In vitro maturation medium for buffalo oocytes containing celastrol and culture method thereof

Abstract

The present invention discloses an in vitro maturation medium for buffalo oocytes containing celastrol, and a culture method using the in vitro maturation medium for buffalo oocytes. The in vitro maturation medium for buffalo oocytes comprises celastrol and a solvent, and the solvent is TCM-199 culture solution containing NaHCO3, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, buffalo estrus bovine serum, yellow cattle follicular fluid, follicle stimulating hormone, luteinizing hormone, and cysteine. The in vitro maturation medium for buffalo oocytes of the present invention can significantly improve the quality of buffalo in vitro matured oocytes and the developmental ability of subsequent embryos. The cost of celastrol is moderate and it is easy to obtain, and it has extremely high popularization value and application prospect in the in vitro production of buffalo embryos.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an in vitro maturation medium for buffalo oocytes containing celastrol and a culture method thereof. Background Art

[0002] With the continuous progress of embryo biotechnology, compared with eggs matured naturally in vivo, obtaining immature eggs from excised ovaries and performing in vitro maturation culture has become an important method. This method can not only obtain a large number of mature eggs, but also greatly promote the research and production applications in subsequent fields such as in vitro fertilization, somatic cell cloning, and embryonic stem cells. However, the current in vitro maturation efficiency of buffalo oocytes is still insufficient, and this bottleneck problem severely restricts the in-depth exploration and wide application of buffalo embryo bioengineering technology.

[0003] Celastrol, as a natural bioactive nucleoside antibiotic, its diverse pharmacological effects have attracted wide attention, including multiple effects such as anti-tumor, anti-virus, antibacterial, anti-inflammatory, and immunomodulation. Particularly noteworthy is that celastrol exhibits significant antioxidant properties, can effectively counteract cancer neovascularization, and shows outstanding effects in the research of anti-cancer, tumor treatment, and the field of cranial nerves.

[0004] During the in vitro maturation of eggs, a large amount of reactive oxygen species (ROS) are generated during the metabolic activities of immature eggs themselves and in vitro operation processes, thereby triggering oxidative stress responses. This phenomenon not only interferes with the normal meiosis process of eggs, but may also lead to maturation arrest, making the maturation paces of the egg cytoplasm and nucleus inconsistent, and ultimately weakening the developmental potential of subsequent embryos. Therefore, how to effectively reduce the damage of oxidative stress to eggs during the in vitro maturation of eggs has always been a research hotspot that scholars in this field are committed to overcoming. Although celastrol shows great potential in multiple biomedical fields, its application in the in vitro maturation process of eggs has not been reported yet, and this blank field urgently needs to be explored and developed. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide an in vitro maturation medium for buffalo oocytes containing celastrol, as well as a culture method using this in vitro maturation medium for buffalo oocytes, which can effectively improve the subsequent developmental ability and embryo quality of in vitro matured buffalo eggs.

[0006] To achieve the above purpose, the technical solutions provided by the present invention are as follows:

[0007] An in vitro maturation medium for buffalo oocytes containing celastrol, wherein the in vitro maturation medium for buffalo oocytes contains celastrol.

[0008] Preferably, the in vitro maturation medium for buffalo oocytes comprises celastrol and a solvent, and the solvent is TCM-199 culture medium containing NaHCO3, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, buffalo estrus bovine serum, yellow cattle follicular fluid, follicle-stimulating hormone, luteinizing hormone, and cysteine.

[0009] Preferably, the molar concentration of celastrol in the solvent is 1 μM.

[0010] Preferably, in the solvent, the molar concentration of NaHCO3 is 26.2 mM, the molar concentration of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid is 5 mM, the mass percentage of buffalo estrus bovine serum is 10%, the mass percentage of yellow cattle follicular fluid is 3%, the concentration of follicle-stimulating hormone is 0.5 μg / mL, the concentration of luteinizing hormone is 5 μg / mL, and the concentration of cysteine is 10 μg / mL.

[0011] The present invention also provides a culture method for culturing buffalo oocytes using the above-mentioned in vitro maturation medium for buffalo oocytes, comprising the following steps: selecting oocytes with a complete cumulus cell coat, washing them first with an egg washing solution, then washing them with the in vitro maturation medium for buffalo oocytes, and then placing the oocytes into the in vitro maturation medium for buffalo oocytes for in vitro maturation culture of the oocytes.

[0012] Preferably, the egg washing solution comprises celastrol and a solvent, and the solvent is TCM-199 culture medium containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, NaHCO3, bovine serum albumin, penicillin, and streptomycin.

[0013] Preferably, the molar concentration of celastrol in the solvent of the egg washing solution is 1 μM.

[0014] Preferably, in the solvent of the egg washing solution, the molar concentration of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid is 20 mM, the molar concentration of NaHCO3 is 5 mM, the mass percentage of bovine serum albumin is 4%, the concentration of penicillin is 0.003 g / mL, and the concentration of streptomycin is 0.005 g / mL.

