CN114847274A - Oocyte cryopreservation reagent and application thereof - Google Patents

Abstract

The invention discloses a cryopreservation reagent for oocyte and application thereof, relating to the technical field of reproductive engineering. The invention provides a cryopreservation solution for oocyte, which comprises a freezing equilibrium solution and a vitrified refrigerating solution, wherein both the freezing equilibrium solution and the vitrified refrigerating solution contain procyanidine B2. The invention also provides a thawing solution for cryopreservation of the oocyte, wherein the thawing solution is PBS (phosphate buffer solution) containing procyanidine B2. The invention also provides a recovery liquid after the oocyte is unfrozen. The procyanidine B2(PCB2) is added into the cryopreservation liquid, the unfreezing liquid and the recovery liquid, so that the cryopreservation damage of oocytes can be reduced, and the effects of improving the survival rate after unfreezing, improving the mitochondrial function and Ca are shown ²⁺ The stable state is maintained, the oxidative stress level is reduced, the cortical tone is restored, the non-integral rate is reduced, the normal promotion of the meiosis process is realized, and the procyanidine B2 can participate in the sugar metabolism regulation and control, so that the subsequent development capability of the frozen oocyte is improved.

Description

Oocyte cryopreservation reagent and application thereof

Technical Field

The invention relates to the technical field of reproductive engineering, in particular to a cryopreservation reagent for oocyte and application thereof.

Background

The oocyte freezing technology is an important reproductive biological technology and is widely applied to germplasm resource preservation, fertility preservation and auxiliary reproduction technologies, but the thawed oocytes have the problems of reduced development potential and the like, and the wide application of the technology is severely restricted by the fact that 20 mature oocytes can be frozen to obtain birth offspring, so that the improvement of the utilization efficiency of the frozen oocytes is a problem to be solved urgently.

The challenges faced in cryopreservation of oocytes include their relatively large size, high water content, unique chromosomal alignment and meiotic spindle properties that make oocytes particularly vulnerable to ice crystal formation during freezing and thawing, and damage to oocytes from freezing stress includes the following:

1. freezing affects oocyte energy supply

The low temperature induced damage mainly includes ATP depletion and reduced enzymatic activity, resulting in an imbalance in metabolism and redox. This is because although mitochondrial production of ATP is significantly reduced at low temperatures, maintaining the basic cellular process still requires constant production of bioenergy, which results in relatively rapid depletion of intracellular ATP. Studies have shown that about 95% of ATP can be hydrolyzed to adenosine monophosphate within 4 hours at 0-4 ℃. The reduced production and rapid depletion of ATP can lead to a series of cellular deleterious events, including significantly reduced high energy phosphate storage levels, membrane depolarization, and disruption of membrane lipid bilayer integrity and cytoskeletal structure, which ultimately lead to disorganization of ion homeostasis, leading to cell swelling and loss of cell viability.

During cryopreservation of oocytes, freezing/thawing also has a significant negative effect on ATP concentration. Slow freezing and vitrification freezing research of oocytes after ovulation of rabbits shows that ATP level of frozen oocytes is obviously lower than that of fresh oocytes, and is irrelevant to a method adopted by freezing. This result was also confirmed in cryopreservation of oocytes from human, mouse, pig and cow.

2. Freezing to affect oocyte bone

Cryopreservation has been shown to induce plasma membrane and cytoskeleton collapse leading to loss of barrier function and abnormalities in ion homeostasis and metabolite supply, thereby accelerating cell death. The interaction between cellular lipids and cytoskeletal components is complex, and hardening of these lipids can lead to deformation and destruction of the cytoskeleton, which can negatively affect cell survival and development, and can also be an intrinsic cause of dehydration and cell morphological changes during cryopreservation.

In oocytes, the cytoskeleton is important for the development and fertilization of the oocyte. Microwires and microtubules, which are the major components of cytoskeleton, are extremely sensitive to low temperatures, and cryopreservation causes depolymerization of microtubules and disorganized distribution of microwires. It has been shown that vitrification can cause the depolymerization of oocyte microtubules, which in turn affects the structure and function of the spindle. The network structure of cortical microfilaments can also be abnormal during cryopreservation, which can induce misarrangement of oocytes chromosomes and eventually form aneuploidy.

In order to solve the problems of cytoskeletal abnormality caused by freezing, etc., some studies have been conducted to cryopreserve cells using membrane stabilizers (e.g., antifreeze proteins and hydrogels). The anti-freeze glycoproteins found in the sera of polar fish and some insects were first used in 1992 for cryopreservation of bovine oocytes and were shown to significantly improve the viability and bioactivity of oocytes after cryopreservation. The L-proline oligomer with polyproline II helix structure the same as that of antifreeze glycoprotein has also been shown to have strong ice growth inhibiting activity, preventing direct damage of large ice crystals to oocytes and increase of osmotic pressure.

3. Freeze-induced oxidative stress of oocytes

Under physiological conditions, the endogenous cellular antioxidant defense system scavenges harmful reactive oxygen species to maintain intracellular redox balance. Oxidative stress is an imbalance between oxidation and resistance to oxidation, which is caused by the production of Reactive Oxygen Species (ROS) in large quantities under extreme conditions. For example, as cryopreservation and warming processes are accompanied by a decrease in the antioxidant capacity of the cells, mitochondrial damage caused by low temperatures can further enhance ROS production, thereby inducing oxidative stress in the cells.

Oxidative stress during cryopreservation can also result from other mechanisms, such as osmotic stress (induced by ice nucleation and dehydration) and increased oxidative metabolism can induce damage to plasma membranes and organelles. The decrease in Glutathione (GSH) levels and the increase in hydrogen peroxide levels observed in porcine oocytes and embryos after vitrification freeze thawing indicate that cryopreservation compromised the antioxidant defense system of the porcine oocytes and embryos.

To mitigate the potential for oxidative damage during cryopreservation, some studies have added antioxidants to conventional cryopreservation solutions. Resveratrol, N-acetylcysteine, melatonin, etc. have been widely used in cryopreservation of oocytes/embryos of humans and livestock, and have a positive effect on the antioxidant defense system of cells.

4. Mechanism of freeze-induced cell damage

Ice crystals, i.e. frozen water, have been shown to be harmful to humans or animals on a macroscopic level and lethal to cells on a microscopic level. In cryobiology, the lethality of ice to cells has been a challenge limiting the development of cryopreservation of mammalian cells. Mazur first proposed the hypothesis of "two cryoinjury", including ice-induced osmotic injury and mechanical injury; in recent years, damage of ROS to cells has also been demonstrated during cryopreservation.

