Use of mangiferin in promoting maturation of animal oocytes

Abstract

The present application relates to the field of embryo engineering, and particularly relates to application of mangiferin in promoting maturation of animal oocytes. The present application discloses that mangiferin can promote maturation of animal oocytes, especially maturation of pig oocytes; can improve in-vitro maturation efficiency of oocytes; can reduce the apoptosis rate of oocytes and improve the subsequent development potential of oocytes. Mangiferin can be used to promote in-vitro maturation of oocytes, and can be used to prepare or directly serve as a drug for improving in-vitro maturation and development of oocytes, thereby opening up a new application direction for mangiferin. The present application uses mangiferin to improve in-vitro maturation efficiency of oocytes, solves the problems of low maturation efficiency and long culture period, and provides more high-quality oocytes for somatic cell nuclear transfer, in-vitro fertilization and transgenic cloning by using the technical scheme of mangiferin in the present application, thereby having a wide application prospect in actual production.

Description

Technical Field

[0001] This invention relates to the field of embryo engineering technology, specifically the application of mangiferin in promoting the maturation of animal oocytes. Background Technology

[0002] In vitro maturation culture (IVM) of oocytes is becoming increasingly important in biomedical and agricultural applications as one of the methods for producing pig embryos. Obtaining high-quality in vitro matured oocytes is the first step in embryo engineering technologies such as in vitro fertilization, somatic cell cloning, and transgenic animal production. However, compared with oocytes matured in vivo, in vitro matured oocytes show significant differences in maturation rate, maturation quality, and developmental potential. Therefore, perfecting the in vitro maturation system of oocytes is a key issue that urgently needs to be addressed in embryo engineering technology.

[0003] In vitro oocyte maturation is influenced by many factors, such as oocyte species, culture conditions, and culture medium composition (proteins, carbohydrates, protein factors, hormone levels, etc.), among which the culture medium composition plays a crucial role in oocyte development. The oocyte maturation microenvironment exhibits variations in oxidative stress levels and abnormal lipid droplet metabolism, as well as excessive production of intracellular reactive oxygen species (ROS). These factors can all lead to abnormal oocyte development; therefore, maintaining a stable oocyte maturation microenvironment is particularly important for in vitro oocyte maturation. Porcine oocyte maturation exhibits its own growth characteristics. Lipid droplets are abundantly distributed in the oocyte cytoplasm, where they are broken down by lipases into fatty acids of varying lengths, which then enter the mitochondria for β-oxidation, maintaining oocyte morphology and normal development.

[0004] Currently, the in vitro development efficiency of porcine oocytes during in vitro culture is low, and the apoptosis rate and subsequent developmental potential are not ideal. Therefore, finding a new component that can improve the in vitro maturation efficiency of porcine oocytes and thus improve the in vitro developmental potential of nuclear transfer embryos is an urgent problem to be solved.

[0005] Mangoes are known as the "King of Tropical Fruits," and Guangxi, with its superior natural environment and climate, is a region renowned for producing high-quality agricultural products. Mangoes are one of Guangxi's signature fruits. Mangiferin is a component extracted from mango leaves. Currently, there are no studies on the combined addition of mangiferin during the in vitro maturation of porcine oocytes. Investigating whether the addition of mangiferin can significantly improve the in vitro maturation efficiency of porcine oocytes, thereby enhancing the in vitro developmental potential of nuclear transfer embryos, is of great significance. Summary of the Invention

[0006] To address the aforementioned issues, this invention proposes that mangiferin can enhance the in vitro maturation rate of oocytes and the in vitro developmental potential of transplanted embryos, as detailed below:

[0007] Application of mangiferin in promoting the maturation of animal oocytes.

[0008] Furthermore, the promotion of animal oocyte maturation specifically refers to the promotion of porcine oocyte maturation.

[0009] Furthermore, the promotion of animal oocyte maturation is to improve the efficiency of oocyte maturation in vitro.

[0010] Furthermore, promoting the maturation of animal oocytes involves reducing the apoptosis rate of oocytes and increasing their subsequent developmental potential.

[0011] Furthermore, the application in promoting the maturation of animal oocytes is specifically used in the preparation of drugs that promote the maturation of porcine oocytes.

[0012] Furthermore, the mangiferin is extracted from mango leaves, peel, and pit.

[0013] Furthermore, the mangiferin has the molecular formula C19H18O11 and a molecular weight of 422.34, and its structure...

[0014] The formula is as follows:

[0015]

[0016] This invention, through research, has discovered that mangiferin can improve the in vitro maturation rate of oocytes and the in vitro development potential of transplanted embryos, thus developing a new application direction for mangiferin and also contributing to the improvement of the in vitro maturation system of oocytes in embryo engineering technology.

