A bovine oocyte cryoprotectant and its application

Abstract

The present invention relates to a bovine oocyte cryoprotectant and application thereof, belonging to the technical field of cell freezing. The method comprises: firstly, immersing the bovine oocyte in a vitrification equilibration solution, transferring the bovine oocyte to a vitrification freezing solution containing CspB protein but not containing DMSO, utilizing the CspB protein to inhibit ice crystals and reduce oxidative damage to the oocyte; then, conventionally cryopreserving the treated oocyte; after thawing, the survival rate of the oocyte is significantly improved, avoiding the toxicity problem caused by DMSO to the oocyte, and exhibiting better cell morphology while showing higher developmental potential.

Description

Technical Field

[0001] The invention belongs to the field of oocyte freezing technology, and particularly relates to a bovine oocyte cryoprotectant and application thereof. Background Art

[0002] Oocyte cryopreservation is key to preserving fertility, but achieving high survival rates after thawing remains challenging due to inherent cell fragility. Currently, effective strategies for cell and tissue cryopreservation include slow freezing and vitrification. Compared with slow freezing, vitrification has faster freezing speed and less damage. DMSO is the most commonly used cryoprotectant (CPA), which inhibits ice nucleation by disrupting water hydrogen bonds. However, high concentrations of DMSO often exhibit cytotoxicity and may cause cell mutations or even death. These limitations highlight the urgent need for alternative cryoprotectants that can mitigate the physical and biochemical damage during vitrification. Therefore, there is an urgent need to find new cryoprotectants to improve traditional cryoprotectants and reduce their toxicity to cells.

[0003] Over the past two decades, the discovery of antifreeze proteins has provided new insights into the search for novel cryoprotectants. Studies have shown that incorporating antifreeze glycoproteins into vitrification solutions significantly improves the morphological integrity of mouse oocytes and two-cell embryos, while adding type III antifreeze proteins to vitrification solutions enhances the survival and developmental rates of mouse oocytes. Despite the potential of antifreeze proteins, their complex structure and high production costs have hindered their clinical application. This has sparked interest in simpler cold-acclimation agents, such as cold shock proteins (CSPs). CSPs are ubiquitous in prokaryotes and eukaryotes, playing key roles in adapting to environmental temperature fluctuations and binding nucleic acids. Researchers at Sejong University in South Korea discovered a psychrotrophic bacterium, KOPRI 22228, in the Arctic. This strain possesses a cold shock protein, CspB, with a disrupted N-terminal domain, which confers remarkable cryoprotective properties. However, whether CspB could replace DMSO for oocyte vitrification by simultaneously addressing ice crystallization and oxidative stress remains unexplored.

[0004] Cryopreservation of biological materials is primarily concerned with physical damage, chemical damage, and various cellular and molecular damages caused by freeze-thaw cycles. Physical damage frequently occurs, primarily due to mechanical disruption of oocyte membranes and subcellular structures by ice crystal nucleation and recrystallization during freeze-thaw cycles. These damages may lead to loss of biological activity in cells or tissues, posing a major challenge to the effective development of cryopreservation techniques. Meanwhile, osmotic shock and cryoprotectant toxicity can elevate reactive oxygen species (ROS), triggering mitochondrial dysfunction, caspase-3 activation, and lipid peroxidation. We hypothesized that CspB has a dual cryoprotective effect: (1) directly inhibiting ice crystal growth by binding to the ice surface, and (2) alleviating oxidative stress by regulating antioxidant pathways. Summary of the Invention

[0005] In response to the toxicity problem caused by DMSO to oocytes, the present invention aims to find a substitute for DMSO. The protective agent is a solution that solves the toxicity problem of oocytes after DMSO cryopreservation by adding CspB protein to the vitrification freezing solution.

[0006] In view of this, the present invention mainly provides the following technical solutions:

[0007] In a first aspect, the present invention provides a bovine oocyte cryoprotectant, wherein the cryoprotectant comprises CspB protein and DMSO-free cryoprotectant;

[0008] Furthermore, the bovine oocyte is a bovine MII stage oocyte;

[0009] Furthermore, in the oocyte cryoprotectant, the concentration of CspB protein used is 1-3 mg / mL.

