Application of rps26 recombinant protein in promoting early embryonic development of animals

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to application of RPS26 recombinant protein in promoting early embryo development of animals. The application experiment finds that, during early in-vitro embryo culture of lamb goats, after RPS26 recombinant protein is added, the blastocyst rate of the lamb goats can be increased to 40.0%; meanwhile, the proportion of inner cell mass cells in the blastocyst can be significantly increased, the proportion of trophoblast cells can be reduced, and the total number of blastocyst cells is not affected, which indicates that the RPS26 recombinant protein can significantly increase the blastocyst development rate and improve the blastocyst quality.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the application of recombinant RPS26 protein in promoting early embryonic development in animals. Background Technology

[0002] Goats are highly adaptable and versatile ruminants, providing a variety of products including meat, milk, hides, and cashmere. However, goats exhibit seasonal estrus, meaning their ovarian function is often dormant during the non-breeding season. This leads to decreased estrus and ovulation rates, resulting in fewer lambs per year, severely restricting population expansion and the stability of meat and milk product supply. It also increases breeding costs and makes market regulation more difficult. Currently, goat breeding technologies such as estrus synchronization, superovulation, in vitro maturation of oocytes, in vitro fertilization, embryo culture, and embryo transfer are widely used. The introduction of Juvenile In Vitro Embryo Transfer (JIVET) technology has made it possible to obtain oocytes on a large scale and fully utilize the reproductive potential of superior female animals. This technology helps shorten generation intervals and accelerate genetic progress, providing a breakthrough means for efficient and low-cost breeding and the establishment of core populations. Although JIVET technology can significantly shorten the breeding cycle, its practical application still faces a core bottleneck—low early embryonic developmental potential and poor blastocyst quality in lambs. Possible reasons for developmental limitations include: firstly, insufficient cytoplasmic maturation of lamb oocytes, inadequate reserves of maternal mRNA and essential proteins, and immature mitochondrial function, making it difficult to support complete embryonic development; secondly, existing in vitro culture systems fail to fully mimic the in vivo microenvironment, potentially leading to abnormal epigenetic modifications such as DNA methylation, which in turn interfere with the expression regulation of key genes. However, the core molecular mechanisms leading to early embryonic developmental defects still require further elucidation.

[0003] Ribosomal protein S26 (RPS26) is a component of the 40S ribosome subunit and, as a structural protein, participates in ribosome assembly and maintains protein translation. In eukaryotic cells, RPS26 is mainly located in the cytoplasm, participating not only in mRNA recognition and translation initiation but also in ribosome quality control, cell cycle regulation, and stress response. Current research on RPS26 largely focuses on its function in disease development. Studies have shown that RPS26 is abnormally highly expressed in various tumors and can promote tumor cell proliferation by enhancing ribosome biosynthesis; other reports indicate that RPS26 can interact with the p53 signaling pathway, affecting the apoptosis process. Furthermore, in terms of metabolic regulation, RPS26 has been found to be related to insulin secretion and glucose homeostasis, and its dysregulation may be involved in the development of type 2 diabetes. However, there are currently no reports on the use of recombinant RPS26 protein in early embryonic studies. Summary of the Invention

[0004] To address the issues of low in vitro development potential and poor blastocyst quality in early goat embryos, this invention provides the application of recombinant RPS26 protein in promoting early animal embryo development. By adding recombinant RPS26 protein during early animal embryo culture, the blastocyst development rate and blastocyst quality in goats can be significantly improved.

[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of this invention provides the application of recombinant RPS26 protein in promoting early embryonic development in animals.

[0006] Furthermore, recombinant RPS26 protein was added during early in vitro embryo culture in animals to promote early embryo development.

[0007] Furthermore, the promotion of early embryonic development refers to promoting blastocyst formation or improving blastocyst quality.

[0008] Furthermore, the improvement in blastocyst quality refers to increasing the proportion of inner cell mass cells or decreasing the proportion of trophectoderm cells.

[0009] Furthermore, the animal is a mouse, goat, sheep, or cow.

[0010] Furthermore, the goat is a lamb or an adult goat, the sheep is a lamb or an adult sheep, and the cow is an adult cow or a calf.

[0011] Furthermore, the lambs mentioned are lambs aged 4 to 8 weeks.

[0012] A second aspect of the present invention provides an embryo culture medium, which is composed of the aforementioned RPS26 recombinant protein and an early embryo in vitro culture medium.

[0013] Furthermore, the concentration of recombinant RPS26 protein in the embryo culture medium is 25 ng / mL to 400 ng / mL.

