Efficient method for generating haploid sperms through bostrichthys sinensis spermatogonium cell in-vitro induction

Abstract

The invention discloses an efficient method for generating haploid sperms through in-vitro induction of bostrichthys sinensis spermatogonium cells, which improves the efficiency of generating functional sperms through in-vitro induction of the spermatogonium cells and comprises the following steps: placing the spermatogonium cells in a three-dimensional non-scaffold type culture material containing a sperm induction culture medium for culture; the sperm induction culture medium is prepared by adding sex hormone, melatonin and an ERK1 / 2 activator into a basic sperm culture medium. The invention further discloses an efficient culture medium for generating haploid sperms through in-vitro induction of bostrichthys sinensis spermatogonium cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an efficient method for inducing haploid sperm from spermatogonia of Channa sinensis in vitro. Background Art

[0002] In most sexually reproducing animals, germ cells undergo meiosis to produce genetically diverse haploid gametes (sperm or eggs). Fertilization then produces a diploid zygote, giving rise to a new individual. Germ cells are the foundation of biological genetic diversity and 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 lies in targeted selection of germ cells, retaining and accumulating favorable traits such as growth, stress resistance, and flavor, while eliminating unfavorable traits such as disease susceptibility, thereby mass-producing offspring with stable traits. The long maturation cycle of many farmed fish species—for example, grass carp at five years, sturgeon at five to seven years, and grouper at four to five years—is a major limitation in the development of superior new varieties. Existing techniques utilize germline stem cell transplantation, where donor germline stem cells are transplanted into closely related, infertile recipient fish with a shorter maturation cycle to produce donor-derived gametes. This is an effective way to shorten breeding cycles. However, donor germline stem cells are easily eliminated by the recipient's immune system, resulting in very few successful cases of producing donor gametes in recipient fish. Therefore, the research on direct in vitro culture and induction of germline stem cells to produce functional gametes has attracted increasing attention from technicians.

[0003] In sexually reproducing animals, germline stem cells (GSCs) are specialized stem cells for reproductive functions. They include three main types: primordial germ cells (PGCs) during the embryonic stage, spermatogonia, and oogonia during the juvenile stage. In higher vertebrates such as mammals, PGCs and oogonia are only present during the embryonic stage, leaving spermatogonia (SSCs) as the only GSCs found in large numbers in most adult animals and capable of continuous proliferation and differentiation. SSCs can sustain self-renewal through mitosis and further differentiate into spermatocytes, which then undergo meiosis to produce haploid sperm. Recently, researchers have isolated cell lines from the testes of several economically important fish species, such as the croaker, grouper, redfin pufferfish, and Yellow River carp, that weakly express several GSC markers. These SSC lines, known as "SSC cell lines," can proliferate indefinitely in vitro and differentiate into different cell types, even forming flagellated "sperm-like" cells induced by sex hormones. However, these SSCs may lack the motility and fertilization abilities of normal sperm. This may be due to the incomplete genome of fish SSCs caused by long-term in vitro culture. Therefore, establishing a stable fish SSC cell line and then inducing SSCs to produce functional sperm may not be effective. Therefore, a more effective method for obtaining breeding sperm is to maintain the integrity of their germline stem cell function through brief primary culture of SSCs, and then induce them to form a "testis-like" organ with supporting cells in vitro using hormones to produce functional sperm.