[0010] Furthermore, the cryoprotectant includes 10~20% (v / v) ethylene glycol, 1~3 mg / mL CspB protein, 10~30% (v / v) fetal bovine serum, 1~3% (v / v) 10 mg / mL penicillin-streptomycin, and 15~20% (w / v) sucrose.

[0011] Furthermore, the cryoprotectant is 15% (v / v) ethylene glycol, 2 mg / mL CspB protein, 20% (v / v) fetal bovine serum, 1% (v / v) 10 mg / mL penicillin-streptomycin, and 17% (w / v) sucrose.

[0012] A second aspect of the present invention is to provide a method for improving the survival rate of bovine oocytes during freezing, the method comprising:

[0013] 1) placing the bovine oocyte in a cryoprotectant containing the cryoprotectant described in the first aspect;

[0014] 2) performing conventional cryopreservation on the processed bovine oocytes;

[0015] Furthermore, the bovine oocyte is a bovine MII stage oocyte;

[0016] Furthermore, before placing the cells in the cryoprotectant, the cells need to be equilibrated in cryoprotectant solution I for 4-7 minutes and in cryoprotectant solution II for 10-25 seconds; wherein cryoprotectant solution I contains 7-8% (v / v) ethylene glycol, 7-8% (v / v) DMSO, 15-25% (v / v) fetal bovine serum, 0.8-1.2% (v / v) penicillin-streptomycin, and 60-68% (v / v) PBS; and cryoprotectant solution II contains 10-20% (v / v) ethylene glycol, 10-20% (v / v) DMSO, 15-25% (v / v) 10 mg / mL penicillin-streptomycin, and 15-20% (w / v) sucrose.

[0017] Furthermore, the cryoprotectant I is 7.5% (v / v) ethylene glycol, 7.5% (v / v) DMSO, 20% (v / v) fetal bovine serum, 1% (v / v) penicillin-streptomycin, and 64% (v / v) PBS; the cryoprotectant II is 15% (v / v) ethylene glycol, 15% DMSO, 20% (v / v) fetal bovine serum, 1% (v / v) 10 mg / mL penicillin-streptomycin, and 17% sucrose (w / v).

[0018] The third aspect of the present invention is to provide the use of CspB protein in preparing a preparation for improving the cryopreservation survival rate of bovine oocytes, wherein the CspB protein replaces DMSO;

[0019] Furthermore, the concentration of CspB is 1-3 mg / mL, preferably 2 mg / mL.

[0020] The beneficial effects of the present invention include:

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] (1) The present invention discovered that immersing non-human animal oocytes in a cryoprotectant containing CspB protein can inhibit ice crystal formation during the freezing and thawing process of oocytes;

[0023] (2) The treated oocytes were subjected to conventional cryopreservation treatment; after thawing, the survival rate of the oocytes was significantly improved, and they showed better cell morphology and reduced cell damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of the ice crystal recrystallization experiment of CspB protein solution showed that at 0, 30 and 60 minutes, the ice crystal size of 2 mg / mL was significantly smaller than that of 0 mg / mL and 1 mg / mL.

[0025] Figure 2 When CspB protein replaced DMSO in vitrification solution, the freezing survival rate of bovine oocytes had no significant difference from that of the 15% CPA group, but was significantly higher than that of the 15% EG group.

