Preparation method of spermatogonial stem cell transplantation receptor and application of spermatogonial stem cell transplantation receptor

Abstract

The invention provides a preparation method and application of a spermatogonial stem cell transplantation receptor, and the preparation method of the spermatogonial stem cell transplantation receptor comprises the following steps: S1, obtaining a zebra fish embryo containing Cycla2 homozygous mutant gene; s2, removing endogenous germ cells of the embryo; s3, culturing the embryos without the endogenous germ cells into seedlings to obtain the spermatogonial stem cell transplantation receptor. According to the method, on the basis of original SSCs enrichment, living body amplification is conducted on the SSCs through a cyp11a2 homozygous mutant, enrichment is conducted after the SSCs reach a very high concentration, the size of a testis at the moment is remarkably larger than that of a wild type, the proportion of the SSCs is very high, and the high-concentration SSCs can be obtained without percoll centrifugation, so that operation steps are reduced, the cost is reduced, and the method is suitable for large-scale popularization and application. And the cell characteristics and functions of the SSCs can be reserved to a greater extent.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a preparation method of a spermatogonial stem cell transplantation receptor and application of the receptor. Background Art

[0002] Spermatogonial stem cells (SSCs) are the basis of spermatogenesis. They originate from primordial germ cells (PGCs) in the embryonic period and contain the genetic information of the diploid genome. They are a type of germline stem cells that exist in the male testis and can both self-renew and maintain their own stable number and can differentiate to produce mature gametes. In the testis of adult male fish, although SSCs only account for 0.02-0.03%, they play a vital role in maintaining male fertility. In germline stem cell transplantation, spermatogonial stem cell transplantation (SSCT) occupies an important position. However, due to the low proportion of SSCs in adult testis, the efficient separation and enrichment of SSCs has always been an important factor restricting the success of SSCT. Summary of the invention

[0003] In view of this, the present invention provides a method for preparing a spermatogonial stem cell transplantation receptor and application of the receptor, which improves the enrichment efficiency of spermatogonial stem cells and further improves the success rate of spermatogonial stem cell transplantation.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a method for preparing a spermatogonial stem cell transplantation recipient, comprising the following steps: S1. Obtain zebrafish embryos containing the homozygous mutant gene of Cyplla2; S2, eliminating the endogenous germ cells of the embryo itself; S3. The embryos with endogenous germ cells removed are cultured into seedlings to obtain spermatogonial stem cell transplantation recipients.

[0005] Preferably, in step S2, the endogenous germ cells of the embryo itself are removed. The method comprises: injecting morpholino antisense oligonucleotide into 1-cell stage embryos; and / or, In step S3, the embryos without endogenous germ cells are cultured into seedlings. The temperature is 28-29°C; and / or, In step S3, the embryos from which endogenous reproductive cells have been removed are cultured into seedlings, and the culture medium used for the culture is 0.3×Danieau's buffer; and / or, In step S3, the embryos without endogenous germ cells are cultured into seedlings. The culture period was 4 days.

[0006] In a second aspect, the present invention provides a use of a spermatogonial stem cell transplantation receptor prepared by the preparation method in amplifying Tg (vasa:gfp)-labeled spermatogonial stem cells.

[0007] Preferably, the spermatogonial stem cell receptor amplifies spermatogonial stem cells labeled with Tg (vasa: gfp) The following steps are involved: SA, obtain spermatogonial stem cells of zebrafish marked with Tg(vasa:gfp), called donor; SB. Transplant the donor into the spermatogonial stem cell recipient for expansion.

[0008] Preferably, in step SA, the method for obtaining the donor is: taking an adult zebrafish The testis is cut into pieces, separated, digested, centrifuged and purified to obtain the donor.

[0009] Preferably, the specific steps of cutting and separating the testis of an adult zebrafish and then digesting it are as follows: The chopped testes were digested in L-15 medium for 2-3 hours. During the digestion process, the cells on the L-15 medium were aspirated every 15 minutes using a 1 mL low-adsorption pipette tip to fully disperse the cells.