Disclosure of Invention

The invention aims to provide an oocyte cryopreservation reagent and application thereof, and aims to solve the problems in the prior art, procyanidine B2 is added into a cryopreservation solution and a thawing solution, so that frozen damage of frozen oocytes is reduced, mitochondrial functions are improved, internal quality of the frozen oocytes is improved, and a new method is provided for improving the cryopreservation effect of the oocytes.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a cryopreservation solution for oocyte, which comprises a freezing equilibrium solution and a vitrified refrigerating solution, wherein both the freezing equilibrium solution and the vitrified refrigerating solution contain procyanidine B2.

Further, the concentration of the procyanidin B2 in the frozen equilibrium liquid and the vitrified freezing liquid is 5 mug/mL.

Further, the frozen equilibrium liquid also contains dimethyl sulfoxide and glycol.

Further, the vitrification refrigerating fluid is PBS fluid containing glycol, dimethyl sulfoxide, sucrose and polysucrose.

The invention also provides a thawing solution for cryopreservation of the oocyte, wherein the thawing solution is PBS (phosphate buffer solution) containing procyanidine B2.

Further, the concentration of the procyanidin B2 in the thawing solution is 5 mug/mL.

The invention also provides a recovery liquid after the oocyte is unfrozen, and the recovery liquid is M2 liquid containing procyanidine B2.

Further, the concentration of the procyanidin B2 in the post-thaw recovery solution is 5 μ g/mL.

The invention also provides a cryopreservation method of the oocyte, which comprises the following steps: placing the oocyte in the freezing equilibrium liquid, transferring the oocyte to the vitrification freezing liquid after equilibrium treatment, then adding liquid nitrogen, and freezing and preserving; and during thawing, placing the cryopreserved oocytes in the thawing solution for thawing, and placing the thawed oocytes in the post-thawing recovery solution for recovery.

The invention also provides application of the procyanidine B2, the cryopreservation liquid, the thawing liquid or the post-thawing recovery liquid in oocyte cryopreservation.

Procyanidins belong to the group of polyphenols, also known as condensed tannins, and are oligomers or polymers of flavan-3-ols [ e.g. (-) -epicatechin or (+) -catechin ] linked by interferon bonds. Procyanidins, one of the most abundant phytochemicals in plants, is widely present in plants such as fruits, vegetables, grains, beans, and tea leaves. Normally, procyanidins containing only epicatechin are called procyanidins. The research finds that the procyanidine has wide regulation effect and can participate in regulation of lipid metabolism, epigenetics and the like, procyanidine B2(dimer procyanidinin B2[4,8' -BI- [ (+) -epicatechin ] ] (PCB2)) is the main procyanidine, and in view of the wide influence of freezing stress on oocytes, the procyanidine is supposed to be helpful for reducing freezing damage. The invention discloses a method for improving the cryopreservation effect of oocytes, which is characterized in that the conventional freezing and thawing reagents in the market are generally permeable and impermeable freezing protective agents, the moisture in the oocytes is replaced, and the formation of ice crystals in the freezing and thawing process is prevented to achieve the purpose of protecting the oocytes, and the addition of a spectrum freeze injury resistant preparation is lacked.

The invention discloses the following technical effects:

the procyanidine B2(PCB2) is added into the cryopreservation liquid and the thawing liquid, so that the cryopreservation damage of oocytes can be reduced, and the effects of improving the survival rate after thawing, improving the mitochondrial function and Ca are shown ²⁺ The stable state is maintained, the oxidative stress level is reduced, the cortical tone is restored, the non-integral rate is reduced, the normal promotion of the meiosis process is realized, and the procyanidine B2 can participate in the sugar metabolism regulation and control, so that the subsequent development capability of the frozen oocyte is improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a graph of the effect of varying concentrations of procyanidin B2 on the survival and development of vitrified frozen oocytes, where A is cleavage and blastogenesis of various groups of oocytes, scale: 100 μm; b is the survival rate of the thawed oocyte; c is the embryo cleavage rate after parthenogenetic activation; d is the blastocyst rate of the embryo after parthenogenetic activation; "n" represents the number of oocytes used in the experiment, all experiments were performed in at least three biological replicates and data are expressed as mean ± sem, × P <0.05, × P < 0.01, ns is no statistical difference;

FIG. 2 is a graph of the effect of procyanidins B2 on oxidative stress of vitrified frozen oocytes, where A is the ROS and GSH levels of each set of oocytes, scale: 100 μm; b, analyzing ROS fluorescence intensity in the oocyte; c, analyzing GSH fluorescence intensity in the oocyte; "n" represents the number of oocytes used in the experiment, all experiments were performed in at least three biological replicates and data are expressed as mean ± sem, × P <0.05, × P < 0.001;

FIG. 3 is a graph showing the effect of procyanidins B2 on the organelle distribution of vitrified frozen oocytes, wherein A is the mitochondrial distribution of each group of oocytes, scale: 50 μm; b is the abnormal distribution proportion of mitochondria of each group of oocytes; c is endoplasmic reticulum distribution of each group of oocytes, scale: 50 μm; d is the distribution ratio of abnormal endoplasmic reticulum of each group of oocytes; "n" represents the number of oocytes used in the experiment, all experiments were performed in at least three biological replicates and data are expressed as mean ± sem, × P <0.05, ns is no statistical difference;

FIG. 4 is a graph of the effect of procyanidins B2 on mitochondrial function of vitrified frozen oocytes, where A is the mitochondrial membrane potential of each group of oocytes, scale: 50 μm; b is the mitochondrial temperature of each group of oocytes, scale: 50 μm; c, analyzing the mitochondrial membrane potential level of the oocyte; d is analysis of the temperature level of mitochondria of the oocyte; e is mitochondrial fusion (Opa1, Mfn1, Mfn2) and division (Drp1) gene expression analysis; "n" represents the number of oocytes used in the experiment, all experiments were performed in at least three biological replicates and data are presented as mean ± sem, × P <0.05, × P <0.001, ns is no statistical difference;

FIG. 5 is a graph of the effect of procyanidin B2 on the calcium homeostasis of vitrified frozen oocytes, where A is the co-localization of specific calcium sensitive probes to organelles, scale: 50 μm; b is the level of cytoplasmic calcium, mitochondrial calcium and endoplasmic reticulum calcium for each set of oocytes, scale: 50 μm; c, analyzing the calcium level of oocyte cytoplasm; d is oocyte mitochondrial calcium level analysis; e is the analysis of the endoplasmic reticulum calcium level of the oocyte; "n" represents the number of oocytes used in the experiment, all experiments were performed in at least three biological replicates and data are expressed as mean ± sem, × P <0.001, ns is not statistically different;

