Application of spermidine in promoting goose follicular granular cells to secrete steroid hormone

Abstract

The invention discloses application of spermidine in promoting goose follicular granule cells to secrete steroid hormones, and relates to the field of animal production practice application. The invention provides application of spermidine in promoting goose follicular granulosa cells to secrete steroid hormones, geese are Sichuan white geese, the steroid hormones comprise estradiol, progesterone and testosterone, the concentration of spermidine in a culture medium is 80 [mu] mol / L or 160 [mu] mol / L, and spermidine promotes the goose follicular granulosa cells to secrete steroid hormones through eIF5A. According to the invention, the problems of follicular atresia of geese and low egg yield of geese are effectively solved, the spermidine can be applied to the poultry breeding industry and is expected to be developed into an additive for promoting poultry production performance, and the poultry production performance, the economic benefit and the production efficiency are improved.

Description

Technical Field

[0001] The invention relates to the field of animal production practice, and in particular to the application of spermidine in promoting the secretion of steroid hormones by granulosa cells of goose ovarian follicles. Background Art

[0002] Studies have shown that there are about 480,000 primordial follicles in the ovaries of poultry, of which more than 99% of the follicles cannot ovulate but gradually degenerate and disappear after atresia. The number of follicles that can normally develop to the ovulation stage in poultry during their lifetime is less than 1% of the primordial follicles, about 500-1000. Poultry egg-laying performance is closely related to follicular development and ovulation. Follicular development involves follicle recruitment, selection and ovulation, which is a complex biological process. Geese are widely welcomed by farmers and consumers for their tolerance to roughage, strong adaptability, fresh and nutritious internal quality. However, the low egg production of geese has seriously hindered the development and growth of the goose industry. Therefore, increasing the egg production of geese has become one of the urgent problems to be solved in the current goose farming industry.

[0003] The growth and atresia of follicles in the ovary are not only regulated systemically by gonadotropins, but also by gonadal steroids, growth factors, cytokines and intracellular proteins. Granulosa cells in particular have been shown to play an important role in determining the fate of follicles due to their communication, synthesis and secretion functions. The level of egg production is mainly determined by the results of follicle development and selection. The steroid hormones secreted by granulosa cells include estradiol and progesterone. Estradiol plays an important role in promoting follicle growth and inhibiting follicle atresia; while progesterone plays an indispensable role in the transformation from primordial follicles to primary follicles, oocyte maturation, ovulation and maintenance of pregnancy. Studies have shown that after injecting estradiol into chicks, the number of growing follicles in chicks increased by 28, and the diameter and area of ​​3 types of follicles and oocytes increased respectively.

[0004] Eukaryotic translation initiation factor 5A (eIF5A) is an essential translation factor for maintaining cell activity and is highly conserved in evolution. eIF5A is the only protein in eukaryotic cells that contains hydroxyputrescine lysine, and this post-translational modification is essential for the activity of eIF5A. Spermidine is the only substrate for the activation of eIF5A. Spermidine is a natural polyamine and a polycationic aliphatic compound widely distributed in organisms. Spermidine can participate in the regulation of physiological and pathological processes such as animal reproduction, aging and cancer by binding and stabilizing DNA and RNA, regulating protein translation and expression, mediating cell proliferation and autophagy, and scavenging reactive oxygen species. In recent years, studies have found that polyamines can participate in the regulation of follicle occurrence, development and atresia by mediating the production of ovarian steroid hormones. However, studies on spermidine in regulating steroid hormones in granulosa cells of goose follicles have not been reported. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides the use of spermidine in promoting the secretion of steroid hormones by granulosa cells of goose ovarian follicles. The present invention effectively solves the problems of atresia of goose ovarian follicles and low egg production in geese. Spermidine can be applied to poultry farming and is expected to be developed as an additive to promote poultry production performance, thereby improving poultry production performance, economic benefits and production efficiency.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is: to provide the application of spermidine in promoting the secretion of steroid hormones by granulosa cells of goose ovarian follicles.

[0007] Furthermore, spermidine is used as or in the preparation of an additive for secreting steroid hormones from goose follicle granulosa cells.

[0008] Furthermore, the goose is a Sichuan white goose.

[0009] Furthermore, goose follicles are hierarchical follicles.