[0004] The technology for inducing fish SSCs was first established in Japanese eels. Specifically, male germ cells from Japanese eels are co-cultured with testicular somatic cells or cell lines. Under the influence of sex hormones such as testosterone, the cells reconstruct into "round spherical masses" resembling testicular organs and undergo meiosis to produce sperm. However, the efficiency of sperm production is extremely low, with a ratio of no more than 10%. Currently, researchers typically induce fish SSCs in vitro in three-dimensional scaffold-type culture materials to improve the induction efficiency. These three-dimensional scaffold-type culture materials use artificial polymer materials (polycaprolactone, polyglycolic acid, etc.) or natural polymer materials (collagen, gelatin, sodium alginate, etc.) to construct porous or fibrous scaffolds that simulate the in vivo cellular environment, provide physical support and attachment surfaces for cells, and facilitate cell access to nutrients. For example, the applicant disclosed in Chinese patent publication number CN114480262A a method for producing functional sperm from 3D in vitro cultured spermatogonia in snakehead snakehead (Snakehead tang) snakehead. This method involves culturing spermatogonia in Transwell-COL, a three-dimensional collagen-based scaffold. Using a culture medium containing sex hormones and melatonin, the method achieved a sperm production rate of 17.13% after four weeks of culture. However, this method still yields a low rate of haploid sperm production in snakehead snakehead spermatogonia, and requires at least two weeks to form a "testis-like" spherical structure exceeding 80 μm in diameter. The formation time and size of these spherical structures are also important indicators of the efficiency of in vitro spermatogenesis induction. Therefore, this method still suffers from low induction efficiency and requires further improvement. Summary of the Invention

[0005] The purpose of the present invention is to provide an efficient method for inducing spermatogonia of Chinese snakehead to produce haploid sperm in vitro, thereby improving the efficiency of inducing spermatogonia to produce functional sperm in vitro.

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

[0007] An efficient method for inducing haploid sperm production from spermatogonia of Channa sinensis in vitro, comprising the following steps:

[0008] The spermatogonia are placed in a three-dimensional non-scaffold culture material containing a sperm induction medium for culture; the sperm induction medium is a basic sperm culture medium supplemented with sex hormones, melatonin and an ERK1 / 2 activator.

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

[0010] In this method, the present invention proposes, for the first time, the use of a three-dimensional, non-scaffold-based culture material, a spherical microplate (SM), for inducing spermatogonia in vitro, replacing commonly used three-dimensional scaffold-based materials. Within this SM, spermatogonia have weak adhesion to the pores of the SM. Spermatogonia secrete cell surface adhesion factors, which act as signaling molecules to attract spermatogonia to migrate and, through covalent bonding, connect spermatogonia to form three-dimensional aggregates, or "testis-like" spherical masses. The present invention utilizes a three-dimensional, non-scaffold-based culture material, the SM, to facilitate spermatogonia's migration and confluence, significantly reducing the time required for "testis-like" spherical mass formation and increasing the proportion of haploid sperm produced. Through experiments, the present invention discovered that spermatogonia of Chinese snakehead snakehead cultured using the three-dimensional non-scaffold culture material SM aggregated to form distinctly dense cell clusters, namely "testis-like" spherical masses, after only one week of culture. This significantly surpasses the more than two weeks required by the three-dimensional scaffold material Transwell-COL (TC), as disclosed in Chinese patent publication number CN114480262A. Moreover, the proportion of haploid sperm produced after two weeks of culture in the non-scaffold SM was 25.34%, which was also significantly higher than the 6.87% haploid sperm produced by culture in the scaffold material TC.

[0011] The present invention also uses a sperm induction medium containing an ERK1 / 2 activator to significantly promote the formation of "testis-like" balls and improve the efficiency of producing haploid sperm. The present invention uses molecular and cell biology experiments to confirm that ERK1 / 2 signals will be activated during the in vitro culture of Chinese black snakehead spermatogonia; and, through experiments, it was found that compared with the basic sperm culture medium, the addition of ERK1 / 2 activators to it increased the proportion of haploid sperm produced by Chinese black snakehead spermatogonia from 0.02% to 6.23% after culturing Chinese black snakehead spermatogonia for 2 weeks. Therefore, ERK1 / 2 activators can significantly promote the production of sperm in vitro by Chinese black snakehead spermatogonia.

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

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

[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-pregnant3-one (DHP).

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

[0016] In the spermatogonial cell culture process of the present invention, half of the fresh sperm induction culture medium is replaced every three days.

[0017] The basal sperm culture medium is DMEM 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] The present invention can adopt existing means to obtain spermatogonia of Channa sinensis, and specifically can adopt the method for isolating spermatogonia of Channa sinensis disclosed in Chinese patent publication number CN114480262A.

[0019] The present invention also provides a highly efficient culture medium for inducing the production of haploid sperm from spermatogonia of Chinese snakehead snakehead in vitro, which can significantly promote the formation of "testis-like" spherical masses and improve the efficiency of producing haploid sperm. The culture medium comprises a basic sperm culture medium and sex hormones, melatonin and ERK1 / 2 activators added thereto.

