An in vitro maturation medium for oocytes of hamsters and a preparation method thereof

Abstract

This invention discloses an in vitro maturation culture medium for ferret oocytes and its preparation method, belonging to the field of animal embryo engineering technology. It comprises the following components: TCM199 basal culture medium, 50 mL fetal bovine serum, 25 IU follicle-stimulating hormone, 25 IU luteinizing hormone, 0.5 mg 17β-estradiol, 0.005 mg epidermal growth factor, 12.1 mg sodium pyruvate, 0.2 mM cysteine, 0.1 mM cysteine, 0.030 g penicillin, 0.025 g streptomycin sulfate, as well as an antioxidant complex, a combination of growth factors, and extracellular matrix components. This invention provides a comprehensive and suitable nutritional environment for ferret oocytes by precisely formulating a composite culture medium containing multiple key components. Specifically, the hormone combination at specific concentrations accurately simulates the in vivo physiological environment, effectively regulating the oocyte maturation process; multiple growth factors work synergistically to promote oocyte growth, development, and differentiation; and the addition of the antioxidant complex effectively eliminates free radicals generated during in vitro culture, reducing oxidative stress damage to oocytes.

Description

Technical Field

[0001] This invention relates to the field of animal embryo engineering technology, and in particular to an in vitro maturation culture medium for ferret oocytes and its preparation method. Background Technology

[0002] Ferrets, as highly valuable model animals, play an irreplaceable role in biomedical research. Due to their high similarity to humans in physiological structure and gene sequence, ferrets are widely used in virology research, providing crucial research models for exploring the pathogenic mechanisms and transmission patterns of pathogens such as influenza and COVID-19, as well as in vaccine development. Simultaneously, in reproductive biology research, ferrets provide important experimental subjects for a deeper understanding of mammalian reproductive processes and embryonic development mechanisms; in ferret-related research, in vitro fertilization and embryonic development techniques are key links, and high-quality oocytes are the foundation for the successful implementation of these techniques.

[0003] Currently, existing in vitro maturation media for ferret oocytes are often not precise or comprehensive enough in terms of composition and formulation. Many media contain only basic nutrients and a small amount of hormones, failing to fully simulate the physiological environment within ferrets. For example, the types and concentrations of hormones in some media cannot precisely regulate the oocyte maturation process, resulting in low oocyte maturation rates. Moreover, the lack of synergistic effects from multiple growth factors restricts oocyte growth, development, and differentiation, leading to insufficient cytoplasmic maturity and incomplete organelle function, which in turn affects subsequent in vitro fertilization and embryo development, making it difficult to achieve ideal nuclear maturation rates.

[0004] During in vitro culture, oocytes are inevitably subjected to oxidative stress. The accumulation of free radicals damages important biomolecules such as the cell membrane, DNA, and proteins, leading to abnormal cell function or even death. However, most existing culture media do not adequately consider antioxidant protection and rarely add effective antioxidants, failing to promptly eliminate free radicals generated in the in vitro culture environment. This increases the risk of oxidative damage to oocytes, further reducing their maturation quality and developmental potential.

[0005] The extracellular matrix plays a crucial role in the mechanical support and biological signal transduction of oocytes during development. It provides a suitable microenvironment for oocytes and regulates cell morphology, function, and metabolism. However, existing culture media often neglect the addition of extracellular matrix components, resulting in oocytes lacking the support and signal guidance found in vivo during in vitro culture. This hinders oocyte development and makes it difficult to reach optimal maturation.

[0006] Due to the lack of standardization in existing culture medium preparation methods and lax quality control, there are component differences between different batches of culture media, leading to unstable performance. Over long-term use, the quality of the culture medium may change, failing to consistently maintain its ability to promote oocyte maturation and embryonic development. Furthermore, in multiple independent experiments, fluctuations in culture medium quality often result in a lack of reproducibility, increasing research costs and risks, and hindering in-depth research on ferret model animals.

[0007] Therefore, we provide an in vitro maturation culture medium for ferret oocytes and its preparation method. Summary of the Invention

[0008] To overcome the shortcomings of the prior art and solve at least one of the technical problems mentioned in the background art, the present invention proposes an in vitro maturation culture medium for ferret oocytes and its preparation method.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: A culture medium for in vitro maturation of ferret oocytes, per 500 mL of basal culture medium, comprises the following components: TCM199 basal culture medium, 50 mL fetal bovine serum, 25 IU follicle-stimulating hormone, 25 IU luteinizing hormone, 0.5 mg 17β-estradiol, 0.005 mg epidermal growth factor, 12.1 mg sodium pyruvate, 0.2 mM cysteine, 0.1 mM cysteine, 0.030 g penicillin, 0.025 g streptomycin sulfate, as well as an antioxidant complex, a combination of growth factors, and extracellular matrix components.

