme Mse I.
  • the primers used for the GDF-9 [787] nucleotide change amplify a 139 bp PCR product were: [787]-DdelR: 5'-CATGGATGATGTTCTGCACCATGGTGTGAACCTGA-3'
  • the annealing temperature was 62°C.
  • the wildtype strand was cleaved by restriction enzyme Dde I.
  • the primers used for the GDF-9B [SI] nucleotide change amplify a 141 bp PCR product were:
  • the annealing temperature was 63 °C.
  • the wildtype strand was cleaved by restriction enzyme Hinfl.
  • the primers used for the GDF-9B [S2] nucleotide change amplify a 153 bp PCR product were:
  • the annealing temperature was 64°C.
  • the wildtype strand was cleaved by restriction enzyme Dde I.
  • the digested fragments were separated on a 4% agarose gel and visualised with ethidium bromide staining. The gels were scored for the presence or absence of the mutations. Homozygous, heterozygous and negative controls were included with each assay.
  • KKPLVPASVNLSEYFC GDF-9
  • SEVPGPSREHDGPESC GDF-9B
  • STM Span- Tween-Marcol
  • estrus day 0
  • ewes were laparascoped to determine ovulation rate and fitted with an indwelling jugular cannula.
  • Ewes were given another injection of Estrumate, at 96h after administration of the antiplasma to induce a follicular phase and ovulation rate was determine by laparoscopy at 10 days after the injection of Estrumate and every 15-18 days thereafter until the end of the breeding season (as assessed by lack of estrous activity in non-experimental sheep). Blood samples were collected from the ewes at 5 minutes, 1 h and 96 h after injection of the antiplasma and thereafter 3 times a week from the 2 nd injection of Estrumate for determination of antibody titers and concentrations of progesterone in plasma.
  • ovulation rate for individual ewes was calculated as the mean of the number of corpora lutea observed at all observations for that ewe when at least 1 corpus luteum (CL) was present (i.e. observations of no CL were excluded from the calculation).
  • CL corpus luteum
  • the Kruskal-Wallis test was used to compare ovulation rates between the KLH-GDF-9B mature protein and the KLH treated groups. No other groups were included in this comparison since none had sufficient numbers of ewes ovulating.
  • the Chi Square test was used to compare the proportion of ewes observed in estrus by the time all the control ewes had been observed in estrus. In addition the Chi Square test was used to compare the proportion of ewes with corpora lutea on their ovaries 3-4 weeks before and at ovarian collection.
  • ovulation rate for individual ewes was calculated as the average of the number of corpora lutea observed at both observations. Data was analysed using the general linear models procedures of SAS. Differences between least-squares means were evaluated by least significant differences. RESULTS andANALYSIS OFRESULTS
  • Sequence of GDF-9 revealed eight single nucleotide polymorphisms across the entire coding region (Table 1, Figure 4).
  • SSCP analysis identified five fragments across the gene which contained conformational differences. These differences correspond to one single nucleotide polymo ⁇ hism (SNP) in exon 1, one SNP in the intron and five SNPs in exon 2.
  • mutation [581] is a G to A nucleotide substitution at coding nucleotide 978 of GDF-9 which corresponds to an unchanged glutamate (Glu) coding residue 326 of the full length unprocessed protein, or residue 8 of the processed mature peptide.
  • nucleotide changes which do not result in an altered amino acid ([74] at nucleotide position 471, [80] at nucleotide 477, and [581] at nucleotide position 978).
  • the five remaining nucleotide changes [324], [597], [714], and [787] gave rise to amino acid changes (Table 1), Figure 1, Figure 4), although three of them were relatively conservative changes.
  • the [El] arginine to histidine change at amino acid residue 87 in exon 1 substituted one basic charged polar group with another, and occurred at a position prior to the furin processing site, so was unlikely to affect the activity of the mature protein.
  • F700 Belclare sheep revealed four polymo ⁇ hisms across the entire coding region (Table 1, Figure 5).
  • Original naming of these mutations (in square brackets [ ]) refers specifically to the leucine deletion [Leu], or for the conservative [422] T to C mutation, the nucleotide position from the start of exon 2.
  • GDF-9B mutations which changed amino acids were named [SI] and [S2].
  • Table 1 shows the relationship between (a) the original numbering system, (b) the coding nucleotide position in the full length coding sequence numbered from the first atg, (c) the position of the coding amino acid residue involved, and (d) the position of the coding residue (if any) within the mature coding sequence, numbering from the first amino acid after the furin protease processing site.
  • mutation [S2] was a G to T nucleotide substitution at coding nucleotide 1100 of GDF-9B which corresponds to an serine (Ser) residue changing to an isoleucine (He) residue at coding residue 367 of the full length unprocessed protein, or residue 99 of the processed mature peptide.
  • the [SI] C to T change at nucleotide 718 introduced a premature stop codon (TAG) in the place of glutamic acid (Q, CAG) at amino acid residue 239 of the unprocessed protein, which presumably resulted in complete loss of GDF-9B function.
