In vitro culture method for inducing follicles from embryonic ovarian cells of primates

Abstract

The present invention provides a method for producing oocytes or follicles, or a reconstructed ovary comprising oocytes or follicles, the method comprising a step of culturing a reconstructed ovary of a primate under suspension culture conditions.

Description

Technical Field

[0001] The present invention relates to a method for inducing ovarian follicles in vitro. More specifically, it relates to a method for producing oocytes or ovarian follicles, or Those A method for reconstructing an ovary comprising an oocyte or an ovarian follicle, the method comprising the step of culturing the reconstructed ovary under suspension culture conditions. Background Art

[0002] Germ cells differentiate into eggs or sperm, which combine to form a fully developed fertilized egg, thus ensuring the persistence and diversity of genetic information across generations. Disorders of germ cell development lead to serious consequences, including infertility and genetic or epigenetic diseases in offspring. Therefore, understanding the mechanisms of germ cell development is a fundamental topic in both biology and medicine.

[0003] In mammals, germ cells appear as primordial germ cells (PGC) in the early stage of embryogenesis.PGC begins epigenetic reprogramming, and after migration, forms colonies in embryonic gonads.In embryonic gonads, PGC is called oogonia in females and gonocytes in males, and shows the same genetic and epigenetic characteristics until obvious sexual differentiation begins.In females, in response to signals from embryonic ovaries, particularly pre-granular cells (pre-granular cells, pre-granular membrane cells), oogonia differentiate into oocytes in early meiotic prophase accompanied by rapid migration, and the oocytes that have ended the first meiotic prophase are covered by a single layer of granular cells (granular cells, granular membrane cells), forming the primordial follicles that act as the source of oogenesis. In contrast, in males, in response to signals from the embryonic testis, particularly the Sertoli cells, the sex globular cells differentiate into pro-spermatogonia and then into spermatogonia / spermatogonial stem cells (SSCs) that function as a source of spermatogenesis. The mechanisms of mammalian germ cell development have been primarily studied using mice as a model organism, and through insights gained from such studies and advances in pluripotent stem cells (PSCs) and reproductive technology, the entire germ cell development process in both females and males in mice has been reconstructed in vitro using mouse PSCs (mPSCs) as starting material.

[0004] For example, it has been reported that primordial germ cell-like cells (PGCLCs) produced by differentiation induction of mouse pluripotent stem cells and embryonic ovarian somatic cells are aggregated to produce a reconstructed ovary, and the reconstructed ovary is cultured using a gas-liquid interface culture method to induce follicles (Non-Patent Document 1). It has also been reported that follicles are induced by culturing embryonic ovarian tissue using a gas-liquid interface culture method (Non-Patent Document 2).

[0005] In addition, the technology of making reconstructed ovaries in mice can also be applied to human PGCLC. The inventors have previously successfully constructed heterogeneous reconstructed ovaries by agglutinating human PGCLC and mouse embryonic ovarian somatic cells, and cultured the reconstructed ovaries for about 3 months using a gas-liquid interface culture method, thereby differentiating them into early oocyte-like cells at the beginning of the first meiotic division (non-patent literature 3). However, the method of culturing the same type of reconstructed ovaries of primates including humans for a long time is unknown, and the in vitro culture method of inducing cells derived from primate embryonic ovaries to follicles (primordial follicles / primary follicles) as more mature structures does not exist yet.

[0006] Prior art literature

[0007] Non-patent literature

[0008] Non-patent document 1: Hayashi K, et al., Nat Protoc. 12(9): 1733-1744 (2017),

[0009] Non-patent document 2: Morohaku K, et al., Nat Protoc. 12(9): 1817-1829 (2017),

[0010] Non-patent literature 3: Yamashiro C, et al., Science. 362(6412): 356-360 (2018). Summary of the invention

[0011] Problems to be solved by the invention

[0012] Therefore, the present invention aims to provide a method for maintaining a reconstructed ovary of a primate including a human for a long period of time (e.g., 7 weeks or more, 9 weeks or more, preferably 12 weeks or more) in an in vitro culture environment. In addition, the present invention aims to provide a method for reproducing the progress of meiosis and follicle formation in the reconstructed ovary by culturing the reconstructed ovary for a long period of time.

[0013] Means for solving problems

[0014] The inventors have attempted to determine the conditions under which the reconstructed ovaries of primates, including humans, can be cultured in vitro for a long time. First, using cynomolgus monkeys as a primate model, after collecting the embryonic ovaries of cynomolgus monkeys, the tissue was lysed with enzymes, etc., and then the cells were (long-term) frozen and stored. After the cells were thawed, a reconstructed ovary (a cell aggregate) was made. The cynomolgus monkey reconstructed ovary (cy reconstructed ovary) was cultured using the gas-liquid interface culture method as a previous method. As a result, as the culture progressed, the reconstructed ovary gradually collapsed, and it was found difficult to maintain its morphology. That is, it is clear that the previous gas-liquid interface culture method is not suitable for long-term in vitro culture of cy reconstructed ovaries. It is believed that this difference between mice and cynomolgus monkeys reflects the difference in the biophysical properties of mouse ovarian cells and primate ovarian cells.

[0015] Therefore, the conditions suitable for long-term culture of primate reconstructed ovaries were verified. After in-depth research, the inventors believed that the main advantage of the gas-liquid interface culture method is that it improves ventilation and oxygen supply to the cultured tissue, but in the ovarian culture of mammals, ventilation and oxygen supply are not very important. Therefore, the gas-liquid interface culture method was not improved, but a completely different culture method was adopted. Specifically, the inventors paid attention to the suspension culture method, and proved that the reconstructed ovary of the crab-eating monkey was actually cultured by the method, so that the reconstructed ovary was maintained for a long time of more than 12 weeks, and by culture, the mitotic oogonia in the reconstructed ovary differentiated into oocytes, completed the first meiotic prophase, and the reconstructed ovary contained a primitive follicle-like structure. The analysis of the expression / intracellular localization of important markers in these cells or the analysis of transcriptome and other analysis methods was used, and the results confirmed that the primitive follicle-like structure obtained by the above-mentioned culture has a property very close to the primitive follicle in the organism (In vivo). In addition, it was found that the in vitro culture method can also be applied to human embryonic ovarian cells. Based on these insights, the inventors further studied repeatedly and completed the present invention.

[0016] That is, the present invention is as follows.

[0017] [1] A method for producing an oocyte or an ovarian follicle, or a reconstructed ovary containing an oocyte or an ovarian follicle, the method comprising the step of culturing a primate reconstructed ovary under suspension culture conditions.

[0018] [2] The method according to [1], wherein the reconstructed ovary is constructed by agglutinating primate germ cells and primate embryonic ovarian somatic cells.

[0019] [3] The method according to [2], wherein the germ cells and embryonic ovarian somatic cells are prepared by dissociation from primate embryonic ovaries by enzyme treatment.

[0020] [4] The method according to [2] or [3], wherein the germ cell is at least one selected from the group consisting of an oogonia, a primordial germ cell, and a primordial germ cell-like cell.

[0021] [5-1] The method according to any one of [2] to [4], wherein at least one of the germ cells and the ovarian somatic cells is derived from pluripotent stem cells.

[0022] [5-2] The method according to [5-1], wherein the pluripotent stem cells are artificial pluripotent stem cells.

[0023] [6-1] The method according to any one of [1] to [5-2], wherein the culture is performed under suspension culture conditions for 9 weeks or more.

[0024] [6-2] The method according to any one of [1] to [5-2], wherein the culture is performed under suspension culture conditions for 12 weeks or more.

[0025] [7-1] The method according to any one of [1] to [6-2], wherein the culture under suspension culture conditions is culture in a medium containing L-glutamine, ascorbic acid, and 2-mercaptoethanol.

[0026] [7-2] The method according to [7-1], wherein the concentration of L-glutamine in the culture medium is 10 μM to 1 M.

[0027] [7-3] The method according to [7-1] or [7-2], wherein the concentration of ascorbic acid in the culture medium is 1 μM to 1 M.

[0028] [7-4] The method according to any one of [7-1] to [7-3], wherein the concentration of 2-mercaptoethanol in the culture medium is 0.1 μM to 1 M.

[0029] [8] The method according to any one of [7-1] to [7-4], wherein the culture medium further contains retinoic acid.

[0030] [9-1] The method according to any one of [7-1] to [8], wherein the culture medium further contains one or more selected from the group consisting of progesterone, cortisol, dehydroepiandrosterone and dehydroepiandrosterone sulfate.

[0031] [9-2] The method according to [9-1], wherein the concentration of retinoic acid in the culture medium is 0.1 nM to 5 μM.

[0032] [9-3] The method according to [9-1] or [9-2], wherein the concentration of progesterone in the culture medium is 0.01 μg / mL to 10 μg / mL.

[0033] [9-4] The method according to any one of [9-1] to [9-3], wherein the concentration of cortisol in the culture medium is 0.01 μg / mL to 10 μg / mL.

[0034] [9-5] The method according to any one of [9-1] to [9-4], wherein the concentration of dehydroepiandrosterone in the culture medium is 0.1 nM to 5 μM.

[0035] [9-6] The method according to any one of [9-1] to [9-5], wherein the concentration of dehydroepiandrosterone sulfate in the culture medium is 0.1 μM to 5 mM.

[0036] [9-7] The method according to any one of [7-1] to [9-6], wherein the culture medium contains serum.

[0037] [9-8] The method according to [9-7], wherein the concentration (v / v) of serum in the culture medium is 0.01% to 50%.

[0038]

[10] The method according to any one of [1] to [9-8], wherein the primate is a human.

[0039]

[11] An oocyte, follicle or reconstructed ovary produced by the method according to any one of [1] to

[10] .

[0040]

[12] A method for producing an egg, the method comprising the step of maturing the oocyte according to

[11] .

[0041] [13-1] Oocyte or follicle, or Those A kit for manufacturing a reconstructed ovary containing oocytes or follicles, the kit comprising a culture container for suspension culture and L-glutamine, ascorbic acid and 2-mercaptoethanol.

[0042] [13-2] The kit according to [13-1], further comprising one or more molecules selected from retinoic acid, progesterone, cortisol, dehydroepiandrosterone and dehydroepiandrosterone sulfate.

[0043] [13-3] The kit according to [13-1] or [13-2], The kit Also contains serum.

[0044]

[14] The kit according to any one of [13-1] to [13-3], The kit Also contains basal medium.

[0045] [15-1] The kit according to any one of [13-1] to

[14] , The kit Also contains retinoic acid.

[0046] [15-2] The kit according to any one of [13-1] to [15-1], The kit The invention further comprises at least one selected from the group consisting of progesterone, cortisol, dehydroepiandrosterone and dehydroepiandrosterone sulfate.

[0047] Effects of the Invention

[0048] According to the present invention, a general reconstructed ovary of primates including humans can be maintained in vitro for a long time (e.g., more than 7 weeks, more than 9 weeks, preferably more than 12 weeks), and germ cells such as primordial germ cells, primordial germ cells, and oogonia can be matured into cells in the meiotic stage, and primordial follicle-like structures can be induced. By adopting this method or the primordial follicle-like structures induced by this method, the differentiation process of primate female germ cells can be observed in vitro, which is useful for understanding the occurrence mechanism of germ cells. Therefore, according to the present invention, an important basis can be provided for both biology and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] [ Figure 1] Ovarian development of cynomolgus monkey embryos. (A) Scheme of female germ cell development in cynomolgus monkeys (cy). wpf: weeks after fertilization, PGC: primordial germ cell. (B) Macroscopic view of the developing cy ovaries at 8-18wpf. Scale bar = 1mm. (C) Hematoxylin and eosin (H&E) staining of cy embryonic ovaries at 8wpf. Arrows indicate oogonia. M: mesonephros. Scale bar = 100μm (top), 40μm (bottom). (D) H&E staining of the ovarian cortex of cy embryos at 10-18wpf. The ovarian cortex is divided into the outer cortex and the inner cortex by a line bisecting the ovarian cortex. Black arrow: oocyte with a large nucleus with condensed filamentous chromatin. White arrow: primordial follicle. Scale bar = 50μm. (E) Immunofluorescence (IF) analysis of oogonial markers (POU5F1, TFAP2C), RA-responsive markers (ZGLP1, STRA8), meiotic markers (γH2AX, SYCP1), and oogenesis markers (ZP3, TP63) in cy embryonic ovarian development. Representative images of each marker are shown. Germ cells are labeled with DDX4. Nuclear DAPI staining is indicated in white. The enlarged image in the upper right corner of each figure shows the expression of important markers co-stained with DDX4. The enlarged image in the lower right corner of each figure shows the expression of DDX4 co-stained with DAPI (white). The number written in the upper left corner indicates the stage of the embryo (wpf). Scale bar = 20μm. (F) Representative images of germ cells obtained by immunostaining SYCP3, DMC1, and DDX4 at each meiotic substage. Nuclear DAPI staining is indicated in white. Meiotic subphases were defined based on the staining patterns of SYCP3, DMC1, and DAPI as described in Materials and Methods. Scale bar = 10 μm. (G) Figure 1 The percentage of germ cells in each meiotic substage as defined in F. The average percentage of the lateral or medial ovarian cortex in each meiotic substage was calculated from ovaries of at least 3 embryos. Colors are shown in the figure.

[0050] [ Figure 2] Histological analysis of cy embryonic ovaries in vivo and in xenotransplanted reconstructed ovaries. (A) Dimensions of developing cy embryos and ovaries at 8-18wpf. The line shows the LOESS curve consistent with the sample population. (B and C) IF of cy embryonic gonads at 8wpf (B) and ovarian cortex of cy embryos at 10-18wpf (C). The results of staining with FOXL2 (granulosa cell marker), DDX4 (germ cell marker) and DAPI (nucleus, white) are shown. The ovarian cortex at 10-18wpf is divided into the outer cortex and the inner cortex by the line that bisects the ovarian cortex. Scale bar = 40μm (B) and 50μm (C). (D) The granulosa membrane / germ cell ratio at each stage of development evaluated by IF data is shown. (E) IF of cell proliferation markers (Ki67), apoptosis markers (truncated PARP, truncated CASPASE 3), extracellular matrix markers (LAMININ), and oogenesis markers in cy embryonic ovarian development (FIGLA, NOBOX, NLRP5). Representative images of each marker are shown. Germ cells were labeled with DDX4. Nuclear DAPI staining is represented by white. The enlarged image in the upper right corner of each figure shows the expression of important markers co-stained with DDX4 except NLRP5. The enlarged image in the lower right corner of each figure shows the expression of DDX4 co-stained with DAPI (white). The number written in the upper left corner indicates the stage of the embryo (wpf). Scale bar = 20μm. (F) DDX4 of IF of the outer cortex / endocortex from cy embryonic ovaries at 8-18wpf + Percentage of cells positive for each marker in germ cells. Mean values ​​from more than 3 biological replicates are shown. (G) H&E staining of transplanted cy-reconstructed ovaries at 3 / 6 / 9 / 12 / 15 weeks post-transplantation (w-ptp). Dashed lines indicate cy-reconstructed ovaries transplanted under the renal capsule of KSN / Slc mice. K: Kidney of mouse. Scale bar = 200 μm.

[0051] [ Figure 3 ] Induction of embryonic gonadal cells into cy follicles by xenotransplantation. (A) Xenotransplantation experimental scheme. Cy reconstructed ovaries made from in vivo ovarian cells at 8wpf were transplanted under the renal capsule of immunodeficient KSN / Slc mice (see <Materials and Methods>). (B) Macroscopic appearance (a), H&E staining (b) and IF for FOXL2 / DDX4 / DAPI (c) of the transplanted cy reconstructed ovaries at 3 / 6 / 9 / 12 / 15 weeks after transplantation (w-ptp). FOXL2 was observed in the reconstructed ovaries at 12w-ptp and 15w-ptp. +Primordial follicle-like complex surrounded by granulosa cells. Arrows indicate reconstructed ovaries. FOXL2: granulosa cell marker, DDX4: germ cell marker. Scale bar = 1 mm (a), 40 μm (b and c). (C) IF of oogonia markers (POU5F1, TFAP2C), meiotic stage markers (SYCP3, SYCP1) and oogenesis markers (ZP3, TP63) in transplanted cy-reconstructed ovaries. Germ cells and nuclei were labeled with DDX4 and DAPI (white), respectively. The enlarged image in the upper right corner of each figure shows the expression of each marker co-stained with DDX4. The enlarged image in the lower right corner of each figure shows the expression of DDX4 co-stained with DAPI (white). The number written in the upper left corner indicates the age in weeks after transplantation (w-ptp). Scale bar = 20 μm. (D) DDX4 from IF of cy-reconstructed ovaries at 3 to 15 w-ptp. + Percentage of cells positive for each marker in germ cells. Mean values ​​are shown together with SD. (E) H&E staining of transplanted cy-reconstructed ovaries at 21 w-ptp. Dashed lines indicate cy-reconstructed ovaries transplanted under the renal capsule of KSN / Slc mice. K: Kidney of mouse. Scale bars = 200 μm (left) and 100 μm (right).

[0052] [ Figure 4 ] In vitro culture system for inducing follicles from embryonic gonadal cells. (A) Scheme of in vitro culture system for inducing cy follicles. Cy reconstructed ovaries made from embryonic gonadal cells at 8wpf were cultured under either gas-liquid interface or suspension conditions (see <Materials and Methods>). (B) Macroscopic appearance (a), H&E staining (b, c) and IF for FOXL2 / DAZL / DAPI (d) of cy reconstructed ovaries cultured under gas-liquid interface conditions at 3 and 6 weeks of in vitro culture (w-ivc). FOXL2: granulosa cell marker, DAZL: germ cell marker, DAPI: nucleus. Scale bars = 200μm (a), 100μm (b) and 40μm (c and d). (C) Quantitative analysis of the size of cy reconstructed ovaries cultured with Advanced MEM or αMEM as the basal medium of P-IVD culture medium. The mean values ​​from more than 3 biological replicates are shown together with SD, except for the αMEM reconstructed ovaries at 15w-ivc (n=1). **P<0.01, Welch's T test. (D) Macroscopic images (a) and H&E stained images (b) of cy reconstructed ovaries cultured in P-IVD medium at 3 / 6 / 9 / 12 / 15w-ivc. Arrows indicate primordial follicle-like structures. Scale bars = 200μm (a) and 40μm (b). (E) Expression of cy in cultured cy reconstructed ovaries. Figure 1IF analysis of important markers described in E. Germ cells and nuclei were labeled with DDX4 and DAPI (white), respectively. The enlarged image in the upper right corner of each figure shows the expression of important markers co-stained with DDX4. The enlarged image in the lower right corner of each figure shows the expression of DDX4 co-stained with DAPI (white). The number written in the upper left corner indicates the age of IVC (w-ivc). Scale bar = 20μm. (F) DDX4 from IF of cy-reconstructed ovaries at 3w-ivc to 15w-ivc. + The percentage of cells positive for each marker in germ cells. The mean value is shown together with the SD. (G) Figure 1 The percentage of germ cells in each meiotic substage as defined in F (see also <Materials and Methods>). The average percentage of each meiotic substage was determined from at least 3 cy reconstructed ovaries.

