AU2017238706B2

AU2017238706B2 - Antibody capable of binding to undifferentiated germ cells of scombridae fish

Info

Publication number: AU2017238706B2
Application number: AU2017238706A
Authority: AU
Inventors: Makoto Hayashi; Kensuke Ichida; Yutaka Takeuchi; Ryosuke Yazawa; Goro Yoshizaki
Original assignee: Tokyo University of Marine Science and Technology NUC
Current assignee: Tokyo University of Marine Science and Technology NUC
Priority date: 2016-03-24
Filing date: 2017-03-24
Publication date: 2024-05-30
Anticipated expiration: 2037-03-24
Also published as: JPWO2017164390A1; WO2017164390A1; JP7006939B2; AU2017238706A1

Abstract

The present invention relates to a monoclonal antibody that specifically recognizes undifferentiated germ cells of scombridae fish, wherein the monoclonal antibody is an antibody against undifferentiated germ cells isolated from a testis or an ovary of scombridae fish. The present invention provides an antibody that can isolate undifferentiated germ cells of scombridae fish.

Description

DESCRIPTION ANTIBODY CAPABLE OF BINDING TO UNDIFFERENTIATED GERM CELLS OF SCOMBRIDAE FISH

Cross-Reference to Related Application

[0001] The present patent application is based on Japanese Patent Application No. 2016-60908 (filing date: March 24, 2016) which is a prior application applied to Japan, and claims priority thereto. The entire disclosure of the prior application is incorporated herein by reference.

BACKGROUND OF INVENTION

[0002] Technical Field The present invention relates to an antibody that specifically recognizes undifferentiated germ cells of scombridae fish, and a method for concentrating undifferentiated germ cells of scombridae fish using the antibody and a method for inducing differentiation of the undifferentiated germ cells, and a method for producing sperms or eggs of scombridae fish and a method for producing scombridae fish based on the above concentration and differentiation induction methods.

[0003] Background Art Scombridae fish including bluefin tuna are important fish with high economic value in the fishery. Meanwhile, as for large-sized scombridae fish such as bluefin tuna and southern bluefin tuna and the like which are highly demanded as high-quality fish, a remarkable decrease in the abundance of natural resources of fisheries due to over catch has become the issue of concern. Therefore, artificial seedling production and the release of artificial seedling (fry) for the purpose of increasing the number of natural resources have been considered. In artificial seedling production, genetic diversity of individual parent fish is required in order to avoid the total eradication due to inbreeding or the development of specific diseases, therefore it is necessary to breed a large number of parent fish. However, for example, as for bluefin tunas, it takes a long time to become parent fish as it is granted as mature only after the body weight of 100 kg or more, the body length of 1 m or more, and the age of 3 to 5 years old, and it requires extremely ample spaces as well to breed them because they swim at a high-speed. In addition, artificially induced maturation is technically difficult because fish is vulnerable to the stress of handling, and the success rate of one-to-one crossbreeding is also low. From such a background, it is extremely difficult to cultivate a large number of parent fish under the artificial management condition and harvest sperms and eggs from the financial and technical points of views including problems of feed and breeding spaces.

[0004] As a technique of artificial seedling production of fish, the inventors of the present invention found that a germ ceil can be induced differentiation to a germline (gamete) by transplanting germ cells derived from a xenogenic (donor fish) species which is different from a host (recipient fish) species into the intraperitoneal cavity of the host individual species before or after hatching. Based on these finding, the inventors succeeded in inducing differentiation into gametes of the desired fish species using xenogenic host species (e.g., refer to Patent Document 1). This technique, also called surrogate broodstock technology or surrogate farming technique, is expected as a technique which enables seedling production at low cost and easily by producing and crossbreeding sperms or eggs of fishes such as bluefin tuna and the like, that is difficult in cultivating, by using xenogenic host fish species which easily breed.

[0005] In this surrogate broodstock technology, it is necessary to separate only undifferentiated germ cells from a gonad, testis or ovary of the donor fish and use them for transplantation to ensure enough transplantation efficiency for commercial mass production. In Patent Document 1, in order to separate only primordial germ cells, transgenic rainbow trout introduced with a GFP (Green Fluorescent Protein) gene into the transcriptional regulatory region of a vasa gene that is specifically expressed in primordial germ cells was used as a donor fish and cells expressing the GFP gene are separated as primordial germ cells. However, sperms and eggs obtained by inducing differentiation of primordial germ cells derived from these transgenic fishes have the GFP gene introduced, and the seedlings produced by utilizing those sperms or eggs also have the GFP gene introduced. Therefore, the seedlings cannot be utilized as food fish or released seedlings and is not suitable for practical production techniques.

[0006] As a method for separation of undifferentiated germ cells such as primordial germ cells from donor fish without the introduction of a reporter gene such as a GFP gene, a separation method of germ cells have been studied using an antibody that specifically binds to germ cells. For example, Patent Document 2 discloses with respect to an anti-tuna vasa antibody that can specifically detect germ cells derived from tuna. However, since the antigen recognition site of this antigen exists in the cytoplasm, the antibody can detect germ cells in fixed cells or tissues, but cannot detect or separate living cells.

[0007] Meanwhile, the inventors of the present invention have confirmed that in salmonidae fish such as rainbow trout and the like, the antibody obtained from mice immunized with living spermatogonia isolated from GFP gene-transferred rainbow trout as an antigen can selectively label spermatogonia and germ cells can be separated by using the antibody obtained even if they are not transgenic fish (cf. refer to Non-Patent Document 1). However, according to the method described in Non-Patent Document 1, spermatogonia as the source of the antigen is originated from transgenic fish and the establishment of transgenic fish is difficult in scombridae fish such as bluefin tuna and the like, therefore, the production of antibody by the method is difficult.

PRIOR ART DOCUMENT Patent Documents

[0008]

[Patent Document 1] Japanese patent 4300287

[Patent Document 2] W02010/035465 Non-Patent Document

[0009]

[Non-Patent Document 1] 10th International Symposium of Reproductive Physiology of Fish (ISRPF), Abstract book, Olhao, Portugal, 25-30, May 2014

SUMMARY OF INVENTION

[0010]

The inventors of the present invention used the antibody of rainbow trout disclosed in Non-Patent Document 1 in order to selectively separate spermatogonia of bluefin tuna. However, while the antibody of rainbow trout was able to detect a signal for the gonad cells of bluefin tuna, the antibody could not detect or separate undifferentiated germ cells such as living spermatogonia of bluefin tuna and the like.

[0011] Thus, there is no fully satisfactory antibody for the practical use in surrogate broodstock technology of scombridae fish such as bluefin tuna and the like until now as long as the inventors are aware of it.

