Method for inhibiting expression of OPA1 activity for reducing quality of oocytes cultured in vitro and application

Abstract

The invention relates to the technical field of animal reproduction, in particular to a method for inhibiting expression of OPA1 activity to reduce the quality of in-vitro cultured oocytes and application. OPA1 is expressed in different stages of meiosis of bovine oocytes, inhibition of OPA1 leads to reduction of first polar body discharge rate of the bovine oocytes and damage of spindle structures, and research on mitochondria finds that inhibition of OPA1 leads to mitochondrial dysfunction and promotion of mitochondrial division, and mitochondrial autophagy is induced through a PINK-1 / Parkin pathway.

Description

Technical Field

[0001] The present application relates to the field of animal reproduction technology, and more specifically, to a method and application of inhibiting the expression of OPA1 activity for reducing the quality of oocytes cultured in vitro. Background Art

[0002] In recent years, the live egg collection-in vitro embryo production (OPU-IVP) technology has been increasingly applied to the breeding and production of beef cattle. The use of OPU-IVP technology can greatly shorten the breeding cycle of beef cattle and greatly improve the breeding efficiency of beef cattle. The basis for obtaining embryos through IVP is the in vitro maturation of oocytes (IVM) technology, but the quality of mature oocytes obtained by IVM is not satisfactory. Therefore, it is very important to improve the quality of IVM of oocytes. Mitochondria provide energy for various biological processes in the maturation of oocytes and play a very important role in the maturation of oocytes. The function of mitochondria is closely related to the quality of oocytes.

[0003] Autophagy is a genetically programmed process that removes and recycles unnecessary or dysfunctional cellular components. Initially, autophagy was defined as a non-selective pathway, but it is now generally accepted that there are two different autophagy pathways: selective and non-selective. When nutrients are scarce, non-selective autophagy is mainly activated to provide nutrients to the cell. Under nutrient-rich conditions, selective autophagy is active and is mainly responsible for the removal of dysfunctional organelles and intracellular pathogens. Cells encounter various stresses that can lead to mitochondrial dysfunction. In order to maintain the homeostasis of the intracellular environment and the health of the mitochondrial network, cells initiate the autophagic mechanism to degrade damaged mitochondria, a process called mitophagy.

[0004] There are two main pathways of mitochondrial autophagy: ubiquitin-dependent pathway and non-ubiquitin-dependent pathway. The key proteins of the ubiquitin-dependent pathway are PINK-1 and Parkin, so it is also called the PINK-1 / Parkin pathway. Mitochondrial damage is manifested by decreased membrane potential. After PINK-1 accumulates and stabilizes on the mitochondrial surface, it is activated by autophosphorylation, and then recruits Parkin in the cytoplasm to the mitochondrial surface and activates the E3 ubiquitin ligase. Subsequently, Parkin ubiquitinates OMM proteins, and the ubiquitin chains are phosphorylated by PINK-1. At this time, autophagy adaptor proteins (NDP52 and OPTN, etc.) recognize the phosphorylated ubiquitin-modified proteins on OMM. NDP52 and OPTN interact with LC3 through their own LC3 interaction regions (LIRs), and finally connect the ubiquitin-labeled mitochondria to the autophagosome to initiate autophagy. The non-ubiquitin-dependent pathway is different from the PINK-1 / Parkin pathway. There are many proteins containing LIR on the OMM, which can directly bind to LC3 to initiate autophagy.

[0005] Oocyte maturation involves multiple processes, such as spindle assembly, chromosome arrangement, and organelle rearrangement, all of which require energy support from mitochondria. It is known that mitochondrial dynamics accompanies the progression of oocyte meiosis, and the IMM protein OPA1 is essential for mitochondrial dynamics. However, there are few reports on the effect of OPA1 on bovine oocyte maturation. Therefore, the expression and localization of OPA1 during bovine oocyte maturation were first explored. Then, the OPA1-specific inhibitor MYLS22 was used to inhibit OPA1 activity to explore the role of OPA1 in mitochondrial function, mitochondrial dynamics, apoptosis, and autophagy during bovine oocyte maturation. Summary of the invention

[0006] The present invention provides a method and application for reducing the quality of in vitro cultured oocytes by inhibiting the expression of OPA1 activity. OPA1 is expressed at different stages of bovine oocyte meiosis. Inhibition of OPA1 leads to a decrease in the first polar body extrusion rate of bovine oocytes and damages the spindle structure. Studies on mitochondria have found that inhibition of OPA1 leads to mitochondrial dysfunction, promotes mitochondrial fission, and induces mitochondrial autophagy through the PINK-1 / Parkin pathway.

[0007] In a first aspect, the present invention provides a method for reducing the quality of oocytes cultured in vitro by inhibiting the expression of OPA1 activity.

