JP2008237204A - Diseased model animal, cell, tissue, orchis, animal for mating, method for producing germ cell, germ cell, cultured cell, screening method, medicinal composition, pregnacy-diagnosing kit, detection method, polynucleotide, polypeptide, and antibody - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a tool useful on the researches of low pregnant or sterile conditions and therapeutic methods. <P>SOLUTION: A Meichroacidin (MCA) homo defective mouse in which the first exon of MCA gene is deleted by a gene targeting technique exhibits azoospermia wherein sperm cells can be seen in the testis, but can not be seen in epididymis. Thus, the MCA homo defective mouse is useful as a model animal for the azoospermia. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a tool such as a disease model animal that is useful for elucidating disease phenomena and establishing treatment methods. The present invention also relates to a technique that enables estimation of fertility and identification of the cause of infertility.

  In recent years, the problem of declining birthrate has been frequently taken up, and various measures are being sought to solve this problem. Needless to say, it is important to rescue couples who have no children due to infertility as a countermeasure to the declining birthrate.

  Non-Patent Document 1 reports that about 15% of couples who want children can not get pregnant even after two years. In recent years, the development of in vitro fertilization (IVF) technology has enabled pregnancy and birth even when sperm activity is low, but the molecular mechanism behind infertility remains unclear. .

  According to Non-Patent Document 2, studies on male infertility in mice have revealed the existence of many genes that affect fertility, and mutations in these genes contribute to male infertility in humans. It may have become.

  Maturation of mammalian male germ cells occurs through a number of structural and functional changes. The process is called spermatogenesis, and is mainly involved in (1) proliferation and differentiation of spermatogonia, (2) meiosis in the early stage of spermatocytes, and (3) differentiation from haploid round sperm cells to sperm. It includes three stages of rapid shape change.

  Meichroacidin (hereinafter referred to as “MCA”) is known as a protein that is specifically expressed in the above-mentioned meiosis phase (2). The MCA gene was first isolated in mice (Non-patent Document 3). The gene product is expressed in the cytoplasm of pachytene spermatocytes, and is known to bind to chromosomes and spindles when the nuclear membrane disappears during meiosis. From this, the above gene product was named Meichroacidin after the initial of Male Meiotic Metaphase Chromosome-Associated Acidic Protein. The MCA protein contains a specific amino acid repetitive sequence, which was later named the MORN motif (Non-patent Document 4).

  Thereafter, the human MCA gene was also isolated, and as a result of analysis, it was revealed that the human MCA protein was expressed in sperm flagella, basal body and acrosome (Non-patent Document 5). It was also revealed that MCA protein is expressed in sperm flagella in mice.

In addition, it is known that the MCA protein is expressed not only in mammals such as humans and mice but also in flagella of carp and squirt sperm (Non-patent Documents 6 and 7).
de Kretser DM et al., J Clin Endocrinol Metab. 1999; 84: 3443-3450. Matzuk MM et al., Nat Cell Biol. 2002; 4 (Supp1): S41-S49 / Nat Med. 2002; 8 (Supp1): S33-S40. Tsuchida J et al., Dev Biol. 1998 May 1; 197 (1): 67-76. Takeshima H et al., Mol Cell. 2000 July; 6 (1): 11-22. Yasuhiro M et al., Reproductive Medicine and Biology. 2005; 4: 213-219. Ju TK and Huang FL., Biol Reprod. 2004 Nov; 71 (5): 1419-1429. Epub 2004 Jun 23. Satouh Y et al., Mol Biol Cell. 2005 Feb; 16 (2): 626-636. Epub 2004 Nov 24.

  However, disease animal models that can be useful tools for studying the pathology and treatment of male infertility have not been sufficiently obtained. The cause of male infertility ranges from the loss of germ cells themselves to the failure of fertilization of sperm. Therefore, in order to establish an effective treatment method according to the disease state, it is desired to obtain various male infertility model animals.

  This invention is made | formed in view of the said subject, The 1st objective is to provide a useful tool in studying the pathological condition and treatment method of low fertility or infertility.

  In addition, an effective genetic diagnosis method for diagnosing male infertility has not been sufficiently established.

  This invention is made | formed in view of the said subject, The 2nd objective is to enable the estimation of the fertility possibility and the identification of the cause of infertility.

  As shown in the Examples described later, the present inventor has found that this mouse becomes infertile as a result of producing an MCA-deficient mouse that is deficient in the MCA gene and does not express MCA, thereby completing the present invention. It was.

  That is, in order to achieve the first object described above, the disease model animal according to the present invention is a male non-human mammal exhibiting low fertility or infertility in which normal MCA protein expression is decreased. It is characterized by.

  Alternatively, the disease model animal according to the present invention may be a male non-human mammal that does not express normal MCA protein.

  Further alternatively, the disease model animal according to the present invention may be a male non-human mammal deficient in the MCA gene.

  This provides a new model animal with low fertility or infertility.

  The disease model animal preferably exhibits low fertility or infertility. The non-human mammal is preferably a mouse. The disease model animal preferably exhibits azoospermia.

  The cell according to the present invention is isolated from the disease model animal described above. Another cell according to the present invention is characterized in that the MCA gene is deficient or mutated and isolated from a human male exhibiting low fertility or infertility. In addition, it is preferable that the said cell is a germ cell.

  The tissue or testis in the testis according to the present invention is characterized by being isolated from the above-mentioned disease model animal or the above-mentioned human male.

  The animal for mating according to the present invention is a non-human mammal that is used for mating to produce the above-mentioned disease model animal and is deficient in part or all of the MCA gene. By using this animal for mating, the above-mentioned disease model animal can be easily produced.

  A germ cell production method according to the present invention is a germ cell production method for producing a low fertility or infertility male germ cell, which is isolated from a cultured cell derived from a male mammal or a male mammal. A gene deletion step of deficient in the MCA gene possessed by the mammalian cell, and a differentiation induction step of differentiating the mammalian cell deficient in the MCA gene in the gene deficiency step into a germ cell. To do.

  The germ cell according to the present invention is a germ cell of a male non-human mammal, and is characterized by being deficient in the MCA gene and exhibiting low fertility or infertility.

  The cultured cell according to the present invention is a cultured cell derived from a human male and is a germ cell that lacks the MCA gene and exhibits low fertility or infertility.

  The screening method according to the present invention is a screening method for screening a candidate compound for a therapeutic drug for hypofertility or infertility, which comprises the above-mentioned disease model animal, cell, tissue or testis, male germ cell, germ cell Or a step of allowing the candidate compound to act on cultured cells and evaluating the result.

  The pharmaceutical composition for the treatment and / or prevention of hypofertility or infertility according to the present invention is characterized by comprising as a main component a compound obtained by the screening method described above.

  In order to achieve the second object, the fertility diagnostic kit according to the present invention includes a reagent for detecting the presence or absence of a human MCA gene deletion or mutation.

  When the adenine encoding the translation initiation codon is the 1st base, the above reagent is a mutation of at least one of the 92nd base and the 121st base of the translation region of the human MCA gene. It is preferable to detect the presence or absence.

  Specifically, the reagent comprises a primer set for amplifying a region containing at least one of the 92nd base and the 121st base of the human MCA gene, and the base sequence of the region And a nucleotide sequence determination reagent for determining.

  Alternatively, the reagent may detect the presence or absence of the mutation by an invader method.

  Alternatively, the reagent may detect the presence or absence of the mutation by the SMMD method.

  In order to achieve the second object, a detection method according to the present invention is a detection method for detecting a gene deficiency or mutation in the human using a biological sample isolated from human, It includes a step of detecting the presence or absence of a defect or mutation in the MCA gene.

  Since the deficiency or mutation of the MCA gene affects male fertility, the fertility diagnostic kit or detection method described above makes it possible to estimate fertility and identify the cause of infertility.

  In order to achieve the first object, the polynucleotide according to the present invention provides a nucleotide sequence of a translation region of a human wild type MCA gene when the adenine encoding the translation initiation codon is the first base. In the base sequence in which the base at position 92 is substituted with cytosine.

  In order to achieve the first object, another polynucleotide according to the present invention has a translation region of a human wild-type MCA gene when an adenine encoding a translation initiation codon is the first base. It consists of a base sequence in which the base at position 121 in the base sequence is substituted with adenine.

  In order to achieve the first object, another polynucleotide according to the present invention provides a translation region of a human wild type MCA gene when an adenine encoding a translation initiation codon is the first base. In the base sequence, the base at position 92 is substituted with cytosine, and the base at position 121 is substituted with adenine.

  In addition, it is preferable that the said polynucleotide is concerned with low fertility or infertility.

  The polypeptide according to the present invention is characterized by comprising an amino acid sequence encoded by any of the polynucleotides described above.

  The above-mentioned polynucleotide or polypeptide can be used as a useful tool for studying the pathology and treatment methods of hypofertility or infertility.

  In order to achieve the second object, the antibody according to the present invention is characterized by binding to the above-mentioned polypeptide and not binding to the human wild-type MCA polypeptide.

  In order to achieve the second object, a detection method according to the present invention is a detection method for detecting a mutation in an MCA polypeptide expressed in the human using a biological sample collected from the human. And a step of detecting a mutation of the MCA polypeptide by the above-mentioned antibody.

  In order to achieve the second object, another fertility diagnostic kit according to the present invention includes an anti-human MCA antibody.

  As described above, since the disease model animal according to the present invention exhibits low fertility or infertility, the individual, cell, tissue, and testis are used for studying the pathophysiology and treatment method of low fertility or infertility. It can be used as a useful tool.

  Further, according to the germ cell production method, germ cells or cultured cells according to the present invention, male fertility cells with low fertility or infertility are provided. It can be used as a useful tool for studying the law.

  In addition, cells, tissues, and testis obtained from human men who are deficient or mutated in the MCA gene and exhibit low fertility or infertility are also useful for studying the pathophysiology and treatment of low fertility or infertility. It can be used as a simple tool.

  In addition, since the polynucleotide according to the present invention has a mutation that is actually found in human infertility patients, the polynucleotide or polypeptide according to the present invention is a pathological condition or treatment of low fertility or infertility. It can be used as a useful tool for studying the law.

  Moreover, according to the fertility diagnostic kit, detection method or antibody according to the present invention, fertility possibility can be estimated and the cause of infertility can be identified.

  Although one embodiment of the present invention is described in detail below, the present invention is not limited to the following description. In this embodiment, the disease model animal according to the present invention, its production method, and its use will be described in detail in this order.

