Application of pyruvic acid in regulation and control of follicular development of mammals

Abstract

The invention discloses application of pyruvic acid in regulation and control of follicular development of mammals. According to the invention, pyruvic acid is taken as a research object, and the influence of pyruvic acid on oestrus day age, follicle development and granular cell proliferation and apoptosis of mammals is researched by adopting molecular and cell biology methods: HE staining and Tunel staining find that the pyruvic acid significantly increases the number of corpus luteum and luteum follicles and reduces the apoptosis of granular cells; qRT-PCR and WB results show that the mRNA and protein levels of the key genes of the proliferation pathway are up-regulated after pyruvic acid treatment and the mRNA and protein levels of the key genes of the apoptosis pathway are down-regulated after pyruvic acid treatment. In conclusion, pyruvic acid can promote the development of ovarian follicles by promoting granular cell proliferation and inhibiting granular cell apoptosis, so as to promote mouse oestrus.

Description

Technical Field

[0001] The present invention belongs to the field of cell engineering, and specifically relates to the application of pyruvate in regulating the follicular development of mammals. Background Art

[0002] In mammals, follicular growth and development is a very complex physiological process. The reproductive performance of livestock is closely related to the growth and development of follicles, and its reproductive efficiency can further affect its production performance. Granulosa cells play a key role in the growth and development of oocytes in follicles. The proliferation of granulosa cells can promote follicular growth and development and thus promote ovulation, and its apoptosis may cause follicular atresia.

[0003] Pyruvate is an important organic small molecule and a very weak organic acid. It has the properties of both carboxylic acid and ketone, and also has the properties of α-keto acid. Pyruvate enters the mitochondria and is oxidized to generate acetyl-CoA, enters the tricarboxylic acid cycle, and is oxidized into carbon dioxide and water to complete the aerobic oxidation energy supply process of glucose. The tricarboxylic acid cycle is the connection point between catabolism and anabolism, and has the basic function of oxidizing nutrients to support cell bioenergetics. Using pathway enrichment analysis, the results show that the tricarboxylic acid cycle is closely related to follicular development.

[0004] A large number of studies have shown that pyruvate is one of the important factors for maintaining follicular growth and development. The oxidative metabolism of pyruvate in mitochondria is the key to maintaining oocyte maturation. Pyruvate is the most basic energy substrate of oocytes. Since oocytes cannot directly utilize glucose as an energy source, but can directly utilize ATP, and ATP is produced in large amounts by pyruvate, the development and maturation of oocytes are inseparable from pyruvate. However, the specific regulatory role and mechanism of pyruvate on follicular development are still unclear. Summary of the Invention

[0005] 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 pyruvate and / or its pharmaceutically acceptable salts in promoting estrus in mammals.

[0006] The second object of the present invention is to provide the application of pyruvate and / or its pharmaceutically acceptable salts in promoting follicular development in mammals.

[0007] The third object of the present invention is to provide the application of pyruvate and / or its pharmaceutically acceptable salts in the culture of ovarian granulosa cells.

[0008] The objects of the present invention are achieved by the following technical solutions:

[0009] The application of pyruvate and / or its pharmaceutically acceptable salts in mammalian estrus is at least one of the following applications 1 and 2:

[0010] Application 1: Use of pyruvic acid and / or its pharmaceutically acceptable salts in promoting estrus in mammals;

[0011] Application 2: Use of pyruvic acid and / or its pharmaceutically acceptable salts in the preparation of a product for promoting estrus in mammals.

[0012] Furthermore, the promotion of estrus in mammals is to shorten the estrus age of mammals.

[0013] Furthermore, the product is any one of a drug, a reagent, a feed, and a feed additive.

[0014] Furthermore, the dosage form of the drug or reagent is any one of an oral dosage form and an injection dosage form.

[0015] Furthermore, the drug or reagent further comprises a pharmaceutically acceptable excipient or carrier.

[0016] Furthermore, the mammal is any one of a human, a mouse, and a pig.