[0012] Until now, the inventors of the present invention have succeeded in the establishment of a gate (fraction) containing a large number of undifferentiated germ cells such as spermatogonia and the like in bluefin tuna without using gene transfer technology. In addition, when a monoclonal antibody was prepared using the fraction obtained as an antigen, the inventors have found that the monoclonal antibody specifically recognized undifferentiated germ cells, particularly the surface of the undifferentiated germ cells. The inventors have also succeeded in specifically separating and concentrating undifferentiated germ cells of scombridae fish by labeling the monoclonal antibody. Furthermore, the undifferentiated germ cells of bluefin tuna concentrated by the monoclonal antibody and dispersed testis cells (gonad cells) containing undifferentiated germ cells of bluefin tuna without concentration by the monoclonal antibody were transplanted into the intraperitoneal cavity of nibe croaker at the same number of cells. Surprisingly, it was confirmed that transplanted cells were engrafted into the host gonad of nibe croaker when undifferentiated cells of bluefin tuna concentrated by the monoclonal antibody were transplanted, while no engraftment of transplanted cells into nibe croaker host gonad at all was observed when the dispersed testis cells of bluefin tuna without concentration by the monoclonal antibody were transplanted. From the above results, the inventors found that the efficiency of engraftment into the host gonad remarkably increased by using undifferentiated germ cells of bluefin tuna concentrated by the monoclonal antibody as transplant cells. The present invention is based on these findings.

[0013] In other words, the present invention aims to provide a novel antibody that specifically recognizes undifferentiated germ cells of scombridae fish, and a concentration method of undifferentiated germ cells of scombridae fish, a differentiation induction method of undifferentiated germ cells of scombridae fish, and a production method of scombridae fish using these methods.

[0014] According to the present invention, following inventions are provided. (1) A monoclonal antibody that specifically recognizes undifferentiated germ cells of scombridae fish, wherein the monoclonal antibody is an antibody to undifferentiated germ cells separated from a testis or ovary of scombridae fish.

(2) The monoclonal antibody according to (1) that specifically recognizes undifferentiated germ cells of scombridae fish, wherein the monoclonal antibody is produced by hybridoma TA-No. 6-28 (NITE ABP-02222) or TA-No.15-1(NITE ABP-02223).

(3) The monoclonal antibody according to (1) or (2), wherein the undifferentiated germ cell is a primordial germ cell, type A spermatogonium or oogonium.

(4) The monoclonal antibody according to any one of (1) to (3), wherein the scombridae fish is a fish selected from genus Thunnus, genus Euthynnus, genus Katsuwonus, and genus Scomber.

(5) The monoclonal antibody according to any one of (1) to (4), wherein the monoclonal antibody specifically recognizes the surface of the undifferentiated germ cells of scombridae fish.

(6) A method for concentrating undifferentiated germs cells of scombridae fish, comprising separating scombridae-derived undifferentiated germ cells from a testis or ovary of scombridae fish containing scombridae-derived undifferentiated germ cells by using the antibody according to any one of (1) to (5).

(7) A differentiation induction method of undifferentiated germ cells into gametes, comprising transplanting undifferentiated germ cells separated from a testis or ovary of scombridae fish into the intraperitoneal cavity of an individual host fish before or after hatching, further comprising: separating and concentrating undifferentiated germ cells of scombridae fish from a testis or ovary of scombridae fish containing scombridae-derived undifferentiated germ cells by using the antibody according to any one of (1) to (5) before transplantation; and transplanting the separated and concentrated undifferentiated germ cells into the intraperitoneal cavity of the individual host fish before or after hatching.

(8) The differentiation induction method of undifferentiated germ cells into gametes according to (7), wherein the scombridae fish that provides the undifferentiated germ cells to be transplanted is xenogenic to the host fish.

(9) The differentiation induction method of undifferentiated germ cells into gametes according to (7) or (8), wherein the scombridae fish that provides the undifferentiated germ cells to be transplanted is larger-sized fish than the host fish.

(10) The differentiation induction method of undifferentiated germ cells into gametes according to any one of (7) to (9), wherein the host fish is a fish selected from scombridae fish, and the scombridae fish that provides the undifferentiated germ cells to be transplanted, is a fish selected from tuna.

(11) A method for producing sperms or eggs of scombridae fish, comprising transplanting undifferentiated germ cells separated from a testis or ovary of scombridae fish into the intraperitoneal cavity of an individual host fish before or after hatching, further comprising: separating and concentrating undifferentiated germ cells of scombridae fish from a testis or ovary of scombridae fish containing scombridae-derived undifferentiated germ cells by using the antibody according to any one of (1) to (5) before transplanting; transplanting the separated and concentrated undifferentiated germ cells into the intraperitoneal cavity of the individual host fish before or after hatching; and inducing differentiation of the transplanted undifferentiated germ cells into gametes to obtain sperms or eggs of scombridae fish.

(12) A production method of scombridae fish, comprising transplanting undifferentiated germ cells separated from a testis or ovary of scombridae fish into the intraperitoneal cavity of an individual host fish before or after hatching, further comprising: separating and concentrating undifferentiated germ cells of scombridae fish from a testis or ovary of scombridae fish containing scombridae-derived undifferentiated germ cells by using the antibody according to any one of (1) to (5) before transplanting; transplanting the separated and concentrated undifferentiated germ cells into the intraperitoneal cavity of an individual host fish before or after hatching; inducing differentiation of the transplanted undifferentiated germ cells into gametes; and crossbreeding the obtained sperms and eggs.

[0015] Undifferentiated germ cells of scombridae fish can be specifically recognized by using the antibody of the present invention. In addition, undifferentiated germ cells of scombridae fish can be separated and concentrated by using the antibody of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

[0016]

[Figure 1] Figure la shows the results of HE staining and in situ hybridization in the paraffin-embedded histological section (testis tissue) of bluefin tuna. Figure lb is the scatter plot of cells contained in a ceil suspension of bluefin tuna obtained by flow cytometry (FCM) analysis. FS represents the size of cells, and SS represents the complexity of the internal structure of cells. Figure 1c shows photographs of shapes (Biological microscope, Olympus) of the cells contained in the gates (fractions) A to G of the scatter plot, respectively.

[Figure 2] Figure 2a shows the scatter plot of cells contained in the cell suspension of bluefin tuna obtained from FCM analysis in immature individual (one-year-old) and the results of in situ hybridization of cells contained in the gates A to G of the scatter plot, respectively. Figure 2b shows the ratio of vasa positive cell count (vasa* ratio) to the total cell count in each gate.

[Figure 3] Figure 3a shows the results of the HE staining and in situ hybridization (vasa) of the testis in a mature individual (3-year-old). Figure 3b shows the results of the scatter plot of cells contained in a partially purified cell suspension of bluefin tuna obtained from FCM analysis. Figure 3c shows the results of the shape and in situ hybridization of the separated cells contained in gate A. Figure 3d shows the ratio of vasa positive cell count (vasa* ratio) to the total cell count in each gate.

[Figure 4] Figure 4 shows the results of immunocytochemistry staining in the secondary screening; Figure 4a shows cells on whose membrane a green fluorescence was clearly observed (a positive signal); Figure 4b shows cells in which nonspecific signals such as blood cells or debris were observed, and Figure 4c shows a example of cells in which no signal was recognized.

[Figure 5] Figure 5a shows a scatter plot obtained from the FCM analysis (FS-SS scatter) of the total cells used in the tertiary screening. Figure 5b shows histograms of antibody-positive cells (an arrow on the right) and antibody-negative cells (an arrow on the left). The horizontal axis represents the signal intensity of an antibody, and the vertical axis represents the number of cells.

Figure 5c shows a scatter plot obtained by FCM analysis (FS-SS scatter) of an antibody-positive cell population.

