CN113151159A - Oocyte in-vitro maturation culture solution additive and application thereof - Google Patents
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Abstract
The invention discloses an oocyte in-vitro maturation culture solution additive and application thereof, and belongs to the technical field of biology. According to the lactoferrin, the first polar body discharge rate of the oocyte can be remarkably improved, the ROS content of the oocyte can be remarkably reduced, the blastocyst rate of the embryo after in vitro fertilization can be remarkably improved, the maturation rate and the quality of the oocyte can be further remarkably improved, and the lactoferrin can be used for preparing an oocyte in vitro maturation culture solution additive or an oocyte in vitro maturation culture solution. The invention can be applied to the embryo engineering technology, and improves the success rate and the quality of the embryo engineering.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an oocyte in-vitro maturation culture solution additive and application thereof.
Background
The in vitro fertilization embryo, the clone embryo, the parthenogenetic embryo and the transgenic embryo of the mammal are widely applied to the technical fields of embryo engineering such as genetic improvement, the research of early development mechanism of the embryo, epigenetic regulation and the like, the in vitro culture quality of the oocyte is a key factor for determining the development of the embryo, but the maturation efficiency and the quality of the in vitro culture of the oocyte are not greatly improved so far. Reactive Oxygen Species (ROS) is an important factor affecting the in vitro culture of oocytes. ROS are a product of biological aerobic metabolism and include oxygen ions, superoxide ions, hydroxyl radicals, hydrogen peroxide, and the like. In equilibrium, ROS play a beneficial role as signaling molecules in physiological processes such as hormonal signaling, intracellular redox regulation, and embryonic development. However, in vitro culture is a static environment, without the exchange of nutrients and metabolites, and is prone to cause the accumulation of ROS, thereby altering their function and impairing cell survival. Therefore, ROS negatively affect oocyte viability, gene expression, protein synthesis and molecular signaling, affecting oocyte maturation and developmental competence.
Disclosure of Invention
The invention aims to provide an oocyte in-vitro maturation culture solution additive and application thereof, so as to solve the problems.
According to one aspect of the invention, the application of lactoferrin in improving the in vitro maturation rate and quality of oocytes is provided, the lactoferrin can obviously improve the first polar body discharge rate of the oocytes, obviously reduce the ROS content of the oocytes, and obviously improve the blastocyst rate of embryos fertilized in vitro, so that the maturation rate and quality of the oocytes can be greatly improved. Based on the method, the lactoferrin also has the application of preparing oocyte in vitro maturation culture solution additives or oocyte in vitro maturation culture solutions.
In one aspect of the present invention, there is provided an additive for a culture solution for in vitro maturation of oocytes, the additive comprising lactoferrin. Therefore, the first polar body discharge rate of the oocyte can be obviously improved, the ROS content of the oocyte can be obviously reduced, the blastocyst rate of the embryo after in vitro fertilization can be obviously improved, and the maturation rate and the quality of the oocyte can be greatly improved.
In certain embodiments, the lactoferrin concentration in the oocyte in vitro maturation medium additive is 50-200 μ g/mL. Therefore, the oocyte cultured by the lactoferrin additive with the concentration range has higher maturation rate and better quality.
In certain embodiments, the lactoferrin concentration of the oocyte in vitro maturation medium additive is 100 μ g/mL. Therefore, the oocytes cultured in vitro with lactoferrin added at a concentration of 100. mu.g/mL had the highest maturation rate and the best quality.
In another aspect of the present invention, there is provided an oocyte in vitro maturation culture solution, the culture solution including lactoferrin; preferably, the culture broth comprises 50-200 μ g/mL lactoferrin; more preferably, the culture medium contains lactoferrin at a concentration of 100. mu.g/mL. Therefore, the oocyte in-vitro maturation rate and the in-vitro maturation quality of the oocytes can be greatly improved by using the oocyte in-vitro maturation culture solution containing lactoferrin and/or lactoferrin of 50-200 mug/mL and/or lactoferrin of 100 mug/mL.
