Use of vitamin d and vitamin d receptor inhibitor in preparation of drug for regulating activation of primordial follicles

Abstract

The use of vitamin D and a vitamin D receptor inhibitor in the preparation of a drug for regulating the activation of primordial follicles. Vitamin D can inhibit the activation of primordial follicles, and a vitamin D receptor inhibitor promotes the activation of primordial follicles, and thus same can be used as a drug for regulating the activation of primordial follicles in patients with premature ovarian insufficiency, and has good application prospects.

Description

Application of vitamin D and vitamin D receptor inhibitors in the preparation of drugs for regulating primordial follicle activation Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of vitamin D and a vitamin D receptor inhibitor in the preparation of a drug for regulating primordial follicle activation. Background Art

[0002] The female ovary contains a finite number of non-renewable primordial follicles, which gradually deplete with aging until they are exhausted. Primordial follicles consist of a quiescent oocyte surrounded by a single layer of flattened pregranulosa cells. Primordial follicle activation involves oocyte growth and differentiation of pregranulosa cells from a flattened to a cuboidal shape. Primordial follicle activation and development are precisely regulated, with numerous growth factors, cytokines, and protein kinases involved in regulating primordial follicle activation. The mammalian target of rapamycin (mTOR) pathway is one of the major signaling pathways. Activation of the mTOR pathway in pregranulosa cells promotes expression of proto-oncogene receptor tyrosine kinase (KIT) ligand (KITL), which binds to KIT to activate the phosphoinositide 3-kinase / protein kinase B (PI3K / Akt) pathway in the oocyte. Subsequently, forkhead box transcription factor 3a (FOXO3a) is phosphorylated and translocated from the nucleus to the cytoplasm, leading to primordial follicle activation. Studies have shown that enhanced glycolysis in pregranulosa cells activates primordial follicles through the mTOR pathway. Furthermore, E-cadherin, mitogen-activated protein kinases (MAPK 3 / 1), histone deacetylase 6 (HDAC6), and CDC42 activate primordial follicles by regulating the aforementioned signaling pathways. This process of primordial follicle activation is irreversible, and therefore, orderly activation of primordial follicles is crucial for a woman's reproductive lifespan. Overactivation or inactivation of primordial follicles can lead to premature ovarian insufficiency (POI).

[0003] The number of patients with POI, caused by genetic or environmental factors, is increasing, often presenting with infertility. Furthermore, declining fertility in older women presents a clinical challenge in reproductive medicine. Approximately three-quarters of POI patients still have a small number of dormant primordial follicles, but these follicles are difficult to activate physiologically, making conventional IVF fertility impossible. Currently, in vitro activation (IVA) is a method for reactivating these dormant primordial follicles, allowing them to be autologously transplanted to achieve conception. However, this method is only applicable to a small number of patients; laparoscopic surgery can also cause physical damage to the patient; in vitro culture of the ovarian cortex can cause tissue damage and necrosis, leading to programmed cell death; and the drugs used for in vitro activation can adversely affect oocyte quality and increase the risk of genetic disorders. Recently, methods have been developed to activate primordial follicles using intraovarian injections of platelet-rich plasma and disruption of the Hippo signaling pathway. This method can also lead to successful pregnancies, but the number of cases is limited, and its effectiveness warrants further study. Furthermore, IVA treatment is complex and expensive, placing a heavy burden on patients and society. Therefore, it is necessary to explore more effective methods, such as oral medications, to regulate the activation rate of primordial follicles, prolong women's reproductive lifespan, and prevent the formation of POI. This approach could also be used to save the fertility of POI patients or older women, avoiding the adverse effects of the aforementioned surgeries and in vitro activation. Ultimately, a strategy for preserving female fertility in vivo could be established to achieve sustainable fertility.