[0053] [ Figure 5 ]Establishment of a new in vitro culture system for primate oocyte differentiation. (A) (Left) Summary table of coating conditions tested for cy-reconstructed ovaries. (Right) Representative images of cy-reconstructed ovaries cultured on membranes at 12w-ivc. The dotted circles indicate the edge of the cy-reconstructed ovary at the starting point of gas-liquid interface culture. Scale bar = 500μm. (B) Macroscopic appearance (a), H&E staining (b), and IF for FOXL2 / DDX4 / DAPI (c) of cy-reconstructed ovaries cultured in 13 different basal media at 6w-ivc. Reconstructed ovaries cultured in Advanced MEM maintained their size, and DDX4 was found within the reconstructed ovaries. + Germ cells and FOXL2 + Granulosa cells. Scale bars = 500 μm (a) and 40 μm (b and c). (C) Cy reconstructed ovaries in culture Figure 2 E IF analysis of important markers recorded. Germ cells and nuclei were labeled with DDX4 and DAPI (white), respectively. The enlarged image in the upper right corner of each figure shows the expression of important markers co-stained with DDX4 except NLRP5. The enlarged image in the lower right corner of each figure shows the expression of DDX4 co-stained with DAPI (white). The number written in the upper left corner indicates the age of IVC (w-ivc). Scale bar = 20μm. (D) IF analysis of FOXL2 (granulosa cells) / DDX4 (germ cells) stained with DAPI (nuclei) in cy-reconstructed ovaries cultured at 9w-ivc. Scale bar = 40μm. (E) Representative images of human reconstructed ovaries cultured on Transwell-COL membranes at 0 and 7w-ivc. Scale bar = 200μm.

[0054] [ Figure 6] Single-cell RNA-seq analysis of cy ovarian cells in vivo and in reconstructed ovaries. (AJ) 10X scRNA-seq-based analysis of cy germ cells. (A) Uniform manifold approximation and projection (UMAP) plot of in vivo cy embryonic ovarian cells colored by 5 major clusters assigned computationally based on the expression of cell-type-specific markers. Colors are shown in the figure. (B) Figure 6 UMAP graph shown in A. Cells at each embryonic stage are represented in black. (C) Overlay Figure 6Expression profiles of important marker genes for germ cells (DDX4), granulosa cells (WT1), interstitial cells (TCF21), endothelial cells (PECAM1), and blood cells (TYROBP) in cytostomy on UMAP of A. (D) UMAP of germ cells in cytostomy represented by each cluster of meiotic substages. Color distinction is as shown in the figure. M: mitosis, PL: preleptotene, L: leptotene, Z: zygotene, P: pachytene, D: diplotene stage, U: unclassified. (E) UMAP of germ cells in cytostomy colored according to embryonic stage. Color distinction is as shown in the figure. (F) Gene expression profiles of oogonia markers (POU5F1, TFAP2C), RA responsive markers (ZGLP1, STRA8), meiotic markers (SYCP3, SYCP1), oogenesis markers (ZP3), and germ cell markers (DDX4) in germ cell development in cytostomy. (G) UMAP of cy in vivo germ cells colored by pseudotime. The color division of pseudotime is shown in the figure. (H) (Left) Normalized expression heatmap of highly variable genes (HVG; 1481 genes) in cy in vivo germ cell subphases sorted by unsupervised hierarchical clustering (UHC). Eight gene clusters were defined based on the UHC dendrogram. Important genes for female germ cell development are represented in black, and 10 genes temporally upregulated in the PL cluster (TMSB4X, PTRF, BAMBI, RND3, SLC47A1, RASD1, CD24, PLAT, CCL3, and GCNT2) are represented in gray. (Right) Representative genes and enrichment of important gene ontologies (GO) are shown. (I) Heatmap of Pearson correlation coefficients of average expression levels of 1481 HVGs in cy in vivo germ cell meiotic subphases. Asterisks indicate unclassified clusters. Color division is shown in the figure. (J) (Left) UMAP of germ cells derived from cy in vivo embryonic ovaries and in vitro cultured cy reconstructed ovaries, shown together with meiotic substages. Color differentiation is as shown in the figure. (Right) UMAP of in vitro cultured cy reconstructed ovary-derived cells with emphasis on 12w-ivc and 15w-ivc. (K and L) Analysis of cy germ cells based on SC3-seq. (K) UHC of cy germ cells of in vivo and reconstructed ovaries with all expressed genes (19747 genes) and heat map of expression levels of selected marker genes. The colored bars below the dendrogram indicate the in vivo stage (1st bar), age of xenotransplantation (2nd bar), age of IVC (3rd bar) and cell type (4th bar). The color differentiation of the heat map is as shown in the figure. The color differentiation of the bars is as shown Figure 6L. (L) Principal component analysis (PCA) of cy germ cells in vivo and reconstructed ovaries based on SC3-seq. Cells are plotted on a two-dimensional plane defined by the values ​​of PC1 and PC2. Color differentiation is shown in the figure.

[0055] [ Figure 7 ]10X scRNA-seq analysis of oogenesis in cy females in vivo. (A) Average number of genes detected in the five major cell types of ovarian embryos in cy at 8-18 wpf. (B) Figure 6 Percentage of germ cells in meiotic prophase substages as defined and shown in D. Colors are as indicated. M: mitosis, PL: pre-leptotene, L: leptotene, Z: zygotene, P: pachytene, D: diplotene. (C) Number of genes, UMI counts, and percentage of mitochondrial genes detected in each germ cell substage. The number of genes and UMI counts were significantly lower in “Unclassified (U)” compared to other germ cell clusters. **P<0.01, Tukey-Kramer test. (D) Figure 6 Characteristic map of marker genes important for oocyte development on the UMAP map shown in D. (E) (left) Figure 6 Normalized expression heatmap of HVGs listed in gene cluster #2 of H. Three gene clusters (Cluster #2-1 / -2 / -3) were defined based on the UHC dendrogram. (Right) Representative genes and GO enrichment are shown.

[0056] [ Figure 8 ]10X scRNA-seq analysis of cy granulosa cells in vivo and in reconstructed ovaries. (A and B) Using Figure 6 UMAP (A) and PAGA plots (B) of RNA velocity in granulosa cells in vivo as defined in A. Granular cells were divided into 12 subclusters according to Louvain clustering. Colors are shown in the figure. (C) Figure 8 In the UMAP diagram shown in A, cells at each embryonic stage are represented in black. (D) (Left) Normalized expression heat map of HVG (712 genes) between subclusters of in vivo cy granulosa cells sorted by UHC. Eight gene clusters were defined based on the UHC dendrogram. (Right) Representative genes and major GO enrichment are shown. (E) Figure 8 Characteristic map of marker genes important for cy granulosa cell development on the UMAP map shown in A. (F) (Left) UMAP map of granulosa cells from cy in vivo embryonic ovaries and in vitro cultured cy reconstructed ovaries represented by sub-clustering of granulosa membranes. Color differentiation is shown in the figure. (Right) UMAP map of cy reconstructed ovarian derived cells cultured in vitro at 12w-ivc and 15w-ivc is emphasized. (G) Figure 8Heat map of the average expression levels of granulosa cell markers, BMP signaling pathway (BMPR2 and ID1 / 2 / 3 / 4) and NOTCH signaling pathway (NOTCH2 / 3, HES1 and HEY1 / 2 / L) genes in sub-clusters #5-12 of granulosa cells defined in F. The percentage of cells from the cultured reconstructed ovaries (% IVC) within each sub-cluster is shown. Color differentiation is as shown.

[0057] [ Fig. 9 ] In vitro induction of human follicles from human oogonia. (A) Macroscopic view (a), H&E staining (b) and IF for FOXL2 / DDX4 / DAPI (c) of human in vivo female gonads (11W, left column) and in vitro cultured reconstructed ovaries at 7w-ivc and 14w-ivc. FOXL2: granulosa cell marker, DDX4: germ cell marker, DAPI: nucleus. Scale bars = 1 mm (a, left), 200 μm (a, center / right), 50 μm (b) and 20 μm (c). ga: gestational age. (B and C) IF of oogonia markers (POU5F1), cell proliferation markers (Ki67), meiotic markers (SYCP3, DMC1, γH2AX, SYCP1) and oogenesis markers (ZP3, TP63) associated with human female gonads in vivo (11W, B) and human in vitro cultured reconstructed ovaries at 14w-ivc (C). Germ cells and nuclei were labeled with DDX4 and DAPI (white), respectively. The enlarged images in the upper right corner of each figure show the expression of important markers co-stained with DDX4. The enlarged images in the lower right corner of each figure show the expression of DDX4 co-stained with DAPI (white). Scale bar = 20μm. (D) DDX4 from IF of human female gonads in vivo (11W) and human cultured reconstructed ovaries at 7w-ivc and 14w-ivc. + Percentage of cells positive for each marker in germ cells. Averages from 2 biological replicates are shown. (E and F) Analysis of human germ cells based on SC3-seq. (E) Heat map of UHC and expression levels of selected marker genes for human germ cells in vivo and reconstructed ovaries using all expressed genes (20,048 genes). The colored bars below the dendrogram represent the in vivo stage (1st bar), the age of IVC (2nd bar) and the cell type (3rd bar). The color division of the heat map is as shown. The color division of the bars is as shown Fig. 9F. (F) PCA of human germ cells in vivo and reconstructed ovaries based on SC3-seq. Cells are plotted on a two-dimensional plane defined by the values ​​of PC1 and PC2. Color differentiation is shown in the figure. (G) PCA diagram of all cy and human SC3-seq data merged based on canonical correlation analysis (CCA). Cell types are defined after CCA and subsequent Louvain clustering. The encoding of colors and traits is shown in the figure.

[0058] [ Fig.10 ] Interspecies comparative analysis of germ cell development in vivo in female embryos in humans, monkeys, and mice. (AF) Analysis based on 10X scRNA-seq. (A) UMAP plots of germ cells from 6 10X scRNA-seq datasets based on CCA integration (2 for humans [this example and (Chitiashvili T, et al., Nat Cell Biol, 22: 1436-1446 (2020))], 1 for monkeys [this example], and 3 for mice [(Ge W, et al., Cell Mol Life Sci, 78: 695-713 (2021), Niu W, and Spradling AC, Proc Natl Acad Sci USA, 117: 20015-20026 (2020), Zhao ZH, et al., FASEB J, 34: 12634-12645 (2020))]). Meiotic substages were defined after CCA and subsequent Louvain clustering. Colors represent computationally assigned meiotic substages defined based on expression levels of important meiotic markers conserved in all three species (see <Materials and Methods>). Color differentiation is as shown in the figure. M: mitosis, PL: pre-leptotene, L: leptotene, Z: zygotene, P: pachytene, D: diplotene. (B) UMAP plots highlighting cells from each dataset. Color differentiation of each stage of occurrence is shown in the figure. (C) The percentage of germ cells belonging to each meiotic substage. Color differentiation of the germ cell cycle is as shown. Fig.10 As shown in A. (D) The distribution of germ cells along species-specific pseudotime trajectories calculated using Monocle alone. The color distinction of the germ cell cycle is as follows Fig.10As shown in A. (E) Heat map of Pearson correlation coefficients of the average expression levels of 237 HVGs (1-1-1 orthologs) during meiotic subphases in humans, monkeys, and mice (see <Materials and Methods>). The color differentiation of the germ cell cycle is shown in the figure. (F) Normalized expression level heat map of selected genes using 10XscRNA-seq data of all 3 species. Genes that show specific expression changes in the development of female germ cells in humans and monkeys, but not in mice, were manually selected from the list of DEGs obtained from SC3-seq analysis of reconstructed ovaries cultured in vitro by cy / human. The color differentiation of the heat map is shown in the figure. The color differentiation of the germ cell subphases is as shown. Fig.10 E. (G and H) IF analysis of PAX6 and CLGN using TFAP2C (oogonia marker) and DDX4 (germ cell marker) in cy in vivo embryonic ovaries at 12wpf (G) and human cultured reconstructed ovaries at 7w-ivc (H). Nuclear DAPI staining is indicated in white. Scale bar = 20μm. (I) IF analysis of CLGN using TP63 (oogenesis marker) and DDX4 (germ cell marker) in oocytes of 13-year-old human primordial follicles. Nuclear DAPI staining is indicated in white. Scale bar = 20μm.

[0059] [ Fig.11 ] Interspecific analysis using 10X scRNA-seq datasets for in vivo embryonic female germ cell occurrence in humans, monkeys, and mice. (A) UMAP plots of embryonic ovarian cells in each 10X scRNA-seq dataset, color-coded by 5 major clusters assigned by computation based on the expression of cell type-specific markers. (B) Number of genes (nGene), UMI counts (nUMI), and percentage of mitochondrial genes (%Mt) detected in each germ cell meiotic subphase of 6 10X scRNA-seq datasets. M: mitosis, PL: pre-leptotene, L: leptotene, Z: zygotene, P: pachytene, D: diplotene. (C) The percentage of average expression of gene clusters (1-1-1 orthologous, species-specific, and other genes) in embryonic oocyte occurrence of 3 animal species is shown together with SD. Color differentiation is shown in the figure.

[0060] [ Fig.12] Interspecies comparative analysis of oocyte development in humans, monkeys, and mice. (A) Normalized heat map of expression levels of selected genes in all three species based on 10X scRNA-seq analysis. Genes showing conserved expression patterns in female germ cell development in all three animal species were manually selected from the list of DEGs obtained from SC3-seq analysis of in vitro cultured reconstructed ovaries of cy / human. Color differentiation is shown in the figure. (B) Gene expression level box plot of selected markers showing primate-specific expression dynamics in embryonic germ cell development (see also Fig.10 F). (C) IF analysis of LEIOMODIN-3 (LMOD3) using TP63 (oogenesis marker) and DDX4 (germ cell marker) in oocytes of cy in vivo embryos at 18wpf (top) and oocytes of 13-year-old human primordial follicles (bottom). Nuclear DAPI staining is represented in white. Scale bar = 20μm. (D) Autosomal: A and X: A ratios (top) and XIST / Xist expression migration (bottom) in female germ cell development in vivo in humans (this example) and mice (Ge W, et al., Cell Mol Life Sci, 78: 695-713 (2021), Zhao ZH, et al., FASEB J, 34: 12634-12645 (2020)) based on 10XscRNA-seq analysis.

[0061] [ Fig.13] X:A ratio and XIST expression in germ cell development in humans, monkeys and mice. (A) Chr.3:A and X:A ratios (top) and Xist expression (bottom) in mouse embryonic / neonatal gonadal cells based on Bulk RNA-seq analysis. Points: individual data, bars: average. The E9.5 / 10.5 samples contain germ cells from male and female embryos (mixed). E: embryonic days, P: postnatal days, Soma: gonadal cells. (B) Chr.15:A and X:A ratios (top) and XIST expression (bottom) in cy female germ cells of reconstructed ovaries in vivo and cultured / transplanted based on SC3-seq analysis. The migration of the Chr.15:A / X:A ratio in the embryonic system and the hypoblast system reported in (Okamoto I, et al., Science, 374:eabd8887 (2021)) is also shown. ESC: embryonic stem cell, ICM: inner cell mass, Pre_EPI: preimplantation epiblast, PostE_EPI: post-implantation early epiblast, PostL_EPI: post-implantation late epiblast, Gast: gastrulating cell, ePGC: early primordial germ cell. Data with small sample size (n≤3) are represented by points (each data) and bars (mean). (C) Chr.10:A and X:A ratios (top) and XIST expression (bottom) in human female germ cells of reconstructed ovaries in vivo and cultured based on SC3-seq analysis. (D) Autosomal:A and X:A ratios (top) and XIST / Xist expression migration (bottom) in female germ cell development in vivo in all three species based on 10X scRNA-seq analysis.

[0062] [ Fig.14 ] Histological images of reconstructed ovaries at week 9 of in vitro culture. The formation of primordial follicles (arrows) was seen only in the steroid hormone-added group. FOXL2: granulosa cell marker, POU5F1: oogonia marker, DDX4: germ cell marker, DAPI: nucleus.

[0063] [ Fig.15 ] Histological images of reconstructed ovaries at 9 weeks of in vitro culture. The formation of primordial follicles (arrows) was seen in the steroid hormone-added group. ZP3 and TP63: oocyte markers of the stage of follicle formation, DDX4: germ cell marker.

[0064] [ Fig.16]Tissue image of the reconstructed ovary at the 6th week of in vitro culture (6w-ivc). In the RA-added group, the SYCP3 / DMC1 positive rate was high at any concentration, promoting the transition to meiosis. SYCP3 and DMC1: meiotic stage markers, DDX4: germ cell marker.

[0065] [ Fig.17 ] Histological images of reconstructed ovaries at 9 weeks of in vitro culture (9w-ivc). In the RA-added group, the AP2γ positivity rate was low at any concentration, promoting the transition to meiosis. FOXL2: granulosa cell marker, AP2γ: oogonia marker, DDX4: germ cell marker.

[0066] [ Fig.18 ]Tissue images of reconstructed ovaries at week 12 of in vitro culture. Compared with the non-added group (a), a large number of primordial follicles (ZP3 / TP63 positive) were produced in the steroid hormone alone group (b) and retinoic acid (RA) alone group (c). In the group with simultaneous addition of steroid hormones and RA (d), more primordial follicles were produced. ZP3 and TP63: Oocyte markers of the stage that constitutes the follicle, DDX4: Germ cell marker, DAPI: Nucleus. DETAILED DESCRIPTION

[0067] 1. Method for preparing oocytes or ovarian follicles, or reconstructed ovaries containing them

[0068] As shown in the following examples, the present inventors have confirmed that by culturing the reconstructed ovary (re-constituted ovary) (also called re-aggregated ovary) of primates under suspension culture conditions, a reconstructed ovary containing a primordial follicle-like structure having properties very close to those of the primordial follicles in vivo can be produced. Therefore, in this specification, unless otherwise specified, the (primordial) follicle-like structure is also included in the "(primordial) follicle". The follicle is a structure having an oocyte surrounded by granulosa cells (also including "pre-granulosa cells". The same below). Oocytes can also be produced by the above-mentioned culture. Granulosa cells are somatic cells surrounding the oocyte, and the flat pre-granulosa cells are cells expressing FOXL2, which differentiate into cubic granulosa cells by activation. In addition, hereinafter, unless otherwise specified, the simple term "reconstructed ovary" refers to an ovary provided for culture under suspension culture conditions (in other words, an ovary as the starting material for culture).

[0069] Therefore, the present invention provides a method for producing oocytes or follicles, or a reconstructed ovary containing oocytes or follicles (hereinafter sometimes referred to as "the method of the present invention"), which includes the step of culturing a primate reconstructed ovary under suspension culture conditions (also referred to as "suspension culture"). The oocytes and follicles obtained by the production method of the present invention can also be separated and recovered from the obtained reconstructed ovary by a method known per se.

[0070] In this specification, "suspension culture conditions" refer to conditions under which cells or cell aggregates are maintained suspended in the culture medium, that is, conditions under which strong cell-substratum junctions are not formed between cells or cell aggregates and the culture container or the extracellular matrix coating the culture container.

