Method for producing male gametes of ricefield eels based on germline stem cell transplantation technology

Abstract

The invention belongs to the technical field of genetic breeding of aquatic animals, and particularly relates to a method for producing male gametes of finless eels based on a germline stem cell transplantation technology. The method comprises the following steps: firstly, obtaining a cell suspension from gonads of one-year-old female ricefield eel, centrifuging to obtain germline stem cells, adding a fluorescent label, injecting the germline stem cells into bodies of receptor fish two-year-old male mastacembelus armatus, checking testis of the mastacembelus armatus by using a fluorescence microscope, selecting the receptor mastacembelus armatus subjected to colonization differentiation of the germline stem cells of the ricefield eel, breeding to a breeding season the male gametes for artificial insemination of the ricefield eels are obtained. According to the method for special germ cell allograft and functional gamete generation of ricefield eel, donor-sourced gamete can be generated in a short time, the problems of long ricefield eel breeding period, breeding difficulty and the like are solved, and the method has important significance on ricefield eel research and large-scale breeding.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal genetic breeding technology, specifically relating to a method for generating male gametes of shad based on reproductive stem cell transplantation technology. Background Technology

[0002] Yellow eel ( Monopterus albus It belongs to the class Osteichthyes, order Synbranchia, family Synbranchidae, and genus Sphaerophora. Its flesh is delicious and highly nutritious, making it one of the important freshwater aquaculture economic fish in my country.

[0003] The swamp eel possesses a unique "female-to-male" sex reversal characteristic, making it the only known freshwater fish species to undergo complete sex reversal naturally in adulthood. However, this sex reversal leads to asynchronous development between males and females. Female parents are smaller and have a limited egg-bearing capacity, while male parents require a longer time to mature, making mating difficult and resulting in a shortage of swamp eel fry for farming. Furthermore, wild swamp eel populations may be declining due to overfishing. Currently, artificial breeding techniques for swamp eels are still immature, with low and unstable spawning, fertilization, and hatching rates. Semen collection is difficult, and semen viability decreases rapidly after ex vivo, affecting fertilization effectiveness. Artificial operations are complex and prone to damaging parent fish, impacting parent fish utilization and offspring health. Therefore, the core challenge in swamp eel breeding lies in the difficulty of obtaining and mating parent fish due to its unique sex reversal characteristic, coupled with inefficient and unstable artificial breeding techniques, severely hindering the development of swamp eel farming.

[0004] Germ cell transplantation technology (GCT) refers to the process of transplanting donor germ cells into an allogeneic or xenogeneic recipient body. The donor germ cells are integrated into the recipient's gonads, where they proliferate, differentiate, and eventually develop into functional gametes. The earliest report of GCT technology in fish was in 2003, using rainbow trout (…). Oncorhynchus mykiss Fluorescently labeled primordial germ cells (PGCs) are isolated from the genital ridges of hatched embryos and transplanted into allogeneic or closely related species of salmon. Oncorhynchus masou The embryos hatch inside the abdominal cavity, eventually producing donor-derived gametes, which are then fertilized to produce rainbow trout offspring (Takeuchi et al., 2003, 2004). Through continuous exploration, the GCT (global cyprinid) of fish has now expanded from the Salmonidae family to include dozens of different species, such as Cyprinidae, Characidae, Cichlididae, Perchidae, and Sturgeonidae.

[0005] However, there are currently no research reports or technical data on successful surrogacy using locust transplantation of eels to produce functional gametes.

[0006] As is well known in the art, in fish xenotransplantation research, the selection of the recipient is crucial for the successful transplantation of germ stem cells (GSCs). First, the recipient should possess advantages such as a mature artificial breeding system, a high egg production rate in the female parent, and readily available sperm from the male parent. Second, the recipient fish should be closely related to the donor fish; distant kinship can lead to transplantation failure. Furthermore, immune rejection of endogenous germ cells can significantly reduce transplantation efficiency. Finally, the similarity between the recipient and the swamp eel in terms of breeding season, habits, and conditions should also be considered. Significant differences in habits will prevent successful transplantation to obtain functional gametes. For swamp eels, finding a suitable recipient fish is one of the keys to the success of GCT.

[0007] Currently, xenotransplantation of swamp eels is limited to transplanting germ cell stem cells (GSCs) into zebrafish (Sun et al., 2022) and carp (Tao et al., 2025) after removing primordial germ cells. The transplanted swamp eel GSCs colonize and differentiate in the recipient gonads, forming chimeric gonads. Furthermore, the acquisition of donor fish germ cell stem cells and the processing methods of the recipient are crucial to the implementation of GCT technology. Regarding swamp eels, only our laboratory has reported a method for isolating and culturing germ cell stem cells from the ovaries, intergonadal glands, and testes of swamp eels in patent CN112251399A, and the applicant has reported a method for germ cell xenotransplantation and the detection of chimeric gonads after transplantation in patent CN114592075A.

