Methods for reconstructing primordial follicles

Abstract

This invention provides a method to improve age-related decline in ovarian function by supplementing quiescent oocytes within primordial follicles. [Solution] The present invention provides a method for inducing mature oocytes, comprising the steps of: preparing a single-cell suspension from the ovary of a mammal other than a human; separating the single-cell suspension into oocytes and ovarian somatic cells (supporting cells) within a primordial follicle using a flow cytometer; re-aggregating the oocytes and ovarian somatic cells within the separated primordial follicle; and maturing the re-aggregated primordial follicle in vitro.

Description

Technical Field

[0001] The present invention can be used to supplement oocytes in order to solve the decline in egg function due to aging. This technology can also contribute to the conservation of endangered species and the improvement of the reproductive efficiency of livestock animals by using cells between different species.

Background Art

[0002] Since the birth of the first girl by in vitro fertilization and embryo transfer in the world in 1978, reproductive medicine has been widely used. In Japan, in 2019, about 1 out of every 14.3 newborns was born using reproductive medical technology. Furthermore, in 2022, insurance became applicable, and the number of users is expected to increase in the future. Until now, new technologies have been developed one after another in reproductive medicine. Among them, intracytoplasmic sperm injection has solved many problems on the sperm side related to fertilization, such as motility and quantity. However, when there are problems with eggs, the fundamental technology to solve them has not yet been established. The function of eggs declines with aging, and the incidence and pregnancy rate decrease and the abortion rate increases as people get older. In recent years, due to changes in social life, the aging of childbirth has progressed, and the decline in egg function associated with aging has become a major problem. In addition, mammalian oocytes enter meiosis during fetal development, so the number does not increase after birth. Therefore, a continuous ovulation cycle is maintained by keeping a limited number of oocytes in a quiescent state in primordial follicles and activating some of them. Thus, the aging changes of eggs have two aspects: qualitative functional decline and quantitative decrease, and primordial follicles are important follicles that are the basis of the ovulation cycle. The problem is to solve the aging problem in reproductive medicine by supplementing these primordial follicles, extend the healthy life span by maintaining the reproductive period, and prevent problems caused by menopause disorders. Methods for differentiating primordial germ cells into primordial follicles in an in vitro culture system (Patent Document 1), or methods for transplanting aggregates of primordial germ cells and somatic cells from the fetal ovary (Non-Patent Document 1). However, the former method differentiates primordial follicles, and it is unclear whether primordial follicles are present in the latter method. Based on these results, it is considered necessary to target primordial follicles and directly utilize resting oocytes within them. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 2019 / 244581 [Non-patent literature]

[0004] [Non-Patent Document 1] Hayashi K, et al., Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice ,Science, 2012; Nov 16;338(6109):971-5. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The present invention aims to provide a method for improving age-related decline in ovarian function by supplementing quiescent oocytes within primordial follicles. Furthermore, this invention aims to contribute to the conservation of endangered species and the improvement of the reproductive efficiency of livestock animals by using cells from different species. [Means for solving the problem]

[0006] As a result of diligent research, the inventors of this invention have discovered that by maturing quiescent oocytes in primordial follicles isolated from the ovary and from pluripotent stem cells by forming aggregates with somatic cells of the ovary, it is possible to provide a method to compensate for the decline in ovarian function associated with aging within the body, and thus have completed the present invention. More specifically, the present invention provides a method for inducing mature oocytes, comprising the steps of: preparing a single-cell suspension from the ovary of a mammal other than a human; separating the single-cell suspension into oocytes and ovarian somatic cells (supporting cells) within a primordial follicle using a flow cytometer; re-aggregating the oocytes and ovarian somatic cells within the separated primordial follicle; and culturing the re-aggregated primordial follicle in vitro. A method is provided for inducing mature oocytes, which are oocytes in primordial follicles and neonatal ovarian somatic cells, from oocytes and ovarian somatic cells within primordial follicles. A method is provided for inducing oocytes and ovarian somatic cells from primordial follicles, as well as oocytes from primordial follicles induced from pluripotent stem cells and mature oocytes, which are neonatal ovarian somatic cells. A method is provided for inducing mature oocytes, which are oocytes within a primordial follicle and ovarian somatic cells derived from an adult. A method is provided for inducing mature oocytes, which are oocytes within primordial follicles induced from pluripotent stem cells and ovarian somatic cells derived from adults. A method is provided for inducing mature oocytes, which are oocytes from a different mammalian species than the oocytes within the primordial follicle, from oocytes and ovarian somatic cells within the primordial follicle. A method for inducing mature oocytes is provided, comprising the steps of: preparing a single-cell suspension from the ovary of a non-human mammal; separating the single-cell suspension into oocytes and ovarian somatic cells (supporting cells) within a primordial follicle using a flow cytometer; transplanting the separated oocytes from the primordial follicle into an adult ovary; forming a primordial follicle within the ovary using the transplanted oocytes from the primordial follicle; and maturing the transplanted oocytes from the primordial follicle within the body. A method is provided for inducing mature oocytes, which are oocytes within primordial follicles induced from pluripotent stem cells. [Brief explanation of the drawing]

