Application of glycine hyodeoxycholic acid in regulation and control of follicular granule cell viability and steroid hormone secretion

Abstract

The invention discloses application of glycine hyodeoxycholic acid in regulation and control of follicular granule cell viability and steroid hormone secretion. According to the application disclosed by the invention, a pig follicular granule cell in-vitro culture technology is utilized, the cell viability and steroid secretion condition of the follicular granule cells after exogenous addition of the GHDCA are detected, and a result shows that the GHDCA can enhance the cell viability of the pig follicular granule cells in a dose-dependent manner, and meanwhile, the GHDCA can promote the secretion of estradiol and inhibit the secretion of progesterone in a dose-dependent manner. The result shows that the GHDCA can regulate and control the activity of follicular granule cells and the secretion of steroid hormones. According to the invention, a foundation is laid for the research of the GHDCA on the molecular mechanism of promoting the secretion of follicular granular cell hormone, and a possible way is also provided for the development and estrus regulation of pig follicles.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the application of glycohyodeoxycholic acid in regulating the activity of follicular granulosa cells and the secretion of steroid hormones. Background Art

[0002] The ovary is the core organ of the mammalian reproductive system, primarily composed of follicles and medulla. As the functional unit of the ovary, the follicle's primary function is to release the oocyte and secrete estradiol. However, during follicular development, follicular cells undergo proliferation and differentiation, and follicle quality depends on the normal growth of granulosa cells (GCs). However, most follicles undergo atresia and degenerate, and only a few follicles mature and ovulate. This follicular atresia can delay the onset of puberty in gilts on large-scale farms, severely impacting the economic performance of pig farms.

[0003] In female animals, estradiol secretion increases with follicular maturation and, by regulating related hormones, promotes follicular maturation and triggers ovulation. During mammalian pregnancy, estradiol has regulatory effects such as promoting uterine growth and development and altering uterine blood flow. Primordial follicles remain dormant in the mammalian ovary for a long time. As follicles grow and develop, they become activated, growing and developing under the influence of estrogen and other regulatory factors. In pigs, estradiol can inhibit oxidative stress-induced apoptosis in granulosa cells.

[0004] Progesterone plays a crucial role in early embryonic development, stimulating the production of uterine secretions and providing essential nutrients for the development and implantation of the conceptus. During pregnancy, progesterone can inhibit uterine contractions, maintain pregnancy stability, and promote breast development. In cultured granulosa cells, increased progesterone secretion inhibits luteinization of granulosa cells and significantly increases progesterone levels in atretic follicles.

[0005] Bile acid is an important substance synthesized by the liver and involved in fat digestion and absorption in the intestine. Studies have shown that bile acid metabolism disorders can lead to ovulation disorders, impaired steroid hormone synthesis and interrupted follicular development, ultimately leading to ovarian damage and decreased fertility. Glycohyodeoxycholic acid (GHDCA) is a product formed by the combination of hyodeoxycholic acid and glycine. Patent CN117562910A discloses the use of glycohyodeoxycholic acid in the preparation of a drug for the treatment of inflammatory bowel disease, and discloses the use of glycohyodeoxycholic acid in the preparation of a drug for reducing intestinal mucosal barrier damage. However, the prior art does not disclose that glycohyodeoxycholic acid can be used to regulate the activity of follicular granulosa cells or the secretion of steroid hormones. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a method for regulating the activity of granulosa cells and / or the secretion of steroid hormones by using glycohyodeoxycholic acid.

[0007] The second object of the present invention is to provide the use of glycohyodeoxycholic acid in the preparation of products for regulating the activity of follicular granulosa cells and / or the secretion of steroid hormones.

[0008] The third object of the present invention is to provide the use of glycohyodeoxycholic acid in the preparation of products for regulating the development of pig follicles.

[0009] The fourth object of the present invention is to provide the use of glycohyodeoxycholic acid in the preparation of products for regulating pig estrus.

