Method for producing mature oocytes

Abstract

To facilitate oocyte maturation.SOLUTION: A method for producing mature oocytes includes a step of contacting oocytes with curcuminoids.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing a mature oocyte. [Background technology]

[0002] When mammalian oocytes resume meiosis during fetal development, they become primordial follicles surrounded by a single layer of flattened follicular epithelial cells. Primitive follicles develop into secondary follicles, which have multiple layers of granulosa cells and a basement membrane surrounding them, via primary follicles. Ovulation is then induced by the transient release of luteinizing hormone (LH) from the pituitary gland (LH surge). However, not all developed follicles lead to ovulation; at each stage, a dominant follicle is selected, and follicles that are not selected regress. The number of oocytes ovulated in one cycle varies by species; more than 30 oocytes begin follicular development at the same time in pigs and about 10 oocytes in humans, but only a dozen oocytes in pigs and only one oocyte in humans actually become dominant and are ovulated.

[0003] In addition, for efficient livestock production, the In Vitro Maturation (IVM) method is used to mature eggs collected from the ovaries to a state where they can be fertilized outside the body. In humans, too, when it is difficult to obtain mature eggs by administering hormones from outside the body, a method of maturing immature eggs using IVM is used. However, when eggs produced by the existing IVM method are used for in vitro fertilization, their developmental ability into blastocyst stage embryos is significantly lower than when eggs matured in vivo are used.

[0004] Non-Patent Documents 1 and 2 cite a decrease in mitochondrial activity caused by reactive oxygen species (ROS) as a factor inhibiting oocyte maturation. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Prasad et al., J Biomed Sci. 2016 Mar 29;23:36 [Non-Patent Document 2] Kirillova et al., Cells.2021 Sep 19;10(9):2484 Summary of the Invention [Problem to be solved by the invention]

[0006] Of the eggs present in the ovaries, the percentage of those that reach ovulation is less than 1%. If eggs derived from atretic follicles that do not reach ovulation could be utilized, it is expected that livestock production will increase and breeding will become more efficient. In humans, it is expected to be useful in infertility treatment using assisted reproductive technology. In addition, by promoting the maturation of eggs in dominant follicles, it is expected that the fertilization rate and implantation rate will be improved. The present invention aims to promote the maturation of eggs. [Means for solving the problem]

[0007] The present inventors discovered that curcumins promote the maturation of oocytes, and thus completed the present invention.

[0008] That is, the present disclosure (1) is a method for producing mature eggs, which includes a step of contacting eggs with curcumins.

[0009] The present disclosure (2) is the production method described in the present disclosure (1), wherein the egg is an egg contained in a cumulus cell-egg complex (COC).

[0010] The present disclosure (3) relates to a production method described in the present disclosure (2), which improves the proliferative activity of granulosa cells contained in cumulus cell-egg complexes (COCs).

[0011] The present disclosure (4) is a composition for promoting oocyte maturation, comprising curcumins.

[0012] The present disclosure (5) relates to a composition for promoting the proliferation of granulosa cells, comprising curcumins. Effect of the Invention

[0013] The method for producing mature oocytes of the present invention can promote oocyte maturation. [Brief description of the drawings]

[0014] [Figure 1A] Fluorescence images of ROS in oocytes retrieved from VF or NVF are shown. [Figure 1B] Fluorescence intensity of ROS in oocytes retrieved from VF or NVF is shown. [Figure 2A] Fluorescent images of JC-1 staining of oocytes retrieved from VF or NVF are shown. [Figure 2B] Fluorescence intensity ratios (red / green) of oocytes retrieved from VF or NVF are shown. [Figure 3A] Fluorescence images of PCNA-positive granular layer cells are shown. [Figure 3B] The percentage of PCNA-positive granular layer cells is shown. [Figure 4A] The morphology of VF or NVF COCs is shown. [Figure 4B] The diameter of the COC of the VF or NVF is shown. [Diagram 5] The percentage of eggs that reached the MII stage is shown. [Figure 6A] Fluorescence images of ROS in cumulus cells recovered from VF or NVF are shown. [Figure 6B] Fluorescence intensity of ROS in cumulus cells recovered from VF or NVF is shown. [Figure 7A] Fluorescent images of JC-1 staining of cumulus cells recovered from VF or NVF are shown. [Figure 7B] The fluorescence intensity ratio (red / green) of cumulus cells recovered from VF or NVF is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] <Method of manufacturing mature eggs> The method for producing a mature oocyte of the present invention is characterized by comprising a step of contacting an oocyte with curcumin or a similar compound.

