WO2004019042A1 - Method for the in vitro diagnostic of infertility - Google Patents

Method for the in vitro diagnostic of infertility Download PDF

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Publication number
WO2004019042A1
WO2004019042A1 PCT/EP2003/007631 EP0307631W WO2004019042A1 WO 2004019042 A1 WO2004019042 A1 WO 2004019042A1 EP 0307631 W EP0307631 W EP 0307631W WO 2004019042 A1 WO2004019042 A1 WO 2004019042A1
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cdcό
protein
nucleic acid
oocytes
antibody
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French (fr)
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Marcel Mechali
Jean-Marc Lemaitre
Stéphane BOCQUET
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Centre National de la Recherche Scientifique CNRS
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Centre National de la Recherche Scientifique CNRS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction

Definitions

  • the present invention relates to a method for the in vitro diagnostic of infertility of a eukaryotic organism.
  • Infertility notably female infertility, arises from various causes. In women the main causes are pelvic inflammatory diseases, endometriosis, hypothalamic-pituitary disorders, polycystic ovarian syndrome, ovarian cysts, premature ovarian failure, luteal phase defect, or benign uterine fibroids. Medical investigations to diagnose infertility and determine its etiology notably rely on in vitro tests, such as hormonal dosages, uterine biopsies, tests for autoimmune diseases, genetic tests, but also on imaging, with techniques such as echography or magnetic resonance imaging; or on physical investigations, such as hysteroscopy or hysterosalpingography.
  • Cdc6 is a protein which is essential for the initiation of DNA replication (Kelly et al. , Annu. Rev. Biochem. (2000) 69:829-880 ; Lei & Tye, J. Cell Sci. (2001) 114:1447-1454).
  • ORC origin of replication complex
  • MCM mini-chromosome maintenance
  • Oocyte maturation to give an unfertilized egg is a specialized stage of the meiotic cell cycle.
  • oocytes are arrested at the prophase stage of the first meiotic division; they are not fertilizable and have lost the ability to replicate DNA.
  • Hormone stimuli induce resumption of meiosis, with completion of meiosis I and arrest in meiosis II at the metaphase stage in vertebrates.
  • the mature oocyte can be fertilized; it has regained the ability to replicate DNA, and embryonic development can initiate.
  • the present invention provides a new method to detect a newly identified cause of sterility.
  • the invention relates in particular to a method for the in vitro diagnostic of infertility of a eukaryotic organism.
  • Another aspect of the invention relates to a pharmaceutical composition useful for curing infertility.
  • a further aspect of the invention relates to the use of a protein for the manufacture of a medicament for the treatment of infertilities.
  • Still another aspect of the invention relates to the use of a compound inhibiting a protein activity for the manufacture of a contraceptive.
  • the present invention relates to a method for the in vitro diagnostic of infertility of a eukaryotic organism, comprising the detection of the presence or the absence of Cdc6 protein or of Cdc ⁇ mRNA in germinal cells of said eukaryotic organism, at a given maturation stage of said germinal cells.
  • the invention more particularly relates to a method for the in vitro diagnostic of infertility of a female eukaryotic organism.
  • infertility refers to the inability of said eukaryotic organism to sexually reproduce.
  • germ cells refers to cells which can yield gametes once differentiated or to the gametes themselves. In a female organism the gametes are called ovules.
  • the expression "ovule” relates to a fertilisable (unfertilized) egg.
  • the expression “maturation stage” refers to a particular step of the process of germinal cell differentiation, or meiotic maturation, to yield a gamete, and in particular to yield an egg from an oocyte.
  • maturing oocytes In a female organism the germinal cells undergoing meiotic maturation are called maturing oocytes. Oocytes not having been through the first meiotic division are called primary oocytes, after having been through the first meiotic division they are called secondary oocytes.
  • the oocyte is called a mature oocyte. Once liberated from the ovary the mature oocyte is called an ovule.
  • infertility can in particular be tested by in vitro fertilization (INF) on an ovule.
  • IFN in vitro fertilization
  • the ovule, and thus the female organism, is said to be infertile if it does not undergo cell multiplication after the entry of the male gamete in the ovule under suitable conditions.
  • the invention relates in particular to a method as defined above, wherein the eukaryotic organism is a multicellular organism, in particular a plant, a fungus, or an animal, such as a mammal, in particular a human.
  • the invention further relates to a method as defined above, wherein the presence of Cdc6 protein is detected in oocytes prior to germinal vesicle breakdown.
  • GNBD germinal vesicle breakdown
  • oocytes prior to germinal vesicle breakdown refers to oocytes which have not yet been through the stage of germinal vesicle breakdown.
  • Oocytes are said to not to have been through GNBD if a nucleus is visible by microscopic observations for instance.
  • Cdc6 in oocytes prior to GNBD can be a cause of infertility.
  • Cdc ⁇ enables oocytes to undertake D ⁇ A replication, which may cause unwanted parthenogenetic development of the oocytes.
  • the invention also relates to the abovementioned method, wherein the absence of Cdc ⁇ protein is detected in oocytes having undergone germinal vesicle breakdown, or in polar globules derived from oocytes having undergone germinal vesicle breakdown, or in ovules.
  • a primary oocyte divides to yield a secondary oocyte and a first polar body (or globule), each containing a set of duplicated chromosomes.
  • a secondary oocyte divides to yield a mature oocyte and a second polar body (or globule), each containing a set of unduplicated chromosomes.
  • the first polar body duplicates.
  • polar globules derived from refers to the first polar globules, duplicated or not, or to the second polar globule, which are yielded in the course of oocyte maturation.
  • the expression "having undergone germinal vesicle breakdown” refers to oocytes in which germinal vesicle breakdown has occurred. Oocytes are said to have been through GNBD if no nucleus is visible by microscopic observations for instance.
  • the present invention relates to a method as defined above, wherein the presence of Cdc ⁇ protein is detected in oocytes prior to germinal vesicule breakdown, comprising a step of detection of the Cdc ⁇ protein with an anti-Cdc ⁇ protein polyclonal or monoclonal antibody.
  • Anti-Cdc ⁇ protein antibodies can be obtained by methods well known to the man skilled in the art, in particular monoclonal antibodies can be obtained according to K ⁇ hler & Milstein Nature (1975) 256:495-497.
  • Detection of the Cdc6 protein with an antibody can be achieved with several immunological methods well known to the man skilled in the art, such as Western blotting, immunocytochemistry, enzyme linked immnuoassay, immunofluorescence or mass spectrometry after proteolytic digestion.
  • the present invention also relates to a method as defined above, wherein the absence of
  • Cdc ⁇ protein is detected in oocytes having undergone germinal vesicle breakdown, or in polar globules derived from oocytes having undergone germinal vesicle breakdown, or in ovules, comprising a step of detection of the Cdc ⁇ protein with an anti-Cdc ⁇ protein polyclonal or monoclonal antibody.
  • the presence or the absence of the Cdc ⁇ protein can be detected in oocytes prior to GVBD, having undergone GNBD, or in ovules, with methods well known to the man skilled in the art. Those methods comprise in particular Western blotting, immunocytochemistry, enzyme linked immnuoassay, immunofluorescence or mass spectrometry.
  • the present invention further relates to a method as defined above, comprising the following steps:
  • the sampling can be performed during an in, vitro fecundation (INF) procedure of an ovule by spermatozoids, or during intracytoplasmic sperm injection procedure (ICSI) into an ovule, before or after contacting sperm and the ovule.
  • INF vitro fecundation
  • ICSI intracytoplasmic sperm injection procedure
  • IDAT refers to Immuno-Detection Amplified by T7 D ⁇ A polymerase, it is in particular described in Zhang et al., Proc. Nat/. Acad. Sci. USA (2001) 98:5497-502.
  • the present invention relates to a kit for carrying out the above mentioned process, comprising an anti-Cdc ⁇ polyclonal or monoclonal antibody, optionally a medium in which the sampled polar globule is extracted for protein preparation, a medium in which the sampled polar globule, or the protein preparation extracted from said polar globule, is contacted with said antibody, a reagent to detect the complex formed between Cdc ⁇ and said antibody, and a control medium containing the Cdc ⁇ protein.
  • the antibody can be labelled, for instance with a fluorescent label, a radioelement, or an enzyme.
  • the protein preparation can be labelled by fluorescent dyes such as Cy5 or Cy3 dyes (Research Organics, Cleveland, USA)
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising as active substance a protein comprising or consisting of the Cdc ⁇ protein or a part thereof, or a nucleic acid comprising or consisting of a nucleic acid encoding the Cdc ⁇ protein or a part thereof, in association with a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers notably comprise liposomes or ChariotTM carriers
  • the pharmaceutical composition comprises the Cdc ⁇ protein as active substance.
  • the invention relates in particular to a pharmaceutical composition as defined above, wherein the nucleic acid is a RNA, in particular a polyadenylated RNA.
  • polyadenylated RNA refers to a RNA which carries a 3' polyadenosine tail.
