A culture medium for efficiently expanding chicken ovary germ stem cells and application thereof in prolonging egg-laying period

Abstract

The application discloses a culture medium for efficiently amplifying chicken ovary germ stem cells and application of the culture medium to prolong an egg production cycle, and belongs to the cross technical field of livestock breeding and stem cell engineering. The application finds and utilizes an H3K27me3 epigenetic modification inhibitor UNC1999, which is added into a chicken OSCS special culture medium, can effectively reduce the H3K27me3 level in cells, and finally significantly promotes in-vitro proliferation of the OSCS. The culture medium and the culture method can improve the proliferation efficiency of aged chicken OSCS by about 2.4 times within 48 hours. The method breaks through the technical bottleneck of in-vitro amplification of the chicken OSCS, not only provides a new solution for efficiently obtaining high-purity OSCS, but also lays a key technical foundation for directly applying to prolonging an egg-laying period of a laying hen, improving ovary function and establishing a long-term preservation system of avian germplasm resources based on a cell bank, and has great industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of animal husbandry and stem cell engineering, specifically relating to a culture medium for efficiently expanding chicken ovarian reproductive stem cells and its application in extending the egg-laying cycle. Background Technology

[0002] Ovarian germline stem cells (OSCs) are a type of germline stem cells found in the ovaries of animals after birth. They possess both self-renewal capacity and meiotic differentiation potential, and can be directed to generate oocytes. They are the core cell population for maintaining ovarian reproductive function and dynamically renewing the follicle pool. Their quantity and activity directly determine the reproductive lifespan and reproductive efficiency of female animals. (Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women) Nature Medicine ).

[0003] The crucial role of oocytes (OSCs) has been fully validated in multiple species, laying a solid theoretical foundation for the application research of OSCs in poultry. In mouse models, OSCs can proliferate and differentiate into functional oocytes. Supplementing with OSCs can not only significantly delay the aging process of the ovary but also restore the fertility of aging mice. Cytotechnology Researchers have successfully isolated and cultured oocyte-like stem cells (OSCs) in livestock such as pigs, cattle, and sheep, demonstrating that they can maintain stem cell characteristics and differentiate into oocyte-like structures over a long period of time. This has opened up new avenues for improving livestock reproductive performance and protecting the germplasm resources of rare breeds. The Journal ofReproduction and Development In the field of human reproduction, OSCs have been shown to persist throughout the entire reproductive cycle (from reproductive age to postmenopause), and their functional decline is closely related to premature ovarian failure and early menopause in women. Targeted regulation technology for OSCs is expected to become a breakthrough solution for treating female infertility (The quest for human ovarian stem cells). Nat MedFurthermore, in lower vertebrates such as fish and amphibians, the high proliferative activity of OSCs is the core mechanism for maintaining their lifelong reproductive capacity, further confirming the evolutionary conservation and central role of OSCs in vertebrate reproductive regulation (Identification of germline stem cells in the ovary of the teleostmedaka). Science ).

[0004] In the poultry industry, the core reason for the irreversible decline in chicken egg production performance with age lies in the depletion and functional decline of ovarian germline stem cells (OSCs). Current methods for extending the egg-laying cycle are limited to nutritional regulation or hormone supplementation, which not only have limited effectiveness but also pose risks of drug residues in eggs, failing to meet the needs of green farming and industry upgrading. The strategy of in vitro expansion of OSCs to replenish reserves and thus extend the egg-laying cycle or preserve germplasm resources has become a current research hotspot. However, existing in vitro culture systems lack effective methods to efficiently and specifically promote the proliferation of chicken OSCs, hindering large-scale expansion.

[0005] Histone modification H3K27me3 is a key epigenetic marker regulating gene expression, and its function exhibits high cell type and species specificity. Currently, the specific function and mechanism of action of H3K27me3 modification in the proliferation of chicken ovarian germ cell (OSC) remains unreported. UNC1999 is a known small-molecule inhibitor that effectively reduces intracellular H3K27me3 levels. However, no existing technology has reported a culture medium or method using UNC1999 as a functional component to promote the in vitro proliferation of chicken OSC. Therefore, developing a novel technology based on targeted regulation of H3K27me3 to efficiently promote the proliferation of chicken OSC and exploring the role of H3K27me3 in this process is a crucial path to fundamentally overcome the bottlenecks in its in vitro expansion technology. Summary of the Invention

