Method for producing self-fertilized fish and self-fertilized eggs

Abstract

The present invention addresses the problem of providing a means of self-fertilizing in fish, and solves said problem by providing a method for producing a fish having the ability to self-fertilize or self-fertilizing eggs, the method including (1) a step for administering a masculinizing agent to a fish exhibiting deficient or reduced function and / or expression of the foxl3 gene in the reproductive cells thereof.

Description

[Technical field]

[0001] The present invention relates to a method for producing fish having self-fertility, a method for producing self-fertilized eggs, and the like. [Background technology]

[0002] Various animals are bred using useful traits as indicators. For example, ornamental fish such as medaka are bred using body color, patterns, eye shape, fin shape, etc. as indicators based on aesthetic viewpoints, and hundreds of varieties of medaka have been reported in recent years. In addition, various varieties of farmed fish for food such as sea bream, yellowtail, and tuna are also bred using growth rate, disease resistance, palatability, etc. as indicators. However, since fertilization of animals is usually cross-fertilization, even if a useful trait is found, it takes time and effort to create a variety in which the useful trait is fixed.

[0003] If self-fertilization were possible, it would be possible to more efficiently produce varieties with fixed useful traits. However, although self-fertilization occurs in some non-vertebrate animals, it does not usually occur in the majority of animals, particularly vertebrates. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2015-126729 A Summary of the Invention [Problem to be solved by the invention]

[0005] In Patent Document 1, it was reported that in the ovaries of fish in which expression of the foxl3 gene in germ cells was suppressed, both fertilizable sperm and fertilizable eggs were produced, but because they were produced in separate compartments within the ovary, they were unable to self-fertilize (Figure 1).

[0006] An objective of the present invention is to provide a technique for self-fertilization in fish. [Means for solving the problem]

[0007] As a result of intensive research conducted by the present inventors in consideration of the above problems, (1) it was discovered that by administering a masculinizing agent to fish in which the function and / or expression of the foxl3 gene in germ cells is lost or reduced, fish with self-fertilization ability can be produced, and furthermore, self-fertilized eggs can be produced. Based on this discovery, the present inventors conducted further research, and as a result, completed the present invention. That is, the present invention encompasses the following aspects.

[0008] Item 1. (1) A method for producing self-fertile fish, comprising the step of administering a masculinizing agent to fish in which the function and / or expression of the foxl3 gene in germ cells is deleted or reduced.

[0009] Item 2. The method according to Item 1, wherein the fish is a female.

[0010] Item 3. The method according to Item 1 or 2, wherein the fish is in the oogenesis stage.

[0011] Item 4. The method according to any one of Items 1 to 3, wherein the masculinizing agent is administered continuously.

[0012] Item 5. The method according to any one of Items 1 to 4, wherein the period of administration of the masculinizing agent is 5 to 100 days.

[0013] Item 6. A self-fertile fish obtained by the production method according to any one of items 1 to 5.

[0014] Item 7. (2) A method for producing self-fertilized eggs, comprising a step of inducing ovulation in the fish according to Item 6.

[0015] Item 8. The method according to Item 7, wherein the ovulation induction treatment is mating with a male fish or administration of an ovulation inducer.

[0016] Item 9. The method according to Item 8, wherein the ovulation induction treatment is mating with a sterile male fish.

[0017] Item 10. (1) A method for producing self-fertilized eggs, comprising the step of administering a masculinizing agent to fish in which the function and / or expression of the foxl3 gene in germ cells is deleted or reduced.

[0018] Item 11. The production method according to Item 10, further comprising the step of (3) subjecting fish in which the function and / or expression of the foxl3 gene in germ cells is deleted or reduced to an ovulation induction treatment.

[0019] Item 12. The method according to Item 11, wherein the step 2 is carried out after the step 1 is completed.

[0020] Item 13. The method according to Item 11 or 12, wherein the ovulation induction treatment is mating with a male fish or administration of an ovulation inducer.

[0021] Item 14. A self-fertilized egg obtained by the production method according to any one of Items 7 to 13.

