In-vitro maturation culture solution for giant panda oocyte and application thereof

Abstract

This invention relates to an in vitro maturation culture medium for giant panda oocytes and its application, belonging to the field of biotechnology. The invention discloses an in vitro maturation culture medium for giant panda oocytes, which simultaneously adds the following three paracrine factors to the basal culture medium: adenosine diphosphate riboylation factor 6 (ARF6); ciliary neurotrophic factor (CNTF); and hepatocyte growth factor (HGF). The invention also provides the application of the aforementioned in vitro maturation culture medium for giant panda oocytes, specifically for the in vitro maturation culture of giant panda oocytes. This invention effectively addresses the shortcomings of existing in vitro oocyte maturation technologies, significantly improves oocyte maturation quality, is simple to operate, and has high safety, providing a key technological reserve for assisted reproduction in giant pandas.

Description

Technical Field

[0001] This invention relates to an in vitro maturation culture medium for oocytes, specifically to an in vitro maturation culture medium for oocytes used in giant pandas and its applications, belonging to the field of biotechnology. Background Technology

[0002] In vitro oocyte maturation technology has undergone long-term development and is now widely used. However, current in vitro oocyte maturation technology generally suffers from the following problems:

[0003] 1. Low maturation rate and asynchronous maturation: Current in vitro oocyte maturation techniques generally suffer from a lack of coordination and synchronization between nuclear and cytoplasmic maturation. This directly leads to impaired oocyte development potential, low embryo development rate after in vitro fertilization, and poor embryo quality. For example, in conventional ovulation induction cycles, 15-20% of oocytes fail to reach metaphase II of meiosis, resulting in wasted oocytes.

[0004] 2. In vitro culture systems struggle to simulate the in vivo microenvironment: Existing in vitro oocyte maturation culture systems are not sufficiently sophisticated and cannot fully mimic the complex microenvironment required for oocyte maturation in vivo. This is especially true for rare and endangered species like the giant panda, whose oocytes possess unique physiological characteristics (such as being rich in yolk granules and yolk vesicles, and exhibiting the unique phenomenon of conjoined eggs), for which a clear and efficient in vitro maturation culture system has not yet been established.

[0005] 3. Lack of efficient and safe paracrine factor supplementation strategies: Studies have shown that paracrine regulation plays a crucial role in oocyte maturation and early embryonic development. While current technologies, such as somatic cell-oocyte co-culture systems, can provide some paracrine signals, the process is cumbersome, requires maintaining optimal cell conditions, and carries the risk of cell contamination. Furthermore, the paracrine factors added to conventional culture media are limited in variety and lack a combined formulation targeting key aspects of oocyte maturation, resulting in limited comprehensive regulatory effects on cumulus cell expansion, oocyte aging, apoptosis, and mitochondrial function. Summary of the Invention

[0006] One objective of this invention is to overcome the aforementioned deficiencies in the prior art and provide a culture medium for the in vitro maturation of giant panda oocytes. This culture medium, through the addition of a specific combination of giant panda-derived paracrine factors, can more effectively simulate the in vivo maturation microenvironment of oocytes, significantly improving the nuclear maturation rate and cytoplasmic maturation quality of oocytes, promoting cumulus cell expansion, reducing oocyte aging and early apoptosis, and increasing mitochondrial membrane potential, thereby enhancing the overall developmental potential of oocytes and providing key technical support for establishing an efficient in vitro maturation system for giant panda oocytes.

[0007] The present invention is implemented as follows:

[0008] This invention provides an in vitro maturation culture medium for giant panda oocytes, in which the following three paracrine factors are simultaneously added to the basal culture medium:

[0009] ADP-ribosylation factor 6 (ARF6).

[0010] Ciliary neurotrophic factor (CNTF).

[0011] Hepatocyte growth factor (HGF).

[0012] The concentration of ARF6 was 10 ng / mL, the concentration of CNTF was 3 ng / mL, and the concentration of HGF was 30 ng / mL.

[0013] The basal culture medium can be a conventional oocyte maturation basal culture medium in the art, such as TCM-199. Of course, those skilled in the art can use other oocyte maturation basal culture media to achieve the purpose of this invention.