[0006] To address the aforementioned limitations of existing technologies, the present invention aims to provide a culture medium for the efficient expansion of chicken ovarian reproductive stem cells and its application in extending the egg-laying cycle. This method achieves precise regulation of chicken OSC proliferation by targeting and regulating the epigenetic modification of histone H3K27me3, thereby activating the YAP1-mediated Hippo signaling pathway. Specifically, the present invention adds the H3K27me3-specific inhibitor UNC1999 to the chicken OSC in vitro culture system. Through the synergistic effect of UNC1999 with the basic nutrients and functional cytokines in the culture system, the in vitro proliferation efficiency of chicken OSCs is significantly improved, providing a novel solution to the technical problem of difficult in vitro proliferation of poultry OSCs.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of the H3K27me3 epigenetic modification inhibitor UNC1999 in the preparation of a culture medium that promotes the proliferation of chicken ovarian germ cell stem cells.

[0008] In a second aspect, the present invention provides a culture medium for promoting the in vitro proliferation of chicken ovarian reproductive stem cells, the culture medium comprising a basal culture medium and an effective dose of the H3K27me3 inhibitor UNC1999, wherein the concentration of UNC1999 in the culture medium is 200 nM-500 nM.

[0009] Preferably, the concentration of UNC1999 in the culture medium is 500 nM.

[0010] Preferably, the basal culture medium is composed by adding the following components to every 50 mL of Knockout DMEM medium: 2-3mL FBS, 90-110μL chicken serum, 400-600μL NEAA (100×), 400-600μL GlutaMax (100×), 400-600μL GS nucleosides (100×), 400-600μL Antibiotic-antimycotic (100×), 500-700μL Sodium Pyruvate, 4-6μL β-mercaptoethanol, 3-5μL Citivin A, 9-11μL bFGF, 100-300μL Sodium heparin.

[0011] In a third aspect, the present invention provides a method for promoting the in vitro proliferation of chicken ovarian reproductive stem cells, comprising the following steps: (1) Chicken ovarian tissue was taken, the follicles and medulla of the ovarian tissue were removed, and the single-cell suspension was obtained after digestion with 0.25% trypsin. Ovarian reproductive stem cells (OSCs) were then enriched.

[0012] (2) The enriched OSCs were inoculated onto a chicken embryo fibroblast feeder layer and cultured for 45-56 h at 39 °C and 5% CO2 using the culture medium described in claim 2.

[0013] Preferably, in step (1), the obtained ovarian reproductive stem cells (OSCs) are purified chicken ovarian reproductive stem cells that have been positively identified by SSEA-1 and / or DDX4.

[0014] In a fourth aspect, the present invention provides the application of the above-described method for promoting the in vitro proliferation of chicken ovarian germ cells in the following (1) or (2): (1) Extend the egg-laying cycle of laying hens; (2) Improve ovarian function in laying hens.

[0015] In a fifth aspect, the present invention provides a method for extending the egg-laying cycle of laying hens, comprising the following steps: (1) Expand ovarian reproductive stem cells in vitro from target laying hens using the above method; (2) The expanded ovarian reproductive stem cells were transplanted into the recipient laying hen.

[0016] In a sixth aspect, the present invention provides a method for preserving superior poultry germplasm resources, comprising the following steps: expanding chicken ovarian germplasm stem cells in vitro using the above-described method for promoting the in vitro proliferation of chicken ovarian germplasm stem cells; and cryopreserving the expanded cells.

[0017] The beneficial effects of this invention are: This invention establishes a highly efficient technique for promoting the in vitro proliferation of chicken ovarian germ cell stem cells (OSCs) by targeting and regulating the epigenetic modification of histone H3K27me3 and activating the YAP1-mediated Hippo signaling pathway with the H3K27me3-specific inhibitor UNC1999. This technique possesses significant theoretical innovation value, industry application value, and economic and social benefits, as detailed below: (1) Breaking through the bottleneck of chicken OSC in vitro proliferation technology and achieving efficient cell expansion. This invention is the first to apply the H3K27me3 inhibitor UNC1999 to the chicken OSC in vitro culture system. By inhibiting the transcriptional repression of H3K27me3, it relieves the silencing of proliferation-related genes (YAP1, TEAD1, CDK6, etc.) and downregulates the expression of cell cycle repressors such as TP53 and CDKN2C, thus precisely activating the OSC proliferation pathway at the epigenetic level. Compared with the traditional unregulated basic culture system, this method can significantly improve the in vitro proliferation efficiency of chicken OSCs. Moreover, after SSEA-1 / DDX4 double positive identification, the positive rate of purified cells is ≥90%, which ensures the stem cell characteristics and differentiation potential of the cells and solves the long-standing technical pain point of chicken OSCs being difficult to proliferate and easily differentiate in vitro.

