In vitro culturing method for inducing ovarian follicles from fetal ovarian cells of primate

Abstract

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

Description

In vitro culture method for inducing follicles from primate fetal ovarian cells

[0001] The present invention relates to a method for inducing follicles etc. in vitro, and more particularly to a method for producing oocytes or follicles, or a reconstituted ovary containing oocytes or follicles, which comprises a step of culturing a reconstituted ovary in a suspension culture.

[0002] Germ cells differentiate into either eggs or sperm, which then combine to form a zygote with full developmental capacity, thereby ensuring the permanence and diversity of genetic information across generations. Disruptions in germ cell development can lead to serious consequences, including infertility and genetic or epigenetic disorders in offspring. Therefore, understanding the mechanisms of germ cell development is a fundamental theme in both biology and medicine.

[0003] In mammals, germ cells arise early in embryonic development as primordial germ cells (PGCs). PGCs initiate epigenetic reprogramming, undergo migration, and colonize the embryonic gonad. In the embryonic gonad, PGCs, termed oogonia in females and gonocytes in males, exhibit similar genetic and epigenetic characteristics until the onset of overt sexual differentiation. In females, in response to cues from the fetal ovary, particularly pre-granulosa cells, oogonia rapidly differentiate into oocytes through early meiotic prophase. After completing the first meiotic prophase, oocytes are surrounded by a single layer of granulosa cells to form primordial follicles, which serve as the source of oogenesis. In contrast, in males, in response to cues from the fetal testis, particularly Sertoli cells, gonocytes differentiate into pro-spermatogonia, which then differentiate into spermatogonia / spermatogonial stem cells (SSCs), which serve as the source of spermatogenesis. The mechanisms of mammalian germ cell development have been studied exclusively using mice as a model organism, and with the insights gained from such studies and advances in pluripotent stem cells (PSCs) and reproductive technologies, the entire process of both female and male germ cell development in mice has been reconstituted in vitro using mouse PSCs (mPSCs) as starting material.

[0004] For example, it has been reported that primordial germ cell-like cells (PGCLCs), which have been produced by inducing differentiation from mouse pluripotent stem cells, are aggregated with fetal ovarian somatic cells to produce a reconstituted ovary, and that the reconstituted ovary is cultured using the air-liquid interface culture method, thereby inducing ovarian follicles (Non-Patent Document 1). Similarly, it has also been reported that follicles are induced by culturing fetal ovarian tissue using the air-liquid interface culture method (Non-Patent Document 2).

[0005] Furthermore, the techniques used to create reconstituted ovaries in mice can also be applied to human PGCLCs. The present inventors previously constructed xenogeneic reconstituted ovaries by aggregating human PGCLCs and mouse fetal ovarian somatic cells. They then successfully differentiated these reconstituted ovaries into early oocyte-like cells at the onset of meiosis I by culturing them for approximately three months using the air-liquid interface culture method (Non-Patent Document 3). However, no method for long-term culture of allogeneic reconstituted ovaries of primates, including humans, is known, and no in vitro culture method exists to induce cells derived from primate fetal ovaries to develop into more mature structures, such as follicles (primordial follicles / primary follicles).

[0006] Hayashi K, et al., Nat Protoc. 12(9): 1733-1744 (2017)Morohaku K, et al., Nat Protoc. 12(9): 1817-1829 (2017)Yamashiro C, et al., Science. 362(6412):356-360 (2018)

[0007] Therefore, an object of the present invention is to provide a method for maintaining reconstituted ovaries of primates, including humans, in an in vitro culture environment for a long period (e.g., 7 weeks or more, 9 weeks or more, and preferably 12 weeks or more).Another object of the present invention is to provide a method for reproducing the progression of meiosis and follicle formation within the reconstituted ovaries by culturing the reconstituted ovaries for a long period of time.

[0008] The present inventors attempted to identify conditions for long-term in vitro culture of reconstituted ovaries of primates, including humans. First, using cynomolgus monkeys as a primate model, fetal ovaries were harvested from cynomolgus monkeys, subjected to tissue dissociation using enzymes, and then cryopreserved (long-term). After thawing, the cells were used to prepare reconstituted ovaries (cell aggregates). When the prepared cynomolgus monkey reconstituted ovaries (cy-reconstituted ovaries) were cultured using the conventional air-liquid interface culture method, they gradually collapsed over time, making it difficult to maintain their morphology. This demonstrated that the conventional air-liquid interface culture method is not suitable for long-term in vitro culture of cy-reconstituted ovaries. This difference between mouse and cynomolgus monkey ovaries is thought to reflect the differences in biophysical properties between mouse and primate ovarian cells.

[0009] Therefore, we investigated conditions suitable for long-term culture of reconstituted primate ovaries. After extensive investigation, we concluded that the main advantage of the air-liquid interface culture method is improved aeration and oxygen supply to the cultured tissue, but that aeration and oxygen supply may not be important in the culture of mammalian ovaries. Therefore, rather than improving the air-liquid interface culture method, we decided to adopt a completely different culture method. Specifically, we focused on suspension culture and demonstrated that culturing reconstituted ovaries from cynomolgus monkeys using this method maintained the reconstituted ovaries for a long period of time (over 12 weeks). Furthermore, we demonstrated that mitotic oogonia in the reconstituted ovaries differentiated into oocytes, completing the first meiotic prophase, and contained primordial follicle-like structures. Analysis of cell morphology and the expression / intracellular localization of important markers in these cells, as well as transcriptome analysis, confirmed that the primordial follicle-like structures obtained by the above culture method have properties very similar to those of in vivo primordial follicles. Furthermore, it was found that this in vitro culture method can also be applied to human fetal ovarian cells. Based on these findings, the inventors conducted further research and completed the present invention.

[0010] That is, the present invention provides the following: [1] A method for producing oocytes or follicles, or a reconstituted ovary containing oocytes or follicles, comprising the step of culturing a reconstituted ovary of a primate under suspension culture conditions. [2] The method described in [1], wherein the reconstituted ovary is constructed by aggregating primate germ cells and primate fetal ovarian somatic cells. [3] The method described in [2], wherein the germ cells and fetal ovarian somatic cells are prepared by dissociating them from a primate fetal ovary by enzymatic treatment. [4] The method described in [2] or [3], wherein the germ cells are at least one type selected from the group consisting of oogonia, primordial germ cells, and primordial germ cell-like cells. [5-1] The method described in any one of [2] to [4], wherein at least one of the germ cells and ovarian somatic cells is derived from a pluripotent stem cell. [5-2] The method described in [5-1], wherein the pluripotent stem cells are induced pluripotent stem cells. [6-1] The method described in any one of [1] to [5-2], wherein the culture under suspension culture conditions is carried out for 9 weeks or longer. [6-2] The method according to any one of [1] to [5-2], wherein the culture under suspension culture conditions is carried out for 12 weeks or more. [7-1] The method according to any one of [1] to [6-2], wherein the culture under suspension culture conditions is in a medium containing L-glutamine, ascorbic acid, and 2-mercaptoethanol. [7-2] The method according to [7-1], wherein the concentration of L-glutamine in the medium is 10 μM to 1 M. [7-3] The method according to [7-1] or [7-2], wherein the concentration of ascorbic acid in the medium is 1 μM to 1 M. [7-4] The method according to any one of [7-1] to [7-3], wherein the concentration of 2-mercaptoethanol in the medium is 0.1 μM to 1 M. [8] The method according to any one of [7-1] to [7-4], wherein the medium further contains retinoic acid. [9-1] The method according to any one of [7-1] to [8], wherein the medium further contains one or more selected from the group consisting of progesterone, cortisol, dehydroepiandrosterone, and dehydroepiandrosterone sulfate. [9-2] The method according to [9-1], wherein the concentration of retinoic acid in the medium is 0.1 nM to 5 μM.[9-3] The method according to [9-1] or [9-2], wherein the concentration of progesterone in the medium is 0.01 μg / mL to 10 μg / mL. [9-4] The method according to any one of [9-1] to [9-3], wherein the concentration of cortisol in the medium is 0.01 μg / mL to 10 μg / mL. [9-5] The method according to any one of [9-1] to [9-4], wherein the concentration of dehydroepiandrosterone in the medium is 0.1 nM to 5 μM. [9-6] The method according to any one of [9-1] to [9-5], wherein the concentration of dehydroepiandrosterone sulfate in the medium is 0.1 μM to 5 mM. [9-7] The method according to any one of [7-1] to [9-6], wherein the medium contains serum. [9-8] The method according to [9-7], wherein the concentration (v / v) of serum in the medium is 0.01% to 50%.

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

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

[10] .

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

[11] . [13-1] A kit for producing an oocyte or follicle, or a reconstituted ovary containing an oocyte or follicle, comprising a culture vessel for suspension culture, and L-glutamine, ascorbic acid, and 2-mercaptoethanol. [13-2] The kit according to [13-1], further comprising one or more molecules selected from the group consisting of retinoic acid, progesterone, cortisol, dehydroepiandrosterone, and dehydroepiandrosterone sulfate. [13-3] The kit according to [13-1] or [13-2], further comprising serum.

[14] The kit according to any one of [13-1] to [13-3], further comprising a basal medium. [15-1] The kit according to any one of [13-1] to

[14] , further comprising retinoic acid. [15-2] The kit according to any one of [13-1] to [15-1], further comprising one or more members selected from the group consisting of progesterone, cortisol, dehydroepiandrosterone, and dehydroepiandrosterone sulfate.

[0011] The present invention enables the maintenance of reconstituted ovaries of general primates, including humans, ex vivo for long periods (e.g., 7 weeks or more, 9 weeks or more, preferably 12 weeks or more), and also enables the maturation of germ cells such as primordial germ cell-like cells, primordial germ cells, and oogonia into cells at the meiotic stage, thereby inducing primordial follicle-like structures. Using such a method and the primordial follicle-like structures induced by this method, it becomes possible to observe the differentiation process of female germ cells in primates ex vivo, which is useful for understanding the mechanisms of germ cell development. Thus, the present invention provides an important foundation for both biology and medicine.

