Culture solution for improving quality of aged oocytes

Abstract

The invention provides a culture solution for improving the quality of aged oocytes. The culture solution is prepared by adding a 5-100nM tissue-type plasminogen activator into an oocyte in-vitro maturation basic culture solution. As the tissue-type plasminogen activator is massively expressed in each organ tissue in vivo, the culture solution is safer. The culture solution can improve the quality of the oocytes of the elderly women and the success rate of an assisted reproduction technology, and has a better application prospect in coping with the oocyte culture of the elderly women with fertility willingness but difficult ovulation promotion.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a culture medium for improving the quality of aged oocytes. Background Art

[0002] Tissue-type plasminogen activator (tPA) is expressed in large quantities in various organs and tissues in the body. It is a key enzyme in the fibrinolytic pathway and is proteolytically processed by plasmin to produce a heterodimeric serine protease. The protease cleaves the zymogen to produce plasmin, which is a protease required to break down fibrin clots. Because of this property, it is currently the only drug approved by the U.S. Food and Drug Administration to treat ischemic stroke caused by cerebral artery obstruction and has been widely used clinically. In addition, tPA is also involved in other biological processes, such as synaptic plasticity, cell migration, and tissue remodeling. Current work has shown that the addition of exogenous tPA can promote the growth of neurons (see: Lee, SH et al. tPA Regulates Neurite Outgrowth by Phosphorylation of LRP5 / 6 in Neural Progenitor Cells. Molecular Neurobiology 49, 199–215 (2014)). Recent work has also shown that adding an appropriate amount of tPA to the culture medium can improve corticosterone-induced apoptosis of mouse mural granular cells and fallopian tube epithelial cells (see: Hua, Q. et al. Role of tPA in Corticosterone-Induced Apoptosis of Mouse Mural Granulosa and Oviductal Epithelial Cells. Cells 12, 455 (2023)).

[0003] The decline in oocyte quality is an important feature of female reproductive aging and one of the main reasons for the decline in fertility in older women. Many literatures have reported that after women reach the age of 35, oocyte quality begins to decline sharply, characterized by increased aneuploidy, increased oxidative stress, decreased mitochondrial quality, increased apoptosis and lipid peroxidation levels, etc., which may lead to adverse consequences such as infertility, spontaneous abortion, and embryo arrest. At present, due to complex factors such as social and economic development, delayed childbearing in women has become an important trend. Therefore, improving the decline in oocyte quality caused by aging has become an important measure to maintain the fertility of older women.

[0004] However, there are still limited methods to improve the quality of aged oocytes. There is no culture medium specifically for in vitro maturation of aged oocytes on the market, and its safety has not been fully proven, so these studies have not been widely used. Therefore, if a safer culture medium specifically for aged oocytes is studied, it will be of great significance to older women who want to have children but have difficulty in ovulation. Summary of the invention

[0005] The purpose of the present invention is to provide a safer culture medium for improving the quality of aged oocytes in view of the above-mentioned deficiencies in the prior art. The culture medium can be applied to oocytes from women over 30 years old or female mice over 8 months old, and can significantly improve the quality of aged oocytes.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A culture medium for improving the quality of aged oocytes in vitro, comprising tissue plasminogen activator with a final concentration of 5-100 nM.

[0008] As a preferred embodiment of the present invention, the final concentration of tissue plasminogen activator in the culture medium is 50 nM.

[0009] As a further preferred embodiment of the present invention, the tissue plasminogen activator is a recombinant protein of active tissue plasminogen activator derived from human or mouse.

[0010] As a preferred embodiment of the present invention, the aged oocytes are derived from either human or mouse.

[0011] As a further preferred embodiment of the present invention, the aged oocytes are derived from human females over 30 years old or female mice over 8 months old.

[0012] As a preferred embodiment of the present invention, the culture medium is obtained by adding a concentrated stock solution of tissue-type plasminogen activator to any one of M2 culture medium, M16 culture medium, G-1TM PLUS and CZB culture medium as a base culture medium.

[0013] As a preferred embodiment of the present invention, the concentrated stock solution of tissue plasminogen activator is prepared by dissolving tPA in a buffer solution containing a carrier protein.