[0020] The ERK1 / 2 activator is mSIRK, and the added 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-pregnant3-one (DHP).

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

[0023] The basal sperm culture medium is DMEM 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 present invention has the following beneficial effects:

[0025] The present invention utilizes a three-dimensional, non-scaffold-based culture material, SM, to culture spermatogonia from the Chinese snakehead fish. Compared to existing three-dimensional scaffold-based materials, this material significantly shortens the formation time of testis-like spherical masses and improves the efficiency of haploid sperm production. Furthermore, the present invention utilizes a sperm induction medium containing an ERK1 / 2 activator, significantly promoting the formation of testis-like spherical masses and increasing the efficiency of haploid sperm production.

[0026] In summary, the present invention can improve the efficiency of inducing the production of functional sperm from spermatogonia of Chinese snakehead snakehead in vitro. Experiments have shown that, compared with the existing technology, the time for the formation of "testis-like" balls in the method of the present invention is shortened from more than 2 weeks to 1 week, and the proportion of haploid sperm produced after 2 weeks of culture is increased from 6.87% to 30.93%; in addition, the fertilization rate of the sperm induced in vitro by the present invention and the fertilization of mature eggs is comparable to that of the existing technology. In other words, the present invention greatly improves the efficiency of in vitro induction of spermatogenesis and is expected to become a new strategy to accelerate genetic breeding and improvement of fish aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The images show the "testis-like" spherical masses and cell morphology formed by spermatogonia of Chinese snakehead snakehead cultured in two 3D culture materials, TC and SM, at different times; A to C are SM plates, and D to F are TC plates. Scale bars: A, B, D, E 100 μm, C, F 10 μm.

[0028] Figure 2 This is a diagram showing the proportion of haploid cells produced by spermatogonia of Chinese snakehead in two three-dimensional culture materials, TC and SM; A is the SM plate and B is the TC plate.

[0029] Figure 3 Middle A shows the melatonin receptor in the culture of spermatogonia of Channa sinensis Mtnr1 、 Mtnr2 and Mtnr3 B~D show the changes in the transcriptional levels of related genes detected by qPCR after 2 weeks of culture and the addition of melatonin MTNR1 / 2 / 3 Gene expression in Chinese snakehead snakehead cells cultured on SM plates containing siRNA; E-H show the proportion of haploid sperm in the above cells analyzed by flow cytometry.

[0030] Figure 4 Showing the changes in the expression of downstream signaling proteins after knocking down or overexpressing melatonin receptors; among them, A and B show the protein expression of related genes in Chinese snakehead spermatogonia transfected with different siRNAs or plasmids by immunoblotting; C and D are the grayscale value calculation results of the relevant immunoblot bands.

[0031] Figure 5 Flow cytometry analysis shows the haploid cell ratio in cells cultured in different culture media; A is basal sperm culture medium, B is basal sperm culture medium supplemented with 1 μM mSIRK, C is sperm induction culture medium containing sex hormones, and D is sperm induction culture medium containing sex hormones supplemented with 1 μM mSIRK.

[0032] Figure 6 The figures show the size of cell clusters ("testis-like" spherical masses) and the proportion of haploids therein after 2 weeks of culture in different culture media; among them, A is the sperm induction medium of comparative example 1, B is the sperm induction medium of embodiment 1, and C is the sperm induction medium of comparative example 2. DF respectively show the proportion of haploids in cell clusters A and C analyzed by flow cytometry. DETAILED DESCRIPTION

[0033] The following examples are only used to illustrate the present invention, and the scope of protection of the present invention is not limited to the following examples. Those skilled in the art can achieve the purpose of the present invention based on the above disclosure of the present invention and the ranges of various parameters.

[0034] 1. Comparative Experiments on 3D Scaffold-Based and Non-Scaffold-Based Culture Materials

[0035] S1: Preparation of testicular cell suspension: Testes of 5- to 6-month-old Channa sinensis were isolated and disinfected in 70% ethanol for 30 seconds. After washing three times with phosphate-buffered saline (PBS), the testes were minced with medical scissors and digested in 1 ml of testicular digestion solution at 37°C for 1 h. The testicular digestion solution consists of L-15 medium supplemented with 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.