FIG. 6 shows proanthocyanidin B2 for improving DNA damage of vitrified frozen oocytes, wherein A is analysis of the level of DNA damage in oocytes on a scale: 50 μm; b is the DNA damage level of the oocytes in each group; "n" represents the number of oocytes used in the experiment, all experiments were performed in at least three biological replicates and data are expressed as mean ± sem,. P < 0.01;

FIG. 7 is a graph of the effect of procyanidin B2 on the early apoptosis and autophagy of vitrified frozen oocytes, where A is the early apoptosis occurrence and autophagy immunofluorescence staining of various groups of oocytes on scale: 50 μm; b is the early apoptosis occurrence proportion in the oocyte; c is oocyte LC3 fluorescence intensity analysis; d is qPCR for detecting mRNA levels of Beclin1, Map1lc3a, Ulk1, Atg14, Lamp1 and Lamp 2; "n" represents the number of oocytes used in the experiment, all experiments were performed in at least three biological replicates and data are presented as mean ± sem, × P <0.05, × P < 0.01, × P <0.001, ns is no statistical difference;

fig. 8 is a cortical tone of procyanidin B2 remodeled frozen oocytes, where a is a representative picture of the pERM immunofluorescence staining of the various groups of oocytes, scale: 100 μm; b is a representative picture of pMRLC immunofluorescent staining of oocytes of each group, scale: 100 μm; c, analyzing the average fluorescence intensity of the pERMs of each group of oocytes; d is the average fluorescence intensity analysis of the oocyte pMRLC of each group; "n" represents the number of oocytes used in the experiment, all experiments were performed in at least three biological replicates and the data are expressed as mean. + -. standard error. P < 0.001;

figure 9 is the effect of cortical tone on mitochondrial function, where a is the detection of mitochondrial membrane potential by JC-1 staining, ConA, PCB2 and ML-7 were added to the recovery medium for 1 hour during thawing, scale: 50 μm; b is a representative image of mitochondrial distribution, oocytes were stained with Mito-Tracker Green, scale: 50 μm; c, analyzing mitochondrial membrane potential of each group of oocytes; d, analyzing the abnormal distribution proportion of mitochondria of each group of oocytes; e is the ATP level of each group of oocytes; "n" represents the number of oocytes used in the experiment, all experiments were performed in at least three biological replicates and data are presented as mean ± sem, × P <0.05, × P < 0.01, × P <0.001, ns is no statistical difference;

FIG. 10 is a graph of procyanidins B2 modulating frozen oocyte cortical tension via mitochondrial electron transport chain, where A is qPCR detection of mRNA levels of Ndufv1, Sdhb, Uqcrc2, Cox1 and Atp5a 1; b is the pERM immunofluorescent color of the mature oocytes, scale: 50 μm; c is the relative pERM fluorescence intensity analysis of each group of oocytes; d is mRNA level of Ndufv1 detected by qPCR; e is qPCR for mRNA levels of Atp5a 1; "n" represents the number of oocytes used in the experiment, all experiments were performed in at least three biological replicates and data are expressed as mean ± sem, × P <0.001, ns is not statistically different;

FIG. 11 is a graph of the effect of procyanidin B2 on the in vitro maturation of vitrified frozen oocytes, where A is the GVBD and PBE occurrence for each set of oocytes, scale: 100 μm; b is the GVBD proportion of the oocyte cultured in vitro for 2 hours; c is PBE proportion of oocytes cultured in vitro for 12 h; "n" represents the number of oocytes used in the experiment, all experiments were performed in at least three biological replicates and data are expressed as mean ± sem, × P <0.05, ns ═ no statistical difference;

fig. 12 is a graph of the effect of procyanidin B2 on frozen oocyte spindle positioning, where a is a representative picture of the set of oocyte spindle positioning, scale: 50 μm; b is the ratio of the distance (length, L) from the spindle pole to the cortex of the oocyte to the diameter (D) of the oocyte; c is a representative picture of actin of each group of oocytes, scale: 50 μm; d is the relative fluorescence intensity of F-actin; "n" represents the number of oocytes used in the experiment, all experiments were performed in at least three biological replicates and data are expressed as mean ± sem,. P < 0.01,. P < 0.001;

fig. 13 is a graph of procyanidins B2 reduced spindle anomaly ratio and aneuploidy rate, where a is a representative picture of spindle morphology for each set of oocytes, scale: 50 μm; b is a representative picture of oocyte euploidy and aneuploidy, scale: 50 μm; c, analyzing the proportion of spindle morphological abnormality of each group of oocytes; d is the proportional analysis of the aneuploidy rate of the oocytes of each group; "n" represents the number of oocytes used in the experiment, all experiments were performed in at least three biological replicates and data are expressed as mean ± sem, × P <0.05, × P < 0.01, × P < 0.001;

fig. 14 is a picture of proanthocyanidin B2 for improving the quality of frozen oocytes in parthenogenetic activation of blastocysts, wherein a is a representative picture of CDX2 and Nanog immunofluorescence staining of blastocysts of each group, and a scale is as follows: 50 μm; b, ICM and TE ratio analysis of each group of oocytes; c, carrying out ICM (ICM: total cell ratio analysis) on the oocytes of each group; "n" represents the number of oocytes used in the experiment, all experiments were performed in at least three biological replicates and data are expressed as mean ± sem, × P < 0.001;

fig. 15 is a schematic diagram of procyanidin B2 mainly involved in regulation of sugar metabolism during embryonic development, wherein a is a schematic diagram of targeted metabonomics sample collection; b is the relative content of L-hydroxyproline in different groups; c is the relative content of L-citrulline in different groups; d is the relative content of the L-kynurenic acid hydrate in different groups; e is the relative content of L-glutamic acid in different groups; f is the average content of arabinose in different groups; g is the average content of fructose in different groups; h is the average content of glucose in the different groups; i is the average content of maltose in the different groups; j is the average content of xylose in the different groups; all experiments were performed in at least three biological replicates. ns is not statistically different.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every intervening value, to the extent any stated value or intervening value in a stated range, and any other stated or intervening value in a stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1 application of procyanidins to MII stage oocyte freezing

First, experiment method

1.1 oocyte Collection

MII stage oocyte retrieval: taking 4-6w ICR mice, injecting 10IU of Pregnant Mare Serum Gonadotropin (PMSG) into the abdominal cavity, injecting 10IU of Human Chorionic Gonadotropin (HCG) into the abdominal cavity after 48h, killing the mice by cervical dislocation after injecting 12-14h of HCG, taking oviducts, scratching a large-swelling part under a dissecting mirror, obtaining cumulus oocyte complexes, and collecting MII-stage oocytes discharged from a first polar body for later use after treating hyaluronidase.

