A culture solution, kit and application for in vitro maturation of aged oocytes

Abstract

The present application relates to the technical field of biological medicine, and more particularly to a culture solution, a kit and an application for in-vitro maturation of aged oocytes. Through in-vitro experiments, the present application adds exogenous stromal cell-derived factor 1, establishes a culture system for in-vitro maturation of aged oocytes, and reveals the molecular mechanism of stromal cell-derived factor 1 playing a role through autophagy, thereby providing a new solution for improving the fertility of aged women and improving the success rate of assisted reproductive technology, and having important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a culture medium, reagent kit, and application for in vitro maturation of elderly oocytes. Background Technology

[0002] Age-related decline in female fertility is a significant clinical challenge in reproductive medicine. Although assisted reproductive technology (ART) provides a pathway to pregnancy, its effectiveness is heavily influenced by the mother's age. Furthermore, oocytes from older mothers face numerous challenges during in vitro development, such as increased oxidative stress, a higher proportion of abnormal spindle structures and chromosome structures, decreased mitochondrial activity, and reduced embryonic developmental potential. Therefore, improving the quality of oocytes from older mothers and enhancing embryonic development under in vitro culture conditions is crucial for improving pregnancy outcomes in women of advanced maternal age.

[0003] In vitro maturation (IVM) of oocytes refers to the technique of retrieving immature oocytes from the ovary and culturing them in vitro until they reach maturity (MII stage). This technique can reduce the dosage and cycle of ovulation-inducing drugs, and lower the risk of ovarian hyperstimulation syndrome, making it particularly suitable for older patients with diminished ovarian reserve and poor response to ovulation-inducing drugs. However, the application of existing IVM systems in older oocytes still faces significant challenges: older oocytes exhibit low in vitro maturation rates, inadequate cytoplasmic maturation, high rates of spindle abnormalities, and difficulty in controlling oxidative stress levels, resulting in significantly lower subsequent fertilization and blastocyst formation rates compared to younger individuals. Therefore, developing novel IVM culture systems that can target the characteristics of older oocytes and improve their in vitro maturation quality has significant clinical translational value.

[0004] Matrix cell-derived factor-1 (SDF-1), also known as CXC motif chemokine 12 (CXCL12), is a chemokine that participates in cell migration, differentiation, and tissue repair by binding to its receptor CXCR4, and is widely expressed in the female reproductive system. Current reports suggest that SDF-1 may have a positive impact on the in vitro maturation outcome of young COCs (cumulus-oocyte complexes).

[0005] However, the specific role of SDF-1 in the in vitro maturation process of older oocytes has not been reported. Whether it can improve the quality of older oocytes by regulating oxidative stress and autophagy levels, as well as its optimal concentration and application in the IVM culture system, are all lacking systematic research. Summary of the Invention

[0006] In view of this, the present invention proposes a culture medium, kit, and application for in vitro maturation of oocytes in older women. Through in vitro experiments, by adding SDF-1, a culture system for in vitro maturation of oocytes in older women was established, and the molecular mechanism by which SDF-1 exerts its effect through regulating autophagy was revealed. This provides a new solution for improving fertility in older women and increasing the success rate of assisted reproductive technologies, and has significant clinical application value.

[0007] The technical solution of this invention is implemented as follows: In a first aspect, a culture medium for in vitro maturation of older oocytes is provided, the composition of which includes stromal cell-derived factor 1.

[0008] The culture medium also includes a basal culture medium, which is a conventional culture medium suitable for in vitro culture of oocytes, including M2, G-1, and G-2 culture media.

[0009] Secondly, a kit for in vitro maturation of older oocytes is provided, comprising the culture medium described above.

[0010] Thirdly, the application of the culture medium or kit described above in improving the quality of older oocytes is provided.

[0011] Based on the above technical solutions, preferably, the concentration of the stromal cell-derived factor 1 is 10~100 ng / mL.

[0012] Based on the above technical solutions, a further preferred embodiment is that the concentration of the stromal cell-derived factor 1 is 10~20 ng / mL.

[0013] Based on the above technical solution, and even more preferably, the concentration of the stromal cell-derived factor 1 is 20 ng / mL.

[0014] In the above technical solutions, the stromal cell-derived factor 1 has the activity of increasing the autophagy level of oocytes.

[0015] Based on the above technical solutions, preferably, the stromal cell-derived factor 1 upregulates the expression of one or more of the autophagy-related genes Lc3, Atg5, Beclin1 or Lamp2, and / or enhances lysosomal activity, and / or increases the expression levels of LC3 protein and / or Beclin1 protein.

[0016] Based on the above technical solutions, preferably, the advanced-age oocytes include female mouse oocytes aged 44-48 weeks or female oocytes aged 40-45 years.

