Use of zeaxanthin in the preparation of products for improving the in vitro maturation deficiency of oocytes caused by diabetes
By preparing a culture medium that mixes zeaxanthin with oocyte basal culture medium, the in vitro maturation defects of oocytes caused by diabetes were resolved, the maturation rate and quality of oocytes were improved, and the abnormality rate was reduced, which has important clinical application value.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANTONG UNIV
- Filing Date
- 2026-02-12
- Publication Date
- 2026-05-29
AI Technical Summary
Current technologies have failed to effectively address the problem of oocyte maturation defects caused by diabetes, leading to reproductive dysfunctions such as infertility and miscarriage.
A culture medium was prepared by mixing zeaxanthin with oocyte basal culture medium to improve oocyte in vitro maturation defects caused by diabetes. The specific concentration was 0.5 μM. The improvement was achieved by increasing the first polar body extrusion rate, reducing the rate of spindle and chromosome alignment abnormalities, and reducing oxidative stress and early apoptosis rate.
It significantly improved the in vitro maturation rate of diabetic oocytes, improved spindle and chromosome alignment abnormalities, reduced oxidative stress and early apoptosis rates, and comprehensively enhanced the developmental potential of oocytes.
Smart Images
Figure CN122104562A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biomedical technology, and in particular to the application of zeaxanthin in the preparation of products that improve oocyte maturation defects caused by diabetes. Background Technology
[0002] In recent years, diabetes has become a global health problem. Type 1 diabetes is an autoimmune disease primarily characterized by the destruction of pancreatic beta cells, leading to an absolute deficiency in insulin secretion. Women with diabetes often experience numerous reproductive problems, such as infertility, miscarriage, and congenital malformations affecting their offspring. Maternal diabetes can cause abnormal spindle formation and chromosomal abnormalities in preovulatory oocytes, resulting in aneuploid oocytes, and can also lead to abnormal cell metabolism, increased oxidative stress, and defects in meiosis.
[0003] Currently, there is no effective treatment specifically for reproductive dysfunction caused by diabetes, nor are there any targeted methods to improve the problem of in vitro maturation defects of oocytes caused by diabetes. Summary of the Invention
[0004] The purpose of this invention is to improve the in vitro maturation defects of oocytes caused by diabetes. Zeaxanthin can significantly increase the first polar body extrusion rate of oocytes in diabetic mice during in vitro maturation, reduce the rate of spindle and chromosome alignment abnormalities, reduce oxidative stress levels and early apoptosis in oocytes, and improve in vitro maturation defects of oocytes.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] Application of zeaxanthin in the preparation of products that improve oocyte maturation defects caused by diabetes.
[0007] Preferably, the product is a culture medium, a culture additive, or a kit.
[0008] This application also provides a culture medium composed of zeaxanthin and oocyte basal culture medium, which is used to improve oocyte in vitro maturation defects caused by diabetes.
[0009] Preferably, the culture medium is prepared as follows:
[0010] S1: Dissolution of zeaxanthin: Weigh out zeaxanthin and dissolve it in an organic solvent to prepare a stock solution.
[0011] S2: Mixing zeaxanthin with culture medium: Dilute the stock solution in the oocyte basal culture medium, mix thoroughly to obtain the culture medium.
[0012] 5. A culture medium according to claim 4, wherein the organic solvent in S1 is dimethyl sulfoxide.
[0013] Preferably, the basal culture medium for oocytes in S2 is M2 culture medium.
[0014] Preferably, the concentration of zeaxanthin in the culture medium is 0.5 μM.
[0015] This application also provides the use of the culture medium described above in products that promote the in vitro maturation of diabetic oocytes.
[0016] Preferably, the culture medium can also be used in products that reduce oxidative stress levels and early apoptosis rates in diabetic oocytes.
[0017] Compared with the prior art, this application has at least the following beneficial effects.
