Method for establishing gobiocypris rarus spermatogonial stem cells and application thereof

Abstract

The application belongs to the technical field of biology and particularly relates to a method for establishing sperm stem cells of Rhinogobio typus and application thereof. The method for establishing sperm stem cells of Rhinogobio typus comprises primary culture and subculture, and the testis tissue of immature male Rhinogobio typus is digested by trypsin, and then DMEM culture medium is added for primary culture; the DMEM culture medium is removed, trypsin is added, and subculture is carried out according to a ratio of 1:2. The sperm stem cells of Rhinogobio typus prepared by the method for establishing sperm stem cells of Rhinogobio typus have good growth, high proliferation activity, and can be used for expression of exogenous genes, allogeneic transplantation and in-vitro induced differentiation, and provide important application value for researches on reproduction and production, germplasm resource preservation and genetic improvement of Rhinogobio typus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the method for establishing spermatogonial stem cells of the big-nosed gudgeon and its application. Background Technology

[0002] In recent years, with the rapid development of molecular biology and in vitro cell culture technology, reproductive stem cells, as a type of cell capable of self-renewal and producing functional gametes, play an important role in the in vitro reproductive development of fish. This has laid a foundation, in particular, for the conservation of germplasm resources of endangered species and the breeding of superior varieties. Currently, due to the abundant quantity of spermatogonial stem cells in fish gonads, their ease of isolation and purification, and their long in vitro culture time, researchers mainly focus on research related to fish spermatogonial stem cells. However, although there is considerable research on fish spermatogonial stem cells, most studies have not achieved long-term in vitro culture and maintained their potential for induced differentiation. Therefore, the in vitro culture techniques for spermatogonial stem cells of different fish species need further optimization.

[0003] With the advanced development of in vitro culture technology for spermatogonial stem cells, transplanting them into fish species that are easy to reproduce and have short maturation cycles through surrogacy holds promise for restoring the genetic lineage of endangered fish species that are large, slow-maturing, and difficult to reproduce. This could significantly advance the genetic improvement process in aquaculture. Furthermore, by optimizing the culture system and adding various inducing factors to simulate the in vivo germ cell microenvironment, it is hoped that spermatogonial stem cells can produce haploid sperm in vitro. Compared to in vivo transplantation, in vitro induction technology for reproductive stem cells can obtain a large number of fertilizable gametes without the need for raising any parent or surrogate fish, thus significantly shortening the effective generation interval in aquaculture breeding programs and improving economic efficiency. Therefore, both in vivo transplantation and in vitro induction technologies for reproductive stem cells have enormous application potential in fish breeding and gene-editing breeding in the future.

[0004] Big-nosed catfish ( Rhinogobio nasutus As an important endangered wild animal, the large-nosed goby is mainly distributed in the middle and upper reaches of the Yellow River and is endemic to Gansu Province and Ningxia Hui Autonomous Region in my country. However, due to habitat destruction, water pollution, and overfishing, the germplasm resources of the large-nosed goby have declined sharply. In 2021, it was listed as a Class II protected wild animal in China, thus bringing the conservation of large-nosed goby resources to the forefront. Furthermore, the large-nosed goby grows relatively slowly, taking approximately three years to reach sexual maturity, thus requiring more time and space for parent fish to reproduce. Coupled with the current immaturity of artificial breeding techniques and the minimal research on reproductive development, its germplasm resources have not been effectively improved.

[0005] Therefore, how to isolate and culture testicular cells in vitro, establish a stable spermatogonial stem cell line from the big-nosed gudgeon, and transplant and induce its differentiation into gametes in vitro is a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the challenges of existing technologies, this invention provides a method for establishing spermatogonial stem cells from the large-nosed gudgeon. The spermatogonial stem cells obtained by this method exhibit high proliferative activity and good cell growth, and have been successfully used for exogenous gene expression, xenografting, and in vitro induced differentiation to prepare sperm cells.

[0007] The technical solution of this invention is a method for establishing spermatogonial stem cells from the large-nosed goby, involving primary culture and passage culture for no less than 75 days, the steps of which include: (1) Primary culture: The testis tissue of immature male big-nosed gudgeon was taken, the fat was removed and digested with trypsin, and the cells were resuspended in DMEM medium to obtain a cell suspension. The cells were cultured at 25°C until they were covered with a monolayer. (2) Subculture: Repeat the following subculture operation: remove DMEM medium, add trypsin to digest, remove trypsin, add DMEM medium to pipette cells, centrifuge to collect cells and add DMEM medium again, and subculture at 25℃ at a ratio of 1:2 until monolayer is formed.

[0008] Step (1) Primary culture: The testis tissue of immature male gudgeon is placed in a PBS solution containing 1%-2% penicillin-streptomycin to remove fat, and then the fat-removed testis tissue is washed with a PBS solution containing 1%-2% penicillin-streptomycin.

[0009] Step (1) Primary culture: After the testis tissue with fat removed is cut into small pieces, it is digested with 0.05% trypsin solution at room temperature for 20-30 minutes.

[0010] Step (1) Primary culture: Add DMEM medium and gently pipette to resuspend the cells to obtain a cell suspension. After filtering the cell suspension through a 40 μm cell sieve, place it in a container covered with 0.1%-0.2% gelatin and culture it at 25℃ until it forms a monolayer.