[0017] A method for improving and examining the efficiency of porcine oocyte in vitro development using mangiferin, specifically comprising the following steps:

[0018] (1) Prepare oocyte maturation culture medium:

[0019] Culture medium A: 9.5 g / L TCM199, 10% volume of porcine follicular fluid, 10% volume of fetal bovine serum, 0.1 g / L cysteine, 0.1 g / L penicillin, 0.075 g / L streptomycin, 20 ng / mL insulin-like growth factor, 100 ng / mL epidermal growth factor, 0.55 g / L glucose, 2.2 g / L NaHCO3, 10 IU / mL pregnant mare serum gonadotropin, 10 IU / mL human chorionic gonadotropin, and different concentrations of mangiferin (50 μM, 100 μM, 150 μM);

[0020] Culture medium B: 9.5 g / L TCM199, 10% volume of porcine follicular fluid, 10% volume of fetal bovine serum, 0.1 g / L cysteine, 0.1 g / L penicillin, 0.075 g / L streptomycin, 20 ng / mL insulin-like growth factor, 100 ng / mL epidermal growth factor, 0.55 g / L glucose, 2.2 g / L NaHCO3, and different concentrations of mangiferin (50 μM, 100 μM, 150 μM);

[0021] (2) Electro-activation solution: 0.5 mmol / L mannitol, 0.1 mmol / L CaCl2·2H2O, 0.1 mmol / L MgSO4·7H2O, 0.5 mmol / L Hepes, 0.01 g / mL PVA, 0.06 g / L penicillin, 0.01 g / L streptomycin;

[0022] (3) Embryo culture medium: PZM3 embryo culture medium was used, with the following specific components: 6.3 g / L NaCl, 0.75 g / L KCl, 0.048 g / L KH2PO4, 0.1 g / L MgSO4·7H2O, 2.1 g / L NaHCO3, 0.022 g / L sodium pyruvate, 0.62 g / L calcium lactate, 0.15 g / L L-glutamine, 0.8 g / L taurine, 20 ml / L essential amino acids, 15 ml / L non-essential amino acids, 0.065 g / L penicillin, 0.05 g / L streptomycin, and 6 g / L BSA;

[0023] (4) Culture of oocytes: Ovaries were collected from the slaughterhouse and placed in physiological saline containing penicillin and streptomycin, and then sent back to the laboratory. 2-6 mm of clear follicular fluid was drawn using a 10 mL syringe with a 12-gauge needle, and allowed to stand in a constant temperature rack for 15 min. The precipitate was mixed and aliquoted into glass dishes. Under a stereomicroscope, cumulus-oocyte complexes (COCs) with three layers of cumulus, uniform cytoplasm, and good refractive properties were picked up with an oocyte-picking needle. The oocytes were transferred to culture medium A from step 1) and cultured for 20-22 h, then transferred to culture medium B from step 1) and cultured for another 20-22 h.

[0024] (5) Donor cell culture: Fetal pig leg tissue was isolated and cut into 2-3 mm pieces. A small amount of DMEM culture medium containing 10% FBS was added, and the pieces were spread evenly. The lid of the dish was marked, and the dish was inverted in an incubator. After 4 hours, a small amount of culture medium was added to prevent the tissue pieces from floating. After 24 hours, the bottom of the dish was filled with culture medium. After 48 hours, the growth of fibroblasts was observed. When the fibroblasts reached about 90% confluence, they were passaged.

[0025] (6) Nuclear transfer embryo production and embryo cell counting: Oocytes cultured for 40–44 h were aspirated into 1 mg / mL hyaluronidase and pipetted until granulosa cells were completely detached. Oocytes with intact first polar bodies, intact zona pellucida, clear perivitelline space, good morphology, and homogeneous cytoplasm were selected and enucleated under an inverted microscope. The oocytes were fixed with a fixation needle at the 9 o'clock position, and the enucleation was performed with a manipulation needle at the 3 o'clock position. After enucleation, a single scattered donor cell was aspirated with the manipulation needle for nuclear injection. After nuclear injection, the reconstituted embryos were electrically activated. The fusion tank was cleaned three times with activation solution equilibrated for 15 min. After washing the constructs three times in the activation solution, they were transferred to the center of a fusion tank filled with electro-activation solution and activated three times with a DC pulse of 1.00 kV / cm and 80 μs. The reconstructed embryos after electro-activation were washed three times in PZM3 embryo culture medium and then transferred to microdroplets of embryo culture medium for further culture. The conditions for continued culture were 5% CO2, saturated humidity, and 39°C. Blastocysts cultured for 144 h were stained with 10 μg / mL Hoechst 33342 for 15 min, then 1-2 drops of glycerol were added to compress and mount the slides, ensuring they were evenly spread on a glass slide. Cell counting was then performed under fluorescence irradiation.

