Method for inducing immature oocytes and method for producing mature oocytes

Abstract

A method for inducing immature oocytes comprises introducing four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2, or their transcripts or expression proteins, into at least one type of cell selected from the group consisting of pluripotent stem cells, epiblast-like cells, and primordial germ cells. A method for producing mature oocytes comprises introducing four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2, or their transcripts or expression proteins, into at least one type of cell selected from the group consisting of pluripotent stem cells, epiblast-like cells, and primordial germ cells, and co-culture the cell and ovarian somatic cells after introduction.

Description

Technology Field

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

[0002] The present application claims priority based on patent application No. 2019-166578 filed in Japan on September 12, 2019, and incorporates the contents thereof herein by reference. Background Technology

[0003] In mammals, pluripotency, defined as the ability to develop from a single cell, is a special property possessed by unicellular organisms. However, the mechanisms for forming this pluripotency are facing difficulties, despite strong social demand in fields such as infertility treatment. The reason for this lies in the inability to reproduce the process of oocyte formation occurring in the fetal ovary in vitro.

[0004] The inventors have 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 humoral factors such as BMP4, and the obtained PGCLCs are mixed with ovarian somatic cells to produce reconstructed ovaries. Subsequently, the period from the PGCLCs to the formation of an egg in metaphase II of meiosis in the reconstructed ovary is divided into three periods: an "in vitro differentiation period," an "in vitro growth period," and an "in vitro maturation period." For each culture period, optimal culture conditions are established to obtain secondary follicles, egg nucleus stage eggs, and egg nucleus stage II of meiosis eggs (see, for example, Non-patent Literature 1, etc.).

[0005] However, in the method described in Non-Patent Literature 1, a period of at least 3 weeks and at least 4 weeks is required to obtain primordial follicular oocytes from mouse pluripotent stem cells, and it is expected that more than 1 year of culture will be required to produce oocytes from pluripotent stem cells in primates or other large mammals in an in vitro culture system. Prior art literature

[0006] Hikabe O et al., “Reconstitution in vitro of the entire cycle of the mouse female germ line.”, Nature, Vol. 539, p299-303, 2016. The problem to be solved

[0007] The present invention, made in consideration of the above circumstances, provides a method for inducing immature oocytes from cells capable of differentiating into oocytes, such as pluripotent stem cells, through a shorter culture period than conventional methods. Furthermore, the invention provides a method for producing mature oocytes from cells capable of differentiating into oocytes, such as pluripotent stem cells, through a shorter culture period than conventional methods. means of solving the problem

[0008] The inventors, having conducted repeated research to achieve the above objective, discovered that it is possible to induce differentiation into immature oocytes by introducing a specific gene involved in the formation of oocytes into pluripotent stem cells and culturing them for a short period of 5 days or more and 10 days or less, and thus completed the present invention.

[0009] That is, the present invention includes the following aspects.

[0010] A method for inducing immature oocytes according to the first aspect of the present invention comprises introducing four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2, or their transcripts or expression proteins, into at least one type of cell selected from the group consisting of pluripotent stem cells, epiblast-like cells, and primordial germ cells.

[0011] The method for inducing immature oocytes according to the first aspect above may include introducing four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2 into the cells.

[0012] The method for inducing immature oocytes according to the first aspect above may include additionally introducing the gene of STAT3, or its transcript or expression protein, into said cells.

[0013] The method for inducing immature oocytes according to the first aspect above may further include introducing one or more genes selected from the group consisting of SOHLH1, SUB1, and DYNLL1, or their transcripts or expression proteins, into said cells.

[0014] The method for inducing immature oocytes according to the first aspect above may include additionally introducing three types of genes consisting of SOHLH1, SUB1, and DYNLL1 into the cells.

[0015] The above cells may be pluripotent stem cells.

[0016] In the method for inducing an immature oocyte according to the first aspect above, the expression of the gene is controlled to be induced by the presence of an expression-inducing substance, or

[0017] The expression of the above gene is controlled to be stabilized by the presence of an expression stabilizing substance, and

[0018] After the above introduction, proliferating the above cells, and,

[0019] After the above proliferation, adding the expression-inducing substance to the medium to induce the expression of the gene, or after the above proliferation, adding the expression-stabilizing substance to the medium to stabilize the expression of the gene.

[0020] You may include additionally.

[0021] A method for producing mature oocytes according to the second aspect of the present invention is,

[0022] Introducing four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2, or their transcripts or expression proteins, into at least one type of cell selected from the group consisting of pluripotent stem cells and primordial germ cells, and

[0023] Co-culturing the above cells and ovarian somatic cells after introduction

[0024] Includes

[0025] The method for producing a mature oocyte according to the second aspect above may include additionally introducing the gene for STAT3, or its transcript or expression protein, into the cell. Effects of the invention

[0026] According to the above method for inducing immature oocytes, immature oocytes can be easily induced from cells capable of differentiating into oocytes, such as pluripotent stem cells, through culture in a shorter period than conventional methods. According to the above method for producing mature oocytes, mature oocytes can be mass-produced from cells capable of differentiating into oocytes, such as pluripotent stem cells, through culture in a shorter period than conventional methods. Brief explanation of the drawing

[0027] Figure 1 shows the bright-field and fluorescence images (magnification: 200x) of immature oocytes induced from mouse ES cells in Example 1. Figure 2 is a fluorescent image (magnification: 8x) of an aggregate composed of pluripotent stem cells and ovarian somatic cells into which oocyte-forming genes were introduced at each culture day in Example 1. In the top, oocytes were visualized by the fluorescence of an enhanced cyan fluorescent protein (ECFP) expressed under the control of expression of Stella, a germ cell and oocyte marker. In the bottom, oocytes were visualized by the fluorescence of a red fluorescent protein mCherry expressed under the control of expression of Nucleoplasmin 2 (Npm2), an activated (mature) oocyte marker. Figure 3 shows the bright-field and fluorescence images (magnification: 8x) of immature oocytes induced from mouse iPS cells in Example 2. Figure 4 is a diagram showing the results of identifying key factors among oocyte-forming genes in Example 3. In the diagram on the left, the vertical axis represents the sample name, and the horizontal axis represents the type of oocyte-forming gene. White eggs indicate that the corresponding gene has not been introduced. The diagram in the center shows the number of oocytes formed from each cell line. The diagram on the right shows the oocyte area calculated from the fluorescence area of ​​the blue fluorescent protein CFP expressed downstream of the Stella gene in each sample. Specific details for implementing the invention

[0028] Methods for Inducing Immature Oocytes

[0029] In one embodiment, the present invention provides a method for inducing immature oocytes, comprising introducing four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2, or transcripts or expression proteins thereof, into at least one type of cell selected from the group consisting of pluripotent stem cells, epiblast-like cells, and primordial germ cells (hereinafter, these cells are collectively referred to as "cells having the ability to differentiate into oocytes").

[0030] In conventional methods, in the case of mice, it takes about one month to induce differentiation from pluripotent stem cells to immature oocytes in vitro, and about 11 days to induce differentiation from primordial germ cells to immature oocytes in vitro. In contrast, in the method for inducing immature oocytes according to the present embodiment, differentiation into immature oocytes can be induced by introducing the four types of genes mentioned above into cells having the ability to differentiate into oocytes, such as pluripotent stem cells, and culturing them for a short period of about 5 days or more and 10 days or less. Furthermore, in the case of humans, it takes more than 9 months to induce differentiation from primordial germ cells to immature oocytes in vivo, but by using the method for inducing immature oocytes according to the present embodiment, this period can be dramatically shortened.

[0031] In addition, in conventional methods, it was necessary to undergo at least two steps with different culture conditions to induce differentiation from pluripotent stem cells into PGCLCs and then induce differentiation of the PGCLCs into immature oocytes. In contrast, the method for inducing immature oocytes according to the present embodiment can directly induce differentiation of pluripotent stem cells into immature oocytes.

[0032] Meanwhile, in this specification, "immature oocyte" refers to a primary oocyte that has not undergone follicular growth. Immature oocytes do not necessarily have to have a follicular structure. In addition, as shown in the examples described below, immature oocytes express some maternal effector genes, such as the Stella gene or the Padi6 gene, which are oocyte markers.