[0010] Preferably, in step SB, the donor is transplanted into the spermatogonial stem cell recipient for During the amplification, the specific steps of transplantation are: firstly anesthetizing the recipient and then transplanting the donor into the recipient; and / or, In step SB, the donor is transplanted into the spermatogonial stem cell recipient for expansion. wherein the donor is transplanted into the recipient at a position between the swim bladder and the intestine of the recipient zebrafish fry close to the original gonad; and / or, In step SB, after the donor is transplanted into the recipient, the culture time is 4 months; and / or, In step SB, the method for isolating the spermatogonial stem cells amplified in the recipient is as follows: the testis of the recipient is cut into pieces, cultured and digested, filtered and washed, and the spermatogonial stem cells of zebrafish marked with Tg (vasa: gfp) are obtained.

[0011] In a third aspect, the present invention further provides a method for detecting spermatogonial stem cells by immunofluorescence, comprising the following steps: Sa, culturing the recipient of the donor amplified in claim 4, removing the gonads, and preparing frozen sections and testicular cell slides; Sb, using a fixative to fix the frozen sections and the testicular cell slides respectively, and then permeabilizing them; Sc, using a blocking solution to block the permeabilized frozen sections and testicular cell slides respectively; Sd, adding a primary antibody that specifically recognizes the target antigen to the frozen sections and testicular cell slides; Se, adding a fluorescently labeled secondary antibody to the frozen sections and testicular cell slides on the basis of step Sd; Sf. On the basis of step Se, using nuclear dye to stain the cells in the frozen sections and testicular cell slides to obtain spermatogonial stem cells with immunofluorescence colors; Sg. The immunofluorescence-stained spermatogonial stem cells were observed using an optical microscope.

[0012] Preferably, in step Sb, the specific steps of fixing are: fixing the frozen sections and testicular cell slides with 4% PFA for 15-30 min respectively; and / or, In step Sb, the permeabilization step is: rinse with 1×PBS for 5 min×3 times, and the sample Permeabilization was performed in 1× PBS containing 0.5% Triton X-100 for 15 min at room temperature.

[0013] Preferably, in step Sd, the primary antibody comprises Vasa, Sycp3, Nanos2; and / or, In step Se, the secondary antibody included rabbit IgG, Alexa Fluor 488.

[0014] Compared with the prior art, the present invention has the following beneficial effects: Based on the original SSCs enrichment, the present invention first utilizes cyp11a2 The homozygous mutants are used to amplify SSCs in vivo, so that SSCs reach a very high concentration and then enriched. Since the volume of the testis is significantly higher than that of the wild type at this time, and the proportion of SSCs is very high, high-concentration SSCs can be obtained without Percoll centrifugation. This reduces the number of operation steps and can retain the cellular characteristics and functions of SSCs to a greater extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The fluorescently labeled donor SSCs provided in Example 1 of the present invention are cyp11a2- / - Colonization map in the receptor; Figure 2 The fluorescently labeled donor SSCs provided in Example 1 of the present invention are cyp11a2 - / - Developmental maps in receptors; Figure 3 A comparison chart of the testis weight after amplification provided in Example 2 of the present invention; Figure 4 A statistical analysis diagram of reproductive cell types of the testis after amplification provided in Example 2 of the present invention; Figure 5 This is a diagram showing the detection of germ cell types in the testicular cells enriched after amplification provided in Example 2 of the present invention; Figure 6 A comparison chart of cell concentrations after amplification provided in Example 3 of the present invention; Figure 7 A comparison chart of the success rate of transplantation of amplified testicular cells provided in Example 4 of the present invention; Figure 8 This is the gfp fluorescence of the F1 embryo after transplantation provided in Example 4 of the present invention. DETAILED DESCRIPTION

[0016] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0017] Spermatogonial stem cells (SSCs) are the basis of spermatogenesis. They originate from primordial germ cells (PGCs) in the embryonic period and contain the genetic information of the diploid genome. They are a type of germline stem cells that exist in the male testis and can both self-renew and maintain their own stable number and can differentiate to produce mature gametes. In the testis of adult male fish, although SSCs only account for 0.02-0.03%, they play a vital role in maintaining male fertility. In germline stem cell transplantation, spermatogonial stem cell transplantation (SSCT) occupies an important position. However, due to the low proportion of SSCs in adult testis, the efficient separation and enrichment of SSCs has always been an important factor restricting the success of SSCT.