[0017] Use of pyruvic acid and / or its pharmaceutically acceptable salts in follicular development is at least one of the following Applications 3 and 4:

[0018] Application 3: Use of pyruvic acid and / or its pharmaceutically acceptable salts in promoting follicular development in mammals;

[0019] Application 4: Use of pyruvic acid and / or its pharmaceutically acceptable salts in the preparation of a product for promoting follicular development in mammals.

[0020] Furthermore, the promotion of follicular development in mammals is to increase the number of corpora lutea and / or antral follicles.

[0021] Furthermore, the product is any one of a drug and a reagent.

[0022] Furthermore, the drug or reagent further comprises a pharmaceutically acceptable excipient or carrier.

[0023] Furthermore, the mammal is any one of a human, a mouse, and a pig.

[0024] Use of pyruvic acid and / or its pharmaceutically acceptable salts in the culture of ovarian granulosa cells is at least one of the following Applications 5 and 6:

[0025] Application 5: Use of pyruvic acid and / or its pharmaceutically acceptable salts in the in vitro culture of ovarian granulosa cells;

[0026] Application 6: Use of pyruvic acid and / or its pharmaceutically acceptable salts in the preparation of an additive for the in vitro culture of ovarian granulosa cells.

[0027] Further, the application 5 is as follows: under in vitro conditions, culturing ovarian granulosa cells with a culture medium containing pyruvate to promote the proliferation and / or viability of ovarian granulosa cells.

[0028] Further, the concentration of the pyruvate is 1 ± 0.5 mM.

[0029] Further, the culturing time is 24 ± 5 h.

[0030] Further, the in vitro culture additive is a proliferation and / or viability promoter.

[0031] Further, the ovarian granulosa cells are any one of human, mouse, and porcine ovarian granulosa cells.

[0032] Further, the pharmaceutically acceptable salts may be: sodium salts, potassium salts, ammonium salts, amino acid salts, lactate salts, hydrochloride salts, phosphate salts, acetate salts, malate salts, citrate salts, or aspartate salts, etc.

[0033] The present invention takes pyruvate as the research object and uses molecular and cell biology methods to study the effects on the estrus age, follicular development, and granulosa cell proliferation and apoptosis of mammals. The verification results of the present invention are as follows:

[0034] 1. Pyruvate promotes mouse estrus in a dose-dependent manner ( Figure 1 );

[0035] 2. Pyruvate promotes the development of mouse ovarian follicles ( Figure 2 );

[0036] 3. Pyruvate inhibits the apoptosis of mouse ovarian granulosa cells ( Figure 3 );

[0037] 4. Pyruvate up-regulates the mRNA levels of key genes in the proliferation pathway in the mouse ovary; down-regulates the mRNA levels of key genes in the apoptosis pathway ( Figure 4 , Figure 5 );

[0038] 5. The optimal time for pyruvate to treat cells is 24 h, and the optimal concentration is 1 nM ( Figure 6 );

[0039] 6. Pyruvate can promote granulosa cell proliferation ( Figure 7 );

[0040] 7. Pyruvate can inhibit granulosa cell apoptosis ( Figure 8 ).

[0041] The present invention has the following advantages and effects compared with the prior art:

[0042] 1. The present invention takes pyruvate as the research object and uses molecular and cell biology methods to study its effects on the estrus age, follicular development, and the proliferation and apoptosis of granulosa cells in mammals. By increasing the level of pyruvate, it is found that increasing pyruvate can promote the proliferation of ovarian granulosa cells, inhibit apoptosis, promote follicular development, and animal estrus. It has good application value for studying the mechanism of ovarian follicular development, etc.

[0043] 2. The present invention uses mice and KGN cells as experimental materials to verify the effects of pyruvate on estrus age, follicular development, and the proliferation and apoptosis of granulosa cells at the in vivo and cell levels.

[0044] 3. The technical solution of the present invention is carefully designed and the results are reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a graph of the estrus age of mice after treatment with different doses of pyruvate.

[0046] Figure 2 It is a HE staining graph of the mouse ovary.

