Method for promoting development of preantral follicles of mice and application of method

Abstract

The invention discloses a method for promoting development of preantral follicles of a mouse and application of the method. The establishment of the follicle in-vitro culture system provides an ideal research model for the research of sodium butyrate; a mouse precavity follicle in-vitro 3D culture system is utilized, and the promoting effect of sodium butyrate on follicle development is fully revealed; the application of the sodium butyrate in promoting the development of the preantral follicles is disclosed for the first time, the ectogenesis rate of the preantral follicles of mice is improved, a theoretical basis is provided for improving the reproductive performance of animals by the sodium butyrate, and a technical support is provided for the application of the sodium butyrate in animal production.

Description

Technical Field

[0001] The invention relates to a method for promoting the development of mouse preantral follicles and application thereof, belonging to the field of biotechnology. Background Art

[0002] The level of animal reproductive performance is directly related to the scale of breeding and economic benefits of the breeding industry. The main solution is to improve the conception rate, birth rate, survival rate and number of births of livestock. The development of ovarian follicles determines the number and quality of animal ovulation, which is related to the conception rate, birth rate, survival rate and number of births. Improving the quality of animal ovaries has become the key to improving animal reproductive performance. There are many factors that affect animal reproductive performance, including animal nutrition level, feeding management, environmental facilities and disease control. Not only can the quality of animal ovaries be improved by optimizing nutrition plans and improving management levels, but also by using feed additives to improve animal reproductive performance.

[0003] Sodium butyrate (NaBu), as an important feed additive, plays an important role in regulating the body's immunity, improving the intestinal microenvironment and promoting growth. The active ingredient of sodium butyrate is butyric acid. As a feed additive, sodium butyrate can effectively improve the production performance of livestock and poultry. Studies have found that, whether healthy or sick animals, sodium butyrate can promote cell proliferation, differentiation and maturation to promote the growth of gastrointestinal epithelial cells, improve the immune defense ability of the intestine, improve gastrointestinal digestibility and feed conversion efficiency, and improve the growth performance of livestock and poultry. Studies have found that sodium butyrate can inhibit the growth of harmful bacteria in the intestine of piglets, promote the digestion and absorption of nutrients by the gastrointestinal tract of piglets, and improve the barrier function of the intestine. Most studies on sodium butyrate focus on its effects on intestinal morphology, flora and intestinal barrier function. Studies have shown that sodium butyrate can significantly increase the feed intake of sows during lactation, increase the number of piglets born alive at the beginning, the number of piglets weaned alive and daily weight gain. In addition, studies on sodium butyrate for gilts have confirmed that the addition of sodium butyrate has a certain promoting effect on the early growth of piglets delivered by gilts. However, there has been no in-depth research on the effect of sodium butyrate on livestock reproductive performance by directly regulating follicle development in livestock ovaries.

[0004] At present, the research on sodium butyrate mainly focuses on the in vitro 2D culture of single oocytes or ovarian granulosa cells. Follicular development is a process of interaction between oocytes and granulosa cells. Simply focusing on oocytes or granulosa cells cannot fully reveal the effect of sodium butyrate on the follicular development process. In addition, the role of sodium butyrate in promoting the development of preantral follicles into mature follicles has not been reported. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for promoting the development of preantral follicles, which can improve the maturation of oocytes.

[0006] Technical solution: To solve the above technical problems, the present invention provides a method for promoting the development of mouse preantral follicles and its application. The method of the present invention uses the follicle as a whole to study the effect of sodium butyrate through the interaction between oocytes and ovarian granulosa cells, accurately and comprehensively analyzes the function of sodium butyrate, provides a theoretical basis for sodium butyrate to improve animal reproductive performance, and provides technical support for the application of sodium butyrate in animal production.

[0007] Wherein, the concentration of sodium butyrate in the application is 0.05-1.0 mM.

[0008] Among them, the applications include improving the follicle development rate, improving the follicle survival rate, promoting the follicle to secrete estrogen, and improving the cavity formation rate of preantral follicles.

[0009] The invention also provides the use of sodium butyrate in improving the oocyte maturation rate.

[0010] Wherein, the concentration of sodium butyrate in the application is 0.05-1.0 mM.

[0011] Among them, the in vitro 3D culture system is used to improve the development rate of follicles and the secretion of estrogen through in vitro 3D culture, and promote the cavity formation rate of mouse preantral follicles and the maturation rate of oocytes.

