Oocyte vitrification cryopreservation method

Abstract

The invention provides an oocyte vitrification cryopreservation method, which comprises the following steps: pre-treating porcine oocytes before freezing, and jointly applying methionine and proline in vitrification freezing liquid, thereby promoting the survival and in-vitro developmental ability of the frozen-unfrozen MII-stage oocytes. The invention also provides an oocyte vitrification cryopreservation solution, which comprises methionine and proline with effective concentration. The method can be used in the fields of animal breeding, embryo engineering and the like, provides powerful technical support for breed conservation and rapid propagation, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of embryo engineering, and in particular relates to an oocyte vitrification cryopreservation method. Background Art

[0002] Ultra-low temperature cryopreservation of gametes and embryos is a key link in in vitro embryo production technology, and is also an important means for breeding of livestock and poultry breeds, protection and utilization of germplasm resources, and sustainable development of animal husbandry. By freezing and preserving oocytes, not only can a rich oocyte resource be provided for embryo engineering technologies such as in vitro fertilization, nuclear transplantation, and transgenic animal research, but also a female animal gene bank can be established, providing a new way for the long-term preservation of female individuals with high genetic value and rare and endangered wild animal genetic resources, so that they are not restricted by time and space.

[0003] Oocyte vitrification technology is an ultra-rapid freezing method that places oocytes in a high-concentration cryoprotectant (Cryoprotectant, CPA) for a short period of time to replace the water in the cells, and then quickly places the oocytes on a freezing carrier and directly immerses them in liquid nitrogen. et al. 2021). Although live piglets were obtained by vitrification of porcine GV-stage oocytes in 2014 (Somfai et al. 2014) and MII-stage oocytes in 2015 (Gajda et al. 2015), the efficiency of porcine GV-stage oocytes (parthenogenetic activation: 0.41%-12%; in vitro fertilization: 0.79%-13.5%) and MII-stage oocytes (parthenogenetic activation: 1.3%-8.3%; in vitro fertilization: 2%-9.2%) developing into blastocysts after vitrification and thawing is significantly lower than that of fresh oocytes, which seriously hinders the industrial application of this technology. Compared with GV stage oocytes, porcine MII stage oocytes have stronger permeability to cryoprotectants and higher survival rates after thawing (Somfai et al. 2010). However, temperature changes during the vitrification process can lead to abnormal distribution of microfilaments and microtubules in porcine MII stage oocytes, and make them difficult to recover after thawing (Egerszegi et al. 2013), which seriously reduces the developmental capacity of oocytes after thawing. Therefore, it is very important to choose a suitable vitrification method for porcine MII stage oocytes.

[0004] Many cryogenic organisms in nature, such as Arctic springtails, polar fish, and some amphibians, will accumulate trehalose, antifreeze proteins, or secrete small-molecule compounds to regulate their own redox balance to resist low-temperature stress under extremely low survival environmental temperatures (Dou et al. 2022). Amino acids are natural small-molecule compounds and also the basic structural units of antifreeze proteins, with certain antioxidant effects. Methionine (Met) is a thiol-containing antioxidant and also a precursor amino acid of glutathione, playing an important role in protecting the functional integrity of cell membranes or cytoplasm (Patra et al. 2001), improving sperm mitochondrial activity (Bucak et al. 2012), and reducing DNA damage (Tuncer et al. 2010). There are also studies showing that among water-soluble natural amino acids, methionine has the strongest ability to lower the freezing point of ice, but adding a high concentration of methionine will cause osmotic damage to cells (Ma et al. 2023). Proline (Pro) is a natural amino acid with an amino-containing non-polar cyclic pyrrolidine side chain (Hayat et al. 2012), which relieves water stress through osmotic regulation ( et al. 2016), and can also bind to folding intermediates, stabilize protein monomers, prevent them from denaturing during cold stress, and contribute to the restoration of the functional conformation of proteins.

[0005] Methionine and proline have shown great application prospects and development potential in improving plant stress resistance, disease treatment, and drug development. However, although methionine and proline have been widely used in many fields, their combined application in vitrification cryopreservation of oocytes has not been reported. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for vitrification cryopreservation of oocytes.

