Construction method of zebra fish reproductive toxicity model, method for evaluating reproductive toxicity of chemical substances and application

Abstract

The invention discloses a construction method of a zebra fish reproductive toxicity model, a method for evaluating reproductive toxicity of chemical substances and application, and relates to the technical field of reproductive development toxicity of chemical substances. According to the method for evaluating the reproductive toxicity of the chemical substance provided by the invention, the change of a diatrix behavior can be concerned by shooting behavioral pictures or videos without sacrifice of an experimental animal or professional fluorescence imaging instrument equipment and tissue slicing equipment; the method is more in line with animal ethics, cost is saved, and continuous observation can be realized by using the same batch of organisms. The test courtship behavior can quickly utilize software to analyze and obtain zebra fish behavior changes. By adopting the evaluation method provided by the invention, both female ovarian malformation and male reproductive toxicity can be detected, and the evaluation method has the advantage of more evaluation dimensions. Based on the method provided by the invention, a zebrafish reproductive toxicity model can be constructed, and the model can be used as a drug screening platform for reproductive function damage of zebrafish.

Description

Technical Field

[0001] The present invention relates to the technical field of reproductive and developmental toxicity of chemical substances, and in particular, to a method for constructing a zebrafish reproductive toxicity model, a method for evaluating the reproductive toxicity of chemical substances, and an application thereof. Background Art

[0002] Currently, the methods for evaluating the reproductive and developmental toxicity of chemical substances mainly include: the real-time fluorescence dynamic tracking method of transgenic animals expressing fluorescent proteins, the evaluation of the sperm production ability of male zebrafish and the egg-laying ability of female zebrafish after anesthetizing zebrafish, and the evaluation of Drosophila ovarian malformations. Among them, the fluorescence imaging technology mainly focuses on tissue development and / or early embryo implantation and / or the developmental toxicity of in vitro cultured embryos, and it is necessary to take biological tissues for section observation, which not only requires corresponding technical equipment, but also requires sacrificing experimental animals to complete. However, there are complex operation problems in anesthetizing zebrafish. After anesthetizing zebrafish, it is necessary to squeeze the gonads to collect sperm and eggs, and manually count them. The processes such as anesthesia and counting take a long time and are prone to introduce accidental errors.

[0003] In view of this, the present invention is specifically proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for evaluating the reproductive toxicity of chemical substances and an application thereof to solve the above technical problems.

[0005] The present invention is implemented as follows:

[0006] In a first aspect, the present invention provides a method for constructing a zebrafish reproductive toxicity model, which includes the following steps:

[0007] Exposing zebrafish in an exposure environment of a chemical substance with reproductive toxicity;

[0008] The chemical substance with reproductive toxicity is selected from methylmercury, alkaloid toxins, bisphenols or pyrethroid insecticides; the concentration of the chemical substance in the exposure environment is at least 0.5 μg / L, and the exposure time in the exposure environment of the chemical substance with reproductive toxicity is 14 - 42 days;

[0009] Preferably, the alkaloid toxin is cylindrospermopsin (CAS: 143545 - 90 - 8).

[0010] In a second aspect, the present invention provides a method for evaluating the reproductive toxicity of chemical substances, which includes the following steps: evaluating the courtship behavior and fertility of zebrafish;

[0011] The courtship behavior evaluation includes: first, exposing female and male zebrafish to an environment exposed to a chemical substance with reproductive toxicity; then placing the exposed female or male target zebrafish in one area of the courtship behavior evaluation, and correspondingly placing the exposed male or female courtship behavior test zebrafish in another area of the courtship behavior evaluation; and the activity areas of the target zebrafish and the test zebrafish do not overlap. Shoot zebrafish ethology videos and / or pictures within a period of time; evaluate the locomotor behaviors of male and female zebrafish during courtship.

[0012] The concentration of the chemical substance in the exposure environment is at least 0.5 μg / L; the concentration of the chemical substance in the courtship behavior evaluation area is the same as that in the exposure environment.

[0013] The locomotor behaviors during courtship are selected from at least one of the following:

[0014] The locomotor distance of the test zebrafish during courtship, the total duration of the test zebrafish entering a certain designated area, the frequency of entering the designated area, the courtship trajectory, and the courtship index; the designated area is the area close to the target fish.

