Use of mitochondrial fusion modulator m1 in the preparation of fertility improvement products

Abstract

The application discloses an application of a mitochondrial fusion regulator M1 in preparation of a fertility improvement product and belongs to the field of biological medicines.The application first proposes and verifies that the mitochondrial fusion regulator M1 can effectively improve the quality of oocytes, improve ovarian function, improve reproductive capacity and prolong reproductive life.The experimental data show that the mitochondrial fusion regulator M1 can improve the defects of spindle assembly disorder, abnormal chromosome arrangement, mitochondrial dysfunction and oxidative damage of aging oocytes in the process of meiosis, effectively improve the oocyte maturation rate, and significantly improve the fertilization capacity of oocytes, and greatly improve the development efficiency of fertilized eggs, which provides a new technical path and drug selection for clinical intervention of fertility improvement of aged women, and has very high practical application value and clinical conversion prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to the application of a mitochondrial fusion regulator M1 in the preparation of fertility improvement products. Background Technology

[0002] With the shift in childbearing age, pregnancies at age 35 and above are generally considered advanced maternal age. Public guidelines indicate that female fertility begins to decline after age 30, and the decline accelerates after age 35; at the same time, advanced maternal age is closely associated with an increased risk of female infertility, pregnancy loss, fetal abnormalities, stillbirth, and obstetric complications.

[0003] Current strategies for improving the quality of aging oocytes largely focus on antioxidants or energy support, such as coenzyme Q10 (CoQ10), methoxytin (PQQ), and rhodioloside. Existing research suggests that CoQ10 can improve oocyte mitochondrial function, PQQ can play a protective role in in vitro oocyte maturation or ovarian dysfunction models, and rhodioloside can also improve the quality of aged oocytes by alleviating oxidative stress and improving mitochondrial function. However, most of these interventions are still at the level of in vitro, animal, or limited clinical observation, and their mechanisms of action are not fully understood. Furthermore, there are significant differences between different models, dosages, timing of administration, and outcome indicators, making it difficult to draw stable and extrapolable definitive conclusions.

[0004] Mitochondrial fusion regulator M1 is a membrane-permeable phenylhydrazone small molecule. This compound was initially reported to promote mitochondrial fusion and restore fragmented mitochondrial networks in mammalian cells. Studies have shown that this molecule remains effective in cells lacking only mitochondrial fusion protein 1 (MFN1) or mitochondrial fusion protein 2 (MFN2), but its effect is limited in cells lacking both MFN1 / 2 and optic atrophy protein 1 (OPA1), indicating that its effect depends on the existing mitochondrial fusion baseline and is not reversible in all injury scenarios. Meanwhile, current research mainly focuses on non-reproductive systems such as myocardial ischemia / reperfusion, optic nerve regeneration, pancreatic β-cells, and airway inflammation. Given that oocyte aging involves multiple couplings between chromosome homeostasis, meiosis, energy metabolism, and mitochondrial dynamics, whether mitochondrial fusion regulator M1 can be used in the reproductive system remains unclear. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide an application of mitochondrial fusion regulator M1 in the preparation of products that improve fertility, especially oocyte quality, ovarian function, reproductive capacity, and reproductive lifespan.

[0006] Technical solution: The application of the mitochondrial fusion regulator M1 described in this invention in the preparation of fertility improvement products.

[0007] Preferably, the CAS number of the mitochondrial fusion regulator M1 is 219315-22-7.

[0008] Preferably, the fertility improvement is applied to mammalian germ cells or mammalian germ cells.

[0009] Preferably, the improvement in fertility includes at least one of the following: (1) improving oocyte quality; (2) improving ovarian function; (3) enhancing reproductive capacity; and (4) prolonging reproductive lifespan.

[0010] Preferably, the product is any of the following non-therapeutic products: (a) a reagent, (b) a food additive, functional food, or health food, and (c) feed or feed additive. Preferably, the product is a food additive, functional food, or health food.

[0011] Preferably, the product is a drug.

[0012] Preferably, the drug contains mitochondrial fusion regulator M1 or its pharmaceutically acceptable solvate or hydrate as an active ingredient, and also contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include any one or more selected from diluents, lubricants, flow aids, wetting agents, emulsifiers or pH buffers.

[0013] Preferably, the dosage form of the drug includes tablets, capsules, granules, oral liquids, syrups, powders, microcapsules, injections, powder injections, infusions, suspensions, implants, transdermal patches, microemulsions, liposomes, nanoparticles, oral instant films, sponges, and capsules.

