Culture solution for improving oocyte in-vitro maturation defect caused by diabetes and application of BGP-15 or culture solution

Abstract

The invention provides a culture solution for improving an oocyte in-vitro maturation defect caused by diabetes and application of BGP-15 or the culture solution, and particularly belongs to the technical field of oocyte culture. The invention provides an application of BGP-15 in preparation of a product for improving an oocyte in-vitro maturation defect caused by diabetes mellitus. The product further comprises an oocyte basal culture medium. BGP-15 is combined with an oocyte basic culture medium, so that the first polar body discharge rate of in-vitro mature oocytes of diabetic mice can be remarkably increased, the oxidative stress level of the oocytes is reduced, the mitochondrial membrane potential of the oocytes is recovered, and the survival rate of the oocytes is increased. The in-vitro maturation rate of the diabetic mouse oocytes is increased, the quality of the in-vitro mature oocytes of the diabetic mouse is improved, and the method has important guiding significance in treatment of diabetic female infertility.

Description

Technical Field

[0001] This invention belongs to the field of oocyte culture technology, specifically relating to a culture medium for improving oocyte in vitro maturation defects caused by diabetes and the application of BGP-15 or culture medium. Background Technology

[0002] In recent years, the incidence of diabetes has been rising globally. Type 1 diabetes can lead to serious complications and adversely affect patients' quality of life and mental health. Among these complications, reproductive dysfunction is a common one in diabetic women. Maternal diabetes negatively impacts oocyte maturation, preimplantation embryonic development, and pregnancy outcomes. Oocytes undergoing meiotic maturation are sensitive to the high-glycemic environment caused by diabetes, making them susceptible to damage during meiotic maturation, leading to decreased oocyte quality and consequently abnormal preimplantation embryonic development and pregnancy outcomes. Current research indicates that adding proanthocyanidins B2, nicotinamide mononucleotide (NMN), and tea polyphenols to the oocyte in vitro maturation culture medium can improve the in vitro maturation rate and quality of oocytes from diabetic mice. However, other products with highly effective methods to improve in vitro maturation defects in diabetic oocytes are still lacking. Summary of the Invention

[0003] The purpose of this invention is to provide a culture medium for improving oocyte maturation defects caused by diabetes and the application of BGP-15 or the culture medium. The combination of BGP-15 with oocyte basal culture medium can significantly increase the first polar body extrusion rate of oocytes maturated in vitro in diabetic mice, reduce the oxidative stress level of oocytes, restore the mitochondrial membrane potential of oocytes, and improve oocyte maturation defects.

[0004] This invention provides the application of BGP-15 in the preparation of products that improve oocyte in vitro maturation defects caused by diabetes; the products also include oocyte basal culture medium.

[0005] Preferably, the oocyte is derived from a mammal.

[0006] Preferably, the product type includes culture medium.

[0007] The present invention also provides a culture medium for improving oocyte in vitro maturation defects caused by diabetes, the culture medium being composed of BGP-15 and oocyte basal culture medium.

[0008] Preferably, the basal culture medium for oocytes includes M2 medium, M16 medium, DMEM medium, or TCM199 medium.

[0009] Preferably, the molar concentration of BGP-15 in the culture medium is 1 μM to 1 mM.

[0010] The present invention also provides a method for preparing the culture medium described in the above technical solution, comprising the following steps: mixing BGP-15 and oocyte basal culture medium.

[0011] This invention also provides the application of BGP-15 or the culture medium described in the above technical solutions or the culture medium prepared by the preparation method described in the above technical solutions in the preparation of products that promote the in vitro maturation of diabetic oocytes.

[0012] This invention also provides the application of BGP-15 or the culture medium described in the above technical solutions or the culture medium prepared by the preparation method described in the above technical solutions in the preparation of products that reduce the oxidative level of diabetic oocytes and / or improve the mitochondrial function of diabetic oocytes.

