JP2006006311A - Model animal of male sterility exhibiting sperm morphogenesis failure - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a model animal causing dominant sperm morphogenesis failure which is conventionally difficult to technically carry out maintenance and passage and reproducing male sterility, to provide a method for maintenance and passage of the model animal of the male sterility, to provide a method for screening a prophylactic agent or a therapeutic agent for the male sterility using the model animal of the male sterility, to provide a method for screening a causative substance of the male sterility and to provide a method, etc., for diagnosing the male sterility. <P>SOLUTION: A nonhuman animal such as a mouse is used as the model animal of the male sterility as follows. In the nonhuman animal, all or a part of an endogenous gene of the nonhuman animal encoding cortactin is inactivated by a genetic mutation such as destruction, deletion or substitution to lower functions expressing the cortactin and one allele is provided with the genetic mutation. For example, an abnormality is caused in an actin filament layer contained in an ES (Sertoli-spermatid ectoplasmic specialization) in a Sertoli cell and morphologic abnormality of an elongated spermatid and exfoliation thereof from the seminiferous epithelium are caused in the model mouse of the male sterility. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention is a model animal capable of reproducing male infertility caused by dominant sperm dysplasia, which has been technically difficult to maintain and pass until now, a method for maintaining and passaging such male infertility model animals, The present invention relates to a screening method for a prophylactic / therapeutic agent for male infertility using such male infertility model animals, a screening method for a causative substance of male infertility, a diagnostic method for male infertility, and the like.

  Reproduction is a very basic phenomenon for living organisms that transmits its own genetic information to the next generation and maintains the continuity of life. In the case of multicellular organisms including mammals, somatic cells differentiate to maintain an individual's life and finish their role for only one generation, whereas germ cells leave the individual and form the next generation individual. Sperm, which is a male gamete, is excreted out of the body and fertilized with an egg, which is a female gamete, but because mammals are embryonic, the place of fertilization is in the female body and the number of eggs ovulated at one time is also limited Is done. Thus, mammalian males have developed mechanisms to continuously produce and deliver many sperm to ensure fertilization opportunities. In order to understand this mechanism, isolation and analysis of genes involved in spermatogenesis have been promoted.

(Male infertility)
In humans, infertility symptoms are seen in about 10 to 15% of couples, and the number is said to be increasing. Infertility was once thought to be a major problem for women, but current knowledge suggests that men and women contribute almost halfway, and the importance of diagnosing and treating male infertility is increasing. The causes of male infertility seen clinically are roughly classified into the following three. Of these, the most common is spermatogenic dysfunction, which accounts for about 70-90% of male infertility.
(1) Impaired dysfunction: Disorders related to the function of producing sperm in the testicle (testis). Depending on the number of sperm in the semen, malformed sperm rate, motility, etc., it is classified into symptoms such as azoospermia, oligospermia, asthenozoospermia, sperm malformation.
(2) Vascular passage obstruction: An obstruction of an organ through which sperm passes, such as the epididymis or urethra. Obstructive azoospermia, epididymis, retrograde ejaculation, etc.
(3) Sexual dysfunction: Disorders related to sexual activity. Erectile dysfunction (ED), ejaculation disorder, etc.

  The currently used treatment of male infertility is a method that mainly uses pharmacological treatments such as hormonal agents and Chinese medicine, and assisted reproductive technology (ART) such as in vitro fertilization and microinsemination. In the case of vagina obstruction, vagina reconstruction may be performed surgically, but in many cases no improvement is seen. The method using ART is the most powerful method at present, and even patients with complete azoospermia who do not have completed sperm in the testis can get children if there are extended sperm cells, especially by intracytoplasmic sperm injection (ICSI). it can. Although microinsemination has been increasingly applied as the “currently the most desirable way to conceive”, problems have been discussed. First, the method using ART does not impose an economic burden on the patient. There are also concerns about artificially leaving behind genetic elements that would otherwise not be passed on to the next generation. There have been reports of cases where boys born after ICSI were also infertile (see, for example, Non-Patent Documents 1 and 2).

(Slight deletion of Y chromosome)
There are congenital dysfunctions such as chromosomal abnormalities and acquired ones due to environmental factors and infectious diseases. However, in practice, it is difficult to determine the cause of spermatogenic dysfunction in clinical settings, and many are diagnosed as “sudden spermatozoa dysfunction” of unknown cause. It is difficult to know exactly what is due to genetic or environmental factors. Several estimates have been made so far, and Lilford et al. Report that 60% of male infertility is a genetic factor, many of which are autosomal mutations. Many of these have not been clarified (see, for example, Non-Patent Document 3).

  The most well-known and studied genetic factor for spermatogenic disorders is the microdeletion of the Y chromosome, which is found in approximately 5% of male infertile patients. To date, four azoospermia factor regions AZFa, AZFb, AZFc, and AZFd have been mapped on the Y chromosome. There are four known genes for AZFa: USP9Y (ubiquitin-specific protease 9, Y chromosome), DBY (dead box on the Y), UTY (ubiquitous TPR motif on the Y), and AZFaT1 (AZFa transcript 1). In AZFb, there are 20 to 50 genes encoding a protein having an RNA binding motif called RBMY (RNA binding motif on Y chromosome) gene family, and more pseudogenes exist. AZFc contains a plurality of genes belonging to the DAZ (deleted in azoospermia) gene family, which also has an RNA binding motif. Detailed analysis of the genes mapped to these AZF regions is ongoing, but the gene responsible for spermatogenic dysfunction is not yet known. It has also been suggested that a plurality of genes in the region may be involved (see, for example, Non-Patent Documents 4 and 5).

(Environmental factors of male infertility / effects of endocrine disruptors)
Acquired male infertility can be caused by various causes such as stress from the living environment and bacterial / viral infections. In recent years, reproductive impairment due to exposure to environmental endocrine disruptors has attracted attention. In the first place, the origin of this problem is a report by Skakkebaek et al. In 1992 that the number of human spermatozoa has been halved in the past 50 years (for example, see Non-Patent Document 6). Furthermore, in 1998, the same group reported a correlation between an increase in the incidence of testicular tumors and a decrease in the number of spermatozoa (for example, see Non-Patent Document 7), in which both these phenomena caused hormone-like effects in the environment. It was pointed out that it may be due to the influence of the substance possessed, that is, the endocrine disrupting substance. Skakkebaek et al.'S research was based on a meta-analysis based on past literature surveys, and discussions were made on its methodology and statistical processing. Since then, various investigations and researches have been conducted, and both positive and negative opinions have been given, but whether human sperm is really decreasing or, if so, whether it is the effect of endocrine disruptors There is no consensus among the parties. Reproductive problems are related to the survival of humankind, and if endocrine disrupting substances contribute to the reduction of human sperm count, social measures such as the regulation of compounds that can cause them are necessary. Let's go. There is a need for objective and scientific risk assessment as the foundation.

(Male reproductive organ)
The testis, the male reproductive organ, consists of a collection of tubes called the seminiferous tubule leading to the outside of the body. The inner wall of the tube is a tissue called seminiferous epithelium, and germ cells are present in the cytoplasm of Sertoli cells, which are supporting cells (see FIG. 1). Germ cells migrate from the outer side of the seminiferous tubule (basal part) toward the inner lumen, and the completed sperm is released into the lumen. The seminiferous tubules gather in the rete testis and mature while passing through the epididymis, the collecting duct. And it is ejected with prostatic fluid and seminal vesicle fluid at the time of mating. Somatic cells called Leydig cells exist in the stroma outside the seminiferous tubule, and are responsible for the regulation of testicular function by hormones such as androgen production.

(Mammalian spermatogenesis)
Primordial Germ Cell (PGC) derived from an extra-embryonic yolk sac in the male fetal stage enters a genital ridge around day 12 of embryonic life. This process is almost the same for males and females. The settlement of male PGCs allows the male gonad primordium to begin to differentiate into the testis for the first time and to be morphologically distinguishable from the ovary. The genital ridge consists of the cortex and medulla, and PGCs that have entered the cortex move to the medulla when differentiated into the testis. PGCs continue to proliferate after reaching the genital ridge, but once enter the resting phase of cell division. In the meantime, seminiferous tubules are formed, and PGCs become cells in the seminiferous tubules together with mesoderm-derived Sertoli cells. From this period, PGC is called spermatogonia (karyotype is 2N). As sexual maturity approaches, spermatogonia resume cell division and begin meiosis. And mature sperm comes to be ejected with sexual maturity. It takes several weeks from the start of meiosis to the completion of mature sperm. In animals (5 to 8 w) with early sexual maturity such as mice and rats, this process starts immediately after birth. Unlike female oogenesis, males continue to grow and differentiate for the rest of their lives. This process from the division of spermatogonia to the completion of sperm is called spermatogenesis. The structure of the mouse seminiferous tubule is shown in FIG.

  There are two types of spermatogonia, type A and type B. Type A cells are stem cells that remain in the basal part (= outermost layer) of the seminiferous tubule. Type A cells divide into type A cells themselves and type B cells that initiate meiosis. B-type cells gradually synthesize in the direction of the fine lumen (= inside) and synthesize DNA to become primary spermatocytes (4N). Primary spermatocytes, like oocytes, have a long prophase in the first meiosis, leptotene, zygotene, zygotene, pachytene, and diplotene. : Double yarn). When the first meiosis is completed, secondary spermatocytes (2N) are obtained. The secondary spermatocytes immediately begin the second meiosis and become spermatids (spermatid: N) in the latter half of meiosis. In gametogenesis, haploid (N) gametes are finally produced through karyotype changes of 2N → 4N → 2N → N, but in the case of oogenesis, meiosis is an unequal division and the polar Because one cell is discarded in the form of polar body release, only one gamete (egg) is made from one egg progenitor cell. On the other hand, in spermatogenesis, four equivalent gametes (sperm) are made from one spermatogonia.

  Sperm cells immediately after the end of meiosis are called round spermatids. Haploid sperm cells after meiosis can be normally ontogenized by direct injection into unfertilized eggs. This indicates that the round sperm cells immediately after meiosis already have sufficient genetic information necessary for ontogeny. However, in order to fertilize with an egg in a living body, it is necessary to change a sperm cell into a functional sperm. Significant morphological and biochemical changes occur from circular sperm cells to the completion of functional sperm. This stage is called spermiogenesis. What happens in this process is the enrichment of nuclei, the acquisition of motor function, the acquisition of fertility (fertilization protein, egg activator). The sperm cells in the middle of the round sperm cell nuclei and reaching the finished sperm are called elongated spermatids.

  Normally, DNA in the cell nucleus combines with histones to form chromosomes, but in sperm, histones are replaced with protamine and are put together in a compact form that is convenient for movement. Nuclear condensation makes the sperm resistant to some physical and chemical changes. On the other hand, gene transcription is thought to be difficult as the nucleus condenses, and some genes involved in sperm morphogenesis are subject to translational regulation. For example, an excessive amount of TATA-binding protein (TBP) is transcribed in sperm cells before nuclear condensation, but the amount translated is relatively small, suggesting the existence of a translational regulatory mechanism. Next, a tail (flagella) is formed for sperm movement. This can be done by extending the sperm of the sperm cell. The flagellar head has a slightly thicker part called a midpiece, with mitochondria wound in a spiral. These mitochondria supply the energy necessary for sperm movement. Some motility of the tail is also seen in sperm in the testis, but it is in the epididymis that acquires the motility inherent in sperm, such as advancement. The sperm head has a part called acrosome. The acrosome contains various proteins necessary for fertilization such as hydrolase. The sperm head also contains a factor that activates the egg. The completed sperm is released into the seminiferous tubule and released outside the body through maturation in the epididymis.

  The spermatogenesis process is very tightly controlled, producing large quantities of sperm efficiently. The testicular environment is important for the control, and in particular, the interaction between sperm cells and Sertoli cells is essential. Sertoli cells are columnar somatic cells that extend from the base of the seminiferous tubule toward the lumen. The nucleus is irregular and has a large nucleolus inside. Sertoli cells not only structurally support sperm cells, but also secrete various proteins (hormones) to control a series of spermatogenesis processes. Connected. Sertoli-germ cell adhesion is thought to play an important role in germ cell differentiation, but the details are not well understood. The Sertoli cell-Sertoli cell junction (tight junction) at the base of the seminiferous tubule is called the blood testis barrier (BTB), and isolates the systemic circulation from the germ cells. In mammals, since spermatogenesis begins after birth, autoimmune tolerance acquired immediately after birth from the fetal stage does not function for sperm-specific proteins. Therefore, anti-sperm antibodies may be present in the blood, and it is considered necessary to isolate the blood circulation and the testicular environment.