[0010] Preferably, the antioxidant complex contains 0.1 mM vitamin C, 0.05 mM vitamin E and 0.01 μM selenium, which are used to enhance the antioxidant capacity of oocytes and reduce oxidative stress damage during in vitro culture.

[0011] Preferably, the growth factor combination comprises insulin-like growth factor-1 (IGF-1) 0.01 μg / mL and platelet-derived growth factor (PDGF) 0.005 μg / mL, which work synergistically with epidermal growth factor (EGF) to promote the in vitro maturation and development of oocytes.

[0012] Preferably, the extracellular matrix components include 0.1 mg / mL hyaluronic acid and 0.05 mg / mL collagen, mimicking the in vivo extracellular matrix environment to provide mechanical support and biological signals for oocytes.

[0013] Preferably, a method for preparing a culture medium for in vitro maturation of ferret oocytes is also provided, comprising the following steps: S1. Prepare TCM199 basic culture medium; add fetal bovine serum, follicle-stimulating hormone, luteinizing hormone, 17β-estradiol, epidermal growth factor, insulin-like growth factor-1, and platelet-derived growth factor in sequence. S2, add sodium pyruvate, cystine and cysteine; S3, Added antioxidant complex; S4. Add extracellular matrix components; S5. Finally, add penicillin and streptomycin sulfate; filter through a 0.22μm microporous membrane for sterilization, then dispense and store.

[0014] Preferably, the antioxidant complex is added as follows: vitamin C, vitamin E and selenium are dissolved in a small amount of sterile deionized water, and then added dropwise to the culture medium while stirring.

[0015] Preferably, the step of adding the extracellular matrix components is as follows: hyaluronic acid and collagen are dissolved in sterile phosphate buffer (PBS), and then slowly added to the culture medium, gently mixed evenly, avoiding the generation of air bubbles.

[0016] Preferably, a method for in vitro maturation culture of ferret oocytes is also provided, wherein the collected ferret oocytes are placed in a culture medium and cultured in an incubator at 38.5°C, 5% CO2, and saturated humidity for 24-48 hours.

[0017] Preferably, after the oocyte culture is completed, the oocyte maturity rate is assessed by morphological observation and nuclear maturation markers, wherein morphological observation includes extrusion of the first polar body and nuclear maturation markers include chromosome condensation.

[0018] Preferably, the application of a culture medium for in vitro maturation of ferret oocytes in ferret model animal research is also provided, characterized in that the culture medium is used to improve the in vitro maturation rate and developmental capacity of ferret oocytes, providing technical support for the application of ferrets in brain development, brain function, spinal cord injury, epilepsy, heart disease, immunity, obesity, oncology, various lung diseases and infectious diseases research.

[0019] The beneficial effects of this invention are: 1. This invention provides a comprehensive and suitable nutritional environment for ferret oocytes by precisely formulating a composite culture medium containing multiple key components. Specifically, a combination of hormones at specific concentrations accurately simulates the in vivo physiological environment, effectively regulating the oocyte maturation process; multiple growth factors work synergistically to promote oocyte growth, development, and differentiation; and the addition of antioxidant complexes effectively eliminates free radicals generated during in vitro culture, reducing oxidative stress damage to oocytes and thus significantly improving the oocyte maturation rate. Simultaneously, the extracellular matrix components provide oocytes with mechanical support and biological signals similar to those in vivo, further optimizing culture conditions and enabling oocytes to complete the maturation process more efficiently in vitro, with a significantly improved nuclear maturation rate.

[0020] 2. Mature ferret oocytes cultured using the medium of this invention exhibited higher fertilization capacity during in vitro fertilization. Because the medium provides a favorable developmental foundation for the oocytes, their cytoplasm matures more fully, and their organelle functions are more complete, enabling them to better combine with sperm and complete the fertilization process, thereby increasing the cleavage rate. In subsequent embryo culture, embryos formed from oocytes treated with the medium of this invention have stronger developmental potential and can develop to the blastocyst stage more smoothly, significantly improving the blastocyst formation rate. This not only provides more high-quality embryo resources for ferret model animal research but also makes experimental results more stable and reliable, reduces experimental errors caused by poor oocyte quality, and strongly promotes in-depth research in related fields.

