CN116574673A - Application of Allylestradiol in Improving the Quality of Embryo Development in Vitro - Google Patents

Abstract

The application discloses an application of allylestrenol as an additive or a culture solution containing allylestrenol in improving the in-vitro development quality of oocytes and embryos thereof and a method for improving the in-vitro development quality of the oocytes and the embryos thereof. By the application or the method, the in vitro maturation quality of the oocyte can be improved. 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, in vitro fertilization, 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.

Description

Application of Allylestradiol in Improving the Quality of Embryo Development in Vitro

technical field

The invention relates to the technical field of molecular biology, in particular to an application of allylestradiol in improving the quality of embryonic development in vitro.

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. As the incidence of infertility has increased year by year in recent years, in vitro maturation of oocytes is a new assisted reproductive technology, which refers to the application of a small amount of gonadotropin or directly obtaining immature oocytes from the ovary without ovulation induction. The eggs, after proper in vitro culture, can promote the maturation of eggs and have the ability to fertilize, which can reduce the high cost of ovulation induction in conventional in vitro fertilization (IVF) due to reduced stimulation or no stimulation, and reduce ovarian damage caused by conventional ovulation induction drugs. The incidence of adverse reactions such as transitional irritation syndrome. At the same time, 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. In addition, in scientific research, in vitro fertilized embryos, parthenogenetic embryos, and somatic cell cloned 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. The in vitro maturation and culture of oocytes is the basis for obtaining embryos in vitro. Therefore, if the quality of in vitro maturation of oocytes can be improved, the quality of embryos obtained and cultured in vitro will also be improved. Animals are used in research in fields such as medicine, life sciences, and animal husbandry to further advance the development of production practices.

Allylestrenol is commonly used in medicine to treat threatened miscarriage, recurrent miscarriage and threatened premature labor. The effect of allylestradiol is several times that of progesterone, which can increase the endocrine activity of placental trophoblast, promote the secretion of endogenous progesterone and hCG, stimulate the placenta with poor function, and normalize the placental function. But there is no report about the relationship between allylestradiol and embryonic development in vitro.

Contents of the invention

The object of the present invention is to provide an application of allylestradiol as an additive or a culture solution containing allylestradiol in improving the quality of embryonic development in vitro and a method for improving the quality of embryonic development in vitro, so as to solve the quality problem of embryonic development in vitro.

According to the first aspect of the present invention, an application of allylestradiol in improving the quality of embryonic development in vitro is provided. As a result, the prepared high-quality embryos and the animals produced can be used for research in the fields of medicine, life sciences, animal husbandry, etc., and can further promote the development of production practices.

In certain embodiments, the embryo is a somatic cell cloned embryo or a parthenogenic embryo or an in vitro fertilized embryo.

In certain embodiments, the embryo is a porcine embryo.

According to the second aspect of the present invention, there is provided a culture solution containing allylestradiol, the components of the culture solution include: based on the TCM-199 basal culture solution, supplemented with 0.6mM cysteine, 0.1 IU/mL human chorionic gonadotropin and 0.1IU/mL pregnant horse serum gonadotropin, 10% fetal bovine serum, 10% follicular fluid and allyl estradiol.

In certain embodiments, the allylestradiol concentration is 10 ^-6 -10 ^-3 mol/L.

In certain embodiments, the allylestradiol concentration is 10 ^-5 mol/L.

According to the third aspect of the present invention, it provides an application of a culture solution containing allylestradiol in in vitro maturation of oocytes.

According to the fourth aspect of the present invention, an application of a culture solution containing allylestradiol in improving the quality of embryonic development in vitro is provided.

In certain embodiments, the embryo is a somatic cell cloned embryo or a parthenogenic embryo or an in vitro fertilized embryo.

According to a fifth aspect of the present invention, there is provided a method for improving the quality of embryonic development in vitro, the method is by using a culture solution containing allylestradiol for in vitro culture of oocytes, and then culturing mature eggs in vitro Mother cells are used in the construction of in vitro embryos. By improving the quality of in vitro cultured oocytes, the quality of embryonic development in vitro can be improved.