[0016] The source of the eggs used in the method for producing mature eggs of the present invention is not particularly limited, and examples thereof include mammals, birds, reptiles, amphibians, and fish, with mammals being preferred. The mammals may be humans or non-human mammals. Non-human mammals include cows, pigs, sheep, goats, horses, donkeys, rabbits, deer, wild boars, reindeer, camels, cats, dogs, mice, and ferrets. When human eggs are used, the obtained mature eggs can be used for assisted reproductive medicine in humans. When non-human mammal eggs are used, the obtained mature eggs can be used for livestock production, production of companion animals such as pets, preservation of endangered animals, and the like. Examples of birds include chickens, quails, geese, turkeys, ducks, and ostriches. Examples of reptiles include turtles, crocodiles, lizards, and snakes. Examples of amphibians include frogs, salamanders, and newts. Fish include eel, salmon, tuna, flounder, sea bream, bonito, yellowtail, mackerel, and horse mackerel.

[0017] The oocytes may be obtained from mammalian ovaries or may be differentiated from pluripotent stem cells, such as iPS cells, ES cells, embryonic germ cells (EG cells), Muse cells, and multipotent germline stem cells (mGS cells).

[0018] In the step of contacting eggs with curcumins, the eggs are preferably eggs contained in cumulus cell egg complexes (COCs). Mature eggs can be obtained by recovering cumulus cell egg complexes (COCs) containing eggs from follicles and contacting the cumulus cell egg complexes (COCs) with curcumins. In addition, contacting the cumulus cell egg complexes (COCs) with curcumins also promotes the proliferation of granulosa cells that secrete activin and the like, thereby synergistically promoting the maturation of eggs. The method of recovering cumulus cell egg complexes (COCs) from follicles is not particularly limited, and can be carried out, for example, by the method described in the Examples.

[0019] When cumulus cell egg complexes (COCs) are collected from follicles, there are two types of follicles: dominant follicles (Vascularized Follicles; VFs) that can lead to ovulation, and non-vascularized follicles (NVFs). Classification of dominant follicles and atretic follicles can be performed, for example, by observing blood vessels formed on the surface of the follicles as described in the Examples, and can also be determined by the concentrations of hormones such as estradiol and progesterone contained in the follicular fluid. Among these, the production method of the present invention can significantly promote oocyte maturation when using cumulus cell egg complexes (COCs) collected from atretic follicles.

[0020] The oocytes, or the follicles or cumulus cell-oocyte complexes (COCs) containing the oocytes, may be cryopreserved and then thawed for use. Examples of cryopreservation methods include a method using a 10% DMSO solution or a freezing agent such as Cellbanker (registered trademark).

[0021] The contact between the egg and the curcumin can be achieved by culturing the egg in a medium containing the curcumin. Curcumin is a yellow polyphenol compound contained in the rhizome of turmeric, and is known to have antitumor, antiamyloid, and anti-inflammatory effects. Examples of the curcumin include curcumin, demethoxycurcumin, and bisdemethoxycurcumin, which may be used alone or in combination of two or more. Among these, curcumin is preferred. The concentration of the curcumin in the medium is preferably 0.1 to 100 μM, more preferably 1 to 50 μM, and even more preferably 5 to 20 μM.

[0022] The medium used for the culture is not particularly limited as long as it is a medium used for culturing oocytes, and examples of the medium include MEM, D-MEM, I-MDM, modified NSCU37, NCSU23, RPMI, M2, KSOM, and Human Tubal Fluid (HTF).

[0023] The medium may contain serum such as human serum, fetal calf serum (FCS), fetal bovine serum (FBS), porcine serum, horse serum, sheep serum, etc. The concentration of serum in the medium is preferably 1 to 20% by volume, more preferably 2 to 15% by volume, and even more preferably 5 to 12% by volume.

[0024] The medium may contain hormones and growth factors such as FSH (follicle-stimulating hormone), EGF (epidermal growth factor), bFGF (fibroblast growth factor), IGF (insulin-like growth factor), G-CSF (granulocyte colony-stimulating factor), LH (luteinizing hormone), hCG (human chorionic gonadotropin), etc. The concentration of these hormones and growth factors in the medium is preferably 0.001 to 5% by weight, more preferably 0.01 to 1% by weight, and even more preferably 0.1 to 0.5% by weight.