[0010] Further, the diameter of graded follicles is 10-40 mm.

[0011] Further, steroid hormones include estradiol, progesterone, and testosterone.

[0012] Further, the concentration of spermidine in the culture medium is 78-82 μmol / L or 155-165 μmol / L.

[0013] Furthermore, the concentration of spermidine in the culture medium was 80 μmol / L or 160 μmol / L.

[0014] Furthermore, spermidine promotes the secretion of steroid hormones by goose follicle granulosa cells through eIF5A.

[0015] Furthermore, steroid hormone secretion-related genes include StAR, 3β-HSD, 17β-HSD, CYP17A1, CYP11A1, and CYP19A1 。

[0016] Furthermore, the method of using the above-mentioned spermidine to promote the secretion of steroid hormones by goose follicular granulosa cells comprises the following steps: after culturing the goose follicular granulosa cells for 23-25 ​​hours, the spermidine stock solution is prepared into a dilution with a concentration of 78-82 μmol / L or 155-165 μmol / L using a complete culture medium; when the primary cells cover 85%-90% of the culture dish, the original culture medium is aspirated and discarded, and the dilution with a spermidine concentration of 78-82 μmol / L or 155-165 μmol / L is used as a new culture medium for culturing.

[0017] Furthermore, the method of using the above-mentioned spermidine to promote the secretion of steroid hormones by goose follicular granulosa cells comprises the following steps: after culturing the goose follicular granulosa cells for 24 hours, the spermidine stock solution is prepared into a dilution with a concentration of 80 μmol / L or 160 μmol / L using a complete culture medium; when the primary cells cover 85%-90% of the culture dish, the original culture medium is aspirated and discarded, and the dilution with a spermidine concentration of 80 μmol / L or 160 μmol / L is used as a new culture medium for culturing.

[0018] Furthermore, the complete culture medium comprises the following components in parts by weight: 44-46 parts of DMEM, 4-6 parts of fetal bovine serum and 0.4-0.6 parts of double antibody.

[0019] Furthermore, the complete culture medium includes the following components in parts by weight: 45 parts of DMEM, 5 parts of fetal bovine serum and 0.5 parts of double antibody.

[0020] Furthermore, 24 h after the culture medium was replaced, RNA samples were collected using Trizol, protein samples were collected using RIPA lysis buffer, and cell culture supernatant was collected for the determination of steroid hormone levels.

[0021] An additive for promoting goose follicle granulosa cells to secrete steroid hormones, comprising the above-mentioned spermidine and pharmaceutically acceptable excipients.

[0022] The present invention has the following beneficial effects:

[0023] 1. The present invention has found a new way to improve the production performance of poultry. The application of spermidine in goose follicle granulosa cells can effectively increase the level of steroid hormones and significantly affect the protein expression of steroid synthesis related genes. Studies have shown that this promoting effect is achieved through eIF5A. In summary, spermidine has the potential to be used as a health product to improve the production performance of poultry.

[0024] 2. After adding 80μmol / L and 160μmol / L spermidine to the cell culture medium, and interfering with eIF5A, the effect of spermidine on the secretion of steroid hormones in goose follicular granulosa cells was investigated. The experimental results showed that interfering with eIF5A had no effect on the activity of goose follicular granulosa cells, but significantly affected the expression of steroid synthesis-related genes and the secretion of steroid hormones. This effect was reversed after interfering with eIF5A and combining it with spermidine. This shows that spermidine can improve the production of steroid hormones in goose follicular granulosa cells, and its promoting effect is achieved through eIF5A.

[0025] 3. The application of spermidine in goose follicle granulosa cells can not only effectively increase the level of steroid hormones, but also significantly affect the protein expression of steroid synthesis-related genes, indicating that spermidine has application prospects as a health product to improve poultry production performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 To investigate the effect of interference with eIF5A on the activity of goose follicle granulosa cells;

[0027] Figure 2 The results show the effects of different concentrations of spermidine on the expression of StAR and NR5A1 proteins in goose follicle granulosa cells;

[0028] Figure 3 The results show the effects of different concentrations of spermidine on the expression of StAR and NR5A1 proteins in goose follicle granulosa cells;

[0029] Figure 4 To investigate the effect of spermidine on StAR protein expression in goose follicle granulosa cells after interfering with eIF5A;

[0030] Figure 5 To investigate the effect of spermidine on StAR protein expression in goose follicle granulosa cells after interfering with eIF5A;

[0031] Figure 6 Effects of spermidine on key genes of steroidogenesis in goose follicle granulosa cells after interference with eIF5A.