Beneficial effects of the application: the use of allylestradiol as an additive or a culture solution containing allylestradiol in improving the quality of embryonic development in vitro and a method for improving the quality of embryonic development in vitro are disclosed. Through the application or the method, the quality of oocyte maturation in vitro can be improved. 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, in vitro fertilization, 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 is the relative ratio of allylestrenol in mature follicular fluid (pMFF) in vivo, immature follicular fluid (pIFF) in vivo, mature medium (pMM) in vitro and immature medium (pIM) in vitro Content, different superscript letters in the figure indicate significant difference at P<0.05.

Detailed ways

Below in conjunction with accompanying drawing, invention is described in further detail.

1. Preparation of porcine oocyte maturation medium in vitro

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

a. Basic mature culture medium (control group): based on TCM-199 basic culture medium, supplemented with 0.6mM cysteine, 0.1IU/mL human chorionic gonadotropin and 0.1IU/mL pregnant horse serum gonadotropin Hormone, 10% (V/V) fetal bovine serum, 10% (V/V) porcine follicular fluid from slaughterhouse.

b. Allylestradiol maturation medium: add 10 ^-6 mol/L, 10 ^-5 mol/L, 10 ^{- 4} mol/L, and 10 ^-3 mol/L allylestradiol to the basal maturation medium .

2. In vitro maturation culture of oocytes

Extract oocytes from 3mm-8mm follicles, select cumulus cell-oocyte complexes (COCs) with uniform cytoplasm and more than three layers of cumulus cells, and place them in culture medium containing oocyte maturation in vitro in a four-well plate, and then transferred to a 38.5°C incubator for culture. 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.

Place fresh pig COCs in in vitro maturation culture medium (such as the culture medium of the control group in "1. Preparation of porcine oocyte maturation culture medium in vitro"), then collect the IVM culture medium (pIM) in this period into a frozen storage tube, and immediately Quenched in liquid nitrogen for 5 minutes and stored at -80°C for later use. After the porcine COCs were cultured in vitro for 44 hours, the IVM medium (pMM) was collected into a centrifuge tube, centrifuged at 3000rpm for 5 minutes, and the supernatant was immediately transferred to a cryovial for quenching with liquid nitrogen for 5 minutes and stored at -80°C for later use. IVM refers to in vitro maturation culture.

3. Collection of porcine follicular fluid

(1) Collection of mature follicular fluid (pMFF) in vivo

A group of gilt Duroc sows after physical maturity (about 10 months old) were orally administered allylgestrin, each sow was fed once a day, 20 mg once, and fed continuously for 18 days. 42 hours later, inject 1000IU hemotropin for injection. After 80 hours, the estrus was checked. When the vulva of the sow treated in the same period of estrus was red and swollen, mucus flowed out from the vulva, and the back was pressed to stand still, the back was arched, and the ears were erected, it indicated that ovulation was about to occur.

The sows were operated on after 24 hours. Anesthetize the sow first, and then use a breathing anesthesia machine to maintain the sow's state. In order to avoid suffocation, the sow's tongue is pulled out with surgical forceps. Make an incision about 15 cm long in the middle of the second pair of teats at the back of the sow. Along both sides of the uterus, fully expose the position of the ovary, and observe whether there is an ovulation point in the ovary.

If there is an ovulation point, it means that the sow is ovulating. At this time, the main blood vessels of the ovary are ligated, and then the ovary is removed and placed on a 10cm petri dish containing DPBS at 37°C. Then put the sow's viscera back in place, suture them and disinfect them with povidone iodine. After fully washing away the blood on the surface of the ovary with DPBS, use a 10 mL pink syringe to extract the follicle fluid from follicles with a diameter of more than 1.0 cm, place it in a 15 mL centrifuge tube, and let it stand on a metal bath at 37 °C for 10 min. Take out the supernatant and place it in a 15mL centrifuge tube, and let it stand on ice for 20 minutes; add 10mL DPBS to resuspend the previous centrifuge tube sediment, and continue to place it on a metal bath at 37°C for later use. Then, the supernatant was taken out again and placed in a 1.5 mL centrifuge tube, and centrifuged at 1500 rpm for 5 min. Finally, the supernatant was sucked out and poured into cryovials, and stored in liquid nitrogen for later use.