[0071] The medium in the suspension culture condition can be prepared by adding a medium additive to a basal medium as needed. Examples of the basal medium include RPMI-1640 medium, Eagle's MEM (EMEM), Dulbecco's modified MEM, Glasgow's MEM (GMEM), α-MEM, 199 medium, IMDM, DMEM, Hybridoma Serumfree medium, KnockOut TM DMEM(KO DMEM), Advanced TMCulture medium (e.g. Advanced MEM, Advanced RPMI, Advanced DMEM / F-12), Chemically Defined Hybridoma Serum Free Medium, Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, DMEM / F-12, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's Medium (e.g. Waymouth's MB752 / 1), CMRL medium (e.g. CMRL-1066), Williams's medium E, Brinster's BMOC-3 Medium, E8 Medium, StemPro 34. MesenPRO RS (all from Thermo Fisher Scientific), ReproFF2, Primate ES Cell Medium, ReproStem (all from ReproCell Co., Ltd.), ProculAD (ROHTO Pharmaceutical Co., Ltd.), MSCBM-CD, MSCGM-CD (all from Lonza), EX-CELL302 medium (SAFC) or EX-CELL-CD-CHO (SAFC), ReproMed TM iPSC Medium (ReproCell Co., Ltd.), Cellartis MSCXeno-Free Culture Medium (TakaraBio Co., Ltd.), TESR-E8 (Veritas Co., Ltd.), StemFit (registered trademark) AK02N, AK03N (Ajinomoto Co., Ltd.) and mixtures thereof, but not limited thereto. Among them, Advanced MEM is preferred. The medium composition of Advanced MEM disclosed on the HP of Thermo Fisher Scientific (https: / / www.thermofisher.com / jp / ja / home / technical-resources / media-formulation.39.html) is shown in Table 1 (translated into Chinese).

[0072] [Table 1-1]

[0073]

[0074] [Table 1-2]

[0075]

[0076] In addition, physiologically active substances and nutritional factors required for cell survival or proliferation can be added to the culture medium as needed. These culture medium additives can be added to the culture medium in advance or added during cell culture. The method of adding during culture can utilize any form such as one solution or a mixed solution of more than two solutions, and can also be continuous or intermittent addition.

[0077] Physiologically active substances include insulin, IGF-1, transferrin, albumin, coenzyme Q10, various cytokines (interleukins (IL-2, IL-7, IL-15, etc.), stem cell factor (SCF), activin, etc.), various hormones, various growth factors (leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), TGF-β, etc.). Nutritional factors include sugars, amino acids, vitamins, pyruvates (e.g., sodium pyruvate), hydrolyzates, or lipids. Sugars include glucose, mannose, fructose, etc., and one or more of them can be used in combination. As amino acids, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, non-essential amino acids (NEAA), etc. can be listed, and one kind or a combination of two or more kinds can be used. As vitamins, ascorbic acid, retinol, retinoic acid, nicotinic acid, biotin, pyridoxine, vitamin B12, D-pantothenic acid, choline, folic acid, myo-inositol, nicotinamide, pyridoxal, riboflavin, thiamine, cyanocobalamin, DL-α-tocopherol, etc. can be listed, and one kind or a combination of two or more kinds can be used. Examples of the hydrolyzate include hydrolyzates obtained by hydrolyzing soybeans, wheat, rice, peas, corn, cottonseed, yeast extracts, etc. Examples of the lipid include cholesterol, linoleic acid, linolenic acid, and the like.

[0078] In addition, antibiotics such as kanamycin, streptomycin, penicillin or hygromycin may be added to the medium as needed. When acidic substances such as sialic acid are added to the medium, it is desirable to adjust the pH of the medium to a neutral range of pH 5 to 9, preferably pH 6 to 8, suitable for cell growth.

[0079] The culture medium used for suspension culture can be a serum-containing medium (e.g., fetal bovine serum (FBS), human serum, horse serum) or a serum-free medium. As the serum, FBS is preferred. From the viewpoint of preventing the mixing of components derived from different species of animals, it is preferred to not contain serum or to use serum derived from the same animal as the cultured cells. Here, serum-free medium refers to a medium that does not contain unregulated or unpurified serum. Serum-free medium may also contain purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0080] The medium used for suspension culture is the same as serum, and the serum substitute may or may not be included. As serum substitutes, for example, albumin, lipoprotein albumin, recombinant albumin and other albumin substitutes, plant starch, dextran, protein hydrolyzate, transferrin or other iron transporters, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol or their equivalents can be listed. As specific examples of serum substitutes, for example, serum substitutes prepared by the method described in International Publication No. 98 / 30679 or commercially available knockout Serum Replacement [KSR] (Life Technologies), Chemically-defined Lipid concentrated (Life Technologies) and L-alanine-L-glutamine dipeptide (e.g. Glutamax (Life Technologies)) can be listed. In addition, as organism-derived factors, platelet-rich plasma (PRP), culture supernatant components of human mesenchymal stem cells, etc. can be listed.

[0081] In addition, it is preferred that more than one vitamin is included in the culture medium. As preferred vitamins, ascorbic acid, retinoic acid, etc. can be listed. "Ascorbic acid" typically refers to (R)-3,4-dihydroxy-5-((S)-1,2-dihydroxyethyl)furan-2(5H)-one (Cas No: 50-81-7), but also includes "ascorbic acid salts" and "ascorbic acid precursors". In addition, "retinoic acid" typically refers to all-trans 3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexene-1-yl)-2,4,6,8-nonatetraenoic acid (Cas No: 302-79-4), but also includes "retinoic acid salts" and "retinoic acid precursors". As examples of ascorbic acid salts used in the present invention, sodium ascorbate, potassium ascorbate, calcium ascorbate, etc. can be listed, but are not limited thereto. Examples of ascorbic acid precursors used in the present invention include trisodium ascorbyl phosphate, magnesium ascorbyl phosphate (VC-PMg), ascorbyl tetrahexyldecanoate (VC-IP), trisodium ascorbyl palmitate phosphate, etc., but are not limited thereto. Examples of retinoic acid salts used in the present invention include sodium retinoic acid, potassium retinoic acid, calcium retinoic acid, etc., but are not limited thereto. Examples of retinoic acid precursors used in the present invention include β-carotene, retinol esters, retinol, retinal, etc., but are not limited thereto.

[0082] In addition, the culture medium preferably contains more than one hormone. As a preferred hormone, for example, steroid hormones can be listed. As specific steroid hormones, for example, progesterone, cortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, etc. can be listed. In addition, vitamins or hormones can also replace artificial or natural compounds with equivalent steroid structures.

[0083] In one embodiment, suspension culture is cultured in a culture medium comprising L-glutamine, ascorbic acid and 2-mercaptoethanol. As shown in the following examples, by adding retinoic acid in the culture medium, the effect of promoting differentiation from oogonia to oocytes, the effect of promoting the start and progress of meiosis can be obtained. In addition, by adding steroid hormones in the culture medium, the differentiation and maturation of oocytes can be promoted. In addition, by retinoic acid and steroid hormones, the manufacturing efficiency of primordial follicles is also shown to rise synergistically. Thus, it is preferred that the culture medium also comprises retinoic acid. In addition, the culture medium comprises more than one (preferably all kinds) selected from progesterone, cortisol, dehydroepiandrosterone and dehydroepiandrosterone sulfate. More preferably, the culture medium comprises retinoic acid and more than one (preferably all kinds) selected from progesterone, cortisol, dehydroepiandrosterone and dehydroepiandrosterone sulfate. In this specification, these additives also include substances (for example, L-alanine-L-glutamine dipeptide, etc., which are generated by decomposition in the culture medium due to cell secretions, etc.). In addition, in another embodiment, serum may also be included in the culture medium.

[0084] The concentration of L-glutamine in the culture medium is not particularly limited, for example, 10μM to 1M, preferably 100μM to 10mM, more preferably 1mM to 3mM (2mM in one embodiment). The concentration of ascorbic acid in the culture medium is not particularly limited, for example, 1μM to 1M, preferably 10μM to 500μM, more preferably 100μM to 200μM (150μM in one embodiment). The concentration of 2-mercaptoethanol in the culture medium is not particularly limited, for example, 0.1μM to 1M, preferably 1μM to 500μM, more preferably 10μM to 200μM (55μM in one embodiment). The concentration of retinoic acid in the culture medium is not particularly limited, for example, 0.1nM to 5μM, preferably 1nM to 1μM, more preferably 10nM to 300nM (especially 10nM). The concentration of retinoic acid in the culture medium can be 10nM to 1μM. The concentration of progesterone in the culture medium is 0.01 μg / mL to 10 μg / mL, preferably 0.05 μg / mL to 1 μg / mL, more preferably 0.1 μg / mL to 0.2 μg / mL (especially 0.2 μg / mL). The concentration of cortisol in the culture medium is 0.01 μg / mL to 10 μg / mL, preferably 0.05 μg / mL to 1 μg / mL, more preferably 0.1 μg / mL to 0.2 μg / mL (especially 0.1 μg / mL). The concentration of dehydroepiandrosterone in the culture medium is not particularly limited, for example, 0.1 nM to 5 μM, preferably 1 nM to 1 μM, more preferably 20 nM to 100 nM (especially 50 nM). The concentration of dehydroepiandrosterone sulfate in the culture medium is not particularly limited, for example, 0.1 nM to 5 μM, 1 nM to 1 μM, 1 nM to 10 nM. Alternatively, the concentration of dehydroepiandrosterone sulfate in the culture medium is 0.1 μM to 5 mM, preferably 1 μM to 1 mM, more preferably 1 μM to 100 μM (particularly 10 μM). The concentration (v / v) of serum such as FBS in the culture medium is not particularly limited, for example, 0.01% to 50%, preferably 0.01% to 20%, more preferably 0.01% to 10% (10% in one embodiment). They can be added to the culture medium at the same time, or they can be added to the culture medium separately with a time difference.

[0085] The culture container used when carrying out suspension culture is not particularly limited as long as it is a container that can "carry out suspension culture", and as long as it is a person skilled in the art, it can be appropriately determined. In order to be able to carry out suspension culture, these culture containers are preferably cell low adhesion or cell non-adhesion. As a culture container for cell low adhesion or cell non-adhesion, the surface of the culture container can be used without artificial treatment (for example, utilizing the coating treatment of extracellular matrix etc.) for improving the adhesion with the cell. In addition, it can be a culture container in which the processing (for example, Nunclon Sphera processing etc.) of the adsorption that hinders the extracellular matrix is ​​implemented on the container surface, or it can be a culture container that does not implement the processing. In addition, it can be a flat bottom container, it can also be a round bottom (for example: U-shaped bottom etc.) container, it can also be other forms. As the culture container, for example, a flask, a flask for tissue culture, a culture dish, a petri dish, a culture dish for tissue culture, a multi-dish, a microplate, a microplate, a microwell, a multi-plate, a multi-well plate, a chamber slide, a culture dish, a culture tube, a tray, a culture bag or a roller bottle can be listed. In addition, as a container for suspension culture, a bioreactor can be exemplified.

[0086] The culture conditions such as temperature, carbon dioxide concentration, oxygen concentration and pH value can be appropriately set based on the technology used in animal cell culture in the past. For example, the culture temperature is not particularly limited, but can be 30 to 40° C., preferably 37° C. The carbon dioxide concentration can be 1 to 10%, preferably 2 to 5%. The oxygen concentration can be 1 to 40%, preferably 1 to 20%.

[0087] In addition, mechanical stress plays an important role in maintaining the dormant state of oocytes in mouse primordial follicles. It is reported that even in in vitro culture, oocytes can be induced into a dormant state by culturing them under pressurized conditions (Nagamatsu G, et al., Sci Adv. 5 (6): eaav9960 (2019)). Thus, culture is typically carried out at 1 atmosphere, but at least part of the culture period can be carried out under reduced pressure or pressurized conditions (e.g., 0 atmosphere to 10 atmospheres, preferably 0 atmosphere to 5 atmospheres, and more preferably 1 atmosphere to 2 atmospheres). Culture under reduced pressure or pressurized conditions can be carried out, for example, using a commercially available gas pressure cell stimulation device (e.g., AGP series manufactured by Strex Co., Ltd.).

[0088] According to the culture method of the preparation method of the present invention, since the reconstructed ovary can be maintained for a long time, as long as the desired oocyte or follicle can be obtained, the culture period is not particularly limited, and can also be appropriately selected according to the type of primate from which the cells are derived. Typically, the culture period is more than 9 weeks (for example: more than 10 weeks, more than 11 weeks, more than 12 weeks, more than 13 weeks, more than 14 weeks). The culture period can also be more than 7 weeks (for example: more than 8 weeks, more than 9 weeks). The upper limit of the culture period is also not particularly limited, and is typically less than 24 weeks (for example: less than 23 weeks, less than 22 weeks, less than 21 weeks, less than 20 weeks, less than 19 weeks).

[0089] In this specification, "reconstructed ovary" refers to a structure constructed by aggregating germ cells and embryonic ovarian somatic cells, and may include cells other than germ cells and embryonic ovarian somatic cells, or may include multiple germ cells or multiple embryonic ovarian somatic cells. The "reconstructed ovary" obtained by the preparation method of the present invention is derived from the above structure, but the types of cells constituting the reconstructed ovary may be different through suspension culture.

[0090] The reconstructed ovary can typically be constructed by performing suspension culture (hereinafter sometimes referred to as "pre-culture") for a specific number of days (e.g., 1 to 3 days, preferably 2 days) in a culture medium containing germ cells and embryonic ovarian somatic cells. Thus, the preparation method of the present invention may include a step of preparing a reconstructed ovary or a step of pre-culturing germ cells and embryonic ovarian somatic cells to obtain a reconstructed ovary. The suspension culture can be performed in the same manner as the suspension culture of the preparation method of the present invention, and the type of basal culture medium used, the type of culture medium additive, the culture container or the culture conditions, etc. can all be cited from the above description.

[0091] In one embodiment, the preculture is a culture in a culture medium containing one or more (preferably all) selected from L-glutamine, sodium pyruvate, 2-mercaptoethanol and a ROCK inhibitor. In addition, in another embodiment, the culture medium may also contain serum. Among them, in the preculture, from the viewpoint of promoting cell aggregation, a ROCK inhibitor is preferably included. As the ROCK inhibitor, for example, Y-27632, Fasudil / HA1077, H-1152, Wf-536, etc. can be listed. When Y-27632 is used as a ROCK inhibitor, the concentration in the preculture culture medium is not particularly limited, for example, 0.1μM to 1mM, preferably 1μM to 100μM, and more preferably 5μM to 20μM (10μM in one embodiment).

[0092] In the pre-culture, the concentration of L-glutamine in the culture medium is not particularly limited, for example, 10μM to 1M, preferably 100μM to 10mM, more preferably 1mM to 3mM (2mM in one embodiment). The concentration of sodium pyruvate in the culture medium is not particularly limited, for example, 10μM to 100mM, preferably 100μM to 10mM, more preferably 500μM to 5mM (1mM in one embodiment). The concentration of 2-mercaptoethanol in the culture medium is not particularly limited, for example, 0.1μM to 1M, preferably 1μM to 500μM, more preferably 10μM to 200μM (100μM in one embodiment). The concentration (v / v) of serum such as FBS in the culture medium is not particularly limited, for example, 0.01% to 50%, preferably 0.01% to 20%, more preferably 0.01% to 10% (10% in one embodiment). They can be added to the culture medium at the same time, or they can be added to the culture medium with a separate time difference.

[0093] In this specification, "germ cell" refers to a cell expressing at least any one of DDX4 and DAZL, preferably a cell expressing at least DDX4. As the germ cells used in the present invention, for example, oogonia, primordial germ cells (PGC), primordial germ cell-like cells (PGCLC) and the like can be cited. "Oogonia" is also called an oogonial progenitor cell, which is a cell in the period when the primordial germ cells in the ovary repeatedly divide and proliferate, and refers to a cell expressing at least any one of NANOG, POU5F1, TFAP2C and PDPN. "Primordial germ cells" are cells with the ability to differentiate into oogonia and spermatogonia, and refer to cells expressing at least any one of INTEGRINα6, EpCAM, BLIMP1 and TFAP2C. "Primordial germ cell-like cells" refer to cells having the same characteristics as PGCs made by differentiation induction of pluripotent stem cells.

[0094] In addition, in this specification, "embryonic ovarian somatic cells" refer to cells other than reproductive cells contained in the embryonic ovary. The embryonic ovary can be any ovary as long as it contains embryonic ovarian somatic cells, and can be an ovary of an embryo of any age, and can also vary depending on the primate species from which the cells are derived, preferably an ovary of an embryo of 6 to 18 weeks of age (6 to 18 wpf) after fertilization.

[0095] Germ cells and embryonic ovarian somatic cells are specifically prepared by, for example, collecting ovaries from organisms by surgery, and dissociating the germ cells and somatic cells constituting the ovaries into single cells by enzyme (e.g., trypsin, etc.) treatment. At this time, it is also possible to separate the germ cells (embryonic ovarian germ cells) inherent in the ovaries of organisms by cell sorting methods such as FACS (fluorescence-activated cell sorting) or MACS (Magnetic activated cellsorting) methods using germ cell markers (e.g., INTEGRINα6, EpCAM, etc. as PGC-specific markers) as indicators, thereby separating embryonic ovarian germ cells and embryonic ovarian somatic cells. Alternatively, it is also possible not to dissociate embryonic ovarian germ cells and embryonic germ somatic cells, but to use them as cell groups containing these cells. These cells or cell groups can also be cryopreserved. It should be noted that in this specification, "cell" includes "cell group" unless otherwise specified. A cell group can be composed of one cell or more than two cells.

[0096] Germ cells and embryonic ovarian somatic cells can use cells produced by differentiation induction of pluripotent stem cells. "Pluripotent stem cells" refer to stem cells that can differentiate into tissues or cells with various morphologies or functions of an organism, and have the ability to differentiate into any lineage cells of the three germ layers (endoderm, mesoderm, ectoderm). As pluripotent stem cells used in the present invention, for example, artificial pluripotent stem cells (induced pluripotent stem cells: iPS cells), embryonic stem cells (embryonic stem cells: ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transplantation (nuclear transfer Embryonic stem cells: ntES cells), pluripotent germline stem cells (multipotent germline stem cells) ("mGS cells"), embryonic germ stem cells (EG cells), preferably iPS cells (more preferably human iPS cells). In the case where the above-mentioned pluripotent stem cells are ES cells or any cells derived from human embryos, the cells may be cells produced by destroying the embryo or cells produced without destroying the embryo, but from an ethical point of view, cells produced without destroying the embryo are preferred.

[0097] ES cells are stem cells with pluripotency and self-replication ability established from the inner cell mass of early embryos (e.g., blastocysts) of mammals such as humans or mice. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and then ES cell lines were also established in primates such as humans and monkeys (JA Tomson et al. (1998), Science 282: 1145-1147; JA Tomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Tomson et al. (1996), Biol. Reprod., 55: 254-259; JA Tomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165). ES cells can be established by removing the inner cell mass from the embryonic cyst of the fertilized egg of the test animal and culturing the inner cell mass on a fibroblast feeder. Alternatively, ES cells can be established using only a single blastomere of an embryo in the cleavage stage before the embryonic cyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or using an embryo that has stopped producing (Zhang X. et al. (2006), Stem Cells 24: 2669-2676.).

[0098] nt ES cells are ES cells derived from cloned embryos produced by nuclear transfer technology, and have almost the same characteristics as ES cells derived from fertilized eggs (Wakayama T. et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936; Byrne J. et al. (2007), Nature, 450: 497-502). In other words, ES cells established from the inner cell mass of the embryonic cyst derived from a cloned embryo obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell are nt ES (nuclear transfer ES) cells. In order to produce nt ES cells, a combination of nuclear transplantation technology (Cibelli JB et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology (described above) can be used (Wakayama Kiyoka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Supplement), pp. 47-52). In nuclear transplantation, the nucleus of a somatic cell can be injected into an enucleated unfertilized mammalian egg and cultured for several hours to initiate the process.

