CN110484494A - A method of improving Porcine In Vitro Maturation Oocytes quality - Google Patents

Abstract

The invention discloses a kind of methods for improving Porcine In Vitro Maturation Oocytes quality, pre-process pig GV phase egg mother cell using methyl-B-cyclodextrin before glass freezing.Preferably, the concentration of methyl-B-cyclodextrin is 3~8mg/mL, and the pretreated time is 5~6.5min.The present invention directlys adopt methyl-B-cyclodextrin and handles immature cumulus oocytes complesxes, then glass freezing is carried out again, In-vitro maturation is carried out after defrosting again, it was found that adding exogenous cholesterol before glass freezing enhances the quality of the porcine oocytes of maturation in vitro after GV phase glass freezing, be conducive to the long-term preservation of porcine oocytes.

Description

A method for improving the quality of in vitro mature porcine oocytes

technical field

The present invention relates to a method for improving the quality of in vitro mature pig oocytes.

Background technique

Animal oocytes are widely used in processes such as in vitro embryo production, nuclear transfer, and gene bank preservation, and as a result, researchers have developed considerable interest in several areas of oocyte cryopreservation. The preservation of pig oocytes has been hindered for the past 20 years. In recent years, several attempts have been made to cryopreserve porcine oocytes with varying results. The successful cryopreservation of porcine oocytes is of great significance to the conservation of genetic resources and assisted reproductive technology. Although porcine oocytes are highly sensitive to low temperature, the development of vitrification techniques in recent years has improved results. Somfai's study showed successful production of piglets from cryopreserved GV-stage oocytes. Gajda's study showed successful production of piglets from cryopreserved MII stage oocytes. Although vitrification of GV-stage porcine oocytes can produce offspring, the rate of embryonic development remains low. Appeltant et al. Therefore, continued efforts must be made to optimize the vitrification process.

During cryopreservation of mammalian oocytes, an important site of cryopreservation damage is the cytoplasmic membrane, especially the lipids in the membrane. Lowering the temperature causes lipids to undergo a transition from a liquid crystal phase to a gel phase (lipid phase transition), which can lead to decreased membrane integrity and cell death. In this regard, the membrane cholesterol:phospholipid (C:P) ratio is an important determinant of membrane fluidity and stability during cryopreservation. Membranes with high cholesterol concentrations (high cholesterol membrane:phospholipid ratio) are more fluid at lower temperatures and are therefore less sensitive to freezing.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to overcome the defect of low survival rate of pig oocytes after freezing in the prior art, and to provide a method for improving the quality of in vitro mature pig oocytes.

In order to solve the above-mentioned technical problems, the present invention provides the following technical solutions:

A method for improving the quality of in vitro mature porcine oocytes by pretreating porcine GV stage oocytes with methyl-β-cyclodextrin prior to vitrification.

Further, the concentration of methyl-β-cyclodextrin is 3-8 mg/mL. Preferably, the concentration of methyl-β-cyclodextrin is 5mg/mL.

Further, the preprocessing time is 5-6.5min.

The beneficial effects achieved by the present invention are as follows: the present invention directly uses methyl-β-cyclodextrin to treat the immature cumulus-oocyte complex, then conducts vitrification and freezing, and then conducts in vitro mature culture after thawing, and finds that The addition of exogenous cholesterol before vitrification enhanced the quality of porcine oocytes matured in vitro after vitrification at the GV stage, which was beneficial for long-term preservation of porcine oocytes.

Detailed ways

The preferred embodiments of the present invention will be described below, and it should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, but not to limit the present invention.

Example

1.1 Preparation of relevant solutions

The minimal medium for vitrification and thawing solutions was HEPES-buffered TCM199 with 20% FBS. The minimal medium was supplemented with different concentrations (0, 0.5, 5 and 10 mg/mL) of CLC. The equilibration solution consisted of 7.5% ethylene glycol (EG), 7.5% dimethyl sulfoxide (DMSO) and TCM199 with 20% FBS. The vitrification solution consisted of TCM199 containing 15% EG, 15% DMSO, 20% FBS, 0.4M sucrose. Thaw I: Add 0.5M sucrose in TCM199 with 20% FBS, Thaw II consist of 0.25M sucrose in TCM199 with 20% FBS. The incubation solution was TCM199 containing 10% FBS.