[0013] This invention also provides the application of BGP-15 or the culture medium described in the above-described technical solutions, or the culture medium prepared by the preparation method described in the above-described technical solutions, in any of the following: ① to ④ ①Prepare products that improve oocyte quality; ②Prepare products for assisted reproductive research in humans or non-human mammals; ③Prepare products for in vitro fertilization; ④ Prepare products for intracytoplasmic sperm injection.

[0014] This invention provides the application of BGP-15 in the preparation of products that improve oocyte in vitro maturation defects caused by diabetes; the product also includes an oocyte basal culture medium. The combination of BGP-15 and the oocyte basal culture medium can significantly improve the first polar body extrusion rate of oocytes maturing in diabetes mice, reduce the oxidative stress level of oocytes, restore the mitochondrial membrane potential of oocytes, improve the in vitro maturation rate of oocytes in diabetes mice, and improve the quality of in vitro matured oocytes in diabetes mice, which has important guiding significance for the treatment of infertility in diabetic women. This invention uses a commercially available oocyte maturation culture medium combined with BGP-15, resulting in a product with clearly defined components and easy quality control, which is of great significance for improving the in vitro maturation of oocytes in diabetic women and the development of reproductive medicine. Experimental results show that the culture medium provided by this invention promotes the maturation of the cell nucleus of oocytes in diabetic mice, helps to improve the first polar body extrusion rate of oocytes, reduces the accumulation of ROS in oocytes, restores the mitochondrial membrane potential of oocytes, and is beneficial to the maturation of oocyte cytoplasm. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The figure shows the effect of BGP-15 provided by this invention on the oocyte maturation rate of diabetic mice; where A is a representative image of three groups of in vitro matured oocytes; and B is a statistical graph of the in vitro maturation rate of the three groups of oocytes. Figure 2 The figure shows the effect of BGP-15 on ROS content in in vitro mature oocytes of diabetic mice provided by this invention; where A is a representative image of ROS fluorescence detection in three groups of in vitro mature oocytes; and B is a statistical graph of ROS fluorescence intensity in three groups of in vitro mature oocytes. Figure 3 The image shows the effect of BGP-15 on the mitochondrial membrane potential of in vitro mature oocytes from diabetic mice, provided by this invention. A represents representative images of mitochondrial membrane potential fluorescence detection in three groups of in vitro mature oocytes; B is a statistical graph of the fluorescence intensity of mitochondrial membrane potential in three groups of in vitro mature oocytes. Figure 4 The BGP-15 provided by this invention targets oxidation-related genes in in vitro matured oocytes of diabetic mice. Sod1 and Sod2 The effect of expression levels is shown in the figure; where A represents the three groups of oxidation-related genes in in vitro matured oocytes. Sod1 Statistical graph of expression levels; B represents three groups of oxidation-related genes in in vitro matured oocytes. Sod2 Statistical charts representing levels of expression. Detailed Implementation

[0017] This invention provides the application of BGP-15 in the preparation of products that improve oocyte in vitro maturation defects caused by diabetes; the product also includes an oocyte basal culture medium. In a specific embodiment, the BGP-15 is purchased from Selleck, catalog number S8370. In a specific embodiment, the oocytes are derived from mammals, i.e., from humans or any non-human mammals. In a specific embodiment, the non-human mammals include non-human mammal models. In a specific embodiment, the oocytes are GV stage oocytes. In a specific embodiment, the oocytes are derived from diabetic females or diabetic female animals, specifically from diabetic females or diabetic mouse models. In a specific embodiment, the oocyte basal culture medium includes M2 medium, M16 medium, DMEM medium, or TCM199 medium, specifically M16 medium. In a specific embodiment, the product type includes culture medium. The product provided by this invention can significantly promote the in vitro maturation of oocytes from diabetic mice, reduce the oxidative level of in vitro matured oocytes, improve mitochondrial function of oocytes, reduce in vitro maturation defects caused by diabetes, and improve oocyte quality. On the one hand, the improved quality oocytes obtained can be used for experimental research on oocyte development. On the other hand, the improved quality oocytes obtained can be used in the field of assisted reproduction in humans or non-human mammals, such as using the obtained high-quality oocytes for in vitro fertilization or intracytoplasmic sperm injection to obtain high-quality embryos.