(Regulation of testicular function by endocrine system)
Mammalian reproductive functions are under the control of the endocrine system. In response to gonadotropin releasing hormone (GnRH) secreted from the hypothalamus at the top of the endocrine regulating system, gonadotropin is secreted from the anterior pituitary gland. Germ cell differentiation in the gonads (testis / ovary) is regulated by gonadotropins (see FIG. 2). The gonadal stimulating hormones secreted from the anterior pituitary include FSH (follicle stimulating hormone) and LH (luteinizing hormone). These hormones are composed of two subunits of glycoproteins. Testicular function is regulated by FSH and LH. Embryonic and neonatal Sertoli cell division is promoted with FSH, which promotes various Sertoli cell functions that support each stage of spermatogenesis. The effect of promoting the production of androgen binding protein (ABP) by FSH on Sertoli cells is important, and it is necessary for the function of androgen (Testosterone) produced by interstitial Leydig cells. It is. The main effect of LH on testicular function is the androgen production promoting action of Leydig cells. The androgen concentration in the testis is higher than in the blood, and this high concentration is essential for spermatogenesis. Since the androgen receptor is not present in germ cells but is present in Sertoli cells, androgen is thought to act via Sertoli cells, not directly to germ cells.

(Sertoli-sperm cell special junction device)
Sertoli cells are in contact with spermatogenic cells at all stages of differentiation and support spermatogenesis. A special structure called a Sertoli-spermatid ectoplasmic specialization (ES) is found in the part of the Sertoli cytoplasm that contacts the extended sperm cell. The ES structure is shown in FIG. The ES structure is composed of three layers, a Sertoli cell plasma membrane, an actin filament layer, and an endoplasmic reticulum, and has a structure in which actin fibers form a layer inside the Sertoli cell membrane. Although there are still many unclear parts in the function of the ES structure, the actin layer of ES is damaged by the treatment of cytochalasin D (Cytochalasin D), a drug that destroys actin fibers, resulting in detachment of sperm cells and Sertoli cells (For example, see Non-Patent Document 8) and administration of a compound having an estrogenic action such as beta-estradiol 3-benzoate (E2B) also damages the ES actin layer, causing sperm cell malformation and sperm epithelium. Examples of testicular toxicity have been reported from studies on testicular toxicity, such as exfoliation occurring (see, for example, Non-Patent Document 9). From these, ES assists morphogenesis of sperm cells, and It is considered necessary for maintaining the binding between Sertoli cells and sperm cells until spermiation at a certain time (see, for example, Non-Patent Document 10).

(Spermatogenesis cycle)
In the seminiferous tubule of the mature testis, combinations of germ cells (stage, step) in a certain differentiation stage are arranged continuously and periodically (see FIG. 4). In the case of a mouse, the stage is divided into 12 stages from I to XII. If the precision tube is cut in a certain place, a section of one stage can be seen, and another stage can be seen if the same precision tube is cut in another place. In certain stages of seminiferous tubules, a combination of germ cells of a certain differentiation stage can be seen. Step is a number representing the differentiation stage of sperm cells after meiosis, and is divided into 16 stages from 1 to 16 in the case of mice. The sperm cells of steps 1-8 are round and are called round spermatids. The sperm cells after step 8 are called extended spermatids, and become complete sperm through changes in the shape of the nucleus and formation of the tail. For example, in the stage VI seminiferous tubule, the type-B spermatogonia is located on the outermost side of the seminiferous tubule, the spermatocyte in the pakiten phase is located on the inside, and the step 6 round sperm cell (Step 6) on the inside. round spermatid), and Step 15 elongated spermatids are observed.

  Rough tubule stage discrimination can be performed by observing the state of sperm cells. Step 16 sperm cells immediately before being released into the lumen are located on the most luminal side of the seminiferous epithelium (outside the seminiferous epithelium). Therefore, it can be determined from the image in which sperm cells are lined up facing the lumen that the seminiferous tubules are stages VII to VIII. Step 15 The sperm cells are almost complete in head form, but the head is thrust into the seminiferous epithelium. Therefore, stages I to VI can be discriminated from an image in which the concentrated sperm head (which can be easily discriminated by nuclear staining) is in the sperm epithelium. Stages IX to XII can be distinguished from the lack of complete sperm cell enrichment and the absence of circular sperm cells. In particular, stage IX tubules immediately after sperm release have step 9 sperm cells, but step 9 sperm cells can be distinguished from the fact that the concentration of nuclei is not complete and nuclear staining is thinner than the subsequent steps. More detailed discrimination (particularly stages I to VI) can be performed by observing acrosomes generated in circular sperm cells by PAS staining or the like.

(Male infertile mouse)
As described above, spermatogenesis in mammals is a higher-order biological phenomenon that requires interaction between somatic cells and germ cells and is controlled by various factors such as hormones and cell growth factors. Many factors required for spermatogenesis are difficult to reproduce in vitro, and spermatogenesis in vitro (in vitro) has not been successful to date. Therefore, research on spermatogenesis is centered on in vivo analysis at the individual level. Among them, knockout mice for genes involved in spermatogenesis have greatly contributed to the elucidation of the mechanism of spermatogenesis. So far, various knockout mice that have abnormalities in each stage of spermatogenesis have been reported. For example, a transcription factor CREM (cyclic AMP-responsive element modulator) is an example of a gene that is expressed in sperm cells after meiosis. CREM knockout mice undergo sperm differentiation arrest and apoptosis, resulting in infertility. From this, it is thought that CREM controls many gene expression in a sperm cell (for example, refer nonpatent literatures 11 and 12). Protamine (Prm-1, -2) knockout mice that bind to DNA in place of histones in sperm show symptoms such as decreased sperm count, sperm malformation, and decreased sperm chemical resistance. A protamine knockout mouse is characterized by symptoms when it has a mutation in one allele (see, for example, Non-Patent Document 13). In addition to these, many knockout mice have been reported in recent years, contributing to the elucidation of the mechanism of spermatogenesis.

  Of course, genes expressed in germ cells such as CREM and protamine are important, but genes functioning in somatic cells such as Sertoli cells are equally important for spermatogenesis. The nectin-2 knockout mouse, which is a cell adhesion molecule, shows malformation of the elongated sperm cell head and decreased sperm motility after meiosis. Since Nectin-2 is expressed in Sertoli cells and not in germ cells, it is considered to be a Sertoli cell-side factor involved in adhesion between sperm cells and Sertoli cells (see, for example, Non-Patent Documents 14 and 15). ). Sertoli cells play a major role in efficient spermatogenesis, and it is thought that there are many other molecules involved in germ cell-Sertoli cell interactions. In addition to the knockout mice as described above, infertile mice due to mutations have been reported and analyzed, but the analysis of mutant mice is delayed compared to the analysis of knockout mice.

(Genetic and phenotypic approaches)
In general, gene function analysis involves gene-driven approaches, such as knockout mice and transgenic mice, and phenotype-preceding methods that isolate mutants using phenotypes as indicators to find the cause. There is a phenotype-driven approach. The former is also called reverse genetics, and the latter is also called “forward” genetics. Both are complementary to each other and should be advanced simultaneously. However, in the field of reproduction, it has been difficult to maintain and pass mutants, and genetic analysis is difficult, so far the phenotype-first approach has been delayed. In particular, there is no example in which a causative gene has been determined so far for dominant infertile mutant mice. However, the development of reproductive engineering has made it possible to obtain offspring even from infertile mice, and the entire genome sequence of mice has been determined, making it easier to identify mutation sites. This has enabled a phenotypic approach. A mouse mutant production project that actually targets germ cells has also begun (see, for example, Non-Patent Document 16). It is expected that the analysis of mutants related to reproduction will progress in the future, and more knowledge will be brought about.

(Coat actin)
Coat actin is an actin-binding protein having a molecular weight of 80 to 85 kD. It contains a repetitive sequence of approximately 36 amino acids called HS1 repeat and has an SH3 domain at the C-terminus. Depending on the number of HS1 repeats, at least three types of splice variants (coat actin-A: 6 times, -B: 5 times, -C: 4 times) have been reported (see FIG. 10). Furthermore, it seems that types with a small number of repetitions (3 and 2) exist with a small amount of expression. The interaction between coat actin and various proteins has been reported, and it is known to bind to Arp2 / 3 complex, N-WASP via N-terminus and F-actin via HS1 repeat. Furthermore, it has been reported to bind to structural proteins such as ZO-1 and Shank3 via the C-terminal SH3 domain. It is known that several Ser, Thr and Tyr residues in proteins are phosphorylated. For these reasons, coat actin is considered to be a protein that receives a phosphorylation signal from the cell and controls the actin cytoskeleton. Coat actin has increased expression due to gene amplification in 15% of human breast cancers and has been reported to be involved in the development of breast cancer. However, no coat actin knockout mice have been reported to date. Although many functions are unknown, there is a high possibility of being involved in cell adhesion and morphological change due to localization to Post-Synaptic Density (PSD) and Tight Junction (see, for example, Non-Patent Documents 17 to 20). .

Kubo H. Sanka to Fujinka (Japanese) 2003; 6: 703-709 Matsumiya K. Sanka to Fujinka (Japanese) 2003; 6: 751-756 Lilford R, Jones AM, Bishop DT, Thornton J, Mueller R. Case-control study of whether subfertility in men is familial.Bmj 1994; 309: 570-573 Buch B, Galan JJ, Lara M, Ruiz R, Segura C, Real LM, Martinez-Moya M, Ruiz A. Scanning of Y-chromosome azoospermia factors loci using real-time polymerase chain reaction and melting curve analysis.Fertil Steril 2003; 80: 907-913. Foresta C, Moro E, Ferlin A. Y chromosome microdeletions andalterations of spermatogenesis. Endocr Rev 2001; 22: 226-239 Carlsen E, Giwercman A, Keiding N, Skakkebaek NE. Evidence for decreasing quality of semen during past 50 years. Bmj 1992; 305: 609-613 Skakkebaek NE, Rajpert-De Meyts E, Jorgensen N, Carlsen E, Petersen PM, Giwercman A, Andersen AG, Jensen TK, Andersson AM, Muller J. Germ cell cancer and disorders of spermatogenesis: an environmental connection.Amis 1998; 106: 3-11; discussion 1 Russell LD, Goh JC, Rashed RM, Vogl AW. The consequences of actin disruption at Sertoli ectoplasmic specialization sites facing spermatids after in vivo exposure of rat testis to cytochalasin D. Biol Reprod 1988; 39: 105-118 Toyama Y, Hosoi I, Ichikawa S, Maruoka M, Yashiro E, Ito H, Yuasa S. beta-estradiol 3-benzoate affects spermatogenesis in the adult mouse. Mol Cell Endocrinol 2001; 178: 161-168 Toyama Y, Maekawa M, Yuasa S. Ectoplasmic specializations in the Sertoli cell: new vistas based on genetic defects and testicular toxicology. Anat Sci Int 2003; 78: 1-16 Blendy JA, Kaestner KH, Weinbauer GF, Nieschlag E, Schutz G. Severe impairment of spermatogenesis in mice lacking the CREM gene.Nature 1996; 380: 162-165 Fimia GM, Morlon A, Macho B, De Cesare D, Sassone-Corsi P. Transcriptional cascades during spermatogenesis: pivotal role of CREM and ACT. Mol Cell Endocrinol 2001; 179: 17-23 Cho C, Willis WD, Goulding EH, Jung-Ha H, Choi YC, Hecht NB, Eddy EM.Haploinsufficiency of protamine-1 or -2 causes infertility in mice. Nat Genet 2001; 28: 82-86 Bouchard MJ, Dong Y, McDermott BM, Jr., Lam DH, Brown KR, Shelanski M, Bellve AR, Racaniello VR. Defects in nuclear and cytoskeletal morphology and mitochondrial localization in spermatozoa of mice lacking nectin-2, a component of cell- cell adherens junctions. Mol Cell Biol 2000; 20: 2865-2873 Mueller S, Rosenquist TA, Takai Y, Bronson RA, Wimmer E. Loss of nectin-2 at sertoli-spermatid junctions leads to male infertility and correlates with severe spermatozoan head and midpiece malformation, impaired binding to the zona pellucida, and oocyte penetration. Biol Reprod 2003; 69: 1330-13 Ward JO, Reinholdt LG, Hartford SA, Wilson LA, Munroe RJ, Schimenti KJ, Libby BJ, O'Brien M, Pendola JK, Eppig J, Schimenti JC. Toward thegenetics of mammalian reproduction: induction and mapping of gametogenesis mutants in mice. Biol Reprod 2003; 69: 1615-1625 Wu H, Parsons JT. Cortactin, an 80 / 85-kilodalton pp60src substrate, is a filamentous actin-binding protein enriched in the cell cortex.J Cell Biol 1993; 120: 1417-1426 Thomas SM, Soriano P, Imamoto A. Specific and redundant roles of Src and Fyn in organizing the cytoskeleton. Nature 1995; 376: 267-271 Weed SA, Parsons JT. Cortactin: coupling membrane dynamicsto cortical actin assembly. Oncogene 2001; 20: 6418- Schafer DA. Coupling actin dynamics and membrane dynamics during endocytosis. Curr Opin Cell Biol 2002; 14: 76-81

  In recent years, the demand for human infertility treatment has been increasing, and progress in reproductive biology as the foundation is desired. However, the molecular mechanism of germ cell formation is still not fully understood. This is partly due to insufficient analysis of mutants related to germ cell formation. In particular, when a male infertility model animal is produced, no offspring can be produced. Therefore, despite the fact that many mutant mice have been reported, there has been no example of identifying the causative gene. An object of the present invention is to provide a model animal capable of reproducing male infertility caused by dominant sperm dysplasia that has been technically difficult to maintain and pass until now, and a method for maintaining and passaging such a male infertility model animal. Another object is to provide a screening method for a prophylactic / therapeutic agent for male infertility using such a male infertility model animal, a screening method for a causative substance of male infertility, a diagnostic method for male infertility, and the like.