[0008] Preferably, the inhibition of OPA1 activity expression specifically induces bovine oocyte mitochondrial autophagy via the PINK-1 / Parkin pathway.

[0009] Preferably, the OPA1 activity is inhibited by the OPA1-specific inhibitor MYLS22, and the oocytes are cultured in an in vitro culture medium containing the inhibitor MYLS22, and the concentration of the OPA1-specific inhibitor MYLS22 in the in vitro culture medium is 0 μM-60 μM.

[0010] In a second aspect, the present invention provides a bovine oocyte in vitro maturation fluid, comprising the OPA1 specific inhibitor MYLS22, wherein the OPA1 specific inhibitor is expressed in mRNA and protein in bovine oocytes at GV, pre-MI, MI and MII stages.

[0011] Preferably, the in vitro culture medium concentration of the OPA1 specific inhibitor MYLS22 is 0 μM-60 μM.

[0012] Preferably, the in vitro culture medium concentration of the OPA1 specific inhibitor MYLS22 is 40 μM.

[0013] Preferably, the bovine oocyte in vitro maturation medium is based on TCM-199 culture medium buffered with NaHCO3, and also contains 10% fetal bovine serum by volume, 1% penicillin and streptomycin mixture, 0.022 g / mL sodium pyruvate, 0.9 mg / mL cysteine, 1 mg / mL estradiol, 10 ng / mL epidermal growth factor, and 0.04 mg / mL follicle stimulating hormone.

[0014] In a third aspect, the present invention provides a method for reducing the quality of oocytes after in vitro maturation, wherein bovine oocytes are added to the oocyte in vitro maturation culture medium for culture.

[0015] Preferably, oocytes are matured in vitro in a CO2 incubator at 38.5°C and 5% CO2 for 22-24 h.

[0016] In summary, this application has the following effects: 1. Inhibition of OPA1 in the present invention affects the maturation of bovine oocytes, impairs mitochondrial function, destroys mitochondrial dynamics homeostasis, and induces the occurrence of mitochondrial autophagy through the PINK-1 / Parkin pathway; 2. In the present invention, inhibition of OPA1 promotes the recruitment of Parkin to mitochondria, and inhibition of OPA1 activates mitochondrial autophagy through the PINK-1 / Parkin pathway. Inhibition of OPA1 increases the expression of LC3 protein and the lysosome content of bovine oocytes; 3. OPA1 of the present invention is essential for bovine oocyte maturation. Inhibition of OPA1 leads to mitochondrial dysfunction in bovine oocytes, induces mitochondrial fission, and induces mitochondrial autophagy through the PINK-1 / Parkin pathway.