[0008] It is evident that the current methods for transplanting eels can only achieve the chimeric gonad stage at most. This chimeric gonad only indicates that the donor-derived reproductive stem cells can survive, colonize, and initially proliferate in the recipient gonad, forming a "mixed" state in which donor and recipient cells coexist. It is far from achieving the ultimate goal of producing mature gametes.

[0009] Based on the currently lacking GCT technology for swamp eels, this paper proposes a method for allogeneic transplantation of swamp eel germ cells that can produce functional gametes, which is of great significance for swamp eel research and large-scale farming. Summary of the Invention

[0010] To address the problems existing in the prior art, this invention provides a method for generating male gametes from eels based on reproductive stem cell transplantation technology.

[0011] The present invention adopts the following technical solution: A method for generating male gametes from loach using reproductive stem cell transplantation technology, with the recipient fish being the spiny loach, includes the following steps: S1. Obtaining germline stem cells from eels: S11. Select 1-year-old female swamp eels for dissection to obtain gonads. After squeezing out eggs from the gonads, clean, cut, digest, filter, and wash them. After washing, resuspend the precipitate in PBS to obtain a cell suspension. S12. The cell suspension is subjected to gradient centrifugation to obtain the desired reproductive stem cells. Fluorescent labels are added to the obtained reproductive stem cells, and the cells are resuspended in eel reproductive stem cell culture medium to obtain a reproductive stem cell suspension. S2. Treatment and transplantation of recipient fish: S21. Two-year-old male spiny loaches were kept in water at 31-33℃. From October to December, they were injected with 15-25 mg / kg of busulfan through the genital opening, twice, with an interval of 12-14 days between injections. S22. After the second injection, the giant spiny loach was anesthetized, and a suspension of reproductive stem cells was injected into its gonads through the genital pore using a syringe. The number of reproductive stem cells injected was 1×10⁻⁶. 7 ~2×10 7 Cells / tail; S23. After injection, add 3 ppm potassium dichromate solution to the water body daily for one week; S3. Selection of recipient fish: Randomly select 1-3% of the total number of recipient spiny loaches from the recipient loaches. If there are fewer than one loach, count it as one. Examine their testes with a fluorescence microscope. After observing the colonization and differentiation of the loach reproductive stem cells, raise the recipient spiny loaches until the following breeding season. Collect semen to obtain male gametes that can be used for artificial insemination of loaches. If colonization is not observed, repeat steps S2 and S3 until male gametes that can be used for artificial insemination of loaches are obtained.

[0012] Preferably, in step S11, after the gonads are minced, 3-4 times the volume of TrypLE Express enzyme is added, and the mixture is digested on a shaker at room temperature for 1-1.5 h. The mixture is then filtered through a 40 μm cell filter membrane, and the filtrate is centrifuged to obtain the precipitate. The precipitate is washed three times with pre-cooled PBS containing 3% penicillin-streptomycin, and then resuspended in PBS to obtain a cell suspension.

[0013] Preferably, in step S12, the reproductive stem cells are enriched using the discontinuous Percoll density gradient centrifugation method. The density gradient is set to three concentration layers from top to bottom: 20%, 40%, and 60%. The cells are centrifuged at 800×g for 40 min, and the number of reproductive stem cells in each concentration layer is counted. The layer with the highest number of enriched reproductive stem cells is selected and centrifuged to obtain the reproductive stem cells.

[0014] Preferably, in step S12, the fluorescent label is PKH26 labeling. The method is as follows: the germline stem cells are resuspended in Diluent C, mixed with an equal volume of pre-prepared 2-4 μM PKH26 dye working solution, incubated at room temperature in the dark for 2-5 min, PBS containing 1% BSA is added to terminate the labeling reaction, the supernatant is removed by centrifugation, the cells are washed with PBS and resuspended in eel germline stem cell culture medium to obtain the labeled germline stem cell suspension.

[0015] Preferably, in step S12, the culture medium for the reproductive stem cells of the yellow eel is DMEM or F12 basal culture medium, with 5-8% FBS, 0-10% yellow eel serum, 10 ng / mL LIF, 20 ng / mL FGF, 15 ng / mL EGF and 0-8% KSR added.

[0016] Preferably, in step S3, the testes are examined by fluorescence microscopy three times, at weeks 3-7, 3-4, and 6-8 after the last injection.