1.2 oocyte freezing

The oocyte vitrification freezing and thawing method refers to the conventional steps, and specifically comprises the following steps: the oocytes were placed in ED solution (frozen equilibrium solution), namely PBS solution containing 10% dimethyl sulfoxide and 10% ethylene glycol, for 30sec, and then transferred to EDFS30 solution (vitrified refrigerating solution), namely PBS solution containing 15% ethylene glycol, 15% dimethyl sulfoxide, 0.5M sucrose and 30% Ficoll (Ficoll), the oocytes were placed at the front end of a carrier rod, liquid nitrogen was added, and the time from the time when the oocytes entered EDFS30 solution to the time when the oocytes were added to liquid nitrogen was controlled within 25 sec. During thawing, the rod to be thawed is quickly taken out of liquid nitrogen, placed in thawing solution (PBS containing 0.5M sucrose) for 5min, and then treated with M2 solution (a modified Krebs-Ringer solution, containing sodium pyruvate 0.036g/L, MgSO. RTM., or PBS) to which the rod is placed ₄ ×7H ₂ O0.293 g/L, streptomycin 5.0g/L, KH ₂ PO ₄ 0.162G/L, 7.5G/L penicillin G, 0.356G/L KCl, 1.0G/L glucose, NaHCO ₃ 0.349g/L, NaCl5.533g/L, 4.349ml/L of 60% sodium lactate, 4.969g/L of Hepes, CaCl ₂ ×2H ₂ 0.252g/L of O and 4.0g/L of BSA4) for standby after being washed for three times.

Grouping oocytes: fresh control group oocyte (CT), vitrified frozen oocyte (VT), PCB2 processing group (PCB2-VT), PCB2-VT group are respectively added with 5 mug/mLPCB 2 in frozen preservation solution (ED solution and EDFS30 solution), thawing solution and post-thawing recovery solution, and the thawed oocyte is cultured in the recovery solution for 1h for standby.

The preparation method of the frozen equilibrium liquid comprises the following steps: adding 10% dimethyl sulfoxide (V: V) and 10% ethylene glycol (V: V) into the PBS solution to obtain ED solution, namely the frozen equilibrium solution.

The preparation method of the vitrified refrigerating fluid comprises the following steps: adding 15% dimethyl sulfoxide (V: V), 15% ethylene glycol (V: V), 0.5M sucrose and 30% Ficoll into PBS solution to obtain EDFS30 liquid, i.e. vitrified refrigerating liquid.

The preparation method of the recovery liquid after thawing comprises the following steps: to the M2 solution was added 5. mu.g/ml of PCB 2.

1.3 oocyte oxidative stress index detection

1.3.1ROS assay

ROS were measured using 2 ', 7' -dichlorofluorescein diacetate (DCHFDA), and oocytes were placed in 10mM drops of DCFHDA and incubated for 20min at 37 ℃ in an incubator. Oocytes were washed three times with DPBS containing 0.1% BSA (bovine serum albumin) and observed under a fluorescent microscope with 460nm excitation light. The experiment was repeated three times.

1.3.2GSH detection

GSH was measured using 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (4-chloromethyl-6,8-difluoro-7-hydroxycoumarin), oocytes were placed in 10. mu. mol/L droplets of GSH dye and incubated for 20min at 37 ℃ in an incubator. Oocytes were washed three times with DPBS containing 0.1% BSA and observed under a fluorescent microscope with excitation light of 370 nm. The experiment was repeated three times.

1.4 analysis of oocyte mitochondrial function

1.4.1 mitochondrial Membrane potential (. DELTA.. psi.m) detection

Mitochondrial membrane potential was detected using fluorescent probe JC-1. Each group of oocytes was collected, washed three times with DPBS containing 0.1% BSA and placed in 10mM JC-1 drops and incubated at 37 ℃ for 30min in an incubator. Oocytes were washed three times with DPBS containing 0.1% BSA and observed under a fluorescent microscope. The maximum excitation wavelength of the JC-1 monomer is 514nm, and the maximum emission wavelength is 529 nm; the maximum excitation wavelength of the polymer is 585nm, and the maximum emission wavelength is 590 nm. The experiment was repeated three times.

1.4.2ATP content detection

Preparing ATP standard product. And adding each group of oocytes into a PCR tube containing ATP lysate, adding ATP detection buffer solution after the lysis is finished, finally adding ATP detection premix solution, fully and uniformly mixing, incubating for 30min at room temperature in a dark place, and detecting by using a chemiluminescence apparatus. And (3) calculating the total ATP amount in each sample to be detected according to the standard substance with known concentration, and finally removing the number of the oocytes to obtain the ATP content in each egg. The experiment was repeated three times.

1.4.3 mitochondrial temperature detection

Mitochondrial temperature was detected using the Mito Thermo Yellow (MTY) probe. Firstly, diluting MTY to 0.5 mu M with M2 solution, preheating the disc in an incubator at 37 ℃ for 15min, and incubating the oocyte and MTY dye solution in the incubator at 37 ℃ for 15min in a dark place; washed 3 times with M2 and placed in M2 microdroplets for photographing under a confocal laser microscope.

1.5 mitochondrial and endoplasmic reticulum distribution detection

And (3) detecting the distribution of mitochondria: washing the oocyte for 3 times by using M2 for later use; oocyte mitochondrial distribution was detected using Mito-Tracker Red or Mito-Tracker Green probes. Firstly, diluting a probe to be 5 mu M by using M2 liquid, and preheating the probe in an incubator at 37 ℃ for 20 min; incubating the oocyte with a Mito-Tracker Red or Mito-Tracker Green dye solution in an incubator at 37 ℃ for 20min in the dark; washing with M2 for 3 times, incubating in preheated H33342 microdroplet, and incubating in 37 deg.C incubator for 10 min; washed 3 times with M2 solution, and placed in microdroplets for observation under a laser confocal microscope. The mitochondria are distributed in the cytoplasm of the oocyte normally; abnormal distribution is the case if mitochondria cluster or present an uneven distribution in the cytoplasm of the oocyte.

And (3) detecting endoplasmic reticulum distribution: washing the oocyte for 3 times by using M2 for later use; the ER-Tracker Red probe was used to detect the endoplasmic reticulum distribution in oocytes. Firstly, diluting a probe to be 5 mu M by using M2 liquid, and preheating the probe in an incubator at 37 ℃ for 20 min; incubating the oocyte and ER-Tracker Red staining solution in an incubator at 37 ℃ for 20min in the dark; m2 was washed 3 times and incubated in a preheated H33342 microdroplet at 37 ℃ for 10min, after incubation was completed, washed 3 times with M2, and placed in M2 microdroplet for photographing under a confocal laser microscope. Counting the distribution abnormality of endoplasmic reticulum: the endoplasmic reticulum is distributed in the cytoplasm of the oocyte in a normal distribution; an abnormal distribution is one in which the endoplasmic reticulum is clustered or shows an uneven distribution in the cytoplasm of the oocyte.