[0026] Figure 3 When CspB protein replaced DMSO in vitrification medium, the cleavage and blastocyst rates of bovine oocytes remained similar to those in the 15% CPA group, but were significantly higher than those in the 15% EG group (A, B). CspB protein increased ROS and DHE levels in post-thaw oocytes and promoted the expression of the antioxidant enzyme GSH (C, D). CspB protein also increased mitochondrial membrane potential (E, F) and mitochondrial content (G, H) in post-thaw oocytes. CspB protein also decreased Caspase 3 (I, J) and Caspase 9 levels (K, L) in post-thaw oocytes. DETAILED DESCRIPTION

[0027] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods described are all conventional methods unless otherwise specified. The materials described can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute undue limitations of the present invention. It should be noted that, unless there is a conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0028] The CspB protein in this example was constructed by plasmid in our laboratory and expressed and purified. The amino acid sequence is:

[0029] MAKSQQTFSKSEKEKKRLKKRQDKQKKMDARKADRDEHGPSGIQFAYVDHNGNLTDTPPDPELKVEYELEDIQISVSKKEDLPEEDPVRKGKVSFFDSDKGFGFIIDMENNEKYFTHVSGLIDDIVENDKVSFELERGMRGMNAVKVTKI(SEQ ID NO:1)

[0030] Unless otherwise stated, all chemicals used in this example were purchased from Sigma-Aldrich (St. Louis, USA).

[0031] The results of the following examples were statistically processed as follows: data obtained from three replicates were reported as mean ± standard error (SEMs). Statistical analysis was performed using SPSS version 26.0 software (SPSS, Chicago, IL, USA) using one-way analysis of variance (ANOVA) and independent sample t-test. GraphPad Prism software package (version 6.01; GraphPad, La Jolla, CA, USA) was used. Mean immunofluorescence intensity was measured and analyzed using Image J software. All statistical tests were performed using P The significance level was <0.05.

[0032] All animal experiments were performed in accordance with the corresponding animal use guidelines approved by the Animal Care and Use Committee of Jilin Agricultural University.

[0033] Example 1 Effect of CspB protein on the survival rate of bovine oocytes during vitrification

[0034] 1. CspB protein ice crystal recrystallization

[0035] Different concentrations of cold shock protein were added to normal saline and observed by the clip recrystallization method. 6 μL of sample was prepared into clips, and then normal crystallization was performed. The quenching temperature was -80°C and the aging temperature was -20°C. Finally, observation was made under a cryomicroscope. The results showed that at 0 min, the ice crystal size of the 2 mg / mL CspB protein solution was significantly reduced (p < 0.001) ( Figure 1 A, B).

[0036] 2. Collection and culture of bovine oocytes

[0037] Source of Bovine Oocytes: Bovine ovaries were collected from a local slaughterhouse and transported to the laboratory. Within 4 hours, the ovaries were stored in a thermos containing sterile saline. In the laboratory, the ovaries were washed thoroughly with heated saline, and bovine follicular fluid was aspirated using a syringe. Cumulus cell complexes (COCs) were selected from the follicular fluid under a microscope using a mouth pipette. COCs encasing three or more layers of cumulus granulosa cells were selected. At this stage, the oocytes were at the GV stage and washed three times in oocyte selection medium before being transferred to in vitro oocyte maturation medium. After incubation for 23 hours in a 38.5°C incubator containing 5% CO₂, the oocytes matured to the MII stage, with distinct polar bodies visible under a microscope. Mature oocytes were selected for subsequent experiments.

[0038] 3. Detection of vitrification survival rate

[0039] Oocyte vitrification uses a two-step loading of cryoprotectants.

[0040] First, oocytes were placed in cryoprotectant solution I containing 7.5% (v / v) EG, 7.5% (v / v) DMSO, 20% (v / v) FBS, 1% (v / v) 10 mg / mL PS, and 64% (v / v) PBS for equilibration for 5 minutes.

[0041] Then, the oocytes were transferred into cryoprotectant solution II containing 15% (v / v) EG, 15% (v / v) DMSO, 20% (v / v) FBS, 1% (v / v) 10 mg / mL PS, and 17% (w / v) sucrose and stored at room temperature for 30 seconds.

[0042] The experiment was divided into 3 groups:

[0043] Control group (15% CPA),

[0044] 15% EG group

[0045] 15% EG + 2 mg / mL CspB protein group (DMSO was removed from cryoprotectant solution II and 2 mg / mL CspB protein was added).

