Application of Kitl in promoting meiosis of germ cells cultured in vitro
By adding Kitl to the germ cell culture medium and combining it with RA and BMP2, the Kit–AKT–mTOR–pS6 signaling axis is activated, which solves the problem of low efficiency in in vitro induced meiosis of germ cells in the prior art and achieves more efficient meiosis simulation and promotion of key events.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-11
- Publication Date
- 2026-03-24
AI Technical Summary
In existing technologies, RA and BMP2 have low efficiency in inducing meiosis in germ cells in vitro, especially in simulating real synapsis and recombination. The role of the Kitl/Kit signaling pathway in the initiation and progression of meiosis is unclear, resulting in poor in vitro induction effects.
Kitl, added to germ cell culture medium, combines with RA and BMP2 to promote the expression of key meiotic proteins by activating the Kit–AKT–mTOR–pS6 signaling axis, thereby increasing the initiation rate of meiosis and the pairing and recombination of homologous chromosomes.
It significantly improved the initiation rate of meiosis, enhanced the pairing and recombination of homologous chromosomes, and improved the realism of in vitro meiosis simulation, especially in terms of pairing and recombination, which are closer to the in vivo process.
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Figure CN121718488A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of reproductive biology and cell engineering technology, and in particular to the application of Kitl in promoting meiosis in in vitro cultured germ cells. Background Technology
[0002] Meiosis is the core process of gamete formation, and its successful initiation and precise progression are crucial for maintaining genetic stability and reproductive health. In female mammals, primordial germ cells (PGCs) migrate into the genital ridge during embryonic development and, guided by somatic cell signals, enter prophase I of meiosis, undergoing a series of critical events such as homologous chromosome pairing, mating, and recombination. Any disruption in this process can lead to the production of aneuploid gametes, which in turn can cause infertility, miscarriage, or congenital genetic diseases.
[0003] For a long time, the retinoic acid (RA) signaling pathway has been considered the "master switch" for initiating female meiosis. This model is mainly based on the phenomenon that exogenous RA can induce germ cells to enter meiosis in vitro. However, recent genetic studies have found that female germ cells can still initiate meiosis after knocking out key enzymes or receptors that synthesize RA in vivo, indicating the existence of RA-independent regulatory pathways. At the same time, signaling proteins such as bone morphogenetic protein (BMP) have also been shown to be involved in this process. Nevertheless, the efficiency of using RA and BMP2 alone to induce primordial germ cell-like cells (PGCLCs) or PGCs to enter meiosis in vitro remains low, and the induced cells show significant differences from the in vivo state in terms of the integrity of homologous chromosome pairing and the fidelity of recombination events, making it difficult to fully simulate the real meiotic process.
[0004] The interaction between somatic cells and germ cells is the cornerstone of precise regulation of meiosis. Studies have found that the initiation of meiosis in germ cells is severely impaired in the absence of gonadal cell co-culture, highlighting the critical role of somatic cell-derived signals. The signaling pathway formed by Kit ligand (Kit1, also known as stem cell factor SCF) and its receptor Kit plays an important role in multiple stages of germ cell development, such as migration, survival, and proliferation. However, the direct regulatory role and molecular mechanism of this pathway in the initiation and progression of meiosis, particularly in the formation of the synaptic complex and homologous recombination, have remained unclear and even controversial. Some argue that Kit signaling may actually inhibit meiosis, leading to its long-term neglect in the in vitro induction of meiosis.
[0005] Therefore, there is an urgent need in this field to discover new, efficient in vitro meiosis induction strategies that can simulate real in vivo processes, especially to identify signaling molecules and specific methods that can promote key meiotic events (such as co-homogeneous synapsis).
[0006] In existing technologies, RA and BMP2 are widely used for in vitro meiosis induction, but their efficiency is limited, especially in simulating real synapsis and recombination. While Kit1 / Kit signaling has been reported in germ cell survival and migration, its specific mechanism of action in meiosis initiation and progression remains unclear. This invention reveals for the first time that Kit1 promotes the expression of key meiotic proteins through the mTOR pathway, providing a new strategy for efficient in vitro meiosis induction. Summary of the Invention
[0007] The purpose of this invention is to address the technical deficiencies in the prior art by providing the application of Kitl in promoting meiosis of germ cells in vitro.