  • the invention more particularly relates to a pharmaceutical composition as defined above, wherein the Cdc ⁇ protein comprises a sequence corresponding to the human Cdc ⁇ protein sequence, said sequence corresponding in particular to SEQ ID NO: 2, or wherein the nucleic acid encoding the Cdc ⁇ protein comprises a sequence corresponding to the human
  • Cdc ⁇ nucleic acid sequence said sequence corresponding in particular to SEQ ED NO: 1.
  • human Cdc ⁇ refers to a Cdc ⁇ protein or a Cdc ⁇ encoding nucleic acid, the sequences of which are identical to the sequences of Cdc ⁇ proteins or Cdc ⁇ encoding nucleic acids which can be found in natural human cells.
  • the present invention relates to the use of a protein comprising or consisting of the Cdc ⁇ protein or a part thereof, or of a nucleic acid comprising or consisting of a nucleic acid encoding the Cdc ⁇ protein or a part thereof, for the manufacture of a medicament for the treatment of infertilities.
  • the Cdc ⁇ protein or a nucleic acid encoding said protein can be administered to an unfertile ovocyte or ovule.
  • the delivery of the Cdc ⁇ protein or the Cdc ⁇ mRNA is carried out by cell microinjection.
  • sperm injection into the ovule is accompanied by Cdc ⁇ protein or Cdc ⁇ protein encoding nucleic acid administration, in particular inj ection, into the ovule.
  • the invention relates in particular to the use as defined above, wherein the nucleic acid is a RNA, in particular a polyadenylated RNA.
  • the invention further relates to the use as defined above, wherein the Cdc ⁇ protein comprises a sequence corresponding to the human Cdc ⁇ protein sequence, said sequence corresponding in particular to SEQ ID NO: 2, or wherein the nucleic acid encoding the Cdc ⁇ protein comprises a sequence corresponding to the human Cdc ⁇ nucleic acid sequence, said sequence corresponding in particular to SEQ TD NO: 1.
  • the present invention relates to the use of a compound inhibiting Cdc ⁇ protein activity, such as a pharmacological compound, an antisense polynucleotide directed against the mRNA encoding the Cdc ⁇ protein, a siRNA inhibiting Cdc ⁇ protein expression, an anti-Cdc ⁇ protein antibody or fragments thereof, such as a Fab fragment or a scFv fragment, for the manufacture of a contraceptive.
  • a compound inhibiting Cdc ⁇ protein activity such as a pharmacological compound, an antisense polynucleotide directed against the mRNA encoding the Cdc ⁇ protein, a siRNA inhibiting Cdc ⁇ protein expression, an anti-Cdc ⁇ protein antibody or fragments thereof, such as a Fab fragment or a scFv fragment, for the manufacture of a contraceptive.
  • antisense polynucleotide refers to a polynucleotide the sequence of which is essentially complementary to a target sequence, said target sequence being in particular located within the sequence of Cdc ⁇ mRNA
  • siRNA relates to a double stranded RNA which can silence the expression of specific genes, advantageously the sequence of said siRNA comprises or consists of at least a part of a sequence of a nucleic acid encoding the Cdc ⁇ protein.
  • Fab fragment relates to antibody fragments which can be obtained by papaine digestion of antibodies.
  • scFv fragment refers to a single chain Fv fragment, it is usually recombinant.
  • the invention more particularly relates to the use as defined above, wherein the Cdc ⁇ protein corresponds to a human Cdc ⁇ protein.
  • the present invention also relates to a phytosanitary composition
  • a phytosanitary composition comprising as active substance a protein comprising or consisting of the Cdc ⁇ protein or a part thereof, or a nucleic acid comprising or consisting of a nucleic acid encoding the Cdc ⁇ protein or a part thereof, or a compound inhibiting Cdc ⁇ activity, such as an antisense nucleotide directed against the mRNA encoding the Cdc ⁇ protein, a siRNA inhibiting Cdc ⁇ protein expression, an anti-Cdc ⁇ protein antibody or fragments thereof, such as a Fab fragment or a scFv fragment.
  • phytosanitary composition relates to a composition useful for the treatment of plants.
  • the invention relates in particular to the above defined phytosanitary composition, wherein the Cdc ⁇ protein corresponds to a plant Cdc ⁇ protein.
  • plant Cdc ⁇ refers to a Cdc ⁇ protein or a Cdc ⁇ encoding nucleic acid, the sequences of which are identical to the sequences of Cdc ⁇ proteins or Cdc ⁇ encoding nucleic acids which can be found in natural plant cells
  • the present invention relates to the use of a protein comprising or consisting of the Cdc ⁇ protein or a part thereof, or of a nucleic acid comprising or consisting of a nucleic acid encoding the Cdc ⁇ protein or a part thereof, or of a compound inhibiting Cdc ⁇ protein activity, such as an antisense polynucleotide directed against the mRNA encoding the Cdc ⁇ protein, a siRNA inhibiting Cdc ⁇ protein expression, an anti-Cdc ⁇ protein antibody or fragments thereof, such as a Fab fragment or a scFv fragment, for the treatment of a plant or parts thereof, or for the inhibition of plant reproduction.
  • the invention relates in particular to the above defined use of a protein comprising or consisting of the Cdc ⁇ protein or a part thereof, or a nucleic acid comprising or consisting of a nucleic acid encoding the Cdc ⁇ protein or a part thereof, for the treatment of plant infertility.
  • the invention more particularly relates to the above defined use of a compound inhibiting Cdc ⁇ protein activity, such as an antisense polynucleotide directed against the mRNA encoding the Cdc ⁇ protein, a siRNA inhibiting Cdc ⁇ protein expression, an anti-Cdc ⁇ protein antibody or fragments thereof, such as a Fab fragment or a scFv fragment, as an herbicide.
  • the expression "herbicide” relates to a compound which inhibits plant growth and/or reproduction, in particular sexual reproduction.
  • the invention further relates to the use as defined above, wherein the Cdc ⁇ protein corresponds to a plant Cdc ⁇ protein.
  • Figure 1 represents immunoblots, revealed with antibodies directed against Xenopus CDC6, of a SDS-PAGE of total, cytoplasmic and germinal vesicle (GN) fractions of Xenopus oocytes, and of CSF (ie containing the cytostatic factor) and interphasic extracts of Xenopus eggs.
  • the bands approximately migrating to 60 kD correspond to Cdc ⁇ .
  • Figure 2 represents the immunoblotting of a SDS-PAGE of extracts from Xenopus oocytes derived from stage 6 oocytes induced to mature by progesterone (PG).
  • GNBD occured 2 h 45 min after progesterone addition (GNBD). Extracts were collected 0, 1, 2, 3, 4.5, 6.5 or 7.5 hours after the addition of progesterone.
  • Some oocytes were also treated with cycloheximide either 30 min before GNBD (cyclo before GNBD), or 1 hour after GNBD (cyclo after GVBD), and collected at the Metaphase I / Metaphase II transition (2h 30 min after GNBD).
  • the immunoblot was revealed with antibodies directed against CDC6, ORC2, MCM4, MCM3 or CDC45.
  • Figure 3 represents a Northern blot of extracts from Xenopus oocytes derived from stage 6 oocytes induced to mature by progesterone (PG) and revealed by a labelled anti-Cdc ⁇ mRNA probe.
  • GNBD occured 2 h 45 min after progesterone addition (GNBD). Extracts were collected 0, 1, 2, 3, 4.5, 6.5 or 7.5 hours after the addition of progesterone.
  • Some oocytes were also treated with cycloheximide either 30 min before GNBD (cyclo before GNBD), or 1 hour after GNBD (cyclo after GNBD), and collected at the Metaphase I / Metaphase II transition (2h 30 min after GNBD).
  • Some oocytes were also treated with Actinomycin D.
  • Figure 4 represents the immunoblotting, revealed with antibodies d ected against Xenopus Cdc ⁇ , of a SDS-PAGE of extracts from stage 6 oocytes, or stage 6 oocytes injected with 50 ng of 25-mer sense Cdc ⁇ oligonucleotide, 50 ng of 25-mer antisense Cdc ⁇ oligonucleotide, or or water (mock), and induced to mature by PG.
  • Matured oocytes were collected 12 hours after PG stimulation
  • Figure 5A represents the D ⁇ A synthesis ability, analyzed by agarose gel electrophoresis, of Xenopus oocytes injected with 25 nl of a 2 mg/ml solution of purified Cdc ⁇ sense oligonucleotides (lane 1), the corresponding antisense oligonucleotides (lanes 2, 3), or water (lane 4) and induced to mature.
  • Rescue experiments were assayed by a second injection performed soon after GNBD that contained 0.25 ⁇ Ci ⁇ 32-PdCTP (lanes 1-4) and either boiled-inactivated (lane 2) or intact purified Cdc ⁇ (lane 3).
  • the arrow on the left marks the migration of the bands corresponding to the newly synthesized D ⁇ A.
  • Figure 5B represents the D ⁇ A synthesis ability, analyzed by agarose gel electrophoresis, of Xenopus oocytes injected with water (lane 1), antisense oligonucleotides (lanes 2, 3) or sense oligonucleotides (lane 4) and fully matured during twelve hours.
