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 technology]

[0002] In mammals, totipotency, defined as the ability to develop into an individual cell from a single cell, is a special property of single cells. However, despite the strong social demand for totipotency in areas such as infertility treatment, the mechanism underlying this has been difficult to elucidate. The reason for this is that the process of oogenesis that occurs 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 liquid factors such as BMP4, and the resulting PGCLCs are mixed with ovarian somatic cells to produce reconstructed ovaries. Next, the period from PGCLCs to the formation of metaphase II oocytes in the reconstructed ovaries is divided into three periods: an "in vitro differentiation period," an "in vitro growth period," and an "in vitro maturation period." The inventors have established optimal culture conditions for each period to obtain secondary follicles, germinal vesicle-stage oocytes, and metaphase II oocytes (see, for example, Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Hikabe O et al., “Reconstitution in vitro of the entire cycle of the mouse female germ line.”, Nature, Vol. 539, p299-303, 2016. Summary of the Invention [Problem 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 oocytes from primordial follicles from mouse pluripotent stem cells, and it is expected that culture for more than one year will be required 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 inducing immature oocytes from cells capable of differentiating into oocytes, such as pluripotent stem cells, in a simple manner through culture for a shorter period of time than conventional methods, and also provides a method for producing mature oocytes from cells capable of differentiating into oocytes, such as pluripotent stem cells, in a simple manner through culture for a shorter period of time than conventional methods. [Means for solving the problem]

[0007] As a result of extensive research to achieve the above-mentioned objective, the inventors discovered that it is possible to induce differentiation into immature oocytes 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, thereby completing the present invention.

[0008] That is, the present invention includes the following aspects. The 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 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 aspect may include introducing four types of genes consisting of FIGLA, NOBOX, LHX8 and TBPL2 into the cells. The method for inducing immature oocytes according to the first aspect may further comprise introducing a STAT3 gene, or a transcript or expressed protein thereof, into the cells. The method for inducing immature oocytes according to the first aspect may further comprise introducing into the cells one or more genes selected from the group consisting of SOHLH1, SUB1 and DYNLL1, or transcripts or expressed proteins thereof. The method for inducing immature oocytes according to the first aspect may further include introducing three types of genes, SOHLH1, SUB1, and DYNLL1, into the cells. The cells may be pluripotent stem cells. In the method for inducing immature oocytes according to the first aspect, expression of the gene is controlled so as to be induced by the presence of an expression inducer, allowing the cells to grow after said introduction; After the growth, adding the expression inducer to the medium to induce expression of the gene. , may further include.

[0009] The method for producing a mature oocyte according to the second aspect of the present invention comprises: Introducing four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2, or transcripts or expressed proteins thereof, into at least one cell selected from the group consisting of pluripotent stem cells and primordial germ cells; co-culturing the cells after the introduction with ovarian somatic cells; Includes: The method for producing a mature oocyte according to the second aspect may further comprise introducing a STAT3 gene, or a transcript or expressed protein thereof, into the cell. [Effects of the Invention]

[0010] According to the method for inducing immature oocytes of the above aspect, immature oocytes can be induced from cells capable of differentiating into oocytes, such as pluripotent stem cells, in a shorter culture period than conventional methods. According to the method for producing mature oocytes of the above aspect, mature oocytes can be produced in large quantities from cells capable of differentiating into oocytes, such as pluripotent stem cells, in a shorter culture period than conventional methods. [Brief explanation of the drawings]

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

[0012] <Method for inducing immature oocytes> In one embodiment, the present invention provides a method for inducing immature oocytes, which comprises introducing four types of 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 (hereinafter, these cells may be collectively referred to as "cells capable of differentiating into oocytes").

[0013] In conventional methods, in the case of mice, it takes just under one month to induce differentiation from pluripotent stem cells to immature oocytes in vitro, and it takes about 11 days to induce differentiation from primordial germ cells to immature oocytes in vitro. In contrast, the method for inducing immature oocytes of the present embodiment introduces the above four types of genes into cells capable of differentiating into oocytes, such as pluripotent stem cells, and then cultures the cells for a short period of time, such as five to ten days, thereby inducing differentiation into immature oocytes. Furthermore, in the case of humans, it takes nine months or more to induce differentiation from primordial germ cells to immature oocytes in vivo, but by using the method for inducing immature oocytes of the present embodiment, this period can be dramatically shortened.

[0014] Furthermore, conventional methods require at least two steps with different culture conditions to induce differentiation from pluripotent stem cells into PGCLCs and then further induce differentiation of the PGCLCs into immature oocytes. In contrast, the method for inducing immature oocytes of the present embodiment allows direct differentiation of pluripotent stem cells into immature oocytes.

[0015] In this specification, the term "immature oocyte" refers to a primary oocyte that has not undergone follicular growth. An immature oocyte does not necessarily have to have a follicular structure. Furthermore, as shown in the Examples below, immature oocytes express some maternal effect genes, such as the Stella gene and Padi6 gene, which are oocyte markers. In addition, in this specification, the term "follicle" refers to a cell that consists of an oocyte and the somatic cells (granulosa cells and theca cells) that surround it. The method for inducing immature oocytes according to this embodiment will be described in detail below.

[0016] [Oocyte formation genes] The oocyte formation genes used for introduction into cells capable of differentiating 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. Specific examples of oocyte formation genes include FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2, and DYNLL1. As shown in the Examples below, four genes, FIGLA, NOBOX, LHX8, and TBPL2, are particularly important for inducing pluripotent stem cells into immature oocytes. Therefore, by introducing at least the four genes, FIGLA, NOBOX, LHX8, and TBPL2, or their transcripts or expressed proteins, among the above-mentioned oocyte formation genes, into cells, cells capable of differentiating into oocytes can be induced to become immature oocytes.