[0014] This invention also provides the application of the aforementioned giant panda oocyte in vitro maturation culture medium, which is used for the in vitro maturation culture of giant panda oocytes. Of course, the specific culture method can refer to conventional mammalian oocyte in vitro maturation culture methods, and this invention does not limit it in this regard.

[0015] Compared with the prior art, the present invention has at least the following outstanding technical effects:

[0016] This invention addresses the shortcomings of existing in vitro maturation (IVM) technology for oocytes by combining three paracrine factors—ARF6, CNTF, and HGF—at specific concentrations for oocyte maturation culture. This overcomes the technical deficiencies of existing culture systems, which rely on single paracrine factors and have incomplete regulatory effects, and achieves multi-target synergistic regulation of both nuclear and cytoplasmic maturation of oocytes.

[0017] Significantly improves oocyte maturation quality: The culture medium of this invention significantly increases the first polar body emission rate of oocytes, enhances the expansion of cumulus cells, reduces oocyte aging and early apoptosis, and lowers the degree of mitochondrial membrane potential depolarization, thus comprehensively improving the developmental potential of oocytes from multiple dimensions.

[0018] Simple to operate and highly safe: This invention uses the method of directly adding paracrine factors with clearly defined functions to the basic culture medium, which avoids the problems of cumbersome operation, need to maintain the optimal state of cells, and the risk of cell contamination in the existing somatic cell-oocyte co-culture system. It has the advantages of simple operation, clear composition, and high safety.

[0019] This invention provides key technological reserves for assisted reproduction in giant pandas: It validated the significant effects of combining paracrine factors using a canine oocyte model. This invention provides a directly applicable technical solution for the in vitro maturation culture of giant panda oocytes, which is of great significance for improving the genetic quality and protecting genetic diversity of captive giant panda populations. Attached Figure Description

[0020] Figure 1 Schematic diagram of the effects of adding different paracrine factors on oocyte maturation; (A) Nuclear phase images of canine oocytes at GV, GVBD, MI, and MII stages. Scale bar: 30 µm. Statistical analysis of PB1 emission rates of COCs with different concentrations of ARF6 (B), CNTF (C), HGF (D), and the optimal concentration of a single factor or a three-factor combination (ACH) (E). Data are shown as mean ± standard deviation; no common letters in the figure indicate statistical differences (P < 0.05). (F) Typical image of oocytes after ACH culture.

[0021] Figure 2 Schematic diagram of cumulus cell expansion detection. (A) Images of three typical cumulus cell expansions, scale bar at 150 µm. (B) Percentage of COCs at different stages of cumulus cell expansion with or without ACH. Data are shown as mean ± standard deviation. **P<0.01, ***P<0.001. (C) Statistical analysis of relative PTGS2 mRNA expression, with GAPDH as the internal reference gene. ***P<0.001.

[0022] Figure 3 Detection of oocyte aging and early apoptosis, schematic diagram of oocyte mitochondrial membrane potential detection, including: (A) Reactive oxygen species (ROS) staining, scale bar 20 µm; Annexin V staining (AXIN-V), scale bar 20 µm. (B) ROS cumulative rate statistics, data show mean ± standard deviation. ***P<0.001. (C) Early apoptosis rate statistics, data show mean ± standard deviation. ***P<0.001. (D) Oocyte mitochondrial membrane potential staining. Monomer, green fluorescence; Diner, red fluorescence, scale bar 20 µm. (E) Mitochondrial high membrane potential rate statistics, data show mean ± standard deviation. ***P<0.001. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0024] Example 1: Isolation of endometrial stem cells from giant pandas and screening of paracrine factors

[0025] To screen for paracrine factors with the potential to promote oocyte maturation, the applicant first isolated and cultured endometrial stem cells from the uterine tissue of a deceased giant panda. Morphological observation, growth curve determination, chromosome karyotype analysis, detection of mesenchymal stem cell surface markers (positive for CD44, CD49f, and CD105, negative for CD31 and CD34), and detection of pluripotent transcription factor expression (high expression of SOX2 and KLF4) confirmed that the obtained cells possessed typical mesenchymal stem cell characteristics and had the potential to differentiate into adipocytes, osteoblasts, neural cells, and hepatocytes.