[0012] Wherein, the in vitro 3D culture system is cultured using a 96-well ultra-low adsorption culture plate.

[0013] Furthermore, the application cultured the preantral follicles isolated from the ovaries of female mice in a follicle growth medium containing sodium butyrate.

[0014] The 96-well ultra-low adsorption culture plate contains follicle growth culture medium; the follicle growth culture medium is a MEM-alpha culture medium containing fetal bovine serum (FBS), insulin-transferrin-selenium (ITS), penicillin, streptomycin, follicle-stimulating hormone (FSH), sodium pyruvate (PNa) and vitamin C (Vc), wherein the mass percentage concentration of FBS is 10%, the mass percentage concentration of ITS is 1%, the concentration of penicillin is 100U / mL, the concentration of streptomycin is 100μg / mL, the concentration of FSH is 0.1IU / mL, the concentration of PNa is 0.33mM, and the concentration of Vc is 50μg / mL.

[0015] Furthermore, the concentration of sodium butyrate in the follicle culture medium is 0.05-1.0 mM.

[0016] The establishment of the in vitro follicle culture system of the present invention provides an ideal research model for the study of sodium butyrate. Therefore, the present invention uses the in vitro 3D culture system of mouse preantral follicles to explore the promoting effect of sodium butyrate on follicle development.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The establishment of an in vitro follicle culture system provides an ideal research model for the study of sodium butyrate; 2. The present invention utilizes an in vitro 3D culture system of mouse preantral follicles to fully reveal the promoting effect of sodium butyrate on follicle development; 3. The application of sodium butyrate in promoting the development of preantral follicles is disclosed for the first time, which improves the in vitro development rate of mouse preantral follicles, provides a theoretical basis for sodium butyrate to improve animal reproductive performance, and provides technical support for the application of sodium butyrate in animal production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The morphology of mouse follicles cultured in vitro in 3D;

[0019] Figure 2 is the diameter of the follicle;

[0020] Figure 3 The follicle survival rate, cavity formation rate and oocyte extrusion rate;

[0021] Figure 4 is the estrogen content in the follicle growth culture medium;

[0022] Figure 5 is the oocyte development rate;

[0023] Figure 6 The spindle morphology of oocytes;

[0024] Figure 7 Distribution of mitochondria in oocytes. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0026] Example

[0027] 1. Materials and Methods

[0028] 1.1 Experimental animals

[0029] The experiment used clean-grade female ICR mice born 12.5 days ago, purchased from the Comparative Medicine Center of Yangzhou University. The mice lived in a controlled environment with temperature (24°C ± 2°C), humidity (50% ± 10%), and a light and dark cycle of 12h:12h. All experimental operations were approved by the Animal Protection and Ethics Committee of Yangzhou University (approval number: 202103322).

[0030] 1.2 Preparation of sodium butyrate

[0031] Sodium butyrate (B5887-250 mg, Sigma-Aldrich) was prepared into a 1.0M stock solution. The solvent can be high-pressure double distilled water or PBS. After filtering the bacteria, it was stored at -20°C in the dark. This experiment used high-pressure double distilled water. Before use, prepare the sodium butyrate working solution, which was prepared and used immediately. A total of 4 sodium butyrate working solution concentrations (0.05, 0.1, 0.5 and 1.0 mM) were set.

[0032] 1.3 Collection of mouse preantral follicles

[0033] Female mice of the ICR strain of clean grade born 12.5 days old were taken out, and after being killed, the abdominal cavity was cut open, and the ovaries were quickly taken out and placed in a circular dish containing L-15 (41300039, Gibco) operating solution, which contained 10% FBS, 100U / mL penicillin and 100μg / mL streptomycin. Under a stereomicroscope, the excess tissue around the ovaries was peeled off, and after cleaning, the ovaries were placed in another circular dish containing L-15 operating solution, and a single follicle was separated using a Gonal-Fin syringe, and the intact preantral follicle (diameter = 100-130μm) was selected using an oviduct. Transfer the follicles to an arc dish containing MEM-alpha (C12571500BT, Gibco) growth medium for later use. MEM-alpha follicle growth medium contains 10% fetal bovine serum + 1% insulin-transferrin-selenium + 100U / mL penicillin + 100μg / mL streptomycin + 0.1IU / mL follicle stimulating hormone + 0.33mM sodium pyruvate + 50μg / mL vitamin C.

