A culture medium for improving in vitro aging of oocytes after ovulation and application thereof

Abstract

The application discloses a culture medium for improving in-vitro aging of oocytes after ovulation and application thereof. The culture medium is prepared by adding 0.1-30 muM of astragaloside IV into an oocyte culture solution. The culture medium is safe, non-toxic and free of side effects, can reduce the oocyte fragmentation rate, active oxygen level and abnormal spindle rate of the oocytes after 24 hours of in-vitro culture, can significantly improve the mitochondrial membrane potential, in-vitro sperm binding capacity, in-vitro fertilization pronucleus rate and cleavage rate of the mature oocytes after 24 hours of in-vitro culture, and can improve the quality of the late embryos. The application of the culture medium provides favorable support for assisted reproductive technology, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of assisted reproductive biology, and particularly relates to a culture medium for improving post-ovulatory oocyte in vitro aging and application thereof. BACKGROUND

[0002] In mammals, mature oocytes after ovulation will always wait for fertilization, and there is an optimal time window for oocyte and sperm fertilization. If the post-ovulatory oocyte is not fertilized for a long time, it will age, and the quality and the ability of the embryo to develop after fertilization will significantly decrease. This type of oocyte aging is not related to age, but to the time of in vitro storage after ovulation. Since there is no visible ovulation in primates, there is no appropriate mechanism to ensure that sperm is delivered to the female reproductive tract synchronously with ovulation, thereby increasing the possibility of fertilization of aged post-ovulatory oocytes and fresh sperm, resulting in decreased quality of late embryos and pregnancy failure. In addition, with the increase of lifestyle, environment and female age, the quality of female oocytes is getting worse. At this time, the emergence of assisted reproductive technology brings hope to many families. In the in vitro fertilization technology in assisted reproduction, the timely combination of the ovulated oocyte with the sperm within the time window to form a zygote is an important link for the success of assisted reproduction. However, a considerable part of mature oocytes cannot be immediately fertilized and need to be cultured in a culture medium for a period of time, and a considerable part of oocytes also need to be remedied after in vitro fertilization failure, i.e. single sperm ooplasm microinjection. However, at this time, the oocyte has undergone in vitro aging for 18-24 hours, resulting in a sharp decrease in the fertilization rate and the embryo development potential after single sperm injection. Therefore, there is an urgent need for a culture medium capable of improving post-ovulatory oocyte in vitro aging to maintain the quality of post-ovulatory oocytes and improve the pregnancy rate.

[0003] Studies have shown that in vitro aging of post-ovulatory oocytes mainly manifests in the increase of reactive oxygen species level, mitochondrial dysfunction, chromosomal segregation errors and in vitro fertilization ability. If some components are added to the in vitro culture medium, the aging of post-ovulatory oocytes can be alleviated, the increase of intracellular reactive oxygen species level can be inhibited, the mitochondrial function can be maintained, and the chromosomal arrangement errors can be reduced, which can greatly improve the assisted reproductive rate. On the other hand, if possible, oral administration of such components can also prolong the time for in vivo oocytes to wait for sperm fertilization, thereby improving the pregnancy rate and the pregnancy rate.

[0004] Astragaloside IV is one of the main active components of Chinese medicine Astragalus, and the molecular formula is C 14 H 68 O 14Astragaloside IV is a lanolin alcohol form of a triterpene saponin extracted from Astragalus membranaceus. It has anti-inflammatory, immunomodulatory, antioxidant, anti-apoptotic, metabolic regulation, anti-fibrosis, tumor inhibition, hypoglycemic and insulin resistance activities, and has protective effects on the kidney, liver, central nervous system, cardiovascular system and gastrointestinal tract. In view of the advantages of Astragaloside IV, more and more researchers are further exploring its new uses to fully utilize the medicinal value of Astragaloside IV. However, it is not certain whether Astragaloside IV can alleviate the in vitro and in vivo aging of oocytes after ovulation and whether it can be applied to assisted reproductive technology. Currently, there is no research report in this regard.

