CN108588011B - Method for improving in vitro fertilization capability of vitrified frozen oocyte - Google Patents
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Abstract
The invention provides a method for improving the fertilization capability of vitrified frozen bovine oocytes. Before vitrification freezing treatment, bovine oocytes are jointly treated by a solution containing gadolinium and CLC, and then vitrification freezing is carried out; after thawing, the cells are treated by methylated-beta-cyclodextrin, and then in vitro fertilization is carried out. The result shows that the method can obviously improve the cleavage rate and blastocyst rate of the vitrified frozen bovine oocyte, is higher than the in vitro fertilization efficiency of fresh bovine oocyte, has wide application prospect and is worthy of popularization.
Description
Technical Field
The invention relates to the field of artificial in vitro fertilization, in particular to a method for improving the in vitro fertilization capability of vitrified frozen oocytes.
Background
The oocyte vitrification freezing technology is an important component of livestock embryo biotechnology, removes the time and space limitation for in vitro embryo production, greatly promotes the cross-space-time efficient utilization of excellent female animal genetic resources, and has important significance for the long-term preservation of rare and endangered animal genetic resources (Zhushien, ever-stated, Wutong, Mengqing, Zhang faithful, Chengyufu. research of vitrification freezing bovine oocytes by OPS method, Chinese agricultural science, 2002; 35(6): 700-. Meanwhile, the technology provides important methodological guarantee for female fertility preservation (Azari M, Kafi M, Ebrahimi B, Fatehi R, Jamalzadeh M. Ocyte mapping, embryo definition and gene expression following two-degree differentiation methods of bone-cavity complexes vision Res Commun.2017; 41 (49-56)), can preserve fertility for females with reproductive diseases such as ovarian cancer, and can also provide fertility opportunity for females with delayed fertility (De Munck N, Petrussa L, Verheyen G, Staessen C, VaskellelY, Borckx J, Stemken G, JacoK, Stoke M, Yeast G, calcium H. expression, blood viscosity, growth H. 535. gene expression, clone DNA of gene expression, growth medium DNA, clone G, chromosome DNA, clone G, growth medium DNA, clone of growth, growth of growth medium, growth of women, growth of women, growth of women, and growth of women, growth of women, growth of growth. Therefore, the technology has very important application value and social significance.
Through more than 30 years of research, researchers have continuously improved vitrification refrigeration sources, carrying tools, refrigerating fluid components, refrigerating procedures and the like, so that the vitrification refrigeration method is developed rapidly. However, during the freezing/thawing process of the oocyte, the oocyte is damaged by freezing due to high concentration of the anti-freezing protective agent, a rapid temperature reduction/rise process, strong osmotic pressure change and the like. Studies have shown that the fertilization capacity of vitrified frozen oocytes is drastically reduced (Fujiwara K, Kamoshita M, Kato T, Ito J, Kashiwazaki N.Generation of rates from transformed oocytes with subauding cumulus cells via visualization with reconstructed study J.2017; 88(1): 180; 184; Amidi F, KhodabandZ, Nori Mogahi MH. Comparison of the effects of visualization on gene expression of texture cells J.fertil.2018; 12(1):61-67. inte. J.fertil.2018; 12, ultralow potential of vitrification oocytes is a bottleneck for vitrification potential, and this is a key to the investigation of cryopreservation potential.
Disclosure of Invention
In view of the problem of the prior art of decreased fertilization ability of vitrified frozen oocytes, it is an object of the present invention to provide the use of gadolinium and CLC to improve the in vitro fertilization ability of vitrified frozen oocytes.
It is another object of the present invention to provide an oocyte treatment solution.
It is a final object of the invention to provide a method for improving the fertilization ability of a vitrified frozen oocyte.
In particular, the invention relates to the use of gadolinium and beta-cyclodextrin cholesterol clathrate (CLC) to treat oocytes prior to freezing of the oocytes, which oocytes are bovine oocytes.
Therefore, the present invention also provides an oocyte treatment fluid, the oocyte treatment fluid containing gadolinium and CLC;
wherein the gadolinium exists in an ionic form at a concentration of 50-300. mu.M, and the CLC is at a concentration of 10-20 mM.
