Application of IL17D in rescuing oocytes damaged by cold stress
By adding IL17D to the oocyte culture medium, the problem of impaired maturation quality and developmental ability of pig oocytes under cold stress was solved, the maturation rate and embryo quality were improved, the level of reactive oxygen species was reduced, and the antioxidant capacity was enhanced.
Patent Information
- Application Number
- CN202511065072.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-31
- Publication Date
- 2025-10-17
AI Technical Summary
Pig oocytes are easily damaged under cold stress conditions, which affects their maturation quality and developmental ability. Existing technologies lack effective rescue methods.
IL17D was added to the oocyte culture medium at a concentration of 50 ng/mL. The culture medium consisted of TCM-199 basal culture medium, gonadotropins, luteinizing hormone-releasing hormone A3, cysteine, serum replacement, insulin-transferrin-selenium liquid culture medium supplement, EGF, FGF2, LIF, IGF, inositol, progesterone and IL17D for in vitro maturation culture.
It significantly improved the maturation rate of oocytes damaged by cold stress and the developmental quality of somatic cell cloned embryos, reduced the level of reactive oxygen species, increased glutathione content and antioxidant gene expression, and rescued the damage to oocytes caused by cold stress.
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Figure CN120796176A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of oocyte culture, and particularly relates to application of IL17D in rescuing oocytes damaged by cold stress. BACKGROUND
[0002] Stress is an adaptive response of organisms to environmental stimuli. This stress response can cause changes in the physiological state, behavioral characteristics and homeostasis of organisms. Factors that can cause stress in animals are collectively referred to as stress factors, which can also be referred to as stressors. Cold, crowding, hunger, sudden changes in environmental conditions, etc. can all be stressors that cause stress in animals. Among them, cold environment as a stressor can affect the physiological immunity, blood indicators, antioxidant performance and expression changes of cold stress-related genes of the organism.
[0003] Pig oocytes have a high fat content and a large volume, and are therefore very sensitive to changes in environmental temperature, which can easily cause cold stress, thereby adversely affecting the maturation quality and development ability of the oocytes. Too low a temperature can cause oxidative damage to the oocytes, damage to the spindle, microfilament, chromosome and mitochondrial structure and function, and thus affect the in vitro development ability of the oocytes. Many studies have reported the effects of cold stress on the development quality of oocytes or oocytes during transportation and culture.
[0004] Therefore, there is a need to research a substance or culture medium or method that can rescue oocytes damaged by cold stress, to provide a solution to the problem of damage to the quality of pig oocytes caused by cold stress in winter.
[0005] Interleukin 17 (IL17) is secreted by CD4+ T cells, and can induce epithelial cells, endothelial cells and fibroblasts to synthesize and secrete IL-6, IL-8, G-CSF and PGE2, and promote the expression of ICAM-1. The members of the interleukin 17 (IL17) family include 6 ligands IL17A-IL17F and 5 receptors IL-17RA-RE. SUMMARY
[0006] The purpose of the present application is to provide application of IL17D in rescuing oocytes damaged by cold stress, to rescue the damage to pig oocytes caused by cold stress, and to provide a solution to the problem of damage to the quality of pig oocytes caused by cold stress in winter.
[0007] According to a first aspect of the present application, there is provided an application of IL17D in rescuing oocytes damaged by cold stress. Thus, by adding IL17D to the oocyte maturation culture solution, the damage of pig oocytes caused by cold stress can be rescued, thereby providing a solution to the problem of damaged quality of pig oocytes caused by cold stress in winter.
[0008] In some embodiments, the application is to rescue the damage of oocytes caused by cold stress by adding IL17D to the oocyte culture solution.
[0009] In some embodiments, the concentration of IL17D is 50 ng / mL.
[0010] According to a second aspect of the present application, there is provided a culture solution capable of rescuing oocytes damaged by cold stress, wherein the culture solution contains IL17D. Thus, by using the culture solution, the damage of pig oocytes caused by cold stress can be rescued, thereby providing a solution to the problem of damaged quality of pig oocytes caused by cold stress in winter.
[0011] In some embodiments, the concentration of IL17D is 50 ng / mL.
[0012] In some embodiments, the culture solution contains the following components: TCM-199 basic culture solution, 10 IU / m blood gonadotropin, 10 IU / mL luteinizing hormone-releasing hormone A3, 0.6 mM cysteine, 5% serum substitute, insulin transferrin selenium liquid culture medium supplement, 10 ng / mL EGF, 40 ng / mL FGF2, 20 ng / mL LIF, 20 ng / mL IGF, 5 μg / mL inositol, 10 nmol / L progesterone and 50 ng / mL IL17D.