[0033] In addition, in this specification, "follicle" is composed of an oocyte and somatic cells (granulosa cells and capsule cells) surrounding it.

[0034] The method for inducing immature oocytes according to the present embodiment is described in detail below.

[0035] [Oocyte formation gene]

[0036] The oocyte-forming genes used for introduction into cells capable of differentiating into oocytes, such as pluripotent stem cells, were identified by the inventors by analyzing the expression dynamics of genes in oocyte lineages through RNA sequencing (RNA-Seq). Specifically, examples of oocyte-forming genes include FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2, and DYNLL1. As shown in the examples described below, four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2 are particularly important for the induction of immature oocytes from pluripotent stem cells. Accordingly, among the aforementioned oocyte-forming genes, by introducing at least four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2, or their transcripts or expression proteins (preferably, at least the four types of genes), into a cell, a cell having the ability to differentiate into an oocyte can be induced into an immature oocyte.

[0037] In addition, it is preferable to further introduce the STAT3 gene, its transcript, or its expression protein in addition to the four types of genes or their transcripts or expression proteins mentioned above, and it is more preferable to further introduce the STAT3 gene. As shown in the examples described below, by introducing STAT3 into cells in addition to FIGLA, NOBOX, LHX8, and TBPL2, the oocyte formation rate can be further improved.

[0038] In addition, in addition to the five types of genes, FIGLA, NOBOX, LHX8, TBPL2, and STAT3, or their transcripts or expression 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 expression proteins; it is more preferable to further introduce three types of genes consisting of SOHLH1, SUB1, and DYNLL1, or their transcripts or expression proteins; and it is even more preferable to further introduce three types of genes consisting of SOHLH1, SUB1, and DYNLL1. As shown in the examples described below, by introducing all eight types of genes, or their transcripts or expression proteins (preferably all eight types of genes), into a cell, cells capable of differentiating into oocytes can be more efficiently induced into immature oocytes.

[0039] Meanwhile, the FIGLA gene encodes a basic helix-loop-helix (bHLH) transcription factor that regulates multiple oocyte-specific genes (including genes involved in follicle 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 gene ontology (GO) annotations of FIGLA include sequence-specific DNA binding and protein dimerization activity. The important paralog of FIGLA is SCX. FIGLA 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.

[0040] Information on the nucleotide sequences of oocyte-forming genes such as the FIGLA gene, the nucleotide sequences of the mRNA of the corresponding genes, and the amino acid sequences of the proteins encoded by the corresponding genes can be obtained from databases such as Genbank.

[0041] The nucleotide sequence of the human FIGLA gene is disclosed, for example, as “Gene ID: 344018” in Genbank. The nucleotide sequence of the mRNA of the human FIGLA gene is disclosed, for example, as accession number NM_001004311 in Genbank. The amino acid sequence of the human FIGLA is disclosed as accession number NP_001004311 in Genbank.

[0042] 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.

[0043] The NOBOX gene codes for a transcription factor involved in oocyte formation. Diseases associated with NOBOX include, for example, premature ovarian failure (Type 5). The GO annotation of NOBOX includes DNA-binding transcription factor activity and DNA binding specific to promoter sequences near RNA polymerase II. Specifically, it preferably binds to nucleotide sequences such as "5'-TAATTG-3'", "5'-TAGTTG-3'", and "5'-TAATTA-3'". An important paralog of NOBOX is UNCX. NOBOX is also referred to as NOBOX Oogenesis Homeobox, Homeobox Protein NOBOX, Newborn Ovary Homeobox-Encoding Gene, Newborn Ovary Homeobox-Encoding, TCAG_12042, OG-2 (OG2), OG2X, and POF5.

[0044] The nucleotide sequence of the human NOBOX gene is disclosed, for example, as “Gene ID: 135935” in Genbank. The nucleotide sequence of the mRNA of the human NOBOX gene is disclosed, for example, as access numbers NM_001080413 and XM_001134420 in Genbank. The amino acid sequence of the human NOBOX is disclosed as access numbers NP_001073882 and XP_001134420 in Genbank.

[0045] The nucleotide sequence of the mouse NOBOX gene is disclosed, for example, as “Gene ID: 18291” in Genbank. The nucleotide sequence of the mRNA of the mouse NOBOX gene 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.

[0046] The SOHLH1 gene is a gonad-specific transcription factor essential for spermatogenesis, oogenesis, and follicle formation, and it codes for 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 hypoplasia. The GO annotations of SOHLH1 include DNA-binding transcription factor activity and protein dimerization activity. An important paralog of the SOHLH1 gene is SOHLH2. SOHLH1, 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, Also called SPATA27, BHLHe80, SPGF32, and ODG5.

[0047] The nucleotide sequence of the human SOHLH1 gene is disclosed, for example, as “Gene ID: 402381” in Genbank. The nucleotide sequence of the mRNA of the human SOHLH1 gene is disclosed, for example, as accession number NM_001012415, XM_497082 in Genbank. The amino acid sequence of human SOHLH1 is disclosed as accession number NP_001012415, XP_497082 in Genbank.

[0048] 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 access numbers NM_001001714 and XM_130180 in Genbank, and the amino acid sequence of mouse SOHLH1 is disclosed as access numbers NP_001001714 and XP_130180 in Genbank.

[0049] LHX8 is a member of the LIM homeobox family of proteins and is involved in the pattern formation and differentiation of various tissues. Proteins of the LIM homeobox family contain, in addition to a DNA-binding homeodomain, a cysteine-rich double zinc finger motif repeated in two tandems, known as the LIM domain. Proteins of the LIM homeobox family are transcription factors involved in tooth morphogenesis, oocyte formation, and neuronal differentiation. Diseases associated with the LHX8 gene include, for example, cleft palate and odontoma. LHX8 is also referred to as LIM Homeobox 8, LIM / Homeobox Protein Lhx8, LIM-Homeodomain Protein Lhx8, LIM Homeobox Protein 8, and LHX7.

[0050] The nucleotide sequence of the human LHX8 gene is disclosed, for example, as “Gene ID: 431707” in Genbank. The nucleotide sequence of the mRNA of the human LHX8 gene is disclosed, for example, as access numbers NM_001001933, XM_086344, NM_001256114, XM_017001316, XM_017001317 in Genbank. The amino acid sequence of the human LHX8 is disclosed as access numbers NP_001001933, XP_086344, NP_001243043, XP_016856805, XP_016856806 in Genbank.

[0051] The nucleotide sequence of the mouse LHX8 gene is disclosed, for example, as “Gene ID: 16875” in Genbank. The nucleotide sequence of the mRNA of the mouse LHX8 gene is disclosed, for example, as accession numbers NM_010713, XM_006501072, XM_017319470 in Genbank. The amino acid sequence of mouse LHX8 is disclosed as accession numbers NP_034843, XP_006501135, XP_017174959 in Genbank.

[0052] The SUB1 gene codes for a transcriptional regulator. SUB1 functions in coordination with TAF and acts as a coactivator mediating functional interactions between upstream activators and general transcriptional functions. Diseases associated with SUB1 include, for example, onychomycosis. The GO annotation of SUB1 includes single-strand DNA binding. SUB1 is also referred to 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.

[0053] The nucleotide sequence of the human SUB1 gene is disclosed, for example, as “Gene ID: 10923” in Genbank. The nucleotide sequence of the mRNA of the human SUB1 gene is disclosed, for example, as access numbers NM_006713, XM_017008986, XM_017008987, and XM_011513944 in Genbank. The amino acid sequence of human SUB1 is disclosed as access numbers NP_006704, XP_016864475, XP_016864476, and XP_011512246 in Genbank.

[0054] The nucleotide sequence of the mouse SUB1 gene is disclosed, for example, as “Gene ID: 20024” in Genbank. The nucleotide sequence of the mRNA of the mouse SUB1 gene is disclosed, for example, as accession number NM_011294, XM_006520042 in Genbank. The amino acid sequence of mouse SUB1 is disclosed as accession number NP_035424, XP_006520105 in Genbank.

