Methods for producing heterologous eggs and heterologous gametes in fish

Abstract

The present invention is a method for producing a reproductive tissue, comprising the steps of: separating down into cell units each of a reproductive tissue of the same species as an immunodeficient fish and a reproductive tissue of a fish of a different species therefrom, and then combining the cells to obtain an aggregate; and the step of transplanting the aggregate under the skin or into an ovary of the immunodeficient fish.

Description

[Technical Field]

[0001] The present invention relates to a method for producing allogeneic eggs and heterogeneous gametes in fish, and more particularly to a method for producing allogeneic eggs and heterogeneous gametes by transplantation into immunodeficient adult fish. This application claims priority based on Japanese Patent Application No. 2020-074968, filed on April 20, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] Given the decline in natural fishery resources, there is a need for technology to produce reproductive tissue using surrogate parent fish. However, even if reproductive tissue from a different individual of the same species or a different species of fish is transplanted into a surrogate parent fish, there is a problem in that the transplanted tissue is difficult to take root and grow in the body of the surrogate parent fish due to immune rejection.

[0003] To address this problem, the inventors discovered that by transplanting allogeneic tissue of the same species subcutaneously into fish in which the rag1 gene is not functional, the transplanted tissue can take root and grow without immune rejection, and that allogeneic sperm can be produced from the transplanted testicular tissue (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6472960 Summary of the Invention [Problem to be solved by the invention]

[0005] In addition to the technology disclosed in Patent Document 1, there is a need for more efficient engraftment and growth of transplanted ovarian tissue from individuals of the same species, and furthermore, reproductive tissue from different species of fish. The present invention has been made in consideration of the above circumstances, and provides a method for producing allogeneic eggs or heterogeneous gametes by efficiently engrafting and growing transplanted ovarian tissue from an individual of the same species or reproductive tissue from a different species of fish. [Means for solving the problem]

[0006] The present invention includes the following aspects. [1] A method for producing reproductive tissue, comprising the steps of isolating homologous reproductive tissue of an immunodeficient fish and heterologous reproductive tissue of a heterologous fish down to the cellular level, then mixing them to obtain an aggregate, and transplanting the aggregate subcutaneously or intraovarianly of the immunodeficient fish. [2] The method for producing reproductive tissue described in [1], further comprising a step of removing corresponding host germ cells within the immunodeficient fish. [3] A method for producing reproductive tissue, comprising the steps of transplanting reproductive tissue of a fish of the same species as an immunodeficient fish into the ovaries of the immunodeficient fish, and removing the corresponding host reproductive cells within the immunodeficient fish. [4] A method for producing reproductive tissue described in any one of [1] to [3], in which the immunodeficient fish has suppressed or lost function of the rag1 gene or its homolog, or the Rag1 protein or its homolog. [5] A method for producing gametes, comprising obtaining gametes from the reproductive tissue obtained by the method for producing reproductive tissue according to any one of [1] to [4]. [Effects of the Invention]