[0018] (2) Extending the egg-laying cycle of chickens and promoting the quality and efficiency of poultry farming. As chickens age, the decline in the number and function of oocytes (OSCs) in the ovary is the core reason for ovarian aging and loss of egg-laying performance. By activating the proliferation of OSCs, it is hoped that the aging process of the ovary can be slowed down, thus extending the egg-laying cycle and improving farming efficiency.

[0019] (3) Optimize the technology system for preserving superior poultry germplasm resources to achieve long-term preservation of genetic resources. Traditional poultry germplasm resource preservation relies on fertilized eggs or live animal preservation. Due to limitations such as preservation time, environmental conditions, and disease risks, fertilized egg preservation is difficult to achieve long-term stable preservation of genetic resources of superior breeds. The efficient propagation method for chicken OSCs established in this invention can be combined with programmed cryopreservation technology: thawed and revived cells can form functional eggs in the gonads of recipient chicken embryos. This breaks through the bottleneck of the difficulty in long-term preservation of fertilized eggs, significantly reduces the cost and space occupation of germplasm resource preservation, and provides an innovative solution for the protection of rare poultry breeds and the efficient propagation of superior genetic traits.

[0020] (4) Enriching the theoretical system of epigenetic regulation of reproductive stem cells and providing a reference for cross-species applications. This invention confirms the core role of H3K27me3 modification in the functional regulation of chicken OSCs, clarifies the proliferation regulation mechanism mediated by the Hippo signaling pathway, and fills the gap in research on epigenetic regulation of poultry reproductive stem cells. At the same time, this regulatory strategy can provide a reference for the in vitro culture of mammalian OSCs, and is expected to promote the technological development in fields such as the treatment of premature ovarian failure in humans and the improvement of reproductive performance in livestock, and has important cross-species theoretical research and translational application value. Attached Figure Description

[0021] Figure 1: In vitro culture and identification of OSCs derived from chicken ovaries at different ages; where SSEA-1: red; DDX4: green; DAPI: blue; scale bar = 200 μm.

[0022] Figure 2: Comparison of in vitro proliferation capacity of OSCs from chickens of different ages; among which, Figure 2 A: EdU staining and DDX4 immunofluorescence staining in cultured OSCs; arrows indicate EdU-DDX4 double-positive cells; Figure 2 B: Percentage of EdU-positive proliferating cells; EdU: red; DDX4: green; Scale bar = 200 μm; P<0.01; C: Comparison of CCK-8 proliferation assays of D60 OSCs and D400 OSCs in vitro; P<0.01.

[0023] Figure 3: Immunofluorescence detection of H3K27me3 modification levels in OSCs derived from chicken ovaries of different ages; Figure 3 A: Representative images of H3K27me3 staining in OSCs of chickens at different laying stages; Figure 3 B: H3K27me3 fluorescence intensity statistics; scale bar = 50 μm; P<0.001.

[0024] Figure 4: Effect of different concentrations of the H3K27me3 inhibitor UNC1999 on the in vitro proliferation of D400OSCs; P<0.05, P<0.01.

[0025] Figure 5: qPCR analysis of gene expression changes related to UNC1999 promoting chicken OSC proliferation; qPCR was performed using 2... −ΔΔCt The method was used for analysis; the error bars represent the standard error (SE) of the biological triplet analysis (n=3); the t-test was used to assess the differences. P<0.05. Detailed Implementation

