Application of deaminotyrosine in preparation of reagent or medicine for improving quality of aged oocytes

Abstract

The invention belongs to the field of reproductive medicine, and particularly relates to application of deaminotyrosine in preparation of a reagent or a medicine for improving the quality of aged oocytes. The invention discloses and provides a mechanism for activating an ERK signal channel by utilizing deaminotyrosine specificity to act on the quality improvement of the aged oocytes for the first time. Experiments prove that the deaminotyrosine can reduce the abnormal form proportion of spindle bodies of oocytes, regulate the transformation of mitochondria to uniform distribution, improve the membrane potential of the mitochondria, reduce the ROS level, improve the maturation rate of the oocytes and improve the development rate of in-vitro fertilized blastocysts. The deaminated tyrosine is an endogenous and symbiotic microbial metabolite of a human body, so that the deaminated tyrosine has extremely high biological safety while improving the quality of aged oocytes, and an effective and safe new means is provided for improving the fertility potential of elderly women and optimizing and assisting a reproductive in-vitro maturation technology.

Description

Technical Field

[0001] This invention belongs to the field of reproductive medicine, specifically relating to the application of deaminotyrosine in the preparation of reagents or drugs for improving the quality of aging oocytes. Background Technology

[0003] Oocyte quality declines significantly with age or ovarian stress, exhibiting characteristics such as increased oxidative stress levels, mitochondrial dysfunction, spindle assembly errors, increased chromosomal aneuploidy, and abnormal epigenetic modifications. Patent searches have revealed that antioxidant mechanisms are a major research direction in improving aging oocytes, with existing research results such as ursodeoxycholic acid and antioxidant nanomaterials. Although they can alleviate aging through antioxidant mechanisms, they still have the following limitations: (1) Single response mechanism: They focus on exerting antioxidant effects by clearing reactive oxygen species (ROS) as an outer characteristic, without addressing the core issues of cell cycle regulation disorders and cytoplasmic maturation defects within aging oocytes; (2) Low safety: The core components are mostly exogenously synthesized or inorganic materials, posing a safety risk for long-term use in the highly sensitive system of germ cells.

[0004] Desaminotyrosine (DAT), also known as p-hydroxyphenylpropionic acid or 4-hydroxyphenylpropionic acid, is an important endogenous functional gut microbial metabolite with well-defined antioxidant, anti-inflammatory, and immunomodulatory activities. Patent searches reveal that no existing technologies utilize desaminotyrosine in in vitro oocyte maturation culture systems, particularly for improving the quality of aging oocytes. Summary of the Invention

[0005] To address the issues of limited mechanisms and low safety in related technologies, this invention first provides the application of deaminotyrosine in the preparation of reagents or drugs that improve the quality of aging oocytes, and briefly introduces its effects.

[0006] The inventors discovered that the activity of the ERK signaling pathway in aging oocytes is typically downregulated or disordered, and that deaminotyrosine can specifically enhance the activity of the ERK signaling pathway in aging oocytes. Based on this discovery, the inventors started from the logic chain of activating or repairing the ERK signaling pathway, and verified the effect of deaminotyrosine on improving quality by targeting the downstream direct manifestations of ERK phosphorylation upregulation, ERK signaling pathway regulation of the cytoskeleton, and the optimization of mitochondrial dynamics and cytoplasmic maturation caused by enhanced ERK pathway activity, namely oocyte maturation rate, spindle morphology and distribution, mitochondrial homogenization and membrane potential enhancement, and endogenous reduction of reactive oxygen species levels. Furthermore, the downstream effects were combined with the improvement in in vitro fertilization embryo development rate to confirm the practical effect and application value of deaminotyrosine in improving the developmental potential of aging oocytes.

[0007] The specific technical solution is as follows: According to one aspect of the present invention, the use of deaminotyrosine in the preparation of reagents or drugs for improving the quality of aging oocytes is first disclosed.

[0008] Furthermore, the improvement in the quality of aging oocytes includes at least one of the following: increasing oocyte maturation rate, reducing the proportion of oocyte spindle morphological abnormalities, regulating the transformation of mitochondria towards uniform distribution, increasing mitochondrial membrane potential, and reducing ROS levels.

[0009] Furthermore, the deaminotyrosine improves cell cycle regulation disorders or cytoplasmic maturation defects caused by oocyte aging by increasing the activity of the ERK signaling pathway in oocytes.