[Figure 6] Figure 6 shows the results of immunocytochemistry staining and in situ hybridization using an antibody produced by hybridoma TA-No. 6-28.

[Figure 7] Figure 7 shows the results of in situ hybridization using (a) an antibody produced by hybridoma TA-No. 6-28 and (b) an antibody produced by hybridoma TA-No. 15-1. The concentration factor were each 85.2% and 78.9%, respectively.

[Figure 8] Figure 8 shows the results of the HE staining in a testicular histological section of bluefin tuna and of immunohistochemical staining using an antibody produced by hybridoma TA-No. 6-28.

[Figure 9] Figure 9 shows the results of the HE staining (left) in a testicular histological section of bluefin tuna and of immunohistochemical staining (right) using an antibody produced by hybridoma TA-No. 6-28 (upper part) and an antibody produced by hybridoma TA-No. 15-1 (lower part).

[Figure 10] Figure 10 shows the results from Flow cytometry analysis of MACS-concentrated undifferentiated germ cells of bluefin tuna by using an antibody produced by hybridoma TA-No. 6-28 (left) and an antibody produced by hybridoma TA-No. 15-1 (right), depicting (a) photos of cellular shapes (Biological microscope, Olympus) of a flow fraction (Flow) and elution fraction (Elute), and (b) photos of vasa in situ hybridization (Biological microscope, Olympus) of a flow fraction (Flow) and elution fraction (Elute).

[Figure 11] Figure 11 shows the photo of laparotomized nibe croaker subjected to transplantation. Photographs show a fluorescence field (Fluorescence biological microscope, Olympus) and bright field.

[Figure 12] Figure 12 shows the engraftment rate of transplanted cells into the host gonad of nibe croaker. The vertical axis represents the ratio ( %) of individuals in which the engraftment of undifferentiated germ cells of bluefin tuna, which are transplanted, was observed to the total nibe croaker hosts under the observation.

DETAILED DESCRIPTION OF INVENTION

[0017] According to one embodiment of the present invention, provided is a monoclonal antibody that specifically recognizes undifferentiated germ cells of scombridae fish, preferably the surface of undifferentiated germ cells of scombridae fish.

[0018] The "scombridae fish" refers to fishes that belong to the order Perciformes, family Scombridae. For example, it includes genus Thunnus, genus Euthynnus affinis, genus Katsuwonus, genus Scomber, genus Scomberomorus, genus Auxis, genus Sarda, genus Gymnosarda, preferably genus Thunnus, genus Euthynnus affinis, genus Katsuwonus or genus Scomber. Representative fish species of the family Scombridae are as follows: the genus Thunnus includes, for example, bluefin tuna (e.g., Pacific bluefin tuna, Atlantic bluefin tuna), southern bluefin tuna, bigeye tuna, yellowfin tuna, albacore tuna, blackfin tuna, longtail tuna; the genus Euthynnus includes, for example, mackerel tuna and little tunny; the genus Katsuwonus includes, for example, skipjack tuna; the genus Scomber includes chub mackerel and blue mackerel (Scomberaustra/asicus). Thefish species in scombridae fish of the present invention are preferably bluefin tuna, southern bluefin tuna, bigeye tuna, yellowfin tuna, albacore tuna, blackfin tuna, or longtail tuna, which may be called tuna as a generic name. More preferably, fish species in scombridae fish of the present invention include bluefin tuna, southern bluefin tuna, blackfin tuna or mackerel tuna for which resource preservation is expected to be improved by the artificial seedling production.

[0019] The term "undifferentiated germ cells" refers to substantially undifferentiated germ cells among those classified as germ cells such as primordial germ cells, oogonia, spermatogonia, oocytes, spermatocytes, spermatids, eggs and sperms. For example, primordial germ cells, spermatogonia, and oogonia are cited. Whether a germ cell is an undifferentiated germ cell or not can be determined by its morphological characteristics. For example, a type A spermatogonium can be identified by histological observation when histological observation is performed because it is a germ cell which is surrounded by somatic cells and independently exists in testicular cells. In addition, oogonia belong to a cell population in which the first meiosis has not started among the smaller germ cells before entering the vitellogenic stage. Therefore, no expression of meiotic marker genes such as Sycp3 and the like can be one of the indicators to identify oogonia. The undifferentiated germ cells of the present invention are preferably undifferentiated germ cells which have engraftability when they are transplanted into the gonad of a host in surrogate broodstock technology and engraftability to the gonad of the host fish. Undifferentiated germ cells having engraftability to the host gonad include primordial germ cells, type A spermatogonia or oogonia. Type A spermatogonia are undifferentiated germ cells coexisting with somatic cells in an undifferentiated testis and the type A spermatogonia are cells to be differentiated into type B spermatogonia, spermatocytes, spermatids, and spermatozoids in the testis which has started maturation or is matured. Oogonia are undifferentiated germ cells coexisting with somatic cells in an undifferentiated ovary and are cells to be differentiated into oogonia and eggs as they become mature. Whether germ cells are undifferentiated germ cells having engraftability to a host gonad or not can be ascertained by the presence or absence of engraftability when transplanted into the host fish in surrogate broodstock technology. Specifically, cells having a high expression of vasa gene and engraftability that is recognized when transplanted into the gonad of a host fish in surrogate broodstock technology can be ascertained as undifferentiated germ cells having engraftability to the host gonad. For example, Non-Patent Document 1 can be referred to.

[0020] The term "to specifically recognize undifferentiated germ cells of scombridae fish" means to specifically bind to the undifferentiated germ cells of scombridae fish and not to specifically bind to differentiated germ cells and somatic cells of scombridae fish, preferably to specifically bind to the cell surface antigen of the undifferentiated germ cells of scombridae fish. The term "to specifically recognize undifferentiated germ cells of scombridae fish" means, preferably, to specifically recognize the surface of the undifferentiated germ cells of scombridae fish, more preferably, to specifically recognize the surface of cell membrane of the germ cells. In the present invention, the term "to specifically bind" includes to preferentially bind.

[0021] A "monoclonal antibody" means an antibody (immunoglobulin molecule) obtained from a clone derived from a single antibody-producing cell, and is not particularly limited to the classes of immunoglobulin, for example, includes IgG, IgM, IgA, IgD and IgE, and preferably IgG.

[0022] The monoclonal antibody of the present invention can be acquired by preparing an antibody-producing hybridoma using undifferentiated germ cells of scombridae fish as an antigen, and detecting an antibody produced by the hybridoma as the antibody that recognizes the undifferentiated germ cells.