[0014] As a further preferred embodiment of the present invention, the concentration of the concentrated stock solution of tissue plasminogen activator is 100 μg / ml-500 μg / ml.

[0015] As further preferred embodiment of the present invention, the carrier protein is 0.1% bovine serum albumin, 5% human serum albumin, 10% fetal bovine serum or 5% trehalose, preferably 0.1% bovine serum albumin.

[0016] As a further preference of the present invention, the buffer is sterile water or phosphate buffer; preferably phosphate buffer.

[0017] Application of tissue plasminogen activator in the preparation of reagents for improving the quality of aged oocytes in vitro.

[0018] Beneficial effects:

[0019] The biological experiment results of the present invention show that: ① After 16 hours of culture in this culture medium, the first polar body extrusion rate of the oocytes of 12-month-old mice increased significantly, indicating that the present invention can significantly improve the in vitro maturation rate of oocytes of old mice. ② After 48 hours of culture in this culture medium, the fragmentation rate of oocytes of 12-month-old mice decreased significantly, indicating that the present invention can reduce the mortality rate of oocytes of old mice. ③ The apoptosis level of oocytes of 12-month-old mice was checked by a cell apoptosis detection kit, and imaging under a confocal fluorescence microscope found that the present invention can effectively reduce the apoptosis level of oocytes of old mice. ④ The lipid peroxidation level of oocytes of 12-month-old mice was checked by a cell lipid peroxidation probe, and imaging under a confocal fluorescence microscope found that the present invention can effectively reduce the lipid peroxidation level of oocytes of old mice. ⑤ The mitochondrial membrane potential of oocytes of 12-month-old mice was checked by a mitochondrial membrane potential analysis kit, and imaging under a confocal fluorescence microscope found that the present invention can effectively restore the membrane potential of mitochondria of oocytes of old mice and improve the quality of mitochondria. ⑥ The intracellular distribution of mitochondria in oocytes of 12-month-old mice was examined by mitochondrial deep red fluorescent probe, and imaging under a confocal fluorescence microscope revealed that the present invention can effectively restore the normal and uniform distribution of mitochondria in oocytes of aged mice. The mitochondrial membrane potential of oocytes of 12-month-old mice was examined by a mitochondrial membrane potential analysis kit, and imaging under a confocal fluorescence microscope revealed that the present invention can effectively restore the membrane potential of mitochondria in oocytes of aged mice and improve the quality of mitochondria. ⑦ The accumulation of reactive oxygen species in oocytes of 12-month-old mice was detected by reactive oxygen fluorescence, and it was found that the present invention can effectively reduce the level of reactive oxygen species in oocytes of aged mice. ⑧ The spindle assembly of oocytes of 12-month-old mice was examined by tubulin staining, and imaging under a confocal fluorescence microscope revealed that the present invention can effectively reduce the incidence of spindle abnormalities in oocytes of aged mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to facilitate understanding by those skilled in the art, the present invention is further described below with reference to the accompanying drawings.

[0021] Figure 1 This is a schematic diagram of the three-dimensional structure of tPA.

[0022] Figure 2This is a schematic diagram of the effect of adding tPA on the first polar body extrusion rate of oocytes in aged mice.

[0023] Figure 3 This is a schematic diagram of the effect of adding tPA on the fragmentation rate of oocytes in aged mice.

[0024] Figure 4 This is a schematic diagram of the effect of adding tPA on the apoptosis level of oocytes in aged mice.

[0025] Figure 5 This is a schematic diagram of the effect of adding tPA on lipid peroxidation levels in aged mouse oocytes.

[0026] Figure 6 This is a schematic diagram of the effect of adding tPA on the mitochondrial membrane potential level of aged mouse oocytes.

[0027] Figure 7 This is a schematic diagram of the effect of adding tPA on the mitochondrial distribution of aged mouse oocytes.

[0028] Figure 8 This is a schematic diagram of the effect of adding tPA on the intracellular reactive oxygen levels in aged mouse oocytes.

[0029] Fig. 9 Schematic diagram of the effect of adding tPA on spindle assembly in aged mouse oocytes. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. It should be noted that the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The reagents or instruments used in the examples of the present invention without indicating the manufacturer are all commercially available conventional reagent products.

