Method for seed production based on transplantation of redfin pufferfish germ stem cells into adult sterile recipients

Abstract

The present invention relates to a method for seed production based on transplanting redfin puffer reproductive stem cells into adult sterile recipients, belonging to the field of aquatic breeding. The method comprises the following steps: high-temperature culturing male redfin puffer fish after the spawning season, and injecting busulfan solution into the male fish gonads for multiple times during the high-temperature culturing stage to obtain sterile redfin puffer fish receptors; transplanting redfin puffer spermatogonial stem cells into the gonads of sterile recipients, and culturing the successfully implanted redfin puffer fish until the gonads mature to produce sperm carrying genes of excellent varieties for seed breeding. The method of the present invention can be used to quickly and efficiently deplete the number of reproductive cells in the gonads of adult male redfin puffer fish in the breeding period, and to perform reproductive stem cell transplantation on sterile recipients; the method provides a new method for preparing adult sterile receptors of marine fish, isotype transplantation, and shortening the germplasm creation cycle.

Description

Technical Field

[0001] The invention belongs to the field of aquatic breeding, and specifically relates to a method for producing seeds based on transplanting redfin pufferfish reproductive stem cells into adult sterile receptors. Background Art

[0002] Reproductive stem cell transplantation technology is a cutting-edge breeding technology for preparing excellent new aquatic germplasm. This technology involves transplanting donor reproductive stem cells into recipients, waiting for the recipients to mature and produce donor gametes, and then artificially inseminating the donor offspring. This technology can significantly shorten the breeding cycle, and because reproductive stem cells have bidirectional differentiation potential, they have good application prospects in sex-controlled breeding.

[0003] The preparation of infertile receptors is one of the key links in reproductive stem cell transplantation technology. At present, the main methods for preparing infertile receptors include: knockdown or knockout of key reproductive genes, triploid induction, and high temperature-chemical drug combined treatment. The first two methods mainly start treatment in the fertilized egg or early embryo period, and can achieve good results, but for most marine farmed fish, the survival rate is affected due to the long sexual maturity period and the susceptibility to adversity, epidemics and other stress factors during the culture process.

[0004] Takifugu rubripes has tender meat and rich nutrition. It is known as the "king of fish" and is an important high-quality breeding object for export and foreign exchange earning in northern my country. The use of reproductive stem cell transplantation technology to create excellent germplasm of Takifugu rubripes is an urgent need for industrial development. The fertilized eggs of Takifugu rubripes are sinking eggs, which are sticky, with thick and opaque egg membranes. Male and female fish need 2 and 3 years to reach sexual maturity, respectively, which limits the application of reproductive key gene knockdown or knockout, triploid induction and other technologies in the preparation of sterile receptors. Traditional busulfan treatment can cause obvious degeneration of the testes of Takifugu rubripes, but reproductive cells of various differentiation periods still remain in some spermatocysts, which will affect the efficiency of reproductive stem cell transplantation. Summary of the invention

[0005] The present invention provides a method for seed production based on transplanting redfin puffer fish reproductive stem cells into adult sterile recipients. The endogenous reproductive stem cells of the sterile recipients obtained by the method are almost completely exhausted, and after the reproductive stem cell transplantation, successful colonization of the exogenous donor can be observed. The redfin puffer fish spermatogonial stem cells with excellent traits such as disease resistance, stress resistance, and fast growth are transplanted into the adult fish sterile recipients, and mature sperm of the parent with excellent traits can be quickly produced, thereby shortening the breeding time.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for seed production based on transplanting redfin pufferfish reproductive stem cells into adult sterile recipients, the method comprising the preparation of sterile recipients, the preparation and transplantation of spermatogonial stem cells;

[0008] The preparation of the sterile receptor comprises the following steps: culturing the male redfin pufferfish at high temperature after the spawning season, and injecting a busulfan solution with a final concentration of 30 mg / kg into the gonad of the male redfin pufferfish for many times during the high temperature culturing stage, thereby obtaining the sterile redfin pufferfish receptor, wherein the male redfin pufferfish is a male redfin pufferfish with fully developed gonads and sperm production;

[0009] Furthermore, the high-temperature culture is to raise the temperature of male redfin puffer fish with fully developed gonads and sperm production by 1°C to 32°C every day for high-temperature culture. After 1 week of adaptation, a busulfan solution with a final concentration of 30 mg / kg is injected. The injection interval is 14 days, and a total of 3 injections are given to obtain redfin puffer fish sterile receptors.

