A preparation for enhancing the proliferation ability of pig spermatogonial stem cells in vitro

Abstract

The application provides a preparation for enhancing the in-vitro proliferation capacity of pig spermatogonial stem cells, and belongs to the technical field of livestock reproduction. The preparation is composed of a basic culture solution, basic maintenance components, supportive growth factors and a core active component polypeptide Japonicin-1Npb. Research shows that under the condition of approaching the physiological temperature (38.5 DEG C) of pigs, the addition of 25-100 mu g / mL of the polypeptide Japonicin-1Npb can significantly improve the EdU positive rate of pig spermatogonial stem cells, promote cell proliferation, specifically up-regulate the expression of stemness genes PLZF, UCHL1 and GFR alpha 1, and inhibit the differentiation gene c-KIT. The application solves the problems of low in-vitro expansion efficiency and easy loss of stemness of pig spermatogonial stem cells, and the cells still retain good meiotic differentiation potential after the removal of the preparation, thereby providing technical support for the development of high-quality genetic resources of pigs and the research of reproductive biology.

Description

Technical Field

[0001] This invention belongs to the field of animal reproductive technology, and in particular relates to a preparation that enhances the in vitro proliferation ability of porcine spermatogonial stem cells. Background Technology

[0002] Spermatogonial stem cells (SSCs) are the basal cell population maintaining spermatogenesis in male animals. They are a type of porcine spermatogonium and possess the ability to self-renew and differentiate into sperm. SSCs have significant applications in animal husbandry breeding, reproductive biology research, and genetic resource preservation. In particular, establishing stable and efficient in vitro culture and expansion systems is crucial for the propagation of superior germplasm, the preparation of gene-edited animals, and the protection of endangered species.

[0003] However, compared with model animals such as mice, the in vitro culture system for porcine spermatogonial stem cells is still imperfect. Existing culture systems suffer from complex culture conditions, poor reproducibility, and limited cell proliferation efficiency. Furthermore, under conditions close to the physiological temperature of pigs (approximately 37–39°C), porcine spermatogonial stem cells often exhibit decreased proliferation capacity, increased apoptosis rate, and difficulty in maintaining stemness, severely limiting their large-scale expansion and application. Therefore, how to effectively promote the proliferation capacity of porcine spermatogonial stem cells while maintaining their stem cell characteristics under near-physiological temperature conditions remains a pressing technical challenge in this field.

[0004] Japonicin-1Npb is a natural short-chain bioactive peptide derived from amphibians, initially isolated from the alpine frog *Nanorana parker*. Previous studies have demonstrated its significant antibacterial activity. However, research on the function of Japonicin-1Npb in mammalian cell systems, particularly in reproductive stem cells (such as spermatogonial stem cells), remains limited, and its potential applications in in vitro cell culture systems have not been fully explored. Therefore, exploring the biological role of Japonicin-1Npb in stem cell culture systems is of great significance for expanding the functional applications of natural antimicrobial peptides and developing novel cell culture regulatory factors. Summary of the Invention

[0005] The purpose of this invention is to provide a formulation that enhances the in vitro proliferation capacity of porcine spermatogonial stem cells, thereby effectively promoting their proliferation capacity while maintaining the stem cell stemness of porcine spermatogonial stem cells.

[0006] In a first aspect, the present invention provides a culture preparation for enhancing the in vitro proliferation capacity of porcine spermatogonial stem cells and maintaining the stemness of spermatogonial stem cells, the culture preparation comprising a basic culture medium, basic maintenance components, supporting growth factors and a core active ingredient; wherein, the core active ingredient is a polypeptide Japonicin-1Npb, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] Preferably, the concentration of the polypeptide Japonicin-1Npb in the culture medium is 25–100 μg / mL. Preferably, the concentration of the polypeptide Japonicin-1Npb is 50 μg / mL.

[0008] Preferably, the basic culture medium is DMEM / F12 culture medium; The basic maintenance components include: L-glutamine 1.5–2 mM, sodium pyruvate 0.5–1 mM, HEPES 8–10 mM, bovine serum albumin 0.2–0.4% w / v, insulin 5–10 μg / mL, transferrin 5–10 μg / mL, sodium selenite 3–5 ng / mL, and penicillin-streptomycin bispecific antibody 1%; The supporting growth factors include: GDNF 8–10 ng / mL and bFGF 2–4 ng / mL.

