Method for separating, purifying and transplanting spermatogonial stem cells of lateolabrax japonicus

Abstract

The invention discloses a method for separating, purifying and transplanting spermatogonial stem cells of lateolabrax japonicus, the spermatogonial stem cells of lateolabrax japonicus are separated and purified, marine fish larvae or freshwater fish larvae are used as receptor larvae, and then the spermatogonial stem cells of lateolabrax japonicus are transplanted to abdominal cavities of the receptor larvae. The spermatogonial stem cells obtained through separation are complete in number, structure and function and low in cell clustering rate. After the transplanted spermatogonial stem cells are subjected to fluorescence labeling, the migration and proliferation processes of donor germ cells can be observed in receptor fishes of marine fish fries or freshwater fish fries, so that the receptor fishes with the chimeric germline are obtained. According to the method, through xenotransplantation of the spermatogonial stem cells, a'belly borrowing reproduction 'breeding mode is provided for the marine fishes, a new way for gamete preservation and rapid propagation is provided for the marine fishes with the long reproductive cycle, and a new method and a new thought are provided for genetic improvement and germplasm resource preservation.

Description

Technical Field

[0001] The invention relates to a cell separation, purification and transplantation method, and in particular to a separation, purification and transplantation method for spermatogonial stem cells of Lateolabrax japonicus. Background Art

[0002] The Lateolabrax maculatus, also known as the Chinese spotted seabass, commonly known as seabass, village seabass, and seven-star seabass, is endemic to Northeast Asia. According to statistics, the national production in 2023 reached 246,900 tons, ranking among the top three marine aquaculture fish species in China. Due to its nutritious meat and high economic value, the Lateolabrax maculatus has become one of my country's most important marine aquaculture fishes in the past decade. Its rapid growth rate, rich nutrition, high economic value, wide temperature and salinity range, and strong resistance to wind and waves make it a preferred species for deep-sea aquaculture in modern marine ranches. In recent years, hybridization of different Lateolabrax species has led to varietal confusion and degradation of germplasm resources. Furthermore, Lateolabrax takes a long time to mature sexually, generally taking three to four years, and its body size is relatively large. Therefore, isolating and preserving high-quality Lateolabrax germplasm resources and providing new methods for gamete preservation and rapid reproduction for fish with long reproductive cycles are of great significance. However, existing methods for isolating and purifying Lateolabrax spermatogonia are not only expensive but also yield suboptimal results. Therefore, it is necessary to propose a method for the separation and purification of Lateolabrax spermatogonia in order to obtain Lateolabrax spermatogonia with high purity and strong cell activity.

[0003] Spermatogonial stem cells in fish testes are germline stem cells that are capable of self-renewal, proliferation, and differentiation. They are also totipotent, capable of forming germ cells at various stages all the way to mature gametes. Fish germline stem cell transplantation, as an emerging technology, has opened up new avenues for the conservation of fish germplasm resources. This technique cleverly exploits the early plasticity of fish gonadal development: before sex is determined, the direction of gonadal development can be manipulated using steroid hormones or exogenous germline stem cells. Furthermore, for donor germ cells, the genital ridge of the recipient larvae has a developmental window during which donor germ cells can successfully migrate and integrate into the recipient genital ridge. Therefore, researchers typically use newly hatched larvae as recipients. For example, experiments with germ cell transplantation in rainbow trout, American shad, and redfin pufferfish have all used newly hatched larvae, and have observed that transplanted germ cells migrate and integrate into the recipient genital ridge, enabling the recipient to produce functional gametes similar to the donor. Fish larvae were chosen as recipients not only because their gonads have not yet differentiated, but also because they have a relatively weak immune system. Newly hatched fish larvae have incompletely developed T cells and thymocytes, lacking mature adaptive immune function, which makes them less likely to reject foreign cells. Summary of the Invention

[0004] The invention provides a method for separating, purifying and transplanting spermatogonial stem cells of Lateolabrax japonicus.

[0005] The present invention adopts the following technical scheme: a method for separating, purifying and transplanting spermatogonial stem cells of Lateolabrax japonicus, separating and purifying spermatogonial stem cells of Lateolabrax japonicus, using marine fish fry or freshwater fish fry as recipient fry, and then transplanting the spermatogonial stem cells of Lateolabrax japonicus into the recipient fry.

