System for promoting in-vitro differentiation of chicken oogonial stem cells and forming blastocysts

Abstract

The invention discloses a system for promoting in-vitro differentiation of chicken oogonial stem cells and forming blastocysts, and belongs to the technical field of stem cell differentiation culture. According to the invention, retinoic acid (RA) and vitamin C (VC) are added on the basis of an oogonial stem cell culture medium to induce cells to start meiosis, and then follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), glial cell-derived neurotrophic factor (GDNF), progesterone (P4) and rosiglitazone are further added on the basis of the culture medium containing RA and VC to promote the recovery of meiosis, so that the effect of promoting meiosis is achieved. A complete system that the chicken oogonial stem cells are differentiated in vitro and form blastocysts is creatively established by inducing formation of secondary oocytes, direct evidence is provided for in-vitro reconstruction of poultry ovum generation, and a technical basis and application prospects are provided for preservation of female germ cells and large-scale production of the chicken oocytes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of stem cell differentiation culture, in particular to a system for promoting in-vitro differentiation of chicken oogonial stem cells and formation of blastula. BACKGROUND

[0002] In sexually reproducing animals, the generation and development of germ cells are of great significance for the propagation and continuation of the species. The traditional view believes that female animals have a limited number of oocytes at birth, and such cells cannot be regenerated after birth (Female mice lack adult germ-line stem cells but sustain oogenesis using stable primordial follicles. PNAS). However, in recent years, research has found that there is still a kind of cells with proliferation and differentiation potential in the ovary of adult female animals, i.e. oogonial stem cells (OSCs). Such cells can differentiate to generate new oocytes, thereby providing a new source of cells for the replenishment of primary follicles (Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature; Identification of oogonial stem cells in chicken ovary. Cell Proliferation). This discovery provides a new idea for in-depth study of the cytological and molecular mechanisms of oogenesis, and lays a theoretical foundation for the maintenance of female fertility and in-vitro oocyte reconstruction.

[0003] Currently, in vitro induction and differentiation system based on oocyte stem cells has shown good prospects in mammals, which not only provides potential treatment ideas for female ovarian function decline and reduced fertility, but also can be used to construct a complete in vitro oogenesis model, providing an experimental platform for mechanism research and drug screening of reproductive development related diseases (Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women. Nature Medicine; Human GV oocytes generated by mitotically active germ cells obtained from follicular aspirates. Scientific Reports. Comparison of different in vitro differentiation conditions formurine female germline stem cells. Cell Proliferation). In addition, OSCs have the advantages of long-term in vitro expansion potential and relatively abundant source, and are expected to realize large-scale and efficient oocyte generation system, which has important practical value in animal breeding, germplasm preservation, and reproductive resource reconstruction.

[0004] In the field of poultry, there is a significant yolk deposition process during the maturation of avian oocytes, which is fundamentally different from mammals. This process is difficult to effectively replicate in vitro, limiting the progress of avian oocyte research in vitro. Currently, pre-ovulatory follicles can be obtained by dissection to isolate oocytes for maturation analysis (Chick production by in vitro fertilization of the fowl ovum. Journal of Reproduction and Fertility; Fertilization and development of quail oocytes after intracytoplasmic sperm injection. Biology of Reproduction), but this method has many limitations such as high cost, difficult operation, fragile yolk structure, and unsustainable "killing chickens for eggs".

[0005] Therefore, there is an urgent need to establish an in vitro culture system that can simulate the in vivo ovarian microenvironment and support the differentiation of germ cells into functional oocytes, in order to obtain oocytes with developmental potential without relying on animal sacrifice. The establishment of such a system not only has significant theoretical value but also broad application prospects in areas such as avian germplasm resource conservation, improving breeding efficiency of breeding poultry, and the conservation of endangered birds. However, there are currently no research reports on the in vitro reconstruction of oocytes from avian oogonial stem cells, and related technologies remain largely undeveloped, requiring urgent technological breakthroughs. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide a system for promoting the in vitro differentiation of chicken oocyte primordial stem cells into blastocysts. This invention innovatively establishes a complete system for the in vitro differentiation of chicken oocyte primordial stem cells into blastocysts, providing direct evidence for the in vitro reconstruction of avian oocyte development, and also providing a technical foundation and application prospect for the preservation of female germ cells and the large-scale production of chicken oocytes.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a culture medium for promoting the in vitro differentiation of chicken oocyte stem cells, the culture medium comprising: a meiosis induction medium and an oocyte maturation induction medium; The meiosis induction medium is a basal medium for oogonial stem cells with added retinoic acid (RA) and vitamin C (VC).

[0008] During embryonic development, RA induces germ cells to enter meiosis in the gonads and initiates oogenesis, while VC ensures the smooth progress of meiosis by regulating DNA methylation reprogramming. The oocyte maturation induction medium is a meiosis induction medium supplemented with follicle-stimulating hormone (FSH), human chorionic gonadotropin (HCG), glial cell-derived neurotrophic factor (GDNF), progesterone (P4), and rosiglitazone.