[0018] 1. This invention is the first to discover and confirm that zeaxanthin has a significant effect on improving oocyte maturation defects caused by diabetes, which not only expands the application field of zeaxanthin, but also fills the relevant research gap in this field.
[0019] 2. The special culture medium of the present invention has the effect of improving oocyte quality in multiple dimensions. It can not only improve the in vitro maturation rate of diabetic oocytes, but also improve multiple key aspects such as oxidative stress, cell apoptosis, and meiotic structural stability, thereby comprehensively enhancing the developmental potential of oocytes.
[0020] 3. The special culture medium of the present invention has a good foundation for industrialization and application. Its components are clear, the preparation method is simple, and the cost can be effectively controlled. Moreover, zeaxanthin is a natural compound with high biological safety, which is easy to realize industrial production. It can be directly applied to the oocyte culture process related to assisted reproduction and in vitro fertilization, and has important translational value for the clinical treatment of infertility in diabetic women.
[0021] 4. The research conclusions of this invention have a solid scientific basis. Rigorous animal experiments were conducted using a streptozotocin-induced type 1 diabetic mouse model. Combined with cell culture and immunofluorescence staining and other detection techniques, the actual effects of zeaxanthin and special culture medium were systematically verified. The experimental design was rigorous, and the results obtained were true and reliable, providing strong scientific support for subsequent practical applications. Attached Figure Description
[0022] Figure 1The figure shows the effect of zeaxanthin on the maturation rate of in vitro mature oocytes in diabetic mice, as provided by this invention. In this figure, A is a representative microscopic image of in vitro mature oocytes from the control group, the diabetic group, and the zeaxanthin-enhanced group (Dia+ZEA). Oocytes marked with a yellow asterisk are mature oocytes that have expelled the first polar body. B is a bar chart showing the statistical histogram of the maturation rate of oocytes in the MII stage in the three groups.
[0023] Figure 2 The present invention provides the effects of zeaxanthin on the spindle and chromosomes of mature oocytes in diabetic mice during the MI stage; wherein, A is a representative immunofluorescence staining image of the spindle (Tubulin) and chromosome (DNA) of three groups of oocytes during the MI stage; B is a statistical bar chart of the abnormality rate of the three groups of spindles; C is a statistical bar chart of the abnormality rate of chromosome arrangement;
[0024] Figure 3 The effect of zeaxanthin on mitochondrial distribution in oocytes during the MI phase of diabetic mice provided by this invention; wherein, A is a typical immunofluorescence map of mitochondrial distribution in three groups of oocytes; B is a statistical bar chart of abnormal mitochondrial distribution in three groups;
[0025] Figure 4 This invention illustrates the effect of zeaxanthin, which enhances the effects of ROS content and early apoptosis in in vitro mature oocytes of diabetic mice. A represents representative images of ROS fluorescence detection in three groups of in vitro mature oocytes; B is a statistical graph of ROS fluorescence intensity in three groups of in vitro mature oocytes; C represents representative images of Annexin V fluorescence detection in three groups of in vitro mature oocytes; and D is a statistical graph of Annexin V fluorescence intensity in three groups of in vitro mature oocytes.
[0026] Figure 5 This is the chemical structural formula of zeaxanthin in this application. Detailed Implementation
[0027] This application provides the use of zeaxanthin in the preparation of products that improve in vitro oocyte maturation defects caused by diabetes, wherein the chemical formula of the zeaxanthin is as follows: Figure 5 As shown.
[0028] Zeaxanthin is a lutein-like substance among oxygenated carotenoids. It is a dihydroxy derivative of β-carotene and an isomer of lutein. Its molecular formula is C0. 40 H 56 O2, with a molecular weight of approximately 568.87, is a pure orange-red crystal. It is highly lipophilic but insoluble in water.
[0029] This application also provides a culture medium composed of zeaxanthin and oocyte basal medium. Preferably, the oocyte basal medium is M2 medium, and the molar concentration of zeaxanthin is 0.5 μM.
