Oocyte culture solution and application thereof

Abstract

The invention discloses an oocyte culture solution and application thereof, and relates to the technical field of biological medicine, and the oocyte culture solution comprises phosphatidylinositol, L-carnitine, a bicarbonate buffer solution, an energy metabolism substrate, polyvinyl alcohol, hormone and growth factors. The oocyte culture solution is beneficial to improving the steady state unbalance phenomenon of oocyte lipid metabolism, the cytoplasm fragment rate and the cleavage fragment rate in the aging process of oocytes after ovulation are reduced, and the effect of relieving aging of the oocytes after ovulation is achieved.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to an oocyte culture medium and its application. Background Art

[0002] During the implementation of assisted reproductive technology (ART), oocytes are often cultured and preserved using oocyte culture medium. During culture, the oocytes will undergo a process in which their developmental potential gradually weakens as the culture time goes by.

[0003] When mammalian oocytes mature in vitro, their lipid metabolism is affected by prolonged exposure to the in vitro environment. As time passes after ovulation, oocytes gradually age, manifesting as an imbalance in lipid metabolism. Furthermore, as in vitro culture time increases, nutrients in the culture medium are gradually consumed by the oocyte, while harmful substances or metabolic waste accumulate in the medium. This dual effect of reduced nutrients and increased harmful substances makes oocytes susceptible to various abnormalities, such as abnormal polar body formation, cytoplasmic fragmentation, and degeneration. Summary of the Invention

[0004] In view of the deficiencies of the above-mentioned related technologies, the present application provides an oocyte culture medium and its application. The oocyte culture medium of the present application is beneficial for improving the steady-state imbalance of oocyte lipid metabolism, reducing the fragmentation rate of oocytes during post-ovulation aging, and achieving the effect of alleviating post-ovulation aging of oocytes.

[0005] On the one hand, the present application provides an oocyte culture medium using the following technical solution: An oocyte culture medium comprises phosphatidylinositol, L-carnitine, bicarbonate buffer, energy metabolism substrate, polyvinyl alcohol, hormone and growth factor.

[0006] Preferably, the concentration of the phosphatidylinositol is 10-50 mM.

[0007] Preferably, the concentration of phosphatidylinositol is 30 mM.

[0008] Preferably, the concentration of L-carnitine is 0.3-0.7 mg / mL.

[0009] Preferably, the concentration of L-carnitine is 0.5 mg / mL.

[0010] Preferably, the energy metabolism substrate comprises 0.9-0.95 mM sodium pyruvate, 3.0-3.1 mM glucose and 0.55-0.6 mM L-cysteine.

[0011] Preferably, the energy metabolism substrate comprises 0.91 mM sodium pyruvate, 3.05 mM glucose and 0.57 mM L-cysteine.

[0012] Preferably, the hormones include 8-12 IU / mL of equine chorionic gonadotropin, 8-12 IU / mL of human chorionic gonadotropin, and 2-3 IU / mL of follicle-stimulating hormone.

[0013] Preferably, the hormones include 10 IU / mL of equine chorionic gonadotropin, 10 IU / mL of human chorionic gonadotropin, and 2.5 IU / mL of follicle-stimulating hormone.

[0014] Preferably, the growth factors include 35-45 ng / mL of fibroblast growth factor 2, 15-25 ng / mL of insulin-like growth factor 1, 15-25 ng / mL of leukemia inhibitory factor, and 8-12 ng / mL of epidermal growth factor.

[0015] Preferably, the growth factors include 40 ng / mL of fibroblast growth factor 2, 20 ng / mL of insulin-like growth factor 1, 20 ng / mL of leukemia inhibitory factor, and 10 ng / mL of epidermal growth factor.

[0016] Preferably, the concentration of the polyvinyl alcohol is 0.9-1.1 mg / mL.

[0017] Preferably, the concentration of the polyvinyl alcohol is 1 mg / mL.

[0018] On the other hand, the present application provides a use of an oocyte culture medium in alleviating post-ovulation oocyte aging.

[0019] Preferably, the method comprises the following steps: stripping the connective tissue attached to the surface of the ovary, disinfecting and then flushing with double-antibody saline; aspirating the follicular fluid in the follicle with a syringe, and screening out the oocyte-cumulus complex that is coated with at least three layers of dense cumulus cells and has uniform cytoplasm and no dark granules or vacuoles; The oocyte-cumulus complex is cultured until the first polar body is discharged, and then the cumulus cells are removed to obtain a mature oocyte, which is then cultured in the oocyte culture medium.

