Method for inducing immature oocytes and method for producing mature oocytes

Abstract

To provide a method for easily inducing, by a culture time period shorter than before, immature oocytes from cells, such as pluripotent stem cells, having differentiation potential to oocytes.SOLUTION: The method for inducing immature oocytes comprises inducing four types of genes consisting of FIGLA, NOBOX, LHX8 and TBPL2, or their transcripts or expressed proteins into at least one cell selected from the group consisting of Pluripotent stem cells, Epiblast-like cells, and Primordial germ cells.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for inducing immature oocytes and a method for producing mature oocytes.

Background Art

[0002] In mammals, totipotency defined as the ability to develop into an individual from a single cell is a special property possessed by single cells. However, the mechanism for forming such totipotency has been difficult, despite strong social demands in fields such as infertility treatment. The reason is that the oogenesis process that progresses in the fetal ovary cannot be reproduced in vitro.

[0003] The inventors have previously developed an in vitro culture system for reconstructing oocytes from mouse pluripotent stem cells. Specifically, mouse ES cells or iPS cells are induced to differentiate into primordial germ cell-like cells (PGCLCs) using a medium containing a humoral factor such as BMP4, and the obtained PGCLCs are mixed with ovarian somatic cells to prepare a reconstructed ovary. Next, the period from PGCLCs to the formation of metaphase II eggs in the reconstructed ovary is divided into three periods: "in vitro differentiation period", "in vitro growth period", and "in vitro maturation period", and optimal culture conditions for obtaining secondary follicles, oocytes at the oocyte nucleus stage, and metaphase II eggs are established in each culture period (see, for example, Non-Patent Document 1, etc.).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, the method described in Non-Patent Document 1 requires a period of approximately 3 to 4 weeks to obtain primordial follicle oocytes from mouse pluripotent stem cells, and it is expected that culturing for more than one year will be necessary to produce oocytes from pluripotent stem cells in an in vitro culture system in primates and other large mammals.

[0006] The present invention has been made in view of the above circumstances, and provides a method for easily inducing immature oocytes from cells having the ability to differentiate into oocytes, such as pluripotent stem cells, in a shorter culture period than conventional methods. Furthermore, it provides a method for easily producing mature oocytes from cells having the ability to differentiate into oocytes, such as pluripotent stem cells, in a shorter culture period than conventional methods. [Means for solving the problem]

[0007] As a result of diligent research to achieve the above objective, the inventors discovered that by introducing specific genes involved in oocyte formation into pluripotent stem cells and culturing them for a short period of time, approximately 5 to 10 days, differentiation into immature oocytes can be induced, thus completing the present invention.

[0008] In other words, the present invention includes the following embodiments. A method for inducing immature oocytes according to a first aspect of the present invention includes introducing four genes, consisting of FIGLA, NOBOX, LHX8, and TBPL2, or their transcripts or expressed proteins, into at least one type of cell selected from the group consisting of pluripotent stem cells, epiblast-like cells, and primordial germ cells. The method for inducing immature oocytes according to the first embodiment described above may include introducing four genes, consisting of FIGLA, NOBOX, LHX8, and TBPL2, into the cells. The method for inducing immature oocytes according to the first embodiment described above may further include introducing the STAT3 gene, or its transcript or expressed protein, into the cells. The method for inducing immature oocytes according to the first embodiment described above may further include introducing one or more genes selected from the group consisting of SOHLH1, SUB1, and DYNLL1, or their transcripts or expressed proteins, into the cells. The method for inducing immature oocytes according to the first embodiment described above may further include introducing three genes consisting of SOHLH1, SUB1, and DYNLL1 into the cells. The aforementioned cells may also be pluripotent stem cells. In the method for inducing immature oocytes according to the first embodiment described above, the expression of the gene is controlled to be induced by the presence of an expression-inducing substance. After the introduction, the cells are made to proliferate, After the aforementioned proliferation, the expression-inducing substance is added to the culture medium to induce the expression of the gene. It may also include ,

[0009] A method for producing mature oocytes according to a second aspect of the present invention is: The process involves introducing four genes, consisting of FIGLA, NOBOX, LHX8, and TBPL2, or their transcripts or expressed proteins, into at least one cell type selected from the group consisting of pluripotent stem cells and primordial germ cells. The cells introduced and the ovarian somatic cells are co-cultured, Includes. The method for producing mature oocytes according to the second embodiment described above may further include introducing the STAT3 gene, or its transcript or expressed protein, into the cells. [Effects of the Invention]

[0010] According to the method for inducing immature oocytes in the above embodiment, immature oocytes can be easily induced from cells with the ability to differentiate into oocytes, such as pluripotent stem cells, in a shorter culture period than conventional methods. According to the method for producing mature oocytes in the above embodiment, a large number of mature oocytes can be easily produced from cells with the ability to differentiate into oocytes, such as pluripotent stem cells, in a shorter culture period than conventional methods. [Brief explanation of the drawing]

[0011] [Figure 1] These are bright-field and fluorescence images (magnification: 200x) of immature oocytes induced from mouse ES cells in Example 1. [Figure 2] The images show fluorescence images (magnification: 8x) of aggregates consisting of pluripotent stem cells into which oocyte formation genes have been introduced and ovarian somatic cells at each culture period in Example 1. In the upper panel, oocytes are visualized by fluorescence of enhanced cyan fluorescent protein (ECFP) expressed under controlled expression of Stella, a germ cell and oocyte marker. In the lower panel, oocytes are visualized by fluorescence of the red fluorescent protein mCherry expressed under controlled expression of Nucleoplasmin 2 (Npm2), an activated (mature) oocyte marker. [Figure 3] These are bright-field and fluorescence images (magnification: 8x) of immature oocytes induced from mouse iPS cells in Example 2. [Figure 4] This figure shows the results of identifying key factors in oocyte formation genes in Example 3. In the left figure, the vertical axis represents the sample name, and the horizontal axis represents the type of oocyte formation gene. White cells indicate that the gene in question was not introduced. The center figure shows the number of oocytes formed from each cell line. The right figure shows the area of ​​oocytes calculated from the fluorescence area of ​​the blue fluorescent protein CFP expressed downstream of the Stella gene in each sample. [Modes for carrying out the invention]

[0012] <Method for inducing immature oocytes> In one embodiment, the present invention provides a method for inducing immature oocytes, which includes introducing four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2, or their transcripts or expressed proteins, into at least one cell selected from the group consisting of pluripotent stem cells, epiblast-like cells, and primordial germ cells (hereinafter, these cells may be collectively referred to as "cells having the ability to differentiate into oocytes").

[0013] In the conventional method, in the case of mice, it took about less than a month to induce differentiation from pluripotent stem cells to immature oocytes in vitro, and it took about 11 days to induce differentiation from primordial germ cells to immature oocytes in vitro. In contrast, in the method for inducing immature oocytes of the present embodiment, by introducing the above four types of genes into cells having the ability to differentiate into oocytes such as pluripotent stem cells and culturing them for a short period of 5 days or more and 10 days or less, it is possible to induce differentiation into immature oocytes. Furthermore, in the case of humans, in vivo, it takes a period of 9 months or more to induce differentiation from primordial germ cells to immature oocytes, but by using the method for inducing immature oocytes of the present embodiment, this period can be dramatically shortened.

[0014] Also, in the conventional method, in order to induce differentiation from pluripotent stem cells to PGCLCs and further induce differentiation of the PGCLCs into immature oocytes, it was necessary to go through at least two steps with different culture conditions. In contrast, in the method for inducing immature oocytes of the present embodiment, pluripotent stem cells can be directly induced to differentiate into immature oocytes.

[0015] In this specification, the "immature oocyte" refers to a primary oocyte that has not undergone follicular growth. Immature oocytes do not necessarily have to have a follicular structure. Also, in immature oocytes, as shown in the examples described later, some maternal effect genes such as the Stella gene and Padi6 gene, which are oocyte markers, are expressed. In addition, in this specification, an "ovum" consists of an oocyte and somatic cells (granulosa cells and theca cells) surrounding it. The method for inducing immature oocytes of the present embodiment will be described in detail below.

