Application of OGDH gene in ovarian granular cells

Abstract

The invention discloses an application of an OGDH gene in ovarian granular cells. According to the invention, OGDH is taken as a research object, and the application of OGDH in ovarian granular cells is researched on living and cellular levels: qRT-PCR (quantitative reverse transcription-polymerase chain reaction) finds that the expression quantities of OGDH genes in follicles with different sizes are obviously different; through overexpression or interference of the expression quantity of the OGDH in human ovarian granule cells, the OGDH is found to be capable of promoting proliferation of the human ovarian granule cells, inhibiting oxidative stress of the cells and promoting development of ovarian follicles of mice so as to promote oestrus of the mice.

Description

Technical Field

[0001] The present invention belongs to the technical fields of cell engineering and genetic engineering, and particularly relates to the application of the OGDH gene in ovarian granulosa cells. Background Art

[0002] The ovary is an important part of the female mammalian reproductive system and plays a key role in mammalian sexual maturity and the growth and development of oocytes. As the basic functional unit of the ovary, the development of follicles is mainly a process of mutual regulation among oocytes, granulosa cells and theca cells. The growth and development of ovarian follicles and the maturation of oocytes depend on the viability of granulosa cells. When the autophagy level of granulosa cells increases, follicular atresia will occur. Oxidative stress is a common cause of follicular atresia induced by autophagy of granulosa cells. As the largest cell population in ovarian follicles, when the redox reaction of granulosa cells loses balance and reactive oxygen species (ROS) accumulate excessively, the function of granulosa cells will be severely damaged, resulting in apoptosis of granulosa cells and thus inhibiting follicular development.

[0003] Oxoglutarate Dehydrogenase (OGDH) is an important component of the α-ketoglutarate dehydrogenase complex (AKGDHC), and its expression increases in apoptotic cells. In addition, a large number of literature reports that OGDH is an effective source of reactive oxygen species (ROS). Follicular development and apoptosis of granulosa cells are both closely related to the level of ROS. Some studies have shown that FRI-1 reduces the activity of OGDH by disrupting mitochondrial redox capacity, thereby inducing apoptosis. Knocking out the OGDH gene in human embryonic stem cells will lead to the obstruction of the tricarboxylic acid cycle process in cells, the reduction of mitochondrial respiratory activity and the total ATP level, and cause the death of embryonic stem cells. In summary, OGDH can alleviate oxidative stress and inhibit apoptosis of cells. However, the connection between the OGDH gene and the oxidative stress state and growth and development of human ovarian granulosa cells has not been reported yet. Summary of the Invention

[0004] In order to overcome the disadvantages and deficiencies of the prior art, the first object of the present invention is to provide the application of the OGDH gene in promoting the estrus of mammals.

[0005] The second object of the present invention is to provide the application of the OGDH gene in promoting the development of mammalian follicles.

[0006] The third object of the present invention is to provide the application of the OGDH gene in the culture of ovarian granulosa cells.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] The application of the OGDH gene in promoting the estrus of mammals is at least one of the following applications 1 and 2:

[0009] Application 1: Application of overexpressing OGDH gene in promoting estrus in mammals;

[0010] Application 2: Application of OGDH gene overexpression vector in the preparation of products for promoting estrus in mammals.

[0011] Furthermore, the promotion of estrus in mammals is to shorten the estrus age of mammals.

[0012] Furthermore, the product is any one of drugs, reagents, feeds, and feed additives.

[0013] Furthermore, the dosage form of the drug or reagent is any one of oral dosage forms and injection dosage forms.

[0014] Furthermore, the drug or reagent also contains pharmaceutically acceptable excipients or carriers.

[0015] Furthermore, the mammal is any one of humans, mice, and pigs.

[0016] The application of OGDH gene in follicular development is at least one of the following Applications 3 and 4:

[0017] Application 3: Application of overexpressing OGDH gene in promoting follicular development in mammals;

[0018] Application 4: Application of OGDH gene overexpression vector in the preparation of products for promoting follicular development in mammals.

[0019] Furthermore, the promotion of follicular development in mammals is to increase the number of corpora lutea and / or antral follicles and reduce the number of preantral follicles.