[0007] According to the present invention, the transplanted reproductive tissue of an individual of the same species or a different species of fish can be efficiently engrafted and grown, and gametes of an individual of the same species or a different species of fish can be produced. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows a method for producing mature eggs by ovarian transplantation from zebrafish ovary-derived germline stem cells or oogonia, or early oocytes into individuals of the same species in Experimental Examples 1 and 2. [Figure 2]These are the results of transplanting germline stem cells or oogonia, or ovarian regions containing early oocytes, derived from the zebrafish ovaries (germinal zones) in Experimental Example 1 into the ovaries of the same immunodeficient rag1 mutant mouse. Six weeks after transplantation, mature oocytes derived from the transplanted cells were observed. [Figure 3] These are the results of an investigation into the efficiency of oocyte removal in the 254A::ntr strain (a strain resulting from the cross between the 254A strain, which expresses GAL4 in early oocytes, and a strain expressing nitroreductase (ntr) under the UAS promoter), by varying the concentration of Metrodinazole in Experimental Example 2. (A) Image confirming GAL4 expression in the 254A strain by EGFP expression. (B) Image of the ovaries of the 254A::ntr strain after two cycles of treatment with 10mM, 5mM, or 2.5mM Metrodinazole for three days followed by normal rearing for four days. The top image shows the ovaries after laparotomy of a zebrafish, and the bottom image shows the excised ovaries. It was found that mature oocytes were removed in a concentration-dependent manner. [Figure 4] FIG. 1 shows that fertilized embryos (indicated by arrows) derived from transplanted zebrafish ovary-derived germline stem cells or oogonia, or early oocytes were obtained from the immunodeficient 254A::ntr strain by metrodinazole treatment in Example 2. [Figure 5] FIG. 1 shows a method for producing heterologous gametes by subcutaneously or intraovarian transplantation of aggregates of germ tissue cells from individuals of the same species and fish of a different species in Examples 3, 4, and 5. [Figure 6] This figure shows that Honmoroko testis tissue and zebrafish vas::egfp testis tissue from Example 3 were each isolated down to the cellular level, then mixed to form an aggregate, which was then subcutaneously transplanted into an immunodeficient zebrafish to obtain Honmoroko sperm. (A) Image of the aggregate two months after transplantation. (B) Image of a section of the aggregate. GFP-negative Honmoroko spermatogonia, spermatocytes, and sperm are observed. [Figure 7]This figure shows that Honmoroko ovarian tissue and zebrafish vas::egfp testicular tissue from Example 4 were each isolated down to the cellular level, then mixed to form an aggregate, which was then subcutaneously transplanted into an immunodeficient zebrafish to obtain Honmoroko sperm. (A) Image of the aggregate two months after transplantation. (B) Image of a section of the aggregate. GFP-negative Honmoroko spermatogonia, spermatocytes, and sperm are observed. [Figure 8] This figure shows that, as in Example 5, Honmoroko testis tissue and zebrafish meioc mutant testis tissue, which do not differentiate into spermatogonial stem cells, were isolated down to the individual cells and then mixed to form an aggregate. This aggregate was then subcutaneously transplanted into an immunodeficient zebrafish, and differentiation into Honmoroko sperm and oocytes was confirmed. (A) A cross-section of the aggregate two months after transplantation. (B) An enlarged view of Honmoroko spermatogonia, spermatocytes, and sperm. (C) An enlarged view of Honmoroko oocytes. DETAILED DESCRIPTION OF THE INVENTION

[0009] <<Method for producing reproductive tissue>> First Embodiment The method for producing reproductive tissue in this embodiment includes step 1 of isolating homologous reproductive tissue of an immunodeficient fish and heterologous reproductive tissue of a heterologous fish down to the cellular level and then mixing them to obtain an aggregate, and step 2 of transplanting the aggregate subcutaneously or into the ovary of the immunodeficient fish. The method for producing reproductive tissues and heterologous gametes of this embodiment preferably further comprises a step 3 of removing corresponding host germ cells in the immunodeficient fish.

[0010] [Process 1] In step 1, allogeneic reproductive tissue from immunodeficient fish and heterogeneous reproductive tissue from heterogeneous fish are isolated down to the cellular level, and then mixed at a ratio of preferably 10:1, more preferably 5:1, such that the allogeneic reproductive tissue is in excess relative to the heterogeneous reproductive tissue to obtain an aggregate. In this step, heterogeneous fish germ cells are encapsulated by cells of the allogeneic reproductive tissue. Although immunodeficient fish (rag1 mutants) reject heterogeneous fish tissue from distant lineages, this prevents contact between host immune cells and heterogeneous fish germ cells, thereby avoiding immune rejection of the heterogeneous fish germ cells.

[0011] Immunodeficient fish are not particularly limited as long as they do not undergo immune rejection when tissue from an allogeneic individual is transplanted into them, and examples include fish treated with immunosuppressants, fish whose immune systems have been destroyed by gamma-ray irradiation, genetically modified fish in which the function of genes involved in the immune system has been suppressed or eliminated, etc. Genetically modified fish include, for example, fish in which the function of the rag1 gene or its homolog, or the function of the Rag1 protein or its homolog, has been suppressed or eliminated.

[0012] "Suppressed Rag1 protein function" refers to a state in which the original function of Rag1 protein is partially lost, and "loss of Rag1 protein function" refers to a state in which the original function of Rag1 protein is completely lost.

[0013] The suppression or loss of function of the Rag1 protein can also occur by suppressing or losing expression of the rag1 gene.

[0014] Suppression of rag1 gene expression means that the amount of rag1 gene product is suppressed in immunodeficient fish compared to wild-type control fish. The expression of the rag1 gene can be suppressed by introducing a nucleic acid sequence that induces the expression of an RNAi-inducing nucleic acid, antisense nucleic acid, aptamer, or ribozyme against the rag1 gene into fish, and then performing gene knockdown, etc.