[0014] Furthermore, the early embryo in vitro culture medium contains 2w / v%~3w / v% essential amino acids, 0.5w / v%~1.5w / v% non-essential amino acids, 18ng / mL~22ng / mL epidermal growth factor, 38ng / mL~42ng / mL fibroblast growth factor 2, 18ng / mL~22ng / mL leukemia inhibitory factor, 18ng / mL~22ng / mL insulin-like growth factor 1, 2mM~3mM taurine, 25μg / mL~50μg / mL gentamicin, 6mg / mL~8mg / mL bovine serum albumin, and supplemented with oviduct synthetic fluid.

[0015] Furthermore, each milliliter of the fallopian tube synthetic solution contains 6.29 mg NaCl, 0.534 mg KCl, 0.162 mg KH2PO4, 0.6 μL sodium lactate, 0.089 mg MgSO4, 2.1 mg NaHCO3, 0.0357 mg sodium pyruvate, and 0.299 mg CaCl2·2H2O.

[0016] A third aspect of the present invention provides the application of the above-described embryo culture medium in promoting early embryonic development in animals: a presumed zygote fertilized for 18 hours is placed in the embryo culture medium for culture to promote early embryonic development.

[0017] Furthermore, the presumed zygote is a presumed zygote washed with in vitro culture medium of an early embryo.

[0018] Furthermore, the culture temperature is 38.5℃, the culture humidity is 100% saturated humidity, and the culture gas environment is 5% CO2 and 5% O2.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides the application of recombinant RPS26 protein in promoting early embryonic development in animals. Studies using goat lambs as an experimental model show that adding recombinant RPS26 protein during early in vitro embryo culture increases the blastocyst rate from 15.24% to 40.0%. Simultaneously, while maintaining the total number of blastocyst cells, this treatment optimizes cell lineage distribution, significantly increasing the inner cell mass ratio from 16.97% to 25.23%, and correspondingly decreasing the trophectoderm cell ratio from 83.03% to 74.77%. This indicates that recombinant RPS26 protein not only promotes blastocyst formation but also helps improve blastocyst quality. Based on these findings, this invention further develops an embryo culture medium containing recombinant RPS26 protein at an effective concentration of 25 ng / mL to 400 ng / mL. Using this embryo culture medium to culture probable zygotes during early in vitro embryo culture in animals can promote early embryonic development.

[0020] This invention provides a clear translational pathway for the fields of agricultural reproduction and reproductive medicine. In animal husbandry, the determination of RPS26 and its effective concentration lays the foundation for developing novel embryo culture medium additives with clearly defined chemical compositions and no animal origin, which is expected to directly improve the efficiency of jivet embryo transfer (JIVET) technology and accelerate the breeding process of superior livestock. In human assisted reproduction, given the conservation of fallopian tube fluid exosomes and the ribosomal proteins they carry across species, RPS26 may serve as a novel biomarker for assessing female fallopian tube function or embryonic developmental potential, with potential clinical applications. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The figure shows the experimental results of the application of recombinant RPS26 protein (hRec-RPS26) in promoting early embryonic development in animals. Figure 1 In the diagram, 'a' represents the pre-pubertal goat in vitro embryo production process and the supplementation of hRec-RPS26. Figure 1 Image b in the image represents the blastocyst stage on day 8 after in vitro fertilization, from three independent experiments involving the control group and hRec-RPS26 treatment; the scale bar is 100 μm; the images in the bottom row are magnified views of the area framed in the images in the top row. Figure 1 In the figure, 'c' represents the proportion of embryos at each developmental stage in the control group (n=53), the 25 ng / mL (n=45), the 100 ng / mL (n=42), and the 400 ng / mL (n=56) hRec-RPS26 treatment groups. Blastocyst rate = number of blastocysts / number of fertilized oocytes × 100%. Figure 1 In the figure, d represents representative immunofluorescence staining images of marker proteins of the inner cell mass (ICM) and trophectoderm (TE) of blastocysts in the control group and the hRec-RPS26-treated group. Blue, DNA; green, Sox2; red, Cdx2; scale bar is 20 μm. Figure 1 In the table, e~g represent the total number of blastocyst cells, ICM, and TE ratio in the control group (n=10) and the hRec-RPS26 treatment group (n=16), respectively. Results are expressed as mean ± standard error, with ns indicating no statistical significance. * represent P <0.05; ** represent P <0.01. Detailed Implementation

[0023] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0024] The recombinant RPS26 protein in this invention is the recombinant human RPS26 protein hRec-RPS26 (P2953, FineTest).