[0022] Item 15. A fish produced by the self-fertilized egg according to Item 14. Effect of the Invention

[0023] According to the present invention, it is possible to provide a self-fertilization technique for fish. Specifically, it is possible to provide a method for producing fish capable of self-fertilization, a method for producing self-fertilized eggs, fish capable of self-fertilization, self-fertilized eggs, fish produced from self-fertilized eggs, etc. [Brief description of the drawings]

[0024] [Figure 1] The cross-sectional schematic diagrams of a normal fish ovary and a fish ovary in which the expression of the foxl3 gene in germ cells is suppressed are shown, along with an enlarged schematic diagram of the area in which germline stem cells are present. [Diagram 2] The upper image shows the ovary of a foxl3 - / - female medaka before the start of mating with a sterile male. The lower image shows a section of the area indicated by the dotted line in the upper image. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0026] 1.Definition In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0027] The "identity" of an amino acid sequence refers to the degree of amino acid sequence agreement between two or more comparable amino acid sequences. Thus, the higher the agreement between two amino acid sequences, the higher the identity or similarity between those sequences. The level of identity of an amino acid sequence can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87: 2264-2268 (1990), Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences" Proc Natl Acad Sci USA. 90: 5873-7 (1993)). A program called BLASTX based on such a BLAST algorithm has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of base sequences is also defined in the same manner as above.

[0028] In this specification, "conservative substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is considered to be conservative substitution. In addition, substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine are also considered to be conservative substitutions.

[0029] 2. Method for producing self-fertile fish In one aspect, the present invention relates to (1) a method for producing fish having self-fertility, comprising a step of administering a masculinizing agent to fish in which the function and / or expression of the foxl3 gene in germ cells is deleted or reduced (also referred to as "production method 1 of the present invention" in this specification). This will be described below.

[0030] The fish is not particularly limited as long as it has the foxl3 gene endogenously. The fish may be fish for various purposes such as experiment or research, food, ornamental, and aquaculture. Specific examples include killifish, zebrafish, goldfish, loach, tilapia, eel, conger eel, carp, crucian carp, Japanese sea bream, red sea bream, salmon, rainbow trout, amago, yellowtail, tuna, pufferfish, flounder, and yamame salmon. The fish may belong to any of the categories of bony fish, cartilaginous fish, and gnathostomatous fish. The present invention is preferably applicable to bony fish, and examples thereof include fish belonging to the genus Oryzias and its close relatives.

[0031] Regarding the sex of fish, in the case of fish having sex chromosomes, it is preferable that the fish is a female chromosome type fish, but in the case of fish not having sex chromosomes (such as sea bream), there is no particular limitation.

[0032] There are no particular limitations on female chromosome fish, so long as the combination of sex chromosomes is female. There are various types of sex determination by sex chromosomes, such as XY type, XO type, modified type, ZW type, and ZO type. For example, in the case of XY type fish, the sex chromosomes are XX, which corresponds to the female chromosome type, and in the case of ZW type fish, the sex chromosomes are ZW, which corresponds to the female chromosome type.

[0033] The foxl3 gene is a gene belonging to a protein family having a forkhead-type DNA binding domain. The foxl3 gene belongs to the same protein family as the foxl2 gene, which is conserved across species. Here, the foxl2 gene generally means a female-specific gene expressed in somatic cells of ovarian follicles, and is widely found in mammals, birds, and fish. In fish, foxl2 is also written as foxl2a. On the other hand, the foxl3 gene is a different gene from foxl2a, and Patent Document 1 reports that both fertilizable sperm and fertilizable eggs are produced in the ovaries of fish in which the function and / or expression of the foxl3 gene in germ cells is lost or reduced. In various fish, the base sequence and amino acid sequence of the foxl3 gene are publicly known, or can be easily determined based on the base sequence and amino acid sequence of the publicly known foxl3 gene (for example, by identity analysis with these). As an example, the foxl3 gene of medaka (Oryzias latipes) is a protein consisting of an amino acid sequence specified by NCBI RefSeq accession number: NP_058673 (a protein consisting of an amino acid sequence encoded by a nucleotide sequence specified by NCBI RefSeq accession number: NM_016977). SEQ ID NO: 1 is the base sequence of the medaka foxl3 gene, and SEQ ID NO: 2 is the amino acid sequence of the protein encoded by the foxl3 gene. Note that the foxl3 gene is sometimes called the "foxl2b gene" depending on the classification or naming method.