[0026] (7) Detection of apoptotic cells in embryos: Nuclear transfer blastocysts developed for 144–168 h were collected for TUNEL assay. The nuclear transfer blastocysts were washed three times with PBS and then fixed in 4% paraformaldehyde for 30 min. After the fixed blastocysts were washed three times with PBS / PVA, they were permeabilized with 1% Triton X-100 in PBS for 1 h, and then incubated in TUNEL staining solution at 38.5℃, 5% CO2, and 100% humidity for 1.5 h. After washing three times with PBS / PVA, the nuclei were stained with 10 μg / mL Hoechst 33342 for 10 min. The processed samples were placed on a glass slide with an anti-quenching agent and sealed with Vaseline, and observed under a fluorescence microscope.

[0027] Furthermore, the glucose used is 0.55 g / L.

[0028] Furthermore, the BSA concentration is 6 g / L.

[0029] Furthermore, the mangiferin is 100 μM.

[0030] Compared with the prior art, the technical effects of this invention are reflected in:

[0031] (1) This invention discloses that mangiferin can promote the maturation of animal oocytes, especially porcine oocytes; it can improve the efficiency of in vitro oocyte maturation; it can reduce the apoptosis rate of oocytes and improve the subsequent developmental potential of oocytes. Mangiferin can be used to promote the in vitro maturation of oocytes, or it can be used to prepare or directly as a drug to improve the in vitro maturation and development of oocytes, opening up new application directions for mangiferin.

[0032] (2) The present invention uses mangiferin to improve the in vitro maturation efficiency of oocytes, which solves the problems of low maturation efficiency and long culture cycle. The technical solution of using mangiferin in the present invention can provide more high-quality oocytes for somatic cell nuclear transfer, in vitro fertilization and transgenic cloning, and has broad application prospects in actual production.

[0033] (3) This invention provides a method for improving the in vitro development rate of porcine oocytes using mangiferin. It is a method for improving the in vitro development efficiency of porcine oocytes using mangiferin, increasing the maturation rate from 76.23% to 85.69%, reducing the apoptosis rate of oocytes, and improving the in vitro development potential of nuclear transfer embryos. Attached Figure Description

[0034] Figure 1 Comparison of oocyte in vitro maturation: Polar body expulsion of COCs in different treatment groups during MII stage (A: 100 μM mangiferin treatment group; B: blank control group. Bar = 50 μm). Detailed Implementation

[0035] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.

[0036] Example 1:

[0037] A method for improving the in vitro development efficiency of porcine oocytes using the phenolic antioxidant mangiferin includes the following steps:

[0038] (1) Prepare the maturation culture medium for oocytes:

[0039] Culture medium A: 9.5 g / L TCM199, 10% volume of porcine follicular fluid, 10% volume of fetal bovine serum, 0.1 g / L cysteine, 0.1 g / L penicillin, 0.075 g / L streptomycin, 20 ng / mL insulin-like growth factor, 100 ng / mL epidermal growth factor, 0.55 g / L glucose, 2.2 g / L NaHCO3, 10 IU / mL pregnant mare serum gonadotropin, 10 IU / mL human chorionic gonadotropin, 50 μM mangiferin;

[0040] Culture medium B: 9.5 g / L TCM199, 10% volume of porcine follicular fluid, 10% volume of fetal bovine serum, 0.1 g / L cysteine, 0.1 g / L penicillin, 0.075 g / L streptomycin, 20 ng / mL insulin-like growth factor, 100 ng / mL epidermal growth factor, 0.55 g / L glucose, 2.2 g / L NaHCO3, 50 μM mangiferin;

[0041] (2) Electro-activation solution: 0.5 mmol / L mannitol, 0.1 mmol / L CaCl2·2H2O, 0.1 mmol / L MgSO4·7H2O, 0.5 mmol / L Hepes, 0.01% (w / v) PVA, 0.06 g / L penicillin, 0.01 g / L streptomycin;

[0042] (3) Embryo culture medium: PZM3 embryo culture medium was used, with the following specific components: 6.3 g / L NaCl, 0.75 g / L KCl, 0.048 g / L KH2PO4, 0.1 g / L MgSO4·7H2O, 2.1 g / L NaHCO3, 0.022 g / L sodium pyruvate, 0.62 g / L calcium lactate, 0.15 g / L L-glutamine, 0.8 g / L taurine, 20 ml / L essential amino acids, 15 ml / L non-essential amino acids, 0.065 g / L penicillin, and 0.05 g / L streptomycin;