[0010] According to another aspect of the invention, an in vitro culture reagent for improving the quality of aging oocytes is provided, the in vitro culture reagent comprising an effective dose of an active ingredient; said active ingredient is deaminotyrosine or a pharmaceutically acceptable salt thereof.

[0011] Furthermore, the concentration of the deaminotyrosine is 0.1-10 mM.

[0012] In some embodiments, the preferred concentration of the deaminotyrosine is 1 mM.

[0013] Finally, the present invention also provides a pharmaceutical composition for improving the quality of aging oocytes, the pharmaceutical composition comprising a therapeutically effective amount of an active ingredient; the active ingredient being deaminotyrosine or a pharmaceutically acceptable salt thereof.

[0014] Furthermore, the pharmaceutical composition is an oral preparation, an injection, or a topical preparation for assisted reproduction, used to improve the quality of oocytes in older women or patients with diminished ovarian reserve.

[0015] Compared with related technologies, the present invention has at least the following beneficial effects: I. High safety: As an endogenous and symbiotic microbial metabolite in the human body, DAT has better biocompatibility and clinical safety than synthetic drugs, and can meet the safety requirements for long-term use.

[0016] II. Novel Uses of Deaminotyrosine: This invention discloses for the first time a novel use of deaminotyrosine in improving aging oocytes. Unlike related technologies that simply scavenge free radicals, this invention enhances the activity of the ERK signaling pathway through DAT, actively regulating repair signals and verifying its characterization.

[0017] III. Comprehensive and Deep Improvement: DAT can significantly increase the proportion of normal spindle morphology in aging oocytes, improve the uniform distribution rate of mitochondria, and increase mitochondrial membrane potential while reducing reactive oxygen species levels and improving oxidative stress. This results in increased maturation rate of aging oocytes and development rate of in vitro fertilized blastocysts, stimulating their developmental potential and effectively improving oocyte aging from multiple aspects. Attached Figure Description

[0018] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0019] Figure 1 The graphs show the optimal concentrations of DAT and galactose in the culture medium, where A represents the optimal concentration of galactose in the culture medium and B represents the optimal concentration of DAT in the culture medium.

[0020] Figure 2 A schematic diagram illustrating how DAT improves abnormal spindle morphology in aging oocytes, where A is a representative fluorescence image of spindle morphology; and B is a statistical graph of spindle morphology.

[0021] Figure 3 A schematic diagram illustrating how DAT improves the mitochondrial distribution pattern in aging oocytes, where A is a representative diagram of mitochondrial distribution; and B is a statistical diagram of mitochondrial distribution patterns.

[0022] Figure 4 A schematic diagram illustrating the effect of DAT on mitochondrial membrane potential in aging oocytes, where A is a representative image stained with JC-1; and B is a statistical graph of mitochondrial membrane potential fluorescence intensity.

[0023] Figure 5 A schematic diagram illustrating how DAT reduces ROS levels in aging oocytes, where A is a representative image of ROS fluorescence; and B is a statistical graph of ROS fluorescence intensity.

[0024] Figure 6DAT activates the ERK signaling pathway in aging oocytes; where A is an immunoblot map; B is a statistical graph of the relative expression levels of p-Erk1 / 2 protein; and C is a statistical graph of the relative expression levels of Erk1 / 2 protein.

[0025] Figure 7 Schematic diagram of DAT improving the maturation rate of aging oocytes and the development rate of blastocysts after in vitro fertilization. A is a statistical graph of in vitro maturation rate; B is a bright field graph of oocytes in the MII stage; C is a statistical graph of blastocyst development rate after in vitro fertilization; and D is a bright field graph of blastocysts. Detailed Implementation

[0026] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0027] It should be noted that the terms "comprising" and "having" and any variations thereof in the specification, claims and accompanying drawings of this invention are intended to cover non-exclusive inclusion, such as a process, method, system, product or device that includes a series of steps or units, which is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products or devices.

[0028] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0029] Example 1: Establishment of a galactose-induced oocyte aging model and screening of DAT concentration 8. Oocyte collection: 6-8 week old female SPF grade ICR mice were used. The mice were injected intraperitoneally with 10 IU of pregnant mare serum gonadotropin. After 48 hours, the female mice were sacrificed and the ovaries were removed. The cumulus-oocyte complex (the cumulus cells were densely packed and the cytoplasm was uniform and transparent) was extracted from the follicles by needle puncture under a stereomicroscope for later use.