[0099] As the ES cell strain used in the present invention, any human ES cell strain may be used, and for example, various human ES cell strains established by the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Developmental Medicine, Cellartis, etc. can be used. Specifically, for example, as human ES cell strains, CHB-1 to CHB-12 strains, RUES1 strains, RUES2 strains, HUES1 to HUES28 strains, etc., distributed from ESI Bio, H1 strains, H9 strains, etc., distributed from WiCell Research, KhES-1 strains, KhES-2 strains, KhES-3 strains, KhES-4 strains, KhES-5 strains, SSES1 strains, SSES2 strains, SSES3 strains, etc., distributed from RIKEN can be cited.

[0100] iPS cells are cells obtained by introducing specific factors (nuclear reprogramming factors) into mammalian somatic cells or undifferentiated stem cells for reprogramming. Currently, there are many types of iPS cells, including human iPSCs established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872.), Nanog-iPSCs established by screening with Nanog expression as an index after the introduction of the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPSCs prepared by a method that does not include c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), and iPSCs established by introducing six factors by a virus-free method (Okita et al. Nat. Methods 2011). May; 8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.) etc. In addition, artificial pluripotent stem cells established by introducing four factors, namely OCT3 / 4, SOX2, NANOG, and LIN28, prepared by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318:1917-1920.), artificial pluripotent stem cells prepared by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451:141-146), artificial pluripotent stem cells prepared by Sakurada et al. (Japanese Patent Application Laid-Open No. 2008-307007), etc. can also be used.

[0101] In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA., et al., Nature Biotechnology, (2008) 26, No Any artificial pluripotent stem cells known in the art and described in patent publications (e.g., Japanese Patent Application Publication No. 2008-307007, Japanese Patent Application Publication No. 2008-283972, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852) can be used.

[0102] As artificial pluripotent stem cell strains, various iPSC strains established by NIH, RIKEN, Kyoto University, etc. can be used. For example, as long as they are human iPSC strains, HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain, etc. of RIKEN, 253G1 strain, 253G4 strain, 1201C1 strain, 1205D1 strain, 1210B2 strain, 1383D2 strain, 1383D6 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 648A1 strain, 1231A3 strain, FfI-01s04 strain, etc. of Kyoto University can be listed, and 1231A3 strain is preferred.

[0103] mGS cells are pluripotent stem cells derived from the testis and are cells that serve as the origin of spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various cell lines, and have the property of producing chimeric mice if transplanted into mouse embryonic blastocysts (Kanatsu-Shinohara M. et al. (2003) Biol. Reprod., 69: 612-616; Shinohara K. et al. (2004), Cell, 119: 1001-1012). They can self-replicate in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and can be obtained by repeated passage under the same culture conditions as ES cells (Takebayashi Masanori et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Supplement), pp. 41-46, Yodosha (Tokyo, Japan)).

[0104] EG cells are cells established from embryonic primordial germ cells that have the same pluripotency as ES cells. They can be established by culturing primordial germ cells in the presence of substances such as LIF, bFGF, and stem cell factor (Matsui Y. et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550-551).

[0105] Differentiation induction from pluripotent stem cells to PGCLC can be performed by a method known per se. Specifically, induction from artificial pluripotent stem cells to PGCLC can be performed by the method described in Hayashi K, et al., Cell 2011 Aug 19; 146(4): 519-32. Alternatively, the method described in WO 2017 / 002888 and WO2022 / 039279 can be used, specifically, (1) culturing pluripotent stem cells in a culture medium containing activin A and a GSK-3β inhibitor to induce early mesoderm-like cells (iMeLC), (2) culturing the iMeLC in the presence of BMP, thereby inducing differentiation into PGCLC.

[0106] As the basal medium for inducing differentiation of pluripotent stem cells into PGCLCs, the basal medium exemplified for use in the above suspension culture can be cited, but is not limited thereto. The basal medium may contain other medium additives known per se, such as those exemplified for use in the above suspension culture.

[0107] The culture medium may be a serum-containing culture medium or a serum-free culture medium (SFM). Preferably, a serum-free culture medium is used. The concentration (v / v) of serum (e.g., FBS, etc.) in the culture medium may be, for example, less than 20%, less than 5%, less than 2%, or 0% (i.e., serum-free). SFM may or may not contain any serum substitute such as KSR.

[0108] The concentration of activin A in the culture medium for inducing differentiation of pluripotent stem cells into iMeLC is, for example, 5 to 100 ng / ml, 10 to 90 ng / ml, 15 to 80 ng / ml, 20 to 70 ng / ml or 30 to 60 ng / ml. Preferably, the concentration of activin A in the culture medium is 50 ng / ml. Examples of GSK-3β inhibitors used when inducing iMeLC from pluripotent stem cells include LiCl, BIO, SB216763, GSK-3β inhibitor VII, L803-mts, CHIR99021, etc., preferably CHIR99021. The concentration of CHIR99021 in the culture medium is not particularly limited, but is preferably 0.1 μM to 50 μM. By incubating pluripotent stem cells at 10 4 ~10 5 Cells / cm 2 , preferably 2 to 6 × 10 4 cells / cm 2 The inoculation density is cultured at 30-40°C for less than 60 hours, preferably 42 hours, to induce differentiation of iMeLCs.

[0109] Examples of BMPs used to induce differentiation of iMeLC to PGCLC include BMP2, BMP4, and BMP7, preferably BMP4. The concentration of BMP in the culture medium is, for example, 50 to 100 ng / ml, 100 to 800 ng / ml, or 150 to 600 ng / ml. Preferably, the concentration of BMP in the culture medium is 200 ng / ml. iMeLC is cultured at a concentration of 1 to 50 × 10 3 cells / cm 2 , preferably 5 to 20 × 10 3 cells / cm 2 The inoculation density is 30-40° C. and cultured for 4-10 days, preferably 5-8 days, more preferably 6 days, to induce differentiation of PGCLCs.

[0110] Oogonia can also be induced to differentiate from PGC or PGCLC, and the differentiation induction can be performed, for example, by the method described in Non-Patent Document 3. Specifically, mouse embryonic somatic cells are isolated from mouse embryonic ovaries, and the cells and PGC or PGCLC are reaggregated to construct a heterogeneous reconstructed ovary, and the ovary is cultured for a specific number of days (e.g., 77 days or more) by a gas-liquid interface culture method, thereby producing oogonia.

[0111] Differentiation induction from pluripotent stem cells to embryonic ovarian somatic cells can be carried out, for example, according to the method of Lin et al. (Yoshino Y, et al., Science, 373 (6552): eabe0237 (2021)). For example, by differentiating pluripotent stem cells into early mesoderm, intermediate mesoderm, and coelomic epithelial state, embryonic ovarian somatic cell-like cells (FOLSC) having the same characteristics as embryonic ovarian somatic cells can be induced. Specifically, in the case of mice, the following method can be illustrated. Activin A and bFGF are used to differentiate pluripotent stem cells into EpiLCs. EpiLCs were cultured in low cell adhesion U-bottom 96-well plates containing 14 μM CHIR99021, 1 ng / ml BMP4 and 50 ng / ml epidermal growth factor (EGF) for 2 days, 3 μM retinoic acid, 30 ng / ml sonic hedgehog, 1 μM PD0325901, 50 ng / ml EGF and 1 ng / ml BMP4 for another 2 days, followed by 20 ng / ml BMP4 and 2 ng / ml FGF9 for another 1 or 2 days.

[0112] As shown in the following examples, by the preparation method of the present invention, a primordial follicle having a primary oocyte that has at least completed the first meiotic prophase (meiotic prophase I) can be obtained. In addition, by culturing for a longer period of time, the oocyte is likely to mature into a secondary oocyte, and the follicle is also likely to mature from the primordial follicle into a primary follicle, a secondary follicle, and a mature follicle. Thus, the oocyte obtained by the preparation method of the present invention can be any one of a primary oocyte (also including cells in the process of maturing into a secondary oocyte.) and a secondary oocyte. The follicle obtained by the preparation method of the present invention can be any one of a primordial follicle (also including follicles in the process of maturing into primary follicles.), a primary follicle (also including follicles in the process of maturing into secondary follicles.), a secondary follicle (also including follicles in the process of maturing into mature follicles.), and a mature follicle.

[0113] In addition, the oocyte obtained by the method of the present invention can also be further matured by a method known per se. Therefore, in another embodiment of the present invention, a method for producing an egg (hereinafter sometimes referred to as "the method for producing an egg of the present invention") is provided, which includes a step of maturing the oocyte obtained by the method of the present invention. As the method, for example, the method of Telfer et al. (McLaughlin M, et al., Mol Hum Reprod, 24 (3): 135-142 (2018)) can be cited. Specifically, (1) the reconstructed ovary is cultured in a serum-free medium for a specific number of days (e.g., 8 days), (2) secondary / multi-layer follicles are separated from the reconstructed ovary, and the separated follicles are cultured alone in a serum-free medium in the presence of activin A, (3) after a specific number of days (e.g., 8 days), a stable pressure is applied to the cultured follicle to recover the cumulus oocyte complex (COC), (4) the recovered complex is cultured on a membrane in the presence of activin A and FSH for a specific number of days (e.g., 4 days), thereby producing an egg.

[0114] Alternatively, the step of maturing the oocyte may be performed by: Those The process of transplanting the reconstructed ovary into the subrenal capsule or subovarian capsule of a primate is carried out. The transplantation method can be a known method per se.

[0115] In this specification, "primate" refers to mammals belonging to the order Primates. The sources of the artificial pluripotent stem cells, germ cells and embryonic ovarian somatic cells mentioned above are not particularly limited as long as they are primates. Primates can be Prosimian animals or Simplorhinus animals. As Prosimian animals, for example, lemurs, aye-aye, slow lorises, etc. can be listed. As Simplorhinus animals, for example, tarsiers, long-tailed monkeys (e.g., cynomolgus monkeys, macaques, Japanese macaques, Taiwan macaques, etc.), gibbons, humans, spider monkeys, capuchins, and thrushes can be listed. Among them, long-tailed monkeys such as cynomolgus monkeys and humans are preferred, and humans are more preferred.

[0116] In addition, in another embodiment, a method for maintaining a reconstructed ovary is also provided, which includes a step of culturing a reconstructed ovary of a primate under suspension culture conditions. The specific culture method of the method, etc. can all be cited from the records about the preparation method of the present invention. Here, "maintaining the reconstructed ovary" means maintaining the morphology of the reconstructed ovary from collapsing, and the types of cells constituting the reconstructed ovary can be different after suspension culture. In addition, in another embodiment, oocytes, follicles or reconstructed ovaries containing them obtained (or obtainable) by the preparation method of the present invention, and eggs obtained (or obtainable) by the preparation method of the egg of the present invention are also provided. It is preferably provided in a state of cryopreservation by a method known per se. They, such as the above-mentioned oocytes or follicles, are not only the starting materials for making eggs, but are also very useful for understanding the mechanism of germ cell development.

[0117] 2. Kit for producing oocytes or ovarian follicles, or reconstructed ovaries containing them

[0118] The present invention also provides an oocyte or ovarian follicle, or Those A kit for producing a reconstructed ovary containing an oocyte or follicle (hereinafter sometimes referred to as "the kit for producing the present invention"), comprising a culture vessel for suspension culture and L-glutamine, ascorbic acid and 2-mercaptoethanol. The kit for producing the present invention may further comprise serum. In addition, the kit for producing the present invention may further comprise retinoic acid and / or one or more molecules (preferably all) selected from progesterone, cortisol, dehydroepiandrosterone and dehydroepiandrosterone sulfate.

[0119] The manufacturing kit of the present invention may further include a basal medium and / or a medium additive such as a physiologically active substance. The definitions or specific examples of the culture container for suspension culture, serum, L-glutamine, ascorbic acid, 2-mercaptoethanol, retinoic acid, progesterone, cortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, basal medium, and medium additives contained in the manufacturing kit of the present invention may all be referred to in the above “1. Oocyte or follicle, or Those The contents described in "Method for preparing reconstructed ovary containing them".

[0120] The present invention will be described in more detail below with reference to Examples, but the present invention is not limited thereto.

[0121] Example

[0122] Example 1: Occurrence of cy embryonic oocytes

[0123] cyPGCs are specialized in the amnion during the development period from around embryonic day (E) 11, migrate through the developing hindgut endoderm and mesentery, and form colonies in the early embryonic gonads from around E30 (Sasaki K, et al., Dev Cell, 39: 169-185 (2016)). In female embryos, the size of the embryonic gonads gradually increases, and at around E40, they migrate to the ovaries. cyPGCs continue to form colonies in the developing gonads and increase in number by mitosis as oogonia (Sasaki K, et al., Dev Cell, 39: 169-185 (2016), Sasaki K, et al., Cell reports, 35: 109075 (2021)). Then, the development of embryonic oocytes is induced in the embryonic ovaries ( Figure 1 A). To establish a benchmark for in vitro reconstruction of cy embryo oogenesis, in vitro cy embryo oogenesis was carefully characterized at 2-week intervals from 8 weeks post fertilization (8wpf: E56-58) to 18wpf (E127 and 128). Figure 1 B. Figure 2 A) The gestation period of cynomolgus monkeys is approximately 22 weeks.

[0124] Histological analysis

[0125] At 8 wpf, the ovaries showed a relatively uniform tissue image with abundant oogonia, and no obvious differences were found between the ovarian cortex and medulla ( Figure 1 C). Oogonia are large in size (approximately 12 μm in diameter), have round nuclei with pale hematoxylin staining and prominent eosinophilic nucleoli (nucleosomes), and form anastomosing cord-like structures interposed with densely stained nuclei and smaller cells with a high nuclear-to-cytoplasmic ratio ( Figure 1 C). Interstitial cells are prominent, displaying a spindle shape with a high nuclear-to-cytoplasmic ratio and may have been embedded in their own synthetic loose connective tissue ( Figure 1 C). By immunofluorescence (IF) analysis, it was confirmed that all oogonia expressed DDX4, an RNA binding protein expressed in oogonia and oocytes (Toyooka Y, et al., Mech Dev, 93: 139-49 (2000)), while small intervening cells expressed FOXL2, an important transcription factor (TF) for granulosa membrane development (Crisponi L, et al., Nat Genet, 27: 159-66 (2001)). This confirmed the identity of granulosa cells ( Figure 2B) At 10 wpf, the ovary acquires a kidney-like shape, produces a medulla composed mainly of interstitial cells, and thus increases in size ( Figure 1 B). The cortex is occupied by abundant oogonia, and the outer and inner cortices do not show obvious histological differences ( Figure 1 D. Figure 2 C). At 12 wpf, oogonia are still abundant, while germ cells with larger nuclei with condensed filamentous chromatin become evident, especially in the inner cortex ( Figure 1 D. Figure 2 C), indicating that the oogonia entered the first meiotic stage and differentiated into oocytes. At 14 wpf, oocytes in the first meiotic stage also dominated the outer cortex, and larger oocytes, namely primordial follicles (, were observed in the inner cortex, differentiated by granulosa cells). Figure 1 D. Figure 2 C). At 16 wpf, primordial follicles were also observed in the outer cortex and at 18 wpf, they predominated in both the outer and inner cortex ( Figure 1 D. Figure 2 C). On the other hand, at 18 wpf, oogonia-like cells with no signs of meiotic transition were observed in the outer cortex ( Figure 1 D. Figure 2 C), which indicates that oogenesis is quite asynchronous. + The cell ratio was measured and the result was about 2 at 8wpf, decreased and remained at about 1 at 10wpf and 12wpf, especially in the inner cortex, and increased sharply after 14wpf, reaching about 6 and about 3 in the inner cortex and outer cortex, respectively, at 18wpf ( Figure 2 D). This finding is consistent with the idea that germ cells increase in number at least until 10 wpf due to mitotic expansion of oogonia, followed by a sharp decrease in cell number after 14 wpf. This may be due to apoptosis associated with meiotic prophase (see below). On the other hand, granulosa cells remain relatively constant in cell number and proliferate slowly during specification.

[0126] Expression of important markers

[0127] According to scRNA-seq analysis, human female embryonic germ cells are divided into four cell types: "mitotic stage" (oogonia), "retinoic acid (RA) responsive", "meiotic stage" and "oogenesis" (hereinafter referred to as "oogenesis") (Li L, et al., Cell Stem Cell, 20: 858-873e4 (2017)). Therefore, the expression of important markers of the four cell types was used to evaluate the occurrence of cy embryonic oocytes. At 8wpf, most DDX4 + Cells express oogonia markers. Almost all DDX4 + The cells were NANOG positive, about 80% were POU5F1, TFAP2C and PDPN positive, and only a few cells in the inner cortex (about 10%) were specialized cells of oogonia and expressed ZGLP1 ( Figure 1 E. Figure 2 E and F) (Li L, et al., Cell Stem Cell, 20: 858-873e4 (2017), Nagaoka S, I., et al., Science 367 (2020)). At 10wpf, although the cells were histologically similar to those at 8wpf, the number of germ cells expressing oogonia markers, especially in the inner cortex, decreased sharply. On the other hand, the number of cells expressing RA-responsive cell markers increased (ZGLP1: about 40-50%, STRA8: about 10-20%), and some (about 10-20%) expressed SYCP3 (an axial component of the synaptonemal complex (SC) that is specifically expressed from the pre-leptotene stage of meiosis) (Yuan L, et al., Science, 296: 1115-8 (2002), Yuan L, et al., Mol Cell 5: 73-83 (2000)) (see below)) ( Figure 1 E. Figure 2 F). NANOG, POU5F1, and TFAP2C, which are oogonial markers, are evenly distributed throughout the oogonial nucleus, but ZGLP1 and STRA8, which are RA-responsive cell markers, are granularly distributed ( Figure 1 E. Figure 2E). At 12wpf, the number of germ cells expressing oogonia markers continued to decrease, while the number of cells expressing RA-responsive and meiotic cell markers (SYCP3, DMC1, γH2AX, SYCP1) continued to increase. At 14wpf, the number of oogonia was small (about 5%), meiotic cells were dominant (about 50%), and cells expressing oogenesis markers (ZP3 and TP63) appeared in both the outer cortex (about 10%) and the inner cortex (about 30%). Figure 1 E. Figure 2 F). γH2AX (Fernandez-Capetillo O, et al., J Cell Biol, 163: 15-20 (2003)), a double-strand break (DSB) marker during meiotic recombination, shows a punctate distribution in the nucleus during meiosis. On the other hand, SYCP1 (Meuwissen RL, et al., EMBO J, 11: 5091-100 (1992)), another axial component of the SC, shows a typical filamentous distribution along the synapsed homologous chromosomes in the nucleus corresponding to the late zygotene or pachytene stage of the first meiotic prophase (see below). At 16 and 18 wpf, oogenic cells (diplotene oocytes of primordial follicles) (see below) are dominant in the inner cortex, and meiotic cells are abundant in the outer cortex ( Figure 1 E. Figure 2 F). In oogenic cells, ZP3 is localized in the plasma membrane, or in the extracellular space between the oocyte and surrounding granulosa cells, suggesting the formation of an early zona pellucida (ZP) ( Figure 1 E). TP63, FIGLA, and NOBOX, which are important TFs for oogenesis (Liang L, et al., Development, 124: 4939-47 (1997), Suh EK, et al., Nature, 444: 624-8 (2006), Suzumori N, et al., Mech Dev, 111: 137-41 (2002)), were shown to be localized in the entire nucleoplasm of oocytes except for the nucleolus. On the other hand, NLRP5, a maternal effect protein indispensable for early embryogenesis (Tong ZB, et al., Nat Genet, 26: 267-8 (2000)), was shown to be punctately localized throughout the cytoplasm ( Figure 1 E. Figure 2E). Laminin, a basement membrane marker, distinguishes the basal side of the squamous epithelial granulosa cells that make up the primordial follicles, marking both single and sometimes multiple primordial follicles ( Figure 2 E).