[0025] To address the issues of low in vitro developmental potential and poor blastocyst quality in early-stage goat embryos, this invention previously integrated oviduct microenvironment proteomics and metabolomics, oviduct-derived exosome proteomics, and transcriptomics and translatomics techniques from adult goats and goat in vitro fertilized embryos to identify RPS26, a key factor in the early development of goat in vitro fertilized embryos. This invention, by adding recombinant RPS26 protein (hRec-RPS26) to the in vitro culture medium of early embryos, found that it improved the blastocyst development rate and significantly improved blastocyst quality, indicating that recombinant RPS26 protein can be used to promote early-stage goat embryo development.

[0026] Example 1: Application of RPS26 recombinant protein in promoting early embryonic development in animals 1. Collection of oocytes from lamb goats 1) Preparation of oocyte extraction solution: 9.5 mg / mL Tissue Culture Medium 199 (TCM-199, 31100035, Gibco) + 10% Fetal Bovine Serum (10099141, Gibco) + 0.01 mg / mL Heparin Sodium (H3149, Sigma) + 0.42 mg / mL NaHCO3 (S5761, Sigma) + 4.766 mg / mL Hydroxyethylpiperazine Ethyl Acetate (H3375, Sigma) + 0.05 mg / mL Penicillin (P7794, Sigma) + 0.065 mg / mL Streptomycin (S1277, Sigma).

[0027] 2) Collection of oocytes from lambs Each goat lamb (4-8 weeks old) received six injections of follicle-stimulating hormone (FSH) (Ningbo Sansheng), 45 IU each time, with 12-hour intervals between injections. 400 IU of pregnant mare serum gonadotropin (MPSG) (Ningbo Sansheng) was administered concurrently with the first FSH injection. Twelve hours after the last FSH injection, cumulonoocyte complexes (COCs) were aspirated from follicles larger than 2 mm in diameter using an 18G syringe containing 2 mL of oocyte aspiration fluid.

[0028] 2. In vitro maturation of oocytes 1) Collection of serum from estrus goats Estrus detection in female goats: Estrus detection in female goats combines mounting by the male goat with observation of the female goat's vulva. Select a healthy adult male goat and test the female goat twice a day, morning and evening. When the male goat approaches the female goat, the female goat will become excited, and her vulva will become red and swollen, accompanied by a discharge of mucus; this indicates that the female goat is in estrus.

[0029] Collecting estrus goat serum: Collect non-anticoagulated whole blood from estrus ewes, let it stand at room temperature for 2 hours, collect the supernatant serum, centrifuge at 3000 rpm for 10 minutes, filter the supernatant and store it at -80℃.

[0030] 2) Formula for preparing oocyte maturation medium: 199 g of tissue culture medium (11150059, Gibco) + 20 v / v% estrous goat serum + 1 mM / L sodium pyruvate (P4562, Sigma) + 0.05 mM / L L-cysteine ​​(C8755, Sigma) + 1% insulin-transferrin-selenium (ITS, I3146, Sigma) + 20 ng / mL epidermal growth factor (EGF, P00033, Solarbio) + 0.5 IU / mL follicle-stimulating hormone (FSH, F8470, Solarbio) + 0.5 IU / mL luteinizing hormone (LH, L8040, Solarbio) + 1 μg / mL estradiol (E2,758, Sigma) + 25 μg / mL gentamicin (G1914, Sigma).

[0031] 3) In vitro maturation of lamb oocytes The COCs collected in step 1 above were washed three times in oocyte extraction fluid, then washed three times with oocyte maturation medium, and then placed in oocyte maturation medium that had been equilibrated in a CO2 incubator for more than 8 hours (four-well plate maturation culture, 600 μL oocyte maturation medium, surface covered with 350 μL liquid mineral oil), with 10 to 30 COCs per well; the maturation culture conditions were 38.5℃, 100% saturated humidity and 5% CO2, and the culture time was 24 hours.

[0032] 3. In vitro fertilization of oocytes 1) Preparation of fertilization and fallopian tube synthesis fluid (H-SOF) Fertilization fluid: Synthetic oviduct fluid (SOF) + 2% essential amino acids (M5550, Sigma) + 1% non-essential amino acids (M7145, Sigma) + 10% fetal bovine serum (10099141, Gibco) + 0.01 g / L heparin sodium (H3149, Sigma) + 6 mg / mL bovine serum albumin (A9418, Sigma).

[0033] Fallopian tube synthesis solution: 6.29 mg / mL NaCl + 0.534 mg / mL KCl + 0.162 mg / mL KH2PO4 + 0.6 μL / mL sodium lactate + 0.089 mg / mL MgSO4 + 2.1 mg / mL NaHCO3 + 0.0357 mg / mL sodium pyruvate + 0.299 mg / mL CaCl2·2H2O.

