Application of L-proline in improving the post-freezing development rate of vitrified germinal vesicle stage oocytes

Abstract

The present invention provides an application of L-proline in improving the post-freezing development rate of vitrified germinal vesicle stage oocytes, which can improve the parthenogenetic embryo development rate after vitrified thawing of oocytes. The present invention also provides a pre-treatment method for improving the maturation and development ability of vitrified thawed oocytes, which includes placing oocytes in a freezing balance solution for balancing, transferring them into a vitrification freezing solution containing L-proline for 30 seconds, and storing them in liquid nitrogen.

Description

Technical Field

[0001] The invention belongs to the technical field of cell preservation reagent application, and particularly relates to an application of L-proline in improving the post-freezing development rate of vitrified germinal vesicle stage oocytes. Background Art

[0002] The existing local pig breed resources are very rich and are an important basis for the study of biological genetic diversity. However, with the development of modern intensive animal husbandry, the number of some excellent local pig populations has decreased sharply. How to effectively protect and rationally utilize these precious genetic resources has brought severe challenges to the development of local pig breeds. The traditional method of living seed preservation has weak ability to resist diseases and natural disasters, and requires a lot of capital and labor investment. With the continuous development of cryobiology, cryopreservation technology has provided a new way to preserve animal genetic resources.

[0003] Oocyte cryopreservation technology is an important part of embryonic bioengineering. By cryopreserving oocytes, a female animal gene bank can be established, which not only provides rich oocyte resources for the research of embryonic biotechnology such as in vitro fertilization and nuclear transplantation, but also can be used for the long-term preservation of genetic resources of excellent livestock and poultry breeds and endangered rare species. In 2014, Somfai et al. successfully preserved porcine germinal vesicle (GV) stage oocytes for the first time using vitrification freezing and obtained live piglets (Somfai et al. 2014). In recent years, the mechanism of freezing damage of porcine oocytes has been continuously elucidated, and the cryopreservation system has been gradually optimized, which has effectively improved the development and recovery ability of porcine oocytes after thawing. However, compared with ruminants such as cattle and sheep, porcine oocytes have richer lipid content and poorer tolerance to low temperatures (McEvoy et al. 2000), and the cryopreservation efficiency is still very low.

[0004] Oocyte vitrification refers to an ultra-fast freezing method in which oocytes are briefly treated in a solution containing high-concentration cryoprotective agents (CPAs) and then directly placed in liquid nitrogen for cryopreservation at a temperature above 0°C. CPAs are mainly composed of permeable CPAs, non-permeable CPAs and buffer media, and are one of the important factors affecting the effect of oocyte vitrification. During the vitrification process, the freezing of extracellular water causes the osmotic pressure of the extracellular fluid to increase. Under the action of the osmotic pressure difference, the intracellular water penetrates outside the cell, and the oocyte shrinks, resulting in impaired functions of macromolecules such as proteins and nucleic acids, causing osmotic damage. Due to the rapid cooling rate, the intracellular water cannot be fully evacuated, and a large number of ice crystals are formed in the cells, resulting in ice crystal damage. Therefore, the selection of appropriate CPAs is crucial to the efficiency of oocyte vitrification.