[0023] An antigen for preparing the monoclonal antibody of the present invention is preferably undifferentiated germ cells separated from the testis or ovary of scombridae fish from the viewpoint that it is easy to ensure enough numbers of the undifferentiated germ cells to be used for transplantation in surrogate broodstock technology. In a preferred embodiment of the invention, a cell population in which only undifferentiated germ cells are separated and concentrated from a cellular suspension also containing somatic cells and the like derived from the testis or ovary can be used as undifferentiated germ cells separated from the testis or ovary. Specifically, in terms of a fraction of a cell population containing a large number of undifferentiated germ cells obtained by flow cytometry analysis (FCM), a fraction with relatively large FS value and relatively small SS value can be used when linear scale is used for FS and SS. More specifically, as shown in Example 1 to be cited later, when FCM analysis was carried out by Epics Altra (Beckman Coulter) equipped with Argon laser at 488 nm to represent a FS-SS plotting under the condition in which all the testis cells scattered in a single state by dispersion of cells are detectable, a cell population appearing in a fraction with relatively large FS value and relatively small SS value, and preferably a fraction with FS value of about higher 1/3 and SS value of about lower 1/3 can be used. Such a cellular population containing a large number of undifferentiated germ cells can be prepared as the gate A by the method of Example 1 mentioned later. Therefore, according to one embodiment of the present invention, an antigen is provided for preparing the antibody of the present invention that specifically recognizes undifferentiated germ cells of scombridae fish. The antigen is preferably contained in the fraction of the cell population containing a large number of undifferentiated germ cells by flow cytometry (FCM) analysis, and the fraction is, when linear scale is used for FS and SS, a fraction with relatively large FS value and relatively small SS value.

[0024] According to a preferred embodiment of the present invention, the monoclonal antibody of the present invention can be acquired by preparing an antibody-producing hybridoma using undifferentiated germ cells separated from a testis or ovary of scombridae fish as an antigen, and detecting an antibody produced by the hybridoma as the antibody that recognizes undifferentiated germ cells. An antibody-producing hybridoma can be prepared according to a conventional method, for example, by administrating an antigen into an animal (e.g., mouse, rat, and rabbit), immunizing, and fusing cells derived from lymph nodes obtained from the animal with myeloma cells. It can be detected, for example, by an RT-PCR method for the vasa gene as well as a Cell ELISA method, immunocytochemistry staining, in situ hybridization and the like according to Example 2 described later that the antibody can recognize undifferentiated germ cells. It can be detected by confirming engraftability into the gonad of the host when transplanted into the gonad of the host in surrogate broodstock technology (refer to Non-Patent Document 1) that the antibody can recognize undifferentiated germ cells having engraftability into the gonad ofthe host.

[0025] According to a preferred embodiment of the present invention, the monoclonal antibody in the present invention that specifically recognizes undifferentiated germ cells of scombridae fish is produced by an antibody-producing hybridoma, TA-No. 6-28 (receipt number: NITE ABP-02222) or TA-No. 15-1 (receipt number: NITE ABP-02223) that is deposited in the incorporated administrative agency, National Institution of Technology and Evaluation, Patent MicroorganismsDepositary.

[0026] TA-No. 6-28 is a hybridoma producing an antibody that specifically recognizes spermatogonia of scombridae fish. The hybridoma is deposited in the incorporated administrative agency, National Institution of Technology and Evaluation, Patent Microorganisms Depositary (2-5-8, Kazusakamatari, Kisarazu, Chiba, Japan) on March 22, 2016 (original depositing date) as a receipt number NITE ABP-02222 (transferred from domestic deposition NITE P-02222) (identification: TA-No. 6-28). TA-No. 6-28 is a hybridoma prepared by fusing myeloma cells with B lymphoid cells which are antibody-producing cells, and has a circular shape and weak adhesive property. The hybridoma proliferates in a Hybridoma-SFM culture medium (Gibco, 12300-067) containing Penicillin-Streptomycin, Liquid (Gibco) at the final concentration of 1% and 10% of fetal bovine serum (FBS) at 37C and 5% CO 2 .

TA-No. 6-28 has a mouse antibody (IgG antibody) production capability, and its production amount is about 1 to 10 pg/ml.

[0027] TA-No. 15-1 is a hybridoma producing an antibody that specifically recognizes spermatogonia of scombridae fish. The hybridoma is deposited in the incorporated administrative agency,

National Institution of Technology and Evaluation, Patent Microorganisms Depositary (2-5-8, Kazusakamatari, Kisarazu, Chiba, Japan) on March 22, 2016 (original deposition date) as a receipt number NITE ABP-02223 (transferred from domestic deposition NITE P-02223) (identification: TA-No. 15-1). TA-No. 15-1 is a hybridoma prepared by fusing myeloma cells with B lymphoid cells which are antibody-producing cells, and has a circular shape and weak adhesive property. TA-No. 15-1 proliferates in a Hybridoma-SFM culture medium (Gibco, 12300-067) containing Penicilin-Streptomycin, Liquid (Gibco) at the final concentration of 1% and 10% of fetal bovine serum (FBS) at 37°C and 5% CO 2

. TA-No. 15-1 has a mouse antibody (IgG antibody) production capability, and its production amount is about 1 to 10 pg/ml.

[0028] The monoclonal antibody of the present invention can separate undifferentiated germ cells of scombridae fish from a testis or ovary of scombridae fish. The separation method of undifferentiated germ cells of scombridae fish using the monoclonal antibody of the present invention is not particularly limited as long as undifferentiated germ cells can be separated alive. Well-known methods can be applied, for example, cell separation (isolation) by flow cytometry analysis using an antibody labeled with fluorescence labeling such as FITC and the like or Magnetic cell separation using the antibody binding to magnetic beads can be used. Preferably, the method for separation is a Magnetic cell separation (MACS) method from the viewpoint of not requiring advanced facilities and techniques. Compared to the cell separation method according to flow cytometry analysis, the Magnetic cell separation method is advantageous from the perspective that even a large number of cells can be easily treated because the cells are separated by magnetic beads. In addition, the MACS method using the monoclonal antibody of the present invention is also advantageous from the perspective that the MACS method is less likely to adversely affect the property of cells because it is not necessary to use a solution with cytotoxicity like density-gradient centrifugation using Percoll. Furthermore, it is also advantageous from the standpoint that a part of the cell populations can be concentrated even if their specific gravities are close.

[0029] Undifferentiated germ cells can be concentrated by collecting undifferentiated germ cells separated (isolated) by the monoclonal antibody of the present invention. Therefore, according to one embodiment of the present invention, provided is a concentration method of undifferentiated germ cells of scombridae fish comprising separating scombridae-derived undifferentiated germ cells by using the antibody of the present invention from a testis or ovary of scombridae fish containing scombridae-derived undifferentiated germ cells. Because the resulting undifferentiated germ cells concentrated by the concentration method of the present invention are living cells, the improved transplant efficiency can be expected by using them as undifferentiated germ cells to be applied for transplant in surrogate broodstock technology. In other words, according to one embodiment of the present invention, provided is a production method of undifferentiated germ cells of scombridae fish, comprising separating scombridae-derived undifferentiated germ cells by using the antibody of the present invention from a testis or ovary of scombridae fish containing scombridae-derived undifferentiated germ cells.

