Use of hydrogen peroxide in constructing oxidative stress model of female and male chicken primordial germ cells

Abstract

The application discloses a use of hydrogen peroxide (H2O2) in constructing an oxidative stress model of female and male chicken primordial germ cells (PGCs); in the application, the H2O2 can achieve the effect of oxidative stress of the female and male chicken PGCs by increasing the ROS content in the female and male chicken PGCs, inhibiting the T-SOD and CAT enzyme activities and increasing the MDA content; the application verifies the effect of the oxidative stress model established by the H2O2 on the female and male chicken PGCs by screening different concentrations of the H2O2 added into the female and male chicken PGCs and then detecting the apoptosis, reproductive markers, mitochondrial functions and autophagy genes of the cells; in the application, the effect of the oxidative stress model constructed by the H2O2 on the female and male chicken PGCs provides new experimental basis for clarifying the molecular mechanism of oxidative stress causing poultry reproductive damage, and provides new ideas and targets for protecting the PGC functions of poultry and improving the reproductive efficiency by regulating the oxidative stress, autophagy or apoptosis pathways in the future, and provides important research and development basis for the selection of compounds resisting oxidative stress.

Description

Technical Field

[0001] This invention relates to the use of hydrogen peroxide (H2O2) in constructing oxidative stress models of primordial germ cells (PGCs) in female and male chickens. Background Technology

[0002] ROS (Reactive Oxygen Species) is a collective term for a class of chemically reactive molecules and ions with high oxidative activity. It is also an important signaling molecule regulating metabolic activities such as cell proliferation, differentiation, and apoptosis. When ROS accumulates excessively in the body, the body suffers oxidative stress, continuously damaging the antioxidant system and leading to an imbalance between the oxidation and antioxidant systems. This results in damage to biomolecules such as nucleic acids, lipids, and proteins, causing inhibited cell proliferation, abnormal programmed apoptosis and autophagy, disordered epigenetic modifications, abnormal genome-wide methylation, and the enrichment of intracellular lipid peroxidation products and DNA oxidative damage products. Germ cells, as a special cell population for the continuation of the species, are more sensitive to oxidative stress. Oxidative stress, by inhibiting autophagy and inducing ferroptosis, damages the function of interstitial cells and spermatogenic cells in boar testes, leading to reduced testosterone synthesis and impaired spermatogenesis. ROS imbalance in the ovarian microenvironment accelerates follicular atresia, induces granulosa cell apoptosis, and reduces the number of preantral and mature follicles. Oxidative stress damages oocyte spindle assembly and chromosome segregation, causing aneuploidy and reducing oocyte quality and fertilization potential.

[0003] Primordial germ cells (PGCs), as the only cell group responsible for transmitting genetic information to the next generation, are the precursor cells of eggs and sperm and occupy a central position in reproductive biology research. Under in vitro culture conditions, the proliferation index of male PGCs is 30%-50% higher than that of females, and the population doubling time is shortened by 4-6 hours. Male PGCs are more tolerant to the culture environment, have a significantly lower apoptosis rate than females, and have a higher BCL2 / BAX ratio; female PGCs proliferate slowly, are more sensitive to oxidative stress, and have a lineage establishment success rate that is only half that of males. Therefore, simulating oxidative stress environments in vitro is of great significance for improving the in vitro proliferation efficiency of male and female chicken PGCs. However, the establishment of oxidative stress models for male and female chicken PGCs is still in the exploratory stage, and the mechanism of action of oxidative stress on primordial germ cells in male and female chickens is very poorly understood. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a novel application of hydrogen peroxide that can stably induce oxidative stress under normoxic conditions, realistically simulate the oxidative stress environment, and be used to construct oxidative stress models of primordial germ cells (PGCs) in female and male chickens, thereby exploring its effects on PGCs.

[0005] The technical solution of the present invention is: the use of hydrogen peroxide as described in the present invention in constructing oxidative stress models of female and male chicken PGCs.

[0006] Furthermore, by adding a certain dose of H2O2, the ROS was increased to construct oxidative stress models of PGCs in female and male chickens.

[0007] Furthermore, the concentration range of the H2O2 is 200-600 μM (200, 400, 600 μM, preferably 600 μM).

[0008] Furthermore, the processing time for the H2O2 is 6 hours.

[0009] Furthermore, the H2O2 is used to construct oxidative stress models of female and male chicken PGCs by increasing MDA content and reducing T-SOD and CAT activity.