[0025] In addition to the components mentioned above, the medium may contain synthetic polymers, polysaccharide polymers, proteins, proteoglycans, antibiotics, and the like.

[0026] Examples of synthetic polymers include polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA). Examples of polysaccharide polymers include dextran, starch derivatives, and cellulose derivatives. Examples of polysaccharide polymers include ficoll and glycosaminoglycan. Examples of proteins include serum albumin. Examples of proteoglycans include chondroitin sulfate proteoglycan. The concentration of these components in the medium is preferably 0.01 to 15% by weight, more preferably 0.1 to 10% by weight.

[0027] The pH of the medium is preferably 6.5 to 8.5, more preferably 7 to 8, and even more preferably 7.2 to 7.8. The pH of the medium can be adjusted to the above range by providing an atmosphere containing CO2 during culture. The atmosphere during culture preferably contains 1 to 15% by volume of CO2, and more preferably contains 3 to 7% by volume.

[0028] The temperature when the egg is contacted with the curcumin is preferably 30 to 40°C, more preferably 35 to 39°C. The time for contacting the egg with the curcumin is preferably 20 to 60 hours, more preferably 30 to 55 hours. The contact between the egg and the curcumin can be carried out in a general culture vessel. Examples of the culture vessel include, but are not limited to, a 24-well plate and a 96-well plate.

[0029] The mature oocytes produced by the method of the present invention preferably have the ability to develop into blastocysts by fertilization with sperm, and more preferably have the ability to develop into individuals capable of growing independently. The mature oocytes are preferably those that have reached the metaphase of meiosis II (MII stage). The arrival at the MII stage can be determined by observing the nuclear phase of the oocytes using the method described in the Examples.

[0030] The mature oocyte preferably has improved mitochondrial activity compared to the oocyte before contact with curcumins. When the concentration of reactive oxygen species (ROS) is decreased or the mitochondrial membrane potential is maintained compared to the oocyte before contact with curcumins, the mitochondrial activity can be judged to be improved. The concentration of reactive oxygen species (ROS) and the mitochondrial membrane potential can be measured by the method described in the Examples.

[0031] When eggs contained in cumulus cell egg complexes (COCs) are used in the step of contacting eggs with curcumins, it is preferable that the mitochondrial activity of the cumulus cells contained in the cumulus cell egg complexes (COCs) is improved compared to before contacting with curcumins. When the concentration of reactive oxygen species (ROS) is decreased or the mitochondrial membrane potential difference is maintained after contacting with curcumins compared to before contacting with curcumins, it can be determined that the mitochondrial activity is improved. The concentration of reactive oxygen species (ROS) and the mitochondrial membrane potential difference can be measured by the method described in the Examples. When eggs contained in cumulus cell egg complexes (COCs) are used in the step of contacting eggs with curcumins, the swelling of the cumulus cells may be promoted through the improvement of the mitochondrial activity of the cumulus cells.

[0032] When eggs contained in cumulus cell-egg complexes (COCs) are used in the step of contacting eggs with curcumins, it is preferable that the proliferation activity of granulosa cells contained in the cumulus cell-egg complexes (COCs) is promoted compared to the eggs before contact with curcumins. When the intensity of immunofluorescence staining using a PCNA antibody of granulosa cells is stronger or the diameter of the cumulus cell-egg complexes (COCs) is larger compared to before contact with curcumins, it can be determined that the proliferation activity of granulosa cells is promoted. The intensity of immunofluorescence staining using a PCNA antibody and the diameter of the cumulus cell-egg complexes (COCs) can be measured by the method described in the Examples.

[0033] After the step of contacting the eggs with curcumins, the eggs or cumulus cell egg complexes (COCs) containing the eggs are separated from the medium containing the curcumins by a conventional method to isolate mature eggs. The obtained mature eggs can be used for in vitro fertilization. In in vitro fertilization, fertilization is promoted by allowing mature eggs and sperm to coexist outside the body. In addition, microinsemination can be performed by injecting sperm into mature eggs under a microscope. As sperm to be used in in vitro fertilization, sperm obtained by sperm collection, microscopic epididymal sperm collection, testicular sperm collection, etc., or sperm obtained by freezing and storing these sperm, and then thawing them, can be used.

[0034] In addition, after the step of contacting the oocyte with curcumin, the oocyte or the cumulus cell-oocyte complex (COC) containing the oocyte can be cryopreserved. Examples of the cryopreservation method include a cryopreservation method using a freezing agent such as a 10% DMSO solution or Cellbanker (registered trademark).

