Methods of reducing lipid content of oocytes or improving oocyte cryosurvival

Abstract

The application discloses a method for reducing the lipid content of oocytes or improving the cryopreservation survival rate of oocytes, and comprises the following steps: (1) preheating an incubation solution; and incubating oocytes in the preheated incubation solution; (2) freezing and thawing the incubated oocytes; wherein the incubation solution is prepared by adding bezafibrate and cholic acid to a culture medium 199 containing 10wt% fetal bovine serum. The incubation solution used in the method provided by the application can significantly reduce the lipid content of oocytes or improve the cryopreservation survival rate of oocytes, compared with the conventional culture medium 199 containing 10wt% fetal bovine serum, because the added bezafibrate and cholic acid have a synergistic lipid-lowering effect. The application has application prospects in reducing the lipid content of oocytes or improving the cryopreservation survival rate of oocytes.

Description

TECHNICAL FIELD

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

[0002] Oocytes are female reproductive cells. Mature porcine oocytes combine with sperm to form zygotes, which implant in the uterus of a sow and develop into a fetal individual. Oocytes are distributed in follicles on the surface of the ovary before ovulation in sows, and can be obtained by surgery or by taking the ovary after slaughter.

[0003] Oocyte cryopreservation technology mainly preserves oocytes at very low temperatures, which can maintain their viability and developmental potential for a long time. It is one of the main components of livestock breeding and livestock germplasm resource protection. Currently, frozen oocytes have been used to produce offspring in several animal species. However, in pigs, the high lipid content in oocytes has limited the effectiveness of pig oocyte cryopreservation. The high lipid content of porcine oocytes is a species-related cellular characteristic. In the cryopreservation of porcine oocytes, the high intracellular lipid content hinders the penetration of cryoprotectants, limits the effect of vitrification, and causes the loss of cell viability and the potential to develop into embryos after fertilization, which is a technical problem in pig breeding and germplasm resource protection. SUMMARY

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

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present application comprises:

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

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

[0008] In a preferred embodiment of the present application, the oocytes in step (1) are mature porcine oocytes.

[0009] In a preferred embodiment of the present application, the method for preheating the incubation solution in step (1) comprises: placing the incubation solution overlaid with mineral oil in a 39℃, 5% carbon dioxide, saturated humidity environment for 1h.

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

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

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

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

[0014] In a preferred embodiment of the present application, the freezing method in step (2) includes transferring the incubated porcine oocytes to vitrification freezing solution I at 39 ℃ for 3 min, then transferring to vitrification freezing solution II at 39 ℃ for 10 s, and freezing in liquid nitrogen; wherein the freezing solution I is a medium obtained by adding 7.5 wt% ethylene glycol, 7.5 wt% dimethyl sulfoxide, and 20 wt% fetal bovine serum to medium 199; and the freezing solution II is a medium obtained by adding 15 wt% ethylene glycol, 15 wt% dimethyl sulfoxide, and 20 wt% fetal bovine serum to medium 199.

[0015] In a preferred embodiment of the present application, the thawing method in step (2) includes taking the freezing carrier out of liquid nitrogen, immediately immersing the loading end of the oocytes in thawing solution I at 39 ℃ to detach the oocytes from the freezing carrier, maintaining the oocytes in the thawing solution I for 5 min, transferring to thawing solution II at 39 ℃ for 5 min, and incubating in medium 199 containing 10 wt% fetal bovine serum and overlaid with mineral oil for 4 h; wherein the incubation conditions include an incubation temperature of 39 ℃, 5% carbon dioxide, and saturated humidity; the thawing solution I is a medium obtained by adding 300 μM sucrose and 20 wt% fetal bovine serum to medium 199; and the thawing solution II is a medium obtained by adding 150 μM sucrose and 20 wt% fetal bovine serum to medium 199.

[0016] The incubation solution used in the method provided by the present application has a synergistic lipid-lowering effect due to the addition of bezafibrate and cholic acid compared with conventional medium 199 containing 10 wt% fetal bovine serum, so that the method can significantly reduce the lipid content of oocytes or improve the cryopreservation survival rate of oocytes. The present application has application prospects in reducing the lipid content of oocytes or improving the cryopreservation survival rate of oocytes. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The lipid-lowering effect of incubating porcine oocytes with incubation solution 1-8 for 4h; wherein, Figure 1 -A is the fluorescence quantitative detection graph of lipid droplets in oocytes; Figure 1 -B is the statistical analysis of the lipid-lowering effect of incubation solutions 1-3 and 8 on porcine oocytes; Figure 1 -C is the statistical analysis of the lipid-lowering effect of incubation solutions 1, 4-8 on porcine oocytes; ****: P < 0.0001;

[0018] Figure 2 The cell survival rate of porcine oocytes incubated with incubation solution 1 for 4h after freezing and thawing; wherein, Figure 2 -A is the graph of live oocytes, oocytes were incubated with diacetate fluorescein dye, and the determination of fluorescence under a fluorescence microscope was live cells; Figure 2 -B is the statistical analysis graph of oocyte survival rate. DETAILED DESCRIPTION

[0019] The advantages and characteristics of the present application will become more apparent from the following detailed description. However, it should be understood that the described embodiments are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present application without departing from the spirit and scope of the present application, and such modifications or substitutions all fall within the protection scope of the present application.