[0016] [Oocyte formation gene] The oocyte formation genes used for introduction into cells having the ability to differentiate into oocytes such as pluripotent stem cells have been identified by the inventors through RNA sequencing (RNA-Seq) analysis of gene expression dynamics in the oocyte lineage. Specifically, examples of oocyte formation genes include FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2, DYNLL1, etc. As shown in the examples described later, four genes consisting of FIGLA, NOBOX, LHX8, and TBPL2 are particularly important for the induction of immature oocytes from pluripotent stem cells. Therefore, among the above-mentioned oocyte formation genes, by introducing at least four genes consisting of FIGLA, NOBOX, LHX8, and TBPL2 or their transcripts or expressed proteins into cells, cells having the ability to differentiate into oocytes can be induced into immature oocytes.

[0017] In addition to the above four genes or their transcripts or expressed proteins, it is preferable to further introduce the STAT3 gene or its transcript or expressed protein, and it is more preferable to further introduce the STAT3 gene. As shown in the examples described later, by introducing STAT3 into cells in addition to FIGLA, NOBOX, LHX8, and TBPL2, the formation rate of oocytes can be further improved.

[0018] Furthermore, in addition to the five genes FIGLA, NOBOX, LHX8, TBPL2, and STAT3, or their transcripts or expressed proteins, it is preferable to further introduce one or more genes selected from the group consisting of SOHLH1, SUB1, and DYNLL1, or their transcripts or expressed proteins, and it is even more preferable to further introduce three genes consisting of SOHLH1, SUB1, and DYNLL1, or their transcripts or expressed proteins. As shown in the examples described later, by introducing all eight of the above genes, or their transcripts or expressed proteins, into cells, cells capable of differentiating into oocytes can be efficiently induced from immature oocytes.

[0019] The FIGLA gene encodes a basic helix-loop-helix (bHLH) transcription factor that regulates several oocyte-specific genes (including genes involved in follicular formation and genes encoding the zona pellucida (ZP1, ZP2, and ZP3)). The transcription factor FIGLA binds to the E-box (5'-CANNTG-3') of the ZP (ZP1, ZP2, and ZP3) promoter. Diseases associated with FIGLA include, for example, premature ovarian failure (type 6) and pseudohermaphroditism. The FIGLA gene ontology (GO) annotation includes sequence-specific DNA binding and protein dimerization activity. An important paralog of FIGLA is SCX. FIGA is also called Folliculogenesis Specific BHLH Transcription Factor, Factor In The Germline Alpha, Folliculogenesis-Specific Basic Helix-Loop-Helix Protein, Transcription Factor FIGa, BHLHC8 (BHLHc8), Folliculogenesis Specific Basic Helix-Loop-Helix, FIGALPHA (FIGalpha), and POF6.

[0020] The nucleotide sequences of oocyte-forming genes such as the FIGLA gene, the mRNA sequences of these genes, and the amino acid sequences of the proteins encoded by these genes can be obtained from databases such as Genbank.

[0021] The nucleotide sequence of the human FIGLA gene is disclosed, for example, as "Gene ID:344018" in Genbank. The nucleotide sequence of the human FIGLA gene mRNA is disclosed, for example, as accession number NM_001004311 in Genbank. The amino acid sequence of human FIGLA is disclosed as accession number NP_001004311 in Genbank.

[0022] The nucleotide sequence of the mouse FIGLA gene is disclosed, for example, as "Gene ID:26910" in Genbank. The nucleotide sequence of the mRNA of the mouse FIGLA gene is disclosed, for example, as accession number NM_012013 in Genbank. The amino acid sequence of mouse FIGLA is disclosed as accession number NP_036143 in Genbank.

[0023] The NOBOX gene encodes a transcription factor involved in oogenesis. Diseases associated with NOBOX include, for example, premature ovarian failure (type 5). The GO annotation of NOBOX includes DNA-binding transcription factor activity and RNA polymerase II-promoter sequence-specific DNA binding. Specifically, it preferably binds to base sequences such as "5'-TAATTG-3'", "5'-TAGTTG-3'", and "5'-TAATTA-3'". An important paralog of NOBOX is UNCX. NOBOX is also known as NOBOX Oogenesis Homeobox, Homeobox Protein NOBOX, Newborn Ovary Homeobox-Encoding Gene, Newborn Ovary Homeobox-Encoding, TCAG_12042, OG-2 (OG2), OG2X, and POF5.

[0024] The nucleotide sequence of the human NOBOX gene is disclosed, for example, as "Gene ID:135935" in Genbank. The nucleotide sequence of the human NOBOX gene mRNA is disclosed, for example, as accession numbers NM_001080413 and XM_001134420 in Genbank. The amino acid sequence of human NOBOX is disclosed as accession numbers NP_001073882 and XP_001134420 in Genbank.

[0025] The nucleotide sequence of the mouse NOBOX gene is disclosed, for example, as "Gene ID:18291" in Genbank. The nucleotide sequence of the mouse NOBOX gene mRNA is disclosed, for example, as accession number NM_130869 in Genbank. The amino acid sequence of mouse NOBOX is disclosed as accession number NP_570939 in Genbank.

[0026] The SOHLH1 gene is a gonad-specific transcription factor essential for spermatogenesis, oogenesis, and follicular formation, and encodes a basic helix-loop-helix (bHLH) transcription factor. SOHLH1 plays a role in regulating oocyte differentiation without affecting meiosis I. Diseases associated with SOHLH1 include, for example, non-obstructive azoospermia and ovarian dysplasia. GO annotations for SOHLH1 include DNA-binding transcription factor activity and protein dimerization activity. An important paralog of the SOHLH1 gene is SOHLH2. SOHLH1 is also known as Spermatogenesis And Oogenesis Specific Basic Helix-Loop-Helix 1, Spermatogenesis-And Oogenesis-Specific Basic Helix-Loop-Helix-Containing Protein 1, Spermatogenesis Associated 27, C9orf157, NOHLH, TEB2, Chromosome 9 Open Reading Frame 157, Newborn Ovary Helix Loop Helix, BA100C15.3, SPATA27, BHLHe80, SPGF32, and ODG5.

[0027] The nucleotide sequence of the human SOHLH1 gene is disclosed, for example, as "Gene ID:402381" in Genbank. The nucleotide sequence of the human SOHLH1 gene mRNA is disclosed, for example, as accession numbers NM_001012415 and XM_497082 in Genbank. The amino acid sequence of human SOHLH1 is disclosed as accession numbers NP_001012415 and XP_497082 in Genbank.

[0028] The nucleotide sequence of the mouse SOHLH1 gene is disclosed, for example, as "Gene ID:227631" in Genbank; the nucleotide sequence of the mRNA of the mouse SOHLH1 gene is disclosed, for example, as accession numbers NM_001001714 and XM_130180 in Genbank; and the amino acid sequence of mouse SOHLH1 is disclosed as accession numbers NP_001001714 and XP_130180 in Genbank.

[0029] LHX8 is a member of the LIM homeobox family of proteins and is involved in pattern formation and differentiation of various tissues. In addition to the DNA-binding homeodomain, LIM homeobox family proteins contain two tandem repeating cysteine-rich double zinc finger motifs known as LIM domains. LIM homeobox family proteins are transcription factors involved in tooth morphogenesis, oogenesis, and neuronal differentiation. Diseases associated with the LHX8 gene include, for example, cleft palate and odontoma. LHX8 is also known as LIM Homeobox 8, LIM / Homeobox Protein Lhx8, LIM-Homeodomain Protein Lhx8, LIM Homeobox Protein 8, and LHX7.