[0015] The fertility evaluation includes the following steps: co-culturing the exposed female and male zebrafish in an environment exposed to a chemical substance with reproductive toxicity, and then conducting at least one of the following evaluations on the zebrafish: evaluating the egg production and / or fertilization rate of the female zebrafish, and detecting the reproductive-related hormone levels of the female zebrafish and / or male zebrafish.

[0016] Preferably, the locomotor distance of the test zebrafish in the designated area during courtship.

[0017] Preferably, the co-culturing time of the exposed female and male zebrafish is 7 - 28 days.

[0018] In a preferred embodiment of the application of the present invention, if the courtship behavior of zebrafish decreases and the fertility of zebrafish decreases in the exposure environment, then evaluate the reproductive toxicity of the chemical substance to zebrafish.

[0019] In a preferred embodiment of the application of the present invention, the exposure time of female and male zebrafish in an environment exposed to a chemical substance with reproductive toxicity is 14 - 42 days.

[0020] In a preferred embodiment of the application of the present invention, the time for shooting zebrafish ethology videos and / or pictures is 10 - 20 min.

[0021] In a preferred embodiment of the application of the present invention, shooting zebrafish ethology videos and / or pictures is carried out under continuous light conditions.

[0022] In a preferred embodiment of the application of the present invention, software is used to analyze zebrafish ethology videos and / or pictures to obtain the locomotor behavior during courtship.

[0023] In a preferred embodiment of the application of the present invention, the courtship index refers to the time when a male zebrafish or a female zebrafish chases another female zebrafish or another male zebrafish.

[0024] In a preferred embodiment of the application of the present invention, the reproduction-related hormones are selected from testosterone, estradiol or vitellin.

[0025] In a preferred embodiment of the application of the present invention, the chemical substances with reproductive toxicity are selected from methylmercury, alkaloid toxins, bisphenols or pyrethroid insecticides. The alkaloid toxin is cylindrospermopsin (CAS: 143545-90-8).

[0026] In a third aspect, the present invention also provides the application of the zebrafish reproductive toxicity model constructed by the above model construction method or the zebrafish reproductive toxicity model constructed by the method for evaluating the reproductive toxicity of chemical substances in screening drugs for preventing and / or treating reproductive function damage. The application includes: evaluating the reproductive toxicity of chemical substances to zebrafish according to the above method for evaluating the reproductive toxicity of chemical substances to obtain a zebrafish reproductive toxicity model.

[0027] In a fourth aspect, the present invention also provides the application of the zebrafish reproductive toxicity model constructed by the above model construction method or the zebrafish reproductive toxicity model constructed by the method for evaluating the reproductive toxicity of chemical substances in screening drugs for improving or inhibiting abnormal courtship behavior of zebrafish. The application includes: evaluating the reproductive toxicity of chemical substances to zebrafish according to the above method for evaluating the reproductive toxicity of chemical substances to obtain a zebrafish reproductive toxicity model.

[0028] The present invention has the following beneficial effects:

[0029] The method for evaluating the reproductive toxicity of chemical substances provided by the present invention can focus on the changes in courtship behavior by taking ethology pictures or videos, without sacrificing experimental animals, and without professional fluorescence imaging instrument equipment and tissue section equipment; and it is more in line with animal ethics, saves costs, can realize continuous observation with the same batch of organisms, and the experimental process is more flexible.

[0030] Testing courtship behavior can quickly obtain the behavior changes of zebrafish by software analysis. Compared with the traditional spawning statistics method, it can greatly save manpower and time, and does not require any anesthesia treatment for the fish; indicating reproductive effects by courtship behavior is also a live observation, which can support long-term tracking or repeated experiments of the same batch of zebrafish.

[0031] Compared with Drosophila, the present invention uses zebrafish, which has 87% similarity with human genes, as a model organism. Its reproductive system has less difference from that of mammals, and the results have higher reference value for clinical and environmental risk assessment. The present invention not only detects ovarian malformations in females but also pays attention to male reproductive toxicity, having the advantage of more evaluation dimensions.