[0015] Preferably, the conditions for in vitro maturation culture of the oocytes are: culturing in an environment of 38.5 °C and 5% CO2 for 22 - 24 h.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides an in vitro maturation medium for buffalo oocytes containing celastrol, and also provides a specific method for in vitro culture of buffalo oocytes using this maturation medium. By applying this in vitro maturation medium for buffalo oocytes containing celastrol to the in vitro maturation process of buffalo oocytes, compared with the traditional in vitro maturation medium for buffalo oocytes without celastrol, the present invention can significantly improve the quality of in vitro matured buffalo oocytes and the developmental ability of subsequent embryos. In addition, the cost of celastrol is moderate and it is easy to obtain, which makes the present invention have extremely high popularization value and application prospect in the actual in vitro production of buffalo embryos. Description of the Drawings

[0018] Figure 1 It is a diagram of the total number of inner cell masses in blastocysts of in vitro matured buffalo oocytes;

[0019] In the figure, A is the control group, B is the 1 μM celastrol group, and C is the 2 μM celastrol group. Detailed Description of the Embodiments

[0020] The following is a detailed description in combination with specific embodiments, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. The raw materials used in the examples are all commercially available unless otherwise specified.

[0021] Example 1 In Vitro Maturation Culture of Buffalo Oocytes

[0022] 1. Preparation of Bovine Follicular Fluid (BFF)

[0023] (1) Prepare 10 ml centrifuge tubes, 0.5 ml EP tubes and 18 ml glass bottles in advance (all sterilized by high temperature or high pressure);

[0024] (2) Use a 10 ml syringe to draw follicular fluid from follicles on the surface of bovine ovaries with a diameter of 2 - 8 mm and collect it in a centrifuge tube;

[0025] (3) After collection, centrifuge at 3500 r / min for 10 min, discard the precipitate, and repeat 2 - 3 times;

[0026] (4) Filter with a 0.22 μm filter membrane and aliquot into pre-sterilized 18 ml glass bottles;

[0027] (5) Aliquot into 0.5 ml EP tubes, 30 μl per tube;

[0028] (6) Label the aliquoted BFF (indicating the date, category, etc.) and store it at -20 °C;

[0029] 2. Preparation of Buffalo Estrus Cow Serum (ECS)

[0030] (1) Prepare 50 ml centrifuge tubes and 10 ml plastic test tubes (both sterilized by autoclaving) in advance, as well as a test tube rack, etc.;

[0031] (2) Use a sterile needle and centrifuge tube to collect blood from the jugular vein of the female water buffalo on the day of estrus, and let it stand for 4 - 5 h;

[0032] (3) After standing, place it in a 4°C refrigerator overnight;

[0033] (4) After all the serum has separated out, centrifuge at 3000 r / min for 15 min, and aspirate the serum. Then centrifuge the separated serum at the same speed for 10 min, and discard the precipitate;

[0034] (5) Inactivate the serum in a 56°C water bath for 30 min, and then let it cool to room temperature naturally;

[0035] (6) Finally, aliquot the inactivated serum into 10 ml test tubes, with about 8 ml in each tube.

[0036] (7) Label the aliquoted serum (indicating the date, category, etc.), cool it in a 4°C refrigerator and then store it at -20°C.

[0037] 3. Preparation of the granulosa cell monolayer culture system

[0038] (1) After dispersing the granulosa cells around the mature water buffalo oocytes on the same day, remove the oocytes and let it precipitate for a while to prepare for making a monolayer;

[0039] (2) Take out several disposable dishes, label them, use a pipette to aspirate 14 - 20 μl to make drops, and then cover with mineral oil;

[0040] (3) Place it in an incubator at 39°C filled with air saturated with 5% CO2 and with saturated humidity for culture;

[0041] (4) After two days, observe the growth situation under a microscope. If it can be used, the granulosa cell monolayer culture system is obtained. 4. Test method

[0042] Use a 10 ml syringe with a 12 - gauge needle to aspirate follicles of 2 - 6 mm from the excised water buffalo ovaries, select oocytes with a complete cumulus cell coat under an ordinary stereomicroscope, wash them twice with the oocyte washing solution first, and then wash them twice with the in vitro maturation culture medium; Place 70 - 80 water buffalo oocytes in a 25 mm × 10 mm glass culture dish, add 1 ml of the in vitro maturation culture medium, and perform in vitro maturation culture of the oocytes for 24 h in an environment of 38.5°C and 5% CO2.

