Application of small molecule compound A-769662 in promoting differentiation of testicular germ cells

Abstract

The invention provides application of a small molecule compound A-769662 in preparation of a medicine for treating male infertility and application of the small molecule compound A-769662 in preparation of a reagent for inducing spermatogenesis in vitro. The medicine has the functions of treating male infertility and preserving fertility by increasing the proportion of haploid spermatids in the process of inducing spermatogenesis in vitro. The invention provides a new way for improving the proportion of haploid sperm cells.

Description

Technical Field

[0001] The present invention relates to the fields of assisted reproduction and male reproductive health, and specifically includes a small molecule compound C 20 H 12 N 2 O 3 The regulatory application of S in testicular germ cell differentiation and in vitro spermatogenesis belongs to a subdivision of germ cell culture and assisted reproduction technology. Background Art

[0002] Currently, approximately 17.5% of the global population is troubled by infertility problems, and male factors account for about 50%. The main causes of male infertility include spermatogenesis disorders, manifested as oligospermia, asthenospermia, teratospermia, and azoospermia. To address male reproductive health problems, scientists have been working on developing a culture system that can support spermatogenesis in vitro. The stable establishment of this system can not only advance the basic theory of human reproductive biology but also provide important application prospects for the evaluation of environmental factors in infertility and fertility preservation technology.

[0003] In recent years, research has shown that androgens and various growth factors play important roles in the proliferation and differentiation of testicular germ cells in a culture system that supports spermatogenesis in vitro. However, spermatogenesis is a multi-step complex process, including the mitosis of spermatogonia differentiated from primordial germ cells, meiosis of spermatocytes, and the transformation of spermatids into tadpole-like sperm with the help of the somatic cell microenvironment. Defects in any of the above processes can lead to spermatogenesis disorders.

[0004] There are literature reports that in the current in vitro spermatogenesis system, the efficiency of forming haploids during testicular germ cell meiosis (i.e., the spermatogonia-spermatocyte-haploid spermatid differentiation stage) is low. Therefore, it is of great significance to explore and screen small molecule compounds with significant effects to increase the haploid ratio and construct a more robust in vitro spermatogenesis system to assist in establishing and maintaining a stable in vitro spermatogenesis system. Summary of the Invention

[0005] To solve the problem of low and unstable haploid formation efficiency in in vitro spermatogenesis, this patent provides the application of the small molecule compound A-769662 in an in vitro spermatogenesis system, aiming to promote the proliferation and differentiation of testicular germ cells, provide a potentially effective means for improving male infertility, and at the same time promote the further development of the in vitro spermatogenesis system.

[0006] The technical solution of the present invention is as follows:

[0007] The first object of the present invention is to provide the application of A-769662 in the preparation of a drug for treating male infertility.

[0008] Further, the drug treats male infertility by increasing the proportion of haploid spermatids during in vitro induction of spermatogenesis.

[0009] The second object of the present invention is to provide the use of A-769662 in the preparation of a reagent for in vitro induction of spermatogenesis.

[0010] Further, the reagent for in vitro induction of spermatogenesis can increase the proportion of haploid spermatids during in vitro induction of spermatogenesis.

[0011] In the use described in the present invention, the structural formula of A-769662 is

[0012] In the use described in the present invention, the drug or reagent further includes an in vitro induction and differentiation medium, and the use is to add A-769662 to the in vitro induction and differentiation medium.

[0013] The in vitro induction and differentiation medium described in this patent refers to a medium that induces spermatogonia to differentiate into spermatocytes and then into spermatids. In a particular embodiment, the in vitro induction and differentiation medium is the TMC medium.

[0014] In the use described in the present invention, the addition amount of A-769662 in the drug or reagent is 2-10 μM.

[0015] In a particular embodiment, the in vitro induction time is not less than 10 days, or not less than 20 days, or not less than 30 days.

[0016] The beneficial effects of the present invention are as follows:

[0017] This patent provides a new way to increase the proportion of haploid spermatids (i.e., round sperm). After adding A-769662, the haploid proportion of in vitro spermatogenesis at 20D increased from about 11% on average in the control group to about 18% on average. The haploid proportion of in vitro spermatogenesis at 30D increased from about 14.4% on average in the control group to about 23.55% on average. Description of the Drawings

[0018] Figure 1 It is an immunofluorescence staining map of the experimental group and the control group taken at 10D, where the experimental group is the experimental group with three concentrations of 2, 5, and 10 μM, and it is taken under a 10-fold microscope;

[0019] Figure 2 It is an immunofluorescence staining map of the experimental group and the control group taken at 10D, where the experimental group is the experimental group with three concentrations of 2, 5, and 10 μM, and it is taken under a 20-fold microscope;

[0020] Figure 3Immunofluorescence staining images of the experimental group and the control group at 20D. The experimental group includes three concentrations of 2, 5, and 10 μM, and the images were taken under a 10× microscope.