[0012] Ovarian development in cynomolgus monkey fetuses. (A) Scheme of female germ cell development in cynomolgus monkeys (cy). wpf: week after fertilization; PGC: primordial germ cell. (B) Macroscopic findings of the developing cy ovary at 8-18 wpf. Scale bar = 1 mm. (C) Hematoxylin and eosin (H&E) staining of the ovary from a cy fetus at 8 wpf. Arrows indicate oogonia. M: mesonephros. Scale bar = 100 μm (top), 40 μm (bottom). (D) H&E staining of the ovarian cortex from a cy fetus at 10-18 wpf. The ovarian cortex was divided into outer and inner cortices by a line that bisects the ovarian cortex. Black arrow: oocyte with a large nucleus with condensed, filamentous chromatin. White arrow: primordial follicle. Scale bar = 50 μm. (E) Immunofluorescence (IF) analysis of oogonia markers (POU5F1, TFAP2C), RA-responsive markers (ZGLP1, STRA8), meiotic markers (γH2AX, SYCP1), and oogenesis markers (ZP3, TP63) during the development of the cy fetal ovary. Representative images of each marker are shown. Germ cells were labeled with DDX4. Nuclear DAPI staining is shown in white. The enlarged images in the upper right corner of each panel show the expression of key markers co-stained with DDX4. The enlarged images in the lower right corner of each panel show the expression of DDX4 co-stained with DAPI (white). Numbers in the upper left corner indicate the embryonic stage (wpf). Scale bar = 20 μm. (F) Representative images of germ cells immunostained for SYCP3, DMC1, and DDX4 at each meiotic substage. Nuclear DAPI staining is shown in white. Meiotic substages were defined based on the staining patterns of SYCP3, DMC1, and DAPI, as described in Materials and Methods. Scale bar = 10 μm. (G) Percentage of germ cells in each meiotic substage defined in Figure 1F. The average percentage of ovarian cortex outside or inside each meiotic substage was calculated from ovaries of at least three fetuses. Color coding is as indicated. Histological analysis of cy fetal ovaries in vivo and in xenografted reconstituted ovaries. (A) Size of developing cy fetuses and ovaries at 8 to 18 wpf. The lines indicate LOESS curves fitted to the sample population. (B and C) IF of cy fetal gonads at 8 wpf (B) and ovarian cortex of cy fetuses at 10 to 18 wpf (C).Staining results for FOXL2 (a granulosa cell marker), DDX4 (a germ cell marker), and DAPI (nuclei, white) are shown. Ovarian cortices from 10 to 18 wpf were divided into outer and inner cortices along the line that bisects the ovarian cortex. Scale bars = 40 μm (B) and 50 μm (C). (D) The granulosa / germ cell ratio at each developmental stage is shown, as assessed from IF data. (E) IF for cell proliferation marker (Ki67), apoptosis markers (cleaved PARP, cleaved CASPASE 3), extracellular matrix marker (LAMININ), and oogenesis markers (FIGLA, NOBOX, and NLRP5) during ovarian development in cytoplasmic fetuses. Representative images for each marker are shown. Germ cells were marked with DDX4. Nuclear DAPI staining is shown in white. The enlarged images in the upper right corner of each panel show the expression of key markers co-stained with DDX4, except for NLRP5. The enlarged images in the lower right corner of each panel show the expression of DDX4 co-stained with DAPI (white). Numbers in the upper left corner indicate the fetal stage (wpf). Scale bar = 20 μm. (F) DDX4 from IF of the outer / inner cortex of cy fetal ovaries at 8-18 wpf. + Percentage of germ cells positive for individual markers. Average values ​​from three or more biological replicates are shown. (G) H&E staining of transplanted cy-reconstituted ovaries at 3, 6, 9, 12, and 15 weeks post-transplantation (w-ptp). The dashed line indicates the cy-reconstituted ovary transplanted under the kidney capsule of KSN / Slc mice. K: Mouse kidney. Scale bar = 200 μm. Induction of cy follicles from fetal gonad cells by xenotransplantation. (A) Scheme of xenotransplantation experiment. cy-reconstituted ovaries generated from in vivo ovarian cells at 8 wpf were transplanted under the kidney 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 transplanted cy-reconstituted ovaries at 3, 6, 9, 12, and 15 weeks post-transplantation (w-ptp). FOXL2 +Primordial follicle-like complexes surrounded by granulosa cells were observed in the reconstituted ovaries at 12 and 15 w-ptp. Arrows indicate the reconstituted ovaries. FOXL2: granulosa cell marker; DDX4: germ cell marker. Scale bars = 1 mm (a) and 40 μm (b and c). (C) IF analysis of oogonia markers (POU5F1, TFAP2C), meiotic markers (SYCP3, SYCP1), and oogenesis markers (ZP3, TP63) in transplanted cy-reconstituted ovaries. Germ cells and nuclei were labeled with DDX4 and DAPI (white), respectively. The enlarged images in the upper right corner of each panel show the expression of each marker co-stained with DDX4. The enlarged images in the lower right corner of each panel show the expression of DDX4 co-stained with DAPI (white). The numbers in the upper left corner indicate the age after transplantation (w-ptp). Scale bars = 20 μm. (D) DDX4 from IF of cy-reconstituted ovaries at 3–15 w-ptp. +Percentage of germ cells positive for individual markers. Mean values ​​with SD are shown. (E) H&E staining of transplanted cy-reconstituted ovaries at 21 wpf. The dashed line indicates a cy-reconstituted ovary transplanted under the kidney capsule of a KSN / Slc mouse. K: Mouse kidney. Scale bar = 200 μm (left) and 100 μm (right). In vitro culture system for inducing follicles from fetal gonad cells. (A) Scheme of the in vitro culture system for inducing cy follicles. cy-reconstituted ovaries prepared from fetal gonad cells at 8 wpf were cultured either at the air-liquid interface or in suspension (see Materials and Methods). (B) Macroscopic appearance (a), H&E staining (b, c), and IF for FOXL2 / DAZL / DAPI (d) of cy-reconstituted ovaries cultured at the air-liquid interface at 3 and 6 weeks in vitro (w-ivc). FOXL2: granulosa cell marker; DAZL: germ cell marker; DAPI: nuclei. Scale bars = 200 μm (a), 100 μm (b), and 40 μm (c and d). (C) Quantitative analysis of the size of cy-reconstituted ovaries cultured in Advanced MEM or αMEM as the basal medium in P-IVD medium. Mean values ​​from three or more biological replicates are shown with SD, except for αMEM-reconstituted ovaries at 15 w-ivc (n = 1). **P < 0.01, Welch's t-test. (D) Macroscopic images (a) and H&E-stained images (b) of cy-reconstituted ovaries cultured in P-IVD medium at 3 / 6 / 9 / 12 / 15 w-ivc. Arrowheads indicate primordial follicle-like structures. Scale bars = 200 μm (a) and 40 μm (b). (E) IF analysis of cultured cy-reconstituted ovaries for key markers listed in Figure 1E. Germ cells and nuclei were marked with DDX4 and DAPI (white), respectively. The enlarged images in the upper right corner of each panel show the expression of key markers co-stained with DDX4. The enlarged images in the lower right corner of each panel show the expression of DDX4 co-stained with DAPI (white). The numbers in the upper left corner indicate the IVC period (w-ivc). Scale bar = 20 μm. (F) DDX4 from IF of cy-reconstituted ovaries at 3 w-ivc to 15 w-ivc. +Percentage of germ cells positive for individual markers. Mean values ​​are shown with SD. (G) Percentage of germ cells at each meiotic substage defined in Figure 1F (see also Materials and Methods). The average percentage of each meiotic substage was determined from at least three cy-reconstituted ovaries. Establishment of a new in vitro culture system for primate oocyte differentiation. (A) (Left) Summary table of coating conditions tested for cy-reconstituted ovaries. (Right) Representative images of cy-reconstituted ovaries cultured on membranes at 12 w-ivc. The dotted circle indicates the edge of the cy-reconstituted ovary at the start of air-liquid interface culture. Scale bar = 500 μm. (B) Macroscopic appearance (a), H&E staining (b), and IF for FOXL2 / DDX4 / DAPI (c) of cy-reconstituted ovaries cultured in 13 different basal media at 6 w-ivc. Reconstituted ovaries cultured in Advanced MEM maintained their size and DDX4 expression. + Germline and FOXL2 +Granulosa cells were found in the reconstituted ovaries. Scale bars = 500 μm (a) and 40 μm (b and c). (C) IF analysis of key markers listed in Figure 2E in cultured cy-reconstituted ovaries. Germ cells and nuclei were marked with DDX4 and DAPI (white), respectively. The enlarged images in the upper right corner of each panel show the expression of key markers co-stained with DDX4, except for NLRP5. The enlarged images in the lower right corner of each panel show the expression of DDX4 co-stained with DAPI (white). The numbers in the upper left corner indicate the IVC period (w-ivc). Scale bar = 20 μm. (D) IF analysis of FOXL2 (granulosa cells) / DDX4 (germ cells) stained with DAPI (nuclei) in cultured cy-reconstituted ovaries at 9 w-ivc. Scale bar = 40 μm. (E) Representative images of human reconstituted ovaries cultured on a Transwell-COL membrane at 0 and 7 w-ivc. Scale bar = 200 μm. Single-cell RNA-seq analysis of cy ovarian cells in vivo and in reconstituted ovaries. (A-J) Analysis of cy germ cells by 10X scRNA-seq. (A) Uniform manifold approximation and projection (UMAP) plot of in vivo cy fetal ovarian cells, colored by five major clusters computationally assigned based on the expression of cell-type-specific markers. Color coding is as indicated. (B) UMAP plot shown in Figure 6A. Cells at each fetal stage are shown in black. (C) Expression plot of key marker genes for cy in vivo germ cells (DDX4), granulosa cells (WT1), stromal cells (TCF21), endothelial cells (PECAM1), and blood cells (TYROBP) overlaid on the UMAP plot in Figure 6A. (D) UMAP plot of cy in vivo germ cells, classified by meiotic substage cluster. Color coding is as indicated. M: mitosis, PL: proleptotene, L: leptotene, Z: zygotene, P: pachytene, D: diplotene, U: unclassified. (E) UMAP plot of in vivo germ cells from cy colored by embryonic stage. Color coding is as indicated.(F) Gene expression plots of oogonia markers (POU5F1, TFAP2C), RA-responsive markers (ZGLP1, STRA8), meiotic markers (SYCP3, SYCP1), oogenesis marker (ZP3), and germ cell marker (DDX4) during in vivo germ cell development in cy. (G) UMAP plot of in vivo germ cells in cy colored by pseudotime. Pseudotime color coding is as indicated. (H) (Left) Heatmap of normalized expression of highly variable genes (HVG, 1,481 genes) during in vivo germ cell substages of cy ordered by unsupervised hierarchical clustering (UHC). Eight gene clusters were defined according to the UHC dendrogram. Key genes for female germ cell development are shown in black, and 10 genes (TMSB4X, PTRF, BAMBI, RND3, SLC47A1, RASD1, CD24, PLAT, CCL3, and GCNT2) that were transiently upregulated in the PL cluster are shown in gray. (Right) Representative genes and significant gene ontology (GO) enrichments are shown. (I) Heatmap of the Pearson correlation coefficient for the average expression levels of 1,481 HVGs among meiotic substages of cy in vivo germ cells. Asterisks indicate unclassified clusters. Color coding is as indicated. (J) (Left) UMAP plot of germ cells from cy in vivo fetal ovaries and in vitro-cultured cy-reconstituted ovaries, along with meiotic substages. Color coding is as indicated. (Right) UMAP plot highlighting cells from in vitro-cultured cy-reconstituted ovaries at 12 w-ivc and 15 w-ivc. (K and L) Analysis of cy germ cells by SC3-seq. (K) Heatmap of UHC and expression levels of selected marker genes in cy germ cells from in vivo and reconstituted ovaries with all expressed genes (19,747 genes). The color bar below the dendrogram indicates the in vivo stage (1. st bar), duration of xenotransplantation (2 nd bars), IVC period (3 rd bar), and cell types (4 th(Bars) are shown. Heatmap color coding is as indicated. Bar color coding is as in Figure 6L. (L) Principal component analysis (PCA) of cy germ cells in in vivo and reconstituted ovaries performed by SC3-seq. Cells were plotted on a two-dimensional plane defined by PC1 and PC2 values. Color coding is as indicated. 10X scRNA-seq analysis of in vivo cy female oocyte development. (A) Average number of genes detected in the five major cell types of cy in vivo fetal ovaries from 8 to 18 wpf. (B) Percentage of germ cells at the indicated meiotic prophase substages defined in Figure 6D. Color coding is as indicated. M: mitosis, PL: preleptotene, L: leptotene, Z: zygote, P: pachytene, D: diplotene. (C) Number of detected genes, UMI counts, and percentage of mitochondrial genes in each germ cell substage. Gene counts and UMI counts were significantly lower in the "Unclassified (U)" germ cell clusters compared to other germ cell clusters. **P < 0.01, Tukey-Kramer test. (D) Feature plot of marker genes important for oocyte development on the UMAP plot shown in Figure 6D. (E) (Left) Heatmap of normalized expression of HVGs listed in gene cluster #2 in Figure 6H. Three gene clusters (cluster #2-1 / -2 / -3) were defined according to the UHC dendrogram. (Right) Representative genes and significant GO enrichments are shown. 10X scRNA-seq analysis of cy granulosa cells in vivo and in reconstituted ovaries. (A and B) UMAP (A) and PAGA graph (B) using RNA velocity of cy in vivo granulosa cells defined in Figure 6A. Granulosa cells were divided into 12 subclusters by Louvain clustering. Color coding is as indicated. (C) Cells from each embryonic stage are indicated in black in the UMAP plot shown in Figure 8A. (D) (Left) Heatmap of normalized expression of HVG (712 genes) among subclusters of in vivo cy granulosa cells ordered by UHC. Eight gene clusters were defined according to the UHC dendrogram. (Right) Representative genes and major GO enrichments are shown.(E) Signature plot of key marker genes for cy granulosa cell development on the UMAP plot shown in Figure 8A. (F) (Left) UMAP plot of granulosa cells derived from cy in vivo fetal ovaries and in vitro-cultured cy-reconstituted ovaries, as indicated by the granulosa subcluster. Color coding is as indicated. (Right) UMAP plot highlighting cells derived from in vitro-cultured cy-reconstituted ovaries at 12 w-ivc and 15 w-ivc. (G) Heatmap 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 the granulosa cell subclusters #5-12 defined in Figure 8F. The percentage of cells derived from cultured reconstituted ovaries within each subcluster is shown (%IVC). Color coding is as indicated. In vitro induction of human oogonia into human follicles. (A) Macroscopic findings (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 reconstituted ovaries at 7 w-ivc and 14 w-ivc. FOXL2: granulosa cell marker; DDX4: germ cell marker; DAPI: nuclei. 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 analysis of human in vivo female gonads (11W, B) and human in vitro cultured reconstituted ovaries at 14 w-ivc (C) for oogonia marker (POU5F1), cell proliferation marker (Ki67), meiosis markers (SYCP3, DMC1, γH2AX, SYCP1), and oogenesis markers (ZP3, TP63). Germ cells and nuclei were marked with DDX4 and DAPI (white), respectively. Enlarged images in the upper right corner of each panel show the expression of key markers co-stained with DDX4. Enlarged images in the lower right corner of each panel show the expression of DDX4 co-stained with DAPI (white). Scale bar = 20 μm. (D) DDX4 from IF analysis of human in vivo female gonads (11W) and human cultured reconstituted ovaries at 7 w-ivc and 14 w-ivc. +Percentage of cells positive for individual markers in germ cells. Average values ​​from two biological replicates are shown. (E and F) Analysis of human germ cells by SC3-seq. (E) Heatmap of expression levels of UHC and selected marker genes for human germ cells in vivo and in reconstituted ovaries using all expressed genes (20,048 genes). The color bar below the dendrogram indicates the in vivo stage (1 st bars), period of IVC (2 nd bar), and cell types (3 rd(Bars) are shown. Heatmap color coding is as indicated. Bar color coding is as in Figure 9F. (F) PCA of human germ cells in vivo and in reconstituted ovaries performed by SC3-seq. Cells are plotted on a 2D plane defined by PC1 and PC2 values. Color coding is as indicated. (G) PCA plot of all cy and human SC3-seq data merged by canonical correlation analysis (CCA). Cell types were defined after CCA followed by Louvain clustering. Color and shape coding are as indicated. Cross-species comparative analysis of in vivo female fetal germ cell development in humans, monkeys, and mice. (AF) Analysis performed by 10X scRNA-seq. (A) UMAP plot of germ cells from six 10X scRNA-seq datasets (two for humans [this example and (Chitiashvili T, et al., Nat Cell Biol, 22: 1436-1446 (2020))], one for monkeys [this example], and 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))]) integrated by CCA. Meiotic substages were defined after CCA followed by Louvain clustering. Colors indicate computationally assigned meiotic substages defined by the expression levels of key meiotic markers conserved across all three species (see Materials and Methods). Color coding is as indicated. M: mitosis, PL: proleotene, L: leptotene, Z: zygotene, P: pachytene, D: diplotene. (B) UMAP plot highlighting cells from each dataset. Color coding for each developmental stage is as indicated. (C) Percentage of germ cells belonging to each meiotic substage. Color coding for germ cell stages is as shown in Figure 10A.(D) Distribution of germ cells along species-specific pseudotime trajectories calculated individually using Monocle. The color coding of germ cell stages is as shown in Figure 10A. (E) Heatmap of the Pearson correlation coefficients of the mean expression levels of 237 HVGs (1-1-1 orthologs) between meiotic substages in humans, monkeys, and mice (see Materials and Methods). The color coding of germ cell stages is as shown. (F) Heatmap of the normalized expression levels of selected genes using 10X scRNA-seq data from all three species. From the list of DEGs obtained from SC3-seq analysis of in vitro-cultured reconstituted ovaries of cy / humans, we manually selected genes that show specific expression changes during female germ cell development in humans and monkeys, but not in mice. The color coding of the heatmap is as shown. The color coding of germ cell substages is as shown in Figure 10E. (G and H) IF analysis of PAX6 and CLGN using TFAP2C (oogonia marker) and DDX4 (germ cell marker) in cy in vivo fetal ovaries at 12 wpf (G) and human cultured reconstituted ovaries at 7 wpf (H). Nuclear DAPI staining is shown in white. Scale bar = 20 μm. (I) IF analysis of CLGN using TP63 (oogenesis marker) and DDX4 (germ cell marker) in oocytes from 13-year-old human primordial follicles. Nuclear DAPI staining is shown in white. Scale bar = 20 μm. Cross-species analysis using 10X scRNA-seq datasets for in vivo fetal female germ cell development in humans, monkeys, and mice. (A) UMAP plots of fetal ovarian cells in each 10X scRNA-seq dataset, color-coded by the five major clusters computationally assigned based on the expression of cell-type-specific markers. (B) Number of genes (nGene), UMI counts (nUMI), and percentage of mitochondrial genes (%Mt) detected at each germ cell meiotic substage for six 10X scRNA-seq datasets: M: mitosis, PL: preleptotene, L: leptotene, Z: zygotene, P: pachytene, D: diplotene.(C) The average expression percentages of gene classes (1-1-1 orthologs, species-specific, and other genes) during fetal oocyte development in three animal species are shown with SD. Color coding is as indicated. Cross-species comparative analysis of oocyte development in humans, monkeys, and mice. (A) Heatmap of normalized expression levels of selected genes in all three species analyzed by 10X scRNA-seq. Genes showing conserved expression patterns across all three animal species during female germ cell development were manually selected from the list of DEGs obtained from SC3-seq analysis of in vitro-cultured reconstituted ovaries of cy / humans. Color coding is as indicated. (B) Boxplots of gene expression levels of selected markers showing primate-specific expression dynamics during fetal germ cell development (see also Figure 10F). (C) IF analysis of LEIOMODIN-3 (LMOD3) using TP63 (oogenesis marker) and DDX4 (germ cell marker) in in vivo fetal oocytes from 18 wpf cy (top) and oocytes from primordial follicles from a 13-year-old human (bottom). Nuclear DAPI staining is shown in white. Scale bar = 20 μm. (D) Autosome:A and X:A ratios (top) and XIST / Xist expression transitions (bottom) during in vivo female germ cell development 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)) analyzed by 10X scRNA-seq. X:A ratio and XIST expression during germ cell development in humans, monkeys, and mice. (A) Chr.3:A and X:A ratios (top) and Xist expression (bottom) in mouse fetal / neonatal gonad cells analyzed by bulk RNA-seq. Dots: individual data; bars: average. E9.5 / 10.5 samples contain germ cells derived from male and female embryos (mixed). E: embryonic day, P: postnatal day, Soma: somatic gonadal cells. (B) Chr.15:A and X:A ratios (top) and XIST expression (bottom) in cy female germ cells from in vivo and cultured / transplanted reconstituted ovaries analyzed by SC3-seq.The transition of Chr.15:A / X:A ratios in the embryonic and hypoblast lineages 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 sizes (n ≤ 3) are indicated by dots (individual data) and bars (average). (C) Chr.10:A and X:A ratios (top) and XIST expression (bottom) in human female germ cells from in vivo and cultured reconstituted ovaries analyzed by SC3-seq. (D) Autosome:A and X:A ratios (top) and XIST / Xist expression transition (bottom) during in vivo female germ cell development in all three species analyzed by 10X scRNA-seq. Histology of reconstituted ovaries after 9 weeks of in vitro culture. Primordial follicle formation (arrows) was observed only in the steroid hormone-supplemented group. FOXL2: granulosa cell marker, POU5F1: oogonia marker, DDX4: germ cell marker, DAPI: nucleus. Histology of reconstituted ovaries after 9 weeks of in vitro culture. Primordial follicle formation (arrows) was observed in the steroid hormone-supplemented group. ZP3 and TP63: oocyte markers at the follicle-forming stage, DDX4: germ cell marker. Histology of reconstituted ovaries after 6 weeks of in vitro culture (6 w-ivc). In the RA-supplemented group, the SYCP3 / DMC1 positivity rate was high at all concentrations, indicating promotion of meiotic transition. SYCP3 and DMC1: meiosis phase markers, DDX4: germ cell marker. Histological image of reconstituted ovaries after 9 weeks of in vitro culture (9 w-ivc). In the RA-added group, the AP2γ positivity rate was low at all concentrations, promoting the transition to meiosis. FOXL2: granulosa cell marker, AP2γ: oogonia marker, DDX4: germ cell. Histological image of reconstituted ovaries after 12 weeks of in vitro culture.Compared to the non-addition group (a), the steroid hormone-only group (b) and the retinoic acid (RA)-only group (c) produced many more primordial follicles (ZP3 / TP63 positive). The simultaneous addition of steroid hormones and RA group (d) produced even more primordial follicles. ZP3 and TP63: oocyte markers at the stage of forming follicles, DDX4: germ cell marker, DAPI: nuclei.

[0013] 1. Method for Producing Oocytes or Follicles, or Reconstituted Ovaries Containing These As shown in the Examples below, the inventors have demonstrated that culturing primate reconstituted ovaries (also referred to as re-aggregated ovaries) under suspension culture conditions enables the production of reconstituted ovaries containing primate primordial follicle-like structures with properties very similar to those of in vivo primordial follicles. Therefore, unless otherwise specified, the term "primordial follicle" also includes (primordial) follicle-like structures in this specification. Follicles are structures containing oocytes surrounded by granulosa cells (including "pre-granulosa cells"; the same applies below). Oocytes can also be produced by the above-described culture. Granulosa cells are somatic cells surrounding oocytes, and flat pre-granulosa cells are FOXL2-expressing cells that differentiate into cuboidal granulosa cells upon activation. Furthermore, hereinafter, unless otherwise specified, the term "reconstituted ovary" refers to an ovary subjected to culture under suspension culture conditions (in other words, an ovary as a starting material for culture).

