Fish masculinization inducer, fish masculinization method, and fish masculinization inhibitor

Abstract

To provide a novel masculinization induction agent for fish and a masculinization method for fish, and a novel masculinization inhibitor and a masculinization inhibition method, capable of safely producing an individual subjected to sex manipulation such as a reverse sex individual in which a genetic sex and a functional sex are opposite each other and a normal sex individual in which the genetic sex and the functional sex match each other, regardless of an environmental factor, instead of a sex manipulation method using a sex manipulation agent and a hormone for fish mainly formed of a prior hormone.SOLUTION: There is provided a masculinization induction agent for fish, mainly formed of a PPAR activator or a β oxidation activator. There is also provided a masculinization method for fish for performing masculinization of fish by administering the PPAR activator or the β oxidation activator to a fish in which a sex is not differentiated for activating the PPAR or β oxidation for masculinization of fish. There is also provided a masculinization inhibitor for fish mainly formed of the PPAR inhibitor or the β oxidation inhibitor. There is also provided a masculinization inhibition method for fish for administering the PPAR inhibitor or the β oxidation inhibitor to a fish in which a sex is not differentiated for deactivating the PPAR or the β oxidation for suppressing masculinization of the fish.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a novel fish masculinization inducer, a fish masculinization method, and a novel fish masculinization inhibitor. [Background technology]

[0002] There are sex-changing fish species in which external environmental factors such as sex disrupting substances and water temperature reverse the inherent genetic sex (hereinafter simply referred to as genetic sex) from the actually expressed traits and functional sex (hereinafter simply referred to as functional sex).

[0003] In addition, since fish generally have different patterns and body sizes depending on whether they are male or female, the commercial value of ornamental fish, edible fish, and other fish products sold in the market varies greatly depending on the sex.

[0004] In other words, from the perspective of the commercial value of fish, a method was needed to artificially control sex in sex-changing fish species by suppressing inadvertent sex changes caused by external environmental factors, known as "sexual fluctuation," and inducing differentiation into the desired sex.

[0005] A widely known method for controlling the sex of such fish is to administer a sex control agent whose main component is a hormone to fish before sex determination, thereby inducing stable differentiation into the desired sex.

[0006] According to this method of fish sex control, it is possible to artificially and stably produce sex-controlled individuals, such as reversed-sex individuals in which genetic and functional sex are reversed, and normal-sex individuals in which genetic and functional sex match, through the action of hormones, without being influenced by environmental factors (see, for example, Patent Document 1). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] J-STAGE Animal Genetics and Breeding Research, 2004, Vol. 31, No. 2, 57-66 Summary of the Invention [Problem to be solved by the invention]

[0008] Incidentally, the hormones used in conventional methods for controlling the sex of fish are steroids such as testosterone, which is an masculinizing hormone that induces masculinization, and estrogens, which is a feminizing hormone that induces feminization. These are known to have direct and strong physiological activity in biological species other than fish, regardless of whether they are juveniles or adults.

[0009] Therefore, conventional methods for sex-controlling fish and the sex-controlled individuals produced by these methods have tended to be avoided due to concerns about the indiscriminate physiological activity of hormones on humans and other organisms other than fish, and concerns about their safety in the market.

[0010] The present invention has been made in view of the above circumstances, and provides a novel fish masculinization inducer and a method for masculinizing fish. The present invention also provides a novel fish masculinization inhibitor and a method for suppressing masculinization in fish. [Means for solving the problem]

[0011] In order to solve the above-mentioned problems, the masculinization inducer for fish according to the present invention comprises: (1) a PPAR activator (However, capsaicin and carotene are excluded.) was determined to be the main component.

[0012] In addition, in the method for masculinizing fish according to the present invention, (2) administering a PPAR activator to sexually undifferentiated fish (However, capsaicin and carotene are excluded.) It is characterized by its administration of the compound to activate PPAR and masculinize fish.

[0013] The fish masculinization inhibitor according to the present invention is characterized in that it contains (3) a PPAR inhibitor as a main component.

[0014] Furthermore, the method for suppressing masculinization in fish according to the present invention is characterized in that (4) a PPAR inhibitor is administered to sexually undifferentiated fish to inactivate PPAR and suppress masculinization of the fish.

[0015] The present invention is also characterized in that (5) the fish masculinization inducer according to (1) above is characterized in that the PPAR activator is a fatty acid.

[0016] The method for masculinizing fish according to the present invention is characterized in that (6) a fatty acid is administered to sexually undifferentiated fish to activate PPAR and masculinize the fish.

[0017] Furthermore, the fish masculinization inducer according to the present invention is characterized in that it contains (7) a β-oxidation activator as a main component.

[0018] The method for masculinizing fish according to the present invention is characterized in that (8) a β-oxidation activator is administered to sexually undifferentiated fish to activate β-oxidation and masculinize the fish.

[0019] Furthermore, the fish masculinization inhibitor according to the present invention is characterized in that it contains (9) a β-oxidation inhibitor as a main component.

[0020] In addition, the method for suppressing masculinization in fish according to the present invention is characterized in that (10) a β-oxidation inhibitor is administered to sexually undifferentiated fish to inactivate β-oxidation and suppress masculinization of the fish. Effect of the Invention

[0021] According to the fish masculinization inducer of the present invention, a PPAR activator (However, capsaicin and carotene are excluded.) Since the main ingredient is PPAR, instead of the masculinizing hormones used up until now, it is possible to locally activate PPAR in fish and induce masculinization from genetic females or genetic males to functional males, thereby producing sex-controlled males such as reverse males or normal males.

[0022] In addition, according to the method for masculinizing fish of the present invention, a PPAR activator is administered to sexually undifferentiated fish. (However, capsaicin and carotene are excluded.) By administering this to activate PPAR and masculinize fish, it is possible to stably produce sex-controlled males according to the sex determination period of each fish species.

[0023] Furthermore, the fish masculinization inhibitor of the present invention contains a PPAR inhibitor as its main ingredient, and therefore, instead of using conventional feminizing hormone agents, it locally inactivates the fish PPAR and suppresses the induction of masculinization in fish so that genetic females become normally functional females, making it possible to stably produce normal female individuals regardless of environmental factors.