[0017] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of protection of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The expression of OPA1 mRNA and protein in bovine oocytes at various stages in the present invention; A. Representative immunofluorescence staining pictures, blue is DNA; red is OPA1; Bar=20 μm; B. Relative quantitative analysis of OPA1 protein expression (GV: n=47, pre-MI: n=38, MI; n=38, MII: n=39); C. Relative quantitative analysis of OPA1 mRNA expression (n=3 / group); Figure 2The effect of inhibiting OPA1 on bovine oocyte maturation in the present invention; wherein, A. Representative images of bovine oocyte spindles, blue is DNA, green is α-tubulin, Bar=10 μm; B. Quantitative analysis of spindle abnormality ratio (n=3 / group); C. Representative images of bovine oocyte CGs staining, blue is DNA, green is CGs, Bar=20 μm; D. Relative quantitative analysis of CGs fluorescence intensity (Control: n=26, 40 μM: n=29); Figure 3 The effects of inhibiting OPA1 on redox homeostasis and mitochondrial function of bovine oocytes in the present invention; wherein, A. Representative images of ROS and GSH staining of bovine oocytes, blue is GSH, green is ROS, Bar=100 μm; B. Relative quantitative analysis of intracellular ROS levels (Control: n=69, 40 μM: n=52); C. Relative quantitative analysis of intracellular GSH levels (Control: n=91, 40 μM: n=77); D. Relative expression levels of antioxidant-related gene mRNA (n=3 / group); E. Representative images of mitochondrial membrane potential staining, red is JC-1 aggregates, green is JC-1 monomer, Bar=100μm; F. Relative quantitative analysis of mitochondrial membrane potential levels (Control: n=46, 40 μM: n=47); G. Representative images of intracellular ATP staining, Bar=100 μm; H. Relative quantitative analysis of intracellular ATP levels (Control: n=36, 40 μM: n=36); I. Representative images of MitoSOX staining, Bar=100 μm; J. Relative quantitative analysis of ROS levels in mitochondria (Control: n=38, 40 μM: n=34); K. Representative images of TOM20 staining, blue is DNA, red is TOM20, Bar=20 μm; L. Relative quantitative analysis of TOM20 fluorescence intensity (Control: n=34, 40 μM: n=30); Figure 4 The effect of inhibiting OPA1 on bovine oocyte apoptosis in the present invention; wherein, A. Representative images of bovine oocyte CB staining, Bar=100 μm; B. Relative quantitative analysis of CB levels in bovine oocytes (Control: n=36, 40 μM: n=37); C. Relative expression levels of apoptosis-related gene mRNA (n=3 / group); Figure 5The effect of inhibiting OPA1 on mitochondrial dynamics in bovine oocytes in the present invention; wherein, A. Representative images of MFN1 immunofluorescence staining, blue for DNA, green for MFN1, Bar=20 μm; B. Relative quantitative analysis of MFN1 fluorescence intensity (Control: n=38, 40 μM: n=40); C. Representative images of Drp1 immunofluorescence staining, blue for DNA, green for Drp1, Bar=20 μm; D. Relative quantitative analysis of Drp1 fluorescence intensity (Control: n=45, 40 μM: n=41); E. Relative quantitative analysis of mRNA expression of genes related to mitochondrial dynamics (n=3 / group); F. Representative images of Drp1 / MFF immunofluorescence colocalization, blue for DNA, green for Drp1, red for MFF, Bar=20 μm; G. Quantification of Drp1 and MFF fluorescence intensities in oocytes of the control group along the straight line shown in Figure F; H. The fluorescence intensity of Drp1 and MFF in oocytes of the treatment group was quantified along the straight line shown in Figure F; I. Pearson correlation coefficient of Drp1 / MFF proteins in oocytes of the two groups (Control: n=25, 40 μM: n=23); J. Representative image of Drp1 / TOM20 immunofluorescence co-localization, blue is DNA, green is Drp1, and red is TOM20. Bar=20μm; K. The fluorescence intensity of Drp1 and TOM20 in oocytes of the control group was quantified along the straight line shown in Figure J; L. The fluorescence intensity of Drp1 and TOM20 in oocytes of the treatment group was quantified along the straight line shown in Figure J; M. Pearson correlation coefficient of Drp1 / TOM20 proteins in oocytes of the two groups (Control: n=22, 40μM: n=20); Figure 6The effect of the present invention on the mitophagy of bovine oocytes by inhibiting OPA1; A. Representative images of Parkin immunofluorescence staining, blue for DNA, green for Parkin, Bar=20μm; B. Relative quantitative analysis of Parkin fluorescence intensity (Control: n=30, 40 μM: n=29); C. Relative quantitative analysis of the mRNA expression of mitophagy-related genes (n=3 / group); D. Representative images of Parkin / TOM20 immunofluorescence colocalization, blue for DNA, green for Parkin, red for TOM20, Bar=20 μm; E. Quantification of the fluorescence intensity of Parkin and TOM20 in the oocytes of the control group along the straight line shown in Figure D; F. Quantification of the fluorescence intensity of Parkin and TOM20 in the oocytes of the treatment group along the straight line shown in Figure D; G. Pearson correlation coefficient of Parkin / TOM20 proteins in the two groups of oocytes (Control: n=20, 40 μM: n=17); H. Representative images of LC3 immunofluorescence staining, blue is DNA, green is LC3, Bar=20 μm; I. Relative quantitative analysis of LC3 fluorescence intensity (Control: n=32, 40 μM: n=30); J. Relative quantitative analysis of mRNA expression of autophagy-related genes (n=3 / group); K. Representative images of lysosome staining, Bar=100 μm; L. Relative quantitative analysis of intracellular lysosome content (Control: n=50, 40 μM: n=40). DETAILED DESCRIPTION

[0019] The present application is further described in detail below in conjunction with the examples. It is particularly noted that if no specific conditions are specified in the following examples, the experiments are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from common commercial sources.

[0020] Example The materials in the examples were obtained from: OPA1 antibody was purchased from NOVUS (1E8-1D9, Shanghai, China); TOM20 (66777-1-Ig), MFN1 (13798-1-AP), Drp1 (12975-1-AP), MFF (66527-1-Ig), Parkin (14060-1-AP) were purchased from Proteintech (Wuhan, China). OPA1 specific inhibitor MYLS22 was purchased from MedChemExpress (HY-136446, Shanghai, China). The remaining chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0021] 1. In vitro maturation of bovine oocytes: The fresh bovine ovaries used in this example were purchased from a local slaughterhouse in Yanji City, and the ovaries were transported to the laboratory within 2 hours using a thermos bottle. Use scissors to remove excess tissue around the ovaries (such as uterine horns, etc.), and then wash three times with saline. Select 2-8 mm follicles, use an 18G disposable syringe to aspirate follicular fluid, and then place the follicular fluid in a centrifuge tube. After 15-20 min, remove the supernatant, transfer the precipitate to a sterile culture dish and mix with 1 mL IVM (Table 1). Under a stereomicroscope, select cumulus oocyte complexes (COCs) with uniform cytoplasm and 3 or more layers of tightly connected cumulus cells, wash in IVM, transfer to a four-well culture plate, and culture in a CO2 incubator at 38.5 ° C and 5% CO2 for 22-24 h, and then count the first polar body (PBI) extrusion rate. In this study, four MYLS22 concentrations were used, namely 0 μM, 20 μM, 40 μM, and 60 μM.