[0014] Therefore, the present invention provides a method for producing oocytes or follicles, or a reconstituted ovary containing oocytes or follicles, which comprises the step of culturing a reconstituted ovary of a primate under suspension culture conditions (this can also be referred to as "suspension culture") (hereinafter, this may be referred to as "the production method of the present invention"). The oocytes and follicles obtained by the production method of the present invention can also be isolated and recovered from the obtained reconstituted ovary by a method known per se.

[0015] As used herein, "suspension culture conditions" refers to conditions under which cells or cell aggregates are maintained suspended in a culture medium, i.e., conditions under which strong cell-substratum junctions are not formed between the cells or cell aggregates and the culture vessel or the extracellular matrix coating the culture vessel.

[0016] The medium for suspension culture can be prepared by adding medium additives 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 Serum-free medium, and KnockOut. TM DMEM (KO DMEM), Advanced TMMedium (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' medium E, Brinster's BMOC-3 Medium, E8 Medium, StemPro 34, MesenPRO RS (Thermo Fisher Scientific), ReproFF2, Primate ES Cell Medium, ReproStem (ReproCELL Co., Ltd.), ProculAD (Rohto Pharmaceutical Co., Ltd.), MSCBM-CD, MSCGM-CD (Lonza), EX-CELL302 medium (SAFC) or EX-CELL-CD-CHO (SAFC), ReproMed TM Examples of suitable media include, but are not limited to, iPSC Medium (ReproCELL, Inc.), Cellartis MSC Xeno-Free Culture Medium (Takara Bio Inc.), TESR-E8 (Veritas, Inc.), StemFit® AK02N, AK03N (Ajinomoto Co., Inc.), and mixtures thereof. Among these, Advanced MEM is preferred. The composition of Advanced MEM, published on the Thermo Fisher Scientific website (https: / / www.thermofisher.com / jp / ja / home / technical-resources / media-formulation.39.html), is shown in Table 1 (translated into Japanese).

[0017]

[0018]

[0019] Furthermore, physiologically active substances and nutritional factors necessary for cell survival or proliferation can be added to the medium as needed. These medium additives may be added to the medium in advance or may be added during cell culture. The method of adding additives during culture may be in any form, such as a single solution or a mixed solution of two or more types, and may be added continuously or intermittently.

[0020] 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), hydrolysates, lipids, etc. Sugars include glucose, mannose, fructose, etc., and may be used alone or in combination of two or more. Amino acids include 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, and non-essential amino acids (NEAAs), which may be used alone or in combination of two or more. Vitamins include ascorbic acid, retinol, retinoic acid, niacin, biotin, pyridoxine, vitamin B12, D-pantothenic acid, choline, folic acid, myo-inositol, niacinamide, pyrodoxal, riboflavin, thiamine, cyanocobalamin, and DL-α-tocopherol, which may be used alone or in combination of two or more. Hydrolyzed foods include those obtained by hydrolyzing soybeans, wheat, rice, peas, corn, cottonseed, yeast extract, etc. Lipids include cholesterol, linoleic acid, linolenic acid, etc.

[0021] Furthermore, antibiotics such as kanamycin, streptomycin, penicillin, or hygromycin may be added to the medium as needed. When an acidic substance such as sialic acid is added to the medium, it is desirable to adjust the pH of the medium to a neutral range suitable for cell growth, between pH 5 and 9, preferably between pH 6 and 8.

[0022] The medium used for suspension culture may be a serum-containing medium (e.g., fetal bovine serum (FBS), human serum, or horse serum) or a serum-free medium. FBS is preferred as the serum. From the viewpoint of preventing contamination with components derived from different animal species, it is preferable that the medium does not contain serum or that serum derived from the same animal species as the cells to be cultured is used. Here, serum-free medium refers to a medium that does not contain unconditioned or unpurified serum. The serum-free medium may contain purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0023] Similar to serum, the medium used for suspension culture may or may not contain serum substitutes. Serum substitutes include albumin, lipid-rich albumin, recombinant albumin, and other albumin substitutes, plant starch, dextran, protein hydrolysates, transferrin or other iron transporters, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, and their equivalents. Specific examples of serum substitutes include those prepared by the method described in WO 98 / 30679, commercially available products such as knockout serum replacement (KSR) (Life Technologies), chemically-defined lipid concentrated (Life Technologies), and L-alanine-L-glutamine dipeptide (e.g., Glutamax (Life Technologies)). Examples of biofactors include platelet-rich plasma (PRP) and components of human mesenchymal stem cell culture supernatant.

[0024] The medium also preferably contains one or more vitamins. Suitable vitamins include ascorbic acid and retinoic acid. "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 "ascorbate" and "ascorbic acid precursor." "Retinoic acid" typically refers to all-trans-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraenoic acid (Cas No. 302-79-4), but also includes "retinoic acid" and "retinoic acid precursor." Examples of ascorbate salts used in the present invention include, but are not limited to, sodium ascorbate, potassium ascorbate, and calcium ascorbate. Examples of ascorbic acid precursors for use in the present invention include, but are not limited to, trisodium ascorbyl phosphate, magnesium ascorbyl phosphate (VC-PMg), ascorbyl tetrahexyldecanoate (VC-IP), trisodium ascorbyl palmitate phosphate, etc. Examples of retinoic acid salts for use in the present invention include, but are not limited to, sodium retinoate, potassium retinoate, calcium retinoate, etc. Examples of retinoic acid precursors for use in the present invention include, but are not limited to, β-carotene, retinol esters, retinol, retinal, etc.

[0025] Furthermore, the medium preferably contains one or more hormones. Suitable hormones include, for example, steroid hormones. Specific examples of steroid hormones include progesterone, cortisol, dehydroepiandrosterone, and dehydroepiandrosterone sulfate. Vitamins and hormones can be replaced with artificial or natural compounds having an equivalent steroid structure.

[0026] In one embodiment, the suspension culture is performed in a medium containing L-glutamine, ascorbic acid, and 2-mercaptoethanol. As shown in the Examples below, adding retinoic acid to the medium promoted differentiation of oogonia into oocytes and the initiation and progression of meiosis. Furthermore, adding steroid hormones to the medium promoted oocyte differentiation and maturation. Furthermore, it has been shown that retinoic acid and steroid hormones synergistically increase the production efficiency of primordial follicles. Therefore, preferably, the medium further contains retinoic acid. Furthermore, the medium also contains one or more (preferably all) selected from the group consisting of progesterone, cortisol, dehydroepiandrosterone, and dehydroepiandrosterone sulfate. More preferably, the medium also contains retinoic acid and one or more (preferably all) selected from the group consisting of progesterone, cortisol, dehydroepiandrosterone, and dehydroepiandrosterone sulfate. In this specification, these additives also include those produced in the medium by decomposition or the like of cell secretions or the like (e.g., L-alanine-L-glutamine dipeptide, which produces L-glutamine). In another embodiment, the medium may contain serum.

[0027] The concentration of L-glutamine in the medium is not particularly limited, but may be, for example, 10 μM to 1 M, preferably 100 μM to 10 mM, and more preferably 1 mM to 3 mM (2 mM in one embodiment). The concentration of ascorbic acid in the medium is not particularly limited, but may be, for example, 1 μM to 1 M, preferably 10 μM to 500 μM, and more preferably 100 μM to 200 μM (150 μM in one embodiment). The concentration of 2-mercaptoethanol in the medium is not particularly limited, but may be, for example, 0.1 μM to 1 M, preferably 1 μM to 500 μM, and more preferably 10 μM to 200 μM (55 μM in one embodiment). The concentration of retinoic acid in the medium is not particularly limited, but may be, for example, 0.1 nM to 5 μM, preferably 1 nM to 1 μM, and more preferably 10 nM to 300 nM (particularly 10 nM). The concentration of retinoic acid in the medium may be 10 nM to 1 μM. The concentration of progesterone in the 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 (particularly 0.2 μg / mL). The concentration of cortisol in the 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 (particularly 0.1 μg / mL). The concentration of dehydroepiandrosterone in the medium is not particularly limited, but may be, for example, 0.1 nM to 5 μM, preferably 1 nM to 1 μM, more preferably 20 nM to 100 nM (particularly 50 nM). The concentration of dehydroepiandrosterone sulfate in the medium is not particularly limited, but may be, for example, 0.1 nM to 5 μM, 1 nM to 1 μM, or 1 nM to 10 nM. Alternatively, the concentration of dehydroepiandrosterone sulfate in the medium is 0.1 μM to 5 mM, preferably 1 μM to 1 mM, and more preferably 1 μM to 100 μM (particularly 10 μM). The concentration (v / v) of serum such as FBS in the medium is not particularly limited, but may be, for example, 0.01% to 50%, preferably 0.01% to 20%, and more preferably 0.01% to 10% (10% in one embodiment). These may be added to the medium simultaneously or separately at staggered times.

[0028] The culture vessel used for suspension culture is not particularly limited as long as it is capable of "suspension culture," and those skilled in the art can appropriately determine the appropriate vessel. These culture vessels are preferably low- or non-cell-adhesive to enable suspension culture. Examples of low- or non-cell-adhesive culture vessels include those whose surfaces have not been artificially treated to improve cell adhesion (e.g., coated with extracellular matrix, etc.). The vessel surface may also be treated to inhibit extracellular matrix adsorption (e.g., Nunclon Sphera treatment, etc.), or may not be treated at all. The vessel may be flat-bottomed, round-bottomed (e.g., U-bottomed), or of other shapes. Examples of such culture vessels include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles. Further, a bioreactor is an example of a vessel for suspension culture.

[0029] Culture conditions such as temperature, carbon dioxide concentration, oxygen concentration, and pH can be appropriately set based on techniques conventionally used for culturing animal cells. 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%.

[0030] Mechanical stress also plays an important role in maintaining the dormant state of oocytes in mouse primordial follicles. It has been reported that culturing oocytes under pressurized conditions in vitro can induce dormancy in oocytes (Nagamatsu G, et al., Sci Adv. 5(6):eaav9960 (2019)). Therefore, although culturing is typically performed under 1 atmosphere, at least a portion of the culture period may be performed under reduced pressure or pressurized conditions (e.g., 0 to 10 atmospheres, preferably 0 to 5 atmospheres, more preferably 1 to 2 atmospheres). Culture under reduced pressure or pressurized conditions can be performed, for example, using a commercially available gas pressure cell stimulator (e.g., the AGP series manufactured by STREX Corporation).

[0031] Because the culture method of the present invention allows for the long-term maintenance of reconstituted ovaries, the culture period is not particularly limited as long as the desired oocytes or follicles are obtained, and is appropriately selected depending on the species of primate from which the cells are derived. Typically, the culture period is 9 weeks or longer (e.g., 10 weeks or longer, 11 weeks or longer, 12 weeks or longer, 13 weeks or longer, or 14 weeks or longer). The culture period may be 7 weeks or longer (e.g., 8 weeks or longer, 9 weeks or longer). The upper limit of the culture period is also not particularly limited, but is typically 24 weeks or shorter (e.g., 23 weeks or shorter, 22 weeks or shorter, 21 weeks or shorter, 20 weeks or shorter, or 19 weeks or shorter).

[0032] As used herein, the term "reconstituted ovary" refers to a structure constructed by aggregating germ cells and fetal ovarian somatic cells, and may contain cells other than germ cells and fetal ovarian somatic cells, or may contain multiple types of germ cells or multiple types of fetal ovarian somatic cells. The "reconstituted ovary" obtained by the production method of the present invention is derived from the above structure, but the types of cells constituting the reconstituted ovary may be different due to suspension culture.

[0033] A reconstituted ovary can typically be constructed by suspension culture (hereinafter, sometimes referred to as "preculture") in a medium containing germ cells and fetal ovarian somatic cells for a specific number of days (e.g., 1 to 3 days, preferably 2 days). Therefore, the production method of the present invention may include a step of preparing a reconstituted ovary, or a step of obtaining a reconstituted ovary by preculturing germ cells and fetal ovarian somatic cells. Such suspension culture can be performed in the same manner as the suspension culture of the production method of the present invention, and the types of basal medium, medium additives, culture vessels, culture conditions, etc. used can all be described above.

[0034] In one embodiment, the preculture is performed in a medium containing one or more (preferably all) selected from the group consisting of L-glutamine, sodium pyruvate, 2-mercaptoethanol, and a ROCK inhibitor. In another embodiment, the medium may contain serum. In particular, a ROCK inhibitor is preferably included in the preculture to promote cell aggregation. Examples of such ROCK inhibitors include Y-27632, Fasudil / HA1077, H-1152, and Wf-536. When Y-27632 is used as the ROCK inhibitor, its concentration in the medium during the preculture is not particularly limited, but is, for example, 0.1 μM to 1 mM, preferably 1 μM to 100 μM, and more preferably 5 μM to 20 μM (10 μM in one embodiment).

[0035] In the preculture, the concentration of L-glutamine in the medium is not particularly limited, but may be, for example, 10 μM to 1 M, preferably 100 μM to 10 mM, and more preferably 1 mM to 3 mM (2 mM in one embodiment). The concentration of sodium pyruvate in the medium is not particularly limited, but may be, for example, 10 μM to 100 mM, preferably 100 μM to 10 mM, and more preferably 500 μM to 5 mM (1 mM in one embodiment). The concentration of 2-mercaptoethanol in the medium is not particularly limited, but may be, for example, 0.1 μM to 1 M, preferably 1 μM to 500 μM, and more preferably 10 μM to 200 μM (100 μM in one embodiment). The concentration (v / v) of serum such as FBS in the medium is not particularly limited, but may be, for example, 0.01% to 50%, preferably 0.01% to 20%, and more preferably 0.01% to 10% (10% in one embodiment). These may be added to the medium at the same time, or may be added separately at different times.

[0036] As used herein, "germ cells" refer to cells that express at least one of DDX4 and DAZL, preferably cells that express at least DDX4. Germ cells used in the present invention include, for example, oogonia, primordial germ cells (PGCs), and primordial germ cell-like cells (PGCLCs). "Oogonia," also known as oogonia, are cells in the ovary at the stage when primordial germ cells repeatedly divide and proliferate, and refer to cells that express at least one of NANOG, POU5F1, TFAP2C, and PDPN. "Primordial germ cells" refer to cells that have the ability to differentiate into oogonia and spermatogonia, and refer to cells that express at least one of INTEGRINα6, EpCAM, BLIMP1, and TFAP2C. "Primordial germ cell-like cells" refer to cells that have properties equivalent to PGCs and are produced by inducing differentiation from pluripotent stem cells.

[0037] Furthermore, as used herein, "fetal ovarian somatic cells" refers to cells contained in a fetal ovary other than germ cells. The fetal ovary may be an ovary of a fetus of any age as long as it contains fetal ovarian somatic cells, and may vary depending on the species of primate from which the cells are derived. However, the ovary of a fetus aged 6 to 18 weeks after fertilization (6 to 18 wpf) is preferred.

[0038] Specifically, germ cells and fetal ovarian somatic cells can be prepared by surgically removing ovaries from a living body and dissociating the germ cells and somatic cells that make up the ovaries into single cells using enzymes (e.g., trypsin, etc.). Germ cells (fetal ovarian germ cells) present in the living body's ovaries can also be isolated by cell sorting methods such as FACS (fluorescence-activated cell sorting) or MACS (magnetic activated cell sorting) using germ cell markers (e.g., PGC-specific markers such as INTEGRINα6 and EpCAM) as indicators, thereby isolating fetal ovarian germ cells and fetal ovarian somatic cells. Alternatively, fetal ovarian germ cells and fetal germ somatic cells can be used as cell populations containing these cells without dissociation. These cells and cell populations can also be cryopreserved. In this specification, unless otherwise specified, the term "cells" includes "cell populations." A cell population may be composed of one type of cell or two or more types of cells.

[0039] Germ cells and fetal ovarian somatic cells may be prepared by inducing differentiation from pluripotent stem cells. "Pluripotent stem cells" refer to stem cells that can differentiate into various tissues and cells with different morphologies and functions in the body and have the ability to differentiate into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm). Examples of pluripotent stem cells used in the present invention include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES cells), multipotent germline stem cells (mGS cells), and embryonic germ stem cells (EG cells). Preferably, iPS cells (more preferably, human iPS cells) are used. When the pluripotent stem cells are ES cells or any cells derived from a human embryo, the cells may be cells produced by destroying an embryo or cells produced without destroying an embryo, but from an ethical point of view, cells produced without destroying an embryo are preferred.

[0040] ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice, and have the ability to proliferate through pluripotency and self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in humans, monkeys, and other primates (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by isolating the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Alternatively, ES cells can be established using only a single blastomere from an embryo at the cleavage stage prior to the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or from a developmentally arrested embryo (Zhang X. et al. (2006), Stem Cells 24: 2669-2676).

[0041] nt ES cells are ES cells derived from cloned embryos produced by nuclear transfer technology and have almost the same properties 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). Specifically, nt ES (nuclear transfer ES) cells are established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nucleus of an unfertilized egg with that of a somatic cell. To generate nt ES cells, nuclear transfer technology (Cibelli JB et al. (1998), Nature Biotechnol., 16:642-646) is combined with ES cell generation technology (see above) (Wakayama Sayaka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized mammalian egg, and the egg is then cultured for several hours to reprogram the embryo.

