CN114908041A - Culture system and method for in vitro maturation of oocyte of mouse - Google Patents
Culture system and method for in vitro maturation of oocyte of mouse Download PDFInfo
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Abstract
The invention relates to the technical field of animal cell culture, in particular to a culture system and a culture method for in vitro maturation of mouse oocytes. The culture system for in vitro maturation of the mouse oocyte can improve the proportion and the quality of the in vitro maturation of the oocyte, so that the embryo development proportion of the oocyte of the mouse in the later stage is improved.
Description
Technical Field
The invention relates to the technical field of animal cell culture, in particular to a culture system and a culture method for in vitro maturation of mouse oocytes.
Background
Meiosis of mammalian oocytes remains in the Germinal Vesicle (GV) phase. The maturation phase of mammalian oocytes is the period from the early stage of meiosis to the metaphase of the second meiosis. In vitro maturation of oocytes is important for scientific research, while during the in vitro maturation of oocytes, the rate and quality of maturation of oocytes is crucial for the subsequent embryo development (Lin, Z et al (2014.) JMY functions as action circulation-promoting factor and mediator for p 53-processed DNA damage in cavities. PLoS One,9(10), e 109385; Wu, J et al (2018.) chromatography analysis in human early stage degradation and regeneration propagation reduction ZGA. Nature). The in vitro maturation technology of mouse oocytes is mature at present, but the quality of the oocytes cannot be detected subsequently only through in vitro maturation. If the quality of the oocytes needs to be detected, the oocytes are generally subjected to in vitro fertilization or parthenogenetic activation and then further subjected to in vitro culture and development to the blastocyst stage. However, the in vitro operation of mouse oocytes and embryos at the present stage is limited to a certain process, and the complete process from in vitro maturation to development cannot be completed, namely in vitro maturation + parthenogenesis activation/in vitro fertilization + in vitro development.
In the prior art, the oocyte in vitro maturation culture solution (MEM (Gibco11095-080) + 10% FBS +50ng/ml EGF) can mature the oocyte, but the mature oocyte is broken after parthenogenetic activation and cannot develop continuously (figure 1). This indicates that although the above-mentioned culture medium is sufficient to support the oocyte to eject the first polar body, and the nuclear maturation occurs, the cytoplasmic maturation of the oocyte is highly likely to be problematic, and thus there is a need for improvement of the basic components in the culture medium for in vitro maturation.
Another commercial oocyte basal medium, CZB (Millipore Sigma, product number MR-019-D), also matures oocytes, but activation results in a poor developmental process and little continued development, with most oocytes being fragmented (FIG. 2). This indicates that although the above-mentioned culture medium can support oocyte maturation, it cannot support its further development. This is most likely the absence of substances in the culture medium that support complete maturation of the oocytes.
Therefore, in order to overcome the defects of the existing in vitro maturation culture solution, a culture system suitable for the in vitro growth and development of the early embryo body of the mouse needs to be established, so that the in vitro embryo development quality is improved. In the field, a new culture solution for in vitro maturation of mouse oocytes is needed to be found according to the nutritional requirements and metabolic characteristics of the mouse oocytes in the developmental stage, so that the in vitro maturation rate of the oocytes is increased, and the embryonic development rate in the later stage is further increased.
Disclosure of Invention
As described above, the conventional culture medium for in vitro maturation of mouse oocytes cannot support further development of oocytes, although oocytes can be matured. Therefore, there is a need in the art for a culture system for in vitro maturation of oocytes in mice, which can increase the rate of in vitro maturation of oocytes and further increase the rate of late embryo development.
Accordingly, in a first aspect, the present invention provides a culture system for in vitro maturation of oocytes in mice, the culture system comprising:
a) a culture solution, wherein the culture solution comprises a basal culture solution, 8% to 12% serum replacement, 0.05IU/mL to 0.1IU/mL follicle stimulating hormone, 0.05IU/mL to 0.1IU/mL luteinizing hormone, and 10ng/mL to 20ng/mL epidermal growth factor;
b)100 μ M to 200 μ M cysteamine and 100 μ M to 200 μ M cysteine;
wherein a) and b) are mixed prior to use.