[0020] Preferably, the oocyte culture medium is conducive to reducing the level of lipids in oocytes.

[0021] Preferably, the oocyte culture medium is beneficial for reducing the fragmentation rate of oocytes during the aging process after ovulation.

[0022] In summary, this application includes at least one of the following beneficial technical effects: 1. The oocyte culture medium of the present application, by adding phosphatidylinositol, combined with L-carnitine, bicarbonate buffer, energy metabolism substrates, polyvinyl alcohol, hormones and growth factors, is beneficial to improving the steady-state imbalance of oocyte lipid metabolism, reducing the fragmentation rate of oocytes during postovulatory aging, and achieving the effect of alleviating postovulatory oocyte aging; 2. Compared with the control group, the maturation rate, cytoplasmic fragmentation rate, cleavage rate, embryo fragmentation rate, and blastocyst rate of oocytes cultured in the oocyte culture medium of the present application are close to the levels of unaged oocytes, especially the cytoplasmic fragmentation rate, cleavage fragmentation rate, and blastocyst rate, showing significant improvement. 3. The levels of lipid (Lipid) and intracellular phosphatidylinositol kinase (PI3KR4) in oocytes cultured in the oocyte culture medium of the present application were significantly reduced to a level close to that of oocytes cultured without aging, indicating that the oocyte culture medium of the present application is beneficial for regulating abnormal lipid metabolism through intracellular phosphatidylinositol kinase, thereby effectively alleviating post-ovulatory aging of oocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 1. It is a comparison chart of the maturation rates of oocytes cultured in the oocyte culture solution of Examples 1-5, oocytes cultured in the oocyte culture solution of Comparative Examples 1-2, and normal mature oocytes cultured without simulated aging; Figure 2 This is a comparison chart of the cytoplasmic fragmentation rates of oocytes cultured in the oocyte culture solutions of Examples 1-5, oocytes cultured in the oocyte culture solutions of Comparative Examples 1-2, and normal mature oocytes that were not cultured with simulated aging; Figure 3 1. It is a comparison chart of the cleavage rates of oocytes cultured in the oocyte culture solutions of Examples 1-5, oocytes cultured in the oocyte culture solutions of Comparative Examples 1-2, and normal mature oocytes cultured without simulated aging at the early embryonic stage; Figure 4 1. It is a comparison chart of the cleavage fragmentation rates of oocytes cultured in the oocyte culture solutions of Examples 1-5, oocytes cultured in the oocyte culture solutions of Comparative Examples 1-2, and normal mature oocytes cultured without simulated aging at the early embryonic stage; Figure 5This is a comparison chart of the blastocyst rates at the early embryonic stage of oocytes cultured in the oocyte culture medium of Examples 1-5, oocytes cultured in the oocyte culture medium of Comparative Examples 1-2, and normal mature oocytes that were not cultured with simulated aging; Figure 6 This is a comparison of fluorescent staining of lipids in oocytes cultured in the oocyte culture medium of Example 1, oocytes cultured in the oocyte culture medium of Comparative Example 1, and normal mature oocytes that were not cultured with simulated aging; Figure 7 1 is a graph comparing the fluorescence intensity of lipids in oocytes cultured in the oocyte culture medium of Example 1, oocytes cultured in the oocyte culture medium of Comparative Example 1, and normal mature oocytes cultured without simulated aging; Figure 8 This is a comparison of fluorescent staining of intracellular phosphatidylinositol kinase in oocytes cultured in the oocyte culture medium of Example 1, oocytes cultured in the oocyte culture medium of Comparative Example 1, and normal mature oocytes that were not cultured with simulated aging; Figure 9 This is a comparison of the fluorescence intensity of intracellular phosphatidylinositol kinase in oocytes cultured in the oocyte culture medium of Example 1, oocytes cultured in the oocyte culture medium of Comparative Example 1, and normal mature oocytes that were not cultured with simulated aging. DETAILED DESCRIPTION

[0024] The present application is described in further detail below in conjunction with Examples. The following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. In the following examples, if specific conditions are not specified, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. The methods used are conventional methods well known in the art unless otherwise specified, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise indicated, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0025] The raw materials used in the examples and comparative examples can all be obtained commercially.

