Method for detecting reproductive toxicity marker by taking zebra fish as model organism

Abstract

The invention discloses a method for detecting a reproductive toxicity marker by taking zebra fish as a model organism, which comprises the following two steps of: measuring the content of ovuprogen (VTG) in blood plasma of the zebra fish and preparing and analyzing gonad slices of the zebra fish. The endocrine disruption effect of CYF is evaluated by taking fish body sex hormone and VTG levels and gamete conditions in gonads as indexes, the increase of vitellogenin detected by the method indicates that the content of estrogen in male zebra fish is increased, the estrogen-like effect of CYF is verified, the male zebra fish also has VTG genes, and the CYF has a good endocrine disruption effect due to the stimulation of estrogen or estrogen-like effect. In the prior art, the VTG in the male fish body can rise, mature sperms and sperm cells are exposed by CYF, the two cells locally change, the sperm concentration degree is reduced, and sperm arrangement in some areas is sparse compared with that in a control group, so that the detection of the change of the VTG in the male fish body can be used as an effective means for evaluating whether a compound has an estrogen effect or not.

Description

Technical Field

[0001] The present invention relates to the technical field of reproductive toxicity detection, and in particular to a method for detecting reproductive toxicity markers using zebrafish as a model organism. Background Art

[0002] Cyflumetofen (CYF) is a new type of acyl acetonitrile acaricide with the molecular formula C24H24F3NO4, relative molecular mass 447.4, and CAS number 400882-07-7. Cyflumetofen was first registered and sold in 2007, and has been widely used since 2010. It is mainly used for the control of mites in agricultural products such as fruit trees and tea trees. For example, it is registered for use on tomatoes, strawberries and citrus fruits. In addition, it can also be used to control harmful mites such as red spider mites in the cultivation of some Chinese medicinal materials and flowers.

[0003] The reproductive behavior of zebrafish is regulated by the brain-gonad-liver (HPGL) axis, and the liver is an important part of the zebrafish reproductive axis. Vitellogenin (VTG) is produced in the liver and plays an important role in gamete maturation. Under normal circumstances, the content of VTG in male fish is relatively low. When male fish are stimulated by estrogen or estrogen-like hormones, the content of VTG will increase with the increase of estrogen content in the fish. Therefore, detecting the changes in VTG content in male fish is an effective means to evaluate whether a compound has an estrogenic effect, that is, an endocrine disrupting effect; in addition, as the gonad is the direct organ for controlling reproduction, the maturation and damage of gametes in the gonad is also one of the ways to evaluate the endocrine disrupting effect of a compound.

[0004] To this end, this project proposes a detection method for reproductive toxicity markers using zebrafish as a model organism. It uses zebrafish as a model and the levels of fish sex hormones and VTG as well as the status of gametes in the gonads as indicators to evaluate the endocrine disrupting effect of CYF on zebrafish. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art mentioned in the background technology, and to propose a method for detecting reproductive toxicity markers using zebrafish as a model organism.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: A method for detecting reproductive toxicity markers using zebrafish as a model organism includes determining the content of vaginogen (VTG) in zebrafish plasma, which is specifically divided into the following steps: S1. 45 five-month-old sexually mature male zebrafish were selected as experimental organisms: S2, the selected 45 zebrafish were randomly divided into 3 groups, and after adapting to the recirculating aquaculture system for 14 days, a 21-day exposure experiment was conducted; S3. After 21 days of exposure, 6 zebrafish were randomly selected from each group, and blood samples from the tail vein of the zebrafish were collected; S4. Centrifuge the blood sample at 7000 x g for 5 min at 4°C and take the supernatant to determine whether the level of vaginogen (VTG) in the plasma of male zebrafish is increased.

[0007] As a further step of the method of the present invention, S2-1, a total of 3 concentration gradients are set from environmental concentration to LC10, L-0.025μg / L (environmentally relevant concentration), M-7.5μg / L, H-2.25mg / L (LD10); S2-2. Replace all volumes of exposure solution regularly every day to maintain appropriate pesticide concentration and water quality.

[0008] As a further step of the method of the present invention, S3-1, randomly select fish from each group and place them on ice for anesthesia, treat the capillary tube with sodium heparin, and then take blood samples from the tail vein of zebrafish, and the blood samples are used to measure the vitellogenin content in the plasma; S3-2. Take tail vein blood samples from 6 fish in the same group and mix them as one sample. Take 3 parallel samples from each group.

[0009] As a further method of the present invention, S4-1, using an enzyme-linked immunosorbent assay (ELISA) kit to determine the zebrafish vitellogenin (VTG) in the supernatant.

[0010] As a further aspect of the method of the present invention, it also includes preparing and analyzing zebrafish gonad slices, specifically preparing zebrafish gonad slices for histological analysis and stereological analysis, as follows: The zebrafish gonads were weighed and placed in a 2.5% glutaraldehyde fixative in a 4°C refrigerator for the preparation of histopathological sections. The gonadal tissue preserved in 2.5% pentanediol was taken out and dehydrated, then embedded in paraffin, cut longitudinally into 6 μm thick sections and fixed on glass slides. After fixation, the samples were stained with hematoxylin and eosin (HE).