[0043] (4) Culture of oocytes: Ovaries were collected from the slaughterhouse and placed in physiological saline containing penicillin and streptomycin, and then sent back to the laboratory. 2-6 mm of clear follicular fluid was drawn using a 10 mL syringe with a 12-gauge needle, and allowed to stand in a constant temperature rack for 15 min. The precipitate was mixed and aliquoted into glass dishes. Under a stereomicroscope, cumulus-oocyte complexes (COCs) with three layers of cumulus, uniform cytoplasm, and good refractive properties were picked up with an oocyte-picking needle. The oocytes were transferred to culture medium A from step 1) and cultured for 20-22 h, then transferred to culture medium B from step 1) and cultured for another 20-22 h.

[0044] (5) Donor cell culture: Fetal pig leg tissue was isolated and cut into 2-3 mm pieces. A small amount of DMEM culture medium containing 10% FBS was added, and the pieces were spread evenly. The lid of the dish was marked, and the dish was inverted in an incubator. After 4 hours, a small amount of culture medium was added to prevent the tissue pieces from floating. After 24 hours, the bottom of the dish was filled with culture medium. After 48 hours, the growth of fibroblasts was observed. When the fibroblasts reached about 90% confluence, they were passaged.

[0045] (6) Nuclear transfer embryo production: Mature oocytes are aspirated into 1 mg / mL hyaluronidase and pipetted until granulosa cells are completely detached. Oocytes with intact first polar bodies, zona pellucida, clear perivitelline space, good morphology, and homogeneous cytoplasm are selected and enucleated under an inverted microscope. The oocytes are fixed with a fixation needle at the 9 o'clock position, and the enucleation is performed with a manipulation needle at the 3 o'clock position. After enucleation, a single scattered donor cell is aspirated with the manipulation needle for nuclear injection. After nuclear injection, the reconstructed embryos are electrically activated. The fusion tank is cleaned three times with activation solution equilibrated for 15 min. The reconstructed embryos are then placed in… After rinsing three times in the activation solution, the embryos were transferred to the center of a fusion tank filled with electro-activation solution and activated three times with a DC pulse of 1.00 kV / cm and 80 μs. The reconstructed embryos after electro-activation were rinsed three times in PZM3 embryo culture medium and then transferred to microdroplets of embryo culture medium for further culture. The conditions for continued culture were 5% CO2, saturated humidity, and 39°C. The blastocysts cultured for 144 h were stained in Hoechst 33342 for 15 min, then 1-2 drops of glycerol were added to the slide, which was then mounted and spread evenly on a glass slide. Cell counting was then performed under fluorescence irradiation.

[0046] (7) Detection of apoptotic cell count in embryos: Nuclear transfer blastocysts developed for 144–168 h were collected for TUNEL assay. The nuclear transfer blastocysts were washed three times with PBS and then fixed in 4% paraformaldehyde for 30 min. After the fixed blastocysts were washed three times with PBS / PVA, they were permeabilized with 1% Triton X-100 PBS for 1 h, and then incubated in TUNEL staining solution at 38.5℃, 5% CO2, and 100% humidity for 1.5 h. After washing three times with PBS / PVA, the nuclei were stained with 10 μg / mL Hoechst 33342 for 10 min. The processed samples were placed on a glass slide with an anti-quenching agent and sealed with Vaseline, and observed under a fluorescence microscope.

[0047] Example 2:

[0048] The method of using mangiferin to improve the in vitro maturation and development rate of porcine oocytes is different from that in Example 1, the amount of mangiferin used in step (1) is 100 μM, and the rest is the same as in Example 1.

[0049] Example 3:

[0050] The method of using mangiferin to improve the in vitro maturation and development rate of porcine oocytes is different from that in Example 1, the amount of mangiferin used in step (1) is 150 μM, and the rest is the same as in Example 1.

[0051] Comparative Example 1:

[0052] Compared with Example 1, no mangiferin was added in step (1), the amount of mangiferin used was 0, and the rest was the same as in Example 1.

[0053] Comparative Example 2:

[0054] Compared with Example 1, no mangiferin was added in step (1), and the amount of mangiferin used was 0. Instead, idebenone was added at a dosage of 100 μM, and the rest was the same as in Example 1.

[0055] The in vitro maturation rate of oocytes, the apoptosis rate of oocytes, and the 2-cell cleavage rate, blastocyst rate, and total number of blastocyst cells of nuclear transfer embryos were measured using each group of culture media.