[0007] [Figure 1] Follicular reconstruction using neonatal mouse oocytes and somatic cells. A: Schematic diagram of the experimental system. A single-cell suspension is prepared from the ovary of a neonatal mouse, and oocytes and supporting cells are separated using a flow cytometer and then re-aggregated. B: Follicular formation after re-aggregation. C: Follicular maturation after follicular formation. D: MII oocyte due to follicular maturation. E: Two-cell stage embryo developed by in vitro fertilization of an MII oocyte. [Figure 2] Follicular reconstruction W using neonatal mouse oocytes and aged mouse support cells represents the age in weeks, and follicular formation can be confirmed even with 30W support cells. [Figure 3] Follicle reconstruction using oocytes from primordial follicles induced from mouse pluripotent stem (ES / iPS) cells and supporting cells from mouse ovaries. A: Schematic diagram of the experimental system. B: Follicle formation after reaggregation. C: MII oocytes due to follicular maturation after follicle formation. [Figure 4] Schematic diagram (top) of a follicle reconstruction experiment using oocytes and somatic cells from mouse and rat ovaries, and a follicle formed by mouse supporting cells and rat oocytes (bottom right). Follicle formation did not occur in primordial follicles with mouse oocytes and rat supporting cells (bottom left). [Figure 5] Schematic diagram of primordial follicle reconstruction by transplanting neonatal mouse oocytes into adult mouse ovaries. [Figure 6] Schematic diagram of primordial follicle reconstruction by transplanting oocytes from primordial follicles induced from mouse pluripotent stem (ES / iPS) cells into adult mouse ovaries. [Modes for carrying out the invention]

[0008] In this invention, as described above, we have found that mature oocytes can be obtained by activating quiescent oocytes within primordial follicles by agglutinating them with ovarian supporting cells. In this invention, when referring to mammals other than humans, the animal species is not particularly limited as long as it is an animal that maintains a resting oocyte within a primordial follicle. Therefore, examples include, but are not limited to, mice, cattle, pigs, goats, sheep, rats, and guinea pigs. To explain a specific aspect of this invention, the following description will focus on the case where a mouse is used as the mammal.

[0009] Isolation of quiescent oocytes and supporting cells from primordial follicles in neonatal mice Ovaries of 7-day-old female mice are harvested, washed in a petri dish containing PBS at room temperature, and then cut into several-millimeter fragments using dissecting scissors. These fragments are then reacted in CTK (1 μM CaCl2, collagenase type IV (0.1 mg / ml), 20% KSR (Invitrogen), and 0.025% trypsin EDTA) at 37°C for 30 minutes, and then transferred to Accutase solution (Nacalai Tesque) at 37°C and reacted for 5 minutes. A single-cell suspension is then obtained by pipetting using a micropipette. This solution is then separated into oocytes and supporting cells using a flow cytometer.

[0010] Isolation of quiescent oocytes from primordial follicles derived from pluripotent stem cells. Primordial follicles are induced from pluripotent stem cells by pressurized culture, and the aggregated follicles are reacted in CTK (1 μM CaCl2, collagenase type IV (0.1 mg / ml), 20% KSR (Invitrogen), and 0.025% trypsin EDTA) at 37°C for 30 minutes. The mixture is then transferred to Accutase solution (Nacalai Tesque) at 37°C and reacted for 5 minutes. A single-cell suspension is then obtained by pipetting using a micropipette. Oocytes are isolated from this solution using a flow cytometer. Isolation can be performed by visually separating individual cells under a microscope, or by using a column with magnetic beads called MACS to separate cells magnetically. Separation can be performed using known isolation methods.

[0011] Creation of Aggregates of Oocytes and Support Cells within Primary Follicles Seed 500 oocytes and 50,000 support cells in a low-attachment 96-well U-bottom plate and culture for 1 day in an incubator at 37 °C and 5% CO2.