[0005] The prior art disclosed by Ren Xuehua et al. that Astragaloside IV can significantly improve the number of superovulation of T1D female mice, inhibit follicular granulosa cell apoptosis, improve the ROS level of oocytes in T1D group, and ultimately improve the damage of T1D to the female reproductive system. This technology is through Astragaloside IV gavage to mice for in vivo experiment. On the one hand, Liu Xiaoya et al. showed that Astragaloside IV has low oral bioavailability as a macromolecule through pharmacokinetics and in vitro and in vivo metabolism. And it is easy to be biotransformed in vivo to generate saponin ring astragalol to exert efficacy. Astragalol has been proven to have antioxidant function in vitro, and can activate telomerase to protect telomeres (Wu J et al., Phytother Res 2020, Wang Y et al., Br J Pharmaco 2019), increase the proliferation ability of cells, and resist cell apoptosis (Seviml-Guet al., JEthnopharroacol 2011). On the other hand, the prior art disclosed by Ren Xuehua et al. that Astragaloside IV gavage inhibits follicular granulosa cell apoptosis to alleviate the reproductive capacity of diabetic female mice also speculates that the in vivo metabolite astragalol plays a role in inhibiting granulosa cell apoptosis and improving ROS level. Therefore, it is not known whether Astragaloside IV plays a role in vivo in this study, or its biotransformation product plays a role, or they play a synergistic role. In this application, for the first time, Astragaloside IV is directly added to the embryo culture medium in vitro to directly study whether Astragaloside IV itself can alleviate the in vitro aging of oocytes after ovulation. In addition, the prior art disclosed by Ren Xuehua et al. is the effect of Astragaloside IV on oocyte maturation in vitro and in vivo after in vivo absorption by gavage. However, the signal transduction, intracellular cytoplasmic changes, structural and functional changes involved in the maturation of oocytes in vitro and in vivo are different from those involved in the aging of mature oocytes after ovulation and their progression to apoptosis. It cannot be concluded from this study that Astragaloside IV can improve the in vitro aging of mature oocytes after ovulation and its specific concentration. SUMMARY

[0006] Technical problem solved:

[0007] The present application solves the technical problems of the current in vitro aging of oocytes after ovulation, mainly reflected in the increase of reactive oxygen species level, mitochondrial dysfunction, chromosome segregation error and decline of in vitro fertilization ability, and provides a culture medium for improving in vitro aging of oocytes after ovulation and application thereof, and a method for improving the quality of oocytes after ovulation in vitro by using the culture medium.

[0008] Technical scheme:

[0009] To achieve the above-mentioned purpose, the present application is realized by the following technical scheme:

[0010] A culture medium for improving in vitro aging of oocytes after ovulation, the culture medium for improving in vitro aging of oocytes after ovulation is composed of KSOM culture medium, FBS, EGF, sodium pyruvate and astragaloside IV; the FBS accounts for 5% of the final system volume; the EGF accounts for 20 ng / ml of the final system volume; the sodium pyruvate accounts for 30 ug / ml of the final system volume, and the final concentration of the astragaloside IV is 0.1-30 μM.

[0011] The present application also discloses application of the culture medium for improving in vitro aging of oocytes after ovulation in protecting the quality of mature oocytes during in vitro storage.

[0012] Further, the final concentration of the astragaloside IV in the culture medium for improving in vitro aging of oocytes after ovulation is 0.3-1 μM.

[0013] Further, the final concentration of the astragaloside IV in the culture medium for improving in vitro aging of oocytes after ovulation is 0.3-0.5 μM.

[0014] Further, the final concentration of the astragaloside IV in the culture medium for improving in vitro aging of oocytes after ovulation is 0.5 μM. The principle of the above-mentioned culture medium for improving in vitro aging of oocytes after ovulation and application thereof lies in that the present application adds FBS, EGF, sodium pyruvate and astragaloside IV on the basis of KSOM culture medium, and the KSOM culture medium and 5% FBS of the final system volume, 20 ng / ml EGF, 30 ug / ml sodium pyruvate and 0.1-30 μM astragaloside IV produce a synergistic effect, which can significantly play the role of antioxidation and anti-apoptosis, improve the mitochondrial membrane potential of mature oocytes after 24 hours of in vitro aging, in vitro sperm-egg binding capacity, in vitro fertilization pronuclear rate and cleavage rate (2-cell rate), and improve the quality of late embryos as a whole.