[0034] The foxl3 gene of interest in the present invention also includes mutants that may occur in nature. The foxl3 gene of interest in the present invention may have base mutations such as substitutions, deletions, additions, and insertions, so long as the properties of the encoded protein (such as DNA binding ability) are not significantly impaired. As the mutation, a mutation that does not result in an amino acid substitution in the protein translated from the mRNA or a mutation that results in a conservative amino acid substitution is preferred.

[0035] The foxl3 gene of interest in the present invention is, for example, a gene in which the amino acid sequence of the protein encoded thereby has identity, for example, of 95% or more, preferably 98% or more, and more preferably 99% or more, to the amino acid sequence of the protein encoded by the wild-type foxl3 gene of the same fish species. Furthermore, the foxl3 gene of interest in the present invention is, for example, a gene in which the amino acid sequence of the protein encoded thereby is identical to the amino acid sequence of the protein encoded by the wild-type foxl3 gene of the same fish species, or in which one or more (for example, 2 to 10, preferably 2 to 5, more preferably 2 to 3, and even more preferably 2) amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence.

[0036] In germ cells of fish that are the subject of the present invention, the function and / or expression of the foxl3 gene that is the subject of the present invention is lost or reduced. In one embodiment of the present invention, a mutation is introduced into the foxl3 gene in fish that are the subject of the present invention so that the function and / or expression of the foxl3 gene that is the subject of the present invention is lost or reduced. Here, the "function" of the foxl3 gene refers to the properties of the foxl3 protein (such as DNA binding ability). Furthermore, the "expression" of the foxl3 gene includes both the expression of foxl3 mRNA and the expression of foxl3 protein, but is preferably the expression of foxl3 protein. "Lack of" refers to the activity and / or expression level of the foxl3 gene being below the detection limit in a sample obtained from a fish that is the subject of the present invention. Furthermore, "reduced" means that the activity and / or expression level of the foxl3 gene in a sample obtained from a fish that is the subject of the present invention is lower (e.g., 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 200, 1 / 500, 1 / 1000, 1 / 2000, 1 / 5000, 1 / 10000 or less) than the activity and / or expression level of the foxl3 gene before the introduction of mutation (the foxl3 gene that is the subject of the present invention). The activity and / or expression level of the foxl3 gene can be measured according to a known method.

[0037] The suppression of gene expression can be evaluated by detecting at least one of the mRNA transcribed from the target gene and the protein encoded by the target gene. Specifically, when evaluating based on mRNA, the amount of mRNA transcribed from the target gene can be measured by Northern hybridization, RT-PCR, quantitative PCR, microarray, etc. using primers or probes specific to the target gene. When evaluating based on protein, the amount of expression of the protein can be measured by immunological methods such as Western blotting and ELISA using an antibody specific to the protein encoded by the target gene.

[0038] The preferred time to control the foxl3 gene in germ cells can be appropriately selected according to the development of the gonads of the target fish. Specifically, in order to differentiate germ cells into sperm, it is preferable to suppress the expression of the foxl3 gene at the time when it is expressed in germ cells. For example, in medaka, the differentiation time of the gonads is around hatching, and it is possible to suppress the expression of the foxl3 gene in germ cells at this time. In addition, since it is thought that stem cell type and cyst type germ cells maintain sexual immaturity or sexual plasticity, it is also possible to control it in the juvenile fish stage after sex differentiation and in the mature gonads (ovaries). In addition, for individuals that are thought to undergo sex differentiation several years after hatching, such as hagfish, the expression of the foxl3 gene in germ cells can be suppressed according to the time of sex differentiation according to the individual.

[0039] When a mutation is introduced into the foxl3 gene, the mutation is not particularly limited as long as it is a mutation that causes a loss or decrease in the function and / or expression of the foxl3 gene. Examples of such mutations include gene deletion, a mutation in a protein coding region, a mutation in a splicing regulatory region, and a mutation in an expression control region (e.g., a promoter, an activator, an enhancer, etc.). Among these, gene deletion is preferable. Furthermore, it is preferable that the fish targeted by the present invention have the mutation in both of a pair of chromosomes.

