Nucleotide and amino acid sequences of oocyte factors for altering ovarian follicular growth in vivo or in vitro

Abstract

Aspects of the present invention relate to nucleotide and amino acid sequences of oocyte factors for altering ovarian follicular growth in vivo or in vitro. The present invention also concerns novel homodimeric and heterodimeric polypeptides and their use for altering mammalian ovarian follicular growth in vivo or in vitro.

1 . An isolated wildtype GDF-9B nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: 
 a) SEQ ID NO: 1;    b) a sequence able to hybridise under stringent conditions to the molecule in (a) above;    c) a sequence which is a functional variant or fragment of the molecule in (a) above;    d) a sequence complementary to the molecule defined in (a), (b) or (c) above; and    e) an anti-sense sequence corresponding to any of the molecules in (a)-(d) above.    
     
     
         2 . An isolated full length mutated GDF-9B nucleic acid molecule comprising a ucleotide sequence selected from the group consisting of: 
 a) SEQ ID NO: 3 or SEQ ID NO: 5;    b) a sequence able to hybridise under stringent conditions to the molecule(s) in (a) above;    c) a sequence which is a functional variant or fragment of the molecule(s) in (a) above;    d) a sequence complementary to the molecule(s) defined in (a), (b) or (c) above; and    e) an anti-sense sequence corresponding to any of the molecule(s) in (a)-(d) above.    
     
     
         3 . An isolated full-length GDF-9B polypeptide comprising an amino acid sequence selected from the group consisting of: 
 a) SEQ ID NO:2, SEQ ID NO:4; or SEQ ID NO:6; and    b) a functional variant or fragment of the sequence(s) in (a) above.    
     
     
         4 . An isolated polypeptide, wherein said peptide is a homodimeric mature GDF-9B polypeptide having subunits comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, a functional fragment thereof, and a variant of said sequence.  
     
     
         5 . An isolated polypeptide, wherein said peptide is a heterodimeric polypeptide having subunits selected from the group consisting of: 
 a) a mature GDF-9B polypeptide comprising an amino acid sequence derived from SEQ ID NO:2, a functional fragment or a variant of said sequence; and    b) a mature GDF-9 polypeptide, a functional variant or a fragment thereof.    
     
     
         6 . A vector comprising the nucleic acid molecule selected from the group consisting of: 
 a) SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5;    b) a sequence able to hybridise under stringent conditions to the molecule(s) in (a) above;    c) a sequence which is a functional variant or fragment of the molecule(s) in (a) above;    d) a sequence complementary to the molecule(s) defined in (a), (b), or (c) above; and    e) an anti-sense sequence corresponding to any of the molecules in (a)-(d) above.    
     
     
         7 . A construct comprising the nucleic acid molecule of  claim 6 .  
     
     
         8 . A host cell, wherein said host cell has been transformed by a vector or construct comprising the nucleic acid molecule of  claim 6 .  
     
     
         9 . A ligand, wherein said ligand binds to a polypeptide derived from polypeptide of  claim 3 , and wherein the polypeptide of  claim 3  is a full-length prepro polypeptide.  
     
     
         10 . A ligand, wherein said ligand binds to the homodimeric polypeptide of  claim 4 .  
     
     
         11 . A ligand, wherein said ligand binds to the heterodimeric polypeptide of  claim 5 .  
     
     
         12 . A ligand, wherein the ligand binds to a homodimeric GDF-9B peptide or a heterodimeric GDF-9B peptide, and wherein the ligand is selected from the group consisting of an antibody, an antibody fragment comprising the antigen-binding domain, a phage display molecule, and in the form of cell surface receptor(s).  
     
     
         13 . The ligand of  claim 12  wherein the ligand is a monoclonal antibody.  
     
     
         14 . The ligand of  claim 12  wherein the ligand is a phage display molecule.  
     
     
         15 . The ligand of  claim 12  wherein the ligand is in the form of cell surface receptor(s).  
     
     
         16 . A method of expressing biologically active processed homodimeric GDF-9B polypeptide comprising: 
 a) generating an expression construct comprising a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, a functional fragment, and a variant of said sequence of the group;    b) transfecting suitable cells with said expression construct of (a) above;    c) selecting stable clones resulting from the transfection of (b) above; and    d) isolating and purifying the expressed polypeptide recovered from the clones of (c) above.    
     
     
         17 . A method of expressing biologically-active processed heterodimeric GDF-9B and GDF-9 polypeptides comprising the steps of: 
 a) generating an expression construct containing a nucleic acid molecule comprising: 
 (i) a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, a functional fragment, and a variant of said sequence; and  
 (ii) a nucleic acid molecule encoding GDF-9, a functional fragment or a variant thereof;  
   b) transfecting suitable cells with said expression construct of (a) above;    c) selecting stable clones resulting from the transfection of (b) above; and    d) isolating and purifying the expressed polypeptide recovered from the clones of (c) above.    
     
     
         18 . A method of adenoviral, retroviral and alphaviral transfer of GDF-9B expression cassettes or GDF-9 expression cassettes to host cells or organisms comprising: 
 transferring into a recipient cell, organ culture or recipient animal, a recombinant adenovirus including an expression cassette comprising a nucleic acid molecule having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, a fuictional fragment, and a variant of said sequence, wherein said nucleic acid molecule is in operative association with an expression control sequence,    whereby said transfer of the expression cassette(s) affects the in vivo expression of GDF-9B homodimers or GDF-9B/GDF-9 heterodimers.    
     