[0022] Table 1 Bovine oocyte in vitro maturation medium (IVM)

[0023] 2. Detection of intracellular ROS and GSH levels After 22-24 h of in vitro culture, 0.01% hyaluronidase (Hy) was used to remove the cumulus cells on COCs, and then washed with 1% BSA-PBS. According to the instructions, the oocytes were placed in a 96-well plate containing DCFH-DA or CMF2HC dye solution and incubated at 37 °C for 20 min. After washing with 1% BSA-PBS, the oocytes were observed and photographed under an inverted fluorescence microscope.

[0024] 3. Detection of intracellular mitochondrial membrane potential (MMP) and ATP levels After 22-24 h of in vitro culture, 0.01% Hy was used to remove the cumulus cells on the COCs, and then washed with 1% BSA-PBS. According to the instructions, the oocytes were placed in a 96-well plate containing JC-1 or ATP dye and incubated at 37 °C for 20 min. After washing with 1% BSA-PBS, they were observed and photographed under an inverted fluorescence microscope.

[0025] 4. Detection of ROS levels in mitochondria After 22-24 h of in vitro culture, cumulus cells on COCs were removed with 0.01% Hy and then washed with 1% BSA-PBS. According to the instructions, oocytes were placed in a 96-well plate containing MitoSOX Red (Invitrogen) dye and incubated at 37 °C for 20 min. After washing with 1% BSA-PBS, they were observed and photographed under an inverted fluorescence microscope.

[0026] 5. Detection of intracellular cathepsin B (CB) levels After 22-24 h of in vitro culture, cumulus cells on COCs were removed with 0.01% Hy and then washed with 1% BSA-PBS. According to the instructions, oocytes were placed in a 96-well plate containing CB (Invitrogen) staining solution and incubated at 37 °C for 20 min. After washing with 1% BSA-PBS, the oocytes were observed and photographed under an inverted fluorescence microscope.

[0027] 6. Detection of intracellular lysosome content After 22-24 h of in vitro culture, cumulus cells on COCs were removed with 0.01% Hy and then washed with 1% BSA-PBS. According to the instructions, oocytes were placed in a 96-well plate containing Lyso-Tracker Red (Beyotime) dye solution and incubated at 37 °C for 20 min. After washing with 1% BSA-PBS, the oocytes were observed and photographed under an inverted fluorescence microscope.

[0028] 7. Immunofluorescence staining After 22-24 h of in vitro culture, cumulus cells on COCs were removed with 0.01% Hy and then washed with 1% BSA-PBS. After fixation with 4% paraformaldehyde at room temperature for 15 min, the cells were washed and blocked with 1% BSA-PBS for 1 h. The cells were incubated with 0.5% Triton X-100 at room temperature for 30 min, washed and blocked with 1% BSA-PBS for 1 h.

[0029] For α-tubulin, MFN1, Drp1, LC3 and Parkin staining, the slides were incubated with anti-α-tubulin (1:100), MFN1 (1:200), Drp1 (1:200), LC3 (1:200) and Parkin (1:200) antibodies at 4°C overnight. After washing and blocking with 1% BSA-PBS for 1 h, the slides were incubated with donkey anti-rabbit IgG conjugated to Alexa Fluor 488-labeled secondary antibody (1:200) at 37°C for 2 h, washed with 1% BSA-PBS and blocked for 1 h. The slides were incubated with Hoechst 33342 at 37°C for 20 min, washed with 1% BSA-PBS and mounted.

[0030] For staining of cortical granules (CGs), LCA-FITC (1:1000) was incubated at 4°C overnight, washed with a mixture of 1% Tween 20 and 0.01% Triton X-100, incubated with Hoechst 33342 at 37°C for 20 min, washed with 1% BSA-PBS and mounted.