[0004] Vitamin D is a compound with a cyclopentahydrophenanthrene ring structure that is soluble in fat solvents. A small amount is obtained from food, but the majority of the vitamin D needed is produced in the skin. Under ultraviolet light, the skin converts the substrate 7-dehydrocholesterol (7-DHC) into cholecalciferol. This cholecalciferol binds to vitamin D-binding protein (DBP) and travels through the bloodstream to the liver, where it is converted by 25-hydroxylase into 25-hydroxyvitamin D (25-OHD), also known as calcifediol. This is then converted by 1α-hydroxylase in the kidneys into 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3, VD3), also known as calcitriol. VD3 is the active form of vitamin D and a high-affinity ligand for the transcription factor vitamin D receptor (VDR). VDR belongs to the nuclear receptor superfamily and is a DNA-binding transcription factor. Ligand-activated VDR binds tightly to the retinoid X receptor (RXR) to form an active signaling complex, VD3-VDR-RXR. This complex recognizes the vitamin D response element (VDRE) in DNA and regulates the transcription of approximately 1,000 downstream target genes. In addition to its crucial role in bone metabolism, vitamin D homeostasis is crucial for female reproductive function. Studies have shown that low vitamin D levels in follicular fluid are associated with adverse assisted reproductive outcomes, including lower oocyte retrieval, fertilization rate, embryo quality, implantation rate, and clinical pregnancy rate. Vitamin D supplementation during assisted reproductive treatment can improve pregnancy and ovulation rates in women with polycystic ovary syndrome (PCOS), while reducing androgen levels, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and early miscarriage rates. In animal studies, VDR knockout mice were found to have hypoplastic uteri at 7 weeks of age, with ovarian follicles arrested at the secondary follicle stage and no mature follicles. These findings suggest a crucial role for vitamin D and VDR in the female reproductive system, but studies have not yet reported their role in regulating primordial follicle activation. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies in the prior art, the primary purpose of the present invention is to provide the use of vitamin D and vitamin D receptor inhibitors in the preparation of drugs for regulating primordial follicle activation.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] Use of vitamin D or vitamin D receptor inhibitors in the preparation of medicines for treating primary ovarian insufficiency.

[0008] Furthermore, the primary ovarian insufficiency includes primary ovarian insufficiency caused by overactivation or inactivation of primordial follicles.

[0009] Furthermore, the application is the application of vitamin D in the preparation of a drug for inhibiting the activation of primordial follicles, or the application of a vitamin D receptor inhibitor in the preparation of a drug for promoting the activation of primordial follicles.

[0010] Furthermore, the vitamin D is 1,25-dihydroxyvitamin D3; and the vitamin D receptor inhibitor is TEI-9647, ZK159222 or MeTC7.

[0011] Furthermore, in the application, the dosage of vitamin D is calculated based on individual body weight, and 0.0006-6 mg / day of vitamin D is administered per 50 kg of body weight; the dosage of vitamin D inhibitors is calculated based on individual body weight, and the dosage range per 50 kg of body weight is: TEI-9647: 3.28-32820 mg / day, ZK159222: 3.94-39440 mg / day, MeTC7: 1.42-14277 mg / day.

[0012] Furthermore, the primordial follicles are mammalian primordial follicles.

[0013] Furthermore, the mammal is a mouse or a human.

[0014] Furthermore, the drug is administered orally or by intravenous injection.

[0015] The invention relates to the use of a vitamin D receptor inhibitor in the preparation of a primordial follicle activation drug, wherein the vitamin D receptor inhibitor is TEI-9647, ZK159222 or MeTC7.

[0016] Furthermore, the primordial follicle in vitro activation drug is a drug that promotes the transformation of primordial follicles into growth follicles.

[0017] An in vitro activation method for primordial follicles for non-disease diagnosis and treatment purposes, comprising culturing ovaries in an in vitro activation reagent; the in vitro activation reagent contains a vitamin D receptor inhibitor; the vitamin D receptor inhibitor is TEI-9647, ZK159222, or MeTC7.

[0018] Furthermore, the concentrations of TEI-9647, ZK159222, and MeTC7 are 500±50nM, 1±0.5μM, and 2±0.5μM, respectively.

[0019] Furthermore, the ovary is a mouse ovary.

[0020] Furthermore, the mice are newborn mice.