[0017] In medical reproductive aging research, female mouse models aged 44-48 weeks are generally considered to be classic animal models simulating reproductive aging in human women over 38 years of age. See Flurkey, Currer, and Harrison. “The mouse in biomedical research” in James G. Fox (ed.), American College of Laboratory Animal Medicine series (Elsevier, AP: Amsterdam; Boston). 2007.

[0018] Fourthly, an in vitro culture method for improving the quality of older oocytes is provided, which includes the step of culturing older oocytes in an in vitro maturation medium containing stromal cell-derived factor 1.

[0019] The culture medium, kit, and application of the present invention for in vitro maturation of advanced oocytes have the following advantages over the prior art: 1. An optimized culture system for in vitro maturation of older oocytes was established by adding SDF-1, and the optimal concentration of SDF-1 was determined. This improved the IVM maturation rate and quality of mature oocytes, enhanced subsequent embryo development, and increased blastocyst quality. This provides a new approach for the in vitro culture and embryo development of older oocytes.

[0020] 2. Transcriptome sequencing and mechanism verification revealed the mechanism of action of SDF-1. SDF-1 reduces the production of total ROS and mitochondrial ROS in mature oocytes of older mice by increasing the level of autophagy in oocytes of older mice, clearing damaged organelles and protein aggregates, thereby improving spindle assembly and chromosome alignment, and improving oocyte quality and subsequent developmental potential.

