Use of EPHA inhibitor in inducing meiosis

Abstract

The present invention relates to the use of an EPHA inhibitor in inducing meiosis. The EPHA inhibitor can be used to effectively induce the differentiation of undifferentiated spermatogonial cells into spermatocytes, and therefore can be used for the establishment of a model for inducing meiosis in vitro, thus having extremely high application value and clinical significance. Moreover, the EPHA inhibitor does not significantly change the apoptosis level within a dosage of 2 μM-200 μM, such that spermatocytes can be obtained safely and efficiently.

Description

Application of EPHA inhibitors in inducing meiosis Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of an EPHA inhibitor in inducing meiosis. Background Art

[0002] The incidence of childhood cancer is 1.0‰ to 1.5‰, and approximately 30% of patients will experience permanent infertility due to radiotherapy or chemotherapy. For fertility preservation prior to childhood radiotherapy / chemotherapy, the use of in vitro spermatogenesis techniques to achieve spermatogenesis in vitro and to reconstruct spermatogenesis in testicular tissue is crucial, as the spermatogenic cells in the retained testicular tissue have not yet entered meiosis and formed mature sperm. However, due to the current immaturity of in vitro spermatogenesis techniques, spermatogenesis often arrests at the spermatogonial level or the rate of differentiation into spermatocytes is low, leading to ineffective or inefficient in vitro spermatogenesis. Therefore, it is crucial to increase the rate of spermatogonial cells entering meiosis.

[0003] Spermatogonial stem cells (SSCs) are undifferentiated spermatogonia that can self-renew, maintain a constant population, and differentiate into spermatocytes. EPHA, a member of the tyrosine kinase receptor family, is involved in tumorigenesis and spermatogenesis. Literature reports that EPHA activates spermatogonia self-renewal, and that inhibition of EPHA may have a positive effect on spermatogonia entering meiosis.

[0004] Therefore, the development of a drug and induction method that can efficiently induce undifferentiated spermatogonia to differentiate into spermatocytes without changing the level of cell apoptosis is of great significance for the establishment of an in vitro induced meiosis model.

[0005] Summary of the Invention

[0006] In view of this, the present invention proposes the use of an EPHA inhibitor in inducing meiosis, preparing a reagent or drug for inducing the differentiation of undifferentiated spermatogonia into spermatocytes, and preparing a reagent or drug for treating meiotic arrest. The inventors found that the EPHA inhibitor can induce undifferentiated spermatogonia to enter meiosis without affecting cell apoptosis, increase the proportion of spermatocytes, promote the initiation of meiosis in germ cells, and improve the efficiency of meiosis in vitro.

[0007] The technical solutions of the present invention are as follows:

[0008] The first object of the present invention is to provide a use of an EPHA inhibitor in preparing a reagent or drug for treating meiotic arrest.

[0009] The second object of the present invention is to provide use of an EPHA inhibitor in preparing a reagent or drug for inducing meiosis.

[0010] The third object of the present invention is to provide the use of an EPHA inhibitor in the preparation of a reagent or drug for inducing spermatogenesis in vitro.

[0011] Based on the first and second purposes above, the present invention differentiates undifferentiated spermatogonia into spermatocytes during meiosis.

[0012] Based on the above three purposes, in a specific embodiment, the spermatogenesis arrest model is an androgen receptor knockout (ARKO) male mouse.

[0013] The androgen receptor (AR) is a member of the nuclear receptor superfamily and plays an important role in male reproductive function. The AR gene consists of eight exons that encode a 110 kD protein containing an N-terminal transactivation domain, a central DNA-binding domain, and a C-terminal ligand-binding domain. Testosterone and its metabolite dihydrotestosterone can bind to AR, forming homodimers and interacting with numerous coregulators to regulate androgen target genes. AR dysfunction can lead to a variety of clinical diseases, such as testicular feminization mutation syndrome, prostate cancer, and Kennedy's disease. The ARKO male mouse is a model of spermatogenesis arrest. In this model, a partial deletion of the exon 1 region of the AR gene is performed in C57BL / 6J mice using CRISPR / CAS9 technology, resulting in a frameshift mutation in the gene, loss of AR protein expression, and spermatogenesis arrest in spermatocytes.

