A method for efficiently inducing haploid spermatids from Chinese ocellated uakari (Sphaeramia nematoptera) spermatogonia in vitro

Abstract

The application discloses a method for efficiently inducing haploid sperms from Chinese Ophiocephalus sertifer primordial cells in vitro, and improves the efficiency of inducing primordial cells to generate functional sperms in vitro, and comprises the following steps: placing the primordial cells in a three-dimensional non-stent type culture material containing a sperm induction culture medium to culture; the sperm induction culture medium is obtained by adding sex hormones, melatonin and ERK1 / 2 activators into a basic sperm culture medium. The application further discloses a culture medium for efficiently inducing haploid sperms from Chinese Ophiocephalus sertifer primordial cells in vitro.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a highly efficient method for in vitro induction of haploid spermatogonia from the spermatogonium of the Chinese goby. Background Technology

[0002] In most sexually reproducing animals, germ cells produce haploid gametes (sperm or egg) with genetic diversity through meiosis. These gametes combine to form diploid zygotes, resulting in new individuals. Germ cells are fundamental to the generation of biological genetic diversity and are crucial for population reproduction and stability. In agriculture, obtaining high-quality gametes is the core foundation for creating superior new varieties. The essence of superior breeding is the directional selection of germ cells, preserving and accumulating superior traits such as growth, stress resistance, and flavor, while eliminating undesirable traits such as susceptibility to disease, thus producing offspring with stable traits in large quantities. The long maturation cycles of many farmed fish, such as grass carp (5 years), sturgeon (5-7 years), and grouper (4-5 years), are a major limiting factor in the cultivation of superior new varieties. Currently, using reproductive stem cell transplantation technology, transplanting donor reproductive stem cells into recipient sterile fish with short maturation cycles and close kinship to produce donor-derived gametes, is an effective way to shorten the breeding cycle. However, donor reproductive stem cells are easily eliminated by the recipient's immune rejection, resulting in very few successful cases of producing donor gametes in recipient fish. Therefore, research on the direct in vitro culture and induction of reproductive stem cells to produce functional gametes has increasingly attracted the attention of technical personnel.

[0003] In sexually reproducing animals, reproductive stem cells are stem cells dedicated to reproductive function, comprising three main categories: primordial germ cells (PGCs) during the embryonic period, spermatogonia and oogonia during the juvenile period. In higher vertebrates such as mammals, PGCs and oogonia exist only during the embryonic period; therefore, spermatogonia (SSCs) are the only reproductive stem cells in most adult animals that are numerous and capable of continuous proliferation and differentiation. SSCs can maintain self-renewal through mitosis and can further differentiate into spermatocytes, which in turn produce haploid sperm through meiosis. In recent years, researchers have isolated cell lines, known as "SSC cell lines," from the testes of some important economic fish species, such as the grouper, redfin pufferfish, and Yellow River carp, which weakly express several reproductive stem cell markers. Although these lines can proliferate indefinitely in vitro, differentiate into different types of cells, and even form flagellated "sperm-like cells" under the induction of sex hormones, these "sperm-like cells" may lack the motility and fertilization capacity of normal sperm. This may be due to the incomplete genome of fish SSCs caused by long-term in vitro culture. Therefore, establishing stable fish SSC cell lines and then inducing SSCs to produce normally functioning sperm may not be effective. Thus, short-term primary culture of SSCs to maintain the integrity of their reproductive stem cell function, followed by in vitro hormone induction to form a "testis-like" organ with Sertoli cells to produce normally functioning sperm, is currently a more effective method for obtaining breeding sperm.

[0004] The earliest known technique for inducing spermatogenic cells (SSCs) in fish was developed using the Japanese eel. Specifically, male germ cells from the Japanese eel are co-cultured with testicular somatic cells or cell lines. Under the influence of sex hormones such as testosterone, the cells reconstruct into "spherical masses" resembling testicular organs and undergo meiosis to produce sperm. However, the sperm production efficiency is extremely low, not exceeding 10%. Currently, researchers typically use three-dimensional scaffold culture materials to induce SSCs in vitro, thereby improving the induction efficiency. These three-dimensional scaffold culture materials utilize artificial polymer materials (such as polycaprolactone and polyglycolic acid) or natural polymer materials (such as collagen, gelatin, and sodium alginate) to construct porous or fibrous scaffolds to simulate the in vivo cellular environment, providing physical support and attachment surfaces for cells and facilitating cell contact with nutrients. For example, the applicant disclosed a method for producing functional sperm from spermatogonia of the Chinese mudskipper in 3D in vitro culture in Chinese patent publication number CN114480262A. This method induces spermatogonia to be cultured in a three-dimensional scaffold-type culture material, Transwell-COL, using collagen as a scaffold, and employs a culture medium containing sex hormones and melatonin. After 4 weeks of culture, the spermatogonia produced a sperm production rate of 17.13%. However, the proportion of haploid sperm produced by this method is still not high, and it takes at least 2 weeks to form a "testis-like" spherical structure with a diameter exceeding 80 μm. The formation time and size of the "testis-like" spherical structure are important indicators of the efficiency of in vitro sperm production induction. Therefore, this method still has the drawback of low induction efficiency and requires further improvement. Summary of the Invention

