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

Abstract

The application discloses an oocyte in-vitro maturation culture solution added with a chemokine CCL16, and application of the culture solution or the chemokine CCL16 in oocyte in-vitro maturation and improvement of cloned embryo quality. Therefore, when the culture solution or the chemokine CCL16 is used for in-vitro maturation culture of the oocyte, the quality of in-vitro maturation culture of the oocyte can be improved, and when the mature oocyte is used for somatic cell cloning, the quality of somatic cell cloning is further improved.

Description

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

Technical Field

The application relates to the field of molecular biology, in particular to application of CCL16 in oocyte in-vitro maturation and improvement of cloned embryo quality.

Background

The oocyte in vitro maturation (in vitro maturation, IVM) technology has very important application value in the fields of life sciences such as agricultural science, human assisted reproductive medicine, biomedicine and the like. In agricultural production, using mature oocytes as recipient cells and somatic cells from living animals as donor cells, a large number of offspring having the same genetic information can be produced by somatic cell nuclear transfer techniques. This means that an excellent breeding animal can produce a large number of cloned individuals in a short time, and the production value is increased. Furthermore, in combination with gene editing technology, particularly CRISPR-Cas9 technology, is used for preparing animal models of human diseases and as a bioreactor for the production of drugs with medical application value. In addition, in scientific research, in vitro fertilized embryo and parthenogenetic embryo are important research materials, and have important roles in understanding the occurrence and reprogramming mechanism of life. Because of limited number of oocytes matured in vivo, high acquisition cost and great technical difficulty, the oocytes can not be applied to practical production and scientific research on a large scale. In contrast, low cost, IVM techniques that allow for the mass availability of mature oocytes are of increasing importance in practical applications. At present, oocytes matured in vitro are dependent on a relatively static microenvironment to support their maturation. But the quality of in vitro matured oocytes is worse than in vivo matured oocytes, and will continue to affect the subsequent developmental capacity of the embryo. This affects the efficiency of scientific research and practical production. Thus, there is a need for improved current oocyte in vitro maturation culture systems. The enhancement of oocyte maturation is facilitated by identifying changes in signal factors of the oocyte maturation microenvironment during in vivo maturation.

CCL16, also known as CC chemokine 4, liver Expressed Chemokine (LEC) and Monostatin-1 (MTN-1), is a small cytokine belonging to the CC chemokine family, a chemokine cluster located on human chromosome 17. CCL16 can affect cells by interacting with the cell surface chemokine receptors CCR1, CCR2, CCR5 and CCR 8. CCL16 is also a ligand for the histamine H4 receptor. CCL16 can chemotactic 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 may also enhance the anticancer effect of cytotoxic T cells and Dendritic Cell (DC) lymphocytes. At present, little research has been done on CCL16 in animals, particularly pigs, for its role in maturation of oocytes or in cloning embryos, even though it has not been reported.

Disclosure of Invention

The application aims to provide an additive 'chemokine CCL 16' and application thereof, so as to solve the problem of low in-vitro maturation culture of oocytes and low development quality of cloned embryos.

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

In certain embodiments, the chemokine CCL 16-added oocyte in vitro maturation medium composition comprises: based on TCM-199 basal medium, 0.6mM cysteine, 0.1IU/mL human chorionic gonadotrophin and 0.1IU/mL pregnant horse serum gonadotrophin, 10% fetal bovine serum, 10% follicular fluid and chemokine CCL16 were added.

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

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

According to a second aspect of the present application there is provided the use of a chemokine CCL16 added to an oocyte in vitro maturation medium for oocyte in vitro maturation culture.

In certain embodiments, the oocyte is a porcine oocyte.

According to a third aspect of the present application there is provided the use of an in vitro maturation medium of oocytes supplemented with the chemokine CCL16 for improving the quality of development of cloned embryos.

In certain embodiments, the oocyte is a porcine oocyte.

According to a fourth aspect of the present application, there is provided a method for improving somatic cell cloning efficiency by using a culture solution added with a chemokine CCL16 for culturing oocytes during in vitro maturation of the oocytes and using the cultured mature oocytes for somatic cell cloning, thereby improving somatic cell cloning efficiency by improving in vitro maturation quality of the oocytes.

According to a fifth aspect of the present application there is provided the use of the chemokine CCL16 in oocyte maturation culture in vitro.

According to a sixth aspect of the present application there is provided the use of the chemokine CCL16 for improving the quality of cloned embryos.

