Application of Izorlisib in improving in-vitro spermatogenesis efficiency

Abstract

The invention relates to an application of Izorlisib in improving in-vitro spermatogenesis efficiency, in particular to an application of Izorlisib in preparing a medicine or a reagent for treating male infertility, the structural formula of Izorlisib is # imgabs0 #, and the application can be as follows: a) increasing the proportion of haploid spermatocytes in in-vitro induction of spermatogenesis, b) increasing the proportion of haploid spermatocytes in vitro induction of spermatogenesis, and c) increasing the proportion of haploid spermatocytes in vitro induction of spermatogenesis; b) promoting the round spermatozoa to deform into long spermatozoa during in-vitro induction of spermatogenesis; and c) increasing the number of spermatogonium in the in-vitro induced spermatogenesis. The Izorlisib can increase the proportion of haploid sperm cells generated in vitro, efficiently promotes establishment of an in-vitro induced meiosis generation model and reconstruction of male fertility, and has extremely high application value and clinical significance.

Description

Technical Field

[0001] The present invention belongs to reproductive medicine and assisted reproductive technology in the field of biomedicine, and specifically relates to an application of Izorlisib in the process of in vitro spermatogenesis to improve the efficiency of germ cell meiosis and maturation. Background Art

[0002] Multiple factors, including environmental and genetic factors, may lead to male infertility. In some patients, germ cells cannot complete meiosis normally in the body, resulting in impaired fertility. To address this problem, scientific research has gradually developed techniques for culturing testicular tissue in vitro to induce meiosis in germ cells and form functional sperm, thereby restoring fertility for patients. However, current in vitro spermatogenesis technology is still in the exploratory stage and has problems with low efficiency, especially in the process of spermatogonia or spermatocytes differentiating into spermatids, which is easily blocked, resulting in a low proportion of sperm cell production.

[0003] In 2011, the SATO team successfully constructed an in vitro spermatogenesis culture system for mouse testicular tissue. After about 30 days of in vitro culture, round spermatids can be observed, and healthy fertile offspring can be produced. However, the proportion of haploid spermatids produced is low, only 1.71%. Therefore, how to increase the proportion of haploid spermatids produced by the current in vitro spermatogenesis system is an urgent problem to be solved. At the same time, the spermatids produced by the current in vitro spermatogenesis system of mice are relatively early round spermatids. How to transform early round spermatids into elongated spermatids is also an urgent problem to be solved.

[0004] To solve these problems, improving the meiotic efficiency and sperm morphological transformation efficiency of in vitro spermatogenesis is of great significance for promoting the treatment of male infertility.

[0005] Therefore, there is an urgent need to develop an innovative culture method that can improve the efficiency of sperm cell production and elongated sperm maturation, so as to further improve the clinical treatment of male infertility. Summary of the invention

[0006] The present invention provides an application of Izorlisib in increasing the proportion of haploid spermatids produced by an in vitro spermatogenesis system and inducing deformed elongated spermatids, as well as in preparing an agent or drug for treating meiotic arrest. The inventors found that Izorlisib can increase the proportion of haploid spermatids in an in vitro meiotic system and promote the efficiency of in vitro meiosis.

[0007] The first object of the present invention is to provide the use of Izorlisib in preparing a drug for treating male infertility.

[0008] Furthermore, the drug treats male infertility by at least one of the following a) to c):

[0009] a) increasing the proportion of haploid sperm cells during induction of spermatogenesis in vitro;

[0010] b) Promoting the transformation of round spermatids into elongated spermatids during spermatogenesis in vitro;

[0011] c) Increase the number of spermatogonia during induction of spermatogenesis in vitro.

[0012] The second object of the present invention is to provide the use of Izorlisib in preparing an in vitro spermatogenesis inducing agent.

[0013] Furthermore, the in vitro spermatogenesis induction reagent has at least one of the following functions a) to c):

[0014] a) increasing the proportion of haploid sperm cells during induction of spermatogenesis in vitro;

[0015] b) Promoting the transformation of round spermatids into elongated spermatids during spermatogenesis in vitro;

[0016] c) Increase the number of spermatogonia during induction of spermatogenesis in vitro.

[0017] In the application of the present invention, the structural formula of Izorlisib is

[0018] In the application of the present invention, the medicine or reagent also includes an in vitro meiosis induction medium;

[0019] Preferably, the in vitro meiosis induction medium is TMC medium.

[0020] In the application of the present invention, the amount of Izorlisib added to the drug or reagent is less than 15 μM.

[0021] In the application of the present invention, the amount of Izorlisib added to the drug or reagent in a) is 1-2 μM.

[0022] Preferably, the amount of Izorlisib added to the drug or reagent in a) is 1 μM or 2 μM.