[0010] Furthermore, the method for preparing a busulfan solution with a final concentration of 30 mg / kg is to dissolve busulfan drug powder in cell culture grade DMSO to prepare a 4 mg / mL stock solution.

[0011] Furthermore, the injection method is to inject into the gonads of the redfin pufferfish through the genital pore.

[0012] Specifically, the male adult redfin pufferfish was given the first busulfan injection, a busulfan solution with a final concentration of 30 mg / kg was injected into the fish body through the genital pore, and then the high temperature culture was continued for 14 days; then the male adult redfin pufferfish was given the second busulfan injection, a busulfan solution with a final concentration of 30 mg / kg was injected into the fish body through the genital pore, and then the high temperature culture was continued for 14 days; then, the male adult redfin pufferfish was given the third busulfan injection, a busulfan solution with a final concentration of 30 mg / kg was injected into the fish body through the genital pore, and then the high temperature culture was continued for 14 days; then, the temperature was lowered to the normal breeding temperature of redfin pufferfish by reducing it by 1°C every day.

[0013] The preparation of the spermatogonial stem cells is carried out by isolating the spermatogonial stem cells from the testicular tissue of the male redfin pufferfish whose gonads have not matured;

[0014] The transplantation is to transplant spermatogonial stem cells into the gonads of infertile recipients through the genital pores. One month after the transplantation, the colonization efficiency is tested, and the successfully colonized redfin pufferfish is cultured until the gonads mature to produce sperm carrying genes of excellent varieties for seed breeding.

[0015] The beneficial effects of the present invention compared with the prior art are:

[0016] The present invention provides a method for rapidly and efficiently preparing sterile receptors of redfin pufferfish, which can be used to rapidly and efficiently deplete the number of reproductive cells in the gonads of adult male redfin pufferfish during the breeding period, and to perform reproductive stem cell transplantation on the sterile receptors; the method provides a new method for preparing adult sterile receptors of marine fish, isotransplantation, and shortening the germplasm creation cycle; specifically:

[0017] The method of the present invention directly injects a busulfan solution into the gonadal part of the redfin pufferfish through the genital pore injection, which can reduce the dosage of the busulfan reagent used, reduce the toxicity to the adult redfin pufferfish, and at the same time ensure a good endogenous reproductive cell depletion effect, and statistically calculate the growth index and survival rate of the redfin pufferfish before and after the treatment;

[0018] The method of the present invention uses adult male redfin pufferfish with mature gonads as experimental subjects, which can be used for reproductive stem cell transplantation immediately after being processed, thus saving the time of culturing to sexual maturity compared with the method of using embryos or fry to prepare sterile recipients.

[0019] In the method of the present invention, all gonads of redfin pufferfish treated with high temperature-busulfan undergo significant degeneration, and the treatment efficiency is high;

[0020] The method of the present invention identified through H&E staining that spermatocytes and sperm in the gonads of redfin pufferfish were depleted, which did not affect the normal survival and proliferation of spermatogonial stem cells, and the gonad state was suitable for receiving exogenous spermatogonial stem cells.

[0021] The present invention uses red fin puffer fish reproductive stem cells purified by Percoll and fluorescently stained as donors for transplantation. One month after the transplantation, the colonization efficiency of the donor reproductive stem cells in the sterile recipient gonads of adult male red fin puffer fish is 75%, indicating that the transplantation effect of the red fin puffer fish reproductive stem cells on the sterile recipient is ideal. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The growth index changes and survival rate changes of different groups of adult redfin pufferfish after different treatments provided in the embodiments of the present invention. The italic numbers represent P value;

[0023] Figure 2 The H&E staining effect diagram of the testis of adult redfin pufferfish after different treatments provided in the embodiment of the present invention; wherein, groups A, B, C, and D are the control group fish, the high temperature group fish, the high temperature-DMSO group fish, and the high temperature-busulfan group fish, respectively. Ps: primary spermatocyte; SC: sperm vesicle; SCN: sperm vesicle without germ cells, the scale bar is 100um;

[0024] Figure 3The colonization test results of the germ stem cells transplanted into the sterile recipient of redfin puffer fish treated with high temperature and busulfan provided in the embodiment of the present invention are shown in red arrows, which are the donor redfin puffer spermatogonial stem cells detected one month after the transplantation, and the scale bar is 50um. DETAILED DESCRIPTION

[0025] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the examples. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technical personnel in the field without creative work are within the scope of protection of the present invention.