Preferably, the concentration of gadolinium is 150. mu.M and the concentration of CLC is 15 mM. Further, the oocyte treatment is IVM liquid added with gadolinium and CLC, and the IVM liquid comprises the following components: TCM199 (basal medium) +0.01IU/ml FSH (follitropin) +0.01IU/ml LH (luteinizing hormone) +1 μ g/ml E2 (estrogen) + 10% FBS (fetal bovine serum). In order to ensure that gadolinium exists in an ionic form, the gadolinium is selected from soluble gadolinium salts, such as gadolinium (III) chloride hexahydrate;
the preparation method of the CLC comprises the following steps: dissolving 1g of methyl-beta-cyclodextrin in 2ml of methanol, dissolving 200mg of cholesterol in 1ml of chloroform, respectively and fully mixing, fully mixing 0.45ml of cholesterol solution with 2ml of methyl-beta-cyclodextrin solution, and then putting the mixed solution on liquid nitrogen steam for fumigation for 24 hours to obtain a solid matter, namely CLC. The oocyte handling solution of the present invention may be used to improve the fertilization ability of vitrified frozen oocytes.
Further, the present invention also provides a method for improving the fertility of a vitrified frozen oocyte, in particular, the method comprising the step of treating the oocyte in a solution comprising gadolinium and CLC.
Further, the method also comprises the steps of vitrifying and freezing the oocytes treated in the solution containing the gadolinium and the CLC, then unfreezing the oocytes, and then treating the oocytes with the methyl-beta-cyclodextrin solution.
Specifically, the method of the invention comprises the following steps:
1) taking oocytes, and treating the oocytes by adopting an oocyte treatment solution containing gadolinium and CLC; wherein the concentration of gadolinium is 50-300. mu.M, the concentration of CLC is 10-20 mM;
2) carrying out vitrification freezing on the oocyte obtained by the step 1), and then unfreezing;
3) processing the oocyte obtained in the step 2) by using in-vitro maturation liquid containing M beta CD (methyl-beta-cyclodextrin) for later use; wherein the concentration of the methyl-beta-cyclodextrin is 2.25-6.25 mM.
Wherein the treatment time in step 1) is 40 minutes.
In step 2), the vitrification freezing and thawing processes can be performed by methods commonly used in the art.
The treatment time in step 3) was 40 minutes.
In the step 3), the in vitro maturation solution can be an in vitro maturation solution commonly used in the field.
The processing of the invention is to place the oocyte in the solution and keep the oocyte still.
Before bovine oocyte vitrification freezing treatment, gadolinium and CLC combined treatment is adopted, so that the problem of decline of fertilization capability of frozen oocytes is solved, the cleavage rate and blastocyst rate of vitrified frozen bovine oocytes can be obviously improved, the in vitro fertilization efficiency of vitrified frozen bovine oocytes is higher than that of fresh bovine oocytes, and the method has wide application prospect and is worthy of popularization.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
1. Bovine oocyte IVM
After the ovaries obtained from the slaughterhouse were thoroughly washed, cumulus-oocyte complexes (COCs) in 2-8mm follicles were aspirated by a vacuum pump, and IVM maturation was carried out in an incubator (38.5 ℃ C., 5% CO2) for 22-24h with intact, compact COCs. The IVM liquid comprises the following components: TCM199+0.01IU/ml FSH +0.01IU/ml LH +1 ug/ml E2+ 10% FBS. After IVM was completed, COCs were digested with 0.1% hyaluronidase for 2-3min to remove granulosa cells, and oocytes with first polar bodies and uniform cytoplasm were selected for subsequent studies.
2. Preparation of gadolinium solutions with different concentrations
Preparing 300 mu M gadolinium solution: dissolving 1.12mg of gadolinium (III) chloride hexahydrate in 10mL of IVM solution, mixing well, filtering, sterilizing, and storing at 4 deg.C.
Preparing 150 mu M and 50 mu M gadolinium solution: diluting 300 μ M gadolinium solution with IVM solution by 2 times and 6 times respectively, mixing well, filtering, and storing at 4 deg.C.