[0032] Based on the method provided by the present invention, a zebrafish reproductive toxicity model can be constructed. This model can be used as a drug screening platform for reproductive function impairment in zebrafish, and potential effective drugs can be screened by observing changes in the courtship behavior of zebrafish. In addition, using the genetic background of zebrafish is beneficial to studying the molecular mechanism of reproductive dysfunction and providing a theoretical basis for future treatment. The proposal of the present invention has good application prospects in the fields of medicine, drug development, and screening of natural active ingredients, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0034] Figure 1 Schematic diagram of the cross-shaped chamber structure for the social behavior test of adult zebrafish;

[0035] Figure 2 Technical roadmap for evaluating the reproductive toxicity of chemical substances;

[0036] Figure 3 Effects of cylindrospermopsin on the courtship distance (A, B) and the frequency of entering the region of approaching (ROA) (C, D) of male and female zebrafish;

[0037] Figure 4 Effect diagram of cylindrospermopsin on the courtship trajectories of male (A) and female zebrafish (B) in the entire area;

[0038] Figure 5 Effects of cylindrospermopsin on the duration of male and female zebrafish entering the ROA (A: Duration and heat map of male fish entering the ROA; B: Duration and heat map of female fish entering the ROA (**p < 0.01; ***p < 0.001));

[0039] Figure 6Results of the effects of cylindrospermopsin on the fecundity of adult zebrafish ((A) Cumulative average number of eggs laid per female during the 14-day pre-exposure; (B) Cumulative average number of eggs laid per female within 14 days of cylindrospermopsin exposure; (C) Fertilization rate after 14 days of pre-exposure and 14 days of cylindrospermopsin exposure);

[0040] Figure 7 Testosterone (T), estradiol (E2), and vitellogenin (VTG) levels in male (A, B, and C) and female (D, E, and F) zebrafish after exposure to cylindrospermopsin. Asterisks indicate significant differences (*, p < 0.05) and highly significant differences (**, p < 0.01) compared to the control group. Detailed implementation mode

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0042] In a first aspect, the present invention provides a method for constructing a zebrafish reproductive toxicity model, which includes the following steps:

[0043] Exposing zebrafish to an exposure environment of a chemical substance with reproductive toxicity;

[0044] The chemical substance with reproductive toxicity is selected from methylmercury, alkaloid toxins, bisphenols, or pyrethroid insecticides; the concentration of the chemical substance in the exposure environment is at least 0.5 μg / L, and the exposure time in the exposure environment of the chemical substance with reproductive toxicity is 14 - 42 days;

[0045] Preferably, the alkaloid toxin is cylindrospermopsin (CAS: 143545 - 90 - 8).

[0046] In a second aspect, the present invention provides a method for evaluating the reproductive toxicity of a chemical substance, which includes the following steps: evaluating the courtship behavior and fecundity of zebrafish;

[0047] The courtship behavior evaluation includes: first, exposing female and male zebrafish to an exposure environment of a chemical substance with reproductive toxicity; then placing the exposed female or male target zebrafish in one area of the courtship behavior evaluation, and correspondingly placing the exposed male or female courtship behavior test zebrafish in another area of the courtship behavior evaluation; and the activity areas of the target zebrafish and the test zebrafish do not overlap, and filming zebrafish ethology videos and / or pictures within a period of time; evaluating the locomotor behavior of male and female zebrafish during courtship;

[0048] The concentration of the chemical substance in the exposure environment is at least 0.5 μg / L; the concentration of the chemical substance in the courtship behavior evaluation area is the same as that in the exposure environment;

[0049] The locomotor behavior during courtship is selected from at least one of the following:

[0050] The swimming distance of the test zebrafish during courtship, the total duration of the test zebrafish entering a certain designated area, the frequency of entering the designated area, the courtship trajectory, and the courtship index; the designated area is the area close to the target fish;

[0051] The fertility evaluation includes the following steps: cohabiting the female and male zebrafish after exposure to the chemical substance with reproductive toxicity in the exposure environment, and then conducting at least one of the following evaluations on the zebrafish: evaluating the egg production and / or fertilization rate of the female zebrafish, and detecting the levels of reproductive-related hormones in the female and / or male zebrafish;

[0052] Preferably, the swimming distance of the test zebrafish in the designated area during courtship;

[0053] Preferably, the cohabitation time of the exposed female and male zebrafish is 7 - 28 days.