[0005] There are many natural osmotic regulating substances in nature, which play an important role in maintaining the osmotic balance of cells. Under low temperature stress, some osmotic regulating substances will be produced in large quantities, such as amino acids, polyols, soluble sugars, etc. Proline is a natural amino acid with a non-polar cyclic pyrrolidine side chain with an amino group. It has the characteristics of small molecular weight and high water solubility (Hayat et al. 2012). Its unique ring structure determines that it can interact with water molecules on one side and macromolecules on the other side, and balance turgor pressure and relieve water stress by penetrating into cells ( et al. 2016). A large number of studies have shown that proline accumulation is an important metabolic adaptive mechanism for plants to resist stress (Khalid et al. 2022). Proline has shown great application prospects and development potential in improving plant stress resistance, pharmaceutical industry, drug therapy and regenerative medicine (Patriarca et al. 2021). At present, patents related to proline are mostly concentrated on its extraction and preparation, production and processing, disease prevention and treatment, and drug development and application. For example, a method for preparing DL-proline (CN111836800B), a method for purifying proline (CN101348453B), a method for producing L-proline (CN108841886B), a method for synthesizing D-proline (CN107827802B), a method for preparing functional amino acid proline (CN101830842B), a method for synthesizing proline-based polymers (CN113501946B), polymers based on hydroxyproline, preparation methods and depolymerization methods thereof (CN111621014B), a compound containing hydroxyproline, preparation method and application thereof (CN109761957B), a method for purifying L-proline by cooling crystallization (CN1 03265467B), application of proline and alanine in the prevention and treatment of wheat scab (CN113854302B), method, preparation and culture medium used for improving the effectiveness of yeast in inhibiting fruit diseases by inducing culture with L-proline (CN109628334B), application of proline in high temperature cultivation of shiitake mushrooms (CN111418443B), application of proline in the prevention and treatment of bee virus infection (CN111481541B), a method for improving the salt resistance of bluegrass using exogenous proline (CN103270894B), application of proline in the preparation of lung cancer treatment drugs (CN114028388A), proline derivatives and their use in the preparation of drugs for the treatment of cardiovascular and cerebrovascular diseases (CN110357800B), application of L-proline in improving early embryonic development and antioxidant capacity of oocytes (CN110205284B), etc. However, there are no reports on the application of proline in oocyte vitrification. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an application of L-proline in improving the post-freezing development rate of vitrified germinal vesicle stage oocytes, which can improve the development rate of parthenogenetic activated embryos of oocytes after thawing.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] Application of L-proline in improving the post-freezing development rate of vitrified germinal vesicle stage oocytes.

[0009] The invention discloses an oocyte vitrification freezing solution, which comprises 1×TCM199 at a final concentration, 20% FBS (fetal bovine serum), 10% EG (ethylene glycol), 7.5% DMSO (dimethyl sulfoxide), 0.4M sucrose and 0.4-1M L-proline.

[0010] A pretreatment method for improving the maturation and developmental ability of oocytes after vitrification and thawing, comprising placing oocytes in a freezing equilibrium solution for equilibrium, transferring them into a vitrification freezing solution containing L-proline for treatment for 30 seconds, and storing them in liquid nitrogen.

[0011] In the above-mentioned pretreatment method, preferably, the freezing balance solution has a final concentration of 1×TCM199, 20% FBS, 7.5% EG and 7.5% DMSO; further, the vitrification freezing solution has a final concentration of 1×TCM199, 20% FBS, 10% EG, 7.5% DMSO, 0.4M sucrose and 0.4-1M L-proline. In the above-mentioned pretreatment method, preferably, the oocyte is a porcine oocyte at the germinal vesicle stage.

[0012] The beneficial effects of the present invention are:

[0013] The present invention provides an application of L-proline in improving the post-freezing development rate of vitrified germinal vesicle stage oocytes, and also provides an application of L-proline in preparing oocyte vitrified freezing preparations, wherein the application can improve the embryonic development rate of parthenogenetic activation of oocytes after thawing.

[0014] (1) The present invention adds L-proline to porcine oocyte vitrification solution for the first time, thereby promoting the survival, in vitro maturation and in vitro developmental capacity of oocytes after freezing and thawing, and providing a new use of L-proline.

[0015] (2) The present invention also provides a pretreatment method for improving the maturation and developmental capacity of vitrified oocytes after thawing, which uses a vitrification freezing solution containing L-proline, wherein L-proline is a natural osmotic regulating substance that can significantly alleviate the osmotic damage of oocytes and significantly improve the maturation and developmental capacity of oocytes after thawing. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results of FDA staining of vitrified porcine germinal vesicle stage oocytes 2 hours after thawing.

[0017] Figure 2 This is the morphology of vitrified oocytes 42 hours after thawing and in vitro maturation.

[0018] Figure 3 This is the development of parthenogenetic activated embryos after thawing of vitrified oocytes. DETAILED DESCRIPTION

[0019] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all belong to the scope of the present invention.