1.6 oocyte calcium content detection

Cytoplasmic calcium ion-specific probe Fluo 3-AM, mitochondrial calcium-specific probe Rhod 2-AM and endoplasmic reticulum calcium-specific probe Fluo 4-AM were used to detect cytoplasmic calcium, mitochondrial calcium and endoplasmic reticulum calcium levels, respectively, in oocytes. Firstly, diluting a probe to be 5 mu M by using M2 liquid, and preheating the probe in an incubator at 37 ℃ for 20 min; washing oocyte with M2 for 3 times, removing zona pellucida with pronase, and washing in M2 for 3 times; and (3) incubating the oocytes and calcium ion probe dye liquor in an incubator at 37 ℃ for 20min in a dark place, cleaning the incubated oocytes for 3 times by using M2, placing the oocytes in M2 microdroplets to take pictures under a laser confocal microscope, keeping the photographing parameters and exposure time consistent among all groups during photographing, and analyzing the fluorescence value by adopting NIS-Elements AR software.

1.7 detection of early apoptosis

Washing the oocyte for 3 times by using M2 for later use; the oocytes were incubated in 100. mu.L of buffer containing 5. mu.L of Lannexin-V FITC for 10min at 37 ℃ in an incubator; after incubation, washing with M2 for 3 times, placing in M2 microdroplets and taking pictures under an inverted fluorescence microscope; counting the occurrence of early apoptosis: the oocyte which does not undergo early apoptosis only has weak green fluorescence at the position of the zona pellucida; oocytes that undergo early apoptosis have green fluorescence at both the zona pellucida and at the membrane of the oocyte.

1.8RNA extraction, reverse transcription and real-time fluorescent quantitative PCR

Collecting oocytes, collecting the oocytes into a 1.5mL centrifuge tube according to 30-50 oocytes per group, absorbing redundant liquid, and storing in a refrigerator at-80 ℃; extracting oocyte RNA by Trizol; the reverse transcription was performed, the reverse transcription system is shown in Table 1, the reaction procedure is shown in Table 2, and after completion of the reverse transcription, the sample cDNA was stored in a refrigerator at-80 ℃ for later use. The reverse transcription cDNA sample is diluted into 5 cDNA templates with different concentrations according to a 10-fold continuous gradient dilution method, the primer efficiency is verified, the PCR reaction system is shown in Table 3, the primer sequence is shown in Table 4, and the qPCR program is shown in Table 5. And (3) observing a dissolution curve after the real-time fluorescent quantitative PCR reaction is finished, and if the dissolution curve is a single peak, determining that the result is credible. And taking the logarithm of the template concentration as an abscissa and the CT value as an ordinate to make a standard curve, and calculating the amplification efficiency of the primer according to the slope. Amplification efficiency of primers>90% and<110% of the primers were used in subsequent experiments. Beta-actin is used as an internal reference gene and 2 ^-△△Ct Respectively calculateRelative expression content of the gene of interest.

TABLE 1 reverse transcription System (20. mu.L)

Table 2 reverse transcription procedure:

table 3 qPCR reaction (20 μ L system):

TABLE 4 primer sequences

TABLE 5 fluorescent quantitative PCR reaction procedure

1.9 immunofluorescence staining

The oocyte is placed in a fixing solution and fixed for more than 1h at room temperature. If the zona pellucida is removed from the oocyte, diluting the fixing solution by using DPBS according to the proportion of 1:1, and then fixing the oocyte for 30 min; the fixed oocytes were washed with PBS-0.1% PVA lotion for 3 times and placed in 0.5% Triton-PBS-0.1% PVA permeation solution, and then the solution was allowed to permeate for 1 hour at room temperature. If the zona pellucida is removed from the oocyte, the oocyte is permeated for 20 min; washing the penetrated oocyte for 3 times by using 0.1% Triton-PBS-0.1% PVA washing liquid, then placing the oocyte in 3% BSA-0.1% Triton-PBS-0.1% PVA sealing liquid, and sealing for 1 hour at room temperature; directly placing the sealed oocyte in a prepared primary antibody, incubating overnight at 4 ℃ or incubating in an incubator at 37 ℃ for 2.5h, and replacing the primary antibody with sealing liquid or immunostaining primary antibody diluent in a negative control group; the oocytes after the primary antibody incubation are washed for 3 times by using 0.1% Triton-PBS-0.1% PVA washing liquor, and then placed in prepared secondary antibodies to be incubated for 1 hour at room temperature; after the secondary antibody incubation is finished, the oocytes are washed for 3 times by using 0.1% Triton-PBS-0.1% PVA washing liquor, and then the oocytes are stained with DAPI for 5 min; the oocytes after completion of staining were transferred to an adhesive slide and observed by pressing with a cover glass. If the oocytes were not pelleted, the oocytes after completion of the staining were washed 3 times with M2, placed in microdroplets on a glass-bottom petri dish and observed covered with paraffin oil. When photographing and observing by using an A1 Confocal laser Confocal microscope and the data needs to be counted on the fluorescence intensity, the imaging parameters and the exposure time between each group need to be controlled to be consistent, and the variables need to be controlled.

Second, experimental results

2.1 PCB2 can improve frozen oocyte survival and blastocyst development rate

The influence of different concentrations of PCB2(1 mug/mL, 5 mug/mL and 25 mug/mL) on the survival of frozen oocytes and the development of blastocysts is analyzed by adding different concentrations of PCB2 to the cryopreservation solution and the thawing solution respectively (FIG. 1A), and the result shows that 5 mug/mLPCB 2 can remarkably improve the survival rate of frozen oocytes (FIG. 1B), and remarkably improve the cleavage rate after parthenogenetic activation (P <0.05, FIG. 1C) and the development rate of blastocysts (P <0.05) (FIG. 1D).

2.2 PCB2 reduces oxidative stress in frozen oocytes

The ROS and GSH contents in fresh, frozen and PCB 2-treated frozen oocytes are respectively detected by adopting an immunofluorescence staining method (figure 2A), and the ROS level (P <0.05, figure 2B) of the frozen oocytes can be remarkably reduced by respectively adding 5 mu g/mL of PCB2 into a frozen preservation solution and a thawing solution, so that the GSH level in the frozen oocytes can be remarkably increased (P <0.001, figure 2C).

2.3 Effect of PCB2 on the distribution of frozen oocyte organelles

As shown in FIG. 3A, the oocytes in the fresh group exhibited a uniform distribution of cytoplasm, while the oocytes in the frozen group exhibited an abnormal distribution of clusters. After the treatment of the PCB2, the abnormal distribution ratio of the mitochondria is obviously reduced (P is less than 0.05, and the figure 3B). Distribution of endoplasmic reticulum as shown in fig. 3C, the abnormal distribution of endoplasmic reticulum was not statistically different between the different groups (P >0.05, fig. 3D).