[0005] The purpose of this invention is to provide a highly efficient method for in vitro induction of haploid spermatogonia from Chinese goby, thereby improving the efficiency of in vitro induction of functional spermatogonia.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A highly efficient method for in vitro induction of haploid spermatogonia from *Cannabis sativa* spermatogonia, comprising the following steps:

[0008] Spermatogonia were cultured in a three-dimensional non-scaffold culture medium containing sperm induction medium; the sperm induction medium was a basal sperm medium supplemented with sex hormones, melatonin and ERK1 / 2 activator.

[0009] The three-dimensional non-scaffolded culture material, as the name suggests, is a porous or fibrous scaffold without artificial or natural polymer materials; the three-dimensional non-scaffolded culture material of the present invention specifically adopts a spherical microplate (SM).

[0010] In the above method, this invention proposes for the first time the use of a three-dimensional non-scaffolded culture material, spherical microplates (SM), for the in vitro induction and culture of spermatogonia, replacing commonly used three-dimensional scaffolded materials. Within the SM, the adhesion between spermatogonia and the pores is weak. Spermatogonia secrete cell surface adhesion factors, which act as signaling molecules to attract spermatogonia to migrate among themselves, and covalently bind spermatogonia to form three-dimensional aggregates, i.e., "testis-like" spheres. In other words, the use of the SM in this invention facilitates the migration and aggregation of spermatogonia, thus significantly reducing the time required for the formation of "testis-like" spheres and increasing the proportion of haploid sperm. This invention, through experiments, found that using the three-dimensional non-scaffold culture material SM to culture spermatogonia of the Chinese mudskipper resulted in the formation of distinct and dense cell clusters, or "testis-like" spheres, within just one week of culture. This is significantly better than the more than two weeks required by the Chinese patent with publication number CN114480262A, which uses the three-dimensional scaffold material Transwell-COL (TC). Furthermore, the proportion of haploid sperm produced after two weeks of culture in the non-scaffold SM was 25.34%, which is also significantly higher than the 6.87% haploid sperm produced in the scaffold material TC.

[0011] This invention also employs a sperm induction medium containing ERK1 / 2 activators, which significantly promotes the formation of "testis-like" spheroids and improves the efficiency of haploid sperm production. This invention utilizes molecular and cell biology experiments to confirm that ERK1 / 2 signaling is activated during the in vitro culture of *Cannabis sativa* spermatogonia; furthermore, experiments showed that compared to basal sperm culture medium, adding ERK1 / 2 activators increased the proportion of haploid sperm produced from *Cannabis sativa* spermatogonia after 2 weeks of culture from 0.02% to 6.23%. Therefore, ERK1 / 2 activators can significantly promote the in vitro sperm production of *Cannabis sativa* spermatogonia.

[0012] The ERK1 / 2 activator is a G protein βγ binding peptide (mSIRK), CAS number 593267-11-9.

[0013] The amount of ERK1 / 2 activator added is 0.5~3μM, preferably 1μM.

[0014] The sex hormones include 5-20 U / ml human chorionic gonadotropin (hCG), 5-20 IU / ml pregnant mare serum gonadotropin (PMSG), 50-200 ng / ml 11-ketotestosterone (11-KT), 50-200 ng / ml testosterone (T), 50-200 ng / ml 17β-estradiol (E2), and 20-70 ng / ml 17α,20β-dihydroxy-4-pregnenol (DHP).

[0015] The amount of melatonin added is 0.1~10 μM, preferably 1 μM.

[0016] In this invention, half of the sperm induction culture medium is replaced with fresh sperm every 3 days during the spermatogonial cell culture process.