[0011] Step (1) Primary culture: During the primary culture process, the culture medium is changed every 2-3 days, and half of the volume is changed each time.

[0012] Step (2) Subculture: After removing the DMEM medium, wash with buffer 2-3 times before adding trypsin digestion. The buffer is PBS solution.

[0013] Step (2) Subculture, add 0.05% trypsin and digest at room temperature for 30 seconds.

[0014] Step (2) Subculture: Replace the culture medium with a completely new one every 2-3 days during the adherent subculture process. Replace all the culture medium with fresh culture medium.

[0015] Step (2) Subculture: Adherent subculture is carried out in a container lined with 0.1%-0.2% gelatin.

[0016] The DMEM culture medium described in this invention is a DMEM culture medium containing Hepes, FBS, medaka embryo extract, large-nosed gudgeon serum or large-nosed gudgeon serum and sea bass serum, bFGF, L-Glu, non-essential amino acids, sodium pyruvate, penicillin-streptomycin, β-mercaptoethanol, and sodium selenite. Preferably, it is a DMEM culture medium containing Hepes, FBS, medaka embryo extract, large-nosed gudgeon serum, bFGF, L-Glu, non-essential amino acids, sodium pyruvate, penicillin-streptomycin, β-mercaptoethanol, and sodium selenite. The preparation includes: adding 13-14 g DMEM and 4.5-5.5 g Hepes to each liter of sterile ddH2O, adjusting the pH to 7.4-7.6 with NaOH to obtain DMEM basal medium; then adding 150 mL FBS, 1 mL medaka embryo extract, 2 mL large-nosed gudgeon serum or 2 mL large-nosed gudgeon serum and 2 mL sea bass serum, 100 µL 0.1 mg / mL basic fibroblast growth factor, 10 mL 2 mM L-glutamate, 10 mL 1 mM non-essential amino acids, 10 mL 1 mM sodium pyruvate, 10 mL 100 ug / mL penicillin-streptomycin, 2 mL 50 mM β-mercaptoethanol, and 1 mL 2 µM sodium selenite to each liter of DMEM basal medium; preferably, adding 150 mL FBS, 1 mL Medaka embryo extract, 2 mL of large-nosed gudgeon serum, 100 µL of 0.1 mg / mL basic fibroblast growth factor, 10 mL of 2 mM L-glutamate, 10 mL of 1 mM non-essential amino acids, 10 mL of 1 mM sodium pyruvate, 10 mL of 100 U ug / mL penicillin-streptomycin, 2 mL of 50 mM β-mercaptoethanol, and 1 mL of 2 µM sodium selenite.

[0017] This invention provides spermatogonial stem cells from the large-nosed gudgeon, prepared by the method described above. These spermatogonial stem cells contain 50 chromosomes, exhibit strong alkaline phosphatase activity, and express germ cell marker genes. vasa , nanog , oct4 , piwi Weak expression of somatic cell marker genes dmrt1,Simultaneously, it highly expresses the germ cell marker protein Vasa and the proliferating cell activity marker protein PCNA. Furthermore, the spermatogonial stem cells of the large-nosed gudgeon of this invention exhibit good cell growth and maintain high proliferative activity even after being continuously cultured in vitro for more than 4 months.

[0018] The spermatogonial stem cells of the big-nosed gudgeon provided by this invention can express exogenous genes in vitro and are used for the expression of exogenous genes.

[0019] This invention provides a method for in vitro expression of exogenous genes in spermatogonial stem cells of the large-nosed gudgeon. The steps include: transferring an exogenous gene vector into the spermatogonial stem cells of the large-nosed gudgeon using a physical method and incubating for expression. The physical method is selected from any one of microinjection, electroporation, and gene gun, such as electroporation parameters: 1400V, 20ms, 3plues.

[0020] The spermatogonial stem cells of the big-nosed gudgeon provided by this invention can be allogeneically transplanted and develop into chimeras as the zebrafish embryos, which serve as recipients, and integrate into the brain, muscles, notochord, skin and gonadal primordia of the zebrafish recipients, maintaining normal proliferation and differentiation capabilities.

[0021] This invention provides a method for allogeneic transplantation of spermatogonial stem cells from gudgeon, comprising the following steps: transplanting gudgeon spermatogonial stem cells cultured in vitro for four months into zebrafish embryos that have developed to the blastocyst stage after their outer membrane has been removed, incubating them in an embryo balancing solution at room temperature, and changing the embryo balancing solution daily. Specifically, the method involves using a microinjection device to transplant the gudgeon spermatogonial stem cells into the zebrafish embryos that have developed to the blastocyst stage after their outer membrane has been removed.

[0022] The preparation of zebrafish embryos developed to the blastocyst stage by removing the outer membrane includes: removing the outer membrane of zebrafish embryos before they reach the blastocyst stage, incubating them in an embryo balancing solution at room temperature until the blastocyst stage, and obtaining zebrafish embryos developed to the blastocyst stage by removing the outer membrane. Further, zebrafish eggs are placed in an embryo balancing solution and incubated at room temperature to obtain zebrafish embryos developed to the blastocyst stage.