2.4 PCB2 can improve the mitochondrial function of frozen oocytes

Mitochondrial membrane potential can intuitively reflect the functions of mitochondria, the detection of the mitochondrial membrane potential is shown in fig. 4A, and the PCB2 can obviously improve the membrane potential reduction of oocytes caused by vitrification freezing (P <0.001, fig. 4C).

MTY is a mitochondrial temperature specific probe whose fluorescence intensity is inversely proportional to mitochondrial temperature, and MTY is shown in FIG. 4B. The mitochondrial temperature of the oocytes increased after freezing, while supplementation with PCB2 significantly decreased the mitochondrial temperature (P <0.05, fig. 4D). Mitochondrial aggregation due to impaired mitochondrial dynamics has also been shown to impair mitochondrial function in oocytes, and mRNA levels of mitochondrial fusion (Opa1, Mfn1, Mfn2) and division (Drp1) genes were examined, respectively. The results show that the expression of Mfn1, Mfn2, Drp1 was significantly reduced in frozen oocytes compared to fresh group oocytes, while supplementation with PCB2 could significantly improve mitochondrial kinetics (fig. 4E).

2.5 Effect of PCB2 on calcium homeostasis of frozen oocytes

Rhod 2-AM and Fluo 4-AM are specific fluorescent probes for mitochondrial calcium and endoplasmic reticulum calcium, respectively, and the result of organelle co-localization shows that: rhod 2-AM co-localized with Mito tracker and Fluo 4-AM co localized with ER tracker (FIG. 5A). As shown in FIG. 5B, the detection of the levels of cytosolic calcium, mitochondrial calcium and endoplasmic reticulum calcium in oocytes, respectively, revealed that: vitrification did not have a significant effect on oocyte cytosolic calcium levels (P >0.05, fig. 5C), but freezing caused an abnormal elevation of oocyte mitochondrial calcium levels (fig. 5D), and supplementation with PCB2 significantly improved mitochondrial calcium overload (P < 0.001) caused by freezing. In contrast to mitochondrial calcium levels, where endoplasmic reticulum calcium was abnormally reduced by vitrification, supplementation with PCB2 could significantly increase the endoplasmic reticulum calcium of frozen oocytes (P <0.001, fig. 5E).

2.6 Effect of PCB2 on frozen oocyte DNA Damage

As shown in FIGS. 6A-B, the oocyte DNA damage was significantly increased after freezing (P < 0.01), and the addition of PCB2 significantly reduced the DNA damage (P < 0.01).

2.7 Effect of PCB2 on early apoptosis and autophagy of frozen oocytes

Early apoptosis and autophagy immunofluorescent staining as shown in fig. 7A, PCB2 significantly reduced the incidence of early apoptosis (P < 0.01, fig. 7B) and autophagy levels (P <0.001, fig. 7C) in vitrified frozen oocytes. Autophagy and lysosome-related gene detection revealed that vitrification resulted in significantly elevated mRNA levels of Mpa1lc3a and Atg14, while PCB2 could significantly reduce its expression levels (fig. 7D).

2.8 Effect of PCB2 on frozen oocyte cortical tension

The function of actin in mediating spindle migration is partly achieved by cortical tone, and pERM and pMRLC are two important corticotropin-regulating proteins whose expression was measured by immunofluorescence staining (fig. 8A-B). The results show that the mean fluorescence intensity of pERM decreased significantly in the frozen group (P <0.001, FIG. 8C), while the fluorescence signal of pMRLC in the cytoplasm increased significantly after vitrification freezing (P <0.001, FIG. 8D). PCB2 was found to remodel the cortical tone of frozen oocytes as evidenced by increased pERM fluorescence intensity (P < 0.001) and decreased pMRLC fluorescence intensity (P < 0.001).

2.9 cortical tone modulation is closely related to mitochondrial function

ConA is a tetravalent lectin, cross-linked to the cell surface by binding to cell membrane glycosylated proteins. ConA treatment is reported to increase the cortical tone of oocytes. Here, we used ConA or Myosin Light Chain Kinase (MLCK) specific inhibitor ML-7 to induce an increase or decrease in cortical tension, respectively, and further investigated their effects on mitochondrial membrane potential (fig. 9A), mitochondrial distribution (fig. 9B) and ATP (fig. 9E). Both PCB2(P < 0.01) and ConA (P < 0.001) significantly increased the mitochondrial membrane potential of oocytes after freezing compared to the frozen group. PCB2 also increased the mitochondrial membrane potential of frozen oocytes after ML-7 treatment (P <0.05, FIG. 9C). Furthermore, both PCB2 and ConA significantly improved the abnormal mitochondrial distribution of frozen oocytes (P < 0.01, fig. 9D). Notably, PCB2 significantly increased ATP levels of oocytes after freezing compared to ConA (P <0.05, panel E). The above results indicate that treatment of PCB2 not only alleviates mitochondrial dysfunction, but also promotes ATP production.

2.10 PCB2 Regulation of frozen oocyte cortical tension by electronic transfer chain

qPCR detection of mitochondrial electron transport chain-associated genes revealed that vitrification resulted in significant decrease in mRNA levels of Ndufv1, Cox1 and Atp5a1, while PCB2 significantly increased its expression levels (FIG. 10A). Frozen oocytes were treated with Rotenone (Rotenone, mitochondrial electron transport chain complex I inhibitor), diphenyliodine (DPI, pentose phosphate pathway inhibitor) and Oligomycin (ATP synthase inhibitor), respectively, and the expression level of each set of perms was examined (fig. 10B). As shown in FIG. 10C, both Rotenone (P < 0.01), DPI (P < 0.001), and oligomycin (P < 0.001) treatments significantly reduced the relative fluorescence intensity of the oocytes pERM as compared to vitrified frozen oocytes. Among them, oligomycin almost eliminated the distribution of pERM in the cortical region of oocytes. When frozen oocytes were treated with PCB2 in combination with the three inhibitors described above, it was further tested whether PCB2 could ameliorate cortical tone abnormalities following inhibitor treatment. The results show that PCB2 can rescue the corticotonia reduction induced by rotenone (P < 0.001) or DPI (P < 0.001), but not oligomycin (P > 0.05). In addition, the mRNA levels of Ndufv1 and Atp5a1 were measured after treatment of PCB2 in combination with rotenone and oligomycin, respectively. As shown in fig. 2-10D and E, the expression level of Ndufv1 in the frozen oocytes was significantly decreased (P <0.05) after rotenone treatment, and the expression level of Ndufv1 was increased (P <0.05) by the combined use of PCB 2; however, both oligomycin and the combined treatment of PCB2 with oligomycin had no effect on the mRNA levels of frozen oocytes Atp5a 1. This suggests that PCB2 can regulate the cortical tone of oocytes via the mitochondrial electron transport chain and pentose phosphate pathway.