[0009] FSH promotes follicle growth and oocyte meiosis; HCG induces ovulation and promotes oocyte maturation; GDNF maintains oocyte microenvironment homeostasis, promoting its growth and development; P4 plays a key regulatory role in meiosis recovery and nuclear maturation; and rosiglitazone improves energy metabolism and cell quality, enhancing oocyte maturation rate and developmental potential. The synergistic effect of these factors effectively promotes oocyte development and maturation.

[0010] The basal culture medium for the oogonial stem cells was KO-DMEM containing 10 ng / mL Acitivin A, 10 ng / mL bFGF, 0.1 mM β-mercaptoethanol, 1.2 mM Sodium Pyruvate, 1X Antibiotic-antimycotic, 100 µg / mL Sodium heparin, 1X GS nucleosides, 1X GlutaMax, 1X NEAA, 0.2% chicken serum, and 5% fetal bovine serum. All concentrations mentioned above are final concentrations in the prepared ordinary chicken oogonial stem cell culture medium.

[0011] Preferably, in the meiosis induction medium, the final concentration of RA added is 0.5-1.5 μM, and the final concentration of VC added is 100-200 μM.

[0012] More preferably, in the meiosis induction medium, the final concentration of RA added is 1 μM, and the final concentration of VC added is 150 μM.

[0013] Preferably, in the oocyte maturation induction medium, the final concentration of FSH is 0.1-0.3 IU / mL, the final concentration of HCG is 1-3 IU / mL, the final concentration of GDNF is 40-60 ng / mL, the final concentration of P4 is 0.5-1.5 ng / mL, and the final concentration of rosiglitazone is 15-25 μM.

[0014] More preferably, in the oocyte maturation induction medium, the final concentration of FSH is 0.2 IU / mL, the final concentration of HCG is 2 IU / mL, the final concentration of GDNF is 50 ng / mL, the final concentration of P4 is 1 ng / mL, and the final concentration of rosiglitazone is 20 μM.

[0015] A second aspect of the present invention provides the use of the above-described culture medium in (1) or (2) as follows: (1) Promote the in vitro differentiation of chicken oocyte stem cells into secondary oocytes; (2) Large-scale production of chicken oocytes.

[0016] A third aspect of the present invention provides a method for promoting the in vitro differentiation of chicken oocyte stem cells into secondary oocytes, comprising the following steps: (1) Chicken oocyte stem cells were seeded into chicken embryo fibroblast feeder layer, and meiosis induction medium was added for culture. The medium was changed every 24 hours. (2) After culturing in meiosis induction medium for 3 days, the meiosis induction medium was replaced with oocyte maturation induction medium and cultured until the first polar body appeared.

[0017] The expulsion of the first polar body is direct evidence that the primary oocyte has completed its first meiotic division (MI). Under in vitro culture conditions, after the oocyte resumes division from prophase I (diplotene), it undergoes processes such as chromosome condensation and separation of homologous chromosomes, ultimately expelling the first polar body. This event indicates that the primary oocyte has broken through MI arrest and transformed into the secondary oocyte stage; therefore, the release of the first polar body is a direct marker of secondary oocyte formation.

[0018] Preferably, in steps (1) and (2), the cultivation conditions are: 39℃ and 5% CO2.

[0019] A fourth aspect of the present invention provides a method for promoting the in vitro differentiation of chicken oocyte stem cells into blastocysts, comprising the following steps: (1) Chicken oocyte stem cells were seeded into chicken embryo fibroblast feeder layer, and meiosis induction medium was added for culture. The medium was changed every 24 hours. (2) After culturing in meiosis induction medium for 3 days, the meiosis induction medium is replaced with oocyte maturation induction medium and cultured until the first polar body appears, forming secondary oocytes; (3) Secondary oocytes were placed in activation solution for parthenogenetic activation. After activation, they were transferred to M199 medium containing 10% FBS for further culture.

[0020] Preferably, in step (3), the activation solution is prepared by adding 10% fetal bovine serum (FBS), 20mM strontium chloride (SrCl2) and 5µg / mL cytochalasin B (CB) to M199 culture medium.

[0021] Preferably, in step (3), the culture conditions for parthenogenetic activation are: incubation at 39°C for 4 hours in a humid environment containing 5% CO2.

[0022] The beneficial effects of this invention are: (1) Female germ cells are precursors to oocytes, and oogenesis is crucial for animal reproduction. This invention develops a complete system for the in vitro differentiation of chicken oocyte stem cells into blastocysts. Oocyte-like cells (OLCs) induced by this system possess meiotic and oocyte characteristics. Activation of these secondary oocytes derived from germ cells with strontium chloride enables them to undergo cleavage and develop into blastocysts. This not only provides direct evidence for the in vitro reconstruction of avian oogenesis but also provides a technical foundation and application prospect for the preservation of female germ cells and the large-scale production of chicken oocytes.

