Separation and transplantation method of zebra fish spermatogonial stem cells

Abstract

The invention discloses a zebra fish spermatogonial stem cell separation and transplantation method, and belongs to the technical field of fish germline stem cell separation. The method comprises the following steps: washing complete sexual glands of zebra fish testis by adopting penicillin / streptomycin double-antibody-containing PBS (Phosphate Buffer Solution), physically shearing, digesting and dissociating, and filtering by a screen, so as to obtain zebra fish testis cell suspension; performing density gradient centrifugation to obtain zebra fish spermatogonial stem cells; dyeing, marking, centrifuging, washing, re-suspending, and carrying out micro-injection transplantation to enter the back of the abdominal cavity of the acceptor zebrafish juvenile fish. According to different physical properties of different cells, spermatogonial stem cells are separated, purified and transplanted from zebra fish testis cell suspension by adopting a density gradient centrifugation method, and migration and proliferation processes of donor germ cells can be observed in receptor fish after fluorescence labeling. And a new technical path is provided for fish germplasm resource preservation, endangered fish protection, economic cultured fish yield increase and new variety cultivation.

Description

Technical Field

[0001] This invention belongs to the field of fish reproductive stem cell isolation technology, specifically relating to a method for isolating and transplanting zebrafish spermatogonial stem cells. Background Technology

[0002] Zebrafish are the most widely studied fish species, named for their distinctive deep blue longitudinal stripes along their sides. Their importance lies primarily in their high reproductive rate, transparent embryos, rapid development facilitating direct observation of organogenesis, and high genome homology with humans, making them an excellent model for studying human disease mechanisms. They have yielded fruitful results in developmental biology, genetics, and toxicology. In fish research, they are an ideal tool for exploring fish physiology, immunity, behavior, and gene function, providing crucial theoretical foundations and experimental evidence for aquaculture breed improvement, disease control, and environmental protection, greatly advancing the development of aquaculture science. Currently, zebrafish have become a vital model organism for life science research, especially in fish studies.

[0003] Reproductive stem cells are a special type of stem cell capable of self-renewal and mitosis, and able to develop into male and female gametes. Fish reproductive stem cell transplantation technology utilizes these characteristics to transplant reproductive stem cells from a donor fish into a recipient fish, enabling the recipient fish to produce gametes derived from the donor fish. The emergence and development of surrogate reproduction technology in fish has significant implications for the selection of superior traits and increased production in economically important fish species, and is equally significant for the population expansion and conservation of endangered fish species. Therefore, research on the isolation, enrichment, and transplantation of reproductive stem cells from the zebrafish model organism is of typical representative significance for the preservation of fish germplasm resources, the protection of endangered fish species, and the increase in production and yield of economically farmed fish. Summary of the Invention

[0004] Based on this, the purpose of this invention is to provide a method for isolating and transplanting zebrafish spermatogonial stem cells. Using zebrafish as a model organism and taking advantage of the different physical properties between different cells, the method employs density gradient centrifugation to isolate, purify, and transplant spermatogonial stem cells from the dissociated zebrafish testis cell suspension. This method is used for the preservation of fish germplasm resources, the protection of endangered fish species, the increase in production and quantity of economically farmed fish species, and the breeding of new varieties.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides a method for isolating and transplanting zebrafish spermatogonial stem cells, comprising the following steps:

[0007] (1) The intact testes of immature or mature zebrafish were washed with PBS containing penicillin / streptomycin, physically sheared, digested and dissociated and filtered through a sieve to obtain a zebrafish testicular cell suspension.

[0008] (2) The zebrafish testis cell suspension described in step (1) was subjected to density gradient centrifugation to obtain zebrafish spermatogonial stem cells;

[0009] (3) The zebrafish spermatogonial stem cells described in step (2) are stained and labeled, centrifuged and washed, and resuspended. Then, they are transplanted into the posterior dorsal side of the abdominal cavity of the recipient zebrafish juveniles by microinjection.