[0047] Figure 3 It is a Tunel staining graph of the mouse ovary.

[0048] Figure 4 It is a graph of the mRNA levels of key genes in the proliferation pathway in the mouse ovary.

[0049] Figure 5 It is a graph of the mRNA levels of key genes in the apoptosis pathway in the mouse ovary.

[0050] Figure 6 It is a graph of the effect of detecting the viability of human ovarian granulosa cells by the CCK8 method.

[0051] Figure 7 It is a graph of the effect of detecting the proliferation of human ovarian granulosa cells by the EdU method; among them, a is the effect of detecting the proliferation of ovarian granulosa cells by the EdU method; b is the effect of pyruvate on the mRNA levels of genes related to the proliferation pathway of granulosa cells; c is the effect of pyruvate on the protein levels of genes related to the proliferation pathway of granulosa cells.

[0052] Figure 8 It is a graph of the effect of detecting the apoptosis of human ovarian granulosa cells by the Annexin V-FITC method; among them, a is the effect of detecting the apoptosis of ovarian granulosa cells by the Annexin V-FITC method; b is the effect of pyruvate on the mRNA levels of genes related to the apoptosis pathway of granulosa cells; c is the effect of pyruvate on the protein levels of genes related to the apoptosis pathway of granulosa cells. DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto. For the experimental methods without specific conditions noted in the following embodiments, they are generally in accordance with conventional conditions. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0054] In the present invention, statistical methods are applied to analyze the results of 3 independent experiments in each embodiment, calculate the "mean ± standard deviation" respectively, and use one-way analysis of variance for significant difference analysis (in the figure, "*" indicates P < 0.05, and "**" indicates P < 0.01).

[0055] Example 1: Effect of pyruvate on the estrus age of mice

[0056] Female C57BL / 6J mice at 3 weeks of age were randomly divided into three groups: a blank group, a control group, and a pyruvate group. Among them, there were 10 mice in the blank group, which were adaptively fed throughout the process; there were 10 mice in the control group, which were adaptively fed for 3 days and then injected with 0.9% NaCl, and each mouse was intraperitoneally injected once a day for 3 weeks; there were 50 mice in the pyruvate group, which were adaptively fed for 3 days and then divided into five subgroups, and were respectively injected with 0.5, 5, 25, 50, and 500 mg / kg sodium pyruvate, and each mouse was intraperitoneally injected once a day for 3 weeks.

[0057] The results are as Figure 1 shown. It can be seen that within the concentration range of 0.5 - 500 mg / kg, after pyruvate injection, the estrus age of mice can be advanced, and it shows a dose-dependent manner.

[0058] Example 2: Effect of pyruvate on the ovarian follicles of mice

[0059] Female C57BL / 6J mice at 3 weeks of age were adaptively fed for 3 days and then injected with 50 mg / kg sodium pyruvate, and each mouse was intraperitoneally injected once a day for 3 weeks. Subsequently, the mice were sacrificed, and their ovaries were collected to make sections; the RNA and proteins of the mouse ovaries were extracted to detect the mRNA and protein levels of key genes in the proliferation and apoptosis pathways. The specific operations are as follows:

[0060] 1. Treatment of mouse ovaries

[0061] (1) HE staining was carried out according to the instructions of Wuhan Sevier Biotechnology Co., Ltd.

[0062] (2) Tunel staining was carried out according to the instructions of Wuhan Sevier Biotechnology Co., Ltd.

