A method for isolating and culturing preovulatory follicles in porcine ovarian cavity

Abstract

The application provides a method for separating and culturing porcine preantral follicles, and belongs to the technical field of preantral follicle separation and culture. The preantral follicles are separated by a mechanical-enzyme compound method, and the preantral follicles are cultured in vitro by a three-dimensional culture method coated by a sodium alginate gel system. The separation method of the porcine preantral follicles comprises the following steps: mixing porcine ovarian tissue fragments with follicle separation solution and digesting for 0.5-2 hours, discarding the supernatant, stopping the digestion, and passing the digested tissue through a 400-micron mesh screen to obtain the preantral follicles. The preantral follicle separation and in-vitro culture system are optimized, the in-vitro separation efficiency of the preantral follicles is improved, and the in-vitro antrum formation culture of the preantral follicles is achieved. Not only can the method provide sufficient egg sources for porcine embryo engineering research and application, but also can provide a reference for further tapping the genetic potential of pigs.

Description

Technical Field

[0001] This invention belongs to the field of precavitary follicle isolation and culture technology, and particularly relates to a method for isolating and culturing precavitary follicles of the porcine ovary. Background Technology

[0002] Follicles are the basic structural and functional units of the mammalian ovary, the center of sex hormone synthesis, and the microenvironment for oocyte growth and maturation. Follicular cavity formation is one of the most critical stages in the development of pre-antral follicles, during which the vast majority of follicles undergo atresia and degeneration. Studies have shown that atresia occurs very rarely during the pre-antral follicle growth phase in the mammalian ovarian cortex. However, over 99% of pre-antral follicles undergo atresia and degeneration during the transition from pre-antral to mature follicles. This high rate of pre-antral follicle atresia and degeneration results in low oocyte utilization efficiency and hinders the selection of high-quality oocytes. Isolating these pre-antral follicles with developmental potential and culturing them in suitable in vitro media can prevent the waste of oocyte resources.

[0003] Establishing an in vitro culture system to culture pre-coelom follicles using a defined culture medium and obtain oocytes capable of fertilization and embryonic development is crucial for perfecting in vitro models of folliculogenesis. Research on porcine follicular matrix tissue has been slow due to problems such as significant adhesion, difficulty in separation, long culture cycles, and inconsistent quality. In 1994, Hirao et al. obtained mature oocytes capable of fertilization by embedding and culturing porcine pre-coelom follicles in vitro for 16 days; however, the fertilized eggs could not form male pronuclei. In 2001, Wu et al. obtained blastocysts for the first time by adhering porcine pre-coelom follicles to a wall after 4 days of culture, followed by IVF and intracytoplasmic sperm injection (ICSI). In 2010, Hashimoto et al. demonstrated that mature oocytes could also be obtained from granulosa-oocyte complexes derived from early porcine follicular follicles through in vitro culture. In 2015, Yang Hongxing et al. isolated pre-coelom granulosa-oocyte complexes in vitro and cultured them adhering to a wall for 18.5 days, achieving a follicular formation rate of 40.0% and an oocyte maturation rate of 26.2%. However, no complete system for isolating and culturing porcine pre-ovarian follicles has been reported to date. Because the connective tissue of the porcine ovary is tightly connected, follicles cannot be isolated through simple dissection. Therefore, the porcine pre-ovarian follicle isolation system needs further improvement, and the culture system needs refinement. In summary, research on the in vitro isolation and culture of porcine follicles still faces challenges such as low isolation efficiency, the inability to culture intact follicles in vitro, and the need to clarify the culture components. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for separating pre-ovarian follicles in pigs, which has the advantage of improving the efficiency of in vitro separation of pre-ovarian follicles.

[0005] Another objective of this invention is to provide a three-dimensional culture method for porcine pre-ovarian follicles, which has the advantage of enabling intact porcine pre-ovarian follicles to form cavities in vitro.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for isolating precavitary follicles from the porcine ovary, comprising the following steps: mixing and digesting porcine ovarian tissue fragments with follicle separation solution for 0.5-2 hours, discarding the supernatant, terminating digestion, and obtaining digested tissue; passing the digested tissue through a 400μm mesh sieve to obtain precavitary follicles; wherein the follicle separation solution is composed of L-15 culture medium, 2mg / ml collagenase I, 200μg / ml DNase I (deoxyribonuclease I), and 1% penicillin-streptomycin solution.