[0017] In addition to the four genes or their transcripts or expressed proteins, it is preferable to further introduce the STAT3 gene or its transcripts or expressed proteins, and it is more preferable to further introduce the STAT3 gene. As shown in the Examples below, the oocyte formation rate can be further improved by introducing STAT3 into cells in addition to FIGLA, NOBOX, LHX8, and TBPL2.

[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 more preferable to further introduce the three genes consisting of SOHLH1, SUB1, and DYNLL1, or their transcripts or expressed proteins. As shown in the Examples below, by introducing all eight of the above genes, or their transcripts or expressed proteins, into cells, it is possible to more efficiently induce cells capable of differentiating into oocytes from immature oocytes.

[0019] The FIGLA gene encodes a basic helix-loop-helix (bHLH) transcription factor that regulates multiple oocyte-specific genes, including genes involved in folliculogenesis and genes encoding the zona pellucida (ZP1, ZP2, and ZP3). The FIGLA transcription factor binds to the E-box (5'-CANNTG-3') in the ZP (ZP1, ZP2, and ZP3) promoters. Diseases associated with FIGLA include premature ovarian failure (type 6) and pseudohermaphroditism. The Gene Ontology (GO) annotation for FIGLA includes sequence-specific DNA binding and protein dimerization activity. An 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.

[0020] Information on the nucleotide sequences of oocyte formation genes such as the FIGLA gene, the nucleotide sequences of the mRNA of said genes, and the amino acid sequences of the proteins encoded by said 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 mRNA of the human FIGLA gene is disclosed, for example, as Genbank accession number NM_001004311. The amino acid sequence of human FIGLA is disclosed, for example, as Genbank accession number NP_001004311.

[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 Genbank accession number NM_012013. The amino acid sequence of mouse FIGLA is disclosed, for example, as Genbank accession number NP_036143.

[0023] The NOBOX gene encodes a transcription factor involved in oogenesis. Examples of NOBOX-related disorders include premature ovarian failure (type 5). The GO annotation of NOBOX includes DNA-binding transcription factor activity and specific DNA binding to RNA polymerase II-proximal promoter sequences. Specifically, it preferentially 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 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 mRNA of the human NOBOX gene is disclosed, for example, as Genbank accession numbers NM_001080413 and XM_001134420. The amino acid sequence of human NOBOX is disclosed, for example, as Genbank accession numbers NP_001073882 and XP_001134420.

[0025] 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 Genbank accession number NM_130869. The amino acid sequence of mouse NOBOX is disclosed, for example, as Genbank accession number NP_570939.

[0026] The SOHLH1 gene encodes a basic helix-loop-helix (bHLH) transcription factor, a gonad-specific transcription factor essential for spermatogenesis, oogenesis, and folliculogenesis. SOHLH1 regulates oocyte differentiation without affecting the first meiotic division. SOHLH1-associated disorders include non-obstructive azoospermia and ovarian hypoplasia. The GO annotation for SOHLH1 includes 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 mRNA of the human SOHLH1 gene is disclosed, for example, as Genbank accession numbers NM_001012415 and XM_497082. The amino acid sequence of human SOHLH1 is disclosed, for example, as Genbank accession numbers NP_001012415 and XP_497082.

[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 Genbank accession numbers NM_001001714 and XM_130180; and the amino acid sequence of mouse SOHLH1 is disclosed, for example, as Genbank accession numbers NP_001001714 and XP_130180.

[0029] LHX8 is a member of the LIM homeobox family of proteins and is involved in the patterning and differentiation of various tissues. In addition to a DNA-binding homeodomain, LIM homeobox family proteins contain two tandemly repeated 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 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, under Genbank's "Gene ID: 431707." The nucleotide sequence of the mRNA of the human LHX8 gene is disclosed, for example, under Genbank's accession numbers NM_001001933, XM_086344, NM_001256114, XM_017001316, and XM_017001317. The amino acid sequence of human LHX8 is disclosed, for example, under Genbank's accession numbers NP_001001933, XP_086344, NP_001243043, XP_016856805, and XP_016856806.

[0031] 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 Genbank accession numbers NM_010713, XM_006501072, and XM_017319470. The amino acid sequence of mouse LHX8 is disclosed, for example, as Genbank accession numbers NP_034843, XP_006501135, and XP_017174959.

[0032] The SUB1 gene encodes a transcriptional regulator. SUB1 functions in cooperation with TAFs as a coactivator, mediating functional interactions between upstream activators and general transcriptional functions. SUB1-associated diseases include onychomycosis. GO annotations for SUB1 include single-stranded 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 mRNA of the human SUB1 gene is disclosed, for example, as Genbank accession numbers NM_006713, XM_017008986, XM_017008987, and XM_011513944. The amino acid sequence of human SUB1 is disclosed, for example, as Genbank accession numbers NP_006704, XP_016864475, XP_016864476, and XP_011512246.

[0034] 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 Genbank accession numbers NM_011294 and XM_006520042. The amino acid sequence of mouse SUB1 is disclosed, for example, as Genbank accession numbers NP_035424 and XP_006520105.