[0023] In the application of the present invention, the amount of Izorlisib added to the reagent in b) is 1-2 μM.

[0024] Preferably, the amount of Izorlisib added to the drug or reagent in b) is 1 μM.

[0025] In the application of the present invention, the amount of Izorlisib added to the drug or reagent in c) is 5 μM ≤ Izorlisib added < 15 μM.

[0026] Preferably, in c), the amount of Izorlisib added to the drug or reagent is 10 μM.

[0027] The beneficial effects of the technical solution of the present invention are:

[0028] Izorlisib increases the proportion of spermatogonia and haploid spermatids in the in vitro spermatogenesis system. Under the culture condition of 1 μM, the proportion of haploid cells induced by Izorlisib increases to 26.7%; in addition, it is observed that Izorlisib induces more round spermatids in the I-II stage that can only be produced by the in vitro spermatogenesis system before, and induces round spermatids and deformed spermatids in the IV-VI stage; under the culture condition of 10 μM, Izorlisib induces an increase in the number of spermatogonia; under the culture condition of 15 μM, culturing for 40 days will lead to an increase in cell apoptosis and a decrease in germ cells. In addition, Izorlisib in the present invention can increase the proportion of haploid spermatids produced in vitro, efficiently promote the establishment of an in vitro induced meiosis model and the reconstruction of male fertility, and has extremely high application value and clinical significance.

[0029] TMC medium: a medium for in vitro meiosis used in the present invention.

[0030] Izorlisib (CH5132799): is a selective class I PI3K inhibitor with a molecular formula of C 26 H 20 F3N7O. The structural formula is as follows:

[0031] DDX4: is a specific marker protein of germ cells.

[0032] PLZF: is a classic undifferentiated spermatogonia-specific marker protein.

[0033] PNA: Peanut acrosome agglutinin, is a specific marker protein of the acrosome of sperm cells.

[0034] TNP1: Transition protein 1, is a specific marker protein for mature and deformed spermatids.

[0035] Hoechst 33342: It is a blue fluorescent dye that can penetrate the cell membrane, has low toxicity to cells, and labels the cell nucleus. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1The results of flow cytometry ploidy sorting of the control group, 1μM and 2μM Izorlisib treatment groups cultured in vitro for 40 days;

[0037] Figure 2 The immunofluorescence results of the control group, 1μM, 2μM, 5μM, 10μM and 15μM Izorlisib treatment groups were cultured in vitro for 20 days;

[0038] Figure 3 The immunofluorescence results of the control group, 1μM, 2μM, 5μM, 10μM and 15μM Izorlisib treatment groups were cultured in vitro for 40 days;

[0039] Figure 4 These are the results of immunofluorescence and HE staining after 48 days of in vitro culture with 1 μM Izorlisib. DETAILED DESCRIPTION

[0040] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0041] Example 1

[0042] ICR male mice born 5 days ago were selected and killed by cervical dislocation. The testicles on both sides were removed, the tunica albuginea was removed, and the testicles were torn into tissue blocks of appropriate size.

[0043] The tissue blocks were divided into six groups and TMC medium containing 1 μM, 2 μM, 5 μM, 10 μM and 15 μM Izorlisib was added respectively (the components of TMC medium include: aMEM accounting for 80-90% of the total volume of complete testis medium, serum replacement accounting for 5-10% of the total volume of complete testis medium, 10-50 ng / ml glial cell line-derived neurotrophic factor, 10-50 ng / ml basic fibroblast growth factor, 10- 50ng / ml epidermal growth factor, 50-100ng / m transforming growth factor β family activin A, 10-50ng / mL stem cell growth factor, 10-50ng / ml bone morphogenetic protein, 10-50mM testosterone, 100-200ng / ml follicle stimulating hormone (FSH) and 50-100mg / m pituitary extract. ) and TMC medium (blank control), cultured at 34°C, 5% CO2 for 40 days. After 40 days of culture, one side of the testicle of the same mouse was used for section staining, and the other side of the testicle was used for flow cytometry sorting for ploidy analysis. Continue to culture until 48 days, and use for section staining to observe cell morphology. Test method:

[0044] (1) Flow cytometry was performed on the cultured mouse testicular tissue cells to analyze the production ratio of haploid sperm cells: the testicular tissues of each group of mice were digested with collagenase IV at 37°C and a carbon dioxide concentration of 5% for 10 minutes, and PBS was added for dilution and termination. The cells were centrifuged at 1200 rpm for 3 minutes, and the supernatant was discarded. 0.25% trypsin was added to digest the cells at 37°C and a carbon dioxide concentration of 5% for 5 minutes. The cells were centrifuged at 1200 rpm for 3 minutes, and the supernatant was discarded. The cells were stained with H33342 and analyzed for ploidy by flow cytometry to explore the specific ratio of haploid sperm cells. The results are as follows: Figure 1 shown.