If there is no ovulation point, return the sow's organs, and disinfect them with povidone iodine after suturing.

(2) Collection of immature follicular fluid (pIFF) in vivo

A group of gilts matured (about 10 months old) were operated on, and the operation procedure was referred to "(1) Collection of follicular fluid (pMFF) during in vivo maturation". After the ovaries were exposed, observe whether the follicles on the ovaries were congested and enlarged. If not, the ovaries were removed and the follicular fluid was obtained according to the procedure of "(1) Collection of follicular fluid (pMFF) during in vivo maturation". Finally stored in liquid nitrogen for later use.

pIM, PMM, pMFF and pIFF were detected by non-targeted metabolomics, and after bioinformatics analysis, the relative expression levels of allylestradiol in pIM, PMM, pMFF and pIFF were detected. The results of the content of allylestradiol in the follicular fluid before and after in vivo maturation and the culture fluid before and after in vitro maturation are shown in Figure 1: it shows that the relative content of allylestradiol in pMFF is significantly higher than that in other groups, indicating that the follicles in the in vivo maturation period Allylestradiol in the liquid will be expressed in large quantities, and it is speculated that allylestradiol can help the maturation of oocytes, and then follow-up experimental research will be carried out.

4. Construction of pig embryos and in vitro culture of embryos

a. Construction of porcine cloned embryos: prepare 1 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. Construction of porcine parthenogenetic embryos: put the obtained in vitro matured oocytes into the electro-activation solution to balance for about 2 minutes, and then put them into the fusion tank with the pre-heated electro-activation solution, 100V, 100μs, 2DC activates the oocyte. Activated oocytes were cultured in PZM embryo culture medium, washed 3 times, and placed in a four-well plate added with PZM culture medium.

The composition of PZM culture solution 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, Na-Pyruvate 0.20mM, Ca-(Lactate) 2.00mM, Hypotaurine5 .00mM, L-glutamine 1.00mM, essential amino acid 20mL/L, non-essential amino acid 10mL/L.

c. Embryo culture in vitro: After the embryos were constructed, the embryos were placed in a four-well plate with PZM culture medium, transferred to a saturated humidity incubator at 38.5°C to continue culturing, and the data were recorded after 48h and 168h of culture respectively.

5. Experimental results

(1) Effects of different concentrations of allylestradiol 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 oocyte maturation experiment in vitro was divided into: the control group (that is, the concentration of allylestradiol is 0), the experimental group (the concentration of allylestradiol is 10 ^-6 , 10 ^-5 , 10 ^-4 , 10 ^-3 mol/ L), the preparation of specific culture medium is shown in "1, the preparation of porcine oocyte maturation culture medium in vitro". The results showed that adding allylestradiol to the culture medium could increase the oocyte maturation rate in vitro, and the optimal concentration group (10 ^{- 4} mol/L) could increase the oocyte maturation rate by 7.76%. Improvement (see Table 1):

Table 1 Oocyte maturation rate of allylestradiol treatment group

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

It can be seen that adding allylestradiol to oocyte maturation medium in vitro can improve oocyte maturation rate in vitro.

(2) Effects of mature oocytes treated with different concentrations of allylestradiol on embryo culture in vitro

Effects of allylestradiol supplementation on oocyte quality during oocyte maturation in vitro. The oocytes were cultured by using the "Allylestrol Maturation Medium (see "1. Preparation of Porcine Oocyte Maturation Medium")" added with allylestrol, and the mature oocytes treated with allylestrol The oocytes are prepared into somatic cell cloned embryos, and the cloned embryos are cultured in vitro. The results showed that compared with the control group, the rate of blastocysts in the 10 ^-5 mol/L allylestradiol group increased by 48.50%, and the average cell number of blastocysts increased by 98.99%. Improve the blastocyst rate and blastocyst cell number of cloned embryos. The detailed results are shown in Table 2 below:

Table 2 The effect of allylestradiol treatment group oocytes on cloned embryos

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

According to the above results, the 10 ^-5 mol/L allylestradiol group is the best concentration group in the experiment of cloning embryos, which can significantly increase the blastocyst rate and blastocyst cell number of cloned embryos. The blastocyst rate and blastocyst cell number are important indicators reflecting the quality of embryo development in vitro: the higher the blastocyst rate, the higher the embryo development efficiency; the higher the blastocyst cell number, the better the embryo development quality.

