FR2918075A1 - CONTROL OF THE FERTILITY OF A HUMAN SEX OF MALE SEX VIA SPATA16 - Google Patents

Abstract

The present application relates to the control of the fertility of the male human being. The inventors demonstrate that the protein SPATA16 is necessary for the formation of the acrosome of human spermatozoa and as such, propose means for the contraception of the male human being, and means for the diagnosis and prognosis of teratozoospermia, more particularly globozoospermia.

Description

TITLE

  Controlling the fertility of a male human being via SPATA16 TECHNICAL FIELD OF THE INVENTION

  The present patent application relates to means for the contraception of a male human being, as well as means for the diagnosis of infertility, or at least insufficient fertility, in a human being. human male. The contraceptive means according to the invention imply a decrease in the amount of functional SPATA16 protein present in a male human, more particularly a negative regulation of the expression of this protein. The diagnostic means of the invention implement the determination of the ability of a male human to express a functional SPATAI6 protein.

  BACKGROUND OF THE INVENTION According to the WHO, about 15% of couples experience the inability to conceive within two years of unprotected sex. In about half of the cases, infertility is due to the fact that the male partner produces insufficient numbers of spermatozoa (oligozoospermia), or with inadequate mobility (asthenozoospermia), or abnormal morphology (teratozoospermia), or because of a combination of these defects. Globozoospermia is a rare but severe teratozoospermia, characterized by ejaculates in which at least 20% of the spermatozoa: have an acrosome malformation that prevents or limits their ability to fertilize an egg, or are totally devoid of acrosome. These sperms then have a round head.

  Globozoospermia can be total (in this case, 100% of the sperm are affected by a malformation of their acrosome or are devoid of acrosome), or partial (generally, in a proportion of 20% to 90% of the spermatozoa).

  Men affected by total globozoospermia are completely infertile: even the application of intracytoplasmic sperm injection (ICSI) has only met with low and disappointing success rates. A reference description of globozoospermia can be found under OMIM 102530 (OMIM = Online Mendelian Inheritance in Man ™, Johns Hopkins University and NCBI). Globozoospermia is a result of disturbed spermiogenesis and, although the underlying cause is still unknown, a genetic contribution appears to be supported by several reports on familial cases and by three recessive mouse models involving CSNK2A2 (OMIM 115442), HRB (OMIM 600862) and GOPC (OMIM 606845). However, no causal gene mutation has been identified at this gene or any other human gene so far. For example, mutations that could be identified in non-human animal models (mice), such as certain mutations affecting the CSNK2A2, HRB and GOPC genes, could not be identified in humans (Pirrello et al. 2005, Human Reproduction 20 (5): 1314-1318, Christensen et al., 2006, Journal of Andrology 27 (1): 11-15). To the inventors' knowledge, the prior art has not been able to identify any gene mutation that would have a clear link with globozoospermia that affects certain human beings of the male sex.

  The inventors provide the demonstration that the human SPATA16 gene is involved in the morphogenesis of human spermatozoa, and more particularly in the formation of their acrosome. A description of the human SPATAI6 gene can be found in OMIM 609856. In particular, it can be seen that prior to the present invention, the knowledge concerning the SPATA16 gene, more particularly the human SPATAI6 gene, was at least limited. At most it was suggested that this gene appeared to be involved in testicular development.

  The inventors provide for their demonstration that in the absence of functional SPATA16 protein, or at least in the presence of an insufficient amount of such a protein, the human spermatozoa produced are, in their entirety, or at all the least in a proportion of at least 20% of them, of abnormal morphology (teratozoospermia), more particularly that they have an acrosome of abnormal morphology or that they are totally devoid of acrosome (globozoospermia). The result of this morphological disorder is a failure, or at least a very diminished ability, to achieve fertilization of an egg, ie infertility, or at the very least an insufficient fertility of the human being. male.

  In the example of the non-functional SPATA16 protein presented by the inventors, all the spermatozoa produced are devoid of acrosome.

  This demonstration allows inventors to propose means for the diagnosis of insufficient fertility in a male human being and means for the contraception of a male human being.

  In the prior art to the present invention, the contraceptive means of the male human being are essentially limited to condoms or vasectomy. Some birth control products have been tested on the male human, but none of these products has given satisfactory results. For example, products intended to interrupt the production of spermatozoa have been tested. The vast majority of these test products act hormonally. This is the case, for example, with: gonadotropin-releasing hormone (GN-RH), which has been administered in a tetanus toxoid-related form, and to which a testosterone substitute administration (7-alpha-methyl-19-nortestosterone) has to be combined , or MENT) so as to compensate for hypoandrogenic symptoms related to blockage of the hypothalamohypophysis axis, excessively administered testosterone derivatives, such as injectable testosterone enanthate (ET), which have an effect on the hypothalamohypophisotesticular axis but induce high-density lipoprotein (HDL) and high coronary risk-associated side effects, androgen-progestin combinations, such as an ET + levonorgestrel combination, whose effects on equilibrium are difficult to control hormones, and for which side effects of acne type and weight gain have been reported.

  Products intended to interrupt sperm production by the non-hormonal route have also been tested, but without success. This is the case, for example, of gossypol, which is extracted from cottonseed oil, for which it has been found to induce side effects such as decreased potassium levels and increased risk of cardiac arrhythmias. .

  Products intended to disturb the spermatic function have, moreover, been tested. For example, nifedipine, which is usually used to treat hypertension and migraines, appears to inhibit the release of enzymes that are necessary for spermatozoa to penetrate the zona pellucida of the oocyte. However, at doses useful for the desired contraceptive effect, nifedipine also appears to induce dangerously low heart rates.

  Mifepristone (or RU 486) has also been tested on males. It would prevent sperm from using calcium, which would disrupt the sperm membrane and induce a decrease in motility. However, mifepristone also interferes with some hormonal functions. Vaccine compositions produced from sperm surface proteins (PH2O, ZP1O, PR34, for example) have also been tested, but to no avail.

  Thus, to the knowledge of the inventors, there is in the prior art no pharmaceutical product that would have given sufficiently satisfactory results in terms of efficacy and safety, so that it can be effectively considered as a contraceptive manageable to the male human being.

  The present invention provides novel means for the contraception of the male human, which can induce a complete inability of human sperm to penetrate an egg. The contraceptive means of the invention are not directed to the hormonal pathways: they are directed at a protein that is specifically expressed in the human testes, namely the SPATA16 protein. The means according to the invention have a molecular target in the human being which is sufficiently specific for the testicular organ so that the degree of harmful side effects that can be envisaged is very low. As a result, the contraceptive means of the invention are likely not to induce undesirable side effects, which have been observed with the prior art pharmaceutical contraceptive products, such as inhibition of secondary sexual characteristics (feminization). , erectile dysfunction, loss of libido, disruption of heart rhythm, increased coronary risk, weight gain. To the inventors' knowledge, the present invention is also the first description of means making it possible to control the formation of the acrosome of spermatozoa, and more particularly of human spermatozoa. SUMMARY OF THE INVENTION

  The present invention describes the identification of a human gene, the expression product of which is necessary for the establishment of a correct sperm function, and more particularly the production of spermatozoa having the capacity to fertilize an egg.

  The inventors demonstrate that the human SPATA16 gene is necessary for human spermatozoa to acquire the ability to fertilize an egg. The inventors demonstrate in particular that a mutation of the human SPATA16 gene leading to the expression of a non-functional SPATA16 protein results in the production of human spermatozoa which have an abnormal morphology, that is to say a teratozoospermia. More particularly, the inventors demonstrate that such a mutation of the human SPATA16 gene can lead to globozoospermia, that is to say to the production of human spermatozoa which, in their totality, or at least in a proportion of at least 20% of them do not have an acrosome, or at least have an acrosome whose morphology is abnormal enough that these sperm can not fertilize an egg. In the example of the non-functional SPATA16 protein presented by the inventors, all the spermatozoa produced are devoid of acrosome.

  The inventors' demonstration allows the development of means for the diagnosis of insufficient fertility, or even infertility, of a male human being. The diagnostic means of the invention comprise the detection of a mutation of the SPATAI6 gene, more particularly a mutation 5 leading to a lack of transcription and / or translation of this gene, and / or the detection of an altered structure of the gene. SPATAI6 protein, such detection being indicative of inferior fertility observed in a healthy human individual, or even infertility.

  The demonstration of the inventors makes it possible especially to propose means for the contraception of a human being of male sex. The contraceptive means of the invention comprise in particular a product which is administrable to the male human being, and which is capable of altering or inhibiting the protein function of SPATAI6 and / or of reducing the amount of functional SPATA16 protein present. in a male human, more particularly capable of inhibiting the expression of the SPATA16 gene. The present application is directed to such a contraceptive product, as well as methods for identifying and / or producing such a contraceptive product.

BRIEF DESCRIPTION OF THE FIGURES

  FIGURES 1A to 1D: Human family, showing globozoospermia and within which a mutation in the SPATAI6 gene has been identified FIG. 1A: Morphology of sperm. Fluorescent acrosin staining (green FITC) acrosomes, and staining of DAPI (blue) nuclei. On the left, a sample from a fertile control. On the right, a sample of a patient with globozoospermia. Sperm morphology and acrosome structures are severely disrupted in the patient's cells. Acrosin staining residues were observed on some deformed sperm cells of the patient, but most of the signals correspond to non-specific acrosin staining at the leukocyte level. FIG. 1B: Chromatograms of the mutation. The sequences of a patient, his father (heterozygote) and a control are presented.

  FIG. 1C: The Ncil recognition site (5'-CCCGG-3 ') is lost due to the G> A mutation at a nucleotide of exon 4. The Hpall recognition site is 5'-CCGG -3 '. This mutation can lead to a substitution of the amino acid R283Q, as well as the rupture of the splice site 5 'of the intron 4.

  FIG. 1D: Genealogy of an Ashkenazi Jewish family who was the subject of this study. The order of ten siblings is arbitrary. The segregation of the mutation was studied by Ncil digestion of a PCR amplification of exon 4 and its flanking sequences. M is the marker corridor; C that of the witness; black symbol = individual affected by globozoospermia; white symbol = unaffected individual. The asterisk indicates the individuals tested. Both parents and two unaffected siblings are heterozygous for the G> A mutation. The third unaffected siblings do not carry this mutation. The three male siblings that are affected by globozoospermia (three black squares) are homozygous for this mutation. The witness does not carry the mutation.

  FIGURES 2A-2C: Splice exon 4 SPATA16 splice site of the mutated donor and SnRNP UI link for the wild type splice site and the mutant donor FIG. 2A: Schematic presentation of minigene constructs that were used to test the splicing of exon 4.

  FIG. 2B: this gel shows that wild-type exon 4 (WT) is invariably included in the final messenger RNA; in sharp contrast, the mutated exon (MT) gives rise to two forms of aberrant splicing. M is the marker track. FIG. 2C: the inventors have analyzed the U1 SnRNP binding by psoralen-mediated UV hybridization experiments; these experiments revealed that the mutant exon 4 is not recognized by the splicing machinery, whereas the wild type exon 4 is clearly recognized; the identity of U1 snRNP was confirmed by treatment of RNAse H using a complementary oligodeoxynucleotide at nucleotide positions 1 to 15 of U1 snRNA.