[0055] STAT3 is a member of the STAT protein family. The STAT protein family is phosphorylated by receptor-associated kinases in response to cytokines and growth factors such as interferon (IFN), epidermal growth factor (EGF), interleukin 5 (IL5), interleukin 6 (IL6), hepatocyte growth factor (HGF), leukemia suppressor factor (LIF), and bone morphogenetic protein 2 (BMP2). Subsequently, they form homodimers or heterodimers, migrate to the cell nucleus where they act as transcriptional activators, and play important roles in many cellular processes, such as cell proliferation and apoptosis. Diseases related to STAT3 include, for example, infantile-onset multi-organ autoimmune diseases and autosomal dominant high-IgE syndromes. The GO annotations of STAT3 include DNA-binding transcriptional factor activity and sequence-specific DNA binding. The important paralog of the STAT3 gene is STAT1. STAT3 is also called 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.

[0056] The nucleotide sequence of the human STAT3 gene is disclosed, for example, as “Gene ID: 6774” in Genbank. The nucleotide sequences of the mRNA of the human STAT3 gene are, for example, Genbank access numbers NM_001369512, NM_001369513, NM_001369514, NM_001369516, NM_001369517, NM_001369518, NM_001369519, NM_001369520, NM_003150, NM_139276, NM_213662, XM_017024973, XM_011525146, XM_011525145, XM_017024972, XM_005257617, XM_005257616, It is disclosed as XM_017024975, XM_024450896, XM_017024974, and XM_017024976. The amino acid sequences of human STAT3 are Genbank accession numbers NP_001356441, NP_001356442, NP_001356443, NP_001356445, NP_001356446, NP_001356447, NP_001356448, NP_001356449, NP_003141, NP_644805, NP_998827, XP_016880462, XP_011523448, XP_011523447, XP_016880461, XP_005257674, XP_005257673, XP_016880464, XP_024306664, It is disclosed as XP_016880463 and XP_016880465.

[0057] The nucleotide sequence of the mouse STAT3 gene is disclosed, for example, as “Gene ID: 20848” in Genbank. The nucleotide sequence of the mRNA of the mouse STAT3 gene is disclosed, for example, as access numbers NM_011486, NM_213659, NM_213660, XM_011248846, XM_017314401 in Genbank. The amino acid sequence of mouse STAT3 is disclosed as access numbers NP_035616, NP_998824, NP_998825, XP_011247148, XP_017169890 in Genbank.

[0058] The TBPL2 gene codes for TATA-Box Binding Protein-Like 2. TBPL2 is a transcription factor that forms a complex with TAF3 to induce the differentiation of myoblasts into myocytes, and this complex replaces TFIID in specific promoters during the early stages of differentiation. Diseases associated with TBPL2 include, for example, retinitis pigmentosa. The GO annotation of TBPL2 includes DNA-binding transcription factor activity. The important paralog of the TBPL2 gene is TBP. TBPL2 is also called 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.

[0059] The nucleotide sequence of the human TBPL2 gene is disclosed, for example, as “Gene ID: 387332” in Genbank. The nucleotide sequence of the mRNA of the human TBPL2 gene 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.

[0060] The nucleotide sequence of the mouse TBPL2 gene is disclosed, for example, as “Gene ID: 227606” in Genbank. The nucleotide sequence of the mRNA of the mouse TBPL2 gene is disclosed, for example, as access numbers NM_001289689 and NM_199059 in Genbank. The amino acid sequence of mouse TBPL2 is disclosed as access numbers NP_001276618 and NP_951014 in Genbank.

[0061] The DYNLL1 gene codes for a protein classified as a light chain among the proteins constituting cytoplasmic dynin, an enzyme complex with a molecular weight of approximately 1200 kDa. Diseases associated with DYNLL1 include, for example, chronic intestinal venous insufficiency. The GO annotations of DYNLL1 include protein homodimerization activity and protein domain-specific binding. An important paralog of the DYNLL1 gene is DYNLL2. DYNLL1 is 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), Also called LC8a and LC8.

[0062] The nucleotide sequence of the human DYNLL1 gene is disclosed, for example, as “Gene ID: 8655” in Genbank. The nucleotide sequence of the mRNA of the human DYNLL1 gene is disclosed, for example, as access numbers NM_001037494, NM_001037495, and NM_003746 in Genbank. The amino acid sequence of human DYNLL1 is disclosed as access numbers NP_001032583, NP_001032584, and NP_003737 in Genbank.

[0063] 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.

[0064] [Cells capable of differentiating into oocytes]

[0065] In the method for inducing immature oocytes according to the present embodiment, the cell having the ability to differentiate into an oocyte is preferably at least one type of cell selected from the group consisting of pluripotent stem cells, epiblast-like cells (EpiLCs), and primordial germ cells.

[0066] (Pluripotent stem cells)

[0067] 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 include, but are not limited to, artificial 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 culture process of establishing GS (Germline Stem) cells from testicular tissue, and Muse cells isolated from myeloid mesenchymal cells. Meanwhile, ES cells may be ES cells formed by nuclear resetting from somatic cells. The pluripotent stem cells listed above can each be obtained by known methods.

[0068] In this specification, "iPS cells" refers to cells that can be reprogrammed into cells of various tissues or organs by introducing several genes into differentiated somatic cells. The iPS cells used in the method for inducing immature oocytes of the present embodiment may be derived from primary culture cells of somatic cells collected from a suitable donor, or may be derived from an established cell line. Since iPS cells can be induced to differentiate into any germ layer cell, the somatic cells used for preparing iPS cells may, in principle, be derived from cells of either the ectoderm or the endoderm. Cells such as skin, hair, gums, and blood, which are low in invasiveness and easy to collect, are suitable as somatic cells for preparing iPS cells. Regarding the method for preparing iPS cells, methods known in the art may be followed. Specifically, for example, "Okita K. et al., "Generation of germline-competent induced pluripotent stem cells.", Nature, Vol. Preparation methods described in known literature, such as “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), may be used.

[0069] The ES cells used in the method for inducing immature oocytes of the present embodiment can be obtained by known methods. For example, they can be established by collecting an internal cell mass from a blastocyst of a fertilized egg of a target animal and culturing said internal cell mass on a feeder cell derived from a fibroblast. In addition, ES cells established by culturing an early embryo produced by nuclear transplanting the nucleus of a somatic cell can also be used. Furthermore, as shown in the examples described below, ES cells can be maintained cultured in a serum-free medium supplemented with 2i (2 inhibitors; PD0325901 and CHIR99021) and LIF (Leukemia Inhibitory Factor) without using a feeder cell (Reference 3: "Ying QL et al., "The ground state of embryonic stem cell self-renewal.", Nature, Vol. 453, No. 7194, p519-523, 2008.").

[0070] (Epiblast-like cells)

[0071] Meanwhile, 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 have characteristics very similar to epiblasts (tissues that differentiate into primordial germ cells in vivo).

[0072] A method for inducing differentiation of EpiLCs from pluripotent stem cells (iPS cells or ES cells) can be carried out by referring to known methods, for example, 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, p519-532, 2011.” (Reference 5).

[0073] (Siwon germ cells)

[0074] In addition, in this specification, "primordial germ cell" refers to a cell that is scheduled to differentiate into a germ cell and ultimately differentiates into an egg or sperm through meiosis. Primordial germ cells may be of in vivo origin or may be primordial germ cell-like cells (PGCLCs) induced from differentiation of pluripotent stem cells. When primordial germ cells are recovered from in vivo, for example, they may be recovered together with gonads from female mouse fetuses (from 11.5 days to 12.5 days of age). Meanwhile, when gonads are recovered from in vivo, they may be recovered in a form accompanied by a mesonephron, or recovered with the mesonephron separated.

[0075] In addition, as previously mentioned, "primordial germ cells" include primordial germ cell-like cells differentiated from pluripotent stem cells. A method for inducing differentiation of PGCLCs from pluripotent stem cells (iPS cells or ES cells) can be carried out by referring to known methods, for example, the above reference 5.