[0034] 1.4 In vitro culture of preantral follicles

[0035] 30 minutes in advance, place 200 μL / well of MEM-alpha follicle growth medium in an ultra-low adsorption 96-well plate and pre-equilibrate in an incubator at 37°C and 5% CO2. Use an oviduct to wash the isolated single preantral follicle with MEM-alpha growth medium and then inoculate it into a 96-well plate for culture. One follicle per well. The day of inoculation is day 0. Replace half of the medium every 2 days, aspirate 100 μL of the old follicle growth medium from each well and add 100 μL of new follicle growth medium. After culturing to day 4, replace the follicle growth medium containing different concentrations of sodium butyrate (0.05, 0.1, 0.5 and 1.0 mM). After culturing to day 6, restore the follicle growth medium containing sodium butyrate to normal follicle growth medium. On the 8th day of culture, the culture medium was changed to oocyte maturation medium (MEM-alpha growth medium + 1.5 IU / mL human chorionic gonadotropin HCG + 10 ng / mL epidermal growth factor EGF). After 16 hours, ovulatory follicles were obtained.

[0036] 1.5 Effect of sodium butyrate on the morphology of preantral follicles during development

[0037] An inverted microscope (CKX53, Olympus) was used to observe the survival of follicles under different concentrations of sodium butyrate and take pictures. The survival rate of follicles, the rate of antral follicle formation and the rate of oocyte extrusion in different treatment groups were calculated. The diameter of follicles was measured using ImageJ software, and their development was evaluated based on the increase in diameter. Follicles were measured from the outer layer of cells, including the measurement of the widest diameter of the follicle and the second measurement perpendicular to the first diameter.

[0038] 1.6ELISA method to detect follicle exocrine hormone levels

[0039] When changing the medium every other day, collect the culture medium (100 μL) of the 2nd, 4th, 6th and 8th day into centrifuge tubes and store them at -80°C (i.e., 4 samples per week) to avoid repeated freezing and thawing. The mouse estradiol (E2) ELISA research kit (F2566-A, Fancovi) was used to determine the E2 content, and the supernatant of every 10 follicles was collected into one tube.

[0040] 1.7 Observation of oocyte spindle morphology

[0041] After sodium butyrate treatment, 30 oocytes were randomly selected from each experimental group and immunofluorescence staining was performed using Tubulin-Tracker Green (C1051S, Beyotime). After washing with PBS, the cells were fixed with 3.7% formaldehyde for 15 minutes, and then washed twice with immunostaining washing solution for 5 minutes each time; then transferred to Tubulin-Tracker Green staining working solution (immunofluorescence staining secondary antibody dilution solution: Tubulin-Tracker Green = 1:500), incubated at room temperature in the dark for 1 hour; washed oocytes with immunostaining washing solution 3 times, 5 minutes each time; finally, DAPI staining for 20 minutes, washed 3 times with PBS, 5 minutes each time, sealed, and photographed and observed using a laser confocal microscope.

[0042] 1.8 Distribution of mitochondria in oocytes

[0043] After sodium butyrate treatment, 30 oocytes were randomly selected from each experimental group and immunofluorescence staining was performed using MitoTracker Red (C1035, Beyotime). MitoTracker Red mitochondrial dye was dissolved in DMSO and prepared into a 1mM storage solution, which was stored at -20°C after aliquoting. The storage solution was diluted to a working concentration of 200nM with follicle culture fluid before use. After the cells were washed with PBS, they were added to a culture dish with MitoTracker Red working solution incubated in advance and incubated in the dark at 37°C for 30 minutes. After staining, the oocytes were washed 3 times with PBS, stained with DAPI for 20 minutes, washed 3 times with PBS for 5 minutes each time, sealed, and photographed and observed using a laser confocal microscope.

[0044] 1.9 Statistical analysis

[0045] GraphPad Prism was used to perform statistical analysis on the experimental data, and the data were expressed as mean ± standard deviation. Dunnett's multiple comparisons test was used for comparison of differences among multiple groups. *P<0.05, **P<0.01, and ***P<0.001; P is a parameter for determining the results of hypothesis testing, and when P<0.05, there is a significant difference.

[0046] 2. Results Analysis

[0047] The morphology of mouse follicles cultured in vitro in 3D Figure 1 As the number of days of culture increases, the volume of the follicles gradually increases, and the number of granulosa cell layers increases significantly; the follicular cavity begins to form on the 4th day, and the ovarian granulosa cells continue to proliferate on the 6th day, the volume of the follicle increases, and the follicular cavity in the follicle becomes larger and larger; when cultured to the 8th day, the follicular cavity continues to increase, and the antral follicles are close to maturity. Adding oocyte maturation fluid can discharge oocytes wrapped with multiple layers of granulosa cells and with complete morphology. There is no significant difference in the growth of follicles in the 0.05, 0.1 and 0.5mM sodium butyrate groups compared with the control group, while in the 1.0mM sodium butyrate group, the oocytes no longer grow on the 6th day, and the follicles appear atretic.