[0032] Example 1

[0033] Prepare 100 μg / ml tPA (MCE, CAS: HY-P701312) concentrated stock solution in a sterile environment according to the following components:

[0034] Element quality BSA 50ng tPA 5μg

[0035] Dissolve the above ingredients in 50 μl ultrapure water, gently shake tPA and BSA to dissolve, blow lightly a few times with the tip of a pipette, and dispense into 5 200 μl EP tubes, 10 μl in each tube, and freeze at -20°C.

[0036] Prepare the culture medium containing 50 nM tPA in a sterile environment according to the following components:

[0037] Element volume M2 medium 993.5μl tPA concentrated stock solution (100 μg / ml) 6.5μl

[0038] Example 2

[0039] 1) 12-month-old female ICR mice were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin.

[0040] Month-old female ICR mice were injected intraperitoneally with 5 IU of pregnant mare serum gonadotropin and killed by dislocation 48 hours later.

[0041] 2) The ovaries of both sides of mice aged 2 and 12 months were removed, the adipose tissue was peeled off and minced with a blade, the oocytes were released into the operating solution, and the oocytes were collected using a homemade capillary glass tube with a diameter of about 150 um.

[0042] 3) After washing the collected cells and removing the granulosa cells attached to the surface, the oocytes from 2-month-old mice were transferred to ordinary M2 culture medium (2-month-old group), and the oocytes from 12-month-old mice were transferred to ordinary M2 culture medium (12-month-old group) and culture medium supplemented with 50 nM tPA (tPA+12-month-old group). The cells were cultured at 37°C and 5% CO2 for 16 hours, and the first polar body extrusion rates of the oocytes in the three groups were counted.

[0043] 4) The oocytes that have not expelled the first polar body are discarded and cultured in an environment of 37°C and 5% CO2 for 32 hours. The fragmentation rate of the oocytes is observed and the fragmented cells are judged as dead.

[0044] The results are as follows Figure 2 and 3 As shown, culture medium containing 50nM tPA can significantly increase the in vitro maturation rate of aged mouse oocytes and reduce the fragmentation rate.

[0045] Example 3

[0046] 1) 12-month-old female ICR mice were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin, and 2-month-old female ICR mice were intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin, and then killed by dislocation 48 hours later.

[0047] 2) The ovaries of both mice were removed, the fat tissue was peeled off and minced with a blade, the oocytes were released into the operating solution, and the oocytes were collected using a homemade capillary glass tube with a diameter of about 150 um.

[0048] 3) After washing the collected cells and removing the granulosa cells attached to the surface, the 2-month-old mouse oocytes were transferred to ordinary M2 culture medium (2-month-old group), and the 12-month-old mouse oocytes were transferred to ordinary M2 culture medium (12-month-old group) and culture medium supplemented with 50nM tPA (tPA+12-month-old group). The cells were cultured at 37°C and 5% CO2 for 16 hours, and the oocytes that did not expel the first polar body were discarded.

[0049] 4) Wash the oocytes three times with phosphate buffered saline.

[0050] 5) Fix the cells with 4% paraformaldehyde for 30 minutes.

[0051] 6) Wash the oocytes three times with phosphate buffer.

[0052] 7) Permeabilize the cells with phosphate buffer containing 0.3% Triton X-100 and incubate at room temperature for 25 minutes.

[0053] 8) Add 50 μl of TUNEL detection solution (Biyuntian, C1086) to the sample and incubate at 37°C in the dark for 60 minutes.

[0054] 9) Wash the oocytes 3 times with phosphate buffer

[0055] 10) After sealing with anti-fluorescence quenching sealing solution, the slides were observed under a fluorescence microscope, and the relative fluorescence intensity of the oocytes of the three groups of mice was calculated.

[0056] The results are as follows Figure 4 As shown, the culture medium containing 50nM tPA can significantly reduce the apoptosis level of aged mouse oocytes.

[0057] Example 4

[0058] 1) 12-month-old female ICR mice were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin, and 2-month-old female ICR mice were intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin, and then killed by dislocation 48 hours later.