[0026] Culture supernatants from giant panda endometrial stem cells, skin fibroblasts, and umbilical cord mesenchymal stem cells were collected for liquid chromatography-mass spectrometry (LC-MS). Results showed that 1073 proteins were upregulated in endometrial stem cells compared to skin fibroblasts, and 1558 proteins were upregulated in endometrial stem cells compared to umbilical cord mesenchymal stem cells. Bioinformatics analysis, combined with KEGG signaling pathway enrichment analysis (PI3K-Akt signaling pathway, mTOR signaling pathway, etc.) and differential protein functional annotation, identified ARF6, CNTF, and HGF—three paracrine factors highly expressed in giant panda endometrial stem cells and closely related to oocyte maturation regulation—as candidate factors.

[0027] Example 2: Single-factor concentration gradient experiment

[0028] To determine the optimal concentration of each candidate factor, canine oocytes were used as an experimental model. Single-factor addition experiments with different concentration gradients were set up in the basal culture medium TCM-199 to detect the effect of each factor on the oocyte first polar body emission rate.

[0029] In the following embodiments, the paracrine factors used are commercially available recombinant human proteins. The catalog numbers and manufacturers of the three paracrine factors are as follows: ARF6 (catalog number: ARF6-369H; manufacturer: Creative BioMart), CNTF (catalog number: 01-195; manufacturer: Sigma-Aldrich), and HGF (catalog number: SRP 6014; manufacturer: Sigma-Aldrich). Of course, those skilled in the art can choose other suitable paracrine factors according to actual needs.

[0030] In the following experiment, the factor concentration was set using the academically recognized three-fold concentration increment test method. After experimental testing, it was found that the optimal concentration had a promoting effect, but excessively high concentrations would have a reverse effect. Therefore, the optimal concentration was determined.

[0031] (1) ARF6 concentration gradient experiment

[0032] ARF6 concentrations of 0 ng / mL (control group), 1 ng / mL, 3 ng / mL, 10 ng / mL, 30 ng / mL, and 100 ng / mL were used to treat canine cumulus-oocyte complexes (COCs). Oocyte maturation stages were determined by nuclear phase staining after culture. Results showed that the first polar body emission rate was highest at a ARF6 concentration of 10 ng / mL, significantly higher than the control group (P < 0.001), and also higher than other concentration groups. Figure 1 B).

[0033] (2) CNTF concentration gradient experiment

[0034] CNTF concentrations were set at 0 ng / mL (control group), 0.3 ng / mL, 1 ng / mL, 3 ng / mL, 10 ng / mL, and 30 ng / mL. Results showed that the first electrode emission rate of CNTF was highest at a concentration of 3 ng / mL, significantly higher than the control group (P < 0.001), and also higher than other concentration groups. Figure 1 C).

[0035] (3) HGF concentration gradient experiment

[0036] HGF concentrations were set at 0 ng / mL (control group), 1 ng / mL, 3 ng / mL, 10 ng / mL, 30 ng / mL, and 100 ng / mL. Results showed that the first electrode emission rate was highest at a concentration of 30 ng / mL, significantly higher than the control group (P < 0.001), and also higher than other concentration groups. Figure 1 D).

[0037] In summary, the optimal single-factor addition concentrations for ARF6, CNTF, and HGF were determined to be 10 ng / mL, 3 ng / mL, and 30 ng / mL, respectively.

[0038] Example 3: Verification of the synergistic effect of combined factors

[0039] Based on the determination of the optimal concentration of each single factor, a three-factor combination addition experiment was further carried out. The experiment was divided into a control group (basal culture medium), an ARF6 single factor group (10 ng / mL), a CNTF single factor group (3 ng / mL), an HGF single factor group (30 ng / mL), and a combination factor group (ACH group, containing ARF6 10 ng / mL, CNTF 3 ng / mL, and HGF 30 ng / mL).

[0040] (1) Detection of oocyte maturation rate

[0041] The maturation stage of oocytes was determined by nuclear phase staining, and the first polar body emission rate was calculated. Figure 1 A). The results showed that the first polar body emission rate of the ACH group was significantly higher than that of the control group (P < 0.001) and also significantly higher than that of each single-factor group (P < 0.05), and the maturity rate was further increased by about 15% compared with the single-factor groups. This result indicates that the three have a synergistic effect at this concentration ratio. Figure 1 E).