[0017] The basic sperm culture medium consisted of DMEM medium supplemented with 10% fetal bovine serum, 2% sea bass serum, 100 ng / ml epidermal growth factor (EGF), 10 ng / ml fibroblast growth factor (bFGF), 100 ng / ml insulin-like growth factor 1 (IFG-I), and 0.1 mM β-mercaptoethanol.

[0018] This invention can obtain spermatogonia of the Chinese mudskipper using existing methods, specifically the method for separating spermatogonia of the Chinese mudskipper disclosed in Chinese Patent No. CN114480262A.

[0019] The present invention also provides a highly efficient culture medium for in vitro induction of haploid spermatogonia from Chinese goby, which can significantly promote the formation of "testis-like" spheres and improve the efficiency of haploid sperm production. It includes a basic sperm culture medium and sex hormones, melatonin and ERK1 / 2 activators added to it.

[0020] The ERK1 / 2 activator is mSIRK, and the addition amount is 0.5~3 μM, preferably 1 μM.

[0021] The sex hormones include 5-20 U / ml human chorionic gonadotropin (hCG), 5-20 IU / ml pregnant mare serum gonadotropin (PMSG), 50-200 ng / ml 11-ketotestosterone (11-KT), 50-200 ng / ml testosterone (T), 50-200 ng / ml 17β-estradiol (E2), and 20-70 ng / ml 17α,20β-dihydroxy-4-pregnenol (DHP).

[0022] The amount of melatonin added is 0.1~10 μM, preferably 1 μM.

[0023] The basic sperm culture medium consisted of DMEM medium supplemented with 10% fetal bovine serum, 2% sea bass serum, 100 ng / ml epidermal growth factor (EGF), 10 ng / ml fibroblast growth factor (bFGF), 100 ng / ml insulin-like growth factor 1 (IFG-I), and 0.1 mM β-mercaptoethanol.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] This invention utilizes a three-dimensional non-scaffold culture medium (SM) to culture spermatogonia of the Chinese mudskipper. Compared to existing three-dimensional scaffold materials, this method exhibits significant advantages in shortening the formation time of "testis-like" spheroids and improving the efficiency of haploid sperm production. Furthermore, this invention employs a sperm induction culture medium containing ERK1 / 2 activators, which significantly promotes the formation of "testis-like" spheroids and enhances the efficiency of haploid sperm production.

[0026] In summary, this invention can improve the efficiency of in vitro induction of functional spermatogenesis from spermatogonia of the Chinese mudskipper. Experiments have shown that, compared to existing technologies, the time for "testis-like" sphere formation in this invention is shortened from more than two weeks to one week, and the proportion of haploid sperm produced after two weeks of culture increases from 6.87% to 30.93%. Furthermore, the fertilization rate of spermatogenesis induced in vitro by this invention with mature eggs is comparable to that of existing technologies. In other words, this invention significantly improves the efficiency of in vitro induced spermatogenesis and holds promise as a new strategy for accelerating genetic breeding and improvement in fish aquaculture. Attached Figure Description

[0027] Figure 1 This image shows the "testis-like" spheres and cell morphology formed by spermatogonia of the Chinese black goby at different times in two 3D culture materials, TC and SM. A-C represent SM plates, and D-F represent TC plates. Scale bars: A, B, D, E 100 μm, C, F 10 μm.

[0028] Figure 2 The diagram shows the proportion of haploid cells produced by spermatogonia of the Chinese black goby in two three-dimensional culture materials, TC and SM; where A is the SM plate and B is the TC plate.

[0029] Figure 3 Chinese A shows the melatonin receptor during spermatogonium culture of the Chinese black goby. Mtnr1 , Mtnr2 and Mtnr3 Changes in transcriptional levels; B~D shows qPCR detection of related genes in melatonin after 2 weeks of culture. MTNR1 / 2 / 3 Gene expression of siRNA in *Cyprinus sinensis* cells cultured in SM plates; E~H show the proportion of haploid sperm in the above cells as analyzed by flow cytometry.

[0030] Figure 4 The images show the changes in the expression of downstream signaling proteins caused by knockdown or overexpression of the melatonin receptor; A and B show the protein expression of related genes detected by Western blot in spermatogonia transfected with different siRNAs or plasmids; C and D show the gray values ​​of the relevant immunoblot bands.

[0031] Figure 5 The diagram shows the proportion of haploid cells in cells under different culture media analyzed by flow cytometry; where A is basal sperm culture medium, B is basal sperm culture medium with 1 μM mSIRK added, C is sperm induction medium containing sex hormones, and D is sperm induction medium containing sex hormones with 1 μM mSIRK added.