[0014] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. This invention is the first to propose and verify that the mitochondrial fusion regulator M1 can effectively improve oocyte quality, ovarian function, reproductive capacity, and prolong reproductive lifespan; 2. Experimental data show that the mitochondrial fusion regulator M1 can improve the defects of spindle assembly disorder, abnormal chromosome arrangement, mitochondrial dysfunction, and oxidative damage in aging oocytes during meiosis, effectively improve the oocyte maturation rate, and thus significantly improve the fertilization capacity of oocytes. At the same time, it greatly improves the development efficiency of fertilized eggs, providing a new technical path and drug selection for clinical intervention to improve fertility in older women, and has extremely high practical application value and clinical translation prospects. Attached Figure Description

[0015] Figure 1 Flowchart of the intervention experiment for mitochondrial fusion regulator M1;

[0016] Figure 2 Figure showing the results of oocyte maturation rate assessment in aging mice after intervention with mitochondrial fusion regulator M1;

[0017] Figure 3 Figure showing the fertility assessment results of aging mice after intervention with the mitochondrial fusion regulator M1;

[0018] Figure 4 Figure showing the evaluation results of in vitro fertilization and embryonic development after intervention with mitochondrial fusion regulator M1;

[0019] Figure 5 Figure showing the evaluation results of oocyte spindle assembly and chromosome alignment after intervention with mitochondrial fusion regulator M1;

[0020] Figure 6 The figure shows the evaluation results of mitochondrial function and oxidative damage in oocytes after intervention with the mitochondrial fusion regulator M1. Detailed Implementation

[0021] The technical solution of the present invention will be further described below.

[0022] Example 1: Evaluation of the effect of intervention with mitochondrial fusion regulator M1 on improving fertility in aging mice

[0023] Mitochondrial fusion regulator M1 (M1, purchased from MedChemExpress LLC., catalog number HY-111475) was dissolved in DMSO solution to a concentration of 25 mg / mL to obtain a stock solution. Subsequent experimental working solutions were prepared by serial dilution of this stock solution using corn oil (CAS number: 8001-30-7), ultimately yielding working solutions of M1 at concentrations of 5 mg / kg, 10 mg / kg, and 20 mg / kg. All working solutions were strictly diluted according to the specified ratio and thoroughly vortexed before use.

[0024] Ten-month-old senescent female ICR mice and eight-week-old young female ICR mice were purchased from Jiangsu Qinglongshan Biotechnology Co., Ltd. After one week of acclimatization, experiments were conducted. All senescent mice were randomly divided into four groups: no treatment (senescent group), 5 mg / kg M1 treatment group (senescent group + 5 mg / kg M1), 10 mg / kg M1 treatment group (senescent group + 10 mg / kg M1), and 20 mg / kg M1 treatment group (senescent group + 20 mg / kg M1), with five mice in each group. Mice in the M1 treatment group were administered the corresponding dose of the aforementioned mitochondrial fusion regulator M1 working solution via gavage using a #9, 60mm curved mouse gavage needle. The needle was left in place for 3 seconds after administration. Mice in the no-treatment group (n=5) and young mice (n=5, young group) were administered corn oil via gavage in the same manner. The gavage volume for all mice was 0.1 mL. Administration was performed daily for 7 consecutive days. During the experiment, mice had free access to food and water, and were kept in an environment with 12-hour light-dark cycles, a temperature of 22±2℃, and a humidity of 50±5%. The experimental procedure was as follows: Figure 1 As shown.

[0025] 1. Assessment of oocyte first polar body expulsion after intervention

[0026] Seven days after intervention, five mice in each group were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin (PMSG, purchased from Ningbo Second Hormone Factory Co., Ltd., trade name: Quick Pregnancy) to induce superovulation. After 48 hours, the mice were euthanized and the ovarian tissue was aseptically isolated. The fat and connective tissue around the ovary were dissected in preheated M2 medium. Under a stereomicroscope, the follicles were gently punctured with a 30 G injection needle. Oocytes with uniform cytoplasm, intact zona pellucida, and complete germinal follicles (GV) were picked and washed three times in fresh M2 medium to remove tissue debris and granulosa cells. They were then transferred to M16 medium and washed once to obtain GV stage oocytes. These oocytes were seeded into M16 medium and covered with sterile mineral oil. They were cultured continuously at 37°C, 5% CO2, and saturated humidity to observe the expulsion of the first polar body.