[0010] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0011] This study utilizes in vitro culture of porcine granulosa cells. By adjusting the concentration of glycohyodeoxycholic acid (GHDCA) in the culture medium, the effects of GHDCA on granulosa cell viability and steroid secretion were examined. The results showed that exogenous addition of GHDCA dose-dependently enhanced granulosa cell viability. Furthermore, ELISA revealed that GHDCA dose-dependently promoted estradiol (E2) secretion and inhibited progesterone (PROG) secretion. RT-qPCR and Western blotting techniques were used to further examine the expression levels of estradiol synthase and progesterone synthase genes in granulosa cells. The results revealed that GHDCA dose-dependently promoted the mRNA expression levels of the estradiol synthase genes STAR, CYP11A1, and CYP19A1, as well as the protein expression levels of CYP11A1 and CYP19A1; it also inhibited the mRNA and protein expression levels of the progesterone synthase gene 3βHSD. This suggests that GHDCA can regulate granulosa cell viability and steroid hormone secretion.

[0012] Therefore, the present invention provides the use of glycohyodeoxycholic acid in regulating the activity of follicular granulosa cells and / or the secretion of steroid hormones.

[0013] The present invention also provides the use of glycohyodeoxycholic acid in preparing products for regulating the activity of follicular granulosa cells and / or the secretion of steroid hormones.

[0014] Furthermore, the steroid hormone is estradiol and / or progesterone.

[0015] Specifically, the regulation is that glycohyodeoxycholic acid positively regulates the activity of granulosa cells and the secretion of estradiol and / or negatively regulates the secretion of progesterone in granulosa cells. That is, the regulation is that glycohyodeoxycholic acid enhances the activity of granulosa cells, promotes the secretion of estradiol in granulosa cells and / or inhibits the secretion of progesterone in granulosa cells.

[0016] Furthermore, the promotion of estradiol secretion in granulosa cells is achieved by promoting the expression of estradiol secretion-related genes STAR, CYP11A1 and CYP19A1.

[0017] Preferably, promoting the secretion of estradiol by granulosa cells is promoting the mRNA expression levels of estradiol synthase genes STAR, CYP11A1 and CYP19A1, and promoting the protein expression levels of CYP11A1 and CYP19A1.

[0018] Furthermore, the inhibition of progesterone secretion in granulosa cells is achieved by inhibiting the expression of progesterone synthase gene 3βHSD.

[0019] Preferably, inhibiting the secretion of progesterone from granulosa cells is inhibiting the mRNA and protein expression levels of the progesterone synthase gene 3βHSD.

[0020] Furthermore, the follicular granulosa cells are porcine follicular granulosa cells.

[0021] During mammalian pregnancy, estradiol has regulatory effects such as promoting uterine growth and development and altering uterine blood flow. In pigs, estradiol can inhibit apoptosis of pig granulosa cells induced by oxidative stress. By regulating estradiol levels, it can also promote follicle maturation and trigger ovulation. Increased progesterone secretion inhibits luteinization of granulosa cells, thereby inhibiting follicle development and preventing animals from entering estrus. When progesterone levels decrease, it promotes gonadotropin secretion, allowing follicles to continue developing and the animals to enter estrus. The present invention's research shows that, in vitro, GHDCA can dose-dependently positively regulate estradiol secretion and negatively regulate progesterone secretion in pig granulosa cells.

[0022] Therefore, the present invention also provides the use of glycohyodeoxycholic acid in preparing products for regulating the development of pig follicles.

[0023] The present invention also provides the use of glycohyodeoxycholic acid in preparing products for regulating pig estrus.

[0024] Furthermore, the product is achieved by enhancing the activity of porcine follicular granulosa cells and / or regulating the secretion of estradiol and progesterone.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides the use of glycohyodeoxycholic acid in regulating the activity of granulosa cells and / or steroid hormone secretion. The present invention utilizes porcine granulosa cells in vitro culture technology to detect the cell activity and steroid secretion of granulosa cells after exogenous addition of GHDCA. The results show that GHDCA can enhance the activity of porcine granulosa cells in a dose-dependent manner. ELISA detection also reveals that GHDCA can promote the secretion of estradiol (E2) and inhibit the secretion of progesterone (PROG) in a dose-dependent manner. Further analysis of the expression levels of estradiol synthase and progesterone synthase genes in granulosa cells using RT-qPCR and Western blotting revealed that GHDCA dose-dependently increased the mRNA expression of the estradiol synthase genes STAR, CYP11A1, and CYP19A1, as well as the protein expression of CYP11A1 and CYP19A1; it also inhibited the mRNA and protein expression of the progesterone synthase gene 3βHSD. This suggests that GHDCA can regulate granulosa cell viability and steroid hormone secretion, particularly by promoting estradiol secretion and inhibiting progesterone secretion in a dose-dependent manner. This study lays the foundation for research on the molecular mechanism of GHDCA's role in promoting steroid hormone secretion in granulosa cells and provides a potential approach for regulating porcine follicular development and estrus. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The effect of different concentrations of GHDCA on the cell viability of porcine granulosa cells.