[0015] Beneficial effects:

[0016] The application provides a culture medium for improving in vitro aging of post-ovulation oocytes and an application thereof, and has the following beneficial effects compared with the prior art:

[0017] 1. Application of astragaloside IV to improve in vitro aging of mature oocytes: the present application first uses a one-step in vitro embryo culture method to apply astragaloside IV to improve in vitro aging of mature oocytes, especially the activity of reactive oxygen species and the function of mitochondria of oocytes aged in vitro for 24 hours, and to improve the quality and potential embryo development ability of mature oocytes after in vitro culture for a period of time. The present technical solution increases the in vitro culture time of mature oocytes, provides people with more time and possibility to perform assisted reproductive technology or natural conception, greatly improves the pregnancy rate and increases the neonatal rate. Of course, the present application can also be used to improve in vitro or in vivo aging of post-mature oocytes within 24 hours, and brings hope to families plagued by infertility.

[0018] 2. Increase of pronucleus and 2-cell rates of mouse mature oocytes after in vitro culture for 24 hours, and improvement of embryo quality: through the in vitro oocyte culture method of the present application, the in vitro pronucleus rate of mature oocytes after in vitro culture for 24 hours is 36.4±0.27%, which is nearly doubled compared with the control group, the 2-cell development rate is 26.23±0.28%, which is significantly increased, the embryo quality is improved, and the potential utilization value of post-ovulation oocytes after in vitro culture for a period of time is improved.

[0019] 3. Support for embryo biotechnology of humans and other mammals: the culture medium of the present application greatly improves the cell quality and potential embryo development ability of post-ovulation oocytes after in vitro culture for a period of time; this achievement provides support for the development of reproductive technology of other viviparous mammals, such as humans, sheep, horses, pigs, pandas and primates.

[0020] 4. Simple operation, safety and non-toxicity: astragaloside IV, as a component of traditional Chinese medicine, has been passed down for thousands of years, has high safety to cells, has good permeability, easily enters cells without damaging the cells, and has no toxicity within an effective dose range. The operation is simple, and only a certain amount of astragaloside IV needs to be added to the conventional post-ovulation oocyte in vitro embryo culture solution. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The chemical formula of astragaloside IV in the present application is as follows:

[0022] Figure 2Figure 1 is a graph of the mitochondrial membrane potential and ROS levels in mature oocytes in the control group and the experimental group of the present application; Figure A is a representative fluorescence graph of the ROS levels in mature oocytes after in vitro aging for 24 hours in the control group (left) and the experimental group (right); Figure B is a graph of the relative intensity of immunofluorescence of the ROS levels in mature oocytes after in vitro aging for 24 hours in the control group and the experimental group; Figure C is a representative graph of the JC-1 red and green fluorescence of mature oocytes after in vitro aging for 24 hours in the control group and the experimental group, the upper left graph in Figure C is a representative graph of the JC1 red fluorescence of the control group, the upper right graph is a representative graph of the JC1 red fluorescence of the experimental group, the lower left graph is a representative graph of the JC1 green fluorescence of the control group, and the lower right graph is a representative graph of the JC1 green fluorescence of the experimental group; and Figure D is a graph of the relative ratio of the mitochondrial membrane potential of mature oocytes after in vitro aging for 24 hours in the control group and the experimental group.

[0023] Figure 3 Figure 2 is a graph of the early apoptosis signals, chromosome arrangement, microfilament distribution, and spindle morphology in mature oocytes in the control group and the experimental group of the present application; Figure A is a graph of the number of early apoptosis occurring in mature oocytes after in vitro aging for 24 hours in the control group and the experimental group; Figure B is a graph of the number of chromosome misarrangement occurring in mature oocytes after in vitro aging for 24 hours in the control group and the experimental group; Figure C is a representative graph of the normal (left) and abnormal (right) distribution of the microfilament skeleton of mature oocytes after in vitro aging for 24 hours; Figure D is a graph of the number of abnormal microfilament distribution occurring in mature oocytes after in vitro aging for 24 hours in the control group and the experimental group; Figure E is a representative graph of the normal (left) and abnormal (right) spindle morphology of mature oocytes after in vitro aging for 24 hours; and Figure F is a graph of the number of abnormal spindle morphology occurring in mature oocytes after in vitro aging for 24 hours in the control group and the experimental group.