[0030] In surrogate broodstock technology, undifferentiated germ cells transplanted can be efficiently induced to differentiate into gametes by using the undifferentiated germ cells separated (isolated) by the monoclonal antibody of the present invention. Herein, inducing differentiation of transplanted undifferentiated germ cells into gametes means that the undifferentiated germ cells that are transplanted into the intraperitoneal cavity migrate and engraft to the gonad of the transplanted host, and have matured to be induced differentiation into gametes in the gonad of the host. Therefore, the success or failure in inducing differentiation of transplanted undifferentiated germ cells into gametes can be ascertained by the presence of the engraftment of the transplanted undifferentiated germ cells in the gonad of the host (refer to; Okutsu, T., Suzuki, K., Takeuchi, Y.,Takeuchi, T & Yoshizaki, G. (2006) Testicular germ cells can colonize sexually undifferentiated embryonic gonad and produce functional eggs in fish. Proceedings of the National Academy of Sciences of the United States of America, 103, cf. 2725-2729). When engraftment of transplanted undifferentiated germ cells in the gonad of the host can be confirmed, it might be said that transplanted undifferentiated germ cells can be induced differentiation into gamete with a high probability. As a result, according to one embodiment of the present invention, in surrogate broodstock technology of scombridae fish, namely a method for inducing differentiation of the undifferentiated germ cells into gametes, comprising transplanting undifferentiated germ cells of scombridae fish into the intraperitoneal cavity of an individual host fish before or after hatching, provided is a differentiation induction method of undifferentiated germ cells into gametes, comprising: separating the undifferentiated germ cells of scombridae fish from a testis or ovary of scombridae fish containing scombridae-derived undifferentiated germ cells by using the antibody of the present invention before transplantation, concentrating, and transplanting the undifferentiated germ cells separated and concentrated into the intraperitoneal cavity of the individual host fish before or after hatching.

[0031] According to one embodiment of the present invention, in surrogate broodstock technology of scombridae fish, namely a method for inducing differentiation of the undifferentiated germ cells of scombridae fish into gametes, comprising transplanting undifferentiated germ cells separated from a testis or ovary of scombridae fish into the intraperitoneal cavity of an individual host fish before or after hatching, provided is a differentiation induction method of undifferentiated germ cells into gametes comprising: separating the undifferentiated germ cells of scombridae fish from the testis or ovary of scombridae fish containing scombridae-derived undifferentiated germ cells by using the antibody of the present invention before transplantation, concentrating, and transplanting the undifferentiated germ cells separated and concentrated into the intraperitoneal cavity of the individual host fish before or after hatching.

[0032] According to one embodiment of the present invention, in the method for inducing differentiation of the present invention, provided is a differentiation induction method of undifferentiated germ cells into gametes comprising: separating undifferentiated germ cells of scombridae fish having engraftability into the gonad of a host from a testis or ovary of scombridae fish containing scombridae-derived undifferentiated germ cells by using the antibody of the present invention before transplantation, concentrating, and transplanting the separated and concentrated undifferentiated germ cells into the intraperitoneal cavity of the individual host fish before or after hatching.

[0033] In the differentiation induction method of the present invention, as a donor fish, scombridae fish that provides undifferentiated germ cells to be transplanted may be the same species as the host fish or different species from the host fish, preferably different species. That the donor fish and the host fish are the same species means that species of scombridae fish that provides undifferentiated germ cells are the same as the species of the host fish. For example, by this method, using a strain having a good trait as the donor fish results in producing a good strain in large quantities for a short period without selection breeding. That the donor fish and host fish are different species means that the species of scombridae fish that provides undifferentiated germ cells are different from the species of the host fish. For example, it includes a case in which species of scombridae fish that provides undifferentiated germ cells are different from the genus of the host fish or other species included in the same genus as the host fish. When they are different species, the combination of the donor fish and the host fish can be determined in consideration of the similarity of their habitat environment and gonadal size.

[0034] Scombridae fish as a donor fish is preferably larger-sized fish than the host fish. For example, large-sized fish of scombridae fish includes bluefin tuna (e.g., Pacific bluefin tuna, Atlantic bluefin tuna) and southern bluefin tuna. Also, parent fish enormously matured is included in large-sized fish as fish grow larger in size with age. Feed and breeding spaces which are problematic for large-sized fish can be reduced by separating germ cells of large-sized fish, transplanting them into the gonad of small-sized host fish and inducing differentiation into gametes.

[0035] According to a more preferred embodiment of the present invention, in the differentiation induction method of the present invention, the host fish is one species chosen among scombridae fish, and the donor fish of scombridae fish that provides undifferentiated germ cells to be transplanted is one species chosen from Thunnus, namely bluefin tuna, southern bluefin tuna, bigeye tuna, yellowfin tuna, albacore tuna, blackfin tuna or longtail tuna. According to a further preferred embodiment of the present invention, the host fish are mackerel or mackerel tuna, and scombridae fish to be used as the donor of the undifferentiated germ cells are bluefin tuna (e.g., Pacific bluefin tuna and blackfin tuna) or southern bluefin tuna. Further preferably, the combination of the host fish and scombridae fish that provides undifferentiated germ cells includes, for example, mackerel and Pacific bluefin tuna, mackerel and southern bluefin tuna, mackerel and Atlantic bluefin tuna, mackerel tuna and Pacific bluefin tuna, mackerel tuna and southern bluefin tuna, mackerel tuna and Atlantic bluefin tuna, considering that it is a combination of fish which are difficult for breeding as the donor, and closely related species to the donor and feasible for seedling production in a small water tank.

[0036] According to one embodiment of the present invention, provided is a production method of sperms or eggs of scombridae fish, comprising the differentiation induction method of the present invention. In other words, according to one embodiment of the present invention, provided is a method for producing sperms or eggs of scombridae fish, comprising transplanting undifferentiated germ cells separated from a testis or ovary of scombridae fish into the intraperitoneal cavity of the individual host fish before or after hatching, and specifically provided is a method for producing sperms or eggs of scombridae fish, comprising: separating undifferentiated germ cells of scombridae fish with engraftability into the gonad of the host from the testis or ovary of scombridae fish containing scombridae-derived undifferentiated germ cells by using the antibody of the present invention before transplantation, concentrating, transplanting the separated and concentrated undifferentiated germ cells into the peritoneal cavity of the host fish before or after hatching, inducing the transplanting undifferentiated germ cells into gametes, and obtaining sperms or eggs of scombridae fish.

[0037] According to another embodiment of the present invention, provided is a production method of scombridae fish, comprising transplanting undifferentiated germ cells separated from a testis or ovary of scombridae fish into the intraperitoneal cavity of the individual host fish before or after hatching, and specifically provided is a production method, comprising: separating undifferentiated germ cells of scombridae fish with engraftability into the gonad of the host from the testis or ovary of scombridae fish containing scombridae-derived undifferentiated germ cells by using the antibody of the present invention before transplantation, concentrating, transplanting the separated and concentrated the undifferentiated germ cells into the intraperitoneal cavity of the individual host fish before or after hatching, inducing differentiation of the transplanted undifferentiated germ cells into gametes, and crossbreeding the sperms and eggs obtained.

[0038] The method for producing the sperms or eggs of scombridae fish and the method for producing scombridae fish in the present invention can be carried out according to the contents described in the present description regarding with the concentration method and differentiation induction method of the present invention.

EXAMPLES

[0039] The present invention will be explained in detail by the following Examples, but the present invention is not limited thereto.