[0008] The technical solution adopted to achieve the purpose of this invention is: Kitl is used to promote meiosis in in vitro cultured germ cells. The amino acid sequence of Kitl is shown in SEQ ID NO:1.
[0009] In the above technical solution, the germ cells are primordial germ cells (PGCs) or primordial germ cell-like cells (PGCLCs) obtained by induction from pluripotent stem cells.
[0010] Another aspect of the present invention includes a culture medium additive for promoting meiosis of germ cells, comprising Kitl, wherein the amino acid sequence of Kitl is shown in SEQ ID NO:1.
[0011] In the above technical solution, the culture medium additive also includes RA and / or BMP2.
[0012] Another aspect of the present invention includes a Kitl-based in vitro germ cell culture system, comprising MF10 medium and Kitl, wherein the amino acid sequence of Kitl is shown in SEQ ID NO:1.
[0013] In the above technical solution, the concentration of Kitl in the in vitro culture system of germ cells is 50~200 ng / mL.
[0014] In the above technical solution, the MF10 culture medium includes 10% v / v FBS, 1 mM L-glutamine, 1% v / v non-essential amino acids, 1% v / v penicillin / streptomycin, 50 μg / ml vitamin C and 10 μM Rock inhibitor, with the balance being M199 (Medium 199).
[0015] In the above technical solution, the in vitro culture system for germ cells also includes RA and / or BMP2.
[0016] In the above technical solution, the concentration of RA in the in vitro culture system of germ cells is 100 nM, and the concentration of BMP2 in the in vitro culture system of germ cells is 300 ng / mL.
[0017] Another aspect of the present invention includes a meiosis induction kit, the kit comprising the culture medium additive or germ cell in vitro culture system and culture instructions.
[0018] Another aspect of the present invention includes a method for promoting the entry and pre-meiotic phase of germ cells cultured in vitro using Kitl, comprising the following steps: Step 1: Obtain primordial germ cells (PGCs) from female gonads, or obtain primordial germ cell-like cells (PGCLCs) induced from pluripotent stem cells. Step 2: Prepare the Kit1-based in vitro germ cell culture system; Step 3: Culture the primordial germ cells (PGCs) or primordial germ cell-like cells (PGCLCs) obtained in Step 1 in the in vitro germ cell culture system prepared in Step 2.
[0019] Compared with the prior art, the beneficial effects of the present invention are: 1. Kitl significantly improves the meiotic initiation rate: Kitl treatment significantly increases the proportion of Stra8⁺ cells.
[0020] 2. Kitl promotes the expression of key meiotic proteins: the levels of proteins such as Stra8, Sycp1, Sycp3, and Vasa are significantly increased.
[0021] 3. Kitl improves homologous chromosome pairing and recombination: enhanced co-localization of Sycp1 and Sycp3 increases the proportion of normally paired cells.
[0022] 4. Kitl enhances the realism of in vitro meiosis simulation: more closely resembles the in vivo meiosis process, especially in terms of synapsis and recombination.
[0023] 5. Mechanism is clear: Kitl promotes the expression of key proteins in meiosis by activating the Kit–AKT–mTOR–pS6 signaling axis. Attached Figure Description
[0024] Figure 1 It is the interaction network of Kitl / Kit signaling between somatic cells and germ cells (single-cell RNA-seq analysis).
[0025] Figure 2 Kitl deficiency leads to decreased meiosis initiation and expression of key proteins.
[0026] Figure 3 Kitl defects affect homologous pairing and recombination (changes in the number of focal points of Rad51, Dmc1, and Mlh1).
[0027] Figure 4 Kitl deficiency leads to downregulation of meiosis-related genes and the mTOR signaling pathway.
[0028] Figure 5 The Kit inhibitor ISCK03 inhibits the meiotic process.
[0029] Figure 6 Activating mTOR can partially salvage meiotic defects caused by Kitl defects.
[0030] Figure 7 Kitl activates the mTOR / pS6 signal via p-AKT.