[0024] Figure 4 Figure 3 is a graph of the number of sperm binding, pronucleus formation rate, and cleavage rate in mature oocytes in the control group and the experimental group of the present application; Figure A is a representative graph of mature oocytes after in vitro aging for 24 hours binding with sperm, and the graph directly below it is a graph of the number of mature oocytes after in vitro aging for 24 hours binding with sperm in the control group and the experimental group; Figure B is a representative graph of mouse pronucleus embryos, and the graph directly below it is a graph of the pronucleus formation rate in the control group and the experimental group; and Figure C is a representative graph of mouse 2-cell embryos, and the graph directly below it is a graph of the cleavage rate in the control group and the experimental group. DETAILED DESCRIPTION

[0025] The following examples are intended to illustrate the present application but not to limit the scope of the present application. Modifications or substitutions of the methods, steps, or conditions of the present application are included in the scope of the present application without departing from the spirit and essence of the present application.

[0026] Unless otherwise specifically indicated, the technical means used in the examples are conventional means well known to those skilled in the art.

[0027] The experimental mice are from the animal feeding center of Nantong University, and astragaloside IV is purchased from Sigma Company in the United States.

[0028] Example 1

[0029] A culture medium for improving in vitro aging of oocytes after ovulation, which is composed of KSOM culture medium, FBS, EGF, sodium pyruvate and astragaloside IV; the FBS accounts for 5% of the final system volume; the EGF accounts for 20 ng / ml of the final system volume; the sodium pyruvate accounts for 30 ug / ml of the final system volume, and the final concentration of the astragaloside IV is 0.1-30 μM.

[0030] The application of the culture medium for improving in vitro aging of oocytes after ovulation in protecting the quality of mature oocytes during in vitro storage, specifically the application in improving the quality of mammalian mature oocytes after 24 hours of in vitro storage, finally reflected in improving the pronuclear rate and / or embryo development rate after in vitro fertilization; the oocytes are in vivo matured oocytes or in vitro cultured oocytes for 24 hours after in vivo maturation; the mammal is a viviparous mammal. The viviparous mammal is a human or a primate, a cow, a sheep, a horse, a mouse, a pig or a panda; the method for improving the quality of mammalian mature oocytes after 24 hours of in vitro storage comprises the following steps:

[0031] Step 1: washing the oocytes after ovulation in the KSOM culture medium containing 5% FBS, 20 ng / ml EGF and 30 ug / ml sodium pyruvate in the final system volume, and then washing in the culture medium for improving in vitro aging of oocytes after ovulation according to the present application;

[0032] Step 2: after washing, placing the oocytes after ovulation in the culture medium for improving in vitro aging of oocytes after ovulation, and culturing in a CO2 incubator at 37℃, 5% CO2 and 100% humidity;

[0033] The number of oocytes: the volume of the culture medium for improving in vitro aging of oocytes after ovulation is 1:3-1:10 μL in the ratio, and the culture is carried out in a culture box at 37℃, 5% CO2 and 100% humidity for 12-24 hours. The preferred culture time is 24 hours.

[0034] The oocytes after ovulation in the first step are mature oocytes collected 12 hours after intraperitoneal injection of luteinizing hormone in mice.

[0035] Example 2

[0036] In vitro culture of mouse oocytes after ovulation

[0037] (1) Collection of mature oocytes

[0038] 8-10 weeks old female mice were injected with PMSG 5 IU intraperitoneally, and 48 hours later, hCG 5 IU was injected intraperitoneally. 12 hours later, the mice were euthanized, and the post-ovulatory oocytes were collected.