[0028] Figure 2 Effects of different concentrations of GHDCA on the secretion of estradiol and progesterone in porcine granulosa cells.

[0029] Figure 3 The effect of different concentrations of GHDCA on the expression level of estradiol synthase mRNA in porcine granulosa cells.

[0030] Figure 4 The effect of different concentrations of GHDCA on the expression level of progesterone synthase 3βHSD mRNA in porcine granulosa cells.

[0031] Figure 5 The effect of different concentrations of GHDCA on the expression level of estradiol synthase protein in porcine granulosa cells.

[0032] Figure 6 The effect of different concentrations of GHDCA on the expression level of progesterone synthase 3βHSD protein in porcine granulosa cells. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0035] 1. Culture of porcine follicular granulosa cells

[0036] (1) Healthy pig ovaries were collected from the slaughterhouse, placed in physiological saline, and quickly transported back to the laboratory in a warm place;

[0037] (2) Cut off the ovarian mesentery, squeeze and wash with 75% alcohol for 30 seconds, and then wash three times with 37℃ normal saline. Use a 10mL syringe to extract follicular fluid from follicles of uniform size. Place the follicles in a 15mL sterile centrifuge tube in a 37℃ water bath for 15 minutes and collect the supernatant.

[0038] (3) Collect the pelleted cells by centrifugation at 1500 g for 5 min, wash twice with PBS, resuspend in 7 mL of complete culture medium (DMEM basal medium + 1% double antibody + 10% fetal bovine serum), transfer the cells to a cell culture dish, and culture overnight in a cell culture incubator at 37°C and 5% CO2;

[0039] (4) Discard the original culture medium, rinse once with PBS, and add complete culture medium to continue culturing;

[0040] (5) When the cell confluence reaches 90%, add 0.25% trypsin to digest for 2 min. Observe under a microscope until most cells float up, and then immediately add an equal amount of complete culture medium to terminate the digestion.

[0041] (6) Use a pipette tip to blow off the cells attached to the wall and blow evenly, transfer the cell suspension to a centrifuge tube, centrifuge at 1500g for 5 minutes, and discard the supernatant;

[0042] (7) After washing twice with PBS, resuspend the cell pellet in complete culture medium, evenly distribute it to the culture plate, add complete culture medium to make up the volume, shake gently, and culture in the incubator;

[0043] (6) After about 12 hours, observe the cell status. When the cell confluence reaches about 50%, replace the culture medium with complete culture medium supplemented with glycohyodeoxycholic acid (GHDCA) in different proportions (0 μM, 100 μM, 200 μM, 300 μM, 400 μM, 600 μM, 800 μM, 1600 μM) and continue culturing.

[0044] Example 1 Detection of cell viability of porcine follicle granulosa cells

[0045] 1. Experimental Methods

[0046] The present invention uses CCK-8 technology to detect cell viability. Referring to the instructions of the CCK-8 kit of Biosharp, the specific operation steps are as follows:

[0047] (1) Discard the original culture medium in the 96-well plate, wash once with PBS, and add 100 μL of new complete culture medium to each well;

[0048] (2) In a dark environment, add 10 μL of CCK-8 solution to each well and gently shake the culture plate to mix the liquid evenly;

[0049] (3) Transfer to an incubator away from light and incubate for 1.5 h;

[0050] (4) After incubation, measure the absorbance of each well at 450 nm using a microplate reader.