In some embodiments, the oocyte in vitro maturation medium comprises: oocyte basic culture fluid, 2mM glutathione, 100 mu M cysteine, 0.3mM sodium pyruvate, 1 mu g/mL estradiol, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10IU/mL pregnant horse serum gonadotropin, 10IU/mL human chorionic gonadotropin and lactoferrin. Therefore, the culture solution can greatly improve the in vitro maturation rate and the in vitro maturation quality of the oocyte.
In some embodiments, the oocyte in vitro maturation medium comprises: oocyte basic culture fluid, 2mM glutathione, 100 MuM cysteine, 0.3mM sodium pyruvate, 1 Mug/mL estradiol, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10IU/mL pregnant horse serum gonadotropin and 10IU/mL human chorionic gonadotropin, and 50-200 Mug/mL lactoferrin. Therefore, the culture solution can greatly improve the in vitro maturation rate and the in vitro maturation quality of the oocyte.
In some embodiments, the oocyte in vitro maturation medium comprises: oocyte basic culture fluid, 2mM glutathione, 100 mu M cysteine, 0.3mM sodium pyruvate, 1 mu g/mL estradiol, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10IU/mL pregnant horse serum gonadotropin and 10IU/mL human chorionic gonadotropin, 100 mu g/mL lactoferrin. Therefore, the culture solution has the best effect on improving the in vitro maturation rate and the in vitro maturation quality of the oocyte.
In another aspect, the invention provides an application of the oocyte in vitro maturation culture solution additive or the oocyte in vitro maturation culture solution in embryo engineering technology. Therefore, the in vitro maturation rate and quality of the oocyte can be greatly improved, and the success rate and quality of the somatic cell cloned embryo can be improved when the oocyte cloning method is applied to a somatic cell cloning technology.
In certain embodiments, the concentration of lactoferrin in the oocyte in vitro maturation culture fluid additive or oocyte in vitro maturation culture fluid applied to the somatic cloning technique is 50-200 μ g/mL. Therefore, the in vitro maturation rate and quality of the oocyte can be greatly improved, and the success rate and quality of the somatic cell cloned embryo can be improved when the oocyte in vitro maturation rate and quality are further applied to a somatic cell cloning technology.
In certain embodiments, the concentration of lactoferrin in the oocyte in vitro maturation medium additive or oocyte in vitro maturation medium applied to the somatic cloning technique is 100 μ g/mL. Therefore, the in vitro maturation rate and quality of the oocyte can be improved with the best effect, and the success rate and quality of the somatic cell cloned embryo can be improved when the oocyte in vitro maturation rate and quality are further applied to a somatic cell cloning technology.
The invention has the beneficial effects that:
1. lactoferrin is added into the oocyte maturation culture solution, the first polar body discharge rate of the oocyte can be obviously improved, the ROS content of the oocyte can be obviously reduced, the embryo blastocyst rate after in vitro fertilization can be obviously improved, and therefore the maturation rate and the quality of the oocyte can be greatly improved.
2. The lactoferrin-containing oocyte maturation culture solution can remarkably improve the first polar body discharge rate of oocytes, remarkably reduce the ROS content of the oocytes, and remarkably improve the blastocyst rate of embryos after in vitro fertilization, so that the maturation rate and the quality of the oocytes can be greatly improved.
3. Lactoferrin is applied to the embryo engineering technology and is used as an additive of oocyte maturation culture solution, the first polar body discharge rate of oocytes can be obviously improved, the ROS content of the oocytes is obviously reduced, the embryo blastocyst rate after in vitro fertilization is obviously improved, the maturation rate and the quality of the oocytes can be greatly improved, and further, the lactoferrin is applied to the embryo engineering technology and can improve the success rate and the quality of embryo engineering.
Drawings
FIG. 1 is a fluorescence plot of the effect of different concentrations of lactoferrin treatment on oocyte ROS levels.
FIG. 2 is a graph of the results of different concentrations of lactoferrin treatment on the ROS level in oocytes.
