Human male fertility control using spata 16

TITLE

0002Controlling the fertility of a male human being via SPATA16

TECHNICAL AREA OF THE INVENTION

0004The present patent application relates to means for the contraception of a male human being, as well as to means for the diagnosis of infertility, or at least of insufficient fertility, in a being human male.

0005The contraceptive means according to the invention implement a reduction in the amount of functional SPATA16 protein present in a male human being, more particularly a negative regulation of the expression of this protein. The diagnostic means of the invention implement the determination of the capacity of a male human being to express a functional SPATA16 protein.

TECHNOLOGICAL BACKGROUND OF THE INVENTION

0007According to IOMS, around 15% of couples face 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 sperm (oligozoospermia), or with inadequate mobility (asthenozoospermia), or with an abnormal morphology (teratozoospermia), or due to 'a combination of these defects.

0008Globozoospermia is a rare but severe teratozoospermia, characterized by ejaculates in which at least 20% of sperm:

0009- have a malformation of the acrosome which prevents or limits their ability to fertilize an ovum, or - are completely devoid of acrosome.

0010These sperm then have a round head. Globozoospermia can be total (in this case, 100% of spermatozoa are affected by a malformation of their acrosome or are devoid of acrosome), or partial (generally, in a proportion of 20% to 90% of sperm). Men affected by total globozoospermia are completely infertile: even the application of intracytoplasmic sperm injection (IICS) has had only low and disappointing success rates. A reference description of globozoospermia can be consulted under OMIM 102530 (OMIM = Online Mendelian Inheritance in Man ™; Johns Hopkins University and NCBI).

0011Globozoospermia arises from 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).

0012However, no causal genetic mutation has been identified in these genes 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).

0013To the knowledge of the inventors, the prior art has not been able to identify any gene mutation which would have a clear link with globozoospermia which affects certain male humans.

0014The inventors demonstrate that the human gene SPATA16 is involved in the morphogenesis of human spermatozoa, and more particularly in the formation of their acrosome. A description of the human SPATA16 gene can be consulted under OMIM 609856. It can in particular be noted there that before the present invention, knowledge concerning the SPATAW gene, more particularly the human SPATA16 gene, was at the very least limited. At most, it was suggested that this gene appeared to be involved in testicular development.

0015The inventors demonstrate that in the absence of a functional SPATA16 protein, or at least in the presence of an insufficient quantity of such a protein, the human sperm 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 completely devoid of acrosome (globozoospermia). The result of this morphological disorder is an incapacity, or at the very least a very diminished capacity, to succeed in fertilizing an ovum, that is to say an infertility, or at least an insufficient fertility of the human being male. In the example of non-functional SPATA16 protein presented by the inventors, all of the sperm produced is free of acrosome.

0016This demonstration allows the 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.

0017In the art prior to the present invention, the means of contraception for the male human being are essentially limited to condoms or vasectomy.

0018A few contraceptive pharmaceuticals have been tested in humans, but none of these have produced satisfactory results. For example, products intended to interrupt the production of sperm have been tested. The vast majority of these test products act hormonally. This is for example the case of:

0019- Gonadoliberine (GN-RH), which has been administered in the form of a tetanus toxoid, and which must be combined with the administration of testosterone substitute (7-alpha methyl-19-nortestoterone, or MENT) so as to compensate symptoms of hypoandrogenism linked to blockage of the hypothalamohypophytes axis,

0020- testosterone derivatives administered in excess, such as injectable testosterone enanthate (ET), which have an effect on the hypothalamohypophisotesticular axis but induce side effects such as decreased high density lipoproteins (HDL) and increased coronary risk ,

0021- androgen-progestogen combinations, such as an ET + levonorgestrel combination, the effects of which on hormonal balance are difficult to control, and for which side effects such as acne and weight gain have also been reported.

0022Products intended to interrupt non-hormonal sperm production have also been tested, but without success. This is for example the case 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 heart rhythm disorders .

0023Products intended to disrupt sperm function have also been tested.

0024For example, nifedipine, which is usually used to treat high blood pressure and migraines, appears to inhibit the release of enzymes that are needed by sperm to enter the pellucid area of ​​the oocyte. However, at doses useful for the desired contraceptive effect, nifedipine also seems to induce dangerously low heart rates. Mifepristone (or RU 486) has also been tested in humans. It would prevent sperm from using calcium, which would disrupt the sperm membrane, and induce a decrease in their motility. However, mifepristone also interferes with certain hormonal functions.

0025Vaccine-based compositions produced from sperm surface proteins (PH20, ZP10, PR34, for example) have also been tested, but without success.

0026Thus, to the knowledge of the inventors, there is in the prior art no pharmaceutical product which would have given sufficiently satisfactory results in terms of efficacy and safety, so that it can be effectively considered as a contraceptive which can be administered at the male human being.

0027The present invention provides new means for the contraception of the male human being, which make it possible to induce a complete inability of human sperm to penetrate an ovum. The contraceptive means of the invention are not directed to the hormonal pathways: they target a protein which is specifically expressed in human testes, namely the protein SPATA16.

0028The 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. Therefore, the contraceptive means of the invention are likely not to induce the undesirable side effects, which have been observed with the contraceptive pharmaceutical products of the prior art, such as inhibition of the secondary sexual characteristics (feminization), erectile dysfunction, loss of libido, disturbed heart rate, increased coronary risk, weight gain. To the knowledge of the inventors, 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

0030The present invention describes the identification of a human gene, the expression product of which is necessary for the establishment of correct sperm function, and more particularly for the production of spermatozoa having the capacity to fertilize an ovum.

0031The inventors demonstrate that the human gene SPATA16 is necessary for the acquisition by human sperm of the capacity to fertilize an egg. The human gene SPATA16 is described under OMIM 609856: it corresponds to positions 174 089 842 to 174 341 700 of the human chromosome 3. A reference sequence is available under ENSG 00000144962 (for example, Ensembl Release 44), or NC_000003. The inventors demonstrate in particular that a mutation in 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 SPATAW 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 the very least have an acrosome whose morphology is sufficiently abnormal for these sperms to be unable to fertilize an egg. In the example of non-functional SPATA16 protein presented by the inventors, all of the sperm produced is free of acrosome. The demonstration of the inventors 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 include the detection of a mutation in the SPATA16 gene, more particularly a mutation leading to a transcription and / or translation defect in this gene, and / or the detection of an altered structure of the protein SPATA16, such detection being indicative of fertility below the average observed in a healthy human individual, or even of infertility.

0032The demonstration of the inventors above all makes it possible to propose means for the contraception of a male human being.

0033The contraceptive means of the invention include in particular a product which can be administered to a male human being, and which is capable of altering or inhibiting the protein function of SPATA16 and / or of decreasing the amount of functional SPATA16 protein present in a male human being, more particularly capable of inhibiting the expression of the SP ATA 16 gene.

0034The present application relates to such a contraceptive product, as well as methods for identifying and / or producing such a contraceptive product.

BRIEF DESCRIPTION OF THE FIGURES

0036FIGURES 1A to 1 D: human family, presenting a globozoospermia and within which a mutation at the level of the SPATAW gene has been identified FIG. 1A: Morphology of sperm. Acrosin fluorescent staining (green FITC) of the acrosomes, and DAPI staining (blue) of the nuclei. On the left, a sample from a fertile witness. Right, from a sample of a patient with globozoospermia. The sperm morphology and acrosome structures are severely disrupted in the patient's cells. Residues of acrosin staining have been observed in some deformed cells of the patient's sperm, but most of the signals correspond to non-specific acrosin staining in leukocytes.

0037FIG. 1 B: Chromatograms of the mutation. The sequences of a patient, his father (heterozygote) and a witness are presented. FIG. 1 C: The recognition site Nci \ (5'-CCCGG-3 ') is lost due to the mutation G> A at the level of a nucleotide of exon 4. The recognition site of HPall is 5' -CCGG-3 '. This mutation can result in a substitution of the amino acid R283Q, as well as the rupture of the 5 'splice site of intron 4. FIG. 1 D: Genealogy of an Ashkenazi Jewish family that was the subject of this study. The order of ten siblings is arbitrary. The segregation of the mutation was studied by estion / c / 1 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 sibling does not carry this mutation. The three male siblings affected by globozoospermia (three black squares) are homozygous for this mutation. The witness does not carry the mutation.

0038FIGURES 2A to 2C: splicing site of exon 4 SPATA16 of the mutated donor and Ul SnRNP binding for the splicing site of wild type and of the mutant donor FIG. 2A: schematic presentation of minigene constructions which 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 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, while the wild type exon 4 is clearly recognized; the identity of the U1 snRNP was confirmed by treatment of RNAse H using an oligodeoxynucleotide complementary to the nucleotide positions 1 to 15 of U1 snRNA.