[0040] Example 1: Acquisition of a fraction containing undifferentiated germ cells of scombridae fish (1) Exenteration of testis A testis was exenterated from one individual bluefin tuna (2 to 3-year-old, 30 to 40 kg in weight, gonad weight was about 100 to 200 g) and minced. To minced testis was added 10 ml of L-15 culture medium (Invitrogen) (5% fetal bovine serum (FBS)) containing collagenase H (Roche) at the final concentration of 2 mg/ml, and disperse II (Godo Shusei Co., Ltd.) at the final concentration of 1.65 mg/mL. While performing physical dispersion by pipetting, the mixture was incubated at 20°C for two hours. Enzyme reactions was stopped by adding 1 ml of L-15 culture medium (Invitrogen). The cell suspension obtained (approximately 10 million cells/ml) was partially purified by Percoll (GE Healthcare) density-gradient centrifugation. The partially purified cell suspension was used for flow cytometry (FCM) analysis and cell separation.

[0041] The degree of maturity of the testis used was confirmed by HE staining and in situ hybridization analysis of the paraffin-embedded histological section (4 pm) of the testis of bluefin tuna fixed with 4% paraformaldehyde (PFA) /PBS fixative.

[0042] In situ hybridization analysis In situ hybridization analysis was carried out according to the method described as follows (Nagasawa, K., Takeuchi, Y., Miwa, M., Higuchi, K., Morita, T., Mitsuboshi, T., & Yoshizaki, G. (2009). cDNA cloning and expression analysis of a vasa-like gene in Pacific bluefin tuna Thunnus orientalis. Fisheries Science, 75(1), 71-79). Specifically, deparaffinization for histological sections was performed by treating with xylene/ethanol series and hydration. The section obtained was post-fixed with 4% paraformaldehyde, endogenous alkaline phosphatase (AP) was deactivated with 1% hydrogen peroxide, and proteins were degraded by protease K (Wako) and later acetylated. After the mixture was standing in a hybridization buffer (50% formamide, 2xSSC, 50 ng/ml yeast tRNA, 50 ng/ml heparin, 0.02% SDS, 10% dextran sulfate) at 65°C for one hour, hybridization was carried out at 65 0 C overnight in the buffer to which bluefin tuna vasa probe: digoxigenin (DIG)-labeled RNA probe was added so as to attain a concentration of 1 ng/pl. A bluefin tuna vasa probe was synthesized according to a standard method of the DIG-labled probe using a DIG Labeling kit (Roche) with a plasmid DNA, as a template, which contained, in an insert site, a 1085 bp fragment amplified by PCR using the primer (vasa F 5'-CAACCAGGGAGCTCATCAACCAGA-3'/vasa R 5'-GCAACTCAGGTCAATGCTGTGTGG-3'), which is specific for bluefin tuna vasa gene.

[0043] After the histological section was washed out thoroughly, the nonspecifically bound probe was removed by RNaseA (Invitrogen).

The histological section was masked by a blocking solution (2/ Blocking reagent (Roche) /TBST) and an AP-labeled anti-DIG antibody solution (Roche) (diluted with a blocking solution to 1:500) was added drop-wise to the histological section. The histological section stood at room temperature for one hour and was visualized using an NBT/BCIP chromogenic substrate. The histological section was washed with xylene/ethanol series and then enclosed with Entellan in slide glasses. The histological section was observed under microscope and the images were digitally recorded.

[0044] Microscopic images of the histological sections are shown in Figure la (immature individual) and Figure 3a (mature individual).

[0045] (2) Flow cytometry (FCM) analysis Flow cytometry analysis was carried out by EpicsAltra (Beckman Coulter) equipped with the 488 nm argon laser. Previous studies have found that spermatogonia of fish are larger than other germ cells and have round shape. Flow cytometry analysis using vasa-GFP transgenic fish clarified that a spermatogonia population can be fractionated into a population having a large size and a simple internal structure. Therefore, assuming that spermatogonia of bluefin tuna is similarly capable of fractionation, a partially purified cell suspension obtained by a Percoll density gradient method were irradiated with a laser and the resulting scattered light was analyzed to measure signals of size of ceil (Forward light scatter: FS) and complexity of internal structure (side light scatter: SS). Based on the scatter plot obtained, each cell population that is densely distributed were fractionated into gates (fractions) A to G, and cell separation was carried out for each gate. The separated cell populations of each gate were analyzed by morphology and in situ hybridization.

[0046] For morphological analysis, each one of cells was irradiated with a laser, and the cells were divided into seven kinds of gates A to G from the characteristic of each cell obtained from the scattered light. Generally, spermatogonia are larger-sized than other testicular cells and round. Therefore, a fraction of cells having a large size and simple internal structure was assumed as a fraction containing a large number of spermatogonia (gate A in Figure 1c).

[0047] According to above "(1) In situ hybridization analysis", in situ hybridization analysis was carried out using cell smear preparation in which cells of each gate were smeared on slide glasses and fixed with 4% of paraformaldehyde (PFA)/PBS in place of a paraffin-embedded histological section.

[0048] The scatter plots are shown in Figure lb (immature individual) and Figure 2a (mature individual). Results of the fractionation are shown in Figures 1c, 2 (immature individual) and 3 (mature individual).

[0049] As shown in Figures 2 and 3, it was confirmed that a large number of undifferentiated germ cells such as spermatogonia were fractionated in the gate A. The ratio of undifferentiated germ cells such as spermatogonia fractionated in the gate A was 80.7±1.5% (standard error) forthetotal numberof cells in immature individuals, and approximately 77.2±4.9% in matured individuals (standard error).

[0050] Example 2: Acquisition of an antibody that recognizes undifferentiated germ cells (1) Preparation of a hybridoma producing monoclonal antibody The cell population fractionated in the gate A obtained in Example 1 above was used as an antigen to prepare a mouse monoclonal antibody. Specifically, four mice (Balb/c strain) were immunized with the ceil population (5x10 6 cells/m) of the gate A in divided five doses. For immunization, the cell population in the gate A was diluted 2-fold with Freund complete adjuvant (Sigma) and PBS, and 100 pi of the mixture was administered to the paws of mice.

[0051] On day 8 after the fifth immunization, enlarged lymph nodes were aseptically enucleated from the foot of five mice mentioned above, and lymph node cells were harvested. The harvested lymph node cells and myeloma cells (P3U1, Tokyo University of Marine Science and Technology) were mixed in the presence of 50% polyethylene glycol (PEG) and cell fusion was carried out. A mixed solution of the obtained fusion cells (hybridoma) was diluted with HAT medium containing 15% FBS, and the diluted mixture was seeded on 12 pieces of 96-well plates.

[0052] (2) Primary screening: Cell ELISA After the incubation (37°C) for 14 days, the formation of 1152 colonies was recognized. Therefore, the screening by Cell ELISA was carried out to determine whether these hybridomas produce the antibody that recognizes undifferentiated germ cells. Specifically, partially purified spermatogonia obtained by Percoll density-gradient centrifugation were seeded (105 cells /well) on a culture plate, and the plate was used to measure the potency of the antibody produced by the hybridomas to the antigen for immunization. A HRP chromogen system was used to measure the potency. A clone with a signal value of 0.100 or more in Cell ELISA was selected as a positive clone with high-titer (high affinity).