[0006] The invention transplants Lateolabrax spermatogonial stem cells into the abdominal cavity of recipient fry through a microinjection instrument.

[0007] In the embodiment of the present invention, the marine fish larvae are medaka larvae, and the freshwater fish larvae are zebrafish larvae. The present invention preferably uses larvae that are 5 days old after hatching.

[0008] The isolation and purification of spermatogonial stem cells of Lateolabrax japonicus includes separation and Percoll gradient purification. During the separation, testis tissue blocks are digested with testis digestion fluid; during the Percoll gradient purification, Percoll solutions are layered from high to low in density, with concentrations of 40%, 30% and 10% in sequence.

[0009] In one embodiment, the testes are digested in a shaker at 37°C, 80 rpm for 1 hour. To prevent cell clumping, trypsin-EDTA is added to a final concentration of 0.25% after digestion. Digestion is continued on a shaker for 1.5 hours. Finally, L-15 medium containing 5% FBS is added to terminate digestion. After completion, the cells are sieved through 200-mesh and 70-mesh sieves, respectively.

[0010] The testis digestion solution is based on L-15 culture medium, supplemented with 2 mg / mL collagenase IV, 0.1% DNase I and 5% FBS, and sterilized by filtration with a 0.22 μm filter membrane. After digestion, 0.5% trypsin-EDTA is added to a final concentration of 0.25% and digestion is continued.

[0011] Percoll gradient solution: 100% Percoll was prepared by 10xPBS:Percoll=1:9, and diluted with 1xPBS to form 10% Percoll solution, 30% Percoll solution and 40% Percoll solution.

[0012] The testes were from 6-month-old Lateolabrax japonicus.

[0013] During transplantation, spermatogonial stem cells collected at the junction of 10% and 30% Percoll solutions were first washed, stained with PKH-26 at a final concentration of 0.5 μM, incubated at room temperature, collected by centrifugation, washed with PBS, and finally resuspended in L-15 culture medium and placed on ice for use.

[0014] Approximately 100 cells were injected into each larva to avoid rejection due to excessive cells or colonization failure due to insufficient cells.

[0015] Beneficial effects

[0016] 1. The ability of spermatogonial stem cells from Lateolabrax japonicus to colonize in medaka or zebrafish demonstrates that xenotransplantation is a conserved mechanism and pathway for germline stem cell migration. However, incompatibility of the gonadal microenvironments between fish species makes xenotransplantation between phylogenetically distant fish challenging for producing surrogate offspring. The success of this invention provides new possibilities for overcoming this challenge and reveals the potential for achieving spermatogonial stem cell transplantation and successful offspring reproduction between different fish species.

[0017] 2. Select 6-month-old Lateolabrax japonicus as the isolation object, with a gonadal index of 0.042% and strong expression of vasa / nanos3 ( Figure 1 ), avoiding the problem of immature reproductive cells in young fish or excessive impurities in adult fish.

[0018] 3. The nest digestion solution uses an enzyme combination: collagenase IV + DNase I + trypsin-EDTA step-by-step digestion for a total of 2.5 hours, which significantly reduces cell clumps ( Figure 2 B), while traditional methods mostly use single enzymes or short digestion times.

[0019] The preferred digestion solution in the present invention is a combination of 2 mg / mL collagenase IV, 0.1% DNase I, and 5% FBS. Furthermore, 2 mg / mL collagenase IV is used in the first digestion step to obtain a higher number of viable spermatogonial stem cells. However, the inventors attempted other enzyme combinations for digestion, but after one hour of digestion, the number of isolated spermatogonial stem cells was low, the number of dead cells increased, and even overdigestion occurred. For example:

[0020] ① The combination of 1mg / ml collagenase IV and 0.25mg / ml collagenase I was used for digestion for 1h, resulting in a small number of spermatogonial stem cells isolated;

[0021] ②1.7mg / ml collagenase IV + 0.25mg / ml collagenase V combination, digestion for 1h, the amount of dead cells in the cell suspension increased;

[0022] ③1mg / ml collagenase IV + 0.25mg / ml collagenase I + 0.25mg / ml collagenase V combination, digestion for 1h, overdigestion;

[0023] ④1mg / ml collagenase IV + 1mg / ml collagenase V + 1mg / ml neutral protease combination, digestion for 1h, over-digestion. 5. Gradient centrifugation innovation: design 10% / 30% / 40% Percoll gradient, efficient enrichment of spermatogonial stem cells with a diameter of 8-15μm at the 10%-30% junction ( Figure 2DE), the cell viability reached 98.37% ( Figure 2 J), far exceeding conventional methods (usually <90%).

