Use of tamoxifen derivatives for the preparation of a medicine for improving fertility

Abstract

The application discloses application of a tamoxifen derivative in preparation of a medicine for improving fertility. The application first proposes and verifies that the tamoxifen derivative can effectively improve fertility of aged mice, delay decrease of ovary reserve in aging, and improve oocyte maturation process and early embryo development potential. Meanwhile, the experimental results show that in-vivo supplement of the tamoxifen derivative can effectively rescue aging oocyte nucleus and cytoplasm maturation damage, partially reverse cytoskeleton disorder caused by aging, improve aging oocyte mitochondrial dysfunction, effectively reduce the level of oxidative stress, and has good application value. The application can be widely applied to the fields of biological medicine, reproductive health and anti-aging health care, provides a new intervention strategy and theoretical basis for delaying ovary aging and improving success rate of assisted reproductive technology, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of tamoxifen derivatives in the preparation of drugs to improve fertility. Background Technology

[0002] With the global trend of population aging and later marriage and childbearing, the age structure of women of childbearing age continues to shift later, the core childbearing age group of 20-35 years old is shrinking, and the average age of women giving birth is significantly delayed. Studies have confirmed that with increasing age, oocyte aging can lead to thickening of the zona pellucida, obstruction of fertilization, and decreased embryo quality, resulting in an increased risk of miscarriage and fetal arrest. Simultaneously, the risk of chromosomal abnormalities increases, directly manifesting as a decrease in female fertility, a significant increase in infertility, miscarriage, and birth defect rates, ultimately leading to reproductive dysfunction and adverse pregnancy outcomes. Current clinical and basic research indicates that the reproductive risks associated with advanced maternal age are not solely caused by abnormalities in fertilization or pregnancy; the core root cause lies in the quality decline of oocytes themselves with age. Therefore, developing novel small-molecule regulators with both physiological activity and safety, starting from the intrinsic regulatory mechanisms of oocyte aging, has significant research value and clinical significance.

[0003] Aging oocytes exhibit simultaneous impairment of both nuclear and cytoplasmic functions. Impaired nuclear maturation primarily manifests as arrested meiosis, disordered chromosome arrangement, and ultimately a significantly increased aneuploidy rate. Impaired cytoplasmic maturation is characterized by disordered organelle dynamics, including abnormal mitochondrial morphology and membrane potential, decreased mitochondrial copy number, calcium homeostasis imbalance, endoplasmic reticulum stress, cytoskeleton abnormalities, and elevated oxidative stress levels. Mitochondrial dysfunction is a key factor limiting oocyte quality; aging oocytes show decreased AMP / ATP and NAD+ / NADH ratios, accelerated telomere shortening, and intracellular mitochondrial dysfunction. Current strategies for mitigating oocyte aging include antioxidant supplementation, improving mitochondrial function, anti-inflammatory treatments, clearing senescent cells, mesenchymal stem cell therapy, follicular cell therapy, and ovarian transplantation. However, these interventions generally have drawbacks such as ethical constraints, immune rejection, and potential safety risks. Against this backdrop, small molecule targeted drug intervention, due to its ease of implementation and clearly defined targets, represents the most direct and efficient technical approach to improving the quality of aging oocytes.

[0004] Tamoxifen derivative (Mitotam) is a mitochondrial-targeting tamoxifen derivative, belonging to the category of specific mitochondrial electron transport chain (ETC) functional inhibitors, possessing both excellent tumor proliferation inhibitory activity and mitochondrial homeostasis regulation function. Tamoxifen derivative can effectively inhibit changes in mitochondrial membrane potential and affect mitochondrial morphology in senescent cells. Studies have shown that tamoxifen derivative can inhibit the formation of respiratory complexes (CI-respiration) and supercomplexes (SCs) in breast cancer cells. Compared with traditional antioxidant and mitochondrial protective small molecule regulators, tamoxifen derivative has significant comprehensive application advantages. Its chemical structure is clear, it precisely targets mitochondria, is low in toxicity and highly effective, and animal and human studies have shown that tamoxifen derivative has high safety, with no obvious toxic side effects or off-target effects, demonstrating excellent biocompatibility and good application value. Currently, there are no reports on the effects of tamoxifen derivative on improving fertility. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide an application of tamoxifen derivatives in the preparation of drugs for improving fertility.

