CN116445396A - New application of WNT2 in improving development efficiency and quality of in vitro fertilized embryo - Google Patents
New application of WNT2 in improving development efficiency and quality of in vitro fertilized embryo Download PDFInfo
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Abstract
The invention provides a new application of WNT2 in improving the development efficiency and quality of in vitro fertilized embryos by enhancing the WNT signaling pathway activity in the in vitro fertilized embryos. Specifically, the obtained fertilized eggs are transferred into a culture solution added with WNT2 with proper concentration for in-vitro culture, so that the IVF blastula rate is effectively improved, the development potential is improved, and the implantation rate and the birth rate after embryo transplantation are improved. The low production efficiency of the in vitro embryo is a key factor for restricting the production of the livestock in vitro embryo and even the development of the human assisted reproduction technology. The WNT2 used in the invention is an endogenous natural protein factor secreted by a mammalian oviduct, has definite action path and target point, and ensures the safety of the factor in IVF application. Therefore, the invention provides an effective, safe and feasible strategy for improving the production efficiency and quality of the livestock and human in-vitro embryos.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a new application of WNT2 in improving development efficiency and quality of an external fertilization embryo.
Background
In vitro embryo production technology for mammals has been widely used in livestock genetic improvement, improved variety propagation, assisted reproduction for humans, etc. for the past decades. However, the in vitro embryo development culture solution has the difficult problems of low in vitro embryo development capacity and poor quality due to imperfect components, and the in vitro embryo production technology is greatly limited. Therefore, optimizing the in vitro embryo culture solution components is the most direct and effective strategy for improving the development efficiency and quality of in vitro embryos and perfecting the in vitro embryo production technology at present. At present, the screening strategy of the exogenous additive factors of the in-vitro embryo culture solution is time-consuming and labor-consuming, and has the problems of high cost, strong blindness and low efficiency.
The invention analyzes the expression pattern of the receptor ligand between the early embryo and the oviduct based on the transcriptome data of the early embryo and the oviduct in vitro and in vivo, and combines the phenotype of insufficient WNT signal pathway activity in the embryo in vitro. The first discovery and demonstration shows that adding WNT2 into the culture solution of in vitro embryo can obviously increase the total cell number and the cell number of inner cell mass of blastula, obviously improve the blastula rate of in vitro embryo, the implantation rate and the live fetal rate after embryo transplantation, and improve the development efficiency and quality of in vitro fertilized embryo.
Prior to the present invention, the beneficial effects of WNT2 on in vitro embryo development have not been found by those skilled in the art.
Disclosure of Invention
In order to solve the technical problems, the invention provides a new application of WNT2 in improving the development efficiency and quality of in vitro fertilization (in vitro fertilization, IVF) embryos.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the use of WNT2 for improving the developmental efficiency and quality of an in vitro fertilized embryo, in particular by enhancing WNT signaling pathway activity in an in vitro fertilized embryo.
For use as described above, preferably, said enhancing WNT signaling pathway activity in an in vitro fertilized embryo is achieved by adding WNT2 protein to the embryo culture broth.
For the applications described above, it is preferred that the in vitro fertilized embryo is cultured using an in vitro culture medium containing WNT2 protein.
For the above application, it is preferable that the WNT2 protein is used at an action concentration of 10 to 100ng/mL.
Further, WNT2, preferably at a working concentration of 50ng/mL, is added to the in vitro embryo culture broth.
For the applications described above, preferably, the in vitro culture broth is KSOM+AA.
As described above, preferably, the culture conditions for in vitro embryos are 37℃and 5% CO 2 And 100% humidity.
For use as described above, preferably the in vitro fertilized embryo is a mammalian in vitro fertilized embryo.
The invention also provides application of the WNT2 protein in improving the development efficiency and quality of in vitro fertilized embryos of mammals.
Use of WNT2 protein in the preparation of an in vitro culture medium for improving the development efficiency and quality of a mammalian in vitro fertilized embryo.