0039FIGURE 3: alignment of SPATA 16 sequences of different animal species (CLUSTAL W (1.81) multiple alignment of sequences) Consensus key

0040^* - single residue, highly conserved

0041: - conservation of strong groups

0042. - conservation of weak groups - no consensus Large gray rectangle = TPR domain.

0043In the human SPATA16 sequence, the TPR domain extends from the amino acid

0044186 (Cys) included with amino acid 281 (AIa) included.

0045Vertical gray stripe = altered amino acid (amino acid R).

0046In the human SPATA16 sequence, the altered amino acid is in position 283 (Arg). It therefore corresponds to the first amino acid in 5 'from the end

0047C-terminal of the TPR domain.

0048Black frame = directly affected part (fully coded by exon 4).

0049In the human SPATA16 sequence, the amino acids which are entirely coded by exon 4, and which are therefore directly affected, extend from position 253 (Arg) included to position 282 (AIa) included. The codon of the amino acid Arg at position 283 overlaps exon 4 and exon 5; it is this codon which is affected by the G848A mutation.

0050FIGURE 4: cDNA sequences of the wild human SPATAW gene (SEQ ID NO: 1), of the CDS of this gene (SEQ ID NO: 2), of the wild human SPATA16 protein (SEQ ID NO: 3), of the CDS of the TRP domain of wild human SPATA16 (SEQ ID NO: 4), and of the wild human TRP polypeptide (SEQ ID NO: 5). FIGURE 5: sequences of the fragments 92041 -92340 (SEQ ID NO: 6), 121561 - 121800 (SEQ ID NO: 7) and 164101 -164340 (SEQ ID NO: 8) of the sequence of the wild human SPATA16 gene.

FIGURE 6:

0052- sequence formed by the splicing of exons 1 to 3 of the wild human SPATA16 gene (SEQ ID NO: 9), and polypeptide sequence encoded by exons 1 to 3 (SEQ ID NO: 10),

0053- sequence formed by the splicing of exons 5 to 1 1 of the wild human SPATA 16 gene (SEQ ID NO: 11), and polypeptide sequence encoded by exons 5 to 1 1 (SEQ ID NO: 12).

FIGURE 7:

0055- sequence of exon 4 of the wild human SPATA16 gene (SEQ ID NO: 13), and polypeptide sequence encoded by this exon 4 (SEQ ID NO: 14),

0056- polypeptide sequence encoded by the splicing of the last two nucleotides of the exon of the wild human SPATA16 gene and of exons 5 to 11 of this gene (SEQ ID NO: 15).

FIGURE 8:

0058- CDS sequence (coding sequence) of the SPA TA 16Δexon4 gene, that is to say of the mutated human gene SPATA 16 observed in globozoospermic siblings (SEQ ID NO: 16),

0059- sequence of the mutant SPATA16 protein, coded by the mutated SPATAW gene {SPATA 16Aexor \ 4) (SEQ ID NO: 17).

DETAILED DESCRIPTION OF THE INVENTION

0061The inventors demonstrate that at least in humans, the protein SPATA16 is necessary for the formation of the acrosome of spermatozoa, more particularly for the formation of an acrosome of sufficiently normal morphology to allow sperm to penetrate a ovum. In the absence of a 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 that does not allow the sperm to penetrate an egg.

0062In the example of non-functional SPATA16 protein presented by the inventors, all of the sperm produced is free of acrosome.

0063The inventors demonstrate that the globozoospermia of certain patients is caused by a mutation in their SPATA16 gene. The mutated SPATA16 gene expresses a mutant SPATA16 protein. An example of such a mutation is described in Example 1 below. In this example of mutation, exon 4 of the mutated SPATA16 gene is no longer spliced ​​correctly, and this leads to a mutated SPATA16 mRNA in which exon 3 is directly spliced ​​on exon 5. The mutant SPATA16 protein produced by patients carrying this mutated SPATA16 gene has lost the polypeptide part encoded by exon 4 of the wild-type gene, which leads to a SPATA16 protein which, unlike the wild-type protein, does not include a functional TRP domain. (tetratricopeptide repeat domain).

0064The inventors thus demonstrate that when the protein SPATA16 is non-functional, the sperm produced have no functional acrosome, that is to say that they are at least provided with an acrosome whose morphology is sufficiently abnormal to prevent them from successfully fertilizing an egg, or that they are entirely devoid of acrosome. The proportion of sperm affected is at least 20%, and can reach 100% in the case of total globozoospermia. In the example of mutation presented in detail in example 1, all of the sperm produced by the patients affected by this mutation is completely free of acrosome.

0065The patients affected by this mutation do not suffer from any other physical or mental anomaly.

0066The inventors thus demonstrate that globozoospermia can, at least in certain cases, be a genetic trait with an autosomal recessive mode of transmission. To the knowledge of the inventors, the present invention provides the first description of a gene directly involved in the pathogenesis of human globozoospermia.

0067As such, the present application relates to mutant polynucleotide, polypeptide, and protein products, as well as to diagnostic and prognostic applications, which comprise the detection of at least one mutation of the human SPATA16 protein and / or of at least one mutation in the gene and / or

0068SPATA 16 human mRNA capable of leading to the expression of a protein

0069SPATA16 human mutant.

0070That said, an essential aspect of the present invention is the demonstration that a human protein exists:

0071- 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 sperm, so that in l absence of this functional protein, or at least in the presence of an insufficient quantity 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 ovum, advantageously is devoid of acrosome, and

0072- 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

0073- which is not a hormone, and which does not interact, or at least not directly, on the patient's hormonal pathways.

0074This human protein is the SPATA16 protein.

0075These remarkable characteristics make this protein a prime target for the development of a contraceptive product intended for the male human being.

0076The present application relates to a mutant SPATA16 protein. A mutant SPATA16 protein of the invention comprises, or consists of an amino acid sequence which is capable of deriving from that of a wild human SPATA16 protein by deletion and / or substitution and / or addition of at least one amino acid.

0077Compared with a wild human SPATA16 protein, a mutant SPATA16 protein of the invention has lost the capacity to participate in, or to induce, the formation of an acrosome, and in particular the formation of a functional acrosome.

0078The expression “functional acrosome” is understood in its ordinary sense in the matter, namely an acrosome which allows the sperm to succeed in penetrating an ovum of the same animal species, more particularly the pellucid zone of this ovum.

0079The inability to participate in, or induce, the formation of a functional acrosome results in the fact that the spermatogenic cell (sperm precursor cell) fails to evolve into a functional sperm structure, i.e. -to say that it fails to develop into a sperm capable of fertilizing an egg.

0080The loss of this capacity can for example be observed by transfecting or infecting a spermatogenic cell (that is to say a sperm precursor cell) by a nucleic acid coding for this mutant protein so that this mutant protein is expressed by the transfected or infected spermatogenic cell.

0081This spermatogenic cell can for example be chosen from spermatogonia, spermatocytes, spermatids, preferably from spermatocytes and spermatids.

0082This spermatogenic cell can be a spermatogenic cell of a non-human animal, in particular a spermatogenic cell of a non-human mammal, such as a mouse spermatogenic cell.

0083This sperm cell can be a sperm cell that has been isolated from the human body. Preferably, this sperm cell is a human sperm cell.

0084The person skilled in the art can choose to implement a spermatogenic cell whose expression of the wild SPATAW gene is blocked, for example by “knockout” of the wild SPATAW gene (for example by homologous recombination), or by transfection of antisense oligonucleotides of the wild SPATAW gene, and transfecting or infecting this silent wild SPATAW gene spermatogenic cell with a nucleic acid which codes for a mutant candidate SPATA16 protein so that this mutant candidate protein is expressed in the spermatogenic cell. The spermatogenic cell thus treated can be placed in 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 being a mutant protein of the invention. The disturbance of the formation of a functional acrosome can alternatively or additionally be observed by observing a disturbance of the intracellular mechanisms which accompany this formation of acrosome, such as an absence or an inhibition of the fusion of the acrosomal vesicles, an absence or a inhibition of acrosome docking on the nuclear surface, an anomaly in the placement of the axoneme.

0085Alternatively or additionally, the person skilled in the art can choose to test a candidate mutant protein at a more mechanistic level of the spermatogenic cell.

0086For example, after transfection or infection of the spermatogenic cell, the person skilled in the art can determine whether the candidate mutant protein has lost at least one of the following capacities: capacity to be localized in the Golgi apparatus of said spermatogenic cell,

0087- ability to be located in the pro-acrosomal vesicles of said spermatogenic cell,

0088- Ability to be located in the acrosome, where appropriate, the candidate mutant protein can be identified as being a mutant protein of the invention.