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0027] As mentioned earlier, the proliferative capacity of oocyte stem cells (OSCs) in the ovaries of laying hens significantly declines during the later stages of egg production, and their in vitro culture efficiency is low, making it difficult to delay ovarian aging and extend the egg production cycle by supplementing OSCs. Therefore, establishing an in vitro treatment method that enables efficient proliferation of chicken OSCs while maintaining stem cell characteristics is a key prerequisite for solving the pain point of "short egg production cycle" in egg-laying hen farming. In view of this, the present invention has conducted an in-depth study on the proliferation mechanism of chicken OSCs. The present invention is the first to discover that the elevated level of H3K27me3 modification is a key factor leading to the decline in the proliferation of chicken OSCs in the late laying period. By targeting and inhibiting this modification with the H3K27me3 inhibitor UNC1999, the in vitro proliferation of chicken OSCs can be significantly promoted while maintaining their reproductive stem cell characteristics, thus proposing the present invention. The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0028] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein: Human Activin-A was purchased from Pipertech Biotechnology Co., Ltd. (catalog number 961201410); Human Basic Fibroblast Growth Factor (bFGF) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (catalog number P00032); β-mercaptoethanol, sodium pyruvate, and sodium heparin were purchased from Beijing Solarbio Science & Technology Co., Ltd. (catalog numbers M8210, S8080, and H1025, respectively); GS nucleosides were purchased from Merck Millipore China Co., Ltd. (catalog number ES-008-D); KO-DMEM Culture media were purchased from Thermo Fisher Scientific (catalog number 10829018); antibacterial-antimycotic agents, glutamine additive (GlutaMax), and non-essential amino acid solution (NEAA) were purchased from Thermo Fisher Scientific (catalog numbers 15240062, 35050061, and 11140050, respectively); defined fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific (catalog number 10270106); chicken serum was purchased from Beijing Solarbio Science & Technology Co., Ltd. (catalog number S9080); and the H3K27me3 inhibitor UNC1999 was purchased from MedChemExpress (catalog number HY-12321), CAS number 1415562-82-1. Example 1: Preparation of basal culture medium for ovarian germline stem cells (OSCs) Take 50 mL of sterile Knockout DMEM and add it to a sterile centrifuge tube. Add each component according to the following dosage and mix gently: 2.5 mL FBS, 100 μL chicken serum, 500 μL NEAA (100×), 500 μL GlutaMax (100×), 500 μL GS nucleosides (100×), 500 μL Antibiotic-antimycotic (100×), 600 μL Sodium Pyruvate, 5 μL β-mercaptoethanol, 4 μL Acitivin A, 10 μL bFGF, 200 μL Sodium heparin; The prepared basal culture medium was filtered and sterilized using a 0.22 μm bacterial filter; the centrifuge tubes were sealed with sealing film and stored in a refrigerator at 4°C.

[0029] The basic culture medium prepared in this embodiment can effectively support the adhesion and cloning of chicken OSCs, and serves as a benchmark system for subsequent functional regulation by adding UNC1999.

[0030] Example 2: Culture of Chicken Embryo Fibroblast Feeder Layer (1) The fertilized eggs of Luhua chickens were incubated for 6 to 8 days at a temperature of 37.8℃ and a humidity of 60%. The eggs were cleaned with 75% alcohol, and the shells were carefully cracked open in a laminar flow hood. The chicken embryos were carefully removed into a sterile culture dish with sterile ophthalmic curved tweezers and washed 3 times with PBS. (2) Place the chicken embryo in a new sterile culture dish and remove the limbs, head, tail, gonads and internal organs of the chicken embryo with sterile ophthalmic pointed forceps. Wash the remaining tissues three times with PBS. (3) Use sterile ophthalmic surgical scissors to repeatedly cut the remaining tissue into minced meat, and then transfer it to a 1.5 mL sterile centrifuge tube; (4) Preheat the 0.25% trypsin-0.04% EDTA digestion solution in a 37°C water bath for 20 minutes. Take 0.5 mL of the preheated 0.25% trypsin-0.04% EDTA digestion solution and add it to the 1.5 mL sterile centrifuge tube in (3) above. Digest at room temperature for 15 minutes, and gently pipette continuously until no obvious precipitate appears. (5) Add an equal volume of RPMI-1640 culture medium containing 10% FBS to stop digestion, and gently pipette to mix the cell suspension. (6) Filter the mixed cell suspension through a 40 μm filter membrane, collect the filtered cell suspension in a 15 mL sterile centrifuge tube, centrifuge at 1,000 rpm / min for 5 min, discard the supernatant, and collect the cell pellet. (7) Resuspend the cell pellet from (6) above in CEF medium at 2×10⁻⁶. 5 10 cells / well were seeded into 24-well plates and incubated at 39°C in a 5% CO2 incubator.

[0031] A successfully prepared CEF feeder layer should exhibit a uniform morphology and well-adhered monolayer of cells. Providing crucial physical support and a suitable nutritional microenvironment for subsequent inoculation of OSCs is one of the key components of this invention for achieving long-term in vitro culture and expansion of chicken OSCs.