[0014] In a specific embodiment, the EPHA inhibitor is NVP-BHG712 isomer.

[0015] NVP-BHG712 isomer is a positional isomer of NVP-BHG712, with the molecular formula C 26 H 20 F3N7O, chemically named 4-methyl-3-(2-methyl-6-pyridin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-4-amino)-N-(3-trifluoromethylphenyl)benzamide, CAS number 2245892-85-5, has a structural formula shown in Formula I. The NVP-BHG712 isomer exhibits conservative non-bonded binding to EPHA2 and EPHB4 and is used in experiments as an EPHA inhibitor.

[0016] Furthermore, the dosage of the EPHA inhibitor is 2 μM-200 μM.

[0017] Furthermore, the dosage of the EPHA inhibitor is 2 μM, 20 μM, 50 μM, 100 μM, and 200 μM;

[0018] More preferably, the dosage of the EPHA inhibitor is 20 μM or 100 μM.

[0019] Furthermore, the EPHA inhibitor inhibits the expression of undifferentiated spermatogonia-specific proteins, promotes the expression of spermatocyte-specific proteins, and ensures the occurrence of meiosis. The undifferentiated spermatogonia-specific proteins are CDH1 and PLZF; the spermatocyte-specific proteins are LY6K and SYCP3.

[0020] CDH1 is a classic undifferentiated spermatogonia-specific membrane protein, whose expression inhibits spermatogonia from entering meiosis. In the present invention, the inventors found that EPHA inhibitors can effectively inhibit the expression of CDH1, indicating that EPHA inhibitors can effectively promote the differentiation of undifferentiated spermatogonia into spermatocytes.

[0021] PLZF is a classic undifferentiated spermatogonia-specific marker protein, and its expression inhibits undifferentiated spermatogonia from entering meiosis. In the present invention, the inventors found that EPHA inhibitors can effectively inhibit the expression of PLZF, indicating that EPHA inhibitors can effectively promote the differentiation of undifferentiated spermatogonia into spermatocytes.

[0022] LY6K is a specific membrane protein of spermatocytes. In the present invention, the inventors found that EPHA inhibitors can effectively promote the expression of LY6K, indicating that EPHA inhibitors can effectively increase the ratio of spermatogonia to spermatocytes.

[0023] SYCP3 is a classic spermatocyte-specific marker protein involved in meiotic chromosome synapsis, recombination, and segregation. In the present invention, the inventors found that EPHA inhibitors can effectively promote the expression of SYCP3, indicating that EPHA inhibitors can effectively increase the ratio of spermatogonia to spermatocytes.

[0024] The beneficial effects of the present invention are:

[0025] 1. The EPHA inhibitor of the present invention can effectively promote the expression of spermatocyte-related genes and improve the efficiency of inducing undifferentiated spermatogonia to differentiate into spermatocytes.

[0026] 2. The EPHA inhibitor of the present invention has no obvious toxic side effects within the dosage range of 2 μM-200 μM, and can efficiently induce undifferentiated spermatogonia to differentiate into spermatocytes without changing the level of cell apoptosis, which is of great significance for the establishment of an in vitro meiotic induction model.

[0027] 3. Clinical application: The EPHA inhibitor of the present invention can repair the spermatogenic function of meiotic arrest and effectively promote the differentiation of spermatogonia into spermatocytes in patients with spermatogonial arrest, and has extremely high application value and clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 shows the percentage of PI and Annexin V-positive cells in isolated testicular tissue from 5-day-old mice treated with different concentrations of EPHA inhibitor by flow cytometry. Testicular cells from 5-day-old mice treated with 10 μM hydrogen peroxide for 30 minutes served as a positive control. The different stages of cell apoptosis are defined as: (1) live cells (Annexin V-PI-), (2) early apoptosis (Annexin V+PI-), (3) late apoptosis (Annexin V+PI+), and (4) necrotic cells (Annexin V-PI+).

[0029] FIG2 shows the TUNEL staining results of isolated testicular tissues of 5-day-old mice treated with different concentrations of EPHA inhibitors.