Detailed Description
The following examples are intended to further illustrate the present invention but should not be construed as limiting the scope of the invention, which is not limited to the examples described below. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
1. Oocyte collection and maturation culture
Sheep ovaries were harvested from slaughter houses, returned to the laboratory in Saline at 34 ℃, rinsed 3 times with Saline supplemented with 200IU of streptomycin, and after aspiration of 2-8mm follicles using a 10mL syringe equipped with an 18G needle, the follicular fluid was collected in a 50mL conical centrifuge tube, allowed to settle for 10min, the supernatant was discarded, the sediment was resuspended in DPBS (Dulbecco's phophate-Buffered Saline, doodle Phosphate buffer) containing 0.1% PVA (Polyvinyl alcohol), cumulocyte complexes (COCs) that were packed above 3 layers and that were homogeneous cytoplasms were collected under a stereomicroscope, after three washes in mature culture fluid, the COCs were transferred to mature culture fluid containing 500 μ L of different groups of mature culture fluid described below and having been treated at 38.5 ℃ with 5% CO250 COCs in a Nunc four-hole culture dish balanced for 2h in an incubator with saturated humidity, the culture dish is placed at 38.5 ℃ and 5% CO2And culturing for 24h in an incubator with saturated humidity. Transferring the cumulus oocyte complex into 0.1% (w/v) hyaluronidase for 5min, repeatedly blowing with a pipette with appropriate caliber, removing cumulus cells, selecting oocytes under a stereoscopic microscope, wherein oocytes with obvious perivitelline space, no impurities, uniform cytoplasm and obviously discharged first polar body are mature oocytesThe cell, without perivitelline space, cytoplasmic divergence was considered a dead oocyte.
The mature culture solution is as follows: TCM-199(Gibco) base solution, added 2mM glutathione, 100. mu.M cysteine, 0.3mM sodium pyruvate, 1. mu.g/mL estradiol, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10IU/mL pregnant horse serum gonadotropin (PMSG), and 10IU/mL human chorionic gonadotropin (hCG). Mature cultures of COCs are divided into five groups: control (mature culture without lactoferrin addition, control), 50. mu.g/mL (mature culture with lactoferrin addition, 50. mu.g/mL), 100. mu.g/mL (mature culture with lactoferrin addition, 100. mu.g/mL), 150. mu.g/mL (mature culture with lactoferrin addition, 150. mu.g/mL), 200. mu.g/mL (mature culture with lactoferrin addition, 200. mu.g/mL). After 24h maturation culture, the discharge rate of the first polar body of the group cells of 100 mu g/mL is obviously improved (P is less than 0.05), and the maturation culture solution added with lactoferrin of 100 mu g/mL can obviously improve the maturation rate of the oocyte (P is less than 0.05), and the results are shown in Table 1.
TABLE 1 Effect of Lactoferrin on oocyte maturation
Remarking: data from 3 replicates were statistically analyzed, with different lower case letters in the same column indicating significant differences (P < 0.05), as follows.
2. Mature oocyte ROS level detection
The ROS level in oocytes was detected using the ROS detection kit (Sigma-Aldrich). Oocytes were incubated in H-SOF (Hepes-synthetic ovoid, Hepes buffered synthetic oviduct fluid, SOF synthetic oviduct fluid) containing 10. mu.MDFH-DA (2, 7-dichloro-hydro fluorescein diacetate) in the absence of light for 30min, 0.3mM sodium pyruvate, 25mM sodium bicarbonate, 5.4mM sodium lactate, 108mM sodium chloride, 7.2mM potassium chloride, 1.2mM potassium dihydrogen phosphate, 1.8mM calcium chloride, 1.5mM magnesium chloride, 1.3. mu.g/mL phenol red, 4mg/mL albumin, and then the oocytes were washed 1-2 times with H-SOF, fluorescence signals were detected and photographed on a confocal laser microscope, and the relative ratio of the fluorescence intensity of the oocytes was analyzed with the software of ageJ. The five oocytes were tested following the same procedure including incubation, washing, imaging. After oocyte maturation for 24h, the maturation medium supplemented with lactoferrin at 100. mu.g/mL (100. mu.g/mL group) reduced the intracellular% of ROS content to the greatest extent as compared to the control group, as shown in FIGS. 1-2.