[0032] Example 3: Isolation, purification, and identification of basic characteristics of OSCs from late-laying chickens 1. Isolation and purification of OSCs OSCs were isolated and cultured from the ovaries of 60-day-old and late-laying (400-day-old) chickens, respectively. The specific isolation and culture methods are as follows: (1) Collect the ovaries of four 400-day-old Luhua chickens and place them in a 60 mm sterile culture dish. Wash them three times with PBS. (2) Place the ovarian tissue into a new sterile culture dish and remove the follicles and medulla of the ovarian tissue with sterile ophthalmic forceps; wash repeatedly with PBS during this period; (3) After washing the remaining ovarian tissue three times in PBS, use sterile ophthalmic surgical scissors to repeatedly cut the remaining ovarian tissue into minced meat until it is minced, and then transfer it to a 4 mL sterile centrifuge tube; (4) Preheat the 0.25% trypsin-0.04% EDTA digestion solution in a 37°C water bath for 20 minutes. Take 1 mL of the 37°C preheated 0.25% trypsin-0.04% EDTA digestion solution and add it to the 4 mL sterile centrifuge tube in (3) above. Digest at room temperature for 15 minutes, and continuously and gently pipette until no obvious tissue block is visible to the naked eye. (5) When most of the tissue has been dispersed into single cells, add an equal volume of OSCs basal culture medium (prepared in Example 1) to terminate digestion; (6) OSCs were enriched by immunomagnetic bead sorting using SSEA-1 antibody conjugated with anti-mouse IgM magnetic beads; (7) The enriched OSCs were inoculated into a 24-well plate that had been pre-laid and grown to a density of about 60% CEF feeder layer (prepared in Example 2), OSC basic culture medium was added, and the 24-well plate was placed in a 39°C, 5% CO2 incubator for culture.

[0033] 2. Identification of stem cell and germ cell characteristics of OSCs The cultured OSCs were subjected to immunofluorescence double staining. Mouse anti-SSEA-1 antibody (stem cell marker) and rabbit anti-DDX4 antibody (germ cell marker) were used as primary antibodies, and the cells were labeled with red and green fluorescent secondary antibodies, respectively. The cell nuclei were counterstained with DAPI.

[0034] Results and Conclusions: Fluorescence microscopy revealed that, regardless of whether the OSCs were from 60-day-old or 400-day-old cells, the vast majority of cells simultaneously exhibited clear positive signals for both SSEA-1 (red) and DDX4 (green), and the signal localization was accurate. Figure 1 The CEF feeder layer cells showed negative staining. The results indicate that the cell population isolated and cultured using this method has high purity and possesses both stem cell and germ cell characteristics.

[0035] 3. Comparison of in vitro proliferation capacity of OSCs of different ages (1) Objective: To compare the differences in basal proliferation capacity of OSCs of chickens of different ages.

[0036] (2) Methods: EdU detection: The two groups of OSCs were co-incubated with the EdU probe, and then DDX4 immunofluorescence staining was performed. The proportion of EdU and DDX4 double positive cells to the total number of DDX4 positive cells was counted.

[0037] CCK-8 assay: Two groups of cells were seeded at the same density, and CCK-8 assay was performed on days 1, 2 and 3 of culture. The absorbance value at 450 nm (OD450) was measured to assess cell number growth.

[0038] (3) Results and conclusions: EdU detection showed that EdU-DDX4 double-positive cells could be observed in both OSCs derived from chicken ovaries with strong proliferative capacity (60 days old) and weak proliferative capacity (400 days old), indicating that the OSCs cultured in vitro were in an active DNA synthesis state and had proliferative capacity (Figure 2A).

[0039] Further statistical analysis of the proportion of EdU-positive cells in the total number of OSCs revealed that the proportion of EdU-positive cells in OSCs derived from 60-day-old chicken ovaries was significantly higher than that in OSCs derived from 400-day-old chickens (P<0.05, Figure 2B), suggesting that the proliferative activity of OSCs in chicken ovaries decreases significantly with increasing age.

[0040] CCK-8 assay results showed that the proliferation rate of OSCs derived from 60-day-old plants was significantly faster than that derived from 400-day-old plants (P<0.01, Figure 2C), further confirming that the in vitro expansion capacity of chicken ovarian germ cells was significantly limited under conditions of weak proliferation.

