An oocyte aging model, a preparation method and application thereof

Abstract

The application belongs to the field of cell model construction and biological medicine technology, and particularly relates to an oocyte aging model and a preparation method and application thereof. The application directly carries out in-vitro culture on fresh separated live complete growth germinal vesicle (GV) mouse oocytes by using a culture medium containing milrinone, can simulate the physiological state of oocytes in the GV arrest period in the ovary in the aging process, and is close to the spontaneous aging process in the physiological state. The oocyte aging model prepared by the application has a decreased mitochondrial membrane potential and reduced ATP content in the oocyte in the GV period, and is accompanied by an increased level of reactive oxygen species (ROS); when cultured to the first meiotic metaphase (MI), the level of chromosomal binding to the adhesion protein REC8 is significantly reduced; when cultured to the second meiotic metaphase (MII), the level of chromosomal arrangement abnormality is significantly increased. The above key indicators all show high similarity to the oocytes from naturally aging mice.

Description

Technical Field

[0001] This invention belongs to the field of cell model construction and biomedical technology, specifically relating to an oocyte aging model and its preparation method and application. Background Technology

[0002] With the increasing postponement of childbearing age among women in modern society, the decline in oocyte quality associated with advanced maternal age has become a significant cause of infertility, early miscarriage, and birth defects. Therefore, in-depth research into the mechanisms of oocyte aging and the exploration of effective anti-aging and rescue strategies are of great importance. In basic biomedical research, due to the limited availability of clinical samples and high individual heterogeneity, a stable and reliable oocyte aging model is urgently needed for studying the mechanisms of oocyte aging and conducting high-throughput drug screening.

[0003] Currently, senescent oocytes used for scientific research mainly come from the following three types of experimental animal models: (1) Natural aging animal models: Taking mice as an example, oocytes are obtained from their ovaries by raising them to more than 12 months of age. (2) Chemical / environmentally induced animal models: Animals are treated with D-galactose, oxidizing agents or environmental toxins to artificially accelerate the aging process of their ovaries. (3) Genetically engineered animal models: Key genes related to DNA repair, oxidative stress or apoptosis are knocked out or knocked down using technologies such as CRISPR / Cas9, thereby inducing premature ovarian failure and a decline in oocyte quality. However, each of the above animal models has its drawbacks: (1) Natural aging animal models usually require a breeding period of more than one year, and the ovarian reserve is nearly exhausted, making it extremely difficult to obtain samples of aged oocytes, which seriously restricts the accumulation of experimental data; (2) Chemical / environmentally induced animal models are often accompanied by systemic toxic interference, and the ovarian aging mechanism they induce is complex and difficult to rule out off-target effects of drugs, deviating from the core biological pathway of oocyte quality degradation in clinically elderly women; (3) Genetically engineered animal models rely on the targeted modification of a single or a few genes to artificially induce premature aging, and cannot truly and comprehensively simulate the complex regulatory network of multiple factors involved in oocyte aging under natural physiological conditions. The drawbacks of the existing model construction methods seriously restrict the efficiency of obtaining target oocytes and related scientific research breakthroughs - the experimental cycle of natural aging and genetic engineering models is long and costly, while induced models and genetic engineering models generally have the problem of single and incomplete aging phenotypes. Summary of the Invention

[0004] The purpose of this invention is to provide an oocyte aging model, its preparation method and application, which simulates the state of oocytes arrested in the GV phase in the ovary for a long time, and obtains an in vitro aging oocyte model with a relatively comprehensive aging phenotype quickly and at low cost.

[0005] This invention provides a method for preparing an oocyte senescence model, comprising the following steps: A model of oocyte senescence was obtained by culturing GV stage oocytes in vitro using a medium containing milrinone.

[0006] Preferably, the in vitro culture time is 24~48 hours; The in vitro culture conditions include: 37°C, 5% CO2.

[0007] Preferably, the culture medium containing milrinone is replaced with fresh medium every 24 hours during the in vitro culture process.