[0024] Furthermore, according to the method for suppressing masculinization in fish of the present invention, a PPAR inhibitor is administered to sexually undifferentiated fish to inactivate PPAR and suppress masculinization of the fish, making it possible to stably produce sex-controlled female individuals according to the sex determination period of each fish species, regardless of environmental factors.

[0025] Furthermore, in the fish masculinization inducer of the present invention, if the PPAR activator is a fatty acid, it has a proven track record of being ingested by living organisms, and it is possible to locally activate PPAR in fish to induce masculinization from genetic females or genetic males to functional males, thereby safely producing sex-controlled male individuals such as reverse males and normal males.

[0026] Furthermore, according to the method for masculinizing fish of the present invention, fatty acids are administered to sexually undifferentiated fish to activate PPAR and masculinize the fish, so that sex-controlled male individuals can be stably produced according to the sex determination period of each fish species using fatty acids that have been proven to have been ingested by living organisms.

[0027] Furthermore, the fish masculinization inducer of the present invention contains a β-oxidation activator as its main component, and therefore, instead of using conventional masculinizing hormones, can locally promote β-oxidation of fatty acids in fish, thereby inducing masculinization from genetic females or genetic males to functional males, and can produce sex-controlled males such as reverse males or normal males.

[0028] Furthermore, according to the method for masculinizing fish of the present invention, a β-oxidation activator is administered to sexually undifferentiated fish to activate β-oxidation and masculinize the fish, making it possible to stably produce sex-controlled male individuals according to the sex determination period for each fish species.

[0029] Furthermore, the fish masculinization inhibitor of the present invention contains a β-oxidation inhibitor as its main component, which locally inhibits the β-oxidation of fatty acids in fish, thereby suppressing the induction of masculinization in fish so that genetic females become normal functional females, and thus making it possible to stably produce normal female individuals regardless of environmental factors.

[0030] Furthermore, according to the method for suppressing masculinization in fish of the present invention, a β-oxidation inhibitor is administered to sexually undifferentiated fish to inactivate β-oxidation and suppress masculinization of the fish, making it possible to stably produce sex-controlled female individuals according to the sex determination period for each fish species, regardless of environmental factors. [Brief description of the drawings]

[0031] [Figure 1] 1 is a microscopic image for verifying the effect of the masculinization inducer according to the present embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The masculinization inducer and masculinization inhibitor of the present invention are novel fish sex control agents that can safely produce sex-controlled individuals, such as reverse-sex individuals in which the genetic sex and functional sex are the opposite of each other, and normal-sex individuals in which the genetic sex and functional sex match, without being affected by environmental factors, instead of conventional fish sex control agents that contain hormones as the main ingredient.

[0033] Conventional hormones as sex control agents are so-called steroids, which show a wide range of physiological activity across different species of organisms, regardless of whether they are young or adults, and therefore have been viewed as problematic in terms of environmental pollution and safety to the human body. In light of these problems, the present inventors have developed a novel masculinization inducer and masculinization inhibitor.

[0034] As described above, the fish species that are the subject of sex control using the sex control agent of the present invention are sex-reversing species in which the genetic sex and functional sex of the individual are reversed due to external environmental factors, such as, but not limited to, medaka, guppy, flounder, and tiger pufferfish.

[0035] The functional sex of adult fish in such sex-changing species is closely related to the number of germ cells in sexually undifferentiated fish.

[0036] In other words, the number of germ cells in a fish at the time of sex determination is an indicator of the sex-determining factor that determines the future functional sex of the fish, and there is a certain number of cells that will differentiate into functional male adult fish or functional female adult fish in the future.

[0037] As a result of extensive research, the inventors have discovered that the activity or inactivity of PPAR (peroxisome proliferator-activated receptor), a nuclear receptor that originally plays an important role in fatty acid metabolism, is deeply involved in the increase or decrease in the number of germ cells, which is an indicator of sex-determining factors in sexually undifferentiated fish.

[0038] That is, the present invention was completed based on the inventors' findings through intensive research that PPAR in the somatic gonadal cells of sexually undifferentiated fish can be targeted and a PPAR activator or PPAR inhibitor can be applied to the PPAR to achieve localized activation or inactivation of the PPAR, thereby enhancing the induction of sexual differentiation into functional males or the inhibition of sexual differentiation.

[0039] Furthermore, the present inventors conducted further intensive research to realize more stable sex control in fish, and as a result, discovered that the activation or inactivation of β-oxidation, a metabolic pathway in fatty acid metabolism that oxidizes the β-position of fatty acids and breaks them down from fatty acyl-coenzyme A complex (acyl-CoA) to acetyl-CoA, is deeply involved in the increase or decrease in the number of germ cells, which is an indicator of sex-determining factors in sexually undifferentiated fish. Two types of β-oxidation have been confirmed: the mitochondrial system and the peroxisome system.

[0040] In other words, the present invention was completed based on the discovery by the inventors, through further intensive research, that by targeting β-oxidation in the somatic gonadal cells of sexually undifferentiated fish and applying a β-oxidation activator or β-oxidation inhibitor, localized activation or inactivation of β-oxidation can be achieved, thereby enhancing the induction of sexual differentiation into functional males or the inhibition of sexual differentiation.

[0041] Here, sexually undifferentiated fish refers to fish that are in the period of sex determination, and refers to individuals in the sex determination period from fertilization to hatching, for example, embryos whose sex is determined from fertilization to hatching, and larvae or adult fish whose sex is determined from hatching to a specified number of days.

[0042] In other words, since the sex differentiation period differs depending on the fish species, the fish masculinization inducer and fish masculinization inhibitor according to the present embodiment are administered for a predetermined period from the start of the sex differentiation period for each fish species. In other words, the administration period of the masculinization inducer and masculinization inhibitor can be appropriately selected within the sex differentiation period.

[0043] The fish masculinization inducer according to this embodiment contains a PPAR activator or a β-oxidation activator as a main component, and the fish masculinization suppressor contains a PPAR inhibitor or a β-oxidation inhibitor as a main component.

[0044] The PPAR activator is not particularly limited as long as it specifically binds to PPAR and activates PPAR. There are three types of PPAR: α, β, and γ. Examples of PPAR activators that specifically bind to these types of PPAR include fibrate drugs such as bezafibrate, clofibrate, and fenofibrate (FF), thiazolidinedione drugs such as troglitazone, pioglitazone, rosiglitazone, balaglitazone, and rivoglitazone, and 15-deoxy-Δ12, 14-prostaglandin J2, nitrolinoleic acid, oxidized LDL, eicosanoids, lysophospholipids, fatty acids, pyrinixic acid, leukotrienes, GW-7647, GW-7641, and mixtures thereof.