  • D ⁇ A replication was analyzed by injection of 0.25 ⁇ Ci ⁇ 32P-dCTP in absence (lanes 1, 2, 4) or presence of recombinant Cdc ⁇ protein (lane 3).
  • the arrow on the left marks the migration of the bands corresponding to the newly synthesized D ⁇ A.
  • Figure 5C represents the D ⁇ A synthesis ability analyzed by agarose gel electrophoresis of Xenopus oocytes induced to mature and treated with 250 ⁇ g/ml cycloheximide added 30 min before GNBD (lane 2) or 1 hour after GNBD (lane 4). Maturing oocytes were injected soon after GNBD with 0.25 ⁇ Ci of ⁇ 32P-dCTP without (lanes 1, 4) or with active (lane 2) or inactivated (lane 3) recombinant Cdc ⁇ protein. The arrow on the left marks the migration of the bands corresponding to the newly synthesized D ⁇ A
  • Figure 6A, Figure 6B and Figure 6C Figure 6A represents the D ⁇ A synthesis ability analyzed by agarose gel electrophoresis of stage 6 Xenopus oocytes induced to mature by PG, either non treated (-cyclo) or treated with 250 ⁇ g/ml cycloheximide 30 min before GNBD (cyclo -30 min), or 1 hour after GNBD (cyclo +lh).
  • Oocytes extracts were collected at the metaphasel/metaphasell (MI/MII) transition (2 hrs 30 min after GNBD).
  • Matured oocytes were also 'collected 12 h after PG addition. Sperm nuclei were added to the extracts and D ⁇ A synthesis was followed by agarose gel electrophoresis, after 2 hours.
  • Figure 6B represents the percentage of D ⁇ A replication after 2 hours (vertical axis) for a metaphase I/II extract from maturing oocytes either treated by: - cycloheximide before GVBD, and water (mock), Cdc ⁇ protein (+ CDC6) or Cdc ⁇ protein and aphidicolin (+ CDC6 Aphi), or - cycloheximide after GNBD and water (mock) or aphidicolin (+ Aphi).
  • Figure 6C represents the percentage of DNA replication after 2 hours (vertical axis) for:
  • Figure 7 represents an immunoblot of a SDS-PAGE of extracts from Xenopus or mouse oocytes in prophase I, metaphase I or metaphase II.
  • the immunoblot was revealed with antibodies directed against Xenopus CDC6 and MCM2, or mouse CDC6 and MCM2.
  • Figure 8 represents an immunoblot of a SDS-PAGE of extracts from virgin female Drosophila melanogaster at stage 14 (lane 1), or activated oocytes (lane 2).
  • the immunoblot was revealed with antibodies directed against Cdc ⁇ (DmCDC ⁇ ), MCM2 (DmMCM2), and tubulin (DmTub).
  • Cdc6 confers competence to replicate in the unfertilized egg of Xenopus laevis
  • Defolliculated oocytes were prepared as described in Menu et al. in Advances in Molecular Biology (1999) Oxford University Press, Ed. J.D. Richter. Cytoplasmic and germinal vesicle fractions were obtained after manual enucleation either from intact oocytes or oocytes fixed by 2 % TCA to prevent possible leaks from the nucleus.
  • Protein extracts were obtained by grinding oocytes in 100 mM KC1, 5 mM MgCl , 0.1 mM CaCl , 10 mM K/Hepes pH 7.7, 50 mM sucrose, 5 ⁇ g/ml leupeptin, 5 ⁇ g/ml pepstatin, 5 ⁇ g/ml aprotinin, 0.5 mM EGTA, 0.3 % XI 00 Triton, centrifuged 5 min at 8 000 g, and further analyzed by SDS- PAGE.
  • GNBD extract germinal vesicle breakdown
  • MI/MII extract MI/MII extract
  • Maturing oocytes extracts were performed as for egg extracts in eppendorf tubes (Menu et al. in Advances in Molecular Biology (1999) Oxford University Press, Ed. J.D. Richter). After removing the excess buffer, oocytes were centrifuged at 8000 g, 5 min at 4°C, and then 12000 g, 2 min at 4°C.
  • Oocytes were injected with 25 nl of 5 mCi/ml 32P-dCTP, collected 5 hours after GNBD, and subjected to D ⁇ A extraction. Samples with equal numbers of total counts were analyzed by 0.8 % agarose gel electrophoresis. D ⁇ A replication assays were also performed by quantification of ⁇ 32P-dCTP incorporation in sperm nuclei used at 1000 nuclei/ ⁇ l of extract. Analysis was both done by TCA precipitation and migration on 0.8 % Agarose (Menu et al. in Advances in Molecular Biology (1999) Oxford University Press, Ed. J.D. Richter). Purified protein was added at 15 ng/ ⁇ l of extract to rescue DNA replication activity.
  • Interphasic extract ie extracts containing the mitotic cytostatic factor
  • CSF extract ie extracts containing the mitotic cytostatic factor
  • Total oocyte RNA was extracted using a LiCl method (Taylor et al, EMBO J. (1986) 5:3563-3570). 10 ⁇ g of total RNA was submitted to electrophoresis in 1 % formaldehyde agarose gel and transferred to Amersham Hybond N+ membrane.
  • Stage 6 oocytes were injected with 25 nl of a 2 mg/ml solution of purified Cdc ⁇ 25 mers sense oligonucleotide (5'-TCCCCACCCAAGCAGTCTCGCAAAG-3') (SEQ ID NO: 3) or 25 mers of the corresponding antisense (5'-CTTTGCGAGACTGCTTGGGTGGGGA- 3') (SEQ ID NO: 4) oligonucleotide, and induced to mature by addition of progesterone. Control oocytes were injected in parallel with water.
  • Xenopus His ⁇ -Cdc ⁇ protein was purified from recombinant baculovirus-infected insect cells. Its activity was monitored by depletion-rescue experiments as follows: 50 ⁇ l of low speed interphasic extract was depleted with Cdc ⁇ specific polyclonal antibody, or mock- depleted with preimmune serum IgG, as previously described (Maiorano et al, Nature (2000) 404:622-625). Cdc ⁇ purified protein was added to the depleted extract and D ⁇ A replication was then measured. The rabbit polyclonal anti-Cdc ⁇ antibodies were obtained by 4 injections of Cdc ⁇ protein. B. Results
  • Cdc ⁇ was absent in the oocyte, a relatively large amount of the protein was detected in the unfertilized egg, 5 ng / unfertilized egg, an estimate comparable with the 3 ng- value previously reported (Coleman et al, Cell (1996) 87:53-63).
  • Cdc ⁇ is an essential factor for replication licensing, the Inventors asked at what point during maturation it was accumulated.
  • Figure 2 the Cdc ⁇ protein becomes detectable soon after germinal vesicle breakdown (GNBD, e.g. 3 hrs after progesterone addition), when MCM4 is phosphorylated by MPF (Coue et al, EMBO J.
  • Cdc ⁇ was sufficient to give competence to replicate in- vivo to the matured oocyte.
  • parthenogenetic activation triggered by a micropipette injection of water for instance induces D ⁇ A replication (Gurdon et al, Proc. Natl. Acad. Sci. U.S.A. (1967) 58:545-552 ; Hara et al., Proc. Natl Acad. Sci. U.S.A. (1980) - 77:462-466 ; Harland & Laskey, Cell (1980) 21:761-771) as shown in Figure 5B (lane 1).
  • Cdc ⁇ is the missing factor synthesized during oocyte maturation sufficient to give (i) replication-induced ability between meiosisl/meiosisll, and (ii), competence to replicate in the unfertilized egg.
  • CDC6 confers competence to replicate in the unfertilized egg of Drosophila melanogaster
  • Drosophila melanogaster provides a particularly potent system to study whether in invertebrate organism Cdc ⁇ appears during meiosis to give the competence to replicate DNA to the egg.
  • female D. melanogaster generate oocytes but store them, arrested and unactivated, in the ovary. After a period of growth, Drosophila oocytes arrest in metaphase of meiosis I, with egg coverings (a vitelline envelope and chorion). Ovulation releases these mature eggs from the ovary and triggers the process known as activation.
  • the Inventors have compared activated eggs laid by a virgin female with stage 14 oocytes blocked at Metaphase I, collected directly from the ovaries. Both were treated to remove the vitelline envelope and chorion. The activation of the oocytes triggers the synthesis of Cdc ⁇ that was absent from the stage 14 (metaphase I oocyte), while MCM2 was already present at stage 14 (Figure 8). Despite several differences in egg formation D. melanogaster accumulates Cdc ⁇ betwen metaphase I and metaphase II, before fertilisation.
  • Cdc ⁇ immunostaining during oocyte growth revealed a positive signal in the cytoplasm of nurse cells and cells covering the oocyte, while MCM2 was mostly nuclear, consistent with cell cycle regulation of Cdc ⁇ function.
  • Cdc ⁇ is absent from prophase I oocyte and translated between Metaphase I and Metaphase II to give to the egg the competence to replicate before fertilisation.