[0007] The present invention has found through research that the combined application of methionine and proline in vitrification cryopreservation of oocytes can effectively improve the survival rate of oocytes after freezing and thawing and their in vitro development level. Accordingly, the solution of the present invention is as follows:

[0008] The present invention first provides the application of methionine and proline in vitrification cryopreservation of oocytes.

[0009] Specifically, it includes the application of methionine and proline in improving the survival rate of vitrified cryopreserved oocytes and the application of methionine and proline in improving the in vitro development ability of vitrified cryopreserved oocytes.

[0010] The present invention also provides the application of methionine and proline in the preparation of vitrification cryopreservation solution.

[0011] The present invention also provides a vitrification cryopreservation solution for oocytes, which contains effective concentrations of methionine and proline. It should be understood in the art that based on the disclosed ideas and experimental methods of the present invention, those skilled in the art can appropriately adjust the concentrations of methionine and proline according to different species to enable the improvement of the survival rate of oocytes after freezing and thawing or the in vitro developmental ability of oocytes after freezing and thawing.

[0012] Preferably, the concentration of methionine is 10 - 40 mM, and the concentration of proline is 0.4 - 2 M. In one embodiment of the present invention, the preferred concentration of methionine is 20 mM, and the preferred concentration of proline is 1 M.

[0013] Furthermore, the cryopreservation solution is based on TCM199 and further includes 20% FBS, 7.5% DMSO, 10% EG, and 0.4 M sucrose. The percentages are by weight.

[0014] The present invention also provides a vitrification cryopreservation method for oocytes, which uses the above-mentioned vitrification cryopreservation solution for oocytes for cryopreservation. Specifically, the cryopreservation method includes the following steps: treating the oocytes in a pretreatment solution for 20 - 40 min, equilibrating in a cryo - equilibration solution, transferring them into the vitrification cryopreservation solution for treatment for 20 - 40 s, and storing them in liquid nitrogen. Preferably, the oocytes are treated in the pretreatment solution for 30 min, equilibrated in the cryo - equilibration solution, transferred into the vitrification cryopreservation solution for treatment for 30 s, and stored in liquid nitrogen.

[0015] In the present invention, the oocytes are preferably MII - stage oocytes. They can be from mammals. In one embodiment of the present invention, the oocytes are porcine oocytes.

[0016] The present invention promotes the survival and in vitro developmental ability of MII - stage oocytes after freezing - thawing by performing pretreatment on porcine oocytes before freezing and jointly applying methionine and proline in the vitrification cryoprotectant. It can be used in fields such as animal reproduction and embryo engineering, providing strong technical support for germplasm conservation and rapid propagation, and having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 . FDA staining of porcine MII - stage vitrified - cryopreserved oocytes 2 h after thawing (methionine).

[0018] Figure 2 . FDA staining of porcine MII - stage vitrified - cryopreserved oocytes 2 h after thawing (proline).

[0019] Figure 3. It shows the development of parthenogenetic activation embryos after thawing vitrified oocytes. Detailed implementation manners

[0020] The following examples further illustrate the content of the present invention, but should not be construed as a limitation to the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0021] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0022] In the examples of the present invention, the experimental ovaries were collected from the slaughterhouse of Beijing No. 5 Meat Joint Processing Factory. The TCM199 and fetal bovine serum used in the present invention were purchased from Gibco, and other reagents were purchased from Sigma-Aldrich unless otherwise specified.

[0023] Example 1

[0024] 1. Collection and in vitro maturation of oocytes

[0025] Ovaries were collected from the slaughterhouse and placed in sterile physiological saline containing double antibiotics at 28 - 32°C, and transported back to the laboratory within 2 hours. The ovaries were washed 2 - 3 times with sterile physiological saline preheated to about 30°C. A 10 mL disposable sterile syringe with an 18-gauge needle was used to aspirate follicles with a diameter of 2 - 6 mm on the ovaries. The follicular fluid was collected in a 15 ml sterile centrifuge tube and washed 3 times with TL-HEPES operating fluid. After the follicular fluid was allowed to precipitate for 5 - 10 minutes, the precipitate at the bottom of the centrifuge tube was observed under a stereomicroscope, and cumulus-oocyte complexes (COCs) with uniform cytoplasm and surrounded by more than 3 layers of dense cumulus cells were selected for in vitro maturation. They were transferred into a four-well plate that had been equilibrated in a CO2 incubator for at least 2 hours (500 μL of maturation medium per well, 100 COCs), and matured in a CO2 incubator at 38.5°C, 5% CO2, and saturated humidity for 42 - 44 hours.