[0013] According to a third aspect of the present application, there is provided an application of the culture solution in rescuing oocytes damaged by cold stress. Thus, by using the culture solution, the damage of pig oocytes caused by cold stress can be rescued, thereby providing a solution to the problem of damaged quality of pig oocytes caused by cold stress in winter.
[0014] According to a fourth aspect of the present application, there is provided an application of the culture solution in improving the quality of somatic cell cloned embryos, wherein the oocytes used in the somatic cell cloning are oocytes damaged by cold stress. Thus, by using the culture solution, the damage of pig oocytes caused by cold stress can be rescued, thereby providing a solution to the problem of damaged quality of pig oocytes caused by cold stress in winter.
[0015] According to a fifth aspect of the present application, there is provided a method for improving the quality of in vitro maturation of oocytes damaged by cold stress, by placing the oocytes damaged by cold stress into the culture solution for in vitro maturation culture. Thus, the damage of pig oocytes caused by cold stress can be rescued by the culture solution, and the quality of in vitro maturation of oocytes can be improved.
[0016] According to a sixth aspect of the present application, there is provided an application of IL17D in reducing the level of reactive oxygen species in oocytes damaged by cold stress.
[0017] According to a seventh aspect of the present application, there is provided an application of IL17D in improving the content of glutathione in oocytes damaged by cold stress.
[0018] According to an eighth aspect of the present application, there is provided an application of IL17D in improving the expression of antioxidant genes in oocytes damaged by cold stress.
[0019] In some embodiments, IL17D can improve the expression levels of BIRC5, CAT, and GPX4 genes in oocytes damaged by cold stress.
[0020] Advantages of the present application:
[0021] The present application discloses an application of IL17D in rescuing oocytes damaged by cold stress, a culture solution for rescuing oocytes damaged by cold stress, and an application of the culture solution in rescuing oocytes damaged by cold stress, an application of the culture solution in improving the quality of somatic cell cloned embryos, a method for improving the quality of in vitro maturation of oocytes damaged by cold stress, and an application of IL17D in reducing the level of reactive oxygen species in oocytes damaged by cold stress, an application of IL17D in improving the content of glutathione in oocytes damaged by cold stress, and an application of IL17D in improving the expression of antioxidant genes in oocytes damaged by cold stress. By adding IL17D to the in vitro maturation culture solution of oocytes, the damage of pig oocytes caused by cold stress can be rescued, and a new solution and idea are provided for solving the problem that oocytes are damaged by cold stress in winter, which affects the quality of in vitro maturation of oocytes and further affects the quality of embryos. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 The results of the effect of IL17D treatment on the ROS level of pig oocytes damaged by cold stress are shown in the following figure: Figure 1 A shows the ROS fluorescence distribution in pig oocytes damaged by cold stress in the control group and the IL17D treatment group, Figure 1B represents the statistical results of the relative fluorescence intensity of ROS in the cold-stressed damaged pig oocytes in the control group and the IL17D treatment group, * represents significant difference P<0.05, Control represents the control group, and IL17D represents the IL17D treatment group;
[0023] Figure 2 The figure shows the effect of IL17D treatment on the GSH level of the cold-stressed damaged pig oocytes, wherein, Figure 2 A represents the GSH fluorescence distribution in the cold-stressed damaged pig oocytes in the control group and the IL17D treatment group, Figure 2 B represents the statistical results of the relative fluorescence intensity of GSH in the cold-stressed damaged pig oocytes in the control group and the IL17D treatment group, * represents significant difference P<0.05, Control represents the control group, and IL17D represents the IL17D treatment group;
[0024] Figure 3 The figure shows the effect of IL17D treatment on the cold-stressed damaged pig oocytes antioxidant-related genes, wherein, * represents significant difference P<0.05, Control represents the control group, and IL17D represents the IL17D treatment group. DETAILED DESCRIPTION
[0025] The application will be further described in detail below with reference to the accompanying drawings.
[0026] 1. Preparation of pig oocyte in vitro maturation culture solution.
[0027] a. Basic oocyte in vitro maturation culture solution (control group): based on TCM-199 basic culture solution, add 10 IU / mL blood gonadotropin, 10 IU / mL luteinizing hormone-releasing hormone A3, 0.6 mM cysteine, 5% serum substitute, insulin transferrin selenium liquid culture medium supplement (1x), 10 ng / mL epidermal growth factor (EGF), 40 ng / mL fibroblast growth factor 2 (FGF2), 20 ng / mL leukemia inhibitory factor (LIF), 20 ng / mL insulin-like growth factor (IGF), 5 μg / mL inositol, 10 nmol / L progesterone.