[0032] Figure 6 The display shows the size of cell clusters ("testis-like" spheres) and the proportion of haploids in them after 2 weeks of culture in different culture media; where A is the sperm induction medium of Comparative Example 1, B is the sperm induction medium of Example 1, C is the sperm induction medium of Comparative Example 2, and DF shows the proportion of haploids in cell clusters A and C respectively, as analyzed by flow cytometry. Detailed Implementation

[0033] The following embodiments are for illustrative purposes only, and the scope of protection of the present invention is not limited to these embodiments. Those skilled in the art can achieve the objectives of the present invention based on the above disclosure and the ranges of the parameters.

[0034] 1. Comparative experiment on three-dimensional scaffold-type and non-scaffold-type culture materials

[0035] S1: Preparation of testicular cell suspension: The testes of 5-6 month old Chinese mudskippers were isolated, disinfected in 70% ethanol for 30 seconds, washed 3 times with phosphate-buffered saline (PBS), and then the testes were cut into small pieces with medical scissors. 1 ml of testicular digestion solution was added, and the mixture was digested at 37°C for 1 h. The components of the testicular digestion solution were: 4 mg / mL type IV collagenase, 0.05 wt% DNase I, 0.25 wt% trypsin, 10 wt% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin were added to L-15 medium.

[0036] S2: Isolation and identification of spermatogonia from the Chinese goby: Preparation of Percoll gradient solution: The solution consisted of 1.5 ml of 25% Percoll and 1.5 ml of 40% Percoll. A single-cell suspension of testes was added to the Percoll gradient solution and centrifuged horizontally at 1500 rpm for 15 minutes. The testes were then re-separated into upper, middle, and lower layers, and spermatogonia were obtained from the middle layer (the junction of the 25% and 40% Percoll layers).

[0037] S3: Culture and induced differentiation of spermatogonia from the Chinese goby:

[0038] Sperm induction medium was prepared by adding 10 U / ml human chorionic gonadotropin, 10 IU / ml pregnant mare serum gonadotropin, 100 ng / ml 11-ketotestosterone, 100 ng / ml testosterone, 100 ng / ml 17β-estradiol, 50 ng / ml 17α,20β-dihydroxy-4-pregnenol, and 1 μM melatonin to the basal sperm culture medium. The basal sperm culture medium consisted of DMEM medium supplemented with 10% fetal bovine serum, 2% sea bass serum, 100 ng / ml epidermal growth factor, 10 ng / ml fibroblast growth factor, 100 ng / ml insulin-like growth factor 1, and 0.1 mM β-mercaptoethanol.

[0039] The obtained spermatogonia of the Chinese black goby were cultured in TC and 96-well SM containing the above-mentioned sperm induction medium, respectively; during the culture process, half of the sperm induction medium was replaced every three days.

[0040] After one week of culture (1 WAC), the cells in the SM initially aggregated into a cell cluster of approximately 80 μm, i.e., a testis-like spherical structure (see...). Figure 1 In A), only a very small portion of the cells in TC aggregate, forming loose cell clumps of approximately 50 μm (see A). Figure 1 (D in the text); after 2 weeks (2WAC), the cell cluster diameter in SM reached 200 μm (see D); Figure 1 (B in the text), and a large number of motile sperm can be observed (see B). Figure 1 In C), the scattered small cell masses in TC aggregate into a cell cluster of about 150 μm (see C). Figure 1 In the E), almost no flagellated sperm are visible (see Figure 1 (F in the text). Cells cultured for 2 weeks were analyzed using flow cytometry, such as... Figure 2 The results showed that the haploid proportion in SM reached 25.34%, while the haploid proportion in TC was only 6.87%.

[0041] 2. Experiments on the role of activators of the ERK1 / 2 signaling pathway in spermatogonial cell culture.

[0042] 1) Analyze the role of melatonin receptors in the in vitro spermatogenesis of spermatogonia.

[0043] After obtaining spermatogonia of the Chinese black goby according to S1~S2 above, they were cultured in SM containing sperm induction medium, which was the same as that in S3 above, and sperm induction medium without melatonin was used as the control group.

[0044] First, spermatogonia cultured for 2 days were used to examine the effect of melatonin in the sperm induction medium on the expression of three melatonin receptors on the surface of the cell membrane. The results showed that three melatonin receptors... Mtnr1 , Mtnr2 and Mtnr3 The transcriptional level increased significantly (see Figure 3 (A in the middle).