[0021] 3. SDF-1 is an endogenous small molecule chemokine with good biocompatibility and low immunogenicity. This invention has demonstrated through in vitro experiments that, within the effective dosage range, SDF-1 significantly improves the development of older oocytes and embryos, without obvious toxic side effects. Furthermore, SDF-1 can be recombinantly expressed and purified using existing biotechnological methods, making production costs controllable and showing promising prospects for clinical translation. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The diagram illustrates the effect of 20 ng / mL SDF-1 on the GVBD rate and maturation rate of oocytes in IVM of aged mice in Example 1 of the present invention. Figure (A) shows the oocyte development during IVM in the control group (0 ng / mL SDF-1) and the SDF-1 group (20 ng / mL SDF-1), including immature GV oocytes, MI stage oocytes, and MII stage oocytes. Scale bar: 100 μm. Figure (B) shows the comparison of the GVBD rate of oocytes in the two groups. Ctrl: control group, SDF-1: SDF-1 group. Figure (C) shows the comparison of the maturation rate of oocytes in the control group and the SDF-1 group. Figure 2 The figures show the ICSI results of mouse oocytes of different ages after IVM was supplemented with 20 ng / mL SDF-1 in Example 1 of the present invention. Figure (A) shows the embryonic development of oocytes in the control group and SDF-1 group of different ages at different time points after ICSI, scale bar: 100 μm; Figure (B) shows the fertilization rate of mature oocytes in the control group and SDF-1 group of different ages after ICSI; Figure (C) shows the cleavage rate of mature oocytes in the control group and SDF-1 group of different ages after ICSI; Figure (D) shows the blastocyst formation rate of mature oocytes in the control group and SDF-1 group of different ages after ICSI. Figure 3 The figures show the ROS levels of blastocysts formed by ICSI in mice of different ages in the control group and SDF-1 group in Example 1 of the present invention. Figure (A) shows the ROS fluorescence staining of blastocysts after ICSI in oocytes of mice of different ages in the control group and SDF-1 group, scale bar: 75 mm. Figure (B) shows the statistical graph of ROS levels of blastocysts after ICSI in oocytes of mice of different ages in the control group and SDF-1 group. Figure 4The diagram shows the detection of apoptosis levels in blastocysts formed by ICSI in mice of different ages in the control group and SDF-1 group of mice in Example 1 of the present invention. Figure (A) shows the TUNEL fluorescence staining of blastocysts after ICSI in oocytes of mice of different ages in the control group and SDF-1 group of mice. Red fluorescence represents apoptotic cells, and blue fluorescence represents cell nuclei. Scale bar: 75 μm. Figure (B) shows the statistical diagram of the proportion of apoptotic cells in blastocysts after ICSI in oocytes of mice of different ages in the control group and SDF-1 group of mice. Figure 5 The diagram shows the effect of SDF-1 on the spindle morphology and chromosome arrangement of IVM oocytes from mice of different ages in Example 1 of the present invention. Figure (A) shows the fluorescence images of spindle and chromosome staining in IVM oocytes of each group, scale bar: 25 μm; Figure (B) shows the statistical chart of the proportion of abnormal spindle morphology in oocytes of each group; Figure (C) shows the statistical chart of the proportion of abnormal chromosome arrangement in oocytes of each group. Figure 6 The diagram shows the effect of SDF-1 on the distribution and fluorescence intensity of cortical granules in mature IVM oocytes of mice of different ages in Example 1 of the present invention. In the diagram (A), the fluorescence staining of cortical granules in IVM oocytes of each group is shown. Green fluorescence represents cortical granules and blue fluorescence represents chromosomes. Scale bar: 25 μm. The diagram (B) shows the proportion of cells with abnormal distribution of cortical granules among each group. The diagram (C) shows the comparison of the immunofluorescence intensity of cortical granules among each group. Figure 7 The diagram shows the effect of SDF-1 on the mitochondrial distribution and fluorescence intensity of mature mouse IVM oocytes of different ages in Example 1 of the present invention. In the diagram (A), the mitochondrial fluorescence staining diagram of IVM oocytes of each group is shown. The green fluorescence represents mitochondria and the blue fluorescence represents chromosomes. The scale bar is 50 μm. The diagram (B) shows the comparison of mitochondrial immunofluorescence intensity among the groups. The diagram (C) shows the proportion of cells with abnormal mitochondrial distribution among the groups. Figure 8 The figure shows the effect of SDF-1 on MMP levels in IVM oocytes of mice of different ages in Example 1 of the present invention. Figure (A) shows the JC-1 fluorescence images of mouse oocytes in the control group and SDF-1 group at different ages, scale bar: 75μm; Figure (B) shows the statistical graph of MMP levels among the groups, where MMP level is recorded as red light intensity / green light intensity. Figure 9 The figure shows the effect of in vitro addition of SDF-1 on the ROS level of mouse oocytes of different ages in Example 1 of the present invention. Figure (A) shows the ROS fluorescence map of mature oocytes in each group, scale bar: 100 μm; Figure (B) shows the statistical graph of ROS level among each group. Figure 10 The figure shows the effect of in vitro addition of SDF-1 on the mitochondrial ROS level of mouse oocytes of different ages in Example 1 of the present invention. Figure (A) shows the Mito Sox fluorescence map of mature oocytes in each group, scale bar: 100 μm; Figure (B) shows the statistical graph of Mito Sox fluorescence intensity among each group. Figure 11 The figures show the results of Smart-seq bioinformatics analysis of mature oocytes from the IVM of aged mice in Example 2 of the present invention, including the control group of aged mice, the SDF-1 group of aged mice, and the young control group. Figure (A) shows the heatmap of differentially expressed genes in the three groups of oocytes; Figure (B) shows the volcano plot of differentially expressed genes between the control group of aged mice and the young control group; and Figure (C) shows the volcano plot of differentially expressed genes between the control group of aged mice and the SDF-1 group of aged mice. Figure 12 The figures show the Smart-seq bioinformatics analysis results of mature oocytes from IVMs in aged mice (control group, SDF-1 group, and young control group) in Example 2 of the present invention. Figure (A) shows the GO analysis of differences between the aged mouse control group and the young mouse control group; Figure (B) shows the GO analysis of differences between the aged mouse control group and the SDF-1 group; Figure (C) shows the KEGG analysis of differences between the aged mouse control group and the young mouse control group; and Figure (D) shows the KEGG analysis of differences between the aged mouse control group and the SDF-1 group. Figure 13 This figure shows the expression of autophagy-related genes in the control group and SDF-1 group of aged mouse oocytes in Example 2 of the present invention; Figure 14 The diagram shows the effect of SDF-1 on the fluorescence staining of lysosomes in oocytes of elderly mice in Example 2 of the present invention. In the diagram (A), the fluorescence staining of lysosomes in oocytes of each group of mice is shown. Scale bar: 50 μm. In the diagram (B), the fluorescence intensity of lysosomes among oocytes of each group is shown. Figure 15 The figure shows the effect of SDF-1 on the expression level of LC3, an autophagy-related gene, in IVM oocytes of aged mice in Example 2 of the present invention. Figure (A) shows the LC3 fluorescence staining of mouse oocytes in each group, scale bar: 50 μm; Figure (B) shows the statistical graph of LC3 fluorescence intensity among oocytes in different groups. Figure 16The figure shows the effect of SDF-1 on the expression level of Beclin1, an autophagy-related gene, in IVM oocytes of aged mice in Example 2 of the present invention. Figure (A) shows the fluorescence staining of Beclin1 in oocytes of different groups of mice, scale bar: 50 μm; Figure (B) shows the statistical graph of Beclin1 fluorescence intensity among oocytes of different groups. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] All animal experimental protocols used in this invention were approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (TJH-202312019). The 6-8 week old young female ICR mice, 44-48 week old older female ICR mice, and 10 week old male ICR mice used were all SPF-grade animals purchased from Shulaibao (Wuhan) Biotechnology Co., Ltd.; the mice were raised in the Animal Experiment Center of the inventor's research building and have been approved by the hospital's Animal Ethics Committee.