[0053] (3) Secondary screening: Immunocytochemistry staining (observation under fluorescence microscope) A screening by immunocytochemistry staining was carried out to determine whether antibodies produced from 384 clones which were positive in the primary screening can recognize the cell surface antigen of undifferentiated germ cells. The testis enucleated from bluefin tuna (2 to 3-year-old, 2 fish) were enzymatically digested with collagenase IV (Sigma) (at 20 0C for 2 hours), and the resulting dispersed cells in the living state were reacted with the antibodies derived from 384 clones described above, and the testis cells of bluefin tuna were indirectly labeled with a secondary antibody Alexa Flour 488 anti-mouse IgG (Life technology) that is an antibody labeled with a green fluorescent substance. Thereafter, the clones were observed under a fluorescence microscope and the clones in which green fluorescence was clearly observed on the cell membrane were selected as the clone that is capable of recognizing the cell surface antigen. The images observed under a fluorescence microscope in the secondary screening are shown in Figure 4.

[0054] (4) Tertiary screening: FCM analysis FCM analysis was carried out for the total cells and antibody-positive cells to clarify whether antibodies produced from 50 clones which were positive in the secondary screening can specifically recognize the cells in the gate A, namely the fraction of undifferentiated germ cells. Specifically, a scatter plot was prepared by FCM analysis (FS-SS scatter) of the total cells (Figure 5a) and the gate A ratio was calculated. A fluorescence-labeled secondary antibody was reacted for the total cells above and the mixture was subjected to FCM analysis to prepare histograms of antibody-positive cell population and the antibody--negative cell population (Figure 5b). A scatter plot was prepared by FCM analysis (FS-SS scatter) of the obtained antibody-positive cell population (Figure Sc) and the gate A ratio was calculated. From the ratio of the gate A in the population of antibody-positive cells obtained (a ratio of the gate A in the antibody-positive fraction) and the ratio of the gate A in the total cells including antibody-negative cells and antibody-positive cells (a ratio of the gate A to the total cells), the concentration factor of the undifferentiated germ cells by the antibody was determined using the following equation.

[0055]

[Equation 1] Concentration factor of germ cells by an antibody = Ratio of the gate A in an antibody-positive fraction/Ratio of the gate A in the total cells

[0056] An antibody with a concentration factor of 1.0 or more was selected as a positive clone. The results are given in Table 1.

[0057]

[Table 1]

Enrichment rate Ab number Ab number Enrichment rate, b number Enrichmentrate 7~ ~ 91 18911118 0.10 14'J2 18 09 175 0.08 383 84~82, 7 1 81 0.05 IA) <>l 377 0J79 1 109 0.05 93_ 7C J257 0,04 t 65 38 06 114 0.04 S0 330 J 0.63 285 0.03 115 85 0.56 294 0 2 20 L0A9 302 0.02 361' 44 132 0.47 121 0 Jl250 i 0.38 31_ _

1~ 27 271 /t 0.36 1 1 0 70.4 12810 304 022281 0 12 0.13. 2 ?0 8 20 147 0.13 144 ost 5 17 129 0.12

[0058] (4) Quaternary screening: In situ hybridization For 20 clones which were positive in the tertiary screening, another screening was carried out by in situ hybridization analysis to determine whether spermatogonia of bluefin tuna can be subjected to cell separation by FCM analysis using antibodies. According to the method of Example 1 (1), the antibodies as primary antibody was bound to partially purified cells in the suspension prepared from the testis of bluefin tuna, and a green fluorescent labeled-antibody as secondary antibody was bound to the primary antibody, and an antibody-positive fraction was isolated by FCM analysis using green fluorescence as a marker. Next, the isolated cells were smeared on a microscope slide glass to prepare a cellular smear specimen. According to the method of Example 2 (2), in situ hybridization analysis was carried out for the cell smear specimen using a vasa RNA probe known as a marker of undifferentiated germ cells, and the positive ratio of vasa-positive cells was determined.

[0059]

Figures 6 and 7 show the vasa-positive images.

[0060] As shown in Figure 7, cell populations separated by antibodies produced from two kinds of the clones obtained (hybridoma TA-No. 6-28 and hybridoma TA-No. 15-1) contain vasa positive cells in the ratio of 85.2% and 78.9%, respectively. In other words, it was confirmed that the undifferentiated germ cells of bluefin tuna could be separated (isolated) and concentrated at a high-concentration by using these antibodies.

[0061] Microscopic images of immunohistochcemical staining using these two kinds of antibodies produced from clones were shown in Figures 8 and 9. Immunohistochemical staining was carried out according to the method described in Example 1 (1).

[0062] As shown in Figures 8 and 9, it was confirmed that these two kinds of antibodies specifically recognized a spermatogonia population, namely the population of undifferentiated germ cells on histological sections. In addition, it was confirmed that the two kinds of antibodies specifically recognized the surface of the undifferentiated germ cells, in particular, the cell membrane surface as the binding of the antibody was confirmed in the process that the antibody was reacted with living cells without fixation or digestion of cell membranes.

[0063] Example 3: Concentration of undifferentiated germ cells of tuna by a MACS (Magnetic cell separation, Magnetic activated cell sorting) method Using two kinds of antibodies obtained from above Example 2, the concentration of the undifferentiated germ cells was carried out from the testis cells of the tuna using the MACS method. Specifically, the procedures are as follows. At first, testis cells of tuna were dispersed with L-15 culture medium (Invitrogen) containing collagenase H (Roche) at the final concentration of 2 mg/ml and disperse II (Godo Shusei Co., Ltd.) at the final concentration of 1.65 mg/m, and the ceil count was adjusted to 3x10 6 cells. As a primary antibody, 100 ul of antibody produced from hybridoma TA-No. 6-28 or hybridoma TA-No. 15-1, which was suspended and biotinylated using a One-step Antibody Biotinlation Kit (Miltenyi Biotech) by reacting at room temperature for 24 hours, was added to the dispersed cells. The mixture was incubated at 40 C for 30 minutes. After the completion of reaction, centrifugal separation (at 200 xg for 5 minutes) was carried out and the obtained cell sediment was washed with L-15 culture medium. Furthermore, the cell sediment was re-suspended in 240 pl of L-15 culture medium and 60 pl of anti-biotin microbeads (Miltenyi Biotech) as a secondary antibody was added to the suspension, and the mixture was incubated at 4°C for 15 minutes. After the completion of reaction, centrifugal separation (200 xg for 5 minutes) was carried out and the obtained cell sediment was washed with L-15 culture medium. Furthermore, the cell sediment was suspended in 500 p] of auto-MACS buffer (Miltenyi Biotech). Cells bound to magnetic beads were collected from the suspension using a magnetic separation apparatus: Mini MACS separator (Miltenyi Biotech) and an MS column (Miltenyi Biotech) to obtain MACS-concentrated undifferentiated germ cells.

[0064] The resulting MACS-concentrated undifferentiated germ cells were subjected to flow cytometry analysis and vasa in situ hybridization analysis according to the method of Example 1. The microscopic images by these analyses are shown in Figure 10.

[0065] As shown in Figure 10, the testis cells concentrated by a MACS method contained a large number of vasa positive cells.