[0031] For OPA1 and TOM20 staining, the slides were incubated with OPA1 (1:50) and TOM20 (1:200) antibodies at 4°C overnight. After washing and blocking with 1% BSA-PBS for 1 h, the slides were incubated with goat anti-mouse IgG conjugated to Alexa Fluor 568-labeled secondary antibody (1:200) at 37°C for 2 h, washed with 1% BSA-PBS and blocked for 1 h. The slides were incubated with Hoechst 33342 at 37°C for 20 min, washed with 1% BSA-PBS and mounted.

[0032] For immunofluorescence colocalization staining, Drp1 (1:200) and MFF (1:200), Drp1 (1:200) and TOM20 (1:200), and Parkin (1:200) and TOM20 (1:200) antibodies were mixed and incubated overnight at 4°C. After washing and blocking with 1% BSA-PBS for 1 h, donkey anti-rabbit IgG conjugated to Alexa Fluor 488-labeled secondary antibodies (1:200) and goat anti-mouse IgG conjugated to Alexa Fluor 568-labeled secondary antibodies (1:200) were mixed and incubated at 37°C for 2 h. After washing and blocking with 1% BSA-PBS for 1 h, Hoechst 33342 was used for incubation at 37°C for 20 min, and the slides were washed with 1% BSA-PBS and mounted.

[0033] Finally, an Olympus laser confocal microscope was used to observe and photograph.

[0034] 8. mRNA extraction and real-time fluorescence quantitative PCR According to the manufacturer's instructions, mRNA was extracted from mature oocytes using the DynaBeads mRNA Direct Kit (Life Technologies, Carlsbad, CA, USA), and then cDNA was synthesized by reverse transcription. The real-time fluorescence quantitative PCR system consisted of 2 μL cDNA, 2 μL primers (primer sequences are shown in Table 2), 10 μL SYBR, 0.2 μL ROX-high, 0.2 μL ROX-low, and 5.6 μL ddH2O. The reaction program was 95 °C for 30 s, 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 20 s, for a total of 40 cycles. 2 -ΔΔCT The relative expression of mRNA was analyzed by the method.

[0035] Table 2 Primer sequences

[0036] Statistical analysis Image J was used to measure the fluorescence intensity of the images. Image J was used to analyze the immunofluorescence colocalization images, and the results were expressed as the Pearson correlation coefficient. SPSS statistical software was used to analyze the data, and the data were all in accordance with normal distribution and homogeneity of variance. The significance of the difference between the two groups was tested by T test, and the significance of the difference between three or more groups was tested by one-way analysis of variance. The data were expressed as mean ± standard deviation. Statistical graphs were prepared using GraphPad Prism. All experiments were repeated at least three times or more. P < 0.05 was considered statistically significant.

[0037] 9. Expression of OPA1 at various stages of bovine oocyte maturation Firstly, the expression of OPA1 mRNA and protein in bovine oocytes at GV, pre-MI, MI and MII stages was analyzed. Figure 1 The mRNA and protein expressions of OPA1 in bovine oocytes at various stages in the present invention are shown in Figure 2, wherein A. Representative images of immunofluorescence staining, blue is DNA; red is OPA1; Bar=20 μm; B. Relative quantitative analysis of OPA1 protein expression (GV: n=47, pre-MI: n=38, MI; n=38, MII: n=39); C. Relative quantitative analysis of OPA1 mRNA expression (n=3 / group).

[0038] The results of immunofluorescence staining are shown in the figure Figure 1 A and Figure 1As shown in B, the expression of OPA1 protein in bovine oocytes at pre-MI, MI and MII stages was significantly higher than that in GV stage (P<0.05). Real-time fluorescence quantitative PCR results showed (P<0.05) that the expression of OPA1 mRNA in bovine oocytes at pre-MI, MI and MII stages was also significantly higher than that in GV stage (P<0.05). The above results indicate that OPA1 plays an important role in the meiotic maturation of bovine oocytes.

[0039] 10. Effect of OPA1 inhibition on bovine oocyte maturation Bovine oocytes were treated with OPA1-specific inhibitor MYLS22 to explore the role of OPA1 in bovine oocyte maturation. First, the PBI excretion rate of bovine oocytes treated with different concentrations of MYLS22 was analyzed. The results showed that the PBI excretion rate in the 40 μM and 60 μM treatment groups was significantly reduced compared with the control group (P<0.05, Table 1). The MYLS22 concentration used in subsequent experiments was 40 μM.

[0040] Figure 2 The effects of inhibiting OPA1 on the nucleocytoplasmic maturation of bovine oocytes in the present invention, A. Representative images of bovine oocyte spindles, blue is DNA, green is α-tubulin, Bar=10 μm; B. Quantitative analysis of spindle abnormality ratio (n=3 / group); C. Representative images of bovine oocyte CGs staining, blue is DNA, green is CGs, Bar=20 μm; D. Relative quantitative analysis of CGs fluorescence intensity (Control: n=26, 40 μM: n=29).