[0128] Next, the expression of Ki67 (Gerdes, Schwab et al., 1983), a cell proliferation marker, and the expression of cleaved poly ADP ribose polymerase (cPARP) and CASPASE3 (cCAS3) (Lazebnik YA, et al., Nature, 371: 346-7 (1994), Tewari M, et al., Cell, 81: 801-9 (1995)), apoptosis markers, were studied. At 8 wpf, most (about 70%) oogonia expressed Ki67, which was mainly localized around the nucleolus ( Figure 2 E and F). Germ cells expressing oogonia markers decrease dramatically after 10 wpf, but Ki67 is observed throughout the validation period. + Cells (about 40%) Figure 2 F). These cells will represent RA-responsive and meiotic cells at the early stages of meiosis (see below). No germ cells showing apoptotic markers were detected at 8 wpf, but at 10 wpf to 14 wpf, which is associated with the onset and progression of meiotic prophase, and at 16 wpf and 18 wpf, which are associated with the formation of primordial follicles, approximately 10% and approximately 15% of the germ cells showed apoptotic markers, respectively ( Figure 2 E and F). Apoptotic primordial follicles showed cCAS3 but not cPARP ( Figure 2 F).

[0129] Progression of the first meiotic prophase

[0130] The progression of the first meiotic prophase was determined based on chromatin structure visualized by DAPI staining and the expression / localization of SYCP3 and DMC1, the latter of which is involved in single-strand invasion of homologous recombination (Bishop DK, et al., Cell, 69:439-56 (1992)). Figure 1F). Meiotic oocytes neither express SYCP3 nor DMC1, and do not show obvious signs of chromosome condensation. Pre-leptotene oocytes begin chromosome condensation, expressing SYCP3 located around the nucleolus, but do not show DMC1. Leptotene oocytes further show chromosome condensation, and in addition to DMC1 located in condensed chromosomes, also express SYCP3. SYCP3 is located on both nucleoli and condensed chromosomes. Zygotene oocytes show signs of chromosome pairing, expressing SYCP3 and DMC1. They are located in paired chromosomes, and DMC1 is strongly stained into multiple dots. Early pachytene oocytes show firm chromosome pairing with clear axis structure, expressing SYCP3 and DMC1. DMC1 is strongly stained into dots, indicating that it may be co-located in the crossover recombination site of homologous chromosomes. Late pachytene oocytes are similar to early pachytene oocytes except that they do not express DMC1. Diplotene oocytes were larger in size, showed signs of diakinesis, and suppressed both SYCP3 and DMC1. This confirmed that in this classification, most germ cells were classified as mitotic before 10 wpf, and the first meiotic division began in the endothelium at 10 wpf, with the diplotene oocytes appearing from 14 wpf onwards and completing prophase, and gradually progressing during cy embryogenesis ( Figure 1 G). In summary, these data update the classic observations of macaques (Macaca Mulatta) (Baker TG, J Anat 100:761-76 (1966)), indicating that the onset of the first meiotic division and obvious morphological changes require about 2 weeks from the suppression of oocyte markers, and 4 to 6 weeks to complete the first meiotic prophase, establishing the cytological basis required for the progression of cy embryonic oocytes.

[0131] Example 2: Development of cy embryonic oocytes in xenotransplanted cy reconstructed ovaries (cy rOvary)

[0132] Next, we investigated whether oogenesis in cy embryos proceeded by xenotransplanting cy re-aggregated ovaries (rOvaries) into immunodeficient mice. Figure 3 A). Cy embryonic ovaries were isolated at 8 wpf and dissociated into single cells (approximately 6.0 × 10 per ovary). 5 About 50,000 cells (about 5,000 germ cells and 45,000 somatic cells) were reaggregated under suspension conditions for 2 days to produce cy-reconstructed ovaries, which were transplanted subepithelially into the surface of the kidneys of immunodeficient mice, and the occurrence of cy oocytes was analyzed at 3-week intervals.

[0133] At 3 weeks post-transplantation (3w-ptp (post-transplantation)), the cy-reconstructed ovary had an integrated structure and reconstructed anastomotic cords interspersed with interstitial cells ( Figure 3 B. Figure 2 G). Most germ cells display an immature morphology representative of oogonia, but a portion display larger nuclei with condensed chromatin indicative of the transition to meiosis ( Figure 3 B). Consistent with these findings, approximately 40% of germ cells expressed oogonial markers (NANOG, POU5F1, TFAP2C, PDPN), approximately 50% expressed ZGLP1, approximately 30% expressed SYCP3, and approximately 10% expressed meiotic markers (DMC1, γH2AX, SYCP1) ( Figure 3 C and D). At 6 w-ptp, oogenesis was further advanced in the cy-reconstructed ovary, with most germ cells showing nuclear morphology of the first meiotic prophase also containing pachytene nuclear morphology with synapsed chromosome pairs (<10% positive for oogonia markers, ~60% positive for SYCP3, ~30% positive for DMC1, γH2AX, SYCP1), but no cells expressing oocyte markers ( Figure 3 BD, Figure 2 G). After 9w-ptp, there are differentiated primordial follicle-like complexes with oocytes expressing ZP3 and TP63 surrounded by a single layer of squamous epithelial granulosa cells expressing FOXL2, and before 15w-ptp, the complexes are dominant ( Figure 3 BD). These findings indicate that upon xenotransplantation into mice, oogenesis and first meiotic prophase of Cy embryos appear to proceed normally, but progress slightly slower than in vivo. Cy reconstructed ovaries were studied at 21 w-ptp and found that, although clear architecture was maintained, only granulosa and interstitial cells survived, with essentially all oocytes degenerating ( Figure 3 E) This indicates that under the current xenotransplantation conditions, the follicles did not undergo further growth.

[0134] Example 3: Reconstruction of cy embryonic oocytes in vitro

[0135] Next, it was verified whether in vitro culture promotes the occurrence of appropriate cy embryonic oocytes in cy reconstructed ovaries. First, the air-liquid interface culture (air-liquid interface culture) that supports the development of both mouse embryonic ovaries and reconstructed ovaries was studied (Hikabe O, et al., Nature, 539: 299-303 (2016), Morohaku K, et al., Proc Natl Acad Sci USA, 113: 9021-6 (2016)). Cy reconstructed ovaries with approximately 50,000 ovarian cells at 8 wpf were made and cultured on Transwell-COL membrane inserts ( Figure 4 A). First, the cytosolic reconstructed ovary on the Transwell-COL membrane showed an integrated structure. Different from the mouse reconstructed ovary, if cultured in vitro earlier, some cells began to proliferate from the cytosolic reconstructed ovary from 1 week (1w-ivc). At 3w-ivc, a large number of cells migrated, and the cytosolic reconstructed ovary became smaller and flatter ( Figure 4 B. Figure 5 A). Then, the cells continued to expand, and at 6 weeks of IVC (invitro culture), the size of the cy-reconstructed ovary was significantly reduced compared to the original size ( Figure 4 B. Figure 5 A) By histological and IF analysis, at 3 weeks-ivc, cy-reconstructed ovaries had ovarian cords, but the regions were limited to a few germ cells, and at 6 weeks-ivc, FOXL2 + Granulosa cells partially clustered, but only a few germ cells with degenerated morphology remained ( Figure 4 B) We investigated whether cy-reconstructed ovaries could be maintained on culture membranes coated with various extracellular matrix components including collagen (types I, III, IV, V, VI), fibronectin, laminin, and combinations thereof. Figure 5 A). In some conditions, such as membranes coated with collagen types I, III, IV, V, VI or iMatrix (laminin 511), cy reconstructed ovaries maintained integrity until 5w-ivc, but began to collapse thereafter. That is, some cells migrated, and in all conditions studied, cy reconstructed ovaries flattened at 12w-ivc ( Figure 5 A) These findings suggest that cytoremodeling of the ovary requires conditions that differ from those of mouse ovaries for proper in vitro development.

[0136] Therefore, it was verified whether cy-reconstructed ovaries could be maintained for a long time under suspension conditions ( Figure 4A). When the ovaries were suspended and proliferated in αMEM containing 10% fetal bovine serum (FBS), which is the basic condition for mouse reconstructed ovary culture (Hikabe O, et al., Nature, 539: 299-303 (2016), Morohaku K, et al., Proc Natl Acad Sci USA, 113: 9021-6 (2016)), at 6 weeks-ivc, the cy-reconstructed ovaries showed an integrated appearance distinguished by multiple layers of squamous epithelial cells, but the central area seemed to be somewhat necrotic, and a relatively large amount of DDX4 + Germ cells and the involvement of FOXL2 + Granulocytes survive together ( Figure 5 B). Thirteen basal culture media including αMEM were studied (αMEM, GMEM, DMEM, KODMEM, Advanced MEM, DMEM / F-12, Waymouth's, McCoy's 5A, CMRL, IMDM, Advanced RPMI, StemPro 34, MesenPRO RS), and it was found that Advanced MEM gave the best results in terms of cy-reconstructed ovarian size and tissue images at 6w-ivc ( Figure 5 B). The cy-reconstructed ovaries may shrink in size at the beginning of culture due to the continuous compression of the aggregate, but gradually recover in size after 1w-ivc ( Figure 4 C). The size of cy reconstructed ovaries cultured in Advanced MEM + 10% FBS remained consistently larger during the 15-week culture period compared to reconstructed ovaries cultured in αMEM containing 10% FBS ( Figure 4 C). Therefore, the occurrence of cy embryonic oocytes in cy reconstructed ovaries cultured under suspension conditions in Advanced MEM + 10% FBS was analyzed at 3-week intervals ( Figure 4 D) At 3w-ivc, most germ cells (about 50%) are oogonia expressing NANOG, POU5F1, TFAP2C and PDPN, but about 30% and about 10% progress to the RA response phase (ZGLP1 + / STRA8 + ) and meiotic period (DMC1 + / γH2AX + / SYCP1 + )( Figure 4 E and F, Figure 5C). Then, the number of oogonia decreased and the number of meiotic cells increased, but the rate was slower than in vivo and xenotransplantation. Nevertheless, oocytes expressing oogenesis markers (ZP3, TP63, FIGLA, NOBOX, NLRP5) in primordial follicle-like structures began to differentiate after 12w-ivc ( Figure 4 E and F, Figure 5 C). The morphology of oogonia and RA-responsive cells, meiotic cells, oogenic cells and granulosa cells, as well as the in vitro expression / intracellular localization of important markers of these cells in cy-reconstructed ovaries were similar to those of the corresponding cell types in vivo ( Figure 4 E. Figure 5 C and D). Use Figure 1 The progress of meiotic prophase was studied by the criteria defined in F, and the results showed that the progress of meiotic prophase in cy-reconstructed ovaries in vitro was slow but steady ( Figure 4 G). It was found that a large fraction of germ cells remained in the proleptotene stage from 6 weeks to 15 weeks. Figure 4 G), which suggests that the transition from pre-leptotene to leptotene under the present conditions may be the rate-limiting stage. These findings indicate that after 12 weeks, mitotic oogonia differentiate into oocytes and complete the early meiotic prophase of the primitive follicle-like structure, thereby carrying out the occurrence of cy embryonic oocytes in vitro.

[0137] Example 4: Transcriptome dynamics of cy embryonic ovarian development in vivo, xenograft, and in vitro

[0138] To comprehensively define cy embryonic ovarian development in vivo as well as in xenografts and in vitro, single-cell RNA sequencing (scRNA-seq) analysis of cy embryonic ovaries and cy reconstructed ovaries was performed (Table 2). Cy / human SC3-seq data information (sample information, sequence summary, and data summary) is omitted.

[0139] [Table 2]

[0140]

[0141]

[0142] First, in order to prepare single-cell cDNA using the 10X Chromium platform, a total of 35,141 single cells isolated from cy embryonic ovaries at 8wpf, 10wpf, 12wpf, 16wpf and 18wpf were processed. By sequencing, 29,127 cells passed the main quality filtering [number of detected genes (nGene), total count of unique molecular identifiers (UMI), proportion of mitochondrial gene number (%)] and were processed based on Scrublet [doublet / multiplet elimination algorithm (Wolock SL, et al., Cell Syst, 8:281-291e9 (2019))] for subsequent analysis (Table 2). The batch effect was removed from the subspace of the principal component analysis (PCA) based on the fast mutual nearest neighbor (fastMNN) algorithm, and the cells were subsequently divided into five cell types through graph-based clustering using the Louvain algorithm: DDX4 + Germ cells (2283 cells), WT1 + Pregranular cells (11986 cells), TCF21 + Interstitial cells (14028 cells), PECAM1 + Endothelial cells (673 cells) and TYROBP + Blood cells (157 cells) Figure 6 AC). At all stages, more genes were detected in germ cells compared with somatic cells ( Figure 7 A). Germ cells were isolated and reanalyzed using PCA, fastMNN, and Louvain clustering. Thus, germ cells were classified into 11 clusters after manually removing putative dimorphic / multimorphic aggregates. Based on the expression of important markers, they were annotated as mitotic phase 1 / 2 / 3, preleptotene 1 / 2 / 3, leptotene, zygotene, pachytene 1 / 2, and unclassified (probably apoptotic cells), and cells in pachytene 2 were manually classified as dimorphic ( Figure 6 D. Figure 7 B) Distribution of cells annotated by scRNA-seq throughout the entire developmental stage, except for a few diplotene cells ( Figure 6 D. Figure 7B) (this is at least in part due to the incompatibility of diplotene cells with the 10X platform due to their large cell size), essentially consistent with the cells annotated by IF analysis. The number of genes detected and the total UMI counts were relatively constant from mitotic cells to zygotene (average of approximately 7,000 and 50,000, respectively), but decreased slightly in pachytene cells and were more variable ( Figure 7 C).

[0143] Mitotic stage 1 / 2 / 3 cells expressed important oogonia markers including POU5F1, TFAP2C, PDPN, and PRDM1, and were mainly composed of cells at 8 wpf, but included cells of all stages ( Figure 6 D, E and F, Figure 7 B and D). It is believed that cells in mitosis phase 1 / 2 / 3 represent cells in the G1, S, and G2 / M phases of the cell cycle, respectively. Figure 7 E). Preleptotene 1 cells weakly expressed POU5F1 and ZGLP1, but did not show typical meiotic markers such as DMC1, PRDM9 and SYCP1, and consisted of cells at 8wpf, 10wpf, 12wpf and 16wpf. They are in the G1 phase of the cell cycle ( Figure 6 D and F, Figure 7 D and E). Although the 2 / 3 cells in the pre-leptotene stage expressed ZGLP1, REC8 and SYCP3, DMC1, PRDM9 and SYCP1 were negative / very weak, and they were composed of cells in all stages of development, and were in the S phase and G2 phase of the cell cycle respectively ( Figure 6 D and F, Figure 7 D and E). Therefore, the cells in the pre-leptotene 2 are cells that undergo meiotic DNA replication. The leptotene and zygotene cells are mainly composed of cells after 12 wpf, and they express REC8, SYCP2, and SYCP3 at higher levels than the pre-leptotene cells. The zygotene cells upregulate DMC1, PRDM9, and SYCP1 ( Figure 6 D and F, Figure 7 D). The cells in the pachytene 1 / 2 stage are mainly composed of cells after 12wpf, which inhibit ZGLP1 and REC8 and express SYCP1 / 2 / 3, DMC1 and PRDM9. The cells in the pachytene 2 stage begin to downregulate these genes and upregulate FIGLA ( Figure 6 D and F, Figure 7 D) Diplotene cells (ZP3 + / GDF9 + / NOBOX + Cells) consisted of cells at 16wpf and 18wpf, downregulated meiotic markers, and expressed important genes for oocyte development such as FIGLA and NLRP5 ( Figure 6 D and F, Figure 7D). The pseudo-time analysis based on the occurrence trajectory of Monocle 3 (Cao J, et al., Nature, 566: 496-502 (2019)) obtained results consistent with the above results ( Figure 6 G).

[0144] Highly variable genes were identified from 11 clusters [results from subtracting genes with low expression levels from the top 2000 HVGs (log(ssUMI+1)≤0.5 of the cluster's average expression): 1481 genes]. List of the identified 1481 genes, their normalized expression values, and GO analysis of HVGs are omitted.

[0145] Unsupervised hierarchical clustering (UHC) divided the genes into 8 clusters ( Figure 6 H). Cluster 1 genes were highly expressed in cells at mitotic stages 1 / 2 / 3 and sharply downregulated after pre-leptotene stage 1, and were enriched for genes with Gene Ontology (GO) functional terms related to "somatic stem cell population maintenance" (LIN28A, DPPA4, NANOG, KLF4, etc.) and "developmental process involved in reproduction" (PRDM1, UTF1, TFAP2C, NANOS3, SOX15, etc.). Cluster 2 genes showed expression characteristics related to the progression of the cell cycle (expressed in mitotic stages 2 / 3 and pre-leptotene 2 / 3), and were actually enriched for "mitotic cell cycle" and "meiotic cell cycle process" (TOP2A, MCM3 / 10, MKI67, CCNA2 / B1 / B2, PCNA, etc.) ( Figure 6 H. Figure 7E). Cluster 3 genes are expressed from preleptotene 1 to zygotene, and are rich in "regulation of transcription, DNA-templated" (STRA8, ZGLP1, ANHX, KDM6B, etc.) and the interesting "pattern specification process" (a large number of HOX genes, SIX1, TBX3, GLI3, PBX1, etc.). Cluster 4 genes are gradually up-regulated after preleptotene 1, and are rich in "meiotic cell cycle" (TDRD1 / 12, RAD50, EHMT2, MSX1, PLD6, etc.) and "reproductive system development" (HOXA9 / A10, SERPINE2 / F1, CHD7, etc.). Genes in clusters 3 and 4, especially those expressed in preleptotene 1 / 2, contain specialized factors for oogenesis. The genes of clusters 5 and 6 are expressed from leptotene to pachytene 2 (cluster 5) or diplotene (cluster 6), and include important genes for meiotic recombination [“synapsis / double-strand break repair / reciprocal DNA recombination / meiotic cell cycle” (cluster 5: SYCP1, SYCE1 / 2 / 3, MSH4 / 5, MEIOB / C, TEX11 / 15, PRDM9, SPO11, MEIOSIN, etc. Cluster 6: SYCP2, ASZ1, MAEL, TEX12, MEIKIN, etc.)]. The genes of clusters 7 and 8 are strongly upregulated in diplotene, and are enriched in “reproductive process” and “oogenesis” (NPM2, BMP5, GDF9, ZP2 / 3, SOHLH1, FIGLA, NOBOX, etc.). By using correlation analysis of the same genes, it was clear that the 11 clusters could be divided into four groups with higher correlation [mitosis 1 to preleptotene 1, preleptotene 1 to zygotene (corresponding to RA responsiveness), zygotene to pachytene 2 (corresponding to meiosis), pachytene 2 to diplotene (corresponding to oogenesis)], and three clusters (preleptotene 1, zygotene, pachytene 2) played a transitional role between the groups ( Figure 6 I), providing a high-resolution transcriptome catalogue of oogenesis in primate embryos.

