CN114908041B - Culture system and method for in vitro maturation of mouse oocytes - Google Patents
Culture system and method for in vitro maturation of mouse oocytes Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
The invention relates to the technical field of animal cell culture, in particular to a culture system and method for in vitro maturation of a mouse oocyte. The in vitro maturation ratio and quality of the oocyte can be improved by the in vitro maturation culture system of the mouse oocyte, so that the embryo development ratio in the later stage of the mouse oocyte is improved.
Description
Technical Field
The invention relates to the technical field of animal cell culture, in particular to a culture system and method for in vitro maturation of a mouse oocyte.
Background
The meiosis of mammalian oocytes remains in the foaming (GV) phase. The maturation stage of a mammalian oocyte refers to the period from the beginning of the pre-meiosis to the middle of the second meiosis. In vitro maturation of oocytes is very important for scientific research, whereas in the in vitro maturation of oocytes the rate of oocyte maturation and the quality of maturation are critical for subsequent embryo development (Lin, Z et al (2014), JMY functions as actin nucleation-promoting factor and mediator for p-mediated DNA damage in porcine oocysts. Plos One,9 (10), e109385; wu, J et al (2018), chromatin analysis in human early development reveals epigenetic transition during zga. Nature). The current in vitro maturation technique of mouse oocytes has been relatively mature, but the quality of subsequent oocytes cannot be detected by in vitro maturation alone. If the quality of the oocyte needs to be detected, in vitro fertilization or parthenogenesis is usually carried out on the oocyte, and in vitro culture development is further carried out until the blastocyst stage. However, the in vitro operation of the oocytes and embryos of the mice at the present stage is limited to a certain process, and the complete in vitro maturation to development process, namely in vitro maturation+parthenogenesis/in vitro fertilization+in vitro development, cannot be completed.
The oocyte in vitro maturation culture medium (MEM (Gibco 11095-080) +10% FBS+50ng/ml EGF) in the prior art can mature the oocyte, but the mature oocyte can be disintegrated after parthenogenesis activation and cannot continue to develop (figure 1). This suggests that although the above culture solution is sufficient to support the discharge of oocytes from the first polar body, nuclear maturation occurs, there is a high probability that oocyte cytoplasmic maturation is problematic, and thus improvement of the basic components in the culture solution for in vitro maturation is required.
Another commercial oocyte basal medium CZB (Millipore Sigma, product number MR-019-D) also matured oocytes, but the oocyte development process was poor after activation, almost no continued development, and most oocytes were disintegrated (FIG. 2). This suggests that although the above culture may support oocyte maturation, it cannot support further development. This is most likely to be the lack of material in the culture that supports complete maturation of the oocyte.
Therefore, in order to solve the defect of the existing in-vitro maturation culture solution, a culture system suitable for in-vitro growth and development of early embryo of mice needs to be established, so that in-vitro embryo development quality of the early embryo of the mice is improved. The field needs to find a new culture solution for in vitro maturation of the oocytes of mice according to the nutrition requirement and metabolic characteristics of the development stage of the oocytes of the mice, and the in vitro maturation proportion of the oocytes is improved while the later embryo development proportion is further improved.
Disclosure of Invention
As described above, the existing culture solution for in vitro maturation of mouse oocytes, although allowing oocytes to mature, cannot support further development thereof. Thus, there is a need in the art for a culture system for in vitro maturation of mouse oocytes that can increase the in vitro maturation rate of oocytes while further increasing the later embryo development rate.
Accordingly, in a first aspect, the present invention provides a culture system for in vitro maturation of mouse oocytes, said culture system comprising:
a) A culture broth, wherein the culture broth comprises a basal broth, 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. Mu.M to 200. Mu.M cysteamine and 100. Mu.M to 200. Mu.M cysteine;
wherein a) and b) are mixed prior to use.
In a second aspect, the present invention provides a method of in vitro maturation of a mouse oocyte, wherein the mouse oocyte is placed in a culture system according to the first aspect of the invention for cultivation.
The beneficial effects of the invention are as follows: provides a culture system and a method for in vitro maturation of a mouse oocyte, which can improve the in vitro maturation proportion and quality of the oocyte, thereby improving the embryo development proportion of the later stage of the mouse oocyte.
Drawings
In order to more clearly illustrate the examples of the invention or the technical solutions of the prior art, the drawings used in the examples will be briefly described below, it being obvious that the drawings in the following description are only examples of the invention and that other embodiments can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a photograph of oocytes matured by culture in vitro maturation medium (MEM (Gibco 11095-080) +10% FBS+50ng/ml EGF) of prior art, and the oocytes disintegrate after parthenogenesis and cannot continue to develop.