[0018] This invention also provides a culture medium for improving oocyte in vitro maturation defects caused by diabetes, the culture medium being composed of BGP-15 and oocyte basal culture medium. In a specific embodiment, the oocyte basal culture medium includes M2 medium, M16 medium, DMEM medium, or TCM199 medium, specifically M16 medium. This invention does not specifically limit the source of the oocyte basal culture medium; conventional commercially available oocyte basal culture media are acceptable, such as M16 medium purchased from Sigma (catalog number M7292); and M2 medium purchased from Sigma (catalog number M7167). In a specific embodiment, the molar concentration of BGP-15 in the culture medium is 1 μM to 1 mM. The culture medium of this invention can be used for oocyte in vitro maturation culture, improving oocyte in vitro maturation defects caused by diabetes. The culture medium of this invention can restore mitochondrial function impairment in oocytes caused by diabetes and improve oocyte quality, which is of great significance for female diabetic patients who wish to conceive but experience difficulty ovulating.

[0019] This invention also provides a method for preparing the culture medium described in the above technical solution, comprising the following steps: mixing BGP-15 and oocyte basal culture medium. In a specific embodiment, the oocyte basal culture medium includes M2 medium, M16 medium, DMEM medium, or TCM199 medium, specifically M16 medium. In a specific embodiment, the preparation method includes mixing concentrated BGP-15 stock solution and oocyte basal culture medium to prepare a culture medium with a BGP-15 concentration of 1 μM to 1 mM. In a specific embodiment, the concentration of BGP-15 in the concentrated BGP-15 stock solution is 500 μM to 100 mM. The preparation method of this invention is simple and can prepare a culture medium that improves the in vitro maturation of oocytes from diabetic patients.

[0020] This invention also provides the application of BGP-15 or the culture medium described in the above-described technical solutions, or the culture medium prepared by the preparation method described in the above-described technical solutions, in the preparation of products that promote the in vitro maturation of diabetic oocytes. Experimental results show that BGP-15 can significantly increase the proportion of MII stage oocytes after in vitro culture of diabetic mouse oocytes.

[0021] This invention also provides the application of BGP-15 or the culture medium described in the above-described technical solutions, or the culture medium prepared by the preparation method described in the above-described technical solutions, in the preparation of products that reduce the oxidative level of diabetic oocytes and / or improve the mitochondrial function of diabetic oocytes. Experimental results show that, compared with normal mice, the ROS intensity of in vitro matured oocytes from diabetic mice is significantly increased. In vitro culture of diabetic mouse oocytes in a medium supplemented with BGP-15 can significantly reduce their ROS level, indicating that the oxidative level of in vitro matured oocytes from diabetic mice is increased, and the medium supplemented with BGP-15 can significantly reduce the oxidative level of in vitro maturation. The mitochondrial function of in vitro matured oocytes from diabetic mice is impaired, and the medium supplemented with BGP-15 can significantly restore its mitochondrial function. Compared with normal mice, antioxidant genes in in vitro matured oocytes from diabetic mice are... Sod1 and Sod2 The mRNA expression level of the antioxidant gene was downregulated, but when diabetic mouse oocytes were matured in vitro in a medium supplemented with BGP-15, the expression level of the antioxidant gene was reduced. Sod1 and Sod2 The mRNA level of the cells could be restored to a level comparable to that of normal mouse oocytes, indicating that the oxidation level of oocytes matured in vitro in diabetic mice is increased, and the culture medium with added BGP-15 can significantly reduce its oxidation level.