[0010] Preferably, in step (1), the operation process includes: under sterile conditions, taking 8 testes of active adult zebrafish, washing them twice in PBS with 3% penicillin / streptomycin antibiotics, cutting the washed zebrafish testes into small pieces, the cutting process being carried out on ice, and keeping the zebrafish testis tissue moist throughout the process; transferring the cut zebrafish testis tissue to 1 mL of dissociation solution, and dissociating it in a shaker at 28℃, 130 rpm / min and under sterile conditions for 2.5 h; adding 100 μL of fetal bovine serum (FBS) stock solution to the dissociated system to terminate the dissociation; and removing the undissociated tissue clumps from the testis tissue through 100 μm and 40 μm cell filters respectively to obtain a zebrafish testis cell suspension.

[0011] More preferably, in step (1), the dissociation solution comprises: L-15 medium containing 0.25% trypsin, 4 mg / mL collagenase H, 5% FBS and 0.05% DNase I.

[0012] Preferably, in step (2), the operation process includes: adding the zebrafish testis cell suspension to the density gradient separation system for density gradient centrifugation at 800×g for 30min; wherein, the preparation process of the density gradient separation system includes: first diluting the percoll stock solution to the physiological environment with 10×PBS, then dissolving the diluted percoll solution with PBS in different proportions, shaking and centrifuging, mixing thoroughly to obtain different density gradient levels, and preparing 1mL of separation solution for each level; aspirating the separation solutions of different density gradient levels layer by layer from the bottom of the centrifuged system, retaining 35% of the level separation solution, transferring the 35% level separation solution into a 15mL centrifuge tube for mixing, adding 6 times the volume of PBS for thorough mixing, centrifuging at 300×g for 15min, discarding the supernatant, and the spermatogonial stem cells aggregated at the bottom of the tube to obtain zebrafish spermatogonial stem cells.

[0013] More preferably, in step (2), the density gradient layers include density gradient layers of 20%, 25%, 30%, 35%, 40%, 50%, and 60%.

[0014] Preferably, in step (3), the operation process includes: adding 1 mL of PBS to the zebrafish spermatogonial stem cells to resuspend the cells, then adding 2 μL of CM-Dil live cell membrane staining solution to stain and label the spermatogonial stem cells; adding 5 mL of PBS to the stained cell suspension and mixing thoroughly, centrifuging at 300×g for 15 min, repeating the above process until the residual CM-Dil live cell membrane staining solution is washed away, adding L-15 culture medium containing 5% FBS and 0.01% DNaseI to obtain a spermatogonial stem cell suspension, which is then transplanted into the peritoneal cavity of the recipient zebrafish juveniles via microinjection.

[0015] As a more preferred embodiment, in step (3), the staining and labeling process of the spermatogonial stem cells includes: staining at 28°C for 15 minutes, then staining at 4°C for 10 minutes, and the staining process is conducted in the dark throughout.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] This invention proposes a method for isolating and transplanting spermatogonial stem cells from zebrafish, a model organism. Using zebrafish as a representative fish, and taking advantage of the different physical properties between different cells, spermatogonial stem cells are isolated, purified, and transplanted from dissociated zebrafish testis cell suspension using density gradient centrifugation. After fluorescent labeling, the migration and proliferation process of donor germ cells can be observed in the recipient fish. This provides a new technical approach for the preservation of fish germplasm resources, the protection of endangered fish species, the increase in production and quantity of economically farmed fish, and the breeding of new varieties. Attached Figure Description

[0018] Figure 1 The images show the morphological results of spermatogonial stem cells at different gradients in zebrafish testis cell suspensions in the examples; the red arrows represent spermatogonial stem cells with a cell diameter of 5-10 μm, and the green arrows represent non-spermatogonial stem cells.

[0019] Figure 2 The PCR detection results of the nanos2 gene, a marker of spermatogonial stem cells, in zebrafish testis cell suspensions at different density gradients are shown in the examples.