  FIGURE 3: alignment of SPATAI6 sequences of different animal species (CLUSTAL W (1.81) multiple sequence alignment) Consensus key * -residu unique, highly conserved: - conservation of strong groups. -conservation of weak groups - no consensus Large gray rectangle = TPR domain. In the human SPATA16 sequence, the TPR domain extends from amino acid 186 (Cys) included to amino acid 281 (Ala) inclusive. Vertical gray band = altered amino acid (R amino acid).

  In the human SPATA16 sequence, the altered amino acid is at position 283 (Arg). It therefore corresponds to the first amino acid at 5 'from the C-terminal end of the TPR domain. Black frame = directly affected part (fully coded by exon 4). In the human SPATA16 sequence, the amino acids that are fully encoded by exon 4, and thus directly affected, range from the included position 253 (Arg) to the included position 282 (Ala). The codon of the Arg amino acid at position 283 overlaps exon 4 and exon 5; it is this codon that is affected by the G848A mutation.

  FIGURE 4: sequences of the cDNA of the wild-type human SPATA16 gene (SEQ ID NO: 1), the CDS of this gene (SEQ ID NO: 2), the wild-type SPATA16 protein (SEQ ID NO: 3), the CDS the wild-type human SPATAI6 TRP domain (SEQ ID NO: 4), and the wild-type human TRP polypeptide (SEQ ID NO: 5). FIGURE 5: Sequences of fragments 92041-92340 (SEQ ID NO: 6), 121561-121800 (SEQ ID NO: 7) and 164101-164340 (SEQ ID NO: 8) of the wild-type human SPATA16 gene sequence. FIGURE 6: Sequence formed by splicing of exons 1-3 of the wild-type human SPATAI6 gene (SEQ ID NO: 9), and polypeptide sequence encoded by exons 1-3 (SEQ ID NO: 10), sequence formed by splicing exons 5 to 11 of the wild-type human SPATA16 gene (SEQ ID NO: 11), and polypeptide sequence encoded by exons 5 to 11 (SEQ ID NO: 12).

  FIGURE 7: sequence of exon 4 of the wild-type human SPATAI6 gene (SEQ ID NO: 13), and polypeptide sequence encoded by this exon 4 (SEQ ID NO: 14), polypeptide sequence encoded by the splicing of the last two nucleotides of the exon of the wild-type human SPATA16 gene and exons 5 to 11 of this gene (SEQ ID NO: 15).

  FIGURE 8: sequence of the CDS (coding sequence) of the SPATA16Aexon4 gene, that is to say the mutated human SPATA16 gene observed in globozoospermic siblings (SEQ ID NO: 16), sequence of the mutant SPATA16 protein coded by the gene SPATAI6 mutated (SPATA16Aexon4) (SEQ ID NO: 17).

  DETAILED DESCRIPTION OF THE INVENTION

  The inventors demonstrate that, at least in humans, the SPATA16 protein is necessary for the formation of the acrosome of spermatozoa, more particularly for the formation of an acrosome of morphology sufficiently normal to allow spermatozoa to penetrate a human body. egg. In the absence of functional SPATA16 protein, the spermatozoa are, at least in a proportion of at least 20% of them, and most generally in their entirety, either devoid of acrosome or provided with an acrosome non-functional, that is to say which does not allow the sperm to penetrate an egg. In the example of non-functional SPATAI6 protein presented by the inventors, all the spermatozoa produced are devoid of acrosome. The inventors demonstrate that the globozoospermia of certain patients originates from a mutation of their SPATAI6 gene. The mutated SPATAI6 gene expresses a mutant SPATA16 protein. An example of such a mutation is described in Example 1 below. In this mutation example, exon 4 of the mutated SPATAI6 gene is no longer correctly spliced, and this leads to a mutated SPATAI6 mRNA in which exon 3 is spliced directly to exon 5. Mutant SPATA16 protein produced by patients carrying this mutated SPATA16 gene lost the polypeptide portion encoded by exon 4 of the wild-type gene, which leads to a SPATAI6 protein which, unlike the wild-type protein, does not include a functional TRP domain (repetition domain tetratricopeptide).

  The inventors thus demonstrate that when the SPATAI6 protein is non-functional, the spermatozoa produced do not have a functional acrosome, that is to say that they are at least provided with an acrosome whose morphology is sufficiently abnormal to prevent them from succeeding in fertilizing an egg, or that they are entirely devoid of acrosome. The proportion of spermatozoa affected is at least 20%, and can reach 100% in the case of total globozoospermia. In the mutation example detailed in Example 1, all the spermatozoa produced by patients affected by this mutation are completely free of acrosome. Patients with this mutation do not suffer from any other physical or mental abnormality. The inventors thus demonstrate that globozoospermia can, at least in some cases, be a genetic trait with an autosomal recessive inheritance pattern. To the inventors' knowledge, the present invention provides the first description of a gene directly involved in the pathogenesis of human globozoospermia. As such, the present application relates to mutant polynucleotide, polypeptide, and protein products, as well as to diagnostic and prognostic applications, which include the detection of at least one mutation of the human SPATAI6 protein and / or at least one mutation of the gene and / or human SPATA16 mRNA capable of leading to the expression of a mutant human SPATA16 protein.

  That being so, an essential aspect of the present invention is the demonstration that there is a human protein: which is expressed specifically in human testicular organs, and which is not expressed in other organs of the human body, which is necessary for the formation of the acrosome of human spermatozoa, so that in the absence of this functional protein, or at least in the presence of an insufficient amount of this protein, a proportion of at least 20%, preferably at least 90%, preferably at least 95%, advantageously 100% of human spermatozoa is not provided with an acrosome of sufficiently normal morphology to succeed in fertilizing an egg, advantageously is acrosome-free, and which can be produced in the human body in such a non-functional mutant form without causing other physical or mental disorders in the patient, and which is not a hormone, and It does not interact, or at least not directly, with the patient's hormonal pathways.

  This human protein is the SPATA16 protein. These remarkable features make this protein a prime target for the development of a contraceptive product for the male human.

  The present application relates to a mutant SPATAI6 protein. A mutant SPATA16 protein of the invention comprises, or consists of, an amino acid sequence which is derivable from that of a wild-type human SPATAI6 protein by deletion and / or substitution and / or addition of at least one amino acid.

  In comparison with a wild-type human SPATAI6 protein, a mutant SPATAI6 protein of the invention has lost the ability to participate in, or induce, the formation of an acrosome, including the formation of a functional acrosome.

  The expression functional acrosome is understood in its ordinary meaning in the field, namely an acrosome that allows the spermatozoon to penetrate an egg of the same animal species, more particularly the zona pellucida of this egg. The inability to participate in, or induce, the formation of a functional acrosome results in the spermatogenic cell (precursor sperm cell) failing to evolve into a functional sperm structure, that is, that is, it can not evolve into a spermatozoon capable of fertilizing an egg.

  The loss of this capacity can for example be observed by transfecting or infecting a spermatogenic cell (i.e. a spermatozoid precursor cell) with a nucleic acid encoding this mutant protein so that this mutant protein is expressed by the transfected or infected spermatogenic cell.

  This spermatogenic cell may for example be chosen from spermatogonia, spermatocytes and spermatids, preferably from spermatocytes and spermatids.

  This spermatogenic cell may be a spermatogenic cell of a non-human animal, especially a spermatogenic cell of a non-human mammal, such as a mouse spermatogenic cell. This spermatogenic cell may be a spermatogenic cell that has been isolated from the human body. Preferably, this spermatogenic cell is a human spermatogenic cell.

  The person skilled in the art may choose to use a spermatogenic cell whose expression of the wild-type SPATA16 gene is blocked, for example by knockout of the wild-type SPATA16 gene (for example by homologous recombination), or by transfection of antisense oligonucleotides of the gene. SPATAI6 wild, and transfect or infect this spermatogenic cell to wild-type SPATAI6 gene silently by a nucleic acid that encodes a candidate mutant SPATAI6 protein so that this candidate mutant protein is expressed in the spermatogenic cell. The spermatogenic cell thus treated can be placed under conditions favorable to the formation of the acrosome. The person skilled in the art can then observe the effects induced by the expression of this candidate mutant protein on spermatogenesis, and more particularly on the formation of a functional acrosome. If no acrosome, or at least no functional acrosome, is formed by this spermatogenic cell, the candidate mutant protein can be identified as a mutant protein of the invention. Disruption of functional acrosome formation may alternatively or complementarily be observed by observing a disruption of the intracellular mechanisms that accompany this acrosome formation, such as absence or inhibition of acrosomal vesicle fusion, absence or an inhibition of the anchoring of the acrosome on the nuclear surface, an anomaly of the placement of the axoneme.

  Alternatively or additionally, those skilled in the art may choose to test a candidate mutant protein at a more mechanistic level of the spermatogenic cell. For example, after transfection or infection of the spermatogenic cell, one skilled in the art can determine whether the candidate mutant protein has lost at least one of the following abilities: ability to be located in the Golgi apparatus of said spermatogenic cell - ability to be localized in the pro-acrosomal vesicles of said spermatogenic cell; - ability to be localized in the acrosome. Where appropriate, the candidate mutant protein can be identified as a mutant protein of the invention.

  In order to carry out the verification tests of the capacity (s) of a mutant SPATA16 protein of the invention, the person skilled in the art may choose to couple it to a marker allowing its detection, such as for example a GFP (Green Fluorescence Protein) molecule. ).

  Preferably, a mutant SPATAI6 protein of the invention comprises, or consists of, an amino acid sequence which is derivable from that of a wild-type human SPATA16 protein by mutation of the TPR domain of the wild-type human SPATAI6 protein, said mutation comprising deletion and / or substitution of at least one amino acid of this TPR domain and / or the addition of at least one amino acid in this TPR domain, said mutation being selected such that mutant SPATAI6 protein which This does not induce the formation of an acrosome, or at least does not induce the formation of a functional acrosome (i.e., an acrosome whose morphology would allow the spermatozoon to penetrate an ovum ), on a human spermatogenic cell whose expression of the wild SPATA16 gene has been blocked.

  Preferably, a mutant SPATA16 protein of the invention comprises, or consists of, an amino acid sequence which is capable of derived from that of a wild-type human SPATAI6 protein by a deletion operation (s) and / or substitution and / or addition of at least one amino acid, which comprises (include) at least the deletion of part of the sequence of the wild-type human SPATA16 protein, advantageously the deletion of all or part of its TPR domain.

  Advantageously, a mutant SPATAI6 protein of the invention comprises, or consists of, an amino acid sequence which is likely to derive from that of a wild-type human SPATA16 protein by deletion of a part of the TPR domain of the wild-type human SPATAI6 protein.

  The portion of the TPR domain that is the subject of the deletion may be any part that causes the resulting mutant protein to lose one of the abilities described above, and / or to exhibit one of the disabilities described above.