[0026] Among them, the TL-HEPES operating fluid was 6.6622 g / L NaCl, 0.2386 g / L KC1, 0.168 g / L NaHCO3, 0.294 g / L CaCl2·2H2O, 0.0463 g / L KH2PO4, 0.1017 g / L MgCl2·6H2O, 2.383 g / L HEPES, 1.1214 g / L sodium lactate, 0.022 g / L sodium pyruvate, 2.1864 g / L sorbitol, 0.065 g / L penicillin, 0.05 g / L streptomycin, 3 g / L BSA.

[0027] The in vitro maturation medium is: TCM199 (1×), 0.0001 g / mL L-cysteine, 10 ng / mL epidermal growth factor, 10% porcine follicular fluid, 10 IU / mL PMSG (Merck Sharp & Dohme PG600), 5 IU / mL hCG (Merck Sharp & Dohme PG600), 75 μg / mL penicillin, 50 μg / mL streptomycin.

[0028] 2. Vitrification, freezing, thawing of oocytes

[0029] (1) Pre-treatment of oocytes before freezing

[0030] Put the COCs matured in vitro for 42 - 44 h into 0.1% hyaluronidase, and gently pipette repeatedly with a pipette gun to remove cumulus cells. Continuously observe the removal situation under a stereomicroscope. After most of the COCs are removed, transfer the oocytes to HN-23 operating fluid, and count the extrusion of the first polar body of the oocytes under a stereomicroscope. Put the in vitro matured oocytes with the first polar body into the pre-treatment solution and incubate for half an hour, then transfer them into HN23 operating fluid after the incubation.

[0031] The pre-treatment solution described in (1) is TCM199 (1×), 20% FBS, 0.25 M sucrose, 1 μM paclitaxel.

[0032] The HN-23 operating fluid is 7.6966 g / L NaCl, 0.168 g / L NaHCO3, 0.356 g / L KC1, 0.162 g / L KH2PO4, 0.293 g / L MgSO4·7H2O, 1.000 g / L glucose, 0.146 g / L glutamine, 1.5012 g / L taurine, 2.383 g / L HEPES, 0.065 g / L penicillin, 0.05 g / L streptomycin, 1 mL phenol red, 4 g / L BSA.

[0033] (2) Vitrification freezing of oocytes

[0034] Take out the equilibration solution and vitrification solution in advance and restore them to room temperature. At room temperature, first put the oocytes into the equilibration solution (ES) for 4 minutes of equilibration, then transfer them into the vitrification solution (VS) for 30 seconds of treatment, immediately load them onto the Cryotop carrier rod and plunge into liquid nitrogen. After putting on the carrier rod cap under liquid nitrogen, transfer them to the liquid nitrogen tank for cryopreservation.

[0035] Among them, the equilibration solution ES described in (2) is TCM199 (1×), 20% FBS, 7.5% EG, and 7.5% DMSO; the cryoprotectant solution VS is TCM199 (1×), 20% FBS, 15% EG, 15% DMSO, and 0.4 M sucrose; the cryoprotectant solution V+M is TCM199 (1×), 20% FBS, 15% EG, 15% DMSO, 0.4 M sucrose, and 10 / 20 / 40 mM methionine; the cryoprotectant solution V+P is TCM199 (1×), 20% FBS, 10% EG, 7.5% DMSO, 0.4 / 1 / 2 M proline; the cryoprotectant solution P+M is TCM199 (1×), 20% FBS, 10% EG, 7.5% DMSO, 0.4 M sucrose, 20 mM methionine, and 1 M proline.