[0021] Figure 4 Immunofluorescence staining images of the experimental group and the control group at 20D. The experimental group includes three concentrations of 2, 5, and 10 μM, and the images were taken under a 20× microscope.

[0022] Figure 5 Immunofluorescence staining images of the experimental group and the control group at 30D. The experimental group has a concentration of 10 μM, and the images were taken under 10× and 20× microscopes.

[0023] Figure 6 Flow cytometry diagrams of the experimental group at 20D; among them, Figure 6 A shows the flow cytometry results of testicular samples from 5dpp mice cultured in vitro for 20D in a system supplemented with A-769662. The haploid ratio is 19.5%. Figure 6 B is a repeated experiment, showing the flow cytometry results of testicular samples from 5dpp mice cultured in vitro for 20D in a system supplemented with A-769662. The haploid ratio is 17.4%. The average haploid ratio of samples cultured in vitro for 20D in the system supplemented with the small molecule is 18.45%.

[0024] Figure 7 Flow cytometry diagrams of the control group at 20D; among them, Figure 7 A shows the flow cytometry results of testicular samples from 5dpp mice cultured in vitro for 20D in the control group system without A-769662. The haploid ratio is 15.4%. Figure 7 B is a repeated experiment, showing the flow cytometry results of testicular samples from 5dpp mice cultured in vitro for 20D in the control group system without A-769662. The haploid ratio is 8.29%. The average haploid ratio of samples cultured in vitro for 20D in the system without the small molecule is 11.85%.

[0025] Figure 8 Flow cytometry diagrams of the experimental group at 30D; among them, Figure 8 A shows the flow cytometry results of testicular samples from 5dpp mice cultured in vitro for 30D in a system supplemented with A-769662. The haploid ratio is 24.0%. Figure 8 B is a repeated experiment, showing the flow cytometry results of testicular samples from 5dpp mice cultured in vitro for 30D in a system supplemented with A-769662. The haploid ratio is 23.1%. The average haploid ratio of samples cultured in vitro for 30D in the system supplemented with the small molecule is 23.55%.

[0026] Figure 9 Flow cytometry diagrams of the control group at 30D; among them, Figure 9A is the flow cytometry result obtained from culturing the testes of 5-day-old postnatal (5dpp) mice in vitro for 30 days in the control group system without adding A-769662, and the haploid ratio is 14.9%. Figure 9 B is a repeated experiment, which is the flow cytometry result obtained from culturing the testes of 5dpp mice in vitro for 30 days in the control group system without adding A-769662, and the haploid ratio is 13.9%. The average haploid ratio of culturing in vitro for 30 days in the system without adding small molecules is 14.4%. Detailed implementation manners

[0027] The following examples are used to further explain the present invention, but the examples do not limit the present invention in any form.

[0028] Example 1

[0029] Take the testes of 5-day-old postnatal (5dpp) mice, remove the capsule, and perform in vitro culture on the nested membrane of a six-well plate. Add A-769662 (AMPK activator) C at concentrations of 2 μM, 5 μM, and 10 μM in the differentiation medium. 15 H 8 N 2 O 3 S, and set up a control group. Except for not adding A-769662 (AMPK activator) C 15 H 8 N 2 O 3 S, the other conditions are the same as those of the experimental group. Sampling is performed at fixed time points (10D, 20D, 30D), and operations such as immunofluorescence staining and flow cytometry analysis are carried out to observe the cell ratio and state and analyze the haploid ratio.

[0030] The formula of the differentiation medium (TMC medium) is as follows: taking 50 mL as a unit: 5 mL KSR (a serum-free culture substitute), 42.5 mL aMEM (α-modified Eagle's minimum essential medium), 500 μL GlutaMax (a high-level cell culture additive), 500 μL NEAA (MEM non-essential amino acid solution), 500 μL double antibody (penicillin + streptomycin), 500 μL sodium pyruvate, 50 μL β-mercaptoethanol. Then add 200 μL BPE (bovine pituitary extract), 10 μL testosterone, mix well, add 20 μL FSH (follicle-stimulating hormone), filter, and then add 2.5 μL BMP4 (bone morphogenetic protein 4), 10 μL ActA (multifunctional cytokine), 10 μL SCF (stem cell factor), and then filter.