[0018] In zebrafish, cyp11a2It is the gene encoding cytochrome cholesterol side chain cleavage enzyme (P450ssc), which converts cholesterol (Cholesterol) into pregnenolone (Pregnenolone, P5), which is the first and rate-limiting step in the synthesis of steroid hormones (Steroidhormone). Our previous studies have shown that cyp11a2 - / - In the testis, the function of testicular somatic cells changes, which causes SSCs to proliferate in large quantities, but they cannot enter the differentiation process and eventually develop into an enlarged testis rich in high-abundance SSCs. cyp11a2 - / - A model is created to infinitely expand SSCs in vivo, and then the SSCs are enriched. This greatly improves the enrichment efficiency of SSCs and thus increases the success rate of SSCT.

[0019] The verification method of the present invention includes the following steps: A. Preparation of donor cells: Obtain zebrafish germ cell-specifically labeled SSCs. Using the germ cell-specifically labeled Tg (vasa: gfp) transgenic strain (provided by the National Zebrafish Resource Center of the National Germplasm Resource Bank), we obtained green fluorescently labeled SSCs by Percoll density gradient centrifugation, which facilitated us to observe their colonization and development in the recipient in subsequent transplantation experiments.

[0020] B. cyp11a2 Preparation of mutant transplant recipients: Obtaining endogenous germ cell deletion cyp11a2 Mutant receptor (this strain has been submitted to the National Germplasm Resource Bank, National Zebrafish Resource Center, with CZ number 1217). We found in previous studies cyp11a2 - / - Due to the dysfunction of testicular somatic cells, SSCs continued to proliferate without differentiation and eventually developed into an enlarged testis, indicating that testicular somatic cells provide a good microenvironment for SSCs proliferation. cyp11a2 - / - In vivo expansion as a transplant recipient. cyp11a2 + / - and cyp11a2 - / - The mated embryos were injected with 100 μM dead end morpholino antisense oligonucleotide (dnd MO) at the 1-cell stage to completely eliminate the recipient's own endogenous germ cells, and cultured in a 28.5°C incubator with 0.3× Danieau's buffer until they developed to 4 dpf (daypost fertilization) in preparation for the next transplantation experiment.

[0021] C. In vivo unlimited expansion of SSCs: SSCs labeled with green fluorescence were transplanted into 4 dpf cyp11a2 In the mutant, on the 5th day after transplantation (5dpt), individuals with and without GFP fluorescence at the genital ridge were selected under a fluorescence microscope and cultured separately. Individuals with GFP fluorescence were considered positive fish, and individuals without GFP fluorescence were considered negative controls. They were cultured under appropriate conditions until the recipient was 4mpf (month post fertilization) to allow donor SSCs to proliferate in the recipient.

[0022] D. Screening of positive fish in the recipients after SSCs proliferation. When the recipients are cultured to 4 mpf, the genotype of the recipient fish is identified and the positive fish are screened. cyp11a2 - / - The fish with the genotype of cyp11a2 + / - Individuals with a small GFP fluorescence area were considered as the control group.

[0023] E. Immunofluorescence detection of the composition of gonadal germ cells after amplification: The gonads of the obtained 4mpf control group and positive fish were anesthetized and dissected out, and the composition of germ cells in the amplified gonads was detected using the germ cell marker Vasa and the meiosis marker Sycp3 antibody, respectively.

[0024] F. Efficient separation and enrichment of SSCs: The gonads of the 4 mpf control and positive fish were anesthetized and dissected out. The testes were dispersed into single cells using 1 mL L-15 medium (Sigma-Aldrich) containing 0.25% trypsin (Invitrogen), 0.26% U collagenase (Roche), 0.05% DNaseI (Roche) and 10% FBS (BioInd), and filtered and washed to prepare a testis single cell suspension.

[0025] G. Immunofluorescence detection of SSCs: The single cell suspension prepared above was subjected to immunofluorescence detection using the germ stem cell marker antibody Nanos2, and the proportion of SSCs was counted and compared.