[0036] S2: Isolation and Identification of Spermatogonia from Channa sinensis: Prepare a Percoll gradient solution consisting of 1.5 ml of 25% Percoll and 1.5 ml of 40% Percoll. Add the single-cell suspension of testes to the Percoll gradient and centrifuge horizontally at 1500 rpm for 15 minutes. The testes will then separate into upper, middle, and lower layers. Spermatogonia will be isolated from the middle layer (at the junction of the 25% and 40% Percoll).

[0037] S3: Culture and differentiation of spermatogonia of Channa sinensis:

[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-pregnant3-one, and 1 μM melatonin to basal sperm culture medium; the basal sperm culture medium was prepared by adding 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 to DMEM medium.

[0039] The obtained spermatogonia of Channa sinensis were placed in TC and 96-well SM containing the above-mentioned sperm induction medium for culture. During the culture process, half of the sperm induction medium was replaced every three days.

[0040] After 1 week of culture (1 WAC), the cells in the SM initially aggregated into a cell mass of about 80 μm, a testis-like spherical structure (see Figure 1 In Figure A), only a few cells in the TC aggregated to form loose cell clumps of approximately 50 μm (see Figure 1 D in the figure); After 2 weeks (2WAC), the diameter of the cell clusters in the SM reached 200 μm (see Figure 1 B in the figure), and a large number of active sperm can be observed (see Figure 1 C in the figure), while the scattered small cell clusters in the TC converge into a cell cluster of about 150 μm (see Figure 1 E in the figure), in which sperm with flagella are barely visible (see Figure 1 F). Cells cultured for 2 weeks were analyzed by flow cytometry. Figure 2 The results showed that the haploid ratio in SM reached 25.34%, while the haploid ratio in TC was only 6.87%.

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

[0042] 1) Analysis of the role of melatonin receptors in spermatogenesis in vitro

[0043] After obtaining spermatogonia from Channa sinensis according to S1-S2 above, they were cultured in SM containing sperm induction medium, where the sperm induction medium was the same as that in S3 above, and sperm induction medium without melatonin was used as a control group.

[0044] First, the spermatogonia were cultured for 2 days and the effect of melatonin in the sperm induction medium on the expression of melatonin receptors on the surface of three cell membranes was detected. Mtnr1 、 Mtnr2 and Mtnr3 The transcript levels of Figure 3 A in ).

[0045] Then, siRNA targeting melatonin receptor was synthesized and transfected into spermatogonia of Channa sinensis 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. Mtnr1 back, cyclin A and cdk1 / 2 There was no significant change in the expression of dmc1 and sycp3 The expression of Figure 3 B in the middle); knock down Mtnr2 back, cyclin A and cdk1 / 2 The expression of dmc1 and sycp3 The expression of Figure 3 C in the middle); knock down Mtnr3 back, cyclin A 、 cdk1 / 2 、 dmc1 and sycp3 were significantly reduced (see Figure 3 D in Figure 1). Flow cytometry analysis showed that the haploid ratio of the control group transfected with control siRNA was 20.117% (see Figure 3 E in), but knock down Mtnr1 、 Mtnr2 and Mtnr3 , resulting in the haploid proportions decreasing to 8.76%, 9.30% and 5.13% respectively (see Figure 3 F~H in it).

[0046] The above results show that melatonin receptors are crucial for inducing haploid sperm production in vitro from spermatogonia of Chinese snakehead.

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

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

[0049] The results showed that knockdown MTNR1 and MTNR3 Significantly reduced the level of phosphorylated ERK1 / 2 (see Figure 4 In contrast, overexpression MTNR1 and MTNR3 Significantly upregulated phosphorylated ERK1 / 2 ( Figure 4 These results indicate that melatonin receptor signaling regulates the phosphorylation of the ERK1 / 2 signaling pathway, which may in turn affect the in vitro spermatogenesis of SSCs in Channa sinensis.