[0041] Example 4: Immunofluorescence detection of H3K27me3 modification level in OSCs of different ages 1. Experimental Objective To investigate whether the level of the epigenetic modification H3K27me3 is associated with the decline in the proliferative capacity of chicken OSCs.

[0042] 2. Cell samples: 60-day-old (D60) and 400-day-old (D400) chicken OSCs were isolated and cultured according to the method in Example 3.

[0043] 3. Experimental Procedure D60 and D400 OSCs were seeded separately into 24-well plates with a CEF feeder layer. The medium was changed daily. After cell purification and depletion of the CEF feeder layer, the cells were confluent at a density of approximately 60%–70% for immunofluorescence staining. (1) Remove the cells from the 39°C, 5% CO2 incubator, discard the culture medium in the cell culture dish (all the following operations are performed in the cell culture dish), and wash with PBS once for 5 min each time; (2) Add 1 mL of 4% paraformaldehyde and fix at room temperature for 10 min, then wash twice with PBS for 5 min each time; (3) Add 500 μL of 0.5% Triton X-100 and permeate at room temperature for 10 min, then wash twice with PBS for 5 min each time; (4) Add 500 μL of 10% goat blocking serum and block at room temperature for 1.5 h; (5) Add 500 μL of H3K27me3 primary antibody (1:500) and incubate overnight at 4°C; wash twice with PBS, 5 min each time; (6) Add 400 μL of Alexa Fluor 488 donkey anti-rabbit IgG secondary antibody (1:500), incubate at room temperature in the dark for 1.5 h, and wash twice with PBS for 5 min each time; (7) Add 300 μL of DAPI to stain the cell nuclei, incubate in a 39℃ incubator for 10 min, wash once with PBS for 5 min each time, and observe with a fluorescence microscope.

[0044] 4. Results and Conclusions To investigate the differences in H3K27me3 epigenetic modification levels in chicken ovarian germ cell stem cells (OSCs) at different proliferative stages, this study performed immunofluorescence staining analysis of H3K27me3 in chicken ovarian germ cell stem cells (OSCs) with strong proliferative capacity (60 days old) and weak proliferative capacity (400 days old) cultured in vitro. The results showed that ( Figure 3In both groups of OSCs, the H3K27me3 signal was localized in the cell nucleus. Compared with D60 OSCs, the intensity of the green fluorescence signal in the nucleus of D400 OSCs was significantly enhanced. Quantitative analysis using image analysis software confirmed that the average fluorescence intensity of H3K27me3 in the D400 group OSCs was significantly higher than that in the D60 group (P<0.001). The conclusions of this experiment indicate that the repressive epigenetic marker H3K27me3 accumulates significantly in late-laying chicken OSCs with declining proliferative capacity. This suggests that abnormally elevated H3K27me3 modification levels may be closely related to the decline in OSC proliferative function, thus establishing it as a potential key target for regulating chicken OSC proliferation.

[0045] Example 5: The promoting effect of UNC1999 on OSC proliferation in late-laying hens and determination of the optimal concentration 1. Experimental Objective To verify whether targeted inhibition of H3K27me3 modification can promote the proliferation of OSCs in late-laying hens, and to determine the optimal concentration of the small molecule inhibitor UNC1999 through dose-effect experiments.

[0046] 2. Experimental Methods 400-day-old chicken OSCs isolated and cultured according to the method in Example 3 were randomly divided into four groups. The basal culture medium was treated with 0 nM (control), 100 nM, 200 nM, and 500 nM UNC1999, respectively. Cell proliferation was assessed using the CCK-8 assay at 24, 48, and 72 hours after treatment, and OD was measured. 450 Value. The experiment was independently repeated 3 times.

[0047] like Figure 4 As shown, the promoting effect of UNC1999 on OSC proliferation was concentration- and time-dependent. At all time points, the 500 nM treatment group showed the most significant proliferative effect. Especially after 48 hours of treatment, the OD of the 500 nM group was significantly higher. 450 The value reached 1.50±0.03, which was 2.4 times that of the control group (0.62±0.02), and the difference was extremely significant (P<0.01).

[0048] These results indicate that the H3K27me3-specific inhibitor UNC1999 can effectively promote the in vitro proliferation of OSCs from late-laying chickens with declining proliferative capacity. Based on comprehensive evaluation, 500 nM was determined to be the optimal concentration of UNC1999 for promoting OSC proliferation in this culture system.