[0026] H. Preparation of wild-type transplant recipients: Obtain wild-type recipients with endogenous germ cells removed. Collect wild-type embryos, inject 100 μM dead end morpholino antisense oligonucleotide (dnd MO) at the 1-cell stage to completely remove the recipient's own endogenous germ cells, and culture them in a 28.5°C incubator with 0.3× Danieau's buffer until they develop to 4 dpf (day post fertilization) in preparation for the next transplantation experiment.

[0027] I. Detect the cellular characteristics and functions of SSCs using spermatogonial stem cell transplantation (SSCT) technology: Transplant the single cell suspension prepared above into wild-type recipients respectively, select GFP-positive individuals for cultivation on the 5th day after transplantation, and detect whether the recipients can produce donor-derived gametes when they are sexually mature.

[0028] cyp11a2 Gene and mutant analysis Cytochrome cholesterol side-chain cleavage enzyme (P450ssc), encoded by Cyp11a1, converts cholesterol to pregnenolone (P5), which is the first and rate-limiting step in the synthesis of steroid hormones. cyp11a1 There are two homologous genes cyp11a1 and cyp11a2 ,in cyp11a1 Maternally expressed, cyp11a2 For zygotic expression, our previous studies have shown that cyp11a2 - / - In the testis, the function of testicular somatic cells changes, causing SSCs to proliferate in large quantities, but they cannot enter the differentiation process, and eventually develop into an enlarged testis rich in high-abundance SSCs. This suggests that we cyp11a2 - / - Testicular somatic cells may provide a good microenvironment for the proliferation of SSCs. Specific embodiments Example 1 In vivo expansion of SSCs (1) Preparation of in vivo expansion receptors for SSCs Preparation of recipient embryos: Collection cyp11a 2- / - and cyp11a 2+ / -For the embryos of the mating offspring, 100 μM dead end morpholino antisense oligonucleotide (dnd MO) was injected into the 1-cell stage embryos to completely eliminate the recipient's own endogenous germ cells, and the embryos were cultured in 0.3× Danieau's buffer at 28.5°C in an incubator until 4 dpf in preparation for the next transplantation experiment.

[0030] (2) Isolation and enrichment of Tg(vasa:gfp) zebrafish spermatogonial stem cells.

[0031] 1) First, five adult zebrafish of Tg(vasa:gfp) were taken and their testes were removed after anesthesia. The testes were cut into pieces in a 2 mL low-adsorption EP tube and digested in the following formula: 2 mL L-15 medium (Sigma-Aldrich) containing 0.25% trypsin (Invitrogen), 0.26 U collagenase (Roche), 0.05% DNaseI (Roche) and 10% FBS (BioInd), digested at room temperature (RT) for 2-3 hours. During the digestion process, a 1 mL low-adsorption pipette tip was used to pipette several times every 15 minutes to fully disperse the cells.

[0032] 2) When no obvious tissue blocks are visible to the naked eye, the resulting testicular cell suspension is passed through 70μm and 40μm cell sieves in turn to remove large tissue fragments, and washed three times with PBS. Finally, the dispersed testicular cells are resuspended in 1 mL of L-15.

[0033] 3) Prepare discontinuous Percoll (GE Healthcare) gradients in a 12 mL transparent centrifuge tube, 20%, 25%, 30%, 35%, 40%, 50%, 60% Percoll / PBS from top to bottom, 1.5 mL for each gradient. The preparation should be as slow as possible so that clear boundaries can be seen between different gradients.

[0034] 4) Slowly add 1 mL of testicular cell suspension to the top layer of the prepared discontinuous Percoll gradient and centrifuge at 4°C and 800g for 30 minutes.

[0035] 5) Remove the purified cells from the gradient layer by layer from top to bottom, mix the spermatogonia obtained from the 30%-35% and 35%-40% Percoll gradient interfaces, transfer them to a 1.5mL EP tube, wash them 2-3 times with PBS, and finally resuspend the dispersed testicular cells in 10μL L-15 culture medium and keep on ice for later use.

[0036] (3) Transplantation and expansion of donor SSCs (SSCT) 1) Prepare the tools needed for transplantation in advance, including transplantation needles and agar plates, etc. Anesthetize zebrafish recipient fry in batches of about 50 fish per batch, place them in a 90 mm dish covered with 1.5% agarose gel, dry them, and place all recipient fry with their heads and tails facing the same direction for easy transplantation.