[0020] Figure 3 The immunohistochemical results of zebrafish testis cell suspensions after centrifugation at different density gradients (35% and 60% density gradient cell smears) are shown in the example (merging of VASA antibody and DAPI nuclear staining, which are markers of reproductive stem cells).

[0021] Figure 4 The following is a fluorescence observation of the whole zebrafish 14 days after the zebrafish reproductive stem cells were injected into the recipient fish in the example (left, middle and right are bright field, fluorescent field and merge, respectively). Detailed Implementation

[0022] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that other embodiments obtained by those skilled in the art without departing from the concept of the present invention are all within the protection scope of the present invention.

[0023] Example 1

[0024] 1) Select 8 well-developed male zebrafish, kill them in an ice bath, soak the whole fish in 70% alcohol for 1 minute, then remove the zebrafish testes with sterile instruments and wash them twice in PBS containing 3% penicillin / streptomycin.

[0025] 2) After removing the zebrafish testes, cut them into small pieces and soak them in 1 mL of tissue dissociation solution for dissociation. Dissociation conditions: 28℃, 2.5h, 130rpm / min; dissociation solution: L-15 medium containing 0.25% trypsin, 4mg / mL collagenase H, 5% FBS and 0.05% DNase I.

[0026] 3) After the cell suspension has been dissociated, it is first passed through cell sieves of different densities (100μm, 40μm) to remove undissociated tissue fragments, and then 100μL of FBS is added to terminate the dissociation.

[0027] 4) The processed cell suspension was added to the density gradient separation system for spermatogonial stem cell separation. The preparation process of the density gradient separation system included: first, diluting Percoll to the working solution at a ratio of 1:9 with 10×PBS, then mixing it with PBS and DNase I in proportion to prepare different density gradient levels (20%, 25%, 30%, 35%, 40%, 50%, 60%), mixing each level, and then adding the dissociation product to the top layer. The density gradient centrifugation process was as follows: the mixed density gradient centrifugation system was centrifuged at room temperature and 800×g for 30 min.

[0028] 5) Carefully separate each cell suspension layer from bottom to top using a pipette. After mixing the cell suspensions of 35% of the layers, add 6 mL of PBS and wash twice (centrifuge at 300×g for 15 min) to remove residual liquid from the density gradient medium.

[0029] 6) The washed spermatogonial stem cells were stained with live cell fluorescent dye. 2 μL CCM-Dil was added to 1 mL PBS cell suspension and incubated at 28℃ for 15 min, then at 4℃ for 10 min, in the dark throughout the process.

[0030] 7) Add 5 mL of PBS to the stained cell suspension and wash twice (centrifuge at 300×g for 15 min) to remove residual live cell fluorescent membrane dye.

[0031] 8) Add a small amount of L-15 medium containing 5% FBS and 0.01% DNaseI to the washed cells, and place the resulting cell suspension on ice to await microinjection.

[0032] 9) Microinjection was performed into the reproductive ridge of the gonadal primordium of the recipient fish, and the injection was continuously monitored using a fluorescence microscope after injection.

[0033] like Figures 1-4 As shown, Figure 1 In the morphological analysis of spermatogonial stem cells at different gradients in zebrafish testis cell suspensions in the examples, red arrows represent spermatogonial stem cells with a cell diameter of 5–10 μm, and green arrows represent non-spermatogonial stem cells. Figure 1 ); PCR detection results of the nanos2 gene, a marker of spermatogonial stem cells, in zebrafish testis cell suspensions at different density gradients are as follows: Figure 2 As shown; Immunohistochemical results of zebrafish testis cell suspensions after centrifugation at different density gradients (merging of VASA antibody and DAPI nuclear staining, both germline stem cell markers) at 35% and 60% density gradients. Figure 3 The image shows the fluorescence observation of the whole zebrafish 14 days after the zebrafish germline stem cell injection recipient fish. From left to right, the images are bright field, fluorescent field, and merge field. Figure 4 As shown.