  • CDC6 confers competence to replicate in the unfertilized egg of Mus musculus
  • Oocyte extracts were prepared as described above and analyzed by SDS-PAGE. Irrimunoblots were revealed with antibodies directed against Xenopus laevis Cdc ⁇ and MCM2, as well as mouse Cdc ⁇ and MCM2. Prophase I oocytes were induced to mature by addition of progesterone (PG). Extracts from 5 X. laevis maturing oocytes or 85 mice oocytes were separated by SDS PAGE and examined by immunoblotting. The results are shown in Figure 7. Mouse Cdc ⁇ is absent from prophase I oocyte and translated between Metaphase I and Metaphase II, which is compatible with its ability to confer competence to replicate to the egg before fertilization.
  • PG progesterone
  • mammalian Cdc ⁇ appears to be a potent target for the development of new contraceptives. Besides, its essential role in the acquisition of the competence to replicate in the egg strongly suggests that it might be involved in several infertilities, which could be overcome by providing Cdc ⁇ protein or Cdc ⁇ mRNA to infertile eggs or oocytes.
  • the following method is used for the detection of the Cdc ⁇ protein, in particular for the detection of the human Cdc ⁇ protein in germinal cells, ovocytes, ovules or polar bodies:
  • Ovulated oocytes or oocyte or polar bodies for instance obtained before or after "in vitro fertilization” (INF) or "intracytoplasmic sperm injection” (ICSI), are transferred to a sterile watch glass containing fixation buffer (20 mM Pipes, ImM MgCl 2 , 0.5 mM EGTA, 1 mM DTT, 1 ⁇ M Taxol, 0.1 % XI 00 triton, 2 % Formaldehyde) at 37°C for 30 minutes.
  • fixation buffer (20 mM Pipes, ImM MgCl 2 , 0.5 mM EGTA, 1 mM DTT, 1 ⁇ M Taxol, 0.1 % XI 00 triton, 2 % Formaldehyde
  • oocyte Tranfer on a microscope slide and add a drop of mounting medium and cover with a glass coverslip. 10) The oocyte can be observed under a fluorescence microscope for Cdc ⁇ immunostaining. A negative signal is indicative of an unfertilizable ovulated oocyte.
  • the detection of the presence or the absence of Cdc ⁇ detected by this method provides the basis for an in vitro diagnostic method of infertilities linked to a deficiency in active Cdc ⁇ .

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Abstract

The present invention relates to a method for the in vitro diagnostic of infertility of a eukaryotic organism, comprising the detection of the presence or the absence of Cdc6 protein or of Cdc6 mRNA in germinal cells of said eukaryotic organism, at a given maturation stage of said germinal cells.

Description

METHOD FOR THE IN VITRO DIAGNOSTIC OF INFERTILITY
The present invention relates to a method for the in vitro diagnostic of infertility of a eukaryotic organism.
Infertility, notably female infertility, arises from various causes. In women the main causes are pelvic inflammatory diseases, endometriosis, hypothalamic-pituitary disorders, polycystic ovarian syndrome, ovarian cysts, premature ovarian failure, luteal phase defect, or benign uterine fibroids. Medical investigations to diagnose infertility and determine its etiology notably rely on in vitro tests, such as hormonal dosages, uterine biopsies, tests for autoimmune diseases, genetic tests, but also on imaging, with techniques such as echography or magnetic resonance imaging; or on physical investigations, such as hysteroscopy or hysterosalpingography.
However, about 15 to 20 % of female infertilities remain unexplained and thus untreated.
Cdc6 is a protein which is essential for the initiation of DNA replication (Kelly et al. , Annu. Rev. Biochem. (2000) 69:829-880 ; Lei & Tye, J. Cell Sci. (2001) 114:1447-1454). In conjunction with Cdctl and the origin of replication complex (ORC) it is responsible for the loading of the mini-chromosome maintenance (MCM) complex onto the chromatin to form a pre-replication complex. This step establishes the license to replicate DNA, enabling the association of a replication complex to chromatin.
Oocyte maturation to give an unfertilized egg is a specialized stage of the meiotic cell cycle. In most species, oocytes are arrested at the prophase stage of the first meiotic division; they are not fertilizable and have lost the ability to replicate DNA. Hormone stimuli induce resumption of meiosis, with completion of meiosis I and arrest in meiosis II at the metaphase stage in vertebrates. At this stage, the mature oocyte can be fertilized; it has regained the ability to replicate DNA, and embryonic development can initiate.
The present invention provides a new method to detect a newly identified cause of sterility. The invention relates in particular to a method for the in vitro diagnostic of infertility of a eukaryotic organism.
Another aspect of the invention relates to a pharmaceutical composition useful for curing infertility. A further aspect of the invention relates to the use of a protein for the manufacture of a medicament for the treatment of infertilities.
Still another aspect of the invention relates to the use of a compound inhibiting a protein activity for the manufacture of a contraceptive. The present invention relates to a method for the in vitro diagnostic of infertility of a eukaryotic organism, comprising the detection of the presence or the absence of Cdc6 protein or of Cdcό mRNA in germinal cells of said eukaryotic organism, at a given maturation stage of said germinal cells.
According to a preferred embodiment, the invention more particularly relates to a method for the in vitro diagnostic of infertility of a female eukaryotic organism.
The expression "infertility" refers to the inability of said eukaryotic organism to sexually reproduce.
The expression "germinal cells" refers to cells which can yield gametes once differentiated or to the gametes themselves. In a female organism the gametes are called ovules.
The expression "ovule" relates to a fertilisable (unfertilized) egg. The expression "maturation stage" refers to a particular step of the process of germinal cell differentiation, or meiotic maturation, to yield a gamete, and in particular to yield an egg from an oocyte. In a female organism the germinal cells undergoing meiotic maturation are called maturing oocytes. Oocytes not having been through the first meiotic division are called primary oocytes, after having been through the first meiotic division they are called secondary oocytes. At the final stage of maturation the oocyte is called a mature oocyte. Once liberated from the ovary the mature oocyte is called an ovule. In a female organism, infertility can in particular be tested by in vitro fertilization (INF) on an ovule. The ovule, and thus the female organism, is said to be infertile if it does not undergo cell multiplication after the entry of the male gamete in the ovule under suitable conditions.
The invention relates in particular to a method as defined above, wherein the eukaryotic organism is a multicellular organism, in particular a plant, a fungus, or an animal, such as a mammal, in particular a human.
The invention further relates to a method as defined above, wherein the presence of Cdc6 protein is detected in oocytes prior to germinal vesicle breakdown.
The expression "germinal vesicle" refers to the nucleus of a non-mature oocyte. At a particular stage of oocyte meiotic maturation, prior to metaphase I and after prophase I, the nucleus is fragmented into vesicles, it is said to undergo germinal vesicle breakdown (GNBD).
The expression "oocytes prior to germinal vesicle breakdown" refers to oocytes which have not yet been through the stage of germinal vesicle breakdown.
Oocytes are said to not to have been through GNBD if a nucleus is visible by microscopic observations for instance.
The presence of Cdc6 in oocytes prior to GNBD can be a cause of infertility. The presence of Cdcό enables oocytes to undertake DΝA replication, which may cause unwanted parthenogenetic development of the oocytes.
The invention also relates to the abovementioned method, wherein the absence of Cdcό protein is detected in oocytes having undergone germinal vesicle breakdown, or in polar globules derived from oocytes having undergone germinal vesicle breakdown, or in ovules. During the first division of meiotic maturation a primary oocyte divides to yield a secondary oocyte and a first polar body (or globule), each containing a set of duplicated chromosomes. During the second division of meiotic maturation a secondary oocyte divides to yield a mature oocyte and a second polar body (or globule), each containing a set of unduplicated chromosomes. Upon fertilization of the mature oocyte the first polar body duplicates. The expression "polar globules derived from" refers to the first polar globules, duplicated or not, or to the second polar globule, which are yielded in the course of oocyte maturation.
The expression "having undergone germinal vesicle breakdown" refers to oocytes in which germinal vesicle breakdown has occurred. Oocytes are said to have been through GNBD if no nucleus is visible by microscopic observations for instance.
The absence of Cdcό in oocytes having undergone GNBD can be a cause of infertility. The absence of Cdc6 impairs the replication of DΝA, and thus impairs embryogenic development. According to another embodiment the present invention relates to a method as defined above, wherein the presence of Cdcό protein is detected in oocytes prior to germinal vesicule breakdown, comprising a step of detection of the Cdcβ protein with an anti-Cdcβ protein polyclonal or monoclonal antibody. Anti-Cdcό protein antibodies can be obtained by methods well known to the man skilled in the art, in particular monoclonal antibodies can be obtained according to Kδhler & Milstein Nature (1975) 256:495-497.
Detection of the Cdc6 protein with an antibody can be achieved with several immunological methods well known to the man skilled in the art, such as Western blotting, immunocytochemistry, enzyme linked immnuoassay, immunofluorescence or mass spectrometry after proteolytic digestion.
The present invention also relates to a method as defined above, wherein the absence of
Cdcό protein is detected in oocytes having undergone germinal vesicle breakdown, or in polar globules derived from oocytes having undergone germinal vesicle breakdown, or in ovules, comprising a step of detection of the Cdcό protein with an anti-Cdcό protein polyclonal or monoclonal antibody.