[0041] Spindle assembly and chromosome arrangement are crucial for oocyte meiosis, so the oocyte spindle morphology was first detected. Figure 2 A and Figure 2 As shown in B, the abnormal spindle ratio in the 40 μM treatment group was significantly increased compared with the control group (P<0.05). An important indicator of oocyte cytoplasmic maturation is the distribution and number of CGs, so the number of CGs in oocytes in different treatment groups was detected. The number of CGs in the 40 μM treatment group was significantly higher than that in the control group (P<0.05). The above results indicate that inhibiting OPA1 will reduce the PBI extrusion rate of oocytes and affect the nuclear maturation of oocytes, but will improve the cytoplasmic maturation of oocytes.

[0042] Table 3 Effect of MYLS22 on PBI extrusion rate of bovine oocytes

[0043] Different letters on the shoulders indicate significant differences (P<0.05). 11. Effects of OPA1 inhibition on oocyte redox homeostasis and mitochondrial function Oocytes are often affected by oxidative stress during in vitro maturation. Therefore, the oxidative stress level of bovine oocytes was detected. Figure 3 The effects of inhibiting OPA1 on redox homeostasis and mitochondrial function of bovine oocytes in the present invention, A. Representative images of ROS and GSH staining of bovine oocytes, blue is GSH, green is ROS, Bar=100 μm; B. Relative quantitative analysis of intracellular ROS levels (Control: n=69, 40 μM: n=52); C. Relative quantitative analysis of intracellular GSH levels (Control: n=91, 40 μM: n=77); D. Relative expression levels of antioxidant-related gene mRNA (n=3 / group); E. Representative images of mitochondrial membrane potential staining, red is JC-1 aggregates, green is JC-1 monomer, Bar=100μm; F. Relative quantitative analysis of mitochondrial membrane potential levels (Control: n=46, 40 μM: n=47); G. Representative images of intracellular ATP staining, Bar=100 μm; H. Relative quantitative analysis of intracellular ATP levels (Control: n=36, 40 μM: n=36); I. Representative images of MitoSOX staining, Bar=100 μm; J. Relative quantitative analysis of ROS levels in mitochondria (Control: n=38, 40 μM: n=34); K. Representative images of TOM20 staining, blue is DNA, red is TOM20, Bar=20 μm; L. Relative quantitative analysis of TOM20 fluorescence intensity (Control: n=34, 40 μM: n=30).

[0044] like Figure 3 A to Figure 3 As shown in C, compared with the control group, the ROS level in the 40 μM treatment group was significantly reduced (P<0.05), and the GSH level was significantly reduced (P<0.05). The results of real-time fluorescence quantitative PCR showed that the mRNA expression levels of GPX4, Sirt3, SOD1, SOD2 and SOD3 in the 40 μM treatment group were significantly higher than those in the control group (P<0.05, Figure 3 D). This result was suspected to be caused by mitochondrial dysfunction, which caused the imbalance of cellular redox homeostasis. Then, the relevant indicators of mitochondrial function were tested. The results of mitochondrial membrane potential test showed that the mitochondrial membrane potential level of the 40 μM treatment group was significantly higher than that of the control group (P<0.05, Figure 3 E and Figure 3F). The decreased mitochondrial membrane potential level indicates impaired mitochondrial function, which directly affects the tricarboxylic acid cycle and ATP production. The ATP content of bovine oocytes was measured ( Figure 3 G and Figure 3 H) and ROS levels in mitochondria ( Figure 3 I and Figure 3 J), compared with the control group, the ATP content in the 40 μM treatment group was significantly reduced (P<0.05), and the level of ROS in the mitochondria was significantly reduced (P<0.05). Finally, the level of mitochondrial marker protein TOM20 was detected by immunofluorescence staining. The results showed that the level of TOM20 in the 40 μM treatment group was significantly lower than that in the control group (P<0.05, Figure 3 K and Figure 3 These results confirm the hypothesis that inhibition of OPA1 leads to an imbalance in cellular redox homeostasis, which is caused by abnormal mitochondrial function.

[0045] 12. Effect of OPA1 inhibition on bovine oocyte apoptosis Abnormal mitochondrial function usually induces apoptosis. Cathepsin B (CB) is an important lysosomal protease that plays an important role in apoptosis. Figure 4 The effect of inhibiting OPA1 on bovine oocyte apoptosis in the present invention, A. Representative images of bovine oocyte CB staining, Bar=100 μm; B. Relative quantitative analysis of CB levels in bovine oocytes (Control: n=36, 40 μM: n=37); C. Relative expression levels of apoptosis-related gene mRNA (n=3 / group).