[0012] Culture of Aggregates Culture the aggregates on a collagen-coated membrane insert (Transwell (Corning)) placed in a 6-well plate. The culture medium is S10 medium (StemPro (Invitrogen) containing 10% fetal bovine serum (FBS), 55 μM 2ME, 1× penicillin / streptomycin, 1× GlutaMAX, and 150 μM ascorbic acid). Replace the culture medium every 2 days.

[0013] Isolation of Secondary Follicles Isolate secondary follicles one by one under a stereomicroscope on the 21st day after culture. Culture the isolated secondary follicles on a membrane insert (Milicell (Millipore)) placed in a 6-well plate. The culture medium is IVG medium (αMEM containing 5% FBS, 1× penicillin / streptomycin, 1× sodium pyruvate, 2% PVP, 0.1 IU / ml FSH (Follistim), 55 μM 2ME, and 150 μM ascorbic acid). Replace the culture medium every 2 days. Add 15 ng / ml BMP15 and 15 ng / ml GDF9 to the above culture medium for the first 2 days.

[0014] Oocyte Maturation On the 14th day after IVG medium culture, COC (cumulus oocyte complex) was collected under a stereomicroscope after culture, and cultured in an incubator at 37 °C and 5% CO2 with IVM medium (αMEM containing 5% FBS, 1× penicillin / streptomycin, 1× sodium pyruvate, 4 ng / ml EGF, 0.1 IU / ml FSH (Follistim), 1.2 IU / ml Gonatropin) for 16 hours.

Example

[0015] The following examples are described for the purpose of further elaborating on the invention. However, these examples are only used to solely explain the invention described in the claims and are not intended to restrict the scope of the present invention. In this example, oocytes and supporting cells of 7-day-old mice were used. Oocytes of neonatal mice were isolated as GFP-positive cells using a flow cytometer with the expression of Stella-GFP as an indicator, using Stella-GFP transgenic mice. On the other hand, fluorescently labeled c-Kit antibody was used for the supporting cells. Since the expression of Stella-GFP and c-Kit in the ovaries of 7-day-old mice was co-positive at a rate of 90% or more, c-Kit-negative cells on the supporting cell side were isolated using a flow cytometer. These cells were mixed and cultured for 1 day in a U-bottom plate to form aggregates. The formed aggregates were transferred onto a membrane insert and cultured. On the 21st day after culture, secondary follicles were isolated one by one under a stereomicroscope. The isolated secondary follicles were further cultured on a membrane insert. On the 14th day after culture, COC (cumulus oocyte complex) was collected under a stereomicroscope and cultured in a maturation medium for 16 hours. The obtained MII oocytes were cultured with sperm for in vitro fertilization. Also, in a similar experimental system, the age of the supporting cells was increased, and follicle formation was also carried out in the supporting cells of mouse ovaries over 30 weeks old. At this time, since there were few immature oocytes as cell preparation, a single cell suspension was used as it was. Furthermore, in a similar experimental system, oocytes were prepared from 7-day-old rats and mixed with supporting cells from 7-day-old mice to induce follicular formation. In this case, the oocytes were prepared by isolating c-Kit-positive cells using a flow cytometer. Furthermore, follicular formation was also performed using oocytes induced from mouse pluripotent stem (ES / iPS) cells in a similar experimental system. In this case, the oocytes were induced using a method for differentiating primordial germ cells into primordial follicles in an in vitro culture system (Patent Document 1), and the oocytes were prepared by isolating them as Stella-GFP positive cells using a flow cytometer. [Examples]

[0016] Following the schematic diagram of the experiment shown in Figure 1A, a single-cell suspension is prepared from the ovary of a neonatal mouse, and oocytes and supporting cells within the primordial follicle are separated using a flow cytometer and then re-aggregated. We successfully reconstructed primordial follicles from single-cell suspensions of neonatal ovaries. These primordial follicles could be induced into mature oocytes through maturation culture, and we confirmed that they retained the ability to be fertilized and develop through in vitro fertilization. Figure 1B shows follicular formation after reaggregation, Figure 1C shows follicular maturation after formation, Figure 1D shows MII oocytes due to follicular maturation, and Figure 1E shows 2-cell stage embryos developed by in vitro fertilization of MII oocytes. [Examples]

[0017] Single-cell suspensions were prepared from the ovaries of neonatal and aged mice, and oocytes and supporting cells within primordial follicles were separated using a flow cytometer and then re-aggregated. We found that ovarian supporting cells from older mice (30 weeks old or older) also possess the ability to reconstruct primordial follicles. Figure 2 shows the process of follicular reconstruction using neonatal mouse oocytes and aged mouse support cells. W represents the age in weeks, and follicular formation can be observed even in supporting cells at 30 weeks. [Examples]