[0023] The spermatogonial stem cells of the large-nosed gudgeon provided by this invention can differentiate into sperm cells in vitro under the induction of sex hormones.

[0024] This invention provides a method for differentiating spermatogonial stem cells of large-nosed gudgeon into sperm cells in vitro under the induction of sex hormones. The steps include: inoculating large-nosed gudgeon spermatogonial stem cells cultured in vitro for four months into DMEM medium containing sex hormones for 14-16 days, and changing the medium containing sex hormones every 3-4 days, replacing half the volume each time.

[0025] The sex hormone-containing DMEM medium is a DMEM medium containing 10 U / mL human chorionic gonadotropin, 10 U / mL pregnant mare serum gonadotropin, 200 ng / mL 11-ketotestosterone, and 50 ng / mL 17α,20β-dihydroxy-4-pregnen-3-one. The DMEM medium is a DMEM medium containing Hepes, FBS, medaka embryo extract, large-nosed gudgeon serum or large-nosed gudgeon serum and sea bass serum, bFGF, L-Glu, non-essential amino acids, sodium pyruvate, penicillin-streptomycin, β-mercaptoethanol, and sodium selenite. Preferably, it is a DMEM medium containing Hepes, FBS, medaka embryo extract, large-nosed gudgeon serum, bFGF, L-Glu, non-essential amino acids, sodium pyruvate, penicillin-streptomycin, β-mercaptoethanol, and sodium selenite. The preparation includes: adding 13-14 g DMEM and 4.5-5.5 g Hepes to each liter of sterile ddH2O, adjusting the pH to 7.4-7.6 with NaOH to obtain DMEM basal medium; then adding 150 mL FBS, 1 mL medaka embryo extract, 2 mL large-nosed gudgeon serum or 2 mL large-nosed gudgeon serum and 2 mL sea bass serum, 100 µL 0.1 mg / mL basic fibroblast growth factor, 10 mL 2 mM L-glutamate, 10 mL 1 mM non-essential amino acids, 10 mL 1 mM sodium pyruvate, 10 mL 100 ug / mL penicillin-streptomycin, 2 mL 50 mM β-mercaptoethanol, and 1 mL 2 µM sodium selenite to each liter of DMEM basal medium; preferably, adding 150 mL FBS, 1 mL Medaka embryo extract, 2 mL of large-nosed gudgeon serum, 100 µL of 0.1 mg / mL basic fibroblast growth factor, 10 mL of 2 mM L-glutamate, 10 mL of 1 mM non-essential amino acids, 10 mL of 1 mM sodium pyruvate, 10 mL of 100 U ug / mL penicillin-streptomycin, 2 mL of 50 mM β-mercaptoethanol, and 1 mL of 2 µM sodium selenite.

[0026] The room temperature described in this invention is 15℃-30℃, preferably 25℃±2℃.

[0027] This invention yields a spermatogonial stem cell line from the large-nosed gudgeon that can stably proliferate and passage in vitro over a long period. Under electroporation conditions, it can be used to study the expression of exogenous genes in vitro. Under in vivo transplantation and in vitro induction conditions, it exhibits pluripotency, which has significant application value for research in the breeding and production, germplasm resource preservation, and genetic improvement of endangered fish species. (See attached figures.) Figure 1Images of the established spermatogonial stem cell line from the large-nosed gudgeon. A shows cells cultured on day 1, B on day 7, C on day 15, D on day 40, E on day 80, and F on day 120.

[0028] Figure 2 Images show the growth of testicular cells from the large-nosed gudgeon at different culture temperatures. AC: cultured at 28℃; DF: cultured at 25℃. Red arrows indicate adherent cells; black boxes indicate vacuoles.

[0029] Figure 3 Images of testicular cells from the bigmouth gudgeon cultured under different fish serums (AC: 0.2% sea bass serum; DF: 0.2% sea bass serum + 0.2% bigmouth gudgeon serum).

[0030] Figure 4 Characterization of RnSSCs (rhinogi trigonella foetida) spermatogonial stem cells. (A) Alkaline phosphatase (AP) staining of RnSSCs cultured in vitro for 80 days. The results showed that the cells stained deep purple, indicating that the cultured cells had strong stem cell characteristics. (B) Karyotype analysis results of RnSSCs from passage 13 (2n=50) (scale bar=10 µm). (C) RT-PCR analysis of germ cell markers (vasa, piwi), stem cell markers (oct4, nanog), and somatic cell markers (dmrt1) in RnSSCs and gonadal tissue. The results showed that the cells expressed germ cell and stem cell marker genes and weakly expressed somatic cell marker genes, indicating that the obtained RnSSCs were Rhinogi trigonella foetida spermatogonial stem cells.

[0031] Figure 5 Immunofluorescence images of VASA (green) and PCNA (red) in the spermatogonial stem cell line of the bigmouth gudgeon. The images show that the spermatogonial stem cells of the bigmouth gudgeon express germ cell marker proteins (VASA) and exhibit proliferative activity (PCNA).

[0032] Figure 6 Image showing pCVpr plasmid transfected into a large-nosed gudgeon spermatogonial stem cell line. It indicates that 40% of the cells successfully received the foreign gene.