[0026] The SDF-1 recombinant protein used in this invention was purchased from Wuhan Aiboteke Biotechnology Co., Ltd., and the other reagents were all commercially available products.

[0027] Example 1: Study on the effect of in vitro SDF-1 supplementation on oocyte quality in aged mice In this embodiment, aged mice (44-48 weeks old) and young mice (6-8 weeks old) were used as the main experimental subjects. SDF-1 was added to the IVM culture system to comprehensively evaluate the effects of adding SDF-1 at the optimal concentration on the quality and developmental potential of mice of different ages, especially aged mice.

[0028] 1. Effects of SDF-1 on in vitro maturation of mouse oocytes (1) Preparation of petri dishes Place the necessary pipettes, centrifuge tubes, mineral oil, culture dishes, pipette tips, and other items in a cell sterilizer and sterilize them with ultraviolet light for at least 30 minutes. Reconstitute 10 μg of SDF-1 powder with PBS solution to a concentration of 0.1 mg / mL according to the instructions. Equilibrate at room temperature for 5 minutes to ensure complete dissolution, then aliquot and store in a -80°C freezer. Use as needed and avoid repeated freeze-thaw cycles. The following culture dishes were then prepared for subsequent experiments: A: 2 mL of sterile PBS solution; B: 2 mL of M2 culture medium + 1 μL of 100 mM IBMX; C: Take liquid from B and prepare several droplets, each droplet being 20 μL, and cover with tissue culture mineral oil to completely cover the culture droplets; D: Prepare several 20 μL droplets of M2 culture medium without SDF-1 or containing 10 ng / mL, 20 ng / mL, 50 ng / mL, or 100 ng / mL SDF-1, and cover with tissue culture mineral oil to completely cover the culture droplets; E: Prepare one 20 μL droplet of M2 culture medium without SDF-1 or containing 10 ng / mL, 20 ng / mL, 50 ng / mL, or 100 ng / mL SDF-1, and cover with tissue culture mineral oil to completely cover the culture droplets. Each mouse was provided with one AC dish, several D dishes, and one E dish. The prepared petri dishes were transferred to a 37°C, 5% CO2 incubator and pre-equilibrated for at least 4 hours.

[0029] (2) Mouse oocyte IVM Preheat the operating board to 37°C using the heating plate. After euthanizing the mouse by cervical dislocation, disinfect the mouse abdomen with 75% alcohol. Make a "Y"-shaped incision in the mouse's abdomen to expose both ovaries. Use surgical forceps to separate the connective tissue around the ovaries, cut off both ovaries, and place them in dish A. Quickly place them on the preheated operating board. Under a stereomicroscope, use a 1 mL syringe to separate the adipose tissue and fallopian tubes around the ovaries in dish A, then transfer both ovaries to dish B. Under a stereomicroscope, repeatedly prick the ovaries in dish B with a syringe needle until they resemble cotton wool, releasing the mouse oocytes into M2 culture medium (this step is limited to 10 minutes). Under a stereomicroscope, repeatedly blow on the immature mouse oocytes (COCs) in dish B using a 90-100µm glass pipette to mechanically remove the granulosa cells from the surface of the COCs. Select the morphologically sound and structurally normal germinal vesicles (Germinal vesicles) from dish B. GV (Gross Vulnerability) mouse naked oocytes were collected using a glass pipette and temporarily stored in droplets in dish C. The dishes were then returned to a 37°C, 5% CO2 incubator. After all mouse GV stage oocytes had been collected into dish C, all naked oocytes were randomly and evenly distributed into droplets of different SDF-1 concentrations in dish D using a glass pipette. The naked oocytes were washed at least five times in each concentration droplet to remove residual IBMX before being transferred to the corresponding concentration droplets in dish E. Dish E was placed in a 37°C, 5% CO2 incubator for further culture. After 2 hours of culture, dish E was removed, and the germinal vesicle breakdown (GVBD) of mouse oocytes was assessed under a stereomicroscope. After 14-16 hours of culture, the expulsion of the first polar body (PB1) of mouse oocytes, i.e., the maturation rate, was observed under a stereomicroscope. The main indicators were calculated as follows: GVBD rate = number of GVBD oocytes / total number of oocytes; maturation rate = number of oocytes expelled from PB1 / total number of oocytes. The results are shown in Table 1.

[0030] Table 1. Effects of different concentrations of SDF-1 on IVM GVBD rate and maturation rate of oocytes from young mice.

[0031] Note: *The difference was statistically significant compared with the control group (0 ng / mL SDF-1). ***P<0.001.