[0020] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The ovaries used in the following examples were collected from the slaughterhouse of the Fifth Meat Processing Plant in Beijing. 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. In the following examples, % refers to mass percentage concentration.

[0021] Example 1

[0022] 1. Oocyte collection

[0023] Ovaries were collected from slaughterhouses, placed in sterile saline containing double antibodies at 28-32°C, and transported back to the laboratory within 2 hours. The ovaries were washed 2-3 times with sterile saline preheated to about 30°C, and follicles with a diameter of 2-6 mm on the ovaries were extracted using a 10mL disposable sterile syringe with an 18-gauge needle. The follicular fluid was collected in a 15ml sterile centrifuge tube and washed 3 times with TL-HEPES operating solution. After the follicular fluid was precipitated for 5-10 minutes, the bottom of the centrifuge tube was precipitated under a stereo microscope, and a mouth pipette was used to select germinal vesicle stage oocytes with uniform cytoplasm and more than 3 layers of dense cumulus cells, i.e., cumulus-oocyte complexes (COCs), for vitrification or in vitro maturation.

[0024] The formula of the TL-HEPES operating solution is 6.6622 g / L NaCl, 0.2386 g / L KCl, 0.168 g / L NaHCO 3 , 0.294 g / L CaCl 2 ·2H 2 O, 0.0463 g / L KH 2 PO 4 , 0.1017g / LMgCl 2 6H 2 O, 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, and 3 g / L BSA.

[0025] 2. Oocyte vitrification, thawing and in vitro maturation

[0026] (1) Oocyte vitrification

[0027] Take out the equilibration solution and vitrification solution in advance and restore them to room temperature. At room temperature, first place the COCs in the equilibration solution (ES) for 5 minutes, then transfer them to the vitrification solution (VS) for 30 seconds, immediately load them into the Cryotop carrier and put them into liquid nitrogen, put the carrier cap on them under liquid nitrogen, and transfer them to the liquid nitrogen tank for freezing.

[0028] The freezing equilibrium solution ES described in (1) is TCM199 (1×), 20% FBS, 7.5% EG and 7.5% DMSO; the vitrification freezing solution VS is TCM199 (1×), 20% FBS, 15% EG, 15% DMSO and 0.4 M sucrose (proline-free) or TCM199 (1×), 20% FBS, 10% EG, 7.5% DMSO, 0.4 M sucrose and L-proline (0.4, 1, 2 M), wherein 0.4 M L-proline is recorded as proline I, 1 M L-proline is recorded as proline II, and 2 M L-proline is recorded as proline proline III.

[0029] (2) Oocyte thawing and in vitro maturation

[0030] Take out the thawing solution in advance and place it in CO 2When thawing, the Cryotop straw was quickly taken out of the liquid nitrogen, and the end containing the oocyte was immediately immersed in the balanced preheated thawing solution I for 2 minutes. Then the oocytes were moved to thawing solution II, thawing solution III and thawing solution IV for 1 minute respectively, and then moved to thawing solution V for 3 minutes. Finally, after washing with in vitro maturation solution 3 times, one part was used for oocyte viability determination, and the other part was moved to a CO 2 The four-well plates (500 μL maturation solution, 100 COCs per well) were equilibrated in an incubator for at least 2 h at 38.5°C and 5% CO 2 , saturated humidity CO 2 Mature culture in incubator for 42 to 44 hours.

[0031] The thawing solution I described in (2) is TCM199 (1×), 20% FBS, and 0.4 M sucrose; the thawing solution II is TCM199 (1×), 20% FBS, and 0.2 M sucrose; the thawing solution III is TCM199 (1×), 20% FBS, and 0.1 M sucrose; the thawing solution IV is TCM199 (1×), 20% FBS, and 0.05 M sucrose; and the thawing solution V is TCM199 (1×), 10% FBS.

[0032] The formula of in vitro maturation fluid is: TCM199 (1×), 0.0001 g / mL L-cysteine, 10 ng / mL epidermal growth factor, 10% porcine follicular fluid, 10 IU / mL PMSG (Merck PG600), 5 IU / mL hCG (Merck PG600), 75 μg / mL penicillin, and 50 μg / mL streptomycin.