(1) Disease model animal according to the present invention The disease model animal according to the present invention is a male animal that exhibits hypofertility or infertility, and is characterized by a decrease in the expression of normal MCA protein. .

  Examples of the animal used as the disease model animal include mammals other than humans (non-human mammals). Specifically, there are various experimental animals such as rats, mice (animals belonging to the genus Mus such as Mus musculus, Mus spretus), guinea pigs, hamsters, rabbits, more preferably rats and mice, and particularly preferable ones. Can mention a mouse. The reason these animals are preferred is that they are easy to rear and can use the accumulated research to date. Next, MCA will be described.

  The deduced amino acid sequence of mouse MCA consists of 284 residues and has a MORN motif (Non-patent Document 3). The mouse MCA transcript is detected from 14 days after birth and is mainly expressed in the testis. Similarly, proteins are mainly expressed in the testis. By tissue immunostaining, the protein is found in the cytoplasm between the spermatocytes of the pachytene phase and the circular sperm cells, and further, during the disappearance of the nuclear membrane during the first and second meiotic phases, the M phase It has been shown to localize around the chromosomes and spindles. Subsequent studies have also shown that mouse MCA is present in sperm flagella and the like.

  MCA is also present in humans, its amino acid sequence is 79.8% identical to that of mouse, and the MORN motif is also conserved (Non-patent Document 5). Human MCA protein is also localized in sperm flagella, basal bodies and acrosomes. From the above, MCA is considered to be a protein that plays an important role in the development of sperm flagella. In addition, since the MCA gene is also present in carp that is a fish and sea squirt that is an invertebrate as described above (Non-Patent Documents 6 and 7), it is considered to be conserved in all mammals. In addition, since those skilled in the art can know details of MCA from these non-patent documents if necessary, detailed description of MCA is omitted in this specification.

  Since the disease model animal according to the present invention exhibits low fertility or infertility due to a decrease in the expression level of MCA protein, as a low fertility model animal or an infertility model animal, elucidation and treatment of infertility It can be used for various purposes such as law establishment. The disease model animal according to the present invention is a useful tool for establishing an effective therapeutic method particularly for a human patient having an abnormality in the MCA gene.

  In addition, it is preferable that the disease model animal which concerns on this invention does not express normal MCA protein. This ensures that the disease model animal exhibits low fertility or infertility.

  The disease model animal according to the present invention may be a male non-human mammal deficient in the MCA gene. If the MCA gene is deficient, normal MCA protein expression decreases and the model animal exhibits hypofertility or infertility. Therefore, it can be suitably used as a model animal for hypofertility or infertility. .

  The deletion of the MCA gene may be a part of the entire gene, but the upstream exon so that the expression of the normal MCA protein is significantly reduced or the amino acid sequence of the normal MCA protein is reduced. It is preferable that (for example, the first exon) is deleted. Of course, it is preferable that the deletion of the MCA gene occurs in both alleles.

  As shown in Examples described later, MCA homo-deficient mice in which the MCA gene is deficient in both alleles and do not express normal MCA protein are particularly preferred as the disease model animals of the present invention. MCA homo-deficient mice have abnormal sperm morphology, and therefore there is almost no change in the amount of germ cells such as spermatocytes and sperm cells in the testis, but there is no sperm in the accessory testis and no sperm It presents an interesting symptom of getting sick. Therefore, MCA homo-deficient mice can be used to treat azoospermia caused by sperm flagellar abnormalities and the like, for example, development of drugs that recover sperm morphological abnormalities and drugs that activate sperm flagellar movement, etc. Very useful.

  In addition, since the role of the majority of genes is common in mammals, even when mammals other than mice are used as disease model animals, it is expected to exhibit similar symptoms. Therefore, even mammals other than mice that do not express normal MCA protein are useful as model animals for hypofertility and infertility (specifically, azoospermia).

  In addition, in the disease model animal according to the present invention, the MCA gene may be mutated instead of being deficient in the MCA gene. Of course, it is preferable that the mutation of the MCA gene occurs in both alleles.

(2) Method for Producing Disease Model Animal According to the Present Invention A typical method for producing the disease model animal according to the present invention includes a method for deleting the MCA gene by gene targeting technology. A known method can be used as the gene targeting technique. In this case, the entire MCA gene may be deleted, but a method of deleting a part of the MCA gene exons (especially the first exon) is simple and preferable.

  An example of a method for producing a disease model animal (here, a mouse) by gene targeting technology is as follows. First, a targeting vector is prepared in which a sequence upstream and downstream of the region to be deleted is linked before and after the drug resistance gene cassette. Note that the MCA gene and its surrounding nucleotide sequence can be obtained from a public database (GenBank, etc.) as necessary by those skilled in the art. Then, this targeting vector is introduced into mouse embryonic stem cells (ES cells), and ES cells are screened using a drug to select ES cells in which homologous recombination has occurred. Then, the selected ES cell is injected into a blastocyst to obtain a chimeric mouse.

  In this chimeric mouse, if ES cells differentiate into germ cells, germ cells lacking the MCA gene in one allele will proliferate. By mating the chimeric mouse with a wild-type mouse, the MCA gene will be in one allele. Deficient F1 mice (hereinafter referred to as “MCA hetero-deficient mice”) can be obtained. By further mating the thus obtained MCA hetero-deficient mice, a mouse in which the MCA gene is deficient in both alleles (hereinafter referred to as “MCA homo-deficient mouse”) can be obtained.

  Thereafter, if female MCA homo-deficient mice and male or female MCA hetero-deficient mice are maintained, MCA homo-deficient mice can be easily obtained again by mating. Accordingly, these mice for producing MCA homo-deficient mice, that is, mating mice in which part or all of the MCA gene is deleted in one or both alleles are also included in the present invention.

  By the way, although the mouse | mouth was demonstrated to the example above, this invention is not limited to this, Mammals other than a mouse | mouth may be sufficient. The MCA gene and its surrounding nucleotide sequences in mammals other than mice can be obtained from a public genome database by homology search using the nucleotide sequences of mouse and human MCA genes. In addition, when producing a disease model animal according to the present invention using an animal for which gene targeting technology has not been established, a known method (for example, ENU mutagenesis) that randomly generates mutations in genomic DNA is used. May be. In this case, a mutation is caused in the genomic DNA of the target animal by ENU treatment or the like, and an individual lacking the MCA gene is selected by screening.

  Moreover, you may produce the disease model animal which concerns on this invention by the gene silencing technique instead of the gene targeting technique. In this case, siRNA targeting MCA may be designed and synthesized, and introduced into the testis of the target animal. Furthermore, an RNAi transgenic animal may be prepared by integrating a nucleobase sequence necessary for expression of this siRNA into the chromosome of the target animal using a transgenic technique. Thereby, siRNA is constantly expressed, and the inhibitory effect of MCA by RNAi lasts for a long time.

(3) Use (usefulness) of a disease model animal according to the present invention
The disease model animal according to the present invention is a novel disease model animal that develops low fertility and infertility (specifically, azoospermia). Many azoospermia have been reported in humans, but no useful model animal has been obtained so far. The disease model animal according to the present invention is particularly useful for elucidating the pathology of hypofertility and infertility (particularly azoospermia) and establishing a treatment method.

  When the disease model animal according to the present invention is viewed as a material (or experimental material), not only the individual itself but also cells, tissues, organs and the like isolated therefrom can be used for the above-described applications. The isolation method of a cell is not specifically limited, A well-known method can be used and it can culture by a well-known method. Similarly, tissues and organs can also be extracted by a known method and cultured by a known method.

  In addition, it is preferable that it is a male germ cell as said cell, and it is more preferable that a differentiation stage is a male germ cell after a spermatocyte. The organ is preferably a male reproductive organ including at least a testis, and the male reproductive organ more preferably includes a secondary testis in addition to the testis. Further, the tissue is preferably a tissue contained in the male reproductive organ, and more preferably an organ contained in the testis. These materials are more suitable for investigating the causes of infertility and developing treatments.

  In addition, in the case of a mouse, the cells, tissues, and organs are preferably isolated from an individual after 14 days of birth. Since the transcript of MCA is detected in mice after 14 days after birth, the above-mentioned material isolated from mice after 14 days is in a state of low fertility or infertility, It is considered to be a state that is about to develop hypofertility or infertility. Therefore, the above-mentioned material isolated from an individual after 14 days after birth can be immediately used for the above-mentioned use without culturing for a long time.

  Below, an example of the isolation method of a male germ cell is demonstrated. Male germ cells can be isolated and cultured by the method described in, for example, Kita K et al., Biol Reprod. 2007 Feb; 76 (2): 211-7. Epub 2006 Oct 25.

  In this method, first, the testis thin film is peeled to expose the seminiferous tubule. Then, the testis tissue is treated at 37 ° C. for 20 minutes with a first digestion solution containing collagenase type 2 (2 mg / ml) and DNAse (10 μg / ml) in PBS. Then, the test solution is centrifuged to precipitate testis tissue, and a second solution containing collagenase type 2 (2 mg / ml), DNAse (10 μg / ml) and hyaluronidase (2 mg / ml) in DMEM (Dulbecco's Modified Eagle's Medium). Treat testicular tissue with digestive fluid at 37 ° C. for 5 minutes. The cell suspension is washed twice with PBS, and then passed through a mesh filter having a pore size of 40 μm to recover male germ cells.

  The method described above is an example of a method suitable for mice and rats, but of course it is not limited to the isolation method described above, and various modifications may be made depending on the animal from which the cells are isolated.

(4) Human cells, tissues and testis Since almost all genes play the same role in mice and humans, it is strongly suggested that human MCA protein plays the same role as mouse MCA protein. Therefore, if the MCA gene is deficient or mutated in humans and the expression level of the MCA protein is decreased or the MCA protein is not expressed, it is considered that hypofertility or infertility is exhibited like the mouse. Therefore, cells, testicular tissue and testis isolated from human males that are deficient or mutated in the MCA gene and exhibit hypofertility or infertility are also considered to be useful, similar to those in mice. Therefore, these cells, testicular tissue, testis are also included in the scope of the present invention. Of course, the cells are preferably germ cells.

  The above-mentioned cells, testicular tissue or testis can be used to find a patient who is deficient or mutated in the MCA gene by genetic diagnosis from among human male patients who are hypofertile or infertile. It can be obtained by isolating cells, testicular tissue or testis from a patient by a surgical technique. The patient is preferably confirmed to have azoospermia, and more preferably morphological abnormality is observed in the flagella or nucleus of the sperm. Moreover, you may isolate said cell from the testis extracted from the patient by the method similar to the non-human mammal mentioned above. These human cells, testicular tissue or testis can also be cultured basically in the same manner as non-human mammals.