[0027] Representative images of the first polar body discharge are shown below. Figure 2 As shown in A, the statistical results of the first polar body discharge rate are as follows: Figure 2 As shown in B, the results showed that the oocyte first polar body expulsion rate after treatment with 10 mg / kg M1 was significantly higher than that of untreated aged mice, and there was no statistical difference compared with young mice, suggesting that 10 mg / kg is the optimal treatment dose, and this dose was used for subsequent fertility tests.

[0028] 2. Post-intervention fertility assessment

[0029] Seven days after intervention, young mice, untreated aged mice, and mice treated with 10 mg / kg M1 (aged group + M1) were placed in cages with 12-week-old male ICR mice (purchased from Jiangsu Qinglongshan Biotechnology Co., Ltd.) at a female:male ratio of 2:1 for 7 days to allow them to mate naturally. Pregnant female mice were raised separately, and the average number of live offspring per litter was recorded after pregnancy and delivery.

[0030] Representative images of suckling mice obtained after delivery, as shown below Figure 3 The left figure shows the statistics of the number of piglets born. Figure 3 As shown in the right figure, compared with young mice, the litter size of aged mice showed a significant downward trend. The average litter size of young mice was 13-14, while that of aged mice was only 6-7. However, after M1 intervention, the litter size of aged mice was significantly increased, averaging 11-12. This result indicates that supplementing with M1 can improve the fertility of mice.

[0031] Example 2: Study on the regulatory function of mitochondrial fusion regulator M1 in the development of oocytes in aging mice

[0032] Ten-month-old senescent female ICR mice and eight-week-old young female ICR mice were purchased from Jiangsu Qinglongshan Biotechnology Co., Ltd. and subjected to experiments after one week of acclimatization. All senescent mice were randomly divided into two groups: an untreated group (Aged) and a 10 mg / kg M1 treatment group, with six mice in each group. Both senescent and young mice (Control) were administered the drug by gavage daily for seven consecutive days as described in Example 1.

[0033] Seven days after intervention, each mouse was injected intraperitoneally with 10 IU of PMSG to induce superovulation. Forty-eight hours later, ovulation was induced by intraperitoneal injection of 10 IU of hCG (purchased from Ningbo Second Hormone Factory Co., Ltd., trade name Duoqingsu). Sixteen hours later, the mice were euthanized, and the ampulla of the oviduct was aseptically isolated and placed in M2 medium pre-warmed at 37°C. The ampulla was gently punctured with a 30 G injection needle to release cumulus-oocyte complexes (COCs). Mature oocytes in the MII stage were obtained after digestion with 1 mg / mL hyaluronidase (purchased from Nanjing Luanchuang Life Science and Technology Co., Ltd., catalog number M08-H) for 5 minutes under 37°C, 5% CO2, and saturated humidity conditions.

[0034] Ten-week-old young male ICR mice were purchased from Jiangsu Qinglongshan Biotechnology Co., Ltd., and sperm from the epididymal tail was collected and placed in TYH sperm capacitation solution (purchased from Nanjing Luanchuang Life Science, catalog number M05-Y) at a constant temperature of 37℃ for 1 h to obtain sperm suitable for in vitro fertilization.

[0035] 4 μL of a concentration of 3 × 105 Capacitated sperm cells were co-cultured with MII stage mature oocytes to complete in vitro fertilization. The fertilization system was incubated at 37℃, 5% CO2, and saturated humidity for 6 h. Afterward, attached granulosa cells and excess sperm were removed, and morphologically normal fertilized eggs were selected and transferred into KSOM embryo culture medium (purchased from Nanjing Luanchuang Life Science and Technology, catalog number M03-AA) for continuous in vitro culture at 37℃, 5% CO2, and saturated humidity.

[0036] 1. Evaluation of fertilization and embryonic development

[0037] Embryonic development was continuously observed, and representative images of fertilized eggs at each developmental stage were collected. Fertilization rate, 2-cell rate, 4-cell rate, 8-cell rate, morula rate, and blastocyst development rate were statistically analyzed. Representative images of fertilized eggs at each developmental stage from each group of mice are shown below. Figure 4 As shown in A, the developmental rates at each developmental stage are as follows: Figure 4 As shown in B~G, the results indicate that M1 treatment can effectively reverse the decline in fertilization rate and early embryonic developmental arrest caused by aging.

[0038] 2. Evaluation of spindle assembly and chromosome arrangement

[0039] After 7 days of gavage supplementation with M1 solution, mice were induced to undergo superovulation by intraperitoneal injection of 10 IU of PMSG. The mice were euthanized 48 h later, and GV-stage oocytes were isolated from the ovaries. These oocytes were washed three times with M2 medium and once with M16 medium, then cultured in M16 medium covered with 5% CO2 mineral oil at 37°C for 8 h to obtain MⅠ-stage oocytes.