[0054] The inventor found that through the above-mentioned courtship behavior evaluation and fertility evaluation of zebrafish, the reproductive toxicity evaluation of chemical substances can be achieved quickly, at low cost, and continuously for the same batch of zebrafish. By taking ethological pictures or videos to observe the changes in courtship behavior, it is not necessary to sacrifice the experimental animals, nor is it necessary to use professional fluorescence imaging equipment and tissue sectioning equipment.

[0055] Testing the courtship behavior can quickly obtain the behavioral changes of zebrafish through software analysis. Compared with the traditional spawning statistics method, it can greatly save manpower and time, and it is not necessary to perform any anesthesia treatment on the fish; the courtship behavior indicating reproductive effects is also an in vivo observation, which can support the long-term tracking or repeated experiments of the same batch of zebrafish. The present invention not only detects ovarian malformations in females but also pays attention to male reproductive toxicity, having the advantage of more evaluation dimensions.

[0056] When testing the courtship behavior of zebrafish, the courtship behavior towards the opposite-sex fish is judged by observing the distance of the test fish approaching the target fish, the appearance frequency and appearance time in the designated area, etc.

[0057] In the same courtship behavior evaluation experimental device or container, the target fish and the test zebrafish are of different genders. For example, when testing the courtship behavior of male zebrafish, the target fish is female, and the male and female are separated by a physical barrier, such as in the form of a partition.

[0058] Based on the method provided by the present invention, a zebrafish reproductive toxicity model can be constructed. This model can be used as a drug screening platform for reproductive function impairment in zebrafish, and potential effective drugs can be screened by observing changes in the courtship behavior of zebrafish. In addition, using the genetic background of zebrafish is conducive to studying the molecular mechanism of reproductive dysfunction and providing a theoretical basis for future treatment. The proposal of the present invention has good application prospects in the fields of medicine, drug development, and screening of natural active ingredients, etc.

[0059] When the concentration of the chemical substance in the exposure environment is at least 0.5 μg / L, it can affect the reproductive function of zebrafish, and the reproductive toxicity is manifested as: the total duration of male or female test zebrafish entering the designated area, the frequency of test zebrafish entering the designated area are significantly reduced, the courtship distance between male and female zebrafish is shortened, the courtship trajectory of test zebrafish entering the designated area is more dispersed, and the courtship index is smaller.

[0060] The concentration of the chemical substance in the exposure environment is, for example, 0.5 μg / L, 1 μg / L, 1.5 μg / L, 2.5 μg / L, 3 μg / L, 3.5 μg / L, 4 μg / L, 4.5 μg / L, 5 μg / L, 5.5 μg / L or 6 μg / L. The experiments of the present invention prove that the exposure concentrations of 0.5 μg / L and 5 μg / L will cause reproductive toxicity in zebrafish, and higher exposure concentrations should also have reproductive toxicity in zebrafish. Therefore, the concentration of the chemical substance in the exposure environment is at least 0.5 μg / L.

[0061] The frequency of entering a certain designated area refers to the frequency of male or female zebrafish entering a certain designated area.

[0062] The "certain designated area" described in the present invention refers to: when evaluating the reproductive toxicity of female zebrafish and / or male zebrafish, a defined area where the test zebrafish (male or female) can pass through and stay, which is set adjacent to the release area of the target fish. The designated area can be one area, or it can refer to the combined area of several sub-areas, or it can refer to multiple areas. For example, the area for evaluating courtship behavior is divided into Area 1, Area 2, Area 3, Area 4, and Area 5. Among them, Area 1 is used for releasing female zebrafish, Area 2 is used for releasing male zebrafish, and Area 3 is used for the "designated area". By evaluating the total duration of female and male fish entering Area 3 and the frequency of entering Area 3, the courtship behavior can be evaluated.

[0063] In this application, the "ROA area" is used to refer to the "certain designated area".

[0064] In a preferred embodiment of the application of the present invention, if in the exposure environment, the courtship behavior of zebrafish decreases and the fertility of zebrafish decreases, then it is evaluated that the alkaloid toxin has reproductive toxicity to zebrafish.

[0065] In a preferred embodiment of the application of the present invention, the exposure time of female and male zebrafish in an environment exposed to a chemical substance with reproductive toxicity is 14 - 42 days.

[0066] In a preferred embodiment of the application of the present invention, the time for shooting zebrafish ethology videos and / or pictures is 10 - 20 min;

[0067] In a preferred embodiment of the application of the present invention, shooting zebrafish ethology videos and / or pictures is carried out under continuous light conditions.