  The present inventors created a transgenic mouse for the purpose of examining the function of the imprinting gene PEG8 / IGF2AS. In the process of producing a transgenic mouse, foreign DNA injected into a fertilized egg is inserted at a random location on the genome. As a result, gene disruption at the insertion site may occur. This is called an insertional mutation. Generally, it is said that infertile mice due to insertion mutations occur in several percent of transgenic mice. When the introduced sequence is a sequence that does not exist in the original mouse genome, the mutation site can be identified using the sequence as a tag. In recent years, the entire genome sequence of mice has been decoded, and information on single nucleotide polymorphisms (SNPs) has become available. As a result, it is possible to examine mutation sites on the genome more easily than before. It became possible. In addition, insertion mutations often involve large-scale genomic abnormalities such as chromosomal translocations and deletions, which can now be analyzed relatively easily by using genomic information.

  We have found an insertion mutant mouse showing sperm morphogenesis in a line of imprinting gene PEG8 / IGF2AS transgenic mice. In order to elucidate the mechanism of spermatogenesis in mammals, we considered that such mutant mice were valuable, and proceeded with the analysis of strains that were insertion mutants. In normal mating, it is not possible to obtain offspring from this male and the strain cannot be maintained. Therefore, in vitro fertilization (IVF) was considered, but there was almost no sperm in the epididymis and IVF. Was impossible. Therefore, when ICSI (intracytoplasmic sperm injection) was performed, in which sperm cells collected from the testis were injected into unfertilized eggs, a litter could be obtained, and the mutation was passaged to maintain this model mouse. Succeeded. Moreover, the gene region deletion of the mutant mice was analyzed by utilizing the SNPs database. As a result, we succeeded in analyzing dominant spermatogenesis-deficient mice that were technically difficult to maintain and pass until now, and in mice lacking the coat actin gene on one side of the chromosome, the expression level of coat actin protein was halved. It was possible to reproduce male infertility that caused sperm dysplasia due to the decrease. This gene was also expressed in the cerebellum, and this model mouse also showed abnormal behavior such as abnormal gait, indicating that it is important as a model mouse for behavioral control research.

As described above, the present inventors found a dominant insertion mutation Dspd (Dominant spermiogenesis defect) in one strain of PEG8 transgenic mice, and revealed the following.
(1) An abnormality has occurred in the actin fiber layer contained in the special junction device (ES) in Sertoli cells, and abnormal morphologies of the elongated sperm cells and detachment from the seminiferous epithelium have occurred.
(2) A translocation occurred between chromosome 7 and chromosome 14. There was a deletion of about 1 Mb genome in the vicinity of chromosome 7 telomere, and the expression level of coat actin mapped there was decreased.
(3) Coat actin was localized around the elongated sperm cells in normal mouse testis, and coincided well with the time when sperm cell abnormalities occurred in Dspd mice. In the Dspd mouse testis, the localization of coat actin protein and actin was disturbed.

  From these results, in Dspd mice, deletion of a coat allin allele and a decrease in the expression level lead to abnormal control of Sertoli cell actin fibers (especially the actin layer of the ES structure), resulting in abnormal sperm cell morphology. The possibility of detachment of sperm cells from the seminiferous epithelium was shown (see FIG. 15). Based on such knowledge, the present invention has been completed.

    That is, the present invention is (1) all or part of the endogenous gene of non-human animal encoding coat actin is inactivated by gene mutation such as destruction, deletion or substitution, and the function of expressing coat actin is reduced. Or a male infertility model animal characterized by comprising a lost non-human animal, or (2) the male infertility model animal according to (1) above, wherein the male allergy has a gene mutation in one allele, (3) The present invention relates to a male infertility model animal as described in (1) or (2) above, which exhibits behavioral abnormalities such as gait abnormalities.

  The present invention also provides (4) a male infertility model animal according to any one of (1) to (3) above, wherein the non-human animal is a mouse, and (5) a mouse sperm cell (independent administrative agency) All of the endogenous genes of the male infertility model animal according to (4) above and (6) a non-human animal encoding coat actin, which is produced using RIKEN BioResource Center deposit number 01136) Alternatively, sperm cells collected from non-human animals that have been partially inactivated by mutations, deletions, substitutions, or other genetic mutations and that have lost or lost the ability to express coat actin are injected into wild-type unfertilized eggs. The present invention relates to a method for maintaining and substituting a male infertility model animal, wherein the cultured 4-8 cell embryo is transplanted into a fallopian tube of a non-human animal pseudopregnant to obtain a litter.

  The present invention also relates to (7) the method for maintaining and substituting male infertility model animals described in (6) above, wherein the non-human animal is a mouse, and (8) mouse sperm cells (independently (9) The method for maintaining and substituting male infertility model animals according to (7) above, characterized in that it is deposited at the Institute of Physical and Chemical Research Bioresource Center (01136), or (9) above (1) to (5) The present invention relates to a screening method for a prophylactic / therapeutic agent for male infertility, characterized by administering a test substance to any male infertility model animal and measuring and evaluating the degree of improvement in symptoms of spermatogenic dysfunction.

The present invention also relates to (10) a DNA encoding a protein consisting of any one of the following base sequences (A) to (F), wherein the reduced expression function or loss of expression function causes male infertility.
(A) a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(B) a base sequence encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having a binding activity to actin;
(C) a base sequence encoding a protein consisting of an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2 and having a binding activity to actin;
(D) the base sequence represented by SEQ ID NO: 1;
(E) a base sequence encoding a protein consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 and having actin-binding activity;
(F) a base sequence encoding a protein that hybridizes with the base sequence shown in SEQ ID NO: 1 under stringent conditions and has a binding activity to actin;
Furthermore, (11) the present invention relates to a protein consisting of an amino acid sequence of any one of (A) to (C) below, whose decrease in expression function or loss of expression function causes male infertility.
(A) the amino acid sequence shown in SEQ ID NO: 2;
(B) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and the protein comprising the amino acid sequence has an activity to bind to actin Sequence;
(C) an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2 and a protein comprising the amino acid sequence having an activity to bind to actin;

  The present invention also relates to (12) a method for diagnosing male infertility, characterized by examining the presence or absence of a human coat actin gene abnormality in a specimen, and (13) examining the presence or absence of a human coat actin protein abnormality in a specimen. And (14) a human coat actin gene comprising, as an active ingredient, a human coat actin gene or a recombinant vector in which a human coat actin gene is integrated into an expression vector Or a human coat actin gene or human characterized by administering to the testis a therapeutic agent for male infertility caused by an abnormality in human coat actin protein or (15) the therapeutic agent for male infertility described in (14) above The present invention relates to a method for treating male infertility caused by abnormalities in coat actin protein.

  Mutation model animals play an extremely important role in elucidating the causes of male infertility. In the present invention, a dominant mutation related to spermatogenesis was subcultured and the mutation site was determined. If microinsemination is performed using a mutant mouse having the coat actin gene mutation of the present invention in one allele, it is possible to maintain a valuable model animal and to advance gene analysis. Moreover, if it becomes clear that it participates in specific structure formation like coat actin, screening of infertility including other structure genes becomes possible. That is, according to the present invention, a model animal capable of reproducing male infertility caused by dominant sperm morphogenesis, which has been difficult to maintain and pass until now, and maintenance and passaging of such male infertility model animals. It is possible to provide a method, a screening method for a prophylactic / therapeutic agent for male infertility using such a male infertility model animal, a screening method for a causative substance of male infertility, a diagnostic method for male infertility, and the like.

  Protamine-1, -2 (protamine-1, -2) has been known as an example of infertility due to one gene haploinsufficiency [Non-patent document 13, Tanaka H, Miyagawa Y, Tsujimura A, Matsumiya K, Okuyama A, Nishimune Y. Single nucleotide polymorphisms in the protamine-1 and -2 genes of fertile and infertile human male populations. Mol Hum Reprod 2003; 9: 69-73], It is expected that there are other such dominant mutations. To elucidate the mechanism of spermatogenesis and to understand human infertility, the analysis of mutant mice such as Dspd, which could not be analyzed so far, is important.

  The male infertility model animal of the present invention is a function in which all or part of the endogenous gene of a non-human animal encoding coat actin is inactivated by gene mutation such as destruction / deletion / replacement and expresses coat actin Is not particularly limited as long as it is a non-human animal that develops male infertility whose function has been reduced or has lost the function to express coat actin, but a non-human animal having a gene mutation in one allele and / or behavior such as abnormal gait Non-human animals exhibiting abnormalities are preferred, and examples of the non-human animals include mice, rats, and dogs, cats, cows, monkeys, birds, fish (zebrafish), and frogs (Xenopus laevis) that are recognized to express coat actin. Examples include fly (Drosophila) and the like, but a mouse is preferred because of its ease of handling. Hereinafter, all or part of the endogenous genes of non-human animals encoding coat actin are inactivated by gene mutations such as destruction, deletion, replacement, etc. The male infertility model mouse possessed by allele will be described.

  A male infertility model mouse having a coat actin gene mutation in one allele is prepared by injecting a transgene into the male pronucleus of a mouse fertilized egg as described in the Examples below, and the treated fertilized egg is a pseudopregnant female. Circular and elongated sperm cells collected from offspring (mutant mice) born from foster mothers after transplanted into the oviduct of mice were injected into wild-type unfertilized eggs and cultured for 48 hours, then 4-8 cell embryos were pseudopregnant It is also possible to obtain offspring by transplanting into the fallopian tube of a female mouse. An example of an elongated sperm cell collected from a litter (mutant mouse) is deposited under the deposit number 01136 at the RIKEN BioResource Center.

  In addition to the methods described in the above examples, it can also be obtained by utilizing a normal method for producing knockout mice. Specifically, a coat actin gene is screened using a gene fragment obtained from a mouse gene library by a method such as PCR, and the coat actin gene thus screened is all or all of the coat actin gene by recombinant DNA technology. A part of the gene is replaced with a marker gene such as a neomycin resistance gene, and a gene such as a diphtheria toxin A fragment (DT-A) gene or a herpes simplex virus thymidine kinase (HSV-tk) gene is introduced at the 5 ′ end. A targeting vector, linearize the prepared targeting vector, introduce it into ES cells by electroporation, etc., perform homologous recombination, and select from the homologous recombinants , G418, Ganciclovia (GANC), etc. Selecting ES cells that are resistant to the raw material. It is preferable to confirm whether or not the selected ES cell is the target recombinant by Southern blotting or the like.

  The recombinant ES cell is microinjected into a blastocyst of a mouse, and the blastocyst is returned to a temporary parent mouse to produce a chimeric mouse. When this chimeric mouse is crossed with a wild-type mouse, a heterozygous mouse having a coat actin gene mutation in one allele can be obtained. Circular and elongated sperm cells collected from this heterozygous mouse were injected into wild-type unfertilized eggs, cultured for 48 hours, and transplanted into the oviduct of a female mouse pseudopregnant with 4-8 cell embryos. You can also get Although this heterozygous mouse can be crossed, a coat actin knockout mouse can be obtained, but a knockout mouse may not be born. For this reason, the Cre-loxP system or the like can be used to conditionally (eg, only Sertoli cells).

  As a method for confirming that the coat actin gene in the mutant mouse (heterozygous mouse) is deficient in a single allele on the chromosome, for example, RNA is isolated from the mouse testis obtained by the above method. Separately, use real-time RT-PCR method and Western blotting method to examine the expression level of coat actin gene and coat actin protein in mouse testis, or to examine protein extracted from mouse sperm cells by immunoblot analysis etc. Etc.