[0031] Figure 8 Kitl, in combination with RA / BMP2, significantly improves the meiotic efficiency and synapsis level of PGCs. Detailed Implementation
[0032] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
[0033] The Kitl in the following examples has the catalog number PeproTech (250-03). The amino acid sequence of Kitl is shown in SEQ ID NO:1, specifically MKEICGNPVT DNVKDITKLV ANLPNDYMIT LNYVAGMDVL PSHCWLRDMVIQLSLSLTTL LDKFSNISEG LSNYSIIDKL GKIVDDLVLC MEENAPKNIKESPKRPETRS FTPEEFFSIFNRSIDAFKDF MVASDTSDCV LSSTLGPEKD SRVSVTKPFM LPPVA.
[0034] Example 1 Single-cell sequencing reveals the presence of the Kit1 / Kit signaling pathway in the embryonic gonad: This embodiment systematically analyzes cell types and their signal communication in the female gonads of mouse embryos, focusing on verifying the potential importance of the Kit1 / Kit pathway in somatic cell-germ cell interactions.
[0035] Samples and Operations: 1. Obtain female gonads from mouse E12.5, E13.5, and E14.5 embryos.
[0036] 2. Prepare single-cell suspensions and perform 10x Genomics single-cell RNA sequencing.
[0037] 3. Use the Seurat software package for cell clustering and annotation to identify cell types such as germ cells, granular cells, and mesenchymal cells.
[0038] 4. Use the CellChat software package to analyze intercellular signaling communication networks.
[0039] Performance verification and results analysis: 1. Cell Atlas Construction: UMAP visualization clearly displays various cell populations in the gonads, including germ cells (such as...). Figure 1 (As shown in a and b).
[0040] 2. Signaling pathway intensity analysis: CellChat analysis showed that KIT signaling was one of the most important signaling pathways connecting somatic cells and germ cells during the E12.5 to E14.5 stages (e.g., KIT signaling). Figure 1 (As shown in c and d).
[0041] 3. Ligand-receptor pair localization: Analysis showed that the Kitl gene is mainly expressed in somatic cells such as granulosa cells that highly express Foxl2, while its receptor Kit is highly expressed in germ cells, constituting a typical ligand-receptor signaling pattern (e.g., Figure 1 (as shown in e).
[0042] 4. Protein level verification: Immunofluorescence staining confirmed that Kitl protein was located at Foxl2 in gonadal sections from E12.5 to E14.5. + The somatic cells and their surroundings, while the Kit protein is in Vasa + Expression on germ cells (e.g.) Figure 1 (as shown in f).
[0043] This embodiment confirms at the transcriptomic and protein levels the existence of an active Kit1 / Kit signaling pathway from somatic cells (senders) to germ cells (receivers) during the critical period of gonadal meiosis initiation in female embryos.
[0044] Example 2 Granulosa cell-specific knockout of Kitl impairs meiotic initiation and progression: Validating in vivo the necessity of granulosa cell-derived Kitl for germ cell meiosis.
[0045] Models and Operations: 1. Construct a mouse model of granulocyte-specific knockout of Kitl (Kitl f / f; Foxl2-Cre, i.e., cKO), with Kitl f / +; Foxl2-Cre as the control (Control / WT).
[0046] 2. Collect female gonads from E14.5 and E16.5 embryos.
[0047] 3. Meiotic phenotypes were analyzed using techniques such as immunofluorescence and Western blot.
[0048] Performance verification and results analysis: 1. For example Figure 1 As shown in a and b, the two key indicators of meiosis, Stra8 and Sycp3, change significantly at E14.5. Therefore, subsequent experiments mainly focused on the E14.5 and E16.5 periods.
[0049] 2. Impaired meiotic initiation: In E14.5, cKO gonads, Stra8 + The number of germ cells was significantly less than that of WT (e.g. Figure 2 (As shown in c and d). Meanwhile, total germ cells (Vasa) + The number of germ cells (Sycp3) entering meiosis and the number of germ cells entering meiosis. + and Sycp1 + The number of all decreased significantly (e.g.) Figure 2 (as shown in e and f).
[0050] 3. Synaptic complex formation defect: In E14.5 and E16.5, the number of cells capable of forming complete Sycp3 protein filaments in cKO gonads is reduced (e.g., Figure 2 As shown in g), the proportion of cells with co-localization of Sycp1 and Sycp3 (indicating normal synapsis) was significantly reduced (e.g. Figure 2 As shown in Figure j), this indicates impaired homologous chromosome pairing.