[0042] As the ES cell line used in the present invention, various human ES cell lines can be used, for example, those established by the University of Wisconsin, NIH, RIKEN, Kyoto University, the National Center for Child Health and Development, Cellartis, etc. Specific examples of human ES cell lines include CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28 strains distributed by ESI Bio, H1 and H9 strains distributed by WiCell Research, and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3 strains distributed by RIKEN.

[0043] iPS cells are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various 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 introducing the above four factors and then selecting using Nanog expression as an indicator (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPSCs produced using 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 using a virus-free method (Okita K et al. Nat. Methods 2011). May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.) and the like can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, produced by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells produced by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), induced pluripotent stem cells produced by Sakurada et al. (JP Patent Publication No. 2008-307007), and the like can also be used.In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, 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. 7, 795-797), or patent publications (e.g., JP 2008-307007 A, JP 2008-283972 A, US 2008-2336610 A, US 2009-047263 A, WO 2007-069666 A, WO 2008-118220 A, WO 2008-124133 A, WO 2008-151058 A, WO 2009-006930 A, WO 2009-006997 A, WO 2009-007852 A) and known in the art can be used.

[0044] As induced pluripotent stem cell lines, various iPSC lines established by NIH, RIKEN, Kyoto University, etc. can be used. For example, human iPSC lines include RIKEN's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 lines, and Kyoto University's 253G1, 253G4, 1201C1, 1205D1, 1210B2, 1383D2, 1383D6, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1, 1231A3, and FfI-01s04 lines, with 1231A3 being preferred.

[0045] mGS cells are pluripotent stem cells derived from the testis and are the source of spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various cell lineages, e.g., when transplanted into mouse blastocysts, chimeric mice can be generated (Kanatsu-Shinohara M. et al. (2003) Biol. Reprod., 69:612-616; Shinohara K. et al. (2004) Cell, 119:1001-1012). They are capable of self-renewal in culture medium containing glial cell line-derived neurotrophic factor (GDNF). Furthermore, germline stem cells can be obtained by repeated passage under culture conditions similar to those for ES cells (Takebayashi M. et al. (2008) Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 41-46, Yodosha, Tokyo, Japan).

[0046] EG cells are derived from embryonic primordial germ cells (PGCs) and have pluripotency similar to that of ES cells. They can be established by culturing PGCs in the presence of LIF, bFGF, stem cell factor, and other substances (Matsui Y. et al. (1992), Cell, 70:841-847; JL Resnick et al. (1992), Nature, 359:550-551).

[0047] Pluripotent stem cells can be differentiated into PGCLCs by known methods. Specifically, induced pluripotent stem cells can be differentiated into PGCLCs by the method described in Hayashi K, et al., Cell 2011 Aug 19;146(4):519-32. Alternatively, the methods described in WO 2017 / 002888 and WO 2022 / 039279 may be used. Specifically, (1) pluripotent stem cells are cultured in a culture medium containing activin A and a GSK-3β inhibitor to induce early mesoderm-like cells (iMeLCs), and (2) the iMeLCs can be cultured in the presence of BMP to induce differentiation into PGCLCs.

[0048] Basal media for inducing differentiation of pluripotent stem cells into PGCLCs include, but are not limited to, the basal media exemplified for use in the suspension culture described above. The basal medium may contain other known medium additives such as those exemplified for use in the suspension culture described above.

[0049] The medium may be a serum-containing medium or a serum-free medium (SFM). Preferably, a serum-free medium is used. The concentration (v / v) of serum (e.g., FBS) in the medium may be, for example, 20% or less, 5% or less, 2% or less, or 0% (i.e., serum-free). SFM may or may not contain any serum substitute, such as KSR.

[0050] The concentration of activin A in the medium for inducing differentiation from pluripotent stem cells to iMeLCs 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 medium is 50 ng / ml. Examples of GSK-3β inhibitors used in inducing iMeLCs from pluripotent stem cells include LiCl, BIO, SB216763, GSK-3β inhibitor VII, L803-mts, and CHIR99021, with CHIR99021 being preferred. The concentration of CHIR99021 in the medium is not particularly limited, but is preferably 0.1 μM to 50 μM. Pluripotent stem cells are cultured in a medium containing 10 4 ~10 5 cells / cm 2 , preferably 2 to 6 × 10 4 cells / cm 2 The iMeLCs can be induced to differentiate by culturing at a seeding density of 1000 μg / ml at 30 to 40° C. for less than 60 hours, preferably 42 hours.

[0051] Examples of BMPs used to induce differentiation of iMeLCs into PGCLCs include BMP2, BMP4, and BMP7, with BMP4 being preferred. The concentration of BMP in the 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 medium is 200 ng / ml. iMeLCs are cultured at a concentration of 1 to 50 × 10 3 cells / cm 2 , preferably 5 to 20 × 10 3 cells / cm 2 The differentiation of PGCLCs can be induced by culturing at a seeding density of 1000 μg / cm2 at 30-40° C. for 4-10 days, preferably 5-8 days, and more preferably 6 days.

[0052] It is also possible to induce differentiation of oogonia from PGCs or PGCLCs, and such differentiation can be performed, for example, by the method described in Non-Patent Document 3. Specifically, mouse fetal somatic cells are isolated from the ovaries of mouse fetuses, and then these cells are reaggregated with PGCs or PGCLCs to construct heterologous reconstituted ovaries, which are then cultured for a specific number of days (e.g., 77 days or more) by the air-liquid interface culture method, thereby producing oogonia.

[0053] Pluripotent stem cells can be differentiated into fetal ovarian somatic cells, for example, according to the method of Hayashi et al. (Yoshino Y, et al., Science, 373(6552):eabe0237 (2021)). For example, fetal ovarian somatic cell-like cells (FOLSCs), which have properties similar to fetal ovarian somatic cells, can be induced by sequentially differentiating pluripotent stem cells into nascent mesoderm, intermediate mesoderm, and coelomic epithelium. Specifically, in the case of mice, the following method is exemplified: Pluripotent stem cells are differentiated into EpiLCs using activin A and bFGF. EpiLCs were cultured in low-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, then cultured for an additional 2 days with 3 μM retinoic acid, 30 ng / ml sonic hedgehog, 1 μM PD0325901, 50 ng / ml EGF, and 1 ng / ml BMP4, followed by 20 ng / ml BMP4 and 2 ng / ml FGF9 for an additional 1 or 2 days.

[0054] As shown in the examples below, the method of the present invention can produce primordial follicles containing primary oocytes that have completed at least meiotic prophase I. Furthermore, by culturing for a longer period of time, the oocytes may mature into secondary oocytes, and the follicles may also mature from primordial follicles into primary, secondary, and mature follicles. Therefore, the oocytes obtained by the method of the present invention may be either primary oocytes (including cells in the process of maturing into secondary oocytes) or secondary oocytes. The follicles obtained by the method of the present invention may be either primordial follicles (including follicles in the process of maturing into primary follicles), primary follicles (including follicles in the process of maturing into secondary follicles), secondary follicles (including follicles in the process of maturing into mature follicles), or mature follicles.

[0055] Furthermore, the oocytes obtained by the method of the present invention may be further matured by a method known per se. Therefore, in another aspect of the present invention, there is provided a method for producing an oocyte (hereinafter, sometimes referred to as the "oocyte production method of the present invention"), which includes a step of maturing the oocytes obtained by the method of the present invention. An example of such a method is the method of Telfer et al. (McLaughlin M, et al., Mol Hum Reprod, 24(3):135-142 (2018)). Specifically, eggs can be produced by (1) culturing reconstituted ovaries in serum-free medium for a specific number of days (e.g., 8 days), (2) isolating secondary / multilayered follicles from the reconstituted ovaries and individually culturing the isolated follicles in serum-free medium in the presence of activin A, (3) retrieving cumulus-oocyte complexes (COCs) by applying gentle pressure to the cultured follicles after a specific number of days (e.g., 8 days), and (4) culturing the retrieved complexes on a membrane in the presence of activin A and FSH for an additional specific number of days (e.g., 4 days).

[0056] Alternatively, the step of maturing oocytes can be carried out by transplanting the oocytes or follicles obtained by the method of the present invention, or a reconstituted ovary containing them, into the kidney capsule or ovarian capsule of a primate. The transplantation method can be a method known per se.

[0057] As used herein, "primate" refers to a mammal belonging to the order Primates. The origin of the above-mentioned induced pluripotent stem cells, germ cells, and fetal ovarian somatic cells is not particularly limited as long as they are primates. Primates may be Strangorhinians or Plagiorhini. Examples of Strangorhinians include lemurs, aye-ayes, and lorises. Examples of Plagiorhini include tarsiers, long-tailed macaques (e.g., cynomolgus monkeys, rhesus monkeys, Japanese macaques, Taiwanese macaques, etc.), gibbons, humans, spider monkeys, capuchin monkeys, and saki monkeys. Among these, long-tailed macaques such as cynomolgus monkeys and humans are preferred, and humans are more preferred.

[0058] In another aspect, a method for maintaining a reconstituted ovary is also provided, comprising culturing a reconstituted ovary of a primate under suspension culture conditions. The specific culture method and other aspects of such a method are fully incorporated herein by reference. Here, "maintaining a reconstituted ovary" means that the morphology of the reconstituted ovary is maintained without disruption, and suspension culture may result in different cell types constituting the reconstituted ovary. In yet another aspect, oocytes, follicles, or reconstituted ovaries containing these obtained (or obtainable) by the method of the present invention, as well as ova obtained (or obtainable) by the method of ova production of the present invention, are also provided. Preferably, they are provided in a cryopreserved state using a method known per se. For example, the oocytes and follicles described above are not only useful as starting materials for producing ova, but are also highly useful for understanding the mechanisms of germ cell development.

[0059] 2. Kit for Producing Oocytes or Follicles, or Reconstituted Ovaries Containing These The present invention also provides a kit for producing oocytes or follicles, or reconstituted ovaries containing oocytes or follicles (hereinafter, sometimes referred to as the "production kit of the present invention"), which comprises a culture vessel for suspension culture, L-glutamine, ascorbic acid, and 2-mercaptoethanol. The production kit of the present invention may also contain serum. The production kit of the present invention may also contain retinoic acid and / or one or more (preferably all) molecules selected from the group consisting of progesterone, cortisol, dehydroepiandrosterone, and dehydroepiandrosterone sulfate.

[0060] The production kit of the present invention may contain a basal medium and / or medium additives such as a physiologically active substance. The definitions and specific examples of the culture vessel 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 production kit of the present invention are all incorporated by reference in the description of "1. Method for producing oocytes or follicles, or reconstituted ovaries containing these."

[0061] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0062] Example 1: Development of cyFetal Oocytes. cyPGCs are specified in the developing amnion starting at approximately embryonic day (E) 11, migrate through the developing hindgut endoderm and mesentery, and colonize the early embryonic gonad starting at approximately E30 (Sasaki K, et al., Dev Cell, 39: 169-185 (2016)). In female embryos, the embryonic gonad gradually increases in size and migrates to the ovary by approximately E40. However, cyPGCs continue to colonize the developing gonad and mitotically increase in number as oogonia (Sasaki K, et al., Dev Cell, 39: 169-185 (2016); Sasaki K, et al., Cell Reports, 35: 109075 (2021)). Subsequently, development of fetal oocytes occurs in the fetal ovary (Figure 1A). To benchmark in vitro reconstitution of cynomolgus oocyte development, we performed careful characterization of cynomolgus oocyte development in vivo at 2-week intervals from 8 weeks post-fertilization (8 wpf: E56-58) to 18 wpf (E127 and E128) (Fig. 1B, Fig. 2A). The gestational period in cynomolgus monkeys is approximately 22 weeks.

[0063] Histological Analysis: At 8 wpf, the ovaries showed a relatively homogeneous histology, with abundant oogonia and no clear distinction between the ovarian cortex and medulla (Fig. 1C). The oogonia were large (approximately 12 μm in diameter), had pale hematoxylin staining, round nuclei with prominent eosinophilic nucleoli, and formed anastomosing cord-like structures interspersed with relatively small cells with densely stained nuclei and a high nuclear-to-cytoplasmic ratio (Fig. 1C). Interstitial cells were evident, exhibiting a spindle shape with a high nuclear-to-cytoplasmic ratio, and were embedded in loose connective tissue, likely composed of their own cells (Fig. 1C). Immunofluorescence (IF) analysis revealed that all oogonia expressed DDX4 (Toyooka Y, et al., Mech Dev, 93: 139-49 (2000)), an RNA-binding protein expressed in oogonia and oocytes, while small interstitial cells expressed FOXL2 (Crisponi L, et al., Nat Genet, 27: 159-66 (2001)), a key transcription factor (TF) for granulosa development, demonstrating their identity as granulosa cells (Figure 2B). At 10 wpf, the ovary acquired a kidney-like shape and grew in size, developing a medulla composed mainly of interstitial cells (Figure 1B). The cortex was dominated by abundant oogonia, and the outer and inner cortices showed no obvious histological differences (Figure 1D, Figure 2C). At 12 wpf, while oogonia were still abundant, germ cells with larger nuclei and condensed filamentous chromatin became apparent, particularly in the inner cortex (Fig. 1D, Fig. 2C), indicating that oogonia were entering meiotic division I and differentiating into oocytes. At 14 wpf, meiotic division I oocytes also predominated in the outer cortex, and relatively large oocytes delineated by granulosa cells, i.e., primordial follicles, began to be observed in the inner cortex (Fig. 1D, Fig. 2C). At 16 wpf, primordial follicles were also observed in the outer cortex, and by 18 wpf, they predominated in both the outer and inner cortices (Fig. 1D, Fig. 2C). Meanwhile, even at 18 wpf, oogonia-like cells without signs of meiotic transition were observed in the outer cortex (Fig. 1D, Fig. 2C), indicating considerable asynchrony in oocyte development. (Pre)granulosa:germ cell ratio (FOXL2 positive)（+） :DDX4 + We measured the germ cell ratio (Germ cell ratio) and found that it was approximately 2 at 8 wpf, decreased to approximately 1 at 10 and 12 wpf, and remained constant. It then rapidly increased after 14 wpf, particularly in the inner cortex, reaching approximately 6 and 3 in the inner and outer cortex, respectively, at 18 wpf (Fig. 2D). This finding is consistent with the idea that germ cells increase by mitotic expansion of oogonia until at least 10 wpf, followed by a dramatic decrease in cell number after 14 wpf, likely due to apoptosis associated with meiotic prophase (see below). Granulosa cells, on the other hand, remain relatively constant in number or proliferate slowly after their fate specification.

[0064] Expression of key markers. Using scRNA-seq analysis, human female fetal germ cells were classified into four cell types: "mitotic" (oogonia), "retinoic acid (RA) responsive," "meiotic," and "oogenesis" (hereafter referred to as "oogenic") (Li L, et al., Cell Stem Cell, 20: 858-873 e4 (2017)). Therefore, the expression of key markers of the four cell types was used to evaluate the development of cy fetal oocytes. At 8 wpf, DDX4 + The majority of cells expressed oogonia markers. Nearly all DDX4 +Although the cells were NANOG-positive, and approximately 80% were POU5F1-, TFAP2C-, and PDPN-positive, only a minority of cells in the inner cortex (approximately 10%) expressed ZGLP1, a determinant of oogonia fate and a marker of RA-responsive cells (Figure 1E, Figure 2E and F) (Li L, et al., Cell Stem Cell, 20: 858-873 e4 (2017); Nagaoka S, I., et al., Science 367 (2020)). At 10 wpf, germ cells expressing oogonia markers were dramatically reduced, especially in the inner cortex, despite their histological similarity to those at 8 wpf. On the other hand, the number of cells expressing RA-responsive cell markers increased (ZGLP1: approximately 40-50%; STRA8: approximately 10-20%), and a portion (approximately 10-20%) of these cells expressed SYCP3, an axial component of the synaptonemal complex (SC) whose characteristic expression begins at the preleptotene stage of meiosis (Yuan L, et al., Science, 296: 1115-8 (2002), Yuan L, et al., Mol Cell 5: 73-83 (2000) (see below)) (Fig. 1E, Fig. 2F). The oogonium markers NANOG, POU5F1, and TFAP2C were uniformly distributed throughout the oogonium nuclei, whereas the RA-responsive cell markers ZGLP1 and STRA8 showed a granular distribution (Fig. 1E, Fig. 2E). At 12 wpf, germ cells expressing oogonia markers continued to decrease, while cells expressing RA-responsive and meiotic markers (SYCP3, DMC1, γH2AX, SYCP1) continued to increase. At 14 wpf, oogonia became a minority (approximately 5%), meiotic cells predominated (approximately 50%), and cells expressing oogenic markers (ZP3 and TP63) appeared in both the outer cortex (approximately 10%) and inner cortex (approximately 30%) (Fig. 1E, Fig. 2F).γH2AX, a marker of double-strand breaks (DSBs) during meiotic recombination (Fernandez-Capetillo O, et al., J Cell Biol, 163: 15-20 (2003)), showed a dot-like distribution in meiotic nuclei, whereas SYCP1, another axial component of the SC (Meuwissen RL, et al., EMBO J, 11: 5091-100 (1992)), showed a typical filament-like distribution along synapsed homologous chromosomes in nuclei corresponding to late zygotene or pachytene stages of meiotic prophase I (see below). At 16 and 18 wpf, oocytes (diplotene oocytes from primordial follicles) (see below) dominated the inner cortex, whereas meiotic cells were abundant in the outer cortex (Figures 1E and 2F). In oocytes, ZP3 was localized to the plasma membrane or the extracellular space between the oocyte and surrounding granulosa cells, indicating early zona pellucida (ZP) formation (Figure 1E). TFs important for oogenesis, such as TP63, FIGLA, and NOBOX (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 all uniformly localized in the nucleoplasm, except for the nucleolus, in oocytes. On the other hand, NLRP5 (Tong ZB, et al., Nat Genet, 26: 267-8 (2000)), a maternal effect protein essential for early embryonic development, showed punctate localization throughout the cytoplasm (Fig. 1E, Fig. 2E). Laminin, a basement membrane marker, delineated the basal side of squamous granulosa cells that constitute primordial follicles, marking both single and sometimes multiple primordial follicles (Fig. 2E).