Therefore, the present invention further verified the influence of oocytes on their parthenogenetic embryos after being treated with 10 ^-5 mol/L allylestradiol. In the parthenogenetic embryo experiment, compared with the control group, the cleavage rate of the 10 ^-5 mol/L allylestradiol group increased by 8.36%, the blastocyst rate of parthenogenetic embryos increased by 11.94%, and the average number of blastocyst cells increased by 28.64%. %. The detailed results are shown in Table 3 below:

Table 3 The effect of allylestradiol treatment group oocytes on parthenogenetic embryos

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

It can be seen that the cleavage rate, blastocyst rate and blastocyst cell number of parthenogenetic embryos prepared from mature oocytes treated with allylestradiol at a concentration of 10 ^-5 mol/L were significantly higher than those in the control group .

In summary, adding allylestradiol to oocyte maturation medium in vitro, and using allylestradiol-treated mature oocytes to prepare cloned embryos or parthenogenetic embryos can improve the quality of embryos . Specifically, different concentrations of allylestradiol can increase the cleavage rate, blastocyst rate and blastocyst cell number of cloned embryos, and when the concentration of allylestradiol is 10 ^{- 5} mol/L, the blastocyst rate of cloned embryos and the number of blastocyst cells were significantly higher than those in the control group; when the concentration of allylestradiol was 10 ^-5 mol/L, the cleavage rate, blastocyst rate and blastocyst cell number of parthenogenetic embryos were significantly higher than those in the control group. It shows that allylestradiol can be used to improve the quality of embryonic development in vitro. The improvement in the quality of embryonic development in vitro is due to the improvement in the quality of in vitro maturation of oocytes used, so allylestradiol can also be used to improve the quality of in vitro maturation of oocytes.

What has been described above are only some implementations of the present invention. Those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all belong to the protection scope of the present invention. In addition, the above-mentioned embodiment of the present invention can not only be used to improve the developmental birth efficiency of pig cloned embryos and the developmental ability of parthenogenetic embryos, but also can be used to improve the embryos of other animals, such as mice, sheep, cows and cats. Development and birth efficiency, these also all belong to the protection scope of the present invention.

Claims (10)

1. The application of allylestradiol in improving the quality of embryonic development in vitro. 2. The application according to claim 1, wherein the embryo is a somatic cell cloned embryo or a parthenogenic embryo or an in vitro fertilized embryo. 3. The application according to claim 2, wherein the embryo is a pig embryo. 4. A culture solution containing allylestradiol, wherein the components of the culture solution include: based on the TCM-199 basal culture solution, supplemented with 0.6mM cysteine, 0.1IU/mL human chorionic gonadotropin Hormone and 0.1IU/mL pregnant horse serum gonadotropin, 10% fetal bovine serum, 10% follicular fluid and allyl estradiol. 5. The culture solution according to claim 4, wherein the concentration of allylestradiol is 10 ^-6 -10 ^-3 mol/L. 6. The culture solution according to claim 5, wherein the concentration of allylestradiol is 10 ^-5 mol/L. 7. The application of the culture solution described in any one of claims 4-6 in the in vitro maturation of oocytes. 8. The application of the culture solution described in any one of claims 4-6 in improving the quality of embryonic development in vitro. 9. The application according to claim 8, wherein the embryo is a somatic cell cloned embryo, a parthenogenic embryo, or an in vitro fertilized embryo. 10. A method for improving embryo in vitro development quality, wherein, the method is by using the culture fluid described in any one of claims 4-6 for the in vitro cultivation of oocytes, and then culturing mature eggs in vitro Mother cells are used in the construction of in vitro embryos. By improving the quality of in vitro cultured oocytes, the quality of embryonic development in vitro can be improved.