The application has the beneficial effects that: an additive "chemokine CCL16" which can be added into oocyte culture solution to improve the in vitro maturation quality of oocyte is disclosed, and then the additive is applied to the in vitro culture of oocyte to improve the in vitro maturation quality of oocyte. The quality of in vitro oocyte culture is improved, and the method is further applied to the fields of in vitro oocyte maturation, somatic cell cloning technology, parthenogenesis embryo, assisted reproduction and the like. On one hand, the method can promote the in vitro maturation of oocytes of various species, can prepare in vitro fertilized embryos and cloned embryos with better quality, and is beneficial to the in vitro maturation culture of oocytes of human assisted reproduction; on the other hand, the prepared high-quality embryo and the animals produced by the embryo are used for researching the fields of medicine, life science, animal husbandry and the like, and further promote the development of production practice.

Drawings

Fig. 1 is a graph showing that the difference in P <0.01 is very significant, showing the relative amounts of CCL16 in mature follicular fluid (pMFF) and immature follicular fluid (pIFF) in vivo.

Detailed Description

The application is described in further detail below.

1. Quantitative proteomic detection of follicular fluid

Quantitative proteomic mass spectrometry was used to detect follicular fluid before and after maturation in vivo in order to find out which factors were significantly changed during follicular maturation. Collecting in vivo mature follicular fluid in the ovaries of the oestrus sow and in vivo immature follicular fluid in the ovaries of the non-oestrus sow in a surgical mode, wherein the immature follicular fluid is selected from follicles with the diameter of 3-8 mm. And then removing high-abundance proteins in the two follicular fluids by using a high-abundance protein removal kit, preparing an on-machine sample, and then carrying out quantitative proteomics mass spectrometry detection, wherein the result shows that: the expression level of chemokine 16 (CCL 16) was extremely significantly up-regulated in mature follicular fluid (pMFF) in vivo, and the relative expression level of CCL16 protein in mature follicular fluid in vivo was 1.75 times that in immature follicular fluid (pIFF) in vivo (results shown in fig. 1). Furthermore, the diameter of mature follicles is 3-5 times that of immature follicles, that is, the absolute contents of CCL16 in both are different by at least 100 times, so that it can be reasonably presumed that CCL16 protein is a substance that plays an important role in the maturation of oocytes.

2. Preparation of in vitro maturation culture solution of pig oocyte

To investigate the role of CCL16 in oocyte maturation in vitro, the following culture solutions were prepared:

a. basal maturation medium (control): based on TCM-199 basal broth, 0.6mM cysteine, 0.1IU/mL human chorionic gonadotrophin and 0.1IU/mL pregnant horse serum gonadotrophin, 10% (V/V) fetal bovine serum, 10% (V/V) slaughterhouse derived pig follicular fluid were added.

Ccl16 maturation broth: the basic maturation medium was supplemented with 5ng/mL, 25ng/mL, and 125ng/mL of CCL16 protein, respectively.

3. In vitro maturation culture of oocytes

Oocytes were withdrawn from 3-8mm follicles, cumulus cell-oocyte complexes having three or more layers of cumulus cells with uniform cytoplasm were selected, placed in four-well plates containing an oocyte in vitro maturation medium, and transferred to a 38.5℃incubator for culture, respectively. After 44h, the cumulus cells-oocyte complex is treated by hyaluronidase, the cumulus cells are discarded, and mature oocytes with polar bodies, uniform cytoplasm and normal shape are selected for standby.

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

a. Construction of porcine cloned embryos: preparing donor cells of a 1-plate 6cm culture dish in advance, sucking and removing the culture solution when the cells grow to 80% -90%, gently cleaning for 2 times by using DPBS (phosphate buffer solution), then adding preheated trypsin digestion solution, adding 1mL of cell culture solution into a hole in a super clean workbench, blowing the bottom of the culture dish for several times by using a pipetting gun, transferring the cell suspension into a 15mL centrifuge tube, and centrifuging at 800rpm for 5min. The supernatant was discarded, 1mL of culture medium was added, and the cells were resuspended by pipetting.

And (3) performing enucleation treatment on the obtained in-vitro mature oocyte, then picking donor cells with good morphology under a microscope, injecting the donor cells into gaps of the oocyte, and performing fusion activation after all enucleated oocytes are injected. Setting parameters of an electrofusion instrument to be 50V, 50 mu s and 2DC, adopting fusion-activating solution to clean the electrofusion tank, adding 500 mu L of fusion-activating solution, transferring 10 nuclear-injected eggs into the electrofusion tank, and rotating the glass fine needle to enable the axis of an egg receptor-nuclear donor to be perpendicular to an electrode for fusion activation.

b. Embryo in vitro culture: after the embryo is constructed, the embryo is placed in a four-hole plate with PZM embryo culture solution, transferred to a saturated humidity incubator with the temperature of 38.5 ℃ for continuous culture, and the data such as the number of ova and the number of blastula are recorded after 48 hours and 168 hours of culture respectively.