[0024] 4. The present invention selects medaka and zebrafish as recipients. As model organisms of seawater and freshwater fish, they have the commonalities of fast reproduction, easy cultivation, and convenient genetic manipulation. Their larvae are transparent within a period of time after hatching, which is convenient for operation and observation. In order to determine whether cells are transplanted into and the migration path after transplantation, the donor cells are visually labeled, and the larvae of medaka and zebrafish meet the relevant requirements. In summary, the present invention has established a method for fish reproductive stem cell transplantation, which is of great significance for the preservation, protection and sustainable development of marine germplasm resources and mariculture fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The macroscopic structure and H&E staining of the testis of 6-month-old Lateolabrax japonicus;

[0026] in, Figure 1 A is the macroscopic structure of the testis of a 6-month-old Lateolabrax japonicus; Figure 1 B is H&E staining of testis of 6-month-old Lateolabrax japonicus; Figure 1 C is Figure 1 Partial enlarged view in B; Figure 1 D shows that these germ cells are spermatogonia.

[0027] Figure 2 To isolate and identify spermatogonia from Lateolabrax japonicus;

[0028] Figure 2 A: Percoll density gradient (10%, 30%, 40%) centrifugation produces cells in different bands;

[0029] Figure 2 B: Single-cell suspension of testis of 6-month-old Lateolabrax japonicus includes cells of various morphologies;

[0030] Figure 2 C: In situ hybridization was used to detect the expression of vasa and nanos3, demonstrating the presence of spermatogonia in the obtained single-cell suspension;

[0031] Figure 2 D-2G, 2I-2J: Cells in the cell layer at the junction of different concentrations of Percoll solutions

[0032] Figure 2 H cell counter analysis showed that the cell viability of the cell suspension was 99.63%, with green representing living cells, red representing dead cells, and the white circle representing cell clusters.

[0033] Figure 3 Spermatogonial stem cells were stained with PKH-26 and DAPI;

[0034] Spermatogonial stem cells were stained with PKH-26 and DAPI ( Figure 3 A); PKH-26-labeled Leptothorax donor cells were observed in the recipients (medaka, zebrafish) 60 days after transplantation, and these cells almost all migrated to the primitive genital ridge. Figure 3 CD), zebrafish ( Figure 3 F), while the control group showed no fluorescence at the same position, medaka ( Figure 3 B), zebrafish ( Figure 3 E). DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be fully described below in conjunction with the drawings in the embodiments of the present application, so as to fully understand the purpose, effect and application prospects of the present invention. The following examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention.

[0036] 1. Reagents Concerning the Present Invention

[0037] Spermatogonial stem cell separation solution, including spermatogonial washing solution, spermatogonial digestion solution and Percoll gradient solution.

[0038] The testis washing solution includes MEM culture medium (51200038, Gbico), fetal bovine serum FBS (10099141C, Gbico), Hepes buffer (15630080, Gbico), and L-glutamine (25030081, Gbico).

[0039] The testis digestion solution included collagenase IV (C8160, Solebro), DNase I (11284932001, Roche), Leibovitz L-15 medium (L-15) (11415064, Gbico), and trypsin-EDTA (0.5%) (15400054, Gbico).

[0040] A Percoll gradient solution consisting of Percoll (P1644, Sigma-Aldrich), 10x PBS (P1022, Solebro), and 1x PBS (10010023, Gbico) was used to isolate and purify L. japonicum spermatogonial stem cells. This separation solution yielded highly pure and active L. japonicum spermatogonial stem cells, which were then stained with PKH-26 (154214-55-8, MCE) red fluorescence for in vivo transplantation experiments.

