CN114847274B - Oocyte cryopreservation reagent and application thereof - Google Patents

Abstract

The invention discloses an oocyte cryopreservation reagent and application thereof, and relates to the technical field of reproductive engineering. The invention provides a cryopreservation solution for oocytes, which comprises a freezing balance solutionAnd vitrification refrigerating fluid, wherein the freezing balance fluid and the vitrification refrigerating fluid both contain procyanidine B2. The invention also provides a thawing solution for freezing and preserving oocytes, wherein the thawing solution is PBS (phosphate buffered saline) solution containing procyanidine B2. The invention also provides a recovery liquid for the oocysts after thawing. The invention adds procyanidine B2 (PCB 2) into the cryopreservation solution, the thawing solution and the recovery solution, which can reduce the oocyte freezing injury, and is characterized by increased survival rate, improved mitochondrial function and Ca after thawing ²⁺ Maintenance of homeostasis, reduction of oxidative stress level, restoration of cortical tension, reduction of non-whole multiplying power, normal promotion of meiosis process, and the procyanidine B2 can participate in sugar metabolism regulation and control, improving the subsequent development ability of frozen oocytes.

Description

Oocyte cryopreservation reagent and application thereof

Technical Field

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

Background

The oocyte freezing technology is an important reproductive biology technology and is widely applied to germplasm resource preservation, fertility preservation and assisted reproduction technology, but the problems of development potential reduction and the like of the thawed oocytes are reported that at least 20 mature oocytes can be frozen to obtain offspring, and the wide application of the technology is severely restricted, so that the improvement of the utilization efficiency of frozen oocytes is a problem to be solved urgently.

Challenges faced by cryopreservation of oocytes include their relatively large size, high water content, unique chromosomal arrangement and meiotic spindles, which make oocytes particularly vulnerable to ice crystal formation during freezing and thawing, damage to oocytes by freezing stress includes the following aspects:

1. freezing affects oocyte energy supply

Cryogenically induced damage mainly involves ATP depletion and reduced enzymatic activity, resulting in metabolic and redox imbalances. This is because, although the production of ATP by mitochondria is significantly reduced at low temperatures, maintaining basic cellular processes still requires continuous production of bioenergy, which results in relatively rapid consumption of intracellular ATP. Studies indicate that about 95% of ATP can be hydrolyzed to adenosine monophosphate in 4 hours at 0-4 ℃. Reduced ATP production and rapid consumption can cause a series of cell adverse 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 disruption of ion homeostasis, leading to cell swelling and loss of cell viability.

During cryopreservation of oocytes, freeze/thaw also has a significant negative effect on the ATP concentration. Slow freezing and vitrification freezing studies of post-ovulation oocytes in rabbits have found that the ATP level of frozen oocytes is significantly lower than that of fresh oocytes and is independent of the method used for freezing. This result was also verified in cryopreservation of oocytes of humans, mice, pigs and cattle.

2. Freezing affects oocyte skeleton

Cryopreservation has been shown to induce plasma membrane and cytoskeletal collapse leading to loss of barrier function and abnormalities in ion homeostasis and metabolite supply, thereby accelerating cell death. Interactions between cellular lipids and cytoskeletal components are complex, and hardening of these lipids can lead to deformation and destruction of the cytoskeleton, which can negatively impact cell survival and development, and can be an inherent cause of dehydration and cell morphology changes during low temperature storage.

In oocytes, the cytoskeleton is important for the development and fertilization of the oocyte. Microwires and microtubules are used as main components of cytoskeleton, are extremely sensitive to low temperatures, and can cause depolymerization of microtubules and disordered distribution of microwires by cryopreservation. It has been demonstrated that vitrification freezing can cause the depolymerization of oocyte microtubules, which in turn affects spindle structure and function. The network of cortical microwires can also be abnormal during cryopreservation, which can induce the oocyte chromosomes to misalign and eventually form aneuploidy.

In order to solve the problems of cytoskeletal abnormalities caused by freezing, some studies have used membrane stabilizers (e.g., antifreeze proteins and hydrogels) to cryopreserve cells. Antifreeze glycoproteins found in the serum of polar fish and some insects were first used in 1992 for cryopreservation of bovine oocytes and demonstrated significant improvements in viability and bioactivity of oocytes after cryopreservation. The L-proline oligomer having the same polyproline II helix structure as the antifreeze glycoprotein was also confirmed to have a strong activity of inhibiting ice growth, preventing direct damage of large ice crystals to oocytes and an increase in osmotic pressure.

3. Freezing induced oocyte oxidative stress

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 antioxidant, which is caused by the massive production of reactive oxygen species (Reactive oxygen species, ROS) under extreme conditions. For example, the low temperature preservation and warming process is accompanied by a decrease in the antioxidant capacity of the cells, and mitochondrial damage caused by low temperature can further enhance ROS production, thereby inducing oxidative stress in the cells.

Oxidative stress during cryopreservation can also be generated by 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 vitrified freeze-thawed porcine oocytes and embryos suggests that cryopreservation compromises the antioxidant defense system of porcine oocytes and embryos.

In order to mitigate the potential oxidative damage during cryopreservation, some studies have added antioxidants to conventional cryopreservation solutions. Resveratrol, N-acetylcysteine, melatonin and the like have been widely used for cryopreservation of oocytes/embryos of humans and domestic animals 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 proven to be harmful to humans or animals on a macroscopic level and to be fatal to cells on a microscopic level. In cryobiology, the lethality of ice to cells has been a challenge in limiting the development of cryopreservation of mammalian cells. Mazur first proposed the hypothesis of "two low temperature lesions", including ice-induced osmotic lesions and mechanical lesions; in recent years, ROS damage to cells has also been demonstrated during cryopreservation.