[0022] This invention also provides the application of BGP-15 or the culture medium described in the above-described technical solutions, or the culture medium prepared by the preparation method described in the above-described technical solutions, in any of the following: ① to ④ ①Prepare products that improve oocyte quality; ②Prepare products for assisted reproductive research in humans or non-human mammals; ③Prepare products for in vitro fertilization; ④ Prepare products for intracytoplasmic sperm injection.

[0023] BGP-15 or the culture medium described in this invention can significantly promote the in vitro maturation of oocytes from diabetic mice, reduce the oxidative level of in vitro matured oocytes, improve mitochondrial function of oocytes, reduce oocyte maturation defects caused by diabetes, and improve oocyte quality. The improved quality oocytes obtained can be used for experimental research on oocyte development; and can also be used in the field of assisted reproduction in humans or non-human mammals, such as using the obtained high-quality oocytes for in vitro fertilization or intracytoplasmic sperm injection to obtain high-quality embryos.

[0024] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a culture medium and BGP-15 or culture medium for improving oocyte maturation defects caused by diabetes, is provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0025] Unless otherwise stated, the scientific terms used in this invention have the same meanings as commonly understood by those skilled in the art, and the technical means used in this invention are conventional means well known to those skilled in the art. The raw materials and reagents involved in this invention are all commercially available.

[0026] Example 1 The feeding of laboratory animals: Five-week-old female ICR mice were purchased from Vital Rivers. Five mice were housed per cage, with free access to water and food. After one week of acclimatization, the mice were randomly divided into two groups: a control group and a diabetic model group. Control group mice received an intraperitoneal injection of sodium citrate buffer after a 5-hour fast, while diabetic model group mice received an intraperitoneal injection of streptozotocin dissolved in sodium citrate buffer at a dose of 200 mg / kg after a 5-hour fast. Two weeks later, fasting blood glucose (FBG) was measured using a Roche glucometer. Mice with an FGB value of 16.7 mmol / L or higher were considered to have successfully established a type 1 diabetic mouse model.

[0027] Main reagents and instruments: The main reagents and instruments included: citrate / sodium citrate buffer, streptozotocin, BGP-15, M2 medium, M16 medium, mineral oil, reactive oxygen species (ROS) detection kit, mitochondrial membrane potential (MMP) detection kit, SuperScript™ IVCellsDirect™ cDNA synthesis kit, PowerUp SYBR Green Master Mix kit, and fluorescence microscope. Specifically: citrate / sodium citrate buffer was purchased from Beijing Solarbio Science & Technology Co., Ltd. (China); streptozotocin, M2 medium, M16 medium, and mineral oil were purchased from Sigma-Aldrich (USA); BGP-15 was purchased from Selleck (Germany); ROS detection kit and mitochondrial membrane potential (MMP) detection kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China); SuperScript™ IVCellsDirect™ cDNA synthesis kit was purchased from Invitrogen (USA); PowerUp SYBR Green Master Mix kit was purchased from Applied Biosystems (USA); and fluorescence microscope was purchased from Olympus (Japan).

[0028] Oocyte isolation and in vitro maturation culture: To collect GV-stage oocytes from control and diabetic mice, cumulus-oocyte complexes (COCs) were rapidly isolated from the ovaries using an injection needle in M2 medium. Cumulus cells were then removed using a pipette to obtain fully grown GV-stage oocytes from normal mice and successfully modeled diabetic mice. GV-stage oocytes from normal mice served as the control group (n=28). GV-stage oocytes from successfully modeled diabetic mice were divided into two groups: a diabetic group (n=28) and a BGP-15 improvement group (n=28). GV-stage oocytes from the control and diabetic groups were placed in equilibrated M16 medium and cultured in vitro at 37°C in a 5% CO2 incubator for maturation. Simultaneously, GV-stage oocytes from the BGP-15 improvement group were placed in M16 medium containing 10 μM BGP-15 and cultured in vitro at 37°C in a 5% CO2 incubator for maturation.

