ISOLATION METHOD AND CULTURE METHOD FOR PREANTRAL FOLLICLES IN PORCINE OVARY
Patent Information
- Application Number
- NL2039184
- Authority / Receiving Office
- NL · NL
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2024-11-06
- Filing Date
- 2024-11-27
- Publication Date
- 2026-06-08
- Estimated Expiration
- 2044-11-26
Smart Images
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Abstract
Description
l ISOLATION METHOD AND CULTURE METHOD FOR PREANTRAL FOLLICLES IN PORCINE OVARY TECHNICAL FIELD [Ol] The present invention belongs to the technical field of preantral follicle isolation and culture, and particularly relates to an isolation method and a culture method for preantral follicles in a porcine ovary. BACKGROUND ART
[02] As a basic structural and functional unit on a mammalian ovary, the follicle is a center for sex hormone synthesis and. a microenvironment for oocyte growth and maturation. Follicular antral formation is one of the most critical stages of preantral follicle development, and the vast majority of follicles will undergo atretic degeneration at this stage. Studies have shown that on a mammalian ovarian cortex, atresia occurs very low in the growth stage before follicle antral formation. However, atretic degeneration occurs in more than 99% of preantral follicles at this stage fron1 a preantral follicle to a mature follicle. A. large number of preantral follicles undergo atretic degeneration, which results in inefficient utilization of oocytes, as well as unfavorable screening of highquality oocytes. If these preantral follicles with developmental potential are isolated and cultured in vitro in. a suitable medium, waste of oocyte resources can. be avoided.
[03] It is crucial for perfecting ea model for ill vitro folliculogenesis studies to establish an in vitro culture system to culture preantral follicles and obtain oocytes with the ability of fertilization and embryonic development by using a defined composition of defined medium. Porcine follicular stromal tissues have been studied. slowly due to problems such. as more adhesions, more difficult isolation, longer culture cycles, and uneven quality. In 1994, Hirao et al. cultured. porcine preantral follicles in vitro in an embedded manner for 16 d, and obtained mature oocytes that were capable of fertilization, but fertilized eggs were unable to form male pronuclei. In 2001, Wu et al. obtained. the first blastocysts by IVF with oocyte intracytoplasmic single sperm. microinjection. of porcine preantral follicles cultured in a 4 d adherent wall. In 2010, Hashimoto et al. demonstrated that mature oocytes can also be obtained from granuleoocyte complexes derived from early porcine antral follicles after in vitro culture. In 2015, Hongxing Yang et al. isolated preantral granulosaoocyte complexes by in vitro for adherence culture for 18.5 d. An antral formation rate was 40.0%, and an oocyte maturation rate reached 26.2%. However, intact isolation and culture systems for porcine preantral follicles have not been reported. Since connective tissue of a porcine ovary is so tight that follicles cannot be isolated by simple stripping, the isolation system for porcine preantral follicles needs to be further improved, and the culture system needs to be perfected. In summary, it can be seen that the study of porcine follicle isolation and culture in vitro still has problems that the isolation method is inefficient, the intact follicle can not be cultured in vitro, and the culture composition is to be clarified, and so on. SUMMARY
[04] In view of this, an objective of the present invention is to provide an isolation method for preantral follicles in a porcine ovary, which has the advantage of improving isolation efficiency of preantral follicles in vitro.
[05] .Another object of the present invention is to provide a threedimensional culture method for preantral follicles in a porcine ovary, which has the advantage of enabling intact porcine preantral follicles to form antra in vitro.
[06] In order to realize the above inventive objective, the present invention provides the technical solutions as follows:
[07] The present invention. provides an isolation. method for preantral follicles in a porcine ovary, including: mixing' porcine ovarian tissue fragments and a follicle isolation solution for digestion for 0.5 h2 h discarding supernatant, terminating digestion, and obtaining digested tissue; and sieving the digested tissue with a 400 um mesh, and obtaining the preantral follicles; where the follicle isolation solution consists of an L15 culture medium, 2 mg / ml collagenase I, 200 ug / ml DNase I (deoxyribonuclease I) and a 1% penicillinstreptomycin solution.
[08] Preferably, a particle size of the porcine ovarian tissue fragments is less than 1 mm2.
[09] Preferably, a volume ratio of the porcine ovarian tissue fragments to the follicle isolation solution is 1:2.
[10] Preferably, a temperature for mixing and digestion is 37.5T, and shaking is performed for 10 seconds every 20 min30 min during mixing and digestion.