Disclosure of Invention

The invention aims to provide an oocyte cryopreservation reagent and application thereof, which are used for solving the problems in the prior art.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides a cryopreservation liquid for oocytes, which comprises a freezing balance liquid and a vitrification freezing liquid, wherein the freezing balance liquid and the vitrification freezing liquid both contain procyanidine B2.

Further, the procyanidine B2 concentration in both the freezing balance liquid and the vitrification frozen liquid is 5 μg/mL.

Further, the freezing balance liquid also contains dimethyl sulfoxide and ethylene glycol.

Further, the vitrification freezing solution is PBS solution containing glycol, dimethyl sulfoxide, sucrose and polysucrose.

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

Further, the concentration of procyanidine B2 in the thawing solution is 5 μg/mL.

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

Further, the concentration of procyanidine B2 in the post-thawing recovery liquid is 5 μg/mL.

The present invention also provides a method for cryopreserving oocytes, the method including: firstly placing oocytes in the freezing balance liquid, transferring the oocytes into the vitrification freezing liquid after the balance treatment, then adding liquid nitrogen, and freezing and preserving; and (3) during thawing, placing the frozen and preserved oocyte in the thawing solution for thawing, and placing the thawed oocyte 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 recovery liquid after thawing in cryopreservation of oocytes.

Procyanidins are polyphenols, also known as condensed tannins, which are oligomers or polymers of flavan-3-ols linked by interferon linkages [ e.g. (-) -epicatechin or (+) -catechin ]. Procyanidins are one of the most abundant phytochemicals in plants, and are widely found in fruits, vegetables, grains, beans, tea leaves, and the like. Normally, procyanidins containing only epicatechin are called procyanidins. The research finds that the procyanidine has wide regulation effect, can participate in lipid metabolism, epigenetic regulation and the like, and procyanidine B2 (dimer procyanidin B [4,8' -BI- [ (+) -epicatechin ] ] (PCB 2)) is the main procyanidine, and in view of the wide influence of freezing stress on oocytes, we speculate that the procyanidine may be helpful for reducing freezing injury. The existing freezing and thawing reagents in the market are generally permeability and non-permeability cryoprotectants, and the aim of protecting oocytes is fulfilled by replacing water in the oocytes and preventing ice crystals from forming in the freezing and thawing process, and the addition of a spectrum anti-freezing injury preparation is lacking.

The invention discloses the following technical effects:

the procyanidine B2 (PCB 2) is added into the cryopreservation solution and the thawing solution, so that the oocyte freezing injury can be relieved, and the characteristics of improved survival rate, mitochondrial function and Ca after thawing are presented ²⁺ Maintenance of homeostasis, reduction of oxidative stress level, restoration of cortical tension, reduction of non-whole multiplying power, normal promotion of meiosis process, and the procyanidine B2 can participate in sugar metabolism regulation and control, improving the subsequent development ability of frozen oocytes.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are 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 other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is the effect of different concentrations of procyanidin B2 on the survival and development of vitrified frozen oocytes, wherein A is the cleavage and blastogenesis of each group of oocytes, scale: 100 μm; b is survival rate of oocytes after thawing; c is embryo cleavage rate after parthenogenesis activation; d is embryo blastula rate after parthenogenesis activation; "n" represents the number of oocytes used in the experiments, all experiments were performed at least three biological replicates, data expressed as mean ± standard error, P < 0.05, P < 0.01, ns = no statistical difference;

FIG. 2 is the effect of procyanidin B2 on oxidative stress of vitrified frozen oocytes, wherein A is the ROS and GSH levels of each group of oocytes, scale: 100 μm; b is the fluorescent intensity analysis of ROS in the oocyte; c is GSH fluorescence intensity analysis in the oocyte; "n" represents the number of oocytes used in the experiments, all experiments were performed at least three biological replicates, and data were expressed as mean ± standard error, P < 0.05, P < 0.001;

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

FIG. 4 is the effect of procyanidin B2 on mitochondrial function of vitrified frozen oocytes, wherein A is the mitochondrial membrane potential of each group of oocytes, scale: 50 μm; b is the oocyte mitochondrial temperature of each group, scale: 50 μm; c is the analysis of the mitochondrial membrane potential level of the oocyte; d is the analysis of the temperature level of the mitochondria of the oocyte; e is mitochondrial fusion (Opa 1, mfn1, mfn 2) and division (Drp 1) gene expression analysis; "n" represents the number of oocytes used in the experiments, all experiments were performed at least three biological replicates, data expressed as mean ± standard error, P < 0.05, P < 0.001, ns = no statistical difference;

FIG. 5 is the effect of procyanidin B2 on vitrified frozen oocyte calcium homeostasis, wherein A is the co-localization of specific calcium sensitive probes with organelles, scale: 50 μm; b is the level of cytoplasmic calcium, mitochondrial calcium and endoplasmic reticulum calcium of each group of oocytes, scale: 50 μm; c is the analysis of the cytoplasmic calcium level of the oocyte; d is analysis of oocyte mitochondrial calcium level; e is the analysis of the calcium level of the endoplasmic reticulum of the oocyte; "n" represents the number of oocytes used in the experiments, all experiments were performed at least three biological replicates, data expressed as mean ± standard error, P < 0.001, ns = no statistical difference;

