Application of wheat germ extract in the preparation of products that improve the quality of aging oocytes

Abstract

This invention belongs to the field of biomedicine and reproductive health technology, specifically relating to the application of wheat germ extract in the preparation of products that improve the quality of aging oocytes. Through in vitro culture and in vivo gavage models, this invention demonstrates for the first time that wheat germ extract can significantly improve the in vitro maturation rate and in vivo superovulation maturation rate of oocytes from aged mice, significantly reduce intracellular reactive oxygen species levels, inhibit early apoptosis, and reduce oocyte mortality. The optimal in vitro concentration of the wheat germ extract is 0.1%–0.2%, and the optimal gavage dose is 5%. At these concentrations, the improving effect of wheat germ extract is significantly superior to that of spermidine. This invention provides a safe, natural, and long-term-accessible reproductive aging intervention strategy with good scientific value and promising prospects for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and reproductive health technology, specifically relating to the application of wheat germ extract in the preparation of products that improve the quality of aging oocytes. Background Technology

[0002] As physiological age increases, the quality and developmental potential of oocytes in female individuals gradually decline. Aging oocytes exhibit adverse physiological changes such as decreased fertilization capacity, polyspermia, parthenogenesis, chromosomal abnormalities, apoptosis, extracellular secretion of some cortical granules (CG), zona pellucida sclerosis, mitochondrial damage, and production of reactive oxygen species (ROS). These changes not only affect the developmental quality of the oocyte itself but also further limit its ability to fertilize and develop into an embryo. Current research on improving age-related oocyte quality decline focuses primarily on chemical supplements or antioxidants, but the safety and edibility of their long-term use remain somewhat limited.

[0003] In the prior art, spermidine has been proven effective as an active ingredient for improving oocyte quality. However, those skilled in the art have not yet realized that wheat germ extract, as a mixture, enriches other synergistic components while retaining spermidine, and can produce technical effects that are significantly superior to those of an equal amount of spermidine. Summary of the Invention

[0004] The purpose of this invention is to provide a new application of wheat germ extract in the preparation of products that improve the quality of aging oocytes, and to provide a safe and effective natural solution for reproductive aging intervention.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: Application of wheat germ extract in the preparation of products that improve the quality of aging oocytes.

[0006] Furthermore, the product applications include both in vitro and in vivo applications.

[0007] Furthermore, in in vitro applications, the product is a culture medium additive.

[0008] Furthermore, the in vitro concentration of the culture medium additive is 0.1% to 0.2%.

[0009] Furthermore, in vivo applications, the product is a drug.

[0010] Furthermore, the drug is a solution dosage form that can be taken orally, by gavage, or by injection.

[0011] Furthermore, the concentration of wheat germ extract in the drug is 40-60 mg / mL.

[0012] Furthermore, the improvement in the quality of aging oocytes includes: (1) Improve the in vitro maturation rate of oocytes and promote the expulsion of the first polar body; (2) Reduces the accumulation of reactive oxygen species in oocytes and improves oxidative stress; (3) Reduce the level of early apoptosis in oocytes and decrease the eversion of phosphatidylserine in the cell membrane; (4) Increase the number of superovulatory oocytes in the body; (5) Reduce oocyte mortality.

[0013] The present invention has the following beneficial effects: This invention is the first to discover that wheat germ extract has a clear protective effect on senescent oocytes; it clarifies the optimal in vitro concentration (0.1%~0.2%) and the optimal in vivo intervention dose (5% by gavage), at which the protective effect of wheat germ extract on senescent oocytes is significantly stronger than that of spermidine; this invention provides a complete chain of in vitro and in vivo experimental evidence, including multiple dimensions such as maturation rate, ROS, apoptosis, and superovulation; wheat germ extract is naturally sourced, inexpensive, and safe for consumption, and has good prospects for clinical translation and industrialization. Attached Figure Description