  Since the male infertility model animal of the present invention develops male infertility, it is useful for analyzing the mechanism of male infertility development, and the male infertility model animal of the present invention is used to prevent male infertility. • Can be used for screening for therapeutic agents. As a screening method for a prophylactic / therapeutic agent for male infertility according to the present invention, a screening method for administering a test substance to the male infertility model animal according to the present invention and measuring / evaluating the degree of improvement in symptoms of spermatogenic dysfunction The measurement and evaluation of the degree of improvement of spermatogenic dysfunction is performed by morphological observation of testis tissue and the number and arrangement of elongated sperm cells, as described in the following examples. A method of measuring the degree of turbulence, Sertoli cell morphology, the number of spermatozoa in the epididymal duct, and comparing with the wild type as a control.

  On the other hand, there are cases in which immature sperm cells are often found in the ejaculated semen of patients with oligospermia. On the other hand, according to the present invention, a decrease in the expression level of coat actin causes dysplasia of the ES structure, leading to spermatogenesis Became clear. Also, abnormalities in the special junction apparatus and detachment of sperm cells from the seminiferous epithelium are phenomena that have been observed mainly in rats and mice with hormonal abnormalities such as estrogen administration and testosterone reduction. This formation failure of the ES structure is also observed in the case of spermatogenesis abnormalities caused by environmental hormones such as dioxins, and therefore the possibility that the target gene of environmental hormones is coat actin has increased. If it can be confirmed that it is a target gene for environmental hormones, the expression analysis of this gene will be very useful for screening for environmental harmful substances. In addition, environmental hormones are thought to cause not only spermatogenesis but also behavioral abnormalities, suggesting a relationship with the increase in sharp children that are currently a problem. Since it is thought that the influence on the nervous system can be seen even in an amount of 1/100 of spermatogenesis, detection of such environmental harmful substances is indispensable in modern society. Coat actin has been observed in the central nervous system (particularly the cerebellum), and gait abnormalities have also been confirmed in model animals.

  Therefore, the screening method for the causative agent of male infertility of the present invention is not particularly limited as long as it is a method of administering a test substance to a non-human animal and examining the presence or absence of abnormalities in the coat actin gene or coat actin protein, Examples of the non-human animals include mice and rats in which the expression of coat actin is recognized, as well as birds, fish, frogs, and the like. Fish such as carp and carp are preferred. The base sequences of these non-human animal coat actin genes include mouse (GenBank accession number: 15030314), rat (GenBank accession number: 11177917), chicken (GenBank accession number: 45382632), zebrafish (GenBank accession). No .: 33417208), Xenopus laevis (GenBank accession number: 17826995), and flies (GenBank accession number: 3896003).

  The test substance is not particularly limited, such as extracts from animals and plants and microorganisms, various peptides / proteins, polysaccharides, and chemically synthesized substances.

  According to the above-mentioned present invention, since a decrease in expression function or loss of expression function of coat actin causes male infertility, it has been found that coat actin is a protein having a function of controlling male infertility. Use of coat actin and coat actin gene, coat actin and coat actin gene focusing on such functions is also encompassed by the present invention. Hereinafter, these will be described.

  The “DNA encoding a protein whose expression function decline or expression function causes male infertility” according to the present invention is (A) a base sequence encoding the amino acid sequence shown in SEQ ID NO: 2 (human coat actin); (B) a base sequence encoding an amino acid sequence consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having a binding activity to actin; C) a nucleotide sequence comprising an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2 and encoding an amino acid sequence having binding activity with actin; (D) shown in SEQ ID NO: 1 Base sequence (DNA encoding human coat actin); (E) In the base sequence shown in SEQ ID NO: 1, A nucleotide sequence consisting of a nucleotide sequence in which several bases are deleted, substituted or added, and encoding a protein having a binding activity to actin; or (F) a stringent condition with the nucleotide sequence shown in SEQ ID NO: 1 The DNA is not particularly limited as long as it is a DNA consisting of any one of the following nucleotide sequences: a base sequence encoding a protein that hybridizes under the above and has a binding activity with actin; As a protein whose loss causes male infertility ”, (A) an amino acid sequence represented by SEQ ID NO: 2; (B) one or several amino acids are deleted or substituted in the amino acid sequence represented by SEQ ID NO: 2 Or an amino acid sequence consisting of an added amino acid sequence and having a binding activity to actin; or (C) the amino acid shown in SEQ ID NO: 2 It is not particularly limited as long as it is a protein consisting of any amino acid sequence consisting of an amino acid sequence having at least 60% homology with a sequence and having an activity to bind to actin. The term “protein having binding activity” means a protein that is involved in some form of binding to actin, and its specific mechanism of action is not particularly limited, and examples thereof include the ability to bind to actin.

  The above “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 5. It means an amino acid sequence in which any number of amino acids are deleted, substituted or added. The “base sequence in which one or several bases have been deleted, substituted or added” is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 It means a base sequence in which ˜5 arbitrary number of bases are deleted, substituted or added.

  For example, DNA (mutant DNA) consisting of a base sequence in which one or several bases are deleted, substituted or added can be obtained by any method known to those skilled in the art such as chemical synthesis, genetic engineering techniques, mutagenesis, etc. Can also be produced. Specifically, the DNA consisting of the base sequence shown in SEQ ID NO: 1 is mutated into these DNAs by using a method of contacting with a mutagen agent, a method of irradiating with ultraviolet rays, a genetic engineering technique, etc. Mutant DNA can be obtained by introducing. Site-directed mutagenesis, which is one of genetic engineering techniques, is useful because it can introduce specific mutations at specific positions. Molecular Cloning: A laboratory Mannual, 2nd Ed., Cold Spring Harbor Laboratory , Cold Spring Harbor, NY., 1989. (hereinafter abbreviated as "Molecular Cloning 2nd Edition"), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997), etc. It can be carried out. By expressing this mutant DNA using an appropriate expression system, a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added can be obtained.

  The “amino acid sequence having at least 60% or more homology with the amino acid sequence shown in SEQ ID NO: 2” is particularly limited as long as the homology with the amino acid sequence shown in SEQ ID NO: 2 is 60% or more. Instead, for example, it means 60% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more.

  The above-mentioned “base sequence that hybridizes under stringent conditions” means that a nucleic acid such as DNA or RNA is used as a probe, and a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like is used. Specifically, using a filter in which DNA derived from colonies or plaques or a fragment of the DNA is immobilized, the DNA is high at 65 ° C. in the presence of 0.7 to 1.0 M NaCl. After hybridization, the filter is washed under conditions of 65 ° C. using an SSC solution of about 0.1 to 2 times (the composition of a 1-fold concentration SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). The DNA which can be identified can be mentioned. Hybridization can be performed according to the method described in Molecular Cloning 2nd edition.

  For example, DNA that can hybridize under stringent conditions can include DNA having a certain degree of homology with the base sequence of the DNA used as a probe, for example, 60% or more, preferably 70%. Preferred examples thereof include DNA having homology of 80% or more, more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more.

  The method for obtaining and preparing the DNA of the present invention is not particularly limited, and appropriate probes and primers based on the amino acid sequence or base sequence information shown in SEQ ID NO: 1 or SEQ ID NO: 2 disclosed in the present specification. And the target gene can be isolated by screening a cDNA library in which the DNA is expected to exist, or can be prepared by chemical synthesis according to a conventional method.

  There are no particular limitations on the method for obtaining and preparing the “protein whose expression function declines or loss of expression function causes male infertility” according to the present invention, and it may be a natural isolated protein, a chemically synthesized protein, or genetic recombination. Any recombinant protein produced by technology may be used. When a naturally derived protein is obtained, the protein of the present invention can be obtained by appropriately combining protein isolation / purification methods from cells or tissues expressing such protein. When preparing a protein by chemical synthesis, for example, the protein of the present invention can be synthesized according to a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method). it can. In addition, the protein of the present invention can be synthesized using various commercially available peptide synthesizers. When a protein is prepared by a gene recombination technique, the protein of the present invention can be prepared by introducing DNA consisting of a base sequence encoding the protein into a suitable expression system. Among these, preparation by a gene recombination technique that can be prepared in a large amount by a relatively easy operation is preferable.

  For example, when preparing “a protein whose decreased expression function or loss of expression function causes male infertility” of the present invention by gene recombination technology, ammonium sulfate can be used to recover and purify the protein from the cell culture. Or known methods including ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography, preferably high performance liquids Chromatography is used. In particular, the column used for affinity chromatography is, for example, a column in which an antibody such as a monoclonal antibody against the protein of the present invention is bound, or when a normal peptide tag is added to the protein of the present invention, By using a column to which an affinity substance is bound, purified products of these proteins can be obtained. In addition, when the protein of the present invention is expressed on the cell membrane, a purified sample can be obtained by carrying out the above purification treatment after allowing the cell membrane degrading enzyme to act.

  Furthermore, it has a homology of 60% or more with a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2. A protein comprising an amino acid sequence can be appropriately prepared or obtained by those skilled in the art based on the information of the base sequence shown in SEQ ID NO: 1 showing an example of the base sequence encoding the amino acid sequence shown in SEQ ID NO: 2. . For example, the DNA having the nucleotide sequence shown in SEQ ID NO: 1 or a part thereof can be used as a probe to isolate a DNA homolog by screening under appropriate conditions from a non-human organism. A protein encoded by the homologous DNA can be produced by cloning the full-length DNA of this homologous DNA and then incorporating it into an animal cell expression vector and expressing it in an appropriate animal cell.

  It was clarified that coat actin is contained in the ES structure important for spermatogenesis, and that intracellular localization was altered in mutant mice. Is likely to be. Even now, in such cases, children can be obtained by microinsemination, but there have already been reported cases of inheriting male infertility that should not occur. By performing fertilized egg diagnosis after micro-fertilization, it is possible to eliminate the risk associated with such medical treatment by returning an individual having a normal gene to the uterus. Thus, by screening for mutations in the human coat actin gene and human coat actin protein, it becomes possible to diagnose male infertility patients in humans. The method for diagnosing male infertility according to the present invention is not particularly limited as long as it is a method for examining the presence or absence of a human coat actin gene abnormality or a human coat actin protein abnormality in a sample. In the genetic diagnosis using the sequencing method, the target human coat actin gene is isolated from sperm cells, amplified and purified, and the nucleotide sequence of the human coat actin gene is determined using a gene sequencing device. Although it can be performed by reading, since the operation requires a huge amount of work, a very long time, and a great running cost, various proposed improved methods (Japanese Patent Application Laid-Open No. 2004-093297, JP 2003-215031 A, JP 2003-159054 A, JP 2003-156476 A, Open 2002-340859, JP-can be used JP 2002-340858, JP 2002-340857, JP 2002-333444 and JP advantageous to see JP 2002-171988). In addition, the presence or absence of abnormality of human coat actin protein can be examined by measuring not only the structural abnormality of the protein but also the abnormal expression level or localization.

  As a therapeutic agent for male infertility caused by abnormality of human coat actin gene or human coat actin protein of the present invention, human coat actin gene or recombinant vector in which human coat actin gene is integrated into an expression vector is contained as an active ingredient Examples of the expression vector include adenoviral vectors (Science, 252, 431) used for transient expression in all cells including non-dividing cells (other than blood cells). -434, 1991), retroviral vectors used for long-term expression in dividing cells (Microbiology and Immunology, 158, 1-23, 1992) and non-pathogenic, non-dividing cells can be introduced for long-term expression. Specific examples include adeno-associated virus vectors used (Curr. Top. Microbiol. Immunol., 158, 97-129, 1992) and liposomes. But it is not limited thereto. Among these, adenovirus vectors capable of gene expression in cell systems and living organs with high efficiency are particularly preferable. The human coat actin gene can be introduced into these expression vectors by a conventional method. For example, an expression vector is constructed by inserting a human coat actin gene or the like downstream of an appropriate promoter in these expression vectors. be able to. The human coat actin gene is delivered to the testis by conventional gene delivery techniques aimed at gene therapy, such as methods that administer plasmid DNA alone, methods that use gene carrier molecules, and gene delivery methods that use dendritic polylysine. Coat actin is expressed.

  These preventive / therapeutic agents can be used for preventing the onset of infertility in male patients, treating male patients who have developed infertility, and the like. When used as a pharmaceutical, it can be administered orally or parenterally, but parenteral administration such as intravenous administration is preferred. The parenteral preparation can be an injection, a transdermal preparation, a suppository, or the like. Oral administration agents can be solid preparations such as powders, granules, capsules and tablets, or liquid preparations such as syrups and elixirs. These preparations can be produced according to a conventional method by adding pharmacologically and pharmaceutically acceptable auxiliaries to the active ingredient. As an auxiliary agent, for example, in oral preparations and mucosal administration agents, light anhydrous silicic acid, excipients such as starch, lactose, crystalline cellulose and lactose calcium, disintegrants such as carboxymethyl cellulose, stearic acid Ingredients for preparations such as lubricants such as magnesium, and for injections, solubilizers or solubilizers such as physiological saline, mannitol, propylene glycol, suspending agents such as surfactants, etc. In the case of the above-mentioned formulation components, the formulation components such as aqueous or oily solubilizers, solubilizers, and adhesives are used. In addition, the dose can be appropriately determined according to the type of target disease, the age, sex, weight, symptom, and dosage form of the patient.