[0009] Preferably, the basic maintenance components include: L-glutamine 2 mM, sodium pyruvate 1 mM, HEPES 10 mM, bovine serum albumin 0.4% w / v, insulin 10 μg / mL, transferrin 10 μg / mL, sodium selenite 5 ng / mL, and penicillin-streptomycin bispecific antibody 1%; The supporting growth factors include: GDNF 10 ng / mL and bFGF 2 ng / mL.

[0010] Secondly, the present invention provides the application of a polypeptide in the preparation of a culture formulation that enhances the in vitro proliferation capacity of porcine spermatogonial stem cells and maintains the stemness of spermatogonial stem cells, wherein the polypeptide is a polypeptide Japonicin-1Npb, and the amino acid sequence of the polypeptide Japonicin-1Npb is shown in SEQ ID NO:1.

[0011] Preferably, maintaining the stemness of spermatogonial stem cells is manifested in maintaining the expression of stemness-related genes PLZF, UCHL1, and GFRα1 and inhibiting the expression of differentiation-related gene c-KIT.

[0012] Thirdly, the present invention provides an in vitro culture method for enhancing the in vitro proliferation capacity of porcine spermatogonial stem cells and maintaining the stemness of spermatogonial stem cells, the culture method comprising the following steps: (1) Obtain a single-cell suspension of porcine testes, separate and purify it by differential adhesion method to obtain porcine spermatogonial stem cells; (2) The porcine spermatogonial stem cells were resuspended using the culture preparation described above and seeded into a culture dish coated with Laminin and cultured at 38.5°C and 5% CO2.

[0013] Preferably, the concentration of Laminin coating in the culture vessel is 20 μg / mL.

[0014] Preferably, the porcine testicular single-cell suspension is derived from the testicular tissue of a 7-day-old Large White pig.

[0015] The beneficial effects of this invention are as follows: Firstly, this invention is the first to apply the peptide Japonicin-1Npb to the culture of porcine spermatogonial stem cells (SSCs). Experiments have shown that at concentrations of 25–100 μg / mL, this peptide significantly increases the rate of EdU-positive cells, with the best effect observed at 50 μg / mL, resulting in a significantly higher proliferation efficiency compared to conventional culture media. This invention maintains excellent performance even at temperatures close to the physiological temperature of pigs (38.5℃), and the proliferative effect of the peptide Japonicin-1Npb is unaffected by minor adjustments to the basic components, overcoming the challenge of decreased proliferation capacity in traditional culture systems at high temperatures.

[0016] Secondly, this formulation can specifically upregulate the expression of stem genes such as PLZF, UCHL1, and GFRα1, while significantly inhibiting the expression of the differentiation-related gene c-KIT. This ensures that cells can maintain their undifferentiated, primitive state for a long period of time while expanding efficiently.

[0017] Finally, after the peptide was removed and the SSCs amplified by this formulation were induced, they were able to rapidly initiate the meiotic program, indicating that the formulation maintained the stemness without impairing their differentiation and development potential, providing efficient and stable technical support for the preservation of pig genetic resources and reproductive biology research. Attached Figure Description

[0018] Figure 1 Microscopic cell morphology diagram of porcine spermatogonial stem cells (pSSCs) prepared in Example 7 of the present invention; Figure 2 This is a comparison of the relative expression levels of stem genes (PLZF(a), GFRα1(b), UCHL1(c)) and differentiation genes (c-KIT(d))) in porcine spermatogonial stem cells and supporting cells obtained by qRT-PCR in Example 8 of the present invention. Figure 3 This is a statistical comparison chart of the proliferation capacity (EdU-positive cell rate) of porcine spermatogonial stem cells after 48 hours of culture in different culture medium groups (Examples 1–6 and Comparative Examples 1–2) in Example 9 of the present invention. Figure 4 This is a graph showing the detection results of the relative expression level of the stemness-related gene PLZF in porcine spermatogonial stem cells after continuous culture for 7 days in different culture medium groups in Example 9 of the present invention; Figure 5 This is a graph showing the detection results of the relative expression level of the stemness-related gene UCHL1 in porcine spermatogonial stem cells after continuous culture for 7 days in different culture medium groups in Example 9 of the present invention; Figure 6 This is a graph showing the detection results of the relative expression level of the stemness-related gene GFRα1 in porcine spermatogonial stem cells after continuous culture for 7 days in different culture medium groups in Example 9 of the present invention; Figure 7 This is a graph showing the detection results of the relative expression level of the differentiation-related gene c-KIT in porcine spermatogonial stem cells after continuous culture for 7 days in different culture medium groups in Example 9 of the present invention; Figure 8 This is a statistical graph showing the expression changes of meiosis-related genes (Stra8, SCP3) and stemness gene (PLZF) in porcine spermatogonial stem cells cultured with the formulation of the present invention under RA-induced conditions in Example 10 of the present invention. Detailed Implementation