[0065] Next, we examined the expression of Ki67, a marker of cell proliferation (Gerdes, Schwab et al., 1983), and the expression of truncated poly(ADP-ribose) polymerase (cPARP) and CASPASE 3 (cCAS3), markers of apoptosis (Lazebnik YA, et al., Nature, 371: 346-7 (1994); Tewari M, et al., Cell, 81: 801-9 (1995)). At 8 wpf, the majority of oogonia (approximately 70%) expressed Ki67, which was primarily localized around the nucleoli (Figure 2E and F). Germ cells expressing oogonia markers dramatically decreased after 10 wpf, but Ki67 expression remained constant throughout the stages examined. + Apoptotic germ cells were observed (approximately 40%) (Figure 2F). These cells likely represent RA-responsive and meiotic cells at the early stage of meiosis (see below). No apoptotic markers were detected at 8 wpf. However, approximately 10% and 15% of germ cells showed apoptotic markers at 10 wpf to 14 wpf, which involve the initiation and progression of meiotic prophase, and at 16 and 18 wpf, which involve the formation of primordial follicles (Figures 2E and F). Apoptotic primordial follicles expressed cCAS3 but not cPARP (Figure 2F).

[0066] Progression of meiotic prophase I. Progression of meiotic prophase I was determined based on chromatin structure visualized by DAPI staining and the expression / localization of SYCP3 and DMC1 (the latter involved in single-strand invasion in homologous recombination (Bishop DK, et al., Cell, 69: 439-56 (1992)) (Figure 1F). Meiotic oogonia expressed neither SYCP3 nor DMC1 and showed no obvious signs of chromosome condensation. Preleptotene oocytes began to condense chromosomes and expressed SYCP3, which was localized around the nucleoli, but not DMC1. Leptotene oocytes showed further chromosome condensation and expressed SYCP3 in addition to DMC1, which was localized to the condensed chromosomes. SYCP3 was localized to both the nucleoli and the condensed chromosomes. Zygotene oocytes showed signs of chromosome pairing and expressed SYCP3 and DMC1. These were localized to paired chromosomes, and DMC1 stained strongly in the form of multiple dots. Early pachytene oocytes showed strong chromosome pairing with a clear axial structure and expressed SYCP3 and DMC1. DMC1 stained strongly in the form of dots, likely colocalizing to sites of crossover recombination between homologous chromosomes. Late pachytene oocytes were similar to early pachytene oocytes, except that they did not express DMC1. Diplotene oocytes were larger in size, showed signs of diakinesis, and repressed both SYCP3 and DMC1. This classification indicates that the majority of germ cells are 10 These data demonstrate that meiotic division I begins in the inner cortex at 10 wpf and progresses gradually during cy fetal development, with diplotene oocytes completing prophase emerging from 14 wpf onward (Figure 1G). Collectively, these data update classic observations in rhesus monkeys (Macaca mulatta) (Baker TG, J Anat 100: 761-76 (1966)) and lay the cytological foundation for the progression of cy fetal oocyte development, showing that the onset of meiotic division I and overt morphological changes take approximately 2 weeks from the suppression of oocyte markers, and that it takes 4-6 weeks for meiotic division I prophase to be completed.

[0067] Example 2: Development of fetal cy oocytes in xenotransplanted cy reconstituted ovaries (cy rOvary) Next, we investigated whether the development of fetal cy oocytes would proceed when cy reconstituted ovaries (re-aggregated ovaries; rOvary) were xenotransplanted into immunodeficient mice (Figure 3A). Fetal cy ovaries were isolated at 8 wpf and dissociated into single cells (approximately 6.0 × 10 per ovary). 5 Approximately 50,000 cells (approximately 5,000 germ cells and 45,000 somatic cells) were reaggregated in suspension for two days to create cy-reconstituted ovaries, which were then transplanted under the surface epithelium of the kidneys of immunodeficient mice, and the development of cy oocytes was analyzed at 3-week intervals.

[0068] At 3 weeks post-transplantation (3 w-ptp), cy-reconstituted ovaries had a unified structure and reconstituted anastomosing cords interspersed with interstitial cells (Fig. 3B, Fig. 2G). Although the majority of germ cells displayed immature morphology, representing oogonia, some exhibited larger nuclei with condensed chromatin, indicative of meiotic transition (Fig. 3B). Consistent with these findings, approximately 40% of germ cells expressed oogonia markers (NANOG, POU5F1, TFAP2C, and PDPN), approximately 50% expressed ZGLP1, approximately 30% expressed SYCP3, and approximately 10% expressed meiotic markers (DMC1, γH2AX, and SYCP1) (Fig. 3C and D). At 6 w-ptp, oocyte development further progressed in cy-reconstituted ovaries, and the majority of germ cells displayed meiotic prophase I nuclear morphology, including pachytene nuclear morphology with synapsed chromosome pairs (<10% positive for oogonia markers, approximately 60% positive for SYCP3, and approximately 30% positive for DMC1, γH2AX, and SYCP1), but no cells expressed oocyte markers (Figures 3B–D, 2G). From 9 w-ptp onward, differentiated primordial follicle-like complexes were present, with oocytes expressing ZP3 and TP63 surrounded by a single layer of squamous granulosa cells expressing FOXL2. By 15 w-ptp, such complexes predominated (Figures 3B–D). These findings indicate that cy-fetal oocyte development and meiotic prophase I proceeded apparently normally upon xenotransplantation into mice, although developmental progression appeared somewhat slower than in vivo. When we examined cy-reconstituted ovaries at 21 w-ptp, we found that although they maintained a distinct structure, only granulosa and stromal cells survived, and essentially all oocytes had degenerated (Fig. 3E), indicating that further follicle development would not proceed under the current xenotransplantation conditions.

[0069] Example 3: Reconstitution of cy fetal oocyte development in vitro. Next, we examined whether in vitro culture promotes proper cy fetal oocyte development in cy reconstituted ovaries. First, we investigated air-liquid interface culture, which supports the development of both mouse embryonic ovaries and reconstituted ovaries (Hikabe O, et al., Nature, 539: 299-303 (2016); Morohaku K, et al., Proc Natl Acad Sci USA, 113: 9021-6 (2016)). cy reconstituted ovaries containing approximately 50,000 ovarian cells at 8 wpf were generated and cultured on Transwell-COL membrane inserts (Figure 4A). First, cy-reconstituted ovaries on Transwell-COL membranes exhibited an integrated structure. However, unlike mouse-reconstituted ovaries, some cells began to expand from the cy-reconstituted ovaries as early as 1 week of in vitro culture (1 w-ivc). By 3 w-ivc, many cells migrated, and the cy-reconstituted ovaries became smaller and flatter (Figures 4B and 5A). Cell expansion continued, and by 6 w-ivc (in vitro culture), the cy-reconstituted ovaries were significantly reduced in size compared with their original size (Figures 4B and 5A). Histological and IF analyses revealed that, at 3 w-ivc, the cy-reconstituted ovaries contained ovarian cords, but these areas were limited to a small number of germ cells. At 6 w-ivc, FOXL2 was not present. +Granulosa cells formed some clusters, but only a few germ cells remained, showing a degenerated morphology (Figure 4B). We investigated whether cy-reconstituted ovaries could be maintained on culture membranes coated with various extracellular matrix components, including collagen (types I, III, IV, V, and VI), fibronectin, laminin, and combinations thereof (Figure 5A). Under some conditions, such as membranes coated with collagen types I, III, IV, V, and VI, or iMatrix (laminin 511), cy-reconstituted ovaries maintained their integrity up to 5 w-ivc, but then began to disintegrate. That is, some cells migrated, and under all conditions examined, cy-reconstituted ovaries flattened at 12 w-ivc (Figure 5A). These findings indicate that cy-reconstituted ovaries require different conditions than mouse-reconstituted ovaries for proper in vitro development.

[0070] Therefore, we investigated whether cy-reconstituted ovaries could be maintained for a long period under suspension conditions (Figure 4A). When grown in suspension in αMEM containing 10% fetal bovine serum (FBS), the basic condition for mouse reconstituted ovary culture (Hikabe O, et al., Nature, 539: 299-303 (2016), Morohaku K, et al., Proc Natl Acad Sci USA, 113: 9021-6 (2016)), cy-reconstituted ovaries showed a unified appearance delineated by several layers of squamous epithelial cells, although their central regions appeared somewhat necrotic and contained a relatively large number of DDX4 cells. + Germ cells mediate FOXL2 +They survived alongside granulosa cells (Figure 5B). We examined 13 basal media, including αMEM (αMEM, GMEM, DMEM, KO DMEM, Advanced MEM, DMEM / F-12, Waymouth's, McCoy's 5A, CMRL, IMDM, Advanced RPMI, StemPro 34, and MesenPRO RS), and found that Advanced MEM provided the best results in terms of size and histology of cy-reconstituted ovaries at 6 w-ivc (Figure 5B). cy-reconstituted ovaries shrank in size at the beginning of culture, likely due to continued compaction of the aggregates, but gradually recovered after 1 w-ivc (Figure 4C). Cy-reconstituted ovaries cultured in Advanced MEM + 10% FBS were consistently larger than those cultured in αMEM with 10% FBS over the 15-week culture period (Figure 4C). Therefore, we analyzed the development of cy fetal oocytes in cy-reconstituted ovaries cultured in Advanced MEM + 10% FBS under suspension conditions at 3-week intervals (Fig. 4D). In 3 w-ivc, the majority of germ cells (approximately 50%) were oogonia expressing NANOG, POU5F1, TFAP2C, and PDPN, whereas approximately 30% and 10% were at the RA-responsive stage (ZGLP1), respectively. + / STRA8 + ) and meiotic (DMC1 + / γH2AX + / SYCP1 +) (Figures 4E and F, 5C). Thereafter, the number of oogonia decreased, and meiotic cells increased, albeit at a slower rate than in vivo and xenotransplantation. Nevertheless, oocytes expressing oogenic markers (ZP3, TP63, FIGLA, NOBOX, and NLRP5) in primordial follicle-like structures differentiated from 12 w-ivc onward (Figures 4E and F, 5C). The morphology of oogonia, as well as that of RA-responsive, meiotic, oogenic, and granulosa cells, as well as the expression and subcellular localization of key markers for these cells in cy-reconstituted ovaries in vitro, were similar to those of the corresponding cell types in vivo (Figures 4E, 5C and D). Examination of meiotic prophase progression using the criteria defined in Figure 1F revealed that meiotic prophase progression in cy-reconstituted ovaries in vitro was slow but steady (Figure 4G). A relatively large fraction of germ cells remained in the preleptotene stage from 6 w-ivc to 15 w-ivc (Figure 4G), suggesting that the preleptotene-to-leptotene transition may be the rate-limiting step under these conditions. These findings demonstrate that over a 12-week period, fetal oocyte development in vitro proceeds by differentiating mitotic oogonia into oocytes and completing early meiotic prophase in primordial follicle-like structures.

[0071] Example 4: Transcriptome dynamics for cy fetal ovarian development in vivo, xenograft, and in vitro To comprehensively define cy fetal ovarian development in vivo, as well as under xenograft and in vitro culture, we performed single-cell RNA sequencing (scRNA-seq) analysis of cy fetal ovaries and cy reconstituted ovaries (Table 2). The cy / human SC3-seq data (sample information, sequence summary, and data summary) are omitted.

[0072]

[0073] We first processed a total of 35,141 single cells isolated from fetal ovaries at 8, 10, 12, 16, and 18 wpf for single-cell cDNA preparation using the 10X Chromium platform. Sequencing revealed that 29,127 cells passed key quality filters (number of detected genes (nGene), total unique molecular identifier (UMI) count, and percent mitochondrial gene count) and were subsequently processed by Scrublet (doublet / multiplet removal algorithm (Wolock SL, et al., Cell Syst, 8: 281-291 e9 (2019)) for analysis (Table 2). Following batch effect removal from the subspace of principal component analysis (PCA) using the fast mutual nearest neighbor (fastMNN) algorithm, followed by graph-based clustering using the Louvain algorithm, we classified these cells into five cell types: DDX4, DDX5, DDX6, DDX7, DDX8, DDX9, DDX10, DDX11, DDX12, DDX13, DDX14, DDX15, DDX16, DDX17, DDX18, DDX19, DDX20, DDX21, DDX22, DDX23, DDX24, DDX25, DDX26, DDX27, DDX28, DDX29, DDX30, DDX31, DDX32, DDX33, DDX34, DDX35, DDX40, DDX41, DDX42, DDX43, DDX44, DDX45, DDX46, DDX47, DDX48, DDX49, DDX50, DDX51, DDX52, DDX53, DDX54, DDX55, DDX56, DDX57, DDX58, DDX59, DDX59, DDX59, DDX59, DDX59, DDX59, DDX59, DDX + Germ cells (2,283 cells), WT1 + Pregranulosa cells (11,986 cells), TCF21 + Stromal cells (14,028 cells), PECAM1 + Endothelial cells (673 cells), and TYROBP +We classified the germ cells into 11 clusters: mitotic 1 / 2 / 3, preleptotene 1 / 2 / 3, leptotene, zygotene, pachytene 1 / 2, and unclassified (probably apoptotic cells) based on the expression of key markers. Pachytene 2 cells were manually classified as diplotene (Figures 6D and 7B). The distribution of cells annotated by scRNA-seq across developmental stages was essentially consistent with that annotated by IF analysis, except for the presence of only a small number of diplotene cells (Figures 6D and 7B), which may be due, at least in part, to the incompatibility of diplotene cells with the 10X platform due to their large cell size. The number of detected genes and total UMI counts remained relatively constant from mitotic to zygotene cells (on average, approximately 7,000 and 50,000, respectively), but decreased somewhat and became more variable in pachytene cells ( Fig. 7C ).

[0074] Mitotic 1 / 2 / 3 cells expressed key oogonia markers, including POU5F1, TFAP2C, PDPN, and PRDM1, and were primarily composed of 8 wpf cells, but included cells from all developmental stages (Figures 6D, E, and F; Figures 7B and D). Mitotic 1 / 2 / 3 cells were thought to represent cells in the G1, S, and G2 / M phases of the cell cycle, respectively (Figure 7E). Prolenotene 1 cells weakly expressed POU5F1 and ZGLP1 but did not display typical meiotic markers, such as DMC1, PRDM9, and SYCP1. They were composed of 8 wpf, 10 wpf, 12 wpf, and 16 wpf cells, which were in the G1 phase of the cell cycle (Figures 6D and F; Figures 7D and E). Preleptotene 2 / 3 cells expressed ZGLP1, REC8, and SYCP3, but negatively or very weakly expressed DMC1, PRDM9, and SYCP1. They were composed of cells from all developmental stages and were in the S and G2 phases of the cell cycle, respectively (Fig. 6D and F, Fig. 7D and E). Thus, preleptotene 2 cells are cells undergoing meiotic DNA replication. Leptotene and zygotene cells, primarily composed of cells after 12 wpf, expressed higher levels of REC8, SYCP2, and SYCP3 than preleptotene cells, and zygotene cells upregulated DMC1, PRDM9, and SYCP1 (Fig. 6D and F, Fig. 7D). Pachytene 1 / 2 cells, primarily composed of cells after 12 wpf, downregulated ZGLP1 and REC8 and expressed SYCP1 / 2 / 3, DMC1, and PRDM9, while pachytene 2 cells initiated downregulation of these genes and upregulation of FIGLA (Fig. 6D and F, Fig. 7D). + / GDF9 + / NOBOX + The oocytes (16 and 18 wpf cells) were composed of meiotic markers downregulated and expressed genes important for oocyte development, such as FIGLA and NLRP5 (Figures 6D and F, Figure 7D). Pseudotime analysis of developmental trajectories using Monocle 3 (Cao J, et al., Nature, 566: 496-502 (2019)) yielded results consistent with those described above (Figure 6G).