[0030] The nucleotide sequence of the human LHX8 gene is disclosed, for example, as "Gene ID:431707" in Genbank. The nucleotide sequence of the human LHX8 gene mRNA is disclosed, for example, as accession numbers NM_001001933, XM_086344, NM_001256114, XM_017001316, and XM_017001317 in Genbank. The amino acid sequence of human LHX8 is disclosed as accession numbers NP_001001933, XP_086344, NP_001243043, XP_016856805, and XP_016856806 in Genbank.

[0031] The nucleotide sequence of the mouse LHX8 gene is disclosed, for example, as "Gene ID:16875" in Genbank. The nucleotide sequence of the mouse LHX8 gene mRNA is disclosed, for example, as accession numbers NM_010713, XM_006501072, and XM_017319470 in Genbank. The amino acid sequence of mouse LHX8 is disclosed as accession numbers NP_034843, XP_006501135, and XP_017174959 in Genbank.

[0032] The SUB1 gene is a gene that encodes a transcription regulator. SUB1 functions in conjunction with TAF, acting as a coactivator that mediates functional interactions between upstream activators and general transcriptional functions. Diseases associated with SUB1 include, for example, onychomycosis (nail fungus). GO annotation for SUB1 includes single-strand DNA binding. SUB1 is also known as SUB1 Homolog, Transcriptional Regulator, Positive Cofactor 4, Activated RNA Polymerase II Transcriptional Coactivator P15, PC4, P14, Activated RNA Polymerase II Transcription Cofactor 4, RPO2TC1, and P15.

[0033] The nucleotide sequence of the human SUB1 gene is disclosed, for example, as "Gene ID:10923" in Genbank. The nucleotide sequence of the human SUB1 gene mRNA is disclosed, for example, as accession numbers NM_006713, XM_017008986, XM_017008987, and XM_011513944 in Genbank. The amino acid sequence of human SUB1 is disclosed as accession numbers NP_006704, XP_016864475, XP_016864476, and XP_011512246 in Genbank.

[0034] The nucleotide sequence of the mouse SUB1 gene is disclosed, for example, as "Gene ID:20024" in Genbank. The nucleotide sequence of the mouse SUB1 gene mRNA is disclosed, for example, as accession numbers NM_011294 and XM_006520042 in Genbank. The amino acid sequence of mouse SUB1 is disclosed as accession numbers NP_035424 and XP_006520105 in Genbank.

[0035] STAT3 is a member of the STAT protein family. The STAT protein family is phosphorylated into receptor-associated kinases in response to cytokines and growth factors such as interferon (IFN), epidermal growth factor (EGF), interleukin-5 (IL-5), interleukin-6 (IL-6), hepatocyte growth factor (HGF), leukemia suppressor (LIF), and bone morphogenetic protein 2 (BMP2). These kinases then form homodimers or heterodimers, which translocate to the cell nucleus to act as transcription activators, playing important roles in many cellular processes such as cell proliferation and apoptosis. Diseases associated with STAT3 include, for example, infantile-onset multi-organ autoimmune diseases and autosomal dominant hyper-IgE syndromes. GO annotations for STAT3 include DNA-binding transcription factor activity and sequence-specific DNA binding. A key paralog of the STAT3 gene is STAT1. STAT3 is also known as Signal Transducer And Activator Of Transcription 3, Acute-Phase Response Factor, APRF, Signal Transducer And Activator Of Transcription 3, DNA-Binding Protein APRF, ADMIO1, ADMIO, and HIES.

[0036] The nucleotide sequence of the human STAT3 gene is disclosed, for example, as "Gene ID:6774" in Genbank. The nucleotide sequence of the human STAT3 gene mRNA is disclosed, for example, as accession numbers NM_001369512, NM_001369513, NM_001369514, NM_001369516, NM_001369517, NM_001369518, NM_001369519, NM_001369520, NM_003150, N It is disclosed as M_139276, NM_213662, XM_017024973, XM_011525146, XM_011525145, XM_017024972, XM_005257617, XM_005257616, XM_017024975, XM_024450896, XM_017024974, and XM_017024976. The amino acid sequences of human STAT3 are listed in Genbank accession numbers NP_001356441, NP_001356442, NP_001356443, NP_001356445, NP_001356446, NP_001356447, NP_001356448, NP_001356449, NP_003141, and NP_6448. It is disclosed as 05, NP_998827, XP_016880462, XP_011523448, XP_011523447, XP_016880461, XP_005257674, XP_005257673, XP_016880464, XP_024306664, XP_016880463, and XP_016880465.

[0037] The nucleotide sequence of the mouse STAT3 gene is disclosed, for example, as "Gene ID:20848" in Genbank. The nucleotide sequence of the mouse STAT3 gene mRNA is disclosed, for example, as accession numbers NM_011486, NM_213659, NM_213660, XM_011248846, and XM_017314401 in Genbank. The amino acid sequence of mouse STAT3 is disclosed as accession numbers NP_035616, NP_998824, NP_998825, XP_011247148, and XP_017169890 in Genbank.

[0038] The TBPL2 gene encodes TATA-box binding protein-like 2. TBPL2 is a transcription factor that forms a complex with TAF3 to induce myoblast differentiation into muscle cells. This complex replaces TFIID in specific promoters during the early stages of differentiation. Diseases associated with TBPL2 include, for example, retinitis pigmentosa. GO annotation for TBPL2 includes DNA-binding transcription factor activity. An important paralog of the TBPL2 gene is TBP. TBPL2 is also known as TATA-Box Binding Protein Like 2, TATA Box-Binding Protein-Related Factor 3, TATA Box-Binding Protein-Like Protein 2, TBP-Related Factor 3, TBP-Like Protein 2, TBP2, and TRF3.

[0039] The nucleotide sequence of the human TBPL2 gene is disclosed, for example, as "Gene ID:387332" in Genbank. The nucleotide sequence of the human TBPL2 gene mRNA is disclosed, for example, as accession number NM_199047 in Genbank. The amino acid sequence of human TBPL2 is disclosed as accession number NP_950248 in Genbank.

[0040] The nucleotide sequence of the mouse TBPL2 gene is disclosed, for example, as "Gene ID:227606" in Genbank. The nucleotide sequence of the mouse TBPL2 gene mRNA is disclosed, for example, as accession numbers NM_001289689 and NM_199059 in Genbank. The amino acid sequence of mouse TBPL2 is disclosed as accession numbers NP_001276618 and NP_951014 in Genbank.

[0041] The DYNLL1 gene encodes a protein classified as a light chain, which is one of the proteins that make up cytoplasmic dynein, an enzyme complex with a molecular weight of approximately 1200 kDa. Diseases associated with DYNLL1 include, for example, chronic enterovenous insufficiency. GO annotation for DYNLL1 includes protein homodimerization activity and protein domain-specific binding. An important paralog of the DYNLL1 gene is DYNLL2. DYNLL1 is also known as Dynein Light Chain LC8-Type 1, Protein Inhibitor Of Neuronal Nitric Oxide Synthase, Dynein, Cytoplasmic, Light Polypeptide 1, Dynein Light Chain 1, Cytoplasmic, 8 kDa Dynein Light Chain, DNCLC1, DNCL1, DLC1, DLC8, PIN, Cytoplasmic Dynein Light Polypeptide, HDLC1 (HDLC1), LC8a, and LC8.

[0042] The nucleotide sequence of the human DYNLL1 gene is disclosed, for example, as "Gene ID:8655" in Genbank. The nucleotide sequence of the human DYNLL1 gene mRNA is disclosed, for example, as accession numbers NM_001037494, NM_001037495, and NM_003746 in Genbank. The amino acid sequence of human DYNLL1 is disclosed as accession numbers NP_001032583, NP_001032584, and NP_003737 in Genbank.

[0043] The nucleotide sequence of the mouse DYNLL1 gene is disclosed, for example, as "Gene ID: 56455" in Genbank. The nucleotide sequence of the mRNA of the mouse DYNLL1 gene is disclosed, for example, as accession number NM_019682 in Genbank. The amino acid sequence of mouse DYNLL1 is disclosed as accession number NP_062656 in Genbank.