[0146] Using 10X Chromium, 937 quality-filtered single-cell transcriptomes (73 germ cells, 702 (pre)granulosa cells, 118 interstitial cells) were generated from 12w-ivc and 15w-ivc cy-reconstructed ovaries and compared with the single-cell transcriptomes of cy-embryonic ovarian cells in vivo. PCA and fastMNN, followed by Louvain clustering, revealed that cy germ cells from cy-reconstructed ovaries in vitro showed transcriptomes consistent with oocyte transcriptomes ranging from pre-leptotene to pachytene ( Figure 6 J). To gain further insights into transcriptome dynamics associated with cy embryonic oogenesis in cy reconstructed ovaries, single cells with germ cell appearance were manually picked from 3 / 6 / 9 / 12 / 15w-ivc and w-ptp cy reconstructed ovaries and 12 / 16 / 18wpf oocytes as well as adult ovaries dissociated from cy reconstructed ovaries and adult ovaries, and scRNA-seq analysis was performed using single-cell mRNA 3' end sequencing (SC3-seq) (Nakamura T, et al., Nucleic Acids Res, 43:e60(2015)) ( Figure 6 K and L). UHC and PCA were performed on 172 DDX4 + Single cells (including 19 germ cells at 6 / 7 wpf (Okamoto I, et al., Science, 374: eabd8887 (2021)) were classified into 4 major groups corresponding to mitotic, RA-responsive, meiotic, and oogenic cells. In vivo, xenografted, and in vitro cells were included in all groups, and transcriptome profiles were mixed within the groups ( Figure 6 K and L). In conclusion, based on these results, it can be concluded that cy embryonic germ cells undergo appropriate transcriptome maturation in cy reconstructed ovaries in vitro.

[0147] In addition, the expression of WT1 in vivo and in vitro + Using the same analysis as for germ cells, the in vivo cytosolic (pre)granulosa cells were divided into 12 clusters ( Figure 8 A). The cluster distribution analysis of the entire occurrence stage showed that most clusters (clusters 1, 2, 3, 4, 5, 6, 7, 9, 11 and 12) already existed at 8wpf, and clusters 8 and 10 appeared after 10wpf and 16wpf respectively ( Figure 8AC). Genes with greatly changed expression in 12 clusters were identified [results obtained by deducting genes with low expression levels from the top 1500 HVGs (average expression of clusters log(ssUMI+1)≤0.5): 788 genes]. The list of the identified 712 genes, their normalized expression values, and GO analysis of HVGs is omitted.

[0148] UHC using these genes confirmed that cells in clusters 2 and 3 are progranulosa cells in the S phase of the cell cycle, and cells in cluster 1 are in the G2 / M phase. In addition, cells in cluster 1 are abundant in the early stages (8 / 10 / 12 wpf), and like cells in cluster 4, genes for “cell migration” and “cytoskeleton organization” are abundant ( Figure 8 Clusters 11 and 12 cells were also abundant at early stages (8 / 10 / 12 wpf) and expressed genes involved in “steroid biosynthetic process” and “reproductive system development”, thus representing a unique pregranulosa cell subtype ( Figure 8 AD). RNA velocity calculation (Bergen V, et al., Nat Biotechnol, 38: 1408-1414 (2020)) and partition-based graphical abstraction (PAGA) (Wolf FA, et al., Genome Biol, 20: 59 (2019)) suggest that the differentiation of anterior granule cells proceeds from cluster 4 to 5, 7, and 9, or from 4 to 5, 6, 8, and 10 ( Figure 8 A and B), cells in cluster 10 that appeared after 16 wpf were enriched in genes for “extracellular matrix organization” (COL1A2 / 3A1 / 4A3 / 6A1 / 6A2 / 14A1, LAMA4 / B1, ITGA1 / A2 / A6, TIMP2, SULF1, etc.) and “Notch signaling pathway” (NOTCH3, JAG1, HEY2, NR1H4, HES1), and therefore represent the granulosa cells that constitute the primordial follicles ( Figure 8 D and E) (Li L, et al., Cell Stem Cell, 20: 858-873e4 (2017)). +Composite analysis of the transcriptome of pregranulosa cells revealed that most WT1 + The cells were close to the cluster of granulosa cells of the primordial follicles at 16wpf and 18wpf (cluster 11) / formed a cluster merged with this cluster (cluster 12). This indicates that the pre-granulosa cells at 8wpf are properly differentiated into granulosa cells of the follicles ( Figure 8 F and G). However, in vitro granulosa cells showed upregulation of ID2 / 3 / 4 (i.e., activation of BMP signaling) as well as upregulation of HEY2 and downregulation of HEY1 / HEYL (i.e., regulation of JAG1-NOTCH2 / 3 signaling) ( Figure 8 G)(Vanorny DA, and Mayo KE, Reproduction, 153: R187-R204 (2017), Zhang Y, et al., Mol Cell, 72: 1021-1034e4 (2018)), which suggests that the granulosa cells in culture may enter the activated primary follicle stage.

[0149] Example 5: Reconstruction of human embryonic oocytes in vitro

[0150] In order to determine the occurrence conditions of cy embryonic oocytes in vitro, it was verified whether it is possible to reconstruct human embryonic oocytes in vitro. Female embryos of two people who underwent artificial abortion at 11 weeks of gestation (11W) were available, and the gonads were separated and processed for histological analysis, human reconstructed ovaries and single-cell transcriptome analysis. Furthermore, primordial follicles were isolated from human adult ovaries, and these oocytes were processed for single-cell transcriptome analysis. All human samples were obtained based on appropriate informed consent (refer to <Materials and Methods>). Consistent with existing reports (Li L, et al., Cell Stem Cell, 20: 858-873e4 (2017)), oogonia with round nuclei accompanied by pale hematoxylin staining and obvious nucleoli were abundant in 11W human embryonic ovaries, but there were also cells with larger nuclei accompanied by a more condensed chromatin appearance ( Fig. 9 A). Therefore, about 85% of DDX4 + Germ cells are POU5F1 + A very small number (about 3%) of them showed expression of early meiotic markers such as SYCP3, γH2AX, and DMC1, but no expression of SYCP1 ( Fig. 9 B and D). This indicates that most of the germ cells in the human ovary at 11 weeks are oogonia.

[0151] Under air-liquid interface conditions, cells migrated from human reconstructed ovaries (about 50,000 ovarian cells), which flattened and collapsed during long-term culture. This is similar to the results of cytosolic reconstructed ovaries, but different from mouse reconstructed ovaries ( Figure 5 E). Therefore, human reconstructed ovaries were cultured under the suspension conditions defined for cy-reconstructed ovaries and their properties were verified at 7 / 8 and 14 w-ivc. At 7 w-ivc, human reconstructed ovaries showed integrated architecture, but similar to cy-reconstructed ovaries, central areas showed a necrotic appearance ( Fig. 9 A). Histological sections revealed that the cortical region was filled with germ cells with oogonia-like or early meiotic oocyte-like morphology ( Fig. 9 A) is very consistent with this result, DDX4 + About 30% of germ cells are POU5F1 + , about 50% is SYCP3 + , about 30% is γH2AX + / DMC1 + , only about 2% is SYCP1 + ( Fig. 9 C and D). At 14 weeks-ivc, human reconstructed ovaries remain integrated, with abundant germ cells in their cortex with condensed filamentous chromatin and, notably, large nuclei containing numerous primordial follicle-like structures ( Fig. 9 A). Consistent with these observations, DDX4 + / POU5F1 + The ratio of cells decreased to about 10%, and about 30% became SYCP1 + , about 3% become ZP3 + / TP63 + ( Fig. 9 C and D). The morphology of meiotic and primordial follicle-like cells and the expression / intracellular localization of important markers in these cells were similar to the results of the corresponding in vivo cell types in cynomolgus monkeys and humans ( Figure 1 E and 9C) (Baker TG, Proc R Soc Lond B Biol Sci, 158:417-33 (1963)).

[0152] Based on the single-cell transcriptome analysis based on SC3-seq of 120 quality-filtered single cells (human embryonic ovary at 11 weeks: 33 cells, human adult ovary: 4 cells, human reconstructed ovary at 8 weeks-ivc: 39 cells, human reconstructed ovary at 14 weeks-ivc: 44 cells), these cells were divided into four cell types, namely mitotic cells, RA-responsive cells, meiotic cells and oogenic cells ( Fig. 9 E and F). Consistent with histological and IF analyses, the majority of single cells picked from human embryonic ovaries dissociated at 11 W were mitotic oogonia (19 / 33) expressing NANOG, POU5F1, TFAP2C, and PRDM1 or RA-responsive cells (14 / 33) expressing ANHX, REC8, and ASB9. On the other hand, single cells from human reconstructed ovaries at 8 weeks-ivc contained all four cell types (mitotic: 16 / 39, RA-responsive: 13 / 39, meiotic: 7 / 39, oogenesis: 3 / 39), and single cells from human reconstructed ovaries at 14 weeks-ivc were mainly meiotic cells expressing DMC1, RAD51AP2, SYCP1 or oogenesis cells expressing ZP3, NLRP5, TP63, and NOBOX (mitotic: 0 / 44, RA-responsive: 3 / 44, meiotic: 21 / 44, oogenesis: 20 / 44) ( Fig. 9 E and F). In particular, oocytes isolated from human reconstructed ovaries cluster closely with mature oocytes from primordial follicles ( Fig. 9 E and F), indicating that human oocytes in human reconstructed ovaries in vitro are properly progressing. To further confirm this, and to supplement the lack of late human embryonic ovarian samples, the SC3-seq data of human germ cells and cy germ cells were merged by canonical correlation analysis (CCA) (Stuart T, et al., Cell, 177: 1888-1902 e21 (2019)), and PCA was performed. As a result, it was clarified that human oocytes in human reconstructed ovaries in vitro, cy oocytes in vivo, and cy reconstructed ovaries in vitro had highly parallel transcriptomic progression ( Fig. 9 G). Taken together, these results indicate that human oogenesis can be reconstructed in vitro at least up to oocytes that have completed the first meiotic prophase of primordial follicles.

[0153] Example 6: Primate-specific procedures for embryonic oogenesis

[0154] In mammals, the occurrence of germ cells is carried out in conservative cell events, including the specialization (PGC specification) of pluripotent embryonic precursor cells to PGC, epigenetic reprogramming, the sex determination of germ cells and the epigenetic programming of meiosis (female case) or meiosis occurring thereafter (male case) for generating sexually dimorphic haploid gametes. On the other hand, the correct cell dynamics and molecular mechanisms underlying the conservative events seem to be significantly different due to species (Saitou M, and Hayashi K, Science, 374: eaaz6830 (2021)). In order to obtain insights into the intrinsic programs of primates related to embryonic oocytes, the transcriptome data sets of association were compared between mice, monkeys and humans. The cynomolgus monkey and human datasets prepared in this example were compared with four public datasets [one for humans (Chitiashvili T, et al., Nat Cell Biol, 22: 1436-1446 (2020)), three for mice (Ge W, et al., Cell Mol Life Sci, 78: 695-713 (2021), Niu W, and Spradling AC, Proc Natl Acad Sci USA, 117: 20015-20026 (2020), Zhao ZH, et al., FASEB J, 34:12634-12645(2020))], Louvain clustering was performed, and then the developmental stages of embryonic oocytes in 15 clusters across 3 species were defined based on the expression of associated genes (mitotic stages 1-4, preleptotene 1-3, leptotene 1-3, zygotene 1 / 2, pachytene 1 / 2, diplotene) ( Fig.10 A, Table 2). The distribution of cells that defined the developmental stage at each developmental time point was consistent with previous observations ( Fig.10 B and C, Fig.11 A) (Chitiashvili T, et al., Nat Cell Biol, 22: 1436-1446 (2020)) (Ge W, et al., Cell MolLife Sci, 78: 695-713 (2021), Niu W, and Spradling AC, Proc Natl Acad Sci USA, 117: 20015-20026 (2020)).

[0155] Through pseudo-time analysis based on Monocle 3, it was clarified that in humans and monkeys, the transition from the mitotic phase to the pre-meiotic phase, i.e., the transition to the meiotic phase, causes a large transcriptomic change (transcriptomicalteration), whereas in mice, the transition is accompanied by small changes, and the subsequent transition causes large changes ( Fig.10 D). HVGs were identified in the development of embryonic oocytes of each species, of which 237 genes were orthologous and shared by all three species. Correlation analysis of the expression of these genes at each development stage clarified the major differences between mitotic cells and pre-leptotene cells in humans and monkeys, but not in mice ( Fig.10 E). In addition, the expression characteristics of each cluster are relatively well conserved between humans and monkeys, but different in mice ( Fig.10 E). Based on previous studies showing that in the meiotic prophase of spermatogenesis in humans, monkeys, and mice, as meiosis progresses, the contribution of 1-to-1-to-1 (1-1-1) orthologous genes to overall expression decreases, while on the other hand, the contribution of species-specific genes to overall expression increases in humans and mice (no clear conclusion can be drawn for monkeys due to the low level of gene annotation), the species-specific specialization of the first meiotic prophase in males was discussed (Lau X, et al., Dev Cell, 54: 548-566e7 (2020)). The same analysis was performed for the first meiotic prophase in females. In stark contrast to the results in males, as meiosis progresses in females, the contribution of 1-1-1 orthologous genes to overall expression gradually increases, while on the other hand, the contribution of species-specific genes to overall expression is always low ( Fig.11 C) Thus, the transition from mitosis to meiosis during oogenesis is accompanied by significant transcriptomic changes in humans and monkeys compared with mice, and the overall transcriptomic differences between the three species arise from the differential usage of orthologous genes.

[0156] Furthermore, we identified primate-specific genes that show dynamic changes during embryonic oogenesis. Fig. 9 G's SC3-seq analysis identified 1-1 orthologous genes that were differentially expressed between 2 consecutive cell types [log2(normalized read count + 1)>3, fold-change>2], and extracted genes shared by humans and monkeys (397 genes), such as Fig.10 The expression changes of this gene in the 10X dataset during embryonic oocyte development were investigated as defined in A. The list of 397 genes and their normalized expression values ​​in the 10X scRNA-seq data of human / monkey / mouse is omitted.

[0157] The expression characteristics of 130 of the 397 genes were well conserved among the three species, and a noteworthy trend was observed. That is, genes expressed in mitotic cells continued to be expressed in pre-leptotene / leptotene cells, and genes expressed in meiotic cells showed upregulation in the relatively late stage of mouse ( Fig.12 A). These genes include PRDM1, TFAP2C, NANOS3, TCL1A, SOX15, DPPA3 (mitosis), HORMAD1, REC8, STAG3, SMC1B (leptotene ~), SYCP1, SYCP2, SYCE3, HORMAD2, MEIOB, MEIOC, TEX12, SPO11 (zygotene ~), NPM2, ZP2, NOBOX, FIGLA, GDF9, NLRP5 and TP63 (pachytene 3 / diplotene), which are known genes involved in embryonic oocyte development ( Fig.12 A). On the other hand, 83 genes showed specific expression changes in humans and monkeys, but not in mice, including PIM2, THY1, MMP2, SOX17, TFCP2L1 (mitotic), PBX1, SKAP2, KDM1B, HOXA3 / B3, PAX6, MORC4 (preleptotene / leptotene), DMRTB1, CLGN, GSAP, SPAG6, UPB1, CABYR (leptotene-), SLCO4A1, CSF1R, TMEM163, KPNA7, ARHGEF33 / GAP18, SERPINF1, LMOD3 (pachytene2 / diplotene) ( Fig.10 F. Fig.12 B). Among them, PAX6 (Jordan T, et al., Nat Genet, 1: 328-32 (1992), Walther C, and Gruss P, Development 113: 1435-49 (1991)), which is a homeobox TF involved in important cell specialization processes including eye development, CLGN, CLGN (Ikawa M, et al., Nature, 387: 607-11 (1997)), which is a chaperone protein specifically expressed in the endoplasmic reticulum (ER) of spermatogenic cells and plays an important role in the binding of sperm to ZP, and LMOD3 (Yuen M, et al., J Clin Invest, 124: 4693-708 (2014)), which is an actin fiber-nucleation protein expressed in skeletal muscle and cardiac muscle, were verified. IF analysis revealed that PAX6 was specifically and strongly expressed in TFAP2C- germ cells at the early stage of meiotic prophase in cynomolgus monkey and human reconstructed ovaries ( Fig.10G and H). CLGN is expressed and localized in the cytoplasm, possibly in the ER, in TFAP2C- germ cells of cynomolgus monkey and human reconstructed ovaries, and in human immature primordial follicles ( Fig.10 GI). LMOD3 showed specific expression in primordial follicles of both cynomolgus monkeys and humans, with a reticular localization in the cytoplasm ( Fig.12 C). Interestingly, LMOD3 showed higher enrichment in the cytoplasm facing the ovarian medulla within human immature primordial follicles ( Fig.12 C).

[0158] Example 7: Conserved activity of chromosome X

[0159] In mammals, female cells have two X chromosomes (Xs), while male cells have one X chromosome and one Y chromosome. In females, one X is randomly inactivated (Xi) in the early embryo after implantation (X chromosome inactivation: XCI), and the amount of X chain genes in males is supplemented (Lyon MF, Nature, 190: 372-3 (1961), Zylicz JJ, and Heard E, Annu Rev Biochem, 89: 255-282 (2020)). In somatic cells, one activated X chromosome (Xa) is overactivated and supplements the X chromosome (autosomal (X: A) gene amount in both females and males (X chromosome upregulation: XCU)) (Deng X, et al., Nat Rev Genet, 15: 367-78 (2014), Ohno S, Springer-Verlag, Berlin (1967)). On the other hand, in female germ cells, Xi is reactivated (X chromosome reactivation: XCR) (Chitiashvili T, et al., Nat Cell Biol, 22: 1436-1446 (2020), Guo F, et al., Cell, 161: 1437-52 (2015), Monk M, et al., J Embryol Exp Morphol, 63: 75-84 (1981), Okamoto I, et al., Science, 374: eabd8887 (2021), Sugimoto M, and Abe K, PLoS Genet, 3: e116 (2007), Tang WW, et al., Cell, 161: 1453-67 (2015), Vertesy A, et al., Nat Commun, 9: 1873 (2018)), in mice and humans, germ cells show non-standard X chromosome amount compensation (Sangrithi MN, et al., Dev Cell, 40: 289-301e3 (2017)). However, other studies have reached different conclusions (Chitiashvili T, et al., Nat Cell Biol, 22: 1436-1446 (2020)).

[0160] Therefore, the X chromosome activity during embryonic oogenesis in the three species was compared. First, the X:A ratio of gene expression levels during mouse germ cell development was investigated based on bulk RNA-seq data, thereby ensuring high quantitative performance. Information on mouse bulk RNA-seq data (sample information, sequence summary, and data summary) is omitted. In gonadal somatic cells, the X:A ratio is slightly lower than 1 (about 0.9), indicating that XCU is generated from a single Xa, albeit incompletely ( Fig.13 A). Information on the X:A ratio analysis using the 10X / SC3-seq / Bulk RNA-seq dataset has been omitted.