[0059] 2) The ovaries of both mice were removed, the fat tissue was peeled off and minced with a blade, the oocytes were released into the operating solution, and the oocytes were collected using a homemade capillary glass tube with a diameter of about 150 um.

[0060] 3) After washing the collected cells and removing the granulosa cells attached to the surface, the 2-month-old mouse oocytes were transferred to ordinary M2 culture medium (2-month-old group), and the 12-month-old mouse oocytes were transferred to ordinary M2 culture medium (12-month-old group) and culture medium supplemented with 50nM tPA (tPA+12-month-old group). The cells were cultured at 37°C and 5% CO2 for 16 hours, and the oocytes that did not expel the first polar body were discarded.

[0061] 4) Wash the oocytes three times with phosphate buffered saline.

[0062] 5) Prepare lipid peroxidation detection working solution: add 1 μl BODIPY 581 / 591-C11 (Biyuntian, S0043M) into 1 ml M2 culture medium, and mix well to obtain 2 μM BODIPY 581 / 591-C11 staining working solution.

[0063] 6) Transfer the oocytes to the working solution and incubate at 37°C and 5% CO2 for 30 minutes.

[0064] 7) Wash the oocytes three times with phosphate buffered saline, 5 minutes each time.

[0065] 8) The red and green fluorescence intensity ratios of oocytes of the three groups of mice were observed under a confocal fluorescence microscope and the results were statistically analyzed.

[0066] The results are as follows Figure 5 As shown, culture medium containing 50 nM tPA can significantly reduce the level of lipid peroxidation in aged mouse oocytes.

[0067] Example 5

[0068] 1) 12-month-old female ICR mice were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin, and 2-month-old female ICR mice were intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin, and then killed by dislocation 48 hours later.

[0069] 2) The ovaries of both mice were removed, the fat tissue was peeled off and minced with a blade, the oocytes were released into the operating solution, and the oocytes were collected using a homemade capillary glass tube with a diameter of about 150 um.

[0070] 3) After washing the collected cells and removing the granulosa cells attached to the surface, the 2-month-old mouse oocytes were transferred to ordinary M2 culture medium (2-month-old group), and the 12-month-old mouse oocytes were transferred to ordinary M2 culture medium (12-month-old group) and culture medium supplemented with 50nM tPA (tPA+12-month-old group). The cells were cultured at 37°C and 5% CO2 for 16 hours, and the oocytes that did not expel the first polar body were discarded.

[0071] 4) Wash the oocytes three times with phosphate buffered saline.

[0072] 5) Prepare the mitochondrial membrane potential detection reagent working solution: dilute the mitochondrial membrane potential detection reagent concentrated stock solution dissolved in dimethyl sulfoxide to 20 μg / ml using M2 culture medium to prepare the working solution.

[0073] 6) Transfer the oocytes to the working solution and incubate at 37°C and 5% CO2 for 30 minutes.

[0074] 7) Wash the oocytes 3 times with M2 culture medium, 5 minutes each time.

[0075] 8) The oocytes of the three groups were observed under a confocal fluorescence microscope and the ratios of red and green fluorescence were counted.

[0076] The results are as follows Figure 6 As shown, culture medium containing 50nM tPA can significantly increase the mitochondrial membrane potential level of aged mouse oocytes and restore mitochondrial quality.

[0077] Example 6

[0078] 1) 12-month-old female ICR mice were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin, and 2-month-old female ICR mice were intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin, and then killed by dislocation 48 hours later.

[0079] 2) The ovaries of both mice were removed, the fat tissue was peeled off and minced with a blade, the oocytes were released into the operating solution, and the oocytes were collected using a homemade capillary glass tube with a diameter of about 150 um.

[0080] 3) After washing the collected cells and removing the granulosa cells attached to the surface, the 2-month-old mouse oocytes were transferred to ordinary M2 culture medium (2-month-old group), and the 12-month-old mouse oocytes were transferred to ordinary M2 culture medium (12-month-old group) and M2 culture medium supplemented with 50nM tPA (tPA+12-month-old group). The cells were cultured at 37°C and 5% CO2 for 16 hours, and the oocytes that did not expel the first polar body were discarded.