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

[0021] (1) Take 10 mL fetal bovine serum and add it to 90 mL of culture medium 199, which is Gibco culture medium 199 purchased from Thermo Fisher Scientific (China) Co., Ltd. with the item number 12340030.

[0022] (2) Take 0.0036 g of bezafibrate and dissolve it in 1 mL of anhydrous cholic acid (10 mM in DMSO).

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

[0024] Test Example 1 Test of lipid-lowering and improving the survival rate of frozen porcine oocytes

[0025] 1 Test method

[0026] 1.1 Incubation solution for testing:

[0027] (1) Incubation solution 1: incubation solution 1 prepared in Example 1; (2) Incubation solution 2: the same as incubation solution 1 prepared in Example 1 except that no cholic acid was added; (3) Incubation solution 3: the same as incubation solution 1 prepared in Example 1 except that no bezafibrate was added; (4) Incubation solution 4: incubation solution obtained by replacing bezafibrate in incubation solution 1 prepared in Example 1 with L-carnitine; (5) Incubation solution 5: incubation solution obtained by replacing bezafibrate in incubation solution 1 prepared in Example 1 with nicotinic acid; (6) Incubation solution 6: incubation solution obtained by replacing cholic acid in incubation solution 1 prepared in Example 1 with L-carnitine; (7) Incubation solution 7: incubation solution obtained by replacing cholic acid in incubation solution 1 prepared in Example 1 with nicotinic acid; (8) Incubation solution 8: incubation solution (culture medium 199 containing 10 wt% fetal bovine serum) prepared in step (1) of Example 1, as a control.

[0028] 1.2 Test method

[0029] (1) Obtaining mature porcine oocytes: the ovary was taken from a sow, a 10 mL syringe was used to puncture the surface of the ovary follicle, follicular fluid was aspirated, and then placed under a body microscope for examination. The cumulus-oocyte complex in the follicular fluid was picked out and cultured 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 repeatedly blown with a pipette to separate the cumulus from the oocyte. The oocyte was transferred to culture medium 199 and observed under a microscope. The mature oocyte was the one that expelled the first polar body.

[0030] (2) Incubation: the incubation solution overlaid with mineral oil was preheated at 39°C, 5% carbon dioxide and saturated humidity for 1 h. The mature porcine oocyte was incubated in the incubation solution for 4 h, and the incubation conditions were 39°C, 5% carbon dioxide and saturated humidity. After 4 h, the oocyte was sequentially transferred to 3 droplets of culture medium 199 to thoroughly wash away the incubation solution, and then frozen.

[0031] (3) Freezing: the oocyte was transferred to glassing freezing solution I at 39°C for 3 min, then transferred to glassing freezing solution II at 39°C for 10 s, loaded onto a freezing carrier, and then put into liquid nitrogen.

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

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

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

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

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

[0037] (5) Lipid-lowering effect evaluation: Nile red fluorescent dye was used to stain the lipid droplets in the oocyte. Laser scanning confocal microscopy was used to take pictures and quantify the lipid droplets in the oocyte. Image J software was used to digitize the image data. IBM SPSS Statistics software was used to perform single factor ANOVA analysis to determine the significance between groups. The results were expressed as mean ± standard deviation, and **** represented P < 0.0001.

[0038] (6) Freezing effect evaluation: fluorescein diacetate was used to stain the oocyte. Fluorescein diacetate can enter the cell and be converted into a green fluorescent metabolite under the action of enzymes in living cells. Fluorescence microscopy was used to take pictures, and green fluorescent cells were determined as living cells. The survival rate of each group was calculated, and IBM SPSS Statistics software was used to perform single factor ANOVA analysis to determine the significance between groups. The results were expressed as mean ± standard deviation, and *** represented P < 0.001, and **** represented P < 0.0001.