[0034] 2) Preparation of fresh semen Semen was collected from Chengdu Ma sheep (4 years old) and immediately diluted appropriately with preheated diluent (Tianyuan Aori); then, high-motile sperm were separated by Percoll gradient centrifugation (SAGE); the sperm precipitate after centrifugation was washed with H-SOF solution and resuspended; finally, the processed sperm (2×10⁻⁶) was collected. 6 Cells / mL ~4×10 6 The cells / mL were transferred into fertilization droplets containing COCs to complete the preparation for in vitro fertilization. The fertilization culture conditions were 38.5℃, 100% saturated humidity, 5% CO2 and 5% O2.

[0035] 3) Oocyte isolation: Hyaluronidase was added to the oocyte extraction solution prepared above to obtain an oocyte extraction solution containing 0.1 w / v% hyaluronidase. Cumulus cells (COCs) that had matured and cultured for 24 hours were transferred to the oocyte extraction solution containing 0.1 w / v% hyaluronidase and gently pipetted to remove most of the cumulus cells, leaving only 1-3 layers. Subsequently, the cells were washed three times with fertilization fluid to obtain oocytes ready for fertilization. These oocytes were then transferred to a fertilization fluid system that had been equilibrated in a tri-gas incubator for at least 8 hours (using a four-well plate, placing 30-40 mature oocytes and 450 µL of fertilization fluid per well, with the surface covered with 300 µL of liquid mineral oil) for in vitro fertilization.

[0036] 4. Early embryo in vitro culture 1) Preparation of in vitro culture medium for early embryos Early embryo in vitro culture medium: SOF + 2 w / v% essential amino acids (M5550, Sigma) + 1 w / v% non-essential amino acids (M7145, Sigma) + 20 ng / mL epidermal growth factor (P00033, Solarbio) + 40 ng / mL fibroblast growth factor 2 (FGF2, P00110, Solarbio) + 20 ng / mL leukemia inhibitory factor (LIF, P0042, Solarbio) + 20 ng / mL insulin-like growth factor 1 (IGF1, P0048, Solarbio) + 2.5 mM taurine (H1384, Sigma) + 25 μg / mL gentamicin (G1914, Sigma) + 6 mg / mL bovine serum albumin (A9418, Sigma).

[0037] Based on a total volume of 1 mL, embryo culture medium A consists of early embryo in vitro culture medium and 25 ng recombinant human RPS26 protein hRec-RPS26 (P2953, Fine Test).

[0038] Embryo culture medium B, with a total volume of 1 mL, consists of early embryo in vitro culture medium and 100 ng hRec-RPS26.

[0039] Embryo culture medium C, with a total volume of 1 mL, consists of early embryo in vitro culture medium and 400 ng hRec-RPS26.

[0040] 2) Early embryo in vitro culture Control group: Probable zygotes were removed 18 hours after fertilization, and the cumulus cells and sperm on the surface were gently removed by agitation. The embryos were washed three times in early embryo culture medium and then placed in early embryo culture medium (4-well plate culture, 500 μL culture medium, surface covered with 400 μL liquid mineral oil) for further culture. In vitro culture conditions were 38.5℃, 100% saturated humidity, 5% CO2, and 5% O2. The time of fertilization was recorded as day zero, and the blastocyst rate was calculated on day eight post-fertilization.

[0041] RPS26 Recombinant Proteome A: Prophetic zygotes were retrieved 18 hours after fertilization, and the cumulus cells and sperm on the surface of the prophetic zygote were gently removed by pipetting. The embryos were washed three times in early embryo culture medium and then placed in embryo culture medium A (four-well plate culture, 500 μL culture medium, surface covered with 400 μL liquid mineral oil) for further culture. In vitro culture conditions were 38.5℃, 100% saturated humidity, 5% CO2, and 5% O2. The time of fertilization was recorded as day zero, and the blastocyst rate was calculated on day eight post-fertilization.

[0042] RPS26 recombinant proteome B: Prophetic zygotes were retrieved 18 hours after fertilization, and the cumulus cells and sperm on the surface of the prophetic zygote were gently removed by pipetting. The embryos were washed three times in early embryo culture medium and then placed in embryo culture medium B (four-well plate culture, 500 μL culture medium, surface covered with 400 μL liquid mineral oil) for further culture. In vitro culture conditions were 38.5℃, 100% saturated humidity, 5% CO2, and 5% O2. The time of fertilization was recorded as day zero, and the blastocyst rate was calculated on day eight post-fertilization.