0089To carry out tests to verify the capacity or capacities of a mutant SPATA16 protein of the invention, the person skilled in the art can choose to couple it to a marker allowing its detection, such as for example a GFP molecule (Green Fluorescence Protein) . Preferably, a mutant SPATA16 protein of the invention comprises, or consists of an amino acid sequence which is capable of deriving from that of a wild human SPATA16 protein by mutation of the TPR domain of the wild human SPATA16 protein, said mutation comprising deletion and / or substitution of at least one amino acid in this TPR domain and / or addition of at least one amino acid in this TPR domain, said mutation being selected such that the resulting mutant SPATA16 protein does not induce the formation of an acrosome, or at the very 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.

0090Preferably, a mutant SPATA16 protein of the invention comprises, or consists of an amino acid sequence which is capable of deriving from that of a wild human SPATA16 protein by one (or more) operation (s) of deletion and / or substitution and / or addition of at least one amino acid, which comprises (comprise) at least the deletion of a part of the sequence of the wild human SPATA16 protein, advantageously the deletion of all or part of its TPR domain.

0091Advantageously, a mutant SPATA16 protein of the invention comprises, or consists of an amino acid sequence which is capable of deriving from that of a wild human SPATA16 protein by deletion of a part of the TPR domain of the wild human SPATA16 protein.

0092The part of the TPR domain subject to the deletion may be any part which leads the resulting mutant protein to lose one of the capacities described above, and / or to exhibit one of the disabilities described above. For example, the part of TPR domain object of the deletion may correspond to all or part of the region of the TPR domain which is coded by exon 2, or to all or part of the region coded by exon 3, or to all or part of the region coded by exon 4, or a combination of at least two of these elements, such as for example a part of the region coded by exon 3 and a part of the region coded by l 'exon 4.

0093Advantageously, the part of the TPR domain which is the subject of the deletion is a part which comprises, or which is, that coded by exon 4.

0094In FIG. 4, a sequence of the cDNA of the wild human SPATA16 gene is presented under SEQ ID NO: 1. The corresponding coding sequence (CDS) is presented there under SEQ ID NO: 2, and the sequence of the wild human SPATA16 protein corresponding under SEQ ID NO: 3.

0095A coding sequence of the TPR domain of the wild human SPATA16 protein is presented in FIG. 4 under SEQ ID NO: 4.

0096This coding sequence extends from positions 686 to 973 of the cDNA sequence of SEQ ID NO: 1, which corresponds to positions 556 to 843 of the CDS sequence of SEQ ID NO: 2 (cf. FIG. 4). The coding sequence of the wild-type TPR domain consists of a 3 ′ part of exon 2, of exon 3 and of almost all of exon 4 (cf. table 6 below, for the positions of exons).

0097A polypeptide sequence of the TPR domain of the wild human SPATA16 protein is presented in Figure 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 (cf. Figure 4 ).

0098In FIG. 4, the part of the TPR domain of the wild human SPATA16 protein which is encoded by exon 4 extends from positions 253 to 282 of the protein sequence of SEQ ID NO: 3. The amino acid sequence of this part of domain TPR is presented in Figure 4 under SEQ ID NO: 14. Its coding sequence is that of exon 4, which is presented in Figure 4 under SEQ ID NO: 13.

0099Advantageously, the sequence of the mutant protein of the invention comprises, or is, that of SEQ ID NO: 17.

0100Advantageously, the mutant nucleic acid sequence of the invention comprises, or is, that of SEQ ID NO: 16.

0101The present application also relates to the nucleic acids which code for such a mutant SPATA16 protein according to the universal genetic code, taking into account the degeneration of this code, and to the nucleic acids whose sequence is complementary to such a nucleic acid coding for the entire length of this encoding nucleic acid.

0102These nucleic acids can 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 human SPATA16 protein by a mutation which comprises the substitution and / or deletion of at least at least one nucleotide, and / or by adding 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 the very least does not induce the formation of a functional acrosome, on a spermatogenic cell (preferably a human spermatogenic cell) whose expression of the wild-type SPATA 16 gene has been blocked.

0103By analogy to the elements described above under the mutant proteins of the invention, a mutant nucleic acid of the invention can for example be characterized in that its sequence comprises, or consists of a sequence which is capable of derive from a nucleotide sequence which comprises a sequence coding for the wild human SPATA16 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

0104deletion of a sequence encoding part of the TPR domain of the wild 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 capacities mentioned above, and / or o has at least one of the above-mentioned disabilities.

0105By analogy to the elements described above under the mutant proteins of the invention, said deletion of a sequence encoding part of the TPR domain of the wild human SPATA16 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 the very least does not induce the formation of a functional acrosome, in a spermatogenic cell whose expression of the wild-type SPATA 16 gene has been blocked.

0106An example of such a sequence encoding a part of the TPR domain which can be deleted is the sequence of exon 2 and / or exon 3 and / or exon 4 of the wild human SPATA16 gene, preferably exon 4 , such as the sequence of SEQ ID NO: 13.

0107An example of such a mutant nucleic acid of the invention which no longer comprises the sequence of exon 4 of the wild human SPATAW 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 human SPATA16 gene (nucleotide G replaced by a nucleotide A). As presented in Example 1 below, a SPATA 16 gene thus mutated undergoes incorrect splicing of exon 4, at the end of which exon 3 is found directly spliced ​​on exon 5. The mRNA or cDNA which results from the transcription of such a mutated SPATA 16 gene has therefore lost exon 4.

0108A mutant nucleic acid of the present invention may therefore comprise, or be made up of a sequence capable of deriving from the genomic DNA sequence of the wild human SPATA 16 gene by a mutation which comprises:

0109the substitution and / or deletion of at least one nucleotide of an exon encoding part of the TPR domain, and / or

0110- the addition of at least one nucleotide in such an exon. Preferably, said at least one exon is exon 4 of the wild human SPATA16 gene. Advantageously, said at least one nucleotide of exon 4 is the last nucleotide of exon 4.

0111This mutation can be selected so that the splicing of an exon encoding part of the TPR domain, such as exon 2 and / or 3 and / or 4, cannot be done in the order observed on the wild human SPATA 16 gene.

0112This is notably the case of a substitution of a nucleotide of exon 4 which leads to the splicing of exon 3 directly onto exon 5, such as the substitution of the last nucleotide of exon 4 of the gene SPATA16 human.

0113A mutant nucleic acid of the invention can be DNA (genomic DNA, cDNA, single-stranded DNA) or RNA (notably mRNA). Preferably, it is a cDNA or an mRNA.

0114Such a nucleic acid may be in free form, or else in the form inserted into a transfection, transformation or infection vector, and in particular into an expression vector, advantageously a vector comprising the regulatory sequences necessary for expression in a prokaryotic cell. such as £. coli, or preferably in a eukaryotic cell such as that HeLa or COS or a spermatid.

0115The 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 can be a genetically modified cell, for example by transfection, transformation or infection.

0116The 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.

0117The present application also relates to any solid support, and in particular to any solid support suitable for the circulation of a microfluidic flux, such as a chip, on which is fixed at least one mutant protein of the invention and / or at least one mutant nucleic acid of the invention.

0118The 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 human SPATA16 protein of SEQ ID

0119NO: 3. Advantageously, such an antibody is specific for one or more mutant protein (s) of the invention.

0120Techniques for producing antibodies by immunizing a mammal are known to those of skill in the art. Techniques for producing monoclonal antibodies are also known to those skilled in the art (Kohler and

0121Milstein (1975) Nature 256: 495-497; US Patent No. 4,376,110; Kosbor et al.

0122(1983) Immunology Today 4:72; CoIe et al. (1983) Proc. Natl. Acad. Sci. USA

012380: 2026-2030, CoIe et al. (1985) Monoclonal Antibodies And Cancer Therapy,

0124Alan R. Liss, Inc., pp. 77-96).

0125The present application also relates to any hybridoma capable of producing a monoclonal antibody of the invention.

0126The present application also relates to means for: - the diagnosis or prognosis of a human teratozoospermia, more particularly of a human globozoospermia, - the diagnosis of infertility of a male human being, or of a fertility of such a human being which is insufficient to allow spontaneous conception by natural means,

0127- determine the existence of a predisposition to human teratozoospermia, and more particularly to human globozoospermia.

0128These means include methods and kits.

0129The means of the invention comprise means for determining whether a sample likely to contain spermatozoa from the patient, such as a sample of sperm or testicular tissue, contains SPATA16 proteins which have a mutant structure and / or a mutant sequence, with respect to the structure and / or sequence of the wild human SPATA16 protein (i.e., the SPATA16 protein produced by fertile male humans), respectively.