[0063] 2. RNA extraction and reverse transcription

[0064] (1) RNA extraction

[0065] ①Extract two types of follicles with diameters < 3 mm and > 3 mm from sows' ovaries. Thoroughly homogenize the samples after adding TRIzol in proportion, let them stand on ice for 10 min, and centrifuge at 4°C and 12,000 rpm for 5 min;

[0066] ②Transfer the supernatant to a new sterile 1.5 mL centrifuge tube, add 200 μL of chloroform, shake and mix well for 30 s, let it stand on ice for 15 min, and centrifuge at 4°C and 12,000 rpm for 15 min;

[0067] ③Transfer the upper aqueous phase to a new sterile 1.5 mL centrifuge tube, add 500 μL of isopropanol, gently mix, let it stand on ice for 15 min, and centrifuge at 4°C and 12,000 rpm for 15 min, then discard the supernatant;

[0068] ④Add 1 mL of pre-cooled 75% ethanol (ethanol:DEPC water = 3:1), resuspend and wash the RNA precipitate, and centrifuge at 4°C and 12,000 rpm for 5 min;

[0069] ⑤Discard the supernatant, leave the RNA precipitate, dry for 5 min, then add 30 μL of DEPC water to resuspend and mix the RNA precipitate evenly. Use a UV spectrophotometer to detect the ratio of OD 260 / OD 280 of the sample for quality detection, and store the qualified RNA in an -80°C refrigerator.

[0070] (2)RNA reverse transcription:

[0071] Configure the system according to the PrimeScript RT Master Mix instruction manual. The reaction system is shown in Table 1.

[0072] Table 1 RNA reverse transcription system

[0073]

[0074] Put the configured system into a PCR instrument and perform the reaction at 37°C for 15 min and 85°C for 5 s. Store the reverse-transcribed cDNA in a -20°C refrigerator for subsequent experiments.

[0075] 3. qRT-PCR

[0076] The qRT-PCR detection in the present invention uses qPCR SYBR Green Master Mix kit. The comparative Ct value method is used in the experiment to detect the content of sample genes. The specific calculation formula is as follows:

[0077] 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﹚〉}

[0078] GAPDH was used as an internal reference for detecting genes, and the qRT-PCR primers used in this invention are shown in Table 2, Table 3, and Table 4.

[0079] Table 2 Primer Sequences

[0080]

[0081]

[0082] Table 3 Primer Sequences

[0083]

[0084] Table 4 Primer Sequences

[0085]

[0086]

[0087] 4. Protein Extraction

[0088] Wash the cells with pre-cooled PBS buffer, add an appropriate amount of protein lysate (containing 1% protease inhibitor), incubate on a shaker at 4°C for 15 min, blow the cells in the wells to fully lyse them, repeat the addition into the same tube, centrifuge at 12,000 rpm and 4°C for 10 min, and transfer the supernatant to a new centrifuge tube.

[0089] The results of HE staining are as Figure 2 shown. Pyruvate significantly increased the number of corpora lutea and antral follicles, promoting ovarian development and ovulation.

[0090] The results of Tunel staining are as Figure 3 shown. The number of apoptotic cells in the pyruvate treatment group was less than that in the control group.

[0091] Extract the RNA and protein of mouse ovaries, and detect the mRNA and protein levels of key genes in the proliferation and apoptosis pathways. The results show that pyruvate significantly increased the mRNA ( Figure 4 in a)) and protein levels ( Figure 4 in b)) of key genes in the proliferation pathway in mouse ovaries, and significantly decreased the mRNA ( Figure 5 in a)) and protein levels ( Figure 5 in b)) of key genes in the apoptosis pathway in mouse ovaries.

[0092] Example 3: Effects of Pyruvate on Human Ovarian Granulosa Cells

[0093] 1. Culture of Ovarian Granulosa Cells

[0094] (1) Cell resuscitation: Wait for the water bath to heat up to 37 °C. Place the cryopreserved KGN cells in the water bath, heat for 1 min, and shake the cryotube to accelerate cell thawing. Add complete medium to a centrifuge tube, add the thawed cell suspension to the centrifuge tube, mix well, and centrifuge at 1,000 rpm for 5 min. Pour off the supernatant, add 5 ml of complete medium to the pellet in the centrifuge tube, resuspend, transfer the cell suspension to a small culture flask, mix the suspension well, and place it in the incubator for culture.