  As a method for treating male infertility resulting from an abnormality in the human coat actin gene or human coat actin protein of the present invention, the therapeutic agent for male infertility resulting from an abnormality in the above human coat actin gene or human coat actin protein, There is no particular limitation as long as it is a method of administration to the testis, and as a method for gene transfer into the living body, there have been reported cases in which Sertoli cell damage was actually rescued by in vivo electroporation [Yomogida K, Yagura Y, Nishimune Y. Electroporated transgene-rescued spermatogenesis in infertile mutant mice with a sertoli cell defect. Biol Reprod 2002; 67: 712-717], in vivo electroporation [Muramatsu T, Shibata O, Ryoki S, Ohmori Y , Okumura J. Foreign gene expression in the mouse testis by localized in vivo gene transfer. Biochem Biophys Res Commun 1997; 233: 45-49, Yamazaki Y, Fujimot o H, Ando H, Ohyama T, Hirota Y, Noce T. In vivo gene transfer to mouse spermatogenic cells by deoxyribonucleic acid injection into seminiferous tubules and subsequent electroporation. Biol Reprod 1998; 59: 1439-1444] It can be preferably exemplified in terms of safety. If only the introduction efficiency is considered, virus vectors such as adeno-associated virus vectors and lentiviruses are effective.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

(Mouse isolation and phenotype analysis)
In one strain of PEG8 / IGF2AS transgenic mice, a mutant mouse strain in which male mice showed severe spermatogenesis and became infertile due to transgene insertion mutations occurred. It has been conventionally known that infertile mice are generated at a rate of several percent when producing transgenic mice, but such mutant mice are difficult to maintain and pass, and are outside the purpose of the original experiment. It was often lost without being analyzed. However, we considered that the analysis of infertile mutant mice is important for reproductive biology, and decided to proceed with the analysis of infertile mice. As a result of phenotypic analysis, it was revealed that abnormal sperm morphogenesis occurred in the testes of infertile mice, so the mutant mice were named Dspd (Dominant spermiogenesis defect).

  Since normal mating does not give birth and analysis cannot proceed, we decided to use ICSI (intracytoplasmic sperm injection) technology that directly injects spermatid nuclei into unfertilized eggs. . ICSI is a reproduction assist technology that has already been applied to humans. Even if there is no completed sperm, it is a very powerful method for the treatment of male infertility because it is possible to obtain offspring if there is an elongated sperm cell, but application to mice can maintain and passaging infertile mutant mice It becomes possible. Since the abnormalities of the Dspd mutant mice were postmeiotic abnormalities, it was possible to maintain the mutations by applying ICSI and perform subsequent analysis.

[experimental method]
1. Production of transgenic mice Human PEG8 / IGF2AS cDNA Full length (GenBank Accession Number: AB030733) [Okutsu T, Kuroiwa Y, Kagitani F, Kai M, Aisaka K, Tsutsumi O, KanekoY, Yokomori K, Surani MA, Kohda T, Kaneko- Ishino T, Ishino F. Expression and imprinting status of human PEG8 / IGF2AS, a paternally expressed antisense transcript from the IGF2 locus, in Wilms' tumors. J Biochem (Tokyo) 2000; 127: 475-483] is incorporated into the pCAGGS expression vector A 4.5 kb fragment excised with restriction enzymes Bam HI and Sal I was used as a construct. This construct, which is designed to express PEG8 / IGF2AS under the influence of the chick triactin promoter and CMV-IE enhancer, is injected into the male pronucleus of mouse fertilized eggs (C57BL / 6xC3H) F ₁ and treated fertilized eggs in foster parents. One pseudopregnant female mouse was transplanted to the fallopian tube. Genomic DNA was collected from the tail of a litter born from a foster parent, the presence or absence of the transgene was confirmed by Southern hybridization using the construct as a probe, and transgenic mice were selected.

2. Confirmation of transgene expression by RT-PCR Tissue (one testis) was homogenized in 1 ml of ISOGEN (Nippon Gene) from the testis of each male strain of the produced transgenic mice. Add 0.2 ml of chloroform and mix vigorously, then centrifuge (15,000 rpm, 10 min, 4 ° C.) to recover the aqueous layer (about 0.5 ml), add an equal volume of isopropanol, mix and centrifuge. Thereafter, the pellet was dissolved in 250 μl of DEPC-treated water, 0.75 ml of ISOGEN-LS was added, and the above operation was repeated again. 10x DNase buffer and DNase were added, and DNase treatment was performed at room temperature for 30 minutes. Phenol extraction was performed twice and chloroform extraction was performed twice to remove proteins. After ethanol precipitation, it was dissolved in 100 μl DEPC water. Total RNA was prepared by the above procedure.

CDNA was synthesized from the prepared RNA, and PCR was performed using the following primers as a template to amplify PEG8 / IGF2AS, thereby confirming the expression of the transgene. The preparation method of cDNA followed SUPERSCRIPT preamplification system (GIBCO BRL).
PEG8-F: 5′-TGGAACACAGCTCTGCCTTG-3 ′ (SEQ ID NO: 3) PEG8-R: 5′-CCTGGGGAATGCTCATTCATG-3 ′ (SEQ ID NO: 4)

3. Microinsemination Sperm cells collected from mutant mice were stored frozen in a preservation solution (7.5% fetal bovine serum (FBS), 7.5% glycerol in PBS (-)). After thawing, round and elongated sperm cells were injected into wild type unfertilized eggs. After 48 hours of culture, 4-8 cell embryos were transplanted into the oviducts of pseudo-pregnant female ICR mice to obtain offspring. [Ogura A, Matsuda J, Asano T, Suzuki O, Yanagimachi R. Mouse oocytes injected with cryopreserved round spermatids can develop into normal offspring. J Assist Reprod Genet 1996; 13: 431-434, Ogura A, Ogonuki N, Inoue K, Mochida K. New microinsemination techniques for laboratory animals. Theriogenology 2003; 59: 87-94]

4). Confirmation of expression of PEG8 / IGF2AS protein by Western blotting method A histidine-tagged PEG8 protein (His-PEG8) was synthesized in E. coli and purified using Probond Nickel-Chelating Resin (Invitrogen). Anti-PEG8 serum was obtained from rabbits immunized with His-PEG8. A cell extract (whole cell extract) is prepared from the testis of each line of the transgenic mouse, an extract containing 10 μg of protein is electrophoresed using 10% SDS-polyacrylamide gel, and the electrophoresed protein is subjected to Hybond-P. Transferred to (Amersham) membrane. His-PEG8 was run simultaneously as a positive control. The protein on the membrane and anti-PEG8 serum were reacted and visualized using the ECL Western blotting detection system (Amersham).

5. Tissue morphology observation Light microscope observation: testis and epididymis were fixed overnight with Bouin's solution (0.9% picric acid, 9% formaldehyde, 5% acetic acid), washed in 70% ethanol, and then routinely Paraffin embedded. Sections having a thickness of 3 to 4 μm were cut out, deparaffinized in xylene, and replaced in an ethanol solution series, followed by hematoxylin-eosin (HE) staining. Fifteen Dspd / wt male mice were observed under an optical microscope, and similar findings were obtained. In order to quantify the decrease in sperm cells, the number of sperm cells on the tissue section was examined with a light microscope. Stages I to VI (step 15) and stages VII to VIII (step 16), the number of sperm cells per seminiferous tubule of each stage was counted. Three normal mice and 10 Dspd / wt mice were examined in 10 tubules per stage, and the average number of sperm cells per seminiferous tube was compared.

  Observation by transmission electron microscope: A small piece of testicular tissue was fixed with 2.5% glutaraldehyde and 2% paraformaldehyde solution in 0.1M phosphate buffer (pH 7.3), and then fixed with 1% osmium tetraoxide. did. After dehydration, the tissue was embedded in Epon / Araldite resin to prepare ultrathin sections. The sections were observed with an electron microscope (JEM-1210, JEOL) after staining with uranyl acetate / lead citrate. Four Dspd / wt male mice were observed with an electron microscope, and similar findings were obtained.

[result]
In order to examine the function of the human imprinting gene PEG8 / IGF2AS, transgenic mice were created. PEG8 / IGF2AS was suggested to be a causative gene for Wilms tumor development, but no tumor was observed in any of the 4 transgenic mice (1L, 11L, 16L, 19L). Of the 4 strains, 1L strain male mice were infertile. No other phenotypic abnormalities were found in other strains of mice. When the expression of the transgene in the male testis of each strain was examined by RT-PCR, the expression at the messenger RNA (mRNA) level of PEG8 was confirmed in all strains (FIG. 5 (C)). 1L, 11L, and 19L showed similar expression levels, and 16L had less expression levels than those. Furthermore, as a result of Western blotting analysis using anti-PEG8 serum, PEG8 protein could not be detected in 1L and 19L testis (FIG. 5 (D)).

  The transgene mRNA is expressed in all strains, and it is unlikely that PEG8 functions as a protein from the results of Western blotting analysis. Therefore, the cause of infertility in the 1L strain is the expression of PEG8 transgene. Rather, we thought that it was highly likely that the mutation was an insertion mutation that destroyed the gene at the insertion site when the transgene was inserted into the genome. A transgenic mouse was created for the purpose of examining the function of the PEG8 gene. However, in order to elucidate the mechanism of spermatogenesis in mammals, such a mutant mouse was considered valuable, and analysis of the 1L strain, an insertion mutant, was advanced. . In normal mating, pups cannot be obtained from 1L males and the strain cannot be maintained. Therefore, in vitro fertilization (IVF) was considered, but there was almost no sperm in the epididymis and IVF. Was impossible. Therefore, when ICSI (intracytoplasmic sperm injection) was performed, in which sperm cells collected from the testis were injected into unfertilized eggs, pups could be obtained. The mutation was passaged by ICSI, and subsequent analysis became possible (see FIG. 5). An example of sperm cells has been deposited at the RIKEN BioResource Center as deposit number 01136.

  All Tg (+) male mice that inherited the transgene were infertile, but no phenotypic abnormalities were observed in Tg (+) females and Tg (-) males. Interestingly, if you have a mutation (transgene) in one allele, you will be infertile, that is, a dominant mutation. From Tg (+) females, pups could be obtained by normal mating, but there was a tendency that the number of single births was smaller than that of normal mice (3.0 on average, SD = 1.1). , N = 12). Subsequent observation of the morphology of the testis tissue in order to investigate the cause of infertility in more detail revealed an abnormality in the spermogenesis process. Therefore, this mutation was named Dspd. The histological images of the testis and epididymis of Dspd mice are shown in FIG.

  In Dspd mice (Dspd / wt) testis, round spermatids were normal, but the number of elongated spermatids was reduced, and the sequence of elongated sperm cells was also disturbed. In some cases, the Sertoli cell morphology was disordered and voids were found in the seminiferous epithelium. The number of step 15 elongated sperm cells per seminiferous tubule (stages I-VI) was approximately 30% of normal individuals. Most of the elongated sperm cells had an abnormally shaped nucleus (head). Step 16 sperm cells located on the most luminal side of the seminiferous epithelium were markedly reduced. The number of step 16 sperm cells per seminiferous tubule (stages VII-VIII) was 7% of normal individuals. Multinucleated sperm cells were also found in the seminiferous epithelium. Leydig cells were morphologically normal. In the observation of the epididymis tail, the epididymal duct of normal mice (wt / wt) was filled with the finished sperm, whereas the epididymal duct of the mutant mouse showed almost no sperm, The few sperm heads also showed morphological abnormalities. Interestingly, there were many remnants of immature sperm cells in the epididymis. This is probably because sperm cells exfoliated from the seminiferous epithelium flowed into the epididymis.