[0040] Oocytes from each group at the MI stage were fixed with 4% paraformaldehyde (PFA) solution for 30 min at room temperature. After fixation, they were permeabilized with PBS containing 0.5% (v / v) Triton X-100 for 20 min at room temperature. Then, they were blocked with PBS containing 1% (m / v) bovine serum albumin (BSA) for 60 min at room temperature. After blocking, the oocytes were incubated overnight at 4°C with anti-α-tubulin-FITC antibody (purchased from Merck KGaA, catalog number F2168) diluted 1:400. They were washed three times with PBS containing 0.1% (v / v) Tween-20 and 0.01% (v / v) Triton X-100 for 3 min each time. After washing, the nuclei were counterstained with Hoechst 33342, washed again, mounted, and observed and images were acquired using a laser confocal microscope. The acquired images were analyzed using ImageJ software to count the proportion of abnormal spindle fibers and the proportion of chromosome misalignment.

[0041] Representative images of immunofluorescence staining are as follows Figure 5As shown in A, the statistical results of the spindle body abnormality rate are as follows: Figure 5 As shown in B, the statistical results of chromosome abnormality rates are as follows: Figure 5 As shown in C, the results indicate that M1 treatment can salvage microtubule disorder and chromosomal abnormalities in aging oocytes.

[0042] 3. Evaluation of mitochondrial function and oxidative damage

[0043] The aforementioned oocytes from the MI stage were retrieved and processed using MitoTracker. TM The Red CMXRos kit (purchased from ThermoFisher Scientific Inc., catalog number M7512) or the reactive oxygen species detection kit (purchased from Beyotime Biotechnology, catalog number S0033S-1) were incubated at 37°C, 5% CO2, and saturated humidity in the dark for 30 min. After incubation, the cells were washed three times with M2 culture medium pre-warmed at 37°C for 3 min each time. The nuclei were then counterstained with Hoechst 33342. After washing again, the cells were observed and images were acquired using a laser confocal microscope. The distribution of mitochondria in the MitoTracker staining images and the fluorescence intensity of reactive oxygen species (ROS) in the ROS detection images were analyzed using ImagJ software.

[0044] To further evaluate ATP production levels, oocytes from the MI stage were harvested and measured using an ATP assay kit (purchased from Merck KGaA, catalog number MAK190).

[0045] Representative images of MitoTracker staining are shown below. Figure 6 As shown in A, the statistical results of mitochondrial distribution abnormality rate are as follows: Figure 6 As shown in B, the statistical results of the relative ATP levels are as follows: Figure 6 As shown in C, representative images of reactive oxygen species detection are as follows: Figure 6 As shown in D, the statistical results of reactive oxygen species levels are as follows: Figure 6 As shown in E, the results indicate that M1 treatment can effectively correct the abnormal mitochondrial distribution in senescent oocytes, restore ATP levels, and alleviate the excessive accumulation of reactive oxygen species.

Claims

1. The application of a mitochondrial fusion regulator M1 in the preparation of fertility-enhancing products.

2. The application according to claim 1, characterized in that, The CAS number of the mitochondrial fusion regulator M1 is 219315-22-7.

3. The application according to claim 1, characterized in that, The fertility improvement targets mammals or mammalian germ cells.

4. The application according to claim 1, characterized in that, The improvement in fertility includes at least one of the following: (1) improving oocyte quality; (2) improving ovarian function; (3) enhancing reproductive capacity; and (4) prolonging reproductive lifespan.

5. The application according to claim 1, characterized in that, The product is any of the following non-therapeutic products: (a) reagents, (b) food additives, functional foods or health foods, (c) feed or feed additives.

6. The application according to claim 1, characterized in that, The product is a medicine.

7. The application according to claim 6, characterized in that, The drug contains mitochondrial fusion regulator M1 or its pharmaceutically acceptable solvate or hydrate as an active ingredient.

8. The application according to claim 6, characterized in that, The drug also contains pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The pharmaceutically acceptable excipients include any one or more selected from diluents, lubricants, flow aids, wetting agents, emulsifiers, or pH buffers.

10. The application according to claim 6, characterized in that, The dosage forms of the drugs include tablets, capsules, granules, oral liquids, syrups, powders, microcapsules, injections, powder injections, infusions, suspensions, implants, transdermal patches, microemulsions, liposomes, nanoparticles, oral instant films, sponges, and capsules.

Images