[0037] 2) After the donor cells were dispersed, they were loaded into the bottom of a 30-50 μm diameter glass capillary tube using a dedicated micropipette tip (Eppendorf, 5242956003) for transplantation, and cell transplantation was performed under a stereomicroscope. With the help of a microinjector, we transplanted the donor cells into the position between the swim bladder and the intestine of the recipient zebrafish larvae, close to the primitive gonads, and injected approximately 0.2 μL of the donor cell suspension into each recipient.

[0038] 3) 3 days after transplantation (3 dpt), all recipients were examined by fluorescence to determine potential reproductive chimeras based on the presence of green fluorescent cells in the genital ridge region ( Figure 1 ) and cultured it until it developed to 4 mpf.

[0039] 4) Identify the genotype of the receptor and screen out cyp11a 2- / - , and the individuals with significantly increased fluorescence area were selected under a fluorescence microscope and regarded as positive fish with SSCs amplification ( Figure 2 ).

[0040] Example 2 Sperm tissue cryosection / sperm cell immunofluorescence (1) Sample preparation 1) Preparation of frozen sections: After the zebrafish was anesthetized with MS-222, the gonads were dissected out, and the fat particles were peeled off as much as possible. The weights were measured separately, and the tissues were washed twice with 1×PBS and rinsed once in 50% OCT embedding medium. The tissues were then placed in an embedding box containing 50% OCT embedding medium under a dissecting microscope and quickly fixed in liquid nitrogen. The sections were sliced ​​with a Leica CM 1950 cryostat, and the thickness of the testis sections was 12 μm. The sections were attached to adhesive slides, and the samples were framed and marked with an immunohistochemistry pen. The slides were baked at 37°C for 15-30 min to promote adhesion.

[0041] 2) Preparation of testicular cell slides: After the zebrafish was anesthetized with MS-222, the testes were dissected out, and the fat particles were stripped as much as possible. After washing twice with 1×PBS, the testes were cut into pieces in a 2mL low-adsorption EP tube and separated and digested in the following formula: 2mL L-15 medium (Sigma-Aldrich) containing 0.25% trypsin (Invitrogen), 0.26 U collagenase (Roche), 0.05% DNaseI (Roche) and 10% FBS (BioInd). Digestion was carried out at room temperature (RT) for 2-3 hours. During the digestion process, a 1mL low-adsorption pipette tip was used to blow and aspirate several times every 15 minutes to fully disperse the cells. Finally, the cells were resuspended in an appropriate amount of L-15 medium to adjust the cell concentration to 1×10 4 / mL, take 50μL and drop it on the adhesion slide.

[0042] (2) Fixation: Fix the sliced ​​samples with 4% PFA for 15-30 minutes.

[0043] (3) Permeabilization: The samples were then rinsed with 1× PBS (pH 7.4) for 5 min × 3 times. The samples were then permeabilized in 1× PBS (pH 7.4) containing 0.5% Triton X-100 (Sigma-Aldrich) for 15 min at room temperature.

[0044] (4) Blocking: Wash with blocking solution (1× PBS (pH 7.4) containing 1% BSA, 0.1% Triton-100 and 1% DMSO) for 5 min × 3 times, with the last wash for no less than 1 hour.

[0045] (5) Primary antibody incubation: dilute the antibodies with the blocking solution to the corresponding multiples. The primary antibodies include Vasa (1:1000); Sycp3 (ab150292, 1:200), Nanos2 (laboratory homemade, 1:500). Add 200 μL of the diluted primary antibody to each slide, cover with a coverslip, place in a humidified box, and incubate overnight at 4°C.

[0046] (6) Washing: Remove the slides, remove the coverslips, and wash with PBST for 5 min x 3 times.

[0047] (7) Secondary antibody incubation: Use an immunohistochemistry pen to reinforce the sample frame, add secondary antibody (anti-rabbit IgG, AlexaFluor 488, dilution ratio of 1:1000) on the glass slide, cover with a coverslip, and incubate at room temperature for 3-4 hours.

[0048] (8) Washing: Wash the slides with PBST for 5 min x 3 times.