  The detachment of sperm cells from the sperm epithelium became more apparent by observation with an electron microscope (see FIG. 7). An image was obtained in which immature sperm cells were detached from the seminiferous epithelium and released into the lumen. The released elongated sperm cells had an abnormally shaped nucleus, and some cells were detached from the epithelium with a cytoplasm presumably of Sertoli cells. A small number of round sperm cells were also released in the lumen, but these were morphologically normal. From these observation results, in the testes of Dspd mice, many cells were detached from the seminiferous epithelium during the process of elongating sperm cells toward mature sperm, resulting in a marked decrease in the number of completed sperm. It is thought that. Sperm cell malformation and detachment from the seminiferous epithelium are phenomena observed in mice administered with E2B (beta-estradiol 3-benzoate), an estrogen compound (see Non-Patent Document 9). A similar phenomenon is also observed in mice administered with BPA (bisphenol A), which is a compound having an estrogenic action. In Sertoli cells of E2B-administered mice, there is an abnormality in the binding structure with sperm cells called Sertoli-spermatid ectoplasmic specialization (ES), which is caused by abnormal sperm cell morphology or sperm epithelium. It is thought that this has led to separation. Since the testicular tissue image of Dspd mice was similar to that of E2B-treated mice, abnormalities in ES structure were suspected. When the ES structure of the mutant mice was examined by electron microscopic observation, those having no actin layer, those partially lacking, those having an abnormal arrangement of actin fibers, etc. were observed (see FIG. 7). This suggests that a phenomenon similar to that in the E2B-administered mouse occurs in this mutant mouse. The mechanism by which ES structure is damaged by estrogen administration is currently unknown at all, and analysis of Dspd mice may contribute to elucidation of this mechanism.

(Genomic analysis of Dspd mice)
Usually, to genetically identify (map) the mutation site in the genome, 1) cross the mutant mouse with a normal mouse, 2) select the offspring that inherited the phenotype, 3) polymorphic markers, etc. It is necessary to repeat generations of the procedure of examining which part of the genome has been inherited from a mutant mouse by using. Although the genome sequence information such as SNPs has been improved, mapping with a much higher accuracy than before has become possible, but the mouse has a gestation period of about 20 days and takes about two months to reach the breeding age. The above cycle requires at least 3 months, and this method takes a lot of time. In addition, it is difficult to perform such analysis for infertility mutations such as Dspd. Point mutations can only be identified by such a method, but in the case of insertion mutations, foreign DNA sequences are used as tags and mutations are made in a relatively short time from one generation (one individual). There is a great advantage that the site can be identified.

  In order to roughly identify the mutation site, first, the chromosome on which the transgene was inserted was examined by FISH (fluorescein in situ hybridization). Subsequently, using the transgene sequence as a probe, a clone containing the mutation site was obtained from the genomic library, and the mutation site was identified from the sequence adjacent to the transgene. As mentioned earlier, insertional mutations are often accompanied by large genomic abnormalities such as chromosomal translocations and deletions. Nakanishi et al. Reported that chromosomal translocation was observed in about 5% of the GFP transgenic mouse strains [Nakanishi T, Kuroiwa A, Yamada S, Isotani A, Yamashita A, Tairaka A, Hayashi. T, Takagi T, Ikawa M, Matsuda Y, Okabe M. FISH analysis of 142 EGFP transgene integration sites into the mouse genome. Genomics 2002; 80: 564-574]. Such chromosomal translocations are often accompanied by large-scale genomic deletions. Conventionally, it has not been easy to analyze particularly large-scale genomic deletions, but it has become possible to determine the size of deletions relatively easily by using SNPs. Therefore, the size of the genomic deletion that occurred at the mutation site was analyzed using the genome database of Celera Genomics, which contains information on single nucleotide polymorphisms (SNPs).

[experimental method]
1. FISH analysis FISH analysis was performed using the full length of the transgene as a probe. Two Tg (+) F ₁ males and one F ₃ female were analyzed, for a total of 2 individuals.

2. Screening of genomic library Genomic DNA collected from Dspd mice was partially digested with restriction enzyme Sau3AI and then ligated with cosmid vector Supercos I (Stratagene). The product was packaged using Gigapack III XL (Stratagene) and infected with E. coli XL1-Blue. The cosmid library thus prepared was screened using PEG8 cDNA as a probe.

3. Analysis of genomic deletion region using SNPs (C57BL / 6xDBA2) F ₁ mice with Dspd mutation were generated by ICSI. Genomic DNA was collected from the obtained F ₁ mice (3 each of Tg (+) and Tg (−)), and a sequence having a polymorphism between DBA2 and C57BL / 6 was amplified by PCR, and the PCR product The presence or absence of the polymorphism was confirmed by reading the sequence by the direct sequencing method. Polymorphism information between mouse strains was obtained from Celera Genomics database (Celera Discovery System, Celera Genomics, HYPERLINK http://www.celera.com/ http://www.celera.com/ ). The polymorphism names (mCV ---) and primer sequences used in the analysis are as follows.
Chromosome 7 mCV24660825:
5′-CATGGGGACTGTGATGGTAC-3 ′ (SEQ ID NO: 5), 5′-GCATGTGTATCTGCTCCAAGG-3 ′ (SEQ ID NO: 6)
mCV24854750:
5'-ACACCGCAGATCCTCTTGTAG-3 '(SEQ ID NO: 7), 5'-ATGTATCTAACAGTGAAGGC-3' (SEQ ID NO: 8)
mCV2370239
5′-GAAGCATGCAAAACACAGC-3 ′ (SEQ ID NO: 9), 5′-GTAAAGTCTGTGGTGCAGTG-3 ′ (SEQ ID NO: 10)
mCV2810840:
5′-GCTGCTTTGAACTTCGCTC-3 ′ (SEQ ID NO: 11), 5′-AACTCTCCCAGGTTTGTT-3 ′ (SEQ ID NO: 12)
mCV24856115:
5′-CCATGTATCAAAGCCCAGG-3 ′ (SEQ ID NO: 13), 5′-GATCTGTCATTGA GTGTACCC-3 ′ (SEQ ID NO: 14);
mCV243317904:
5'-TCGGAGCAGTAACTCTTGTG-3 '(SEQ ID NO: 15), 5'-GGAGTACAGGTACTGCAGGG-3' (SEQ ID NO: 16)
mCV22532403:
5′-GCAGGAGGTCACCACTAGAG-3 ′ (SEQ ID NO: 17), 5′-AGTAAATGCAGAAAATAGCTG-3 ′ (SEQ ID NO: 18)
mCV 22989836:
5′-CCAAGGTTTCCAACAAAAG-3 ′ (SEQ ID NO: 19), 5′-ACCACCACCCTATGTGATCAG-3 ′ (SEQ ID NO: 20)
Chromosome 14 mCV24975960:
5′-CAACTGTGTCACAGTATGG-3 ′ (SEQ ID NO: 21), 5′-GGTTTCTGGTGATTGTGG-3 ′ (SEQ ID NO: 22);
mCV22764767:
5′-CTCCCTGCTGTGATTGATCTC-3 ′ (SEQ ID NO: 23), 5′-TGCAGAGGATGAAAGATTGG-3 ′ (SEQ ID NO: 24);
mCV249986264:
5'-TGTGCACACTGTTAACAAACC-3 '(SEQ ID NO: 25), 5'-GCACTGTTTTGCATTTGTCC-3' (SEQ ID NO: 26);
mCV24616564:
5'-CTGCAGCCATAAAACTTCC-3 '(SEQ ID NO: 27), 5'-CAAGGGTCTTTATCACCAC-3' (SEQ ID NO: 28);
mCV23859822:
5'-TTCAGGTTGCCTTGTAAGCC-3 '(SEQ ID NO: 29), 5'-TAAGCAGGAGGTGATCTG-3' (SEQ ID NO: 30)

[result]
As a result of FISH analysis, it was revealed that translocation occurred in the chromosome of Dspd mice. A long fused chromosome in which the CE region of chromosome 14 was bound near the telomere of chromosome 7 and a short chromosome containing the proximal portion of chromosome 14 were observed. The transgene was inserted at the junctions of fusion chromosomes 7 and 14 (FIG. 8A). The two individuals examined ( _one each of F ₁ and F ₃ ) had the same karyotype. This suggests that two abnormal chromosomes resulting from the translocation are inherited in pairs. The tendency for the number of females with a single Dspd mutation to be small as mentioned in the previous chapter (average: 3.0) is likely due to the formation of eggs that do not occur due to abnormal chromosome combinations during meiosis. . Sequences flanking the transgene isolated from the genomic library were mapped to chromosomes 7 and 14. This is consistent with the FISH results. Subsequently, in order to examine whether or not a genomic deletion occurred at the transgene insertion site, the presence or absence of allele was examined using SNPs between two mouse strains (C57BL / 6 and DBA2). The mapping information and names of SNPs were based on the Celera genomics database. The results of the SNPs analysis are summarized in FIG.

  For chromosome 14, all C57BL / 6 (B6) and DBA2 (D2) alleles were detected for all markers examined. The physical distance from the junction of the transgene and mouse genome (Tg-genome junction) to the nearest marker, mCV24616564, is approximately 1 megabase (Mb). This indicates that no deletion of more than 1 Mb has occurred in this region. Thus, the closest mapped gene Sftpd is aligned with both alleles. 1 Mb from Tg-genome junction to mCV24616564 cannot be analyzed at present. This is because this region contains many repetitive sequences and the genome sequence is incomplete. However, based on the results of examining the draft sequence of this region, it is unlikely that there is a functioning gene. On the other hand, at the mutation site of chromosome 7, only D2 allele was detected on the distal side (telomere side) from the Tg-genome junction. Both alleles were detected in the marker in the vicinity of the gene Dhcr7 located on the proximal side of the junction. All markers from mCV24854750 to mCV22532403 had a deletion of the B6 allele (ie, the abnormal allele in which the translocation occurred). The distance from the junction to mCV22532403 (the furthest marker missing) is approximately 1.2 Mb. B6 allele was detected again with the marker mCV22989836 closer to the telomere. This indicates that the region exceeding 1 Mb from the junction to mCV22532403 is deleted on chromosome 7. No gene is mapped between mCV22532403 and mCV22989836. Based on these data, mutation sites in Dspd mice are summarized in FIG. Chromosome 7 is linked to the chromosome 14 region through the transgene. The proximal part of chromosome 14 appears to be associated with the telomere of chromosome 7. The region exceeding 1 Mb in the vicinity of chromosome 7 telomere is deleted. No deletion larger than at least 1 Mb is found on chromosome 14.

  In the deletion region of chromosome 7, there are 6 known genes (Cttn, Fadd, Fgf3, Fgf4, Fgf15, Ccnd1) and at least 5 function unknown genes (LOC2339777, AU040576, 2210010N10Rik, BC025890, BC019711) . Among the known genes, five genes except for Cttn (Fadd, Fgf3, Fgf4, Fgf15, Ccnd1) have been reported in knockout mice, and no abnormality related to spermatogenesis has been reported [Yeh WC, Pompa JL, McCurrach ME, Shu HB, Elia AJ, Shahinian A, Ng M, Wakeham A, Khoo W, Mitchell K, El-Deiry WS, Lowe SW, Goeddel DV, Mak TW.FADD: essential for embryo development and signaling from some, but not Science 1998; 279: 1954-1958, Zhang J, Cado D, Chen A, Kabra NH, Winoto A. Fas-mediated apoptosis and activation-induced T-cell proliferation are defective in mice lacking FADD / Mort1 Nature 1998; 392: 296-300, Mansour SL, Goddard JM, Capecchi MR.Mice homozygous for a targeted disruption of the proto-oncogene int-2 have developmental defects in the tail and inner ear.Development 1993; 117: 13- 28 、 Moon AM, Boulet AM, Capecchi MR.Normal limb development in conditional mutants of Fgf4.Development 2000; 127: 989-996, Ornitz DM, Itoh N. Fibroblast growth factors.Genome Biol 2001; 2: REVIEWS3005, Sicinski P, Donaher JL, Parker SB, Li T, Fazeli A, Gardner H, Haslam SZ Bronson RT, Elledge SJ, Weinberg RA. Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell 1995; 82: 621-630] (see Table 1).

  Therefore, these 5 gene deletions, at least alone, cannot explain the Dspd phenotype. However, it cannot be denied that the deletion of multiple genes may cause spermatogenesis abnormalities. Fdd, Fgf4, and Fgf15 homozygous mutant mice have been reported to be lethal during fetal life (but no abnormality in heterozygosity), so the Dspd homozygous mutant, if made, is fetal lethal It is expected that. The possibility that the other five unknown genes contribute to the phenotype cannot be excluded at present. Among these, from the amino acid sequence, it was predicted that LOC2339797 is a receptor type protein tyrosine phosphatase, BC025890 is a six-transmembrane ion channel protein, and BC0197711 is a G protein coupled receptor. The distal part of mouse chromosome 7 where deletion was observed has high homology with the long arm of human chromosome 11 (11q13), and the gene sequence is almost completely preserved. Therefore, even in humans, deletion of 11q13 can cause gene deletion in the same combination as in Dspd mice. However, no association between 11q13 and human male infertility has been reported to date. Although various possibilities remain as responsible genes for the Dspd phenotype, the Cttn gene is considered to be the most promising candidate because of its localization and expected function. Cttn encodes coat actin which is an actin binding protein. Coat actin is considered to be a protein that receives a phosphorylation signal from the cell and controls the actin cytoskeleton.