[0020] Furthermore, the product is any one of drugs and reagents.

[0021] Furthermore, the drug or reagent also contains pharmaceutically acceptable excipients or carriers.

[0022] Furthermore, the mammal is any one of humans, mice, and pigs.

[0023] The application of OGDH gene in ovarian granulosa cell culture is at least one of the following Applications 5 and 6:

[0024] Application 5: Application of overexpressing OGDH gene in in vitro culture of ovarian granulosa cells;

[0025] Application 6: Application of OGDH gene overexpression vector in the preparation of additives for in vitro culture of ovarian granulosa cells.

[0026] Further, the application 5 is as follows: under an in vitro environment, overexpressing the OGDH gene in ovarian granulosa cells to promote the proliferation of ovarian granulosa cells and reduce the oxidative stress level of ovarian granulosa cells.

[0027] Further, the in vitro culture additive is at least one of a proliferation promoter and an oxidative stress inhibitor.

[0028] Further, the ovarian granulosa cells are any one of human, mouse, and porcine ovarian granulosa cells.

[0029] Further, the full-length genomic sequence of the human OGDH gene is as shown in the sequence with the NCBI accession number GeneID: 4967 (NG_023260.1), the cDNA is as shown in the sequence with the NCBI accession number NM_002541.4, and the CDS region sequence is as shown in the sequence with the NCBI accession number CCDS: CCDS34627.1.

[0030] Further, the full-length genomic sequence of the mouse OGDH gene is as shown in the sequence with the NCBI accession number GeneID: 18293, and the cDNA sequence is as shown in the sequence with the NCBI accession number NM_001252282.1.

[0031] Further, the basic plasmid used for the overexpression vector is pcDNA3.1.

[0032] Application of the OGDH gene as a marker for ovarian follicle growth and development.

[0033] The verification results of the present invention are as follows:

[0034] 1. The relative expression level of OGDH in follicles with a diameter of >5 mm is significantly higher than that in follicles with a diameter of 3 - 5 mm, and is significantly higher than that in follicles with a diameter of <3 mm ( Figure 1 ).

[0035] 2. Design primers for the target gene OGDH: Search for the sequence of the target gene OGDH (NCBI Gene ID: 4967) on NCBI, determine the restriction enzyme sites BamHI and EcoRI, use Primer Premier 5.0 software for primer design, specifically amplify, purify, and digest the target fragment by PCR, then ligate the pcDNA3.1 vector, and finally construct the overexpression vector pcDNA3.1-OGDH of the target gene OGDH. Subsequently, by transfecting the overexpression vector at different concentrations (100, 200, and 500 ng / mL) into ovarian granulosa cells and detecting the expression level of OGDH by qRT-PCR and Western Blot, it was found that the transfection efficiency was better when transfected with the 200 ng / mL overexpression vector, and there were significant differences. In subsequent studies, 200 ng / mL was selected as the transfection concentration of pcDNA3.1-OGDH( Figure 2 ).

[0036] 3. Synthesize 3 pairs of OGDH small interfering fragments / controls (si-OGDH / si-NC), screen and detect their interference efficiency. Transfect the gene small interfering fragments into human ovarian granulosa cells, and finally screen si-OGDH 1 with better interference effect for subsequent experiments by qRT-PCR and Western Blot methods( Figure 3 ).

[0037] 4. Transfect pcDNA3.1-OGDH or si-OGDH 1 into human ovarian granulosa cells respectively, and use qRT-PCR, Western Blot, EdU, and reactive oxygen species detection kits to detect the effects of OGDH on cell proliferation and oxidative stress. The results showed that the proliferation rate of the pcDNA3.1-OGDH group was significantly higher than that of the control group; the proliferation rate of the si-OGDH 1 group was significantly lower than that of the control group si-NC. The detection results of oxidative stress showed that the reactive oxygen species level of the pcDNA3.1-OGDH group was significantly lower than that of the control group; the reactive oxygen species level of the si-OGDH1 group was significantly higher than that of the control group si-NC. In summary, OGDH can promote the proliferation of human ovarian granulosa cells and inhibit cell oxidative stress( Figure 4 , Figure 5 ).