[0033] 3. Oocyte Viability Assessment

[0034] The oocytes were transferred to 5 μg / mL fluorescein diacetate (FDA) dye 2 h after thawing and 42 h after thawing and maturation, respectively, and incubated at 38.5°C in the dark for 5 min. They were then washed three times with 0.1% PVA / DPBS and images were collected under a fluorescence microscope. Figure 1 and Figure 2 Living oocytes produce bright green fluorescence, while dead oocytes do not emit light or fluoresce weakly.

[0035] 4. Parthenogenetic activation and embryo culture in vitro

[0036] Place COCs matured in vitro for 42-44 hours in 0.1% hyaluronidase, and use a pipette to blow gently and repeatedly to remove cumulus cells, and constantly observe the removal under a stereoscope. After most COCs are removed, transfer the oocytes to HN-23 operating solution, and count the discharge of the first polar body of the oocytes under a stereoscope. The results are shown in Figure 3 .

[0037] Preheat the activation solution in advance. Wash the oocytes that have expelled the first polar body three times with the activation solution, then transfer them to a fusion tank (electrode width 500μm, American BTX) covered with the activation solution, and use a 1.4kv / cm, 35μs DC pulse for electrical activation. After activation, move the oocytes into the chemical activation solution for chemical activation. After 4 to 6 hours, take out the oocytes, wash them three times with PZM3 culture solution, and then move them into PZM-3 culture droplets at 38.5℃, 5% CO 2 , and continued to culture under saturated humidity. The cleavage rate and blastocyst rate were counted at 48h and 168h respectively.

[0038] The formula of the HN-23 operating fluid is 7.6966 g / L NaCl, 0.168 g / L NaHCO 3 , 0.356g / LKC1, 0.162g / L KH 2 PO 4 , 0.293 g / L MgSO 4 7H 2 O, 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.

[0039] The activation solution is formulated as 0.25 mM mannitol, 0.1 mM CaCl 2 ·2H 2 O, 0.1 mM MgCl 2 6H 2 O, 0.5mM HEPES, 0.01% PVA (w / v).

[0040] The formula of PZM-3 culture medium is 6.312g / L NaCl, 2.106g / L NaHCO 3 , 0.746g / LKCl, 0.048g / LKH 2 PO 4 , 0.098 g / L MgSO 4 7H 2O, 0.022 g / L sodium pyruvate, 0.616 g / L hemicalcium lactate, 0.146 g / L L-glutamine, 0.066 g / L penicillin, 0.05 g / L streptomycin, 1 mL phenol red, 0.546 g / L sodium hypotaurine, 50× essential amino acids, 100× non-essential amino acids, and 3 g / L BSA.

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

[0042] 5. Data Analysis

[0043] Each experiment was repeated at least three times. SPSS software was used to perform one-way analysis of variance (ANOVA) and Duncan's test to determine the significance of the differences between different treatments, and P < 0.05 was considered significant.

[0044] 6. Conclusion

[0045] 1) 0.4 M (proline I), 1 M (proline II) and 2 M (proline III) L-proline were selected to optimize the vitrification solution of porcine GV stage oocytes. FDA staining was performed 2 h after thawing and 42 h after thawing and maturation. The results of statistical oocyte survival rate are shown in Tables 1 and 2.

[0046] Table 1 Effect of L-proline on the survival of porcine GV oocytes 2h after vitrification and thawing

[0047]

[0048] Note: Oocyte survival rate = number of surviving oocytes × 100 / total number of oocytes; Different letters in the same column (a, b) represent significant differences (P < 0.05).

[0049] As shown in Table 1, the survival rate of oocytes in the proline I and proline II groups 2 hours after thawing was not significantly different from that in the proline-free group, but was significantly higher than that in the proline III group 2 hours after thawing (P < 0.05).

[0050] Table 2 Effect of L-proline on the survival of porcine GV oocytes 42 hours after vitrification and thawing

[0051]

[0052]

[0053] Note: Oocyte survival rate = number of surviving oocytes × 100 / total number of oocytes; Different letters in the same column (a-d) represent significant differences (P < 0.05).