In a second aspect, the present invention provides a method for in vitro maturation of a mouse oocyte, wherein the mouse oocyte is cultured in the culture system of the first aspect of the invention.
The invention has the beneficial effects that: provides a culture system and a method for in vitro maturation of mouse oocytes, which can improve the proportion and the quality of the in vitro maturation of the oocytes so as to improve the proportion of the later-stage embryo development of the mouse oocytes.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other embodiments can be obtained according to the drawings without inventive labor.
FIG. 1 is a picture of oocytes matured by culturing in vitro oocyte maturation culture medium (MEM (Gibco11095-080) + 10% FBS +50ng/ml EGF) in the prior art, wherein the oocytes are fragmented after parthenogenetic activation, and can not be developed continuously.
FIG. 2 is a photograph of an oocyte matured by culturing with a prior art CZB (Millipore Sigma, product number MR-019-D), the development of the oocyte is progressed poorly and development hardly continues.
FIG. 3 is a picture of oocytes from adult mice in the blastocyst stage.
FIG. 4 is a diagram showing the development ratio of embryos at different stages under different oocyte in vitro maturation culture systems.
FIG. 5 is a picture of an embryo at different developmental stages under the oocyte in vitro maturation culture system of the present invention.
Detailed Description
The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. It is to be understood that the described embodiments are merely a subset of the present invention and not all embodiments. All other embodiments, which can be derived from the embodiments of the present invention by those skilled in the art, are within the scope of the present invention.
As described above, although the conventional culture solution for in vitro maturation of mouse oocytes can mature the surface of oocytes, the mature oocytes have poor quality, cannot be activated by parthenogenesis, have no capability of further developing into embryos, and prove that the maturation quality is poor. Therefore, the present invention aims to provide an improved culture system for in vitro maturation of mouse oocytes, which supports the oocytes to complete in vitro maturation, and the mature oocytes can be activated and have the ability to further develop into embryos.
The embryo in vitro maturation technology is related to three factors, namely embryo species, culture conditions and culture solution components, wherein the improvement of the culture solution components is the basis and key link of the in vitro culture technology, and directly influences the in vitro culture effect and the quality of in vitro embryo production.
The current research shows that Epidermal Growth Factor (EGF) plays an important role in follicular development, oocyte maturation and embryonic development, and EGF can promote the maturation of oocytes of various animals in vitro. Further research shows that EGF has a promoting effect on in vitro maturation and fertilization of mouse oocytes and can improve in vitro maturation rate and blastocyst hatching rate. Furthermore, hormones are also important in the maturation of mammalian oocytes, where Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) are required for both follicle maturation and the production of ovarian gonadal steroids. In vivo FSH initiates follicular growth, while activation of downstream signaling pathways by LH acting on follicular granulosa cells induces cytoplasmic and nuclear maturation, ovulation and formation of post-ovulatory luteal cells in the anaphase of oocyte growth. FSH and LH can obviously shorten the time of oocyte In Vitro Maturation (IVM), improve the maturation and development potential of the oocyte and improve the pregnancy rate.
Glutathione (GSH) plays an important role in the maturation of oocytes and the development of embryos, and the content of GSH in the cells can be used for evaluating the quality of the oocytes. The synthesis of Glutathione (GSH) and the embryo development ability can be enhanced by adding Cysteamine (Cysteamine) and cysteine (Cysteamine) in an in vitro culture system.
Serum is the most effective and commonly used culture component in natural culture media, and it contains many unknown components essential for maintaining cell growth and reproduction and maintaining cell biological properties. The addition of serum is beneficial to embryo development, but the source, manufacturer and batch of serum all cause the culture effect to be far from each other. Therefore, the selection of serum is also critical for culture.
In conclusion, the inventor optimizes the mature culture solution outside the embryo body of the mouse by properly adding hormone, EGF and amino acid and selecting a serum substitute to replace serum according to the specific signal path of the mouse by referring to the in-vitro oocyte mature solution system of large mammals such as pigs, cattle and sheep, so as to further improve the in-vitro maturation proportion and quality of the oocyte of the mouse, and finally improve the capability of the oocyte to develop into an embryo in vitro.