[0040] Mutation introduction into the foxl3 gene can be achieved by, for example, gene knockout using artificial nucleases (TALEN, Cas protein), gene knockout using ES cells or the like into which a targeting vector containing at least a part of the foxl3 gene has been introduced by homologous recombination, gene knockdown using shRNA, antisense, ribozyme, or the like against the foxl3 gene, irradiation, administration of a chemical mutagen, introduction of a transposon, etc. Those skilled in the art can apply conventionally known methods to construct targeting vectors used for gene knockout, gene knockdown, and gene mutation introduction, design and synthesis of shRNA, antisense, ribozyme, etc., introduction method of DNA into ES cells, unfertilized eggs, fertilized eggs, embryos, primordial germ cells, etc., irradiation, administration of a chemical mutagen, introduction of a transposon, etc.

[0041] As described above, "fish in which the function and / or expression of the foxl3 gene is lost or reduced" used in the production method of the present invention include foxl3 gene knockout fish (foxl3 gene-deficient fish), foxl3 gene knockdown fish, fish with introduced foxl3 gene mutations, etc., but foxl3 gene knockout fish are preferred.

[0042] In the ovaries of fish in which the function and / or expression of the foxl3 gene in germ cells is absent or reduced, only sperm are produced initially, but then eggs are also produced. This can be determined by a method known to those skilled in the art, for example, by observing stained images of ovarian sections, to determine whether or not eggs as well as sperm are produced. The fish to be subjected to step 1 are preferably in the stage of egg formation (oogenesis stage: the stage in which the presence of eggs can be confirmed in the ovaries). In the case of medaka, the oogenesis stage is, for example, 90 to 120 days after hatching. By employing fish at such a stage, the production efficiency of fish having self-fertility can be improved.

[0043] The masculinizing agent is not particularly limited as long as it is a substance that causes masculinization, and examples thereof include substances that promote masculinization such as androgens, and substances that suppress feminization such as antiestrogens. Examples of androgens include 11-ketotestosterone, adrenosterone, testosterone, dihydrotestosterone, dehydroepiandrosterone, and the like. Examples of antiestrogens include estrogen receptor inhibitors, aromatase inhibitors, and the like. The masculinizing agent is preferably androgen. The masculinizing agent may be one type alone or two or more types in combination.

[0044] The method of administration of the masculinizing agent is not particularly limited, and an appropriate method can be adopted depending on the type of fish and the rearing method, etc. Examples of the administration method include oral administration, transdermal administration, intraperitoneal administration, intravenous administration, subcutaneous administration, etc.

[0045] In one embodiment of the present invention, for example, the masculinizing agent can be administered simply and reliably by dissolving it in the rearing water.

[0046] In one aspect of the present invention, for example, the masculinizing agent-containing feed can be used as the feed, so that the masculinizing agent can be administered simply and reliably. The form of the feed is not particularly limited as long as it is in a form that can be ingested by the target fish. Examples of the form of the feed include powder, granules, crumbles, pellets, cubes, paste, liquid, etc. Masculinizing agent-containing feed contains, in addition to the masculinizing agent, other substances and ingredients that constitute the feed, such as fish meal; plant-based feed ingredients such as soybean meal, concentrated soy protein, corn gluten meal, corn distillers' grains, and rapeseed oil cake; animal-based feed ingredients such as meat meal, meat and bone meal, feather meal, and blood meal; microalgae ingredients; single-celled organism ingredients such as yeast and bacteria; binders (e.g., sodium carboxymethylcellulose (CMC), guar gum, sodium alginate, sodium polyacrylate, sodium caseinate, propylene glycol, etc.), grain flours (e.g., wheat flour, starch, etc.), minerals, vitamins (e.g., vitamin C, vitamin B1, vitamin A, vitamin D, vitamin E, etc.), amino acids (e.g., lysine, methionine, histidine, etc.), pigments (e.g., β-carotene, astaxanthin, canthaxanthin, etc.), and liver oils (e.g., cod liver oil, etc.).

[0047] Administration of a masculinizing agent induces some sperm to migrate into the ovarian cavity where mature eggs are released (see Test Example 2).

[0048] The dosage of the masculinizer is not particularly limited as long as it is an amount sufficient to cause the transfer of sperm to the ovarian cavity, and can be appropriately set using the presence or absence of the phenomenon as an indicator. For example, when the masculinizer is dissolved in water in which fish are bred, the concentration of the masculinizer in the water is, for example, 1 to 1000 ng / ml, preferably 5 to 500 ng / ml, more preferably 10 to 200 ng / ml, and even more preferably 20 to 100 ng / ml. For example, when feeding the fish with a masculinizer-containing feed, the content of the masculinizer in the feed is, for example, 0.00001 to 50% by mass. The upper and lower limits of the content can be, for example, 0.0001% by mass, 0.001% by mass, 0.01% by mass, 0.1% by mass, 0.2% by mass, 0.5% by mass, 1% by mass, 2% by mass, 5% by mass, 10% by mass, 20% by mass, 30% by mass, 40% by mass, etc.