[0081] 4) Wash the oocytes three times with phosphate buffered saline.

[0082] 5) Preparation of Mito-Tracker Red CMXRos stock solution: Dissolve 50 μl of Mito-Tracker Red CMXRos solution (Biyuntian, C1049B) in 420 μl of dimethyl sulfoxide, mix thoroughly, and obtain a Mito-Tracker Red CMXRos stock solution with a concentration of 200 μM. After aliquoting, store at -20°C in the dark.

[0083] 6) Preparation of Mito-Tracker Red CMXRos working solution: add 1 μl of Mito-Tracker Red CMXRos stock solution to 1 ml of M2 culture medium and vortex to mix.

[0084] 7) The oocytes from 2-month-old and 12-month-old mice were transferred into Mito-Tracker Red CMXRos working solution and incubated at 37° C. and 5% CO 2 for 30 minutes.

[0085] 8) Wash the oocytes three times with phosphate buffered saline, 5 minutes each time.

[0086] The results are as follows Figure 7 As shown, the culture medium containing 50nM tPA can significantly restore the normal distribution of mitochondria in aged mouse oocytes and reduce the abnormal aggregation of mitochondria.

[0087] Example 7

[0088] 1) 12-month-old female ICR mice were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin, and 2-month-old female ICR mice were intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin, and then killed by dislocation 48 hours later.

[0089] 2) The ovaries of both mice were removed, the fat tissue was peeled off and minced with a blade, the oocytes were released into the operating solution, and the oocytes were collected using a homemade capillary glass tube with a diameter of about 150 um.

[0090] 3) After washing the collected cells and removing the granulosa cells attached to the surface, the 2-month-old mouse oocytes were transferred to ordinary M2 culture medium (2-month-old group), and the 12-month-old mouse oocytes were transferred to ordinary M2 culture medium (12-month-old group) and M2 culture medium supplemented with 50nM tPA (tPA+12-month-old group). The cells were cultured at 37°C and 5% CO2 for 16 hours, and the oocytes that did not expel the first polar body were discarded.

[0091] 4) Wash the oocytes three times with phosphate buffered saline.

[0092] 5) Prepare the working solution of active oxygen detection reagent: dilute 1 μl DCFH-DA (Yeason, 50101ES01) with 1 ml M2 culture medium to prepare a working solution with a final concentration of 10 μM.

[0093] 6) The oocytes of the above three groups of mice were transferred into the working solution of the reactive oxygen species detection reagent and incubated in an environment of 37° C. and 5% CO 2 for 30 minutes.

[0094] 7) Wash the oocytes three times with phosphate buffered saline, 5 minutes each time.

[0095] 8) The relative fluorescence intensity of oocytes in the three groups of mice was observed under a confocal fluorescence microscope and counted.

[0096] The results are as follows Figure 8As shown, culture medium containing 50 nM tPA can significantly reduce the level of oxidative stress in aged mouse oocytes.

[0097] Example 8

[0098] 1) 12-month-old female ICR mice were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin, and 2-month-old female ICR mice were intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin, and then killed by dislocation 48 hours later.

[0099] 2) The ovaries of both mice were removed, the fat tissue was peeled off and minced with a blade, the oocytes were released into the operating solution, and the oocytes were collected using a homemade capillary glass tube with a diameter of about 150 um.

[0100] 3) After washing the collected cells and removing the granulosa cells attached to the surface, the 2-month-old mouse oocytes were transferred to ordinary M2 culture medium (2-month-old group), and the 12-month-old mouse oocytes were transferred to ordinary M2 culture medium (12-month-old group) and M2 culture medium supplemented with 50nM tPA (tPA+12-month-old group). The cells were cultured at 37°C and 5% CO2 for 16 hours, and the oocytes that did not expel the first polar body were discarded.

[0101] 4) The oocytes were washed three times with phosphate buffer and fixed with 4% paraformaldehyde for 30 minutes.

[0102] 5) The oocytes were permeabilized with 0.1% Triton X-100 for 20 minutes.

[0103] 6) Wash the oocytes with 1% bovine serum albumin three times, 5 minutes each time.

[0104] 7) Block with 1% bovine serum albumin for 1 hour.