[0068] In a preferred embodiment of the application of the present invention, software is used to analyze zebrafish ethology videos and / or pictures to obtain the locomotor behavior during courtship.

[0069] In a preferred embodiment of the application of the present invention, the courtship index refers to the time when a male zebrafish or a female zebrafish chases another female zebrafish or another male zebrafish.

[0070] In a preferred embodiment of the application of the present invention, the reproductive-related hormones are selected from testosterone (T), estradiol (E2), or vitellogenin (VTG). If the level of reproductive-related hormones in zebrafish is significantly decreased compared to the control group (i.e., the group not exposed to alkaloid toxins), it indicates that alkaloid toxins have an impact on the fertility of zebrafish.

[0071] The chemical substance with reproductive toxicity is selected from methylmercury, alkaloid toxins, bisphenols, or pyrethroid pesticides. The alkaloid toxin is cylindrospermopsin (CAS: 143545 - 90 - 8). In other embodiments, it can also be other compounds with similar effects.

[0072] In a third aspect, the present invention also provides the application of the zebrafish reproductive toxicity model constructed by the method for constructing a zebrafish reproductive toxicity model or the zebrafish reproductive toxicity model constructed by the method for evaluating the reproductive toxicity of a chemical substance in screening drugs for preventing and / or treating reproductive function impairment. The application includes: evaluating the reproductive toxicity of a chemical substance to zebrafish according to the above method for evaluating the reproductive toxicity of a chemical substance to obtain a zebrafish reproductive toxicity model.

[0073] In a fourth aspect, the present invention also provides the application of the zebrafish reproductive toxicity model constructed by the method for constructing a zebrafish reproductive toxicity model or the zebrafish reproductive toxicity model constructed by the method for evaluating the reproductive toxicity of a chemical substance in screening drugs for improving or inhibiting abnormal courtship behavior of zebrafish. The application includes: evaluating the reproductive toxicity of a chemical substance to zebrafish according to the above method for evaluating the reproductive toxicity of a chemical substance to obtain a zebrafish reproductive toxicity model.

[0074] The features and properties of the present invention are further described in detail below in conjunction with examples.

[0075] Example 1

[0076] This example provides a method for evaluating the reproductive toxicity of chemical substances. The technical route is referred to Figure 2 as shown. Specifically, zebrafish with 87% similarity to human genes are used as model organisms to establish an animal model that indicates reproductive toxicity by zebrafish courtship behavior, which has a short modeling time, high success rate, and is simple and convenient.

[0077] 1. Conduct the exposure experiment according to the following method

[0078] Prepare the stock solution of cylindrospermopsin using acetone as the solvent, and then further dilute the stock solution to a final concentration of 0.5 μg / L. In this experiment, a blank control group and a treatment group (0.5 μg / L cylindrospermopsin) are set up. Each 30-L glass tank contains 18 adult zebrafish (male and female zebrafish are placed separately) and 20 L of the test solution. Each treatment is exposed 3 times for 42 days. During the exposure period, the exposure solution is changed every 24 hours, and during this period, adult brine shrimp are fed normally 2 times a day and de-shelled brine shrimp feed is given 1 time.

[0079] 2. Social behavior test of adult zebrafish

[0080] Conduct the following social behavior test of adult zebrafish 5 minutes after the adult zebrafish adapt to the test environment:

[0081] As Figure 1 shown, conduct the social behavior experiment of adult zebrafish in the chamber with the structure shown in Figure 1 shown. Figure 1 It is a cross-shaped chamber. In the four directions, there are Area 1 (Arena1), Area 2 (Arena2), Area 3 (Arena3), and Area 4 (Arena4) respectively, and in the middle is Area 5 (Arena5). Area 1 (Arena1) has an extension end, and Arena A is at the extension end. In this example, Arena A (i.e., Area A) is the area for placing female fish (target fish), Area 1 (Arena1) is the designated area, i.e., the ROA area, and Area 5 (Arena5) is the area for placing male fish (test fish). Each chamber contains the culture solution for zebrafish, and the culture solution contains 0.5 μg / L of cylindrospermopsin.