TCM 199 (with 10% FBS) supplemented with 3.05mM D-glucose, 0.91mM sodium pyruvate, 25.07mM sodium bicarbonate, 0.1% polyvinyl alcohol, 75ug/ml penicillin, 50μg/ml streptomycin, 10IU/ml hCG , 10IU/ml eCG and 10ng/ml EGF for oocyte maturation culture. ZP dissolving solution: dissolve 0.06 g of sodium dihydrogen phosphate in deionized water, dilute to 100 ml, prepare 5 mM sodium dihydrogen phosphate buffer, and adjust to pH 2.5. The blocking solution for western blotting contained 5 g of nonfat dry milk dissolved in 100 mL of Tris-buffered saline Tween (10×TBS) and stored at 4°C until use.

Sperm capacitation fluid refers to García et al.: 96 mM NaCl, 4.7 mM KCl, 0.4 mM MgSO ₄ , 0.3 mM NaH ₂ PO ₄ , 5.5 mM glucose, 1 mM sodium pyruvate, 21.6 mM sodium lactate, 0.5 mM CaCl ₂ , 10 mM NaHCO ₃ , 20 mM HEPES (pH 7.45) and 3 mg/ml BSA. Embryo medium (NCSU23): 0.6355g NaCl, 0.2105g _NaHCO3 , 0.0356g _KCl , _{0.0162g KH2PO4} , _{0.0142g MgSO4} , 0.025g CaCl2.2H2O, 0.100g Glucose, 0.0146gGlutamine, 0.0875g Taurine, 0.0545g Hypotaurine, 0.0065g Penicilin G sodiumsalt, 0.005g Streptomycin sulfate were dissolved in 100 ml deionized water (consisting of North Carolina State University 23 Development Medium (NCSU-23) supplemented with 0.4% BSA).

1.2 Collection of cumulus-oocyte complexes

The ovaries of the sows used in this experiment were taken from the slaughterhouse in Yanji City. After the ovaries were taken out of the sows, they were immediately placed in a thermos bottle filled with 0.9% normal saline at about 38°C and transported back to the laboratory. Dissection procedures for isolation of cumulus-oocyte complexes (COCs) were performed on a slide warmer platform set at 38.5°C. After the saline was aspirated, follicles 3-6 mm in each ovary were selected and the follicular fluid was individually aspirated using a 10 ml disposable syringe with an 18G needle and then gently transferred to a sterile 50 ml tube. After 30-60 min incubation in a 38°C water bath, the COCs and other pellets at the bottom of the tube were aspirated with a Pasteur pipette and then transferred to the TCM wash solution (at 38,5°C). Thereafter, high-quality COCs showing a consistent, uniform dark color and surrounded by three or more layers of cumulus granulosa cells were selected under an inverted microscope.

1.3 Incubation of cultured GV-stage oocytes with NBD-labeled nitrobenzoxadiazole (NBD)-labeled CLC

Cumulus cells were removed with 0.1% (w/v) hyaluronidase. To detect cholesterol binding to unvitrified GV-stage oocytes, oocytes were incubated in oocytes supplemented with 20% (v/v) FBS and 20% 22-N-(7-nitrobenzo-2-oxo) Hetero-1,3-diazol-4-yl)amino-23,24diyn-5-indol-3b-ol-labeled cholesterol (NBD-CLC) was incubated in TCM199 for 1 hr. NBD-CLCs were prepared as described by Horvath and Seidel et al. After 1 h, oocytes were washed in DPBS and 0.1% (w/v) BSA, fixed (4% paraformaldehyde fixative), and COCs and COCs were photographed using a Nikon Eclipse Epifluorescence E800 microscope with a 11001v2 blue filter. oocyte.