[0014] Figure 1 Figure showing the results of wheat germ extract improving the in vitro maturation rate of oocytes in aged mice. Figure 2 Figure showing the results of wheat germ extract reducing ROS accumulation in in vitro cultured oocytes from aged mice. Figure 3 Figure showing the results of wheat germ extract reducing early apoptosis levels in in vitro cultured oocytes from aged mice. Figure 4 Figure showing the results of improving in vivo development of senescent oocytes through gavage supplementation with wheat germ extract; Figure 5 Figure showing the results of reducing ROS accumulation in oocytes of aged mice by gavage supplementation with wheat germ extract; Figure 6 The results of administering wheat germ extract via gavage to reduce the level of early apoptosis in senescent oocytes. Detailed Implementation

[0015] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0016] The wheat germ extract used in this embodiment of the invention was purchased from Shaanxi Panier Biotechnology Co., Ltd., batch number PB250313.

[0017] Example 1: Wheat germ extract improves the in vitro maturation rate of oocytes from aged mice. 1. Reagent preparation (1) Wheat germ extract concentrated stock solution: Weigh 1 g of wheat germ extract powder and dissolve it in 9 mL of M2 culture medium to prepare a concentrated stock solution of 100 mg / mL. Place the mixture in a shaker at 4℃ and shake continuously for 24 h, then centrifuge at 4500 rpm for 20 min, collect the supernatant and filter it through a 0.22 μm filter membrane. Aliquot the obtained filtrate into sterile cryovials and store at -20℃ for later use. Dilute with M2 culture medium to the required mass concentration (0.02%, 0.05%, 0.1% or 0.2%) before the experiment.

[0018] (2) Preparation of spermidine: Dissolve spermidine in Sigma ultrapure sterile water to prepare a 100 mM concentrated stock solution, and store at -20℃ for later use. Dilute to 50 μM with M2 culture medium before the experiment as a positive control.

[0019] 2. Oocyte collection Young (6-8 weeks old) and older (12-14 months old) female C57 mice were euthanized by cervical dislocation, and both ovaries were removed and placed in M2 operating medium pre-equilibrated in a CO2 incubator. The ovaries were chopped up with a sharp blade, and an appropriate amount of MEM culture medium was added and mixed well. Under a stereomicroscope, normal-shaped and sized GV-stage oocytes were picked up with a pipette.

[0020] 3. In vitro maturation culture Preheat the M2 culture medium at 37°C for 2-3 hours. Add three 50 μL drops of mature culture medium to a petri dish, cover with paraffin oil and seal, then place in an incubator containing 5% CO2 at 37°C for preheating and equilibration.

[0021] Oocytes were divided into six groups and transferred to equilibrated M2 culture drops. The culture medium consisted of: 50 μM spermidine (positive control), blank control (0), and treatment groups containing 0.02%, 0.05%, 0.1%, and 0.2% wheat germ extract, respectively. After 13 h of culture, the in vitro maturation of oocytes was assessed. The extrusion of the first polar body (PB1) was observed under a microscope, and the maturation rate was recorded.

[0022] 4. Results like Figure 1The results showed that the developmental capacity of oocytes from aged C57 mice was assessed under in vitro culture conditions. Compared with the young group, the extrusion rate of the first polar body (PB1) in aged oocytes was significantly reduced, exhibiting a clear phenotype of impaired maturation. As a positive control, the addition of 50 μM spermidine (SPD) significantly improved the maturation rate of aged oocytes. Further analysis of different concentrations of wheat germ extract (WGE) revealed that it had a certain restorative effect on the maturation of aged oocytes. The PB1 extrusion rate was significantly increased in the 0.1% and 0.2% WGE treatment groups, and the improvement effect was significantly stronger than that in the positive control spermidine group. These results suggest that WGE can promote the in vitro maturation process of aged oocytes to a certain extent.

[0023] Example 2: Wheat germ extract reduces ROS accumulation in in vitro cultured oocytes from aged mice. 1. Reactive oxygen species staining Using M2 culture medium as a diluent, ROS dye was diluted at a volume ratio of 1:800 to prepare ROS staining working solution. 50 μL of the staining working solution was added dropwise to a culture dish, covered with paraffin oil, and placed in a 37℃, 5% CO2 incubator for preheating and equilibration.