[0076] Meanwhile, when using PGCLCs derived from pluripotent stem cells as primordial germ cells, it is desirable to remove undifferentiated cells from a population of differentiated pluripotent stem cells beforehand. Such a method is known, for example, by introducing a nucleic acid encoding a fusion protein in which Blimp1, a marker gene for primordial germ cells, and a reporter protein are bound, into pluripotent stem cells, the differentiated PGCLCs and undifferentiated cells from pluripotent stem cells can be easily separated by methods such as Fluorescence-activated cell sorting (FACS).

[0077] In addition, "primordial germ cells" include cells in which the genes of in vivo primordial germ cells or primordial germ cell-like cells derived from pluripotent stem cells have been modified using genetic engineering methods. As a method for modifying the genes of in vivo primordial germ cells and primordial germ cell-like cells derived from pluripotent stem cells, the introduction of the target nucleic acid or vector, etc., can be performed 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 methods. Examples of methods for introducing nucleic acid or vector, etc., include microinjection, electroporation, lipofection, and nucleic acid introduction methods using viral vectors. Furthermore, the method for introducing foreign genes or foreign nucleic acid fragments is not limited to the methods listed above, provided that the genetically modified primordial germ cells can differentiate into functional oocytes by the method of the present embodiment. Meanwhile, genetic modification of primordial germ cells can be performed at an appropriate timing during the culture period of the primordial germ cells. For example, in mice, it can be performed during the period from 11.5 days of age to 12.5 days of age. In addition, when using primordial germ cell-like cells derived from pluripotent stem cells, pluripotent stem cells prior to differentiation into primordial germ cell-like cells may be genetically modified by a known method.

[0078] Among them, pluripotent stem cells are preferred as the cells capable of differentiating into oocytes used in the method for inducing immature oocytes of the present embodiment. In particular, when ES cells are used, a large number of immature oocytes can be obtained due to their high proliferative capacity.

[0079] In addition, cells capable of differentiating into oocytes may be derived from mammals. Mammals may 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, and guinea pigs; pets and laboratory animals, but are not limited thereto.

[0080] [Introduction Process]

[0081] In the method for inducing immature oocytes according to the present 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.

[0082] In addition, instead of the oocyte-forming gene, its transcript mRNA or its expression protein may be introduced. As the mRNA of the oocyte-forming gene and its expression protein, the mRNA consisting of a nucleotide sequence represented by the accession number of the above Genbank and the expression protein consisting of an amino acid sequence may be used.

[0083] Among these, it is preferable to introduce the above oocyte-forming gene.

[0084] The introduction method is not particularly limited and can be appropriately selected depending on the target cell and the type of introduction material (whether it is nucleic acid or protein, etc.).

[0085] 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 the lipofection method, microinjection method, DEAE dextran method, gene gun method, electroporation method, calcium phosphate method, etc.

[0086] The method for introducing mRNA of an oocyte-forming gene into a cell is not particularly limited, and known methods may be appropriately selected and used. Specifically, examples include methods using commercially available RNA transfection reagents such as Lipofectamine (registered trademark) and MessengerMAX (manufactured by Life Technologies).

[0087] The method for introducing the expression protein of the oocyte-forming gene into the cell is not particularly limited, and known methods may be appropriately selected and used. Specifically, examples include methods using protein introduction reagents, methods using protein introduction domain (PTD) fusion proteins, and microinjection methods.

[0088] The oocyte-forming gene may be introduced in the form of an expression vector into at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells and expressed transiently, or the oocyte-forming gene may be incorporated into the chromosome of at least one cell 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 selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells in order to stably express the gene.

[0089] When introducing an oocyte-forming gene into at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells in the form of an expression vector, an expression vector comprising the nucleotide sequence of the oocyte-forming gene and a promoter that controls the expression of the nucleotide sequence of the oocyte-forming gene may be used. In the expression vector, the nucleotide sequence of the oocyte-forming gene is functionally linked to the promoter. Furthermore, among the eight types of oocyte-forming genes mentioned above, all genes used may be incorporated into a single expression vector, or they may be incorporated into different vectors one by one; however, from the perspective of introduction efficiency, it is preferable to incorporate all genes used into a single expression vector.

[0090] As for the promoter, there are no specific limitations; it may be active in target cells, or it may be an expression-inducing promoter capable of inducing activity by drugs, etc.

[0091] Examples of promoters that are active in target cells include the cytomegalovirus promoter (CMV promoter) and the CMV early enhancer / chicken beta actin (CAG promoter), which have strong promoter activity in almost all cells.

[0092] Examples of expression-inducing promoters include doxycycline-inducing promoters (TetO promoters) that can artificially control promoter activity.

[0093] In addition to the nucleotide sequence of an oocyte-forming gene and a promoter, the expression vector may, if desired, include an enhancer, a poly-A addition signal, a marker gene, a replication initiation site, a gene encoding a protein that binds to the replication initiation site and controls replication, etc. "Marker gene" refers to a gene that enables cell selection or selection by introducing the corresponding marker gene into a cell. Specific examples of marker genes include, for instance, drug resistance genes, fluorescent protein genes, bioluminescent enzyme genes, chromogenic enzyme genes, etc. These may be used individually or in combination of two or more types. Specific examples of the above-mentioned drug resistance genes include, for instance, puromycin resistance genes, geneticin resistance genes, neomycin resistance genes, tetracycline resistance genes, kanamycin resistance genes, zeocin resistance genes, hygromycin resistance genes, chloramphenicol resistance genes, etc. Specific examples of the above fluorescent protein gene include, for example, the green fluorescent protein (GFP) gene, the yellow fluorescent protein (YFP) gene, the red fluorescent protein (RFP) gene, etc. Specific examples of the above luminescent enzyme gene include, for example, the luciferase gene, etc. Specific examples of the above chromogenic enzyme gene include, for example, the β-galactosidase gene, the β-glucuronidase gene, the alkaline phosphatase gene, etc.

[0094] As an expression vector incorporating an oocyte-forming gene, any known expression vector may be used without being particularly limited. Examples of expression vectors include plasmid vectors and viral vectors.

[0095] The plasmid vector is not particularly limited as long as it is a plasmid vector capable of being expressed in at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells. For example, a plasmid vector that is generally used as a mammalian cell expression vector may be used. Examples of mammalian cell expression plasmid vectors include pX459, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc., but are not limited to these.

[0096] Examples of virus vectors include retrovirus (including lentivirus) vectors, adenovirus vectors, adeno-associated virus vectors, Sendai virus vectors, herpes virus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, silvis virus vectors, rhabdovirus vectors, paramyxovirus vectors, orthomyxovirus vectors, etc.

[0097] Among them, plasmid vectors are preferred as expression vectors.

[0098] When incorporating an oocyte-forming gene into a chromosome, this can be done using a known knock-in system. Examples of known knock-in systems include a method of cutting a chromosome using a known genome editing method such as a CRISPR / Cas system, Transcription Activator-Like Effector Nucleases (TALEN), or zinc finger nucleases, and performing homologous recombination using a donor vector for homologous recombination, or a method using a transposon vector system.

[0099] The donor vector includes a nucleotide sequence adjacent to the target region as a homology arm. The donor vector may include a nucleotide sequence of an oocyte-forming gene (hereinafter referred to as the "knock-in sequence") between the 5' arm and the 3' arm. In addition, in order to stably express the oocyte-forming gene, it is preferable to set the target region within the safe harbor region.

[0100] The donor vector may be a circular DNA vector (e.g., a plasmid vector) or a linear DNA vector. In addition to the homology arm and knock sequence, the donor vector may contain other sequences. Examples of other sequences include marker genes, replication initiation sites, and genes encoding proteins that bind to replication initiation sites and control replication. Examples of marker genes include those mentioned above.

[0101] The method of introducing the donor vector is not particularly limited and can be appropriately selected depending on the target cell. Examples of methods for introducing the donor vector into cells include lipofection, microinjection, DEAE dextran method, gene gun method, electroporation method, calcium phosphate method, etc.