The presence or the absence of the Cdcό protein can be detected in oocytes prior to GVBD, having undergone GNBD, or in ovules, with methods well known to the man skilled in the art. Those methods comprise in particular Western blotting, immunocytochemistry, enzyme linked immnuoassay, immunofluorescence or mass spectrometry.
The present invention further relates to a method as defined above, comprising the following steps:
- sampling a polar globule from an egg, fertilized or not; - contacting the sampled polar globule or extracts thereof with an anti-Cdcό polyclonal or monoclonal antibody,
- detecting the presence or the absence of complexes between the Cdcό protein and the anti-Cdcό polyclonal or monoclonal antibody.
Advantageously, the sampling can be performed during an in, vitro fecundation (INF) procedure of an ovule by spermatozoids, or during intracytoplasmic sperm injection procedure (ICSI) into an ovule, before or after contacting sperm and the ovule.
Detection of the Cdcό protein can be carried out in particular with the ID AT method. The expression IDAT refers to Immuno-Detection Amplified by T7 DΝA polymerase, it is in particular described in Zhang et al., Proc. Nat/. Acad. Sci. USA (2001) 98:5497-502. According to another embodiment the present invention relates to a kit for carrying out the above mentioned process, comprising an anti-Cdcό polyclonal or monoclonal antibody, optionally a medium in which the sampled polar globule is extracted for protein preparation, a medium in which the sampled polar globule, or the protein preparation extracted from said polar globule, is contacted with said antibody, a reagent to detect the complex formed between Cdcό and said antibody, and a control medium containing the Cdcό protein.
Advantageously the antibody can be labelled, for instance with a fluorescent label, a radioelement, or an enzyme. Alternatively, the protein preparation can be labelled by fluorescent dyes such as Cy5 or Cy3 dyes (Research Organics, Cleveland, USA)
The present invention also relates to a pharmaceutical composition comprising as active substance a protein comprising or consisting of the Cdcό protein or a part thereof, or a nucleic acid comprising or consisting of a nucleic acid encoding the Cdcό protein or a part thereof, in association with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers notably comprise liposomes or Chariot™ carriers
(Active Motif, Carlsbad, USA) for instance.
Advantageously the pharmaceutical composition comprises the Cdcό protein as active substance.
The invention relates in particular to a pharmaceutical composition as defined above, wherein the nucleic acid is a RNA, in particular a polyadenylated RNA.
The expression "polyadenylated RNA" refers to a RNA which carries a 3' polyadenosine tail.
The invention more particularly relates to a pharmaceutical composition as defined above, wherein the Cdcό protein comprises a sequence corresponding to the human Cdcό protein sequence, said sequence corresponding in particular to SEQ ID NO: 2, or wherein the nucleic acid encoding the Cdcό protein comprises a sequence corresponding to the human
Cdcό nucleic acid sequence, said sequence corresponding in particular to SEQ ED NO: 1.
The expression human Cdcό refers to a Cdcό protein or a Cdcό encoding nucleic acid, the sequences of which are identical to the sequences of Cdcό proteins or Cdcό encoding nucleic acids which can be found in natural human cells.
Human Cdcό is in particular described in Williams et al., Proc. Natl. Acad. Sci. U.S.A. (1997) 94:142-147.
According to another embodiment the present invention relates to the use of a protein comprising or consisting of the Cdcό protein or a part thereof, or of a nucleic acid comprising or consisting of a nucleic acid encoding the Cdcό protein or a part thereof, for the manufacture of a medicament for the treatment of infertilities.
The Cdcό protein or a nucleic acid encoding said protein, can be administered to an unfertile ovocyte or ovule. According to a preferred embodiment the delivery of the Cdcό protein or the Cdcό mRNA is carried out by cell microinjection.
According to another preferred embodiment in the case of ICSI, sperm injection into the ovule is accompanied by Cdcό protein or Cdcό protein encoding nucleic acid administration, in particular inj ection, into the ovule.
The invention relates in particular to the use as defined above, wherein the nucleic acid is a RNA, in particular a polyadenylated RNA.
The invention further relates to the use as defined above, wherein the Cdcό protein comprises a sequence corresponding to the human Cdcό protein sequence, said sequence corresponding in particular to SEQ ID NO: 2, or wherein the nucleic acid encoding the Cdcό protein comprises a sequence corresponding to the human Cdcό nucleic acid sequence, said sequence corresponding in particular to SEQ TD NO: 1.
According to another embodiment the present invention relates to the use of a compound inhibiting Cdcό protein activity, such as a pharmacological compound, an antisense polynucleotide directed against the mRNA encoding the Cdcό protein, a siRNA inhibiting Cdcό protein expression, an anti-Cdcό protein antibody or fragments thereof, such as a Fab fragment or a scFv fragment, for the manufacture of a contraceptive.
The expression "antisense polynucleotide" refers to a polynucleotide the sequence of which is essentially complementary to a target sequence, said target sequence being in particular located within the sequence of Cdcό mRNA
The expression "siRNA" relates to a double stranded RNA which can silence the expression of specific genes, advantageously the sequence of said siRNA comprises or consists of at least a part of a sequence of a nucleic acid encoding the Cdcό protein.
The expression "Fab fragment" relates to antibody fragments which can be obtained by papaine digestion of antibodies.
The expression "scFv fragment" refers to a single chain Fv fragment, it is usually recombinant.
The expression "contraceptive" refers to a compound which blocks sexual reproduction.
The invention more particularly relates to the use as defined above, wherein the Cdcό protein corresponds to a human Cdcό protein.
The present invention also relates to a phytosanitary composition comprising as active substance a protein comprising or consisting of the Cdcό protein or a part thereof, or a nucleic acid comprising or consisting of a nucleic acid encoding the Cdcό protein or a part thereof, or a compound inhibiting Cdcό activity, such as an antisense nucleotide directed against the mRNA encoding the Cdcό protein, a siRNA inhibiting Cdcό protein expression, an anti-Cdcό protein antibody or fragments thereof, such as a Fab fragment or a scFv fragment.
The expression "phytosanitary" composition relates to a composition useful for the treatment of plants. The invention relates in particular to the above defined phytosanitary composition, wherein the Cdcό protein corresponds to a plant Cdcό protein.
The expression "plant Cdcό" refers to a Cdcό protein or a Cdcό encoding nucleic acid, the sequences of which are identical to the sequences of Cdcό proteins or Cdcό encoding nucleic acids which can be found in natural plant cells According to another embodiment the present invention relates to the use of a protein comprising or consisting of the Cdcό protein or a part thereof, or of a nucleic acid comprising or consisting of a nucleic acid encoding the Cdcό protein or a part thereof, or of a compound inhibiting Cdcό protein activity, such as an antisense polynucleotide directed against the mRNA encoding the Cdcό protein, a siRNA inhibiting Cdcό protein expression, an anti-Cdcό protein antibody or fragments thereof, such as a Fab fragment or a scFv fragment, for the treatment of a plant or parts thereof, or for the inhibition of plant reproduction.
The invention relates in particular to the above defined use of a protein comprising or consisting of the Cdcό protein or a part thereof, or a nucleic acid comprising or consisting of a nucleic acid encoding the Cdcό protein or a part thereof, for the treatment of plant infertility. The invention more particularly relates to the above defined use of a compound inhibiting Cdcό protein activity, such as an antisense polynucleotide directed against the mRNA encoding the Cdcό protein, a siRNA inhibiting Cdcό protein expression, an anti-Cdcό protein antibody or fragments thereof, such as a Fab fragment or a scFv fragment, as an herbicide. The expression "herbicide" relates to a compound which inhibits plant growth and/or reproduction, in particular sexual reproduction.
The invention further relates to the use as defined above, wherein the Cdcό protein corresponds to a plant Cdcό protein.
DESCRIPTION OF THE FIGURES
Figure 1
Figure 1 represents immunoblots, revealed with antibodies directed against Xenopus CDC6, of a SDS-PAGE of total, cytoplasmic and germinal vesicle (GN) fractions of Xenopus oocytes, and of CSF (ie containing the cytostatic factor) and interphasic extracts of Xenopus eggs. The bands approximately migrating to 60 kD correspond to Cdcό.
Figure 2 Figure 2 represents the immunoblotting of a SDS-PAGE of extracts from Xenopus oocytes derived from stage 6 oocytes induced to mature by progesterone (PG). GNBD occured 2 h 45 min after progesterone addition (GNBD). Extracts were collected 0, 1, 2, 3, 4.5, 6.5 or 7.5 hours after the addition of progesterone. Some oocytes were also treated with cycloheximide either 30 min before GNBD (cyclo before GNBD), or 1 hour after GNBD (cyclo after GVBD), and collected at the Metaphase I / Metaphase II transition (2h 30 min after GNBD). The immunoblot was revealed with antibodies directed against CDC6, ORC2, MCM4, MCM3 or CDC45.