0130If the mutant SPATA16 proteins detected have a structure and / or a sequence sufficiently different from that of wild-type human SPATA16 protein not to be functional, for example sufficiently different to have lost the capacity to induce the formation of an acrosome on a spermatogenic cell (preferably a human spermatogenic cell) whose expression of the wild SPATA16 gene has been blocked:

0131- a diagnosis of human teratozoospermia, and more particularly of human globozoospermia, and / or

0132- a diagnosis of infertility, or of insufficient fertility to allow spontaneous conception by natural means, and / or

0133- the existence of a predisposition to human teratozoospermia, and more particularly to human globozoospermia, can (can) be pronounced.

0134The means of the invention may include means for determining whether a sample capable of containing sperm from the patient, such as a sample of sperm or testicular tissue, contains at least one protein

0135SPATA16 mutant of the invention.

0136A positive determination being indicative:

0137- human teratozoospermia, and more particularly human globozoospermia, and / or

0138- infertility, or insufficient fertility to allow spontaneous conception by natural means, and / or

0139- a predisposition to human teratozoospermia, and more particularly to human globozoospermia.

0140Alternatively or additionally, the means of the invention may include means for determining whether a sample likely to contain spermatozoa from the patient, such as a sample of sperm or testicular tissue: - does not contain a wild human SPATA16 protein, that is to say does not contain a SPATA16 protein whose sequence and / or tertiary structure would be that (would be those) of a wild human SPATA16 protein (such as the protein wild human of SEQ ID NO: 3),

0141- contains such a wild human SPATA16 protein in an amount less than the amount normally observed in a fertile male subject.

0142A positive determination is indicative:

0143- 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 conception spontaneously via the natural route within two years of stopping all contraception), and / or - a predisposition to human teratozoospermia, and more particularly to human globozoospermia. To determine whether a sample does not contain a wild human SPATA16 protein, or if it contains insufficient protein, the person skilled in the art can use any suitable detection reagent (in particular an antibody, more particularly a monoclonal antibody) specific for the protein SPATA16 wild human.

0144Such a reagent must be sufficiently specific for the wild human SPATA16 protein, for example the protein of SEQ ID NO: 3, not to exhibit a cross-reaction with a mutant SPATA16 protein of the invention, in particular with a mutant SPATA16 protein of the invention which has lost all or part of the TPR domain, for example the mutant protein of SEQ ID NO: 17.

0145The present application therefore relates to such a detection reagent for its use as a reagent for determining the existence of: - a human teratozoospermia, and more particularly a human globozoospermia, and / or

0146- infertility, or insufficient fertility to allow spontaneous conception by the natural way (or at least insufficient fertility to allow spontaneous conception by the natural way in the two years following the end of any contraception), and or

0147- a predisposition to human teratozoospermia, and more particularly to human globozoospermia.

0148To determine whether 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 protein ( s) mutant (s) of the invention. Such a reagent must be sufficiently specific for the mutant protein (s) of the invention (such as one or more SPATA16 protein (s) which has (have) lost all or part of its TPR domain, by example a protein of SEQ ID NO: 17) so as not to exhibit a cross-reaction with a wild human SPATA16 protein (such as a protein of SEQ ID NO: 3). The present application therefore relates to such a detection reagent for its use as a reagent for determining the existence of: - a human teratozoospermia, and more particularly a human globozoospermia, and / or

0149- infertility, or insufficient fertility to allow spontaneous conception by the natural way (or at least insufficient fertility to allow spontaneous conception by the natural way in the two years following the end of any contraception), and or

0150- a predisposition to human teratozoospermia, and more particularly to human globozoospermia.

0151A kit of the invention may in particular comprise at least one specific detection reagent for a wild human SPATA16 protein and / or at least one specific detection reagent for a mutant protein of the invention. This or these reagents can (can) 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.

0152This or these reagents can (can) be in free form, or else fixed to a solid support such as for example fixed to an ELISA plate or to a transfer membrane.

0153A diagnosis of this type can, alternatively or additionally, be established via a nucleic acid encoding the human SPATA16 protein. Tests for the diagnosis of human astenozoospermia, and more particularly of human globozoospermia, may thus usefully include means for determining whether the SPATAW gene un'a male human being and / or if SPATA16 mRNA of such a human being has a mutant sequence with respect to the gene and / or I¹SPATA16 wild-type human mRNA (i.e. in relation to the SPATA 16 gene and / or mRNA of fertile male humans). If the detected mutation is likely to lead to the expression of mutant SPATA16 proteins, whose structure and / or sequence differs sufficiently from that (s) of the wild human SPATA16 protein not to be functional, or if the detected mutation is likely to lead to an absence of expression of the protein SPATA16, a diagnosis of human teratozoospermia, and more particularly of human globozoospermia, can be pronounced.

0154The means of the invention can therefore include means for determining whether the patient, or at least a biological sample from said patient:

0155does not have a SPATA16 gene and / or a SPATA16 mRNA whose sequence and / or structure would be that of the wild human SPATA16 gene and / or of the wild human 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

0156- Has a mutant nucleic acid of the invention, in particular a mutant SPATA16 gene and / or an mRNA of the invention. An example of a mutant gene of the invention comprises for example a gene whose sequence is capable of deriving from that of the wild human SPATA16 gene by a mutation which comprises the substitution and / or the deletion of at least one nucleotide of an exon encoding a part of the TPR domain, for example of 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 incorrect splicing of the resulting SPATAW gene, such as for example splicing of exon 3 directly on exon 5.

0157An example of a mutant mRNA of the invention comprises an mRNA whose sequence is capable of deriving from that of wild human SPATA16 mRNA by a mutation which comprises the deletion of all or part of at least one exon among exons 2 , 3 and 4, preferably at least exon 4. An example of mutant DNA of the invention comprises a DNA whose sequence is capable of deriving from that of the wild human SPATA16 gene by a mutation which includes the substitution of the last nucleotide of exon 4 of the wild human SPATA16 gene (nucleotide G ), for example to replace this nucleotide with nucleotide A.

0158A positive determination is indicative:

0159- 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 conception spontaneously via the natural route within two years of stopping all contraception), and / or - a predisposition to human teratozoospermia, and more particularly to human globozoospermia.

0160A positive determination is also also indicative of an infertility of said patient, or of a fertility of said patient which is insufficient to allow spontaneous conception by natural means (or at least spontaneous conception by natural means within a time two years after stopping all contraception).

0161To determine whether the patient, or at least a biological sample from the patient, does not contain nucleic acid encoding a wild human SPATA16 protein, such as for example a gene and / or a wild human SPATA16 mRNA, and optionally which quantity (for example which quantity of wild human SPATA16 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 complementary to said coding nucleic acid over the entire length of the sequence of said coding nucleic acid. Advantageously, such a specific reagent is a nucleic acid probe.

0162Preferably, 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

0163NO: 1 or 2, or of the sequence which is its complementary over the entire length of this sequence of SEQ ID NO: 1 or 2.

0164Such a specific reagent does not cross-hybridize with a mutant nucleic acid of the invention nor with a nucleic acid whose sequence is complementary to a mutant nucleic acid of the invention over the entire length of this mutant nucleic 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 the complementary thereof over the entire length of this sequence of SEQ ID NO: 1 or 2, and which does not show a cross-reaction with the mutant nucleic acid of SEQ ID NO: 16, or with the sequence which is its complementary over the entire length of this sequence of SEQ ID NO: 16.

0165The 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:

0166- human teratozoospermia, and more particularly human globozoospermia, and / or

0167- infertility, or insufficient fertility to allow spontaneous conception by the natural way (or at least insufficient fertility to allow spontaneous conception by the natural way in the two years following the end of any contraception), and or

0168- a predisposition to human teratozoospermia, and more particularly to human globozoospermia, and / or to determine the existence of infertility of said patient, or of fertility of said patient which is insufficient to allow spontaneous conception by the natural routes (or at least spontaneous conception by natural routes within two years of stopping all contraception).

0169To determine whether the patient, or at least a biological sample from the patient, contains a mutant nucleic acid of the invention, such as for example a DNA and / or a mutant mRNA of the invention, the person skilled in the art can use any reagent allowing the specific detection of such a mutant nucleic acid or of a sequence which is complementary to such a mutant nucleic acid over the entire 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.

0170The person skilled in the art can 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 over the entire length of this sequence of SEQ ID NO: 16 . Such a specific reagent does not cross-hybridize with a nucleic acid coding for a wild human SPATA16 protein or with the sequence which is the complement of this coding sequence over the entire sequence of this coding sequence. For example, such a specific reagent is a probe which is specific for a nucleic acid comprising the sequence of SEQ ID NO: 16 or of the sequence which is the complementary thereof over the entire length of this sequence of SEQ ID NO: 16, and which does not exhibit cross-hybridization with the nucleic acid of SEQ ID NO: 2 or with the sequence which is the complement of 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:

0171- human teratozoospermia, and more particularly human globozoospermia, and / or

0172- infertility, or insufficient fertility to allow spontaneous conception by the natural way (or at least insufficient fertility to allow spontaneous conception by the natural way in the two years following the end of any contraception), and or

0173- a predisposition to human teratozoospermia, and more particularly to human globozoospermia, and / or to determine the existence of infertility of said patient, or of fertility of said patient which is insufficient to allow spontaneous conception by the natural routes (or at least spontaneous conception by natural routes within two years of stopping all contraception).