[0051] 4. Decreased levels of key proteins: Western blot analysis confirmed that the protein levels of Stra8, Vasa, Sycp1, and Sycp3 in cKO gonads were significantly lower than those in WT gonads (e.g., ...). Figure 2 (As shown in k and l).
[0052] Conclusion: Kitl derived from granulosa cells is crucial for the successful initiation of meiosis in germ cells, the expression of key meiotic proteins, and the completion of homologous chromosome pairing.
[0053] Example 3 Kitl deficiency leads to abnormal homologous recombination repair and crossover junction formation: Investigating the impact of Kitl deficiency on homologous recombination, a core event in meiosis.
[0054] Models and Operations: Using E16.5 cKO and WT gonads from Example 2, chromosome slices were prepared and subjected to high-resolution immunofluorescence analysis.
[0055] Performance verification and results analysis: 1. Meiotic arrest: Chromosome spread analysis showed that more germ cells in the cKO gonads were arrested in the zygotene stage and had difficulty entering the pachytene stage (e.g., Figure 3 (As shown in b and c).
[0056] 2. Abnormal synapsis: Sycp1 protein is significantly reduced in cKO germ cells, exhibiting abnormal synapsis morphology (e.g., Figure 3 As shown in b and d), STEDCON super-resolution microscopy further confirmed the decreased pairing tightness and anomalous morphology (e.g., as shown in b and d). Figure 3 (as shown in e).
[0057] 3. Abnormal DSB repair markers: Compared with WT, the number of Rad51 focal points, representing DSB repair, was abnormally increased in cKO germ cells ( Figure 3 f), while the number of focal points for the key protein Dmc1 is reduced (e.g. Figure 3 As shown in g), this indicates an abnormal DSB repair process.
[0058] 4. Reduced crossing knot formation: The number of Mlh1 foci, a marker of crossing knot maturation, is significantly reduced in cKO germ cells (e.g., Figure 3 As shown in h), this demonstrates that the final genetic exchange event was impaired.
[0059] Conclusion: The Kitl signaling pathway is essential for the successful completion of homologous recombination repair during meiosis and for the formation of sufficient crossing junctions.
[0060] Example 4 Kit inhibitors reproduce the Kitl deficiency phenotype in vitro: Kit receptor function was validated in vitro using pharmacological methods, and the cKO phenotype was simulated.
[0061] operate: 1. Culture the gonads of wild-type female E12.5 mice in vitro for 2 or 4 days.
[0062] 2. The experimental group was given the Kit inhibitor ISCK03, while the control group used DMSO solvent.
[0063] Performance verification and results analysis: 1. Inhibition of meiosis initiation: After 2 days of culture, Stra8 cells in the ISCK03 treatment group... + / Vasa + The proportion of cells was significantly lower than that of the control group (e.g., Figure 5 (As shown in b and c).
[0064] 2. Inhibition of synaptic protein expression and synapsis: After 4 days of culture, Sycp3 in the ISCK03-treated group... + and Sycp1 + Cell number (e.g.) Figure 5 (as shown in d and e) and the number of normally synapsed cells (as shown in d and e) Figure 5 (as shown in f) all decreased significantly.
[0065] 3. Arrest of meiosis: Chromosome preparations showed that ISCK03 treatment caused more cells to arrest in zygote phase, and the proportion of cells in pachytene phase decreased (e.g., Figure 5 (As shown in g and h).
[0066] 4. Decreased levels of key proteins: Western blot confirmed that ISCK03 treatment reduced the protein levels of Stra8, Sycp1, Sycp3, and Vasa (e.g., Figure 5 As shown in i).
[0067] Disruption of homologous recombination: ISCK03 treatment resulted in an increase in Rad51 focal groups, a decrease in Dmc1 focal groups, and a decrease in Mlh1 focal groups (e.g., Figure 5 As shown in the middle jl), it is highly consistent with the in vivo phenotype of cKO.
[0068] Conclusion: Pharmacological inhibition of Kit receptor function can completely reproduce the meiotic defect phenotype of genetically knocked-out Kitl in vitro, which in turn verifies the importance of this pathway.