[0030] The method for preparing the culture medium is as follows:
[0031] S1: Dissolution of zeaxanthin: Weigh out zeaxanthin and dissolve it in an organic solvent to prepare a stock solution. Preferably, the organic solvent is dimethyl sulfoxide.
[0032] S2: Mixing zeaxanthin with culture medium: Add the stock solution to the oocyte basal culture medium and dilute to a final concentration of zeaxanthin of 0.5 μM.
[0033] This application also provides the application of the above-mentioned culture medium in products that promote the in vitro maturation of diabetic oocytes, and the culture medium can also be used in products that reduce the oxidative stress level and early apoptosis rate of diabetic oocytes.
[0034] The above content will be explained in conjunction with specific verification experiments:
[0035] I. Experimental Materials and Sources
[0036] The main reagents and instruments used in this application include: citrate / sodium citrate buffer, streptozotocin, zeaxanthin, M2 medium, mineral oil, Tubulin antibody, reactive oxygen species (ROS) detection kit, MitoTracker Red, apoptosis detection kit, and fluorescence microscope; wherein: citrate / sodium citrate buffer was purchased from Shanghai Maclean Biotechnology Co., Ltd., China; streptozotocin was purchased from Beyotime Biotechnology Co., Ltd., China; zeaxanthin was purchased from MCE Biotechnology Co., Ltd., USA; M2 medium and mineral oil were purchased from Nanjing Aibei Biotechnology Co., Ltd., USA; Tubulin antibody was purchased from Sigma-Aldrich, USA; ROS detection kit was purchased from Nanjing Jiancheng Bioengineering Institute; MitoTracker Red was purchased from Thermo Fisher Scientific, USA; apoptosis detection kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; and fluorescence microscope was purchased from Zeiss, Germany.
[0037] II. Verification Experiment and Results:
[0038] Example 1: Establishment of a streptozotocin-induced mouse model of diabetes and in vitro culture of its oocytes
[0039] 1.1 Establishment of a streptozotocin-induced diabetic mouse model
[0040] Three-week-old female ICR mice were purchased from the Experimental Animal Center of Nantong University and housed in the SPF-grade animal facility of the center. The animals were housed in a constant temperature environment of 23±2℃ and humidity of 40±10%, with a 12-hour day-night cycle. The diet used was standard irradiated feed (AIN-93). Intervention protocol: Three-week-old female ICR mice were randomly divided into two groups (n=6 per group). Each group received a single injection of streptozotocin dissolved in sodium citrate buffer. The streptozotocin dose was 200 mg / kg, serving as the model group. The healthy control group received a single intraperitoneal injection of the same volume of sodium citrate buffer based on mouse weight. Four days later, fasting blood glucose (FBG) was measured using a Sinocare blood glucose meter. Mice with an FGB value of 16.7 mmol / L or higher were considered to have successfully established a type 1 diabetes mouse model and were used for subsequent studies 15 days later.
[0041] 1.2 Collection and in vitro culture of GV stage oocytes
[0042] Mice were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin (PMSG). 48 hours later, ovarian cumulus-oocyte complexes (COCs) were rapidly isolated in M2 culture medium. Then, hyaluronidase was used to digest the COCs in M2 culture medium to obtain gestational age (GV) stage oocytes. GV stage oocytes from the control and diabetic groups were placed in normal M2 culture medium, while GV stage oocytes from the zeaxanthin-enhanced group were placed in M2 culture medium containing 0.5 μM zeaxanthin. In vitro maturation was performed at 37°C in a 5% CO2 incubator.
[0043] 1.3 Observation and statistics of oocyte maturation:
[0044] After culturing oocytes in an incubator for 12-14 hours, the microscope field of view and pupil distance were adjusted to observe and select oocytes in the mature stage (MII stage), and the proportion of MII stage oocytes was counted.