[0046] Oocytes were rapidly loaded onto a cryotop (Kato, Japan) and plunged into liquid nitrogen. Half an hour later, they were thawed using a vitrified thawing kit (VT102, Kato, Japan), which contains thawing solution (TS), diluent (DS), washing solution 1 (WS1), and washing solution 2 (WS2). Thawing solution was preheated to 37°C, and the remaining solutions were kept at room temperature. Thawing was performed on a 37°C hot plate, with each step lasting 1 minute in TS, 3 minutes in DS, 5 minutes in WS1, and 5 minutes in WS2. The oocytes were then incubated again. After 2 hours of incubation in a 37.5°C, 5% CO2 incubator, the appearance of the oocyte cytoplasm and plasma membrane was observed under a stereomicroscope to assess oocyte survival. Oocytes with a clear zona pellucida, small perivitelline spaces, and normal size and color were considered viable.

[0047] The results showed that the freezing survival rate of oocytes in the CspB-added group was not significantly different from that in the control group, but was significantly higher than that in the 15% EG group. Control: 85.9%±1.48% VS 15% EG: 45.3%±2.88% VS 15% EG + 2mg / mL CspB: 87.2%±2.31% ( Figure 2 ).

[0048] In order to evaluate the effect of CspB protein on oocyte development, bovine oocytes were parthenogenetically activated. The frozen bovine oocytes were transferred into parthenogenetic activation medium. The activation medium was Ca 2+ -free CZB, supplemented with 1 mol / L SrCl2 and 1 mg / mL Cytochalasin B, cultured in parthenogenetic initiation medium for 6 h, transferred to KSOM embryo culture medium, washed three times, transferred to KSOM culture medium, and cultured at 37.5°C, 5% CO2, and 100% humidity. The cleavage rate and blastocyst rate were calculated 24 h and 96 h after activation.

[0049] The results showed that the freezing survival rate of oocytes in the CspB-added group was not significantly different from that in the control group, but was significantly higher than that in the 15% EG group. The cleavage rate was Control: 47.3%±2.88% VS 15% EG: 22.7%±2.74% VS 15% EG + 2mg / mL CspB: 46.1%±2.31% ( Figure 3 AB). Blastocyst rate Control: 19.5%±3.77% VS 15% EG: 3.1%±2.99% VS 15% EG + 2mg / mL CspB: 19.1%±3.64% ( Figure 3 AB).

[0050] 4. Measurement of ΔΨm, DHE, ROS, and GSH Levels

[0051] To assess ΔΨm, oocytes were incubated with 2 μM JC-1 (Biyuntian, China) at 37°C in the dark for 30 minutes. ΔΨm in oocytes was assessed using red fluorescence intensity / green fluorescence intensity and analyzed using ImageJ software. Fluorescence intensity in the resulting oocytes was analyzed using a fluorescence microscope (Olympus, Tokyo, Japan). Reactive oxygen species were detected using the fluorescent probe DCFH-DA (Biyuntian, China). Oocytes were incubated in PBS-PVP containing 10 μM DCFH-DA for 15 minutes at 37°C in the dark. Oocytes were then stained with CMF2HC (Invitrogen, C12881, USA). Oocytes were incubated in PBS-PVP containing 10 μM CMF2HC dye for 15 minutes at 37°C in the dark, and fluorescence intensity was observed under a fluorescence microscope (Olympus, Tokyo, Japan).

[0052] The results are as follows Figure 3 As shown in the results, the CspB protein can significantly increase the mitochondrial membrane potential of thawed oocytes compared with the control group ( Figure 3EH), indicating that CspB protein significantly improved mitochondrial dysfunction caused by vitrification. In addition, CspB protein can significantly reduce the levels of ROS and DHE and promote the expression of antioxidant enzyme GSH ( Figure 3 C, D), indicating that CspB protein significantly improved the oxidative stress induced by vitrification.