[0032] As shown in Table 1, when the SDF-1 concentration was in the range of 0-20 ng / mL, the GVBD rate and in vitro maturation rate of young mouse oocytes gradually increased with increasing SDF-1 concentration. When the SDF-1 addition reached 20 ng / mL, the oocyte GVBD rate was not statistically different from the control group (0 ng / mL SDF-1), but the maturation rate was statistically different from the control group (P<0.001). When the SDF-1 concentration continued to increase, both the GVBD rate and maturation rate of young mouse oocytes decreased. Therefore, 20 ng / mL was determined as the experimental concentration for subsequent experiments in this study.

[0033] 20 ng / mL SDF-1 was applied to IVM in oocytes of aged mice. After 2 hours of IVM, the GVBD status of the aged mice was observed. The results are as follows: Figure 1 As shown, the GVBD rate of oocytes in the SDF-1 group was slightly higher than that in the control group (0 ng / mL SDF-1), but the difference was not statistically significant (90.9±0.7% vs. 85.6±2.9%, P=0.216). After 14-16 hours of culture, the IVM maturation rate of oocytes in the SDF-1 group was significantly higher than that in the control group, with a statistically significant difference (84.3±2.0% vs. 72.6±1.0%, P=0.012).

[0034] 2. Effects of SDF-1 on ICSI of oocytes (1) Preparation of working dish A: Capacitance dish: 80 μL G-IVF culture medium, covered with tissue culture mineral oil to completely cover the culture medium droplets; B: Operation dish: several 5 μL G-MOPS culture medium droplets and one row of 10% PVP culture medium droplets, covered with tissue culture mineral oil to completely cover the culture medium droplets; C: Cleavage embryo culture dish: several 20 μL G-1 culture medium droplets, covered with tissue culture mineral oil to completely cover the culture medium droplets; D: Blastocyst culture dish: several 20 μL G-2 culture medium droplets, covered with tissue culture mineral oil to completely cover the culture medium droplets; The prepared culture dishes are placed in a 37℃, 6% CO2, 5% O2 and 89% N2 three-gas incubator and equilibrated overnight.

[0035] (2) Prepare sperm Ten-week-old male mice were euthanized by cervical dislocation. The mice's abdomens were disinfected with 75% alcohol. A "Y"-shaped incision was made in the abdomen to expose both epididymis. Surgical forceps were used to separate the connective tissue surrounding the epididymis, and both epididymis were cut off and placed in mineral oil in dish A. A 1 mL syringe was used to puncture the epididymis, and the clumps of sperm were pulled into a G-IVF droplet. Sperm motility was observed; highly motile sperm diffused rapidly in a cloud-like pattern. The collected sperm were subjected to repeated freeze-thaw cycles at -80℃ and 37℃ for more than five times to decompose the sperm tails. After incubation for 1 hour in a 37℃ tri-gas incubator, 2.5 μL of sperm was added to a PVP droplet in dish B. The heating stage was turned on, and after installing the injection and fixation needles on the machine, dish B was placed on the heating stage. Ten mature mouse oocytes from IVM were collected each time and placed in G-MOPS droplets. Before injection, suitable sperm heads were selected and loaded into the injection needle. One mature IVM oocyte was fixed in place with a fixation needle held in the left hand, while the injection needle was inserted into the oocyte with the right hand, injecting the sperm head into the oocyte cytoplasm. The injection needle was gently withdrawn under slight negative pressure to release the oocyte, which was then transferred to dish C and incubated in a 37°C tri-gas incubator. Pronucleus formation was observed 7 hours after injection, fertilization 24 hours later, cleavage 48 hours later, and the embryos were transferred to dish D. Blastocyst formation was observed 108 hours later. The fertilization rate, cleavage rate, and blastocyst formation rate were calculated. The main indicators were calculated as follows: Fertilization rate = Number of two-cell embryos / Number of surviving oocytes; Cleavage rate = Number of cleaved embryos / Number of two-cell embryos; Blastocyst formation rate = Number of blastocysts / Number of two-cell embryos.

[0036] like Figure 2 As shown, in young mice, the fertilization rate and cleavage rate were slightly higher in the SDF-1 group compared with the control group, but there was no statistical difference (fertilization rate: 72.2±5.0% vs. 68.4±2.0%, P=0.595; cleavage rate: 95.4±1.9% vs. 89.5±4.4%, P=0.379). The blastocyst formation rate in the SDF-1 group was significantly higher than that in the control group (70.6±1.3% vs. 60.8±1.0%, P=0.008). In older mice, all ICSI outcomes were lower than in younger mice. Treatment with SDF-1 slightly increased the fertilization and cleavage rates of oocytes in older mice compared to the untreated group, but the differences were not statistically significant (fertilization rate: 63.5±1.5% vs. 62.5±2.3%, P=0.781; cleavage rate: 85.8±7.1% vs. 79.2±3.8%, P=0.536). The blastocyst formation rate was significantly higher in the SDF-1 group than in the control group (63.2±2.8% vs. 48.4±0.7%, P=0.014).