[0006] Technical solution: This invention provides the use of tamoxifen derivatives in the preparation of drugs for improving fertility, wherein the structural formula of the tamoxifen derivatives is as follows: .

[0007] The improvement of fertility includes one or more of the following: i) Increase the number of piglets born; ii) Improve the quality of oocyte maturation; iii) Improve the quality of early embryonic development; iv) Delaying aging and slowing down ovarian reserve; v) Improve mitochondrial dysfunction in aging oocytes; vi) Reduce the level of oxidative stress in oocytes; vii) Reduce oocyte cytoskeleton abnormalities.

[0008] Preferably, the ovary, oocyte, and early embryo are derived from a mammal. The mammal is a human or a mouse.

[0009] The drug further contains pharmaceutically acceptable excipients, carriers, or diluents. Preferably, the drug is an oral or injectable formulation. Preferably, the oral or injectable dose of the drug is 0.5-2 mg / kg per day.

[0010] This invention demonstrates through in vivo animal experiments that supplementation with tamoxifen derivatives can effectively restore the fertility of aged mice, significantly increase their litter size, and improve the follicular development status of aged ovaries, thereby enhancing ovarian reserve. Aged oocytes generally exhibit maturation disorders and a significant decline in early embryonic development potential; tamoxifen derivatives can effectively correct maturation abnormalities in aging oocytes, increase the number of ovulations, and significantly improve oocyte fertilization rate and blastocyst formation rate.

[0011] This invention also provides the use of tamoxifen derivatives in the preparation of medicaments for improving the developmental potential of aging oocytes, wherein the structural formula of the tamoxifen derivatives is as follows: .

[0012] This invention, through in vivo animal experiments, demonstrates that tamoxifen derivatives can target mitochondria to exert their function, significantly repairing mitochondrial dysfunction in aging oocytes and improving mitochondrial structure and energy metabolism abnormalities. Live cell staining results show that supplementing with tamoxifen derivatives effectively improves abnormal mitochondrial arrangement in aging oocytes, significantly reducing the proportion of abnormal mitochondrial aggregation. Simultaneously, reactive oxygen species (ROS) level detection results show that, compared to young oocytes, aging oocytes exhibit excessive ROS accumulation and significantly upregulated oxidative stress levels. Tamoxifen derivative intervention effectively clears excess intracellular ROS, alleviating oxidative stress damage in oocytes. The precise assembly and morphological maintenance of the spindle are the core structural basis for the normal progress of meiosis in oocytes and the successful extrusion of the first polar body. Immunofluorescence assays showed that the spindle apparatus of normal young oocytes has a regular barrel-shaped structure, and chromosomes are neatly anchored at the equatorial plate of the spindle. In contrast, aging oocytes exhibit frequent spindle morphological abnormalities, chromosome misalignment, and abnormal segregation, indicating meiotic defects. Supplementation with tamoxifen derivatives effectively repaired the abnormal cytoskeleton assembly in aging oocytes and corrected the pathological disorder of the spindle apparatus and chromosomes. In conclusion, in vivo supplementation with tamoxifen derivatives can simultaneously rescue the nucleus and cytoplasm of aging oocytes from synchronous damage, thus improving the quality of aging oocytes.