[0045] The results showed that the haploid ratio of 1μM Izorlisib was 26.7%, and the haploid ratio of 2μM Izorlisib was 10.2%. Compared with the haploid ratio of 3.25% in the control group, the haploid ratio induced in the experimental group was greatly improved.

[0046] (2) Immunofluorescence detection was performed on the cultured mouse testicular tissue cells to analyze the levels of germ cell and sperm cell related proteins: the testicular tissue of each group was fixed with 4% PFA at room temperature for 1 hour, and then transferred to gradient alcohol for gradual dehydration, 70%, 80%, 90% and 100% for 40 minutes each, 100% alcohol: xylene (1:1) for 8 minutes, xylene for 30 seconds, and then the tissue was wax-embedded and sectioned. Immunofluorescence staining was performed with germ cell-specific protein DDX4 and sperm cell-specific protein PNA, and nuclear DNA was stained with Hoechst 33342. Films were taken for observation and statistics. The results are as follows Figure 2 and Figure 3 shown.

[0047] The results showed that by culturing the testicular tissue of newborn mice from 5 days old to 20 days old, adding 10 μM Izorlisib to the culture system can promote the expression of spermatogonia marker protein PLZF, and the number of spermatogonia induced in the experimental group was greatly increased ( Figure 2 ); The experiment of culturing the testicular tissue of newborn mice from 5 days old to 40 days showed that adding 1μM Izorlisib to the culture system can promote the expression of sperm cell marker protein PNA. In addition, more round spermatids in stages IV-VI with acrosome angles of 120°-180° were observed, indicating that adding Izorlisib to the culture system can greatly improve the efficiency of the in vitro spermatogenesis system. Adding 15μM Izorlisib to the culture system showed a decrease in germ cells, indicating that adding high concentrations of Izorlisib to the culture system may damage the activity of germ cells ( Figure 3 ).

[0048] (3) HE staining was performed on the cultured mouse testicular tissue cells to observe the morphology of germ cells and sperm cells: the testicular tissues of each group were fixed with MDF at room temperature for 1 hour, and then transferred to gradient alcohol for gradual dehydration, 70%, 80%, 90% and 100% for 40 minutes each, 100% alcohol: xylene (1:1) for 8 minutes, xylene for 30 seconds, and then the tissues were wax-embedded and sectioned. The testicular tissues were stained with hematoxylin and eosin, and the films were taken for observation. The results are as follows Figure 4 shown.

[0049] The results showed that the addition of 1 μM Izorlisib to the culture system could promote the expression of Tnp1, a marker protein for mature spermatids, by culturing testicular tissue from newborn mice for 48 days. Figure 4 A), which causes round spermatids to further develop into deformed spermatids ( Figure 4 B), Izorlisib can promote the progress of in vitro induced spermatogenesis system.

[0050] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of Izorlisib in the preparation of a drug for treating male infertility.

2. The use according to claim 1, characterized in that: The drug treats male infertility by at least one of the following a) to c): a) increasing the proportion of haploid sperm cells during induction of spermatogenesis in vitro; b) Promoting the transformation of round spermatids into elongated spermatids during spermatogenesis in vitro; c) Increase the number of spermatogonia during induction of spermatogenesis in vitro.

3. Application of Izorlisib 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 has at least one of the following functions a) to c): a) increasing the proportion of haploid sperm cells during induction of spermatogenesis in vitro; b) Promoting the transformation of round spermatids into elongated spermatids during spermatogenesis in vitro; c) Increase the number of spermatogonia during induction of spermatogenesis in vitro.

5. The use according to any one of claims 1 to 4, characterized in that: The structural formula of Izorlisib 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 meiosis induction medium. Preferably, the in vitro meiosis induction medium is TMC medium.

7. The use according to any one of claims 1 to 4, characterized in that ,,Izorlisib was added to the drug or reagent at an amount lower than 15 μM.

8. The use according to claim 7, characterized in that: The amount of Izorlisib added to the reagent in a) is 1-2 μM. Preferably, the amount of Izorlisib added to the drug or reagent in a) is 1 μM or 2 μM.

9. The use according to claim 7, characterized in that: The amount of Izorlisib added to the drug or reagent in b) is 1 to 2 μM. Preferably, the amount of Izorlisib added to the drug or reagent in b) is 1 μM.

10. The use according to claim 7, characterized in that: In c), the amount of Izorlisib added to the drug or reagent is 5 μM≤Izorlisib added<15 μM. Preferably, the amount of Izorlisib added to the drug or reagent in c) is 10 μM.

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