[0028] b. IL17D (interleukin 17D) oocyte in vitro maturation culture solution (IL17D treatment group): supplement 50 ng / mL of IL17D in the basic oocyte in vitro maturation culture solution.
[0029] 2. Collection and in vitro culture of oocytes.
[0030] Pig ovaries were collected from slaughterhouse in winter, and washed with physiological saline supplemented with penicillin and streptomycin. The temperature of the physiological saline was measured. If the temperature was lower than 22℃, the ovary was considered to be under cold stress. Because of the low temperature in winter, especially in northern China, the ovaries were often under cold stress (temperature lower than 22℃) during transportation, which seriously affected the quality of in vitro maturation of oocytes and further affected the quality of somatic cell cloned embryos.
[0031] To facilitate experimental research, the oocytes were subjected to cold stress in winter by placing the ovaries in physiological saline at 20℃ for 4h. Then the ovaries were moved to physiological saline at 37℃. Pig ovaries with follicles of 3-6mm in diameter were selected, and oocytes were extracted from the follicles. The cumulus cells-oocyte complexes (COCs) with uniform cytoplasm and at least three layers of compact cumulus cells were selected. After all the oocytes were selected, they were placed in the in vitro maturation medium of the control group and the IL17D treatment group, respectively, and then cultured in an incubator at 38.5℃ for 44h. After in vitro maturation, the cumulus cells-oocyte complexes were treated with hyaluronidase, and the cumulus cells were discarded. The maturation rate of oocytes was determined by whether the first polar body was discharged. The oocytes that discharged the polar body, had intact cytoplasm, and had a round cell shape were selected to construct embryos.
[0032] 3. Preparation and culture of somatic cell cloned embryos.
[0033] Construction of pig cloned embryos: The donor cells were prepared in advance in a 6cm culture dish. When the cells grew to 80%-90%, the culture medium was aspirated, and the cells were gently washed twice with DPBS. Then, 1mL of preheated trypsin digestion solution was added to the well, and the culture dish was blown several times with a pipette gun. Then, the cell suspension was transferred to a 15mL centrifuge tube and centrifuged at 800rpm for 5min. The supernatant was discarded, and 1mL of culture medium was added to resuspend the cells for later use.
[0034] Preparation of recombinant embryos: The in vitro matured oocytes were subjected to enucleation, and then good-quality donor cells were selected under a microscope and injected into the gap of the oocytes. After all the enucleated oocytes were injected, fusion and activation were performed to obtain recombinant embryos. The parameters of the electric fusion instrument were set as 50V, 50μs, and 2DC. The electric fusion groove was washed with fusion-activation solution, 500μL of fusion-activation solution was added, 10 enucleated oocytes were transferred to the electric fusion groove, and the oocyte recipients-nuclear donors were rotated with a glass needle to make the axis perpendicular to the electrodes for fusion and activation.
[0035] The PZM culture solution components are as follows: NaCl 108.00 mM, KCl 10.00 mM, KH2PO40.35 mM, MgSO4·7H2O 0.40 mM, NaHCO325.07 mM, sodium pyruvate (Na-Pyruvate) 0.20 mM, calcium lactate 2.00 mM, Hypotaurine 5.00 mM, L-glutamine 1.00 mM, essential amino acids 20 mL / L, and non-essential amino acids 10 mL / L.
[0036] In vitro culture of embryos: After the construction of the recombinant embryos, the embryos were placed in a four-well plate containing PZM culture solution and transferred to a 38.5°C saturated humidity incubator for further culture. Data were recorded at 48 h and 168 h, respectively.
[0037] 4. Detection of ROS level (active oxygen level) and glutathione (GSH) content in oocytes
[0038] The ROS level in oocytes was detected using an active oxygen detection kit (Yeasen, 50101ES01). First, the cumulus cells were completely removed using hyaluronidase, and then the DCHF-DA concentrated stock solution was diluted to a working solution concentration of 10 μmol / L (1:1000) according to the instructions, and a ROS staining disc was prepared. The oocytes were transferred into the ROS staining disc, incubated in the dark for 30 min, and observed and photographed using a fluorescence microscope. The operation parameters of each group were kept consistent during photographing.