[0102] In a transposon vector system, as shown in the examples described below, a transposon vector containing the nucleotide sequence of an oocyte-forming gene can be introduced into a cell and easily incorporated into the cell's chromosome by applying a transposase. Furthermore, by applying a transposase again, the nucleotide sequence of the oocyte-forming gene incorporated into the chromosome can be cut out from the chromosome and removed without leaving a trace. Examples of transposons include piggyBac (registered trademark), Hyperactive version of piggyBac (registered trademark), transposase (hyPBase), Sleeping Beauty, Tol II, mariner, etc.

[0103] The method for inducing immature oocytes according to the present embodiment may include any process in addition to the introduction process. Any process may include, for example, a process for proliferating cells (proliferation process), a process for selecting cells into which the introduced oocyte-forming gene, its transcript, or its expression protein has been introduced (selection process), and a process for culturing cells after the introduction process in a state in which the oocyte-forming gene is expressed within the cell or the expression protein of the oocyte-forming gene is present (culture process). Furthermore, in the introduction process, when introducing the oocyte-forming gene, the method may include a process for inducing the expression of the introduced oocyte-forming gene (expression induction process) or a process for stabilizing the expression of the introduced oocyte-forming gene (expression stabilization process).

[0104] [Proliferation Process]

[0105] 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.

[0106] In the proliferation process, at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells can be proliferated by culturing it in a proliferation medium. As a proliferation medium, known media for culturing ES cells, iPS cells, EpiLCs, primordial germ cells, etc. may be used, but are not limited thereto; any medium suitable for culturing ES cells, iPS cells, EpiLCs, and primordial germ cells may be used. Specifically, as a proliferation medium, examples include serum-free media supplemented with 2i (2 inhibitors; PD0325901 and CHIR99021) and LIF (Leukemia Inhibitory Factor), as shown in the examples described below.

[0107] As for the culture conditions in the proliferation process, the process may be carried out under known conditions 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 may be set to 30°C or higher and 37°C or lower. In the case of mice, the culture period is not specifically limited, but for example, it may be set to 1 day or higher and 10 days or lower, 3 days or higher and 7 days or lower, or 5 days. Furthermore, a person skilled in the art may set an appropriately desirable culture period depending on the animal species from which it originates.

[0108] The proliferation process may be either before or after the introduction process described above. On the other hand, if it is after the introduction process, proliferation is stopped because differentiation into immature oocytes is initiated from at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, if the oocyte-forming gene is expressed or the expression protein of the oocyte-forming gene is present. Therefore, as shown in the expression induction process / expression stabilization process described below, if the expression of the oocyte-forming gene is controlled to be induced by the presence of an expression induction substance or if the expression of the oocyte-forming gene is controlled to be stabilized by the presence of an expression stabilization substance, the proliferation process can be performed after the introduction process in the absence of the expression induction substance and the expression stabilization substance.

[0109] In addition, when introducing an oocyte-forming gene in the form of a transient expression vector, or when introducing a transcript or expression protein of an oocyte-forming gene, it is preferable to perform a proliferation process prior to the introduction process.

[0110] [Selection Process]

[0111] In the selection process, cells into which an oocyte-forming gene, its transcript, or its expression protein has been introduced are selected.

[0112] In the selection process, for example, by using a reporter gene, cells into which an oocyte-forming gene, its transcript, or its expression protein has been introduced can be selected. Specifically, for example, when an oocyte-forming gene is introduced into a cell in the form of an expression vector, the reporter gene can be included in the expression vector so that the reporter gene is expressed within the cell together with the expression of the oocyte-forming gene or independently of the expression of the oocyte-forming gene, thereby allowing cells to be selected. When an oocyte-forming gene is incorporated into the chromosome of at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, a construct in which the reporter gene is functionally linked upstream or downstream of the oocyte-forming gene can be incorporated so that the reporter gene is expressed together with the expression of the oocyte-forming gene or independently of the expression of the oocyte-forming gene, thereby allowing cells to be selected. When introducing a transcript of an oocyte-forming gene, cells can be selected by introducing into cells a construct in which a transcript of a reporter gene is functionally linked upstream or downstream of the transcript of the oocyte-forming gene, thereby expressing the reporter gene along with the expression of the oocyte-forming gene. When introducing an expression protein of the oocyte-forming gene into cells, cells can be selected by introducing into cells a fusion protein of the expression protein of the oocyte-forming gene and the expression protein of the reporter gene. As the reporter gene, the marker gene exemplified in the description of the "introduction process" above may be used.

[0113] [Expression Induction Process / Expression Stabilization Process]

[0114] In the above introduction process, if the expression of the oocyte-forming gene is controlled to be induced by the presence of an expression-inducing substance, the expression of the oocyte-forming gene is induced by adding the expression-inducing substance to the culture medium. Alternatively, in the above introduction process, if the expression of the oocyte-forming gene is controlled to be stabilized by the presence of an expression-stabilizing substance, the expression of the oocyte-forming gene is stabilized by adding the expression-stabilizing substance to the culture medium. On the other hand, in the absence of an expression-stabilizing substance, the expression protein derived from the expressed oocyte-forming gene is degraded by the proteasome. When differentiation into immature oocytes is induced from at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, cell proliferation is stopped. Therefore, in order to obtain a larger quantity of immature oocytes, it is preferable to perform a proliferation process before performing an expression induction process or an expression stabilization process. That is, when introducing an oocyte-forming gene into a cell in the form of an expression vector, it is preferable to perform the proliferation process, the introduction process, and the expression induction process or expression stabilization process in this order. Meanwhile, when incorporating an oocyte-forming gene into the chromosome of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, it is preferable to perform the introduction process, the proliferation process, and the expression induction process or expression stabilization process in this order.

[0115] Inducing the expression of oocyte-forming genes can be described, for example, by introducing the oocyte-forming gene into a cell in a form functionally linked to an expression-inducing promoter (e.g., a doxycycline-inducing promoter (TetO promoter)) and expressing the oocyte-forming gene by adding an expression-inducing substance (e.g., doxycycline) to the culture medium.

[0116] Alternatively, the stabilization of the expression of oocyte-forming genes may be achieved by using a ProteoTuner (registered trademark) system (manufactured by CloneTech, etc.), for example, as shown in the examples described below. Specifically, by introducing a construct into a cell in which a sequence encoding a destabilizing domain (DD, 12 kDa) is functionally linked to the upstream or downstream of the oocyte-forming gene, the fusion protein expressed by the said construct is rapidly degraded by the proteasome in the absence of an expression stabilization substance. On the other hand, by adding Shield1 (membrane-permeable small molecule, 750 Da), a small molecule that protects against degradation by the proteasome, to the medium as an expression stabilization substance, the oocyte-forming gene can be stably expressed and accumulated within the cell.

[0117] As for the amount of expression-inducing substance or expression-stabilizing substance added, it should be at a concentration that results in the desired amount of expression of the oocyte-forming gene, and there are no specific limitations. For example, if the expression-inducing substance is doxycycline, the concentration in the medium can be, for example, 1 nM (1 nmol / L) or more and 10 μM (10 μmol / L) or less. For example, when the expression stabilizing substance is Shield1, the concentration in the medium can be, for example, 10 nM (10 nmol / L) or more and 10 μM (10 μmol / L) or less, 100 nM (100 nmol / L) or more and 5 μM (5 μmol / L) or less, 300 nM (300 nmol / L) or more and 3 μM (3 μmol / L) or less, 400 nM (400 nmol / L) or more and 1 μM (1 μmol / L) or less, and 500 nM (500 nmol / L).

[0118] As a medium used in the expression induction process or expression stabilization process, the medium exemplified as a proliferation medium in the above proliferation process may be used.

[0119] [Cultivation Process]

[0120] In the culture process, the cells after the above introduction process are cultured in a state where an oocyte-forming gene is expressed within the cell or where the expression protein of the oocyte-forming gene is present.

[0121] As for the culture conditions, the process may be carried out under known conditions 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 may be set to 30°C or higher and 37°C or lower. In the case of mice, the culture period may be set to 1 day or higher and 10 days or lower, and may be set to 3 days or higher and 7 days or lower.