Example 2 application of procyanidin B2 to freezing of GV stage oocytes

Firstly, an experimental method:

1.1GV stage oocyte retrieval: 10IU of Pregnant horse Serum Gonadotropin (PMSG) is injected into the abdominal cavity of the mouse, and the mouse is killed by cervical dislocation after 46-48 h. The ovaries were removed and placed in an HX tray equilibrated in the incubator in advance, and the follicles on the surface of the ovaries were completely punctured with a 1mL syringe needle to obtain Cumulus-Oocyte complexes (COCs). Removing granular cells on the surface of the oocyte, and allowing the obtained GV-stage oocyte to hatch into M16 culture solution (containing CaCl) balanced in the culture box in advance ₂ ×2H ₂ O 0.25137g/L，MgSO ₄ 0.1649 g/L，KCl 0.35635g/L，KH ₂ PO ₄ 0.162 g/L，NaHCO ₃ 2.101G/L, NaCl 5.53193G/L, BSA4.0G/L, D-glucose 1.0G/L, phenol red sodium 0.0106G/L, sodium pyruvate 0.0363G/L, DL-sodium lactate 2.95G/L, penicillin G potassium 0.06G/L, streptomycin sulfate 0.05G/L), 37 ℃ and 5% CO ₂ The GVBD occurrence rate was counted after 2 hours of culture in the incubator, and the polar body discharge rate was counted after 12 hours of culture.

1.2 oocyte freezing method as in example 1.

1.3 immunofluorescent staining method As in example 1, different antibodies were selected according to the purpose of the experiment.

1.4 chromosome plating

Washing the oocyte for 3 times by using M2 liquid for later use; preparing nocodazole: diluting nocodazole with M2 solution to obtain working concentration of 2mg/mL, and making into microdroplet; incubating the oocyte in nocodazole microdroplet for 20min at 37 ℃, then eluting the oocyte by using M2 liquid, and removing a zona pellucida by using pronase; washing the zona pellucida removed oocyte in M2 for 3 times, and hypotonic treating with 1% sodium citrate solution for about 5min until the oocyte is swelled without rupture; on the in situ hybridization slide glass, processing by using a hydrophobic pen, then dripping about 20 mu L of slide paving liquid into the circle, and blowing the expanded oocyte from the top of the liquid surface of the slide paving liquid to ensure that the oocyte freely falls onto the slide glass until the oocyte is completely dissolved; and naturally drying the glass slide at room temperature, dripping DAPI into a circle of the glass slide after air drying, covering a cover glass, taking a picture under a laser confocal microscope, and counting the karyotype of the oocyte.

Secondly, the result is:

2.1 Effect of PCB2 on meiosis of frozen oocytes

To investigate whether PCB2 could alleviate meiotic injury to mouse oocytes caused by vitrification freezing, 5. mu.g/mL of PCB2 was added during in vitro maturation and the GVBD, PBE ratio of oocytes in each group was evaluated (FIG. 11A). As shown in FIG. 11B, freezing significantly reduced the GVBD (P <0.05), PBE (P <0.05) ratios, while PCB2 significantly increased the frozen oocyte polar body discharge rate (P <0.05), but had no effect on the development of GVBD (P > 0.05).

2.2 Effect of PCB2 on frozen oocyte spindle migration

Spindle positioning can be quantified by the distance (length, L) of the spindle pole to the oocyte cortex and the oocyte diameter (D). Wherein the rate of spindle migration to the cortex is reflected by the ratio of L/D. As shown in FIGS. 12A-B, the spindle of the oocyte of the frozen group was still located at the center of the cell when the spindle of the oocyte of the fresh group migrated to the cortical region. In addition, L/D reflects that the spindle migration rate of oocytes in the frozen group after 9h of in vitro maturation was significantly higher than that in the fresh group (P < 0.001). The replenishment of PCB2 can save the wrong positioning of the spindle (P < 0.01). To further explore the cause of spindle localization defects, actin distribution and expression in MI oocytes were examined. As shown in FIGS. 12C-D, F-actin relative fluorescence intensity of oocytes in frozen group was significantly decreased compared to fresh group, and supplementation with PCB2 could rescue this phenomenon (P < 0.001).

2.3 Effect of PCB2 on frozen oocytes spindle morphology and aneuploidy Rate

As shown in FIG. 13A, C, the spindle form of fresh oocyte in MII stage was measured, and the spindle of frozen oocyte lost normal form, and the abnormal form ratio of the frozen oocyte spindle was significantly increased (P < 0.001) compared with the fresh oocyte, and the supplement of PCB2 significantly improved the phenomenon (P < 0.01). Abnormal morphology of meiotic metaphase spindles is often accompanied by high aneuploidy. As shown in FIG. 13B, D, the non-integral rate of oocytes after freezing was significantly higher than that of fresh group (P < 0.001), and the supplementation of PCB2 could decrease the non-integral rate of oocytes in frozen group (P < 0.05).

Example 3 Effect of procyanidins on the subsequent developmental competence of frozen oocytes

The first experiment method comprises the following steps:

1.1 oocyte retrieval the same as in example 1.

1.2 oocyte freezing method as in example 1.

1.3 parthenogenetic activation of oocytes

Balancing the embryonic development disc: the KSOM culture medium (EDTA 0.38mg/100mL, sodium pyruvate 2.2mg/100mL, glucose 3.6mg/100mL, KH) is added one night in advance ₂ PO ₄ 4.75mg/100mL，MgSO ₄ ×7H ₂ O4.95 mg/100mL, streptomycin 5.0mg/100mL, penicillin G6.3 mg/100mL, KCl 18.5mg/100mL, glutamine 14.5mg/100mL, NaHCO ₃ 210.0 mg/100mL, NaCl 559.5mg/100mL, 60% sodium lactate 174. mu.L/100 mL, essential amino acids × 501000. mu.L/100 mL, non-essential amino acids × 100500. mu.L/100 mL, phenol red 50. mu.L/100 mL, CaCl ₂ ×2H ₂ O25 mg/100mL, fetal bovine serum 100mg/100mL) was prepared and placed at 37 ℃ in a 5% CO atmosphere ₂ Equilibrating in an incubator overnight; preparing and activating a mixed solution A: taking 890 μ L of HTF liquid without calcium, adding 100 μ L of SrCl ₂ And 10. mu.L of CB, mixed well. The solution A is made into a plate and placed at 37 ℃ and 5% CO ₂ Equilibrate in the incubator for 10 minutes. After balancing, the oocytes in the MII stage are washed by the solution A for 3 times and then are placed in the solution A for activation for 2.5 hours; preparing and activating mixed solution B: 990 μ L of HTF solution containing calcium was added with 10 μ L of CB, and mixed well. Placing the solution B on a plate at 37 deg.C and 5% CO ₂ Equilibrate in the incubator for 10 minutes. After balancing, the oocyte activated by the solution A is washed by the solution B for 3 times and then is placed in the solution B for activation for 3.5 hours; counting pronucleus and embryo culture: and (5) cleaning the pronucleus activated by the B solution for 3 times by using a KSOM culture solution which is well balanced one night in advance, and then counting the pronucleus occurrence rate. Place pronucleus in KSOM at 37 ℃ with 5% CO ₂ And performing subsequent embryo culture in the incubator. At this time, the development of the embryo is recorded as 0hAnd counting the number of cleavage and blastocysts after 24h and 96h respectively.