[0045] The fatty acid is intended to mean a fatty acid in a broad sense including organic acids that specifically bind to PPAR and activate PPAR, and is, for example, various fatty acids represented by the rational formula CH3-(R)-CO2H. Specifically, it may be butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, lauric acid, myristic acid, pentadecylic acid, palmitic acid, palmitoleic acid, margaric acid, stearic acid, oleic acid, vaccenic acid, linoleic acid, linolenic acid, eleostearic acid, arachidic acid, mead acid, arachidonic acid, behenic acid, lignoceric acid, nervonic acid, cerotic acid, montanic acid, melissic acid, or a mixture thereof.

[0046] The β-oxidation activator is a broad term including enzymes that induce β-oxidation in peroxisomes or mitochondria, their substrates, and intermediate products, and is not particularly limited as long as it activates β-oxidation. As for the enzymes that constitute the β-oxidation system, examples of the β-oxidation activator include acyl-CoA synthetase, carnitine palmitoyltransferase, acyl-CoA dehydrogenase, 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, carnitine octanoyltransferase, acyl-CoA oxidase, two-headed enzyme, and mixtures thereof.

[0047] In addition, the β-oxidation activator can be carnitine (L-carnitine), a transporter that binds to fatty acyl-CoA to generate fatty acylcarnitine when passing through the mitochondrial membrane. Using carnitine as the β-oxidation activator is preferable from the viewpoints of economy and convenience, which is cheap and easy to obtain, workability such as reagent preparation, and safety, which has a proven track record of dietary intake, and can facilitate control of β-oxidation activation, stabilizing the reproducibility of fish masculinization.

[0048] Similarly to the PPAR activators, the PPAR inhibitors are not particularly limited as long as they specifically bind to PPAR and inactivate PPAR, and for example, GW-6471 can be used.

[0049] The β-oxidation inhibitor is not particularly limited as long as it inactivates β-oxidation, but for example, an inhibitor of each enzyme constituting the β-oxidation system, specifically etomoxir, which inhibits the activity of carnitine palmitoyltransferase, can be used as the aforementioned β-oxidation activator. If etomoxir is used as the β-oxidation inhibitor, it is preferable from the viewpoints of convenience, cheapness and easy availability, and workability such as reagent preparation, as with carnitine as the aforementioned β-oxidation activator, and it makes it easier to control the inactivation of β-oxidation, stabilizing the reproducibility of masculinization inhibition in fish.

[0050] Furthermore, the method for masculinizing fish or the method for inhibiting masculinization of fish according to this embodiment is a method in which a masculinization inducer having as its main component the above-mentioned PPAR activator or β-oxidation activator, or a masculinization inhibiting agent having as its main component a PPAR inhibitor or β-oxidation inhibitor, is administered to sexually undifferentiated fish, thereby activating or inactivating PPAR or β-oxidation in the administered individual, thereby inducing or inhibiting masculinization of the fish.

[0051] The component concentration of the PPAR activator or β-oxidation activator, which is the main component of the masculinization inducer of the present invention, or the PPAR inhibitor or β-oxidation inhibitor, which is the main component of the masculinization inhibitor of the present invention, i.e., the concentration to be administered to undifferentiated fish, may be a "substantial concentration" that activates PPAR or β-oxidation at the sex determination period of undifferentiated fish, thereby enabling induction of masculinization, or that inactivates or enables inhibition of masculinization, and differs depending on the species of fish and the sex determination period.

[0052] As an example of this "substantial concentration," in the case of undifferentiated fish larvae whose sex determination period is a certain number of days after hatching and the main component of the masculinization inducer is a PPAR activator, the component concentration of the PPAR activator is 1 x 10 -7 M~1×10 -4 By setting the concentration at M (M = mol / L), it is possible to activate PPAR and induce masculinization in fish.

[0053] Specifically, when the main component of the masculinizing agent is, for example, the aforementioned fibrate drug, GW-7647, thiazolidinedione drug, or fatty acid, the component concentration is 9 × 10 -6M~1×10 -4 It's M.

[0054] Specifically, when the main ingredient of the masculinizing agent is a fibrate drug or GW-7647, the concentration of the ingredient is 9 × 10 -6 M~1×10 -6 M, preferably 7 x 10 -6 M~3×10 -6 M. In addition, when the main component of the masculinizing agent is a thiazolidinedione drug or a fatty acid, the component concentration is 1×10 -6 M~1×10 -4 M, more preferably 2×10 -5 M~9×10 -4 It's M.

[0055] In addition, when the undifferentiated fish are larvae whose sex determination period is from hatching to a certain number of days, and the main ingredient of the masculinization inhibitor is a PPAR inhibitor, the concentration of the PPAR inhibitor is 1 × 10 -6 M~1×10 -4 By adjusting the concentration to M (M = mol / L), PPAR can be inactivated, thereby suppressing masculinization in fish.

[0056] Specifically, when the main component of the masculinization inhibitor is GW-6471, the component concentration is 1×10 -7 M~1×10 -5 M, more preferably 2×10 -6 M~9×10 -5 It's M.

[0057] In addition, when the undifferentiated fish are larvae whose sex determination period is from hatching to a certain number of days, and the main component of the masculinization inducer is a β-oxidation activator, specifically, the above-mentioned carnitine, the component concentration of the β-oxidation activator is 1 × 10 -5 M~1×10 -4 By setting the concentration at M (M = mol / L), β-oxidation can be activated, thereby inducing masculinization in fish.

[0058] In addition, when the undifferentiated fish are larvae whose sex determination period is from hatching to a certain number of days, and the main component of the masculinization inhibitor is a β-oxidation inhibitor, specifically, the aforementioned etomoxir, the component concentration of the β-oxidation inhibitor is 1 × 10 -6 By setting the concentration at M (M = mol / L), β-oxidation can be inactivated, thereby suppressing masculinization in fish.

[0059] If the concentrations of the components of these masculinization inducers and masculinization inhibitors fall below their respective lower limits, there is a risk that they will not be able to sufficiently activate or inactivate PPAR or β-oxidation during the period of sexual differentiation, and therefore will not be able to achieve the effects of masculinization induction or masculinization inhibition.