(5) Disease model cells Male or non-human mammal male germ cells (especially sperm cells and sperm) that are deficient in the MCA gene and exhibit symptoms of low fertility or infertility are used to study low fertility and infertility. Although it has already been mentioned that it becomes an important tool, such germ cells can also be produced by the following method.

  That is, the germ cell production method according to the present invention is a germ cell production method for producing a low fertility or infertility male germ cell, and is obtained from a cultured cell derived from a male mammal or a male mammal. It includes a gene deficiency step in which the MCA gene possessed by the isolated mammalian cell and a differentiation induction step in which the mammalian cell deficient in the MCA gene by the gene deficiency step is differentiated into a germ cell. In the above-described germ cell production method, mammals include both human and non-human mammals.

  In the gene deficiency step, as the cells to be deficient in the MCA gene, it is preferable to use human or mouse-derived male ES cells in the case of cultured cells. Since ES cells are totipotent, they can be easily differentiated into germ cells. Hereinafter, an example using male ES cells will be described.

  First, in the gene deletion step, the MCA gene possessed by male ES cells can be deleted by the gene targeting technique described above. At this time, when it is desired to delete the MCA gene in both alleles, the above-described targeting vector introduction and screening may be further performed on ES cells in which the MCA gene is deleted in one allele.

  In the differentiation induction step, ES cells deficient in the MCA gene are transformed into germ cells (spermatogonia and gametes by the method described in Nayernia et al., Developmental Cell, 2006, 11, 125-132, etc. ). In this method, a construct in which a reporter gene (green fluorescent protein or red fluorescent protein gene) is linked to the promoter of Stra8 gene and protamine 1 gene is introduced into mouse ES cells, and ES cells are differentiated while germ cells are differentiated. Only those differentiated into lineages are selected by FACS. Although this document describes mice, it is expected that humans can be induced to differentiate by the same technique.

  In addition, although the example using ES cells was described above, various stem cells and differentiated cells having pluripotency may be used instead of ES cells. These stem cells and differentiated cells may be established cultured cells or primary cultured cells obtained from humans or non-human mammals. These cells can also be deficient in the MCA gene using the gene targeting technique described above. Then, these cells deficient in the MCA gene are once dedifferentiated to be in an undifferentiated state and then differentiated into germ cells, whereby low fertility or infertile male germ cells can be obtained.

  As described above, human or mouse male germ cells lacking the MCA gene and exhibiting low fertility or infertility can be obtained. The male germ cells obtained in this way also exhibit the same traits as germ cells obtained from the disease model animals according to the present invention, and are considered to be useful tools for studying hypofertility and infertility. It is done.

  As described above, (i) a germ cell of a male non-human mammal that is deficient in the MCA gene and exhibits low fertility or infertility, or (ii) a cultured cell derived from a human male The cultured cells that are germ cells that are deficient in the MCA gene and exhibit low fertility or infertility can be produced by various methods. Included in the range.

(6) Method for Screening Candidate Compound for Treatment Drug or Method for Evaluating Pharmacological Action of Candidate Compound Next, a specific application example of the disease model animal according to the present invention will be described.

<Application Example 1: Drug screening method>
As a typical use example of the disease model animal according to the present invention, use for screening a candidate compound of a therapeutic drug for infertility can be mentioned. The disease model animal according to the present invention develops azoospermia due to abnormal sperm flagella, and therefore can be suitably used for screening a therapeutic drug for azoospermia.

  The specific configuration of the screening method is not particularly limited, and a known method can be suitably used. Specifically, the screening method according to the present invention includes (a) a step of administering a candidate compound to the disease model animal according to the present invention, and (b) infertility in the disease model animal administered with the candidate compound (details). (E) a process of evaluating changes in symptoms of azoospermia and sperm morphology, and (c) mitigating infertility (specifically, azoospermia and sperm morphology) of disease model animals based on the evaluation results And a step of selecting a compound to be processed. Of course, other steps may be included as necessary.

<Use Example 2: Evaluation Method of Pharmacological Action>
The disease model animal according to the present invention can also be used for the evaluation of the pharmacological action of candidate compounds for therapeutic agents for infertility. That is, even if a candidate compound is found by the above screening method or other methods, it is necessary to evaluate whether or not the candidate compound can withstand practical use. It can be suitably used for applications.

  The specific configuration of the evaluation method is not particularly limited, and a known method can be suitably used. Specifically, for example, (a) a candidate compound is administered to the disease model animal according to the present invention, and (b) infertility in the disease model animal administered with the candidate compound (specifically, azoospermia or abnormal sperm morphology) It is sufficient to go through each step of observing changes in the symptoms of) and confirming the effectiveness of pharmacological action and the presence or absence of side effects. Moreover, you may include another process as needed.

  In both of the above-mentioned usage examples 1 and 2, an individual of the disease model animal according to the present invention may be used, and cells, tissues or organs isolated from this individual may be used as necessary. In addition, cells, tissues, or organs isolated from the above-described human MCA-deficient patients may be used, or germ cells that are produced in vitro by gene targeting technology or the like and lack the MCA gene may be used. By using human cells, tissues or organs, screening of candidate compounds and evaluation of pharmacological effects are more reliable.

  In addition, when screening for candidate compounds as described above or evaluating pharmacological action using non-human mammals or human sperm, evaluation may be performed based on whether or not morphological abnormalities of sperm are recovered. The evaluation may be performed based on whether or not the athletic performance of the patient is improved.

  Thus, screening is performed using the disease model animal (or its cells, tissues, organs) according to the present invention, cells, tissues or organs isolated from human MCA deficient patients, germ cells produced in In Vitro, and the like. If the evaluated compound is the main component, it becomes possible to produce a pharmaceutical composition for the treatment and / or prevention of infertility (especially azoospermia caused by abnormal sperm morphology).

(7) Detection method and fertility diagnostic kit according to the present invention As described above, deletion or mutation of the MCA gene is considered to cause hypofertility or infertility even in humans. Therefore, it is possible to estimate fertility and identify the cause of infertility by collecting a biological sample from a subject and examining whether the subject's MCA gene is defective or mutated using this biological sample. Become.

  That is, the detection method according to the present invention is a detection method for detecting a gene deficiency or mutation in the human using a biological sample collected from a human, and detecting the presence or absence of a human MCA gene deficiency or mutation It is characterized by including.

  Moreover, the fertility diagnostic kit according to the present invention includes a reagent used in the detection method. The fertility diagnostic kit may include an instruction manual describing how to use it.

  The biological sample collected from the subject is not particularly limited, and examples thereof include blood and a mucous membrane inside the cheek. In addition, the method for detecting a deficiency or mutation of the human MCA gene is not particularly limited, and various known methods, for example, a nucleotide sequence determination method based on the Sanger method, an invader method, a SMMD method (simultaneous multiple mutation detection system) , PCR-RFLP method, MASA method, base extension method, Taqman probe method, a modified method thereof, and the like can be used.

  The human MCA gene and the surrounding nucleotide sequences can be easily obtained by those skilled in the art from, for example, NCBI Human Genome Resources (http://www.ncbi.nlm.nih.gov/genome/guide/human/). Can be obtained. In the NCBI human genome resource, MCA is also referred to as TSGA2. Primers, probes, and the like for detecting a deficiency or mutation in the human MCA gene can be designed based on the human MCA gene and its surrounding nucleotide sequences.

  If a mutation is found in the MCA gene by the above detection method or fertility diagnostic kit, the subject is estimated to have a low fertility potential. Moreover, if a test subject is an infertility patient, it turns out that the cause is the defect | deletion or mutation of a MCA gene. Therefore, the disease can be cured at an early stage by applying an appropriate treatment to such a subject. That is, it can be said that the detection method of the present invention is a diagnostic method for the purpose of estimating fertility and identifying the cause of infertility.

  SEQ ID NO: 8 is obtained by extracting only the translation region from the base sequence of the transcription product of the wild-type human MCA gene registered under the accession number AB006536 in GeneBank. As shown in SEQ ID NO: 8, the wild type human MCA gene has a translation region consisting of 930 bp. SEQ ID NO: 9 shows the deduced amino acid sequence of wild-type human MCA protein.

  Wild-type human MCA protein has been reported to be particularly strongly expressed in mature male testicular germ cells, as described above. FIG. 8 is a schematic diagram showing the structure of the wild type human MCA gene. According to the human genome resource constructed by the Human Genome Project, the human MCA gene is located on the long arm of human chromosome 21. The transcript of this gene is about 1.4 kb.

  As a result of the investigation shown in Examples described later, the inventors of the present application have found that the adenine at position 92 in the translation region of the human MCA gene represented by SEQ ID NO: 8 (hereinafter simply referred to as “A” )) Is replaced with cytosine (hereinafter simply referred to as “C”) in one allele, and guanine at position 121 (hereinafter simply referred to as “G”) is replaced with A in one allele. Found that there are patients. On the other hand, the base substitutions at positions 92 and 121 were not observed at all in the group consisting of men with confirmed fertility. These substitution sites are all in the second exon of the MCA gene as shown in FIG.

  When the above-mentioned substitution mutation occurs in the human MCA gene, these sites that originally encoded histidine and glycine will encode proline and arginine, so that normal proteins cannot be expressed. Therefore, the substitution mutations at the 92nd and 121st positions of the human MCA gene, even when occurring only in one allele, due to a decrease in the translation amount of normal MCA protein, or It is speculated that the mutant MCA protein is likely to cause infertility by acting in a dominant negative manner.

  From the above, the detection method according to the present invention was isolated from genomic DNA contained in a biological sample (blood, mucous membrane inside cheek, etc.) collected from a subject (human) or from the subject's biological sample. It includes a step of detecting the presence or absence of a mutation that causes a functional defect in the human MCA gene in the genomic DNA.

  In addition, the fertility diagnostic kit according to the present invention is a mutation that causes a functional defect in the human MCA gene in genomic DNA contained in a biological sample collected from a subject or in genomic DNA isolated from a biological sample of the subject. Reagents for detecting the presence or absence of

  The above-described detection method and fertility diagnostic kit are not particularly limited as long as they detect the presence or absence of a mutation that causes a functional defect in the human MCA gene. For example, the detection method and the fertility diagnostic kit may detect that the human MCA gene of the subject contains a mutation that causes a loss of function of the gene, or the human MCA gene of the subject , It may be detected that a mutation causing a functional defect of the gene is not included.