Preparation of CLC, M beta CD solution
1g M beta CD was dissolved in 2ml of methanol and 200mg of cholesterol was dissolved in 1ml of chloroform, and the solutions were mixed well. 0.45ml of cholesterol solution and 2ml of M beta CD are fully and uniformly mixed, and then the mixed solution is placed on liquid nitrogen steam to fumigate for 24 hours, so that the obtained solid is CLC. The CLC molecular weight is 10866.66 g/mol.
Preparation of 20mM CLC solution: 2.17g CLC is dissolved in 10ml IVM solution, and after mixing well, filtered and sterilized, and stored at 4 ℃.
Preparation of 15mM, 10mM CLC solution: diluting 20mM CLC solution with IVM solution by 1.33 times and 2 times respectively, mixing well, filtering, and storing at 4 deg.C.
6.25mM, 4.25, 2.25mM M β CD solution preparation: respectively weighing 81.88mg, 55.68mg and 29.48mg of M beta CD, dissolving in IVM solution, mixing well, filtering, sterilizing, and storing at 4 deg.C.
OPS vitrification Freeze-thaw procedure
The cryoprotectant formulation and vitrification freezing procedure was modified slightly with reference to the method of Zhuston et al (Anim Biotechnol, 2005; 16: 153-.
I. Preparing an anti-freezing protective agent:
10％EG：EG、FBS、DPBS(-Ca2+、Mg2+) Prepared according to the ratio of 1:2:7 (v/v).
10％DMSO：DMSO、FBS、DPBS(-Ca2+、Mg2+) Prepared according to the ratio of 1:2:7 (v/v).
10％EG+10％DMSO：EG、DMSO、FBS、DPBS(-Ca2+、Mg2+) Prepared according to the ratio of 1:1:2:6 (v/v).
EDFSF 40: EG. DMSO and FSF are prepared according to the ratio of 2:2:6 (v/v).
And (3) FSF: adding 0.5M sucrose into 300g/l sucrose, and adding DPBS (-Ca)2+、Mg2+) After the solution was dissolved, 20% FBS was added to prepare an FSF solution.
Vitrification freezing procedure
Freezing procedure: the room temperature was adjusted to 25. + -. 1 ℃ to fully balance the test device and the reagents. The test was carried out on a 38-39 ℃ thermostatic bench. The oocytes are transferred into a pretreatment solution (10% EG + 10% DMSO) by an OPS tube for balancing for 30s, then transferred into a freezing solution (EDFSF40) for balancing for 25s, and then the oocytes are sucked into the OPS tube and directly put into liquid nitrogen for freezing and preservation.
And (3) unfreezing procedure: after the OPS tube was removed from the liquid nitrogen, the oocytes were immediately placed in a petri dish containing 0.25M sucrose thawing solution and equilibrated for 1min, then transferred to 0.15M sucrose thawing solution and equilibrated for 5 min. The oocytes are washed for 2 times by using the IVM solution, and the thawed oocytes with normal morphology (according to membrane integrity and cell glossiness) are judged to be alive and transferred into the IVM solution for standby.
5. Oocyte cytoplasm calcium ion, endoplasmic reticulum calcium ion and mitochondria calcium ion level detection
And (3) detecting cytoplasmic calcium ions: after the oocytes were washed with DBPS, they were incubated in 1. mu.M Fluo-3/AM for 30min in the dark. After the staining was completed, the oocytes were washed with DBPS and then pelleted, and then photographed under a fluorescent microscope.
The endoplasmic reticulum calcium ion and mitochondrial calcium ion concentrations in oocytes were detected by staining using a staining kit purchased from jimet corporation of shanghai (fluorescence detection kit for mitochondrial calcium ion concentration, GMS 10153; fluorescence detection kit for endoplasmic reticulum calcium ion concentration, GMS 10267.1). The staining procedure is as follows, after the oocyte is washed, the oocyte is respectively put into the calcium ion staining working solution in the mitochondria and the calcium ion staining working solution in the endoplasmic reticulum which are provided by the kit to be stained in a dark place for 60 min. After staining, the oocytes were washed and then pelleted, and photographed under a confocal laser microscope. When the mitochondrial calcium ions are photographed, the laser microscope sets an excitation wavelength of 550nm and an emission wavelength of 590nm, and the mitochondrial calcium ions are red. When the endoplasmic reticulum calcium ions are photographed, the laser microscope sets the excitation wavelength of 490nm and the emission wavelength of 525nm, and the endoplasmic reticulum calcium ions are green.