[0041] In the following experiments, sea bass were purchased from China Resources Supermarket in Guangzhou, Guangdong Province. 0.4% trypan blue solution (T8154, Sigma-Aldrich), MS-222 anesthetic (E10521, Sigma-Aldrich) (400 mg of MS-222 was dissolved in 90 mL of deionized water, and 2.1 mL of Tris (1 M, pH 9.0) was added to adjust the pH to 7.0, and the volume was made up to 100 mL and stored at 4°C. When used, 4.2 mL of MS-222 stock solution was diluted in 100 mL of deionized water and prepared before use), penicillin-streptomycin (double antibody) (15140122, Gbico), and Calcein-AM / PI live cell / dead cell double staining reagent (CA1630, Solebol).

[0042] Spermatogonial stem cell isolation formula:

[0043] Sperm washing solution: Use MEM medium as the base solution, add 5% FBS, 25 mM Hepes and 2 mM L-glutamine, and filter sterilize through a 0.22 μm filter membrane.

[0044] Sperm digestion solution: Use L-15 culture medium as the base, add 2 mg / mL collagenase IV, 0.1% DNase I and 5% FBS, filter and sterilize with a 0.22 μm filter membrane, and add 0.5% trypsin-EDTA to a final concentration of 0.25% to continue digestion.

[0045] Percoll gradient solution: 100% Percoll was prepared by 10xPBS:Percoll=1:9, and diluted with 1xPBS to form 10% Percoll solution, 30% Percoll solution and 40% Percoll solution.

[0046] 2. Isolation of Spermatogonial Stem Cells

[0047] 1) The testes of 6-month-old 500-600 g Lateolabrax japonicus (gonadal index: 0.042 ± 0.013%) were removed. Excess gonadal fat and blood were removed using scissors and sharp forceps in pre-chilled PBS. The cleaned testes were disinfected by soaking in disinfectant (75% ethanol) for 10 seconds and washed five times with pre-chilled PBS containing 1% penicillin-streptomycin (double antibody).

[0048] 2) After washing, transfer the gonads into pre-cooled testis washing solution and cut the testis into 1mm pieces with scissors. 3 Wash the tissue blocks with about 3 volumes of spermatozoa washing solution, centrifuge at 1500 rpm at 4°C for 5 minutes, and repeat twice.

[0049] 3) Add spermatid digestion solution to the precipitate obtained after washing and centrifugation, and digest for 1 hour in a 37°C shaking table (80 rpm). To prevent cell agglomeration, continue to add trypsin-EDTA to a final concentration of 0.25% to the original spermatid digestion solution after digestion, continue digestion in the shaking table for 1.5 hours, and finally add L-15 culture medium containing 5% FBS to terminate digestion. After completion, the cells are respectively passed through 200 mesh and 70 mesh sieves. In the present invention, it is also possible to blow with a pipette once every 15 minutes during digestion in a water bath, which can further reduce the cell agglomeration rate during digestion, make the cell digestion in the tissue block uniform, and reduce the contact of the cell digestion solution with air, thereby reducing pollution. During the operation, step-by-step digestion is adopted to ensure the structural and functional integrity of spermatogonial stem cells and reduce cell agglomerations in the cell suspension while improving cell survival rate.

[0050] 4) After harvesting the cells, centrifuge at 1500 rpm for 5 minutes at room temperature, then resuspend and wash twice with L-15 medium. After centrifugation, resuspend the cells in 2 mL of L-15 medium to obtain a single-cell suspension of testes. Place on ice until ready to use.

[0051] 5) The testicular single cell suspension was placed in a Percoll gradient solution and collected after low-speed centrifugation to obtain Lateolabrax spermatogonial stem cells.

[0052] 3.Percoll Gradient Purification of Spermatogonial Stem Cells

[0053] 1) Prepare Percoll stock solution with 10xPBS, with a ratio of 10xPBS:Percoll = 1:9 to make 100% Percoll. Dilute the desired gradient Percoll with 1xPBS.

[0054] 2) Prepare 10%, 30%, and 40% Percoll solutions. Add a small amount of trypan blue solution (final concentration 0.01%) to the 10% and 40% Percoll solutions as an indicator. This allows for clear differentiation of the different Percoll interfaces, pinpointing the interface where the target cells are located, and allowing for the collection of spermatogonia of higher purity.