[0046] like Figure 4 A and Figure 4 As shown in B, there was no significant difference in the CB content in the control group and the 40 μM treatment group. Real-time fluorescence quantitative PCR results showed that ( Figure 4 C). The expression levels of BAX, Caspase3 and Bcl-2 in the 40 μM treatment group were significantly higher than those in the control group (P<0.05), but there was no significant difference in the expression of BAX / Bcl-2 between the two groups.

[0047] 13. Effects of OPA1 inhibition on oocyte mitochondrial dynamics OPA1 is a protein located on the IMM that mediates IMM fusion. It is speculated that inhibition of OPA1 will disrupt mitochondrial dynamics homeostasis and induce mitochondrial fission. Figure 5The effect of inhibiting OPA1 on mitochondrial dynamics in bovine oocytes in the present invention, A. Representative images of MFN1 immunofluorescence staining, blue is DNA, green is MFN1, Bar=20 μm; B. Relative quantitative analysis of MFN1 fluorescence intensity (Control: n=38, 40 μM: n=40); C. Representative images of Drp1 immunofluorescence staining, blue is DNA, green is Drp1, Bar=20 μm; D. Relative quantitative analysis of Drp1 fluorescence intensity (Control: n=45, 40 μM: n=41); E. Relative quantitative analysis of mRNA expression of genes related to mitochondrial dynamics (n=3 / group); F. Representative images of Drp1 / MFF immunofluorescence colocalization, blue is DNA, green is Drp1, red is MFF, Bar=20 μm; G. Quantification of Drp1 and MFF fluorescence intensities in oocytes of the control group along the straight line shown in Figure F; H. Quantification of Drp1 and MFF fluorescence intensities in oocytes of the treatment group along the straight line shown in Figure F; I. Pearson correlation coefficient of Drp1 / MFF proteins in two groups of oocytes (Control: n=25, 40 μM: n=23); J. Representative images of Drp1 / TOM20 immunofluorescence co-localization, blue is DNA, green is Drp1, and red is TOM20. Bar=20μm; K. Quantification of Drp1 and TOM20 fluorescence intensity in oocytes of the control group along the straight line shown in Figure J; L. Quantification of Drp1 and TOM20 fluorescence intensity in oocytes of the treatment group along the straight line shown in Figure J; M. Pearson correlation coefficient of Drp1 / TOM20 proteins in two groups of oocytes (Control: n=22, 40μM: n=20).

[0048] First, the expression levels of OMM fusion protein MFN1 and mitochondrial fission protein Drp1 were detected. Figure 5 A to Figure 5 D. Compared with the control group, the expression of MFN1 protein in the 40 μM treatment group was significantly decreased (P<0.05), and the expression of Drp1 protein was significantly increased (P<0.05). The results of fluorescence quantitative PCR showed that ( Figure 5 E), the expression levels of MFN1 and Drp1 in the 40 μM treatment group were significantly higher than those in the control group (P<0.05). Drp1 is located in the cytoplasm, and its mitochondrial receptor protein MFF recruits it to the mitochondria and activates mitochondrial fission. The co-localization of Drp1 and MFF was analyzed by immunofluorescence co-localization method. The trends of the fluorescence intensity changes of Drp1 and MFF in the two groups of oocytes were similar ( Figure 5 F to Figure 5 H), Pearson correlation coefficient shows ( Figure 5There was no significant difference in the co-localization of Drp1 and MFF in the two groups of oocytes. At the same time, the co-localization of mitochondrial marker protein TOM20 and Drp1 was analyzed. The results are as follows Figure 5 J to Figure 5 There was no significant difference in the correlation between Drp1 and TOM20 in the two groups of oocytes ( Figure 5 M).

[0049] 14. Effect of inhibiting OPA1 on mitochondrial autophagy in bovine oocytes As shown in the previous results, inhibition of OPA1 activity can induce mitochondrial fission. In addition, inhibition of OPA1 activity can also activate mitochondrial autophagy, so indicators related to mitochondrial autophagy were detected. Figure 6 The effect of the present invention on the mitophagy of bovine oocytes by inhibiting OPA1, A. Representative images of Parkin immunofluorescence staining, blue is DNA, green is Parkin, Bar=20μm; B. Relative quantitative analysis of Parkin fluorescence intensity (Control: n=30, 40 μM: n=29); C. Relative quantitative analysis of the mRNA expression of mitophagy-related genes (n=3 / group); D. Representative images of Parkin / TOM20 immunofluorescence colocalization, blue is DNA, green is Parkin, red is TOM20, Bar=20 μm; E. Quantification of the fluorescence intensity of Parkin and TOM20 in the oocytes of the control group along the straight line shown in Figure D; F. Quantification of the fluorescence intensity of Parkin and TOM20 in the oocytes of the treatment group along the straight line shown in Figure D; G. Pearson correlation coefficient of Parkin / TOM20 proteins in the two groups of oocytes (Control: n=20, 40 μM: n=17); H. Representative images of LC3 immunofluorescence staining, blue is DNA, green is LC3, Bar=20 μm; I. Relative quantitative analysis of LC3 fluorescence intensity (Control: n=32, 40 μM: n=30); J. Relative quantitative analysis of mRNA expression of autophagy-related genes (n=3 / group); K. Representative images of lysosome staining, Bar=100 μm; L. Relative quantitative analysis of intracellular lysosome content (Control: n=50, 40 μM: n=40).