[0036] (3) Oocyte thawing and in vitro embryo culture

[0037] Take out the thawing solution in advance and preheat it in a CO2 incubator. During thawing, quickly take out the Cryotop carrier rod from liquid nitrogen, immediately immerse the oocyte-containing end in the pre-equilibrated and preheated thawing solution I and incubate for 1 min, then sequentially transfer the oocytes into thawing solution II, thawing solution III, and thawing solution IV and incubate for 3, 5, and 5 min respectively. Finally, after washing 3 times with thawing solution IV, part of them are used for the determination of oocyte viability for 2 h, and the other part is parthenogenetically activated after 2 h of recovery. Wash the oocytes 3 times with the activation solution, then transfer them to a fusion chamber (electrode width 1 mm, BTX, USA) filled with the activation solution, and perform electroactivation with a direct current pulse of 1.2 kv / cm and 70 μs. After activation, transfer the oocytes into the chemical activation solution for chemical activation. After 4 - 6 h, take out the oocytes, wash them 3 times with PZM3 culture medium, and then transfer them into a PZM-3 culture droplet and continue to culture at 38.5°C, 5% CO2, and saturated humidity. Count the cleavage rate, morula rate, and blastocyst rate at 48 h, 144 h, and 168 h respectively.

[0038] (3) The thawing solution I described is TCM199 (1×), 20% FBS, 1 M sucrose; the thawing solution II is TCM199 (1×), 20% FBS, 0.5 M sucrose; the thawing solution III is TCM199 (1×), 20% FBS, 0.25 M sucrose; the thawing solution IV is TCM199 (1×), 20% FBS.

[0039] The activation solution is 0.25 mM mannitol, 0.1 mM CaCl2·2H2O, 0.1 mM MgCl2·6H2O, 0.5 mM HEPES, 0.01% PVA (w / v).

[0040] The PZM-3 culture medium consists of 6.312 g / L NaCl, 2.106 g / L NaHCO3, 0.746 g / L KCl, 0.048 g / L KH2PO4, 0.098 g / L MgSO4·7H2O, 0.022 g / L sodium pyruvate, 0.616 g / L semi-calcium lactate, 0.146 g / L L-glutamine, 0.066 g / L penicillin, 0.05 g / L streptomycin, 0.0586 g / L betaine, 1 mL phenol red, 0.546 g / L sodium taurinate, 50× essential amino acids, 100× non-essential amino acids, and 3 g / L BSA.

[0041] The chemical activation solution is PZM-3, 7.5 μg / mL cytochalasin B, and 10 μg / mL cycloheximide.

[0042] 3. Oocyte viability assessment

[0043] Transfer the oocytes 2 h after thawing to a 5 μg / mL fluorescein diacetate (FDA) dye, incubate in the dark at 38.5 °C for 5 min, wash 3 times with 0.1% PVA / DPBS, and perform image acquisition under a fluorescence microscope. As Figure 1 and 2 shown, live oocytes produce bright green fluorescence, while dead oocytes do not emit light or emit weak fluorescence.

[0044] 4. Data analysis

[0045] Each experiment was repeated at least 3 times biologically. The experimental results were analyzed by one-way analysis of variance (ANOVA) and Duncan's test using SPSS software to determine the significance of differences between different treatments. A P value < 0.05 was considered significant.

[0046] 5. Methionine and proline significantly improve the survival rate of porcine MII oocytes 2 h after vitrification and thawing

[0047] Optimize the vitrification solution of porcine MII oocytes with 10 mM, 20 mM, and 40 mM methionine respectively. After thawing for 2 h, perform FDA staining and count the oocyte survival rate (Table 1). As shown in Table 1, the survival rate of oocytes in the 20 mM methionine group 2 h after thawing was significantly higher than that of oocytes in other groups (P < 0.05). Optimize the vitrification solution of porcine MII oocytes with 0.4 M, 1 M, and 2 M proline respectively. After thawing for 2 h, perform FDA staining and count the oocyte survival rate (Table 2). As shown in Table 2, the survival rate of oocytes in the 1 M proline group 2 h after thawing was significantly higher than that of oocytes in other groups (P < 0.05).

[0048] Table 1 Effects of Methionine on the Survival of Porcine Metaphase II Oocytes after Vitrification and Thawing for 2 h

[0049]

[0050] Note: Oocyte survival rate = (number of surviving oocytes) × 100 / total number of oocytes; different letters (a, b, c) in the same column indicate significant differences (P < 0.05).