[0031] The specific steps are as follows:

[0032] 1. Cell culture

[0033] Decapitate 5-day-old postnatal (5dpp) mice, remove the testes, and remove the tunica albuginea. Wash the testes three times with washing solution. Use forceps to divide one intact testis into five equal pieces of tissue, place them on the nested membrane of a six-well plate, and culture them in an incubator at 34 °C and 5% CO₂. Add 800 μL of differentiation medium (supplemented with small molecule compounds at concentrations of 2 μM / 5 μM / 10 μM) to each well.

[0034] 2. Immunofluorescence staining

[0035] 1) At 10 days in vitro (10D), 20 days in vitro (20D), and 30 days in vitro (30D), collect the samples, add 4% PFA, and fix at room temperature for 2 h. Remove the liquid, and add 70% alcohol overnight.

[0036] 2) Dehydrate the fixed tissue sequentially in 1 mL of 80%, 90%, and 100% alcohol for 1 h each (pour the alcohol along the wall of the tube into the EP tube).

[0037] 3) Discard the liquid, add an equal volume (500 μL) of 1:1 100% ethanol and xylene, and incubate for 6 - 8 min. At this time, the tissue becomes semi-transparent and white.

[0038] 4) Discard the liquid, add xylene, and immediately infiltrate with paraffin after 30 - 40 s. Then place it in an oven at 65 °C for 1 h.

[0039] 5) Turn on the paraffin embedding instrument 40 - 50 min in advance to fully preheat the paraffin and the dropper. Immerse the embedding frame + small container in paraffin and then take it out. Adjust the position of the tissue so that it is in the center of the small container. Use a dropper to add paraffin along the edge to cover the embedding frame, transfer it to an ice table, and freeze it on the ice table for 10 min.

[0040] 6) Cut sections (5 μm thick). Place them in an oven at 65 °C overnight (at least 4 h).

[0041] 7) After the slides have cooled, dewax them by soaking them twice in xylene, with each soak for 15 - 20 min.

[0042] 8) Then place them sequentially in 100% (repeat twice, 1 - 2 min each), 90%, 80%, and 70% alcohol for 1 - 2 min each.

[0043] 9) Take them out and place them in distilled water for 5 min.

[0044] 10) After dewaxing, place them in an acidic repair solution and heat them in a microwave oven at high power for 3 min and low power for 7 min.

[0045] 11) Let them cool. Block with 5% blocking solution for 1 h and incubate with the primary antibody overnight at 37 °C.

[0046] 12) Immerse them in PBS for 5 min, three times. Incubate with the secondary antibody for 4 h.

[0047] 3. Streaming analysis

[0048] 1) Place one tissue in each well of a six-well plate, add 100 μl collagenase IV and 900 μl PBS, tear off the peripheral tissue five or six times, and incubate at 37 degrees for 20 minutes.

[0049] 2) Transfer to a 15 ml centrifuge tube, pipette a few times until no large flocculent tissues are left, centrifuge to remove the supernatant, add two ml of 25 pancreatin, pipette a few times, and incubate at 37 degrees for three to five minutes. (Check under a microscope to see if it is a single cell)

[0050] 3) Add 2 ml of 10G, centrifuge and take the supernatant.

[0051] 4) Add 500 μl of antibody (mixed with 10G at a ratio of 1:2000) and incubate at 37°C for 10-15 minutes in the dark.

[0052] 5) Centrifuge and take the supernatant, add 500 μl of antibody LY6K (1:50).

[0053] 6) Centrifuge and remove the supernatant, resuspend in 300 μl 10G, filter, and observe under a microscope.

[0054] 7) Get on the computer.

[0055] Figure 1 The immunofluorescence staining images of the experimental group and the control group were taken at 10 days. The experimental group was the experimental group with three concentrations of 2, 5, and 10uM, and was taken under a 10x microscope. It can be seen that when the testicular tissue was cultured in vitro for ten days, a large number of germ cells were visible in the lumen, and most of them were spermatocytes (because spermatocyte marker-SYCP3 was positive), and there were spermatogonia in the seminiferous epithelium of the lumen (because spermatogonia maeker-PLZF was positive). At this time, there was no significant difference between the experimental group and the control group.