For the application described above, the concentration of the WNT2 protein in the in vitro medium is 10-100ng/mL.
The invention has the beneficial effects that:
the invention provides a new application of WNT2 in improving development efficiency and quality of in vitro fertilized embryos, which is to culture in vitro fertilized embryos in an in vitro culture solution containing WNT2, enhance WNT signal pathway activity of the in vitro embryos and obviously improve development efficiency and quality of the in vitro embryos.
The invention provides a new application of WNT2, in particular to an application in preparing an in vitro culture medium for improving the development efficiency and quality of in vitro fertilized embryos of mammals. WNT2 is an endogenous protein factor secreted by oviduct, has definite action path and target point, and can be used as an additive component of embryo in-vitro culture medium with safety, high efficiency and low cost.
Drawings
FIG. 1 is a heat map of the WNT signaling pathway in embryo and oviduct and uterine tissues.
FIG. 2 is a graph showing the comparison of GSVA scores of WNT signaling pathways in early in vivo and in vitro embryos.
FIG. 3 is a statistical result of the effect on embryo development rate after WNT2 addition.
FIG. 4 is a graph showing the statistical results of the effect on total blasts, inner cell mass cells and trophoblasts after WNT2 addition.
Detailed Description
The invention discovers that WNT2 can improve the development efficiency and quality of in vitro embryos through a ligand expression pattern between the early embryo and the oviduct based on transcriptome data of the early embryo and the oviduct.
The following examples serve to further illustrate the invention, which is described in more detail and should not be construed as limiting the invention. Modifications and substitutions made to the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. In the examples, the culture broth KSOM+AA was purchased from Millipore, WNT2 protein was purchased from Abnova, china, accession number H00007472-P01, ICR mice were purchased from St Bei Fu (Beijing) biotechnology Co., ltd, and M2 medium was purchased from Michaelis (Beijing) technology Co., ltd.
Example 1WNT2 enhances the developmental efficiency and quality of mouse in vitro embryos
1. Early embryo, oviduct and uterine tissue sequencing in mice
RNA of early embryo, oviduct and uterus tissue samples of mice are respectively extracted according to the Trizol method, and then sent to a BGI (Shenzhen) platform for transcriptome sequencing. The final expression value is presented as RPKM.
2. Analysis of WNT Signal pathway Gene expression in early embryo, oviduct and uterus of mice
WNT ligands act by activating their receptors, and for the study of WNT signaling pathway activity during early embryo development, cluster analysis of the receptor and LRP6 ligand expression levels of WNT signaling pathway was performed using the pheeatmap package in R software based on transcriptome data of early embryo and oviduct and uterus corresponding to its development period, see fig. 1 for specific results. Panel A shows the expression levels of WNT signaling pathway receptor mRNA in the oviduct and uterus of an early embryo and the oviduct and uterus corresponding to its developmental stage; b is the expression level of WNT signaling pathway LRP6 ligand mRNA in the fallopian tube and uterus corresponding to its developmental stage in early embryo.
The results show that WNT signal receptor genes Fzd2, fzd9 and Lrp6 are expressed in early embryo of mice, the Lrp6 gene is expressed in each period and the expression level is high, and the activation of WNT signal in embryo is probably mainly mediated by LRP6 receptor. In addition, WNT ligands that activate LRP6 receptors are highly expressed in the fallopian tube and uterus. Thus, oviduct and uterus may activate LRP6 receptors on embryos by secreting WNT ligands, activating WNT signaling, promoting early normal development.