[0038] 5. Statistically analyze the estrus age of mice. The results showed that compared with the control group, knocking down OGDH could delay the age of first estrus in mice, while overexpressing OGDH advanced the age of first estrus in mice( Figure 6 ).

[0039] 6. The expression of OGDH in mouse ovaries was detected by qRT-PCR and Western Blot. The results showed that compared with the control group, the LV-OGDH group significantly increased the mRNA and protein expression levels of the OGDH gene; while the sh-OGDH group significantly decreased the mRNA and protein expression levels of OGDH. It indicated that the constructed mouse model could overexpress and knockdown the OGDH gene in the ovary and could be used for subsequent experiments( Figure 7 ).

[0040] 7. Photographs of HE sections of mouse ovaries were taken to detect the development of ovarian follicles in mice. The results showed that compared with the control group, the proportion of preantral follicles in the ovaries of mice in the LV-OGDH group was significantly decreased, and the proportion of corpora lutea was significantly increased (P<0.01); while in the sh-OGDH group, the proportion of preantral follicles was significantly increased, and the proportion of corpora lutea was significantly decreased (P<0.05). This indicated that OGDH could promote the development of ovarian follicles in mice( Figure 8 )

[0041] 8. The apoptosis of GCs in ovarian follicles of mice was detected by TUNEL assay. The results showed that compared with the control group, there were fewer apoptotic GCs in the ovaries of mice in the LV-OGDH group; while the apoptosis level of GCs in ovarian follicles of mice in the sh-OGDH group was higher than that in the control group, which indicated that OGDH could inhibit the apoptosis of GCs in mouse ovaries( Figure 9 )

[0042] 9. To further study the role of OGDH in mouse ovarian development, qRT-PCR and Western Blot were used to detect the expression of key genes in the mouse ovarian function pathway. The results showed that OGDH significantly inhibited the expression of key genes in the oxidative stress pathway in mouse ovaries, inhibited the expression of apoptosis pathway genes, and promoted the expression of proliferation pathway genes( Figure 10 )

[0043] The present invention has the following advantages and effects compared with the prior art:

[0044] 1. The present invention used three kinds of porcine follicles with diameters of <3 mm, 3 - 5 mm and >5 mm as experimental materials. Through qRT-PCR, it was found that with the increase of the diameter of porcine follicles, the expression level of the gene OGDH gradually increased.

[0045] 2. The present invention took OGDH as the research object and adopted in vivo and cell biology methods to study its application in mouse and human ovarian granulosa cells. By overexpressing or interfering with OGDH, it was found that OGDH could promote cell proliferation and inhibit the level of cell oxidative stress. It has good application value for studying ovarian follicle development, etc.

[0046] 3. The technical solution of the present invention is carefully designed and the results are reliable. Description of the Drawings

[0047] Figure 1 It is a graph of the relative expression levels of OGDH in follicles of different sizes.

[0048] Figure 2 It is a graph for detecting the efficiency of the overexpression vector pcDNA3.1-OGDH.

[0049] Figure 3 It is a graph for detecting the efficiency of the interfering fragment si-OGDH.

[0050] Figure 4 It is a graph for detecting the effect of overexpressing or interfering with OGDH on the proliferation of ovarian granulosa cells by the EdU method; among them, a is the effect of overexpressing and knocking down OGDH on the proliferation of ovarian granulosa cells detected by the EdU method; b is the expression of key genes in the proliferation pathway of the mouse ovary detected by qRT-PCR after overexpressing and knocking down OGDH; c is the expression of proliferation-related proteins in the mouse ovary detected by Western Blot after overexpressing and knocking down OGDH.

[0051] Figure 5 It is a graph for detecting the effect of overexpressing or interfering with OGDH on the oxidative stress of granulosa cells using a reactive oxygen species detection kit; among them, a is the effect of overexpressing OGDH on the level of reactive oxygen species in granulosa cells; b is the expression of key genes in the oxidative stress pathway of the mouse ovary detected by qRT-PCR after overexpressing and knocking down OGDH; c is the expression of oxidative stress-related proteins in the mouse ovary detected by Western Blot after overexpressing and knocking down OGDH; d is the final reverse verification by CoIP.