3. In vitro fertilization, in vitro culture and blastocyst cell counting of oocytes
Five groups of matured oocytes were washed 3 times in the receptor fluid and transferred into a four-well plate previously placed in an incubator to be pre-equilibrated for 2 hours, containing 500. mu.L/well of receptor fluid (SOF + 2% oestrous sheep serum +2mM sodium pyruvate + 10. mu.g/mL heparin +1mM caffeine), and 40 matured oocytes were placed in each well. The frozen sperm of the Dorper sheep is adopted, and the viable sperm is separated by gradient centrifugation with 90 percent Percoll and 45 percent Percoll (diluted by fertilization fluid), and is centrifuged for 15 minutes at 2100 rpm. Diluting sperm at the bottom of the centrifugal tube with a fertilization solution, and adding the diluted sperm into the sperm receiving hole to enable the sperm density in the sperm receiving hole to reach 1 × 106one/mL. Placing the fertilization well plate at 38.5 deg.C and 5% CO2、5％O2The fertilization is carried out for 18-24h in a saturated humidity incubator.
The embryos after 24H fertilization are removed from the fertilization solution and placed in H-SOF, and blowing and beating are repeated to remove the sperms attached to the surfaces of the embryos. After 3 washes in culture medium (SOF +4mg/mL BSA + 2% essential amino acid (Thermo Fisher) + 1% non-essential amino acid (Sigma Aldrich) +2mM glutathione +1mM sodium pyruvate), they were transferred to a petri dish containing 7 drops of 60. mu.L each, covered with mineral oil, 6-10 embryos and placed at 38.5 ℃ in a petri dish of 5% CO2、5％O2An incubator saturated with humidity. Cleavage was observed 48 hours after fertilization, and blastocyst rate was observed until day 7 (day 0 for fertilization) after half-way replacement on days 3, 5, and 7 (10% FBS was added to the culture medium).
Selecting in vitro fertilization embryos which have developed to blastocysts, fixing the embryos by 4% paraformaldehyde for 10min, then staining the embryos by 10mg/mL Hoechst33342 for 10min, and observing and recording the number of blastocyst cells under a fluorescence microscope after tabletting. The lactoferrin group of 100 ug/mL was added to the mature culture medium to culture the mature oocytes, and the blastocyst rate after in vitro fertilization was significantly higher than that of the control group (P < 0.05), and the results are shown in Table 2.
TABLE 2 development and quality of Lactoferrin supplemented group culture of mature oocytes in vitro fertilized embryos
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (9)
1. Application of lactoferrin in improving oocyte in-vitro maturation rate and quality.
2. Application of lactoferrin in preparation of oocyte in vitro maturation culture solution additive.
3. Application of lactoferrin in preparation of oocyte in-vitro maturation culture solution.
4. An additive for a culture solution for in vitro maturation of oocyte, which is characterized in that: the additive comprises lactoferrin.
5. An oocyte in-vitro maturation culture solution, which is characterized in that: the culture solution contains the additive according to claim 4.
6. The culture solution according to claim 5, wherein: the culture solution comprises: oocyte basic culture fluid, 2mM glutathione, 100 mu M cysteine, 0.3mM sodium pyruvate, 1 mu g/mL estradiol, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10IU/mL pregnant horse serum gonadotropin, 10IU/mL human chorionic gonadotropin and lactoferrin.
7. The use of the oocyte in vitro maturation culture medium additive according to claim 4 or the oocyte in vitro maturation culture medium according to claim 5 or 6 in embryo engineering technology.
8. The use according to any one of claims 1 to 3, the oocyte in vitro maturation medium additive according to claim 4, the oocyte in vitro maturation medium additive according to claim 5 or 6, or the use according to claim 7, wherein: the concentration of the lactoferrin is 50-200 mug/mL.
9. The use according to any one of claims 1 to 3, the oocyte in vitro maturation medium additive according to claim 4, the oocyte in vitro maturation medium additive according to claim 5 or 6, or the use according to claim 7, wherein: the concentration of the lactoferrin is 100 mug/mL.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114410573A (en) * | 2021-12-27 | 2022-04-29 | 内蒙古大学 | Oocyte in-vitro maturation culture solution additive and application thereof |
| CN114410573B (en) * | 2021-12-27 | 2024-09-27 | 内蒙古大学 | Oocyte in-vitro maturation culture solution additive and application thereof |
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