[0037] We used standard methods to perform immunofluorescence staining on oocyte chromosomes and spindle fibers, mitochondrial staining on oocytes, and ROS staining on oocytes / blastocysts. We then assessed blastocyst quality by detecting ROS and apoptosis levels. The results are as follows: Figure 3 As shown, in both young and old mice, the ROS level of blastocysts derived from oocytes in the SDF-1 group was significantly lower than that in the control group, and the difference was statistically significant (young group: 9.2±0.1 vs. 11.6±0.6, P=0.026; old group: 11.9±0.9 vs. 16.5±0.5, P=0.020).

[0038] TUNEL staining was performed using a one-step terminal deoxynucleotidyl transferase-mediated nick-and-end mark (TUNEL) apoptosis detection kit (TMR red fluorescence) to detect apoptosis levels in blastocysts. The procedure was performed according to the kit instructions. The apoptotic cell ratio was calculated as: number of TUNEL-positive cells / total number of embryonic cells. Results are as follows: Figure 4 As shown, the proportion of apoptotic cells in the young mouse control group was approximately 18.7±2.7%, while it decreased significantly in the SDF-1 group (6.6±2.0%), with a statistically significant difference (P=0.043). In older mice, the proportion of apoptotic cells in the control group was significantly higher than that in young mice, at 34.2±2.8%, while the proportion of apoptotic cells in oocytes cultured with SDF-1 decreased significantly after ICSI in the blastocysts, at 12.4±0.9% (P=0.004).

[0039] The results suggest that adding 20 ng / mL SDF-1 to the in vitro culture system can significantly improve the maturation rate of oocytes from mice of different ages, and improve the subsequent ICSI outcome and blastocyst quality. To explore the reasons for this, we further statistically analyzed the proportion of abnormal spindle morphology and abnormal chromosome arrangement in oocytes matured after IVM in mice of different ages.

[0040] The results are as follows Figure 5As shown, in young mice, the proportion of abnormal spindle morphology decreased compared to the control group, but the difference was not statistically significant (17.1±0.6% vs. 18.6±0.5%, P=0.168), while the proportion of abnormal chromosome arrangement decreased significantly compared to the control group (18.4±0.7% vs. 23.5±1.0%, P=0.031). In 44-48 week old mice, the addition of 20 ng / mL SDF-1 to the IVM system significantly reduced the proportions of abnormal spindle morphology and abnormal chromosome arrangement in mature oocytes compared to the control group (proportion of abnormal spindle morphology: 20.5±0.8% vs. 26.0±1.2%, P=0.035; proportion of abnormal chromosome arrangement: 27.7±0.7% vs. 33.4±1.4%, P=0.039).

[0041] Fluorescent staining of cortical granules in IVM mature oocytes was performed using conventional methods. The proportion of cells with abnormal cortical granule distribution and the fluorescence intensity of cortical granules between different groups were statistically analyzed. Under a fluorescence microscope, the cortical granules of normal mature oocytes typically showed clear green fluorescence, distributed in a monolayer beneath the cell membrane, indicating that the oocyte cytoplasm was mature. Abnormal oocytes, however, exhibited abnormal cortical granule distribution and insufficient number, showing uneven green fluorescence distribution and reduced fluorescence intensity under the microscope. The results are as follows: Figure 6 As shown: In mature oocytes of IVMs from both young and old mice, the proportion of abnormal cortical granules in the SDF-1 group was significantly lower than that in the control group (young group: 20.0±0.5% vs. 25.8±1.1%, P=0.017; old group: 31.4±0.7% vs. 38.3±0.8%, P=0.006). In old mice, the fluorescence intensity of cortical granules in the SDF-1 group was significantly higher than that in the control group (5.0±0.4 vs. 2.8±0.1, P=0.012). In young mice, the addition of SDF-1 also slightly increased the fluorescence intensity of cortical granules in oocytes, but no statistical difference was observed (4.6±0.6 vs. 2.9±0.4, P=0.071).