[0045] Then, melatonin receptor siRNA was synthesized and transfected into spermatogonia of the Chinese goby cultured in the above sperm induction medium for one week; cells were collected 48 hours after transfection for Q-PCR detection and flow cytometry analysis. (Knockdown...) Mtnr1 back, cyclin A and cdk1 / 2 The expression did not change significantly, but dmc1 and sycp3 The expression of was significantly reduced ( Figure 3 (B in the middle); knockdown Mtnr2 back, cyclin A and cdk1 / 2 The expression of was significantly increased. dmc1 and sycp3 The expression of was significantly reduced ( Figure 3 (C in the middle); knockdown Mtnr3 back, cyclin A , cdk1 / 2 , dmc1 and sycp3 All significantly reduced (see) Figure 3 (D in the text). Flow cytometry analysis showed that the proportion of haploids in the control group transfected with control siRNA was 20.117% (see D). Figure 3 (E in the middle), but knockdown Mtnr1 , Mtnr2 and Mtnr3 This resulted in a decrease in the proportion of haploids to 8.76%, 9.30%, and 5.13%, respectively (see...). Figure 3 (F~H in the middle).

[0046] The results above show that the melatonin receptor is crucial for the in vitro induction of haploid sperm from spermatogonia of the Chinese goby.

[0047] 2) Analyze the regulatory role of melatonin receptors on downstream signaling pathway proteins.

[0048] The melatonin receptor gene was fused into the pCMV expression vector; the melatonin receptor expression plasmid or siRNA was transfected into SSC cells of *Culter alburnus* cultured in SM using Lipo8000 transfection reagent; 48 hours after transfection, cells were collected for immunoblotting experiments to detect β-actin and ERK1 / 2 proteins.

[0049] The results showed that knockdown MTNR1 and MTNR3 Significantly reduced the level of phosphorylated ERK1 / 2 (see) Figure 4 (A, C in the text). Conversely, overexpression MTNR1 and MTNR3 Significantly upregulated phosphorylation of ERK1 / 2 ( Figure 4 (B and D in the above results). The above results indicate that melatonin receptor signaling regulates the phosphorylation of the ERK1 / 2 signaling pathway, which may in turn affect in vitro spermatogenesis in SSCs of the Chinese mudskipper.

[0050] 3) Testing the effect of ERK1 / 2 signaling pathway activators

[0051] Based on the above results, it can be inferred that melatonin can activate the ERK1 / 2 signaling pathway to regulate the production of haploid sperm by *Cyprinus sinensis* SSCs in vitro. Therefore, *Cyprinus sinensis* SSCs were cultured with 1 μM ERK1 / 2 signaling pathway activator added to both basal sperm culture medium and sex hormone-containing sperm induction medium to track cell ploidy composition.

[0052] Specifically, 1 μM of the ERK1 / 2 activator G protein βγ-binding peptide mSIRK was added to the basal sperm culture medium as the experimental group, and the basal sperm culture medium served as the control group. Then, spermatogonia of the Chinese black goby were cultured in SM containing this medium. After 2 weeks of culture, flow cytometry analysis showed that the haploid rate in the control group was 0.02% (see...). Figure 5 (A) in the study, while the haploid proportion in the experimental group with added mSIRK was 6.23% (see A). Figure 5 (B in the middle).

[0053] Furthermore, 10 U / ml human chorionic gonadotropin, 10 IU / ml pregnant mare serum gonadotropin, 100 ng / ml 11-ketotestosterone, 100 ng / ml testosterone, 100 ng / ml 17β-estradiol, and 50 ng / ml 17α,20β-dihydroxy-4-progesterone were added to the basal sperm culture medium to prepare a sex hormone-containing sperm induction medium. 1 μM mSIRK was added to this medium as the experimental group, and the sex hormone-containing sperm induction medium was used as the control group. Then, spermatogonia from the Chinese black goby were cultured in SM containing this medium. After 2 weeks of culture, flow cytometry analysis showed that the haploid rate in the control group was 8.86% (see...). Figure 5 The haploid proportion in the group with added mSIRK was 11.23% (see C in the original text). Figure 5 (D in the text). This demonstrates that, regardless of whether it is in basal sperm culture medium or sperm induction medium containing sex hormones, the addition of the ERK1 / 2 activator mSIRK can significantly increase the proportion of haploid sperm produced by spermatogonia of the Chinese goby.

