Method for reducing lipid content of oocytes or improving freezing survival rate of oocytes

Abstract

The invention discloses a method for reducing the lipid content of an oocyte or increasing the freezing survival rate of the oocyte. The method comprises the following steps: (1) preheating an incubation solution; incubating the oocytes in the preheated incubation liquid; (2) freezing and unfreezing the incubated oocytes; wherein the incubation liquid is prepared by adding bezafibrate and cholic acid into a culture medium 199 containing 10wt% of fetal calf serum. Compared with a conventional culture medium 199 containing 10 wt% of fetal calf serum, the incubation liquid used in the method provided by the invention has the advantages that bezafibrate and cholic acid which are added have a synergistic lipid-lowering effect, so that the lipid content of the oocytes can be remarkably reduced or the freezing survival rate of the oocytes can be increased by the method. The method has application prospects in the aspects of reducing the lipid content of the oocytes or improving the freezing survival rate of the oocytes and the like.

Description

Technical Field

[0001] The present invention relates to a method for reducing the lipid content of animal cells or improving the freezing survival rate of animal cells, and in particular to a method for reducing the lipid content of oocytes or improving the freezing survival rate of oocytes, and belongs to the field of cryopreservation of porcine oocytes. Background Art

[0002] Oocytes are reproductive cells of female animals. Mature pig oocytes combine with sperm to form fertilized eggs, which implant in the sow's uterus and develop into fetuses. Oocytes are distributed in the follicles on the surface of the ovaries of sows before ovulation. They can be obtained by surgery or by removing the ovaries after slaughter.

[0003] Oocyte cryopreservation technology mainly preserves oocytes under extremely low temperature conditions to maintain their vitality and developmental potential for a long time. It is one of the main components of livestock breeding and livestock germplasm resource protection. At present, frozen oocytes have been used to obtain surviving offspring in the reproduction of multiple animal species. However, in pigs, a large amount of intracellular lipids restricts the effect of porcine oocyte cryopreservation. The high lipid content of porcine oocytes is a species-related cell characteristic. During the cryopreservation of porcine oocytes, a large amount of intracellular lipids hinder the penetration of cryoprotectants and limit the vitrification effect, resulting in the loss of cell activity after freezing and thawing and the potential to develop into embryos after fertilization. This is a technical problem in pig reproduction and germplasm resource protection. Summary of the invention

[0004] The main purpose of the present invention is to provide a method for reducing the lipid content of oocytes or improving the freezing survival rate of oocytes.

[0005] To achieve the above object, the technical solution adopted by the present invention includes:

[0006] A method for reducing the lipid content of oocytes or improving the survival rate of oocytes during freezing, comprising: (1) preheating an incubation solution; incubating oocytes in the preheated incubation solution; and (2) freezing and thawing the incubated oocytes.

[0007] In a preferred embodiment of the present invention, the incubation solution in step (1) is prepared by adding bezafibrate and bile acid to a basal cell culture medium.

[0008] In a preferred embodiment of the present invention, the oocyte in step (1) is a mature porcine oocyte.

[0009] In a preferred embodiment of the present invention, the method for preheating the incubation solution in step (1) comprises: preheating the incubation solution covered with mineral oil in an environment of 39° C., 5% carbon dioxide, and saturated humidity for 1 hour.

[0010] In a preferred embodiment of the present invention, the basal cell culture medium in step (1) is culture medium 199 containing 10 wt % fetal bovine serum.

[0011] In a preferred embodiment of the present invention, the concentrations of bezafibrate and bile acid in the incubation solution in step (1) are both 100 μM.

[0012] In a preferred embodiment of the present invention, the incubation time in step (1) is 3-5 hours; preferably, the incubation time is 4 hours.

[0013] In a preferred embodiment of the present invention, the incubation conditions in step (1) include: an incubation temperature of 37-39.5°C, 4-6% carbon dioxide, and saturated humidity; preferably, the incubation conditions are an incubation temperature of 39°C, 5% carbon dioxide, and saturated humidity.

[0014] In a preferred specific embodiment of the present invention, the freezing method described in step (2) comprises: transferring the incubated porcine oocytes to a vitrified freezing solution I at 39°C for 3 minutes, then transferring to a vitrified freezing solution II at 39°C for 10 seconds, and freezing in liquid nitrogen; wherein the freezing solution I is a culture medium obtained by adding 7.5wt% ethylene glycol, 7.5wt% dimethyl sulfoxide and 20wt% fetal bovine serum to a culture medium 199; and the freezing solution II is a culture medium obtained by adding 15wt% ethylene glycol, 15wt% dimethyl sulfoxide and 20wt% fetal bovine serum to a culture medium 199.