[0039] The GSH level in oocytes was detected using Cell Tracker Blue CMF2HC reagent (Invitrogen, C12881). The Cell Tracker Blue CMF2HC powder was dissolved in DMSO to obtain a concentrated stock solution (10 mmol / L), and then 1 μL of the concentrated stock solution was diluted to a working solution concentration of 10 μmol / L using the maturation culture solution, and a GSH staining disc was prepared. After the granulosa cells were removed, the oocytes were transferred into the GSH staining disc and incubated for 30 min. The oocytes were observed and photographed under a fluorescence microscope, and the fluorescence parameters of two groups were kept consistent. The whole experiment was carried out in the dark.
[0040] 5. Micro-reverse of porcine oocytes and real-time fluorescence quantitative PCR
[0041] Each group collected 100 mature cultured oocytes, and the granulosa cells on the surface were digested. According to the instructions of the total RNA extraction kit (Magen, R4125-02), RNA extraction was performed, the RNA concentration was measured, and a part of the sample without RNA degradation was reverse transcribed.
[0042] The reverse transcription process was mainly based on One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, AT311-02) instructions. The reverse transcription process is shown in Table 1.
[0043] The cDNA obtained by micro-reverse transcription was used as a template, and PowerUp TM SYBR TM Green Master Mix kit (Thermo Fisher, A25742) instructions for RT-qPCR (Real Time Quantitative PCR, Real-time fluorescent quantitative PCR) to detect sample gene expression. The reaction system is as shown in Table 2. The reaction conditions are 50°C for 2 min, 95°C for 10 min, 95°C for 10 sec, 60°C for 1 min, and the sample is cycled 45 times during PCR. The β-actin gene is used as the internal reference gene for oocyte quantitative PCR, and the primer sequences of other genes are shown in Table 3. The relative expression amount of the gene is calculated by using the calculation formula R=2-[ △Ctsample-△Ctcontrol ] and the primer sequences are obtained from the NCBI website and synthesized by Beijing Lihe Huada Gene Technology Co., Ltd.
[0044] Table 1 Reverse transcription process
[0045]
[0046] Table 2 qPCR reaction system
[0047]
[0048] Table 3 qPCR reaction primer sequence
[0049]
[0050] 6、Experimental results
[0051] (1) The effect of IL17D on the in vitro maturation of pig oocytes and early embryonic development under cold stress.
[0052] The ovary was placed in 20°C physiological saline for 4h, then the oocyte was extracted, and the oocyte was divided into IL17D treatment group (50ng / mL IL17D) and control group (0ng / mL IL17D), the control group used the basic oocyte in vitro maturation culture fluid, and the IL17D treatment group used the oocyte in vitro maturation culture fluid added with 50ng / mL IL17D (50ng / mL IL17D was supplemented in the basic oocyte in vitro maturation culture fluid). Then the oocytes in the two groups were cultured in vitro, and the first polar body discharge rate, i.e. the maturation rate, was counted, and the results are shown in Table 4: compared with the control group, the addition of 50ng / mL IL17D in the treatment group can significantly improve the maturation rate of oocytes (56.62% vs. 46.25%, P<0.05), which indicates that IL17D can rescue the cold-stressed damaged oocytes and improve the maturation rate of the cold-stressed damaged oocytes.
[0053] Table 4 Effect of supplementing IL17D on the maturation rate of cold-stressed damaged pig oocytes
[0054]
[0055] Note: Values with different superscripts in the same column represent P<0.05.
[0056] The mature oocytes in the two groups were subjected to somatic cell cloning, and then the development efficiency of the embryos was counted, and the results are shown in Table 5: compared with the control group, the cleavage rate (66.27% vs. 37.96%, P<0.05) and the blastocyst rate of the embryos in the treatment group were significantly higher than those in the control group (26.16% vs. 7.41%, P<0.05). It is shown that IL17D can significantly improve the in vitro maturation rate of cold-stressed damaged pig oocytes, and the cleavage rate and blastocyst rate of somatic cell cloned embryos. It is further shown that IL17D can rescue the quality of cold-stressed damaged oocytes, improve the maturation rate of cold-stressed damaged oocytes, and further improve the quality of somatic cell cloned embryos.