[0054] Example 1

[0055] S1: Preparation of testicular cell suspension: The testes of 5-6 month old Chinese mudskippers were isolated, disinfected in 70% ethanol for 30 seconds, washed 3 times with phosphate-buffered saline (PBS), and then the testes were cut into small pieces with medical scissors. 1 ml of testicular digestion solution was added, and the mixture was digested at 37°C for 1 h. The components of the testicular digestion solution were: 4 mg / mL type IV collagenase, 0.05 wt% DNase I, 0.25 wt% trypsin, 10 wt% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin were added to L-15 medium.

[0056] S2: Isolation and identification of spermatogonia from the Chinese goby: Preparation of Percoll gradient solution: The solution consisted of 1.5 ml of 25% Percoll and 1.5 ml of 40% Percoll. A single-cell suspension of testes was added to the Percoll gradient solution and centrifuged horizontally at 1500 rpm for 15 minutes. The testes were then re-separated into upper, middle, and lower layers, and spermatogonia were obtained from the middle layer (the junction of the 25% and 40% Percoll layers).

[0057] S3: Culture and induced differentiation of spermatogonia from the Chinese goby:

[0058] Sperm induction medium was prepared by adding 10 U / ml human chorionic gonadotropin, 10 IU / ml pregnant mare serum gonadotropin, 100 ng / ml 11-ketotestosterone, 100 ng / ml testosterone, 100 ng / ml 17β-estradiol, 50 ng / ml 17α,20β-dihydroxy-4-pregnenol, 1 μM melatonin, and 1 μM mSIRK to the basal sperm culture medium (all added amounts are based on the volume of the basal sperm culture medium). The basal sperm culture medium consisted of DMEM medium supplemented with 10% fetal bovine serum, 2% sea bass serum, 100 ng / ml epidermal growth factor, 10 ng / ml fibroblast growth factor, 100 ng / ml insulin-like growth factor 1, and 0.1 mM β-mercaptoethanol.

[0059] The spermatogonia obtained from S2 were cultured in 96-well SM medium containing the above-mentioned sperm induction medium; during the culture process, half of the sperm induction medium was replaced every three days.

[0060] After 2 weeks of cultivation, if Figure 6 As shown in B, the spermatogonia of the Chinese black goby form testis-like spheres with a diameter of approximately 300 μm, while... Figure 6 As shown in Figure A (i.e., Comparative Example 1), spermatogonia of the Chinese black goby formed testis-like spheres with a diameter of approximately 150 μm when cultured without mSIRK; in other words, mSIRK promotes the formation of spermatogonia of the Chinese black goby.

[0061] Flow cytometry analysis of cells cultured for 2 weeks showed that the proportion of haploid sperm produced was 30.93%, which was higher than the proportion of haploid sperm produced without mSIRK (see...). Figure 6 (E in the table). The fertilization rate of sperm produced in vitro and mature oocytes was 47.48 ± 2.12% (see Table 1).

[0062] Example 2

[0063] The operation steps in this embodiment are the same as those in Example 1, except that: 10 U / ml human chorionic gonadotropin, 10 IU / ml pregnant mare serum gonadotropin, 100 ng / ml 11-ketotestosterone, 100 ng / ml testosterone, 100 ng / ml 17β-estradiol, 50 ng / ml 17α,20β-dihydroxy-4-pregn-3-one, 1 μM melatonin, and 0.5 μM mSIRK are added to the basic sperm culture medium to prepare the sperm induction culture medium.

[0064] After two weeks of culture, the spermatogonia in the SM plate initially aggregated into a cell cluster of approximately 200 μm. Flow cytometry analysis of the cells after two weeks of culture showed that the proportion of haploid sperm in the SM plate was 27.52%. The sperm produced in vitro were then used to fertilize mature oocytes, and the fertilization rate was 43.50 ± 3.51% (see Table 1).

[0065] Example 3

[0066] The operation steps in this embodiment are the same as those in Example 1, except that: 10 U / ml human chorionic gonadotropin, 10 IU / ml pregnant mare serum gonadotropin, 100 ng / ml 11-ketotestosterone, 100 ng / ml testosterone, 100 ng / ml 17β-estradiol, 50 ng / ml 17α,20β-dihydroxy-4-pregn-3-one, 1 μM melatonin, and 3 μM mSIRK are added to the basic sperm culture medium to prepare the sperm induction culture medium.

[0067] After two weeks of culture, the spermatogonia in the SM plate initially aggregated into a cell cluster of approximately 245 μm. Flow cytometry analysis of the cells after two weeks of culture showed that the proportion of haploid sperm in the SM plate was 28.31%. The sperm produced in vitro were then used to fertilize mature oocytes, and the fertilization rate was 43.28 ± 3.59% (see Table 1).