[0026] Example 1 Example 1 of the present application discloses an oocyte culture medium comprising the following components: TCM-199 bicarbonate buffer (Gibco, product number 11150059) was used as the base solution, and 30 mM phosphatidylinositol (MCE, HY-W020658), 1 mg / mL polyvinyl alcohol (PVA, Sigma-Aldrich, USA, product number P8136), 0.5 mg / mL L-carnitine (Sigma-Aldrich, USA, product number 840092), 0.91 mM sodium pyruvate (Sigma-Aldrich, product number SLCJ1780), 3.05 mM glucose (Sigma-Aldrich, product number G6152), and 0.57 mM L-cysteine ​​(Sigma-Aldrich, Catalog No. 168149), 10 IU / mL equine chorionic gonadotropin (eCG, Ningbo Second Hormone Factory, Cixi, China), 10 IU / mL human chorionic gonadotropin (hCG, Ningbo Second Hormone Factory), 2.5 IU / mL follicle-stimulating hormone (FSH, Ningbo Second Hormone Factory), 40 ng / mL fibroblast growth factor 2 (FGF-2, Sino Biological, Beijing, China, Catalog No. 10014-HNAE), 20 ng / mL insulin-like growth factor 1 (IGF-1, Sino Biological, Catalog No. 10598-HNAE), 20 ng / mL leukemia inhibitory factor (LIF, Sino Biological, Catalog No. 14890-HNAH), and 10 ng / mL epidermal growth factor (EGF, Gibco, Catalog No. PHG0311).

[0027] Example 2 Example 2 of the present application discloses an oocyte culture medium. The difference between Example 2 and Example 1 is that the concentration of phosphatidylinositol in Example 2 is 10 μM.

[0028] Example 3 Example 3 of the present application discloses an oocyte culture medium. The difference between Example 3 and Example 1 is that the concentration of phosphatidylinositol in Example 3 is 20 μM.

[0029] Example 4 Example 4 of the present application discloses an oocyte culture medium. The difference between Example 4 and Example 1 is that the concentration of phosphatidylinositol in Example 4 is 40 μM.

[0030] Example 5 Example 5 of the present application discloses an oocyte culture medium. The difference between Example 5 and Example 1 is that the concentration of phosphatidylinositol in Example 5 is 50 μM.

[0031] Comparative Example 1 Comparative Example 1 of the present application discloses an oocyte culture medium. The difference between Comparative Example 1 and Example 1 is that in Comparative Example 1, inositol is used instead of phosphatidylinositol.

[0032] Comparative Example 2 Comparative Example 2 of the present application discloses an oocyte culture medium. The difference between Comparative Example 2 and Example 1 is that phosphatidylinositol is not added in Comparative Example 2.

[0033] Test and Inspection (1) After obtaining the porcine ovaries from the slaughterhouse, they were immediately placed in 37°C preheated double-antibiotic saline (containing penicillin-streptomycin) and transported to the laboratory within 2-4 hours. The connective tissue attached to the ovarian surface was stripped off and the surface was disinfected with 75% ethanol for 30 seconds. Then, the ovaries were rinsed 2-3 times with 37°C double-antibiotic saline to remove residual impurities. Follicular fluid from follicles with a diameter of 3-8 mm was punctured and aspirated using an 18-20G syringe. Oocyte-cumulus complexes (COCs) with at least three layers of dense cumulus cells, uniform cytoplasm, and no dark granules or vacuoles were selected under a stereomicroscope (40× magnification).

[0034] The oocyte-cumulus complex was placed in a maturation medium covered with mineral oil and cultured in an incubator containing 5% CO2, 38.5°C, and saturated humidity for 44-46 hours. After the first polar body was excluded, the oocyte-cumulus complex was moved into 0.1% hyaluronidase, and the cumulus cells were removed by repeated blowing to obtain normal mature oocytes.

[0035] Normal mature oocytes were cultured in an incubator containing 5% CO2, 38.5°C and saturated humidity for 24 hours using the oocyte culture solutions of Examples 1-5 and Comparative Examples 1-2, respectively, to simulate post-ovulation aging of oocytes.