[0052] Figure 6 It is a statistical graph of the estrus age of mice after overexpressing / interfering with OGDH.

[0053] Figure 7 It is a graph for detecting the efficiency of overexpressing and knocking down OGDH in the mouse ovary by qRT-PCR and Western Blot.

[0054] Figure 8 It is a HE section graph of the ovaries of mice overexpressing and knocking down OGDH.

[0055] Figure 9 It is a graph for detecting the apoptosis of ovarian granulosa cells in mice after overexpressing and knocking down OGDH by the TUNEL method.

[0056] Figure 10 It is a graph for detecting the expression of key genes in the ovarian function pathway of mice after overexpressing and knocking down OGDH by qRT-PCR and Western Blot. Detailed implementation methods

[0057] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the implementation modes of the present invention are not limited thereto. For the experimental methods without specific conditions noted in the following embodiments, they are generally carried out under conventional conditions. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0058] In the present invention, statistical methods are applied to analyze the results of 3 independent experiments in each embodiment, and the "mean ± standard deviation" is calculated respectively. One-way ANOVA is used for significant difference analysis (in the figure, "*" indicates P < 0.05, and "**" indicates P < 0.01).

[0059] Example 1: Detection of relative expression level of OGDH in follicles of different sizes

[0060] (1) RNA extraction

[0061] ① Three types of follicles with diameters < 3 mm, 3 - 5 mm, and > 5 mm were extracted from the ovaries of sows (healthy commercial sows from Guangzhou Kongwangji Slaughterhouse), and the samples after adding TRIzol in proportion were thoroughly homogenized;

[0062] ② Left standing on ice for 10 min, and the supernatant was transferred after centrifugation at 12000 rpm for 5 min;

[0063] ③ Chloroform was added, shaken and mixed evenly, left standing on ice for 15 min, and centrifuged at 12000 rpm at 4℃ for 15 min;

[0064] ④ The upper aqueous phase was transferred to a new de-enzymed 1.5 mL centrifuge tube, isopropanol was added, gently mixed evenly, left standing on ice for 15 min, and centrifuged at 12000 rpm at 4℃ for 15 min. The supernatant was discarded, and the RNA precipitate was retained;

[0065] ⑤ Pre-cooled 75% ethanol was added to resuspend and wash the RNA precipitate, centrifuged at 12000 rpm at 4℃ for 15 min, the supernatant was discarded, and the precipitate was retained;

[0066] ⑥ DEPC water was added to resuspend and dissolve the precipitate, the RNA concentration was measured, and it was stored at -80℃.

[0067] (2) RNA reverse transcription, referring to the PrimeScript RT Master Mix instruction manual, the reaction system is shown in Table 1:

[0068] Table 1 RNA reverse transcription system

[0069]

[0070] Note: The reaction conditions are 15 min at 37℃ and 5 s at 85℃.

[0071] (3) qRT-PCR

[0072] In the present invention, qRT-PCR detection is performed using qPCR SYBR Green Master Mix kit. The comparative Ct value method is used to detect the content of sample genes in the experiment. The specific calculation formula is as follows:

[0073] Relative gene expression = 2 -{〈﹙实验组目的基因Ct值﹚-﹙实验组内参基因Ct值﹚〉-〈﹙对照组目的基因Ct值﹚-﹙对照组内参基因Ct值﹚〉}

[0074] GAPDH is used as an internal reference for the detected gene. The qRT-PCR primers used in the present invention are:

[0075] qRT-PCR-OGDH Forward: 5′-ATTTTCCACGTGAACTCAGATG-3′;

[0076] Reverse: 5′-GTAACACACCAAATCGACAACC-3′;

[0077] qRT-PCR-GAPDH Forward: 5′-TGTTCGTCATGGGTGTGAAC-3′;

[0078] Reverse: 5′-ATGGCATGGACTGTGGTCAT-3′.

[0079] The results are as Figure 1 shown. With the increase in the diameter of porcine follicles, the expression level of gene OGDH gradually increases.