[0010] Furthermore, the H2O2-based oxidative stress model for female and male chicken PGCs increases the expression levels of HO-1 and NRF2 genes and HO-1 protein, while decreasing the expression levels of SOD1, SOD2, CAT genes and SOD2 protein.

[0011] Furthermore, the H2O2-based oxidative stress model of female and male chicken PGCs resulted in a decrease in the expression of PGC biomarkers KIT, DDX4, and OCT4.

[0012] Furthermore, the H2O2-based oxidative stress model for female and male chicken PGCs exacerbates cell apoptosis, increases CASPASE3 / 9 gene expression, and decreases BCL2, BCL2L1 gene and BCL2 protein expression.

[0013] Furthermore, the H2O2-based oxidative stress model for male and female chicken PGCs increases the expression level of the MFN1 gene and decreases the expression levels of the MFN2, SIRT1, and SIRT3 genes, thereby damaging mitochondrial function.

[0014] Furthermore, the H2O2-based oxidative stress model for male and female chicken PGCs inhibits autophagy in chicken PGCs and upregulates the expression levels of MAP1LC3A, MAP1LC3B, ATG9, P62, and PARKIN genes and the expression levels of proteins P62 and LC3-II.

[0015] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: In the present invention, H2O2 can increase the ROS content in female and male chicken PGCs, inhibit the activity of T-SOD and CAT enzymes, and increase the MDA content, thereby achieving the effect of oxidative stress in female and male chicken PGCs; The present invention verifies the effect of the oxidative stress model established by H2O2 on female and male chicken PGCs by screening different concentrations of H2O2 added to female and male chicken PGCs and then detecting apoptosis, reproductive markers, mitochondrial function and autophagy genes in the cells; The effect of H2O2-induced oxidative stress on female and male chicken PGCs provides new experimental evidence for elucidating the molecular mechanism of oxidative stress-induced reproductive damage in poultry, and also provides new ideas and targets for subsequent protection of poultry PGC function and improvement of reproductive efficiency by regulating oxidative stress, autophagy or apoptosis pathways, and provides important research and development basis for the selection of compounds to develop anti-oxidative stress. Attached Figure Description

[0016] Figure 1 These are sex identification and cell state diagrams of female and male chicken PGCs in embodiments of the present invention; Figure 2 This is a graph showing the changes in oxidative stress-related indicators of PGCs in female and male chickens under H2O2 treatment in this embodiment of the invention. Figure 3 This is a diagram illustrating the effect of H2O2 treatment on PGCs marker genes in female and male chickens in an embodiment of the present invention. Figure 4 This is a graph showing the changes in apoptosis-related indicators of PGCs in female and male chickens after H2O2 treatment in an embodiment of the present invention. Figure 5 This is a diagram illustrating the effect of H2O2 treatment on the mitochondrial genes of female and male chicken PGCs in an embodiment of the present invention. Figure 6 This is a graph showing the changes in autophagy-related indicators of PGCs in female and male chickens after H2O2 treatment in an embodiment of the present invention. Detailed Implementation

[0017] The specific technical solution of the present invention will be further described in detail below with reference to specific examples.

[0018] The present invention describes the application of hydrogen peroxide (H2O2) in constructing an oxidative stress model of primordial germ cells (PGCs) in female and male chickens. By adding a certain dose of H2O2, the invention affects the reproduction of PGCs in female and male chickens and induces apoptosis, oxidative damage, mitochondrial damage and autophagy activation in chicken PGCs.

[0019] The concentration of H2O2 is 200, 400, or 600 μM.

[0020] The oxidative stress of the female and male chicken PGCs increased ROS and MDA content, decreased T-SOD and CAT activity, increased HO-1 and NRF2 gene and HO-1 protein expression levels, and decreased SOD1, SOD2, and CAT gene and SOD2 protein expression levels.

[0021] The oxidative stress in the PGCs of both male and female chickens was caused by a decrease in cellular reproductive marker genes due to H2O2.

[0022] The H2O2-induced apoptosis in female and male chicken PGCs was achieved by increasing the expression level of Caspase3 / 9 genes and decreasing the expression levels of BCL2, BCL2L1 genes and BCL2 protein.

[0023] The H2O2 damage to the mitochondria of PGCs in both male and female chickens increases the expression level of the MFN1 gene and decreases the expression levels of the MFN2, SIRT1, and SIRT3 genes.

[0024] The H2O2 inhibition of autophagy in male and female chicken PGCs significantly upregulated the expression levels of MAP1LC3A, MAP1LC3B, ATG9, P62, and PARKIN genes and the expression levels of proteins P62 and LC3-II.