[0105] 8) Incubate with Tubblin mouse antibody at 4°C overnight.

[0106] 9) Wash 3 times, 5 minutes each time.

[0107] 10) Incubate with anti-mouse secondary antibody at room temperature for 1 hour.

[0108] 11) Wash three times, 5 minutes each time.

[0109] 12) Use DAPI dye to stain the nucleus at room temperature for 10 minutes, and wash the cells three times with Wash, each time for 5 minutes.

[0110] 13) Use antifade agent to seal the slides, observe under a confocal fluorescence microscope, and count the abnormal proportion of spindles.

[0111] The results are as follows Fig. 9As shown, culture medium containing 50 nM tPA can significantly reduce the proportion of spindle abnormalities in aged mouse oocytes.

[0112] Example 9 Preparation of 10 nM tPA Culture Medium

[0113] Prepare the culture medium containing 10 nM tPA in a sterile environment according to the following components:

[0114] Element volume M2 medium 998.7μl tPA concentrated stock solution (100 μg / ml) 1.3μl

[0115] Mix gently with a pipette and store at 4°C.

[0116] Example 1040 nM tPA culture medium

[0117] Prepare the culture medium containing 40 nM tPA in a sterile environment according to the following components:

[0118] Element volume M2 medium 994.8μl tPA concentrated stock solution (100 μg / ml) 5.2μl

[0119] Mix gently with a pipette and store at 4°C.

[0120] Example 1160 nM tPA culture medium

[0121] Prepare the culture medium containing 60 nM tPA in a sterile environment according to the following components:

[0122] Element volume M2 medium 992.2μl tPA concentrated stock solution (100 μg / ml) 7.8μl

[0123] Mix gently with a pipette and store at 4°C.

[0124] Example 1270 nM tPA culture medium

[0125] Prepare the culture medium containing 70 nM tPA in a sterile environment according to the following components:

[0126]

[0127]

[0128] Mix gently with a pipette and store at 4°C.

[0129] Example 1380 nM tPA culture medium

[0130] Prepare the culture medium containing 80 nM tPA in a sterile environment according to the following components:

[0131] Element volume M2 medium 989.6μl tPA concentrated stock solution (100 μg / ml) 10.4μl

[0132] Mix gently with a pipette and store at 4°C.

[0133] Example 14 100 nM tPA culture medium

[0134] Prepare the culture medium containing 100 nM tPA in a sterile environment according to the following components:

[0135] Element volume M2 medium 987μl tPA concentrated stock solution (100 μg / ml) 13μl

[0136] Mix gently with a pipette and store at 4°C.

Claims

1. A culture medium for improving the quality of aged oocytes in vitro, characterized in that: Contains tissue plasminogen activator at a final concentration of 5-100 nM.

2. The culture solution according to claim 1, characterized in that: The final concentration of tissue plasminogen activator in the culture medium is 50 nM.

3. The culture solution according to claim 1, characterized in that: The tissue plasminogen activator is a recombinant protein of active tissue plasminogen activator derived from human or mouse.

4. The culture solution according to claim 1, characterized in that: Aged oocytes can be derived from either human or mouse.

5. The culture solution according to claim 4, characterized in that: Aged oocytes were derived from human females aged 30 years or older or female mice aged 8 months or older.

6. The culture solution according to claim 1, characterized in that: The culture medium is obtained by adding a concentrated stock solution of tissue-type plasminogen activator to any one of M2 culture medium, M16 culture medium, G-1TM PLUS and CZB culture medium as a basic culture medium.

7. The culture solution according to claim 1, characterized in that: The concentrated stock solution of tissue plasminogen activator is prepared by dissolving tPA in a buffer solution containing a carrier protein.

8. The culture solution according to claim 7, characterized in that: The carrier protein is 0.1% bovine serum albumin, 5% human serum albumin, 10% fetal bovine serum or 5% trehalose, preferably 0.1% bovine serum albumin.

9. The culture solution according to claim 7, characterized in that: The buffer is sterile water or phosphate buffer; preferably phosphate buffer.

10. Use of tissue plasminogen activator in the preparation of reagents for improving the quality of aged oocytes in vitro.

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