CN114350600B

CN114350600B - Culture solution for improving in-vitro culture quality of frozen oocytes and application of culture solution

Info

Publication number: CN114350600B
Application number: CN202210097053.4A
Authority: CN
Inventors: 傅祥伟; 李俊; 杜幸柱; 颛清芮; 侯云鹏
Original assignee: FIRST HOSPITAL OF HEBEI MEDICAL UNIVERSITY; China Agricultural University
Current assignee: FIRST HOSPITAL OF HEBEI MEDICAL UNIVERSITY; China Agricultural University
Priority date: 2022-01-26
Filing date: 2022-01-26
Publication date: 2023-08-18
Anticipated expiration: 2042-01-26
Also published as: CN114350600A

Abstract

The invention relates to a culture solution for improving the in vitro culture quality of frozen oocytes and application thereof. According to the culture solution for improving the in-vitro culture quality of the frozen oocytes, concanavalin A is added into the M16 culture solution, and the mechanical properties of the frozen oocytes are recovered by adjusting the cortical tension, so that the in-vitro culture quality of the frozen oocytes is improved. The invention provides a technical route for improving the in-vitro culture quality of the frozen oocyte by adjusting the mechanical characteristics of the frozen oocyte and the method for improving the in-vitro culture quality of the frozen oocyte by adjusting the cortical tension for the first time. The present invention also gives an addition concentration of ConA in the M16 broth of 1-100. Mu.g/ml, preferably 10. Mu.g/ml.

Description

Culture solution for improving in-vitro culture quality of frozen oocytes and application of culture solution

Technical Field

The invention relates to the technical field of propagation, in particular to a culture solution for improving the in-vitro culture quality of frozen oocytes and application thereof.

Technical Field

The oocyte freezing technology is an important reproductive biology technology, is widely applied to germplasm resource preservation, fertility preservation and auxiliary reproductive technology, but has the problems of development potential reduction and the like after thawing, and is reported to obtain offspring by freezing at least 20 mature oocytes, so that the wide application of the technology is severely restricted, the improvement of the utilization efficiency of frozen oocytes is a problem to be solved urgently, and the deep analysis of thawing damage mechanisms is a precondition for improving the effective utilization of the frozen oocytes.

Oocyte development underwent meiosis from GV, GVHD (2.5 h after the beginning of in vitro maturation of oocytes) to MII (12 h after the beginning of in vitro maturation of oocytes), and our earlier studies found that the aneuploidy proportion was significantly increased after in vitro maturation of frozen GV-stage oocytes, and kinetochore-microtubule (KT-MT) misconnection could lead to incorrect chromosome segregation. The cortex is located under the plasma membrane and plays an important role in maintaining cell shape and mechanical properties. In vitrification freezing, oocytes undergo shrink-expansion morphological change in volume in the dehydration-rehydration process, and severe volume change of the oocytes is likely to cause change of cell mechanical force, and research indicates that freezing has the greatest damage to cytoskeletons and mitochondrial functions of the oocytes, but the influence of cortical mechanical force change on the quality of frozen oocytes is not yet reported at present.

Cortical tension is a highly sensitive reading of the contractility of the cortical cytoskeleton, reflecting the biochemical and structural characteristics of the cortex. Oocyte cortical tension is mainly mediated by ERM family proteins, myosin-II, actin. The Ezrin/Radixin/Moesin (ERM) family and the non-muscle myosin II are two important proteins that regulate the cortical tension of oocytes, and participate in the establishment of cell polarity by regulating mechanical properties to promote the meiosis process. Phosphorylated ERM (phosphor-ERMs, pERM) is an active form of ERM, whereas phosphorylated myosin-regulated light chain proteins (phosphorylation of myosin regulatory light chain, pMRLC) can regulate myosin II activity, where the level of pERM is positively correlated with cortical tension and the level of distribution of pMRLC in the cytoplasm is negatively correlated with cortical tension. Remodeling of cortical areas and changes in mechanical properties during oocyte development are important for the completion of meiosis, fertilization, and embryo development.

At present, the existing freezing and thawing agents in the market are generally permeability and non-permeability cryoprotectants, and by replacing moisture in oocytes, ice crystal formation in the freezing and thawing process is prevented so as to achieve the purpose of protecting the oocytes, and no cryopreservation system for adjusting cortical tension to improve the quality of frozen oocytes is available.

Disclosure of Invention

In order to solve the problems, the invention provides a culture solution for improving the in-vitro culture quality of frozen oocytes and application thereof.