  For example, the deletion portion of the TPR domain may correspond to all or part of the region of the TPR domain that is encoded by exon 2, or all or part of the region encoded by exon 3, or to all or part of the region encoded by exon 4, or a combination of at least two of these, such as for example part of the region encoded by exon 3 and part of the region coded by Exon 4. Advantageously, the part of the TPR domain which is the subject of the deletion is a part which comprises, or is that, that encoded by exon 4.30 In FIG. 4, a sequence of the cDNA of the wild-type human SPATA16 gene is presented under SEQ ID NO: 1. The corresponding coding sequence (CDS) is presented under SEQ ID NO: 2, and the sequence of the corresponding wild-type human SPATA16 protein under SEQ ID NO: 3.

  A coding sequence of the TPR domain of the wild-type human SPATA16 protein is presented in FIG. 4 under SEQ ID NO: 4. This coding sequence extends from positions 686 to 973 of the cDNA sequence of SEQ ID NO: 1, which corresponds to the positions 556 to 843 of the CDS sequence of SEQ ID NO: 2 (see Figure 4). The coding sequence of the wild-type TPR domain consists of a 3 'part of exon 2, exon 3 and almost all exon 4 (see Table 6 below, for exon positions).

  A polypeptide sequence of the TPR domain of the wild-type human SPATAI6 protein is shown in FIG. 4 under SEQ ID NO: 4. This amino acid sequence extends from positions 186 to 281 of the protein sequence of SEQ ID NO: 3 (see FIG. ).

  In Figure 4, the portion of the TPR domain of the wild-type human SPATA16 protein that is encoded by exon 4 extends from positions 253-282 of the protein sequence of SEQ ID NO: 3. The amino acid sequence of this domain portion TPR is presented in FIG. 4 under SEQ ID NO: 14. Its coding sequence is that of exon 4, which is presented in FIG. 4 under SEQ ID NO: 13.

  Advantageously, the sequence of the mutant protein of the invention comprises, or is that of SEQ ID NO: 17. Advantageously, the sequence of the mutant nucleic acid of the invention comprises, or is that of SEQ ID NO: 16.30 The present application also relates to the nucleic acids which encode such a mutant SPATAI6 protein according to the universal genetic code, taking into account the degeneracy of this code, and to the nucleic acids whose sequence is complementary to such a nucleic acid coding throughout the length of this coding nucleic acid.

  These nucleic acids may be defined as comprising or consisting of a nucleotide sequence which is capable of deriving from a nucleotide sequence comprising a sequence encoding the wild-type human SPATA16 protein by a mutation which comprises the substitution and / or the deletion of minus one nucleotide, and / or by addition of at least one nucleotide. A mutant nucleic acid of the invention has a nucleotide sequence such that the mutant SPATA16 protein capable of being encoded by such a mutant nucleic acid does not induce the formation of an acrosome, or at least does not induce the formation of an acrosome. formation of a functional acrosome on a spermatogenic cell (preferably a human spermatogenic cell) whose expression of the wild-type SPATA16 gene has been blocked.

  By analogy with the elements described above as mutant proteins of the invention, a mutant nucleic acid of the invention may for example be characterized in that its sequence comprises, or consists of a sequence that is likely to deriving from a nucleotide sequence which comprises a sequence encoding the wild-type human SPATAI6 protein, by deletion of the sequence encoding the TPR domain of the wild-type SPATA16 protein, such as deletion of the sequence of SEQ ID NO: 4, or deletion of a sequence coding part of the TPR domain of the wild-type human SPATA16 protein, said deletion being such that the mutant SPATA16 protein encoded by such a mutant nucleic acid: o has lost at least one of the abovementioned capacities, and / or o present at least one of the above-mentioned disabilities.

  By analogy with the elements described above as mutant proteins of the invention, said deletion of a sequence coding part of the TPR domain of the wild-type human SPATAI6 protein is advantageously such that the mutant SPATA16 protein encoded by such a nucleic acid mutant does not induce the formation of an acrosome, or at least does not induce the formation of a functional acrosome, in a spermatogenic cell whose expression of wild-type SPATAI6 gene has been blocked. An example of such a sequence encoding a portion of the TPR domain that can be deleted is the exon 2 and / or exon 3 and / or exon 4 sequence of the wild-type human SPATA16 gene, preferably exon 4 , such as the sequence of SEQ ID NO: 13.

  An example of such a mutant nucleic acid of the invention which no longer includes the sequence of exon 4 of the wild-type human SPATAI6 gene is a nucleic acid which comprises, or consists of, the sequence of SEQ ID NO: 16 ( cDNA sequence, single-stranded DNA sequence or corresponding mRNA sequence). This nucleic acid of SEQ ID NO: 16 is the coding sequence which results from the splicing of a mutated SPATA16 gene, which derives from the substitution of the last nucleotide of exon 4 of the wild-type human SPATAI6 gene (nucleotide G replaced by a nucleotide A). As shown in example 1 below, a mutated SPATAI6 gene undergoes an incorrect splicing of exon 4, at the end of which exon 3 is directly spliced on exon 5. The resulting mRNA or cDNA transcription of such a mutated SPATAI6 gene has thus lost exon 4.

  A mutant nucleic acid of the present invention may therefore comprise, or consist of, a sequence capable of deriving from the sequence of the genomic DNA of the wild-type human SPATAI6 gene by a mutation which comprises: the substitution and / or the deletion of at least one nucleotide of an exon encoding a portion of the TPR domain, and / or the addition of at least one nucleotide in such an exon.

  Preferably, said at least one exon is exon 4 of the wild-type human SPATAI6 gene. Advantageously, said at least one nucleotide of exon 4 is the last nucleotide of exon 4. This mutation can be selected so that the splicing of an exon coding for a part of the TPR domain, such as the Exon 2 and / or 3 and / or 4 can not be done in the order observed on the wild-type human SPATA16 gene. This is particularly the case of a substitution of a nucleotide of exon 4 which leads to the splicing of exon 3 directly on exon 5, such as the substitution of the last nucleotide of exon 4 of human SPATAI6 gene.

  A mutant nucleic acid of the invention may be a DNA (genomic DNA, cDNA, single-stranded DNA) or an RNA (mRNA in particular). Preferably, it is a cDNA or an mRNA. Such nucleic acid may be in free form, or in an inserted form in a vector for transfection, transformation or infection, and in particular on an expression vector, advantageously a vector comprising the regulatory sequences necessary for expression in a prokaryotic cell. such as E. coli, or preferably in a eukaryotic cell such as that of HeLa or COS or spermatide.

  The present application also relates to any cell which is in isolated form, and which contains at least one nucleic acid according to the invention. This isolated cell may be a genetically modified cell, for example by transfection, transformation or infection. The present application also relates to any non-human transgenic animal which comprises at least one mutant protein according to the invention, or a mutant nucleic acid according to the invention.

  The present application also relates to any solid support, and in particular to any solid support adapted to the circulation of a microfluidic stream, such as a chip, on which at least one mutant protein of the invention is attached and / or at least one mutant nucleic acid of the invention.

  The present application also relates to any antibody, more particularly any monoclonal antibody, which binds to a mutant protein of the invention, without binding to a wild-type human SPATA16 protein of SEQ ID NO: 3. Advantageously, such an antibody is specific of one or more mutant protein (s) of the invention. Techniques for producing antibodies by immunizing a mammal are known to those skilled in the art. Techniques for producing monoclonal antibodies are also known to those skilled in the art (Kohler and Milstein (1975) Nature 256: 495-497, US Patent No. 4,376,110, Kosbor et al (1983) Immunology Today 4:72; et al (1983) Proc Natl Acad Sci USA 80: 2026-2030, Cole et al (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

  The present application also relates to any hybridoma capable of producing a monoclonal antibody of the invention.

  The present application also relates to means for: the diagnosis or prognosis of a human teratozoospermia, more particularly of a human globozoospermia, the diagnosis of the infertility of a male human being, or a fertility of such a human being which is insufficient to allow a spontaneous conception by natural means, to determine the existence of a predisposition to a human teratozoospermia, and more particularly to a human globozoospermia. These means include methods and kits.

  The means of the invention comprise means for determining whether a sample which may contain spermatozoa of the patient, such as a sample of sperm or testicular tissue, contains SPATAI6 proteins which have a mutant structure and / or a mutant sequence, relative to the structure and / or sequence of the wild-type human SPATAI6 protein (i.e., the SPATA16 protein produced by fertile male humans), respectively.

  If the detected mutant SPATA16 proteins have a structure and / or sequence sufficiently different from that of the wild-type human SPATA16 protein to be non-functional, for example sufficiently different (s) for having lost the capacity to induce the formation of an acrosome on a spermatogenic cell (preferably a human spermatogenic cell) whose expression of the wild-type SPATA16 gene has been blocked: a diagnosis of a human teratozoospermia, and more particularly of human globozoospermia, and / or a diagnosis of infertility, or insufficient fertility to allow spontaneous conception by natural means, and / or the existence of a predisposition to a human teratozoospermia, and more particularly to human globozoospermia, may be pronounced (s) ).

  The means of the invention may comprise means for determining whether a sample capable of containing spermatozoa of the patient, such as a sample of sperm or testicular tissue, contains at least one mutant SPATAI6 protein of the invention.

  A positive determination being indicative of: - a human teratozoospermia, and more particularly a human globozoospermia, and / or infertility, or insufficient fertility to allow a spontaneous conception by natural means, and / or a predisposition to a human teratozoospermia, and more particularly to a human globozoospermia.

  Alternatively or additionally, the means of the invention may comprise means for determining whether a sample likely to contain spermatozoa of the patient, such as a sample of sperm or testicular tissue: - does not contain wild-type SPATA16 protein, c that is, does not contain SPATAI6 protein whose sequence and / or tertiary structure would be that (would be those) of a wild-type human SPATAI6 protein (such as the wild-type human protein of SEQ ID NO: 3), contains such a wild-type human SPATA16 protein in less than the amount normally seen in a fertile male subject. A positive determination is indicative: of a human teratozoospermia, and more particularly of a human globozoospermia, and / or infertility, or insufficient fertility to allow a spontaneous conception by natural means (or at least an insufficient fertility to allow a spontaneous conception by natural means within two years after the cessation of any contraception), and / or a predisposition to a human teratozoospermia, and more particularly to a human globozoospermia.