  For example, in the case of detecting the presence of the mutation, the detection method and fertility diagnostic kit need only be able to detect that the mutation causing the functional defect occurs in at least one allele. On the other hand, when detecting the absence of the mutation, the detection method and the fertility diagnostic kit preferably detect that the mutation causing the functional defect does not occur in both alleles.

  The presence or absence of the mutation is examined by the detection method or the fertility diagnostic kit. As a result, if no mutation is detected, it can be estimated that there is a high possibility that there is no problem with fertility. Moreover, when a test subject is an infertility patient, it can be determined that there is a high possibility that the cause of infertility is due to something other than the functional defect of the MCA gene.

  On the other hand, if a mutation is detected, it can be presumed that there is a high possibility of not being low fertility or infertility. Moreover, when a test subject is an infertility patient, it can be determined that the cause of infertility is highly likely due to a functional defect in the MCA gene. Therefore, in this case, it is possible to present an appropriate selection of an existing treatment method such as TSE-ICSI (Testicular sperm extraction-intracytoplasmic sperm injection).

  Thus, according to the detection method and fertility diagnostic kit, it is possible to estimate the possibility of fertility and identify the cause of infertility. That is, according to the detection method or fertility diagnostic kit, information useful for diagnosing infertility is provided. Therefore, it can be said that the step of detecting the presence or absence of mutation in the detection method is preferably used in a method for diagnosing hypofertility or infertility.

  The detection method and the fertility diagnostic kit have the above-mentioned positions 92 and 121 of the translation region of the human MCA gene when the adenine encoding the translation initiation codon is the first base. It is preferable to detect the presence or absence of a substitution mutation in at least one base.

  Furthermore, in the detection method and fertility diagnostic kit, the 92nd base of the human MCA gene is replaced with cytosine, or the 121st base is replaced with adenine. It is preferable to detect this. Thus, by narrowing down the location where mutation is detected and the type of mutation to be detected, the presence or absence of a mutation causing low fertility or infertility can be efficiently examined.

  However, mutations that occurred in the human MCA gene (including substitution mutations, insertion mutations, and deletion mutations) are likely to cause infertility even if they are other than the substitution mutations at positions 92 and 121 described above. Therefore, the present invention is not limited to the detection of the substitution mutation at the 92nd position or the 121st position.

  In one embodiment, the detection method and the fertility diagnostic kit include at least one of the 92nd position and the 121st position (hereinafter referred to as “mutation detection site”) among the regions in the human MCA gene. The presence or absence of the above substitution mutation may be detected by determining the base sequence of the region containing the base. For the determination of the base sequence, the Sanger method or various known techniques applying the Sanger method can be used.

  In this case, it is preferable that the fertility diagnostic kit includes a primer set designed so as to sandwich the mutation detection site in order to amplify the region including the mutation detection site of the human MCA gene. More specifically, the primer set includes a primer having a part or all of the base sequence upstream from the mutation detection site of the human MCA gene and a base sequence downstream from the mutation detection site of the human MCA gene. And a primer having a base sequence complementary to part or all.

  When PCR (polymerase chain reaction) is performed using genomic DNA contained in a biological sample collected from a subject as a template and the above primer set, the region containing the base of the mutation detection site of the human MCA gene is amplified as a DNA fragment. Furthermore, if the nucleotide sequence is determined using one or both primers of the above primer set, the presence or absence of a substitution mutation can be reliably and easily detected. The base sequence can be easily determined by using, for example, ABI-PRISM 310 Genetic Analyzer (Applied Biosystems Inc.) according to the instruction manual. The primer used for determining the base sequence may be different from the primer used for PCR for amplification of the DNA fragment.

  Each primer preferably has at least 10 bases, more preferably 15 bases or more, and more preferably 18 bases or more, the same base sequence as the MCA gene or its periphery, or a complementary base sequence. It is preferable to have 20 bases or more. By lengthening the length of the same (or complementary) base sequence of the MCA gene or its surroundings, only the target region including at least one of positions 92 and 121 of the MCA gene is reliably amplified. Can be made.

  Further, in the above primer pair, the length of the region sandwiched between the primers is preferably 5 kb or less, more preferably 3 kb or less, further preferably 2 kb or less, and 1.5 kb or less. It is particularly preferred that By shortening the length of the region sandwiched between the primers, the region including the base of the mutation detection site can be effectively amplified.

  The primer used for determining the base sequence is preferably set at a position within 2 kb from the base of the mutation detection site, preferably set at a position within 1.5 kb, and within 1.0 kb. More preferably, the position is set to a position within 0.7 kb. With the above configuration, the base sequence can be accurately determined.

  Furthermore, the primer used for determining the base sequence is preferably set at a position separated by 20 bp or more from the base of the mutation detection site, more preferably set at a position separated by 30 bp or more, and separated by 40 bp or more. It is more preferable that the position is set, and it is particularly preferable that the position is set at a position separated by 50 bp or more. Moreover, it is preferable that the said primer group amplifies the area | region 20 bp or more containing the base of the mutation detection site | part of a human MCA gene. In the vicinity of the primer, the determination result of the base sequence tends to be inaccurate, but the base sequence can be accurately determined by the above configuration.

  As the above primer set, for example, primers (MCA-P3 and MCA-P4R in FIG. 1) having the nucleotide sequences shown in SEQ ID NOs: 16 and 17, respectively, can be used. Primers that can be used in the present invention It goes without saying that the base sequence is not limited to this sequence. A person skilled in the art can easily obtain the base sequence of the human MCA gene and the surrounding base sequence from the above-mentioned human genome resources and GeneBank. Therefore, based on the obtained base sequence, Oligo (registered) Primers can be designed using trademarks such as National Bioscience Inc.) or GENETYX (software development).

  Each primer can be synthesized by a known method such as a phosphoramidite method, and can be easily synthesized by using, for example, an Applied Biosystems Inc. type 392 synthesizer according to the instruction manual.

  Each primer described above may have a fluorescent label. When a fluorescent label is attached to the primer, the base sequence of the amplification region can be determined by the die primer method without using a radioisotope.

  On the other hand, when the fluorescent label is not attached to each primer, the base sequence can be determined by the dye terminator method. In this case, the fertility diagnostic kit includes deoxyribonucleic acid composed of monomers A, T, G, and C, fluorescently labeled A, T, G, and C, in addition to the above primer set. A dideoxyribonucleic acid (so-called dye terminator) comprising a monomer, a polymerase, and the like may further be included.

  In one embodiment, the detection method and fertility diagnostic kit may detect presence or absence of a base substitution mutation at the mutation detection site of the human MCA gene by an invader method. Details of the invader method are described in a paper by Lyamichev et al. (Lyamichev V et al., Nat biotechnol. 17, 292, 1999), which is incorporated herein by reference.

  FIG. 9 is a schematic diagram for explaining the principle of the invader method. In the invader method, an invader probe 2 and a signal probe 3 that are non-fluorescently labeled probes and a fret probe 5 that is a fluorescently labeled probe are used.

  The invader probe 2 has a base sequence complementary to the target gene 1 and is designed so that the base at the 3 'end corresponds to the mutation (or single nucleotide polymorphism) detection site 1a of the target gene 1. On the other hand, the signal probe 3 has a flap 3b which is a base sequence unrelated to the target gene 1 and a base sequence complementary to the target gene 1, and the base at the 5 'end is a mutation (or a target gene 1). Single nucleotide polymorphism) A nucleotide 3a designed to correspond to the detection site 1a is linked. Here, the flap 3b is linked to the 5 'end of the nucleotide 3a.

  When the probes 2 and 3 designed in this way are hybridized with the target gene 1, the target gene 1, the invader probe 2 and the signal probe 3 form a triple chain at the mutation detection site 1a. Then, the flap endonuclease (also called “cleavase”) 4 recognizes this triple chain and cleaves the signal probe 3 on the 3 ′ side of the triple chain. As a result, a flap free body 3c in which the flap 3b and the mutation detection site are linked is generated.

  The fret probe 5 is composed of a nucleotide 5a capable of hybridizing with itself on the 5 'end side and hybridizing with the flap 3b on the 3' end side, a fluorescent dye 5b, and a quencher (luminescence inhibitor) 5c. Here, nucleotide 5a has a base sequence complementary to the mutation detection site of flap educt 3c at the 5 'end. The fluorescent dye 5b is bonded to the 5 'end of the nucleotide 5a. However, in the fret probe 5, the fluorescence of the fluorescent dye 5b is suppressed by the quencher 5c.

  When the flap free body 3c hybridizes to such a fret probe 5, a triple chain is formed again at the mutation detection site of the flap free body 3c. As a result, the above-described flap endonuclease 4 recognizes this triple chain again, and the 5 'end portion of the fret probe 5 is cleaved. As a result, the fluorescent dye 5b bonded to the 5 'end of the fret probe 5 is released, and fluorescence is generated.

  Therefore, when the fertility diagnostic kit detects the presence or absence of the above substitution mutation by the invader method, the fertility diagnostic kit may include the invader probe 2, the signal probe 3, the fret probe 5, and the like. preferable.

  When fluorescence is generated when there is a substitution mutation, the invader probe 2 has a base sequence complementary to the human MCA gene, and the 3 ′ terminal base is the above-mentioned 92nd position of the mutant MCA gene. It is designed to correspond to the base (C) or the 121st base (A). The nucleotide 3a of the signal probe 3 has a base sequence complementary to the human MCA gene, and the base at the 5 ′ end is the above-mentioned 92nd base (C) or 121st position of the mutant MCA gene It is designed to correspond to the base (A). The fret probe 5 detects the flap free body 3c that is cleaved by the flap endonuclease 4 when the invader probe 2 and the signal probe 3 are hybridized with the mutant MCA gene to form a triple chain structure. Designed to be able to.

  With the above configuration, when the 92nd or 121st base of the subject MCA gene is substituted, the 3 ′ end of nucleotide 3a and the 5 ′ end of invader probe 2 overlap. Triple chains are formed and fluorescence occurs. On the other hand, when the subject's MCA gene is a wild type, no triple chain is formed at the terminal portion of the invader probe 2 and thus no fluorescence occurs.

  In the above example, fluorescence is generated when the subject's MCA gene is a substitution mutation type. However, the present invention is not limited to this, and fluorescence occurs when the subject's MCA gene is a wild type. In the case of the substitution mutant type, the fluorescence may be suppressed, and various modifications can be made.