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

FIG. 7 is the effect of procyanidin B2 on early apoptosis and autophagy of vitrified frozen oocytes, wherein A is the early apoptosis occurrence and autophagy immunofluorescent staining of each group of oocytes, scale: 50 μm; b is the occurrence proportion of early apoptosis in oocytes; c is the analysis of LC3 fluorescence intensity of the oocyte; d is qPCR to detect mRNA levels of Beclin1, map1lc3a, ulk1, atg14, lamp1 and Lamp 2; "n" represents the number of oocytes used in the experiments, all experiments were performed at least three times, the data are expressed as mean ± standard error, P < 0.05, P < 0.01, P < 0.001, ns = no statistical difference;

FIG. 8 is the cortical tension of procyanidin B2 remodelled frozen oocytes, wherein A is a representative picture of pERM immunofluorescence staining of each group of oocytes, scale: 100 μm; b is a representative picture, scale bar, of pMRLC immunofluorescent staining of each group of oocytes: 100 μm; c is the average fluorescence intensity analysis of each group of oocytes pERM; d is the average fluorescence intensity analysis of each group of oocyte pMRLC; "n" represents the number of oocytes used in the experiments, all experiments were performed at least three biological replicates and the data are expressed as mean ± standard error. * P < 0.001;

fig. 9 is the effect of cortical tension on mitochondrial function, where a is the detection of mitochondrial membrane potential by JC-1 staining, conA, PCB2 and ML-7 were added to recovery medium during thawing for 1 hour, scale: 50 μm; b is a representative image of mitochondrial distribution, oocytes were stained with Mito-Tracker Green, scale: 50 μm; c is the mitochondrial membrane potential analysis of each group of oocytes; d is analysis of abnormal mitochondrial distribution proportion of oocytes in each group; e is the ATP level of the oocytes of each group; "n" represents the number of oocytes used in the experiments, all experiments were performed at least three times, the data are expressed as mean ± standard error, P < 0.05, P < 0.01, P < 0.001, ns = no statistical difference;

FIG. 10 is a graph showing that procyanidin B2 modulates the cortical tension of frozen oocytes by mitochondrial electron transport chains, wherein A is qPCR for detecting mRNA levels of Ndefv 1, sdhb, uqcrc2, cox1 and Atp a 1; b is the pecm immunofluorescence of mature oocytes, scale: 50 μm; c is the relative pERM fluorescence intensity analysis of each group of oocytes; d is qPCR to detect the mRNA level of Ndefv 1; e is qPCR to detect mRNA levels of Atp a 1; "n" represents the number of oocytes used in the experiments, all experiments were performed at least three biological replicates, data expressed as mean ± standard error, P < 0.001, ns = no statistical difference;

FIG. 11 is the effect of procyanidin B2 on in vitro maturation of vitrified frozen oocytes, wherein A is the occurrence of GVBD and PBE for each group of oocytes, scale: 100 μm; b is GVBD proportion of oocyte in vitro culture for 2 h; c is the PBE proportion of oocyte in vitro culture for 12 hours; "n" represents the number of oocytes used in the experiments, all experiments were performed at least three biological replicates, data expressed as mean ± standard error, P <0.05, ns = no statistical difference;

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

FIG. 13 is a graph of procyanidin B2 decreasing spindle abnormality ratio and aneuploidy rate, where A is a representative picture of spindle morphology of each group of oocytes, scale: 50 μm; b is a representative picture of oocyte aneuploidy and aneuploidy, scale: 50 μm; c is the proportion analysis of the morphological abnormality of the spindle bodies of the oocytes in each group; d is the proportional analysis of the aneuploidy rate of each group of oocytes; "n" represents the number of oocytes used in the experiments, all experiments were performed at least three biological replicates, and data were expressed as mean ± standard error, P < 0.05, P < 0.01, P < 0.001;

FIG. 14 shows that procyanidine B2 improves the quality of frozen oocyte parthenogenetic activated blasts, wherein A is a representative picture of CDX2 and Nanog immunofluorescent staining of blasts of each group, scale: 50 μm; b is ICM to TE ratio analysis of each group of oocytes; c is ICM of each group of oocytes, total cell ratio analysis; "n" represents the number of oocytes used in the experiments, all experiments were performed at least three biological replicates, and data are expressed as mean ± standard error, P < 0.001;

FIG. 15 is a schematic diagram showing that procyanidine B2 is mainly involved in the regulation of glycometabolism during embryo 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 L-canine uric 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 glucose content in the different groups; i is the average content of maltose in different groups; j is the average content of xylose in the different groups; all experiments were performed in at least three biological replicates. ns = no statistical difference.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions 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. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, 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 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 invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

Example 1 application of procyanidine in MII-stage oocyte freezing

1. Experimental method

1.1 oocyte collection

MII phase oocyte retrieval: 4-6w ICR mice are taken, pregnant horse serum gonadotropin (PMSG) 10IU is injected into the abdominal cavity, human Chorionic Gonadotrophin (HCG) 10IU is injected into the abdominal cavity after 48h, the mice are killed by a cervical dislocation method after 12-14h of HCG injection, oviducts are taken, an expanding part is scratched under an anatomical lens, a cumulus oocyte complex is obtained, and a MII-stage oocyte discharged from a first polar body is collected for standby after hyaluronidase treatment.