  In order to determine whether a sample does not contain wild-type SPATAI6 protein, or if it contains insufficiently, the person skilled in the art can use any suitable detection reagent (especially an antibody, more particularly a monoclonal antibody) specific for the protein. SPATAI6 wild human. Such a reagent must be sufficiently specific for the wild-type human SPATA16 protein, for example the protein of SEQ ID NO: 3, so as not to cross-react with a mutant SPATA16 protein of the invention, in particular with a mutant SPATAI6 protein of the invention which has lost all or part of the TPR domain, for example the mutant protein of SEQ ID NO: 17. The present application therefore relates to such a detection reagent for its use as a reagent to determine the existence : human teratozoospermia, and more particularly human globozoospermia, and / or infertility, or insufficient fertility to allow spontaneous conception by natural means (or at least insufficient fertility to allow natural spontaneous conception within two years after stopping any contraception), and / or a predisposition to human teratozoospermia, and more especially to a human globozoospermia. In order to determine if a sample contains a mutant protein of the invention, and possibly in what quantity, the person skilled in the art can use any suitable detection reagent (in particular an antibody, more particularly a monoclonal antibody) specific for one or more proteins. (s) mutant (s) of the invention. Such a reagent should be sufficiently specific for the mutant protein (s) of the invention (such as one or more SPATAI6 protein (s) which has (have) lost all or part of its TPR domain, for example a protein of SEQ ID NO: 17) not to cross-react with a wild-type human SPATA16 protein (such as a protein of SEQ ID NO: 3). The present application therefore relates to such a detection reagent for use as a reagent for determining the existence of: human teratozoospermia, and more particularly human globozoospermia, and / or infertility, or insufficient fertility to allow spontaneous conception by natural means (or at least insufficient fertility to allow spontaneous conception by natural routes within two years of stopping any contraception), and / or - predisposition to human teratozoospermia, and more particularly to human globozoospermia.

  A kit of the invention may in particular comprise at least one detection reagent specific for a wild-type human SPATAI6 protein and / or at least one detection reagent specific for a mutant protein of the invention. This or these reagents may (for example) be placed in an ELISA kit, which for example contains a microwell plate, or in a kit intended for carrying out a western blot. This or these reagents may (may) be in free form, or attached to a solid support such as for example attached to an ELISA plate or a transfer membrane.

  A diagnosis of this type may, alternatively or additionally, be established via a nucleic acid encoding the human SPATA16 protein. The diagnostic tests for human asthenozoospermia, and more particularly for human globozoospermia, can thus usefully include means for determining whether the SPATAI6 gene of a male human being and / or the SPATA16 mRNA of such a human being. has a mutant sequence to the wild-type human SPATAI6 gene and / or mRNA (i.e., to the SPATAI6 gene and / or mRNA of male humans that are fertile). If the detected mutation is likely to lead to the expression of mutant SPATA16 proteins, whose structure and / or sequence differs sufficiently from that of the wild-type human SPATA16 protein to be non-functional, or if the mutation detected is which may lead to a lack of expression of the SPATA16 protein, a diagnosis of human teratozoospermia, and more particularly of human globozoospermia, can be pronounced.

  The means of the invention may therefore comprise means for determining whether the patient, or at least a biological sample from said patient: - does not have a SPATAI6 gene and / or a SPATAI6 mRNA whose sequence and / or structure would be that of the wild-type human SPATAI6 gene and / or wild-type SPATA16 mRNA respectively, such as the sequence of a gene and / or an mRNA encoding a protein of SEQ ID NO: 3, and / or if the patient shows a mutant nucleic acid of the invention, in particular a gene and / or a mutant SPATA16 mRNA of the invention. An example of a mutant gene of the invention comprises, for example, a gene whose sequence may be derived from that of the wild-type human SPATAI6 gene by a mutation which comprises the substitution and / or the deletion of at least one nucleotide of an exon. coding part of the TPR domain, for example at least one nucleotide of exon 4, and / or the addition of at least one nucleotide in such an exon, said mutation being selected so as to lead to a incorrect splicing of the resulting SPATAI6 gene, such as, for example, splicing of exon 3 directly to exon 5. An example of a mutant mRNA of the invention comprises mRNA whose sequence is likely to be derived from that of Wild-type human SPATAI6 mRNA by a mutation which comprises deleting all or part of at least one exon from exons 2, 3 and 4, preferably at least exon 4. An example of mutant DNA of invention comprises a DNA whose sequence is likely to derive from this 1 of the wild-type human SPATA16 gene by a mutation which comprises the substitution of the last nucleotide of exon 4 of the wild-type human SPATAI6 gene (nucleotide G), for example to replace this nucleotide with nucleotide A.

  A positive determination is indicative of: - a human teratozoospermia, and more particularly a human globozoospermia, and / or infertility, or insufficient fertility to allow spontaneous conception by natural routes (or at least one insufficient fertility to allow spontaneous conception by the natural routes within two years following the cessation of all contraception), and / or a predisposition to a human teratozoospermia, and more particularly to a human globozoospermia.

  A positive determination is furthermore also indicative of an infertility of said patient, or a fertility of said patient which is insufficient to allow a spontaneous conception by the natural pathways (or at least a spontaneous conception by the natural pathways within a given time period). two years after stopping any contraception).

  To determine whether the patient, or at least one biological sample from the patient, does not contain nucleic acid encoding a wild-type human SPATAI6 protein, such as for example a wild-type human SPATA16 gene and / or mRNA, and possibly what quantity (for example, how much wild-type human SPATAI6 mRNA), the person skilled in the art can use any reagent allowing the specific detection of such a coding nucleic acid, or the specific detection of the nucleic acid whose sequence is the complementary one said nucleic acid encoding the entire length of the sequence of said encoding nucleic acid. Advantageously, such a specific reagent is a nucleic acid probe.

  Preferably, such a probe hybridizes to its target (s) under conditions of high stringency. For example, the person skilled in the art can use a probe allowing the specific detection of a nucleic acid comprising the sequence of SEQ ID NO: 1 or 2, or of the sequence which is its complementary throughout the length of this SEQ sequence. ID NO: 1 or 2.

  Such a specific reagent does not cross-hybridize with a mutant nucleic acid of the invention or with a nucleic acid whose sequence is complementary to a mutant nucleic acid of the invention throughout the length of this mutant nuléic acid. . An example of such a specific reagent is a probe which is specific for a nucleic acid comprising the sequence of SEQ ID NO: 1 or 2, or of the sequence which is complementary thereto along the entire length of this SEQ ID sequence. NO: 1 or 2, which does not cross-react with the mutant nucleic acid of SEQ ID NO: 16, or with the sequence which is complementary thereto along the entire length of this sequence of SEQ ID NO: 16. The present application therefore relates to such a nucleic acid probe, and in particular to such a nucleic acid probe as a reagent for determining the existence of: a human teratozoospermia, and more particularly a human globozoospermia, and / or infertility, or insufficient fertility to allow spontaneous conception by natural means (or at least insufficient fertility to allow a spontaneous conception by natural means within two years after the arrest). and any contraception), and / or a predisposition to human teratozoospermia, and more particularly to a human globozoospermia, and / or to determine the existence of infertility of said patient, or a fertility of said patient who is insufficient to allow spontaneous conception by natural routes (or at least spontaneous conception by natural routes within two years of stopping any contraception).

  To determine whether the patient, or at the very least a biological sample from the patient, contains a mutant nucleic acid of the invention, such as for example a mutant DNA and / or mRNA of the invention, the person skilled in the art may use any reagent for the specific detection of such a mutant nucleic acid or a sequence that is complementary to such a mutant nucleic acid throughout the length of the sequence of this mutant nucleic acid. Advantageously, such a specific reagent is a nucleic acid probe. Preferably, such a probe hybridizes to its target (s) under conditions of high stringency.

  The person skilled in the art may, for example, use a probe allowing the specific detection of a nucleic acid comprising the sequence of SEQ ID NO: 16 or of the sequence which is complementary thereto along the entire length of this sequence of SEQ ID NO: 16. Such a specific reagent does not cross-hybridize with a nucleic acid encoding a wild-type SPATA16 protein or with the sequence that is complementary to this coding sequence throughout the sequence of this coding sequence. For example, such a specific reagent is a probe that is specific for a nucleic acid comprising the sequence of SEQ ID NO: 16 or the sequence that is complementary thereto along the entire length of this sequence of SEQ ID NO: 16 , and which does not cross-hybridize with the nucleic acid of SEQ ID NO: 2 nor with the sequence which is complementary to the sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. The present application therefore relates to such a nucleic acid probe, and in particular to such a nucleic acid probe as a reagent for determining the existence of: a human teratozoospermia, and more particularly a human globozoospermia, and / or infertility, or insufficient fertility to allow spontaneous conception by natural means (or at least insufficient fertility to allow a spontaneous conception by natural means within two years after the judgment any contraception), and / or a predisposition to a human teratozoospermia, and more particularly to a human globozoospermia, and / or to determine the existence of an infertility of said patient, or a fertility of said patient which is insufficient to allow spontaneous conception by natural means (or at least spontaneous conception by natural means within two years of stopping any contraception).

  A kit of the invention may in particular comprise at least one nucleic acid probe specific for a nucleic acid encoding a wild-type human SPATA16 protein or nucleic acid complementary to this coding nucleic acid, and / or at least one specific nucleic acid probe. a mutant nucleic acid of the invention or nucleic acid complementary to this mutant nucleic acid.

  Such probes are intended to be placed in contact with the nucleic acids of the sample under conditions favorable to their hybridization with their target sequence (s). This bringing into contact may for example be carried out according to Northern (Northern blot), Northern reverse (reverse Northern blot) or Southern (Southern blot) transfer techniques. Alternatively or additionally, such probes can be fixed on a solid support, in particular a solid support adapted to the circulation of a microfluidic flow, such as a nucleic acid chip. If desired or required, the nucleic acids can be purified from the sample prior to contact with either or both of the two types of probes.

  Alternatively or additionally, the nucleic acids of the sample can be amplified by amplification reaction using primers, for example by PCR (polymerase chain reaction). Advantageously, such primers are primers which hybridize to the human SPATA16 gene at positions such that they make it possible to amplify by polymerization the SPATAI6 sequence that these primers frame. Such primers thus make it possible to amplify the sequence of the human SPATAI6 gene present in the sample tested, or at least one fragment of this gene. The amplified sequence can then be directly sequenced so as to determine whether this sequence is that of a wild-type human SPATA16 gene or gene fragment, or that of a gene or mutant gene fragment according to the present invention. If the primers have been chosen so as to frame the target sequence of one of the probes of the invention, the primers can then be used in association with this probe, so as to detect with this probe the product of amplification likely to be obtained with said primers. The primers and the probe can be used successively, or simultaneously, for example in the context of a real-time PCR.

  The present application thus relates to pairs of amplification primers whose sequences hybridize to the sequence of the wild-type human SPATA16 gene or mRNA or to the complementary sequence of this gene or mRNA, as well as to amplification primers whose sequences hybridize to the sequence of a mutant nucleic acid of the invention or to the complementary sequence of this mutant nucleic acid. Preferably, said primers hybridize to their target (s) under conditions of high stringency. Such primers may be specific to their target (s), or may not be specific (s). Preferably, however, such primers are specific for the wild-type and mutated forms of the human SPATAI6 mRNA or gene, i.e. they hybridize to the human SPATAI6 gene or mRNA sequence. or on the complementary sequence of this gene, and that they also hybridize on the sequence of a mutant nucleic acid of the invention or on the complementary sequence of this mutant nucleic acid, without however hybridizing to a other human nucleic acid.

  These primers can be used alone, or in combination with at least one probe of the invention.