[0082] Randomly place the female fish (or male fish) in the blank group into Figure 1In the terminal chamber (Arena A) with a cross groove shown, Arena A (i.e., Area A) is separated from Area 1 (Arena1) by a transparent Plexiglas plate (acrylic plate). The total area for testing fish consists of Area 1, Area 2, Area 3, Area 4, and Area 5. Male fish are placed in the central chamber (Arena 5) with a water depth of 8 cm of reconstituted water according to the blank treatment group for courtship behavior determination.

[0083] When evaluating the social behavior of female fish, male fish are used as target fish and placed in Arena A, while female fish are used as test fish and placed in Area 5 (Arena5). Observe the behavior of female fish in Area 1 (i.e., the designated area - ROA area) close to male fish.

[0084] This determination is to shoot a zebrafish behavioral video under continuous light for 10 minutes, and the camera model is logiHD1080p.

[0085] Record the following locomotor behaviors of zebrafish during courtship:

[0086] The locomotor distance entered the ROA area during courtship, the total duration of female and male zebrafish entering the ROA area, the frequency of entering the ROA area, the courtship trajectory, and the courtship index.

[0087] Among them, the time in the ROA area: the total time (moving time and non - moving time) that zebrafish spend in Arena 1. Area 1 is the area closest to the opposite - sex fish. The EthoVision XT 15 software is used to extract the time when zebrafish enter the ROA area from the behavioral video.

[0088] The locomotor distance during courtship, in this embodiment, specifically refers to the total distance of zebrafish in Area 1 of Arena. This parameter is related to the degree of individual zebrafish participating in courtship behavior. The X software is used to extract the total distance of zebrafish entering Area 1 of Arena from the behavioral video.

[0089] The frequency in the ROA: the total frequency of zebrafish entering Arena 1. The X software is used to extract the total frequency of zebrafish entering Area 1 of Arena from the behavioral video.

[0090] The courtship index: the time (moving time) that male zebrafish (or female zebrafish) chase another female zebrafish (or another male zebrafish).

[0091] 3. Evaluation of the fertility of the F0 generation

[0092] Female and male zebrafish were exposed separately according to Step 1; the exposed female and male zebrafish were co-cultured for 14 days in an exposure environment of cylindrospermopsin with reproductive toxicity. Three parallel groups were set for each exposure concentration, and 8 zebrafish were placed in each parallel group. Then, the following evaluations were carried out on the zebrafish: the fecundity and fertilization rate of female zebrafish were evaluated, and the reproductive-related hormone levels of female and male zebrafish were detected;

[0093] (1) Manual statistics of the fecundity and fertilization rate of female zebrafish.

[0094] (2) Determine the levels of related reproductive hormones to verify the reproductive effects of chemicals on zebrafish.

[0095] Example 2

[0096] Compared with Example 1, the difference is only that the cylindrospermopsin solution with an exposure concentration of 5 μg / L in the exposure environment was diluted to a cylindrospermopsin solution with a final concentration of 5 μg / L with acetone. Other steps are the same as those in Example 1.

[0097] Experimental Example 1

[0098] An evaluation experiment on the courtship behavior of zebrafish was carried out on the methods for evaluating the reproductive toxicity of chemicals provided in Example 1 and Example 2.

[0099] The results showed that compared with the control group, the 5 μg / L cylindrospermopsin treatment in Example 2 reduced the moving distance of male and female zebrafish in the ROA ( Figure 3 A and B therein) and the frequency of entering the ROA area ( Figure 3 C and D therein).

[0100] The 0.5 μg / L cylindrospermopsin treatment in Example 1 reduced the number of times males entered the ROA and the moving distance of female zebrafish in the ROA area ( Figure 3 C and B therein).

[0101] Similarly, the trajectory diagram ( Figure 4 ) further showed the effects of exposure to cylindrospermopsin on the locomotor ability of males and females. Compared with the control group, after exposure to cylindrospermopsin, males ( Figure 4 A therein) and females ( Figure 4 B therein) of zebrafish no longer concentrated on moving in the ROA area but were more dispersed in other areas outside the ROA area, and this phenomenon showed an obvious dose relationship.