  In one embodiment, the detection method and fertility diagnostic kit may detect the presence or absence of a base substitution mutation at the mutation detection site of the human MCA gene by the SMMD method. Details of the SMMD method are described in a paper by Wakai et al. (Wakai J et al., Nucleic Acids Res., 2004. 32 (18), e141.), Which is incorporated herein by reference.

  FIG. 10 is a schematic diagram for explaining the principle of the SMMD method. In addition, this figure has shown the example in the case of detecting the presence or absence of the substitution mutation of the said 92nd base among the variation | mutations in a human MCA gene. The SMMD method detects single nucleotide polymorphisms, substitution mutations, deletion mutations, or insertion mutations using an electrochemical reaction. First, as shown in FIG. 10, in order to amplify a region including a mutation detection site using genomic DNA 11 of a subject as a template, PCR is performed using a counter primer 13 and a special primer 12 at a ratio of 1: 4. (Process A). Here, the special primer 12 includes an oligomer 12a having a base sequence complementary to a region downstream of the mutation detection site 11a of the gene 11 whose mutation is to be detected, and a tag 12b. The tag 12b has a base sequence complementary to the region from the base one base downstream of the mutation detection site 11a of the gene 11 to the base one base upstream of the base to which the oligomer 12a is hybridized. It is linked to the 5 'end of 12a.

  As a result of the asymmetric PCR, the PCR product 14 shown in Step B is generated (Step B). Among the PCR products 14, the single-stranded asymmetric PCR product 14 including the special primer 12 includes the tag 12b, and thus forms a self-loop to form a special target (steps C and D).

  Next, the mutant type probe 20 and the wild type probe 30 are prepared. The probes 20 and 30 both have the same base sequence as the gene 11 whose mutation is to be detected, and an oligomer having the mutation detection sites 11a and 11b as the 3 ′ end is fixed to the gold electrode 25 on the 5 ′ end side. It is a thing. Here, in the mutant probe 20, the 3 ′ end of the oligomer is a mutant base (C), whereas in the wild type probe 30, the 3 ′ end of the oligomer is a wild type base (A). ing. These mutant probe 20 and wild type probe 30 can hybridize with the special target 15 because the oligomer has a base sequence complementary to the 3 ′ end of the special target 15 described above. A detailed method for immobilizing the oligomer on the gold electrode 25 is described in a paper by Takenaka et al. (Takenaka S et al., Anal Chem., 2000, 72, 1334-1341). This is used.

Before the probes 20 and 30 are hybridized with the special target 15, a background response is made in advance in a solution containing ferrocenylnaphthalene diimide (hereinafter abbreviated as “FND”), and the current I ₀ is measured. (Step E).

  Then, hybridization and ligation of the special target 15 with the mutant probe 20 and the wild type probe 30 are performed. Here, when the special target 15 is a mutant type (that is, when the subject has a mutant gene), the special target 15 is ligated because the nucleotide sequence is completely identical to the mutant probe 20. On the other hand, since the base sequence is different from that of the wild-type probe 30, the ligation is not performed (step F).

  Thereafter, when a denaturation treatment is performed, the mutant special target 15 is dissociated from the wild type probe 30 in the combination of the wild type probe 30 and the mutant special target 15. On the other hand, in the set of the mutant probe 20 and the mutant special target 15, since ligation is performed, dissociation does not occur (step G).

  Then, each sample is washed to remove excess DNA, FND is added, and the electrochemical current I1 of the FND molecule is measured (step H). Here, in the electrode having the double-stranded DNA, the current I1 is larger than that of the electrode having the single-stranded DNA (Step I).

  Thereby, it can determine based on the electric current value which measured whether a test subject's genomic DNA was a mutant type or a wild type. In the SMMD method, a large number of mutations can be detected simultaneously by forming the mutant probe 20 and the wild-type probe 30 as an ECA (Electrochemical array) chip in an array.

  Therefore, when the fertility diagnostic kit according to the present invention detects the presence or absence of the above substitution mutation by the SMMD method, the fertility diagnostic kit includes a special primer 12, a counter primer 13, a mutant probe 20, and It is preferable that the wild-type probe 30 and the like are included. Only one of the mutant probe 20 and the wild-type probe 30 may be used.

  The special primer 12 includes an oligomer 12a having a base sequence complementary to a predetermined region downstream from the mutation detection site of the human MCA gene, and a tag 12b. The tag 12b has a base sequence complementary to the region from the mutation detection site of the human MCA gene to the site where the oligomer 12a hybridizes, and is linked to the 5 'end of the oligomer 12a. On the other hand, the counter primer 13 has the same base sequence as the predetermined region upstream from the mutation detection site of the human MCA gene.

  The mutant probe 20 is an oligomer having the same base sequence as the human mutant MCA gene and having the 92nd base (C) or the 121st base (A) at the 3 ′ end. Designed to have The wild-type probe 30 has the same base sequence as the human wild-type MCA gene, and the above-mentioned base 92 (A) or base 121 (G) is the 3 ′ end of the oligomer. Designed to have

  According to the above configuration, when the subject's MCA gene is a substitution mutant type, the special target 15 can be ligated with the mutant probe 20 but cannot be ligated with the wild type probe 30. On the other hand, when the subject's MCA gene is wild-type, the special target 15 can be ligated with the wild-type probe 30 but cannot be ligated with the mutant probe 20.

  In each of the above methods, the configuration for determining the presence or absence of a substitution mutation by examining the sense strand of the MCA gene (codon-encoding strand) has been described in detail, but the same can be said by examining the antisense strand. Needless to say, it is possible to make a judgment.

(8) Modification of Detection Method and Fertility Diagnostic Kit According to the Present Invention In the above, the fertility possibility and the cause of infertility were identified by examining the genomic DNA of a sample collected from a human. It is possible to estimate fertility and identify the cause of infertility at the protein level as well.

  That is, in one embodiment, the detection method and fertility diagnostic kit according to the present invention may determine whether normal human MCA protein is expressed in humans. More specifically, the detection method includes a step of determining whether or not the human MCA protein is expressed in a biological sample collected from a human using an anti-human MCA antibody that binds to the human MCA protein. You may go out. The fertility diagnostic kit may contain an anti-human MCA antibody as a reagent.

  In the above detection method and fertility diagnostic kit, the method for determining the presence or absence of human MCA protein expression using an anti-human MCA antibody is not particularly limited, and known Western blotting, ELISA (Enzyme-linked Immunosorbent Assay) and the like are known. Can be mentioned. Examples of biological samples collected from humans include testicular tissue (eg, seminiferous tubules), sperm cells, or seminal plasma collected from a subject by testicular biopsy. The seminal plasma is considered to contain a protein expressed in the sperm cell, which is released when the sperm cell is destroyed, even if it does not contain the sperm cell. Moreover, as an anti-human MCA antibody, the polyclonal antibody etc. which were disclosed by the nonpatent literature 3 mentioned above etc. can be used, for example. The polyclonal antibody described in the above document is prepared using a mouse antigen, but it has also been confirmed to bind to human MCA protein. Of course, other anti-human MCA antibodies may be used.

  When the fertility diagnostic kit uses Western blotting or ELISA, the fertility diagnostic kit includes anti-globulin labeled with a specific enzyme such as peroxidase necessary for the color reaction in addition to the anti-human MCA antibody. An antibody may be included.

  It is determined whether or not human MCA protein is expressed using an anti-human MCA antibody. If it is determined that human MCA protein is not expressed in a subject, the human MCA gene may be deleted or mutated. At the same time, it can be estimated that the subject is less likely to be fertile or likely to be infertile. Moreover, when a test subject is infertility, it can be estimated that the cause of infertility is due to the fact that normal human MCA protein is not expressed.

  In another embodiment, the detection method and fertility diagnostic kit according to the present invention are encoded by a human mutant MCA gene containing a base substitution of at least one of positions 92 and 121 described above. An antibody that binds to the human mutant MCA protein and does not bind to the human wild type MCA protein is used to determine whether the human mutant MCA protein is expressed in a biological sample collected from humans. There may be. Details of the antibody used in this case will be described later.

  Whether or not the human mutant MCA protein is expressed using the antibody is determined, and if it is determined that the human mutant MCA protein is expressed in the subject, the substitution mutation described above has occurred in the human MCA gene. At the same time, it can be estimated that the subject is less likely to be fertile or likely to be infertile. Moreover, when a test subject is infertile, it can be estimated that the cause of infertility originates in abnormality of a human MCA gene.

(9) Polynucleotide, polypeptide, expression vector and expression kit according to the present invention Next, the polynucleotide according to the present invention will be described. The polynucleotide according to the present invention is a nucleotide sequence in which the 92nd base is substituted with C in the base sequence of the translation region of the human wild type MCA gene when the adenine encoding the translation initiation codon is the 1st base. It consists of In addition, another polynucleotide according to the present invention provides that the base at position 121 is A in the base sequence of the translation region of the human wild-type MCA gene when adenine encoding the translation initiation codon is the base at position 1. It consists of a substituted base sequence. Furthermore, in another polynucleotide according to the present invention, when the adenine encoding the translation initiation codon is the first base, the 92nd base is C in the base sequence of the translation region of the human wild type MCA gene. And a base sequence in which the base at position 121 is substituted with A.

  In one embodiment, the polynucleotide according to the present invention may consist of the base sequence shown in any one of SEQ ID NOs: 10 to 12. However, the polynucleotide according to the present invention is not limited to the nucleotide sequence shown in any one of SEQ ID NOs: 10 to 12. In the polynucleotide according to the present invention, not only the base sequence shown in any one of SEQ ID NOs: 10 to 12, but also one or several bases are deleted in the base sequence shown in any one of SEQ ID NOs: 10 to 12, A polynucleotide comprising a substituted or added polynucleotide and involved in hypofertility or infertility is also included. In particular, in the nucleotide sequence shown in any one of SEQ ID NOs: 10 to 12, 1 or Those having a base sequence in which several bases are substituted are also included.

  Here, the term “several” means a number that can be made by a person skilled in the art without performing trial and error or complicated advanced experiments exceeding the level that can be expected by those skilled in the art. More preferably, it is 10 or less, and more preferably 5 or less.

  As used herein, “polynucleotide” is synonymous with “gene”, “nucleic acid”, or “nucleic acid molecule” and refers to a polymer of nucleotides. In the present specification, “base sequence” is synonymous with “nucleic acid sequence” or “nucleotide sequence” and is shown as a sequence of deoxyribonucleotides (abbreviated as A, G, C, and T).