[0015] In a preferred embodiment of the present invention, the thawing method described in step (2) comprises: taking the freezing carrier out of liquid nitrogen, immediately immersing the oocyte loading end in thawing solution I at 39°C to separate the oocyte from the freezing carrier, maintaining the oocyte in thawing solution I for 5 minutes, transferring to thawing solution II at 39°C and maintaining it for 5 minutes, and transferring to a culture medium 199 covered with mineral oil and containing 10wt% fetal bovine serum and incubating for 4 hours; wherein the incubation conditions include: incubation temperature of 39°C, 5% carbon dioxide, and saturated humidity; the thawing solution I is a culture medium obtained by adding 300μM sucrose and 20wt% fetal bovine serum to the culture medium 199; the thawing solution II is a culture medium obtained by adding 150μM sucrose and 20wt% fetal bovine serum to the culture medium 199.

[0016] Compared with the conventional culture medium 199 containing 10wt% fetal bovine serum, the incubation solution used in the method provided by the present invention can significantly reduce the lipid content of oocytes or improve the freezing survival rate of oocytes because the added bezafibrate and bile acid have a synergistic lipid-lowering effect. The present invention has application prospects in reducing the lipid content of oocytes or improving the freezing survival rate of oocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The lipid-lowering effect of incubating porcine oocytes in incubation medium 1-8 for 4 hours; Figure 1 -A is a fluorescence quantitative detection diagram of lipid droplets in oocytes; Figure 1 -B is the statistical analysis of the lipid-lowering effect of incubation medium 1-3, 8 on porcine oocytes; Figure 1 -C is the statistical analysis of the lipid-lowering effect of incubation medium 1, 4-8 on porcine oocytes; ****: P<0.0001;

[0018] Figure 2 The cell survival rate of porcine oocytes incubated in incubation solution 1 for 4 hours after freezing and thawing is detected; wherein, Figure 2 -A is a live cell image of an oocyte. The oocyte was incubated with fluorescein diacetate dye, and the cells that showed color under a fluorescence microscope were considered live cells. Figure 2 -B is a statistical analysis chart of oocyte survival rate. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0020] Example 1 Preparation of incubation medium for porcine oocytes

[0021] (1) 10 mL of fetal bovine serum was added to 90 mL of culture medium 199. The culture medium 199 was Gibco culture medium 199 with catalog number 12340030 purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0022] (2) Dissolve 0.0036 g of bezafibrate in 1 mL of anhydrous bile acid (10 mM in DMSO).

[0023] (3) Mix the above two solutions to obtain incubation solution 1.

[0024] Experimental Example 1 Experiment on reducing lipid in porcine oocytes and improving the survival rate of porcine oocytes frozen

[0025] 1 Test method

[0026] 1.1 Incubation medium for the test:

[0027] (1) Incubation Solution 1: Incubation Solution 1 prepared in Example 1; (2) Incubation Solution 2: Incubation Solution 1 prepared in Example 1, except for the absence of bile acid, other components and preparation methods are exactly the same as those of Incubation Solution 1 prepared in Example 1; (3) Incubation Solution 3: Incubation Solution 1 prepared in Example 1, except for the absence of bezafibrate, other components and preparation methods are exactly the same as those of Incubation Solution 1 prepared in Example 1; (4) Incubation Solution 4: Incubation Solution 1 prepared in Example 1, bezafibrate is replaced by L-carnitine of the same concentration; (5) Incubation Solution 5: Incubation Solution 1 prepared in Example 1, bezafibrate is replaced by L-carnitine of the same concentration; An incubation solution obtained by replacing bezafibrate in the incubation solution 1 prepared in Example 1 with nicotinic acid of the same concentration; (6) Incubation solution 6: an incubation solution obtained by replacing bile acid in the incubation solution 1 prepared in Example 1 with L-carnitine of the same concentration; (7) Incubation solution 7: an incubation solution obtained by replacing bile acid in the incubation solution 1 prepared in Example 1 with nicotinic acid of the same concentration; (8) Incubation solution 8: the incubation solution prepared in step (1) of Example 1 (culture medium 199 containing 10 wt% fetal bovine serum), as a control group.