[0035] <Composition for promoting egg maturation> The composition of the present invention is a composition for promoting oocyte maturation, containing curcumins. The composition of the present invention may contain the above-mentioned medium, serum, hormones, growth factors, synthetic polymers, polysaccharide-based polymers, proteins, proteoglycans, antibiotics, etc., in addition to containing curcumins. The concentration of each component in the composition of the present invention is not particularly limited, and for example, the concentration of curcumins is preferably 0.1 to 10 mM, more preferably 1 to 5 mM, and even more preferably 2 to 5 mM. In addition, the concentration of components other than curcumins is preferably 0.001 to 5 wt%, more preferably 0.01 to 1 wt%, and even more preferably 0.1 to 0.5 wt%. However, the concentration is not limited to these concentrations, and may be appropriately adjusted to a suitable concentration when promoting oocyte maturation. By culturing oocytes in the composition of the present invention or a dilution of the composition of the present invention with a medium or the like, the oocytes and curcumins are brought into contact with each other, and the maturation of the oocytes can be promoted. The culture conditions are as described above. The oocytes may be oocytes contained in cumulus cell-oocyte complexes (COCs).

[0036] <Composition for promoting proliferation of granulosa cells> The composition of the present invention is a composition for promoting the proliferation of granulosa cells, which contains curcumins. The composition of the present invention may contain the above-mentioned medium, serum, hormones, growth factors, synthetic polymers, polysaccharide polymers, proteins, proteoglycans, antibiotics, etc., in addition to containing curcumins. The concentration of each component in the composition of the present invention is not particularly limited, and may be appropriately adjusted to a suitable concentration when promoting the proliferation of granulosa cells. Granulosa cells are cultured in the composition of the present invention or a dilution of the composition of the present invention with a medium or the like, thereby contacting the granulosa cells with curcumins, and promoting the proliferation of granulosa cells. The culture conditions are as described above.

[0037] It is preferable to contact granulosa cells with oocytes in a state where they are contained in cumulus cell-oocyte complexes (COCs) with curcumins, which promotes oocyte maturation by the effects of activin and other substances secreted by the proliferated granulosa cells. EXAMPLES

[0038] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples. In the following, "parts" and "%" mean "parts by weight" and "% by weight", respectively, unless otherwise specified. The experimental materials and methods are described below.

[0039] <Experiments 1-6> Experiment 1. Effect of curcumin on ROS production in NVF-derived oocytes COCs derived from VF and NVF were cultured for 48 hours in a control group without curcumin or a +Curcumin group containing curcumin. After culture, ROS activity in the oocytes was detected by green fluorescent images using a total ROS activity measurement kit.

[0040] Figure 1A shows the fluorescence images of ROS in oocytes retrieved from VF or NVF. Figure 1B shows the fluorescence intensity of ROS in oocytes retrieved from VF or NVF. In Figure 1A-B, 0h is the ROS in COC oocytes immediately after retrieval from VF or NVF. Values ​​are the mean ± SEM of three independent experiments (n = 3). * indicates significant difference (p < 0.05).

[0041] No ROS activity was observed in VF at 0 hours, but some ROS production was observed in NVF. High ROS activity was observed in the control group in VF, but this value was completely suppressed in the +Curcumin group. Furthermore, NVF also showed a significantly high ROS activity in the control group, but this value was halved in the +Curcumin group, and ROS production was suppressed.

[0042] Experiment 2. Effect of curcumin on mitochondrial activity in NVF-derived oocytes VF- and NVF-derived COCs were cultured for 48 hours in either the control or +Curcumin group. After culture, oocyte mitochondrial activity was measured as the ratio of the fluorescence intensity of JC-1 aggregates (indicating high mitochondrial activity) to the fluorescence intensity of JC-1 monomer (indicating low mitochondrial activity) (green).

[0043] Figure 2A shows the fluorescence images of JC-1 staining of oocytes retrieved from VF or NVF. Figure 2B shows the fluorescence intensity ratio (red / green) of oocytes retrieved from VF or NVF. Time 0 is the test result using oocytes immediately after retrieval from VF or NVF. Values ​​are the mean ± SEM of three independent experiments (n = 3). * indicates a significant difference between the VF control group, the NVF control group, and the NVF + Curcumin group (p < 0.05).