[0035] STAT3 is a member of the STAT protein family. 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 inhibitory factor (LIF), and bone morphogenetic protein 2 (BMP2), STAT proteins are phosphorylated by receptor-associated kinases (RISK) and subsequently form homo- or heterodimers. These RISK-associated STATs then translocate to the nucleus, where they act as transcriptional activators, playing an important role in many cellular processes, including cell proliferation and apoptosis. STAT3-associated diseases include childhood-onset multisystemic autoimmune disease and autosomal dominant hyper-IgE syndrome. The GO annotation for STAT3 includes 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 mRNA of the human STAT3 gene is disclosed, for example, under Genbank accession numbers NM_001369512, NM_001369513, NM_001369514, NM_001369516, NM_001369517, NM_001369518, NM_001369519, NM_001369520, NM_003150, NM_0031511, NM_0031512, NM_0031513, NM_0031514, NM_0031515, NM_0031516, NM_0031517, NM_0031518, NM_0031519, NM_0031519, NM_0031520, NM_0031521, NM_0031522, NM_0031523, NM_0031524, NM_0031525, NM_0031526, NM_0031527, NM_0031528, NM_0031529, NM_0031530, NM_0031540, NM_0031555, NM_0031560, NM_0031570, NM_0031580, NM_0031590, NM_00315910, NM_00315921, NM_00315930, NM_00 Disclosed as M_139276, NM_213662, XM_017024973, XM_011525146, XM_011525145, XM_017024972, XM_005257617, XM_005257616, XM_017024975, XM_024450896, XM_017024974, XM_017024976 The amino acid sequence of human STAT3 is listed in Genbank under accession numbers NP_001356441, NP_001356442, NP_001356443, NP_001356445, NP_001356446, NP_001356447, NP_001356448, NP_001356449, NP_003141, and NP_003148 05, NP_998827, XP_016880462, XP_011523448, XP_011523447, XP_016880461, XP_005257674, XP_005257673, XP_016880464, XP_024306664, XP_016880463, 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 mRNA of the mouse STAT3 gene is disclosed, for example, as Genbank accession numbers NM_011486, NM_213659, NM_213660, XM_011248846, and XM_017314401. The amino acid sequence of mouse STAT3 is disclosed, for example, as Genbank accession numbers NP_035616, NP_998824, NP_998825, XP_011247148, and XP_017169890.

[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 retinitis pigmentosa. The 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 mRNA of the human TBPL2 gene is disclosed, for example, as Genbank accession number NM_199047. The amino acid sequence of human TBPL2 is disclosed, for example, as Genbank accession number NP_950248.

[0040] 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 Genbank accession numbers NM_001289689 and NM_199059. The amino acid sequence of mouse TBPL2 is disclosed, for example, as Genbank accession numbers NP_001276618 and NP_951014.

[0041] The DYNLL1 gene encodes a light chain protein that constitutes the cytoplasmic dynein enzyme complex, which has a molecular weight of approximately 1200 kDa. DYNLL1-associated diseases include chronic intestinal venous insufficiency. The GO annotation for DYNLL1 includes protein homodimerization activity and protein domain-specific binding. The key 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 mRNA of the human DYNLL1 gene is disclosed, for example, as Genbank accession numbers NM_001037494, NM_001037495, and NM_003746. The amino acid sequence of human DYNLL1 is disclosed, for example, as Genbank accession numbers NP_001032583, NP_001032584, and NP_003737.

[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 Genbank accession number NM_019682. The amino acid sequence of mouse DYNLL1 is disclosed, for example, as Genbank accession number NP_062656.

[0044] [Cells capable of differentiating into oocytes] The cells capable of differentiating into oocytes used in the method for inducing immature oocytes of 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) As used herein, "pluripotent stem cells" refer to undifferentiated cells that possess both the "self-renewal ability" (ability to proliferate while maintaining an undifferentiated state) and the "pluripotency" (ability to differentiate into all three germ layer lineages). Examples of pluripotent stem cells include, but are not limited to, 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 of GS (germline stem) cells from testicular tissue, and Muse cells isolated from bone marrow mesenchymal cells. ES cells may also be those derived from somatic cells by nuclear reprogramming. The pluripotent stem cells listed above can be obtained by known methods.

[0046] As used herein, "iPS cells" refer to differentiated somatic cells that can be reprogrammed into cells of various tissues or organs by introducing several genes into them. The iPS cells used in the method for inducing immature oocytes of this embodiment may be derived from primary cultured somatic cells collected from an appropriate donor, or may be derived from an established cell line. Because iPS cells can be induced to differentiate into any germ layer, somatic cells used to prepare iPS cells may, in principle, be derived from either ectodermal or endodermal germ layer cells. Cells from sources such as skin, hair, gums, and blood, which are less invasive and easier to collect, are suitable somatic cells for use in preparing iPS cells. Methods known in the art may be used to prepare iPS cells. Specifically, preparation methods can be used that are described in known publications 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).

[0047] The ES cells used in the method for inducing immature oocytes of this embodiment can be obtained by known methods. For example, they can be established by collecting an inner cell mass from a blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on feeder cells derived from fibroblasts. Alternatively, ES cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei can also be used. Furthermore, as shown in the Examples below, ES cells can be maintained and cultured in a serum-free medium supplemented with 2i (2 inhibitor; PD0325901 and CHIR99021) and LIF (leukemia inhibitory factor) without the use of feeder cells (Reference 3: Ying QL et al., "The ground state of embryonic stem cell self-renewal," Nature, Vol. 453, No. 7194, pp. 519-523, 2008).

[0048] (epiblast-like cells) As used herein, "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 similar to epiblasts (tissues that differentiate into primordial germ cells in vivo). Methods for inducing differentiation of EpiLCs from pluripotent stem cells (iPS cells or ES cells) can be performed with reference to known methods, such as those described in JP-A-2013-538038 (Reference 4) and 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, as used herein, the term "primordial germ cells" refers to cells that are destined to differentiate into germ cells and ultimately differentiate into eggs or sperm through meiosis. Primordial germ cells may be derived from a living organism, or may be primordial germ cell-like cells (PGCLCs) that have been induced to differentiate from pluripotent stem cells. When recovering primordial germ cells from a living organism, they can be recovered together with the gonads from, for example, female mouse fetuses (11.5 to 12.5 days old). When recovering gonads from a living organism, they may be recovered together with the mesonephros, or the mesonephros may be isolated and recovered.

[0050] As mentioned above, "primordial germ cells" also 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 performed by known methods, such as those described in Reference 5 above.

[0051] When PGCLCs derived from pluripotent stem cells are used as primordial germ cells, it is preferable to remove undifferentiated cells from a differentiation-induced pluripotent stem cell population in advance. Such methods are known. For example, by introducing a nucleic acid encoding a fusion protein combining Blimp1, a primordial germ cell marker gene, with a reporter protein into pluripotent stem cells, PGCLCs induced to differentiate from pluripotent stem cells and undifferentiated cells can be easily separated by fluorescence-activated cell sorting (FACS) or other methods.