[0021] Furthermore, the culture is carried out at a constant temperature of 37±2°C and 5%±1% CO2 for 4 days.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] POI is a relatively serious ovarian disease clinically. The disease is showing a trend of becoming younger, and the incidence rate is constantly increasing, which seriously affects women's fertility and physical health. In order to ensure that female mammals (including humans) have mature eggs available throughout their reproductive cycle, the activation of primordial follicles in their primordial follicle reservoir must be precisely regulated. Excessive activation of primordial follicles or primordial follicle activation disorders under physiological conditions can lead to POI. In the present invention, we found that vitamin D can inhibit the activation of primordial follicles, and vitamin D receptor inhibitors TEI-9647, ZK159222, and MeTC7 have the effect of promoting the activation of primordial follicles in the ovaries of newborn mice. Vitamin D, as a common supplement, can be administered orally and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 shows immunofluorescence staining of vitamin D receptor VDR in the ovaries of mice at 1 day (1 dpp), 4 days (4 dpp), and 7 days (7 dpp) after birth (a), and morphological observation and follicle counting results of the ovaries of 3 dpp newborn mice cultured for 4 days with the addition of the active form of vitamin D VD3 of the present invention, vitamin D receptor inhibitors TEI-9647, ZK159222, and MeTC7 (b-c); among them, (b) is the statistical count of primordial follicles and growing follicles; (c) is a comparison of the ovarian histological morphology of the control group and the VD3, vitamin D receptor inhibitors TEI-9647, ZK159222, and MeTC7 groups (scale: 50 μm). DETAILED DESCRIPTION

[0025] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0026] In the following examples, experimental methods without specific experimental conditions were generally performed under conventional experimental conditions or those recommended by the manufacturer. Materials and reagents used were commercially available unless otherwise specified.

[0027] Example 1

[0028] 1. Experimental Materials

[0029] 1. Preparation of Reagents Related to In Vitro Culture

[0030] 1.1 Preparation of drug solution

[0031] Weigh appropriate amounts of VD3, vitamin D receptor inhibitor TEI-9647, ZK159222, and MeTC7 powders, prepare a storage solution using DMSO as solvent, filter with a 0.2 μm filter after the solids are fully dissolved, seal with parafilm, and store at -20°C.

[0032] 1.2 Preparation of DMEM / F12 culture medium (Table 1)

[0033] Table 1. DMEM / F12 culture medium

[0034] After all the solids are dissolved, filter with a 0.22 μm filter, operate in a clean bench, seal with sealing film, and store at 4°C.

[0035] 1.3 Preparation of phosphate buffered solution (PBS) (Table 2)

[0036] Table 2. PBS buffer

[0037] Autoclave and store at 4°C.

[0038] 2. Preparation of immunofluorescence-related reagents

[0039] 2.1 Preparation of 10% blocking serum

[0040] Donkey serum and PBS were prepared at a ratio of 1:10 as needed.

[0041] 2.2 Primary Antibody Preparation

[0042] Corresponding primary antibody: 10% blocking serum at a ratio of 1:200, prepared as needed.

[0043] 2.3 Secondary Antibody Preparation

[0044] Prepare the corresponding secondary antibody in PBS at a ratio of 1:200 according to the species of the primary antibody.

[0045] 2.4 Preparation of DAPI working solution

[0046] DAPI storage solution: PBS at a ratio of 1:200, prepared as needed.

[0047] 2.5 Preparation of citric acid buffer working solution (Table 3, Table 4, Table 5)

[0048] Table 3. 0.1M citric acid (Solution A)

[0049] Table 4. 0.1M Sodium Citrate (Solution B)

[0050] Table 5. Citric acid working solution

[0051] 2. Isolation and in vitro culture of mouse ovaries

[0052] 1. Isolation of Mouse Ovaries

[0053] 1.1 Surgical instruments (ophthalmic straight scissors, 10 cm toothed forceps, pointed forceps, 1 mL syringe), 90 mm culture dishes, six-well plates, etc. were placed in a clean bench and irradiated with UV light for 30 minutes. Intercellular UV light was used for 30 minutes.

[0054] 1.2 In this experiment, 3-day-old ICR female mice were sacrificed by cervical dislocation. The abdomen was disinfected with 70% alcohol, and the abdominal cavity was opened through a V-shaped incision at the umbilicus. The mice were divided into the head and tail along the upper edge of the kidneys, and the tail was placed in pre-cooled PBS.

[0055] 1.3 Under a stereomicroscope, remove the mouse ovary along with the surrounding connective tissue and use a 1 mL syringe needle to peel off the connective tissue surrounding the ovary.