(Analysis of coat actin in which one allele was deleted in Dspd mouse)
The Cttn gene in which deletion of a single allele was observed in Dspd mice and its coat actin protein were analyzed. In addition, it has been reported that coat actin is localized around sperm cells in rat testis [Chapin RE, Wine RN, Harris MW, Borchers CH, Haseman JK. Structure and control of a cell-cell adhesion complex associated with spermiation in rat seminiferous epithelium. J Androl 2001; 22: 1030-1052], and coat actin was considered to be a strong candidate as a causative gene for Dspd. From the phenotypic analysis by electron microscope, it is considered that the interaction between Sertoli cells and sperm cells is abnormal in Dspd mice, and from this, coat actin, which is a regulator of the actin cytoskeleton, is involved. There is a high possibility. Therefore, first, the expression levels of Cttn gene and coat actin protein in mouse testis were quantified by Real-time RT-PCR method and Western blotting method. Next, the localization of coat actin protein and actin in the mouse testis was examined, and normal mice and Dspd mice were compared.

[experimental method]
1. Real-time RT-PCR
RNA was extracted from the testis by the method described in Example 1, and cDNA was synthesized from the total RNA. RT-PCR was performed by the following procedure using ABI PRISM 7700 system, and the copy number of Cttn and Dhcr7 in the cDNA sample was calculated from the obtained amplification curve. Gapdh was used as an internal control.

  Two Tg (+) (Dspd / wt) individuals and 10 Tg (−) (wt / wt) individuals at 10 weeks of age were analyzed. Cttn, Dhcr7 and Gapdh were measured four times per individual, and the measured value (copy number) of Cttn and Dhcr7 was divided by the average value of the measured value (copy number) of Gapdh. The expression level of one normal mouse (wt / wt_1) was taken as 1, and the relative value of each individual was shown in the graph. From the Cttn / Gapdh value (relative value) of each individual, a significant difference between Dspd / wt and wt / wt was tested by an unpaired t-test (significance level 0.01) assuming equal variance.

2. Real-time PCR (using ABI PRISM 7700 Sequence Detector)
(1) A dilution series (2 × 10 ⁵ , 2 × 10 ⁴ , 2 × 10 ³ , 2 × 10 ² , 2 × 10 ¹ copy / μl) of standard DNA (Cttn, Gapd) was prepared. (The standard DNA used was a plasmid DNA obtained by TA cloning the PCR product and purified by cesium density gradient centrifugation.)
(2) The prepared cDNA was diluted to 1 ng / μl.
(3) Reaction solution preparation: 5 μl of template (therefore, standard DNA is 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² copies, cDNA is 5 ng), primer (100 pmol / μl) each 0. 125 μl, SYBR Green PCR Master Mix (Applied Biosystems) 12.5 μl, ultrapure water 7.5 μl
(4) Denaturation was carried out at 95 ° C. for 10 minutes, and PCR was carried out under the conditions of repeating 40 cycles of 95 ° C. for 15 seconds, 65 ° C. for 30 seconds and 72 ° C. for 1 minute to obtain an amplification curve.
Primer used Cttn_F: 5′-TCTGTGACCCTCTCATGAC-3 ′ (SEQ ID NO: 31)
Cttn_R: 5′-GCATTGATCCCTCATGTATCCCC-3 ′ (SEQ ID NO: 32)
Dhcr7_F: 5′-AGAACGTATCACCAGTCAGGTGG-3 ′ (SEQ ID NO: 33)
Dhcr7_R: 5′-CCTGCAAGGCTATAGATAAGCC-3 ′ (SEQ ID NO: 34)
Gapdh_F: 5′-CACTCTTCCCACTTCGATGC-3 ′ (SEQ ID NO: 35)
Gapdh_R: 5′-CTCTTGCTCAGTGTCCTTGC-3 ′ (SEQ ID NO: 36)

3. Coat actin protein quantification by Western blotting An extract was prepared from 10-week-old mouse testis by the following method. The extracted mouse testis was homogenized in 1 ml of extraction buffer (25 mM HEPES, 100 mM KCl, 12 mM MgCl ₂ , 0.5 mM EDTA, 2 mM DTT, 17% glycerol). Centrifugation was performed at 15,000 rpm and 4 ° C. for 10 minutes, and the supernatant (soluble fraction) was recovered. This prepared testis extract was subjected to SDS-PAGE using 10% SDS-polyacrylamide gel, transferred to a membrane, and then the protein was detected by the following procedure. DNA ligase III was used as an internal control for the amount of protein. DNA ligase III is expressed in testis in spermatocytes in the pachytene stage [Chen J, Tomkinson AE, Ramos W, Mackey ZB, Danehower S, Walter CA, Schultz RA, Besterman JM, Husain I. Mammalian DNA ligase III: Molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination.Mol Cell Biol 1995; 15: 5412-5422, Mackey ZB, Ramos W, Levin DS, Walter CA, McCarrey JR, Tomkinson AE.An alternative splicing event which occurs in mouse pachytene spermatocytes generates a form of DNA ligase III with distinct biochemical properties that may function in meiotic recombination. Mol Cell Biol 1997; 17: 989-998]. Since the spermatocytes of Dspd mice are normal, there is no problem as a control.

4). Western blotting After performing a blocking reaction at room temperature for 1 hour in TBS-T containing 5% skim milk, TBS-T washing (15 minutes × 3) was performed. Anti-coat actin antibody (Rabbit, polyclonal, Cell Signaling # 3502) 1: 500 and DNA ligase III antibody (Mouse, polyclonal, GeneTex MS-LIG30UP50) at a dilution of 1: 1000 in 5 ml PBS-MLK (3% skimmed milk) The solution was diluted with PBS containing PBS, placed on a membrane, subjected to primary antibody reaction at room temperature for 3 hours, and then washed with TBS-T (15 minutes × 4). After the secondary antibody reaction with a secondary antibody (Horse radish peroxidase binding) that binds to each primary antibody, TBS-T washing (15 minutes × 4) was performed. Subsequently, proteins were detected using ECL-Plus Western blotting detection system (Amersham).

5. Analysis of Coat Actin Localization by Antibody Staining Organs were excised from mice under anesthesia with Nembutal anesthetic solution (Dynabot) diluted 10-fold. The cut surface was quickly put with a razor, immersed in a fixative (20% formalin, 5% sucrose), and fixed with shaking overnight while changing the fixative once every few hours. Paraffin embedding was performed according to a conventional method, and a section (3 to 4 μm thickness) was prepared. Sections were deparaffinized in xylene and replaced in ethanol solution, and then stained with antibody by the following procedure.
(1) Antigen activation: After heating at 98 ° C. for 10 minutes in 1 mM EDTA (pH 8.0), the mixture was allowed to stand at room temperature for 30 minutes. Thereafter, it was washed with PBS.
(2) Inhibition of endogenous peroxidase: After treatment in 1% H ₂ O ₂ at room temperature for 10 minutes, the plate was washed with PBS.
(3) Normal serum treatment: Treated with 5% normal goat serum (NGS) for 5 minutes at room temperature. (Without PBS washing)
(4) Primary antibody reaction: Anti-coat actin antibody (rabbit polyclonal, commercially available from Cellsignaling, # 3502) was diluted 50-fold with a 0.1% BSA / PBS solution. The primary antibody solution was placed on the tissue, reacted at room temperature for 1 hour, and then washed with PBS.
(5) Secondary antibody reaction: anti-rabbit ENVISION Plus (DAKO Glostrup) was used as the secondary antibody. After treatment with 5% NGS again, the secondary antibody solution was placed on the tissue, reacted at room temperature for 1 hour, and then washed with PBS.
(6) Color development (DAB reaction): Reaction was performed in a color development solution (100 ml of 0.05 M Tris-Cl (pH 7.6) +20 mg DAB (3,3-diaminobenzidine, DOTITE) +65 mg sodium azide) for 3 minutes. It was.
(7) Nuclear staining / encapsulation: Nuclear staining was performed with methyl green and encapsulated by a conventional method.

6). Localization analysis of actin by rhodamine-phalloidin staining The testis was removed from the mouse under anesthesia, the face was cut with a razor, and then fixed overnight in 4% paraformaldehyde (PFA), 5% sucrose / PBS. (Fixing solution was changed twice in the middle). Subsequently, the PFA was washed by shaking in a sucrose / PBS solution. The solution was changed every few hours, and the sucrose concentration was gradually increased (10%, 15%, 20%, 25%: twice each). Thereafter, the tissue was embedded in an OCT compound in isopentane (= dimethylbutane) cooled with liquid nitrogen. The prepared frozen block was sliced (5 to 10 μm thick) to prepare a section. The fixed frozen section thus prepared was stained according to the following procedure.

7). Rhodamine-phalloidin staining The sections were washed with PBS, then treated with PBS (-) containing 0.1% NP40 for 5 minutes at room temperature, washed again with PBS, and rhodamine-phalloidin staining solution (5 μM Rhodamine-Phalloidin (Molecular Probes, Inc., Cat # R-415) + 5% BSA / PBS) for 30 minutes at room temperature (light-shielded), then washed with PBS and treated with nuclear stain (PBS containing 1 μg / ml DAPI) for 2 hours at room temperature (Shaded), washed with PBS, sealed with anti-fading mounting medium (0.05 M Tris-Cl (pH 9.0), 10% polyvinyl alcohol, 5% glycerol, 2.5% DABCO), and then fluorescent Observed with a microscope.

[result]
As a result of quantification by the real-time RT-PCR method, the expression level of the Cttn gene was significantly reduced in the testes of Dspd mice compared to normal individuals (P <0.01) (FIG. 11 (A)). There was no decrease in the Dhcr7 gene. Further, it was confirmed by Western blotting analysis using an anti-coat actin antibody that the coat actin protein was decreased (FIG. 11B). This result supports the result of the SNPs analysis that the Cttn gene is missing one allele. As a result of antibody staining of the coat actin protein (FIG. 12), in the normal mouse seminiferous tubule, a relatively weak positive image was observed at the peripheral part of the seminiferous tubule in all stages. This seems to be included in the Tight junction between the celtry celtris. Step 8-15 A moderate positive image was also observed around the elongated sperm cells. Furthermore, a strong dot-like positive image was observed in the vicinity of Step 16 mature sperm cells (FIG. 13 (A, inset)). Localization around these elongated sperm cells suggests that coat actin functions in the process of sperm morphogenesis. Coat actin is localized in Sertoli cells, not sperm cells, because the head of Step 16 sperm cells having almost no cytoplasm and the dot-like positive image do not necessarily overlap. . In organs other than the testis, characteristic localization was observed, particularly around the cerebellar granule layer and kidney tubules. Coat actin is ubiquitously expressed in almost all tissues at the mRNA level, but at the protein level, it has a different localization in each tissue, suggesting that it has a different role in each tissue.

  In the seminiferous tubules of Dspd mice, although weak positive images were seen throughout the seminiferous tubule, no strong localization around the sperm cells was observed. A small number of Step 16 mature sperm cells were also found in Dspd mouse seminiferous tubules, but no dot-like localization of coat actin was observed in the vicinity of them. This result shows that the localization of coat actin is abnormal in the Dspd mouse testis. It is unclear whether this localization abnormality is caused by a single allele deletion and decreased expression level of coat actin itself. No abnormalities were found in localization in the cerebellum and kidney. The localization of coat actin around elongated sperm cells was in good agreement with the time when sperm cell abnormalities (abnormal nuclear morphology, separation from epithelium) occurred in Dspd mice. Furthermore, the dot-like signal in the vicinity of Step 16 sperm cells suggests that coat actin forms some structure. This structure plays an important role in adhesion between sperm cells and Sertoli cells, and it is considered that adhesion between Sertoli cells and sperm cells is insufficient because the structure is not formed in the Dspd testis.

  Subsequently, rhodamine-phalloidin staining was performed on normal mouse testis, and the localization of actin varied depending on the stage. In the stage III-IV seminiferous tubule, strong actin concentration was observed around the elongated sperm cells (step 15) present in the seminiferous epithelium. This is an actin contained in a special joining device. Actin concentration was also observed in the vicinity of the sperm cells of stage VII-VIII (step 16). Actin signals were also obtained on the acrosomes of round sperm cells. This suggests that actin functions in the formation of acrosomes. In all stages, actin was localized at the Sertoli-Sertoli junction near the base (blood testis barrier, BTB). These observations indicate that actin plays an important role from sperm cell deformation to complete sperm release. In the Dspd seminiferous tubule, a decrease in the number of elongated sperm cells and disorder of the sperm cell arrangement were observed. In addition, voids were conspicuous in the seminiferous epithelium. It is thought that the void is generated because the sperm cells are separated from the place where the sperm cells should be. The concentration of actin was observed around the few elongated sperm cells (often abnormal in shape) as in the normal testis. However, an abnormal concentration of actin was also observed at a position that was not normally observed, which was not associated with the elongated sperm cells. The disturbance of actin seems to reflect that the morphology of Sertoli cells is disturbed. The causal relationship is unclear whether this is due to a decrease in coat actin, but coat actin is involved in the control of the Sertoli cytoskeleton, and it is possible that Sertoli cells are unable to maintain their normal shape due to the decrease. Conceivable.