  • the [S2] G to T change at nucleotide 1100 changed the serine residue at amino acid 99 of the mature active protein (residue 367 of the unprocessed protein) to an isoleucine, thereby substituting an uncharged polar group with a nonpolar group.
  • the nucleotide and amino acid changes are illustrated in Figure 2 and Figure 5. Screening for mutations in more animals
  • Homozygous mutations relate to sterility
  • FIG 3a illustrates a F700 Belclare pedigree.
  • the sire R830 carried the GDF-9B [S2] mutation on his X chromosome and a single copy of the GDF-9 [787] mutation on chromosome 5, but did not have the GDF-9B [SI] mutation.
  • Dam 9704 carried a single copy of the GDF-9B [SI] mutation on her X chromosome and their two female offspring (930458 and 930459) were sterile since they had inherited mutated copies of GDF-9B from both parents.
  • Dam 8783 carried a single copy of the GDF-9 [787] mutation on chromosome 5 and the female offspring of her mating with sire R830 were infertile and were homozygous for the GDF-9 [787] mutation. Their infertility cannot be explained by GDF-9B mutations. Offspring 930810 and 948302 were not homozygous for any of these mutations and hence were fertile. All three functional mutations ([SI], [S2], and [787]) were seen in the F700 Belclare flock (Table 2).
  • FIG. 3b illustrates two Cambridge pedigrees.
  • the sire 962101 carried the GDF-9B [SI] mutation on his X chromosome and a single copy of the GDF-9 [787] mutation on chromosome 5, but did not have the GDF-9B [S2] mutation.
  • Dam 962152 carried a single copy of the [SI] mutation on her X chromosome and a single copy of the [787] mutation on chromosome 5.
  • Their two female offspring (997634 and 997635) were sterile and had inherited mutated copies of both GDF-9B ([SI]) and GDF-9 ([787]) from both parents.
  • Dam 976234 only carried a single copy of the [SI] mutation and one female offspring (997553) was infertile, having inherited mutated copies of GDF-9B ([SI]) from both parents, whereas 997552 was fertile.
  • Sire 930142 was homozygous for the GDF-9 [787] mutation and carried the GDF-9B [SI] mutation on his X chromosome, whereas dam 8874 was only heterozygous for the GDF-9 [787] mutation and carried no GDF-9B mutation.
  • Their daughter (948093) had inherited two copies of the GDF-9 [787] mutation and was sterile even though she was also heterozygous for the GDF-9B [SI] mutation which she inherited from her sire.
  • the [S2] mutation was not seen in any animals tested from the Cambridge flock (Table 2).
  • TGF ⁇ superfamily may provide information about the likely effects of each of the three mutations ([SI], [S2] and [787]) on the biological activity of GDF-9 and GDF-9B, and hence explain the association with sterility. Structures have been reported for TGF- ⁇ l (Hinck et al, 1996); TGF- ⁇ 2 (Daopin et al, 1992), TGF- ⁇ 3 (Mittl et al, 1996), BMP7/OP1 (Griffith et al, 1996) and BMP2 (Scheufler et al, 1999).
  • Receptor binding structures have also been reported for BMP2 with the BRIA receptor binding ectodomain (Kirsch et al, 2000) and for TGF- ⁇ 3 with the T ⁇ R2 receptor binding ectodomain (Hart et al, 2002).
  • the [SI] mutation resulted in premature termination of GDF- 9B protein prior to the mature active protein processing site. It is thus expected that this mutation would result in no mature protein being translated, and appears to be an even more severe effect than the Hanna mutation (Galloway et al., 2000) which results in infertility in sheep.
  • the GDF-9B [S2] mutation changed an uncharged polar serine residue (residue 99 of mature GDF-9B) which is conserved across most members of the TGF ⁇ superfamily, to a non-polar isoleucine (Figure 6). This serine (and the nearby conserved leucine) has been shown to be essential for receptor binding by structural and site-directed mutagenesis studies of BMP2.
  • GDF-9 [787] mutation changed an uncharged polar serine residue (residue 77 of mature GDF-9) to a non-polar phenylalanine in a region of the molecule which appears to be involved in dimerisation. This change occurred only three residues away from a conserved histidine (H80) of the mature GDF-9 peptide ( Figure 6). In BMP7 this conserved histidine exhibits hydrogen bonding to three residues of the paired molecule in the BMP7 dimer (Griffith et al, 1996) and TGF ⁇ 3 (Mittl et al, 1996).
  • GDF-9 lacks the interchain disulphide bond which forms a covalent link between both monomers of the biologically active dimer that is found in most other members of the TGF ⁇ superfamily. It is therefore likely that in GDF-9 the hydrogen bonds between monomers would be even more critical for maintaining dimer stability, so that the GDF-9 [787] mutation possibly abolished biological activity by disrupting dimerisation.
  • Figure 8 also shows data for passive immunisation with GDF-9B/(BMP15) for comparison.
  • the present invention provides compositions and methods for modulating the ovulation rate and therefore fertility in female mammals including humans.
  • mutation [581] is a G to A nucleotide substitution at coding nucleotide 978 of GDF-9 which corresponds to an unchanged glutamate (Glu) coding residue 326 of the full length unprocessed protein, or residue 8 of the processed mature peptide.
  • Polymo ⁇ hisms associated with infertility and ovulation rate traits are in bold.
  • Genotypes were determined by specific SNP assay and/or sequencing.
  • GDF-9 novel growth differentiation factor-9