[11] Preferably, a manner of terminating the digestion is to add a follicle washing solution consisting of an L15 culture medium, 10% fetal bovine serum. (FBS) and a 1% penicillinstreptomycin solution.
[12] The present invention further provides a culture method for preantral follicles in a porcine ovary. Based on the isolation method, the method further includes: mixing the obtained preantral follicles with a sodium alginate solution, then mixing with a CaClz solution, and mixing the coated preantral follicles with a culture medium for culture.
[13] Preferably, a concentration. of the sodium. alginate solution is 1%.
[14] Preferably, a concentration of the CaClz solution is 100 mM.
[15] Preferably, a mixing time with the CaClz solution is 1 min2 min.
[16] Preferably, the culture medium for culture consists of a DMEM / F12 medium, 10% FBS, 1 ug / mL 10 >< insulin transferrinselenium. (ITSG), a 100 mIU / mL follicle stimulating hormone (FSH) and a 1% penicillinstreptomycin solution.
[17] The present invention has the beneficial effects as follows:
[18] According to the isolation method and culture method for preantral follicles in a porcine ovary provided in the present invention, porcine preantral follicle isolation and in vitro culture systems are optimized, and remarkable technical effects of improving in vitro isolation efficiency of the porcine preantral follicles and implementing in vitro antral formation culture of the intact porcine preantral follicles are achieved. Not only a sufficient egg source can be provided for porcine embryo engineering research and application, but also reference is provided for further exploring porcine genetic potential. BRIEF DESCRIPTION OF THE DRAWINGS
[19] FIG. 1 shows an isolation process for preantral follicles in a porcine ovary, where A represents fragments of an aovarian cortex cut through a mechanical method, B represents obtained preantral follicles, and C represents a micrograph of the preantral follicles; and
[20] FIG. 2 shows a culture process for preantral follicles coated through a sealing membrane method, where A represents preantral follicles isolated in Example 1, B represents washing twice in a follicle washing solution, C represents dripping' of a sodium. alginate solution. on a sealing membrane, D represents placing the sealing membrane upside down into a calcium chloride solution, E represents preantral follicles successfully coated, and F represents porcine follicles cultured into antra in vitro. DETAILED DESCRIPTION OF THE EMBODIMENTS
[21] The present invention. provides an isolation. method for preantral follicles in a porcine ovary, including: porcine ovarian tissue fragments and a follicle isolation solution were mixed for digestion for 0.5 h2h discarding supernatant, digestion was terminated, and digested tissue was obtained; and the digested tissue was sieved with a 400 um mesh, and the preantral follicles were obtained. The follicle isolation solution consisted of an L15 culture medium, 2 mg / ml collagenase I, 200 ug / ml DNase I and a 1% penicillinstreptomycin solution.
[22] In the present invention, it was preferable to puncture antral follicles in the ovary first, and. then crush the ovarian tissue. A specific method for crushing was not particularly limited in the present invention. A particle size of the tissue fragments was preferably less than 1 mm?. In the present invention, a volume ratio of the porcine ovarian tissue fragments to the follicle isolation solution was preferably 1: 2. According to the present invention, specific sources of components in the follicle isolation solution are not particularly limited, and conventional commercial products in the field can be used. In the present invention, a temperature for mixing and digestion was preferably 37.5%L shaking needed to be performed for preferably 10 seconds every 20 min30 min during mixing and digestion, and a deing and digestion time was preferably 1 h. In the present invention, after the mixing' and. digestion. was completed, the supernatant was discarded. preferably by centrifugation, and a centrifugation condition was preferably 600 g for 3 min. In the follicle isolation solution of the present invention, the 1% penicillinstreptomycin solution referred to that a final volume percentage of the penicillinstreptomycin solution in the follicle isolation solution was 1%.
[23] In the present invention, a manner of terminating the digestion was preferably to add a follicle washing solution preferably consisting of an L15 culture medium, 10% FBS and. the 1% penicillinstreptomycin. solution. In the follicle washing' solution. of the present invention, the 10% FBS referred to that a final volume percentage of the FBS in the follicle washing solution was 10%, and the 1% penicillinstreptomycin solution referred to that a final volume percentage of the penicillinstreptomycin solution in the follicle washing solution was 1%. According to the present invention, specific sources of components in the follicle washing solution are not particularly limited, and conventional commercial products in the field can be used. In the present invention, preantral follicles with intact theca and no antrum were preferably selected after sieving with a 400 um mesh for subsequent culture.