[0029] Observation and statistics of oocyte maturation: After culturing oocytes in an incubator for 14–16 h, the microscope's field of view and pupillary distance were adjusted to observe and select oocytes in the mature stage (MII). The proportion of MII oocytes was then calculated. Results are as follows: Figure 1 As shown in Table 1, Figure 1 The figure shows the effect of BGP-15 on the oocyte maturation rate of diabetic mice; where A is a representative image of in vitro matured oocytes from the three groups; and B is a statistical graph of the in vitro maturation rate of oocytes from the three groups. Note, * p <0.05,**p <0.01, *** p <0.001, **** p <0.0001. The results showed that BGP-15 could significantly increase the proportion of MII stage oocytes after in vitro culture of diabetic mouse oocytes.

[0030] Table 1 Results of oocyte maturation rate

[0031] Detection of ROS levels in oocytes: ROS levels in oocytes were detected using a reactive oxygen species detection kit (Beyotime Biotechnology, China), following the kit's instructions. The procedure was as follows: Mature oocytes were selected from the control group, diabetes group, and BGP-15 improvement group. After washing three times in PBS solution supplemented with 0.1% PVP, the oocytes were transferred to DCFH-DA staining drops and incubated at 37°C in the dark for 30 min. They were then washed three times with PBS solution supplemented with 0.1% PVP and 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. Results are shown below. Figure 2 As shown, Figure 2 The figure shows the effect of BGP-15 on ROS content in in vitro mature oocytes of diabetic mice; where A is a representative image of ROS fluorescence detection in in vitro mature oocytes of the three groups; B is a statistical graph of ROS fluorescence intensity in in vitro mature oocytes of the three groups. Note, * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001. The results showed that, compared with the control group mice, the ROS intensity of in vitro matured oocytes of diabetic mice was significantly increased. In vitro culture of oocytes from diabetic mice in a medium supplemented with BGP-15 significantly reduced their ROS levels, indicating that the oxidation level of in vitro matured oocytes from diabetic mice was increased, and the medium supplemented with BGP-15 could significantly reduce the oxidation level of their in vitro maturation.

[0032] Detection of mitochondrial membrane potential in oocytes: Mitochondrial membrane potential in oocytes was detected using the JC-1 Mitochondrial Membrane Potential Detection Kit (Beyotime Biotechnology, China), following the kit's instructions. The procedure was as follows: Mature oocytes were selected from the control group, diabetes group, and BGP-15 improvement group. After washing three times in PBS solution supplemented with 0.1% PVP, the cells were transferred to working solution and incubated at 37°C in the dark for 30 min. Then, the cells were washed three times with PBS solution supplemented with 0.1% PVP and observed and photographed under a fluorescence microscope. The fluorescence intensity of aggregates and monomers was analyzed using Image-J software. After importing the images into Image-J software, thresholds were adjusted, parameters were set, and measurements were taken. The ratio of fluorescence intensity of aggregates to monomers was used to quantify the mitochondrial membrane potential, and Prism software was used for plotting. Results are shown below. Figure 3 As shown, Figure 3 The image shows the effect of BGP-15 on the mitochondrial membrane potential of mature oocytes from diabetic mice in vitro. A represents representative images of mitochondrial membrane potential fluorescence detection in the three groups of mature oocytes in vitro; B is a statistical graph of the fluorescence intensity of mitochondrial membrane potential in the three groups of mature oocytes in vitro. Note: * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001. The results showed that, compared with the control group mice, the mitochondrial membrane potential of in vitro matured oocytes of diabetic mice was significantly decreased. In vitro culture of oocytes from diabetic mice in a medium supplemented with BGP-15 could significantly restore their mitochondrial membrane potential, indicating that the mitochondrial function of in vitro matured oocytes from diabetic mice was impaired, and the medium supplemented with BGP-15 could significantly restore their mitochondrial function.