[0056] Figure 2 The immunofluorescence staining images of the experimental group and the control group were taken at 10 days. The experimental group was the experimental group with three concentrations of 2, 5, and 10uM, and was taken under a 20x microscope. It can be seen that when the testicular tissue was cultured in vitro for ten days, a large number of germ cells were visible in the lumen, and most of them were spermatocytes (because spermatocyte marker-SYCP3 was positive), and there were spermatogonia in the seminiferous epithelium of the lumen (because spermatogonia maeker-PLZF was positive). At this time, there was no significant difference between the experimental group and the control group.

[0057] Figure 3The immunofluorescence staining images of the experimental group and the control group were taken at 20 days. The experimental group was the experimental group with three concentrations of 2, 5, and 10uM, and was taken under a 10x microscope. It can be seen that when the testicular tissue was cultured in vitro for 20 days, germ cells were visible in the lumen, and round spermatozoa appeared (because the sperm cell acrosome marker-PNA was positive). At this time, it can be seen from the fluorescence image that the lumen of the experimental group was filled with a large number of round spermatozoa.

[0058] Figure 4 The immunofluorescence staining images of the experimental group and the control group were taken at 20 days. The experimental group was the experimental group with three concentrations of 2, 5, and 10uM, and was taken under a 20x microscope. It can be seen that when the testicular tissue was cultured in vitro for 20 days, germ cells were visible in the lumen, and round sperm appeared (because the sperm cell acrosome marker-PNA was positive). At this time, it can be seen from the fluorescence image that the lumen of the experimental group was filled with a large number of round sperm.

[0059] Figure 5 The immunofluorescence staining images of the experimental group and the control group were taken at 30 days. The experimental group was the 10uM experimental group, which was taken under 10x and 20x microscopes. It can be seen that when the testicular tissue was cultured in vitro for 30 days, there were germ cells and round sperm in the lumen (because the sperm cell acrosome marker-PNA was positive). At this time, it can be seen from the fluorescence image that the lumen of the experimental group was filled with a large number of round sperm and was significantly more than that of the control group.

[0060] Streaming graph data such as Figures 6 to 9 As shown, they are the data of 20D experimental group ( Figure 6 ) and 20D control group data ( Figure 7 ), there are three peaks in each of the two figures, namely 1N, 2N, and 4N, which mean haploid, diploid, and tetraploid. Figure 6 The haploid ratio is 18.45%. Figure 7 The haploid ratio of the control group was 11.85%, and the flow cytometry graph showed that the haploid ratio of the experimental group with the addition of the A-769662 small molecule was increased.

[0061] Mouse testicles were cultured in vitro for 30 days in a system supplemented with A-769662. Figure 8 ) and mouse testis were cultured in vitro for 30 days in a control group system without A-769662. Figure 9 ) showed that the average haploid ratio of the system with small molecules added after 30 days of in vitro culture was 23.55%; the average haploid ratio of the system without small molecules added after 30 days of in vitro culture was 14.4%. The experimental group with the addition of A-769662 small molecules increased the haploid ratio.

[0062] In summary, A-769662 can be used to prepare drugs for treating meiotic arrest for clinical use, which helps to establish an in vitro spermatogenesis model and reconstruct male fertility.

[0063] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Use of A-769662 in the preparation of drugs for treating male infertility.

2. The use according to claim 1, characterized in that: The drug is used to treat male infertility by increasing the proportion of haploid sperm cells during induction of spermatogenesis in vitro.

3. Application of A-769662 in the preparation of in vitro spermatogenesis induction reagents.

4. The use according to claim 3, characterized in that: The in vitro spermatogenesis induction reagent can increase the proportion of haploid sperm cells during in vitro spermatogenesis induction.

5. The use according to any one of claims 1 to 4, characterized in that ,, the structural formula of A-769662 is 6. The use according to any one of claims 1 to 4, characterized in that The medicine or reagent also includes an in vitro differentiation medium for inducing spermatogonia to differentiate into spermatocytes and spermatids, and the application is to add A-769662 to the in vitro differentiation medium.

7. The use according to any one of claims 1 to 4, characterized in that ,,The amount of A-769662 added to the drug or reagent is 2 to 10 μM.

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