Sperm factor sequences

Abstract

A nucleic acid sequence, including an isolated, purified or recombinant nucleic acid sequence, includes: (a) a nucleic acid sequence encoding a polypeptide for stimulating embryonic development, namely, a PLC-zeta; PLCζ amino acid sequence, capable of triggering calcium oscillations in oocytes; (b) a sequence substantially homologous to or that hybridizes to sequence (a) under stringent conditions; (c) a sequence substantially homologous to or that hybridizes to the sequences (a) or (b) but for degeneracy of the genetic code; and (d) an oligonucleotide specific for any of the sequences (a), (b) or (c) above.

What is claimed is: 
     
         1 . An isolated, purified or recombinant nucleic acid molecule capable of triggering cytoplasmic calcium oscillations (CCOs) when introduced into a mammalian or a non-mammalian oocyte, said molecule comprising a nucleic acid molecule encoding a PLC-zeta (PLCζ) polypeptide or protein, a molecule substantially homologous thereto, or a sequence which hybridizes thereto under stringent conditions. 
     
     
         2 . The isolated, purified or recombinant nucleic acid molecule according to  claim 1 , having a sequence comprising a nucleotide sequence selected from one of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 10, a sequence substantially homologous thereto, or a sequence which hybridizes thereto under stringent conditions. 
     
     
         3 . An isolated, purified or recombinant PLC-zeta (PLCζ) polypeptide or protein capable of triggering cytoplasmic calcium oscillations (CCOs) when introduced into a mammalian or a non-mammalian oocyte, encoded by a nucleic acid molecule according to  claim 1 . 
     
     
         4 . The isolated, purified or recombinant polypeptide or protein according to  claim 3 , having a sequence comprising an amino acid sequence selected from one of SEQ ID NO: 1, SEQ ID NO.: 2, or SEQ ID NO.: 11, or a sequence substantially homologous thereto. 
     
     
         5 . The PLC-zeta polypeptide or protein according to  claim 3 , characterized by the following properties:
 (a) an amino acid sequence comprising in the range of from 600 to 720, preferably 600 to 699, more preferably 600 to 650, amino acid residues;   (b) a domain sequence comprising the EF hand, X, Y, and C2 domains but absent the PH domain; and   (c) at least five consecutive amino acid residues from a conserved region, which region is selected from:   
       
         
           
                 
                 
               
                     
                   (i) 
                 
                     
                   QDDFRGGKI (11-19); 
                 
                     
                     
                 
                     
                   (ii) 
                 
                     
                   LLEKLD (27-32); 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (iii) 
                 
                     
                   QGRIT (52-56) in the EF1 domain; 
                 
                     
                     
                 
                     
                   (iv) 
                 
                     
                   ENRKIL (82-87); 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (v) 
                 
                     
                   FLTQEQY (95-101) in the EF2 domain; 
                 
                     
                     
                 
                     
                   (vi) 
                 
                     
                   YQQFNE (403-408) in the Y domain; 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (vii) 
                 
                     
                   TLTIR (516-520); 
                 
                     
                     
                 
                     
                   (viii) 
                 
                     
                   ISGIQLP (522-528);  
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (ix)  
                 
                     
                   LCMNKGYRR (609-617) in the C2 domain, 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein the residues are denoted by their conventional single letter codes and the numbers in parentheses refer to the AB070108 (monkey A) reference sequence. 
       
     
     
         6 . The polypeptide or protein according to  claim 5 , having a molecular weight in the range of from 70 to 75 kD, as determined by mass spectrometry. 
     
     
         7 . A method for the preparation of a polypeptide or protein according to  claim 3 , comprising:
 expressing a nucleic acid molecule encoding the polypeptide or protein; and   optionally, isolating and/or purifying the expressed polypeptide or protein;   wherein said step of expressing requires use of one or more of the following oligonucleotides:   
       
         
           
                 
                 
               
                     
                   Forward human primer: 
                 
                     
                   (SEQ ID NO.: 12; 28mer) 
                 
                     
                   5′ CAG CGA GCT CTT ATC  TGA  CGT ACC AAA 
                 
                     
                     
                 
                     
                   C 3′; 
                 
                     
                     
                 
                     
                   Reverse TriplEx primer: 
                 
                     
                   (SEQ ID NO.: 13; 26mer) 
                 
                     
                   5′ CTC GGG AAG CGC GCC ATT GTG TTG GT 3′; 
                 
                     
                     
                 
                     
                   Forward mouse primer: 
                 
                     
                   (SEQ ID NO.: 14; 28mer) 
                 
                     
                   5′ GCT AAC GCG TCA GTT ACA TGC G TC   A CT 
                 
                     
                     