[0044] [Cells capable of differentiating into oocytes] The cells having the ability to differentiate into oocytes used in the method for inducing immature oocytes in this embodiment are preferably at least one type of cell selected from the group consisting of pluripotent stem cells, epiblast-like cells (EpiLCs), and primordial germ cells.

[0045] (pluripotent stem cells) In this specification, "pluripotent stem cells" refers to undifferentiated cells that possess "self-renewal ability," which allows them to proliferate while maintaining an undifferentiated state, and "differentiation pluripotency," which allows them to differentiate into all three germ layer lineages. Examples of pluripotent stem cells, though not limited to the following, include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic germ cells (EG cells) derived from primordial germ cells, multipotent GS cells (mGS cells) isolated during the establishment and culture process of GS (Germline Stem) cells from testicular tissue, and Muse cells isolated from bone marrow mesenchymal cells. Note that ES cells may also be ES cells produced by nuclear reprogramming from somatic cells. The pluripotent stem cells listed above can each be obtained by known methods.

[0046] In this specification, "iPS cells" refers to cells that have been differentiated into somatic cells and have been reprogrammed into cells of various tissues and organs by introducing several genes into them. The iPS cells used in the method for inducing immature oocytes in this embodiment may be derived from primary cultured somatic cells collected from a suitable donor, or from an established cell line. Since iPS cells can be differentiated into any germ cell line, the somatic cells used to prepare iPS cells may, in principle, be derived from either ectoderm or endoderm cells. Cells such as skin, hair, gums, and blood are suitable as somatic cells for preparing iPS cells because they are less invasive and easier to collect. The method for preparing iPS cells may be any method known in the art. Specifically, preparation methods described in known literature such as "Okita K. et al., "Generation of germline-competent induced pluripotent stem cells.", Nature, Vol.448, p313-317, 2007." (Reference 1) and "Hamanaka S. et al., "Generation of germline-competent rat induced pluripotent stem cells.", PLoS One, Vol.6, Issue 7, e22008, 2011." (Reference 2) can be used.

[0047] The ES cells used in the method for inducing immature oocytes in this embodiment can be obtained by known methods. For example, they can be established by collecting the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on feeder cells derived from fibroblasts. In addition, ES cells established by culturing early embryos created by nuclear transfer of somatic cell nuclei can also be used. Furthermore, as shown in the examples described later, ES cells can be maintained and cultured in serum-free medium supplemented with 2i (2 inhibitor; PD0325901 and CHIR99021) and LIF (Leukemia Inhibitory Factor) without using feeder cells (Reference 3: "Ying QL et al., "The ground state of embryonic stem cell self-renewal.", Nature, Vol.453, No.7194, p519-523, 2008.").

[0048] (Epiblast-like cells) In this specification, "epiblast-like cells (EpiLCs)" are cells differentiated from pluripotent stem cells (e.g., iPS cells, ES cells, etc.) under specific culture conditions, and possess characteristics very similar to epiblasts (tissues that differentiate into primordial germ cells in vivo). Methods for differentiating EpiLCs from pluripotent stem cells (iPS cells or ES cells) can be carried out by referring to known methods, such as Japanese Patent Publication No. 2013-538038 (Reference 4) or "Hayashi K. et al., "Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells," Cell, Vol. 146, No. 4, pp. 519-532, 2011." (Reference 5).

[0049] (Primordial germ cells) Furthermore, in this specification, "primordial germ cells" refer to cells that are intended to differentiate into germ cells and will eventually differentiate into eggs or sperm after meiosis. Primordial germ cells may be of living origin or may be primordial germ cell-like cells (PGCLCs) differentiated from pluripotent stem cells. When primordial germ cells are collected from living organisms, for example, they can be collected together with the gonads from female mouse fetuses (from 11.5 days to 12.5 days of age). When collecting gonads from living organisms, they may be collected together with the mesonephrone, or the mesonephrone may be detached and collected separately.

[0050] Furthermore, as mentioned above, "primordial germ cells" include primordial germ cell-like cells differentiated from pluripotent stem cells. Methods for inducing differentiation of PGCLCs from pluripotent stem cells (iPS cells or ES cells) can be carried out by known methods, such as referring to Reference 5 above.

[0051] Furthermore, when using PGCLCs derived from pluripotent stem cells as primordial germ cells, it is preferable to remove undifferentiated cells from the differentiated pluripotent stem cell population beforehand. Such methods are well known; for example, by introducing nucleic acids encoding a fusion protein in which the primordial germ cell marker gene Blimp1 and a reporter protein are bound into pluripotent stem cells, PGCLCs differentiated from pluripotent stem cells and undifferentiated cells can be easily separated by methods such as fluorescence-activated cell sorting (FACS).

[0052] Furthermore, "primordial germ cells" also include cells whose genes have been modified using genetic engineering techniques, such as primordial germ cells derived from living organisms or primordial germ cell-like cells derived from pluripotent stem cells. Methods for modifying the genes of primordial germ cells derived from living organisms and primordial germ cell-like cells derived from pluripotent stem cells include the introduction of target nucleic acids or vectors using known genome editing methods such as the CRISPR system, methods using Transcription Activator-Like Effector Nucleases (TALEN), methods using zinc finger nucleases, and homologous recombination. Examples of methods for introducing nucleic acids or vectors include microinjection, electroporation, lipofection, and nucleic acid introduction using viral vectors. Moreover, the methods for introducing foreign genes or foreign nucleic acid fragments are not limited to those listed above, as long as the genetically modified primordial germ cells can differentiate into functional oocytes by the method of this embodiment. Note that genetic modification of primordial germ cells can be performed at an appropriate timing during the culture period of primordial germ cells. For example, in mice, this can be performed between 11.5 and 12.5 days of age. Furthermore, when using primordial germ cell-like cells derived from pluripotent stem cells, the pluripotent stem cells can be genetically modified using known methods before differentiation into primordial germ cell-like cells.

[0053] In particular, pluripotent stem cells are preferred as the cells with the ability to differentiate into oocytes used in the method for inducing immature oocytes in this embodiment. Especially when ES cells are used, a large number of immature oocytes can be obtained due to their high proliferative capacity.

[0054] Furthermore, cells capable of differentiating into oocytes may be derived from mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, and other primates; domestic animals such as dogs, cats, rabbits, horses, sheep, goats, cattle, pigs, rats (including nude rats), mice (including nude mice and skid mice), hamsters, guinea pigs, and other domestic animals, pets, and laboratory animals.

[0055] [Introduction process] In the method for inducing immature oocytes according to this embodiment, the oocyte-forming gene is introduced into at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells.

[0056] Alternatively, instead of the oocyte-forming gene, its transcript, mRNA, or its expression protein may be introduced. As the mRNA and expression protein of the oocyte-forming gene, mRNA consisting of the nucleotide sequence indicated by the Genbank accession number and expression protein consisting of the amino acid sequence can be used.

[0057] The method of introduction is not particularly limited and can be appropriately selected depending on the target cells and the type of introduction material (whether it is nucleic acid or protein, etc.).

[0058] The method for introducing oocyte-forming genes into cells is not particularly limited, and known methods can be appropriately selected and used. Specifically, examples include lipofection, microinjection, DEAE dextran, gene gun, electroporation, and calcium phosphate.

[0059] The method for introducing mRNA of oocyte-forming genes into cells is not particularly limited, and known methods can be appropriately selected and used. Specifically, for example, methods using commercially available RNA transfection reagents such as Lipofectamine® MessengerMAX (manufactured by Life Technologies) can be used.

[0060] The method for introducing the expression protein of the oocyte-forming gene into cells is not particularly limited, and known methods can be appropriately selected and used. Specifically, examples include methods using protein delivery reagents, methods using protein delivery domain (PTD) fusion proteins, and microinjection methods.

[0061] The oocyte-forming gene may be introduced in the form of an expression vector into at least one cell type selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells and transiently expressed, or the oocyte-forming gene may be incorporated into the chromosome of at least one cell type selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells. Among these, it is preferable to incorporate the oocyte-forming gene into the chromosome of at least one cell type selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells because it allows for stable gene expression.