[0056] The experimental results are shown in Tables 1 and 2:

[0057] Table 1. Effects of different treatment groups on in vitro maturation efficiency of porcine oocytes

[0058] Group oocyte count First polarity yield (%) Apoptosis rate (%) Example 1 156 <![CDATA[80.37±1.15 b ]]> <![CDATA[11.36±0.26 b ]]> Example 2 174 <![CDATA[85.49±1.23 a ]]> <![CDATA[9.61±1.33 c ]]> Example 3 168 <![CDATA[83.03±0.96 a ]]> <![CDATA[11.47±0.83 b ]]> Comparative Example 1 143 <![CDATA[76.23±1.81 c ]]> <![CDATA[13.15±1.14 a ]]> Comparative Example 2 16 <![CDATA[81.49±1.02 b ]]> <![CDATA[11.82±1.53 b ]]>

[0059] Note: Different letters in the superscript of data in the same column indicate significant differences (P<0.05), while no letter or the same letter indicates no significant differences (P>0.05).

[0060] Table 2. Effects of different treatment groups on in vitro development of porcine nuclear transfer embryos.

[0061] Group Number of reconstructed embryos 2-cell cleavage rate Blastocyst rate (%) Total number of blastocyst cells Example 1 98 <![CDATA[87.09±1.41 a ]]> <![CDATA[33.34±1.59 b ]]> <![CDATA[42.67±2.13 b ]]> Example 2 119 <![CDATA[88.15±1.69 a ]]> <![CDATA[37.11±1.41 a ]]> <![CDATA[49.53±3.21 a ]]> Example 3 107 <![CDATA[86.63±0.72 a ]]> <![CDATA[34.75±0.88 b ]]> <![CDATA[39.78±1.52 b ]]> Comparative Example 1 90 <![CDATA[81.41±1.35 b ]]> <![CDATA[29.27±1.22 c ]]> <![CDATA[33.82±0.87 c ]]> Comparative Example 2 122 <![CDATA[82.73±0.99 b ]]> <![CDATA[33.96±2.37 b ]]> <![CDATA[39.08±1.29 b ]]>

[0062] Note: Different letters in the superscript of data in the same column indicate significant differences (P<0.05), while no letter or the same letter indicates no significant differences (P>0.05).

[0063] As shown in Tables 1 and 2, compared with the basal culture medium group, the culture medium provided by this invention can effectively improve the in vitro maturation rate of porcine oocytes, reduce the apoptosis rate, increase the 2-cell cleavage rate and blastocyst rate of porcine nuclear transfer embryos, and increase the total number of blastocyst cells, thereby promoting the in vitro developmental potential of nuclear transfer embryos. This indicates that the culture medium with added mangiferin can better improve the in vitro maturation rate of oocytes and the in vitro developmental potential of transferred embryos.

[0064] Compared with the embodiments, no mangiferin was added to the culture medium in Comparative Example 1. The results showed that, compared with the embodiments, the oocyte in vitro maturation rate, the 2-cell cleavage rate, blastocyst rate and total number of blastocyst cells of the porcine embryos cultured in vitro were all reduced in Comparative Example 1, while the apoptosis rate of oocytes and embryos was increased. This indicates that mangiferin can improve the oocyte in vitro maturation rate and the in vitro developmental potential of transplanted embryos.

[0065] Compared with Example 2, Comparative Example 2 added another drug, idebenone, which can improve embryonic development. The results showed that the oocyte in vitro maturation rate, the 2-cell cleavage rate, blastocyst rate, and the total number of blastocyst cells of the in vitro cultured pig embryos all decreased, while the apoptosis rate of oocytes and embryos increased. This indicates that mangiferin can improve the oocyte in vitro maturation rate and the in vitro developmental potential of transplanted embryos.

[0066] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. Use of mangiferin for promoting maturation of porcine oocytes, characterized in that, The application promotes the maturation of pig oocytes, improves the efficiency of in-vitro maturation of oocytes or reduces the apoptosis rate of oocytes in-vitro; the application in promoting the maturation of pig oocytes is specifically a method of adding 50 μM or 100 μM or 150 μM mangiferin to a maturation culture solution of pig oocytes, and then culturing pig oocytes with the maturation culture solution.

2. Use according to claim 1, characterized in that, The mangiferin is extracted from mango leaves, peels and kernels.

3. Use according to claim 1, characterized in that, The molecular formula of the mangiferin is C 19 H 18 O 11 .

4. Use according to claim 3, characterized in that, The mangiferin has a molecular weight of 422.

34.

5. Use according to claim 3, characterized in that, The mangiferin has the following structural formula: 。

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