[0008] Preferably, the particle size of the porcine ovarian tissue fragments is less than 1 mm. 2 .

[0009] Preferably, the volume ratio of the porcine ovarian tissue fragments to the follicle separation fluid is 1:2.

[0010] Preferably, the temperature of the mixed digestion is 37.5°C, and the mixture is shaken for 10 seconds every 20-30 minutes during the mixed digestion process.

[0011] Preferably, the digestion is terminated by adding a follicle washing solution, which is composed of L-15 culture medium, 10% FBS (fetal bovine serum), and 1% penicillin-streptomycin solution.

[0012] The present invention also provides a method for culturing precavitary follicles of porcine ovaries, which, based on the above separation method, further includes the following steps: mixing the obtained precavitary follicles with sodium alginate solution, then mixing with CaCl2 solution, and then mixing the coated precavitary follicles with culture medium for culture.

[0013] Preferably, the concentration of the sodium alginate solution is 1%.

[0014] Preferably, the concentration of the CaCl2 solution is 100 mM.

[0015] Preferably, the mixing time with the CaCl2 solution is 1-2 minutes.

[0016] Preferably, the culture medium consists of DMEM / F12 medium, 10% FBS, 1 μg / mL 10× insulin-transferrin-selenium (ITS-G), 100 mIU / mL follicle-stimulating hormone (FSH), and 1% penicillin-streptomycin solution.

[0017] The beneficial effects of this invention are:

[0018] The method for isolating and culturing pre-ovarian follicles in pigs provided by this invention optimizes the isolation and in vitro culture system for pre-ovarian follicles, achieving significant technical effects such as improving the efficiency of pre-ovarian follicle isolation and enabling intact pre-ovarian follicles to form cavities in vitro. This not only provides a sufficient source of eggs for pig embryo engineering research and applications but also offers a reference for further exploring the genetic potential of pigs. Attached Figure Description

[0019] Figure 1 This image shows the process of separating pre-cavity follicles from a pig ovary. In the image, A represents fragments of the ovarian cortex cut by mechanical means, B represents the obtained pre-cavity follicles, and C represents a microscopic image of the pre-cavity follicles.

[0020] Figure 2 The process of culturing precavitary follicles coated with a sealing film is described below. In this process, A represents the precavitary follicles isolated in Example 1, B represents the follicles being washed twice in follicle washing solution, C represents the addition of sodium alginate solution to the sealing film, D represents the sealing film being inverted and placed in calcium chloride solution, E represents the successfully coated precavitary follicles, and F represents the porcine follicles that have formed a cavity in vitro. Detailed Implementation

[0021] This invention provides a method for isolating precavitary follicles from the porcine ovary, comprising the following steps: mixing and digesting porcine ovarian tissue fragments with follicle separation solution for 0.5-2 hours, discarding the supernatant, terminating digestion, and obtaining digested tissue; passing the digested tissue through a 400μm mesh sieve to obtain precavitary follicles; wherein the follicle separation solution is composed of L-15 culture medium, 2mg / ml collagenase I, 200μg / ml DNase I, and 1% penicillin-streptomycin solution.

[0022] In this invention, it is preferable to first puncture the cavitary follicles in the ovary, and then break up the ovarian tissue. This invention does not have a specific limitation on the specific method of breaking up the tissue fragments, but the particle size of the tissue fragments is preferably less than 1 mm. 2 In this invention, the preferred volume ratio of the porcine ovarian tissue fragments to the follicle separation fluid is 1:2. This invention does not specifically limit the source of each component in the follicle separation fluid; any commercially available product in the art can be used. In this invention, the preferred temperature for the mixing and digestion is 37.5°C, and during the mixing and digestion process, it is preferably shaken for 10 seconds every 20-30 minutes. The preferred time for the mixing and digestion is 1 hour. In this invention, after the mixing and digestion is completed, the supernatant is preferably discarded by centrifugation, preferably at 600g for 3 minutes. In the follicle separation fluid of this invention, 1% penicillin-streptomycin solution refers to a final volume percentage of 1% of the penicillin-streptomycin solution in the follicle separation fluid.

