CN107603944B - Rosmarinic acid-added oocyte in-vitro maturation culture solution and application thereof - Google Patents
Rosmarinic acid-added oocyte in-vitro maturation culture solution and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of cell culture, in particular to an oocyte in-vitro maturation culture solution added with rosmarinic acid and application thereof. The in vitro maturation culture solution is characterized in that the components comprise rosmarinic acid; the concentration range of the rosmarinic acid is 2-200 mu M. According to the invention, the rosmarinic acid is added into the mature culture solution of the porcine oocyte, and the synergistic effect of other components is matched, so that the mature quality of the porcine oocyte is improved, and the porcine embryo development is promoted well. The result shows that the in vitro maturation culture solution provided by the invention can improve the antioxidant capacity of the oocyte, and the rosmarinic acid with proper concentration can effectively improve the maturation quality of the oocyte, so that the in vitro development potential of the porcine oocyte is improved. And the in vitro maturation culture solution is simple to prepare, convenient to operate and has a wide application prospect.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to an oocyte in-vitro maturation culture solution added with rosmarinic acid and application thereof.
Background
In vitro maturation of mammalian oocytes is a key precursor to embryo engineering, such as in vitro production of animal embryos and transgenic cloning. In the in vitro microenvironment of oocyte maturation, if some active oxygen substances generated by oxygen participating in metabolism are not cleared in time, the phenomenon of oxidative stress can be induced. Research shows that oxidative stress is one of the main factors influencing the quality of oocytes and the development potential of embryos, and a great deal of oxidative stress can cause the change of the morphology and the function of mitochondria in the oocytes, further influence the synthesis of Adenosine Triphosphate (ATP), interfere meiosis in the oocytes, increase the number of aneuploid chromosomes in the oocytes, cause the damage of embryo DNA and the early development arrest, and finally cause poor pregnancy outcome. In vitro culture, the adverse effects of oxidative stress are often antagonized by the addition of exogenous antioxidants or free radical scavengers, as well as by establishing and maintaining a hypoxic environment required for oocyte and embryo development. Therefore, optimizing the oocyte in vitro maturation culture condition has important significance for improving the in vitro embryo production efficiency.
Disclosure of Invention
The invention provides an oocyte in-vitro maturation culture solution added with rosmarinic acid and application thereof.
The first object of the present invention is to provide an oocyte in vitro maturation culture solution supplemented with rosmarinic acid, the components of which include rosmarinic acid. Preferably, the concentration of rosmarinic acid is 2-200. mu.M.
Further preferably, the concentration range of the rosmarinic acid is 25-100 mu M, and 50 mu M is the optimal range.
According to the invention, rosmarinic acid is added into the oocyte in-vitro maturation culture solution, so that the oocyte maturation quality is improved, and the oocyte in-vitro maturation culture solution has a good effect of promoting embryo development.
Preferably, the components also comprise conventional culture solution of oocyte, glutamine, gentamicin, pregnant mare serum gonadotropin, human chorionic gonadotropin, pig follicular fluid and serum;
preferably, the volume ratio of the conventional oocyte culture solution is 70-80%, the concentration of glutamine is 0.02-0.06g/L, the concentration of gentamicin is 0.1-0.2g/L, the concentration of pregnant mare serum gonadotropin is 20000-25000U/L, the concentration of human chorionic gonadotropin is 10000-15000U/L, the volume ratio of the pig follicular fluid concentration is 8-12%, and the volume ratio of the serum concentration is 8-12%.
Regarding the conventional oocyte culture solution, preferably, the conventional oocyte culture solution is one of Medium199, DMEM, TCM. Further preferably Medium 199.
With respect to serum, preferably, the serum is one of calf serum, fetal bovine serum or neonatal bovine serum.
The invention provides a relatively preferable oocyte in-vitro maturation culture solution, which comprises the following formula: medium199 concentration volume ratio of 75-80%, glutamine concentration of 0.02-0.06g/L, gentamicin concentration of 0.12-0.16g/L, pregnant mare serum gonadotropin concentration of 20000-25000U/L, human chorionic gonadotropin concentration of 10000-15000U/L, pig follicular fluid concentration volume ratio of 8-12%, serum concentration volume ratio of 8-12%, and rosmarinic acid concentration of 25-80 μ M.