[0062] When introducing an oocyte-forming gene into at least one cell type selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells in the form of an expression vector, an expression vector can be used that includes the nucleotide sequence of the oocyte-forming gene and a promoter that controls the expression of the nucleotide sequence of the oocyte-forming gene. In this expression vector, the nucleotide sequence of the oocyte-forming gene is functionally linked to the promoter. Furthermore, all of the eight oocyte-forming genes to be used may be incorporated into a single expression vector, or they may be incorporated into different vectors one by one, but from the viewpoint of introduction efficiency, it is preferable to incorporate all of the genes to be used into a single expression vector.

[0063] There are no particular limitations on the promoter; it may be one that is active in the target cells, or it may be an expression-inducing promoter whose activity can be induced by drugs or the like.

[0064] Examples of promoters that exhibit activity in target cells include the cytomegalovirus promoter (CMV promoter) and CMV early enhancer / chicken beta actin (CAG promoter), which exhibit strong promoter activity in almost all cells.

[0065] Examples of expression-inducing promoters include doxycycline-inducible promoters (TetO promoters), which allow for the artificial control of promoter activity.

[0066] The expression vector may optionally include, in addition to the nucleotide sequence and promoter of the oocyte-forming gene, enhancers, poly(A) addition signals, marker genes, replication origins, genes encoding proteins that bind to replication origins and regulate replication. A "marker gene" refers to a gene that, when introduced into cells, enables cell sorting or selection. Specific examples of marker genes include drug resistance genes, fluorescent protein genes, luminescent enzyme genes, and chromogenic enzyme genes. These may be used individually or in combination of two or more. Specific examples of drug resistance genes include puromycin resistance genes, genetisin resistance genes, neomycin resistance genes, tetracycline resistance genes, kanamycin resistance genes, zeosin resistance genes, hygromycin resistance genes, and chloramphenicol resistance genes. Specific examples of fluorescent protein genes include green fluorescent protein (GFP) genes, yellow fluorescent protein (YFP) genes, and red fluorescent protein (RFP) genes. Specific examples of luminescent enzyme genes include luciferase genes. Specific examples of the aforementioned chromogenic enzyme genes include, for example, the β-galactosidase gene, the β-glucuronidase gene, and the alkaline phosphatase gene.

[0067] The expression vector into which the oocyte-forming gene is incorporated is not particularly limited, and any known expression vector can be used. Examples of expression vectors include plasmid vectors and viral vectors.

[0068] Plasmid vectors are not particularly limited as long as they are plasmid vectors that can be expressed in at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells. For example, plasmid vectors commonly used for mammalian cell expression can be used. Examples of mammalian cell expression plasmid vectors include, but are not limited to, pX459, pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo.

[0069] Examples of viral vectors include retrovirus (including lentivirus) vectors, adenovirus vectors, adeno-associated virus vectors, Sendai virus vectors, herpesvirus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, sylvisvirus vectors, rhabdovirus vectors, paramyxovirus vectors, and orthomyxovirus vectors.

[0070] Among these, plasmid vectors are preferred as expression vectors.

[0071] When incorporating oocyte-forming genes into chromosomes, this can be done using known knock-in systems. Examples of known knock-in systems include methods that involve cutting chromosomes using known genome editing methods such as the CRISPR / Cas system, Transcription Activator-Like Effector Nucleases (TALEN), and zinc finger nucleases, followed by homologous recombination using a donor vector for homologous recombination, as well as methods using transposon vector systems.

[0072] The donor vector contains a nucleotide sequence adjacent to the target region as a homology arm. The donor vector may contain the nucleotide sequence of the oocyte-forming gene (hereinafter sometimes referred to as the "knock-in sequence") between the 5' arm and the 3' arm. Furthermore, in order to stably express the oocyte-forming gene, it is preferable to set the target region within the safe harbor region.

[0073] The donor vector may be a circular DNA vector (e.g., a plasmid vector) or a linear DNA vector. In addition to homology arms and knock-in sequences, the donor vector may contain other sequences. Examples of other sequences include marker genes, replication origins, and genes encoding proteins that bind to replication origins and regulate replication. Examples of marker genes are the same as those mentioned above.

[0074] The method of introducing the donor vector is not particularly limited and can be appropriately selected depending on the target cells. Examples of methods for introducing the donor vector into cells include lipofection, microinjection, DEAE dextran method, gene gun method, electroporation method, and calcium phosphate method.

[0075] In the transposon vector system, as shown in the examples described later, a transposon vector incorporating the nucleotide sequence of an oocyte-forming gene can be introduced into cells and easily incorporated into the cell's chromosomes by acting on it with a transposase. Furthermore, by acting on it again with a transposase, the nucleotide sequence of the oocyte-forming gene incorporated into the chromosome can be excised from the chromosome and removed without leaving any trace. Examples of transposons include piggyBac®, Sleeping Beauty, Tol II, and mariner.

[0076] The method for inducing immature oocytes in this embodiment may include optional steps in addition to the introduction step described above. Optional steps include, for example, a step of cell proliferation (proliferation step), a step of selecting cells into which the introduced oocyte-forming gene, its transcript, or its expression protein has been introduced (selection step), and a step of culturing the cells after the introduction step in a state where the oocyte-forming gene is expressed within the cell or the expression protein of the oocyte-forming gene is present (cultivation step). Furthermore, if the oocyte-forming gene is introduced in the introduction step, a step of inducing the expression of the introduced oocyte-forming gene (expression induction step) may be included.

[0077] [Proliferation process] In the proliferation process, in order to obtain a larger quantity of immature oocytes, at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells is proliferated.

[0078] In the proliferation process, for example, cells can be proliferated by culturing at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells in a proliferation medium. As the proliferation medium, known media for culturing ES cells, iPS cells, EpiLCs, primordial germ cells, etc., can be used, but are not limited to these, and any medium suitable for culturing ES cells, iPS cells, EpiLCs, and primordial germ cells may be used. Specifically, as shown in the examples described later, examples of proliferation media include serum-free media supplemented with 2i (2 inhibitor; PD0325901 and CHIR99021) and LIF (Leukemia Inhibitory Factor).

[0079] The culture conditions in the proliferation process can be those known for culturing at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells. Specifically, for example, the culture temperature can be set to approximately 30°C to 37°C. While there are no specific limitations on the culture period in the case of mice, it can be, for example, between 1 and 10 days, or between 3 and 7 days. Furthermore, those skilled in the art can appropriately set a preferred culture period depending on the animal species from which the cells originate.

[0080] The proliferation step may be performed before or after the introduction step described above. If the proliferation step is performed after the introduction step, if the oocyte formation gene is expressed or the expression protein of the oocyte formation gene is present, differentiation induction into immature oocytes will begin from at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, and proliferation will stop. Therefore, as shown in the expression induction step described later, if the expression of the oocyte formation gene is controlled to be induced by the presence of an expression inducer, the proliferation step can be performed after the introduction step in the absence of the expression inducer.

[0081] Furthermore, when introducing oocyte-forming genes in the form of transient expression vectors, or when introducing transcripts or expressed proteins of oocyte-forming genes, it is preferable to perform the proliferation step before the introduction step.

[0082] [Selection Process] In the selection process, cells into which oocyte-forming genes, their transcripts, or their expressed proteins have been introduced are selected.

