Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of storing cells in a viable state
Reexamination Certificate
1999-10-12
2002-11-26
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of storing cells in a viable state
C435S006120, C435S007200, C435S007210, C435S069500, C435S260000, C530S350000, C436S501000, C514S002600, C514S04400A, C536S023500, C424S001210
Reexamination Certificate
active
06485972
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to reproductive biology.
Wnt polypeptides are secreted cysteine-rich glycosylated polypeptides that play a role in the development of a wide range of organisms. The Wnt family of polypeptides contains at least 16 mammalian members which bind to an extracellular domain of a family of cell surface proteins called Frizzled receptors. Wnt polypeptides may play a role in embryonic induction, generation of cell polarity, and specification of cell fate.
SUMMARY OF THE INVENTION
The invention is based on the discovery that Wnt signalling is involved in the development of female reproductive organs in the embryo and in oocyte development in adult animals. Accordingly, the invention features a method of contraception which is carried out by administering to a female primate an antagonist of a Wnt polypeptide. The antagonist inhibits Wnt-signalling which plays a role in oocyte development and, in turn, inhibits oocyte development. Preferably, the Wnt polypeptide is Wnt-1 class polypeptide such as Wnt-1, Wnt-2, Wnt-3a, Wnt-4, Wnt-7a, or Wnt-7b polypeptide.
A Wnt antagonist is a composition which inhibits the physiological activity of a Wnt polypeptide. For example, the activity is preferably Wnt signalling by a Wnt-1 class polypeptide. A Wnt antagonist inhibits oocyte development in an adult female animal and is therefore useful as a contraceptive. A Wnt antagonist binds to a Wnt polypeptide or to its receptor or to another component of the Wnt-1 class signal transduction pathway but fails to potentiate Wnt-signalling. For example, the Wnt antagonist is a dominant negative N-cadherin mutant, a dominant negative &bgr;-catenin mutant, or a Frizzled polypeptide. Antagonistic polypeptides may contain a cysteine-rich domain, e.g., a peptide having an amino acid sequence with at least 5%, preferably at least 7%, and most preferably at least 8%, cysteines. The polypeptide preferably contains at least 5 cysteines, more preferably at least 8 cysteines, and most preferably at least 10 cysteines. For example, the polypeptide is about 120 amino acids in length and contains 10 cysteines. Preferably, the polypeptide contains the amino acid consensus sequence of CX
7-9
CX
36-42
C
8
CX
6
CX
6-10
CX
3
CX
6-7
CX
12-27
CX
8-13
(SEQ ID NO: 1) or CXPXXXXXXX CXXXXYXXXX XPNXXXHXXX XXXXXXXXXX XXXXXXXLXX XXCSXXXXXF LCXXXXPXCX XXXXXXXXXX PCRXXCEXXX XXXCXXXXXX XXXXXXXXXX XXXXXWPXXX XCXXXPXXXX XXXXXC (SEQ ID NO:2). Preferably, the polypeptide has at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 75% identity to the cysteine-rich domain of a Frizzled receptor. Sequence identity is determined using the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), with the default parameters as specified therein. Other Wnt antagonists include Wnt-specific antibodies, e.g., an antibody which binds to a Wnt polypeptide and inhibits oocyte development, and Wnt-5a class polypeptides.
The invention also includes a contraceptive vaccine. The vaccine contains a Wnt polypeptide, e.g., Wnt-1, Wnt-2, Wnt-3a, Wnt-4, Wnt-7a, or Wnt-7b. The Wnt polypeptide is characterized as binding to an antibody that inhibits Wnt-signalling, and thereby, oocyte development.
Wnt polypeptides, e.g., Wnt-4 and Wnt-7a, promote oocyte development. Thus, a method of promoting maturation of an immature oocyte by contacting the oocyte with a substantially pure preparation of a Wnt polypeptide, e.g., Wnt-1, Wnt-2, Wnt-3a, Wnt-4, Wnt-7a, or Wnt-7b polypeptide, is also within the invention. Polypeptides or other compounds are said to be “substantially pure” when they are within preparations that are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity is measured by any appropriate standard method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
Wnt polypeptides are also used to enhance viability of an oocyte, e.g., an oocyte to be used in an in vitro fertilization (IVF) procedure. The oocyte is contacted with w Wnt polypeptide either in vivo prior to retrieval or in vitro prior to the IVF procedure. For example, the viability of a cryopreserved oocyte is enhanced by contacting the oocyte with a Wnt polypeptide during and/or subsequent to thawing. Wnt polypeptides are also used to increase the number of mature oocytes by culturing a sample containing immature oocytes in a culture medium which includes a Wnt polypeptide. The oocytes are cultured in the presence of a Wnt polypeptide for at least one hour and up to several days. Following culture, the number of mature oocytes in the sample cultured in the presence of said Wnt polypeptide is at least 50% greater than the number of mature oocytes cultured in the absence of the Wnt polypeptide. Preferably, the increase in the number of mature oocytes in the sample is at least 75%, more preferably 100%, and most preferably 200% greater than the number of mature oocytes cultured in the absence of the Wnt polypeptide.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
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MacLean et
McMahon Andrew P.
Parr Brian A.
Vaino Seppo
Beattie Ingrid A.
Brannock Michael
Eyler Yvonne
Mintz Levin Cohn Ferris Glovsky & Popeo P.C.
President and Fellows of Harvard College
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