[0033] Figure 7 The colonization of RnSSCs at different stages in zebrafish embryos after transplantation. (A) Results of RnSSCs after PKH26 staining. (BG) Chimeras observed by fluorescence microscopy at the blastocyst stage (B), outer embryo stage (C), 2d (DD″), 4d (EE″), 6d (F), and 8d (G) after transplantation. (BG) are superimposed images of bright field and fluorescence. (D′-E′) are magnified images of the white boxes in images D and E. (D″-E″) are magnified images of the red fluorescence of the white boxes in images D and E.

[0034] Figure 8 This describes the differentiation of spermatogonial stem cells from the large-nosed gudgeon into spermatozoites under the induction of various sex hormones. (A) Image without sex hormones, (B) Embryoid bodies appear after 2 days of induction with sex hormones, (C) Spermatocyte-like cells appear after 5 days of induction, (D) A tetrad formed by four round spermatocytes connected by cytoplasmic bridges (indicated by red arrows), (E) Round spermatocytes and spermatocytes with extended cytoplasm appear after 10 days of induction, (FI) Spermatocytes further differentiate into spermatozoites; spd, spermatocyte; spm, spermatozoite.

[0035] Figure 9 Images showing the induced differentiation of spermatogonial stem cell lines from the big-nosed gudgeon for PCR identification. Detailed Implementation

[0036] Experimental materials Preparation of culture medium: Add 13.37 g DMEM and 4.77 g Hepes (hydroxyethylpiperazine ethanethiolic acid) to every 1 L of sterile ddH2O, and adjust the pH to 7.5 with NaOH to obtain the basic culture medium DMEM. The following components were added sequentially to each 1 L of basal culture medium: 150 mL fetal bovine serum (FBS), 1 mL medaka embryo extract, 2 mL large-nosed gudgeon serum, 100 µL basic fibroblast growth factor (bFGF, 0.1 mg / mL), 10 mL L-glutamate (L-Glu, 100×, 2 mM), 10 mL non-essential amino acid (100×, 1 mM), 10 mL sodium pyruvate (Na-pyruvate, 100×, 1 mM), 10 mL penicillin-streptomycin (Penicillin-streptomycin, 100×, 100 ug / mL), 2 mL β-mercaptoethanol (β-mercaptoethanol, 50 mM), and 1 mL sodium selenite (Na-selenite, 1000×, 2 µM). The prepared culture medium was filtered through a 0.22 µm filter and stored at 4 °C. Unless otherwise specified, this culture medium shall be used in all subsequent experiments.

[0037] Preparation of medaka embryo extract: Medaka embryos developed to 7 days were collected, ground, and the ground material was collected. The mixture was repeatedly frozen and thawed three times in liquid nitrogen and a 37°C water bath. The mixture was centrifuged at 3500 g for 30 min at 4°C and the supernatant was collected. The mixture was then centrifuged at 18000 g for 30 min at 4°C to form a three-phase homogenate. The middle green phase was collected. The embryo extract was diluted with PBS at pH 7.4 at a ratio of 1 mL per 1000 embryos, aliquoted, and stored at -20°C for later use.

[0038] Preparation of serum from bignostos gudgeon: Blood from bignostos gudgeon was collected via the tail vein on ice and allowed to stand at room temperature for 1-4 hours. Then, it was transferred to 4°C for overnight incubation and centrifuged at 3500 g for 30 min at 4°C. The supernatant was collected, aliquoted, and stored at -20°C for later use.

[0039] All equipment used in the experiment was sterilized before use.

[0040] Example 1: Culture of spermatogonial stem cell line from big-nosed gudgeon 1. Isolation and digestion of testicular cells from the large-nosed gudgeon Immature male gudgeon were collected, their bodies were wiped with 75% alcohol, and washed three times with PBS containing 2% penicillin-streptomycin (w / v) before dissection. The abdomen of the gudgeon was cut open with scissors, and the testis tissue was carefully removed with forceps and placed in a culture dish containing PBS containing 2% penicillin-streptomycin. The fat on the surface of the testis tissue was carefully peeled off with forceps, and then washed three times with PBS containing 1% penicillin-streptomycin. The testis tissue was then transferred to a 1.5 mL EP tube containing 0.05% trypsin (w / v), and minced with sterile scissors. After digestion at room temperature for 0.5 h, 1 mL of culture medium was added to stop the digestion. The tissue pellet was gently dispersed with a pipette, and the cell suspension was filtered through a 40 μm cell sieve and transferred to a 96-well plate coated with 0.1% gelatin (w / w). The plate was then incubated at 25°C.

[0041] 2. Culture and passage of testicular cells from the large-nosed gudgeon The next day, the cell adhesion was observed under a microscope to check for contamination. If no contamination was found, half of the culture medium was replaced, and the cells were placed in the incubator for further culture. Subsequent medium changes were made based on cell growth until the cells completely covered the entire well.

[0042] Once the cells have formed a monolayer, passage them at a 1:2 ratio. First, discard the culture medium in the wells, wash three times with 100 μL PBS, then add one drop of 0.05% trypsin and digest for 30 seconds. Discard the digestion solution, add culture medium, pipette and agitate the cells, centrifuge, then add fresh culture medium to divide the cells into two wells. Transfer the cells to 96-well plates coated with 0.1% gelatin (w / w) and continue incubation at 25°C.