[0069] Example 5 Single-cell transcriptomics reveals the molecular mechanism by which Kitl regulates meiosis through the mTOR pathway: This study elucidates the molecular mechanism by which Kitl deficiency leads to meiotic defects at the transcriptomic level.
[0070] operate: 1. 10x single-cell RNA sequencing was performed on cKO and WT gonads of E14.5.
[0071] 2. Separate the germ cells for in-depth analysis.
[0072] Performance verification and results analysis: 1. Signaling pathway alterations: CellChat analysis showed a significant decrease in the strength of the Kit signaling pathway in cKO gonads (e.g., Figure 4 (As shown in b).
[0073] 2. Downregulation of meiosis-related pathways: GSEA analysis showed that pathways such as meiosis and homologous recombination were significantly enriched in the downregulated gene set in cKO germ cells (e.g., Figure 4 (as shown in e).
[0074] 3. Decreased expression of key genes: The mRNA levels of key meiotic genes such as Meioc, Ythdc2, and Spo11 were significantly downregulated in cKO germ cells (e.g., Figure 4 (as shown in f).
[0075] 4. Significantly impaired mTOR signaling pathway: GSEA analysis showed that the mTOR signaling pathway is one of the most significantly downregulated signaling pathways in cKO germ cells (e.g., Figure 4 As shown in i).
[0076] Conclusion: Kitl deletion leads to extensive alterations in the germ cell transcriptome, among which the transcriptional downregulation of the core meiotic program and related signaling pathways (especially the mTOR pathway) is an important molecular basis for phenotype generation.
[0077] Example 6 Activation of mTOR can rescue meiotic defects caused by Kitl deficiency: verify whether mTOR is a key downstream effector of Kitl and explore its therapeutic potential.
[0078] operate: 1. Take cKO and WT gonads from E12.5 and culture them in vitro.
[0079] 2. Treatment group: The cKO group was given either Kitl protein or mTOR agonist 3BDO.
[0080] Performance verification and results analysis: 1. Rescue of Meiosis Initiation: After 2 days of culture, the addition of Kitl or 3BDO significantly increased the Vasa function of germ cells in cKO gonads. + Quantity (e.g.) Figure 6 (As shown in b and c) Stra8 + Cell number (e.g.) Figure 6 (as shown in b and d) and Stra8 protein levels (as shown in...) Figure 6 (as shown in e and f).
[0081] 2. Rescuing Synaptic Protein Expression: After 4 days of culture, the addition of Kitl or 3BDO significantly increased Sycp3 expression in cKO gonads. +and Sycp1 + Cell number (e.g.) Figure 6 (as shown in gi).
[0082] 3. Partial restoration of homologous synapses: Treatment with Kitl or 3BDO can partially restore the proportion of normally synapsed cells in cKO gonads (e.g., Figure 6 (As shown in j).
[0083] Conclusion: Direct activation of the mTOR pathway can largely bypass the signal interruption caused by Kitl deficiency, thus rescuing the initiation and progression of meiosis, proving that mTOR is a key signaling node downstream of Kitl that performs its function.
[0084] Example 7 Kitl promotes the mTOR / pS6 signaling pathway by activating p-AKT: This study directly verifies at the molecular level whether Kitl / Kit signaling regulates the downstream mTOR / pS6 pathway by activating AKT.
[0085] Models and Operations: In vivo validation: Kitl cKO and gonads of WT female mice were extracted from E14.5 and subjected to Western blot analysis.
[0086] In vitro pharmacological validation: E12.5 wild-type gonads were cultured in vitro for 2 days, and Kit inhibitor ISCK03 and mTOR inhibitor rapamycin were added respectively.
[0087] PGCs were isolated and cultured, and Kitl protein was added to the culture medium.
[0088] p-AKT specific activator SC79 was added to the E12.5 wild-type gonad culture system.
[0089] Salvage experiment verification: Kitl or mTOR agonist 3BDO was added to the Kitl cKO gonad in vitro culture system, respectively.
[0090] Performance verification and results analysis: Kitl deficiency inhibits the AKT-mTOR-pS6 axis: Western blot results showed that, compared with WT, the ratios of p-AKT / AKT, p-mTOR / mTOR, and pS6 / S6, as well as the Stra8 protein level, were significantly decreased in the gonads of Kitl cKO (e.g., Figure 7 (as shown in a).