[0083] In the selection process, for example, by using a reporter gene, cells into which an oocyte-forming gene, its transcript, or its expressed protein has been introduced can be selected. Specifically, for example, when introducing an oocyte-forming gene into cells in the form of an expression vector, by including a reporter gene in the expression vector, cells can be selected by expressing the reporter gene in conjunction with the expression of the oocyte-forming gene, or independently of the expression of the oocyte-forming gene. When incorporating an oocyte-forming gene into the chromosome of at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, cells can be selected by incorporating a construct in which the reporter gene is functionally linked upstream or downstream of the oocyte-forming gene, thereby expressing the reporter gene in conjunction with the expression of the oocyte-forming gene, or independently of the expression of the oocyte-forming gene. When introducing a transcript of an oocyte-forming gene, cell selection can be achieved by introducing a construct into cells in which a transcript of a reporter gene is functionally linked upstream or downstream of the transcript of the oocyte-forming gene. This allows for the expression of both the oocyte-forming gene and the reporter gene. When introducing the expression protein of an oocyte-forming gene into cells, cell selection can be achieved by introducing a fusion protein of the expression protein of the oocyte-forming gene and the expression protein of the reporter gene into the cells. As the reporter gene, those exemplified as marker genes in the description of the "introduction process" above can be used.

[0084] [Facial Expression Induction Process] In the above introduction step, if the expression of oocyte-forming genes is controlled to be induced by the presence of an expression-inducing substance, the expression of oocyte-forming genes is induced by adding the expression-inducing substance to the culture medium. When at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells is differentiated into immature oocytes, cell proliferation stops. Therefore, in order to obtain a larger number of immature oocytes, it is preferable to perform the proliferation step before the expression induction step. That is, when introducing oocyte-forming genes into cells in the form of an expression vector, it is preferable to perform the proliferation step, introduction step, and expression induction step in this order. On the other hand, when incorporating oocyte-forming genes into the chromosomes of at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, it is preferable to perform the introduction step, proliferation step, and expression induction step in this order.

[0085] Induction of oocyte formation gene expression can be achieved, for example, by introducing a oocyte formation gene into cells in which it is functionally linked to an expression-inducing promoter (e.g., a doxycycline-inducing promoter (TetO promoter)), and then adding an expression-inducing substance (e.g., doxycycline) to the culture medium to express the oocyte formation gene. Alternatively, as shown in the examples described later, one can use the ProteoTuner® system (manufactured by Klontech). Specifically, by introducing a construct into cells in which a sequence encoding a destabilizing domain (DDT, 12kDa) is functionally linked upstream or downstream of the oocyte formation gene, the fusion protein expressed by this construct is rapidly degraded by the proteasome in the absence of the expression-inducing substance. On the other hand, by adding Shield1 (a membrane-permeable low-molecular-weight compound, 750Da), which protects against degradation by the proteasome as an expression-inducing substance, to the culture medium, the oocyte formation gene can be stably expressed and accumulated in the cells.

[0086] The amount of expression-inducing substance added can be any concentration that results in the desired level of expression of the oocyte-forming gene; there are no specific limitations. For example, if the expression-inducing substance is doxycycline, the concentration in the culture medium can be, for example, between 1 nM and 10 μM. If the expression-inducing substance is Shield1, the concentration in the culture medium can be, for example, between 10 nM and 10 μM.

[0087] As the culture medium used in the expression induction step, the culture medium exemplified in the growth step described above can be used.

[0088] [Culture process] In the culture process, the cells following the introduction step are cultured in a state in which the oocyte-forming gene is expressed within the cell or in which the expression protein of the oocyte-forming gene is present.

[0089] The culture conditions can be those of known cells selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells. Specifically, for example, the culture temperature can be set to approximately 30°C to 37°C. The culture period for mice can be approximately 1 to 10 days, or 3 to 7 days.

[0090] As the culture medium used in the culture process, the culture medium exemplified in the growth process described above may be used. If the expression of oocyte-forming genes is controlled to be induced by the presence of an expression-inducing substance, the expression-inducing substance should be added to the culture medium to induce expression of oocyte-forming genes within the cells during cultivation.

[0091] A preferred embodiment of the method for inducing immature oocytes involves controlling the expression of oocyte-forming genes so that it is induced by the presence of an expression-inducing substance, and includes the following 1) to 5). 1) Introducing one or more oocyte-forming genes selected from the group consisting of FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2, and DYNLL1 into the chromosome of at least one type of cell (particularly preferably pluripotent stem cells) selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells; 2) Proliferate the cells after introduction; 3) Select cells from the proliferated cells in which the oocyte-forming gene has been introduced into the chromosome; 4) In the selected cells, induce the expression of oocyte-forming genes by adding an expression-inducing substance to the culture medium; 5) Culture the cells after expression induction in a state where the oocyte formation gene is expressed within the cell.

[0092] In the method for inducing immature oocytes of this embodiment, as described above, at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells can be differentiated into immature oocytes within a short culture period of approximately 5 to 10 days from the introduction of an oocyte-forming gene, its transcript, or its expressed protein.

[0093] The differentiation of at least one cell type selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells into immature oocytes can be confirmed by the expression of a known oocyte marker gene (e.g., Stella). Specifically, as shown in the examples described later, by pre-introducing nucleic acids encoding a fusion protein in which the oocyte marker gene Stella and a reporter protein (e.g., Enhanced cyan fluorescent protein; ECFP) are bound into the chromosomes of at least one cell type selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, differentiation into immature oocytes can be confirmed by the fluorescence detected by the expression of Stella-ECFP. Furthermore, based on the expression of Stella-ECFP, undifferentiated cells can be removed from the differentiated cell population using methods such as Fluorescence-activated cell sorting (FACS), and immature oocytes can be easily isolated.

[0094] <Method for producing mature oocytes> In one embodiment, the present invention provides a method for producing mature oocytes, comprising introducing one or more genes selected from the group consisting of FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2, and DYNLL1, or their transcripts or expressed proteins, into at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, and co-culturing the introduced cells with ovarian somatic cells.

[0095] In the method for producing mature oocytes according to this embodiment, the introduction of oocyte-forming genes (introduction step) is the same as the introduction step described in the "Method for inducing immature oocytes" above, so the explanation is omitted.

[0096] In this specification, "mature oocyte" refers to an egg in metaphase II of meiosis, also known as a secondary oocyte. Furthermore, as shown in the examples described later, mature oocytes express the Npm2 gene, which is an activated oocyte marker.

[0097] [Agglomerate formation process] The method for producing mature oocytes in this embodiment includes a step of co-culturing at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step described in the "Method for Inducing Oocytes" above, and ovarian somatic cells to form aggregates (aggregate formation step).

[0098] The ovarian somatic cells used in the aggregate formation process are somatic cells collected from the ovaries of living organisms. These cells differentiate into granulosa cells and theca cells that constitute follicles when co-cultured with at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells.

[0099] Furthermore, the method for collecting somatic cells from the ovaries of living organisms can be carried out according to the method described in Reference 5 above.

[0100] Specifically, for example, a method for collecting somatic cells from the ovary involves surgically removing the ovary from a living organism and dissociating the somatic cells constituting the ovary by trypsin treatment or the like. It is preferable to remove germ cells inherent in the living organism's ovary at this time. Methods for removing germ cells inherent in the ovary can be known; for example, germ cells can be removed by magnetic activated cell sorting using anti-SSEA1 antibody or anti-CD31 antibody. Here, since ovarian somatic cells are to be collected, it is preferable to use ovaries derived from a fetus. In the case of mice, for example, gonads (ovaries) derived from mouse fetuses at 12.5 days of gestation (also called "embryonic age") can be used. Furthermore, those skilled in the art can select gonads (ovaries) at a suitably preferred stage depending on the animal species from which they originate, based on this disclosure and common technical knowledge in the art.

[0101] In the aggregate formation step, it is preferable to co-culture at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells, thereby forming aggregates consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells.

[0102] A method for producing aggregates consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells, can be carried out, for example, as shown in the examples described later, by mixing and agglutinating at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step with ovarian somatic cells in S10 medium (StemPro®-34 SFM, manufactured by Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FCS), 150 μM ascorbic acid, 1 × Glutamax, 1 × penicillin / streptomycin, and 55 μM mercaptoethanol, and culturing. If the expression of oocyte formation genes is controlled to be induced by the presence of an expression-inducing substance, the expression-inducing substance is added to the medium and culture is performed in a state where oocyte formation genes are expressed. It is preferable to use a low-adhesion culture dish (e.g., a 96-well plate with low cell adhesion U-bottom) for culture.