[0023] (2) This invention provides an in vitro induced differentiation system for oogonial stem cells from the ovaries of avian animals. This system addresses the structural characteristic of avian oocytes during maturation, which involves the deposition of a large amount of yellow yolk, overcoming the technical bottleneck of artificially simulating yolk accumulation in existing technologies. Currently, assessing the developmental capacity of avian oocytes typically relies on obtaining oocytes from pre-ovulatory follicles. This method requires ovarian dissection, which is not only costly but also complex and prone to failure due to the fragility and instability of the yolk structure. This invention achieves efficient induced differentiation in oogonial stem cells derived from adult chicken ovaries for the first time, successfully supporting their in vitro differentiation into functional oocytes. The induction system established by this invention provides a feasible technical path for the in vitro construction of avian oocytes, with promising application prospects, particularly suitable for germ cell preservation, reproductive performance research, and the protection of endangered avian resources.

[0024] (3) In this invention, we successfully established the first in vitro parthenogenesis activation system for oocytes derived from avian germ cells, and demonstrated that strontium ion treatment can effectively activate induced oocyte-like cells and promote their subsequent development into blastocyst-like structures. Since the embryos produced by parthenogenesis contain only maternal genetic material, this system provides a unique method for directly preserving the maternal genome and helps to create a "pure maternal gene bank." This method provides a new technical strategy for the protection and genetic improvement of endangered or rare bird species. Attached Figure Description

[0025] Figure 1 Isolation, culture and identification of chicken oocyte stem cells. Figure 1 Figure A shows the EdU-DDX4 co-staining identification of cultured chicken oocyte stem cells. In this culture system, chicken oocyte stem cells maintain stable proliferation capacity. Scale bar = 25 μm. Figure 1 Figure B in the image shows the co-staining identification of cultured chicken oocyte stem cells with SSEA1-DDX4. In this culture system, chicken oocyte stem cells continuously express the pluripotent stem cell marker SSEA-1 and the germ cell-specific marker DDX4. Scale bar = 25 μm.

[0026] Figure 2 Retinoic acid (RA) and vitamin C (VC) induce oogonial stem cells to develop into oocyte-like cells and arrest them in prophase I of meiosis. Figure 2 Figure A shows oocyte-like cells (OLCs) with a diameter greater than 22µm appearing after the addition of RA and VC to the culture medium. The red arrows point to oocyte-like cells, and the white arrows point to undifferentiated oogonial stem cells. Scale bar = 20μm. Figure 2 Figure B shows that the expression levels of meiosis marker genes (STRA8, SCP3, SpO11, and DMC1) are increased after cell induction. Figure 2 Figure C in the diagram shows the identification of induced oocyte-like cells co-stained with γH2AX-DDX4 in the induction system. Scale bar = 5μm. Figure 2 Figure D in the diagram shows the SCP3-DDX4 co-staining identification of induced oocyte-like cells in the induction system. Scale bar = 5 μm.

[0027] Figure 3 Hormone-induced meiosis resumes and develops to the secondary oocyte stage. Figure 3 Figure A shows that after replacing the meiosis induction medium with an oocyte maturation induction medium, the expression levels of oocyte marker genes (NOBOX, GDF9, BMP15, and LHX8) increased. Figure 3 Figure B in the diagram shows the NOBOX-DDX4 co-staining identification of induced oocyte-like cells after the meiosis induction medium was replaced with an oocyte maturation induction medium. Scale bar = 5 μm. Figure 3 Figure C in the figure shows the identification of BMP15-DDX4 co-stained oocyte-like cells after the meiosis induction medium was replaced with oocyte maturation induction medium. Scale bar = 5 μm. Figure 3 In Figure D, after replacing the meiosis induction medium with the oocyte maturation induction medium, the induced cells showed the appearance of the first polar body. The white arrow points to the first polar body. Scale bar = 5 μm.

[0028] Figure 4 Secondary oocytes derived from germ cells develop to the blastocyst stage after parthenogenetic activation. Representative images of cleavage cells after parthenogenetic activation induced by strontium chloride (SrCl2), scale bar = 5 μm. Detailed Implementation

[0029] 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.

[0030] As mentioned earlier, in sexually reproducing animals, the generation and development of germ cells are crucial for the reproduction and continuation of the species. Avian oocytes exhibit a distinct yolk deposition process during maturation, fundamentally different from that in mammals. This process is difficult to replicate effectively in vitro, limiting the progress of in vitro oocyte research in birds.