[0026] Example 1: A highly efficient method for preparing sterile recipients for redfin pufferfish reproductive stem cell transplantation comprises the following steps:

[0027] 1. Cultivation of adult male redfin pufferfish

[0028] Adult male redfin pufferfish entering the breeding period were selected as experimental subjects and divided into a control group of 7, a high-temperature group of 7, a high-temperature-DMSO group of 7 and a high-temperature-busulfan group of 7. The water temperature was 18℃±1℃, and they were fed with granular bait twice a day, and fresh seawater was replaced once in the morning and evening.

[0029] 2. High temperature-busulfan treatment of adult male redfin pufferfish

[0030] The water temperature of the redfin pufferfish in the high temperature group, high temperature-DMSO group, and high temperature-busulfan group was raised to 32°C by 1°C per day, and the redfin pufferfish was adapted to the 32°C water temperature for 1 week. The busulfan stock solution was prepared with DMSO at a concentration of 4 mg / mL.

[0031] The redfin pufferfish in the high temperature-busulfan group were anesthetized with MS-222 (200 mg / L) and then weighed. The required volume of busulfan solution was calculated to make the final concentration of 30 mg / kg, and the busulfan solution was injected into the fish through the genital pore to complete the first injection. The required volume of injection solution in the high temperature-DMSO group was calculated according to the above method, and the same volume of DMSO was injected into the fish through the genital pore, and then the high temperature culture was continued for 14 days.

[0032] Afterwards, the redfin pufferfish in the high temperature-busulfan group were injected with busulfan for the second time according to the above method, and the high temperature-DMSO group was injected with the same amount of DMSO, and then the high temperature culture was continued for 14 days;

[0033] Afterwards, the redfin pufferfish in the high temperature-busulfan group were injected with busulfan for the third time according to the above method, and the high temperature-DMSO group was injected with the same amount of DMSO, and then the high temperature culture was continued for 14 days;

[0034] On the 14th day after the third injection, the culture water temperature (32°C) of the redfin pufferfish in the high temperature group, the high temperature-DMSO group and the high temperature-busulfan group was reduced by 1°C per day to 18°C ​​to complete the preparation of sterile receptors.

[0035] 3. Statistics on growth indicators and survival rate of redfin pufferfish

[0036] The initial body weight (i.e., body weight before the experiment) and final body weight (i.e., body weight after the experiment) of adult male redfin pufferfish in the control group, high temperature group, high temperature-DMSO group, and high temperature-busulfan group were tested, and the survival rate after the experimental operation was recorded. The data of each group were statistically analyzed by One-Way ANOVA, with P<0.05 as the standard of significant difference. Figure 1 .

[0037] Depend on Figure 1 It can be seen that there was no significant difference between the initial and final weights of adult male redfin pufferfish in each group, and there was no mortality, indicating that high temperature-busulfan treatment has little effect on the fish body and is a relatively safe method.

[0038] 4. Observation on the depletion of endogenous germ cells in the gonadal tissue of Takifugu rubripes

[0039] Three redfin pufferfish were randomly selected from each of the control group, high temperature group, high temperature-DMSO group, and high temperature-busulfan group. After being anesthetized in a 200 mg / L MS-222 solution for 3-4 minutes, the testicular tissue was removed by dissection. After being washed with PBS, the tissue was transferred to an animal in situ hybridization fixative (4% paraformaldehyde) for preservation. The effect of endogenous germ cell depletion in redfin pufferfish was observed by tissue sectioning and H&E staining. The steps are as follows:

[0040] (1) Tissue processing: Fix the testis tissue of redfin pufferfish with fixative for 24 hours. Take the tissue out of the fixative and trim it flat with a scalpel in a fume hood. Place the trimmed tissue and the corresponding label in a dehydration box;

[0041] (2) Dehydration and wax dipping: Place the dehydration box in a dehydrator for gradient alcohol dehydration, in order: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, alcohol benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, paraffin I melted at 65°C for 1 hour, paraffin II melted at 65°C for 1 hour, paraffin III melted at 65°C for 1 hour;

[0042] (3) Embedding: Embed the wax-soaked tissue in an embedding machine. First, place the melted wax in the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and place it in the embedding frame according to the requirements of the embedding surface and attach the corresponding label. Cool it in a -20℃ freezer. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block;