[0030] Figure 3 shows the expression of PLZF, a protein marker for undifferentiated spermatogonia, in isolated testicular tissue from 5-day-old mice treated with different concentrations of EPHA inhibitor. Large scale, 200 μm; small scale, 20 μm.

[0031] Figure 4 shows the expression of SYCP3, a protein marker, in spermatocytes from isolated testicular tissue from newborn 5-day-old mice treated with different concentrations of EPHA inhibitor. Large scale, 200 μm; small scale, 20 μm.

[0032] FIG5 shows the expression percentages of CDH1, a surface marker of undifferentiated spermatogonia, and LY6K, a surface marker of spermatocytes, in isolated testicular tissues of newborn mice treated with different concentrations of EPHA inhibitors 5 days after birth, as determined by flow cytometry.

[0033] FIG6 shows the expression of PLZF, a protein marker for undifferentiated spermatogonia, in the testicular tissue of ARKO mice treated with 20 μM EPHA inhibitor 5 days after birth.

[0034] FIG7 shows the situation of treating SYCP3, a protein marker of spermatocytes in testicular tissue of newborn 5-day-old ARKO mice with 20 μM inhibitor. DETAILED DESCRIPTION

[0035] In order to make the invention purpose, technical solution and technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the present invention and are not intended to limit the present invention.

[0036] Unless otherwise specified, the experimental materials and reagents used are consumables and reagents that can be obtained from conventional commercial channels.

[0037] Experimental Materials

[0038] The EPHA inhibitors in the following examples are all NVP-BHG712 isomers.

[0039] NVP-BHG712 isomer, chemical name: 4-methyl-3-(2-methyl-6-pyridin-3-yl)-2H-pyrazolo[3,4-d]pyrimidin-4-amino)-N-(3-trifluoromethylphenyl)benzamide, CAS No.: 2245892-85-5, structural formula: shown in Formula I.

[0040] The following examples utilize NVP-BHG712 isomer as an example of an EPHA inhibitor to demonstrate the effects of EPHA inhibitors. It should be understood that the following examples utilize NVP-BHG712 isomer as a representative example and do not limit the scope of applicable EPHA inhibitors to NVP-BHG712 isomer. Those skilled in the art may select other EPHA inhibitors based on their specific needs.

[0041] Example 1 Cytotoxicity assay of EPHA inhibitors

[0042] The specific experimental steps are:

[0043] (1) Isolate mouse testicles:

[0044] The testes of 5-day-old normal newborn mice were removed from the capsule and used as the in vitro research object. The culture medium used was complete testis culture medium.

[0045] (2) The decapsulated mouse testes were divided into six groups, and 2 μM, 20 μM, 50 μM, 100 μM, and 200 μM NVP-BHG712 isomer and DMSO (blank control) were added, respectively, and cultured at 34°C, 5% CO2 for 5 days.

[0046] (3) Detection of cultured mouse testicular tissue cells to analyze the level of cell apoptosis:

[0047] In order to ensure the accuracy of the test results and experimental conclusions, the inventors used two methods to detect the level of cell apoptosis.

[0048] The first method for detecting cell apoptosis level is:

[0049] The testicular tissues of each group of mice after culture were digested with collagenase IV at 37°C and 5% carbon dioxide for 10 minutes, diluted and terminated by adding PBS, centrifuged at 1200 rpm for 3 minutes, and the supernatant discarded. 0.25% trypsin was added and digested at 37°C and 5% carbon dioxide for 3 minutes, and culture medium containing 10% fetal bovine serum and 90% DMEM was added to terminate the digestion. Centrifuged at 1200 rpm for 3 minutes, the supernatant discarded, and stained with Annexin V-FITC / PI. Apoptosis was detected by flow cytometry to analyze whether high concentration of NVP-BHG712 isomer affects cell apoptosis.

[0050] The second method for detecting cell apoptosis level is:

[0051] Testicular tissue from each group was fixed with 4% PFA at room temperature for 1 hour. The tissue was then transferred to a gradient of alcohol for dehydration: 70%, 80%, 90%, and 100% for 40 minutes each, followed by 100% alcohol:xylene (1:1) for 8 minutes, and xylene for 30 seconds. The tissue was then paraffin-embedded and sectioned. TUNEL staining was performed. The images were photographed and analyzed.