[0011] Compared with the prior art, the present invention has the following beneficial effects: 1. This method can verify the suspected endocrine disrupting effect of CYF on non-target organisms.

[0012] 2. VTG content is sensitive to endocrine disruptors, and changes in content in fish can be detected by exposure to environmentally relevant concentrations in this method.

[0013] 3. The mature sperm count can be obtained through the pathological observation of gonadal tissue in this method, which can more accurately determine the estrogen-like interference effect of CYF on male zebrafish. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a schematic diagram of the changes in VTG content in male zebrafish; Figure 2 This is a schematic diagram of the gonadal tissue section of male fish; Figure 3 Schematic diagram of mature sperm area counting. DETAILED DESCRIPTION

[0015] The content of the present invention can be more easily understood by selecting the following detailed description of the preferred implementation method of the present invention and the embodiments included. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. When there is a conflict, the definition in this specification shall prevail.

[0016] As used herein, the term "prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0017] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0018] When amount, concentration or other value or parameter is expressed as range, preferred range or a series of upper preferred value and lower preferred value limit range, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when disclosing range "1 to 5", described range should be interpreted as including range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5" etc. When numerical range is described in this article, unless otherwise stated, the range is intended to include its end value and all integers and fractions within the range.

[0019] Singular forms include plural references unless the context clearly indicates otherwise. "Optional" or "either" means that the subsequently described event or incident may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

[0020] Approximate terms in the specification and claims are used to modify quantities, indicating that the present invention is not limited to the specific quantity, but also includes acceptable and modified parts close to the quantity without causing changes in the relevant basic functions. Accordingly, the use of "about", "approximately", etc. to modify a numerical value means that the present invention is not limited to the exact numerical value. In some examples, the approximate terms may correspond to the accuracy of the instrument for measuring the numerical value. In the specification and claims of this application, range limitations can be combined and / or interchanged, and if not otherwise stated, these ranges include all subranges contained therein.

[0021] In addition, the indefinite articles "a" and "an" before the elements or components of the present invention have no restrictions on the quantity requirements (i.e. the number of occurrences) of the elements or components. Therefore, "a" or "an" should be interpreted as including one or at least one, and the elements or components in the singular form also include the plural form, unless the number is obviously intended to be in the singular form.

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0023] A method for detecting reproductive toxicity markers using zebrafish as a model organism, comprising determining the content of vaginogen (VTG) in zebrafish plasma and preparing and analyzing zebrafish gonadal slices; 1. Determination of the content of ovalbumin (VTG) in zebrafish plasma 1) 45 five-month-old sexually mature male zebrafish were randomly divided into three groups. After 14 days of adaptation in the recirculating aquaculture system, a 21-day exposure experiment was conducted. In the exposure experiment, three concentration gradients were set from environmental concentration to LC10: L-0.025μg / L (environmentally relevant concentration), M-7.5μg / L, and H-2.25mg / L (LD10); 2) Expose for 21 days, and replace all volumes of exposure solution regularly every day to maintain appropriate pesticide concentration and water quality; 3) After the 21-day exposure, 6 zebrafish were randomly selected from each group and placed on ice for anesthesia. The capillary tubes were treated with sodium heparin and then blood samples were collected from the tail vein of the zebrafish. The blood samples were used to measure the vitellogenin content in their plasma. 4) Take the tail vein blood samples of 6 fish in the same group and mix them as one sample. Take 3 parallel samples from each group. Centrifuge the blood samples at 7000xg for 5 minutes at 4℃ and take the supernatant for testing. 5) Use an enzyme-linked immunosorbent assay (ELISA) kit to measure zebrafish vitellogenin (VTG) in the supernatant. 2. Determination of the content of ovalbumin (VTG) in zebrafish plasma 1) Take the zebrafish gonads, weigh them, and place them in 2.5% glutaraldehyde fixative in a 4°C refrigerator for the preparation of histopathological sections; 2) Take out the gonadal tissue preserved in 2.5% pentanediol and dehydrate it; 3) After dehydration, the cells were embedded in paraffin and cut into 6 μm thick sections along the gonadal longitudinal direction and fixed on a glass slide; 4) After fixation, the samples were stained with hematoxylin and eosin (HE staining), and the slices were prepared for histological and stereological analysis; 5) The germ cells in the testis at different stages were counted, and the area occupied by the germ cells in each stage was statistically analyzed using Image-proplus6.0 software to evaluate the development level of the testis. The germ cells in the testis include spermatogonia (Spermatogonium, Sg), spermatocytes (Spermatocyte, Sc), spermatids (Spermatids, St) and mature sperm (Spermatozoa, Sz).