[0066] Example 4: Transplantation of concentrated undifferentiated germ cells of bluefin tuna into nibe croaker (host) MACS-concentrated undifferentiated germ cells of bluefin tuna obtained in Example 3 were transplanted into the intraperitoneal cavity of nibe croaker and engraftability of the transplanted cells into the gonad of nibe croaker was confirmed. Specifically, the procedures are as follows.

[0067] As the host nibe croaker, 246 individuals of nibe croaker (Tokyo University of Marine Science and Technology, produced in Tateyama station) of 2 weeks of age were used. MACS-concentrated undifferentiated germ cells of bluefin tuna obtained in Example 3 was stained with PKH26 (SIGMA). The stained undifferentiated germ cells of bluefin tuna concentrated by MACS were transplanted into the intraperitoneal cavity of nibe croaker host according to a microinjection method (refer to Yazawa, R., Takeuchi, Y., Higuchi, K., Yatabe, T., Kabeya, N & Yoshizaki, Goro (2010) Chub mackerel gonads support colonization, survival, and proliferation of intraperitoneally transplanted xenogenic germ cells. Biology of reproduction, 82, 896-904) so as to be approximately 7000 to 10000 cells/fish. Laparotomy and fluorescence analysis were carried out 20 days after the transplantation. As control (without concentration), testis cells of bluefin tuna without concentration were dispersed with L-15 culture medium (Invitrogen) containing collagenase H (Roche) at the final concentration of 2 mg/ml and disperse II (Godo Shusei Co., Ltd.) at the final concentration of 1.65 mg/ml, and the obtained dispersed testis cells were used. The microscopic images by fluorescence analysis are shown in Figure 11.

[0068] As shown in Figure 11, in nibe croaker which was transplanted with MACS-concentrated undifferentiated germ cells of bluefin tuna, positive cells in the gonad were confirmed in the fluorescence field, more specifically, it was confirmed that MACS-concentrated undifferentiated germ cells of bluefin tuna transplanted into the gonad of nibe croaker were engrafted. Meanwhile, no positive reaction was confirmed at all in the control (without concentration). Figure 12 shows the percentage of each engrafted individuals as engraftment rate. Engraftment rate was calculated based on the following equation.

[0069]

[Equation 2] Engraftment rate (%) =Number of individuals engrafted with transplanted cells / Number of individuals performed transplantation and laparotomy

[0070] As shown in Figure 12, the engraftment rate of MACS-concentrated undifferentiated germ cells of bluefin tuna remarkably increased as compared to that of the undifferentiated germ cells without concentration.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

36A

SEQUENCE LISTING SEQUENCE LISTING

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<210> <210> 11 <211> <211> 24 24 <212> <212> DNA DNA <213> ArtificialSequence <213> Artificial Sequence

<220> <220> <223> Primer <223> Primer <400> 11 <400> caaccaggga gctcatcaac caaccaggga gctcatcaac caga caga 24 24

<210> <210> 22 <211> <211> 24 24 <212> <212> DNA DNA <213> ArtificialSequence <213> Artificial Sequence

<220> <220> <223> Primer <223> Primer <400> <400> 22 gcaactcagg tcaatgctgt gtgg gcaactcagg tcaatgctgt gtgg 24 24

1

Claims

[1] A monoclonal antibody that specifically recognizes a surface of undifferentiated germ cells of scombridae fish, wherein the monoclonal antibody is an antibody to undifferentiated germ cells separated from a testis or ovary of scombridae fish, and wherein the undifferentiated germ cells separated from a testis or ovary of scombridae fish are a cell population that appears in a fraction with forward scatter (FS) value of about higher 1/3 and side scatter (SS) value of about lower 1/3 in a flow cytometry (FCM) analysis with argon laser at 488nm of a cellular suspension containing undifferentiated germ cells and somatic cells derived from the testis or ovary, wherein the FCM analysis represents a FS-SS plot under a condition in which all the testis or ovary cells scattered in a single state by dispersion of cells are detectable.
[2] The monoclonal antibody according to claim 1 that specifically recognizes undifferentiated germ cells of scombridae fish, wherein the monoclonal antibody is produced by hybridoma TA-No. 6-28 (NITE ABP 02222) or TA-No.15-1 (NITE ABP-02223).
[3] The monoclonal antibody according to claim 1 or 2, wherein the undifferentiated germ cell is a primordial germ cell, type A spermatogonium or oogonium.
[4] The monoclonal antibody according to any one of claims 1 to 3, wherein the scombridae fish is a fish selected from genus Thunnus, genus Euthynnus, genus Katsuwonus, and genus Scomber.
[5] A method for concentrating undifferentiated germs cells of scombridae fish, comprising separating scombridae-derived undifferentiated germ cells from a testis or ovary of scombridae fish containing scombridae-derived undifferentiated germ cells by using the antibody according to any one of claims 1 to 4.
[6] A differentiation induction method of undifferentiated germ cells into gametes, comprising transplanting undifferentiated germ cells separated from a testis or ovary of scombridae fish into the intraperitoneal cavity of an individual host fish before or after hatching, further comprising: separating and concentrating undifferentiated germ cells of scombridae fish from a testis or ovary of scombridae fish containing scombridae-derived undifferentiated germ cells by using the antibody according to any one of claims 1 to 4 before transplantation; and transplanting the separated and concentrated undifferentiated germ cells into the intraperitoneal cavity of the individual host fish before or after hatching.
[7] The differentiation induction method of undifferentiated germ cells into gametes according to claim 6, wherein the scombridae fish that provides the undifferentiated germ cells to be transplanted is xenogenic to the host fish.
[8] The differentiation induction method of undifferentiated germ cells into gametes according to claim 6 or 7, wherein the scombridae fish that provides the undifferentiated germ cells to be transplanted is larger-sized fish than the host fish.
[9] The differentiation induction method of undifferentiated germ cells into gametes according to any one of claims 6 to 8, wherein the host fish is a fish selected from scombridae fish, and the scombridae fish that provides the undifferentiated germ cells to be transplanted, is a fish selected from tuna.
[10] A method for producing sperms or eggs of scombridae fish, comprising transplanting undifferentiated germ cells separated from a testis or ovary of scombridae fish into the intraperitoneal cavity of an individual host fish before or after hatching, further comprising: separating and concentrating undifferentiated germ cells of scombridae fish from a testis or ovary of scombridae fish containing scombridae-derived undifferentiated germ cells by using the antibody according to any one of claims 1 to 4 before transplanting; transplanting the separated and concentrated undifferentiated germ cells into the intraperitoneal cavity of the individual host fish before or after hatching; and inducing differentiation of the transplanted undifferentiated germ cells into gametes to obtain sperms or eggs of scombridae fish.
[11] A production method of scombridae fish, comprising transplanting undifferentiated germ cells separated from a testis or ovary of scombridae fish into the intraperitoneal cavity of an individual host fish before or after hatching, further comprising: separating and concentrating undifferentiated germ cells of scombridae fish from a testis or ovary of scombridae fish containing scombridae-derived undifferentiated germ cells by using the antibody according to any one of claims 1 to 4 before transplanting; transplanting the separated and concentrated undifferentiated germ cells into the intraperitoneal cavity of an individual host fish before or after hatching; inducing differentiation of the transplanted undifferentiated germ cells into gametes; and crossbreeding the obtained sperms and eggs.