[0015] Loss of rag1 gene expression means that the rag1 gene product is lost in fish. Loss of function of the gene product, Rag1 protein, can be caused, for example, by introducing a mutation into the rag1 gene to disrupt the rag1 gene. Mutations can be generated by partial or complete deletion, substitution, or insertion of any sequence in the rag1 gene or the gene expression regulatory region. These mutations can be introduced using techniques such as treatment with mutagens, ultraviolet irradiation, gene targeting using homologous recombination technology, gene knockout, and conditional knockout. Genome editing technology may also be used for gene targeting and gene knockout.

[0016] In zebrafish, rag1 mutants have been isolated and reported to be unable to form mature T and B cells (BMC Immunol. 2009 Feb 3;10:8. doi: 10.1186 / 1471-2172-10-8. Characterization of rag1 mutant zebrafish leukocytes. Petrie-Hanson L1, Hohn C, Hanson L.).

[0017] The fish to be made immunodeficient is not particularly limited, and is selected from the viewpoints of ease of genetic manipulation, being a closely related species to the fish that retains the reproductive tissue to be transplanted, etc. Examples include zebrafish, medaka, goldfish, rainbow trout, pufferfish, mackerel, anchovy, etc.

[0018] The allogeneic reproductive tissue used in step 1 may be either an allogeneic or heterogeneic reproductive tissue, as long as it can encase the heterogeneous fish reproductive cells and protect them from host immune cells. Examples of reproductive tissues in the same species of fish include testes, testicular epithelium, ovaries, and ovarian epithelium.

[0019] The heterologous fish reproductive tissue used in step 1 is reproductive tissue derived from a fish belonging to a species different from the immunodeficient fish host, and serves as the source of the gametes to be produced in this embodiment. From the viewpoint of transplantation efficiency, it is preferable that the heterologous fish from which the heterologous fish reproductive tissue is derived and the immunodeficient fish host are closely related species. Examples of reproductive tissues in heterologous fish include testis, testicular epithelium, ovary, and ovarian epithelium.

[0020] In step 1, the reproductive tissue of an individual of the same species as the immunodeficient fish and the reproductive tissue of a different species of fish are each isolated to the cellular level and then mixed together. Methods for isolating reproductive tissues down to the cellular level include conventional methods, such as collagenase treatment, etc. The isolated cells include undifferentiated germ cells such as spermatogonia and oogonia, differentiated germ cells such as spermatocytes and oocytes, and somatic cells derived from the gonad. As will be described later in the Examples, when undifferentiated germ cells obtained by isolating germ tissue from a heterologous fish are mixed with gonad somatic cells obtained by isolating germ tissue from the same individual, the transplanted undifferentiated germ cells derived from the germ tissue of the heterologous fish are induced to differentiate depending on the somatic cells derived from the germ tissue of the same individual and the transplantation site. For example, even if the undifferentiated germ cells obtained by isolating germ tissue from a heterologous fish are ovarian germline stem cells, if the cells obtained by isolating germ tissue from the same individual are testicular somatic cells and transplanted subcutaneously, the ovarian germline stem cells will be induced to differentiate into sperm after transplantation. In step 1, the cells are mixed together and then brought into contact with each other by centrifugation or the like, and then cultured to allow the cells to adhere to each other and obtain an aggregate.

[0021] [Process 2] Step 2 is a step of transplanting the aggregate mass subcutaneously or intraovarianly into an immunodeficient fish. Adult immunodeficient fish can be used as transplant hosts. For example, in the case of zebrafish, it is preferable to use fish with a body length of 3 cm or more or fish that are 60 days or older. Subcutaneous transplantation can be achieved, for example, by cutting the epidermis on the side of an anesthetized host with a scalpel, inserting tweezers through the incision between the epidermis and muscle to create a space into which the aggregate mass can be transplanted. Ovarian transplantation can be achieved, for example, by using a host with a body length of 3 cm or more whose abdomen has expanded due to ovarian growth. The abdomen of an anesthetized host is preferably incised to expose the ovary, an incision is made on the surface of the ovary, and the aggregate mass or ovarian tissue is inserted through the incision with tweezers.

[0022] The immunodeficient fish receiving the transplanted mass are preferably reared in a buffer solution containing an antibiotic in the dark without food for a certain period of time to allow the wound to heal. After the wound has healed, the fish may be reared in the usual manner.