Accordingly, in a first aspect, the present invention provides a culture system for in vitro maturation of oocytes in mice, the culture system comprising:
a) a culture solution, wherein the culture solution comprises a basal culture solution, 8% to 12% serum replacement, 0.05IU/mL to 0.1IU/mL follicle stimulating hormone, 0.05IU/mL to 0.1IU/mL luteinizing hormone, and 10ng/mL to 20ng/mL epidermal growth factor;
b)100 μ M to 200 μ M cysteamine and 100 μ M to 200 μ M cysteine;
wherein a) and b) are mixed prior to use.
Energy metabolism is the basic vital activity of oocytes during in vitro development, and thus energy substances are important components in cell culture solutions. Sodium pyruvate and glucose are used as energy substances, the action mechanisms of the sodium pyruvate and the glucose are different, and the amount of the sodium pyruvate and the amount of the glucose required by different animal oocytes in vitro maturation are also different. In some embodiments, the culture fluid further comprises 3mM to 4mM D-glucose and 0.8mM to 1mM sodium pyruvate.
The inventor finds that the serum substitute is adopted to replace animal serum in the culture solution, so that the influence of uncertain factors in the serum on the maturation and development of oocytes can be eliminated, and a better culture effect can be obtained. In one embodiment, the serum replacement is knockout TM Serum Replacement (KSR). In a preferred embodiment, the serum replacement is provided at a concentration of 10%.
In one embodiment, the basal medium is TCM-199.
During the development of the oocyte, the nucleus of the oocyte in the germinal vesicle stage has increased nucleoli, active synthesis and enlarged cell nucleus, and the rupture of the germinal vesicle marks the maturation of the oocyte. Thus, in some embodiments of the present application, oocytes in the blastocyst stage are used for in vitro maturation.
In a second aspect, the present invention provides a method for in vitro maturation of a mouse oocyte, wherein the mouse oocyte is placed in the culture system of the first aspect of the present invention for culturing, for example, for 14 to 16 hours. In one embodiment, the oocyte is an oocyte in the blastocyst stage.
Examples
Test materials and methods
1. Source and Collection of oocytes
ICR and C57BL/6J mice were selected, superovulated (PMSG (5IU, Ningbo second hormone plant, cat # 110254564) injected at 14: 00-16: 00 in the afternoon, mice were sacrificed by cervical dislocation after 46-48 hours, ovaries were dissected, and then dissected by 26-gauge needle to obtain oocytes, and healthy and intact oocytes in the blastocyst stage were selected under a microscope (FIG. 3).
2. In vitro maturation of oocytes
The rinsed mouse oocytes were transferred to different in vitro maturation culture systems (35 oocytes per group) for in vitro maturation culture using 50 to 70 μ L droplets of culture medium, each droplet culturing 30 to 100 oocytes, covering the droplets with mineral oil (Sigma, cat # M8410) and 5% CO at 37 deg.C 2 And the culture is carried out in an incubator with 100 percent of humidity, and the liquid does not need to be changed in the mature culture process.
The in vitro maturation culture systems respectively comprise a culture solution, 100 mu M cysteamine and/or 200 mu M cysteine, wherein the culture solution comprises a basal culture solution, 5% -10% of serum substitute, 3.05mM D-glucose, 0.91mM sodium pyruvate, 0.05IU/mL follicle stimulating hormone, 0.05IU/mL luteinizing hormone and 20ng/mL epidermal growth factor; wherein the culture broth is mixed with cysteamine and/or cysteine prior to use. The different compositions or ratios in different oocyte in vitro maturation culture systems are shown in table 1.
TABLE 1 different oocyte in vitro maturation culture systems
Note: knock out TM Serum replacement (KSR, purchased from Thermo Fisher); ultroser G serum replacement (Ultro, from PALL); other ingredients are also commercially available.