[0021] 3. This invention discloses in detail the precise preparation methods and strict quality control standards for each component of the culture medium, ensuring the consistency and stability of components between different batches of the medium. Long-term stability verification experiments show that the culture medium maintains its performance in promoting oocyte maturation and embryonic development throughout its specified shelf life, providing a reliable guarantee for experiments. Simultaneously, repeatability verification experiments show that multiple independent experiments using the same or different batches of the culture medium yield similar results, demonstrating the excellent repeatability of the culture medium of this invention. This allows researchers to conduct large-scale experiments without worrying about the impact of fluctuations in culture medium quality on experimental results, reducing research costs and risks, and improving research efficiency. Attached Figure Description

[0022] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings: Figure 1 This is a microscopic image showing an in vitro maturation culture medium for ferret oocytes proposed in this invention. Figure 2 This is a flowchart illustrating a method for preparing an in vitro maturation culture medium for ferret oocytes, as proposed in this invention. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 are within the scope of protection of the present invention.

[0024] Specific implementation examples are given below.

[0025] Example 1: Preparation of basal culture medium TCM199 basic culture medium: Take commercially available TCM199 powder, dissolve it in deionized water according to the instructions, prepare 500mL basic solution, adjust the pH to 7.2-7.4, filter and sterilize before use.

[0026] Fetal bovine serum (FCS): Take 50 mL of fetal bovine serum and add it to the above TCM199 basic culture medium. Mix gently until homogeneous.

[0027] Hormone addition: Add 25 IU of follicle-stimulating hormone (FSH), 25 IU of luteinizing hormone (LH), and 0.5 mg of 17β-estradiol in sequence, and gently mix well after each addition.

[0028] Growth factor supplementation: Add 0.005 mg epidermal growth factor (EGF), 0.01 μg / mL insulin-like growth factor-1 (IGF-1), and 0.005 μg / mL platelet-derived growth factor (PDGF). The amounts of IGF-1 and PDGF added are calculated using the following formula: Required IGF-1 mass (mg) = 0.01 μg / mL × 500 mL × 10⁻³ = 0.005 mg Required PDGF mass (mg) = 0.005 μg / mL × 500 mL × 10⁻³ = 0.0025 mg (In actual operation, since the PDGF concentration may be high, it can be diluted according to the ratio before addition) Antioxidant complex added: Vitamin C: 0.1mM × 500mL × 176.12g / mol (molecular weight of vitamin C) × 10⁻³ = 8.806mg Vitamin E: 0.05mM × 500mL × 430.71g / mol (molecular weight of vitamin E) × 10⁻³ = 10.768mg Selenium: 0.01μM × 500mL × 78.96g / mol (atomic weight of selenium) × 10⁻⁶ = 0.00395mg Dissolve the above components in a small amount of sterile deionized water, and then add them dropwise to the culture medium while stirring.

[0029] Addition of extracellular matrix components: Hyaluronic acid: 0.1 mg / mL × 500 mL = 50 mg Collagen: 0.05mg / mL × 500mL = 25mg Dissolve hyaluronic acid and collagen in sterile phosphate-buffered saline (PBS), then slowly add them to the culture medium, gently mixing to ensure even distribution and avoid creating air bubbles.

[0030] Antibiotic addition: Finally, add 0.030g of penicillin and 0.025g of streptomycin sulfate, and gently mix well.

[0031] Sterilization by filtration: Sterilize by filtration through a 0.22μm microporous membrane, dispense into sterile culture flasks, and store at 4℃ for later use.

[0032] Example 2: Collection and Culture of Oocytes Oocyte collection: Oocytes were collected from the ovaries of adult female ferrets, and oocytes with normal morphology and uniform cytoplasm were selected for in vitro culture.

[0033] Culture conditions: Place the collected oocytes in the prepared culture medium, adding an appropriate amount of medium to each well (or bottle) to ensure sufficient infiltration of the oocytes. Culture in an incubator at 38.5℃, 5% CO2, and saturated humidity for 24-48 hours.

[0034] Evaluation of cultivation effectiveness: Morphological observation: After culture, the morphology of oocytes was observed using an inverted microscope, and the number of oocytes expelled from the first polar body was recorded.