[0044] At 0 hours before incubation, mitochondrial activity was low in both VF and NVF, but in VF, the values ​​were high in the control and +Curcumin groups. In NVF, the control group showed a low value similar to that at 0 hours, but the +Curcumin group showed a significantly higher value.

[0045] Experiment 3. Effect of curcumin on proliferation of NVF-derived granulosa cells Granulosa cells derived from NVF were cultured for 10 hours in a medium containing FSH and EGF in either a control group or a +Curcumin group. PCNA-positive cells in the granulosa cells after culture were detected by immunofluorescence staining using PCNA antibody, and proliferation activity was examined. PCNA-positive cells in granulosa cells derived from VF were detected as a control group.

[0046] Figure 3A shows a fluorescent image of PCNA-positive granular layer cells. Figure 3B shows the percentage of PCNA-positive granular layer cells. In Figures 3A-B, 0 h is the result using granular layer cells immediately after recovery from VF or NVF. Values ​​are the mean ± SEM of three independent experiments (n = 3). * indicates significant difference (p < 0.05).

[0047] In the control group, a large number of PCNA-positive cells were detected in VF-derived granulosa cells, and the percentage of PCNA-positive cells was approximately 25%, which was as high as that in the +Curcumin group. On the other hand, in NVF, the control group showed a low percentage, but the +Curcumin group showed a significantly higher percentage.

[0048] Experiment 4. Effect of curcumin on NVF-derived COC diameter and oocyte maturation VF- and NVF-derived COCs were cultured for 48 hours in the control and +Curcumin groups. The diameters of the COCs after culture are shown in Figures 4A-B. The percentage of oocytes that reached the MII stage is shown in Figure 5.

[0049] Figure 4A shows the morphology of COCs from VF or NVF. Figure 4B shows the diameter of COCs from VF or NVF. In Figures 4A-B, 0h is the result of using COCs immediately after recovery from VF or NVF. Values ​​are the mean ± SEM of three independent experiments (n = 3). * indicates significant difference (p < 0.05).

[0050] In Figures 4A-B, no swelling of cumulus cells was observed in the COC before culture in either the VF or NVF. In the VF, the COC diameter was approximately 400 mm regardless of whether curcumin was added or not, and no significant difference was observed (Control group: 379.3 ± 20.7 mm; +Curcumin group: 406.17 ± 24.6 mm). On the other hand, in the NVF, the COC diameter in the control group without curcumin was 209.85 ± 16.81 mm, but in the +Curcumin group, it was significantly higher at 296.28 ± 23.95 mm.

[0051] In Figure 5, no significant difference was observed in the MII rate of oocytes between the control and +Curcumin groups in VF (Control group: 56.25±18.88%; +Curcumin group: 68.13±5.25%). In NVF, no MII oocytes were observed in the control group, but in the +Curcumin group, the rate had recovered to 41.03%. Values ​​are the mean ± SEM of three independent experiments (n=3).

[0052] Experiment 5. Effect of curcumin on ROS production in cumulus cells derived from NVF VF- and NVF-derived COCs were cultured for 10 hours in either a control group containing no curcumin or a curcumin-containing group. After culture, ROS activity in cumulus cells was detected by green fluorescent images using a total ROS activity measurement kit.

[0053] Figure 6A shows the fluorescence image of ROS in cumulus cells retrieved from VF or NVF. Figure 6B shows the fluorescence intensity of ROS in cumulus cells retrieved from VF or NVF. In Figures 6A-B, 0h is the ROS in cumulus cells of COCs immediately after retrieval from VF or NVF. Values ​​are the mean ± SEM of three independent experiments (n = 3). * indicates significant difference (p < 0.05).

[0054] At 0 hours, slight ROS production was observed in VF and NVF. In VF, there was no significant difference in ROS production between the control and curcumin groups. In NVF, the control group showed significantly high ROS activity, but this value was significantly reduced in the curcumin group, indicating that ROS production was suppressed.

[0055] Experiment 6. Effect of curcumin on mitochondrial activity in NVF-derived cumulus cells VF- and NVF-derived COCs were cultured for 10 hours in either the control or curcumin group. After culture, mitochondrial activity in cumulus cells was measured as the ratio of the fluorescence intensity of JC-1 aggregates (red, indicating high mitochondrial activity) to the fluorescence intensity of JC-1 monomers (green, indicating low mitochondrial activity).