[0051] 2. Experimental Results

[0052] The cell viability of porcine follicle granulosa cells treated with different concentrations of GHDCA was detected by CCK-8 technology. Figure 1 As shown in the results, when the concentration of GHDCA in the complete culture medium reached 300 μM, the cell viability could be significantly enhanced (P < 0.01). As the concentration of GHDCA increased, the cell viability also gradually increased.

[0053] Example 2 Detection of estradiol and progesterone content in porcine follicle granulosa cells

[0054] 1. Experimental Methods

[0055] The present invention uses ELISA technology to detect estradiol and progesterone secretion. Referring to the instructions of the PROG and E2 ELISA kits of Huamei Company, the specific operating steps are as follows:

[0056] 1. Estradiol (E2):

[0057] (1) Equilibrate each reagent at room temperature for 1 h; prepare the washing solution at a ratio of 1:20 between concentrated washing solution and deionized water;

[0058] (2) Collect the culture medium from the culture plate, centrifuge at 12000 rpm for 10 min at 4°C, and transfer the supernatant to a new EP tube;

[0059] (3) Remove the ELISA plate, set up a blank control well without adding any liquid; set up two wells for each standard point in sequence, and add 50 μL of the corresponding standard to each well; add 50 μL of the sample to be tested to each of the remaining test wells, and set up three counting replicates;

[0060] (4) Prepare the enzyme conjugate mixture at a volume ratio of 1:10, add 50 μL to each well (no addition to the blank control well), mix thoroughly, attach a self-adhesive sealant, and incubate at 37°C for 1 h.

[0061] (5) Discard the liquid in the wells, fill each well with washing solution, let it stand for 10 seconds to dry, and then pat it dry on kitchen paper. Repeat three times;

[0062] (6) Add 50 μL of color developer A and 50 μL of color developer B to each well, shake and mix thoroughly, then color develop at 37°C in the dark for 15 min. Add 50 μL of stop solution to each well.

[0063] (7) Use an enzyme-labeled instrument to measure the OD value of each well at a wavelength of 450 nm.

[0064] (8) In the ELISA calculation software, the standard curve is fitted with the concentration and OD value of the standard. 2 The closer the value is to 1, the better the fitting effect is. Then substitute the OD value of the sample to be tested into the formula of the standard to calculate the concentration of the sample to be tested.

[0065] 2. Progesterone (PROG):

[0066] Step (4) is to add 50 μL of enzyme conjugate to each well (no addition to blank control wells), then add 50 μL of antibody in the same order, mix thoroughly, attach self-adhesive sealant, and incubate at 37°C for 1 hour. The remaining steps are the same as for estradiol.

[0067] 2. Experimental Results

[0068] The culture medium containing different concentrations of GHDCA and the control was added to the porcine follicle granulosa cells by changing the medium and cultured for 24 hours. The secretion levels of estrogen and progesterone in the culture medium were detected by ELISA kit. The results are as follows: Figure 2 As shown, when the concentration of GHDCA in the complete culture medium reached 100 μM, it could significantly promote the secretion of estradiol and inhibit the secretion of progesterone (P < 0.0001), and it was dose-dependent.

[0069] Example 3 Detection of mRNA expression levels of estradiol synthase and progesterone synthase in porcine follicle granulosa cells

[0070] 1. Experimental Methods

[0071] 1. RNA extraction from porcine follicular granulosa cells

[0072] Refer to the Vazyme FastPure Cell / Tissue Total RNA Isolation Kit V2 operating instructions. The specific steps are as follows:

[0073] (1) After adding different concentrations of GHDCA to the culture medium of porcine follicular granulosa cells and culturing for 24 h, the culture medium was discarded, the cells were washed once with PBS, and 500 μL of Buffer RL was added to each well of a 12-well plate to lyse the cells;

[0074] (2) Place on ice for 10 min to allow for complete lysis, transfer the lysate to FastPure gDNA-Filter Column III, centrifuge at 12,000 rpm for 30 s, and collect the filtrate;

[0075] (3) Transfer the entire mixture to FastPure RNA Column III, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate;

[0076] (4) Add 700 μL of Buffer RW1, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate;