[0030] 9. Induction of aging model: A galactose concentration gradient was set up from low to high: 0, 10, 20, 30, 40, 50 and 100 mM were added to M16 medium. Then, cumulus-oocyte complexes were sequentially inoculated into the above galactose-containing M16 medium and cultured at 37℃, 5% CO2 and saturated humidity for 12-14 h to simulate the natural aging process of oocytes in vivo. The results were observed.

[0031] Experimental results are as follows Figure 1 As shown in Figure A, when the galactose concentration is 30 mM, the oocyte maturation rate is significantly reduced; when the galactose concentration is higher than 30 mM, oocyte fragmentation and death are significantly increased. It can be seen that 30 mM galactose has the best effect on inducing oocyte aging. Therefore, 30 mM was selected as the effective galactose concentration for the subsequent aging model construction.

[0032] It should be noted that the formula for M16 medium is as follows: calcium chloride dihydrate 0.25137 g / L, magnesium sulfate heptahydrate 0.293 g / L, potassium chloride 0.35635 g / L, potassium dihydrogen phosphate 0.162 g / L, sodium bicarbonate 2.101 g / L, sodium chloride 5.53193 g / L, bovine serum albumin 4 g / L, D-glucose 1 g / L, phenol red 0.0106 g / L, sodium pyruvate 0.0363 g / L, sodium lactate 2.95 g / L, and gentamicin 0.05 g / L.

[0033] 10. DAT concentration gradient selection: Set DAT concentration gradients of 0, 0.2, 0.5, 1, 5 and 10 mM and add them to M16 medium containing 30 mM galactose. Then, inoculate cumulus-oocyte complexes into the above medium sequentially and culture them at 37℃, 5% CO2 and saturated humidity for 12-14 h and observe the results.

[0034] Experimental results are as follows Figure 1 As shown in Figure B, the maturation rate of aged oocytes remained almost unchanged in the range of low DAT concentrations (0-0.5 mM), but the maturation rate increased significantly when the DAT concentration was 1 mM. It can be seen that adding 1 mM DAT to the culture system is the optimal concentration to improve the maturation rate of aged oocytes, and this concentration was used in subsequent experiments.

[0035] Example 2: DAT improves abnormal spindle morphology in aging oocytes 11. Grouping and treatment: The collected mouse cumulus-oocyte complexes were randomly divided into 3 groups and inoculated into the following 3 culture media: control group (M16 medium); model group (D-Gal, M16 medium induced by 30 mM galactose); experimental group (D-Gal+DAT, M16 medium induced by 30 mM galactose supplemented with 1 mM DAT) and cultured at 37℃, 5% CO2 and saturated humidity for 12-14 h.

[0036] 12. Fluorescent staining: After collecting and fixing oocytes cultured to the MII stage and washing them with PBS, dilute Tubulin-Tracker Green (Beyotime, C1051S) 1:50 according to the instructions and add it to the cell sample. Incubate at room temperature (25℃) for 60 min. After incubation, transfer the oocytes to DAPI staining solution and stain in the dark for 10 min. Observe the spindle morphology of the oocytes using a laser confocal microscope.

[0037] Experimental results are as follows Figure 2 As shown, abnormal spindle morphology increased significantly in aging oocytes induced by galactose. Since the ERK pathway regulates spindle assembly, the addition of DAT significantly increased the proportion of normal spindle morphology, which is a downstream manifestation of ERK signaling pathway activation. This also confirms the activation and repair function of DAT on ERK pathway activity.

[0038] Example 3: DAT improves mitochondrial distribution patterns in aging oocytes 13. Grouping: The collected mouse cumulus-oocyte complexes were randomly divided into 3 groups and inoculated into the following 3 culture media: control group (M16 medium); model group (D-Gal, M16 medium induced by 30 mM galactose); experimental group (D-Gal+DAT, M16 medium induced by 30 mM galactose supplemented with 1 mM DAT) and cultured at 37℃, 5% CO2 and saturated humidity for 12-14 h.

[0039] 14. Fluorescent Staining: Following the manufacturer's instructions, dilute 200 μM Mito-Tracker Red CMXRos stock solution (Beyotime, C1035) 1:1000 with M16 medium to prepare Mito-Tracker Red CMXRos working solution, and pre-incubate at 37°C. Collect oocytes cultured to the MII stage, remove the cell culture medium, add Mito-Tracker Red CMXRos working solution, incubate at 37°C for 30 min, remove the Mito-Tracker Red CMXRos working solution, and observe mitochondrial distribution using a laser confocal microscope.