[0056] Figure 7A shows the fluorescence image of JC-1 staining of cumulus cells recovered from VF or NVF. Figure 7B shows the fluorescence intensity ratio (red / green) of cumulus cells recovered from VF or NVF. Time 0 is the test result using cumulus cells immediately after recovery from VF or NVF. Values ​​are the mean ± SEM of three independent experiments (n = 3). There was a significant difference between the VF Curcumin group and the NVF Curcumin group (p < 0.05).

[0057] At 0 hours before incubation, mitochondrial activity was low in both VF and NVF, but in VF, the control group showed a high value and the curcumin group showed an even higher value. In NVF, the control group showed a low value similar to that at 0 hours, but the curcumin group showed a higher value than that.

[0058] Materials and Methods 1. Preparation of Reagents FSH FSH (National Institute of Digestive and Kidney Disease (KIDDK)) was dissolved at 1 mg / ml in Medium 199 (Life technologies CA, USA) and stored at -20°C. When used, it was diluted to the final concentration (1 μg / ml) in maturation medium (NCSU37) and used for culture.

[0059] EGF EGF (Sigma Aldrich) was dissolved in dimethyl sulfoxide (DMSO) (Wako Japan) at 100 μg / ml, and the final concentration was adjusted to 100 ng / ml in the maturation medium prior to use.

[0060] Curcumin Curcumin (Yaegaki Fermentation Engineering) was dissolved in DMSO to 1 M, diluted 100-fold with Medium 199 to make 10 mM, and then diluted 1000-fold with maturation medium to adjust to the final concentration (10 μM) before use.

[0061] Phosphate Buffered Saline (PBS) PBS(-)×10 was prepared by dissolving 40 g of NaCl (Fujifilm Wako), 1 g of KCl (Nacalai tesque), 14.05 g of Na2HPO4·12H2O (Nacalai tesque), and 1 g of KH2PO4 (Wako) in 441 ml of ultrapure water. PBS(+)×10 was prepared by dissolving 0.2 g of CaCl2 (Hayashi Pure Chemical Ind., Ltd., Osaka, Japan), 0.2 g of MgCl2·6H2O (Katayama Chemical Industries Co, Ltd., Japan), and 2 g of D-glucose (Nacalai tesque) in 200 ml of ultrapure water. Phosphate Buffered Saline (PBS) was prepared using the prepared PBS(-) and PBS(+) as follows. 27 ml of PBS(-) x 10 was added to 243 ml of ultrapure water and mixed thoroughly using a stirrer. Then, 30 ml of PBS(+) x 10 was added, 6 ml was removed, and 2.4 ml of ultrapure water, 3 ml of 5% polyvinylpyrrolidone (PVP) (5 g of PVP (Sigma Aldrich, MO, USA) dissolved in 100 ml of ultrapure water), and 0.6 ml of Penicillin-Streptomycin (Nacalai tesque, Japan) were mixed to prepare a total of 300 ml of PBS.

[0062] 2. Preparation of culture medium for granulosa cells, oocytes and cumulus cells Granulosa cells were cultured in D-MEM (Low Glucose) with L-Glutamine and Phenol Red (Fujifilm Wako) supplemented with 10% Fatal Calf Serum (FCS), FSH (1μg / ml), EGF (100ng / ml), and 1% Penicillin-Streptomycin. At least 3 hours before culture, 100 or 300 ml of medium was placed in the wells of a 96-well plate (ThermoFisher Scientific) or 48-well plate (VIOLAMO), and the space between the wells was filled with maturation medium. The pH of the maturation medium at the start of use was adjusted to 7.4 by gas equilibration in the incubator.

[0063] For the culture of COCs, modified NCSU37 medium supplemented with 10% Fatal Calf Serum (FCS), FSH (1 μg / ml), EGF (100 ng / ml), and 1% Penicillin-Streptomycin was used (Petter and Reed, 1991). At least 3 hours before culture, 100 ml of medium was placed in the wells of a 96-well plate (ThermoFisher Scientific), and the space between the wells was filled with maturation medium. The pH of the maturation medium at the start of use was adjusted to 7.4 by gas equilibration in the incubator.