[0102] In addition, compared with the control group, both the 5 μg / L in Example 2 and the 0.5 μg / L cylindrospermopsin in Example 1 significantly reduced males ( Figure 5 A therein) and female zebrafish ( Figure 5B) Total time staying in the ROA. Compared with the control group, after exposure to cylindrospermopsin, the activity of male ( Figure 5 A) and female ( Figure 5 B) outside the ROA gradually increased, and this phenomenon was most obvious in the 5 μg / L cylindrospermopsin treatment group.

[0103] Experimental Example 2

[0104] Perform a zebrafish reproductive impact test on the methods for evaluating the reproductive toxicity of chemicals provided in Example 1 and Example 2.

[0105] Statistical analysis of the egg production and fertilization rate of female zebrafish, and determination of relevant reproductive hormone levels to verify the reproductive impact of chemicals on zebrafish. The zebrafish testosterone (T) ELISA kit was purchased from Jiangsu Jingmei, product number JM-08003F2; the zebrafish estradiol (E2) ELISA kit was purchased from Jiangsu Jingmei, product number JM-07997F2 48T; the zebrafish vitellogenin (VTG) ELISA kit was purchased from Jiangsu Jingmei, product number JM-07790F2 48T.

[0106] Figure 6 The results showed that during the 14 days before exposure (i.e., under non-exposed conditions), no differences were observed in terms of spawning and fertilization ( Figure 6 A and C). After 14 days of exposure to cylindrospermopsin, the cumulative average number of eggs laid by female fish in the 0.5 and 5 μg / L cylindrospermopsin treatment groups was significantly lower than that of the control group ( Figure 6 B and C). The spawning and fertilization rate of the 14-day pre-exposure group was 97.59%-98.42%, and there was no difference among the groups. However, after 14 days of exposure, the spawning and fertilization rates of the 0.5 and 5 μg / L cylindrospermopsin treatment groups were significantly reduced.

[0107] For male fish after exposure to cylindrospermopsin, both testosterone and estrogen increased significantly. For female fish, the levels of testosterone and estrogen did not change significantly, but the VTG level decreased significantly. These results indicate that cylindrospermopsin acts as an estrogen in zebrafish. The decrease in VTG concentration affects mature oocytes and the number of eggs laid. Therefore, it is speculated that long-term exposure to cylindrospermopsin may affect reproduction by interfering with the sex hormone levels of fish, thereby reducing the fertilization rate and hatching success rate. Therefore, the reproductive ability of zebrafish after exposure to cylindrospermopsin is significantly affected.

[0108] In summary, under the exposure condition of cylindrospermopsin at 0.5 - 5 μg / L, the courtship behavior of zebrafish was significantly affected. To verify the model that courtship behavior can indicate the reproductive effects on zebrafish, under the same concentration condition, the effects of the above-mentioned chemical substances on the reproductive function of zebrafish were verified by traditional methods, namely, by detecting the egg production, fertilization rate and reproductive hormones of zebrafish. The results showed that under the exposure condition of 0.5 - 5 μg / L, the reproductive function of zebrafish was significantly affected. Thus, a model for evaluating the reproductive toxicity of chemical substances by the courtship behavior of zebrafish can be established.

[0109] The experimental results of the present invention show that after adult zebrafish were exposed to cylindrospermopsin, the total duration of ROA and the frequency of entering ROA of male and female zebrafish decreased significantly, so the courtship behavior decreased significantly. The frequency and time of entering the spawning area are key parameters for evaluating the reproductive behavior of fish. After exposure to environmental pollution-related substances, the egg production and fertilization rate of zebrafish decreased significantly, indicating that cylindrospermopsin affects the reproductive function of zebrafish. Therefore, it can cause damage to the reproductive function of zebrafish by affecting the courtship behavior of zebrafish. Therefore, drugs for preventing and treating reproductive function damage or abnormal courtship behavior can be screened through this reproductive toxicity model. The method for evaluating the reproductive toxicity of chemical substances provided by the present invention can be used in the fields of medicine, drug development and screening of natural active ingredients, etc.

[0110] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for constructing a zebrafish reproductive toxicity model, characterized in that: It includes the following steps: Expose zebrafish to chemicals that are reproductively toxic; The chemical substance with reproductive toxicity is selected from methylmercury, alkaloid toxins, bisphenols or pyrethroid insecticides; the concentration of the chemical substance in the exposure environment is at least 0.5 μg / L, and the exposure time in the exposure environment of the chemical substance with reproductive toxicity is 14-42 days; Preferably, the alkaloid toxin is cylindrospermopsin (CAS: 143545-90-8).