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

[0051] Based on these results, we hypothesize that melatonin can activate the ERK1 / 2 signaling pathway to regulate haploid sperm production in vitro by SSCs of the Chinese snakehead. Therefore, we cultured SSCs of the Chinese snakehead with 1 μM of an ERK1 / 2 signaling pathway activator in basal sperm culture medium and in a sperm induction medium containing sex hormones, respectively, to track their ploidy.

[0052] Specifically, 1 μM of 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 was used as the control group. Then, the spermatogonia of Chinese snakehead were cultured in SM containing this culture medium. After 2 weeks of culture, flow cytometric analysis showed that the haploid ratio of the control group was 0.02% (see Figure 5 A in the figure), while the haploid ratios of the experimental groups with added mSIRK were 6.23% (see Figure 5 B in ).

[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-pregnant3-one were added to the basal sperm culture medium to prepare a sex hormone-containing sperm induction medium. 1 μM mSIRK was added to the medium as the experimental group, and the sex hormone-containing sperm induction medium was used as the control group. Then, spermatogonia of Channa sinensis were cultured in SM containing the medium. After 2 weeks of culture, flow cytometric analysis showed that the haploid ratio of the control group was 8.86% (see ). Figure 5 C in the figure), while the haploid ratio of the experimental group with added mSIRK was 11.23% (see Figure 5 D in the figure). This shows that the addition of the ERK1 / 2 activator mSIRK can significantly increase the proportion of haploid sperm produced by spermatogonia in S. sinensis, whether in the basal sperm culture medium or the sperm induction medium containing sex hormones.

[0054] Example 1

[0055] S1: Preparation of testicular cell suspension: Testes of 5- to 6-month-old Channa sinensis were isolated and sterilized in 70% ethanol for 30 seconds. After washing three times with phosphate-buffered saline (PBS), the testes were minced with medical scissors and digested in 1 ml of testicular digestion solution at 37°C for 1 hour. The testicular digestion solution consists of L-15 medium supplemented with 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.

[0056] S2: Isolation and Identification of Spermatogonia from Channa sinensis: Prepare a Percoll gradient solution consisting of 1.5 ml of 25% Percoll and 1.5 ml of 40% Percoll. Add the single-cell suspension of testes to the Percoll gradient and centrifuge horizontally at 1500 rpm for 15 minutes. The testes will then separate into upper, middle, and lower layers. Spermatogonia will be isolated from the middle layer (at the junction of the 25% and 40% Percoll).

[0057] S3: Culture and differentiation of spermatogonia of Channa sinensis:

[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-pregnant3-one, 1 μM melatonin, and 1 μM mSIRK to basal sperm culture medium (the above amounts are based on the volume of basal sperm culture medium). The basal sperm culture medium was prepared by adding 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 to DMEM medium.

[0059] The spermatogonia of Channa sinensis obtained in S2 were cultured in a 96-well SM 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 culture, Figure 6 As shown in Figure B, the spermatogonia of the Chinese snakehead form a "testis-like" ball with a diameter of about 300 μm, while Figure 6 As shown in A in FIG (i.e., comparative example 1), in the absence of mSIRK, spermatogonia of S. sinensis were cultured to form "testis-like" spherical masses with a diameter of about 150 μm; in other words, mSIRK promoted the formation of spermatogonia of S. sinensis.

[0061] Flow cytometric analysis of cells after 2 weeks of culture showed that the proportion of haploid sperm was 30.93%, which was higher than the proportion of haploid sperm produced in the absence of mSIRK (see Figure 6 The sperm produced by in vitro culture were inseminated with mature eggs, and the fertilization rate was 47.48 ± 2.12% (see Table 1).

[0062] Example 2

[0063] The operating procedures of this example are the same as those of 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 and 50 ng / ml 17α,20β-dihydroxy-4-pregnant3-one, 1 μM melatonin and 0.5 μM mSIRK were added to the basal sperm culture medium to prepare a sperm induction medium.

[0064] After two weeks of culture, spermatogonia in the SM plates initially aggregated into clusters approximately 200 μm in size. Flow cytometric analysis of cells after two weeks of culture revealed a haploid sperm ratio of 27.52%. Insemination of mature eggs with sperm produced in vitro resulted in a fertilization rate of 43.50 ± 3.51% (Table 1).