[0024] Results and conclusions: Figure 1 It can be seen that after exposure to all concentrations of CYF, the level of vitellogenin (VTG) in the plasma of male zebrafish increased significantly (P<0.05); VTG is a phospholipid protein present in the serum of sexually mature zebrafish animals and is the liver-derived yolk protein precursor in all oviparous hard bones. Under normal circumstances, the level of vitellogenin in the serum of male fish is lower than that of female fish; the synthesis and secretion of VTG are strictly controlled by estrogen. Once synthesized, VTG is released into the blood and transported to the gonads to promote gonadal maturation.

[0025] In this scheme, the increase of vitellogenin indicates that the estrogen content in male zebrafish is increased, which verifies the estrogen-like effect of CYF. The VTG gene also exists in male fish. Due to the stimulation of estrogen or estrogen-like hormones, the VTG in male fish will increase. Therefore, detecting the changes in VTG in male fish can be used as an effective means to evaluate whether a compound has an estrogenic effect.

[0026] Depend on Figure 2 As shown in the figure, zebrafish testes contain a large number of spermatids (St) and mature sperm (Sz), while the number of spermatogonia (Sg) and spermatocytes (Sc) is relatively small; compared with the control group, the proportion of mature sperm in the testes of male zebrafish in the three CYF concentration treatment groups was significantly reduced (P<0.05), as shown in the figure. Figure 3 shown.

[0027] In addition to the reduction in the proportion of mature sperm, Figure 2 and 3 It can be clearly found that the distribution of mature sperm and sperm cells in the testicles in the control group is relatively uniform and concentrated, but after exposure to CYF, these two types of cells undergo local changes, and the degree of sperm concentration decreases; however, in some areas, the sperm arrangement becomes sparser than in the control group.

[0028] The examples referred to herein are merely illustrative and are used to explain some features of the method of the present invention. The appended claims are intended to claim the widest possible range that can be imagined, and the embodiments presented herein are only illustrations of selected implementations according to the combination of all possible embodiments. Therefore, it is the applicant's intention that the appended claims are not limited by the selection of examples that illustrate the features of the present invention. Some numerical ranges used in the claims also include sub-ranges within them, and changes in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A method for detecting reproductive toxicity markers using zebrafish as a model organism, characterized in that: The method includes the determination of the ovalbumin content in zebrafish plasma, which is specifically divided into the following steps: S1. 45 five-month-old sexually mature male zebrafish were selected as experimental organisms: S2, the selected 45 zebrafish were randomly divided into 3 groups, and after adapting to the recirculating aquaculture system for 14 days, a 21-day exposure experiment was conducted; S3. After 21 days of exposure, 6 zebrafish were randomly selected from each group, and blood samples from the tail vein of the zebrafish were collected; S4. Centrifuge the blood sample at 7000 x g for 5 min at 4°C and take the supernatant to determine whether the ovalbumin content in the plasma of male zebrafish is increased.

2. The method for detecting reproductive toxicity markers using zebrafish as a model organism according to claim 1, characterized in that: S2-1. Set three concentration gradients from environmental concentration to LC10: L-0.025μg / L, M-7.5μg / L, H-2.25mg / L.

3. The method for detecting reproductive toxicity markers using zebrafish as a model organism according to claim 2, characterized in that: S2-2. Replace all volumes of exposure solution regularly every day to maintain appropriate pesticide concentration and water quality.

4. The method for detecting reproductive toxicity markers using zebrafish as a model organism according to claim 1, characterized in that: S3-1. Randomly select fish from each group and place them on ice for anesthesia. Treat the capillary tube with sodium heparin before taking blood samples from the tail vein of zebrafish.

5. The method for detecting reproductive toxicity markers using zebrafish as a model organism according to claim 4, characterized in that: S3-2. Take tail vein blood samples from 6 fish in the same group and mix them as one sample. Take 3 parallel samples from each group.

6. The method for detecting reproductive toxicity markers using zebrafish as a model organism according to claim 1, characterized in that: S4-1. Use an ELISA kit to measure the zebrafish vitellogenin in the supernatant.

7. The method for detecting reproductive toxicity markers using zebrafish as a model organism according to claim 1, characterized in that: The invention also includes the preparation and analysis of zebrafish gonad slices, specifically the preparation of zebrafish gonad slices for histological analysis and stereological analysis.

8. The method for detecting reproductive toxicity markers using zebrafish as a model organism according to claim 7, characterized in that: The zebrafish gonads were weighed and placed in a 2.5% glutaraldehyde fixative in a 4°C refrigerator.

9. The method for detecting reproductive toxicity markers using zebrafish as a model organism according to claim 8, characterized in that: The gonadal tissue preserved in 2.5% pentanediol was removed and dehydrated, then embedded in paraffin, cut longitudinally into 6 μm thick sections and fixed on slides.

10. The method for detecting reproductive toxicity markers using zebrafish as a model organism according to claim 9, characterized in that: After fixation, the samples were stained with hematoxylin and eosin.

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