[0122] As the medium used in the culture process, the medium exemplified as a proliferation medium in the proliferation process above may be used. Meanwhile, if the expression of the oocyte-forming gene is controlled to be induced by the presence of an expression-inducing substance, the expression-inducing substance is added to the medium, and the cells are cultured in a state where the oocyte-forming gene is expressed within the cells. Alternatively, if the expression of the oocyte-forming gene is controlled to be stabilized by the presence of an expression-stabilizing substance, the expression-stabilizing substance is added to the medium, and the cells are cultured in a state where the oocyte-forming gene is expressed within the cells.

[0123] A method for inducing immature oocytes according to a preferred embodiment is such that the expression of an oocyte-forming gene is controlled to be induced by the presence of an expression-inducing substance, or the expression of an oocyte-forming gene is controlled to be stabilized by the presence of an expression-stabilizing substance, and includes the following 1) to 5).

[0124] 1) Introducing four types of oocyte-forming genes, FIGLA, NOBOX, LHX8, and TBPL2 (preferably five types of oocyte-forming genes, FIGLA, NOBOX, LHX8, TBPL2, and STAT3; more preferably eight types of oocyte-forming genes, FIGLA, NOBOX, LHX8, TBPL2, STAT3, SOHLH1, SUB1, and DYNLL1) into the chromosomes of at least one cell type selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells (particularly preferably pluripotent stem cells);

[0125] 2) Proliferating cells after introduction;

[0126] 3) Selecting cells from the cells after proliferation in which the oocyte-forming gene has been introduced into the chromosome;

[0127] 4) In the cells after selection, adding an expression-inducing substance to the medium to induce the expression of an oocyte-forming gene, or in the cells after selection, adding an expression-stabilizing substance to the medium to stabilize the expression of an oocyte-forming gene;

[0128] 5) Culturing cells after expression induction or expression stabilization in a state where oocyte-forming genes are expressed within the cells.

[0129] A more preferred embodiment of a method for inducing immature oocytes comprises controlling the expression of oocyte-forming genes by a ProteoTuner (registered trademark) system and including the following 1) to 5).

[0130] 1) Introducing into the chromosome of a pluripotent stem cell a construct having a sequence encoding a destabilizing domain (DD, 12kDa) functionally linked upstream or downstream of four types of oocyte-forming genes, FIGLA, NOBOX, LHX8, and TBPL2 (preferably five types of oocyte-forming genes, FIGLA, NOBOX, LHX8, TBPL2, and STAT3; more preferably eight types of oocyte-forming genes, FIGLA, NOBOX, LHX8, TBPL2, STAT3, SOHLH1, SUB1, and DYNLL1);

[0131] 2) Proliferating cells after introduction;

[0132] 3) Selecting cells from the cells after proliferation in which the oocyte-forming gene has been introduced into the chromosome;

[0133] 4) In the cells after selection, adding Shield1 to the medium to stabilize the expression of oocyte-forming genes;

[0134] 5) Culturing cells after expression stabilization in a state where the oocyte-forming gene is expressed within the cell.

[0135] In the method for inducing immature oocytes according to the present 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 induced to differentiate into immature oocytes with a short culture period of 5 days or more and 10 days or less from the introduction of an oocyte-forming gene, or its transcript or its expression protein.

[0136] It can be confirmed from the expression of a known oocyte marker gene (e.g., Stella, etc.) that at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells has been induced to differentiate into an immature oocyte. Specifically, as shown in the examples described below, by introducing a nucleic acid encoding a fusion protein formed by combining the oocyte marker gene Stella with a reporter protein (e.g., Enhanced cyan fluorescent protein (ECFP)) into the chromosome of at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, it can be confirmed that differentiation into an immature oocyte has been induced from the fluorescence detected by the expression of Stella-ECFP. Furthermore, from the expression of Stella-ECFP, immature oocytes can be easily isolated by removing undifferentiated cells from the differentiated cell population using methods such as Fluorescence-activated cell sorting (FACS).

[0137] Method for producing mature oocytes

[0138] In one embodiment, the present invention,

[0139] Introducing four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2, or their transcripts or expression proteins, into at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, and

[0140] Co-culturing the above cells and ovarian somatic cells after introduction

[0141] A method for producing mature oocytes including

[0142] In the method for producing mature oocytes of the present embodiment, the introduction of the oocyte-forming gene (introduction process) is identical to the introduction process described in the "method for inducing immature oocytes" above, so the explanation is omitted.

[0143] Meanwhile, in this specification, "mature oocyte" refers to an egg in the metaphase of the second meiotic division and is also called a secondary oocyte. In addition, as shown in the examples described below, mature oocytes express the Npm2 gene, which is an activated oocyte marker.

[0144] [Aggregation Formation Process]

[0145] The method for producing mature oocytes according to the present embodiment includes a process of co-culturing at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction process described in the “method for inducing oocytes” and ovarian somatic cells to form an aggregate (aggregate formation process).

[0146] The ovarian somatic cells used in the aggregation process are somatic cells collected from the ovaries of a living organism, and are cells that differentiate into granulosa cells or capsular cells constituting the follicle by co-culturing with at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells.

[0147] Meanwhile, the method of collecting somatic cells from the ovaries of a living organism can be carried out according to the method described in Reference 5 above.

[0148] Specifically, for example, a method for collecting somatic cells from an ovary involves surgically collecting the ovary from a living organism and dissociating the somatic cells constituting the ovary through trypsin treatment, etc. Meanwhile, it is desirable to remove germ cells present in the ovary derived from the living organism. The method for removing germ cells present in the ovary can be performed by known methods, and for example, germ cells present can be removed by a magnetic activated cell sorting method using an anti-SSEA1 antibody or an anti-CD31 antibody. Here, in order to collect ovarian somatic cells, it is preferable that the ovary be of fetal origin. In the case of a mouse, for example, a gonad (ovary) derived from a mouse fetus with a fetal age (also called "embryo age") of 12.5 days may be used. Furthermore, a person skilled in the art can select a gonad (ovary) of an appropriately desirable time according to the animal species from which it is derived, based on the present disclosure and technical common sense in the relevant technical field.

[0149] In the process of forming an aggregation, it is preferable to form an aggregation consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction process, and ovarian somatic cells by co-culturing them.

[0150] A method for producing an aggregate composed of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after an introduction process, and ovarian somatic cells, can be carried out by mixing and aggregating and culturing at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after an introduction process, and ovarian somatic cells in S10 medium (StemPro (registered trademark)-34 SFM, manufactured by Life Technologies) to which 10% fetal bovine serum (FCS), 150 μM ascorbic acid, 1×Glutamax, 1×penicillin / streptomycin, and 55 μM mercaptoethanol have been added, for example, as shown in the examples described below. Meanwhile, if the expression of an oocyte-forming gene is controlled to be induced by the presence of an expression-inducing substance or stabilized by the presence of an expression-stabilizing substance, the expression-inducing substance or the expression-stabilizing substance is added to the medium and cultured in a state where the oocyte-forming gene is expressed. For culture, it is preferable to use a low-adhesion culture dish (e.g., a cell-low adhesion U-bottom 96-well plate).

[0151] For example, in the case of mice, the culture period for producing aggregates can be set to be between 2 and 3 days, and 2 days is preferred. In addition, a person skilled in the art can set an appropriately preferred culture period depending on the animal species from which it originates.

[0152] In addition, the ratio of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction process to ovarian somatic cells is not limited as long as the produced aggregate forms a mature follicle, but for example, in the case of a mouse, it is preferable to make the ratio of the number of cells of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction process to ovarian somatic cells about 2:1.

[0153] In addition, at least one 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, may be used in a cryopreserved state. The cryopreservation method may be carried out by a known method. For example, cryopreservation of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction process, or ovarian somatic cells, may be carried out by a slow freezing method using a 10% DMSO solution or a commercially available freezing agent (Cell Banker (registered trademark), etc.).

[0154] Meanwhile, the ovarian somatic cell used in the method for producing mature oocytes according to the present embodiment may be derived from a mammal of the same species as at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells constituting the aggregate, or it may be derived from a mammal of a different species, but it is preferable to use one derived from a mammal of the same species. As for the mammal, the same as those exemplified in the description of the cell having the ability to differentiate into an oocyte can be cited.