[0043] Microscopic examination revealed that a very small number of cells adhered to the culture medium one day after inoculation, and a large number of cells began to proliferate after one week. Subsequently, the culture medium was changed every two days, and the cells filled the wells in approximately two weeks. Once a monolayer of cells was formed, the cells were passaged at a ratio of 1:2. Figure 1 Currently, the spermatogonial stem cell line has been cultured in vitro for more than 4 months, and the cells are growing well and still maintaining high proliferative activity, indicating that the spermatogonial stem cell line of the big-nosed gudgeon has been basically established and can be used for subsequent identification and other experiments.

[0044] Example 2: Investigation of Culture Temperature of the Large-nosed Gizzard Spermatogastrocytic Stem Cell Line The operation in this embodiment is the same as in Embodiment 1, except that the incubation temperature is different; two groups were set up: a 25℃ group and a 28℃ group. The results are shown below. Figure 2 When cultured at 28℃, a small number of cells adhered and grew after 2 days, but a large number of small vacuoles appeared on the 4th day. As time went on, the vacuoles gradually enlarged, and the cells entered an apoptotic state. Figure 2 (AC). Subsequently, when the culture temperature was changed to 25℃, a small number of cells adhered and grew after 2 days. The cell count increased over time, and no vacuoles were observed. Figure 2 (DF). Therefore, a culture temperature of 25℃ is recommended.

[0045] Example 3: Investigation of Culture Medium for the Spermatogastrodon Spermatogastrodon Stem Cell Line The operation of this embodiment is the same as that of embodiment 1, except that the culture medium used is different: (1) 2 mL of large-nosed gudgeon serum in the culture medium used in embodiment 1 is replaced with 2 mL of sea bass serum, and it is set as an experimental group; (2) 2 mL of sea bass serum (2 mL of large-nosed gudgeon serum + 2 mL of sea bass serum) is added to the culture medium used in embodiment 1, and it is set as another experimental group.

[0046] The results are as follows Figure 3 As shown, when cultured with 0.2% sea bass serum, cell growth was slow. The first passage was performed after 30 days of in vitro culture. Although the passaged cells adhered to the culture vessel, their growth was poor, and even apoptosis occurred. Figure 3 (AC). Subsequently, 0.2% sea bass serum and 0.2% large-nosed gudgeon serum were added to the culture medium. After 3 days, cell growth improved significantly, and after 7 days, the cells covered the entire well plate and were passaged. Figure 3 (DF).

[0047] Primary culture of bigmouth gudgeon testis cells using only bigmouth gudgeon serum yielded the results shown in Example 1. Figure 1 The results showed that a large number of cells adhered and began to proliferate after one week.

[0048] The present invention uses Example 1 as a preferred embodiment for the culture of the spermatogonial stem cell line of the big-nosed gudgeon to prepare the spermatogonial stem cell line of the big-nosed gudgeon for subsequent identification and application research.

[0049] Example 4: Identification of spermatogonial stem cell lines from the big-nosed gudgeon 1. Alkaline phosphatase staining to identify the pluripotency of cultured gudgeon testis cells. Cells cultured in vitro for 80 days were seeded into 24-well plates and incubated at 25°C until they reached 60-70% confluence. Cells were washed three times with PBS, and fixed with 500 μL of 4% paraformaldehyde fixative for 10 min at room temperature. Cells were washed three times with PBS. Cells were covered with 500 μL of BCIP / NPT matrix solution and incubated at 25°C in the dark for 12 h. The matrix solution was discarded, and cells were washed twice with PBS. Cells were covered with 100 μL of neutral resin and mounted.

[0050] Microscopic observation revealed that the cells were stained deep purple. Figure 4 (A) indicates that the cultured cells have strong alkaline phosphatase activity and pluripotency.

[0051] 2. Karyotype analysis to identify chromosomes in testicular cells of the large-nosed gudgeon. Cells cultured in vitro for 90 days were seeded into 6-well plates and cultured at 25°C until the cell confluence reached approximately 70%. Then, colchicine solution with a final concentration of 0.2 µg / mL was added, and the cells were cultured for another 4 hours. After washing twice with PBS, the cells were collected into centrifuge tubes. A hypotonic solution of 75 mM KCl was added, and the cells were incubated at room temperature for 30 minutes. Then, 1 mL of freshly prepared fixative (methanol:acetone = 3:1) was added to terminate the hypotonic reaction, and the cells were resuspended in 1 mL of fixative for 1 hour. After centrifugation, the cells were resuspended in 300 µL of fixative and added to a chilled, moistened glass slide. After air-drying, the cells were stained with 5% Giemsa stain at room temperature for 15 minutes, followed by slow rinsing with distilled water. The cells were observed and photographed using a 100× oil immersion microscope.

[0052] Microscopic observation revealed that the testicular cells of the big-nosed gudgeon contain 50 chromosomes. Figure 4 (B)

[0053] 3. RT-PCR identification of the type of cultured gudgeon testis cells To identify the type of cultured snout gudgeon testis cells, cellular RNA was extracted and reversed into cDNA for RT-PCR detection.