1.4 Vitrification of GV-stage porcine oocytes

The vitrification freeze equilibration process step was performed under a microscope at 38.5°C. The COCs were first placed in equilibration solution for 5 min, then transferred to vitrification solution, and after 30 s incubation, oocytes were loaded onto OPS and plunged into liquid nitrogen (LN2). The total exposure time of COCs transferred to the vitrification solution until they are thrown into liquid nitrogen should be less than 90 s.

Thawing and maturation culture of 1.5 GV stage porcine oocytes

The vitrified oocytes were placed in Thaw I for 5 min and then transferred to Thaw II for 5 min. Subsequently, oocytes were washed three times in 0.1% PBS-PVA and TCM199 containing 10% FBS, then transferred to oocyte maturation medium and cultured in 5% CO ₂ (38.5°C, 95% maximum saturated humidity environment) Incubate for 46-48 hours.

1.6 Assessing oocyte maturation and survival

After 46 hours in vitro, oocytes were treated with 0.1% hyaluronidase until most of the cumulus cells were removed. Oocyte maturation, such as expulsion of the first polar body, was measured under a microscope (400x).

1.7 Measurement of oocyte mitochondrial membrane potential

After removal of cumulus cells, MII oocytes matured in vitro were washed in PBS buffer and then placed in a mixture of 1 ml cell culture medium (maturation solution) and 1 ml JC-1 staining working solution (configured according to the kit instructions) . The staining medium was mixed well and then incubated in a 37°C incubator (5% CO ₂ , 95% maximum saturated humidity environment) for 20 minutes. Aspirate the supernatant, then wash twice in pre-chilled JC-1 staining buffer. Finally, the stained oocytes were mixed with 2 ml of cell culture medium and observed under a fluorescence microscope in the dark.

1.8 Measurement of pro-apoptotic gene mRNA expression

After 46 hours of oocyte IVM treatment and removal of cumulus cells, use Oocytes were lysed by SuperTotal RNAExtraction Kit (Promega, Shanghai, China). DNAse I incubation buffer was then added to remove any genomic DNA contamination, and the wash was repeated with RNA wash buffer until high purity RNA was obtained. The extracted RNA was reverse transcribed from cDNA according to the instructions of TaKaRa's Prime Script ^™ RT Master Mix kit (catalog number: RR036A). Determine the cDNA concentration of the samples. The cDNA is used as a template to amplify the target gene, and the resulting product is amplified.

1.9 Lysis of the zona pellucida of oocytes

Samples containing 200 to 300 MII oocytes were washed three times in 0.1% PVA-PBS and then transferred to 0.6 ml centrifuge tubes. After removing excess buffer, add 20 μl of ZP lysis solution to each tube to dissolve the zona pellucida, then incubate in a 70° C. water bath for 90 minutes. When observed under a microscope, it was confirmed that the zona pellucida was completely dissolved. Samples were collected and centrifuged at 5000 xg for 5 minutes. The supernatant was then aliquoted and stored at -20°C until analysis.

1.10 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (kit, P0012A)

15% SDS-PAGE separating gels were prepared in-house. Protein samples were mixed with sample loading buffer and then denatured at 99°C for 5 minutes. After cooling to room temperature, 20-25 μl of protein sample was loaded into each well of the gel. Electrophoresis was performed at 100 V and stopped when the bromophenol blue dye front was approximately 2 cm from the bottom of the gel.

1.11 Western blotting

After SDS-PAGE, PVDF membranes (Millipore ISEQ00010) were cut to the size of SDS-PAGE gels, immersed in methanol for 5 minutes, and then immersed in membrane transfer buffer (P0021B) for 3-5 minutes. The proteins in the gel were transferred to PVDF membranes at 25 V for 90 min using a semi-dry membrane transfer apparatus.