Fluorescence intensities of cytosolic calcium ions, endoplasmic reticulum calcium ions, and mitochondrial calcium ions were analyzed using Nikon EZ-C1FreeViewer software for subsequent statistics.
6. Detection of protein expression quantity of JUNO and CD9 in oocyte
The collected bovine oocytes were placed in DPBS solution and stored at-80 ℃. During detection, a sample is taken out, put into a lysate for lysis, subjected to gel electrophoresis after high-temperature denaturation, subjected to membrane transfer after the current of 300mA is kept for 2.5 hours at the concentration of 12 percent of glue, the membrane is sealed in skimmed milk powder (5 percent) for 1 hour, and then incubated with primary antibody (JUNO, 1:1000, MAB6124-SP, R & D; CD9, 1; 1000, ab28094, abcam) at 4 ℃ overnight, and finally incubated with secondary antibody for 1 hour. Results were detected using ECL detection kit.
7. Oocyte CGs staining
The oocytes were digested with pronase (5mg/mL) to remove the zona pellucida and then incubated in 4% paraformaldehyde for 30 min. After thorough washing, the oocytes were washed 3 times with DBPS blocking solution containing 3% BSA, then placed in 0.1% Triton X-100 for 5min of permeation treatment at room temperature, and washed 3 times for 5min each time with blocking solution. Finally, oocytes were incubated in 20. mu.g/mL FITC-LCA dye at 38.5 ℃ for 30min in the dark and the DBPS solution was washed 3 times. After oocyte pelleting, CGs morphology (circular distribution, discontinuous circular distribution, uniform cytoplasmic distribution and complete release) was analyzed by confocal microscopy, wherein circular distribution was judged as normal distribution.
8. Oocyte fertilization ability detection
Thawing frozen semen in 37 deg.C water bath, thawing, placing in 7ml BO semen washing solution, mixing gently, and centrifuging at 1800rpm for 5min 2 times. Centrifuging, removing supernatant, and adjusting sperm density to 5 × 10 with fertilization solution6One per ml. Adding 20 μ l semen into 80 μ l semen containing 20-30 oocytes at 38.5 deg.C with 5% CO2IVF was performed in an incubator. After 8h of IVF, the oocytes were treated with pronase to remove zona pellucida and further stained with Hoechst 33342 at 10. mu.g/mL for 10 min. After dyeing is finished, the oocytes without the transparent band are tabletted and then observed by adopting a fluorescence microscope, and the oocytes are divided into three types: unfertilized eggs, normal fertilization, and polyspermic fertilization.
9. Bovine oocyte IVF
Thawing frozen semen in 37 deg.C water bath, thawing, placing in 7ml BO semen washing solution, mixing gently, and centrifuging at 1800rpm for 5min 2 times. Centrifuging, removing supernatant, and adjusting sperm density to 5 × 10 with fertilization solution6One per ml. Adding 20 μ l semen into 80 μ l semen containing 20-30 oocytes at 38.5 deg.C with 5% CO2IVF was performed in an incubator. 18-20h after IVF, culturing in CR1aa, and replacing culture medium (CR1aa + 10% FBS) after culturing for 48 h. After in vitro culture for 7d, in vitro fertilization blastocysts were obtained. And (5) counting the cleavage rate and the blastocyst rate.
10. Data statistics
And (3) analyzing the experimental data by adopting SAS software, performing inverse-normal rotation conversion during percentage comparison, and then performing variance analysis, wherein the result is expressed by the mean according to the standard deviation, and P <0.05 is the difference significance standard. Each treatment group was repeated at least 3 times.