According to the culture solution for improving the in-vitro culture quality of the frozen oocytes, concanavalin A is added into the M16 culture solution, and the mechanical properties of the frozen oocytes are recovered by adjusting the cortical tension, so that the in-vitro culture quality of the frozen oocytes is improved.

Further, the concentration of concanavalin A is 1-100 mug/ml.

Further, the concentration of concanavalin A added was 10. Mu.g/ml.

The invention also provides a method for improving the in vitro culture quality of frozen oocytes, which comprises the following steps:

s1, collecting oocytes in the GV period and placing the oocytes in M2 liquid drops for standby;

s2, transferring the oocyte into a balance liquid for 30 seconds, transferring the oocyte into a vitrification refrigerating liquid for 20-30 seconds, rapidly placing the oocyte at the front end of a carrier rod, and adding liquid nitrogen;

s3, when thawing, rapidly taking the carrier rod of S2 out of liquid nitrogen, placing the carrier rod into PBS containing 0.5M sucrose for 5min, and then cleaning the carrier rod with M2 liquid for three times for later use;

s4, placing the oocyte obtained in the S3 into the novel in-vitro maturation culture solution for improving the quality of frozen oocyte culture, and covering paraffin oil in an incubator for in-vitro maturation culture.

Further, in the step S2, the time from the start of entering the vitrification frozen solution to the time of throwing in the liquid nitrogen is controlled within 25 seconds.

Further, the equilibration solution was PBS solution containing 10% DMSO and 10% EG.

Further, the vitrification frozen solution is PBS solution containing 15% EG, 15% DMSO, 0.5M sucrose and 30% Ficoll.

The beneficial effects of the invention are as follows:

1. the technical route for improving the in vitro culture quality of the frozen oocytes by adjusting the cortical tension to recover the mechanical characteristics of the frozen oocytes is first proposed. The invention clearly can recover the mechanical characteristics of the frozen oocyte by adjusting the cortical tension, enhance the activity of the spindle body assembly test point, reduce the aneuploidy proportion and further improve the internal quality of the in vitro culture of the frozen oocyte. The invention is based on the recognition that cytoprotective agent toxicity and cytoplasmic osmotic damage occurring in vitrification freezing can cause damage to oocytes, especially cytoskeleton; the oocyte volume undergoes the process of shrink-re-expansion during the freeze-thaw process, and the cell shape and mechanical mechanics change, so that the mechanical properties of the frozen oocyte can be recovered by adjusting the cortical tension. But the cortical tension plays an important role in the meiosis of the oocyte of the mouse, the cortical tension is reduced by 6 times during the development process of the first meiosis period and the second meiosis period (MII period), and the cortical tension is increased by 1.6 times after fertilization, which indicates that the reduction of the cortical tension in the MII period is necessary for the normal meiosis of the oocyte, and the excessive or the low cortical tension can cause the abnormal spindle migration, and the artificial increase of the cortical tension of the oocyte can cause the abnormal arrangement of chromosomes, the abnormal separation of chromosomes and aneuploidy. The correct arrangement and accurate separation of chromosomes are the key to ensuring oocyte maturation, and the spindle assembly inspection point can ensure the accurate separation of chromosomes. The chromosome is separated by spindle traction composed of microtubules, the chromosome is connected with the spindle through an kinetochore protein complex, and kinetochores are assembled on centromere chromatin. If the kinetochore does not bind to microtubules, the late promoting complex is inhibited, preventing the meiosis process. Spindle assembly checkpoint proteins Mad2, bubR1, etc. can monitor kinetochore-microtubule ligation, and only after all kinetochore-microtubules are correctly joined will meiosis be initiated, whereas decreased checkpoint protein activity can result in increased aneuploidy. An increase in the frozen oocyte aneuploidy proportion is a significant cause of reduced oocyte quality. At present, no report of adjusting the cortical tension applied to the in vitro maturation culture of frozen oocytes is yet seen. In combination with the phenotype of cortical tension reduction in the development of frozen oocytes, a mouse oocyte is taken as a model, a cortical tension enhancer ConA is added on the basis of a traditional defreezed culture solution for culturing the frozen oocytes, the mechanical characteristics of the frozen oocytes are recovered, the activity of a spindle assembly test point is enhanced, the aneuploidy proportion is reduced, and the internal quality of the oocytes is further improved.