[0019] Example 1 Porcine spermatogonial stem cell culture medium A consists of the following basic culture medium, basic maintenance components, supporting growth factors, and core active ingredients; Basic culture medium: DMEM / F12 basic culture medium; Basic maintenance ingredients: L-glutamine 2 mM, sodium pyruvate 1 mM, HEPES 10 mM, bovine serum albumin 0.4% (w / v), insulin 10 μg / mL, transferrin 10 μg / mL, sodium selenite 5 ng / mL, and penicillin-streptomycin antibiotic 1%; Supportive growth factors: GDNF 10ng / mL, bFGF 2 ng / mL; Key active ingredients: The polypeptide Japonicin-1Npb 50 μg / mL, with the amino acid sequence FVLPLVMCKILRKC, SEQ ID NO:1, was synthesized by GL Biochem (Shanghai) with a polypeptide purity >98%.

[0020] Example 2 Porcine spermatogonial stem cell culture medium B consists of the following basic culture medium, basic maintenance components, supporting growth factors, and core active ingredients; Basic culture medium: DMEM / F12 basic culture medium; Basic maintenance ingredients: L-glutamine 2 mM, sodium pyruvate 1 mM, HEPES 10 mM, bovine serum albumin 0.4% (w / v), insulin 10 μg / mL, transferrin 10 μg / mL, sodium selenite 5 ng / mL, and penicillin-streptomycin antibiotic 1%; Supportive growth factors: GDNF 10 ng / mL, bFGF 2 ng / mL; Key active ingredients: Polypeptide Japonicin-1Npb 25 μg / mL.

[0021] Example 3 The porcine spermatogonial stem cell culture medium C consists of the following basic culture medium, basic maintenance components, supporting growth factors, and core active ingredients; Basic culture medium: Composition of DMEM / F12 basal culture medium and basal maintenance components; Basic maintenance ingredients: L-glutamine 2 mM, sodium pyruvate 1 mM, HEPES 10 mM, bovine serum albumin 0.4% (w / v), insulin 10 μg / mL, transferrin 10 μg / mL, sodium selenite 5 ng / mL, and penicillin-streptomycin antibiotic 1%; Supportive growth factors: GDNF 10 ng / mL, bFGF 2 ng / mL; Key active ingredients: Polypeptide Japonicin-1Npb 100 μg / mL.

[0022] Example 4 The porcine spermatogonial stem cell culture medium D consists of the following basic culture medium, basic maintenance components, supporting growth factors, and core active ingredients; Basic culture medium: Composition of DMEM / F12 basal culture medium and basal maintenance components; Basic maintenance ingredients: L-glutamine 2 mM, sodium pyruvate 1 mM, HEPES 10 mM, bovine serum albumin 0.4% (w / v), insulin 10 μg / mL, transferrin 10 μg / mL, sodium selenite 5 ng / mL, and penicillin-streptomycin antibiotic 1%; Supportive growth factors: GDNF 10 ng / mL, bFGF 2 ng / mL; Key active ingredients: Polypeptide Japonicin-1Npb 10 μg / mL.

[0023] Example 5 The porcine spermatogonial stem cell culture medium E consists of the following basic culture medium, basic maintenance components, supporting growth factors, and core active ingredients; Basic culture medium: Composition of DMEM / F12 basal culture medium and basal maintenance components; Basic maintenance ingredients: L-glutamine 1.5 mM, sodium pyruvate 0.5 mM, HEPES 8 mM, bovine serum albumin 0.2% (w / v), insulin 5 μg / mL, transferrin 5 μg / mL, sodium selenite 3 ng / mL, and penicillin-streptomycin bispecific antibody 1%; Supportive growth factors: GDNF 10 ng / mL, bFGF 2 ng / mL; Key active ingredients: Polypeptide Japonicin-1Npb 50 μg / mL.