[0080] Example 2: Effects of overexpressing or interfering with OGDH on ovarian granulosa cells

[0081] 1. Construction of overexpression vector and interference vector for OGDH gene

[0082] (1) Search for the target human OGDH gene (Gene ID: 4967, NG_023260.1, NM_002541.4, CCDS: CCDS34627.1) on NCBI, and use Primer Premier 5.0 software for primer design. Extract the total RNA of KGN granulosa cells, and then reverse transcribe it into cDNA using a kit as a template for amplification. Purify and recover the amplified fragment, ligate it to the pMD18T vector (Takara), transform it, and after detecting the bacterial liquid and performing sequencing identification correctly, extract the ordinary plasmid and name it T-OGDH.

[0083] The primer sequences are as follows:

[0084] F: 5′-atgtttcatttaaggacttgtg-3′;

[0085] R: 5′-ctacgagaagttcttgaagacg-3′.

[0086] (2) Add BamHI and EcoRI restriction site sequences to the upstream and downstream primers respectively. Using the common plasmid T-OGDH as a template, perform PCR amplification; recover and purify the target fragment, double digest the pcDNA3.1 vector (Invitrogen, catalog number V79020) and the target fragment, ligate pcDNA3.1, transform, screen, and sequence for identification. After confirmation, extract endotoxin-free plasmid (the endotoxin-free plasmid miniprep kit is purchased from Magen, USA), and name it pcDNA3.1-OGDH. The restriction enzyme primer sequences for the OGDH gene are as follows:

[0087] F: 5′-CTTGGTACCGAGCTCGGATCCGCCACCatgtttcatttaaggacttgtg-3′;

[0088] R: 5′-TGCTGGATATCTGCAGAATTCctacgagaagttcttgaagacg-3′.

[0089] (3) Entrust Guangzhou Dongze Biotechnology Co., Ltd. to synthesize small interfering fragments / control for OGDH (si-OGDH / si-NC):

[0090] si-OGDH1: 5’-CTGGTGTGTTATCGACGAAAT-3’.

[0091] 2. Culture of ovarian granulosa cells

[0092] Resuscitation of KGN cell line: Take out the cryopreserved cells from liquid nitrogen, thaw in a 37°C water bath, add the suspension to an appropriate amount of complete medium, centrifuge at 1000 rpm for 5 min, and discard the supernatant; resuspend the cells with 5 mL of complete medium, transfer to a small culture flask, and mix well by the cross method. Incubate in a 37°C, 5% CO2 cell culture incubator. Change the medium after about 48 h and continue to culture for 24 h before subculturing.

[0093] Cell subculture and transfection: When the cell confluence reaches 80%, discard the medium, wash twice with PBS; add 0.25% trypsin and digest at 37°C for 5 min, terminate the digestion with complete medium; pipette the cells, transfer to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and wash the cells twice with PBS; resuspend the cells with an appropriate amount of complete medium, evenly transfer to a culture flask or culture plate, and incubate in a cell culture incubator. Observe the cell status. When the cell confluence reaches 80%, refer to the 3000 kit instruction manual for transfection.

[0094] 3. Detection of ovarian granulosa cell proliferation

[0095] The EdU method was used to detect the proliferation of ovarian granulosa cells. The experimental steps refer to the instruction manual of Ribobio EdU kit:

[0096] (1) Seed granulosa cells in 48-well plates. When the cell confluence reaches 80%, perform transfection or drug treatment, with at least 3 replicates in each group;

[0097] (2) After 24 h, add 200 μL of pre-prepared 50 μM EdU medium to each well, incubate in a cell culture incubator for 2 h, discard the medium, and wash twice with PBS;

[0098] (3) Add 200 μL of cell fixative (80% acetone diluted with PBS) to each well, incubate at room temperature for 30 min, and wash twice with PBS;

[0099] (4) Add 200 μL of permeabilization solution (PBS containing 0.5% Triton X-100) to each well, incubate on a shaker for 5 min for permeabilization, and wash twice with PBS;

[0100] (5) Add 200 μL of pre-prepared 1× Apollo staining solution to each well, incubate in the dark at room temperature for 30 min, and wash twice with PBS;

[0101] (6) Permeabilize the cells again, repeat step (4);

[0102] (7) Add 200 μL of pre-prepared DAPI reaction solution to each well, incubate in the dark at room temperature for 30 min, and wash twice with PBS;

[0103] (8) Add 100 μL of PBS to each well for preservation, take pictures under a fluorescence microscope, and save the pictures.