  These primers are, according to the present invention, intended to be used to determine the existence of: a human teratozoospermia, and more particularly a human globozoospermia, and / or infertility, or insufficient fertility to allow a conception natural spontaneous (or at least insufficient fertility to allow spontaneous conception by natural means within two years after stopping any contraception), and / or a predisposition to a human teratozoospermia, and more particularly to a human globozoospermia, and / or to determine the existence of an infertility of said patient, or a fertility of said patient which is insufficient to allow a spontaneous conception by natural means (or at least a spontaneous conception by natural means within two years of stopping any contraception).

  A kit of the invention preferably comprises at least one probe of the invention and at least one pair of primers of the invention. Advantageously, a kit of the invention comprises at least one pair of primers 20 of the invention, and at least one probe of each of the two types of probes of the invention.

  The hybridization conditions of a probe or a primer of the invention are preferably hybridization conditions of high stringency. Such conditions of high stringency can be adjusted by those skilled in the art, in particular depending on the hybridization technique used. By way of illustration, an example of high stringency conditions includes transfer membrane hybridization in 5xSSC, sodium dodecyl sulfate (SDS) 2%, 100 micrograms / mL of single-stranded nucleic acid at 55-65 ° C. 8 hours, and wash in 0.2xSSC and 0.2% SDS at 60-65 C for thirty minutes.

  By way of illustration, an example of conditions of high stringency more particularly adapted to amplification primers comprises hybridization in the presence of polymerase at an annealing temperature of 15 to 5 C below the melting temperature (Tm) of the primers tested. .

  The present invention also provides novel means for the contraception of the male human. The contraceptive means of the invention have the effect of inducing the inability of human spermatozoa to penetrate an egg. More specifically, the means of the invention are means which block, inhibit or alter the formation of the acrosome of human spermatozoa to an extent sufficient to induce the desired contraceptive effect.

  The contraceptive means of the invention act at the level of the human SPATAI6 protein, more particularly at the level of its expression. The contraceptive means of the invention are means which: - inhibit or block the expression of the human SPATA16 gene, and / or - induce an incorrect splicing of the human SPATAI6 gene (or pro-RNA), and / or 20 -inhibit or block the intracellular translocation of the human SPATA16 protein within a human spermatogenic cell (said human spermatogenic cell being preferably a human spermatocyte and / or human spermatide), and / or - inhibit or block the activity of human SPATA16 protein, by binding to the TPR domain of this protein and / or by inducing the lysis of this protein.

  The contraceptive means of the invention are not directed to the hormonal pathways: their mode of action is a protein which is specifically expressed in the human testes, and more particularly in the adult germ cells of the human testes, namely the SPATAI6 protein.

  The means according to the invention have a molecular target in humans (SPATAI6) which is sufficiently specific for the testicular organ, so that the degree of harmful side effects that can be envisaged is very low. Therefore, the contraceptive means of the invention are likely not to induce undesirable side effects, which have been observed with the prior art pharmaceutical contraceptives, such as inhibition of secondary sexual characteristics (feminization), erectile dysfunction, loss of libido, disruption of heart rhythm, increased coronary risk, weight gain.

  The method of the invention is to select and / or produce a compound that disrupts the normal expression of the wild-type human SPATA16 gene to a sufficient extent that at least a proportion of at least 20%, preferably at least 90%, preferably at least 95%, advantageously 100% of human spermatocytes are devoid of acrosome, or are provided with an acrosome of abnormal morphology, which do not allow them to fertilize a woman's egg.

  The present application thus relates to a method for selecting and / or producing a candidate compound likely to have a contraceptive effect on the male human, which comprises the selection and / or production of a compound - which inhibits , block or alter the expression of the human SPATAI6 gene, and / or - which induces an incorrect splicing of the human SPATAI6 gene (or pro-RNA), and / or - which inhibits, blocks or alters the transcription of the human SPATAI6 gene, and / or - which inhibits, blocks or alters the translation of the human SPATAI6 gene, and / or - which inhibits or blocks the intracellular translocation of the human SPATA16 protein within a human spermatogenic cell (said cell human spermatogen preferably being a human spermatocyte and / or a human spermatide), and / or - which inhibits or blocks the activity of the human SPATA16 protein, by binding to the TPR domain of this protein and / or by inducing the lysis of this protein ine.

  According to the present invention, a compound which alters the expression of the human SPATAI6 gene, that is to say a compound which alters the expression of the human SPATA16 gene to a sufficient extent to alter the expression, can be selected and / or produced. formation of the acrosome of a spermatozoon, preferably a human sperm. Such a compound may in particular have the effect of altering the formation of the acrosome, preferably preventing it, when placed in contact with at least one human spermatogenic cell, preferably with at least one human spermatocyte and / or or at least one human spermatide. Such a compound may for example be a compound that alters the transcription and / or translation of the human SPATAI6 gene.

  Preferably, it is a compound that induces an incorrect splicing of the human SPATAI6 gene. The human SPATA16 gene comprises eleven exons. At the correct splicing of the human SPATAI6 gene, exons 1 to 11 are spliced in an order that corresponds to the numbering assigned to them (exon 1 is spliced on exon 2, exon 2 is spliced on exon 3, exon 3 is spliced on exon 4, exon 4 is spliced on exon 5, etc.). An incorrect splice is a splice that does not respect this order, for example by splicing exon 3 directly to exon 5, which has the effect of eliminating exon 4 from the continuation of the spliced exons, or any other splicing that leads to the loss of exon 2 and / or exon 3 and / or exon 4, that is to say the loss of all or part of the sequence which, within the human SPATAI6 gene, encodes the TPR domain of the human SPATA16 protein.

  According to the present invention, a compound which blocks or inhibits the expression of the human SPATAI6 gene, more particularly which blocks or inhibits its translation, that is to say the translation of SPATAI6 mRNA, can be selected and / or produced. human SPATA16 protein.

  Such a compound may advantageously be a DNA or RNA interferant, that is to say a DNA or RNA molecule which has the capacity to bind to the DNA or mRNA of the human SPATA16 gene, or which contains an element capable of binding to such DNA or mRNA, and which: - interferes with the transcription of the human SPATA16 gene, that is to say which blocks, inhibits or impairs the transcription of the human SPATAI6 gene, and / or interferes with the translation of human SPATAI6 gene mRNA, that is, that blocks, inhibits or impairs the translation of this mRNA. An anti-human SPATA16 DNA or RNA (DNA or RNAi) interferer inhibits or blocks the production of the SPATAI6 protein by a human spermatogenic cell (said human spermatogenic cell preferably being a human spermatocyte and / or human spermatide). According to the present invention, such a DNA or RNA interferant leads to an absence of acrosome, or to the formation of an acrosome of morphology sufficiently abnormal not to allow the penetration of a woman's egg. An example of such a compound is an RNA interferant that cleaves or degrades human SPATAI6 gene mRNA within a human spermatogenic cell, and / or repressing the translation of that mRNA. Such an RNA interfering agent may for example be a human anti-SPATA16 (small interfering RNA) siRNA, or a human anti-SPATA16 shRNA (short hairpin RNA). Examples of such DNA or RNA interferants are described in more detail below.

  In accordance with the present invention, a compound that inhibits or blocks the intracellular translocation of human SPATA16 protein within a human spermatogenic cell can be selected and / or produced (said human spermatogenic cell preferably being a human spermatocyte and / or or human spermatide). An example of such a compound is a compound that inhibits or blocks the translocation of the human SPATA16 protein to the Golgi apparatus and / or its translocation from the Golgi apparatus to the acrosome formation site.

  An exemplary embodiment comprises: the implementation of a cell which comprises a Golgi apparatus but which does not express SPATA16, the transfection, transformation or infection of this cell by the human SPATAI6 gene, preferably a SPATA16 gene wild type, so that the human SPATA16 protein encoded by this gene can be expressed in this cell and transferred to the Golgi apparatus of this cell, bringing this transfected, transformed or infected cell into contact with at least one candidate compound, preferably with a library of candidate compounds, so as to determine whether this candidate compound, or any of the candidate compounds of said library, blocks, inhibits or modifies the translocation of the human SPATA16 protein to the Golgi apparatus of said cell, a positive determination being indicative of the fact that the or each candidate compound (s) concerned is (are) a compound likely to have a coeffective effect. ntraceptive about the male human being. Preferred candidate compounds are human SPATA16 protein ligands, preferably compounds that are ligands specific for human SPATA16 protein.

  In accordance with the present invention, a compound that inhibits or blocks the activity of human SPATAI6 protein can be selected and / or produced by binding to the TPR domain of this protein and / or by inducing lysis of this protein. An example of such a compound is a compound that specifically inhibits or blocks the activity of this protein without altering the activity of another human protein.

  Compounds that bind to human SPATA16 protein, particularly its TPR domain, can be identified by those skilled in the art. Preferably, these ligands are ligands specific for the human SPATAI6 protein.

  According to a preferred embodiment of the invention, said selected or produced compound is a human SPATA16 gene ligand and / or human SPATA16 mRNA and / or human SPATAI6 protein, or is a compound that induces formation. of such a ligand, once administered in a mammal (non-human or human mammal, preferably male or male).

  The present application also relates to a method for selecting a compound having a contraceptive effect on the male human, which comprises: selecting and / or producing at least one candidate compound capable of having a contraceptive effect on the male human according to the method of selection and / or production of the candidate compound (s) of the invention, the bringing into contact of this, or each of these candidate compound (s) ) with at least one human spermatogenic cell (said human spermatogenic cell being preferably a human spermatocyte and / or a human spermatide), so as to determine whether this candidate compound induces inhibition or blockage of acrosome formation on this at least one human spermatogenic cell, - the selection of at least one candidate compound which has the ability to induce such an inhibition or blocking, such a compound being a contraceptive effect compound on the male human being.

  The present application also relates to a method for producing a contraceptive effect product on a male human, which comprises selecting a compound having a contraceptive effect according to the method of the invention, and the formulation of this compound in a form administrable to humans.

  The present application also relates to a product or compound having a contraceptive effect on the male human, which comprises at least one human DNA or RNA SPATAI6 interferer, i.e. DNA or RNA that interferes with the transcription of the human SPATA16 gene into mRNA and / or with the translation of human SPATA16 mRNA into protein. These molecules will be called DNAi or RNAi depending on whether they interfere with the SPATA16 gene DNA or with the mRNA of the human SPATAI6 gene.

  Such human SPATAI6 DNA or RNA interferants are intended to hybridize to a target region of the human SPATAI6 gene or to the mRNA of the human SPATAI6 gene, in a form coupled to the RISC enzyme complex, of to induce the degradation or cleavage of human SPATAI6 DNA or mRNA, or to induce post-translational repression of the SPATAI6 protein produced by the cell.

  For example, a DNA interferant can target an intron region of the human SPATAI6 gene.

  Such an RNA interferant targets an exonic region of the human SPATA16 gene. An RNA interfering agent can cleave or degrade the mRNA of the human SPATAI6 gene, and / or repress the translation of this mRNA. Such an RNA interfering agent may for example be a human TA 16 anti-SPA small interfering RNA or a human anti-SPATAI6 shRNA (short hairpin RNA).

  The present application also relates to such RNA or DNA interferants as such, and to their uses as a contraceptive agent for the male human.