     
         19 . A transgenic animal which has been transformed by the vector of  claim 6  or a construct comprising the nucleic acid molecule according to  claim 6 .  
     
     
         20 . A method of altering ovarian follicular growth, in vivo, in a female mammal or other female vertebrate, said method comprising: 
 transforming mammalian and other vertebrate ovarian host cells with GDF-9B and GDF-9 expression cassettes,    whereby the transforming of the ovarian host cells allows over-expression of GDF-9B homodimers and GDF-9B/GDF-9 heterodimers.    
     
     
         21 . A method of altering ovarian follicular growth, in vitro, in a female mammal or other female vertebrate, said method comprising: 
 transforming mammalian and other vertebrate ovarian host cells with GDF-9B and GDF-9 expression cassettes,    whereby he transforming of the ovarian host cells allows over-expression of GDF-9B homodimers and GDF-9B/GDF-9 heterodimers.    
     
     
         22 . A method as claimed in  claim 20  or  21  wherein said mammal is selected from the group consisting of sheep, cattle, goats, deer, humans, pigs, horses, camelids, and possums, cats and dogs and any other commercially important species.  
     
     
         23 . A method as claimed in claims  20  or  21  wherein said vertebrate is selected from the group consisting of chickens, ducks, geese, salmon and any other commercially important species.  
     
     
         24 . A composition comprising an effective amount of an agent selected from the group consisting of: 
 a) a homodimeric polypeptide having subunits comprising a GDF-9B polypeptide, a functional fragment or a variant thereof with or without homodimeric polypeptide having subunits comprising GDF-9 polypeptide, a functional fragment or variant thereof; and    b) a heterodimeric polypeptide having subunits comprising a GDF-9B polypeptide and a GDF-9 polypeptide, functional fragments or variants of said GDF-9B or GDF-9 polypeptides;    together with a pharmaceutically or veterinarily acceptable carrier, adjuvant or diluent.    
     
     
         25 . The composition of  claim 24 , wherein the composition further comprises supplementary gonadotrophins.  
     
     
         26 . The composition of  claim 24 , wherein the composition further comprises an ovarian growth factor selected from the group consisting of IFG-1, kit ligand (stem cell factor), epidermal growth factor and TGFβ agonsists/antagonsists.  
     
     
         27 . The composition of  claim 25 , wherein the composition further comprises an ovarian growth factor selected from the group consisting of IGF-1, kit ligand (stem cell factor), epidermal growth factor and TGFβ agonists/antagonists.  
     
     
         28 . A method for altering ovarian follicular growth, in vitro or in vivo, in a female mammal or other female vertebrate comprising administering a composition selected from the group consisting of: 
 a) the composition of  claim 24;     b) the composition of  claim 24 , wherein the composition further comprises supplementary gonadotrophins;    c) the composition of  claim 24 , wherein the composition further comprises an ovarian growth factor selected from the group consisting of IGF-1, kit ligand (stem cell factor), epidermal growth factor and TGFβ agonsists/antagonists; and    d) the composition of c) above, wherein the composition further comprises supplementary gonadotrophins,    whereby the administered composition alters ovarian follicular growth.    
     
     
         29 . A method for altering isolated ovarian cell growth or maturation in vitro comprising administering to isolated ovarian cells a composition selected from the group consisting of: 
 a) the composition of  claim 24;     b) the composition of  claim 24 , wherein the composition further comprises supplementary gonadotrophins;    c) the composition of  claim 24 , wherein the composition further comprises an ovarian growth factor selected from the group consisting of IGF-1, kit ligand (stem cell factor), epidermal growth factor and TGFβ agonsists/antagonists; and    d) the composition of c) above, wherein the composition further comprises supplementary gonadotrophins,    whereby the administered composition alters isolated ovarian cell growth or maturation.    
     
     
         30 . A method for assessing the activity of GDF-9B homodimers, GDF-9B/GDF-9 heterodimers, or the combination thereof, comprising: 
 a) adding an effective amount of a GDF-9B homodimeric polypeptide, a GDF9B/GDF-9 heterodimeric polypeptide, or the combination thereof to an ovarian cell or organ culture; and    b) conducting a bioassay on said cell or organ culture to assess the biological activity of said homodimeric and heterodimeric polypeptides.    
     
     
         31 . A method of altering follicular growth comprising introducing a ligand to a mammal or other vertebrate to alter follicular growth in ovaries of a mammal or other vertebrate either in vivo or in vitro or to alter isolated ovarian cell growth/maturation in vitro, wherein said ligand binds to a molecule which binds to a polypeptide selected from the group consisting of: 
 a) a full length GDF-9B polypeptide;    b) a homodimeric mature GDF-9B polypeptide;    c) a heterodimeric mature GDF-9B/GDF-9 polypeptide; and    d) a functional fragment or variant of (a) or (c) above.    
     
     
         32 . An isolated functional variant polypeptide comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18.  
     
     
         33 . An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, a functional fragment thereof and a variant of said sequences.  
     
     
         34 . An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, a functional fragment thereof and a variant of said sequences.