FIG. 2 is a photograph of oocytes matured by culture with CZB (Millipore Sigma, product number MR-019-D) of the prior art, and the development progress of the oocytes is poor and almost impossible to continue.
FIG. 3 is a photograph of oocytes taken from adult mice during the foaming phase.
FIG. 4 is a graph showing the ratio of embryo development at different stages in different oocyte in vitro maturation culture systems.
FIG. 5 is a photograph of embryos 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 will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments that can be obtained by a person skilled in the art based on the embodiments of the present invention are within the scope of the present invention.
As described above, the conventional culture solution for in vitro maturation of mouse oocytes can mature the surface of the oocyte, but the mature oocyte has poor quality, cannot be activated by parthenogenesis, does not have the ability to further develop into an embryo, and proves that the maturation quality is poor. It is therefore an object of the present invention 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 develop further into embryos.
The in vitro maturation technology of embryo is related to the factors of three aspects, namely embryo type, culture condition and culture solution composition, wherein the improvement of culture solution composition 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 existing research shows that the Epidermal Growth Factor (EGF) plays an important role in follicular development, oocyte maturation and embryo development, and EGF can promote the in vitro maturation of egg cells of various animals. Also, the research shows that EGF has promotion effect on in vitro maturation and fertilization of the mouse oocyte, and can improve in vitro maturation rate and blastocyst hatchability. In addition, hormones are also important substances for mammalian oocyte maturation, with Follicle Stimulating Hormone (FSH), luteinizing Hormone (LH) being necessary for both follicle maturation and ovarian gonadal steroid production. In vivo FSH initiates follicular growth, whereas LH acts on follicular granulosa cells to activate downstream signaling pathways that induce cytoplasmic and nuclear maturation in the late growth phase of oocytes, ovulation and formation of post-ovulatory luteal cells. FSH and LH can significantly shorten the In Vitro Maturation (IVM) time of oocytes, improve the potential for oocyte maturation and development, and improve pregnancy rate.
Glutathione (GSH) plays an important role in oocyte maturation and embryo development, and the content of GSH in cells is often used to evaluate the quality of oocytes. The addition of Cysteamine (cystamine) and cysteine (cysteine) to in vitro culture systems can enhance Glutathione (GSH) synthesis and embryogenic capacity.
Serum is the most effective and most commonly used culture component in natural media and contains many unknown components that are essential for maintaining cell growth and reproduction and maintaining cell biological properties. Serum addition is beneficial to embryo development, but the culture effect is far from different sources, manufacturers and batches of serum. Thus, the choice of serum is also critical for culture.
In summary, the present inventors refer to in vitro oocyte maturation liquid systems of large mammals such as pigs, cattle and sheep, and optimize in vitro maturation culture liquid of mouse embryo by properly adding hormone, EGF and amino acid and selecting serum substitute instead of serum for specific signaling pathway of mouse, so as to further improve in vitro maturation proportion and quality of mouse oocyte, thereby finally improving in vitro embryo development ability.
Accordingly, in a first aspect, the present invention provides a culture system for in vitro maturation of mouse oocytes, said culture system comprising:
a) A culture broth, wherein the culture broth comprises a basal broth, 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. Mu.M to 200. Mu.M cysteamine and 100. Mu.M to 200. Mu.M cysteine;
wherein a) and b) are mixed prior to use.
Energy metabolism is the fundamental vital movement of oocytes during in vitro development, and therefore energy substances are important components in cell culture fluids. Sodium pyruvate and glucose are used as energy materials, the action mechanisms of the sodium pyruvate and the glucose are different, and the amounts of sodium pyruvate and glucose required for in vitro maturation of oocytes of different animals are also different. In some embodiments, the culture broth further comprises 3mM to 4mM D-glucose and 0.8mM to 1mM sodium pyruvate.
The inventor finds that the serum replacement is adopted to replace animal serum in the culture solution, so that the influence of uncertain factors in the serum on oocyte maturation and development can be eliminated, and a better cultivation effect can be obtained. In one embodiment, the serum replacement is knockout TM Serum Replacement (KSR). In a preferred embodiment, the serum replacement is at a concentration of 10%.
In one embodiment, the basal medium is TCM-199.
During the development of oocytes, the nucleoli of the oocyte in the foaming stage are increased, synthesis is active, the nucleus is enlarged, and the germinal vesicle rupture marks the maturation of the oocyte. Thus, in some embodiments of the present application, oocytes in the foaming phase are used for in vitro maturation.
In a second aspect, the invention provides a method of in vitro maturation of a mouse oocyte, wherein the mouse oocyte is placed in a culture system according to the first aspect of the invention for cultivation, e.g. for 14 to 16 hours. In one embodiment, the oocyte is an oocyte in the foaming stage.