[0075] Highly variable genes were identified from 11 clusters (the top 2,000 HVGs minus genes with low expression levels (log(ssUMI + 1) of the cluster's mean expression ≤ 0.5): 1,481 genes). The list of the 1,481 identified genes, their normalized expression values, and GO analysis of the HVGs is omitted here.

[0076] Unsupervised hierarchical clustering (UHC) classified these genes into eight clusters (Figure 6H). Genes in cluster 1 were highly expressed in mitotic phases 1 / 2 / 3 and rapidly downregulated after preleptotene phase 1. They were enriched for genes with gene ontology (GO) functional terms related to "somatic stem cell population maintenance" (e.g., LIN28A, DPPA4, NANOG, KLF4) and "developmental process involved in reproduction" (e.g., PRDM1, UTF1, TFAP2C, NANOS3, SOX15). Genes in cluster 2 showed an expression profile related to cell cycle progression (expressed at mitotic 2 / 3 and proleotene 2 / 3) and were indeed enriched for genes involved in the mitotic cell cycle and meiotic cell cycle processes (e.g., TOP2A, MCM3 / 10, MKI67, CCNA2 / B1 / B2, and PCNA) (Fig. 6H, Fig. 7E). Genes in cluster 3, expressed from proleotene 1 to zygotene, were enriched for genes involved in the regulation of transcription, DNA-templated (e.g., STRA8, ZGLP1, ANHX, and KDM6B) and, interestingly, the pattern specification process (e.g., many HOX genes, SIX1, TBX3, GLI3, and PBX1). Genes in cluster 4 were gradually upregulated from preleptotene stage 1 onwards and were enriched for "meiotic cell cycle" (e.g., TDRD1 / 12, RAD50, EHMT2, MSX1, PLD6) and "reproductive system development" (e.g., HOXA9 / A10, SERPINE2 / F1, CHD7). Genes in clusters 3 and 4, especially those expressed at preleptotene stage 1 / 2, include oogenic fate determiners.Genes in clusters 5 and 6 were expressed from leptotene to pachytene 2 (cluster 5) or diplotene (cluster 6) and included genes important in 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.)]. Genes in clusters 7 and 8 were strongly up-regulated at diplotene and enriched for genes involved in "reproductive process" and "oogenesis" (cluster 8: NPM2, BMP5, GDF9, ZP2 / 3, SOHLH1, FIGLA, NOBOX, etc.). Correlation analysis using the same genes revealed that the 11 clusters could be divided into four groups with higher correlations [mitotic stage 1 to preleukocyte stage 1, preleukocyte stage 1 to zygote (corresponding to RA responsiveness), zygote stage to pachytene stage 2 (corresponding to meiosis), and pachytene stage 2 to diplotene stage (corresponding to oogenesis)], with three clusters (preleukocyte stage 1, zygote stage, and pachytene stage 2) playing transitional roles between the groups (Figure ​(Figure6I). 6I), providing a high-resolution transcriptome classification for primate embryonic oocyte development.

[0077] We used 10X Chromium to generate quality-filtered single-cell transcriptomes (73 germ cells, 702 (pre)granulosa cells, and 118 interstitial cells) from 12 w-ivc and 15 w-ivc cy-reconstituted ovaries and compared them with those of in vivo cy-reconstituted fetal ovarian cells. PCA and fastMNN, followed by Louvain clustering, revealed that cy-reconstituted germ cells in in vitro cy-reconstituted ovaries exhibited transcriptomes consistent with those of oocytes ranging from preleptotene to pachytene (Figure 6J). To gain further insight into the transcriptome dynamics associated with the development of cy fetal oocytes in cy-reconstituted ovaries, we manually picked single cells with germ cell appearance dissociated from cy-reconstituted ovaries at 3 / 6 / 9 / 12 / 15 w-ivc and w-ptp, and oocytes at 12 / 16 / 18 wpf, as well as from adult ovaries, and performed scRNA-seq analysis using single-cell mRNA 3'-end sequencing (SC3-seq) (Nakamura T, et al., Nucleic Acids Res, 43: e60 (2015)) (Figure 6K and L). UHC and PCA were performed on 172 quality-filtered DDX4 genes. + Single cells (including 19 germ cells at 6 / 7 wpf (Okamoto I, et al., Science, 374: eabd8887 (2021)) were classified into four major groups corresponding to mitotic, RA-responsive, meiotic, and oogenic cells. In vivo, xenograft, and in vitro cells were represented in all groups, with mixed transcriptome profiles within the groups (Figure 6K and L). Collectively, these results indicate that cy fetal germ cells undergo appropriate transcriptome maturation in in vitro cy-reconstituted ovaries.

[0078] In addition, WT1 in vivo and in vitro +We investigated the development of pre-cy granulosa cells. Using the same analysis as for germ cells, we classified in vivo pre-cy granulosa cells into 12 clusters after manually removing putative doublets / multiplets (Figure 8A). Analysis of cluster distribution across developmental stages showed that the majority of clusters (clusters 1, 2, 3, 4, 5, 6, 7, 9, 11, and 12) were present as early as 8 wpf, while clusters 8 and 10 appeared at 10 wpf and 16 wpf, respectively (Figure 8A-C). We identified genes with highly variable expression within the 12 clusters (the top 1,500 HVGs minus genes with low expression levels (log(ssUMI + 1) of the cluster's mean expression ≤ 0.5): 788 genes. The list of the 712 identified genes, their normalized expression values, and GO analysis of the HVGs is omitted here.

[0079] UHC using these genes revealed that cells in clusters 2 and 3 were pre-granulosa cells in the S phase of the cell cycle, while cells in cluster 1 were in the G2 / M phase. Furthermore, cells in cluster 1 were enriched in early stages (8 / 10 / 12 wpf) and, like cells in cluster 4, were enriched in genes involved in "cell migration" and "cytoskeleton organization" (Fig. 8A-D). Cells in clusters 11 and 12 were also enriched in early stages (8 / 10 / 12 wpf) and expressed genes involved in "steroid biosynthetic process" and "reproductive system development," thereby representing a unique pre-granulosa cell subtype (Fig. 8A-D). RNA velocity calculation (Bergen V, et al., Nat Biotechnol, 38: 1408-1414 (2020)) and partition-based graph abstraction (PAGA) (Wolf FA, et al., Genome Biol, 20: 59 (2019)) suggested that differentiation of pregranulosa cells progressed from cluster 4 to cluster 5, 7, and 9, or from cluster 4 to cluster 5, 6, 8, and 10 (Figure 8A and B). Cluster 10 cells, which appeared after 16 wpf, were found to be involved in the extracellular matrix organization (e.g., COL1A2 / 3A1 / 4A3 / 6A1 / 6A2 / 14A1, LAMA4 / B1, ITGA1 / A2 / A6, TIMP2, SULF1, etc.) and the Notch signaling pathway. The genes for the "cytogenetic pathway" (NOTCH3, JAG1, HEY2, NR1H4, HES1) were enriched, and therefore likely represent granulosa cells that constitute primordial follicles (Figure 8D and E) (Li L, et al., Cell Stem Cell, 20: 858-873 e4 (2017)). WT1 in cytogenetic pathway-reconstituted ovaries in vitro +Combined analysis of the transcriptome of pre-granulosa cells revealed that the majority of WT1 in 12 w-ivc and 15 w-ivc cells + It was revealed that the cells formed a cluster (cluster 12) close to / merged with the cluster of granulosa cells (cluster 11) of primordial follicles at 16 and 18 wpf, indicating that pre-granulosa cells at 8 wpf had properly differentiated into follicular granulosa cells (Fig. 8F and G). However, in vitro follicular granulosa cells exhibited characteristics of activated granulosa cells, including upregulation of ID2 / 3 / 4 (i.e., activation of BMP signaling) and upregulation of HEY2 and downregulation of HEY1 / HEYL (i.e., modulation of JAG1-NOTCH2 / 3 signaling) (Figure 8G) (Vanorny DA, and Mayo KE, Reproduction, 153: R187-R204 (2017); Zhang Y, et al., Mol Cell, 72: 1021-1034 e4 (2018)), suggesting that granulosa cell development in culture may progress to the activated primary follicle stage.

[0080] Example 5: Reconstitution of Human Fetal Oocyte Development In Vitro Having established the conditions for in vitro cytogenetic oocyte development, we examined whether human fetal oocyte development could also be reconstituted in vitro. Two female fetuses were obtained through artificial abortion at 11 weeks gestation (11W). The gonads were isolated and processed for histological analysis, human reconstituted ovaries, and single-cell transcriptome analysis. Additionally, primordial follicles were isolated from adult human ovaries, and their oocytes were processed for single-cell transcriptome analysis. All human samples were obtained with appropriate informed consent (see Materials and Methods). Consistent with previous reports (Li L, et al., Cell Stem Cell, 20: 858-873 e4 (2017)), 11W human fetal ovaries contained abundant oogonia with pale hematoxylin staining and round nuclei with prominent nucleoli, but also cells with larger nuclei with a more condensed chromatin appearance (Figure 9A). Therefore, DDX4 + Approximately 85% of germ cells contain POU5F1 + A small proportion of these (approximately 3%) expressed early meiotic markers such as SYCP3, γH2AX, and DMC1, but none expressed SYCP1 (Fig. 9B and D), indicating that the majority of germ cells in the human ovary at 11 wk are oogonia.

[0081] Under air-liquid interface conditions, cells migrated from the human reconstituted ovaries (approximately 50,000 ovarian cells), which flattened and collapsed over long-term culture. This was similar to the results for cy-reconstituted ovaries but different from mouse reconstituted ovaries (Fig. 5E). Therefore, we cultured human reconstituted ovaries under the suspension conditions defined for cy-reconstituted ovaries and verified their characteristics at 7 / 8 and 14 w-ivc. At 7 w-ivc, the human reconstituted ovaries showed an integrated structure, but similar to cy-reconstituted ovaries, the central region showed a necrotic appearance (Fig. 9A). Histological sections revealed that the cortical region was filled with germ cells with oogonia-like or early meiotic oocyte-like morphology (Fig. 9A). In good agreement with this result, DDX4+ Approximately 30% of germ cells contain POU5F1 + , and approximately 50% are SYCP3 + , and approximately 30% is γH2AX + / DMC1 + However, only about 2% were SYCP1 + In 14 w-ivc, the reconstituted human ovaries remained united, and their cortices were rich in germ cells with condensed, filamentous chromatin and, notably, large nuclei with numerous primordial follicle-like structures (Fig. 9A). Consistent with these observations, DDX4 + / POU5F1 + The proportion of cells decreased to about 10%, and about 30% were SYCP1 + Approximately 3% are ZP3 + / TP63 + (Fig. 9C and D). The morphology of meiotic and primordial follicle-like cells and the expression / subcellular localization of key markers in these cells were similar to those of the corresponding in vivo cell types in cynomolgus monkeys and humans (Fig. 1E and 9C) (Baker TG, Proc R Soc Lond B Biol Sci, 158: 417-33 (1963)).

[0082] Single-cell transcriptome analysis by SC3-seq of 120 quality-filtered single cells (human fetal ovaries at 11W: 33 cells, human adult ovaries: 4 cells, human reconstituted ovaries at 8W-ivc: 39 cells, and human reconstituted ovaries at 14W-ivc: 44 cells) classified the cells into four cell types: mitotic cells, RA-responsive cells, meiotic cells, and oogenic cells (Fig. 9E and F). Consistent with histological and IF analyses, the majority of single cells picked from dissociated human fetal ovaries at 11W 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 reconstituted ovaries at 8 w-ivc contained all four cell types (mitotic: 16 / 39; RA-responsive: 13 / 39; meiotic: 7 / 39; oogenesis: 3 / 39), whereas single cells from human reconstituted ovaries at 14 w-ivc were primarily meiotic cells expressing DMC1, RAD51AP2, and SYCP1, or oogenic cells expressing ZP3, NLRP5, TP63, and NOBOX (mitotic: 0 / 44; RA-responsive: 3 / 44; meiotic: 21 / 44; oogenesis: 20 / 44) (Figures 9E and F). Notably, oocytes isolated from human reconstituted ovaries were closely clustered with mature oocytes from primordial follicles (Figures 9E and F), indicating the proper progression of human oocyte development in human reconstituted ovaries in vitro. To further substantiate this point and compensate for the lack of human fetal ovarian samples at later stages, we merged the SC3-seq data from human germ cells and cy germ cells using canonical correlation analysis (CCA) (Stuart T, et al., Cell, 177: 1888-1902 e21 (2019)) and performed PCA.This revealed highly parallel transcriptomic progression in human oocytes in in vitro human-reconstituted ovaries, as well as in in vivo cy oocytes and in vitro cy reconstituted ovaries (Figure 9G). Collectively, these results demonstrate that human oocyte development can be reconstituted in vitro, at least up to the point where oocytes complete the first meiotic prophase of primordial follicles.

[0083] Example 6: Primate-specific Program for Fetal Oocyte Development In mammals, germ cell development proceeds through conserved cellular events, including PGC specification from pluripotent embryonic precursors, epigenetic reprogramming, germ cell sex determination, and epigenetic programming accompanied (in females) or followed (in males) by meiosis to generate sexually dimorphic haploid gametes. However, the precise cellular dynamics and molecular mechanisms underlying these conserved events appear to vary significantly between species (Saitou M, and Hayashi K, Science, 374: eaaz6830 (2021)). To gain insight into the primate-specific program associated with fetal oocyte development, we compared relevant transcriptome datasets between mice, monkeys, and humans. The cynomolgus monkey and human datasets created in this example were compared using CCA with four publicly available 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))], performed Louvain clustering, and defined 15 clusters of fetal oocyte developmental stages across three species based on the expression of associated genes: mitosis 1-4, proleptotene 1-3, leptotene 1-3, zygotene 1 / 2, pachytene 1 / 2, and diplotene (Figure 10A, Table 2).The distribution of stage-defined cells at each developmental time point was consistent with previous observations (Figures 10B and C, 11A) (Chitiashvili T, et al., Nat Cell Biol, 22: 1436-1446 (2020)) (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)).

[0084] Pseudotime analysis using Monocle 3 revealed that in humans and monkeys, the transition from mitosis to premeiosis, i.e., the meiotic transition, induced relatively large transcriptomic alterations, whereas in mice, this transition was accompanied by minor changes, but subsequent transitions induced significant changes (Fig. 10D). HVGs were identified during fetal oocyte development in each species, of which 237 genes were orthologous and shared among all three species. Correlation analysis of their expression at each developmental stage revealed major differences between mitotic and premeiotic cells in humans and monkeys, but not in mice (Fig. 10E). Furthermore, the expression profiles of each cluster were relatively conserved between humans and monkeys, but diverged in mice (Fig. 10E). Previous studies have shown that during meiotic prophase of spermatogenesis in humans, monkeys, and mice, the contribution of 1-1-1 orthologous genes to global expression decreases as meiosis progresses, whereas the contribution of species-specific genes to global expression increases in humans and mice (though no clear conclusions could be reached for monkeys due to the low level of gene annotation), arguing for species-specific specialization during male meiotic prophase (Lau X, et al., Dev Cell, 54: 548-566 e7 (2020)). We also performed the same analysis for female meiotic prophase. The results clearly differed from those in males; as meiosis progressed in females, the contribution of 1-1-1 orthologs to global expression gradually increased, whereas the contribution of species-specific genes to global expression remained consistently low (Figure 11C). Thus, in humans and monkeys, the transition from mitosis to meiosis during oogenesis is accompanied by more dramatic transcriptomic changes than in mice, and the overall transcriptomic differences between the three species result from differential usage of orthologous genes.

[0085] Furthermore, we identified primate-specific genes that exhibit dynamic changes during fetal oocyte development. Based on the SC3-seq analysis in Figure 9G, we identified differentially expressed 1-1 orthologs between the two consecutive cell types [log2(normalized read count + 1) > 3, fold change > 2], extracted genes shared between humans and monkeys (397 genes), and examined their expression changes in the 10X dataset during fetal oocyte development, as defined in Figure 10A. The list of 397 genes and their normalized expression values ​​in the 10X scRNA-seq data for humans, monkeys, and mice is omitted.