[0043] (4) Sectioning: Place the trimmed wax block in a -20℃ freezer to cool, then place the cooled wax block in a paraffin slicer to slice at a thickness of 4μm. Float the slices on 40℃ warm water on the slicer to flatten the tissue, pick up the tissue with a glass slide, and bake the slices in a 60℃ oven;

[0044] (5) H&E staining: paraffin sections were dewaxed in xylene twice, 10 minutes each time, and then rehydrated in gradient alcohol of 100%, 95%, 85%, and 75%, respectively, for 3 minutes in each gradient, and finally immersed in distilled water for 2 minutes; stained with hematoxylin solution for 10 minutes, and the floating color was washed off with distilled water; differentiated with differentiation solution for 30 seconds, and immersed in distilled water twice, 3 minutes each time; treated with eosin solution for 1 minute, and the excess staining solution was removed; immersed in 75%, 85%, 95%, and 100% alcohol for 2 seconds each; immersed in 100% ethanol for 1 minute, transparentized in xylene twice, 1 minute each time, and mounted with neutral gum, and the tissue structure and cell morphology were observed under a microscope (see Figure 2 ).

[0045] Depend on Figure 2 It can be seen that in the control group, the testes developed into spermatocysts, and the main cell type in the spermatocysts was primary spermatocytes ( Figure 2 2(A); In the high temperature group, the number of germ cells in the spermatocyst was greatly reduced, but some germ cells still existed in the spermatocyst, indicating that the 32℃ treatment temperature can promote the apoptosis of germ cells in the testis (2(B); In the high temperature-DMSO group, most of the germ cells in the spermatocyst of the redfin pufferfish also apoptotic (2(C); In the high temperature-busulfan group, the germ cells in the spermatocyst were basically non-existent, forming a cavity in the spermatocyst (2(D), indicating that the high temperature of 32℃ and busulfan treatment have a significant effect on the apoptosis of endogenous germ cells in the testis of the redfin pufferfish.

[0046] 5. Observation on the effect of germ stem cell transplantation and infertility recipient colonization in redfin pufferfish

[0047] (1) Take a healthy male redfin pufferfish, anesthetize it in 200 mg / L MS-222 for 2-3 minutes, wipe the fish body with 75% alcohol, dissect the testis tissue, wash the testis with PBS 3-4 times, and use scissors to remove non-testis tissue during the washing process;

[0048] (2) The trimmed testicular tissue was transferred to a culture dish, cut into small pieces, washed once with PBS, and centrifuged at 300 × g for 5 minutes to remove the supernatant;

[0049] (3) 1 mL of digestion mixture was added for every 0.1 g of testis tissue (the ratio of each 1 mL of digestion mixture was 0.25% trypsin: fetal bovine serum: DNase I = 900 μL: 50 μL: 50 μL), and digested at 25°C for 3 hours, mixing every 30 minutes; after 3 hours, the digested tissue was filtered through 100 μm and 40 μm filters in turn to obtain cells;

[0050] (4) Prepare 50%, 30% and 10% Percoll density gradients according to the Percoll instructions and add them to a 15 mL centrifuge tube in sequence;

[0051] (5) Add 2 mL of cell suspension to a centrifuge tube and centrifuge at 800 × g for 30 min to separate the cells. Aspirate the cell band at 30% gradient and wash twice with PBS to adjust the cell concentration.

[0052] (6) Use trypan blue staining to count the number of live cells, and use PKH26 to fluorescently stain the cells. According to the reagent instructions, 0.4mL diluent C + 4μL dye working solution can stain 10 8 cells, transfer the dye solution into the cells and incubate for 5 minutes, add an equal amount of fetal bovine serum and incubate for 1 minute to terminate the reaction, wash twice with L-15, and add 10 μL of penicillin-streptomycin per milliliter of cell suspension to prepare a cell suspension;

[0053] (7) The infertile recipients were anesthetized in a 200 mg / L MS-222 solution. A 1 mL syringe was used to probe 2-3 cm below the genital opening (the location of the gonads). 2 μL of the cell suspension was injected into the gonads. After transplantation, the recipient fish were continuously injected with 3 mg / L penicillin.

[0054] (8) One month after transplantation, the recipient redfin pufferfish was selected and anesthetized with 200 mg / L MS-222 solution for 2 minutes. The gonadal tissue was separated and cut into pieces. The fluorescence state of the donor cells in the gonad was observed under a fluorescence microscope (see Figure 3 ).