[0008] Preferably, the culture medium containing milrinone is preheated before the in vitro culture. The preheating treatment temperature and time is 4~6 hours; The preheating conditions include: 37°C and 5% CO2.

[0009] Preferably, the concentration of milrinone in the culture medium is 1.0~10.0 μM; The culture medium includes 10-20% fetal bovine serum, 1-2% penicillin-streptomycin antibiotics, and the remainder MEM-α medium.

[0010] Preferably, the method for preparing GV stage oocytes includes the following steps: The ovary, which is developing and maturing from the follicle, is placed in a basal medium containing milrinone to release the cumulus-oocyte complex; the granulosa cells surrounding the cumulus-oocyte complex are removed to obtain the GV stage oocytes.

[0011] Preferably, the concentration of milrinone in the basal culture medium is 1.0~10.0 μM; The basal culture medium includes 1-2% penicillin-streptomycin antibiotics and the remainder in M2 medium.

[0012] Preferably, the ovary comprises a mouse ovary, obtained by intraperitoneal injection of pregnant mare serum gonadotropin into a female mouse to obtain the follicle-developed and mature ovary.

[0013] This invention provides an oocyte senescence model obtained by the preparation method described in the above technical solution.

[0014] This invention provides the application of the oocyte senescence model described above in drug screening and / or efficacy evaluation; The drugs mentioned include those for the prevention, relief, or treatment of ovarian aging diseases.

[0015] Beneficial effects: This invention utilizes a culture medium containing milrinone to directly culture freshly isolated, live, fully grown vesicle-stage (GV) mouse oocytes in vitro. This simulates the physiological state of oocytes in the GV arrest phase within the ovary during the aging process, closely approximating their spontaneous aging process. The oocyte aging model prepared by this invention exhibits decreased mitochondrial membrane potential and reduced intraocular ATP content during the GV phase, accompanied by an increase in reactive oxygen species (ROS) levels. At metaphase I (MI), the level of chromosome-binding adhesion protein REC8 is significantly reduced; and at metaphase II (MII), the level of chromosome alignment abnormalities is significantly increased. This invention can induce oocyte aging models with varying degrees of aging through different in vitro treatment times (24 or 48 hours), showing high similarity to oocytes derived from naturally aging mice in several key indicators, providing a possibility for evaluating the effectiveness of drug treatment at different aging stages. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The results show the mitochondrial membrane potential detection results for each oocyte model; where A is a representative image of mitochondrial membrane potential fluorescence staining in C57BL / 6J mouse oocytes; B is the quantitative result of mitochondrial membrane potential fluorescence staining in C57BL / 6J mouse oocytes; and C is the quantitative result of mitochondrial membrane potential fluorescence staining in ICR mouse oocytes (oocytes were derived from the ICR mouse strain). Figure 2 The ATP levels of oocytes in each oocyte model are shown; where A represents C57BL / 6J mouse oocytes and B represents ICR mouse oocytes. Figure 3 The levels of reactive oxygen species (ROS) in oocytes of various oocyte models are shown in Figure A; A is a representative image of ROS probe staining in oocytes of C57BL / 6J mice; B is the quantitative result of ROS levels in oocytes of C57BL / 6J mice. Figure 4 The levels of chromosome-binding adhesion protein REC8 in oocytes during the MI stage of various oocyte models are shown in Figure A. A is a representative immunofluorescence staining image of chromosome-binding REC8 in C57BL / 6J mouse oocytes; B is the relative quantitative result of chromosome-binding REC8 in C57BL / 6J mouse oocytes. Figure 5 The levels of chromosome arrangement abnormalities in MII stage of each oocyte model are shown in Figure A; A is a representative diagram of chromosome arrangement in different states in MII stage oocytes of C57BL / 6J mice; B is the analysis results of chromosome arrangement status in MII stage oocytes of C57BL / 6J mice. Detailed Implementation

[0018] This invention provides a method for preparing an oocyte senescence model, comprising the following steps: A model of oocyte senescence was obtained by culturing GV stage oocytes in vitro using a medium containing milrinone.