3. Comparison of WNT Signal pathway Activity in vivo and in vitro early embryo
The in vitro culture environment of the embryo is culture solution with definite chemical components, and compared with in vivo embryo, in vitro embryo lacks ligand of WNT signal channel secreted by oviduct and uterus. To further explore the role of WNT signaling pathway in early embryo development, we first drew the mouse WNT signaling pathway from KEGG and then compared the activity of the in vitro and in vivo embryo WNT signaling pathway by GSVA package analysis in R software, see figure 2 for specific results. As can be seen from the figure, the WNT signaling pathway activity of in vitro developing embryos is significantly lower than that of in vivo developing embryos, particularly morula stage to blastula stage. Thus, insufficient WNT signaling pathway activity in an in vitro developing embryo was suggested. It was shown that LRP6 and WNT2 may play important roles in embryonic development.
4. WNT2 improves the development efficiency and quality of mouse in vitro embryos
(1) Collection of mouse in vitro fertilized embryos
Taking 8-week-old young female mice as experimental material, injecting 5IU pregnant horse serum gonadotropin (PMSG) into each female mouse abdominal cavity, injecting 5IU human chorionic gonadotrophin (hCG) into each female mouse abdominal cavity after 48 hours, killing female mice after 12 hours, taking out oviduct, placing in preheated 37 ℃ M2, drawing COCs (cumulus-oocyte complex) on the ampulla of oviduct into fertilized drop with 1ml injector, and placing CO 2 The incubator is balanced for 30 minutes, then the neck of the young rat with the age of 8 weeks is sacrificed, epididymis is sheared, sperms are gently squeezed into capacitation drops from the epididymis by forceps, and CO is put in 2 The incubator was allowed to harvest for 1 hour. After sperm capacitation, a proper amount of semen is sucked by a pipetting gun and added into fertilized drops containing COCs, so that the sperm and the COCs are incubated for 4 hours, and then fertilized eggs are selected for the step (2) according to the polar body discharge condition.
(2) Culture of mouse in vitro fertilized embryo
Washing fertilized ovum obtained in step (1) in fertilized solution for 3 times, and placing into pre-balanced mouse embryo in vitro culture solution at 37deg.C under 5% CO 2 100% humidity, and the culture time is 96 hours. The development rates of 2-cell, 4-cell, 8-cell, morula and blastocyst were recorded during this period.
The in vitro culture fluid of the mouse embryo comprises 2 components: the in vitro culture solution of the mouse embryo of the control group is KSOM+AA; the in vitro culture solution of the WNT2 group mouse embryo is added with 50ng/mL WNT2 for KSOM+AA.
(3) Blastocyst immunofluorescent staining
The blastula obtained in step (2) was washed three times with 0.1% PBS-PVA, the zona pellucida was removed with acidic Tyrode solution (T1788, sigma Germany), the embryos were transferred to 4% paraformaldehyde fixative for 1 hour at room temperature, then permeabilized with DPBS (Dulbecce phosphate buffer) containing 0.5% Triton X-100 for 1 hour at room temperature, then embryos were blocked with DPBS containing 1% BSA for 1 hour at room temperature, and incubated overnight at 4 ℃. The next day was washed three times with DPBS containing 0.5% Triton X-100 at room temperature, followed by incubation with secondary antibody for 1 hour at room temperature. Finally, the nuclei were stained by incubation with DAPI (4', 6-diamidino-2-phenylindole) for 15 minutes, fluorescent signals were observed under a BX51 microscope, photographed, and the cell numbers were counted.
Wherein the primary antibodies used were as follows: NANOG antibodies (1:500 dilution, ab80892, abcam, uk), CDX2 antibodies (1:500 dilution, MU392A-UC, biogex laboratories, usa), secondary antibodies used were as follows: alexa fluor 594 goat anti-rabbit antibody (1:1000 dilution, invitrogen, U.S. A-11034), alexa fluor 488 goat anti-mouse antibody (1:1000 dilution, invitrogen, U.S. A-11030).