The in vitro maturation culture solution provided by the invention can improve the antioxidant capacity of the oocyte, and the synergistic effect of the active ingredients can effectively improve the maturation quality of the oocyte, so that the in vitro development potential of the oocyte is improved. And the in vitro maturation culture solution is simple to prepare, convenient to operate and has a wide application prospect.
The invention also provides application of the in vitro maturation culture solution in culturing oocytes of animals.
The application of the oocyte in-vitro maturation liquid to culture the oocyte comprises the step of placing the oocyte in the in-vitro maturation culture liquid of the oocyte added with the rosmarinic acid to culture.
Specifically, the culture is carried out according to the following steps:
placing the oocyte in the balanced oocyte in-vitro maturation culture solution, and then placing the oocyte in CO2And (3) mature culturing for 42-44 hours in an environment with the volume concentration of 4-6% to obtain the culture medium.
Preferably, the balance is in particular: equilibrating the oocyte in vitro maturation medium for at least 3 hours.
Preferably, the oocyte is a porcine oocyte and the temperature of the maturation culture is 38-39 ℃.
Preferably, the oocytes are Cumulus Oocyte Complexes (COCs).
Drawings
FIG. 1 is a graphical representation of the effect of rosmarinic acid on Glutathione (GSH) and Reactive Oxygen Species (ROS) levels in mature oocytes
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The pig ovaries were supplied from the slaughterhouse, placed in a 37 ℃ thermos bottle in 0.9% saline and transported back to the laboratory within 1 h.
Serum was bovine serum, purchased from Gibico, pregnant mare serum gonadotropin for injection and human chorionic gonadotropin for injection were purchased from Ningbo second hormone plant, and other materials were purchased from Sigma.
Rosmarinic acid stock solution: dissolving 10mg rosmarinic acid powder in 2.77ml sterile water to a final concentration of 10mM, filtering with 0.22 filter, packaging, and storing in refrigerator at-20 deg.C in dark place.
Physiological saline: 9g of NaCl was dissolved in 1L of sterile water and sterilized by autoclaving at high temperature.
PBS egg wash: 1G of PVA, 9.55G of PBS powder, 0.066G of penicillin G sodium and 0.05G of streptomycin are dissolved in sterile water, the volume is determined to be 1L, and the mixture is sterilized.
IVM mature liquid: the volume ratio of the M199 concentration is 78%, Glutamine is 0.04g/L, Gentamicin-stock is 0.146g/L, pregnant mare serum gonadotropin concentration is 20000U/L, human chorionic gonadotropin concentration is 10000U/L, pig follicular fluid is 10%, and bovine serum is 10%. The osmotic pressure is adjusted to about 280mOsm, and the pH is adjusted to 7.2-7.4. Filtering with 0.22 filter, packaging, and storing at-20 deg.C.
HAE hyaluronidase solution: 20mg of hyaluronidase was dissolved in 20mL of PBS, filtered through a 0.22 filter and dispensed, and stored at-20 ℃ until use.
T2 in vitro procedure fluid: NaHCO 230.168mg/mL, 0.22mg/mL of pyruvic acids (Na Salt), 3.6mg/mL of HEPES (Na Salt), 2.63mg/mL of HEPES (acid), 10% of M199(10 ×), 0.1461mg/mL of L-Glutamine, 2% of FBS, osmotic pressure adjusted to about 280mOsm, pH adjusted to 7.2-7.4.0.22, filter, subpackaged, and stored at-20 ℃ for later use.
P/A electric activation liquid: mannitol 0.25M, CACl2·2H2O 0.1mM，MgCl2·6H2O0.1 mM, HEPES (Na salt)0.5mM, PVA 0.1 g/L. Filtering with 0.22 filter, packaging, and storing at-20 deg.C.