[0053] 5. Immunofluorescence

[0054] Oocytes were washed three times in PBS-PVP containing 0.1% dapoxetine (PBS-PVP), fixed in 4% formaldehyde for 1 hour, and permeabilized with 0.2% Triton X-100 in PBS-PVP for 30 minutes at room temperature. Oocytes were then blocked in PBS-PVP containing 3% BSA for 2 hours. Next, oocytes were incubated with monoclonal antibodies against caspase 3 and caspase 9 (1:100, Fitzgerald, USA) at 4°C overnight, followed by incubation with goat anti-rabbit IgG (H+L) fluor488-conjugated antibody (1:200, Invitrogen, USA) at 37°C for 2 hours. DAPI (Saiwei, China) was used for DNA staining. Fluorescence intensity of oocytes was observed using a fluorescence microscope (Olympus Tokyo, Japan).

[0055] The results are as follows Figure 3 As shown in Figure 3, CspB protein significantly reduced vitrification-induced oocyte apoptosis compared with the control group ( Figure 3 IL), suggesting that CspB protein significantly improved vitrification-induced oocyte apoptosis and improved the quality of cryopreserved bovine oocytes.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

Claims

1. A bovine oocyte cryoprotectant, characterized in that The cryoprotectant comprises CspB protein and DMSO-free cryoprotectant solution.

2. The bovine oocyte cryoprotectant according to claim 1, wherein The bovine oocyte is a bovine MII stage oocyte.

3. The bovine oocyte cryoprotectant according to claim 2, wherein In the bovine oocyte cryoprotectant, the concentration of CspB protein is 1-3 mg / mL.

4. The bovine oocyte cryoprotectant according to claim 1, characterized in that The bovine oocyte cryoprotectant comprises 10-20% v / v ethylene glycol, 1-3 mg / mL CspB protein, 10-30% v / v fetal bovine serum, 1-3% v / v 10 mg / mL penicillin-streptomycin, and 15-20% w / v sucrose.

5. The bovine oocyte cryoprotectant according to claim 1, characterized in that The cryoprotectant is 15% v / v ethylene glycol, 2 mg / mL CspB protein, 20% v / v fetal bovine serum (Fetal Bovine Serum), 1% v / v 10 mg / mL penicillin-streptomycin, and 17% w / v sucrose.

6. A method for improving the survival rate of bovine oocytes during freezing, characterized in that: The method includes: 1) placing bovine oocytes in a cryoprotectant according to any one of claims 1 to 5; 2) The processed bovine oocytes are subjected to conventional cryopreservation treatment.

7. The method according to claim 6, characterized in that The bovine oocyte is a bovine MII stage oocyte.

8. The method according to claim 6, characterized in that Before placing the cells in the cryoprotectant, the cells need to be equilibrated in cryoprotectant solution I for 4-7 minutes and then placed in cryoprotectant solution II for 10-25 seconds; wherein cryoprotectant solution I contains 7-8% v / v ethylene glycol, 7-8% v / v DMSO, 15-25% v / v fetal bovine serum, 0.8-1.2% v / v 10 mg / mL penicillin-streptomycin, and 60-68% v / v PBS; the cryoprotectant solution II contains 10-20% v / v ethylene glycol, 10-20% v / v DMSO, 15-25% v / v fetal bovine serum, 0.8-1.2% v / v 10 mg / mL penicillin-streptomycin, and 15-20% w / v sucrose.

9. The method according to claim 8, characterized in that The cryoprotectant I is 7% v / v ethylene glycol, 7.5% v / v DMSO, 20% v / v fetal bovine serum, 1% v / v 10 mg / mL penicillin-streptomycin, and 64% v / v PBS; the cryoprotectant II is 15% v / v ethylene glycol, 15% DMSO, 20% v / v fetal bovine serum, 1% v / v 10 mg / mL penicillin-streptomycin, and 17% w / v sucrose.

10. Use of CspB protein in preparing a preparation for improving the survival rate of bovine oocytes during cryopreservation, characterized in that: The CspB protein replaces DMSO, and the concentration of CspB is 1-3 mg / mL.

Images