[0023] [Process 3] Step 3 is a step of removing corresponding host germ cells in the immunodeficient fish. In this embodiment, "corresponding host germ cells" refers to host germ cells corresponding to the reproductive tissue to be produced. For example, when the reproductive tissue to be produced is the testis, host spermatogonia, spermatocytes, and sperm cells are removed in step 3. When the reproductive tissue to be produced is the ovary, host oogonia and oocytes are removed in step 3. Methods for removing host germ cells are not particularly limited, and include, for example, methods that conditionally induce cell death in a specific cell-specific manner in the host through genetic modification. Specifically, as described below in the Examples, a method is exemplified in which fish that express nitroreductase specifically in germ cells are constructed and the fish are reared in an aquarium containing metrodinazole, whereby metrodinazole is decomposed and becomes toxic, thereby removing germ cells. Furthermore, by using fish that express nitroreductase specifically in germ cells as the germ tissue of individual fishes when preparing aggregates, gametes of heterologous fish germ tissues can be efficiently obtained from the aggregates. Other methods for removing host germ cells include injecting a deadend morpholino into a one-cell embryo to prevent the development of host germ cells in advance, and surgically removing the host gonads. In this embodiment, it is preferred that the corresponding host germ cells are removed by 10% or more, more preferably by 20% or more, even more preferably by 50% or more, and particularly preferably by 100%. If the number of corresponding host germ cells is large compared to the transplanted cells, the transplanted cells may lose out in the proliferation competition and be selected out. Therefore, by reducing the number of host germ cells in step 3, the engraftment and growth of the transplanted cells can be promoted, and heterologous gametes can be produced.

[0024] Second Embodiment The method for producing reproductive tissue in this embodiment includes step 4 of transplanting reproductive tissue from a fish of the same species as the immunodeficient fish into the ovaries of the immunodeficient fish, and step 5 of removing the corresponding host reproductive cells within the immunodeficient fish.

[0025] [Step 4] Step 4 is a step of transplanting reproductive tissue from an individual of the same species as the immunodeficient fish into the ovaries of the immunodeficient fish. This is the same as step 2, except that the transplant target is reproductive tissue from an individual of the same species as the immunodeficient fish.

[0026] [Step 5] Step 5 is a step of removing the corresponding host germ cells in the immunodeficient fish. Step 5 is similar to step 3, and by reducing the number of host germ cells, it is possible to promote the engraftment and growth of the reproductive tissue of the transplanted allogeneic fish and produce eggs derived from the transplanted cells.

[0027] <<Gamete production method>> In this embodiment, gametes are obtained from the reproductive tissue obtained by the above-described method for producing reproductive tissue. Examples of gametes include eggs and sperm.

[0028] <<Immunodeficient fish>> The immunodeficient fish of this embodiment is a fish having transplanted fish reproductive tissue. Examples of the fish reproductive tissue to be transplanted include reproductive tissue from a different fish species and reproductive tissue from an individual of the same species. From the viewpoint of promoting the engraftment and growth of the transplanted germ cells, it is preferable that 10% or more of the corresponding host germ cells have been removed, more preferably 20% or more, even more preferably 50% or more, and particularly preferably 100%. In other words, the immunodeficient fish of this embodiment preferably has 90% or less of the corresponding host germ cells, more preferably 80% or less, even more preferably 50% or less, and particularly preferably no germ cells at all, compared to before transplantation. Furthermore, in the immunodeficient fish of this embodiment, the function of the rag1 gene or a homolog thereof, or the function of the Rag1 protein or a homolog thereof is preferably suppressed or lost. The method for producing immunodeficient fish of this embodiment is not particularly limited, but examples thereof include the method of the first or second embodiment of the above-mentioned <<Method for producing reproductive tissue>>. [Example]

[0029] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0030] [Experimental Example 1] The experimental procedure is shown in Figure 1. Tissue fragments containing oogonia and early oocytes were isolated from the ovaries of sox17::egfp individuals expressing GFP in early germ cells and transplanted into the ovaries of immunodeficient rag1 mutant females. Six weeks later, we confirmed the development of oocytes derived from the transplanted sox17::egfp oogonia (Figure 2). However, ovulation of oocytes derived from these cells was not confirmed. This suggests that the transplanted cells were unable to grow sufficiently due to competition with the endogenous host oocytes.

[0031] [Experimental Example 2] We isolated the 254A line, which expresses GAL4 specifically in early oocytes, from a zebrafish gene-enhancer trap line (Figure 3A). The resulting line (254A::ntr line) was obtained by crossing the 254A line with a line carrying the nitroreductase (ntr) gene downstream of the UAS promoter. The 254A line expresses nitroreductase specifically in early oocytes. Metrodinazole is degraded by nitroreductase and becomes toxic. Specifically, when metrodinazole is added to the rearing tank, its degradation products become toxic due to the nitroreductase specifically expressed in early oocytes, resulting in oocyte elimination. We examined the oocyte elimination efficiency of the 254A line by varying the concentration of metrodinazole added. As shown in Figure 3B, oocyte elimination was confirmed in a concentration-dependent manner.