[0028] 1.2 Test methods

[0029] (1) Obtaining mature porcine oocytes: The ovaries were removed from the sows, and a 10 mL syringe was used to penetrate the follicles on the surface of the ovaries, and the follicular fluid was aspirated. The follicular fluid was placed under a stereomicroscope for microscopic examination, and the cumulus-oocyte complex in the follicular fluid was picked out and placed in a conventional porcine oocyte in vitro maturation culture medium for 44 h. The culture conditions were 39° C., 5% carbon dioxide and saturated humidity. After 44 h, the cumulus-oocyte complex was placed in a 0.1% hyaluronidase solution for about 1 min, and was repeatedly blown with a pipette to separate the cumulus and the oocyte. The oocyte was transferred to culture medium 199 and observed under a microscope. The mature oocyte was the oocyte that discharged the first polar body.

[0030] (2) Incubation: The incubation solution covered with mineral oil was preheated for 1 h at 39°C, 5% carbon dioxide, and saturated humidity. The mature porcine oocytes were incubated in the incubation solution for 4 h at 39°C, 5% carbon dioxide, and saturated humidity. After 4 h, the oocytes were transferred to three drops of medium 199 in sequence to fully wash away the incubation solution and then frozen.

[0031] (3) Freezing: The oocytes were transferred to vitrification solution I at 39°C for 3 min, then transferred to vitrification solution II at 39°C for 10 s, loaded onto a cryostat, and immersed in liquid nitrogen.

[0032] Freezing medium I: Medium 199 containing 7.5 wt% ethylene glycol, 7.5 wt% dimethyl sulfoxide and 20 wt% fetal bovine serum.

[0033] Freezing medium II: Medium 199 containing 15 wt % ethylene glycol, 15 wt % dimethyl sulfoxide and 20 wt % fetal bovine serum.

[0034] (4) Thawing: The freezing straw was taken out of liquid nitrogen and the oocyte loading end was immediately immersed in thawing solution I at 39°C to separate the oocyte from the freezing straw. The oocyte was maintained in thawing solution I for 5 min, transferred to thawing solution II at 39°C and maintained for 5 min, and then transferred to medium 199 covered with mineral oil and containing 10 wt% fetal bovine serum and incubated for 4 h. The incubation conditions were: 39°C, 5% carbon dioxide, and saturated humidity.

[0035] Thawing solution I: Medium 199 containing 300 μM sucrose and 20 wt% fetal bovine serum.

[0036] Thawing solution II: Medium 199 containing 150 μM sucrose and 20 wt% fetal bovine serum.

[0037] (5) Evaluation of lipid-lowering effect: Nile red fluorescent dye was used to stain lipid droplets in oocytes. Laser scanning confocal microscopy was used to photograph and quantify lipid droplets in oocytes. Image J software was used to convert images into data. IBM SPSS Statistics software was used to perform one-way ANOVA analysis to determine the significance between groups. The results are expressed as mean ± SD. **** represents the difference between groups P < 0.0001.

[0038] (6) Evaluation of freezing effect: The oocytes were stained with fluorescein diacetate. Fluorescein diacetate can enter the cells and be converted into metabolites with green fluorescence under the action of enzymes in living cells. The cells showing green fluorescence were photographed under a fluorescence microscope and were determined to be living cells. The survival rate of each group was calculated. IBM SPSS Statistics software was used to perform one-way ANOVA analysis to determine the significance between groups. The results were expressed as mean ± standard deviation. *** represents the difference between groups P < 0.001, and **** represents the difference between groups P < 0.0001.

[0039] 2 Test results

[0040] 2.1 Experimental results on reducing lipid content in porcine oocytes

[0041] Depend on Figure 1 -A, Figure 1 -B shows that incubation solution 1 incubated oocytes for 4 hours, and compared with incubation solution 8 (control group), the lipid content in oocytes was significantly reduced. Incubation solution 2 or incubation solution 3 incubated oocytes for 4 hours, the intracellular lipid droplet content was significantly higher than that of oocytes incubated in incubation solution 1, that is, the lipid-lowering effect of a single lipid-lowering agent was significantly weaker than the combined application of two lipid-lowering agents. Therefore, the combined application of bezafibrate and bile acid in incubation solution 1 prepared in Example 1 has a synergistic effect on lipid-lowering in pig oocytes.