[0031] In view of this, the present invention provides an in vitro induction system that can effectively support the differentiation of avian germ cells, especially oogonial stem cells, into functional oocytes with developmental potential. This system, without relying on animal sacrifice, simulates the key signals and microenvironment required for oocyte development, promoting the formation of oocyte-like cells from germ cells, and providing a reliable technical pathway for the in vitro reconstruction of avian oocytes. Therefore, based on the above background and practical needs, the present invention proposes and establishes an innovative and practical in vitro induced differentiation culture system for avian germ cells, filling a gap in related technologies in this field.

[0032] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. If specific experimental conditions are not specified in the embodiments, they are generally based on conventional conditions or conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments, unless otherwise specified, can be obtained commercially. Wherein: Human activator A (Acitivin A) was purchased from Pipertech Biotechnology Co., Ltd.; GS nucleosides (nucleotide supplement) and retinoic acid (RA) were purchased from Merck Millipore China Co., Ltd.; chicken serum, sodium heparin, human basic fibroblast growth factor (bFGF), vitamin C (VC), β-mercaptoethanol, sodium pyruvate, follicle-stimulating hormone (FSH), human chorionic gonadotropin (HCG), strontium chloride (SrCl2), and cytochalasin B were purchased from Beijing Solarbio Science & Technology Co., Ltd.; KO-DMEM medium, antibiotic-antimycotic, 1640 medium, GlutaMax (glutamine additive), and NEAA were purchased from Thermo Fisher Scientific Co., Ltd.; fetal bovine serum was purchased from Shanghai Wenren Biotechnology Co., Ltd.; progesterone (P4) was purchased from Shanghai Maclean Biotechnology Co., Ltd.; and glial cell-derived neurotrophic factor (GDNF) was purchased from Shanghai Baisai Biotechnology Co., Ltd.

[0033] Example 1: Preparation of culture medium for promoting in vitro differentiation of chicken oocyte stem cells 1. Basic culture medium for oogonial stem cells: The basal culture medium for oogonial stem cells was KO-DMEM containing 10 ng / mL Acitivin A, 10 ng / mL bFGF, 0.1 mM β-mercaptoethanol, 1.2 mM Sodium Pyruvate, 1X Antibiotic-antimycotic, 100 µg / mL Sodium heparin, 1X GS nucleosides, 1X GlutaMax, 1X NEAA, 0.2% (v / v) chicken serum and 5% (v / v) fetal bovine serum.

[0034] The above concentration parameters are all final concentrations in the prepared chicken oocyte stem cell culture medium.

[0035] 2. Meiosis induction medium: Meiosis induction medium is prepared by adding RA to the basal medium of oogonial stem cells at a final concentration of 1 µM and VC at a final concentration of 150 µM.

[0036] 3. Oocyte maturation induction culture medium The oocyte maturation induction medium was prepared by adding FSH to a final concentration of 0.2 IU / mL, HCG to a final concentration of 2 IU / mL, GDNF to a final concentration of 50 ng / mL, P4 to a final concentration of 1 ng / mL, and rosiglitazone to a final concentration of 20 μM to the meiosis induction medium.

[0037] Example 2: Culture of Chicken Embryo Fibroblast Feeder Layer (1) After fertilized eggs have been incubated for 6-8 days, wash the eggs with 75% alcohol, crack open the eggshell, remove the embryo with curved tweezers and place it in a culture dish, and wash with PBS 3 times. (2) Remove the head, tail, limbs and internal organs of the chicken embryo, and wash the remaining tissue three times with PBS; (3) Use ophthalmic surgical scissors to thoroughly cut the remaining tissue into a uniform paste, and transfer it to a 1.5 mL centrifuge tube; (4) Preheat 0.25% trypsin in a 37°C water bath for 10 min, take 0.5 mL of the preheated 0.25% trypsin and add it to a centrifuge tube. Digest at room temperature for 15 min, while intermittently and gently pipetting until there is no obvious precipitate in the suspension. (5) Add an equal volume of RPMI 1640 culture medium containing 10% fetal bovine serum (FBS) to terminate the enzymatic reaction, and gently pipette to mix again to form a cell suspension; (6) Filter the cell suspension through a 40 μm cell sieve, collect the effluent, and then centrifuge at 1,000 rpm for 5 min. Discard the supernatant and collect the cell pellet. (7) Resuspend the cell pellet in RPMI-1640 medium containing 10% FBS, seed it in 24-well plates, and continue to culture at 39°C in a 5% CO2 incubator.