[0024] Mature oocytes from Example 1 (treated with 0.2% wheat germ extract) were transferred to staining drops and incubated at 37°C in a 5% CO2 incubator for 30 min. After staining, the cells were washed three times with M2 culture medium to remove unbound dye and ensure stable imaging background. After washing, the oocytes were transferred to live staining dishes for subsequent immunofluorescence signal detection.

[0025] 2. Image Acquisition and Analysis Place the staining dish under a laser confocal microscope and use confocal microscopy (LSM 900 META, Zeiss) to image and observe the prepared oocytes. Invert the slide containing the oocytes onto the stage of the confocal microscope, select appropriate eyepiece and objective magnification, and adjust the field of view using the coarse / fine adjustment knobs on both sides of the stage. Simultaneously, move the oocytes to the center of the appropriate field of view by adjusting the stage position. Finally, scan the mouse oocytes of each group using an appropriate laser channel and laser intensity. After confocal microscopy, label and save the images on the computer interface for later analysis and processing.

[0026] During the observation and recording process using laser confocal microscopy, appropriate values ​​were set for different parameters in the ZEN software according to the strength of the positive signal. To ensure the comparability of the results between the control and treatment groups, the same parameters were maintained when observing and recording different groups of samples. The obtained images were analyzed for fluorescence using ImageJ software. When measuring the fluorescence intensity in the cortical area, the Threshold color function module was used for analysis. Finally, statistical analysis was performed using GraphPad Prism 9 software.

[0027] 3. Results Age-related mitochondrial dysfunction can lead to the accumulation of excessive reactive oxygen species (ROS) in oocytes, thereby disrupting cytoplasmic redox homeostasis. To assess the effects of wheat germ extract on oxidative stress in aged oocytes, the ROS levels in each group were measured using the Beyotime reactive oxygen species assay kit.

[0028] like Figure 2 As shown, the fluorescence signal of oocytes in the young group was weak, indicating a low level of ROS content; the fluorescence signal in the older group was significantly enhanced, indicating a large accumulation of ROS. Treatment with 0.1%-0.2% wheat germ extract significantly reduced the green fluorescence intensity of older oocytes, indicating a significant alleviation of abnormal ROS accumulation, and the effect was significantly stronger than that of spermidine treatment. The results indicate that wheat germ extract can effectively reduce the excessive accumulation of ROS in older oocytes and improve their oxidative stress state.

[0029] Example 3: Wheat germ extract reduces early apoptosis levels in oocytes from aged mice in vitro. 1. Annexin-V staining Using the Binding Buffer provided with the kit as a diluent, the Annexin-V stock solution was diluted at a ratio of 1:10 to prepare the Annexin-V staining working solution. 50 μL of the staining working solution was added dropwise to a culture dish, covered with paraffin oil, and preheated to equilibration in a 37°C, 5% CO2 incubator.

[0030] Oocytes (the group treated with 0.2% wheat germ extract in Example 1) were transferred to staining drops and incubated for 30 min. After staining, the cells were washed and observed under a laser confocal microscope.

[0031] 2. Results Age-related mitochondrial damage and increased oxidative stress not only disrupt cytosol homeostasis but also induce early apoptosis-related events such as phospholipid eversion in oocyte membranes. To assess whether this age-related membrane damage can be improved by wheat germ extract, this study used Annexin-V staining to detect the phospholipid exposure levels in oocytes from different groups. Annexin-V specifically binds to phosphatidylserine (PS) exposed on the outer surface of the cell membrane, and its fluorescence signal intensity reflects the tendency of oocytes to undergo early apoptosis.

[0032] like Figure 3 The results showed that the Annexin-V fluorescence signal in young oocytes was weak, indicating intact cell membrane structure; the fluorescence signal in older oocytes was significantly enhanced, and the PS eversion level was significantly increased, indicating that aging exacerbates cell membrane damage and early apoptosis phenotype. After treatment with wheat germ extract, the Annexin-V fluorescence intensity of older oocytes decreased significantly, and the membrane eversion signal was restored to some extent. Quantitative fluorescence results further showed that wheat germ extract can effectively reduce the excessively elevated Annexin-V signal in older oocytes and alleviate their apoptosis-related characteristics.