[0017] The beneficial effects of this invention are as follows: Currently, most transplantation techniques for swamp eels only reach the chimeric gonad stage. The inventors, after analysis, believe the main reason lies in the inappropriate selection of recipients. These methods use species that are distantly related to swamp eels (such as zebrafish and carp belonging to different orders). This distant relationship between species leads to incompatibility between the recipient's gonadal microenvironment (such as supporting cell signaling and hormonal regulation) and the developmental conditions required by the donor swamp eel's reproductive cells. This prevents the latter from receiving the correct differentiation instructions and nutritional support, resulting in the transplanted cells' development being "stuck" in the early stages, unable to complete the final maturation process.

[0018] This application proposes for the first time that the spiny loach be used as the recipient fish for the GCT technology of swamp eel. Both spiny loach and swamp eel belong to the order Synbranchia, and spiny loach has mature aquaculture techniques. Its breeding season and conditions highly overlap with those of swamp eel. Furthermore, spiny loach and swamp eel are reproductively isolated, meaning spiny loach sperm cannot fertilize swamp eel eggs. Moreover, the average egg-bearing capacity of spiny loach is 3-5 times that of swamp eel, meeting the requirements for recipient selection, such as easy sperm acquisition and high parent stock utilization.

[0019] Current transplantation techniques lack methods to efficiently and specifically eliminate endogenous germ cells from recipient fish. Existing methods can only partially deplete germ cells, and the remaining endogenous stem cells compete with donor cells for ecological niches and nutrient resources. Even if colonization is successful, it is difficult to produce donor-derived gametes.

[0020] To address the transplant rejection problem between different species and to reduce or eliminate the immune response caused by the recipient's own germ cells, this application proposes a specialized treatment method for the giant spiny loach. By increasing the culture temperature and combining it with the injection of busulfan, the germ cells of the recipient giant spiny loach can be depleted, thus preserving the gonadal cell support function to the maximum extent.

[0021] This application provides a comprehensive technical system for maintaining and promoting the colonization, proliferation, and directed differentiation of donor stem cells in xenobiotic environments before and after transplantation, and provides a set of operational guidelines for maintaining high activity of stem cells during transplantation. Experiments have demonstrated that gonads capable of producing functional gametes in swamp eels were obtained from the spiny loach. Swamp eel reproductive stem cells not only complete the entire complex differentiation process from stem cells to fully mature gametes within the spiny loach, but these gametes also possess normal genetic material, morphology, viability, and fertilization capacity, ultimately enabling the production of healthy offspring through fertilization.

[0022] This application establishes a method for allogeneic transplantation of reproductive cells specifically for swamp eels, which can produce functional gametes. Adult fish are used as recipients, and high-precision instruments and techniques for incubation are not required. By selecting "surrogate" parent fish with mature breeding systems and short breeding cycles, gametes from donor sources can be produced in a relatively short period of time (3-6 months), solving the problems of long breeding cycles and breeding difficulties in swamp eels. This method is of great significance to the research and large-scale farming of swamp eels. Attached Figure Description

[0023] Figure 1 For the selection of reproductive stem cell donors from swamp eels, Figure A shows a comparison of the gonadal indices of 1-year-old and 2-year-old swamp eels. It can be seen that the gonads of 1-year-old swamp eels are well-developed. Figure B is an H&E staining image, which shows that there are more female reproductive stem cells in the gonads of 1-year-old swamp eels. Therefore, 1-year-old swamp eels can be selected as reproductive stem cell donors.

[0024] Figure 2 The results of Percoll gradient centrifugation are shown in the figure. A shows the morphology of germline stem cells dispersed in the cell suspension (left) and the cell stratification after centrifugation (right). B shows the expression of specific stem cell marker genes in each Percoll stratum obtained by qPCR.

[0025] Figure 3 To validate and label female germline stem cells from loach donors, Figure A shows the proportion of Vasa-positive cells in the PBS group and 40% Percoll stratification, as indicated by flow cytometry results. Count represents the cell number, and FITC-A represents the fluorescence intensity of the FITC channel, used for indirect reaction. ddx4(Expression levels of stem cell marker genes); B shows the expression of Vasa protein in 40% Percoll layer cells detected by immunofluorescence. The left side shows the Vasa signal (green fluorescence) of germ cells; the stronger the germ cell stemness, the stronger the Vasa signal. The middle shows the Dapi fluorescence signal (blue fluorescence), used to label the cell nucleus, facilitating observation of cell morphology and precise location of germ cells. The right side shows the Merge image, which is a superimposed and fused image of the Dapi and Vasa channels. Scale bar: 10 μm. C shows a representative fluorescence image of PKH26-labeled female germ cells. All experiments were repeated at least twice.

[0026] Figure 4 The results of the treatment of the recipient, the giant spiny loach, are shown in Figure A, which is an H&E staining image of a testis section, showing the effect of combined high temperature and busulfan treatment. SPT: sperm cells. Figure B is an immunofluorescence image showing the expression of Vasa protein in the testes of treated and untreated giant spiny loach. Scale bar: 50 μm.