[0038] Example 3: Isolation, Culture and Identification of Chicken Oocyte Stem Cells 1. Isolation and culture of chicken oocyte progenitor stem cells: The chicken breed used in the experiment was a 60-day-old Lufeng chicken (purchased from Jining, Shandong; this invention is not limited to this breed). After harvesting the chicken ovaries, they were rinsed 5 times in PBS buffer containing secondary antibodies. The ovarian tissue was then minced and added to 0.25% trypsin solution, and digested at 37°C for 15-20 min, gently agitating the tissue several times every 5 min to promote thorough dispersion. After digestion, the reaction was immediately stopped and the cells were centrifuged. The supernatant was discarded, and the cell pellet was retained. The pellet was resuspended in magnetic bead-activated cell sorting buffer, and Anti-SSEA1 antibody (stage-specific embryonic antigen-1, SSEA-1, purchased from Santa Cruz Antibody Company, sc-21702) was added and thoroughly mixed, then incubated for 10-20 min. Secondary antibody magnetic beads (Anti-mouse IgM Dynabeads, 11039D, purchased from Invitrogen) were then added, and the pellet was incubated for a total of 20-30 min. The pellet was then eluted with magnetic bead-activated cell sorting buffer. Finally, the sorted cells were resuspended in chicken oocyte stem cell basal culture medium, collected and spread on chicken embryo fibroblast feeder layer (prepared in Example 2), and cultured in a 39°C, 5% CO2 incubator.

[0039] 2. Identification of chicken oocyte progenitor stem cells: (1) Proliferation detection: The isolated chicken oogonial stem cells were transferred into 24-well plates with a chicken embryo fibroblast feeder layer, and oogonial stem cell basal culture medium was added. The plates were then cultured at 39°C in a 5% CO2 incubator. The cell status of the in vitro cultured oogonial stem cells was observed under a microscope. When the cells were in good condition, EdU (purchased from Thermo Fisher Scientific) was added to the oogonial stem cell ordinary culture medium at a final concentration of 10 μM. The plates were then incubated at 39°C in a 5% CO2 incubator for 6-10 h.

[0040] After EdU incubation, cells were fixed with 4% paraformaldehyde solution for 15 min at room temperature, followed by washing three times with phosphate-buffered saline (PBS). The fixed cells were then permeabilized with 0.5% Triton X-100 at room temperature for 10 min, followed by washing three times with PBS to remove residue. After permeabilization, 10% goat serum blocking solution was added, and the cells were blocked at room temperature for 1.5 h. Then, primary antibody DDX4 (purchased from Abogen Biosciences, catalog number ab13840, dilution 1:200) was added, and the cells were incubated overnight at 4°C. The next day, after washing three times with PBS, secondary antibody Alexa Fluor 488-labeled donkey anti-rabbit IgG (purchased from Wuhan Saiwei Biotechnology Co., Ltd., dilution 1:400) was added, and the cells were incubated at room temperature in the dark for 1 h. After incubation, the cells were washed three times with PBS, EdU dye was added, and the cells were incubated at room temperature for 30 min, followed by three washes with PBS. Finally, DAPI staining solution was added to stain the cell nuclei for 10 min, followed by washing with PBS for 5 min. Finally, PBS was added to cover the cell layer for subsequent fluorescence microscopy observation.

[0041] Co-immunofluorescence staining with DDX4 and EdU can be used to identify and label proliferating chicken oocyte primordial stem cells (see [link to relevant documentation]). Figure 1 A).

[0042] (2) Immunofluorescence staining identification: Immunofluorescence staining was performed on the cultured, normally proliferating oogonial stem cells for identification, as detailed below: Cells were fixed with 4% paraformaldehyde solution at room temperature for 15 min, followed by washing three times with PBS. The fixed cells were then permeabilized with 0.5% Triton X-100 at room temperature for 10 min, followed by washing three times with PBS to remove any residue. After permeabilization, cells were blocked with 10% goat serum blocking solution at room temperature for 1.5 h. Then, primary antibody DDX4 (ab13840, 1:200 dilution) and SSEA-1 (sc-21702, 1:200 volume ratio, Santa Cruz Antibody) were added for co-staining, and the cells were incubated overnight at 4°C. The next day, after washing three times with PBS, donkey anti-rabbit secondary antibody 488 (1:400 dilution, Wuhan Saiwei Biotechnology Co., Ltd.) and goat anti-mouse secondary antibody 555 (1:400 volume ratio, Thermo Fisher Scientific Co., Ltd.) were added, and the cells were incubated at room temperature in the dark for 1 h. After incubation, the cells were rinsed three times with PBS, and then the nuclei were stained with DAPI for 10 minutes, followed by rinsing with PBS for 5 minutes. Finally, PBS was added to cover the cell layer for subsequent fluorescence microscopy observation.

[0043] The test results are shown in Figure 1B. It was observed that the cell membrane of chicken oocyte progenitor cells isolated and cultured in vitro expressed the germ cell-specific marker DDX4, exhibiting green fluorescence, and also expressed the stem cell-specific marker SSEA-1, exhibiting red fluorescence. The cell nucleus was stained with DAPI. In this experiment, chicken embryo fibroblasts (CEF) were used as a negative control for immunofluorescence staining, and the results showed no expression of the relevant proteins. Figure 1 The term "Fibroblast" indicates that chicken oocyte progenitor cells isolated and cultured in vitro can maintain their biological characteristics well.