  Compounds that are known as siRNAs or shRNAs are single-stranded or double-stranded DNA or RNA molecules.

  SiRNAs are 14 to 200 bp, preferably 14 to 25 bp, double-stranded DNA or RNA molecules, one of whose strands (antisense strand, or negative strand) has a sufficiently complementary sequence for hybridizing to mRNA of the human SPATAI6 gene, at least under the conditions of the intracellular medium of a human spermatogenic cell (said human spermatogenic cell preferably being a human spermatocyte and / or human spermatide). SiRNAs can be administered to the patient directly as small double-stranded DNA or RNA molecules, e.g. 14 to 25 bp, or as larger double-stranded DNA or RNA molecules. strand, for example from 50 to 200 bp, which will then be cleaved into small double-stranded DNA or RNA, for example from 14 to 25 bp, by the DICER enzyme complex within the spermatogenic cell.

  The shRNAs are single-stranded DNA or RNA molecules of 14 to 25 nucleotides, which may have a stem-loop structure, and which have a sufficiently complementary sequence to hybridize to the gene mRNA. SPATA16 human, at least under the conditions of the intracellular medium of a human spermatogenic cell (said human spermatogenic cell being preferably a human spermatocyte and / or human spermatide).

  A sufficiently complementary sequence will generally have an identity of at least 85%, preferably at least 90%, with the sequence complementary to the human SPATA16 gene mRNA, over the entire length of this strand.

  Preferably, the antisense strand sequence of the siRNA, or, if appropriate, the shRNA strand sequence, is perfectly complementary to the sequence of the human SPATAI6 gene mRNA along the entire length of this strand (100% identity ).

  The present application is thus more particularly related to any anti-human SPATAI6 RNA or DNA interferant, more particularly to any human anti-SPATAI6 RNA interfering agent, which comprises a strand of hybridized antisense nucleic acid to a strand of sense nucleic acid, said antisense strand and said sense strand being: - either not covalently bonded to each other, thus forming a siRNA type structure, or covalently connected to each other via a loop nucleic acid, thus forming a shRNA loop stem structure, said antisense strand consisting of 14 to 200 nucleotides, and said antisense strand comprising (or consisting of) a nucleotide sequence of 14 to 25 nucleotides which has a at least 85% identity, preferably at least 90%, advantageously at least 95%, very preferably 100%, with a nucleotide sequence that is included in the sequence of the gene and / or mRNA Human SPATAI6, preferentially in the sequence of at least one exon of the human SPATAI6 gene (or in the sequence of human SPATAI6 mRNA), very preferably in the sequence of exon 2 and / or 3 and / or 4 of the gene Human SPATAI6 (or in the sequence of human SPATA16 mRNA), for example in exon 4 of the human SPATAI6 gene (or in the sequence of human SPATA16 mRNA). Said percentage value of sequence identity is calculated over the entire length of said sequence of 14 to 25 nucleotides.

  Preferably, the antisense strand sequence of the siRNA, or, where appropriate, the strand of the shRNA, targets at least one exon of SPATAI6 selected from exons 2, 3 and 4, that is to say the exons which comprise the sequences encoding the TPR domain of SPATAI6. Such a shRNA or shRNA thus comprises a strand of DNA or antisense RNA which: - is composed of 14 to 200 nucleotides, and which - comprises, or consists of a sequence of 14 to 25 nucleotides which has an identity of at least 85%, preferably at least 90%, advantageously at least 95%, very preferably 100%, with the sequence of exon 2 and / or 3 and / or 4 of the human SPATA16 gene, said identity value being calculated over the entire length of this strand. Preferably, the sequence of the antisense strand of shRNA, or where appropriate shRNA, targets exon 4 of SPATA16.

  Examples of such RNA or DNA interferants include, inter alia, RNA or DNA interferants, whose antisense strand comprises, or consists of, the sequence of SEQ ID NO: 18 and / or of SEQ ID NO: 19 and / or SEQ ID NO: 20.

SEQ ID NO: 18 = CCACCTAACATCCTGGAAA

  SEQ ID NO: 19 = GCTGAAATAGTAGACCCTT SEQ ID NO: 20 = GCTAAATTGGACACGACTT

  Preferably, the sequence of the antisense strand of shRNA, or if appropriate, shRNA, has a G + C content greater than 50%. Preferably, the antisense strand of siRNA or shRNA is hybridized to the sense strand of this siRNA. or shRNA so that they each have, 2 relative to each other, 2 nucleotides in overflow or overhang in 3 '.

  The portion of the sense fragment of siRNA or shRNA that is hybridized to the antisense strand is preferably perfectly complementary to the antisense strand sequence portion to which it is hybridized (100% identity with the complementary sequence).

  Preferably, the antisense strand sequence of siRNA, or, where appropriate, shRNA, has no identity of more than 70% with respect to the DNA or RNA sequence of a human gene other than SPATAI6 (identity value calculated along the entire length of the strand).

  The present application also relates to such RNA or DNA interferants as a contraceptive agent for the male human.

  The term "comprising", with which "including" or "containing" is synonymous, is an open term, and does not exclude the presence of one or more element (s), ingredient (s) or step (s) of additional method (s) that would not be explicitly stated, while the term "consistent" or "constituted" is a closed term, which excludes the presence of any additional element, step, or ingredient that does not would not be explicitly exposed. The term "substantially consisting of" or "essentially consisting of" is a partially open term, which does not exclude the presence of one or more element (s), ingredient (s) or additional step (s) in the to the extent that such element (s), ingredient (s) or additional step (s) do not materially affect the basic properties of the invention. Therefore, the term "comprising" (or "comprises (include)") includes the terms "consisting", "consisting", as well as the terms "consisting essentially" and "essentially consisting".

  The present invention is illustrated by the following examples, which are given for purely illustrative purposes.

EXAMPLES Example 1

  Here is the example of a family comprising three siblings affected by globozoospermia, in which we have identified a homozygous mutation in the SPATAI6 gene (OMIM 609856) specific for spermatogenesis. To the knowledge of the inventors, this is the first example of a non-syndromic male infertility state in humans due to a unique genetic defect. The inventors analyzed an Ashkenazi Jewish family comprising six brothers 20 (three affected, three healthy) and four sisters, who were identified at the Center for Reproductive Medicine of the Dutch Speaking Brussels Free University. The three unaffected brothers were respectively fathers of 7, 6 and 5 children, but the three affected brothers were childless and had a fertility disorder due to oligoasthenoterazoospermia, showing the characteristics of total globozoospermia such as sperm without acrosome ( round-headed), as shown by acrosin staining (OMIM 102480) in Figure 1A. No known consanguinity was reported, although the family belonged to an isolated Jewish population. The karyotype appeared normal and no microdeletion of the Y chromosome was found. In two of the 30 affected brothers, intracytoplasmic sperm injections (ICSI) were performed, but the fertilization obtained was poor and no pregnancy occurred. The inventors screened the extent of the genome for each of the six siblings using a 10K Arine Single Nucleotide Polymorphism (Affymetrix GeneChip Human Mapping, Affymetrix, Santa Clara, California, U.S.A.). Homozygosity regions were defined by the presence of more than 25 consecutive homozygous SNPs. Large areas of homozygosity were observed in all six individuals (Tables 1 and 2), indicating second or third degree consanguinity in the family.

  Table 1: Chromo Position SNP ID Brother Brother Brother some affected affected affected n 1 n 2 n 3 3 165727121 SNP A-1508753 AA AA AA 3 167054711 SNP_A-1512676 AB AB AB 3 167631594 SNP_A-1508627 AA AA AA 3 167801377 SNP A- 1519252 YY AA YY 3 169050879 SNP_A-1511126 BB BB BB 3 169140975 SNP_A-1509483 YY AA YY 3 169748882 SNP_A-1518417 BB BB BB 3 170222552 SNP_A-1518965 BB BB BB 3 170229669 SNP_A-1509719 BB BB BB 3 170266790 SNP_A-1519387 BB BB BB 3 170341708 SNP_A-1513150 BB BB BB 3 170440150 SN P_A-1513458 AA AA AA 3 170815817 SNP_A-1516975 AA AA AA 3 171062673 SNP_A-1514614 - - AA 3 171141972 SNP_A-1508020 BB BB BB 3 172368188 SNP_A-1510308 AA AA AA 3 172643136 SNP_A-1517656 BB BB BB 3 172771949 SNP_A-1509479 AA AA AA 3 172933454 SNP_A-1509435 AA AA AA 3 172933529 SNP_A-1509382 BB BB BB 3 173575998 SNP A-1516388 BB BB BB 3 173580444 SNP_A-1514288 BB BB BB 3 SNP_A-1507368 AA YY 3 AAA 3 AAA 3 AAA 3 AAA AAA 3 174680729 SNP_A-1507368 SNP_A-1508795 BB BB BB 3 176537318 SNP_A-15117 79 AA AA AA 3 176610712 SNP_A-1509801 BB BB BB 3 176827123 SNP_A-1511813 AA AA AA 3 177333023 SNP_A-1517824 AA AA AA 3 177621541 SNP_A-1518682 BB BB BB 3 177708137 SNP_A-1510834 AA AA AA 3 177723240 SNP_A-1516780 BB BB BB 3 178105781 SNP_A-1516425 AA AA AA 3 178105781 SNP_A-1515959 - - BB 3 179324469 SNP_A-1517000 - - AA 3 179597123 SNP_A-1516215 AA AA AA 3 179597352 SNP_A-1516746 AA AA AA 3 179636158 SNP_A-1512810 BB BB BB 3 179706441 SNP_A-1510950 BB BB BB 3 179839122 SNP_A-1514173 BB BB BB 3 180413909 SNP_A-1511671 AA AA AA 3 180729255 SNP_A-1508156 BB BB BB 3 181028753 SNP_A-1512457 BB BB BB 3 181180943 SNP_A-1507868 BB BB BB 3 181251555 SNP_A-1512336 AA AA AA 3 181298402 SNP_A-1513747 AA AA AA 3 181506449 SNP_A-1517808 BB BB BB 3 181552274 SNP A-1509494 BB BB BBTable 2: Shared Haplotype Shared Homozygosity Chrom #SNP 1 Start Stop Length j #SNP Start Stop Length 1 15 33955677 36556426 2600749 1 11 117399167 119296010 1896843 6 117866019 119296010 1429991 1 11 151489481 156376556 4887075 11 151489481 156376556 4887075 1 14 160643582 162290883 1647301 1 10 194965564 198362174 33966102 55 19716714 34386124 14669410 2 14 54483803 57217291 2733488 2 11 59155900 65801963 6646063 2 91 65801963 107509848 41707885 2 12 113469814 115959977 2490163 2 10 212719583 215032622 2313039 2 16 224103332 226044921 1941589 2 14 234216839 239151790 4934951 8 234719101 239151790 4432689 3 12 653347 3558063 2904716 3 10 100634082 103786422 3152340 3 48 113839242 127152417 13313175 48 3 49 165655532 184087390 18431858 47 167054711 184087390 17032679 3 26 184087390 190712906 6625516 4 10 173190930 176925273 3734343 8 173530324 176925273 3394949 4 48 181334851 191091333 9756482 11 120723042 122283900 1560858 7 121110284 122283900 1173616 4829725 5 15 134671015 139500740 6 10 28766533 31094058 2327525 6 29479394 31094058 1614664 6 14 46333713 47986997 1653284 5 46824038 47986997 1162959 6 12 96879221 102207593 5328372 6 10 105174572 108446020 3271448 6 169 108446020 5 2383480 43937460 7 169 77627148 131407468 53780320 8 15 53838124 57959516 4121392 8 10 72728798 76200122 3471324 9 85 507715 13460671 12952956 6 12445236 13460671 1015435 9 10 72760108 75532057 2771949 7 73265909 75532057 2266148 9 56 75532057 90138831 14606774 10 63884288 66164664 2280376 10 10 66164664 68113302 1948638 10 47 91501439 105835217 14333778 14 20 19490525 22620727 3130202 14 10 31508337 33265230 1756893 49 14 10 36021565 37647284 1625719 13 21490270 23325412 1835142 15 127 23325412 59077153 35751741 15 12 79723090 84387340 4664250 16 24 22705353 50026393 27321040 17 10 28942222 34693022 5750800 9 29183029 34693022 5509993 17 10 66657008 72151125 5494117 18 13 63746898 66906214 3159316 9 64766169 66906214 2140045 10 38790879 43185196 4394317 21 11 18764912 21113081 2348169 11 18764912 21113081 2348169 21 12 29925652 31842275 1916623 21 10 36234195 37974748 1740553 7 36447405 37974748 1527343 On the left side of Table 2, a selection ( > 9 SNPs) haplotype zones shared is presented. In the right-hand part of Table 2, the regions of shared homozygosity (> 4 SNPs) that extend inside are shown.