[0051] Table 2 Effects of Proline on the Survival of Porcine Metaphase II Oocytes after Vitrification and Thawing for 2 h

[0052]

[0053] Note: Oocyte survival rate = (number of surviving oocytes) × 100 / total number of oocytes; different letters (a, b, c) in the same column indicate significant differences (P < 0.05).

[0054] 6. Methionine combined with proline significantly improves the survival rate of porcine metaphase II oocytes after vitrification and thawing for 2 h

[0055] 20 mM methionine, 1 M proline, and the combined addition of methionine and proline were respectively selected to optimize the vitrification solution for porcine metaphase II oocytes. After thawing for 2 h, FDA staining was used to count the oocyte survival rate (Table 3). As shown in Table 3, the survival rate of oocytes in the 1 M proline group and the combined addition group was significantly higher than that of oocytes in other groups (P < 0.05).

[0056] Table 3 Effects of Proline on the Survival of Porcine Metaphase II Oocytes after Vitrification and Thawing for 2 h

[0057]

[0058] Note: Oocyte survival rate = (number of surviving oocytes) × 100 / total number of oocytes; different letters (a, b, c) in the same column indicate significant differences (P < 0.05).

[0059] 7. Methionine combined with proline significantly improves the parthenogenetic activation development rate of porcine metaphase II oocytes after vitrification and thawing

[0060] By counting the cleavage rate, morula rate and blastocyst rate of parthenogenetically activated embryos, the effect of methionine combined with proline on the development of parthenogenetically activated embryos of vitrified-thawed oocytes was analyzed. The results are shown in Table 4. The cleavage rate, morula rate and blastocyst rate of parthenogenetically activated embryos of oocytes in each freezing group were significantly lower than those in the fresh group (P < 0.05). Compared with the VS group, the cleavage rate, morula rate and blastocyst rate of parthenogenetically activated embryos of oocytes in the P+M group were significantly increased (P < 0.05). There was no significant difference in the cleavage rate and morula rate of parthenogenetically activated embryos of oocytes between the V+M and V+P groups and the VS group (P > 0.05), but the blastocyst rate of parthenogenetically activated embryos in both groups was significantly higher than that in the VS group (P < 0.05).

[0061] Table 4 Effects of methionine combined with proline on the development of parthenogenetically activated embryos of porcine MII oocytes after vitrification and thawing

[0062]

[0063]

[0064] 8. Conclusion

[0065] From the above results, it can be seen that the freezing method of the present invention can significantly improve the vitrification and thawing effect of porcine MII oocytes, increase the survival rate of vitrified-thawed oocytes, and improve the in vitro development level of vitrified-thawed oocytes. It has potential application value in the development of vitrification solutions for porcine oocytes.

Claims

1. Application of methionine and proline in vitrification cryopreservation of oocytes.

2. Application of methionine and proline in improving the survival rate of vitrified oocytes.

3. Application of methionine and proline in improving the in vitro developmental ability of vitrified oocytes.

4. Application of methionine and proline in the preparation of vitrification cryopreservation solution.

5. A vitrification cryopreservation solution for oocytes, which contains effective concentrations of methionine and proline.

6. The preservation solution according to claim 5, characterized in that, The concentration of methionine is 10 - 40 mM, and the concentration of proline is 0.4 - 2 M.

7. The preservation solution according to claim 6, wherein, The concentration of methionine is 20 mM, and the concentration of proline is 1 M.

8. The preservation liquid according to any one of claims 5 to 7, characterized in that, The preservation solution is based on TCM199 and also includes 20% FBS, 7.5% DMSO, 10% EG and 0.4 M sucrose.

9. A method for vitrification cryopreservation of oocytes, which uses the vitrification cryopreservation solution for oocytes according to any one of claims 5 - 8 for cryopreservation.

10. The method according to claim 9, wherein The cryopreservation includes the following steps: treating the oocytes in a pretreatment solution for 20 - 40 min, equilibrating in a cryoprotectant solution, transferring them into the vitrification cryopreservation solution for 20 - 40 s, and storing them in liquid nitrogen. Preferably, the oocytes are treated in the pretreatment solution for 30 min, equilibrated in the cryoprotectant solution, transferred into the vitrification cryopreservation solution for 30 s, and stored in liquid nitrogen.

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