[0052] The term "primordial germ cells" also includes cells in which the genes of primordial germ cells derived from living organisms or primordial germ cell-like cells derived from pluripotent stem cells have been modified using genetic engineering techniques. Methods for modifying the genes of primordial germ cells derived from living organisms or primordial germ cell-like cells derived from pluripotent stem cells include 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, to introduce the desired nucleic acid, vector, etc. Examples of methods for introducing nucleic acids, vectors, etc. include microinjection, electroporation, lipofection, and nucleic acid introduction methods using viral vectors. Furthermore, the method for introducing an exogenous gene or exogenous nucleic acid fragment is not limited to the methods listed above, as long as the genetically modified primordial germ cells can be differentiated into functional oocytes by the method of this embodiment. Genetic modification of primordial germ cells can be performed at an appropriate time during the culture period of the primordial germ cells. For example, in mice, this can be performed between days 11.5 and 12.5 of age. When using primordial germ cell-like cells derived from pluripotent stem cells, the pluripotent stem cells can be genetically modified by known methods before being induced to differentiate into primordial germ cell-like cells.

[0053] 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 proliferation ability.

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

[0055] [Introduction process] In the method for inducing immature oocytes of this embodiment, the oocyte formation 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 formation gene, its transcript, mRNA, or its expressed protein may be introduced. As the mRNA of the oocyte formation gene and its expressed protein, the mRNA consisting of the base sequence and the expressed protein consisting of the amino acid sequence indicated by the above GenBank accession number can be used.

[0057] The introduction method is not particularly limited and can be appropriately selected depending on the target cells and the type of material to be introduced (such as nucleic acid or protein).

[0058] The method for introducing the oocyte formation gene into cells is not particularly limited, and any known method can be appropriately selected and used, such as lipofection, microinjection, DEAE-dextran, gene gun, electroporation, calcium phosphate, etc.

[0059] The method for introducing mRNA of an oocyte formation gene into cells is not particularly limited, and any known method can be appropriately selected and used.Specific examples include methods using commercially available RNA transfection reagents such as Lipofectamine (registered trademark) MessengerMAX (manufactured by Life Technologies).

[0060] The method for introducing the expressed protein of the oocyte formation gene into cells is not particularly limited, and any known method can be appropriately selected and used, such as a method using a protein introduction reagent, a method using a protein transduction domain (PTD) fusion protein, or microinjection.

[0061] The oocyte formation 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 transiently expressed, or the oocyte formation gene may be integrated into the chromosome of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells. Of these, integration of the oocyte formation gene into the chromosome of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells is preferred, as it allows stable gene expression.

[0062] When an oocyte formation gene is 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, an expression vector containing the nucleotide sequence of the oocyte formation gene and a promoter that controls the expression of the nucleotide sequence of the oocyte formation gene can be used. In the expression vector, the nucleotide sequence of the oocyte formation gene is operably linked to the promoter. Furthermore, among the above eight oocyte formation genes, all of the genes to be used may be incorporated into a single expression vector, or each of the genes may be incorporated into a different vector. However, from the viewpoint of introduction efficiency, it is preferable to incorporate all of the genes to be used into a single expression vector.

[0063] The promoter is not particularly limited, and may be one that has activity in target cells, or may be an expression-inducible promoter whose activity can be induced by a drug or the like.

[0064] Examples of promoters 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.

[0065] Examples of expression-inducible promoters include the doxycycline-inducible promoter (TetO promoter), whose promoter activity can be artificially controlled.

[0066] In addition to the nucleotide sequence and promoter of the oocyte formation gene, the expression vector may optionally contain an enhancer, a poly(A) addition signal, a marker gene, a replication origin, a gene encoding a protein that binds to the replication origin and controls replication, and the like. The term "marker gene" refers to a gene that enables cell sorting or selection by introducing the marker gene into cells. Specific examples of marker genes include drug resistance genes, fluorescent protein genes, luciferase genes, and chromogenic enzyme genes. These may be used alone or in combination. Specific examples of drug resistance genes include puromycin resistance genes, geneticin resistance genes, neomycin resistance genes, tetracycline resistance genes, kanamycin resistance genes, zeocin 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 luciferase genes include luciferase genes. Specific examples of the chromogenic enzyme gene include the β-galactosidase gene, the β-glucuronidase gene, and the alkaline phosphatase gene.

[0067] The expression vector into which the oocyte formation gene is incorporated is not particularly limited, and any known expression vector can be used, including, for example, a plasmid vector and a viral vector.

[0068] The plasmid vector is not particularly limited as long as it can be expressed in at least one cell type selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells. For example, a plasmid vector 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 retroviral (including lentiviral) vectors, adenoviral vectors, adeno-associated viral vectors, Sendai viral vectors, herpes viral vectors, vaccinia viral vectors, pox viral vectors, polio viral vectors, Silvis viral vectors, rhabdoviral vectors, paramyxoviral vectors, and orthomyxoviral vectors.

[0070] Among these, a plasmid vector is preferred as the expression vector.

[0071] Oocyte formation genes can be integrated into chromosomes using known knock-in systems, such as those that involve cleaving 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, or those that use a transposon vector system.

[0072] The donor vector contains nucleotide sequences flanking the target region as homology arms. The donor vector can also contain the nucleotide sequence of an oocyte formation gene (hereinafter sometimes referred to as a "knock-in sequence") between the 5' and 3' arms. Furthermore, it is preferable to set the target region within the safe harbor region to ensure stable expression of the oocyte formation gene.

[0073] The donor vector may be a circular DNA vector (e.g., a plasmid vector) or a linear DNA vector. The donor vector may contain other sequences in addition to the homology arms and knock-in sequence. Examples of other sequences include a marker gene, a replication origin, and a gene encoding a protein that binds to the replication origin and controls replication. Examples of marker genes include those described above.