[0032] Figure 7 The effect of different concentrations of spermidine on the levels of E2, P4 and T hormones in granulosa cells of goose ovarian follicles;

[0033] Figure 8 To study the effect of spermidine on the levels of E2, P4 and T hormones in goose follicle granulosa cells after interfering with eIF5A. DETAILED DESCRIPTION

[0034] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0035] The experimental materials used in the following examples are as follows:

[0036] Experimental animals: 60 Sichuan white geese (provided by the Waterfowl Breeding Farm of Sichuan Agricultural University) at the peak of egg-laying period (35-40 weeks) under the same environment were selected and killed by bleeding from the neck. Granulosa cells in follicles of different grades (10-40 mm) were quickly collected for subsequent experiments. This experiment was completed at the College of Animal Science and Technology of Sichuan Agricultural University, and all feeding and killing requirements were approved by the Animal Operation Specifications and Welfare Management Committee of Sichuan Agricultural University.

[0037] Main experimental reagents: SDS, APS and spermidine (S2626-25, Sigma-Aldrich); Trizol RNA extraction kit, reverse transcription kit, DL2000 DNA marker, 6× loading buffer and TB Green TM Premix ExTaqTMⅡ were purchased from Bao Biotechnology (Dalian) Co., Ltd.; DMEM / F12 was purchased from Hyclone (USA); fetal bovine serum was purchased from Gibco (USA); penicillin-streptomycin, PBS powder, GoldView I type nucleic acid dye, 30% gel preparation solution, Tris-base and Tris-HCL were purchased from Solebao; fluorescent quantitative PCR consumables and PVDF membrane were purchased from BIO-RAD (USA); BCA kit, ECL colorimetric solution, goat anti-rabbit secondary antibody, goat anti-mouse secondary antibody, primary antibody diluent, secondary antibody diluent and RIPA were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.; E2, P4 and T kits were purchased from Shanghai ELISA Biotechnology Co., Ltd.

[0038] The main instruments include: constant temperature cell culture incubator (Thermo, USA); multifunctional microplate reader (Thermo, USA); -80℃ ultra-low temperature refrigerator (Thermo, USA); gel imaging system (Bio-Rad, USA); fluorescence inverted microscope (Olympus, USA); vertical electrophoresis instrument (Bio-Rad, USA); membrane transfer instrument (Bio-Rad, USA); fluorescence quantitative PCR instrument (Thermo, USA).

[0039] Example 1 Isolation and culture of goose follicle granulosa cells

[0040] Collection of follicles: Healthy Sichuan white geese at the peak of egg-laying period were killed by bleeding from the neck, and the abdomen was sprayed with 75% alcohol for disinfection. The abdominal cavity was opened with surgical scissors to remove the graded follicles and placed in a beaker filled with PBS buffer, and then brought to the clean bench for isolation and culture of follicular granulosa cells.

[0041] Isolation and culture of granulosa cells: Use forceps to peel off the connective tissue and vascular network on the surface of the follicle, cut the blade along the opposite side of the follicle handle, and clamp one side of the opening with forceps after 2 / 3 of the yolk flows out. Shake slightly in PBS to separate the granulosa cell layer. Wash the granulosa cell layer in PBS 4-5 times, collect it in a 50mL beaker, transfer it to the clean bench and use ophthalmic scissors to mechanically separate it for 3-5 minutes, add an equal volume of collagenase, digest it at 37℃ for 5-10 minutes, and then terminate the digestion with 2-3 drops of fetal bovine serum. Centrifuge, sieve, resuspend the cells, and inoculate them into a cell culture plate. Observe the cell morphology and density under a microscope and culture them in a 37℃ incubator containing 5% CO2.