[0103] In the case of mice, for example, the culture period for producing aggregates can be set to approximately 2 to 3 days, with 2 days being preferable. Furthermore, those skilled in the art can appropriately set a preferred culture period depending on the animal species from which the product originates.

[0104] Furthermore, the ratio of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step to ovarian somatic cells when mixed is not limited as long as the resulting aggregate forms mature follicles. However, in the case of mice, for example, it is preferable to set the ratio of the number of cells to ovarian somatic cells to at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step to approximately 2:1.

[0105] Furthermore, at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction process, or ovarian somatic cells, or an ovary containing ovarian somatic cells, can also be used from cryopreserved cells. Cryopreservation can be carried out by known methods. For example, cryopreservation of at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction process, or ovarian somatic cells, can be carried out by slow freezing using a 10% DMSO solution or a commercially available cryotherapy agent (such as Cellbanker®).

[0106] The ovarian somatic cells used in the method for producing mature oocytes in this embodiment may be derived from at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells that constitute the aggregate, or from the same species of mammal, or from a different species of mammal; however, it is preferable to use cells derived from the same species of mammal. Examples of mammals include those exemplified in the description of cells having the ability to differentiate into oocytes.

[0107] The method for producing mature oocytes in this embodiment may include any step after the introduction step and before the aggregate formation step. Examples of optional steps include the proliferation step and selection step described in the "Method for Inducing Immature Oocytes" above.

[0108] [Culture process] Furthermore, the method for producing mature oocytes in this embodiment may include a culture step after the aggregate formation step described above.

[0109] In the culture process, it is preferable to transfer the aggregates formed after the aggregate formation process onto a collagen membrane and culture them there.

[0110] The culture conditions can be those known for culturing aggregates. Specifically, for example, the culture temperature can be set to approximately 30°C to 37°C. The culture period can be approximately 7 to 35 days for mice, or 8 to 30 days. Furthermore, those skilled in the art can appropriately set a preferred culture period depending on the animal species from which the material is derived.

[0111] As the culture medium used in the culture process, the culture medium exemplified in the growth process described above may be used. If the expression of oocyte-forming genes is controlled to be induced by the presence of an expression-inducing substance, the expression-inducing substance should be added to the culture medium to induce expression of oocyte-forming genes within the cells during cultivation.

[0112] The aggregated cells after the culture process have a secondary follicular structure, with oocytes surrounded by multilayered granulosa cells. Furthermore, a theca folic membrane is formed surrounding the multilayered granulosa cells, and the theca cells that make up the inside of the theca membrane express luteinizing hormone receptors, while the granulosa cells express follicle-stimulating hormone receptors.

[0113] Next, mature oocytes can be produced by culturing the follicles obtained in the culture step using the method described in Non-Patent Document 1. The process for obtaining mature oocytes can be divided into a growth step and a maturation step.

[0114] [Growth process] In the growth stage, the follicles obtained in the culture stage are isolated into individual follicles and cultured using a growth medium.

[0115] The culture conditions can be those described in Non-Patent Document 1. Specifically, for example, the culture temperature can be set to approximately 30°C to 37°C. The culture period for mice can be approximately 7 to 15 days, or 8 to 13 days. Furthermore, those skilled in the art can appropriately set a preferred culture period depending on the animal species from which the organisms are derived.

[0116] As a growth medium, a medium with the composition described in Non-Patent Document 1 can be used. Specifically, for example in the case of mice, for the first two days from the start of culture, follicles are cultured using α-MEM containing 5% fetal bovine serum (FCS), 2% polyvinylpyrrolidone (all from Sigma), 150 μM ascorbic acid, 1× GlutaMAX, 1× penicillin / streptomycin, 100 μM 2-mercaptoethanol, 55 μg / mL sodium pyruvate (all from Nacalai Tesque), 0.1 IU / mL follicle-stimulating hormone (Follistim®, MSD), 15 ng / mL BMP15 (Bone morphogenetic protein 15), and 15 ng / mL GDF9 (Growth differentiation factor 9) (all from R&D Systems). Next, on the second day after the start of culture, the α-MEM is replaced with the above composition excluding BMP15 and GDF9, and the follicles are incubated in 0.1% Type IV collagenase (MP Biomedicals). Then, after washing several times with 5% FCS-containing α-MEM, the follicles are cultured in α-MEM with the above composition excluding BMP15 and GDF9 until the 11th day after the start of culture.

[0117] After the growth stage, the follicle has a follicular structure and forms a cumulus-oocyte complex containing an egg in the ovarian vesicle stage.

[0118] [Maturity process] In the maturation process, the follicles obtained in the growth process are cultured using a maturation medium.

[0119] The culture conditions can be those described in Non-Patent Document 1. Specifically, for example, the culture temperature can be set to approximately 30°C to 37°C. The culture period for mice can be approximately 7 to 15 days, or 8 to 13 days. Furthermore, those skilled in the art can appropriately set a preferred culture period depending on the animal species from which the organisms are derived.

[0120] For maturation, a culture medium with the composition described in Non-Patent Literature 1 can be used. Specifically, for example, in the case of mice, α-MEM containing 5% FCS, 25 μg / mL sodium pyruvate, 1× penicillin / streptomycin, 0.1 IU / mL follicle-stimulating hormone, 4 ng EGF (Epidermal Growth Factor), and 1.2 IU / mL hCG (Human chorionic gonadotropin, abbreviated as gonadotropin, manufactured by ASKA) can be used.

[0121] After the maturation process, the follicle has matured to the stage of metaphase II of meiosis (secondary oocyte). The presence of an egg in metaphase II of meiosis (secondary oocyte) can be evaluated, for example, by visual inspection using a microscope, based on the release of the first polar body.

[0122] The mature oocytes (secondary oocytes) obtained are suitably used in infertility treatment. In other words, in one embodiment, the present invention provides an infertility treatment method using mature oocytes obtained by the above method. Furthermore, mature oocytes obtained using the method for producing mature oocytes of this embodiment are suitably used for the efficient breeding of farm animals and the reproduction of endangered animals. That is, in one embodiment, the present invention provides a method for breeding farm animals or a method for reproducing endangered animals using mature oocytes obtained by the above method. Furthermore, mammals are preferred as the animals to which the above applies. Examples of mammals are those similar to those exemplified above. Furthermore, mature oocytes obtained using the method for producing mature oocytes according to this embodiment can be used to investigate the causes of infertility and elucidate the mechanisms of menopausal disorders. [Examples]

[0123] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0124] [Example 1] (Vector construction) The CAG promoter and destabilization domain (DD) were cloned from the CAG-DD-hTFAP2C plasmid (Reference 6: "Kobayashi T et al., "Principles of early human development and germ cell program from conserved model systems.", Nature, Vol.546, No.7658, p416-420, 2017."), and inserted into the conventionally used PiggyBAC vector (Reference 7: "Shimamoto S et al., "Hypoxia induces the dormant state in oocytes through expression of Foxo3", PNAS, https: / / doi.org / 10.1073 / pnas.1817223116, 2019.") to create the PB-CAG-DD vector. Next, cDNAs of eight genes—FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2, and DYNLL1—were amplified from cDNA in the ovaries of female mice 13.5 days post-fertilization and cloned into PB-CAG-DD vectors using the Infusion HD Cloning Kit (Takara Bio Inc.). Amplification of each cDNA was performed by PCR using KOD Fx Neo or KOD Plus Neo DNA polymerase (Toyobo Inc.) according to the manufacturer's protocol.

