CN111778204A

CN111778204A - Oocyte in-vitro maturation culture solution additive and application thereof

Info

Publication number: CN111778204A
Application number: CN202010531982.2A
Authority: CN
Inventors: 石俊松; 吴珍芳; 周荣; 麦然标; 罗绿花; 蔡更元; 纪红美; 余婉娴
Original assignee: Wens Foodstuff Group Co Ltd
Current assignee: Wens Foodstuff Group Co Ltd
Priority date: 2020-06-11
Filing date: 2020-06-11
Publication date: 2020-10-16
Anticipated expiration: 2040-06-11
Also published as: CN111778204B

Abstract

The invention discloses an additive of an oocyte in-vitro maturation culture solution, which comprises ceruloplasmin. Therefore, the first polar body discharge rate of the oocyte can be obviously improved, the ROS content of the oocyte can be obviously reduced, the embryo blastocyst rate after parthenogenetic activation can be obviously improved, and the maturation rate and the quality of the oocyte can be greatly improved.

Description

Oocyte in-vitro maturation culture solution additive and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an oocyte in-vitro maturation culture solution additive and application thereof.

Background

The in vitro fertilization embryo, the clone embryo, the parthenogenetic embryo and the transgenic embryo of the mammal are widely applied to the technical fields of embryo engineering such as genetic improvement, the research of early development mechanism of the embryo, epigenetic regulation and the like, the in vitro culture quality of the oocyte is a key factor for determining the development of the embryo, but the maturation efficiency and the quality of the in vitro culture of the oocyte are not greatly improved so far. Reactive Oxygen Species (ROS) is an important factor affecting the in vitro culture of oocytes. ROS are a product of biological aerobic metabolism and include oxygen ions, superoxide ions, hydroxyl radicals, hydrogen peroxide, and the like. In equilibrium, ROS play a beneficial role as signaling molecules in physiological processes such as hormonal signaling, intracellular redox regulation, and embryonic development. However, in vitro culture is a static environment, without the exchange of nutrients and metabolites, and is prone to cause the accumulation of ROS, thereby altering their function and impairing cell survival. Therefore, ROS negatively affect oocyte viability, gene expression, protein synthesis and molecular signaling, affecting oocyte maturation and developmental competence.

Disclosure of Invention

The invention aims to provide an oocyte in-vitro maturation culture solution additive and application thereof, so as to solve the problems.

According to one aspect of the present invention, there is provided an additive for a culture solution for in vitro maturation of oocytes, the additive comprising ceruloplasmin. Therefore, the first polar body discharge rate of the oocyte can be obviously improved, the ROS content of the oocyte can be obviously reduced, the embryo blastocyst rate after parthenogenetic activation can be obviously improved, and the maturation rate and the quality of the oocyte can be greatly improved.

In certain embodiments, the chalcocyanin concentration of the oocyte in vitro maturation medium additive is 1.25-10 μ g/ml. Therefore, the maturative rate of the oocytes cultured by the ceruloplasmin additive in the concentration range is higher, and the quality is better.

In certain embodiments, the chalcocyanin concentration of the oocyte in vitro maturation medium additive is 2.5 μ g/ml. Therefore, the oocytes cultured in vitro with the addition of ceruloplasmin at a concentration of 2.5. mu.g/ml had the highest maturation rate and the best quality.

According to another aspect of the present invention, there is provided a culture solution for in vitro maturation of oocytes, the culture solution comprising ceruloplasmin; or 1.25-10 μ g/ml ceruloplasmin; or 2.5. mu.g/ml ceruloplasmin is included. Therefore, the oocyte in-vitro maturation culture solution containing ceruloplasmin/or 1.25-10 mu g/ml ceruloplasmin/or 2.5 mu g/ml ceruloplasmin is used, so that the oocyte in-vitro maturation rate and the in-vitro maturation quality can be greatly improved.

In certain embodiments, the oocyte in vitro maturation medium includes: basic solution, 0.6mM cysteine, 0.91mM sodium pyruvate, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10% volume fraction follicular fluid, 10IU/mL pregnant mare serum gonadotropin, 10IU/mL human chorionic gonadotropin and ceruloplasmin. Therefore, the culture solution can greatly improve the in vitro maturation rate and the in vitro maturation quality of the oocyte.

In certain embodiments, the oocyte in vitro maturation medium includes: basic solution, 0.6mM cysteine, 0.91mM sodium pyruvate, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10% volume fraction follicular fluid, 10IU/mL pregnant mare serum gonadotropin, 10IU/mL human chorionic gonadotropin and 1.25-10 mug/mL ceruloplasmin. Therefore, the culture solution can greatly improve the in vitro maturation rate and the in vitro maturation quality of the oocyte.

In certain embodiments, the oocyte in vitro maturation medium includes: basic solution, 0.6mM cysteine, 0.91mM sodium pyruvate, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10% volume fraction follicular fluid, 10IU/mL pregnant mare serum gonadotropin and 10IU/mL human chorionic gonadotropin, 2.5 mug/mL ceruloplasmin. Therefore, the culture solution has the best effect on improving the in vitro maturation rate and the in vitro maturation quality of the oocyte.