[0019] In one embodiment, the present invention places the ovaries of adult mice in a basal medium containing milrinone to release the cumulus-oocyte complex; granulosa cells surrounding the cumulus-oocyte complex are then removed to obtain GV-stage oocytes. In one embodiment, the concentration of milrinone in the basal medium is 1.0–10.0 μM; in another embodiment, the concentration is 2.0–5.0 μM; and in yet another embodiment, the concentration is 2.5 μM. The present invention obtains GV-stage oocytes by placing the ovaries in a basal medium containing milrinone, and efficiently and reversibly arrests the meiotic process of oocytes by specifically inhibiting the activity of phosphodiesterase type 3 (PDE3).

[0020] In one embodiment, the basic culture medium of the present invention includes 1-2% penicillin-streptomycin antibiotics and the remainder M2 culture medium; in another embodiment, the present invention includes 1% penicillin-streptomycin antibiotics and the remainder M2 culture medium.

[0021] In one embodiment, the ovary described in this invention comprises a mouse ovary. Female mice are intraperitoneally injected with pregnant mare serum gonadotropin (PMS) to obtain the ovary after follicular development and maturation. In one embodiment, the PMS injection dose is 5 IU to promote synchronous follicular development. In one embodiment, the mouse described in this invention is an ICR mouse or a C57BL / 6J mouse. In one embodiment, the mouse described in this invention is a 6-8 week old mouse. Oocytes retrieved from mice aged 6-8 weeks are numerous and of uniform quality, solving the problems of high long-term feeding costs and low oocyte count in naturally aging mouse models.

[0022] After obtaining the GV stage oocytes, the present invention uses a medium containing milrinone to culture the GV stage oocytes in vitro to obtain an oocyte senescence model.

[0023] In one embodiment, the concentration of milrinone in the culture medium of the present invention is 1.0~10.0 μM; in another embodiment, the concentration of milrinone in the culture medium of the present invention is 2.0~5.0 μM; in yet another embodiment, the concentration of milrinone in the culture medium of the present invention is 2.5 μM. The present invention utilizes milrinone to culture GV-stage oocytes in vitro. Milrinone specifically inhibits phosphodiesterase type 3 (PDE3), thereby maintaining a high concentration of cyclic adenosine monophosphate (cAMP), simulating the physiological state of oocytes in the GV arrest phase within the ovary during the aging process, closely approximating their spontaneous aging process under physiological conditions. This method promotes aging without the need for specific, single chemical inducing agents (such as oxidants), thus constructing an oocyte aging model.

[0024] In one embodiment, the culture medium of the present invention comprises 10-20% fetal bovine serum, 1-2% penicillin-streptomycin antibiotics and the remainder MEM-α culture medium; in another embodiment, the culture medium of the present invention comprises 10% fetal bovine serum, 1% penicillin-streptomycin antibiotics and the remainder MEM-α culture medium.

[0025] As one implementation method, the in vitro culture time described in this invention is 24-48 hours. By using different in vitro treatment times (24 or 48 hours), oocyte senescence models with varying degrees of senescence can be induced, providing a possibility for evaluating the effectiveness of drug treatment at different senescence stages.

[0026] As one embodiment, the in vitro culture conditions described in this invention include: 37°C and 5% CO2. As another embodiment, the culture medium containing milrinone is replaced with fresh medium every 24 hours during the in vitro culture process.

[0027] In one embodiment, the culture medium containing milrinone is preheated before in vitro culture according to the present invention. In one embodiment, the preheating time is 4-6 hours; in another embodiment, the preheating time is 5 hours. In one embodiment, the preheating conditions include: 37°C and 5% CO2.

[0028] This invention provides an oocyte senescence model obtained by the preparation method described in the above technical solution.