PZM-3 in vitro embryo development solution: NaCl 108mM, NaHCO325.07mM，KCl 100mM，KH2PO40.35mM，MgSO40.4mM，Ca-Lactate·5H2O2 mM, Na-Pyvate 0.2mM, Myo-inositol 2.78mM, PhenolRed 10mM, L-Glutamine 1mM, Hypotaurin 5mM, EAA (50 ×) 2%, NEAA (100 ×) 1%, Gentamicin 0.05mg/mL, BSA 3g/L osmotic pressure adjusted to about 280mOsm, pH adjusted to 7.2-7.4.0.22, filter and split charging, and storing at-20 ℃ for later use.
CC auxiliary activating liquid: dimethylsulfoxide was used to lyse cytochalasin B and cycloheximide into 5. mu.L/mL and 1mg/mL stock solutions, respectively. To 1mL of PZM-3, 5. mu.L of cytochalasin B and 10. mu.L of cycloheximide stock solution were added to a working solution concentration.
Example 1
This example provides a rosmarinic acid-supplemented oocyte in vitro maturation medium (RA50) with the following formulation: medium199 is 78%, glutamine is 0.04g/L, gentamicin concentration is 0.146g/L, pregnant mare serum gonadotropin concentration is 20000U/L, human chorionic gonadotropin concentration is 10000U/L, pig follicular fluid concentration is 10%, serum concentration is 10% and rosmarinic acid concentration is 50 μ M.
Example 2
This example provides an oocyte in vitro maturation medium (RA2) supplemented with rosmarinic acid, differing from example 1 only in that the concentration of rosmarinic acid was 2. mu.M.
Example 3
This example provides an oocyte in vitro maturation medium (RA200) supplemented with rosmarinic acid, differing from example 1 only in that the concentration of rosmarinic acid was 200. mu.M.
Example 4
This example provides an oocyte in vitro maturation medium (RA25) supplemented with rosmarinic acid, differing from example 1 only in that the concentration of rosmarinic acid was 25. mu.M.
Example 5
This example provides an oocyte in vitro maturation medium (RA100) supplemented with rosmarinic acid, differing from example 1 only in that the concentration of rosmarinic acid was 100. mu.M.
Comparative example 1
This comparative example provides a culture solution (blank control) for in vitro maturation of oocytes, differing from example 1 only in that the composition does not include rosmarinic acid.
Test examples
The purpose of the test is as follows: verifying the effect of the in vitro maturation culture solution of the oocyte added with the rosmarinic acid;
test subjects: porcine oocytes;
and (3) test operation:
1. oocyte collection and in vitro maturation culture
Extraction of immature oocytes was performed in the embryo laboratory. Evacuating the follicle with the diameter of 3-8mm by connecting a vacuum pump with an acquisition tube, and pouring the follicle fluid containing the ovum into a50 ml centrifuge tube; precipitating for 30min, removing supernatant, washing with ovum-washing solution for 2 times at an interval of 15 min; adding ovum-washing solution in the 3 rd time, shaking up gently, pouring into a culture dish of 100mm, and picking out Cumulus Oocyte Complexes (COCs) by using a self-drawn glass Pasteur tube under a stereoscopic microscope; washing the picked COCs in the pre-balanced oocyte maturation solution for three times, putting the washed COCs into a 24-pore plate for oocyte maturation, adding 500 mu L of the in-vitro maturation culture solution prepared in the examples 1-5 and the comparative example 1 into each hole of the 24-pore plate, covering the upper layer with 300 mu L of mineral oil, and putting 75 COCs into each hole; 38.5 ℃ and 5% CO2And (5) maturing for 42h in an incubator. Three replicates were used as parallel groups.
2. Preparation of oocytes
Transferring Cumulus Oocyte Complexes (COCs) cultured for 42h into a 1.5ml centrifuge tube filled with 500 mul of hyaluronidase solution, blowing and beating the Cumulus Oocyte Complexes (COCs) for about 65 times by a200 mul pipette gun, and removing granular cells; washing the oocyte in vitro operating fluid for three times, and transferring the oocyte in vitro operating fluid into 50 mu l of operating fluid microdroplets coated with mineral oil; oocytes with the first polar body in the space between eggs were picked up in microdroplets and counted. After discarding oocytes with diffuse cytoplasm, incomplete cytoplasm, unclear perivitelline space and unclear cell membrane, the number of remaining oocytes was counted.