Example 1 Effect of gadolinium treatment on the distribution of cytoplasmic calcium, mitochondrial calcium, endoplasmic reticulum calcium, CG of vitrified frozen bovine oocytes
Bovine oocytes are respectively treated with 50 mu M, 150 mu M and 300 mu M gadolinium for 40min, then vitrified and frozen, and the influence of different gadolinium treatments on the distribution of oocyte cytoplasmic calcium, mitochondrial calcium, endoplasmic reticulum calcium and CG is compared.
As shown in table 1, the concentration of cytosolic calcium ions and the concentration of mitochondrial calcium ions in the gadolinium-treated group were significantly lower than those in the vitrified frozen group, the concentration of endoplasmic reticulum calcium ions in the gadolinium-treated group was significantly higher than those in the vitrified frozen group, and the concentration of intracellular calcium ions and the concentration of mitochondrial calcium ions in the 150 μ M gadolinium-treated group were not significantly different from those in the fresh group.
Meanwhile, the normal distribution of CG in 50 μ M, 150 μ M and 300 μ M gadolinium-treated groups (51.43 ± 5.02%, 74.36 ± 6.31% and 78.13 ± 7.46%) is significantly higher than that in vitrification freezing group (29.63 ± 2.84%), and there is no significant difference between 150 μ M gadolinium-treated group and 300 μ M gadolinium-treated group and fresh group (75.00 ± 7.17%).
TABLE 1 Effect of gadolinium on the distribution of cytoplasmic calcium, mitochondrial calcium, endoplasmic reticulum calcium, CG of vitrified frozen bovine oocytes
a, b, c, d, e: the same column data are marked differently to show significant difference (P <0.05)
Example 2 Effect of gadolinium treatment on the fertilization Capacity of vitrified frozen bovine oocytes
Bovine oocytes were treated with 50. mu.M, 150. mu.M, and 300. mu.M gadolinium, respectively, for 40min, and then vitrified and frozen. And (4) selecting the viable oocytes after thawing for IVF, and performing fertilization capability detection on the oocytes after IVF for 8 h. Further comparing the effect of different gadolinium treatments on bovine oocyte fertilization ability.
As shown in table 2, the normal fertilization rate (64.29 ± 6.28%) was significantly higher in the 150 μ M gadolinium-treated group than in the vitrified frozen group (47.22 ± 4.39%), but still lower than in the fresh group (78.13 ± 6.72%). Meanwhile, the non-fertilization rate (26.19 ± 2.41%) of the 150 μ M gadolinium-treated group was significantly lower than that of the vitrified frozen group (38.89 ± 3.71%), and still significantly higher than that of the fresh group (15.63 ± 1.30%). The polyspermia rate of the 150 μ M gadolinium-treated group (9.52 ± 0.62%) was not significantly different from that of the fresh group (6.25 ± 0.47%).
TABLE 2 Effect of gadolinium on the fertilization Capacity of vitrified frozen bovine oocytes
Example 3 Effect of gadolinium treatment on fertilization efficiency of vitrified frozen bovine oocytes
Bovine oocytes were treated with 50. mu.M, 150. mu.M, and 300. mu.M gadolinium, respectively, for 40min, and then vitrified and frozen. And after thawing, selecting the surviving oocytes for IVF, counting the cleavage rate and blastocyst rate, and further comparing the influence of different gadolinium treatments on the fertilization efficiency of the bovine oocytes.
As shown in Table 3, the 150 μ M gadolinium-treated group showed significantly higher cleavage rate, blastocyst rate (62.20. + -. 5.63%, 23.53. + -. 2.31%) than the vitrified frozen group (40.85. + -. 3.82%, 13.79. + -. 1.05%), but significantly lower than the fresh group (79.03. + -. 7.62%, 36.73. + -. 3.28%).
TABLE 3 Effect of gadolinium on the efficiency of in vitro fertilization of vitrified frozen bovine oocytes
Example 4 Effect of CLC treatment on the amount of JUNO, CD9 protein in vitrified frozen bovine oocytes
Bovine oocytes were treated with 10. mu.M, 15. mu.M, and 20. mu.M CLC for 40min, respectively, and then vitrified and frozen. After thawing, the frozen oocytes were treated with 2.25mM, 4.25mM, 6.25mM M.beta.CD for 40 min. And (3) carrying out JUNO and CD9 protein detection on the survival frozen oocytes, and further comparing the influence of different CLC treatments on the protein amounts of JUNO and CD9 of the vitrified frozen bovine oocytes.