[0040] Experimental results are as follows Figure 3 As shown, mitochondria in the model group were mostly abnormally non-uniformly distributed. After the addition of DAT, the mitochondria in the experimental group were restored to a healthy and uniform distribution (the proportion of nonuniform distribution was significantly reduced and the proportion of dyffused was significantly increased). It can be seen that the mitochondrial dysfunction caused by oocyte aging was effectively reversed by DAT.

[0041] Example 4: DAT increases mitochondrial membrane potential in aging oocytes 15. Grouping: The collected mouse cumulus-oocyte complexes were randomly divided into 3 groups and inoculated into the following 3 culture media: control group (M16 medium); model group (D-Gal, M16 medium induced by 30 mM galactose); experimental group (D-Gal+DAT, M16 medium induced by 30 mM galactose supplemented with 1 mM DAT) and cultured at 37℃, 5% CO2 and saturated humidity for 12-14 h.

[0042] 16. Fluorescent Staining: According to the instructions, take an appropriate amount of JC-1 (200X) (Beyotime, C2003S) and dilute JC-1 with 1 mL of JC-1 staining buffer per 5 µL of JC-1 (200X) to obtain the JC-1 staining working solution. Collect oocytes cultured to the MII stage, resuspend them in 0.5 mL of M16 medium, add 0.5 mL of JC-1 staining working solution, mix by inverting several times, and incubate at 37℃ for 30 min. Observe the mitochondrial membrane potential using a laser confocal microscope.

[0043] Experimental results are as follows Figure 4 As shown, the green fluorescence in the model group was significantly enhanced, and the mitochondrial membrane potential decreased. At this time, the JC-1 red / green ratio was at a low level, and the oocytes were in the early stage of apoptosis. After adding DAT, the green fluorescence intensity in the experimental group decreased, and the JC-1 red / green ratio significantly increased, returning to the control group level. At this time, the red fluorescence intensity increased, the mitochondrial membrane potential returned to normal, and the oocyte state returned to normal.

[0044] Example 5: DAT reduces ROS levels in aging oocytes 17. Grouping and treatment: The collected mouse cumulus-oocyte complexes were randomly divided into 3 groups and inoculated into the following 3 culture media: control group (M16 medium); model group (D-Gal, M16 medium induced by 30 mM galactose); experimental group (D-Gal+DAT, M16 medium induced by 30 mM galactose supplemented with 1 mM DAT) and cultured at 37℃, 5% CO2 and saturated humidity for 12-14 h.

[0045] 18. Fluorescent staining: Dilute DCFH-DA (Beyotime, S0033S) 1:1000 with PBS solution according to the manufacturer's instructions to a final concentration of 10 μM / L. Add the collected MII stage oocytes to the diluted DCFH-DA, incubate at 37°C for 30 min, wash with PBS solution, and observe the reactive oxygen species level using a fluorescence microscope.

[0046] Experimental results are as follows Figure 5As shown, the fluorescence intensity of the model group was significantly enhanced, and the ROS activity was significantly increased. After the addition of DAT to the experimental group, the ROS activity level was reduced. This indicates that the ROS production mechanism was effectively inhibited after the mitochondrial function was repaired, which is a manifestation of the effect of DAT improving the upstream pathway.

[0047] Example 6: DAT activates the ERK signaling pathway in aging oocytes 1. Grouping and treatment: The collected mouse cumulus-oocyte complexes were randomly divided into 3 groups and inoculated into the following 3 culture media: control group (M16 medium); model group (D-Gal, M16 medium induced by 30 mM galactose); experimental group (D-Gal+DAT, M16 medium induced by 30 mM galactose supplemented with 1 mM DAT). They were cultured at 37℃, 5% CO2 and saturated humidity for 12-14 h, and oocytes in the MII stage were collected.

[0048] 2. Protein sample preparation: MII oocytes were added to RIPA lysis buffer containing protease phosphatase inhibitor and lysed on ice for 20 minutes. Then, 5×SDS protein sample buffer was added, mixed well, and heated in a 100℃ water bath for 5 minutes. The protein samples were stored at -80℃ for later use.

[0049] 3. Western Blot Validation: After electrophoresis, transfer, and blocking, the protein samples were diluted 1:500 with antibody dilution buffer according to the antibody instructions for p-Erk (Cell Signaling Technology, 4377) and Erk (Cell Signaling Technology, 4695). The PVDF membrane was then immersed in the diluted primary antibody and incubated overnight at 4°C. The secondary antibody (Proteintech, SA00001-2) was diluted 1:500 with antibody dilution buffer according to the antibody instructions for PVDF. After washing the PVDF membrane three times with TBST wash buffer, it was incubated in the diluted secondary antibody at room temperature (25°C) for 2 hours. ECL chemiluminescence buffer was prepared, and protein bands were detected using a chemiluminescence imaging system.