According to another aspect of the invention, the application of the additive of the culture solution for the in vitro maturation of the oocyte in the somatic cell cloning technology is provided. Therefore, the in vitro maturation rate and quality of the oocyte can be greatly improved, and the success rate and quality of the somatic cell cloned embryo can be improved when the oocyte cloning method is applied to a somatic cell cloning technology.

In certain embodiments, the oocyte in vitro maturation medium additive used in the somatic cloning technology is 1.25-10 μ g/ml ceruloplasmin. Therefore, the in vitro maturation rate and quality of the oocyte can be greatly improved, and the success rate and quality of the somatic cell cloned embryo can be improved when the oocyte in vitro maturation rate and quality are further applied to a somatic cell cloning technology.

In certain embodiments, the oocyte in vitro maturation medium additive applied to the somatic cloning technique is 2.5 μ g/ml ceruloplasmin. Therefore, the in vitro maturation rate and quality of the oocyte can be improved with the best effect, and the success rate and quality of the somatic cell cloned embryo can be improved when the oocyte in vitro maturation rate and quality are further applied to a somatic cell cloning technology.

The invention has the beneficial effects that:

1. the ceruloplasmin is added into the oocyte maturation culture solution, so that the first polar body discharge rate of the oocyte can be obviously improved, the ROS content of the oocyte can be obviously reduced, the embryo blastocyst rate after parthenogenetic activation can be obviously improved, and the maturation rate and the quality of the oocyte can be greatly improved.

2. The maturative culture solution for the oocytes containing the ceruloplasmin can obviously improve the first polar body discharge rate of the oocytes, obviously reduce the ROS content of the oocytes and obviously improve the blastocyst rate of embryos after parthenogenetic activation, so that the maturative rate and the quality of the oocytes can be greatly improved.

3. The ceruloplasmin is applied to the somatic cell cloning technology and is used as an additive of an oocyte maturation culture solution, the first polar body discharge rate of the oocyte can be obviously improved, the ROS content of the oocyte can be obviously reduced, the embryo blastocyst rate after parthenogenesis activation can be obviously improved, the maturation rate and the quality of the oocyte can be greatly improved, and the ceruloplasmin is further applied to the somatic cell cloning technology and can improve the success rate and the quality of the embryo cloned by the somatic cell.

Drawings

FIG. 1 is a fluorescence plot of the effect of control treatment on oocyte ROS levels;

FIG. 2 is a fluorescence plot of the effect of 1.25. mu.g/ml of ceruloplasmin treatment on oocyte ROS levels;

FIG. 3 is a fluorescence plot of the effect of 2.5. mu.g/ml of ceruloplasmin treatment on oocyte ROS levels;

FIG. 4 is a fluorescence plot of the effect of 5. mu.g/ml of ceruloplasmin treatment on oocyte ROS levels;

FIG. 5 is a fluorescence plot of the effect of 10. mu.g/ml ceruloplasmin treatment on oocyte ROS levels;

FIG. 6 is a graph showing the results of different concentrations of ceruloplasmin treatment on the ROS level in oocytes.

Detailed Description

The invention is described in further detail below with reference to the accompanying drawings.

1. Oocyte collection and maturation culture

Ovaries of young sows were harvested from a slaughterhouse, returned to the laboratory in 32 ℃ saline, washed 3 times with saline supplemented with antibiotics, 2-6mm follicles were aspirated by a 10mL syringe equipped with an 18G needle, the follicular fluid was collected in a 50mL conical centrifuge tube, the supernatant was discarded after natural sedimentation for 10min, the sediment was resuspended in DPBS containing 0.05% (wt/vol) PVA, Cumulus oophorus-oocyte complexes (COCs) that wrapped more than 3 layers of Cumulus cells and homogenized cytoplasm were collected under a stereomicroscope, and after three washes in maturation medium, the COCs were transferred to four-well Nunc dishes containing 500 μ L of maturation medium and equilibrated overnight in an incubator at 39 ℃, 5% CO2, saturated humidity. 50 COCs are put into a culture dish with one hole, the temperature of the culture dish is 39 ℃, and 5 percent CO is added₂And culturing for 44h in an incubator with saturated humidity. Mixing the mature cultured COCs with 0.1% hyaluronidase, repeatedly sucking and spitting by using a pipette to remove cumulus cells, and selecting oocytes under a stereoscopic microscope, wherein the oocytes with obvious perivitelline gaps, no impurities, uniform cytoplasm and obviously discharged first polar bodies are mature oocytes, and the oocytes without perivitelline system and cytoplasm divergence are regarded as dead oocytes.

The mature culture solution is as follows: TCM-199(Gibco) base solution, added with 0.6mM cysteine, 0.91mM sodium pyruvate, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10% volume fraction follicular fluid, 10IU/mL pregnant horse serum gonadotropin (PMSG), and 10IU/mL human chorionic gonadotropin (hCG).