[0077] (5) Add 700 μL of Buffer RW2 (with anhydrous ethanol added), centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate;

[0078] (6) Add 500 μL of Buffer RW2 (with anhydrous ethanol added), centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate;

[0079] (7) Centrifuge at 12,000 rpm for 2 min, discard the collection tube, and transfer the adsorption column to a new 1.5 mL EP tube;

[0080] (8) After blowing with strong wind for 2 minutes, 20 μL of RNase-free ddH2O was added to the adsorption column and the column was placed on ice for 5 minutes to dissolve the RNA. The column was centrifuged at 12,000 rpm for 2 minutes to collect the filtrate.

[0081] (9) The synthesis of the first-strand cDNA was performed with reference to the ABScript Neo RT Master Mix for qPCR with gDNA Remover kit from ABclonal.

[0082] 2. RT-qPCR detection of mRNA expression

[0083] Refer to the BrightCycle Universal SYBR Green qPCR Mix with UDG instructions from ABclona. The specific steps are as follows:

[0084] (1) Add 0.4 μL of each upstream and downstream primer (10 μM) to each well; the primer sequences are shown in Table 1 below;

[0085] Table 1 Primer sequence list

[0086]

[0087]

[0088] (2) Add 2 μL of template cDNA to each well;

[0089] (3) Add 10 μL of BrightCycle Universal SYBR Green qPCR Mix with UDG to each well in the dark;

[0090] (4) Add ddH2O to make the volume up to 20 μL;

[0091] (5) After thorough mixing, the plate was sealed with a film and placed in a real-time fluorescence quantitative PCR instrument (Shenzhen Qianhai Roche Gene Technology Co., Ltd.). The reaction procedure was as follows: 37°C UDG reaction for 2 min, 95°C pre-denaturation for 3 min; 95°C denaturation for 5 s, 60°C annealing for 30 s, 40 cycles.

[0092] 2. Experimental Results

[0093] Real-time quantitative PCR was used to detect the expression level of estradiol synthase mRNA in porcine granulosa cells treated with different concentrations of GHDCA. Figure 3 As shown, when the concentration of GHDCA in the complete culture medium reached 300 μM, it significantly upregulated the mRNA expression levels of the estrogen synthases STAR and CYP19A1 (P < 0.01). When the concentration of GHDCA in the complete culture medium reached 100 μM, it significantly upregulated the mRNA expression level of the estrogen synthase CYP11A1 (P < 0.01). As the concentration of GHDCA increased, the mRNA expression levels of estrogen synthases also gradually increased.

[0094] Real-time quantitative PCR was used to detect the expression level of progesterone synthase 3βHSD mRNA in porcine follicle granulosa cells treated with different concentrations of GHDCA. Figure 4 As shown in the results, when the concentration of GHDCA in the complete culture medium reached 300 μM, it could significantly down-regulate the mRNA expression level of progesterone synthase 3βHSD (P<0.01), and the degree of down-regulation was proportional to the GHDCA concentration.

[0095] Example 4 Extraction of porcine follicle granulosa cell protein

[0096] 1. Experimental Methods

[0097] 1. Extraction of porcine follicle granulosa cell protein

[0098] (1) Discard the liquid in the culture dish, rinse once with PBS, and after absorbing the liquid, add 500 μL of RIPA (Biyuntian) containing 1% PMSF to each dish and collect the lysate;

[0099] (2) Place the lysate on ice for 30 min, mixing by inverting several times.

[0100] (3) After quick freezing in liquid nitrogen, remove the sample and thaw naturally at room temperature. Repeat this process 3-4 times.

[0101] (4) After centrifugation at 12000 rpm for 10 min at 4°C, the supernatant was collected and 5× loading buffer (Solarbio) was added at 1 / 4 the volume of the supernatant and mixed thoroughly.

[0102] (5) Boil in water for 10 minutes, cool and store at -20℃.

[0103] 2. Western blot test to detect protein expression

[0104] (1) Thaw the protein sample at room temperature, mix thoroughly by inversion, and centrifuge immediately;

[0105] (2) Install the electrophoresis gel plate and add an appropriate amount of electrophoresis solution to the electrophoresis tank. After standing for a few minutes and observing that there is no leakage, slowly pull out the comb vertically to fill the gel loading wells with electrophoresis solution.