[0055] 3) Layer different Percoll solutions in descending order of density, and add 2 mL of 40%, 30%, and 10% Percoll solutions to a 15 mL centrifuge tube to prepare the Percoll gradient centrifugation working solution.

[0056] 4) Carefully add 2 mL of the prepared testis single-cell suspension to the top layer of 6 mL of Percoll gradient centrifugation working solution. Centrifuge at 2000 rpm (acceleration 2, deceleration 4) in a swing-out centrifuge (3-18KS, Sigma) at room temperature for 30 minutes. Remove the suspension slowly and steadily after centrifugation. Accelerate and decelerate smoothly throughout the entire process to avoid intercellular diffusion and blurring.

[0057] 5) Slowly aspirate and collect the cells from the different Percoll layers, dilute the collected cells with 10 volumes of PBS solution, centrifuge at 1500 rpm for 5 minutes at room temperature, discard the supernatant, and resuspend the spermatogonial stem cells in PBS.

[0058] 4. Spermatogonial stem cell transplantation

[0059] 1) The spermatogonial stem cells collected at the junction of 10% and 30% Percoll solutions were precipitated to obtain a cell concentration of 10 7 Spermatogonial stem cells at a concentration of 5 cells / mL were washed twice with PBS, centrifuged at 1500 rpm at room temperature for 5 minutes, and stained with PKH-26 at a final concentration of 0.5 μM. After incubation at room temperature for 15 minutes, the cells were centrifuged and washed twice with PBS. Finally, the cells were resuspended in L-15 medium and placed on ice for cell transplantation experiments.

[0060] 2) Pre-transplantation preparation. Prepare a microinjection glass needle with a tip diameter of 40-50 μm. Connect the microinjection system to a dissecting microscope. Use a pipette tip to transfer the prepared cell suspension into the capillary needle, which is then mounted on the microinjector. Prior to injection, 5-day-old post-embryonic recipient fish (medaka and zebrafish) were anesthetized with 0.001% MS-222 in seawater and freshwater, respectively, and then transferred to a 2% agarose-coated culture dish for microinjection. A control group of juvenile fish was not transplanted and was maintained under the same conditions.

[0061] 3) Under a dissecting microscope, microinject the cells into the abdominal cavity of recipient larvae near the genital ridge using a microinjector, injecting approximately 100 spermatogonial stem cells per larvae. Once completed, return the larvae to the experimental tank for continued culture.

[0062] 4) 60 days after transplantation, observation under a Leica DMi8 fluorescence inverted microscope (Leica) revealed that the spermatogonial stem cells of the donor sea bass had successfully integrated into the recipient gonads, demonstrating that the spermatogonial stem cells possessed the characteristics of germline stem cells.

[0063] 5. Results

[0064] 1) Analysis of the testis structure of Lateolabrax japonicus:

[0065] Six-month-old Lateolabrax japonicus were dissected and the testes removed. The testes were fixed overnight at 4°C in in situ hybridization fixative. The fixed testicular tissue was sent to Wuhan Sevier Biotechnology Co., Ltd. for hematoxylin and eosin (H&E) staining and in situ hybridization. The probes used in the in situ hybridization were also synthesized by Sevier.

[0066] Macroscopic structure of testis of 6-month-old Lateolabrax japonicus ( Figure 1 A); H&E staining of testis of 6-month-old Lateolabrax japonicus ( Figure 1 B) Some of the cells with lighter nuclear staining have typical spermatogonia: clear nuclear-cytoplasmic boundaries, large and round nuclei, and 1-2 nucleoli. Figure 1 Partial enlargement of B ( Figure 1 C). At the same time, the expression of vasa and nanos3, the specific markers of germ cells in the testes of 6-month-old japonicus, showed that there were germ cells with strong vasa and nanos3 positive expression in the testes of 6-month-old japonicus, proving that these germ cells were spermatogonia ( Figure 1 D). Therefore, the testes of 6-month-old Lateolabrax japonicus can be used as a good target for spermatogonia isolation.

[0067] 2) Preparation of Lateolabrax japonicus testis single cell suspension:

[0068] Prepare a cell suspension according to the method from steps 1) to 3) in the above-mentioned step 2 of isolating spermatogonial stem cells.