[0045] The results are as follows Figure 1 As shown, zeaxanthin can improve the maturation rate of oocytes in diabetic mice. The third group, indicated by a yellow asterisk, had significantly more MII stage oocytes than the second group. Figure 1 A), and the statistical data after three repeated experiments also proved this point. Figure 1 B).
[0046] Example 2: Immunofluorescence staining technique
[0047] 2.1 Tubulin immunofluorescence staining method for oocytes:
[0048] After culturing oocytes in vitro for 8 hours, they were fixed with 4% paraformaldehyde for 30 min, then permeabilized with PBS containing 0.5% Triton X-100 for 20 min at room temperature. Oocytes were blocked with 1% bovine serum albumin for 1 h, and then incubated overnight with a 1:200 solution of α-Tubulin antibody with green fluorescence. The next day, after washing three times with washing buffer, they were stained with Hoechst 3334 for 10 min. Finally, the oocytes were fixed on slides and imaged under a microscope. All three groups were scanned under an upright fluorescence microscope with the same parameters and analyzed using ImageJ software.
[0049] The results are as follows Figure 2 As shown, compared with the control group, the rate of spindle morphological abnormalities (such as spindle breakage and asymmetry) and chromosome arrangement abnormalities (such as chromosome dispersion and uneven aggregation) in oocytes at the MI stage were significantly increased in the diabetic group; the above abnormalities were significantly reduced in the maize xanthine improvement group, and the spindle structure and chromosome arrangement were closer to those of the control group. Figure 2 A). After three repeated experiments, statistical data also proved that zeaxanthin can improve spindle assembly abnormalities caused by diabetes. Figure 2 B) and chromosome misalignment ( Figure 2 C).
[0050] 2.2 Staining methods for mitochondria in oocytes
[0051] Mitochondrial analysis of oocytes was performed using the Mito Tracker. The procedure was as follows: Oocytes developed to the MI stage were selected from the control group, diabetes group, and zeaxanthin-improved group. They were transferred into 0.5 μM staining drops, sealed with paraffin oil, and incubated at 37°C in the dark for 30 min. Then, they were washed three times with PBS solution supplemented with 0.1% PVP, stained with Hoechst 3334 for 10 min, and finally washed twice with PBS solution supplemented with 0.1% PVP. The samples were then observed and photographed under a fluorescence microscope. Fluorescence intensity was analyzed using Image-J software. After importing the images into Image-J software, the threshold was adjusted, parameters were set, and measurements were taken. The measured data were exported and plotted using Prism software.
[0052] The results are as follows Figure 3 As shown, compared with the control group, the MitoTracker fluorescence intensity of oocytes in the diabetic group was significantly reduced, and mitochondria clustered together; while the MitoTracker fluorescence intensity in the zeaxanthin-improved group was significantly increased compared with the diabetic group, and the mitochondrial distribution tended to be normal. Figure 3 A), and the fluorescence intensity statistics also proved that zeaxanthin can effectively improve diabetes-induced oocyte mitochondrial reduction (A). Figure 3 B).
[0053] 2.3 Methods for detecting ROS in oocytes:
[0054] ROS levels in oocytes were detected using a reactive oxygen species detection kit, following the kit's instructions. The procedure was as follows: Mature oocytes were selected from the control group, diabetes group, and zeaxanthin-improved group, transferred into DCFH-DA staining drops, sealed with paraffin oil, and incubated at 37°C in the dark for 30 min. After washing three times with PBS solution containing 0.1% PVP, the oocytes were observed and photographed under a fluorescence microscope. Fluorescence intensity was analyzed using Image-J software. Images were imported into Image-J software, thresholds were adjusted, parameters were set, and measurements were performed. The measured data were then exported and plotted using Prism software.