[0106] (3) Add protein samples to each well in sequence, add 4 μL marker to each well before and after the sample, and run the gel at 120 V constant voltage for 1 h;

[0107] (4) Cut the gel to the appropriate size as needed, cut the NC membrane and transfer filter paper of the same size, and transfer the membrane using the semi-dry transfer method. The ratio of the transfer solution is distilled water: methanol: 10× the volume of the transfer solution = 7:2:1. The current is 1.5 times the membrane area, and the constant current transfer is 45 minutes.

[0108] (5) The NC membrane was washed three times with TBST and blocked with 5% skim milk at room temperature for 2 h;

[0109] (6) Rinse the NC membrane twice with TBST, place it in an incubation cell containing the prepared primary antibody, and incubate it overnight at 4°C; the information of the primary antibody used is shown in Table 2;

[0110] Table 2 Antibody information

[0111]

[0112] (7) Recover the primary antibody in a clean centrifuge tube, rinse the NC membrane three times with TBST, and incubate with the secondary antibody at room temperature for 1 h;

[0113] (8) Rinse the NC membrane three times with TBST. In a dark environment, prepare the developer with ECL luminescent solution A and solution B in a ratio of 1:1. Soak the NC membrane in the developer for 1 min and then use the BIO-Rad gel imaging system to develop the color. The results are analyzed by ImageJ.

[0114] 2. Experimental Results

[0115] Western Blot was further used to detect the expression level of estradiol synthase protein in porcine follicle granulosa cells treated with different concentrations of GHDCA. Figure 5 As shown in the results, when the concentration of GHDCA in the complete culture medium reached 300 μM, the protein expression level of estrogen synthase CYP11A1 was significantly upregulated (P < 0.05), and it was not concentration-dependent; while the protein expression level of CYP19A1 was significantly upregulated when the concentration of GHDCA in the complete culture medium reached 200 μM (P < 0.01), and it was not concentration-dependent.

[0116] Western Blot was further used to detect the protein expression level of progesterone synthase 3βHSD in pig follicle granulosa cells treated with different concentrations of GHDCA. Figure 6 As shown in the results, when the concentration of GHDCA in the complete culture medium reached 400 μM, it could significantly down-regulate the protein expression level of progesterone synthase gene 3βHSD (P<0.05), and it was not concentration-dependent.

[0117] In summary, it was shown that GHDCA could enhance the activity of porcine granulosa cells, promote the secretion of estradiol and inhibit the secretion of progesterone in granulosa cells.

[0118] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Application of glycohyodeoxycholic acid in regulating granulosa cell activity and / or steroid hormone secretion.

2. Application of glycohyodeoxycholic acid in the preparation of products for regulating follicular granulosa cell activity and / or steroid hormone secretion.

3. The application according to any one of claims 1 or 2, characterized in that: The steroid hormones are estradiol and / or progesterone.

4. The use according to any one of claims 1 or 2, characterized in that: The regulation is to enhance the activity of granulosa cells, promote the secretion of estradiol by granulosa cells and / or inhibit the secretion of progesterone by granulosa cells.

5. The application according to claim 4, characterized in that: The promotion of estradiol secretion in granulosa cells is achieved by promoting the expression of estradiol secretion-related genes STAR, CYP11A1 and CYP19A1.

6. The application according to claim 4, characterized in that: The inhibition of progesterone secretion in granulosa cells of follicles is achieved by inhibiting the expression of progesterone synthase gene 3βHSD.

7. The use according to claim 1, characterized in that The follicular granulosa cells are porcine follicular granulosa cells.

8. Application of glycohyodeoxycholic acid in the preparation of products for regulating porcine follicle development.

9. Application of glycohyodeoxycholic acid in the preparation of products for regulating pig estrus.

10. The use according to any one of claims 8 or 9, characterized in that: The product is achieved by enhancing the activity of pig follicle granulosa cells and / or regulating the secretion of estradiol and progesterone.

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