[0056] 2. In vitro culture of mouse ovaries

[0057] 2.1 Add 3 mL of DMEM / F12 medium to each well of a six-well plate, then add various drugs to make the final concentrations of VD3: 50 ng / mL, vitamin D receptor inhibitor TEI-9647: 500 nM, ZK159222: 1 μM, and MeTC7: 2 μM, respectively. Mix well by pipetting, place a 0.44 μm culture membrane on each well, and incubate in a 37°C incubator for 30 minutes.

[0058] 2.2 Place the isolated ovaries on a culture membrane and culture in a 37°C, 5% CO2 incubator;

[0059] The medium was changed every two days and cultured for 4 days.

[0060] 3. Hematoxylin staining

[0061] 1. Paraffin Embedding of Ovaries

[0062] 1.1 Sample fixation: Mouse ovaries cultured for 4 days were fixed in 4% paraformaldehyde at 4°C for 12-16 hours;

[0063] 1.2 Alcohol concentration gradient dehydration and transparency: 70% ethanol, 80% ethanol, 95% ethanol / eosin staining solution, 95% ethanol, anhydrous ethanol, anhydrous ethanol / xylene (volume 1:1), xylene, each gradient for 5 minutes, and the xylene in the last step was dried with filter paper.

[0064] 1.3 Wax immersion and embedding: Wax the tissue in a 60℃ water bath for 3 hours, transfer the tissue to an iron paraffin box, cover the box, take it out and let it solidify at room temperature.

[0065] 2. Slicing

[0066] 2.1 Turn on the microtome, adjust the slice thickness to 5 μm, and start continuous sectioning after fixing the wax block.

[0067] 2.2 Spreading: After the wax strip is fully stretched in a 42℃ water bath, pick it up with a glass slide.

[0068] 2.3 Bake the slices at 42℃ overnight.

[0069] 3. Hematoxylin staining

[0070] 3.1 Dewaxing and Rehydration: Place the slides with tissue attached in a series of xylene, xylene, absolute alcohol, absolute alcohol, 95% ethanol, 80% ethanol, and 70% ethanol steps. Each step lasts for 5 minutes, and the slides are then rinsed with deionized water.

[0071] 3.2 Staining: Stain with hematoxylin solution for 1 minute and 30 seconds, then rinse with deionized water.

[0072] 3.3 Dehydration and mounting: The slides were sequentially dehydrated with 70% ethanol, 80% ethanol, 95% ethanol, anhydrous ethanol, anhydrous ethanol, xylene, and xylene, each gradient for 3 minutes, and then mounted with neutral resin.

[0073] IV. Immunofluorescence staining

[0074] 1. Section, dewax, and rehydrate the ovarian tissue wax blocks.

[0075] 2. Antigen retrieval: Place the slices in citric acid buffer working solution and microwave on high heat for 4 minutes and low heat for 4 minutes × 3 times, then cool naturally to room temperature.

[0076] 3. Wash slides: PBS for 5 minutes x 2.

[0077] 4. Blocking: Block with 10% donkey serum at room temperature for 1 hour.

[0078] 5. Incubate with primary antibody: overnight at 4°C.

[0079] 6. Wash slides: PBS for 5 minutes x 6.

[0080] 7. Incubate with secondary antibody at 37°C for 1 hour. From this step onwards, operate in the dark.

[0081] 8. Wash slides: PBS 5 minutes x 6.

[0082] 9. DAPI: Incubate for 1 minute.

[0083] 10. Wash slides: PBS for 5 minutes x 3.

[0084] 11. Sealing: Add 20 μL of anti-fluorescence quencher and cover with a coverslip.

[0085] 12. Take pictures using a laser confocal microscope.

[0086] 5. Ovarian follicle count

[0087] 1. Follicle Counting by Hematoxylin Staining

[0088] Based on their morphological characteristics, follicles can be divided into the following categories: primordial follicles: small oocytes surrounded by three to five flat granulosa cells; growing follicles: larger oocytes surrounded by one or more layers of cuboidal granulosa cells, or a mixture of cuboidal and flat granulosa cells; and atretic follicles: pyknotic oocytes and / or one or more pyknotic granulosa cells. Counts were performed on hematoxylin-stained serial sections, with one count taken every five sections. The final result was the sum of the counts multiplied by five.

[0089] 6. Results

[0090] 1. As shown in Figure 1a, VDR is expressed in both oocytes and granulosa cells at 1 dpp, and is mainly expressed in granulosa cells at 7 dpp.