[0104] 4. Detection of oxidative stress level in ovarian granulosa cells

[0105] The experimental steps refer to the instruction manual of Beyotime Reactive Oxygen Species Assay Kit:

[0106] (1) Seed granulosa cells in 96-well plates. When the cell confluence reaches 80%, perform transfection or drug treatment, with at least 3 replicates in each group;

[0107] (2) After culturing for 24 h, discard the medium, wash the cells 3 times with PBS, add 100 μl of prepared DCFH-DA reaction solution (diluted 1:1000 with serum-free medium) to each well, and incubate in a cell culture incubator for 20 min.

[0108] (3) Wash 3 times with serum-free medium, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value at a wavelength of 488 nm (in the dark);

[0109] (4) Observe the cells under a fluorescence microscope and take pictures for preservation.

[0110] Detect the overexpression efficiency of OGDH by qRT-PCR. The results are as Figure 2 shown. The transfection efficiency of the overexpression vector at 200 ng / mL is better and there are significant differences. For subsequent studies, 200 ng / mL is selected as the transfection concentration of pcDNA3.1-OGDH ( Figure 2 ).

[0111] Synthesize 3 small interfering fragments / controls of OGDH (si-OGDH / si-NC), screen and detect their interference efficiency. Transfect the 3 small interfering fragments into ovarian granulosa cells, and finally screen si-OGDH1 with better interference effect for subsequent experiments by qRT-PCR means ( Figure 3 ).

[0112] Transfect the above-mentioned control groups pcDNA3.1 and pcDNA3.1-OGDH, and the control groups si-NC and si-OGDH1 into ovarian granulosa cells respectively. Use the EdU method and the reactive oxygen species detection kit to detect the effects of OGDH on the proliferation and oxidative stress of ovarian granulosa cells respectively. Use qRT-PCR and Western Blot to detect the changes in the expression levels of genes related to proliferation and oxidative stress in the mouse ovaries after overexpressing and knocking down OGDH. The results show that the proliferation rate of the pcDNA3.1-OGDH group is significantly higher than that of the control group pcDNA3.1, and the proliferation rate of the si-OGDH1 group is significantly lower than that of the control group si-NC ( Figure 4 ). Another part of the results shows that the reactive oxygen species level of the pcDNA3.1-OGDH group is significantly lower than that of the control group pcDNA3.1, and the reactive oxygen species level of the si-OGDH1 group is significantly higher than that of the control group si-NC ( Figure 5 ).

[0113] Example 3: Effects of overexpressing or interfering with OGDH on mouse estrus and ovarian follicle development

[0114] 1. Lentivirus construction

[0115] Entrust Guangzhou Dongze Biotechnology Co., Ltd. to package the lentivirus for overexpression and knockdown of OGDH. Among them, the lentivirus for overexpression is constructed by inserting the OGDH gene (NM_001252282.1) into the pLVX-C-FLAG-PGK-Puro plasmid through the XhoI+MluI restriction enzyme sites, importing it into 293T cells, and generating a lentivirus with high titer containing the target gene, named LV-OGDH; the lentivirus for knockdown is constructed by inserting the shRNA used to knockdown OGDH (target sequence: 5‘-CTGGTGTGTTATCG ACGAAAT-3’) into the pLKO.1-puro plasmid through the BamHI+EcoRI restriction enzyme sites, importing it into 293T cells, and generating a lentivirus with high titer containing the target gene, named sh-OGDH. Among them, the inserted fragment sequences are as follows:

[0116]

[0117] 2. Mouse Breeding and Lentivirus Treatment

[0118] Thirty-two 21-day-old C57BL / 6J female mice were selected and randomly divided into four groups: LV-NC group, LV-OGDH group, sh-NC group, and sh-OGDH group, with 8 replicates in each group. After 1 week of adaptive breeding in the Experimental Animal Center of South China Agricultural University, the lentiviral vector was injected into the mice by intraperitoneal injection, and the injection titer was 1×10 7 , once a week for three consecutive weeks. The mice were weighed every day, and the estrus situation was observed and recorded. The mice were sacrificed at 42 days of age, and the ovaries were taken.