[0027] Figure 5 The results show the survival, proliferation, and differentiation of donor female germline stem cells in the recipient testes. Figure A is a representative H&E staining image, showing PKH26-labeled female germline stem cells present in the recipient testes shortly after transplantation. Figure B is an immunofluorescence image using Vasa antibody, showing the co-localization of PKH26-labeled female germline stem cells and Vasa-positive germline stem cells in the recipient testes at different time points (white arrows). All experiments were repeated at least twice.

[0028] Figure 6 For molecular identification results, A in the figure is an agarose gel electrophoresis image of the dcps genomic DNA fragment amplified from the specified sample using the loach-specific primers, and B and D are agarose gel electrophoresis images of the genomic DNA fragment amplified from the specified sample using three pairs of swamp eel-specific primers, respectively. In the figure, ZE represents the loach, RE represents the swamp eel, and M is the molecular weight standard.

[0029] Figure 7 The results of embryo testing are shown in the figure. A shows the embryo morphology at different times for the specified hybridization combination. B shows the embryo morphology obtained after fertilizing the swamp eel eggs with sperm produced by the recipient. C shows representative images of normally developing surrogate embryos and control RE×RE embryos. D shows the image of the embryo in the figure in C after hatching from the membrane 7 days after fertilization. In the figure, ZE represents spiny loach and RE represents swamp eel. Scale bar: 1 mm.

[0030] Figure 8For the genetic analysis of surrogate offspring, the results were obtained from the genomic DNA fragments amplified from the embryos 3 hours after fertilization of loach eggs by sperm produced by the recipient. The corresponding amplification products were detected using loach-specific primers (Figure A) and spiny loach-specific primers (Figure B). The PCR experiment was repeated at least three times. The figures show representative data.

[0031] Figure 9 The results are from karyotype analysis. WT indicates wild-type yellow eel. Detailed Implementation

[0032] To facilitate understanding, the technical solution of the present invention will be described in more detail below with reference to specific embodiments.

[0033] Unless otherwise stated, the terms used herein have the meanings commonly understood by those skilled in the art.

[0034] 1. Obtaining germline stem cells (fGSCs) from swamp eels

[0035] Moray eels exhibit sex reversal, being female before becoming male. The vast majority of moray eels are born female, gradually transforming into intersex individuals after spawning, and then into males. Therefore, one-year-old moray eels are all female, while males appear at two or three years of age or later. (See [link to relevant documentation]). Figure 1 One-year-old females have the most reproductive stem cells.

[0036] Select one-year-old female swamp eels, dissect and obtain the gonads, place them in pre-cooled PBS containing 3% penicillin-streptomycin, and under a microscope, try to remove the membranes and visceral fragments adhering to the gonads as cleanly as possible. Squeeze out mature gametes to avoid affecting subsequent separation. The processed gonads are placed in a laminar flow hood, briefly disinfected with 75% alcohol, and then placed in new pre-cooled PBS containing 3% penicillin-streptomycin.

[0037] The gonads were minced to 1 mm using sterilized microscissors and forceps. 3 Transfer the small pieces into a 15 mL centrifuge tube, centrifuge at 2500 r / min for 3 min, discard the supernatant, add 3-4 times the volume of TrypLE Express enzyme, seal with sealing film, and digest on a shaker at room temperature for 1-1.5 h.

[0038] After digestion, return the cells to the laminar flow hood, add PBS to terminate digestion, filter through a 40 μm cell filter membrane, centrifuge the filtrate at 1000 rpm for 2 min, and discard the supernatant. Wash three times with pre-cooled PBS containing 3% penicillin-streptomycin, 2 min each time. After washing, resuspend the cells in 1 mL of PBS to obtain a cell suspension.

[0039] Discontinuous Percoll density gradient centrifugation: First, prepare a 100% SIP solution by mixing nine parts of Percoll stock solution with one part of 1.5M NaCl. Then, dilute the solution to three concentrations (20%, 40%, and 60%) with 0.15M NaCl. Moisten 15 mL centrifuge tubes with fetal bovine serum. After moistening, remove the serum and add it layer by layer from high to low concentration, adhering to the tube surface slowly. Finally, add the cell suspension. The entire gradient centrifugation process requires slow incline and slow deceleration. Set the centrifuge to slow acceleration and slow deceleration modes (both acceleration and deceleration are 1). Centrifuge at 800×g for 40 min at room temperature. After centrifugation, count the number of cells in each concentration layer. Select the layer with the highest enrichment and centrifuge to obtain germline stem cells.