[0155] The method for producing mature oocytes according to the present embodiment may include any process after the introduction process and before the aggregation formation process. Any process may include the proliferation process, selection process, etc., described in the "Method for Inducing Immature Oocytes" above.

[0156] [Cultivation Process]

[0157] In addition, the method for producing mature oocytes according to the present embodiment may include a culture process after the aggregation formation process.

[0158] In the culture process, it is preferable to transfer the aggregates after the aggregate formation process onto a collagen membrane and culture them.

[0159] As for the culture conditions, the process can be carried out under known conditions for culturing aggregates. Specifically, for example, the culture temperature can be set to 30°C or higher and 37°C or lower. In the case of mice, the culture period can be set to 7 days or higher and 35 days or lower, and 8 days or higher and 30 days or lower. Furthermore, a person skilled in the art can set an appropriately desirable culture period depending on the animal species from which it originates.

[0160] As a medium used in the culture process, the medium exemplified as a proliferation medium in the proliferation process above may be used. Meanwhile, if the expression of the oocyte-forming gene is controlled to be induced by the presence of an expression-inducing substance, or if the expression of the oocyte-forming gene is controlled to be stabilized by the presence of an expression-stabilizing substance, the expression-inducing substance or the expression-stabilizing substance is added to the medium, and the cells are cultured in a state where the oocyte-forming gene is expressed within the cells.

[0161] The aggregate after the culture process has a secondary follicular structure, in which the oocyte is surrounded by multilayered granulosa cells, and additionally, a theca folic is formed surrounding the multilayered granulosa cells, and the theca cells constituting the theca interna express luteinizing hormone receptors, and the granulosa cells express follicle-stimulating hormone receptors.

[0162] Next, mature oocytes can be produced by culturing the follicles obtained from the culture process using the method described in Non-Patent Literature 1. Specifically, the process up to obtaining mature oocytes can be divided into a growth process and a maturation process.

[0163] [Growth Process]

[0164] In the growth process, the follicles obtained from the culture process are isolated into individual follicles and cultured using a growth medium.

[0165] As for the culture conditions, they may be carried out under the conditions described in Non-Patent Literature 1. Specifically, for example, the culture temperature may be set to 30°C or higher and 37°C or lower. In the case of mice, the culture period may be set to 7 days or higher and 15 days or lower, and may be set to 8 days or higher and 13 days or lower. Furthermore, a person skilled in the art may set an appropriately desirable culture period depending on the animal species from which it originates.

[0166] As a growth medium, a medium of the composition described in Non-Patent Literature 1 may be used. Specifically, for example, in the case of mice, for 2 days from the start of culture, follicles are cultured using α-MEM containing 5% fetal bovine serum (FCS), 2% polyvinylpyrrolidone (Sigma), 150 μM ascorbic acid, 1×GlutaMAX, 1×penicillin / streptomycin, 100 μM 2-mercaptoethanol, 55 μg / mL sodium pyruvate (Nacalai Test), 0.1 IU / mL follicle-stimulating hormone (Polystim (registered trademark), MSD), 15 ng / mL BMP15 (Bone morphogenetic protein 15), and 15 ng / mL GDF9 (Growth differentiation factor 9) (R&D Systems). Next, on the second day from the start of culture, the α-MEM is replaced with one excluding BMP15 and GDF9 from the above composition, and follicles are cultured in 0.1% Type IV collagenase (manufactured by MP Biomedicals). Then, after washing several times with α-MEM containing 5% FCS, the follicles are cultured using α-MEM excluding BMP15 and GDF9 from the above composition until the 11th day from the start of culture.

[0167] The follicle after the growth process has a vesicular follicle structure and forms a cumulus oocyte complex containing an egg cell at the nucleus stage.

[0168] [Maturity Process]

[0169] In the maturation process, the follicles obtained from the growth process are cultured using a maturation medium.

[0170] As for the culture conditions, they may be carried out under the conditions described in Non-Patent Literature 1. Specifically, for example, the culture temperature may be set to 30°C or higher and 37°C or lower. In the case of mice, the culture period may be set to 7 days or higher and 15 days or lower, and may be set to 8 days or higher and 13 days or lower. Furthermore, a person skilled in the art may set an appropriately desirable culture period depending on the animal species from which it originates.

[0171] As a maturation medium, a medium of the composition described in Non-Patent Literature 1 may be used. Specifically, for example, in the case of mice, an 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) may be used.

[0172] After the maturation process, the follicle matures into an egg at the metaphase of the second meiotic division (secondary oocyte).

[0173] The presence of an egg in the metaphase of the second meiotic division (secondary oocyte) can be evaluated, for example, by visual inspection using a microscope, using the release of the first polar body as an indicator.

[0174] The obtained mature oocytes (secondary oocytes) are suitable for use in infertility treatment. That is, in one embodiment, the present invention provides a method for treating infertility using mature oocytes obtained by the above method.

[0175] Furthermore, the mature oocytes obtained using the method for producing mature oocytes of the present embodiment are suitablely used for the efficient breeding of industrial animals and the breeding of rare animals. That is, in one embodiment, the present invention provides a method for breeding industrial animals or a method for breeding rare animals using mature oocytes obtained by the above method.

[0176] Meanwhile, mammals are preferred as the animals to which the above applies. Examples of mammals include those exemplified above.

[0177] In addition, the mature oocytes obtained using the method for producing mature oocytes of the present embodiment can be helpful in identifying the causes of infertility and elucidating the mechanisms of menopausal diseases.

[0178] Examples

[0179] The present invention will be explained below by way of examples, but the present invention is not limited to the following examples.

[0180] [Example 1]

[0181] (Construction of vectors)

[0182] 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 construct the PB-CAG-DD vector. Subsequently, the cDNAs of eight genes, FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2, and DYNLL1, were amplified from the cDNAs of female mouse ovaries 13.5 days old after modification and cloned into the PB-CAG-DD vector using the Infusion HD Cloning Kit (Takara Bio). Amplification of each cDNA was performed by PCR using KOD Fx Neo or KOD Plus Neo DNA polymerase (TOYOBO) according to the manufacturer's protocol.

[0183] (Vector Transfection)

[0184] ES cells were previously maintained in serum-free medium supplemented with 2i and LIF without using feeder cells (see Reference 3 above). In addition, to monitor differentiation into immature oocytes and mature oocytes, mouse ES cells (Blimp1-mVenus: Stella-ECFP: Npm2-mCherry (BVSCNmC)) were used in which a gene encoding mVenus (membrane-targeted Venus), a variant of yellow fluorescent protein (YFP) under the control of expression of Blimp1, an important determinant of germ cell lineage, a gene encoding enhanced cyan fluorescent protein (ECFP), under the control of expression of Stella, a germ cell and oocyte marker, and a gene encoding mCherry (membrane-targeted Cherry), a red fluorescent protein under the control of expression of Nucleoplasmin 2 (Npm2), an activated (mature) oocyte marker, were inserted on the chromosomes. The PB-CAG-DD vector containing the eight constructed genes was simultaneously transfected with the hyperactive PBase (hypBase) plasmid using Lipofectamine 2000. After proliferation in serum-free medium supplemented with 2i and LIF at 37°C for 5 days, a single colony was obtained by puromycin selection.

[0185] (Induction of differentiation into immature oocytes)

[0186] Subsequently, in a low-adhesion U-bottom 96-well plate dispensed with S10 medium (StemPro-34 SFM, Life Technologies) supplemented with 10% fetal bovine serum (FCS), 150 μM ascorbic acid, 1 × Gluamax, 1 × penicillin / streptomycin, and 55 μM mercaptoethanol, mixed with 0.5 μM Shield1 (Clonetech) (hereinafter referred to as "oocyte differentiation induction medium"), 1 × 10⁶ after puromycin selection 5Can ES cells were transferred and cultured at 37°C for 5 days to induce differentiation into immature oocytes. The results observed under a confocal microscope (Carl Zeiss, model number: Zeiss LSM 700) are shown in Fig. 1. In Fig. 1, "oocyte-forming genes" refers to eight genes: FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2, and DYNLL1. "Oocyte-forming gene expression OFF" refers to cells before the addition of a medium containing Shield1, and "Oocyte-forming gene expression ON" refers to cells after adding a medium containing Shield1 and culturing for 5 days.