[0125] (Transfection of vectors) ES cells were pre-cultured in serum-free medium supplemented with 2i and LIF without the use of feeder cells (see Reference 3 above). Furthermore, to monitor differentiation into immature and mature oocytes, mouse ES cells (Blimp1-mVenus:Stella-ECFP:Npm2-mCherry(BVSCNmC)) were used, in which the following genes were inserted onto the chromosome: a gene encoding mVenus (membrane-targeted Venus), a variant of yellow fluorescent protein (YFP) under the control of Blimp1 expression, an important determinant of germline; a gene encoding enhanced cyan fluorescent protein (ECFP) under the control of Stella expression, a germ cell and oocyte marker; and a gene encoding mCherry (membrane-targeted Cherry), a red fluorescent protein under the control of Nucleoplasmin 2 (Npm2) expression, an activated (mature) oocyte marker. The PB-CAG-DD vector, containing the eight constructed genes, was transfected simultaneously with a hyperactive PBase (hypBase) plasmid using Lipofectamine 2000. Single colonies were grown in serum-free medium supplemented with 2i and LIF for 5 days after puromycin selection.

[0126] (Induction of differentiation into immature oocytes) Next, 1 × 10⁵ ES cells were transferred to a 96-well plate with a low cell adhesion U-bottom, dispensed with a medium (hereinafter referred to as "oocyte differentiation induction medium") consisting of S10 medium (StemPro®-34 SFM, Life Technologies Inc.) supplemented with 10% fetal bovine serum (FCS), 150 μM ascorbic acid, 1 × Glutamax, 1 × penicillin / streptomycin, and 55 μM mercaptoethanol, mixed with 0.5 μM Shield1 (Clontech Inc.). The cells were cultured for 5 days to induce differentiation into immature oocytes. The results observed under a confocal microscope (Carl Zeiss, model: Zeiss LSM 700) are shown in Figure 1. In Figure 1, "oocyte formation genes" refer to the eight genes FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2, and DYNLL1. "Oocyte formation gene expression OFF" refers to cells before the addition of the culture medium containing Shield1, while "Oocyte formation gene expression ON" refers to cells after the addition of the culture medium containing Shield1 and culturing for 5 days.

[0127] Figure 1 shows that in cells where the expression of the oocyte-forming gene was OFF, colonies were formed, and there was almost no fluorescence of ECFP expressed under the control of Stella expression (Stella-ECFP). In contrast, in cells where the expression of the oocyte-forming gene was ON, the cells were separated, and strong fluorescence of Stella-ECFP was detected, suggesting that differentiation into immature oocytes was induced.

[0128] (Production of mature oocytes) Next, after transfection, the cells were cultured for 5 days, and single colonies of ES cells (1 × 10⁵ cells) selected with puromycin were mixed with 3 × 10⁴ ovarian somatic cells from 12.5-day-old female mice in the oocyte differentiation induction medium described above to create aggregates, which were then cultured for 2 days. The ovarian somatic cells were obtained by first excising the ovaries of 12.5-day-old female mice using the method described in Non-Patent Document 1, etc., and then isolating them from the ovaries. Subsequently, the aggregates were transferred onto a Transwell-COL membrane (Coaster) and cultured in the oocyte differentiation induction medium described above for 28 days. Figure 2 shows the results of observing the expression of each marker in the aggregates on days 2, 6, 8, 10, and 12 from the start of culture under a confocal microscope (Carl Zeiss, model: Zeiss LSM 700). In Figure 2, the upper panel visualizes oocytes using the fluorescence of Stella-ECFP, a germ cell and oocyte marker. In the lower panel, oocytes were visualized by fluorescence from the red fluorescent protein mCherry (Npm2-mCherry), which is expressed under the control of Nucleoplasmin 2 (Npm2), a marker for activated (mature) oocytes.

[0129] As shown in Figure 2, Stella-ECFP fluorescence was continuously observed in cells throughout the culture period. On the other hand, Npm2-mCherry fluorescence was observed on day 8 from the start of culture (see the arrow in the lower part of "Day 8" in Figure 2), indicating that oocyte maturation was progressing.

[0130] Furthermore, on day 28 from the start of culture, individual follicles were manually isolated using a sharpened tungsten needle. The isolated follicles had a secondary follicular structure. These follicles were cultured under the conditions of the "in vitro growth period" and "in vitro maturation period" described in Non-Patent Literature 1, and matured through antral follicles to metaphase II oocytes.

[0131] [Example 2] We investigated the differentiation induction into oocytes using mouse iPS cells, similar to the process used with ES cells.

[0132] (Transfection of vectors) The iPS cells used were mouse BVSC iPS cells produced by the virus buster established in Non-Patent Document 1. The PB-CAG-DD vector, containing the five genes constructed in Example 1, was transfected into mouse BVSC iPS cells simultaneously with a hyperactive PBase (hypBase) plasmid using Lipofectamine 2000. Single colonies were grown in serum-free medium supplemented with 2i and LIF for 5 days after puromycin selection.

[0133] (Induction of differentiation into immature oocytes) iPS cells transfected with the vector were cultured for 5 days using the same method as in Example 1 to induce differentiation into immature oocytes.

[0134] (Production of mature oocytes) Next, after transfection, the cells were cultured for 5 days and selected with puromycin. These iPS cells (1 × 10⁵ cells) were mixed with 3 × 10⁴ ovarian somatic cells from 12.5-day-old female mice in the oocyte differentiation induction medium described above to form aggregates, which were cultured for 2 days. The aggregates were then transferred to a Transwell-COL membrane (Coaster) and cultured in the oocyte differentiation induction medium described above for 21 days. Figure 3 shows the results of observing Stella-ECFP expression in the aggregates 21 days after the start of culture under a confocal microscope (Carl Zeiss, model: Zeiss LSM 700). In Figure 3, the image on the left is a bright-field image, and the image on the right is a fluorescence image in which oocytes are visualized by the fluorescence of Stella-ECFP, a germ cell and oocyte marker.

[0135] Figure 3 shows that Stella-ECFP fluorescence was observed in cells 21 days after the start of culture. This confirms that iPS cells could be differentiated into oocytes in the same way as ES cells.

[0136] [Example 3] (Identification of key factors in oocyte formation genes) To identify the important genes among the oocyte formation genes, a total of 26 vectors were constructed using the same method as in Example 1, so as to match the gene combinations shown in the left diagram of Figure 4. Next, mouse ES cells (Blimp1-mVenus:Stella-ECFP:Npm2-mCherry (BVSCNmC)) were transfected with each vector using the same method as in Example 1. The transfected ES cells were cultured for 5 days using the same method as in Example 1 to induce differentiation into immature oocytes. Next, single-cell derived ES cells (1 × 10⁵ cells) selected with puromycin after 5 days of transfection were mixed with 3 × 10⁴ ovarian somatic cells from 12.5-day-old female mice in the oocyte differentiation induction medium to create aggregates, which were cultured for 2 days. Next, the obtained aggregates were cultured for 21 days using the same method as in Example 1. The number of oocytes formed from each cell line was measured after culture. Furthermore, the area of ​​oocytes was calculated from the fluorescence area of ​​the blue fluorescent protein CFP expressed downstream of the Stella gene. These results are shown in Figure 4.

[0137] Figure 4 reveals that four genes—FIGLA, NOBOX, LHX8, and TBPL2—are important factors in inducing differentiation from pluripotent stem cells to immature oocytes and in oocyte maturation. Furthermore, it was found that introducing the STAT3 gene in addition to these four genes further improves the efficiency of oocyte formation. [Industrial applicability]

[0138] According to the method for inducing immature oocytes in this embodiment, immature oocytes can be easily induced from cells with the ability to differentiate into oocytes, such as pluripotent stem cells, in a shorter culture period than conventional methods. According to the method for producing mature oocytes in the above embodiment, a large number of mature oocytes can be easily produced from cells with the ability to differentiate into oocytes, such as pluripotent stem cells, in a shorter culture period than conventional methods.

Claims

1. Immature oocytes obtained by introducing genes containing at least FIGLA, NOBOX, LHX8, and TBPL2 into pluripotent stem cells, wherein the genes are incorporated into the chromosomes.

2. Mature oocytes obtained by co-culturing immature oocytes described in claim 1 with ovarian somatic cells.

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