[0049] The number of times the masculinizing agent is administered is not particularly limited, but the masculinizing agent is preferably administered continuously, i.e., the masculinizing agent is preferably administered continuously or at regular intervals (e.g., 1 to 3 times a day, once every 2 to 3 days, etc.).

[0050] The administration period of the masculinizing agent is not particularly limited as long as it is a period during which spermatozoa migrate to the ovarian cavity, and can be appropriately set using the presence or absence of the phenomenon as an indicator. The period is, for example, 1 to 200 days, preferably 5 to 100 days, more preferably 10 to 50 days, and even more preferably 20 to 40 days. This period can vary depending on the period until the fish matures.

[0051] After administration of the masculinizing agent, the animals can be reared in an environment where no masculinizing agent is administered, if necessary. This can increase the transfer of sperm to the ovarian cavity. The rearing period is not particularly limited, and is, for example, 0.1 to 10, preferably 0.3 to 5, and more preferably 0.7 to 3, relative to 1 period of administration of the masculinizing agent. More specifically, the rearing period is, for example, 1 to 400 days, preferably 5 to 200 days, more preferably 10 to 100 days, and even more preferably 20 to 80 days.

[0052] The fish obtained by Production Method 1 of the present invention (sometimes referred to as "fish of the present invention" in this specification) have the ability to self-fertilize. That is, because some of the sperm have migrated out of the ovarian parenchyma (for example, into the ovarian cavity or body cavity), when a mature egg migrates out of the ovarian parenchyma, it can be fertilized with its own sperm.

[0053] 3. Method A for producing self-fertilized eggs In one aspect, the present invention relates to (2) a method for producing a self-fertilized egg, which comprises a step of subjecting the fish of the present invention to an ovulation induction treatment (also referred to as "production method 2A of the present invention" in this specification). This will be described below.

[0054] The ovulation induction treatment is not particularly limited, and examples thereof include mating with male fish, administration of an ovulation inducer, etc. In order to obtain only self-fertilized eggs, it is preferable that the male fish is a sterile male fish. Sterile male fish can be produced, for example, by mutation of a gene involved in sterility (for example, nanos gene). Examples of ovulation inducers include clomiphene, gonadotropin, chorionic gonadotropin, GnRH agonist, GnRH antagonist, etc.

[0055] By the ovulation induction treatment, fertilization with the self-sperm occurs during ovulation, and self-fertilized eggs can be obtained. The obtained self-fertilized eggs can be cultured and developed under an appropriate environment to obtain individual fish.

[0056] 4. Method B for producing self-fertilized eggs In one aspect, the present invention relates to a method for producing a self-fertilized egg, which comprises (1) administering a masculinizing agent to a fish in which the function and / or expression of the foxl3 gene in germ cells is deleted or reduced (also referred to as "production method 2B of the present invention" in this specification). This will be described below.

[0057] The "fish lacking or having reduced function and / or expression of the foxl3 gene in germ cells" and the "administration of a masculinizing agent" are the same as in Production Method 1 of the present invention.

[0058] By administering the masculinizing agent, some of the sperm are transferred to the ovarian cavity, and when the mature eggs are transferred to the ovarian cavity, they are fertilized by the sperm of the mother. This allows for self-fertilized eggs to be obtained.

[0059] It is preferable that the production method 2B of the present invention further includes a step (3) of subjecting fish in which the function and / or expression of the foxl3 gene in germ cells is deleted or reduced to an ovulation induction treatment. This step makes self-fertilization more likely to occur, and can increase the production efficiency of self-fertilized eggs. The ovulation induction treatment is the same as in the production method 2A of the present invention.