0174A kit of the invention can in particular comprise at least one nucleic acid probe specific for a nucleic acid coding for a wild human SPATA16 protein or the nucleic acid complementary to this coding nucleic acid, and / or at least one specific nucleic acid probe of a mutant nucleic acid of the invention or of the nucleic acid complementary to this mutant nucleic acid.

0175Such probes are intended to be placed in contact with the nucleic acids of the sample under conditions favorable to their hybridization to their target sequence (s).

0176This contacting can for example be done according to the transfer techniques of Northern ("Northern blot"), Northern reverse ("reverse Northern blot") or Southern ("Southern blot"). Alternatively or additionally, such probes can be fixed on a solid support, in particular a solid support suitable for the circulation of a microfluidic flow, such as a nucleic acid chip.

0177If desired or required, the nucleic acids can be purified from the sample before being contacted with one and / or the other of the two types of probes.

0178Alternatively or additionally, the nucleic acids in 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 polymerase the SPATA16 sequence which these primers frame. Such primers thus make it possible to amplify the sequence of the human SPATAW gene present in the test sample, or at least a fragment of this gene. The amplified sequence can then be directly sequenced so as to determine whether this sequence is that of a gene or of a wild human SPATA16 gene fragment, or else that of a mutant gene or gene fragment in accordance with 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 combination with this probe, so as to detect using this probe the product of amplification capable of being obtained with said primers. The primers and the probe can be used successively, or simultaneously, for example in the context of real-time PCR.

0179The present application thus relates to pairs of amplification primers, the sequences of which hybridize to the sequence of the gene or of I¹Wild human SPATA16 mRNA or on the sequence complementary to 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 sequence complementary to 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 not be specific (s). Preferably, however, such primers are specific to wild-type and mutated forms of the human SPATA16 gene or mRNA, i.e., they hybridize to the sequence of the human SPATA16 gene or mRNA wild-type 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 hybridizing to a other human nucleic acid.

0180These primers can be used alone, or else in combination with at least one probe of the invention.

0181These primers are, in accordance with the present invention, intended to be used to determine the existence of: - human teratozoospermia, and more particularly human globozoospermia, and / or

0182- infertility, or insufficient fertility to allow spontaneous conception by the natural way (or at least insufficient fertility to allow spontaneous conception by the natural way in the two years following the end of any contraception), and or

0183- a predisposition to human teratozoospermia, and more particularly to human globozoospermia, and / or to determine the existence of infertility of said patient, or of fertility of said patient which is insufficient to allow spontaneous conception by the natural routes (or at least spontaneous conception by natural routes within two years of stopping all contraception).

0184A 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 of the invention, and at least one probe of each of the two types of probes of the invention.

0185The conditions for hybridization of a probe or a primer of the invention are preferably hybridization conditions of high stringency.

0186Such high stringency conditions can be adjusted by the person skilled in the art, in particular depending on the hybridization technique used.

0187As an illustration, an example of high stringency conditions includes hybridization on a transfer membrane in 5xSSC, 2% sodium dodecyl sulfate (SDS), 100 micrograms / mL of 55- single-stranded nucleic acid.

018865 ° C for 8 hours, and washing in 0.2xSSC and SDS 0.2% at 60-65⁰C for thirty minutes.

0189By way of illustration, an example of conditions of high stringency more particularly suited to amplification primers comprises hybridization in the presence of polymerase at an annealing temperature of 15 to 5 ° C. lower than the melting temperature (Tm) of the primers tested.

0190The present invention also proposes new means for the contraception of the male human being.

0191The contraceptive means of the invention have the effect of inducing the inability of human spermatozoa to penetrate an ovum. More specifically, the means of the invention are means which block, inhibit or alter the formation of the acrosome of human sperm to a sufficient extent to induce the desired contraceptive effect.

0192The contraceptive means of the invention act at the level of the human SPATA16 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 SPATAW gene, and / or - induce an incorrect splicing of the human SPATAW gene (or of the pro-RNA), and / or

0193- inhibit or block the intracellular translocation of the human SPATA16 protein inside a human spermatogenic cell (said human spermatogenic cell preferably being a human spermatocyte and / or a human spermatid), and / or

0194- inhibit or block 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.

0195The contraceptive means of the invention are not directed towards the hormonal pathways: their mode of action targets a protein which is specifically expressed in human testes, and more particularly in the adult germ cells of human testes, namely the protein SPATA16. The means according to the invention have a molecular target in human beings (SPATA16) 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 the undesirable side effects, which could have been observed with the contraceptive pharmaceutical products of the prior art, such as inhibition of the secondary sexual characteristics (feminization), erectile dysfunction, loss of libido, disturbed heart rate, increased coronary risk, weight gain.

0196The method of the invention aims to select and / or produce a compound which disturbs the normal expression of the wild human SPATA16 gene to an extent sufficient for at least a proportion of at least 20%, preferably of at least 90%, preferably at least 95%, advantageously 100% of the human spermatozoa are devoid of acrosome, or are provided with an acrosome of abnormal morphology, which do not allow them to fertilize a female ovum. The present application thus relates to a method for selecting and / or producing a compound capable of having a contraceptive effect on the male human being, and / or for selecting and / or producing a compound capable of inhibiting or blocking the formation of the acrosome of a human spermatozoon, said method comprising the selection and / or the production of a compound:

0197- which inhibits, blocks or alters the expression of the human SPATA 16 gene, and / or

0198- which induces an incorrect splicing of the human SPATA16 gene (or of the pro-RNA), and / or - which inhibits, blocks or alters the transcription of the human SPATA16 gene, and / or

0199- which inhibits, blocks or alters the translation of the human SPATA16 gene, and / or

0200- which inhibits or blocks the intracellular translocation of the human SPATA16 protein inside a human spermatogenic cell (said human spermatogenic cell preferably being a human spermatocyte and / or a human spermatid), and / or

0201- 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.

0202According to the present invention, one can select and / or produce a compound which alters the expression of the human SPATA16 gene, i.e. a compound which alters the expression of the human SPATA16 gene to an extent sufficient to alter the formation of the acrosome of a sperm, preferably a human sperm. Such a compound can in particular have the effect of altering the formation of the acrosome, preferably of preventing it, when it is placed in contact with at least one human spermatogenic cell, preferably with at least one human spermatocyte and / or at least one human spermatid.

0203Such a compound is for example a compound which alters the transcription and / or translation of the human SPATA 16 gene. Preferably, it is a compound which induces incorrect splicing of the human SPATA16 gene. The human SPATA16 gene includes eleven exons. When the human SPATAW gene is correctly spliced, 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 splicing is a splicing which does not respect this order, for example by splicing exon 3 directly on exon 5, which has the effect of eliminating exon 4 from the series of spliced ​​exons, or any other splicing which 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 SPATA16 gene, encodes the TPR domain of the human SPATA16 protein.

0204A compound selected and / or produced by the method according to the invention blocks or inhibits the formation of the acrosome of a human spermatozoon, advantageously without otherwise disturbing spermatogenesis.

0205In accordance with the present invention, it is possible to select and / or produce a compound which blocks or inhibits the expression of the human SPATA16 gene, more particularly which blocks or inhibits its translation, that is to say the translation of

0206Human SPATA16 mRNA (cDNA sequence or CDS sequence) in protein

0207SPATA16 human.

0208Such 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 to mRNA of the human SPATA16 gene, or which contains an element capable of binding to such DNA or mRNA, and which:

0209- interferes with the transcription of the human SPATA16 gene, that is to say which blocks, inhibits or alters the transcription of the human SPATAW gene, and / or

0210- interferes with the translation of the mRNA of the human SPATA W gene, that is to say which blocks, inhibits or alters the translation of this mRNA (sequence

0211CDNA or CDS sequence). A DNA or RNA interferant (iDNA or RNAi) ar \\\ - human SPATA 16 inhibits or blocks the production of the protein SPATA16 by a human spermatogenic cell (said human spermatogenic cell preferably being a human spermatocyte and / or a spermatid human). 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 sufficiently abnormal morphology not to allow penetration of a female ovum. An example of such a compound is an RNA interferant which cleaves or degrades PARNm of the human SPATA16 gene within a human spermatogenic cell, and / or by repressing the translation of this mRNA. Such an RNA interferant can for example be a human anti \ -SPATA16 “small interfering RNA”, or a human anti-SP> AL416 “shRNA” (short hairpin RNA). Examples of such DNA or RNA interferers are described in more detail below.