[0161] In female germ cells, the X:A ratio is below 1 (about 0.9) before E12.5 during mitotic amplification, exceeds 1 (about 1.1) from E13.5 to E18.5 during the first meiotic prophase, and is downregulated to about 1 (about 1.2) at postnatal day (P) 0 and 1 in primordial follicles. Fig.13 A). Considering that both X chromosomes are gradually activated in germ cells before E14.5 (Sugimoto M, and Abe K, PLoS Genet, 3:e116 (2007)), this suggests a mild XCU in meiotic prophase and its erasure in primordial follicles. In male germ cells, the X:A ratio is about 0.83 at E11.5, decreases to about 0.65 at E12.5, and stays at about 0.7 ( Fig.13 A), suggesting that XCU counteraction also occurs in males. Interestingly, a long noncoding RNA that induces a mild upregulation of Xist (X inactivation-specific transcript), a cascade of XCI inhibitory events in mice, has been detected in primordial follicles (Brockdorff, Ashworth et al., 1992, Marahrens, Panning et al., 1997, Penny, Kay et al., 1996), but is substantially suppressed in male germ cells ( Fig.13A). The timing of the occurrence and offset of mild XCU in females is different from that of XCU offset in males, but is consistent with previous studies (Sangrithi MN, et al., Dev Cell, 40:289-301e3 (2017)). Next, based on the SC3-seq dataset, the X:A ratio in cy and human embryonic oocytes in vivo, xenotransplantation, and in vitro was studied. In female cy embryogenesis, XCI occurs simultaneously with XCU of somatic cells, and cy germ cells begin XCR based on their specialization, which is completed before about E50 (Okamoto I, et al., Science, 374:eabd8887 (2021)). In vivo, despite XCR, the X:A ratio was found to be lower than 1 in both mitotic cells and RA-responsive cells (about 0.90 and about 0.68, respectively), increased to about 1 in meiotic cells, and then decreased to below 1 in oogenic cells (about 0.71) ( Fig.13 B) XIST is highly expressed in mitotic germ cells, repressed in RA-responsive cells, upregulated in meiotic cells, and repressed again in oogenic cells ( Fig.13 B) The kinetics of X:A ratio and XIST expression are similar in xenotransplanted and in vitro embryonic oocytes, and in vivo and in vitro human embryonic oocytes ( Fig.13 B and C). Next, the dynamics of the X:A ratio of the three species were analyzed based on the 10X data set, which revealed that the three species showed the same dynamics of the X:A ratio, although the dynamic range became narrower. The ratio was below 1 in oocytes during mitosis and early meiosis (preleptotene), increased to about 1 in oocytes during late meiosis (zygotene / pachytene), and decreased to below 1 in oocytes of primordial follicles (diplotene oocytes). Fig.13 D. Fig.12 D) XIST shows the same expression profile in humans and monkeys. That is, XIST is expressed at a significant level in mitotic cells (same as gonadal somatic cells) and then downregulated. On the other hand, in mice, XIST is substantially suppressed in mitotic cells and meiotic cells and shows a mild upregulation in diplotene oocytes ( Fig.13 D. Fig.12D). These findings are consistent with the findings of Chitiashvili et al. (Chitiashvili T, et al., Nat Cell Biol, 22: 1436-1446 (2020)), who reported X: A dynamics in human embryonic oocytes, but are different from the findings of Sangrithi et al. (Sangrithi MN, et al., Dev Cell, 40: 289-301e3 (2017)), who reported mild XCU in human mitotic oogonia. Based on the collective data obtained in this example, it can be concluded that, although there is a certain degree of quantitative fluctuation depending on the measurement method, the amount of X chain genes is regulated in the same pattern in humans, monkeys and mice in general, and the occurrence of embryonic oocytes is carried out through two Xa, which is basically unrelated to XCU.

[0162] Example 8: Evaluation of the effects of adding steroid hormones to the culture medium

[0163] An experiment was conducted in which steroid hormones were added to the above culture medium. The steroid hormones here refer to progesterone, cortisol, dehydroepiandrosterone and dehydroepiandrosterone sulfate, which are sulfate conjugates thereof, and whose concentrations can be measured in cynomolgus monkey embryo blood. In the experiment, 200 ng / mL of progesterone, 100 ng / mL of cortisol, 50 nM of dehydroepiandrosterone, and 10 μM of dehydroepiandrosterone sulfate were used as concentrations equivalent to the physiological concentrations of humans or cynomolgus monkeys, and all of them were added. On the other hand, in the culture medium to which steroid hormones were not added, none of the hormones was added. The same is true in the following examples.

[0164] In the case of culture in a medium without steroid hormones, it takes 12 weeks of culture for primordial follicles to form, and no primordial follicles were found in the reconstructed ovaries after 9 weeks of culture. On the other hand, in the group with steroid hormones, primordial follicles were found in the 9th week of culture. It is believed that by adding steroid hormones, the differentiation and maturation of oocytes can be promoted, and primordial follicles can be generated in a short time ( Fig.14 , 15 ).

[0165] Example 9: Evaluation of the effects of adding retinoic acid (RA) to the culture medium

[0166] A comparison between the retinoic acid (RA) non-addition group and the RA addition group was performed without FBS addition. The RA addition group was tested at 10nM / 100nM / 500nM / 1μM RA concentrations. In the RA addition group, meiotic markers were positively expressed and oogonia markers were negatively expressed at early stages compared to the non-addition group at any concentration ( Fig.16 , 17). That is, it is believed that by adding RA to this culture system, the effect of promoting differentiation from oogonia to oocytes and the effect of promoting the initiation and progress of meiosis can be obtained. This effect is equivalent at any concentration, but as the concentration increases, it hinders the increase in the size of the reconstructed ovary, so it is believed that 10nM, which can obtain a sufficient effect, is suitable as the concentration when adding RA.

[0167] Example 10: Evaluation of the effects of simultaneous addition of steroid hormones and retinoic acid (RA) to the culture medium

[0168] In this example, it was confirmed that primordial follicles were effectively generated in the steroid hormone-added group and the retinoic acid (RA)-free group (10 nM / FBS(-)) compared to the steroid hormone-free group and the retinoic acid (RA)-free group. Fig.18 ). In addition, when steroid hormones and RA (10nM / FBS(-)) were added simultaneously, more primordial follicles were produced ( Fig.18 ). This suggests that the two factors synergistically improve the efficiency of primordial follicle production.

[0169] <Materials and Methods>

[0170] (animal)

[0171] All animal experiments were performed after ethical review by Kyoto University and Shiga University of Medical Science. ICR and immunodeficient KSN / Slc mice were purchased from SLC (Shizuoka, Japan). C57BL / 6N, mvh-RFP Tg / Tg (VR) and Dppa3(Stella)-EGFP Tg / TgTransgenic mice were established as described previously (Imamura M, et al., Mol Reprod Dev, 77: 802-11 (2010), Miyauchi H, et al., EMBO J, 36: 3100-3119 (2017), Payer B, et al., Genesis, 44: 75-83 (2006), Seki Y, et al., Development, 134: 2627-38 (2007)). Mice were housed in a specific pathogen-free animal facility under a 14-hour light period / 10-hour dark period cycle. For animal experiments using cynomolgus monkeys (cy), the steps of oocyte collection, intracytoplasmic sperm injection, preimplantation embryo culture, and embryo transfer to foster mothers were as described previously (Yamasaki J, et al., Theriogenology, 76: 33-8 (2011)). Pregnancy was confirmed by transabdominal embryo echography at 4 weeks post-fertilization (wpf), and the plasma of the pregnant monkey was collected to extract the cell-free DNA of the embryo. In order to minimize the number of cy embryos used, the cell-free DNA in maternal plasma was purified with QIAamp DNA Mini Kit (QIAGEN, 51304), and the sex of the embryo was determined by real-time PCR targeting SRY and DYS14 genes (Yasmin L, et al., Comp Med, 65: 70-6 (2015)). In order to collect cy embryos, cesarean section was performed under general anesthesia based on isoflurane and ketamine. The sex of the embryo was further confirmed by confirming the naked eye of the gonads or the PCR of the UTX / UTY gene (Villesen P, and Fredsted T, BMC Ecol, 6: 8 (2006)).

[0172] (Collection of human embryos and ovarian samples)

[0173] After obtaining the informed consent of the participants who received artificial abortion or gynecological surgery, human embryos or adult ovaries were collected. All experimental procedures were approved by the Ethics Committee of Kyoto University (license numbers G1047 and G1192). The sex of the embryo was determined by sex-specific PCR (Josef B, and Adam K, Folia Zoologica, 52: 269-274 (2003)) for ZFX / ZFY and SRY loci.

[0174] (Histological Analysis)

[0175] In immunohistochemical analysis, embryonic ovaries or reconstructed ovaries were fixed in 4% paraformaldehyde / PBS (Sigma-Aldrich, 158127) at 4°C for 30 minutes or overnight, embedded in paraffin (Leica, 39601006) or OCT compound (Sakura Finetek, 4583), and sections were prepared at a thickness of 5 μm or 8 μm. After deparaffinization, the sections were pretreated with antigen activation reagent HistVT One (Nacalai Tesque, 06380-76) at 90°C for 40 minutes. The sections were treated with blocking solution (1% bovine serum albumin (Sigma-Aldrich, A7030), 5% normal donkey serum (JIR, 017-000-121), 0.1% Tween20 and 1× phosphate buffered saline) at room temperature for 30 minutes and incubated with the primary antibody in the blocking solution at 4°C overnight. The sections were further incubated with the secondary antibody conjugated with Alexa Fluor. Fluorescence images were obtained using a laser scanning confocal microscope (OLYMPUS, FV1000; ZEISS, LSM780). For quantitative analysis of each marker, germ cells marked by the expression of DDX4 were analyzed. In order to clarify the "inner" and "outer" cortices of the embryonic ovary at 10-18wpf, the ovarian cortex was divided into two parts in the center of the cortex and stained with hematoxylin and eosin using standard procedures.

[0176] (Classification of meiotic stages in ovarian sections)

[0177] The germ cells expressing DDX4 in embryonic ovarian sections are divided into 7 stages according to the nuclear staining patterns of DAPI, SYCP3 and DMC1. The stages are defined as follows. Mitotic stage: cells without SYCP3 and DMC1 signals. Pre-leptotene: a large number of short chromatin knots (short nuclear filaments) with weak SYCP3 signals. Leptotene: a large number of short chromatin knots with weak SYCP3 and DMC1 signals. Zygotene: a large number of long filaments stained with SYCP3 with a large number of DMC1 points. Early pachytene: thick filaments stained with SYCP3 with a large number of DMC1 foci. Late pachytene: thick filaments stained with SYCP with less than 1 DMC1 point on each chromosome. Diplotene: a large number of short chromatin knots with weak or no SYCP3 signals. Diplotene cells are distinguished from pre-leptotene cells according to their size and histological morphology.

[0178] (Dissociation of cy / human embryonic ovarian tissue and cy / human reconstructed ovaries)

[0179] The embryonic ovaries collected from cy embryos at 8-18wpf were carefully dissected in cold PBS. The ovaries of embryos at 8-14wpf were minced with scissors in 0.05% trypsin / EDTA / PBS solution (GIBCO, 15400-054) and incubated at 37°C for 15 minutes, with gentle pipetting every 5 minutes. Additional dissociation medium [DMEM (GIBCO, 10313021) containing 10% FBS, 2mM L-glutamine (GIBCO, 25030081), 1X penicillin / streptomycin (GIBCO, 15070063) and 100μg / mL DNaseI (MERCK, DN25)] was added for further dissociation. The dissociated cells were used for single-cell RNA sequencing analysis or resuspended in CELLBANKER 1plus (ZENOGEN PHARMA, CB021) and stored at -80°C. Human embryonic ovaries at 11 weeks of gestation were also isolated using the same procedure and cryopreserved.

[0180] In the case of embryonic ovaries at 16wpf and 18wpf, the minced tissue was incubated in Accumax (Innovative Cell Technologies, AM105) at 37°C for 40 minutes, with gentle pipetting every 5 minutes. Dissociation medium was added for further dissociation. In SC3-seq analysis, cells were selected under an inverted microscope (OLYMPUS, IX71) to remove multiplet cells. In 10X experiments, the cell suspension was further treated with TryPLE Express (1x) (GIBCO, 12604021) at 37°C for 5 minutes to prepare a single cell suspension.

[0181] To dissociate cy / human reconstructed ovaries, tissues were incubated with TryPLE Express (1x) (GIBCO, 12604021) for 5 minutes. After adding dissociation medium, cells were processed for single cell analysis.

[0182] (In vitro culture of cy / human reconstructed ovaries under gas-liquid interface culture conditions)

[0183] 8wpf cy embryonic ovarian cells (50,000 cells / well) were seeded into the wells of Nunclon Sphera-treated U-bottom 96-well plates (NUNC, 174925) and cultured under suspension conditions in αMEM medium containing 10% FBS, 2mM L-glutamine (GIBCO, 25030081), 1×NEAA (GIBCO, 11140050), 1mM sodium pyruvate (GIBCO, 11360070), 1× penicillin / streptomycin (GIBCO, 15070063), 100μM 2-mercaptoethanol (GIBCO, 21985023) and 10μM Y-27632 dihydrochloride (TOCRIS, 1254). After 2 days, the cy reconstructed ovaries were transferred to Transwell-COL membrane inserts (Corning, 3495) immersed in αMEM (GIBCO, 12571063) containing 10% FBS, 2mM L-glutamine (GIBCO, 25030081), 150μM ascorbic acid (Sigma-Aldrich, A4403), 1× penicillin / streptomycin (GIBCO, 15070063) and 55μM 2-mercaptoethanol (GIBCO, 21985023) using glass capillaries. Half of the culture medium was replaced every 2 days. The reconstructed ovaries were cultured at 37°C in air and 5% CO2 atmosphere. Human reconstructed ovaries were made from human embryonic ovarian cells at 11 weeks of gestation and cultured under the same conditions except that AdvancedMEM was used as the basal medium.

[0184] To evaluate the coating status, pre-coated VECELL membranes (VECELL, PSVC12-10) were coated with human collagen type I (Millipore, CC050), type III (Corning, 354244), type IV (Corning, 354245), type V (Corning, 354246) and type VI (Corning, 354261), human fibronectin (Corning, 354008), iMatrix (Nippi, 892014), laminin (111 / 121 / 211 / 221 / 411 / 421 / 332 / 521) (VERITAS, BLA-LNKT-0201), growth factor-reduced matrix gel (Corning, 356230), and their combinations before IVC according to the manufacturer's coating protocol.

[0185] (In vitro culture of cy / human reconstructed ovaries under suspension conditions)

[0186] Cy-reconstructed ovaries were made from embryonic ovarian cells at 8 wpf (50,000 cells / well) under suspension conditions in Advanced MEM (GIBCO, 12492013) containing 10% FBS, 2 mM L-glutamine (GIBCO, 25030081), 1×NEAA (GIBCO, 11140050), 1 mM sodium pyruvate (GIBCO, 11360070), 1× penicillin / streptomycin (GIBCO, 15070063), 100 μM 2-mercaptoethanol (GIBCO, 21985023) and 10 μM Y-27632 dihydrochloride (TOCRIS, 1254). After 2 days, the cy reconstructed ovaries were transferred to other wells of a U-bottom culture plate using a glass capillary and cultured under suspension culture conditions in Advanced MEM (GIBCO, 12492013) [in vitro differentiation medium for primates: P-IVD medium] containing 10% FBS, 2mM L-glutamine (GIBCO, 25030081), 150μM ascorbic acid (Sigma-Aldrich, A4403), 1× penicillin / streptomycin (GIBCO, 15070063) and 55μM 2-mercaptoethanol (GIBCO, 21985023). The medium was replaced every 2 days by transferring the cy reconstructed ovaries to other wells containing freshly prepared medium. The cy reconstructed ovaries were analyzed at 3 / 6 / 9 / 12 / 15w-ivc (days 23 / 44 / 65 / 86 / 107). Human reconstructed ovaries were prepared from human embryonic ovarian cells at 11 weeks of gestation and cultured under the same conditions. In the examples where steroid hormones (progesterone (Sigma, P6149), cortisol (also known as hydrocortisone) (Sigma, H0135), dehydroepiandrosterone (also known as trans-dehydroandrostenone) (Sigma, D4000) and dehydroepiandrosterone sulfate (Cayman chemical, 15873) of its sulfate conjugate) and / or retinoic acid (Sigma, R2625) were added, the compounds were added to the culture medium from the start of in vitro culture.

[0187] (Xenotransplantation experiment)

[0188] The cy reconstructed ovaries prepared by the above culture method were transplanted under the renal capsule of immunodeficient KSN / Slc mice. The transplantation step was performed under mixed anesthesia (midazolam, medetomidine and butorphanol), and the cy reconstructed ovaries were configured under the renal capsule through fine glass capillaries. Histology and SC3-seq analysis were performed at 3 / 6 / 9 / 12 / 15 weeks after transplantation (post-transplantation, ptp).

[0189] (Isolation of mouse gonadal / ovarian cells)

[0190] To isolate mouse gonadal / ovarian cells at embryonic day (E) 11.5 for bulk RNA-seq analysis, VR females were cross-linked with Dppa3(Stella)-EGFP. Tg / Tg Male mating. Noon on the day when the vaginal plug was confirmed was designated as E0.5. The sex of the embryo at E11.5 was determined by sex-specific PCR of the Ube1 locus. In order to separate the gonadal / ovarian cells of mice at E12.5-postnatal day (P) 1, ICR females were mated with VR males and the sex of the embryos was determined by naked eye. The gonads / ovaries were incubated with 0.05% trypsin-EDTA (GIBCO, 15400-054) at 37°C for 10 minutes, separated into single cells, and quenched with an equal amount of DMEM containing 10% FBS. Large cell clusters were removed using a nylon cell screen (FALCON, 352350) and the cells were centrifuged at 200rcf for 5 minutes. The cell pellet was resuspended in DMEM / F12 medium (GIBCO, 1130-082) containing 0.1% BSA fraction V (Gibco, 15260037) and sorted into VR using a fluorescence activated cell sorter (BD Biosciences, FACS Aria III). + Germ cells and VR - Somatic cells.

[0191] (10X Genomics single-cell RNA-seq)

[0192] Cells dissociated from cy embryonic ovaries and cy reconstructed ovaries were stained with DRAQ7 to distinguish dead cells. All DRAQ7 were recovered using a fluorescence activated cell sorter (BD Biosciences, FACS Aria III). - Surviving cells. The subsequent 10X scRNA-seq library preparation (10X Genomics, Single Cell 3'Kit v.3 and v.3.1) and sequencing on the NovaSeq6000 platform (Illumina) were performed according to the manufacturer's instructions. The raw files were processed with the "mkfastq" command of Cell Ranger (v.5.0.1) and mapped to the reference genome of cynomolgus monkey (MacFas5.0 in the case of cynomolgus monkey and GRCh38.p12 in the case of human).