2. The application of Concanavalin A (ConA) in improving the in vitro culture quality of frozen oocytes is clearly proposed. Concanavalin A (ConA), a tetravalent lectin, binds to membrane glycosylated proteins and crosslinks on the cell surface, conA increases the cortical tension of the MII stage oocyte by 69%. According to the invention, conA with proper concentration is added into a culture solution for in-vitro culture of frozen oocytes, so that the cortical tension of the oocytes is regulated, the correct positioning of spindle bodies is facilitated, the protein activity of a spindle body assembly test point is enhanced, the accurate connection of a kinetochore-microtubule is ensured, the accurate separation of chromosomes is promoted, the aneuploidy proportion of the oocytes is reduced, the subsequent development of the oocytes is improved, the quality of the oocytes is improved, and the utilization efficiency of the frozen oocytes is improved.

3. The appropriate concentration of ConA addition in the in vitro maturation medium was determined. The present invention gives ConA at an addition concentration of 1-100. Mu.g/ml, preferably 10. Mu.g/ml, in the M16 broth.

4. The effect of ConA on the in vitro maturation quality of frozen oocytes was clarified. As can be seen in the examples of the present invention, freezing can significantly reduce oocyte quality: the occurrence rate of PBE (P < 0.01) of frozen oocytes in GV period after in vitro maturation culture is obviously lower than that of fresh oocytes, and the cleavage rate and blastula development rate after parthenogenesis activation are obviously reduced, which is shown in the following conditions: the Mad2/CREST (P < 0.001) and BubR1/CREST (P < 0.001) decreased significantly, the proportion of unconnected KT-MT increased significantly (P < 0.001), resulting in a significant increase in aneuploidy ratio (P < 0.01), and a significant decrease in post-developmental capacity such as cleavage rate (P < 0.001) and blastocyst rate (P < 0.001). The addition of 10 μg/ml ConA to conventional culture solution M16 can promote in vitro maturation of frozen oocytes in GV stage, increase spindle checkpoint protein activity (P < 0.001), reduce kinetochore-microtubule misconnection (P < 0.001), and increase whole ploidy ratio (P < 0.01) of frozen oocytes.

Drawings

FIG. 1 is a graph comparing the effect of ConA on oocyte maturation and development in vitro, wherein:

a1 is an in vitro maturation demonstration graph of oocytes in GV phase in different treatment groups;

b1 is an example graph of in vitro development of oocytes in MII phase of different treatment groups;

c1 is a PBE rate analysis contrast graph of different treatment groups;

d1 is a parthenogenesis activated cleavage rate analysis contrast chart of different treatment groups;

e1 is a parthenogenesis activated blastula rate analysis contrast chart of different treatment groups;

FIG. 2 is a graph showing the effect of freezing on the cortical tension of oocytes at the GV stage, wherein:

a2 is an exemplary graph of petm immunofluorescence staining from different treatment groups;

b2 is an exemplary graph of pMRLC immunofluorescence staining for different treatment groups;

c2 is a graph of analysis and comparison of relative fluorescence intensity of petm in different treatment groups;

d2 is a comparative graph of pMRLC relative fluorescence intensity analysis for different treatment groups;

FIG. 3 is a graph showing the effect of freezing on the cortical tension of oocytes at stage MII, wherein;

a3 is an exemplary graph of petm immunofluorescence staining for different treatment groups;

b3 is an exemplary graph of pMRLC immunofluorescence staining for different treatment groups;

c3 is a graph of analysis and comparison of relative fluorescence intensity of petm in different treatment groups;

d3 is a comparative graph of pMRLC relative fluorescence intensity analysis for different treatment groups;

FIG. 4 is a graph comparing the effect of ConA on the cortical tension of frozen oocytes, wherein;

a4 is an exemplary graph of petm immunofluorescence staining from different treatment groups;

b4 is an exemplary graph of pMRLC immunofluorescence staining for different treatment groups;

c4 is a graph of analysis and comparison of relative fluorescence intensity of petm in different treatment groups;

d4 is a comparative graph of pMRLC relative fluorescence intensity analysis for different treatment groups;

FIG. 5 is a graph comparing the effect of ConA on oocyte spindle positioning;

a5 is an exemplary diagram of spindle positioning of oocytes from different treatment groups;

b5 is a relative distance comparison analysis chart of oocyte spindles and cortex of different treatment groups;

FIG. 6 is a graph comparing the effect of ConA on the activity of an oocyte spindle assembly checkpoint protein, wherein:

a6 is an exemplary diagram of immunofluorescence staining of the spindle assembly checkpoint protein Mad2 of oocytes of different treatment groups;

b6 is an exemplary diagram of immunofluorescence staining of the spindle assembly checkpoint protein BubR1 of oocytes of different treatment groups;

c6 is a comparison chart of analysis of the ratio of the immune fluorescence intensity of the oocytes of different treatment groups, namely, mad 2/CREST;

d6 is an analysis and comparison chart of the ratio of the immunofluorescence intensities of the oocytes of different treatment groups, namely BubR 1/CREST;