[0187] From Figure 1, in cells where the expression of the oocyte-forming gene was OFF, colonies were formed, and the fluorescence of ECFP (Stella-ECFP), which is expressed under the control of Stella expression, was barely visible. In contrast, in cells where the expression of the oocyte-forming gene was ON, the cells were scattered apart, and strong fluorescence of Stella-ECFP was detected, suggesting that differentiation into immature oocytes was induced.

[0188] (Production of mature oocytes)

[0189] Subsequently, in the above (transfection of the vector), after transfection, a single colony of ES cells (1×10⁶) selected with puromycin is cultured and proliferated at 37°C for 5 days. 5 (dogs) in the above oocyte differentiation induction medium, 3×10 4Aggregates were prepared by mixing ovarian somatic cells derived from female mice aged 12.5 days post-fertilization with the aggregates and cultured at 37°C for 2 days. Ovarian somatic cells were used by extracting the ovaries from female mice aged 12.5 days post-fertilization using the method previously described in Non-Patent Literature 1, etc., and isolating the cells from the said ovaries. Subsequently, the aggregates were transferred onto a Transwell-COL membrane (Coaster) and cultured in the oocyte differentiation induction medium 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 No.: Zeiss LSM 700). In Figure 2, oocytes were visualized in the upper section by the fluorescence of Stella-ECFP, a germ cell and oocyte marker. At the bottom, oocytes were visualized by the fluorescence of the red fluorescent protein mCherry (Npm2-mCherry), which is expressed under the control of the expression of Nucleoplasmin 2 (Npm2), an activated (mature) oocyte marker.

[0190] From Figure 2, fluorescence of Stella-ECFP in the cells was continuously observed throughout the culture period. Meanwhile, on the 8th day from the start of culture, fluorescence of Npm2-mCherry was observed (see the arrow at the bottom of "Day 8" in Figure 2), indicating that oocyte maturation was progressing.

[0191] In addition, on the 28th day from the start of culture, individual follicles were manually isolated using a sharpened tungsten needle. The isolated follicles had a secondary follicle structure. By culturing the corresponding follicles under the culture conditions of the “in vitro growth period” and “in vitro maturation period” described in Non-Patent Literature 1, they were matured through the vesicular follicles into metaphase II meiotic eggs.

[0192] [Example 2]

[0193] Using mouse iPS cells, we investigated the induction of differentiation into oocytes, similar to ES cells.

[0194] (Vector Transfection)

[0195] Mouse BVSC iPS cells produced by the virus buster established in the paper of Non-Patent Literature 1 were used. The PB-CAG-DD vector containing the 8 genes constructed in Example 1 was transfected into mouse BVSC iPS cells simultaneously with the hyperactive PBase (hypBase) plasmid using Lipofectamine 2000. After proliferation by incubation at 37°C for 5 days in serum-free medium supplemented with 2i and LIF, a single colony was obtained by puromycin selection.

[0196] (Induction of differentiation into immature oocytes)

[0197] iPS cells selected after puromycin were cultured at 37°C for 5 days using the same method as in Example 1 to induce differentiation into immature oocytes.

[0198] (Production of mature oocytes)

[0199] Subsequently, in the above (transfection of the vector), after transfection, a single colony of iPS cells (1×10⁶) selected with puromycin is cultured and proliferated at 37°C for 5 days. 5 (dogs) in the above oocyte differentiation induction medium, 3×10 4Aggregates were prepared by mixing ovarian somatic cells derived from female mice 12.5 days after fertilization with the aggregates and cultured at 37°C for 2 days. Subsequently, the aggregates were transferred onto a Transwell-COL membrane (Coaster) and cultured in the oocyte differentiation induction medium for 21 days. Figure 3 shows the results of observing the expression of Stella-ECFP in the aggregates on the 21st day from the start of culture under a confocal microscope (Carl Zeiss, model number: Zeiss LSM 700). In Figure 3, the image on the left is a bright-field image, and the image on the right is a fluorescent image in which oocytes are visualized by the fluorescence of Stella-ECFP, a germ cell and oocyte marker.

[0200] From Figure 3, fluorescence of Stella-ECFP was observed in cells on the 21st day from the start of culture. From this, it was confirmed that differentiation into oocytes could be induced in iPS cells as well as in ES cells.

[0201] [Example 3]

[0202] (Identification of key factors among oocyte formation genes)

[0203] To identify genes that are important factors among oocyte formation genes, a total of 26 types of vectors were constructed using the same method as in Example 1, such that the combinations of genes listed in the diagram on the left side of Fig. 4 were formed. Subsequently, each vector was transfected into mouse ES cells (Blimp1-mVenus: Stella-ECFP: Npm2-mCherry(BVSCNmC)) using the same method as in Example 1. After transfection, the cells were cultured and proliferated at 37°C for 5 days, and selected single-cell derived ES cells (1×10⁶) were treated with puromycin. 5 (dogs) in the above oocyte differentiation induction medium, 3×10 4Aggregates were prepared by mixing ovarian somatic cells derived from female mice 12.5 days old after fertilization with the cells, and cultured at 37°C for 2 days. Subsequently, the obtained aggregates were cultured for 21 days using the same method as in Example 1. After culture, the number of oocytes formed from each cell line was counted. In addition, the oocyte area 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.

[0204] From Figure 4, it was evident that among the oocyte formation genes, four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2 are important factors in inducing differentiation from pluripotent stem cells into immature oocytes and in maturation of oocytes. In addition, it was evident that the efficiency of oocyte formation is further improved by introducing the STAT3 gene in addition to the four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2. Industrial applicability

[0205] According to the method for inducing immature oocytes of the present embodiment, immature oocytes can be easily induced from cells capable of differentiating into oocytes, such as pluripotent stem cells, through culture in a shorter period than conventional methods. According to the method for producing mature oocytes of the above embodiment, mature oocytes can be produced in large quantities from cells capable of differentiating into oocytes, such as pluripotent stem cells, through culture in a shorter period than conventional methods.

Claims

Claim 1 A method for inducing immature oocytes in vitro, comprising introducing four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2, or their transcripts or expression proteins, into at least one type of cell selected from the group consisting of pluripotent stem cells and epiblast-like cells. Claim 2 A method for inducing immature oocytes in vitro, comprising introducing four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2 into the cell according to claim 1. Claim 3 A method for inducing immature oocytes in vitro, comprising, in addition to introducing the gene of STAT3, or its transcript or expression protein, into said cell in accordance with claim 1 or 2. Claim 4 A method for inducing an immature oocyte in vitro, comprising, in addition to introducing one or more genes selected from the group consisting of SOHLH1, SUB1, and DYNLL1, or their transcripts or expression proteins, into the cell according to claim 1 or 2. Claim 5 A method for inducing immature oocytes in vitro, comprising additionally introducing three types of genes consisting of SOHLH1, SUB1, and DYNLL1 into the cell according to claim 4. Claim 6 A method for inducing immature oocytes in vitro, wherein, in claim 1 or 2, the cell is a pluripotent stem cell. Claim 7 A method for inducing immature oocytes in vitro, wherein, in claim 1 or 2, the expression of the gene is controlled to be induced by the presence of an expression-inducing substance or the expression of the gene is controlled to be stabilized by the presence of an expression-stabilizing substance, and further comprising: proliferating the cell after the introduction; adding the expression-inducing substance to a culture medium to induce the expression of the gene after the proliferation; or adding the expression-stabilizing substance to a culture medium to stabilize the expression of the gene after the proliferation. Claim 8 A method for producing mature oocytes in vitro, comprising introducing four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2, or their transcripts or expression proteins, into at least one type of cell selected from the group consisting of pluripotent stem cells and epiblast-like cells, and co-culturing said cell and ovarian somatic cell after introduction. Claim 9 A method for producing a mature oocyte in vitro, comprising, in addition to introducing the gene of STAT3, or its transcript or expression protein, into the cell according to claim 8.

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