[0043] RPS26 recombinant proteome C: Prophetic zygotes were retrieved 18 hours after fertilization, and the cumulus cells and sperm on the surface of the prophetic zygote were gently removed by pipetting. The embryos were washed three times in early embryo culture medium and then placed in embryo culture medium C (four-well plate culture, 500 μL culture medium, surface covered with 400 μL liquid mineral oil) for further culture. In vitro culture conditions were 38.5℃, 100% saturated humidity, 5% CO2, and 5% O2. The time of fertilization was recorded as day zero, and the blastocyst rate was calculated on day eight post-fertilization.

[0044] like Figure 1 As shown in figures a-c, in early embryo culture systems, the addition of 25 ng / mL and 100 ng / mL hRec-RPS26 significantly promoted blastocyst formation in goat lamb embryos, while the higher concentration treatment group (400 ng / mL) showed no significant effect. P The concentration >0.05 indicates that hRec-RPS26 has a clear promoting effect on embryonic development. The blastocyst rates corresponding to final hRec-RPS26 concentrations of 0 ng / mL, 25 ng / mL, 100 ng / mL, and 400 ng / mL were 15.24%, 40.00%, 27.12%, and 26.69%, respectively. This indicates that the optimal concentration range for hRec-RPS26 is 25 ng / mL to 100 ng / mL.

[0045] 5. CDX2 and SOX2 immunofluorescence staining Embryos were fixed with 4% paraformaldehyde for 1 hour, then permeated with PBS containing 1% Triton X-100 and 0.1% Tween 20 for 30 min, followed by blocking with 3% bovine serum albumin for 30 min. The blocked embryos were then incubated overnight at 4°C with the following primary antibodies: Mouse anti-CDX2 (BioGenex, AM392-5MSG) and Rabbit anti-SOX2 (1:200, CST, 23064). After washing with PBS, the embryos were incubated with the following secondary antibodies: CoraLite488-conjugated donkey anti-rabbit IgG (1:400, Proteintech, SA00013-6) and CoraLite594-conjugated donkey anti-mouse IgG (1:400, Proteintech, SA00013-7) at room temperature in the dark for 2 hours. After washing with PBS again, nuclear staining and mounting were performed using mounting medium containing DAPI (Vector Laboratories Inc., Burlingame, CA, USA), and finally images were acquired using a laser confocal microscope (FEV4000-IX83, Olympus, Japan).

[0046] like Figure 1 As shown in d~g, hRec-RPS26 treatment also significantly improved blastocyst quality: specifically, the proportion of inner cell mass increased from 16.97% to 25.23%, the proportion of trophectoderm cells decreased from 83.03% to 74.77%, while the total number of blastocyst cells was not significantly affected. These results indicate that RPS26 mainly improves blastocyst development quality by optimizing cell lineage allocation.

[0047] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0048] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of RPS26 recombinant protein in promoting early embryonic development in animals in vitro, characterized in that, The recombinant RPS26 protein is the product of Fine Test, catalog number P2953; the animal is a lamb goat.

2. The application according to claim 1, characterized in that, Adding recombinant RPS26 protein during early in vitro embryo culture in animals promotes early embryo development.

3. The application according to claim 2, characterized in that, The promotion of early embryonic development refers to promoting blastocyst formation or improving blastocyst quality.

4. The application according to claim 3, characterized in that, Improving blastocyst quality means increasing the proportion of inner cell mass cells or decreasing the proportion of trophectoderm cells.

5. An embryo culture medium for promoting early embryonic development in goats, characterized in that, The embryo culture medium is composed of the RPS26 recombinant protein as described in claim 1 and an early embryo in vitro culture medium; the RPS26 recombinant protein is a protein with the product number P2953 from Fine Test.

6. The embryo culture medium according to claim 5, characterized in that, The concentration of recombinant RPS26 protein in the embryo culture medium was 25 ng / mL to 400 ng / mL.

7. The embryo culture medium according to claim 5, characterized in that, The early embryo in vitro culture medium contains 2w / v%~3w / v% essential amino acids, 0.5w / v%~1.5w / v% non-essential amino acids, 18ng / mL~22ng / mL epidermal growth factor, 38ng / mL~42ng / mL fibroblast growth factor 2, 18ng / mL~22ng / mL leukemia inhibitory factor, 18ng / mL~22ng / mL insulin-like growth factor 1, 2mM~3mM taurine, 25μg / mL~50μg / mL gentamicin, 6mg / mL~8mg / mL bovine serum albumin, and supplemented with oviduct synthetic fluid.

8. The application of the embryo culture medium according to claim 5 in promoting early embryonic development in animals in vitro, characterized in that, The presumed zygotes, fertilized for 18 hours, were cultured in the embryo culture medium to promote early embryonic development.

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