[24] The present invention further provides a culture method for preantral follicles in a porcine ovary. Based on the isolation method, the method further includes: the obtained preantral follicles were mixed with a sodium alginate solution, then mixed with a CaClz solution, and the coated preantral follicles were mixed with a culture medium for culture.
[25] In the present invention, subsequent in vitro culture of the isolated preantral follicles was carried out preferably by coating the isolated preantral follicles with a sodium alginate gel system. A concentration of the sodium alginate solution was preferably 1%, and a concentration of the CaClz solution was preferably 100 mM. In the present invention, a process for preferably using the sealing membrane method to coat the preantral follicles and forming antra specifically included: the preantral follicles obtained by isolation were washed in the follicle washing solution, then. the sodium. alginate solution was dropped on a sealing membrane, the preantral follicles were transferred into sodium alginate solution drops by using a mouth pipette, and the sealing membrane was placed upside down into the CaClz solution for solidification, and was mixed with the CaClz solution preferably for 1. min2 min. The specific composition of the follicle washing solution referred to herein is the same as above, which is repeated herein. In the present invention, when the coated preantral follicles were mixed with the culture medium for culture, the culture medium for culture preferably consisted of a DMEM / F12 medium, 10% FBS, 1 ug / mL 10 x ITSG, a 100 mIU / mL FSH and a 1% penicillinstreptomycin solution. The percent sign in the 10% FBS and the 1% penicillinstreptomycin solution refers to a final volume percentage. According to the present invention, specific sources of components in the culture medium are not particularly limited, and conventional commercial products in the field can be used. The culture was preferably carried out in a 96well plate. The amount of culture medium contained in each well was preferably 150 uL, and each well preferably contained one coated preantral follicle microcapsule. In the present invention, it was preferable to change the solution every other day when culture. Antra were formed on the third day of culture through the culture method of the present invention. The method. can form. intact porcine follicles into antra in vitro. An antra formation rate is 22.67% i 2.4%.
[26] The technical solutions provided by the present invention are described. in detail below in combination with examples, but should not be interpreted as limiting the scope of protection of the present invention.
[27] The following examples are conventional unless otherwise specified.
[28] The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
[29] Example 1
[30] A.porcine mesoarium was fixed with forceps in a large culture dish containing 10 ml PBS, and an antral follicle was punctured with a 10 ml syringe. An ovary was dissected with a No. 10 scalpel blade until tissue fragments were less than 1 mm?. The tissue fragments were collected into a 15 ml centrifuge tube, and 10 ml of a follicle isolation solution (L15 culture medium + 2 mg / ml collagenase I + 200 ug / ml DNase I 4- 1% penicillinstreptomycin solution) was added. The tissue fragments were put in a 37.5°C water bath, shaken for 10 s every 15min, digested for 1 h. Centrifugation was performed at 600 g for 3 min at room temperature, and supernatant was discarded. Then a follicle washing solution (L15 culture medium + 10% FBS + 1% penicillinstreptomycin solution) was added to terminate digestion, and digested tissue was obtained. The digested. tissue was sieved through. a 400 um. mesh, and preantral follicles with intact theca and no antrum were picked up under a stereomicroscope by using a mouth pipette. Results are shown in FIG. 1.
[31] Comparative Example 1
[32] Insulin needle isolation method: a porcine ovary was cut into pieces and placed in a large dish containing PBS. Under a stereo microscope, follicles were isolated by using tips of two insulin needles.
[33] Comparative Example 2
[34] Mesh sieving method: an entire porcine ovary was rubbed. on a 400 um. mesh. to make tissue fragments pass through the mesh, the tissue fragments were collected, and follicles were searched under a stereomicroscope.
[35] Example 2
[36] A number of follicles obtained through the isolation method described. in Example 1 was compared. with those obtained through the different isolation methods described in Comparative Example 1 and Comparative Example 2. Example 1 was subjected. to three parallel experiments. Results are expressed as mean i standard deviation. Comparative Example 1 and Comparative Example 2 were subjected. to only one experiment. Results are shown in Table 1.
[37] Table 1 Number of follicles obtained. through different isolation methods Method Number of follicles Number of intact obtained / PCs follicles / PCs Comparative Example 1 12 (insulin needle isolation method) Comparative Example 2 (mesh sieving method) Example 1 (mechanicalenzyme 181.00 i 8.00 119.67 i 4.44 compound method)
[38] It can be seen that the isolation method for preantral follicles in. a porcine ovary provided. by the present invention. can isolate and. obtain.:more jpreantral follicles from a single ovary, and the number of intact follicles obtained by isolation accounts for a larger proportion.