[0054] Primers for germ cell marker genes vasa, nanog, oct4, piwi and somatic cell marker gene dmrt1 were designed using Primer5 (primer sequence information is shown in Table 1). Cellular RNA was extracted and reversed into cDNA. RT-PCR was used to identify the type of testis cells of the cultured gudgeon.

[0055] Table 1 RT-PCR primer sequence information Cells from a six-well plate were digested with trypsin and resuspended in fresh culture medium in a 1.5 mL EP tube. The cells were centrifuged at 1000 rpm for 5 min, washed once with PBS, and the supernatant was carefully discarded. RNA was extracted from the cells following these steps: First, 1 mL of Trizol was added to the EP tube containing the cell pellet, and the mixture was thoroughly mixed by pipetting. The mixture was then incubated on ice for 5 min. Next, 200 μL of chloroform was added, and the mixture was vigorously vortexed for 15 s. The mixture was incubated on ice for 2-3 min, and centrifuged at 12,000 rpm at 4°C for 15 min. The supernatant was carefully transferred to a new 1.5 mL EP tube, and an equal volume of isopropanol was added. The mixture was gently mixed and centrifuged at 12,000 rpm at 4°C for 10 min. The supernatant was discarded, and the pellet was washed twice with 1 mL of 75% ethanol. The pellet was centrifuged at 12,000 rpm at 4°C for 5 min. The supernatant was discarded, and the mixture was incubated at room temperature for 5 min to allow the ethanol to evaporate completely. Finally, 20 μL of DEPC water was added, and the pellet was repeatedly pipetted to dissolve it. The obtained RNA can be stored at -80°C for an extended period.

[0056] cDNA was synthesized according to the ABclonal reverse PCR instructions and used as a template for RT-PCR detection. The reaction system and procedure were as follows: 0.5 μL each of forward and reverse primers, 2 μL of template, 6 μL of Mixase, and ddH2O was added to bring the volume to 12 μL; pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 45 s, 36 cycles, and a final extension at 72℃ for 7 min, with β-actin as an internal control.

[0057] The results showed that the testicular cells of the large-nosed gudgeon expressed germ cell marker genes and weakly expressed somatic cell marker genes, such as... Figure 4 As shown in C. This indicates that the cultured testicular cells from the large-snout gudgeon are spermatogonial stem cells, not somatic cells.

[0058] 4. Immunofluorescence analysis of VASA and PCNA protein expression in testicular cells of the large-snout gudgeon. The vasa gene is a marker gene for germline stem cells. Therefore, immunofluorescence analysis was used to analyze the distribution of vasa protein in large-snout gudgeon spermatogonial stem cells, thereby identifying the purity of the cultured large-snout gudgeon spermatogonial stem cells. PCNA is a protein that marks cell proliferation status, so immunofluorescence was used to reflect the proliferative activity of large-snout gudgeon spermatogonial stem cells.

[0059] Large-nosed goat spermatogonial stem cells were seeded onto 24-well cell slides. Once cell confluence reached 70%, the culture medium was discarded, and the cells were washed twice with PBS. Cells were fixed in 4% paraformaldehyde for 10 min, followed by three washes with PBS. Cells were permeabilized with 0.3% Triton-X 100 for 10 min, followed by three washes with PBS. Cells were blocked with 4% bovine serum albumin (BSA) for 30 min, followed by three washes with PBS. 200 µL of VASA antibody and PCNA antibody (1:200 dilution) were incubated overnight at 4°C, followed by three washes with PBS. Then, 200 µL of goat anti-rabbit-FITC and goat anti-mouse-TRITC (1:100 dilution) were added, and the cells were incubated in the dark for 2 hours, followed by three washes with PBS. Finally, 200 µL of DAPI (1:500 dilution) was added, and the cells were treated at room temperature in the dark for 15 min, followed by three washes with PBS. The slides were then mounted with an anti-fluorescence quencher.

[0060] Microscopic observation revealed strong expression of PCNA in the testis cells of *Gizzard magna*, indicating high cell proliferation activity. Simultaneously, VASA showed positive expression in *Gizzard magna* testis cells, suggesting that the cultured testis cells were likely spermatogonial stem cells (SPCs). Figure 5 ).

[0061] Example 5: Study on the in vitro expression of exogenous genes in the rhinoceros gudgeon spermatogonial stem cell line. Cells were seeded into 12-well plates and cultured at 25°C until cell confluence reached 70-90%. Cells were then digested with trypsin, centrifuged at 1000 rpm for 5 min, and washed once with PBS. Cells were resuspended in 10 µl of Buffer R and then transferred to the cathode cup of an electroporator for electroporation (electroporation parameters 1400 V, 20 ms, 3 pluets). After electroporation, cells were seeded into 12-well plates for further culture.

[0062] After 48 hours of culture, fluorescence microscopy revealed that some cells carried red fluorescent markers, proving that these cells had been successfully transferred with the foreign gene. Figure 6 ).