(4) Mouse blastula uterus transplantation
On the day of in vitro fertilization, young female mice in natural estrus were selected for mating with ligature young males with vasectomy. The next morning, the female with the vaginal plug was considered as pseudopregnant recipient and day 0.5. Embryo transplantation is carried out on the 4.5 th day blastula obtained in the step (2), and 6 blastula with good development are selected to be transplanted to uterine horn positions at two sides of a pseudo-pregnancy receptor. On day 19.5 of embryo (14 days of embryo transfer), fetuses were obtained to assess the developmental capacity of each embryo, as follows: the mother mice are sacrificed by cervical fracture, the uterus is taken out, the number of implantation sites is counted, then the fetuses and the placenta are taken out respectively, and the number of live fetuses is recorded.
The results of the above steps (2, 3, 4) are shown in FIG. 3, FIG. 4, and Table 1, respectively, in which the abscissa of the "control group" represents the use of KSOM+AA to culture in vitro embryos, and the "control group +50ng/mL WNT2" represents the addition of 50ng/mL WNT2 protein on the basis of KSOM+AA to culture in vitro embryos.
FIG. 3 is a statistical result of the effect on embryo development rate after WNT2 addition: zygate (fertilized egg), morula (Morula), blastctst (blastocyst), fragmented (fragmentation),
as can be seen from the figure, the blastocyst rate after adding WNT2 to the in vitro embryo culture solution (65.56.+ -. 1.57%) was significantly higher than that of the control group without WNT2 (40.93.+ -. 2.58%) (p < 0.05) after 96 hours of fertilization.
Fig. 4 is a statistical result of the effect of the total number of blasts, the number of inner cell mass cells and the number of trophoblasts after WNT2 addition, and it can be seen from the figure that the total number of blasts (71.8±10.0) after WNT2 addition in the in vitro embryo culture solution is not significantly different from the control group (71.8±12.1) (p=0.997), the number of inner cell mass cells (13.72±3.69) is significantly higher than the control group (16.33±4.96) without WNT2 addition (p < 0.05), and the ratio of the number of inner cell mass cells to the number of trophoblasts (31.50±13.39) is significantly higher than the control group (24.44±7.79%) without WNT2 addition (p < 0.05).
Table 1 shows the statistical results of the effect of WNT2 addition on the ability of embryos to develop after embryo transfer
As can be seen from Table 1, the blastocysts obtained after adding WNT2 to the in vitro embryo culture solution, after embryo transfer, had significantly higher implantation rates (82.87.+ -. 15.33%) and live fetal rates (39.81.+ -. 17.25%) than those of the control group without WNT2 (66.13.+ -. 23.37%) and (30.11.+ -. 13.50%) (p < 0.05).
In conclusion, the method for activating the WNT signal channel by adding the WNT2 protein into the in vitro culture solution effectively improves the blastocyst rate of the in vitro fertilized embryo, improves the development potential of the in vitro fertilized embryo, improves the implantation rate and the fetal survival rate of the in vitro fertilized embryo, and effectively improves the production efficiency of the in vitro embryo.
Claims (9)
1. Use of WNT2 for improving the development efficiency and quality of an in vitro fertilized embryo by enhancing WNT signaling pathway activity in the in vitro fertilized embryo.
2. The use according to claim 1, wherein said enhancing WNT signaling pathway activity in an in vitro fertilized embryo is achieved by adding WNT2 protein to the embryo culture broth.
3. Use according to claim 2, wherein the embryo after in vitro fertilization is cultivated using an in vitro culture medium comprising WNT2 protein.
4. The use according to claim 3, wherein the WNT2 protein has an action concentration of 10-100ng/mL.
5. The use according to claim 3, wherein the in vitro culture medium is ksom+aa.
6. The use according to claim 1, wherein the in vitro fertilized embryo is a mammalian in vitro fertilized embryo.
Use of wnt2 protein for improving the developmental efficiency and quality of a mammalian in vitro fertilized embryo.
Use of wnt2 protein in the preparation of an in vitro culture medium for improving the development efficiency and quality of a mammalian in vitro fertilized embryo.
9. The use according to claim 7, wherein the WNT2 protein is present in the in vitro medium at a concentration of 10-100ng/mL.
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