[0013] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: By supplementing aging mice with tamoxifen derivatives, experiments have shown that in vivo supplementation with tamoxifen derivatives can effectively improve the fertility of aging mice, delay the decline in ovarian reserve due to aging, and enhance the maturation process of oocytes and the potential for early embryonic development. Simultaneously, combined with immunofluorescence technology, it was found that in vivo supplementation with tamoxifen derivatives can effectively salvage the damaged nucleus and cytoplasm maturation of aging oocytes, partially reverse aging-induced cytoskeleton disorders, improve mitochondrial dysfunction in aging oocytes, and effectively reduce oxidative stress levels, demonstrating significant application value. Attached Figure Description

[0014] Figure 1 The optimal concentration and treatment duration of tamoxifen derivatives for treating aging mice are shown in Figure A. A represents the first polar body extrusion morphology of oocytes in the young group, the old group, and the tamoxifen derivative treatment group. B represents the statistical data on the number of oocytes, nuclear membrane rupture rate, and first polar body extrusion rate in the young group, the old group, and the tamoxifen derivative treatment group.

[0015] Figure 2 Tamoxifen derivatives can improve the fertility of aging mice; A shows representative photos of the number of offspring in the young group, old group, and tamoxifen derivative treatment group; B shows statistical data on the number of offspring in the young group, old group, and tamoxifen derivative treatment group.

[0016] Figure 3 Tamoxifen derivatives can improve ovarian reserve in aging mice.

[0017] Figure 4 Tamoxifen derivatives can improve the early embryonic development potential of aging mice; A shows representative images of 2-cell embryos and blastocysts in the young group, old group, and tamoxifen derivative treatment group; B shows statistical data on ovulation number, fertilization rate, 2-cell rate, and blastocyst rate in the young group, old group, and tamoxifen derivative treatment group.

[0018] Figure 5 Tamoxifen derivatives can improve mitochondrial dysfunction in oocytes of aged mice; A shows representative fluorescence images of mitochondrial distribution in the young group, the aged group, and the tamoxifen derivative treatment group; B shows statistical data on the proportion of abnormal mitochondrial distribution in the young group, the aged group, and the tamoxifen derivative treatment group.

[0019] Figure 6 Tamoxifen derivatives can improve the level of oxidative stress in oocytes of aging mice; A shows representative fluorescence images of reactive oxygen species (ROS) levels in the young group, the old group, and the tamoxifen derivative treatment group; B shows statistical data on ROS levels in the young group, the old group, and the tamoxifen derivative treatment group.

[0020] Figure 7 Tamoxifen derivatives can improve abnormal cytoskeleton in oocytes of aging mice; A shows representative fluorescence images of abnormal spindle distribution and chromosomal disorder in the young group, old group, and tamoxifen derivative treatment group; B shows statistical data on the proportion of abnormal spindle distribution in the young group, old group, and tamoxifen derivative treatment group; C shows statistical data on the proportion of chromosomal disorder in the young group, old group, and tamoxifen derivative treatment group. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0022] Example 1

[0023] Effects of tamoxifen derivatives on fertility in reproductively aged mice (1) Preparation of intraperitoneal injection solution of tamoxifen derivative Twelve-month-old aged mice were treated with intraperitoneal injections of tamoxifen derivative solution at doses of 0.5 mg / kg, 1 mg / kg, and 2 mg / kg for 7 days, with each mouse weighing 40 g. The aged mice received an equal volume of saline solution without tamoxifen derivative daily. After superovulation, mature oocytes were retrieved from the ampulla of the ovary and counted. Oocyte count, GVBD ratio, and PB1 ratio were compared with those of 6-8 week old ICR mice. The optimal tamoxifen derivative treatment dose was determined based on the experimental results.

[0024] The structural formula of the tamoxifen derivative is as follows: .

[0025] Twelve-month-old aged mice were treated with the optimal concentration of tamoxifen derivative for 10 and 14 days, respectively, with each mouse weighing 40 g. The aged group received an equal volume of saline solution without tamoxifen derivative daily. After superovulation, mature oocytes were retrieved from the ampulla of the ovary and counted. The number of oocytes, the nuclear membrane rupture rate, and the first polar body expulsion rate were compared with those of 6-8 week old ICR mice. The optimal number of days of tamoxifen derivative treatment was determined based on the experimental results.