[0042] Example 2 Cell activity detection

[0043] Detect the activity of granulocytes in the 96-well plate after the corresponding treatment, and prepare a culture medium solution containing 10% CCK-8 with DMEM / F12. Discard the original culture medium in the 96-well plate, add 100μL of DMEM / F12 containing 10% CCK-8 to each well, and finally seal the blank wells with DMEM / F12 without CCK-8 (blank wells), shake gently to mix, and incubate in a 37℃ incubator with 5% CO2 for 2h. Detect the absorbance of each well at a wavelength of 450nm, and calculate the cell activity by using the following formula:

[0044] Cell viability = (OD 处理组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )

[0045] The results are as follows Figure 1 shown.

[0046] Depend on Figure 1 It can be seen that interference with eIF5A1 for 48 h had no significant effect on cell viability (P>0.05)

[0047] Example 3 Protein expression detection

[0048] Western blotting (WB) was used to detect the expression of eIF5A and steroidogenesis-related proteins: granulosa cells were treated with 0, 20, 40, 80 and 160 μmol / L spermidine for 24 h, then the culture medium was discarded and the cells were washed with 4°C PBS for 30 s each time for 5 times. 120 μL RIPA was evenly added to each cell culture dish and placed in a 4°C refrigerator for 15 min. The lysed granulosa cells were scraped off with a cell scraper and placed in a 1.5 mL EP tube. The cells were fully disrupted using a cell disruptor. After centrifugation at 15000 r / min for 10 min at 4°C in a high-speed refrigerated centrifuge, the supernatant was placed in a new EP tube. The protein concentration was normalized after the BCA method was used to detect the protein concentration. The protein was separated by 12% SDS-PAGE separation gel and 5% After separation by SDS-PAGE concentrated gel, the membrane was transferred to PVDF membrane and transferred at 400 mA constant flow for 25 min. The PVDF membrane was blocked with 5% skim milk for 2 h, and the PVDF membrane was incubated with primary antibodies to β-actin, β-tubulin, StAR, NR5A1, and eIF5A overnight. The membrane was washed 3 times with TBST, each time for 10 min. The PVDF membrane was incubated with secondary antibody (goat anti-rabbit) for 2 h, and after washing 3 times with TBST, the PVDF membrane was soaked with supersensitive ECL chemiluminescent liquid and placed in the sample placement area of ​​the chemiluminescent imaging system for development and imaging. The results are shown in Figure 2 The results of the effects of different concentrations of spermidine on the expression of StAR and NR5A1 proteins are shown in Figure 3 shown.

[0049] Depend on Figure 2 and Figure 3 It can be seen that 80 μmol / L spermidine can significantly increase the expression of StAR protein, and 80 μmol / L and 160 μmol / L spermidine can significantly increase the expression of SF-1 protein; the above results indicate that spermidine is involved in regulating the expression of steroidogenic proteins.

[0050] The results of the effect of spermidine on StAR protein expression in goose follicle granulosa cells after interference with eIF5A are as follows Figure 4 and Figure 5 shown.

[0051] Depend on Figure 4 and Figure 5 It can be seen that the expression of StAR protein was significantly reduced after interfering with eIF5A, and compared with the interference group, the expression of StAR protein was significantly increased after adding spermidine, indicating that spermidine affects the production of steroid hormones through eIF5A.

[0052] Example 4 Gene Expression Detection

[0053] 1. qRT-PCR detection of steroid hormone production-related gene expression: According to the instructions of the reverse transcription kit, the extracted total RNA was reversed into cDNA and then fluorescent quantification was performed according to the PCR system shown in Table 1. The optimal temperature was determined by the amplification efficiency and the melting curve. GAPDH was used as the internal reference. The reaction conditions were: 95°C, 5 min; 95°C, 30 s; 55-68°C, 30 s; 72°C, 30 s; 39 cycles, and then the melting curve was drawn. The relative expression of the target gene was calculated using the 2-ΔΔCt method. The results are shown in the figure. Figure 6 As shown, Figure 6 In the figure, from top to bottom and from left to right are the results of steroid hormone production-related genes StAR, 3β-HSD, 17β-HSD, CYP17A1, CYP11A1, and CYP19A1.