[0091] 2. Figure 1 (c) shows the comparison of ovarian morphology between the control group and the groups supplemented with vitamin D3, vitamin D receptor inhibitors TEI-9647, ZK159222, and MeTC7. (b) shows the counts of primordial and growing follicles. As can be seen, compared with the control group, the number of growing follicles in the vitamin D supplementation group was significantly decreased (control group: 367.50±37.17; vitamin D group: 241.25±14.31), while the number of growing follicles in the vitamin D receptor inhibitor supplementation group was significantly increased, with TEI-9647 (517.50±12.50), ZK159222 (500.00±12.75), and MeTC7 (515.00±23.18). There was no significant difference in the number of primordial follicles between the groups and the control group. Scale bar: 50 μm. **P<0.01, ***P<0.005.

[0092] The above results indicate that vitamin D receptor is expressed and located in the mouse ovary; VD3 inhibits the activation of primordial follicles, while vitamin D receptor inhibitors TEI-9647, ZK159222, and MeTC7 promote the activation of primordial follicles and have a regulatory effect on the activation of primordial follicles.

[0093] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of vitamin D or vitamin D receptor inhibitors in the preparation of drugs for treating primary ovarian insufficiency.

2. Use of vitamin D or a vitamin D receptor inhibitor according to claim 1 in the preparation of a medicament for treating primary ovarian insufficiency, characterized in that: The primary ovarian insufficiency includes primary ovarian insufficiency caused by overactivation or inactivation of primordial follicles.

3. Use of vitamin D or a vitamin D receptor inhibitor according to claim 2 in the preparation of a medicament for treating primary ovarian insufficiency, characterized in that: The application is the application of vitamin D in the preparation of a drug for inhibiting the activation of primordial follicles, or the application of a vitamin D receptor inhibitor in the preparation of a drug for promoting the activation of primordial follicles.

4. Use of vitamin D or a vitamin D receptor inhibitor according to any one of claims 1 to 3 in the preparation of a medicament for treating primary ovarian insufficiency, characterized in that: The vitamin D is 1,25-dihydroxyvitamin D3; the vitamin D receptor inhibitor is TEI-9647, ZK159222 or MeTC7.

5. Use of vitamin D or a vitamin D receptor inhibitor according to any one of claims 1 to 3 in the preparation of a medicament for treating primary ovarian insufficiency, characterized in that: In the application, the dosage of vitamin D is calculated based on the individual body weight, and vitamin D is administered in an amount of 0.0006-6 mg / day for every 50 kg body weight; the dosage of vitamin D inhibitors is calculated based on the individual body weight, and the dosage range for every 50 kg body weight is: TEI-9647: 3.28-32820 mg / day, ZK159222: 3.94-39440 mg / day, MeTC7: 1.42-14277 mg / day; The primordial follicles are primordial follicles of mammals; the mammals are mice or humans; The drug is administered orally or by intravenous injection.

6. Use of a vitamin D receptor inhibitor in the preparation of a drug for activating primordial follicles in vitro, characterized in that: The vitamin D receptor inhibitor is TEI-9647, ZK159222 or MeTC7.

7. Use of the vitamin D receptor inhibitor according to claim 6 in the preparation of a drug for activating primordial follicles in vitro, characterized in that: The primordial follicle in vitro activation drug is a drug that promotes the transformation of primordial follicles into growth follicles.

8. An in vitro method for activating primordial follicles for non-disease diagnosis and treatment purposes, characterized in that: The ovaries are cultured in an in vitro activation reagent; the in vitro activation reagent contains a vitamin D receptor inhibitor; the vitamin D receptor inhibitor is TEI-9647, ZK159222 or MeTC7.

9. The in vitro activation method of primordial follicles for non-disease diagnosis and treatment purposes according to claim 8, characterized in that: The concentrations of TEI-9647, ZK159222, and MeTC7 are 500±50nM, 1±0.5μM, and 2±0.5μM, respectively.

10. The in vitro activation method of primordial follicles for non-disease diagnosis and treatment purposes according to claim 8 or 9, characterized in that: The ovary is a mouse ovary; The culture is carried out at a constant temperature of 37±2°C and 5%±1% CO2 for 4 days.

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