  The polynucleotide may be in the form of RNA (for example, mRNA) or DNA (for example, synthetic DNA, cDNA, or genomic DNA). Furthermore, in the case of DNA, it may be either double-stranded or single-stranded. Furthermore, the single-stranded DNA or RNA may be a coding strand (also known as a sense strand) or a non-coding strand (also known as an antisense strand).

  The polynucleotide may be linked to a polynucleotide encoding a tag label (tag sequence or marker sequence) at the 5 'end or 3' end.

  Examples of the method for obtaining the polynucleotide include a method using PCR. Specifically, it can be obtained by PCR using genomic DNA of a person having a mutant MCA gene as a template, or by performing PCR using a primer with a mutation, from a wild type MCA gene. It can also be obtained.

  In addition, the polynucleotide may be inserted into a recombinant vector. This recombinant vector may be a vector used for in vitro translation or a vector used for recombinant expression.

  The specific type of vector is not particularly limited, and a vector that can be expressed in a host cell can be appropriately selected. That is, according to the type of the host cell, an appropriate promoter sequence is selected appropriately for the expression of the human mutant MCA protein from the above polynucleotide, and a vector incorporating this and the above polynucleotide in various plasmids is used as an expression vector. Use it.

  The expression vector contains an expression control region (for example, a promoter, an enhancer, a terminator, and / or an origin of replication) depending on the type of host to be introduced. Conventional promoters (eg, trc promoter, tac promoter, lac promoter, etc.) are used as promoters for bacterial expression vectors, and examples of yeast promoters include glyceraldehyde 3-phosphate dehydrogenase promoter, PH05 promoter, etc. Examples of the promoter for filamentous fungi include amylase and trpC. Examples of animal cell host promoters include viral promoters (eg, SV40 early promoter, SV40 late promoter, etc.). The expression vector can be prepared according to a conventional technique using a restriction enzyme and / or ligase. Transformation of the host with the expression vector can also be performed according to conventional techniques.

  A host transformed with the above expression vector is cultured, cultivated or bred, and then subjected to conventional techniques (eg, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography) from the culture. Etc.), the mutant MCA protein can be recovered and purified.

  A method for introducing the expression vector into a host cell, that is, a transformation method is not particularly limited, and a conventionally known method such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method can be suitably used. . In addition, for example, when a polypeptide encoded by the polynucleotide is expressed in insects, an expression system using baculovirus may be used.

  By using the recombinant vector, the polynucleotide can be introduced into an organism or cell, and as a result, a mutant MCA protein can be expressed in the organism or cell. Furthermore, if the above recombinant vector is used in a cell-free protein synthesis system, a mutant MCA protein can be selectively synthesized.

  Moreover, you may make it the form of the expression kit used in order to express the mutant MCA protein mentioned above in a cell including the recombinant vector mentioned above.

  The expression kit may contain a reagent other than the above recombinant vector. Other reagents include, for example, an introduction reagent for introducing a recombinant vector into a host cell, the host cell, a probe group for testing whether the recombinant vector has been introduced into the host cell, and the like. However, it is not limited to this. That is, any reagent may be included as long as the reagent supports the expression experiment of the mutant MCA protein in a reliable and simple manner. By using the above expression kit, the mutant MCA protein can be reliably and easily expressed in the target host cell.

  The polypeptide according to the present invention consists of an amino acid sequence encoded by the above-described polynucleotide.

  In one embodiment, the polypeptide may consist of the amino acid sequence shown in any of SEQ ID NOs: 13 to 15. However, the polypeptide according to the present invention is not limited to the amino acid sequence shown in any of SEQ ID NOs: 13 to 15. In the polypeptide according to the present invention, not only the amino acid sequence shown in any of SEQ ID NOs: 13 to 15, but also one or several amino acids in the amino acid sequence shown in any of SEQ ID NOs: 13 to 15 are deleted, A polypeptide comprising an amino acid sequence that is substituted or added and that is involved in hypofertility or infertility is also included. In particular, in the amino acid sequence shown in any of SEQ ID NOs: 13 to 15, 1 or An amino acid sequence in which a plurality of amino acids are substituted is also included.

  Here, the term “several” means a number that can be made by a person skilled in the art without performing trial and error or complicated advanced experiments exceeding the level that can be expected by those skilled in the art. More preferably, it is 10 or less, and more preferably 5 or less.

  In the present specification, “polypeptide” is synonymous with “peptide” or “protein”. The polypeptide according to the present invention may be isolated from a natural source or chemically synthesized.

  An “isolated” polypeptide refers to a polypeptide that has been removed from its natural environment. For example, a recombinantly produced polypeptide expressed in a host cell is considered isolated, as are natural or recombinant polypeptides that have been substantially purified by any suitable technique. The polypeptide according to the present invention can be obtained according to the method for using the polynucleotide according to the present invention described above.

  Polypeptides according to the present invention include natural purified products, products of chemical synthesis procedures, and prokaryotic or eukaryotic hosts (eg, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells). ) Including products produced by recombinant techniques. The polypeptide may be glycosylated or non-glycosylated depending on the host used in the recombinant production procedure. Furthermore, the polypeptide may have a modified methionine residue that indicates translation initiation as a result of a host-mediated process.

  According to the polynucleotide, expression vector, or polypeptide of the present invention, a mutant MCA protein found in human infertility patients can be obtained. Therefore, these inventions are intended to develop a drug and a therapeutic method for the treatment of hypofertility or infertility caused by a substitution mutation in at least one of the base positions of positions 92 and 121 of the human MCA gene. It is a very useful tool and can be sold to research sites. Moreover, industrial applicability improves further by making these into kits.

(10) Antibody according to the present invention The antibody according to the present invention binds to a human mutant MCA polypeptide (that is, the above-described polypeptide according to the present invention) and does not bind to a human wild-type MCA polypeptide. . Using the antibody according to the present invention, as described above, estimation of fertility and identification of the cause of infertility can be detected at the protein level.

  In one embodiment, the antibody according to the present invention is preferably one that specifically binds to a polypeptide encoded by a polynucleotide comprising the nucleotide sequence shown in any one of SEQ ID NOs: 10 to 12. In other words, the antibody according to the present invention preferably binds specifically to a polypeptide consisting of the amino acid sequence shown in any one of SEQ ID NOs: 13 to 15.

  The antibody according to the present invention can be produced by immunizing an experimental animal with a human mutant MCA polypeptide containing the above-mentioned amino acid substitution mutation or a fragment thereof containing a mutation part as an antigen. As a specific antibody production procedure, a conventionally known method can be used. See, for example, Chow, M. et al., Proc. Natl. Acad. Sci. USA 82: 910-914; and Bittle, FJ et al, J. Gen. Virol. 66: 2347-2354 (1985). .

  In general, animals can be immunized with free peptides. However, it may be possible to efficiently immunize by coupling the free peptide to a macromolecular carrier (eg, keyhole limpet hemocyanin (KLH) or tetanus toxoid). For example, a peptide containing cysteine can be coupled to a carrier by a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). On the other hand, other peptides can be coupled to the carrier using more common linking agents such as glutaraldehyde.

  Animals such as rabbits, rats and mice can be immunized by intraperitoneal and / or intradermal injection of emulsions containing free or carrier-coupled peptides and Freund's adjuvant. In order to obtain an anti-peptide antibody with a useful titer that can be detected by ELISA, booster injections are preferably performed at intervals of about 2 weeks.

  The antibody can be obtained by collecting blood from the immunized animal and extracting the IgG fraction from the serum. Further, the antibody titer is improved by purifying the antibody by a known technique.

  The antibody according to the present invention binds to the above-described human mutant MCA polypeptide, but does not bind to human wild-type MCA polypeptide. Therefore, the antibody according to the present invention can be obtained by selecting antibodies that do not bind to human wild-type MCA protein from the antibodies obtained by the above procedure. In order to facilitate the above selection, it is preferable to select an oligopeptide having about 10 residues consisting of the mutated portion of the human mutant MCA polypeptide and the surrounding amino acid sequence as the antigenic peptide to be injected into the animal.

  In the present specification, the base sequence shown in SEQ ID NO: 8 is exemplified as a representative base sequence of the translation region of the human wild-type MCA gene. However, the “human MCA gene” and “human” described in the claims are exemplified. The translation region of “wild-type MCA gene” is not limited to the nucleotide sequence shown in SEQ ID NO: 8. This is because there are many single nucleotide polymorphisms in human genes, and human MCA genes are no exception. Therefore, the “human MCA gene” and “human wild-type MCA gene” described in the claims include not only the nucleotide sequence represented by SEQ ID NO: 8, but also one or several nucleotide sequences represented by SEQ ID NO: 8. Having a translation region in which the base of is deleted, substituted or added, and involved in fertility (that is, a function equivalent to a polypeptide whose amino acid sequence is represented by the amino acid sequence shown in SEQ ID NO: 9) In particular, a polynucleotide having a base sequence in which one or several bases are substituted by a single nucleotide polymorphism in the base sequence shown in SEQ ID NO: 8 as a translation region is also included.

  Here, the term “several” means a number that can be made by a person skilled in the art without performing trial and error or complicated advanced experiments exceeding the level that can be expected by those skilled in the art. More preferably, it is 10 or less, and more preferably 5 or less. The same applies to the human mutant MCA gene.

  Hereinafter, an embodiment of the present invention will be described with reference to FIGS. 2 to 5, but the present invention is not limited thereto. In this example, a disease model animal according to the present invention was prepared by deleting the MCA gene of a mouse (Mus musculus). In each of the following steps, the treatment was performed according to a conventional method or an instruction manual attached to the kit unless otherwise specified.

[Preparation of MCA homo-deficient mice]
FIG. 1 is a physical map of the first exon of mouse MCA gene and its surroundings. The base sequence corresponding to the region shown in FIG. The base sequence shown in SEQ ID NO: 1 is the base sequence from positions 17582317 to 176222316 of chromosome 17 registered in the NCBI Mouse Genome Resource. In the NCBI Mouse Genome Resource, MCA is also referred to as Tsga2. In the figure, the region labeled “first exon” (corresponding to the nucleotide sequence from position 17848 to position 17991 of SEQ ID NO: 1) corresponds to the first exon of the MCA gene, and the right side is the MCA gene. Upstream and downstream on the left. In this example, this first exon was replaced with a neomycin resistance gene cassette (including promoter and translation region) so that normal MCA protein was not expressed.