[0119] 3. Detection of Follicular Development in Mouse Ovaries

[0120] The development of ovarian follicles in mice was observed by HE staining of mouse ovaries.

[0121] (1) When the mice were sacrificed at 42 days of age, the ovaries were taken and fixed with 4% paraformaldehyde;

[0122] (2) The specific experimental steps were carried out according to the HE staining instruction manual of Sevier: dehydration, infiltration, embedding, and sectioning; the paraffin sections were dewaxed to water, stained with hematoxylin and eosin, and finally dehydrated and sealed;

[0123] (3) The sections were placed under a white light microscope and photographed to save the pictures.

[0124] 4. Detection of Apoptosis of Granulosa Cells in Mouse Ovaries

[0125] The apoptosis of granulosa cells in mouse ovaries was detected by TUNEL.

[0126] (1) The wax blocks of mouse ovaries embedded with HE staining were selected, and paraffin sections were made;

[0127] (2) Refer to the TUNEL kit instruction manual of Sevier;

[0128] (3) The sections were placed under an inverted fluorescence microscope and photographed to save the pictures.

[0129] 5. Co-Immunoprecipitation

[0130] The experimental steps refer to the rProtein A / G Magnetic IP / Co-IP Kit instruction manual:

[0131] (1) Wash the cells twice with pre-cooled PBS, and add 1 mL of lysis buffer (add 1% protease inhibitor) to every 1×10 7 cells;

[0132] (2) Incubate at 4°C on a shaker for 20 min, centrifuge at 13,000 x g at 4°C for 10 min, transfer the supernatant to a new 1.5 mL RNase-free centrifuge tube, and measure the protein concentration;

[0133] (3) Wash the magnetic beads with 1× Lysis / Wash Buffer (Enhanced);

[0134] (4) Add 2 - 10 μL of antibody to the washed magnetic beads, supplement the volume to 500 μL with 1× Lysis / Wash Buffer, mix well vertically at room temperature for 30 min, adsorb the magnetic beads with a magnetic stand, aspirate the supernatant, and save the sample for detection;

[0135] (5) Add 500 μL of 1× Lysis / Wash Buffer to the centrifuge tube, vortex gently for 1 min, adsorb the magnetic beads with a magnetic stand, discard the supernatant, and repeat twice;

[0136] (6) Add 500 μL of cell protein lysate sample (total protein amount is 500 - 1000 μg) to the centrifuge tube, mix well vertically at 4°C for 4 h, adsorb the magnetic beads with a magnetic stand, aspirate the supernatant, and save the sample for detection;

[0137] (7) Add 500 μL of 1× Lysis / Wash Buffer (Enhanced) to the centrifuge tube, vortex gently for 1 min, adsorb the magnetic beads with a magnetic stand, discard the supernatant, and repeat once;

[0138] (8) Add 500 μL of 1× Lysis / Wash Buffer (Enhanced) to the centrifuge tube, transfer it to a new EP tube together with the magnetic beads, vortex gently for 1 min, adsorb the magnetic beads with a magnetic stand, and discard the supernatant;

[0139] (9) Add 50 μL of 1× electrophoresis loading buffer to the centrifuge tube, incubate in a 99°C water bath for 10 min, adsorb the magnetic beads with a magnetic stand, retain the loading buffer containing the target antigen, and store the sample at -20°C.

[0140] 6. For other experiments, refer to Example 1 and Example 2.

[0141] The results of counting the estrus day age of mice showed that compared with the control group, the first estrus day age of mice in the OGDH overexpression group was significantly advanced; while the first estrus day age of mice was delayed after OGDH knockdown ( Figure 6 ).