[0035] Nuclear maturation marker assessment: By fixing oocytes and performing nuclear staining (such as Hoechst 33342 staining), the chromosome condensation is observed and the nuclear maturation rate is assessed.

[0036] Calculation formula: Maturation rate calculation: Maturation rate = (Number of oocytes expelled from the first polar body / Total number of oocytes) × 100% Nuclear maturation rate calculation: Nuclear maturation rate = (number of oocytes with chromosomal condensation / total number of oocytes) × 100%.

[0037] Example 3: Application of culture medium in ferret model animal research The prepared culture medium was then applied to ferret model animal studies, and the specific steps are as follows: Experimental design: Healthy adult female ferrets were selected and randomly divided into an experimental group and a control group, with at least 3 ferrets in each group.

[0038] Oocyte collection and culture: Oocytes were collected and cultured according to the method in Example 2. The experimental group used the culture medium prepared in this invention, while the control group used the conventional culture medium.

[0039] Analysis of experimental results: Compare the maturation rate and nuclear maturation rate of oocytes in the experimental group and the control group.

[0040] Mature oocytes were further subjected to in vitro fertilization or parthenogenesis to observe embryo development and assess the impact of culture medium on embryo development potential.

[0041] Data statistics and analysis: Statistical software was used to process the experimental data, compare the differences between the experimental group and the control group, and evaluate the superiority of the culture medium of the present invention.

[0042] Example 4: Comparative Experiment on Optimization of Culture Medium Components Experimental Objective To investigate the effects of different concentrations of growth factor combinations on the in vitro maturation quality of ferret oocytes and their subsequent embryonic development potential, and to further optimize the culture medium formulation.

[0043] Experimental Groups Three experimental groups were set up: experimental group A, experimental group B, and control group.

[0044] Experimental Group A: Based on the basal culture medium formulation of Example 1, the amount of growth factors added was adjusted. The amount of epidermal growth factor (EGF) added was 0.01 mg, the amount of insulin-like growth factor-1 (IGF-1) added was 0.02 μg / mL, and the amount of platelet-derived growth factor (PDGF) added was 0.01 μg / mL. After determining the required mass according to the calculation method of growth factor addition in Example 1, the addition was carried out. Other components and preparation steps were the same as in Example 1.

[0045] Experimental Group B: Based on the same basal culture medium formulation as in Example 1, the growth factor addition amounts were adjusted as follows: EGF 0.0025 mg, IGF-1 0.005 μg / mL, and PDGF 0.0025 μg / mL. The required mass was calculated and added, and the remaining components and preparation process were the same as in Example 1.

[0046] Control group: using the conventional culture medium prepared in Example 1.

[0047] oocyte collection Oocytes were collected from the ovaries of adult female ferrets. Oocytes with normal morphology and uniform cytoplasm were selected for the experiment to ensure that there was no significant difference in the initial quality of the oocytes used in each group of experiments.

[0048] Oocyte culture The collected oocytes were placed in the culture media of experimental group A, experimental group B, and control group, respectively. An appropriate amount of culture medium was added to each well (or each bottle) to ensure that the oocytes were fully infiltrated. The culture conditions were set at 38.5℃, 5% CO2, and saturated humidity in an incubator for 36 hours.

[0049] Evaluation of cultivation effect Morphological observation: After culture, the morphology of oocytes was carefully observed using an inverted microscope, and the number of oocytes expelled from the first polar body was recorded in detail.

[0050] Nuclear maturation marker assessment: By fixing oocytes, the chromosome condensation was observed using the Hoechst 33342 staining method to assess the nuclear maturation rate.

[0051] Formulas for calculating maturity rate and nuclear maturity rate Maturation rate calculation: Maturation rate = (Number of oocytes expelled from the first polar body / Total number of oocytes) × 100% Nuclear maturation rate calculation: Nuclear maturation rate = (Number of oocytes with chromosome condensation / Total number of oocytes) × 100% In vitro fertilization and embryo development assessment In vitro fertilization (IVF) procedure: Mature oocytes are fertilized in vitro with capacitated ferret sperm. The fertilization process is carried out in a specially designed fertilization culture medium, with strict control over fertilization time and environmental conditions.

[0052] Embryo culture: After fertilization, the fertilized eggs are transferred to embryo culture medium for further culture, and the embryo development is observed and the cleavage rate and blastocyst formation rate are recorded.