[0049] (9) Nuclear staining: DAPI was prepared with PBST at 1 μg / mL and stained at room temperature for 30 min. The slides were then washed with PBST for 5 min × 3 times and sealed with 75% glycerol containing anti-fluorescence quencher. The samples were photographed under an SP8 confocal microscope (Leica, Germany), and the images were subsequently processed using ImageJ.

[0050] (10) The results show that cyp11a 2- / - After expansion, the testis weight obtained was significantly higher than that of the control group ( Figure 3 ); The proportion of SSCs cells was significantly higher than that of the control group, and the proportion of germ cells in meiosis was significantly lower than that of the control group ( Figure 4 After testicular lysis, without Percoll purification, the proportion of SSCs obtained in the amplification group was significantly higher than that in the control group ( Figure 5 ).

[0051] Example 3 Purification and transplantation of testicular cells (TC) To further confirm that the enlarged testes were rich in SSCs, we transplanted the testicular cells directly without percoll centrifugation after separation and tested the transplantation success rate. Since the weight of the expanded testes was significantly greater than that of the wild type, we compared the efficiency of testicular cell transplantation in the 1-tail expansion group with that in the wild type control group.

[0052] (1) Preparation of transplant recipients Preparation of recipient embryos: Embryos of wild-type zebrafish mating offspring were collected, and 100 μM dead end morpholino antisense oligonucleotide (dnd MO) was injected into 1-cell embryos to completely eliminate the recipient's own endogenous germ cells. The embryos were then cultured in 0.3× Danieau's buffer at 28.5°C in an incubator until 4 dpf in preparation for the next transplantation experiment.

[0053] (2) Isolation and purification of testicular cells.

[0054] 1) First, a 4-month-old zebrafish from the control group and the SSCs expansion group was taken, and the testis was removed after anesthesia. The testis of the two groups were cut into pieces in 2 mL low-adsorption EP tubes and digested in the following formula: 2 mL L-15 medium (Sigma-Aldrich) containing 0.25% trypsin (Invitrogen), 0.26 U collagenase (Roche), 0.05% DNaseI (Roche) and 10% FBS (BioInd), digested at room temperature (RT) for 2-3 hours, and a 1 mL low-adsorption pipette tip was used to pipette several times every 15 minutes during the digestion process to fully disperse the cells.

[0055] 2) When no obvious tissue blocks can be seen with the naked eye, the resulting testicular cell suspension is passed through 70μm and 40μm cell sieves in turn to remove large tissue fragments, and washed three times with PBS. Finally, the dispersed testicular cells are resuspended in 10μL L-15.

[0056] 3) Count the number of GFP-positive testicular cells using a hemocytometer under a fluorescence microscope and calculate their concentration ( Figure 6 ).

[0057] (3) Transplantation of testicular cells The transplantation operation method is the same as SSCT.

[0058] Example 4 Comparative analysis of transplantation success rates between the SSCs expansion group and the control group (1) After the transplanted amplification group and control group recipients were cultured until adulthood, GFP fluorescence was detected under a fluorescence microscope, and individuals with green fluorescence in the gonadal position were selected. The positive rates of the amplification group and the control group were calculated separately.

[0059] (2) The obtained GFP-positive male fish were naturally mated with wild-type female fish, the fertilization rate of the offspring was counted, and the 4 dpf embryos were tested under a fluorescence microscope to see whether they had fluorescence derived from donor cells.

[0060] The results showed that all GFP-positive individuals were male, and the transplantation success rate of the expansion group was significantly higher than that of the control group ( Figure 7 ), when developing to 4 dpf, such as Figure 8 As shown in the figure, GFP green fluorescence can be observed at the genital ridge under a fluorescence microscope, indicating that wild-type recipients can efficiently produce functional sperm from donors. In summary, the enlarged testes of the expansion group are rich in SSCs with high abundance and still have the potential for differentiation.

[0061] The above results show that it can be used as a model for unlimited expansion of zebrafish SSCs, thereby solving the bottleneck problem of efficient enrichment of SSCs in SSCs transplantation and improving the success rate of transplantation.