[0024] Example 6 The porcine spermatogonial stem cell culture medium F consists of the following basic culture medium, basic maintenance components, supporting growth factors, and core active ingredients; Basic culture medium: Composition of DMEM / F12 basal culture medium and basal maintenance components; Basic maintenance ingredients: L-glutamine 2 mM, sodium pyruvate 1 mM, HEPES 10 mM, bovine serum albumin 0.4% (w / v), insulin 10 μg / mL, transferrin 10 μg / mL, sodium selenite 5 ng / mL, and penicillin-streptomycin antibiotic 1%; Supportive growth factors: GDNF 8 ng / mL, bFGF 4 ng / mL; Key active ingredients: Polypeptide Japonicin-1Npb 50 μg / mL.

[0025] Comparative Example 1 The culture medium for ordinary porcine spermatogonial stem cells consists of the following basic culture medium, basic maintenance components, and supporting growth factors; Basic culture medium: Composition of DMEM / F12 basal culture medium and basal maintenance components; Basic maintenance ingredients: L-glutamine 2 mM, sodium pyruvate 1 mM, HEPES 10 mM, bovine serum albumin 0.4% (w / v), insulin 10 μg / mL, transferrin 10 μg / mL, sodium selenite 5 ng / mL, and penicillin-streptomycin antibiotic 1%; Supportive growth factors: GDNF 10 ng / mL, bFGF 2 ng / mL.

[0026] Comparative Example 2 The porcine spermatogonial stem cell culture medium G consists of the following basic culture medium, basic maintenance components, supporting growth factors, and core active ingredients; Basic culture medium: Composition of DMEM / F12 basal culture medium and basal maintenance components; Basic maintenance ingredients: L-glutamine 2 mM, sodium pyruvate 1 mM, HEPES 10 mM, bovine serum albumin 0.4% (w / v), insulin 10 μg / mL, transferrin 10 μg / mL, sodium selenite 5 ng / mL, and penicillin-streptomycin antibiotic 1%; Supportive growth factors: GDNF 10 ng / mL, bFGF 2 ng / mL; Key active ingredients: The polypeptide Japonicin-1Npa 50 μg / mL has the amino acid sequence FLLFPLMCKIQGKC, SEQ ID NO:2.

[0027] Example 7 1. Collect testicular tissue from 7-day-old Large White pigs, place it in a sterile centrifuge tube, disinfect the surface of the testes with 75% alcohol, place the disinfected testicular tissue in pre-cooled PBS, and transfer it to the laboratory for further processing within 30 minutes.

[0028] 2. In a clean bench, place the testis in a sterile culture dish, use a sterile scalpel to cut open the tunica albuginea on the outside of the testis, and peel off and remove the testicular parenchyma.

[0029] 3. Rinse the tissue three times with PBS buffer to remove blood and impurities, then cut the tissue into pieces of approximately 2–3 mm. 3 A tissue block of a certain size.

[0030] 4. Add 3 times the volume of collagenase IV solution (concentration 1 mg / mL) to the shredded tissue, and digest in a 37℃ incubator for 25 min, gently mixing by pipetting during the process. When the seminiferous tubules are clearly observed to be released under a microscope, the digestion is basically complete.

[0031] 5. Add trypsin (final concentration 0.25%) and DNase I (final concentration 30 μg / mL) to the digestion product above, and continue digestion in a 37℃ incubator for 20 min. Gently pipette to promote tissue dispersion.

[0032] 6. When the seminiferous tubule structure is observed to have basically disappeared and the cells are in a monodisperse state, add 3 times the volume of serum-containing DMEM / F12 medium (containing 10% FBS) to terminate the enzyme digestion reaction.

[0033] 7. Pass the digestive fluid through a 200-mesh cell sieve and a 400-mesh cell sieve in sequence, collect the filtrate, and obtain a single-cell suspension of testes.

[0034] 8. Cover the bottom of the culture dish with 0.1% gelatin solution, incubate at 37°C for 30 min for coating, and rinse 3 times with PBS to remove excess gelatin.