                 
                     
                   C 3′; 
                 
                     
                     
                 
                     
                   Reverse T7 primer: 
                 
                     
                   (SEQ ID NO.: 15; 22mer) 
                 
                     
                   5′ GTA ATA CGA CTC ACT ATA GGG C 3′;  
                 
                     
                     
                 
                     
                   Forward human primer: 
                 
                     
                   (SEQ ID NO.: 16; 28mer) 
                 
                     
                   5′ CAG CGA GCT CTT ATC TGA CGT ACC AAA 
                 
                     
                     
                 
                     
                   C 3′; 
                 
                     
                     
                 
                     
                   Reverse human primer: 
                 
                     
                   (SEQ ID NO.: 17; 24mer) 
                 
                     
                   5′ ATG AAA CT A   TG G AAA TGA GAT GGT 3′; 
                 
                     
                     
                 
                     
                   Forward mouse primer: 
                 
                     
                   (SEQ ID NO.: 18; 28mer) 
                 
                     
                   5′ GCT AAC GCG TCA GTT ACA TGC GTC ACT 
                 
                     
                     
                 
                     
                   C 3′;  
                 
                     
                   and 
                 
                     
                     
                 
                     
                   Reverse mouse primer: 
                 
                     
                   (SEQ ID NO.: 19; 19mer) 
                 
                     
                   5′ ATC  ATG  GAA AGC CAA CTT C 3′. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         8 . A vector comprising a recombinant construct comprising a nucleic acid molecule according to  claim 1 . 
     
     
         9 . A cultured host cell transformed or transfected by a vector according to  claim 8 . 
     
     
         10 . A cell, plasmid, virus, live organism or other vehicle that has been genetically- or protein-engineered to produce a polypeptide or protein according to  claim 3 . 
     
     
         11 . The cell, plasmid, virus, live organism, or other vehicle of  claim 10 , having incorporated expressibly therein a nucleic acid molecule according to  claim 1 . 
     
     
         12 . A pharmaceutical formulation for stimulating embryonic development of an oocyte, comprising:
 a polypeptide or protein according to  claim 3 ; and   a pharmaceutically acceptable carrier therefor.   
     
     
         13 . The pharmaceutical formulation according to  claim 12  wherein the pharmaceutical is formulated so that the amount of PLC-zeta polypeptide or protein delivered is between 150-450 fg per oocyte. 
     
     
         14 . A pharmaceutical formulation for stimulating embryonic development of an oocyte, comprising:
 an expressible nucleic acid molecule according to  claim 1 ; and   a pharmaceutically acceptable carrier therefore.   
     
     
         15 . The pharmaceutical formulation according to  claim 14  wherein the pharmaceutical is formulated so that the amount of PLC-zeta polypeptide or protein expressed is between 150-450 fg per oocyte. 
     
     
         16 . A method for stimulating embryonic development of an aged oocyte, comprising administering thereto an isolated, purified or recombinant molecule capable of triggering cytoplasmic calcium oscillations (CCOs) when introduced into said aged oocyte, said molecule comprising a nucleic acid molecule encoding a PLC-zeta (PLCζ) polypeptide or protein, a molecule substantially homologous thereto, or a sequence which hybridizes thereto under stringent conditions. 
     
     
         17 . A method for stimulating embryonic development of an aged oocyte, comprising administering thereto an isolated, purified or recombinant molecule capable of triggering cytoplasmic calcium oscillations (CCOs) when introduced into said aged oocyte, said molecule comprising a PLC-zeta (PLCζ) polypeptide or protein. 
     
     
         18 . The method according to  claim 16  wherein said nucleic acid molecule encodes a nucleotide sequence selected from one of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 10 
     
     
         19 . The method according to  claim 17  wherein or said PLC-zeta (PLCζ) polypeptide or protein is selected from one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 11. 
     
     
         20 . A pharmaceutical formulation for stimulating embryonic development of an aged oocyte, comprising:
 an isolated, purified or recombinant PLC-zeta (PLCζ) polypeptide or protein according to  claim 3 , said polypeptide or protein being capable of triggering cytoplasmic calcium oscillations (CCOs) and stimulating embryonic development when introduced into said aged oocyte; and   a pharmaceutically acceptable carrier.   
     
     
         21 . A pharmaceutical formulation for stimulating embryonic development of an aged oocyte, comprising:
 an isolated, purified or recombinant molecule capable of triggering cytoplasmic calcium oscillations (CCOs) and stimulating embryonic development when introduced into said aged oocyte, said molecule comprising a nucleic acid molecule according to  claim 1 ;   an expression vector comprising said isolated, purified or recombinant molecule; and   a pharmaceutically acceptable carrier.