As shown in table 4, the expression level of JUNO protein and CD9 protein in the 15mM CLC +4.25mM M β CD-treated group is significantly higher than that in the frozen group, and has no significant difference from the fresh group.
TABLE 4 influence of CLC treatment on the amount of JUNO, CD9 protein in vitrified frozen bovine oocytes
Example 5 Effect of CLC treatment on fertilization efficiency of vitrified frozen bovine oocytes
Bovine oocytes were treated with 10. mu.M, 15. mu.M, and 20. mu.M CLC for 40min, respectively, and then vitrified and frozen. After thawing, the frozen oocytes were treated with 2.25mM, 4.25mM, 6.25mM M.beta.CD for 40 min. And carrying out IVF on the surviving frozen oocytes, and counting the cleavage rate and blastocyst rate so as to compare the influence of different CLC treatments on the fertilization efficiency of the bovine oocytes.
As shown in table 5, the cleavage rate and blastocyst rate (65.56 ± 5.68%, 23.73 ± 2.02%) of the 15mM CLC +4.25mM M β CD-treated group were significantly higher than those of the vitrified frozen group (53.13 ± 4.85%, 11.76 ± 1.06%), but were significantly lower than those of the fresh group (83.33 ± 7.46%, 33.33 ± 3.16%).
TABLE 5 Effect of CLC treatment on the efficiency of in vitro fertilization of vitrified frozen bovine oocytes
Example 6 Effect of optimal concentration treatment of gadolinium and CLC on fertilization efficiency of vitrified frozen bovine oocytes
Before vitrification freezing, bovine oocytes are respectively treated by the optimal gadolinium treatment concentration and the CLC treatment concentration independently, and after thawing the CLC treatment group, the optimal M beta CD concentration is adopted for treatment. And carrying out IVF on the surviving oocytes, counting the cleavage rate and blastocyst rate, and further comparing the influence of the single treatment of the optimal concentration of gadolinium and CLC on the fertilization efficiency of the bovine oocytes.
As shown in Table 6, the cleavage rate and blastocyst rate (63.73 + -5.32%, 23.08 + -2.14%) of the 150 μ M gadolinium-treated group were not significantly different from those of the 15mM CLC +4.25mM Mb CD-treated group (67.02 + -6.17%, 25.40 + -2.45%), which were significantly higher than those of the vitrified frozen group (54.29 + -4.68%, 12.28 + -1.35%, P <0.05), but significantly lower than those of the fresh group (85.87 + -7.46%, 35.44 + -2.86%, P < 0.05).
TABLE 6 Effect of optimal concentration treatment of gadolinium and CLC on the efficiency of in vitro fertilization of vitrified frozen bovine oocytes
Example 7 Effect of gadolinium in combination with CLC on the efficiency of fertilization of vitrified frozen bovine oocytes
Before vitrification freezing, the bovine oocytes are treated by adopting the optimal gadolinium treatment concentration and CLC treatment concentration simultaneously, and the thawed bovine oocytes are treated by adopting the optimal M beta CD. And carrying out IVF on the surviving oocytes, counting the cleavage rate and blastocyst rate, and further comparing the influence of gadolinium and CLC combined treatment on the fertilization efficiency of the bovine oocytes.
As shown in Table 7, the cleavage rate and blastocyst rate (92.50. + -. 8.41%, 45.95. + -. 4.21%) of 150. mu.M gadolinium +15mM CLC +4.25mM Mss CD treated group were significantly higher than those of the vitrified frozen group (52.17. + -. 5.14%, 12.50. + -. 1.14%) and the fresh group (80.95. + -. 7.63%, 32.35. + -. 3.18%).