[0068] Comparative Example 1

[0069] The comparative example follows the same procedure as Example 1, except that 10 U / ml human chorionic gonadotropin, 10 IU / ml pregnant mare serum gonadotropin, 100 ng / ml 11-ketotestosterone, 100 ng / ml testosterone, 100 ng / ml 17β-estradiol, 50 ng / ml 17α,20β-dihydroxy-4-pregn-3-one, and 1 μM melatonin were added to the basic sperm culture medium to prepare the sperm induction medium, which is mSIRK-free.

[0070] like Figure 6 As shown in Figure A, after 2 weeks of culture, spermatogonia in the SM plate formed cell clusters with a diameter of approximately 150 μm; flow cytometry analysis of the cells after 2 weeks of culture showed... Figure 6 The proportion of haploid sperm in the D-scan of the SM plate was 25.48%. The sperm produced in vitro were then used to fertilize mature oocytes, and the fertilization rate was 42.20 ± 4.61% (see Table 1).

[0071] Comparative Example 2

[0072] The comparative example follows the same procedure as Example 1, except that 10 U / ml human chorionic gonadotropin, 10 IU / ml pregnant mare serum gonadotropin, 100 ng / ml 11-ketotestosterone, 100 ng / ml testosterone, 100 ng / ml 17β-estradiol, 50 ng / ml 17α,20β-dihydroxy-4-pregn-3-one, 1 μM melatonin, and 5 μM mSIRK were added to the basal sperm culture medium to prepare the sperm induction medium.

[0073] After two weeks of culture, the spermatogonia in the SM plate initially aggregated into a cell cluster of approximately 220 μm. Flow cytometry analysis of the cells after two weeks of culture showed that the proportion of haploid sperm in the SM plate was 25.47%. The sperm produced in vitro were then used to fertilize mature oocytes, with a fertilization rate of 42.54 ± 3.06% (see Table 1).

[0074] Comparative Example 3

[0075] The comparative example follows the same procedure as Example 1, except that 10 U / ml human chorionic gonadotropin, 10 IU / ml pregnant mare serum gonadotropin, 100 ng / ml 11-ketotestosterone, 100 ng / ml testosterone, 100 ng / ml 17β-estradiol, 50 ng / ml 17α,20β-dihydroxy-4-progesterone, 1 μM melatonin, and 1 μM ERK1 / 2 inhibitor KO-947 were added to the basal sperm culture medium to prepare the sperm induction medium.

[0076] After two weeks of culture, the cells in the SM plate initially aggregated into a loose cell cluster of about 100 μm (see...). Figure 6 (C in the text). Flow cytometry analysis of cells after 2 weeks of culture showed that the proportion of haploid sperm in the SM plate was 8.33% (see...). Figure 6 F in the figure). The sperm produced in vitro was fertilized with mature oocytes, and the fertilization rate was 9.78 ± 1.29% (see Table 1).

[0077] Table 1. Analysis of fertilization rate of sperm induced in vitro in different culture media.

[0078]

[0079] The following are the test methods used in the above experiments, examples, and comparative examples:

[0080] 1. Flow cytometry analysis of haploid composition: Cultured cells were digested into a single-cell suspension, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and pre-chilled 70% ethanol was added. The cells were then fixed on ice for 1 h. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and the cells were washed twice with PBS. The cells were then resuspended in PBS containing 0.1% BSA. Subsequently, 10 μg / ml DAPI staining solution was added, and the cells were incubated on ice for 10 min in the dark. The cells were then loaded onto a flow cytometer for analysis.

[0081] 2. Immunoblot assay: Cells were collected 48 hours after transfection, sonicated in lysis buffer, and centrifuged at 12,000×g for 15 minutes at 4°C. The supernatant was collected. 20 μg of total protein was mixed with 5×SDS loading buffer and denatured at 95°C for 5 minutes. The mixture was then added to the wells of a 10% or 15% protein gel and electrophoresed at 80 V for 30 minutes, then increased to 120 V until bromophenol blue reached the bottom of the gel. The PVDF membrane was fully activated in methanol for 30 seconds and then transferred to the anode buffer for at least 15 minutes. After electrophoresis, the protein gel was washed in deionized water for 2 minutes and then transferred to the cathode buffer for 5 minutes. The membrane was then transferred using the S-TRANS rapid multichannel semi-dry transfer instrument. The order of placement was: anode - filter paper (fully soaked in anode buffer) - PVDF membrane - protein gel - filter paper (fully soaked in cathode buffer). The transfer procedure was as follows: constant voltage of 26 V, current of 1.5 A, and time of 600 s; after the transfer, the membrane was placed in 5% skim milk-TBST solution and blocked at room temperature for 1 hour; after blocking, the PVDF membrane was transferred to 4°C and incubated with primary antibody overnight; the membrane was washed with TBST 3-5 times, 10 min each time, and incubated with the corresponding secondary antibody at room temperature for 1 h; the PVDF membrane was treated with ECL chemiluminescence reagent, exposed and the signal was acquired using a chemiluminescence imaging system, and standardized analysis was performed using β-actin as an internal control.