0212According to the present invention, it is possible to select and / or produce a compound which inhibits or blocks the intracellular translocation of the human SPATA16 protein inside a human spermatogenic cell (said human spermatogenic cell preferably being a human spermatocyte and / or a human spermatid). An example of such a compound is a compound which 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,

0213- the transfection, transformation or infection of this cell with the human SPATA16 gene, preferably a wild human SPATA16 gene, so that the human SPATA16 protein encoded by this gene can be expressed in this cell and transferred to the Golgi 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 one of the candidate compounds from said library, blocks, inhibits or modifies the translocation of the human SPATA16 protein towards the Golgi apparatus of said cell,

0214a positive determination being indicative of the fact that the or each of the candidate compounds concerned is (are) a compound capable of having a contraceptive effect on the human being of the male sex. Preferred candidate compounds are ligands for human SPATA16 protein, preferably compounds which are specific ligands for human SPATA16 protein.

0215According to the present invention, it is possible to select and / or produce a 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, for example an antibody or antibody fragment having retained the capacity for binding and / or inducing lysis. An example of such a compound is a compound which specifically inhibits or blocks the activity of this protein, without altering the activity of another human protein.

0216Compounds which bind to the human SPATA16 protein, more particularly to its TPR domain, such as antibodies or fragments of conservative antibodies, can be identified by the person skilled in the art. Preferably, these ligands are ligands specific for the human SPATA16 protein, for example a monoclonal antibody or a preservative fragment of such an antibody which has retained the capacity for binding and / or inducing lysis.

0217According to a preferred embodiment of the invention, said selected or produced compound is a ligand of the human SPATA16 gene and / or of I¹Human SPATA16 mRNA and / or human SPATA16 protein, or is a compound which induces the formation of such a ligand, once administered in a mammal (non-human or human mammal, preferably male or male).

0218The present application also relates to a method for selecting a compound with contraceptive effect on humans, which comprises:

0219- the selection and / or production of at least one compound capable of having a contraceptive effect on the male human being and / or capable of inhibiting or blocking the formation of the pacrosome of a human spermatozoon in accordance with a method of selection and / or production of compound (s) of the invention (inhibition, blocking or alteration of the transcription, translation, expression, translocation and / or activity of SPATA16, as described above ),

0220bringing this, or each of these candidate compound (s) into contact with at least one human spermatogenic cell (said human spermatogenic cell preferably being a human spermatocyte and / or a human spermatid), so as to determining whether this candidate compound induces an inhibition or a blocking of the formation of the acrosome on this at least one human spermatogenic cell, - The selection of at least one candidate compound which has the capacity to induce such inhibition or blocking, such a compound being a compound with contraceptive effect on the male human being.

0221A compound selected and / or produced according to a method of the invention inhibits or blocks the formation of the acrosome of a human sperm, advantageously without otherwise disturbing human spermatogenesis.

0222The present application also relates to a method for manufacturing a product with contraceptive effect on a male human being, which comprises the selection of a compound with contraceptive effect in accordance with the method of the invention, and the formulation of this compound in a form which can be administered to humans.

0223Such a contraceptive product inhibits or blocks the formation of the acrosome of a human sperm, advantageously without otherwise disturbing human spermatogenesis.

0224The present application also relates to a product or a compound having a contraceptive effect on humans, which comprises at least one interfering agent of human SPATA16 DNA or RNA, i.e. a molecule d DNA or RNA which 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 ADNi or RNAi depending on whether they interfere with the DNA of the SPATAI 6 gene or with FARNm of the human SPATAI 16 gene.

0225Such human SPATA16 DNA or RNA interferers are intended to hybridize to a target region of the DNA of the human SPATA16 gene or of the mRNA of the human SPATA16 gene, in a form coupled to the RISC enzyme complex, of so as to induce degradation or cleavage of human SPATA 16 DNA or mRNA, or to induce post-translational repression of the SPATA16 protein produced by the cell.

0226A DNA interferer can, for example, target an intronic region of the human SPATA 16 gene.

0227Such an RNA interferer targets an exonic region of the human SPATA16 gene. An RNA interferant can cleave or degrade the mRNA of the human SPATA 16 gene, and / or repress the translation of this mRNA. Such an RNA interferant can for example be a human anti \ -SPATA16 “small interfering RNA”, or a human ani-SPATA16 “shRNA” (short hairpin RNA). The present application also relates to these interferants of RNA or DNA as such, and to their uses as a contraceptive agent intended for the male human being.

0228The compounds which are known as "siRNA" or "shRNA" are single-stranded or double-stranded DNA or RNA molecules.

0229The "siRNAs" are DNA or double-stranded RNA molecules of 14 to 200 bp, preferably of 14 to 25 bp, one of the strands of which (antisense strand, or negative strand) has a sufficiently complementary sequence to hybridize to mRNA of the human SPATA 16 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 a human spermatid). The "siRNAs" can be administered to the patient directly in the form of small DNA or double-stranded RNA molecules, for example from 14 to 25 bp, or in the form of larger DNA or RNA molecules double-stranded, for example from 50 to 200 bp, which will then be cleaved into DNA or small double-stranded RNA, for example from 14 to 25 bp, by the enzymatic complex DICER within the spermatogenic cell.

0230"ShRNAs" are DNA or single-stranded RNA molecules of 14 to 25 nucleotides, which may have a rod-loop structure, and which have a sequence sufficiently complementary to hybridize to the mRNA of the gene. Human SPATA16, 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 a human spermatid).

0231A “sufficiently complementary” sequence will generally have an identity of at least 85%, preferably at least 90%, with the complementary sequence of mRNA of the human SPATA16 gene, over the entire length of this strand.

0232Preferably, the sequence of the antisense strand of “siRNA”, or if necessary, the sequence of the strand of “shRNA”, is perfectly complementary to the sequence of the mRNA of the human SPATA16 gene over the entire length of this strand (identity 100%).

0233The present application thus relates to any interfering agent for RNA or anti-SPATA16 human DNA, more particularly to any interfering agent for anti-SPATA16 human RNA, which inhibits or blocks the formation of the acrosome of a human spermatozoon.

0234Advantageously, an anti-human SPATA16 RNA or DNA interferant of the invention, more particularly a human anti-SPATA16 RNA interferant of the invention, inhibits or blocks the acrosome formation of a human sperm. , without otherwise disrupting human spermatogenesis.

0235The present application thus relates more particularly to any interfering agent for RNA or anti-SPATA 16 human DNA, more particularly to any interfering agent for human anW-SPATA 16 RNA, which comprises a strand of antisense nucleic acid hybridized to a sense nucleic acid strand, said antisense strand and said sense strand being:

0236- either not covalently linked to each other, thus forming a structure of the “siRNA” type,

0237- either covalently linked to each other via a nucleic acid loop, thus forming a stem-loop structure of the “shRNA” type,

0238- said antisense strand consisting of 14 to 200 nucleotides, and - said antisense strand comprising (or consisting of) a nucleotic 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 a nucleotide sequence which is included in the sequence of the gene and / or of human SPATA 16 mRNA (cDNA sequence or CDS sequence), preferably in the sequence of at least one exon of the human SPATA16 gene (or in the sequence of I¹Human SPATA16 mRNA), very preferably in the sequence of exon 2 and / or 3 and / or 4 of the human SPATA16 gene (or in the sequence of human SPATA16 mRNA), for example in exon 4 of the human SPATAW gene ( or in the sequence of human SPATA 16 mRNA). Said percentage sequence identity value is calculated over the entire length of said sequence from 14 to 25 nucleotides.

0239Preferably, the sequence of the antisense strand of “siRNA”, or if appropriate, of the strand of “shRNA”, targets at least one exon of SPA TA16 chosen from exons 2, 3 and 4, that is to say exons which include the sequences encoding the TPR domain of SPATA16.

0240Such a "siRNA" or "shRNA" therefore comprises a strand of DNA or antisense RNA which:

0241- consists 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 minus 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 “siRNA”, or if appropriate, of the

0242"ShRNA", targets exon 4 of SPATA16.

0243A representative example of the mRNA sequence of the human SPATA16 gene is presented in SEQ ID NO: 1 (cDNA sequence) and

0244SEQ ID NO: 2 (CDS sequence); cf. Figure 4. These sequences are reproduced from the public sequence accessible under AF345909.

0245Examples of such RNA or DNA interferers include in particular RNA or DNA interferers, the antisense strand of which comprises, or consists of, the sequence of SEQ ID NO: 18 and / or of SEQ ID NO: 19 and / or SEQ ID NO: 20.