1.4 blastocyst quality detection

Fixing the blastocyst in fixing liquid at room temperature for 30min, then permeating the permeation liquid for 1h at room temperature by using 0.5 percent TritonX-100-DPBS, washing the blastocyst with 0.1 percent TritonX-100-0.1 percent PVA-DPBS washing liquid for three times, transferring the blastocyst into immunofluorescence blocking liquid for blocking for 1h at room temperature, and then incubating the blastocyst in primary antibodies of Nanog (1:1000) and CDX2(1:500) at 4 ℃ overnight; after incubation in FITC-488 and Alex-594 secondary antibody for 1h at room temperature, the cells were stained for 5min with DAPI and visualized by compression. The ratio of the inner cell mass to the total blastocyst cell count was counted by counting the inner cell mass and trophoblast cells separately. The experiment was repeated three times.

1.5 blastocyst Medium Metabonomics detection

Taking out each group of embryos, leaving the embryo culture solution, sucking the culture solution by using a 1mL syringe and transferring the culture solution into a 1.5mL centrifuge tube, and repeatedly taking 200 mu L of the culture solution every time and storing the culture solution in a refrigerator at minus 80 ℃; after all samples are collected, the amino acid and carbohydrate in the blastocyst culture solution are subjected to targeted metabonomics sequencing.

And (3) detecting saccharides: slowly thawing the sample at 4 ℃, adding 300 mu L of 80% methanol water into 0.1mL of the sample, and uniformly mixing by vortex; ultrasonic oscillating at 4 deg.C for 30min, and standing at 4 deg.C for 60 min; centrifuging at 12000rpm for 10min at 4 deg.C, and sampling supernatant for LC-MS/MS analysis.

And (3) amino acid detection: slowly thawing the sample at 4 ℃, and placing 50 mu L of sample into a 1.5mL centrifugal tube; adding 450 mu L (containing internal standard 100ng/mL) of glacial methanol, and fully shaking for 1 min; standing at 4 deg.C for 30min, centrifuging at 12000rpm for 10min, and collecting supernatant and detecting on a machine.

Second, experimental results

2.1 Effect of PCB2 on frozen oocytes parthenogenetically activated embryo development

Immunofluorescence staining of CDX2 (cell line specific marker of Trophoblast (TE)) and Nanog (cell line specific marker of Inner Cell Mass (ICM)) as shown in fig. 14A, it can be found that the total ICM to TE ratio (P < 0.001) and ICM to total cell number ratio (P < 0.001) of oocytes parthenogenically activated blastocysts after freezing are both significantly reduced compared to the fresh group, and the quality of blastocysts after PCB2 treatment is greatly improved (fig. 14B-C). The ICM to TE ratio (P < 0.001) and ICM to total cell number ratio (P < 0.001) were both significantly increased in the PCB2 treatment, indicating a significant improvement in blastocyst quality.

2.2 Effect of PCB2 on frozen oocytes parthenogenetically activated embryo metabolism

The culture medium of fresh group, frozen group and PCB2 treated group frozen oocytes on day 4 after parthenogenetic activation was subjected to targeted metabonomic analysis, and sample collection was shown in FIG. 15A. The results of the targeted metabolome of amino acids showed (FIGS. 15B-E) that the relative contents of L-hydroxyproline, L-citrulline, and L-kynurenine hydrate in the blastocyst culture fluid of the frozen group were all significantly reduced. But only the relative content of L-glutamic acid was significantly affected in the PCB2 treatment group. As shown in fig. 15F-J, the addition of PCB2 had a significant effect on the metabolic levels of arabinose, fructose, glucose, maltose, and xylose, suggesting that PCB2 is primarily involved in sugar metabolism to regulate embryonic development.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (10)

1. The cryopreservation liquid for the oocyte is characterized by comprising a frozen equilibrium liquid and a vitrified refrigerating liquid, wherein both the frozen equilibrium liquid and the vitrified refrigerating liquid contain procyanidine B2.

2. The cryopreservation solution for oocytes according to claim 1, wherein the concentration of procyanidin B2 in the frozen equilibration solution and the vitrified freezing solution is 5 μ g/mL.

3. The cryopreservation solution for oocytes according to claim 1, wherein the cryo-equilibration solution further comprises dimethyl sulfoxide and ethylene glycol.

4. The cryopreservation solution for oocytes according to claim 1, wherein the vitrified cryopreservation solution is a PBS solution containing ethylene glycol, dimethyl sulfoxide, sucrose and polysucrose.

5. An unfreezing liquid for cryopreservation of oocyte, which is a PBS liquid containing procyanidine B2.

6. The thawing solution of claim 5, wherein said procyanidin B2 is present at a concentration of 5 μ g/mL in said thawing solution.

7. A recovery liquid after oocyte thawing is characterized in that the recovery liquid is M2 liquid containing procyanidine B2.

8. The recovery fluid of claim 7, wherein the concentration of procyanidin B2 in the recovery fluid is 5 μ g/mL.

9. A method for cryopreservation of oocytes, the method comprising: placing the oocyte in the freezing equilibrium liquid described in any one of claims 1 to 4, transferring the oocyte to the vitrification freezing liquid described in any one of claims 1 to 4 after equilibrium treatment, then throwing liquid nitrogen into the oocyte, and freezing and preserving the oocyte; when thawing, the cryopreserved oocytes are thawed by being placed in the thawing solution according to claim 5 or 6, and then thawed and then placed in the recovery solution according to claim 7 or 8 for recovery.

10. Use of procyanidin B2, a cryopreservation solution as defined in any one of claims 1 to 4, a thawing solution as defined in claim 5 or 6 or a post-thawing recovery solution as defined in claim 7 or 8 for cryopreservation of oocytes.

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