[0025] Example: A novel application of hydrogen peroxide in constructing oxidative stress models of primordial germ cells in female and male chickens. I. Experimental Instruments and Materials Instruments and equipment: Microplate reader (BioTek, EPOCH2, Nanjing, China); Chemiluminescence imaging system (Qinxiang, ChemiScope 6100, Shanghai, China); Fluorescence inverted microscope (Olympus, IX73, Japan); Flow cytometer (BD, FACSLyric, New Jersey, USA). Reagents and materials: H2O2, fertilized eggs, PGCs culture medium, fetal bovine serum, real-time quantitative PCR primers (B-ACTIN, SOD2, SOD1, CAT, HO-1, NRF2, KIT, DDX4, OCT4, BCL2, BCL2L1, CASPASE9, CASPASE3, MFN1, MFN2, SIRT1, SIRT3, MAP1LC3A, MAP1LC3B, ATG9, P62), BCL2 (Proteintech), SOD2 (Proteintech), HO-1 (Proteintech), P62 (Proteintech), LC3 (Proteintech), malondialdehyde (MDA), total superoxide dismutase (T-SOD), catalase (CAT), ROS, and Annexin-FITC apoptosis detection kit were all purchased from the market. II. Sex determination of female and male chicken embryos 1. Experimental Method: Chicken embryos hatched for 5.5 days were harvested aseptically, and embryonic tissue and genital ridges approximately 0.5 mm in length and width were collected. Based on the genomic sequences of the homologous genes CHD1 (NM_204941.2) and CHD1W (NM_001396597.1) on the chicken sex chromosome Z / W in NCBI, primers CHD-F (CTGCGAGAACGTGGCAACAGAGT) and CHD-R (ATTGAAATGATCCAGTGCTTG) were designed and used for PCR amplification to identify the sex of the chicken embryos. After collecting genital ridges of the same sex, 1000 μL of trypsin was added for digestion for 3 min. When no tissue was visible to the naked eye, an equal volume of complete culture medium (DMEM / F12 containing 10% FBS) was added to terminate the digestion. After centrifugation and discarding the supernatant, 3000 μL of PGCs medium was added to each tube and inoculated into 6 cm culture dishes. After incubation at 37℃ and 5% CO2 for 1 h, the cells were purified by differential purification and transferred to new 6 cm culture dishes. 2. Experimental Results and Analysis: The experimental results are shown in Figure 1 To accurately collect PGCs from chicken embryos of different sexes, the CHD1 / CHD1W gene genomic fragment was amplified using PCR, and the collected genital ridge tissue was used for sex identification. Gel electrophoresis results showed that in the same lane, there were 434bp and 580bp bands indicating females (ZW), while only the 580bp band indicated males (ZZ). Figure 1 A); Gonads of the same sex were subsequently isolated, purified, and cultured as PGCs; after 48 hours of culture, typical characteristics of PGCs—large volume, large and centrally located nuclei, and a distinct halo—could be observed under an inverted microscope. Figure 1 B); III. Effects of H2O2 on the establishment of oxidative stress models of PGCs in female and male chickens 1. Experimental Method: Chicken PGCs at 1×10 6 Chicken PGCs were seeded into 12-well plates and cultured. When the cell density was about 80%, the cells were divided into a control group, a 200 μM H2O2 group, a 400 μM H2O2 group, and a 600 μM H2O2 group, and cultured at 37 ℃ in a 5% carbon dioxide incubator for 6 h. The DCFH-DA in the reactive oxygen species detection kit was diluted 1:1000 with serum-free medium to a final concentration of 10 µM. After treating with H2O2 for 6 h, cells were collected and resuspended in the diluted DCFH-DA probe. The cells were incubated at 37 °C for 20 min, inverting every 5 min to ensure thorough contact between the probe and cells. After low-speed centrifugation, the DCFH-DA probe was discarded, and the cells were washed three times with serum-free medium to remove excess probe. The cells were then resuspended in 500 µL of PBS and observed and photographed under a fluorescence inverted microscope. Cells were collected after H2O2 treatment for 6 h. After washing the collected cells with PBS, 100 µL of PBS was added to resuspend the cells. The cells were then placed in a 1.5 mL centrifuge tube containing the cells in an ice-water bath and sonicated to disrupt the cells. The cells were centrifuged at 12000 r / min and 4 ℃ for 10 min. The supernatant was collected, and the protein concentration was determined using the BCA Protein Assay Kit. The changes in MDA, T-SOD, and CAT of PGCs were detected according to the kit instructions. Total RNA was extracted from the samples using TRIzol reagent, and then reverse transcribed into cDNA using the Primer Script RT regent kit with gDNA Eraser. Subsequently, RT-qPCR was performed using the NovoStart® SYBR qPCR SuperMix Plus reagent prepared for the CFX96 Touch RTPCR detection system; β-ACTIN was used as an internal control. -△△Ct The relative expression levels of SOD1, SOD2, CAT, HO-1, and NRF2 genes were calculated. 100 µL of RIPA cell lysis buffer containing 1 mM protease inhibitor was added to each tube to extract total protein, and the protein concentration was analyzed using a BCA kit. An equal volume of denatured protein sample was added to the wells of a Future PAGE™ protein precast gel, and the protein samples were separated by electrophoresis at 130 V for 60 min. The samples were then rapidly transferred to a PVDF membrane and blocked with 5% skim milk powder for 2 h. The membrane was then incubated overnight at 4 ℃ with SOD2, HO-1, and β-ACTIN, respectively. The primary antibody was recovered, and the samples were washed three times with TBST. The membrane was then incubated with secondary antibody on a shaker at room temperature for 1 h, washed three times with TBST again, and ECL chromogenic solution was added. The samples were then imaged using a chemiluminescence imaging system, and the protein bands were analyzed for grayscale using ImageJ software. β-ACTIN was used as an internal control protein. 