TABLE 7 Effect of gadolinium + CLC treatment on the efficiency of in vitro fertilization of vitrified frozen bovine oocytes
The results show that the bovine oocyte is jointly processed for 40min by adopting 150 mu M gadolinium and 15mM CLC before vitrification and is processed for 40min by adopting 4.25mM Mbeta CD after thawing, the cleavage rate and blastocyst rate of the vitrified frozen bovine oocyte can be obviously improved, and the efficiency of the bovine oocyte is higher than the efficiency of in vitro fertilization of fresh bovine oocytes.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. Use of gadolinium and CLC in the treatment of an oocyte to improve the capacity of a vitrified frozen oocyte for in vitro fertilization for non-therapeutic purposes, wherein the oocyte is a bovine oocyte and the CLC is prepared by the following method: dissolving 1g of methyl-beta-cyclodextrin in 2ml of methanol, dissolving 200mg of cholesterol in 1ml of chloroform, respectively and fully mixing, fully mixing 0.45ml of cholesterol solution with 2ml of methyl-beta-cyclodextrin solution, and then putting the mixed solution on liquid nitrogen steam for fumigation for 24 hours to obtain a solid matter, namely CLC.
2. An oocyte treatment solution, wherein the solution contains gadolinium and CLC; wherein the gadolinium exists in an ionic form, the concentration of gadolinium is 50-300. mu.M, and the concentration of CLC is 10-20 mM;
the solution is IVM solution containing gadolinium and CLC, and the IVM solution comprises the following components: TCM199+0.01IU/ml follitropin +0.01IU/ml lutein +1 μ g/ml estrogen + 10% fetal calf serum; the preparation method of the CLC comprises the following steps: dissolving 1g of methyl-beta-cyclodextrin in 2ml of methanol, dissolving 200mg of cholesterol in 1ml of chloroform, respectively and fully mixing, fully mixing 0.45ml of cholesterol solution with 2ml of methyl-beta-cyclodextrin solution, and then putting the mixed solution on liquid nitrogen steam for fumigation for 24 hours to obtain a solid matter, namely CLC.
3. The oocyte handling solution of claim 2, wherein: the gadolinium concentration is 150. mu.M, and the CLC concentration is 15 mM.
4. Use of an oocyte treatment solution according to claim 2 or 3 to improve the fertilisation capacity of a vitrified frozen oocyte for in vitro fertilisation for non-therapeutic purposes.
5. A method of improving the fertilisation capacity of a vitrified frozen oocyte, comprising the steps of treating the oocyte in a solution comprising gadolinium and CLC; it is in vitro fertilization for non-therapeutic purposes;
which comprises the following steps:
1) taking oocytes, and treating the oocytes by adopting IVM solution containing gadolinium ions and CLC; wherein the concentration of gadolinium is 50-300. mu.M, the concentration of CLC is 10-20 mM; the IVM solution comprises the following components: TCM199+0.01IU/ml follitropin +0.01IU/ml lutein +1 μ g/ml estrogen + 10% fetal calf serum;
the preparation method of the CLC comprises the following steps: dissolving 1g of methyl-beta-cyclodextrin in 2ml of methanol, dissolving 200mg of cholesterol in 1ml of chloroform, respectively and fully mixing, fully mixing 0.45ml of cholesterol solution with 2ml of methyl-beta-cyclodextrin solution, and then putting the mixed solution on liquid nitrogen steam for fumigation for 24 hours to obtain a solid matter, namely CLC; wherein the concentration of the methyl-beta-cyclodextrin is 2.25-6.25 mM;
2) carrying out vitrification freezing on the oocyte obtained by the step 1), and then unfreezing;
3) treating the oocyte obtained in the step 2) by using in-vitro maturation liquid containing methyl-beta-cyclodextrin for later use.
6. The method of claim 5, wherein the treatment time in step 1) is 40 minutes.
7. The method of claim 5, wherein the treatment time in step 3) is 40 minutes.
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| CN108588011B (en) * | 2018-05-08 | 2022-04-05 | 中国农业科学院北京畜牧兽医研究所 | Method for improving in vitro fertilization capability of vitrified frozen oocyte |
| CN110484494A (en) * | 2019-09-27 | 2019-11-22 | 延边大学 | A method of improving Porcine In Vitro Maturation Oocytes quality |
| CN114214270B (en) * | 2021-12-17 | 2023-11-24 | 中国农业科学院北京畜牧兽医研究所 | Method for regulating and controlling developmental capacity of frozen bovine oocytes and application thereof |
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