FIG. 7 is a graph comparing the effect of ConA on the connection of oocyte kinetochore-microtubule (KT-MT), wherein;

a7 is an exemplary diagram of the connection of the kinetochore to the microtubules of oocytes in different treatment groups;

b7 is an analysis and comparison chart of the proportion of unconnected animal granules of oocytes in different treatment groups;

c7 is a chromosome proportion analysis comparison chart of the oocytes of different treatment groups which are not orderly arranged;

d7 is an analysis and comparison graph of abnormal proportions of the spindle body shape of the frozen oocyte after ConA treatment of the oocyte in different treatment groups;

FIG. 8 is a graph of the effect of ConA on oocyte aneuploidy versus graph, wherein:

a8 is an exemplary diagram of an oocyte euploid and an aneuploidy;

b8 is an analysis and comparison graph of the aneuploidy and the aneuploidy of the oocytes of different treatment groups;

FIG. 9 is a graph showing the effect of ConA on the expression of the F-actin and Mos pathway genes in oocytes, wherein:

a9 is an exemplary diagram of oocytes in different treatment groups;

b9 is a relative fluorescence intensity analysis contrast chart of cytoplasmic F-actin in oocytes of different treatment groups;

c9 is a graph for comparing the relative fluorescence intensity analysis of cortical F-actin in oocytes of different treatment groups;

d9 is a comparison graph of the mRNA level analysis of oocytes in different treatment groups;

e9 is a comparison graph of analysis of mRNA levels of oocytes Arpc2 of different treatment groups;

f9 is a comparison graph of analysis of the level of RhoA mRNA in oocytes of different treatment groups;

g9 is a comparison graph of the analysis of the mRNA level of the oocyte Myosin5b of different treatment groups.

Detailed Description

The invention is further illustrated by the following examples.

In the following experiments, the culture solutions of M2 and M16 are both traditional gamete and embryo in-vitro culture reagents, and are commercial reagents.

The M2 broth is a modified Krebs-Ringer solution with the following composition: 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, naCl 5.533g/L,60% sodium lactate 4.349ml/L, hepes 4.969g/L, caCl ₂ ×2H ₂ O 0.252g/L，BSA 4.0g/L。

M16 is an embryo in vitro culture reagent, and comprises the following components: caCl (CaCl) ₂ ×2H ₂ O 0.25137g/L，MgSO ₄ 0.1649g/L，KCl 0.35635g/L，KH ₂ PO ₄ 0.162g/L，NaHCO ₃ 2.101G/L NaCl 5.53193G/L, BSA 4.0G/L, D-glucose 1.0G/L, sodium phenolate red 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.

Example 1: oocyte collection and freezing

1.1 oocyte collection

GV phase oocyte collection: mice were intraperitoneally injected with 10IU of pregnant mare serum gonadotropin and after 48 hours, the ovarian tissue of the mice was collected. Ovaries were placed in M2 solution containing 2 μm milrinone and the follicles were pricked under a microscope using a 1mL syringe needle. The released GV-stage oocytes were collected and placed in a 35mm dish in 100. Mu.L of M2 droplets containing 2. Mu.M milrinone for later use.

MII phase oocyte collection: mice were intraperitoneally injected with 10IU of pregnant mare serum gonadotropin, 48h later, 10IU of HCG was intraperitoneally injected, 12-14h later, the mice were sacrificed by cervical dislocation, oviducts were taken out, the enlarged part was lacerated under a dissecting scope, the cumulus oocyte complex was obtained, and the MII-stage oocyte discharged from the first polar body was collected for standby after hyaluronidase treatment.

1.2 freezing of oocytes

The oocyte vitrification freezing and thawing method refers to the conventional steps of the subject group, and specifically comprises the following steps: oocytes were placed in ED solution, i.e., PBS solution containing 10% DMSO and 10% EG for 30sec, and then transferred to EDFS30 solution, i.e., PBS solution containing 15% EG, 15% DMSO, 0.5M sucrose and 30% Ficoll, and placed in front of a carrier rod, and liquid nitrogen was added thereto, and the time from the start of entering the EDFS30 solution to the addition of liquid nitrogen was controlled within 25 sec.

1.3 thawing frozen oocytes

During thawing, the carrying rod to be thawed is quickly taken out from liquid nitrogen, placed in PBS containing 0.5M sucrose for 5min, and then washed three times with M2 liquid for standby.