[0069] The collected testis single cell suspension was examined and observed under a microscope (Leica fluorescence inverted microscope DMi8), photographed, and the expression of vasa and nanos3 in the cell suspension was detected by in situ hybridization. The cell viability was calculated using an automatic cell analyzer (Countstar Mira BF, Ruiyu Biotechnology, Shanghai) after trypan blue staining.

[0070] 3) Isolation and identification of spermatogonia from Lateolabrax japonicus

[0071] Semi-quantitative RT-PCR was used to detect the expression of germ cell genes in the isolated cells, and live / dead double staining was used to observe the cell activity.

[0072] Semi-quantitative RT-PCR: RNA was extracted from testicular single-cell suspensions, spermatogonial stem cells at the interface of 10% and 30% Percoll solutions, and cell pellets at the bottom of the testes according to the aforementioned method. First-strand cDNA was synthesized using the FastKing One-Step Genomic Removal Kit (KR118, Tiangen) according to the manufacturer's instructions. Gene-specific primers (Table 1) were designed to amplify vasa (649 bp), nanos3 (329 bp), and dnd (377 bp), respectively. β-actin (114 bp) was used as an internal control. The concentrations of each template were adjusted, and RT-PCR conditions were optimized. PCR was performed using the 2xRapid Taq Master Mix Kit (P222, Norvegian). The optimized PCR amplification protocol in cells was as follows: 95°C pre-denaturation for 3 minutes; 95°C denaturation for 15 seconds, 60°C annealing for 15 seconds, and 72°C extension for 15 seconds for vasa, nanos3, and dnd genes (30 cycles); and β-actin (25 cycles); and 72°C extension for 5 minutes. The reaction products were subjected to 1.5% agarose gel electrophoresis, stained with ethidium bromide (EB), and photographed using a Bio-Rad gel imaging system.

[0073] Table 1 Semi-quantitative primers for vasalsenos3 / dad1β-actin

[0074]

[0075] Calcein-AM / PI live / dead cell double staining method: Collect the spermatogonial stem cell pellet at the junction of the 10% and 30% Percoll solutions. Resuspend the cells in 1× Assay Buffer according to the kit instructions. Add 2 μL of Calcein-AM per 1 mL of cells, mix thoroughly by pipetting, and incubate at 37°C in the dark for 20 minutes. Add 3 μL of PI stock solution to the stained cells and continue staining at room temperature in the dark for 5 minutes. After incubation, centrifuge the cells at 1500 rpm for 5 minutes at room temperature to remove the staining solution. Wash the cells with PBS, centrifuge, resuspend the cells, and examine both live cells (yellow-green) and dead cells (red) under a fluorescence microscope using a 490 nm excitation filter.

[0076] Percoll density gradient (10%, 30%, 40%) centrifugation produces cells in different bands ( Figure 2 A); Microscopic examination results showed that the single cell suspension of the testis of 6-month-old Lateolabrax japonicus included cells of various morphologies ( Figure 2 B); The expression of vasa and nanos3 was detected by in situ hybridization, proving the presence of spermatogonia in the obtained single cell suspension ( Figure 2C); Cell counting instrument analysis showed that the cell viability of the cell suspension was 99.63% (green represents live cells, red represents dead cells, and the white circle represents cell clusters) Figure 2 H).

[0077] The cells in the cell layer at the junction of different concentrations of Percoll solution were collected and detected. The results showed that there were a large number of spermatogonia at the junction of 10% and 30% Percoll solution, with a diameter of about 8-15μm ( Figure 2 D) In ​​situ hybridization was used to detect the expression of Vasa and Nanos3, proving that the junction of 10% and 30% Percoll solutions was the target cell and that a large number of spermatogonial stem cells were present ( Figure 2 E). In the bottom layer of 40% Percoll solution, there are blood cells and cell debris ( Figure 2 F), In situ hybridization detected the lack of expression of Vasa and Nanos3 ( Figure 2 G); RT-PCR detection of the expression of germ cell genes vasa, nanos3, and dnd in cells in different bands revealed that the three germ cell genes were expressed to varying degrees in the uncentrifuged cell suspension and the two cell bands, but were strongly expressed in spermatogonial stem cells ( Figure 2 I). Spermatogonia at the junction of 10% and 30% Percoll solutions were observed and photographed under a fluorescence microscope. Live / dead cell double staining results showed that the cell viability was 98.37% ( Figure 2 J).