  Chrom: chromosome. #SNP: The amount of SNP that forms the shared haplotype zone or homozygosity. Departure: departure location. Stop: stop location.

Length: fragment length.

  Therefore, the inventors considered that this family was consanguineous, and deduced that the pathology was recessive autosomal. The inventors identified a single region of identical haplotypic homozygosity shared by all affected brothers, and for which the parents and two of the healthy brothers were heterozygous. The smallest area of overlap extended over 17 Mb of chromosome 3q26 (Chr3: 167054711-184087390). This region contains some 50 known genes in the UCSC Genome Browser. The analysis conducted led the inventors to select the SPATAI6 gene (associated spermatogenesis 16, also known as NYD-SP12) as a candidate gene. The sequence of the human SPATAI6 gene, that of its CDS (coding sequence), and that of the human SPATA16 protein are available on the databases, under accession number AF345909. SPATAI6 is composed of 11 exons encoding a highly conserved protein of 65 kDa (569 aa), which contains a tetratricopeptide repetition domain (TPR (OMIM 602259)). The sequence alignment (Clustel W 1.81, see Figure 3) shows that SPATA16 is highly conserved across mammals with an identity level ranging from 77% (mouse) to 96% (chimpanzee) (cf. Table 3).

  Table 3: Species Identity (%): Positive (%) Breach (%) human gap SPATA16 TPR SPATAI6 TPR SPATA16 TPR Homo Sapiens 100 100 100 100 0 0 Bos Taurus 87 94 92 97 0 0 Canis familiaris 83 97 89 97 0 0 Macaca fascicularis 95 97 97 97 0 0 Mus musculus 77 92 86 96 0 0 isoform 1 Mus musculus 78 92 87 96 0 0 isoform 2 Pan troglodytes 96 98 98 98 0 0 Battus norvegicus 80 93 87 96 0 0 Table 3 shows the percentage of sequence identity for SPATA16 in other species compared to humans, as well as for the TPR domain.

  It can thus be noted that the sequence conservation is even greater for the TPR domain, a proteinprotein interaction domain currently but exclusively found in cochaperone proteins (92 / U and 98% respectively in mice and chimpanzees).

  Sequence analysis of one of the affected sons revealed a homozygous sequence variation at exon 4 (c.848G> A), which breaks an Ncil or Hpall recognition site (see Figure 1C). The restriction enzyme analysis revealed that the three affected brothers are in fact homozygous, and that both parents and the two healthy brothers are heterozygous for the mutation. The third unaffected brother appeared to be homozygous for the wild-type sequence (Figure ID). The nucleotide variation c.848G> A is not known in any SNP database, and has not been identified in the 231 controls tested, including 151 random controls of both sexes and 80 fertile males.

  The mutation identified could result in an amino acid change from a highly conserved residue (p.R283Q) located at the C-terminus of the conservatively conserved TPR domain. In addition, the c.848G> A mutation affects the last nucleotide of exon 4 (Figure 1B), and therefore can disrupt the 5 'splice site of intron 4. Three different models of site prediction splicing predicted that the mutation disrupts this splice site (see Table 4).

  Table 4: WT MT Website http://www.cbs.dtu.dk/services/netgene2 0.80 <0.50 http://www.genet.sickkids.on.ca/uali/splicesitefinder.html 0.805 0.681 http://www.fruitfly.org/seq_tools/splice.html 0.97 <0.40 It can be seen that the various predictions of in-line splice sites all indicate irregular relationships between the wild-type (WT) sequence. and the mutated sequence (MT). Unfortunately, the SPATA16 protein has testicular-restricted expression and we have not been allowed to use fresh sperm cells, nor to perform a biopsy in these patients in order to verify the aberrant splicing predicted in vivo. As a result, minigene constructs have been produced; they included two constitutive 13-globin exons surrounding a 420 bp fragment containing either the wild type or the mutated form of exon 415 and flanking the intron sequences of SPATA16 (see Figure 2A). These minigenes were transfected into COS1 or HeLa cells, and the transcripts were analyzed by RT-PCR (reverse transcription polymerase chain reaction) 24 hours after transfection. As shown in Figure 2B, the wild-type exon 4 is invariably included in the final messenger RNA, as confirmed by the sequencing of the PCR product. In sharp contrast, the mutated exon gives rise to two aberrant splice forms, as shown by the cloning and sequencing of these PCR products. The product that appears larger and stronger is the result of using a splice site located in the [3-globin intron used for minigene construction. The product that appears smaller and weaker corresponds to the use of a cryptic splice site located 18 bp upstream of the normal splice site. These aspecific products are probably due to very small and short intron sequences in minigene construction. Such products are often observed in exon trap experiments in the absence of a true splice site and are indicative of the occurrence of exon skips due to the mutation. Notably, we could not detect any transcripts containing the correct junctions from the mutated exon 4, proving that the mutation blocks the normal splicing of the gene.

  The first critical step of exon inclusion is the binding of splice factor U1 snRNP (small nuclear ribonucleoprotein) to the 5 'splice sites. In order to confirm that the mutated SPATAI6 exon 4 is not recognized by the splicing machinery, the inventors verified its binding to U1 snRNP by psoralen mediated UV hybridization. While binding of U1 snRNP to wild type DNA was easily detected, this was not observed when DNA carrying the c.848G> A mutation was used as a template (Figure 2C). The identity of U1 snRNP was confirmed by the HseRNA treatment using oligodeoxynucleotide complementary to positions 1 to 15 of U1 snRNA. Therefore, the bioinformatic prediction results, the minigene, and the U1 binding all indicate that the c.848G> A mutation leads to the inappropriate splicing of exon 4, and therefore to TPR domain disruption.

  The inventors also analyzed SPATA16 in 29 globozoospermic patients including 6 familial cases (14 patients), including 12 with total globozoospermia and 17 with partial globozoospermia. None of them showed any sequence variation, except for 3 known polymorphisms and 2 point mutations that are not segregated to the disease (see Table 5). Table 5: Variations Segregation Patient Globozoospermia Variation of exon I SNP Parents I Brides I Partial / total population known 1 Partial 2 c.232G> A p.E78K Yes European c.397A> G p.M133V Yes European 2 Partial 2 c.232G> A p.E78K Yes European c.397A> G p.M133V Yes European c.1526 C> T p.A509V No Mother Absent at affected European brother c.1577 T> C p.M526T No Mother Absent at affected European brother 3-4 Partial 2 c.232G> A p.E78K Yes European c.397A> G p.M133V Yes European 5 Partial 2 c.232G> A p.E78K Yes Absent at North African Affected Brother c. 397A> G p.M133V Yes c.440G> A p.G147E Yes 56 7-8 Partial 2 c.232G> A p.E78K Yes These patients are European 9 Total 2 c.397A> G p.M133V Yes European 10 Total 2 c.232G> A p.E78K Yes North African 2 c.397A G p.M133V Yes c.440G> A p.G147E Yes In Table 5, all non-synonymous coding variations, which were discovered in 9 of 28 patient They are shown. Patients 1 to 5 and 7 to 8 suffer from partial globozoospermia, while patients 9 to 10 suffer from total globozoospermia. Three known SNPs were identified, located near two unknown but unsegregated variations. In patients 6 and 11 to 28, no non-synonymous, coding variation was identified.

  To the knowledge of the inventors, this is the first description of a gene involved in the pathogenesis of human globozoospermia.

  In Figure 4 is reproduced the cDNA sequence of the wild-type human SPATAI6 gene (SEQ ID NO: 1).

  Figure 4 also shows the coding sequence (CDS, SEQ ID NO: 2) of the wild-type human SPATAI6 protein, as well as the sequence of the wild-type human SPATAI6 protein (SEQ ID NO: 3). This data is reproduced from the available sequence data under accession number AF 345909.

  The positions of the exons within, or in relation to, these sequences are as follows:

  Table 6: Exon Position Position Position Position within Breast Breast of SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 (cDNA) (CDS) (Protein) 1 112 1-112 - - 2,630 / 612 113-742 1-612 1-204 3 146 743-888 613-758 205-253 4 90 889-978 759-848 253-28225 5 85 979-1063 849-933 282-311 6 148 1064-1211 934 -1081 312-361 7 147 1212-1358 1082-1228 361- 410 8 110 1359-1468 1229-1338 410-446 9 165 1469-1633 1339-1503 447-501 10 84 1634-1717 1504-1587 502-529 11 333/123 1718-2050 1588-1710 530-569 For the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, the position of an exon is defined by indicating the position of the first nucleotide forming part of this exon, and the position of the last nucleotide that is part of this exon.

  For the protein sequence of SEQ ID NO: 3, the position indicated is that of the amino acids. In Table 6 above, when the codon of an amino acid overlaps two exons, the position of this amino acid is indicated for each of the two exons; this is the case of: the amino acid at position 253, whose codon overlaps exon 3 and exon 4, of the amino acid at position 282, whose codon overlaps exon 4 and the exon 5, of the amino acid at position 361 (Asp), whose codon overlaps exon 6 and exon 7, of the amino acid at position 410 (Glu), whose codon overlaps exon 7 and Exon 8.

  The fragment 92041-92340 (SEQ ID NO: 6), the fragment 121561-121800 (SEQ ID NO: 7) and the fragment 164101-164340 (SEQ ID NO: 8) which are presented in FIG. 5 are the fragments 92041-92340. , 121561-121800 and 164101-164340, respectively of the wild-type SPATAI6 gene sequence (assembled sequence of the wild-type SPATA16 gene, available at http: //www.ensembl/orq, under the accession number ENSG00000144962).