1.2 freezing of oocytes

The oocyte vitrification freezing and thawing method refers to the conventional steps, and specifically comprises the following steps: oocytes were placed in ED solution (freezing balance solution), i.e., PBS solution containing 10% dimethyl sulfoxide and 10% ethylene glycol, for 30sec, and then transferred to EDFS30 solution (vitrification freezing solution), i.e., PBS solution containing 15% ethylene glycol, 15% dimethyl sulfoxide, 0.5M sucrose, 30% polysucrose (Ficoll), and placed in front of a carrier rod, liquid nitrogen was added, and the time from the start of entering EDFS30 solution to the addition of liquid nitrogen was controlled within 25 sec. In thawing, the rod to be thawed is rapidly removed from 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) ₄ ×7H ₂ O0.293 g/L, streptomycin 5.0g/L，KH ₂ PO ₄ 0.162G/L, penicillin G7.5G/L, KCl 0.356G/L, glucose 1.0G/L, naHCO ₃ 0.349g/L, naCl5.533g/L,60% sodium lactate 4.349ml/L, hepes 4.969g/L, caCl ₂ ×2H ₂ O0.252 g/L and BSA4.0 g/L) for three times.

Grouping oocytes: fresh control group oocyte (CT), vitrified frozen oocyte (VT), PCB2 treatment group (PCB 2-VT), wherein 5 mug/mLPCB 2 is respectively added into cryopreservation liquid (ED liquid and EDFS30 liquid), thawing liquid and restoring liquid after thawing, and the thawed oocyte is cultured in the restoring liquid for 1h for standby.

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

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

The preparation method of the restoration liquid after thawing comprises the following steps: 5. Mu.g/mLPCB 2 was added to the M2 solution.

1.3 detection of oxidative stress index of oocyte

1.3.1ROS assay

ROS were measured using 2',7' -dichlorofluorescein diacetate (2 ',7' -dichlorofluorescin diacetate, DCHFDA), oocytes were taken and placed in 10mM DCFHDA drops and incubated for 20min in an incubator at 37 ℃. The oocytes were washed three times with DPBS containing 0.1% BSA (bovine serum albumin) and then observed under a fluorescence microscope with an excitation light of 460nm. 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), and oocytes were taken and placed in 10. Mu. Mol/L GSH dye droplets and incubated in an incubator at 37℃for 20min. The oocytes were washed three times with DPBS containing 0.1% BSA and then observed under a fluorescence microscope, and excitation light was 370nm. The experiment was repeated three times.

1.4 analysis of mitochondrial function in oocytes

1.4.1 mitochondrial Membrane potential (. DELTA.ψm) 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, placed in 10mM JC-1 drop, and incubated in an incubator at 37℃for 30min. The oocytes were washed three times with DPBS containing 0.1% BSA and then observed under a fluorescence microscope. The maximum excitation wavelength of JC-1 monomer is 514nm, and the maximum emission wavelength is 529nm; the maximum excitation wavelength of the polymer is 585nm, and the maximum emission wavelength is 590nm. The experiment was repeated three times.

1.4.2ATP content detection

ATP standard was prepared. And adding each group of oocytes into a PCR tube containing ATP lysate, adding ATP detection buffer solution after the completion of the lysis, finally adding ATP detection premix solution, fully mixing, incubating for 30min at room temperature in a dark place, and detecting by using a chemiluminescent instrument. And 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 oocytes to obtain the ATP content in each egg. The experiment was repeated three times.

1.4.3 mitochondrial temperature detection

Mitochondrial temperatures were detected using a Mito Thermos Yellow (MTY) probe. Firstly, diluting MTY to 0.5 mu M by using M2 solution, placing the diluted MTY into a 37 ℃ incubator for preheating for 15min, and incubating oocytes and MTY dye solution in the 37 ℃ incubator for 15min in a dark place; washed 3 times with M2 and photographed under a laser confocal microscope in M2 droplets.

1.5 mitochondrial and endoplasmic reticulum distribution detection

Mitochondrial distribution detection: washing oocyte with M2 for 3 times for standby; oocyte mitochondrial distribution was detected using Mito-Tracker Red or Mito-Tracker Green probes. Firstly, diluting a probe to 5 mu M by using M2 liquid, making a disc, and placing the disc into a 37 ℃ incubator to be preheated for 20min; incubating the oocyte with Mito-Tracker Red or Mito-Tracker Green dye solution at 37 ℃ in an incubator in the absence of light for 20min; washing 3 times with M2, incubating in a preheated H33342 microdroplet, and incubating in an incubator at 37 ℃ for 10min; washed 3 times with M2 solution and placed in a droplet for observation under a confocal laser microscope. Mitochondria are evenly distributed in cytoplasm of oocyte as normal distribution; mitochondria clustered or otherwise present an uneven distribution in the cytoplasm of the oocyte are abnormal distributions.

Endoplasmic reticulum distribution detection: washing oocyte with M2 for 3 times for standby; the ER-Tracker Red probe was used to detect the distribution of the endoplasmic reticulum of oocytes. Firstly, diluting a probe to 5 mu M by using M2 liquid, making a disc, and placing the disc into a 37 ℃ incubator to be preheated for 20min; incubating the oocyte and ER-Tracker Red dye liquor in a 37 ℃ incubator for 20min in the dark; after 3 times of M2 washing, the mixture is incubated in a preheated H33342 micro-drop for 10min at 37 ℃ in an incubator, and after the incubation is completed, the mixture is washed 3 times by M2 and is placed in the M2 micro-drop for photographing under a laser confocal microscope. And (3) counting abnormal endoplasmic reticulum distribution: the endoplasmic reticulum is evenly distributed in the cytoplasm of the oocyte as normal distribution; endoplasmic reticulum clusters or exhibits an uneven distribution in the cytoplasm of oocytes as an abnormal distribution.