[0057] Table 5 Effect of supplementing IL17D on the subsequent development ability of cold-stressed damaged pig oocytes
[0058]
[0059] Note: Values with different superscripts in the same column represent P<0.05; cleavage rate, blastocyst rate are (mean ± S.E.). (2) Effect of supplementing IL17D during in vitro maturation process on ROS level and GSH content of cold-stressed pig oocytes
[0060] Cold stress leads to excessive intracellular ROS level, which is an important factor for damaging the quality of oocytes. For example, the ROS level of cold-stressed oocytes is significantly higher than that of normal oocytes, and the GSH content of cold-stressed oocytes is significantly lower than that of normal oocytes. Figure 1As shown in A, oocytes in the control group showed stronger green fluorescence, while the green fluorescence in the IL17D-treated group was weaker. The stronger the green fluorescence, the higher the ROS level. Using ImageJ software to calculate the fluorescence signal, it was found that the fluorescence signal level in the IL17D-treated group was significantly lower than that in the control group (P<0.05) ( Figure 1 B) This indicates that IL17D can significantly reduce ROS levels in porcine oocytes damaged by cold stress.
[0061] Intracellular GSH is an important mediator for scavenging ROS. By comparing the fluorescence intensity after staining ( Figure 2 A), it can be found that the fluorescence intensity of the IL17D-treated group is stronger than that of the control group. Quantitative analysis of the fluorescence signal using ImageJ software revealed that the fluorescence intensity of the IL17D-treated group was significantly higher than that of the control group (P<0.05) ( Figure 2 B) The above data indicate that IL17D can increase the GSH content in porcine oocytes damaged by cold stress.
[0062] Through the study of the effect of supplementing IL17D during in vitro maturation on the ROS level and GSH content of cold-stressed porcine oocytes, it is further demonstrated that IL17D can rescue the damage to oocytes caused by cold stress and improve the in vitro maturation quality of oocytes damaged by cold stress.
[0063] (3) Effect of IL17D supplementation during in vitro maturation on the expression of antioxidant-related genes in cold-stressed porcine oocytes
[0064] The RT-qPCR method was used to detect the effect of IL17D treatment on the transcriptional expression levels of anti-oxidative stress-related genes in cold-stressed porcine oocytes. Figure 3 The results showed that compared with the control group, the expression levels of anti-oxidative stress-related genes BIRC5, CAT, and GPX4 in the IL17D-treated group were significantly increased (P < 0.05), further indicating that IL17D can rescue the damage caused by cold stress to oocytes and improve the quality of in vitro maturation of cold-stress-damaged oocytes.
[0065] The above-mentioned content is only some enforcements of the present invention.For those of ordinary skill in the art, without departing from the inventive conception, some distortions and improvements can also be made, and these all belong to protection scope of the present invention. In addition, the above-mentioned embodiments of the present invention not only can be used for improving the developmental birth efficiency of pig in vitro embryos and the developmental ability of parthenogenetic embryos, can also be used for improving other animals, and for example the developmental birth efficiency of embryos such as mice, sheep, cattle and cats, these also all belong to protection scope of the present invention.
Claims
1. Application of IL17D in rescuing oocytes damaged by cold stress.
2. The use according to claim 1, wherein The application is to rescue the damage of cold stress to oocytes by adding IL17D to oocyte culture medium.
3. The use according to claim 2, wherein: The concentration of IL17D was 50 ng / mL.
4. A culture medium capable of rescuing oocytes damaged by cold stress, wherein: The culture medium contains IL17D.
5. The culture solution according to claim 4, wherein The concentration of IL17D was 50 ng / mL.
6. The culture solution according to claim 4, wherein The culture medium contains the following components: TCM-199 basal culture medium as a basis, supplemented with 10 IU / mL blood gonadotropin, 10 IU / mL luteinizing hormone-releasing hormone A3, 0.6 mM cysteine, 5% serum replacement, insulin transferrin selenium liquid culture medium supplement, 10 ng / mL EGF, 40 ng / mL FGF2, 20 ng / mL LIF, 20 ng / mL IGF, 5 μg / mL inositol, 10 nmol / L progesterone and 50 ng / mL IL17D.
7. Use of the culture medium according to any one of claims 4 to 6 in rescuing oocytes damaged by cold stress.
8. Use of the culture medium according to any one of claims 4 to 6 in improving the quality of somatic cell cloned embryos, wherein: The oocytes used in the somatic cell cloning are oocytes damaged by cold stress.
9. A method for improving the in vitro maturation quality of oocytes damaged by cold stress, wherein: The method comprises placing the oocyte damaged by cold stress into the culture medium according to any one of claims 4 to 6 for in vitro maturation culture.
10. Use of IL17D in reducing the level of reactive oxygen species in oocytes damaged by cold stress, or in increasing the content of glutathione in oocytes damaged by cold stress, or in increasing the expression of antioxidant genes in oocytes damaged by cold stress.