0246SEQ ID NO: 18 = CCACCTAACATCCTGGAAA

0247SEQIDNO: 19 = GCTGAAATAGTAGACCCTT

0248SEQ ID NO: 20 =

GCTAAATTGGACACGACTT

0250Preferably, the sequence of the antisense strand of “siRNA”, or if appropriate, of “shRNA”, has a G + C content greater than 50%.

0251Preferably, 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 overflowing or overhanging (“overhang”) in 3 '. The part of the sense fragment of the “siRNA” or “shRNA” which is hybridized to the antisense strand is preferably perfectly complementary to the part of the antisense strand sequence to which it is hybridized (100% identity with the complementary sequence).

0252Preferably, the sequence of the antisense strand of “siRNA”, or if appropriate, of “shRNA”, does not exhibit any identity of more than 70% relative to the DNA or RNA sequence of a human gene other than SPATA16 (value of identity calculated over the entire length of the strand).

0253The present application also relates to these RNA or DNA interferers, as a contraceptive agent intended for the male human being, and / or as an agent (more particularly a contraceptive agent) which inhibits or blocks the formation of the acrosome of a human sperm, advantageously without otherwise disturbing human spermatogenesis.

0254The 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 stage (s) of additional method (s) which would not be explicitly indicated, while the term "consistent" or "constituted" is a closed term, which excludes the presence of any other additional element, stage, or ingredient which does not would not be explicitly stated. The term "consisting essentially" or "essentially constituted" 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 measure that this (s) element (s), ingredient (s) or additional step (s) does not materially affect the basic properties of the invention.

0255Therefore, the term "comprising" (or "comprises (include)") includes the terms "consistent", "incorporated", as well as the terms "consisting essentially" and "essentially constituted".

0256The content of the documents referred to in this application (such as scientific articles, patents or patent applications) is incorporated by reference into the content of this patent application.

0257The present invention is illustrated by the examples which follow, which are given for purely illustrative purposes. EXAMPLES

0258Example 1

0259Here is presented here the example of a family comprising three brothers affected by globozoospermia, in which we have identified a homozygous mutation in the gene SPATA16 (OMIM 609856) specific for spermatogenesis.

0260To the knowledge of the inventors, this is the first example of a state of non-syndromic male infertility in human beings due to a single genetic defect.

0261The inventors analyzed an Ashkenazi Jewish family comprising six brothers (three affected, three healthy) and four sisters, who was identified at the Center for Reproductive Medicine of the Dutch-Speaking Brussels Free University. The three unaffected brothers were fathers of 7, 6 and 5 children respectively, but the three affected brothers were childless and had a fertility disorder due to oligoasthenoteratatozoospermia, showing the characteristics of total globozoospermia such as sperm without acrosome (at round head), as shown by acrosin staining (OMIM 102480) in Figure 1A. No known consanguinity was reported, although the family belongs to an isolated Jewish population. The karyotype appeared normal and no microdeletion of the Y chromosome was found. In two of the brothers affected, intracytoplasmic sperm injections (ICSI) were carried out, but the fertilization obtained was poor and no pregnancy occurred.

0262The inventors carried out a screening on the extent of the genome for each of the six brothers, using a chip with SNP (Single Nucleotide Polymoφhism) 1OK (Affymetrix GeneChip® Human Mapping 1OK Array, Affymetrix, Santa Clara, California, USA). The regions of homozygosity were defined by the presence of more than 25 consecutive homozygous SNPs. Large regions of homozygosity were observed in all six individuals (Tables 1 and 2), indicating second or third degree consanguinity in the family.

0263Table 1

0264<img file="WO2009013405A2_D0001.tif" /><img file="WO2009013405A2_D0002.tif" />

0265Table 2:

0266<img file="WO2009013405A2_D0003.tif" />

0267<img file="WO2009013405A2_D0004.tif" />

0268<img file="WO2009013405A2_D0005.tif" />

0269On the left side of Table 2, a selection (> 9 SNPs) of the shared haplotype zones is presented. In the right part of Table 2, the regions of shared homozygosity (> 4 SNPs) which extends inside are shown. Chrom: chromosome.

0270#SNP: quantity of SNP which forms the area of ​​shared haplotype or homozygosity.

0271Departure: departure location.

0272Stop: stop location. Length: length of the fragment.

0273Consequently, the inventors considered that this family was inbred, and deduced therefrom that the pathology was autosomal recessive.

0274The inventors identified a unique region of identical haplotypic homozygosity shared by all the affected brothers, and for which the parents and two of the healthy brothers were heterozygous.

0275The smallest overlap region spanned 17 Mb of chromosome 3q26 (Chr3: 167054711 -184087390). This region contains some 50 genes known in PUCSC Genome Browser. The analysis conducted led the inventors to select the SPA TA16 gene (associated spermatogenesis 16, also known as NYD-SP12) as the candidate gene.

0276The sequence of the human SPATA16 gene, that of its CDS (coding sequence), and that of the human SPATA16 protein are available on the databases, under the access number AF345909.

0277SPATA16 is composed of 11 exons encoding a highly conserved protein of 65 kDa (569 aa), which contains a tetratricopeptide repeat domain

0278(TPR (OMIM 602259)).

0279The sequence alignment (Clustel W 1.81; cf. Figure 3) shows that SPATA16 is strongly conserved across mammals showing an identity rate varying from 77% (mouse) to 96% (chimpanzee) (cf. table 3). Table 3:

0280<img file="WO2009013405A2_D0006.tif" />

0281Table 3 shows the percentage of sequence identity for SPATA16 in other species compared to humans, as well as for the TPR domain.

0282We can thus see that the sequence conservation is even higher for the TPR domain, a protein-protein interaction domain commonly but exclusively found at the level of co-chaperone proteins (92% and 98% respectively in mice and chimpanzees) .

0283The sequence analysis of one of the affected sons revealed a variation in homozygous sequence at the level of exon 4 (c.848G> A), which breaks a recognition site Ncil or Hpall (cf. Figure 1C). Restriction enzyme analysis revealed that the three affected brothers are in fact homozygous, and that both parents and two healthy brothers are heterozygous for the mutation. The third unaffected brother appeared to be homozygous for the wild-type sequence (Figure 1 D). The nucleotide variation c.848G> A is not known in any SNP database, and was not identified in the 231 controls tested, including 151 random controls of both sexes and 80 fertile males.

0284The identified mutation could lead to an amino acid change of a highly conserved residue (p.R283Q) located at the C-terminal end of the highly 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 would disrupt this splicing site (see Table 4).

0285Table 4:

0286<img file="WO2009013405A2_D0007.tif" />

0287It is found that the various predictions of online splicing sites all indicate irregular relationships between the wild type sequence (WT) and the mutated sequence (MT).

0288Unfortunately, the SPATA16 protein has expression restricted to the testes and we have not been allowed to use fresh sperm cells or to perform a biopsy in these patients to verify the aberrant splicing predicted in vivo.

0289Consequently, minigene constructions were produced; they included two exons of constitutive β-globin, surrounding a 420 bp fragment containing either the wild type or the mutated form of exon 4 and flanking the intronic sequences of SPATA16 {cf. Figure 2A). These minigene constructs were transfected into COS1 or HeLa cells, and the transcripts were analyzed by RT-PCR (polymerase chain reaction by reverse transcription) 24 hours after transfection. As shown in Figure 2B, wild-type exon 4 is invariably included in final messenger PARN, as confirmed by sequencing of the PCR product. In sharp contrast, the mutated exon gives rise to two aberrant forms of splicing, as shown by the cloning and sequencing of these PCR products. The product that appears larger and stronger is the result of the use of a splicing site located in the β-globin intron used for the minigene construction. The product which appears smaller and weaker corresponds to the use of a cryptic splicing site located 18 bp upstream of the normal splicing site. These non-specific products are probably due to very small and short intron sequences in the minigene construction. Such products are often observed in exon trap experiments in the absence of a true splicing site and are indicative of the appearance of exon jumps due to the mutation. Notably, we were unable to detect any transcript containing the correct junctions from the mutated exon 4, which proves that the mutation blocks normal splicing of the gene.

0290The first critical step in including exon is the binding of the splicing factor U1 snRNP (small nuclear ribonucleoprotein) to the 5 'splicing sites. In order to confirm that the mutated SPATA 16 exon 4 is not recognized by the splicing machinery, the inventors verified its binding to LM snRNP by UV hybridization mediated by psoralen. While binding of U1 snRNP to wild type DNA was readily detected, this was not observed when DNA carrying the c.848G> A mutation was used as a model (Figure 2C). The identity of U1 snRNP was confirmed by treatment with HseRNA using Poligodeoxynucleotide complementary to positions 1 to 15 of U1 snRNA. Consequently, the results of the bioinformatics, minigene and LM binding prediction all indicate that the c.848G> A mutation led to the inappropriate splicing of exon 4, and consequently to the disruption of the TPR domain.