[0029] This invention provides the application of the oocyte aging model described above in drug screening and / or efficacy evaluation; the drugs include those for the prevention, relief or treatment of ovarian aging diseases.

[0030] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides an oocyte aging model, its preparation method, and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 1. Experimental reagents (1) First basic culture medium: M2 culture medium (M7167, Sigma-Aldrich) + 1% antibiotic (penicillin-streptomycin; 15140122, Gibco), used for short-term in vitro manipulation of oocytes in a constant temperature environment of 37℃.

[0032] (2) Second basic culture medium: MEM-α culture medium (12561049, Gibco) + 10% fetal bovine serum (16140071, Gibco) + 1% antibiotic (penicillin-streptomycin; 15140122, Gibco), used for long-term culture in a 37℃, 5% CO2 incubator environment.

[0033] (3) Milrinone (M4659, Sigma-Aldrich): General structural formula: 1,6-dihydro-2-methyl-6-oxo-[3,4'-bipyridine]-5-carboxynitrile.

[0034] (4) Mineral oil (M8410, Sigma-Aldrich): After adding the culture medium to the petri dish, quickly cover the culture medium with a layer of mineral oil to prevent the culture medium from evaporating.

[0035] (5) Pregnant mare serum gonadotropin (PMSG).

[0036] 2. Laboratory animals Female ICR mice and C57BL / 6J mice aged 6-8 weeks.

[0037] 3. Experimental Methods (1) Ovulation induction in mice Female mice aged 6-8 weeks were intraperitoneally injected with PMSG (5 IU) to promote synchronous follicle development.

[0038] (2) Collection of oocytes during the GV stage Mice were euthanized 48 hours after PMSG injection. Ovaries were removed and placed in basal culture medium containing 2.5 μM milrinone. Follicles on the surface of the ovary were punctured with a 23G needle to release cumulus-oocyte complexes (COCs). Under a stereomicroscope, COCs were repeatedly blown and aspirated using a capillary tube with an opening slightly larger than the diameter of the oocyte to mechanically remove the granulosa cells surrounding the oocyte, thus obtaining GV stage oocytes. The entire process was performed under a constant temperature of 37°C.

[0039] (3) Preparation of oocyte senescence model by inhibiting the culture of GV stage oocytes The obtained GV-stage oocytes were transferred to a second basal medium containing 2.5 μM milrinone and cultured continuously at 37°C in a 5% CO2 incubator to obtain an oocyte senescence model. The second basal medium containing 2.5 μM milrinone was pre-incubated at 37°C in a 5% CO2 incubator for 4-6 hours (pH equilibration). Fresh second basal medium containing 2.5 μM milrinone, pre-incubated for 4-6 hours, was replaced every 24 hours. Each female mouse yielded 20-40 GV-stage oocytes; after inhibition culture, more than 80% of the oocytes could be used as a senescence cell model.

[0040] 4. Evaluation of aging model indicators (1) Mitochondrial membrane potential Freshly isolated GV stage oocytes from step 3, oocyte senescence models obtained by inhibited culture for 24 h and 48 h respectively, were used for detection using the JC-1 probe in the Enhanced Mitochondrial Membrane Potential Detection Kit (C2003S, Beyotime) according to the instructions.

[0041] Detection principle: The JC-1 probe enters the mitochondrial matrix and forms polymers, emitting red light under a fluorescence microscope. When mitochondria are damaged, the membrane potential depolarizes (decreases), and the JC-1 probe cannot enter the mitochondria, remaining as monomers in the cytoplasm and emitting green light. The ratio of red to green (polymer / monomer) fluorescence intensity is typically used to quantify changes in mitochondrial membrane potential. A lower ratio indicates a more severe decrease in mitochondrial membrane potential.