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Abstract

L'invention concerne des compositions et des procédés permettant de moduler la vitesse d'ovulation et, par là même, la fertilité chez des mammifères femelles, et notamment chez la femme. Cette invention concerne également de nouvelles mutations dans les gènes GDF-9 et GDF-9B, lesquels sont associés à des modifications de la fertilité.
PCT/NZ2003/000109 2002-05-30 2003-05-30 Nouvelles sequences gdf-9 et gdf-9b (bmp-15) destinees a modifier la fonction ovarienne et la vitesse d'ovulation chez des mammiferes WO2003102199A1 (fr)

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BR0311465-1A BR0311465A (pt) 2002-05-30 2003-05-30 Novas sequências gdf-9 e gdf-9b (bmp-15) para alteração da função ovariana e da taxa de ovulação de mamìferos
AU2003235525A AU2003235525B2 (en) 2002-05-30 2003-05-30 New GDF-9 and GDF-9B (BMP-15) sequences for altering mammalian ovarian function and ovulation rate
EP03723538A EP1551973A4 (fr) 2002-05-30 2003-05-30 Nouvelles sequences gdf-9 et gdf-9b (bmp-15) destinees a modifier la fonction ovarienne et la vitesse d'ovulation chez des mammiferes
CA002490051A CA2490051A1 (fr) 2002-05-30 2003-05-30 Nouvelles sequences gdf-9 et gdf-9b (bmp-15) destinees a modifier la fonction ovarienne et la vitesse d'ovulation chez des mammiferes
JP2004510436A JP2006510345A (ja) 2002-05-30 2003-05-30 哺乳動物の排卵機能および排卵頻度を改変する新しいｇｄｆ−９およびｇｄｆ−９ｂ（ｂｍｐ−１５）配列

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WO2006059914A1 (fr) * 2004-12-02 2006-06-08 Agresearch Limited Modulation de l'ovulation
WO2006059913A1 (fr) * 2004-12-02 2006-06-08 Agresearch Limited Modulation de l'ovulation
WO2006130022A1 (fr) * 2005-05-30 2006-12-07 Agresearch Limited Modulation de l'ovulation au moyen d'agonistes et d'antagonistes de bmprii
EP1947195A1 (fr) * 2005-09-13 2008-07-23 Neocodex, S.L. Methode de detection in vitro de la predisposition a developper des alterations de la fonction ovarienne
WO2009065204A1 (fr) * 2007-11-23 2009-05-28 Empresa Brasileira De Pesquisa Agropecuária - Embrapa Procédé d'identification de la prolificité chez des mammifères
ES2338960A1 (es) * 2007-11-23 2010-05-13 Carnes Oviaragon S.C.L. Procedimiento de mejora de la productividad en ganado ovino.
WO2014018404A1 (fr) * 2012-07-21 2014-01-30 Baylor College Of Medicine Hétérodimères gdf9/bmp15 pour augmenter la fertilité
CN104774836A (zh) * 2015-04-15 2015-07-15 兰州大学 一种提高绵羊产羔数多基因聚合早期选种的方法
WO2019213690A1 (fr) 2018-05-09 2019-11-14 Monash University Agent et procédé d'amélioration de la fertilité
CN110914686A (zh) * 2017-02-01 2020-03-24 新南创新私人有限公司 配子分泌的生长因子