3. Determination of Glutathione (GSH) and Reactive Oxygen Species (ROS) levels in oocytes
20 mature oocytes were washed in PBS 3 times, placed in M199 containing 10. mu. M H2DCFDA (for ROS detection, Blue light excitation) or CellTracker Blue CMF2HC (for GSH detection, ultraviolet light excitation), and stained in an incubator at 38.5 ℃ for 20 min. After staining, oocytes were washed 3 times with PBS and then placed in 10. mu.L drops of PBS, images were collected under a fluorescence microscope and fluorescence intensity analysis was performed using Image J software.
4. Parthenogenetic activation of oocytes
The fusion tank was washed 3 times with the fusion solution, 75 μ l of the fusion solution was added to the fusion tank, and the oocytes were first equilibrated in the fusion solution for 1min and then placed in the fusion tank to which the fusion solution was added. Two direct current pulses of 80 mus at an interval of 100ms and a voltage of 1.5kV/cm are applied to activate the oocytes in a CF-150/B fusion instrument, the reconstructed embryos are picked up and washed 3 times by PZM-3, and the washed reconstructed embryos are placed in an auxiliary activating solution for treatment for 4 h.
5. Embryo culture
And taking out the oocytes from the auxiliary activating solution, removing dead eggs, washing for 3 times by using a PZM-3 solution, transferring into a culture solution PZM-3 which is well balanced in advance, and culturing in a low-oxygen culture environment (a low-oxygen incubator or sealed culture by filling a low-oxygen mixed gas distribution bag). Culturing with micro-drop or four-well plate under gas phase condition of 7% O2、88％N2And 5% CO2The culture temperature of the mixed gas of (3) was 38.5 ℃ and the humidity was 100%. Cleavage rate and blastocyst formation rate were observed and recorded at 48 and 168 hours of activation culture, respectively.
6. Blastocyst cell count
Collecting blastocysts formed by culturing embryos for 168 hours, fixing with 4% formaldehyde for 30min, breaking membranes with 1% Triton-100, staining with 0.3mM DAPI (stained nuclei) for 20min, performing conventional tabletting treatment on the blastocysts, exciting with ultraviolet light by a fluorescence microscope, and collecting images to count the number of blastocyst cells.
7. Data processing
And (3) carrying out single-factor variance analysis on the maturation rate of each group of oocytes, the levels of GSH and ROS in the oocytes, the cleavage rate after parthenogenetic activation, the blastocyst formation rate and the number of blastocyst cells by using DPS7.0 statistical software, wherein P <0.05 has statistical significance for the difference.
Test results and analysis:
1. effect of Rosmarinic acid on GSH and ROS levels in mature porcine oocytes in vitro
The effects of the different dosages of rosmarinic acid of examples 1-5 on the levels of ROS and GSH in porcine mature oocytes in vitro were observed using comparative example 1 as a blank control and different dosages of rosmarinic acid as test groups, respectively, and the results are shown in fig. 1. As can be seen from the figure, rosmarinic acid can significantly reduce the ROS level in mature oocytes and increase the GSH level, wherein the RA50 group has the best effect, and thus rosmarinic acid has a protective effect on in vitro maturation of porcine oocytes.
2. Effects of rosmarinic acid on in vitro maturation of porcine oocyte and early development of parthenogenetic embryo
The influence of the rosmarinic acid with different dosages in examples 1-5 on the in vitro development effect of the early embryo in the in vitro fertilization stage of the pig was observed by using the comparative example 1 as a blank control group and adding different dosages of rosmarinic acid as a test group, and the results are shown in table 1. The result proves that the rosmarinic acid does not influence the maturation rate and the cleavage rate of the porcine oocyte, but can obviously improve the blastocyst rate and the blastocyst cell number of the embryo after parthenogenetic activation. And the blastocyst rate and the number of blastocyst cells of the RA50 group are higher than those of other groups, which indicates that the effect of adding 50 μ M rosmarinic acid into IVM on the promotion of the early development of pig embryos is best.