2. A method for evaluating the reproductive toxicity of a chemical substance, characterized in that: The method comprises the following steps: evaluating courtship behavior and fertility of zebrafish; The courtship behavior evaluation comprises: first exposing female and male zebrafish to a chemical exposure environment with reproductive toxicity; then taking the exposed female or male target zebrafish and placing it in one of the courtship behavior evaluation areas, and correspondingly taking the exposed male or female courtship behavior test zebrafish and placing it in another area of ​​courtship behavior evaluation; and the activity areas of the target zebrafish and the test zebrafish do not overlap, and taking zebrafish behavioral videos and / or pictures within a period of time; and evaluating the movement behavior of male zebrafish and female zebrafish during the courtship period; The concentration of the chemical substance in the exposure environment is at least 0.5 μg / L; the concentration of the chemical substance in the courtship behavior evaluation area is the same as that in the exposure environment; The motor behavior during the courtship period is selected from at least one of the following: The movement distance of the test zebrafish during the courtship period, the total duration of the test zebrafish entering a certain designated area, the frequency of entering the designated area, the courtship trajectory and the courtship index; the designated area is an area close to the target fish; The fertility evaluation comprises the following steps: co-culturing female and male zebrafish exposed to a chemical substance with reproductive toxicity, and then performing at least one of the following evaluations on the zebrafish: evaluating the egg-laying amount and / or fertilization rate of the female zebrafish, and detecting the reproductive-related hormone levels of the female zebrafish and / or the male zebrafish; Preferably, the movement distance of the test zebrafish in the designated area during courtship; Preferably, the exposed female and male zebrafish are co-housed for 7-28 days.

3. The method for evaluating the reproductive toxicity of a chemical substance according to claim 2, characterized in that: If the zebrafish's courtship behavior and fertility decrease in the exposure environment, the chemical is assessed to have reproductive toxicity to the zebrafish.

4. The method for evaluating the reproductive toxicity of a chemical substance according to claim 2, characterized in that: Female and male zebrafish were exposed to reproductively toxic chemicals for 14-42 days.

5. The method for evaluating the reproductive toxicity of a chemical substance according to claim 4, characterized in that: The time for shooting the zebrafish behavioral video and / or picture is 10-20 minutes; Preferably, the shooting of zebrafish behavioral videos and / or pictures is performed under continuous lighting conditions; Preferably, the zebrafish behavioral videos and / or pictures are analyzed using software to obtain the movement behavior during courtship.

6. The method for evaluating the reproductive toxicity of a chemical substance according to claim 2, characterized in that: The courtship index refers to the time a male zebrafish or a female zebrafish chases another female zebrafish or another male zebrafish.

7. The method for evaluating the reproductive toxicity of a chemical substance according to claim 2, characterized in that: The reproduction-related hormone is selected from testosterone, estradiol or vitellogenin.

8. The method for evaluating the reproductive toxicity of a chemical substance according to any one of claims 2 to 7, characterized in that: Chemicals with reproductive toxicity are selected from methylmercury, alkaloid toxins, bisphenols or pyrethroid insecticides; Preferably, the alkaloid toxin is cylindrosporin.

9. Use of the zebrafish reproductive toxicity model constructed according to claim 1 or the zebrafish reproductive toxicity model constructed according to the method for evaluating the reproductive toxicity of a chemical substance according to any one of claims 2 to 8 in screening drugs for preventing and / or treating reproductive function damage, characterized in that: The application comprises: evaluating the reproductive toxicity of the chemical substance to zebrafish according to the method for evaluating the reproductive toxicity of a chemical substance according to any one of claims 2 to 8, and obtaining a zebrafish reproductive toxicity model.

10. Use of the zebrafish reproductive toxicity model constructed according to claim 1 or the zebrafish reproductive toxicity model constructed according to the method for evaluating the reproductive toxicity of a chemical substance according to any one of claims 2 to 8 in screening drugs for improving or inhibiting abnormal courtship behavior of zebrafish, characterized in that: The application comprises: evaluating the reproductive toxicity of the chemical substance to zebrafish according to the method for evaluating the reproductive toxicity of a chemical substance according to any one of claims 2 to 8, and obtaining a zebrafish reproductive toxicity model.

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