[0060] On the other hand, if the component concentration of the masculinization inducer or masculinization inhibitor exceeds the respective upper limit, it may encourage over-intake by the fish to be masculinized, causing deterioration of the growth environment of embryos and larvae, or showing toxicity, which may result in the death of the fish to be masculinized, and the number of hatched fish and the number of adult fish may become unstable. In this regard, since there is a track record of dietary intake of L-carnitine and fatty acids (oleic acid) as masculinization inducers, it is presumed that at least the above lower limit value will be able to induce masculinization while stabilizing the number of hatched fish and the number of adult fish.

[0061] Furthermore, the method of administering the masculinization inhibitor or masculinization inducer to undifferentiated fish is not particularly limited as long as it involves directly or indirectly contacting and acting a PPAR activator or β-oxidation activator, or a PPAR inhibitor or β-oxidation inhibitor on the reproductive tract of the undifferentiated fish.

[0062] Methods for administering the masculinization inhibitor or masculinization inducer to undifferentiated fish include, for example, mixed water administration, in which the masculinization inhibitor or masculinization inducer is dissolved in breeding water at a certain concentration and allowed to be freely ingested by the fish, mixed in feed administration, in which the masculinization inhibitor or masculinization inducer is mixed in feed at a certain concentration and allowed to be freely ingested by the fish, etc. As an example, in the case of embryos whose sex is determined from fertilization to hatching or larvae up to a certain period after hatching, mixed water administration, in which the masculinization inhibitor or masculinization inducer is allowed to be freely contacted via breeding water, is preferred.

[0063] In this way, the sex control method consisting of the masculinization method and masculinization suppression method of the present invention exposes genetically sexual individuals in a sexually undifferentiated state to the above-mentioned masculinization inducers or masculinization suppressors, thereby activating or inactivating PPAR or β-oxidation in gonadal cells, thereby inducing or suppressing masculinization, and producing sex-controlled individuals such as reverse-sex individuals or normal-sex individuals.

[0064] The fish masculinization method and masculinization inhibition method according to the present embodiment will be described in detail below with reference to preparation examples and test results of the masculinization inducer and masculinization inhibitor. The following description will be specifically made in the following order. 1. Preparation of masculinizing agent: 2. Verification test of the effect of masculinizing agents: 3. Preparation of masculinization inhibitor: 4. Verification test of the effect of masculinization inhibitors:

[0065] 1. Preparation of masculinizing agent Four types of PPAR activators, fenofibrate (FF), GW-7647, oleic acid (fatty acid), and rosiglitazone, were used as the main components of the fish masculinization inducer, and L-carnitine was used as the main component of the fish masculinization inducer.

[0066] After preparing each masculinization inducer to a certain composition, it was mixed with the control rearing water X for administration to the sexually undifferentiated fish, and rearing waters A to G containing the masculinization inducer were prepared. Examples of the preparations are shown in Tables 1 to 3. [Table 1] [Table 2] [Table 3]

[0067] [Breeding Water X] For the control and as a standard for the various masculinizing inducers, rearing water X was used, which had a composition of 17 mM NaCl, 0.4 mM KCl, 0.27 mM CaCl2·2H2O, 0.66 mM MgSO4, and a pH of 7.

[0068] [FF breeding water A] Fenofibrate (FF: Wako, 060-05361) was used as a masculinizing agent. Fenofibrate was dissolved in dimethyl sulfoxide (Sigma-Aldrich Japan, D2650) to a concentration of 0.1 M to obtain a fenofibrate solution. The same solution was diluted with 5×10 -6 The masculinization inducer was added to rearing water X so that the concentration became M, and rearing water A containing the masculinization inducer was prepared.

[0069] [GW-7647 Breeding Water B] GW-7647 (Tocris, 1677) was used as a masculinizing agent. GW-7647 was dissolved in dimethyl sulfoxide (Sigma-Aldrich Japan, D2650) to a concentration of 0.1 M to obtain a GW-7647 solution. The same solution was then diluted with water to a concentration of 1 × 10 -6 The masculinization inducer was added to the breeding water X so that the concentration became M, and breeding water B containing the masculinization inducer was prepared.

[0070] [Oleic acid breeding water C] Oleic acid (Wako, 152-03433) was used as a masculinizing agent. Oleic acid was added at a concentration of 1×10 -5 The masculinization inducer was added to the breeding water X so that the concentration became M, and breeding water C containing the masculinization inducer was prepared.

[0071] [Rosiglitazone breeding water D] Rosiglitazone (Wako, 180-02653) was used as a masculinizing agent. Rosiglitazone was dissolved in dimethyl sulfoxide (Sigma-Aldrich Japan, D2650) to a concentration of 0.1 M to obtain a rosiglitazone solution. The same solution was diluted with rosiglitazone at a concentration of 1 × 10 -5 The masculinization inducer was added to the breeding water X so that the concentration became M, and breeding water D containing the masculinization inducer was prepared.

[0072] [Carnitine (10μ) breeding water E] L-carnitine (Wako, 359-44361) was used as a masculinizing agent. L-carnitine was dissolved in dimethyl sulfoxide (Sigma-Aldrich Japan, D2650) to a concentration of 0.1 M to obtain an L-carnitine solution. The same solution was diluted with water to obtain an L-carnitine concentration of 1 × 10 -5 The masculinization inducer was added to the breeding water X so that the concentration became M, and breeding water E containing the masculinization inducer was prepared.

[0073] [Carnitine (50μ) breeding water F] L-carnitine (Wako, 359-44361) was used as a masculinizing agent. L-carnitine was dissolved in dimethyl sulfoxide (Sigma-Aldrich Japan, D2650) to a concentration of 0.1 M to obtain an L-carnitine solution. The same solution was diluted with water to obtain an L-carnitine solution with a concentration of 5 × 10 -5 The masculinization inducer was added to the breeding water X so that the concentration became M, and breeding water F containing the masculinization inducer was prepared.

[0074] [Carnitine (100μ) breeding water G] L-carnitine (Wako, 359-44361) was used as a masculinizing agent. L-carnitine was dissolved in dimethyl sulfoxide (Sigma-Aldrich Japan, D2650) to a concentration of 0.1 M to obtain an L-carnitine solution. The concentration of L-carnitine was 1 × 10 -4 The masculinization inducer was added to the breeding water X so that the concentration became M, and breeding water G containing the masculinization inducer was prepared.