[0065] Example 3

[0066] The operating procedures of this example are the same as those of 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 and 50 ng / ml 17α,20β-dihydroxy-4-pregnant3-one, 1 μM melatonin and 3 μM mSIRK were added to the basal sperm culture medium to prepare a sperm induction medium.

[0067] After two weeks of culture, spermatogonia in the SM plates initially aggregated into a cluster of approximately 245 μm. Flow cytometric analysis of cells after two weeks of culture revealed a haploid sperm ratio of 28.31%. Insemination of mature eggs with sperm produced in vitro resulted in a fertilization rate of 43.28 ± 3.59% (Table 1).

[0068] Comparative Example 1

[0069] The operating procedures of this comparative example are the same as those of 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-pregnant3-one, and 1 μM melatonin were added to the basal sperm culture medium to prepare a sperm induction medium, i.e., the sperm induction medium did not contain mSIRK.

[0070] like Figure 6 As shown in Figure A, spermatogonia in the SM plate formed cell clusters with a diameter of about 150 μm after culturing for 2 weeks. Flow cytometry analysis of cells after culturing for 2 weeks showed that Figure 6 Figure D shows that the proportion of haploid sperm in the SM plate was 25.48%. Insemination of mature eggs with sperm produced in vitro resulted in a fertilization rate of 42.20 ± 4.61% (Table 1).

[0071] Comparative Example 2

[0072] The operating procedures of this comparative example are the same as those of 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 and 50 ng / ml 17α,20β-dihydroxy-4-pregnant3-one, 1 μM melatonin and 5 μM mSIRK were added to the basal sperm culture medium to prepare a sperm induction medium.

[0073] After two weeks of culture, spermatogonia in the SM plates initially aggregated into a cluster of approximately 220 μm. Flow cytometric analysis of cells after two weeks of culture revealed a haploid sperm ratio of 25.47%. Insemination of mature eggs with sperm produced in vitro resulted in a fertilization rate of 42.54 ± 3.06% (Table 1).

[0074] Comparative Example 3

[0075] The operating procedures of this comparative example are the same as those of 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 and 50 ng / ml 17α,20β-dihydroxy-4-pregnant3-one, 1 μM melatonin and 1 μM ERK1 / 2 inhibitor KO-947 were added to the basal sperm culture medium to prepare a sperm induction medium.

[0076] After 2 weeks of culture, the cells in the SM plate initially aggregated into a loose cell mass of about 100 μm (see Figure 6 C in Figure 1). 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 1). Figure 6 F in the figure). The fertilization rate of in vitro cultured sperm and mature eggs was 9.78 ± 1.29% (Table 1).

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

[0078]

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

[0080] 1. Flow cytometric analysis of haploid composition: After digesting cultured cells into a single-cell suspension, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add pre-chilled 70% ethanol, and fix on ice for 1 hour. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, wash twice with PBS, and resuspend the cells in PBS containing 0.1% BSA. Subsequently, add 10 μg / ml DAPI staining solution and incubate on ice for 10 minutes in the dark. Load the sample on a flow cytometer for analysis.

[0081] 2. Immunoblotting: 48 hours after transfection, cells were harvested, ultrasonically disrupted in lysate, 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, denatured at 95°C for 5 minutes, and loaded onto the wells of a 10% or 15% protein gel. Electrophoresis was performed at 80 V for 30 minutes, then switched 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 anode buffer for at least 15 minutes. After electrophoresis, the gel was rinsed in deionized water for 2 minutes and then transferred to cathode buffer for 5 minutes. Transfer was performed using an S-TRANS rapid multichannel semi-dry transfer apparatus in the following order: anode - filter paper (soaked in anode buffer) - PVDF membrane - gel - filter paper (soaked in cathode buffer). The transfer procedure was as follows: constant voltage of 26 V, current of 1.5 A, time of 600 s; the transfer was completed 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 the primary antibody overnight; the membrane was washed with TBST 3-5 times, each time for 10 minutes, and incubated with the corresponding secondary antibody at room temperature for 1 hour; the PVDF membrane was treated with ECL chemiluminescence reagent, exposed and collected on a chemiluminescence imaging system, and β-actin was used as an internal reference for normalization analysis.