[0050] Immunofluorescence staining revealed that the Parkin expression in the 40 μM treatment group was significantly higher than that in the control group ( Figure 6 A and Figure 6 B, P<0.05). Real-time fluorescence quantitative PCR results showed that ( Figure 6 C), compared with the control group, the expression levels of mitochondrial autophagy-related genes PINK-1 and Parkin were significantly increased (P<0.05).

[0051] Mitochondrial autophagy recruits Parkin to mitochondria through PINK-1, so the co-localization of Parkin and TOM20 was detected by immunofluorescence co-localization. Figure 6 D to Figure 6 As shown in F, in the 40 μM treatment group ( Figure 6 D and Figure 6 F), the changing trends of Parkin and TOM20 fluorescence were similar, while in the control group ( Figure 6 D and Figure 6 E), Parkin and TOM20 have different changing trends. Combined with the Pearson correlation coefficient ( Figure 6 G), and found that after 40 μM MYLS22 treatment, the correlation between Parkin and TOM20 in bovine oocytes increased significantly (P<0.05). Then the expression level of LC3 in bovine oocytes was detected. The results are as follows Figure 6 H and Figure 6 As shown in I. Compared with the control group, the expression level of LC3 protein in the 40 μM treatment group was significantly increased (P<0.05). Real-time fluorescence quantitative PCR results showed that ( Figure 6 The expression levels of LC3, ATG5 and ATG6 in the 40 μM treatment group were significantly higher than those in the control group (P<0.05). Finally, the lysosome content in bovine oocytes was detected ( Figure 6 K and Figure 6 Compared with the control group, the level of lysosomes in bovine oocytes treated with 40 μM was significantly increased (P<0.05). The above results indicate that inhibition of OPA1 can induce mitochondrial autophagy in bovine oocytes.

[0052] The above is only an exemplary embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present disclosure should be included in the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be based on the protection scope of the claims.

Claims

1. Application of inhibiting the expression of OPA1 activity in the preparation of a method for reducing the quality of oocytes cultured in vitro.

2. The use according to claim 1, characterized in that: The inhibition of OPA1 activity expression specifically induces bovine oocyte mitochondrial autophagy via the PINK-1 / Parkin pathway.

3. The use according to claim 1, characterized in that: The expression of OPA1 activity is inhibited by the OPA1-specific inhibitor MYLS22. The oocytes are cultured in an in vitro culture medium containing the inhibitor MYLS22. The concentration of the in vitro culture medium of the OPA1-specific inhibitor MYLS22 is 0 μM-60 μM.

4. A bovine oocyte in vitro maturation solution, characterized in that: The invention contains the OPA1 specific inhibitor MYLS22 according to claim 3, wherein the OPA1 specific inhibitor is expressed in mRNA and protein in the GV, pre-MI, MI and MII stages of bovine oocytes.

5. The bovine oocyte in vitro maturation solution according to claim 4, characterized in that: The in vitro culture medium concentration of the OPA1 specific inhibitor MYLS22 is 0 μM-60 μM.

6. The bovine oocyte in vitro maturation solution according to claim 4, characterized in that: The in vitro culture medium concentration of the OPA1 specific inhibitor MYLS22 is 40 μM.

7. The bovine oocyte in vitro maturation solution according to claim 4, characterized in that: The bovine oocyte in vitro maturation medium is based on TCM-199 culture medium buffered with NaHCO3, and also contains 10% fetal bovine serum, 1% penicillin and streptomycin mixture, 0.022 g / mL sodium pyruvate, 0.9 mg / mL cysteine, 1 mg / mL estradiol, 10 ng / mL epidermal growth factor, and 0.04 mg / mL follicle stimulating hormone in a volume fraction.

8. A method for reducing the quality of oocytes after in vitro maturation, characterized in that: The bovine oocytes are added to the oocyte in vitro maturation culture medium according to any one of claims 4 to 7 for culture.

9. The method according to claim 8, characterized in that Oocytes need to be matured in vitro in a CO2 incubator at 38.5°C and 5% CO2 for 22-24 hours.

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