summary

Through the research progress of in vitro maturation (IVM) of mouse oocytes, including oocyte acquisition, in vitro maturation culture system, influencing factors, and the application and prospect of IVM technology in reproductive medicine and biology research. Methods of oocyte collection from the ovaries, such as mechanical methods, enzymatic hydrolysis methods and their respective advantages and disadvantages; The selection of basal medium and the role of additives (such as hormones, growth factors, serum, etc.) in the in vitro maturation culture system were introduced. The effects of mouse strains, ovarian collection time, and culture conditions (temperature, gas environment, humidity, etc.) on the maturation of oocytes in vitro were analyzed. This paper discusses the application status and problems of IVM technology in assisted reproduction, embryo engineering, transgenic animal preparation, etc., so as to provide a theoretical reference for further optimizing the in vitro maturation technology of mouse oocytes and promote its development in related fields.

Keywords: mouse oocytes; in vitro maturation; cultivation system; influencing factors; Application prospects

introduction

In vitro maturation of oocytes is an important research content in the field of reproductive biology and assisted reproductive technology ^[1]. In vitro oogenesis experiments have led to a clearer understanding of ovarian function ^[2]. As a commonly used model organism, the study of in vitro maturation of oocytes in mice is of great significance for understanding the mechanism of oocyte development, optimizing human assisted reproductive technology, and carrying out embryo engineering and transgenic animal research. By establishing a stable and efficient in vitro maturation system for mouse oocytes, it can not only provide a large number of experimental materials for basic scientific research, but also provide theoretical basis and technical support for solving human reproductive disorders and promoting the development of biotechnology ^[3].

Acquisition of mouse oocytes

Ovarian collection

Two adult female mice are selected at a time by injection of 100 μL of gonadotropin (PMSG) in pregnant horses. Promote follicle development in female mice. After 48 hours, the mice were sacrificed to remove the ovaries.

1.2 Oocyte collection

1.2.1 Mechanical Law

The mechanical method is to physically remove the ovaries from the female mice, and then use forceps to remove the fat, blood and some impurities attached around the ovaries in the M2 culture medium under the microscope. The syringe needle is then used to continuously cut the follicles on the surface of the ovary so that the oocytes in the follicular fluid are released. The oocytes are then aspirated with a needle drawn from a glass tube (there are hand grip and mouth suction methods). This method is relatively simple and fast, with less chemical damage to oocytes, but it may cause some oocyte morphological damage due to mechanical manipulation, and the number of oocytes obtained is relatively limited, especially for oocytes in smaller follicles.

1.2.2 Enzymatic hydrolysis

Enzymatic hydrolysis usually uses digestive enzymes such as collagenase to break down the extracellular matrix in the ovarian tissue, dispersing the follicles and thus releasing the oocytes. This method is able to obtain a large number of oocytes, including some in smaller follicles, which is advantageous for studying oocytes at different stages of follicular development. However, the enzymatic hydrolysis process may cause a certain degree of damage to the plasma membrane and organelles of oocytes, affecting their subsequent maturation and development ability, and factors such as enzyme types, concentrations and digestion time need to be precisely controlled, otherwise it is easy to lead to the problem of overdigestion or insufficient digestion.

1.3 Selection of oocytes

There are two types of oocytes collected, namely bare eggs and eggs with attached granulosa cells (COCs), so oocytes with normal morphology, uniform cytoplasm, and no obvious damage should be selected for in vitro maturation ^{culture [4].} We make a preliminary judgment under the microscope based on the characteristics of the oocyte, such as the size, shape, presence or absence of vesicles, and the integrity of the surrounding granulosa cells. Oocytes in the germinal vesicle (GV) stage surrounded by an intact and compact layer of granule cells are considered to be suitable starting materials for in vitro maturation ^{culture [5],} and these oocytes have a high maturation potential under suitable culture conditions, can complete meiosis and develop into mature MII. stage oocytes, laying the foundation for subsequent fertilization and embryonic development^[6]。

1.3.1 Degranulation

After the COC is matured in vitro, it needs to be degranulated to remove the granule cells from the surface. We add 50 μL of hyaluronidase to the EP tube, then remove the mature COC with an oocyte needle and put it into the EP tube, and gently pipette with a pipette to detach the granule cells on the surface of the COC.

In vitro maturation culture of mouse oocytes

2.1 Culture medium

Commonly used basal media include M16, MEM, etc. These media contain a variety of amino acids, vitamins, inorganic salts and other nutrients, which can provide the basic material basis for the survival and metabolism of oocytes. Different basal media differ slightly in composition and proportion, as well as their ability to support in vitro maturation of oocytes. Therefore, we use M2 medium for ovarian cleaning and egg retrieval in the early stage, and M16 medium for culture. M16 medium is widely used in mouse embryo culture and has a good maintenance effect on the early development of oocytes.