[0044] Example 4: Promoting the in vitro differentiation of chicken oocyte stem cells into secondary oocytes The chicken oogonial stem cells isolated in Example 3 were transferred into 24-well plates with a chicken embryo fibroblast feeder layer, meiosis induction medium was added, and the plates were cultured at 39°C in a 5% CO2 incubator. The medium was replaced every 24 hours. After 48 hours, a large number of round, large cells with a diameter exceeding 22 μm appeared in the culture dish, approximately twice the diameter of normally cultured oogonial stem cells (see Example 3). Figure 2 A).

[0045] After 3 days of culture, the meiosis induction medium was replaced with an oocyte maturation induction medium, and culture continued until the appearance of the first polar body was observed (see...). Figure 3 D).

[0046] Example 5: Promoting the in vitro differentiation of chicken oocyte stem cells into blastocysts Parthenogenetic activation of secondary oocytes obtained in Example 4 was performed using strontium chloride (SrCl2). The activation solution was prepared by adding 10% fetal bovine serum (FBS), 20 mM strontium chloride (SrCl2), and 5 µg / mL cytochalasin B (CB) to M199 medium and pre-equilibrating at 39°C for 4 hours. Subsequently, secondary oocytes were placed in the pre-equilibrated activation solution and incubated at 39°C for 4 hours under a humidified environment containing 5% CO2. After activation, the secondary oocytes were washed three times with pre-equilibrated M199 medium and then transferred to M199 medium containing 10% FBS for further culture.

[0047] Cleavage cells were stained with Hoechst 33342 working solution for 15 min, followed by washing with PBS for 5 min. Finally, PBS was added to cover the cell layer for subsequent fluorescence microscopy observation.

[0048] The test results are shown in Figure 4This invention reveals that activation with 20mM SrCl2 can significantly induce a large number of secondary oocytes to undergo typical discoid incomplete cleavage, with the cleavage stage progressing to the two-cell, four-cell, and even sixteen-cell stages. Bright-field microscopy observation revealed structures similar to yolk lipids in the dividing cells, suggesting their potential for further development into early embryos.

[0049] Comparative Example 1: The chicken oocyte protostem cells isolated in Example 3 were transferred into a 24-well plate with a chicken embryo fibroblast feeder layer, meiosis induction medium was added, and the plate was cultured at 39°C in a 5% CO2 incubator, with the medium being replaced every 24 h. After 3 days of culture, the cells were cultured again on meiosis induction medium.

[0050] Comparative Example 2: The chicken oocyte protostem cells isolated in Example 3 were transferred into a 24-well plate with a chicken embryo fibroblast feeder layer, meiosis induction medium was added, and the plate was cultured at 39°C in a 5% CO2 incubator, with the medium being replaced every 24 h. After 3 days of culture, the meiosis induction medium was replaced with medium A, which consisted of meiosis induction medium with the addition of FSH to a final concentration of 0.2 IU / mL and HCG to a final concentration of 2 IU / mL, and the culture was continued.

[0051] Comparative Example 3: The chicken oocyte protostem cells isolated in Example 3 were transferred into a 24-well plate with a chicken embryo fibroblast feeder layer, meiosis induction medium was added, and the plate was cultured at 39°C in a 5% CO2 incubator, with the medium being replaced every 24 h. After 3 days of culture, the meiosis induction medium was replaced with medium B, which consisted of GDNF at a final concentration of 50 ng / mL, P4 at a final concentration of 1 ng / mL, and rosiglitazone at a final concentration of 20 μM, and the culture was continued.

[0052] Experimental example: The in vitro induction effects of Example 4 and Comparative Examples 1-3 were investigated, as follows: 1. Proportion of cells entering different stages of meiosis after induction The cells were induced in vitro according to the methods of Example 4 and Comparative Examples 1-3, and the proportions of cells entering different stages of meiosis are shown in Table 1.

[0053] Table 1: Proportion of cells entering different stages of meiosis after induction Following the induction treatment protocol of Comparative Example 1, cells could enter prophase I of meiosis, and the proportion of induced cells was not significantly different compared to other protocols. However, under these conditions, cells could not progress to subsequent stages, and cell membrane collapse and death occurred on day 5 of induction.

[0054] Following the induction treatment protocol of Comparative Example 2, approximately 14% of the cells were able to enter meiosis II. However, when parthenogenetic activation was performed as described in Example 5, no cleavage was observed, and the cleavage rate was 0%.

[0055] Following the induction treatment protocol of Comparative Example 3, approximately 12% of the cells were able to enter meiosis II. However, when parthenogenetic activation was performed as described in Example 5, no cleavage was observed, and the cleavage rate was 0%.

[0056] Using the induction treatment scheme of Example 4, the proportion of cells entering meiosis II phase was significantly increased to 35%. Further induction through parthenogenesis resulted in approximately 5% of these cells undergoing cleavage.