TABLE 1 Effect of different concentrations of rosmarinic acid on oocyte maturation and parthenogenesis in vitro
Note: the same column is labeled with different lower case letters indicating a significant difference P < 0.05.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. An in vitro maturation culture solution for porcine oocytes is characterized by consisting of rosmarinic acid, oocyte basic culture solution, glutamine, gentamicin, pregnant mare serum gonadotropin, human chorionic gonadotropin, porcine follicular fluid and serum; the concentration range of the rosmarinic acid is 2-200 mu M.
2. The in vitro maturation culture solution of claim 1, wherein in said oocyte in vitro maturation culture solution, the volume ratio of the concentration of said oocyte basic culture solution is 70-80%, the concentration of said glutamine is 0.02-0.06g/L, the concentration of said gentamicin is 0.1-0.2g/L, the concentration of said pregnant mare serum gonadotropin is 20000-25000U/L, the concentration of said human chorionic gonadotropin is 10000-15000U/L, the volume ratio of the concentration of said porcine follicular fluid is 8-12%, and the volume ratio of the concentration of said serum is 8-12%.
3. The in vitro maturation culture Medium according to claim 1 or 2, wherein said oocyte basic culture Medium is one of Medium199, DMEM, TCM.
4. The in vitro maturation culture medium of claim 1 or 2, wherein the serum is one of calf serum, fetal bovine serum or neonatal bovine serum.
5. The in vitro maturation culture medium of claim 3, wherein the serum is one of calf serum, fetal bovine serum or neonatal bovine serum.
6. The in vitro maturation culture medium according to any one of claims 1, 2 and 5, wherein the formulation is as follows:
medium199 is 75-80% by volume, glutamine is 0.02-0.06g/L, gentamicin concentration is 0.12-0.16g/L, pregnant mare serum gonadotropin concentration is 20000-25000U/L, human chorionic gonadotropin concentration is 10000-15000U/L, pig follicular fluid concentration is 8-12% by volume, serum concentration is 8-12% by volume, and rosmarinic acid concentration is 25-80 μ M.
7. The in vitro maturation culture solution of claim 3, wherein the formulation is as follows:
medium199 is 75-80% by volume, glutamine is 0.02-0.06g/L, gentamicin concentration is 0.12-0.16g/L, pregnant mare serum gonadotropin concentration is 20000-25000U/L, human chorionic gonadotropin concentration is 10000-15000U/L, pig follicular fluid concentration is 8-12% by volume, serum concentration is 8-12% by volume, and rosmarinic acid concentration is 25-80 μ M.
8. Use of the porcine oocyte maturation culture medium of any one of claims 1 to 7 to culture oocytes,
placing the oocyte in the balanced oocyte in-vitro maturation culture solution, and placing the oocyte in CO2And (3) mature culturing for 42-44 hours in an environment with the volume concentration of 4-6% to obtain the culture medium.
9. Use according to claim 8, wherein the balancing is in particular: equilibrating the oocyte in vitro maturation medium for at least 3 hours.
10. Use according to claim 8 or 9, wherein the temperature of the maturation culture is 38-39 ℃.
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| CN110184233B (en) * | 2019-05-15 | 2023-07-25 | 湖北省农业科学院畜牧兽医研究所 | Oocyte in-vitro maturation culture solution additive and application thereof |
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| CN101429492A (en) * | 2008-12-12 | 2009-05-13 | 深圳华大基因科技有限公司 | Mature method for in vitro culture of porcine oocyte |
| CN105104890A (en) * | 2015-09-25 | 2015-12-02 | 四川农业大学 | Composition for promoting meiotic resumption of oocytes of sows and application of composition |
| CN106831403A (en) * | 2017-01-18 | 2017-06-13 | 河南佳尚农业科技发展有限公司 | It is a kind of that fat-soluble and water soluble antioxidant method is extracted from rosemary |
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| CN101429492A (en) * | 2008-12-12 | 2009-05-13 | 深圳华大基因科技有限公司 | Mature method for in vitro culture of porcine oocyte |
| CN105104890A (en) * | 2015-09-25 | 2015-12-02 | 四川农业大学 | Composition for promoting meiotic resumption of oocytes of sows and application of composition |
| CN106831403A (en) * | 2017-01-18 | 2017-06-13 | 河南佳尚农业科技发展有限公司 | It is a kind of that fat-soluble and water soluble antioxidant method is extracted from rosemary |
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