[0052] The results are shown in Figure 1-2.

[0053] As shown in Figures 1 and 2, it can be found that treatment of mouse testicular tissue with NVP-BHG712 isomer at concentrations between 2 μM and 200 μM does not cause massive apoptosis of mouse testicular tissue cells, and there is no significant difference compared with the blank control. No significant difference is also shown in the flow cytometry graph, indicating that NVP-BHG712 isomer at concentrations between 2 μM and 200 μM has no effect on the apoptosis of mouse testicular tissue cells and is non-toxic to the cells.

[0054] Example 2 EPHA inhibitors can inhibit the expression of spermatogonial specific marker proteins and promote the expression of spermatocyte-related proteins

[0055] The specific experimental steps are:

[0056] (1) Isolate mouse testicles:

[0057] The testes of 5-day-old normal newborn mice were removed from the capsule and used as the in vitro research object. The culture medium used was complete testis culture medium.

[0058] (2) The decapsulated mouse testes were divided into six groups, and 2 μM, 20 μM, 50 μM, 100 μM, and 200 μM NVP-BHG712 isomer and DMSO (blank control) were added, respectively, and cultured at 34°C, 5% CO2 for 5 days.

[0059] (3) Immunofluorescence detection was performed on the cultured mouse testicular tissue cells to analyze the levels of spermatogonia and spermatocyte-related proteins:

[0060] Testicular tissue from each group was fixed with 4% PFA at room temperature for 1 hour. The tissue was then transferred to a gradient of alcohol for dehydration: 70%, 80%, 90%, and 100% for 40 minutes each, followed by 100% alcohol:xylene (1:1) for 8 minutes, and xylene for 30 seconds. The tissue was then paraffin-embedded and sectioned. Immunofluorescence staining was performed with the undifferentiated spermatogonia-specific protein PLZF and the spermatocyte-specific protein SYCP3, and nuclear DNA was stained with Hoechst 33342. The images were photographed and analyzed.

[0061] The results are shown in Figures 3 and 4.

[0062] Experiments on 5-day-old mouse testicular tissue showed that adding the EPHA receptor inhibitor NVP-BHG712 isomer to the culture system can inhibit the expression of the undifferentiated spermatogonial marker protein PLZF and promote the expression level of the spermatocyte SYCP3 gene, indicating that the EPHA inhibitor can effectively increase the proportion of spermatogonia that differentiate into spermatocytes. Example 3: The promoting effect of EPHA inhibitor on the induction of spermatogonia to differentiate into spermatocytes

[0063] The specific experimental steps are:

[0064] (1) Isolate mouse testicles:

[0065] The testes of 5-day-old normal newborn mice were removed from the capsule and used as the in vitro research object. The culture medium used was complete testis culture medium.

[0066] (2) The decapsulated mouse testes were divided into six groups, and 2 μM, 20 μM, 50 μM, 100 μM, and 200 μM NVP-BHG712 isomer and DMSO (blank control) were added, respectively, and cultured at 34°C, 5% CO2 for 5 days.

[0067] (3) The testicular tissues of the mice in each group were digested with collagenase IV at 37°C and a carbon dioxide concentration of 5% for 10 minutes, diluted and terminated by adding PBS, centrifuged at 1200 rpm for 3 minutes, and the supernatant was discarded. The tissues were then digested with 0.25% trypsin at 37°C and a carbon dioxide concentration of 5% for 3 minutes, terminated by adding culture medium containing 10% fetal bovine serum and 90% DMEM, centrifuged at 1200 rpm for 3 minutes, and the supernatant was discarded. The undifferentiated spermatogonia surface marker CDH1 and spermatocyte surface marker LY6K were stained at room temperature for 10 minutes, and the stained single cells were then subjected to flow cytometry.

[0068] The results are shown in Figure 5.

[0069] As shown in Figure 5, it can be found that when mouse testicular tissue is treated with NVP-BHG712 isomer at concentrations between 2 μM and 200 μM, the expression level of CDH1, a surface marker of undifferentiated spermatogonia, decreases, while the expression level of LY6K, a surface marker of spermatocytes, increases, indicating that undifferentiated spermatogonia differentiate into spermatocytes.