[0075] 2. Verification test of the effect of masculinizing agents The samples used in this test were medaka (FLFII medaka strain) whose sex determination period is from 0 days after fertilization to 5 days after hatching. This verification test was carried out by dividing the test into two parts: verifying the effect of the masculinization inducer of this example on embryos to larvae (1. Verification test of masculinization induction during hatching), and verifying the effect on larvae to adult fish (2. Verification test of masculinization induction during adult fish). The specific experimental procedures and results are as follows:

[0076] (1. Verification test for masculinization induction during hatching) First, we performed a test to verify sex differentiation during the hatching period. As mentioned above, the number of germ cells in fish at the time of sex determination is the first indicator of the sex determining factor that determines the future functional sex of the fish. Normally, XY individuals have around 50 germ cells, and XX individuals have around 100 germ cells.

[0077] In this test, the sex determination period was set to the period from 0 days after fertilization to hatching, and the administration period was set to the same period. Medaka eggs were reared in each of the rearing waters A to D and rearing water X at a water temperature of 26°C.

[0078] Then, the larvae immediately after hatching (0 days after hatching) were used as samples for verifying masculinization induction during the hatching period, and were designated as test groups A1 to D1 and control group X1, respectively.

[0079] The genetic sex of each of the test plots A1 to D1 and the control plot X1 was determined by visually checking the presence or absence of leucophores expressed as a trait in the larvae immediately after hatching. In the following, the genetic female individuals determined by visual inspection are referred to as XX individuals, and the genetic male individuals are referred to as XY individuals.

[0080] The functionality of each of the test groups A1 to D1 and the control group X1 was determined by microscopic observation of tissue sections stained with Hematoxylin-Eosin (HE) for XX and XY individuals, respectively, to count the number of germ cells. The stained tissue sections for each of the test groups A1 to D1 and the control group X1 were prepared according to the following procedures (a) to (d).

[0081] (a) Fixation of tissue Each individual from each test group A1 to D1 and each control group X1 was fixed using Bouin's fixative and left to stand overnight at 4°C. Then, it was immersed in 70% ethanol for 10 minutes, washed twice with 70% ethanol for 30 minutes, immersed in 80% ethanol for 1 hour, immersed in 90% ethanol for 30 minutes, immersed in 95% ethanol for 30 minutes, immersed in 100% ethanol for 5 minutes, immersed in methyl benzoate for 30 minutes, and stored in fresh methyl benzoate at 4°C. Bouin's fixative was prepared by mixing saturated picric acid solution: formaldehyde solution: acetic acid in a ratio of 15:5:1.

[0082] (b) Embedding of tissue The fixed specimens were immersed in xylene for 1 hour twice, immersed in xylene / paraffin (1:1) heated to 64°C for 30 minutes, and placed in paraffin in an embedding dish for 60 minutes. The embedding dish was then removed and placed at room temperature, and the tissue was embedded in paraffin.

[0083] (c) Preparation of tissue slices The embedded specimens were trimmed to 5 μm thick ultrathin sections using a microtome (Leica RM2125RT). The sections were stretched on a slide glass with water on a 42°C stretcher (Sakura Finetech Japan, Tissue-Tek Slide Warmer PS-53), dried for 3 hours, and stored at 4°C to prepare tissue sections.

[0084] (d) HE staining of tissue sections The tissue pieces obtained from each test group A1 to D1 and each control group X1 were left to stand at 64°C for 10 minutes with the slide glass, and then placed in xylene for 15 minutes to deparaffinize. The tissue pieces were then immersed in 99%, 95%, 90%, 80%, and 70% ethanol for 1 minute each to dexyleneize and immerse in water, and then washed with running water for 10 minutes. The tissue pieces were then stained by placing them in hematoxylin solution with the slide glass for 3 minutes, and washed with running water for 5 minutes. The tissue pieces were then stained by immersing them in eosin solution for 10 seconds. Finally, the tissue pieces were dehydrated by immersing them in 70%, 80%, 90%, 95%, and 99% ethanol for 1 minute each, immersed in xylene for 10 minutes, and sealed with Canada balsam to obtain stained tissue pieces. The hematoxylin solution was prepared by dissolving 0.1 g of hematoxylin monohydrate, 5 g of potassium aluminum sulfate, 0.02 g of sodium iodate, and 0.1 g of citric acid in 100 mL of distilled water, while the eosin solution was prepared by mixing 25 mL of eosin alcohol solution prepared by mixing 40 mL of 95% ethanol, 0.5 g of eosin Y, and 10 mL of distilled water, 75 mL of 80% ethanol, and 0.5 mL of acetic acid.

[0085] The stained tissue sections of the XX and XY individuals in each of the test groups A1 to D1 and the control group X1 were observed under a microscope, and the number of germ cells was counted to verify masculinization induction. The results are shown in Table 4. [Table 4]

[0086] As shown in Table 4, the number of germ cells in the control group X1 was about 50 for XY individuals and about 100 for XX individuals. In other words, the relationship between the number of germ cells in XX individuals scheduled for normal sex differentiation that were not treated with masculinizing agents and the number of germ cells in XY individuals was approximately twice as high in XX individuals as in XY individuals.

[0087] On the other hand, the number of germ cells in the XY individuals in the test groups A1 to D1 administered with the masculinization inducer of this example was approximately 50, which was almost the same as that of the XY individuals in the control group X1, whereas the number of germ cells in the XX individuals was approximately 50, which was approximately half that of the XX individuals in the control group X1.

[0088] In other words, the XX individuals administered with the masculinizing agent of this example had almost the same number of germ cells as the XY individuals as functional males. In addition, no significant difference was observed between the number of germ cells of the XX individuals in the test group A1 of fenofibrate, which binds to PPARα, and the test group D1 of rosiglitazone, which binds to PPARγ.

[0089] In particular, the fatty acid (oleic acid) test group C1 showed results equivalent to those of masculinization inducers that specifically bind to specific receptors, such as fenofibrate-based agents, despite being a highly safe test group with a proven track record of dietary intake. In other words, the masculinization inducer shown in test group C1 can promote stable masculinization without unintentionally losing the individuals targeted for masculinization, suggesting that it is possible to produce male fish with improved reproducibility of sex control.

