CN116590221A - Application of CCL16 in oocyte in-vitro maturation and improvement of cloned embryo quality - Google Patents

Abstract

The invention discloses an in vitro maturation culture solution of oocytes added with chemokine CCL16, and the application of the culture solution or the chemokine CCL16 in the in vitro maturation of oocytes and the improvement of the quality of cloned embryos. Thus, when the culture solution or the chemokine CCL16 is used for in vitro maturation of oocytes, the quality of in vitro maturation of oocytes can be improved, and when mature oocytes are used for somatic cell cloning, it can further improve the quality of oocytes. Quality of cell clones.

Description

Application of CCL16 in in vitro maturation of oocytes and improvement of cloned embryo quality

technical field

The invention relates to the field of molecular biology, in particular to an application of CCL16 in in vitro maturation of oocytes and improvement of the quality of cloned embryos.

Background technique

Oocyte in vitro maturation (in vitro maturation, IVM) technology has very important application value in agricultural science, human assisted reproductive medicine and biomedicine and other life science fields. In agricultural production, mature oocytes are used as recipient cells, somatic cells from living animals are used as donor cells, and a large number of offspring with the same genetic information can be produced through somatic cell nuclear transfer technology. This means that excellent breeding animals can produce a large number of cloned individuals in a short period of time, expanding the production value. Not only that, combined with gene editing technology, especially CRISPR-Cas9 technology, is used to prepare animal models of human diseases and as a bioreactor to produce drugs with medical application value. In addition, in scientific research, in vitro fertilized embryos and parthenogenetic embryos are very important research materials, which play an important role in understanding the occurrence of life and the mechanism of reprogramming. Due to the limited number of mature oocytes in vivo, high acquisition cost and technical difficulty, it cannot be applied on a large scale in actual production and scientific research. In contrast, IVM technology, which is low-cost and can obtain a large number of mature oocytes, is becoming more and more important in practical applications. Currently, oocytes matured in vitro rely on a relatively static microenvironment to support their maturation and development. However, compared with oocytes matured in vivo, the quality of oocytes matured in vitro is worse, which will continue to affect the subsequent development of embryos. This affects the efficiency of scientific research and actual production. Therefore, it is necessary to improve the current oocyte maturation culture system in vitro. Changes in signaling factors in the oocyte maturation microenvironment during in vivo maturation can help improve oocyte maturation.

CCL16, also known as CC chemokine 4, liver-expressed chemokine (LEC) and monotactin-1 (MTN-1), is a small cytokine belonging to the CC chemokine family and is located on the chemokine cluster on human chromosome 17. CCL16 can affect cells by interacting with cell surface chemokine receptors CCR1, CCR2, CCR5 and CCR8. CCL16 is also a ligand for the histamine H4 receptor. CCL16 can chemoattract monocytes and lymphocytes, but not neutrophils. Recent studies have shown that CCL16 increases tumor rejection, antigen presentation by macrophages, and angiogenic activity of vascular endothelial cells. On the other hand, CCL16 can also enhance the anticancer effect of cytotoxic T cells and dendritic cell (DC) lymphocytes. At present, there are very few studies on CCL16 in animals, especially in pigs, and there is no report on its role in maturation of oocytes or in cloning embryos.

Contents of the invention

The purpose of the present invention is to provide an additive "chemokine CCL16" and its application to solve the problems of in vitro maturation and culture of oocytes and low development quality of cloned embryos.

According to the first aspect of the present invention, a culture medium for in vitro maturation of oocytes added with chemokine CCL16 is provided. Therefore, when the culture solution is used for in vitro maturation and culture of oocytes, the quality of in vitro maturation and culture of oocytes can be improved, and when mature oocytes are used for somatic cell cloning, the quality of somatic cell cloning can be further improved.

In some embodiments, the oocyte in vitro maturation culture medium components added with chemokine CCL16 include: based on TCM-199 basal culture medium, 0.6mM cysteine, 0.1IU/mL human chorion Gonadotropin and 0.1IU/mL pregnant horse serum gonadotropin, 10% fetal bovine serum, 10% follicular fluid and chemokine CCL16.

In certain embodiments, the concentration of the chemokine CCL16 is 5-125 ng/mL.

In certain embodiments, the concentration of the chemokine CCL16 is 25 ng/mL.

According to the second aspect of the present invention, it provides an application of oocyte in vitro maturation culture medium added with chemokine CCL16 in oocyte in vitro maturation culture.

In certain embodiments, the oocyte is a porcine oocyte.

According to the third aspect of the present invention, it provides an application of oocyte in vitro maturation culture medium added with chemokine CCL16 to improve the developmental quality of cloned embryos.

In certain embodiments, the oocyte is a porcine oocyte.

According to the fourth aspect of the present invention, a method for improving the efficiency of somatic cell cloning is provided. The method is to use the culture medium added with the chemokine CCL16 during the in vitro maturation of oocytes when performing somatic cell cloning. It is used for culturing oocytes and using the mature oocytes for somatic cell cloning. By improving the quality of in vitro maturation of oocytes, the efficiency of somatic cell cloning can be improved.