[0036] The maturation rate and cytoplasmic fragmentation rate of the oocytes cultured in the oocyte culture medium of Examples 1-5 and Comparative Examples 1-2 and the normal mature oocytes cultured without aging were detected respectively, and the comparison diagrams between the groups were obtained as follows: Figure 1-2 The oocytes cultured in the oocyte culture medium of Examples 1-5 and Comparative Examples 1-2 and the normal mature oocytes cultured without aging were fertilized and cultured in embryo culture medium for 7 days to enter the early embryo stage. The cleavage rate, cleavage fragment rate and blastocyst rate were calculated, and the blastocyst rate comparison charts of the oocytes cultured in the oocyte culture medium of Examples 1-5 and Comparative Examples 1-2 and the normal mature oocytes cultured without aging were obtained as shown in the figure below. Figure 3-5 shown.

[0037] (2) Fluorescence staining of lipids (DIPI labeled cell nuclei, stained with fluorescent neutral lipid dye (BODIPY 493 / 503, Molecular Probes, Invitrogen) for 1 hour, washed with dye and incubated with DAPI for 8-10 minutes) was performed on oocytes cultured in the oocyte culture medium of Example 1 and Comparative Example 2, as well as normal mature oocytes of the control group that were not subjected to aging treatment. The fluorescence staining comparison diagram was obtained as shown in FIG. Figure 6 As shown in the fluorescence intensity comparison diagram Figure 7 As shown. Fluorescence staining of intracellular phosphatidylinositol kinase (PI3KR4) of oocytes cultured in the oocyte culture medium of Example 1 and Comparative Example 2 and the normal mature oocyte control group without aging treatment was performed (the cell nucleus was labeled with DIPI, the collected samples were fixed with 4% paraformaldehyde at 4°C overnight, and then permeabilized with Triton X-100. After washing, the samples were blocked with immunofluorescence blocking solution at room temperature for 2-3 hours, washed, incubated at 4°C for 14 hours to bind to PI3KR4 (primary antibody), washed, and transferred to the secondary antibody in the dark at room temperature for incubation for 2 hours. After washing, incubated with DAPI for 8-10 minutes), and the fluorescence staining comparison diagram was obtained as shown in the figure below. Figure 8 As shown in the fluorescence intensity comparison diagram Figure 9 shown.

[0038] Figures 1 to 9 In the figure, the test data of the oocytes cultured in the oocyte culture medium of Examples 1-5 are marked as 30mM PI, 10mM PI, 20mM PI, 40mM PI and 50mM PI, respectively. The test data of the oocytes cultured in the oocyte culture medium of Comparative Examples 1-2 are marked as Aging 24h and 30mM INS, respectively. The test data of the normal mature oocytes without aging treatment are marked as Control.

[0039] Result Analysis Reference Figure 1-5Compared with the oocyte culture medium of Comparative Example 2, the oocytes cultured in the oocyte culture medium of Examples 1-5 had increased maturation rates, decreased cytoplasmic fragmentation rates, increased cleavage rates, decreased embryo fragmentation rates, and increased blastocyst rates. In particular, the cytoplasmic fragmentation rates, cleavage fragmentation rates, and blastocyst rates were significantly improved, approaching the levels of the normal mature oocyte control group. This indicates that the oocyte culture medium of the present application is conducive to the developmental phenotype of oocytes approaching that of normal mature oocytes and alleviates post-ovulatory oocyte aging.

[0040] Taking Comparative Example 1 as a control, the cytoplasmic fragmentation rate and the cleavage fragmentation rate of the oocytes cultured in the oocyte culture solution of Example 1 are much lower than the cytoplasmic fragmentation rate and the cleavage fragmentation rate of the oocytes cultured in the oocyte culture solution of Comparative Example 1, and the blastocyst rate of the oocytes cultured in the oocyte culture solution of Example 1 is much higher than the blastocyst rate of the oocytes cultured in the oocyte culture solution of Comparative Example 1. This indicates that the use of phosphatidylinositol in the oocyte culture solution of the present application is beneficial to reducing the cytoplasmic fragmentation rate and the cleavage fragmentation rate of the oocyte, and improving the blastocyst rate of the oocyte, compared with the use of inositol.