  The targeting vector includes (a) a 5 ′ arm sequence consisting of the base sequence from positions 19218 to 23000 of SEQ ID NO: 1, and (b) a base sequence from positions 10021 to 16250 of SEQ ID NO: 1. And (c) a neomycin resistance gene cassette (neo cassette) sandwiched between the 5 ′ arm sequence and the 3 ′ arm sequence. The neo cassette was inserted in such a direction that the left side in the figure was upstream and the right side was downstream (that is, opposite to the MCA gene). FIG. 2 is a schematic diagram showing the structure of a targeting vector.

  Next, the prepared targeting vector of FIG. 2 was digested with a restriction enzyme to change the targeting vector from circular to linear. The linear targeting vector was then introduced into W9.5 ES cells by electroporation. After the introduction, ES cells into which the targeting vector was introduced were cultured in a culture solution to which G418 ganciclovir was added to obtain a G418 gancyclovir resistant clone.

And about the obtained resistant clone, it was investigated by the following three methods whether homologous recombination was performed without a problem (whether targeting was performed). In the first method, a neo1746f primer (SEQ ID NO: 2: 5′-cctgccataggcctcagttactc-3 ′) consisting of a base sequence present in the 3 ′ end region of the neomycin resistance gene and 23250r outside the 5 ′ arm sequence shown in FIG. PCR was performed using a pair with a primer (SEQ ID NO: 5: 5′-gcagagggaggcgaggctcacacaggg-3 ′) to examine whether the targeting on the 5 ′ end side was normally performed. At this time, The Expand Long Template PCR System manufactured by Roche was used as the reagent, and the reaction conditions were as follows.
(1) 94 ° C for 5 minutes (2) 94 ° C for 30 minutes, 58-61 ° C for 1 minute, 68 ° C for 5 minutes, 35 cycles (3) 68 ° C for 10 minutes In a sample of the cloned clone, a fragment of about 4.2 kb was detected.

  In the second method, it was examined whether or not the targeting was normally performed by Southern analysis. At this time, the genomic DNA of the clone was digested with XbaI, and a 5 'probe (see FIG. 1) consisting of the nucleotide sequence from position 23051 to position 23750 of SEQ ID NO: 1 was used as the probe. This 5 ′ probe was obtained by using a wild-type mouse genomic DNA as a template and using 23051F primer (SEQ ID NO: 5: 5′-gttggcccagggaagactcttttctc-3 ′) and 23750R primer (SEQ ID NO: 5: 5′-taacgctgttagcccaatagg-3 ′). It was prepared by performing PCR. As a result of Southern analysis, a band of about 12.6 kb was detected in the sample of the wild type clone, and a band of about 16.5 kb was detected in the sample of the clone being targeted.

  In the third method, a neo6r consisting of a 9725f primer (SEQ ID NO: 6: 5′-tcctgcctcatcctgtttttaatgg-3 ′) outside the 3 ′ arm sequence shown in FIG. 1 and a base sequence present in the 5 ′ end region of the neomycin resistance gene. PCR was performed using a pair with a primer (SEQ ID NO: 7: 5′-catagccgaatagccctctcc-3 ′) to examine whether targeting on the 3 ′ end side was normally performed. The PCR conditions were basically the same as those in the first method described above, but the time for the extension step ((2) treatment at 68 ° C.) was 7 minutes. As a result, a fragment of about 7.1 kb was detected from the targeted clones.

  As described above, ES cell clones confirmed to be targeted were injected into C57BL / 6J blastocysts to obtain male chimeric mice. The obtained chimeric mouse was mated with a wild-type female mouse to obtain an MCA hetero-deficient mouse (F1). The identification of MCA hetero-deficient mice was performed by the above-described first method using PCR. FIG. 3 shows representative screening results. As indicated by the arrows in the figure, in some lanes (lanes 11, 12, and 15), a band of about 4.2 kb indicating that MCA hetero-deficient mice were obtained was detected. The obtained MCA hetero-deficient mice were crossed to obtain MCA homo-deficient mice.

  Next, Western blot was performed to examine the expression of MCA protein in the testis. The anti-MCA antibody described in Non-Patent Document 3 was used for Western blotting. This antibody was obtained by immunizing a fusion protein of mouse full-length MCA protein and GST protein described in Non-Patent Document 3, and was found to react specifically with MCA in mammalian cells. It is what. The detailed procedure of Western blot is as follows.

First, about 40 μg of testis protein was developed by SDS-PAGE, and then transferred to a polyvinyl difluoride (PVDF) filter by semi-dry method (80 min at 1 mA / cm ² ). The filter was blocked with 5% skim milk and treated overnight at 4 ° C. with the anti-MCA antibody diluted 1000 times with TBS (25 mM Tris-HCl pH = 7.4, 150 mM NaCl, 50 mM KCl). Next, the treatment of washing the filter with TBS for 5 minutes was repeated three times, and the filter was washed with anti-rabbit antibody (DAKO, A / S, Denmark) conjugated with peroxidase (POD) diluted 750 times with TBS at room temperature. Time processed. After the treatment, the treatment of washing the filter with TBS for 5 minutes was repeated three times, and the signal was detected with a POD immunostain set (Wako, Osaka, Japan).

  FIG. 4 is a diagram showing the results of Western blotting. In the figure, “+ / +” indicates a wild type mouse, “+/−” indicates an MCA hetero-deficient mouse, and “− / −” indicates an MCA homo-deficient mouse. As shown in FIG. 4, it was shown that MCA protein was not expressed at all in MCA homo-deficient mice.

  Northern blots were also performed to examine the expression of MCA transcripts in the testis. Northern blotting was performed by the following procedure using the full length MCA cDNA as a probe.

  About 5 mm square each tissue (brain, heart, kidney, liver, lung, muscle, spleen, testis, salivary gland) removed from a mature mouse is placed in a 1.5 mL microtube and 1 ml of TRISOL (registered trademark) reagent (GIBCO BRL). , NY, USA), homogenized on ice, and left at room temperature for 5 minutes. Thereafter, 200 μl of chloroform was added and stirred vigorously, allowed to stand at room temperature for 5 minutes, and then centrifuged at 4 ° C. and 13500 rpm for 10 minutes. Next, the upper layer was transferred to a new tube, 1 ml of 100% ethanol was added and stirred, and centrifuged at 4 ° C. and 13500 rpm for 10 minutes. The supernatant was discarded, 1 ml of 70% ethanol was added and stirred, centrifuged at 4 ° C. and 13500 rpm for 10 minutes, the supernatant was discarded again, the precipitate was lightly dried, and then dissolved in DEPC-treated water. Thereby, total RNA was obtained.

  Next, 20 μg of total RNA was denatured at 65 ° C. for 20 minutes, and then electrophoresed on a 1% agarose gel containing 0.66 M formaldehyde. The gel was stained with acridine orange to confirm 18S and 28S ribosomal RNA, and then RNA was transferred to a Zeta-Probe (registered trademark) Blotting Members filter (BIO-RAD, CA, USA) by a capillary transfer method. Thereafter, the filter was treated with 2 × SSC for 5 minutes and baked at 80 ° C. to fix the RNA to the filter.

Subsequently, the filters were prehybridized for 1 hour in PerfectHyb (registered trademark) Hybridization Solution (TOYOBO, Osaka, Japan) at 65 ° C. Subsequently, the full length MCA fragment was labeled with ³² P using BcaBest (registered trademark) random primer kit (Takara, Osaka, Japan) to prepare a probe, and the probe was added to PerfectHyb (registered trademark) Hybridization Solution at 60 ° C. The filter was immersed in the hybridization solution of No. 2 and hybridization was performed for 2 hours. The filter was washed with 1 × SSC containing 1% SDS at room temperature for 10 minutes. Thereafter, the filter was washed twice with 0.2% SSC containing 0.1% SDS at 60 ° C. for 30 minutes, and a signal was detected with the bioimaging analyzer system BAS1000 (Fuji Film, Japan).

  FIG. 5 is a diagram showing the results of Northern blotting for testis samples. “+ / +”, “+/−”, and “− / −” in the figure are the same as those in the western blot in FIG. As shown in FIG. 5, in the MCA homo-deficient mice, no MCA transcript was observed, confirming that the gene was surely deleted.

[Analysis of MCA-deficient mice]
Next, the characteristics of the obtained MCA-deficient mice were analyzed. Specifically, testis and accessory testis were excised from each of MCA hetero-deficient mice and MCA homo-deficient mice, and section samples were prepared. Then, the prepared section specimen was stained and observed with a microscope. The preparation of the section specimen and the staining method are as follows.

  The testis and accessory testis were removed from a mature mouse, the excised tissue was fixed overnight at 4 ° C. with a Buan solution and embedded in paraffin, and the extracted tissue was not fixed and Tissue-Tek Compound (Sakura Finetechnical Co. Ltd, Tokyo, Japan), and a section having a thickness of 7 μm was prepared from what was embedded and cooled to −20 ° C. Next, an unfixed tissue embedded in Tissue-Tek Compound was fixed with 100% alcohol or paraformaldehyde, and then subjected to an antibody reaction. Specifically, it was treated overnight at 4 ° C. with an anti-MCA antibody diluted 1000 times with PBS, and washing with PBS at room temperature for 5 minutes was repeated three times. Thereafter, the POD-conjugated anti-rabbit antibody (Amersham) diluted 750 times with PBS was treated for 4 hours at room temperature. Finally, washing with PBS for 5 minutes at room temperature was repeated three times, followed by coloring with diaminobenzimide (DAB) and counterstaining with hematoxylin.

  FIG. 6 is a diagram showing microscopic images of testis, accessory testis tail, and accessory testis head extracted from MCA hetero-deficient mice, and FIG. 7 shows testis, accessory testicular tail, accessory testis head extracted from MCA homo-deficient mice. It is a figure which shows a microscope image. As shown in FIG. 6, in the MCA hetero-deficient mice, no abnormality was observed in any of the testis, accessory testicular tail, and accessory testicular head. There was no particular problem with fertility of MCA hetero-deficient mice. On the other hand, in the MCA homo-deficient mouse, as shown in FIG. 7, no remarkable abnormality was observed in the testis, but no sperm was present in the accessory testicular tail and accessory testis head. For this reason, MCA homo-deficient mice showed azoospermia and were infertile.

  As a result of examining the testis weight of MCA homo-deficient mice, it was suggested that sperm cell differentiation after meiosis was advanced because there was no significant change compared to the wild type. As a result of detailed observation of testicular sperm cells, the number of sperm cells after meiosis was almost the same as that of wild-type mice. However, sperm flagella folds and nuclei did not extend like rods like wild-type mice and remained in a non-uniform circle, indicating that spermatogenic dysplasia in the testis occurred. It was. From this, it is considered that spermatozoa with incomplete flagellum formation in the testis did not migrate well to the subtestis after spermatogenesis, and therefore no sperm was found in the subtestis. The sperm morphology is currently being further analyzed with an electron microscope.