[0082] 3. Preparation and process of PCR reaction system: PCR reaction was performed using SYBR green mix on Roche LightCycle 480 II (primer sequences are shown in SEQ ID NO:1-18).

[0083] (1) PCR reaction system: see Table 2.

[0084] Table 2 PCR reaction system

[0085]

[0086] (2) Reaction conditions:

[0087] The PCR reaction procedure is as follows:

[0088] 1): 95℃ for 30 seconds;

[0089] 2): 95℃ for 10 seconds; 60℃ for 10 seconds; 72℃ for 10 seconds; repeat 40 times;

[0090] 3): 40℃ for 2 minutes.

[0091] 4. Testing the fertilization capacity of sperm produced in vitro:

[0092] 1) Obtaining mature eggs of *Channa argus*: Normally developed female fish with swollen abdomens and deep red genital papillae were selected and injected intramuscularly with 13 μg / kg luteinizing hormone-releasing hormone (LHRHA2). After maturation in the dark for 72 hours, 5000 units / kg human chorionic gonadotropin (HCG) and 13 μg / kg HCG were injected intramuscularly. 48-60 hours after the second intramuscular injection, the mature eggs were collected by gently squeezing the abdomen of the female fish.

[0093] 2) Fertilization capacity test of cultured sperm: Cells collected from each example and comparative example were resuspended in physiological saline. Dry in vitro fertilization was then performed. Eggs from *Cannabis sativa* were squeezed into clean petri dishes, incubated with sperm for 1 minute, and then activated with 25‰ saline solution. The proportion of embryos entering the somnolence stage was used as the fertilization rate. Three sets of tests were performed for each example and comparative example, and the average fertilization rate was calculated.

[0094] This invention can be summarized in other specific forms that do not depart from the spirit or main features of the invention. The above embodiments of the invention are merely illustrative and not restrictive. Therefore, any minor modifications, equivalent variations, and alterations made to the above embodiments based on the essential technology of this invention fall within the scope of the invention's technical solution.

Claims

1. A method for efficiently inducing haploid sperm from Chinese Oxyeleotris marmoratus spermatogonial cells in vitro, characterized in that, The method comprises the following steps: culturing the spermatogonia in a three-dimensional non-scaffold culture material containing a sperm induction medium; the three-dimensional non-scaffold culture material is a spherical microwell plate; the adhesion between the spermatogonia and the spherical microwell plate is weak in the spherical microwell plate; the sperm induction medium is a basic sperm culture medium added with sex hormones, melatonin and an ERK1 / 2 activator; the ERK1 / 2 activator is a G protein βγ binding peptide, and the added amount is 0.5-3 μM; the sex hormones include 5-20 U / ml human chorionic gonadotropin, 5-20 IU / ml pregnant mare serum gonadotropin, 50-200 ng / ml 11-ketotestosterone, 50-200 ng / ml testosterone, 50-200 ng / ml 17β-estradiol and 20-70 ng / ml 17α, 20β-dihydroxy-4-pregn-3-one; the added amount of the melatonin is 0.1-10 μM; the basic sperm culture medium is a DMEM medium added with 10% fetal bovine serum, 2% sea bass serum, 100 ng / ml epidermal growth factor, 10 ng / ml fibroblast growth factor, 100 ng / ml insulin-like growth factor 1 and 0.1 mM β-mercaptoethanol.

2. The method for in vitro inducing the production of haploid spermatozoa from Chinese Oxyeleotris marmoratus spermatogonial cells according to claim 1, characterized in that, The added amount of the ERK1 / 2 activator is 1 μM.

3. The method of claim 1, wherein the Chinese Fire Belly Newt spermatogonial cells are induced to produce haploid sperm in vitro, and the haploid sperm are collected and used for the production of haploid embryos. During the culture of the spermatogonia, the half of the fresh sperm induction medium is replaced every 3 days.

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