[0035] 9. Inoculate the cell suspension into gelatin-coated culture dishes and incubate at 37°C in a 5% CO2 incubator for 2 h. Collect the non-adherent cells from the supernatant and transfer them to new culture dishes.

[0036] 10. The collected non-adherent cells were cultured for another 4 h, and then collected again. After centrifugation, the cells were resuspended in ordinary porcine spermatogonial stem cell culture medium (with 10% FBS added) and seeded into laminin-coated culture dishes (concentration 20 μg / mL). The cells were then incubated at 37℃ in a 5% CO2 incubator until the cells were stably adhered, thus obtaining porcine spermatogonial stem cells. The cell morphology results are shown in the figure below. Figure 1 As shown.

[0037] Example 8 1. Collect cultured porcine spermatogonial stem cells and porcine testicular supporting cells (obtained after liquid nitrogen resuscitation), wash twice with PBS, add 1 mL of TRIzol reagent, and lyse thoroughly.

[0038] 2. Total RNA was extracted using the TRIzol extraction procedure, which involved chloroform extraction, isopropanol precipitation, and washing with 75% ethanol.

[0039] 3. After determining the RNA concentration and purity using NanoDrop, 1 μg of total RNA was reverse transcribed using the Transcriptor FirstStrand cDNA Synthesis Kit to obtain cDNA.

[0040] 4. The following reaction system and procedure were used to detect the PLZF gene (promyelocytic leukemia zinc finger protein), UCHL1 gene (ubiquitin carboxyl-terminal hydrolase L1), GFRα1 (glial cell-derived neurotrophic factor receptor α1), and c-KIT gene (stem cell factor receptor). The reaction system was: 10 μL SYBR Green Master Mix, 1 μL cDNA template, 0.5 μL forward primer, 0.5 μL reverse primer, and 8 μL ddH2O. Reaction program: 95℃ for 10 min (pre-denaturation), 40 cycles: 95℃ for 15 s (denaturation), 60℃ for 30 s (annealing / extension), melt curve analysis (60–95℃, 0.5℃ / s).

[0041] 5. After exporting the Ct value using the instrument software, calculate ΔCt = Ct (target gene). Ct (internal reference gene), calculate ΔΔCt = ΔCt (porcine spermatogonial stem cell group) ΔCt (Supporting cell group), relative expression level = 2^ ΔΔCt (with the supporting cell group as the control, set to 1).

[0042] from Figure 2 The results showed that the expression of PLZF, GFRα1, and UCHL1 mRNA in porcine spermatogonial stem cells was significantly higher than that in testicular Sertoli cells, proving that the obtained cells possessed stem cell characteristics. Conversely, the expression of c-KIT was significantly lower than that in testicular Sertoli cells, indicating that the obtained cells had not differentiated. Combined with the cell morphology results, this demonstrates that the present invention successfully obtained porcine spermatogonial stem cells.

[0043] Example 9 1. Take 20 μL of 1 mg / mL Laminin stock solution and add it to 980 μL of pre-cooled diluent (calcium- and magnesium-free PBS), and gently mix by pipetting.

[0044] 2. In a clean bench, add the diluted Laminin working solution to a 24-well culture dish, and gently shake the dish to ensure that the liquid evenly covers the entire culture surface.

[0045] 3. After incubating overnight in a 4°C refrigerator, aspirate excess Laminin liquid, wash twice with sterile PBS, and obtain coated culture dishes.

[0046] 4. The porcine spermatogonial stem cells obtained in Example 7 were prepared into cell suspensions (2×10⁻⁶) using the porcine spermatogonial stem cell culture medium AF used in Examples 1-6, the ordinary spermatogonial stem cell culture medium of Comparative Example 1, and the porcine spermatogonial stem cell culture medium G of Comparative Example 2. 4 (1 per well) is inoculated into the coated culture dish.

[0047] 5. Incubate the coated culture dishes in an incubator at 38.5℃ and 5% CO2 for 48 hours. 6. After the culture is complete, add the prepared 10 μM EdU working solution, put the culture dish back into the incubator, and continue to incubate for 2 hours.

[0048] 7. Carefully aspirate the culture medium, gently wash the cells twice with PBS, add 4% paraformaldehyde, and fix at room temperature for 15 min.