1.6 detection of calcium content of oocyte

Cytoplasmic calcium, mitochondrial calcium and endoplasmic reticulum calcium levels in oocytes were detected using cytoplasmic calcium ion specific probes Fluo 3-AM, mitochondrial calcium specific probes Rhod 2-AM and endoplasmic reticulum calcium specific probes Fluo 4-AM, respectively. Firstly, diluting a probe to 5 mu M by using M2 liquid, making a disc, and placing the disc into a 37 ℃ incubator to be preheated for 20min; washing oocyte with M2 for 3 times, removing zona pellucida with pronase, and washing in M2 for 3 times for standby; incubating the oocyte and the calcium ion probe dye solution for 20min in a 37 ℃ incubator in the dark, washing the incubated oocyte for 3 times by using M2, placing the oocyte and the calcium ion probe dye solution in M2 microdroplet for photographing under a laser confocal microscope, keeping photographing parameters and exposure time between groups consistent during photographing, and analyzing fluorescence values by using NIS-Elements AR software.

1.7 detection of early apoptosis occurrence

Washing oocyte with M2 for 3 times for standby; the oocytes were incubated in 100. Mu.L buffer containing 5. Mu.LAnnexin-V FITC for 10min in an incubator at 37 ℃; after incubation, the incubation is completed, the incubation is washed 3 times by M2, and the incubation is placed in M2 microdroplets to take pictures under an inverted fluorescence microscope; counting early apoptosis occurrence: oocytes that did not undergo early apoptosis had only weak green fluorescence at the zona pellucida; an oocyte that undergoes early apoptosis has green fluorescence both at the zona pellucida and at the membrane of the oocyte.

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

Collecting oocytes, namely collecting the oocytes into a 1.5mL centrifuge tube according to 30-50 oocytes in each group, sucking out redundant liquid, and storing the oocytes in a refrigerator at the temperature of minus 80 ℃; extracting oocyte RNA by Trizol; 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 use. According to a 10-fold serial gradient dilution method, the cDNA sample after reverse transcription is subjected to gradient dilution into 5 cDNA templates with different concentrations, primer efficiency verification is carried out, a PCR reaction system is shown in table 3, a primer sequence is shown in table 4, and a qPCR program is shown in table 5. After the real-time fluorescent quantitative PCR reaction is finished, observing a dissolution curve, and if the dissolution curve is unimodal, considering that the result is reliable. And taking the logarithm of the template concentration as an abscissa, taking the CT value as an ordinate as a standard curve, and calculating the primer amplification efficiency according to the slope. Amplification efficiency of primer>90% and<110% of the primers were used in the subsequent experiments. Beta-actin is used as reference gene and 2 ^-△△Ct The relative expression content of the target genes is calculated respectively.

TABLE 1 reverse transcription system (20. Mu.L)

Table 2 reverse transcription procedure:

table 3 qPCR reaction (20 μl system):

TABLE 4 primer sequences

TABLE 5 fluorescent quantitative PCR reaction procedure

1.9 immunofluorescent staining

Placing the oocyte in a fixing solution to be fixed for more than 1h at room temperature. If the oocyte is stripped of zona pellucida, diluting the fixing solution with DPBS according to the ratio of 1:1, and then fixing the oocyte for 30min; the fixed oocytes were washed 3 times with PBS-0.1% PVA washing solution and then placed in 0.5% Triton-PBS-0.1% PVA permeation solution, and allowed to permeate at room temperature for 1 hour. If the oocyte is removed from the zona pellucida, the oocyte is penetrated for 20min; the permeabilized oocytes were washed 3 times with 0.1% Triton-PBS-0.1% PVA wash and then blocked in 3% BSA-0.1% Triton-PBS-0.1% PVA blocking solution for 1h at room temperature; directly placing the blocked oocyte in the prepared primary antibody, and incubating at 4 ℃ overnight or in a 37 ℃ incubator for 2.5 hours, wherein the negative control group replaces the primary antibody with blocking solution or immunostaining primary antibody diluent; the oocyte after the primary antibody incubation is washed 3 times with 0.1% Triton-PBS-0.1% PVA washing solution, then placed in the prepared secondary antibody, and incubated for 1h at room temperature; oocytes after completion of secondary antibody incubation were washed 3 times with 0.1% triton-PBS-0.1% pva wash followed by DAPI for 5min; the oocytes after completion of the nucleus staining were transferred onto an adhesive slide glass, and subjected to pellet observation with a cover glass. If the oocyte is not subjected to tabletting, the oocyte after nuclear dyeing is washed 3 times by using M2, placed in a micro-drop of a glass bottom culture dish, and covered with paraffin oil for observation. Photographing and observing by using an A1 Confocal laser Confocal microscope, and controlling imaging parameters and exposure time to be consistent among groups and controlling variables if the data need to count fluorescence intensity.