[0095] (2) Cell passage: Pour off the medium in the flask, wash the culture flask twice with PBS, add 3 ml of trypsin, digest in the incubator for 5 min, and use an equal volume of complete medium to end the digestion. Transfer the cell suspension to a centrifuge tube, centrifuge at 1,000 rpm for 5 min. Pour off the supernatant, wash twice with PBS, pour off the supernatant, resuspend the cells with 2 ml of complete medium, transfer the suspension to a large culture flask, mix the suspension well, and place it in a 37 °C incubator for culture.

[0096] 2. Treatment of ovarian granulosa cells with pyruvate

[0097] (1) When the cell density in the flask grows to about 80%, pour off the medium in the flask, wash the culture flask twice with PBS, add 3 ml of trypsin, digest in the incubator for 5 min, and use an equal volume of complete medium to end the digestion. Transfer the cell suspension to a centrifuge tube, centrifuge at 1,000 rpm for 5 min. Pour off the supernatant, wash twice with PBS, pour off the supernatant, and resuspend the cells with 2 ml of complete medium;

[0098] (2) Divide the cells into two culture flasks, culture for 24 h first, then pour off the medium in the flasks, wash the culture flasks twice with PBS, treat one flask with 1 mM pyruvate, and one group is the blank group. After culturing for 24 h, carry out subsequent experiments.

[0099] 3. Detection of ovarian granulosa cell viability

[0100] This experiment was carried out according to the instruction manual of the CCK8 cell viability / cytotoxicity detection kit. The specific operation steps are as follows:

[0101] (1) Digest the cells, count and inoculate them on a 96-well cell culture plate for culture, and transfect the cells according to the purpose.

[0102] (2) When the cell density reaches 70%, add 10 μL of CCK8 reagent to each well at 12 h, 24 h, 36 h, and 48 h respectively, and continue to incubate in the cell culture incubator for 2 h.

[0103] (3) After incubation, use a multifunctional microplate reader to detect the absorbance value of the cells at 450 nm.

[0104] (4) Calculate the viability of cells at different time periods according to the calculation formula.

[0105] 4. Detection of ovarian granulosa cell proliferation

[0106] The present invention uses the EdU method to detect cell proliferation. Referring to the instruction manual of the Cell-Light EdU Apollo 567 In vitro Kit of Guangzhou Ribobio Co., Ltd., the specific operation steps are as follows: TM EdU Apollo 567 In vitro Kit detection kit instruction manual, the specific operation steps are as follows:

[0107] (1) Dilute the EdU solution with complete medium to a final concentration of 50 μM. Add 150 μL of the EdU dilution to each well, place the culture plate in the incubator for 2 h, discard the medium, and wash the cells 2 times with PBS solution;

[0108] (2) Add 150 μL of fixative (PBS containing 80% acetone) to each well, incubate at room temperature for 30 min, discard the fixative, and wash 2 times with PBS;

[0109] (3) Add 150 μL of PBS containing 0.5% Triton X to each well to permeabilize the cells for 10 min, and wash the cells 3 times with PBS;

[0110] (4) Prepare 1× Apollo staining reaction solution according to the instruction manual, add 150 μL to each well, incubate at room temperature in the dark for 30 min, aspirate the reaction solution, and wash 3 times with PBS;

[0111] (5) Prepare DAPI staining solution, add 150 μL to each well, incubate at room temperature in the dark for 30 min, aspirate the reaction solution, and wash 3 times with PBS;

[0112] (6) Add 150 μL of PBS solution to each well to avoid drying at the bottom of the well. Take pictures with a fluorescence microscope. The cells stained blue in the picture are the cells on the cell culture plate before fixation, and the cells stained red are the newly proliferated cells on the cell culture plate within 30 min of incubation. Finally, use ImageJ to count the number of granulosa cells and analyze the proliferation rate of granulosa cells.