After protein transfer, PVDF membranes were washed three times (10 min each) with TBST buffer (Tris buffered saline, Tween-20) and then incubated in blocking solution with shaking at room temperature for 2 hours. After this time, the blocking solution was removed and the membrane was washed three times (10 minutes) with TBST. The PVDF membrane was then placed in a petri dish containing a solution of rabbit polyclonal antibody (AB1690) against ubiquitin protein (diluted at 1:2000) and incubated at 4°C for 12 hours. The antibody was poured and the membrane was washed three times (10 minutes) with TBST. Membranes were then incubated with rabbit IgG peroxidase secondary antibody (A0545) (1:500 dilution) for 2 hours at room temperature, followed by three washes (10 minutes) in TBST. Image J was used to quantify the expression levels of different proteins and the chemiluminescence intensity of each protein band. The ubiquitination level of ZP protein was normalized by the amount of GAPDH protein.

1.12 In vitro fertilization and embryo culture

Transfer the mature oocytes into the capacitation medium, put 15 cells in each droplet, and then inject 50μl of capacitated 1x10 ⁶ cell/ml semen with a pipette, and put them in 38.5°C, 5% CO2, 95 Fertilize for 4-6 hours in an incubator with % maximum saturated humidity. After 4-6 hours, oocytes were washed 4 times in 0.1% PBS-PVA and fixed in 5% paraformaldehyde for 30 minutes at room temperature. Thereafter, oocytes were incubated with 5 μg/ml Hoechst 33342 for 15 min in the dark, washed twice in 0.1% PBS-PVA solution, and then observed under a nu-425-600e fluorescence microscope (Leica, Germany) (UV ): excitation wavelength: 330nm-400nm, emission wavelength: 425nm) to determine the number of spermatozoa attached to the zona pellucida per oocyte. The fertilized oocytes were transferred into embryo medium, 10 per droplet, and cultured in an incubator at 38.5° C., 5% CO ₂ , and 95% maximum saturated humidity. After 48 hours, the cleavage was checked, and then transferred to a new embryo medium to continue culturing until the seventh day.

1.13 Statistical analysis

Experiments were repeated three times, Western blot band density was determined using Image J software, all data were analyzed using ANOVA with SPSS19, Duncan's multiple comparison test was used to compare means, and P<0.05 was considered significant.

2 results

2.1 Assessment of cholesterol fluorescence levels

The results showed that oocytes that were not vitrified and incubated with NBD-CLC showed no fluorescence; oocytes that were not vitrified and incubated with NBD-CLC showed strong fluorescence; those that were vitrified and incubated with NBD-CLC showed strong fluorescence. Oocytes show weak fluorescence.

2.2 In vitro maturation rate of GV stage oocytes

When porcine GV-stage oocytes were treated with different concentrations of CLC (0.5, 5 and 10 mg/ml) in vitrification solution, the in vitro maturation rate of vitrified oocytes was slightly higher than that in the unfrozen treatment group, where 5 and 10 mg/ml The maturity rate of CLC treatment group was significantly higher than 0 and 0.5mg/ml CLC vitrification treatment groups (P<0.05).

2.3 The effect of CLC on the mitochondrial membrane potential of porcine oocytes vitrified at GV stage after maturation

The red fluorescence intensity of oocytes in the 5mg/ml CLC-treated group was significantly higher than that in the 0.5mg/ml, 10mg/ml and 0mg/ml CLC groups in the vitrification-treated group, although the fluorescence was significantly lower than that in the non-vitrified group shown in comparison. The weakest fluorescent signal in the control group indicated some damage to the mitochondrial membrane of these oocytes, resulting in reduced mitochondrial activity. The 5mg/ml CLC treatment group also showed stronger red-green fluorescence than the other two treatment groups (P<0.05). Taken together, these results indicate that 5 mg/ml CLC treatment provided the highest level of protection against oocyte damage during vitrification, and thus these cells were able to maintain the highest level of mitochondrial activity.

2.4 mRNA expression levels of pro-apoptotic genes in porcine oocytes vitrified at GV stage after maturation

The expression levels of apoptosis genes in oocytes in the vitrification treatment group were significantly higher than those in the non-vitrification treatment group, among which, the gene expressions of Caspase3, Caspase8 and Caspase9 in oocytes in the 5 and 10 mg/ml CLC treatment groups were significantly lower than 0. and 0.5mg/ml CLC treatment group (P<0.01).