[0091] Inhibition of the Kit receptor produces a similar effect: in wild-type gonads, ISCK03 treatment also reduced protein levels of p-AKT / AKT, p-mTOR / mTOR, pS6 / S6, and Stra8 (e.g., Figure 7 (As shown in b).
[0092] Exogenous Kitl activates this pathway: In isolated PGC culture systems, the addition of Kitl directly increases the protein levels of p-AKT / AKT, p-mTOR / mTOR, pS6 / S6, and Stra8 (e.g., ...). Figure 7 (As shown in c).
[0093] Activation of AKT can mimic the Kitl effect: Treatment of the gonads with the p-AKT activator SC79 can effectively increase p-AKT levels, subsequently leading to upregulation of p-mTOR / mTOR and pS6 / S6 (e.g., Figure 7 As shown in Figure d), this demonstrates that AKT is a key upstream kinase of mTOR in this pathway.
[0094] mTOR is a key downstream pathway: rapamycin treatment inhibited the phosphorylation of mTOR and pS6 and reduced Stra8 levels, but did not affect the Kit protein itself (e.g., Figure 7 (As shown in e). In Kitl cKO gonads, the addition of Kitl or direct activation of mTOR (3BDO) effectively increased the protein levels of p-mTOR / mTOR, pS6 / S6, and Stra8 (as shown in e). Figure 7 (as shown in f).
[0095] Conclusion: This embodiment fully demonstrates the linear signal transduction pathway of Kitl / Kit → p-AKT → mTOR / pS6 → key meiotic proteins (such as Stra8) through genetic, pharmacological, and rescue experiments, elucidating the core principle of Kitl promoting meiosis from a molecular mechanism perspective.
[0096] Example 8 Kitl combined with RA / BMP2 significantly improves the efficiency of meiosis in PGCs in vitro: This study verified the synergistic effect of adding Kitl, especially in combination with RA / BMP2, to the in vitro culture system of PGCs on improving meiosis efficiency.
[0097] operate: 1. PGCs were isolated from the gonads of female E12.5 mice.
[0098] 2. Experimental groups: control group, RA group, BMP2 group, Kitl group, RAB2 group (RA+BMP2), RAB2+Kitl group (RA+BMP2+Kitl).
[0099] Control group culture medium: 10% v / v FBS, 1 mM L-glutamine, 1% v / v non-essential amino acids, 1% v / v penicillin / streptomycin, 50 μg / ml vitamin C and 10 μM Rock inhibitor, balance M199 (Medium199).
[0100] RA group culture medium: 100 nM RA was added to the control group culture medium.
[0101] BMP2 group culture medium: 300 ng / mL of BMP2 was added to the control group culture medium.
[0102] Kitl group culture medium: 100 ng / mL Kitl was added to the control group culture medium.
[0103] RAB2 group culture medium: 100 nM RA and 300 ng / mL BMP2 were added to the control group culture medium.
[0104] RAB2+Kitl group culture medium: 100 nM RA, 300 ng / mL BMP2, and 100 ng / mL Kitl in the control group culture medium.
[0105] 3. After 4 days of culture, the effect was evaluated by immunofluorescence and Western blot.
[0106] Figure 8 The diagram clearly demonstrates the entire design process of the in vitro experiment, including the sorting, grouping and processing of PGCs, and the detection time points. Figure 8 The study provided crucial morphological evidence that the combination of Kitl and RAB2 can improve the in vitro growth status of PGCs, making them more closely resemble their real developmental environment.
[0107] Performance verification and results analysis: 1. Promotes the formation of the synaptic complex: Chromosome spreading showed that the RAB2+Kitl group could form the most complete and clear Sycp1 and Sycp3 co-localized protein filaments, indicating the highest homologous synapsis efficiency (e.g., Figure 8 (As shown in c).
[0108] 2. Significantly increased proportion of synaptic cells: Statistical analysis showed that the percentage of homologous synaptic cells in the RAB2+Kitl group was significantly higher than that in other groups (e.g., Figure 8 (as shown in d).
[0109] 3. Synergistic enhancement of key protein expression: Western blot confirmed that RAB2+Kitl treatment most effectively increased the protein levels of Stra8, Sycp1, and Sycp3 (e.g., Figure 8(as shown in e and f).