[0078] 4) Spermatogonial stem cell transplantation

[0079] Figure 3 Spermatogonial stem cells were stained with PKH-26 and DAPI ( Figure 3 A). PKH-26-labeled Lateolabrax donor cells were observed in recipients (medaka, zebrafish) 60 days after transplantation, and these cells almost all migrated to the primitive genital ridge. Figure 3 CD), zebrafish ( Figure 3 F), while the control group showed no fluorescence at the same position, medaka ( Figure 3 B), zebrafish ( Figure 3 E).

[0080] The above embodiment merely represents one embodiment of the present invention. While the description is relatively specific and detailed, it should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and all such modifications and improvements fall within the scope of protection of the present invention.

Claims

1. A method for isolating, purifying and transplanting spermatogonial stem cells from Lateolabrax japonicus, characterized in that: The spermatogonial stem cells of Lateolabrax japonicus are isolated and purified, marine fish larvae or freshwater fish larvae are used as recipient larvae, and then the spermatogonial stem cells of Lateolabrax japonicus are transplanted into the recipient larvae.

2. The method for isolating, purifying and transplanting spermatogonial stem cells of Lateolabrax japonicus according to claim 1, wherein: The Lateolabrax spermatogonial stem cells are transplanted into the abdominal cavity of the recipient fish larvae.

3. The method for isolating, purifying and transplanting spermatogonial stem cells of Lateolabrax japonicus according to claim 1, wherein: The marine fish larvae are medaka larvae, and the freshwater fish larvae are zebrafish larvae.

4. The method for isolating, purifying and transplanting spermatogonial stem cells of Lateolabrax japonicus according to claim 1, 2 or 3, wherein: The marine fish larvae or freshwater fish larvae are larvae that are 5 days old after emerging from the membrane.

5. The method for isolating, purifying and transplanting spermatogonial stem cells of Lateolabrax japonicus according to claim 1, wherein: The isolation and purification of Lateolabrax spermatogonial stem cells includes separation and Percoll gradient purification, wherein the testis tissue blocks are digested with the testis digestion fluid during the separation; In the Percoll gradient purification, the Percoll solutions are placed layer by layer in descending order of density, with the concentrations being 40%, 30% and 10% in sequence.

6. The method for isolating, purifying and transplanting spermatogonial stem cells of Lateolabrax japonicus according to claim 1, wherein: Percoll gradient solution: Prepare 100% Percoll by 10xPBS:Percoll=1:9, and dilute with 1xPBS to 10% Percoll solution, 30% Percoll solution and 40% Percoll solution.

7. The method for isolating, purifying and transplanting spermatogonial stem cells of Lateolabrax japonicus according to claim 5 or 6, characterized in that: During transplantation, spermatogonial stem cells were collected at the junction of 10% and 30% Percoll solutions, washed, stained with PKH-26 at a final concentration of 0.5 μM, incubated at room temperature, collected by centrifugation, washed with PBS, and finally resuspended in L-15 medium and placed on ice for use.

8. The method for isolating, purifying and transplanting spermatogonial stem cells of Lateolabrax japonicus according to claim 1, 2 or 3, characterized in that: The testis digestion solution is based on L-15 culture medium, supplemented with 2 mg / mL collagenase IV, 0.1% DNase I and 5% FBS, and sterilized by filtration with a 0.22 μm filter membrane. After digestion, 0.5% trypsin-EDTA is added to a final concentration of 0.25% to continue digestion.

9. The method for isolating, purifying and transplanting spermatogonial stem cells of Lateolabrax japonicus according to claim 1, 2 or 3, characterized in that: The testis was digested in a shaker at 37°C and 80 rpm for 1 hour. After digestion, trypsin-EDTA was added to a final concentration of 0.25%, and digestion was continued in the shaker for 1.5 hours. Finally, L-15 culture medium containing 5% FBS was added to terminate the digestion. After completion, the cells were sieved through 200-mesh and 70-mesh sieves respectively.

10. The method for isolating, purifying and transplanting spermatogonial stem cells of Lateolabrax japonicus according to claim 1, wherein: The testis was from a 6-month-old Lateolabrax japonicus.

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