2. Experimental results

2.1 PCB2 can improve the survival rate of frozen oocytes and the development rate of blastula

PCB2 (1 mug/mL, 5 mug/mL and 25 mug/mL) with different concentrations is added into the freezing preservation solution and the thawing solution respectively, and the influence of the PCB2 on the survival of frozen oocytes and the development of blastula is analyzed (figure 1A), so that the result shows that 5 mug/mLPCB 2 can remarkably improve the survival rate of frozen oocytes (figure 1B), and remarkably improve the cleavage rate after parthenogenesis (P <0.05, figure 1C) and the development rate of blastula (P < 0.05) (figure 1D).

2.2 PCB2 can reduce oxidative stress of frozen oocytes

The immune fluorescent staining method is adopted to detect the ROS and GSH content in the frozen oocytes of fresh, frozen and PCB2 treated groups (figure 2A), and the addition of 5 mug/mL PCB2 to the frozen preservation solution and the thawing solution can obviously reduce the ROS level of the frozen oocytes (P <0.05, figure 2B) and can obviously raise the GSH level in the frozen oocytes (P <0.001, figure 2C).

2.3 Effects of PCB2 on frozen oocyte organelle distribution

As shown in fig. 3A, fresh groups of oocyte mitochondria exhibited a uniform distribution of cytoplasm, while frozen groups of oocyte mitochondria exhibited a clustered abnormal distribution. After treatment with PCB2, the proportion of abnormal mitochondrial distribution was significantly reduced (P <0.05, fig. 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 mitochondrial function of frozen oocytes

Mitochondrial membrane potential can intuitively reflect mitochondrial function, and detection of mitochondrial membrane potential is shown in fig. 4A, and PCB2 can significantly improve oocyte membrane potential reduction 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 detection is shown in FIG. 4B. After freezing the oocyte mitochondrial temperature increased, whereas supplementation with PCB2 can significantly lower mitochondrial temperature (P < 0.05, fig. 4D). Studies have shown that mitochondrial aggregation due to impaired mitochondrial dynamics also impairs mitochondrial function in oocytes, and that mRNA levels of mitochondrial fusion (Opa 1, mfn 2) and division (Drp 1) genes were examined, respectively. The results showed that the expression levels of Mfn1, mfn2, drp1 were significantly reduced in frozen oocytes compared to fresh group oocytes, whereas supplementation with PCB2 could significantly improve mitochondrial dynamics (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 results of co-localization of organelles show that: rhod 2-AM was co-located with Mito tracker and Fluo 4-AM was co-located with ER tracker (FIG. 5A). As shown in fig. 5B, the cytoplasmic calcium, mitochondrial calcium and endoplasmic reticulum calcium levels in the oocytes were measured, respectively, and the results showed that: vitrification freezing has no obvious effect on cytoplasmic calcium levels of oocytes (P >0.05, fig. 5C), but freezing can cause abnormal elevation of mitochondrial calcium levels of oocytes (fig. 5D), and supplementation of PCB2 can significantly improve mitochondrial calcium overload caused by freezing (P < 0.001). Contrary to mitochondrial calcium levels, endoplasmic reticulum calcium was abnormally decreased after vitrification freezing, and supplementation of PCB2 could significantly raise endoplasmic reticulum calcium of frozen oocytes (P < 0.001, fig. 5E).

2.6 Effects of PCB2 on DNA damage of frozen oocytes

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

2.7 Effects of PCB2 on frozen oocytes early apoptosis and autophagy

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 lysosomal related gene testing found that vitrification freezing could result in significantly elevated mRNA levels of Mpa1lc3a and Atg14, while PCB2 could significantly reduce its expression level (fig. 7D).

2.8 Effects of PCB2 on frozen oocyte cortical tension

The functional part of actin in mediating spindle migration is achieved by cortical tonicity, and petm and pMRLC are two important cortical tonicity regulating proteins whose expression levels were detected by immunofluorescent staining (fig. 8A-B). The results showed a significant decrease in the average fluorescence intensity of pERM in the frozen group (P < 0.001, FIG. 8C), while the fluorescence signal of pMRLC in the cytoplasm after vitrification freezing was significantly increased (P < 0.001, FIG. 8D). The study found that PCB2 remodelled the cortical tension of frozen oocytes, which was manifested in increased pERM fluorescence intensity (P < 0.001) and decreased pMRLC fluorescence intensity (P < 0.001).

2.9 cortical tone and mitochondrial function are closely related

ConA is a tetravalent lectin that is crosslinked to the surface of cells by binding to cell membrane glycosylated proteins. ConA treatment has been reported to increase the cortical tension of oocytes. Here, we induced an increase or decrease in cortical tension using ConA or Myosin Light Chain Kinase (MLCK) specific inhibitors ML-7, respectively, and further studied their effect 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 the frozen oocytes compared to the frozen group. PCB2 can also raise the mitochondrial membrane potential of frozen oocytes after ML-7 treatment (P < 0.05, FIG. 9C). In addition, both PCB2 and ConA significantly improved mitochondrial distribution abnormalities in 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 adjusts frozen oocyte cortical tension through electron transfer chain