[0110] Conclusion: Adding Kitl to the in vitro culture system of PGCs, especially in combination with RA and BMP2, can synergistically and significantly promote the initiation of meiosis, the expression of key proteins, and the correct pairing of homologous chromosomes.
[0111] Based on the clear mechanism and significant effects revealed by this invention in mouse models, this technical solution has broad prospects for translational medicine, especially in the fields of human assisted reproductive technology and reproductive medicine: In Vitro Gametogenesis (IVG): This invention provides key technical support for the directed induction of functional gametes from human pluripotent stem cells (including embryonic stem cells and induced pluripotent stem cells). By adding Kitl to PGCLCs at the critical stage of meiotic differentiation, the initiation efficiency and completion rate of meiosis can be significantly improved, resulting in more chromosomally normal mature gametes, bringing new hope to patients who have lost gametes due to infertility, cancer radiotherapy and chemotherapy, etc.
[0112] As a novel additive to enhance in vitro fertilization (IVF) culture systems: Current human oocyte in vitro maturation (IVM) technology has room for improvement in efficiency. Given that this study found Kitl to be a strong signal driving meiosis, we hypothesize that its introduction into the IVM culture system could provide a key initiation signal for oocytes that have difficulty maturing spontaneously in vitro, potentially increasing the proportion and quality of mature oocytes. This application concept is based on a pathway innovation of the core regulatory mechanism of meiosis, and has not been reported before.
[0113] To improve the function of germ cells after cryopreservation and thawing: Based on Kitl's known cell survival support function and its role in maintaining the meiotic process demonstrated in this study, we propose its application in thawing culture as a potential novel cell function protectant.
[0114] Reproductive disease model construction and drug screening: This method can efficiently and synchronously obtain human germ cells at various stages of meiosis in vitro, providing an ideal cell model for studying the pathogenesis of human meiotic abnormalities (such as aneuploidy, premature ovarian failure, etc.), and can be used to screen drugs that can correct meiotic errors and improve germ cell quality.
[0115] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. The application of Kitl in promoting meiosis in in vitro cultured germ cells, characterized in that, The amino acid sequence of Kitl is shown in SEQ ID NO:
1.
2. A culture medium additive that promotes meiosis in germ cells, characterized in that, It contains Kitl, whose amino acid sequence is shown in SEQ ID NO:
1.
3. The culture medium additive as described in claim 2, characterized in that, The culture medium additive also includes RA and / or BMP2.
4. A Kit1-based in vitro germ cell culture system, characterized in that, The contents include the culture medium and Kitl, whose amino acid sequence is shown in SEQ ID NO:
1.
5. The in vitro culture system for germ cells as described in claim 4, characterized in that, The concentration of Kitl in the in vitro germ cell culture system is 50~200 ng / mL.
6. The in vitro culture system for germ cells as described in claim 4, characterized in that, The culture medium is MF10 medium, which contains 10% v / v FBS, 1 mM L-glutamine, 1% v / v non-essential amino acids, 1% v / v penicillin / streptomycin, 50 μg / ml vitamin C and 10 μM Rock inhibitor, with the balance being M199.
7. The in vitro culture system for germ cells as described in claim 4, characterized in that, The in vitro culture system for germ cells also includes RA and / or BMP2.
8. The in vitro culture system for germ cells as described in claim 7, characterized in that, The concentration of RA in the in vitro germ cell culture system is 100 nM, and the concentration of BMP2 in the in vitro germ cell culture system is 300 ng / mL.
9. A meiosis induction kit, characterized in that, The kit includes the culture medium additive as described in claim 2 or the in vitro culture system for germ cells as described in claim 4, and a culture instruction manual.
10. A method for promoting the entry and completion of promeiosis in in vitro cultured germ cells using Kit1, characterized in that, Includes the following steps: Step 1: Obtain primordial germ cells from female gonads, or induce primordial germ cell-like cells from pluripotent stem cells; Step 2: Prepare the Kit1-based in vitro germ cell culture system as described in any one of claims 4 to 8; Step 3: Culture the primordial germ cells or primordial germ cell-like cells obtained in Step 1 in the in vitro germ cell culture system prepared in Step 2.