[0062] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0063] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a spermatogonial stem cell transplantation recipient, characterized in that: The following steps are involved: S1. Get the content Cyplla2 Zebrafish embryos homozygous for the mutant gene; S2, eliminating the endogenous germ cells of the embryo itself; S3. Cultivate embryos with endogenous germ cells removed into seedlings to obtain spermatogonial stem cell transplantation Implant receptors.

2. The method according to claim 1, characterized in that In step S2, the specific method of eliminating the endogenous germ cells of the embryo itself is: injecting morpholino antisense oligonucleotide into the 1-cell stage embryo; and / or, In step S3, the embryos without endogenous germ cells are cultured into seedlings. The temperature is 28-29°C; and / or, In step S3, the embryos from which endogenous reproductive cells have been removed are cultured into seedlings, and the culture medium used for the culture is 0.3×Danieau's buffer; and / or, In step S3, the embryos from which endogenous reproductive cells have been removed are cultured into seedlings, and the embryo culture time is 4 days.

3. Use of the spermatogonial stem cell transplantation receptor prepared according to the preparation method according to any one of claims 1 to 2 in amplifying Tg (vasa:gfp)-labeled spermatogonial stem cells.

4. The use according to claim 3, characterized in that: The spermatogonial stem cell receptor amplification Tg (vasa: gfp) marked spermatogonial stem cells comprises the following steps: SA, obtain spermatogonial stem cells of zebrafish marked with Tg(vasa:gfp), called donor; SB. Transplant the donor into the spermatogonial stem cell recipient for expansion.

5. The use according to claim 4, characterized in that: In step SA, obtain The donor method is as follows: the testis of an adult zebrafish is cut into pieces, separated, digested, centrifuged and purified to obtain the donor.

6. The use according to claim 5, characterized in that: The specific steps of cutting and separating the testes of adult zebrafish are as follows: digesting the cut testes in L-15 medium for 2-3 hours, and using a 1 mL low-adsorption pipette tip to blow and aspirate the cells on the L-15 medium every 15 minutes during the digestion process to fully disperse the cells.

7. The use according to claim 4, characterized in that: In step SB, transplanting the donor into the spermatogonial stem cell recipient for expansion, the specific steps of transplantation are: first anesthetizing the recipient, and then transplanting the donor into the recipient; and / or, In step SB, the donor is transplanted into the spermatogonial stem cell recipient for expansion. wherein the donor is transplanted into the recipient at a position between the swim bladder and the intestine of the recipient zebrafish fry close to the original gonad; and / or, In step SB, after the donor is transplanted into the recipient, the culture time is 4 months; and / or, In step SB, the method for isolating the spermatogonial stem cells amplified in the recipient is as follows: the testis of the recipient is cut into pieces, cultured and digested, filtered and washed, and the spermatogonial stem cells of zebrafish marked with Tg (vasa: gfp) are obtained.

8. A method for detecting spermatogonial stem cells by immunofluorescence, characterized in that: The following steps are involved: Sa, culturing the recipient of the donor amplified in claim 4, removing the gonads, and preparing frozen sections and testicular cell slides; Sb, using a fixative to fix the frozen sections and the testicular cell slides respectively, and then permeabilizing them; Sc, using a blocking solution to block the permeabilized frozen sections and testicular cell slides respectively; Sd, adding a primary antibody that specifically recognizes the target antigen to the frozen sections and testicular cell slides; Se, adding a fluorescently labeled secondary antibody to the frozen sections and testicular cell slides on the basis of step Sd; Sf. On the basis of step Se, using nuclear dye to stain the cells in the frozen sections and testicular cell slides to obtain spermatogonial stem cells with immunofluorescence colors; Sg. The immunofluorescence-stained spermatogonial stem cells were observed using an optical microscope.

9. The method according to claim 8, characterized in that In step Sb, the specific steps of fixation are: fix the frozen sections and testicular cell slides with 4% PFA for 15-30 minutes respectively; and / or, In step Sb, the permeabilization step is: rinse with 1×PBS for 5 min×3 times, and the sample Permeabilization was performed in 1× PBS containing 0.5% Triton X-100 for 15 min at room temperature.

10. The method according to claim 8, characterized in that In step Sd, the primary antibody comprises Vasa, Sycp3, Nanos2; and / or, In step Se, the secondary antibody included rabbit IgG, Alexa Fluor 488.

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