[0113] 5. Detection of ovarian granulosa cell apoptosis

[0114] The present invention uses the Annexin V-FITC technique to detect cell apoptosis. Referring to the instruction manual of the Annexin V-FITC Apoptosis Detection Kit of BioVision company, the specific operation steps are as follows:

[0115] (1) Sow ovarian granulosa cells were inoculated into 6-well plates, and the cells were cultured until the confluence reached 50 - 80%, and then the cells were washed with PBS solution;

[0116] (2) Trypsin without EDTA (ethylenediaminetetraacetic acid) was used to digest the cells, and the cells were washed with 2 mL of PBS solution;

[0117] (3) Take 0.5 mL of cell suspension (about 5×10 5 cells), and add 500 μL of 1×Binding Buffer;

[0118] (4) Add 5 μL of Annexin V-FITC and 5 μL of PI (Propidium Iodide) at room temperature, and incubate in the dark at room temperature for 5 min;

[0119] (5) Immediately detect and analyze with a flow cytometer.

[0120] 6. qRT-PCR Detection and Protein Extraction

[0121] RNA and proteins of mouse ovarian granulosa cells were extracted to detect the mRNA and protein levels of key genes in the proliferation and apoptosis pathways.

[0122] For the specific operation, refer to Example 2, and the primers used are shown in Tables 2, 3, and 4.

[0123] Cells were treated with pyruvate, and the results showed that the proliferation rate of cells in the pyruvate group was significantly higher than that in the control group ( Figure 7 in a)), and the mRNA ( Figure 7 in b)) and protein level ( Figure 7 in c)) of key genes in the proliferation pathway in granulosa cells were significantly increased.

[0124] Flow cytometry results showed that the apoptosis rate (early apoptosis + late apoptosis) of cells in the pyruvate group was significantly lower than that in the control group ( Figure 8 in a)), and the mRNA ( Figure 8 in b)) and protein level of key genes in the apoptosis pathway in granulosa cells were also significantly decreased ( Figure 8 in c)).

[0125] In summary, pyruvate treatment can promote the proliferation of ovarian granulosa cells, inhibit cell apoptosis, thereby promoting follicle development, and further promoting mouse estrus.

[0126] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by 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. Use of pyruvic acid and / or its pharmaceutically acceptable salts in mammalian estrus, characterized in that: For at least one of the following applications 1 and 2: Application 1: Use of pyruvic acid and / or a pharmaceutically acceptable salt thereof in promoting estrus in mammals; Application 2: Use of pyruvic acid and / or a pharmaceutically acceptable salt thereof 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.

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 mammal is any one of a human, a mouse, and a pig.

4. The application according to claim 3, wherein: 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.

5. Use of pyruvic acid and / or its pharmaceutically acceptable salts in follicular development, characterized in that For at least one of the following applications 3 and 4: Application 3: Use of pyruvic acid and / or a pharmaceutically acceptable salt thereof in promoting follicular development in mammals; Application 4: Use of pyruvic acid and / or a pharmaceutically acceptable salt thereof in the preparation of a product for promoting follicular development in mammals.

6. The application according to claim 5, wherein: The promotion of follicular development in mammals is to increase the number of corpora lutea and / or antral follicles.

7. The application according to claim 5, wherein: The product is any one of a drug and a reagent; The drug or reagent further comprises a pharmaceutically acceptable excipient or carrier; The mammal is any one of a human, a mouse, and a pig.

8. Use of pyruvic acid and / or its pharmaceutically acceptable salts in ovarian granulosa cell culture, characterized in that: For at least one of the following applications 5 and 6: Application 5: Use of pyruvic acid and / or a pharmaceutically acceptable salt thereof in the in vitro culture of ovarian granulosa cells; Application 6: Use of pyruvic acid and / or a pharmaceutically acceptable salt thereof in the preparation of an additive for the in vitro culture of ovarian granulosa cells.

9. The application according to claim 8, wherein: The Application 5 is: In an in vitro environment, ovarian granulosa cells are cultured with a medium containing pyruvic acid to promote the proliferation and / or viability of ovarian granulosa cells; The in vitro culture additive is a proliferation and / or viability promoter.

10. The application according to claim 9, wherein: The concentration of the pyruvic acid is 1 ± 0.5 mM; The culture time is 24 ± 5 h; The ovarian granulosa cells are any one of human, mouse, and pig ovarian granulosa cells.

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