[0039] (2) In vitro culture of mature oocytes

[0040] A culture medium for improving in vitro aging of post-ovulatory oocytes was prepared, which consisted of KSOM medium, FBS, EGF, sodium pyruvate, and astragaloside IV; the FBS accounted for 5% of the final system volume; the EGF accounted for 20 ng / ml of the final system volume; the sodium pyruvate accounted for 30 ug / ml of the final system volume, and the final concentration of the astragaloside IV was 0.5 uM; the culture medium for improving in vitro aging of post-ovulatory oocytes was equilibrated in a CO2 incubator for 2-3 hours.

[0041] The post-ovulatory oocytes with normal cell morphology and good refractive properties were selected and washed 2-3 times in the KSOM medium containing 5% FBS, 20 ng / ml EGF, and 30 ug / ml sodium pyruvate in the final system volume, and then washed in a 35 mm culture dish containing the culture medium for improving in vitro aging of post-ovulatory oocytes.

[0042] After washing, 50-65 uL of the culture medium for improving in vitro aging of post-ovulatory oocytes was added to each well, which was covered with mineral oil, and 15-20 post-ovulatory oocytes were dropped into each well. Finally, the culture dish was placed in an incubator for culture, and the culture conditions were 37℃, 5% CO2, and 100% humidity, and the culture time was 24 hours.

[0043] After 24 hours of maturation culture, the post-ovulatory oocytes were detected for abnormal conditions such as fragmentation, deformity, and abnormal cell morphology, and the abnormal rate was calculated (taking the post-ovulatory oocytes cultured in the KSOM medium containing 5% FBS, 20 ng / ml EGF, and 30 ug / ml sodium pyruvate in the final system volume without the addition of astragaloside IV as a control). The results are shown in Table 1. The following control group and experimental group were the same in other culture conditions except for the addition of astragaloside IV.

[0044] Table 1 Effect of the addition of astragaloside IV on the cell morphology of mature oocytes cultured in vitro for 24 hours

[0045] Grouping Oocyte number Abnormal rate (%) Control group 180 27.5 ± 3.13 a ]] Experimental group 165 8.7 ± 2.12 b ]]

[0046] Note: The above table was statistically analyzed by t-test. Different letters in the same column represent significant differences (P<0.05), and the same below.

[0047] Table 1 shows that after culturing oocytes in a medium that improves in vitro aging after ovulation for 24 hours, the abnormality rate was significantly lower than that in KSOM medium containing 5% FBS, 20 ng / ml EGF and 30 ug / ml sodium pyruvate in the conventional final volume, and the difference was statistically significant (P < 0.05).

[0048] (3) Levels of reactive oxygen species and mitochondrial membrane potential after 24 hours of in vitro culture of mature oocytes

[0049] Further experiments were conducted on post-ovulatory oocytes cultured in the aforementioned culture medium that improves the in vitro aging of post-ovulatory oocytes.

[0050] Reactive oxygen species (ROS) levels: Mature oocytes cultured for 24 hours were incubated with the oxidation-sensitive fluorescent probe DCFH-DA (1:250) at 37°C in a 5% CO2 incubator for 30 minutes. Observation under a fluorescence inverted microscope revealed that the addition of astragaloside IV effectively reduced the accumulation of ROS in oocytes after ovulation. Figure 2 A, 2B). (Passed) Figure 2 A and 2B indicate that astragaloside IV can alleviate oxidative damage during the in vitro aging process of mature oocytes. High levels of ROS induce mitochondrial damage. Using JC-1 dye to detect the mitochondrial membrane potential of the oocytes, the results showed that the mitochondrial membrane potential of oocytes in the astragaloside IV-added group was significantly higher than that in the unadded group, thus alleviating the effects of aging on mature oocytes after in vitro culture. Figure 2 C, 2D). Figure 2 C, 2D indicates that astragaloside IV has a protective effect on the mitochondrial membrane during the in vitro aging process of mature oocytes.

[0051] (4) Early apoptosis and chromosome alignment of mature oocytes after 24 hours of in vitro culture

[0052] Further experiments were conducted on post-ovulatory oocytes cultured in the aforementioned culture medium that improves the in vitro aging of post-ovulatory oocytes.