[0086] The expression profiles of 130 of the 397 genes were well conserved across the three species, with notable trends observed: genes expressed in mitotic cells continued to be expressed in preleptotene / leptotene cells, while genes expressed in meiotic cells showed upregulation at relatively later stages in mouse (Fig. S1A). These genes included well-known genes involved in fetal oocyte development, such as PRDM1, TFAP2C, NANOS3, TCL1A, SOX15, DPPA3 (mitotic stage), HORMAD1, REC8, STAG3, SMC1B (leptotene and later), SYCP1, SYCP2, SYCE3, HORMAD2, MEIOB, MEIOC, TEX12, SPO11 (zygote and later), NPM2, ZP2, NOBOX, FIGLA, GDF9, NLRP5, and TP63 (pachytene 3 / diplotene stage) (Fig. S1A). On the other hand, 83 genes showed differential expression changes in humans and monkeys, but not in mice. These genes included, among others, PIM2, THY1, MMP2, SOX17, and TFCP2L1 (mitosis), PBX1, SKAP2, KDM1B, HOXA3 / B3, PAX6, and MORC4 (preleptotene / leptotene), DMRTB1, CLGN, GSAP, SPAG6, UPB1, and CABYR (leptotene to leptotene), and SLCO4A1, CSF1R, TMEM163, KPNA7, ARHGEF33 / GAP18, SERPINF1, and LMOD3 (pachytene 2 / diplotene) (Fig. 10F and Fig. 12B). Among these, we examined PAX6, a homeobox TF involved in key cell fate specification processes, including eye development (Jordan T, et al., Nat Genet, 1: 328-32 (1992), Walther C, and Gruss P, Development, 113: 1435-49 (1991)); CLGN, a chaperone specifically expressed in the endoplasmic reticulum (ER) of spermatogenic cells that plays an important role in sperm binding to the ZP (Ikawa M, et al., Nature, 387: 607-11 (1997)); and LMOD3, an actin filament-nucleating protein expressed in skeletal and cardiac muscle (Yuen M, et al., J Clin Invest, 124: 4693-708 (2014)).IF analysis revealed that PAX6 inhibited TFAP2C at early meiotic prophase stages in cynomolgus monkey and human reconstituted ovaries. - CLGN was found to be specifically and strongly expressed in germ cells (Fig. 10G and H). CLGN was expressed in the cytoplasm, possibly the ER, and TFAP2C in cynomolgus monkey and human reconstituted ovaries. - LMOD3 was expressed and localized in germ cells and young human primordial follicles (Fig. 10G-I). LMOD3 showed specific expression in both cynomolgus monkey and human primordial follicles, with a reticular pattern in the cytoplasm (Fig. 12C). Interestingly, LMOD3 was more enriched in the cytoplasm facing the ovarian medulla in young human primordial follicles (Fig. 12C).

[0087] Example 7: Conserved Activity of the X Chromosome In mammals, female cells have two X chromosomes (Xs), while male cells have one X and one Y chromosome. In females, one X is randomly inactivated (Xi) in the early embryo after implantation (XCI), complementing the male X-linked gene dosage (Lyon MF, Nature, 190: 372-3 (1961); Zylicz JJ, and Heard E, Annu Rev Biochem, 89: 255-282 (2020)). In somatic cells, one active X chromosome (Xa) is hyperactivated and compensates for the autosomal (X:A) gene dosage 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: 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)), and in mice and humans, germ cells have been shown to exhibit non-canonical X chromosome dosage compensation (Sangrithi MN, et al., Dev Cell, 40: 289-301 e3 (2017)), although another study reached a different conclusion (Chitiashvili T, et al., Nat Cell Biol, 22: 1436-1446 (2020)).

[0088] Therefore, we decided to compare X chromosome activity during fetal oocyte development in three species. First, we examined the X:A ratio of gene expression levels during mouse germ cell development based on bulk RNA-seq data, which ensures high quantitative accuracy. Information on the mouse bulk RNA-seq data (sample information, sequence summary, and data summary) is omitted. In gonadal somatic cells, the X:A ratio was slightly below 1 (approximately 0.9), indicating that XCU occurs, albeit not completely, on a single Xa (Figure 13A). Information on the X:A ratio analysis using the 10X / SC3-seq / bulk RNA-seq dataset is omitted.

[0089] In female germ cells, the X:A ratio was less than 1 (approximately 0.9) during mitotic expansion until E12.5, exceeded 1 (approximately 1.1) during meiotic prophase I from E13.5 to E18.5, and was downregulated to approximately 1 at postnatal days (P) 0 and 1 in primordial follicles (Figure ​(Figure13A). 13A). Given that both X chromosomes are progressively activated in germ cells by E14.5 (Sugimoto M, and Abe K, PLoS Genet, 3: e116 (2007)), this indicates a mild XCU during meiotic prophase and its erasure in primordial follicles. In male germ cells, the X:A ratio was approximately 0.83 at E11.5, decreased to approximately 0.65 at E12.5, and remained at approximately 0.7 at mitotic arrest (Figure ​(Figure13A). 13A), indicating that XCU erasure also occurs in males. Interestingly, we detected mild upregulation of Xist (an X-inactive specific transcript) in primordial follicles, a long non-coding RNA that triggers a cascade of suppressive events in mouse XCI (Brockdorff, Ashworth et al., 1992; Marahrens, Panning et al., 1997; Penny, Kay et al., 1996), while it was substantially suppressed in male germ cells (Figure ​(Figure13A). 13A). The timing of the mild XCU development and elimination in females differed from that of XCU elimination in males, but was consistent with previous studies (Sangrithi MN, et al., Dev Cell, 40: 289-301 e3 (2017)). Next, based on the SC3-seq dataset, we examined the X:A ratio during in vivo, xenotransplantation, and in vitro development of cytoplasmic and human fetal oocytes. During female cy embryogenesis, XCI occurs simultaneously with XCU in somatic cells, and cy germ cells initiate XCR based on their fate commitment, completing it by approximately E50 (Okamoto I, et al., Science, 374: eabd8887 (2021)). In vivo, we found that despite XCR, the X:A ratio remains below 1 in mitotic and RA-responsive cells (approximately 0.90 and 0.68, respectively), increases to approximately 1 in meiotic cells, and then decreases to below 1 in oogenic cells (approximately 0.71) (Figure ​(Figure13B).XIST was highly expressed in mitotic germ cells, repressed in RA-responsive cells, upregulated in meiotic cells, and repressed again in oocytes (Fig. 13B). The dynamics of the X:A ratio and XIST expression were similar in xenotransplantation and in vitro fetal oocyte development, as well as in vivo and in vitro human fetal oocyte development (Fig. 13B and C). Next, we analyzed the dynamics of the X:A ratio of the three species based on the 10X data set, revealing that the three species exhibited similar, albeit narrower, dynamic ranges. The ratio was less than 1 in oocytes during mitosis and early meiosis (preleptotene), increased to approximately 1 in oocytes during late meiosis (zygote / pachytene), and decreased to less than 1 in oocytes of primordial follicles (diplotene oocytes) (Fig. 13D, Fig. 12D). XIST showed a similar expression profile in humans and monkeys: it was significantly expressed in mitotic cells (as in somatic gonadal cells) and then down-regulated, whereas in mice, it was substantially suppressed in mitotic and meiotic cells and showed a mild up-regulation in diplotene oocytes (Figure S13D, Figure S12D). These findings are consistent with those of Chitiashvili et al., who reported X:A dynamics during human fetal oocyte development (Chitiashvili T, et al., Nat Cell Biol, 22: 1436-1446 (2020)), but differ from those of Sangrithi et al., who reported a mild XCU in human mitotic oogonia (Sangrithi MN, et al., Dev Cell, 40: 289-301 e3 (2017)). Based on the collective data obtained in this example, we conclude that although there is some quantitative variation depending on the measurement method, overall, X-linked gene dosage is regulated in a similar manner in humans, monkeys, and mice, and that fetal oocyte development proceeds via two Xa and is largely independent of XCU.

[0090] Example 8: Evaluation of the effects of adding steroid hormones to the culture medium. An experiment was conducted in which steroid hormones were added to the above-mentioned culture medium. Here, steroid hormones refer to progesterone, cortisol, dehydroepiandrosterone, and their sulfate conjugates, dehydroepiandrosterone sulfate, whose concentrations in fetal cynomolgus monkey blood can be measured. For the experiment, the following concentrations were added: progesterone (200 ng / mL); cortisol (100 ng / mL); dehydroepiandrosterone (50 nM); and dehydroepiandrosterone sulfate (10 μM), which correspond to physiological concentrations in humans and cynomolgus monkeys. On the other hand, none of these hormones were added to the medium without steroid hormones. This also applies to the following examples.

[0091] When cultured in a medium without steroid hormones, it took 12 weeks for primordial follicles to form, and no primordial follicles were observed in the reconstituted ovaries after 9 weeks of culture. On the other hand, in the steroid hormone-added group, primordial follicles were observed after 9 weeks of culture. Therefore, it is thought that the addition of steroid hormones promotes oocyte differentiation and maturation, resulting in the production of primordial follicles in a short period of time (Figures 14 and 15).

[0092] Example 9: Evaluation of the Effects of Adding Retinoic Acid (RA) to Culture Media. A comparison of RA-treated and non-treated groups was performed under FBS-free conditions. The RA-treated groups were tested at RA concentrations of 10 nM, 100 nM, 500 nM, and 1 μM. At all RA concentrations, meiotic markers became positive and oogonia markers became negative earlier than the control group (Figures 16 and 17). Thus, the addition of RA to this culture system appears to promote differentiation of oogonia into oocytes and the initiation and progression of meiosis. While this effect was comparable at all concentrations, increasing the RA concentration inhibited the growth of reconstituted ovaries, suggesting that 10 nM RA is the optimal concentration for sufficient effect.

[0093] Example 10: Evaluation of the effect of simultaneous addition of steroid hormones and retinoic acid (RA) to the culture medium. In this example, it was confirmed that primordial follicles were produced more efficiently in the steroid hormone-added group and the retinoic acid (RA)-free group (10 nM / FBS(-)) than in the steroid hormone- and retinoic acid-free group (Figure 18). Furthermore, when steroid hormones and RA (10 nM / FBS(-)) were added simultaneously, even more primordial follicles were produced (Figure 18). This suggests that the two agents synergistically enhance the efficiency of primordial follicle production.

[0094] Materials and Methods: Animals. All animal experiments were performed with ethical approval from 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 previously described (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 / 10-hour dark cycle. For animal experiments using cynomolgus monkeys (cynomolgus monkeys), the procedures for oocyte collection, intracytoplasmic sperm injection, preimplantation embryo culture, and embryo transfer into foster mothers were as previously described (Yamasaki J, et al., Theriogenology, 76: 33-8 (2011)). Pregnancy was confirmed by transabdominal fetal ultrasound at 4 weeks post-fertilization (wpf). Plasma was collected from pregnant monkeys, and fetal cell-free DNA was extracted. To minimize the number of cy fetuses used, cell-free DNA from maternal plasma was purified using the QIAamp DNA Mini Kit (QIAGEN, 51304). Fetal sex was determined by real-time PCR targeting the SRY and DYS14 genes (Yasmin L, et al., Comp Med, 65: 70-6 (2015)). Caesarean section was performed under isoflurane- and ketamine-based general anesthesia to collect cy fetuses. Fetal sex was further confirmed by gross gonad observation or by PCR of the UTX / UTY genes (Villesen P, and Fredsted T, BMC Ecol, 6: 8 (2006)).

[0095] (Collection of human embryo and ovarian samples) Human fetuses or adult ovaries were collected after informed consent from participants undergoing induced abortion or gynecological surgery. All experimental procedures were approved by the Kyoto University Ethics Committee (approval numbers G1047 and G1192). Fetal sex was determined by sex-specific PCR for the ZFX / ZFY and SRY loci (Josef B, and Adam K, Folia Zoologica, 52: 269-274 (2003)).

[0096] For immunohistochemical analysis, fetal or reconstituted ovaries were fixed in 4% paraformaldehyde / PBS (Sigma-Aldrich, 158127) at 4°C for 30 minutes or overnight, embedded in paraffin wax (Leica, 39601006) or OCT compound (Sakura Finetek, 4583), and sectioned at 5 or 8 μm thickness. After deparaffinization, sections were pretreated with HistVT One (Nacalai Tesque, 06380-76) antigen retrieval reagent at 90°C for 40 minutes. Sections were treated with blocking solution (1% bovine serum albumin (Sigma-Aldrich, A7030), 5% normal donkey serum (JIR, 017-000-121), 0.1% Tween 20, and 1× phosphate-buffered saline) at room temperature for 30 minutes and then incubated with primary antibodies in blocking solution at 4°C overnight. Slides were further incubated with Alexa Fluor-conjugated secondary antibodies. Fluorescent images were captured using a laser scanning confocal microscope (OLYMPUS, FV1000; ZEISS, LSM780). For quantitative analysis of each marker, germ cells marked by DDX4 expression were analyzed. To clarify the "inner" and "outer" cortices of fetal ovaries at 10–18 wpf, ovarian cortices were divided into two halves at the center of the cortex. Hematoxylin and eosin staining was performed using standard procedures.

[0097] (Meiotic Staging in Ovarian Sections) DDX4-expressing germ cells in fetal ovarian sections were classified into seven stages based on the patterns of nuclear staining for DAPI, SYCP3, and DMC1. The stages were defined as follows: Mitosis: Cells without SYCP3 or DMC1 signals. Preleptonetene: Numerous short nuclear filaments with low SYCP3 signals. Leptonetene: Numerous short nuclear filaments with low SYCP3 and DMC1 signals. Zygotene: Numerous long, thin SYCP3-stained filaments with many DMC1 dots. Early pachytene: Thick SYCP3-stained filaments with many DMC1 foci. Late pachytene: Thick SYCP3-stained filaments with one or fewer DMC1 dots on each chromosome. Diplotene: Numerous short nuclear filaments with weak or no SYCP3 signals. Diplotene cells were distinguished from preleptonetene cells by their size and histological morphology.

[0098] Dissociation of cy / human fetal ovarian tissue and cy / human reconstituted ovaries. Fetal ovaries collected from cy fetuses at 8–18 wpf were carefully dissected in chilled PBS. 8–14 wpf fetal ovaries were finely minced with scissors in 0.05% trypsin / EDTA / PBS solution (GIBCO, 15400-054) and incubated at 37°C for 15 min with gentle pipetting every 5 min. Dissociation medium [DMEM (GIBCO, 10313021) containing 10% FBS, 2 mM L-glutamine (GIBCO, 25030081), 1X penicillin / streptomycin (GIBCO, 15070063), and 100 μg / mL DNase I (MERCK, DN25)] was added for further dissociation. Dissociated cells were used for single-cell RNA-seq analysis or resuspended in CELLBANKER 1 plus (ZENOGEN PHARMA, CB021) and stored at −80°C. Human fetal ovaries from 11 weeks of gestation were also isolated and cryopreserved using the same procedure.

[0099] For ovaries from 16 and 18 wpf fetuses, 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. For SC3-seq analysis, cells were picked under an inverted microscope (OLYMPUS, IX71) and multiplet cells were removed. For 10x experiments, the cell suspension was further treated with TryPLE Express (1x) (GIBCO, 12604021) at 37°C for 5 minutes to obtain a single cell suspension.

[0100] To dissociate the cy / human reconstituted ovaries, the tissue was incubated with TryPLE Express (1x) (GIBCO, 12604021) for 5 minutes. After adding dissociation medium, the cells were processed for single-cell analysis.

[0101] In vitro culture of cy / human reconstituted ovaries under air-liquid interface conditions. 8 wpf cy fetal ovarian cells (50,000 cells / well) were seeded into wells of a Nunclon Sphera-treated U-bottom 96-well plate (NUNC, 174925) and cultured in suspension in αMEM medium 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 two days, the cy-reconstituted ovaries were transferred using a glass capillary to Transwell-COL membrane inserts (Corning, 3495) soaked in αMEM (GIBCO, 12571063) containing 10% FBS, 2 mM L-glutamine (GIBCO, 25030081), 150 μM ascorbic acid (Sigma-Aldrich, A4403), 1× penicillin / streptomycin (GIBCO, 15070063), and 55 μM 2-mercaptoethanol (GIBCO, 21985023). Half of the medium was replaced every two days. The reconstituted ovaries were cultured at 37°C in an atmosphere of 5% CO2 in air. Human reconstituted ovaries were generated from human fetal ovarian cells from 11 weeks of gestation and cultured under the same conditions, except that Advanced MEM was used as the basal medium.

[0102] 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 Matrigel (Corning, 356230), and combinations thereof prior to IVC according to the manufacturer's coating protocol.

[0103] In vitro culture of cy / human reconstituted ovaries in suspension. cy / human reconstituted ovaries were generated from fetal ovarian cells (50,000 cells / well) at 8 wpf in suspension 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 two days, the cy-reconstituted ovaries were transferred to separate wells of a U-bottom culture plate using a glass capillary and cultured in suspension in Advanced MEM (GIBCO, 12492013) containing 10% FBS, 2 mM L-glutamine (GIBCO, 25030081), 150 μM ascorbic acid (Sigma-Aldrich, A4403), 1× penicillin / streptomycin (GIBCO, 15070063), and 55 μM 2-mercaptoethanol (GIBCO, 21985023). Medium changes were performed every two days by transferring the cy-reconstituted ovaries to separate wells containing freshly prepared medium. The cy-reconstituted ovaries were analyzed at 3, 6, 9, 12, and 15 days (Day 23, 44, 65, 86, and 107). Reconstituted human ovaries were prepared from 11-week-pregnant human fetal ovarian cells and cultured under the same conditions. In examples where steroid hormones (progesterone (Sigma, P6149), cortisol (also known as hydrocortisone) (Sigma, H0135), dehydroepiandrosterone (also known as trans-dehydroandrosterone) (Sigma, D4000, and its sulfate conjugate, dehydroepiandrosterone sulfate (Cayman Chemical, 15873)) and / or retinoic acid (Sigma, R2625) were added, the compounds were added to the culture medium from the start of in vitro culture.