3. Parthenogenetic activation of mature oocytes
Transferring the mature oocyte into parthenogenetic activating solution Ca 2+ /Mg 2+ -free CZB(mg/100ml)：NaCl(498.8mg)、KCl(36mg)、KH 2 PO 4 (16mg), disodium EDTA (4mg), NaHCO 3 (211mg), sodium lactate (443. mu.l), sodium pyruvate (3mg), gentamicin (10mg/mL), phenol red (10mg/mL) and PVA (cold water soluble, MW 30K-70K), 5. mu.g/mL Cytochalasin B (CB) and 2.5mM SrCl were added to the parthenogenetic activating solution 2 Activating for 4-6 hours to obtain the mouse parthenogenetic activated embryo.
4. In vitro development of embryos
The embryos were then transferred to KSOM medium (Millipore, cat # MR-121-D) supplemented with 10% KSR at 37 ℃ with 5% CO 2 Culturing in 100% humidity incubator for 3-4 days, and observing the subsequent development status of embryo. The culture process does not need to change the liquid.
5. Statistics and calculation of development proportion of embryo in different stages
The embryos are continuously cultured for 120 hours, and the development and apoptosis of the embryos in each group are counted at 24 hours (2-cell stage), 48 hours (4-cell stage), 72 hours (morula stage), 96 hours (blastocyst stage) and 120 hours (blastocyst stage) (fig. 4, from left to right, the culture systems 1 to 6 are respectively used), so that the proportion of the embryos developing at different periods is calculated.
Results of the experiment
After the inventors continuously adjust the composition and proportion of the oocyte in vitro maturation culture system, and perform parthenogenetic activation and further in vitro culture on the mature oocytes by using the above experimental method, the inventors finally found that significantly higher embryo development rate (the embryo development rate reaches 75%) can be obtained from oocytes in vitro maturation culture by using the culture system 3 in table 1 (table 2 and fig. 4).
Experimental results show that the selection and concentration of the serum substitute and the addition of amino acid can have remarkable influence on the later development of the oocyte, wherein the culture system 3 can support the oocyte to complete in vitro maturation and improve the capability of further developing the oocyte into an embryo. The embryonic development patterns of the oocytes cultured for in vitro maturation by the culture system 3 at different stages are shown in FIG. 5.
TABLE 2 comparison of embryo development rates after parthenogenetic activation in different oocyte in vitro maturation culture systems
Claims (9)
1. A culture system for in vitro maturation of oocytes in mice, the culture system comprising:
a) a culture solution comprising a basal culture solution, 8% to 12% serum replacement, 0.05IU/mL to 0.1IU/mL follicle stimulating hormone, 0.05IU/mL to 0.1IU/mL luteinizing hormone, and 10ng/mL to 20ng/mL epidermal growth factor;
b)100 μ M to 200 μ M cysteamine and 100 μ M to 200 μ M cysteine;
wherein a) and b) are mixed prior to use.
2. The culture system of claim 1, wherein the culture liquid further comprises 3mM to 4mM D-glucose and 0.8mM to 1mM sodium pyruvate.
3. The culture system of claim 2, wherein the culture system comprises a culture broth comprising basal medium, 8% to 12% serum replacement, 3.05mM D-glucose, 0.91mM sodium pyruvate, 0.05IU/mL follicle stimulating hormone, 0.05IU/mL luteinizing hormone, 20ng/mL epidermal growth factor, 100 μ M cysteamine, and 200 μ M cysteine; wherein the culture broth is mixed with cysteamine and cysteine prior to use.
4. The culture system of any one of claims 1-3, wherein the basal medium is TCM-199.
5. The culture system of any one of claims 1-4, wherein the serum replacement is knockkout TM A serum replacement.
6. The culture system of any one of claims 1-5, wherein the serum replacement is provided at a concentration of 10%.
7. A culture system according to any of claims 1-6, wherein the oocyte is an oocyte in the blastocyst stage.
8. A method for in vitro maturation of mouse oocytes, wherein the mouse oocytes are cultured in a culture system according to any one of claims 1 to 7, e.g. for 14 to 16 hours.
9. The method of claim 8, wherein the oocyte is an oocyte in a blastocyst stage.
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| CN101560494A (en) * | 2009-05-31 | 2009-10-21 | 山东农业大学 | Mouse denuded oocyte in vitro maturation technology |
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| 孙韬, 李裕强: "卵母细胞的体外成熟培养研究进展", 草食家畜, no. 02, pages 30 - 34 * |
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