[0039] 2 Test results

[0040] 2.1 Test results of reducing the lipid content in pig oocytes

[0041] From Figure 1 -A, Figure 1 -B, it can be seen that incubating oocytes in incubation solution 1 for 4 h significantly reduced the lipid content in oocytes compared with incubation solution 8 (control group). Incubating oocytes in incubation solution 2 or incubation solution 3 for 4 h significantly increased the lipid droplet content in the cells compared with incubation in incubation solution 1, i.e., the lipid-lowering effect of a single lipid-lowering agent was significantly weaker than the combined use of two lipid-lowering agents. Therefore, the combined use of bezafibrate and cholic acid in the incubation solution 1 prepared in Example 1 has a synergistic effect on lipid reduction in pig oocytes.

[0042] From Figure 1 -A, Figure 1-C As can be seen, when the drug combination for lowering lipid content was bezafibrate and cholic acid, the lipid content in the oocytes after incubation was significantly lower than that in the oocytes incubated in the various alternative combinations (P<0.0001). Compared with incubation solutions 4-7, incubation solution 1 had the highest lipid-lowering effect. When either bezafibrate or cholic acid was replaced, the lipid content in the oocytes after incubation was not significantly different from that in the fresh control, and the lipid-lowering effect was not as good as that of incubation solution 1 prepared in Example 1.

[0043] 2.2 Test results for improving the cryopreservation survival rate of porcine oocytes

[0044] The oocytes incubated in incubation solution 1 or the oocytes incubated in incubation solution 8 (control group) were subjected to freezing, thawing and survival rate detection, respectively. Figure 2 -A, Figure 2 -B As can be seen, the survival rate of the oocytes incubated in incubation solution 8 (control group) after freezing was significantly reduced, confirming that freezing can cause a large proportion of oocytes to die or lose biological activity; and the survival rate of the oocytes incubated in incubation solution 1 after freezing was significantly higher than that of the oocytes incubated in incubation solution 8 and then frozen. Therefore, incubation solution 1 significantly improves the cryopreservation survival rate of porcine oocytes by reducing the lipid content of the oocytes.

Claims

1. A method of reducing lipid content or improving cryosurvival of oocytes, characterized by, The application relates to a method for preparing mature porcine oocytes. The method comprises the following steps: (1) preheating an incubation solution, and incubating oocytes in the preheated incubation solution; (2) freezing and thawing the incubated oocytes; wherein the incubation solution is prepared by adding bezafibrate and cholic acid into a basic cell culture medium; and the oocytes are mature porcine oocytes. The preheating method of the incubation solution in step (1) comprises the following steps: placing the incubation solution covered with mineral oil in a 39 DEG C, 5% carbon dioxide and saturated humidity environment for preheating for 1 h.

2. The method of claim 1, wherein, The basic cell culture medium is a culture medium 199 containing 10wt% fetal bovine serum.

3. The method of claim 1, wherein, The concentration of bezafibrate and cholic acid in the incubation solution is 100 muM.

4. The method of claim 1, wherein, The incubation time in step (1) is 3-5 h.

5. The method of claim 1, wherein, The incubation time is 4 h.

6. The method of claim 5, wherein, The incubation condition in step (1) comprises the following steps: incubation temperature is 37-39.5 DEG C, 4-6% carbon dioxide and saturated humidity.

7. The method of claim 1, wherein, The incubation condition in step (1) is: incubation temperature is 39 DEG C, 5% carbon dioxide and saturated humidity.

8. The method of claim 7, wherein, The freezing method in step (2) comprises the following steps: transferring the incubated porcine oocytes into 39 DEG C vitrification freezing solution I for 3 min, then transferring the oocytes into 39 DEG C vitrification freezing solution II for 10 s, and freezing the oocytes in liquid nitrogen; the vitrification freezing solution I comprises 7.5wt% ethylene glycol, 7.5wt% dimethyl sulfoxide and 20wt% fetal bovine serum culture medium 199; and the vitrification freezing solution II comprises 15wt% ethylene glycol, 15wt% dimethyl sulfoxide and 20wt% fetal bovine serum culture medium 199.

9. The method of claim 1, wherein, The thawing method in step (2) comprises the following steps: taking out the freezing carrier from the liquid nitrogen, immediately immersing the loading end of the oocytes into 39 DEG C thawing solution I to make the oocytes separate from the freezing carrier, maintaining the oocytes in the thawing solution I for 5 min, transferring the oocytes into 39 DEG C thawing solution II for 5 min, and incubating the oocytes in 10wt% fetal bovine serum culture medium 199 covered with mineral oil for 4 h; wherein the incubation condition comprises the following steps: incubation temperature is 39 DEG C, 5% carbon dioxide and saturated humidity; the thawing solution I comprises 300 muM sucrose and 20wt% fetal bovine serum culture medium 199; and the thawing solution II comprises 150 muM sucrose and 20wt% fetal bovine serum culture medium 199.

10. The method of claim 1, wherein, ​

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