[0040] Fluorescent labeling: To ensure the visual tracking of donor cells after transplantation, the isolated and purified eel germline stem cells need to be fluorescently labeled with PKH26. The specific steps are as follows: Resuspend the cell pellet in Diluent C, mix it with an equal volume of pre-prepared 2-4 μM PKH26 dye working solution, and incubate at room temperature in the dark for 2-5 min; then add PBS containing 1% BSA to terminate the labeling reaction, centrifuge and discard the supernatant, then wash thoroughly with pre-cooled PBS 2-3 times to completely remove unbound dye, and finally resuspend the cells in PBS to obtain the labeled germline stem cell suspension. Throughout the process, it is necessary to keep the cells in the dark and operate quickly to maintain cell viability.

[0041] Reproductive stem cells can be resuspended in a specific culture medium to maintain their dryness. The culture medium is DMEM or F12 basal medium supplemented with 5-8% FBS, 0-10% eel serum, 10 ng / mL LIF, 20 ng / mL FGF, 15 ng / mL LEGF, and 0-8% KSR.

[0042] See Figures 2-3 RT-qPCR was used to detect marker genes of germline stem cells in each layer. ddx4, nanog, ktf4 nanos2 The expression of germline stem cell marker genes was found to be significantly higher in the 40% Percoll layer than in other layers. Further analysis using flow cytometry, alkaline phosphatase staining, and VASA antibody immunofluorescence revealed that the 40% Percoll layer... ddx4 Positive cells were much higher than those in the PBS layer, accounting for 72.52%, and were successfully stained with alkaline phosphatase and labeled with VASA antibody. Therefore, it was proved that the 40% concentration layer was an enrichment layer of swamp eel reproductive stem cells, and the enriched swamp eel reproductive stem cells were fluorescently labeled with PKH26.

[0043] 2. Treatment and transplantation of recipient fish

[0044] Two-year-old spiny loach were used as recipient fish. The loach were raised at a relatively high temperature (31-33℃) for 30 days, and then injected twice with 18 mg / kg busulfan via the genital opening. The injections were spaced 14 days apart. This group was designated as the thermochemical treatment group. The control group was raised at 30℃ for 30 days without any treatment.

[0045] Germ cell transplantation was performed 14 days after the second busulfan injection. Recipient giant spiny loaches were anesthetized with MS-222 for 2–3 minutes and then transferred to the surgical platform. Under anesthesia, a suspension of PKH26-labeled fGSCs was gently injected into the gonads through the genital pore using a 1 mL syringe. The number of germ cell stem cells injected was 1.5 × 10⁻⁶. 7 Cells / tail

[0046] After injection, 3 ppm of potassium dichromate was sprayed into the entire pond of the giant spiny loach farming area daily for one week to complete the transplantation.

[0047] After transplantation, the giant spiny loaches were artificially reared until the following breeding season (June). During this period, the testes and semen of the recipients were observed using a fluorescence microscope to determine whether they were marked with PKH26. The recipient giant spiny loaches with fluorescent markers were selected, and their semen was collected for artificial insemination of the yellow eel.

[0048] Specifically, recipient fish representing 1-3% of the total number of recipient spiny loaches are randomly selected for examination at weeks 3-7, 3-4, and 6-8 after the last transplant (less than one fish is counted as one). Only when colonization is observed in all three examinations will the recipient spiny loaches continue to be raised until the following breeding season to collect semen. Otherwise, it means the transplant has failed and a new transplant is required.

[0049] In this transplantation embodiment, the following tests are performed: 1) One to two transplanted samples were collected from both the experimental and control groups. The gonads were dissected and removed, fixed overnight in 4% PFA at 4°C, and then dehydrated and embedded in paraffin. Gonad sections were stained with hematoxylin and eosin (HE) to examine the depletion of germ cells in the recipient *Solanum lycopersicum* gonads. Furthermore, in conjunction with immunofluorescence (IF) experiments, the fixed gonads were dehydrated, embedded in OCT, frozen, and sectioned, followed by Vasa antibody immunofluorescence assays to further examine the depletion of germ cells in the recipient gonads.

[0050] See results Figure 4 Figure A shows an H&E staining image of a testis section, illustrating the effect of combined high-temperature and busulfan treatment. Figure B shows an immunofluorescence image, illustrating the expression of Vasa protein in the testes of treated and untreated giant spiny loach. Compared to the control group, most germ cells disappeared in the testes of the treated group. Immunofluorescence (IF) experiments showed that, compared to the untreated control group, the treated group had fewer Vasa-positive germ cell stem cells but more Vasa-positive cells.