[0049] 8. Discard the fixative, wash 3 times with PBS, add 0.3% Triton X-100 to each well, permeate at room temperature for 15 min, and then wash with PBS to remove the permeation solution.

[0050] 9. Add Click reaction mixture and incubate at room temperature in the dark for 30 min. After the reaction is complete, wash three times with PBS to remove unbound reaction solution.

[0051] 10. Add DAPI staining solution, stain at room temperature in the dark for 5 minutes, wash with PBS to remove excess DAPI staining, take pictures of 6 random fields of view using a fluorescence microscope, count the total number of cells and the number of EdU positive cells, and calculate the EdU positive cell rate.

[0052] from Figure 3 The results showed that in Comparative Example 1 without the peptide, the EdU positivity rate of porcine spermatogonial stem cells was only 20.35±1.77%, while in Comparative Example 2 with the addition of the structurally similar peptide Japonicin-1Npb, it was only 22.01±1.57%. However, the addition of the peptide Japonicin-1Npb of this invention significantly enhanced cell proliferation. Specifically, at concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL, the EdU positivity rate increased to 45.67±1.96%, 63.81±2.80%, and 60.24±2.62%, respectively, indicating that Japonicin-1Npb can significantly promote the proliferation of porcine spermatogonial stem cells in the range of 25–100 μg / mL, with 50 μg / mL showing the best effect.

[0053] Furthermore, in Example 5, where the concentration of the basal maintenance component was reduced, the EdU positivity rate still reached 56.18 ± 2.38%; and in Example 6, where the ratio of supporting growth factors was changed, the EdU positivity rate still reached 59.88 ± 2.77%. These results demonstrate that the proliferative effect of the present invention's polypeptide Japonicin-1Npb has good system adaptability and is not limited to a single basal maintenance component or supporting growth factor condition.

[0054] Example 10 1. Take 20 μL of 1 mg / mL Laminin stock solution and add it to 980 μL of pre-cooled diluent (calcium- and magnesium-free PBS), and gently mix by pipetting.

[0055] 2. In a clean bench, add the diluted Laminin working solution to a 6-well culture dish, and gently shake the dish to ensure that the liquid evenly covers the entire culture surface.

[0056] 3. After incubating overnight in a 4°C refrigerator, aspirate excess laminin liquid, wash twice with sterile PBS, and obtain coated culture dishes.

[0057] 4. The porcine spermatogonial stem cells obtained in Example 7 were prepared into cell suspensions (1×10⁻⁶) using porcine spermatogonial stem cell culture medium AC of Examples 1-3, ordinary spermatogonial stem cell culture medium of Comparative Example 1, and porcine spermatogonial stem cell culture medium G of Comparative Example 2. 5 (1 per well) is inoculated into the coated culture dish.

[0058] 5. Place the coated culture dish in an incubator at 38.5℃ and 5% CO2, and change half of the medium every two days for 7 consecutive days.

[0059] 6. On day 7 of culture, collect cells from each group, wash twice with PBS, add 1 mL of TRIzol reagent, and lyse thoroughly.

[0060] 7. Total RNA was extracted using the TRIzol extraction procedure, which involved chloroform extraction, isopropanol precipitation, and washing with 75% ethanol.

[0061] 8. After determining the RNA concentration and purity using NanoDrop, 1 μg of total RNA was reverse transcribed using the Transcriptor FirstStrand cDNA Synthesis Kit to obtain cDNA.

[0062] 9. Detect the PLZF gene, UCHL1 gene, GFRα1 and c-KIT gene according to the following reaction system and procedure. The reaction system is: 10 μL SYBR Green Master Mix, 1 μL cDNA template, 0.5 μL forward primer, 0.5 μL reverse primer, and 8 μL ddH2O. Reaction program: 95℃ for 10 min (pre-denaturation), 40 cycles: 95℃ for 15 s (denaturation), 60℃ for 30 s (annealing / extension), melt curve analysis (60–95℃, 0.5℃ / s).

[0063] 10. The relative expression level was calculated using the 2^-ΔΔCt method, with Comparative Example 1 as the control and the relative expression level set to 1.