2.2 Additions

2.2.1 Hormones

Gonadotropins: such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are key hormones that regulate oocyte maturation. The addition of appropriate amounts of FSH and LH to the in vitro culture system can mimic the hormonal environment in vivo and promote meiosis, recovery, and maturation of oocytes^[7].。 FSH mainly acts on granulosa cells, and indirectly affects the development of oocytes by binding to receptors on the surface of granulosa cells to regulate the proliferation, differentiation and secretion of granulosa cells. LH plays a role in the late stage of oocyte maturation, promoting the completion of the first meiosis and the transition of the oocyte to the MII. stage. The sensitivity and requirement of oocytes to FSH and LH may vary between different strains of mice, and the concentration and timing of hormone addition need to be optimized experimentally^[8].

Estrogen: Estrogen also plays an important role in the growth and maturation of oocytes. It can promote the growth and differentiation of granulosa cells and increase the expression of FSH and LH receptors on granulosa cells, thereby enhancing the effect of gonadotropins on oocytes. Adding an appropriate amount of estrogen to the in vitro culture system can improve the maturity rate and fertilization ability of oocytes, but excessive concentrations of estrogen may have negative effects on oocytes, such as causing chromosomal abnormalities and other problems, so it is necessary to precisely control the amount of estrogen added ^[9].

2.2.2 Growth factors

Epidermal Growth Factor (EGF): EGF can promote the growth and development of oocytes and improve the quality of oocyte maturation. It activates intracellular signaling pathways by binding to EGF receptors on the surface of oocytes and granulosa cells, regulating processes such as cell proliferation, differentiation, and metabolism. The addition of EGF to the in vitro culture system can increase the mitochondrial activity of oocytes, improve the cytoplasmic maturation state, and increase the developmental potential of subsequent embryos.

Insulin-like growth factor (IGF): The IGF family includes members such as IGF-I and IGF-II, which play an important role in the maturation of oocytes. IGF can promote the proliferation and survival of granulosa cells, enhance the communication between granulosa cells and oocytes, and regulate glucose metabolism and protein synthesis in oocytes, thereby promoting the maturation and development of oocytes. Studies have shown that there is a synergistic effect between IGF and gonadotropins and other growth factors that jointly regulate the in vitro maturation process of oocytes ^[10].

2.2.3 Serum

Serum is rich in nutrients, hormones, growth factors, and proteins, which can provide nutritional support and regulatory signals for oocytes that are closer to the in vivo environment. Commonly used serums include fetal bovine serum (FBS) and newborn bovine serum (NCS). The addition of serum can improve the maturation rate of oocytes and the ability of embryonic development after fertilization, but the complexity of serum composition, batch-to-batch variation, and potential risk of pathogen contamination may affect the stability and reproducibility of experimental results. Therefore, in recent years, some serum-free culture systems have gradually attracted attention, and researchers have tried to replace serum by adding growth factors and nutrients with clear components to improve the stability and controllability of the culture system.

2.3 Cultivation conditions

2.3.1 Temperature

The suitable temperature for in vitro maturation culture of mouse oocytes is generally 37°C, which is similar to the normal body temperature of mice, which can ensure the normal enzyme activity and metabolic processes in oocytes. For example, too high a temperature may lead to protein denaturation and metabolic disorders of oocytes, thereby affecting their fertilization ability and embryonic development potential; If the temperature is too low, the metabolism of the oocyte will be slowed down, the development process will be blocked, and even the cell may die.

2.3.2 Gas atmosphere

The gas atmosphere in the culture system is typically 5% CO₂, 5% O₂, and 90% N₂. The main role of CO₂ is to maintain a stable pH of the medium by interacting with the buffer system in the medium to keep the pH of the medium within the range suitable for oocyte growth and development (typically 7.2 - 7.4) ^[11]. 。 Moderate amounts of O₂ are necessary for aerobic respiration and energy metabolism in oocytes, but excessive concentrations of O₂ may produce too many oxygen radicals, causing oxidative damage to oocytes and affecting their quality and developmental ability. Therefore, precise control of the gas composition and proportion in the culture system is essential for in vitro maturation of mouse oocytes.

2.3.3 Humidity

The humidity inside the incubator is generally maintained at 95% - 100% to prevent evaporation of the medium and changes in osmotic pressure. If the humidity is insufficient, the water in the medium will gradually be lost, resulting in an increase in the osmotic pressure of the medium, which will cause osmotic stress to the oocyte, affecting its normal physiological function and development process; Excessive humidity may lead to the growth of bacteria and mold in the incubator, contaminating the culture system, which is also not conducive to the in vitro maturation of oocytes.