qPCR testing of mitochondrial electron transport chain related genes revealed that vitrification freezing could result in a significant decrease in mRNA levels of Ndufv1, cox1 and Atp a1, while PCB2 could significantly increase its expression level (fig. 10A). Frozen oocytes were treated with Rotenone (Rotenone, mitochondrial electron transport chain complex I inhibitor), diphenyliodide (DPI, pentose phosphate pathway inhibitor) and Oligomycin (Oligomycin, ATP synthase inhibitor) respectively and the expression levels of the pERM were examined for each group (fig. 10B). As shown in FIG. 10C, the relative fluorescence intensity of oocyte pERM was significantly reduced by both Rotenone (P < 0.01), DPI (P < 0.001), and oligomycin (P < 0.001) treatments compared to vitrified frozen oocytes. Among these, oligomycin almost eliminates the distribution of pERM in the cortical areas of oocytes. When the frozen oocytes were treated with PCB2 in combination with the three inhibitors described above, it was further examined whether PCB2 could improve the cortical dystonia after inhibitor treatment. The results show that PCB2 can rescue the reduction in cortical tension induced by rotenone (P < 0.001) or DPI (P < 0.001), but cannot rescue the reduction in cortical tension caused by oligomycin (P > 0.05). In addition, mRNA levels of Ndufv1 and Atp a1 after the combined treatment of PCB2 with rotenone and oligomycin were measured, respectively. As shown in FIGS. 2-10D and E, after rotenone treatment, the expression level of the frozen oocyte Ndefv 1 is remarkably reduced (P < 0.05), and the combined use of the PCB2 can improve the expression level of the Ndefv 1 (P < 0.05); however, the combined treatment of oligomycin and PCB2 with oligomycin had no effect on the mRNA level of the frozen oocyte Atp a 1. This suggests that PCB2 can regulate the cortical tension of oocytes via the mitochondrial electron transport chain and the pentose phosphate pathway.

Example 2 application of procyanidine B2 in GV-stage oocyte freezing

1. The experimental method comprises the following steps:

1.1GV phase oocyte retrieval: mice were intraperitoneally injected with 10IU of pregnant mare serum gonadotropin (Pregnant Mare Serum Gonadotropin, PMSG), and after 46-48h, the mice were sacrificed by cervical dislocation. The ovaries were removed and placed in HX trays equilibrated in advance in an incubator, and the follicles on the surface of the ovaries were all punctured with a 1mL syringe needle to obtain cumulus-oocyte complexes (Cumulus Oocyte Complex, COCs). After removing granular cells on the surface of oocyte, the obtained GV-stage oocyte is hatched into M16 culture solution (CaCl 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 2.101 G/L, naCl 5.53193G/L, BSA 4.0G/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), at 37℃and 5% CO ₂ Counting GVBD after 2h of culture in an incubatorAnd (5) counting the discharge rate of the polar body after the culturing for 12 hours.

1.2 oocyte freezing method was the same as in example 1.

1.3 immunofluorescent staining procedure the same as in example 1, different antibodies were selected according to the purpose of the experiment.

1.4 chromosome spreads

Washing oocyte with M2 solution for 3 times for later use; preparing nocodazole: diluting nocodazole with M2 solution to a working concentration of 2mg/mL, and making microdroplets; incubating the oocyte in a nocodazole microdroplet at 37 ℃ for 20min, eluting with M2 solution, and removing zona pellucida with pronase; after 3 washes of the zona pellucida-removed oocytes in M2, hypotonic treatment with 1% sodium citrate solution for about 5min until the oocytes swelled but did not rupture; treating the in situ hybridization glass slide by using a hydrophobic pen, then dripping about 20 mu L of spreading liquid into the pen, and blowing the expanded oocyte from the top end of the liquid level of the spreading liquid to enable the oocyte to freely fall onto the glass slide until the oocyte is completely dissolved; naturally air-drying the glass slide at room temperature, dropwise adding DAPI into a glass slide circle after air-drying, covering a cover glass, photographing under a laser confocal microscope, and counting the karyotype of the oocyte.

2. Results:

2.1 Effects of PCB2 on meiosis of frozen oocytes

To investigate whether PCB2 could alleviate meiotic damage caused by vitrification freezing to mouse oocytes, the proportion of GVBD, PBE was assessed for each group of oocytes with 5. Mu.g/mL of PCB2 added during in vitro maturation (FIG. 11A). As shown in FIG. 11B, freezing can significantly reduce GVBD (P < 0.05) and PBE (P < 0.05) ratios, while PCB2 can significantly increase the polar body discharge rate (P < 0.05) of oocytes in frozen groups, but has no effect on the occurrence of GVBD (P > 0.05).

2.2 Effect of PCB2 on frozen oocyte spindle migration

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

2.3 Effects of PCB2 on frozen oocyte spindle morphology and aneuploidy rate

MII phase spindle morphology detection As shown in FIG. 13A, C, fresh group oocyte spindles were in a regular barrel shape, while frozen oocyte spindles lost normal shape, compared with fresh oocytes, frozen oocyte spindles had significantly increased abnormal morphology ratio (P < 0.001), and PCB2 supplementation could significantly improve this phenomenon (P < 0.01). Abnormal morphology of the meiotic second metaphase spindle is often accompanied by high-frequency aneuploidy. As shown in FIG. 13B, D, the non-whole magnification of oocytes after freezing was significantly increased (P < 0.001) compared to fresh groups, and supplementation with PCB2 reduced the non-whole magnification of oocytes in frozen groups (P < 0.05).

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

1. The experimental method comprises the following steps:

1.1 oocyte retrieval the procedure of example 1 is followed.

1.2 oocyte freezing method was the same as in example 1.