[0051] 2) Detection of donor cell colonization, proliferation, and migration

[0052] To investigate the fate of donor cells transplanted into the recipient gonads, 1-2 samples from both the experimental and control groups were collected for immunofluorescence experiments at 15, 25, and 55 days after the last germline stem cell injection. Specifically, recipient fish were anesthetized. The gonads were dissected and removed, fixed overnight in PFA at 4°C, then dehydrated, embedded in OCT, and frozen sections were prepared. Immunofluorescence assays were then performed, and Vasa antibody and PKH26 were used to detect donor cell colonization, proliferation, and migration. Sections were dried at room temperature for 30 min, then fixed in a slide washing tank with 4% PFA for 20 min. The sections were gently removed and washed twice with 1×PBS for 10 min each time, avoiding vigorous shaking to prevent detachment from the slide. The tissue sections were circled with an immunohistochemical pen, and an appropriate amount of blocking solution was added within the circle. The sections were incubated at room temperature for 2 h.

[0053] Primary antibody incubation: Dilute the antibody according to the antibody instructions. The usual antibody concentration is 250 ng / mL. The antibody used in the experiment is diluted at a ratio of primary antibody / 1×blocking buffer = 1 / 500. Remove the blocking solution from the tissue slide, add an appropriate amount of primary antibody dilution buffer, and incubate overnight at 4°C. Recover the primary antibody (it can be reused approximately 5 times), and wash three times with 1×PT for 15 min each time. Secondary antibody incubation: Dilute the secondary antibody at a ratio of secondary antibody / 1×blocking buffer = 1 / 500. Add the secondary antibody dilution buffer to the tissue area of ​​the slide and incubate at room temperature for 2 hours, avoiding light. Depending on the thickness of the section, overnight incubation at 4°C is also possible. After the secondary antibody binding reaction is complete, wash three times with 1×PT for 15 min each time. DAPI counterstaining: Prepare the stock solution (1 mg / mL) according to the DAPI reagent instructions. Dilute with 1×PBS (1 μg / mL) before staining. Add the solution to the slide and stain for 2-5 hours; the thicker the section, the longer the staining time. Wash three times with 1×PT, 15 min each time. Aspirate excess liquid, keeping the sample moist throughout the process. Mount the slide with an anti-fluorescence quencher and take photos for record-keeping.

[0054] Figure 5The image shows the survival, proliferation, and differentiation of donor female germline stem cells in the recipient's testes. Figure A is a representative H&E staining image showing PKH26-labeled female germline stem cells present in the recipient's testes shortly after transplantation (day 3). Figure B is an immunofluorescence image using Vasa antibody, showing the co-localization of PKH26-labeled female germline stem cells and Vasa-positive germline stem cells in the recipient's testes at different time points; white arrows indicate PKH26-labeled cells.

[0055] As can be seen, PKH26-labeled cells were observed in the testes of the recipient giant spiny loach on day 25 post-transplantation. On day 55 post-transplantation, PKH26-labeled cells of varying sizes were observed. However, most of the PKH26-labeled cells were not labeled with Vasa antibody immunostaining, indicating that they were differentiating into mature sperm (such as spermatocytes, cytokines, etc.).

[0056] Insemination experiment

[0057] Since the testes of the recipient giant spiny loach contain both endogenous germ cells and donor-derived germ cells, artificial insemination was performed on wild yellow eel eggs using sperm produced by the recipient. Simultaneously, wild yellow eel eggs were fertilized with sperm from both wild yellow eels and wild giant spiny loach, serving as positive and negative controls, respectively.

[0058] Semen was collected using a 1 mL sterile syringe and frozen before fertilization. Sperm were activated with 0.5 mL of sperm activation solution, stirred for 20 seconds, and then a suitable amount of eggs (sperm:egg = 5000:1) were added. The fertilized eggs were then collected in a dedicated flowing-water hatching tank, maintaining a constant water temperature of 26℃, supplemented with continuous oxygenation or micro-flow circulation to ensure sufficient dissolved oxygen (>5 mg / L) and clean water quality. The hatching water required thorough aeration, with a neutral pH, and should be kept in the dark and static during the entire incubation process. Under these conditions, the fertilized eggs underwent developmental stages including cleavage, blastocyst, and gastrula. The embryo gradually formed a tail bud and began to wriggle, eventually hatching into fry approximately 5-7 days (120-170 hours) after fertilization. Throughout the process, unfertilized white or moldy eggs were promptly removed to prevent the spread of fungus, thus significantly improving the hatching success rate.

[0059] Results: In the positive control group, the self-fertilization rate of the swamp eel was 78.77% and the hatching rate was 21.23%, indicating that the swamp eel eggs and sperm used in the experiment were viable. In the negative control group, both the fertilization rate and hatching rate were 0, verifying the reproductive isolation between the spiny loach and the swamp eel, and that the spiny loach sperm could not fertilize the swamp eel eggs. Therefore, the interference of the spiny loach can be ruled out in subsequent experiments. When using a mixed semen containing both spiny loach sperm and swamp eel sperm produced by surrogate spiny loach for fertilization, a fertilization rate of 3.42% and a hatching rate of 1.75% were obtained.