According to the fifth aspect of the present invention, an application of chemokine CCL16 in in vitro maturation of oocytes is provided.

According to the sixth aspect of the present invention, an application of chemokine CCL16 in improving the quality of cloned embryos is provided.

Beneficial effects of the present application: It discloses an additive "chemokine CCL16" that can improve the quality of oocyte maturation in vitro when added to oocyte culture fluid, and then apply this to oocyte culture in vitro to improve oocyte maturation. In vitro maturation quality of mother cells. By improving the quality of in vitro culture of oocytes, it can be applied in the fields of in vitro maturation of oocytes, somatic cell cloning technology, parthenogenetic embryos, and assisted reproduction. On the one hand, it can promote the in vitro maturation of oocytes of various species, and can prepare better-quality in vitro fertilized embryos and cloned embryos, which is helpful for the in vitro maturation and culture of oocytes for human assisted reproduction; on the other hand, the prepared high-quality embryos and their production Animals are used for research in medicine, life sciences, animal husbandry and other fields, further promoting the development of production practices.

Description of drawings

Figure 1 shows the relative content of CCL16 in mature follicular fluid (pMFF) and immature follicular fluid (pIFF) in vivo, where ** in the figure indicates a very significant difference at P<0.01.

Detailed ways

The invention will be described in further detail below.

1. Quantitative proteomic detection of follicular fluid

Quantitative proteomics mass spectrometry was used to examine follicular fluid before and after in vivo maturation in order to find out which factors were significantly changed during follicular maturation. The mature follicle fluid in the ovary of the estrus sow and the immature follicle fluid in the ovary of the non-estrus sow are collected by operation. The immature follicles are follicles with a diameter of 3-8mm. After that, the high-abundance protein in the two kinds of follicular fluid was removed with the high-abundance protein kit, and the samples were prepared on the machine, and then quantitative proteomics mass spectrometry detection was performed. The results showed that: the chemokine in mature follicular fluid (pMFF) in vivo The expression of 16 (CCL16) was extremely significantly up-regulated, and the relative expression of CCL16 protein in mature follicular fluid in vivo was 1.75 times that of immature follicular fluid (pIFF) in vivo (results are shown in Figure 1). Moreover, the diameter of mature follicles is 3-5 times that of immature follicles, which means that the absolute content of CCL16 in the two is at least 100 times different. important substances.

2. Preparation of porcine oocyte maturation medium in vitro

In order to study the role of CCL16 in the in vitro maturation of oocytes, the following culture medium was prepared:

a. Basal maturation medium (control group): Based on TCM-199 basal medium, add 0.6mM cysteine, 0.1IU/mL human chorionic gonadotropin and 0.1IU/mL pregnant horse serum gonadotropin , 10% (V/V) fetal bovine serum, 10% (V/V) porcine follicular fluid from slaughterhouse.

b. CCL16 maturation medium: 5 ng/mL, 25 ng/mL, and 125 ng/mL of CCL16 protein were added to the basal maturation medium.

3. In vitro maturation culture of oocytes

Extract oocytes from 3mm-8mm follicles, select cumulus cell-oocyte complexes with uniform cytoplasm and more than three layers of cumulus cells, and place them in four wells containing oocyte maturation medium in vitro plate, and then transferred to a 38.5°C incubator for cultivation. After 44 hours, the cumulus cell-oocyte complex was treated with hyaluronidase, the cumulus cells were discarded, and mature oocytes with polar bodies, uniform cytoplasm, and normal shape were selected for use.

4. Construction of porcine cloned embryos and in vitro culture of embryos

a. Construction of porcine cloned embryos: prepare a plate of donor cells in a 6cm culture dish in advance, when the cells grow to 80%-90%, discard the culture medium, gently wash twice with DPBS cleaning solution, and then add preheated For trypsin digestion solution, add 1mL cell culture solution to the well in the ultra-clean workbench, blow the bottom of the culture dish several times with a pipette gun, then transfer the cell suspension to a 15mL centrifuge tube, and centrifuge at 800rpm for 5min. Discard the supernatant, add 1 mL of culture medium, and pipette to resuspend the cells for later use.

The obtained in vitro matured oocytes are enucleated, and then the donor cells with better morphology are selected under the microscope and injected into the gap between the oocytes. After all the enucleated oocytes are injected, the fusion activation is carried out. Set the parameters of the electrofusion instrument to 50V, 50μs, and 2DC, use the fusion-activation solution to clean the electrofusion tank, add 500 μL of fusion activation solution, transfer 10 nucleated eggs to the electrofusion tank, and use a glass fine needle to rotate the egg receptor- The nuclear donor axis is perpendicular to the electrode for fusion activation.

b. Embryo culture in vitro: After the embryos are constructed, place the embryos in a four-well plate with PZM embryo culture medium, transfer them to a saturated humidity incubator at 38.5°C to continue the culture, and record the cleavage after 48 hours and 168 hours of culture respectively number and number of blastocysts.