[0042] Test results as follows Figure 1 As shown, strong red fluorescence (JC-1 multimer) was mainly observed in the oocytes of the control group (0h), while weak green fluorescence was observed, indicating that the mitochondrial membrane potential was at a high level. However, in the aging model group constructed in this invention, the red fluorescence of the oocytes was significantly weakened. Quantitative fluorescence analysis showed that the red / green fluorescence intensity ratio of the model group was significantly lower than that of the control group, indicating a significant decrease in mitochondrial membrane potential in the aging model. The reduction effect was more pronounced with the extension of the inhibition culture time. The oocytes showed high similarity to those derived from naturally aging mice in the prior art (Salidroside Improves Oocyte Competence of Reproductively Old Mice by Enhancing Mitophagy; 2025; Aging Cell).

[0043] (2) Adenosine triphosphate (ATP) level Freshly isolated GV-stage oocytes from step 3, oocyte senescence models obtained by 24h and 48h of inhibited culture, were used for detection using an enhanced ATP assay kit (S0027, Beyotime) according to the manufacturer's instructions. The results are as follows: Figure 2 As shown.

[0044] according to Figure 2 As can be seen, compared with the control group (0h), the ATP level in the oocyte aging model prepared by this invention was significantly reduced, and the reduction effect was more obvious with the extension of inhibition culture time. It showed a high degree of similarity to oocytes derived from naturally aging mice in existing technologies (Salidroside Improves Oocyte Competence of Reproductively Old Mice by Enhancing Mitophagy; 2025; Aging Cell.).

[0045] (3) Reactive oxygen species (ROS) level Freshly isolated GV stage oocytes from step 3, oocyte senescence models obtained by inhibited culture for 24 hours and 48 hours were used for detection using the H2DCFDA probe (D399, Invitrogen).

[0046] Detection principle: The H2DCFDA probe can freely penetrate the cell membrane and enter living cells. It is hydrolyzed by intracellular esterases into non-fluorescent DCFH and remains inside the cell. The probe is then oxidized by reactive oxygen species (ROS) to generate DCF with strong green fluorescence. The fluorescence intensity is directly proportional to the intracellular ROS level. The oxidative stress state in living cells can be quantitatively assessed by fluorescence detection.

[0047] Test results as follows Figure 3 As shown, under a fluorescence microscope, only a weak green fluorescence signal was observed in the control group (0h) oocytes, while a strong green fluorescence was observed in the oocyte aging model group constructed in this invention. Quantitative analysis of fluorescence intensity showed that the relative fluorescence intensity of the model group increased significantly with the extension of the inhibition culture time, and was significantly higher than that of the control group. This model exhibits a high degree of similarity to oocytes derived from naturally aging mice in existing technologies (The lipid peroxidation product 4-hydroxynonenal contributes to oxidative stress-mediated deterioration of the ageing oocyte; 2017; Scientific Reports.).

[0048] (4) Chromosome-binding REC8 protein level Freshly isolated GV-stage oocytes from step 3 and oocytes cultured for 24 hours to inhibit senescence were taken separately. They were washed three times sequentially with milrinone-free basal medium to completely remove the inhibitory effect of milrinone on GV oocytes, and then placed in milrinone-free basal medium for further culture at 37°C. After 2 hours of culture, oocytes still arrested in the GV stage were discarded. Oocytes undergoing germinal follicle rupture (GVBD) were cultured for another 5 hours to obtain oocytes in metaphase I (MI) of meiosis. Chromosome slides were then prepared for REC8 immunofluorescence staining and quantitative analysis of the fluorescence signal. The results are as follows: Figure 4 As shown.

[0049] according to Figure 4 As can be seen, in the control group (0h), the fluorescence signal (green) of REC8, a component of the adhesion protein, on the oocyte chromosomes was strong and clear, clearly localized to DNA and tightly connecting sister chromosomes. However, in the aging model group constructed in this invention, the fluorescence signal of REC8 on the chromosomes was significantly weakened. Quantitative analysis of fluorescence intensity showed that the relative fluorescence intensity of REC8 on the chromosomes of oocytes in the model group was significantly lower than that in the control group. This demonstrates a high degree of similarity to oocytes derived from naturally aging mice in existing techniques (Age-Related Meiotic Segregation Errors in Mammalian Oocytes Are Preceded by Depletion of Cohesin and Sgo2; 2014; Current Biology.).