[0074] The method for introducing the donor vector into cells 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, gene gun, electroporation, and calcium phosphate.

[0075] In the transposon vector system, as shown in the examples below, a transposon vector incorporating the base sequence of an oocyte formation gene is introduced into a cell, and then transposase is applied to easily integrate the gene into the chromosome of the cell. Furthermore, by applying transposase again, the base sequence of the oocyte formation gene integrated into the chromosome can be excised from the chromosome and removed without leaving any trace. Examples of transposons include piggyBac (registered trademark), Sleeping Beauty, Tol II, and mariner.

[0076] The method for inducing immature oocytes of this embodiment may include any optional step in addition to the introduction step. Examples of the optional step include a step of growing cells (growth step), a step of selecting cells into which the introduced oocyte formation gene, or its transcript or its expressed protein, has been introduced (selection step), and a step of culturing the cells after the introduction step in a state in which the oocyte formation gene is expressed or the expressed protein of the oocyte formation gene is present in the cells (culture step). Furthermore, when an oocyte formation gene is introduced in the introduction step, the method may include a step of inducing expression of the introduced oocyte formation gene (expression induction step).

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

[0078] In the proliferation step, for example, 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. The proliferation medium may be any known medium for culturing ES cells, iPS cells, EpiLCs, primordial germ cells, etc., but is not limited thereto. Any medium suitable for culturing ES cells, iPS cells, EpiLCs, and primordial germ cells may be used. Specific examples of proliferation media include serum-free media supplemented with 2i (2 inhibitor; PD0325901 and CHIR99021) and LIF (leukemia inhibitory factor), as shown in the Examples below.

[0079] The culture conditions in the proliferation step can be 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 can be about 30°C or higher and about 37°C or lower. The culture period is not particularly limited in the case of mice, but can be, for example, about 1 day to about 10 days, or about 3 days to about 7 days. Furthermore, those skilled in the art can appropriately determine a preferable culture period depending on the animal species from which the cells are derived.

[0080] The proliferation step may be performed before or after the introduction step. If the proliferation step is performed after the introduction step, the expression of the oocyte formation gene or the presence of an expressed protein of the oocyte formation gene will cause 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, thereby terminating proliferation. Therefore, as described in the expression induction step described below, if the expression of the oocyte formation gene is controlled so as 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 the oocyte formation gene is introduced in the form of a transient expression vector, or when a transcript or expressed protein of the oocyte formation gene is introduced, it is preferable to carry out a proliferation step before the above-mentioned introduction step.

[0082] [Selection process] In the selection step, cells into which the oocyte formation gene, its transcript, or its expressed protein has been introduced are selected.

[0083] In the selection step, for example, a reporter gene can be used to select cells into which an oocyte formation gene, or its transcript or its expressed protein, has been introduced. Specifically, for example, when an oocyte formation gene is introduced into cells in the form of an expression vector, the reporter gene can be included in the expression vector, allowing the reporter gene to be expressed in the cells together with or independently of the expression of the oocyte formation gene, thereby enabling cell selection. When an oocyte formation gene is integrated into the chromosome of at least one cell type selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, a construct in which a reporter gene is operably linked upstream or downstream of the oocyte formation gene can be integrated, allowing the reporter gene to be expressed together with or independently of the expression of the oocyte formation gene, thereby enabling cell selection. When introducing a transcript of an oocyte formation gene, a construct in which a transcript of a reporter gene is functionally linked upstream or downstream of the transcript of the oocyte formation gene is introduced into cells, thereby expressing the reporter gene along with the expression of the oocyte formation gene, and allowing cells to be selected. When introducing an expression protein of an oocyte formation gene into cells, a fusion protein of the expression protein of the oocyte formation gene and the expression protein of the reporter gene is introduced into cells, allowing cells to be selected. As the reporter gene, those exemplified as marker genes in the explanation of the above "introduction step" can be used.

[0084] [Expression induction step] In the above-mentioned introduction step, when the expression of the oocyte formation gene is controlled so as to be induced by the presence of an expression inducer, the expression of the oocyte formation gene is induced by adding the expression inducer to the culture medium. When at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells is induced to differentiate into immature oocytes, cell proliferation stops. Therefore, in order to obtain a larger number of immature oocytes, it is preferable to perform a proliferation step before the expression induction step. That is, when the oocyte formation gene is introduced 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 the oocyte formation gene is integrated 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 step, proliferation step, and expression induction step in this order.

[0085] For example, the expression of an oocyte formation gene can be induced by introducing the oocyte formation gene into cells in a form operably linked to an inducible promoter (e.g., a doxycycline-inducible promoter (TetO promoter)) and adding an expression inducer (e.g., doxycycline) to the culture medium to express the oocyte formation gene. Alternatively, as shown in the Examples below, a method using the ProteoTuner (registered trademark) system (manufactured by Clontech) can be used. Specifically, by introducing into cells a construct in which a sequence encoding a destabilizing domain (DD, 12 kDa) is operably linked upstream or downstream of the oocyte formation gene, the fusion protein expressed by the construct is rapidly degraded by the proteasome in the absence of an expression inducer. On the other hand, by adding to the culture medium the low-molecular-weight compound Shield1 (membrane-permeable low-molecular-weight compound, 750 Da), which protects the oocyte formation gene from degradation by the proteasome, as an expression inducer, the oocyte formation gene can be stably expressed and accumulated in the cells.

[0086] The amount of the expression inducer to be added is not particularly limited, as long as it is a concentration that results in the desired expression level of the oocyte formation gene. For example, if the expression inducer is doxycycline, the concentration in the medium can be, for example, about 1 nM to 10 μM. If the expression inducer is Shield1, the concentration in the medium can be, for example, about 10 nM to 10 μM.

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

[0088] [Culture process] In the culturing step, the cells after the introduction step are cultured in a state in which the oocyte formation gene is expressed or the expressed protein of the oocyte formation gene is present in the cells.