[0023] In this invention, the preferred method for terminating digestion is the addition of a follicle washing solution, which is preferably composed of L-15 culture medium, 10% FBS, and 1% penicillin-streptomycin solution. In this invention, 10% FBS refers to a final volume percentage of 10% for FBS in the follicle washing solution, and 1% penicillin-streptomycin solution refers to a final volume percentage of 1% for the penicillin-streptomycin solution in the follicle washing solution. This invention does not have specific limitations on the specific sources of the components in the follicle washing solution; commercially available products commonly used in the art can be used. In this invention, after passing through a 400μm mesh, precavitary follicles with intact follicular membranes and no cavities are preferably collected for subsequent culture steps.

[0024] The present invention also provides a method for culturing precavitary follicles of porcine ovaries, which, based on the above separation method, further includes the following steps: mixing the obtained precavitary follicles with sodium alginate solution, then mixing with CaCl2 solution, and then mixing the coated precavitary follicles with culture medium for culture.

[0025] In this invention, the precavitary follicles obtained by separation are preferably cultured using a sodium alginate gel system. The concentration of the sodium alginate solution is preferably 1%, and the concentration of the CaCl2 solution is preferably 100 mM. In this invention, the precavitary follicles are preferably coated and cultured using a sealing film method. Specifically, the separated precavitary follicles are washed in follicle washing solution, then sodium alginate solution is added dropwise to the sealing film. The precavitary follicles are transferred into the sodium alginate solution droplets using a pipette. The sealing film is then inverted and placed in CaCl2 solution for solidification. The mixing time with the CaCl2 solution is preferably 1-2 minutes. The specific composition of the follicle washing solution is the same as above and will not be repeated here. In this invention, when the coated precavitary follicles are cultured with a culture medium, the culture medium preferably consists of DMEM / F12 medium, 10% FBS, 1 μg / mL 10×ITS-G, 100 mIU / mLFSH, and 1% penicillin-streptomycin solution. The percentages in the 10% FBS and 1% penicillin-streptomycin solutions refer to final volume percentages. This invention does not specifically limit the source of each component in the culture medium; any commercially available product in the art can be used. The culture is preferably carried out in a 96-well plate, with each well preferably containing 150 μL of culture medium and one coated precavitary follicle microcapsule per well. In this invention, the medium is preferably changed every other day during culture, and according to the culture method of this invention, follicles form cavities on day 3. The method of this invention can form cavities in vitro from intact porcine follicles, with a cavitation rate of 22.67% ± 2.4%.

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Unless otherwise specified, the following embodiments are all conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] Example 1

[0030] The porcine ovarian mesentery was held in place with forceps and placed in a large culture dish containing 10 ml of PBS. The cavitary follicles were punctured using a 10 ml syringe. The ovary was then fragmented using a No. 10 scalpel blade until the tissue fragments were less than 1 mm. 2 Tissue fragments were collected into 15ml centrifuge tubes, and 10ml of follicle separation medium (L-15 medium + 2mg / ml collagenase I + 200ug / ml DNase I + 1% penicillin-streptomycin solution) was added. The tubes were placed in a 37.5℃ constant temperature water bath, and the mixture was shaken for 10s every 15min for 1h for digestion. The tubes were then centrifuged at 600g for 3min at room temperature, and the supernatant was discarded. Follicle washing solution (L-15 medium + 10% FBS + 1% penicillin-streptomycin solution) was added to terminate digestion, yielding the digested tissue. The digested tissue was passed through a 400μm mesh sieve, and pre-cavity follicles with intact follicular membranes and no cavities were collected under a stereomicroscope using an oral pipette. Results are as follows: Figure 1 As shown.

[0031] Comparative Example 1

[0032] Insulin needle separation method: The pig ovary is cut into pieces and placed in a large dish containing PBS. The follicles are separated under a stereomicroscope using the tips of two insulin needles.

[0033] Comparative Example 2

[0034] Mesh sieving method: The intact pig ovary is rubbed on a 400μm mesh sieve to allow tissue fragments to pass through the sieve. The tissue fragments are collected and follicles are searched under a stereomicroscope.

[0035] Example 2

[0036] The number of follicles obtained by different separation methods described in Example 1 was compared with those of Comparative Examples 1 and 2. Example 1 was performed in three parallel experiments, and the results are expressed as mean ± standard deviation. Comparative Examples 1 and 2 were performed in only one experiment. The results are shown in Table 1.