[0018] Oocytes from primordial follicles induced from mouse pluripotent stem (ES / iPS) cells and supporting cells from mouse ovaries are separated using a flow cytometer and then re-aggregated. We successfully reconstructed primordial follicles in vitro from oocytes within primordial follicles induced from mouse pluripotent stem (ES / iPS) cells. Figure 3 shows the follicular reconstruction process by oocytes in primordial follicles induced from mouse pluripotent stem (ES / iPS) cells and supporting cells in mouse ovaries. Figure 3A is a schematic diagram of the experimental system, Figure 3B shows follicular formation after reaggregation, and Figure 3C shows the appearance of MII oocytes after follicular maturation following follicular formation. [Examples]

[0019] Single-cell suspensions were prepared from mouse and rat ovaries, and oocytes and supporting cells within primordial follicles were separated using a flow cytometer and then re-aggregated. We successfully reconstructed primordial follicles using mouse supporting cells and rat oocytes. However, we were unable to reconstruct primordial follicles using mouse oocytes and rat supporting cells. Figure 4 shows the process of follicular reconstruction using oocytes and somatic cells from mouse and rat ovaries. The top of Figure 4 is a schematic diagram of the experiment, the bottom right of Figure 4 shows follicles formed by mouse supporting cells and rat oocytes, and the bottom left shows that follicular formation did not occur in primordial follicles with mouse oocytes and rat supporting cells. [Examples]

[0020] A single-cell suspension is prepared from the ovaries of neonatal mice, and oocytes from primordial follicles are isolated using a flow cytometer and transplanted into the ovaries of adult mice. In adult mouse ovaries, primordial follicles are reconstructed from transplanted oocytes to obtain mature oocytes. Figure 5 shows a schematic diagram of primordial follicle reconstruction by transplanting neonatal mouse oocytes into adult mouse ovaries. [Examples]

[0021] Oocytes from primordial follicles induced from mouse pluripotent stem (ES / iPS) cells are isolated using a flow cytometer and transplanted into adult mouse ovaries. In adult mouse ovaries, primordial follicles are reconstructed from oocytes derived from pluripotent stem (ES / iPS) cells that have been transplanted, and mature oocytes are obtained. Figure 6 shows a schematic diagram of primordial follicle reconstruction by transplanting oocytes from primordial follicles induced from mouse pluripotent stem (ES / iPS) cells into adult mouse ovaries.

Claims

1. The process of preparing a single-cell suspension from the ovaries of non-human mammals, The process involves separating a single-cell suspension into oocytes and ovarian somatic cells (supporting cells) within a primordial follicle using a flow cytometer, The process involves re-aggregating oocytes and ovarian somatic cells within the primordial follicles after separation, The process involves culturing the re-aggregated primordial follicles in vitro to achieve maturation, A method for inducing mature eggs consisting of the following:

2. The method for inducing mature oocytes according to claim 1, wherein the oocyte and ovarian somatic cells within the primordial follicle are the oocyte and neonatal ovarian somatic cells within the primordial follicle.

3. The method for inducing mature oocytes according to claim 1, wherein the oocytes and ovarian somatic cells within the primordial follicle are oocytes within a primordial follicle induced from pluripotent stem cells and neonatal ovarian somatic cells.

4. The method for inducing mature oocytes according to claim 1, wherein the oocyte and ovarian somatic cells within the primordial follicle are oocytes within the primordial follicle and adult-derived ovarian somatic cells.

5. The method for inducing mature oocytes according to claim 1, wherein the oocytes and ovarian somatic cells within the primordial follicle are oocytes within a primordial follicle induced from pluripotent stem cells and adult-derived ovarian somatic cells.

6. The method for inducing mature oocytes according to claim 1, wherein the oocyte and ovarian somatic cells within the primordial follicle are ovarian somatic cells of a different mammalian species than the oocyte within the primordial follicle.

7. The process of preparing a single-cell suspension from the ovaries of non-human mammals, The process involves separating a single-cell suspension into oocytes and ovarian somatic cells (supporting cells) within a primordial follicle using a flow cytometer, The process involves transplanting oocytes from the primordial follicles after sorting into the adult ovary, The process involves the formation of primordial follicles in the ovary by oocytes within the transplanted primordial follicles, The process of maturing oocytes within the transplanted primordial follicle in the body, A method for inducing mature eggs consisting of the following:

8. The method for inducing mature oocytes according to claim 7, wherein the oocytes within the primordial follicle are oocytes within the primordial follicle induced from pluripotent stem cells.

Images