[0060] Molecular identification

[0061] Using a DNA extraction kit, extract total genomic DNA from sperm, embryos, larvae, and fins according to the manufacturer's instructions. Sperm should be extracted immediately for total genomic DNA or frozen at -80°C to avoid repeated freeze-thaw cycles that could degrade the DNA. Since these are pure reproductive cells with high DNA content, but the sample size is small, cross-contamination must be prevented. Select embryos from the gastrula to heartbeat stages, wash thoroughly with PBS, and completely remove the yolk sac (yolk is rich in lipids and proteins, which are major inhibitors of downstream DNA extraction and PCR reactions and must be removed). Take intact individuals or heads within 7 days of hatching, avoiding the eyes and yolk sac. Larvae are small and are often ground whole, avoiding the pigmented eyes and residual yolk sacs (again to avoid PCR inhibitors). Fins are muscle and connective tissue with high DNA yield and quality, making them ideal material for genotyping. Cut 2-3 mm² tail fin rays from 3-5 month old eels, fix with ethanol, or freeze fresh.

[0062] This study used species-specific primers designed based on NCBI's Primer-Blast technique to clearly distinguish the DNA origins of the donor (eel, RE) and recipient (spiny loach, ZE) using PCR technology. For the spiny loach, primers playing a key role in sex differentiation were selected. gsdf Genes, involved in epigenetic regulation kdm6b Using genes and the dcps gene, which serves as a housekeeping gene, as specific targets, these primers can effectively detect the recipient's own genetic background. The longer dcps fragment (1084 bp) can also be used to assess DNA integrity. Accordingly, homologous but sequence-specific primers were designed for the swamp eel. gSdf and kdm6b Gene primers serve as molecular markers of donor origin; simultaneously, extremely short amplification fragments (94 bp) are introduced. amh Gene primers, crucial for spermatogenesis, are designed for highly sensitive detection even from partially degraded samples such as sperm or fixed tissue, serving as an internal quality control measure. This primer set together constitutes a reliable identification system capable of accurately determining the species origin of chimeric gonads, semen, and offspring, providing key molecular evidence for the success of xenogeneic germ cell transplantation.

[0063] Primer information is shown in Table 1. Species specificity was verified using total genomic DNA obtained from semen, embryos, larvae, and fins.

[0064] Table 1. Specific primers used for amplification

[0065] PCR: PCR was performed using 20 μL of reaction solution (6 μL ddH₂O₂, 10 μL 2 × Taq Master Mix), 1 μL of forward primer (1 μM), 1 μL of reverse primer (1 μM), and 2 μL of DNA template. The reaction program was as follows: initial denaturation at 95℃ for 5 min, denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 1 min, repeated 35 times at the denaturation step, and a final extension at 72℃ for 3 min. The product from the first PCR was used as a template for a second PCR. The obtained PCR products were detected by 1% agarose gel electrophoresis to determine the origin of gametes or progeny.

[0066] See Figure 6 PCR results showed that two eel-specific molecules could be amplified to 450 bp in the receptor. gsdf This fragment demonstrates that the donor germ cells successfully developed in the recipient. (914bp) dcps The fragments can also be amplified, indicating that both receptors' own germ cells are functioning normally and can produce donor and receptor-derived sperm. kdm6b Similar results were obtained with primers.

[0067] See Figure 7 In this experiment, no blastocysts formed by the sperm of the giant spiny loach and the eggs of the yellow eel were observed at 3 h embryonic development (hpf). Since giant spiny loach eggs can easily self-fertilize (ZE×ZE), sperm quality issues could be ruled out, indicating a strict reproductive isolation between the giant spiny loach and the yellow eel. Artificial fertilization was performed using sperm from surrogate giant spiny loach with yellow eel eggs. The results showed that approximately 1 / 3 to 1 / 4 of the eggs were fertilized at 3 hpf, and about 1 / 3 to 1 / 4 of the embryos survived and had normal morphology on day 3. The embryos successfully hatched 6-7 days after fertilization (dpf), and their morphology was indistinguishable from that of the control yellow eel embryos. See [link to relevant documentation]. Figure 8 PCR analysis was performed on embryos hatched at 7 days post-hatching (dpf) using primers specific to both spiny loach and swamp eel. Analysis of the PCR products showed that each embryo amplified only the swamp eel-specific primers. kdm6b The fragment (1003 bp) did not amplify the specificity of the giant spiny loach. kdm6b、gsdf Gene fragments proved that the embryo's genetic material came entirely from the yellow eel, with no genetic components from the giant spiny loach.