[0042] (2) Detection of the extent of cumulus cell expansion

[0043] The extent of cumulus cell expansion is the gold standard for evaluating oocyte maturation. Based on the morphology of cumulus cell expansion, it is classified into three categories: unexpanded, partially expanded, and fully expanded. Figure 2 A). The results showed that the proportion of cumulus cells with complete expansion in the ACH group was significantly higher than that in the control group (P < 0.001). Figure 2 B). Simultaneously, the mRNA expression level of the cumulus expansion marker gene PTGS2 was detected using real-time quantitative PCR. The results showed that the PTGS2 expression level in the ACH group was significantly higher than that in the control group (P < 0.001). Figure 2 C).

[0044] (3) Detection of oocyte aging and early apoptosis

[0045] The degree of oocyte aging was detected by reactive oxygen species (ROS) staining. Figure 3 The results (first column A) showed that the ROS accumulation in oocytes of the ACH group was significantly lower than that of the control group (P < 0.001). Figure 3 B). Early apoptosis was detected using annexin V (AXIN-V) staining. Figure 3 (Column A, second column), the results showed that the fluorescence intensity of the ACH group was significantly weaker than that of the control group (P < 0.001). Figure 3 C) indicates a lower incidence of early apoptosis.

[0046] (4) Detection of mitochondrial membrane potential in oocytes

[0047] Mitochondrial membrane potential was detected using immunofluorescence staining (red fluorescence indicates dimer form, representing high membrane potential; green fluorescence indicates monomer form, representing low membrane potential). Figure 3 D). The results showed that the ACH group had stronger red fluorescence intensity and weaker green fluorescence intensity. Statistical analysis showed that the proportion of mitochondrial membrane potential dimers in oocytes of the ACH group was higher (P < 0.001). Figure 3 E) indicates that the oocytes are of higher quality.

[0048] Example 4: A method for preparing one culture medium according to the present invention

[0049] Take TCM-199 basal culture medium, add conventional components, then add ARF6 to a final concentration of 10 ng / mL, CNTF to a final concentration of 3 ng / mL, and HGF to a final concentration of 30 ng / mL. Mix thoroughly to obtain the in vitro maturation culture medium for giant panda oocytes described in this invention. The selection and dosage of conventional components can be determined by those skilled in the art based on actual needs and do not constitute a limitation of this invention.

[0050] As a more specific embodiment of the present invention, the conventional components added to the TCM-199 basal culture medium include: 10% (volume concentration) fetal bovine serum (FBS), luteinizing hormone (LH, 10 μg / mL), follicle-stimulating hormone (FSH, 10 μg / mL), cysteine ​​(Cysteine, 1 mM), sodium pyruvate (Sodium pyruvate, 0.2 mM), β-estradiol (β-estradiol, 1 μg / mL), and 1% (volume concentration) penicillin / streptomycin.

[0051] Although the present invention has been described herein with reference to illustrative embodiments, the above embodiments are merely preferred embodiments of the present invention, and the implementation of the present invention is not limited to the above embodiments. It should be understood that those skilled in the art can devise many other modifications and implementations, which will fall within the scope and spirit of the principles disclosed in this application.

Claims

1. An in vitro maturation culture medium for giant panda oocytes, characterized in that: The following three paracrine factors were added simultaneously to the basal culture medium: Adenosine diphosphate ribosylation factor 6, also known as ARF6; Ciliary neurotrophic factor, i.e., CNTF; Hepatocyte growth factor, or HGF.

2. The in vitro maturation culture medium for giant panda oocytes according to claim 1, characterized in that: The concentration of ARF6 was 10 ng / mL, the concentration of CNTF was 3 ng / mL, and the concentration of HGF was 30 ng / mL.

3. The in vitro maturation culture medium for giant panda oocytes according to claim 1 or 2, characterized in that: The basal culture medium is a conventional oocyte maturation basal culture medium: TCM-199.

4. The application of the in vitro maturation culture medium for giant panda oocytes as described in any one of claims 1 to 3, characterized in that: It is used for in vitro maturation culture of giant panda oocytes.

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