[0057] 2. Identification by immunofluorescence staining: (1) Cells induced by meiosis in Comparative Example 1 were identified by immunofluorescence staining: Cells were fixed with 4% paraformaldehyde solution at room temperature for 15 min, followed by rinsing with PBS 3 times. The fixed cells were permeabilized with 0.5% Triton X-100 at room temperature for 10 min, and then rinsed with PBS 3 times to remove residue. The permeabilized cells were then blocked with 10% goat serum blocking solution at room temperature for 1.5 h. Then, primary antibody DDX4 (purchased from Abogen Biosciences, catalog number ab13840, dilution ratio 1:200) and γH2AX (purchased from Abogen Biosciences, ab26350, used volume ratio 1:200) were co-stained, as were primary antibody DDX4 (purchased from Boster Biologics, catalog number M02448-1, dilution ratio 1:200) and SCP3 (purchased from Thermo Fisher Scientific, NB30231A350, used volume ratio 1:200) and incubated overnight at 4°C. The following day, after rinsing three times with PBS, the cells were co-stained with donkey anti-rabbit secondary antibody 488 (purchased from Wuhan Saiwei Biotechnology Co., Ltd., diluted 1:400) and goat anti-mouse secondary antibody 555 (purchased from Thermo Fisher Scientific Co., Ltd., used at a volume ratio of 1:400). Co-staining was also performed with monkey anti-mouse secondary antibody 488 (purchased from Wuhan Saiwei Biotechnology Co., Ltd., diluted 1:400) and monkey anti-rabbit secondary antibody 555 (purchased from Thermo Fisher Scientific Co., Ltd., used at a volume ratio of 1:400). The cells were incubated at room temperature in the dark for 1 hour. After incubation, the cells were rinsed three times with PBS, and DAPI staining solution was added to stain the cell nuclei for 10 minutes, followed by washing with PBS for 5 minutes. Finally, PBS was added to cover the cell layer for subsequent fluorescence microscopy observation.

[0058] The test results are shown in Figure 2 It was observed that these oocyte-like cells (OLCs) induced two days after the addition of retinoic acid and vitamin C entered the meiotic phase. We examined the expression of classic meiotic markers, namely γH2AX and SCP3 proteins, related to chromosome recombination and synaptic complex formation. Immunochromatographic staining showed that the induced oocyte-like cells (OLCs) were positive for γH2AX and SCP3. In this experiment, uninduced oogonial stem cells were used as a negative control for immunofluorescence staining, and the results showed no expression of these proteins. Figure 2 (in "OSCs").

[0059] (2) Immunofluorescence staining was performed on the cells induced in Example 4: The cells were fixed with 4% paraformaldehyde solution at room temperature for 15 min, and then washed three times with PBS. The fixed cells were permeabilized with 0.5% Triton X-100 at room temperature for 10 min, and then washed three times with PBS to remove residue. The permeabilized cells were then blocked with 10% goat serum blocking solution at room temperature for 1.5 h. Then, primary antibody DDX4 (purchased from Abogen Biosciences, catalog number ab13840, dilution 1:200) and NOBOX (purchased from Santa Cruz Antibody, sc-514178, used volume ratio 1:200) were added for co-staining, as were primary antibody DDX4 (purchased from Abogen Biosciences, catalog number ab13840, dilution 1:200) and BMP15 (purchased from Santa Cruz Antibody, sc-271824, used volume ratio 1:200) for co-staining. The mixture was incubated overnight at 4°C. The next day, after washing three times with PBS, donkey anti-rabbit secondary antibody 488 (purchased from Wuhan Saiwei Biotechnology Co., Ltd., dilution 1:400) and goat anti-mouse secondary antibody 555 (purchased from Thermo Fisher Scientific, used volume ratio 1:400) were added for co-staining. The mixture was incubated at room temperature in the dark for 1 hour. After incubation, the cells were rinsed three times with PBS, and then the nuclei were stained with DAPI for 10 minutes, followed by rinsing with PBS for 5 minutes. Finally, PBS was added to cover the cell layer for subsequent fluorescence microscopy observation.

[0060] The test results are shown in Figure 3 To further assess the development of oocyte-like cells (OLCs), we used oocyte-specific markers BMP15 and NOBOX for staining. The staining results showed that OLCs induced by additional supplementation with 0.2 IU / mL FSH, 2 IU / mL HCG, 50 ng / mL GDNF, 1 ng / mL P4, and 20 µM rosiglitazone were positive for these oocyte markers, while cells treated with only RA and VC were negative. This indicates that after the initiation of meiosis, FSH, HCG, GDNF, P4, and rosiglitazone promote the development and maturation of oocytes.

[0061] 3. Quantitative detection of marker genes in meiosis and oocytes: Total RNA was extracted from cells induced in Example 4 and Comparative Example 1 using the PicoPure RNA Isolation Kit, and cDNA was synthesized using a DNase-free reverse transcription kit. Finally, real-time quantitative PCR (qPCR) was performed using the primer sequences listed in Table 2 to analyze the expression levels of relevant genes.