[0063] Example 6: Identification of the allogeneic transplantation capacity of the rhinoceros gudgeon spermatogonium stem cell line 1. Preparation of donor cells and recipients Spermatoglyphic stem cells from large-nosed goat, cultured in vitro for four months, were seeded into 6-well plates and cultured at 25°C until the cell confluence reached approximately 80%. Cells were stained with PKH26 live cell staining according to the manufacturer's instructions to obtain a red fluorescent label. Before transplantation, the cells were washed twice with PBS, digested with trypsin, and collected into 1.5 mL EP tubes. The tubes were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 50 µL of transplantation buffer to prepare a cell suspension, which was then kept on ice until needed.

[0064] Male and female zebrafish in their breeding season were separated one day before the experiment at a ratio of 1:1 or 2:1. On the day of the experiment, they were placed together in a breeding tank. After spawning, the eggs were collected and placed in a culture dish containing embryo balancing solution for incubation at room temperature. Before the zebrafish embryos developed to the blastocyst stage, the outer membrane of the embryo was peeled off using forceps, leaving only the membrane-free embryo. Microsurgical transfer was then performed when the embryos reached the blastocyst stage.

[0065] 2. Microsurgical grafting procedure A 1mm glass capillary was fixed to a needle puller, and the cell transplantation-specific glass needle was pulled out under parameters of Heat=530, Pull=60, Vol=60, and Time=200. After purging the air from the microinjector, the injection needle was attached to the injector, and a 30µm oblique incision was made on the needle with a blade to allow cells to pass through smoothly. Spermatoglyphic stem cells from large-snout gudgeon were aspirated using negative pressure and then transplanted into decapitated zebrafish blastocysts. Care must be taken not to damage the zebrafish embryo structure during the transplantation process to ensure the zebrafish survival rate. The transplanted zebrafish embryos were cultured in embryo balance medium and observed, with the medium being changed daily.

[0066] After transplantation, it was found that the spermatogonial stem cells of the large-nosed gudgeon did not exhibit rejection in zebrafish. These cells formed chimeras as the zebrafish embryos developed and proliferated and differentiated normally within the zebrafish. Of the 60 transplanted zebrafish embryos, 37 survived (survival rate 61.7%), while the remaining embryos died due to external damage during the transplantation process. Among the surviving individuals, 30 showed a positive reaction (positive rate 50%). Of the positive embryos, the transplanted cells in 27 embryos were integrated into the zebrafish brain, muscle, notochord, and skin, while the transplanted cells in 3 embryos migrated to the zebrafish gonadal primordia (…). Figure 7 This experiment confirmed that the spermatogonial stem cells of the large-nosed gudgeon can proliferate and differentiate into various tissues in zebrafish embryos of different genera without cell rejection, and also possess the characteristics of autologous reproductive stem cells that integrate into the gonads.

[0067] Example 7 Identification of the in vitro differentiation potential of the large-nosed gudgeon spermatogonial stem cell line 1. Sex hormone-induced spermatogonial stem cell line from large-nosed gudgeon Spermatoglyphic stem cells from *Gnaphalium affine* cultured in vitro for four months were seeded into 12-well plates. The control group was cultured in the same *Gnaphalium affine* spermatogonium culture medium as described above; the experimental group was cultured in a hormone-containing medium (10 U / mL human chorionic gonadotropin, 10 U / mL pregnant mare serum gonadotropin, 200 ng / mL 11-ketotestosterone, and 50 ng / mL 17α,20β-dihydroxy-4-pregnen-3-one). Half of the hormone-containing medium was replaced every 3 days. Embryoids appeared in the experimental group after 2 days of induction, and their volume increased with the extension of culture time; on day 8 of induction, tetrads formed by four round spermatocytes connected by cytoplasmic bridges appeared; on day 10 of induction, some spermatoid cells gradually changed from round to spindle-shaped; on day 14 of induction, a small number of flagellated spermatocytes with weak motility were produced; on day 16 of induction, spermatocytes were further differentiated into sperm with condensed heads and elongated flagella. Figure 8 In the control group, the cell count increased significantly with prolonged culture time, and some cells detached due to lack of growth space.

[0068] To identify the ability of large-nosed gudgeon spermatogonial stem cells to be induced, cellular RNA was extracted and reverse-engineered into cDNA for RT-PCR detection. Primers for the meiotic marker genes rec8 and dmc1 were designed using Primer5 (primer sequence information is shown in Table 2).

[0069] Table 2 RT-PCR primer sequence information Cells from the control group and the sex hormone-induced group were digested with trypsin, resuspended in serum-containing culture medium into 1.5 mL EP tubes, centrifuged at 1000 rpm for 5 min, washed once with PBS, and the supernatant was carefully discarded. Cell RNA was extracted according to the following steps. First, add 1 mL of Trizol to the EP tube containing the cell pellet, mix thoroughly by pipetting, and incubate on ice for 5 min. Add 200 μL of chloroform, vortex vigorously for 15 s, incubate on ice for 2-3 min, and centrifuge at 12,000 rpm at 4°C for 15 min. Carefully transfer the supernatant to a new 1.5 mL EP tube, add an equal volume of isopropanol, mix gently, and centrifuge at 12,000 rpm at 4°C for 10 min. Discard the supernatant, wash the pellet twice with 1 mL of 75% ethanol, and centrifuge at 12,000 rpm at 4°C for 5 min. Discard the supernatant and incubate at room temperature for 5 min to allow the ethanol to evaporate completely. Add 20 μL of DEPC water and repeatedly pipet to dissolve the pellet. The obtained RNA can be stored at -80°C for an extended period.