[0026] (2) Fertility testing After determining the optimal treatment duration and dosage of tamoxifen derivatives, treatment was administered using tamoxifen derivatives. The control group was injected with the same dose of saline. After the intervention, healthy ICR male mice aged 8-10 weeks were selected as breeding males. A cage ratio of "2 females and 1 male" was adopted, and female mice in each group were placed in the same cage with healthy male mice. The number of offspring was counted.

[0027] (3) Ovarian section staining Ovaries were obtained from mice in the young, old, and rescue groups. After fixation, dehydration, clearing, embedding, sectioning and mounting, dewaxing, and staining, the ovarian sections were observed using optical microscopes (100×, 200×, and 400× magnification), with a focus on analyzing the morphology and number of follicles.

[0028] (4) In vitro fertilization Mature MII oocytes were obtained from the young group, the old group, and the rescue group. In vitro fertilization technology was used to observe the fertilization rate, cleavage rate, and blastocyst rate among the different groups.

[0029] The experiment was conducted using the above methods, and the results are as follows: (1) Treatment days and dosage regimen of tamoxifen derivatives Figure 1 The results in A and B indicate that treatment of aging mice with a dose of 1 mg / kg for 7 days yielded the best results. Compared with the aging group, in vivo supplementation with tamoxifen derivatives could effectively salvage the number of oocytes and the proportion of first polar body expulsion in aging mice.

[0030] (2) Fertility assessment Figure 2 Results A and B showed that compared with the younger group, the litter size of aged mice was significantly reduced, while the litter size of aged mice supplemented with tamoxifen derivatives was significantly increased, indicating that supplementation with tamoxifen derivatives can improve the fertility of aged female mice.

[0031] (3) Ovarian development assessment Figure 3 The results showed that, compared with the ovarian sections of young mice, the ovaries of aged mice exhibited degeneration and a decrease in the number of follicles. Supplementation with tamoxifen derivatives could improve the development of some follicles.

[0032] (4) Early embryonic developmental potential assessment Figure 4 The results showed that compared with the young group, the number of oocytes, fertilization rate and blastocyst rate of mice in the old group were significantly reduced. Supplementation with tamoxifen derivatives could effectively salvage the above-mentioned undesirable phenotypes. Compared with the old group, the salvage group increased the number of oocytes, fertilization rate and blastocyst rate by 67.94%, 8.71% and 56.20%, respectively.

[0033] Example 2

[0034] Effects of tamoxifen derivatives on oocyte developmental potential in reproductively aged mice In this embodiment, the experimental reagents are as follows: α-tubulin-FITC, Hoechst, Mito-Tracker, ROS, M2, M16 culture medium, fixative, permeabilization solution, blocking solution, and washing solution. (1) Oocyte collection Mice in the young, aged, and rescue groups were intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin (PMSG). Forty-eight hours later, the mice were euthanized by cervical dislocation, and ovarian tissue was dissected and collected. Germinal vesicle (GV) stage oocytes were obtained using a stereomicroscope and oral pipette. The oocytes were cultured in vitro at 37 ℃ in a 5% CO2 incubator for 8 hours to allow them to develop to metaphase I (MI) for subsequent experiments.

[0035] (2) Immunofluorescence technique Oocytes in the MI stage were collected and fixed in 4% histocellular fixative at room temperature for 30 min. Cells were then transferred to permeabilization buffer containing 0.5% Triton X-100 and permeabilized at room temperature for 20 min. After permeabilization, cells were blocked in blocking buffer at room temperature for 1 h, then transferred to primary antibody working solution and incubated overnight at 4 °C. After primary antibody incubation, cells were washed three times with washing buffer for 5 min each time; then, secondary antibody conjugated with 488, 594 / TRITC fluorescent groups was added and incubated at room temperature in the dark for 1 h. After incubation, cells were washed three more times with washing buffer for 5 min each time. Finally, nuclei were stained with Hoechst 33342 dye at room temperature for 10 min, and the cells were mounted for fluorescence observation.