[39] Example 3
[40] .A difference from. Example 1 was that a digestion reaction time was 0.5 h, and the remaining is the same as Example 1.
[41] Example 4
[42] .A difference from. Example 1 was that a digestion reaction time was 2 h, and the remaining is the same as Example 1.
[43] Example 5
[44] A number of follicles obtained in a digestion reaction time in Example 1 was compared with those obtained in different digestion reaction times in Example 3 and Example 4. Example 1, Example 3 and Example 4 were each subjected to three parallel experiments. Results are expressed as mean i standard deviation as shown in Table 2. As can be seen from Table 2, the number of follicles obtained in different digestion times varies.
[45] Table 2 Number of follicles obtained by isolation in different digestion times Time Number of follicles Number of intact obtained / PCs follicles / PCs
[46] Example 6
[47] A.process for using a sealing membrane method to coat the preantral follicles and forming antra is shown in FIG. 2. The preantral follicles isolated. in Example 1 were washed twice ill a follicle washing solution (L15 4 10% FBS + 1% penicillinstreptomycin). 21 1% sodium. alginate solution was dropped onto a sealing membrane, and the preantral follicles were transfered into sodium alginate solution drops with a mouth pipette. The sealing membrane was placed. upside down into a 100 mM calcium. chloride solution for 1. min. The coated preantral follicles were collected and cultured in a 96well plates containing 150 uL culture medium (one microcapsule per well) (the culture medium consisted of DMEM / F12 culture medium + 10% FBS + 1 ug / mL 10 >< ITS-G + 100 mIU / mL FSH and 1% penicillin streptomycin solution). The solution was changed every other day. Photos were taken. On a third day of culture, the follicles formed antra. An antral formation rate was calculated. The antrum formahtion rate (%) = (the number of formed. antra / the total number* of jpreantral follicles transferred to the sodium alginate solution drops) X 100.
[48] Five parallel experiments were carried. out in the example. Results were expressed as mean i standard deviation. The antral formation rate is 22.67% i 2.4%.
[49] The above descriptions are merely preferred embodiments of the present invention. It should be noted that a person of ordinary skill in the art may further make several improvements and. modifications without departing from the principle of the present invention, but such improvements and Hbdifications should 1%; deemed as falling within the scope of protection of the present invention.
Claims
1. Isolation procedure for preantral follicles in a porcine ovary, comprising: the mixing of porcine ova sodium tissue fragments and a follicle isolation solution for digestion for 0.5 hours 2 hours, removal of supernatant, the completion of digestion, and the obtaining of digested tissue; and the sieving of the digest star-shaped tissue with a mesh size of 400 µm and the obtaining of the preantral follicles; whereby the follicles Cell isolation solution consists of an L15 culture medium, 2 mg / ml collagenase I, 200 µg / ml DNase I and a 1% penicil linestreptomycin solution.
2. Insulation method according to claim 1, whereby a particle size of the porcine ovarian tissue fragments is smaller than 1 mm².
3. Insulation method according to claim 1, whereby a volume ratio of the porcine ovarian tissue fragments is 1:2 compared to the follicle isolation solution.
4. Insulation method in accordance with claim 1, whereby a temperature for mixing and digestion is 37.5°C, and shake for 10 seconds every 20 min 30 min becomes carried out during mixing and digestion.
5. Insulation method in accordance with claim 1, whereby a method of ending digestion the addition of a follicle wax solution consisting of an L15 culture medium dium, 10% fetal bovine serum (FBS) and a 1% penicillin streptomycin solution is.
6. Culture method for preantral follicles in a porcine ovary, based on the isolation method according to one of the claims 15, further comprising: mixing of the obtained preantral follicles with a sodium alginate solution, subsequently mixing with a CaCl₂ solution, and the mixing of the coated preantral follicles kels with a culture medium for cultivation.
7. Cultivation method in accordance with claim 6, whereby a the concentration of the sodium alginate solution is 1%.
8. Cultivation method in accordance with claim 6, whereby a the concentration of the CaClz solution is 100 mM.
9. Cultivation method in accordance with claim 6, whereby a The mixing time with the CaClz solution is 1 min 2 min.
10. Cultivation method in accordance with claim 6, whereby the culture medium. for culture consists of a DMEM / F12 medium, 10% FBS, 1 µg / mL 10 x insulin transferrinselenium, a 100 mlU / mL follicle-stimulating hormone and a 1% penicill linestreptomycin solution. ooo FIG.1 FIG.2