2.3.4 Paraffin oil covering

When the purity of paraffin oil is 100%, the in vitro culture of oocytes works best. 100% paraffin oil effectively reduces the content of peroxides and endotoxins. Thus, in vitro maturation of oocytes can be promoted ^[1, ^2]. At 0 h, 4 h, 8 h, and 14 h of culture, oocytes corresponded to the GV stage, GVBD stage, MI stage, and MII stage, respectively ^[1]. ^3]。

Oocyte in vitro culture results

In the process of in vitro culture of oocytes, we judge the maturity rate of oocytes by judging the expulsion rate of polar bodies. The figure below shows the excretion of polar bodies of oocytes after 20 hours of in vitro culture. Figure 1 shows the in vitro culture maturity rate of bare eggs, Figure 2 shows the in vitro culture maturity rate of COC degranulation, and Figure 3 shows the in vitro culture maturity rate of COC.

Factors influencing in vitro maturation of mouse oocytes

4.1 Mouse strains

There are differences in genetic background, physiological characteristics, and reproductive performance among different strains of mice, which affect the in vitro maturation ability of oocytes. Due to the influence of gene modification, the development and maturation process of oocytes of some transgenic mouse strains may be changed, such as the knockout or overexpression of some genes may lead to problems such as abnormal meiosis and organelle dysfunction of oocytes, thus affecting their in vitro maturation effect.

4.2 Stability of culture conditions

Small fluctuations in culture conditions can have a significant impact on the in vitro maturation of mouse oocytes. In addition to the above-mentioned factors such as temperature, gas environment, and humidity, the frequency of media changes, aseptic technique during operation, and the quality of culture utensils can also affect the stability of culture conditions. Frequent change of medium may cause physical stress and changes in the chemical environment of oocytes, which is not conducive to their stable growth and maturation; Contamination during the procedure can introduce bacteria, fungi or other pathogens, interfere with the normal development of oocytes, and even lead to culture failure; However, if the material, surface treatment, and cleanliness of the culture utensils do not meet the requirements, they may adsorb nutrients or growth factors in the culture medium, or release harmful substances, affecting the living environment and development process of oocytes ^[14].

Application of in vitro maturation technology for mouse oocytes

5.1 Application in assisted reproduction

In vitro maturation of mouse oocytes provides an important model and reference for the study of human assisted reproductive technology ^[15]. Through an in-depth understanding of the in vitro maturation process of mouse oocytes, we can better explore the maturation mechanisms and influencing factors of human oocytes in vitro environment, optimize the in vitro maturation culture system of human oocytes, and improve the success rate of assisted reproductive technologies such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) ^[16].

5.2 Application in embryo engineering

5.2.1 Embryonic stem cell research

Embryonic stem cells (ES cells) have the ability to self-renew and differentiate into multiple cell types, and have great potential for applications in regenerative medicine and basic biology research. In vitro maturation of mouse oocytes provides a stable source of embryos for the study of embryonic stem cells. Embryonic stem cell lines can be isolated and cultured from embryos generated by in vitro fertilization or parthenogenetic activation, and these ES cell lines can be used to study cell differentiation mechanisms, gene function validation, and drug screening ^[17]. At the same time, the use of gene editing technology to genetically modify mature oocytes or early embryos in vitro, and then establish the corresponding embryonic stem cell line, can provide a powerful tool for studying the role of specific genes in embryonic development and cell differentiation, which is helpful to understand the mysteries of life development and overcome some major diseases.

5.2.2 Application in the preparation of transgenic animals

In vitro maturation of mouse oocytes plays a key role in the preparation of transgenic animals. As mentioned earlier, transgenic mice carrying foreign genes can be obtained by introducing exogenous genes into mature oocytes or fertilized eggs in vitro through methods such as microinjection to integrate them into the genome, and after embryonic development and individual birth. This approach enables precise modification and modification of the mouse genome for the study of gene function, the establishment of disease models, and the production of animals with specific economic traits. For example, by introducing human disease-related genes into mouse oocytes, a mouse model of human disease was constructed, which provides an important in vivo research model for studying the pathogenesis and pathological process of disease and drug development. At the same time, in the field of agricultural biotechnology, genes with excellent production traits can be introduced into the oocytes of livestock, and new genetically modified livestock varieties with high yield, high quality and disease resistance can be cultivated to promote the development of animal husbandry.

discuss

Remarkable progress has been made in the development of mouse oocyte in vitro maturation technology over the years, from the method of oocyte acquisition to the optimization of in vitro maturation culture system, as well as the in-depth study of influencing factors, which have laid a solid foundation for the application of this technology in the fields of reproductive medicine, embryo engineering, and transgenic animal research ^[18].。 However, there are still some issues and challenges that need to be further addressed. In the future, with the continuous development of biotechnology, such as gene editing technology, single-cell sequencing technology, stem cell technology and the progress of biomaterials science, it is expected to provide new ideas and methods for the optimization and innovation of in vitro maturation technology of mouse oocytes. Through a deeper understanding of the biological characteristics and maturation mechanism of oocytes, combined with new technical means, a more personalized, efficient and safe in vitro maturation culture system will be developed, which will promote the further development of mouse oocyte maturation technology in vitro maturation technology in basic research and application fields, and make greater contributions to solving human reproductive health problems and promoting the progress of biomedical research and agricultural biotechnology.