[0060] The obtained self-fertilized eggs can be cultured and developed under an appropriate environment to obtain individual fish. EXAMPLES

[0061] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0062] Reference Example 1. Production of sterile male medaka Sterile male nanos homozygous mutant (nanos - / -) medaka, in which both alleles of the nanos gene were disrupted, were generated using TALEN technology as follows. TALEN target sites in the nanos3 gene were searched for using the TALEN Targeter program (https: / / tale-nt.cac.cornell.edu / node / add / talen). TALEN plasmids were linearized by NotI digestion and used as templates for in vitro RNA synthesis using the mMESSAGE mMACHINE® T7 Transcription Kit (Thermo Fisher). TALEN mRNA (250 ng / μl left and right) was injected into 1-cell or 2-cell stage embryos. F0 founders were mated with wild-type medaka. The resulting nanos heterozygous mutant (nanos + / -) males and females were mated to obtain sterile male nanos homozygous mutant (nanos - / -) medaka.

[0063] Test Example 1. Production of self-fertilized fish and self-fertilized eggs Medaka of foxl3 heterozygous mutant (foxl3 + / -) in which one allele of the foxl3 gene was destroyed (Patent Document 1) were crossed with each other to obtain female chromosome type (XX type) medaka of foxl3 homozygous mutant (foxl3 - / -). The foxl3 - / - medaka were raised in an aquarium according to a conventional method, and from 90 to 120 days after hatching, they were raised for 30 days in an aqueous masculinizer solution containing a masculinizer (11-ketotestosterone) at a concentration of 50 ng / ml. During this time, the aqueous masculinizer solution was replaced once every 24 hours. After raising in the aqueous masculinizer solution, they were raised for at least 30 days (30 to 60 days) in normal water that did not contain the masculinizer. Sterile male medaka (Reference Example 1) was placed in the aquarium, raised for 30 days, and mated. During this period, the eggs that were laid were collected, and the presence or absence of fertilization was judged based on the activation of the chorion membrane and normal development up to the blastocyst stage.

[0064] As a result, for individual 1, 33 eggs were laid, of which 8 were fertilized, and of these, 2 eggs hatched normally. For individual 2, 29 eggs were laid, of which 8 were fertilized, and of these, 2 eggs hatched normally. On the other hand, when the test was conducted in the same manner except that the animals were reared in water containing no masculinizer instead of in the aqueous masculinizer solution, no fertilized eggs were confirmed.

[0065] Test Example 2. Histological analysis of the ovary The gonads of foxl3 - / - female medaka before the start of mating with the sterile male medaka of Test Example 1 were dissected and fixed in Buin's solution overnight at 4°C. Plastic sections 4 µm thick were prepared using Technovit (registered trademark) 8100 (Heraeus Kulzer). The sections were stained with periodic acid-Schiff and hematoxylin according to standard methods.

[0066] The results are shown in Figure 2. The presence of sperm (black dots within the dotted line) was observed in the ovarian cavity where mature eggs are ovulated.

Claims

1. (1) A method for producing self-fertile fish, comprising step 1 of administering a masculinizing agent to fish in which the function and / or expression of the foxl3 gene in germ cells is lost or reduced.

2. The method of claim 1 , wherein the fish is female.

3. The method according to claim 1 or 2, wherein the fish is in the oogenesis stage.

4. The method according to any one of claims 1 to 3, wherein the masculinizing agent is administered continuously.

5. The method according to any one of claims 1 to 4, wherein the period of administration of the masculinizing agent is 5 to 100 days.

6. A fish having a lack or reduced function and / or expression of the foxl3 gene in germ cells and having the ability to self-fertility, obtained by the production method according to any one of claims 1 to 5.

7. (2) A method for producing self-fertilized eggs, comprising step 2 of subjecting the fish described in claim 6 to an ovulation induction treatment.

8. The method according to claim 7, wherein the ovulation induction treatment is mating with a male fish or administration of an ovulation induction agent.

9. The method according to claim 8, wherein the ovulation induction treatment is mating with a sterile male fish.

10. (1) A method for producing self-fertilized eggs, comprising step 1 of administering a masculinizing agent to fish in which the function and / or expression of the foxl3 gene in germ cells is lost or reduced.

11. The method for producing according to claim 10, further comprising the step (3) of subjecting fish in which the function and / or expression of the foxl3 gene in germ cells is deleted or reduced to an ovulation induction treatment.

12. The method according to claim 11 , wherein the step 3 is carried out after the step 1 is completed.

13. The method according to claim 11 or 12, wherein the ovulation induction treatment is mating with a male fish or administration of an ovulation induction agent.

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