[0064] 3. Collection and transportation of porcine ovaries and recovery of granulosa cells and COCs Ovaries were collected from prepubertal pigs slaughtered at the Hiroshima City Meat Hygiene Inspection Station, stored in a thermos containing 0.85% (w / v) physiological saline (4.25 g of NaCl (Fujifilm Wako) and 1 ml of Penicillin-Streptomycin (Nacalai tesque, Japan) dissolved in 500 ml of ultrapure water) and brought home within 2 hours. The ovaries were washed several times with 0.85% (w / v) physiological saline in a laboratory kept at 25°C until no blood came out from inside the ovaries, and then placed in a beaker containing fresh physiological saline and stored. The ovaries were then wiped with a paper towel to remove the physiological saline, and classified into dominant follicles (VF) and atretic follicles (NVF) based on the presence or absence of vascularization. The tissue and fluid in the follicle were aspirated using a 10 ml syringe (TERUMO) with an 18 G needle (TERUMO, Tokyo, Japan) and collected in a centrifuge tube, then centrifuged at 1000 rpm for 10 seconds and the supernatant was discarded. PBS was added to the tube, and the tube was centrifuged at 1000 rpm for 10 seconds and the supernatant was discarded. This washing was repeated three times, after which the tissue was suspended in PBS and collected using a glass tube under a stereomicroscope.

[0065] 4. In Vitro Culture of Granulosa Cells and In Vitro Maturation of COCs The collected granulosa cells from the VF and NVF and COCs were washed with culture medium. Granulosa cells were cultured in maturation medium without or with curcumin for 10 h in an incubator at 39°C and 5% CO2. COCs were cultured in maturation medium without or with curcumin for 10 or 48 h in an incubator at 39°C and 5% CO2. To prevent evaporation of the medium, the space between the wells was filled with an equal amount of medium.

[0066] 5. Measurement of ROS (reactive oxygen species) ROS was detected using a total ROS detection kit (Dojindo, Kumamoto, Japan) as follows. For oocyte measurement, 1% hyaluronidase (Sigma-Aldrich) was added to the medium after 48 hours of COC culture, and after 5 minutes of incubation, cumulus cells were removed from the COCs using a glass tube to obtain denuded oocytes. The denuded oocytes were placed in a 96-well plate containing 100 μl of Highly Sensitive DCFH-DA working solution per well. For cumulus cell measurement, 1% hyaluronidase (Sigma-Aldrich) was added to the medium after 10 hours of COC culture, and after 5 minutes of incubation, the COCs were collected. The COCs were placed in a 96-well plate containing 100 μl of Highly Sensitive DCFH-DA working solution per well. Cumulus cell measurement was performed on the COCs collected by the above method. Oocytes and COCs were then incubated at 37°C for 30 minutes. After incubation, the oocytes and COCs were washed twice with medium and then transferred to wells containing 100 μl of working solution per well. The green fluorescence indicating intracellular ROS was then observed and photographed using a BZ-X700 at an excitation wavelength of 470 nm and an absorption wavelength of 525 nm. The green fluorescence intensity was measured using the Hybrid Cell Count of the BZ-Xanalyzer, and the green fluorescence intensity was converted into numerical values ​​and graphed.

[0067] 6. Measurement of Mitochondrial Membrane Potential of Oocytes and Cumulus Cells The mitochondrial membrane potential of oocytes and cumulus cells was measured using a mitochondrial membrane potential detection kit (Dojindo, Kumamoto, Japan) as follows. For oocyte measurement, 1% hyaluronidase (Sigma-Aldrich) was added to the medium after 48 hours of COC culture, and after 5 minutes of incubation, cumulus cells were removed from the COCs using a glass tube to obtain denuded oocytes. The denuded oocytes were placed in a 96-well plate containing 100 μl of JC-1 working solution per well. For cumulus cell measurement, 1% hyaluronidase (Sigma-Aldrich) was added to the medium after 10 hours of COC culture, and after 5 minutes of incubation, the COCs were collected. The COCs were placed in a 96-well plate containing 100 μl of Highly Sensitive DCFH-DA working solution per well. For cumulus cell measurement, the COCs collected by the above method were measured. The oocytes and cumulus cells were then incubated at 37°C for 30 minutes. After incubation, the oocytes and cumulus cells were washed twice with medium and then transferred to wells containing 100 μl of Imaging Buffer solution per well. The red fluorescence, which indicates high mitochondrial activity, was observed with a BZ-X700 at an excitation wavelength of 560 nm and an absorption wavelength of 630 nm, and the green fluorescence, which indicates low mitochondrial activity, was observed with an excitation wavelength of 470 nm and an absorption wavelength of 525 nm, and images were taken. The concentration of red relative to green was quantified and graphed using the hybrid cell count of the BZ-Xanalyzer.