  Fragment 92041-92340 (SEQ ID NO: 6) contains exon 3 (bold type), surrounded 5 'with intron 2-3 and 3' with intron 3-4 .

  The fragment 121561-121800 (SEQ ID NO: 7) contains exon 4 (bold type), surrounded 5 'by a 3-4 intron portion and 3' by an intron 4-5 portion. . The fragment 164101-164340 (SEQ ID NO: 8) contains exon 5 (bold type), surrounded in 5 'with a 4-5 intron portion and 3' with a 5-6 intron portion. .

  The last two nucleotides of exon 4 (see SEQ ID NO: 7) are CG (underlined). The first nucleotide of exon 5 (see SEQ ID NO: 8) is G (underlined). In the wild form of the SPATAI6 gene, the last two nucleotides of exon 4 and the first nucleotide of exon 5 form codon CGG, which encodes the amino acid Arg (first amino acid from end C- end of the TPR domain of wild-type SPATA16 protein, see Figure 3).

  A mutation, which has been detected by the inventors in globozoospermal siblings, affects the last nucleotide of exon 4, that is, nucleotide G, which in Figure 5 is presented in a larger character than the other nucleotides of SEQ ID NO: 7. This nucleotide G corresponds to nucleotide G at position 978 of SEQ ID NO: 1 (wild-type SPATA16 cDNA) and at position 848 of SEQ ID NO: 2 (wild-type SPATAI6 CDS). In the mutation detected by the inventors as being segregant of 20 patients with globozoospermia within the studied family, this nucleotide G is mutated in nucleotide A. This mutation prevents the correct splicing of exon 4, which leads to a loss of exon 4, and a direct splicing of exon 3 on exon 5.

  The G> A mutation observed by the inventors in globozoospermic siblings leads to a mutant SPATA16 protein, which has lost the polypeptide portion encoded by exon 4. The resulting mutated SPATAI6 protein is therefore encoded by exons 1-3. and 5-11 of the SPATAI6 gene. The sequence of this mutant protein, and a sequence encoding it, are shown in Figure 8 (SEQ ID NO: 17 and SEQ ID NO: 16, respectively). The sequence encoding this mutant SPATA16 protein results from the direct splicing of exon 3 to exon 5 (CDS of SEQ ID NO: 16). In this CDS sequence of SEQ ID NO: 16, an AGG codon is presented in bold and underlined characters. The first two nucleotides of this codon (AG nucleotides) are the last two nucleotides of exon 3, and the last nucleotide this codon (G) is the first nucleotide of exon 5. The result is a mutant human SPATAI6 protein. of SEQ ID NO: 17, which has 539 amino acids (instead of 569 aa of the wild-type protein): the first 252 amino acids are identical to those of the wild-type human SPATAI6 protein (amino acids coded by exons 1-3) the other 287 amino acids result from the translation of the nucleotide sequence consisting of the last two nucleotides of exon 3 (nucleotides AG) followed by exons 5 to 11 (nucleotide sequence starting with G AGT GCC etc. to AGG TAG ). If the sequence of this mutant human SPATA16 protein is aligned with that of the sequence of the wild-type human SPATA16 protein, a deletion of the 29 amino acid sequence which, in the wild-type SPATA16 protein, extends 254 positions is observed. at 282 (sequence SIVLNPAYFRNHLRQATVFRCLERYSEAA, SEQ ID NO: 14), that is, the polypeptide sequence encoded by exon 4 of SPATA16. The sequence of the mutant human SPATAI6 protein (SEQ ID NO: 17) therefore corresponds to SEQ ID NO: 10 + SEQ ID NO: 15, and a sequence that codes for it (SEQ ID NO: 16) corresponds to SEQ ID NO: 9 + SEQ ID NO: 11.

  The data obtained by the inventors strongly indicate that the homozygous mutation identified in SPATAI6 causes globozoospermia in three of the six brothers of the presented family, which allows the inventors to declare that globozoospermia may be a genetic trait with an autosomal recessive mode of transmission .

  The SPATA16 protein is localized in the Golgi apparatus and in pro-acrosomal vesicles that are transported to the acrosome in round and elongated spermatids during spermiogenesis. The present results support the fact that SPATA16 plays a crucial role in the formation of the acrosome. The strongest conservation within the sequence of this protein is observed in the TPR domain, and the inventors demonstrate that this TPR domain is broken in the case of globozoospermia presented here. The TPR domain is known to mediate protein-protein interactions and assembly of multiprotein complexes. The study of X-ray structure revealed that the wild-type TPR domain adopts a helix-turn-helix arrangement with the ability to associate with other alpha-helical structures. The molecule (s) of interaction may (for example) be: the mouse model GOPC gene, which encodes a protein associated with the Golgi complex, and which contains alpha helices of coiled-coil type motif, and / or the gene of the HIV-1 rev binding protein (HRB), which is also localized at the level of the Golgi complex. Finally, it is important to note that SPATA16 contains six casein kinase II phosphorylation sites, and casease kinase 11a is the most abundant casein kinase in the testes, whose knockout model shows acrosomal defects, as well as other morphological defects. In addition, the existence of several candidate genes suggests the genetic heterogeneity in human globozoospermia, which could be a reason why the inventors have so far not found other patients with gene alteration in SPATAI6.

  It is also important to note that heterozygous mice of the above-mentioned models have no abnormalities in sperm. This indicates that mutation carriers should have normal fertility. In the human family subject to the present study, this seems to be the case, as the father and two heterozygote brothers were respectively fathers of 10, 7, and 6 children. In two of the affected brothers, the inventors performed an ICSI to induce fertilization and pregnancy, but without success. This is consistent with the literature, which shows that ICSI allows fertilization of oocytes, but with low fertilization rates in about half of the cases.

  Insofar as male infertility does not respect the canonical rules of genetics, the determination of heredity patterns and the clarification of genetic causes are complicated. Several genetic factors have been described which affect male fertility, but these give rise to more complex phenotypes. However, the patients in this study did not present any mental or physical abnormalities, and in particular no andrological abnormalities, apart from the aberrant analyzes of their seeds.

  The mutation of SPATA16 that the inventors have here identified presents SPATA16 as a human gene in which mutations give rise to male infertility without any other associated abnormality.

  Additional studies of other patients may lead to identifying other actors involved in the formation of the acrosome, allowing fine dissection of the mechanisms involved in establishing such a specialized cellular organelle. The defects of SPATAI6 influence spermiogenesis, but meiosis is not disturbed. Thus, modulation of SPATAI6 function, or that of other components in the same pathway, offers an innovative reversible approach to male contraception, which has the advantage of not being based on the control of the hormonal pathway of production. sperm.

  Available Internet Resources: UCSC Genome Browser (March 2006 release), http://genome.cse.ucsc.edu/; SDSC Biology Workbench 3.2, Clustel W 1.81, http://workbench.sdsc.edu; NCBI SNP Reference Assembly Online, http://www.ncbi.nlm.nih.gov/SNP/; NCBI protein blast online, http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi ?; Mendelian Inheritance in Man (OMIM) online, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=OMIM; Prediction of splice site, http://www.cbs.dtu.dk/services/NetGene2; Prediction of splicing site, http://www.genet.sickkids.on.ca/ùali/splicesitefinder.html; Prediction of the splice site, http://www.fruitfly.org/seq_tools/splice.html.

Claims (13)

  A method for selecting or producing a candidate compound capable of having a contraceptive effect on the male human, characterized in that it comprises selecting and / or producing a compound which: inhibits, blocks or alters the expression of the human SPATA16 gene, and / or - induces an incorrect splicing of the human SPATAI6 gene, and / or - inhibits, blocks or alters the transcription of the human SPATA16 gene, and / or - inhibits, blocks or alters the translation of the human SPATA16 gene, and / or - inhibits or blocks the intracellular translocation of the human SPATAI6 protein within a spermatocyte and / or human spermatide, and / or - inhibits or blocks the activity of the human SPATA16 gene. human SPATA16 protein, by binding to the TPR domain of this protein and / or by inducing the lysis of this protein.   2. The method according to claim 1, characterized in that said compound induces the loss of an exon during the splicing of the human SPATA16 gene, more particularly the loss of exon 2 and / or 3 and / or 4.   3. The method according to claim 2, characterized in that said compound blocks or inhibits the translation of the human SPATA16 gene.   4. The method according to claim 3, characterized in that said compound is an interfering RNA (RNAi).   5. The method according to any one of claims 1 to 4, characterized in that said compound is a compound which inhibits or blocks the translocation of the human SPATA16 protein to the Golgi apparatus and / or its translocation of the apparatus from Golgi to the acrosome formation site.   6. The method according to claim 1, characterized in that said compound which inhibits or blocks the activity of the human SPATA16 protein, by binding to the TPR domain of this protein and / or by inducing the lysis of this protein, is a A compound which specifically inhibits or blocks the activity of this protein without altering the activity of another human protein.   The method according to any one of claims 1 to 6, characterized in that said compound is a human SPATAI6 gene ligand and / or human SPATAI6 mRNA and / or human SPATA16 protein, or a compound which induces the formation of such a ligand.   8. A method for selecting a compound having a contraceptive effect on a human being, characterized in that it comprises: selecting and / or producing at least one candidate compound according to the method of the invention any of claims 1 to 7, contacting said or each of said candidate compound (s) with at least one human spermatozoid precursor cell, so as to determine whether said candidate compound induces a inhibiting or blocking the formation of the acrosome on this at least one human spermatozoid precursor cell, selecting at least one candidate compound which has the ability to induce such inhibition or blocking, such a compound being a 20 compound for contraceptive effect on the male human being.   9. A method for making a contraceptive effect product on a male human, characterized in that it comprises selecting a contraceptive compound according to the method of claim 8, and the formulation of this compound in a form administrable to humans.   10. A human anti-SPATA16 RNAi, which comprises an antisense nucleic acid strand hybridized to a sense nucleic acid strand, said antisense strand and said sense strand being either unconnected or covalently linked to one another. to one another covalently via a nucleic acid loop, characterized in that: - said antisense strand is comprised of 14 to 200 nucleotides, and 65 - said antisense strand comprises a nucleotide sequence of 14 at 25 nucleotides which has an identity of at least 90% with a nucleotide sequence which is included in the sequence of the gene and / or human SPATAI6 mRNA.   11. The anti-human SPATA16 RNAi of claim 10, characterized in that said antisense strand comprises a nucleotide sequence of 14 to 25 nucleotides which has an identity of at least 90% with a nucleotide sequence which is included in the sequence at least one exon of the human SPATAI6 gene selected from exons 2, 3, 4 of the human SPATAI6 gene.   The human anti-SPATA16 RNAi of claim 10 or 11, characterized in that said antisense strand comprises the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO: 19 or SEQ ID NO: 20.   13. The anti-human SPATA16 RNAi according to any one of claims 10 to 12 as a contraceptive agent for the human male.