Examples
Experimental materials and methods
1. Oocyte source and collection
ICR and C57BL/6J mice were selected for superovulation (PMSG (5 IU, ningbo second hormone works, cat# 110254564) injected at 14:00-16:00 PM) and cervical dislocation sacrificed after 46 to 48 hours, ovaries were isolated, and then the isolated ovaries were dissected through a 26 gauge needle to obtain oocytes, and healthy and intact oocytes in the foaming phase were selected under a microscope (FIG. 3).
2. In vitro maturation of oocytes
Transferring the rinsed mouse oocyte into different in vitro maturationsIn vitro maturation culture was performed in a culture system (35 oocytes per group) using 50. Mu.L to 70. Mu.L droplets of culture medium, each droplet culturing 30 to 100 oocytes, covering the droplet with mineral oil (Sigma, cat# M8410) and culturing at 37℃with 5% CO 2 Culturing in an incubator with 100% humidity, and no liquid change is needed in the maturation culture process.
The in-vitro maturation culture system comprises 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 replacement, 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 before use. The different compositions or ratios of the different oocyte in vitro maturation culture systems are shown in Table 1.
TABLE 1 in vitro maturation culture systems for different oocytes
Note that: knockout TM Serum replacement (KSR, available from Thermo Fisher); ultraser G serum substitutes (ultraro, available 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 (16 mg), EDTA disodium (4 mg), naHCO 3 (211 mg), sodium lactate (443. Mu.l), sodium pyruvate (3 mg), gentamicin (10 mg/mL), phenol red (10 mg/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 activator 2 The parthenogenesis activated embryos of mice are obtained from 4 hours to 6 hours of activation.
4. In vitro development of embryos
The embryos are then transferred to KSOM broth (Millipore, cat. No.)MR-121-D) at 37℃in 5% CO 2 Culturing in 100% humidity incubator for 3-4 days, and continuously observing embryo subsequent development condition. The culture process does not need to change liquid.
5. Statistics and calculation of embryo development ratio at different periods
Embryos were continuously cultured for 120 hours, and the embryo development and apoptosis conditions of each group were counted at 24 hours (2 cell stage), 48 hours (4 cell stage), 72 hours (morula stage), 96 hours (morula stage) and 120 hours (morula stage) (fig. 4, left to right, culture systems 1 to 6, respectively), to calculate the ratio of development of embryos at different periods.
Experimental results
The inventors have finally found that significantly higher embryo development rates (embryo development rates up to 75%) can be obtained for oocytes that were matured in vitro by culture system 3 in table 1, by continuously adjusting the composition and ratio of the oocyte in vitro maturation culture system, and parthenogenetically activating and further in vitro culturing the mature oocytes using the above experimental methods (tables 2 and 4).
Experimental results indicate that the selection and concentration of serum substituents and the addition of amino acids have a significant impact on the later development of oocytes, wherein the culture system 3 can support the oocytes to complete in vitro maturation and enhance their ability to further develop into embryos. An embryo development diagram of the different stages of oocytes subjected to in vitro maturation culture by the culture system 3 is shown in FIG. 5.
TABLE 2 embryo development Rate comparison after parthenogenesis activation in different oocyte in vitro maturation culture systems
Claims (6)
1. A culture system for in vitro maturation of mouse oocytes, the culture system comprising:
a) A culture broth comprising a basal broth, 10% 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, 3mM to 4mM D-glucose and 0.8mM to 1mM sodium pyruvate, wherein the serum replacement is knockout TM Serum replacement, wherein the basal culture solution is TCM-199;
b) 100. Mu.M to 200. Mu.M cysteamine and 100. Mu.M to 200. Mu.M cysteine;
wherein a) and b) are mixed prior to use.
2. The culture system of claim 1, wherein the culture system comprises a culture broth comprising a basal culture broth, 10% 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 μΜ cysteamine, and 200 μΜ cysteamine.
3. The culture system of any one of claims 1-2, wherein the oocyte is an oocyte in a foaming stage.
4. A method of in vitro maturation of a mouse oocyte, wherein the mouse oocyte is placed in a culture system according to any of claims 1-3 for culture.
5. The method of claim 4, wherein the culturing is performed for 14 to 16 hours.
6. The method according to claim 4 or 5, wherein the oocyte is an oocyte in the foaming stage.
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| KR20190052542A (en) * | 2017-11-08 | 2019-05-16 | 서울대학교산학협력단 | A follicular fluid replacement medium for in vitro Maturation of oocytes and The Use thereof |
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