[0034] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for isolating and transplanting zebrafish spermatogonial stem cells, characterized in that, Includes the following steps: (1) The intact testes of immature or mature zebrafish were washed with PBS containing penicillin / streptomycin, physically sheared, digested and dissociated and filtered through a sieve to obtain a zebrafish testicular cell suspension. (2) The zebrafish testis cell suspension was subjected to density gradient centrifugation to obtain zebrafish spermatogonial stem cells; (3) The zebrafish spermatogonial stem cells were stained, centrifuged, washed, and resuspended. They were then transplanted into the posterior dorsal side of the abdominal cavity of the recipient zebrafish juveniles via microinjection.

2. The method for isolating and transplanting zebrafish spermatogonial stem cells according to claim 1, characterized in that, In step (1), the operation process includes: under sterile conditions, taking the testes of live adult zebrafish, washing them in PBS with 3% penicillin / streptomycin antibiotics, cutting the washed zebrafish testes into small pieces, and performing the cutting process on ice, keeping the zebrafish testes moist throughout the process; transferring the cut zebrafish testes into 1 mL of dissociation solution, dissociating them for 2.5 h at 28 ℃, 130 rpm / min and under sterile conditions, and then adding 100 μL of FBS stock solution to the dissociated system to terminate the dissociation, and passing the cells through 100 μm and 40 μm cell filters to obtain zebrafish testes cell suspension.

3. The method for isolating and transplanting zebrafish spermatogonial stem cells according to claim 2, characterized in that, In step (1), the dissociation solution comprises L-15 medium containing 0.25% trypsin, 4 mg / mL collagenase H, 5% FBS and 0.05% DNase I.

4. The method for isolating and transplanting zebrafish spermatogonial stem cells according to claim 1, characterized in that, In step (2), the operation process includes: adding the zebrafish testis cell suspension to the density gradient separation system for density gradient centrifugation at 800×g for 30min; wherein, the preparation process of the density gradient separation system includes: first diluting the Percoll stock solution to the physiological environment with 10×PBS, dissolving the diluted Percoll solution in PBS at different ratios, shaking and centrifuging to mix thoroughly to obtain different density gradient levels, and preparing 1mL of separation solution for each level; aspirating the separation solutions of different density gradient levels layer by layer from the bottom of the centrifuged system, retaining 35% of the level separation solution, transferring the 35% level separation solution into a 15mL centrifuge tube for mixing, adding 6 times the volume of PBS for thorough mixing, centrifuging at 300×g for 15min, discarding the supernatant, and the spermatogonial stem cells aggregated at the bottom of the tube to obtain high-purity zebrafish spermatogonial stem cells.

5. The method for isolating and transplanting zebrafish spermatogonial stem cells according to claim 4, characterized in that, In step (2), the density gradient levels include density gradient levels of 20%, 25%, 30%, 35%, 40%, 50%, and 60%.

6. The method for isolating and transplanting zebrafish spermatogonial stem cells according to claim 1, characterized in that, In step (3), the operation process includes: adding 1 mL of PBS to the zebrafish spermatogonial stem cells to resuspend the cells, then adding 2 μL of CM-Dil live cell membrane staining solution to stain and label the spermatogonial stem cells; adding 5 mL of PBS to the stained cell suspension and mixing thoroughly, centrifuging at 300×g for 15 min, repeating the above process until the residual CM-Dil live cell membrane staining solution is washed away, adding L-15 culture medium containing 5% FBS and 0.01% DNase I to obtain a spermatogonial stem cell suspension, which is then transplanted into the peritoneal cavity of the recipient zebrafish juveniles via microinjection.

7. The method for isolating and transplanting zebrafish spermatogonial stem cells according to claim 6, characterized in that, In step (3), the staining and labeling process of the spermatogonial stem cells includes: staining at 28℃ for 15 minutes and then staining at 4℃ for 10 minutes, with the staining process conducted in the dark.

Images