Example 2 comparative experiments of different concentration of cortical tension enhancer ConA on quality of in vitro culture of GV-stage frozen oocytes

2.1 Gv stage frozen oocyte in vitro maturation

And (3) respectively placing the thawed GV-stage oocytes into M16 solutions containing the cortical tension enhancers ConA with different concentrations, and covering paraffin oil in an incubator for in-vitro maturation.

The concentration of ConA in M16 solution was: 1. Mu.g/ml, 10. Mu.g/ml, 100. Mu.g/ml.

2.2 PBE observation method

PBE excretion was counted 12h after the GV stage oocyte began to mature in vitro.

2.3 data statistics method

All experiments were performed in at least three biological replicates, and all values were presented as mean ± standard error. All experimental data were subjected to unpaired two-tailed t-test using GraphPad Prism 8 software (GraphPad Software inc., san Diego, CA, USA), P <0.05 believes that the data were statistically different.

Results:

ConA can improve in vitro maturation rate of GV-stage frozen oocytes

Fresh oocyte: the incidence of PBE was 97.22%.

Freezing the oocyte: the incidence of PBE was 78.95%.

ConA addition group at a concentration of 1. Mu.g/ml: the incidence of PBE was 77.78%.

ConA addition group at a concentration of 10. Mu.g/ml: the incidence of PBE was 92.31%.

ConA addition group at a concentration of 100. Mu.g/ml: the PBE incidence was 69.23%.

As can be seen from the above, freezing can lead to a decrease in the proportion of PBE in the GV-stage oocyte, and 10 mug/ml ConA can effectively increase the in vitro maturation proportion of the GV-stage frozen oocyte.

EXAMPLE 3 comparative experiment of ConA addition on the influence of in vitro maturation and developability of frozen oocytes

3.1 thawing the frozen oocytes of example 1

3.2 In vitro maturation of GV-stage oocytes

Dividing GV oocytes into three groups, namely a fresh group, namely a fresh obtained GV oocyte, a frozen group, namely a thawed frozen oocyte obtained in the step 3.1, culturing the fresh group and the frozen group in an M16 solution, and a dosing group, namely the thawed frozen oocyte obtained in the step 3.1, in the M16 solution added with 10 mug/ml ConA, covering paraffin oil in an incubator for in vitro maturation.

3.3 Oocyte thawing recovery in MII stage

Thawing the MII-stage oocytes in example 1, putting the thawed MII-stage oocytes into M16 solutions containing 0 mug/ml and 10 mug/ml ConA for recovery culture for 1h, and carrying out parthenogenetic activation experiments with fresh MII-stage oocytes as a freezing group and a dosing group respectively.

3.4 Parthenogenesis of oocytes at MII stage

Placing the oocytes of the fresh group, the frozen group and the dosing group in the step 3.3 respectively in the Ca-free state ²⁺ Containing 10mM SrCl ₂ HTF solution of 5. Mu.g/mL CB at 37deg.C in 5% CO ₂ Incubation for 2.5h, transfer to HTF solution containing 5. Mu.g/mL CB after washing and incubation for 3.5h, activated oocytes in KSOM solution at 37℃in 5% CO ₂ And (4) culturing in the environment, and counting the cleavage rate and the blastocyst development rate at 24 hours and 96 hours respectively.

3.5 data statistics method

All experiments were performed in at least three biological replicates, and all values were presented as mean ± standard error. All experimental data were unpaired, double-tailed t-test using GraphPad Prism 8 software (GraphPad Software inc., san Diego, CA, USA) and plotted. P <0.05 considered the data statistically different.

3.6 results

3.6.1 ConA can improve in vitro maturation rate of GV-stage frozen oocytes

FIG. 1A1 is a graph showing in vitro maturation of GV-stage oocytes, wherein the occurrence rate of PBE of GV-stage frozen oocytes after conventional in vitro maturation culture is 83.99%, the occurrence rate of PBE of GV-stage fresh oocytes after conventional in vitro maturation culture is 97.22%, and the occurrence rate of PBE of GV-stage frozen oocytes after 10 μg/ml ConA is added during in vitro culture is 91.94%, as shown in FIG. 1C 1. It was demonstrated that the proportion of PBE in GV-stage frozen oocytes was significantly lower than in GV-stage fresh oocytes (P < 0.01), but ConA at 10. Mu.g/ml was effective in enhancing the in vitro maturation of GV-stage frozen oocytes (P < 0.05).