[0049] Example 6: UNC1999 affects H3K27me3 modification levels and proliferation-related gene expression in chicken OSCs by regulating the YAP1-mediated Hippo signaling pathway. To further clarify whether changes in H3K27me3 modification levels regulate OSC proliferation by affecting YAP1, a key regulator of the Hippo signaling pathway, this study performed qPCR analysis on D400 OSCs treated with UNC1999.

[0050] 1. Experimental Methods After treating 400-day-old chicken OSCs with 500 nM UNC1999 for 48 hours, total RNA was extracted from the cells and reverse transcribed into cDNA. Quantitative PCR was used, with GAPDH as an internal control, to detect the expression levels of core components of the Hippo signaling pathway and cell cycle-related genes.

[0051] 2. Experimental Results qPCR results showed that inhibition of H3K27me3 modification significantly upregulated the expression level of YAP1, a core transcriptional regulator of the Hippo signaling pathway. Simultaneously, the expression levels of its synergist TEAD1 and upstream regulatory genes STRN, WWC3, and STK24 were all increased to varying degrees. Consistent with the upregulation of YAP1 expression, the expression of cell cycle progression-related genes CDK6 and Cyclin D significantly increased, while the expression of cell cycle repressors TP53, CDKN2C, and CDKN1C significantly decreased (Figure 5).

[0052] The above results indicate that the H3K27me3 inhibitor UNC1999, by relieving the transcriptional repression of YAP1, reactivates YAP1 as a core hub of the Hippo signaling pathway, thereby synergistically regulating the expression of downstream cell cycle-related genes and ultimately promoting the in vitro proliferation of chicken ovarian reproductive stem cells.

[0053] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted. Equivalent substitutions and improvements should all be included within the scope of protection of this application.

Claims

1. Application of H3K27me3 epigenetic modification inhibitor UNC1999 in the preparation of culture medium that promotes the proliferation of chicken ovarian germ cell stem cells.

2. A culture medium for promoting the in vitro proliferation of chicken ovarian reproductive stem cells, characterized in that, The culture medium contains a basal culture medium and an effective dose of the H3K27me3 inhibitor UNC1999, wherein the concentration of UNC1999 in the culture medium is 200 nM-500 nM.

3. The culture medium according to claim 2, characterized in that, The concentration of UNC1999 in the culture medium was 500 nM.

4. The culture medium according to claim 2, characterized in that, The basal culture medium is composed by adding the following components to every 50 mL of Knockout DMEM medium: 2-3mL FBS, 90-110μL chicken serum, 400-600μL NEAA (100×), 400-600μL GlutaMax (100×), 400-600μL GS nucleosides (100×), 400-600μL Antibiotic-antimycotic (100×), 500-700μL Sodium Pyruvate, 4-6μL β-mercaptoethanol, 3-5μL Acitivin A, 9-11μL bFGF, 100-300μL Sodium heparin.

5. A method for promoting the in vitro proliferation of chicken ovarian reproductive stem cells, characterized in that, Includes the following steps: (1) Chicken ovarian tissue was taken, the follicles and medulla of the ovarian tissue were removed, and the single-cell suspension was obtained after digestion with 0.25% trypsin. Ovarian reproductive stem cells (OSCs) were then enriched. (2) The enriched OSCs were inoculated onto a chicken embryo fibroblast feeder layer and cultured for 45-56 h at 39 °C and 5% CO2 using the culture medium described in claim 2.

6. The method according to claim 5, characterized in that, In step (1), the obtained ovarian reproductive stem cells (OSCs) are purified chicken ovarian reproductive stem cells that have been positively identified by SSEA-1 and / or DDX4.

7. The application of the method of claim 5 in either (1) or (2): (1) Extend the egg-laying cycle of laying hens; (2) Improve ovarian function in laying hens.

8. A method for extending the egg-laying cycle of laying hens, characterized in that, Includes the following steps: (1) Expanding ovarian reproductive stem cells in vitro from target laying hens using the method described in claim 5; (2) The expanded ovarian reproductive stem cells were transplanted into the recipient laying hen.

9. A method for preserving superior poultry germplasm resources, characterized in that, Includes the following steps: Chicken ovarian reproductive stem cells were expanded in vitro using the method described in claim 5; the expanded cells were then cryopreserved.

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