[0068] Karyotype analysis

[0069] Five wild-type swamp eels and swamp eels that produced gametes through surrogacy (60 days post-fertilization) were selected and injected three times with 2 μL / g hemagglutinin (PHA) at 12-hour intervals. Three hours after the last PHA injection, 0.2 μL / g colchicine was injected. Two hours later, the swamp eels were dissected, their kidneys were collected, and the cells were minced. The cells were hypotonic with 0.75% NaCl solution for 70 minutes to induce cell swelling and chromosome dispersion. Cell morphology was fixed overnight at 4°C using a fixative solution (a 3:1 mixture of methanol and glacial acetic acid). The fixed cell suspension was then dropped onto pre-cooled glass slides, baked for 2-3 hours, stained with Giemsa stain, and finally photographed and counted under a microscope.

[0070] See Figure 9 It can be seen that the offspring of surrogate gametes have the same chromosome number as wild-type eels (2n=24).

[0071] In conclusion, the surrogate spiny loach produces sperm derived from the yellow eel, and healthy offspring can be obtained by fertilizing the eggs of the yellow eel with this sperm.

[0072] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for generating male gametes from swamp eels based on reproductive stem cell transplantation technology, characterized in that, The recipient fish is the spiny loach, and the following steps are included: S1. Obtaining germline stem cells from eels: S11. Select 1-year-old female swamp eels for dissection to obtain gonads. After squeezing out eggs from the gonads, clean, cut, digest, filter, and wash them. After washing, resuspend the precipitate in PBS to obtain a cell suspension. S12. The cell suspension is subjected to gradient centrifugation to obtain the desired reproductive stem cells. Fluorescent labels are added to the obtained reproductive stem cells, and the cells are resuspended in eel reproductive stem cell culture medium to obtain a reproductive stem cell suspension. S2. Treatment and transplantation of recipient fish: S21. Two-year-old male spiny loaches were kept in water at 31-33℃. From October to December, 15-25 mg / kg of busulfan was injected through the genital opening twice, with an interval of 12-14 days between injections. S22. After the second injection, the giant spiny loach was anesthetized, and a suspension of reproductive stem cells was injected into its gonads through the genital pore using a syringe. The number of reproductive stem cells injected was 1×10⁻⁶. 7 ~2×10 7 Cells / tail; S23. After injection, add 3 ppm potassium dichromate solution to the water body daily for one week; S3. Selection of recipient fish: Randomly select 1-3% of the total number of recipient spiny loaches from the recipient loaches. If there are fewer than one loach, count it as one. Examine their testes with a fluorescence microscope. After observing the colonization and differentiation of the loach reproductive stem cells, raise the recipient spiny loaches until the following breeding season. Collect semen to obtain male gametes that can be used for artificial insemination of loaches. If colonization is not observed, repeat steps S2 and S3 until male gametes that can be used for artificial insemination of loaches are obtained.

2. The method for generating male eel gametes based on reproductive stem cell transplantation technology as described in claim 1, characterized in that, In step S11, after the gonads are minced, 3-4 times the volume of TrypLE Express enzyme is added, and the mixture is digested on a shaker at room temperature for 1-1.5 hours. The mixture is then filtered through a 40 μm cell filter membrane, and the filtrate is centrifuged to obtain the precipitate. The precipitate is washed three times with pre-cooled PBS containing 3% penicillin-streptomycin, and then resuspended in PBS to obtain a cell suspension.

3. The method for generating male eel gametes based on reproductive stem cell transplantation technology as described in claim 1, characterized in that, In step S12, the germline stem cells are enriched using the discontinuous Percoll density gradient centrifugation method. The density gradient is set to three concentration layers from top to bottom: 20%, 40%, and 60%. The cells are centrifuged at 800×g for 40 min. The number of germline stem cells in each concentration layer is counted. The layer with the highest number of enriched germline stem cells is selected and centrifuged to obtain the germline stem cells.

4. The method for generating male eel gametes based on reproductive stem cell transplantation technology as described in claim 1, characterized in that, In step S12, the fluorescent labeling is PKH26 labeling. The method is as follows: the germline stem cells are resuspended in Diluent C, and an equal volume of 2-4 μM PKH26 dye working solution is mixed. After incubation at room temperature in the dark for 2-5 min, PBS containing 1% BSA is added to terminate the labeling reaction. The supernatant is removed by centrifugation, the cells are washed with PBS and resuspended in eel germline stem cell culture medium to obtain the labeled germline stem cell suspension.

5. The method for generating male eel gametes based on reproductive stem cell transplantation technology as described in claim 1, characterized in that, In step S12, the culture medium for the reproductive stem cells of the swamp eel is DMEM or F12 basal medium, with 5-8% FBS, 0-10% swamp eel serum, 10 ng / mL LIF, 20 ng / mL FGF, 15 ng / mL EGF and 0-8% KSR added.

6. The method for generating male eel gametes based on reproductive stem cell transplantation technology as described in claim 1, characterized in that, In step S3, the testes were examined three times using a fluorescence microscope, at weeks 3-7, 3-4, and 6-8 after the last injection.

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