[0062] Table 2: qPCR primer sequences The experimental results of Comparative Example 1 are shown in Figure 2 B. qPCR analysis of meiotic marker genes (including STRA8, SCP3, SpO11, and DMC1) was found to be upregulated in retinoic acid and vitamin C-induced cells. Therefore, we confirm that meiosis has been initiated in retinoic acid and vitamin C-induced cells.

[0063] The test results of Example 4 are shown below. Figure 3 A. This invention found that treatment with 0.2 IU / mL FSH, 2 IU / mL HCG, 50 ng / mL GDNF, 1 ng / mL P4, and 20 µM rosiglitazone significantly upregulated the expression levels of oocyte-specific marker genes (including NOBOX, GDF9, BMP15, and LHX8) in induced cells, suggesting that these cells acquired the molecular characteristics of oocytes.

[0064] In summary, this invention establishes an in vitro induction system that enables oogonial stem cells to differentiate into oocyte-like cells, and further develop into blastocysts through parthenogenetic activation. This is the first time that oocytes have been obtained in vitro from reproductive stem cells in chickens. It provides a valuable model for exploring oocyte development in birds and offers a new strategy for the in vitro generation of oocytes.

[0065] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A culture medium for promoting the in vitro differentiation of chicken oocyte stem cells, characterized in that, The culture medium includes: meiosis induction medium and oocyte maturation induction medium; The meiosis induction medium is a basal culture medium for oogonial stem cells with added RA and VC; The oocyte maturation induction medium is a meiosis induction medium supplemented with FSH, HCG, GDNF, P4 and rosiglitazone; The basal culture medium for the oogonial stem cells was KO-DMEM medium containing 10 ng / mL Acitivin A, 10 ng / mL bFGF, 0.1 mM β-mercaptoethanol, 1.2 mM Sodium Pyruvate, 1X Antibiotic-antimycotic, 100 µg / mL Sodium heparin, 1X GS nucleosides, 1X GlutaMax, 1X NEAA, 0.2% chicken serum and 5% fetal bovine serum.

2. The culture medium according to claim 1, characterized in that, In the meiosis induction medium, the final concentration of RA added is 0.5-1.5 μM, and the final concentration of VC added is 100-200 μM. In the oocyte maturation induction medium, the final concentration of FSH is 0.1-0.3 IU / mL, the final concentration of HCG is 1-3 IU / mL, the final concentration of GDNF is 40-60 ng / mL, the final concentration of P4 is 0.5-1.5 ng / mL, and the final concentration of rosiglitazone is 15-25 μM.

3. The culture medium according to claim 2, characterized in that, In the meiosis induction medium, the final concentration of RA added is 1 μM, and the final concentration of VC added is 150 μM. In the oocyte maturation induction medium, the final concentration of FSH was 0.2 IU / mL, the final concentration of HCG was 2 IU / mL, the final concentration of GDNF was 50 ng / mL, the final concentration of P4 was 1 ng / mL, and the final concentration of rosiglitazone was 20 μM.

4. The use of the culture medium according to any one of claims 1-3 in either (1) or (2) below: (1) Promote the in vitro differentiation of chicken oocyte stem cells into secondary oocytes; (2) Large-scale production of chicken oocytes.

5. A method for promoting the in vitro differentiation of chicken oocyte primordial stem cells into secondary oocytes, characterized in that, Includes the following steps: (1) Chicken oocyte stem cells were seeded into chicken embryo fibroblast feeder layer, and meiosis induction medium was added for culture. The medium was changed every 24 hours. (2) After culturing in meiosis induction medium for 3 days, the meiosis induction medium was replaced with oocyte maturation induction medium and cultured until the first polar body appeared.

6. The method according to claim 5, characterized in that, In steps (1) and (2), the cultivation conditions are: 39℃ and 5% CO2.

7. A method for promoting the in vitro differentiation of chicken oocyte stem cells into blastocysts, characterized in that, Includes the following steps: (1) Chicken oocyte stem cells were seeded into chicken embryo fibroblast feeder layer, and meiosis induction medium was added for culture. The medium was changed every 24 hours. (2) After culturing in meiosis induction medium for 3 days, the meiosis induction medium is replaced with oocyte maturation induction medium and cultured until the first polar body appears to form a secondary oocyte. (3) Secondary oocytes were placed in activation solution for parthenogenetic activation. After activation, they were transferred to M199 medium containing 10% FBS for further culture.

8. The method according to claim 7, characterized in that, In step (3), the activation solution is prepared by adding 10% fetal bovine serum, 20mM strontium chloride and 5µg / mL cytochalasin B to M199 culture medium.

9. The method according to claim 7, characterized in that, In step (3), the culture conditions for parthenogenetic activation are: incubation at 39°C for 4 hours in a humid environment containing 5% CO2.

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