[0193] (Analysis of cy scRNA-seq data (10X))

[0194] According to the online tutorial (https: / / satijalab.org / seurat / index.html), the gene barcode matrix was analyzed using the "Seurat" R package (v.3.2.2) (Satija R, et al., Nat Biotechnol, 33: 495-502 (2015)). Low-quality cells, putative double cells / multiple cell aggregates, and putative stripped nuclei were excluded from downstream analysis (Table 2). The raw counts of each cell were divided by the size factor calculated by the "scran" R package (v.1.14.1), followed by logarithmic transformation [log2 (size-scaled (ss) UMI+1)] for further analysis. Double cells were removed using the "Scrublet" python package (v.0.2.1) according to the reported steps (Wolock SL, et al., Cell Syst, 8: 281-291e9 (2019)). The double cell estimation threshold was determined based on a bimodal distribution calculated by default parameters. Next, batch correction was implemented using the "RunFastMNN" function of the Seurat R package under default settings. Cell clusters were characterized and visualized using the "RunUMAP" function of Seurat. The resolution of the "FindClusters" function was set to 0.5 in the overall cell analysis and to 0.8 in the analysis of reproductive / granulosa cells. Clusters were annotated by confirming the expression patterns of previously characterized marker genes in embryonic ovarian cells of humans and mice. The sources and identification numbers of other R or Python packages are recorded in Table 3.

[0195] [Table 3-1]

[0196] Reagents and Tools List

[0197]

[0198] [Table 3-2]

[0199]

[0200] [Table 3-3]

[0201] DMEM / F12 GIBCO 11330057 MEMa, nucleoside GIBCO 12571063 Advanced MEM GIBCO 12492013 CMRL medium, without glutamine GIBCO 11530037 DMEM / F-12, without glutamine GIBCO 21331020 IMDM GIBCO 12440053 KnockOut DMEM GIBCO 10829018 McCoy's 5A (Modified) Medium GIBCO 16600082 MesenPRO RS Medium GIBCO 12746012 StemPro-34 SFM(1×) GIBCO 10639011 Waymouth medium GIBCO 11220035 Glasgow's MEM (GMEM) GIBCO 11710035 Advanced RPMI 1640 GIBCO 12633012 Fetal bovine serum GIBCO 10437028 BSA Fraction V GIBCO 15260037 Penicillin-Streptomycin (5000U / mL) GIBCO 15070063 MEM non-essential amino acid solution (100×) GIBCO 11140-050 Sodium pyruvate (100 mM) GIBCO 11360070 L-Glutamine (200 mM) GIBCO 25030081 L-Ascorbic Acid Sigma-Aldrich A4403 2-Mercaptoethanol GIBCO 21985023 Y-27632 dihydrochloride TOCRIS 1254 Trypsin-EDTA (0.5%), without phenol red GIBCO 15400054 TryPLE Express GIBCO 12604021 Accumax Innovative Cell Technologies AM105 DNase I MERCK DN25 CELLBANKER 1 plus ZENOGEN PHARMA CB021 Human collagen type I Millipore CC050 Human collagen type III Corning 354244 Human collagen type IV Corning 354245 Human collagen type V Corning 354246 Human collagen type VI Corning 354261

[0202] [Table 3-4]

[0203]

[0204] [Table 3-5]

[0205]

[0206] [Table 3-6]

[0207] CCD Camera Olympus DP70 Cryostat Leica CM1850

[0208] (cy germ cell analysis (10X))

[0209] Further analysis of DDX4 + The cells in the germ cell cluster were divided into 13 clusters using the "FindClusters" function. After removing two small clusters consisting of putative two-cell / multi-cell aggregations that co-expressed other cell type markers, the 11 substages of the first meiotic phase were defined as "Mitosis 1 / 2 / 3", "Pre-leptotene 1 / 2 / 3", "Leptotene", "Zygotene", "Pachytene 1 / 2" and "Unclassified" based on the expression patterns of important germ cell markers. Cells expressing GDF9 / NOBOX / ZP3 were manually selected as other clusters defined as "Diplotene". In order to identify highly variable genes (HVGs) in the occurrence of cy female germ cells, the "FindVariableFeatures(selection.method="vst,"nfeatures="2000)" function of the Seurat R package was implemented across 11 substages (mitosis-diplotene), and 2000 HVGs were detected. Among them, genes with low expression levels [cluster average of log(ssUMI+1) ≤ 0.5: 519 genes] were removed, and 1481 genes were used for heat map visualization, GO enrichment analysis (see below), and calculation of correlation coefficients for each meiotic subphase ( Figure 6 H and I). Pseudotime trajectory analysis was performed using the "Monocle 3" R package (v.0.2.1). Cy germ cells from cultured reconstructed ovaries at 12 / 15w-ivc were also treated in the same way.

[0210] (Cy granulocyte analysis (10X))

[0211] Further analysis of FOXL2 +Cells of granulocyte clusters. The count matrix of spliced ​​transcripts and unspliced ​​transcripts was created by implementing the commands "count--include-introns" and "count" in Cell Ranger (v.5.0.1). The resolution of the "FindClusters" function was set to 0.8, and the cells were divided into 12 clusters after removing two small clusters consisting of putative bicellular / multicellular aggregations that co-expressed other cell type markers. RNA velocity analysis was performed using the "scvelo.pp.moments(n_pcs=30, n_neighbors=30)" and "scvelo.tl.velocity(mode='stochastic')" functions of the scVelopython package (v.0.2.0) (Bergen V, et al., Nat Biotechnol, 38:1408-1414 (2020)). According to the online tutorial (https: / / scanpy-tutorials.readthedocs.io / en / latest / paga-paul15.html), using the "scanpy.tl.paga" function of Scanpy (v.1.2.2) with Python (v.3.7.7), in Figure 8 The partition-based graph abstraction (PAGA) graph (Wolf FA, et al., Genome Biol, 20:59 (2019)) was calculated on the clusters marked in A, and the threshold of cluster connection was set to 0.03. In order to identify HVGs from 12 clusters, 1500 HVGs were detected using the "FindVariableFeatures(selection.method="vst", "nfeatures="1500)" function of the Seurat R package. Among them, genes with low expression levels [cluster average of log(ssUMI+1) ≤ 0.5: 712 genes] were removed, and 788 genes were used for heat map visualization and GO enrichment analysis (see below) ( Figure 8 D) Cy granulosa cells from cultured reconstructed ovaries at 12 / 15w-ivc were also treated in the same manner.

[0212] (cy / SC3-seq of human germ cells)

[0213] Under a stereomicroscope (Leica, S8 APO), glass capillaries were used to manually select germ cells dissociated from cy / human embryonic ovaries and cy / human reconstructed ovaries in vivo. cDNA synthesis and amplification methods were as previously described (Kurimoto K, et al., Nucleic Acids Res, 34: e42 (2006), Kurimoto K, et al., Nature protocols, 2: 739-52 (2007), Nakamura T, et al., Nature, 537: 57-62 (2016), Nakamura T, et al., Nucleic Acids Res, 43: e60 (2015)). The quality of the amplified cDNA was determined by SYBR real-time PCR targeting PPIA, GAPDH, NANOG, DDX4, DAZL, SYCP3, SYCP1, ZP3 and FOXL2 genes. Primer sequences are shown in Table 4. The SC3-seq library was constructed as previously described (Nakamura T, et al., Nature, 537:57-62 (2016), Nakamura T, et al., Nucleic Acids Res, 43:e60 (2015), Okamoto I, et al., Science, 374:eabd8887 (2021)) and sequenced on the Illumina NextSeq500 / 550 platform using NextSeq 500 / 550 high output kit v.2 (75 cycles) (FC-404-2005, Illumina).

[0214] [Table 4]

[0215] Primers used in this example

[0216] Macaca fascicularis

[0217]

[0218] Homo sapiens

[0219]

[0220] (Bulk RNA-seq analysis of mouse gonadal / ovarian cells)

[0221] According to the manufacturer's instructions, mouse gonadal / ovarian cells were dissolved and total RNA was purified using NucleoSpin RNA XS (TakaraBio, U0902A). 1 ng of total RNA from each sample was used for cDNA synthesis and amplification. cDNA synthesis and library construction were as previously described (Ishikura Y, et al., Cell reports, 17: 2789-2804 (2016), Nakamura T, et al., Nucleic Acids Res, 43: e60 (2015), Okamoto I, et al., Science, 374: eabd8887 (2021)). Sequencing was performed on the Illumina NextSeq 500 / 550 platform using NextSeq 500 / 550 high output kit v.2 (75 cycles) (FC-404-2005, Illumina).

[0222] (Bulk RNA-seq / SC3-seq read mapping and conversion to gene expression levels)

[0223] Download the genomic sequence and transcript annotations from the NCBI ftp site (GRCm38.p6 for mice, GRCh38.p12 for humans, and MacFas5.0 for cynomolgus monkeys). Since the expression level is calculated as a gene (Entrez gene) rather than mRNA, Bulk RNA-seq / SC3-seq only reads the 3' end of the transcript. Read segment trimming, mapping, and expression level evaluation were performed as previously described (Ishikura Y, et al., Cell reports, 17: 2789-2804 (2016), Nakamura T, et al., Nature, 537: 57-62 (2016)).

[0224] (Orthologous genes among 3 species)

[0225] In order to perform interspecific comparative analysis of SC3-seq and 10X scRNA-seq data, a list of orthologous genes of three species was obtained from Ensembl BioMart (Ensembl 97: Jul2019). The interspecies integration of SC3-seq data used 1 to 1 (1-1) orthologous genes between human and cy (16734 genes). The interspecies integration of 10X data used 1 to 1 to 1 (1-1-1) orthologous genes across three species (15007 genes). Since the list of orthologous genes from Ensembl BioMart is relatively incomplete, the LiftOver tool was used to make a coordinate comparison list of the genome as previously reported (Nakamura T, et al., Nature, 537: 57-62 (2016), Sasaki K, et al., Cell Stem Cell, 17: 178-94 2015). Listings of orthologous genes are omitted.

[0226] (cy / SC3-seq data analysis of human germ cells)

[0227] SC3-seq data analysis was performed using R software (v.4.0.2). The read counts were normalized to 200,000 reads, followed by logarithmic transformation (log2 (normalized read count + 1)). "All expressed genes" were defined as genes with a log2 (normalized read count + 1) value of more than 2 in at least one sample [more than about 10 to 20 copies per cell] (Nakamura T, et al., Nucleic Acids Res, 43: e60 (2015)). Unsupervised hierarchical clustering (UHC) was performed using the "hclust" function based on Euclidean distance and Ward's method (ward.D2). Principal component analysis (PCA) was performed using the "prcomp" function without scaling.

[0228] In the interspecific analysis between monkeys and humans, 1-1 orthologous genes were used between humans and monkeys, and the data were processed using the "Seurat" R package (v.4.0.4). The cy SC3-seq data set was integrated with the human data set using the Seurat CCA algorithm, i.e., "FindIntegrationAnchors" (parameter "anchor.features" = 5000) and the default "IntegrateData" function. The integrated (integrated) germ cells were divided into 4 clusters using Seurat's "FindClusters" function (parameter "resolution" = 0.4). PCA was performed on the corrected count matrix obtained after adjusting for species differences. Gene expression levels were compared between 2 consecutive clusters, and DEGs were defined as having more than 2-fold changes between clusters and an average gene expression level [log2 (normalized counts + 1)] greater than 3.

[0229] (Gene Ontology Enrichment Analysis)

[0230] Gene ontology (GO) enrichment analysis for HVG / DEG was implemented using DAVID web tool (Huang da W, et al., Nature protocols, 4: 44-57 (2009)). Since the annotation of cynomolgus monkey genes is relatively incomplete, the annotation of people corresponding to the annotation of cynomolgus monkeys is used in GO analysis. Genes missing from the 1-1 orthologous gene list from Ensembl BioMart are supplemented according to the orthologous gene list made with LiftOver tool.

[0231] (Interspecific analysis among three animal species using the 10X public dataset)

[0232] For interspecies analysis between humans, monkeys, and mice, 10X scRNA-seq datasets for human in vivo germ cells (Chitiashvili T, et al., Nat Cell Biol, 22: 1436-1446 (2020)) and three datasets for mouse in vivo germ cells (Ge W, et al., Cell Mol Life Sci, 78: 695-713 (2021), Niu W, and Spradling AC, Proc Natl Acad Sci USA, 117: 20015-20026 (2020)) were obtained. The downloaded raw files were processed with the "mkfastq" command of Cell Ranger and mapped to the reference genome sequence (GRCm38.p6 in the case of mice and GRCh38.p12 in the case of humans). Low-quality cells and putative double cells were excluded from downstream analysis. Since the thresholds for these cells depend on the quality of each dataset (e.g., the reagent version and reference genome used), the number of genes, UMIs, and mitochondrial genes detected in each dataset was carefully evaluated to define the thresholds. Each dataset was processed by the same method as described above for the cy 10X scRNA-seq data analysis, and DDX4 / Ddx4 +Germ cell clustering is used for the following interspecies comparative analysis. The cy / human 10XscRNA-seq dataset produced in this example was integrated with 4 public datasets. The size of each dataset was scaled to 10000UMI (ss10kUMI) and processed using the CCA algorithm, i.e., the "FindIntegrationAnchors" (parameter "anchor.features" = 2000) and "IntegrateData" functions of the "Seurat" R package (v.3.2.2). PCA was performed on the corrected count matrix obtained after adjusting for species differences, and the "RunUMAP" function was used to visualize UMAP in 30 dimensions. The integrated germ cells were divided into 15 clusters using the "FindClusters" function of Seurat (parameter "resolution" = 0.8). Based on the expression pattern of important germ cell markers, each cluster was defined as "Mitosis 1 / 2 / 3 / 4", "Pre-leptotene 1 / 2 / 3", "Leptotene 1 / 2 / 3", "Zygotene 1 / 2", "Pachytene 1 / 2" or "Diplotene". In order to confirm the accuracy of the merged clusters, pseudo-time trajectory analysis was performed on each data set using the "Monocle 3" R package (v.0.2.1). Among the 1-1-1 orthologous genes supplemented with the LiftOver tool, common HVGs (237 genes) were obtained across 3 data sets / species, and the Pearson correlation coefficient of the average HVG expression level during the meiotic subphases of the three animal species was calculated.

[0233] (Primate-specific variable genes conserved in female meiosis)

[0234] Among the DEGs identified by interspecific comparative analysis based on the above SC3-seq analysis, the common and variable 1-1 orthologous genes were defined as genes conserved in humans and monkeys. The average expression levels of these genes in the 10X dataset from the three animal species were verified using clusters defined by the 10X interspecific analysis. Genes that also changed in mice were manually selected as genes conserved among the three species (130 genes, Fig.12 A), genes that showed no expression changes in mice were selected as genes that showed specific changes in primates (83 genes, Fig.10 F).

[0235] (Chromosome Expression Rate Analysis)

[0236] For Bulk RNA-seq / SC3-seq data, the autosome (chromosome (chr.) 10 in the case of humans, chr. 15 in the case of monkeys, and chr. 3 in the case of mice) : total autosome ratio and chr. X (X chromosome): total autosome ratio (X: A ratio) were calculated by the reported method (Okamoto I, et al., Science, 374: eabd8887 (2021)). In short, the 75th percentile log2 (normalized read count + 1) value of the genes expressed in the autosomes and chr. X in each sample / cell was calculated [the maximum log2 (normalized read count + 1) value in the analyzed cells exceeded 2] and was used as the representative expression value of the sample / cell. In the case of humans, 19307 / 811 / 728 genes were used for total autosomes / Chr.10 / Chr.X, in the case of monkeys, 20576 / 849 / 841 genes were used for total autosomes / Chr.15 / Chr.X, and in the case of mice, 12399 / 618 / 503 genes were used for total autosomes / Chr.3 / Chr.X.

[0237] For 10X scRNA-seq data, the average log2(ss10kUMI+1) value of all expressed genes in autosomes (chr.10 in humans, chr.15 in monkeys, and chr.3 in mice), total autosomes, and chr.X that were expressed in more than two cells was used to calculate the ratios of autosomes:A and X:A. In the case of humans, 17190 / 682 / 725 genes are used for total autosomes / Chr.10 / Chr.X (Chitiashvili T, et al., Nat Cell Biol, 22: 1436-1446 (2020)), in the case of monkeys, 18066 / 736 / 791 genes are used for total autosomes / Chr.15 / Chr.X, and in the case of mice, 17698 / 896 / 744 genes are used for total autosomes / Chr.3 / Chr.X (Niu W, and Spradling AC, Proc Natl Acad Sci USA, 117: 20015-20026 (2020)).

[0238] (Statistical Analysis)

[0239] All statistical analyses were performed using R software (v.4.0.2). Tukey-Kramer test and T-test were performed using the "TukeyHSD" and "t.test" functions with default settings.

[0240] (Availability of data and code)

[0241] The accession number for the data in this study is GSE194266 (GEO database). R and Python codes used to reproduce the analyses and figures are available upon request.

[0242] Industrial Applicability

[0243] By using the ovarian follicles induced according to the present invention, the maturation process of female germ cells of primates can be observed in vitro, which is useful for understanding the mechanism of germ cell development. Therefore, according to the present invention, an important foundation can be provided for both biology and medicine.

[0244] This application is based on Japanese Patent Application No. 2022-117665 (filing date: July 25, 2022) filed in Japan, the contents of which are all incorporated herein by reference.

Claims

1. A method for producing an oocyte or an ovarian follicle, or a reconstructed ovary containing an oocyte or an ovarian follicle, the method comprising the step of culturing a primate reconstructed ovary under suspension culture conditions.

2. The method according to claim 1, wherein: The reconstructed ovary is constructed by agglutinating primate germ cells and primate embryonic ovarian somatic cells.

3. The method according to claim 2, wherein: The germ cells and embryonic ovarian somatic cells are prepared by dissociation from primate embryonic ovaries by enzymatic treatment.

4. The method according to claim 2 or 3, wherein: The germ cell is at least one selected from the group consisting of an oogonia, a primordial germ cell, and a primordial germ cell-like cell.

5. The method according to any one of claims 2 to 4, wherein: At least one of the germ cells and the ovarian somatic cells is derived from pluripotent stem cells.

6. The method according to any one of claims 1 to 5, wherein: The cultures were cultured in suspension conditions for more than 9 weeks.

7. The method according to any one of claims 1 to 6, wherein: The culture under the suspension culture condition is culture in a medium containing L-glutamine, ascorbic acid and 2-mercaptoethanol.

8. The method according to claim 7, wherein: The culture medium also contains retinoic acid.

9. The method according to claim 7, wherein: The culture medium further comprises one or more selected from the group consisting of progesterone, cortisol, dehydroepiandrosterone and dehydroepiandrosterone sulfate.

10. The method according to any one of claims 1 to 9, wherein: Primates are humans.

11. An oocyte, an ovarian follicle or a reconstructed ovary produced by the method according to any one of claims 1 to 10.

12. A method for producing an egg, the method comprising the step of maturing the oocyte according to claim 11.

13. A kit for producing an oocyte or an ovarian follicle, or a reconstructed ovary containing an oocyte or an ovarian follicle, the kit comprising a culture container for suspension culture, and L-glutamine, ascorbic acid, and 2-mercaptoethanol. The kit of claim 13 , further comprising a basal medium. 15 . The kit according to claim 13 or 14 , further comprising one or more selected from the group consisting of retinoic acid, progesterone, cortisol, dehydroepiandrosterone and dehydroepiandrosterone sulfate.

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