[0082] 3. Preparation and process of PCR reaction system: PCR reaction was performed using SYBR green mix on a Roche LightCycle 480 II (primer sequences are shown in SEQ ID NOs: 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℃ 30 seconds;

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

[0090] 3): 40℃ 2 minutes.

[0091] 4. Detection of fertilization ability of sperm produced by in vitro culture:

[0092] 1) Obtaining mature eggs from Chinese snakehead snakehead: Female fish with normal development, an enlarged abdomen, and dark red genital papillae were injected intramuscularly with 13 μg / kg of luteinizing hormone-releasing hormone (LHRHA2). After 72 hours of dark ripening, 5000 units / kg of human chorionic gonadotropin (HCG) and 13 μg / kg of LHRHA2 were injected intramuscularly. 48–60 hours after the second intramuscular injection, the female's abdomen was gently squeezed to collect mature eggs.

[0093] 2) Testing the Fertilization Capacity 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 the Chinese snakehead snakehead were squeezed into a clean dish and incubated with sperm for 1 minute. The eggs were then activated by adding 25‰ saline to allow fertilization. The fertilization rate was determined by the percentage of embryos reaching the somite stage. Three tests were performed for each Example and Comparative Example, and the average fertilization rate was calculated.

[0094] The present invention may be summarized in other specific forms that do not violate the spirit or main features of the present invention. The above embodiments of the present invention are only to be considered as illustrative and not restrictive of the present invention. Therefore, any minor modifications, equivalent variations, and modifications made to the above embodiments based on the essential technology of the present invention are within the scope of the technical solution of the present invention.

Claims

1. A highly efficient method for inducing haploid sperm production from spermatogonia of Channa sinensis in vitro, characterized by: The following steps are involved: The spermatogonia are placed in a three-dimensional non-scaffold culture material containing sperm induction medium for culture; The three-dimensional non-scaffold culture material is a spherical microporous plate; The sperm induction medium is prepared by adding sex hormones, melatonin and ERK1 / 2 activator to the basic sperm medium.

2. The method for inducing haploid spermatogenesis in vitro from spermatogonia of Channa sinensis according to claim 1, wherein: The ERK1 / 2 activator is a G protein βγ binding peptide, and the added amount is 0.5~3 μM.

3. The method for inducing haploid spermatogenesis in vitro from spermatogonia of Channa sinensis according to claim 2, wherein: The added amount of the ERK1 / 2 activator was 1 μM.

4. The method for inducing haploid spermatogenesis in vitro from spermatogonia of Channa sinensis according to claim 2 or 3, wherein: 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-pregnant3-one; the added amount of melatonin is 0.1-10 μM.

5. The method for inducing haploid spermatogenesis in vitro from spermatogonia of Channa sinensis according to claim 4, wherein: The basic sperm culture medium is 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.

6. The method for inducing haploid sperm production from spermatogonia of Channa sinensis in vitro according to claim 5, wherein: During the spermatogonial cell culture, half of the sperm induction medium was replaced with fresh sperm every 3 days.

7. A culture medium for efficiently inducing haploid spermatogenesis in vitro from spermatogonia of Channa sinensis, characterized by: It includes basic sperm culture medium and sex hormones, melatonin and ERK1 / 2 activator added thereto.

8. The culture medium for inducing haploid sperm production in vitro from spermatogonia of Channa sinensis according to claim 7, characterized in that: The ERK1 / 2 activator is a G protein βγ binding peptide, and the added amount is 0.5~3 μM.

9. The culture medium for inducing haploid sperm production in vitro from spermatogonia of Channa sinensis according to claim 8, characterized in that: The added amount of the ERK1 / 2 activator was 1 μM.

10. The culture medium for inducing haploid sperm production in vitro from spermatogonia of Channa sinensis according to claim 8 or 9, characterized in that: 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-pregnant3-one; The amount of melatonin added is 0.1~10 μM; The basal sperm culture medium is 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.

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