[0070] cDNA was synthesized according to the ABclonal reverse PCR instructions and used as a template for RT-PCR detection. The reaction system and procedure were as follows: 0.5 μL each of forward and reverse primers, 2 μL of template, 6 μL of Mixase, and ddH2O was added to bring the volume to 12 μL; pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s, annealing at 56℃ for 15 s, extension at 72℃ for 45 s, 36 cycles, and a final extension at 72℃ for 7 min, with β-actin as an internal control.

[0071] The results showed that no expression of meiotic marker genes was detected in the control group; however, the induced group showed significant expression of meiotic marker genes, such as... Figure 9 As shown in the figure. This indicates that the spermatogonial stem cell line of the large-nosed gudgeon has the potential for induced differentiation.

Claims

1. A method for establishing spermatogonial stem cells from the large-nosed gudgeon, characterized in that, Primary and subcultures lasting at least 75 days are performed, including the following steps: (1) Primary culture: The testis tissue of immature male big-nosed gudgeon was taken, the fat was removed and digested with trypsin, and the cells were resuspended in DMEM medium to obtain a cell suspension. The cells were cultured at 25°C until they were covered with a monolayer. (2) Subculture: Repeat the following subculture operation: remove DMEM medium, add trypsin to digest, remove trypsin, add DMEM medium to pipette cells, centrifuge to collect cells and add DMEM medium again, and subculture at 25℃ at a ratio of 1:2 until a monolayer is formed. The DMEM medium is a DMEM medium containing Hepes, FBS, medaka embryo extract, large-nosed gudgeon serum or large-nosed gudgeon serum and sea bass serum, bFGF, L-Glu, non-essential amino acids, sodium pyruvate, penicillin-streptomycin, β-mercaptoethanol, and sodium selenite.

2. The method for establishing according to claim 1, characterized in that, The preparation of the DMEM medium includes: adding 13-14 g of DMEM and 4.5-5.5 g of Hepes to each liter of sterile ddH2O, adjusting the pH to 7.4-7.6 with NaOH to obtain the DMEM basal medium; then adding 150 mL of FBS, 1 mL of medaka embryo extract, 2 mL of large-nosed gudgeon serum or 2 mL of large-nosed gudgeon serum and 2 mL of sea bass serum, 100 µL of 0.1 mg / mL basic fibroblast growth factor, 10 mL of 2 mM L-glutamate, 10 mL of 1 mM non-essential amino acids, 10 mL of 1 mM sodium pyruvate, 10 mL of 100 ug / mL penicillin-streptomycin, 2 mL of 50 mM β-mercaptoethanol, and 1 mL of 2 µM sodium selenite to each liter of DMEM basal medium.

3. The method for establishing according to claim 1, characterized in that, Step (1) Primary culture: The cell suspension is placed in a container lined with gelatin and cultured at 25°C until it forms a monolayer.

4. The method for establishing according to claim 1, characterized in that, Step (2) Subculture: Adherent subculture is carried out in a container lined with gelatin.

5. The application of the large-nosed gudgeon spermatogonial stem cells prepared by the method described in claim 1 in expressing exogenous genes.

6. A method for in vitro expression of exogenous genes by spermatogonial stem cells of large-nosed gudgeon, characterized in that the steps are as follows: include: The exogenous gene vector was transferred into the spermatogonial stem cells of the large-nosed gudgeon prepared by the method described in claim 1 using a physical method and then incubated for expression.

7. The application of the large-nosed gudgeon spermatogonial stem cells prepared by the method described in claim 1 in allogeneic transplantation.

8. A method for allogeneic transplantation of spermatogonial stem cells from large-nosed gudgeon, characterized by the following steps: include: The spermatogonial stem cells of the large-nosed gudgeon, cultured in vitro for four months using the method described in claim 1, were transplanted into zebrafish embryos that had developed to the blastocyst stage after their outer membranes were removed, and then incubated at room temperature in an embryo balancing solution.

9. The application of the spermatogonial stem cells of the large-nosed gudgeon prepared by the method described in claim 1, which differentiate into sperm cells in vitro under the induction of sex hormones.

10. A method for differentiating spermatogonial stem cells of the large-nosed gudgeon into sperm cells in vitro under the induction of sex hormones, characterized in that the steps are as follows: include: The spermatogonial stem cells of the large-nosed goby, cultured in vitro for four months using the method described in claim 1, were seeded into a DMEM medium containing sex hormones for further culture. The DMEM medium containing sex hormones was a DMEM medium containing 10 U / mL human chorionic gonadotropin, 10 U / mL pregnant mare serum gonadotropin, 200 ng / mL 11-ketotestosterone, and 50 ng / mL 17α,20β-dihydroxy-4-pregnen-3-one. The DMEM medium was a DMEM medium containing Hepes, FBS, medaka embryo extract, large-nosed goby serum or large-nosed goby serum and sea bass serum, bFGF, L-Glu, non-essential amino acids, sodium pyruvate, penicillin-streptomycin, β-mercaptoethanol, and sodium selenite.

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