[0064] from Figures 4-7 The results showed that, compared with Comparative Example 1 (without the peptide), treatment with the present invention's peptide Japonicin-1Npb significantly upregulated the expression of stemness-related genes PLZF, UCHL1, and GFRα1 in porcine spermatogonial stem cells. Specifically, at a concentration of 50 μg / mL, the expression of PLZF, UCHL1, and GFRα1 increased by 3.41-fold, 2.90-fold, and 4.08-fold, respectively, representing the highest levels among all groups; indicating that the peptide has a stable stemness-maintaining effect within the range of 25–100 μg / mL.

[0065] Meanwhile, the differentiation-related gene c-KIT was significantly downregulated in the treatment groups of this invention, with the 50 μg / mL group showing a decrease to 0.32, indicating that cell differentiation was inhibited.

[0066] Furthermore, the treatment group with the structurally similar polypeptide Japonicin-1Npa in the comparative example had little effect on the expression of the above-mentioned genes, further demonstrating that the polypeptide of the present invention has a specific regulatory effect.

[0067] The above results indicate that the culture medium of the present invention not only promotes the proliferation of porcine spermatogonial stem cells, but also effectively maintains their undifferentiated state and stem cell characteristics.

[0068] Example 11 1. Take 20 μL of 1 mg / mL Laminin stock solution and add it to 980 μL of pre-cooled diluent (calcium- and magnesium-free PBS), and gently mix by pipetting.

[0069] 2. In a clean bench, add the diluted Laminin working solution to a 6-well culture dish, and gently shake the dish to ensure that the liquid evenly covers the entire culture surface.

[0070] 3. After incubating overnight in a 4°C refrigerator, aspirate excess laminin liquid, wash twice with sterile PBS, and obtain coated culture dishes.

[0071] 4. The porcine spermatogonial stem cells obtained in Example 7 were prepared into cell suspensions (1×10⁻⁶) using porcine spermatogonial stem cell culture medium AC of Examples 1-3, ordinary spermatogonial stem cell culture medium of Comparative Example 1, and porcine spermatogonial stem cell culture medium G of Comparative Example 2. 5 (1 per well) is inoculated into the coated culture dish.

[0072] 5. Place the coated culture dish in an incubator at 38.5℃ and 5% CO2, and change half of the medium every two days for 7 consecutive days.

[0073] 6. After digesting the pSSCs cultured for 7 days, use 5 × 10⁻⁶ ppm... 4 Re-inoculate at a density of cells / well in Laminin-coated 6-well plates and continue to use the culture medium from Example 1 to allow them to adhere and recover for 24 hours.

[0074] 7. The maintenance group was given the complete culture medium of Example 1, and the induction group was given ordinary porcine spermatogonial stem cell culture medium containing 1.5 μM RA retinoic acid. The cells were continuously induced in a 38.5℃, 5% CO2 incubator for 72 h. The medium was completely replaced on the second day.

[0075] 8. After the culture is completed, collect the cells from each group, wash twice with PBS, add 1 mL of TRIzol reagent, and lyse thoroughly.

[0076] 9. Total RNA was extracted using the TRIzol extraction procedure, which involved chloroform extraction, isopropanol precipitation, and washing with 75% ethanol.

[0077] 10. After determining the RNA concentration and purity using NanoDrop, 1 μg of total RNA was reverse transcribed using the Transcriptor FirstStrand cDNA Synthesis Kit to obtain cDNA.

[0078] 11. Detect the Stra8 gene, SCP3 gene, and PLZF gene according to the following reaction system and procedure. The reaction system is: 10 μL SYBR Green Master Mix, 1 μL cDNA template, 0.5 μL forward primer, 0.5 μL reverse primer, and 8 μL ddH2O. Reaction program: 95℃ for 10 min (pre-denaturation), 40 cycles: 95℃ for 15 s (denaturation), 60℃ for 30 s (annealing / extension), melt curve analysis (60–95℃, 0.5℃ / s).

[0079] 12. The relative expression level was calculated using the 2^-ΔΔCt method, with the maintenance group as the control and the relative expression level set to 1.

[0080] Figure 8 The experimental results showed that the expression level of Stra8, a key marker of meiosis initiation, was significantly increased by 14.27 times in the induced group compared to the maintenance group. Simultaneously, the expression of the SCP3 gene, reflecting the entry of germ cells into prophase I of meiosis, was also upregulated by 9.11 times. This indicates that after removing peptide intervention and applying induction signals, cells can rapidly sense and respond to differentiation stimuli, successfully initiating the meiotic process. This result demonstrates that porcine spermatogonial stem cells obtained by amplification using the formulation of this invention not only possess good proliferative capacity but also maintain complete reproductive and developmental potential under differentiation-induced conditions.