[0089] The culture conditions 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 about 30°C to 37°C. The culture period can be about 1 day to 10 days, or about 3 days to 7 days, in the case of mice.

[0090] The medium used in the culture step can be any of the mediums exemplified as the growth medium in the growth step. When the expression of the oocyte formation gene is controlled so as to be induced by the presence of an expression inducer, the expression inducer is added to the medium, and the cells are cultured in a state in which the oocyte formation gene is expressed in the cells.

[0091] In a preferred embodiment, the method for inducing immature oocytes includes controlling the expression of oocyte formation genes so that they are induced by the presence of an expression inducer, and includes the following methods 1) to 5). 1) introducing one or more oocyte formation genes selected from the group consisting of FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2, and DYNLL1 into the chromosomes of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells (particularly preferably, pluripotent stem cells); 2) Proliferating the cells after transfection; 3) Selecting cells from the multiplied cells that have the oocyte formation gene introduced into their chromosomes; 4) adding an inducer to the medium to induce the expression of oocyte formation genes in the selected cells; 5) After induction of expression, the cells are cultured in a state where the oocyte formation gene is expressed within the cells.

[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 induced to differentiate into immature oocytes within a short culture period of approximately 5 to 10 days after the introduction of an oocyte formation gene, or its transcript or its expressed protein.

[0093] The induction of 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 below, a nucleic acid encoding a fusion protein combining the oocyte marker gene Stella with a reporter protein (e.g., enhanced cyan fluorescent protein (ECFP)) is introduced into the chromosome of at least one cell type selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells. The induction of differentiation into immature oocytes can be confirmed by the fluorescence detected by the expression of Stella-ECFP. Furthermore, the expression of Stella-ECFP allows for the removal of undifferentiated cells from the differentiation-induced cell population, and the easy isolation of immature oocytes by, for example, fluorescence-activated cell sorting (FACS).

[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 transcripts or expressed proteins thereof, into at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, and co-culturing the cell after the introduction with ovarian somatic cells.

[0095] In the method for producing mature oocytes of this embodiment, the introduction of the oocyte formation gene (introduction step) is the same as the introduction step described in the above "method for inducing immature oocytes," and therefore, description thereof will be omitted.

[0096] As used herein, the term "mature oocyte" refers to an egg in the metaphase of meiosis II, also known as a secondary oocyte. As will be described in the Examples below, mature oocytes express the Npm2 gene, which is a marker for activated oocytes.

[0097] [Agglomerate formation process] The method for producing mature oocytes of 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 above "Method for inducing oocytes," and ovarian somatic cells to form an aggregate (aggregate formation step).

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

[0099] The somatic cells can be collected from the ovaries of a living organism according to the method described in Reference 5 above.

[0100] Specifically, for example, somatic cells can be collected from ovaries by surgically removing ovaries from a living organism and dissociating the somatic cells that make up the ovaries using trypsin treatment or the like. It is preferable to remove germ cells present in the ovaries derived from the living organism at this time. Methods for removing germ cells present in the ovaries can be performed using known methods, for example, magnetic activated cell sorting using anti-SSEA1 antibodies or anti-CD31 antibodies. Here, fetal-derived ovaries are preferred for collecting ovarian somatic cells. In the case of mice, for example, gonads (ovaries) from mouse fetuses at 12.5 days of fetal age (also referred to as "embryonic age") can be used. Furthermore, a person skilled in the art can select gonads (ovaries) of a suitable age depending on the animal species from which the ovaries are derived, based on the present disclosure and common general technical knowledge in the relevant technical field.

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

[0102] A method for preparing aggregates consisting of at least one cell type selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step and ovarian somatic cells can be performed, for example, as described in the Examples below. The method involves mixing, aggregating, and culturing at least one cell type 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, Life Technologies) supplemented with 10% fetal calf serum (FCS), 150 μM ascorbic acid, 1× Glutamax, 1× penicillin / streptomycin, and 55 μM mercaptoethanol. If the expression of an oocyte formation gene is regulated so that it is induced by the presence of an expression inducer, the expression inducer is added to the medium and the cells are cultured under conditions in which the oocyte formation gene is expressed. It is preferable to use a low-adhesion culture dish (e.g., a low-cell-adhesion U-bottom 96-well plate) for the culture.

[0103] In the case of mice, for example, the culture period for producing aggregates can be about 2 to 3 days, preferably 2 days. Furthermore, those skilled in the art can appropriately determine a preferable culture period depending on the animal species from which the cells are derived.

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

[0105] Furthermore, the at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, ovarian somatic cells, or ovaries containing ovarian somatic cells after the introduction step can also be cryopreserved and used. Cryopreservation methods can be known. For example, cryopreservation of the at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, or ovarian somatic cells after the introduction step can be performed by slow freezing using a 10% DMSO solution or a commercially available freezing agent (e.g., Cellbanker (registered trademark)).

[0106] The ovarian somatic cells used in the method for producing mature oocytes of this embodiment may be derived from the same mammalian species as the at least one cell type selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells that constitute the aggregates, or may be derived from a different mammalian species, but it is preferable to use cells derived from the same mammalian species. Examples of mammalian cells include those exemplified in the description of the cells capable of differentiating into oocytes above.

[0107] The method for producing a mature oocyte of this embodiment may include an optional step after the introduction step and before the aggregate formation step. Examples of the optional step include the proliferation step and selection step described in the above "Method for inducing immature oocytes."

[0108] [Culture process] Furthermore, the method for producing a mature oocyte of this embodiment can include a culture step after the aggregate formation step.

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

[0110] Culture conditions can be those known for culturing aggregates. Specifically, for example, the culture temperature can be about 30°C or higher and 37°C or lower. In the case of mice, the culture period can be about 7 days or higher and 35 days or lower, or about 8 days or higher and 30 days or lower. Furthermore, those skilled in the art can appropriately determine a preferable culture period depending on the animal species from which the cells are derived.