[0142] The expression of OGDH in mouse ovaries was detected by qRT-PCR and Western Blot. Compared with the control group, the mRNA and protein expression levels of OGDH gene were significantly increased in the LV-OGDH group; while the mRNA and protein expression levels of OGDH were significantly decreased in the sh-OGDH group. It indicates that the constructed mouse model can overexpress and knockdown the OGDH gene in the ovary and can be used for subsequent experiments( Figure 7 ).

[0143] Photographs of HE sections of mouse ovaries were taken and the data were analyzed quantitatively. The results showed that compared with the control group, the proportion of preantral follicles in the ovaries of mice in the LV-OGDH group was significantly decreased, and the proportion of corpora lutea was significantly increased (P<0.01); while the proportion of preantral follicles in the sh-OGDH group was significantly increased, and the proportion of corpora lutea was significantly decreased (P<0.05); this indicates that OGDH can promote the development of ovarian follicles in mice( Figure 8 ).

[0144] The apoptosis of GCs in mouse ovarian follicles was detected by TUNEL assay. Compared with the control group, there were fewer apoptotic GCs in the ovaries of mice in the LV-OGDH group; while the apoptosis level of GCs in the ovarian follicles of mice in the sh-OGDH group was higher than that in the control group, which indicates that OGDH can inhibit the apoptosis of GCs in mouse ovaries( Figure 9 ).

[0145] The expression of key genes in the mouse ovarian function pathway was detected by qRT-PCR and Western Blot. The experimental results showed that OGDH significantly inhibited the expression of key genes in the oxidative stress pathway in mouse ovaries, inhibited the expression of apoptosis pathway genes, and promoted the expression of proliferation pathway genes( Figure 10 ).

[0146] In summary, OGDH can promote the proliferation of ovarian granulosa cells, inhibit cellular oxidative stress, promote the development of ovarian follicles in mice, and thus promote estrus in mice.

[0147] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. The application of the OGDH gene in mammalian estrus, characterized in that: For at least one of the following applications 1 and 2: Application 1: Application of overexpressing the OGDH gene in promoting estrus in mammals; Application 2: Application of the OGDH gene overexpression vector in the preparation of a product for promoting estrus in mammals.

2. The application according to claim 1, wherein: The promotion of estrus in mammals is to shorten the estrus age of mammals; The mammal is any one of human, mouse, and pig.

3. The application according to claim 1, wherein: The product is any one of a drug, a reagent, a feed, and a feed additive; The dosage form of the drug or reagent is any one of an oral dosage form and an injection dosage form; The drug or reagent further comprises a pharmaceutically acceptable excipient or carrier.

4. The application of the OGDH gene in follicular development is for at least one of the following applications 3 and 4: Application 3: Application of overexpressing the OGDH gene in promoting follicular development in mammals; Application 4: Application of the OGDH gene overexpression vector in the preparation of a product for promoting follicular development in mammals.

5. The application according to claim 4, wherein: The promotion of follicular development in mammals is to increase the number of corpora lutea and / or antral follicles and reduce the number of preantral follicles; The mammal is any one of human, mouse, and pig.

6. The application according to claim 4, wherein: The product is any one of a drug and a reagent; The drug or reagent further comprises a pharmaceutically acceptable excipient or carrier.

7. The application of the OGDH gene in the culture of ovarian granulosa cells is for at least one of the following applications 5 and 6: Application 5: Application of overexpressing the OGDH gene in the in vitro culture of ovarian granulosa cells; Application 6: Application of the OGDH gene overexpression vector in the preparation of an additive for the in vitro culture of ovarian granulosa cells.

8. The application according to claim 7, wherein: Application 5 is: Under an in vitro environment, overexpressing the OGDH gene in ovarian granulosa cells to promote the proliferation of ovarian granulosa cells and reduce the oxidative stress level of ovarian granulosa cells; The in vitro culture additive is at least one of a proliferation promoter and an oxidative stress inhibitor; The ovarian granulosa cells are any one of human, mouse, and pig ovarian granulosa cells.

9. The application according to claim 8, wherein: The basic plasmid used for the overexpression vector is pcDNA3.

1.

10. Application of the OGDH gene as a marker for ovarian follicle growth and development.

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