[0041] Reference Figure 6-7 The lipid level in the oocytes cultured with the oocyte culture medium of Comparative Example 2 was significantly increased compared with the lipid level in the normal mature oocytes of Comparative Example 1 that had not been aged, while the lipid level in the oocytes cultured with the oocyte culture medium of Example 1 was significantly decreased compared with the lipid level in the oocyte cultured with the oocyte culture medium of Comparative Example 2, indicating that the oocyte culture medium of Example 1 of the present application is beneficial to alleviating the post-ovulation aging of oocytes.

[0042] Reference Figure 8-9 The intracellular phosphatidylinositol kinase level in the oocytes cultured with the oocyte culture medium of Comparative Example 2 was significantly increased compared with the intracellular phosphatidylinositol kinase level in the normal mature oocytes without aging treatment in Comparative Example 1, while the intracellular phosphatidylinositol kinase level in the oocytes cultured with the oocyte culture medium of Example 1 was significantly decreased compared with the intracellular phosphatidylinositol kinase level in the oocytes cultured with the oocyte culture medium of Comparative Example 2, indicating that the oocyte culture medium of Example 1 of the present application is beneficial for alleviating post-ovulatory oocyte aging by regulating abnormal lipid metabolism through intracellular phosphatidylinositol kinase.

[0043] Unless otherwise defined, the technical or scientific terms used in this application shall have the usual meanings understood by persons of ordinary skill in the field to which this application belongs. The words "first", "second", "third" and similar terms used in the specification and claims of this application do not indicate any order, quantity or importance, but are only used to distinguish different components. Words such as "one" or "a" do not indicate a quantity limitation, but rather indicate the existence of at least one. Words such as "include" or "comprise" mean that the elements or objects appearing before "include" or "comprises" cover the elements or objects listed after "include" or "comprises" and their equivalents, and do not exclude other elements or objects. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0044] The above are all optional embodiments of the present application, and are not intended to limit the scope of protection of the present application. Therefore, any equivalent changes made based on the structure, shape, and principle of the present application should be included in the scope of protection of the present application.

Claims

1. An oocyte culture medium, characterized in that: These include phosphatidylinositol, L-carnitine, bicarbonate buffer, energy metabolism substrates, polyvinyl alcohol, hormones, and growth factors.

2. An oocyte culture medium according to claim 1, characterized in that: The concentration of the phosphatidylinositol is 10-50 mM.

3. An oocyte culture medium according to claim 1, characterized in that: The concentration of the L-carnitine is 0.3-0.7 mg / mL.

4. An oocyte culture medium according to claim 1, characterized in that: The energy metabolism substrates include 0.9-0.95 mM sodium pyruvate, 3.0-3.1 mM glucose and 0.55-0.6 mM L-cysteine.

5. The oocyte culture medium according to claim 1, wherein: The hormones include 8-12 IU / mL of equine chorionic gonadotropin, 8-12 IU / mL of human chorionic gonadotropin, and 2-3 IU / mL of follicle-stimulating hormone.

6. An oocyte culture medium according to claim 1, characterized in that: The growth factors include 35-45 ng / mL of fibroblast growth factor 2, 15-25 ng / mL of insulin-like growth factor 1, 15-25 ng / mL of leukemia inhibitory factor and 8-12 ng / mL of epidermal growth factor.

7. Use of the oocyte culture medium according to any one of claims 1 to 6 in alleviating post-ovulatory aging of oocytes.

8. The use according to claim 7, characterized in that: The following steps are involved: The connective tissue attached to the ovarian surface was stripped away and rinsed with double-antibiotic saline after disinfection. The follicular fluid in the follicle was aspirated with a syringe, and oocyte-cumulus complexes with at least three layers of dense cumulus cells, uniform cytoplasm, and no dark granules or vacuoles were selected. The oocyte-cumulus complex is cultured until the first polar body is discharged, and then the cumulus cells are removed to obtain a mature oocyte, which is then cultured in the oocyte culture medium.

9. The use according to claim 7, characterized in that: The oocyte culture medium is beneficial for reducing the lipid level in the oocyte.

10. The use according to claim 7, characterized in that: The oocyte culture medium is beneficial for reducing the cytoplasmic fragmentation rate and the cleavage fragmentation rate of the oocyte during the aging process after ovulation.

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