1.3 parthenogenesis of oocytes

Balance embryo development disc: KSOM culture solution (EDTA 0.38mg/100mL, sodium pyruvate 2.2mg/100mL, glucose 3.6mg/100mL, KH) was 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 X501000. Mu.L/100 mL, non-essential amino acids X100500. Mu.L/100 mL, phenol red 50. Mu.L/100 mL, caCl ₂ ×2H ₂ O25 mg/100mL, fetal bovine serum 100mg/100 mL) was placed at 37℃in 5% CO ₂ Overnight equilibration in incubator; preparing and activating a mixed solution A: taking 890 μl of calcium-free HTF solution, adding 100 μl of SrCl ₂ And 10. Mu.L of CB were thoroughly mixed. The solution A was placed in a tray at 37℃with 5% CO ₂ Equilibrated in the incubator for 10 minutes. After balancing, the oocyte in MII stage is washed 3 times by A solution and then is placed in A solution for activation for 2.5 hours; preparing and activating a mixed solution B: taking 990 μl of HTF solution containing calcium, adding 10 μl of CB, and mixing thoroughly. The solution B was placed at 37℃in a tray with 5% CO ₂ Equilibrated in the incubator for 10 minutes. After balancing, the oocyte after the activation of the solution A is washed 3 times by the solution B, and then is placed in the solution B for activation for 3.5 hours; counting procaryon and embryo culture: and (3) after the prokaryotic cells activated by the liquid B are washed for 3 times by using the KSOM culture solution balanced in advance and in the evening, the prokaryotic occurrence rate is counted. The prokaryotes were placed in KSOM at 37℃with 5% CO ₂ Subsequent embryo culture is performed in an incubator. This time was counted as embryo development for 0h, and the number of blastomeres and blastula were counted after 24h and 96h, respectively.

1.4 blastocyst quality detection

The blastula is fixed in a fixing solution at room temperature for 30min, then is penetrated for 1h by using 0.5% Triton X-100-DPBS penetrating solution at room temperature, is transferred into an immunofluorescence sealing solution for sealing for 1h after being washed three times by using 0.1% Triton X-100-0.1% PVA-DPBS washing solution, and is then incubated overnight at 4 ℃ in Nanog (1:1000) and CDX2 (1:500) primary antibodies; incubation in FITC-488 and Alex-594 secondary antibody for 1h at room temperature was visualized by 5min after DAPI staining. The proportion of inner cell mass to total blasts was counted by counting inner cell mass and trophoblast cells separately. The experiment was repeated three times.

1.5 Metabolic histology detection of blastocyst culture

Taking out each group of embryos, leaving embryo culture solution, sucking the culture solution by a 1mL syringe, transferring the culture solution into a 1.5mL centrifuge tube, repeatedly taking 200 mu L of culture solution each time, and preserving the culture solution in a refrigerator at-80 ℃; after all samples are collected, amino acid and saccharide targeted metabonomics sequencing in blastula culture solution is carried out.

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

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

2. Experimental results

2.1 Influence of PCB2 on parthenogenesis of frozen oocytes

Immunofluorescent staining of CDX2 (cell line specific marker for Trophoblast (TE)) and Nanog (cell line specific marker for Inner Cell Mass (ICM)) as shown in FIG. 14A, it was found that the ratio of total ICM to TE (P < 0.001) and the ratio of ICM to total cell number (P < 0.001) of the parthenogenetic activated blastula of the frozen oocyte were significantly lower than in the fresh group, and the blastula quality after PCB2 treatment was greatly improved (FIGS. 14B-C). In the PCB2 treatment, both the ICM to TE ratio (P < 0.001) and the ICM to total cell number ratio (P < 0.001) were significantly increased, indicating a significant improvement in blastocyst quality.

2.2 Influence of PCB2 on metabolism of frozen oocytes in parthenogenesis of embryo

The samples were collected as shown in fig. 15A for the media-line targeted metabonomics analysis on day 4 after parthenogenetic activation of the frozen oocytes in the fresh, frozen and PCB2 treated groups. The results of the targeted metabolome of amino acids showed that the relative content of L-hydroxyproline, L-citrulline, and L-kynurenine in blastula cultures of frozen groups was significantly reduced (FIGS. 15B-E). But only the relative content of L-glutamic acid was significantly affected in the PCB2 treated 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 was primarily involved in sugar metabolism to regulate embryo development.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (5)

1. A cryopreservation solution for an oocyte in an MII stage, which is characterized by comprising a freezing balance solution and a vitrification freezing solution, wherein the freezing balance solution and the vitrification freezing solution both contain procyanidine B2;

the concentration of the procyanidine B2 in the freezing balance liquid and the vitrification freezing liquid is 5 mug/mL;

the freezing balance liquid also contains dimethyl sulfoxide and ethylene glycol;

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

2. The thawing solution for cryopreservation of the MII-stage oocyte is characterized by being PBS (phosphate buffered saline) solution containing procyanidine B2, wherein the concentration of procyanidine B2 in the thawing solution is 5 mug/mL.

3. The recovery liquid after the MII-stage oocyte is thawed is M2 liquid containing procyanidine B2, and the concentration of procyanidine B2 in the recovery liquid is 5 mug/mL.

4. A method of cryopreserving an MII-stage oocyte, the method comprising: firstly placing the oocyte in the freezing balance liquid according to claim 1, transferring the oocyte into the vitrification freezing liquid according to claim 1 after the balancing treatment, then adding liquid nitrogen, and freezing and preserving; in thawing, the cryopreserved oocyte is thawed in the thawing solution according to claim 2, and recovered in the recovering solution according to claim 3 after thawing.

5. Use of procyanidine B2, the cryopreservation solution of claim 1, the thawing solution of claim 2 or the post-thawing recovery solution of claim 3 in the cryopreservation of stage MII oocytes.

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