[0050] Experimental results are as follows Figure 6 As shown, the phosphorylation level of Erk in the model group was reduced, while the phosphorylation level of Erk in the experimental group was significantly increased after the addition of DAT, which directly proved the activation effect of DAT on the ERK signaling pathway at the cellular level.

[0051] Example 7: DAT improves the maturation rate of aging oocytes and the development rate of in vitro fertilized blastocysts. 19. Grouping: The collected mouse cumulus-oocyte complexes were randomly divided into 3 groups and inoculated into the following 3 culture media: control group (M16 medium); model group (D-Gal, M16 medium induced by 30 mM galactose); experimental group (D-Gal+DAT, M16 medium induced by 30 mM galactose supplemented with 1 mM DAT). They were cultured at 37℃, 5% CO2 and saturated humidity for 12-14 h. The number of oocytes extruding the first polar body was counted. The number of oocytes extruding the first polar body / the total number of oocytes was the maturation rate.

[0052] 20. After grouping and culturing oocytes for 12-14 h, co-culture them with capacitated sperm for 6 h, then transfer the fertilized eggs into preheated CZB culture medium in a 37℃ incubator. Observe the blastocyst formation at E4 after fertilization, count the number of blastocysts, and the blastocyst rate is calculated as the number of blastocysts / total number of embryos.

[0053] Experimental results are as follows Figure 7 As shown, by Figure 7 As shown in Figure A, the first polar body (PB1) expulsion rate of oocytes in the control group remained at a high level, while the PB1 expulsion rate in the model group was significantly reduced, indicating cell cycle arrest in aging oocytes. Galactose successfully induced the aging model. The significant increase in PB1 expulsion rate in the experimental group after the addition of DAT indicates that DAT broke the meiotic arrest caused by oocyte aging, improved the spindle assembly dynamics and chromosome alignment stability in aging cells by activating the ERK signaling pathway, and confirmed the repair effect of DAT on the cytoskeleton system, overcoming the limitation of traditional antioxidants that can only maintain cell survival. Figure 7 C represents the statistical graph of blastocyst development rate in in vitro fertilization of aged oocytes. It is evident that the application of DAT significantly increased the blastocyst development rate in the experimental group compared to the model group, improving endogenous material reserves and organelle homeostasis in aged oocytes by activating the ERK pathway. This improvement can continuously influence the cleavage and blastocyst formation stages after fertilization, giving DAT promising prospects for clinical application in assisted reproduction.

[0054] The above description represents the preferred embodiments of the present invention. It should be noted that all reagents and materials used in the embodiments of the present invention, unless otherwise specified, are standardized products that can be obtained through conventional commercial channels. However, it should be understood that those skilled in the art may choose equivalent products from other suppliers, which does not depart from the scope of protection of the present invention. For those skilled in the art, several improvements and modifications can be made without departing from the principles described in the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of deaminotyrosine in the preparation of reagents or drugs to improve the quality of aging oocytes.

2. The application according to claim 1, characterized in that, The improvement in the quality of aging oocytes includes at least one of the following: reducing the proportion of oocyte spindle morphological abnormalities, regulating the transformation of mitochondria towards uniform distribution, increasing mitochondrial membrane potential, reducing ROS levels, increasing oocyte maturation rate, and increasing in vitro fertilization blastocyst development rate.

3. The application according to claim 1, characterized in that, The deaminotyrosine improves cell cycle regulation disorders or cytoplasmic maturation defects caused by oocyte aging by increasing the activity of the ERK signaling pathway in oocytes.

4. An in vitro culture reagent for improving the quality of aging oocytes, characterized in that, The in vitro culture reagent contains an effective dose of the active ingredient; the active ingredient is deaminotyrosine or a pharmaceutically acceptable salt thereof.

5. The in vitro culture reagent according to claim 4, characterized in that, The concentration range of the deaminotyrosine is 0.1-10 mM.

6. The in vitro culture reagent according to claim 5, characterized in that, The concentration of the deaminotyrosine is 1 mM.

7. A pharmaceutical composition for improving the quality of aging oocytes, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of an active ingredient; the active ingredient is deaminotyrosine or a pharmaceutically acceptable salt thereof.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is an oral preparation, an injection, or a topical preparation for assisted reproduction, used to improve the quality of oocytes in elderly women or patients with diminished ovarian reserve.

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