[0033] Example 4: Gavage supplementation with wheat germ extract to improve in vivo development of senescent oocytes 1. Reagent preparation (1) Wheat germ extract gavage solution: Weigh an appropriate amount of wheat germ extract powder and prepare a concentrated stock solution of 100 mg / mL using Beyotime cell culture water as solvent. Shake at 4°C for 24 h. After aliquoting, store at -20°C. Dilute before administration to prepare gavage working solutions of 100 mg / mL (10%), 50 mg / mL (5%), and 25 mg / mL (2.5%).

[0034] (2) Superovulation hormones: Take one tube each of lyophilized human chorionic gonadotropin (HCG) and pregnant mare serum gonadotropin (PMSG) (each tube containing 1000 IU), add 10 mL of sterile physiological saline to dissolve them thoroughly, and prepare a stock solution with a final concentration of 100 IU / mL. Aliquot the prepared HCG and PMSG solutions into centrifuge tubes and store them at −20℃ for later use.

[0035] 2. Animal handling and over-discharge The experimental mice were divided into a young group, an old group, and a wheat germ extract gavage group (2.5%, 5%, and 10%). The young and old groups were gavaged with 0.2 mL of cell culture water daily; the treatment groups were gavaged with the corresponding concentration of wheat germ extract working solution for 10 consecutive days.

[0036] On the afternoon of day 8, mice were injected intraperitoneally with 10 IU PMSG, followed by 10 IU HCG 48 hours later. Mice were sacrificed 13-14 hours after the HCG injection to collect oocytes.

[0037] 3. Oocyte Collection and Evaluation Thirteen to fourteen hours after HCG injection, mice in the young, old, and wheat germ extract gavage groups were euthanized by cervical dislocation. The abdomens of the mice were disinfected with 75% alcohol. The abdominal skin and lining were opened with ophthalmic scissors to expose the ovaries and fallopian tubes. The ampulla was held in place with forceps, and the connection between the ovary, uterine horn, and both ends of the fallopian tube ampulla was severed to obtain the fallopian tube ampulla. The fallopian tube ampulla was placed in an M2 manipulation droplet containing 0.1% hyaluronidase. Under a stereomicroscope, the enlarged ampulla was torn open with pointed forceps, allowing contents containing a large number of cocci (COCs) to flow out. Subsequently, the cumulus cells and oocytes were separated. MII-stage oocytes were collected using an oral pipette and cleaned for subsequent experiments.

[0038] The ampulla of the oviduct was placed in preheated M2 manipulation solution. Under a microscope, the ampulla was gently pressed to expel the cumulus-oocyte complex (COCs). The COCs were then removed with M2 manipulation solution containing hyaluronidase to obtain naked oocytes. Morphology was observed under a stereomicroscope: oocytes that expelled the first polar body (PB1) and had normal morphology were classified as mature oocytes; oocytes that did not expel PB1 and had largely intact morphology were classified as immature oocytes; and oocytes with cytoplasmic shrinkage, darkening, or fragmentation were classified as dead oocytes. The total number of oocytes, the number of mature oocytes, and the number of dead oocytes were recorded for each mouse. The expulsion rate and mortality rate were calculated. Typical oocyte morphological images for each group were taken for results presentation.

[0039] 4. Results To verify whether wheat germ extract also improves the quality of senescent oocytes in vivo, this study further employed a gavage combined with superovulation protocol to evaluate the number of superovulated oocytes, the expulsion rate of the first polar body (PB1), and the mortality rate in different treatment groups. After gavage treatment, oocytes were collected, and the total number of superovulated oocytes, maturation rate, and mortality rate were recorded.