[0068] 7. Immunofluorescence staining of granulosa cells using anti-PCNA antibody After culturing, the granulosa cells were smeared on a slide glass and air-dried. The air-dried slide glass was washed three times with PBS(-). Then, 0.1% Triton-X (Sigma-Aldrich) / PBS(-) was placed on the slide glass and left at room temperature for 30 minutes for membrane permeabilization. After washing three times with PBS(-), 5% BSA (Sigma-Aldrich) / PBS(-) was placed on the slide glass and left at room temperature for 90 minutes for blocking. Then, the primary antibody (PCNA (PC10) Mouse mAb (Cell Signaling Technology, MA, USA): 5% BSA / PBS(-) = 1:100) was placed on the plate and left to stand at room temperature for 4 hours. After washing three times with PBS(-), the plate was placed on the plate with a secondary antibody (Anti-Mouse IgG (whole molecule) F(ab')2 Fragment-Cy3 antibody produced in sheep (Sigma-Aldrich): 5% BSA / PBS(-) = 1:100) and left to stand at room temperature for 2 hours in the dark. After washing the glass-bottom petri dishes three times with PBS(-) to remove excess water droplets, the nuclei were stained with DAPI (VECTAHIELD Mounting Medium with DAPI) (Vector Laboratories, Inc., CA, USA) and observed under a fluorescence microscope (Keyence, BX-X700, Osaka, Japan) at an excitation wavelength of 550 nm and an absorption wavelength of 570 nm for Cy3, which indicates PCNA-positive cells, and at an excitation wavelength of 360 nm and an absorption wavelength of 460 nm for DAPI, which indicates nuclei, and photographed. The ratio of PCNA-positive cells to DAPI-stained cells was measured using the hybrid cell count of the BZ-Xanalyzer and graphed.

[0069] 8. Measuring Cumulus Cell Swelling After In Vitro Culture Cumulus cell swelling was measured according to the report by Shimada et al. (2004) . Photographs of COCs were taken with an ocular micrometer under an inverted phase-contrast microscope (Olympus, IX73, Tokyo, Japan) before and after 48 h of culture, and the diameters of COCs before and after culture were measured.

[0070] 9. Preparation of Egg Whole Mount Preparations For the observation of the nuclear phase of the oocytes, 1% hyaluronidase was added to the culture medium for the COCs after culture. After 5 minutes, the oocytes were moved in and out with a Pasteur pipette adjusted to the oocyte diameter to remove the cumulus cells. The oocytes from which the cumulus cells had been removed were transferred to a 50 nm plastic petri dish and washed with PBS. After washing, the oocytes were placed on a slide glass with a small amount of PBS, and a cover glass with Vaseline paraffin attached to the four corners was placed over the oocytes, and the oocytes were sandwiched between the slide glass and the cover glass. 50% acetic acid ethanol (acetic acid (Nakalai tesque):ethanol (Fijifilm Wako) = 1:3) was poured between the oocytes sandwiched between the slide glass and the cover glass to remove the lipids from the oocytes, and the oocytes were left in the dark for 2 days. After 2 days, the eggs were further delipidated with Carnoy's solution (acetic acid:chloroform (Nakalai tesque):ethanol = 1:3:4) and stained with acetolacmoid staining solution (10 ml of 45% acetic acid + 0.1 g of Lacmoid (Sigma-Aldrich)). Excess staining solution was removed with Carnoy's solution, and the eggs were mounted with mounting medium (acetic acid:glycerin:ultrapure water = 1:1:3) and the nuclear phase of the eggs was determined.

[0071] 10. Observation of the nuclear phase of nuclei The nuclear phase of the eggs was observed using an upright phase-contrast microscope (Olympus, CE). The presence of egg capsules indicated that the eggs were in the germinal vesicle (GV) stage, and the shape of the first polar body and the chromosomes aligned at the equator indicated that the eggs were in the second metaphase (MII) stage.

Claims

1. A method for producing mature eggs, comprising a step of contacting eggs with curcumins.

2. The method of claim 1, wherein the egg is an egg contained in a cumulus cell-egg complex (COC).

3. Improves the proliferation activity of granulosa cells contained in cumulus cell-egg complexes (COCs); The method according to claim 2 .

4. A composition for promoting oocyte maturation comprising curcumins.

5. A composition for promoting the proliferation of granulosa cells, comprising curcumins.

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