2.5 Effect of CLC on ZP protein ubiquitination after maturation of vitrified porcine oocytes at GV stage

Western blotting results showed that the three ZP proteins of 61kDa (ZP1), 80kDa (ZP2) and 106kDa (ZP3) underwent different degrees of protein ubiquitination during maturation. Compared with 0.5mg/ml CLC, 10mg/ml CLC and vitrification control group, 5mg/ml CLC-treated oocytes had higher levels of total ZP protein ubiquitination, especially ZP3 protein. These results suggest that these ZP proteins undergo protein ubiquitination during in vitro maturation of porcine oocytes.

2.6 The effect of CLC on embryonic development of pig oocytes

This indicated that the binding values of sperm and oocytes in the 0.5, 5 and 10 mg/mL CLC-treated groups were significantly higher than those in the vitrified control group (P<0.01). The blastocyst rate of the oocytes in the vitrified group was significantly lower than that in the non-vitrified group, and the blastocyst rate of the oocytes in the CLC-treated group during vitrification was slightly higher than that in the non-vitrified group.

3.1 The effect of CLC on oocyte survival and maturation rate and embryonic development

The results of this study showed that the survival rate of oocytes in the vitrification treatment group was lower than that in the unfrozen control group, and the maturation rate of oocytes in the 5 mg/mL and 10 mg/mL LC vitrification treatment groups was slightly higher than that in the unfrozen treatment group . However, the embryonic development rate of oocytes in the vitrified group was significantly lower than that in the non-vitrified group, suggesting that CLC could improve the maturation rate of vitrified GV oocytes, but not the embryonic development rate.

3.2 The effect of CLC on oocyte mitochondrial membrane potential

Pig oocytes are particularly sensitive to low temperature. It turns out that they basically lose their survival rate when exposed to low temperatures above 15°C for a short period of time. The high lipid content in porcine oocytes is considered to be one of the main reasons for their sensitivity to low temperature. Other causes, such as mitochondrial damage, may affect the survival or apoptosis of vitrified porcine oocytes.

Mitochondria are major cellular components associated with energy supply and organelle migration, therefore, mitochondrial damage or changes in mitochondrial distribution may have a strong impact on oocyte development. Jc-1 is widely used for the detection of mitochondrial ΔΨm, which can accumulate in the mitochondrial matrix and form aggregates from monomers and can be stained red under a fluorescence microscope. The ratio of red to green fluorescence intensities is often used to indicate the functional state of mitochondria.

The mitochondrial membrane potential ΔΨm is susceptible to damage during cryopreservation and thawing. When the mitochondrial transmembrane potential is low, green fluorescence should be generated throughout the cytoplasm. The results showed that the mitochondrial membrane potential of vitrified thawed oocytes was lower than that of unvitrified thawed oocytes. This result is consistent with that of Dai et al. Compared with other vitrification treatment groups, the mitochondrial ΔΨm of oocytes treated with 5 mg/ml CLC was significantly increased, indicating that exogenous CLC can relatively reduce cold-induced mitochondrial membrane damage in oocytes after infiltrating the cytoplasm. Mitochondria maintain normal activity.

3.3 The effect of CLC on the mRNA expression levels of pro-apoptotic genes

Vallorani et al. studied vitrified porcine oocytes by freezing method and found that vitrification can activate caspase activity, loss of mitochondrial membrane potential and induce apoptosis. Vitrified MII stage porcine oocytes are characterized by reduced developmental capacity associated with activation of apoptotic pathways Elisa et al. Huang et al pointed out that adding antioxidants can reduce oxidative damage and reduce the rate of apoptosis.

In the death receptor-mediated extrinsic apoptotic pathway, a death ligand binds to its receptor and activates caspase 8 through autocatalysis, which in turn cleaves and activates caspase 3 or 7 through autocatalysis to stimulate apoptosis. As the initiator of extrinsic apoptosis pathway, caspase 8 plays an important role in extrinsic apoptosis. After vitrification, the activity of caspase 8 was greatly increased. When a caspase 8 inhibitor was applied, not only the activity of caspase 8, but also the activity of caspase 3 was inhibited. These results suggest that the death receptor-mediated extrinsic apoptotic pathway promotes the occurrence of apoptosis after vitrification.