[0040] like Figure 4 The results showed that the number of superovulated oocytes and the PB1 expulsion rate were significantly lower in the aged group than in the younger group, and the mortality rate was significantly higher. The group receiving 5% wheat germ extract via gavage showed a significant increase in the number of superovulated oocytes, a marked improvement in the PB1 expulsion rate, and a significant decrease in mortality. No similar improvements were observed in other concentration groups. This indicates that gavage administration of an appropriate dose (5%) of wheat germ extract can effectively improve the in vivo developmental quality of oocytes in aged mice.

[0041] Example 5: Gavage supplementation with wheat germ extract reduced ROS accumulation in oocytes of aged mice Oocytes obtained from the 5% gavage group in Example 4 were subjected to ROS staining detection according to the method in Example 2.

[0042] Age-related metabolic disorders lead to increased reactive oxygen species (ROS) production in oocytes, exhibiting typical characteristics of oxidative stress. To further verify whether wheat germ extract can alleviate oxidative damage in aged oocytes under in vivo intervention conditions, this study stained oocytes obtained by gavage superovulation with ROS to detect changes in ROS content in different groups.

[0043] like Figure 5 The results showed that the green fluorescence signal of oocytes in the young group was weak, indicating that the ROS level remained in a low range; the fluorescence intensity of oocytes in the older group was significantly increased, indicating that their oxidative stress level was significantly increased. Notably, after treatment with 5% wheat germ extract by gavage, the fluorescence signal of older oocytes decreased significantly, and the ROS content decreased significantly, approaching the level of the young group.

[0044] The above results indicate that long-term gavage supplementation with wheat germ extract can effectively alleviate the excessive accumulation of ROS in senescent oocytes and improve their redox homeostasis, further supporting the significant antioxidant protective effect of WGE on senescent oocytes.

[0045] Example 6: Gavage supplementation with wheat germ extract reduced the level of early apoptosis in senescent oocytes. Oocytes obtained from the 5% gavage group in Example 4 were subjected to Annexin-V staining detection according to the method in Example 3.

[0046] During aging, oocytes often exhibit early apoptotic phenotypes such as increased phospholipid eversion of the cell membrane, leading to a decline in oocyte quality. To assess whether in vivo intervention with wheat germ extract can alleviate aging-related membrane damage, this study performed Annexin-V staining on oocytes obtained by gavage superovulation to detect phosphatidylserine (PS) exposure levels in different groups. Annexin-V specifically binds to everted PS, and its fluorescence signal intensity reflects the early apoptotic tendency of oocytes.

[0047] like Figure 6 The results showed that the Annexin-V fluorescence signal was weaker in the young group of oocytes, while the fluorescence intensity was significantly enhanced in the older group, and the proportion of PS eversion increased. After treatment with wheat germ extract by gavage, the Annexin-V fluorescence signal of older oocytes was significantly reduced, and the level of early apoptosis was significantly decreased.

[0048] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0049] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. Application of wheat germ extract in the preparation of products that improve the quality of aging oocytes.

2. The application according to claim 1, characterized in that, The product applications include both in vitro and in vivo applications.

3. The application according to claim 2, characterized in that, In in vitro applications, the product is a culture medium additive.

4. The application according to claim 3, characterized in that, The in vitro concentration of the culture medium additive is 0.1% to 0.2%.

5. The application according to claim 2, characterized in that, In vivo application, the product is a drug.

6. The application according to claim 5, characterized in that, The drug is a solution dosage form that can be taken orally, by gavage, or by injection.

7. The application according to claim 6, characterized in that, The concentration of wheat germ extract in the drug is 40-60 mg / mL.

8. The application according to claim 1, characterized in that, The improvement in the quality of aging oocytes includes: (1) Improve the in vitro maturation rate of oocytes and promote the expulsion of the first polar body; (2) Reduces the accumulation of reactive oxygen species in oocytes and improves oxidative stress; (3) Reduce the level of early apoptosis in oocytes and decrease the eversion of phosphatidylserine in the cell membrane; (4) Increase the number of superovulatory oocytes in the body; (5) Reduce oocyte mortality.

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