3.6.2 Effect of ConA on MII-stage frozen oocyte developmental Capacity

FIG. 1B1 is a diagram showing an example of the in vitro development of an MII stage oocyte, as shown in FIGS. 1D1 and E1, the parthenogenesis activation rate of the frozen oocyte in the MII stage is remarkably reduced (P < 0.001), the blastocyst development rate is remarkably reduced (P < 0.001), and the result shows that the freezing can cause the reduction of the development capacity of the oocyte in the MII stage. Compared with the frozen group, the frozen oocyte cleavage rate (P <0.001, figure 1D 1) and blastula development rate (P <0.01, figure 1E 1) of the dosing group are obviously improved, which suggests that ConA can obviously improve the subsequent development capacity of the oocyte in the MII stage.

Example 4 comparative experiment of the influence of freezing on the cortical tension of oocytes

4.1 thawing of frozen oocytes in GV and MII phases of example 1

4.2 cortical tension detection

The method for respectively detecting the cortical tension of the frozen oocytes in the GV stage and the MII stage comprises the following specific steps: the oocyte was fixed in 4% (w/v) paraformaldehyde at room temperature for 40min,0.5%Triton X-100 membrane permeation for 1h, blocked in 3% BSA at room temperature for 1h, incubated with primary antibody (anti-pERM, 1:600; anti-pMRLC) overnight at 4 ℃, thoroughly washed, incubated with secondary antibody at room temperature for 1h, finally stained with DAPI dye for 5min, tabletted, and observed under a laser confocal microscope.

4.3 statistical method of data

4.4 results:

4.4.1 freezing can significantly reduce the cortical tension of the oocyte in GV stage

As shown in fig. 2A2 and C2, the fluorescence intensity of the petm was significantly reduced (P < 0.001) after freezing of the GV stage oocyte, and as shown in fig. 2B2 and D2, the enrichment degree of the pMRLC in the GV stage frozen oocyte was significantly reduced (P < 0.01).

4.4.2 freezing can significantly reduce the cortical tension of the oocyte in MII stage

As shown in fig. 3A3 and C3, the fluorescence intensity of the pecm was significantly decreased (P < 0.001) after freezing of the MII stage oocyte, and as shown in fig. 3B3 and D3, the fluorescence intensity of pMRLC was significantly increased (P < 0.001) in the cytoplasm of the MII stage oocyte.

The results show that freezing can cause the cortical tension of frozen oocytes in GV and MII phases to be obviously reduced.

Example 5 comparative experiment of ConA effect on the cortical tension of frozen oocytes

5.1 thawing the GV-stage frozen oocytes of example 1

5.2 in vitro maturation of oocytes at GV stage

Dividing GV oocytes into three groups, namely a fresh group, namely fresh GV oocytes obtained, a frozen group, namely the thawed frozen oocytes obtained in the step 5.1, culturing the fresh group and the frozen group in an M16 solution, and a dosing group, namely the thawed frozen oocytes obtained in the step 5.1, in the M16 solution added with 10 mug/ml ConA, covering paraffin oil in an incubator for in vitro maturation.

5.3 cortical tension detection

The oocyte is subjected to in vitro maturation culture for about 2.5 hours and then is subjected to germinal vesicle rupture (GVBD), the oocyte which is continuously cultured for 6 hours (GVBD+6h) after germinal vesicle rupture is placed in 4% (w/v) paraformaldehyde for fixation at room temperature for 40min,0.5%Triton X-100 membrane permeation for 1 hour, the oocyte is sealed for 1 hour at room temperature in 3% BSA, the oocyte and primary antibody (anti-pERM, 1:600; anti-pMRLC) are incubated at 4 ℃ overnight, fully cleaned and then are incubated with secondary antibody at room temperature for 1 hour, finally, the oocyte is subjected to compression after being dyed with DAPI dye for 5 minutes, and the oocyte is observed under a laser confocal microscope.

5.4 data statistics method

5.5 results

5.5.1 Effect of ConA on expression of pERM in frozen oocytes

The oocyte pERM immunofluorescence staining assay is shown in FIG. 4A 4. The addition of 10 μg/ml ConA to the dosing group significantly increased the pERM fluorescence intensity of the frozen oocytes compared to the frozen group (P <0.01, FIG. 4C 4).

2. Effect of ConA on expression of frozen oocytes pMRLC

The oocyte pMRLC immunofluorescence staining assay is shown in FIG. 4B 4. The addition of 10 μg/ml ConA to the dosing group significantly reduced the fluorescence intensity of the frozen oocyte pMRLC in the cytoplasm compared to the frozen group (P <0.01, fig. 4D 4).

The results suggest that ConA can effectively restore cortical tension in the in vitro maturation process of frozen oocytes.