[0055] Depend on Figure 3 One month after the transplantation of reproductive stem cells, red fluorescent signals were detected in the testes of the sterile recipient redfin pufferfish through fluorescence observation, indicating that the donor redfin pufferfish spermatogonial stem cells have successfully colonized in the sterile recipient testes of the redfin pufferfish. By detecting the colonization status of the recipient gonads of 4 redfin pufferfish, it was found that 3 recipient gonads had donor cells, and the colonization rate reached 75%.

[0056] According to the above method, the individuals that are successfully colonized are obtained again and cultured until the gonads mature, which can quickly produce mature sperm and then be used for seed cultivation, which can effectively shorten the cultivation cycle of male parents.

[0057] This method can be applied to individuals or groups of redfin pufferfish with excellent traits such as disease resistance, stress resistance, and fast growth, and their spermatogonial stem cells can be transplanted into the infertile recipient gonads of adult fish. This can quickly obtain mature sperm containing genetic material with excellent traits, significantly shorten the breeding cycle, and promote the development of the breeding of excellent redfin pufferfish varieties.

[0058] The present invention is not limited to the above specific embodiments, which are only for the purpose of describing the use of the present invention in detail, and production methods and technical details with the same functions also belong to a part of the present invention. In fact, those skilled in the art can find different adjustment schemes according to the above description, and these adjustments are also within the scope of the claims attached hereto.

Claims

1. A method for seed production based on transplanting redfin pufferfish germ stem cells into adult sterile recipients, characterized in that: The method includes the preparation of an infertile recipient, the preparation and transplantation of spermatogonial stem cells; The preparation of the sterile receptor comprises the following steps: culturing the male redfin pufferfish at high temperature after the spawning season, and injecting a busulfan solution with a final concentration of 30 mg / kg into the gonad of the male redfin pufferfish for many times during the high temperature culturing stage, thereby obtaining the sterile redfin pufferfish receptor, wherein the male redfin pufferfish is a male redfin pufferfish with fully developed gonads and sperm production; The preparation of the spermatogonial stem cells is carried out by isolating the spermatogonial stem cells from the testicular tissue of the male redfin pufferfish whose gonads have not matured; The transplantation is to transplant spermatogonial stem cells into the gonads of infertile recipients through the genital pores. One month after the transplantation, the colonization efficiency is tested, and the successfully colonized redfin pufferfish is cultured until the gonads mature to produce sperm carrying genes of excellent varieties for seed breeding.

2. The method for seed production based on transplanting redfin pufferfish germ stem cells into adult sterile recipients according to claim 1, characterized in that: The high temperature culture is to raise the temperature of male redfin puffer fish with fully developed gonads and sperm production by 1°C to 32°C every day for high temperature culture, and inject a busulfan solution with a final concentration of 30 mg / kg after adaptation for 1 week, with an injection interval of 14 days and a total of 3 injections to obtain redfin puffer fish sterile receptors.

3. The method for seed production based on transplanting redfin pufferfish germ stem cells into adult sterile recipients according to claim 1, characterized in that: The method for preparing a busulfan solution with a final concentration of 30 mg / kg is to dissolve busulfan drug powder in cell culture grade DMSO to prepare a 4 mg / mL stock solution.

4. The method for seed production based on transplanting redfin pufferfish germ stem cells into adult sterile recipients according to claim 1, characterized in that: The injection method is to inject into the gonads of the redfin pufferfish through the genital pore.

5. The method for seed production based on transplanting redfin pufferfish germ stem cells into adult sterile recipients according to claim 4, characterized in that: The first busulfan injection was performed on the adult male redfin pufferfish, and a busulfan solution with a final concentration of 30 mg / kg was injected into the fish body through the genital pore, and then the high temperature culture was continued for 14 days; then the second busulfan injection was performed on the adult male redfin pufferfish, and a busulfan solution with a final concentration of 30 mg / kg was injected into the fish body through the genital pore, and then the high temperature culture was continued for 14 days; then, the third busulfan injection was performed on the adult male redfin pufferfish, and a busulfan solution with a final concentration of 30 mg / kg was injected into the fish body through the genital pore, and then the high temperature culture was continued for 14 days; then, the temperature was lowered to the normal breeding temperature of redfin pufferfish by reducing it by 1°C every day.

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