[0111] The medium used in the culture step can be any of the mediums exemplified as the growth medium in the growth step. When the expression of the oocyte formation gene is controlled so as to be induced by the presence of an expression inducer, the expression inducer is added to the medium, and the cells are cultured in a state in which the oocyte formation gene is expressed in the cells.

[0112] The aggregates after the culture process have a secondary follicle structure, with the oocytes surrounded by multilayered granulosa cells, and the theca folic, which surrounds the multilayered granulosa cells, is further formed. The theca cells that make up the inside of the theca (theca interna) express luteinizing hormone receptors, and 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 of obtaining mature oocytes can be divided into a growth step and a maturation step.

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

[0115] Culture conditions can be those described in Non-Patent Document 1. Specifically, for example, the culture temperature can be about 30°C or higher and 37°C or lower. In the case of mice, the culture period can be about 7 days or higher and 15 days or lower, or about 8 days or higher and 13 days or lower. Furthermore, those skilled in the art can appropriately determine a preferable culture period depending on the animal species from which the cells are derived.

[0116] The growth medium may have the composition described in Non-Patent Document 1. Specifically, in the case of mice, for example, for two days after the start of culture, follicles are cultured using α-MEM containing 5% fetal calf serum (FCS), 2% polyvinylpyrrolidone (Sigma), 150 μM ascorbic acid, 1× GlutaMAX, 1× penicillin / streptomycin, 100 μM 2-mercaptoethanol, 55 μg / mL sodium pyruvate (Nacalai Tesque), 0.1 IU / mL follicle-stimulating hormone (Follistim (registered trademark), MSD), 15 ng / mL BMP15 (Bone morphogenetic protein 15), and 15 ng / mL GDF9 (Growth differentiation factor 9) (R&D Systems). On the second day after the start of culture, the medium was replaced with α-MEM minus BMP15 and GDF9, and the follicles were incubated in 0.1% Type IV collagenase (MP Biomedicals). After washing several times with α-MEM containing 5% FCS, the follicles were cultured in α-MEM minus BMP15 and GDF9 until the 11th day after the start of culture.

[0117] After the growth process, the follicle has an antral follicle structure and forms a cumulus oocyte complex containing an egg at the germinal vesicle stage.

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

[0119] Culture conditions can be those described in Non-Patent Document 1. Specifically, for example, the culture temperature can be about 30°C or higher and 37°C or lower. In the case of mice, the culture period can be about 7 days or higher and 15 days or lower, or about 8 days or higher and 13 days or lower. Furthermore, those skilled in the art can appropriately determine a preferable culture period depending on the animal species from which the cells are derived.

[0120] The maturation medium may have the composition described in Non-Patent Document 1. Specifically, for mice, an example of such a medium is 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).

[0121] After the maturation process, the follicle matures into an egg in the metaphase of the second meiotic division (secondary oocyte). Whether an egg is in the metaphase of the second meiotic division (secondary oocyte) can be evaluated, for example, by visual observation using a microscope or the like, using the release of the first polar body as an indicator.

[0122] 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 infertility treatment using the mature oocytes obtained by the above-mentioned method. Furthermore, mature oocytes obtained using the method for producing mature oocytes of this embodiment are suitable for efficient breeding of industrial animals and propagation of rare animals. That is, in one embodiment, the present invention provides a method for breeding industrial animals or a method for propagating rare animals, using mature oocytes obtained by the above-mentioned method. The animals to which the present invention is applied are preferably mammals, and examples of mammals include those exemplified above. Furthermore, mature oocytes obtained using the method for producing mature oocytes of this embodiment can be used to investigate the causes of infertility and to elucidate the mechanisms of menopausal disorders. [Example]

[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, pp. 416-420, 2017). The PB-CAG-DD vector was then inserted into the 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). Next, cDNAs for eight genes (FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2, and DYNLL1) were amplified from ovarian cDNA of 13.5-day-old female mice and cloned into the PB-CAG-DD vector using the Infusion HD Cloning Kit (Takara Bio Inc.). Each cDNA was amplified by PCR using KOD Fx Neo or KOD Plus Neo DNA polymerase (Toyobo) according to the manufacturer's protocol.

[0125] (Vector transfection) ES cells were maintained in serum-free medium supplemented with 2i and LIF without feeder cells (see Reference 3). To monitor differentiation into immature and mature oocytes, we used mouse ES cells (BVSCNmC) containing a chromosomal insertion of a gene encoding mVenus (membrane-targeted Venus), a variant of yellow fluorescent protein (YFP) under the control of Blimp1, a key germline determinant; a gene encoding enhanced cyan fluorescent protein (ECFP) under the control of Stella, a germ cell and oocyte marker; and a gene encoding mCherry (membrane-targeted Cherry) under the control of Nucleoplasmin 2 (Npm2), a marker for activated (mature) oocytes. The constructed PB-CAG-DD vector containing the eight genes was co-transfected with the 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 × 10 ES cells were transferred to a low-attachment U-bottom 96-well plate containing S10 medium (StemPro®-34 SFM, Life Technologies) supplemented with 10% fetal calf serum (FCS), 150 μM ascorbic acid, 1× Glutamax, 1× penicillin / streptomycin, and 55 μM mercaptoethanol, mixed with 0.5 μM Shield1 (Clontech) (hereafter referred to as "oocyte differentiation induction medium"). The cells were then cultured for 5 days to induce differentiation into immature oocytes. The results of observation under a confocal microscope (Carl Zeiss, model number: Zeiss LSM 700) are shown in Figure 1. In Figure 1, "oocyte formation genes" refers to eight genes: FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2, and DYNLL1. "Oocyte formation gene expression OFF" refers to cells before the addition of medium containing Shield1, and "oocyte formation gene expression ON" refers to cells after the addition of medium containing Shield1 and culturing for 5 days.