TABLE 1 comparison of data on ConA effects on the cortical tension and development of frozen oocytes in examples 3-5

Index (I)	Fresh group	Freezing group	Dosing group
				Gv stage pecm	1±0.07	0.70±0.03	/
GV-stage pMRLC	1.05±0.03	0.93±0.02	/
				GVBD+6h pERM	1±0.07	0.70±0.03	1.03±0.08
GVBD+6h pMRLC	1±0.04	1.16±0.05	0.98±0.03
				pERM in MII phase	1±0.04	0.68±0.04	/
MII phase pMRLC	1±0.03	1.59±0.04	/
				PBE(％)	97.22±1.61	83.99±1.38	91.94±1.80
Cleavage Rate (%)	99±1.00	86.72±0.82	95.27±0.32
				Blastula rate (%)	70.72±2.80	24.44±2.65	45.07±2.54

EXAMPLE 6 comparative experiment of ConA addition on the meiotic Effect of frozen oocytes

6.1 thawing the GV-stage frozen oocytes of example 1

6.2 In vitro maturation of GV-stage oocytes

Dividing GV oocytes into three groups, namely a fresh group, namely a fresh obtained GV oocyte, a frozen group, namely a thawed frozen oocyte obtained in the step 6.1, culturing the fresh group and the frozen group in an M16 solution, and a dosing group, namely the thawed frozen oocyte obtained in the step 6.1, in the M16 solution added with 10 mug/ml ConA, covering paraffin oil in an incubator for in vitro maturation.

Performing spindle body positioning analysis on the oocyte which is subjected to in vitro maturation culture for 9 hours (GV+9h), and performing chromosome spread detection on the oocyte which is subjected to in vitro maturation culture for 12 hours (GV+12h) and enters the MII stage; and (3) performing a granulosa microtubule ligation test on the oocytes entering the first meiosis metaphase, which are continuously cultured for 3 hours (GVBD+3h) after the germinal vesicle rupture, by performing a spindle checkpoint protein activity analysis on the oocytes which are continuously cultured for 6 hours (GVBD+6h) after the germinal vesicle rupture, wherein the germinal vesicle rupture occurs after the germinal vesicle rupture for about 2.5 hours.

6.3 immunofluorescent staining of oocytes

The oocytes were fixed in 4% (w/v) paraformaldehyde at room temperature for 40min,0.5%Triton X-100 permeabilization for 1h, blocked in 3% BSA at room temperature for 1h, and incubated with primary antibody (anti- α -tubulin,1:8000; anti-Mad2,1:200; anti-BubR1,1:100; nti-centromere, 1:200) overnight at 4℃for 1h after extensive washing (F-actin staining and F-actin incubation overnight at 4 ℃) and finally pressed into tablets after staining with DAPI dye for 5min and observation under a laser confocal microscope.

6.4 spread of oocyte chromosomes

Oocytes which have entered MII stage after the above in vitro maturation culture for 12h (GV+12h) were treated in M2 solution containing 0.5% pronase for 5min, and zona pellucida was digested. Preparing a paving fixing solution: 1% paraformaldehyde, 0.15% Triton X100 and 3mM DTT were dissolved in PBS and the pH was adjusted to 9.2 with NaOH solution. A square with a side length of 1cm was drawn on the slide glass with a hydrophobic pen, and 100. Mu.l of a fixing solution was dropped into the square. The oocytes were gently placed from above the drop and then left to air dry overnight at 4 ℃. Then DAPI is added into the square, and the square is placed for 5 minutes at room temperature and in dark place, and then the square is photographed under an inverted fluorescence microscope.

6.5 qPCR

The GVBD+3h oocytes were collected, RNA was extracted, reverse transcribed, and the sample cDNA was stored in a-80℃refrigerator for use, and synthetic primers were designed (primer sequences are shown in Table 2).

TABLE 2 primer sequences

RT-qPCR analysis was performed under standard conditions with a real-time PCR detection system using SYBR Premix Ex TaqTM (gold full). For example, 10. Mu.L of the reaction system was mixed with the reagents in an ice bath at 0℃according to Table 3.

TABLE 3 RT-qPCR reaction System

After mixing evenly, centrifuging, carrying out real-time fluorescence PCR amplification, and the procedure is as shown in Table 4:

TABLE 4 RT-qPCR reaction procedure

After the PCR reaction is finished, observing whether the curve is unimodal, and taking Ct values of three samples. GAPDH was used as an internal gene, 2 ^-△△Ct The relative expression content of the target genes is calculated respectively.

6.6 data statistics method

6.7 results

6.7.1 ConA effect on frozen oocyte spindle positioning

Spindle localization of oocytes was detected by immunofluorescent staining (fig. 5 A5). As shown in fig. 5B5, the relative distance of the frozen oocyte spindle from the cortex increases significantly (P < 0.01), while increasing the cortical tension decreases the relative distance of the spindle from the cortex significantly (P < 0.01).