PZM embryo culture fluid comprises the following components: 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 2.00mM, taurine (Hypotaurine) 5.00mM, L-glutamine 1.00mM, essential amino acids 20mL/L, and non-essential amino acids 10mL/L.

5. Experimental results

(1) Effect of different concentrations of CCL16 on oocyte maturation in vitro

For in vitro maturation of oocytes, the maturation rate is one of the important criteria for measuring the quality of oocytes. The maturation rate is the rate at which oocytes are expelled from the first polar body. The application also correspondingly completes the verification of the experiment. The oocyte in-vitro maturation culture experiment is divided into: the preparation of specific culture solutions is shown as '2' and 'preparation of in vitro maturation culture solutions of porcine oocytes', namely, CCL16 concentration is 0) and experimental groups (CCL 16 concentration is 5, 25 and 125ng/mL respectively). The results showed that the optimal concentration group (5 ng/mL) of CCL 16-added experimental group culture solution increased the oocyte maturation rate by 9.82% and other concentrations were also increased to different degrees (see Table 1):

note that: the same column of different superscripts indicates that P <0.05 is significantly different.

Thus, CCL16 can be added into the oocyte in-vitro maturation culture solution to improve the in-vitro maturation rate of the oocyte.

(2) Effect of mature oocytes treated with different concentrations of CCL16 on in vitro embryo culture when preparing cloned embryos

Effect of CCL16 addition on oocyte quality in relation to oocyte maturation in vitro. Oocytes were cultured using "CCL16 maturation medium" supplemented with CCL16, and CCL16 treated mature oocytes were prepared into cloned embryos, which were subjected to in vitro culture. The results show that: compared with a control group, the cleavage rate of the CCL16 group with 25ng/mL is improved by 11.19 percent, the blastocyst rate is improved by 25.10 percent, and the average cell number of the blastocyst is improved by 75.54 percent; in addition, the addition of additional concentrations of CCL16 also increased embryo blastocyst rate and blastocyst cell number to varying degrees. The detailed results are shown in Table 2 below:

TABLE 2 results of influence of CCL16 treatment of group oocytes on cloned embryos

Note that: the same column of different superscripts indicates that P <0.05 is significantly different.

Therefore, after CCL16 is added to culture the oocyte, the oocyte is used for somatic cell cloning, so that the blastomeres, the blastocysts and the blastocysts of the embryo can be improved, the blastocysts are important indexes for the development quality of the response cloned embryo, and the higher the number of embryo cells at the same time, the better the development quality of the embryo. Thus, culturing oocytes via CCL16 for somatic cloning can improve the quality and efficiency of cloned embryos, which also demonstrates that CCL16 can improve the quality of oocyte maturation in vitro. Chemokines, however, are polypeptide molecules that regulate cellular functions, primarily by activating G-protein coupled receptors. Chemokine activity is regulated by a number of interleukins to increase extracellular matrix secretion by cells. The extracellular matrix is beneficial in promoting expansion of the cumulus cell-oocyte complex, which means that CCL16 may enhance the quality of the oocyte maturation rate cells of the fine egg white table 1CCL16 treated group by promoting expansion of the cumulus cell-oocyte complex. By improving the in vitro maturation quality of oocytes, the development quality of embryos can be improved when the oocytes are further used for preparing in vitro embryos.

What has been described above is merely some embodiments of the present application. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit of the application.

Claims (10)

1. An oocyte in vitro maturation medium added with chemokine CCL16.

2. The culture broth of claim 1, wherein the broth components comprise: based on TCM-199 basal medium, 0.6mM cysteine, 0.1IU/mL human chorionic gonadotrophin and 0.1IU/mL pregnant horse serum gonadotrophin, 10% fetal bovine serum, 10% follicular fluid and chemokine CCL16 were added.

3. The culture broth of claim 2, wherein the chemokine CCL16 concentration is 5-125ng/mL.

4. The culture broth of claim 3, wherein the chemokine CCL16 concentration is 25ng/mL.

5. Use of the culture broth according to any of claims 1-4 for in vitro maturation of oocytes.

6. Use of the culture broth according to any of claims 1-4 for improving the developmental quality of cloned embryos.

7. The use according to claim 5 or 6, wherein the oocyte is a porcine oocyte.

8. A method for improving the cloning efficiency of somatic cells, wherein the method is to use the culture solution according to any one of claims 1 to 4 for culturing oocytes during in vitro maturation of oocytes and to use the mature oocytes for somatic cell cloning, thereby improving the cloning efficiency of somatic cells by improving the in vitro maturation quality of oocytes.

9. Use of the chemokine CCL16 in oocyte maturation culture in vitro.

10. Use of the chemokine CCL16 for improving the quality of cloned embryos.