[0055] The results are as follows Figure 4 As shown, compared with the control group, the ROS intensity of in vitro mature oocytes from diabetic mice was significantly increased. In vitro culture of diabetic mouse oocytes in a medium supplemented with zeaxanthin significantly reduced their ROS levels. Figure 4 A), statistical results of fluorescence intensity showed that the level of oxidative stress in in vitro matured oocytes of diabetic mice was increased, and the culture medium supplemented with zeaxanthin could significantly reduce the level of oxidative stress in in vitro maturation. Figure 4 B).
[0056] 2.4 Methods for detecting early oocyte apoptosis:
[0057] Annexin V-FITC dye and the binding solution in the kit were mixed at a 1:10 ratio. After removing the zona pellucida from three groups of oocytes, they were transferred into the staining solution and incubated at 37°C in the dark for 30 min. After rinsing three times with the binding solution, the cells were observed and photographed under an upright fluorescence microscope. The fluorescence intensity was analyzed using Image-J software.
[0058] The results are as follows Figure 4 As shown, compared with the control group mice, the Annexin V level in in vitro matured oocytes of diabetic mice was significantly increased. In vitro culture of diabetic mouse oocytes in a medium supplemented with zeaxanthin significantly reduced their Annexin V levels. Figure 4 C), statistical results of fluorescence intensity indicated that early apoptosis occurred in in vitro mature oocytes of diabetic mice, and the culture medium supplemented with zeaxanthin significantly reduced oocyte apoptosis. Figure 4 D).
[0059] In summary, this application is the first to discover and demonstrate that zeaxanthin has a significant ameliorative effect on oocyte maturation defects caused by diabetes, expanding the application field of zeaxanthin and filling a research gap in this area. The specialized culture medium of this invention not only improves the in vitro maturation rate of diabetic oocytes but also improves oocyte quality from multiple key aspects such as oxidative stress, apoptosis, and meiotic structural stability, comprehensively enhancing their developmental potential. The specialized culture medium has clearly defined components, a simple preparation method, and controllable cost. Furthermore, zeaxanthin is a natural compound with high biosafety, making it easy to achieve industrial-scale production. It can be directly applied to oocyte culture processes related to assisted reproduction and in vitro fertilization, and has significant translational value for the clinical treatment of infertility in diabetic women. This invention systematically verifies the effects of zeaxanthin and the specialized culture medium through rigorous animal experiments, cell culture, and immunofluorescence staining techniques. The experimental design is rigorous, and the results are reliable, providing a solid scientific basis for its application.
Claims
1. Application of zeaxanthin in the preparation of products that improve oocyte maturation defects caused by diabetes.
2. The application of zeaxanthin according to claim 1 in the preparation of products that improve oocyte in vitro maturation defects caused by diabetes, characterized in that: The product is a culture medium, culture additive, or reagent kit.
3. A culture medium, characterized in that: The culture medium consists of zeaxanthin and oocyte basal culture medium, and is used to improve oocyte in vitro maturation defects caused by diabetes.
4. The culture medium according to claim 3, characterized in that: The culture medium is prepared as follows: S1: Dissolution of zeaxanthin: Weigh out zeaxanthin and dissolve it in an organic solvent to prepare a stock solution. S2: Mixing zeaxanthin with culture medium: Dilute the stock solution in the oocyte basal culture medium, mix thoroughly to obtain the culture medium.
5. The culture medium according to claim 4, characterized in that: The organic solvent in S1 is dimethyl sulfoxide.
6. The culture medium according to claim 5, characterized in that: The basal culture medium for oocytes in S2 is M2 medium.
7. The culture medium according to claim 6, characterized in that: The concentration of zeaxanthin in the culture medium was 0.5 μM.
8. The use of the culture medium as described in any one of claims 2-5 in a product for promoting the in vitro maturation of diabetic oocytes.
9. The application according to claim 8, characterized in that: The culture medium can also be used in products that reduce oxidative stress levels and early apoptosis rates in diabetic oocytes.