0291The 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 presented any sequence variation, except for 3 known polymorphisms and 2 point mutations which are not segregated to the disease {cf. table 5).

0292Table 5:

0293<img file="WO2009013405A2_D0008.tif" />

0294<img file="WO2009013405A2_D0009.tif" />

0295In Table 5, all non-synonymous coding variations, which have been discovered in 9 of the 28 patients 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 have been identified, located near two unknown but non-segregating variations. In patients 6 and 11 to 28, no non-synonymous, coding variation was identified.

0296To the knowledge of the inventors, this is the first description of a gene involved in the pathogenesis of human globozoospermia.

0297In Figure 4, the cDNA sequence of the wild human SPATA16 gene is reproduced (SEQ ID NO: 1).

0298Figure 4 also shows the coding sequence (CDS; SEQ ID NO: 2) of the wild human SPATA16 protein, as well as the protein sequence

0299SPATA16 wild human (SEQ ID NO: 3).

0300These data are reproduced from the sequence data available under the accession number AF345909.

0301The positions of the exons within, or with respect to, these sequences are as follows:

0302Table 6:

0303<img file="WO2009013405A2_D0010.tif" /><img file="WO2009013405A2_D0011.tif" />

0304For 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 forming 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 under each of the two exons; this is the case of :

0305- amino acid at position 253, the codon of which overlaps exon 3 and exon 4,

0306- amino acid at position 282, the codon of which overlaps exon 4 and exon 5,

0307- amino acid at position 361 (Asp), the codon of which overlaps exon 6 and exon 7,

0308- amino acid in position 410 (Glu), the codon of which overlaps exon 7 and exon 8.

0309The 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 Figure 5 are the fragments 92041 -92340 , 121561-121800 and 164101-164340, respectively of the wild SPATA16 gene sequence (assembled sequence of the wild SPATA16 gene, available on the website http: //www.ensembl/orq, under the accession number ENSG00000144962).

0310The fragment 92041-92340 (SEQ ID NO: 6) contains exon 3 (bold type), surrounded in 5 'by a part of intron 2-3 and in 3' by a part of intron 3-4 . The fragment 121561-121800 (SEQ ID NO: 7) contains exon 4 (bold type), surrounded in 5 'by a part of intron 3-4 and in 3' by a part of intron 4-5 . The fragment 164101 -164340 (SEQ ID NO: 8) contains exon 5 (bold characters), surrounded in 5 'by a part of intron 4-5 and in 3' by a part of intron 5-6 .

0311The last two nucleotides of exon 4 (cf. SEQ ID NO: 7) are CG (underlined). The first nucleotide of exon 5 {cf. SEQ ID NO: 8) is G (underlined). In the wild form of the SPATA 16 gene, the last two nucleotides of exon 4 and the first nucleotide of exon 5 form the codon CGG, which codes for the amino acid Arg (first amino acid from the C end -terminal of the TPR domain of the wild SPATA16 protein; cf. FIG. 3).

0312A mutation, which has been detected by the inventors in globozoospermic siblings, affects the last nucleotide of exon 4, that is to say the nucleotide G, which in FIG. 5 is presented in larger character than the other nucleotides. of SEQ ID NO: 7. This nucleotide G corresponds to nucleotide G in position 978 of SEQ ID NO: 1 (cDNA of wild SPATA 16) and in position 848 of SEQ ID NO: 2 (CDS of wild SPATA 16).

0313In the mutation detected by the inventors as being segregating from patients with globozoospermia within the family studied, this nucleotide

0314G is mutated to nucleotide A.

0315This mutation prevents correct splicing of exon 4, which leads to a loss of exon 4, and to direct splicing of exon 3 on exon 5.

0316The G> A mutation observed by the inventors in globozoospermic siblings leads to a mutant SPATA16 protein, which has lost the polypeptide part encoded by exon 4.

0317The resulting mutated SPATA16 protein is therefore encoded by exons 1 to 3 and 5 to 11 to the SPATAW gene. The sequence of this mutant protein, and a sequence which encodes it, are presented 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 onto 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 of this codon (G) is the first nucleotide of exon 5.

0318The result is a mutant human protein SPATA16 of SEQ ID NO: 17, which has 539 amino acids (instead of the 569 aa of the wild protein): the first 252 amino acids are identical to those of the wild human SPATA16 protein (amino acids coded by exons 1 to 3), the other 287 amino acids result from the translation of the nucleotide sequence constituted by the last two nucleotides of exon 3 (nucleotides AG) followed by exons 5 to 11 (nucleotide sequence starting with G AGT GCC etc. up to AGG TAG). If the sequence of this mutant human SPATA16 protein is aligned with that of the sequence of the wild human SPATA16 protein, a deletion of the sequence of 29 amino acids is observed which, in the wild SPATA16 protein, extends from positions 254 to 282 (sequence SIVLNPAYFRNHLRQATVFRCLERYSEAA; SEQ ID NO: 14), that is to say the polypeptide sequence encoded by exon 4 of SPATA16. The sequence of the mutant human SPATA16 protein (SEQ ID NO: 17) therefore corresponds to SEQ ID NO: 10 + SEQ ID NO: 15, and a sequence which codes it (SEQ ID NO: 16) corresponds to SEQ ID NO: 9 + SEQ ID NO: 11.

0319The data obtained by the inventors strongly indicate that the homozygous mutation identified in SPATA16 causes globozoospermia in three of the six brothers of the family presented, which allows the inventors to declare that globozoospermia can be a genetic trait with an autosomal recessive mode of transmission. .

0320The protein SPATA16 is localized in the Golgi apparatus and in the pro-acrosomal vesicles which are transported to the acrosome in round and elongated spermatids during spermiogenesis. The present results support the fact that SP477l76 plays a crucial role in the formation of the acrosome.

0321The highest 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 for mediating protein-protein interactions and assembling multiprotein complexes. The study of the X-ray structure revealed that the wild TPR domain adopts a helix-turn-helix arrangement with the ability to associate with other alpha-helical structures. The interaction molecule (s) can, for example, be:

0322- The mouse model GOPC gene, which codes for a protein associated with the Golgi complex, and which contains alpha helices of two-spiral type motif, and / or

0323- the gene for the HIV-1 rev binding protein (HRB), which is also located at the Golgi complex.

0324Finally, it is important to note that SPATAW contains six casein kinase II phosphorylation sites, and that casein kinase lia is the most abundant casein kinase in the testes, of which the knockout model shows acrosome defects, as well than other morphological defects. In addition, the existence of several candidate genes suggests genetic heterogeneity in human globozoospermia, which could be a reason why the inventors have not yet found other patients with a gene alteration in SPATA16.

0325It is also important to note the fact that the heterozygous mice of the above-mentioned models show no abnormality in the sperm. This indicates that the mutation carriers should have normal fertility. In the human family subject to the present study, this seems to be the case, insofar as the father and two heterozygous brothers were respectively fathers of 10, 7, and 6 children. In two of the affected brothers, the inventors carried out an ICSI to induce fertilization and pregnancy, but without success. This is in agreement with the documentation, which shows that ICSI allows the fertilization of oocytes, but with low fertilization rates in about half of the cases.

0326Insofar as male infertility does not respect the canonical rules of genetics, the determination of inheritance patterns and the clarification of genetic causes are complicated. Several genetic factors have been described that affect male fertility, but these give rise to more complex phenotypes. However, the patients subject to the present study did not present any mental or physical anomaly, and in particular no andrological anomaly, apart from the aberrant analyzes of their seeds.

0327The SPATAW mutation that the inventors have identified here presents SPATA16 as a human gene in which mutations give rise to male infertility without any other associated abnormality.

0328Additional studies of other patients may lead to the identification of other actors involved in the formation of the acrosome, allowing fine dissection of the mechanisms involved in the establishment of such a specialized cellular organelle. The defects of SPATA16 influence spermiogenesis, but meiosis is not disturbed.

0329Thus, the modulation of the SPATAW function, or that of other components in the same way, offers an innovative reversible approach to male contraception, which has the advantage of not being based on the control of the hormonal production pathway. sperm.

0330Internet resources available:

0331UCSC Genome Navigator (March 2006 version), http://genome.cse.ucsc.edu/;

0332SDSC Biology Workbench 3.2, Clustel W 1.81, http://workbench.sdsc.edu;

0333NCBI SNP Reference Assembly online, http://www.ncbi.nlm.nih.gov/SNP/;

0334NCBI 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&amp;DB=OMIM;

0335Splicing site prediction, http://www.cbs.dtu.dk/services/NetGene2;

0336Splicing site prediction, http://www.genet.sickkids.on.ca/~ali/splicesitefinder.html; Splicing site prediction, http: //www.fruitfly.0rg/seqJ: 00ls / splice.htrnl.