[0033] Oocyte oxidation-related genes Sod1 and Sod2 Detection of expression levels: Total RNA was extracted and reverse transcribed into cDNA using the SuperScript™ IV CellsDirect™ cDNA Synthesis Kit (Invitrogen, USA). qPCR was performed using the PowerUp SYBR Green Master Mix kit (Applied Biosystems, USA). The specific steps for the qPCR reaction were as follows: pre-denaturation at 50°C for 2 min, activation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 60°C for 1 min, extension at 72°C for 15 s, for 45 cycles; gene expression levels were calculated using Formula 2. -Δ Δ CT The primers were synthesized by Shanghai Sangon Biotech Co., Ltd., China. Table 2 lists the primer sequence information. The results are as follows: Figure 4 As shown, Figure 4 BGP-15 targets oxidation-related genes in in vitro matured oocytes of diabetic mice. Sod1 and Sod2 The effect of expression levels is shown in the figure; where A represents the three groups of oxidation-related genes in in vitro matured oocytes. Sod1 Statistical graph of expression levels; B represents three groups of oxidation-related genes in in vitro matured oocytes. Sod2 A statistical graph of expression levels. Note: * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001. qPCR results showed that, compared with control mice, the antioxidant gene in mature oocytes of diabetic mice was significantly higher. Sod1 and Sod2 The mRNA expression level of the antioxidant gene was downregulated, but when diabetic mouse oocytes were matured in vitro in a medium supplemented with BGP-15, the expression level of the antioxidant gene was reduced. Sod1 and Sod2 The mRNA level of the cells could be restored to a level comparable to that of normal mouse oocytes, indicating that the oxidation level of oocytes matured in vitro in diabetic mice is increased, and the culture medium with added BGP-15 can significantly reduce its oxidation level.

[0034] Table 2 Primer sequence information

[0035] The results of immunofluorescence staining and qPCR above indicate that, compared with M16 medium without BGP-15, M16 medium with BGP-15 can significantly reduce the oxidation level of in vitro matured oocytes from diabetic mice, restore the mitochondrial membrane potential of in vitro matured oocytes from diabetic mice, thereby restoring the mitochondrial function of in vitro matured oocytes from diabetic mice and improving oocyte quality.

[0036] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of BGP-15 in the preparation of products that improve oocyte maturation defects caused by diabetes; said products also include oocyte basal culture medium.

2. The application according to claim 1, characterized in that, The oocytes are derived from mammals.

3. The application according to claim 1, characterized in that, The types of products include culture media.

4. A culture medium for improving oocyte maturation defects caused by diabetes, characterized in that, The culture medium consists of BGP-15 and oocyte basal culture medium.

5. The culture medium according to claim 4, characterized in that, The basal culture medium for oocytes includes M2 medium, M16 medium, DMEM medium, or TCM199 medium.

6. The culture medium according to claim 4, characterized in that, The molar concentration of BGP-15 in the culture medium is 1 μM to 1 mM.

7. A method for preparing the culture medium according to any one of claims 4 to 6, comprising the following steps: Mix BGP-15 with oocyte basal culture medium.

8. The use of BGP-15 or the culture medium according to any one of claims 4 to 6 or the culture medium prepared by the preparation method according to claim 7 in the preparation of products that promote the in vitro maturation of diabetic oocytes.

9. The use of BGP-15 or the culture medium according to any one of claims 4 to 6 or the culture medium prepared by the preparation method according to claim 7 in the preparation of products that reduce the oxidative level of diabetic oocytes and / or improve the mitochondrial function of diabetic oocytes.

10. The use of BGP-15 or the culture medium according to any one of claims 4 to 6 or the culture medium prepared by the preparation method according to claim 7 in any one of the following: ①Prepare products that improve oocyte quality; ②Prepare products for assisted reproductive research in humans or non-human mammals; ③Prepare products for in vitro fertilization; ④ Prepare products for intracytoplasmic sperm injection.

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