[0104] (Xenotransplantation Experiment) cy-reconstituted ovaries prepared by the above culture method were transplanted under the kidney capsule of immunodeficient KSN / Slc mice. The transplantation procedure was performed under mixed anesthesia (midazolam, medetomidine, and butorphanol), and the cy-reconstituted ovaries were placed under the kidney capsule using a thin glass capillary. Histological and SC3-seq analyses were performed at 3, 6, 9, 12, and 15 weeks post-transplantation (ptp).

[0105] (Isolation of Mouse Gonadal / Ovarian Cells) To isolate mouse gonad / ovarian cells at embryonic day (E) 11.5 for bulk RNA-seq analysis, VR females were transfected with Dppa3(Stella)-EGFP mice. Tg / Tg The females were mated with males. The noon on the day a vaginal plug was confirmed was designated E0.5. The sex of the embryos at E11.5 was determined by sex-specific PCR of the Ube1 locus. To isolate mouse gonad / ovarian cells at E12.5–postnatal day (P) 1, ICR females were mated with VR males, and the sex of the embryos was determined by gross observation. The gonads / ovaries were isolated into single cells by incubation with 0.05% trypsin-EDTA (GIBCO, 15400-054) at 37°C for 10 minutes and quenched with an equal volume of DMEM containing 10% FBS. Large cell clumps were removed using a nylon cell strainer (FALCON, 352350), and the cells were centrifuged at 200 rcf for 5 minutes. The cell pellet was resuspended in DMEM / F12 medium (GIBCO, 1130-082) containing 0.1% BSA Fraction V (Gibco, 15260037) and analyzed by VR sorting using a fluorescence-activated cell sorter (BD Biosciences, FACS Aria III). + Germ cells and VR - Somatic cells were sorted.

[0106] (10X Genomics single-cell RNA-seq). Dissociated cells from cy fetal ovaries and cy reconstituted ovaries were stained with DRAQ7 to distinguish dead cells. All DRAQ7 -Viable cells were collected using a fluorescence-activated cell sorter (BD Biosciences, FACS Aria III). Subsequent 10X scRNA-seq library preparation (10X Genomics, Single Cell 3'Kit v. 3 and v. 3.1) and sequencing on the NovaSeq 6000 platform (Illumina) were performed according to the manufacturer's instructions. Raw files were processed using the "mkfastq" command in Cell Ranger (v. 5.0.1) and mapped to the cynomolgus monkey reference genome (MacFas5.0 for cynomolgus monkey and GRCh38.p12 for human).

[0107] Analysis of cy scRNA-seq Data (10X) Gene-barcode matrices were analyzed using the "Seurat" R package (v. 3.2.2) (Satija R, et al., Nat Biotechnol, 33: 495-502 (2015)) according to the online tutorial (https: / / satijalab.org / seurat / index.html). Low-quality cells, putative doublets / multiplets, and putative stripped nuclei were excluded from downstream analysis (Table 2). Raw counts for each cell were divided by the size coefficient calculated by the "scran" R package (v. 1.14.1) and then log-transformed for further analysis [log2(size-scaled(ss)UMI + 1)]. Doublet cells were removed using the "Scrublet" Python package (v. 0.2.1) according to a previously published procedure (Wolock SL, et al., Cell Syst, 8: 281-291 e9 (2019)). A putative doublet threshold was determined based on a bimodal distribution calculated with default parameters. Batch correction was then performed using the "RunFastMNN" function of the Seurat R package with 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 for whole-cell analysis and 0.8 for germ / granulosa cell analysis. Clusters were annotated by confirming the expression patterns of marker genes previously characterized in human and mouse fetal ovarian cells. The source and identification numbers of other R or Python packages are listed in Table 3.

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] (cy Germline Analysis (10X)) DDX4 + Cells within the germ cell cluster were further analyzed and divided into 13 clusters using the "FindClusters" function. After removing two small clusters consisting of putative doublets / multiplets that coexpressed markers for other cell types, 11 substages of meiosis I were defined according to the expression patterns of key germ cell markers: "mitosis 1 / 2 / 3," "proleptotene 1 / 2 / 3," "leptotene," "zygotene," "pachytene 1 / 2," and "unclassified." Cells expressing GDF9 / NOBOX / ZP3 were manually selected into a separate cluster defined as "diplotene." To identify highly variable genes (HVGs) during female germ cell development, we ran the "FindVariableFeatures (selection.method= "vst," nfeatures=2,000)" function in the Seurat R package across the 11 substages (mitosis-diplotene) and found 2,000 HVGs. Of these, 1,481 genes were excluded from the analysis (519 genes with low expression levels [log(ssUMI + 1) cluster mean ≤ 0.5]) and were used for heatmap visualization, GO enrichment analysis (see below), and calculation of correlation coefficients between each meiotic substage (Figure 6H and I). Pseudotime trajectory analysis was performed using the "Monocle 3" R package (v. 0.2.1). Cy germ cells from reconstituted ovaries cultured in 12 / 15w-ivc were also treated in the same manner.

[0115] (Granulosa cell analysis (10X)) FOXL2 +Cells from the granulosa cell cluster were further analyzed. A count matrix of spliced ​​and unspliced ​​transcripts was generated by running the commands "count--include-introns" and "count" in Cell Ranger (v. 5.0.1). After removing two small clusters consisting of putative doublets / multiplets that co-expressed markers for other cell types using the "FindClusters" function with a resolution of 0.8, cells were divided into 12 clusters. RNA velocity analysis was performed using the "scvelo.pp.moments(n_pcs = 30, n_neighbors = 30)" and "scvelo.tl.velocity(mode = 'stochastic')" functions in the scVelo python package (v. 0.2.0) (Bergen V, et al., Nat Biotechnol, 38: 1408-1414 (2020)). Following the online tutorial (https: / / scanpy-tutorials.readthedocs.io / en / latest / paga-paul15.html), a partition-based graph abstraction (PAGA) graph (Wolf FA, et al., Genome Biol, 20: 59 (2019)) was calculated on the annotated clusters in Figure 8A using the "scanpy.tl.paga" function in Scanpy (v. 1.2.2) in Python (v. 3.7.7). The cluster connectivity threshold was set to 0.03. To identify HVGs from the 12 clusters, 1,500 HVGs were found using the "FindVariableFeatures(selection.method="vst," nfeatures=1,500)" function in the Seurat R package.Of these, 788 genes were excluded from the analysis (712 genes with low expression levels [cluster mean log(ssUMI + 1) ≤ 0.5]) and used for heatmap visualization and GO enrichment analysis (see below) (Fig. 8D). Cy granulosa cells from reconstituted ovaries cultured in 12 / 15w-ivc were also treated in the same manner.

[0116] (SC3-seq of cy / human germ cells) Germ cells dissociated from in vivo cy / human fetal ovaries and cy / human reconstituted ovaries were manually picked using a glass capillary under a stereomicroscope (Leica, S8 APO). 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 measured by SYBR real-time PCR targeting the PPIA, GAPDH, NANOG, DDX4, DAZL, SYCP3, SYCP1, ZP3, and FOXL2 genes. Primer sequences are listed 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 sequencing was performed on the Illumina NextSeq500 / 550 platform using the NextSeq 500 / 550 high output kit v. 2 (75 cycles) (FC-404-2005; Illumina).

[0117]

[0118] (Bulk RNA-seq analysis of mouse gonad / ovarian cells) Mouse gonad / ovarian cells were lysed and total RNA was purified using NucleoSpin RNA XS (Takara Bio, U0902A) according to the manufacturer's instructions. 1 ng of total RNA from each sample was used for cDNA synthesis and amplification. cDNA synthesis and library construction were performed 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 NextSeq500 / 550 platform using the NextSeq 500 / 550 high output kit v. 2 (75 cycles) (FC-404-2005; Illumina).

[0119] (Mapping of bulk RNA-seq / SC3-seq reads and conversion to gene expression levels) Genome sequences and transcript annotations (GRCm38.p6 for mouse, GRCh38.p12 for human, and MacFas5.0 for cynomolgus monkey) were downloaded from the NCBI ftp site. To calculate expression levels as genes (Entrez genes) rather than mRNAs, bulk RNA-seq / SC3-seq reads were obtained only from the 3' end of the transcripts. Read trimming, mapping, and evaluation of expression levels were performed as previously described (Ishikura Y, et al., Cell Reports, 17: 2789-2804 (2016); Nakamura T, et al., Nature, 537: 57-62 (2016)).

[0120] Orthologous Genes Among Three Species: For cross-species comparative analysis of SC3-seq and 10X scRNA-seq data, we obtained lists of orthologous genes for the three species from Ensembl BioMart (Ensembl 97: Jul 2019). For the interspecies integration of SC3-seq data, we used the one-to-one (1-1) orthologous genes (16,734 genes) between human and cy. For the interspecies integration of 10X data, we used the one-to-one (1-1-1) orthologous genes across the three species (15,007 genes). Because the orthologous gene lists from Ensembl BioMart were relatively incomplete, we created a genomic coordinate comparison list using the LiftOver tool, as previously reported (Nakamura T, et al., Nature, 537: 57-62 (2016); Sasaki K, et al., Cell Stem Cell, 17: 178-94 2015). The list of orthologous genes is omitted.

[0121] (Analysis of SC3-seq Data from Human Germ Cells) SC3-seq data analysis was performed using R software (v. 4.0.2). Read counts were normalized to 200,000 reads and then log-transformed (log2(normalized read count + 1)). "All expressed genes" were defined as genes with a log2(normalized read count + 1) value greater than 2 [approximately 10-20 copies per cell] in at least one sample (Nakamura T, et al., Nucleic Acids Res, 43: e60 (2015)). Unsupervised hierarchical clustering (UHC) was performed using the "hclust" function with Euclidean distance and Ward's method (ward.D2). Principal component analysis (PCA) was performed using the "prcomp" function without scaling.

[0122] For cross-species analysis between monkeys and humans, we used 1-1 orthologous genes between humans and monkeys and processed the data with the "Seurat" R package (v. 4.0.4). The cySC3-seq dataset was integrated with the human dataset using the Seurat CCA algorithm, i.e., "FindIntegrationAnchors" (parameter "anchor.features" = 5,000) and the "IntegrateData" function with default settings. The integrated germline was divided into four 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 two consecutive clusters, and DEGs were defined as those with a >2-fold change between clusters and a mean gene expression level [log2(normalized counts + 1)] >3.

[0123] Gene Ontology Enrichment Analysis: Gene ontology (GO) enrichment analysis for HVGs / DEGs was performed using the DAVID web tool (Huang da W, et al., Nature protocols, 4: 44-57 (2009)). Because annotations of cynomolgus macaque genes were relatively incomplete, human annotations corresponding to cynomolgus macaque annotations were used for GO analysis. Missing genes in the 1-1 orthologous gene list from Ensembl BioMart were complemented by an orthologous gene list created with the LiftOver tool.

[0124] Cross-species analysis among three animal species using 10X public datasets. To perform cross-species analysis among humans, monkeys, and mice, we obtained the 10X scRNA-seq dataset 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)). The downloaded raw files were processed using the "mkfastq" command in Cell Ranger and mapped to the reference genome sequences (GRCm38.p6 for mouse and GRCh38.p12 for human). Low-quality cells and putative doublets were excluded from downstream analysis. The threshold for these cells depended on the quality of each dataset (e.g., the reagent version and reference genome used), and was defined by carefully evaluating the number of genes, UMIs, and mitochondrial genes detected in each dataset. Each dataset was processed in the same manner as described above for the analysis of cy 10X scRNA-seq data, and DDX4 / Ddx4 +Germ cell clusters were extracted for the following interspecies comparative analysis. The cy / human 10X scRNA-seq dataset created in this example was integrated with four public datasets. Each dataset was size-scaled to 10,000 UMIs (ss10kUMI) and processed using the CCA algorithm, i.e., the "FindIntegrationAnchors" parameter ("anchor.features" = 2,000) and the "IntegrateData" function in the "Seurat" R package (v. 3.2.2). PCA was performed on the corrected count matrix obtained after adjusting for species differences, and UMAP visualization was performed on 30 dimensions using the "RunUMAP" function. The integrated germ cells were divided into 15 clusters using the "FindClusters" function in Seurat (parameter "resolution" = 0.8). Each cluster was defined as "mitosis 1 / 2 / 3 / 4," "preleptotene 1 / 2 / 3," "leptotene 1 / 2 / 3," "zygotene 1 / 2," "pachytene 1 / 2," or "diplotene" based on the expression patterns of key germ cell markers. To confirm the accuracy of the merged clusters, pseudotime trajectory analysis was performed for each dataset using the "Monocle 3" R package (v. 0.2.1). Among the 1-1-1 orthologous genes imputed with the LiftOver tool, common HVGs (237 genes) across the three datasets / species were obtained, and the Pearson correlation coefficients of the average HVG expression levels between meiotic substages of the three animal species were calculated.

[0125] Conserved Primate-Specific Variable Genes During Female Meiosis Among the DEGs identified by the interspecies comparison analysis using SC3-seq described above, commonly variable 1-1 orthologous genes were defined as genes conserved between humans and monkeys. The clusters defined by the 10X cross-species analysis were used to verify the average expression levels of these genes in the 10X dataset from the three animal species. Genes that were similarly altered in mice were manually selected as conserved genes among the three species (130 genes; Figure ​Figure12A). Genes that did not show expression changes in mice were selected as genes specifically altered in primates (83 genes; Figure ​Figure10F).

[0126] Chromosome expression analysis. For bulk RNA-seq / SC3-seq data, the autosome (human chromosome (chr.) 10, monkey chr. 15, mouse chr. 3):total autosome ratio and chr. X (X chromosome):total autosome ratio (X:A ratio) were calculated using a previously published method (Okamoto I, et al., Science, 374: eabd8887 (2021)). Briefly, the 75th percentile log2(normalized read count + 1) value [the maximum log2(normalized read count + 1) value among the analyzed cells > 2] of genes expressed on autosomes and chr. X in each sample / cell was calculated and used as the representative expression value for the sample / cell. For humans, 19,307 / 811 / 728 genes for total autosomes / Chr.10 / Chr.X were used, for monkeys 20,576 / 849 / 841 genes for total autosomes / Chr.15 / Chr.X, and for mice 12,399 / 618 / 503 genes for total autosomes / Chr.3 / Chr.X.

[0127] For 10X scRNA-seq data, the average log2(ss10kUMI + 1) value of all expressed genes expressed in two or more cells on autosomes (human chr. 10, monkey chr. 15, mouse chr. 3), all autosomes, and chr. X was used to calculate the autosome:A and X:A ratios. In humans, 17,190 / 682 / 725 genes for total autosomes / Chr.10 / Chr.X (Chitiashvili T, et al., Nat Cell Biol, 22: 1436-1446 (2020)), in monkeys, 18,066 / 736 / 791 genes for total autosomes / Chr.15 / Chr.X, and in mice, 17,698 / 896 / 744 genes for total autosomes / Chr.3 / Chr.X (Niu W, and Spradling AC, Proc Natl Acad Sci USA, 117: 20015-20026 (2020)) were used.

[0128] Statistical Analysis All statistical analyses were performed using R software (v. 4.0.2). Tukey-Kramer tests and t-tests were performed using the "TukeyHSD" and "t.test" functions with default settings.

[0129] Availability of data and code: Data from this study are accessed under the accession number GSE194266 (GEO database). R and Python code to reproduce the analyses and figures is available upon request.

[0130] The use of follicles induced by the present invention makes it possible to observe the maturation process of female germ cells in primates in vitro, which is useful for understanding the mechanism of germ cell development. Thus, the present invention provides an important foundation for both biology and medicine.

[0131] This application is based on patent application No. 2022-117665 filed in Japan (filing date: July 25, 2022), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing oocytes or follicles, or a reconstructed ovary containing oocytes or follicles, comprising the step of culturing a reconstructed primate 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 fetal ovarian somatic cells.

3. The method according to claim 2, wherein the germ cells and fetal ovarian somatic cells are prepared by dissociating from the fetal ovary of a primate by enzymatic treatment.

4. The method according to claim 2, wherein the germ cells are at least one selected from the group consisting of oogonia, primordial germ cells, and primordial germ cell-like cells.

5. The method according to claim 2, wherein at least one of the germ cells and ovarian somatic cells is derived from pluripotent stem cells.

6. The method according to claim 1, wherein the culture is performed under suspension culture conditions for 9 weeks or more.

7. The method according to claim 1, wherein the culture under suspension conditions is culture in a medium containing L-glutamine, ascorbic acid, and 2-mercaptoethanol.

8. The method according to claim 7, wherein the culture medium further comprises 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 claim 1, wherein the primate is a human.

11. Oocytes, follicles, or reconstituted ovaries produced by the method described in any one of claims 1 to 10.

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

13. A kit for producing oocytes or follicles, or reconstituted ovaries containing oocytes or follicles, comprising a culture vessel for suspension culture and L-glutamine, ascorbic acid, and 2-mercaptoethanol.

14. The kit according to claim 13, further comprising a basal culture 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.