6.7.2 Effect of ConA on frozen oocyte spindle assembly checkpoint protein expression

FIGS. 6A6 and B6 are diagrams showing examples of immunofluorescence staining of the frozen oocyte spindle assembly checkpoint protein Mad2 and BubR1 after fresh, frozen and dosed groups. As shown in FIGS. 6C6 and D6, the expression of Mad2 (P < 0.001), bubR1 (P < 0.001) was significantly decreased in frozen oocytes, and ConA treatment significantly increased the expression of Mad2 (P < 0.001) and BubR1 (P < 0.001) in frozen oocytes, indicating that freezing could cause meiosis in frozen oocytes.

6.7.3 Effect of ConA on frozen oocyte kinetochore-microtubule ligation

The ratio of unligated kinetochores (P <0.001, FIG. 7B 7), the ratio of misaligned chromosomes (P <0.001, FIG. 7C 7), the ratio of abnormal spindle shapes (P <0.001, FIG. 7D 7) and the ratio of unjoined kinetochores (P <0.001, FIG. 7B 7), the ratio of misaligned chromosomes (P <0.001, FIG. 7C 7) and the ratio of abnormal spindle shapes (P <0.001, FIG. 7D 7) in frozen oocytes were significantly increased by the dosing group as shown in FIG. 7A 7.

6.7.4 ConA Effect on frozen oocyte aneuploidy

Oocyte aneuploidy and aneuploidy analysis as shown in fig. 8A8, freezing can significantly increase the proportion of oocyte aneuploidy (P < 0.01), and ConA treatment can significantly decrease the proportion of frozen oocyte aneuploidy (P < 0.01), as shown in fig. 8B 8.

6.7.5 Effect of ConA on frozen oocyte F-actin and Mos pathway

The F-actin immunofluorescence staining of oocytes is shown in FIG. 9A 9. Statistical analysis of cytoplasmic and cortical F-actin fluorescence intensities revealed that freezing resulted in a significant decrease in cytoplasmic F-actin levels (P <0.05, fig. 9B 9), but no change in cortical F-actin levels (P >0.05, fig. 9C 9). qPCR examined mRNA levels of Mos pathway-associated genes, and it was found that freezing and increasing cortical tension did not affect mRNA levels of Mos (P >0.05, FIG. 9D 9) and Arpc2 (P >0.05, FIG. 9E 9). Freezing can result in significant upregulation of RhoA expression levels associated with myosin II phosphorylation, while increasing cortical tension can significantly decrease RhoA expression (P <0.05, fig. 9F 9). In addition, mRNA levels of Myosin5b associated with spindle migration were not affected by freezing and elevated cortical tension (P >0.05, fig. 9G 9).

From the above results, it was found that freezing can cause meiosis of the oocyte in GV stage, and the aneuploidy ratio was significantly increased. The addition of 10 mug/ml ConA in the M16 culture solution can enhance the cortical tension, facilitate the correct positioning of the spindle body, enhance the protein activity of the spindle body assembly inspection point, reduce the wrong connection proportion of kinetochore and microtubule and obviously reduce the occurrence rate of aneuploidy.

In summary, after GV-stage oocytes and MII-stage oocytes are frozen, the cortical tension is reduced, and after thawing, a cortical tension activator with a certain concentration is added into the culture solution, for example, 10 mu g/ml ConA can effectively improve the reduction of cortical tension caused by freezing, thereby reducing meiosis abnormality and improving the quality of frozen oocytes.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.

Claims

1. A culture solution for improving the in-vitro culture quality of frozen oocytes is characterized in that concanavalin A is added into an M16 culture solution, and the mechanical properties of the frozen oocytes are recovered by adjusting the cortical tension so as to improve the in-vitro culture quality of the frozen oocytes, wherein the adding concentration of the concanavalin A is 10 mug/ml.

2. A method for improving the quality of in vitro culture of frozen oocytes using the culture medium of claim 1, comprising the steps of:

s4, placing the oocyte obtained in the S3 into the culture solution for improving the in-vitro culture quality of the frozen oocyte, and covering paraffin oil in an incubator for in-vitro maturation culture.

3. The method of improving the quality of frozen oocyte in vitro culture according to claim 2, wherein said equilibration solution is a PBS solution containing 10% DMSO and 10% EG.

4. The method for improving the quality of in vitro culture of frozen oocytes according to claim 2, wherein the vitrification frozen solution is PBS solution containing 15% EG, 15% DMSO, 0.5M sucrose and 30% Ficoll.