[0053] The level of early apoptosis in mature oocytes cultured in vitro for 24 hours was detected using Annexin V-FITC. The results showed that the level of oocyte apoptosis was significantly reduced in the group with added astragaloside IV (experimental group). Figure 3 A). Chromosome alignment was assessed by DAPI staining of cell nuclei, revealing that the addition of Astragalus membranaceus (AIV) to the cell nucleus alleviated chromosomal misalignment. Figure 3 B).

[0054] (5) Distribution of spindle fibers and microfilaments in oocytes cultured in vitro for 24 hours after ovulation

[0055] The post-ovulatory oocytes cultured in the medium for improving the in vitro aging of post-ovulatory oocytes are used for further experiments.

[0056] The post-ovulatory oocytes cultured in vitro for 24 hours are fixed, permeated, blocked and incubated with tubulin-FITC or phalloidin-FITC at room temperature for 1.5 hours, which mark the spindle and microfilament respectively. The distribution of microfilament and the morphology of spindle are observed under the fluorescence confocal microscope. The results show that the abnormal loss of microfilament and the abnormal morphology of spindle can be improved in the Astragaloside IV added group Figure 3 C, 3D, 3E, 3F).

[0057] (6) The number of in vitro sperm binding, pronuclear rate and 2-cell rate of the post-ovulatory oocytes cultured in vitro for 24 hours.

[0058] The post-ovulatory oocytes cultured in the medium for improving the in vitro aging of post-ovulatory oocytes are used for further experiments.

[0059] After the post-ovulatory oocytes cultured in vitro for 24 hours are inseminated in vitro for 6 hours, the excess sperm not bound to the oocytes is washed away by a glass needle operated by a mouth pipette, and then the oocytes are fixed, permeated and stained, and the number of sperm bound to the oocytes is counted. At the same time, all the sperm around the oocytes is washed away by a thinner glass needle, and then the oocytes are cultured in the final system of KSOM medium containing 5% FBS, 20 ng / ml EGF and 30 ug / ml sodium pyruvate for 6 hours and 24 hours, and the pronuclear rate and 2-cell (cleavage) rate are counted respectively. It is found that the post-ovulatory oocytes in the Astragaloside IV added group have stronger sperm binding rate, higher pronuclear rate and cleavage rate after being cultured in vitro for 24 hours compared with the control group Figure 4 A, 4B, 4C). These results all show that Astragaloside IV can obviously improve the fertilization ability and early embryonic development ability of post-ovulatory oocytes cultured in vitro for a period of time.

[0060] Figures 1-4 The significances in the table are the significances (p<0.05) obtained by comparing the experimental group with the control group.

[0061] The above is an exemplary description of the present application, and it should be noted that any simple modification, change or equivalent replacement without creative labor of those skilled in the art without departing from the core of the present application falls within the protection scope of the present application.

Claims

1. Use of a culture medium that ameliorates in vitro aging of postovulatory oocytes to preserve the quality of mature oocytes during in vitro storage, characterized in that: The medium for improving in vitro aging of oocytes after ovulation is composed of KSOM medium, FBS, EGF, sodium pyruvate and astragaloside IV; the FBS accounts for 5% of the final system volume; the EGF accounts for 20 ng / ml of the final system volume; the sodium pyruvate accounts for 30 ug / ml of the final system volume, and the final concentration of the astragaloside IV is 0.1-30 μM.

2. Use of the culture medium according to claim 1 for improving the in vitro aging of oocytes after ovulation for preserving their quality during in vitro storage of mature oocytes, characterized in that: The final concentration of the astragaloside IV in the medium for improving in vitro aging of oocytes after ovulation is 0.3-1 μM.

3. Use of the culture medium according to claim 1 for improving the in vitro aging of oocytes after ovulation for preserving their quality during in vitro storage of mature oocytes, characterized in that: The final concentration of the astragaloside IV in the medium for improving in vitro aging of oocytes after ovulation is 0.3-0.5 μM.

4. Use of the culture medium according to claim 1 for improving the in vitro aging of oocytes after ovulation for preserving their quality during in vitro storage of mature oocytes, characterized in that: The final concentration of the astragaloside IV in the medium for improving in vitro aging of oocytes after ovulation is 0.5 μM.

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