[0127] As shown in Figure 1, cells in which the expression of oocyte formation genes was OFF formed colonies, and the fluorescence of ECFP (Stella-ECFP), which is expressed under the control of Stella expression, was hardly observed. In contrast, cells in which the expression of oocyte formation genes was ON existed separately, and strong fluorescence of Stella-ECFP was detected, suggesting that they had been induced to differentiate into immature oocytes.

[0128] (Mature oocyte production) Next, after transfection, single colony ES cells (1 × 10 cells) were cultured for 5 days and selected with puromycin. These cells were mixed with 3 × 10 cells from ovarian somatic cells derived from 12.5-day-old female mice in the oocyte differentiation-inducing medium to form aggregates, which were then cultured for 2 days. The ovarian somatic cells were isolated from ovaries previously removed from 12.5-day-old female mice using a method described in Non-Patent Document 1. The aggregates were then transferred onto a Transwell-COL membrane (Coaster) and cultured for 28 days in the oocyte differentiation-inducing medium. The expression of each marker in the aggregates on days 2, 6, 8, 10, and 12 after the start of culture was observed under a confocal microscope (Carl Zeiss, model number: Zeiss LSM 700). The upper panel of Figure 2 visualizes oocytes using the fluorescence of Stella-ECFP, a germ cell and oocyte marker. In the bottom panel, oocytes were visualized by the fluorescence of the red fluorescent protein mCherry (Npm2-mCherry) expressed under the expression control of Nucleoplasmin 2 (Npm2), a marker for activated (mature) oocytes.

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

[0130] Furthermore, on day 28 after the start of culture, individual follicles were manually isolated using a sharpened tungsten needle. The isolated follicles had a secondary follicle structure. The follicles were cultured under the culture conditions for the "in vitro growth period" and "in vitro maturation period" described in Non-Patent Document 1, and were allowed to mature into antral follicles and then into metaphase II meiotic oocytes.

[0131] [Example 2] Using mouse iPS cells, we investigated the induction of differentiation into oocytes, similar to that of ES cells.

[0132] (Vector transfection) The iPS cells used were mouse BVSC iPS cells generated using the Virus Buster method established in Non-Patent Document 1. The PB-CAG-DD vector containing the five genes constructed in Example 1 was co-transfected into mouse BVSC iPS cells using Lipofectamine 2000 along with the hyperactive PBase (hypBase) plasmid. 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) The 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] (Mature oocyte production) Next, after transfection, iPS cells (1 × 10 cells) were cultured for 5 days and selected with puromycin. These cells were mixed with 3 × 10 cells from ovarian somatic cells derived from 12.5-day-old female mice in the oocyte differentiation-inducing medium described above to form aggregates, which were then cultured for 2 days. The aggregates were then transferred onto a Transwell-COL membrane (Coaster) and cultured in the oocyte differentiation-inducing medium described above for 21 days. The expression of Stella-ECFP in the aggregates on day 21 of culture was observed under a confocal microscope (Carl Zeiss, model number: Zeiss LSM 700). 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.

[0135] As shown in Figure 3, Stella-ECFP fluorescence was observed in the cells on day 21 after the start of culture. This confirmed that iPS cells could be induced to differentiate into oocytes, just like ES cells.

[0136] [Example 3] (Identification of key factors in oocyte formation genes) To identify genes that are important factors in oocyte formation, a total of 26 types of vectors were constructed using the same method as in Example 1, with the gene combinations shown in the left diagram of Figure 4. Next, using the same method as in Example 1, mouse ES cells (Blimp1-mVenus:Stella-ECFP:Npm2-mCherry (BVSCNmC)) were transfected with each vector. The transfected ES cells were cultured for 5 days to induce differentiation into immature oocytes using the same method as in Example 1. Next, single-cell-derived ES cells (1 × 10 cells) that had been cultured for 5 days and selected with puromycin after transfection were mixed with 3 × 10 cells of ovarian somatic cells from a 12.5-day-old female mouse in the oocyte differentiation-inducing medium described above to prepare aggregates, which were then cultured for 2 days. The resulting aggregates were then cultured for 21 days using the same method as in Example 1. The number of oocytes formed from each cell line after culture was counted. Furthermore, the area of the oocyte was calculated from the fluorescent area of the blue fluorescent protein CFP, which was expressed downstream of the Stella gene. These results are shown in Figure 4.

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

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

Claims

1. A method for inducing immature oocytes, comprising introducing four types of 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.

2. 2. The method for inducing immature oocytes according to claim 1, comprising introducing four types of genes consisting of FIGLA, NOBOX, LHX8 and TBPL2 into said cells.

3. The method for inducing immature oocytes according to claim 1 or 2, further comprising introducing a STAT3 gene, or a transcript or expressed protein thereof, into the cells.

4. The method for inducing immature oocytes according to any one of claims 1 to 3, further comprising introducing into the cells one or more genes selected from the group consisting of SOHLH1, SUB1 and DYNLL1, or transcripts or expressed proteins thereof.

5. The method for inducing immature oocytes according to claim 4, further comprising introducing three types of genes consisting of SOHLH1, SUB1 and DYNLL1 into the cells.

6. The method for inducing immature oocytes according to any one of claims 1 to 5, wherein the cells are pluripotent stem cells.

7. The expression of the gene is controlled so as to be induced by the presence of an expression inducer, allowing the cells to grow after said introduction; The method for inducing immature oocytes according to any one of claims 1 to 6, further comprising adding the expression inducer to the culture medium after the proliferation to induce expression of the gene.

8. Introducing four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2, or transcripts or expressed proteins thereof, into at least one cell selected from the group consisting of pluripotent stem cells, epiblast-like cells, and primordial germ cells; co-culturing the cells after the introduction with ovarian somatic cells; A method for producing a mature oocyte, comprising:

9. The method for producing a mature oocyte according to claim 8, further comprising introducing a STAT3 gene, or a transcript or expressed protein thereof, into the cell.

Images