[0090] Thus, it was shown that genetic female XX individuals exposed to masculinizing agents from day 0 after fertilization until hatching during the incubation period had the proliferation of germ cells suppressed by the activation of PPAR, and were induced to masculinize into functional males immediately after hatching.

[0091] (2. Verification test for masculinization induction in adult fish) Next, a verification test for masculinization induction in adult fish was conducted. In this test, the sex determination period was set to the period from 0 to 5 days after hatching, and the administration period was also set to the same period. The larvae of the killifish were reared in each of the rearing waters A to G and rearing water X at a water temperature of 26°C.

[0092] Thereafter, the larvae raised in each of the rearing waters A to G and X were transferred to rearing water consisting of filtered water, and reared in an environment with a water temperature of 26°C for two months after hatching until they became adults.

[0093] Then, adult fish two months after hatching were used as samples for verifying masculinization induction at the adult stage, and were designated as test plots A2 to G2 and control plot X2.

[0094] In addition, in this test, PPARα knockout medaka were used separately as samples and were reared in rearing water A and rearing water X under the same conditions as described above, and test area AN2 and control area XN2 were used as samples to verify masculinization induction at the adult stage.

[0095] The genetics of each test group (A2-G2, AN2) and the control groups (X2, XN2) were determined by visually inspecting the body color expressed as a trait in adult fish, in accordance with the above-mentioned (1. Verification test of masculinization induction during hatching).

[0096] In addition, the functionality of each test group (A2-G2, AN2) and control group (X2, XN2) was determined by microscopic observation of tissue sections stained with Hematoxylin-Eosin (HE) from XX and XY individuals, respectively, to assess whether they had differentiated into testes (males) or ovaries (females), in accordance with the above-mentioned (1. Verification test for masculinization induction during hatching).

[0097] The sex reversal rate (%) in each test group and control group was calculated as XX individuals (male) / XX individuals (male)+XX individuals (female)(total number of XX individuals)×100. The results are shown in Tables 5 to 7. [Table 5] [Table 6] [Table 7]

[0098] In addition, when a large number of spermatogonia (SG), spermatocytes (SC), spermatids (ST), and sperm (SZ) were confirmed in the microscopic images (100μ scale), it was evaluated as having differentiated into testes, and when a large number of oocytes (O) and ovarian cavity (OC) were confirmed, it was evaluated as having differentiated into ovaries. As an example, the microscopic images of test areas A2 and B2 are shown in Figure 1.

[0099] As shown in Table 5, in all XX individuals from the control group X2, the PPARα knockout medaka test group AN2, and the control group XN2, many oocytes (O) and ovarian cavities (OC) were visible in the microscopic images, and it was confirmed that they had differentiated into ovaries.

[0100] In other words, in the XX individuals in the control group X2, where no masculinizing agent was administered, or in the test group AN2, where PPAR was not activated even after administration of the agent, it was evaluated that masculinization was not induced and the individuals differentiated into females that matched their genetic and functional sex.

[0101] On the other hand, in some of the XX individuals in test groups A2 to D2, to which a PPAR activator was administered as a masculinizing agent, numerous spermatogonia (SG), spermatocytes (SC), spermatids (ST), and sperm (SZ) were visible in the microscopic images, as shown in Figure 1, and it was confirmed that they had differentiated into testes.

[0102] That is, the sex reversal rate to functional males in XX individuals administered with test groups A2 to D2 was 25% in test group A2, which was administered with a masculinizing inducer mainly composed of fenofibrate, 14.3% in test group B2, which was administered with a masculinizing inducer mainly composed of GW-7647, 25% in test group C2, which was administered with a masculinizing inducer mainly composed of oleic acid, and 20% in test group D2, which was administered with a masculinizing inducer mainly composed of rosiglitazone, as shown in Table 6. In addition, no significant difference was observed between the sex reversal rate to functional males in XX individuals in test group A2, which was administered with fenofibrate that binds to PPARα, and test group D2, which was administered with rosiglitazone that binds to PPARγ.

[0103] In addition, in some of the XX individuals in test groups E2 to G2, which were administered a β-oxidation activator as a masculinizing inducer, numerous spermatogonia (SG), spermatocytes (SC), spermatids (ST), and sperm (SZ) were visible in microscopic images, similar to those in the case of the PPAR activator, and it was confirmed that they had differentiated into testes.

[0104] In particular, the sex reversal rate to functional males in XX individuals in test groups E2 to G2, in which carnitine was administered as a β-oxidation activator, was generally equal to or greater than the sex reversal rate when a PPAR activator was administered.

[0105] Specifically, as shown in Table 7, the percentage was 46.2% in test group E2, where a masculinizing inducer containing carnitine (10 μ) as the main component was administered, 25.0% in test group F2, where a masculinizing inducer containing carnitine (50 μ) as the main component was administered, and 29.4% in test group G2, where a masculinizing inducer containing carnitine (100 μ) was administered.

[0106] These results show that when the masculinization inducer of this example is not administered, or when it is administered but PPAR or β-oxidation is not activated, masculinization induction does not occur and sex reversal to functional males does not occur. On the other hand, when the masculinization inducer of this example is administered to activate PPAR or β-oxidation, masculinization is induced in sexually undifferentiated fish.

[0107] In particular, L-carnitine, the masculinizing agent that showed the most stable and highest rate of sex reversal, is water-soluble, has a proven track record of being taken orally or intravenously, and is relatively inexpensive and easily available.

[0108] That is, the fatty acid (oleic acid) test group C2 and the beta-oxidation activator L-carnitine test groups E2 to G2 show the same results as fenofibrate-based agents and other masculinization inhibitors that specifically bind to specific receptors, despite the fact that they are both highly safe test groups with a proven track record of dietary intake. In particular, water-soluble L-carnitine shows a certain degree of masculinization regardless of the concentration. Thus, it was suggested that the use of L-carnitine as a masculinization inducer allows the preparation of reagents to be carried out efficiently and economically, and that sex-controlled male individuals can be produced in large quantities more stably.

[0109] 3. Preparation of masculinization inhibitor GW-6471 was used as a PPAR inhibitor as the main ingredient of the fish masculinization inhibitor, and etomoxir was used as a β-oxidation inhibitor as the main ingredient of the fish masculinization inducer.