(Introduction of coat actin gene into Dspd mouse testis)
A plasmid vector that co-expresses coat actin-A or -B expressed in normal mouse testis and EGFP as a marker was prepared and introduced into Dspd mouse testis by in vivo electroporation. The expression of EGFP and coat actin was confirmed (one week after introduction) (see FIG. 14).

(Discussion)
As described in Examples 2 and 3 above, in the Dspd mouse, the region containing the Cttn gene of chromosome 7 was deleted by one allele, the expression level of Cttn was reduced by half, and the coat actin protein in the testis and Abnormal localization of actin was observed.

  As described above, sperm cells after meiosis become complete sperm through a large morphological change. Sertoli cells change their morphology simultaneously with changes in sperm cells. Although there is not yet sufficient knowledge of how Sertoli cell movement is involved in sperm cell morphogenesis, studies of testicular toxicity have shown that Sertoli cell degeneration leads to sperm cell abnormalities. I came. In the process of sperm morphogenesis, Sertoli cells need to change their shape greatly along with deformation of sperm cells and movement into the lumen. Coat actin localization is likely necessary to control the dynamic changes of the Sertoli cells.

The localization of coat actin is similar to that of espin, one of the constituent proteins of ES [Bartles JR, Wierda A, Zheng L. Identification and characterization of espin, an actin-binding protein localized to the F- actin-rich junctional plaques of Sertoli cell ectoplasmic specializations. J Cell Sci 1996; 109 (Pt 6): 1229-1239]. Coat actin has also been reported to interact with β1-integrin contained in ES in the testis [31]. Furthermore, coat actin is a Src protein kinase
Although it is known that phosphorylation is caused by Fyn belonging to the family, Fyn knockout mice have been reported to show abnormalities in ES structure at 3 to 4 weeks of age [Maekawa M, Toyama Y, Yasuda M, Yagi T, Yuasa S. Fyn tyrosine kinase in Sertoli cells is involved in mouse spermatogenesis. Biol Reprod 2002; 66: 211-221].
These findings suggest the involvement of coat actin in the ES structure, indicating that abnormalities in coat actin lead to abnormalities in ES.

  It is interesting that Dspd is a dominant mutation. Deletions of genes often appear as phenotypes only when homozygous, but some structural proteins and complex components are known to show phenotypes due to a decrease in expression due to single allele deletion. (Haploinsufficiency). For example, desmoplakin, the major constituent protein of desmosomal plaque, has been shown to cause palmoplantar keratoderma by deletion of a single allele [Armstrong DK, McKenna KE, Purkis PE, Green KJ, Eady RA, Leigh IM, Hughes AE. Haploinsufficiency of desmoplakin causes a striate subtype of palmoplantar keratoderma. Hum Mol Genet 1999; 8: 143-148]. Moreover, shank3 haploinsufficiency, which is a component of post-synaptic density and interacts directly with coat actin, is thought to cause mental retardation [Boeckers TM, Bockmann J, Kreutz MR, Gundelfinger ED. ProSAP / Shank proteins-a family of higher order organizing molecules of the posts ynaptic density with an emerging role in human neurological disease.J Neurochem 2002; 81: 903-910, Wilson HL, Wong AC, Shaw SR, Tse WY, Stapleton GA, Phelan MC, Hu S, Marshall J, McDermid HE. Molecular characterization of the 22q13 deletion syndrome supports the role of haploinsufficiency of SHANK3 / PROSAP2 in the major neurological symptoms. J Med Genet 2003; 40: 575-584]. Similarly to these, coat actin may be affected by haploinsufficiency.

  If the structure is dynamic, that is, if the molecules contained in the structure are changing at a fast rate, a certain protein concentration may be required in the cell to maintain the structure. . Actin filaments are dynamic structures that maintain an equilibrium state where actin molecules continuously polymerize to one end and dissociate from the other end, even when the filament length does not seem to change. (Treadmill state). The polymerization rate of actin depends on the concentration of actin molecules in the cell, and at a certain concentration, the polymerization and dissociation rates become equal and reach equilibrium.

From the results of immunostaining, it is considered that coat actin controls actin filaments at the contact point between Sertoli cells and sperm cells, and that the structure is dynamic in response to the movement of actin filaments. It is done. To maintain it, a coat actin concentration above a certain threshold in Sertoli cells may be required. Several other possibilities remain that may explain the dominance of Dspd. 1) When only a part of a gene having a transgene insertion or genome deletion is destroyed, a dominant negative type protein that antagonistically inhibits the function of a normal gene product may be generated. To verify this possibility, genes were searched for at the end of the transgene insertion site and deletion region, but no such gene has been found to date. 2) In addition, chromosomal translocations and transgenes (including enhancers) may affect the expression of surrounding genes. It was confirmed by real-time RT-PCR that at least Dhcr7 was expressed at the same level as normal individuals (see FIG. 10). The genes (Sftpd, Rbmxrt) existing around other mutation sites were all expressed in normal individual testis by normal RT-PCR, and it was confirmed that the expression level was almost the same level in Dspd testis. 3) Genomic imprinting is a phenomenon in which functional differences occur in the genomes derived from fathers and mothers, and mutations in single alleles tend to appear in the phenotype for genes that are imprinted. The mutation site of Dspd was close to the imprinting region at the distal part of chromosome 7, suggesting the involvement of imprinting. However, this possibility could be eliminated because the Dspd mutation shows the same phenotype regardless of whether it is inherited from the father or mother.

It is a figure which shows the structure of a mouse fine tube. It is a schematic diagram showing the regulation of testicular function by the endocrine system. It is the schematic which shows a mouse | mouth Sertoli-sperm cell special joining apparatus. A special structure (ectoplasmic) including an actin layer is observed between Sertoli cells and elongated sperm cells. There is an actin layer inside the Sertoli cell membrane and surrounds the acrosome (Ac), which is the structure on the sperm cell side. Although the function of the ES structure is unknown, it is believed to be necessary to support sperm cell morphological changes and to maintain Sertoli cell-sperm cell adhesion until release at the appropriate time. It is a figure which shows a mouse | mouth spermatogenesis cycle (partly modified from Lonnie D.Russel et al., Evaluation of the Testis). In the case of a mouse, the stage of the fine tube is divided into 12 stages up to I-XII. A cross section of a certain seminiferous tube shows a combination of types of cells arranged vertically. After spermatogonia self-proliferation and differentiation into spermatocytes (blue vaccinia) and spermatocyte meiosis (green vaccinia), it becomes haploid spermatids. Sperm cells undergo morphological changes to complete sperm (red vaccinia). This process is called spermiogenesis. The differentiation stage of sperm cells is represented by numbers from Step 1 to Step 16. It is a figure which shows the family of a PEG8 transgene and a PEG8 transgenic mouse. It is a figure which shows the Dspd mouse testis and epididymis. In Dspd mouse seminiferous tubules, the number of elongated sperm cells was decreased. Sperm cell malformation was also observed. (A), (C) Normal mouse epididymis was filled with completed sperm (F), (H), but the number of spermatozoa was reduced in Dspd mouse epididymis. (E), (G) A, B, E, G Bar = 100 μm, C, D, G, H Bar = 200 μm, insets Bar = 5 μm It is a figure which shows ES structure of a Dspd mouse | mouth. It is an enlarged view showing the mutation site of Dspd mouse. The red arrow represents the Tg and genomic junction cloned from the genomic library. A long fused chromosome of chromosome 7 and chromosome 14 is represented by a pink line, and a short chromosome including the proximal part of chromosome 14 is represented by a blue line. The blue dotted line represents the region where the genome sequence was not completed and could not be analyzed. Chromosome 7 is linked to the chromosome 14 region through the transgene. The proximal part of chromosome 14 appears to be associated with the telomere of chromosome 7. The region exceeding 1 Mb in the vicinity of chromosome 7 telomere is deleted. No deletion larger than at least 1 Mb is found on chromosome 14. The gene Sftpd closest to the chromosome 14 mutation site is not deleted. It is an enlarged view showing the structure and function of coat actin. It is a figure which shows the expression level of coat actin in a Dspd mouse testis. It is a figure which shows the expression level of coat actin in a Dspd mouse testis. It is a figure which shows the localization of actin in a seminiferous tubule. In normal mouse seminiferous tubules, strong actin localization was observed around the elongated sperm cells. In particular, strong actin concentration was observed around the sperm cells in Step 15 (A)-(C). This suggests that actin plays an important role in the process of spermiogenesis. Abnormal concentration of actin was observed in the Dspd testis (arrows I and L). Concentration of actin was observed in the sperm head (H arrow) as in the normal testis. It is a figure which shows the result of a coat actin antibody dyeing | staining. It is a figure which shows gene introduction | transduction to the mouse testis by the in vivo electroporation method. It is a figure which shows a Dspd mouse | mouth disease state model. Due to the decrease in the expression level of coat actin, a structure containing coat actin cannot be produced normally. As a result, the control of actin fibers is disturbed, abnormalities of the ES structure occur, and sperm cell malformation and separation from the sperm epithelium occur.

Claims (15)

  Non-human animals in which all or part of the endogenous genes of non-human animals encoding coat actin have been inactivated by genetic mutations such as disruption, deletion or substitution, and the function of expressing coat actin has been reduced or lost A male infertility model animal characterized by comprising:   The male infertility model animal according to claim 1, which has a gene mutation in one allele.   The male infertility model animal according to claim 1 or 2, which exhibits behavioral abnormalities such as gait abnormalities.   The male infertility model animal according to any one of claims 1 to 3, wherein the non-human animal is a mouse.   The male infertility model animal according to claim 4, wherein the male infertility model animal is produced using mouse sperm cells (Deposit Number 01136, RIKEN BioResource Center).   Non-human animals in which all or part of the endogenous genes of non-human animals encoding coat actin have been inactivated by genetic mutations such as disruption, deletion or substitution, and the function of expressing coat actin has been reduced or lost A male infertility characterized by injecting sperm cells collected from a wild-type unfertilized egg and transplanting the cultured 4-8 cell embryo into the oviduct of a non-human animal pseudopregnant to obtain a litter To maintain and pass the disease model animal.   The method for maintaining / passaging a male infertility model animal according to claim 6, wherein the non-human animal is a mouse.   The method for maintaining and subculturing male infertility model animals according to claim 7, wherein the sperm cells are mouse sperm cells (deposit number 01136 at RIKEN BioResource Center, an independent administrative agency).   A test substance is administered to the male infertility model animal according to any one of claims 1 to 5, and the degree of improvement in symptoms of spermatogenic dysfunction is measured and evaluated, thereby preventing and treating male infertility Agent screening method. DNA consisting of any one of the following base sequences (A) to (F), which encodes a protein whose reduced expression function or loss of expression function causes male infertility:
(A) a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(B) a base sequence encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having a binding activity to actin;
(C) a base sequence encoding a protein consisting of an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2 and having a binding activity to actin;
(D) the base sequence represented by SEQ ID NO: 1;
(E) a base sequence encoding a protein consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 and having actin-binding activity;
(F) a base sequence encoding a protein that hybridizes with the base sequence shown in SEQ ID NO: 1 under stringent conditions and has a binding activity to actin; A protein consisting of an amino acid sequence of any of the following (A) to (C), whose reduced expression function or loss of expression function causes male infertility.
(A) the amino acid sequence shown in SEQ ID NO: 2;
(B) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and the protein comprising the amino acid sequence has an activity to bind to actin Sequence;
(C) an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2 and a protein comprising the amino acid sequence having an activity to bind to actin;   A method for diagnosing male infertility, characterized by examining the presence or absence of an abnormality in a human coat actin gene in a specimen.   A method for diagnosing male infertility, characterized by examining the presence or absence of an abnormality in human coat actin protein in a specimen.   A therapeutic agent for male infertility caused by an abnormality in human coat actin gene or human coat actin protein, comprising as an active ingredient a human coat actin gene or a recombinant vector in which a human coat actin gene is integrated into an expression vector .   A method for treating male infertility caused by an abnormality in a human coat actin gene or a human coat actin protein, comprising administering the therapeutic agent for male infertility according to claim 14 to a testis.