Figure 3 Figure 3 represents a Northern blot of extracts from Xenopus oocytes derived from stage 6 oocytes induced to mature by progesterone (PG) and revealed by a labelled anti-Cdcό mRNA probe. GNBD occured 2 h 45 min after progesterone addition (GNBD). Extracts were collected 0, 1, 2, 3, 4.5, 6.5 or 7.5 hours after the addition of progesterone. Some oocytes were also treated with cycloheximide either 30 min before GNBD (cyclo before GNBD), or 1 hour after GNBD (cyclo after GNBD), and collected at the Metaphase I / Metaphase II transition (2h 30 min after GNBD). Some oocytes were also treated with Actinomycin D.
Figure 4
Figure 4 represents the immunoblotting, revealed with antibodies d ected against Xenopus Cdcό, of a SDS-PAGE of extracts from stage 6 oocytes, or stage 6 oocytes injected with 50 ng of 25-mer sense Cdcό oligonucleotide, 50 ng of 25-mer antisense Cdcό oligonucleotide, or or water (mock), and induced to mature by PG. Matured oocytes were collected 12 hours after PG stimulation
Figure 5A, Figure 5B and Figure 5C
Figure 5A represents the DΝA synthesis ability, analyzed by agarose gel electrophoresis, of Xenopus oocytes injected with 25 nl of a 2 mg/ml solution of purified Cdcό sense oligonucleotides (lane 1), the corresponding antisense oligonucleotides (lanes 2, 3), or water (lane 4) and induced to mature. Rescue experiments were assayed by a second injection performed soon after GNBD that contained 0.25 μCi α32-PdCTP (lanes 1-4) and either boiled-inactivated (lane 2) or intact purified Cdcό (lane 3). The arrow on the left marks the migration of the bands corresponding to the newly synthesized DΝA.
Figure 5B represents the DΝA synthesis ability, analyzed by agarose gel electrophoresis, of Xenopus oocytes injected with water (lane 1), antisense oligonucleotides (lanes 2, 3) or sense oligonucleotides (lane 4) and fully matured during twelve hours. DΝA replication was analyzed by injection of 0.25 μCi α32P-dCTP in absence (lanes 1, 2, 4) or presence of recombinant Cdcό protein (lane 3). The arrow on the left marks the migration of the bands corresponding to the newly synthesized DΝA.
Figure 5C represents the DΝA synthesis ability analyzed by agarose gel electrophoresis of Xenopus oocytes induced to mature and treated with 250 μg/ml cycloheximide added 30 min before GNBD (lane 2) or 1 hour after GNBD (lane 4). Maturing oocytes were injected soon after GNBD with 0.25 μCi of α32P-dCTP without (lanes 1, 4) or with active (lane 2) or inactivated (lane 3) recombinant Cdcό protein. The arrow on the left marks the migration of the bands corresponding to the newly synthesized DΝA
Figure 6A, Figure 6B and Figure 6C Figure 6A represents the DΝA synthesis ability analyzed by agarose gel electrophoresis of stage 6 Xenopus oocytes induced to mature by PG, either non treated (-cyclo) or treated with 250 μg/ml cycloheximide 30 min before GNBD (cyclo -30 min), or 1 hour after GNBD (cyclo +lh). Oocytes extracts were collected at the metaphasel/metaphasell (MI/MII) transition (2 hrs 30 min after GNBD). Matured oocytes were also 'collected 12 h after PG addition. Sperm nuclei were added to the extracts and DΝA synthesis was followed by agarose gel electrophoresis, after 2 hours.
Figure 6B represents the percentage of DΝA replication after 2 hours (vertical axis) for a metaphase I/II extract from maturing oocytes either treated by: - cycloheximide before GVBD, and water (mock), Cdcό protein (+ CDC6) or Cdcό protein and aphidicolin (+ CDC6 Aphi), or - cycloheximide after GNBD and water (mock) or aphidicolin (+ Aphi). Figure 6C represents the percentage of DNA replication after 2 hours (vertical axis) for:
- a GNBD extract trated by water (mock), Cdcό protein (+ CDC6) or Cdcό protein and aphidicolin (+ CDC6 Aphi), or
- a metaphase I/II extracts treated by water (mock) or aphidicolin (+ Aphi).
Figure 7
Figure 7 represents an immunoblot of a SDS-PAGE of extracts from Xenopus or mouse oocytes in prophase I, metaphase I or metaphase II. The immunoblot was revealed with antibodies directed against Xenopus CDC6 and MCM2, or mouse CDC6 and MCM2.
Figure 8
Figure 8 represents an immunoblot of a SDS-PAGE of extracts from virgin female Drosophila melanogaster at stage 14 (lane 1), or activated oocytes (lane 2). The immunoblot was revealed with antibodies directed against Cdcό (DmCDCό), MCM2 (DmMCM2), and tubulin (DmTub).
EXAMPLES
Example 1
Cdc6 confers competence to replicate in the unfertilized egg of Xenopus laevis
A. Materials and methods
1. Fractionation of oocytes
Defolliculated oocytes were prepared as described in Menu et al. in Advances in Molecular Biology (1999) Oxford University Press, Ed. J.D. Richter. Cytoplasmic and germinal vesicle fractions were obtained after manual enucleation either from intact oocytes or oocytes fixed by 2 % TCA to prevent possible leaks from the nucleus. Protein extracts were obtained by grinding oocytes in 100 mM KC1, 5 mM MgCl , 0.1 mM CaCl , 10 mM K/Hepes pH 7.7, 50 mM sucrose, 5 μg/ml leupeptin, 5 μg/ml pepstatin, 5 μg/ml aprotinin, 0.5 mM EGTA, 0.3 % XI 00 Triton, centrifuged 5 min at 8 000 g, and further analyzed by SDS- PAGE.
2. Oocytes extracts
Low Speed Extracts of maturing oocytes were performed at GNBD (germinal vesicle breakdown) (GNBD extract) or between MI/MII, 2h30 after GNBD (MI/MII extract). GVBD, judged by the appearance of a white spot at the animal pole, started at 2 hrs 45 - 3 hrs 30 min. Maturing oocytes extracts were performed as for egg extracts in eppendorf tubes (Menu et al. in Advances in Molecular Biology (1999) Oxford University Press, Ed. J.D. Richter). After removing the excess buffer, oocytes were centrifuged at 8000 g, 5 min at 4°C, and then 12000 g, 2 min at 4°C. The upper phase was collected and centrifuged again at 12000 g for 2 min at 4°C. Extracts were collected and supplemented with energy mix (Menu et al. in Advances in Molecular Biology (1999) Oxford University Press, Ed. J.D. Richter).
3. DΝA replication assays
Oocytes were injected with 25 nl of 5 mCi/ml 32P-dCTP, collected 5 hours after GNBD, and subjected to DΝA extraction. Samples with equal numbers of total counts were analyzed by 0.8 % agarose gel electrophoresis. DΝA replication assays were also performed by quantification of α32P-dCTP incorporation in sperm nuclei used at 1000 nuclei/μl of extract. Analysis was both done by TCA precipitation and migration on 0.8 % Agarose (Menu et al. in Advances in Molecular Biology (1999) Oxford University Press, Ed. J.D. Richter). Purified protein was added at 15 ng/μl of extract to rescue DNA replication activity. Interphasic extract (Int) and CSF extract (ie extracts containing the mitotic cytostatic factor) were prepared as described in Menu et al. in Advances in Molecular Biology (1999) Oxford University Press, Ed. J.D. Richter and Lemaϊtre et al, J. Cell Biol. (1998) 142:1159-1166.
4. Northern blot analysis
Total oocyte RNA was extracted using a LiCl method (Taylor et al, EMBO J. (1986) 5:3563-3570). 10 μg of total RNA was submitted to electrophoresis in 1 % formaldehyde agarose gel and transferred to Amersham Hybond N+ membrane.
5. Intracellular injections of nucleotides and proteins
Stage 6 oocytes were injected with 25 nl of a 2 mg/ml solution of purified Cdcό 25 mers sense oligonucleotide (5'-TCCCCACCCAAGCAGTCTCGCAAAG-3') (SEQ ID NO: 3) or 25 mers of the corresponding antisense (5'-CTTTGCGAGACTGCTTGGGTGGGGA- 3') (SEQ ID NO: 4) oligonucleotide, and induced to mature by addition of progesterone. Control oocytes were injected in parallel with water. For rescue experiments a 25 nl second injection containing 5 mCi/ml dCTP and 300 μg/ml purified Cdcό protein was injected after GNBD or in 12 hours-matured oocyte. Maturation was followed by appearance of a clear white spot at the animal hemisphere as well as manual dissection to check for germinal vesicle breakdown.