[0037] Table 1. Number of follicles obtained by different methods of follicle separation

[0038] method Number of follicles obtained / Number of complete follicles / Comparative Example 1 (Insulin Injection Separation Method) 12 0 Comparative Example 2 (Mesh Screening Method) 0 0 Example 1 (Mechano-enzyme combined method) 181.00±8.00 119.67±4.44

[0039] It is evident that the method for separating precavitary follicles from a single ovary provided by this invention can obtain a greater number of precavitary follicles from a single ovary, and the proportion of intact follicles obtained is large.

[0040] Example 3

[0041] The difference from Example 1 is that the digestion reaction time is 0.5 hours, while the rest are the same as in Example 1.

[0042] Example 4

[0043] The difference from Example 1 is that the digestion reaction time is 2 hours, while the rest are the same as Example 1.

[0044] Example 5

[0045] The number of follicles obtained at different digestion reaction times described in Examples 1, 3, and 4 was compared. Examples 1, 3, and 4 were each performed in triplicate. The results are expressed as mean ± standard deviation, and are shown in Table 2. Table 2 shows that the number of follicles obtained at different digestion times varied.

[0046] Table 2. Number of follicles obtained from follicles isolated at different digestion times.

[0047] time Number of follicles obtained / Number of complete follicles / Example 3 (0.5h) 28.00±5.33 20.33±3.56 Example 1 (1h) 181.00±8.00 119.67±4.44 Example 4 (2h) 211.67±10.89 90.67±1.56

[0048] Example 6

[0049] The process of using a sealing film to encapsulate the precavitary follicle and create the cavity is as follows: Figure 2 As shown, the pre-cavity follicles isolated in Example 1 were washed twice in follicle washing solution (L-15 + 10% FBS + 1% penicillin-streptomycin). A 1% sodium alginate solution was added to the sealing film, and the pre-cavity follicles were transferred into the sodium alginate solution droplet using an oral pipette. The sealing film was inverted and immersed in 100mM calcium chloride solution for 1 min. The coated pre-cavity follicles were collected and cultured in 96-well plates containing 150 μL of culture medium (one microcapsule per well) (the culture medium consisted of DMEM / F12 medium + 10% FBS + 1 μg / mL 10×ITS-G + 100mIU / mLFSH and 1% penicillin-streptomycin solution). The medium was changed and photographed every other day. Follicles formed cavities on the 3rd day of culture, and the cavitation rate was calculated as follows: Cavity rate (%) = (Number of cavities / Total number of pre-cavity follicles transferred to the sodium alginate solution droplet) × 100.

[0050] This embodiment involved 5 parallel experiments, and the results are expressed as mean ± standard deviation. The cavity formation rate was 22.67% ± 2.4%.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for isolating and culturing precocious follicles of the porcine ovary, characterized in that, The process includes the following steps: mixing and digesting fragments of porcine ovarian tissue with follicle separation fluid for 0.5-2 hours, discarding the supernatant, terminating digestion, and obtaining digested tissue; passing the digested tissue through a 400μm mesh sieve to obtain precavitary follicles; The follicle separation medium consisted of L-15 medium, 2 mg / ml collagenase I, 200 μg / ml DNase I, and 1% penicillin-streptomycin solution; The obtained pre-cavity follicles were mixed with sodium alginate solution, then mixed with CaCl2 solution, and the coated pre-cavity follicles were mixed with culture medium for culture. The concentration of the sodium alginate solution is 1%; The concentration of the CaCl2 solution is 100 mM; The culture medium used for the culture consists of DMEM / F12 medium, 10% FBS, 1 μg / mL 10× insulin-transferrin-selenium, 100 mIU / mL follicle-stimulating hormone and 1% penicillin-streptomycin solution.

2. The separation and culturing method according to claim 1, characterized in that, The particle size of the porcine ovarian tissue fragments is less than 1 mm. 2 .

3. The separation and cultivation method according to claim 1, characterized in that, The volume ratio of the porcine ovarian tissue fragments to the follicle separation fluid was 1:

2.

4. The separation and cultivation method according to claim 1, characterized in that, The temperature for the mixed digestion was 37.5°C, and the mixture was shaken for 10 seconds every 20-30 minutes during the mixed digestion process.

5. The separation and culturing method according to claim 1, characterized in that, The digestion is terminated by adding follicle washing solution, which consists of L-15 culture medium, 10% FBS and 1% penicillin-streptomycin solution.

6. The separation and culturing method according to claim 1, characterized in that, The mixing time with the CaCl2 solution is 1-2 minutes.

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