[0070] Example 4 EPHA inhibitors can promote the expression of spermatocyte-related proteins in the testis of the meiotic arrest model

[0071] This example uses NVP-BHG712 isomer as an example of an EPHA inhibitor to demonstrate the effects of EPHA inhibitors. It should be understood that the following examples use NVP-BHG712 isomer as a representative example and do not limit the scope of applicable EPHA inhibitors to NVP-BHG712 isomer. Those skilled in the art may select other EPHA inhibitors based on actual application needs.

[0072] The spermatogenesis arrest models in the following examples were all ARKO male mice.

[0073] ARKO male mice are a model of spermatogenesis arrest. CRISPR / CAS9 technology is used to knock out a fragment of the exon 1 region of the AR gene in C57BL / 6J mice, resulting in a frameshift mutation in the gene, the disappearance of AR protein expression, and spermatogenesis arrest in spermatocytes.

[0074] The following examples use ARKO mice as a model of spermatogenesis arrest to demonstrate the effects of EPHA inhibitors in this model. It should be understood that the following examples use ARKO male mice as a representative model and do not limit the applicable spermatogenesis arrest model to ARKO male mice. Those skilled in the art may select other spermatogenesis arrest models based on their specific needs.

[0075] The specific experimental steps are:

[0076] (1) Isolate mouse testicles:

[0077] The testes of 5-day-old ARKO male mice were removed from the capsule and used as the in vitro research object. The culture medium used was complete testis culture medium.

[0078] (2) The decapsulated mouse testes were divided into two groups, and 20 μM NVP-BHG712 isomer and DMSO (blank control) were added to each group and cultured at 34°C, 5% CO2 for 5 days.

[0079] (3) Immunofluorescence detection was performed on the cultured mouse testicular tissue cells to analyze the levels of spermatogonia and spermatocyte-related proteins:

[0080] Testicular tissue from each group was fixed with 4% PFA at room temperature for 1 hour. The tissue was then transferred to a gradient of alcohol for dehydration: 70%, 80%, 90%, and 100% for 40 minutes each, followed by 100% alcohol:xylene (1:1) for 8 minutes, and xylene for 30 seconds. The tissue was then paraffin-embedded and sectioned. Immunofluorescence staining was performed with the spermatocyte-specific protein SYCP3, and nuclear DNA was stained with Hoechst 33342. The images were photographed and analyzed.

[0081] The results are shown in Figures 6 and 7.

[0082] Experiments on the testicular tissue of newborn 5-day-old ARKO mice showed that adding the EPHA receptor inhibitor NVP-BHG712 isomer to the culture system promoted the expression level of the spermatocyte SYCP3 gene, indicating that the EPHA inhibitor can effectively increase the proportion of undifferentiated spermatogonia differentiating into spermatocytes.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of EPHA inhibitors in the preparation of reagents or drugs for treating meiotic arrest.

2. Use of EPHA inhibitors in the preparation of reagents or drugs for inducing meiosis.

3. Use of EPHA inhibitors in the preparation of reagents or drugs for inducing spermatogenesis in vitro.

4. The use according to claim 1 or 2, characterized in that: The meiotic division causes undifferentiated spermatogonia to differentiate into spermatocytes.

5. The use according to any one of claims 1 to 3, characterized in that: The EPHA inhibitor is NVP-BHG712 isomer.

6. The use according to any one of claims 1 to 3, characterized in that: The dosage of the EPHA inhibitor is 2 μM-200 μM.

7. The use according to any one of claims 1 to 3, characterized in that: The dosage of the EPHA inhibitor is 2 μM, 20 μM, 50 μM, 100 μM, and 200 μM.

8. The use according to any one of claims 1 to 3, characterized in that: The dosage of the EPHA inhibitor was 20 μM and 100 μM.

9. The use according to any one of claims 1 to 3, characterized in that: The EPHA inhibitor inhibits the expression of undifferentiated spermatogonia-specific proteins, and the undifferentiated spermatogonia-specific proteins are CDH1 and PLZF.

10. The use according to any one of claims 1 to 3, characterized in that: The EPHA inhibitor promotes the expression of spermatocyte-specific proteins, and the spermatocyte-specific proteins are LY6K and SYCP3.

Images