[0081] Correspondingly, PLZF, a core transcription factor maintaining the undifferentiated state of stem cells, significantly decreased in the induced group to 28% of that in the maintenance group, indicating that cell stemness was effectively relieved, gradually exiting the self-renewal state and entering the directed differentiation pathway. The downregulation of PLZF and the upregulation of Stra8 and SCP3 showed a good inverse regulatory relationship, further verifying that the transition from "stemness maintenance" to "meiosis initiation" has a clear molecular basis and consistent regulation. Therefore, this invention not only achieves efficient in vitro expansion of porcine spermatogonial stem cells but also ensures the integrity of their reproductive function, providing a reliable cellular basis and technical support for subsequent applications such as spermatogenesis research, genetic improvement, and germplasm resource preservation.

Claims

1. A culture preparation for enhancing the in vitro proliferation capacity of porcine spermatogonial stem cells and maintaining their stemness, characterized in that, The culture preparation consists of a basic culture medium, basic maintenance components, supporting growth factors, and a core active ingredient; wherein, the core active ingredient is a polypeptide Japonicin-1Npb, and the amino acid sequence of the polypeptide Japonicin-1Npb is shown in SEQ ID NO:

1.

2. The culture preparation according to claim 1, characterized in that, The concentration of the polypeptide Japonicin-1Npb in the culture medium is 25–100 μg / mL.

3. The culture preparation according to claim 2, characterized in that, The concentration of the polypeptide Japonicin-1Npb was 50 μg / mL.

4. The culture preparation according to claim 3, characterized in that, The basic culture medium is DMEM / F12 culture medium; The basic maintenance components include: L-glutamine 1.5–2 mM, sodium pyruvate 0.5–1 mM, HEPES 8–10 mM, bovine serum albumin 0.2–0.4% w / v, insulin 5–10 μg / mL, transferrin 5–10 μg / mL, sodium selenite 3–5 ng / mL, and penicillin-streptomycin bispecific antibody 1%; The supporting growth factors include: GDNF 8–10 ng / mL and bFGF 2–4 ng / mL.

5. The culture preparation according to claim 4, characterized in that, The basic maintenance components include: L-glutamine 2mM, sodium pyruvate 1mM, HEPES 10mM, bovine serum albumin 0.4% w / v, insulin 10 μg / mL, transferrin 10 μg / mL, sodium selenite 5 ng / mL, and penicillin-streptomycin bispecific antibody 1%; The supporting growth factors include: GDNF 10 ng / mL and bFGF 2 ng / mL.

6. The application of a polypeptide in the preparation of a culture formulation that enhances the in vitro proliferation capacity of porcine spermatogonial stem cells and maintains the stemness of spermatogonial stem cells, characterized in that, The polypeptide is a polypeptide Japonicin-1Npb, and the amino acid sequence of the polypeptide Japonicin-1Npb is shown in SEQ ID NO:

1.

7. The application according to claim 6, characterized in that, Maintaining the stemness of spermatogonial stem cells is manifested in maintaining the expression of stemness-related genes PLZF, UCHL1, and GFRα1 and inhibiting the expression of differentiation-related gene c-KIT.

8. A method for in vitro culture that enhances the in vitro proliferation capacity of porcine spermatogonial stem cells and maintains the stemness of spermatogonial stem cells, characterized in that, The cultivation method includes the following steps: (1) Obtain a single-cell suspension of porcine testes, separate and purify it by differential adhesion method to obtain porcine spermatogonial stem cells; (2) The porcine spermatogonial stem cells were resuspended using the culture preparation described in any one of claims 1–5 and seeded into a culture dish coated with Laminin and cultured at 38.5°C and 5% CO2.

9. The in vitro culture method according to claim 8, characterized in that, The laminin coating concentration of the culture vessel was 20 μg / mL.

10. The cultivation method according to claim 9, characterized in that, The porcine testicular single-cell suspension was derived from the testicular tissue of a 7-day-old Large White pig.

Images