[0042] Furthermore, the distribution and activity of mitochondria in mature IVM oocytes were observed and analyzed by fluorescent staining. In immature oocytes, mitochondria are distributed around the cell periphery. As the oocyte matures, mitochondria migrate from the cytoplasm to the cytoplasm, becoming evenly distributed around the nucleus, and their number increases, providing a good foundation for oocyte metabolism and energy supply. The fluorescent staining results are shown below. Figure 7As shown, adding SDF-1 to IVM culture medium increased the mitochondrial fluorescence intensity of IVM oocytes from young mice (21.6±0.3 vs. 18.8±0.5, P=0.020), while there was no statistically significant difference in the proportion of abnormal mitochondria between the two groups (12.0±0.8% vs. 12.0±1.2%, P=0.999). In older mice, the mitochondrial fluorescence intensity was significantly weaker than that of younger mice (15.8±0.4 vs. 18.8±0.5, P=0.016), and the proportion of abnormal mitochondria was also higher in older mice (29.6±1.2% vs. 12.0±1.2%, P=0.0004). After the addition of SDF-1, the mitochondrial fluorescence intensity of mature oocytes in IVMs of older mice was significantly increased (15.8±0.4 vs. 18.6±0.6, P=0.032), and the proportion of abnormal mitochondria was significantly reduced (29.6±1.2% vs. 24.1±0.5%, P=0.016).

[0043] To assess mitochondrial activity, this invention also performed fluorescent staining on the mitochondrial membrane potential (MMP) levels of mature oocytes from IVMs of different ages, using the conventional JC-1 staining method. The results are as follows: Figure 8 As shown, regardless of whether they were young mice aged 6-8 weeks or older mice aged 44-48 weeks, the MMP level in mature oocytes of IVM was significantly increased after treatment with SDF-1 (young group: 2.0±0.2 vs. 1.0±0.1, P=0.023; older group: 1.5±0.1 vs. 0.8±0.1, P=0.029).

[0044] The level of mitochondrial ROS in mature oocytes was detected using the known MitoSOX staining method, and the results are as follows: Figure 9 As shown, the in vitro addition of 20 ng / mL SDF-1 significantly reduced the ROS fluorescence intensity in mouse oocytes of different ages (young group: 12.1±0.2 vs. 14.4±0.4, P=0.009; older group: 11.6±0.2 vs. 15.1±0.5, P=0.007). The ROS level in mitochondria, as shown... Figure 10As shown, in mature oocytes of young mouse IVMs, the mitochondrial Mito Sox fluorescence intensity in the SDF-1 group was significantly lower than that in the control group (5.5±0.5 vs. 8.4±0.7, P=0.048). In older mice, the Mito Sox fluorescence intensity was significantly higher than that in younger mice (27.6±0.5 vs. 8.4±0.7, P<0.0001), and the addition of SDF-1 significantly reduced the Mito Sox fluorescence intensity (21.8±0.9 vs. 27.6±0.5, P=0.011).

[0045] Follicular fluid provides a suitable ovarian microenvironment for oocyte development and maturation. This study investigated the effects of in vitro SDF-1 supplementation on oocyte quality and subsequent developmental potential in aged mice, simulating the in vivo follicular fluid environment. Results showed that in vitro supplementation of 20 ng / mL SDF-1 reduced total ROS and mitochondrial ROS levels in mature oocytes from both young and aged mice, optimized spindle assembly and chromosome alignment, promoted cytoplasmic maturation, and enhanced mitochondrial activity, thereby improving oocyte quality and subsequent embryonic developmental potential.

[0046] Example 2: Exploring the mechanism by which SDF-1 improves the quality of older oocytes by upregulating autophagy levels To further explore the molecular mechanism by which SDF-1 improves oocyte quality in aged mice, single-cell transcriptome sequencing was performed on mature oocytes from IVMs of aged control, aged SDF-1 group, and young control mice. Bioinformatics analysis showed that after SDF-1 treatment, differentially expressed genes in oocytes of aged mice were significantly enriched in autophagy-related pathways, suggesting that SDF-1 may exert its effect by regulating autophagy levels, such as... Figure 11 and Figure 12 As shown.

[0047] To validate the sequencing results, real-time quantitative PCR was used to detect the expression levels of autophagy-related genes. The results showed that, compared with the aged control group, the mRNA expression levels of autophagy-related genes Lc3, Atg5, Beclin1, and Lamp2 in mature oocytes of the IVM from aged mice in the SDF-1 group were significantly increased (P<0.05). Figure 13 (As shown). Simultaneously, lysosomal fluorescence staining showed that the lysosomal fluorescence intensity in the SDF-1 group of older oocytes was significantly enhanced compared to the control group (129.2±6.5 vs. 70.8±7.7, P<0.0001) (as shown). Figure 14As shown in the figure, SDF-1 can promote lysosomal activity. Immunofluorescence staining further confirmed that the fluorescence intensity of key autophagy proteins LC3 (33.3±0.7 vs. 28.7±0.9, P=0.0002) and Beclin1 (109.4±4.4 vs. 89.7±6.6, P=0.027) in older oocytes of the SDF-1 group was significantly higher than that in the control group (as shown in the figure). Figure 15 and Figure 16 (As shown).