[0042] Depend on Figure 1 -A, Figure 1-C shows that when the drug combination that exerts a lipid-lowering effect is bezafibrate and cholic acid, the lipid content in the oocytes after incubation is significantly lower than that in the various replacement combination incubation solution groups (P<0.0001), and compared with incubation solutions 4-7, incubation solution 1 has the highest lipid-lowering effect. When any one of bezafibrate and cholic acid is replaced, the lipid content of the pig oocytes after incubation is not significantly different from that of the fresh control, and the lipid-lowering effect is not as good as that of incubation solution 1 prepared in Example 1.

[0043] 2.2 Experimental results on improving the survival rate of porcine oocyte cryopreservation

[0044] The oocytes incubated in incubation solution 1 or the oocytes incubated in incubation solution 8 (control group) were frozen, thawed and tested for survival rate. Figure 2 -A, Figure 2 -B shows that the survival rate of oocytes incubated with incubation solution 8 (control group) after freezing is significantly reduced, confirming that freezing will cause a large proportion of oocytes to die or lose biological activity; while the survival rate of oocytes incubated with incubation solution 1 after freezing is significantly higher than that of oocytes frozen after incubation with incubation solution 8. Therefore, incubation solution 1 significantly improves the freezing survival rate of oocytes by reducing the lipid content of porcine oocytes.

Claims

1. A method for reducing the lipid content of oocytes or improving the survival rate of oocytes during freezing, characterized in that: include: (1) preheating an incubation solution; incubating oocytes in the preheated incubation solution; (2) Freezing and thawing the incubated oocytes; wherein the incubation solution is prepared by adding bezafibrate and bile acid to a basal cell culture medium.

2. The method according to claim 1, characterized in that The oocyte described in step (1) is a mature porcine oocyte.

3. The method according to claim 1, characterized in that The method for preheating the incubation solution described in step (1) comprises: placing the incubation solution covered with mineral oil in an environment of 39° C., 5% carbon dioxide, and saturated humidity and preheating for 1 hour.

4. The method according to claim 1, characterized in that: The basic cell culture medium is medium 199 containing 10 wt % fetal bovine serum.

5. The method according to claim 1, characterized in that The concentrations of bezafibrate and bile acid in the incubation solution were both 100 μM.

6. The method according to claim 1, characterized in that The incubation time in step (1) is 3-5 hours; preferably, the incubation time is 4 hours.

7. The method according to claim 1, characterized in that The incubation conditions described in step (1) include: incubation temperature 37-39.5° C., 4-6% carbon dioxide, and saturated humidity.

8. The method according to claim 7, characterized in that The incubation conditions described in step (1) are: incubation temperature 39° C., 5% carbon dioxide, and saturated humidity.

9. The method according to claim 1, characterized in that: The freezing method described in step (2) comprises: transferring the incubated pig oocytes to a vitrified freezing solution I at 39°C for 3 minutes, then transferring to a vitrified freezing solution II at 39°C for 10 seconds, and freezing in liquid nitrogen; wherein the freezing solution I is a culture medium obtained by adding 7.5wt% ethylene glycol, 7.5wt% dimethyl sulfoxide and 20wt% fetal bovine serum to the culture medium 199; and the freezing solution II is a culture medium obtained by adding 15wt% ethylene glycol, 15wt% dimethyl sulfoxide and 20wt% fetal bovine serum to the culture medium 199.

10. The method according to claim 1, characterized in that The thawing method described in step (2) comprises: taking the freezing carrier out of liquid nitrogen, immediately immersing the oocyte loading end in 39°C thawing solution I to separate the oocyte from the freezing carrier, maintaining the oocyte in thawing solution I for 5 minutes, transferring to 39°C thawing solution II for 5 minutes, and transferring to culture medium 199 covered with mineral oil and containing 10wt% fetal bovine serum for incubation for 4 hours; wherein the incubation conditions include: incubation temperature of 39°C, 5% carbon dioxide, and saturated humidity; the thawing solution I is a culture medium obtained by adding 300μM sucrose and 20wt% fetal bovine serum to culture medium 199; the thawing solution II is a culture medium obtained by adding 150μM sucrose and 20wt% fetal bovine serum to culture medium 199.

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