The composition of PZM embryo culture medium is as follows: NaCl 108.00mM, KCl 10.00mM, KH ₂ PO ₄ 0.35mM, MgSO ₄ 7H ₂ O 0.40mM, NaHCO ₃ 25.07 mM, sodium pyruvate (Na-Pyruvate) 0.20mM, calcium lactate 2.00 mM, taurine (Hypotaurine) 5.00mM, L-glutamine 1.00mM, essential amino acid 20mL/L, non-essential amino acid 10mL/L.

5. Experimental results

(1) Effects of different concentrations of CCL16 on in vitro maturation of oocytes

For in vitro maturation of oocytes, the maturation rate is one of the important criteria to measure the quality of oocytes. The so-called maturation rate refers to the ratio of oocytes expelled from the first polar body. The present invention also completes the verification of this part of the experiment accordingly. The in vitro maturation culture experiment of oocytes was divided into: the control group (that is, the concentration of CCL16 was 0), and the experimental group (the concentrations of CCL16 were 5, 25, and 125 ng/mL respectively). For the preparation of the specific culture medium, see "2. Preparation of maturation medium". The results showed that the optimal concentration group (5ng/mL) could increase the oocyte maturation rate by 9.82% in the culture medium of the experimental group added with CCL16, and other concentrations were also improved to varying degrees (see Table 1):

Note: Different superscripts in the same column indicate significant difference at P<0.05.

It can be seen that adding CCL16 to the in vitro maturation medium of oocytes can increase the in vitro maturation rate of oocytes.

(2) Effects on in vitro culture of embryos when using mature oocytes treated with different concentrations of CCL16 to prepare cloned embryos

Effects of adding CCL16 on the quality of oocytes during in vitro maturation of oocytes. The "CCL16 maturation medium" added with CCL16 was used to culture oocytes, and the mature oocytes treated with CCL16 were prepared into cloned embryos, and the cloned embryos were cultured in vitro. The results showed that: compared with the control group, the cleavage rate of the 25ng/mL CCL16 group increased by 11.19%, the rate of blastocysts increased by 25.10%, and the average cell number of blastocysts increased by 75.54%. Increase the blastocyst rate and blastocyst cell count of embryos. The detailed results are shown in Table 2 below:

Table 2 Effect of CCL16 treatment group oocytes on cloned embryos

Note: Different superscripts in the same column indicate significant difference at P<0.05.

It can be seen that after adding CCL16 to culture oocytes, and then using the oocytes for somatic cell cloning, the cleavage rate, blastocyst rate and blastocyst cell number of embryos can be improved, and the number of blastocyst cells is the response to cloned embryos. An important indicator of developmental quality, the more embryonic cells in the same period, the better the developmental quality of the embryo. Therefore, when oocytes are cultured with CCL16 for somatic cell cloning, the developmental quality and efficiency of cloned embryos can be improved, which also proves that CCL16 can improve the quality of in vitro maturation of oocytes. Chemokines are polypeptide molecules that regulate cell functions mainly by activating G protein-coupled receptors. Chemokine activity is regulated by several interleukin factors to increase cell secretion of extracellular matrix. The extracellular matrix is beneficial to promote the expansion of the cumulus cell-oocyte complex, which means that CCL16 may enhance the oocyte maturation rate of the oocyte by promoting the expansion of the cumulus cell-oocyte complex Table 1 CCL16 treatment group cell quality. By improving the quality of in vitro maturation of oocytes, and further using it to prepare in vitro embryos, the development quality of embryos can be improved.

What have been described above are only some embodiments of the present invention. For those skilled in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention.

Claims (10)

1. A culture medium for in vitro maturation of oocytes added with chemokine CCL16. 2. The culture fluid according to claim 1, wherein the culture fluid components include: based on the TCM-199 basal culture fluid, add 0.6mM cysteine, 0.1IU/mL human chorionic gonadotropin And 0.1IU/mL pregnant horse serum gonadotropin, 10% fetal bovine serum, 10% follicular fluid and chemokine CCL16. 3. The culture fluid according to claim 2, wherein the concentration of the chemokine CCL16 is 5-125 ng/mL. 4. The culture fluid according to claim 3, wherein the concentration of the chemokine CCL16 is 25 ng/mL. 5. The application of the culture solution described in any one of claims 1-4 in oocyte maturation in vitro. 6. The application of the culture solution described in any one of claims 1-4 in improving the developmental quality of cloned embryos. 7. The application according to claim 5 or 6, wherein the oocyte is a porcine oocyte. 8. A method for improving somatic cell cloning efficiency, wherein the method is to use the culture solution according to any one of claims 1-4 in the process of in vitro maturation of oocytes when carrying out somatic cell cloning Cultivate oocytes and use the cultured mature oocytes for somatic cell cloning. By improving the quality of in vitro maturation of oocytes, the efficiency of somatic cell cloning can be improved. 9. The application of chemokine CCL16 in in vitro maturation of oocytes. 10. The application of chemokine CCL16 in improving the quality of cloned embryos.