  MCA protein has been reported to be expressed during meiosis as described above. Thus, MCA homo-deficient mice were examined for abnormalities in the meiosis phase of the testis and an increase in the number of sperm cells causing apoptosis. As a result, it became clear that the number of sperm cells causing apoptosis increased. However, because spermatogenesis was observed and there was no significant difference in testis weight, normal testicular haploid sperm cells were not formed due to flagellar formation defects during the spermatogenesis stage, and sperm cells were phagocytosed by Sertoli cells. As a result, it is thought that the spermatozoon was not present in the accessory testis, resulting in azoospermia.

  Hereinafter, another embodiment of the present invention will be described, but the present invention is not limited thereto. In this example, substitution mutations in the human MCA gene were analyzed. In each of the following steps, each treatment was performed according to a conventional method or an instruction manual attached to the kit unless otherwise specified.

〔participant〕
Infertile Japanese male subjects (N = 245) were divided into subgroups based on the degree of spermatogenesis deficiency. Of these infertile subjects, 97 (40%) had non-occlusive azoospermia and 148 (60%) had non-occlusive severe hypospermia (<5 × 10 ⁶ cells / mL). It was. When examined for genetic factors, all of these subjects had primary idiopathic infertility (Birmingham A et al. Fertil Steril 2004; 9: 2313-2317).

  On the other hand, the father of a child born to a pregnant woman in the obstetrics and gynecology clinic was selected as a control group (N = 172) of males with confirmed fertility.

  The donor agreed to use blood for analysis of genomic DNA in this study.

[Identification of MCA gene mutation by direct sequencing of PCR amplified DNA]
In each of the following steps, unless otherwise specified, the treatment was performed according to a conventional method or an instruction manual attached to the kit. Genomic DNA was isolated from blood samples by protease treatment and phenol extraction (Sambrook J et al., Isolation of DNA from Mammalian Cells. New York: Cold Spring Harbor Press. 1989: pp. 9.16-9.21). Moreover, in order to amplify the region containing the second exon and the third exon of the MCA gene, MCA-P3 and MCA-P4R were designed as primer pairs used for PCR (see FIG. 8). As shown in FIG. 8, MCA-P3 consists of the same base sequence (SEQ ID NO: 16: 5′-TAGCATAGTGGGATGAACTCAAGGG-3 ′) as the upstream region of the second exon of the MCA gene. MCA-P4R is composed of a base sequence complementary to the downstream region of the third exon of the MCA gene (SEQ ID NO: 17: 5′-AACTTCTGTAACACGAAAATCTGC-3 ′).

  PCR was performed under the following conditions: denaturation at 98 ° C. for 10 seconds, annealing at 60 ° C. for 5 seconds, and extension at 72 ° C. for 30 seconds as one cycle for 40 cycles. The PCR amplified fragment was purified using a SUPREC PCR spin column (Takara Bio).

  The fragments amplified by the primer set consisting of MCA-P3 and MCA-P4R were sequenced using the same MCA-P3 and MCA-P4R, respectively. At this time, the reaction was performed using a thermal cycle sequencing kit (Applied Biosystems), and the reaction product was analyzed by ABI-PRISM 310 Genetic Analyzer (Applied Biosystems). The base sequence was determined by comparing the determination results in both directions.

〔result〕
When the nucleotide sequences of exons 2 and 3 of the MCA gene were analyzed for infertile subjects and fertility confirmed subjects, adenine at position 92 in the translation region of MCA gene was replaced with cytosine in one allele. 3 subjects were found among 245 infertile subjects. Of these subjects, two had azoospermia and one had severe hypospermia. On the other hand, this substitution mutation was not found at all in fertile confirmed subjects.

  Furthermore, one test subject in which guanine at position 121 in the translation region of the MCA gene was substituted with adenine in one allele was found among 245 infertile subjects. This subject had severe hypospermia. On the other hand, this substitution mutation was not found at all in fertile confirmed subjects.

  When the adenine base at position 92 in the translation region of the human MCA gene shown in SEQ ID NO: 8 is replaced with a cytosine base, this site encodes proline. In addition, when the guanine base at position 121 is substituted with an adenine base, this site encodes arginine.

  Because the MCA gene is widely conserved in mammals and the protein is particularly strongly expressed in testicular haploid germ cells, mutations in the MCA protein are thought to cause (or are likely to cause) male infertility. A subject who has found the above-mentioned substitution mutation has an insufficient function of the MCA protein because the translation amount of the normal MCA protein is reduced or the mutant MCA protein acts in a dominant negative manner. Presumed to have been infertile.

  In this study, single nucleotide polymorphisms were found in the MCA gene in addition to the substitution mutations described above, but these single nucleotide polymorphisms were observed in infertile and fertility confirmed subjects. There was no significant difference between the two, and it was not found to affect fertility in particular.

  The present invention is not limited to the embodiments and examples described above, and various modifications are possible within the scope of the claims. That is, embodiments obtained by combining technical means appropriately changed within the scope of the claims are also included in the technical scope of the present invention.

  According to the present invention, a tool useful for elucidating the phenomenon of infertility, establishing a treatment method, and diagnosing infertility is provided. Therefore, the present invention can be suitably used in the pharmaceutical field and the medical field.

It is a figure which shows the 1st exon of the MCA gene in a mouse | mouth, and the physical map of the periphery. It is a figure which shows the structure of the targeting vector used for preparation of a MCA deficient mouse. It is a figure which shows the example of the result of PCR performed in order to investigate the genotype of a mouse | mouth. It is a figure which shows the result of the Western blot performed in order to confirm the expression of MCA protein. It is a figure which shows the result of the Northern blot performed in order to confirm the expression of a MCA transcription product. It is a figure which shows the microscope image of the testis of the MCA hetero-deficient mouse, the accessory testicular tail, and the accessory testicular head. It is a figure which shows the microscope image of the testis of the MCA homo-deficiency mouse | mouth, the accessory testicular tail, and the accessory testicular head. It is a physical map which shows the structure of a human MCA gene. It is a figure explaining the principle of an invader method. It is a figure explaining the principle of SMMD method.

Explanation of symbols

1 target gene 1a mutation detection site 2 invader probe 3 signal probe 3a nucleotide 3b flap 3c flap free body 4 flap endonuclease 5 fret probe 5a nucleotide 5b fluorescent dye 5c quencher 11 gene 11a / 11b mutation detection site 12 special primer 12a oligomer 12b Tag 13 Counter primer 14 PCR product 15 Special target 20 Mutant probe 25 Gold electrode 30 Wild type probe

Claims (23)

  A disease model animal, wherein the animal is a male non-human mammal that has reduced normal MCA protein expression and exhibits low fertility or infertility.   A disease model animal, which is a male non-human mammal that does not express normal MCA protein.   A disease model animal, which is a male non-human mammal deficient in the MCA gene.   The disease model animal according to any one of claims 1 to 3, wherein the non-human mammal is a mouse.   A cell isolated from the disease model animal according to any one of claims 1 to 3.   A cell characterized in that the MCA gene is deficient or mutated and isolated from a human male exhibiting low fertility or infertility.   A testis tissue or testis isolated from the disease model animal according to any one of claims 1 to 3 or the human male according to claim 6.   A cross used for mating to produce the disease model animal according to any one of claims 1 to 3, wherein the mating is a non-human mammal deficient in part or all of the MCA gene. For animals. A germ cell production method for producing low fertile or infertile male germ cells comprising:
A gene deletion step of deleting the MCA gene possessed by a mammalian cell which is a cultured cell derived from a male mammal or a cell isolated from a male mammal;
A method of producing a germ cell, comprising a differentiation induction step of differentiating a mammalian cell deficient in the MCA gene into a germ cell by the gene deletion step. A germ cell of a male non-human mammal,
A germ cell that is deficient in MCA gene and exhibits low fertility or infertility. A cultured cell derived from a human male,
A cultured cell characterized by being a germ cell deficient in the MCA gene and exhibiting low fertility or infertility. A screening method for screening candidate compounds for therapeutic agents for hypofertility or infertility, comprising:
A disease model animal according to any one of claims 1 to 3, a cell according to claim 5 or 6, a tissue or testis according to claim 7, and a germ cell production method according to claim 9. A screening method comprising the step of allowing the candidate compound to act on a male germ cell, the germ cell according to claim 10, or the cultured cell according to claim 11, and evaluating the result. .   A fertility diagnostic kit comprising a reagent for detecting the presence or absence of a deficiency or mutation in the human MCA gene.   The above-mentioned reagent detects the presence or absence of a mutation in the 92nd position of the translation region of the human MCA gene when the adenine encoding the translation initiation codon is the 1st position. Item 14. The fertility diagnostic kit according to Item 13.   The reagent is for detecting the presence or absence of a mutation in the 121st base of the translation region of the human MCA gene when the adenine encoding the translation initiation codon is the 1st base. Item 14. The fertility diagnostic kit according to Item 13. A detection method for detecting a gene defect or mutation in a human using a biological sample collected from a human,
A detection method comprising a step of detecting the presence or absence of a deficiency or mutation in a human MCA gene.   A polymorphism comprising a base sequence in which the base at position 92 is replaced with cytosine in the base sequence of the translation region of the human wild-type MCA gene when adenine encoding the translation initiation codon is defined as the base at position 1. nucleotide.   A polymorphism comprising a base sequence in which the base at position 121 is replaced with adenine in the base sequence of the translation region of the human wild-type MCA gene when adenine encoding the translation initiation codon is defined as the base at position 1. nucleotide.   When adenine encoding the translation initiation codon is the first base, the 92nd base is replaced with cytosine in the translation region of the human wild-type MCA gene, and the 121st base is adenine. A polynucleotide comprising a substituted base sequence.   A polypeptide comprising an amino acid sequence encoded by the polynucleotide according to any one of claims 17 to 19.   21. An antibody that binds to the polypeptide of claim 20 and does not bind to a human wild type MCA polypeptide. A detection method for detecting a mutation in an MCA polypeptide expressed in the human using a biological sample collected from the human,
22. A detection method comprising a step of detecting a mutation in an MCA polypeptide with the antibody according to claim 21.   A fertility diagnostic kit comprising an anti-human MCA antibody.