[0054] After 42 hours of in vitro maturation of the thawed oocytes (see Table 2), the survival rate of oocytes in the proline I group was significantly higher than that in the proline-free group (P < 0.05), and there was no significant difference in the survival rate of oocytes in the proline II and proline III groups compared with the proline-free group (P>0.05), but the survival rate of oocytes in the frozen group was significantly lower than that in the fresh group (P < 0.05).

[0055] The above results show that L-proline significantly improves the survival rate of porcine GV stage oocytes 42 hours after vitrification and thawing.

[0056] 2) The effect of L-proline on in vitro maturation of oocytes after vitrification and thawing was analyzed by counting the first polar body extrusion rate of oocytes after vitrification and thawing. The results are shown in Table 3.

[0057] Table 3 Effect of L-proline on the first polar body extrusion rate of porcine GV stage oocytes after vitrification and thawing

[0058]

[0059] Note: First polar body extrusion rate = number of oocytes extruding the first polar body × 100 / number of oocytes; Different letters in the same column (a-d) represent significant differences (P < 0.05).

[0060] Table 3 shows that the oocyte maturation rate of the frozen group was significantly lower than that of the fresh group, but the first polar body extrusion rate of the oocytes in the proline I group was significantly higher than that of the other three frozen groups (P < 0.05). There was no significant difference in the first polar body extrusion rate of the oocytes in the proline II group and the proline-free group, but both were significantly higher than the oocyte maturation rate in the proline III group (P < 0.05).

[0061] The results showed that L-proline significantly improved the in vitro maturation rate of porcine GV stage oocytes after vitrified thawing.

[0062] 3) The effect of L-proline on the development of parthenogenetically activated embryos from frozen oocytes was analyzed by statistically analyzing the cleavage rate and blastocyst rate of parthenogenetically activated embryos. The results are shown in Table 4.

[0063] Table 4 Effect of L-proline on the development of parthenogenetic activated embryos in porcine GV stage oocytes after vitrification and thawing

[0064]

[0065] The cleavage rate and blastocyst rate of parthenogenetically activated embryos in the frozen group were significantly lower than those in the fresh group (P<0.05). Compared with the proline-free group, the blastocyst rate of parthenogenetically activated embryos in the proline I group was significantly increased (P<0.05), while there was no significant difference in the cleavage rate (P>0.05). The cleavage rate of parthenogenetically activated embryos in the proline II and proline III groups was not significantly different from that in the proline-free group, but the blastocyst rate of parthenogenetically activated embryos in the proline II and proline III groups was significantly lower than that in the proline-free group (P<0.05). The results showed that L-proline significantly increased the parthenogenetically activated development rate of porcine GV stage oocytes after vitrified freezing and thawing.

[0066] From the above results, it can be seen that 0.4M L-proline can significantly improve the vitrification and thawing effect of porcine GV stage oocytes, and has potential application value in the development of porcine oocyte vitrification freezing solution.

Claims

1. A vitrification freezing solution for improving the embryonic development rate of parthenogenetic activation of thawed porcine oocytes, It is characterized in that It includes final concentrations of 1×TCM199, 20% FBS, 10% EG, 7.5% DMSO, 0.4 M sucrose and 0.4 M L-proline.

2. A pretreatment method to improve the maturation and developmental capacity of vitrified thawed oocytes, It is characterized in that The method comprises placing the oocyte in a freezing equilibrium solution for equilibrium, transferring the oocyte into a vitrification freezing solution containing L-proline for 30 seconds, and storing the oocyte in liquid nitrogen; the freezing equilibrium solution has a final concentration of 1×TCM199, 20% FBS, 7.5% EG and 7.5% DMSO; The vitrification freezing medium has a final concentration of 1×TCM199, 20% FBS, 10% EG, 7.5% DMSO, 0.4 M sucrose and 0.4 M L-proline.

3. The pre-treatment method according to claim 2, It is characterized in that The oocyte is a pig oocyte in the germinal vesicle stage.

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