[0110] In addition, in order to verify the masculinization inhibitory effect of the masculinization inhibitor of this embodiment, fenofibrate (FF) and L-carnitine were used as simulated sex-disrupting substances (simulated environmental factors) that artificially reproduce the phenomenon of "sexual fluctuation" caused by the external environment.

[0111] In [1. Preparation of masculinization inducer samples], rearing water A containing FF was used as rearing water XA, and rearing water F containing carnitine (10μ) was used as rearing water XF, and these were used as controls under the condition of simulated sex disruptors only. In addition, the masculinization inhibitor was mixed into rearing water X according to [1. Preparation of masculinization inducer samples] to prepare rearing waters H to I containing simulated sex disruptors + masculinization inhibitor. Preparation examples are shown in Tables 8 and 9. [Table 8] [Table 9]

[0112] [FF+GW-6471 Breeding Water H] GW-6471 (Cayman, 11697) was used as a masculinization inhibitor. GW-6471 was dissolved in dimethyl sulfoxide (Sigma-Aldrich Japan, D2650) to a concentration of 0.1 M to obtain a GW-6471 solution. In addition, fenofibrate (FF) was dissolved in dimethyl sulfoxide as a simulated sex disruptor to a concentration of 0.1 M to obtain a FF solution. Each solution was diluted with 100 mL of FF at a concentration of 5 × 10 -6 M, the concentration of GW-6471 is 1 × 10 -6 Each of these was added to rearing water X so that the concentration of masculinization inhibitors became M, and rearing water H containing the masculinization inhibitor was prepared.

[0113] [L-Carnitine + Etomoxir Breeding Water I] Etomoxir (Wako, E933500) was used as a masculinization inhibitor. Etomoxir was dissolved in dimethyl sulfoxide (Sigma-Aldrich Japan, D2650) to a concentration of 0.1 M to obtain an etomoxir solution. In addition, L-carnitine was dissolved in dimethyl sulfoxide as a simulated sex disruptor to a concentration of 0.1 M to obtain an L-carnitine solution. Each solution was prepared so that the concentration of etomoxir was 1×10 -6 M, L-carnitine concentration is 1×10 -5 Each of these was added to rearing water X so that the concentration of masculinization inhibitors became M, and rearing water I containing the masculinization inhibitor was prepared.

[0114] 4. Verification test of the effect of masculinization inhibitors As in the above-mentioned 2. Effect verification test of masculinization inducer, medaka fish with sex determination period of 0 days after fertilization to 5 days after hatching were used as samples for this test. This verification test was carried out in two parts: verifying the effect of the masculinization inhibitor of this example on embryos to larvae (1. Verification test of masculinization suppression during hatching), and verifying the effect on larvae to adult fish (2. Verification test of masculinization suppression during adult fish stage). The specific experimental procedure and results are as follows.

[0115] (1. Verification test of masculinization inhibition during hatching) In this test, in accordance with the above-mentioned (1. Test to verify masculinization induction during the hatching period) in <2. Test to verify the effectiveness of masculinization inducers>, medaka eggs with the sex determination period set to the period from 0 days after fertilization to hatching and the administration period set to the same period were reared in rearing water H at a water temperature of 26°C, and the larvae immediately after hatching (0 days after hatching) were used as test group H1.

[0116] In addition, the test group A1 in (1. Verification test of masculinization induction during hatching period) of <2. Verification test of the effect of masculinization inducers> was used as the control group XA1.

[0117] The genetics of the test group H1 and the control group XA1 were determined as XX or XY individuals by visually checking the presence or absence of leucophores expressed as a trait in the larvae immediately after hatching.

[0118] The functionality of the test group H1 and the control group XA1 was evaluated by measuring the number of germ cells in tissue sections stained with Hematoxylin-Eosin (HE) from XX and XY individuals, respectively, through microscopic observation. The results are shown in Table 10. [Table 10]

[0119] As mentioned above, the number of germ cells in the control group XA1 was about 50 in the XY individuals and about 50 in the XX individuals. In other words, the proliferation of germ cells was suppressed in the XX individuals administered each masculinization inhibitor, and differentiation was induced into functional males.

[0120] On the other hand, in the test group H1, where FF was administered together with a masculinization inhibitor containing GW-6471 as the main ingredient, the number of germ cells in the XY individuals was around 50, which was almost the same as that of the XY individuals in the control groups Y1 and Z1, while the number of germ cells in the XX individuals increased to around 100.

[0121] In other words, it was suggested that the masculinization inhibitor of this example antagonizes the simulated sex disrupting substance to inactivate PPAR in genetic female fish during the sex determination period, thereby suppressing inadvertent induction of masculinization due to environmental factors.

[0122] Thus, it was demonstrated that genetic female XX individuals exposed to the masculinization inhibitor of the present invention during the sex determination period, i.e., from the day of fertilization to hatching, were induced to normally differentiate into functional females through the suppression of masculinization, which antagonizes the inhibition of germ cell proliferation.

[0123] (2. Verification test of masculinization inhibition in adult fish) Next, a test to verify the suppression of masculinization in adult fish was conducted. In this test, medaka larvae were reared in rearing water H and rearing water I at a water temperature of 26°C, with the sex determination period set to 0 to 5 days after hatching and the administration period set to the same period, in accordance with the above-mentioned (2. Test to verify the effect of masculinization inducers). The larvae were then transferred to rearing water consisting of filtered water and reared in a water temperature environment of 26°C. The adult fish two months after hatching were designated as test areas H2 and I2.

[0124] In addition, test area A2 and test area E2 in (2. Verification test of masculinization induction at adult stage) of <2. Verification test of the effect of masculinization inducers> were designated as control area XA2 and control area XE2, respectively.

[0125] The genetics of the test areas H2, I2, and the controls XA2 and XE2 were determined by visually inspecting the body color expressed as a trait in adult fish, in accordance with the procedure described above (1. Verification test of masculinization induction during hatching).