2. Experimental Results and Analysis: To investigate the application of hydrogen peroxide in constructing oxidative stress models of rooster and female broiler chicken charcoal (PGCs), oxidative stress-related indicators were detected in different treatment groups. The results showed that the ROS content levels of rooster and female PGCs were significantly increased. Figure 2 A), the content of MDA increased significantly, while the activities of T-SOD and CAT decreased significantly. Figure 2 B); SOD1, SOD2, and CAT mRNA expression levels decreased, while HO-1 and NRF2 expression levels increased ( Figure 2 C), the downregulation of antioxidant enzyme genes in female chicken PGCs was greater than that in males, while the compensatory upregulation of HO-1 and NRF2 was weaker. Simultaneously, SOD2 protein expression decreased in both female and male chicken PGCs, but the difference in SOD2 expression was not significant in females. HO-1 expression was significantly increased in both female and male chicken PGCs. Figure 2 D) indicates that H2O2 induction may have caused oxidative stress in the PGCs of both female and male chickens; IV. Effects of H2O2 on PGCs marker genes in female and male chickens: 1. Experimental Method: Cells were treated as above, and the mRNA expression levels of KIT, DDX4, and OCT4 were detected. 2. Experimental Results and Analysis: To further investigate the effects of oxidative stress on reproductive marker genes (PGCs) in female and male chickens, reproductive marker genes were detected in PGCs of female and male chickens after stimulation with different concentrations of H2O2 for 6 hours. The results showed that H2O2 significantly reduced the mRNA expression of OCT4, DDX4, and KIT in PGCs of female and male chickens. Figure 3 ); V. Effects of H2O2 on apoptosis of PGCs in female and male chickens: 1. Experimental Method: Cell treatment was the same as above. The mRNA expression levels of BCL2, BCL2L1, CASPASE3, and CASPASE9 were detected, and the protein expression level of BCL2 was detected. Apoptosis was detected using the Annexin-FITC apoptosis detection kit. After H2O2 treatment for 6 h, cells were collected, gently resuspended in PBS, centrifuged, and then gently resuspended in 195 µL of Annexin V-FITC binding buffer. 5 µL of Annexin V-FITC was added and gently mixed. 10 µL of propidium iodide (PI) staining solution was added and gently mixed. Blank groups, Annexin V-FITC single-labeled groups, and PI single-labeled groups were prepared. Cells were incubated at room temperature in the dark for 10-20 min, then placed in an ice bath and wrapped in aluminum foil to protect from light. Flow cytometry was used for analysis. 2. Experimental Results and Analysis: To investigate the regulatory effect of H2O2 on cell apoptosis, cells were treated with different concentrations of H2O2, and the changes in cell apoptosis rate and the expression of apoptosis-related genes and proteins were detected. The results showed that the cell apoptosis rate increased after H2O2 treatment, with the 400 μM and 600 μM treatment groups showing significantly higher apoptosis rates in both male and female chicken PGCs. Figure 4 (A) Compared with the control group, after treatment with various concentrations of H2O2, the expression of BCL2 and BCL2L1 in female chicken PGCs was significantly downregulated, while the expression of CASPASE3 and CASPASE9 was significantly upregulated. After treatment with 400 μM and 600 μM, the expression of BCL2 and BCL2L1 in male chicken PGCs decreased significantly, but the overall decrease was lower than that in female PGCs stimulated with the corresponding concentrations of H2O2. CASPASE3 and CASPASE9 were also significantly upregulated. Figure 4 B), while BCL2 protein expression decreased ( Figure 4 (C) The results showed that H2O2-induced oxidative stress promoted cell apoptosis; VI. Effects of H2O2 on mitochondria of PGCs in female and male chickens: 1. Experimental Method: As above, the mRNA expression levels of MFN1, MFN2, SIRT1, and SIRT3 were detected; 2. Experimental Results and Analysis: Because H2O2 treatment increased ROS levels in both male and female chicken progenygate cells (PGCs), it was inferred that H2O2 treatment might cause mitochondrial damage. Therefore, the mRNA expression levels of mitochondrial-related genes were detected. The results showed that the mRNA expression level of MFN1 in male chicken PGCs first increased and then decreased, while MFN2, SIRT1, and SIRT3 were significantly downregulated. In female chicken PGCs, the mRNA expression levels of MFN2, SIRT1, and SIRT3 generally showed a downward trend. After treatment with 400 μM H2O2, the expression level of MFN1 increased slightly, but the difference was not significant. Figure 5 This indicates that H2O2 treatment may cause mitochondrial damage in cells; VII. Effects of H2O2 on autophagy of PGCs in female and male chickens: 1. Experimental Method: As above, the mRNA expression levels of MAP1LC3A, MAP1LC3B, ATG9, P62 and PARKIN were detected, and the protein expression levels of P62 and LC3-II were detected. 2. Experimental Results and Analysis: To further clarify whether autophagy occurs in female and male chicken PGCs under oxidative stress, changes in autophagy-related genes were detected in female and male chicken PGCs after stimulation with different concentrations of H2O2 for 6 h. The results showed that after H2O2 treatment, the mRNA expression levels of MAP1LC3A, MAP1LC3B, ATG9, P62, and PARKIN in both female and male chicken PGCs were upregulated, with significant differences observed between treatments with 400 μM and 600 μM H2O2. Figure 6 A), and at the same time, the protein expression levels of P62 and LC3-II were both upregulated ( Figure 6 (B) This indicates that H2O2-induced oxidative stress activates the autophagy process, but the autophagic flux may be impeded to some extent, leading to the accumulation of autophagy-related proteins.