We analyzed the mRNA expression of pro-apoptotic genes in mature oocytes, and the results showed that the expression of apoptotic genes in oocytes in the vitrified group was significantly higher than that in the non-vitrified group. In the vitrification treatment group, the gene expression after 5 and 10 mg/ml CLC treatment was significantly lower than other groups (P<0.01). These observations suggest that the addition of CLC during vitrification modulates the apoptotic process and improves the resistance of porcine oocytes to cryopreservation-induced damage. This result is similar to that of Elisa et al.

3.4 The effect of CLC on the ubiquitination of ZP protein

Western blot analysis of porcine oocytes revealed that three ZP proteins of 61 kDa (ZP1), 80 kDa (ZP2), and 106 kDa (ZP3) underwent varying degrees of protein ubiquitination in response to CLC treatment. In the 5 mg/ml CLC-treated group, the total ubiquitination level of ZP proteins was significantly higher than that observed in the other groups, and among these proteins, ZP3 showed the highest level of ubiquitination modification. Our results suggest that exogenous CLCs infiltrating the zona pellucida prior to vitrification protected these tissues. Subsequently, thawed oocytes were able to undergo normal ubiquitination during the culture phase.

Observation of frostbite in oocytes, including damage to the zona pellucida, DNA, and aneuploid embryos, is helpful in assessing the quality of vitrified oocytes. The zona pellucida acts as a material exchange channel between the oocyte and the internal environment and plays an important role in sperm-egg fusion and embryonic development. It has been reported that vitrification enhanced the hardness of the zona pellucida, increased multiple sperm fertilization, and decreased in vitro fertilization fertilization rates.

The zona pellucida surrounding mammalian oocytes functions in various aspects of fertilization. Previous studies have shown that (1) the proteasome is present on the acrosome and the acrosome surface of mammalian sperm, and (2) ubiquitinated proteins are present in mammalian egg cell membranes. Ubiquitinated ZP proteins are hydrolyzed by the sperm-associated proteasome and are involved in sperm penetration of the zona pellucida matrix. Vitrification of oocytes induces biochemical changes related to the secondary structure of protein and carbohydrate residues.

In mammals, ZP proteins are ubiquitinated during oogenesis, sperm receptors are degraded during fertilization, and sperm-carried proteasomes have been shown to degrade ZP-associated sperm receptor proteins in mammals. We therefore propose that oocytes undergo ubiquitination during maturation culture and that ubiquitinated ZP3 may act as a receptor for sperm. Although ubiquitinated ZP proteins have been observed to be hydrolyzed by sperm-associated proteasomes and involved in porcine sperm penetration through the zona pellucida, there are no reports of exogenous cholesterol binding to the zona pellucida and its role in conferring oocytes during slow freezing Cell hypothermia resistance. We speculate that the amount of cholesterol used has a significant effect on these processes, so we intend to conduct further studies aimed at developing an implementation procedure for oocyte cryopreservation.

Finally, it should be noted that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, the The technical solutions described in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (4)

1. a kind of method for improving Porcine In Vitro Maturation Oocytes quality, which is characterized in that use methyl-before glass freezing Beta-cyclodextrin pre-processes pig GV phase egg mother cell.

2. improving the method for Porcine In Vitro Maturation Oocytes quality as described in claim 1, which is characterized in that methyl-β-ring The concentration of dextrin is 3~8mg/mL.

3. improving the method for Porcine In Vitro Maturation Oocytes quality as described in claim 1, which is characterized in that methyl-β-ring The concentration of dextrin is 5mg/mL.

4. the method as described in any one of claims 1-3 for improving Porcine In Vitro Maturation Oocytes quality, which is characterized in that pre- The time of processing is 5~6.5min.