[0048] To clarify the role of autophagy in improving the quality of older oocytes using SDF-1, the autophagy inhibitor chloroquine (CQ) was used for intervention. The CQ group (50 μM CQ), the Mix group (20 ng / mL SDF-1 and 50 μM CQ), and the SDF-1 group (20 ng / mL SDF-1) were all administered. The control group received neither SDF-1 nor CQ inhibitors. However, because CQ is soluble in DMSO, the final CQ group culture medium contained approximately 5‰ DMSO. Therefore, 5‰ DMSO was also added to the culture media of the control and SDF-1 groups to control for potential influencing factors. Results showed:

[0049] (1) CQ significantly inhibited the maturation rate of oocytes in older mice. The maturation rate of the CQ group was significantly lower than that of the control group (50.6±1.6% vs. 66.0±0.3%, P=0.002). The maturation rate of the Mix group was also significantly lower than that of the control group (61.5±1.2% vs. 66.0±0.3%, P=0.041) and the SDF-1 group (61.5±1.2% vs. 75.0±1.5%, P=0.004), but significantly higher than that of the CQ group (61.5±1.2% vs. 50.6±1.6%, P=0.012). (2) Increased the proportion of abnormal spindles and chromosomes. Compared with the control group, the proportion of abnormal spindle morphology (35.3±1.2% vs. 26.0±1.2%, P=0.010) and abnormal chromosome arrangement (39.1±0.9% vs. 33.4±1.4%, P=0.047) in the CQ group after inhibiting autophagy was significantly increased. However, after adding SDF-1, the proportion of abnormal spindle morphology (29.6±0.8% vs. 35.3±1.2%, P=0.033) and abnormal chromosome arrangement (33.0±0.7% vs. 39.1±0.9%, P=0.014) in the Mix group was lower than that in the CQ group, similar to the control group, but still higher than that in the SDF-1 group, which showed statistical difference. (3) ROS levels were increased. The ROS level in the CQ group was significantly higher than that in the control group (34.5±6.3 vs. 11.6±0.9, P=0.001). In the Mix group, which was supplemented with both SDF-1 and CQ, the ROS level of oocytes was significantly lower than that in the CQ group (19.7±2.3 vs. 34.5±6.3, P=0.047), but still higher than that in the control group (19.7±2.3 vs. 11.6±0.9, P=0.005). The differences in mitochondrial ROS levels in oocytes from different groups were also detected using Mito Sox fluorescence staining. It was found that the Mito Sox fluorescence intensity in the CQ group was significantly higher than that in the control group (41.6±1.5 vs. 27.6±0.6, P=0.001). In the Mix group, which simultaneously added SDF-1 and CQ, the Mito Sox fluorescence intensity in oocytes was significantly lower than that in the CQ group (30.5±1.1 vs. 41.6±1.5, P=0.004), but still higher than that in the control group, with no statistically significant difference (30.5±1.1 vs. 27.6±0.6, P=0.079).

[0050] In summary, this embodiment demonstrates through Smart-seq technology and experimental verification that SDF-1 reduces the production of total ROS and mitochondrial ROS in mature oocytes of older mice by increasing autophagy levels in oocytes, clearing damaged organelles and protein aggregates, thereby improving spindle assembly and chromosome alignment, and enhancing oocyte quality and subsequent developmental potential.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A culture medium for in vitro maturation of aging oocytes, characterized in that: The culture medium contains stromal cell-derived factor 1.

2. A kit for in vitro maturation of older oocytes, characterized in that: It contains the culture medium as described in claim 1.

3. The application of the culture medium as described in claim 1 or the kit as described in claim 2 in improving the quality of older oocytes.

4. The application as described in claim 3, characterized in that: The concentration of the stromal cell-derived factor 1 is 10~100 ng / mL.

5. The application as described in claim 4, characterized in that: The concentration of the stromal cell-derived factor 1 is 10-20 ng / mL.

6. The application as described in claim 3, characterized in that: The stromal cell-derived factor 1 upregulates the expression of one or more autophagy-related genes, such as Lc3, Atg5, Beclin1, or Lamp2, and / or enhances lysosomal activity, and / or increases the expression levels of LC3 and / or Beclin1 proteins.

7. The application as described in claim 3, characterized in that: The term "advanced age oocytes" includes female mouse oocytes aged 44-48 weeks or female oocytes aged 38 years or older.

8. An in vitro culture method for improving the quality of older oocytes, characterized in that: This includes the step of culturing older oocytes in an in vitro maturation medium containing stromal cell-derived factor 1.

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