Claims

1. The use of hydrogen peroxide in constructing oxidative stress models of female and male rooster PGCs.

2. The use according to claim 1, characterized in that, By adding a certain amount of H2O2, the ROS level was increased to construct oxidative stress models of PGCs in female and male chickens.

3. The use according to claim 2, characterized in that, The concentration range of H2O2 is 200-600 μM.

4. The use according to claim 2, characterized in that, The treatment time for H2O2 is 6 hours.

5. The use according to claim 1, characterized in that, The H2O2-based oxidative stress model for male and female chicken PGCs was constructed by increasing MDA content and decreasing T-SOD and CAT activity.

6. The use according to claim 1, characterized in that, The H2O2-based oxidative stress model for male and female chicken PGCs was designed to increase the expression levels of HO-1 and NRF2 genes and HO-1 protein, while decreasing the expression levels of SOD1, SOD2, CAT genes and SOD2 protein.

7. The use according to claim 1, characterized in that, The H2O2-based oxidative stress model of female and male chicken PGCs resulted in a decrease in the expression of PGC biomarkers KIT, DDX4, and OCT4.

8. The use according to claim 1, characterized in that, The H2O2-based oxidative stress model for female and male chicken PGCs exacerbates cell apoptosis, increases CASPASE3 / 9 gene expression, and decreases BCL2, BCL2L1 gene and BCL2 protein expression.

9. The use according to claim 1, characterized in that, The H2O2-based oxidative stress model for male and female chicken PGCs increases the expression level of the MFN1 gene and decreases the expression levels of the MFN2, SIRT1, and SIRT3 genes, thereby damaging mitochondrial function.

10. The use according to claim 1, characterized in that, The H2O2-based oxidative stress model for male and female chicken PGCs inhibits autophagy in chicken PGCs and upregulates the expression levels of MAP1LC3A, MAP1LC3B, ATG9, P62, and PARKIN genes and the expression levels of proteins P62 and LC3-II.

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