[0050] (5) Level of chromosome alignment abnormalities: Freshly isolated GV stage oocytes from step 3, oocyte senescence models cultured for 24 hours and 48 hours under inhibition were respectively taken and cultured for an additional 8 hours according to step (4) to obtain oocytes in metaphase II (MII) of meiosis that have extruded the first polar body. After in situ fixation according to existing techniques, chromosomes were marked by α-tubulin immunofluorescence staining and DAPI staining. The chromosome arrangement and level in MII stage oocytes were analyzed, and the results are as follows: Figure 5 As shown.

[0051] according to Figure 5As can be seen, in the control group (0h), the chromosomes (blue) of MII-stage oocytes were neatly and tightly arranged on the equatorial plate of the spindle (green), exhibiting a typical normal MII-stage conformation. However, in the aging model group constructed in this invention, the chromosomes of oocytes were disordered, clumped together, or with some chromosomes free outside the equatorial plate, scattered randomly. Based on the degree of disorder, they could be divided into mild and severe cases. Qualitative classification and statistical analysis of the arrangement revealed that the proportion of oocytes with abnormal chromosome arrangement in the model group was significantly higher than that in the control group. This shows a high degree of similarity to oocytes derived from naturally aging mice in existing techniques (Kinetochore microtubule establishment is defective in oocytes from aged mice; 2014; CellCycle.).

[0052] The oocyte aging model prepared by this invention shows a high degree of similarity to oocytes derived from naturally aging mice in several key indicators, providing important material resources for basic research on aging and clinical anti-aging exploration. It can be used for screening drugs and evaluating efficacy for the prevention, relief or treatment of ovarian aging diseases.

[0053] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing an oocyte senescence model, characterized in that, Includes the following steps: A model of oocyte senescence was obtained by culturing GV stage oocytes in vitro using a medium containing milrinone.

2. The preparation method according to claim 1, characterized in that, The in vitro culture time is 24-48 hours; The in vitro culture conditions include: 37°C, 5% CO2.

3. The preparation method according to claim 1, characterized in that, During the in vitro culture process, the culture medium containing milrinone is replaced with fresh medium every 24 hours.

4. The preparation method according to claim 1, characterized in that, Before the in vitro culture, the culture medium containing milrinone is preheated. The preheating treatment temperature and time is 4~6 hours; The preheating conditions include: 37°C and 5% CO2.

5. The preparation method according to claim 1, characterized in that, The concentration of milrinone in the culture medium is 1.0~10.0 μM; The culture medium includes 10%–20% fetal bovine serum, 1%–2% penicillin-streptomycin antibiotics, and the remainder MEM-α medium.

6. The preparation method according to any one of claims 1 to 5, characterized in that, The method for preparing GV-stage oocytes includes the following steps: The ovary, which is developing and maturing from the follicle, is placed in a basal medium containing milrinone to release the cumulus-oocyte complex; the granulosa cells surrounding the cumulus-oocyte complex are removed to obtain the GV stage oocytes.

7. The preparation method according to claim 6, characterized in that, The concentration of milrinone in the basal culture medium is 1.0~10.0 μM; The basal culture medium includes 1% to 2% penicillin-streptomycin double antibiotics and the remainder M2 culture medium.

8. The preparation method according to claim 6, characterized in that, The ovary includes a mouse ovary, obtained by intraperitoneal injection of pregnant mare serum gonadotropin into a female mouse to obtain the follicle-developed and mature ovary.

9. The oocyte senescence model obtained by the preparation method according to any one of claims 1 to 8.

10. The application of the oocyte senescence model according to claim 9 in drug screening and / or efficacy evaluation; The drugs mentioned include those for the prevention, relief, or treatment of ovarian aging diseases.

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