[0054] Table 1 mRNA quantitative PCR reaction system

[0055] Reagents Reagent mixing system SYBR Green Supermix 5μL PCR Forward Primer 0.2μL PCR Reverse Primer 0.2μL cDNA 0.5μL <![CDATA[RNase-free H2O]]> 4.1μL

[0056] Depend on Figure 6 It can be seen that compared with the control group, the expression levels of steroid synthesis key genes 3β-HSD and CYP19A1 were significantly increased, the expression levels of 17β-HSD, CYP17A1 and CYP11A1 were significantly decreased, and StAR had no significant change after eIF5A interference. After spermidine combined with eIF5A interference treatment, StAR, 17β-HSD and CYP17A1 were significantly higher than the interference group, 3β-HSD was significantly lower than the interference group (P<0.05), and the expression levels of CTP11A1 and CYP19A1 were not significantly different from the interference group (P>0.05), indicating that spermidine regulates the expression of steroidogenesis-related genes through eIF5A.

[0057] Example 5 Detection of steroid hormone levels in goose follicle granulosa cells

[0058] .ELISA kits were used to measure the levels of steroid hormone estradiol (E2), progesterone (P4) and androgen testosterone (T) in cell culture medium. The results of the effects of different concentrations of spermidine on the contents of E2, P4 and T in goose follicle granulosa cells are shown in Figure 2. Figure 7 As shown, Figure 7 In the figure, from left to right are the results of E2, P4, and T.

[0059] Depend on Figure 7 It can be seen that treatment of goose follicle granulosa cells with 80μmol / L spermidine can significantly increase the content of E2 and P4 hormones, and 160μmol / L spermidine can significantly reduce the level of T hormone.

[0060] The results of the effects of spermidine on the levels of E2, P4 and T hormones in goose follicle granulosa cells after interference with eIF5A are as follows Figure 8 As shown, Figure 8 In the figure, from left to right are the results of E2, P4, and T.

[0061] Depend on Figure 8 It can be seen that compared with the control group, the levels of steroid hormones E2 and P4 were significantly increased after eIF5A interference. After spermidine combined with eIF5A interference, the E2 level was significantly lower than the interference group, while the P4 level was significantly higher than the interference group (P<0.05), and there was no significant change in the T hormone level (P>0.05).

[0062] In summary, interfering with eIF5A will affect the expression of steroidogenesis-related genes and proteins in goose follicle granulosa cells, and will also affect the levels of steroid hormones. The addition of spermidine will alleviate the effects of the interference, suggesting that spermidine-mediated steroid hormone production is achieved through eIF5A.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of spermidine in promoting the secretion of steroid hormones by granulosa cells of goose ovarian follicles.

2. The use according to claim 1, characterized in that The goose is a Sichuan white goose.

3. The use according to claim 1, characterized in that The goose follicle is a graded follicle, and the diameter of the graded follicle is 10-40 mm.

4. The use according to claim 1, characterized in that The steroid hormones include estradiol, progesterone and testosterone.

5. The use according to claim 1, characterized in that The concentration of spermidine in the culture medium is 78-82 μmol / L or 155-165 μmol / L.

6. The use according to claim 1, characterized in that The spermidine promotes the secretion of steroid hormones by goose follicle granulosa cells through eIF5A.

7. The use according to claim 1, characterized in that The steroid hormone secretion-related genes include StAR, 3β-HSD, 17β-HSD, CYP17A1, CYP11A1 and CYP19A1.

8. A method for promoting goose follicle granulosa cells to secrete steroid hormones using spermidine as claimed in claim 1, characterized in that: The following steps are involved: After culturing goose follicle granulosa cells for 23-25 ​​hours, use complete culture medium to prepare the spermidine stock solution into a dilution with a concentration of 78-82μmol / L or 155-165μmol / L. When the primary cells cover 85%-90% of the culture dish, aspirate and discard the original culture medium, and use the dilution with a spermidine concentration of 78-82μmol / L or 155-165μmol / L as the new culture medium for culture.

9. The method for promoting goose follicle granulosa cells to secrete steroid hormones using spermidine as claimed in claim 8, characterized in that: The complete culture medium comprises the following components in parts by weight: 44-46 parts of DMEM, 4-6 parts of fetal bovine serum and 0.4-0.6 parts of double antibody.

10. An additive for promoting the secretion of steroid hormones by goose follicle granulosa cells, characterized in that: The invention comprises the spermidine as claimed in claim 1 and pharmaceutically acceptable excipients.

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