[0043] The test is divided into a control group, a 1 μM celastrol group, and a 2 μM celastrol group. The formulations of the oocyte washing solution and the in vitro maturation culture medium used in each group are shown in Tables 1 - 6:

[0044] Table 1 Formula of the oocyte washing solution for the control group

[0045]

[0046]

[0047] Table 2 Formula of the in vitro maturation culture solution for the control group

[0048]

[0049] Table 3 Formula of the oocyte washing solution for the 1 μM celastrol group

[0050]

[0051] Table 4 Formula of the in vitro maturation culture solution for the 1 μM celastrol group

[0052]

[0053] Table 5 Formula of the oocyte washing solution for the 2 μM celastrol group

[0054]

[0055] Table 6 Formula of the in vitro maturation culture solution for the 2 μM celastrol group

[0056]

[0057] After the culture was completed, in vitro matured buffalo oocytes were obtained. Oocytes with uniform cytoplasm and extrusion of the first polar body were selected for the evaluation of the subsequent developmental potential of the oocytes. They were treated in 5 μM ionomycin for 5 min, then incubated in 2.5 mM 6-dimethylaminopurine for 4 h, and then cultured in a buffalo granulosa cell monolayer co-culture system. 15 - 20 embryos were placed in 20 μl of each drop of the buffalo granulosa cell monolayer co-culture system, and the culture medium was changed by half every 48 h. The cleavage rate of the embryos was observed on the 2nd day after parthenogenetic activation, and the blastocyst formation rate was observed on the 7th day. The results are shown in Table 7.

[0058] For the total number counting of the inner cell mass of the blastocysts formed on the 7th day after parthenogenetic activation, first, the blastocysts were placed in 10 mg / ml Hoechst 33342 for light-avoiding staining for 5 min, then washed 2 - 3 times with PBS, and then squashed. Observation was carried out under a common fluorescence microscope at a magnification of 200 times, and the bright spots emitting blue fluorescence in the field of view were counted. The results are shown in Table 8.

[0059] 2. Test results

[0060] The results showed that when 1 μM celastrol was added to the in vitro maturation medium of buffalo oocytes and chemically activated, the cleavage rate on the 2nd day was 81.11%, which was significantly higher than 62.35% of the control group, an increase of 18.76% compared to the control group, and also significantly higher than 66.20% of the 2 μM celastrol group. The blastocyst formation rate on the 7th day was 30.14%, which was significantly higher than 13.21% of the control group, an increase of 16.93% compared to the control group, and also significantly higher than 9.15% of the 2 μM celastrol group (see Table 7).

[0061] Table 7 Comparison of the quality of in vitro matured buffalo oocytes

[0062]

[0063] Note: Different letters in the same column indicate significant differences (P < 0.05)

[0064] The results showed that when 1 μM celastrol was added to the in vitro maturation medium of buffalo oocytes and chemically activated, the total number of inner cell masses in the blastocysts formed on the 7th day was 86.63, which was significantly higher than 57.41 of the control group and also higher than 79.27 of the 2 μM celastrol group, indicating that the quality of the blastocysts obtained in the 1 μM celastrol group was higher than that of the control group (see Figure 1 and Table 8). The in vitro maturation medium of buffalo oocytes containing 1 μM celastrol can increase the total number of inner cell masses of buffalo blastocysts.

[0065] Table 8 Comparison of the total number of inner cell masses of blastocysts of in vitro matured buffalo oocytes

[0066] Group Number of embryos Number of inner cell masses Control group 13 57.41b 1 μM celastrol group 11 86.63a 2 μM celastrol group 15 79.27ab

[0067] The foregoing description of specific exemplary embodiments of the invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many changes and variations are possible in light of the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the invention and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the invention, as well as various different selections and changes. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. An in vitro maturation medium for buffalo oocytes containing celastrol, characterized in that: Among them, The in vitro maturation medium for buffalo oocytes contains celastrol and a solvent, and the solvent is TCM-199 culture medium containing NaHCO3, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, buffalo estrus bovine serum, yellow cattle follicular fluid, follicle-stimulating hormone, luteinizing hormone, and cysteine; the molar concentration of celastrol in the solvent is 1 μM; Among them, in the solvent, the molar concentration of NaHCO3 is 26.2 mM, the molar concentration of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid is 5 mM, the mass percentage of buffalo estrus bovine serum is 10%, the mass percentage of yellow cattle follicular fluid is 3%, the concentration of follicle-stimulating hormone is 0.5 μg / mL, the concentration of luteinizing hormone is 5 μg / mL, and the concentration of cysteine is 10 μg / mL.

2. A method for culturing buffalo oocytes using the in vitro maturation medium for buffalo oocytes of claim 1, characterized in that, It includes the following steps: Select oocytes with a complete cumulus cell coat, wash them first with an oocyte washing solution, then wash them with the in vitro maturation medium for buffalo oocytes, and then place the oocytes in the in vitro maturation medium for buffalo oocytes for in vitro maturation culture of the oocytes.

3. The cultivation method according to claim 2, characterized in that, The oocyte washing solution contains celastrol and TCM-199 culture medium containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, NaHCO3, bovine serum albumin, penicillin, and streptomycin.

4. The cultivation method according to claim 3, characterized in that: The molar concentration of celastrol in the oocyte washing solution is 1 μM.

5. The cultivation method according to claim 3, characterized in that: In the solvent, the molar concentration of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid is 20 mM, the molar concentration of NaHCO3 is 5 mM, the mass percentage of bovine serum albumin is 4%, the concentration of penicillin is 0.003 g / mL, and the concentration of streptomycin is 0.005 g / mL.

6. The cultivation method according to claim 4, characterized in that, The conditions for in vitro maturation culture of the oocytes are: culture at 38.5 °C in an environment of 5% CO2 for 22 - 24 h.

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