[0126] In addition, the functionality of the test group H2, the test group I2, and the control groups XA2 and XE2 was evaluated by microscopic observation of tissue sections stained with Hematoxylin-Eosin (HE) for XX individuals and XY individuals, respectively, to see whether they had differentiated into testes or ovaries, in accordance with the above-mentioned (1. Verification test for masculinization induction during hatching). The results are shown in Tables 11 and 12. [Table 11] [Table 12]

[0127] In some of the XX individuals in the control group XA2 and control group XE2, spermatogonia (SG), spermatocytes (SC), spermatids (ST), and sperm (SZ) were visible in the microscopic images as described above, and it was confirmed that they had differentiated into testes. In other words, as shown in Table 11, the sex reversal rate to functional males in the XX individuals in the control group XA2 and control group XE2 was 25% and 46.2%, respectively.

[0128] On the other hand, the XX individuals in test group H1, which was administered FF together with an antimasculinizing agent containing GW-6471 as its main ingredient, and in test group I1, which was administered carnitine together with an antimasculinizing agent containing etomoxir as its main ingredient, were found to have oocytes (O) and ovarian cavities (OC) in microscopic images, and all of them were found to have differentiated into ovaries.

[0129] In other words, the sex reversal rates to functional males in XX individuals in test groups H2 and I2, where the masculinization inhibitor was administered, were all 0%, as shown in Table 12, and were significantly lower than the sex reversal rates in the control groups XA2 and XE2, where only the masculinization inducer was added, as shown in Table 10.

[0130] These results indicate that even in an environment where masculinization induction is inadvertently induced by environmental factors, masculinization of genetically undifferentiated female fish can be suppressed by administering a masculinization inhibitor to inactivate PPAR or β-oxidation.

[0131] As described above, the fish masculinization inducer of the present invention contains a PPAR activator as a main component, and therefore, instead of using conventional masculinizing hormone agents, it can locally activate PPAR in fish to induce masculinization from genetic females or genetic males to functional males, thereby producing sex-controlled male individuals such as reverse males or normal males.

[0132] Furthermore, according to the method for masculinizing fish of the present invention, a PPAR activator is administered to sexually undifferentiated fish to activate PPAR and masculinize the fish, making it possible to stably produce sex-controlled male individuals according to the sex determination period for each fish species.

[0133] Furthermore, the fish masculinization inhibitor of the present invention contains a PPAR inhibitor as its main ingredient, and therefore, instead of using conventional feminizing hormone agents, it locally inactivates the fish PPAR and suppresses the induction of masculinization in fish so that genetic males or genetic females become functional females, making it possible to produce sex-controlled female individuals such as reversed females or normal females.

[0134] Furthermore, according to the method for suppressing masculinization in fish of the present invention, a PPAR inhibitor is administered to sexually undifferentiated fish to inactivate PPAR and suppress masculinization of the fish, making it possible to stably produce sex-controlled female individuals according to the sex determination period for each fish species.

[0135] Furthermore, in the fish masculinization inducer of the present invention, if the PPAR activator is a fatty acid, it has a proven track record of being ingested by living organisms, and it is possible to locally activate PPAR in fish to induce masculinization from genetic females or genetic males to functional males, thereby safely producing sex-controlled male individuals such as reverse males and normal males.

[0136] Furthermore, according to the method for masculinizing fish of the present invention, fatty acids are administered to sexually undifferentiated fish to activate PPAR and masculinize the fish, so that sex-controlled male individuals can be stably produced according to the sex determination period of each fish species using fatty acids that have been proven to have been ingested by living organisms.

[0137] Furthermore, the fish masculinization inducer of the present invention contains a β-oxidation activator as its main component, and therefore, instead of using conventional masculinizing hormones, can locally promote β-oxidation of fatty acids in fish, thereby inducing masculinization from genetic females or genetic males to functional males, and can produce sex-controlled males such as reverse males or normal males.

[0138] Furthermore, according to the method for masculinizing fish of the present invention, a β-oxidation activator is administered to sexually undifferentiated fish to activate β-oxidation and masculinize the fish, making it possible to stably produce sex-controlled male individuals according to the sex determination period for each fish species.

[0139] In addition, since the fish masculinization inhibitor of the present invention contains a β-oxidation inhibitor as its main component, it can locally suppress the β-oxidation of fatty acids in fish, thereby suppressing the induction of masculinization in fish so that genetic males or genetic females become functional females, and can produce sex-controlled female individuals such as reversed females or normal females.

[0140] Furthermore, according to the method for suppressing masculinization in fish of the present invention, a β-oxidation inhibitor is administered to sexually undifferentiated fish to inactivate β-oxidation and suppress masculinization of the fish, making it possible to stably produce sex-controlled female individuals according to the sex determination period for each fish species.

[0141] In other words, the masculinization inducer and masculinization inhibitor of the present invention have the effect of replacing conventional fish sex control agents that contain hormones as the main ingredient in fish, and are not influenced by environmental factors, and are capable of stably producing sex-controlled individuals, such as pseudo-sex individuals in which the genetic and functional sex are the opposite of each other, or normal-sex individuals in which the genetic and functional sex are the same.

[0142] Finally, the above-mentioned description of each embodiment is merely an example of the present invention, and the present invention is not limited to the above-mentioned embodiments. It goes without saying that various modifications are possible depending on the design, etc., as long as they do not deviate from the technical idea of ​​the present invention.

Claims

1. A fish masculinization inducer whose main ingredient is a PPAR activator (excluding capsaicin and carotene).

2. A method for masculinizing fish, comprising administering a PPAR activator (excluding capsaicin and carotene) to sexually undifferentiated fish to activate PPAR and masculinize the fish.

3. A fish masculinization inhibitor whose main ingredient is a PPAR inhibitor.

4. A method for suppressing masculinization in fish by administering a PPAR inhibitor to sexually undifferentiated fish to inactivate PPAR and suppress masculinization in the fish.

5. The masculinization inducer for fish according to claim 1, characterized in that the PPAR activator is a fatty acid.

6. A method for masculinizing fish by administering fatty acids to sexually undifferentiated fish to activate PPAR and masculinize the fish.

7. A fish masculinization inducer whose main ingredient is a beta-oxidation activator.

8. A method for masculinizing fish, comprising administering a β-oxidation activator to sexually undifferentiated fish to activate β-oxidation and masculinize the fish.

9. A fish masculinization inhibitor whose main ingredient is a beta-oxidation inhibitor.

10. A method for inhibiting masculinization in fish, comprising administering a β-oxidation inhibitor to sexually undifferentiated fish to inactivate β-oxidation and inhibit masculinization in the fish.

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