6. Cdcό protein and antibodies
Xenopus Hisό-Cdcό protein was purified from recombinant baculovirus-infected insect cells. Its activity was monitored by depletion-rescue experiments as follows: 50 μl of low speed interphasic extract was depleted with Cdcό specific polyclonal antibody, or mock- depleted with preimmune serum IgG, as previously described (Maiorano et al, Nature (2000) 404:622-625). Cdcό purified protein was added to the depleted extract and DΝA replication was then measured. The rabbit polyclonal anti-Cdcό antibodies were obtained by 4 injections of Cdcό protein. B. Results
1. Detection of the Cdcό protein
The analysis of replication initiation factors present in the oocyte revealed that Cdcό, a critical factor involved in the assembly of the MCM helicase complex at DNA replication origins (Kelly et al, Annu. Rev. Biochem. (2000) 69:829-880 ; Lei & Tye, J. Cell Sci. (2001) 114:1447-1454), was undeiectable in fully grown oocytes but present in mature oocytes or in activated eggs (Figure 1). This was in contrast to other initiation proteins analyzed that were present in the oocyte, either nearly exclusively in the nucleus (MCM subunits, RPA70, RPA34), or also detected in the cytoplasm (geminin, cdtl, cdc7, ORC, cdc45). Cdcό electrophoretic migration shifts down in inteφhasic egg extract (Figure 1), in agreement with previous observations (Coleman et al, Cell (1996) 87:53-63).
Although Cdcό was absent in the oocyte, a relatively large amount of the protein was detected in the unfertilized egg, 5 ng / unfertilized egg, an estimate comparable with the 3 ng- value previously reported (Coleman et al, Cell (1996) 87:53-63). As Cdcό is an essential factor for replication licensing, the Inventors asked at what point during maturation it was accumulated. During maturation by progesterone (Figure 2 the Cdcό protein becomes detectable soon after germinal vesicle breakdown (GNBD, e.g. 3 hrs after progesterone addition), when MCM4 is phosphorylated by MPF (Coue et al, EMBO J. (1996) 15:1085- 1097 ; Pereverzeva et al, Mol Cell Biol (2000) 20:3667-3676). Inhibition of protein synthesis by cycloheximide added before GNBD, but not 1 hr afterwards, results in the absence of Cdcό in the egg (Figure 2). These data show that Cdcό protein is missing in the oocyte and starts to accumulate during maturation.
2. Detection of Cdcό mRΝA Although Cdcό protein is absent in oocytes, Northern blots show that Cdcό mRNA was already stored as a maternal mRNA during oogenesis (Figure 3). In addition, a shift in electrophoretic mobility of the Cdcό mRNA was detected soon after GNBD, consistent with a polyadenylation process during maturation (Figure 3Y This shift is still detected if cycloheximide is added 1 hour after GNBD, but is extremely reduced if cycloheximide is added 30 min before GNBD, consistent with the respective absence and presence of Cdcό protein in maturing oocyte (Figure 2). Actinomycin D, which prevents DΝA synthesis, present during maturation did not inhibit this shift (Figure 3), suggesting that Cdcό synthesis was regulated at the translational level, by polyadenylation of a maternal Cdcό mRΝA pool. 3. Cdcό synthesis inhibition by injection of antisense oligonucleotides
In addition, Cdcό protein accumulation in the egg is blocked by injection of antisense cdcό oligonucleotides in the oocyte, before maturation, in contrast to oocytes injected with the corresponding sense oligonucleotides (Figure 4). These results suggest that synthesis of Cdcό protein during oocyte maturation relies on translational control of Cdcό mRNA stored in the oocyte.
4. Injection of recombinant Cdcό in the egg
To address if Cdcό was the missing replication factor, sufficient to confer the competence for DNA replication to the egg, the hypothesis according to which recombinant Cdcό was sufficient to provide this competence, was tested. A recombinant Cdcό produced from baculovirus-infected cells was purified and was shown to be active in rescuing DNA replication in Xenopus egg extracts that were Cdcό-depleted using a purified specific polyclonal Cdcό antibody (see Materials and Methods section). The purified recombinant protein was also used in the in vivo rescue experiments described below.
5. Inhibition of replication by injection of antisense oligonucleotides and rescue by Cdcό injection
The activity of the missing DNA synthesis inducer that is synthetized during maturation is normally repressed by MPF to prevent DNA replication between meiosisl/meiosisll (Furuno et al, EMBO J. (1994) 13:2399-2410). Addition of cycloheximide
1 hour after GNBD prevents reaccumulation of MPF and consequently induces unscheduled
DΝA replication between meiosisl/meiosisll (Furuno et al, EMBO J. (1994) 13:2399-2410).
To address if this induced ability to replicate required Cdcό synthesis during maturation, the prevention of unscheduled replication by Cdcό-mRΝA depletion using a Cdcό antisense oligonucleotide was first tested. Figure 5A shows that addition of cycloheximide one hour after GNBD induces DΝA replication between meiosisl/meiosisll, as reported (Furuno et al,
EMBO J. (1994) 13:2399-2410), but this induction is inhibited by Cdcό antisense oligonucleotides. In addition, injection of recombinant Cdcό (Figure 5A) rescues induction of unscheduled replication.
The inventors further asked if Cdcό was sufficient to give competence to replicate in- vivo to the matured oocyte. In vivo, after maturation, parthenogenetic activation triggered by a micropipette injection of water for instance induces DΝA replication (Gurdon et al, Proc. Natl. Acad. Sci. U.S.A. (1967) 58:545-552 ; Hara et al., Proc. Natl Acad. Sci. U.S.A. (1980) - 77:462-466 ; Harland & Laskey, Cell (1980) 21:761-771) as shown in Figure 5B (lane 1).
Injection of Cdcό antisense oligonucleotides in the oocyte before maturation inhibits DNA replication (Figure 5B, lane 2), but microinjection of Cdcό protein after maturation is sufficient to rescue DNA replication (lane 3) with an efficiency similar to control oocytes treated with sense oligonucleotides (Figure 5B. lane 4).
The inventors concluded that Cdcό is the missing factor synthesized during oocyte maturation sufficient to give (i) replication-induced ability between meiosisl/meiosisll, and (ii), competence to replicate in the unfertilized egg.
Previous work has shown that maturing oocytes can replicate DNA after meiosis I by cycloheximide treatment 60 min after GNBD, but not 30 min before GNBD (Furuno et al, EMBO J. (1994) 13:2399-2410). These findings were reproduced by the Inventors, confirming that competence for DΝA replication in vivo is acquired shortly after GNBD, and is dependent on translation (Figure 5C, compare lane 1 and 4). In addition, the Inventors have shown show that microinjection of Cdcό after translation inhibition 30 min before GNBD is sufficient to rescue induced-replication ability between meiosis I and meiosis II (Figure 5C, lanes 1, 2, 3).
6. In vitro observations
This set of in vivo observations could be reproduced in vitro, in oocyte extracts prepared between meiosisl/meiosisll (Blon et al, Cell (1986) 47:577-587 ; Menu et al. in Advances in Molecular Biology (1999) Oxford University Press, Ed. J.D. Richter, and Methods). Oocytes were matured in vitro and cycloheximide was added 30 min before or 60 min after GNBD. Extracts were made 2 hrs 30 min post-GNBD, to which sperm nuclei were added to follow DΝA replication ability. If cycloheximide is added 60 min after GNBD, DΝA replication occurs, as expected (Figure 6A and 6B , in a reaction entirely aphidicolin- sensitive (Figure 6B . If cycloheximide is added 30 min before GNBD, Cdcό is not translated (Figure 2B). and extracts are incompetent to replicate (Figure 6A and 6B). However, addition of recombinant Cdcό to the extract is sufficient to rescue replication, in an aphidicolin-sensitive reaction (Figure 6B). Moreover, the simple addition of recombinant Cdcό to oocyte extracts made at GNBD is sufficient to confer DΝA replication competence, with an efficiency close to that obtained in egg extracts (Figure 6C . These results further demonstrate that Cdcό is the missing DΝA replication factor starting to be translated at GNBD, necessary and sufficient to confer to the egg the ability to replicate embryonic DΝA after fertilization, when the activity MPF is suppressed. Example 2
CDC6 confers competence to replicate in the unfertilized egg of Drosophila melanogaster
Of the established model organisms, Drosophila melanogaster provides a particularly potent system to study whether in invertebrate organism Cdcό appears during meiosis to give the competence to replicate DNA to the egg. Like many other animals, female D. melanogaster generate oocytes but store them, arrested and unactivated, in the ovary. After a period of growth, Drosophila oocytes arrest in metaphase of meiosis I, with egg coverings (a vitelline envelope and chorion). Ovulation releases these mature eggs from the ovary and triggers the process known as activation.
Although D. melanogaster mature virgin females spontaneously ovulate at a very low rate (~1 egg/day), nutritional supplementation causes increased germ cell proliferation resulting in increased ovulation rates (Drummond-Barbosa and Spradling, Dev. Biol. (2001) 231:265-278).
The Inventors have compared activated eggs laid by a virgin female with stage 14 oocytes blocked at Metaphase I, collected directly from the ovaries. Both were treated to remove the vitelline envelope and chorion. The activation of the oocytes triggers the synthesis of Cdcό that was absent from the stage 14 (metaphase I oocyte), while MCM2 was already present at stage 14 (Figure 8). Despite several differences in egg formation D. melanogaster accumulates Cdcό betwen metaphase I and metaphase II, before fertilisation. Moreover, Cdcό immunostaining during oocyte growth revealed a positive signal in the cytoplasm of nurse cells and cells covering the oocyte, while MCM2 was mostly nuclear, consistent with cell cycle regulation of Cdcό function. Thus, in the invertebrate D. melanogaster, like in the vertebrate X laevis, Cdcό is absent from prophase I oocyte and translated between Metaphase I and Metaphase II to give to the egg the competence to replicate before fertilisation.