[0048] The diameter of the follicle Figure 2 Initial follicles with a diameter of 100-130 μm (n=80) were selected for culture. There was no significant difference in the diameter of the initial follicles among the groups. There was no significant difference in the average diameter of the follicles on days 2, 4, 6, and 8 between the 0.05, 0.1, and 0.5 mM sodium butyrate groups and the control group (P>0.05). The average diameter of the follicles in the 1.0 mM sodium butyrate group was significantly smaller than that in the control group (P<0.001).

[0049] The follicle survival rate, cavity formation rate and oocyte extrusion rate are shown in Figure 3 The follicle formation rate on the 8th day in the 0.1mM sodium butyrate group was significantly higher than that in the control group (P<0.05), but the follicle survival rate and ovulation rate were not significantly different from those in the control group (P>0.05); the follicle survival rate, follicle formation rate and ovulation rate in the 0.05 and 0.5mM sodium butyrate groups were not significantly different from those in the control group (P>0.05); the follicle survival rate, follicle formation rate and ovulation rate in the 1.0mM sodium butyrate group were significantly lower than those in the control group (P<0.001).

[0050] Estrogen content in follicle growth culture medium Figure 4 The secretion level of follicle estrogen on the 6th and 8th day after 0.1mM sodium butyrate treatment was significantly higher than that of the control group (P<0.01; P<0.05); there was no significant difference between the 0.05 and 0.5mM sodium butyrate groups and the control group (P>0.05); 0.1mM sodium butyrate significantly promoted the secretion of estrogen by follicles.

[0051] Oocyte development rate Figure 5 There was no significant difference in the proportion of oocytes in GV, GVBD, MI and MII stages between the 0.05 and 0.5 mM sodium butyrate groups and the control group (P>0.05); the proportion of oocytes in MΙΙ stage in the 0.1 mM sodium butyrate group was significantly higher than that in the control group (P<0.05); but there was no significant difference in the proportion of oocytes in GV, GVBD and MI stages between the 0.05 and 0.5 mM sodium butyrate groups and the control group (P>0.05); the proportion of oocytes in GV, GVBD, MI and MII stages in the 1.0 mM sodium butyrate group was significantly lower than that in the control group (P<0.001; P<0.05; P<0.001; P<0.001); 0.1 mM sodium butyrate significantly increased the oocyte maturation rate.

[0052] Oocyte spindle morphology Figure 6 The spindles of the oocytes in the control group showed a typical barrel-shaped structure, and the chromosomes were well arranged on the equatorial plate; the spindles in the oocytes at the GVBD stage in the 0.1 mM sodium butyrate group were evenly distributed around the nucleus and in the cytoplasm.

[0053] The distribution of mitochondria in oocytes is shown in Figure 7 The mitochondria in the oocytes of the 0.1 mM sodium butyrate group and the control group were evenly distributed, with no significant difference.

Claims

1. Application of sodium butyrate in promoting the development of preantral follicles in mice.

2. The application according to claim 1, characterized in that: The concentration of sodium butyrate in the application is 0.05-1.0 mM.

3. The application according to claim 1, characterized in that: The application includes any one or more of increasing the follicle development rate, increasing the follicle survival rate, promoting the follicle to secrete estrogen, increasing the antral follicle formation rate or the oocyte maturation rate.

4. Application of sodium butyrate in improving oocyte maturation.

5. The application according to claim 4, characterized in that: The concentration of sodium butyrate in the application is 0.05-1.0 mM.

6. The use according to any one of claims 1 to 5, characterized in that: Utilize in vitro 3D culture system.

7. The use according to claim 6, characterized in that: The in vitro 3D culture system is cultured using a 96-well ultra-low adsorption culture plate.

8. The use according to claim 7, characterized in that: The 96-well ultra-low adsorption culture plate contains follicle growth culture medium; the follicle growth culture medium is MEM-alpha culture medium containing fetal bovine serum, insulin-transferrin-selenium, penicillin, streptomycin, follicle-stimulating hormone, sodium pyruvate and vitamin C.

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