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WO2001096393A2 (fr) * 2000-06-15 2001-12-20 Agresearch Limited Sequences de nucleotides et d'acides amines de facteurs derives d'ovocytes permettant de modifier la croissance folliculaire ovarienne in vivoet in vitro

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WO2001085926A2 (fr) * 2000-05-05 2001-11-15 Agresearch Limited Sequences nucleotidiques associees a l'augmentation ou a la reduction du taux d'ovulation mammalien
WO2001096393A2 (fr) * 2000-06-15 2001-12-20 Agresearch Limited Sequences de nucleotides et d'acides amines de facteurs derives d'ovocytes permettant de modifier la croissance folliculaire ovarienne in vivoet in vitro

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VITT U.A. ET AL.: "In vivo treatment with GDF-9 stimulates primordial and primary follicle progression and theca cell marker CYP17 in ovaries of immature rats", ENDOCRINOLOGY, vol. 141, no. 10, 2000, pages 3814 - 3820, XP002999769 *
YAN C. ET AL.: "Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function", MOLECULAR ENDOCRINOLOGY, vol. 15, no. 6, June 2001 (2001-06-01), pages 854 - 866, XP001057927 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006059913A1 (fr) * 2004-12-02 2006-06-08 Agresearch Limited Modulation de l'ovulation
WO2006059914A1 (fr) * 2004-12-02 2006-06-08 Agresearch Limited Modulation de l'ovulation
WO2006130022A1 (fr) * 2005-05-30 2006-12-07 Agresearch Limited Modulation de l'ovulation au moyen d'agonistes et d'antagonistes de bmprii
EP1947195A4 (fr) * 2005-09-13 2009-11-04 Neocodex S L Methode de detection in vitro de la predisposition a developper des alterations de la fonction ovarienne
EP1947195A1 (fr) * 2005-09-13 2008-07-23 Neocodex, S.L. Methode de detection in vitro de la predisposition a developper des alterations de la fonction ovarienne
ES2338960A1 (es) * 2007-11-23 2010-05-13 Carnes Oviaragon S.C.L. Procedimiento de mejora de la productividad en ganado ovino.
WO2009065204A1 (fr) * 2007-11-23 2009-05-28 Empresa Brasileira De Pesquisa Agropecuária - Embrapa Procédé d'identification de la prolificité chez des mammifères
EP2225395A1 (fr) * 2007-11-23 2010-09-08 Empresa Brasileira de Pesquisa Agropecuária - EMBRAPA Procédé d'identification de la prolificité chez des mammifères
EP2225395A4 (fr) * 2007-11-23 2011-01-26 Brasil Pesquisa Agropec Procédé d'identification de la prolificité chez des mammifères
ES2338960B1 (es) * 2007-11-23 2011-04-19 Carnes Oviaragon S.C.L. Procedimiento de mejora de la productividad en ganado ovino.
WO2014018404A1 (fr) * 2012-07-21 2014-01-30 Baylor College Of Medicine Hétérodimères gdf9/bmp15 pour augmenter la fertilité
CN104774836A (zh) * 2015-04-15 2015-07-15 兰州大学 一种提高绵羊产羔数多基因聚合早期选种的方法
CN110914686A (zh) * 2017-02-01 2020-03-24 新南创新私人有限公司 配子分泌的生长因子
WO2019213690A1 (fr) 2018-05-09 2019-11-14 Monash University Agent et procédé d'amélioration de la fertilité
US11702457B2 (en) 2018-05-09 2023-07-18 Monash University Agent and method for enhancing fertility

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AU2003235525B2 (en) 2008-09-11
CA2490051A1 (fr) 2003-12-11
AU2003235525A1 (en) 2003-12-19
NZ519330A (en) 2004-12-24

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