[0053] Cleavage rate calculation: Cleavage rate = (Number of cleaved embryos / Number of fertilized eggs) × 100% Blastocyst formation rate calculation: Blastocyst formation rate = (Number of embryos forming blastocysts / Number of fertilized eggs) × 100% Data Statistics and Analysis The experimental data were processed using professional statistical software to compare the differences in oocyte maturation rate, nuclear maturation rate, cleavage rate, and blastocyst formation rate among experimental groups A, B, and the control group. The effects of different growth factor concentration combinations on in vitro maturation and embryonic development potential of ferret oocytes were evaluated using methods such as significance testing, providing a scientific basis for further optimization of culture medium formulations.

[0054] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A culture medium for in vitro maturation of ferret oocytes, characterized in that, Each 500 mL of basal culture medium contains the following components: TCM199 basal culture medium, 50 mL fetal bovine serum, 25 IU follicle-stimulating hormone, 25 IU luteinizing hormone, 0.5 mg 17β-estradiol, 0.005 mg epidermal growth factor, 12.1 mg sodium pyruvate, 0.2 mM cysteine, 0.1 mM cysteine, 0.030 g penicillin, 0.025 g streptomycin sulfate, as well as antioxidant complex, growth factor combination and extracellular matrix components.

2. The culture medium for in vitro maturation of ferret oocytes according to claim 1, characterized in that, The antioxidant complex contains 0.1 mM vitamin C, 0.05 mM vitamin E, and 0.01 μM selenium, which are used to enhance the antioxidant capacity of oocytes and reduce oxidative stress damage during in vitro culture.

3. The culture medium for in vitro maturation of ferret oocytes according to claim 1, characterized in that, The growth factor combination contains insulin-like growth factor-1 (IGF-1) 0.01 μg / mL and platelet-derived growth factor (PDGF) 0.005 μg / mL, which work synergistically with epidermal growth factor (EGF) to promote the in vitro maturation and development of oocytes.

4. The culture medium for in vitro maturation of ferret oocytes according to claim 1, characterized in that, The extracellular matrix components include 0.1 mg / mL hyaluronic acid and 0.05 mg / mL collagen, which simulate the in vivo extracellular matrix environment and provide mechanical support and biological signals for oocytes.

5. A method for preparing an in vitro maturation culture medium for ferret oocytes, the method being applicable to the in vitro maturation culture medium for ferret oocytes as proposed in claims 1-4, characterized in that, Includes the following steps: S1. Prepare TCM199 basic culture medium; add fetal bovine serum, follicle-stimulating hormone, luteinizing hormone, 17β-estradiol, epidermal growth factor, insulin-like growth factor-1, and platelet-derived growth factor in sequence. S2, add sodium pyruvate, cystine and cysteine; S3, Added antioxidant complex; S4. Add extracellular matrix components; S5. Add penicillin and streptomycin sulfate; filter through a 0.22μm microporous membrane for sterilization, then dispense and store.

6. A method for preparing a culture medium for in vitro maturation of ferret oocytes according to claim 5, characterized in that, The steps for adding the antioxidant complex are as follows: dissolve vitamin C, vitamin E and selenium in a small amount of sterile deionized water, and then add them dropwise to the culture medium while stirring.

7. The method for preparing a culture medium for in vitro maturation of ferret oocytes according to claim 5, characterized in that, The steps for adding the extracellular matrix components are as follows: Hyaluronic acid and collagen are dissolved in sterile phosphate-buffered saline (PBS), and then slowly added to the culture medium, gently mixed evenly, avoiding the generation of air bubbles.

8. A method for in vitro maturation culture of ferret oocytes, characterized in that, Using a culture medium, the collected ferret oocytes were placed in the culture medium and cultured in an incubator at 38.5℃, 5% CO2, and saturated humidity for 24-48 hours.

9. The method for in vitro maturation culture of ferret oocytes according to claim 8, characterized in that, After the oocyte culture is completed, the oocyte maturity rate is assessed by morphological observation and nuclear maturation markers, including first polar body extrusion and chromosome condensation.

10. The application of the culture medium for in vitro maturation of ferret oocytes as described in any one of claims 1 to 4 in ferret model animal research, characterized in that, The culture medium is used to improve the in vitro maturation rate and developmental capacity of ferret oocytes, providing technical support for the application of ferrets in research on brain development, brain function, spinal cord injury, epilepsy, heart disease, immunity, obesity, oncology, various lung diseases, and infectious diseases.

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