[0032] The 254A::ntr strain was crossed with the immunodeficient rag1 mutant strain to establish recipient individuals for transplantation. As in Experiment 1, tissue fragments containing oogonia and early oocytes were isolated from the ovaries of sox17::egfp mice and transplanted into the ovaries of 254A::ntr+rag1 mutant females. Starting 7 days later, the mice were treated with 10 mM Metrodinazole for 3 days and then reared normally for 4 days. After mating with males, GFP-positive embryos were confirmed in 2 of 147 mice (see Figure 4).

[0033] [Experimental Example 3] The experimental procedure is shown in Figure 5. Enlarged testes from vas::egfp zebrafish were isolated to individual cells using collagenase. Testes from Honmoroko were also isolated and isolated to individual cells using collagenase. These two types of testicular cells were mixed at a ratio of 5:1 (zebrafish:honmoroko), centrifuged, and cultured to obtain aggregates. These aggregates were then subcutaneously transplanted into male rag1 mutants. The recipients were kept in 0.4x PBS containing 10 μg / mL gentamicin in the dark for four days without food. They were then kept in normal water. Two months after transplantation, the transplanted mass was recovered and immunostained using an anti-GFP antibody. The results are shown in Figure 6. As shown in Figure 6, the presence of GFP-negative Honmoroko spermatogonia and spermatocytes, and the presence of sperm within the same lobule, confirmed that sperm were being formed from Honmoroko testicular germline stem cells.

[0034] [Experimental Example 4] Enlarged testes were isolated from vas::egfp zebrafish and dissociated into individual cells using collagenase. Ovarian epithelium was also isolated from Honmoroko and dissociated into individual cells using collagenase. These two types of cells were mixed at a ratio of 5:1 (zebrafish:honmoroko), centrifuged, and cultured to obtain aggregates. These aggregates were then subcutaneously transplanted into male rag1 mutants (see Figure 5). The recipients were kept in 0.4x PBS containing 10 μg / mL gentamicin in the dark for 4 days without food. They were then kept in normal water. Two months after transplantation, the transplanted mass was recovered and immunostained using an anti-GFP antibody. The results are shown in Figure 7. As shown in Figure 7, the presence of GFP-negative Honmoroko spermatogonia and spermatocytes, and the presence of sperm within the same lobule, confirmed that sperm were being formed from Honmoroko ovarian germline stem cells.

[0035] [Experimental Example 5] Testes were isolated from meioc mutant zebrafish, which lack germline stem cell differentiation, and dissociated into individual cells using collagenase. Testes were also isolated from Honmoroko (Japanese dace) and dissociated into individual cells using collagenase. These two types of cells were mixed at a ratio of 5:1 (zebrafish:ko), centrifuged, and cultured to obtain aggregates. These aggregates were then subcutaneously transplanted into male rag1 mutants (see Figure 5). The recipients were kept in 0.4x PBS containing 10 μg / mL gentamicin in the dark for four days without food. They were then kept in normal water. Two months after transplantation, the transplanted mass was recovered and tissue observation was performed. The results are shown in Figure 8. As shown in Figure 8, it was confirmed that the sperm and oocytes of the Honmoroko had differentiated. [Industrial Applicability]

[0036] According to the present invention, a method can be provided for producing allogeneic eggs or heterogeneous gametes by efficiently engrafting and growing transplanted ovarian tissue from an individual of the same species or reproductive tissue from a different species of fish.

Claims

1. A method for producing reproductive tissue, comprising the steps of isolating homologous reproductive tissue of an immunodeficient fish and heterologous reproductive tissue of a heterologous fish down to the cellular level, and then mixing them to obtain an aggregate, and transplanting the aggregate subcutaneously or into the ovaries of the immunodeficient fish.

2. The method for producing reproductive tissue according to claim 1 , further comprising the step of removing corresponding host germ cells in the immunodeficient fish.

3. A method for producing reproductive tissue, comprising the steps of transplanting reproductive tissue of a fish of the same species as an immunodeficient fish into the ovaries of the immunodeficient fish, and removing corresponding host reproductive cells within the immunodeficient fish.

4. The method for producing reproductive tissue according to any one of claims 1 to 3, wherein the immunodeficient fish has a suppressed or lost function of the rag1 gene or Rag1 protein.

5. The reproductive tissue obtained by the method for producing reproductive tissue according to any one of claims 1 to 4. Obtaining gametes, a method for producing gametes.

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