[0036] (3) Live cell staining Oocytes in the MI stage were collected from each group and transferred to a live-cell fluorescent dye working solution. They were then incubated at 37 ℃ in a 5% CO2 incubator for 30 min in the dark. After incubation, the cells were washed 2-3 times with fresh culture medium to remove free dye. All procedures were performed in the dark.

[0037] The experiment was conducted using the above methods, and the results are as follows: (1) Tamoxifen derivatives improve mitochondrial dysfunction in aging oocytes Mitochondrial arrangement is a crucial structural basis for maintaining the normal physiological function and developmental potential of oocytes. For example... Figure 5 As shown in A and B, mitochondria in young oocytes are clustered around the spindle and there is no abnormal aggregation in the cytoplasm. In contrast, aging oocytes show obvious mitochondrial distribution disorder and abnormal aggregation. After treatment with tamoxifen derivatives, the spatial arrangement pattern of mitochondria in aging oocytes was effectively reshaped, the abnormal aggregation of mitochondria was significantly alleviated, and the aging-induced mitochondrial distribution defects were effectively reversed.

[0038] (2) Tamoxifen derivatives improve oxidative stress levels in aging oocytes Mitochondria are core organelles in eukaryotic cells, responsible for oxidative phosphorylation, ATP synthesis, and energy metabolism. They are also the primary site of intracellular reactive oxygen species (ROS) generation. Live-cell staining results showed that, compared to young oocytes, aging oocytes accumulated significantly more ROS, with markedly increased fluorescence intensity, indicating an abnormal upregulation of oxidative stress levels. However, intervention with tamoxifen derivatives effectively cleared excess ROS from aging oocytes, significantly reducing ROS fluorescence intensity. This significantly inhibited aging-mediated excessive activation of oxidative stress, effectively alleviating the toxic damage of oxidative stress to oocytes and restoring redox homeostasis in aging oocytes. Figure 6 (A and B in the text).

[0039] (3) Tamoxifen derivatives improve abnormal cytoskeleton in aging oocytes The integrity of the spindle apparatus and the precise arrangement of chromosomes are core guarantees for normal meiosis in oocytes. For example... Figure 7 As shown in Figure A, the spindle of a normal young oocyte exhibits a typical, regular barrel-shaped structure during metaphase of meiosis, with chromosomes neatly arranged on the equatorial plate. In contrast, the spindle of an aged oocyte is morphologically abnormal and structurally disordered, accompanied by defects such as disordered, misplaced, and disordered chromosome arrangement. Tamoxifen derivative intervention can effectively improve these abnormal phenotypes. Statistical analysis further confirms that tamoxifen derivative intervention can significantly reduce the incidence of spindle abnormalities and chromosomal disorder in aged oocytes, effectively repair aging-induced cytoskeleton damage in oocytes, and ensure the orderly progress of meiosis in oocytes. Figure 7 (B and C in the text).

Claims

1. The use of tamoxifen derivatives in the preparation of drugs for improving fertility, characterized in that, The structural formula of the tamoxifen derivative is as follows: ； The improvement in fertility includes one or more of the following: i) Increase the number of piglets born; ii) Improve the quality of oocyte maturation; iii) Improve the quality of early embryonic development; iv) Delaying aging and slowing down ovarian reserve; v) Improve mitochondrial dysfunction in aging oocytes; vi) Reduce the level of oxidative stress in oocytes; vii) Reduce oocyte cytoskeleton abnormalities.

2. The application according to claim 1, characterized in that, The ovaries, oocytes, and early embryos described herein are derived from mammals.

3. The application according to claim 2, characterized in that, The mammals referred to are humans or mice.

4. The application according to claim 1, characterized in that, The drug also contains a pharmaceutically acceptable carrier.

5. The application according to claim 1, characterized in that, The drug is an oral or injectable preparation.

6. The application according to claim 5, characterized in that, The oral or intraperitoneal dose of the drug is 0.5-2 mg / kg per day.

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