Example 3
CDC6 confers competence to replicate in the unfertilized egg of Mus musculus
Oocyte extracts were prepared as described above and analyzed by SDS-PAGE. Irrimunoblots were revealed with antibodies directed against Xenopus laevis Cdcό and MCM2, as well as mouse Cdcό and MCM2. Prophase I oocytes were induced to mature by addition of progesterone (PG). Extracts from 5 X. laevis maturing oocytes or 85 mice oocytes were separated by SDS PAGE and examined by immunoblotting. The results are shown in Figure 7. Mouse Cdcό is absent from prophase I oocyte and translated between Metaphase I and Metaphase II, which is compatible with its ability to confer competence to replicate to the egg before fertilization. Thus mammalian Cdcό appears to be a potent target for the development of new contraceptives. Besides, its essential role in the acquisition of the competence to replicate in the egg strongly suggests that it might be involved in several infertilities, which could be overcome by providing Cdcό protein or Cdcό mRNA to infertile eggs or oocytes.
Example 4
Detection of the presence or the absence of the CDC6 protein by immunocytochemistry methods
The following method is used for the detection of the Cdcό protein, in particular for the detection of the human Cdcό protein in germinal cells, ovocytes, ovules or polar bodies:
1) Ovulated oocytes or oocyte or polar bodies, for instance obtained before or after "in vitro fertilization" (INF) or "intracytoplasmic sperm injection" (ICSI), are transferred to a sterile watch glass containing fixation buffer (20 mM Pipes, ImM MgCl2, 0.5 mM EGTA, 1 mM DTT, 1 μM Taxol, 0.1 % XI 00 triton, 2 % Formaldehyde) at 37°C for 30 minutes.
2) Transfer oocytes for three washes in 0.1 % Normal Goat Serum in PBS at 37°C for 5 minutes each.
3) Tranfer oocytes to 10% Normal Goat Serum for one hour (Oocytes can be stored in 10% Normal Goat Serum at 4)C prior to immunostaining). 4) Tranfer oocytes to anti-Cdcό antibody diluted in 5% Normal goat serum and place them at 37°C for 1 hour.
5) Transfer oocytes through 3 washes of 10 % Normal Goat Serum for 5 minutes each.
6) Transfer oocytes to appropriate secondary antibody FITC-conjugated and place them at 37°C for 1 hour. 7) Transfer oocytes through three washes in 10 % Normal Goat Serum for 5 minutes each.
8) Transfer oocytes to Hoescht 33258 500ng/ml in PBS for 2 minutes to stain DNA.
9) Tranfer on a microscope slide and add a drop of mounting medium and cover with a glass coverslip. 10) The oocyte can be observed under a fluorescence microscope for Cdcό immunostaining. A negative signal is indicative of an unfertilizable ovulated oocyte.
The detection of the presence or the absence of Cdcό detected by this method provides the basis for an in vitro diagnostic method of infertilities linked to a deficiency in active Cdcό.

Claims

1. A method for the in vitro diagnostic of infertility of a eukaryotic organism, comprising the detection of the presence or the absence of Cdcό protein or of Cdcό mRNA in germinal cells of said eukaryotic organism, at a given maturation stage of said germinal cells.
2. A method according to claim 1, wherein the eukaryotic organism is a multicellular organism, in particular a plant, a fungus, or an animal, such as a mammal, in particular a human.
3. A method according to claim 1 or 2, wherein the presence of Cdcό protein is detected in oocytes prior to germinal vesicle breakdown.
4. A method according to claim 1 or 2, wherein the absence of Cdcό protein is detected in oocytes having undergone germinal vesicle breakdown, or in polar globules derived from oocytes having undergone germinal vesicle breakdown, or in ovules.
5. A method according to the method of claim 3, comprising a step of detection of the Cdcό protein with an anti-Cdcό protein polyclonal or monoclonal antibody.
6. A method according to the method of claim 4, comprising a step of detection of the Cdcό protein with an anti-Cdcό protein polyclonal or monoclonal antibody.
7. A method according to claim 6, comprising the following steps:
- sampling a polar globule from an egg, fertilized or not;
- contacting the sampled polar globule or extracts thereof with an anti-Cdcό polyclonal or monoclonal antibody, - detecting the presence or the absence of complexes between the Cdcό protein and the anti-Cdcό polyclonal or monoclonal antibody.
8. A kit for carrying out the process of claim 7, comprising an anti-Cdcό polyclonal or monoclonal antibody, optionally a medium in which the sampled polar globule is extracted for protein preparation, a medium in which the sampled polar globule, or the protein preparation extracted from said polar globule, is contacted with said antibody, a reagent to detect the complex formed between Cdcό and said antibody, and a control medium containing the Cdcό protein.
9. A pharmaceutical composition comprising as active substance a protein comprising or consisting of the Cdcό protein or a part thereof, or a nucleic acid comprising or consisting of a nucleic acid encoding the Cdcό protein or a part thereof, in association with a pharmaceutically acceptable carrier.
10. A pharmaceutical composition according to claim 9, wherein the nucleic acid is a RNA, in particular a polyadenylated RNA.
11. A pharmaceutical composition according to claim 9 or 10, wherein the Cdcό protein comprises a sequence corresponding to the human Cdcό protein sequence, said sequence corresponding in particular to SEQ ID NO: 2, or wherein the nucleic acid encoding the Cdcό protein comprises a sequence corresponding to the human Cdcό nucleic acid sequence, said sequence corresponding in particular to SEQ ID NO: 1.
12. The use of a protein comprising or consisting of the Cdcό protein or a part thereof, or of a nucleic acid comprising or consisting of a nucleic acid encoding the Cdcό protein or a part thereof, for the manufacture of a medicament for the treatment of infertilities.
13. The use according to claim 12, wherein the nucleic acid is a RNA, in particular a polyadenylated RNA.
14. The use according to claim 12 or 13, wherein the Cdcό protein comprises a sequence corresponding to the human Cdcό protein sequence, said sequence corresponding in particular to SEQ TD NO: 2, or wherein the nucleic acid encoding the Cdcό protein comprises a sequence corresponding to the human Cdcό nucleic acid sequence, said sequence corresponding in particular to SEQ ID NO: 1.
15. The use of a compound inhibiting Cdcό protein activity, such as a pharmacological compound, an antisense polynucleotide directed against the mRNA encoding the Cdcό protein, a siRNA inhibiting Cdcό protein expression, an anti-Cdcό protein antibody or fragments thereof, such as a Fab fragment or a scFv fragment, for the manufacture of a contraceptive.
16. The use according to claim 15, wherein the Cdcό protein corresponds to a human Cdcό protein.
17. A phytosanitary composition comprising as active substance a protein comprising or consisting of the Cdcό protein or a part thereof, or a nucleic acid comprising or consisting of a nucleic acid encoding the Cdcό protein or a part thereof, or a compound inhibiting Cdcό activity, such as an antisense nucleotide directed against the mRNA encoding the Cdcό protein, a siRNA inhibiting Cdcό protein expression, an anti-Cdcό protein antibody or fragments thereof, such as a Fab fragment or a scFv fragment.
18. A phytosanitary composition according to claim 13, wherein the Cdcό protein corresponds to a plant Cdcό protein.
19. The use of a protein comprising or consisting of the Cdcό protein or a part thereof, or of a nucleic acid comprising or consisting of a nucleic acid encoding the Cdcό protein or a part thereof, or of a compound inhibiting Cdcό protein j activity, such as an antisense polynucleotide directed against the mRNA encoding the Cdcό protein, a siRNA inhibiting Cdcό protein expression, an anti- Cdcό protein antibody or fragments thereof, such as a Fab fragment or a scFv fragment, for the treatment of a plant or parts thereof, or for the inhibition of plant reproduction.
20. The use according to claim 19, of a protein comprising or consisting of the Cdcό protein or a part thereof, or a nucleic acid comprising or consisting of a nucleic acid encoding the Cdcό protein or a part thereof, for the treatment of plant infertility.
21. The use according to claim 19, of a compound inhibiting Cdcό protein activity, such as an antisense polynucleotide directed against the mRNA encoding the Cdcό protein, a siRNA inhibiting Cdcό protein expression, an anti-Cdcό protein antibody or fragments thereof, such as a Fab fragment or a scFv fragment, as an herbicide.
22. The use according to any of claims 19 to 21, wherein the Cdcό protein corresponds to a plant Cdcό protein.
PCT/EP2003/007631 2002-08-14 2003-07-15 Method for the in vitro diagnostic of infertility Ceased WO2004019042A1 (en)

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Citations (2)

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WO2000026242A2 (en) * 1998-10-30 2000-05-11 Cold Spring Harbor Laboratory Dna replication-regulating genes, antibodies thereto and diagnostic applications thereof
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