Mature cultures of COCs are divided into five groups: control group (mature culture without adding ceruloplasmin), 1.25. mu.g/ml group (mature culture with 1.25. mu.g/ml ceruloplasmin added), 2.5. mu.g/ml group (mature culture with 2.5. mu.g/ml ceruloplasmin added), 5. mu.g/ml group (mature culture with 5. mu.g/ml ceruloplasmin added), 10. mu.g/ml group (mature culture with 10. mu.g/ml ceruloplasmin added). After the maturation culture for 44h, the discharge rate of the first polar body of the group cells of 2.5 mu g/ml is obviously improved (P is less than 0.05), and the maturation rate of the oocyte can be obviously improved (P is less than 0.05) by adding the maturation culture solution of Ceruloplastin (CP) of 2.5 mu g/ml, and the results are shown in the table 1.

TABLE 1 influence of ceruloplasmin on oocyte maturation

Group of	Number of eggs cultured	Mortality of oocytes	First pole body discharge rate
				Control group	725	126(17.38％)	441(60.83％)^a
1.25 μ g/ml group	530	95(20.00％)	338(63.77％)^ab
				2.5 μ g/ml group	589	87(14.77％)	389(66.04％)^b
5 μ g/ml group	559	88(18.60)	351(62.79％)^ab
				10 μ g/ml group	599	111(21.65％)	361(60.27％)^a

Remarking: data from 5 replicates were statistically analyzed and different lower case letters in the same column indicated significant differences (P < 0.05), as follows.

2. Mature oocyte ROS level detection

The ROS level in oocytes was detected using the ROS detection kit (Sigma-Aldrich). After incubation of oocytes in serum-free medium containing 10 μ M DCFH-DA ((2,7-Dichlorodi-hydrofluorescein diacetate) for 1h without light, the oocytes were washed 1-2 times with serum-free medium, fluorescence signals were detected and photographed on a Leeka fluorescence microscope, and the relative ratio of fluorescence intensity of the oocytes was analyzed using ImageJ software.

3. Oocyte parthenogenetic activation, in vitro culture and blastocyst cell counting

Five groups of matured oocytes were transferred to an activating solution (0.25mM mannitol, 0.05mM MgCl2, 0.05mM CaCl2 and 0.5mM Hepes, 0.01% PVA (w/v)) for equilibration for 1min, then transferred to a chamber filled with the activating solution between two electrodes spaced 0.5cm apart, and the oocytes were activated for 80 μ s with electrical stimulation of 80kV/cm direct current pulses using a BLS fusion apparatus. The activated oocytes were transferred to PZM-3 medium and cultured in 5% CO2, 5% O2, saturated humidity incubator for 7 days. Cleavage rate and blastocyst formation rate were measured at 48h and 168h, respectively. Selecting parthenogenetic embryo which has developed into blastocyst, fixing with 4% paraformaldehyde for 10min, staining in 10mg/L Hoechst33342 for 10min, tabletting, and observing under fluorescent microscope to record blastocyst cell number. The mature oocytes are cultured by adding 2.5 mu g/ml ceruloplasmin group in the mature culture solution, the blastocyst rate of the embryo after parthenogenetic activation is obviously higher than that of a control group (P is less than 0.05), and the results are shown in Table 2.

TABLE 2 development and quality of maturated oocyte parthenogenetic embryos cultured with ceruloplasmin-supplemented groups

What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept herein, and it is intended to cover all such modifications and variations as fall within the scope of the invention.

Claims

1. An oocyte in vitro maturation culture solution additive, wherein the additive comprises ceruloplasmin.

2. The oocyte in vitro maturation culture medium additive according to claim 1, wherein the ceruloplasmin concentration is 1.25-10 μ g/ml.

3. The oocyte in vitro maturation medium additive according to claim 2, wherein said ceruloplasmin concentration is 2.5 μ g/ml.

4. An oocyte in vitro maturation culture solution, wherein the culture solution comprises the additive of any one of claims 1 to 3.

5. The culture solution of claim 4, wherein the culture solution comprises: basic solution, 0.6mM cysteine, 0.91mM sodium pyruvate, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10% volume fraction follicular fluid, 10IU/mL pregnant mare serum gonadotropin, 10IU/mL human chorionic gonadotropin and ceruloplasmin.

6. The culture solution of claim 5, wherein the culture solution comprises: basic solution, 0.6mM cysteine, 0.91mM sodium pyruvate, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10% volume fraction follicular fluid, 10IU/mL pregnant mare serum gonadotropin, 10IU/mL human chorionic gonadotropin and 1.25-10 mug/mL ceruloplasmin.

7. The culture solution of claim 6, wherein the culture solution comprises: basic solution, 0.6mM cysteine, 0.91mM sodium pyruvate, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10% volume fraction follicular fluid, 10IU/mL pregnant mare serum gonadotropin and 10IU/mL human chorionic gonadotropin, 2.5 mug/mL ceruloplasmin.

8. The application of oocyte in vitro maturation culture solution additive in somatic cell cloning technology.

9. The use according to claim 8, wherein the additive is ceruloplasmin at 1.25-10 μ g/ml.

10. Use according to claim 9, wherein the additive is ceruloplasmin at 2.5 μ g/ml.